Novel Information on Amyoyrophic Lateral Sclerosis and Spinal Muscular Atrophic

**75**

**Chapter 5**

*Junling Wang*

**Abstract**

genetic therapy

**1. Introduction**

Studies on ALS

Novel Aspects on Motor Neuron

At present, with the advanced affordable genetic testing, the rate of discovering amyotrophic lateral sclerosis (ALS)-related genes rapidly increases. These genetic findings provide new insights into therapies that target genetic subset of ALS. However, the research on the genetic and environmental causes of ALS is still in the early stage. In this chapter, we review the current understanding of ALS-related genes and summarize the worldwide ALS distribution feature by the frequency of occurrence in different regions. We summarize the advances in genetic testing and counseling for ALS. Based on the increase in genetic testing, we believe that the ALS

patients and families would be benefited from our studies in the near future.

**Keywords:** ALS, genetic frequency features, GWAS, genetic testing, genetic counseling,

Amyotrophic lateral sclerosis (ALS) is an adult onset and generally fatal neurodegenerative disease characterized by progressive weakness and atrophy of voluntary skeletal muscles due to dysfunction and death of upper and lower motor neurons. Onset typically occurs between 60 and 69 years of age, with wide-range severity. About 90% of cases are sporadic amyotrophic lateral sclerosis (SALS), while familial amyotrophic lateral sclerosis (FALS) accounts for the remaining 10% cases [1]. The pathogenesis of ALS remains obscure, but genetic mutations have been accounted for several impaired cellular and molecular mechanisms and, thus,

The superoxide dismutase 1 (*SOD1*), identified in 1993, was the first gene discovered to be associated with ALS. Subsequently, several gene mutations that have been identified to cause ALS. Till 2018, more than 180 genes have been identified as causative genes or related genes of ALS. Many of these genes are related to metabolism, trafficking of RNA, and chromatin, including *C9orf 72*, *TDP43*, *FUS*, *TAF15*, *ELP3*, *ANG*, *hnRNPA1*, and *hnRNPA2B1* [2]. Some genes are involved in conformational instability and aggregation of proteins, such as *SOD1*, *VCP*, *OPTN*, and *UBQLN2*; others are related to axonal and cytoskeletal biology, such as *PFN1*,

In most cases, FALS is inherited in the dominant pattern and the penetrance is associated with age. It has been observed that the differences of age onset and disease progression within and between ALS families are significant. In addition, some ALS

provide clues for potential therapeutic strategies.

*DCTN1*, *TUBA4A*, and *EPHA4* [2].

Disease: The Recent Genetic

#### **Chapter 5**

## Novel Aspects on Motor Neuron Disease: The Recent Genetic Studies on ALS

*Junling Wang*

### **Abstract**

At present, with the advanced affordable genetic testing, the rate of discovering amyotrophic lateral sclerosis (ALS)-related genes rapidly increases. These genetic findings provide new insights into therapies that target genetic subset of ALS. However, the research on the genetic and environmental causes of ALS is still in the early stage. In this chapter, we review the current understanding of ALS-related genes and summarize the worldwide ALS distribution feature by the frequency of occurrence in different regions. We summarize the advances in genetic testing and counseling for ALS. Based on the increase in genetic testing, we believe that the ALS patients and families would be benefited from our studies in the near future.

**Keywords:** ALS, genetic frequency features, GWAS, genetic testing, genetic counseling, genetic therapy

#### **1. Introduction**

Amyotrophic lateral sclerosis (ALS) is an adult onset and generally fatal neurodegenerative disease characterized by progressive weakness and atrophy of voluntary skeletal muscles due to dysfunction and death of upper and lower motor neurons. Onset typically occurs between 60 and 69 years of age, with wide-range severity. About 90% of cases are sporadic amyotrophic lateral sclerosis (SALS), while familial amyotrophic lateral sclerosis (FALS) accounts for the remaining 10% cases [1]. The pathogenesis of ALS remains obscure, but genetic mutations have been accounted for several impaired cellular and molecular mechanisms and, thus, provide clues for potential therapeutic strategies.

The superoxide dismutase 1 (*SOD1*), identified in 1993, was the first gene discovered to be associated with ALS. Subsequently, several gene mutations that have been identified to cause ALS. Till 2018, more than 180 genes have been identified as causative genes or related genes of ALS. Many of these genes are related to metabolism, trafficking of RNA, and chromatin, including *C9orf 72*, *TDP43*, *FUS*, *TAF15*, *ELP3*, *ANG*, *hnRNPA1*, and *hnRNPA2B1* [2]. Some genes are involved in conformational instability and aggregation of proteins, such as *SOD1*, *VCP*, *OPTN*, and *UBQLN2*; others are related to axonal and cytoskeletal biology, such as *PFN1*, *DCTN1*, *TUBA4A*, and *EPHA4* [2].

In most cases, FALS is inherited in the dominant pattern and the penetrance is associated with age. It has been observed that the differences of age onset and disease progression within and between ALS families are significant. In addition, some ALS

is recessive inherited, such as *OPTN*, *SPG11*, *FUS*, and *SOD1* (definite, Asp90Ala homozygous mutation), and *UBQLN2 associated ALS* is X-linked dominant inherited. Besides causative genes, multiple genetic variants interact simultaneously to increase ALS susceptibility. Considering oligogenic manner of ALS described by some researchers, many ALS patients may not appear to be familial in a conventional Mendelian manner. Therefore, the oligogenic manner may underlie the apparently sporadic form of the disease [3].

Some ALS caused by specific genetic mutations exhibit unique clinical characteristics. For example, ALS associated with *SPG11* and *ALS3* has clinical features of early onset and slow progression. *SPG11* mutations were identified in autosomal recessive juvenile ALS [4], and the patients with *ALS3* mutations have an early onset of approximately 45 years and the average disease duration about 5 years.

Recently, progress in gene discovery and technology has both complicated and empowered the process of genetic testing options, which may help neurological clinicians and ALS patients to understand the pathogenesis of ALS, and then provide genetic counseling for family members, allow accurate risk assessment, and open the door for genotype-specific treatments. As the genetic basis of the remainder of FALS, and potentially SALS, is unraveled, genetic testing and counseling will become increasingly vital and should be incorporated into the routine management of ALS [4–6].

#### **2. Recent advances in ALS gene map**

#### **2.1** *SOD1*

The superoxide dismutase 1 (*SOD1*), located in 21q22, was discovered in 1993. Up to date, more than 180 mutations have been described to be associated with ALS, while most of these mutations are missense mutations. Its mutation probability accounts for 20% of FALS cases and 1–2% of SALS cases [1, 2, 7, 8].

#### **2.2** *C9orf 72*

The *C9orf 72*, located in 9p21.2, was discovered in 2011. The protein encoded by *C9orf 72* is mainly related to autophagy, endosomal transport, and immune function. According to statistics, about 40–50% of FALS and 10% of SALS carried the *C9orf 72* expanded alleles. The pathogenic alleles of *C9orf 72* may have hundreds or even thousands of the GGGGCC hexanucleotide repeats. A large number of clinical investigations have shown that about 700–1600 GGGGCC hexanucleotide repeats are inserted into the intron located between the two untranslated optional exons 1a and 1b of the *C9orf 72* gene [1–3, 9, 10].

#### **2.3** *FUS*

The FUS RNA-binding protein (*FUS*) gene, discovered in 2009, was mapped in 16p11.2.

Mutations in *FUS* are observed in 4% of FALS and 1% of SALS. At present, more than 79 mutations have been described, predominantly in the 3′ region encoding an arginine/glycine-rich region and a NLS domain (nuclear localization signal). FUS protein, essentially localized in the nucleus, regulates RNA processing, splicing, and mRNA trafficking. Mutant FUS localized to cytoplasmic stress granules (SGs) and interacted with the stress granule protein PABP in an RNA-dependent manner resulting in mislocalization of the wildtype protein to stress granules [7–9, 11].

**77**

*Novel Aspects on Motor Neuron Disease: The Recent Genetic Studies on ALS*

*CCNF*, located in 16p13.3, encoding the cyclin F, was first reported in 2016. The mutation of *CCNF* accounts for approximately 4% of FALS and 2% of SALS [7]. CCNF protein is a kind of kithe cell cyclin, involved in the regular of cell cycle transitions by activating cyclin-dependent protein kinases. Furthermore, it is the substrate recognition component of the Skp1-cullin-F-box E3 ubiquitin ligase complex. The neurons of the over-expressed mutant CCNF showed an increase in protein-labeled proteins, including TDP43. It indicates that mutated-CCNF protein interfere the proteasome degradation pathway by using traversing protein abnormalities to mark all proteins or inhibiting transferring transprotein-labeled proteins to proteasome complexes. This finding suggests that the *CCNF* mutation may cause abnormal protein homeostasis, which may be exacerbated by TDP43 protein disease. Therefore, enhancing protein removal or reducing ubiquitin may be a feasible

The *TIA1* gene, located in 2p13.3, encodes an RNA-binding protein involved in splicing regulation and translational repression. The mutations in *TIA1* were identified in 2.2% of FALS and 0.4% of SALS [7]. TIA1 protein is a key component of SGs, cytoplasmic foci sequester untranslated mRNAs upon different types of cellular stress, and the low complexity domain (LCD) region of TIA1 plays a central role in promoting SGs assembly. A heterozygous founder mutation (E384K) in the LCD was first reported in Swedish/Finnish patients as the cause of Welander distal myopathy (WDM). Recently, a mutation (p.P362L) in *TIA1* affecting a highly conserved residue in the LCD was identified as one cause of ALS/ALS-FTD [12].

The TANK-binding kinase 1 (*TBK1*) gene, located in 12q14.2, was discovered in 2015 [7]. The protein encoded by this gene is similar to the IκB kinase and can mediate NFκb activation in response to certain growth factors. The *TBK1* mutations were found in approximately 1% of FALS and 1% of SALS. The clinical phenotypes associated with the *TBK1* mutation are heterogeneous, with different ages at onset,

Some patients also reported with extrapyramidal, ataxia, or psychosis. Neuropathological examination of central nervous system (CNS) tissues from patients with *TBK1* mutations revealed that SQSTM1/p62- and TDP-43-positive inclusion bodies which can indicate abnormalities in TDP-43 protein aggregation

The *TARDBP* (trans-activation element DNA-binding protein), located in

The *TARDBP* gene mutation was found in 5% of FALS cases and 1% in SALS cases. Till now, more than 50 different mutations have been identified [9]. Except for D169G, a majority of these mutations are located in the 3′ region encoding a glycine-rich domain in its product, TDP-43. ALS patients carrying *TARDBP* gene mutations normally exhibit a classical ALS phenotype and rare dementia, they also have earlier disease onset, with upper limb onset being more common and compatible with a longer life. Most of TDP-43 is expressed in nucleus, involved in RNA

different progressions, and irregular survival times.

and protein clearance pathways [1, 2, 4, 5].

1p36.22, was discovered in 2008 [5].

*DOI: http://dx.doi.org/10.5772/intechopen.82085*

**2.4** *CCNF*

treatment [1, 3].

**2.5** *TIA1*

**2.6** *TBK1*

**2.7** *TARDBP*

*Novel Aspects on Motor Neuron Disease: The Recent Genetic Studies on ALS DOI: http://dx.doi.org/10.5772/intechopen.82085*

#### **2.4** *CCNF*

*Novel Aspects on Motor Neuron Disease*

sporadic form of the disease [3].

management of ALS [4–6].

**2.1** *SOD1*

**2.2** *C9orf 72*

**2.3** *FUS*

16p11.2.

**2. Recent advances in ALS gene map**

and 1b of the *C9orf 72* gene [1–3, 9, 10].

is recessive inherited, such as *OPTN*, *SPG11*, *FUS*, and *SOD1* (definite, Asp90Ala homozygous mutation), and *UBQLN2 associated ALS* is X-linked dominant inherited. Besides causative genes, multiple genetic variants interact simultaneously to increase ALS susceptibility. Considering oligogenic manner of ALS described by some researchers, many ALS patients may not appear to be familial in a conventional Mendelian manner. Therefore, the oligogenic manner may underlie the apparently

Some ALS caused by specific genetic mutations exhibit unique clinical characteristics. For example, ALS associated with *SPG11* and *ALS3* has clinical features of early onset and slow progression. *SPG11* mutations were identified in autosomal recessive juvenile ALS [4], and the patients with *ALS3* mutations have an early onset of approximately 45 years and the average disease duration about 5 years.

Recently, progress in gene discovery and technology has both complicated and empowered the process of genetic testing options, which may help neurological clinicians and ALS patients to understand the pathogenesis of ALS, and then provide genetic counseling for family members, allow accurate risk assessment, and open the door for genotype-specific treatments. As the genetic basis of the remainder of FALS, and potentially SALS, is unraveled, genetic testing and counseling will become increasingly vital and should be incorporated into the routine

The superoxide dismutase 1 (*SOD1*), located in 21q22, was discovered in 1993. Up to date, more than 180 mutations have been described to be associated with ALS, while most of these mutations are missense mutations. Its mutation probability

The *C9orf 72*, located in 9p21.2, was discovered in 2011. The protein encoded by *C9orf 72* is mainly related to autophagy, endosomal transport, and immune function. According to statistics, about 40–50% of FALS and 10% of SALS carried the *C9orf 72* expanded alleles. The pathogenic alleles of *C9orf 72* may have hundreds or even thousands of the GGGGCC hexanucleotide repeats. A large number of clinical investigations have shown that about 700–1600 GGGGCC hexanucleotide repeats are inserted into the intron located between the two untranslated optional exons 1a

The FUS RNA-binding protein (*FUS*) gene, discovered in 2009, was mapped in

Mutations in *FUS* are observed in 4% of FALS and 1% of SALS. At present, more than 79 mutations have been described, predominantly in the 3′ region encoding an arginine/glycine-rich region and a NLS domain (nuclear localization signal). FUS protein, essentially localized in the nucleus, regulates RNA processing, splicing, and mRNA trafficking. Mutant FUS localized to cytoplasmic stress granules (SGs) and interacted with the stress granule protein PABP in an RNA-dependent manner resulting in mislocalization of the wildtype protein to stress granules [7–9, 11].

accounts for 20% of FALS cases and 1–2% of SALS cases [1, 2, 7, 8].

**76**

*CCNF*, located in 16p13.3, encoding the cyclin F, was first reported in 2016. The mutation of *CCNF* accounts for approximately 4% of FALS and 2% of SALS [7].

CCNF protein is a kind of kithe cell cyclin, involved in the regular of cell cycle transitions by activating cyclin-dependent protein kinases. Furthermore, it is the substrate recognition component of the Skp1-cullin-F-box E3 ubiquitin ligase complex. The neurons of the over-expressed mutant CCNF showed an increase in protein-labeled proteins, including TDP43. It indicates that mutated-CCNF protein interfere the proteasome degradation pathway by using traversing protein abnormalities to mark all proteins or inhibiting transferring transprotein-labeled proteins to proteasome complexes. This finding suggests that the *CCNF* mutation may cause abnormal protein homeostasis, which may be exacerbated by TDP43 protein disease.

Therefore, enhancing protein removal or reducing ubiquitin may be a feasible treatment [1, 3].

#### **2.5** *TIA1*

The *TIA1* gene, located in 2p13.3, encodes an RNA-binding protein involved in splicing regulation and translational repression. The mutations in *TIA1* were identified in 2.2% of FALS and 0.4% of SALS [7]. TIA1 protein is a key component of SGs, cytoplasmic foci sequester untranslated mRNAs upon different types of cellular stress, and the low complexity domain (LCD) region of TIA1 plays a central role in promoting SGs assembly. A heterozygous founder mutation (E384K) in the LCD was first reported in Swedish/Finnish patients as the cause of Welander distal myopathy (WDM). Recently, a mutation (p.P362L) in *TIA1* affecting a highly conserved residue in the LCD was identified as one cause of ALS/ALS-FTD [12].

#### **2.6** *TBK1*

The TANK-binding kinase 1 (*TBK1*) gene, located in 12q14.2, was discovered in 2015 [7]. The protein encoded by this gene is similar to the IκB kinase and can mediate NFκb activation in response to certain growth factors. The *TBK1* mutations were found in approximately 1% of FALS and 1% of SALS. The clinical phenotypes associated with the *TBK1* mutation are heterogeneous, with different ages at onset, different progressions, and irregular survival times.

