Identification of mutations causing inherited retinal degenerations in the israeli and palestinian populations using homozygosity mapping.

PURPOSE: The Israeli and Palestinian populations are known to have a relatively high level of consanguineous marriages, leading to a relatively high frequency of autosomal recessive (AR) diseases. Our purpose was to use the homozygosity mapping approach, aiming to prioritize the set of genes and identify the molecular genetic causes underlying AR retinal degenerations in the Israeli and Palestinian populations. METHODS: Clinical analysis included family history, ocular examination, full-field electroretinography (ERG), and funduscopy. Molecular analysis included homozygosity mapping and mutation analysis of candidate genes. RESULTS: We recruited for the study families with AR nonsyndromic retinal degenerations, including mainly retinitis pigmentosa (RP), cone-rod degeneration (CRD), and Leber congenital amaurosis (LCA). With the aim to identify the causative genes in these families, we performed homozygosity mapping using whole genome single nucleotide polymorphism (SNP) arrays in 125 families. The analysis revealed the identification of 14 mutations, 5 of which are novel, in 16 of the families. The mutations were identified in the following eight genes: RDH12, PROM1, MFRP, TULP1, LCA5, CEP290, NR2E3, and EYS. While most patients had a retinal disease that is compatible with the causing gene, in some cases new clinical features are evident. CONCLUSIONS: Homozygosity mapping is a powerful tool to identify genetic defects underlying heterogeneous AR disorders, such as RP and LCA, in consanguineous and nonconsanguineous patients. The identification of significant and large homozygous regions, which do not include any known retinal disease genes, may be a useful tool to identify novel disease-causing genes, using next generation sequencing.

Mutations in CRB1 are a relatively common cause of autosomal recessive early-onset retinal degeneration in the Israeli and Palestinian populations.

PURPOSE: We evaluated the role of Crumbs homolog 1 (CRB1) in autosomal recessive (AR) retinal diseases in the Israeli and Palestinian populations using homozygosity mapping. METHODS: Clinical analysis included family history, ocular examination, full-field electroretinography (ERG), and funduscopy. Molecular analysis included homozygosity mapping using whole genome single nucleotide polymorphism (SNP) arrays and mutation analysis of CRB1. RESULTS: We recruited over 400 families with AR nonsyndromic retinal degenerations, including retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). SNP array analysis was performed on 175 index cases, eight of whom carried a homozygous region on chromosome 1 harboring CRB1. A subsequent CRB1 mutation analysis of the eight families, followed by screening of candidate founder mutations in the whole cohort of patients, revealed a total of 13 mutations, six of which are novel, in 15 families. Nine mutations were family-specific, and four were founder mutations identified in patients of Arab-Muslim origin, and Jews originated from Iraq and Kurdistan. Interestingly, a null mutation on at least one of the two mutated CRB1 alleles results in the LCA diagnosis, whereas patients carrying missense mutations were diagnosed with either RP or LCA. The average age at which CRB1 patients were referred to ERG testing was young (11 years). Of the 30 identified CRB1 patients, five had Coats-like exudative vasculopathy. CONCLUSIONS: Our data show that CRB1 mutations are a relatively frequent cause of AR early-onset retinal degeneration in the Israeli and Palestinian populations (10% of LCA families), and causes severe retinal degeneration at an early age.

Exome sequencing identifies a founder frameshift mutation in an alternative exon of USH1C as the cause of autosomal recessive retinitis pigmentosa with late-onset hearing loss.

We used a combined approach of homozygosity mapping and whole exome sequencing (WES) to search for the genetic cause of autosomal recessive retinitis pigmentosa (arRP) in families of Yemenite Jewish origin. Homozygosity mapping of two arRP Yemenite Jewish families revealed a few homozygous regions. A subsequent WES analysis of the two index cases revealed a shared homozygous novel nucleotide deletion (c.1220delG) leading to a frameshift (p.Gly407Glufs*56) in an alternative exon (#15) of USH1C. Screening of additional Yemenite Jewish patients revealed a total of 16 homozygous RP patients (with a carrier frequency of 0.008 in controls). Funduscopic and electroretinography findings were within the spectrum of typical RP. While other USH1C mutations usually cause Usher type I (including RP, vestibular dysfunction and congenital deafness), audiometric screening of 10 patients who are homozygous for c.1220delG revealed that patients under 40 years of age had normal hearing while older patients showed mild to severe high tone sensorineural hearing loss. This is the first report of a mutation in a known USH1 gene that causes late onset rather than congenital sensorineural hearing loss. The c.1220delG mutation of USH1C accounts for 23% of RP among Yemenite Jewish patients in our cohort.

