**5.1. ISCA1**

Iron-sulfur cluster assembly 1 (ISCA1) is one of the mitochondrial proteins required for the biogenesis and assembly of ISC [20]. This protein functions in the late stages of the ISC biogenesis and act as an iron binding molecule that may serve as a chaperone for biogenesis of Fe-S clusters [21]. It is believed that the molecule plays its pivotal role through its interaction with IOP1 (iron-only hydrogenase-like protein)/NARFL (nuclear prelamin A recognition factorlike). Knockdown of *Isca1* causes reduced activity of succinate dehydrogenase, mitochondrial aconitase, and cytosolic aconitase; hence, involving in both cytosolic and mitochondrial Fe-S protein biogenesis [22].

According to GenAtlas [23, 24], the gene has four exons and produces 14 kDa protein with 129 amino acids, which is known as mitochondrial Fe-S cluster assembly 1 homolog or otherwise HESB like domain containing 2. The gene is mapped to chromosome 9q21.33, and sits on genomic coordinates: 88.879.463–88.897.490. It is 2012 base pair long, generates four transcripts (splice variants) and highly expressed heart, esophagus, bladder, uterus, and cervix. Moreover, ISCA1 is a member of consensus coding sequence (CCDS:35056.1) which are manually checked protein annotations on the reference mouse and human genomes that ensures


AR: autosomal recessive; IM: inheritance mode; LN: locus number; MIM: Mendelian inheritance in man; MMDS: mitochondrial dysfunction syndromes; PT#: phenotype number.

**Table 1.** Genes and related mitochondrial dysfunction syndromes.

consistent representation of the tracks of NCBI, Ensembl, and UCSC Genome Browsers. The gene has several synonyms such as hIsca, HBLD2, and ISA1, and localizes to mitochondria as well as cytoplasm.

Effect of depletion of ISC-related proteins on the maturation of cytosolic 4Fe-4S proteins showed that some mitochondrial Fe/S proteins such as mitochondrial aconitase, SDH, several proteins of complex I, and Rieske Fe/S protein were decreased with the deficiency of ISCA1. On the other hand, cellular heme content and mitochondrial 2Fe-2S ferrochelatase were unaffected by the depletion. This implies that ISCA1 is crucial in the maturation of mitochondrial 4Fe-4S proteins [25]. In another study, *ISCA1* was found to be associated with multiple mitochondrial dysfunctions syndrome-5. A homozygous missense mutation at a conserved residue in the Fe-S biogenesis domain (c.259G>A, p.Glu87Lys) was identified in two unrelated Indian families. This mutation destabilizes the protein subsequently causing the syndrome [19].

#### **5.2. ISCA2**

iron-sulfur scaffold protein. The changes in this gene result in severe myopathy and lactic acidosis ("ISCU Fe-S Cluster Assembly Enzyme [*Homo sapiens* (Human)] - Gene - NCBI") Complexes 1, 2, and 3 contain Fe-S clusters. They function in electron transport by transfer of one electron in redox processes [16]. The assembly of the clusters is recently studied in Yeast. In photosynthetic organisms, the iron-sulfur clusters play role in chloroplast processes and

Yeast frataxin, Isu1, and Nfs1 (cysteine desulfurase) take part in *de novo* synthesis of ISC. Many genes encode ISC assembly factors such as *BOLA3*, *NFU1*, *GLRX5*, *NUBPL*, *LYRM4*, *IBA57*, *ISCA1*, and *ISCA2*. These molecules have significant role in mitochondria. They are essential cofactors in the assembly of cluster. Deficiency of these genes leads to different diseases, for instance, GLRX5 deficiency causes sideroblastic anemia, whereas NUBPL mutations lead to respiratory chain complex 1 deficiency. On the other hand, some of these deficiencies are

As the names imply, multiple mitochondrial dysfunction syndromes are disease conditions affecting mitochondria and usually lead to reduced function of more than one stages of energy production in the organelle [18]. The genetic factors causing these disorders are associated with the biogenesis of cellular ISC and currently these are the following genes: *ISCA2*, *NFU1*, *IBA57*, and *BOLA3*. More recently, *ISCA1* is also reported to lead a disease resembling MMDS and suggested to be a member of the group [19]. Interestingly, MMDS members appear to be

Iron-sulfur cluster assembly 1 (ISCA1) is one of the mitochondrial proteins required for the biogenesis and assembly of ISC [20]. This protein functions in the late stages of the ISC biogenesis and act as an iron binding molecule that may serve as a chaperone for biogenesis of Fe-S clusters [21]. It is believed that the molecule plays its pivotal role through its interaction with IOP1 (iron-only hydrogenase-like protein)/NARFL (nuclear prelamin A recognition factorlike). Knockdown of *Isca1* causes reduced activity of succinate dehydrogenase, mitochondrial aconitase, and cytosolic aconitase; hence, involving in both cytosolic and mitochondrial Fe-S

