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Title: Whole exome sequencing in children with rare endocrine disorders
Author: Giri Harirambapu, Dinesh
ISNI:       0000 0004 7428 4405
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Background: Congenital Hyperinsulinism (CHI) is characterized by unregulated secretion of insulin in the presence of hypoglycaemia. Mutations in eleven different genes ABCC8, KCNJ11, GLUD1, GCK, HADH, UCP2, HNF4A, HNF1A, MCT1, HK1 and PGM1 have been associated with genetic forms of CHI. However, the genetic cause for many CHI patients (nearly 50%) remains elusive. Mutations in transcription factors such as HESX1, PROP1, POU1F1, LHX3, LHX4, PITX1, PITX2, OTX2, SOX2 and SOX3 have been associated with congenital hypopituitarism(CH) in mouse and humans. However, these mutations account only for a small proportion with the majority of patients having an unknown genetic cause for their symptoms. The use of next generation sequencing in children with undiagnosed or unidentified syndromic disorders is becoming more popular in recent years, increasing the diagnostic ability and discovery of novel genes and mutations contributing to novel clinical phenotypes. Aims: 1. To identify novel genetic mechanisms in patients with rare endocrine disorders. 2. To functionally characterize a transcription factor associated with pituitary and pancreatic development and to characterize a novel gene associated with CHI. Patients Six patients with varied phenotypes such as CHI and CH(n=1), CHI(n=1), primary IGF1 deficiency with dysmorphic features (n=1), severe short stature with dysmorphic features(n=1), severe short stature(n=1), hypercalcemia and glomerular disease(n=1) were recruited into this study. Methods: Whole exome sequencing(WES) was performed on the genomic DNA in trios (patient and the biological parents) for 5 patients. In one of the patients where the DNA sample was not possible to obtain from the biological father, WES was performed on the genomic DNA from the patient and the biological mother. mRNA expression during murine embryogenesis was studied using in situ hybridization and the protein expression in human embryos was demonstrated by immunohistochemistry. The pathogenic effect of the variant on protein function was further demonstrated by transcriptional activation assays using luciferase and quantification of protein expression using western blot. Results: 1. WES identified a de novo heterozygous mutation in FOXA2 (c.505T > C, p.S169P) in a highly conserved residue within the DNA binding domain in a patient with CHI and CH. A strong expression of Foxa2 mRNA was found in the developing hypothalamus, pituitary, pancreas, lungs and oesophagus of mouse embryos using in situ hybridization. Expression profiling on human embryos by immunohistochemistry showed strong expression of hFOXA2 in the neural tube, third ventricle, diencephalon and in the pancreas. Transient transfection of HEK293T cells with Wt (Wild type) hFOXA2 or mutant hFOXA2 showed an impairment in transcriptional reporter activity by the mutant hFOXA2. Further analyses using western blot assays showed that the FOXA2 p.(S169P) variant is pathogenic resulting in lower expression levels when compared with Wt hFOXA2. 2. A novel compound heterozygous mutation (p.R989G, p.K1026N) in ASXL3 contributing to Bainbridge Ropers syndrome in association with primary IGF1 deficiency was found in a patient with severe short stature with dysmorphism. 3. A de novo heterozygous frameshift mutation (p.G539fs*4) was found in CaMKK2 isoform-7 in a patient with persistent CHI, negative for mutations in known genes. On expressing the pG539fs*4 mutant in COS7 cells a significantly higher basal and Ca2+-CaM dependent kinase activity was noted when compared with WT (Wild Type) isoform-7. Both isoform-7 and the pG539fs*4 mutant have elevated basal kinase activity compared with isoform-1, the major CaMKK2 isoform expressed in most tissues. 4. A heterozygous splicing mutation in B3GAT3 was found in a patient with short stature, congenital heart defects, facial dysmorphism and skeletal abnormalities. 5. A homozygous mutation in the promoter region of GH1 was identified in a patient with severe short stature and low IGF1. In one patient, no relevant genetic variant segregating with the phenotype was identified. Conclusions: FOXA2 mutation can cause a complex congenital syndrome with hypopituitarism, and hyperinsulinism. Frameshift mutation in CaMKK2 is a potential novel cause of persistent CHI.WES has helped to identify the underlying genetic etiology in 5 out of 6 families recruited in the study.
Supervisor: Senniappan, Senthil ; McNamara, Paul ; Peak, Matthew Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral