Cloning, characterization, and chromosome mapping of the human GlcAT-S gene

 We report on the structure, map location, and tissue expression of the human GlcAT-S gene. The gene covers approximately 85 Kb on chromosome 6 (6q13) between the D6S455 and D6S1673 markers. GlcAT-S is composed of four exons and encodes a 324-amino-acid protein, which shows 89% homology with the rat glcat-s protein and is involved in the biosynthesis of the HNK-1 carbohydrate epitope on glycoproteins. Although GlcAT-S was considered an interesting candidate gene for the RP25 locus, the absence of any pathogenic mutations in probands of RP25-linked families ruled out that candidacy. Received: May 23, 2002 / Accepted: September 6, 2002     

Molecular cloning and characterization of human RAB23, a member of the group of Rab GTPases

The RAB small G protein family is composed of approximately 40 members. Many of them are ubiquitous and are expressed and participate in transport processes, such as endocytosis and exocytosis, whereas others are expressed only within a specific cell group carrying out specific functions. In the current study, we present the molecular characterisation and chromosomal location of the human RAB23 gene, a new member of the RAB family. This gene, expressed in retina, is composed of 7 exons spanning 34 kb of genomic DNA and located in the pericentromeric region of chromosome 6 between microsatellite markers D6S257 and D6S1695, within the critical region of RP25. Since proteins belonging to the Rab family have already been related to retinal degeneration we considered RAB23 an interesting candidate for the RP25 locus. However the absence of pathogenic variations after molecular analysis of the coding sequence in the index patients of RP25 linked families would be consistent with the exclusion of RAB23 as responsible for RP25 phenotype.     

Mutation screening of three candidate genes, ELOVL5, SMAP1 and GLULD1 in autosomal recessive retinitis pigmentosa

Retinitis pigmentosa (RP) is the most common form of retinal dystrophy. It is featured by a great clinical and genetic heterogeneity. Different patterns of inheritance exist, such as autosomal dominant and recessive, X-linked and digenic. RP25, a locus for autosomal recessive retinitis pigmentosa (arRP), the most frequently inherited form of RP, was mapped to chromosome 6q between D6S257 and D6S1644 microsatellite markers. ELOVL5, SMAP1 and GLULD1 were selected on the basis of their location, tissue expression and/or function. ELOVL5 is implicated in the elongation of long chain fatty acids, including docosahexanoic acid (DHA), which constitutes 50% of the fatty acids of the outer segment of the photoreceptor. SMAP1 (stromal membrane associated protein 1) was found to be located within RP25 locus and is expressed in retina. GLULD1, glutamate-ammonia ligase (glutamine synthase) domain containing 1, plays a key role in the uptake and metabolism of glutamate in the retina. The absence of pathogenic mutations after molecular analysis argues against the implication of ELOVL5, SMAP1 and GLULD1 in the development of RP25 phenotype. Nevertheless, we could not rule them out as good candidates for other retinal degeneration mapping to the same chromosomal region.     

Linkage Validation of RP25 Using the 10K GeneChip Array and Further Refinement of the Locus by New Linked Families

Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous group of retinal dystrophies, characterised by rod photoreceptor cell degeneration with autosomal recessive RP (arRP) as the commonest form worldwide. To date, a total of 26 loci have been reported for arRP, each having a prevalence of 1–5%, except for the RP25 locus which was identified as the genetic cause of 14% of arRP cases in Spain. In order to validate the original linkage of RP25 , we undertook a total genome scan using the 10K GeneChip mapping array on three of the previously linked families. The data obtained supported the initial findings of linkage. Additionally, linkage analysis in 18 newly ascertained arRP families was performed using microsatellite markers spanning the chromosome 6p12.1-q15 interval. Five out of the 18 families showed suggestive evidence of linkage to RP25 , hence supporting the high prevalence of this locus in the Spanish population. Furthermore, the finding of a crossover in one of these families is likely to have refined the disease interval from the original 16 cM to only a 2.67 cM region between D6S257 and D6S1557 .     

