European Journal of Endocrinology (2008) 158 473–477 ISSN 0804-4643 CLINICAL STUDY Constitutional delay of growth and puberty is not commonly associated with mutations in the acid labile subunit gene I Banerjee1, D Hanson2, R Perveen2, A Whatmore3, G C Black2 and P E Clayton1,3 1Department of Paediatric Endocrinology, Royal Manchester Children’s Hospital, Pendlebury, Manchester M27 4HA, UK, 2Academic Unit of Medical Genetics and 3Endocrine Science Research Group, University of Manchester, Manchester M13 9NT, UK (Correspondence should be addressed to I Banerjee; Email: [email protected]) Abstract Objectives: 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. Results:NoIGFALS 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. Conclusions: IGFALS sequence variants are unlikely to be a common association with pubertal delay in children with CDGP. European Journal of Endocrinology 158 473–477 Introduction genes investigated for association with CDGP, poly- morphic markers in the genes for GnRH and GnRH-R Constitutional delay of growth and puberty (CDGP) is a show only weak association (3). Common polymorphisms common reason for referral to a paediatric endocrinolo- in the leptin and leptin receptor genes are not associated gist and it is estimated that genetic factors account for with CDGP, although a specific leptin genotype does 50–80% of the variability in pubertal delay (1, 2).CDGPis influence the CDGP phenotype; homozygotes for the long a complex trait with variable degrees of growth allele for the CT repeat in the 30 UTR have a greater body retardation and pubertal delay, and is likely to be related mass index (BMI) SDS than heterozygotes or short allele to a number of genes (3). Nevertheless, in pedigree homozygotes (4). Furthermore, there is no association analysis of CDGP children, transmission may follow an between a polymorphic adenine deletion in the cocaine apparently autosomal dominant pattern with variable and amphetamine-regulated transcript (CART)gene, penetrance, as well as autosomal recessive and X-linked which modulates the action of leptin on GnRH secretion patterns of inheritance. This implies that single-gene and CDGP (PE Clayton, unpublished observations). defects may play a part in some cases of CDGP (1). The growth hormone (GH) – insulin-like growth Mutations in gonadotrophin-releasing hormone (GnRH), factor-I (IGF-I) axis influences the onset and progression GnRH receptor (GnRHR), Kallmann syndrome (KAL1), of puberty (5, 6) and therefore has a role in its temporal fibroblast growth factor receptor 1 (FGFR1) and G regulation. Thus, a genetic abnormality in this axis could protein-coupled receptor 54 (GPR54) can all cause be associated with the CDGP phenotype (7). The acid labile hypogonadotrophic hypogonadism with markedly subunit (ALS) is a GH-dependent peptide that binds to delayed or absent puberty. However, no pathogenic gene IGF-I and IGF-binding protein-3 (IGFBP-3) forming a mutations have been clearly associated with the CDGP stable ‘ternary’ complex that carries IGF-I around in the phenotype that is characterised by a slowdown in height circulation. A recent report noted that a frameshift velocity and tempo of pubertal development. Of the single mutation in the ALS gene (IGFALS) in nucleotide q 2008 Society of the European Journal of Endocrinology DOI: 10.1530/EJE-07-0769 Online version via www.eje-online.org Downloaded from Bioscientifica.com at 09/25/2021 05:32:10PM via free access 474 I Banerjee and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2008) 158 positions 1334 through to 1338 in exon 2 led to a growth charts were reviewed to confirm that each child complete absence of circulating ALS in a child with severe with CDGP demonstrated a slowdown in height gain and pubertal delay but only a modest degree of growth failure subsequent catch up with progression of puberty. Height (8). Another study reported a point mutation in IGFALS at and BMI were converted to SDS using British 1990 nucleotide position 1318 in a child presenting with short normative data (14, 15).Boneagewasmeasuredbythe stature and relatively mild pubertal delay (9, 10). Three Greulich–Pyle method and bone age delay noted as the other children, two with pubertal delay and one with difference between skeletal and chronological ages. Target