Maria Isabel Lopez-Molina

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.

Whole Exome Sequencing Reveals ZNF408 as a New Gene Associated With Autosomal Recessive Retinitis Pigmentosa with Vitreal Alterations

Retinitis pigmentosa (RP) is a group of progressive inherited retinal dystrophies that cause visual impairment as a result of photoreceptor cell death. RP is heterogeneous, both clinically and genetically making difficult to establish precise genotype-phenotype correlations. In a Spanish family with autosomal recessive RP (arRP), homozygosity mapping and whole-exome sequencing led to the identification of a homozygous mutation (c.358_359delGT; p.Ala122Leufs*2) in the ZNF408 gene. A screening performed in 217 additional unrelated families revealed another homozygous mutation (c.1621C>T; p.Arg541Cys) in an isolated RP case. ZNF408 encodes a transcription factor that harbors 10 predicted C2H2-type fingers thought to be implicated in DNA binding. To elucidate the ZNF408 role in the retina and the pathogenesis of these mutations we have performed different functional studies. By immunohistochemical analysis in healthy human retina, we identified that ZNF408 is expressed in both cone and rod photoreceptors, in a specific type of amacrine and ganglion cells, and in retinal blood vessels. ZNF408 revealed a cytoplasmic localization and a nuclear distribution in areas corresponding with the euchromatin fraction. Immunolocalization studies showed a partial mislocalization of the p.Arg541Cys mutant protein retaining part of the WT protein in the cytoplasm. Our study demonstrates that ZNF408, previously associated with Familial Exudative Vitreoretinopathy (FEVR), is a new gene causing arRP with vitreous condensations supporting the evidence that this protein plays additional functions into the human retina.

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.

Novel GUCA1A Mutations Suggesting Possible Mechanisms of Pathogenesis in Cone, Cone-Rod, and Macular Dystrophy Patients

Here, we report two novel GUCA1A (the gene for guanylate cyclase activating protein 1) mutations identified in unrelated Spanish families affected by autosomal dominant retinal degeneration (adRD) with cone and rod involvement. All patients from a three-generation adRD pedigree underwent detailed ophthalmic evaluation. Total genome scan using single-nucleotide polymorphisms and then the linkage analysis were undertaken on the pedigree. Haplotype analysis revealed a 55.37 Mb genomic interval cosegregating with the disease phenotype on chromosome 6p21.31-q15. Mutation screening of positional candidate genes found a heterozygous transition c.250C>T in exon 4 of GUCA1A, corresponding to a novel mutation p.L84F. A second missense mutation, c.320T>C (p.I107T), was detected by screening of the gene in a Spanish patients cohort. Using bioinformatics approach, we predicted that either haploinsufficiency or dominant-negative effect accompanied by creation of a novel function for the mutant protein is a possible mechanism of the disease due to c.250C>T and c.320T>C. Although additional functional studies are required, our data in relation to the c.250C>T mutation open the possibility that transacting factors binding to de novo created recognition site resulting in formation of aberrant splicing variant is a disease model which may be more widespread than previously recognized as a mechanism causing inherited RD.

Application of Whole Exome Sequencing in Six Families with an Initial Diagnosis of Autosomal Dominant Retinitis Pigmentosa: Lessons Learned

This study aimed to identify the genetics underlying dominant forms of inherited retinal dystrophies using whole exome sequencing (WES) in six families extensively screened for known mutations or genes. Thirty-eight individuals were subjected to WES. Causative variants were searched among single nucleotide variants (SNVs) and insertion/deletion variants (indels) and whenever no potential candidate emerged, copy number variant (CNV) analysis was performed. Variants or regions harboring a candidate variant were prioritized and segregation of the variant with the disease was further assessed using Sanger sequencing in case of SNVs and indels, and quantitative PCR (qPCR) for CNVs. SNV and indel analysis led to the identification of a previously reported mutation in PRPH2. Two additional mutations linked to different forms of retinal dystrophies were identified in two families: a known frameshift deletion in RPGR, a gene responsible for X-linked retinitis pigmentosa and p.Ser163Arg in C1QTNF5 associated with Late-Onset Retinal Degeneration. A novel heterozygous deletion spanning the entire region of PRPF31 was also identified in the affected members of a fourth family, which was confirmed with qPCR. This study allowed the identification of the genetic cause of the retinal dystrophy and the establishment of a correct diagnosis in four families, including a large heterozygous deletion in PRPF31, typically considered one of the pitfalls of this method. Since all findings in this study are restricted to known genes, we propose that targeted sequencing using gene-panel is an optimal first approach for the genetic screening and that once known genetic causes are ruled out, WES might be used to uncover new genes involved in inherited retinal dystrophies.

