APOE and TGF-[beta]1 genes are associated with obesity phenotypes.(Letter to JMG)

J Med Genet 2003;40:918-924

Obesity, defined as a body mass index (BMI) of greater than 30 kg/[m.sup.2], has become a worldwide public health problem. (1) About 250 million adults, roughly 7% of the world adult population, are considered obese and two or three times as many may be overweight with BMI of 25-30 kg/[m.sup.2]. (2) A recent analysis shows that the health care cost of obesity is probably between 0.89% and 4.32% of the national expenditure in the United States. (3) Obesity is associated with many diseases such as type 2 diabetes mellitus, hypertension, coronary heart disease, and certain forms of cancer. (1) As a complex disease, obesity is determined by multiple genetic and environmental factors, including physiological, behavioural, and sociocultural factors. (4-7) Numerous molecular genetics studies have been launched to search for the genes underlying the variations of obesity phenotypes, resulting in a host of candidate genes and potentially important genomic regions. (8)

Apolipoprotein E (APOE), coding a glycoprotein that plays a central role in lipid metabolism, is considered as a prominent candidate gene for obesity. APOE binds with high affinity to the low density lipoprotein (LDL) receptor and facilitates endocytosis of the associated lipoprotein particle. (9) In addition, APOE mediates lipoprotein interactions with the LDL receptor related protein, very low-density lipoprotein receptor, and other lipoprotein receptors. (9) (10) Some studies have reported positive associations between APOE genotypes and some obesity phenotypes, (11-14) whereas negative results were observed for the other obesity phenotypes. (13-15) The transforming growth factor beta 1 (TGF-[beta]1) gene codes a multifunctional cytokine that controls proliferation, differentiation, and other functions in many cell types, including adipocyte precursor cells. (16) Increased TGF-[beta]1 expression was associated with BMI and abdominal adipose tissue in morbid obesity. (17) A recent study suggested an association between TGF-[beta]1 polymorphism and both BMI and abdominal obesity in Swedish men. (18)

The relevance of these two genes to obesity has been suggested by evidence from linkage studies. Several studies have supported linkage to obesity phenotypes at chromosome 19q13, where the two genes are located. Saar et al (19) got a peak LOD score on 19q12, a region very close to 19q13, for obesity in German children and adolescents. Recently, a whole genome linkage study conducted by our group has provided some suggestive evidence of linkage for obesity phenotypes on chromosomal 19q13 (Liu et al to be submitted). In mouse models, a number of QTLs for obesity phenotypes have been found with the homologous location for human chromosome 19q13, further highlighting the importance of this region to obesity. (8)

In the present study, using the tests implemented in the statistical software package QTDT, quantitative transmission disequilibrium test, (20) we tested the linkage and association of these two genes with obesity phenotypes by investigating single nucleotide polymorphisms (SNPs). Our study sample contains 405 white nuclear families comprising 1873 subjects.

MATERIALS AND METHODS

Subjects

The study subjects came from an expanding database being created for studies to search for genes underlying the risk to osteoporosis and obesity at the Osteoporosis Research Center of Creighton University. The study was approved by the Creighton University institutional review board. All subjects were white Europeans. Only healthy people were included with the exclusion criteria that were detailed elsewhere. (21) For each study subject, information on age, sex, medical history and family history was acquired. A total of 405 nuclear families were recruited with 1873 subjects, including 740 parents, 744 daughters and 389 sons. Among these, 341 families were composed of both parents and at least one offspring. In the remaining 64 families, there were at least two children with either one or no parent. The average family size was 4.62 (1.78) (mean (SD)), ranging from 3 to 12, and there were 1512 sibling pairs in total.

Measurement

Fat mass and lean mass were measured by dual energy X-ray absorptiometry (DXA) with a Hologic 2000+ or 4500 scanner (Hologic Inc., Bedford, MA, USA). Measurements of BMD on the two types of machines agreed within 1%, but the body mass measurements differed in an unsystematic way. Both machines were calibrated daily. The body composition bar was used on every whole body scan on the Hologic 2000+ scanner. On the Hologic 4500 scanner, the bar was not needed for the body scans; instead, it was scanned every week. Percentage fat mass (PFM) is the ratio of fat mass to body weight (the sum of fat mass plus lean mass plus bone mineral content). Weight was measured in light indoor clothing, using a calibrated balance beam scale, and height was measured using a calibrated stadiometer at the same visit as for the body scan. The measurement precision of BMI as reflected by the coefficient of variation was 0.2%. The coefficients of variation for fat mass, PFM, and lean mass were 2.2%, 2.2%, and 1.0%, respectively, for measurements obtained on the Hologic 2000+ scanner, and were 1.2%, 1.1%, and 0.7%, respectively, for measurements on the Hologic 4500 scanner. Members of the same nuclear family were usually measured on the same type of machine.

Genotyping

After searching SNP databases, including dbSNP (www.ncbi.nlm.nih.gov/SNP), JSNP (http://snp.ims.utokyo.ac.jp), HGVbase (http://hgvbase.cgb.ki.se), and OMIM (www.ncbi.nlm.nih.gov/omim), as well as reviewing previously published studies, four SNPs in each gene were selected. Our selection criteria were as follows: a) functional relevance and importance; b) degree of heterozygosity--that is, allele frequencies; c) position in or around the gene; and d) their use in previous genetic epidemiology studies. For presentational conveniences, the selected SNPs were coded as SNP1-8 as indicated in table 1. Among these eight SNPs, SNP6 is an insertion/deletion (+/-) polymorphism of pyrimidine C and the others are nucleotide substitution.

DNA was extracted from whole blood using a commercial isolation kit (Gentra Systems, Minneapolis, MN, USA) following the procedure detailed in the kit. The genotyping procedure for all SNPs was similar, involving polymerase chain reaction (PCR) and invader assay reaction (Third Wave Technology, Madison, WI, USA). PCR was performed in a 10 [micro]1 reaction volume with 30 cycles. After amplification, an invader reaction was performed in a 7.5 [micro]1 reaction volume and the fluorescence intensity for both colours was read using a …