Monogenic diabetes characteristics in a transnational multicenter study from Mediterranean countries.

BACKGROUND: Diagnosis of monogenic diabetes has important clinical implications for treatment and health expenditure. However, its prevalence remains to be specified in many countries, particularly from South Europe, North Africa and Middle-East, where non-autoimmune diabetes in young adults is increasing dramatically. AIMS: To identify cases of monogenic diabetes in young adults from Mediterranean countries and assess the specificities between countries. METHODS: We conducted a transnational multicenter study based on exome sequencing in 204 unrelated patients with diabetes (age-at-diagnosis: 26.1 ± 9.1 years). Rare coding variants in 35 targeted genes were evaluated for pathogenicity. Data were analyzed using one-way ANOVA, chi-squared test and factor analysis of mixed data. RESULTS: Forty pathogenic or likely pathogenic variants, 14 of which novel, were identified in 36 patients yielding a genetic diagnosis rate of 17.6%. The majority of cases were due to GCK, HNF1A, ABCC8 and HNF4A variants. We observed highly variable diagnosis rates according to countries, with association to genetic ancestry. Lower body mass index and HbA1c at study inclusion, and less frequent insulin treatment were hallmarks of pathogenic variant carriers. Treatment changes following genetic diagnosis have been made in several patients. CONCLUSIONS: Our data from patients in several Mediterranean countries highlight a broad clinical and genetic spectrum of diabetes, showing the relevance of wide genetic testing for personalized care of early-onset diabetes.

Genetic Causes of Severe Childhood Obesity: A Remarkably High Prevalence in an Inbred Population of Pakistan.

Monogenic forms of obesity have been identified in ≤10% of severely obese European patients. However, the overall spectrum of deleterious variants (point mutations and structural variants) responsible for childhood severe obesity remains elusive. In this study, we genetically screened 225 severely obese children from consanguineous Pakistani families through a combination of techniques, including an in-house-developed augmented whole-exome sequencing method (CoDE-seq) that enables simultaneous detection of whole-exome copy number variations (CNVs) and point mutations in coding regions. We identified 110 (49%) probands carrying 55 different pathogenic point mutations and CNVs in 13 genes/loci responsible for nonsyndromic and syndromic monofactorial obesity. CoDE-seq also identified 28 rare or novel CNVs associated with intellectual disability in 22 additional obese subjects (10%). Additionally, we highlight variants in candidate genes for obesity warranting further investigation. Altogether, 59% of cases in the studied cohort are likely to have a discrete genetic cause, with 13% of these as a result of CNVs, demonstrating a remarkably higher prevalence of monofactorial obesity than hitherto reported and a plausible overlapping of obesity and intellectual disabilities in several cases. Finally, inbred populations with a high prevalence of obesity provide unique, genetically enriched material in the quest of new genes/variants influencing energy balance.

Pathogenic variants in actionable MODY genes are associated with type 2 diabetes.

Genome-wide association studies have identified 240 independent loci associated with type 2 diabetes (T2D) risk, but this knowledge has not advanced precision medicine. In contrast, the genetic diagnosis of monogenic forms of diabetes (including maturity-onset diabetes of the young (MODY)) are textbook cases of genomic medicine. Recent studies trying to bridge the gap between monogenic diabetes and T2D have been inconclusive. Here, we show a significant burden of pathogenic variants in genes linked with monogenic diabetes among people with common T2D, particularly in actionable MODY genes, thus implying that there should be a substantial change in care for carriers with T2D. We show that, among 74,629 individuals, this burden is probably driven by the pathogenic variants found in GCK, and to a lesser extent in HNF4A, KCNJ11, HNF1B and ABCC8. The carriers with T2D are leaner, which evidences a functional metabolic effect of these mutations. Pathogenic variants in actionable MODY genes are more frequent than was previously expected in common T2D. These results open avenues for future interventions assessing the clinical interest of these pathogenic mutations in precision medicine.

Analysis of novel risk loci for type 2 diabetes in a general French population: the D.E.S.I.R. study.