Involvement of LCA5 in Leber Congenital Amaurosis and Retinitis Pigmentosa in the Spanish Population

OBJECTIVE We aimed to identify novel genetic defects in the LCA5 gene underlying Leber congenital amaurosis (LCA) in the Spanish population and to describe the associated phenotype. DESIGN Case series. PARTICIPANTS A cohort of 217 unrelated Spanish families affected by autosomal recessive or isolated retinal dystrophy, that is, 79 families with LCA and 138 families with early-onset retinitis pigmentosa (EORP). A total of 100 healthy, unrelated Spanish individuals were screened as controls. METHODS High-resolution homozygosity mapping was performed in 44 patients with LCA using genome-wide single nucleotide polymorphism (SNP) microarrays. Direct sequencing of the LCA5 gene was performed in 5 patients who showed homozygous regions at chromosome 6 and in 173 unrelated individuals with LCA or EORP. The ophthalmic history of 8 patients carrying LCA5 mutations was reviewed and additional examinations were performed, including electroretinography (ERG), optical coherence tomography (OCT), and fundus photography. MAIN OUTCOME MEASURES Single nucleotide polymorphism genotyping, identity-by-descent (IBD) regions, LCA5 mutations, best-corrected visual acuity, visual field assessments, fundus appearance, ERG, and OCT findings. RESULTS Four novel and 2 previously reported LCA5 mutations have been identified in 6 unrelated families with LCA by homozygosity mapping or Sanger sequencing. Thus, LCA5 mutations have a frequency of 7.6% in the Spanish population. However, no LCA5 mutations were found in 138 patients with EORP. Although most of the identified LCA5 mutations led to a truncated protein, a likely pathogenic missense variant was identified for the first time as a cause of LCA, segregating in 2 families. We also have characterized a novel splicing site mutation at the RNA level, demonstrating that the mutant LCA5 transcript was absent in a patient. All patients carrying LCA5 mutations presented nystagmus, night blindness, and progressive loss of visual acuity and visual field leading to blindness toward the third decade of life. Fundoscopy showed fundus features of pigmentary retinopathy with atrophic macular lesions. CONCLUSIONS This work reveals a higher frequency of LCA5 mutations in a Spanish LCA cohort than in other populations. This study established gene-specific frequencies and the underlying phenotype of LCA5 mutations in the Spanish population.

Identification of two novel mutations in CDHR1 in consanguineous Spanish families with autosomal recessive retinal dystrophy

Inherited retinal dystrophies present extensive phenotypic and genetic heterogeneity, posing a challenge for patients’ molecular and clinical diagnoses. In this study, we wanted to clinically characterize and investigate the molecular etiology of an atypical form of autosomal recessive retinal dystrophy in two consanguineous Spanish families. Affected members of the respective families exhibited an array of clinical features including reduced visual acuity, photophobia, defective color vision, reduced or absent ERG responses, macular atrophy and pigmentary deposits in the peripheral retina. Genetic investigation included autozygosity mapping coupled with exome sequencing in the first family, whereas autozygome-guided candidate gene screening was performed by means of Sanger DNA sequencing in the second family. Our approach revealed nucleotide changes in CDHR1; a homozygous missense variant (c.1720C > G, p.P574A) and a homozygous single base transition (c.1485 + 2T > C) affecting the canonical 5’ splice site of intron 13, respectively. Both changes co-segregated with the disease and were absent among cohorts of unrelated control individuals. To date, only five mutations in CDHR1 have been identified, all resulting in premature stop codons leading to mRNA nonsense mediated decay. Our work reports two previously unidentified homozygous mutations in CDHR1 further expanding the mutational spectrum of this gene.

