A homozygosity-based search for mutations in patients with autosomal recessive retinitis pigmentosa, using microsatellite markers

PURPOSE: To identify possible mutations in known candidate genes in patients with autosomal recessive (ar) and simplex retinitis pigmentosa (RP), by using an established strategy of flexible, multiplexed, microsatellite-based homozygosity mapping. METHODS: A total of 78 microsatellite markers corresponding to 16 genes known to be responsible for arRP were selected and used in 18 multiplex amplifications, followed by genotyping. Twelve consanguineous probands and 47 nonconsanguineous probands (59 patients with arRP or simplex RP) agreed to the screening. RESULTS: Of the 59 probands examined, 24 had a mean of 1.4 genes showing homozygosity for all markers within the corresponding gene region. Subsequent direct sequencing revealed three homozygous mutations. Two of them were novel mutations in the genes TULP1 (c.1145T-->C, F382S) and CNGB1 (c.3444 + 1G-->A). The other was a mutation in RPE65 (c.1543C-->T, R515W), which is known to cause Leber's congenital amaurosis. The clinical features of each patient, together with the cosegregation analysis, strongly support the pathogenicity of these mutations. CONCLUSIONS: This systematic approach facilitated the identification of genes that cause arRP, and the results provide a widened spectrum of the mutation severity associated with a broader range of phenotypic manifestations of arRP.     

Microarray-based mutation detection and phenotypic characterization of patients with Leber congenital amaurosis

PURPOSE: To test the efficiency of a microarray chip as a diagnostic tool in a cohort of northwestern European patients with Leber congenital amaurosis (LCA) and to perform a genotype-phenotype analysis in patients in whom pathologic mutations were identified. METHODS: DNAs from 58 patients with LCA were analyzed using a microarray chip containing previously identified disease-associated sequence variants in six LCA genes. Mutations identified by chip analysis were confirmed by sequence analysis. On identification of one mutation, all protein coding exons of the relevant genes were sequenced. In addition, sequence analysis of the RDH12 gene was performed in 22 patients. Patients with mutations were phenotyped. RESULTS: Pathogenic mutations were identified in 19 of the 58 patients with LCA (32.8%). Four novel sequence variants were identified. Mutations were most frequently found in CRB1 (15.5%), followed by GUCY2D (10.3%). The p.R768W mutation was found in 8 of 10 GUCY2D alleles, suggesting that it is a founder mutation in the northwest of Europe. In early childhood, patients with AIPL1 or GUCY2D mutations show normal fundi. Those with AIPL1-associated LCA progress to an RP-like fundus before the age of 8, whereas patients with GUCY2D-associated LCA still have relatively normal fundi in their mid-20s. Patients with CRB1 mutations present with distinct fundus abnormalities at birth and consistently show characteristics of RP12. Pathogenic GUCY2D mutations result in the most severe form of LCA. CONCLUSIONS: Microarray-based mutation detection allowed the identification of 32% of LCA sequence variants and represents an efficient first-pass screening tool. Mutations in CRB1, and to a lesser extent, in GUCY2D, underlie most LCA cases in this cohort. The present study establishes a genotype-phenotype correlation for AIPL1, CRB1, and GUCY2D.     

Advantage of Whole Exome Sequencing over Allele-Specific and Targeted Segment Sequencing in Detection of Novel TULP1 Mutation in Leber Congenital Amaurosis

Background: Leber congenital amaurosis (LCA) is a severe form of retinal dystrophy with marked underlying genetic heterogeneity. Until recently, allele-specific assays and Sanger sequencing of targeted segments were the only available approaches for attempted genetic diagnosis in this condition. A broader next-generation sequencing (NGS) strategy, such as whole exome sequencing, provides an improved molecular genetic diagnostic capacity for patients with these conditions.

Materials and Methods: In a child with LCA, an allele-specific assay analyzing 135 known LCA-causing variations, followed by targeted segment sequencing of 61 regions in 14 causative genes was performed. Subsequently, exome sequencing was undertaken in the proband, unaffected consanguineous parents and two unaffected siblings. Bioinformatic analysis used two independent pipelines, BWA-GATK and SOAP, followed by Annovar and SnpEff to annotate the variants.

