Randomized, double-blind, placebo-controlled dose-finding study of the dipeptidyl peptidase-4 inhibitor linagliptin in pediatric patients with type 2 diabetes.

OBJECTIVE: To identify the dose of the dipeptidyl peptidase-4 (DPP-4) inhibitor linagliptin in pediatric patients with type 2 diabetes (T2D). METHODS: Double-blind, randomized, controlled parallel group study comparing linagliptin 1 and 5 mg once daily, with placebo in 39 patients with T2D aged 10 to below 18 years. The primary efficacy endpoint was the change from baseline in glycated hemoglobin (HbA1c) after 12 weeks of treatment. The key pharmacodynamic endpoint was DPP-4 inhibition during steady-state. RESULTS: Compared to placebo, there was a dose-dependent reduction in mean HbA1c of 0.48% and 0.63% with linagliptin 1 and 5 mg, respectively, associated with corresponding declines in mean fasting plasma glucose (FPG) of 5.6 and 34.2 mg/dL. Median DPP-4 inhibition was 38% with linagliptin 1 mg and 79% with linagliptin 5 mg. Geometric mean trough levels of linagliptin were 3.80 and 7.42 nmol/L in the 1 and 5 mg groups, respectively; levels that were slightly higher than in adult patients with T2D that were most likely caused by higher plasma DPP-4 concentrations in the study population. There were no drug-related adverse events during treatment with either dose of linagliptin. CONCLUSIONS: Linagliptin was well tolerated and induced dose-dependent DPP-4 inhibition that was accompanied by corresponding reductions in HbA1c and FPG levels in young people with T2D. The results are consistent with the clinical efficacy and safety profile that have been reported for linagliptin in adult patients with T2D, favoring linagliptin 5 mg over 1 mg.

Relationship between salivary/pancreatic amylase and body mass index: a systems biology approach.

BACKGROUND: Salivary (AMY1) and pancreatic (AMY2) amylases hydrolyze starch. Copy number of AMY1A (encoding AMY1) was reported to be higher in populations with a high-starch diet and reduced in obese people. These results based on quantitative PCR have been challenged recently. We aimed to re-assess the relationship between amylase and adiposity using a systems biology approach. METHODS: We assessed the association between plasma enzymatic activity of AMY1 or AMY2, and several metabolic traits in almost 4000 French individuals from D.E.S.I.R. longitudinal study. The effect of the number of copies of AMY1A (encoding AMY1) or AMY2A (encoding AMY2) measured through droplet digital PCR was then analyzed on the same parameters in the same study. A Mendelian randomization analysis was also performed. We subsequently assessed the association between AMY1A copy number and obesity risk in two case-control studies (5000 samples in total). Finally, we assessed the association between body mass index (BMI)-related plasma metabolites and AMY1 or AMY2 activity. RESULTS: We evidenced strong associations between AMY1 or AMY2 activity and lower BMI. However, we found a modest contribution of AMY1A copy number to lower BMI. Mendelian randomization identified a causal negative effect of BMI on AMY1 and AMY2 activities. Yet, we also found a significant negative contribution of AMY1 activity at baseline to the change in BMI during the 9-year follow-up, and a significant contribution of AMY1A copy number to lower obesity risk in children, suggesting a bidirectional relationship between AMY1 activity and adiposity. Metabonomics identified a BMI-independent association between AMY1 activity and lactate, a product of complex carbohydrate fermentation. CONCLUSIONS: These findings provide new insights into the involvement of amylase in adiposity and starch metabolism.

Rfx6 directs islet formation and insulin production in mice and humans.

