Epigenome Wide Association and Stochastic Epigenetic Mutation Analysis on Cord Blood of Preterm Birth.

Preterm birth (PTB) can be defined as the endpoint of a complex process that could be influenced by maternal and environmental factors. Epigenetics recently emerged as an interesting field of investigation since it represents an important mechanism of regulation. This study evaluates epigenetic impact of preterm birth on DNA methylation. Genome-wide DNAm was measured using the Illumina 450K array in cord blood samples obtained from 72 full term and 18 preterm newborns. Lymphocyte composition was calculated based on specific epigenetic markers that are present on the 450k array. Differential methylation analysis was performed both at site and region level; moreover, stochastic epigenetic mutations (SEMs) were also evaluated. The study showed significant differences in blood cell composition between the two groups. Moreover, after multiple testing correction, statistically significant differences in DNA methylation levels emerged between the two groups both at site and region levels. Results obtained were compared to those reported by previous EWAS, leading to a list of more consistent genes associated with PTB. Finally, the SEMs analysis revealed that the burden of SEMs resulted significantly higher in the preterm group. In conclusion, PTB resulted associated to specific epigenetic signatures that involve immune system. Moreover, SEMs analysis revealed an increased epigenetic drift at birth in the preterm group.

Rare melanocortin-3 receptor mutations with in vitro functional consequences are associated with human obesity.

In contrast to the melanocortin 4 receptor, the possible role of the melanocortin 3 receptor (MC3R) in regulating body weight is still debated. We have previously reported three mutations in the MC3R gene showing association with human obesity, but these results were not confirmed in a study of severe obese North American adults. In this study, we evaluated the entire coding region of MC3R in 839 severely obese subjects and 967 lean controls of Italian and French origin. In vitro functional analysis of the mutations detected was also performed. The total prevalence of rare MC3R variants was not significantly different in obese subjects when compared with controls (P= 0.18). However, the prevalence of mutations with functional alterations was significantly higher in the obese group (P= 0.022). In conclusions, the results of this large study demonstrate that in the populations studied functionally significant MC3R variants are associated with obesity supporting the current hypothesis that rare variants might have a stronger impact on the individual susceptibility to gain weight. They also underline the importance of detailed in vitro functional studies in order to prove the pathogenic effect of such variants. Further investigations in larger cohorts will be needed in order to define the specific phenotypic characteristics potentially correlated with reduced MC3R signalling.

Phenotypes Associated With MEN1 Syndrome: A Focus on Genotype-Phenotype Correlations.

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominant inherited tumor syndrome, associated with parathyroid, pituitary, and gastro-entero-pancreatic (GEP) neuroendocrine tumors (NETs). MEN1 is usually consequent to different germline and somatic mutations of the MEN1 tumor suppressor gene, although phenocopies have also been reported. This review analyzed main biomedical databases searching for reports on MEN1 gene mutations and focused on aggressive and aberrant clinical manifestations to investigate the potential genotype-phenotype correlation. Despite efforts made by several groups, this link remains elusive to date and evidence that aggressive or aberrant clinical phenotypes may be related to specific mutations has been provided by case reports and small groups of MEN1 patients or families. In such context, a higher risk of aggressive tumor phenotypes has been described in relation to frameshift and non-sense mutations, and predominantly associated with aggressive GEP NETs, particularly pancreatic NETs. In our experience a novel heterozygous missense mutation at c.836C>A in exon 6 was noticed in a MEN1 patient operated for macro-prolactinoma, who progressively developed recurrent parathyroid adenomas, expanding gastrinomas and, long after the first MEN1 manifestation, a neuroendocrine uterine carcinoma. In conclusion, proof of genotype-phenotype correlation is limited but current evidence hints at the need for long-term interdisciplinary surveillance in patients with aggressive phenotypes and genetically confirmed MEN1.

Unexpected endocrine features and normal pigmentation in a young adult patient carrying a novel homozygous mutation in the POMC gene.

CONTEXT: Proopiomelanocortin (POMC) is the precursor to five biologically active peptides, including ACTH produced in the anterior pituitary and alpha-MSH produced in the hypothalamus. Mutations that inactivate the POMC gene have been described in children, causing a pleiotropic syndrome that includes secondary hypocortisolism, severe obesity, and variable changes in skin and hair pigmentation. OBJECTIVE: We describe a female patient of North African ancestry, homozygous for a frameshift mutation in the POMC gene (6922InsC) that impairs the production of all melanocortin peptides, and that is associated with novel clinical features. Repeated clinical investigations from birth to age 18 yr are presented. RESULT: ACTH deficiency was diagnosed at birth. Hyperphagia and obesity became apparent before 2 yr of age and rapidly progressed [body mass index (BMI) Z-score, +7 sd at 2 yr, +9.7 sd at 13 yr; BMI, 50 kg/m(2) at 18 yr). At puberty, the patient developed alterations in the somatotropic, gonadotropic, and thyroid axes necessitating hormonal replacement. Surprisingly, there were no obvious pigmentary features; neither the hair color nor measurements of skin reflectance distinguished between the patient and unaffected family members. However, chemical analysis of hair pigment revealed increased production of both pheomelanin and eumelanin. CONCLUSION: Molecular genetic abnormalities of POMC should always be considered in patients with early onset adrenal insufficiency and obesity, even in the presence of normal pigmentation and multiple pituitary hormone anomalies.