Recently, Genome Wide Association (GWA) studies identified novel single nucleotide polymorphisms (SNPs), highly associated with type 2 diabetes (T2D) in several case-control studies of European descent. However, the impact of these markers on glucose homeostasis in a population-based study remains to be clarified. The French prospective D.E.S.I.R. study (N = 4,707) was genotyped for 22 polymorphisms within 14 loci showing nominal to strong association with T2D in recently published GWA analyses (CDKAL1, IGFBP2, CDKN2A/2B, EXT2, HHEX, LOC646279, SLC30A8, MMP26, KCTD12, LDLR, CAMTA1, LOC38776, NGN3 and CXCR4). We assessed their effects on quantitative traits related to glucose homeostasis in 4,283 normoglycemic middle-aged participants at baseline and their contribution to T2D incidence during 9 years of follow-up. Individuals carrying T2D risk alleles of CDKAL1 or SLC30A8 had lower fasting plasma insulin level (rs7756992 P = 0.003) or lower basal insulin secretion (rs13266634 P = 0.0005), respectively, than non-carriers. Furthermore, NGN3 and MMP26 risk alleles associated with higher fasting plasma glucose levels (rs10823406 P = 0.01 and rs2499953 P = 0.04, respectively). However, for these SNPs, only modest associations were found with a higher incidence of T2D: hazard ratios of 2.03 [1.00-4.11] for MMP26 (rs2499953 P = 0.05) and 1.33 [1.02-1.73] for NGN3 (rs10823406 P = 0.03). We confirmed deleterious effects of SLC30A8, CDKAL1, NGN3 and MMP26 risk alleles on glucose homeostasis in the D.E.S.I.R. prospective cohort. However, in contrast to TCF7L2, the contribution of novel loci to T2D incidence seems only modest in the general middle-aged French population and should be replicated in larger cohorts.

Contribution of rare coding mutations in CD36 to type 2 diabetes and cardio-metabolic complications.

We sequenced coding regions of the cluster of differentiation 36 (CD36) gene in 184 French individuals of European ancestry presenting simultaneously with type 2 diabetes (T2D), arterial hypertension, dyslipidemia, and coronary heart disease. We identified rare missense mutations (p.Pro191Leu/rs143150225 and p.Ala252Val/rs147624636) in two heterozygous cases. The two CD36 mutation carriers had no family history of T2D and no clustering of cardio-metabolic complications. While the p.Pro191Leu mutation was found in 84 heterozygous carriers from five ethnic groups from the genome aggregation database (global frequency: 0.0297%, N = 141,321), only one European carrier of the p.Ala252Val mutation was identified (global frequency: 0.00040%, N = 125,523). The Pro191 and Ala252 amino acids were not conserved (74.8% and 68.9% across 131 animal species, respectively). In vitro experiments showed that the two CD36 mutant proteins are expressed and trafficked to the plasma membrane where they bind modified low-density-lipoprotein (LDL) cholesterol as normal. However, molecular modelling of the recent CD36 crystal structure showed that Pro191 was located at the exit/entrance gate of the lipid binding chamber and Ala252 was in line with the chamber. Overall, our data do not support a major contribution of CD36 rare coding mutations to T2D and its cardio-metabolic complications in the French population.

Loss-of-function mutations in MRAP2 are pathogenic in hyperphagic obesity with hyperglycemia and hypertension.

The G-protein-coupled receptor accessory protein MRAP2 is implicated in energy control in rodents, notably via the melanocortin-4 receptor1. Although some MRAP2 mutations have been described in people with obesity1-3, their functional consequences on adiposity remain elusive. Using large-scale sequencing of MRAP2 in 9,418 people, we identified 23 rare heterozygous variants associated with increased obesity risk in both adults and children. Functional assessment of each variant shows that loss-of-function MRAP2 variants are pathogenic for monogenic hyperphagic obesity, hyperglycemia and hypertension. This contrasts with other monogenic forms of obesity characterized by excessive hunger, including melanocortin-4 receptor deficiency, that present with low blood pressure and normal glucose tolerance4. The pleiotropic metabolic effect of loss-of-function mutations in MRAP2 might be due to the failure of different MRAP2-regulated G-protein-coupled receptors in various tissues including pancreatic islets.

CoDE-seq, an augmented whole-exome sequencing, enables the accurate detection of CNVs and mutations in Mendelian obesity and intellectual disability.

