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Purpose: To describe the clinical phenotype and genetic basis of non-syndromic retinitis pigmentosa (RP) in one family and two sporadic cases with biallelic mutations in the transcription factor neural retina leucine zipper ( NRL) ., Methods: Exome sequencing was performed in one affected family member. Microsatellite genotyping was used for haplotype analysis. PCR and Sanger sequencing were used to confirm mutations in and screen other family members where they were available. The SMART tool for domain prediction helped us build the protein schematic diagram., Results: For family MM1 of Pakistani origin, whole-exome sequencing and microsatellite genotyping revealed homozygosity on chromosome 14 and identified a homozygous stop-loss mutation in NRL , NM_006177.5: c.713G>T, p.*238Lext57, which is predicted to add an extra 57 amino acids to the normal protein chain. The variant segregated with disease symptoms in the family. For case RP-3051 of Spanish ancestry, clinical exome sequencing focusing on the morbid genome highlighted a homozygous nonsense mutation in NRL , c.238C>T, p.Gln80*, as the most likely disease candidate. For case RP-1553 of Romanian ethnicity, targeted-exome sequencing of 73 RP/LCA genes identified a homozygous nonsense mutation in NRL , c.544C>T, p.Gln182*. The variants were either rare or absent in the gnomAD database., Conclusions: NRL mutations predominantly cause dominant retinal disease, but there have been five published reports of mutations causing recessive disease. Here, we present three further examples of recessive RP due to NRL mutations. The phenotypes observed are consistent with those in the previous reports, and the observed mutation types and distribution further confirm distinct patterns for variants in NRL causing recessive and dominant diseases., (Copyright © 2022 Molecular Vision.)

Purpose: The aim of the present work is the molecular diagnosis of three patients with deafness and retinal degeneration., Methods: Three patients from two unrelated families were initially analyzed with custom gene panels for Usher genes, non-syndromic hearing loss, or inherited syndromic retinopathies and further investigated by means of clinical or whole exome sequencing., Results: The study allowed us to detect likely pathogenic variants in PEX6 , a gene typically involved in peroxisomal biogenesis disorders (PBDs). Beside deaf-blindness, both families showed additional features: Siblings from Family 1 showed enamel alteration and abnormal peroxisome. In addition, the brother had mild neurodevelopmental delay and nephrolithiasis. The case II:1 from Family 2 showed intellectual disability, enamel alteration, and dysmorphism., Conclusions: We have reported three new cases with pathogenic variants in PEX6 presenting with milder forms of the Zellweger spectrum disorders (ZSD). The three cases showed distinct clinical features. Thus, expanding the phenotypic spectrum of PBDs and ascertaining exome sequencing is an effective strategy for an accurate diagnosis of clinically overlapping and genetically heterogeneous disorders such as deafness-blindness association., (Copyright © 2020 Molecular Vision.)

Purpose: This study aimed to test a newly devised cost-effective multiplex PCR assay for the molecular diagnosis of autosomal dominant retinitis pigmentosa (adRP), as well as the use of whole-exome sequencing (WES) to detect disease-causing mutations in adRP., Methods: Genomic DNA was extracted from peripheral blood lymphocytes of index patients with adRP and their affected and unaffected family members. We used a newly devised multiplex PCR assay capable of amplifying the genetic loci of RHO, PRPH2, RP1, PRPF3, PRPF8, PRPF31, IMPDH1, NRL, CRX, KLHL7, and NR2E3 to molecularly diagnose 18 index patients with adRP. We also performed WES in affected and unaffected members of four families with adRP in whom a disease-causing mutation was previously not found., Results: We identified five previously reported mutations (p.Arg677X in the RP1 gene, p.Asp133Val and p.Arg195Leu in the PRPH2 gene, and p.Pro171Leu and p.Pro215Leu in the RHO gene) and one novel mutation (p.Val345Gly in the RHO gene) representing 33% detection of causative mutations in our adRP cohort. Comparative WES analysis showed a new variant (p.Gly103Arg in the COL6A6 gene) that segregated with the disease in one family with adRP. As this variant was linked with the RHO locus, we sequenced the complete RHO gene, which revealed a deletion in intron 4 that encompassed all of exon 5 and 28 bp of the 3'-untranslated region (UTR)., Conclusions: The novel multiplex PCR assay with next-generation sequencing (NGS) proved effective for detecting most of the adRP-causing mutations. A WES approach led to identification of a deletion in RHO through detection of a new linked variant in COL6A6. No pathogenic variants were identified in the remaining three families. Moreover, NGS and WES were inefficient for detecting the complete deletion of exon 5 in the RHO gene in one family with adRP. Carriers of this deletion showed variable clinical status, and two of these carriers had not previously been diagnosed with RP.