Sporadic mutations in melanocortin receptor 3 in morbid obese individuals.

Several mutations in the melanocortin receptor 4 gene have been identified in humans and account for 3-6% of morbid obesity. In contrast, strong evidence of a causative role for melanocortin receptor 3 (MC3R) mutations are still lacking. In MC3R knockout mice, high feed efficiency rather than hyperphagia seems to contribute to increased fat mass. On the basis of this evidence, the objective of the present study was to investigate the presence of MC3R mutations in a group of 290 obese subjects (mean BMI 44.2+/-5.9 kg/m2). As a control, a group of 215 normal-weight subjects (mean BMI 22.4+/-2.7 kg/m2) was also screened. Three novel mutations in the MC3R gene (A293T, I335S and X361S) were identified among the obese patients. The mutations segregated with obesity in the members of the families studied. In vitro expression studies of each mutation demonstrated a loss of function of the I335S-mutated receptor. These findings suggest that, in humans, MC3R mutations may be a cause of a dominantly inherited form of obesity. However, this association as well as the specific phenotypic characteristics resulting from these mutations need to be further evaluated in larger series of obese subjects.

Recurrent transient global amnesia as presenting symptoms of CADASIL.

Despite transient global amnesia is considered unusual in Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and causal relation is still unclear, this report suggests to consider CADASIL in those patients with recurrent transient global amnesia, especially when MRI shows multifocal hyperintensities affecting the cerebral white matter or when it is followed by cognitive decline.

Episodic ataxia and SCA6 within the same family due to the D302N CACNA1A gene mutation.

Several dominant mutations of CACNA1A gene were associated with at least three different allelic disorders: spino-cerebellar ataxia type 6 (SCA6), episodic ataxia type 2 (EA2), and familial hemiplegic migraine-1 (FHM1). It is generally thought that loss-of-function mutations are associated with EA2, gain-of-function missense mutations with FHM1, and abnormal CAG expansions with SCA6. But, overlapping features, atypical symptoms and co-occurrence of distinct phenotypes within the same family were reported. We describe a four generation family showing different phenotypes ranging from EA2 to SCA6 and carrying the p.D302N CACNA1A gene mutation. In our family the phenotypes maintained separate and gender differences corresponding to different phenotypes were observed.

A novel missense mutation in the signal peptide of the human POMC gene: a possible additional link between early-onset type 2 diabetes and obesity.

Rare mutations in several genes have a critical role in the control of homeostatic mechanisms such as food-intake, energy balance and glucose metabolism. In this study, we performed a mutational screening in a 58-year-old woman presenting early-onset type 2 diabetes and central obesity. The entire coding regions of MC4R, MC3R, HNF1A, GCK and POMC (pro-opiomelanocortin) genes were analyzed by direct sequencing. A new missense mutation was identified within the POMC gene signal peptide sequence, resulting in a heterozygous substitution of an arginine for a glycine at codon 15 (p.A15G) that was excluded in 300 healthy normal weight controls. The mutation segregated in the family and was associated with overweight, type 2 diabetes, hypertension and coronary heart disease in the carriers. Functional studies demonstrated that POMC protein was not detectable in ß-TC3 cells transfected with A15G-POMC vector as well as in their culture media, despite POMC mRNA levels were comparable for amount and stability to those of wild-type-transfected cells. In silico RNA folding prediction indicated that the mutation gives rise to a different RNA secondary structure, suggesting that it might affect translation and protein synthesis. To the best of our knowledge, this is the first report addressing the functional consequences of a mutation in the signal peptide of POMC. These findings further support the hypothesis that POMC-derived peptides might have a role in the control of peripheral glucose metabolism and suggest that disruption of central POMC secretion might represent an additional link between type 2 diabetes and obesity.

Combined evaluation of resting IGF1, N-terminal propeptide of type III procollagen and C-terminal cross-linked telopeptide of type I collagen levels might be useful for detecting inappropriate GH administration in female athletes.

OBJECTIVE: To detect exogenous recombinant human GH (rhGH) abuse in female athletes. DESIGN: GH-dependent markers were assayed in serum of 100 female athletes (control group) and in a subgroup of nine female subjects treated with rhGH (0.09 IU/kg body weight, 6 days/week for 3 weeks). METHODS: Cut-off values (mean+2 s.d.) for IGF1, N-terminal propeptide of type III procollagen (PIIINP) and C-terminal telopeptide of type I collagen (ICTP) were calculated and arbitrary scores (1.5 or 2.0) were assigned to abnormal markers. By using the sum of individual marker scores, positive (> or =3) or negative (<3) scores were obtained. RESULTS: None of the control group obtained a positive score (> or =3). Abnormal IGF1, PIIINP and ICTP levels were found in 61.4, 54.5 and 11.4% samples of the treated group. Overall, positive cases were present in 43.2% blood samples drawn in subjects treated with rhGH and in 26% of samples after rhGH withdrawal. The sensitivity of the detection approach was 66.6% at the end of 3-week rhGH treatment and 11.1% at the 15th day of rhGH withdrawal, while the specificity was 100%. CONCLUSION: Detection test for rhGH administration appears less sensitive in female (66.6%) than in male athletes (previous observation, 100% after 3 weeks of comparable rhGH dose), but shows a similar specificity (98.5-100%). Since athletes supposedly use very high doses and long-term administration of rhGH for doping purposes, it is foreseen that the here-in detection test would in future increase its strength.