A missense mutation in DHDDS, encoding dehydrodolichyl diphosphate synthase, is associated with autosomal-recessive retinitis pigmentosa in Ashkenazi Jews.

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerations caused by mutations in at least 50 genes. Using homozygosity mapping in Ashkenazi Jewish (AJ) patients with autosomal-recessive RP (arRP), we identified a shared 1.7 Mb homozygous region on chromosome 1p36.11. Sequence analysis revealed a founder homozygous missense mutation, c.124A>G (p.Lys42Glu), in the dehydrodolichyl diphosphate synthase gene (DHDDS) in 20 AJ patients with RP of 15 unrelated families. The mutation was not identified in an additional set of 109 AJ patients with RP, in 20 AJ patients with other inherited retinal diseases, or in 70 patients with retinal degeneration of other ethnic origins. The mutation was found heterozygously in 1 out of 322 ethnically matched normal control individuals. RT-PCR analysis in 21 human tissues revealed ubiquitous expression of DHDDS. Immunohistochemical analysis of the human retina with anti-DHDDS antibodies revealed intense labeling of the cone and rod photoreceptor inner segments. Clinical manifestations of patients who are homozygous for the c.124A>G mutation were within the spectrum associated with arRP. Most patients had symptoms of night and peripheral vision loss, nondetectable electroretinographic responses, constriction of visual fields, and funduscopic hallmarks of retinal degeneration. DHDDS is a key enzyme in the pathway of dolichol, which plays an important role in N-glycosylation of many glycoproteins, including rhodopsin. Our results support a pivotal role of DHDDS in retinal function and may allow for new therapeutic interventions for RP.

Homozygosity mapping reveals null mutations in FAM161A as a cause of autosomal-recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal degenerations caused by mutations in at least 45 genes. Using homozygosity mapping, we identified a ∼4 Mb homozygous region on chromosome 2p15 in patients with autosomal-recessive RP (arRP). This region partially overlaps with RP28, a previously identified arRP locus. Sequence analysis of 12 candidate genes revealed three null mutations in FAM161A in 20 families. RT-PCR analysis in 21 human tissues revealed high levels of FAM161A expression in the retina and lower levels in the brain and testis. In the human retina, we identified two alternatively spliced transcripts with an intact open reading frame, the major one lacking a highly conserved exon. During mouse embryonic development, low levels of Fam161a transcripts were detected throughout the optic cup. After birth, Fam161a expression was elevated and confined to the photoreceptor layer. FAM161A encodes a protein of unknown function that is moderately conserved in mammals. Clinical manifestations of patients with FAM161A mutations varied but were largely within the spectrum associated with arRP. On funduscopy, pallor of the optic discs and attenuation of blood vessels were common, but bone-spicule-like pigmentation was often mild or lacking. Most patients had nonrecordable electroretinographic responses and constriction of visual fields upon diagnosis. Our data suggest a pivotal role for FAM161A in photoreceptors and reveal that FAM161A loss-of-function mutations are a major cause of arRP, accounting for ∼12% of arRP families in our cohort of patients from Israel and the Palestinian territories.

Novel null mutations in the EYS gene are a frequent cause of autosomal recessive retinitis pigmentosa in the Israeli population.

