Whole Exome Sequencing in Inherited Endocrine Disorders

tout ensemble Exome Sequencing in Inherited Endocrine DisordersBackgroundMolecular diagnosis is important in the management of various paediatric endocrine gland disorders including disorders of return, metabolism, bone, hypoglycaemia and sexual development. Traditional PCR-based Sanger sequencing is the mainstay format for molecular interrogation in paediatric cases. However, the large number of gene defects associated with the various endocrine disorders renders gene-by-gene testing increasingly expensive and unattractive. The large number of potentially relevant genes makes it challenging for hospital molecular diagnostic laboratories to offer gene-based testing of all candidates. Given the gamy costs associated with single-gene tests, the selection of candidates for single-gene sequencing tends to be sequential rather than inclusive and parallel. In practice, different genes may be outsourced to different clinical or in whatever cases academic research laboratories which a dds to the complexity. Using new high-throughput sequencing technologies, safe and sound genomes, whole exomes or candidate-gene panels (targeted gene sequencing) can now be cost-effectively sequenced for endocrine patient ofs. In the near future, protocols involving next- times sequencing would believably be considered as an appropriate component of routine clinical diagnosis for relevant patients.Defects of pituitary endocrine glands lead to abnormalities in growth (e.g., short altitude), sexual development, fertility, tension response and other metabolic processes. A number of genes coding for arrangement factors fix been identified, mutations in which cause medical disorders in humans associated with pituitary deficiencies 1-2. any(prenominal) of these factors, such as PROP1, TPIT, POU1F1, LHX3 and LHX4, play roles in the normal embryological development of the anterior pituitary. Mutations in these genes can lead to multiple pituitary hormone deficiencies and/or syndro mic hypopituitarism 3.The transcription factors such as HESX1, OTX2, SHH, SOX2 and SOX3 argon involved in midline development. Mutations in these can cause septo-optic dysplasia or holoprosencephaly, both of which may include pituitary hormone deficiencies 4. Other genes encode the precursors to pituitary hormones (growth hormone, ACTH through processing of POMC, gonadotropic-luteinizing hormone and follicle-stimulating hormone, and thyroid-stimulating hormone). Mutations in these genes lead to phenotypes characteristic of individual hormone deficiency. The pituitary secretory cells themselves respond to signals originating in the hypothalamus, some of which are also peptide hormones with proper(postnominal) receptors expressed on the responding cells mutations in these genes or their cognate receptors can also cause combined or circumstantial pituitary deficiencies 1. However, many cases of congenital hypopituitarism still remain unexplained and most are presumably due to other c auses, either mutations in other developmental genes or epi hereditary influences during embryogenesis.Short stature is a common presentation to the paediatric endocrinology clinics. However, no cause is identified in a large proportion of patients who are classified as having idiopathic short stature 5, 6, 7. It is estimated that the underlying cause for short stature remains unknown in approximately 80% of patients 8. In a large-scale pooled Next-Generation Sequencing study to identify transmissible causes of short stature, 4928 genetic variants in 1077 genes were present in patients but not in control subjects 9. big sequencing efforts have the potential to rapidly identify genetic aetiologies of short stature. In another study, seeking to identify known and genetic causes of short stature by conducting whole exome sequencing of the patients with severe short stature and their family members, genetic cause of short stature was found in 5 out of the 14 recruited patients 10. Rar e genetic defects in the GH/IGF-1 axis have been found to cause short stature. A higher frequency of rare CNVs (common number variants) has been reported in patients with short stature 8, 11. A recent study to define genetic characterisation of a cohort of children clinically labelled as Growth Hormone or IGF1 unresponsive found that whole exome sequencing contributed to the diagnosis of children with suspected growth hormone and IGF1 insensitivity, particularly in the Growth hormone insensitive subjects with low serum IGF1 SDS and height SDS 12. It may be now possible to identify likely genetic causes of short stature by implementing genomic investigative techniques like whole exome sequencing in many of these children who have unknown reasons for their poor linear growth.Congenital Hyperinsulinism (qi) is the most common cause of persistent and recurrent hypoglycaemia in infancy 13. It is the result of unregulated insulin secernment from the pancreatic -cells leading to severe h ypoglycaemia 13, 14. This condition has been reported in nearly all major ethnic groups and affects at least 1/50,000 children of European descent 14. CHI is caused by genetic defects in key genes regulating insulin secretion. The genetic basis of CHI involves mutations in nine different genes (ABCC8, KCNJ11, GLUD1, GCK, HADH, SLC16A1, HNF4A, HNF1A and UCP2), which regulate insulin secretion from the pancreatic -cells 14,15. The most common molecular cause of CHI is the dysfunction of the pancreatic KATP run encoded by the sulfonylurea receptor gene (ABCC8) and the inward rectifying potassium channel gene (KCNJ11) 14,15.CHI can also be secondary to risk factors like birth asphyxia, intra-uterine growth retardation, Rh isoimmunisation and maternal diabetes mellitus or associated with various developmental syndromes 16. Histologically, CHI can be associated either with diffuse insulin secretion or with focal