Unusual phenotypic features in a patient with a novel splice mutation in the GHRHR gene

Abstract

Isolated growth hormone deficiency (IGHD) may be of genetic origin. One of the few genes involved in that condition encodes the growth hormone releasing hormone receptor (GHRHR) that, through its ligand (GHRH), plays a pivotal role in the GH synthesis and secretion by the pituitary. Our objective is to describe the phenotype of two siblings born to a consanguineous union presenting with short stature (IGHD) and Magnetic Resonance Imaging (MRI) abnormalities, and to identify the molecular basis of this condition. Our main outcome measures were clinical and endocrinological investigations, MRI of the pituitary region, study of the GHRHR gene sequence and transcripts. In both patients, the severe growth retardation (-5SD) was combined with anterior pituitary hypoplasia. In addition to these classical phenotypic features for IGHD, one of the patients had a Chiari I malformation, an arachnoid cyst, and a dysmorphic anterior pituitary. A homozygous sequence variation in the consensus donor splice site of intron 1 (IVS1 + 2T > G) of the GHRHR gene was identified in both patients. Using in vitro transcription assay, we showed that this mutation results in abnormal splicing of GHRHR transcripts. In this report, which broadens the phenotype associated with GHRHR defects, we discuss the possible role of the GHRHR in the proper development of extrapituitary structures, through a mechanism that could be direct or secondary to severe GH deficiency.

Figure 1

(A) Pedigree of the IGHD studied family with affected children represented by filled symbols. Heights are those measured at first examination for affected subjects. SD is evaluated according to the French standards of Children’s International Center and Sempé (41). (B) Height curves for patients II.3 and II.4. The duration of GH treatment is depicted as follows: ↑beginning, ↓ end.

Figure 2

Cerebral MRI. (A) Sagittal medial section focused on the pituitary region of control (a), patient II.3 (b), and patient II.4 (c), showing an hypoplasia of anterior pituitary (ap). The pituitary stalk (s) is seen and the posterior pituitary (pp) is in normal position. (B) Coronal sections focused on pituitary region for control (a), patients II.3 (b), and patient II.4 (c) showing the anterior pituitary hypoplasia. Note the dysmorphy of the anterior pituitary (b, c). (C) Sagittal sections for patient II.3 (b) showing a Chiari type I malformation (herniated cerebellar tonsils through the foramen magnum showed by an arrow), and a left retrocerebellar arachnoid cyst (asterisk), (a) control MRI.

Figure 3

(A) Electrophoregrams of the normal and mutated GHRHR sequences. Sequence analysis of portion of exon 1 and intron 1 of the GHRHR gene from an unaffected individual (control) and from the proband II.3 (patient). The homozygous T > G transversion (IVS1 + 2 T > G) is indicated by an arrow and the exon 1/intron 1 junction by a dotted vertical line. (B) Segregation analysis of the splice-donor-site mutation of GHRHR in the studied family. A 481-bp GHRHR genomic DNA fragment was digested by Taa-I restriction enzyme that recognizes only the wild type allele (underlined under the electrophoregram), then analyzed on a gel electrophoresis (3% nusieve agarose). This confirmed that the two patients (II.3 and II.4) are homozygous for the mutated allele (481-bp). The parents and the healthy brother (I.1, I.2, and II.2) are heterozygous (three fragments: 418, 330, and 151-bp), the sister (II.1) bears two wild type alleles, and the control (C) has two wild type alleles. L: “1Kb plus” ladder (Invitrogen); ND: not digested, bp: base pair.

Figure 4

Transcription assay. (A) RT-PCR amplification of the GHRHR transcripts obtained from cells transfected with plasmids pcDNA3.1-GHRHR_WT (wild type minigene, amplified between P1–P2) (Lane wt) and pcDNA3.1-GHRHR_mut (mutated minigene, amplified between P1–P2) (Lane mut) using two internal exonic primers (P3 and P4), indicated by arrows in B (a, c). (B), (b) Schematic representation of the genomic GHRHR region where the IVS1 + 2T > G mutation at the donor splice site (gtgagta > gggagta) and the cryptic 5′ splice site (gtaag) (428bp downstream the first nucleotide of intron 1) are in bold characters. The 290-bp product corresponds to the normal transcript and the 718-bp fragment to the mutated transcript with a retention of a part of intron 1 because of activation of the intronic cryptic donor splice site (bold characters). (a) and (c): schematic representation of mutated and wild type partial cDNAs, respectively.