OBJECTIVE: The molecular diagnosis of extreme forms of obesity, in which accurate detection of both copy number variations (CNVs) and point mutations, is crucial for an optimal care of the patients and genetic counseling for their families. Whole-exome sequencing (WES) has benefited considerably this molecular diagnosis, but its poor ability to detect CNVs remains a major limitation. We aimed to develop a method (CoDE-seq) enabling the accurate detection of both CNVs and point mutations in one step. METHODS: CoDE-seq is based on an augmented WES method, using probes distributed uniformly throughout the genome. CoDE-seq was validated in 40 patients for whom chromosomal DNA microarray was available. CNVs and mutations were assessed in 82 children/young adults with suspected Mendelian obesity and/or intellectual disability and in their parents when available (ntotal = 145). RESULTS: CoDE-seq not only detected all of the 97 CNVs identified by chromosomal DNA microarrays but also found 84 additional CNVs, due to a better resolution. When compared to CoDE-seq and chromosomal DNA microarrays, WES failed to detect 37% and 14% of CNVs, respectively. In the 82 patients, a likely molecular diagnosis was achieved in >30% of the patients. Half of the genetic diagnoses were explained by CNVs while the other half by mutations. CONCLUSIONS: CoDE-seq has proven cost-efficient and highly effective as it avoids the sequential genetic screening approaches currently used in clinical practice for the accurate detection of CNVs and point mutations.

TCF7L2 rs7903146 variant does not associate with smallness for gestational age in the French population.

BACKGROUND: In adults, the TCF7L2 rs7903146 T allele, commonly associated with type 2 diabetes (T2D), has been also associated with a lower body mass index (BMI) in T2D individuals and with a smaller waist circumference in subjects with impaired glucose tolerance. METHODS: The present association study aimed at analyzing the contribution of the rs7903146 SNP to smallness for gestational age (SGA) and metabolic profiles in subjects with SGA or appropriate for gestational age birth weight (AGA). Two groups of French Caucasian subjects were selected on birth data: SGA (birth weight < 10th percentile; n = 764), and AGA (25th < birth weight < 75th percentile; n = 627). Family-based association tests were also performed in 3,012 subjects from 628 SGA and AGA pedigrees. RESULTS: The rs7903146 genotypic distributions between AGA (30.7%) and SGA (29.0%) were not statistically different (allelic OR = 0.92 [0.78-1.09], p = 0.34). Family association-based studies did not show a distortion of T allele transmission in SGA subjects (p = 0.52). No significant effect of the T allele was detected on any of the metabolic parameters in the SGA group. However, in the AGA group, trends towards a lower insulin secretion (p = 0.03) and a higher fasting glycaemia (p = 0.002) were detected in carriers of the T allele. CONCLUSION: The TCF7L2 rs7903146 variant neither increases the risk for SGA nor modulates birth weight and young adulthood glucose homeostasis in French Caucasian subjects born with SGA.

Low-frequency variants in HMGA1 are not associated with type 2 diabetes risk.

It has recently been suggested that the low-frequency c.136-14_136-13insC variant in high-mobility group A1 (HMGA1) may strongly contribute to insulin resistance and type 2 diabetes risk. In our study, we attempted to confirm that HMGA1 is a novel type 2 diabetes locus in French Caucasians. The gene was sequenced in 368 type 2 diabetic case subjects with a family history of type 2 diabetes and 372 normoglycemic control subjects without a family history of type 2 diabetes. None of the 41 genetic variations identified were associated with type 2 diabetes. The lack of association between the c.136-14_136-13insC variant and type 2 diabetes was confirmed in an independent French group of 4,538 case subjects and 4,015 control subjects and in a large meta-analysis of 16,605 case subjects and 46,179 control subjects. Finally, this variant had no effects on metabolic traits and was not involved in variations of HMGA1 and insulin receptor (INSR) expressions. The c.136-14_136-13insC variant was not associated with type 2 diabetes in individuals of European descent. Our study emphasizes the need to analyze a large number of subjects to reliably assess the association of low-frequency variants with the disease.

Estimation of newborn risk for child or adolescent obesity: lessons from longitudinal birth cohorts.

