Michela Godi

A Recurrent Signal Peptide Mutation in the Growth Hormone Releasing Hormone Receptor with Defective Translocation to the Cell Surface and Isolated Growth Hormone Deficiency

CONTEXT Mutations in the GHRH receptor (GHRHR) have been detected in the familial type-IB isolated GH deficiency (IGHD-IB) inherited as an autosomal recessive disorder and characterized by a low but detectable serum GH level and good response to substitutive GH therapy. OBJECTIVE The aim of our study was the identification of mutations in sporadic patients with a IGHD-IB phenotype. SUBJECTS AND METHODS The GHRHR gene was systematically screened by DHPLC in 134 IGHD patients with no family history of the disorder or declared parental consanguinity. RESULTS We identified a novel variation, Val10Gly, within the signal peptide at the heterozygous state in three patients and in one of 1084 controls (P = 0.004), suggesting that it might contribute to IGHD. The functional analysis showed that the signal peptide is not cleaved from the mutant GHRHR, which in turn is not translocated to the cellular surface, demonstrating that 10Gly drastically affects the receptor correct processing. Because 10Gly was also present in normal-stature relatives of the patients as well as in a control, it is likely that it exerts its effects in the context of other genetic and environmental susceptibility factors. CONCLUSION At difference from previous papers reporting GHRHR mutations in familial cases with a clear recessive mode of inheritance, our study was conducted on a large sample of sporadic patients and allowed to discover a novel mechanism of the disease caused by a recurrent dominant mutation in the GHRHR signal peptide associated with incomplete penetrance.

A variation in a Pit-1 site in the growth hormone gene (GH1) promoter induces a differential transcriptional activity

The proximal promoter of the human growth hormone gene (GH1) is highly polymorphic. We tested if promoter haplotypes differing at possibly functional sites, namely -278T/G (in the NF1 binding site), -75A/G (in the proximal Pit-1 binding site) and -57G/T (in the VDR binding site), induced a different luciferase activity when transfected in a rat pituitary cell line. The presence of a G instead of an A at position -75 induced a more than two-fold reduced activity (p<0.0001). In accordance with this findings the electrophoretic mobility shift assay demonstrated a reduced affinity of the -75G for the pituitary transcription factor Pit-1. Despite the strong effect of this polymorphism in vitro, the -75G variation was not associated to an impairment of the GH secretion in vivo.

Growth Hormone (GH)-Releasing Hormone Increases the Expression of the Dominant-Negative GH Isoform in Cases of Isolated GH Deficiency due to GH Splice-Site Mutations

An autosomal dominant form of isolated GH deficiency (IGHD II) can result from heterozygous splice site mutations that weaken recognition of exon 3 leading to aberrant splicing of GH-1 transcripts and production of a dominant-negative 17.5-kDa GH isoform. Previous studies suggested that the extent of missplicing varies with different mutations and the level of GH expression and/or secretion. To study this, wt-hGH and/or different hGH-splice site mutants (GH-IVS+2, GH-IVS+6, GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GC-GHRHR). Upon GHRH stimulation, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed reduced hGH secretion and intracellular GH content when compared with cells expressing only wt-hGH, confirming the dominant-negative effect of 17.5-kDa isoform on the secretion of 22-kDa GH. Furthermore, increased amount of 17.5-kDa isoform produced after GHRH stimulation in cells expressing GH-splice site mutants reduced production of endogenous rat GH, which was not observed after GHRH-induced increase in wt-hGH. In conclusion, our results support the hypothesis that after GHRH stimulation, the severity of IGHD II depends on the position of splice site mutation leading to the production of increasing amounts of 17.5-kDa protein, which reduces the storage and secretion of wt-GH in the most severely affected cases. Due to the absence of GH and IGF-I-negative feedback in IGHD II, a chronic up-regulation of GHRH would lead to an increased stimulatory drive to somatotrophs to produce more 17.5-kDa GH from the severest mutant alleles, thereby accelerating autodestruction of somatotrophs in a vicious cycle.

Rare variants in the TREX1 gene and susceptibility to autoimmune diseases

TREX1 (DNase III) is an exonuclease involved in response to oxidative stress and apoptosis. Heterozygous mutations in TREX1 were previously observed in patients with systemic lupus erythematosus (SLE) and Sjögrens syndrome (SS). We performed a mutational analysis of the TREX1 gene on three autoimmune diseases: SLE (210 patients) and SS (58 patients), to confirm a TREX1 involvement in the Italian population, and systemic sclerosis (SSc, 150 patients) because it shares similarities with SLE (presence of antinuclear antibodies and connective tissue damage). We observed 7 variations; two of these are novel nonsynonymous variants (p.Glu198Lys and p.Met232Val). They were detected in one SS and in one SSc patient, respectively, and in none of the 200 healthy controls typed in this study and of the 1712 published controls. In silico analysis predicts a possibly damaging role on protein function for both variants. The other 5 variations are synonymous and only one of them is novel (p.Pro48Pro). This study contributes to the demonstration that TREX1 is involved in autoimmune diseases and proposes that the spectrum of involved autoimmune diseases can be broader and includes SSc. We do not confirm a role of TREX1 variants in SLE.

