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.     

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.     

Mutational analysis and clinical correlation in Leber congenital amaurosis

Leber congenital amaurosis (LCA, MIM 204001) is a clinically and genetically heterogeneous retinal disorder characterized by severe visual loss from birth, nystagmus, poor pupillary reflexes, retinal pigmentary or atrophic changes, and a markedly diminished electroretinogram (ERG). Purpose: To examine 100 consecutive patients with LCA in order to assess the relative burden of the three known genes involved in LCA, namely retinal guanylyl cyclase ( GUCY2D ), retinal pigment epithelium protein ( RPE65 ), and the cone-rod homeobox ( CRX ), and to define their clinical correlates. Methods: Mutational analysis and detailed clinical examinations were performed in patients diagnosed with LCA at the Johns Hopkins Center for Hereditary Eye Diseases and the Montreal Children’s Hospital. Results: Mutations were identified in 11% of our patients: GUCY2D mutations accounted for 6%, while RPE65 and CRX gene mutations accounted for 3% and 2%, respectively. The clinical presentation was variable; however, the visual evolution in patients with mutations in GUCY2D and CRX remained stable, while individuals with mutations in the RPE65 gene showed progressive visual loss. Conclusions: This study suggests that molecular diagnosis of Leber congenital amaurosis could provide important information concerning prognosis and course of treatment.     

Clinical and molecular genetics of Leber's congenital amaurosis: a multicenter study of Italian patients

PURPOSE: To identify the molecular basis of Leber's congenital amaurosis (LCA) in a cohort of Italian patients and to perform genotype-phenotype analysis. METHODS: DNA samples from 95 patients with LCA were analyzed by using a microarray chip containing disease-associated sequence variants in eight LCA genes. In addition, all patients in whom no mutations were identified by microarray were subjected to sequence analysis of the CEP290 gene. Patients with mutations identified underwent a detailed ophthalmic evaluation. RESULTS: Disease-causing mutations were identified in 28% of patients, and twelve novel variants were identified. Mutations occurred more frequently in the RPE65 (8.4%), CRB1 (7.4%), and GUCY2D (5.2%) genes. Mutations in CEP290 were found in only 4.2% of the patients analyzed. Clinical assessment of patients carrying RPE65 or CRB1 mutations revealed the presence of retained visual capabilities in the first decade of life. RPE65 mutations were almost always associated with normal macular thickness, as assessed by optical coherence tomography (OCT), whereas CRB1 mutations were associated with reduced retinal thickness and a coarsely laminated retina. Fundus autofluorescence was mostly observed in patients with RPE65 and GUCY2D mutations and was not elicitable in patients carrying CRB1. CONCLUSIONS: RPE65 gene mutations represented a significant cause of LCA in the Italian population, whereas GUCY2D and CEP290 mutations had a lower frequency than that found in other reports. This finding suggests that the genetic epidemiology of LCA in Italy is different from that reported in the United States and in northern European countries. Autofluorescence in patients with RPE65 mutations was more frequently associated with preserved retinal thickness, which suggests that these mutations are not associated with progression of retinal degeneration. Therefore, normal retinal thickness (identified with OCT) and fundus autofluorescence may be the means with which to identify patients with LCA who carry RPE65 mutations, which are expected to be a potential gene therapy target in the near future.     

Clinicopathologic effects of mutant GUCY2D in Leber congenital amaurosis

PURPOSE: To study the retinal degeneration in an 11 -year-old patient with Leber congenital amaurosis (LCA) caused by mutation in GUCY2D. STUDY DESIGN: Comparative human tissue study. PARTICIPANTS: Two subjects with LCA; postmortem eye from one LCA patient and three normal donors. METHODS: Clinical and visual function studies were performed between the ages of 6 and 10 years in the LCA eye donor and at age 6 in an affected sibling. Genomic DNA was screened for mutations in known LCA genes. The retina of the 11 -year-old subject with LCA was compared with normal retinas from donors age 3 days, 18 years, and 53 years. The tissues were processed for histopathologic studies and immunofluorescence with retinal cell-specific antibodies. RESULTS: Vision in both siblings at the ages examined was limited to severely impaired cone function. Mutation in the GUCY2D gene was identified in both siblings. Histopathologic study revealed rods and cones without outer segments in the macula and far periphery. The cones formed a monolayer of cell bodies, but the rods were clustered and had sprouted neurites in the periphery. Rods and cones were not identified in the midperipheral retina. The inner nuclear layer appeared normal in thickness throughout the retina, but ganglion cells were reduced in number. CONCLUSIONS: An 11-year-old subject with LCA caused by mutant GUCY2D had only light perception but retained substantial numbers of cones and rods in the macula and far periphery. The finding of numerous photoreceptors at this age may portend well for therapies designed to restore vision at the photoreceptor level.     