significant short stature, have been reported in a family height was calculated for each individual and parent- with novel compound heterozygous mutations in exon 2 adjusted height was calculated as (height SDS – parental of IGFALS (11). There are also recent reports of IGFALS target height SDS). mutations in three males with idiopathic short stature The IGFALS gene is located on chromosome 16 and (12) and in two other males with short stature and consists of two exons. PCR amplification of the coding osteoporosis (13). In most of these cases, the pubertal sequences and exon–intron boundaries was undertaken development and the pubertal growth spurt were delayed, using PCR Express thermal cycler (Hybaid) on buccal a pattern consistent with that occurring in children with DNA extracted by standard procedures. The following CDGP (Table 1). We hypothesised that pathogenic forward (f) and reverse (r) primer pairs were designed: sequence variants in IGFALS could be present in children with CDGPand have investigated a cohort of children with f50–acgagggggttaacagagc–30,r50-agactggccaccccta- CDGP to determine their frequency. gagt–30 (exon 1) f50-gacctcgagcctcagttcac-30,r50-cctgctcagacggttgt- tg-30 (exon 2, fragment 1) f50-ctgggcctagagaacctgtg-30,r50-tgcggtcatgaacctc- Subjects and methods tct-30 (exon 2, fragment 2) f50-ggcaacaggctggcctac-30,r50-ctccaggtgcaggctg- The study was approved by the local research ethics tg-30 (exon 2, fragment 3) committee. We recruited 90 children (80 males) with f50-ggcggtcatgaacctctct-30,r50-aaggtccgcagtgagtt- CDGP from among those attending Paediatric Endo- gtt-30 (exon 2, fragment 4) crinology outpatient clinics. The diagnosis of CDGP was f50-ctggacgtctcgcacaac-30,r50-catgccttccaccccatc-30 established by a paediatric endocrinologist and case notes (exon 2, fragment 5). were reviewed for confirmation. The following inclusion Fragments 4 and 5 in exon 2 were amplified from a criteria were used (3, 4): a) evidence of delayed puberty – larger fragment spanning both regions, derived from lack of breast development (Tanner stage 2) by the age of primary PCR with the following primer pairs: 12 years in girls and testicular volume !4 ml by the age f50-aactctacctggaccgcaac-30,r50-tgagacagggtgttg- of 13 years in boys (1 S.D. criteria), b) evidence of slowdown ctctg-30. in height velocity as documented on growth charts and c) absence of other identifiable causes of delayed puberty. The sizes of the fragments amplified by PCR were 287, Children with delayed puberty were further subdivided 595, 400, 591, 600 and 480 bp respectively. into: a) moderate pubertal delay (1–2 S.D. below mean) To screen for sequence variants in IGFALS, amplicons and b) severe pubertal delay (R2 S.D. below mean) – lackof from patients were heteroduplexed with those from breast development in girls R13 years or testicular control DNA using the PCR Express thermal cycler. volume !4mlinboys O14 years (3, 4).Individual Control DNA was obtained from three individuals tested Table 1 Auxological characteristics and age at pubertal onset in cases reported with insulin-like growth factor acid labile subunit (IGFALS) mutations. Bone age Near final Age at onset Case Patient Height SDS at delay height of puberty Pubertal number Reference number Sex presentation (years) SDS (years) delaya 1 Domene et al. (8) IMK2.0 2 0.9 O14.6 2 2 Hwa et al. (9) II M K2.9 2 K2.1 13 1 3 Domene et al. (11) III1 M K2.0 2.1 K0.5 16.9 2 4 Domene et al. (11) III2 M– –K0.5 16 2 5 Domene et al. (11) III3 F– –K1.0 Menarche 13 No delay 6 Campos-Barros et al. (12) IV1 M K2.2 1 – 13 1 7 Campos-Barros et al. (12) IV2 M K3.8 1 – 12 No delay 8 Campos-Barros et al. (12) IV3 M K3.0 3 – 15 2 9 Duyvenvoorde van et al. (13) V1 M K4.2 0 K4.2 14.6 2 10 Duyvenvoorde van et al. (13) V2 M– –K4.3 Not known Not reported M, male; F, female. aPubertal delay: 1Zpubertal delay by relaxed (1 SD) criteria and 2Zpubertal delay by strict criteria (2 SD) (3, 4). www.eje-online.org Downloaded from Bioscientifica.com at 09/25/2021 05:32:10PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2008) 158 ALS mutations and pubertal delay 475 for cystic fibrosis mutations, not identified as CDGP and Table 3 Patient characteristics in 90 children with constitutional sequenced to confirm the absence of any known delay of growth and puberty (CDGP).