Chromosome Xp11 Research Articles

Cloning, genomic organization, alternative splicing and expression analysis of the human gene WNK3 ( PRKWNK3)

We report the isolation of a full length coding WNK3 cDNA from human fetal brain. The WNK3 transcript has an open reading frame of 5403 nucleotides and encodes a putative protein of 1800 amino acids. The human WNK3 gene comprises 24 exons and lies within a 559 kb genomic segment on chromosome Xp11.22 which has conserved synteny with a 705 kb genomic segment of human chromosome 9q22.31 which contains WNK2. The WNK3 transcript is expressed in several human fetal and adult tissues and has at least two splice isoforms generated by the alternative splicing of exon 18 and exon 22 which maintain the open reading frame. Usage of exon 18b is restricted to brain and introduces an additional 47 amino acids into the predicted protein. The predicted WNK3 protein has a similar structural organization to the other human WNK kinases. Significant homology between these proteins is confined to three conserved regions of their amino acid sequences which we have designated CR1, CR2 and CR3. CR1 and CR3 contain highly conserved residues which have been shown to be important for the normal function of WNK1 and WNK4, and CR2 contains a highly conserved 22 amino acid motif specific to chordate species. WNK3 lies within the critical linkage interval for several human monogenic disorders, including X-linked mental retardation. The function of mammalian WNK3 kinase remains to be investigated.

Cytogenetic analysis of Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) with Xyp sex determination system using standard staining, C-bands, NOR and synaptonemal complex microspreading techniques.

The mitotic and meiotic chromosomes of the beetles Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) were analysed using standard staining, C-banding and silver impregnation techniques. We determine the diploid and haploid chromosome numbers, the sex determination system and describe the chromosomal morphology, the C-banding pattern and the chromosome(s) bearing NORs (nucleolar organizer regions). Both species shown 2n = 20 chromosomes, the chromosomal meioformula 9 + Xyp, and regular chromosome segregation during anaphases I and II. The chromosomes of E. atomaria are basically metacentric or submetacentric and P. dermestoides chromosomes are submetacentric or subtelocentric. In both beetles the constitutive heterochromatin is located in the pericentromeric region in all autosomes and in the Xp chromosome; additional C-bands were observed in telomeric region of the short arm in some autosomes in P. dermestoides. The yp chromosome did not show typical C-bands in these species. As for the synaptonemal complex, the nucleolar material is associated to the 7th bivalent in E. atomaria and 3rd and 7th bivalents in P. dermestoides. Strong silver impregnated material was observed in association with Xyp in light and electron microscopy preparations in these species and this material was interpreted to be related to nucleolar material.

Refinement of the X-linked cataract locus (CXN) and gene analysis for CXN and Nance-Horan syndrome (NHS)

The X-linked congenital cataract (CXN) locus has been mapped to a 3-cM (approximately 3.5 Mb) interval on chromosome Xp22.13, which is syntenic to the mouse cataract disease locus Xcat and encompasses the recently refined Nance-Horan syndrome (NHS) locus. A positional cloning strategy has been adopted to identify the causative gene. In an attempt to refine the CXN locus, seven microsatellites were analysed within 21 individuals of a CXN family. Haplotypes were reconstructed confirming disease segregation with markers on Xp22.13. In addition, a proximal cross-over was observed between markers S3 and S4, thereby refining the CXN disease interval by approximately 400 Kb to 3.2 Mb, flanked by markers DXS9902 and S4. Two known genes (RAI2and RBBP7) and a novel gene (TL1) were screened for mutations within an affected male from the CXN family and an NHS family by direct sequencing of coding exons and intron-exon splice sites. No mutations or polymorphisms were identified, therefore excluding them as disease-causative in CXN and NHS. In conclusion, the CXN locus has been successfully refined and excludes PPEF1as a candidate gene. A further three candidates were excluded based on sequence analysis. Future positional cloning efforts will focus on the region of overlap between CXN, Xcat, and NHS.

NDP gene mutations in 14 French families with Norrie disease.

