Tiina Alitalo

X-linked cone-rod dystrophy (locus COD1): identification of mutations in RPGR exon ORF15.

X-linked cone-rod dystrophy (COD1) is a retinal disease that primarily affects the cone photoreceptors; the disease was originally mapped to a limited region of Xp11.4. We evaluated the three families from our original study with new markers and clinically reassessed all key recombinants; we determined that the critical intervals in families 2 and 3 overlapped the RP3 locus and that a status change (from affected to probably unaffected) of a key recombinant individual in family 1 also reassigned the disease locus to include RP3 as well. Mutation analysis of the entire RPGR coding region identified two different 2-nucleotide (nt) deletions in ORF15, in family 2 (delAG) and in families 1 and 3 (delGG), both of which result in a frameshift leading to altered amino acid structure and early termination. In addition, an independent individual with X-linked cone-rod dystrophy demonstrated a 1-nt insertion (insA) in ORF15. The presence of three distinct mutations associated with the same disease phenotype provides strong evidence that mutations in RPGR exon ORF15 are responsible for COD1. Genetic heterogeneity was observed in three other families, including the identification of an in-frame 12-nt deletion polymorphism in ORF15 that did not segregate with the disease in one of these families.

X linked cone-rod dystrophy, CORDX3, is caused by a mutation in the CACNA1F gene

Background: X linked cone-rod dystrophy (CORDX) is a recessive retinal disease characterised by progressive dysfunction of photoreceptors. It is genetically heterogeneous, showing linkage to three X chromosomal loci. CORDX1 is caused by mutations in the RPGR gene (Xp21.1), CORDX2 is located on Xq27.2-28, and we recently localised CORDX3 to Xp11.4-q13.1. We aimed to identify the causative gene behind the CORDX3 phenotype. Methods: All 48 exons of the CACNA1F gene were screened for mutations by DNA sequencing. RNA from cultured lymphoblasts and peripheral blood activated T lymphocytes was analysed by RT-PCR and sequencing. Results: A novel CACNA1F mutation, IVS28-1 GCGTC>TGG, in the splice acceptor site of intron 28 was identified. Messenger RNA studies indicated that the identified mutation leads to altered splicing of the CACNA1F transcript. Aberrant splice variants are predicted to result in premature termination and deletions of the encoded protein, Cav1.4 α1 subunit. Conclusion:CACNA1F mutations cause the retinal disorder, incomplete congenital stationary night blindness (CSNB2), although mutations have also been detected in patients with divergent diagnoses. Our results indicate that yet another phenotype, CORDX3, is caused by a mutation in CACNA1F. Clinically, CORDX3 shares some features with CSNB2 but is distinguishable from CSNB2 in that it is progressive, can begin in adulthood, has no nystagmus or hyperopic refraction, has only low grade astigmatism, and in dark adaptation lacks cone threshold and has small or no elevation of rod threshold. Considering all features, CORDX3 is more similar to other X chromosomal cone-rod dystrophies than to CSNB2.

Thirty distinct CACNA1F mutations in 33 families with incomplete type of XLCSNB and Cacna1f expression profiling in mouse retina

X-linked CSNB patients may exhibit myopia, nystagmus, strabismus and ERG abnormalities of the Schubert-Bornschein type. We recently identified the retina-specific L-type calcium channel α1 subunit gene CACNA1F localised to the Xp11.23 region, which is mutated in families showing the incomplete type (CSNB2). Here, we report comprehensive mutation analyses in the 48 CACNA1F exons in 36 families, most of them from Germany. All families were initially diagnosed as having the incomplete type of CSNB, except for two which have been designated as Åland Island eye disease (ÅIED)-like. Out of 33 families, a total of 30 different mutations were identified, of which 24 appear to be unique for the German population. The mutations, 20 of which are published here for the first time, were found to be equally distributed over the entire gene sequence. No mutation could be found in a classic ÅIED family previously shown to map to the CSNB2 interval. Cacna1f expression in photoreceptor-negative mice strains indicate that the gene is expressed in the outer nuclear, the inner nuclear, and the ganglion cell layer. Such a distribution points to the central role of calcium regulation in the interaction of retinal cells that mediate signal transmission.

Characterization of two unusual RS1 gene deletions segregating in Danish retinoschisis families.