Genetic heterogeneity of Usher syndrome type II: localisation to chromosome 5q

Usher syndrome is a group of autosomal recessive disorders that includes retinitis pigmentosa (RP) with hearing loss. Usher syndrome type II is defined as moderate to severe hearing loss with RP. The USH2A gene at 1q41 has been isolated and characterised. In 1993, a large Usher II family affected with a mild form of RP was found to be unlinked to 1q41 markers. Subsequent linkage studies of families in our Usher series identified several type II families unlinked to USH2A and USH3 on 3q25. After a second unlinked family with many affected members and a mild retinal phenotype was discovered, a genome search using these two large families showed another Usher II locus on 5q (two point lod = 3.1 at D5S484). To date, we have identified nine unrelated 5q linked families (maximum combined multipoint lod = 5.86) as well as three Usher II families that show no significant linkage to any known Usher loci. Haplotype analysis of 5q markers indicates that the new locus is flanked by D5S428 and D5S433. Review of ophthalmological data suggests that RP symptoms are milder in 5q linked families; the RP is often not diagnosed until patients near their third decade. Enamel hypoplasia and severe, very early onset RP were observed in two of the three unlinked families; dental anomalies have not been previously described as a feature of Usher type II.     

Autosomal recessive retinitis pigmentosa locus RP28 maps between D2S1337 and D2S286 on chromosome 2p11-p15 in an Indian family

Retinitis pigmentosa (RP) is a group of clinically and genetically heterogeneous disorders characterised by night blindness, constriction of visual field, and dystrophic changes of the retina. Previous genetic studies have shown extensive allelic and non-allelic genetic heterogeneity of RP. Here we describe an Indian family with multiple consanguineous marriages and a total of four patients with autosomal recessive (AR) RP. The homozygosity mapping strategy was successfully used and indicated close linkage between the disease locus and D2S380, D2S441, D2S291, and D2S1394 with maximum lod scores between 1.51-3.07 at theta=0.00. The analysis of multiply informative meioses maps the locus (RP28) for ARRP in this family between D1S1337 and D2S286 on 2p11-p15. The involvement of visinin (VSNL1), a promising candidate gene assigned to chromosome 2p by previous studies, has been excluded by the absence of linkage.     

A nonsense mutation in a novel gene is associated with retinitis pigmentosa in a family linked to the RP1 locus.

Retinitis pigmentosa (RP) represents a group of inherited human retinal diseases which involve degeneration of photoreceptor cells resulting in visual loss and often leading to blindness. In order to identify candidate genes for the causes of these diseases, we have been studying a pool of photoreceptor-specific cDNAs isolated by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. One of these cDNAs was of interest because it mapped to proximal mouse chromosome 1 in a region homo-logous to human 8q11-q13, the locus of autosomal dominant RP1. Therefore, using the mouse cDNA as probe, we cloned the human cDNA (hG28) and its corresponding gene and mapped it near to D8S509, which lies in the RP1 locus. This gene consists of four exons with an open reading frame of 6468 nt encoding a protein of 2156 amino acids with a predicted mass of 240 kDa. Given its chromosomal localization, we screened this gene for mutations in a large family affected with autosomal dominant RP previously linked to the RP1 locus. We found an R677X mutation that co-segregated with disease in the family and is absent from unaffected members and 100 unrelated controls. This mutation is predicted to lead to rapid degradation of hG28 mRNA or to the synthesis of a truncated protein lacking approximately 70% of its original length. Our results suggest that R677X is responsible for disease in this family and that the gene corresponding to hG28 is the RP1 gene.     