Norrie disease is a rare X-inked recessive condition characterized by congenital blindness and occasionally deafness and mental retardation in males. This disease has been ascribed to mutations in the NDP gene on chromosome Xp11.1. Previous investigations of the NDP gene have identified largely sixty disease-causing sequence variants. Here, we report on ten different NDP gene allelic variants in fourteen of a series of 21 families fulfilling inclusion criteria. Two alterations were intragenic deletions and eight were nucleotide substitutions or splicing variants, six of them being hitherto unreported, namely c.112C>T (p.Arg38Cys), c.129C>G (p.His43Gln), c.133G>A (p.Val45Met), c.268C>T (p.Arg90Cys), c.382T>C (p.Cys128Arg), c.23479-1G>C (unknown). No NDP gene sequence variant was found in seven of the 21 families. This observation raises the issue of misdiagnosis, phenocopies, or existence of other X-linked or autosomal genes, the mutations of which would mimic the Norrie disease phenotype.

Further indications for genetic heterogeneity of euthyroid familial goiter.

Iodine deficiency is the most important etiological factor for euthyroid endemic goiter. However, family and twin pair studies also strongly indicate a genetic prediposition. In euthyroid goiters molecular defects in the thyroglobulin (TG), and Na+/I- symporter (NIS) gene have been identified. Numerous mutations in the Pendrin (PDS) gene have been found in families with PDS characterized by deafness and euthyroid goiter. Moreover, family studies indicated two major candidate loci MNG-1 on chromosome 14q31 and Xp22. However, all previous linkage studies investigated only one family. To clarify the general relevance of these previously identified two major candidate loci for the etiology of euthyroid goiter we investigated four families with a total number of 74 family members by linkage analysis with microsatellite markers. Moreover, we analyzed the thyroid candidate genes TG, thyroperoxidase (TPO), NIS, TSH receptor, and PDS. In a further family with 12 members in whom we have previously demonstrated linkage to the MNG-1 locus we investigated the Xp22 locus and the PDS gene in addition to our initial study. Linkage analysis results of our study are not significant enough to definitely exclude or confirm linkage to the investigated candidate genes and loci. Nevertheless, we obtained very weak indications for possible linkage to Xp22 in one family by a maximal multipoint LOD score of 1.15, and cosegregation of haplotypes among affected family members. Moreover, in another family linkage to PDS was indicated by a maximal multipoint LOD score of 1.87 as well as cosegregation of haplotypes. However, sequencing of the PDS gene did not reveal germline mutations. A significant total NPL score of 6.5 for PDS over all families most likely indicated linkage to a genomic region close to PDS. Furthermore, the likelihood of linkage to MNG-1 and Xp22 is reduced, because multipoint LOD scores were below 1 or negative. In all families there was no significant evidence for linkage for the thyroid candidate genes TG, TPO, NIS, or the TSH receptor. In conclusion, a general role of MNG-1 and Xp22 for the etiology of euthyroid goiter is unlikely but cannot clearly excluded. The multipoint parametric and nonparametric LOD scores further suggest genetic heterogeneity in the etiology of familial euthyroid goiter. To identify other susceptibility loci it is necessary to perform genome-wide linkage analysis studies with more families.