Mutational analysis of the pro-opiomelanocortin gene in French obese children led to the identification of a novel deleterious heterozygous mutation located in the alpha-melanocyte stimulating hormone domain.

The pro-opiomelanocotin (POMC) plays a key role in body weight regulation, where its derived peptides mediate leptin action via the hypothalamic melanocortin 4 receptor (MC4R). The pathogenic effects of POMC mutations have been challenged in obesity. Our aim was to assess the relevance of POMC mutations in a cohort of French obese and nonobese children. Direct sequencing of the POMC gene was performed in 322 obese and 363 control unrelated children. Functional studies for the novel Phe144Leu mutation included the response to alpha-melanocyte stimulating hormone (alphaMSH) and a competitive binding assay. POMC mutations were identified in 3.72% of obese [95% confidence interval (CI): 1.66-5.80] and 2.20% of control (95% CI: 0.69-3.71) subjects. The novel mutation located in the alphaMSH region of the POMC gene (Phe144Leu) was found in one obese child and was transmitted by the obese father. Functional studies showed that MC4R activation in response to Leu144alphaMSH was almost completely abolished due to a dramatically altered binding of Leu144alphaMSH to MC4R. The frequency of POMC mutations is not significantly different between obese and control children in our cohort. The novel heterozygous mutation Phe144Leu leading to the absence of melanocortin signaling was associated with early-onset obesity suggesting its pathogenic role.

Deep subcutaneous adipose tissue: a distinct abdominal adipose depot.

OBJECTIVE: Abdominal visceral (VAT) and subcutaneous adipose tissue (SAT) display significant metabolic differences, with VAT showing a functional association to metabolic/cardiovascular disorders. A third abdominal adipose layer, derived by the division of SAT and identified as deep subcutaneous adipose tissue (dSAT), may play a significant and independent metabolic role. The aim of this study was to evaluate depot-specific differences in the expression of proteins key to adipocyte metabolism in a lean population to establish a potential physiologic role for dSAT. RESEARCH METHODS AND PROCEDURES: Adipocytes and preadipocytes were isolated from whole biopsies taken from superficial SAT (sSAT), dSAT, and VAT samples obtained from 10 healthy normal weight patients (7 women and 3 men), with a mean age of 56.4 +/- 4.04 years and a mean BMI of 23.1 +/- 0.5 kg/m2. Samples were evaluated for depot-specific differences in insulin sensitivity using adiponectin, glucose transport protein 4 (GLUT4), and resistin mRNA and protein expression, glucocorticoid metabolism by 11beta-hydroxysteroid dehydrogenase type-1 (11beta-HSD1) expression, and alterations in the adipokines leptin and tumor necrosis factor-alpha (TNF-alpha). RESULTS: Although no regional differences in expression were observed for adiponectin or TNF-alpha, dSAT whole biopsies and adipocytes, while intermediary to both sSAT and VAT, reflected more of the VAT expression profile of 11beta-HSD1, leptin, and resistin. Only in the case of the intracellular pool of GLUT4 proteins in whole biopsies was an independent pattern of expression observed for dSAT. In an evaluation of the homeostatic model, dSAT 11beta-HSD1 protein (r = 0.9573, p = 0.0002) and TNF-alpha mRNA (r = 0.8210, p = 0.0236) correlated positively to the homeostatic model. DISCUSSION: Overall, dSAT seems to be a distinct abdominal adipose depot supporting an independent metabolic function that may have a potential role in the development of obesity-associated complications.

A fluorescent method for detecting low-grade 11patUPD mosaicism in Beckwith-Wiedemann syndrome.

The quantitative evaluation of mosaicism for uniparental disomy (UPD) involving a restricted chromosomal region requires the availability of a sensitive and reproducible method that is capable of detecting even a small percentage of disomic cells and avoiding false positive and false negative results. The occurrence of UPD is usually monitored by means of the parent-proband segregation analysis of microsatellites mapping to the target region. We here describe the quantitative blood cell evaluation of segmental mosaic UPD11, a marker of Beckwith-Wiedemann syndrome, by means of the segregation analysis of 11p15 microsatellites using both radioactive and fluorescence-based techniques. As the greater amplification efficiency of the shorter allele in heterozygous subjects may bias the correct evaluation of disomy, the mean short/long allele ratio was established at three loci of each of 30 normal heterozygous subjects, as well as the peak As/Al area in the presence of 50% of each allele. The interval was defined using a 5% level of significance. The results show that the fluorescence-based technique is superior to radioactivity in detecting the subtle allelic imbalances present in low-grade mosaicism conditions.