Some patients also reported with extrapyramidal, ataxia, or psychosis. Neuropathological examination of central nervous system (CNS) tissues from patients with *TBK1* mutations revealed that SQSTM1/p62- and TDP-43-positive inclusion bodies which can indicate abnormalities in TDP-43 protein aggregation and protein clearance pathways [1, 2, 4, 5].

#### **2.7** *TARDBP*

The *TARDBP* (trans-activation element DNA-binding protein), located in 1p36.22, was discovered in 2008 [5].

The *TARDBP* gene mutation was found in 5% of FALS cases and 1% in SALS cases. Till now, more than 50 different mutations have been identified [9]. Except for D169G, a majority of these mutations are located in the 3′ region encoding a glycine-rich domain in its product, TDP-43. ALS patients carrying *TARDBP* gene mutations normally exhibit a classical ALS phenotype and rare dementia, they also have earlier disease onset, with upper limb onset being more common and compatible with a longer life. Most of TDP-43 is expressed in nucleus, involved in RNA

metabolism in many ways—transcriptional regulation, splicing, mRNA stabilization (including its own transcripts), and microRNA processing. TDP43 also regulates axonal transport and neuronal plasticity. In ALS, TDP-43 is often observed in cytoplasm. The pathogenesis of TDP-43 mainly includes cytoplasm construction of high phosphorylation TDP-43 and clearance of nuclear TDP-43 [1, 2, 7, 8].

#### **2.8 Pathogenesis of ALS-related genes**

The cellular processes, including RNA processing, protein degradation pathways, ubiquitin-proteasome system (UPS), autophagy, and so on, are all reported related to ALS pathogenesis [1–3, 7–22]. Sorted by the various processes, we summarize the causative genes and genes might increase susceptibility of ALS which impact physiological activities mentioned above (**Figure 1**).

**Figure 1.**

*Dysfunction cellular processes and related genes contributed to the pathogenesis of ALS.*

#### **Figure 2.**

*The worldwide frequency of ALS-related genes. The x-axis is the time when genes discovered. The y-axis is the logarithms of the mutation frequency of genes in ALS. The mutation frequency of C9orf 72, CHCHD10, CCNF, KIF5A, and ANXA11 are only within FALS. Where gene frequency was not available (ALS2, SETX, SIGMAR1, and PDIA1), one "circle size" equivalent to 1% is given for illustrative purposes.*

**79**

*Novel Aspects on Motor Neuron Disease: The Recent Genetic Studies on ALS*

*DOI: http://dx.doi.org/10.5772/intechopen.82085*

**2.9 Advances on gene discovery and frequency**

**3. GWAS on ALS**

*of different mutation genes.*

**Figure 3.**

applied to the research of ALS.

the risk factors of ALS [25–27].

**3.1 SNPs and GWAS**

We describe the worldwide ALS distribution feature by frequency of occurrence in different regions (**Figure 2**) [1–3, 7–9, 11, 14, 23, 24]. We summarize ALS mutation genes frequency according to the researches in Mainland China (**Figure 3**).

*ALS mutation genes frequency among FALS and SALS in Mainland China. The y-axis means the percentage* 

Recently, the studies on ALS have shown the development trend of the blowout with the technology improving, however, no longer limited by technical condition, the number of newly discovered ALS-related gene did not meet expectation. It indicates a shift in the genetic pathway that multiple genetic variants and environmental factors may interact simultaneously to increase ALS susceptibility. On the basis of the fact that sporadic form apparently accounts for high rate of ALS and the hypothesis that ALS may not appear to be familial in a conventional Mendelian manner, a new research method, genome-wide association study (GWAS), is

Since GWAS was applied to complex diseases, remarkable achievements have been made in certain fields. It is also hoped that in this way, we will be able to find

The International HapMap Project, which began in 2002, mapped the singlenucleotide polymorphisms (SNPs) haplotypes of the human genome from four major populations in the world and promoted the development of GWAS. At the same time, the rapid development of high-density and high-throughput genotyping technology, which can detect hundreds of thousands of SNPs in a single reaction, makes it possible to systematically screen mutations associated with complex diseases throughout the genome. Unlike previous candidate gene studies, GWAS does not need to build any assumptions based on disease pathophysiology prior to the study and can relatively be an unbiased screen for almost all common mutations in the genome. At present, some risk factors of complex diseases, such as age-related macular degeneration, diabetes mellitus, breast cancer, and so on, have been initially identified by GWAS.

*Novel Aspects on Motor Neuron Disease: The Recent Genetic Studies on ALS DOI: http://dx.doi.org/10.5772/intechopen.82085*

#### **Figure 3.**

*Novel Aspects on Motor Neuron Disease*

**2.8 Pathogenesis of ALS-related genes**

metabolism in many ways—transcriptional regulation, splicing, mRNA stabilization (including its own transcripts), and microRNA processing. TDP43 also regulates axonal transport and neuronal plasticity. In ALS, TDP-43 is often observed in cytoplasm. The pathogenesis of TDP-43 mainly includes cytoplasm construction of

The cellular processes, including RNA processing, protein degradation pathways, ubiquitin-proteasome system (UPS), autophagy, and so on, are all reported related to ALS pathogenesis [1–3, 7–22]. Sorted by the various processes, we summarize the causative genes and genes might increase susceptibility of ALS which

*The worldwide frequency of ALS-related genes. The x-axis is the time when genes discovered. The y-axis is the logarithms of the mutation frequency of genes in ALS. The mutation frequency of C9orf 72, CHCHD10, CCNF, KIF5A, and ANXA11 are only within FALS. Where gene frequency was not available (ALS2, SETX,* 

*SIGMAR1, and PDIA1), one "circle size" equivalent to 1% is given for illustrative purposes.*

*Dysfunction cellular processes and related genes contributed to the pathogenesis of ALS.*

high phosphorylation TDP-43 and clearance of nuclear TDP-43 [1, 2, 7, 8].

impact physiological activities mentioned above (**Figure 1**).

**78**

**Figure 2.**

**Figure 1.**

*ALS mutation genes frequency among FALS and SALS in Mainland China. The y-axis means the percentage of different mutation genes.*

#### **2.9 Advances on gene discovery and frequency**

We describe the worldwide ALS distribution feature by frequency of occurrence in different regions (**Figure 2**) [1–3, 7–9, 11, 14, 23, 24]. We summarize ALS mutation genes frequency according to the researches in Mainland China (**Figure 3**).

#### **3. GWAS on ALS**

Recently, the studies on ALS have shown the development trend of the blowout with the technology improving, however, no longer limited by technical condition, the number of newly discovered ALS-related gene did not meet expectation. It indicates a shift in the genetic pathway that multiple genetic variants and environmental factors may interact simultaneously to increase ALS susceptibility. On the basis of the fact that sporadic form apparently accounts for high rate of ALS and the hypothesis that ALS may not appear to be familial in a conventional Mendelian manner, a new research method, genome-wide association study (GWAS), is applied to the research of ALS.

Since GWAS was applied to complex diseases, remarkable achievements have been made in certain fields. It is also hoped that in this way, we will be able to find the risk factors of ALS [25–27].

#### **3.1 SNPs and GWAS**

The International HapMap Project, which began in 2002, mapped the singlenucleotide polymorphisms (SNPs) haplotypes of the human genome from four major populations in the world and promoted the development of GWAS. At the same time, the rapid development of high-density and high-throughput genotyping technology, which can detect hundreds of thousands of SNPs in a single reaction, makes it possible to systematically screen mutations associated with complex diseases throughout the genome. Unlike previous candidate gene studies, GWAS does not need to build any assumptions based on disease pathophysiology prior to the study and can relatively be an unbiased screen for almost all common mutations in the genome. At present, some risk factors of complex diseases, such as age-related macular degeneration, diabetes mellitus, breast cancer, and so on, have been initially identified by GWAS.

#### **3.2 ALS GWAS boot**

Schymiek et al. firstly reported GWAS in SALS in February 2007 [28]. A total of 276 patients and 271 controls with white American ancestry were recruited in this research. They used a chip to detect 555,352 SNPs and found 34 of the most relevant SNPs by association analysis. For negative results, they elucidated that SALS might contain a group of diseases with similar clinical manifestations like FALS, each of which is determined by different mutation sites, and that different diseases and mutation sites may interfere with GWAS's shooting out of susceptible genes. Among the 34 SNPs, there was an overexpression of genes associated with cytoskeletal actin regulation. For example, the *KIAAl721* gene of rs11099864 and the *FMN2* gene of rs1037666 had a homologous region that played an important role in the regulation of cytoskeletal actin. The most closely related rs4363506 of 34 SNPs was located in the *DOCKl* gene, which plays an important role in nerve growth. Although no disease-susceptible gene was found, the study identified possible SNPs and published all the results, which facilitated subsequent largescale SALS GWAS studies.

Recently, a series of ALS GWAS studies have been published and found several potential risk genes [29–32]. However, the results of these studies are different with the same ideas and methods. The biological process of some candidate genes is unclear, and the evidence needs to be supplemented.

#### **4. Genetic testing**

Gene testing helps ALS patients and families enhance their understanding on the condition and information requested in genotype-specific treatments. Although ALS patients desire access to genetic testing, genetic advances have been slow to reach the clinical care of the ALS patient. In recent years, the landscape of genetic testing and genetic counseling for ALS has been rapidly transformed with the identification of novel genes and the advent of next-generation sequencing technology.

#### **4.1 Genetic testing options**

As with all clinical testing, genetic risk assessment, including family history and pedigree analysis, and pretest counseling, helping patients anticipate the possible impact of genetic testing on themselves and their family members, are necessary for patients before genetic testing.

Currently available genetic testing options for ALS include Sanger sequencing for traditional simple mutations, assays for the *C9orf 72* repeat expansion, next-generation sequencing panels, whole-exome sequencing, and whole-genome sequencing.

#### **4.2 Post-test counseling**

Regarding the positive result, specific mutation, genotype-phenotype correlations, family history, and inheritance pattern should be thoroughly analyzed. Meanwhile, implications and risks for family members, including offspring and siblings, and theories about why the disease occurs also should be reviewed and addressed. For the reported definite pathogenic mutations, clinicians should provide information and hope about the potential genetic therapies in the future.

**81**

*Novel Aspects on Motor Neuron Disease: The Recent Genetic Studies on ALS*

To increase certainty, make health or lifestyle choices, and make decisions about family planning, the presymptomatic testing could be conducted. According to the guidelines for presymptomatic genetic testing in other neurodegenerative diseases such as Huntington disease and Alzheimer disease, ALS should be tailored as following: pretest genetic counseling, baseline neurologic and cognitive assessment, psychological evaluation, in-person disclosure, presence of support person, and posttest genetic counseling. Most of important, presymptomatic testing should be offered to adult first-degree relatives of ALS patients with established mutations

With the exception of riluzole, an anti-glutamatergic agent which was shown to prolong survival for 2–3 months by blocking the presynaptic release of glutamate, and edaravone, an antioxidant which was shown to decrease the rate of patient immobility, no effective treatment is currently available for ALS that can stop or

Gene therapy is a promising therapeutic approach for ALS since it can be used to deliver "gene drugs," encoding for blocking the novel gene expression, antiapoptotic proteins, and for neurotrophic factors, to the motor neurons crossing the bloodbrain barrier specifically to prevent further motor neuron degeneration and to

Here, we are to illustrate some examples of each therapeutic strategy for describ-

The neurotoxicity of mutant SOD1 is related to the dose of the toxic protein through multiple pathological mechanisms. A potential therapeutic approach to SOD1 related ALS is to block the expression of the toxic SOD1 that could cause motor neuron degeneration [33]. This therapy option which is more worthy of attention is that it possibly avoids and decreases potential influence in downstream pathological cascades. Recently, the studies mostly focus on antisense oligonucleotides and RNA interference which are both to block gene expression through enhancing the degradation of RNA.

In animal models of *SOD1*-associated ALS, antisense oligonucleotide treatment significantly delayed disease onset, improved neuromuscular function, and pro-

The first clinical trial of antisense oligonucleotide treatment in human beings had favorable safety outcomes, and now the clinical trial to assess the safety, tolerability, and pharmacokinetics of a second generation SOD1 antisense oligonucle-

According to literature, in *SOD1G93A* mice, reduction of human *SOD1* expression can significantly slow ALS progression and extend survival by using a single

*DOI: http://dx.doi.org/10.5772/intechopen.82085*

after written informed consent obtained [4].

preserve the function of remaining motor neurons.

*5.1.1 Antisense oligonucleotides in models and human*

otide is in progress (Clinical Trials.Gov, NCT02623699) [34].

*5.1.2 Short hairpin RNA (shRNA) treatment in mutant SOD1 ALS models*

ing the present status and advance of gene therapy treatment.

**4.3 Presymptomatic testing**

**5. Gene therapy**

**5.1 SOD1**

longed survival.

reverse the disease progression.

#### **4.3 Presymptomatic testing**

*Novel Aspects on Motor Neuron Disease*

Schymiek et al. firstly reported GWAS in SALS in February 2007 [28]. A total of 276 patients and 271 controls with white American ancestry were recruited in this research. They used a chip to detect 555,352 SNPs and found 34 of the most relevant SNPs by association analysis. For negative results, they elucidated that SALS might contain a group of diseases with similar clinical manifestations like FALS, each of which is determined by different mutation sites, and that different diseases and mutation sites may interfere with GWAS's shooting out of susceptible genes. Among the 34 SNPs, there was an overexpression of genes associated with cytoskeletal actin regulation. For example, the *KIAAl721* gene of rs11099864 and the *FMN2* gene of rs1037666 had a homologous region that played an important role in the regulation of cytoskeletal actin. The most closely related rs4363506 of 34 SNPs was located in the *DOCKl* gene, which plays an important role in nerve growth. Although no disease-susceptible gene was found, the study identified possible SNPs and published all the results, which facilitated subsequent large-

Recently, a series of ALS GWAS studies have been published and found several potential risk genes [29–32]. However, the results of these studies are different with the same ideas and methods. The biological process of some candidate genes is

Gene testing helps ALS patients and families enhance their understanding on the condition and information requested in genotype-specific treatments. Although ALS patients desire access to genetic testing, genetic advances have been slow to reach the clinical care of the ALS patient. In recent years, the landscape of genetic testing and genetic counseling for ALS has been rapidly transformed with the identification of novel genes and the advent of next-generation sequencing

As with all clinical testing, genetic risk assessment, including family history and pedigree analysis, and pretest counseling, helping patients anticipate the possible impact of genetic testing on themselves and their family members, are necessary for

Currently available genetic testing options for ALS include Sanger sequencing for traditional simple mutations, assays for the *C9orf 72* repeat expansion, next-generation sequencing panels, whole-exome sequencing, and whole-genome

Regarding the positive result, specific mutation, genotype-phenotype correlations, family history, and inheritance pattern should be thoroughly analyzed. Meanwhile, implications and risks for family members, including offspring and siblings, and theories about why the disease occurs also should be reviewed and addressed. For the reported definite pathogenic mutations, clinicians should provide information and hope about the potential genetic therapies in the future.

**3.2 ALS GWAS boot**

scale SALS GWAS studies.

**4. Genetic testing**

technology.

sequencing.

**4.1 Genetic testing options**

patients before genetic testing.

**4.2 Post-test counseling**

unclear, and the evidence needs to be supplemented.

**80**

To increase certainty, make health or lifestyle choices, and make decisions about family planning, the presymptomatic testing could be conducted. According to the guidelines for presymptomatic genetic testing in other neurodegenerative diseases such as Huntington disease and Alzheimer disease, ALS should be tailored as following: pretest genetic counseling, baseline neurologic and cognitive assessment, psychological evaluation, in-person disclosure, presence of support person, and posttest genetic counseling. Most of important, presymptomatic testing should be offered to adult first-degree relatives of ALS patients with established mutations after written informed consent obtained [4].

#### **5. Gene therapy**

With the exception of riluzole, an anti-glutamatergic agent which was shown to prolong survival for 2–3 months by blocking the presynaptic release of glutamate, and edaravone, an antioxidant which was shown to decrease the rate of patient immobility, no effective treatment is currently available for ALS that can stop or reverse the disease progression.

Gene therapy is a promising therapeutic approach for ALS since it can be used to deliver "gene drugs," encoding for blocking the novel gene expression, antiapoptotic proteins, and for neurotrophic factors, to the motor neurons crossing the bloodbrain barrier specifically to prevent further motor neuron degeneration and to preserve the function of remaining motor neurons.

Here, we are to illustrate some examples of each therapeutic strategy for describing the present status and advance of gene therapy treatment.