Over 100 distinct retinoschisis gene (RS1) mutations, of which approximately 10% are single exon deletions, have been described to date. In this paper we have characterized in detail two dissimilar RS1 gene deletions which are accountable for RS in one‐third of Danish patients. First, a 136 kb deletion, spanning from the 5′ region of the RS1 gene to intron 3, was identified. Unexpectedly this large deletion abolishes exons of three adjacent genes: serine‐threonine phosphatase gene (PPEF‐1)/serine‐threonine protein phosphatase gene (PP7), retinoschisis gene (RS1), and serine‐threonine kinase gene (STK9). We demonstrate that the RS1 and STK9 genes are partly overlapping and the sequences of the PP7 and PPEF‐1 genes are identical. This is the first study which reports of retinoschisis patients who also suffer from deletions in genes adjacent to RS1. The 136 kb deletion is also the first gross deletion of the retinoschisis gene deleting three exons. It results from a recombination between two repetitive sequences of the Alu family, one in 5′ region of the RS1 gene and the other in RS1 intron 3. The second alteration, the actual Danish RS founder mutation, is a 4.4 kb noncontiguous two‐part deletion composed of two deleted 1.5 and 2.9 kb segments, separated by an intact 1.2 kb segment. It extends from the 5′ flanking region of the retinoschisis gene to RS intron 1. RS1 gene deletions of this type have not been identified previously. Despite these two unique deletions, which either lead to severely defective transcription or total absence of the retinoschisin and PPEF‐1 protein, all the patients have a typical retinoschisis phenotype. Hum Mutat 16:307–314, 2000.

Prevalence of the fragile X syndrome in four birth cohorts of children of school age

SummaryThe prevalence of the fragile X syndrome among 12,882 children (6594 boys and 6288 girls) born during the years 1969–1972 in Kuopio province in eastern central Finland has been studied retrospectively. Mentally retarded children were selected from normal schools by using school achievement tests and from registers of mentally retarded individuals. In the present study fragile X syndrome was found in 6/111 mentally retarded children (5.4%), in 4/61 boys and in 2/50 girls, respectively. It was not detected in the control group of 85 healthy children. The corrected prevalence of fragile X syndrome among boys in four successive birth cohorts was estimated to the 1 in 1210 or 0.8/1000, and that among girls, 1 in 2418 or 0.4/1000. The overall prevalence was calculated to be 1 in 1612 or 0.6/1000 children.

Chromosomal Localization of ZFX-A Human Gene That Escapes X Inactivation -and Its Murine Homologs

The ZFY gene, found in the sex-determining region of the human Y chromosome, encodes a zinc-finger protein that may be the pivotal sex-determining signal. A closely related gene, ZFX, is found on the human X chromosome, and it may also function in sex determination. ZFX is one of a few genes on the human X chromosome that are known to escape X inactivation. We report the localization of ZFX, by meiotic linkage analysis and physical mapping, distal to POLA but proximal to DXS41 (p99-6), near the boundary of bands Xp21.3 and Xp22.1. (Our results suggest the following order of loci in Xp21-p22: cen-DMD-[GK,AHC]-DXS67 (pB24)-POLA-ZFX-[DXS41 (p99-6), DXS274 (CRI-L1391)]-DXS43 (pD2)-pter.) These findings contradict the model that escape from X inactivation is limited to genes near the short-arm telomere (i.e., in Xp22.3). Instead, escape from X inactivation is likely a property of several noncontiguous segments of the X chromosome. Curiously, in mouse, the homologous Zfx gene maps to X chromosome band D, near the center from which an X-inactivating signal is thought to spread. As judged by comparative mapping, it appears that an X-chromosomal segment that spans the ZFX and DMD genes has remained grossly intact during the divergence of mouse and human from a common ancestor. Conservation of this chromosomal segment may extent to marsupials, where homologs of the ZFX and DMD genes have been observed in proximity, but on an autosome. While autosomal homologs of ZFX have not been observed in other placental mammals, a locus derived from a processed Zfx transcript is found on mouse chromosome 10 band B3 or B4.

