Effects of Perilipin (PLIN) Gene Variation on Metabolic Syndrome Risk and Weight Loss in Obese Children and Adolescents

Context: Genetic polymorphisms at the perilipin (PLIN) locus have been investigated for their potential utility as markers for obesity and metabolic syndrome (MS). We examined in obese children and adolescents (OCA) aged 7-14 yr the association of single-nucleotide polymorphisms (SNP) at the PLIN locus with anthropometric, metabolic traits, and weight loss after 20-wk multi-disciplinary behavioral and nutritional treatment without medication. Design: A total of 234 OCA [body mass index (BMI = 30.4 +/- 4.4 kg/m(2); BMI Z-score = 2.31 +/- 0.4) were evaluated at baseline and after intervention. We genotyped four SNPs (PLIN1 6209T -> C, PLIN4 11482G -> A, PLIN5 13041A -> G, and PLIN6 14995A -> T). Results: Allele frequencies were similar to other populations, PLIN1 and PLIN4 were in linkage disequilibrium (D` = 0.999; P < 0.001). At baseline, no anthropometric differences were observed, but minor allele A at PLIN4 was associated with higher triglycerides (111 +/- 49 vs. 94 +/- 42 mg/dl; P = 0.003), lower high-density lipoprotein cholesterol (40 +/- 9 vs. 44 +/- 10 mg/dl; P = 0.003) and higher homeostasis model assessment for insulin resistance (4.0 +/- 2.3 vs. 3.5 +/- 2.1; P +/- 0.015). Minor allele A at PLIN4 was associated with MS risk (age and sex adjusted) hazard ratio 2.4 (95% confidence interval = 1.1-4.9) for genotype GA and 3.5 (95% confidence interval = 1.2-9.9) for AA. After intervention, subjects carrying minor allele T at PLIN6 had increased weight loss (3.3 +/- 3.7 vs. 1.9 +/- 3.4 kg; P = 0.002) and increased loss of the BMI Z-score (0.23 +/- 0.18 vs. 0.18 +/- 0.15; P +/- 0.003). Due to group size, risk of by-chance findings cannot be excluded. Conclusion: The minor A allele at PLIN4 was associated with higher risk of MS at baseline, whereas the PLIN6 SNP was associated with better weight loss, suggesting that these polymorphisms may predict outcome strategies based on multidisciplinary treatment for OCA. (J Clin Endocrinol Metab 93: 4933-4940, 2008)

Analysis of the PTPN11 gene in idiopathic short stature children and Noonan syndrome patients

Background Mutations in the PTPN11 gene are the main cause of Noonan syndrome (NS). The presence of some NS features is a frequent finding in children with idiopathic short stature (ISS). These children can represent the milder end of the NS clinical spectrum and PTPN11 is a good candidate for involvement in the pathogenesis of ISS. Objective To evaluate the presence of mutations in PTPN11 in ISS children who presented NS-related signs and in well-characterized NS patients. Patients and methods We studied 50 ISS children who presented at least two NS-associated signs but did not fulfil the criteria for NS diagnosis. Forty-nine NS patients diagnosed by the criteria of van der Burgt et al. were used to assess the adequacy of these criteria to select patients for PTPN11 mutation screening. The coding region of PTPN11 was amplified by polymerase chain reaction (PCR), followed by direct sequencing. Results No mutations or polymorphisms were found in the coding region of the PTPN11 gene in ISS children. Nineteen of the 49 NS patients (39%) presented mutations in PTPN11. No single characteristic enabled us to distinguish between NS patients with or without PTPN11 mutations. Conclusion Considering that no mutations were found in the present cohort with NS-related signs, it is unlikely that mutations would be found in unselected ISS children. The van der Burgt et al. criteria are adequate in attaining NS diagnosis and selecting patients for molecular studies. Mutations in the PTPN11 gene are commonly involved in the pathogenesis of NS but are not a common cause of ISS.

