Mutation of BSND causes Bartter syndrome with sensorineural deafness and kidney failure.

Antenatal Bartter syndrome (aBS) comprises a heterogeneous group of autosomal recessive salt-losing nephropathies. Identification of three genes that code for renal transporters and channels as responsible for aBS has resulted in new insights into renal salt handling, diuretic action and blood-pressure regulation. A gene locus of a fourth variant of aBS called BSND, which in contrast to the other forms is associated with sensorineural deafness (SND) and renal failure, has been mapped to chromosome 1p. We report here the identification by positional cloning, in a region not covered by the human genome sequencing projects, of a new gene, BSND, as the cause of BSND. We examined ten families with BSND and detected seven different mutations in BSND that probably result in loss of function. In accordance with the phenotype, BSND is expressed in the thin limb and the thick ascending limb of the loop of Henle in the kidney and in the dark cells of the inner ear. The gene encodes a hitherto unknown protein with two putative transmembrane alpha-helices and thus might function as a regulator for ion-transport proteins involved in aBS, or else as a new transporter or channel itself.

A new gene involved in X-linked mental retardation identified by analysis of an X;2 balanced translocation.

X-linked forms of mental retardation (MR) affect approximately 1 in 600 males and are likely to be highly heterogeneous. They can be categorized into syndromic (MRXS) and nonspecific (MRX) forms. In MRX forms, affected patients have no distinctive clinical or biochemical features. At least five MRX genes have been identified by positional cloning, but each accounts for only 0.5%-1.0% of MRX cases. Here we show that the gene TM4SF2 at Xp11.4 is inactivated by the X breakpoint of an X;2 balanced translocation in a patient with MR. Further investigation led to identification of TM4SF2 mutations in 2 of 33 other MRX families. RNA in situ hybridization showed that TM4SF2 is highly expressed in the central nervous system, including the cerebral cortex and hippocampus. TM4SF2 encodes a member of the tetraspanin family of proteins, which are known to contribute in molecular complexes including beta-1 integrins. We speculate that through this interaction, TM4SF2 might have a role in the control of neurite outgrowth.

DNA sequence and comparative analysis of chimpanzee chromosome 22.

Human-chimpanzee comparative genome research is essential for narrowing down genetic changes involved in the acquisition of unique human features, such as highly developed cognitive functions, bipedalism or the use of complex language. Here, we report the high-quality DNA sequence of 33.3 megabases of chimpanzee chromosome 22. By comparing the whole sequence with the human counterpart, chromosome 21, we found that 1.44% of the chromosome consists of single-base substitutions in addition to nearly 68,000 insertions or deletions. These differences are sufficient to generate changes in most of the proteins. Indeed, 83% of the 231 coding sequences, including functionally important genes, show differences at the amino acid sequence level. Furthermore, we demonstrate different expansion of particular subfamilies of retrotransposons between the lineages, suggesting different impacts of retrotranspositions on human and chimpanzee evolution. The genomic changes after speciation and their biological consequences seem more complex than originally hypothesized.

IXDB, an X chromosome integrated database (update).

Chromosome specific databases are an important research tool as they integrate data from different directions, such as genetic and physical mapping data, expression data, sequences etc. They supplement the genome-wide repositories in molecular biology, such as GenBank, Swiss-Prot or OMIM, which usually concentrate on one type of information. The Integrated X Chromosome Database (IXDB, http://ixdb.mpimg-berlin-dahlem.mpg.de/) is a repository for physical mapping data of the human X chromosome and aims at providing a global view of genomic data at a chromosomal level. We present here an update of IXDB which includes schema extensions for storing submaps and sequence information, additional links to external databases, and the integration of an increasing number of physical and transcript mapping data. The gene data was completely updated according to the approved gene symbols of the HUGO Nomenclature Committee. IXDB receives over 1000 queries per month, an indication that its content is valuable to researchers seeking mapping data of the human X chromosome.

ACRC codes for a novel nuclear protein with unusual acidic repeat tract and maps to DYT3 (dystonia parkinsonism) critical interval in xq13.1.

We searched for novel genes as candidates of X-linked dystonia parkinsonism (XDP) in the critical interval of Xq13.1 that harbors the disease locus (DYT3). A gene, ACRC (acidic repeat containing), was discovered by a combination of in silico and "wet" experiments. ACRC is composed of at least 12 exons and 11 introns. It is expressed in all tissues tested, including skeletal muscle, liver, kidney, pancreas, heart, lung, and brain. Highest levels of expression are found in skeletal muscle. The ACRC protein is characterized by a previously undescribed acidic repeat tract of 21 units of 8-10 amino acids. The N-terminal portion of the protein is highly acidic (pI=3.2), and the C-terminal region is basic (pI=10.2). There are nuclear localization signals in its C-terminal portion. Extensive mutation analysis of the transcribed region of the gene, including intron-exon boundaries and the 5' and 3' untranslated intervals, did not reveal a mutation in XDP patients. Exclusion of a mutation in the transcribed portion of this and all other known genes within the DYT3 critical interval suggests that XDP is most likely caused by a mutation in a regulatory region of a gene within the critical interval, or by a structural rearrangement.

