Genome wide association studies (GWAS) have revealed genomic variants strongly associated with most common disorders; indeed there is general consensus on these findings from positive replication outcomes by independent groups. The clear leader to date, with respect to strength of association, is the FTO
; this association with BMI and obesity has now been widely replicated by multiple independent groups. Common variants of MC4R
have also been discovered to be strongly associated with BMI and related traits2
, complementing the already described rare coding mutations in this gene involved in monogenic obesity3
; more than 150 missense and nonsense mutations have already been reported in MC4R 4-7
but have not been implicated as a frequent cause of human obesity5, 8
. A variant located approximately 30kb downstream of TMEM18
has also been consistently and strongly associated with BMI in GWAS reports 9
To date, most GWAS reports have resulted from investigations of populations of European origin. Indeed, like many of the other replication efforts, FTO
shows the strongest association with BMI in our large European American pediatric cohort10
. However, the role of the FTO
locus in influencing BMI and obesity predisposition in populations of African ancestry has been previously less clear, but consensus is emerging from large cohort studies, both in adults11
and in our own pediatric cohort12
that a common SNP can capture the association in both ethnicities. The picture is substantially less clear for MC4R
, where further work in other ethnicities is required to fully understand their associations with BMI and obesity13
Investigators have hypothesized that loci revealed by GWAS may not only harbor the common variants conferring modest risk that led them there, but may also harbor rarer variants that confer substantially higher risk of the same disease. A precedent for this has already been set in this regard, where a study of ten candidate genes associated with type 1 diabetes led to the discovery of rare variants associated with the disease in the interferon induced with helicase C domain 1 (IFIH1
and more recently an extensive sequencing effort of inflammatory bowel disease GWAS-implicated genes revealed such variants15
A French sequencing effort in Caucasians (primarily adults) has already reported a set of exonic mutations in FTO
; however, due to the lack of significant differences in the frequencies of these variants between lean and obese individuals, this study was largely negative16
. In addition, sequencing efforts to date on MC4R
have been mainly limited to extreme obesity4, 5, 7
We reasoned that such rare disease-conferring but highly penetrant genetic variants at these loci could be easier to determine in children, where the relative environmental exposure time is substantially less. Added to that we were also in a position to investigate this issue in African American children i.e. African ancestry represents the greatest haplotype diversity so we should be able to determine the maximum number of existing exonic variants; indeed this is the same cohort that we first established the distillation of the trans-ethnic association between obesity and FTO12
. In addition we elected to investigate the next two most strongly associated loci resulting from GWAS, namely MC4R
in a comparable fashion.
From our Sanger sequencing effort of the nine exons of the FTO
gene at the ends of the BMI distribution of our defined cohort (200 cases and 200 lean controls) we identified a total of seven variants, three of which were synonymous (T6T, I334I and D394D) and four were non-synonymous [A163T, G182A, M400V and A405V]. G182A, D394D and M400V were not previously reported by the French study of Caucasian cases16
. The most notable observation from this initial sequencing phase was with A405V, which was present in eleven obese (BMI≥95th
percentile) cases and only four lean (BMI≤5th
percentile) subjects i.e. almost three times more frequent in cases ().
Repertoire and frequency of exonic mutations in FTO, MC4R and TMEM18 in African Americans from the initial sequencing effort of 200 childhood obesity (BMI≥95th percentile) cases and 200 lean (BMI≤5th percentile) controls.
A similar sequencing approach for the single exon of MC4R
using the same cohort revealed thirteen variants (), seven of which were synonymous [G8, A135, Q156, I198 and C271, C279, L322] and six were non-synonymous [V103I, N123S, S136A, F202L, N240S and I251L]. Among the non-synonymous variants, four of them had been reported previously5
[V103I, F202L, N240S and I251L], with the remaining two being novel [N123S and S136A].
Finally, sequencing of TMEM18 revealed only four exonic variants, two of which were novel and synonymous [V17 and L51] and two which were non-synonymous [P2S and V113L], with the latter having already been recorded in publically available databases (rs11370572 and 1KG2669666, respectively).
We elected to follow-up all non-synonymous variants detected in these three genes to investigate the possible extent of their role in the pathogenesis of childhood obesity in African Americans in an additional 768 obese (BMI≥95th percentile) and 768 lean (BMI≤50th percentile) individuals using TaqMan genotyping; however it should be noted that we could not generate a successful genotyping assay for S136A in MC4R.
Analysis of the resulting genotyping data revealed that there were no significant differences in the frequency of these variants between cases and controls, including A405V in FTO which had looked initially promising from the sequencing outcomes, except for N240S in MC4R (Fisher's Exact P = 0.0001) ().
Table 2 Distribution of the four missense variants uncovered through the exonic sequencing of FTO, MC4R and TMEM18 in African Americans through the genotyping of 768 obese (BMI≥95th percentile) and 768 lean (BMI≤50th percentile) children of the (more ...)
Our work complements recent work carried out in the French study of Caucasians16
. We also found that missense variants in FTO
did not play a substantial role in conferring risk for obesity in our cohort but interestingly, two of the missense variants had not been detected in that Caucasian sequencing effort i.e. G182A and M400V.
Furthermore, our sequencing effort of MC4R and TMEM18 revealed variants that had not been previously published. Two novel non-synonymous variants were uncovered within MC4R i.e. N123S and S136A, both in the transmembrane domain. The two non-synonymous variants in TMEM18 were P2S and V113L; P2S is located on the very N-terminus of the protein, while V113L is located in the transmembrane domain of the protein. Again, however, these variants did not turn out to be associated with childhood obesity in African Americans, except for the N240S variant in MC4R.
N240S missense variant is an already known loss-of-function mutation, but which has also been observed in non-obese subjects previously17
. Although the follow-up genotyping effort indicated an exclusive presence of the rare G allele in cases only (), when combined with the discovery sequenced dataset, where there was one case and one control harboring the same allele (), the result does not strictly remain significant. As such, in order to fully resolve the role of this variant in obesity in African Americans, further studies are warranted.
So why do we not uncover more disease-conferring missense mutations in these known obesity associated loci? Apart from limited statistical power issues at the discovery stage (detection of variants only >0.5% frequency with the current strategy), it could well be that these loci only harbor a common variant that confers modest risk for common childhood obesity; on the other hand, if we had sequenced all our cases and controls, we would have been powered to detect variants down to >0.1% frequency which could confer substantial risk but we were unable to assess due to our study design. Alternatively, causative variants could be intronic or somewhat further from the initial signal than originally thought and detected via synthetic association18
; indeed, there is still debate whether the neighboring locus to FTO
, i.e. RPGRIP1
, is in fact the culprit gene. Our findings should help inform future studies of these loci.
In summary, we have shown that moderately rare missense variants observed in the exons of the three genes discovered from GWAS, i.e. FTO, MC4R
, do not confer risk of common childhood obesity in African Americans, except for a degree of evidence with the known N240S variant in MC4R
. Furthermore our FTO
findings agree with the prior studies from similar analyses in subjects of European ancestry5, 8