Association Study and Mutation Analysis of Adiponectin Shows Association of Variants in APM1 with Complex Obesity in Women

We performed an association study and mutation analysis of the adiponectin ( APM1 ) gene to study its involvement in the development of obesity. We also studied the interaction with peroxisome proliferator-activated receptor γ ( PPAR γ). 223 obese women and 87 healthy female control subjects were used for association analysis. Mutation analysis was done on 95 morbidly obese adults and 123 overweight and obese children and adolescents. We selected 6 haplotype tagging SNPs in APM1 and the Pro12Ala variant (rs1805192) in PPAR γ to study the interaction. The G allele of rs2241766 was more common in controls (cases 10.8% vs. controls 18.4%, nominal p = 0.011; OR = 0.57, nominal p = 0.018). The rs2241766/rs3774261 haplotype was also associated with obesity (nominal p = 0.004). Only the latter association remained significant after controlling for the False Discovery Rate. Resequencing of exon 2, exon 3 and intron 2 in 95 individuals did not reveal any SNPs in high linkage disequilibrium with rs2241766. No interaction with the Pro12Ala variant in PPAR γ was detected. Mutation analysis of APM1 did not identify mutations. In conclusion, we found an association of an APM1 haplotype with obesity and found that APM1 mutations are not a common cause of monogenic obesity in our cohort.     

The adiponectin gene is associated with adiponectin levels but not with characteristics of the insulin resistance syndrome in healthy Caucasians

Low concentrations of adiponectin, the protein product of the APM1 gene, have been reported to be associated with obesity and insulin resistance. However, contrasting results have been described on the genetic variability in APM1 and characteristics of the metabolic syndrome and adiponectin serum concentrations. In the present study, we investigated the association of the two most well-known SNPs of APM1 (+45T>G and +276G>T) and their haplotypes, with serum adiponectin concentrations, metabolic parameters and intima-media thickness of the carotid arteries in 1,745 well-phenotyped asymptomatic unrelated Caucasian subjects of the SAPHIR cohort. The common T-allele (88.5%) of SNP +45T>G and the common G-allele (70.5%) of SNP +276G>T were associated with significantly lower serum adiponectin levels (P = 0.0008 and P = 0.00005, respectively). The most frequent haplotype TG (59.0%) defined by both loci showed a strong association with lower serum adiponectin concentrations (P = 0.000000002). A clear effect per copy of the respective haplotype was observed. This association was most pronounced in lean and insulin-sensitive subjects. The two less common haplotypes TT (29.5%) and GG (11.5%) were associated with higher serum adiponectin levels in a dose-dependent association. Interestingly, no significant association between the adiponectin 45-276 haplotypes and the majority of parameters of the metabolic syndrome or intima-media thickness of the carotid arteries was found in our study. In summary, we replicated a strong association of the adiponectin 45-276 genotypes and haplotypes with adiponectin levels in healthy Caucasians. However, we could not confirm an association of this gene locus with metabolic parameters of the insulin resistance syndrome.     

Adiponectin gene variation associates with the increasing risk of type 2 diabetes in non-diabetic Japanese subjects

Adiponectin is an adipocyte-secreted protein that is known to modulate insulin sensitivity and glucose homeostasis. A number of genetic variations have been studied. Among them, two single-nucleotide polymorphisms (SNP45T>G, SNP276G>T) showed an association with type 2 diabetes in the Japanese population. In this study, we examined the association between these SNPs and risk factors of type 2 diabetes in 194 non-diabetic Japanese subjects. SNP45 was associated with insulin sensitivity (determined by HOMA-IR, p=0.046) and obesity (body mass index; BMI, p=0.043). SNP276 showed a stronger association with HOMA-IR (p=0.018) and BMI (p=0.017). However, neither SNP had an association with insulin secretion (insulinogenic index) and plasma lipid levels. Moreover, a linkage dis-equilibrium was observed between SNP45 and SNP276. Carriers with SNP45G-SNP276G haplotype had higher BMI (p=0.034) and carriers with SNP45T-SNP276T haplotype had lower BMI (p=0.005) and HOMA-IR (p=0.037). Adiponectin gene variations showed an association with obesity and insulin sensitivity, and adiponectin genotypes may predict the increasing risk for type 2 diabetes in non-diabetic subjects.     

