Genomic organization and cloning of the human homologue of murine Sipa-1.

Murine Sipa-1 (signal-induced proliferation associated protein) is a mitogen induced GTPase activating protein (GAP). While mapping candidate genes for multiple endocrine neoplasia type 1 (MEN1) at 11q13, we cloned the human homologue of Sipa-1. Herein, we report the complete cDNA sequence, expression, and genomic organization of SIPA-1. SIPA-1 consists of 16 exons with highly conserved exon-intron boundaries. The predicted SIPA-1 protein is highly homologous to the mouse protein, particularly in the region of the GAP-related domain at the amino terminus and the leucine zipper at the carboxy terminus. It is widely expressed, including in fetal tissues, but is most highly expressed in lymphoid organs. During the course of cloning SIPA-1, the MEN1 gene was identified, thus excluding human SIPA-1 as a candidate for this disease.

North Carolina macular dystrophy (MCDR1) locus: a fine resolution genetic map and haplotype analysis.

PURPOSE: We previously reported linkage of North Carolina macular dystrophy in a single isolated family to a broad region on chromosome 6q16. In order to refine the localization of the MCDR1 gene (North Carolina macular dystrophy), additional families with this disease and new markers were studied. METHODS: We ascertained 10 families with the North Carolina macular dystrophy phenotype (MCDR1). These families were of various ethnic and geographic origins such as Caucasian, Mayan Indian, African-American, French, British, German, and American of European decent. Two hundred thirty-two individuals in these families underwent comprehensive ophthalmic examinations and blood was collected for genotyping. One hundred seventeen were found to be affected. Linkage simulation studies were performed. Two-point linkage, haplotype analysis, and multipoint linkage was performed using VITESSE and FASTLINK. HOMOG was used to test for genetic heterogeneity. RESULTS: The clinical features were consistent with the diagnosis of North Carolina macular dystrophy in all families. Multipoint linkage analysis indicates that the MCDR1 gene is in the interval between D6D249 and D6S1671 with a maximum LOD score of 41.52. There was no evidence of genetic heterogeneity among the families studied. Families 765, 768, 772, 1193, and 1292 shared the same chromosomal haplotype in this region. CONCLUSIONS: This is the largest single data set of families with the MCDR1 phenotype. The single large family from North Carolina continues to be informative for the closest flanking markers and alone supports the minimal candidate region as suggested by previous studies. There remains no evidence of genetic heterogeneity in this disease. Most of the American families appear to have descended from the same ancestral mutation. The remaining families could each represent independent origins of the mutation in the MCDR1 gene.

NS22: a highly polymorphic complex microsatellite marker within the ATM gene.

We have found a complex repeat sequence (NS22) that is highly polymorphic and located within intron 45 of the ataxia-telangiectasia gene (ATM). Sequencing this region from various individuals demonstrated two different polymorphic repeating units adjacent to one another. The fact that the sequence is located within the ATM gene provides a unique opportunity to follow segregation of affected and unaffected haplotypes for prenatal diagnosis of ataxia-telangiectasia. The high degree of polymorphism observed with this marker will also aid in evaluating loss of heterozygosity (LOH) across this region of the genome and may prove valuable in assessing the role of the ATM gene in susceptibility to cancer.

North Carolina macular dystrophy: clinicopathologic correlation.

PURPOSE: To describe the clinical and histopathologic findings of a 72-year-old female with North Carolina macular dystrophy. METHODS: Observational case report with histopathologic correlation. Clinical examination includes slit-lamp biomicroscopy, indirect ophthalmoscopy, color fundus photography, and focal electroretinography. Histopathologic examination of the enucleated left eye performed with light microscopy. RESULTS: Light microscopy demonstrated a discrete macular lesion characterized by focal absence of photoreceptor cells and retinal pigment epithelium with attenuation of the Bruch membrane and focal atrophy of the choriocapillaris. Adjacent to the macular lesion, some lipofuscin was identified in the retinal pigment epithelium. CONCLUSION: North Carolina macular dystrophy has both clinical and microscopic appearances of a well-demarcated lesion confined to the macula, which involves the retina, pigment epithelium, and choriocapillaris.