Insulin from the beta-cells of the pancreatic islets of Langerhans controls energy homeostasis in vertebrates, and its deficiency causes diabetes mellitus. During embryonic development, the transcription factor neurogenin 3 (Neurog3) initiates the differentiation of the beta-cells and other islet cell types from pancreatic endoderm, but the genetic program that subsequently completes this differentiation remains incompletely understood. Here we show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurog3. Mice lacking Rfx6 failed to generate any of the normal islet cell types except for pancreatic-polypeptide-producing cells. In human infants with a similar autosomal recessive syndrome of neonatal diabetes, genetic mapping and subsequent sequencing identified mutations in the human RFX6 gene. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors that coordinate pancreatic islet development in both mice and humans. Rfx6 could prove useful in efforts to generate beta-cells for patients with diabetes.

Contribution of the low-frequency, loss-of-function p.R270H mutation in FFAR4 (GPR120) to increased fasting plasma glucose levels.

BACKGROUND: We previously reported that the low-frequency, loss-of-function variant p.R270H in FFAR4 encoding the lipid sensor GPR120 was associated with obesity. Gpr120-deficient mice develop obesity and both impaired fasting glucose and glucose intolerance under a high-fat diet. We aimed to assess the contribution of p.R270H to type 2 diabetes (T2D) risk and the variation of glucose-related traits. METHODS: We genotyped p.R270H in 8996 non-diabetic individuals (among whom 4523 had an oral glucose tolerance test (OGTT)) and in a T2D case-control study including 4725 cases and 4339 controls. The regression models were adjusted for age, sex and body mass index (BMI). RESULTS: We found a significant association between p.R270H and increased fasting glucose levels (ß=0.092±0.05 mmol/L; p=4.13×10(-4)). Furthermore, p.R270H nominally contributed to decreased homeostasis model of pancreatic ß-cell function (HOMA-B; ß=-0.090±0.06; p=6.01×10(-3)). Despite a high statistical power, we did not find any significant association between p.R270H and T2D risk or the variation of fasting insulin levels, the homeostasis model of insulin resistance or OGTT-derived indices. CONCLUSIONS: These results suggest that the low-frequency p.R270H variant which inhibits GPR120 activity might influence fasting glucose levels in a normal physiological range. This study does not exclude that other coding mutations in FFAR4 with stronger functional effect than p.R270H may be associated with T2D.

Variation in FTO contributes to childhood obesity and severe adult obesity.

We identified a set of SNPs in the first intron of the FTO (fat mass and obesity associated) gene on chromosome 16q12.2 that is consistently strongly associated with early-onset and severe obesity in both adults and children of European ancestry with an experiment-wise P value of 1.67 x 10(-26) in 2,900 affected individuals and 5,100 controls. The at-risk haplotype yields a proportion of attributable risk of 22% for common obesity. We conclude that FTO contributes to human obesity and hence may be a target for subsequent functional analyses.

Monocytes of Patients with Type 1 Diabetes Harbour Enterovirus RNA.

BACKGROUND: Intracellular enterovirus (EV) RNA was detected in blood of patients with type 1 diabetes (T1D). The presence of EV RNA in subsets of peripheral blood mononuclear cells (PBMCs) of patients, and the in vitro infection of these cells with an EV, was investigated. MATERIALS AND METHODS: Blood was collected from 42 patients with T1D, PBMCs were isolated and monocytes were purified. Interferon alpha (IFNα) mRNA and EV RNA were investigated using RT-PCR. Levels of IFNα in plasma were measured using an immunoassay. Cells were inoculated with Coxsackievirus B4 (CBV4) in vitro, and infection was assessed by indirect immunofluorescence (IFI). RESULTS: Interferon alpha mRNA was detected in blood and in monocytes of 12 of 42 patients with T1D, but not in monocyte-depleted PBMCs of the same individuals. Significant plasma levels of IFNα (≥ 5 IU/mL) were found in six patients. EV RNA was detected in whole blood and in monocytes of seven patients and negative-strand EV RNA was found in monocytes of 6 of them. When monocytes of patients with IFNα and/or EV RNA in their blood were inoculated with CVB4, the proportion of cells stained by an anti-VP1 antibody was 8.8 ± 1%, whereas no VP1 was detected in the monocytes of IFNα, EV RNA negative patients. Nevertheless, when CBV4 was mixed with plasma, VP1 was detected in monocytes of all patients with T1D (staining ranging from 12 to 36%). CONCLUSIONS: Our data indicate that monocytes of patients with T1D can harbor EV RNA and IFNα mRNA and can be infected with an EV in vitro.

