Genotype/Phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease.

Mutation scanning and direct DNA sequencing of all 50 exons of ABCR were completed for 150 families segregating recessive Stargardt disease (STGD1). ABCR variations were identified in 173 (57%) disease chromosomes, the majority of which represent missense amino acid substitutions. These ABCR variants were not found in 220 unaffected control individuals (440 chromosomes) but do cosegregate with the disease in these families with STGD1, and many occur in conserved functional domains. Missense amino acid substitutions located in the amino terminal one-third of the protein appear to be associated with earlier onset of the disease and may represent misfolding alleles. The two most common mutant alleles, G1961E and A1038V, each identified in 16 of 173 disease chromosomes, composed 18.5% of mutations identified. G1961E has been associated previously, at a statistically significant level in the heterozygous state, with age-related macular degeneration (AMD). Clinical evaluation of these 150 families with STGD1 revealed a high frequency of AMD in first- and second-degree relatives. These findings support the hypothesis that compound heterozygous ABCR mutations are responsible for STGD1 and that some heterozygous ABCR mutations may enhance susceptibility to AMD.     

Evaluation of the ELOVL4 gene in a Chinese family with autosomal dominant STGD3-like macular dystrophy

Stargardt disease-3 (STGD3) is an autosomal dominant juvenile-onset macular dystrophy characterized by progressive decreasing visual acuity, bilateral atrophic changes in the macula and absence of characteristic dark choroids. We identified a STGD3-like macular dystrophy pedigree by clinical examination. To explore whether the STGD3-like phenotype in the kindred is linked to ELOVL4 gene or associated with any other identified STGD gene, we extracted genomic DNA from leukocytes of peripheral blood from the available family members and 50 normal controls for mutation analysis. Then the exons of ELOVL4, RDS and the three exons of ABCR were amplified by polymerase chain reaction (PCR). All PCR products were screened for mutations by combination of denaturing high-performance liquid chromatography (DHPLC) analysis and DNA sequencing. No mutation was found in the exons of three candidate genes, but we obtained three non-pathogenic polymorphisms, IVS5–2533T A in ELOVL4, 558C T (Val106Val) and 1150G C (Glu304Gln) in RDS. And IVS5–2533T A is never shown in the previous references. These data suggested that there exist other unknown genes responsible for the STGD3-like phenotype in the pedigree.     

A new locus for autosomal dominant stargardt-like disease maps to chromosome 4

Stargardt disease (STGD) is the most common hereditary macular dystrophy and is characterized by decreased central vision, atrophy of the macula and underlying retinal-pigment epithelium, and frequent presence of prominent flecks in the posterior pole of the retina. STGD is most commonly inherited as an autosomal recessive trait, but many families have been described in which features of the disease are transmitted in an autosomal dominant manner. A recessive locus has been identified on chromosome 1p (STGD1), and dominant loci have been mapped to both chromosome 13q (STGD2) and chromosome 6q (STGD3). In this study, we describe a kindred with an autosomal dominant Stargardt-like phenotype. A genomewide search demonstrated linkage to a locus on chromosome 4p, with a maximum LOD score of 5.12 at a recombination fraction of.00, for marker D4S403. Analysis of extended haplotypes localized the disease gene to an approximately 12-cM interval between loci D4S1582 and D4S2397. Therefore, this kindred establishes a new dominant Stargardt-like locus, STGD4.     

The 2588G-->C mutation in the ABCR gene is a mild frequent founder mutation in the Western European population and allows the classification of ABCR mutations in patients with Stargardt disease

In 40 western European patients with Stargardt disease (STGD), we found 19 novel mutations in the retina-specific ATP-binding cassette transporter (ABCR) gene, illustrating STGD's high allelic heterogeneity. One mutation, 2588G-->C, identified in 15 (37.5%) patients, shows linkage disequilibrium with a rare polymorphism (2828G-->A) in exon 19, suggesting a founder effect. The guanine at position 2588 is part of the 3' splice site of exon 17. Analysis of the lymphoblastoid cell mRNA of two STGD patients with the 2588G-->C mutation shows that the resulting mutant ABCR proteins either lack Gly863 or contain the missense mutation Gly863Ala. We hypothesize that the 2588G-->C alteration is a mild mutation that causes STGD only in combination with a severe ABCR mutation. This is supported in that the accompanying ABCR mutations in at least five of eight STGD patients are null (severe) and that a combination of two mild mutations has not been observed among 68 STGD patients. The 2588G-->C mutation is present in 1 of every 35 western Europeans, a rate higher than that of the most frequent severe autosomal recessive mutation, the cystic fibrosis conductance regulator gene mutation DeltaPhe508. Given an STGD incidence of 1/10,000, homozygosity for the 2588G-->C mutation or compound heterozygosity for this and other mild ABCR mutations probably does not result in an STGD phenotype.     