Expression of decorin in human tissues and cell lines and defined chromosomal assignment of the gene locus (DCN)

Earlier studies had shown that the expression of the gene coding for one eminent connective tissue proteoglycan, decorin (DCN), is deficient in the fibroblasts of 3 out of 15 Marfan patients (Pulkkinen et al., 1990). To obtain more information on the expression of this gene, various human tissues and cell lines were studied. High mRNA levels of decorin were detected in aorta, lung, skin, kidney, smooth muscle, and placenta, whereas significantly lower mRNA levels were found in the rest of the tissues analyzed. Two sizes of transcripts were observed in all tissues. The two transcripts of decorin most probably do not represent two different genes, since in situ hybridization gave only one strong signal, placing the gene in 12q21----q22. No tissue-specific differences in the two mRNA species of decorin were detected. This is in contrast to the gene of versican, another connective tissue proteoglycan gene, that was analyzed as a control; high expression of a longer transcript of the versican gene was found in brain and smooth muscle, whereas the shorter transcript was predominant in all other tissues studied. DCN was actively transcribed in cultured mesenchymal cells, whereas in cells of endothelial or epithelial origin, the transcription level was undetectable. These tissue- and cell type-specific variations in the expression of DCN may help to explain the complex phenotypic variation typical of individuals with Marfan syndrome.

Determination of the breakpoints of 1;7 translocations in myelodysplastic syndrome by in situ hybridization using chromosome-specific α satellite DNA from human chromosomes 1 and 7

A whole-arm translocation involving the short arm of chromosome 7 and the long arm of chromosome 1 occurs nonrandomly in myelodysplastic syndrome and acute nonlymphocytic leukemia. In situ hybridization, using alpha satellite DNA specific for the centromeric regions of chromosomes 1 (probe pSD1-1) and 7 (probe p21-4), was performed to determine the exact breakpoints of the translocation. Both probes hybridized to the centromeric region of the translocation chromosome in metaphases from two patients with myelodysplastic syndrome. Both probes hybridized with approximately equal strength to either chromosome 1 or 7 and to the 1;7 translocation chromosome, suggesting that the t(1;7) had retained the chromosome-specific alpha satellite DNA from both chromosomes. These studies permit us to propose a new description, t(1;7)(cen;cen), for this translocation.

The gene encoding human low-molecular weight insulin-like growth-factor binding protein (IGF-BP25): regional localization to 7p12-p13 and description of a DNA polymorphism

SummaryThe low-molecular weight insulin-like growth-factor binding protein (IGF-BP25) is synthesized by human liver, secretory endometrium and decidua, and is also present in human serum. It binds insulin-like growth factors IGF-I and IGF-II with high affinity, and is proposed to act as a paracrine regulator of cell growth. In situ hybridization studies with a cDNA encompassing the entire protein coding region of IGF-BP25 localized the gene to bands p12–p13 on chromosome 7. Southern blot analysis with the enzyme BglII revealed a common restriction fragment length polymorphism: the presence of the polymorphic BglII site results in the formation of two fragments 4.6 kb and 1.6 kb in size whereas its absence produces a single 6.2 kb fragment. The frequencies of the two alleles were 0.73 and 0.27, respectively. IGF-BP25 constitutes a useful genetic marker for the proximal short arm of chromosome 7.

Molecular characterization of a Y;15 translocation segregating in a family

SummaryWe have used Y-specific and Y-derived DNA probes for in situ hybridization and Southern blotting analysis to characterize a Y;15 translocation showing normal Mendelian inheritance in a family. Cytogenetically there appeared to be an unbalanced translocation of Yqh to 15p; this translocation may be considered as a prototype of those translocations between Yq and the short arm of an acrocentric chromosome which have a population incidence of approximately 1 in 2,000. Our molecular studies showed that, in all probability, the breakpoints were near the border between Yq11.23 and Yq12, and in 15p11, respectively; the translocation is abbreviated t(Y;15)(q12;p11). Using the Y-specific probe pY431 in a quantitative Southern hybridization assay, normal females had no hybridization, female carriers and normal men had the same amount, and male carriers had twice that amount. Cytogenetic analysis and quantitative in situ hybridization using probes pY431 and pY3.4 were consistent with the hypothesis that the portion of Yq translocated to 15p comprised all of Yq12 and none of Yq11. The absence of Southern hybridization with probes specific for Yp and Yq11 confirmed this observation. Even though the family was ascertained through two brothers who both had schizophrenia and were carriers of the translocation, the clinical evaluation of a total of nine individuals with the translocation and five without it did not suggest its association with an abnormal phenotype.