Cases were obese women (BMI ≥ 35 kg/m²) with a lifetime BMI >40 kg/m². Independent controls were selected who had a current and lifetime BMI ≤ 25. All cases and controls were non-Hispanic Caucasians. Sample characteristics are summarized in Table 1. The individuals in the samples were of approximately the same age but differed in average BMI by 29 kg/m². As would be expected for extreme obesity, most cases had an early onset. The median age at obesity onset was 12 and 90% had an onset prior to age 26. This result is consistent with the observation of Frayling et al. 3 of an FTO association in children as young as seven years.

All subjects in the three samples gave informed consent, and the protocol was approved by the Committee on Studies Involving Human Beings at the University of Pennsylvania.

A subset of the case sample had extremely discordant sisters. Characteristics of the sister pairs are presented in Table 1. All thin sisters had BMI < 25 kg/m² and had never been obese (BMI ≥ 27 kg/m²), while obese sisters had BMI ≥ 35 kg/m². The sisters were similar in age but differed markedly in BMI by an average of 26 kg/m². This difference is more than double the average BMI of the thin sisters, making the comparison comparable in magnitude to some animal models (Table 1). The percentage body fat of normal weight and obese sisters were 26.41 ± 5.58 and 49.36 ± 5.18, respectively.

The three SNPs, rs1421085, rs17817449, and rs9939609, are located within 19.6 kb in intron 1 of the FTO gene and are in strong LD (pairwise r²>0.97) based on our samples. There are two primary haplotypes, C-G-A (42.0 %) and T-T-T (55.5 %). The T-C-T haplotype was observed at 1.7 % and 5 others had very low frequencies. Pairwise linkage disequilibrium measures used Proc Allele and haplotype analyses the Proc Haplotype EM algorithm 11 in SAS 9.1.

DNA was extracted from whole blood or lymphoblastoid cell lines using a high salt method. PCR reactions and genotyping were completed using an Applied Biosystems (ABI, Foster City, CA) TaqMan 7300 system. SNP primers and probes were provided by ABI Assays-on-Demand. Detailed conditions are available on request.

Descriptive statistics, allele and genotype frequencies were calculated using SPSS 15.0. Tests of Hardy-Weinberg (HW) equilibrium were based on a Chi Square goodness-of-fit test programmed in Excel. The program Structure was used to test for possible population structure. For this purpose, more than 300 SNP markers were available for cases, while a subset of 37 were available for both cases and controls. Association analyses were completed using the Case Control procedure in SAS 9.1, with the overall association with genotype based on the Armitage trend test and odds ratios based on allele counts, reflecting additive effects.

For discordant sister pairs, mean values for BMI and percent fat were compared using one-way ANOVA and analysis of covariance used General Linear Models in SPSS 15.0. Association analyses for discordant sisters used the sibling transmission disequilibrium test (S-TDT) 12

For the case/control comparisons, power was estimated using parameters from the original Frayling et al. 3 study, since that study had the largest sample size. Values reported by Dina et al. 4 were similar. Assuming a risk allele frequency of .45, obesity prevalence = 0.2 (BMI > 35 kg/m²), linkage disequilibrium (D') = 0.80, relative risk (RR) = 1.67, p < .05, and additive, dominant or recessive expression, we have greater than 80% power with 520 unpaired cases and controls, the minimum number of individuals with genotypes. This estimate considers only the risk for the upper extreme phenotype, and conservatively assumes a population prevalence based on current BMI>35 rather than lifetime BMI>40. Power is higher still if risk and protection are considered in the respective tails of the distribution (analyses not shown).

Genotype frequencies for controls, cases and the combined sample were all in Hardy-Weinberg equilibrium, and there was no evidence of structure (analyses not shown). Genotype and allele frequencies for cases and controls are given in the Table 2. Obese cases have consistently higher minor allele frequencies that are near or above 0.5, while those for controls were about 0.13 lower. The genotype frequencies were similar for controls and thin sisters and minor allele frequencies were 9–12% lower than their obese sibling. Significance for case-control and thin-obese sister allele frequency comparisons are given in the right column. All reach statistical significance except for rs1421085 in discordant sisters.

The results from the association analyses for cases and controls are presented in Table 3. Results were highly significant (p < 10^-9) for all three SNPs. The strongest association was with rs1421985 (p = 3.04 × 10^-10, OR = 1.75, CI = 1.47–2.08) (Table 3). Considering all 8 observed haplotypes the overall χ² for association with case status was 50.69 (df = 7, p = 1.07 × 10^-8). For the two common haplotypes C-G-A and T-T-T the values were 36.47 (df = 7, p = 1.55 × 10^-9) and 36.50 (df = 7, p = 1.53 × 10^-9), respectively.

Results from association analyses for discordant sister pairs are presented in Table 4. All associations are significant and parallel those for allele frequencies (Table 2) and associations in cases and controls (Table 3).

These results suggest common variability in FTO is associated with increased obesity risk or resistance and may in part account for differences between closely related individuals.

BMI Body mass index

SNP Single nucleotide polymorphism

FTO Homolog of mouse fatso (fused toes) gene

LD Linkage disequilibrium

The author(s) declare that they have no competing interests.

RAP is PI of the projects that accrued the samples and performed the phenotyping. For this specific study, he conducted statistical analyses and drafted the manuscript. WDL participated in data collection, performed data analyses, conducted genotyping, and made contributions to the manuscript. HZ analyzed data, advised on statistical analyses, and contributed to the manuscript. All authors have read and approved the final manuscript.