Laura Oksanen

Melanocortin-3-receptor gene variants in morbid obesity

BACKGROUND: Linkage and knock-out mice studies suggest that the melanocortin-3-receptor (MC3R) is a candidate gene for obesity.OBJECTIVE: To evaluate whether MC3R mutations underlie morbid obesity.SUBJECTS AND METHODS: MC3R coding and 5′-flanking regions were sequenced in 48 subjects and the detected variants genotyped in 252 morbidly obese (BMI≥40 kg/m2) Finns. Gel shifts were used to examine whether a mutation in the putative promoter alters GATA-factor binding.RESULTS: Three common MC3R variants were found: a 17C>A variant, changing Thr6→Lys in 16%, a 241G>A variant changing Val81→Ile in 15%, and a −239A>G substitution in the GATA binding site in 21% of the subjects. Four other variants were detected in the 5′ flanking region. Frequencies of the three common variants did not differ between obese and contol subjects. Among the obese, the 17C>A and 241G>A variants were coinherited and associated with increased insulin–glucose ratios (P<0.05) and leptin levels (P<0.05). GATA-4 bound efficiently to wild type oligonucleotide, but only weakly to the oligonucleotide with the −239A>G mutation.CONCLUSIONS: MC3R gene variants are common and do not explain human morbid obesity. These variants associated with subtle changes in onset of weight gain, hyperleptinemia and insulin–glucose ratios. The −239A>G mutation abolishes binding of GATA-4 to the MC3R promoter region.

Novel polymorphism of the human ob gene promoter in lean and morbidly obese subjects

OBJECTIVE: Leptin, the circulating product of the human ob gene, may play role in control of appetite and metabolic rate. We screened the proximal promoter area of the ob gene for mutations and common polymorphisms in order to find out whether genetic variation in the regulatory area of this gene plays a role in human obesity. DESIGN: The technique of single‐strand‐conformation polymorphism (SSCP) was applied to screen for the promoter area of the ob gene for genetic alterations in morbidly obese patients. SUBJECTS: A total of 249 morbidity obese (present or past body mass index [BMI;[ge;40 kg/m2) and 141 lean (BMI≤25 kg/m2) subjects. MEASUREMENTS: DNA analysis was carried out using a single-strand conformation polymorphism (SSCP) technique and PCR followed by digestion with the restriction enzyme BssHII. Leptin was determined by radioimmunoassay in obese subjects. Serum lipids, glucose and insulin concentrations in the obese subjects were also determined. RESULTS: A new polymorphism C(-188)A was identified in the promoter region of the ob gene. This polymorphism was detected with allelic frequencies of 0.06 in morbidly obese subjects and 0.09 in lean controls (P=0.28). Initial studies failed to show an association of this polymorphism with serum leptin, response to treatment for obesity, history and extent of weight gain, or serum insulin, glucose of lipid concentrations. CONCLUSIONS: We have identified a common polymorphism in the promoter area of the human ob gene which appears to be very useful for genetic association and linkage studies. Further studies are needed to elucidate its potential role in the regulation of the human ob gene and in human metabolic disorders.

A common pentanucleotide polymorphism of the 3'-untranslated part of the leptin receptor gene generates a putative stem-loop motif in the mRNA and is associated with serum insulin levels in obese individuals

OBJECTIVE: To find out whether genetic alterations of the leptin receptor gene underlie human forms of obesity.DESIGN: Among 249 morbidly obese adults (body mass index, BMI≥40 kg/m2), we screened 30 patients with the highest serum leptin levels for alterations of their leptin receptor gene by single-strand conformation polymorphism (SSCP) technique.SUBJECTS: 249 severely obese subjects (present or past BMI≥40 kg/m2) and 138 lean controls (BMI≤25 kg/m2).MEASUREMENTS: DNA analysis was carried out using SSCP technique, sequencing and polymerase chain reaction (PCR) followed by digestion with the restriction enzyme RsaI. Serum leptin, glucose, insulin and lipid concentrations were determined in obese subjects.RESULTS: We were able to detect a pentanucleotide insertion (CTTTA) in the 3′-untranslated region of the leptin receptor gene. The presence of this pentanucleotide insert generates a putative stem-loop structure in the mRNA. Association studies were carried out on this variant. The frequency of the insertion allele did not differ between 249 obese (12.4%) and 138 lean (12.0%) subjects. There was no association of serum leptin, glucose or lipid levels with the pentanucleotide genotype in the obese individuals. However, when subjects without medication affecting insulin or glucose levels were considered, serum insulin levels were found to be lower in the heterozygous carriers of the insertion allele (15.1±9.2 mU/l) than in the subjects homozygous for the deletion allele (21.8±13.7 mU/l, P=0.0035).CONCLUSIONS: We were able to confirm the presence of a frequent insertion/deletion polymorphism close to the 3′-end of the leptin receptor gene. We also showed that serum insulin levels in morbidly obese subjects are associated with 3′-UTR variant genotype.

