Very small deletions within the NESP55 gene in pseudohypoparathyroidism type 1b

Pseudohypoparathyroidism (PHP) is caused by reduced expression of genes within the GNAS cluster, resulting in parathormone resistance. The cluster contains multiple imprinted transcripts, including the stimulatory G protein α subunit (Gs-α) and NESP55 transcript preferentially expressed from the maternal allele, and the paternally expressed XLas, A/B and antisense transcripts. PHP1b can be caused by loss of imprinting affecting GNAS A/B alone (associated with STX16 deletion), or the entire GNAS cluster (associated with deletions of NESP55 in a minority of cases). We performed targeted genomic next-generation sequencing (NGS) of the GNAS cluster to seek variants and indels underlying PHP1b. Seven patients were sequenced by hybridisation-based capture and fourteen more by long-range PCR and transposon-mediated insertion and sequencing. A bioinformatic pipeline was developed for variant and indel detection. In one family with two affected siblings, and in a second family with a single affected individual, we detected maternally inherited deletions of 40 and 33 bp, respectively, within the deletion previously reported in rare families with PHP1b. All three affected individuals presented with atypically severe PHP1b; interestingly, the unaffected mother in one family had the detected deletion on her maternally inherited allele. Targeted NGS can reveal sequence changes undetectable by current diagnostic methods. Identification of genetic mutations underlying epigenetic changes can facilitate accurate diagnosis and counselling, and potentially highlight genetic elements critical for normal imprint settin

Waist circumference predicts clustering of cardiovascular risk factors in older South Africans

Objecti~'eand design. A cross-sectional analytical study to determine the cardiovascular risk factor profile of older residents of fishing villages on the West Coast of South Africa, and to det.ermine which anthropometric measures are associated with risk factors. Subjects. A convenient community-based sample of 152 subjects of mixed ancestry aged 55 years and over was recruited door-to-door using an address list of ageeligible subjects provided by the local public health care clinics. Methods. Cardiovascular relationships were im'estigated between: (i) number of risk factors (hypertension, hypercholesterolaemia, diabetes) and body mass index (8MI), waist-to-hip ratio (WHR), and waist circumference; and (ii) continuous cardiovascular risk factor variables and physical activity, smoking, dietary intake, and 24-hour urinary sodium and potassium concentrationS. Results. The prevalence of hypertension (~ 160/95 mmHg) was 74.3% (95% CI: 67.2 - 81.4%). Neither 24- hour urinary sodium nor potassium concentrations was associated with blood pressure (BP). Past, but not present, moderate-intensity physical activity, particularly that associated with occupation, was negatively associated with systolic BP (r = -0.24, P < 0.05). The prevalence of diabetes and hypercholesterolaemia (serum cholesterol ~ 6.5 mmolll) was 24.6% (95% CI: 17.2 - 32%) and 40% (95% CI: 31.8 - 48.2%),respectively. The percentage of subjects with 0, 1, or 2 or more cardiovascular risk factors was 13.4%,44.1% and 42.5%, respectively. Subjects with a waist circumference ~ 92 em had a significantly higher number of cardiovascular risk factors than those with a waist circumference < 92 cm (Xl =9.29, P < 0.01), and this association remained significant even after controlling for age, sex and smoking (P < 0.05). Neither 8MI tertiles according to sex, nor a 8MI cut-point ~ 30, was significantly associated with a clustering of risk factors. COllclusioll. In a sample of older South Africans of ntlxed ancestry at high risk of cardiovascular disease, waist circumference ~ 92 em predicts clustering of risk factors, Independentl)' of BMI. This simple, populationspecific reference value may provide a useful screening tool to identify at-risk Individuals for targeted prevention for coronary heart disease and associated metabolic disorders

Congenital imprinting disorders: EUCID.net - a network to decipher their aetiology and to improve the diagnostic and clinical care

Imprinting disorders (IDs) are a group of eight rare but probably underdiagnosed congenital diseases affecting growth, development and metabolism. They are caused by similar molecular changes affecting regulation, dosage or the genomic sequence of imprinted genes. Each ID is characterised by specific clinical features, and, as each appeared to be associated with specific imprinting defects, they have been widely regarded as separate entities. However, they share clinical characteristics and can show overlapping molecular alterations. Nevertheless, IDs are usually studied separately despite their common underlying (epi) genetic aetiologies, and their basic pathogenesis and long-term clinical consequences remain largely unknown. Efforts to elucidate the aetiology of IDs are currently fragmented across Europe, and standardisation of diagnostic and clinical management is lacking. The new consortium EUCID.net (European network of congenital imprinting disorders) now aims to promote better clinical care and scientific investigation of imprinting disorders by establishing a concerted multidisciplinary alliance of clinicians, researchers, patients and families. By encompassing all IDs and establishing a wide ranging and collaborative network, EUCID.net brings together a wide variety of expertise and interests to engender new collaborations and initiatives

