exomeSuite: Whole exome sequence variant filtering tool for rapid identification of putative disease causing SNVs/indels.

Exome and whole-genome analyses powered by next-generation sequencing (NGS) have become invaluable tools in identifying causal mutations responsible for Mendelian disorders. Given that individual exomes contain several thousand single nucleotide variants and insertions/deletions, it remains a challenge to analyze large numbers of variants from multiple exomes to identify causal alleles associated with inherited conditions. To this end, we have developed user-friendly software that analyzes variant calls from multiple individuals to facilitate identification of causal mutations. The software, termed exomeSuite, filters for putative causative variants of monogenic diseases inherited in one of three forms: dominant, recessive caused by a homozygous variant, or recessive caused by two compound heterozygous variants. In addition, exomeSuite can perform homozygosity mapping and analyze the variant data of multiple unrelated individuals. Here we demonstrate that filtering of variants with exomeSuite reduces datasets to a fraction of a percent of their original size. To the best of our knowledge this is the first freely available software developed to analyze variant data from multiple individuals that rapidly assimilates and filters large data sets based on pattern of inheritance.

eyeGENE®: a vision community resource facilitating patient care and paving the path for research through molecular diagnostic testing.

Molecular genetics and genomics are revolutionizing the study and treatment of inherited eye diseases. In recognition of the impact of molecular genetics on vision and ophthalmology, the National Eye Institute established the National Ophthalmic Disease Genotyping and Phenotyping Network (eyeGENE®) as a multidirectional research initiative whereby a clinical component for patients diagnosed with inherited eye disease fosters research into the causes and mechanisms of these ophthalmic diseases. This is accomplished by broadening access to genetic diagnostic testing and maintaining a repository of DNA samples from clinically characterized individuals and their families to allow investigations of the causes, interventions, and management of genetic eye disorders. The eyeGENE® Network currently includes Clinical Laboratory Improvement Amendments (CLIA)-certified diagnostic laboratory partners, over 270 registered clinical organizations with 500 registered users from around the United States and Canada, and is now testing approximately 100 genes representing 35 inherited eye diseases. To date, the Network has received 4400 samples from individuals with rare inherited eye diseases, which are available for access by the vision research community. eyeGENE® is a model partnership between the U.S. federal government, eye health care providers, CLIA-approved molecular diagnostic laboratories, private industry, and scientists who represent a broad research constituency.

Identification and characterization of C6orf37, a novel candidate human retinal disease gene on chromosome 6q14.

We have identified a novel human gene, chromosome 6 open reading frame 37 (C6orf37), that is expressed in the retina and maps to human chromosome 6q14, a genomic region that harbors multiple retinal disease loci. The cDNA sequence contains an open reading frame of 1314 bp that encodes a 437-amino acid protein with a predicted molecular mass of 49.2 kDa. Northern blot analysis indicates that this gene is widely expressed, with preferential expression observed in the retina compared to other ocular tissues. The C6orf37 protein shares homology with putative proteins in R. norvegicus, M. musculus, D. melanogaster, and C. elegans, suggesting evolutionary conservation of function. Additional sequence analysis predicts that the C6orf37 gene product is a soluble, globular cytoplasmic protein containing several conserved phosphorylation sites. Furthermore, we have defined the genomic structure of this gene, which will enable its analysis as a candidate gene for chromosome 6q-associated inherited retinal disorders.

A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy.

Stargardt-like macular dystrophy (STGD3, MIM 600110) and autosomal dominant macular dystrophy (adMD) are inherited forms of macular degeneration characterized by decreased visual acuity, macular atrophy and extensive fundus flecks. Genetic mapping data suggest that mutations in a single gene may be responsible for both conditions, already known to bear clinical resemblance. Here we limit the minimum genetic region for STGD3 and adMD to a 0.6-cM interval by recombination breakpoint mapping and identify a single 5-bp deletion within the protein-coding region of a new retinal photoreceptor-specific gene, ELOVL4, in all affected members of STGD3 and adMD families. Bioinformatic analysis of ELOVL4 revealed that it has homology to a group of yeast proteins that function in the biosynthesis of very long chain fatty acids. Our results are therefore the first to implicate the biosynthesis of fatty acids in the pathogenesis of inherited macular degeneration.