Association of the human adiponectin gene and insulin resistance

Adiponectin is an adipocyte-secreted protein that modulates insulin sensitivity and whose low circulating concentration is associated with insulin resistance. In the present study, we analysed the association between two single-nucleotide polymorphisms (SNPs) in the adiponectin gene and insulin resistance in 253 nondiabetic subjects. In addition, we investigated whether this association is modulated by body mass index (BMI) levels. The SNPs +45T>G and +276G>T in the human adiponectin gene were detected in real-time PCR with LightCycler. No association was found with the +45T>G SNP. The +276G>T SNP was associated with higher BMI (P<0.01), plasma insulin (P<0.02) and HOMA(IR) (P<0.02). To analyse the possible interaction between BMI and the adiponectin gene on insulin resistance, the study group was divided into two subgroups, according to the BMI below or above the median of 26.2 kg/m(2). In both subgroups, subjects carrying the +276G>T SNP had higher HOMA(IR); however, the difference was highly significant among leaner (P<0.001), but not among heavier individuals, indicating that BMI status and the adiponectin gene interact in modulating insulin resistance. Among individuals with BMI <26.2 kg/m(2), the relative risk of insulin resistance was 9.7 (CI: 1.32-87.7, P<0.035). In a subgroup of 67 subjects, carriers of the +276G>T SNP had significantly (P<0.05) lower mean serum adiponectin levels (25.7 ng/ml) compared to noncarriers (37.0 ng/ml), suggesting a possible influence of the +276G>T SNP on adiponectin levels. In summary, we observed an association between the +276G>T SNP in the adiponectin gene and insulin resistance. In particular, among leaner individuals, the adiponectin gene appears to determine an increased risk to develop insulin resistance.     

Common polymorphisms (single-nucleotide polymorphisms SNP+45 and SNP+276) of the adiponectin gene regulate serum adiponectin concentrations and blood pressure in young Finnish men

Epidemiologic studies have shown that serum level of adiponectin, a circulating protein secreted by adipocytes, predicts the risk of type 2 diabetes and cardiovascular events. Two single-nucleotide polymorphisms (SNPs) at the adiponectin locus (T45G or G276T) of the adiponectin gene (APM1) have been associated with insulin resistance, low serum adiponectin levels, and diabetes. In the present study, the association of these polymorphisms with serum adiponectin level and insulin resistance-associated risk factors was investigated. To this aim, SNP+45 and SNP+276 of APM1 were genotyped in 252 young Finnish men. Serum adiponectin level (p < 0.001) and diastolic blood pressure (p = 0.031) were significantly higher in subjects with the T276T genotype of APM1 compared to those with the G276T or G276G genotypes. Mean diastolic blood pressure among the T276T subjects was 80 mmHg and that in subjects with the G276G and G276T genotypes below 75 mmHg. An interaction between triglycerides, diastolic blood pressure, quantitative insulin sensitivity check index, and SNP276 with regard to serum adiponectin level was found. After adjustment for other covariates, the interaction between triglycerides and SNP276 remained statistically significant (p = 0.009). Among subjects with the T276T genotype, an increase in triglyceride level was associated with a decrease in adiponectin concentration. This result was not observed in other genotype groups. SNP+45 was not significantly related to serum adiponectin concentration, but high-density lipoprotein (HDL) cholesterol tended to be higher in subjects with the T45T genotype (p = 0.051) compared to subjects with the X45G genotype. In conclusion, the T276T genotype of the adiponectin gene was associated with elevated serum adiponectin level and diastolic blood pressure among young Finnish men.     

Non-founder mutations in the MEFV gene establish this gene as the cause of familial Mediterranean fever (FMF)

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurring attacks of fever and serositis. It affects primarily North African Jews, Armenians, Turks and Arabs, in which a founder effect has been demonstrated. The marenostrin-pyrin-encoding gene has been proposed as a candidate gene for the disease ( MEFV ), on the basis of the identification of putative mutations clustered in exon 10 (M680V, M694I, M694V and V726A), each segregating with one ancestral haplotype. In a search for additional MEFV mutations in 120 apparently non-founder FMF chromosomes, we observed eight novel mutations in exon 2 (E148Q, E167D and T267I), exon 5 (F479L) and exon 10 (I692del K695R, A744S and R761H). Except for E148Q and K695R, all mutations were found in a single chromosome. Mutation E148Q was found in all ethnic groups studied and in association with a novel ancestral haplotype in non- Ashkenazi Jews (S2). Altogether, these new findings definitively establish the marenostrin/pyrin-encoding gene as the MEFV locus.      