Results: No disease-causing variants were found using the allele-specific or targeted segment Sanger sequencing assays. Analysis of variants in the exome sequence data revealed a novel homozygous nonsense mutation (c.1081C?>?T, p.Arg361*) in TULP1, a gene with roles in photoreceptor function where mutations were previously shown to cause LCA and retinitis pigmentosa. The identified homozygous variant was the top candidate using both bioinformatic pipelines.

Conclusions: This study highlights the value of the broad sequencing strategy of exome sequencing for disease gene identification in LCA, over other existing methods. NGS is particularly beneficial in LCA where there are a large number of causative disease genes, few distinguishing clinical features for precise candidate disease gene selection, and few mutation hotspots in any of the known disease genes.     

Diverse clinical phenotypes associated with a nonsense mutation in FAM161A

Purpose:

Mutations in the FAM161A gene have been reported in association with autosomal recessive retinitis pigmentosa (arRP) in several ethnic populations. This study aimed to assess the prevalence of FAM161A-related retinopathy in a British cohort and to characterise the phenotype associated with mutations in this gene.

Methods:

The FAM161A coding region and intron–exon boundaries were screened by Sanger sequencing in 120 retinitis pigmentosa (RP) patients (with likely autosomal recessive inheritance) in whom mutations in other known major RP genes have been ruled out by commercially available testing. Homozygosity mapping was performed in one consanguineous family, and high-throughput sequencing of candidate genes was performed to identify disease-associated changes. Clinical assessment of affected individuals included perimetry testing, fundus autofluorescence imaging, and optical coherence tomography.

Results:

Two patients of British origin with a homozygous mutation in FAM161A (c.1309A>T, p.Arg437*) were identified by Sanger sequencing. Homozygosity mapping and subsequent high-throughput sequencing analysis identified a further family of Pakistani origin with the same genotype. Clinical examination of affected members of these families revealed that this mutation was associated with a diverse clinical phenotype, ranging from mild disease with preservation of central acuity to severe visual impairment.

Conclusions:

Homozygosity for the c.1309A>T, p.Arg437* variant in FAM161A is a relatively common cause of arRP. The mutation occurs in diverse ethnic populations, associated with typical retinitis pigmentosa with disease onset usually in the second or third decade of life.     

A novel mutation of the RP1 gene (Lys778ter) associated with autosomal dominant retinitis pigmentosa

BACKGROUND: Besides the three known genes (RHO, RDS/Peripherin, NRL) involved in autosomal dominant retinitis pigmentosa (adRP), a fourth gene, RP1, has been recently identified. Initial reports suggest that mutations in the RP1 gene are the second most frequent cause of adRP. The clinical findings were described in a family with adRP and a novel mutation in the RP1 gene. METHOD: Index patients from 15 independent families with adRP in which RHO mutations had been excluded in previous examinations were screened for mutations in the RP1 gene by means of direct DNA sequencing. Evaluation of the RP1 phenotype in patients included funduscopy, kinetic perimetry, dark adapted final threshold test, standard electroretinography and, in one case, multifocal electroretinography. RESULTS: One novel nonsense mutation (Lys778ter) in one of these 15 patients was detected. Cosegregation of the mutation with the disease phenotype could be established in the index patient's family. The phenotype comprises variable expression of clinical disease probably including one case of incomplete penetrance, a onset of symptoms beginning in adulthood, and evidence of regionally varying retinal function loss. CONCLUSION: The Lys778ter mutation localises inside the critical region harbouring all mutations described so far. The ophthalmic findings support previous observations that variation of disease expression appears as a typical feature of the RP1 phenotype.     

Identification of a Novel LCA6 Mutation in an Emirati Family

ABSTRACT

Purpose: To determine the cause of Leber congenital amaurosis (LCA) in a consanguineous Emirati family.