Purpose: The aim of the present work was to identify and characterize large rearrangements involving the USH2A gene in patients with Usher syndrome and nonsyndromic retinitis pigmentosa., Methods: The multiplex ligation-dependent probe amplification (MLPA) technique combined with a customized array-based comparative genomic hybridization (aCGH) analysis was applied to 40 unrelated patients previously screened for point mutations in the USH2A gene in which none or only one pathologic mutation was identified., Results: We detected six large deletions involving USH2A in six out of the 40 cases studied. Three of the patients were homozygous for the deletion, and the remaining three were compound heterozygous with a previously identified USH2A point mutation. In five of these cases, the patients displayed Usher type 2, and the remaining case displayed nonsyndromic retinitis pigmentosa. The exact breakpoint junctions of the deletions found in USH2A in four of these cases were characterized., Conclusions: Our study highlights the need to develop improved efficient strategies of mutation screening based upon next generation sequencing (NGS) that reduce cost, time, and complexity and allow simultaneous identification of all types of disease-causing mutations in diagnostic procedures.

Purpose: Mutations in genes encoding proteins from the tri-snRNP complex of the spliceosome account for more than 12% of cases of autosomal dominant retinitis pigmentosa (adRP). Although the exact mechanism by which splicing factor defects trigger photoreceptor death is not completely clear, their role in retinitis pigmentosa has been demonstrated by several genetic and functional studies. To test for possible novel associations between splicing factors and adRP, we screened four tri-snRNP splicing factor genes (EFTUD2, PRPF4, NHP2L1, and AAR2) as candidate disease genes., Methods: We screened up to 303 patients with adRP from Europe and North America who did not carry known RP mutations. Exon-PCR and Sanger methods were used to sequence the NHP2L1 and AAR2 genes, while the sequences of EFTUD2 and PRPF4 were obtained by using long-range PCRs spanning coding and non-coding regions followed by next-generation sequencing., Results: We detected novel missense changes in individual patients in the sequence of the genes PRPF4 and EFTUD2, but the role of these changes in relationship to disease could not be verified. In one other patient we identified a novel nucleotide substitution in the 5' untranslated region (UTR) of NHP2L1, which did not segregate with the disease in the family., Conclusions: The absence of clearly pathogenic mutations in the candidate genes screened in our cohort suggests that EFTUD2, PRPF4, NHP2L1, and AAR2 are either not involved in adRP or are associated with the disease in rare instances, at least as observed in this study in patients of European and North American origin.

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.

Purpose: To identify the genetic defect in Spanish families with Usher syndrome (USH) and probable involvement of the CLRN1 gene., Methods: DNA samples of the affected members of our cohort of USH families were tested using an USH genotyping array, and/or genotyped with polymorphic markers specific for the USH3A locus. Based on these previous analyses and clinical findings, CLRN1 was directly sequenced in 17 patients susceptible to carrying mutations in this gene., Results: Microarray analysis revealed the previously reported mutation p.Y63X in two unrelated patients, one of them homozygous for the mutation. After CLRN1 sequencing, we found two novel mutations, p.R207X and p.I168N. Both novel mutations segregated with the phenotype., Conclusions: To date, 18 mutations in CLRN1 have been reported. In this work, we report two novel mutations and a third one previously identified in the Spanish USH sample. The prevalence of CLRN1 among our patients with USH is low.