Analysis of microsatellite markers and single nucleotide polymorphisms in candidate genes for susceptibility to bipolar affective disorder in the chromosome 12Q24.31 region.

Previous results from our genetic analyses using pedigrees from a French Canadian population suggested that the interval delimited by markers D12S86 and D12S378 on chromosome 12 was the most probable genomic region to contain a susceptibility gene for affective disorders. Here we present a more detailed genetic analysis of a 7.7 Mb genomic region located on 12q24.31. This region was saturated with 20 microsatellite markers to refine the candidate region and linkage analysis performed in 41 families from the Saguenay-Lac-St-Jean (SLSJ) region of Quebec. The results of two point parametric analysis using MFLINK supported the presence of a susceptibility locus on chromosome 12q24.31. Association studies with microsatellite markers using a case/control sample from the same population (n = 401) and analyzed with CLUMP revealed significant allelic associations between the bipolar phenotype and markers NBG6 (P = 0.008) and NBG12 (P < 10(-3)). According to these results, we investigated candidate genes in the NBG12 area. We analyzed 32 genes for the presence of polymorphisms in coding sequences and intron/exon junctions and genotyped 22 non-synonymous SNPs in the SLSJ case/control sample. Two uncommon polymorphisms (minor allele frequency < or = 0.03) found in KIAA1595 and FLJ22471 genes, gave P-values below 0.05 with the T1 statistic. Moreover, using haplotype analysis, a nearly significant haplotypic association was observed at the HM74 gene. These results do not give strong support for a role in the susceptibility to bipolar disorder of any of these genes analyzed. However, the significance of rare polymorphisms should be explored by further analyses.     

Vesicle-associated membrane protein 4, a positional candidate gene on 1q24-q25, is not associated with type 2 diabetes in the Old Order Amish

OBJECTIVE: The vesicle-associated membrane protein-4 (VAMP4) gene is an excellent type 2 diabetes (T2DM) positional candidate gene. It is located on chromosome 1q24-q25, a region of linkage to T2DM in the Amish and several other populations. VAMP4 is expressed in liver and skeletal muscle and participates in intracellular trafficking of secreted and membrane-associated proteins. DESIGN AND METHODS: We sequenced VAMP4 in 20 Amish subjects. Polymorphisms in and around VAMP4 were genotyped in 65 Amish subjects with T2DM, 64 subjects with impaired glucose homeostasis (IGH), and 126 normal glucose tolerant controls, as well as in an expanded set of 749 participants of the Amish Family Diabetes Study for whom glucose and insulin levels during an oral glucose tolerance test (OGTT) and other quantitative traits related to diabetes were available. Case-control and quantitative trait association analyses were performed. RESULTS: We found three common non-coding intragenic polymorphisms: a 23bp insertion/deletion (I/D) in the 5' untranslated region (UTR) in exon 1 at position 73127, and G35319T and C335296T single nucleotide polymorphisms (SNPs) in the 3' UTR (NCBI Accession No. Z98751). The two 3' UTR SNPs were in complete linkage disequilibrium (LD) and both were in strong LD with the exon 1 I/D polymorphism (|D'|=0.82). Similarly, three extragenic flanking SNPs (rs978985, rs203255, and rs1023479) showed moderate LD with the neighboring intragenic SNPs (|D'|=0.23-0.69). None of the SNPs individually nor any of the 2-, 3-, 4-, or 5-polymorphism haplotypes were associated with T2DM or IGH. The exon 1 I/D polymorphism was not associated with significant differences in mean fasting or stimulated glucose or insulin levels during an OGTT or other diabetes-related quantitative traits in the expanded set of 749 subjects. CONCLUSION: Variation in VAMP4 does not significantly influence risk of T2DM or IGH in the Amish.     