Evaluation of the ELOVL4 gene in patients with age-related macular degeneration.

Stargardt-like macular degeneration (STGD(3)) and autosomal dominant macular degeneration (adMD) share phenotypic characters with atrophic age-related macular degeneration (AMD). Mutations in a photoreceptor cell-specific factor involved in the elongation of very long chain fatty acids (ELOVL(4)) were shown to be associated with STGD(3), adMD, and pattern dystrophy. We screened 778 patients with AMD and 551 age-matched controls to define the role of sequence variants in the ELOVL(4) gene in age-related macular degeneration. We detected three sequence variants in the non-coding region and eight variants in the coding region. No statistically significant association was observed between sequence variants in the ELOVL(4) gene and susceptibility to AMD. However, for the detection of modest effects of multiple alleles in a complex disease, the analysis of larger cohorts of patients may be required.

Autosomal dominant Stargardt-like macular dystrophy segregating in a large Canadian family.

BACKGROUND: Inherited macular dystrophies account for a major fraction of the cases of retinal degenerative disease that lead to permanent blindness. We describe the clinical and genetic findings in a Canadian family with a form of macular dystrophy resembling autosomal dominant Stargardt-like macular dystrophy. METHODS: Standard ophthalmologic examinations were performed in members of a single five-generation Alberta family. Tests of visual acuity and colour vision, fundus photography, fluorescein angiography and electroretinography were performed in 15 affected people. Blood was collected from 24 family members, and DNA was extracted for genotyping. Genetic linkage analysis was performed using polymorphic short tandem repeat microsatellite markers located on chromosome 6q, a region containing loci for several macular disorders. RESULTS: Affected family members display clinical characteristics resembling autosomal dominant Stargardt-like macular dystrophy, previously assigned to chromosome 6q (STGD3). Linkage analysis generated a peak lod score of 5.50 at an estimated recombination fraction of 0.00 for marker locus D6S300. INTERPRETATION: The family described has an autosomal dominant macular dystrophy that resembles Stargardt-like macular dystrophy. The disease locus for this family maps to an interval on chromosome 6q that overlaps that for STGD3 and other retinal dystrophy loci. These findings provide further evidence that human chromosome 6q represents a "hot spot" for retinal disorders.

Spectrum of color gene deletions and phenotype in patients with blue cone monochromacy.

Blue cone monochromacy (BCM) is an X-linked ocular disease characterized by poor visual acuity, nystagmus, and photodysphoria in males with severely reduced color discrimination. Deletions, rearrangements and point mutations in the red and green pigment genes have been implicated in causing BCM. We assessed the spectrum of genetic alterations in ten families with BCM by Southern blot, polymerase chain reaction, and sequencing analysis, and the phenotype was characterized by ophthalmoscopy, fluorescein angiography, and a battery of tests to assess color vision in addition to routine ophthalmological examination. All families showed clinical features associated with BCM. Acuities were reduced in all affected males, and photopic b-wave was reduced by more than 90% in seven families. In three families, however, the photopic b-wave response showed uncharacteristic relative preservation of 30-80% (of the clinical low-normal value). The color vision was unusually preserved in two affected males, but this was not correlated with photopic electroretinography retention. Progressive macular atrophy was observed in affected members of two BCM families while the rest of the families presented with normal fundus. In nine families deletions were identified in the gene encoding the red-sensitive photopigment and/or in the region up to 17.8 kb upstream of the red gene which contains the locus control region and other regulatory sequences. In the same nine families the red pigment gene showed a range of deletions from the loss of a single exon to loss of the complete red gene. In one family no mutation was found in the exons of the red gene or the locus control region but showed loss of the complete green gene. No association was observed between the phenotypes and genotypes in these families.

Phenotypic expression of juvenile X-linked retinoschisis in Swedish families with different mutations in the XLRS1 gene.