Variants of ENPP1 are associated with childhood and adult obesity and increase the risk of glucose intolerance and type 2 diabetes.

We identified a locus on chromosome 6q16.3-q24.2 (ref. 1) associated with childhood obesity that includes 2.4 Mb common to eight genome scans for type 2 diabetes (T2D) or obesity. Analysis of the gene ENPP1 (also called PC-1), a candidate for insulin resistance, in 6,147 subjects showed association between a three-allele risk haplotype (K121Q, IVS20delT-11 and A-->G+1044TGA; QdelTG) and childhood obesity (odds ratio (OR) = 1.69, P = 0.0006), morbid or moderate obesity in adults (OR = 1.50, P = 0.006 or OR = 1.37, P = 0.02, respectively) and T2D (OR = 1.56, P = 0.00002). The Genotype IBD Sharing Test suggested that this obesity-associated ENPP1 risk haplotype contributes to the observed chromosome 6q linkage with childhood obesity. The haplotype confers a higher risk of glucose intolerance and T2D to obese children and their parents and associates with increased serum levels of soluble ENPP1 protein in children. Expression of a long ENPP1 mRNA isoform, which includes the obesity-associated A-->G+1044TGA SNP, was specific for pancreatic islet beta cells, adipocytes and liver. These findings suggest that several variants of ENPP1 have a primary role in mediating insulin resistance and in the development of both obesity and T2D, suggesting that an underlying molecular mechanism is common to both conditions.

Two new Loci for body-weight regulation identified in a joint analysis of genome-wide association studies for early-onset extreme obesity in French and german study groups.

Meta-analyses of population-based genome-wide association studies (GWAS) in adults have recently led to the detection of new genetic loci for obesity. Here we aimed to discover additional obesity loci in extremely obese children and adolescents. We also investigated if these results generalize by estimating the effects of these obesity loci in adults and in population-based samples including both children and adults. We jointly analysed two GWAS of 2,258 individuals and followed-up the best, according to lowest p-values, 44 single nucleotide polymorphisms (SNP) from 21 genomic regions in 3,141 individuals. After this DISCOVERY step, we explored if the findings derived from the extremely obese children and adolescents (10 SNPs from 5 genomic regions) generalized to (i) the population level and (ii) to adults by genotyping another 31,182 individuals (GENERALIZATION step). Apart from previously identified FTO, MC4R, and TMEM18, we detected two new loci for obesity: one in SDCCAG8 (serologically defined colon cancer antigen 8 gene; p = 1.85x10(-8) in the DISCOVERY step) and one between TNKS (tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase gene) and MSRA (methionine sulfoxide reductase A gene; p = 4.84x10(-7)), the latter finding being limited to children and adolescents as demonstrated in the GENERALIZATION step. The odds ratios for early-onset obesity were estimated at approximately 1.10 per risk allele for both loci. Interestingly, the TNKS/MSRA locus has recently been found to be associated with adult waist circumference. In summary, we have completed a meta-analysis of two GWAS which both focus on extremely obese children and adolescents and replicated our findings in a large followed-up data set. We observed that genetic variants in or near FTO, MC4R, TMEM18, SDCCAG8, and TNKS/MSRA were robustly associated with early-onset obesity. We conclude that the currently known major common variants related to obesity overlap to a substantial degree between children and adults.

The p.Pro2232Leu variant in the ChEL domain of thyroglobulin gene causes intracellular transport disorder and congenital hypothyroidism.