Purpose: To describe the clinical and genetic findings in 11 Spanish patients with confirmed (n=5) or suspected (n=6) Alström syndrome (AS)., Methods: Patients underwent clinical evaluation, and were screened for variations in Alström syndrome 1 gene (ALMS1) using a genotyping microarray from Asper Ophthalmics and by direct sequencing of coding exons 8, 10, and 16 of ALMS1. Furthermore, we analyzed the presence of the A229T variant of retinitis pigmentosa GTPase regulator-interacting protein 1-like gene (RPGRIP1L) with direct sequencing of coding exon 6., Results: A great phenotypic variability was observed in our patients. Four mutations in ALMS1-two novel nonsense mutations in one family (p.Y1715X and p.S616X), one previously described mutation in homozygous state in another family (p.V3597Efs*4), and a likely pathogenic missense variation p.P1822L in a third family-were identified with direct sequencing. All patients were homozygous for 229A allele of RPGRIP1L, with the exception of a p.A229T heterozygous patient., Conclusions: Our findings expand the spectrum of ALMS1 mutations causing Alström syndrome. The phenotypic differences between patients could be attributed to interactions with other genes inherited independently from the ALMS1 gene or with environmental factors. A clear understanding of the phenotypic spectrum in AS will be important to unravel the molecular mechanisms underlying this syndrome.

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.

Purpose: Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive loss of vision. The aim of this study was to identify the causative mutations in 272 Spanish families using a genotyping microarray., Methods: 272 unrelated Spanish families, 107 with autosomal recessive RP (arRP) and 165 with sporadic RP (sRP), were studied using the APEX genotyping microarray. The families were also classified by clinical criteria: 86 juveniles and 186 typical RP families. Haplotype and sequence analysis were performed to identify the second mutated allele., Results: At least one-gene variant was found in 14% and 16% of the juvenile and typical RP groups respectively. Further study identified four new mutations, providing both causative changes in 11% of the families. Retinol Dehydrogenase 12 (RDH12) was the most frequently mutated gene in the juvenile RP group, and Usher Syndrome 2A (USH2A) and Ceramide Kinase-Like (CERKL) were the most frequently mutated genes in the typical RP group. The only variant found in CERKL was p.Arg257Stop, the most frequent mutation., Conclusions: The genotyping microarray combined with segregation and sequence analysis allowed us to identify the causative mutations in 11% of the families. Due to the low number of characterized families, this approach should be used in tandem with other techniques.

Purpose: Bardet-Biedl syndrome (BBS, OMIM 209900) is a rare multi-organ disorder in which BBS patients manifest a variable phenotype that includes retinal dystrophy, polydactyly, mental delay, obesity, and also reproductive tract and renal abnormalities. Mutations in 14 genes (BBS1-BBS14) are found in 70% of the patients, indicating that additional mutations in known and new BBS genes remain to be identified. Therefore, the molecular diagnosis of this complex disorder is a challenging task., Methods: In this study we show the use of the genome-wide homozygosity mapping strategy in the mutation detection of nine Caucasian BBS families, eight of them consanguineous and one from the same geographic area with no proven consanguinity., Results: We identified the disease-causing mutation in six of the families studied, five of which had novel sequence variants in BBS3, BBS6, and BBS12. This is the first null mutation reported in BBS3. Furthermore, this approach defined homozygous candidate regions that could harbor potential candidate genes for BBS in three of the families., Conclusions: These findings further underline the importance of homozygosity mapping as a useful technology for diagnosis in small consanguineous families with a complex disease like BBS.

Purpose: To analyze the contributions of cytochrome P4501B1 (CYP1B1) mutations to primary congenital glaucoma (PCG) in Spanish patients., Methods: We analyzed, by polymerase chain reaction (PCR) DNA sequencing, the presence of promoter (-1 to -867) and exon CYP1B1 mutations in 38 unrelated Spanish probands affected by PCG. Functional analysis of nine identified mutations was performed measuring ethoxyresorufin O-deethylation activity and CYP1B1 stability in transiently transfected human embryonic kidney 293T (HEK-293-T) cells., Results: We found a total of 16 different mutations in 13 (34.2%) index cases. The identified mutations included nine missense and three nonsense nucleotide changes, three small deletions, and a short duplication. Eleven probands were compound heterozygotes and two were heterozygotes. Six of the identified mutations were novel (A106D, E173X, F261L, E262X, W341X, and P513_K514del). Mutations T404fsX30 and R355fsX69 were the most prevalent among index cases and were detected in six (23.0%) and three (11.5%) patients, respectively. Functional analysis showed that the three nonsense mutants assayed (E173X, E262X, and W341X) and F261L were null alleles. Of the remaining mutants, four (P52L, G61E, Y81N, and E229K) showed catalytic activities ranging from 20% to 40% of wild-type CYP1B1 and high protein instability. Mutation P400S showed normal catalytic activity and moderate instability. These five mutants were classified as hypomorphic alleles. Patients carrying two null alleles showed severe phenotypes featured by very early PCG onset usually at birth or in the first month of life (0.6+/-0.9 months). Incomplete penetrance was detected in patients carrying hypomorphic alleles., Conclusions: Our data indicate that approximately one-third of Spanish patients with PCG carry loss-of-function CYP1B1 and show that null alleles are associated with the most severe phenotypes. Hypomorphic alleles may contribute to some cases of incomplete penetrance.