Analysis of Osteocalcin as a Candidate Gene for Type 2 Diabetes (T2D) and Intermediate Traits in Caucasians and African Americans

Recent studies in mice and human identified osteocalcin (OCN) as a bone-derived hormone that modulates insulin secretion and insulin sensitivity. OCN is synthesized by the bone gamma-carboxyglutamate protein (BGLAP) gene located in the well replicated region of type 2 diabetes (T2D) linkage on chromosome 1q22. We resequenced BGLAP gene in 192 individuals with T2D and performed case-control studies in 766 Caucasian (461 T2D and 305 controls) and 563 African American individuals (371 T2D and 192 controls). Metabolic effects of BGLAP variants were examined in 127 nondiabetic members of Caucasian T2D families and in 498 unrelated nondiabetic African American and Caucasian individuals. BGLAP expression was tested in transformed lymphocytes from 60 Caucasian individuals. We identified 17 single nucleotide polymorphisms (SNPs) in African Americans, but observed only the two known SNPs in Caucasians. No SNP was associated with T2D. Promoter SNP rs1800247 was not associated with metabolic traits including insulin sensitivity (S_I) or fasting glucose in either population, but nonsynonymous SNP rs34702397 (R94Q) was nominally associated with S_I (uncorrected p = 0.05) and glucose-mediated glucose disposal (S_G; uncorrected p = 0.03) in African Americans. No SNP altered measures of insulin secretion or obesity, nor was BGLAP expression associated with rs1800247. Our study was sufficiently powered to exclude BGLAP variants as a major risk factor (OR > 1.5) for T2D in Caucasians, but coding variants in exon 4 may alter glucose homeostasis and diabetes risk in African Americans.     

Human genetics of adiponectin in the metabolic syndrome

Adiponectin, an adipose-derived plasma protein, has been well established to be an important biomarker for metabolic syndrome and its complications after exhausted studies in humans. Animal and cell culture experiments also support most claims from human observations of its roles in the metabolic syndrome. Reproducible results of human genetic studies of diverse ethnic origin and by different investigators may provide the evidence for its causative roles in the pathogenesis of the metabolic syndrome and further insight into the genetic constitutions of the metabolic syndrome. Some of the common polymorphisms in the promoter region, exon and intron 2, and the rare nonsynonymous mutations in exon 3 of the human adiponectin gene were repeatedly shown in many studies from many different ethnic populations to associate with the phenotypes related to body weight, glucose metabolism, insulin sensitivity, and risk of type 2 diabetes mellitus and coronary artery disease. The association of adiponectin genetic variations with dyslipidemia and blood pressure was less explored. The common polymorphisms and rare mutations of the human adiponectin gene itself were demonstrated to associate with differential expression of adiponectin at the plasma protein level and mRNA level in adipose tissue. The PPARγ2 Pro12Ala variants were also shown to influence insulin sensitivity in interaction with adiponectin genotype or to influence plasma adiponectin levels. However, the results were not consistent. Three genome-wide scans for the loci that regulate plasma adiponectin concentration suggest further exploration on chromosomes 5, 9, 14, 15, and 18 is required. These human genetic studies on adiponectin and the metabolic syndrome strongly suggest that adiponectin is one of the causative factors in its pathogenesis and provide significant insights into the genetic makeup of the metabolic syndrome. Extension from these studies may accelerate the discovery of new molecular targets for future therapeutic interventions.     

Identification of single-nucleotide polymorphisms in the human LPIN1 gene

 Because mutations in the murine analog of human LPIN1 cause lipodystrophy in mice, LPIN1 is a candidate gene for human lipodystrophy syndromes. To identify possible disease mutations and/or common single-nucleotide polymorphisms (SNPs), we developed primer pairs to amplify the 21 exons of LPIN1. We used these primer pairs to sequence LPIN1 in lipodystrophy patients who had no mutations in known lipodystrophy genes, and also in normal control subjects. We found no rare LPIN1 coding sequence variants that were exclusive to patients with lipodystrophy. However, we found four silent SNPs, namely, +17C>T in exon 3, 935C>T in exon 5, and 1040G>A and 1079G>C in exon 6, and one nonsynonymous SNP, namely, 2211C>T (P616S) in exon 15. The findings suggest that LPIN1 mutations are not commonly seen in patients with lipodystrophy who had no mutations in known disease genes. However, the identification of amplification primers and SNPs provides tools to further investigate LPIN1 for association with other phenotypes. Received: January 10, 2002 / Accepted: March 15, 2002     

