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.

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.

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.

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.

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.