Purpose: To resolve the spectrum of causative retina-specific ATP-binding cassette transporter gene (ABCA4) gene mutations in Portuguese Stargardt (STGD) patients and compare allele frequencies obtained in this cohort with those of previous population surveys., Methods: Using a microarray technique (ABCR400 gene chip), we screened all previously reported ABCA4 gene mutations in the genomic DNA of 27 patients from 21 unrelated Stargardt families whose phenotypes had been clinically evaluated using psychophysics and electrophysiological measurements. Furthermore, we performed denaturing high performance liquid chromatography whenever one or both mutant alleles failed to be detected using the ABCR gene chip., Results: A total of 36 mutant alleles (out of the 54 tested) were identified in STGD patients, resulting in a detection rate of 67%. Two mutant alleles were present in 12 out of 21 STGD families (57%), whereas in four out of 21 (19%) of the families, only one mutant allele was found. We report the presence of 22 putative pathogenic alterations, including two sequence changes not found in other populations, c.2T>C (p.Met1Thr) and c.4036_4037delAC (p.Thr1346fs), and two novel disease-associated variants, c.400C>T (p.Gln134X) and c.4720G>T (p.Glu1574X). The great majority of the mutations were missense (72.7%). Seven frameshift variants (19.4%), three nonsense mutations (8.3%), and one splicing sequence change (2.7%) were also found in STGD chromosomes. The most prevalent pathologic variant was the missense mutation p.Leu11Pro. Present in 19% of the families, this mutation represents a quite high prevalence in comparison to other European populations. In addition, 23 polymorphisms were also identified, including four novel intronic sequence variants., Conclusions: To our knowledge, this study represents the first report of ABCA4 mutations in Portuguese STGD patients and provides further evidence of different mutation frequency across populations. Phenotypic characterization of novel putative mutations was addressed.

Purpose: Leber congenital amaurosis (LCA) is one of the most severe inherited retinal dystrophies with the earliest age of onset. Mutations in the Crumbs homologue 1 (CRB1; OMIM 600105) gene explain 10%-24% of cases with LCA depending on the population. The aim of the present work was to study a fetal mutation associated to LCA in maternal plasma by a new methodology in the noninvasive prenatal diagnosis field: the denaturing High Performance Liquid Chromatography (dHPLC)., Methods: This study presents the case of a compound heterozygous fetus for two mutations in CRB1 (1q3.1-q32.2). dHPLC and automated DNA sequencing were used to detect the paternally inherited fetal mutation in a maternal plasma sample collected at the 12th week of gestation. To test the detection limit of dHPLC, we made serial dilutions of paternal DNA in control DNA., Results: We were able to detect the presence of the paternally inherited fetal CRB1 mutation in maternal plasma by dHPLC. Moreover, by comparing chromatograms of serial dilutions to the plasma sample, we could ascertain that the percentage of fetal DNA in maternal plasma was at least 2%. However, the detection of the fetal mutation was not possible by automated DNA sequencing., Conclusions: dHPLC seems to be sensitive enough to detect small amounts of fetal DNA in maternal plasma samples. It could be a useful tool for the noninvasive prenatal detection of paternally inherited point mutations associated with retinopathies.

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.