Further evidence for an association of ABCR alleles with age-related macular degeneration. The International ABCR Screening Consortium

Age-related macular degeneration (AMD) accounts for >50% of the registered visual disability among North American and Western European populations and has been associated both with environmental factors, such as smoking, and with genetic factors. Previously we have reported disease-associated variants in the ABCR (also called ABCA4) gene in a subset of patients affected with this complex disorder. We have now tested our original hypothesis, that ABCR is a dominant susceptibility locus for AMD, by screening 1,218 unrelated AMD patients of North American and Western European origin and 1,258 comparison individuals from 15 centers in North America and Europe for the two most frequent AMD-associated variants found in ABCR. These two sequence changes, G1961E and D2177N, were found in one allele of ABCR in 40 patients ( approximately 3.4%), and in 13 control subjects ( approximately 0.95%). Fisher's two-sided exact test confirmed that these two variants are associated with AMD at a statistically significant level (P<.0001). The risk of AMD is elevated approximately threefold in D2177N carriers and approximately fivefold in G1961E carriers. The identification of a gene that confers risk of AMD is an important step in unraveling this complex disorder.     

TIMP3 mutation in Sorsby's fundus dystrophy: molecular insights

Sorsby's fundus dystrophy (SFD) is a rare autosomal dominant disorder that results in degeneration of the macular region of the retina, with onset usually in the fourth to fifth decade of life. It leads to the rapid loss of central vision, often followed by further loss of peripheral vision. SFD shares several pathological features commonly found in the 'wet' or exudative form of age-related macular degeneration (AMD), the most common cause of blindness in the elderly in developed countries. These phenotypic similarities have led to SFD being proposed as an acceptable genetic model for AMD. Whereas AMD appears to have a complex aetiology, with both genetic and environmental factors playing a role, SFD has been shown to be a single-gene disorder, linked to mutations in exon 5 of the tissue inhibitor of metalloproteinases 3 (TIMP3) gene on chromosome 22q12-q13. This review confines itself to a discussion of the known biochemical properties of the wild-type and SFD TIMP3 proteins and attempts to relate these to the pathology encountered in SFD patients. We also discuss briefly how, despite the lack of inherited mutations in the structural gene, the TIMP3 protein might play a role in the onset and progression of AMD.     

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. Received: 14 February 1996 / Revised: 25 April 1996     

Mutational scanning of the ABCR gene with double-gradient denaturing-gradient gel electrophoresis (DG-DGGE) in Italian Stargardt disease patients

Mutations in the retina-specific ABC transporter (ABCR) gene are responsible for autosomal recessive Stargardt disease (arSTGD). Mutation detection efficiency in ABCR in arSTGD patients ranges between 30% and 66% in previously published studies, because of high allelic heterogeneity and technical limitations of the employed methods. Conditions were developed to screen the ABCR gene by double-gradient denaturing-gradient gel electrophoresis. The efficacy of this method was evaluated by analysis of DNA samples with previously characterized ABCR mutations. This approach was applied to mutation detection in 44 Italian arSTGD patients corresponding to 36 independent genomes, in order to assess the nature and frequency of the ABCR mutations in this ethnic group. In 34 of 36 (94.4%) STGD patients, 37 sequence changes were identified, including 26 missense, six frameshift, three splicing, and two nonsense variations. Among these, 20 had not been previously described. Several polymorphisms were detected in affected individuals and in matched controls. Our findings extend the spectrum of mutations identified in STGD patients and suggest the existence of a subset of molecular defects specific to the Italian population. The identification of at least two disease-associated mutations in four healthy control individuals indicates a higher than expected carrier frequency of variant ABCR alleles in the general population. Genotype-phenotype analysis in our series showed a possible correlation between the nature and location of some mutations and specific ophthalmoscopic features of STGD disease.     