Thyroglobulin (TG), the predominant glycoprotein of the thyroid gland, functions as matrix protein in thyroid hormonegenesis. TG deficiency results in thyroid dyshormonogenesis. These variants produce a heterogeneous spectrum of congenital goitre, with an autosomal recessive mode of inheritance. The purpose of this study was to identify and functionally characterize new variants in the TG gene in order to increase the understanding of the molecular mechanisms responsible for thyroid dyshormonogenesis. A total of four patients from two non-consanguineous families with marked alteration of TG synthesis were studied. The two families were previously analysed in our laboratory, only one deleterious allele, in each one, was detected after sequencing the TG gene (c.2359 C > T [p.Arg787*], c.5560 G > T [p.Glu1854*]). These findings were confirmed in the present studies by Next-Generation Sequencing. The single nucleotide coding variants of the TG gene were then analyzed to predict the possible variant causing the disease. The p.Pro2232Leu (c.6695 C > T), identified in both families, showing a low frequency population in gnomAD v2.1.1 database and protein homology, amino acid prediction, and 3D modeling analysis predict a potential pathogenic effect of this variant. We also transiently express p.Pro2232Leu in a full-length rat TG cDNA clone and confirmed that this point variant was sufficient to cause intracellular retention of mutant TG in HEK293T cells. Consequently, each family carried a compound heterozygous for p.Arg787*/p.Pro2232Leu or p.Glu1854*/p.Pro2232Leu variants. In conclusion, our results confirm the pathophysiological importance of altered TG folding as a consequence of missense variants located in the ChEL domain of TG.

Barriers to Physical Activity in Children and Adults Living With Type 1 Diabetes: A Complex Link With Real-life Glycemic Excursions.

OBJECTIVES: Ever since the first research on barriers to physical activity (PA) highlighting fear of hypoglycemia as a major barrier, many studies have attempted to understand their demographic and behavioural determinants. However, no research has been conducted on whether these perceived barriers toward PA are based on real-life-experienced adverse glycemic effects of exercise. METHODS: Sixty-two adults and 53 children/adolescents living with type 1 diabetes, along with their parents, completed the Barriers to Physical Activity in Type 1 Diabetes-1 (BAPAD-1) questionnaire on barriers to PA. Continuous glucose-monitoring data were collected during 1 week of everyday life for 26 adults and 33 children/adolescents. Multiple linear regressions were used to explore links between BAPAD-1 scores and glycemic excursions experienced during and after everyday-life self-reported PA sessions, controlling for behavioural (accelerometry) and demographic confounders. RESULTS: In children/adolescents, the more time spent in hypoglycemia on nights after PA sessions, the more they reported hypoglycemic risk as a barrier (ß=+0.365, p=0.034). Conversely, in adults, the higher the proportion of PA sessions accompanied by a drop in blood glucose, the less hypoglycemia was a barrier (ß=-0.046, p=0.004). In parents, BAPAD-1 scores were unrelated to children/adolescents' everyday-life exercise-induced hypo/hyperglycemia. CONCLUSIONS: In children/adolescents, fear of hypoglycemia was predominant in those exposed to nocturnal hypoglycemia associated with PA sessions. In adults, fewer barriers may mean they accept a bigger drop in their glycemia during PA. This shows the importance of finding and promoting age-specific solutions to prevent exercise-induced hypoglycemia.

New insights into the pathogenesis of Beckwith-Wiedemann and Silver-Russell syndromes: contribution of small copy number variations to 11p15 imprinting defects.

