Heritability of young- and old-onset ischaemic stroke.

BACKGROUND AND PURPOSE: Although the genetic contribution to stroke risk is well known, it remains unclear if young-onset stroke has a stronger genetic contribution than old-onset stroke. This study aims to compare the heritability of ischaemic stroke risk between young and old, using common genetic variants from whole-genome array data in population-based samples. METHODS: This analysis included 4050 ischaemic stroke cases and 5765 controls from six study populations of European ancestry; 47% of cases were young-onset stroke (age < 55 years). To quantify the heritability for stroke risk in these unrelated individuals, the pairwise genetic relatedness was estimated between individuals based on their whole-genome array data using a mixed linear model. Heritability was estimated separately for young-onset stroke and old-onset stroke (age ≥ 55 years). RESULTS: Heritabilities for young-onset stroke and old-onset stroke were estimated at 42% (±8%, P < 0.001) and 34% (±10%, P < 0.001), respectively. CONCLUSIONS: Our data suggest that the genetic contribution to the risk of stroke may be higher in young-onset ischaemic stroke, although the difference was not statistically significant.

Influence of intermediate and uninterrupted FMR1 CGG expansions in premature ovarian failure manifestation.

BACKGROUND: Studies attempting to precisely define the range of fragile mental retardation 1 (FMR1) expansions and its inf luence in premature ovarian failure (POF) manifestation are partially lacking. To this aim, we evaluated a large cohort of POF patients for the size and, in selected cases, for the sequence of the CGG expansion. Furthermore, the correlation between POF and X-inactivation was investigated in FRAXA families. METHODS: By fluorescent PCR, 190 POF and 200 control women were sized for the CGG tract; some subjects were also characterized by sequencing and for the FMR1 activation ratio. RESULTS AND CONCLUSION: We found a significant association (19/190, 10%, P < 1 x 10(-6)) between POF and FMR1 premutation (range 63-163 repeats) and a significant enrichment (9/190, 4.7%, P = 0.021) of POF carriers of intermediate expansions (range 41-58 repeats). Interestingly, intermediate alleles were entirely composed of CGG repeats. Furthermore, the analysis of three pairs of siblings with similar FMR1 expansions and discordant for the POF phenotype showed a direct correlation between the expression of the intermediate/premutated allele and POF manifestation. The results obtained strengthen the correlation between FMR1 expansion and POF and suggest that the manifestation of the ovarian dysfunction could be influenced both by the pattern of interruption of the CGG repeat and by X-inactivation.

Mutation analysis of two candidate genes for premature ovarian failure, DACH2 and POF1B.

BACKGROUND: Balanced X;autosome translocations interrupting the 'critical region' of the long arm of the human X chromosome are often associated with premature ovarian failure (POF). However, the mechanisms leading to X-linked ovarian dysfunction are largely unknown, as the majority of the X chromosome breakpoints have been mapped to gene-free genomic regions. A few genes have been found to be interrupted, but their role has never been clarified. METHODS AND RESULTS: By fine mapping of the X chromosome breakpoint of an X;autosome balanced translocation, we identified a new interrupted gene, POF1B. We performed a mutation analysis of POF1B and of another gene previously identified, DACH2, localized approximately 700 kb distal in Xq21, in a cohort of >200 Italian POF patients. Rare mutations were found in patients in both genes. CONCLUSIONS: Our findings could not demonstrate any involvement of POF1B, but suggest that rare mutations in the DACH2 gene may have a role in the POF phenotype.

A mutation in the X-linked Emery-Dreifuss muscular dystrophy gene in a patient affected with conduction cardiomyopathy.

A screening for mutation in the X-linked Emery-Dreifuss muscular dystrophy (X-EMD) gene was performed among patients affected with severe heart rhythm defects and/or dilated cardiomyopathy. Patients were selected from the database of the Department of Cardiology of the University Hospital Brno. One patient presented a mutation in the X-EMD gene and no emerin in his skeletal muscle. The patient had a severe cardiac disease but a very mild muscle disorder that had not been diagnosed until the mutations was found. This case shows that mutations in X-EMD gene, as it was shown for autosomal-dominant EMD, can cause a predominant cardiac phenotype.

Unusual expression of emerin in a patient with X-linked Emery-Dreifuss muscular dystrophy.

We report on a patient with the typical clinical findings of Emery-Dreifuss muscular dystrophy due to a mutation in the emerin gene that should have produced a higher molecular weight protein. Immunohistochemical analysis showed emerin localized only in the cytoplasm of muscle fibres and lymphoblastoid cells. The emerin molecule contained the nucleoplasmic domain and the transmembrane domain responsible for nuclear membrane targeting, so its incorrect localization and lack of function could be due to abnormal folding resulting in rapid degradation or inability to bind other nuclear proteins.