A North Carolina macular dystrophy phenotype in a Belizean family maps to the MCDR1 locus.

PURPOSE: To describe the clinical findings of an autosomal dominant macular dystrophy in a family of Mayan Indian ancestry in Belize, Central America, and to determine its molecular genetic relationship with the original North Carolinian family. METHODS: We performed comprehensive ophthalmic examinations on 56 members of a single family living in Chicago, Illinois, and Belize, Central America. Fundus photography and fluorescein angiography were performed on 17 affected subjects and six affected family members were serially examined over a 12-year period. Blood was collected from 26 individuals, and DNA was extracted for genotyping. Two-point linkage, multipoint linkage, and haplotype analysis was performed. RESULTS: In 17 affected individuals, the clinical features were consistent with the diagnosis of North Carolina macular dystrophy. Multipoint linkage analysis generated a peak lod score of 5.6 in the MCDR1 region. The haplotype associated with the disease was, however, different from that of the original North Carolinian family. CONCLUSIONS: This family has an autosomal dominant macular dystrophy that is clinically indistinguishable from North Carolina macular dystrophy (MCDR1). Our findings indicate that the mutated gene in this Belizean family maps precisely to the same region as that of the North Carolina macular dystrophy (MCDR1) locus. This study provides evidence that MCDR1 occurs in various ethnic groups and that there is no evidence of genetic heterogeneity.

Genetic haplotyping of ataxia-telangiectasia families localizes the major gene to an approximately 850 kb region on chromosome 11q23.1.

The genotyping data given localize the major A-T gene to an approximately 850 kb region. They also localize the group A A-T gene (ATA) to a region that contains the approximately 850 kb region. They are compatible with linking A-TFresno to 11q22-23. NBS-V2 does not link to this region. Four non-linking families contain only single affecteds, suggesting that these may be spontaneous mutations rather than evidence for an A-T gene outside the 11q22-23 region. Finally, two other non-linking families contain recombinant haplotypes that are compatible with a second A-T gene at 11q22-23, slightly distal to the approximately 850 kb region. However, convincing evidence for a second gene is still lacking.

An allelic variant at the ATM locus is implicated in breast cancer susceptibility.

We have tested a simple procedure, disease association by locus stratification, for identifying breast cancer patients with pathogenetic allelic variants at several candidate loci. The strategy was based on the assumption of epistatic interactions of the candidates. We analyzed 66 independent cases from sib pairs affected with breast cancer that had previously been collected during an investigation of pathogenetic-allele-sharing at the HRAS1 mini-satellite locus. An exon 24 polymorphism of ATM, substituting arginine for proline was associated with breast cancer in these cases with an overall odds ratio of 4.5 (95% confidence interval, 1.2-20.5, nominal p = 0.02, 2-tail Fisher exact test). In the presence of a rare HRAS1 allele, the odds ratio increased to 6.9 (95% CI, 1.2-38.3, p = 0.03). Thus, our procedure identified at least one allelic variant of ATM associated with breast cancer, and indicated that the ATM locus may interact with HRAS1.

Sodium selenite induced changes during ripening in post-harvest banana (Musa sapientum) fruits.

The metabolic role of selenium, if any, in plant systems has received little attention, as opposed to animal and other systems where many studies have been performed. Post-harvest banana (Musa sapientum) fruits treated with 1 mM solutions of sodium selenite show an increase in the rate of ripening. Visually, typical ripening changes in colour, aroma and tissue softness were observed to occur earlier in sodium selenite-treated fruits as compared to controls. These results were substantiated biochemically, enzymologically and physiologically, showing a strong relationship of this phenomenon with the oxidative changes.

[Ataxia telangiectasia and genetic predisposition to cancer]. / Ataxie télangiectasie et prédisposition génétique aux cancers.