Exclusion of malignant hyperthermia susceptibility (MHS) from a putative MHS2 locus on chromosome 17q and of the alpha 1, beta 1, and gamma subunits of the dihydropyridine receptor calcium channel as candidates for the molecular defect.

Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disease with autosomal dominant inheritance triggered by exposure to commonly used inhalational anaesthetics or depolarising muscle relaxants. A MHS locus has been identified on human chromosome 19q12-q13.2 and the gene for the skeletal muscle calcium release channel of sarcoplasmic reticulum (ryanodine receptor) (RYR1) is considered a candidate for the molecular defect. However, MH has been shown to be genetically heterogeneous, and in the ensuing search for other MHS genes, a locus on chromosome 17q has been proposed, and the gene for the adult muscle sodium channel (SCN4A) was suggested as a candidate. We performed linkage studies using polymorphic microsatellite markers for subunits of the skeletal muscle dihydropyridine (DHP) receptor, CACNL1A3 mapped to chromosome 1q, as well as C-ACNLB1 and CACNLG, the latter two localised on chromosome 17q11.2-q24 in proximity to the proposed MHS2 and the SCN4A loci, and we also included markers for the loci D17S250, D17S579, NM23 (NME1), GH1, and SCN4A from that region. Our results exclude the alpha 1, beta 1 and gamma subunit of the DHP receptor as well as the SCN4A locus from that region. Our results exclude the alpha 1, beta 1, and gamma subunit of the DHP receptor as well as the SCN4A locus as candidates for the molecular defect in MHS for these pedigrees where also the RYR1 on chromosome 19q13.1 has been excluded. A multipoint analysis excludes the disease from the entire 84 cM interval containing the proposed MHS locus on chromosome 17q.(ABSTRACT TRUNCATED AT 250 WORDS)

Incidental prenatal detection of an Xp deletion using an anonymous primer pair for fetal sexing.

We report on the incidental prenatal detection of an interstitial X-chromosomal deletion in a male fetus and his mother by fetal sexing with a primer pair recognizing an X-Y homologous locus (DXYS19), formerly unassigned on the X chromosome. The proband asked for prenatal diagnosis because of her elevated age and risk of Duchenne muscular dystrophy (DMD). Prior to molecular genetic testing for DMD, fetal sexing was carried out on DNA prepared from cultured amniocytes. PCR analysis revealed the expected Y-chromosomal product, but did not show the constitutive X-chromosomal fragment. The absence of the X-chromosomal fragment in the fetus and on one X chromosome of the mother was confirmed by Southern hybridization of HindIII restricted DNA with probe pJA1165 (DXYS19). DXYS19X was mapped to Xp22.3 by combining several approaches, including: (1) analysis of somatic cell hybrid lines containing different fragments of the human X chromosome; (2) Southern hybridization of a yeast artificial chromosome (YAC)-filter panel provided by the Resource Center/Primary Database (RZPD); (3) FISH analysis; and (4) re-evaluation of two patients with interstitial deletions in Xp22.3. The extent of the deletion in the fetus was estimated by further markers from Xp22.3 and found to include the STS gene. Mental retardation could not be excluded since some mentally retarded patients exhibit overlapping deletions.

Mapping of a further malignant hyperthermia susceptibility locus to chromosome 3q13.1.

Malignant hyperthermia (MH) is a potentially lethal pharmacogenetic disease for which MH susceptibility (MHS) is transmitted as an autosomal dominant trait. A potentially life-threatening MH crisis is triggered by exposure to commonly used inhalational anesthetics and depolarizing muscle relaxants. The first malignant hyperthermia susceptibility locus (MHS1) was identified on human chromosome 19q13.1, and evidence has been obtained that defects in the gene for the calcium-release channel of skeletal muscle sarcoplasmic reticulum (ryanodine receptor; RYR1) can cause some forms of MH. However, MH has been shown to be genetically heterogeneous, and additional loci on chromosomes 17q and 7q have been suggested. In a collaborative search of the human genome with polymorphic microsatellite markers, we now found linkage of the MHS phenotype, as assessed by the European in vitro contracture test protocol, to markers defining a 1-cM interval on chromosome 3q13.1. A maximum multipoint lod score of 3.22 was obtained in a single German pedigree with classical MH, and none of the other pedigrees investigated in this study showed linkage to this region. Linkage to both MHS1/RYR1 and putative loci on chromosome 17q and 7q were excluded. This study supports the view that considerable genetic heterogeneity exists in MH.

Discordance, in a malignant hyperthermia pedigree, between in vitro contracture-test phenotypes and haplotypes for the MHS1 region on chromosome 19q12-13.2, comprising the C1840T transition in the RYR1 gene.