The imprinted 11p15 region is organized in two domains, each of them under the control of its own imprinting control region (ICR1 for the IGF2/H19 domain and ICR2 for the KCNQ1OT1/CDKN1C domain). Disruption of 11p15 imprinting results in two fetal growth disorders with opposite phenotypes: the Beckwith-Wiedemann (BWS) and the Silver-Russell (SRS) syndromes. Various 11p15 genetic and epigenetic defects have been demonstrated in BWS and SRS. Among them, isolated DNA methylation defects account for approximately 60% of patients. To investigate whether cryptic copy number variations (CNVs) involving only part of one of the two imprinted domains account for 11p15 isolated DNA methylation defects, we designed a single nucleotide polymorphism array covering the whole 11p15 imprinted region and genotyped 185 SRS or BWS cases with loss or gain of DNA methylation at either ICR1 or ICR2. We describe herein novel small gain and loss CNVs in six BWS or SRS patients, including maternally inherited cis-duplications involving only part of one of the two imprinted domains. We also show that ICR2 deletions do not account for BWS with ICR2 loss of methylation and that uniparental isodisomy involving only one of the two imprinted domains is not a mechanism for SRS or BWS.

Identification by array-Comparative Genomic Hybridization (array-CGH) of a large deletion of luteinizing hormone receptor gene combined with a missense mutation in a patient diagnosed with a 46,XY disorder of sex development and application to prenatal diagnosis.

This paper reports the case of an infant presenting with sexual ambiguity at birth. The child presented with labia majora synechia, thready genital tubercle and perineal hypospadias. The karyotype was 46,XY. Low testosterone levels with no response to hCG administration, associated with high LH level for her age, high FSH level, high inhibin B levels and normal AMH indicated a lack of LH receptivity and prompted us to screen the LHCGR gene for mutations. A previously described missense mutation (p.Cys131Arg) was identified at homozygous state in the propositus and at heterozygous state in the mother. This variation, however, was not found in the father. Our attention was drawn by the presence of several single nucleotide polymorphisms (SNPs), identified at homozygous state without any paternal contribution from exon 1 to exon 10 of LHCGR, suggesting a paternal deletion. Array DNA analysis was performed revealing a large deletion extending from 61,493 to 135,344 bp and including the LHCGR gene. Adequate genetic counselling was provided. This paper describes the first application of prenatal diagnosis in LHCGR deficiency for 46,XY disorders of sex development with the subsequent delivery of a normal baby.

SHP2 drives inflammation-triggered insulin resistance by reshaping tissue macrophage populations.

Insulin resistance is a key event in type 2 diabetes onset and a major comorbidity of obesity. It results from a combination of fat excess-triggered defects, including lipotoxicity and metaflammation, but the causal mechanisms remain difficult to identify. Here, we report that hyperactivation of the tyrosine phosphatase SHP2 found in Noonan syndrome (NS) led to an unsuspected insulin resistance profile uncoupled from altered lipid management (for example, obesity or ectopic lipid deposits) in both patients and mice. Functional exploration of an NS mouse model revealed this insulin resistance phenotype correlated with constitutive inflammation of tissues involved in the regulation of glucose metabolism. Bone marrow transplantation and macrophage depletion improved glucose homeostasis and decreased metaflammation in the mice, highlighting a key role of macrophages. In-depth analysis of bone marrow-derived macrophages in vitro and liver macrophages showed that hyperactive SHP2 promoted a proinflammatory phenotype, modified resident macrophage homeostasis, and triggered monocyte infiltration. Consistent with a role of SHP2 in promoting inflammation-driven insulin resistance, pharmaceutical SHP2 inhibition in obese diabetic mice improved insulin sensitivity even better than conventional antidiabetic molecules by specifically reducing metaflammation and alleviating macrophage activation. Together, these results reveal that SHP2 hyperactivation leads to inflammation-triggered metabolic impairments and highlight the therapeutical potential of SHP2 inhibition to ameliorate insulin resistance.

R125W coding variant in TBC1D1 confers risk for familial obesity and contributes to linkage on chromosome 4p14 in the French population.