Ataxia telangiectasia (AT) is a genetic disorder with an autosomic recessive transmission. Occurring during childhood, it affects different organs and/or systems. Physiopathology is still unclear. The first clinical signs are evident early in childhood and evolution always leads to death. The secondary cause of mortality in 10 to 15% of the affected is the development of cancers. Genetic predisposition to cancer for homozygotes, as well as for heterozygotes, is one of the most remarkable aspects of this disease. For heterozygotes the risk of cancer is three times that of the norm. The gene responsible for the disease has been cloned. Its function may resolve some questions, and provide the link between degenerative process, cancer susceptibility and immunodeficiency evident in AT patients.

North Carolina macular dystrophy: clinicopathologic correlation.

PURPOSE: To describe the clinical and histopathologic findings in a 72-year-old woman with North Carolina macular dystrophy. METHODS: Clinical examination was performed by slit-lamp biomicroscopy, indirect ophthalmoscopy, color fundus photography, and focal electroretinography. Histopathologic examination of the enucleated left eye consisted of light microscopy. RESULTS: Light microscopy demonstrated a discrete macular lesion characterized by focal absence of photoreceptor cells and retinal pigment epithelium. Bruch's membrane was attenuated in the center of the lesion and associated with marked atrophy of the choriocapillaris. Adjacent to the central lesion, some lipofuscin was identified in the retinal pigment epithelium. CONCLUSIONS: North Carolina macular dystrophy has both clinical and microscopic appearances of a well-demarcated retinal and pigment epithelial lesion confined to the macula. This is consistent with the clinical impression that it is a focal macular dystrophy.

Hydrogen peroxide causes mitochondrial DNA damage in corneal epithelial cells.

PURPOSE: To study the effects of hydrogen peroxide exposure on mitochondrial DNA (mtDNA) in cultured human corneal epithelial cells. In addition, we compared the integrity of mtDNA found in epithelial cells isolated from keratoconus (KC) and normal (NL) corneas. METHODS: Telomerase immortalized human corneal epithelial cell line (hTCEpi) were cultured at pH 7.0 or pH 5.0 with or without 200 microM hydrogen peroxide (H2O2). Immunohistochemistry with a marker for oxidative damage, 8-hydroxy-2'-deoxyguanosine (8-OH-dG), was performed on KC and NL corneas (n = 10). Epithelial cells were isolated from KC corneas (n = 5) and NL corneas (n = 7). Total DNA was extracted, and the mtDNA was analyzed by long extension polymerase chain reaction (LX-PCR). The ratios of mtDNA to nuclear DNA were measured by PCR. The mtDNA control regions were PCR amplified and sequenced. RESULTS: In the epithelial cell cultures, the full-length LX-PCR mtDNA decreased 54% and 44% in the H2O2 + pH7 cultures and H2O2 + pH5 cultures, respectively. 8-OH-dG was present in all layers of KC epithelial cells but only in superficial layers of NL epithelial cells. The isolated KC and NL epithelial cells had comparable levels of full-length LX-PCR mtDNA (16.2 kb) and smaller sized mtDNA bands (4.3 +/- 0.99 vs 4.0 +/- 0.83 bands per individual, respectively). There were no significant differences in the control region nucleotide sequences in KC and NL epithelia. CONCLUSIONS: Hydrogen peroxide can significantly degrade LX-PCR mtDNA in vitro. Although the KC epithelium showed a higher degree of oxidative damage, the levels of mtDNA damage in NL and KC epithelial cells were similar to each other.

North Carolina macular dystrophy (MCDR1) in Texas.