Imprinting disorders: a group of congenital disorders with overlapping patterns of molecular changes affecting imprinted loci

Congenital imprinting disorders (IDs) are characterised by molecular changes affecting imprinted chromosomal regions and genes, i.e. genes that are expressed in a parent-of-origin specific manner. Recent years have seen a great expansion in the range of alterations in regulation, dosage or DNA sequence shown to disturb imprinted gene expression, and the correspondingly broad range of resultant clinical syndromes. At the same time, however, it has become clear that this diversity of IDs has common underlying principles, not only in shared molecular mechanisms, but also in interrelated clinical impacts upon growth, development and metabolism. Thus, detailed and systematic analysis of IDs can not only identify unifying principles of molecular epigenetics in health and disease, but also support personalisation of diagnosis and management for individual patients and families

Congenital imprinting disorders: EUCID.net - a network to decipher their aetiology and to improve the diagnostic and clinical care.

Imprinting disorders (IDs) are a group of eight rare but probably underdiagnosed congenital diseases affecting growth, development and metabolism. They are caused by similar molecular changes affecting regulation, dosage or the genomic sequence of imprinted genes. Each ID is characterised by specific clinical features, and, as each appeared to be associated with specific imprinting defects, they have been widely regarded as separate entities. However, they share clinical characteristics and can show overlapping molecular alterations. Nevertheless, IDs are usually studied separately despite their common underlying (epi)genetic aetiologies, and their basic pathogenesis and long-term clinical consequences remain largely unknown. Efforts to elucidate the aetiology of IDs are currently fragmented across Europe, and standardisation of diagnostic and clinical management is lacking. The new consortium EUCID.net (European network of congenital imprinting disorders) now aims to promote better clinical care and scientific investigation of imprinting disorders by establishing a concerted multidisciplinary alliance of clinicians, researchers, patients and families. By encompassing all IDs and establishing a wide ranging and collaborative network, EUCID.net brings together a wide variety of expertise and interests to engender new collaborations and initiatives

Effectiveness of continuous glucose monitoring in pregnant women with diabetes: randomised clinical trial

Objective To evaluate the effectiveness of continuous glucose monitoring during pregnancy on maternal glycaemic control, infant birth weight, and risk of macrosomia in women with type 1 and type 2 diabetes

Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli

Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, which can select against recombinant clones containing open reading frames or repetitive DNA. Conversely, transcription from cloned promoters can interfere with plasmid stability. We have therefore developed a novel Escherichia coli cloning vector (termed ‘pJAZZ’ vector) that is maintained as a linear plasmid. Further, it contains transcriptional terminators on both sides of the cloning site to minimize transcriptional interference between vector and insert. We show that this vector stably maintains a variety of inserts that were unclonable in conventional plasmids. These targets include short nucleotide repeats, such as those of the expanded Fragile X locus, and large AT—rich inserts, such as 20-kb segments of genomic DNA from Pneumocystis, Plasmodium, Oxytricha or Tetrahymena. The pJAZZ vector shows decreased size bias in cloning, allowing more uniform representation of larger fragments in libraries

Mutations in the DNA methyltransferase gene DNMT3A cause an overgrowth syndrome with intellectual disability

Overgrowth disorders are a heterogeneous group of conditions characterized by increased growth parameters and other variable clinical features such as intellectual disability and facial dysmorphism. To identify new causes of human overgrowth, we performed exome sequencing in ten proband-parent trios and detected two de novo DNMT3A mutations. We identified 11 additional de novo mutations by sequencing DNMT3A in a further 142 individuals with overgrowth. The mutations alter residues in functional DNMT3A domains, and protein modeling suggests that they interfere with domain-domain interactions and histone binding. Similar mutations were not present in 1,000 UK population controls (13/152 cases versus 0/1,000 controls; P < 0.0001). Mutation carriers had a distinctive facial appearance, intellectual disability and greater height. DNMT3A encodes a DNA methyltransferase essential for establishing methylation during embryogenesis and is commonly somatically mutated in acute myeloid leukemia. Thus, DNMT3A joins an emerging group of epigenetic DNA- and histone-modifying genes associated with both developmental growth disorders and hematological malignancies