PURPOSE: To characterize the role of EYS, a recently identified retinal disease gene, in families with inherited retinal degenerations in the Israeli and Palestinian populations. METHODS: Clinical and molecular analyses included family history, ocular examination, full-field electroretinography (ERG), perimetry, autozygosity mapping, mutation detection, and estimation of mutation age. RESULTS: Autozygosity mapping was performed in 171 consanguineous Israeli and Palestinian families with inherited retinal degenerations. Large homozygous regions, harboring the EYS gene, were identified in 15 of the families. EYS mutation analysis in the 15 index cases, followed by genotyping of specific mutations in an additional 121 cases of inherited retinal degenerations, revealed five novel null mutations, two of which are founder mutations, in 10 Israeli and Palestinian families with autosomal recessive retinitis pigmentosa (arRP). The most common mutation identified was a founder mutation in the Moroccan Jewish subpopulation. The ESTIAGE program produced an estimate that the age of the most recent common ancestor was 26 generations. The retinal phenotype in most patients was typical yet relatively severe RP, with an early age of onset and nonrecordable ERGs on presentation. CONCLUSIONS: The results demonstrate that EYS is currently the most commonly mutated arRP gene in the Israeli population, mainly due to founder mutations. EYS mutations were associated with an RP phenotype in all patients. The authors concluded that the gene plays only a minor role in causing other retinal phenotypes.

Variable retinal phenotypes caused by mutations in the X-linked photopigment gene array.

PURPOSE: To examine the involvement of the long (L) and middle (M) wavelength-sensitive cone opsin genes in cone-dominated phenotypes. METHODS: Clinical and molecular analyses included family history, color vision testing, full-field electroretinography (ERG), linkage analysis, and mutation detection. RESULTS: Eighteen families were recruited that had X-linked retinal disease characterized by cone impairment in which affected males usually had nystagmus, reduced visual acuity, normal to subnormal rod ERG, and reduced or extinguished cone ERG responses. A search for mutations in the L-M pigment gene array revealed disease-causing mutations in six families. In two of them, novel mutations were identified: a large deletion affecting both opsin genes and a single L opsin gene harboring a likely pathogenic mutation, p.Val120Met. A third family carried a single hybrid gene with the p.Cys203Arg mutation. Patients from the three remaining families carried a single opsin gene harboring two similar rare haplotypes. Although the phenotype of members in one of the families was compatible with blue cone monochromacy (BCM), patients from the two other families, who shared an identical haplotype, had only reduced or even normal full-field cone ERGs, but maculopathy was evident. CONCLUSIONS: Novel and known mutations affecting the L-M opsin gene array were identified in families with X-linked cone-dominated phenotypes. The results show that different mutations in this gene array can cause a variety of phenotypes, including BCM, cone dystrophy, and maculopathy. Males with X-linked cone-dominated diseases should be routinely analyzed for mutations in the L-M opsin gene array.

The spectrum of retinal diseases caused by NR2E3 mutations in Israeli and Palestinian patients.

OBJECTIVES: To evaluate the involvement of NR2E3 in inherited retinal degenerative diseases in the Israeli and Palestinian populations and to study phenotypic variability in patients who are homozygous for the same mutation. METHODS: Patients from 35 families underwent clinical evaluation, including a full ophthalmologic examination and electroretinography. Genetic analyses included direct sequencing of polymerase chain reaction products and haplotype reconstruction. RESULTS: We recruited 6 consanguineous Muslim families and 2 Jewish families with enhanced S-cone syndrome. Patients from 4 of the Muslim families were homozygous for the same NR2E3 mutation, c.119-2A>C, but showed considerable variability in fundus appearance and retinal function, even among patients of comparable ages. Both Jewish patients were compound heterozygotes for the c.932G>A mutation in combination with c.194-202del9bp or a novel splice-site mutation, c.747+1G>C. Homozygosity analysis in 27 consanguineous families with retinitis pigmentosa revealed a homozygous mutation, c.932G>A, in 2 families. The electroretinographic responses in these patients were compatible with retinitis pigmentosa and did not show the characteristic enhanced S-cone syndrome pattern. CONCLUSION: Our results demonstrate the involvement of NR2E3 in enhanced S-cone syndrome and retinitis pigmentosa phenotypes in our populations. CLINICAL RELEVANCE: Patients with NR2E3 mutations may manifest variable phenotypes. Moreover, patients who are homozygous for the same NR2E3 mutation have variable expression of retinal disease, suggesting the involvement of modifier genes.

A pilot study of topical treatment with an alpha2-agonist in patients with retinal dystrophies.