Stone et al. previously reported an association between the TBC1D1 gene variant R125W (rs35859249) and severe obesity in women from US pedigrees. We attempted to replicate this result in 9714 French Caucasian individuals, combining family-based and general population studies. We confirmed an association with familial obesity (defined as body mass index (BMI) > or = 97th percentile) in women from 1109 obesity-selected pedigrees (Z-score = 2.70, P = 0.008). Analysis of 16 microsatellite markers on chromosome 4 restricted to the 42 pedigrees carrying the TBC1D1 R125W variant allele also revealed a suggestive evidence of linkage with obesity (maximum likelihood binomial LOD of 2.73, P = 0.0002) on chromosome 4p14, where resides TBC1D1. In contrast, R125W variant was neither associated with BMI nor with obesity in a large population-based cohort. These results confirm a putative role of TBC1D1 R125W variant in familial obesity predisposition.

Craniopharyngioma and hypothalamic obesity in children.

BACKGROUND AND PURPOSE: Obesity is a major concern in children treated for craniopharyngioma and is caused by hypothalamic damage. The role of aggressive surgical removal has been questioned, leading some authors to recommend a minimalist approach. In order to test this hypothesis, we decided to study obesity in craniopharyngioma and the factors related to it. MATERIALS AND METHODS: We reviewed retrospectively our series of pediatric craniopharyngiomas operated since 1981. The body-mass index (BMI) was calculated for each patient pre- and at several intervals postoperatively and expressed as standard deviations (SD) adjusted for age and gender. RESULTS: We operated on 45 cases, which were followed up for a mean duration of 11.0 years. Initial resection was total in 25 cases (55.6%). No patient died because of surgery or tumor progression; two died with delay presumably because of endocrine failure. At last control, 28 patients (62%) had obesity (BMI over +2SD). Hypothalamic involvement was significantly correlated with preoperative and postoperative BMI. Subtotal tumor resection was significantly associated with obesity at last control. Reoperation for tumor recurrence was associated with a significantly higher BMI. CONCLUSIONS: Our results suggest that obesity results from hypothalamic lesions caused by the tumor rather than by surgery. The postoperative weight gain appears to result from the continued impact of preoperative hypothalamic damage. The high rate of tumor recurrence in children, with the risk of additional damage to the hypothalamus, incites us to recommend total resection whenever it appears safe during initial surgery.

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.

Molecular analysis of thyroglobulin mutations found in patients with goiter and hypothyroidism.

Thyroid dyshormonogenesis due to thyroglobulin (TG) gene mutations have an estimated incidence of approximately 1 in 100,000 newborns. The clinical spectrum ranges from euthyroid to mild or severe hypothyroidism. Up to now, one hundred seventeen deleterious mutations in the TG gene have been identified and characterized. The purpose of the present study was to identify and characterize new mutations in the TG gene. We report eight patients from seven unrelated families with goiter, hypothyroidism and low levels of serum TG. All patients underwent clinical, biochemical and image evaluation. Sequencing of DNA, genotyping, as well as bioinformatics analysis were performed. Molecular analyses revealed three novel inactivating TG mutations: c.5560G>T [p.E1835*], c.7084G>C [p.A2343P] and c.7093T>C [p.W2346R], and four previously reported mutations: c.378C>A [p.Y107*], c.886C>T [p.R277*], c.1351C>T [p.R432*] and c.7007G>A [p.R2317Q]. Two patients carried homozygous mutations (p.R277*/p.R277*, p.W2346R/p.W2346R), four were compound heterozygous mutations (p.Y107*/p.R277* (two unrelated patients), p.R432*/p.A2343P, p.Y107*/p.R2317Q) and two siblings from another family had a single p.E1835* mutated allele. Additionally, we include the analysis of 48 patients from 31 unrelated families with TG mutations identified in our present and previous studies. Our observation shows that mutations in both TG alleles were found in 27 families (9 as homozygote and 18 as heterozygote compound), whereas in the remaining four families only one mutated allele was detected. The majority of the detected mutations occur in exons 4, 7, 38 and 40. 28 different mutations were identified, 33 of the 96 TG alleles encoded the change p.R277*. In conclusion, our results confirm the genetic heterogeneity of TG defects and the pathophysiological importance of the predicted TG misfolding and therefore thyroid hormone formation as a consequence of truncated TG proteins and/or missense mutations located within its ACHE-like domain.

