Isolation of a candidate gene for Norrie disease by positional cloning.

The gene for Norrie disease, an X-linked disorder characterized by progressive atrophy of the eyes, mental disturbances and deafness, has been mapped to chromosome Xp11.4 close to DXS7 and the monoamine oxidase (MAO) genes. By subcloning a YAC with a 640 kilobases (kb) insert which spans the DXS7-MAOB interval we have generated a cosmid contig which extends 250 kb beyond the MAOB gene. With one of these cosmids, microdeletions were detected in several patients with Norrie disease. Screening of cDNA libraries has enabled us to isolate and sequence a likely candidate gene for Norrie disease which is expressed in retina, choroid and fetal brain. No homologous sequences were found in DNA and protein databases indicating that this cDNA is part of a gene encoding a 'pioneer' protein.

Yeast artificial chromosome cloning of the Xq13.3-q21.31 region and fine mapping of a deletion associated with choroideremia and nonspecific mental retardation.

Microscopically detectable deletions and X;autosome translocations have previously facilitated the construction of a high-resolution interval map of the Xq21 region. Here, we have generated three yeast artificial chromosome contigs spanning approximately 7 megabases of the Xq13.3-q21.31 region. In addition, a novel deletion associated with choroideremia and mental retardation was identified and mapped in detail. The proximal deletion endpoint was positioned between the loci DXS995 and DXS232, which enabled us to confirm the critical region for a locus involved in mental retardation. The distal deletion endpoint is situated in the Xq21.33 band, which allowed us to refine the order of several markers in this region.

Derivation of clones from the choroideremia locus by preparative field inversion gel electrophoresis.

By making use of preparative field inversion gel electrophoresis, we have constructed a lambda ZAP library that is highly enriched for sequences from the choroideremia locus. In vivo excision of pBluescript SK(-) constructs from lambda ZAP obviates the subcloning of DNA inserts and allows for rapid processing of several hundred recombinants. From a 625 kb Sfil fragment we isolated 7 clones that were physically mapped using microdeletions associated with the disease. One of these clones is located within, or just telomeric to, the choroideremia gene and detects two restriction fragment length polymorphisms (RFLPs). Another clone detects a RFLP which maps centromeric to the disease locus. Together these probes should improve the reliability of linkage analysis in choroideremia families and should pave the way for the isolation of the choroideremia gene.

An interstitial duplication of the X chromosome in a male allows physical fine mapping of probes from the Xq13-q22 region.

An insertional translocation into the proximal long arm of the X chromosome in a boy showing muscular hypotony, growth retardation, psychomotor retardation, cryptorchidism, and Pelizaeus-Merzbacher disease (PMD) was identified as a duplication of the Xq21-q22 segment by employing DNA probes. With densitometric scanning for quantitation of hybridization signals, 15 Xq probes were assigned to the duplicated region. Analysis of the duplication allowed us to dissect the X-Y homologous region physically at Xq21 and to refine the assignments of the loci for DXYS5, DXYS12, DXYS13, DXS94, DXS95, DXS96, DXS111, and DXS211. Furthermore, we demonstrated the presence of two different DXYS13 and DXS17 alleles in genomic DNA of our patient, suggesting that the duplication resulted from a meiotic recombination event involving the two maternal X chromosomes.

Deletion of the DXS165 locus in patients with classical choroideremia.

Using various probes from the Xq21 region which is known to carry the choroideremia (tapetochoroideal dystrophy, TCD) locus, we have screened the DNAs from eight unrelated male choroidermia patients for microdeletions. In two of these patients, but not in any of 45 males tested as controls, lack of hybridization signals with probe plbD5 suggested a deletion encompassing the DXS165 locus and (part of) the TCD gene. Absence of additional clinical features in these patients and the fact that two closely linked, and probably flanking, TCD markers (DXYS1 and DXS72) are not deleted may indicate that the physical distance between the DXS165 locus and the TCD gene is small.

Genetic fine mapping of the gene for recessive Stargardt disease.

Stargardt disease (STGD) is one of the most frequent causes of macular degeneration in childhood. Linkage analysis in families with recessive STGD has recently shown genetic homogeneity and a location of the underlying gene at 1p22-p21 in a 4-cM interval. Haplotype analysis in seven Dutch STGD families with 11 highly polymorphic markers spanning the critical region has enabled us to refine the location of the underlying gene to a 2-cM region flanked by the loci D1S406 and D1S236. We have identified one 45-year-old nonpenetrant individual who carries two disease alleles. In another family, an affected individual inherited the paternal but not the maternal disease chromosome, suggesting genetic heterogeneity or a different mechanism leading to the disease in this family.

Complete exon-intron structure of the retina-specific ATP binding transporter gene (ABCR) allows the identification of novel mutations underlying Stargardt disease.