Erythroblast differentiation at spleen in Q137E mutant ribosomal protein S19 gene knock-in C57BL/6J mice

We recently found that erythroblast-like cells derived from human leukaemia K562 cells express C5a receptor (C5aR) and produce its antagonistic and agonistic ligand ribosomal protein S19 (RP S19) polymer, which is cross-linked between K122 and Q137 by tissue transglutaminases. RP S19 polymer binds to the reciprocal C5aRs on erythroblast-like cells and macrophage-like cells derived from human monocytic THP-1 cells and promotes differentiation into reticulocyte-like cells through enucleation in vitro. To examine the roles of RP S19 polymer in mouse erythropoiesis, we prepared Q137E mutant RP S19 gene knock-in C57BL/6J mice. In contrast to wild-type mice, erythroblast numbers at the preliminary stage (CD71^high/TER119^low) in spleen based on transferrin receptor (CD71) and glycophorin A (TER119) values and erythrocyte numbers in orbital artery bloods were not largely changed in knock-in mice. Conversely, erythroblast numbers at the early stage (CD71^high/TER119^high) were significantly decreased in spleen by knock-in mice. The reduction of early erythroblast numbers in spleen was enhanced by the phenylhydrazine-induced pernicious anemia model knock-in mice and was rescued by a functional analogue of RP S19 dimer S-tagged C5a/RP S19. These data indicated that RP S19 polymer plays the roles in the early erythroblast differentiation of C57BL/6J mouse spleen.     

Genetic Analysis of FAM46A in Spanish Families with Autosomal Recessive Retinitis Pigmentosa: Characterisation of Novel VNTRs

Retinitis pigmentosa (RP) is a group of retinal dystrophies characterised primarily by rod photoreceptor cell degeneration. Exhibiting great clinical and genetic heterogeneity, RP be inherited as an autosomal dominant (ad) and recessive (ar), X-linked (xl) and digenic disorder. RP25 , a locus for arRP, was mapped to chromosome 6p12.1-q14.1 where several retinal dystrophy loci are located. A gene expressed in the retina, FAM46A , mapped within the RP25 locus, and computational data revealed its involvement in retinal signalling pathways. Therefore, we chose to perform molecular evaluation of this gene as a good candidate in arRP families linked to the RP25 interval. A comprehensive bioinformatic and retinal tissue expression characterisation of FAM46A was performed, together with mutation screening of seven RP25 families.
Herein we present 4 novel sequence variants, of which one is a novel deletion within a low complexity region close to the initiation codon of FAM46A . Furthermore, we have characterised for the first time a coding tandem variation in the Caucasian population.
This study reports on bioinformatic and moleculardata for the FAM46A gene that may give a wider insight into the putative function of this gene and its pathologic relevance to RP25 and other retinal diseases mapping within the 6q chromosomal interval.     

Refinement of the RP17 locus for autosomal dominant retinitis pigmentosa, construction of a YAC contig and investigation of the candidate gene retinal fascin

The RP17 locus for autosomal dominant retinitis pigmentosa has previously been mapped to chromosome 17q by linkage analysis. Two unrelated South African families are linked to this locus and the identification of key recombination events assigned the RP17 locus to a 10 cM interval on 17q22. The work reported here refines the mapping of the locus from a 10 cM to a 1 cM interval between the microsatellite markers D17S1604 and D17S948. A physical map of this interval was constructed using information from the Whitehead/MIT YAC contig WC 17.8. Sequence-tagged site (STS) content mapping of seven overlapping YACs from this contig was employed in order to build the map. A BAC library was screened to cover a gap in the YAC contig and two positive BACs were identified. Intragenic polymorphisms in the retinal fascin gene provided evidence for the exclusion of this candidate as the RP17 disease gene.     

The roles of a ribosomal protein S19 polymer in a mouse model of carrageenan-induced acute pleurisy

