**2. Role of sequence variations in the etiology and treatment of psychosis**

A growing body of evidence suggests the significance of genetic variants in the etiology of mental health disorders, including schizophrenia. For example, the Psychiatric Genomics Consortium identified several SNPs that are associated with major psychiatric disorders including schizophrenia [6], which included *CACNA1C* variants that have been previously associated with autism [7]. Also, angiotensin‐converting enzyme (ACE) gene insertion/ deletion polymorphism was reported to be associated with schizophrenia susceptibility as well as the severity of schizophrenia depressive symptoms in a Chinese population [8]. According to the schizophrenia‐working group of the Psychiatric Genetic Consortium, the expression of the C4 gene was affected by SNPs in the gene resulting in putative synapse elimination in schizophrenia patients [9]. It has recently been reported that 15 of 48 schizophrenia cases were found to carry rare or novel missense coding variants in four signaling genes studied. These findings suggest that single genes harboring *de novo* mutations in individuals with psychosis as compared to healthy controls may play a critical role in influencing the phenotypes of psychosis and hence may be potential targets for developing treatment strategies. A study from our lab reported that copy number variants (CNVs) in monozygotic twins discordant for schizophrenia could be an important underlying factor in the discordance of the twins for the disease [3]. The findings identified several CNVs and genes, in four of the six twin pairs studied, that were previously implicated in mental health disorders. These findings suggest a role for CNVs in the discordance of twins for schizophrenia.

Similarly, unpublished studies from our lab on complete genomes of two pairs of monozygotic twins discordant for schizophrenia showed multiple individual sequence‐specific differences between cotwins. The observed differences included small nucleotide changes (single nucleo‐ tide variation, block substitutions, and small indels), copy number variations, and structural variations that were unique to either the affected or healthy cotwins. Also, by comparing the sequence differences between cotwins with that of their parents, it was possible to identify *de novo* variants. The study revealed several genes and gene‐networks that may have predisposed the affected cotwins to the disease corroborating the fact that *de novo* variations between cotwins may be an underlying factor for their discordance to the disease.

Due to the fact that there has been no single gene identified that is responsible for causing the disorder, it is imperative that future research be focused on the polygenetic nature of schizo‐ phrenia, as well as the networks and pathways relevant to neurodevelopment and function. A recent study identified genes and pathways associated with psychosis in 22q11.2 deletion syndrome subjects [10]. The study revealed specific pathways affected in 22q11 deletion Syndrome carriers with psychosis and autistic spectrum disorders. Expression changes associated with psychosis symptoms in 22q11 deletion syndrome was also associated with pathways involved in transcriptional regulation. In addition, schizophrenia was reported to be the only psychiatric disorder observed at a higher rate in 22q11 deletion syndrome com‐ pared to other neurodevelopmental disorders [11, 12].

The 22q11.2 deletion represents one of the most established genetic risk factors for the development of psychiatric disorders. Our lab also reported the effect of DNA methylation in the promoter regions of genes located in the microdeletion region on chromosome 22. Accordingly, from a set of genes located in the 22q11.2 microdeletion region that has been previously implicated in psychosis, 29 genes showed increased DNA methylation in their promoters, following olanzapine treatment [13]. In that study, the effect of the antipsychotic drug was revealed through significantly increased (*p* < 0.01) DNA methylation of genes affecting several networks including neurological disease, inflammatory disease, inflamma‐ tory response, cancer, tumor morphology, and cell death and survival.

An increased number of studies suggest that rare genetic variations play an important role in the genetic etiology of schizophrenia. However, the existence of rare genetic variations may not always lead to the predisposition of schizophrenia. For example, a rare missense varia‐ tion in *UCL13B* was identified by whole‐exome sequencing, which was present in five of six schizophrenia‐affected individuals but not in eight unaffected individuals [14]. In a follow‐up case‐control study of two independent Japanese populations, there was no significant associ‐ ation between this missense variation and schizophrenia [14].


Note: G/H/F: stands for healthy cotwin, affected cotwin, and their mom, respectively; Chr: chromosome.

**Table 1.** Mitochondrial single nucleotide variations detected in the discordant twins as well as their mother.

In addition to our analysis of nuclear DNA sequence variation, we have also conducted mitochondrial DNA sequence analysis involving a pair of monozygotic twins discordant for schizophrenia and their mother. Ingenuity Variant Analysis (Ingenuity System Inc, CA, USA) identified no difference in the sequence variations between the discordant twins as well as their mother. All of the biologically relevant variations detected were single nucleotide variations (SNVs) in exonic regions of the *MT‐ATP6* and *MT‐ND4* genes (**Table 1**). The translational impact of the variation found in *MT‐ATP6* was predicted to be *missense* while that of *MT‐ND4* was predicted to be *synonymous*. All of the single nucleotide variations found in the mitochon‐ dria were detected in both of the twins and their mother. As we will discuss in the subsequent sections, despite the identification of these biologically relevant sequence variations in all samples, their interaction with epigenetic signatures including DNA methylation may differ between individuals and lead to differences in susceptibility to disease.