According to GenAtlas [23, 24], the gene has four exons and produces 14 kDa protein with 129 amino acids, which is known as mitochondrial Fe-S cluster assembly 1 homolog or otherwise HESB like domain containing 2. The gene is mapped to chromosome 9q21.33, and sits on genomic coordinates: 88.879.463–88.897.490. It is 2012 base pair long, generates four transcripts (splice variants) and highly expressed heart, esophagus, bladder, uterus, and cervix. Moreover, ISCA1 is a member of consensus coding sequence (CCDS:35056.1) which are manually checked protein annotations on the reference mouse and human genomes that ensures

are important for plastid functioning [17].

classified under a unique category such as MMDS.

**5. Genetic factors of mitochondrial dysfunction syndromes**

inherited in autosomal recessive mode of inheritance (**Table 1**).

**5.1. ISCA1**

240 Mitochondrial Diseases

protein biogenesis [22].

ISCA2 stands for iron-sulfur cluster assembly 2 protein and the gene encodes for A-type ironsulfur cluster protein. Fe-S clusters are inorganic cofactors, mostly found in metalloproteins. The gene is located on chromosome 14 and expressed from the plus strand. According to Ensembl, this gene generates 4 different transcripts and has 96 orthologues. ISCA2 is a regulatory protein found in mitochondria as well as extra mitochondrial sites such as cytosol and nucleus. The protein takes part in assembly of Fe-S clusters in mitochondria which further take part in oxidation reduction (especially in complex 1 and 2), substrate activation, iron/sulfur storage, regulation of gene expression, and enzyme activity. Alternative name for ISCA2 is "HESB-like-domain-containing protein 1" for humans. First human mutation of *ISCA2*

(c.229G>A; p.Glu77Ser) identified in the patients from five consanguineous families was a homozygous ancestral founder mutation that leaded to neurodegeneration, developmental, failure to thrive, quadriplegia, truncal hypotonia, optic atrophy, and leukoencephalopathy [26]. Later, additional 10 cases with the same founder mutation were also described [27]. Recently, two other patients with the same mutation were also studied with detailed functional experiments revealing complex 2 and 4 deficiencies [28]. Interestingly these patients were all Arab descent. Most recently, a second mutation, a compound heterozygous variant (a single basepair deletion causing frameshift with a premature stop codon: mutation: c.295delT; p.Phe99Leufs\*18 and a missense mutation c.334A>G; p.Ser112Gly) in *ISCA2* was reported in a 2-month-old girl from Italy [29]. These mutations causes disorder of energy metabolism which results in respiratory failure, severe hypotonia, nystagmus, lactic acidosis, poor neurologic development, hyperglycemia, leukodystrophy of the brainstem with longitudinally extensive spinal cord involvement, and mtDNA deficiency ultimately leading to death [26].

of [4Fe-4S] proteins. Moreover, potential diseases caused by mutations in these genes are expected to cause defects in mitochondrial respiration and in lipoic acid-dependent proteins [25]. Another study reported two siblings from consanguineous parents died with a condition characterized by generalized hypotonia, respiratory insufficiency, arthrogryposis, microcephaly, congenital brain malformations, and hyperglycinemia. Catalytic activities of the mitochondrial respiratory complexes I and II were deficient in skeletal muscle, a finding suggestive of an inborn error in mitochondrial biogenesis. Homozygosity mapping identified IBA57 located in the largest homozygous region on chromosome 1 as a culprit candidate gene. Their analysis of IBA57 revealed the homozygous mutation c.941A>C, p.Gln314Pro in

