Differentially methylated CpGs in response to growth hormone administration in children with idiopathic short stature.

BACKGROUND: Recombinant human growth hormone (rhGH) has shown a great growth-promoting potential in children with idiopathic short stature (ISS). However, the response to rhGH differs across individuals, largely due to genetic and epigenetic heterogeneity. Since epigenetic marks on the methylome can be dynamically influenced by GH, we performed a comprehensive pharmacoepigenomics analysis of DNA methylation changes associated with long-term rhGH administration in children with ISS. RESULTS: We measured DNA methylation profiles before and after GH treatment (with a duration of ~ 18 months in average) on 47 healthy children using customized methylC-seq capture sequencing. Their changes were compared and associated with changes in plasma IGF1 by adjusting sex, age, treatment duration and estimated blood proportions. We observed a considerable inter-individual heterogeneity of DNA methylation changes responding to GH treatment. We identified 267 response-associated differentially methylated cytosines (DMCs) that were enriched in promoter regions, CpG islands and blood cell-type-specific regulatory elements. Furthermore, the genes associated with these DMCs were enriched in the biology process of "cell development," "neuron differentiation" and "developmental growth," and in the TGF-beta signaling pathway, PPAR Alpha pathway, endoderm differentiation pathway, adipocytokine signaling pathway as well as PI3K-Akt signaling pathway, and cAMP signaling pathway. CONCLUSION: Our study provides a first insight in DNA methylation changes associated with rhGH administration, which may help understand mechanisms of epigenetic regulation on GH-responsive genes.

Long-Term Disease Prevention with a Gene Therapy Targeting Oligodendrocytes in a Mouse Model of Adrenomyeloneuropathy.

Adrenomyeloneuropathy (AMN) is a late-onset axonopathy of spinal cord tracts caused by mutations of the ABCD1 gene that encodes adrenoleukodystrophy protein (ALDP), a peroxisomal transporter of very long-chain fatty acids (VLCFA). Disturbed metabolic interaction between oligodendrocytes (OL) and axons is suspected to play a major role in AMN axonopathy. To develop a vector targeting OL, the human ABCD1 gene driven by a short 0.3 kb part of the human myelin-associated glycoprotein (MAG) promoter was packaged into an adeno-associated viral serotype 9 (rAAV9). An intravenous injection of this vector on postnatal day 10 in Abcd1-/- mice, a model of AMN, allowed a near normal motor performance to persist for 24 months, while age-matched untreated mice developed major defects of balance and motricity. Three weeks postvector, 50-54% of spinal cord white matter OL was expressing human ALDP (hALDP) at the cervical level, and only 6-7% after 24 months. In addition, 29-32% of cervical spinal cord astrocytes at 3 weeks and 16-19% at 24 months also expressed ALDP. C26:0-lysoPC, a sensitive VLCFA marker of AMN, was lower by 41% and 50%, respectively, in the spinal cord and brain of vector-treated compared with untreated mice. In a nonhuman primate, the intrathecal injection of the rAAV9-MAG vector induced abundant ALDP expression at 3 weeks in spinal cord OL (43%, 29%, and 26% at cervical, thoracic, and lumbar levels) and cerebellum OL (35%). In addition, 33-41% of spinal cord astrocytes expressed hALDP, and 27% of cerebellar astrocytes. To our knowledge, OL targeting had not been obtained before in primates with other vectors or promoters. The current results thus provide a robust proof-of-concept not only for the gene therapy of AMN but also for other central nervous system diseases, where the targeting of OL with the rAAV9-MAG vector may be of interest.

Correction of a knock-in mouse model of acrodysostosis with gene therapy using a rAAV9-CAG-human PRKAR1A vector.

