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.     

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.     

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.     

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.     

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.     

Lack of fundus autofluorescence to 488 nanometers from childhood on in patients with early-onset severe retinal dystrophy associated with mutations in RPE65

PURPOSE: Fundus autofluorescence is due to accumulation of lipofuscin in the retinal pigment epithelium (RPE) resulting from incomplete digestion of N-retinylidene-phosphatidyl-ethanolamine from shed photoreceptor outer segment discs. Alteration in autofluorescence reflects changes in lipofuscin content of the RPE. Mutations on both alleles of RPE65 result in absent or largely decreased formation of rhodopsin, due to a defect in all-trans retinol isomerization in the RPE. Autofluorescence could therefore be altered. This study was conducted to evaluate fundus autofluorescence in patients with early-onset severe retinal dystrophy (EOSRD, or early-onset rod-cone dystrophy) associated with mutations on both alleles of RPE65. DESIGN: Case series. PARTICIPANTS AND CONTROLS: Ten 10- to 55-year-old patients with EOSRD and compound heterozygous or homozygous mutations in RPE65. For comparison, 6 heterozygous parents and 2 patients with other forms of EOSRD were examined. METHODS: Participants underwent, in addition to standard clinical and electrophysiological examination, autofluorescence imaging using a confocal scanning laser ophthalmoscope. Three of the patients were also examined by optical coherence tomography (OCT) to evaluate the status of retinal degeneration. Mutations in 7 patients have been reported previously; the other patients were investigated by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing for mutations in RPE65 and lecithin retinol acyltransferase (LRAT). MAIN OUTCOME MEASURES: Fundus autofluorescence and OCT. RESULTS: Absent or minimal autofluorescence was found in all patients with compound heterozygous or homozygous RPE65 mutations. Autofluorescence was normal in the heterozygous parents. Autofluorescence was present in 2 children with EOSRD not associated with mutations in RPE65 or LRAT, another gene involved in retinol recycling. Optical coherence tomography in younger patients revealed an intraretinal appearance similar to that of their healthy, heterozygous parents. CONCLUSIONS: Lack of autofluorescence in patients with EOSRD associated with mutations in RPE65 is in accordance with the biochemical defect and can be used as a clinical marker of this genotype. Optical coherence tomography results in younger patients would indicate still viable photoreceptors despite the absence of autofluorescence.     

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.     

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.     

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.     

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.     

Evaluation of genotype-phenotype associations in leber congenital amaurosis

PURPOSE: To describe the clinical phenotypes associated with various genotypes known to cause Leber congenital amaurosis (LCA). METHODS: One hundred ten LCA patients were screened for various probable disease-causing gene sequence variations. Those patients with a probable disease-causing sequence variation in one of six genotypes were recalled for a follow-up examination. Evaluations included assessment of visual acuity, slit-lamp biomicroscopy, and dilated fundus examination. When possible, Goldmann perimetry was also performed. RESULTS: Of the 37 LCA patients with suspected disease-causing sequence variations, 7 had an AIPL1 variation, 8, a CRB1 variation, 2, a CRX variation, 4, a GUCY2D variation, 11, an RPE65 variation, and 5, an RPGRIP1 variation. Across the 6 genotypes, we observed a wide range of visual acuities from 20/40 to no light perception. The widest range of vision was noted for patients with a CRB1 or RPE65 variation. Younger patients with an AIPL1 or RPGRIP1 variation were found to have severely reduced vision. Drusenlike deposits were more selectively observed in patients with mutations in the AIPL1, CRB1, RPE65, and RPGRIP1 genes, whereas focal regions of peripheral chorioretinal atrophy were observed only in patients with AIPL1 or RPE65 variations. Neurologic, intellectual, or psychomotor developmental delay was noted in 8.1% of our cohort. CONCLUSIONS: There was considerable overlap of phenotypic expression in six genetic subtypes in our LCA cohort. However, phenotypic trends were noted in our patients' visual acuities and posterior segment findings within genotypes. These findings have practical value for genetic screening strategies for LCA patients based upon phenotype as well as for counseling patients on their visual prognosis.     