#### **5.1 SOD1**

The neurotoxicity of mutant SOD1 is related to the dose of the toxic protein through multiple pathological mechanisms. A potential therapeutic approach to SOD1 related ALS is to block the expression of the toxic SOD1 that could cause motor neuron degeneration [33]. This therapy option which is more worthy of attention is that it possibly avoids and decreases potential influence in downstream pathological cascades. Recently, the studies mostly focus on antisense oligonucleotides and RNA interference which are both to block gene expression through enhancing the degradation of RNA.

#### *5.1.1 Antisense oligonucleotides in models and human*

In animal models of *SOD1*-associated ALS, antisense oligonucleotide treatment significantly delayed disease onset, improved neuromuscular function, and prolonged survival.

The first clinical trial of antisense oligonucleotide treatment in human beings had favorable safety outcomes, and now the clinical trial to assess the safety, tolerability, and pharmacokinetics of a second generation SOD1 antisense oligonucleotide is in progress (Clinical Trials.Gov, NCT02623699) [34].

#### *5.1.2 Short hairpin RNA (shRNA) treatment in mutant SOD1 ALS models*

According to literature, in *SOD1G93A* mice, reduction of human *SOD1* expression can significantly slow ALS progression and extend survival by using a single peripheral injection of an adeno-associated virus serotype 9 (AAV9) encoding shRNA [35].

While, in a recent study reported, SOD1 expression in the motor cortex of P70 *SOD1G93A* models was selectively silenced by delivery of AAV9-SOD1-shRNA. As a result, not only the ALS progression was slowed and the survival was extended significantly, but also the survival of spinal motor neurons was significantly enhanced [36].

#### *5.1.3 miRNA treatment in mutant SOD1 ALS models and healthy nonhuman primates*

In a study, scientists reported a new method that systemically delivered drug based on an artificial microRNA. In the *SOD1G93A* mice, this drug delayed ALS onset, prolonged the survival, and significantly preserved muscle strength and motor and respiratory functions. Notably, the research of this drug has been conducted in nonhuman primates, and the result showed that *SOD1* expression in lower motor neurons was safely blocked [37].

#### **5.2 C9orf 72**

Similar therapeutic approach targeting C9orf72 for ALS is also in development. The toxicity of mutated C9orf72 is imparted by the formation of nucleolar RNA foci that sequester important RNA-binding proteins and by the generation of toxic dipeptide repeat (DPR) proteins.

The C9orf72 hexanucleotide repeat expansion (HRE) of GGGGCC DNA and RNA enables the formation of complex structures including G-quadruplexes. Because the Guanosine-rich DNA and RNA sequences are prone to formation of G-quadruplexes, a stable four-stranded structure present within ribosomal DNA sequences, transcription start sites, the promoter and untranslated regions of mRNA, human telomeric DNA sequences. It may play an important role in various cellular processes such as telomere maintenance, ribosome biogenesis, gene replication, transcription, and translation. Therefore, both C9orf72 HRE DNA and RNA may contribute to the pathogenesis of ALS/FTD disease through a mechanism associated with their structure polymorphism. Presently, based on the above mechanism, two strategies including antisense-mediated interventions and smallmolecule-based approach have been developed to interfere with neurodegenerative diseases associated with G-quadruplexes [38].

#### *5.2.1 Antisense oligonucleotides in mutant C9orf72 ALS models*

A recent study reported that the RNA foci and DPR proteins were reduced significantly in mutant C9orf72 ALS mice by a single-dose injection of antisense oligonucleotides to reduce C9orf72 RNA repeats, and after 6 months of treatment, the motor function was also preserved [39].

#### *5.2.2 Small-molecule ligands targeting the G-quadruplex structure in mutant C9orf72 cells*

The small-molecule ligands, such as porphyrin, acridine, pentacridium, telomestatin, naphthalene diamide, and bisquinolium, directly target and bind to the G-quadruplex structure and selectively modulated the function of the G-quadruplex. For example, TMPyP4, a cationic porphyrin, can bind and disrupt the secondary structures of C9orf72 HRE and even damage its interactions with hnRNPA1 and ASF/SF2 proteins [40].

**83**

provided the original work is properly cited.

\*Address all correspondence to: wjling8002@126.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Department of Neurology, Xiangya Hospital, Central South University, Changsha,

*Novel Aspects on Motor Neuron Disease: The Recent Genetic Studies on ALS*

Similarly, some studies also showed that three small-molecule ligands can bind G-quadruplex and decreased RNA foci and RNA translation in both cultured cells

TBK1 is a key regulatory molecule upstream of OPTN, SQSTM1/p62, and IRF3 in the autophagy and neuroinflammatory pathways that are implicated in ALS. Manipulation of TBK1 might potentially compensate for defects caused by other ALS-associated proteins in these pathways—for example, VCP and UBQLN2. NEK1 and C21orf2 are known to interact at the protein level and, in addition to TUBA4A, PFN1, NEFH, and PRPH, they represent the building blocks of the cellular scaffold. Administration of small molecules that enhance cytoskeletal integrity could represent a viable therapy for stopping progression or reversing the disease

Nearly a decade ago, the only way to test ALS-related gene was SOD1 sequencing, whereas clinicians now have a wide availability of testing options already. Whole-exome sequencing is current standard in most related searches. However, the factors such as high rate of incomplete penetrance in ALS, few large pedigrees, and short survival of patients lead to the discovery of ALS-related genes worse than

Although many known ALS-related genes' structural characteristics and roles have been discovered, which have highlighted critical processes, pathways, and intracellular localizations of dysregulation, there are still many reported variants with uncertain significance. Further functional studies are needed to clarify the pathogenesis of these genes. Till now, more people believe that ALS is the result of interaction between multiple genes that each increases the susceptibility of the disease, but does not initiate the pathogenesis alone. So, we need to do more research on oligogenic ALS cases. Most importantly, with the improvement of understanding of ALS genetics, we will have more opportunities to develop meaningful therapies.

expected and the identification of susceptible mutations limited.

*DOI: http://dx.doi.org/10.5772/intechopen.82085*

course in patients with these mutations [4–6].

and patient-derived neurons [41].

**5.3 Others**

**6. Conclusion**

**Author details**

Junling Wang

Hunan, China

*Novel Aspects on Motor Neuron Disease: The Recent Genetic Studies on ALS DOI: http://dx.doi.org/10.5772/intechopen.82085*

Similarly, some studies also showed that three small-molecule ligands can bind G-quadruplex and decreased RNA foci and RNA translation in both cultured cells and patient-derived neurons [41].

#### **5.3 Others**

*Novel Aspects on Motor Neuron Disease*

motor neurons was safely blocked [37].

dipeptide repeat (DPR) proteins.

diseases associated with G-quadruplexes [38].

the motor function was also preserved [39].

hnRNPA1 and ASF/SF2 proteins [40].

*C9orf72 cells*

*5.2.1 Antisense oligonucleotides in mutant C9orf72 ALS models*

shRNA [35].

enhanced [36].

**5.2 C9orf 72**

peripheral injection of an adeno-associated virus serotype 9 (AAV9) encoding

*5.1.3 miRNA treatment in mutant SOD1 ALS models and healthy nonhuman primates*

In a study, scientists reported a new method that systemically delivered drug based on an artificial microRNA. In the *SOD1G93A* mice, this drug delayed ALS onset, prolonged the survival, and significantly preserved muscle strength and motor and respiratory functions. Notably, the research of this drug has been conducted in nonhuman primates, and the result showed that *SOD1* expression in lower

Similar therapeutic approach targeting C9orf72 for ALS is also in development. The toxicity of mutated C9orf72 is imparted by the formation of nucleolar RNA foci that sequester important RNA-binding proteins and by the generation of toxic

The C9orf72 hexanucleotide repeat expansion (HRE) of GGGGCC DNA and RNA enables the formation of complex structures including G-quadruplexes. Because the Guanosine-rich DNA and RNA sequences are prone to formation of G-quadruplexes, a stable four-stranded structure present within ribosomal DNA sequences, transcription start sites, the promoter and untranslated regions of mRNA, human telomeric DNA sequences. It may play an important role in various cellular processes such as telomere maintenance, ribosome biogenesis, gene replication, transcription, and translation. Therefore, both C9orf72 HRE DNA and RNA may contribute to the pathogenesis of ALS/FTD disease through a mechanism associated with their structure polymorphism. Presently, based on the above mechanism, two strategies including antisense-mediated interventions and smallmolecule-based approach have been developed to interfere with neurodegenerative

A recent study reported that the RNA foci and DPR proteins were reduced significantly in mutant C9orf72 ALS mice by a single-dose injection of antisense oligonucleotides to reduce C9orf72 RNA repeats, and after 6 months of treatment,

*5.2.2 Small-molecule ligands targeting the G-quadruplex structure in mutant* 

The small-molecule ligands, such as porphyrin, acridine, pentacridium, telomestatin, naphthalene diamide, and bisquinolium, directly target and bind to the G-quadruplex structure and selectively modulated the function of the G-quadruplex. For example, TMPyP4, a cationic porphyrin, can bind and disrupt the secondary structures of C9orf72 HRE and even damage its interactions with

While, in a recent study reported, SOD1 expression in the motor cortex of P70 *SOD1G93A* models was selectively silenced by delivery of AAV9-SOD1-shRNA. As a result, not only the ALS progression was slowed and the survival was extended significantly, but also the survival of spinal motor neurons was significantly

**82**

TBK1 is a key regulatory molecule upstream of OPTN, SQSTM1/p62, and IRF3 in the autophagy and neuroinflammatory pathways that are implicated in ALS. Manipulation of TBK1 might potentially compensate for defects caused by other ALS-associated proteins in these pathways—for example, VCP and UBQLN2. NEK1 and C21orf2 are known to interact at the protein level and, in addition to TUBA4A, PFN1, NEFH, and PRPH, they represent the building blocks of the cellular scaffold. Administration of small molecules that enhance cytoskeletal integrity could represent a viable therapy for stopping progression or reversing the disease course in patients with these mutations [4–6].

#### **6. Conclusion**

Nearly a decade ago, the only way to test ALS-related gene was SOD1 sequencing, whereas clinicians now have a wide availability of testing options already. Whole-exome sequencing is current standard in most related searches. However, the factors such as high rate of incomplete penetrance in ALS, few large pedigrees, and short survival of patients lead to the discovery of ALS-related genes worse than expected and the identification of susceptible mutations limited.

Although many known ALS-related genes' structural characteristics and roles have been discovered, which have highlighted critical processes, pathways, and intracellular localizations of dysregulation, there are still many reported variants with uncertain significance. Further functional studies are needed to clarify the pathogenesis of these genes. Till now, more people believe that ALS is the result of interaction between multiple genes that each increases the susceptibility of the disease, but does not initiate the pathogenesis alone. So, we need to do more research on oligogenic ALS cases. Most importantly, with the improvement of understanding of ALS genetics, we will have more opportunities to develop meaningful therapies.

#### **Author details**

Junling Wang Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, China

\*Address all correspondence to: wjling8002@126.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Novel Aspects on Motor Neuron Disease: The Recent Genetic Studies on ALS DOI: http://dx.doi.org/10.5772/intechopen.82085*

sclerosis: An update. Molecular Neurodegeneration. 2013;**8**(1):28

[18] Therrien M, Dion PA, Rouleau GA. ALS: Recent developments from genetics studies. Current Neurology & Neuroscience Reports. 2016;**16**(6):1-12

[19] White MA, Sreedharan J. Amyotrophic lateral sclerosis: Recent genetic highlights. Current Opinion in Neurology. 2016;**29**(5):557

[20] Norris FH, Sang UK, Denys EH, et al. Amyotrophic lateral sclerosis. The New England Journal of Medicine. 2017;**53**(8):162-172

[21] Corcia P, Couratier P, Blasco H, et al. Genetics of amyotrophic lateral sclerosis. La Presse Médicale. 2014;**43**(5):555-562

[22] Tripolszki K, Török D, Goudenège D, et al. High-throughput sequencing revealed a novel SETX mutation in a Hungarian patient with amyotrophic lateral sclerosis. Brain and Behavior: A Cognitive Neuroscience Perspective. 2017;**7**(4):e00669

[23] Smith BN, Topp SD, Fallini C, et al. Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis. Science Translational Medicine. 2017;**9**(388):eaad9157

[24] Zhang K, Liu Q, Liu K, et al. ANXA11 mutations prevail in Chinese ALS patients with and without cognitive dementia. 2018;**4**(3):e237

[25] Ozaki K, Ohnishi Y, Iida A, et al. Functional SNPs in the lymphotoxinalpha gene that are associated with susceptibility to myocardial infarction. Nature Genetics. 2002;**32**(4):650

[26] Klein RJ, Caroline Z, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science. 2005;**308**(5720):385-389

[27] Macarthur J, Bowler E, Cerezo M, et al. The new NHGRI-EBI catalog of published genome-wide association studies (GWAS Catalog). Nucleic Acids Research. 2017;**45**(Database issue):D896-D901

[28] Schymick JC, Scholz SW, Fung H-C, et al. Genome-wide genotyping in amyotrophic lateral sclerosis and neurologically normal controls: First stage analysis and public release of data. The Lancet Neurology. 2007;**6**(4):291-292

[29] Travis D, Huentelman MJ, Craig DW, et al. Whole-genome analysis of sporadic amyotrophic lateral sclerosis. New England Journal of Medicine. 2007;**357**(8):775

[30] Michael A van ES, Paul WJ van Vught, Blawu HM, et al. Genetic variation in DPP6 is associated with susceptibility to amyotrophic lateral sclerosis. Nature Genetics. 2008;**40**(1):29-31

[31] Kawam AA, Alshawaqfeh M, Cai JJ, et al. Simulating variance heterogeneity in quantitative genome wide association studies. BMC Bioinformatics. 2018;**19**(Suppl 3):72

[32] Broce I, Karch CM, Wen N, et al. Immune-related genetic enrichment in frontotemporal dementia: An analysis of genome-wide association studies. PLoS Medicine. 2018;**15**(1):e1002487

[33] van Zundert B, Brown RH Jr. Silencing strategies for therapy of SOD1-mediated ALS. Neuroscience Letters. 2017;**636**:32-39

[34] Bishop KM. Progress and promise of antisense oligonucleotide therapeutics for central nervous system diseases. Neuropharmacology. 2017;**120**:56

[35] Foust KD, Salazar DL, Shibi L, et al. Therapeutic AAV9-mediated suppression of mutant SOD1 slows

**84**

*Novel Aspects on Motor Neuron Disease*

[1] Recabarren-Leiva D, Alarcon M. New insights into the gene expression associated to amyotrophic lateral

[2] Ghasemi M, Brown RJ. Genetics of amyotrophic lateral sclerosis. Cold Spring Harbor Perspectives in Medicine. 2018;**8**(5). DOI: 10.1101/cshperspect.

DOI: 10.1016/j.lfs.2017.12.016

[3] Volk AE, Weishaupt JH, Andersen PM, et al. Current knowledge and recent insights into the genetic basis of amyotrophic lateral sclerosis. Medizinische Genetik. 2018;**30**(2):

252-258. DOI: 10.1007/ s11825-018-0185-3

[4] Roggenbuck J, Quick A, Kolb SJ.

[5] Vajda A, Mclaughlin RL, Heverin M, et al. Genetic testing in ALS. Neurology.

[6] Picher-Martel V, Valdmanis PN, Gould PV, et al. From animal models to human disease: A genetic approach for personalized medicine in ALS. Acta Neuropathologica Communications.

[7] Chia R, Chiò A, Traynor BJ. Novel genes associated with amyotrophic lateral sclerosis: Diagnostic and clinical implications. Lancet Neurology.

[8] Al Sultan A, Waller R, Heath P, et al. The genetics of amyotrophic lateral sclerosis: Current insights. Degenerative Neurological & Neuromuscular Disease.

Genetic testing and genetic counseling for amyotrophic lateral sclerosis: An update for clinicians. Genetics in Medicine Official Journal of the American College of Medical

Genetics. 2016;**19**(3):267

2017;**88**(10):991-999

2016;**4**(1):70

2017;**17**(1)

2016:**6**(1):49-64

sclerosis. Life Science. 2018;**193**:110-123.

[9] Maurel C, Dangoumau A, Marouillat S, et al. Causative genes in amyotrophic lateral sclerosis and protein degradation pathways: A link to neurodegeneration.

