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

Abstract

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.357C3GOA) 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.357C3GOA 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.

Identification of HESX1 mutations in Kallmann syndrome

“…However, this same change was seen once in 192 controls and reported previously in controls (5), indicating it represents a polymorphism. Four additional patients contained a heterozygous c.374A>G in HESX1 resulting in a p.N125S missense substitution, a previously reported benign Afro-Caribbean polymorphism (14).…”

“…Mutations in the HESX1 gene were first identified in patients with SOD, a clinical disorder with a strong resemblance to the phenotype of the Hesx1 knockout mouse (1). Homozygous HESX1 mutations generally result in more severe human phenotypes, such as SOD (1), life-threatening neonatal conditions (8) and panhypopituitarism (9–10, 15), whereas published heterozygous mutations seem to display variable expressivity and are more often associated with milder phenotypes such as IGHD (4–5, 12). …”

“…The ten heterozygous mutations included six missense (4–5, 7, 12, 14–15), two frameshift (11, 13), one splice variant (5), and a nonsense mutation (15) (Table 2). The phenotypes consisted of CPHD (n = 6), IGHD (n = 2), or IGHD associated with optic nerve atrophy (n = 1) or SOD (n =1).…”

“…Six of the 10 mutations demonstrated functional effects in vitro , but four were not located within either of the two functional domains. Some of the heterozygous mutations (p.S170L, p.T181A, p.Q6H, p.E149K) have all been seen in at least one unaffected parent, suggesting autosomal dominant inheritance with reduced penetrance (4–5, 7, 12). …”

“…In fact, the first characterized human HESX1 mutation in an SOD patient was a homozygous p.R160C point mutation resulting in the loss of HESX1 DNA binding (1). Additional human mutations have been described in other disorders such as isolated growth hormone deficiency (IGHD) (4–5) and combined pituitary hormone deficiency (CPHD) (6–7), which consists of a deficiency in GH and at least one additional pituitary hormone. At least 16 different human HESX1 mutations, both homozygous (1, 3, 8–10) and heterozygous (4–7, 11–15) have been identified in humans with pituitary disorders.…”