OBJECTIVE: To describe the clinical phenotype of juvenile X-linked retinoschisis in patients with different mutations in the XLRS1 gene. METHODS: Thirty patients with 7 different XLRS1 mutations were examined. The genotype was determined by molecular genetics, which identified 6 known and 1 novel mutation (exon 5, 489 G-->T). Ophthalmologic examination included full-field electroretinogram (ERG) recordings. RESULTS: The fundus appearance showed marked variations between, as well as within, families with different XLRS1 mutations. The ERG demonstrated typical reduction of B-wave amplitude, with relative A-wave preservation, causing a reduced B-A ratio in all affected males. The implicit time of the 30-Hz flicker ERG was prolonged in all patients examined. In a large family with a deletion of exon 1 and the promoter region, 12 affected males showed a phenotype ranging from moderate to severe vision impairment and a broad range of ERG abnormality, suggesting that additional factors may contribute to the disease severity. CONCLUSIONS: Juvenile retinoschisis shows a wide variability in the phenotype between, as well as within, families with different genotypes. The ERG findings show reduced B-A ratios of dark-adapted recordings and prolonged implicit times of 30-Hz flicker response, which provide a useful clinical marker to confirm the clinical diagnosis. CLINICAL RELEVANCE: This study describes the wide variability in the phenotype in patients with juvenile retinoschisis and different mutations in the XLRS1 gene. The study emphasizes the importance of complementing the ophthalmologic examination with full-field ERG and molecular genetics in boys with visual failure of unknown etiology to determine the diagnosis early in the course of the disease. Arch Ophthalmol. 2000;118:1098-1104

A 5-base insertion in the gammaC-crystallin gene is associated with autosomal dominant variable zonular pulverulent cataract.

A seven-generation family with 30 members affected by highly variable autosomal dominant zonular pulverulent cataracts has been previously described. We have localized the cataracts to a 19-cM interval on chromosome 2q33-q35 including the gamma-crystallin gene cluster. Maximum lod scores are 4.56 (theta=0.02) with D2S157, 3.66 (theta=0.12) with D2S72, and 3.57 (theta=0.052) with CRYG. Sequencing and allele-specific oligonucleotide analysis of the pseudo gammaE-crystallin promoter region from individuals in the pedigree suggest that activation of the gammaE-crystallin pseudo gene is unlikely to cause the cataracts in the family. In addition, base changes in the TATA box but not the Sp1-binding site have been found in unaffected controls and can be excluded as a sole cause of cataracts. In order to investigate the underlying genetic mechanism of cataracts in this family further, exons of the highly expressed gammaC- and gammaD-crystallin genes have been sequenced. The gammaD-crystallin gene shows no abnormalities, but a 5-bp duplication within exon 2 of the gammaC-crystallin gene has been found in one allele of each affected family member and is absent from both unaffected family members and unaffected controls. This mutation disrupts the reading frame of the gammaC-crystallin coding sequence and is predicted to result in the synthesis of an unstable gammaC-crystallin with 38 amino acids of the first "Greek key" motif followed by 52 random amino acids. This finding suggests that the appropriate association of mutant betagamma-crystallins into oligomers is not necessary to cause cataracts and may give us new insights into the genetic mechanism of cataract formation.

Autosomal dominant macular atrophy at 6q14 excludes CORD7 and MCDR1/PBCRA loci.

PURPOSE: Localization of the gene responsible for autosomal dominant atrophic macular degeneration (adMD) in a large pedigree UM:H785. METHODS: Standard ophthalmologic examinations were performed. Microsatellite markers were used to map the disease gene by linkage and haplotype analyses. RESULTS: The macular degeneration in this family is characterized by progressive retinal pigment epithelial atrophy in the macula without apparent peripheral involvement by ophthalmoscopy or functional studies. Acuity loss progressed with age and generally was worse in the older affected individuals. The rod and cone function remained normal or nearly normal in all tested affected members up to 61 years of age. The phenotype in our family has characteristics similar to Stargardt-like macular degeneration with some differences. Haplotype analysis localized the disease gene in our adMD family to an 8-cM region at 6q14, which is within the 18-cM interval of STGD3 but excludes cone-rod dystrophy 7 (CORD7; centromeric) and North Carolina macular degeneration and progressive bifocal chorioretinal atrophy (MCDR1/PBCRA; telomeric). The mapping interval overlaps with that of recessive retinitis pigmentosa (RP25). CONCLUSIONS: These results implicate at least three genetically distinct loci for forms of macular degeneration that lie within a 30-cM interval on chromosome 6p11-6q16: CORD7, adMD, and MCDR1/PBCRA. Because the critical interval for the adMD family studied overlaps with STGD3 and RP25, these loci could be allelic.