Leber congenital amaurosis: genes, proteins and disease mechanisms

Leber congenital amaurosis (LCA) is the most severe retinal dystrophy causing blindness or severe visual impairment before the age of 1 year. Linkage analysis, homozygosity mapping and candidate gene analysis facilitated the identification of 14 genes mutated in patients with LCA and juvenile retinal degeneration, which together explain approximately 70% of the cases. Several of these genes have also been implicated in other non-syndromic or syndromic retinal diseases, such as retinitis pigmentosa and Joubert syndrome, respectively. CEP290 (15%), GUCY2D (12%), and CRB1 (10%) are the most frequently mutated LCA genes; one intronic CEP290 mutation (p.Cys998X) is found in approximately 20% of all LCA patients from north-western Europe, although this frequency is lower in other populations. Despite the large degree of genetic and allelic heterogeneity, it is possible to identify the causative mutations in approximately 55% of LCA patients by employing a microarray-based, allele-specific primer extension analysis of all known DNA variants. The LCA genes encode proteins with a wide variety of retinal functions, such as photoreceptor morphogenesis (CRB1, CRX), phototransduction (AIPL1, GUCY2D), vitamin A cycling (LRAT, RDH12, RPE65), guanine synthesis (IMPDH1), and outer segment phagocytosis (MERTK). Recently, several defects were identified that are likely to affect intra-photoreceptor ciliary transport processes (CEP290, LCA5, RPGRIP1, TULP1). As the eye represents an accessible and immune-privileged organ, it appears to be uniquely suitable for human gene replacement therapy. Rodent (Crb1, Lrat, Mertk, Rpe65, Rpgrip1), avian (Gucy2D) and canine (Rpe65) models for LCA and profound visual impairment have been successfully corrected employing adeno-associated virus or lentivirus-based gene therapy. Moreover, phase 1 clinical trials have been carried out in humans with RPE65 deficiencies. Apart from ethical considerations inherently linked to treating children, major obstacles for the treatment of LCA could be the putative developmental deficiencies in the visual cortex in persons blind from birth (amblyopia), the absence of sufficient numbers of viable photoreceptor or RPE cells in LCA patients, and the unknown and possibly toxic effects of overexpression of transduced genes. Future LCA research will focus on the identification of the remaining causal genes, the elucidation of the molecular mechanisms of disease in the retina, and the development of gene therapy approaches for different genetic subtypes of LCA.     

Infantile and childhood retinal blindness: a molecular perspective (The Franceschetti Lecture)

Much progress has been made in the past five years in the understanding of Leber congenital amaurosis (LCA) and allied early-onset retinal dystrophies, various forms of stationary sensory retinal blindness, and genes that are involved in the development of the retina. Uncomplicated Leber congenital amaurosis has been associated with mutations of six genes: GUCY2D (encoding RetGC-1) at 17p13.1, RPE65 at 1q31, CRX at 19q13.3, AIPLI at 17p13.1, CRB1 at 1q31-3, and RPGRIP at 14q11. A similar early-onset severe retinal degeneration phenotype has been associated with mutation of TULP1 at 6p21.3. Leber appreciated that the condition he described merged with the phenotypes of early childhood-onset severe retinal degenerations. This insight has been confirmed at the molecular level for mutations of GUCY2D, RPE65, CRX, AIPL1, and CRB1, which cause not only LCA, but also early-childhood and even adult-onset retinal degenerations. This paper reviews the new finding of LCA from mutations of CRB1 and discusses the molecular basis of X-linked blue monochromacy, autosomal recessive congenital achromatopsia from mutations of the genes for ACHM2 (CNGA3) and ACHM3 (CNGB3), X-linked congenital stationary night blindness (CSNB) from mutations of CACNA1F (incomplete CSNB) and NYX (complete CSNB), and the enhanced S-cone syndrome from mutation of the developmental gene, NR2E3 at 15q23, which appears to regulate the development of M- and L-cones from S-cones. These discoveries have opened new areas of cellular and developmental biology for future research into the causes of retinal blindness.     

