A Comprehensive Analysis of Choroideremia: From Genetic Characterization to Clinical Practice.

Choroideremia (CHM) is a rare X-linked disease leading to progressive retinal degeneration resulting in blindness. The disorder is caused by mutations in the CHM gene encoding REP-1 protein, an essential component of the Rab geranylgeranyltransferase (GGTase) complex. In the present study, we evaluated a multi-technique analysis algorithm to describe the mutational spectrum identified in a large cohort of cases and further correlate CHM variants with phenotypic characteristics and biochemical defects of choroideremia patients. Molecular genetic testing led to the characterization of 36 out of 45 unrelated CHM families (80%), allowing the clinical reclassification of four CHM families. Haplotype reconstruction showed independent origins for the recurrent p.Arg293* and p.Lys178Argfs*5 mutations, suggesting the presence of hotspots in CHM, as well as the identification of two different unrelated events involving exon 9 deletion. No certain genotype-phenotype correlation could be established. Furthermore, all the patients´ fibroblasts analyzed presented significantly increased levels of unprenylated Rabs proteins compared to control cells; however, this was not related to the genotype. This research demonstrates the major potential of the algorithm proposed for diagnosis. Our data enhance the importance of establish a differential diagnosis with other retinal dystrophies, supporting the idea of an underestimated prevalence of choroideremia. Moreover, they suggested that the severity of the disorder cannot be exclusively explained by the genotype.

Clinical aspects of Usher syndrome and the USH2A gene in a cohort of 433 patients.

IMPORTANCE: A new statistical approach is needed to describe the clinical differences between type I and type II Usher syndrome and between the 2 most frequent mutations in the USH2A gene. OBJECTIVES: To describe the primary phenotypic characteristics and differences between type I and type II Usher syndrome and to establish a phenotype-genotype correlation for the 2 most frequent mutations in the USH2A gene. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional study at a genetics department, in which clinical evaluations were performed for 433 patients (297 unrelated families) who were classified as having type I, II, III, atypical, or unclassified Usher syndrome according to their clinical history, pedigree data, results from ophthalmological studies, and audiological, neurophysiological, and vestibular test results. Molecular studies were performed for 304 patients (256 unrelated families). The Mann-Whitney U test or the χ2 test was used for calculating the differences between mean values for the analyzed parameters. MAIN OUTCOMES AND MEASURES: Age at diagnosis; age at onset of night blindness, visual field loss, visual acuity loss, and cataracts; and severity and age at diagnosis of hearing loss. RESULTS: The comparison between patients with type I Usher syndrome and those with type II Usher syndrome revealed P < .001 for most items analyzed. The most frequent mutations in the USH2A gene were the p.Glu767Serfs*21 and p.Cys759Phe mutations, with an allelic frequency of 23.2% (63 of 272 alleles) and 8.1% (22 of 272 alleles), respectively. The phenotypic analysis for patients carrying p.Cys759Phe showed P < .001 for most items analyzed when compared with patients carrying p.Glu767Serfs*21 and when compared with patients carrying other mutations in the USH2A gene. None of the p.Cys759Phe patients exhibited a severe hearing loss phenotype, and more than 60% had only mild hearing loss. Most patients carrying the p.Glu767Serfs*21 mutation (72.1%) were moderately deaf. CONCLUSIONS AND RELEVANCE: Our study presents the clinical differences between type I and type II Usher syndrome and between the 2 most frequent mutations in the USH2A gene. Detailed genotype-phenotype correlations, as presented in our study, allow for a better correlation of clinical signs with a known genotype and can improve the clinical management, genetic counseling, and risk assessment of patients with Usher syndrome because an estimated prognosis of their disease can be made.

Prevalence of Rhodopsin mutations in autosomal dominant Retinitis Pigmentosa in Spain: clinical and analytical review in 200 families.