Autosomal dominant hemorrhagic macular dystrophy not associated with the TIMP3 gene.

OBJECTIVE: To describe the ophthalmic and genetic findings of a large kindred (UM:H389) with autosomal dominant hemorrhagic macular dystrophy. METHODS: The disease state of family members was documented by dilated fundus examination, electroretinography, color vision tests, fluorescein angiography, measurement of visual fields, biomicroscopy, gonioscopy, and intraocular pressure measurement. Linkage and haplo-type analyses were carried out with markers flanking the Sorsby fundus dystrophy TIMP3 (tissue inhibitor of metalloproteinase 3) gene locus, and mutation analysis was carried out by screening exon 5 of the TIMP3 gene. RESULTS: This 4-generation pedigree with autosomal dominant hemorrhagic macular degeneration has visual symptoms beginning in the sixth decade of life. Several family members developed choroidal neovascular membrane formation in the macula of both eyes. The phenotype overlaps that of Sorsby fundus dystrophy. Some of the affected members have unusual zonularlike radial striations on the anterior lens capsule surface, and glaucoma or ocular hypertension has developed in 2 of them. Involvement of the TIMP3 gene was excluded by linkage, haplotype, and mutation analyses. CONCLUSIONS: The phenotype of this family with autosomal dominant macular dystrophy overlaps that of Sorsby fundus dystrophy. Exclusion of the TIMP3 gene in this family indicates genetic heterogeneity for hemorrhagic macular dystrophy. Anterior segment anomalies may occur with this condition, but cosegregation has not yet been established. CLINICAL RELEVANCE: This study broadens the spectrum of hemorrhagic macular dystrophy by identifying a family in which the TIMP3 gene is not involved. Once the gene is cloned, we are eager to learn whether this gene may be involved in age-related macular degeneration.

Novel mutations in XLRS1 causing retinoschisis, including first evidence of putative leader sequence change.

Juvenile retinoschisis is an X-linked recessive disease caused by mutations in the XLRS1 gene. We screened 31 new unrelated patients and families for XLRS1 mutations in addition to previously reported mutations for 60 of our families (Retinoschisis Consortium, Hum Mol Genet 1998;7:1185-1192). Twenty-three different mutations including 12 novel ones were identified in 28 patients. Mutations identified in this study include 19 missense mutations, two nonsense mutations, one intragenic deletion, four microdeletions, one insertion, and one intronic sequence substitution that is likely to result in a splice site defect. Two novel mutations, c.38T-->C (L13P) and c.667T-->C (C223R), respectively, present the first genetic evidence for the functional significance of the putative leader peptide sequence and for the functional significance at the carboxyl terminal of the XLRS1 protein beyond the discoidin domain. Mutations in 25 of the families were localized to exons 4-6, emphasizing the critical functional significance of the discoidin domain of the XLRS1 protein.

Bilateral macular atrophy in blue cone monochromacy (BCM) with loss of the locus control region (LCR) and part of the red pigment gene.

PURPOSE: To describe unusual macular abnormalities in a family with blue cone monochromacy (BCM, or X-linked incomplete achromatopsia) and deletion of about 9.5 kb comprising part of the red pigment gene and the region upstream of the red pigment gene. METHODS: The molecular structure of the red and green pigment genes and the locus control region (LCR) upstream of the red gene were studied for deletions, rearrangements and point mutations by Southern blot analysis and PCR. Four affected males (ages 33, 45, 51, and 59) and a carrier female (age 58) were examined by funduscopy and fluorescein angiography. Extensive color vision testing as well as rod and cone electroretinography (ERG) were performed on two of them. RESULTS: Analysis showed that the 6 kb proximal red gene region, exon 1 and about 3.1 kb of intron 1 of the red gene are deleted in this family. Exons 2-6 of the red gene, all the exons of the green gene and the Tex 28 gene were present. Four affected males had bilateral macular changes, including three with overt atrophy. All had visual acuity of 20/200 and their color vision was typical for BCM, with the absence of long- and middle-wavelength sensitive cone function. The ERG showed normal rod responses, whereas the photopic cone and 30-Hz flicker responses were >95% reduced. CONCLUSIONS: We report the unusual association between macular atrophy and BCM resulting from the loss of an approximately 9.5 kb region encompassing the LCR, proximal red gene promoter elements and exon 1 of the red gene. However, loss of the LCR and promoter is not sufficient to explain the phenotype since we have observed other BCM families with similar deletions who do not exhibit macular changes.

