Nitin Udar

A pooled case-control study of the apolipoprotein E (APOE) gene in age-related maculopathy

Age-related maculopathy (ARM) is a multifactorial disorder known to have a substantial genetic component. The e4 allele of the apolipoprotein E gene (APOE-4) has previously been reported to have a protective effect on ARM risk, while the APOE-2 allele may increase disease risk. This study combined four independent data sets (three US and one European) of Caucasian ARM patients and controls in order to obtain better statistical power to examine the role of APOE in ARM. APOE genotype and allele frequencies were compared for 617 ARM cases and 1260 controls, adjusting for age and sex differences between the two groups via multiple logistic regression. The protective effect of the APOE-4 allele on ARM risk was confirmed (age- and sex-adjusted odds ratio (OR) for APOE-4 carriers 0.54, 95% confidence interval (CI) 0.41–0.70, p < 0.0001). The effect of APOE-4 did not differ significantly between males and females and was observed consistently for both atrophic and neovascular ARM. Evidence for an increased risk of ARM due to the APOE-2 allele was found for men, but not for women (OR for men 1.54, 95% CI 0.97–2.45; OR for women 0.74, 95% CI 0.52–1.06, p = 0.01 for interaction of sex and APOE-2 carrier status). These data confirm that the APOE-4 allele, or an allele in linkage disequilibrium with it, reduces the risk of ARM. They also suggest that the effect of the APOE-2 allele may vary by gender, and that APOE-2 may confer an increased risk only to males.

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.

The pathogenesis of ataxia-telangiectasia. Learning from a Rosetta Stone

ConclusionMany issues in A-T research remain unresolved. However, certain common denominators are becoming clear that were not obvious just a few years ago, i.e., the relationship between double-strand break rejoining, radiosensitivity, and immunodeficiency. The downstream pathways that are activated by ATM phosphorylating p53, c-abl, chk2, Brca1, nibrin, RPA, and other proteins that contribute to cell-cycles checkpoints and DNA repair complexes, are helping to understand the cancer susceptibility of A-T patients, and perhaps of A-T heterozygotes. The identification and categorization of mutations in the ATM, NBS and Mre11 genes now allow more definitive diagnoses. Developmental studies are rapidly identifying early sites of pathogenesis that can perhaps be targeted for neural stem cell implantation therapy. If over-whelmed oxidative stress responses underlie the neurological degeneration of A-T patients, maintaining adequate therapeutic levels of free-radical scavengers, such as Vitamin E, alpha-lipoic acid, aspirin, and Coenzyme Q10, may be of some therapeutic benefit.

North Carolina Macular Dystrophy Is Caused by Dysregulation of the Retinal Transcription Factor PRDM13.

PURPOSE To identify specific mutations causing North Carolina macular dystrophy (NCMD). DESIGN Whole-genome sequencing coupled with reverse transcription polymerase chain reaction (RT-PCR) analysis of gene expression in human retinal cells. PARTICIPANTS A total of 141 members of 12 families with NCMD and 261 unrelated control individuals. METHODS Genome sequencing was performed on 8 affected individuals from 3 families affected with chromosome 6-linked NCMD (MCDR1) and 2 individuals affected with chromosome 5-linked NCMD (MCDR3). Variants observed in the MCDR1 locus with frequencies <1% in published databases were confirmed using Sanger sequencing. Confirmed variants absent from all published databases were sought in 8 additional MCDR1 families and 261 controls. The RT-PCR analysis of selected genes was performed in stem cell-derived human retinal cells. MAIN OUTCOME MEASURES Co-segregation of rare genetic variants with disease phenotype. RESULTS Five sequenced individuals with MCDR1-linked NCMD shared a haplotype of 14 rare variants spanning 1 Mb of the disease-causing allele. One of these variants (V1) was absent from all published databases and all 261 controls, but was found in 5 additional NCMD kindreds. This variant lies in a DNase 1 hypersensitivity site (DHS) upstream of both the PRDM13 and CCNC genes. Sanger sequencing of 1 kb centered on V1 was performed in the remaining 4 NCMD probands, and 2 additional novel single nucleotide variants (V2 in 3 families and V3 in 1 family) were identified in the DHS within 134 bp of the location of V1. A complete duplication of the PRDM13 gene was also discovered in a single family (V4). The RT-PCR analysis of PRDM13 expression in developing retinal cells revealed marked developmental regulation. Next-generation sequencing of 2 individuals with MCDR3-linked NCMD revealed a 900-kb duplication that included the entire IRX1 gene (V5). The 5 mutations V1 to V5 segregated perfectly in the 102 affected and 39 unaffected members of the 12 NCMD families. CONCLUSIONS We identified 5 rare mutations, each capable of arresting human macular development. Four of these strongly implicate the involvement of PRDM13 in macular development, whereas the pathophysiologic mechanism of the fifth remains unknown but may involve the developmental dysregulation of IRX1.

