Potassium overdose using an oral solution: Potassium element expressed in grams as a packaging flaw contributing to medication error.

Drug packaging contributes to the harm-benefit balance of a treatment. Poorly designed packaging can lead to drug misuse with serious consequences. We report a potassium double dose medication error concerning an oral solution. It was triggered by a packaging flaw related to the dosage indicated on the box, expressed for both potassium salt and elemental potassium. Standardizing the units used on packaging and for prescriptions, by using millimoles, could be a solution to avoid this type of medication error.

Mutations in LHX3 result in a new syndrome revealed by combined pituitary hormone deficiency.

Combined pituitary hormone deficiency (CPHD) has been linked with rare abnormalities in genes encoding transcription factors necessary for pituitary development. We have isolated LHX3, a gene involved in a new syndrome, using a candidate-gene approach developed on the basis of documented pituitary abnormalities of a recessive lethal mutation in mice generated by targeted disruption of Lhx3 (ref. 2). LHX3, encoding a member of the LIM class of homeodomain proteins, consists of at least six exons located at 9q34. We identified a homozygous LHX3 defect in patients of two unrelated consanguineous families displaying a complete deficit in all but one (adrenocorticotropin) anterior pituitary hormone and a rigid cervical spine leading to limited head rotation. Two of these patients also displayed a severe pituitary hypoplasia, whereas one patient presented secondarily with an enlarged anterior pituitary. These LHX3 mutations consist of a missense mutation (Y116C) in the LIM2 domain at a phylogenetically conserved residue and an intragenic deletion predicting a severely truncated protein lacking the entire homeodomain. These data are consistent with function of LHX3 in the proper development of all anterior pituitary cell types, except corticotropes, and extrapituitary structures.

RPGR is mutated in patients with a complex X linked phenotype combining primary ciliary dyskinesia and retinitis pigmentosa.

INTRODUCTION: Primary ciliary dyskinesia (PCD) is a rare disease classically transmitted as an autosomal recessive trait and characterised by recurrent airway infections due to abnormal ciliary structure and function. To date, only two autosomal genes, DNAI1 and DNAH5 encoding axonemal dynein chains, have been shown to cause PCD with defective outer dynein arms. Here, we investigated one non-consanguineous family in which a woman with retinitis pigmentosa (RP) gave birth to two boys with a complex phenotype combining PCD, discovered in early childhood and characterised by partial dynein arm defects, and RP that occurred secondarily. The family history prompted us to search for an X linked gene that could account for both conditions. RESULTS: We found perfect segregation of the disease phenotype with RP3 associated markers (Xp21.1). Analysis of the retinitis pigmentosa GTPase regulator gene (RPGR) located at this locus revealed a mutation (631_IVS6+9del) in the two boys and their mother. As shown by study of RPGR transcripts expressed in nasal epithelial cells, this intragenic deletion, which leads to activation of a cryptic donor splice site, predicts a severely truncated protein. CONCLUSION: These data provide the first clear demonstration of X linked transmission of PCD. This unusual mode of inheritance of PCD in patients with particular phenotypic features (that is, partial dynein arm defects and association with RP), which should modify the current management of families affected by PCD or RP, unveils the importance of RPGR in the proper development of both respiratory ciliary structures and connecting cilia of photoreceptors.

A naturally occurring growth hormone receptor mutation: in vivo and in vitro evidence for the functional importance of the WS motif common to all members of the cytokine receptor superfamily.

To obtain an animal model for studying the role of the GH receptor (GHR) in growth and development, we analyzed a sex-linked dwarf (SLD) chicken strain (Leghorn) which exhibits phenotype similarities with a human genetic growth disorder, an autosomal recessive GH resistance condition (Laron dwarfism). Having previously demonstrated the responsibility of the human GHR gene in the Laron phenotype, we focused our analysis on the corresponding gene in SLD chickens. Sequencing of the whole coding region of the chicken GHR cDNA identified a G-to-T transversion segregating with the SLD phenotype and generating an isoleucine instead of a serine at position 199 within a highly conserved region close to the junction between the extracellular and transmembrane domains. This defect involves the last invariant amino acid of the WS-like motif (amino acid sequence WSXWS) common to all members of the cytokine receptor superfamily. Transfection of a mutated GHR cDNA containing this mutation into eukaryotic cells led to the synthesis of a receptor protein that displayed impaired plasma membrane expression and binding activity. These data define the molecular basis for the SLD phenotype and identify this strain as an interesting model for studying Laron dwarfism in humans; this animal model may also represent a system in which therapeutic strategies to promote growth can be evaluated. Finally, the nature of the molecular defect identified provides direct evidence for the functional importance of the WS motif in GHRs and related receptors.

Expression and binding properties of two isoforms of the human growth hormone receptor.

Two isoforms of the human growth hormone receptor mRNA, one containing exon 3 (encoding an extracellular domain of the receptor), hGHR, and one excluding exon 3, hGHRd3, have been described. To study the cellular distribution of the two types of messengers we have analysed a panel of tissues. Both isoforms were expressed independently or simultaneously depending on the tissue studied. To investigate the binding properties of hGHRd3 we have cloned its cDNA in a eukaryotic expression vector; transient expression in COS-7 cells showed that the receptor without exon 3 was expressed on the plasma membrane and was able to bind human growth hormone (hGH) with the same high affinity as hGHR. Human lactogen (hCS) removed 125I-hGH bound to the full-length and exon 3-excluding receptors to the same extent. These results show that hGHR and hGHRd3 have tissue-specific expression and share identical binding properties for hGH and hCS and leave open the possibility that exon 3 might influence receptor signalling.