Progress in Monoamine Oxidase (MAO) Research in Relation to Genetic Engineering

Monoamine oxidase (MAO) is an enzyme that oxidizes various physiologically and pathologically important monoamine neurotransmitters and hormones such as dopamine, noradrenaline, adrenaline, and serotonin. Two types of MAO, i.e. type A (MAO-A) and type B (MAO-B), were first discovered pharmacologically. MAO-A is inhibited by clorgyline; and MAO-B, by deprenyl. cDNAs MAO-A and MAO-B were cloned and their structures determined. MAO-A and MAO-B are made of similar but different polypeptides and encoded by different nuclear genes located on the X chromosome (Xp11.23). MAO-A and MAO-B genes consist of 15 exons with identical intron–exon organization, suggesting that they were derived from a common ancestral gene. Both enzymes require a flavin cofactor, flavin adenine dinucleotide (FAD), which binds to the cysteine residue of a pentapeptide sequence (Ser–Gly–Gly–Cys–Tyr). Both enzymes exist on the outer membrane of mitochondria of various types of cells in various tissues including the brain. In humans, MAO-A is abundant in the brain and liver, whereas the liver, lungs and intestine are rich in MAO-B. MAO-A oxidizes noradrenaline and serotonin; and MAO-B, mainly β-phenylethylamine. In the human brain, MAO-A exists in catecholaminergic neurons, but MAO-B is found in serotonergic neurons and glial cells. MAO-A knockout mice exhibit increased serotonin levels and aggressive behavior, whereas MAO-B knockout mice show little behavioral change. The gene knockout mice of MAO-A or MAO-B, together with the observation that some humans lack MAO-A, MAO-B, or both have contributed to our understanding of the function of MAO-A and MAO-B in health and disease. MAO-A and MAO-B may be closely related to various neuropsychiatric disorders such as depression and Parkinson’s disease, and inhibitors of them are the subject of drug development for such diseases.

Identification and characterization of mouse SSX genes: a multigene family on the X chromosome with restricted cancer/testis expression☆

Human SSX was first identified as the gene involved in the t(X;18) translocation in synovial sarcoma. SSX is a multigene family, with 9 complete genes on chromosome Xp11. Normally expressed almost exclusively in testis, SSX mRNA is expressed in various human tumors, defining SSX as a cancer/testis antigen. We have now cloned the mouse ortholog of SSX. Mouse SSX genes can be divided into Ssxa and Ssxb subfamilies based on sequence homology . Ssxa has only one member, whereas 12 Ssxb genes, Ssxb1 to Ssxb12, were identified by cDNA cloning from mouse testis and mouse tumors. Both Ssxa and Ssxb are located on chromosome X and show tissue-restricted mRNA expression to testis among normal tissues. All putative human and mouse SSX proteins share conserved KRAB and SSX-RD domains. Mouse tumors were found to express some, but not all, Ssxb genes, similar to the SSX activation in human tumors.

A functional polymorphism in the promoter/enhancer region of the FOXP3/Scurfin gene associated with type 1 diabetes.

FOXP3/Scurfin, a member of forkhead/winged-helix proteins, is involved in the regulation of T-cell activation, and essential for normal immune homeostasis. The FOXP3/Scurfin gene is located on chromosome Xp11.23, which includes one of the type 1 diabetes susceptible loci. Therefore, we investigated whether the human FOXP3/Scurfin gene might be a new candidate gene for type 1 diabetes. We first screened the human FOXP3/Scurfin gene for microsatellite and single nucleotide polymorphisms. Next, we performed an association study between the FOXP3/Scurfin gene and type 1 diabetes. Then, the evaluation of promoter/enhancer activity of the intron with (GT)(n) polymorphism was performed by dual luciferase reporter assay. We demonstrated two regions contained microsatellite polymorphisms; one was (GT)(n), located on intron zero and the other (TC)(n) on intron 5, which were under linkage-disequilibrium. The (GT)(15) allele showed a significantly higher frequency in patients with type 1 diabetes than in controls (43.1% vs 32.6%, P=0.0027). The genotype frequencies of (GT)(15)/(GT)(15) in female patients and of (GT)(15) in male patients tended to be higher than those in female ( P=0.064) and male ( P=0.061) controls, respectively. A significant difference in the enhancer activity between (GT)(15) and (GT)(16) dinucleotide repeats was detected. In conclusion, the FOXP3/Scurfin gene appears to confer a significant susceptibility to type 1 diabetes in the Japanese population.

