Novel Adiponectin Variants Identified in Type 2 Diabetic Patients Reveal Multimerization and Secretion Defects

ADIPOQ, encoding adiponectin, is a candidate gene for type 2 diabetes (T2D) identified by genome-wide linkage analyses with supporting evidence showing the protein function in sensitizing insulin actions. In an endeavor to characterize candidate genes causing T2D in Thai patients, we identified 10 novel ADIPOQ variations, several of which were non-synonymous variations observed only in the patients. To examine the impact of these non-synonymous variations on adiponectin structure and biochemical characteristics, we conducted a structural analysis of the wild-type and variant proteins by in silico modeling and further characterized biochemical properties of the variants with predicted structural abnormalities from the modeling by molecular and biochemical studies. The recombinant plasmids containing wild-type and variant ADIPOQ cDNAs derived from the variations identified by our study (R55H, R112H, and R131H) and previous work (G90S and R112C) were constructed and transiently expressed and co-expressed in cultured HEK293T cells to investigate their oligomerization, interaction, and secretion. We found that the novel R55H variant impaired protein multimerization but it did not exert the effect over the co-expressed wild-type protein while novel R131H variant impaired protein secretion and also affected the co-expressed wild-type protein in a dominant negative fashion. The R131H variant could traffic from the endoplasmic reticulum to the Golgi, trans-Golgi network, and early endosome but could not be secreted. The R131H variant was likely to be degraded through the lysosomal system and inhibition of its degradation rescued the variant protein from secretion defect. We have shown the possibility of using in silico modeling for predicting the effect of amino acid substitution on adiponectin oligomerization. This is also the first report that demonstrates a dominant negative effect of the R131H variant on protein secretion and the possibility of using protein degradation inhibitors as therapeutic agents in the patients carrying adiponectin variants with secretion defect

The Functional Polymorphism of DDAH2 rs9267551 Is an Independent Determinant of Arterial Stiffness

Background: The association of circulating asymmetric dimethylarginine (ADMA) levels with cardiovascular risk and arterial stiffness has been reportedly demonstrated, although the causal involvement of ADMA in the pathogenesis of these conditions is still debated. Dimethylaminohydrolase 2 (DDAH2) is the enzyme responsible for ADMA hydrolysis in the vasculature, and carriers of the polymorphism rs9267551 C in the 5 '-UTR of DDAH2 have been reported to have higher DDAH2 expression and reduced levels of serum ADMA.Approach and Results: We genotyped rs9267551 in 633 adults of European ancestry and measured their carotid-femoral pulse wave velocity (cfPWV), the gold-standard method to estimate arterial stiffness. cfPWV resulted significantly lower in rs9267551 C allele carriers (Delta = -1.12 m/s, P < 0.01) after correction for age, sex and BMI, and a univariate regression showed that the presence of rs9267551 C variant was negatively associated with cfPWV (beta = -0.110, P < 0.01). In a multivariable regression model, subjects carrying the rs9267551 C allele manifested significantly lower cfPWV than GG carriers (beta = -0.098, P = 0.01) independently from several potential confounders. We measured circulating ADMA levels in a subset of 344 subjects. A mediation analysis revealed that the effect of DDAH2 rs9267551 genotype on cfPWV was mediated by the variation in ADMA levels.Conclusions: These evidences hint that the presence of rs9267551 C allele may explain, at least in part, a reduction in vessel rigidity as measured by cfPWV, and support the attribution of a causative role to ADMA in the pathogenesis of arterial stiffness

Association between Human Prothrombin Variant (T165M) and Kidney Stone Disease

<div><p>We previously reported the association between <em>prothrombin</em> (<em>F2</em>), encoding a stone inhibitor protein - urinary prothrombin fragment 1 (UPTF1), and the risk of kidney stone disease in Northeastern Thai patients. To identify specific <em>F2</em> variation responsible for the kidney stone risk, we conducted sequencing analysis of this gene in a group of the patients with kidney stone disease. Five intronic SNPs (rs2070850, rs2070852, rs1799867, rs2282687, and rs3136516) and one exonic non-synonymous single nucleotide polymorphism (nsSNP; rs5896) were found. The five intronic SNPs have no functional change as predicted by computer programs while the nsSNP rs5896 (c.494 C>T) located in exon 6 results in a substitution of threonine (T) by methionine (M) at the position 165 (T165M). The nsSNP rs5896 was subsequently genotyped in 209 patients and 216 control subjects. Genotypic and allelic frequencies of this nsSNP were analyzed for their association with kidney stone disease. The frequency of CC genotype of rs5896 was significantly lower in the patient group (13.4%) than that in the control group (22.2%) (<em>P = </em>0.017, OR 0.54, 95% CI 0.32–0.90), and the frequency of C allele was significantly lower in the patient group (36.1%) than that in the control group (45.6%) (<em>P = </em>0.005, OR 0.68, 95% CI 0.51–0.89). The significant differences of genotype and allele frequencies were maintained only in the female group (<em>P = </em>0.033 and 0.003, respectively). The effect of amino-acid change on UPTF1 structure was also examined by homologous modeling and <em>in silico</em> mutagenesis. T165 is conserved and T165M substitution will affect hydrogen bond formation with E180. In conclusion, our results indicate that prothrombin variant (T165M) is associated with kidney stone risk in the Northeastern Thai female patients.</p> </div

<i>F2</i> gene structure, SNP rs5896, alignment of prothrombin amino acid sequences, and 3D structure of UPTF1.