Acrodysostosis is a rare skeletal dysplasia caused by loss-of-function mutations in the regulatory subunit of protein kinase A (PRKAR1A). In a knock-in mouse model (PRKAR1Awt/mut) expressing one copy of the recurrent R368X mutation, we tested the effects of a rAAV9-CAG-human PRKR1A (hPRKAR1A) vector intravenously administered at 4 weeks of age. Caudal vertebrae and tibial diaphyses contained 0.52 ± 0.7 and 0.13 ± 0.3 vector genome per cell (VGC), respectively, at 10 weeks of age and 0.22 ± 0.04 and 0.020 ± 0.04 at 16 weeks while renal cortex contained 0.57 ± 0.14 and 0.26 ± 0.05 VGC. Vector-mediated hPRKAR1A expression was found in growth plate chondrocytes, osteoclasts, osteoblasts, and kidney tubular cells. Chondrocyte architecture was restored in the growth plates. Body length, tail length, and body weight were improved in vector treated PRKAR1Awt/mut mice, not the bone length of their limbs. These results provide one of the few proofs for gene therapy efficacy in a mouse model of chondrodysplasia. In addition, the increased urinary cAMP of PRKAR1Awt/mut mice was corrected almost to normal. In conclusion, gene therapy with hPRKAR1A improved skeletal growth and kidney dysfunction, the hallmarks of acrodysostosis in R368X mutated mice and humans.

Correction to: Fetal growth is associated with CpG methylation in the P2 promoter of the IGF1 gene.

[This corrects the article DOI: 10.1186/s13148-018-0489-9.].

Fetal growth is associated with CpG methylation in the P2 promoter of the IGF1 gene.

Background: There are many reasons to think that epigenetics is a key determinant of fetal growth variability across the normal population. Since IGF1 and INS genes are major determinants of intrauterine growth, we examined the methylation of selected CpGs located in the regulatory region of these two genes. Methods: Cord blood was sampled in 159 newborns born to mothers prospectively followed during their pregnancy. A 142-item questionnaire was filled by mothers at inclusion, during the last trimester of the pregnancy and at the delivery. The methylation of selected CpGs located in the promoters of the IGF1 and INS genes was measured in cord blood mononuclear cells collected at birth using bisulfite-PCR-pyrosequencing. Results: Methylation at IGF1 CpG-137 correlated negatively with birth length (r = 0.27, P = 3.5 × 10-4). The same effect size was found after adjustment for maternal age, parity, and smoking: a 10% increase in CpG-137 methylation was associated with a decrease of length by 0.23 SDS. Conclusion: The current results suggest that the methylation of IGF1 CpG-137 contributes to the individual variation of fetal growth by regulating IGF1 expression in fetal tissues.

Mutations causing acrodysostosis-2 facilitate activation of phosphodiesterase 4D3.

Type 2 acrodysostosis (ACRDYS2), a rare developmental skeletal dysplasia characterized by short stature, severe brachydactyly and facial dysostosis, is caused by mutations in the phosphodiesterase (PDE) 4D (PDE4D) gene. Several arguments suggest that the mutations should result in inappropriately increased PDE4D activity, however, no direct evidence supporting this hypothesis has been presented, and the functional consequences of the mutations remain unclear. We evaluated the impact of four different PDE4D mutations causing ACRDYS2 located in different functional domains on the activity of PDE4D3 expressed in Chinese hamster ovary cells. Three independent approaches were used: the direct measurement of PDE activity in cell lysates, the evaluation of intracellular cAMP levels using an EPAC-based (exchange factor directly activated by cAMP) bioluminescence resonance energy transfer sensor , and the assessment of PDE4D3 activation based on electrophoretic mobility. Our findings indicate that PDE4D3s carrying the ACRDYS2 mutations are more easily activated by protein kinase A-induced phosphorylation than WT PDE4D3. This occurs over a wide range of intracellular cAMP concentrations, including basal conditions, and result in increased hydrolytic activity. Our results provide new information concerning the mechanism whereby the mutations identified in the ACRDYS2 dysregulate PDE4D activity, and give insights into rare diseases involving the cAMP signaling pathway. These findings may offer new perspectives into the selection of specific PDE inhibitors and possible therapeutic intervention for these patients.

Knock-In of the Recurrent R368X Mutation of PRKAR1A that Represses cAMP-Dependent Protein Kinase A Activation: A Model of Type 1 Acrodysostosis.