93rd ENMC international workshop: non-5q-spinal muscular atrophies (SMA) – clinical picture (6–8 April 2001, Naarden, The Netherlands)

This 93rd SMA workshop followed the European Neuromuscular Centre (ENMC) tradition to combine clinical and molecular topics with the aim of not only exchanging current knowledge but also initiating collaborative studies. Despite important milestones in research on autosomal recessive SMA with localisation on chromosome 5q (SMA 5q) in the past 10 years, we are just beginning to understand the molecular basis and phenotypic spectrum of non-5q-SMA entities. The possibility of analysing the SMN gene allowed the delineation of the diagnostic criteria of ‘classical’ proximal SMA and its clinical variability. On the other hand, different entities of SMA could be defined that show atypical features and proved to be unlinked to chromosome 5q. The field of interest has therefore been focussed on further genes responsible for anterior horn cell loss. This will contribute to further understand the underlying pathogenetic pathways, the complex protein interactions and genotype–phenotype correlations in various types of motor neuron disorders. The workshop was structured in a first part, briefly reviewing the current status of SMA 5q including animal models which have been generated. The second part included different sessions on non-5q-SMA forms; clinical and diagnostic criteria were discussed and adapted to current knowledge. These were summarized by invited chairpersons (named in brackets) and followed by contributions of the participants. The following short summaries cover the essentials of the reviews, the discussion and the consensus achieved. 1. Update on SMA 5q

Refinement of the NHS locus on chromosome Xp22.13 and analysis of five candidate genes.

Nance-Horan syndrome (NHS) is an X-linked condition characterised by congenital cataracts, dental abnormalities, dysmorphic features, and mental retardation in some cases. Previous studies have mapped the disease gene to a 2 cM interval on Xp22.2 between DXS43 and DXS999. We report additional linkage data resulting from the analysis of eleven independent NHS families. A maximum lod score of 9.94 (theta=0.00) was obtained at the RS1 locus and a recombination with locus DXS1195 on the telomeric side was observed in two families, thus refining the location of the gene to an interval of around 1 Mb on Xp22.13. Direct sequencing or SSCP analysis of the coding exons of five genes (SCML1, SCML2, STK9, RS1 and PPEF1), considered as candidate genes on the basis of their location in the critical interval, failed to detect any mutation in 12 unrelated NHS patients, thus making it highly unlikely that these genes are implicated in NHS.

Mutational analysis of Peroxiredoxin IV: exclusion of a positional candidate for multinodular goitre

Multinodular goitre (MNG) is a common disorder characterised by an enlargement of the thyroid, occurring as a compensatory response to hormonogenesis impairment. The incidence of MNG is dependent on sex (female:male ratio 5:1) and several reports have documented a genetic basis for the disease. Last year we mapped a MNG locus to chromosome Xp22 in a region containing the peroxiredoxin IV (Prx-IV) gene. Since Prx-IV is involved in the removal of H2O2 in thyroid cells, we hypothesize that mutations in Prx-IV gene are involved in pathogenesis of MNG. Four individuals (2 affected, 2 unrelated unaffected) were sequenced using automated methods. All individuals were originated from the original three-generation Italian family described in previous studies. A Southern blot analysis using a Prx-IV full-length cDNA as a probe was performed in order to exclude genomic rearrangements and/or intronic mutations. In addition a RT-PCR of PRX-IV was performed in order to investigate expression alterations. No causative mutations were found. Two adjacent nucleotide substitutions were detected within introns 1 and 4. These changes were also detected in unaffected individuals, suggesting that they were innocuous polymorphisms. No gross genomic rearrangements and/or restriction fragment alterations were observed on Southern analysis. Finally, using RT-PCR from tissue-specific RNA, no differences of PRX-IV expression-levels were detected between affected and unaffected samples. Based on sequence and genomic analysis, Prx-IV is very unlikely to be the MNG2 gene.

Identification and Characterization of a Novel Cancer/Testis Antigen Gene CAGE

We applied serological analysis of cDNA expression library technique to identify cancer-associated genes. We screened cDNA expression libraries of human testis and gastric cancer cell lines with sera of patients with gastric cancers. We identified a gene whose expression is testis-specific among normal tissues. We cloned and characterized this novel gene. It contains D-E-A-D box domain and encodes a putative protein of 630 amino acids with possible helicase activity. It showed wide expression in various cancer tissues and cancer cell lines. The corresponding gene was named cancer-associated gene (CAGE). PCR of human × hamster Radiation Hybrids showed localization of CAGE on the human chromosome Xp22. Transient transfection of CAGE showed predominantly nuclear localization. Both Western blot and plaque assay indicated seroreactivity of CAGE protein. We found that demethylation played a role in the activation of CAGE in some cancer cell lines that do not express it. Cell synchronization experiments showed that the expression of CAGE was related with cell cycle. This suggests that CAGE might play a role in cellular proliferation. Because CAGE is expressed in a variety of cancers but not in normal tissues except testis, this gene can be a target of antitumor immunotherapy.