Purpose: Leber congenital amaurosis (LCA) is the most severe inherited retinopathy with the earliest age of onset. To date, eleven genes have been reported to cause the non-syndromic LCA phenotype. The CEP290 gene has been shown to account for Joubert and Senior-Loken syndromes and to represent a frequent cause of non-syndromic LCA. The aim of the present study was to establish the prevalence of CEP290 c.2991_1655A>G in non-syndromic Spanish patients having LCA or early-onset retinitis pigmentosa (RP)., Methods: We used automated sequencing to examine 49 non-syndromic Spanish families with LCA and 126 Spanish families with early-onset RP for the CEP290 c.2991_1655A>G mutation. As a control, we recruited 50 unrelated Spanish healthy individuals., Results: The frequencies of mutated alleles were 6% in LCA cases and 0% in early-onset RP and healthy individual controls. These results were compared to other populations., Conclusions: The CEP290 c.2991_1655A>G mutation frequency in Spanish non-syndromic LCA families is lower than that of other countries.

Purpose: Mutations in the peripherin/retinal degeneration slow (RDS) gene are a known cause of various types of central retinal dystrophies. The purpose of this study was to determine the prevalence of mutations in the peripherin/RDS gene in Spanish patients with different types of autosomal dominant macular dystrophy., Methods: Ophthalmic and electrophysiological examination was performed in patients from 61 unrelated autosomal dominant macular dystrophy (adMD) Spanish families. Screening for mutations in the peripherin/RDS gene by denaturing gradient gel electrophoresis (DGGE) and direct genomic sequencing was performed in index patients and extended to the family when positive., Results: We report four novel mutations in peripherin/RDS and a relatively high frequency (23%) of mutations in this gene in families with adMD. Thirteen different mutations were found in fifteen adMD families. Three novel missense, four nonsense and a cis-acting splicing mutation IVS2+2T>C, were found in a Spanish population while five more missense mutations were also reported in other populations. The Arg142Trp and Arg172Trp mutations, present in several populations, were both detected in two independent Spanish families. All the missense mutations produce an amino acid substitution in the second intradiscal loop of the peripherin, while the nonsense mutations presumably generate a truncated protein., Conclusions: A high frequency (23%) of mutations in the peripherin/RDS gene was found in a cohort of 61 unrelated patients with various types of autosomal dominant central retinal dystrophies as compared with a low prevalence (1.3%) of mutations in this gene causing retinitis pigmentosa in a Spanish population. Different macular dystrophy phenotypes according to the mutations in peripherin/RDS are shown. However, a limited phenotype variation was observed for these mutations within the family.

Purpose: Stargardt disease (STGD) is the most common juvenile macular dystrophy, characterized by central visual impairment. All recessively inherited cases are thought to be due to mutations in the ABCA4 gene, mapped to 1p21-p13., Methods: To describe a form of non-mendelian inheritance in a patient with STGD identified through the course of a conventional mutational screening performed on 77 STGD families. DNA from the patient and relatives was analyzed for variants in all 50 exons of the ABCA4 gene by screening on the ABCR400 microarray; results were confirmed by direct sequencing. Haplotype analyses, standard and high-resolution (HR) karyotypes, and multiplex ligation-dependent probe amplification (MLPA) were also performed., Results: A patient with STGD caused by the homozygous p.Arg1129Leu mutation in the ABCA4 gene was found to be the daughter of a noncarrier mother and a father who was heterozygous for this change. Haplotype analysis suggested that no maternal ABCA4 allele was transmitted to the patient. Microsatellite markers spanning the entire chromosome 1 identified a homozygous region of at least 4.4 Mb, involving the ABCA4 gene. The cytogenetic study revealed normal female karyotype. Further evaluation with MLPA showed the patient had a normal dosage for both copies of the ABCA4 gene, thus suggesting partial paternal isodisomy but not a maternal microdeletion., Conclusions: We report that recessive STGD can rarely be inherited from only one unaffected carrier parent in a non-mendelian manner. This study also demonstrates that genomic alterations contribute to only a small fraction of disease-associated alleles for ABCA4.