OBJECTIVES: Prevention of obesity should start as early as possible after birth. We aimed to build clinically useful equations estimating the risk of later obesity in newborns, as a first step towards focused early prevention against the global obesity epidemic. METHODS: We analyzed the lifetime Northern Finland Birth Cohort 1986 (NFBC1986) (N = 4,032) to draw predictive equations for childhood and adolescent obesity from traditional risk factors (parental BMI, birth weight, maternal gestational weight gain, behaviour and social indicators), and a genetic score built from 39 BMI/obesity-associated polymorphisms. We performed validation analyses in a retrospective cohort of 1,503 Italian children and in a prospective cohort of 1,032 U.S. children. RESULTS: In the NFBC1986, the cumulative accuracy of traditional risk factors predicting childhood obesity, adolescent obesity, and childhood obesity persistent into adolescence was good: AUROC = 0·78[0·74-0.82], 0·75[0·71-0·79] and 0·85[0·80-0·90] respectively (all p<0·001). Adding the genetic score produced discrimination improvements ≤1%. The NFBC1986 equation for childhood obesity remained acceptably accurate when applied to the Italian and the U.S. cohort (AUROC = 0·70[0·63-0·77] and 0·73[0·67-0·80] respectively) and the two additional equations for childhood obesity newly drawn from the Italian and the U.S. datasets showed good accuracy in respective cohorts (AUROC = 0·74[0·69-0·79] and 0·79[0·73-0·84]) (all p<0·001). The three equations for childhood obesity were converted into simple Excel risk calculators for potential clinical use. CONCLUSION: This study provides the first example of handy tools for predicting childhood obesity in newborns by means of easily recorded information, while it shows that currently known genetic variants have very little usefulness for such prediction.

Genome-wide association study for early-onset and morbid adult obesity identifies three new risk loci in European populations.

We analyzed genome-wide association data from 1,380 Europeans with early-onset and morbid adult obesity and 1,416 age-matched normal-weight controls. Thirty-eight markers showing strong association were further evaluated in 14,186 European subjects. In addition to FTO and MC4R, we detected significant association of obesity with three new risk loci in NPC1 (endosomal/lysosomal Niemann-Pick C1 gene, P = 2.9 x 10(-7)), near MAF (encoding the transcription factor c-MAF, P = 3.8 x 10(-13)) and near PTER (phosphotriesterase-related gene, P = 2.1 x 10(-7)).

Post genome-wide association studies of novel genes associated with type 2 diabetes show gene-gene interaction and high predictive value.

BACKGROUND: Recently, several Genome Wide Association (GWA) studies in populations of European descent have identified and validated novel single nucleotide polymorphisms (SNPs), highly associated with type 2 diabetes (T2D). Our aims were to validate these markers in other European and non-European populations, then to assess their combined effect in a large French study comparing T2D and normal glucose tolerant (NGT) individuals. METHODOLOGY/PRINCIPAL FINDINGS: In the same French population analyzed in our previous GWA study (3,295 T2D and 3,595 NGT), strong associations with T2D were found for CDKAL1 (OR(rs7756992) = 1.30[1.19-1.42], P = 2.3x10(-9)), CDKN2A/2B (OR(rs10811661) = 0.74[0.66-0.82], P = 3.5x10(-8)) and more modestly for IGFBP2 (OR(rs1470579) = 1.17[1.07-1.27], P = 0.0003) SNPs. These results were replicated in both Israeli Ashkenazi (577 T2D and 552 NGT) and Austrian (504 T2D and 753 NGT) populations (except for CDKAL1) but not in the Moroccan population (521 T2D and 423 NGT). In the overall group of French subjects (4,232 T2D and 4,595 NGT), IGFBP2 and CXCR4 synergistically interacted with (LOC38776, SLC30A8, HHEX) and (NGN3, CDKN2A/2B), respectively, encoding for proteins presumably regulating pancreatic endocrine cell development and function. The T2D risk increased strongly when risk alleles, including the previously discovered T2D-associated TCF7L2 rs7903146 SNP, were combined (8.68-fold for the 14% of French individuals carrying 18 to 30 risk alleles with an allelic OR of 1.24). With an area under the ROC curve of 0.86, only 15 novel loci were necessary to discriminate French individuals susceptible to develop T2D. CONCLUSIONS/SIGNIFICANCE: In addition to TCF7L2, SLC30A8 and HHEX, initially identified by the French GWA scan, CDKAL1, IGFBP2 and CDKN2A/2B strongly associate with T2D in French individuals, and mostly in populations of Central European descent but not in Moroccan subjects. Genes expressed in the pancreas interact together and their combined effect dramatically increases the risk for T2D, opening avenues for the development of genetic prediction tests.