Hereditary Disorders and Human Mutations of Iron-Sulfur Assembly Genes

http://dx.doi.org/10.5772/intechopen.78006

243

BOLA3 is another essential protein in the Fe-S clusters production and involves in the normal maturation of lipoate-containing 2-oxoacid dehydrogenases. Another critical role of the molecule is to facilitate the assembly of the respiratory chain complexes. *BOLA3* was identified in the year 2008 during a search for similar sequences for bacterial BolA and cloned together with *BOL1* and *BOL2* [32]. According to Ensembl, the gene has five different transcripts and two isoforms. The main isoform is longer and localizes to mitochondria while the shorter isoform lacking exon 2 is restrained in the cytosplasm [18, 33]. The main transcript (ENST00000327428.9) has four exons comprising 68 variations [33]. The mRNA is nearly ubiquitously expressed in human tissues. The protein has seven domains including two low complexity segments. The main BolA domain consists of a helix-turn-helix structure close to its C terminus. The gene has three published mutations (c.123dupA; p.Glu42Argfs; c.200T>A, p.Ile67Asn; c.136C>T, p.Arg46Ter) [18, 34–36] in addition to a 5 bp deletion [37] and a single basepair insertion [18]. These mutations were identified in ethnically different families. The first patients were initially described in 2001 in a mapping study [38] that included a singleton from a consanguineous family as well as three siblings from a nonconsanguineous family. Since all the patients from two different families had similar metabolic abnormalities, a mapping strategy was employed to identify the genetic interval for the causative gene. This approach located the gene on chromosome 2. Further positional cloning studies on the subjects yielded a single significant interval on p arm extending ~5 centiMorgan region and excluding the region positioned on the q arm. Interestingly, both of these families were utilized in a follow-up study that yielded deficiencies of two ISC-related genes in each family. While the larger family with three siblings were identified to harbor splice site mutation in *NFU1* (c.545G>A) [18], the singleton had a single nucleotide duplication leading to a frame shift and eventually a premature stop codon (p.Glu42Argfs\*13) in *BOL3* [18]. Later on, a few more follow-up studies revealed additional mutations in the gene. The first follow-up study focused on two patients (male and female) with quite similar clinical course appeared with hypotonia, severe neonatal lactic acidosis, and intractable cardiomyopathy [35]. A missense mutation (c.200T>A, p.Ile67Asn) was identified in the patients' DNA using exome sequencing. The other studies [34, 37] provided two additional missense mutations in *BOLA3*. Interestingly, while c.287A>G (p.His96Arg) causes a lethal infantile mitochondrial disorder [37], c.136C>T (p.Arg46\*), a severe truncation mutation,

leads to nonketotic hyperglycinemia [34] in the affected individuals.

those two patients [15].

**5.5. BOLA3**

#### **5.3. NFU1**

NFU1 is one of the human mitochondrial components that is involved in the assembly of the Fe-S protein cluster. It helps in the transfer of [4Fe-4S] clusters to specific protein targets and facilitates their maturation [30]. *NFU1* is mapped on the 2p13-p15 chromosomal region and codes for the NFU1 protein. During Fe-S assembly, two NFU1 monomers are needed to assemble one 4Fe-4S. Complex I, II, and III of oxidative phosphorylation have multiple Fe-S clusters. Therefore, any deficiency in these clusters causes dysfunctions of respiratory chain complexes [31]. Previous studies showed that the function of the NFU1 has been associated by the fatal mitochondrial disease, multiple mitochondrial dysfunctions syndrome 1 (MMDS1) [30]. Patients with NFU1 mutations usually manifest feeding difficulty, weakness, lethargy, and decreasing responsiveness within a few days after birth and a few had epileptic seizures [31]. It has been shown that the patients with mutations in the NFU1 gene have similar biochemical features to that seen in patients with lipoic acid defects. Thus, NFU1 mutation appears to have some effect on Fe-S enzyme lipoic acid synthase (LAS). In conclusion, NFU1 is an ISC assembly protein, and there is strong evidence that LAS deficiency is important in NFU1 mutation-related disease [31].

#### **5.4. IBA57**

IBA57 is a member of the Fe-S cluster assembly group. It is known as putative transferase CAF17 and Fe-S cluster assembly factor homolog. *IBA57* is located on 1q42.13 and codes for the IBA57 protein that is located in the mitochondrion. This protein functions in the late stages of the biosynthesis of mitochondrial 4Fe-4S proteins. Any deficiency in *IBA57* can cause an autosomal recessive spastic paraplegia-74 or multiple mitochondrial dysfunctions syndrome 3. In a previous study, it was found that the depletion of IBA57 in cell culture caused striking alterations in mitochondrial morphology, including a vast enlargement of the organelles and a loss of cristae membranes. It is also found that the function of IBA57 protein is conserved from bacteria to human, according to a study that provides an evidence for the requirement of bacterial and yeast relatives of human IBA57 for efficient maturation of [4Fe-4S] proteins. Moreover, potential diseases caused by mutations in these genes are expected to cause defects in mitochondrial respiration and in lipoic acid-dependent proteins [25]. Another study reported two siblings from consanguineous parents died with a condition characterized by generalized hypotonia, respiratory insufficiency, arthrogryposis, microcephaly, congenital brain malformations, and hyperglycinemia. Catalytic activities of the mitochondrial respiratory complexes I and II were deficient in skeletal muscle, a finding suggestive of an inborn error in mitochondrial biogenesis. Homozygosity mapping identified IBA57 located in the largest homozygous region on chromosome 1 as a culprit candidate gene. Their analysis of IBA57 revealed the homozygous mutation c.941A>C, p.Gln314Pro in those two patients [15].