Clinical and genetic studies of an autosomal dominant cone-rod dystrophy with features of Stargardt disease

Much progress has been made in the past five years in the understanding of Leber congenital amaurosis (LCA) and allied early-onset retinal dystrophies, various forms of stationary sensory retinal blindness, and genes that are involved in the development of the retina. Uncomplicated Leber congenital amaurosis has been associated with mutations of six genes: GUCY2D (encoding RetGC-1) at 17p13.1, RPE65 at 1q31, CRX at 19q13.3, AIPLI at 17p13.1, CRB1 at 1q31-3, and RPGRIP at 14q11. A similar early-onset severe retinal degeneration phenotype has been associated with mutation of TULP1 at 6p21.3. Leber appreciated that the condition he described merged with the phenotypes of early childhood-onset severe retinal degenerations. This insight has been confirmed at the molecular level for mutations of GUCY2D , RPE65 , CRX , AIPL1 , and CRB1 , which cause not only LCA, but also early-childhood and even adult-onset retinal degenerations. This paper reviews the new finding of LCA from mutations of CRB1 and discusses the molecular basis of X-linked blue monochromacy, autosomal recessive congenital achromatopsia from mutations of the genes for ACHM2 ( CNGA3 ) and ACHM3 ( CNGB3 ), X-linked congenital stationary night blindness (CSNB) from mutations of CACNA1F (incomplete CSNB) and NYX (complete CSNB), and the enhanced S-cone syndrome from mutation of the developmental gene, NR2E3 at 15q23, which appears to regulate the development of M- and L-cones from S-cones. These discoveries have opened new areas of cellular and developmental biology for future research into the causes of retinal blindness.     

Progression of phenotype in Leber's congenital amaurosis with a mutation at the LCA5 locus

BACKGROUND: Leber's congenital amaurosis (LCA) accounts for 5% of inherited retinal disease and is usually inherited as an autosomal recessive trait. Genetic and clinical heterogeneity exist. Mutations have been described in the RPE65, CRB1, RPGRIP1, AIPL1, GUCY2D, and CRX genes and other pedigrees show linkage to the LCA3 and LCA5 loci. The latter is a new locus which maps to 6q11-q16. The ocular findings and the evolution of the macula staphyloma are described in five members of a Pakistani family with consanguinity and a mutation in the LCA5 gene. METHODS: 13 family members including five affected individuals consented to DNA analysis and ocular examination including fundal photography. RESULTS: Ocular abnormalities are described. The most striking feature was the progression of macula abnormalities in three brothers resulting in a colobomatous appearance in the eldest compared to only mild atrophy in the youngest. The phenotypic pattern of this mutation in this Pakistani family contrasts with the "Old Order River Brethren" who were of Swiss descent, in whom the mutation was first described. CONCLUSION: The evolution of a new phenotypic picture is presented to a mutation in LCA5.     

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先天性黑矇六家系临床表现和遗传分析

目的:观察分析Leber先天性黑矇（LCA）致病基因类型及临床表型特征。方法:回顾性临床研究。基因检测确诊的LCA患者6例及其家系成员18名纳入研究。患者分别来自6个无血缘关系家系。采集所有受检者外周静脉血,提取全基因组DNA。应用包含463个致病基因的遗传眼病捕获芯片进行靶向捕获富集高通量测序,对 TUL...     

Analysis of three genes in Leber congenital amaurosis in Indonesian patients

PURPOSE: To assess the frequency, the pattern of disease causing mutations, and phenotypic variations in patients with Leber congenital amaurosis (LCA) from Indonesia. PATIENTS AND METHODS: Twenty-one unrelated index cases with a clinical diagnosis of LCA were screened for mutations in the coding sequence of RetGC1, RPE65 and AIPL1 gene with single strand conformation polymorphism analysis followed by direct sequencing and restriction enzyme digestion. RESULTS: Four novel disease causing mutations were identified: Three in the RPE65 gene (106del9bp, G32V and Y435C) in two of 21 index cases and one in the AIPL1 (K14E). Two of them were homozygous and one was compound-heterozygous. No disease causing mutation was identified in RetGC1. CONCLUSIONS: The four novel disease causing mutations identified in this study confirmed the diagnosis of LCA which has not been recognized before in Indonesia. The frequency of RPE65 mutations was 9.5%; and of AIPL1 mutations 4.8%. This was in general accordance with previous studies reported from other countries. Unlike in those studies, no disease causing RetGC1 mutations could be identified in our patients. Phenotypically, the RPE65 and AIPL1 mutations identified in this study caused nearly total blindness by the second decade of life, but had a different onset of symptoms. The patients with the RPE65 mutations retained some useful visual function until the end of the first decade, which progressed to total blindness during the second decade of life, whereas the (homozygous) AIPL1 mutation was associated with nearly total blindness from infancy on. Therefore, RPE65 mutations have to be considered to cause early onset severe retinal degeneration (EOSRD), and AIPL1 mutations a form of LCA.     