Bestrophin gene mutations in patients with Best vitelliform macular dystrophy.

Best vitelliform macular dystrophy (VMD2) is an autosomal dominant dystrophy with a juvenile age of onset. Mutations in the Bestrophin gene were shown in patients affected with VMD2. In a mutation study, we made three new and interesting observations. First, we identified possible mutation hotspots within the gene, suggesting that particular regions of the protein have greater functional significance than others. Second, we described a 2-bp deletion in a part of the gene where mutations have not previously been reported; this mutation causes a frameshift and subsequent premature termination of the protein. Finally, we have evidence that some mutations are associated with variable expression of the disease, suggesting the involvement of other factors or genes in the disease phenotype.

Juvenile retinoschisis: a model for molecular diagnostic testing of X-linked ophthalmic disease.

BACKGROUND AND PURPOSE: X-linked juvenile retinoschisis (RS) provides a starting point to define clinical paradigms and understand the limitations of diagnostic molecular testing. The RS phenotype is specific, but the broad severity range is clinically confusing. Molecular diagnostic testing obviates unnecessary examinations for boys at-risk and identifies carrier females who otherwise show no clinical signs. METHODS: The XLRS1 gene has 6 exons of 26-196 base-pair size. Each exon is amplified by a single polymerase chain reaction and then sequenced, starting with exons 4 through 6, which contain mutation "hot spots." RESULTS: The 6 XLRS1 exons are sequenced serially. If alterations are found, they are compared with mutations in our > 120 XLRS families and with the > 300 mutations reported worldwide. Point mutations, small deletions, or rearrangements are identified in nearly 90% of males with a clinical diagnosis of RS. XLRS1 has very few sequence polymorphisms. Carrier-state testing produces 1 of 3 results: (1) positive, in which the woman has the same mutation as an affected male relative or known in other RS families; (2) negative, in which she lacks the mutation of her affected male relative; and (3) uninformative, in which no known mutation is identified or no information exists about the familial mutation. CONCLUSIONS: Molecular RS screening is an effective diagnostic tool that complements the clinician's skills for early detection of at-risk males. Useful outcomes of carrier testing depend on several factors: (1) a male relative with a clear clinical diagnosis; (2) a well-defined inheritance pattern; (3) high disease penetrance; (4) size and organization of the gene; and (5) the types of disease-associated mutations. Ethical questions include molecular diagnostic testing of young at-risk females before the age of consent, the impact of this information on the emotional health of the patient and family, and issues of employability and insurance coverage.

Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon.

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.

Construction of a YAC contig encompassing the Usher syndrome type 1C and familial hyperinsulinism loci on chromosome 11p14-15.1.

The Usher syndrome type 1C (USH1C) and familial hyperinsulinism (HI) loci have been assigned to chromosome 11p14-15.1, within the interval D11S419-D11S1310. We have constructed a yeast artificial chromosome (YAC) contig, extending from D11S926 to D11S899, which encompasses the critical regions for both USH1C and HI and spans an estimated genetic distance of approximately 4 cM. A minimal set of six YAC clones constitute the contig, with another 22 YACs confirming the order of sequence-tagged sites (STSs) and position of YACs on the contig. A total of 40 STSs, including 10 new STSs generated from YAC insert-end sequences and inter-Alu PCR products, were used to order the clones within the contig. This physical map provides a resource for identification of gene transcripts associated with USH1C, HI, and other genetic disorders that map to the D11S926-D11S899 interval.