Molecular Neurobiology. 2018;

[10] Woollacott IOC, Simon M. The C9ORF72 expansion mutation: Gene structure, phenotypic and diagnostic issues. Acta Neuropathologica.

[11] Ticozzi N, Vance C, Leclerc AL, et al. Mutational analysis reveals the FUS homolog TAF15 as a candidate gene for familial amyotrophic lateral sclerosis. American Journal of Medical Genetics. Part B, Neuropsychiatric Genetics. 2011;**156B**(3):285-290. DOI:

[12] Yuan Z, Jiao B, Hou L, et al. Mutation analysis of the TIA1 gene in Chinese patients with amyotrophic lateral sclerosis and frontotemporal dementia.

[13] Morgan S, Orrell RW. Pathogenesis of amyotrophic lateral sclerosis. British Medical Bulletin. 2016;**119**(1):87-98

[14] Yohei I, Masahisa K, Kensuke I, et al. Amyotrophic lateral sclerosis: An update on recent genetic insights. Journal of Neurology. 2013;**260**(11):2917-2927

[15] Ji AL, Zhang X, Chen WW, et al. Genetics insight into the amyotrophic lateral sclerosis/frontotemporal

dementia spectrum. Journal of Medical

[16] Ajroud-Driss S, Siddique T. Sporadic and hereditary amyotrophic lateral sclerosis (ALS). Biochimica et

Biophysica Acta. 2015;**1852**(4):679-684

[17] Chen S, Sayana P, Zhang X, Le W. Genetics of amyotrophic lateral

Genetics, 2017;**54**(3):145-154

Neurobiology of Aging. 2018:64

(Pt 2):1-20

2014;**127**(3):319-332

10.1002/ajmg.b.31158

**References**

a024125

disease progression and extends survival in models of inherited ALS. Molecular Therapy. 2013;**21**(12):2148-2159

[36] Thomsen GM, Gowing G, Latter J, et al. Delayed disease onset and extended survival in the SOD1G93A rat model of amyotrophic lateral sclerosis after suppression of mutant SOD1 in the motor cortex. Journal of Neuroscience the Official Journal of the Society for Neuroscience. 2014;**34**(47):15587

[37] Borel F, Gernoux G, Cardozo B, et al. Therapeutic rAAVrh10 mediated SOD1 silencing in adult SOD1(G93A) mice and nonhuman primates. Human Gene Therapy. 2016;**27**(1):19-31

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[40] Zamiri B, Reddy K, Macgregor RB Jr, Pearson CE. TMPyP4 porphyrin distorts RNA G-quadruplex structures of the disease-associated r(GGGGCC)n repeat of the C9orf72 gene and blocks interaction of RNAbinding proteins. Journal of Biological Chemistry. 2014;**289**(8):4653-4659

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**87**

update.

**Chapter 6**

*Wei Li*

**Abstract**

this SMA protein.

**1. Setting the scene up**

Structural and Functional

SMA-Linked Missense Mutations

Genetically linked to the survival motor neuron 1 gene *SMN1*, spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease with dysfunctional α-motor neurons. As the product of the *SMN1* gene, the survival motor neuron protein (SMN) plays an essential role in the molecular pathogenesis of SMA. On 1 June 2017, a PLoS ONE article reported a set of computational structural analysis to illustrate how do SMA-linked mutations of *SMN1* lead to structurally/functionally deficient variants of SMN. Following this article, this chapter provides a brief update of the structural and functional consequences of the missense mutations of

**Keywords:** spinal muscular atrophy, survival motor neuron protein, missense

On 1 June 2017, *PLoS ONE* published an original research article (**Figure 1**) [1] with a title 'How do SMA-linked mutations of *SMN1* lead to structural/functional deficiency of the SMA protein?', of which this chapter aims to provide a brief

SMA is an autosomal recessive neuromuscular disease with α-motor neuron (anterior horn of the spinal cord) dysfunction and muscular atrophy [2]. SMA is caused by loss (∼95% of SMA cases) or mutation (∼5% of SMA cases) of the survival motor neuron gene 1 *SMN1* (telomeric *SMN*, *telSMN* or *SMN1*, GenBank: U18423, the 5q13 region of human chromosome) [3]. In the 5q13 region of the human chromosome, there is also a nearly identical *survival motor neuron 2* gene *SMN2* (centromeric *SMN*, *cenSMN* or *SMN2*, GenBank: NM\_022875) [3]. The two genes (*SMN1* and *SMN2*) have been extensively characterised, and their roles in

mutation, structural consequence(s), functional consequence(s)

**1.1 The genetics of SMA: a brief introduction**

SMA have been reviewed in detail [2–8].

of the Survival Motor Neuron

Consequences of the

Protein: A Brief Update

### **Chapter 6**

*Novel Aspects on Motor Neuron Disease*

disease progression and extends survival in models of inherited ALS. Molecular Therapy. 2013;**21**(12):2148-2159

[36] Thomsen GM, Gowing G, Latter J, et al. Delayed disease onset and

extended survival in the SOD1G93A rat model of amyotrophic lateral sclerosis after suppression of mutant SOD1 in the motor cortex. Journal of Neuroscience the Official Journal of the Society for Neuroscience. 2014;**34**(47):15587

[37] Borel F, Gernoux G, Cardozo B, et al. Therapeutic rAAVrh10 mediated SOD1 silencing in adult SOD1(G93A) mice and nonhuman primates. Human Gene Therapy. 2016;**27**(1):19-31

[38] Kumar V, Kashav T, Islam A, et al. Structural insight into C9orf72 hexanucleotide repeat expansions: Towards new therapeutic targets in FTD-ALS. Neurochemistry International. 2016;**100**:11-20

[39] Jiang J, Zhu Q, Gendron T, et al. Gain of toxicity from ALS/FTD-linked repeat expansions in C9ORF72 is alleviated by antisense oligonucleotides targeting GGGGCC-containing RNAs.

Macgregor RB Jr, Pearson CE. TMPyP4 porphyrin distorts RNA G-quadruplex structures of the disease-associated r(GGGGCC)n repeat of the C9orf72 gene and blocks interaction of RNAbinding proteins. Journal of Biological Chemistry. 2014;**289**(8):4653-4659

[41] Su Z, Zhang Y, Gendron T, et al. Discovery of a biomarker and lead small molecules to target r(GGGGCC)-

associated defects in c9FTD/ ALS. Neuron. 2014;**83**(5):1043-1050

Neuron. 2016;**90**(3):535-550

[40] Zamiri B, Reddy K,

**86**

## Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival Motor Neuron Protein: A Brief Update

*Wei Li*

### **Abstract**

Genetically linked to the survival motor neuron 1 gene *SMN1*, spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease with dysfunctional α-motor neurons. As the product of the *SMN1* gene, the survival motor neuron protein (SMN) plays an essential role in the molecular pathogenesis of SMA. On 1 June 2017, a PLoS ONE article reported a set of computational structural analysis to illustrate how do SMA-linked mutations of *SMN1* lead to structurally/functionally deficient variants of SMN. Following this article, this chapter provides a brief update of the structural and functional consequences of the missense mutations of this SMA protein.

**Keywords:** spinal muscular atrophy, survival motor neuron protein, missense mutation, structural consequence(s), functional consequence(s)

### **1. Setting the scene up**

On 1 June 2017, *PLoS ONE* published an original research article (**Figure 1**) [1] with a title 'How do SMA-linked mutations of *SMN1* lead to structural/functional deficiency of the SMA protein?', of which this chapter aims to provide a brief update.

#### **1.1 The genetics of SMA: a brief introduction**

SMA is an autosomal recessive neuromuscular disease with α-motor neuron (anterior horn of the spinal cord) dysfunction and muscular atrophy [2]. SMA is caused by loss (∼95% of SMA cases) or mutation (∼5% of SMA cases) of the survival motor neuron gene 1 *SMN1* (telomeric *SMN*, *telSMN* or *SMN1*, GenBank: U18423, the 5q13 region of human chromosome) [3]. In the 5q13 region of the human chromosome, there is also a nearly identical *survival motor neuron 2* gene *SMN2* (centromeric *SMN*, *cenSMN* or *SMN2*, GenBank: NM\_022875) [3]. The two genes (*SMN1* and *SMN2*) have been extensively characterised, and their roles in SMA have been reviewed in detail [2–8].

#### **Figure 1.**

*A flow chart for the computational analysis of structural/functional consequences of clinically identified genetic diseases-linked missense mutation(s) of key gene(s) and protein(s). In [1], SMA was shown as an example of the computational analysis as illustrated here in this figure (http://biomedical-advances.org/ep-20182-14/).*

#### **1.2 The survival motor neuron protein and its role in SMA**

The survival motor neuron (SMN) protein is the product of *SMN1*, the SMAdetermining survival motor neuron gene [2, 3]. As a result, SMN is also called the SMA protein. In fact, the 38-kD SMN is the actually affected protein in SMA [9–11], and is a cytoplasmic protein that also occurs in dot-like nuclear structures called gems, which is why SMN is formerly termed Gemin1 [3, 12], too.

In the molecular pathogenesis of SMA, of particular interest is an exon 7-skipping splicing defect identified in the pre-mRNA editing of the *SMN2* gene [5]. Due to this splicing defect, *SMN2* predominantly produces exon 7-skipped transcripts, which encode a truncated isoform of the SMN protein (SMNΔ7 or SMN2 with 282 residues), in comparison with the full-length SMN protein with 294 residues (SMN1 or FL-SMN).

In pre-mRNA editing, spliceosome is the major functional unit, and spliceosomal small nuclear ribonucleoproteins (snRNPs) are essential components of the nuclear pre-mRNA processing machinery [13–17]. In the pathogenesis of SMA, the SMN protein plays a critical role in pre-mRNA processing, because the biogenesis of spliceosomal snRNPs is promoted by the SMN complex [14, 18, 19], which consists of SMN (Gemin1), Gemin2–8 and UNR-interacting protein (UNRIP) [13, 16, 20]. In the formation of the SMN complex, SMN forms oligomers and directly interacts via its N-terminus with Gemin2 and via its tudor domain with spliceosomal (Sm) proteins [13, 21, 22]. A key component of the SMN complex, SMN first assembles the essential SMN/Gemin complex, which in turn mediates the formation of the Sm core domain of the spliceosomal snRNPs [13, 21, 22].

#### **2. Structural and functional consequences of the SMA-linked missense mutations of SMN**

In general, genetic mutation includes missense, nonsense, insertion and deletion mutations. A nonsense mutation is a point mutation in a DNA sequence that results in a premature stop codon, or a nonsense codon in the transcribed mRNA, and in a truncated, incomplete and usually functionally deficient protein product. In contrast, a missense mutation involves substitution of one single amino acid residue, and therefore is able to provide unique access to residue-specific structural insights

**89**

**Table 2**.

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival…*

A set of point mutations (missense and nonsense mutations) have been previously summarised in [1], including A2G [23], nonsense mutation Q15X [24], D30N [25], D44V [25–27], V94G [28], G95R [25], Y130C [29], nonsense mutation Q157X [30], A188S [31], nonsense mutation W190X [32], nonsense mutation L228X [33], P245L [34], L260S [28], S262G and S262I [4, 25], M263T [32], S266P [29], Y272C [4, 35, 36], H273R [29], T274I [4, 35, 36], G275S [32], G279C and G279V [4, 35, 37, 38]. As of 25 September 2018, eight more missense mutations of SMN were summarised and reported, including A2V, Y109C, Y130C, Y130H, P221L, S230L, P244L

**2.2 An update of experimentally determined SMN-related structures**

In [1], 11 SMN-related structures were retrieved from the PDB database [40] with 2 search parameters (text search for: survival motor neuron protein and molecule: survival motor neuron protein). In a new search of the PDB database (accessed 25 September 2018) [40] with the same parameters, 14 PDB entries were retrieved, including **1G5V**, **1MHN**, **2LEH**, **4A4E**, **4A4G**, **4GLI**, **4QQ6**, **4V98**, **5XJL**, **5XJQ**, **5XJR**, **5XJS**, **5XJT** and **5XJU**. In a comparison with the PDB entries in [1], during the past 16 months, six new SMN-related structures were deposited in the Protein Data Bank, including **5XJL** (to supersede **3S6N** [41]), **5XJQ** [42], **5XJR** [42], **5XJS** [42], **5XJT** [42] and **5XJU** [42]. While the six PDB entries do contain a set of different yet functionally related protein molecules, including snRNP Sm-D1, snRNP Sm-D2, snRNP E, snRNP F and snRNP G, they also contain a fragment of the survival motor neuron protein (SMN residues 26–62), according to the fasta

**2.3 An update of the structural and functional consequences of the missense** 

In light of the six new experimentally determined SMN-related structures (**Table 1**), a new set of computational structural analysis, as previously described in detail in [1], is within the reach of this chapter to provide an update of it. Two aspartates (Asp 35 and Asp44) of SMN stood out in the structural analysis of both intramolecular and intermolecular salt bridges for this SMA protein, as listed in

Asp44 is in the exon 2a of *SMN1* (the Gemin2-binding domain), and involved in an SMA-linked Asp44Val (D44V) missense mutation [25], which involves a substitution of Asp44's charged side chain by Val44's hydrophobic side chain. Of extraordinary functional significance is that SMN's Gemin2-binding activity is totally suppressed by the D44V mutation in *SMN1* [41]. Moreover, the D44V SMN (SMND44V) mutant's snRNP assembly activity is lower than that of the wild-type

into the role of the residue in the structure and function of the target protein, provided that the three-dimensional structure of the target protein is experimentally determined and deposited in the Protein Data Bank. Thus, this chapter focuses on SMA-linked missense mutations of SMN and aims to provide a brief update of their structural and functional consequences with a set of computational structural

**2.1 An update of SMA-linked missense mutations of SMN**

*DOI: http://dx.doi.org/10.5772/intechopen.81887*

format data of the six PDB entries [42].

*2.3.1 Asp44 in the Gemin2-binding domain of SMN*

**mutations of SMN**

SMN (FL-SMN or SMN1) [27].

analysis as described in [1].

and R288S [39].

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival… DOI: http://dx.doi.org/10.5772/intechopen.81887*

into the role of the residue in the structure and function of the target protein, provided that the three-dimensional structure of the target protein is experimentally determined and deposited in the Protein Data Bank. Thus, this chapter focuses on SMA-linked missense mutations of SMN and aims to provide a brief update of their structural and functional consequences with a set of computational structural analysis as described in [1].

#### **2.1 An update of SMA-linked missense mutations of SMN**

A set of point mutations (missense and nonsense mutations) have been previously summarised in [1], including A2G [23], nonsense mutation Q15X [24], D30N [25], D44V [25–27], V94G [28], G95R [25], Y130C [29], nonsense mutation Q157X [30], A188S [31], nonsense mutation W190X [32], nonsense mutation L228X [33], P245L [34], L260S [28], S262G and S262I [4, 25], M263T [32], S266P [29], Y272C [4, 35, 36], H273R [29], T274I [4, 35, 36], G275S [32], G279C and G279V [4, 35, 37, 38]. As of 25 September 2018, eight more missense mutations of SMN were summarised and reported, including A2V, Y109C, Y130C, Y130H, P221L, S230L, P244L and R288S [39].

#### **2.2 An update of experimentally determined SMN-related structures**

In [1], 11 SMN-related structures were retrieved from the PDB database [40] with 2 search parameters (text search for: survival motor neuron protein and molecule: survival motor neuron protein). In a new search of the PDB database (accessed 25 September 2018) [40] with the same parameters, 14 PDB entries were retrieved, including **1G5V**, **1MHN**, **2LEH**, **4A4E**, **4A4G**, **4GLI**, **4QQ6**, **4V98**, **5XJL**, **5XJQ**, **5XJR**, **5XJS**, **5XJT** and **5XJU**. In a comparison with the PDB entries in [1], during the past 16 months, six new SMN-related structures were deposited in the Protein Data Bank, including **5XJL** (to supersede **3S6N** [41]), **5XJQ** [42], **5XJR** [42], **5XJS** [42], **5XJT** [42] and **5XJU** [42]. While the six PDB entries do contain a set of different yet functionally related protein molecules, including snRNP Sm-D1, snRNP Sm-D2, snRNP E, snRNP F and snRNP G, they also contain a fragment of the survival motor neuron protein (SMN residues 26–62), according to the fasta format data of the six PDB entries [42].