Genetische und klinische Heterogenität bei LCA-Patienten

BACKGROUND: Leber congenital amaurosis (LCA) usually describes patients with severely reduced vision due to a retinal dystrophy in early childhood. METHODS: In 135 families in a case series with severely reduced vision due to a retinal dystrophy in early childhood a complete ophthalmologic examination was extended by two-color threshold perimetry, fundus autofluorescence (FAF), und optical coherence tomography (OCT). Mutation screening included AIPL1, CRB1, CRX, GUCY2D, LRAT, RPE65, RPGRIP, and TULP1. RESULTS: GUCY2D mutations caused the most severe phenotype with severely reduced vision from birth but unremarkable fundus appearance. RPE65 mutations were correlated with an obvious lack of FAF. CRB1 mutations showed a significantly thickened retina on OCT. CRX mutations were associated with a progressive form of cone-rod dystrophy. CONCLUSION: A genotype-phenotype correlation for selected genes allows an optimized strategy for the molecular genetic work-up.     

An assessment of the apex microarray technology in genotyping patients with Leber congenital amaurosis and early-onset severe retinal dystrophy

PURPOSE: Leber congenital amaurosis (LCA) and early-onset severe retinal dystrophy (EOSRD) are genetically heterogeneous, with 11 genes currently implicated. The LCA chip may be used to interrogate many variants in one hybridization reaction. The purpose of this study was to assess the utility of this technology. METHODS: One hundred fifty-three patients with LCA and EOSRD were screened using an array (Asper Ophthalmics, Tartu, Estonia) containing 344 published disease-causing variants and polymorphisms in eight genes: AIPL1, GUCY2D, CRB1, CRX, RPGRIP1, RPE65, MERTK, and LRAT. One hundred thirty-six probands underwent bidirectional sequencing of the full coding region of the RPE65 gene. The same technique was also used to confirm CRB1 and AIPL1 mutations initially identified with the Apex chip (Asper Ophthalmics). Single nucleotide polymorphism (SNP) analysis within control populations was performed for two variants, P701S and W21R, on the chip for GUCY2D. RESULTS: Of the possible 109,392 interrogations, 3,346 (3.06%) failed on one strand whereas 259 (0.47%) failed on both. The chip reported mutations in 68 (44%) patients; 26 patients had two alleles identified (17%). Direct sequencing of RPE65 showed no discrepancies, whereas sequencing of AIPL1 and CRB1 revealed seven samples called erroneously. The SNP analysis of both GUCY2D variants revealed equal prevalence in the EOSRD panel and the normal population. Subsequent reanalysis, after excluding these polymorphisms, revealed one (18.3%) or two (11.7%) mutations identified in 46 patients. When evaluated by diagnosis, 46% of patients with LCA had one or two mutations identified, compared with 24% of patients with EOSRD. CONCLUSIONS: This approach is a rapid and reasonably low-cost technique for identifying both previously identified mutations and common polymorphisms. The addition of further genes and mutations to the chip will improve its utility, though it is advised that all results be checked by direct sequencing.     

