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Weedon M.N.,University of Exeter | Cebola I.,Institute dInvestigacions Biomediques August Pi i Sunyer | Cebola I.,Research Center Biomedica En Red Of Diabetes fermedades Metabolicas | Cebola I.,Imperial College London | And 22 more authors.
Nature Genetics | Year: 2014

The contribution of cis-regulatory mutations to human disease remains poorly understood. Whole-genome sequencing can identify all noncoding variants, yet the discrimination of causal regulatory mutations represents a formidable challenge. We used epigenomic annotation in human embryonic stem cell (hESC)-derived pancreatic progenitor cells to guide the interpretation of whole-genome sequences from individuals with isolated pancreatic agenesis. This analysis uncovered six different recessive mutations in a previously uncharacterized ∼400-bp sequence located 25 kb downstream of PTF1A (encoding pancreas-specific transcription factor 1a) in ten families with pancreatic agenesis. We show that this region acts as a developmental enhancer of PTF1A and that the mutations abolish enhancer activity. These mutations are the most common cause of isolated pancreatic agenesis. Integrating genome sequencing and epigenomic annotation in a disease-relevant cell type can thus uncover new noncoding elements underlying human development and disease. © 2014 Nature America, Inc. Source

Christesen H.T.,University of Southern Denmark | Brusgaard K.,University of Southern Denmark | Hussain K.,London Center for Paediatric Endocrinology and Metabolism | Hussain K.,Institute of Child Health
Clinical Endocrinology | Year: 2012

Objective: Hypoglycaemia-associated autonomic failure (HAAF) with impaired neurogenic and neuroglycopaenic responses occurs in adults following recent, repeated hypoglycaemia. We aimed to evaluate whether HAAF also occurs in patients with infant-onset congenital hyperinsulinism (CHI). Design, patients: A controlled fast was performed in (i) seven CHI infants with initial symptomatic hypoglycaemia and three recent episodes of spontaneous recurrent hypoglycaemia each lasting <5 min and in (ii) seven infants with idiopathic ketotic hypoglycaemia for control. Measurements: At the time of hypoglycaemia (blood glucose <3 mmol/l or clinical signs), blood was drawn for serum insulin, cortisol, glucagon, adrenalin and nor-adrenalin. Signs of hypoglycaemia were documented. In CHI patients, the ABCC8 and KCNJ11 genes were analysed by denaturing high performance liquid chromatography (DHPLC) and/or direct bidirectional sequencing. Results: Two CHI patients had a paternal ABCC8 mutation, five had no mutations. When repeated hypoglycaemia was provoked, all CHI patients exhibited a complete loss of clinical signs of hypoglycaemia, along with a global blunting of the counter-regulatory hormones cortisol, glucagon, growth hormone, adrenalin and noradrenalin responses (median values 256 nmol/l, 23 pmol/l, 5.6 mU/l, 390 pmol/l and 2.9 nmol/l, respectively), irrespective of mutational status. In the controls, hypoglycaemia was always clinically overt with normal counter-regulatory cortisol, glucagon, adrenalin and nor-adrenalin responses (530 nmol/l, 60, 920 pmol/l and 4.0 nmol/l, respectively). Conclusion: Recurrent hyperinsulinaemic hypoglycaemia even of short duration blunts the autonomic, neuroglycopaenic and glucose counter-regulatory hormonal responses in patients with infant-onset CHI resulting in clinically silent hypoglycaemia. Tight, or continuous, glucose monitoring is therefore recommended, especially in conservatively treated patients. © 2012 Blackwell Publishing Ltd. Source

Khalid J.M.,University College London | Oerton J.M.,University College London | Dezateux C.,University College London | Hindmarsh P.C.,London Center for Paediatric Endocrinology and Metabolism | And 2 more authors.
Archives of Disease in Childhood | Year: 2012

Objectives: To estimate the incidence of clinically diagnosed congenital adrenal hyperplasia (CAH), clinical features and age at first presentation. To assess the potential benefit of newborn screening for CAH. Design: Active surveillance through the British Paediatric Surveillance Unit of all children aged under 16 years with newly diagnosed CAH, undertaken prospectively between August 2007 and August 2009. Twelve laboratories testing for CAH reported new diagnoses between August 2007 and January 2009. Reporting clinicians completed clinical questionnaires. Setting: England, Wales and Scotland. Results: 144 children with CAH were reported, of whom 132 (92%) had 21-hydroxylase deficiency. Thirty-six (25%) children were Asian and 62 (43%; 95% CI 35% to 51%) were boys. Incidence of new diagnoses in children ≤16 years was 0.60 (95% CI 0.50 to 0.71) per 100 000. Eighty-six (59%; 36 boys) children were diagnosed in the first year of life (estimated birth prevalence 5.48 (95% CI 4.42 to 6.81) per 100 000), most (77; 89%) of whom presented in the first month of life. Virilised genitalia were found in three-quarters of girls. Twenty-seven newborns first presented with salt-wasting crises, of whom 18 (67%; 16 boys) presented on or after 14 days of age. Conclusions: Approximately one child in every 18 000 born in Great Britain has CAH. Similar numbers of boys and girls present clinically in the first year of life, but boys present with more severe manifestations, such as salt-wasting crises. Around 70% of newborns who first present with salt-wasting crisis would be detected earlier through newborn screening. Source

Knowles R.L.,University College London | Khalid J.M.,University College London | Oerton J.M.,University College London | Hindmarsh P.C.,London Center for Paediatric Endocrinology and Metabolism | And 2 more authors.
Archives of Disease in Childhood | Year: 2014

Objectives: To describe the clinical presentation and sequelae, including salt-wasting crises of newly-diagnosed congenital adrenal hyperplasia (CAH) in children aged over 1 year in a contemporary population without screening. To appraise the potential benefit of newborn screening for late-presenting CAH. Design: Active national surveillance undertaken in Great Britain prospectively from 2007-2009 through the British Paediatric Surveillance Unit. Setting: England, Wales and Scotland. Patients: Children first presenting aged 1-15 years with clinical features of CAH and elevated 17-hydroxyprogesterone. Results: Fifty-eight children (26 [45%] boys) aged 1-15 years were reported; 50 (86%) had 21-hydroxylase deficiency. Diagnosis was precipitated by secondary sexual characteristics (n=38 [66%]; median age 5.8 [IQR] 4.8, 7.6) years, genital virilisation (8 girls; 3.2 [IQR 1.3, 7.3] years) or an affected sibling (n=8; 10.0 [IQR 7.4, 13.3] years). At least 33 (57%) children had advanced bone age and 13 (30%) were obese (body mass index ≥95th centile). No child had experienced a salt-wasting crisis. Conclusions: In Great Britain, 30 children aged 1-15 years present annually for the first time with CAH. Older children frequently manifest prematurely advanced epiphyseal and pubertal maturation and genital virilisation, which are often irreversible and likely to have long-lasting consequences for adult health and wellbeing. Almost one-third of affected children are obese before commencing steroid therapy. Newborn screening offers the potential to avoid serious clinical manifestations in older children with unrecognised CAH; however, it may also detect some children who would otherwise remain asymptomatic and for whom the benefit from treatment is uncertain. Source

Flanagan S.E.,University of Exeter | Xie W.,University of Exeter | Caswell R.,University of Exeter | Damhuis A.,Royal Devon and Exeter National Health Service Foundation Trust | And 20 more authors.
American Journal of Human Genetics | Year: 2013

Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ∼10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals' fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease. © 2013 The American Society of Human Genetics. Source

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