North Carolina macular dystrophy is assigned to chromosome 6.

North Carolina macular dystrophy (NCMD) is an autosomal dominant macular dystrophy causing impaired central vision at an early age, is completely penetrant, and is present in a single large family. With the development of the hypervariable microsatellite (CA repeats) markers in the human genome, it was possible to relatively rapidly screen most of the genome for linkage to the NCMD gene. After utilizing 124 genetic markers, which excluded over 95% of the human genome, three Marshfield microsatellites located at 6q13-q21 were linked to the NCMD locus. Marshfield marker (MFD) 131 gave a lod score of Z(theta) = 4.36 at theta = 0.137; MFD 171 gave a Z(theta) = 8.42 at theta = 0.004; and MFD 97 gave a Z(theta) = 13.10 at theta = 0.017. Other retinal diseases have been reported on 6q stressing the importance of this region and possibly suggesting that these diseases may be allelic or located in part of a large macular gene family. Locating and characterizing the NCMD gene may be an important step in understanding this group of maculopathies as well as age-related macular degeneration (AMD), a common cause of blindness in the elderly.     

Evidence for a major gene (RP10) for autosomal dominant retinitis pigmentosa on chromosome 7q: linkage mapping in a second,unrelated family

Retinitis pigmentosa is a genetically heterogeneous form of retinal degeneration, which has X-linked, autosomal recessive and autosomal dominant forms. The disease genes in families with autosomal dominant retinitis pigmentosa (adRP) have been linked to six loci, on 3q, 6p, 7p, 7q, 8q and 19q. In a large American family with late-onset adRP, microsatellite markers were used to test for linkage to the loci on 3q, 6p, 7p, 7q and 8q. Linkage was found to 7q using the marker D7S480. Additional microsatellite markers from 7q were then tested. In total, five markers, D7S480, D7S514, D7S633, D7S650 and D7S677, show statistically significant evidence for link-age in this family, with a maximum two-point lod score of 5.3 at 0% recombination from D7S514. These results confirm an earlier report of linkage to an adRP locus (RP10) in an unrelated family of Spanish origin and indicate that RP10 may be a significant gene for inherited retinal degeneration. In addition, we used recently reported microsatellite markers from 7q to refine the linkage map of the RP10 locus.     

A comprehensive survey of sequence variation in the ABCA4 (ABCR) gene in Stargardt disease and age-related macular degeneration

Stargardt disease (STGD) is a common autosomal recessive maculopathy of early and young-adult onset and is caused by alterations in the gene encoding the photoreceptor-specific ATP-binding cassette (ABC) transporter (ABCA4). We have studied 144 patients with STGD and 220 unaffected individuals ascertained from the German population, to complete a comprehensive, population-specific survey of the sequence variation in the ABCA4 gene. In addition, we have assessed the proposed role for ABCA4 in age-related macular degeneration (AMD), a common cause of late-onset blindness, by studying 200 affected individuals with late-stage disease. Using a screening strategy based primarily on denaturing gradient gel electrophoresis, we have identified in the three study groups a total of 127 unique alterations, of which 90 have not been previously reported, and have classified 72 as probable pathogenic mutations. Of the 288 STGD chromosomes studied, mutations were identified in 166, resulting in a detection rate of approximately 58%. Eight different alleles account for 61% of the identified disease alleles, and at least one of these, the L541P-A1038V complex allele, appears to be a founder mutation in the German population. When the group with AMD and the control group were analyzed with the same methodology, 18 patients with AMD and 12 controls were found to harbor possible disease-associated alterations. This represents no significant difference between the two groups; however, for detection of modest effects of rare alleles in complex diseases, the analysis of larger cohorts of patients may be required.     

Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy

The photoreceptor cell-specific ATP-binding cassette transporter gene (ABCA4; previously denoted "ABCR") is mutated, in most patients, with autosomal recessive (AR) Stargardt disease (STGD1) or fundus flavimaculatus (FFM). In addition, a few cases with AR retinitis pigmentosa (RP) and AR cone-rod dystrophy (CRD) have been found to have ABCA4 mutations. To evaluate the importance of the ABCA4 gene as a cause of AR CRD, we selected 5 patients with AR CRD and 15 patients from Germany and The Netherlands with isolated CRD. Single-strand conformation-polymorphism analysis and sequencing revealed 19 ABCA4 mutations in 13 (65%) of 20 patients. In six patients, mutations were identified in both ABCA4 alleles; in seven patients, mutations were detected in one allele. One complex ABCA4 allele (L541P;A1038V) was found exclusively in German patients with CRD; one patient carried this complex allele homozygously, and five others were compound heterozygous. These findings suggest that mutations in the ABCA4 gene are the major cause of AR CRD. A primary role of the ABCA4 gene in STGD1/FFM and AR CRD, together with the gene's involvement in an as-yet-unknown proportion of cases with AR RP, strengthens the idea that mutations in the ABCA4 gene could be the most frequent cause of inherited retinal dystrophy in humans.     

Altersabhängige Makuladegeneration

Age-related macular degeneration (AMD) is a complex disorder of the central retina with readily increasing socio-economic impact on the societies of industrialized countries. With a prevalence of about 12% in the population over 80 years of age, advanced forms of AMD are nowadays the most common cause of blindness in the elderly. The risk of developing AMD is influenced by exogenous and endogenous factors. Although smoking is a well-established environmental component, the first clues to genetic influences resulted from twin studies and familial aggregation analyses. Recent work has identified genetic variants in two genomic regions at 1q32 and 10q26, which independently confer high risks for developing AMD. While association signals at 1q32 were shown to culminate over the complement factor H (CFH) gene, implicating innate immunity and inflammation in the etiology of AMD, the functional contribution of the LOC387715/HTRA1 (HtrA serine peptidase 1) gene locus still remains to be elucidated. In coming years it is to be expected, however, that our knowledge of the genetic factors and their cellular roles in the retina will further deepen and therefore will fundamentally change patient management.     

Mechanistic Studies of ABCR, the ABC Transporter in Photoreceptor Outer Segments Responsible for Autosomal Recessive Stargardt Disease

ABCR is an ABC transporter that is found exclusively in vertebrate photoreceptor outer segments. Mutations in the human ABCR gene are responsible for autosomal recessive Stargardt disease, the most common cause of early onset macular degeneration. In this paper we review our recent work with purified and reconstituted ABCR derived from bovine retina and from cultured cells expressing wild type or site-directed mutants of human ABCR. These experiments implicate all-trans-retinal (or Schiff base adducts between all-trans-retinal and phosphatidylethanolamine) as the transport substrate, and they reveal asymmetric roles for the two nucleotide binding domains in the transport reaction. A model for the retinal transport reaction is presented which accounts for these experimental observations.     

Molecular Diagnosis of Putative Stargardt Disease by Capture Next Generation Sequencing

Stargardt Disease (STGD) is the commonest genetic form of juvenile or early adult onset macular degeneration, which is a genetically heterogeneous disease. Molecular diagnosis of STGD remains a challenge in a significant proportion of cases. To address this, seven patients from five putative STGD families were recruited. We performed capture next generation sequencing (CNGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Seven disease-causing mutations in ABCA4 and two in PROM1 were identified by CNGS, which provides a confident genetic diagnosis in these five families. We also provided a genetic basis to explain the differences among putative STGD due to various mutations in different genes. Meanwhile, we show for the first time that compound heterozygous mutations in PROM1 gene could cause cone-rod dystrophy. Our findings support the enormous potential of CNGS in putative STGD molecular diagnosis.     

Autosomal Recessive Transmission of MYBPC3 Mutation Results in Malignant Phenotype of Hypertrophic Cardiomyopathy

Background

Hypertrophic cardiomyopathy (HCM) due to mutations in genes encoding sarcomere proteins is most commonly inherited as an autosomal dominant trait. Since nearly 50% of HCM cases occur in the absence of a family history, a recessive inheritance pattern may be involved.

Methods

A pedigree was identified with suspected autosomal recessive transmission of HCM. Twenty-six HCM-related genes were comprehensively screened for mutations in the proband with targeted second generation sequencing, and the identified mutation was confirmed with bi-directional Sanger sequencing in all family members and 376 healthy controls.

Results

A novel missense mutation (c.1469G>T, p.Gly490Val) in exon 17 of MYBPC3 was identified. Two siblings with HCM were homozygous for this mutation, whereas other family members were either heterozygous or wild type. Clinical evaluation showed that both homozygotes manifested a typical HCM presentation, but none of others, including 5 adult heterozygous mutation carriers up to 71 years of age, had any clinical evidence of HCM.