Methods: The clinical diagnosis was made on the basis of medical history, ophthalmoscopy and standard ERG. The diagnosis was confirmed by molecular genetic analysis of known LCA genes by Next-Generation Sequencing (NGS). The latter was performed by Bioscientia Institut, Germany (as a clinical service for Latifa Hospital, Dubai).

Results: The next generation sequencing of known LCA genes revealed a homozygous 1bp-insertion c.2608_2609insA in exon 16 of the RPGRIP1 gene. This mutation, which was confirmed by conventional Sanger sequencing, leads to a frameshift, resulting in a premature stop codon (p.Leu870TyrfsX7) and subsequently in a degradation of the m-RNA or in a truncation of the RPGRIP1 protein. The segregation analysis of the identified mutation was performed for the parental samples. Both parents carry the frameshift mutation in a heterozygous state.

Conclusion: We report a novel RPGRIP1 mutation causing LCA in a consanguineous Emirati family. To the best of our knowledge, this alteration has not been described in the literature so far.     

Novel deletion of the RPGR gene in a Chinese family with X-linked retinitis pigmentosa

Purpose: To characterize a Chinese family with inherited retinitis pigmentosa (RP). Methods: Linkage studies and haplotype analysis were used for gene mapping, and single-strand conformation polymorphism (SSCP) analysis and direct DNA sequence analysis were used for identifying the responsible mutation. Results: Pedigree analysis suggests that RP in the Chinese family RP002 is inherited either as an autosomal recessive trait or as an X-linked trait. Linkage analysis of RP002 excluded all known autosomal recessive RP loci. Further analysis with 17 polymorphic markers covering the entire X chromosome localized the RP gene in RP002 between markers GATA175D03 and GATA144D04 on Xp11.4, a region where the RP3 gene ( RPGR ) is found. Mutation analysis of the RPGR gene in RP002 revealed a novel 28-bp deletion in exon 7. This deletion resulted in an in-frame stop codon that eliminates the C-terminal two-thirds of the RPGR protein. The 28-bp deletion co-segregated with the disease in the family and was not present in 100 normal Chinese individuals. Female carriers of the deletion were affected with myopia and had ERG abnormalities and mild constriction of visual field. Conclusions: A novel 28-bp deletion in the RPGR gene identified in an X-linked Chinese RP family causes severe RP in male patients as well as myopia and ERG abnormalities in female carriers. The deletion represents the largest microdeletion identified in RPGR to date, and expands the spectrum of RPGR mutations causing XLRP.     

Novel RDH12 mutations associated with Leber congenital amaurosis and cone-rod dystrophy: biochemical and clinical evaluations

The purpose of this study was to determine the role of the retinol dehydrogenase 12 (RDH12) gene in patients affected with Leber congenital amaurosis (LCA), autosomal recessive retinitis pigmentosa (arRP) and autosomal dominant/recessive cone-rod dystrophies (CORD). Changes in the promoter region, coding regions and exon/intron junctions of the RDH12 gene were evaluated using direct DNA sequencing of patients affected with LCA (n=36 cases), RP (n=62) and CORD (n=21). The allele frequency of changes observed was assessed in a multiethnic control population (n=159 individuals). Detailed biochemical and structural modeling analysis of the observed mutations were performed to assess their biological role in the inactivation of Rdh12. A comprehensive clinical assessment of retinal structure and function in LCA patients carrying mutations in the RDH12 gene was completed. Of the six changes identified, three were novel including a homozygous C201R change in a patient affected with LCA, a heterozygous A177V change in patients affected with CORD and a heterozygous G46G change in a patient affected with LCA. A novel compound heterozygote T49M/A269fsX270 mutation was also found in a patient with LCA, and both homozygous and heterozygous R161Q changes were seen in 26 patients affected with LCA, CORD or RP. These R161Q, G46G and the A177V sequence changes were shown to be polymorphic. We found that Rdh12 mutant proteins associated with LCA were inactive or displayed only residual activity when expressed in COS-7 and Sf9 cells, whereas those mutants that were considered polymorphisms were fully active. Thus, impairment of retinal structure and function for patients carrying these mutations correlated with the biochemical properties of the mutants.     