#### **2.3 An update of the structural and functional consequences of the missense mutations of SMN**

#### *2.3.1 Asp44 in the Gemin2-binding domain of SMN*

In light of the six new experimentally determined SMN-related structures (**Table 1**), a new set of computational structural analysis, as previously described in detail in [1], is within the reach of this chapter to provide an update of it. Two aspartates (Asp 35 and Asp44) of SMN stood out in the structural analysis of both intramolecular and intermolecular salt bridges for this SMA protein, as listed in **Table 2**.

Asp44 is in the exon 2a of *SMN1* (the Gemin2-binding domain), and involved in an SMA-linked Asp44Val (D44V) missense mutation [25], which involves a substitution of Asp44's charged side chain by Val44's hydrophobic side chain. Of extraordinary functional significance is that SMN's Gemin2-binding activity is totally suppressed by the D44V mutation in *SMN1* [41]. Moreover, the D44V SMN (SMND44V) mutant's snRNP assembly activity is lower than that of the wild-type SMN (FL-SMN or SMN1) [27].

*Novel Aspects on Motor Neuron Disease*

**1.2 The survival motor neuron protein and its role in SMA**

gems, which is why SMN is formerly termed Gemin1 [3, 12], too.

core domain of the spliceosomal snRNPs [13, 21, 22].

The survival motor neuron (SMN) protein is the product of *SMN1*, the SMAdetermining survival motor neuron gene [2, 3]. As a result, SMN is also called the SMA protein. In fact, the 38-kD SMN is the actually affected protein in SMA [9–11], and is a cytoplasmic protein that also occurs in dot-like nuclear structures called

*A flow chart for the computational analysis of structural/functional consequences of clinically identified genetic diseases-linked missense mutation(s) of key gene(s) and protein(s). In [1], SMA was shown as an example of the computational analysis as illustrated here in this figure (http://biomedical-advances.org/ep-20182-14/).*

In the molecular pathogenesis of SMA, of particular interest is an exon 7-skipping splicing defect identified in the pre-mRNA editing of the *SMN2* gene [5]. Due to this splicing defect, *SMN2* predominantly produces exon 7-skipped transcripts, which encode a truncated isoform of the SMN protein (SMNΔ7 or SMN2 with 282 residues), in comparison with the full-length SMN protein with 294 residues (SMN1

In pre-mRNA editing, spliceosome is the major functional unit, and spliceosomal small nuclear ribonucleoproteins (snRNPs) are essential components of the nuclear pre-mRNA processing machinery [13–17]. In the pathogenesis of SMA, the SMN protein plays a critical role in pre-mRNA processing, because the biogenesis of spliceosomal snRNPs is promoted by the SMN complex [14, 18, 19], which consists of SMN (Gemin1), Gemin2–8 and UNR-interacting protein (UNRIP) [13, 16, 20]. In the formation of the SMN complex, SMN forms oligomers and directly interacts via its N-terminus with Gemin2 and via its tudor domain with spliceosomal (Sm) proteins [13, 21, 22]. A key component of the SMN complex, SMN first assembles the essential SMN/Gemin complex, which in turn mediates the formation of the Sm

**2. Structural and functional consequences of the SMA-linked missense** 

In general, genetic mutation includes missense, nonsense, insertion and deletion mutations. A nonsense mutation is a point mutation in a DNA sequence that results in a premature stop codon, or a nonsense codon in the transcribed mRNA, and in a truncated, incomplete and usually functionally deficient protein product. In contrast, a missense mutation involves substitution of one single amino acid residue, and therefore is able to provide unique access to residue-specific structural insights

**88**

or FL-SMN).

**Figure 1.**

**mutations of SMN**


#### **Table 1.**

*A list of new (compared with those summarised in [1]) experimentally determined SMN-related structures as of 25 September 2018 [40].*

In a solid alignment with the computational analysis in [1], a set of salt bridges were structurally identified between SMN's Asp44 (M\_Asp\_44) and Gemin2's Arg213 (2\_Arg\_213), as shown in **Table 2**. In particular, four intermolecular salt bridges were identified between the buried side chains (**Table 3**) of these two charged residues, i.e. according to the coordinates data in the PDB entry **5XJL** [42], as shown in **Figure 2**.

Taken together, it is conceivable that the buried side chains of SMN's Asp44 and Gemin2's Arg213 form a salt bridge, which constitutes a favourable electrostatic energy contribution to the SMN-Gemin2 complex structural stability [41], and highlights the functionally indispensable roles of the two residues' charged side chains, considering the experimental observation that the SMN-Gemin2 binding is abrogated by the D44V mutation [41], resulting in a functionally deficient SMAlinked D44V SMN mutant.

In addition to the intermolecular salt bridges formed between SMN's Asp44 and Gemin2's Arg213, a set of intramolecular salt bridges were also identified between side chains of SMN's Asp35 and Lys41 (**Table 2**), which was reported in [1], too, where 15 salt bridges were identified between the side chains of SMN's Asp35 and Lys41 in the salt bridge analysis of the NMR-determined SMN-Gemin2 complex ensemble (PDB ID: 2LEH) [22, 41]. In SMN, Lys41 is a positively charged residue and also a neighbouring residue of Asp44. Functionally different to the SMA-linked D44V mutation, a Lys41Ala (K41A) mutation (not SMA-linked) does not affect SMN-Gemin2 binding [41]. Thus, in another solid agreement with the structural analysis in [1], the structural analysis highlights that the salt bridges between SMN's Asp35 and Lys41 are intramolecular, i.e. within the apo SMN protein, instead of intermolecular, i.e. at the SMN-Gemin2 complex structure interface, which help to explain why the Lys41Ala (K41A) mutation is not SMA-linked [41].

Overall, there is a solid agreement between the old [1] and the new (this chapter) sets of computational structural analysis for both NMR and X-ray SMN-related structures, reflecting the technical maturity of the two main biophysical tools for biomolecular structure determination, particularly in light of the booming number of cryo-electron microscopy (cryo-EM) images uploaded to the Electron

**91**

**Table 2.**

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival…*

**PDB ID SBnum Residue A Atom A Residue B Atom B Distance (Å)** 5XJL 4 M\_ASP\_44 OD1 2\_ARG\_213 NH1 2.946 (Yellow) 5XJL 4 M\_ASP\_44 OD1 2\_ARG\_213 NH2 3.579 (Red) 5XJL 4 M\_ASP\_44 OD2 2\_ARG\_213 NH1 3.236 (Brown) 5XJL 4 M\_ASP\_44 OD2 2\_ARG\_213 NH2 3.848 (Blue) 5XJQ 3 M\_ASP\_44 OD1 2\_ARG\_213 NH1 2.760 5XJQ 3 M\_ASP\_44 OD1 2\_ARG\_213 NH2 3.593 5XJQ 3 M\_ASP\_44 OD2 2\_ARG\_213 NH1 2.968 5XJR 2 M\_ASP\_44 OD1 2\_ARG\_213 NH1 2.385 5XJR 2 M\_ASP\_44 OD2 2\_ARG\_213 NH1 2.871 5XJS 3 M\_ASP\_44 OD1 2\_ARG\_213 NH1 3.078 5XJS 3 M\_ASP\_44 OD1 2\_ARG\_213 NH2 3.670 5XJS 3 M\_ASP\_44 OD2 2\_ARG\_213 NH1 2.631 5XJT 3 M\_ASP\_44 OD1 2\_ARG\_213 NH1 2.335 5XJT 3 M\_ASP\_44 OD1 2\_ARG\_213 NH2 3.386 5XJT 3 M\_ASP\_44 OD2 2\_ARG\_213 NH1 3.067 5XJU 2 M\_ASP\_44 OD1 2\_ARG\_213 NH1 2.302 5XJU 2 M\_ASP\_44 OD2 2\_ARG\_213 NH1 2.989 5XJQ 1 M\_ASP\_35 OD1 M\_LYS\_41 NZ 3.921 5XJS 2 M\_ASP\_35 OD1 M\_LYS\_41 NZ 3.670 5XJS 2 M\_ASP\_35 OD2 M\_LYS\_41 NZ 3.803 5XJT 2 M\_ASP\_35 OD1 M\_LYS\_41 NZ 2.416 XJT 2 M\_ASP\_35 OD2 M\_LYS\_41 NZ 2.931 5XJU 1 M\_ASP\_35 OD1 M\_LYS\_41 NZ 3.274 *In this table, the residue naming scheme is* **Chain ID\_residue name\_residue number***, SBnum represents the number of salt bridges computationally identified from the PDB entries listed in this table. In the top four rows for PDB entry*  **5XJL***, Yellow, Red, Brown and Blue represent the colouring scheme for Figure 2. Distance represents the distance* 

Microscopy Data Bank (EMDB), where a long way is there to go still for cryo-EM to match NMR spectroscopy and X-ray crystallography in terms of technical maturity

*A summary of salt bridge analysis of the six new SMN-related structures as of 25 September 2018 [40].*

Although not located in the structurally determined region of the six new structures (**Table 1**), Gly95 is a residue in the SMN tudor domain, and it is involved in a Gly95Arg (G95R) mutation [25]. This G95R mutation significantly reduces SMN's ability to bind Sm proteins, such as Sm-B and Sm-D1 [25], confirming that tudor

In a further inspection of the computational analysis as reported in [1], no salt bridge or hydrogen bond was identified for Gly95. Nonetheless, in the SMN tudor domain NMR ensemble [46], between the side chains of Asp96 and Lys93, 1 salt bridge was found for PDB ID **1G5V** [46] with 10 structure models, 18 salt bridges

and the urgent need of tools for structural model quality validation [45].

domain is the essential binding site of SMN to Sm proteins.

*2.3.2 Gly95 in the SMN tudor domain*

*between two oppositely charged groups/atoms in Å.*

*DOI: http://dx.doi.org/10.5772/intechopen.81887*


*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival… DOI: http://dx.doi.org/10.5772/intechopen.81887*

*In this table, the residue naming scheme is* **Chain ID\_residue name\_residue number***, SBnum represents the number of salt bridges computationally identified from the PDB entries listed in this table. In the top four rows for PDB entry*  **5XJL***, Yellow, Red, Brown and Blue represent the colouring scheme for Figure 2. Distance represents the distance between two oppositely charged groups/atoms in Å.*

#### **Table 2.**

*Novel Aspects on Motor Neuron Disease*

human

human

as shown in **Figure 2**.

*of 25 September 2018 [40].*

**Table 1.**

linked D44V SMN mutant.

In a solid alignment with the computational analysis in [1], a set of salt bridges were structurally identified between SMN's Asp44 (M\_Asp\_44) and Gemin2's Arg213 (2\_Arg\_213), as shown in **Table 2**. In particular, four intermolecular salt bridges were identified between the buried side chains (**Table 3**) of these two charged residues, i.e. according to the coordinates data in the PDB entry **5XJL** [42],

**PDB ID Structure title Method Release** 

5XJL Crystal structure of the Gemin2-binding domain of SMN,

5XJQ Crystal structure of the Gemin2-binding domain of SMN,

5XJR Crystal structure of the Gemin2-binding domain of SMN,

5XJS Crystal structure of the Gemin2-binding domain of SMN,

5XJT Crystal structure of the Gemin2-binding domain of SMN,

5XJU Crystal structure of the Gemin2-binding domain of SMN,

Gemin2 in complex with SmD1/D2/F/E/G from human

Gemin2 in complex with SmD1(1–82)/D2/F/E/G from human

Gemin2dN39 in complex with SmD1(1-82)/D2/F/E/G from human

Gemin2dN39 in complex with SmD1(1-82)/D2/F/E from human

Gemin2dN39 in complex with SmD1(1-82)/D2.R61A/F/E/G from

*In this table, X-ray represents X-ray crystallography as a biophysical tool for biomolecular structure determination.*

*A list of new (compared with those summarised in [1]) experimentally determined SMN-related structures as* 

Gemin2 in complex with SmD1(1-82)/D2.R61A/F/E/G from

**date**

2018

2018

2018

2018

2018

2018

X-ray 2 May

X-ray 4 July

X-ray 4 July

X-ray 4 July

X-ray 4 July

X-ray 4 July

Taken together, it is conceivable that the buried side chains of SMN's Asp44 and Gemin2's Arg213 form a salt bridge, which constitutes a favourable electrostatic energy contribution to the SMN-Gemin2 complex structural stability [41], and highlights the functionally indispensable roles of the two residues' charged side chains, considering the experimental observation that the SMN-Gemin2 binding is abrogated by the D44V mutation [41], resulting in a functionally deficient SMA-

In addition to the intermolecular salt bridges formed between SMN's Asp44 and Gemin2's Arg213, a set of intramolecular salt bridges were also identified between side chains of SMN's Asp35 and Lys41 (**Table 2**), which was reported in [1], too, where 15 salt bridges were identified between the side chains of SMN's Asp35 and Lys41 in the salt bridge analysis of the NMR-determined SMN-Gemin2 complex ensemble (PDB ID: 2LEH) [22, 41]. In SMN, Lys41 is a positively charged residue and also a neighbouring residue of Asp44. Functionally different to the SMA-linked D44V mutation, a Lys41Ala (K41A) mutation (not SMA-linked) does not affect SMN-Gemin2 binding [41]. Thus, in another solid agreement with the structural analysis in [1], the structural analysis highlights that the salt bridges between SMN's Asp35 and Lys41 are intramolecular, i.e. within the apo SMN protein, instead of intermolecular, i.e. at the SMN-Gemin2 complex structure interface, which help to

Overall, there is a solid agreement between the old [1] and the new (this chapter) sets of computational structural analysis for both NMR and X-ray SMN-related structures, reflecting the technical maturity of the two main biophysical tools for biomolecular structure determination, particularly in light of the booming number of cryo-electron microscopy (cryo-EM) images uploaded to the Electron

explain why the Lys41Ala (K41A) mutation is not SMA-linked [41].

**90**

*A summary of salt bridge analysis of the six new SMN-related structures as of 25 September 2018 [40].*

Microscopy Data Bank (EMDB), where a long way is there to go still for cryo-EM to match NMR spectroscopy and X-ray crystallography in terms of technical maturity and the urgent need of tools for structural model quality validation [45].

#### *2.3.2 Gly95 in the SMN tudor domain*

Although not located in the structurally determined region of the six new structures (**Table 1**), Gly95 is a residue in the SMN tudor domain, and it is involved in a Gly95Arg (G95R) mutation [25]. This G95R mutation significantly reduces SMN's ability to bind Sm proteins, such as Sm-B and Sm-D1 [25], confirming that tudor domain is the essential binding site of SMN to Sm proteins.

In a further inspection of the computational analysis as reported in [1], no salt bridge or hydrogen bond was identified for Gly95. Nonetheless, in the SMN tudor domain NMR ensemble [46], between the side chains of Asp96 and Lys93, 1 salt bridge was found for PDB ID **1G5V** [46] with 10 structure models, 18 salt bridges


*In this table, SASA, SASA-intrinsic and SASA-ratio represent for SMN's Asp44 and Gemin2's Arg213 the average SASA value calculated by DSSP [43], the intrinsic SASA value [44] and the ratio of SASA divided by SASA-intrinsic, respectively. In this table, the residue naming scheme is* **Chain ID\_residue name\_residue number***.*

#### **Table 3.**

*Solvent accessible surface area (SASA) values of SMN's Asp44 and Gemin2's Arg213 (PDB ID:* **5XJL***) [42].*

#### **Figure 2.**

*Four salt bridges formed between the buried side chains of SMN's Asp44 (M\_Asp\_44 in red text) and Gemin2's Arg213 (2\_Arg\_213 in white text). In this figure, the residue naming scheme is* **Chain ID\_residue name\_residue number***. In this figure, Asp44's side chain oxygens are coloured red, and Arg213's nitrogen atoms are coloured blue, while all hydrogen atoms are coloured in white, the four dotted lines in four colours represent the four side chain salt bridges formed between the two oppositely charged residues, where the colouring scheme is described in Table 2.*

were found for PDB ID **4A4E** [47] with 20 structure models (**Figure 3**) and 16 salt bridges were found for PDB ID **4A4G** [47] with 20 structure models. Similarly, 15 salt bridges were also identified between the side chains of Glu147 and Lys97 of SMN (PDB ID: **4A4G** [47], with 20 structure models), with the distance between 2 oppositely charged groups being 2.93 ± 0.39 Å.