Conclusions

Our data identified a MYBPC3 mutation in HCM, which appeared autosomal recessively inherited in this family. The absence of a family history of clinical HCM may be due to not only a de novo mutation, but also recessive mutations that failed to produce a clinical phenotype in heterozygous family members. Therefore, consideration of recessive mutations leading to HCM is essential for risk stratification and genetic counseling.     

A locus for autosomal dominant colobomatous microphthalmia maps to chromosome 15q12-q15

Congenital microphthalmia is a common developmental ocular disorder characterized by shortened axial length. Isolated microphthalmia is clinically and genetically heterogeneous and may be inherited in an autosomal dominant, autosomal recessive, or X-linked manner. Here, we studied a five-generation family of Sephardic Jewish origin that included 38 members, of whom 7 have either unilateral or bilateral microphthalmia of variable severity inherited as an autosomal dominant trait with incomplete penetrance. After exclusion of several candidate loci, we performed a genome-scan study and demonstrated linkage to chromosome 15q12-q15. Positive LOD scores were obtained with a maximum at the D15S1007 locus (maximum LOD score 3.77, at recombination fraction 0.00). Haplotype analyses supported the location of the disease-causing gene in a 13.8-cM interval between loci D15S1002 and D15S1040.     

A novel mutation in <Emphasis Type="Italic">RDH5</Emphasis> gene causes retinitis pigmentosa in consanguineous Pakistani family

Retinitis pigmentosa (RP) is the most frequent genetically and clinically heterogeneous inherited retinal degeneration. To date, more than 80 genes have been identified that cause autosomal dominant, autosomal recessive and X linked RP. However, locus and allelic heterogeneity of RP has not been fully captured yet. This heterogeneity and lack of an accurate genotype phenotype correlation makes molecular dissection of the disease more difficult. The present study was designed to characterize the underlying pathogenic variants of RP in Pakistan. For this purpose, a large consanguineous family with RP phenotype showing autosomal recessive mode of inheritance was selected after a complete ophthalmological examination. Next generation sequencing was used for the identification of molecular determinant followed by Sanger-sequencing for confirmation. After sequence analysis a novel homozygous missense mutation, (c.602 C?>?T) in exon 4 of the RDH5 gene (MIM: 601617) was identified. This mutation resulted in substitution of phenyl alanine for serine at amino acid 201 (p.Ser201Phe) of the RDH5 gene. The same mutation was not detected in the 200 ethnically-matched control samples by Sanger sequencing. The identified mutant allele segregated in homozygous fashion in all the affected individuals of pedigree. Identification of this mutation reveals the allelic heterogeneity of RDH5 in patients with RP phenotype. The findings of this study demonstrate the clinical significance of next generation sequencing to understand the molecular basis of diseases and would help to reveal new proteins and their function in visual cycle will pave the way for early diagnosis, genetic counseling and better therapeutic inventions.     

Autosomal recessive juvenile parkinsonism maps to 6q25.2-q27 in four ethnic groups: detailed genetic mapping of the linked region.

Parkinson disease (PD) is a common neurodegenerative condition associated with degeneration of dopaminergic neurons in the zona compacta of the substantia nigra. There is increasing evidence that genetic factors play a role in the etiology of PD, although genetic heterogeneity is likely. An autosomal dominant syndrome with many similarities to sporadic PD has been mapped to 4q21-22 in a large Italian pedigree and has been found to be due to mutation of the alpha-synuclein gene. However, this gene appears to account for only a minority of PD, and a susceptibility locus for autosomal dominant parkinsonism has recently been mapped, on 2p13. Autosomal recessive juvenile parkinsonism (JP), which shows marked clinical similarity to PD, maps to 6q25.2-q27. We found linkage to this region in a group of 15 families from four distinct ethnic backgrounds. A full genomic screen excluded other candidate regions. We have constructed a detailed genetic map of the linked region and have mapped the position of the manganese superoxide dismutase gene (SOD2). Recombination events restricted the JP locus to a 6.9-cM region and excluded SOD2. The apparent homozygosity for null alleles at D6S955 in one family suggested a deletion and finer localization of the JP locus.