Accentuated osteoclastic response to parathyroid hormone undermines bone mass acquisition in osteonectin-null mice

Matricellular proteins play a unique role in the skeleton as regulators of bone remodeling, and the matricellular protein osteonectin (SPARC, BM-40) is the most abundant non-collagenous protein in bone In. the absence of osteonectin, mice develop progressive low turnover osteopenia, particularly affecting trabecular bone. Polymorphisms in a regulatory region of the osteonectin gene are associated with bone mass in a subset of idiopathic osteoporosis patients, and these polymorphisms likely regulate osteonectin expression. Thus it is important to determine how osteonectin gene dosage affects skeletal function. Moreover, intermittent administration of parathyroid hormone (PTH) (1-34) is the only anabolic therapy approved for the treatment of osteoporosis, and it is critical to understand how modulators of bone remodeling, such as osteonectin, affect skeletal response to anabolic agents. In this study, 10 week old female wild type, osteonectin-haploinsufficient, and osteonectin-null mice (C57Bl/6 genetic background) were given 80 mu g/kg body weight/day PTH(1-34) for 4 weeks. Osteonectin gene dosage had a profound effect on bone microarchitecture. The connectivity density of trabecular bone in osteonectin-haploinsufficient mice was substantially decreased compared with that of wild type mice, suggesting compromised mechanical properties. Whereas mice of each genotype had a similar osteoblastic response to PTH treatment, the osteoclastic response was accentuated in osteonectin-haploinsufficient and osteonectin-null mice. Eroded surface and osteoclast number were significantly higher in PTH-treated osteonectin-null mice, as was endosteal area. In vitro studies confirmed that PTH induced the formation of more osteoclast-like cells in marrow from osteonectin-null mice compared with wild type. PTH treated osteonectin-null bone marrow cells expressed more RANKL mRNA compared with wild type. However, the ratio of RANKL:OPG mRNA was somewhat lower in PTH treated osteonectin-null cultures. Increased expression of RANKL in response to PTH could contribute to the accentuated osteoclastic response in osteonectin(-/-) mice, but other mechanisms are also likely to be involved. The molecular mechanisms by which PTH elicits bone anabolic vs. bone catabolic effects remain poorly understood. Our results imply that osteonectin levels may play a role in modulating the balance of bone formation and resorption in response to PTH. (c) 2008 Elsevier Inc. All rights reserved.

The common p450 oxidoreductase variant A503V is not a modifier gene for 21-hydroxylase deficiency

Context: 21-hydroxylase deficiency (21OHD) is a common genetic disorder caused by mutations in the CYP21A2 gene, which encodes the adrenal 21-hydroxylase, microsomal P450c21. CYP21A2 gene mutations generally correlate well with impaired P450c21 enzymatic activity and the clinical findings in 21OHD, but occasional discrepancies between genotype and phenotype suggest the effects of modifier genes. Mutations in P450 oxidoreductase (POR), the protein that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate to all microsomal P450s, can ameliorate the 21OHD phenotype and, therefore, could be a modifier gene. Objectives: We sought to identify POR variants in patients with 21OHD having discordant phenotype and genotype, and to evaluate their effect on 21-hydroxylase activity. Patients and Methods: We determined the CYP21A2 genotypes of 313 Brazilian patients with 21OHD and correlated the genotype and phenotype. The POR gene was sequenced in 17 patients with discordant genotype and phenotype. Wild-type and A503V POR, and P450c21 were expressed in bacteria and reconstituted in vitro. Activities were assayed by conversion of [C-14] progesterone to deoxycorticosterone and [H-3]17-hydroxyprogesterone to 11-deoxycortisol, and assessed by thin layer chromatography and phosphorimaging. Results: The A503V POR variant was found in 10 of 30 alleles, the same ratio as in the normal population. There were no significant differences in Michaelis constant, maximum velocity and maximum velocity/Michaelis constant of 21-hydroxylase activity supported by wild-type and A503V POR. Conclusion: The only POR missense polymorphism found in atypical 21OHD patients was A503V. Although A503V reduces P450c17 enzymatic activity, it does not influence P450c21 activity, indicating that POR A503V does not modify the 21OHD phenotype.