Mitochondrial DNA haplogroups confer differences in risk for age-related macular degeneration: a case control study

BackgroundAge-related macular degeneration (AMD) is the leading cause of vision loss in elderly, Caucasian populations. There is strong evidence that mitochondrial dysfunction and oxidative stress play a role in the cell death found in AMD retinas. The purpose of this study was to examine the association of the Caucasian mitochondrial JTU haplogroup cluster with AMD. We also assessed for gender bias and additive risk with known high risk nuclear gene SNPs, ARMS2/LOC387715 (G > T; Ala69Ser, rs10490924) and CFH (T > C; Try402His, rs1061170).MethodsTotal DNA was isolated from 162 AMD subjects and 164 age-matched control subjects located in Los Angeles, California, USA. Polymerase chain reaction (PCR) and restriction enzyme digestion were used to identify the J, U, T, and H mitochondrial haplogroups and the ARMS2-rs10490924 and CFH-rs1061170 SNPs. PCR amplified products were sequenced to verify the nucleotide substitutions for the haplogroups and ARMS2 gene.ResultsThe JTU haplogroup cluster occurred in 34% (55/162) of AMD subjects versus 15% (24/164) of normal (OR = 2.99; p = 0.0001). This association was slightly greater in males (OR = 3.98, p = 0.005) than the female population (OR = 3.02, p = 0.001). Assuming a dominant effect, the risk alleles for the ARMS2 (rs10490924; p = 0.00001) and CFH (rs1061170; p = 0.027) SNPs were significantly associated with total AMD populations. We found there was no additive risk for the ARMS2 (rs10490924) or CFH (rs1061170) SNPs on the JTU haplogroup background.ConclusionsThere is a strong association of the JTU haplogroup cluster with AMD. In our Southern California population, the ARMS2 (rs10490924) and CFH (rs1061170) genes were significantly but independently associated with AMD. SNPs defining the JTU mitochondrial haplogroup cluster may change the retinal bioenergetics and play a significant role in the pathogenesis of AMD.

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.

Genomic Organization of Human DLG4, the Gene Encoding Postsynaptic Density 95

Abstract : We have determined the exon‐intron organization and characterized the 5′‐flanking promoter region of DLG4. Encompassing ~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.

Keratoconus--no association with the transforming growth factor beta-induced gene in a cohort of American patients.

Purpose Keratoconus is a noninflammatory, corneal thinning disorder leading to mixed myopic and irregular astigmatism and implicated as a major reason for cornea transplantations in the Western world. Genetic factors have been suggested as a cause of keratoconus. The levels of transforming growth factor &bgr;–induced (TGFBI) protein have been reported to be altered in keratoconus tissues. Mutations in this gene are responsible for causing various corneal dystrophies. Given this strong evidence of the involvement of this gene in corneal dystrophies, we investigated possible mutations within this gene in 15 probands of families with keratoconus. Methods All patients and control individuals had complete ophthalmological examination by a corneal specialist to determine their affectation status. The entire transcript of the TGFBI gene was analyzed by direct sequencing from patient DNA. Results We found 8 sequence variations within the gene, none of which was protein-altering changes. These changes were also observed in control individuals, and 4 are previously known polymorphisms. Conclusions We concluded that the TGFBI gene is not responsible for causing keratoconus in these 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. Bruchs 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.

CAND3: A ubiquitously expressed gene immediately adjacent and in opposite transcriptional orientation to the ATM gene at 1lq23.1

Using a magnetic beads-mediated cDNA selection procedure and a fetal brain expression library, we identified a transcriptional unit within a cosmid positive for the marker D11S384. Pursuit of its full-length cDNA led to the cloning of the third candidate gene (CAND3) we studied in our quest for the ataxiatelangiectasia (A-T) gene, ATM. CAND3 spans ~140 kb of genomic DNA and is located immediately centrimeric to ATM, with 544 bp of DNA separating the two genes. CAND3 encodes two ubiquitously expressed transcripts of ~5.8 kb and ~4.6 kb that are divergently transcribed from a promoter region common to ATM. Nucleotide sequence was determined for one of its alternately spliced transcripts. The predicted protein has 1175 amino acids and is novel in sequence, with only weak homologies to transcriptional factors, nucleoporin protein, and protein kinases, including members of the phosphatidylinositol 3-kinase (PI-3 kinase) family. Although neither homology to ATM nor any mutation of CAND3 in A-T patients has been found, the head-to-head arrangement of CAND3 and ATM, with expression of both housekeeping genes from a common stretch of 544 bp intergenic DNA, suggests a bi-directional promoter possibly for co-regulation of biologically related functions. YACs, BACs, cosmids, and STSs are defined to aid in the further study of this gene.