An Integrated, Functionally Annotated Gene Map of the DXS8026–ELK1 Interval on Human Xp11.3–Xp11.23: Potential Hotspot for Neurogenetic Disorders

Human chromosome Xp11.3–Xp11.23 encompasses the map location for a growing number of diseases with a genetic basis or genetic component. These include several eye disorders, syndromic and nonsyndromic forms of X-linked mental retardation (XLMR), X-linked neuromuscular diseases and susceptibility loci for schizophrenia, type 1 diabetes, and Graves' disease. We have constructed an ∼2.7-Mb high-resolution physical map extending from DXS8026 to ELK1, corresponding to a genetic distance of ∼5.5 cM. A combination of chromosome walking and sequence-tagged site (STS)-content mapping resulted in an integrated framework and transcript map, precisely positioning 10 polymorphic microsatellites (one of which is novel), 16 ESTs, and 12 known genes (RP2, PCTK1, UHX1, UBE1, RBM10, ZNF157, SYN1, ARAF1, TIMP1, PFC, ELK1, UXT). The composite map is currently anchored with 89 STSs to give an average resolution of ∼1 STS every 30 kb. By a combination of EST database searches and in silico detection of UniGene clusters within genomic sequence generated from this template map, we have mapped several novel genes within this interval: a Na+/H+ exchanger (SLC9A7), at least two zincfinger transcription factors (KIAA0215 and Hs.68318), carbohydrate sulfotransferase-7 (CHST7), regucalcin (RGN), inactivation-escape-1 (INE1), the human ortholog of mouse neuronal protein 15.6, and four putative novel genes. Further genomic analysis enabled annotation of the sequence interval with 20 predicted pseudogenes and 21 UniGene clusters of unknown function. The combined PAC/BAC transcript map and YAC scaffold presented here clarifies previously conflicting data for markers and genes within the Xp11.3–Xp11.23 interval and provides a powerful integrated resource for functional characterization of this clonally unstable, yet gene-rich and clinically significant region of proximal Xp.

Sideroblastic anaemias.

Sideroblastic anaemias.

A locus for isolated cataract on human Xp

Purpose: To genetically map the gene causing isolated X linked cataract in a large European pedigree. Methods: Using the patient registers at Birmingham Women's Hospital, UK, we identified and examined...

New insights into ocular albinism type 1 (OA1): Mutations and polymorphisms of the OA1 gene.

Albinism ocular type 1 (OA1) is an X-linked type of albinism that mainly effects pigment production in the eye, resulting in hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers, and reduced visual acuity. The OA1 gene is located on chromosome Xp22.32 and the coding sequence is divided into nine exons. The protein is an integral transmembrane protein that has weak similarities to G protein-coupled receptors. A total of 25 missense, two nonsense, nine frameshift, and five splicing mutations have been reported in the OA1 gene associated with OA1. There are also several deletions of some or all exons of the OA1 gene with deletions of exon 2 resulting from unequal crossing-over, due to flanking Alu repeats. Mutation and polymorphism data on this gene is available from the International Albinism Center - Albinism Database web site (http://www.cbc.umn.edu/tad).