In humans, activating mutations in the PRKAR1A gene cause acrodysostosis 1 (ACRDYS1). These mutations result in a reduction in PKA activation caused by an impaired ability of cAMP to dissociate mutant PRKAR1A from catalytic PKA subunits. Two striking features of this rare developmental disease are renal resistance to PTH and chondrodysplasia resulting from the constitutive inhibition of PTHR1/Gsa/AC/cAMP/PKA signaling. We developed a knock-in of the recurrent ACRDYS1 R368X PRKAR1A mutation in the mouse. No litters were obtained from [R368X]/[+] females (thus no homozygous [R368X]/[R368X] mice). In [R368X]/[+] mice, Western blot analysis confirmed mutant allele heterozygous expression. Growth retardation, peripheral acrodysostosis (including brachydactyly affecting all digits), and facial dysostosis were shown in [R368X]/[+] mice by weight curves and skeletal measurements (µCT scan) as a function of time. [R368X]/[+] male and female mice were similarly affected. Unexpected, however, whole-mount skeletal preparations revealed a striking delay in mineralization in newborn mutant mice, accompanied by a decrease in the height of terminal hypertrophic chondrocyte layer, an increase in the height of columnar proliferative prehypertrophic chondrocyte layer, and changes in the number and spatial arrangement of proliferating cell nuclear antigen (PCNA)-positive chondrocytes. Plasma PTH and basal urinary cAMP were significantly higher in [R368X]/[+] compared to WT mice. PTH injection increased urinary cAMP similarly in [R368X]/[+] and WT mice. PRKACA expression was regulated in a tissue (kidney not bone and liver) manner. This model, the first describing the germline expression of a PRKAR1A mutation causing dominant repression of cAMP-dependent PKA, reproduced the main features of ACRDYS1 in humans. It should help decipher the specificity of the cAMP/PKA signaling pathway, crucial for numerous stimuli. In addition, our results indicate that PRKAR1A, by tempering intracellular cAMP levels, is a molecular switch at the crossroads of signaling pathways regulating chondrocyte proliferation and differentiation. © 2016 American Society for Bone and Mineral Research.

Modulation of signaling through GPCR-cAMP-PKA pathways by PDE4 depends on stimulus intensity: Possible implications for the pathogenesis of acrodysostosis without hormone resistance.

In acrodysostosis without hormone resistance, a disease caused by phosphodiesterase (PDE)-4D mutations, increased PDE activity leads to bone developmental defects but with normal renal responses to PTH. To identify potential mechanisms for these disparate responses, we compared the effect of PDE activity on hormone signaling through the GPCR-Gsα-cAMP-PKA pathway in cells from two lineages, HEK-293 cells stably overexpressing PTH1R (HEKpthr) and human dermal fibroblasts, including studies evaluating cAMP levels using an Epac-based BRET-sensor for cAMP (CAMYEL). For ligand-induced responses inducing strong cAMP accumulation, the inhibition of PDE4 activity resulted in relatively small further increases. In contrast, when ligand-induced cAMP accumulation was of lesser intensity, the inhibition of PDE4 had a more pronounced effect. Similar results were obtained evaluating downstream events (cellular CREB phosphorylation and CRE-luciferase activity). Thus, the ability of PDE4 to modulate signaling through GPCR-cAMP-PKA pathways can depend on the cell type and stimulus intensity.

Functional Characterization of PRKAR1A Mutations Reveals a Unique Molecular Mechanism Causing Acrodysostosis but Multiple Mechanisms Causing Carney Complex.

The main target of cAMP is PKA, the main regulatory subunit of which (PRKAR1A) presents mutations in two genetic disorders: acrodysostosis and Carney complex. In addition to the initial recurrent mutation (R368X) of the PRKAR1A gene, several missense and nonsense mutations have been observed recently in acrodysostosis with hormonal resistance. These mutations are located in one of the two cAMP-binding domains of the protein, and their functional characterization is presented here. Expression of each of the PRKAR1A mutants results in a reduction of forskolin-induced PKA activation (measured by a reporter assay) and an impaired ability of cAMP to dissociate PRKAR1A from the catalytic PKA subunits by BRET assay. Modeling studies and sensitivity to cAMP analogs specific for domain A (8-piperidinoadenosine 3',5'-cyclic monophosphate) or domain B (8-(6-aminohexyl)aminoadenosine-3',5'-cyclic monophosphate) indicate that the mutations impair cAMP binding locally in the domain containing the mutation. Interestingly, two of these mutations affect amino acids for which alternative amino acid substitutions have been reported to cause the Carney complex phenotype. To decipher the molecular mechanism through which homologous substitutions can produce such strikingly different clinical phenotypes, we studied these mutations using the same approaches. Interestingly, the Carney mutants also demonstrated resistance to cAMP, but they expressed additional functional defects, including accelerated PRKAR1A protein degradation. These data demonstrate that a cAMP binding defect is the common molecular mechanism for resistance of PKA activation in acrodysosotosis and that several distinct mechanisms lead to constitutive PKA activation in Carney complex.

