Michael B. Gorin

Susceptibility Genes for Age-Related Maculopathy (ARM) on Chromosome 10q26

On the basis of genomewide linkage studies of families affected with age-related maculopathy (ARM), we previously identified a significant linkage peak on 10q26, which has been independently replicated by several groups. We performed a focused SNP genotyping study of our families and an additional control cohort. We identified a strong association signal overlying three genes, PLEKHA1, LOC387715, and PRSS11. All nonsynonymous SNPs in this critical region were genotyped, yielding a highly significant association (P < .00001) between PLEKHA1/LOC387715 and ARM. Although it is difficult to determine statistically which of these two genes is most important, SNPs in PLEKHA1 are more likely to account for the linkage signal in this region than are SNPs in LOC387715; thus, this gene and its alleles are implicated as an important risk factor for ARM. We also found weaker evidence supporting the possible involvement of the GRK5/RGS10 locus in ARM. These associations appear to be independent of the association of ARM with the Y402H allele of complement factor H, which has previously been reported as a major susceptibility factor for ARM. The combination of our analyses strongly implicates PLEKHA1/LOC387715 as primarily responsible for the evidence of linkage of ARM to the 10q26 locus and as a major contributor to ARM susceptibility. The association of either a single or a double copy of the high-risk allele within the PLEKHA1/LOC387715 locus accounts for an odds ratio of 5.0 (95% confidence interval 3.2-7.9) for ARM and a population attributable risk as high as 57%.

The major intrinsic protein (MIP) of the bovine lens fiber membrane: Characterization and structure based on cDNA cloning

Synthetic oligonucleotide probes have been used to identify two overlapping cDNA clones that represent the entire coding region of the mRNA for the major intrinsic protein (MIP) of bovine lens cell membrane. Hybridization studies indicate that bovine MIP is encoded by a single-copy gene. The cDNA hybridizes to the rat genome, but MIP mRNA is not detected in rat liver. Analysis of the deduced amino acid sequence provides support for the potential role of MIP as a junctional protein. The structure predicted for MIP suggests that it traverses the lipid bilayer six times with both carboxy and amino termini on the cytoplasmic side, and that it has at least one amphiphilic transmembrane segment, as expected if the protein were to participate in the formation of an aqueous channel.

Genetic variants near TIMP3 and high-density lipoprotein–associated loci influence susceptibility to age-related macular degeneration

We executed a genome-wide association scan for age-related macular degeneration (AMD) in 2,157 cases and 1,150 controls. Our results validate AMD susceptibility loci near CFH (P < 10−75), ARMS2 (P < 10−59), C2/CFB (P < 10−20), C3 (P < 10−9), and CFI (P < 10−6). We compared our top findings with the Tufts/Massachusetts General Hospital genome-wide association study of advanced AMD (821 cases, 1,709 controls) and genotyped 30 promising markers in additional individuals (up to 7,749 cases and 4,625 controls). With these data, we identified a susceptibility locus near TIMP3 (overall P = 1.1 × 10−11), a metalloproteinase involved in degradation of the extracellular matrix and previously implicated in early-onset maculopathy. In addition, our data revealed strong association signals with alleles at two loci (LIPC, P = 1.3 × 10−7; CETP, P = 7.4 × 10−7) that were previously associated with high-density lipoprotein cholesterol (HDL-c) levels in blood. Consistent with the hypothesis that HDL metabolism is associated with AMD pathogenesis, we also observed association with AMD of HDL-c—associated alleles near LPL (P = 3.0 × 10−3) and ABCA1 (P = 5.6 × 10−4). Multilocus analysis including all susceptibility loci showed that 329 of 331 individuals (99%) with the highest-risk genotypes were cases, and 85% of these had advanced AMD. Our studies extend the catalog of AMD associated loci, help identify individuals at high risk of disease, and provide clues about underlying cellular pathways that should eventually lead to new therapies.

Interpretation of Genetic Association Studies: Markers with Replicated Highly Significant Odds Ratios May Be Poor Classifiers

Recent successful discoveries of potentially causal single nucleotide polymorphisms (SNPs) for complex diseases hold great promise, and commercialization of genomics in personalized medicine has already begun. The hope is that genetic testing will benefit patients and their families, and encourage positive lifestyle changes and guide clinical decisions. However, for many complex diseases, it is arguable whether the era of genomics in personalized medicine is here yet. We focus on the clinical validity of genetic testing with an emphasis on two popular statistical methods for evaluating markers. The two methods, logistic regression and receiver operating characteristic (ROC) curve analysis, are applied to our age-related macular degeneration dataset. By using an additive model of the CFH, LOC387715, and C2 variants, the odds ratios are 2.9, 3.4, and 0.4, with p-values of 10−13, 10−13, and 10−3, respectively. The area under the ROC curve (AUC) is 0.79, but assuming prevalences of 15%, 5.5%, and 1.5% (which are realistic for age groups 80 y, 65 y, and 40 y and older, respectively), only 30%, 12%, and 3% of the group classified as high risk are cases. Additionally, we present examples for four other diseases for which strongly associated variants have been discovered. In type 2 diabetes, our classification model of 12 SNPs has an AUC of only 0.64, and two SNPs achieve an AUC of only 0.56 for prostate cancer. Nine SNPs were not sufficient to improve the discrimination power over that of nongenetic predictors for risk of cardiovascular events. Finally, in Crohns disease, a model of five SNPs, one with a quite low odds ratio of 0.26, has an AUC of only 0.66. Our analyses and examples show that strong association, although very valuable for establishing etiological hypotheses, does not guarantee effective discrimination between cases and controls. The scientific community should be cautious to avoid overstating the value of association findings in terms of personalized medicine before their time.

Age-related maculopathy: a genomewide scan with continued evidence of susceptibility loci within the 1q31, 10q26, and 17q25 regions.