Susceptibility to pre-eclampsia in Finnish women is associated with R485K polymorphism in the factor V gene, not with Leiden mutation

This study determines whether genetic variability in the gene-encoding factor V contributes to differences in pre-eclampsia susceptibility. Allele and genotype frequencies of three single-nucleotide polymorphisms (SNPs) in the factor V gene leading to nonsynonymous changes (M385T in exon 8, and R485K and R506Q (Leiden mutation) in exon 10) were studied in 133 Caucasian women with pre-eclampsia and 112 healthy controls. Single-point analysis was expanded to haplotype analysis, and haplotype frequencies were estimated using an expectation-maximization algorithm. Comparison of single-point allele and genotype distributions of SNPs in exons 8 and 10 of the factor V gene revealed statistically significant differences in R485K allele (P=0.003) and genotype (P=0.03) frequencies between the patients and the control subjects. The A allele of SNP R485K was over-represented among the patients (12%) vs the control subjects (4%), at an odds ratio (OR) of 2.8 (95% confidence interval (CI) 1.2-6.2) for combined A genotypes (GA+AA vs GG). Allele and genotype differences between the patients and control subjects as regards M385T and Leiden mutation were not significant. In haplotype estimation analysis, there was a significantly elevated frequency of haplotype T-A-G encoding the M385-K485-R506 variant in the pre-eclamptic group vs the control group (P=0.01), at an OR of 2.6 (95% CI 1.2-5.5). We conclude that the T-A-G haplotype was more frequent among the patient group than in the control group, and genetic variations in the factor V gene other than the Leiden mutation may play a role in disease susceptibility.     

NPC1: Complete genomic sequence,mutation analysis,and characterization of haplotypes

Niemann‐Pick type C disease (NP‐C) is a rare, autosomal recessive lipid storage disorder. At least 96% of all NP‐C patients link to NPC1 which encodes for a lysosomally‐targeted protein. We describe the complete genomic sequence of 57,052 kb corresponding to the transcribed region of human NPC1 including several exonic and intronic single nucleotide polymorphisms (SNPs). Sequencing of all exons, splice sites, and the promoter region of NPC1 in 12 unrelated Caucasian NP‐C patients revealed nine novel and four known most likely disease‐causing mutations. Ten unique mutations found only once in 24 disease alleles were observed in patients being compound heterozygous for two different mutations. Two of the three missense mutations identified more than once were observed in a total of four patients homozygous for the respective mutation along with homozygosity for the underlying haplotype. The patients were offspring of most likely nonconsanguineous couples. Based upon genotyping exonic SNPs c.2572A>G (I858V; g.45020A>G) and c.2793C>T (N931N; g.45686C>T) and segregation analysis we characterized the haplotype of all 24 NPC1 alleles and of 138 alleles of healthy Caucasian control subjects. All four permutations between the two SNPs were identified in the control alleles: 2572A‐2793C (50%), 2572G‐2793T (41%), 2572G‐2793C (5%), and 2572A‐2793T (4%). These data are suggestive for an ancestral intragenic recombination within a genomic fragment of <666 bp. While 17 of 24 NP‐C alleles (71%) shared haplotype 2572G‐2793T, this haplotype accounted for only 41% in the controls (p=0.007; 2‐sided Fisher exact test) suggesting the possibility of an influence of the haplotypic background on expression of missense mutations in NPC1. Hum Mutat 19:30–38, 2002. © 2001 Wiley‐Liss, Inc.     

HSPD1 is not a major susceptibility gene for rheumatoid arthritis in the French Caucasian population

The heat shock 60-kDa protein 1 (HSP60) is involved in immune and inflammatory reactions, which are hallmarks of rheumatoid arthritis (RA). HSP60 is encoded by the HSPD1 gene located on 2q33, one of the suggested RA susceptibility loci in the French Caucasian population. Our aim was to test whether HSPD1 is a major susceptibility gene by studing families from the French Caucasian population. Three single nucleotide polymorphisms (SNPs) were studied in 100 RA trio families, and 100 other families were used for replication. Genetic analyses were performed by comparing allelic frequencies, by applying the transmission disequilibrium test, and by assessing the genotype relative risk. We observed a significant RA association for the C/C genotype of rs2340690 in the first sample. However, this association was not confirmed when the second sample was added. The two other SNPs and the haplotype analysis did not give any significant results. We conclude that HSPD1 is not a major RA susceptibility gene in the French Caucasian population.     