A point mutation in the gene encoding the skeletal muscle calcium release channel (RYR1) has been proposed as the probable cause of malignant hyperthermia (MH) in swine, where it segregates with the disease in all MH-prone strains investigated. The same C-to-T exchange in nucleotide position 1840 of the human RYR1 cDNA sequence was found in a few human MH pedigrees. We report a German MH pedigree where in vitro contracture test (IVCT) results and haplotypes of markers for the MHS1/RYR1 region including this base transition have yielded several discrepancies. The MH-susceptible phenotype was defined by IVCT performed according to the European standard protocol. Haplotypes were constructed for markers for the MHS1/RYR1 region on chromosome 19 and include the C1840T base exchange. Discussing the probabilities for a number of hypotheses to explain these data, we suggest that our results may challenge the causative role of this mutation--and possibly the role of the RYR1 gene itself--in human MH susceptibility, at least in some cases.

X chromosome-specific cDNA arrays: identification of genes that escape from X-inactivation and other applications.

Mutant alleles are frequently characterized by low expression levels. Therefore, cDNA array-based gene expression profiling may be a promising strategy for identifying gene defects underlying monogenic disorders. To study the potential of this approach, we have generated an X chromosome-specific microarray carrying 2423 cloned cDNA fragments, which represent up to 1317 different X-chromosomal genes. As a prelude to testing cell lines from patients with X-linked disorders, this array was used as a hybridization probe to compare gene expression profiles in lymphoblastoid cell lines from normal males, females and individuals with supernumerary X chromosomes. Measurable hybridization signals were obtained for more than half of the genes represented on the chip. A total of 53 genes showed elevated expression levels in cells with multiple X chromosomes and many of these were found to escape X-inactivation. Moreover, the detection of a male-viable deletion encompassing three genes illustrates the utility of this array for the identification of small unbalanced chromosome rearrangements.

Characterization of a highly complex region in Xq13 and mapping of three isodicentric breakpoints associated with preleukemia.

The chromosomal abnormality represented by an isodicentric X chromosome [idic(X)(q13)] is associated with a subset of acute myeloid leukemia (AML) and preleukemia observed in elderly females. A previous study localized the breakpoints of two acquired isodicentric X chromosomes associated with myelodysplasia to a 450-kb region proximal to the XIST gene. Here we report the construction and extensive characterization of a reliable 1-Mb P1 artificial chromosome and bacterial artificial chromosome contig covering a highly problematic region in Xq13 that includes the previously described isodicentric breakpoint region. In addition to mapping of the brain-specific gene (NAP1L2) and the phosphoglyceryl kinase alpha subunit 1 gene (PHKA1) and generation and mapping of a large number of STSs throughout the contig, we have mapped a putative transcriptional regulatory protein (HDACL1), and 35 ESTs. Sequencing data, Southern blot analysis, and fiber-FISH analysis have permitted characterization of extensive region-specific duplications and triplications in addition to an unusually high concentration of long interspersed repeat elements, both of which could be implicated in isodicentric chromosome formation and other Xq13 chromosome aberrations. FISH analysis of metaphase chromosomes from two previously unpublished AML patients and one preleukemic patient using cosmid clones and selected subclones allowed mapping of the idic(X)(q13) breakpoints to a 100-kb interval, consistent with the involvement of an X-linked gene in the genesis of this form of preleukemia, disruption of which may represent a preliminary step in progression to AML. Assembly and physical mapping of this complex 1-Mb contig establish a foundation for ongoing sequencing and gene identification projects in the region.

Hailey-Hailey disease is caused by mutations in ATP2C1 encoding a novel Ca(2+) pump.

Hailey-Hailey disease (HHD) is an autosomal dominant skin disorder characterized by suprabasal cell separation (acantholysis) of the epidermis. Previous genetic linkage studies localized the gene to a 5 cM interval on human chromosome 3q21. After reducing the disease critical region to <1 cM, we used a positional cloning strategy to identify the gene ATP2C1, which is mutated in HHD. ATP2C1 encodes a new class of P-type Ca(2+)-transport ATPase, which is the homologue for the rat SPLA and the yeast PMR1 medial Golgi Ca(2+)pumps and is related to the sarco(endo)plasmic calcium ATPase (SERCA) and plasma membrane calcium ATPase (PCMA) families of Ca(2+)pumps. The predicted protein has the same apparent transmembrane organization and contains all of the conserved domains present in other P-type ATPases. ATP2C1 produces two alternative splice variants of approximately 4.5 kb encoding predicted proteins of 903 and 923 amino acids. We identified 13 different mutations, including nonsense, frameshift insertion and deletions, splice-site mutations, and non-conservative missense mutations. This study demonstrates that defects in ATP2C1 cause HHD and together with the recent identification of ATP2A2 as the defective gene in Darier's disease, provide further evidence of the critical role of Ca(2+)signaling in maintaining epidermal integrity.