#### **5.5. BOLA3**

(c.229G>A; p.Glu77Ser) identified in the patients from five consanguineous families was a homozygous ancestral founder mutation that leaded to neurodegeneration, developmental, failure to thrive, quadriplegia, truncal hypotonia, optic atrophy, and leukoencephalopathy [26]. Later, additional 10 cases with the same founder mutation were also described [27]. Recently, two other patients with the same mutation were also studied with detailed functional experiments revealing complex 2 and 4 deficiencies [28]. Interestingly these patients were all Arab descent. Most recently, a second mutation, a compound heterozygous variant (a single basepair deletion causing frameshift with a premature stop codon: mutation: c.295delT; p.Phe99Leufs\*18 and a missense mutation c.334A>G; p.Ser112Gly) in *ISCA2* was reported in a 2-month-old girl from Italy [29]. These mutations causes disorder of energy metabolism which results in respiratory failure, severe hypotonia, nystagmus, lactic acidosis, poor neurologic development, hyperglycemia, leukodystrophy of the brainstem with longitudinally extensive spinal cord involvement, and mtDNA deficiency ultimately leading to death [26].

NFU1 is one of the human mitochondrial components that is involved in the assembly of the Fe-S protein cluster. It helps in the transfer of [4Fe-4S] clusters to specific protein targets and facilitates their maturation [30]. *NFU1* is mapped on the 2p13-p15 chromosomal region and codes for the NFU1 protein. During Fe-S assembly, two NFU1 monomers are needed to assemble one 4Fe-4S. Complex I, II, and III of oxidative phosphorylation have multiple Fe-S clusters. Therefore, any deficiency in these clusters causes dysfunctions of respiratory chain complexes [31]. Previous studies showed that the function of the NFU1 has been associated by the fatal mitochondrial disease, multiple mitochondrial dysfunctions syndrome 1 (MMDS1) [30]. Patients with NFU1 mutations usually manifest feeding difficulty, weakness, lethargy, and decreasing responsiveness within a few days after birth and a few had epileptic seizures [31]. It has been shown that the patients with mutations in the NFU1 gene have similar biochemical features to that seen in patients with lipoic acid defects. Thus, NFU1 mutation appears to have some effect on Fe-S enzyme lipoic acid synthase (LAS). In conclusion, NFU1 is an ISC assembly protein, and there is strong evidence that LAS deficiency is important in

IBA57 is a member of the Fe-S cluster assembly group. It is known as putative transferase CAF17 and Fe-S cluster assembly factor homolog. *IBA57* is located on 1q42.13 and codes for the IBA57 protein that is located in the mitochondrion. This protein functions in the late stages of the biosynthesis of mitochondrial 4Fe-4S proteins. Any deficiency in *IBA57* can cause an autosomal recessive spastic paraplegia-74 or multiple mitochondrial dysfunctions syndrome 3. In a previous study, it was found that the depletion of IBA57 in cell culture caused striking alterations in mitochondrial morphology, including a vast enlargement of the organelles and a loss of cristae membranes. It is also found that the function of IBA57 protein is conserved from bacteria to human, according to a study that provides an evidence for the requirement of bacterial and yeast relatives of human IBA57 for efficient maturation

**5.3. NFU1**

242 Mitochondrial Diseases

**5.4. IBA57**

NFU1 mutation-related disease [31].