Mutations in exons 2 and 3 of the FOLR1 gene in demented and non-demented elderly subjects

We have previously reported six novel mutations in the 5'-UTR of the gene for folate receptor-alpha (FOLR1). In our search for additional mutations we screened patients, referred for investigation of suspected dementia (DGM subgroup) by SSCP and DNA sequencing from the end of exon 1 to the first bases of intron 3. We found 4 sequence variations, FOLR1 g.1314G>A, g.1816delC, g.1841G>A, and g.1928C>T. Pyrosequencing genotyping assays were developed for all of them, and 389 active seniors (AS subgroup) and the 202 DGM patients were genotyped for these mutations. The frequency q of the mutated allele was, among the AS subjects, 0.068, 0.0026, 0.0026, and 0.024 respectively, and among the DGM subjects, 0.067, 0.0076, 0.0078, and 0.023. The g.1816delC and g.1841G>A mutations thus were more frequent in the DGM than in the AS subgroup, but the difference did not reach statistical significance. The mutated alleles, FOLR1 1816(-) and 1841A, always occurred together in the same subjects, suggestive of a rare double-mutant haplotype. The two common polymorphisms, FOLR1 g. 1314G>A and g.1928C>T seemed not to raise tHcy plasma levels, whereas the double-mutated g.1816(-)-g.1841A haplotype may possibly have a slight tHcy-raising effect. Thus, so far 8 novel rare FOLR1 mutations with a combined prevalence of approximately 1.3% in Whites as well as two common polymorphisms with 5% and 13%, respectively, have been demonstrated. Only a few of the rare mutations may potentially be associated with raised plasma tHcy concentrations. No association with dementia was found.     

Association study of +45G15G(T/G) and +276(G/T) polymorphisms in the adiponectin gene in patients with polycystic ovary syndrome

The polycystic ovary syndrome (PCOS) is considered as one of the most common endocrine and reproductive dysfunctional diseases. Recent research for genetic variants has identified genetic influences between the polymorphisms of the adiponectin gene and the metabolic syndromes. The aim of our study was to investigate the relationship between single nucleotide polymorphisms (SNPs) in the adiponectin gene and PCOS. Two SNPs, +45G15G(T/G) and +276(G/T), which are found in exon 2 and intron 2, respectively, of the adiponectin gene, were genotyped by PCR-RFLP. Out of 303 women studied for the +45G15G(T/G) and +276(G/T) SNPs, 144 had PCOS and 159 were healthy controls. No association was found between the +45(T/G) SNP and PCOS (P=0.3558, OR=0.83, 95% confidence interval), per contra to the association between +276(G/T) SNP and PCOS (P=0.0126, OR=0.60, 95% confidence interval). These results indicate that the SNP of +276(G/T) is strongly associated with PCOS. However, the +45(T/G) SNP is not associated with PCOS.     

Specific haplotypes of the RET proto-oncogene are over-represented in patients with sporadic papillary thyroid carcinoma

Background: Papillary thyroid carcinoma (PTC), which may be sporadic (95%) or familial (5%), has a prevalence adjusted for age in the general population of 1:100 000. Somatic rearrangements of the RET proto-oncogene are present in up to 66% of sporadic tumours, while they are rarely found in familial cases.

Purpose: In order to determine if some variants of this gene, or a combination of them, might predispose to PTC, we looked for an association of RET haplotype(s) in PTC cases and in controls from four countries matched for sex, age, and population.

Methods: Four single nucleotide polymorphisms (SNPs) across the RET coding sequence were typed and haplotype frequencies were estimated. Genotype and haplotype distributions were compared among these cases and controls.

Results: Ten haplotypes were observed, the seven most frequent of which have been previously described in sporadic Hirschsprung patients and controls. The single locus analyses suggested association of exon 2 and exon 13 SNPs with sporadic PTC. The haplotype analysis showed over-representation of one haplotype in French and Italian sporadic PTC, whereas a different haplotype was significantly under-represented in French familial PTC.

Conclusions: Our data suggest that some variants of RET and some specific haplotypes may act as low penetrance alleles in the predisposition to PTC.