A novel mutation disrupting the cytoplasmic domain of CRB1 in a large consanguineous family of Palestinian origin affected with Leber congenital amaurosis

Leber congenital amaurosis (LCA) is a genetically heterogeneous autosomal recessive condition responsible for congenital blindness or greatly impaired vision since birth. Eight LCA loci have been mapped, but only six out of eight genes have been hitherto identified. A genome-wide screen for homozygosity was conducted in a large consanguineous family originating from Palestine, for which no mutation was found in any of the six known LCA genes and that excluded the LCA3 and LCA5 loci. Evidence for homozygosity, however, was found in all affected patients of the family on chromosome 1q31, a region in which the human homologue of the Drosophila melanogaster crumbs gene (CRB1) has been mapped. Consequently, we proposed a hypothesis that the disease-causing mutation in this family might lie in an unexplored region of this LCA gene. As a matter of fact, while no mutation was found in any of the 11 CRB1 exons originally reported, we identified a 10-bp (del 4121-4130) deletion segregating with the disease in a later reported 12th exon lying in the 3' end of the gene. Interestingly, this deletion disrupts an amino acid sequence that was shown to be crucial for the function of the protein in the Drosophila counterpart (CRB).     

Whole-exome sequencing identifies novel mutations in genes responsible for retinitis pigmentosa in 2 nonconsanguineous Chinese families

AIM: To detect the pathogenetic mutations responsible for nonsyndromic autosomal recessive retinitis pigmentosa (RP) in 2 nonconsanguineous Chinese families. METHODS: The clinical data, including detailed medical history, best corrected visual acuity (BCVA), slit-lamp biomicroscope examination, fundus photography, optical coherence tomography, static perimetry, and full field electroretinogram, were collected from the members of 2 nonconsanguineous Chinese families preliminarily diagnosed with RP. Genomic DNA was extracted from the probands and other available family members; whole-exome sequencing was conducted with the DNA samples provided by the probands, and all mutations detected by whole-exome sequencing were verified using Sanger sequencing in the probands and the other available family members. The verified novel mutations were further sequenced in 192 ethnicity matched healthy controls. RESULTS: The patients from the 2 families exhibited the typical symptoms of RP, including night blindness and progressive constriction of the visual field, and the fundus examinations showed attenuated retinal arterioles, peripheral bone spicule pigment deposits, and waxy optic discs. Whole-exome sequencing revealed a novel nonsense mutation in FAM161A (c.943A>T, p.Lys315*) and compound heterozygous mutations in RP1L1 (c.56C>A, p.Pro19His; c.5470C>T, p.Gln1824*). The nonsense c.5470C>T, p.Gln1824* mutation was novel. All mutations were verified by Sanger sequencing. The mutation p.Lys315* in FAM161A co-segregated with the phenotype, and all the nonsense mutations were absent from the ethnicity matched healthy controls and all available databases. CONCLUSION: We identify 2 novel mutations in genes responsible for autosomal recessive RP, and the mutation in FAM161A is reported for the first time in a Chinese population. Our result not only enriches the knowledge of the mutation frequency and spectrum in the genes responsible for nonsyndromic RP but also provides a new target for future gene therapy.     

A G1103R mutation in CRB1 is co-inherited with high hyperopia and Leber congenital amaurosis

PURPOSE: To identify the genetic basis of recessive inheritance of high hyperopia and Leber congenital amaurosis (LCA) in a family of Middle Eastern origin. MATERIALS AND METHODS: The patients were examined using standard ophthalmic techniques. DNA samples were obtained and genetic linkage was carried out using polymorphic markers flanking the known genes and loci for LCA. Exons were amplified and sequenced. RESULTS: All four members of this family affected by LCA showed high to extreme hyperopia, with average spherical refractive errors ranging from +5.00 to +10.00. Linkage was obtained to 1q31.3 with a maximal LOD score of 5.20 and a mutation found in exon 9 of the CRB1 gene, causing a G1103R substitution at a highly conserved site in the protein. CRB1 is a vertebrate homolog of the Drosophila crumbs gene, which is required for photoreceptor morphogenesis, and has been associated with either retinitis pigmentosa (RP) or LCA. This sequence variant has previously been reported as a compound heterozygote in one sporadic LCA patient. CONCLUSION: Although hyperopia has been associated with LCA, it is typically moderate and variable between patients with the same mutation. In addition, some CRB1 mutations can be associated with either RP or LCA. We have shown that hyperopia and LCA are linked to the mutant CRB1 gene itself and are not dependent on unlinked modifiers.     