Mutation screening of 299 Spanish families with retinal dystrophies by Leber congenital amaurosis genotyping microarray

PURPOSE: Leber Congenital Amaurosis (LCA) is one of the most severe inherited retinal dystrophies with the earliest age of onset. This study was a mutational analysis of eight genes (AIPL1, CRB1, CRX, GUCY2D, RPE65, RPGRIP1, MERTK, and LRAT) in 299 unrelated Spanish families, containing 42 patients with initial diagnosis of LCA: 107 with early-onset autosomal recessive retinitis pigmentosa (ARRP; onset <10 years of age) and 150 with non-early-onset ARRP (onset, >10 years of age). METHODS: Samples were studied by using a genotyping microarray (Asper Biotech, Ltd., Tartu, Estonia) followed by a family study in cases with potential digenism/triallelism. RESULTS: The frequencies of alleles carrying disease-causing mutations found in the authors'cohort using the chip were 23.8% (20/84) for LCA with 13 families carrying mutations, 6.1% (13/214) for early-onset ARRP with 12 families carrying mutations, and 4.3% (13/300) for non-early-onset ARRP with 12 families carrying mutations. CRB1 was the most frequently found mutated gene in affected Spanish families. Five families with anticipated digenism or triallelism were further studied in depth. Digenism could be discarded in all these cases; however, triallelism could not be ruled out. CONCLUSIONS: CRB1 is the main gene responsible for LCA in the Spanish population. Sequence changes p.Asp1114Gly (RPGRIP1), p.Pro701Ser (GUCY2D), and p.Tyr134Phe (AIPL1) were found at similar frequencies in patients and control subjects. The authors therefore suggest that these changes be considered as polymorphism or modifier alleles, rather than as disease-causing mutations. The LCA microarray is a quick and reasonably low-cost first step in the molecular diagnosis of LCA. The diagnosis should be completed by conventional laboratory analysis as a second step. This stepwise proceeding permits detection of novel disease-causing mutations and identification of cases involving potential digenism/triallelism. Previous accurate ophthalmic diagnosis was found to be indispensable.     

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.     

Spectrum of Disease Severity in Nonsyndromic Patients With Mutations in the CEP290 Gene: A Multicentric Longitudinal Study

PurposeThe purpose of this study was to perform a detailed longitudinal phenotyping and genetic characterization of 32 Italian patients with a nonsyndromic retinal dystrophy and mutations in the CEP290 gene.MethodsWe reviewed the clinical history and examinations of 32 patients with a nonsyndromic retinal dystrophy due to mutations in the CEP290 gene, followed up (mean follow-up: 5.9 years) at 3 Italian centers. The clinical examinations included: best corrected visual acuity (BCVA), optical coherence tomography (OCT), and full-field electroretinogram (ERG).ResultsPatients (mean age = 19.0 ± 3.4 years) had a mean BCVA of 1.73 ± 0.20 logMAR. Longitudinal analysis of BCVA showed a nonsignificant decline. Central retinal thickness (CRT) declined significantly with age at an exponential rate of 1.0%/year (P = 0.001). At disease onset, most patients (19/32; 49.4%) had nystagmus. The absence of nystagmus was significantly associated with better BCVA and more preserved CRT (P < 0.05). ERG showed undetectable responses in most patients (64.0%), whereas reduced scotopic and photopic responses were observed in four patients (16.0%) who had no nystagmus. We identified 35 different variants, among which 12 were novel. Our genotype-phenotype correlation analysis shows a significantly worse BCVA in patients harboring a loss-of-function mutation and the deep-intronic variant c.2991+1655A>G.ConclusionsOur study highlights a mild phenotype of the disease, characterized by absence of nystagmus, good visual acuity, considerably preserved retinal morphology, and recordable ERG, confirming the wide spectrum of CEP290-related retinal dystrophies. Finally, in our cohort, the deep intronic variant c.2991+1655A>G was associated with a more severe phenotype.     

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.     

Leber congenital amaurosis: comprehensive survey of genetic heterogeneity. A clinical definition update

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies, responsible for congenital blindness. Disease-associated mutations have been hitherto reported in seven genes. These genes are all expressed preferentially in the photoreceptor cells or the retinal pigment epithelium, but they are involved in strikingly different physiologic pathways, resulting in an unforeseeable pathophysiologic variety. This broad genetic and physiologic heterogeneity, which could greatly increase in the coming years, hinders molecular diagnosis in LCA patients. Genotyping is, however, required to establish genetically defined subgroups of patients ready for therapy. Here we report a comprehensive mutational analysis of all the known genes in 179 unrelated LCA patients, including 52 familial and 127 sporadic (27/127 consanguineous) cases. Mutations were identified in 47.5% of patients. GUCY2D accounted for by far the largest part of the LCA cases in our series (21.2%), followed by CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), AIPL1 (3.4%), TULP1 (1.7%) and CRX (0.6%). The clinical history of all patients with mutations was carefully revisited in the search for phenotype variations. Genotype-phenotype correlations were found that made it possible to divide patients into two main groups. The first one includes patients whose symptoms fit the traditional definition of LCA, i.e., congenital or very early cone-rod dystrophy, while the second group gathers patients affected with severe yet progressive rod-cone dystrophy. In addition, objective ophthalmologic data subdivided each group into two subtypes. Based on these findings, we have drawn decisional flowcharts directing the molecular analysis of LCA genes in a given case. These flowcharts will hopefully lighten the onerous task of genotyping new patients, but only if the most precise clinical history since birth is available.     