<p>A: Gene structure of <i>F2</i>. Exons 1–14 and intervening sequences (introns) are indicated by boxes and line, respectively. Fragment numbers 1 to 12 represent expected PCR products. B: DNA sequencing profile showing a SNP (rs5896, c.494 C>T) in exon 6, resulting in a substitution of threonine (T) by methionine (M) at position 165 (T165M). Vertical arrows indicate the positions of nucleotide variations. SNP genotypes are indicated by bold capital letters above the vertical arrows. C: Multiple alignment of amino acid sequences in a highly conserved region of prothrombin (F2), residues 152–185 (human sequence numbering) from eleven species. The symbols “*” and “:” under the alignment indicate the positions with conserved and conservative-changed amino acids, respectively. The T165 position is indicated by an arrow. D: Three dimensional (3D) structure of urinary prothrombin fragment 1 (UPTF1), showing an amino acid alteration, T165M. Wild-type T165 is indicated as green, which is superimposed by the altered amino acid, M165, indicated as gray. Wild-type residue is shown as green letters while the variant residue is presented as black letters. The dash line implies the predicted H-bond between T165 and E180 residues. The oxygen and sulfur atoms are shown with red and yellow, respectively.</p

Genotype and allele frequencies of SNP rs5896 in <i>F2</i> in male group (77 patients with kidney stone disease and 90 control subjects).

<p>CI = confidence interval; OR = odd ratio.</p

Genotype and allele frequencies of SNP rs5896 in <i>F2</i> in female group (132 patients with kidney stone disease and 126 control subjects).

<p>CI = confidence interval; OR = odd ratio.</p

Analysis of rs5896 (c.494 C>T) by polymerase chain reaction - restriction fragment length polymorohism (PCR-RFLP).

<p>A: Locations of SNP rs5896 and restriction sites of <i>Nco</i>I on amplified DNA fragment (733 bp), containing exons 5 and 6. The expected RFLP patterns for genotyping of SNP rs5896 are indicated as lines with the lengths of 426, 140, 93 and 74 bp for allele T, and 426, 233 and 74 bp for allele C. B: <i>Nco</i>I-digested DNA patterns separated by electrophoresis on 3% agarose: lane 1, 100-bp ladder markers; lane 2, undigested PCR product (733 bp); lane 3, homozygote TT (426, 140, 93 and 74 bp); lane 4, heterozygote CT (426, 233, 140, 93 and 74 bp), and lane 5, homozygote CC (426, 233 and 74 bp).</p

Genotype and allele frequencies of SNP rs5896 in <i>F2</i> in 209 patients with kidney stone disease and 216 control subjects.

<p>CI = confidence interval; OR = odd ratio.</p

Gain of function of Malate Dehydrogenase 2 (MDH2) and familial hyperglycemia

Objective: We set out to identify the genetic cause of hyperglycemia in multigenerational families with an apparent autosomal dominant form of adult-onset diabetes not due to mutations in known monogenic diabetes genes. Methods: Existing Whole Exome Sequencing (WES) data were used to identify exonic variants segregating with diabetes in 60 families from the US and Italy. Functional studies were carried out in vitro (transfected MIN6-K8 cells) and in vivo (Caenorhabditis elegans) to assess the diabetogenic potential of two variants in the Malate Dehydrogenase 2 (MDH2) gene linked with hyperglycemia in two of the families. Results: A very rare mutation (p.Arg52Cys) in MDH2 strongly segregated with hyperglycemia in one family from the US. An infrequent MDH2 missense variant (p.Val160Met) also showed disease co-segregation in a family from Italy, although with reduced penetrance. In silico, both Arg52Cys and Val160Met were shown to affect MDH2 protein structure and function. In transfected HepG2 cells, both variants significantly increased MDH2 enzymatic activity, thereby decreasing the NAD+/NADH ratio - a change known to affect insulin signaling and secretion. Stable expression of human wild type MDH2 in MIN6-K8 cell lines enhanced glucose- and GLP-1-stimulated insulin secretion. This effect was blunted by the Cys52 or Met160 substitutions. Nematodes carrying equivalent changes at the orthologous positions of the mdh-2 gene showed impaired glucose-stimulated insulin secretion. Conclusions: Our findings suggest a central role of MDH2 in human glucose homeostasis and indicate that gain of function variants in this gene may be involved in the etiology of familial forms of diabetes