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.

Human Prop-1: cloning, mapping, genomic structure. Mutations in familial combined pituitary hormone deficiency.

Prop-1 is a newly isolated pituitary-specific paired-like homeodomain transcription factor whose cDNA sequence is well known in mouse. To study its involvement in human combined pituitary hormone deficiency (CPHD), we have isolated the human cDNA ortholog and determined the exon/intron organization and chromosomal localization of the human gene. A Prop-1 defect was characterized in three CPHD families. One missense mutation (R73C) involves a residue conserved in 95% of the more than 400 homeodomain proteins so far identified; in vitro splicing assays demonstrated the functional importance of the second defect, whereas the remaining mutation is a frameshift. Given the disease phenotype documented in the patients, these data, which will facilitate molecular investigations in other patients, demonstrate the crucial role of Prop-1 in the proper development of somatotrophs, lactotrophs, thyreotrophs and gonadotrophs.

Evolutionary divergence of the truncated growth hormone receptor isoform in its ability to generate a soluble growth hormone binding protein.

The soluble growth hormone binding protein (GHBP), which is encoded by the GH receptor (GHR) gene, is generated by several mechanisms. In rabbits (rb) and humans (h), it is derived by proteolytic cleavage of the full-length membrane-bound receptor molecules (GHR-fl), whereas in rats (r) and mice, it results from an alternative splice excluding the transmembrane domain. Furthermore, in all these species, alternative splicing in the cytoplasmic domain results in a truncated isoform (GHR-tr), that, in humans, produces large amounts of GHBP through proteolysis. To further characterize the species specificity of the mechanism underlying GHBP generation, rbGHR-tr and rGHR-tr expressed in COS-7 cells were assayed for their ability to produce a GHBP in comparison with the corresponding full-length receptors. Large amounts of GHBP were secreted by cells expressing the rabbit constructs, the rbGHR-tr isoform being more efficient in GHBP generation than rbGHR-fl. In contrast, no GHBP was detected from cells expressing rGHR-tr, the cytoplasmic deletion having no effect on GHBP release from membrane receptors. These data further demonstrate evolutionary divergence in the mechanism by which GHBP is generated and provide new clues to decipher the molecular process underlying the cleavage step.

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.

Heterozygous mutation in the WSXWS equivalaent motif of the growth hormone receptor in a child with poor response to growth hormone therapy.

Besides complete GH insensitivity syndrome (GHIS) described by Laron, clinical and molecular evidences have accumulated concerning partial GHIS. We studied GH receptor (GHR) gene in children who show poor response to GH treatment and detected a patient with a heterozygous mutation in exon 7 leading to the Y222H substitution. This missense mutation, located in the YGEFS motif of the GHR equivalent to the WSXWS motif highly conserved throughout all members of the cytokine receptor family, has not been described so far. Although we cannot conclude on the deleterious effect of this mutation, there are several lines of evidence suggesting that it could account for the partial GH insensitivity: (i) hormonal data including IGF-I generation test; (ii) molecular data - no other mutation was identified in the coding sequence, the father who has the same mutation is short, the brother did not inherit the mutated allele and was of normal height.

Phenotype: genotype relationships in growth hormone insensitivity syndrome.

GH insensitivity syndrome (GHIS) is associated with many different mutations of the GH receptor (GHR) gene. We examined the phenotypic and biochemical features in 82 GHIS patients from 23 countries, each fulfilling diagnostic criteria of GHIS. There were 45 males and 37 females [mean age, 8.25 yr; mean height, -6.09 SD score, and mean insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3), -7.99 SD score]. Sixty-three were GH-binding protein (GHBP) negative; 19 were GHBP positive (> 10% binding). The mean height in GHBP-negative subjects was -6.5 SD score, and that in GHBP-positive patients was -4.9 SD score (P = < 0.001). Clinical and biochemical heterogeneity was demonstrated by the wide range of height (-2.2 to -10.4 SD score) and IGFBP-3 (-1.4 to -14.7 SD score) values, which were positively correlated (r2 = 0.45; P = < 0.001). This contrasted with the lack of correlation between mean parental height SD score and height SD score (r2 = 0.01). Fifteen different GH receptor gene mutations were identified in 27 patients. All had homozygous defects, except 1 who had a compound heterozygous defect. The mutations were 5 nonsense, 2 frame shift, 4 splice, 4 missense, and 1 compound heterozygote. There was no relationship between mutation type or exon of the GHR gene involved and height or IGFBP-3 SD score. In conclusion, GHIS is associated with wide variation in the severity of clinical and biochemical phenotypes. This variation cannot clearly be accounted for by defects in the GHR gene. Other genetic and/or environmental factors must, therefore, contribute to phenotype in GHIS.

A membrane-fixed, truncated isoform of the human growth hormone receptor.