Stargardt disease, an autosomal recessive macular dystrophy of childhood, leading to severe visual impairment, is caused by mutations in the retina-specific ATP binding transporter gene (ABCR). Previously, the ABCR cDNA and part of the exon-intron structure were described. We have determined the complete ABCR exon-intron structure by exon-exon PCR. The ABCR gene encompasses 50 exons, 29 of which are first described here with their corresponding intron-exon boundaries. The discovery of a splicing mutation (571: 2A-->G) and missense mutations in the newly identified exons (R18W, R212C) gives additional support to the broad allelic heterogeneity of Stargardt disease.

Cloning of the breakpoints of a deletion associated with choroidermia.

In order to characterize a previously described submicroscopic deletion encompassing (part of) the choroideremia (tapetochoroidal dystrophy: TCD) gene, we have cloned a 10.5-kb EcoRI fragment from the patient's DNA; this fragment carries the junction between both deletion endpoints ("junction fragment"). The distal portion of this fragment defines a new marker within, or just distal to, the TCD gene. This marker has been employed to confirm the diagnosis in several affected family members, and to rule out carriership in a female at risk with conspicuous clinical signs.

Molecular analysis of male-viable deletions and duplications allows ordering of 52 DNA probes on proximal Xq.

While performing a systematic search for chromosomal microdeletions in patients with clinically complex X-linked syndromes, we have observed that large male-viable deletions and duplications are clustered in heterochromatic regions of the X chromosome. Apart from the Xp21 band, where numerous deletions have been found that encompass the Duchenne muscular dystrophy gene, an increasing number of deletions and duplications have been observed that span (part of) the Xq21 segment. To refine the molecular and genetic map of this region, we have employed 52 cloned single-copy DNA sequences from the Xcen-q22 segment to characterize two partly overlapping tandem duplications and two interstitial deletions on the proximal long arm of the human X chromosome. Together with a panel of somatic cell hybrids that had been described earlier, these four rearrangements enabled us to order the 52 probes into nine different groups and to narrow the regional assignment of several genes, including those for tapetochoroidal dystrophy and anhidrotic ectodermal dysplasia.

Molecular basis of choroideremia (CHM): mutations involving the Rab escort protein-1 (REP-1) gene.

Choroideremia (CHM) is an X-linked recessive eye disease that results from mutations involving the Rab escort protein-1 (REP-1) gene. In 18 patients deletions of different sizes have been found. Two females suffering from CHM were reported to have translocations that disrupt the REP-1 gene. In 22 patients, small mutations have been identified. Interestingly, these are all nonsense, frameshift or splice-site mutations; with one possible exception, missense mutations have not been found. This comprises all the known mutations in the disease.

Generation and characterization of radiation reduced cell hybrids and isolation of probes from the proximal short arm of the human X chromosome.

Employing a modified Goss-Harris irradiation fusion protocol, we have generated a panel of somatic cell hybrids containing various overlapping fragments of the Xcen-Xp11.4 interval. This region of the human X chromosome is known to carry genes for several hereditary eye diseases including retinitis pigmentosa (RP2), congenital stationary night blindness (CSNB-1) and Norrie disease. These hybrid cell lines were employed to isolate 17 new DNA probes by making use of the Alu polymerase chain reaction (PCR) method and subsequent cloning of the PCR products in a plasmid vector. With these probes, we have characterized two previously described microdeletions spanning the Norrie locus; these deletions have enabled us to subdivide the Xp11.4-p11.3 region into three defined intervals.

Detection and characterization of point mutations in the choroideremia candidate gene by PCR-SSCP analysis and direct DNA sequencing.

By making use of positional cloning strategies we recently isolated a candidate gene for choroideremia (CHM), which is transcribed in retina, choroid, and/or retinal pigment epithelium. The gene contains an open reading frame that is structurally altered in 10 CHM patients with sizable deletions and in a female patient with a balanced translocation involving the Xq21 band. Employing PCR-SSCP analysis and direct DNA sequencing we have now detected and characterized different point mutations in five patients with CHM. Each of these mutations introduces a termination codon into the open reading frame of the CHM candidate gene, thereby predicting a distinct truncated protein product. Together these findings provide convincing evidence for the candidate gene being identical with the choroideremia gene.

Cloning, characterization, and mRNA expression analysis of novel human fetal cochlear cDNAs.

To identify novel genes that are expressed specifically or preferentially in the cochlea, we constructed a cDNA library enriched for human cochlear cDNAs using a suppression subtractive hybridization technique. We analyzed 2640 clones by sequencing and BLAST similarity searches. One hundred and fifty-five different cDNA fragments mapped in nonsyndromic hearing impairment loci for which the causative gene has not been cloned yet. Approximately 30% of the clones show no similarity to any known human gene or expressed sequence tag (EST). Clones mapping in nonsyndromic deafness loci and a selection of clones that represent novel ESTs were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) of RNA derived from 12 human fetal tissues. Our data suggest that a quarter of the novel genes in our library are preferentially expressed in fetal cochlea. These may play a physiologically important role in the hearing process and represent candidate genes for hereditary hearing impairment.