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Sohan Punia

Sohan Punia
Mayo Clinic, Rochester, Minnesota, United States
Worked on novel gene identification in Polycystic liver disease using exome sequencing. Polycystic liver disease is a rare inherited disease characterized by fluid filled cysts of various sizes scattered throughout the liver. Abdominal pain is the most prominent symptom secondary to local pressure on the stomach and duodenum due to enlarged cystic liver. The enlarged polycystic liver might also cause nausea, vomiting and early satiety, resulting in weight loss, anorexia, shortness of breath, discomfort and sleep apnea. No treatment is available except for surgical removal of enlarged cysts and Liver transplantation. So finding the genetic basis of disease may help in finding the drug treatment and drug targets for this disease. Mutations in PRKCSH and SEC63 genes have been found to cause only 30-40% of the PCLD cases. So we sought to identify mutations in new genes in remaining 60% patients. We used the traditional linkage and exome sequencing to find new genes responsible for PCLD. Genomic DNA was prepared from whole blood and sent to Keck core Facility at Yale University for WES. NimbleGen 2.1M human exome array was used to capture patients genomic DNA (~3 ug) following the manufacturer's protocols with modifications. Captured libraries were sequenced on the Illumina genome analyzer 75-bp paired-end reads, following the manufacturer's protocols and 6 samples were multiplexed per lane (as per Choi et al 2009 and Bilgüvar et al 2010). Sequence reads were mapped to the reference genome (hg19) followed by variant calling, annotation and filtration (as per Choi et al 2009 and Bilgüvar et al 2010). Unique or low MAF variants in NCBI SNP, Yale control database and NHLBI ESP were identified. Variants in many genes were selected to carry out the segregation analysis in families followed by resequencing in replication patients cohort. Many genes segregated in families were not found to have mutations in replication patient cohort. But we observed two unique variants in a new gene ALG8 from the already known endoplasmic pathway in two PCLD families. This new gene was prioritized for Sanger sequencing in sixty more patients and the mutations in this gene were confirmed in additional patients. Subsequently additional patients not having mutations in the known PCLD genes were subjected to exome sequencing. We discovered the heterozygous mutations in the PKHD1 gene in PCLD patients. Normally mutations in PKHD1gene are known to cause ARPKD. (JCI 2017). Human ALG8 gene was cloned in mammalian expression and lentiviral vectors with Myc and GFP tags for functional study. I also made the lentiviral shRNA vectors to knockdown this gene in cell lines. I designed the splice site assay to prove if splice site mutations truly results in disruption of splice site in this gene. These functional analysis experiments revealed that mutations in this gene cause cystic disease by defective maturation and trafficking of master cyst regulator PC1 (JCI 2017). Finally exome sequencing was performed in our complete PCLD cohort and observed the mutations in the GANAB, and SEC61 genes. All the four gene were inactivated in the cell lines using the CRISPR/CAS9. Functionally each gene resulted in defective maturation and trafficking of polycystin-1, the central determinant of cyst pathogenesis (JCI 2017).
Research Interest
neurodevelopmental disorders (autism, intellectual disability, epilepsy, and Rett syndrome-like phenotypes). Identified 15q 11.2 deletion (spanning five genes CYFIP1, TUBGCP5, WHAMML1, NIPA1 and NIPA2)

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