BOLA3 is another essential protein in the Fe-S clusters production and involves in the normal maturation of lipoate-containing 2-oxoacid dehydrogenases. Another critical role of the molecule is to facilitate the assembly of the respiratory chain complexes. *BOLA3* was identified in the year 2008 during a search for similar sequences for bacterial BolA and cloned together with *BOL1* and *BOL2* [32]. According to Ensembl, the gene has five different transcripts and two isoforms. The main isoform is longer and localizes to mitochondria while the shorter isoform lacking exon 2 is restrained in the cytosplasm [18, 33]. The main transcript (ENST00000327428.9) has four exons comprising 68 variations [33]. The mRNA is nearly ubiquitously expressed in human tissues. The protein has seven domains including two low complexity segments. The main BolA domain consists of a helix-turn-helix structure close to its C terminus. The gene has three published mutations (c.123dupA; p.Glu42Argfs; c.200T>A, p.Ile67Asn; c.136C>T, p.Arg46Ter) [18, 34–36] in addition to a 5 bp deletion [37] and a single basepair insertion [18]. These mutations were identified in ethnically different families. The first patients were initially described in 2001 in a mapping study [38] that included a singleton from a consanguineous family as well as three siblings from a nonconsanguineous family. Since all the patients from two different families had similar metabolic abnormalities, a mapping strategy was employed to identify the genetic interval for the causative gene. This approach located the gene on chromosome 2. Further positional cloning studies on the subjects yielded a single significant interval on p arm extending ~5 centiMorgan region and excluding the region positioned on the q arm. Interestingly, both of these families were utilized in a follow-up study that yielded deficiencies of two ISC-related genes in each family. While the larger family with three siblings were identified to harbor splice site mutation in *NFU1* (c.545G>A) [18], the singleton had a single nucleotide duplication leading to a frame shift and eventually a premature stop codon (p.Glu42Argfs\*13) in *BOL3* [18]. Later on, a few more follow-up studies revealed additional mutations in the gene. The first follow-up study focused on two patients (male and female) with quite similar clinical course appeared with hypotonia, severe neonatal lactic acidosis, and intractable cardiomyopathy [35]. A missense mutation (c.200T>A, p.Ile67Asn) was identified in the patients' DNA using exome sequencing. The other studies [34, 37] provided two additional missense mutations in *BOLA3*. Interestingly, while c.287A>G (p.His96Arg) causes a lethal infantile mitochondrial disorder [37], c.136C>T (p.Arg46\*), a severe truncation mutation, leads to nonketotic hyperglycinemia [34] in the affected individuals.


**6. Conclusion**

Iron-sulfur clusters are indispensable inorganic cofactors for biological function and involve in numerous cellular processes such as respiration and DNA repair. The cluster's assembly is complex and requires sophisticated protein machinery for its maturation and insertion into apoproteins. Since mitochondria is the main site for ISC biogenesis in human, any defect disturbing the biogenesis leads to a pathological outcome mostly appears as an mitochondrial entity in human. Currently, genetic alterations in several genes involving in ISC assembly and maturation have been linked to autosomal recessive mitochondrial human diseases known as multiple mitochondrial dysfunction syndromes. It is expected that more genes and alterations

**Gene Mutation type Mutation Disease and phenotype References** *NFU1* Missense c.568G>A; p.Gly190Arg NFU1 deficiency Ahting et al.

*NFU1* Missense c.629G>T; p.Cys210Phe Leukoencephalopathy Invernizzi

*NFU1* Splice c.302+3A>G; p.Val56Glyfs\* NFU1 deficiency Ahting et al.

*NFU1* Microdeletion c.146delC; p.Pro49LeufsX8 Spastic paraplegia Tonduti et al.

*NFU1* Missense c.572A>T; p.Asp191Val Multiple mitochondrial

*NFU1* Missense c.622G>T; p.Gly208Cys Fatal infantile encephalopathy

*NFU1* Splice c.545G>A; Skipping exon 6 Deficiency of the 2-oxoacid

*NFU1* Splice c.545+5G>A; Skipping exon 6 Fatal infantile encephalopathy,

*NFU1* Microdeletion c.90delC; p. Tyr30\* Multiple mitochondrial

*ISCA1* Missense c.259G>A; p. Glu87Lys Multiple mitochondrial

*ISCA2* Missense c.229G>A; p.Glu77Ser Multiple mitochondrial

p.Phe99Leufs\*18 and p.Ser112Gly

*NFU1* Large deletion 55.6 kb region covering exons: 4–8

*ISCA2* Deletion and Missense c.295delT and c.334A>G;

**Table 2.** Published mutations in some Fe-S cluster genes.

[42]

http://dx.doi.org/10.5772/intechopen.78006

Bai et al. [46]

245

Navarro-Sastre et al. [47]

et al. [44]

Cameron et al.

Navarro-Sastre et al. [47]

Bai et al. [46]

Shukla et al. [19]

Al-Hassnan et al. [26] and others [27, 28]

Toldo et al. [29]

[42]

[18]

[45]

[42]

NFU1 deficiency Ahting et al.

dysfunctions syndrome

Hereditary Disorders and Human Mutations of Iron-Sulfur Assembly Genes

dehydrogenases accompanied by respiratory chain defects

pulmonary hypertension

dysfunctions syndrome

dysfunctions syndrome

dysfunctions syndrome

Multiple mitochondrial dysfunctions syndrome

and/or pulmonary hypertension

**Table 2** consists of previously published mutations in some Fe-S cluster genes.


**Table 2.** Published mutations in some Fe-S cluster genes.