adenomatous hyperplasia. Positron emission tomography scan using Fluorine-18 L-3, 4-dihydroxyphenylalanine (18-fluoro DOPA-TC-PET-scan) has been used to tick focal from diffuse forms. Medical treatments of CHI include diazoxide (KATP channel activator), somatostatin analogue (octreotide) injections, and appropriate diet. The surgical treatment with subtotal pancreatectomy is required in diffuse CHI when medical treatment and dietary therapies are ineffective, whereas focal CHI can be cured with resection of the focal area of adenomatous hyperplasia 14, 15, 16. Recently, mammalian target of rapamycin (mTOR) inhibitor sirolimus has been used in treatment of persistent severe CHI not amenable to medical therapies 18. CHI has been described as an associated finding in various syndromes like Beckwith-Wiedemann, Kabuki, Trisomy 13, Mosaic Turner, Sotos, Usher, Timothy, Costello, Central Hypoventilation syndrome and Leprechaunism (Insulin Resistance Syndrome) 17. However, in many patients, with clinically defined syndromic features and with hypoglycaemia, no ide ntifiable genetic cause contributing to hyperinsulinism is found. In a large serial of 300 patients, genetic diagnosis was made only in 45.3% of the patients and mutations in ABCC8 were the commonest identifiable cause 19. The vast majority of patients with Diazoxide-responsive CHI (77.6%) had no identifiable mutations, suggesting other genetic mechanisms 19. Molecular diagnosis can be very important for clinicians to manage the patients more effectively and to counsel parents on the prognosis and disease recurrence. Whole Exome sequencing can be advantageous in these groups of patients to identify the molecular defects and to assess the coding variants that may be pathogenic in these patients 20.AimsTo identify novel genetic causes of rare inherited endocrine disorders in children with a focus on congenital hyperinsulinism, short stature of unknown etiology and IGF1 abnormalities by using whole exome sequencing.Experimental Design and MethodsPatient RecruitmentPatients with a diag nosis of CHI referred to alder tree Hey Childrens Hospital, which is a national referral centre for CHI, provide be recruited into the study. A written informed parental consent will be obtained. These patients will be biochemically confirmed as CHI using the following criteria snag glucose concentration of less than 3.0 mmol/l with detectable insulin and/or C-peptideGlucose requirement 8mg/kg/minLow levels of ketones and fatty acids during the episode of hypoglycaemiaClinical and biochemical selective information will be collated from referral letter or by case note review. Patients with a secondary cause of CHI such as perinatal asphyxia, intra-uterine growth restriction, Rhesus isoimmunisation, infants of diabetic mothers and infants with Beckwith Wiedemann syndrome will be excluded from the study.Patients are considered to be unresponsive to medical treatment if recurrent hypoglycaemia episodes (Patients attending the Paediatric Endocrinology clinic at Alder Hey Childrens Hosp ital with severe short stature (3 SDS below mean) for age and sex in whom the standard clinical work up has not revealed a diagnosis for their short stature will be recruited into the study. Patients referred or evaluated for growth hormone insensitivity (growth failure, low serum IGF1 and normal/elevated serum GH) or IGF1 insensitivity (pre- and postnatal growth failure associated with relatively high IGF1 levels) will also be recruited into the study.A written parental informed consent will be obtained prior to the recruitment.Whole Exome Sequencing (WES)WES will be performed at the Centre for Genomic Research (CGR) based at the University of Liverpool. The test will be ordered after(prenominal) explaining the risks and benefits of testing to the patient and obtaining written informed consent. Each patient (and their parents or guardians) will be advised of the potential disclosure of conditions unrelated to the indication for testing that might warrant treatment or additional me dical surveillance for the patient and possibly other family members.Peripheral-blood samples will be obtained to isolate DNA from the patient and both parents where possible. High-throughput sequencing will be performed using Illumina HiSeq2500. The genomic DNA samples from probands will be fragmented, ligated to Illumina multiplexing and amplified by means of a polymerase-chain-reaction assay with the use of primers with sequencing barcodes. Variants that were deemed clinically significant will be confirmed by means of Sanger sequencing. Parental samples, if available, will also be analysed by means of Sanger sequencing. Further functional analysis will be undertaken to establish the pathogenicity of the identified variant by utilising the in-house lab facilities at Institute of Child Health.Relevance of the proposed projectDespite the advances in understanding the molecular pathogenesis, specific genetic determinants are not known in nearly 50% of patients with CHI and 80% of chi ldren with short stature. Whole exome sequencing in this group of patients will serve well to understand and identify the potential causative mutations in genes implicated in insulin regulation and growth. This will help the clinicians to provide optimal treatment and to counsel patients on disease progression and recurrence risk. Identification of novel genetic aetiology has the potential to identify novel therapeutic strategies for these patients. The applicant will spend season initially at Alder Hey Childrens Hospital to recruit patients and then(prenominal) at Cincinnati Childrens Hospital to learn the techniques of WES, bioinformatics and functional analysis. The applicant will then return to Alder Hey Childrens Hospital Institute of Child Health, University of Liverpool to complete the study and strengthen the local research expertise relevant to next generation sequencing.