Genetic screening of leber congenital amaurosis in a large consanguineous Iranian family

The molecular defect of one large consanguineous Iranian kindred with Leber Congenital Amaurosis (LCA) is presented. The phenotype mapped to 17p13.1 (LCA1) and excluded from five other LCA loci. Sequence analysis of the GUCY2D gene identified a novel homozygous missense mutation (I816S) that segregated with the inherited disease-haplotype in six affected, eight parents, and two normal gene carriers. This mutation was absent in three other normal family members and 92 normal control subjects. In silico analysis predicted that alteration of the highly conserved isoleucine residue at position 816 to serine is deleterious by affecting secondary structure of the GUCY2D protein.     

Leber's congenital amaurosis with anterior keratoconus in Pakistani families is caused by the Trp278X mutation in the AIPL1 gene on 17p

BACKGROUND: Leber's congenital amaurosis (LCA) represents the earliest and severest form of retinal dystrophy leading to congenital blindness. A total of 20% of children attending blind schools have this disease. LCA has a multigenic basis and is proving central to our understanding of the development of the retina. We describe the clinical and molecular genetic features of four inbred pedigrees from neighbouring remote villages in northern Pakistan, in which some of the affected members have concurrent keratoconus. METHODS: History-taking and physical and eye examinations were performed in the field. Venipuncture, DNA extraction, studies of linkage to known LCA genes, automated sequencing and polymorphism analyses for haplotype assessments were done. RESULTS: We examined 12 affected and 15 unaffected family members. By history, there were an additional nine blind people in the four pedigrees. In each pedigree a consanguineous marriage was evident. We found a homozygous nonsense mutation in the AIPL1 gene, which replaces a tryptophan with a stop codon (Trp278X). The phenotype is severe and variable, despite the common molecular genetic etiology in each family. Affected patients had hand motion to no light perception vision and fundus findings ranging from maculopathy to diffuse pigmentary retinopathy. Three affected members had definite keratoconus, and two were suspects based on mild cone formation in the cornea of at least one eye. INTERPRETATION: We have identified four Pakistani families with a severe form of LCA that is associated with severe keratoconus in some affected members. The molecular etiology in all four families is a homozygous nonsense mutation, Trp278X, in the photoreceptor-pineal gene AIPL1. To our knowledge, this is one of the first phenotype-genotype correlations of AIPL1-associated LCA.     

Exclusion of LCA5 locus in a consanguineous Turkish family with macular coloboma-type LCA

BACKGROUND: Leber's congenital amaurosis (LCA) is an inherited retinal dystrophy, which causes severe visual impairment in early childhood. Recent molecular genetic studies have linked 11 loci (AIPL1, CRB1, CRX, GUCY2D, RPE65, RDH12, RPGRIP1, TULP1, LCA3, LCA5, and LCA9) to LCA. LCA5 is a new locus, which maps to the 6q11-q16 chromosomal region and was found to be associated with macular coloboma-type LCA in a Pakistani family. Herein, we describe the molecular genetic features of a consanguineous Turkish family in which four children have macular coloboma-type LCA. METHODS: Haplotype analysis was performed on the DNA of the family members using microsatellite markers against GUCY2D, RPE65, and LCA5. Genomic DNA was screened for mutations by means of single-strand conformational polymorphism (SSCP) analysis in exons of the RPE65 and CRX genes. RESULTS: In haplotype analysis, no linkage to LCA5 or GUCY2D loci was detected. None of the tested markers showed homozygosity or segregation between affected siblings. PCR-SSCP mutation analysis revealed no mutations in the screened RPE65 and CRX genes. CONCLUSION: We excluded LCA5 as the genetic cause of macular coloboma-type LCA in this Turkish family. Macular coloboma-type LCA shows genetic heterogeneity and it is not possible to establish a phenotype-genotype correlation with LCA5 and macular coloboma.     