PURPOSE: The aim of this study was to assess the neuroprotective effect of a topical alpha2-agonist in patients with retinal dystrophies. METHODS: This study was a prospective, placebo-controlled, double-masked, randomized clinical trial. A total of 26 patients with retinal dystrophies were included. One (1) randomly selected eye was treated with brimonidine tartrate 0.2% twice-daily, while the fellow eye received artificial tears. Disease progression parameters tested at 6-8-month intervals throughout the study included Goldmann visual fields, contrast sensitivity, color vision, and fullfield electroretinography. RESULTS: Seventeen (17) of the 26 recruited patients completed the study. Except for 1 patient with an 18-month follow-up, all patients were followed up for 24-36 months (mean, 29). At the conclusion of the study, there were no differences detected in visual acuity, color vision, and contrast sensitivity between the treated and control eyes. There was a trend, however, toward a lesser degree of visual field loss in the brimonidine-treated eyes. There was also a delay in the time required to reach a 25% visual field loss in the treated eyes. These differences were more pronounced in a subgroup of patients diagnosed as retinitis pigmentosa and with visual fields of 5 cm2 or more at baseline. CONCLUSIONS: The findings of this pilot study suggest a trend for slower progression in the eyes of patients with retinal dystrophy when treated with brimonidine, according to one of the parameters that was studied (visual field loss). Further studies that include a larger number of patients and a longer follow-up period are needed to clarify and confirm the potential neuroprotective effect of alpha2-agonists in human retinal dystrophies.

Comparative genomics and gene expression analysis identifies BBS9, a new Bardet-Biedl syndrome gene.

Bardet-Biedl syndrome (BBS) is an autosomal recessive, genetically heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, and hypogenitalism. Eight BBS genes representing all known mapped loci have been identified. Mutation analysis of the known BBS genes in BBS patients indicate that additional BBS genes exist and/or that unidentified mutations exist in the known genes. To identify new BBS genes, we performed homozygosity mapping of small, consanguineous BBS pedigrees, using moderately dense SNP arrays. A bioinformatics approach combining comparative genomic analysis and gene expression studies of a BBS-knockout mouse model was used to prioritize BBS candidate genes within the newly identified loci for mutation screening. By use of this strategy, parathyroid hormone-responsive gene B1 (B1) was found to be a novel BBS gene (BBS9), supported by the identification of homozygous mutations in BBS patients. The identification of BBS9 illustrates the power of using a combination of comparative genomic analysis, gene expression studies, and homozygosity mapping with SNP arrays in small, consanguineous families for the identification of rare autosomal recessive disorders. We also demonstrate that small, consanguineous families are useful in identifying intragenic deletions. This type of mutation is likely to be underreported because of the difficulty of deletion detection in the heterozygous state by the mutation screening methods that are used in many studies.

Wiping microkeratome blades with sterile 100% alcohol to prevent diffuse lamellar keratitis after laser in situ keratomileusis.

PURPOSE: To report our experience in preventing diffuse lamellar keratitis (DLK) after laser in situ keratomileusis (LASIK) by wiping the microkeratome blade with sterile 100% alcohol. SETTING: Enaim Refractive Surgery Center, Jerusalem, Israel. METHODS: Laser in situ keratomileusis was performed in 24 patients (48 eyes) on the same day by the same surgeon. The environment and instruments were identical in all cases. A new blade was used in both eyes of each patient. All blades were from the same batch and were randomly assigned to 2 groups. In the alcohol group, 12 blades were wiped with a Merocel surgical spear (Medtronic Solan) dipped in 100% alcohol and rinsed with balanced salt solution (BSS) before they were mounted in a Hansatome microkeratome (Bausch & Lomb Co.); in the control group, 12 blades were wiped with a Merocel surgical spear dipped in BSS and rinsed with BSS. All patients were examined on the first postoperative day. RESULTS: Grade I to II DLK was diagnosed in 7 eyes of 4 patients in the control group (29%). No DLK was diagnosed in the alcohol group. CONCLUSION: Wiping the microkeratome blade with 100% alcohol before mounting may remove a substance from the manufacturing or sterilization process that can cause DLK.