The actual incidence of congenital adrenal hyperplasia in Brazil may not be as high as inferred - An estimate based on a public neonatal screening program in the state of Goias

The incidence of 21-hydroxylase deficiency (CYP21 D) congenital adrenal hyperplasia (CAH) in Brazil is purportedly one of the highest in the world (1:7,533). However, this information is not based on official data. The aim of this study was to determine the incidence of CYP21 D CAH in the state of Goias, Brazil, based on the 2005 results of government-funded mandatory screening. Of the live births during this period, 92.95% were screened by heel-prick capillary 17 alpha-hydroxyprogesterone (17-OHP). Of these, 82,343 were normal, 28 were at high risk for CAH and 232 at low risk for CAH. Eight cases, all from the high risk group, were confirmed. Eight asymptomatic children at 6-18 months of age still have high 17-OHP levels and await diagnostic definition. Based on the number of confirmed CYP21 D CAH cases among the 82,603 screened, the estimated annual incidence of the disease was 1:10,325, lower than the previously reported rate in Brazil.

Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild-type, and B, C or D also known as O) were evaluated using real-time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme-linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0.022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0.04) and to the control group (P = 0.011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non-responsiveness to pginterferon and ribavirin treatment (P = 0.023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.

Spectrum of MMACHC mutations in Italian and Portuguese patients with combined methylmalonic aciduria and homocystinuria, cblC type

Methylmalonic aciduria (MMA) and homocystinuria, cblC type (MIM 277400) is the most frequent inborn error of vitamin B-12. The recent identification of the disease gene, MMACHC, has permitted preliminary genotype-phenotype correlations. We studied 24 Italian and 17 Portuguese patients with cblC defect to illustrate the spectrum of mutations in a southern European population and discuss the impact that mutation identification has on routine diagnostic procedures. Since the metabolic defect raises the serum levels of homocysteine, we also tested if variants in MTHFR-playing a key role in homocysteine remethylation pathway-could act as genetic modifier in cblC defect. We found that the c.271 dupA (accounting for 55% of the MMA CH alleles in our cohort) followed by c.394C > T (16%) and c.331C > T (9%) were the most frequent mutations. In our study we also identified a novel mutation (c.544T > C). On the other hand, the MTHFR genotype did not appear to influence age at onset, the clinical phenotype and outcome of patients with cblC defect. This study shows that mutation screening for the most common MMACH mutations occurring in early-onset forms (c.271dupA and c.331C > T) seems to have a high diagnostic yield in a southern European population with cblC defect. Although the identification of the gene defect per se does not predict completely time and severity of disease appearance, our data corroborate the importance of a molecular testing to offer accurate prenatal diagnosis to couples at high risk of having affected children. (C) 2007 Elsevier Inc. All rights reserved.

The degree of external genitalia virilization in girls with 21-hydroxylase deficiency appears to be influenced by the CAG repeats in the androgen receptor gene

Background Women with 21-hydroxylase deficiency present much variability in external genitalia virilization, even among those with similar impairments of 21-hydroxylase (21OH) activity. Objective To evaluate if the number of CAG (nCAG) repeats of the androgen receptor gene influences the degree of external genitalia virilization in women with CYP21A2 mutations, grouped according to impairment of 21OH activity. Patients The nCAG was determined in 106 congenital adrenal hyperplasia (CAH) patients and in 302 controls. The patients were divided, according to their CYP21A2 genotypes, into Groups A and B, which confer total and severe impairment of 21OH activity, respectively. Methods The inactivation pattern of the X-chromosome was studied through genomic DNA digestion with Hpa II. The CAG repeat region was amplified by polymerase chain reaction (PCR) and analysed by GeneScan. Results The nCAG and the frequency of severe skewed X-inactivation did not differ between normal women and patients. The nCAG median in genotype A was 20.7 (IQR 2.3) for Prader I + II, 22.5 (3.6) for Prader III and 21 (2.9) for Prader IV + V (P < 0.05 for Prader III and Prader IV + V). The nCAG median in genotype B was 21.3 (1.1) for Prader I + II, 20.5 (2.9) for Prader III and 22 (2.8) for Prader IV + V (P > 0.05). A significant difference was found regarding the nCAG median in patients presenting Prader III from genotypes A and B. Conclusions We observed great variability in the degree of external genitalia virilization in both CYP21A2 genotypes, and we showed that the CAG repeats of the androgen receptor gene influences this phenotypic variability.