Genetics and phenotypes of RPE65 mutations in inherited retinal degeneration

PURPOSE: To characterize the spectrum of RPE65 mutations present in 453 patients with retinal dystrophy with an interest in understanding the range of functional deficits attributable to sequence variants in this gene. METHODS: The 14 exons of RPE65 were amplified by polymerase chain reaction (PCR) from patients' DNA and analyzed for sequence changes by single-strand conformation polymorphism (SSCP) and direct sequencing. Haplotype analysis was performed using RPE65 intragenic polymorphisms. Patients were examined clinically and with visual function tests. RESULTS: Twenty-one different disease-associated DNA sequence changes predicting missense or nonsense point mutations, insertions, deletions, and splice site defects in RPE65 were identified in 20 patients in homozygous or compound heterozygous form. In one patient, paternal uniparental isodisomy (UPD) of chromosome 1 resulted in homozygosity for a probable functional null allele. Eight of the disease-associated mutations (Y79H, E95Q, E102X, D167Y, 669delCA, IVS7+4a-->g, G436V, and G528V) and one mutation likely to be associated with disease (IVS6+5g-->a) have not been reported previously. The most commonly occurring sequence variant identified in the patients studied was the IVS1+5g-->a mutation, accounting for 9 of 40 (22.5%) total disease alleles. This splice site mutation, as well as R91W, the most common missense mutation, exists on at least two different genetic backgrounds. The phenotype resulting from RPE65 mutations appears to be relatively uniform and independent of mutation class, suggesting that most missense mutations (15 of 40 disease alleles [37.5%]) result in loss of function. At young ages, this group of patients has somewhat better subjective visual capacity than is typically associated with Leber congenital amaurosis (LCA) type I, with a number of patients retaining some useful visual function beyond the second decade of life. CONCLUSIONS: RPE65 mutations account for a significant percentage (11.4%) of disease alleles in patients with early-onset retinal degeneration. The identification and characterization of patients with RPE65 mutations is likely to represent an important resource for future trials of rational therapies for retinal degeneration.     

Novel membrane frizzled‐related protein gene mutation as cause of posterior microphthalmia resulting in high hyperopia with macular folds

Purpose: We present a genetic and clinical analysis of two sisters, 3 and 4 years of age, with nanophthalmos and macular folds. Methods: Ophthalmological examination, general paediatric examination and molecular genetic analysis of the MFRP gene were performed in both affected siblings. Results: Clinical analysis showed high hyperopia (+11 D and +12 D), short axial lengths (15 mm) and the presence of macular folds and optic nerve head drusen. Autofluorescence of the retina was generally normal with subtle macular abnormalities. Sequence analysis showed compound heterozygosity for severe MFRP mutations in both sisters: a previously reported p.Asn167fs (c.498dupC) and a novel stop codon mutation p.Gln91X (c.271C>T). Conclusion: These are the youngest nanophthalmos patients in the literature identified with severe loss of MFRP function, showing already the known structural abnormalities for this disease. Adult patients affected by homozygous or compound heterozygous MFRP mutations generally show signs of retinal dystrophy, with ERG disturbances and RPE abnormalities on autofluorescence imaging. ERG examination could not be performed in these children, but extensive RPE abnormalities were not seen at this young age.     

LCA2型基因治疗临床试验研究进展

Leber先天性黑矇(Leber''s congenital amaurosis,LCA)是一种遗传性致盲性眼病,在婴儿早期出现严重的视力低下或丧失丧失。该疾病的LCA2型与RPE65的突变相关。既往对于LCA2在内的遗传性视网膜疾病无有效治疗方法。近年来,随着基因治疗技术的进步,遗传性视网膜疾病的治疗进展取得了巨大进步,其中最成功的便是LCA2的基因治疗。本文简要介绍了LCA2基因治疗的发展,并对既往LCA2临床试验中的注射剂型、剂量、注射方式、测量方法、治疗效果与年龄的相关性和治疗效果的稳定性进行综述,为LCA2基因治疗进入我国临床工作提供参考及临床治疗经验。     

Novel complex GUCY2D mutation in Japanese family with cone-rod dystrophy

PURPOSE: All mutations in the retinal guanylate cyclase gene (GUCY2D) that causes autosomal dominant cone-rod dystrophy (CORD) are associated with an amino acid substitution in codon 838. A novel heterozygous complex missense mutation of I915T and G917R in the GUCY2D gene was found in a Japanese family with autosomal dominant CORD. The clinical features associated with this mutation were described. METHODS: Blood samples were collected from 27 patients with cone-rod or cone dystrophies and from 11 patients with macular dystrophy. Genomic DNA was extracted from peripheral leukocytes. All 18 coding exons of the GUCY2D gene were directly sequenced. The PCR product carrying a novel mutation was subcloned, and each allele was sequenced. A complete ophthalmologic examination was performed in members of the family with the novel mutation. RESULTS: A novel heterozygous complex missense mutation of T2817C and G2822C that would predict I915T and G917R amino acid substitutions, respectively, was found in an autosomal dominant CORD family. The two nucleotide changes were located on the same allele, and segregated with the disease. Two other known missense mutations of R838H and R838C were found in two other CORD families. The clinical phenotype associated with the novel mutation was similar to that with the Arg838 mutations. CONCLUSIONS: A heterozygous complex mutation of I915T and G917R in the GUCY2D gene caused autosomal dominant CORD, indicating that a heterozygous mutation that does not include a codon 838 substitution can lead to this ocular phenotype.     

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.     

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.