PURPOSE: We aimed to determine the prevalence of mutations in the RHO gene in Spanish families with autosomal dominant Retinitis Pigmentosa (adRP), to assess genotype-phenotype correlations and to establish an accurate diagnostic algorithm after 23 years of data collection. PATIENTS AND METHODS: Two hundred patients were analysed through a combination of denaturing gradient gel electrophoresis, single-strand conformation polymorphism, genotyping microarray and Sanger sequencing of the RHO gene. RESULTS: Overall, 42 of 200 Spanish adRP families were mutated for RHO (21.0%). Twenty-seven different RHO mutations were detected; seven of them were novel. A genotype-phenotype correlation was established with clinical data from 107 patients. The most prevalent p.Pro347Leu mutation, responsible for 4.5% (9/200) of all mutated adRP families, was associated with a phenotype of early onset and severe course diffuse RP. CONCLUSIONS: This retrospective study provides a wide spectrum of mutations in the RHO gene in Spanish patients with adRP. Also, the prevalence of mutations is similar to that reported in European population. Genotyping microarray followed by RHO sequencing is proposed as a first step in molecular diagnosis of adRP Spanish families. An increasing understanding of causal RHO alleles in adRP facilitates disease diagnosis and prognosis, especially for the prevalent p.Pro347Leu mutation.

Outcome of ABCA4 disease-associated alleles in autosomal recessive retinal dystrophies: retrospective analysis in 420 Spanish families.

OBJECTIVE: To provide a comprehensive overview of all detected mutations in the ABCA4 gene in Spanish families with autosomal recessive retinal disorders, including Stargardt's disease (arSTGD), cone-rod dystrophy (arCRD), and retinitis pigmentosa (arRP), and to assess genotype-phenotype correlation and disease progression in 10 years by considering the type of variants and age at onset. DESIGN: Case series. PARTICIPANTS: A total of 420 unrelated Spanish families: 259 arSTGD, 86 arCRD, and 75 arRP. METHODS: Spanish families were analyzed through a combination of ABCR400 genotyping microarray, denaturing high-performance liquid chromatography, and high-resolution melting scanning. Direct sequencing was used as a confirmation technique for the identified variants. Screening by multiple ligation probe analysis was used to detect possible large deletions or insertions in the ABCA4 gene. Selected families were analyzed further by next generation sequencing. MAIN OUTCOME MEASURES: DNA sequence variants, mutation detection rates, haplotypes, age at onset, central or peripheral vision loss, and night blindness. RESULTS: Overall, we detected 70.5% and 36.6% of all expected ABCA4 mutations in arSTGD and arCRD patient cohorts, respectively. In the fraction of the cohort where the ABCA4 gene was sequenced completely, the detection rates reached 73.6% for arSTGD and 66.7% for arCRD. However, the frequency of possibly pathogenic ABCA4 alleles in arRP families was only slightly higher than that in the general population. Moreover, in some families, mutations in other known arRP genes segregated with the disease phenotype. CONCLUSIONS: An increasing understanding of causal ABCA4 alleles in arSTGD and arCRD facilitates disease diagnosis and prognosis and also is paramount in selecting patients for emerging clinical trials of therapeutic interventions. Because ABCA4-associated diseases are evolving retinal dystrophies, assessment of age at onset, accurate clinical diagnosis, and genetic testing are crucial. We suggest that ABCA4 mutations may be associated with a retinitis pigmentosa-like phenotype often as a consequence of severe (null) mutations, in cases of long-term, advanced disease, or both. Patients with classical arRP phenotypes, especially from the onset of the disease, should be screened first for mutations in known arRP genes and not ABCA4.

Genotyping microarray: mutation screening in Spanish families with autosomal dominant retinitis pigmentosa.

PURPOSE: Presently, 22 genes have been described in association with autosomal dominant retinitis pigmentosa (adRP); however, they explain only 50% of all cases, making genetic diagnosis of this disease difficult and costly. The aim of this study was to evaluate a specific genotyping microarray for its application to the molecular diagnosis of adRP in Spanish patients. METHODS: We analyzed 139 unrelated Spanish families with adRP. Samples were studied by using a genotyping microarray (adRP). All mutations found were further confirmed with automatic sequencing. Rhodopsin (RHO) sequencing was performed in all negative samples for the genotyping microarray. RESULTS: The adRP genotyping microarray detected the mutation associated with the disease in 20 of the 139 families with adRP. As in other populations, RHO was found to be the most frequently mutated gene in these families (7.9% of the microarray genotyped families). The rate of false positives (microarray results not confirmed with sequencing) and false negatives (mutations in RHO detected with sequencing but not with the genotyping microarray) were established, and high levels of analytical sensitivity (95%) and specificity (100%) were found. Diagnostic accuracy was 15.1%. CONCLUSIONS: The adRP genotyping microarray is a quick, cost-efficient first step in the molecular diagnosis of Spanish patients with adRP.