Neurological manifestations and ATP7B mutations in Wilson`s disease

Wilson`s disease (WD) is a rare inborn metabolic error characterized by deficient biliary copper excretion secondary to ATP7B gene mutations. Neurological presentations are variable in respect to both pattern and age of onset; commonly a movement disorder presents in the second or third decade. The aim of this study was to ascertain genotype correlations with distinct neurological manifestations in 41 WD patients in a Brazilian center for WD. A total of 23 distinct mutations were detected, and the frameshift 3402de1C had the highest allelic frequency (31.7%). An association between 3402de1C and dysphagia was detected (p = 0.01) but the limited number of patients is insufficient to allow one to draw conclusions. Both clinical studies analyzing larger cohorts and basic research on ATP7B protein function could potentially shed more light on our understanding of WD. (c) 2007 Elsevier Ltd. All rights reserved.

Risk Profiles and Penetrance Estimations in Multiple Endocrine Neoplasia Type 2A Caused by Germline RET Mutations Located in Exon 10

Multiple endocrine neoplasia type 2 is characterized by germline mutations in RET. For exon 10, comprehensive molecular and corresponding phenotypic data are scarce. The International RET Exon 10 Consortium, comprising 27 centers from 15 countries, analyzed patients with RET exon 10 mutations for clinical-risk profiles. Presentation, age-dependent penetrance, and stage at presentation of medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism were studied. A total of 340 subjects from 103 families, age 4-86, were registered. There were 21 distinct single nucleotide germline mutations located in codons 609 (45 subjects), 611 (50), 618 (94), and 620 (151). MTC was present in 263 registrants, pheochromocytoma in 54, and hyperparathyroidism in 8 subjects. Of the patients with MTC, 53% were detected when asymptomatic, and among those with pheochromocytoma, 54%. Penetrance for MTC was 4% by age 10, 25% by 25, and 80% by 50. Codon-associated penetrance by age 50 ranged from 60% (codon 611) to 86% (620). More advanced stage and increasing risk of metastases correlated with mutation in codon position (609-620) near the juxtamembrane domain. Our data provide rigorous bases for timing of premorbid diagnosis and personalized treatment/prophylactic procedure decisions depending on specific RET exon 10 codons affected. Hum Mutat 32:51-58, 2011. (C) 2010 Wiley-Liss, Inc.

Superior discriminating value of ACTH-stimulated serum 21-deoxycortisol in identifying heterozygote carriers for 21-hydroxylase deficiency