C5-deficient mice usually present moderate neutrophil activation during the initiation phase of acute inflammation. Conversely, C5a receptor (C5aR)-deficient mice show unusually excessive activation of neutrophils. We identified the ribosomal protein S19 (RP S19) polymer, which is cross-linked at Lys122 and Gln137 by transglutaminases in apoptotic neutrophils, as a second C5aR ligand during the resolution phase of acute inflammation. The RP S19 polymer promotes apoptosis via the neutrophil C5aR and phagocytosis via the macrophage C5aR. To confirm the roles of the RP S19 polymer, we employed a carrageenan-induced acute pleurisy mouse model using C57BL/6J mice with a knock-in of the Gln137Glu mutant RP S19 gene and replaced the RP S19 polymer with either an S-tagged C5a/RP S19 recombinant protein or the RP S19^122–145 peptide monomer and dimer (as functional C5aR agonists/antagonists) and the RP S19^122–145 peptide trimer (as a functional C5aR antagonist). Neutrophils and macrophages were still present in the thoracic cavities of the knock-in mice at 24 h and 7 days after carrageenan injection, respectively. Knock-in mice showed structural organization and severe hemorrhaging from the surrounding small vessels of the alveolar walls in the lung parenchyma. In contrast to the RP S19^122–145 peptide monomer and trimer, the simultaneous presence of S-tagged C5a/RP S19 and the RP S19^122–145 peptide dimer completely improved the physiological and pathological acute inflammatory cues. The RP S19 polymer, especially the dimer, appears to play a role at the resolution phase of carrageenan-induced acute pleurisy in C57BL/6J model mice.     

Guinea pig S19 ribosomal protein as precursor of C5a receptor-directed monocyte-selective leukocyte chemotactic factor

Objective: To reveal the C5a receptor-mediated monocyte-selective chemoattraction of the homo-dimer of guinea pig S19 ribosomal protein (RP S19), and to study the topological relationship between the RP S19 and C5a receptor genes.Methods: cDNA cloning and nucleotide sequencing, leukocyte chemotaxis measurement, and fluorescent in situ hybridization (FISH) were performed in the guinea pig.Results: The amino acid sequence of the guinea pig RP S19 deduced from the cDNA nucleotide sequence was identical to the human protein. The dimer of a recombinant RP S19 attracted guinea pig monocytes but suppressed neutrophil chemotactic movement. Both effects were C5a receptor-mediated. In the FISH analysis, the signals denoting the guinea pig RP S19 gene and C5a receptor gene completely overlapped each other.Conclusions: The guinea pig RP S19 dimer possessed a dual ligand effect, agonistic to the monocyte C5a receptor and antagonistic to the neutrophil receptor. The RP S19 and C5a receptor genes co-localized on the same chromosome.Received 24 April 2004; returned for revision 14 June 2004; accepted by M. Katori 21 June 2004     

Localizaion of the gene for dominant cystoid macular dystrophy on chromosome 7p

In a family with autosomal dominant cystoid macular dystrophy(DCMD) lInkage was detected with the dinucleotide marker D7S435on the short arm of chromosome 7. With markers flanking D7S435,the DCMD locus could be asigned to the interval D7S493-D7S526at 7p15-p21, which spans approximately 20 cM. Three-points lInkageyIelded a maximal lod score of 9.46 and location score of 43.5and suggested that DCMD is 5, 5 cM proximal to D7S493. Recently,a retinitis plgmentosa (RP7) locus has been mapped in roughlythe same area of chromosome 7. Genetic data of both studiesdescribed below, allow a region of overlap between the locationof the DCMD and the RP7 gene between D7S435 and D7S526. Bothgenes being one and the same will further substantiate the closerelationship between macular degeneration and retinitis pIgmentosa.     

Correlation of mismatch repair genes immunohistochemistry and microsatellite instability status in HNPCC-associated tumours

AIM: The aim of this study was to assess the performance of immunohistochemistry using antibodies for MLH1, MSH2, MSH6 and PMS2 mismatch repair gene proteins against microsatellite instability (MSI) testing. METHODS: Tumour samples included in this study were derived from referred patients for screening for hereditary non-polyposis colorectal cancer (HNPCC) and patients who had resections for colorectal cancer that were examined at our institution. MSI was assessed at nine loci (BAT25, BAT26, BAT40, D2S123, D10S197, D17S579, D18S34, D5S346 and D17S250) in all cases. Immunohistochemistry for MLH1 and MSH2 was performed in all cases. Staining for MSH6 and PMS2 was performed in selected cases only. RESULTS: There were 742 tumours including 661 colorectal lesions and 81 extracolonic tumours of the HNPCC spectrum. Among the 555 MSI-negative tumours, 554 showed an intact protein expression. Amongst the 187 MSI-positive tumours, 126 showed abnormal expression of MLH1 gene protein, 41 showed abnormal expression of MSH2 gene, three showed abnormal expression of MSH6 only, one showed abnormal expression of PMS2 gene protein only and one case showed abnormal expression of all four proteins. CONCLUSION: Immunohistochemistry offers an alternative method for assessment of MSI status which is fast and relatively inexpensive compared with MSI testing. We achieved a sensitivity rate of 92% and specificity of 99.8% for immunohistochemistry testing assessed against the MSI testing. It has to be accepted that a small fraction of MSI-positive cases will be missed by testing with immunohistochemistry alone.     