Expanding the phenotype of PRPS1 syndromes in females: neuropathy, hearing loss and retinopathy

BackgroundPhosphoribosyl pyrophosphate synthetase (PRS) I deficiency is a rare medical condition caused by missense mutations in PRPS1 that lead to three different phenotypes: Arts Syndrome (MIM 301835), X-linked Charcot-Marie-Tooth (CMTX5, MIM 311070) or X-linked non-syndromic sensorineural deafness (DFN2, MIM 304500). All three are X-linked recessively inherited and males affected display variable degree of central and peripheral neuropathy. We applied whole exome sequencing to a three-generation family with optic atrophy followed by retinitis pigmentosa (RP) in all three cases, and ataxia, progressive peripheral neuropathy and hearing loss with variable presentation.MethodsWhole exome sequencing was performed in two affecteds and one unaffected member of the family. Sanger sequencing was used to validate and segregate the 12 candidate mutations in the family and to confirm the absence of the novel variant in PRPS1 in 191 controls. The pathogenic role of the novel mutation in PRPS1 was assessed in silico and confirmed by enzymatic determination of PRS activity, mRNA expression and sequencing, and X-chromosome inactivation.ResultsA novel missense mutation was identified in PRPS1 in the affected females. Age of onset, presentation and severity of the phenotype are highly variable in the family: both the proband and her mother have neurological and ophthalmological symptoms, whereas the phenotype of the affected sister is milder and currently confined to the eye. Moreover, only the proband displayed a complete lack of expression of the wild type allele in leukocytes that seems to correlate with the degree of PRS deficiency and the severity of the phenotype. Interestingly, optic atrophy and RP are the only common manifestations to all three females and the only phenotype correlating with the degree of enzyme deficiency.ConclusionsThese results are in line with recent evidence of the existence of intermediate phenotypes in PRS-I deficiency syndromes and demonstrate that females can exhibit a disease phenotype as severe and complex as their male counterparts.

Analysis of the PRPF31 Gene in Spanish Autosomal Dominant Retinitis Pigmentosa Patients: A Novel Genomic Rearrangement

Purpose The aim was to determine the prevalence of PRPF31 mutations in a cohort of Spanish autosomal dominant retinitis pigmentosa (adRP) families to deepen knowledge of the pathogenic mechanisms underlying the disease and to assess genotype-phenotype correlations. Methods A cohort of 211 adRP patients was screened for variants in PRPF31 by using a combined strategy comprising next-generation sequencing approaches and copy-number variation (CNV) analysis. Quantitative RT-PCR and CNV analysis of the regulatory MSR1 element were also performed to assess PRPF31 gene expression. Phenotype was assessed by using ophthalmologic examination protocols. Results Fifteen different causative mutations and genomic rearrangements were identified, revealing five novel mutations. Prevalence of PRPF31 mutations, genomic rearrangements, and lack of penetrance were 7.6%, 1.9%, and 66.7%, respectively. Interestingly, we identified a tandem duplication and a partial PRPF31 deletion in different affected individuals from the same family. PRPF31 gene expression was significantly decreased in symptomatic cases carrying either PRPF31 duplication or deletion as compared to controls. The 4 MSR1 allele in cis with the PRPF31 wild-type allele was apparently a protective factor. The mutated phenotype varied from no symptoms to typical retinitis pigmentosa with variable onset and course depending on the kind of mutation, with the duplication case the most severe. Conclusions In view of the high genetic heterogeneity of PRPF31 mutations, the screening must include the entire gene, as well as CNV assays, to detect large rearrangements. This is the first report of a variable phenotype correlation as well as a gross duplication and deletion within the same family.

Late onset retinitis pigmentosa.