Syndrome carriers with psychosis and autistic spectrum disorders. Expression changes associated with psychosis symptoms in 22q11 deletion syndrome was also associated with pathways involved in transcriptional regulation. In addition, schizophrenia was reported to be the only psychiatric disorder observed at a higher rate in 22q11 deletion syndrome com‐

The 22q11.2 deletion represents one of the most established genetic risk factors for the development of psychiatric disorders. Our lab also reported the effect of DNA methylation in the promoter regions of genes located in the microdeletion region on chromosome 22. Accordingly, from a set of genes located in the 22q11.2 microdeletion region that has been previously implicated in psychosis, 29 genes showed increased DNA methylation in their promoters, following olanzapine treatment [13]. In that study, the effect of the antipsychotic drug was revealed through significantly increased (*p* < 0.01) DNA methylation of genes affecting several networks including neurological disease, inflammatory disease, inflamma‐

An increased number of studies suggest that rare genetic variations play an important role in the genetic etiology of schizophrenia. However, the existence of rare genetic variations may not always lead to the predisposition of schizophrenia. For example, a rare missense varia‐ tion in *UCL13B* was identified by whole‐exome sequencing, which was present in five of six schizophrenia‐affected individuals but not in eight unaffected individuals [14]. In a follow‐up case‐control study of two independent Japanese populations, there was no significant associ‐

pared to other neurodevelopmental disorders [11, 12].

30 Schizophrenia Treatment - The New Facets

tory response, cancer, tumor morphology, and cell death and survival.

ation between this missense variation and schizophrenia [14].

**Variation type** 

**Gene regions** 

Note: G/H/F: stands for healthy cotwin, affected cotwin, and their mom, respectively; Chr: chromosome.

**Table 1.** Mitochondrial single nucleotide variations detected in the discordant twins as well as their mother.

In addition to our analysis of nuclear DNA sequence variation, we have also conducted mitochondrial DNA sequence analysis involving a pair of monozygotic twins discordant for schizophrenia and their mother. Ingenuity Variant Analysis (Ingenuity System Inc, CA, USA) identified no difference in the sequence variations between the discordant twins as well as their mother. All of the biologically relevant variations detected were single nucleotide variations (SNVs) in exonic regions of the *MT‐ATP6* and *MT‐ND4* genes (**Table 1**). The translational

M 8701 A G SNV Exonic MT‐ATP6 255 600/680/611 Missense M 8860 A G SNV Exonic MT‐ATP6 255 336/367/322 Missense M 10819 A G SNV Exonic MT‐ND4 255 729/714/667 Synonymous M 10873 T C SNV Exonic MT‐ND4 255 613/662/573 Synonymous M 11719 G A SNV Exonic MT‐ND4 255 544/652/562 Synonymous

**Gene symbol**  **Sample call quality**  **Sample read depth in G/H/F** 

**Translation impact** 

**Sample allele** 

**Chr Position Reference** 

**allele** 

**Figure 2.** The dotted line in purple shows the mean sequencing coverage of the mitochondrial genome for each sample. (A) Healthy cotwin. (B) Schizophrenia‐affected cotwin. (C) Mother of the twins.


**Table 2.** Homoplasmies detected in the discordant twins and their mother.

We used mtDNA‐server [15] to identify heteroplasmies. All sites with a log likelihood ratio (LLR) of ≥5 were considered as heteroplasmic sites. This analysis did not identify any hetero‐ plasmies in our samples. Among other reasons, the sequencing coverage may have affected our ability to detect heteroplasmies in the present samples (**Figure 2**). Coverage of ≥10× fold per strand is required on both the forward and reverse strand to accurately identify hetero‐ plasmic sites [15]. We believe that better coverage would more accurately identify heteroplas‐ mies that may have a role in the etiology of schizophrenia. Furthermore, analyzing larger samples may help investigate heteroplasmies and their association with schizophrenia; as it is possible that each patient could signify a specific etiology and pathophysiological manifesta‐ tion of the disease via his/her unique genetic makeup and epigenomic signature. In another study, novel and rare nonsynonymous mutations were identified in mtDNA genes (*ND6*, *ATP6*, *CYTB*, and *ND2*) in subjects with psychiatric disorders [16]. The authors also reported mtDNA heteroplasmy at a locus that was known to be associated with schizophrenia (*T16519C*). The homoplasmies detected in the aforementioned sample of a pair of twins discordant for schizophrenia and their mother are presented in **Table 2**.

**Position Mutation Locus** A>G MT‐ND3 A>G MT‐ND4 T>C MT‐ND4 G>A MT‐ND4 C>T MT‐ND5 T>C MT‐CYB A>G MT‐RNR1 C>T MT‐CYB G>A MT‐CYB C>T MT‐DLOOP2 T>C MT‐DLOOP2 G>A MT‐CYB A>G MT‐CYB T>C MT‐DLOOP1 A>C MT‐DLOOP1 T>C MT‐DLOOP1 C>T MT‐DLOOP1 T>C MT‐TV T>C MT‐DLOOP1 T>C MT‐DLOOP2 T>C MT‐RNR2 A>G MT‐DLOOP2 A>G MT‐RNR2 G>A MT‐ND1 A>G MT‐ND2 C>T MT‐C01 A>G MT‐DLOOP2 A>G MT‐RNR1 A>G MT‐ATP6 A>G MT‐ATP6 T>C MT‐C03

32 Schizophrenia Treatment - The New Facets

**Table 2.** Homoplasmies detected in the discordant twins and their mother.