Diamond-Blackfan anemia,ribosome and erythropoiesis

Diamond-Blackfan anemia is a rare inherited bone marrow failure syndrome (five to seven cases per million live births) characterized by an aregenerative, usually macrocytic anemia with an absence or less than 5% of erythroid precursors (erythroblastopenia) in an otherwise normal bone marrow. The platelet and the white cell counts are usually normal but neutropenia, thrombopenia or thrombocytosis have been noted at diagnosis. In 40 to 50% of DBA patients, congenital abnormalities mostly in the cephalic area and in thumbs and upper limbs have been described. Recent analysis did show a phenotype/genotype correlation. Congenital erythroblastopenia of DBA is the first human disease identified to result from defects in ribosomal biogenesis. The first ribosomal gene involved in DBA, ribosomal protein (RP) gene S19 (RPS19 gene), was identified in 1999. Subsequently, mutations in 12 other RP genes out of a total of 78 RP genes have been identified in DBA. All RP gene mutations described to date are heterozygous and dominant inheritance has been documented in 40 to 45% of affected individuals. As RP mutations are yet to be identified in approximately 50% of DBA cases, it is likely that other yet to be identified genes involved in ribosomal biogenesis or other pathways may be responsible for DBA phenotype.     

Large-scale Molecular Analysis of a 34 Mb Interval on Chromosome 6q: Major Refinement of the RP25 Interval

A large scale bioinformatics and molecular analysis of a 34 Mb interval on chromosome 6q12 was undertaken as part of our ongoing study to identify the gene responsible for an autosomal recessive retinitis pigmentosa (arRP) locus, RP25. Extensive bioinformatics analysis indicated in excess of 110 genes within the region and we also noted unfinished sequence on chromosome 6q in the Human Genome Database, between 58 and 61.2 Mb. Forty three genes within the RP25 interval were considered as good candidates for mutation screening. Direct sequence analysis of the selected genes in 7 Spanish families with arRP revealed a total of 244 sequence variants, of which 67 were novel but none were pathogenic. This, together with previous reports, excludes 60 genes within the interval (∼55%) as disease causing for RP. To investigate if copy number variation (CNV) exists within RP25, a comparative genomic hybridization (CGH) analysis was performed on a consanguineous family. A clone from the tiling path array, chr6tp-19C7, spanning ∼100-Kb was found to be deleted in all affected members of the family, leading to a major refinement of the interval. This will eventually have a significant impact on cloning of the RP25 gene.     

A ninth locus (RP18) for autosomal dominant retinitis pigmentosa maps in the pericentromeric region of chromosome 1

We studied a large Danish family of seven generations in which autosomal dominant retinitis pigmentosa (adRP), a heterogeneous genetic form of retinal dystrophy, was segregating. After linkage had been excluded to all known adRP loci on chromosomes 3q, 6p, 7p, 7q, 8q, 17p, 17q and 19q, a genome screening was performed. Positive lod scores suggestive of linkage with values ranging between Z = 1.58-5.36 at theta = 0.04-0.20 were obtained for eight loci on proximal 1p and 1q. Close linkage without recombination and a maximum lod score of 7.22 at theta = 0.00 was found between the adRP locus (RP18) in this family and D1S498 which is on 1q very near the centromere. Analysis of multiply informative meioses suggests that in this family D1S534 and D1S305 flank RP18 in interval 1p13-q23. No linkage has been found to loci from this chromosomal region in six other medium sized adRP families in which the disease locus has been excluded from all known chromosomal regions harbouring an adRP gene or locus suggesting that there is (at least) one further adRP locus to be mapped in the future.      