Purpose: To evaluate, in a pool of affected families, the mutation spectrum in Stargardt patients from Spain, using the ABCR400 microarray that contains described sequence variants in the gene encoding for the photoreceptor specific ATP-binding cassette transporter (ABCA4)., Methods: We analyzed 76 Spanish patients with STGD1 for a population-specific survey on the sequence variations in the ABCA4 gene, using the ABCR400 microarray., Results: Potential disease-associated alleles were identified in 91 of the 152 STGD1 chromosomes studied, resulting in a detection rate of 60%. The two mutant alleles were found in 33/76 patients (43%), whereas in 25/76 cases (33%) only one allele could be identified. In the remaining 18 patients no mutations were found. In total, we identified 40 sequence variations that could be related to the disease. The vast majority of these substitutions (35/40) were missense mutations. Three frameshift mutations and two splicing variants were also found., Conclusions: We identified a major disease-associated allele, R1129L, which accounted for 24% of the mutated alleles detected, and a high frequency (12%) of complex alleles.

Purpose: Only one mutation in the retinal fascin gene (FSCN2) has so far been associated with autosomal dominant retinitis pigmentosa (adRP) and macular dystrophy (adMD), in a Japanese population. Our study was designed to identify mutations in the FSCN2 gene among Spanish persons with adRP or adMD., Methods: Denaturing gradient gel electrophoresis and direct genomic sequencing were used to evaluate the complete coding region and flanking intronic sequences of the FSCN2 gene for mutations in 150 unrelated adRP and 15 adMD index patients, and in 50 sporadic cases of retinitis pigmentosa, together with 50 controls. Ophthalmic and electrophysiological examination of retinitis pigmentosa patients and their relatives was carried out according to pre-existing protocols., Results: Sixteen nucleotide substitutions were detected in the coding sequence of the index patients. Nine of these, His7Tyr, Ala122Thr, Ser126Phe, His138Tyr, Arg149Gln, Ala240Thr, Ala323Thr, Asn331His, and Phe367Leu are missense mutations, one is a nonsense mutation (Lys302Stop), and six are silent mutations. Co-segregation of the mutations in the families showed no direct relation between mutation and disease., Conclusions: The photoreceptor-specific FSCN2 gene showed a relatively high number of sequence variations. The mutation 208delG in FSCN2, the only mutation so far associated with adRP or adMD, and which presumably causes a null allele, was not detected in these Spanish families. The nonsense mutation, Lys302Stop, detected in one adRP Spanish family is not the cause of the disease. These findings support the fact that the kind and frequency of the mutations depend on the ethnic population.

Purpose: Norrie disease (OMIM 310600) is a rare X-linked disorder characterized by congenital blindness in males. Approximately 40 to 50% of the cases develop deafness and mental retardation. X-linked familial exudative vitreoretinopathy (XL-FEVR) is a hereditary ocular disorder characterized by a failure of peripheral retinal vascularization. Both X-linked disorders are due to mutations in the NDP gene, which encodes a 133 amino acid protein called Norrin, but autosomal recessive (AR) and autosomal dominant (AD) forms of FEVR have also been described. In this study, we report the molecular findings and the related phenotype in five Spanish families affected with Norrie disease or XL-FEVR due to mutations of the NDP gene., Methods: The study was conducted in 45 subjects from five Spanish families. These families were clinically diagnosed with Norrie disease or similar conditions. The three exons of the NDP gene were analyzed by automatic DNA sequencing. Haplotype analyses were also performed., Results: Two new nonsense mutations, apart from other mutations previously described in the NDP gene, were found in those patients affected with ND or X-linked FEVR., Conclusions: An important genotype-phenotype variation was found in relation to the different mutations of the NDP gene. In fact, the same mutation may be responsible for different phenotypes. We speculate that there might be other molecular factors that interact in the retina with Norrin, which contribute to the resultant phenotypes.

Purpose: Secreted Frizzled Related Proteins (SFRPs) are soluble molecules capable of modulating Wnt signalling. Different lines of evidence indicate that SFRP activity is related with the development and function of the retina photoreceptor cells as well as with their apoptotic degeneration associated with the onset of different cases of retinal dystrophy (RD). Because the genetic causes of many retinal dystrophies still need to be determined, we have asked whether mutations in the SFRP genes might be associated with retinal dystrophies., Methods: Here we describe the genomic structure of SFRP1, SFRP2, and SFRP5 and a mutational screening of SFRP1 in 325 individuals affected by various non X-linked forms of inherited retinal disorders., Results: Three polymorphic variants were identified., Conclusions: Our data, so far, exclude SFRP1 as a molecular cause of RD, since two out of three genetic variants of the gene were present in both RD patients and normal population.