P>Background Congenital adrenal hyperplasia caused by classic 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder with a high prevalence of asymptomatic heterozygote carriers (HTZ) in the general population, making case detection desirable by routine methodology. HTZ for classic and nonclassic (NC) forms have basal and ACTH-stimulated values of 17-hydroxyprogesterone (17OHP) that fail to discriminate them from the general population. 21-Deoxycortisol (21DF), an 11-hydroxylated derivative of 17OHP, is an alternative approach to identify 21OHD HTZ. Objective To determine the discriminating value of basal and ACTH-stimulated serum levels of 21DF in comparison with 17OHP in a population of HTZ for 21OHD (n = 60), as well as in NC patients (n = 16) and in genotypically normal control subjects (CS, n = 30), using fourth generation tandem mass spectrometry after HPLC separation (LC-MS/MS). Results Basal 21DF levels were not different between HTZ and CS, but stimulated values were increased in the former and virtually nonresponsive in CS. Only 17 center dot 7% of the ACTH-stimulated 21DF levels overlapped with CS, when compared to 46 center dot 8% for 17OHP. For 100% specificity, the sensitivities achieved for ACTH-stimulated 21DF, 17OHP and the quotient [(21DF + 17OHP)/F] were 82 center dot 3%, 53 center dot 2% and 87%, using cut-offs of 40, 300 ng/dl and 46 (unitless), respectively. Similar to 17OHP, ACTH-stimulated 21DF levels did not overlap between HTZ and NC patients. A positive and highly significant correlation (r = 0 center dot 846; P < 0 center dot 001) was observed between 21DF and 17OHP pairs of values from NC and HTZ. Conclusion This study confirms the superiority of ACTH-stimulated 21DF, when compared to 17OHP, both measured by LC-MS/MS, in identifying carriers for 21OHD. Serum 21DF is a useful tool in genetic counselling to screen carriers among relatives in families with affected subjects, giving support to molecular results.

High Frequency of Lamivudine Resistance Mutations in Brazilian Patients Co-Infected With HIV and Hepatitis B

This study analyzed the genotype distribution and frequency of lamivudine (LAM) and tenofovir (TDF) resistance mutations in a group of patients co-infected with HIV and hepatitis B virus (HBV). A cross-sectional study of 847 patients with HIV was conducted. Patients provided blood samples for HBsAg detection. The load of HBV was determined using an ""in-house"" real-time polymerase chain reaction. HBV genotypes/subgenotypes, antiviral resistance, basal core promoter (BCP), and precore mutations were detected by DNA sequencing. Twenty-eight patients with co-infection were identified. The distribution of HBV genotypes among these patients was A (n = 9; 50%), D (n = 4; 22.2%), G (n = 3; 16.7%), and F (n = 2; 11.1%). Eighteen patients were treated with LAM and six patients were treated with LAM plus TDF. The length of exposure to LAM and TDF varied from 4 to 216 months. LAM resistance substitutions (rtL180M + rtM204V) were detected in 10 (50%) of the 20 patients with viremia. This pattern and an accompanying rtV173L mutation was found in four patients. Three patients with the triple polymerase substitution pattern (rtV173L+ rtL180M + rtM204V) had associated changes in the envelope gene (sE164D + sl195M). Mutations in the BCP region (A1762T, G1764A) and in the precore region (G1896A, G1899A) were also found. No putative TDF resistance substitution was detected. The data suggest that prolonged LAM use is associated with the emergence of particular changes in the HBV genome, including substitutions that may elicit a vaccine escape phenotype. No putative TDF resistance change was detected after prolonged use of TDF. J. Med. Virol. 82:1481-1488, 2010. (C) 2010 Wiley-Liss, Inc.

The role of physiological understanding in plant breeding; From a breeding perspective