A new locus for autosomal dominant retinitis pigmentosa on the short arm of chromosome 17

Retinitis pigmentosa (RP) is a group of genetically and clinicallyheterogeneous retinopathies, some of which have been shown toresult from mutations in two different known retinal genes,rhodopsin (3q) and peripherin-rds (6p). Three additional anonymousloci at 7p, 7q and pericentric 8 have been implicated by linkagestudies. There are still, however, a few families in which allknown loci have been excluded. In this report we present dataindicating a location, on the short arm of chromosome 17, forthe autosomal dominant RP (ADRP) locus in a large South African(SA) family of British ancestry. Positive two-point lod scoreshave been obtained for nine markers (D17S938, Z = 5.43; D17S796,Z = 4.82; D17S849, Z = 3.6; D17S786, Z = 3.55; TP53, Z = 3.55;D17S578, Z = 3.29; D17S960, Z = 3.16; D17S926, Z = 1.51; D17S804,Z = 0.47 all at      

Mutations of the retinal specific ATP binding transporter gene (ABCR) in a single family segregating both autosomal recessive retinitis pigmentosa RP19 and Stargardt disease: evidence of clinical heterogeneity at this locus

Stargardt disease (STGD) is an autosomal recessive macular dystrophy of childhood characterised by bilateral loss of central vision over a period of several months. STGD has been mapped to chromosome 1p22.1 and recently ascribed to mutations in the retinal specific ATP binding transporter gene (ABCR). The fundus flavimaculatus with macular dystrophy (FFM), an autosomal recessive condition responsible for gradual loss of visual acuity in adulthood (second to third decade) has also been mapped to the same locus. However, a gene for autosomal recessive retinitis pigmentosa with distinctive features of choriocapillaris atrophy at an advanced stage (RP19) has been mapped to the genetic interval encompassing the STGD gene on chromosome 1p (D1S435-D1S236), raising the question of whether, despite striking differences in clinical course and presentation, RP19 and STGD might be allelic disorders at the ABCR locus. In a family segregating RP and STGD in two first cousins, we found that heterozygosity for a splicing mutation in the ABCR gene (1938-1 G-->A) resulted in STGD while hemizygosity for this splice mutation resulted in RP, and when studying the RP patient's parents, we found a maternal non-contribution with apparent segregation of a null allele ascribed to a partial deletion of the ABCR gene. The present study shows that, despite striking clinical differences, RP19 and STGD are allelic disorders at the ABCR locus.     

一个视网膜色素变性家系致病基因定位和CA4基因突变分析

目的通过对一个常染色体显性视网膜色素变性(autosomal dominant retinitis pigmentosa,adRP)家系致病基因的定位和基因突变分析,以确定该家系的致病基因及其突变形式。方法对15个已知的常染色体显性视网膜色素变性致病基因所在染色体位点进行连锁分析,以确定该家系与疾病连锁的染色体区域,对该区域附近候选基因进行直接序列分析。结果连锁分析提示在D17S701和D17S1604为正的连锁值(logofodds,LOD),分别为Zmax=2.107和Zmax=1.806。其余14个adRP染色体位点的微卫星标记两点LOD值均为负数。单倍型分析进一步将该家系致病基因定位于微卫星标记D17S916和D17S794之间的RP17位点,该位点adRP候选基因碳酸酐酶Ⅳ(carbonic anhydrase4,CA4)直接序列分析在其编码区未发现基因突变。结论将一个中国人常染色体显性视网膜色素变性家系的致病基因定位于RP17位点,但未发现该位点内的CA4基因突变,该家系是否存在CA4基因复杂突变或RP17位点是否存在新的视网膜色素变性致病基因有待于进一步研究。