Different mutations in the LMNA gene cause autosomal dominant and autosomal recessive Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EMD) is a condition characterized by the clinical triad of early-onset contractures, progressive weakness in humeroperoneal muscles, and cardiomyopathy with conduction block. The disease was described for the first time as an X-linked muscular dystrophy, but autosomal dominant and autosomal recessive forms were reported. The genes for X-linked EMD and autosomal dominant EMD (AD-EMD) were identified. We report here that heterozygote mutations in LMNA, the gene for AD-EMD, may cause diverse phenotypes ranging from typical EMD to no phenotypic effect. Our results show that LMNA mutations are also responsible for the recessive form of the disease. Our results give further support to the notion that different genetic forms of EMD have a common pathophysiological background. The distribution of the mutations in AD-EMD patients (in the tail and in the 2A rod domain) suggests that unique interactions between lamin A/C and other nuclear components exist that have an important role in cardiac and skeletal muscle function.

X chromosome genes and premature ovarian failure.

Premature ovarian failure (POF) is a disorder characterized by lack of ovulation and elevated levels of serum gonadotropins before the age of 40. The etiology of POF is not known but different environmental and genetic factors are involved, suggesting high heterogeneity of the disorder. The involvement of X-linked genes in the etiology of POF was hypothesized on the basis of its frequent association with chromosomal rearrangements and monosomies. In recent years a number of genes were described. Two genes, FRAXA and POF1B, have been formally demonstrated to be responsible for POF. Other genes have been proposed as candidates, but their role remains to be demonstrated.

A human homologue of the Drosophila melanogaster diaphanous gene is disrupted in a patient with premature ovarian failure: evidence for conserved function in oogenesis and implications for human sterility.

Premature ovarian failure (POF) is a defect of ovarian development and is characterized by primary or secondary amenorrhea, with elevated levels of serum gonadotropins, or by early menopause. The disorder has been attributed to various causes, including rearrangements of a large "critical region" in the long arm of the X chromosome. Here we report identification, in a family with POF, of a gene that is disrupted by a breakpoint. The gene is the human homologue of the Drosophila melanogaster diaphanous gene; mutated alleles of this gene affect spermatogenesis or oogenesis and lead to sterility. The protein (DIA) encoded by the human gene (DIA) is the first human member of the growing FH1/FH2 protein family. Members of this protein family affect cytokinesis and other actin-mediated morphogenetic processes that are required in early steps of development. We propose that the human DIA gene is one of the genes responsible for POF and that it affects the cell divisions that lead to ovarian follicle formation.

The X-linked gene G4.5 is responsible for different infantile dilated cardiomyopathies.

Barth syndrome (BTHS) is an X-linked disorder characterized clinically by the associated features of cardiac and skeletal myopathy, short stature, and neutropenia. The clinical manifestations of the disease are, in general, quite variable, but cardiac failure as a consequence of cardiac dilatation and hypertrophy is a constant finding and is the most common cause of death in the first months of life. X-linked cardiomyopathies with clinical manifestations similar to BTHS have been reported, and it has been proposed that they may be allelic. We have recently identified the gene responsible for BTHS, in one of the Xq28 genes, G4.5. In this paper we report the sequence analysis of 11 additional familial cases: 8 were diagnosed as possibly affected with BTHS, and 3 were affected with X-linked dilated cardiomyopathies. Mutations in the G4.5 gene were found in nine of the patients analyzed. The molecular studies have linked together what were formerly considered different conditions and have shown that the G4.5 gene is responsible for BTHS (OMIM 302060), X-linked endocardial fibroelastosis (OMIM 305300), and severe X-linked cardiomyopathy (OMIM 300069). Our results also suggest that very severe phenotypes may be associated with null mutations in the gene, whereas mutations in alternative portions or missense mutations may give a "less severe" phenotype.

X-linked Emery-Dreifuss muscular dystrophy can be diagnosed from skin biopsy or blood sample.

We have raised an anti-emerin polyclonal antibody against a fusion protein encompassing most of the hydrophilic portion of emerin. Using this antibody, we have analyzed emerin expression in Emery-Dreifuss muscular dystrophy (EDMD) patients and controls, by immunocytochemistry, in skeletal muscle and skin, and by immunoblot, in peripheral blood mononuclear cells and lymphoblasts. Emerin was localized on the surfaces of nuclei in control skeletal muscle and skin but was absent or reduced in patient skeletal muscle, was absent from the skin of patients, and was expressed only in a few nuclei in a patient's mother. Immunoblot of peripheral blood cells from EDMD patients showed absence of the emerin band, altered-size emerin, or a protein of normal molecular mass but slightly reduced quantity. The diagnosis of X-linked EDMD is normally confirmed by genetic analysis of the STA gene coding for emerin. We propose immunocytochemical evaluation of emerin expression in skin biopsies as a sensitive and more convenient tool for diagnosing X-linked EDMD and, in particular, for distinguishing it from the autosomal dominant form. This technique may be applied to suspected EDMD patients, especially sporadic cases or those with incomplete clinical phenotype, and also suspected carriers. Immunoblot of peripheral blood cells is also useful, but it may not unequivocally identify carriers and some patients.