Quite interestingly, Gly95 sits right between the two oppositely charged neighbouring residues (Asp96 and Lys93), which are the only two charged residues in the

#### **Figure 3.**

*Two salt bridges formed between the side chains of SMN's Asp96 and Lys93 (shown as sticks here) according to a salt bridge analysis of the third structural model of the NMR ensemble (PDB ID* **4A4E***) [47]. In this figure, Asp96's side chain oxygens are coloured red, and Lys93's side chain nitrogen is coloured blue, while all hydrogen atoms are coloured in white.*

**93**

**Table 4.**

**4A4E***).*

of 111.1 ± 4.18 Å2

*SASA* value at 212.7 Å2

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival…*

tudor domain that are in the spatial proximity of Gly95. Thus, it is conceivable that a G95R mutation disrupts the Asp96-Lys93 salt bridge and/or builds another one (possibly even stronger) between the side chains of Lys95 and Asp96, which either perturbs the structure-stabilising activity of the Asp96-Lys93 salt bridge, and/or makes it energetically more unfavourable for Asp96's side chain to orient towards positively charged side chains in Sm proteins and thereby affect the binding of SMN to Sm proteins. While the potential local electrostatic interaction disruption mechanism here for this SMA-linked G95R mutation is similar to that of the E134K and the Q136E mutations of SMN [1], the former mechanism is dependent on the occurrence of energetically unfavourable electrostatic interaction(s), but the latter mechanism is dependent on the loss of energetically favourable electrostatic interaction(s) for local structural stability of the SMN tudor domain, the essential part of SMN for the Sm protein-binding, which can help explain the reduced Sm core assembly activity of the two SMA-linked SMNE134K and SMNQ136E mutants.

Among the eight SMN residues with SMA-linked missense mutations [39], only Y109 and Y130 are located in the structurally determined region of SMN [1], according to the updated list of SMN-related structures as of 25 September 2018. Although Y109C, Y130C and Y130H are not located in the structurally determined region of the six new structures, the three missense mutations are located in the structurally determined region of the experimentally determined structures [1]. Tyr130 is a tudor domain hydrophobic residue with a Tyr130Cys (Y130C) mutation [29]. In the computational analysis in [1], no salt bridge or hydrogen bond was identified for Tyr130. Nonetheless, Tyr130 is ∼50% buried, with an *SASA* value

compared with its standard *SASA* value at 212.7 Å2

mutations highlights the potential significance of the deeply buried hydrophobic

0.pdb OD2, A\_ASP\_105 OH, A\_TYR\_109 HH, A\_TYR\_109 2.73 1.80 13.75 3.pdb OD2, A\_ASP\_105 OH, A\_TYR\_109 HH, A\_TYR\_109 2.69 1.77 15.61 4.pdb OD2, A\_ASP\_105 OH, A\_TYR\_109 HH, A\_TYR\_109 2.67 1.72 10.96 5.pdb OD2, A\_ASP\_105 OH, A\_TYR\_109 HH, A\_TYR\_109 2.77 1.86 17.26 6.pdb OD2, A\_ASP\_105 OH, A\_TYR\_109 HH, A\_TYR\_109 2.71 1.78 15.09 8.pdb OD2, A\_ASP\_105 OH, A\_TYR\_109 HH, A\_TYR\_109 2.78 1.87 16.14 12.pdb OD2, A\_ASP\_105 OH, A\_TYR\_109 HH, A\_TYR\_109 2.83 1.95 20.70 14.pdb OD2, A\_ASP\_105 OH, A\_TYR\_109 HH, A\_TYR\_109 2.71 1.78 13.76 18.pdb OD2, A\_ASP\_105 OH, A\_TYR\_109 HH, A\_TYR\_109 2.71 1.76 10.91 19.pdb OD2, A\_ASP\_105 OH, A\_TYR\_109 HH, A\_TYR\_109 2.63 1.70 13.36 *In this table, the names of the PDB files correspond to the single NMR structural model split from the NMR ensemble (PDB entry* **4A4E***) by a tcl script [1], the residue naming scheme is* **Chain ID\_residue name\_residue number***,* 

. Taken together, the *SASA* analysis of the three SMA-linked

, while Tyr109

**∠***ADH***(∗)**

compared with its standard

**H-A (Å)**

**(Å)**

*DOI: http://dx.doi.org/10.5772/intechopen.81887*

*2.3.3 Y109C, Y130C and Y130H in the SMN tudor domain*

is deeply buried, with an *SASA* value of 61.1 ± 8.43 Å2

side chains of Tyr109 and Tyr130 in the SMN tudor domain.

**PDB File Acceptor (A) Donor (D) Hydrogen (H) D-A** 

∠*ADH represents the angle formed by acceptor (A), donor (D) and hydrogen (H) (*∠*ADH).*

*The hydrogen bonds formed between the residue side chains between SMN's Tyr109 and Asp105 (PDB entry* 

#### *Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival… DOI: http://dx.doi.org/10.5772/intechopen.81887*

tudor domain that are in the spatial proximity of Gly95. Thus, it is conceivable that a G95R mutation disrupts the Asp96-Lys93 salt bridge and/or builds another one (possibly even stronger) between the side chains of Lys95 and Asp96, which either perturbs the structure-stabilising activity of the Asp96-Lys93 salt bridge, and/or makes it energetically more unfavourable for Asp96's side chain to orient towards positively charged side chains in Sm proteins and thereby affect the binding of SMN to Sm proteins. While the potential local electrostatic interaction disruption mechanism here for this SMA-linked G95R mutation is similar to that of the E134K and the Q136E mutations of SMN [1], the former mechanism is dependent on the occurrence of energetically unfavourable electrostatic interaction(s), but the latter mechanism is dependent on the loss of energetically favourable electrostatic interaction(s) for local structural stability of the SMN tudor domain, the essential part of SMN for the Sm protein-binding, which can help explain the reduced Sm core assembly activity of the two SMA-linked SMNE134K and SMNQ136E mutants.

#### *2.3.3 Y109C, Y130C and Y130H in the SMN tudor domain*

Among the eight SMN residues with SMA-linked missense mutations [39], only Y109 and Y130 are located in the structurally determined region of SMN [1], according to the updated list of SMN-related structures as of 25 September 2018. Although Y109C, Y130C and Y130H are not located in the structurally determined region of the six new structures, the three missense mutations are located in the structurally determined region of the experimentally determined structures [1].

Tyr130 is a tudor domain hydrophobic residue with a Tyr130Cys (Y130C) mutation [29]. In the computational analysis in [1], no salt bridge or hydrogen bond was identified for Tyr130. Nonetheless, Tyr130 is ∼50% buried, with an *SASA* value of 111.1 ± 4.18 Å2 compared with its standard *SASA* value at 212.7 Å2 , while Tyr109 is deeply buried, with an *SASA* value of 61.1 ± 8.43 Å2 compared with its standard *SASA* value at 212.7 Å2 . Taken together, the *SASA* analysis of the three SMA-linked mutations highlights the potential significance of the deeply buried hydrophobic side chains of Tyr109 and Tyr130 in the SMN tudor domain.


*In this table, the names of the PDB files correspond to the single NMR structural model split from the NMR ensemble (PDB entry* **4A4E***) by a tcl script [1], the residue naming scheme is* **Chain ID\_residue name\_residue number***,*  ∠*ADH represents the angle formed by acceptor (A), donor (D) and hydrogen (H) (*∠*ADH).*

#### **Table 4.**

*The hydrogen bonds formed between the residue side chains between SMN's Tyr109 and Asp105 (PDB entry*  **4A4E***).*

*Novel Aspects on Motor Neuron Disease*

**Residue SASA (Å2**

**92**

**Figure 3.**

**Figure 2.**

**Table 3.**

*atoms are coloured in white.*

*Two salt bridges formed between the side chains of SMN's Asp96 and Lys93 (shown as sticks here) according to a salt bridge analysis of the third structural model of the NMR ensemble (PDB ID* **4A4E***) [47]. In this figure, Asp96's side chain oxygens are coloured red, and Lys93's side chain nitrogen is coloured blue, while all hydrogen* 

were found for PDB ID **4A4E** [47] with 20 structure models (**Figure 3**) and 16 salt bridges were found for PDB ID **4A4G** [47] with 20 structure models. Similarly, 15 salt bridges were also identified between the side chains of Glu147 and Lys97 of SMN (PDB ID: **4A4G** [47], with 20 structure models), with the distance between 2

*Four salt bridges formed between the buried side chains of SMN's Asp44 (M\_Asp\_44 in red text) and Gemin2's Arg213 (2\_Arg\_213 in white text). In this figure, the residue naming scheme is* **Chain ID\_residue name\_residue number***. In this figure, Asp44's side chain oxygens are coloured red, and Arg213's nitrogen atoms are coloured blue, while all hydrogen atoms are coloured in white, the four dotted lines in four colours represent the four side chain salt bridges formed between the two oppositely charged residues, where the colouring scheme is described in Table 2.*

**) SASA-intrinsic (Å2**

2\_Arg\_213 57 238.76 0.238 M\_Asp\_44 67 140.39 0.477 *In this table, SASA, SASA-intrinsic and SASA-ratio represent for SMN's Asp44 and Gemin2's Arg213 the average SASA value calculated by DSSP [43], the intrinsic SASA value [44] and the ratio of SASA divided by SASA-intrinsic,* 

*Solvent accessible surface area (SASA) values of SMN's Asp44 and Gemin2's Arg213 (PDB ID:* **5XJL***) [42].*

*respectively. In this table, the residue naming scheme is* **Chain ID\_residue name\_residue number***.*

**) SASA-Ratio**

Quite interestingly, Gly95 sits right between the two oppositely charged neighbouring residues (Asp96 and Lys93), which are the only two charged residues in the

oppositely charged groups being 2.93 ± 0.39 Å.

What is more, in the computational analysis in [1], 10 side chain hydrogen bonds (**Table 4**) were identified between SMN's Tyr109 and Asp105 in the PDB entry **4A4E** [47], with the donor-acceptor distances (*D* − *A* in **Table 4**) at 2.72 ± 0.06 Å and ∠*ADH* at 14.75 ± 2.93, no salt bridge was identified for Asp105, and no further hydrogen bonds were identified for Tyr109 and Asp105 for all experimentally determined SMN-related structures as of 25 September 2018.

Taken together, the computational findings here indicate that SMN's Tyr109 and Asp105 contribute to the structural stability of SMN through hydrogen bonding between their side chains, as it is quite clear that if Tyr109 is replaced by Cys109, then the side chain hydrogen bond (**Figure 4**, **Table 4**) will disappear, and that the negatively charged side chain of Asp105 will gain more geometric freedom due to the disappearance of the hydrogen bond, which can cause a potential disruption of the (either intramolecular and/or intermolecular) electrostatic interaction network, not to mention the possibility of a disrupted disulphide bonding network within the SMN protein, the SMN complex or even the snRNP assembly, which is critical to ensure that pre-mRNA editing of the *SMN1* gene does not go wrong and that its product is the FL-SMN protein, instead of its truncated functionally deficient counterpart.

#### *2.3.4 A structural analysis of the hydrogen bonds formed within the six new SMN-related structures*

In light of the six new experimentally determined SMN-related structures (**Table 1**), a new set of hydrogen bonding analysis is conducted according to the details in [1], the result of which is briefly summarised in **Table 5**.

**Table 5** shows the four hydrogen bonds formed between snRNP Sm-D2's Asp93 and Gemin2's Arg235 and Arg239. Functionally, Gemin2 is closely linked to SMN (formerly known as Gemin1), and NMR spectroscopy was used to experimentally determine a Gemin1-Gemin2 complex structure (PDB ID: 2LEH) [22, 41], making a closer visual inspection worthwhile of the SMN-related structures (PDB IDs: 5XJS, 5XJT and 5XJU [42], **Table 1**).

From **Figure 5** (PDB ID:5XJS), it is quite clear that the three charged residues (snRNP Sm-D2's Asp93 and Gemin2's Arg235 and Arg239) sit right at the structural interface between Sm-D2 (pink) and Gemin2 (green), with their oppositely

#### **Figure 4.**

*The hydrogen bond (Table 4) formed between the side chains of SMN's Tyr109 and Asp105 in the PDB entry*  **4A4E** *[47]. In this figure, SMN's Tyr109 and Asp105 are shown in sticks, all side chain oxygens are coloured red, and side chain nitrogen is coloured blue, while all hydrogen atoms are coloured in white, and all atoms are labelled with their names nearby. The blue dotted line between OD2 of Asp105 and HH of Tyr109 represents the hydrogen bond formed between SMN's Tyr109 and Asp105.*

**95**

below and illustrated in **Figure 6**.

structure formation [1].

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival…*

5XJR OE1, A\_GLN\_24 NH2, B\_ARG\_94 HH21, B\_ARG\_94 3.00 1.99 1.79 5XJR OD2, B\_ASP\_104 NH1, B\_ARG\_102 HH12, B\_ARG\_102 2.98 2.07 20.93 5XJS OD1, B\_ASP\_93 NE, 2\_ARG\_235 HE, 2\_ARG\_235 2.94 1.96 11.86 5XJS OD1, B\_ASP\_93 NH2, 2\_ARG\_239 HH21, 2\_ARG\_239 2.98 1.98 5.80 5XJS OD2, B\_ASP\_60 ND2, B\_ASN\_64 HD22, B\_ASN\_64 2.99 2.13 25.53 5XJT OD1, B\_ASP\_93 NE, 2\_ARG\_235 HE, 2\_ARG\_235 2.65 1.75 21.43 5XJU OD1, B\_ASP\_93 NE, 2\_ARG\_235 HE, 2\_ARG\_235 2.98 2.08 23.00 5XJU OD2, B\_ASP\_60 ND2, B\_ASN\_64 HD21, B\_ASN\_64 2.80 1.99 29.63 *In this table, the residue naming scheme is* **Chain ID\_residue name\_residue number***,* ∠*ADH represents the angle* 

*The hydrogen bonds formed between the residue side chains within the six new experimentally determined* 

**(Å)**

**H-A (Å)**

**∠***ADH***(∗)**

**PDB ID Acceptor (A) Donor (D) Hydrogen (H) D-A** 

charged side chains closely facing each other, similar to the situation as reported by [1], where the deeply buried side chains of SMN's Lys45 and Asp36 act as two electrostatic clips at the SMN-Gemin2 interface via interactions with both the side chains and the backbone of Gemin2's Gln105, Gln109, His120, His123 and Trp124. In the subsequent computational salt bridge analysis of the six new SMN-related

*residues are shown in sticks and labelled with red and blue texts nearby.*

*Crystal structure of the Gemin2-binding domain of SMN, Gemin2 in complex with SmD1/D2/F/E (PDB ID:*  **5XJS** *[42]). In this figure, the whole structure is shown in cartoon and coloured by chain using PyMol [48], where green and pink represent Gemin2 and snRNP Sm-D2, respectively. In this figure, three amino acid* 

structures, it turned out that the three charged residues did form salt bridges between their closely facing oppositely charged side chains, as listed in **Table 6**

Collectively, snRNP Sm-D2's Asp93 and Gemin2's Arg235 and Arg239 are three structurally important residues which help stabilise the structural interface through intermolecular electrostatic interactions, including both salt bridges and also hydrogen bonds, similar to the way SMN's Asp44, Gemin2's Arg213 and the two SMN residues (Lys45 and Asp36) play stabilising roles in the SMN-Gemin2 complex

Considering the intimate functional relationship between Gemin2 and SMN, a further set of structural analysis was conducted for the hydrogen bond and the salt bridge for Arg235 and Arg239 of PDB entry 2LEH [22, 41], and it turned out that

*DOI: http://dx.doi.org/10.5772/intechopen.81887*

*formed by acceptor (A), donor (D) and hydrogen (H) (*∠*ADH).*

**Table 5.**

**Figure 5.**

*SMN-related structures.*

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival… DOI: http://dx.doi.org/10.5772/intechopen.81887*


*In this table, the residue naming scheme is* **Chain ID\_residue name\_residue number***,* ∠*ADH represents the angle formed by acceptor (A), donor (D) and hydrogen (H) (*∠*ADH).*

#### **Table 5.**

*Novel Aspects on Motor Neuron Disease*

counterpart.

*SMN-related structures*

5XJT and 5XJU [42], **Table 1**).