Previously, we reported the identification of a new human GH receptor (hGHR) messenger RNA species that encodes a smaller hGHR isoform, termed hGHRtr. Its messenger RNA is expressed in several human tissues and predicts a severely truncated GHR protein that lacks 97.5% of the intracellular domain. Because these two hGHR isoforms, which display similar binding affinity, are coexpressed in several tissues, they may reside side by side and, therefore, interrelate. To further characterize the biological properties of hGHRtr in comparison with hGHR, we generated Chinese hamster ovary (CHO) cell lines stably expressing each of these hGHR isoforms. Cross-linking of [125I]hGH to CHO/hGHRtr cells revealed a majored specific complex with apparent Mr of approximately 100 kDa, which would indicate the hGHRtr to be in molecular mass form of about 80 kDa. When compared with CHO/hGHR, CHO/hGHRtr cells secreted higher amounts of soluble GH-binding protein (GHBP). In contrast to CHO/hGHR cells, CHO/hGHRtr cells did not exhibit any GH-induced receptor down-regulation, and internalization was markedly reduced. Analysis of the constitutive turnover of cellular hGHR and soluble GHBP showed that incubation of CHO/hGHR cells with cycloheximide caused parallel disappearance of hGHR and GHBP. This contrasted with the stability of GHRtr, which showed no decline after cycloheximide treatment for up to 4 h, suggesting that the bulk GHRtr and GHBP may be derived from preformed proteins. Thus, in contrast to hGHR, hGHRtr is fixed at the cell membrane; it undergoes minimal internalization, no down-regulation by hGH, no constitutive turnover for as long as 4 h, but increased capacity to generate a soluble GHBP. Because hGHRtr failed to undergo ligand-induced internalization, the source of the continuous, undisturbed GHBP released into the medium may be from an intracellular storage pool. The relative abundance of these two hGHR isoforms, through regulation of splicing, could be of critical importance in modulating the biological effects of GH.

Intronic mutation in the growth hormone (GH) receptor gene from a girl with Laron syndrome and extremely high serum GH binding protein: extended phenotypic study in a very large pedigree.

Laron syndrome (LS) is a hereditary form of GH resistance due to molecular defects in the GH receptor (GHR). Most of the identified mutations are located in the extracellular domain of the receptor, resulting in a lack of serum GHBP in the majority of LS patients. We present an LS patient with supranormal levels of serum GHBP, in addition to 35 of her relatives. The proband is a 3.5 year-old Druse girl with severe short stature (height SDS -5.1), high GH (250 micrograms/l), low IGF-I (2.7 nmol/l) and IGFBP-3 (410 micrograms/l), both unresponsive to exogenous GH. The binding capacity of the serum GHBP was 22 nM (adult reference serum, 0.7 nM), with an affinity constant Ka = 1.9 x 10(9) M-1 comparable to that of normal sera (Ka = 1.7-2.1 x 10(9) M-1). The apparent MW of the GHBP was approximately 60-80 kDa, similar to that of control sera. In the proband's sister, parents, grandparents and uncles, extremely high GHBP values were observed (43.0 +/- 4.8 RSB, n = 10) compared with normal adults (0.81 +/- 0.06 RSB) (p << 0.001). The remaining subjects had normal or moderately elevated GHBP levels. Serum GH in adults with high GHBP was significantly elevated above control values (6.0 +/- 0.9 micrograms/l vs 0.76 +/- 0.13 microgram/l, p < 0.001). Serum IGF-I and IGFBP-3 levels were normal in all the subjects, with the exception of an aunt (IGF-I 3.9 nmol/l) and the proband's sister (IGFBP-3 460 micrograms/l). All the subjects' heights were within the normal range. Analysis of the GHR gene performed in the proband revealed an as yet undescribed homozygous intronic point mutation. It consists of a G-->T substitution at nucleotide 785-1 preceding exon 8, a sequence that encodes the transmembrane domain. This mutation, which destroys the invariant dinucleotide of the splice acceptor site, is expected to alter GHR mRNA splicing and to be responsible for skipping exon 8. The resulting truncated protein that retains GH binding activity is probably no longer anchored in the cell membrane, affecting signal transmission in the homozygous patient and causing high GHBP levels in the heterozygous relatives.

Nine novel growth hormone receptor gene mutations in patients with Laron syndrome.

The GH receptor (GHR) is a member of the cytokine receptor superfamily; GH binding protein is the solubilized extracellular domain of the GHR. Abnormalities in the GHR produce an autosomal recessive form of GH resistance, the Laron syndrome, characterized by growth failure and the clinical appearance of severe GH deficiency despite elevated circulating GH levels. In 13 unrelated patients with undetectable levels of GH binding protein, we characterized nine novel mutations in the GHR gene. These molecular defects comprise three nonsense mutations (Q65X, W80X, and W157X), one frameshift (36delC), two splice defects (G-->A at 70 + 1, C-->T at 723), and three missense mutations (C38S, S40L, and W50R) located in the extracellular domain of the receptor, and thus would be expected to interfere with GH binding activity. These results further confirm the broad heterogeneity of mutations underlying this rare GH resistance syndrome.

