The human dynein intermediate chain 2 gene (DNAI2): cloning, mapping, expression pattern, and evaluation as a candidate for primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterized by chronic sinusitis and bronchiectasis, and usually associated with hypofertility. Half of the patients present a situs inversus, defining the Kartagener's syndrome. This phenotype results from axonemal abnormalities of respiratory cilia and sperm flagella, i.e., mainly an absence of dynein arms. Recently, a candidate-gene approach, based on documented abnormalities of immotile strains of Chlamydomonas reinhardtii, allowed us to identify the first gene involved in PCD. Following the same strategy, we have characterized DNAI2, a human gene related to Chlamzydomonas IC69, and evaluated its possible involvement in a PCD population characterized by an absence of outer dynein arms. DNAI2, which is composed of 14 exons located at 17q25, is highly expressed in trachea and testis. No mutation was found in the DNAI2 coding sequence of the twelve patients investigated. However, ten intragenic polymorphic sites and an EcoRI RFLP have been identified, allowing the exclusion of DNAI2 in three consanguineous families.

The human growth hormone (GH) receptor and its truncated isoform: sulfhydryl group inactivation in the study of receptor internalization and GH-binding protein generation.

The human GH receptor (hGHR) contains nine intracellular and seven extracellular cysteines, of which six are linked by disulfide bonds and one, at position 241 proximal to the membrane, is free. Recently, an alternatively spliced GHR isoform has been isolated; it encodes a truncated receptor lacking most of the cytoplasmic domain (hGHRtr). In the present study, we have examined the effect of sulfhydryl group(s) inactivation on receptor internalization and GH binding-protein (GHBP) generation from the human (h) and rabbit (rb) full-length GHR, as well as from hGHRtr and a mutant of the free extracellular cysteine (hGHRtr-C241A), expressed in Chinese hamster ovary (CHO) cells. In CHO/rbGHR and CHO/hGHR cells, permeable sulfhydryl-reactive agents, like N-ethylmaleimide (NEM) and iodacetamide (IA), inhibited GHR internalization and induced an immediate dose-dependent loss of cellular GHR, associated with a concomitant marked increase in released GHBP. In contrast, the membrane impermeable IA derivative A-484 had no effect on either GHBP release or on GHR internalization. NEM exposure of CHO cells, expressing hGHRtr, resulted in a dose-dependent increase in GHBP generation, but only a moderate decrease in cellular hGHRtr. The importance of the only unpaired cysteine in these processes was evaluated in CHO/hGHRtr-C241A cells. hGHRtr-C241A was similar to hGHRtr in its impaired internalization and enhanced GHBP release by NEM. Taken together, these data suggest that intracellular sulfhydryl groups, within membranal endocytic vesicles, that do not belong to the GHR molecule, are involved in receptor internalization and GHBP generation. In addition, the present study demonstrates that despite impaired hGHR internalization/down-regulation, the inducible release of GHBP was not affected, further suggesting that GHR endocytosis is not a prerequisite for GHBP generation.

Extensive phenotypic analysis of a family with growth hormone (GH) deficiency caused by a mutation in the GH-releasing hormone receptor gene.

GH secretion and release are complex phenomena depending on activation of several genes, including those encoding GH, GHRH, and its receptor (GHRH-R). The GH gene, which is the most extensively analyzed sequence in patients with familial GH deficiency (GHD), represents the main known target of mutations. To test the involvement of the GHRH-R gene in this disease phenotype, we investigated one candidate Tamoulean family originating from Sri Lanka. Two brothers, with extremely short stature (< -4 SD) and no dysmorphy, were diagnosed as having complete GHD, unresponsive to exogenous GHRH and associated with PRL levels within the lower normal range. Magnetic resonance imaging examination showed anterior pituitary hypoplasia with a normal pituitary stalk. Both patients increased their growth rate while under GH therapy. Molecular investigations revealed a homozygous GHRH-R gene mutation that introduces a stop codon at residue 72. This mutation, which predicts a severely truncated receptor lacking the seven membrane spanning domains, is identical to that recently reported in one Indian Moslem family, raising the possibility of a founder effect. There was no clear evidence for height reduction in the three heterozygous individuals studied. This observation, which underlines the phenotypic criteria associated with a loss of GHRH-R function, raises the question of the frequency of GHRH-R abnormalities among GHD patients.

An exon-skipping mutation in the btk gene of a patient with X-linked agammaglobulinemia and isolated growth hormone deficiency.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency disease associated with a block in differentiation from pre-B to B cells. The XLA gene encodes a 659 amino acids cytoplasmic protein tyrosine kinase named btk (Bruton's tyrosine kinase). The few btk gene alterations so far reported in XLA patients are heterogenous and distributed in all domains of the btk protein. They appear to be responsible for a range of B cell immunodeficiency disorders of variable severity. Rare families in which XLA is inherited together with isolated growth hormone deficiency (IGHD) have been reported. Genetic analysis has shown that this disease association maps to the same region of the X chromosome as XLA, but whether the two phenotypes are caused by a common or different developmental or biochemical mechanism is unknown. We have analyzed the btk gene of a patient with XLA and IGHD. RT-PCR analysis of btk transcripts, sequencing data obtained from cDNA and genomic DNA and in vitro splicing assays showed that an intronic point mutation (1882 + 5G-->A) is responsible for skipping of an exon located in the tyrosine kinase domain. This exon-skipping event results in a frameshift leading to a premature stop codon 14 amino acids downstream, and in the loss of the last 61 residues of the carboxy-terminal end of the protein. Although we studied a sporadic case, the results suggest that an alteration of the btk gene might cause this unusual phenotype.

Spectrum of growth hormone receptor mutations and associated haplotypes in Laron syndrome.

Laron syndrome is a rare autosomal recessive disorder characterized by resistance to growth hormone (GH). In 10 patients of different ethnic origins, we have analyzed all the GH receptor (GHR)-coding exons along with their splice junctions and 6 intragenic polymorphic sites defining several GHR gene haplotypes. This allowed us to identify the mutations in the 20 chromosomes studied and to describe a new GHR haplotype. Eleven different mutations associated with various GHR haplotypes were observed; they included 3 nonsense mutations, 3 splice defects and 5 missense mutations. Of the 11 mutations, 8 were novel. All the mutations involved the exoplasmic domain of the receptor and all the missense mutations were clustered in a short polypeptide segment. Most of the missense mutations affected residues conserved among GHRs from different species and the related molecules that belong to the cytokine receptor superfamily. Adding to the 5 mutations so far described, these findings illustrate the allelic heterogeneity of this syndrome and document the independent origin of the molecular defects, all features of clinical relevance for genetic counselling.