Methylation and transcripts expression at the imprinted GNAS locus in human embryonic and induced pluripotent stem cells and their derivatives.

Data from the literature indicate that genomic imprint marks are disturbed in human pluripotent stem cells (PSCs). GNAS is an imprinted locus that produces one biallelic (Gsα) and four monoallelic (NESP55, GNAS-AS1, XLsα, and A/B) transcripts due to differential methylation of their promoters (DMR). To document imprinting at the GNAS locus in PSCs, we studied GNAS locus DMR methylation and transcript (NESP55, XLsα, and A/B) expression in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) derived from two human fibroblasts and their progenies. Results showed that (1) methylation at the GNAS locus DMRs is DMR and cell line specific, (2) changes in allelic transcript expression can be independent of a change in allele-specific DNA methylation, and (3) interestingly, methylation at A/B DMR is correlated with A/B transcript expression. These results indicate that these models are valuable to study the mechanisms controlling GNAS methylation, factors involved in transcript expression, and possibly mechanisms involved in the pathophysiology of pseudohypoparathyroidism type 1B.

In obese and non-obese adults, the cis-regulatory rs361072 promoter variant of PIK3CB is associated with insulin resistance not with type 2 diabetes.

We recently reported that rs361072, a promoter C/T variant of p110beta, the catalytic subunit of PI3-kinase, was associated with a protection from insulin resistance (IR) in Caucasian adolescents in proportion of their body mass. We tested if this cis-regulatory QTL is associated with IR and type 2 diabetes in 7885 middle-aged obese and non-obese adults of European ancestry. We genotyped rs361072 in 1139 non-diabetic obese (NDO) European adults, in whom IR was estimated by the HOMA-IR index. We also studied 427 type 2 diabetic obese adults (DO) and 424 diabetic non-obese (DNO) adults to test whether their disease status was associated with a decreased prevalence of the protective variant. The prevalence of rs361072 and association with IR was also examined in 5895 non-obese non-diabetic adults (NDNO). rs361072 was associated with HOMA-IR (p=4.10(-4)) in NDO, so that C/C patients had a 17% decrease of this index (p=0.002). A statistical trend (p=1.1.10(-2)) for the same genotypic differences was also observed in NDNO adults, but of insignificant magnitude (4.2%). The distribution of rs361072 genotype was comparable in NDO, DO, DNO and NDNO individuals. Allele C of rs361072 is associated with a protection from IR in obese and non-obese adults, but has no significant effect, however, on diabetes risk in obese or non-obese Europeans.

Endocrine manifestations of the rapid-onset obesity with hypoventilation, hypothalamic, autonomic dysregulation, and neural tumor syndrome in childhood.

CONTEXT: Rapid-onset obesity with hypoventilation, hypothalamic, autonomic dysregulation, and neural tumor (ROHHADNET) is a newly described syndrome that can cause cardiorespiratory arrests and death. It mimics several endocrine disorders or genetic obesity syndromes during early childhood and is associated with various forms of hypothalamic-pituitary endocrine dysfunctions that have not yet been fully investigated. OBJECTIVE: The current report aspires to facilitate the earlier recognition and appropriate treatment of the ROHHADNET syndrome when children present with various endocrine manifestations, such as early obesity, growth failure, pseudo-Cushing's syndrome, glucocorticoid insufficiency, congenital hypopituitarism, or adrenal tumors. A more widespread knowledge of the syndrome will help characterize its molecular origin. DESIGN: Endocrine studies were performed in six patients admitted for seemingly common early-onset obesity associated with growth failure in five of them. The six patients later showed distinctive features of the ROHHADNET syndrome. RESULTS: Abnormalities of the pituitary adrenal axis ranged from a true Cushing-like profile (one of six), to glucocorticoid deficiency with normal ACTH (two of six). Complete GH deficiency with low IGF-I was observed in four of six, hypogonadotropic hypogonadism in four of six, hyperprolactinemia in six of six, and various degrees of TSH/T(4) abnormalities in five of five patients. All had increased natremia without diabetes insipidus. Five children had unilateral macroscopic adrenal ganglioneuroma. Two patients died at 8.5 and 12 yr of age. CONCLUSIONS: Various hypothalamic-pituitary endocrine dysfunctions are associated with ROHHADNET, carrying a risk of misdiagnosis until other elements of the syndrome make it more easily recognizable. Given its severity, ROHHADNET syndrome should be considered in all cases of isolated, rapid, and early obesity.