Age-related maculopathy (ARM), or age-related macular degeneration, is one of the most common causes of visual impairment in the elderly population of developed nations. In a combined analysis of two previous genomewide scans that included 391 families, containing up to 452 affected sib pairs, we found linkage evidence in four regions: 1q31, 9p13, 10q26, and 17q25. We now have added a third set of families and have performed an integrated analysis incorporating 530 families and up to 736 affected sib pairs. Under three diagnostic models, we have conducted linkage analyses using parametric (heterogeneity LOD [HLOD] scores under an autosomal dominant model) and nonparametric (Sall statistic) methods. There is ongoing evidence of susceptibility loci within the 1q31, 10q26, and 17q25 regions. If we treat the third set of families as a replication set, then two regions (10q26 and 17q25) are replicated, with LOD scores >1.0. If we pool all our data together, then four regions (1q31, 2q14.3, 10q26, and 17q25) show HLOD or Sall scores > or =2.0. Within the 1q31 region, we observed an HLOD of 2.72 (genomewide P=.061) under our least stringent diagnostic model, whereas the 17q25 region contained a maximal HLOD of 3.53 (genomewide P=.007) under our intermediate diagnostic model. We have evaluated our results with respect to the findings from several new independent genomewide linkage studies and also have completed ordered subset analyses (OSAs) with apolipoprotein E alleles, smoking history, and age at onset as stratifying covariates. The OSAs generate the interesting hypothesis that the effect of smoking on the risk of ARM is accentuated by a gene in the 10q26 region--a region implicated by four other studies.

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.

Central nervous system involvement in Von Hippel‐Lindau disease

Fifty individuals with Von Hippel-Lindau disease (VHL) were studied with gadolinium-enhanced magnetic resonance imaging (MRI) to determine the frequency and distribution of CNS lesions. The associated clinical features were also reviewed. Thirty-six (72%) of the 50 had 1 or more CNS tumors. The most frequently affected sites in the CNS excluding the retina were the cerebellum (52%), spinal cord (44%), and brainstem (18%). New regional predilections for the craniocervical junction and conus medullaris were demonstrated by this study. Forty-one percent of all VHL patients with CNS tumors were neurologically asymptomatic: cerebellar tumors (50%), spinal cord tumors (50%), and brainstem tumors (44%) were often without clinical signs or symptoms. Multiple lesions were common. The mean age of all VHL patients (34.5 years) was similar to the mean age of all CNS VHL patients (34.4 years), suggesting a lack of age association. CNS lesions commonly occurred in the 2nd decade of life. All patients at risk for VHL, should be evaluated using gadolinium-enhanced MRI after 10 years of age, although ophthalmic examination should be initiated within the 1st 2 years of life. Enhanced MRI is particularly useful in the detection of CNS tumors in patients with the VHL gene.

Age-related maculopathy: an expanded genome-wide scan with evidence of susceptibility loci within the 1q31 and 17q25 regions.

PURPOSE We seek to identify genetic loci that contribute to age-related maculopathy susceptibility. METHODS Families consisting of at least two siblings affected by age-related maculopathy were ascertained using eye care records and fundus photographs. Additional family members were used to increase the power to detect linkage. Microsatellite genotyping was conducted by the National Heart, Lung and Blood Institute Mammalian Genotyping Service and the National Institutes of Health Center for Inherited Disease Research. Linkage analyses were conducted with parametric (autosomal dominant; heterogeneity lod score) and nonparametric methods (S(all) statistic) using three diagnostic models. False-positive rates were determined from simulations using actual pedigrees and genotyping data. RESULTS Under our least stringent diagnostic model, model C, 860 affected individuals from 391 families (452 sib pairs) were genotyped. Sixty-five percent of the affected individuals had evidence of exudative disease. Four regions, 1q31, 9p13, 10q26, and 17q25, showed multipoint heterogeneity lod scores or S(all) scores of 2.0 or greater (under at least one model). Under our most stringent diagnostic model, model A, the 1q31 heterogeneity lod score was 2.46 between D1S1660 and D1S1647. Under model C, the 17q25 heterogeneity lod score at D17S928 was 3.16. Using a threshold of 1.5, additional loci on chromosomes 2 and 12 were identified. CONCLUSIONS The locus on chromosome 1q31 independently confirms a report by Klein and associates mapping an age-related maculopathy susceptibility gene to this region. Simulations indicate that the 1q31 and 17q25 loci are unlikely to be false positives. There was no evidence that other known macular or retinal dystrophy candidate gene regions are major contributors to the genetics of age-related maculopathy.

A Juvenile-Onset, Progressive Cataract Locus on Chromosome 3q21-q22 Is Associated with a Missense Mutation in the Beaded Filament Structural Protein–2

Juvenile-onset cataracts are distinguished from congenital cataracts by the initial clarity of the lens at birth and the gradual development of lens opacity in the second and third decades of life. Genomewide linkage analysis in a multigenerational pedigree, segregating for autosomal dominant juvenile-onset cataracts, identified a locus in chromosome region 3q21.2-q22.3. Because of the proximity of the gene coding for lens beaded filament structural protein-2 (BFSP2) to this locus, we screened for mutations in the coding sequence of BFSP2. We observed a unique C-->T transition, one that was not observed in 200 normal chromosomes. We predicted that this led to a nonconservative R287W substitution in exon 4 that cosegregated with cataracts. This mutation alters an evolutionarily conserved arginine residue in the central rod domain of the intermediate filament. On consideration of the proposed function of BFSP2 in the lens cytoskeleton, it is likely that this alteration is the cause of cataracts in the members of the family we studied. This is the first example of a mutation in a noncrystallin structural gene that leads to a juvenile-onset, progressive cataract.

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.