A novel X-linked gene, G4.5. is responsible for Barth syndrome.

Barth syndrome is a severe inherited disorder, often fatal in childhood, characterized by cardiac and skeletal myopathy, short stature and neutropenia. The disease has been mapped to a very gene-rich region in distal portion of Xq28. We now report the identification of unique mutations in one of the genes in this region, termed G4.5, expressed at high level in cardiac and skeletal muscle. Different mRNAs can be produced by alternative splicing of the primary G4.5 transcript, encoding novel proteins that differ at the N terminus and in the central region. The mutations introduce stop codons in the open reading frame interrupting translation of most of the putative proteins (which we term 'tafazzins'). Our results suggest that G4.5 is the genetic locus responsible for the Barth syndrome.

A family of transmembrane proteins with homology to the MET-hepatocyte growth factor receptor.

In hunting for unknown genes on the human X chromosome, we identified a cDNA in Xq28 encoding a transmembrane protein (SEX) of 1871 amino acids. SEX shares significant homology with the extracellular domain of the receptors encoded by the oncogenes MET, RON, and SEA [hepatocyte growth factor (HGF) receptor family]. Further screenings of cDNA libraries identified three additional sequences closely related to SEX: these were named SEP, OCT, and NOV and were located on human chromosomes 3p, 1, and 3q, respectively. The proteins encoded by these genes contain large cytoplasmic domains characterized by a distinctive highly conserved sequence (SEX domain). Northern blot analysis revealed different expression of the SEX family of genes in fetal tissues, with SEX, OCT, and NOV predominantly expressed in brain, and SEP expressed at highest levels in kidney. In situ hybridization analysis revealed that SEX has a distinctive pattern of expression in the developing nervous system of the mouse, where it is found in postmitotic neurons from the first stages of neuronal differentiation (9.5 day postcoitus). The SEX protein (220 kDa) is glycosylated and exposed at the cell surface. Unlike the receptors of the HGF family, p220SEX, a MET-SEX chimera or a constitutively dimerized TPR-SEX does not show tyrosine kinase activity. These data define a gene family (SEX family) involved in the development of neural and epithelial tissues, which encodes putative receptors with unexpected enzymatic or binding properties.

An X chromosome-linked gene encoding a protein with characteristics of a rhoGAP predominantly expressed in hematopoietic cells.

An increasingly large number of proteins involved in signal transduction have been identified in recent years and shown to control different steps of cell survival, proliferation, and differentiation. Among the genes recently identified at the tip of the long arm of the human X chromosome, a novel gene, C1, encodes a protein that appears to represent a newly discovered member of the group of signaling proteins involved in regulation of the small GTP binding proteins of the ras superfamily. The protein encoded by C1, p115, is synthesized predominantly in cells of hematopoietic origin. It is characterized by two regions of similarity to motifs present in known proteins: GAP and SH3 homologous regions. Its localization in a narrow cytoplasmic region just below the plasma membrane and its inhibitory effect on stress fiber organization indicate that p115 may down regulate rho-like GTPases in hematopoietic cells.

Identification of new mutations in the Emery-Dreifuss muscular dystrophy gene and evidence for genetic heterogeneity of the disease.

The Emery-Dreifuss Muscular Dystrophy (EDMD) is an X-linked recessive muscular disorder characterized by early contractures of the elbows, Achilles tendons and postcervical muscles, slowly progressing muscle wasting and weakness and a cardiomyopathy characterized by conduction defects. Heart block is a frequent cause of death. Finding of mutations in one of the transcripts in the critical region in distal Xq28 led to the identification of the gene responsible for the disease. We now report the sequence of the gene which is 2100 bp long and the development of a set of primers to amplify and sequence the gene from patients' DNA. Eight unrelated X-linked familial cases were studied and they all carried different mutations, showing that lack of emerin in cardiac and skeletal muscle is the cause of the X-linked disease. No mutations were found in a family where the female carrier was affected nor in a sporadic case with a well established diagnosis of EDMD. Our findings suggest genetic heterogeneity of EDMD, and that at least two genes, the X-linked STA gene and one unidentified autosomal gene, are responsible for the disease.