Common nonsynonymous variants in PCSK1 confer risk of obesity.

Mutations in PCSK1 cause monogenic obesity. To assess the contribution of PCSK1 to polygenic obesity risk, we genotyped tag SNPs in a total of 13,659 individuals of European ancestry from eight independent case-control or family-based cohorts. The nonsynonymous variants rs6232, encoding N221D, and rs6234-rs6235, encoding the Q665E-S690T pair, were consistently associated with obesity in adults and children (P = 7.27 x 10(-8) and P = 2.31 x 10(-12), respectively). Functional analysis showed a significant impairment of the N221D-mutant PC1/3 protein catalytic activity.

Akt phosphorylation in lymphocytes provides an index of in vitro insulin-like growth factor I sensitivity associated with growth hormone-induced growth.

CONTEXT: Because IGF-I is the main mediator of GH action on osteogenic cells, individual differences in IGF-I sensitivity are expected to contribute to the variations of GH effects on growth. In GH-treated children, the variable responses in growth rates at a specific IGF-I target level indicate heterogeneity of responses to serum IGF-I exposures. OBJECTIVES: This study tested a cell-based assay as an index of individual IGF-I sensitivity that could help dissect GH pharmacogenetics. DESIGN: Akt phosphorylation (P-Akt) was quantified in response to IGF-I in fresh lymphocytes from 50 short children (25 with idiopathic short stature and 25 born short for gestational age) whose growth parameters were being prospectively monitored during the first year of GH therapy (86 +/- 20 mug/kg.d). RESULTS: Intra-individual triplicate measurements of IGF-I-stimulated P-Akt were reasonably consistent (0.11 < or = sd; mean < or = 0.23). Among the 50 children, the distribution of P-Akt in lymphocytes stimulated by 125 ng/ml IGF-I was closely associated with the growth response to GH administration (univariate P = 0.001). Both GH dosage (P = 0.006) and the fold increase in IGF-I levels (P = 0.04) in response to GH (P = 0.04) were also correlated with the growth response. CONCLUSION: Lymphocytes are the only IGF-I target cells that can be easily studied in clinical research. IGF-I-stimulated P-Akt in these cells was found to be a predictor of GH efficacy, supporting a significant role of the first steps of IGF-I signaling in the individual variability of GH effects on growth.

A single-nucleotide polymorphism in the p110beta gene promoter is associated with partial protection from insulin resistance in severely obese adolescents.

OBJECTIVE: Severe juvenile obesity causes metabolic and cardiovascular complications in adulthood. The catalytic p110beta subunit of phosphatidyl-inositol-3 kinase is a major effector of insulin action. We studied the p110beta gene as a candidate gene for association with insulin resistance (IR) and fasting glycemia in severely obese children. METHODS: We conducted an association study in 580 severely obese European children (body mass index > 99.6th centile) and 606 nonobese control children, in whom glucose and insulin were measured in the fasting state. The homeostasis model assessment insulin resistance index was used to estimate IR. RESULTS: We found that a single-nucleotide polymorphism (rs361072) located in the promoter of the p110beta gene was associated with fasting glucose (P = 0.0002), insulin (P = 2.6 10(-8)), and homeostasis model assessment insulin resistance index (P =1 10(-9)) in the severely obese children. The effect of rs361072 was marginal or not significant in nonobese children. CONCLUSIONS: The C allele of rs361072 attenuates IR in superobese children.

Association analysis indicates that a variant GATA-binding site in the PIK3CB promoter is a Cis-acting expression quantitative trait locus for this gene and attenuates insulin resistance in obese children.