What is more, in the computational analysis in [1], 10 side chain hydrogen bonds

Taken together, the computational findings here indicate that SMN's Tyr109 and Asp105 contribute to the structural stability of SMN through hydrogen bonding between their side chains, as it is quite clear that if Tyr109 is replaced by Cys109, then the side chain hydrogen bond (**Figure 4**, **Table 4**) will disappear, and that the negatively charged side chain of Asp105 will gain more geometric freedom due to the disappearance of the hydrogen bond, which can cause a potential disruption of the (either intramolecular and/or intermolecular) electrostatic interaction network, not to mention the possibility of a disrupted disulphide bonding network within the SMN protein, the SMN complex or even the snRNP assembly, which is critical to ensure that pre-mRNA editing of the *SMN1* gene does not go wrong and that its product is the FL-SMN protein, instead of its truncated functionally deficient

(**Table 4**) were identified between SMN's Tyr109 and Asp105 in the PDB entry **4A4E** [47], with the donor-acceptor distances (*D* − *A* in **Table 4**) at 2.72 ± 0.06 Å and ∠*ADH* at 14.75 ± 2.93, no salt bridge was identified for Asp105, and no further hydrogen bonds were identified for Tyr109 and Asp105 for all experimentally

determined SMN-related structures as of 25 September 2018.

*2.3.4 A structural analysis of the hydrogen bonds formed within the six new* 

details in [1], the result of which is briefly summarised in **Table 5**.

In light of the six new experimentally determined SMN-related structures (**Table 1**), a new set of hydrogen bonding analysis is conducted according to the

**Table 5** shows the four hydrogen bonds formed between snRNP Sm-D2's Asp93 and Gemin2's Arg235 and Arg239. Functionally, Gemin2 is closely linked to SMN (formerly known as Gemin1), and NMR spectroscopy was used to experimentally determine a Gemin1-Gemin2 complex structure (PDB ID: 2LEH) [22, 41], making a closer visual inspection worthwhile of the SMN-related structures (PDB IDs: 5XJS,

From **Figure 5** (PDB ID:5XJS), it is quite clear that the three charged residues (snRNP Sm-D2's Asp93 and Gemin2's Arg235 and Arg239) sit right at the structural interface between Sm-D2 (pink) and Gemin2 (green), with their oppositely

*The hydrogen bond (Table 4) formed between the side chains of SMN's Tyr109 and Asp105 in the PDB entry*  **4A4E** *[47]. In this figure, SMN's Tyr109 and Asp105 are shown in sticks, all side chain oxygens are coloured red, and side chain nitrogen is coloured blue, while all hydrogen atoms are coloured in white, and all atoms are labelled with their names nearby. The blue dotted line between OD2 of Asp105 and HH of Tyr109 represents* 

*the hydrogen bond formed between SMN's Tyr109 and Asp105.*

**94**

**Figure 4.**

*The hydrogen bonds formed between the residue side chains within the six new experimentally determined SMN-related structures.*

#### **Figure 5.**

*Crystal structure of the Gemin2-binding domain of SMN, Gemin2 in complex with SmD1/D2/F/E (PDB ID:*  **5XJS** *[42]). In this figure, the whole structure is shown in cartoon and coloured by chain using PyMol [48], where green and pink represent Gemin2 and snRNP Sm-D2, respectively. In this figure, three amino acid residues are shown in sticks and labelled with red and blue texts nearby.*

charged side chains closely facing each other, similar to the situation as reported by [1], where the deeply buried side chains of SMN's Lys45 and Asp36 act as two electrostatic clips at the SMN-Gemin2 interface via interactions with both the side chains and the backbone of Gemin2's Gln105, Gln109, His120, His123 and Trp124.

In the subsequent computational salt bridge analysis of the six new SMN-related structures, it turned out that the three charged residues did form salt bridges between their closely facing oppositely charged side chains, as listed in **Table 6** below and illustrated in **Figure 6**.

Collectively, snRNP Sm-D2's Asp93 and Gemin2's Arg235 and Arg239 are three structurally important residues which help stabilise the structural interface through intermolecular electrostatic interactions, including both salt bridges and also hydrogen bonds, similar to the way SMN's Asp44, Gemin2's Arg213 and the two SMN residues (Lys45 and Asp36) play stabilising roles in the SMN-Gemin2 complex structure formation [1].

Considering the intimate functional relationship between Gemin2 and SMN, a further set of structural analysis was conducted for the hydrogen bond and the salt bridge for Arg235 and Arg239 of PDB entry 2LEH [22, 41], and it turned out that


*In this table, the residue naming scheme is* **Chain ID\_residue name\_residue number***, SBnum represents the number of salt bridges computationally identified from the PDB entries listed in this table. Distance represents the distance between two oppositely charged groups/atoms in Å.*

#### **Table 6.**

*A summary of salt bridge analysis of the six new SMN-related structures as of 25 September 2018 [40].*

the two arginines did not form any intermolecular electrostatic interaction with SMN, neither salt bridge nor hydrogen bond. Instead, the 2 arginines of Gemin2 only formed 2 hydrogen bonds with Gln272 and His231 of Gemin2, and 1 stable salt bridge with Asp274 of Gemin2, where 16 salt bridges were identified for the 32 NMR structural models (**Table 7**), according to the structural analysis of PDB entry **2LEH** [22, 41].

**97**

**Table 7.**

**3. Concluding remarks**

*of salt bridges computationally identified.*

*A summary of salt bridge analysis of PDB entry 2LEH [22, 41].*

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival…*

*Crystal structure of the Gemin2-binding domain of SMN, Gemin2 in complex with SmD1/D2/F/E (PDB ID:*  **5XJS***) [42]. In this figure, the yellow dotted lines represent two examples of the hydrogen bonds formed between Asp93 and Arg235, while the blue dotted line represents an example of the salt bridge formed between Asp93* 

**PDB ID SBnum Residue A Atom A Residue B Atom B Distance (Å)** 24.pdb 2 A\_ASP\_274 OD1 A\_ARG\_239 NH2 3.797 24.pdb 2 A\_ASP\_274 OD2 A\_ARG\_239 NH2 2.738 01.pdb 1 A\_ASP\_274 OD2 A\_ARG\_235 NH2 3.577 02.pdb 1 A\_ASP\_274 OD2 A\_ARG\_235 NH2 3.164 04.pdb 1 A\_ASP\_274 OD2 A\_ARG\_235 NH2 3.195 09.pdb 1 A\_ASP\_274 OD2 A\_ARG\_235 NH2 2.692 16.pdb 1 A\_ASP\_274 OD2 A\_ARG\_235 NH1 3.871 17.pdb 1 A\_ASP\_274 OD2 A\_ARG\_235 NH1 2.868 19.pdb 1 A\_ASP\_274 OD2 A\_ARG\_235 NH1 3.718 21.pdb 2 A\_ASP\_274 OD2 A\_ARG\_235 NH1 3.457 21.pdb 2 A\_ASP\_274 OD2 A\_ARG\_235 NH2 3.429 23.pdb 1 A\_ASP\_274 OD2 A\_ARG\_235 NH2 3.798 24.pdb 1 A\_ASP\_274 OD2 A\_ARG\_235 NH2 2.888 26.pdb 3 A\_ASP\_274 OD1 A\_ARG\_235 NH2 3.770 26.pdb 3 A\_ASP\_274 OD2 A\_ARG\_235 NH1 3.836 26.pdb 3 A\_ASP\_274 OD2 A\_ARG\_235 NH2 2.442 31.pdb 1 A\_ASP\_274 OD2 A\_ARG\_235 NH2 3.403 *In this table, the residue naming scheme is* **Chain ID\_residue name\_residue number***, SBnum represents the number* 

Given SMN's critical role in the maturation of snRNP and in the development of SMA [2, 6, 11], it is necessary for the structure-activity relationship (SAR) characterisation to continue for the SMA protein. With various biophysical tools available for structural determination, for SMN-related proteins and biological complexes,

*DOI: http://dx.doi.org/10.5772/intechopen.81887*

**Figure 6.**

*and Arg239 (Table 6).*

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival… DOI: http://dx.doi.org/10.5772/intechopen.81887*

#### **Figure 6.**

*Novel Aspects on Motor Neuron Disease*

**PDB ID SBnum Residue A Atom A Residue B Atom B Distance (Å)** 5XJL 3 B\_ASP\_93 OD1 2\_ARG\_239 NH1 3.734 5XJL 3 B\_ASP\_93 OD1 2\_ARG\_239 NH2 3.052 5XJL 3 B\_ASP\_93 OD2 2\_ARG\_239 NH2 3.052 5XJQ 3 B\_ASP\_93 OD1 2\_ARG\_239 NH1 3.817 5XJQ 3 B\_ASP\_93 OD1 2\_ARG\_239 NH2 3.059 5XJQ 3 B\_ASP\_93 OD2 2\_ARG\_239 NH2 3.004 5XJR 3 B\_ASP\_93 OD1 2\_ARG\_239 NH1 3.811 5XJR 3 B\_ASP\_93 OD1 2\_ARG\_239 NH2 3.022 5XJR 3 B\_ASP\_93 OD2 2\_ARG\_239 NH2 2.938 5XJS 3 B\_ASP\_93 OD1 2\_ARG\_239 NH1 3.688 5XJS 3 B\_ASP\_93 OD1 2\_ARG\_239 NH2 2.983 5XJS 3 B\_ASP\_93 OD2 2\_ARG\_239 NH2 3.092 5XJT 2 B\_ASP\_93 OD1 2\_ARG\_239 NH2 3.251 5XJT 2 B\_ASP\_93 OD2 2\_ARG\_239 NH2 3.163 5XJU 3 B\_ASP\_93 OD1 2\_ARG\_239 NH1 3.634 5XJU 3 B\_ASP\_93 OD1 2\_ARG\_239 NH2 3.084 5XJU 3 B\_ASP\_93 OD2 2\_ARG\_239 NH2 3.089 5XJL 2 B\_ASP\_93 OD1 2\_ARG\_235 NH2 3.657 5XJL 2 B\_ASP\_93 OD2 2\_ARG\_235 NH2 3.475 5XJQ 2 B\_ASP\_93 OD1 2\_ARG\_235 NH2 3.686 5XJQ 2 B\_ASP\_93 OD2 2\_ARG\_235 NH2 3.647 5XJR 2 B\_ASP\_93 OD1 2\_ARG\_235 NH2 3.847 5XJR 2 B\_ASP\_93 OD2 2\_ARG\_235 NH2 3.800 5XJS 2 B\_ASP\_93 OD1 2\_ARG\_235 NH2 3.548 5XJS 2 B\_ASP\_93 OD2 2\_ARG\_235 NH2 3.379 5XJT 2 B\_ASP\_93 OD1 2\_ARG\_235 NH2 3.258 5XJT 2 B\_ASP\_93 OD2 2\_ARG\_235 NH2 3.766 5XJU 1 B\_ASP\_93 OD1 2\_ARG\_235 NH2 3.996 *In this table, the residue naming scheme is* **Chain ID\_residue name\_residue number***, SBnum represents the number of salt bridges computationally identified from the PDB entries listed in this table. Distance represents the distance* 

**96**

**Table 6.**

**2LEH** [22, 41].

*between two oppositely charged groups/atoms in Å.*

the two arginines did not form any intermolecular electrostatic interaction with SMN, neither salt bridge nor hydrogen bond. Instead, the 2 arginines of Gemin2 only formed 2 hydrogen bonds with Gln272 and His231 of Gemin2, and 1 stable salt bridge with Asp274 of Gemin2, where 16 salt bridges were identified for the 32 NMR structural models (**Table 7**), according to the structural analysis of PDB entry

*A summary of salt bridge analysis of the six new SMN-related structures as of 25 September 2018 [40].*

*Crystal structure of the Gemin2-binding domain of SMN, Gemin2 in complex with SmD1/D2/F/E (PDB ID:*  **5XJS***) [42]. In this figure, the yellow dotted lines represent two examples of the hydrogen bonds formed between Asp93 and Arg235, while the blue dotted line represents an example of the salt bridge formed between Asp93 and Arg239 (Table 6).*


*In this table, the residue naming scheme is* **Chain ID\_residue name\_residue number***, SBnum represents the number of salt bridges computationally identified.*

#### **Table 7.**

*A summary of salt bridge analysis of PDB entry 2LEH [22, 41].*

#### **3. Concluding remarks**

Given SMN's critical role in the maturation of snRNP and in the development of SMA [2, 6, 11], it is necessary for the structure-activity relationship (SAR) characterisation to continue for the SMA protein. With various biophysical tools available for structural determination, for SMN-related proteins and biological complexes,

such as the SMN complex and snRNPs, their structure determination and functional characterisation will undoubtedly continue to advance, which will be helpful both in further understanding of SMN's role in SMA from a molecular structural point of view. In practice, however, advancements do not come easy. For instance, although both full-length structures of FL-SMN (with 294 residues) and SMNΔ7 (with 282 residues) were already experimentally determined using X-ray crystallography and deposited in the database (PDB IDs: 4NL6 and 4NL7), they were subsequently withdrawn by the author because the sample used for the structure determination was wrong. Otherwise, these two full-length SMN structures would constitute the very first step towards a comprehensive picture of the structural and functional insights into SMN's role in the molecular pathogenesis SMA.

As of 25 September 2018, there is still no full-length SMN (or the SMN complex or the snRNP assembly) structure deposited in the wwPDB website [40], although it contains six new experimentally determined SMN-related structures, in addition to those reported in [1]. In terms of amino acid sequence, those SMN-related structures are still only SMN fragments, ranging from Gly26 to Lys51, and from Asn84 to Glu147. In between, there is still structurally not-determined-yet regions (referred to as **structural gaps** below) consisting of 204 SMN residues. Sixteen months have passed since the publication of [1], the structural gaps still remain, literally zero progress has been made to bridge them in spite of the six newly deposited structures, calling **again** [1] for further comprehensive structural determination and functional research for this SMA protein.

#### **4. A residue-specific distributional analysis of the structural gaps in the Protein Data Bank**

As a 38-kD protein, SMN is essentially a small one in terms of molecular weight, in comparison with all proteins whose structures have been deposited in the Protein Data Bank (PDB), a primary database for experimentally determined structures of biological molecules [40]. As discussed above, even for a protein as small as SMN, experimental structure determination does not seem simple or easy, especially when it has to be done in a full-length and gapless manner. Therefore, to test whether any residue-specific statistical pattern (not known yet before this chapter) exists in the structural gaps in the whole Protein Data Bank (accessed 25 September 2018), this chapter presents a set of residue-specific distributional analysis of all structural gaps throughout PDB.

While the number of experimentally determined protein structures keeps increasing in the PDB, with the number of cryo-EM structures [49] on the rise, X-ray crystallography and NMR spectroscopy remain to date the two main (**Table 8**) supplementary biophysical tools in structural biology, both with strengths and weaknesses [50, 51].

In PDB-format data, the atomic coordinates presented in ATOM records in a PDB file may not exactly match the sequence in the SEQRES records. However, these amino acids will often be included in the SEQRES records, since the portion of the chain was present during the experiment. In these cases, a 'REMARK 465' entry will be included in the header of the PDB file to identify each missing residue. For X-ray crystallography data, the ends of chains and mobile loops are often not observed in crystallographic experiments, and as a result, atomic coordinates are not included as ATOM records in the file, leading to the occurrence of gaps for structure determined by X-ray crystallography. Among currently available biophysical tools, NMR spectroscopy is able to provide unique access to atomic-level structural dynamic behaviour of protein molecules in solution under physiological conditions (such as temperature, pH, etc.). As a result, this chapter focuses on

**99**

**Table 9.**

*September 2018.*

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival…*

**Experimental method Proteins Nucleic acids Protein/NA complex Other Total** X-ray 121,081 1958 6257 10 129,306 NMR 10,848 1256 250 8 12,362 Electron microscopy 1750 31 623 0 2404 Other 244 4 6 13 267 Multi-method 117 5 2 1 125

the structural gaps within protein structures determined by NMR spectroscopy, and aims to test whether any residue-specific statistical pattern exists in them. Here, structural gaps are defined as protein fragments with residues which exist in the originally studied molecule as shown in the SEQRES records, but not in the

*A summary of the number of experimentally determined biomolecular structures in PDB as of 25 September 2018.*

As of 20 September 2018, 10,844 NMR-determined protein structures have been deposited in the Protein Data Bank, according to a structure search with two parameters (molecule type = protein, experimental method = NMR). After the 10,844 PDB files were downloaded from the PDB website, the numbers of the total and the missing amino acid residues were extracted with an in-house python script

**Residue Missing no. Total no. Ratio = Missing no./Total no.**

*The numbers of the total and the missing amino acid residues in NMR-determined protein structures as of 25* 

A 1782 75,627 0.023 C 152 25,777 0.00589673 E 1811 79,729 0.022 D 1390 59,908 0.023 G 3321 81,347 0.041 F 615 38,993 0.015 I 640 55,070 0.011 H 5146 26,182 0.196 K 1442 75,766 0.019 M 1203 23,652 0.050 L 1439 90,833 0.015 N 831 43,080 0.019 Q 1273 44,594 0.028 P 1518 46,205 0.032 S 3159 73,904 0.042 R 1234 52,761 0.023 T 1112 56,787 0.019 W 138 13,211 0.010 V 980 70,252 0.013 Y 626 32,905 0.019 Sum 29,812 1,066,583 0.027

*DOI: http://dx.doi.org/10.5772/intechopen.81887*

observed structure/atomic coordinates.