A missense mutation in GUCY2D acts as a genetic modifier in RPE65-related Leber Congenital Amaurosis

Leber congenital amaurosis (LCA) is a clinically and genetically heterogeneous severe retinal dystrophy presenting in infancy. To explain the phenotypical variability observed in two affected siblings of a consanguineous pedigree diagnosed with LCA and establish a genotype-phenotype correlation, we screened GUCY2D, RPE65, CRX, AIPL1, and RPGRIP1 for mutations. The more severely affected sibling carried a heterozygous missense mutation in the GUCY2D gene (Ile539Val), which did not segregate with the disease phenotype. Subsequently, a homozygous nonsense mutation (Glu102STOP) in the RPE65 gene was identified in both affected siblings, thus identifying the causative gene. This data provides evidence for the presence of genetic modulation in LCA. It appears that the heterozygous GUCY2D mutation further disrupts the already compromised photoreceptor function resulting in more severe retinal dysfunction in the older sibling. We suggest that the unusual phenotypic variability in these two siblings with LCA is caused by the modifying effect of a heterozygous GUCY2D mutation observed against the disease background of a homozygous RPE65 mutation.     

Mutation screening of Pakistani families with congenital eye disorders

To map the disease loci several Pakistani families suffering from autosomal recessive retinitis pigmentosa with preserved para-arteriolar retinal pigment epithelium and Leber congenital amaurosis (LCA) were analyzed. Analysis revealed close genetic linkage between the disease phenotype of some of the families (3330RP, 111RP and 010LCA) and the microsatellite markers on chromosome 1q31. Mutation screening of the candidate gene CRB1 revealed a G to A transversion in exon 7 in arRP family 330RP and a T to C substitution in another arRP family, 111RP. In exon 9 of the CRB1 gene a T to C transversion was found in the family suffering from LCA (010LCA).The LCA phenotype of another family (011LCA) in which the CRB1 locus was excluded, showed linkage with microsatellite markers D17S1294 and D17S796 on chromosome 17p13.1. The association of the candidate gene GUCY2D (17p13.1) with the disease phenotype was excluded as no disease-associated mutation was found in any of its exons. Mutation screening of another candidate gene, AIPL1 located in the same region, showed a novel homozygous C to A substitution in exon 2. These sequence changes are unique for the Pakistani families and some of these have not been reported previously.     

Mutation screen of the TUB gene in patients with retinitis pigmentosa and Leber congenital amaurosis

TUB is the first identified member of the TULP family of four proteins with unknown function. A spontaneous mutation in murine tub causes retinal degeneration, obesity, and deafness. Mutations in another member of the TULP family, TULP1, are a cause of autosomal recessive retinitis pigmentosa (RP). These findings prompted us to investigate TUB as a candidate gene for RP and Leber congenital amaurosis (LCA). A mutation screen of the entire coding region of the TUB gene in 159 unrelated patients with autosomal recessive RP, 114 unrelated patients with simplex RP, and 21 unrelated patients with LCA uncovered 18 sequence variations. Of these, seven were missense mutations, six were isocoding changes, and five were intronic polymorphisms. All seven missense mutations were identified as heterozygous changes and no defect could be found in the other allele. None of the isocoding variants or intronic polymorphisms are predicted to create or destroy splice donor or acceptor sites based on splice-site prediction software. Although variant alleles of the TUB gene were found, none could be definitively associated with a specific retinal disease.     

