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Objective: Schizophrenia (Sch) appears to be a multi-factorial disorder with a strong genetic predisposition. Accumulating evidence from human genetics and animal studies suggest that neuronal nitric oxide synthase (nNOS) might contribute to susceptibility for Sch. Information accrued from human genetics studies indicates nNOS variants are highly associated with Schin many populations such as Ashkenazi Jewish (AJ), European, Asian, Irish and German. In these populations, the variant of intron 9 in nNOS leads to IQ, episodic memory, and working memory impairment. Also, the variants in alternative exon 1 affect the continuous performance test and increase the positive score in PANSS (6). These studies highly suggest that variants in nNOS play critical roles in phenotypes associated with Sch.
Methods: We studied 600 Sch and 1056 healthy control for identifying novel Sch-associated rare mutations in nNOS gene. We utilized next generation sequencing(NGS) to analyze 384 samples (192 for cases, 192 for controls) from the AJ population. All nNOSexonal variants were targeted for PCR amplification with specific primers designed by primer 3 software. Targeted regions include the coding region (CDR; 3’-end of exon 2 ~ 5’-end exon 29) and untranslated region (UTR; alternated exon 1, 5’-end of exon 2 and 3’-end of exon 29). Given that the UTR of 3’-end of exon 29 is difficult to amplify with a single PCR reaction due to the long length of its exon, specific regions of exon 29, including the regions which have a greater potential of micro RNA binding sites, transcription binding sites, and a highly conservation region based on the USCS genome browser and target scan database, were selectively amplified. Since the previous studies indicate that exon 1 has multiple alternative variants (7,8), we designed 10 primer pairs to cover all alternatives of exon 1. In summary, 49 pairs of primers were designed for the present project, namely, 34 for CDR, 10 for alternative exon 1 and 5 for UTR of exon 29. Possible rare unreported variants identified by Next generation sequencing were then verified by Sanger sequencing (SS). The occurrence of identified mutations was examined in additional cohort (408 Sch and 864 controls) by PCR-RFLP (Restriction Fragment Length Polymorphism) method followed by SS.
Results: We found high incidence of genetic variants of nNOS gene occurs in Sch samples compared to controls. Interestingly, we found possible rare variants in the coding region of nNOS gene by NGS data analysis using IGV software. 4 CDR variants which were in unreported Sch group, was verified by SS. One missense mutation was found within these 4 CDR variants. According to RFLP results carrying this mutation was found in another patient with Sch.
Conclusion: We currently characterize biological effects of nNOS rare variants associated with Sch in vitro cell model as well as in animal model, including nNOS knockout mice and mice with knockdown of nNOS expression by RNA interference approach during brain development. We hope to further address molecular and physiological mechanisms of how nNOS variations affect brain maturation, which may in turn, contribute to understanding of etiopathologies of Sch.