**Table 8.**

for all proteins, as listed in **Table 9**.

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival… DOI: http://dx.doi.org/10.5772/intechopen.81887*


#### **Table 8.**

*Novel Aspects on Motor Neuron Disease*

functional research for this SMA protein.

**Protein Data Bank**

weaknesses [50, 51].

such as the SMN complex and snRNPs, their structure determination and functional characterisation will undoubtedly continue to advance, which will be helpful both in further understanding of SMN's role in SMA from a molecular structural point of view. In practice, however, advancements do not come easy. For instance, although both full-length structures of FL-SMN (with 294 residues) and SMNΔ7 (with 282 residues) were already experimentally determined using X-ray crystallography and deposited in the database (PDB IDs: 4NL6 and 4NL7), they were subsequently withdrawn by the author because the sample used for the structure determination was wrong. Otherwise, these two full-length SMN structures would constitute the very first step towards a comprehensive picture of the structural and

functional insights into SMN's role in the molecular pathogenesis SMA.

As of 25 September 2018, there is still no full-length SMN (or the SMN complex or the snRNP assembly) structure deposited in the wwPDB website [40], although it contains six new experimentally determined SMN-related structures, in addition to those reported in [1]. In terms of amino acid sequence, those SMN-related structures are still only SMN fragments, ranging from Gly26 to Lys51, and from Asn84 to Glu147. In between, there is still structurally not-determined-yet regions (referred to as **structural gaps** below) consisting of 204 SMN residues. Sixteen months have passed since the publication of [1], the structural gaps still remain, literally zero progress has been made to bridge them in spite of the six newly deposited structures, calling **again** [1] for further comprehensive structural determination and

**4. A residue-specific distributional analysis of the structural gaps in the** 

As a 38-kD protein, SMN is essentially a small one in terms of molecular weight, in comparison with all proteins whose structures have been deposited in the Protein Data Bank (PDB), a primary database for experimentally determined structures of biological molecules [40]. As discussed above, even for a protein as small as SMN, experimental structure determination does not seem simple or easy, especially when it has to be done in a full-length and gapless manner. Therefore, to test whether any residue-specific statistical pattern (not known yet before this chapter) exists in the structural gaps in the whole Protein Data Bank (accessed 25 September 2018), this chapter presents a set of residue-specific distributional analysis of all structural gaps throughout PDB. While the number of experimentally determined protein structures keeps increasing in the PDB, with the number of cryo-EM structures [49] on the rise, X-ray crystallography and NMR spectroscopy remain to date the two main (**Table 8**) supplementary biophysical tools in structural biology, both with strengths and

In PDB-format data, the atomic coordinates presented in ATOM records in a PDB file may not exactly match the sequence in the SEQRES records. However, these amino acids will often be included in the SEQRES records, since the portion of the chain was present during the experiment. In these cases, a 'REMARK 465' entry will be included in the header of the PDB file to identify each missing residue. For X-ray crystallography data, the ends of chains and mobile loops are often not observed in crystallographic experiments, and as a result, atomic coordinates are not included as ATOM records in the file, leading to the occurrence of gaps for structure determined by X-ray crystallography. Among currently available biophysical tools, NMR spectroscopy is able to provide unique access to atomic-level structural dynamic behaviour of protein molecules in solution under physiological conditions (such as temperature, pH, etc.). As a result, this chapter focuses on

**98**

*A summary of the number of experimentally determined biomolecular structures in PDB as of 25 September 2018.*

the structural gaps within protein structures determined by NMR spectroscopy, and aims to test whether any residue-specific statistical pattern exists in them. Here, structural gaps are defined as protein fragments with residues which exist in the originally studied molecule as shown in the SEQRES records, but not in the observed structure/atomic coordinates.

As of 20 September 2018, 10,844 NMR-determined protein structures have been deposited in the Protein Data Bank, according to a structure search with two parameters (molecule type = protein, experimental method = NMR). After the 10,844 PDB files were downloaded from the PDB website, the numbers of the total and the missing amino acid residues were extracted with an in-house python script for all proteins, as listed in **Table 9**.


#### **Table 9.**

*The numbers of the total and the missing amino acid residues in NMR-determined protein structures as of 25 September 2018.*

In total, the 10,844 protein structures contains 1,066,583 amino acid residues, ∼2.8% of which (29812) are missing, i.e. the atomic positions of the 29,841 residues were not experimentally determined by NMR spectroscopy, although they were present in the NMR sample during the structural determination process.

From **Figure 7**, it can be seen that for 19 residues (excluding histidine), the missing ratio is well below or pretty close to 5%, while the missing ratio is 19.6% for histidine, as shown by the blue sharp peak on **Figure 7**. In a statistical one sample t-test analysis of the 19 missing ratios, it turned out 100% acceptable (*P*=1) that the average of ratio is 0.0231, and that the fitness between the 19 missing ratios and the red horizontal line (**Figure 8**) is 100% acceptable (*P*=1), according to a statistical Chi-square test, as revealed by **Figure 8**.

While a missing ratio of 5% might be considered statistically insignificant, a missing ratio of 19.6% is clearly not to be ignored here, raising one obvious question: what on earth is so special about histidine that makes it so special among the 20 naturally occurring amino acids in this residue-specific distributional analysis of the structural gaps?

Similar to the other 19, histidine is a naturally occurring amino acid that is used in the biosynthesis of proteins. Also similar to the other 19, it contains an amino group (which is in the protonated ▬NH3+ form under biological conditions) and a carboxylic acid group (which is in the deprotonated ▬COO<sup>−</sup> form under biological conditions). In particular, histidine has an imidazole side chain (which is partially protonated), classifying it as a positively charged amino acid at physiological pH (∼7.4). That is, among the 20 naturally occurring amino acids, five (Arg, Lys, His, Glu and Asp) possess ionisable side chains. Among the five, histidine is the only one whose side chain has an ionisable (with an intrinsic pKa at 6.04) [52, 53] imidazole ring structure, which can exist in two inter-convertible tautomeric states. While at a pH of 7.0, the imidazole ring is mostly deprotonated (proton occupancy = 9.88%), at a pH of 6.0, the imidazole ring is largely protonated (proton

#### **Figure 7.**

*A residue-specific distribution of the missing residues in NMR-determined protein structures as of 25 September 2018. In this figure, x-axis represents the one-letter codes for amino acid residues, and y-axis represents the residue-specific ratio of missing versus total residues in those NMR structures. The red vertical line highlights histidine as a particular residue with an outstanding missing ratio.*

**101**

**Figure 8.**

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival…*

occupancy = 52.30%), as defined by the classical Henderson-Hasselbalch equation [50], where the positively charged imidazole ring bears two NH bonds and has a positive electric charge, which is equally distributed between both nitrogens. As the pH increases, the imidazole ring loses the positive charge, and the remaining proton of the neutral imidazole ring can reside on either nitrogen, giving rise to two

*A residue-specific scatter plot of the missing residues in NMR-determined protein structures as of 25 September 2018. In this figure, x-axis represents the one-letter codes for amino acid residues, and y-axis represents the residue-specific ratio of missing versus total residues in those NMR structures. The red horizontal line represents* 

To sum up, it is probable that the missing ratio of histidine is much higher than the other 19 because it has a special side chain with special dynamic structural and physicochemical properties (such as stacking interaction [56]), and with a special imidazole ring in constant protonation-deprotonation equilibrium [57] and two tautomeric states [52, 54, 55], making its NMR-observables (chemical shift for instance) difficult to be experimentally observed and measured by NMR spectroscopy and structurally calculated by NMR-related software in the structural determination of proteins. To address this issue of PDB-wide structural gaps, selective isotope labelling of histidine residues (the side chains in particular) can be a useful approach in biomolecular structural determination by NMR spectroscopy, not just alone, but also in collaboration with other biophysical tools, not just for the special histidine, but also for its 19 siblings in the fundamental building block of life.

tautomeric states of the histidine side chain [52, 54, 55].

*the average missing ratio level of the 19 residues.*

*DOI: http://dx.doi.org/10.5772/intechopen.81887*

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival… DOI: http://dx.doi.org/10.5772/intechopen.81887*

#### **Figure 8.**

*Novel Aspects on Motor Neuron Disease*

Chi-square test, as revealed by **Figure 8**.

the structural gaps?

In total, the 10,844 protein structures contains 1,066,583 amino acid residues, ∼2.8% of which (29812) are missing, i.e. the atomic positions of the 29,841 residues were not experimentally determined by NMR spectroscopy, although they were

From **Figure 7**, it can be seen that for 19 residues (excluding histidine), the missing ratio is well below or pretty close to 5%, while the missing ratio is 19.6% for histidine, as shown by the blue sharp peak on **Figure 7**. In a statistical one sample t-test analysis of the 19 missing ratios, it turned out 100% acceptable (*P*=1) that the average of ratio is 0.0231, and that the fitness between the 19 missing ratios and the red horizontal line (**Figure 8**) is 100% acceptable (*P*=1), according to a statistical

While a missing ratio of 5% might be considered statistically insignificant, a missing ratio of 19.6% is clearly not to be ignored here, raising one obvious question: what on earth is so special about histidine that makes it so special among the 20 naturally occurring amino acids in this residue-specific distributional analysis of

Similar to the other 19, histidine is a naturally occurring amino acid that is used in the biosynthesis of proteins. Also similar to the other 19, it contains an amino group (which is in the protonated ▬NH3+ form under biological conditions) and a carboxylic acid group (which is in the deprotonated ▬COO<sup>−</sup> form under biological conditions). In particular, histidine has an imidazole side chain (which is partially protonated), classifying it as a positively charged amino acid at physiological pH (∼7.4). That is, among the 20 naturally occurring amino acids, five (Arg, Lys, His, Glu and Asp) possess ionisable side chains. Among the five, histidine is the only one whose side chain has an ionisable (with an intrinsic pKa at 6.04) [52, 53] imidazole ring structure, which can exist in two inter-convertible tautomeric states. While at a pH of 7.0, the imidazole ring is mostly deprotonated (proton occupancy = 9.88%), at a pH of 6.0, the imidazole ring is largely protonated (proton

present in the NMR sample during the structural determination process.

**100**

**Figure 7.**

*A residue-specific distribution of the missing residues in NMR-determined protein structures as of 25 September 2018. In this figure, x-axis represents the one-letter codes for amino acid residues, and y-axis represents the residue-specific ratio of missing versus total residues in those NMR structures. The red vertical* 

*line highlights histidine as a particular residue with an outstanding missing ratio.*

*A residue-specific scatter plot of the missing residues in NMR-determined protein structures as of 25 September 2018. In this figure, x-axis represents the one-letter codes for amino acid residues, and y-axis represents the residue-specific ratio of missing versus total residues in those NMR structures. The red horizontal line represents the average missing ratio level of the 19 residues.*

occupancy = 52.30%), as defined by the classical Henderson-Hasselbalch equation [50], where the positively charged imidazole ring bears two NH bonds and has a positive electric charge, which is equally distributed between both nitrogens. As the pH increases, the imidazole ring loses the positive charge, and the remaining proton of the neutral imidazole ring can reside on either nitrogen, giving rise to two tautomeric states of the histidine side chain [52, 54, 55].

To sum up, it is probable that the missing ratio of histidine is much higher than the other 19 because it has a special side chain with special dynamic structural and physicochemical properties (such as stacking interaction [56]), and with a special imidazole ring in constant protonation-deprotonation equilibrium [57] and two tautomeric states [52, 54, 55], making its NMR-observables (chemical shift for instance) difficult to be experimentally observed and measured by NMR spectroscopy and structurally calculated by NMR-related software in the structural determination of proteins. To address this issue of PDB-wide structural gaps, selective isotope labelling of histidine residues (the side chains in particular) can be a useful approach in biomolecular structural determination by NMR spectroscopy, not just alone, but also in collaboration with other biophysical tools, not just for the special histidine, but also for its 19 siblings in the fundamental building block of life.

*Novel Aspects on Motor Neuron Disease*

#### **Author details**

#### Wei Li

Institute of Nautical Medicine, Nantong University, Nantong City, Jiangsu Province, China P. R.

\*Address all correspondence to: liweiqidong@stu.edu.cn

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**103**

*Structural and Functional Consequences of the SMA-Linked Missense Mutations of the Survival…*

motor neurons protein. EMBO Journal.

[10] Carvalho T, Fcalapez AA, Lafarga M, Berciano M, Carmo FM. The spinal muscular atrophy disease gene product, SMN: A link between snRNP biogenesis and the Cajal (coiled) body. Journal of Cell Biology. 1999;**147**(4):715-727

[11] Young PJ, Le TT, Dunckley M, Nguyen TM, Burghes AH, Morris GE. Nuclear gems and Cajal (coiled) bodies in fetal tissues: Nucleolar distribution of the spinal muscular atrophy protein, SMN. Experimental Cell Research. 2001;**265**(2):252-261

[12] Young P, Ntlorson MC, Le T, Androphy E, Burghes A, Morris G. The exon 2b region of the spinal muscular atrophy protein, SMN, is involved in self-association and SIP1 binding. Human Molecular Genetics.

[13] Seng CO, Magee C, Young PJ, Lorson CL, Allen JP. The SMN structure

reveals its crucial role in snRNP assembly. Human Molecular Genetics.

[14] Grimm C, Chari A, Pelz JP, Kuper J, Kisker C, Diederichs K, et al. Structural basis of assembly chaperone-mediated snRNP formation. Molecular Cell.

[15] Pellizzoni L, Charroux B, Dreyfuss G. SMN mutants of spinal muscular atrophy patients are defective in binding to snRNP proteins. Proceedings of the National Academy of Sciences.

2000;**9**(19):2869-8277

2015;**24**(8):2138-2146

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[16] Pellizzoni L. Essential role for the SMN complex in the specificity of snRNP assembly. Science. 2002;**298**(5599):1775-1779

[17] Workman E, Saieva L, Carrel TL, Crawford TO, Liu D, Lutz C, et al. A

1996;**15**(14):3555-3565

*DOI: http://dx.doi.org/10.5772/intechopen.81887*

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[6] Burghes AH, Beattie CE. Spinal muscular atrophy: Why do low levels of survival motor neuron protein make motor neurons sick? Nature Reviews Neuroscience. 2009;**10**(8):597-609

[7] Hua Y, Sahashi K, Hung G, Rigo F, Passini MA, Bennett CF, et al. Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model. Genes & Development.

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*Novel Aspects on Motor Neuron Disease*

**102**

**Author details**

Wei Li

China P. R.

provided the original work is properly cited.

\*Address all correspondence to: liweiqidong@stu.edu.cn

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Institute of Nautical Medicine, Nantong University, Nantong City, Jiangsu Province,

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**106**

*Edited by Humberto Foyaca Sibat and Lourdes de Fátima Ibañez-Valdés*

Dedicated to our readers, we include novel information (not reported in IntechOpen's books before) about new contributions of aberrant astrocytes to MND damage and death in the SOD1G93A rat experimental model of ALS; novel genetic studies on ALS; an update of the structural and functional consequences of the spinal muscular atrophy-linked mutations of the survival motor neuron protein; stem cell therapy for MND; and the novel treatment for SMA and ALS in the introductory chapter. This book contains selected peer-reviewed chapters written by international researchers. In this publication, the readers will find a compilation of state-of-the-art reviews about etiology, therapies, investigations, the molecular basis of disease progression and clinical manifestations, and the genetic familial ALS, as well as novel therapeutic modalities. We look forward with confidence and pride to the remarkable role that this book will play for a new vision and mission.

Published in London, UK © 2020 IntechOpen © Dr\_Microbe / iStock

Novel Aspects on Motor Neuron Disease

Novel Aspects on

Motor Neuron Disease

*Edited by Humberto Foyaca Sibat* 

*and Lourdes de Fátima Ibañez-Valdés*