The role of physiological understanding in improving the efficiency of breeding programs is examined largely from the perspective of conventional breeding programs. Impact of physiological research to date on breeding programs, and the nature of that research, was assessed from (i) responses to a questionnaire distributed to plant breeders and physiologists, and (ii) a survey of literature abstracts. Ways to better utilise physiological understanding for improving breeding programs are suggested, together with possible constraints to delivering beneficial outcomes. Responses from the questionnaire indicated a general view that the contribution by crop physiology to date has been modest. However, most of those surveyed expected the contribution to be larger in the next 20 years. Some constraints to progress perceived by breeders and physiologists were highlighted. The survey of literature abstracts indicated that from a plant breeding perspective, much physiological research is not progressing further than making suggestions about possible approaches to selection. There was limited evidence in the literature of objective comparison of such suggestions with existing methodology, or of development and application of these within active breeding programs. It is argued in this paper that the development of outputs from physiological research for breeding requires a good understanding of the breeding program(s) being serviced and factors affecting its performance. Simple quantitative genetic models, or at least the ideas they represent, should be considered in conducting physiological research and in envisaging and evaluating outputs. The key steps of a generalised breeding program are outlined, and the potential pathways for physiological understanding to impact on these steps are discussed. Impact on breeding programs may arise through (i) better choice of environments in which to conduct selection trials, (ii) identification of selection criteria and traits for focused introgression programs, and (iii) identifying traits for indirect selection criteria as an adjunct to criteria already used. While many breeders and physiologists apparently recognise that physiological understanding may have a major role in the first area, there appears to be relatively Little research activity targeting this issue, and a corresponding bias, arguably unjustified, toward examining traits for indirect selection. Furthermore, research on traits aimed at crop improvement is often deficient because key genetic parameters, such as genetic variation in relevant breeding populations and genetic (as opposed to phenotypic) correlations with yield or other characters of economic importance, are not properly considered in the research. Some areas requiring special attention for successfully interfacing physiology research with breeding are discussed. These include (i) the need to work with relevant genetic populations, (ii) close integration of the physiological research with an active breeding program, and (iii) the dangers of a pre-defined or narrow focus in the physiological research.

Insertion/deletion polymorphism of the angiotensin converting enzyme gene and loss of the insertion allele in aldosterone-producing adenoma

The genetic mechanisms responsible for the formation of adrenocortical adenomas which autonomously produce aldosterone are largely unknown, The adrenal renin-angiotensin system has been implicated in the pathophysiology of these tumours, Angiotensin-converting enzyme (ACE) catalyses the generation of angiotensin II, and the insertion/deletion (I/D) polymorphism of the ACE gene regulates up to 50% of plasma and cellular ACE variability in humans. We therefore examined the genotypic and allelic frequency distributions of the ACE gene I/D polymorphism in 55 patients with aldosterone-producing adenoma, APA, (angiotensin-unresponsive APA n = 28, angiotensin-responsive APA n = 27), and 80 control subjects with no family history of hypertension, We also compared the ACE gene I/D polymorphism allelic pattern in matched tumour and peripheral blood DNA in the 55 patients with APA, The frequency of the D allele was 0.518 and 0.512 and the I allele was 0.482 and 0.488 in the APA and control subjects respectively, Genotypic and allelic frequency analysis found no significant differences between the groups, Examination of the matched tumour and peripheral blood DNA samples revealed the loss of the insertion allele in four of the 25 patients who were heterozygous for the ACE I/D genotype. The I/D polymorphism of the ACE gene does not appear to contribute to the biochemical and phenotypic characteristics of APA, however, the deletion of the insertion allele of the ACE gene I/D polymorphism in 16% of aldosterone-producing adenomas may represent the loss of a tumour suppressor gene/s or other genes on chromosome 17q which may contribute to tumorigenesis in APA.

The shoot controls zeatin riboside export from pea roots. Evidence from the branching mutant rms4

The rms4 mutant of pea (Pisum sativum L.) was used in grafting studies and cytokinin analyses of the root xylem sap to provide evidence that, at least for pea, the shoot can modify the import of cytokinins from the root. The rms4 mutation, which confers a phenotype with increased branching in the shoot, causes a very substantial decrease (down to 40-fold less) in the concentration of zeatin riboside (ZR) in the xylem sap of the roots. Results from grafts between wild-type (WT) and rms4 plants indicate that the concentration of cytokinins in the xylem sap of the roots is determined almost entirely by the genotype of the shoot. WT scions normalize the cytokinin concentration in the sap of rms4 mutant roots, whereas mutant scions cause WT roots to behave like those of self-grafted mutant plants. The mechanism whereby rms4 shoots of pea cause a down-regulation in the export of cytokinins from the roots is unknown at this time. However, our data provide evidence that the shoot transmits a signal to the roots and thereby controls processes involved in the regulation of cytokinin biosynthesis in the root.