Familial ovarian dysgerminomas (Swyer syndrome) in females associated with 46 XY-karyotype

An asymptomatic woman (age 38 years) with a family history of ovarian malignancies was referred for presymptomatic genetic testing of mutations in the BRCA genes. A familial Swyer syndrome with theoccurrence of dysgerminomas is the most likely diagnosis. However, in our case, all known causes of this heterogeneous disorder have been excluded pointing to the existence of another yet unknown geneticlocus.The family history revealed three affected paternal aunts. Two of them developed ovarian malignancies at 13 and 15 years of age, and died at ages 19 and 20. The third aunt, 82 years old, wasaffected by this disease at the age of 35. She underwent hormonal treatment for 3 years starting at the age of 15 because of primary amenorrhea. Under this treatment she developed nearly complete secondarysexual characteristics. Karyotype analysis revealed a normal male karyotype (46 XY, QFQ). Pelvic ultrasound showed an uterus of normal size, incompatible with an androgen resistance syndrome or a defectin testosterone biosynthesis.We excluded a mutation in the sex-determining region on chromosome Y (SRY) by direct sequencing of the SRY gene. An involvement of the subtelomeric region of chromosome9p (9p24.3) recently reported to be involved in XY-sex reversal phenotypes was excluded by molecular testing for loss of heterozygosity as well as fluorescence in situ hybridization studies. Analysesof the DAX1 gene in the dosage sensitive sex reversal locus on chromosome Xp21 by Southern blot analysis showed no duplications.

Structure, Function, and Tissue Expression Pattern of Human SN2, a Subtype of the Amino Acid Transport System N

We have cloned a new subtype of the amino acid transport system N from a human liver cell line. This transporter, designated SN2, consists of 472 amino acids and exhibits 62% identity with human SN1 at the level of amino acid sequence. SN2-specific transcripts are expressed predominantly in the stomach, brain, liver, lung, and intestinal tract. The sizes of the transcripts vary in different tissues, indicating tissue-specific alternative splicing of the SN2 mRNA. In contrast, SN1 is expressed primarily in the brain and liver and there is no evidence for the presence of multiple transcripts of varying size for SN1. When expressed in mammalian cells, the cloned human SN2 mediates Na+-coupled transport of system N-specific amino acid substrates (glutamine, asparagine, and histidine). In addition, SN2 also transports serine, alanine, and glycine. Anionic amino acids, cationic amino acids, imino acids, and N-alkylated amino acids are not recognized as substrates by human SN2. The SN2-mediated transport process is Li+-tolerant and highly pH-dependent. The Michaelis-Menten constant for histidine uptake via human SN2 is 0.6 ± 0.1 mM. The gene coding for SN2 is located on human chromosome Xp11.23. Successful cloning of SN2 provides the first molecular evidence for the existence of subtypes within the amino acid transport system N in mammalian tissues.

Evidence for a Graves' Disease Susceptibility Locus at Chromosome Xp11 in a United Kingdom Population

Evidence for a Graves' Disease Susceptibility Locus at Chromosome Xp11 in a United Kingdom Population

Mutations in the X-linked RSK2 gene (RPS6KA3) in patients with Coffin-Lowry syndrome.

RSK2 is a growth factor-regulated serine-threonine protein kinase, acting in the Ras-Mitogen-Activated Protein Kinase (MAPK) signaling pathway. Mutations in the RSK2 gene (RPS6KA3) on chromosome Xp22.2, have been found to cause Coffin-Lowry syndrome (CLS), an X-linked disorder characterized by psychomotor retardation, characteristic facial and digital abnormalities, and progressive skeletal deformations. By screening of 250 patients with clinical features suggestive of Coffin-Lowry syndrome, 71 distinct disease-associated RSK2 mutations have been identified in 86 unrelated families. Thirty-eight percent of mutations are missense mutations, 20% are nonsense mutations, 18% are splicing errors, and 21% are short deletion or insertion events. About 57% of mutations result in premature translation termination, and the vast majority are predicted to cause loss of function of the mutant allele. These changes are distributed throughout the RSK2 gene and show no obvious clustering or phenotypic association. However, some missense mutations are associated with milder phenotypes. In one family, one such mutation was associated solely with mild mental retardation. It is noteworthy that nine mutations were found in female probands, with no affected male relatives, ascertained through learning disability and mild but suggestive facial and digital dysmorphisms.