OBJECTIVE: In search of functional polymorphisms associated with the genetics of insulin resistance, we studied a variant in the promoter of PIK3CB, the gene coding for the catalytic p110beta subunit of phosphatidylinositol (PI) 3-kinase, a major effector of insulin action. RESEARCH DESIGN AND METHODS: The rs361072 C/T variant was selected among single nucleotide polymorphisms of the PIK3CB region because we suspected that its common C allele (allelic frequency approximately 50% in Europeans) could create a GATA-binding motif and was genotyped in five cohorts of obese (n = 1,876) and two cohorts of nonobese (n = 1,490) European children. To estimate insulin resistance in these children, the homeostasis model assessment for insulin resistance (HOMA-IR) index was measured in strict nutritional conditions. GATA-binding and functional effects of rs361072 were explored in transfected cell lines and in lymphocytes from obese children. RESULTS: The rs361072 C/T variant was associated with HOMA-IR in the obese children cohorts (1.7 x 10(-12) < P < 2 x 10(-4) for C/C vs. T/T using regression analysis). HOMA-IR averaged 3.3 +/- 0.1 in C/C and 4.5 +/- 0.2 in T/T obese children (P = 4.5 x 10(-6) by ANOVA). C/T patients had intermediate values. As shown by the interaction between BMI and genotype (P = 2.1 x 10(-9)), the association of rs361072 with HOMA-IR depended on BMI and was only marginal in nonobese children (P = 0.04). At the molecular level, the C allele of rs361072 was found to create a GATA-binding site able to increase transcription of PIK3CB. CONCLUSIONS: We postulate that the C allele of rs361072 is a causal variant capable of attenuating insulin resistance in obese children through increased expression of p110beta.

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.

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.

Heterogeneity of class I INS VNTR allele association with insulin secretion in obese children.

On the basis of the near-complete linkage disequilibrium of the insulin variable number of tandem repeats (INS VNTR) allele with the neighboring -23Hph1 A/T single-nucleotide polymorphism, previous studies have documented the association of class I ("short") and class III ("long") INS VNTR alleles with metabolic parameters, including circulating insulin levels. Using a new method to sequence class I alleles, we revisited this association in 346 obese children. Class I alleles are made of several types of repeats, whose repartition determines subclasses IC and ID. Fasting insulin was found to be higher in obese children with ID/ID genotypes (135 +/- 12 pmol/l, n = 64) than with ID/IC or IC/IC genotypes (91 +/- 5 pmol/l, n = 97, P = 0.0005). In response to oral glucose, peak insulin levels and insulin-to-glucose area under the curve ratios were higher in ID/ID (872 +/- 122 pmol/l and 109 +/- 15, respectively) than in ID/IC or IC/IC patients (586 +/- 42 pmol/l and 76 +/- 5, P = 0.02 and P = 0.04, respectively). Fasting and postglucose insulin levels were comparable in carriers of IC and of class III alleles. Our results support that the molecular structure of the VNTR allele, not only its overall length, is associated with variations of insulin secretion. ID/ID homozygosity appears responsible for the increased insulin levels previously attributed to the whole class I VNTR group. It will be important to test the ramifications of this observation for class I association with Type 1 (susceptibility) and Type 2 diabetes (protection).

A homozygous null mutation delineates the role of the melanocortin-4 receptor in humans.

As a mediator of the effects of leptin, the melanocortin-4 receptor (MC4R) is an essential component of the central regulation of long-term energy homeostasis. Heterozygous mutations in this receptor are the most frequent genetic cause of severe obesity in children. The very rare described carriers of homozygous MC4R mutations for whom clinical data were available had a residual receptor activity thus not allowing for the description of the full extent of the role of MC4R in humans. Here, we present the clinical and biological features of a patient with complete absence of MC4R activity and compare the clinical and endocrine characteristics of this patient with those previously observed in leptin receptor-deficient patients. Our data suggest that in humans, the MC4R mediates most of the anorectic effects of leptin in early childhood. In contrast, MC4R does not mediate the effect of leptin on linear growth and other endocrine axes. In addition, complete MC4R deficiency is not a cause of relative hyperinsulinemia as recently observed in children with heterozygous MC4R mutations.