Ⅱ型Usher综合征或39型视网膜色素变性患者的临床表型、致病基因与新突变位点分析

目的观察Ⅱ型Usher综合征(USH2)或39型视网膜色素变性患者的临床表型,分析患者的致病基因与新突变位点。方法收集临床诊断为USH2或39型视网膜色素变性患者的相关资料和样本,进行全外显子测序,对测序结果进行分析后锁定致病基因与突变位点,并用Sanger测序验证其突变位点。结果本研究共收集5例患者,来自于3个不同家系,测序数据解读结果显示USH2A基因为5例患者的致病基因,在USH2A基因上共检出3个纯合突变。先证者1(F1-Ⅱ1)及其患病的弟弟(F1-Ⅱ2)在USH2A基因上均存在c.8232G>C (p.W2744C)(M1)纯合突变。先证者2(F2-Ⅱ1)和其患病的妹妹(F2-Ⅱ2)则在USH2A基因上均存在c.8559-2A>G(M2)纯合突变。先证者3(F3-Ⅱ1)在USH2A基因上发现c.12778C>T(p.Q4260X)(M3)纯合突变。其中所发现的M3突变为首次报道。结论 USH2A基因突变是导致USH2和39型视网膜色素变性的主要致病原因,USH2A基因突变所致疾病具有典型的遗传异质性和临床异质性,不同的突变所致疾病表型存在差异。     

CRB1 heterozygotes with regional retinal dysfunction: implications for genetic testing of leber congenital amaurosis

PURPOSE: To test human CRB1 heterozygotes for possible clinical or functional retinal changes and to evaluate whether a patient with Leber congenital amaurosis (LCA) with CRB1 mutations not consistent with previously described CRB1 phenotypes carried a modifier allele in another LCA gene. METHODS: Seven unrelated heterozygous carriers of CRB1 mutations underwent phenotyping by full eye examinations (indirect ophthalmoscopy and slit lamp biomicroscopy) and functional testing (standard full-field electroretinography [ERG] and multifocal ERG). For genotyping of the LCA patients and their parents, denaturing high-performance liquid chromatography (dHPLC) analyses were performed, followed by sequence analysis of CRB1, followed by sequence analysis of the AIPL1 and CRX genes to identify a putative modifier effect in a patient with an atypical CRB1 phenotype. RESULTS: Reduced full-field ERG b-wave amplitudes were observed with scotopic -2 dB flash (140 microV; P < 0.05), normal full-field cone ERGs, and significant regional retinal dysfunction on mfERG in five of seven carriers of CRB1 mutations. A known AIPL1 mutation (p. R302L) was identified as a potential modifier allele in a patient with LCA carrying two CRB1 mutations and with a prominent maculopathy. CONCLUSIONS: In human heterozygotes of CRB1 mutations (parents of offspring with LCA), distinctive regional retinal dysfunctions were found by multifocal ERG measurements that were consistent with the focal histologic abnormalities reported for the two CRB1 knockout mice models. This phenotypic finding may identify CRB1 carriers and point to the causal gene defect in affected LCA offspring, significantly facilitating the molecular diagnostic process. Evidence suggests a modifier allele in AIPL1 in a patient with LCA with prominent atrophic macular lesions and homozygous defects in CRB1.     

Normal range of hearing associated with myocilin Thr377Met

Leber congenital amaurosis (LCA) is a clinically and genetically heterogeneous severe retinal dystrophy presenting in infancy. To explain the phenotypical variability observed in two affected siblings of a consanguineous pedigree diagnosed with LCA and establish a genotype-phenotype correlation, we screened GUCY2D, RPE65, CRX, AIPL1, and RPGRIP1for mutations. The more severely affected sibling carried a heterozygous missense mutation in the GUCY2Dgene (Ile539Val), which did not segregate with the disease phenotype. Subsequently, a homozygous nonsense mutation (Glu102STOP) in the RPE65gene was identified in both affected siblings, thus identifying the causative gene. This data provides evidence for the presence of genetic modulation in LCA. It appears that the heterozygous GUCY2D mutation further disrupts the already compromised photoreceptor function resulting in more severe retinal dysfunction in the older sibling. We suggest that the unusual phenotypic variability in these two siblings with LCA is caused by the modifying effect of a heterozygous GUCY2D mutation observed against the disease background of a homozygous RPE65mutation.