Dear Editor: Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous set of progressive retinal disorders. To date, mutations in 53 genes have been described to cause RP (http://www.sph.uth.tmc.edu/Retnet/ accessed June 22, 2011). However, these genes account for disease in a little over half of all patients. Also, it is well known that several genes cause distinct or partially overlapping clinical phenotypes. Mutations in the SPATA7 gene have been associated with a severe congenital retinal dystrophy (Leber congenital amaurosis [LCA]) and a form of Juvenile RP. Therefore, a correlation between the severity of mutant alleles in SPATA7 and the clinical phenotype has been established. We performed a genome-wide linkage analysis, using the Illumina HumanCyto-12 SNP array, followed by homozygosity mapping in the 2 affected individuals from a consanguineous Spanish family that revealed 3 regions with a maximum logarithm of the odds (LOD) score of 2.8. The third largest region (3.7 Mb) in chromosome 14 encompassed SPATA7. Sequence analysis allowed identifying a homozygous nonsense mutation (c.253C T; p.Arg85ter) in SPATA7. The homozygous mutation segregated with the disease in the family. Both affected members of the family were clinically evaluated and were diagnosed as typical autosomal recessive retinitis pigmentosa (arRP). The index case complained of night blindness at the age of 25. At that time, the visual field was normal. At the age of 29 she was re-examined due to loss of peripheral visual field and the night blindness. The fundus showed narrowed retinal vessels and bone-spicule pigmentations in the peripheral retina, and the rod and mixed electroretinogram (ERG) showed subnormal responses of the rods in both eyes. Two years later (at 31 years old), the visual field was limited to central 10° and the ERG showed severe decreased of the a and b components in terms of the photopic and scotopic stimulation. This phenotype fits exactly the signs of typical arRP. To date the affected sibling did not complain of noticeable symptoms, night blindness, or visual field constriction; however, the mixed ERG showed delay in the aand b-waves latency and a moderate reduction in the amplitude of the a-wave in both eyes at the age of the 26 years old. Previous studies have established a correlation between the severity of SPATA7 mutant alleles and the phenotype of the patients. LCA has been associated with frameshift and nonsense mutations, which truncate the last half of the SPATA7 protein, which are predicted to be degraded by nonsense-mediated decay (NMD) mechanism. However, childhood-onset RP has been only associated with mutations that truncate a small part of the protein, which should be protected from the NMD mechanism. We report a previously described nonsense mutation in the middle of the coding region. This mutation will cause a truncated protein, which will be degraded by the NMD echanism. According to this hypothesis, the patients carying this mutation should have a severe retinal dystrophy ith a congenital or childhood presentation as previously eported. However, the affected individuals of the studied amily showed a non-early onset retinal dystrophy with a apid progression. Thus, our results contradict the published ypothesis of correlation between the severity of the PATA7 mutations and the phenotypes. These variable phenotypes could not be explained by the urrent understanding of the genetic mechanism. In addiion, this same mutation was found in 3 families from ifferent origins affected with LCA and severe early onset P, in contrast to the phenotype reported here. This phenotypic variability could be explained by the resence of modifier alleles contributing to the penetrance nd expressivity, or intronic variants influencing the splicng accuracy or efficiency of the different transcripts, could enerate an altered splicing product, encoding a stable rotein with residual function. Thus, we hypothesize that ntronic variants in this family may influence the severity f the phenotype associated with the p.Arg85ter mutation n this family. Further genomic studies of this uncharacterzed part of the noncoding region of SPATA7 combined with unctional analysis at the RNA level could help to better lucidate this hypothesis. In conclusion, our data established the first linkage asociation of a loss-of-function mutation in the SPATA7 gene ith a typical RP phenotype and not with LCA or early nset RP. inancial Support: This study was supported by the folowing research grants: FIS PI09/90047, FIS PI09/00459, IBERER INTRA/07/704.1, FUNDALUCE 2010 and NCE 2011. ALMUDENA ÁVILA-FERNÁNDEZ, PHD MARTA CORTÓN, PHD MARÍA I. LÓPEZ-MOLINA, MD ESTHER MARTÍN-GARRIDO DIEGO CANTALAPIEDRA, PHD RUTH FERNÁNDEZ-SÁNCHEZ, PHD FIONA BLANCO-KELLY, MD ROSA RIVEIRO-ÁLVAREZ, PHD SORINA D. TATU, PHD MARÍA J. TRUJILLO-TIEBAS, PHD BLANCA GARCÍA-SANDOVAL, MD, PHD CARMEN AYUSO, MD, PHD Madrid, Spain