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.     

Leber congenital amaurosis: a genetic paradigm

Leber congenital amaurosis (LCA; estimated prevalence 1 : 50,000-100,000) is an early-onset inherited cause of childhood blindness characterized by a severe retinal dystrophy immediately after birth. Variants in at least six genes, AIPL1, CRB1, CRX, GUCY2D, RPE65, and RPGRIP1, have been associated with a diagnosis consistent with LCA or early-onset retinitis pigmentosa and together account for less than 50% of all LCA cases. Genetically heterogeneous inheritance has complicated the molecular analysis of LCA cases, especially sporadic ones where conventional methods are of limited value. Until recently, the management of patients with LCA relied mainly on clinical examination, electrophysiology, and other ancillary tests. Genotyping, i.e., determining the exact genetic defect causing LCA in each specific case, was not routinely performed since the comprehensive screening of six genes by SSCP and/or direct sequencing is relatively inefficient and cost-prohibitive. Patients, therefore, were often left with no specific information on their disease status. Recent advances in genotyping technologies have allowed the introduction of comprehensive and affordable screening procedures to determine causal genetic variation, resulting in precise molecular diagnosis, more accurate visual prognosis, and suggestions towards treatment options.     

Genotyping microarray (disease chip) for Leber congenital amaurosis: detection of modifier alleles

PURPOSE: Leber congenital amaurosis (LCA) is an early-onset inherited disorder of childhood blindness characterized by visual impairment noted soon after birth. Variants in at least six genes (AIPL1, CRB1, CRX, GUCY2D, RPE65, and RPGRIP1) have been associated with a diagnosis consistent with LCA or early-onset retinitis pigmentosa (RP). Genetically heterogeneous inheritance complicates the analyses of LCA cases, especially in patients without a family history of the disorder, and conventional methods are of limited value. METHODS: To overcome these limitations, arrayed primer extension (APEX) technology was used to design a genotyping microarray for early-onset, severe retinal degenerations that includes all of the >300 disease-associated variants currently described in eight genes (in addition to the six just listed, the early-onset RP genes LRAT and MERTK were added). The resultant LCA array allows simultaneous detection of all known disease-associated alleles in any patient with early-onset RP. The array was validated by screening 93 confirmed patients with LCA who had known mutations. Subsequently, 205 novel LCA cases were screened on the array, followed by segregation analyses in families, if applicable. RESULTS: The microarray was >99% effective in determining the existing genetic variation and yielded at least one disease-associated allele in approximately one third of the novel patients. More than two (expected) variants were discovered in a substantial fraction (22/300) of the patients, suggesting a modifier effect from more than one gene. In support of the latter hypothesis, the third allele segregated with a more severe disease phenotype in at least five families. CONCLUSIONS: The LCA genotyping microarray is a robust and cost-effective screening tool, representing the prototype of a disease chip for genotyping patients with a genetically heterogeneous condition. Simultaneous screening for all known LCA-associated variants in large LCA cohorts allows systematic detection and analysis of genetic variation, facilitating prospective diagnosis and ultimately predicting disease progression.     

Identification of novel mutations in patients with Leber congenital amaurosis and juvenile RP by genome-wide homozygosity mapping with SNP microarrays

PURPOSE: Leber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) cause severe visual impairment early in life. Thus far, mutations in 13 genes have been associated with autosomal recessive LCA and juvenile RP. The purpose of this study was to use homozygosity mapping to identify mutations in known LCA and juvenile RP genes. METHODS: The genomes of 93 consanguineous and nonconsanguineous patients with LCA and juvenile RP were analyzed for homozygous chromosomal regions by using SNP microarrays. This patient cohort was highly selected, as mutations in the known genes had been excluded with the LCA mutation chip, or a significant number of LCA genes had been excluded by comprehensive mutation analysis. Known LCA and juvenile RP genes residing in the identified homozygous regions were analyzed by sequencing. Detailed ophthalmic examinations were performed on the genotyped patients. RESULTS: Ten homozygous mutations, including seven novel mutations, were identified in the CRB1, LRAT, RPE65, and TULP1 genes in 12 patients. Ten patients were from consanguineous marriages, but in two patients no consanguinity was reported. In 10 of the 12 patients, the causative mutation was present in the largest or second largest homozygous segment of the patient's genome. CONCLUSIONS: Homozygosity mapping using SNP microarrays identified mutations in a significant proportion (30%) of consanguineous patients with LCA and juvenile RP and in a small number (3%) of nonconsanguineous patients. Significant homozygous regions which did not map to known LCA or juvenile RP genes and may be instrumental in identifying novel disease genes were detected in 33 patients.