Alternatively spliced forms in the cytoplasmic domain of the human growth hormone (GH) receptor regulate its ability to generate a soluble GH-binding protein.

The mechanism underlying the generation of soluble growth hormone binding protein (GHBP) probably differs among species. In rats and mice, it involves an alternatively spliced mRNA, whereas in rabbits, it involves limited proteolysis of the membrane-bound growth hormone receptor (GHR). In humans, this latter mechanism is favored, as no transcript coding for a soluble GHR has been detected so far. To test this hypothesis, we analyzed COS-7 cells transiently expressing the full-length human (h) GHR and observed specific GH-binding activity in the cell supernatants. Concomitantly, an alternatively spliced form in the cytoplasmic domain of GHR, hGHR-tr, was isolated from several human tissues. hGHR-tr is identical in sequence to hGHR, except for a 26-bp deletion leading to a stop codon at position 280, thereby truncating 97.5% of the intracellular domain of the receptor protein. When compared with hGHR, hGHR-tr showed a significantly increased capacity to generate a soluble GHBP. Interestingly, this alternative transcript is also expressed in liver from rabbits, mice, and rats, suggesting that, in these four species, proteolysis of the corresponding truncated transmembrane GHR is a common mechanism leading to GHBP generation. These findings support the hypothesis that GHBP may at least partly result from alternative splicing of the region encoding the intracellular domain and that the absence of a cytoplasmic domain may be involved in increased release of GHBP.

Molecular basis of inherited growth hormone resistance in childhood.

The growth hormone receptor (GHR), a member of the cytokine receptor superfamily that gives rise to a soluble and circulating counterpart (GHBP), is the main target of Laron syndrome (LS), a severe autosomal recessive dwarfism characterized by complete GH insensitivity. Genetic and mutation analyses have attested to the high molecular heterogeneity of this syndrome, and, to date, more than 30 different GHR mutations including deletion, frameshift, nonsense, missense and splicing defects have been described. However, among them, missense mutations are of particular interest in potentially providing critical information on the structure-function relationship of the GHR and related molecules. The study of the recently described forms of atypical LS is now very promising. These patients display detectable plasma GH binding activity associated with complete or partial GH insensitivity. Molecular analysis of such a phenotype with positive GHBP and complete GH insensitivity has revealed the existence of a missense mutation abolishing receptor homodimerization, thereby providing in vivo evidence for the critical role of the dimerization process in the growth-promoting action of GH. Similarly, mutations in the cytoplasmic region, which are expected to be associated with normal GH binding activity, should contribute to the identification of other functionally important domains. Partial GH insensitivity syndromes may theorically encompass a wide range of distinct phenotypes with variable degrees of GH resistance. Missense GHR mutations and a quantitative GHR mRNA defect have been identified in some cases belonging to this heterogeneous group. Interestingly, exclusion of linkage between the Laron phenotype and the GHR locus was demonstrated in one affected family. This latter situation may indicate the existence of other genes controlling GHR expression or required at different steps of the signal transduction pathway. In this regard, the availability of a possible animal model for LS should offer new prospects in the identification of GH-inducible genes.

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.

A single amino acid substitution in the exoplasmic domain of the human growth hormone (GH) receptor confers familial GH resistance (Laron syndrome) with positive GH-binding activity by abolishing receptor homodimerization.

Growth hormone (GH) elicits a variety of biological activities mainly mediated by the GH receptor (GHR), a transmembrane protein that, based on in vitro studies, seemed to function as a homodimer. To test this hypothesis directly, we investigated patients displaying the classic features of Laron syndrome (familial GH resistance characterized by severe dwarfism and metabolic dysfunction), except for the presence of normal binding activity of the plasma GH-binding protein, a molecule that derives from the exoplasmic-coding domain of the GHR gene. In two unrelated families, the same GHR mutation was identified, resulting in the substitution of a highly conserved aspartate residue by histidine at position 152 (D152H) of the exoplasmic domain, within the postulated interface sequence involved in homodimerization. The recombinant mutated receptor protein was correctly expressed at the plasma membrane. It displayed subnormal GH-binding activity, a finding in agreement with the X-ray crystal structure data inferring this aspartate residue outside the GH-binding domain. However, mAb-based studies suggested the critical role of aspartate 152 in the proper folding of the interface area. We show that a recombinant soluble form of the mutant receptor is unable to dimerize, the D152H substitution also preventing the formation of heterodimers of wild-type and mutant molecules. These results provide in vivo evidence that monomeric receptors are inactive and that receptor dimerization is involved in the primary signalling of the GH-associated growth-promoting and metabolic actions.

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.