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.

Isolation of several human axonemal dynein heavy chain genes: genomic structure of the catalytic site, phylogenetic analysis and chromosomal assignment.

Dynein heavy chains (DHCs) are the main components of multisubunit motor ATPase complexes called dyneins. Axonemal dyneins provide the driving force for ciliary and flagellar motility. Recent molecular studies demonstrated that multiple DHC isoforms are produced by separate genes. We describe the isolation of five human axonemal DHC genes. Analysis of the human genomic clones revealed the existence of intronic sequences that were used to demonstrate that human axonemal DHC genes are located on different chromosomes. The cloned human DHC sequences were integrated into an evolutionary approach based on phylogenetic analysis. Tissue expression studies showed that these human axonemal DHCs are expressed in testis and/or trachea, two tissues with axonemal structures that can be altered in primary ciliary dyskinesia, making DHC genes strong candidates in the genesis of these human diseases.

[Molecular basis of the primary ciliary dyskinesias]. / Bases moléculaires des dyskinésies ciliaires primitives.

INTRODUCTION: The primary ciliary dyskinesias (PCD) are rare diseases characterised by infection of the airways due to impaired muco-ciliary clearance. Half the patients have situs inversus making up Kartagener's syndrome. STATE OF THE ART: Primary cilia play a role in development. In the adult ciliated cells occur mainly in the airways and the genital tract. The axoneme, the internal structure of the cilia, is made up of a central pair of microtubules surrounded by peripheral doublets carrying the inner and outer dynein arms. These multiprotein complexes are composed of chains of dynein whose ATPase activity is the basis of ciliary movement. Structural and functional abnormalities of the respiratory ciliated cells are the cause of PCD, diseases that are heterogeneous at both the genetic and ultrastructural levels. PERSPECTIVES: There are more than two hundred axonemal proteins. The synthesis and assembly of these proteins are controlled by transcription factors and intraflagellar transport molecules respectively. The genes that code for these proteins are as numerous as candidate genes for PCD. CONCLUSIONS: To date only two dynein genes, DNA11 and DNAH5, have been implicated and only in individuals suffering from PCD with absence of outer dynein arms.

[Molecular genetics of growth hormone resistance syndrome]. / Génétique moléculaire des syndromes de résistance à l'hormone de croissance.

The cloning of a putative growth hormone receptor (GH-R) cDNA has opened new approaches for the understanding of the molecular basis of GH insensitivity in humans. This molecule belongs to a new class of transmembrane receptors including prolactin, granulocyte-macrophage colony stimulating factor, erythropoietin and some interleukin receptors. Although the domains responsible for signal transduction have not yet been identified, the molecular study of a GH-resistance syndrome described by Laron et al. should provide insight into the structure-function relationships of the GH-R and related receptors. This autosomal recessive disorder is characterized by very low serum levels of Insulin-Like Growth Factor I (IGF-I), despite increased secretion of GH with normal activity. Two approaches can be used to test the involvement of the GH-R in this syndrome. The first one, which is indirect, is performed through linkage analysis between GH-R and Laron phenotype; this allowed us to incriminate the GR-R gene in this syndrome. The second approach consists in the identification of molecular defects in the GH-R gene of patients with Laron syndrome; this allowed the detection of a partial gene deletion and different point mutations. The short stature of the Pygmee population could be related to the Laron syndrome because individuals from this population are also resistant to GH therapy. Therefore, it seems interesting to search for molecular variations of the GH-R gene in this population. Nevertheless, preliminary results indicate that the GH-R gene is not directly involved in this particular short stature condition.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

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.

Loss-of-function mutations in a human gene related to Chlamydomonas reinhardtii dynein IC78 result in primary ciliary dyskinesia.

Primary ciliary dyskinesia (PCD) is a group of heterogeneous disorders of unknown origin, usually inherited as an autosomal recessive trait. Its phenotype is characterized by axonemal abnormalities of respiratory cilia and sperm tails leading to bronchiectasis and sinusitis, which are sometimes associated with situs inversus (Kartagener syndrome) and male sterility. The main ciliary defect in PCD is an absence of dynein arms. We have isolated the first gene involved in PCD, using a candidate-gene approach developed on the basis of documented abnormalities of immotile strains of Chlamydomonas reinhardtii, which carry axonemal ultrastructural defects reminiscent of PCD. Taking advantage of the evolutionary conservation of genes encoding axonemal proteins, we have isolated a human sequence (DNAI1) related to IC78, a C. reinhardtii gene encoding a dynein intermediate chain in which mutations are associated with the absence of outer dynein arms. DNAI1 is highly expressed in trachea and testis and is composed of 20 exons located at 9p13-p21. Two loss-of-function mutations of DNAI1 have been identified in a patient with PCD characterized by immotile respiratory cilia lacking outer dynein arms. In addition, we excluded linkage between this gene and similar PCD phenotypes in five other affected families, providing a clear demonstration of locus heterogeneity. These data reveal the critical role of DNAI1 in the development of human axonemal structures and open up new means for identification of additional genes involved in related developmental defects.