Markers for the gene ob and serum leptin levels in human morbid obesity

Abstract Leptin, the product of the ob gene, reduces body fat in genetically obese animals and circulates in elevated concentrations in the blood of obese patients. Polymorphic markers situated in the proximity of the human ob gene have recently been suggested to be linked to morbid obesity. We have studied the possible association between the microsatellite markers near the ob gene and morbid obesity in 252 morbidly obese patients with a mean body mass index (BMI) of 43 ± 7 kg/m2, and 151 lean controls with a mean BMI of 22 ± 2 kg/m2, and searched for linkage of these gene markers to obesity in 76 affected sib-pairs (BMI ≥ 32). No significant association was observed between any of the eight microsatellite markers and morbid obesity, and affected-sib-pair analysis failed to show linkage of three selected ob gene markers to obesity in the sibships. There was a strong positive correlation between serum leptin levels and BMI in morbidly obese patients; a carrier status for either of the two most prevalent alleles of the microsatellite marker D7S530 in the vicinity of the ob gene was associated with serum leptin levels in the obese subjects. Two of the markers (D7S2519, D7S649) showed a significant relation to the weight-losing response to a 16-week very-low-calorie dietary intervention. We have thus been able to confirm a tight relationship between serum leptin and body mass but have found no evidence for genetic linkage of the ob gene markers to morbid obesity in a population considered to represent a genetic isolate and to be an ideal model for studies of complex disorders.

Further Evidence For the Role of ENPP1 in Obesity: Association With Morbid Obesity in Finns

The aim of this study was to investigate a series of single‐nucleotide polymorphisms (SNPs) in the genes MC2R, MC3R, MC4R, MC5R, POMC, and ENPP1 for association with obesity. Twenty‐five SNPs (2–7 SNPs/gene) were genotyped in 246 Finns with extreme obesity (BMI ≥ 40 kg/m2) and in 481 lean subjects (BMI 20–25 kg/m2). Of the obese subjects, 23% had concomitant type 2 diabetes. SNPs and SNP haplotypes were tested for association with obesity and type 2 diabetes. Allele frequencies differed between obese and lean subjects for two SNPs in the ENPP1 gene, rs1800949 (P = 0.006) and rs943003 (P = 0.0009). These SNPs are part of a haplotype (rs1800949 C‐rs943003 A), which was observed more frequently in lean subjects compared to obese subjects (P = 0.0007). Weaker associations were detected between the SNPs rs1541276 in the MC5R, rs1926065 in the MC3R genes and obesity (P = 0.04 and P = 0.03, respectively), and between SNPs rs2236700 in the MC5R, rs2118404 in the POMC, rs943003 in the ENPP1 genes and type 2 diabetes (P = 0.03, P = 0.02 and P = 0.02, respectively); these associations did not, however, remain significant after correction for multiple testing. In conclusion, a previously unexplored ENPP1 haplotype composed of SNPs rs1800949 and rs943003 showed suggestive evidence for association with adult‐onset morbid obesity in Finns. In this study, we did not find association between the frequently studied ENPP1 K121Q variant, nor SNPs in the MCR or POMC genes and obesity or type 2 diabetes.

Functional analysis of the C(–188)A polymorphism of the human leptin promoter

Mutational analysis of the promoter region of the leptin gene in morbidly obese Finnish subjects had revealed a previously unidentified C(–188)A polymorphism in the proximal promoter that showed a weak association with elevated serum leptin levels in obese male carriers of the variant (–188A) allele. In this study we demonstrated that neither expression of reporter gene constructs driven by wild-type (–188C) or variant (–188A) proximal promoter regions, nor assay of binding of cellular proteins reveal a genotype-related difference in promoter activity.