A comparative transcriptional map of a region of 250 kb on the human and mouse X chromosome between the G6PD and the FLN1 genes.

The transcriptional organization of the region of the mouse X chromosome between the G6pd and the Fln1 genes was studied in detail, and it was compared with the syntenic region of the human chromosome. A cosmid contig of 250 kb was constructed by screening mouse cosmid libraries with probes for human genes and with whole cosmids. Overlapping cosmids were aligned by comparing EcoRI and rare-cutter restriction enzyme digestions. The gene order and the orientation of transcription were determined by hybridization with fragments from the 5' and 3' moieties of each cDNA. Our work demonstrates that all of the new genes identified in human are present in the mouse. The size of the region, 250 kb, is also very similar, as are gene order and gene organization: the transcriptional organization in "domains" described in human is found to be identical in the mouse. The major difference detected is the much lower content in rare-cutter restriction sites, which is related to the lower G+C and CpG content of mouse DNA. The very high conservation that we have described suggests that a potent selective pressure has contributed to such conservation of gene organization.

Sequence and gene content in 52 kb including and centromeric to the G6PD gene in Xq28.

A cosmid containing 36.4 kb of high GC human DNA centromeric to the G6PD gene has been analyzed. The sequence was 99.9% precise, based on the comparison of 4.3 kb that overlaps an earlier analysis of 20.1 kb containing G6PD. Properties of the entire 52 kb region that may be characteristic of high GC portions of the genome include a very high density of sixty-two half or full Alu sequences, or 1.2/kb, and an absence of L1 sequences. Other highly repetitive sequences include 11 MER sequences, one of them interrupted at two positions by groups of 3 Alu elements. In segments of unique sequence, computer-aided analysis predicted three possible genes, one of which has thus far been confirmed by the recovery of a corresponding cDNA, both by a direct hybridization method and by a PCR-based method based on a primer pair inferred from the genomic sequence. The cDNA has been sequenced, and is completely concordant with counterpart genomic sequence; it has no resemblance to any previously described gene.

Identification of a novel X-linked gene responsible for Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked recessive disorder characterized by slowly progressing contractures, wasting of skeletal muscle and cardiomyopathy. Heart block is a frequent cause of death. The disease gene has been mapped to distal Xq28. Among many genes in this region, we selected eight transcripts expressed at high levels in skeletal muscle, heart and/or brain as the best candidates for the disease. We now report, in all five patients studied, unique mutations in one of the genes, STA: these mutations result in the loss of all or part of the protein. The EDMD gene encodes a novel serine-rich protein termed emerin, which contains a 20 amino acid hydrophobic domain at the C terminus, similar to that described for many membrane proteins of the secretory pathway involved in vesicular transport.

Isolation of new genes in distal Xq28: transcriptional map and identification of a human homologue of the ARD1 N-acetyl transferase of Saccharomyces cerevisiae.

In this paper, we describe the physical and transcriptional organization of a region of 140 kb in Xq28, 5' to the L1CAM gene. By isolation and mapping of CpG islands to the physical map of the region, isolation of cDNAs, determination of partial nucleotide sequences and study of the pattern of expression and of the orientation of the transcripts identified we have established a transcriptional map of this region. In this map, previously identified genes (L1CAM, V2R, HCF1 and RnBP) have been positioned as well as 3 new genes. All genes in the region are rather small, ranging in size from 2 to 30 kb, and very close to one another. With the exception of the V2R gene, they are housekeeping, have a CpG island at their 5' end and the same orientation of transcription. This kind of organization is consistent with the one previously described for the more distal portion of Xq28, between the Color Vision (CV) and the G6PD genes and indicates that genes with housekeeping and tissue specific pattern of expression are interspersed in the genome but they are probably found in different 'transcriptional domains'. Among the new genes, TE2 demonstrated 40% identity with the protein N-acetyl transferase ARD1 of S. cerevisiae: TE2 may be the human homologue of the S. cerevisiae gene.

Transcriptional organization of a 450-kb region of the human X chromosome in Xq28.

In this paper, we report the transcriptional organization of a 450-kb gene cluster in Xq28, flanked by the glucose-6-phosphate dehydrogenase and the color vision genes. CpG islands previously identified and mapped to distal Xq28 have helped in construction of a continuous contig of cosmids and in identification of cDNAs corresponding to eight transcripts. Thirteen to 16 small genes with CpG islands are clustered in a region of 250-300 kb. Many are highly expressed in muscle or brain and may be the genes responsible for muscle or neurological disorders mapped to distal Xq28. Our analysis indicates that, in this region of the genome, genes not related in sequence are organized in transcriptional domains of 100 kb and that this organization may be important for establishing and regulating gene expression in relation to tissue distribution and X chromosome inactivation.