Discussion
Considering severe early onset obesity might enrich more low-frequency
deleterious variants, we performed whole-exome sequencing and
association study on obesity in Chinese children. We found two missense
mutations (rs1059491 and rs189326455) are significantly associated with
common obesity. The rs1059491 is not only associated with BMI, but also
in association with FMI, FMP, FFMI, and LDL. It might result in obesity
by affecting the expression level of target genes other than alternating
protein structure because it is predictively unharmful to protein
structure but acts as an eQTL that can dramatically change the
expression of several obesity-related genes evidenced by transcriptome
sequencing.
Previous GWAS studies have identified multiple SNVs associated with
increased BMI(Cotsapas et al., 2009; Locke et al., 2015; Speliotes et
al., 2010; Thorleifsson et al., 2009). We noted that our most
significant SNV, rs1059491, was located in a previously reported obesity
locus with lead SNV of rs7359397 which was identified in
European(Speliotes et al., 2010). However, a fine mapping study on the
locus failed to find that rs1059491 was in association with obesity in
European children and adolescents(Volckmar et al., 2015). Explanations
for such contradiction are as follows three reasons. Firstly, although
rs1059491 is highly linked (r2 > 0.75) to
rs7359397 in European, no linkage disequilibrium (r2 =
0.00029) was observed between them in Chinese. Secondly, the allele
frequency of rs1059491 varies drastically among different populations,
which has been exemplified in 1,000 Genomes Project (1KG)
(Supplementary Figure 2 ). Minor allele frequency was 0.3434 in
European while was around 0.1 in Asian populations. In our findings,
minor allele frequency was 0.0421 in the control group, which was
similar to that of 0.0571 in southern Han Chinese in 1KG. Thirdly, our
conditional analysis found that rs1059491 (conditional P=7.13E-21) was independently associated with obesity in Chinese children
when controlling rs7359397. Taken together, rs1059491 was a population
specific obesity-associated SNV and significantly associated with
children obesity in Chinese.
The rs1059491 was a conserved missense mutation and was predicted as
deleterious (PolyPhen score = 1), while our findings show that it might
result in obesity through altering gene expression other than affecting
protein structure. Chip-seq have revealed that rs1059491 resided in
transcription factors binding sites of PPARγ2 and RXRA in ENCODE
datasets(Kheradpour & Kellis, 2014). Irregular behavior of PPARγ2 and
RXRA can result in abnormal lipid metabolism which lead to
obesity(Akyurek et al., 2013; Lima et al., 2013; Stossi et al., 2019;
Takahashi, Morita, Yokoyama, Suzuki, & Yamamoto, 2012). Therefore,
rs1059491 might affect the binding affinity to obesity-related
transcription factors and subsequently alter obesity-related gene’s
expression. Our transcriptome data and GETx data have shown that
rs1059491 is an eQTL affecting the expression of many genes, such asSULT1A2 , ATXN2L , and TUFM . It’s worthy to note thatSULT1A2 and ATXN2L were strongly implicated in
obesity(Voisin et al., 2015; Volckmar et al., 2015).SULT1A2is up-regulated in adipose tissues of individuals carrying G allele of
rs1059491, and high expression level of SULT1A2 is a
characteristic not only in lipid uptake tissues as duodenum and
intestine, but also in fat tissues (GTEx database). On the other hand,
rs1059491 regulates ATXN2L on translational level, and
subsequently affects its interaction partner ATXN2 , the
down-regulation of which would likely lead to increased body size and
fat levels in Caenohabditis elegans (Bar et al., 2016; Kaehler et
al., 2012), and ATXN2 knock-out mice show adult-onset
obesity(Kiehl et al., 2006; Lastres-Becker et al., 2008; Meierhofer,
Halbach, Sen, Gispert, & Auburger, 2016). In brief, the most likely
mechanism of rs1059491 association with obesity is expression regulation
of obesity-related genes on the transcription level and
post-transcription level (Figure 4 ).
We also found that rs189326455 was another variant significantly
associated with obesity. It located in the SH3 domain of MAP3K21, which
was responsible for controlling or regulating protein-protein
interactions in the signal transduction pathways, such as cytoplasmic
signaling(Koch, Anderson, Moran, Ellis, & Pawson, 1991; Schlessinger,
1994). MAP3K21 is a member of the mixed lineage kinases family that acts
as a specific modulator to inhibit lipopolysaccharide (LPS)-induced
activation of c-Jun N-terminal kinase (JNK), or extracellular
signal-regulated kinase (ERK)(Craige, Reif, & Kant, 2016), or as a
negative regulator of Toll-like receptor 4 (TLR4) signaling(Seit-Nebi,
Cheng, Xu, & Han, 2012). It’s well known that JNK, ERK, and TLR4 are
all implicated with obesity through elevating gene expression
level(Ahmad et al., 2012; Hirosumi et al., 2002), promoting insulin
resistance(Hirosumi et al., 2002; Ozaki et al., 2016; Pal et al., 2012;
Solinas & Becattini, 2017), or stimulating adipogenic
differentiation(Gu et al., 2015). It’s worthy to note that rs189326455
is also suggestively (P = 5.63E-07) associated with blood
triacylglycerol levels which is highly associated with insulin
resistance(Ormazabal et al., 2018). Thus, we speculate that rs189326455
might alter the affinity of SH3 domain and subsequently affects
interaction between MAP3K21 and mis-activating down-stream signaling
pathway proteins, such as JNK, ERK, and TLR4 (Figure 4 ), and
results in insulin resistance and obesity(Seit-Nebi et al., 2012).
In this study, we investigated causal variants in severe early onset
obesity by taking advantage of that child obesity is less affected by
cumulative environmental factors and the heritability is higher compared
with adulthood obesity. We identified two potential variants related to
obesity with whole exome sequencing. We found that rs1059491 was an eQTL
which might result in obesity through altering the expression of
obesity-related genes, and rs189326455 might affect the regulation of
MAP3k21 to subsequent obesity-related genes. Although the speculation
should be proved by more functional experiments, our study is among the
first to use exome sequencing technology to identify functional variants
of both common obesity and abdominal obesity in Chinese children.
Chinese people are more likely to develop abdominal obesity, which has
been associated with a higher risk of developing type 2 diabetes and
cardiovascular disease(Thomas et al., 2004). Therefore, identification
of the SNVs associated with abdominal obesity is of great impact on
prevention and control of adiposity-based chronic diseases.
Acknowledge. This work was supported by grants from the Beijing
Natural Science Foundation Program and Scientific Research Key Program
of Beijing Municipal Commission of Education(KZ202010025039) and
National Natural Science Foundation of China (81973110, 81502872, and
31671312)
Author Contributions. K.M., M.Z., and J.C. wrote the
manuscript, researched, analysed and interpreted the data, contributed
to the discussion, and reviewed/edited the manuscript. J.M., and Y.Z.
designed the study, and made critical revisions of the manuscript. C.M.
revised the figures. M.Z., X.Z., L.G., and L. F. were involved in the
sample collection, selection and preparation. H.C. collected the
phenotype data. W.L., and Z.Z. designed the bioinformatics and
experimental sections, coordinated the data collection, maintained
project procedure and carried out bioinformatics analysis. M.Z. and L.G.
performed the genotyping analysis. X.X and X.S. made critical revisions
of the manuscript. All the authors contributed to the final paper.
Duality of Interest. The authors have nothing to declare.
Data Availability Statement. The data that support the findings
of this study are available from the corresponding author upon
reasonable request.