Mutation analysis at codon 838 of the Guanylate Cyclase 2D gene in Spanish families with autosomal dominant cone, cone-rod, and macular dystrophies.

PURPOSE: Heterozygous mutations around codon 838 of the guanylate cyclase 2D (GUCY2D) gene have recently been associated with more than a third of autosomal dominant macular dystrophy patients. The aim of our study was to evaluate the prevalence of these mutations in Spanish families with autosomal dominant cone, cone-rod, and macular dystrophies. METHODS: Mutation analysis was performed by PCR amplification of exon 13 of GUCY2D and subsequent restriction analysis. To confirm the results, automatic sequencing analysis was also performed. RESULTS: Among the 22 unrelated Spanish families included in the study, we found two associated disease mutations at codon 838 of the GUCY2D gene, one of which had not been previously described (p.R838P). This novel mutation exhibited phenotypic variability. CONCLUSIONS: The prevalence of mutations around codon 838 of GUCY2D in our group of families (9.09%) is lower than that previously reported in other populations. However, the discovery of a novel mutation at codon 838 further suggests that this locus is a mutation hotspot within the GUCY2D gene, and confirms the importance of analyzing this codon to characterize molecularly these autosomal dominant retinal disorders.

CERKL mutations and associated phenotypes in seven Spanish families with autosomal recessive retinitis pigmentosa.

PURPOSE: Retinitis pigmentosa (RP) is a genetically heterogeneous group of inherited retinopathies. Up to now, 39 genes and loci have been implicated in nonsyndromic RP, yet the genetic bases of >50% of the cases, particularly of the recessive forms, remain unknown. A novel gene (CERKL) has been described as associated with RP26. It encodes a ceramide kinase that is assumed to be involved in sphingolipid-mediated apoptosis in the retina. This is a report of the phenotypes and genotypes of persons carrying disease-causing mutations in CERKL. METHODS: Two hundred ten unrelated Spanish families with nonsyndromic autosomal recessive RP were analyzed for sequence variations. Seven of these families presented a mutation in CERKL. Nine affected persons of these families were clinically investigated, including visual field, electrophysiology, and fundus examination. RESULTS: The mutation p.Arg257ter was identified in the homozygous state in all seven affected families. The patients with this variation in CERKL presented a common phenotype with characteristic macular and peripheral lesions. CONCLUSIONS: This study presents the first genotype-phenotype correlation for persons carrying p.Arg257ter mutation and provides clues for a characteristic phenotype of these mutations among persons with autosomal recessive cases.

Molecular analysis of ABCA4 and CRB1 genes in a Spanish family segregating both Stargardt disease and autosomal recessive retinitis pigmentosa.

PURPOSE: Stargardt disease (STGD), characterized by central visual impairment, is the most common juvenile macular dystrophy. All recessively inherited cases are thought to be due to mutations in the ABCA4 gene. Early-onset autosomal recessive retinitis pigmentosa (arRP) is a severe retinal degeneration that presents before the patient is ten years old. It has been associated with mutations in different genes, including CRB1. The aim of this study was to determine the genetic causes for two different retinal dystrophies, STGD and early-onset arRP, both segregating in one Spanish family. METHODS: Mutational analyses were performed using the ABCR400 and Leber congenital amaurosis (LCA) genotyping microarrays. Additional scanning for mutations was conducted by denaturing high performance liquid chromatography (dHPLC); results were confirmed by direct sequencing. RESULTS: A patient, who exhibited a STGD phenotype, was found to be homozygous for the p.Asn1805Asp (c.5413A>G) mutation in ABCA4. However, his affected sister, who had the arRP phenotype, was found to be heterozygous for this allele; no other sequence change could be found in ABCA4. Analysis using the LCA chip revealed the p.Cys948Tyr mutation in CRB1 in heterozygous state. A second mutation (p.Trp822ter) was found in the CRB1 gene in the affected female by denaturing high performance liquid chromatography (dHPLC) and direct sequencing. CONCLUSIONS: Two distinct retinal dystrophies with mutations affecting two different genes cosegregated in this family. The presence of two different phenotypes associated with mutations in two distinct genes in one single family must be considered especially when dealing with retinal dystrophies which bear high carrier frequencies in general population.

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.