PURPOSE: To map the gene responsible for causing a macular degeneration in a Texan family that appears clinically similar to the North Carolina macular dystrophy (MCDR1) phenotype. METHODS: A single family in Texas had all the typical clinical features of the North Carolina macular dystrophy phenotype. Of 23 family members examined, 10 were affected. Blood was collected from all 23 members and fundus photographs were obtained on those affected. A detailed family history consisting of nine generations was obtained. Genotyping and likelihood analysis was performed using the closest linked MCDR1 markers. RESULTS: The genealogic data showed no relation with the original North Carolina macular dystrophy pedigree. The dinucleotide repeat marker D6S283 yielded the highest 2-point LOD score with a Zmax = 4.1 at theta = 0. The peak LOD score generated from multipoint analysis was 6.0. CONCLUSIONS: The linkage results indicate that the macular degeneration in this Texan family is due to a mutation in the same genomic region as that causing North Carolina macular dystrophy. Furthermore, haplotype analysis suggests that the original North Carolina family and the Texan family have the same mutation and a common founder.

Genomic organization of human DLG4, the gene encoding postsynaptic density 95.

We have determined the exon-intron organization and characterized the 5'-flanking promoter region of DLG4. Encompassing approximately 30 kb, the DLG4 locus is composed of 22 exons that range in size from 28 to 1,218 nucleotides. All splice sites conform to the GT-AG rule, except for the splice acceptor site of intron 5, which is TG instead of AG. Three different exons of DLG4 were found to be alternatively spliced in a subset of tissues. Two of these variants result in altered postsynaptic density 95 (PSD95) isoforms that dramatically truncate the protein. The third splicing variant represents an extension of exon 4 that encodes an additional 33-amino acid segment. Analysis of the core promoter region for DLG4 suggests that the expression of this gene is controlled by a TATA-less promoter using a single transcriptional start site embedded within a CpG island. DLG4 maps to a region on chromosome 17p13.1 known to contain a locus for autosomal dominant cone dystrophy 5. Scanning for mutations in the DLG4 coding region and splice sites was performed in 15 cone dystrophy patients, including probands from five families showing linkage to the DLG4 region. No disease-causing mutations were identified in any patients, suggesting that DLG4 is not the causative gene for this genetic eye disorder.

Ataxia-telangiectasia: mutations in ATM cDNA detected by protein-truncation screening.

We have examined the distal half of the ataxia-telangiectasia (A-T) gene transcript for truncation mutations in 48 A-T affecteds. We found 21 mutations; 4 of the mutations were seen in more than one individual. Genotyping of the individuals sharing mutations, by using nearby microsatellite markers, established that three of the four groups shared common haplotypes, indicating that these were probably founder effects, not public mutations. The one public mutation was found in two American families, one of Ashkenazi Jewish background and the other not. Most truncations deleted the PI3-kinase domain, although some exceptions to this were found in patients with typical A-T phenotypes. All patients not previously known to be consanguineous were found to be compound heterozygotes when mutations could be identified--that is, normal and abnormal protein segments were seen on SDS-PAGE gels. All 48 patients gave RT-PCR products, indicating the presence of relatively stable mRNAs despite their mutations. These results suggest that few public mutations or hot spots can be expected in the A-T gene and that epidemiological studies of A-T carrier status and associated health risks will have to be designed around populations with frequent founder-effect mutations, despite the obvious limitations of this approach.

MAGE Xp-2: a member of the MAGE gene family isolated from an expression library using systemic lupus erythematosus sera.

Two regions of the genome contain members of the MAGE gene family; Xq27-qter and Xp21.3. We isolated a transcript, MAGE Xp-2, by screening a cDNA library from the human epithelial carcinoma cell line, HEp-2, using autoantibodies from patients with systemic lupus erythematosus (SLE). The open reading frame (ORF) of MAGE Xp-2 is entirely contained in exon 4, a signature feature of the MAGE gene family. While MAGE Xp-2 shares genomic homology with MAGE Xp-1, the predicted proteins are quite divergent. Specific primers were designed to reliably distinguish between MAGE Xp-1 and MAGE Xp-2 expression. MAGE Xp-2 is expressed in testis, but not in other normal tissues. It is also expressed strongly in two of seven melanoma cell lines and one of four breast carcinomas. MAGE gene expression may be important not only for tumor recognition and cancer therapy, but, because it is the apparent target of autoantibodies in SLE sera, it may also play a role in autoimmune diseases.