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.

Genetic study of the melanin-concentrating hormone receptor 2 in childhood and adulthood severe obesity.

CONTEXT: The melanin-concentrating hormone receptor 2 (MCHR2) is a G protein-coupled receptor for melanin-concentrating hormone, a neuropeptide that plays an important role in feeding behaviors. MCHR2 maps on chromosome 6q16.3, in a susceptibility locus for childhood obesity. OBJECTIVE: The aim of this study was to investigate the association between MCHR2 variation and human obesity. DESIGN: Case control and family-based studies were performed. PARTICIPANTS: A total of 141 obese children and 24 nonobese adult subjects was sequenced, and case-control analyses were conducted using 628 severely obese children and 1,401 controls. RESULTS: There were 11 single nucleotide polymorphisms (SNPs) identified. We showed nominal association among -38,245 ATG A/G SNP (P = 0.03; 95% confidence interval 1.02-1.34; odds ratio 1.17), A76A T/C SNP (P = 0.03; 95% confidence interval 0.58-0.97; odds ratio 0.75), and childhood obesity. Analysis of 645 trios with childhood obesity supported further the A76A T/C association, showing an overtransmission to obese children of the at risk T allele (59.0%; P = 0.01), especially in children with most severe forms of obesity (Z score of body mass index > 4) (67.0%; P = 0.003). The A76A at risk T allele was also associated with overeating during meals (P = 0.02) in an additional group of 102 nonobese children. None of the MCHR2 variants, including the A76A SNP, showed association with adult severe obesity, although a trend for association of the T allele of this variant with food disinhibition (P = 0.06) and higher hunger (P = 0.09) was found. This variant was not associated with childhood obesity in an independent case-control study, including 1,573 subjects (P = 0.98). Moreover, the A76A SNP did not explain the linkage on the 6q locus. CONCLUSION: Our results altogether suggest that MCHR2 is not a major contributor to polygenic obesity and support a modest effect of the A76A SNP on food intake abnormalities in childhood.

No contribution of angiotensin-converting enzyme (ACE) gene variants to severe obesity: a model for comprehensive case/control and quantitative cladistic analysis of ACE in human diseases.

Candidate gene analyses are often inconclusive owing to genetic or phenotypic heterogeneity, low statistical power, selection of nonfunctional SNPs, and inadequate statistical analysis of the genetic architecture. Angiotensin-converting enzyme (ACE) is involved in adipocyte growth and function and the ACE-processed angiotensin II inhibits adipocyte differentiation. Associations between body mass index (BMI) and ACE polymorphisms have been reported in general populations, but the contribution to severe obesity of this gene, which is located under an obesity genome-scan linkage peak on 17q23, is unknown. ACE is one of the most studied genes and markers responsible for variation in circulating ACE enzyme levels have been extensively characterised. Eight of these variants were genotyped in 1054 severely obese cases and 918 nonobese controls, as well as 116 nuclear families from the genome scan (n=447), enabling the known clades to be inferred. Qualitative analysis of individual single-nucleotide polymorphisms (SNPs), haplotypes, clades, and diploclades demonstrated no significant associations (P<0.05) after minimal correction for multiple testing. Quantitative analysis of clades and diploclades for BMI, waist-to-hip ratio, or ZBMI in children were also not significant. This rigorous, large-scale study of common, well-defined, severe polygenic obesity provides strong evidence that functionally relevant sequence variation in ACE, whether it is defined at the level of SNPs, haplotypes, or clades, is not associated with severe obesity in French Caucasians. Such a study design exemplifies the strategy needed to clearly define the contribution of the ACE gene to the plethora of complex genetic diseases where weak associations have been previously reported.