Targeted next-generation sequencing and fine linkage disequilibrium mapping reveals association of PNPLA3 and PARVB with the severity of nonalcoholic fatty liver disease.

The genomic regions containing PNPLA3, SAMM50 and PARVB are susceptibility loci for the development and progression of nonalcoholic fatty liver disease (NAFLD). In order to search for all common variations in this region, we amplified the genomic DNA of 28 NAFLD patients by long-range PCR, covering the entire susceptibility region and sequenced the DNA using indexed multiplex next-generation sequencing. We found 329 variations, including four novel variations. Fine mapping of variations including insertion/deletions was performed for 540 NAFLD patients (488 with nonalcoholic steatohepatitis (NASH) and 52 with simple steatosis) and 1012 control subjects. HaploView analysis showed that linkage disequilibrium (LD) block 1 and 2 occurred in PNPLA3, block 3 in SAMM50 and block 4 in PARVB. Variations in LD blocks 1-4 were significantly associated with NAFLD as compared with control subjects (P<1 × 10(-8)). Variations in LD block 2 were significantly associated with the NAFLD activity score (NAS), aspartate aminotransferase and alanine aminotransferase. Variations in LD block 1 were significantly associated with the fibrosis stage. The strongest associations were observed for variations in LD block 4, with NASH as compared with simple steatosis (P=7.1 × 10(-6)) and NAS (P=3.4 × 10(-6)). Our results suggested that variations, including insertion/deletions, in PARVB, as well as those in PNPLA3, are important in the progression of NAFLD.