Identification of BPESC1, a novel gene disrupted by a balanced chromosomal translocation, t(3;4)(q23;p15.2), in a patient with BPES.

The blepharophimosis syndrome (BPES) is a rare genetic disorder characterized by blepharophimosis, ptosis, epicanthus inversus, and telecanthus. In type I, BPES is associated with female infertility, while in type II, the eyelid defect occurs by itself. The BPES syndrome has been mapped to 3q23. Previously, we constructed a YAC-, PAC-, and cosmid-based physical map surrounding the 3q23 translocation breakpoint of a t(3;4)(q23;p15.2) BPES patient, containing a 110-kb PAC (169-C 10) and a 43-kb cosmid (11-L 10) spanning the breakpoint. In this report, we present the identification of BPESC1 (BPES candidate 1), a novel candidate gene that is disrupted by the translocation on chromosome 3. Cloning of the cDNA has been performed starting from a testis-specific EST, AI032396, found in cosmid 11-L 10. The cDNA sequence of BPESC1 is 3518 bp in size and contains an open reading frame of 351 bp. No significant similarities with known proteins have been found in the sequence databases. BPESC1 contains three exons and spans a genomic fragment of 17.5 kb. Expression of BPESC1 was observed in adult testis tissue. We performed mutation analysis in 28 unrelated familial and sporadic BPES patients, but, apart from the disruption by the translocation, found no other disease-causing mutations. These data make it unlikely that BPESC1 plays a major role in the pathogenesis of BPES.

A transcript map encompassing the multiple endocrine neoplasia type-1 (MEN1) locus on chromosome 11q13.

A transcription map of a 1200-kb region encompassing the MEN1 locus was constructed by direct cDNA selection and mapping ESTs. A total of 29 genes were mapped. Ten transcripts were identified by cDNA selection of a focused 300-kb genomic region telomeric to the MEN1 consensus region. Since many of the sequences cloned by cDNA selection also identified ESTs from the region, 19 additional RH-mapped ESTs were mapped to the entire contig region by PCR amplification of genomic clones. Nine known genes, 2 putative human homologues to mouse genes, and 18 novel transcripts map to the region. Transcripts that map to the MEN1 interval PYGM-D11S449 include SGC35223, IB1256, AA147620, ZFM1, FAU, and CAPN1. The latter 3 known genes have already been excluded as candidate MEN1 genes. The 2 putative human homologues of mouse genes Ltbp2 and Spa-1 may be candidate tumor suppressor genes, but they map telomeric to D11S449. Although both of these genes map outside the MEN1 consensus region they may play a role in sporadic endocrine tumors independent of the MEN1 gene or in other tumors, such as breast cancer, that have loss of heterozygosity within this region.

Ataxia-telangiectasia: identification and detection of founder-effect mutations in the ATM gene in ethnic populations.

To facilitate the evaluation of ATM heterozygotes for susceptibility to other diseases, such as breast cancer, we have attempted to define the most common mutations and their frequencies in ataxia-telangiectasia (A-T) homozygotes from 10 ethnic populations. Both genomic mutations and their effects on cDNA were characterized. Protein-truncation testing of the entire ATM cDNA detected 92 (66%) truncating mutations in 140 mutant alleles screened. The haplotyping of patients with identical mutations indicates that almost all of these represent common ancestry and that very few spontaneously recurring ATM mutations exist. Assays requiring minimal amounts of genomic DNA were designed to allow rapid screening for common ethnic mutations. These rapid assays detected mutations in 76% of Costa Rican patients (3), 50% of Norwegian patients (1), 25% of Polish patients (4), and 14% of Italian patients (1), as well as in patients of Amish/Mennonite and Irish English backgrounds. Additional mutations were observed in Japanese, Utah Mormon, and African American patients. These assays should facilitate screening for A-T heterozygotes in the populations studied.