A novel HESX1 splice mutation causes isolated GH deficiency by interfering with mRNA processing.

OBJECTIVE Mutations in HESX1 represent a rare cause of GH deficiency (GHD) associated with a broad spectrum of other anomalies. We searched for causative mutations in a cohort of 244 Italian patients affected by combined and isolated GHD (IGHD). METHODS The HESX1 gene-coding region and exon-intron boundaries were screened by denaturing HPLC scanning. RESULTS A novel mutation adjacent to the invariant donor splice site of intron 2 (c.357+3G>A) was identified at the heterozygous state in an IGHD patient. The in vitro and in vivo mRNA analysis of the wild-type HESX1 allele revealed the presence of the whole cDNA and two isoforms lacking exon 2 and exons 2-3 respectively. The mutant HESX1 allele yielded only two splicing products, the whole cDNA and the cDNA missing exons 2-3, whereas the mRNA lacking exon 2 was absent. An in vitro assay demonstrated that the exon 2-deleted mRNA, predicting a prematurely truncated protein, is subjected to nonsense-mediated mRNA decay (NMD). CONCLUSIONS The c.357+3G>A mutation prevents the generation of one of the alternative isoforms normally produced by the wild-type allele, predicting a truncated HESX1 protein. The mutation is likely to cause IGHD in the heterozygous patient by interfering with the downregulation of HESX1 expression mediated by alternative splicing and NMD. Our results open new insight into the mechanism of HESX1 regulation suggesting that the coupling of alternative splicing and NMD might play a fundamental role in directing the HESX1 expression, and that the alteration of this process might lead to severe consequences.

A novel recessive splicing mutation in the POU1F1 gene causing combined pituitary hormone deficiency

Background: Mutations in the gene encoding the pituitary transcription factor POU1F1 (Pit-1, pituitary transcription factor-1) have been described in combined pituitary hormone deficiency (CPHD). Aim: The aim of this study was the characterisation of the molecular defect causing CPHD in a patient born to consanguineous parents. Subject and methods: The case of a 12.5-yr-old girl presenting with severe growth failure at diagnosis (−3 SD score at 3 months) and deficiency of GH, PRL, and TSH was investigated for the presence of POU1F1 gene mutations by denaturing high performance liquid chromatography analysis. Results: A novel mutation adjacent to the IVS2 splicing acceptor site (IVS2-3insA) was identified in the patient at the homozygous state. Analysis of patient’s lymphocyte mRNA and an in vitro splicing assay revealed the presence of 2 aberrant splicing products: a) deletion of the first 71 nucleotides of exon 3, altering the open reading frame and generating a premature stop codon, b) total exon 3 skipping resulting in an in frame deleted mRNA encoding a putative protein lacking part of the transactivation domain and of the POU-specific homeodomain. Notably, the patient’s relatives heterozygous for the mutation had PRL levels under the normal range with no evident clinical symptoms. Conclusions: The IVS2-3insA mutation, responsible for CPHD at the homozygous state, causes the presence of 2 aberrant splicing products encoding non-functional products. In the heterozygotes one normal allele might not guarantee a complete pituitary function.

Functional SNPs within the Intron 1 of the PROP1 Gene Contribute to Combined Growth Hormone Deficiency (CPHD)

CONTEXT Mutations within the PROP1 gene represent one of the main causes of familial combined pituitary hormone deficiency (CPHD). However, most of the cases are sporadic with an unknown genetic cause. OBJECTIVE The aim of this study was the search for low penetrance variations within and around a conserved regulatory element in the intron 1 of PROP1, contributing to a multifactorial form of the disease in sporadic patients. METHODS AND PATIENTS A fragment of 570 bp encompassing the conserved region was sequenced in 107 CPHD patients and 294 controls, and an association study was performed with the four identified variants, namely c.109+435G>A (rs73346254), c.109+463C>T (rs4498267), c.109+768C>G (rs4431364), and c.109+915_917ins/delTAG (rs148607624). The functional role of the associated polymorphisms was evaluated by luciferase reporter gene expression analyses and EMSA. RESULTS A statistically significant increased frequency was observed in the patients for rs73346254A (P = 5 × 10(-4)) and rs148607624delTAG (P = 0.01) alleles. Among all the possible allele combinations, only the haplotype bearing both risk alleles showed a significantly higher frequency in the patients vs. controls (P = 4.7 × 10(-4)) and conferred a carrier risk of 4.19 (P = 1.2 × 10(-4)). This haplotype determined a significant decrease of the luciferase activity in comparison with a basal promoter and the other allelic combinations in GH4C and MCF7 cells (P = 4.6 × 10(-6); P = 5.5 × 10(-4), respectively). The EMSA showed a differential affinity for nuclear proteins for the alternative alleles of the two associated variations. CONCLUSIONS Variations with a functional significance conferring susceptibility to CPHD have been identified in the PROP1 gene, indicating a multifactorial origin of this disorder in sporadic cases.