Abstract 48: A newly identified rare variant (chr11:47227430) with possible functional activity is associated with fasting insulin at the chromosome 11p11.2-NR1H3 locus in the Cohorts for Heart and Aging Research in Genetic Epidemiology Targeted Sequencing Study (CHARGE-TSS).
Authors: Dauriz Marco , Cornes Belinda K , Brody Jennifer A , Nikpoor Naghmeh , Morrison Alanna C , Wang Shuai , Dupuis Josée , Psaty Bruce M , Boerwinkle Eric , Rotter Jerome I , David Siscovick , Sladek Robert , Meigs James B ,   for the CHARGE Targeted Sequencing Study Diabetesworking group
Published: July 2021

Aim:
Common variation at the polygenic 11p11.2 locus has been associated with fasting glucose (FG) and insulin (FI) in genome-wide association studies. Further insights into the genetic pathways involved in glucose homeostasis and type 2 diabetes pathogenesis might rely on discovery of functional variants in genes or regulatory regions.

Hypothesis:
We hypothesized that high-throughput next-generation deep sequencing at the polygenic 11p11.2 locus might identify additional rare, potentially functional variants influencing FG and/or FI levels.

Methods:
We deeply sequenced (mean depth 38X) 16.1kb across the 11p11.2 locus in 3,566 non-diabetic individuals enrolled in the CHARGE Consortium (http://web.chargeconsortium.com/). We analyzed rare variants (minor allele frequency [MAF] <1%) in five gene regions, including
MADD
,
ACP2
,
NR1H3
,
MYBPC3
and
SPI1
, with FI or FG using Sequence Kernel Association Test (SKAT). Predicted regulatory variants were then analyzed by conditioning in SKAT on two previously known variants at
MADD
locus (rs7944584 and rs10838687 associated, respectively, with FG and FI). All analyses were adjusted for age, sex and study design variables. FI (adjusted for BMI) was naturally log-transformed to improve normality. Further functional studies were performed in human HepG2 hepatoma cells to unravel possible mechanistic pathways linked to functional variants.

Results:
We identified 653 allelic variants (including the known rs7944584 and rs10838687), 79.9% of which were rare and novel. At
NR1H3,
53 rare variants were jointly associated with FI (
p
=2.7 x 10
-3
); of these, seven were predicted to have regulatory function. Conditional analysis suggested more than two independent signals at 11p11.2-
MADD
locus. One predicted regulatory variant, chr11:47227430 (hg18; MAF=0.0007), contributed 20.6% to the overall SKAT score at
NR1H3,
and lies in intron 2 of
NR1H3
, a predicted binding site of the FOXA1 enhancer, a transcription factor associated with insulin regulation. Functional studies in HepG2 cells showed that the chr11:47227430 variant disrupts FOXA1 binding and significantly reduces FOXA1-dependent transcriptional activity.

Conclusions/interpretation:
We confirmed known common FI-associated variants near
MADD
gene and identified rare variation in an intron of
NR1H3
associated with FI. Functional
in vitro
studies showed that the rare A allele of the chr11:47227430 variant at the
NR1H3
locus might theoretically affect insulin regulation by interfering with transcription factor FOXA1 binding and, consequently, FOXA1-dependent transcriptional activity. Our targeted deep resequencing approach proved valuable in identifying new rare functional variants; quantitation of their actual impact on glucose homeostasis needs further confirmation.