I Banerjee, D Hanson, R Perveen, A Whatmore, G C Black and P E Clayton
Constitutional delay of growth and puberty (CDGP) is a common clinical condition that may be inherited as an autosomal dominant, recessive or X-linked trait. However, single-gene defects underlying CDGP have not yet been identified. A small number of children (to date 10) with modest growth failure and in the majority delayed puberty, a phenotype similar to that of CDGP, have been reported to carry mutations in the IGF acid labile subunit (IGFALS) gene which encodes the ALS, a part of the ternary complex carrying IGF-I in the circulation. The aim of our study was to screen a well-characterised CDGP cohort exhibiting a range of growth retardation and pubertal delay for pathogenic sequence variants in IGFALS.
Design and methods
We used denaturing high performance liquid chromatography (dHPLC) to screen for IGFALS mutations in DNA samples from 90 children (80 males) with CDGP of predominantly White European origin. DNA fragments generating abnormal waveforms were directly sequenced.
No IGFALS mutation was identified in the coding sequences or exon–intron boundaries in our CDGP cohort. One abnormal waveform pattern in dHPLC in 15 children with CDGP was found to represent a recognised synonymous single-nucleotide polymorphism of the coding transcript in the second exon in residue 210 of IGFALS.
IGFALS sequence variants are unlikely to be a common association with pubertal delay in children with CDGP.
I Banerjee, M Skae, S E Flanagan, L Rigby, L Patel, M Didi, J Blair, S Ehtisham, S Ellard, K E Cosgrove, M J Dunne and P E Clayton
In children with congenital hyperinsulinism (CHI), KATP channel genes (ABCC8 and KCNJ11) can be screened rapidly for potential pathogenic mutations. We aimed to assess the contribution of rapid genetic testing to the clinical management of CHI.
Follow-up observational study at two CHI referral hospitals.
Clinical outcomes such as subtotal pancreatectomy, 18F-Dopa positron emission tomography–computed tomography (PET–CT) scanning, stability on medical treatment and remission were assessed in a cohort of 101 children with CHI.
In total, 32 (32%) children had pathogenic mutations in KATP channel genes (27 in ABCC8 and five in KCNJ11), of which 11 (34%) were novel. In those negative at initial screening, other mutations (GLUD1, GCK, and HNF4A) were identified in three children. Those with homozygous/compound heterozygous ABCC8/KCNJ11 mutations were more likely to require a subtotal pancreatectomy CHI (7/10, 70%). Those with paternal heterozygous mutations were investigated with 18F-Dopa PET–CT scanning and 7/13 (54%) had a focal lesionectomy, whereas four (31%) required subtotal pancreatectomy for diffuse CHI. Those with maternal heterozygous mutations were most likely to achieve remission (5/5, 100%). In 66 with no identified mutation, 43 (65%) achieved remission, 22 (33%) were stable on medical treatment and only one child required a subtotal pancreatectomy.
Rapid genetic analysis is important in the management pathway of CHI; it provides aetiological confirmation of the diagnosis, indicates the likely need for a subtotal pancreatectomy and identifies those who require 18F-Dopa PET–CT scanning. In the absence of a mutation, reassurance of a favourable outcome can be given early in the course of CHI.
P G Murray, A Read, I Banerjee, A J Whatmore, L E Pritchard, R A Davies, J Brennand, A White, R J Ross and P E Clayton
Leptin deficiency caused by mutations within the leptin gene (LEP) results in severe early onset obesity, hypogonadism, pubertal delay and immune system abnormalities. Constitutional delay in growth and puberty (CDGP) is a common condition seen in paediatric clinics, in which children present with delayed growth and puberty but usually also have a slim body habitus. We hypothesized that LEP variants may play a role in the phenotype seen in CDGP.
To screen a group of children with CDGP for pathogenic sequence variants in LEP.
Patients and methods
Denaturing HPLC was used to screen for LEP sequence variants in DNA samples from 78 children with CDGP (predominantly white males) and 112 control subjects. DNA fragments with a WAVE pattern deviant from wild type were directly sequenced. A STAT3 luciferase reporter assay in human embryonic kidney (HEK293) cells transiently transfected with the leptin receptor was used to test activity of mutant leptin.
One child with CDGP was identified to be heterozygous for a novel missense variant (c.68C>G), which results in a proline to arginine substitution (p.P23R). This sequence variant was not identified in any of the other control subjects, but was identified in his mother who shared a similar phenotype of slim body habitus, reduced appetite and pubertal delay (menarche aged 15 years). The leptin variant showed similar stability in serum compared with wild type and did not demonstrate increased activity in an in vitro reporter gene assay.
This is the first report of a sequence variant within the LEP gene associated with reduced body mass index rather than obesity. We hypothesize that this variant has increased bioactivity in vivo.
S E Flanagan, R R Kapoor, G Mali, D Cody, N Murphy, B Schwahn, T Siahanidou, I Banerjee, T Akcay, O Rubio-Cabezas, J P H Shield, K Hussain and S Ellard
The phenotype associated with heterozygous HNF4A gene mutations has recently been extended to include diazoxide responsive neonatal hypoglycemia in addition to maturity-onset diabetes of the young (MODY). To date, mutation screening has been limited to patients with a family history consistent with MODY. In this study, we investigated the prevalence of HNF4A mutations in a large cohort of patients with diazoxide responsive hyperinsulinemic hypoglycemia (HH).
Subjects and methods
We sequenced the ABCC8, KCNJ11, GCK, GLUD1, and/or HNF4A genes in 220 patients with HH responsive to diazoxide. The order of genetic testing was dependent upon the clinical phenotype.
A genetic diagnosis was possible for 59/220 (27%) patients. KATP channel mutations were most common (15%) followed by GLUD1 mutations causing hyperinsulinism with hyperammonemia (5.9%), and HNF4A mutations (5%). Seven of the 11 probands with a heterozygous HNF4A mutation did not have a parent affected with diabetes, and four de novo mutations were confirmed. These patients were diagnosed with HI within the first week of life (median age 1 day), and they had increased birth weight (median +2.4 SDS). The duration of diazoxide treatment ranged from 3 months to ongoing at 8 years.
In this large series, HNF4A mutations are the third most common cause of diazoxide responsive HH. We recommend that HNF4A sequencing is considered in all patients with diazoxide responsive HH diagnosed in the first week of life irrespective of a family history of diabetes, once KATP channel mutations have been excluded.