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Achromatopsia (ACHM) is a congenital cone photoreceptor disorder characterized by impaired color discrimination, low visual acuity, photosensitivity, and nystagmus. To date, six genes have been associated with ACHM (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6), the majority of these being implicated in the cone phototransduction cascade. CNGA3 encodes the CNGA3 subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors and is one of the major disease-associated genes for ACHM. Herein, we provide a comprehensive overview of the CNGA3 variant spectrum in a cohort of 1060 genetically confirmed ACHM patients, 385 (36.3%) of these carrying "likely disease-causing" variants in CNGA3. Compiling our own genetic data with those reported in the literature and in public databases, we further extend the CNGA3 variant spectrum to a total of 316 variants, 244 of which we interpreted as "likely disease-causing" according to ACMG/AMP criteria. We report 48 novel "likely disease-causing" variants, 24 of which are missense substitutions underlining the predominant role of this mutation class in the CNGA3 variant spectrum. In addition, we provide extensive in silico analyses and summarize reported functional data of previously analyzed missense, nonsense and splicing variants to further advance the pathogenicity assessment of the identified variants., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Biallelic mutations in G-Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB). The purpose of this study was to identify disease causing GRK1 variants and use in-depth bioinformatic analyses to evaluate how their impact on protein structure could lead to pathogenicity. Patients' genomic DNA was sequenced by whole genome, whole exome or focused exome sequencing. Disease associated variants, published and novel, were compared to nondisease associated missense variants. The impact of GRK1 missense variants at the protein level were then predicted using a series of computational tools. We identified twelve previously unpublished cases with biallelic disease associated GRK1 variants, including eight novel variants, and reviewed all GRK1 disease associated variants. Further structure-based scoring revealed a hotspot for missense variants in the kinase domain. In addition, to aid future clinical interpretation, we identified the bioinformatics tools best able to differentiate disease associated from nondisease associated variants. We identified GRK1 variants in Oguchi disease patients and investigated how disease-causing variants may impede protein function in-silico., (© 2020 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Inactivating variants in the centrosomal CEP78 gene have been found in cone-rod dystrophy with hearing loss (CRDHL), a particular phenotype distinct from Usher syndrome. Here, we identified and functionally characterized the first CEP78 missense variant c.449T>C, p.(Leu150Ser) in three CRDHL families. The variant was found in a biallelic state in two Belgian families and in a compound heterozygous state-in trans with c.1462-1G>T-in a third German family. Haplotype reconstruction showed a founder effect. Homology modeling revealed a detrimental effect of p.(Leu150Ser) on protein stability, which was corroborated in patients' fibroblasts. Elongated primary cilia without clear ultrastructural abnormalities in sperm or nasal brushes suggest impaired cilia assembly. Two affected males from different families displayed sperm abnormalities causing infertility. One of these is a heterozygous carrier of a complex allele in SPAG17, a ciliary gene previously associated with autosomal recessive male infertility. Taken together, our data indicate that a missense founder allele in CEP78 underlies the same sensorineural CRDHL phenotype previously associated with inactivating variants. Interestingly, the CEP78 phenotype has been possibly expanded with male infertility. Finally, CEP78 loss-of-function variants may have an underestimated role in misdiagnosed Usher syndrome, with or without sperm abnormalities., (© 2020 The Authors. Human Mutation published by Wiley Periodicals, Inc.)

Inherited retinal disorders (IRD) represent clinically and genetically heterogeneous diseases. To date, pathogenic variants have been identified in ~260 genes. Albeit that many genes are implicated in IRD, for 30-50% of the cases, the gene defect is unknown. These cases may be explained by novel gene defects, by overlooked structural variants, by variants in intronic, promoter or more distant regulatory regions, and represent synonymous variants of known genes contributing to the dysfunction of the respective proteins. Patients with one subgroup of IRD, namely incomplete congenital stationary night blindness (icCSNB), show a very specific phenotype. The major cause of this condition is the presence of a hemizygous pathogenic variant in CACNA1F. A comprehensive study applying direct Sanger sequencing of the gene-coding regions, exome and genome sequencing applied to a large cohort of patients with a clinical diagnosis of icCSNB revealed indeed that seven of the 189 CACNA1F-related cases have intronic and synonymous disease-causing variants leading to missplicing as validated by minigene approaches. These findings highlight that gene-locus sequencing may be a very efficient method in detecting disease-causing variants in clinically well-characterized patients with a diagnosis of IRD, like icCSNB., (© 2019 Wiley Periodicals, Inc.)

Biallelic PDE6C mutations are a known cause for rod monochromacy, better known as autosomal recessive achromatopsia (ACHM), and early-onset cone photoreceptor dysfunction. PDE6C encodes the catalytic α'-subunit of the cone photoreceptor phosphodiesterase, thereby constituting an essential part of the phototransduction cascade. Here, we present the results of a study comprising 176 genetically preselected patients who remained unsolved after Sanger sequencing of the most frequent genes accounting for ACHM, and were subsequently screened for exonic and splice site variants in PDE6C applying a targeted next generation sequencing approach. We were able to identify potentially pathogenic biallelic variants in 15 index cases. The mutation spectrum comprises 18 different alleles, 15 of which are novel. Our study significantly contributes to the mutation spectrum of PDE6C and allows for a realistic estimate of the prevalence of PDE6C mutations in ACHM since our entire ACHM cohort comprises 1,074 independent families., (© 2018 Wiley Periodicals, Inc.)

Achromatopsia is a rare autosomal recessive cone disorder characterized by color vision defects, photophobia, nystagmus, and severely reduced visual acuity. The disease is caused by mutations in genes encoding crucial components of the cone phototransduction cascade (CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H) or in ATF6, involved in the unfolded protein response. CNGB3 encoding the beta subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors is the major achromatopsia gene. Here, we present a comprehensive spectrum of CNGB3 mutations and their prevalence in a cohort of 1074 independent families clinically diagnosed with achromatopsia. Of these, 485 (45.2%) carried mutations in CNGB3. We identified a total of 98 different potentially disease-causing CNGB3 variants, 58 of which are novel. About 10% of patients with CNGB3 mutations only harbored a single heterozygous variant. Therefore, we performed quantitative real-time PCR in 43 of such single heterozygotes in search of the missing allele, followed by microarray-based comparative genomic hybridization and breakpoint mapping. We discovered nine different heterozygous copy number variations encompassing one to 10 consecutive exons in 16 unrelated patients. Moreover, one additional patient with a homozygous CNGB3 deletion encompassing exons 4-18 was identified, highlighting the importance of CNV analysis for this gene., (© 2017 Wiley Periodicals, Inc.)

Leber congenital amaurosis (LCA) is a severe autosomal-recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9-associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5'UTR variant, c.-70A>T. Moreover, an adjacent 5'UTR variant, c.-69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5'UTR variants resulted in decreased NMNAT1 mRNA abundance in patients' lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE-1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu-rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1-associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat-mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness., (© 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.)

The release of benchtop next-generation sequencing (NGS) instruments has paved the way to implement the technology in clinical setting. The need for flexible, qualitative, and cost-efficient workflows is high. We used singleplex-PCR for highly efficient target enrichment, allowing us to reach the quality standards set in Sanger sequencing-based diagnostics. For the library preparation, a modified NexteraXT protocol was used, followed by sequencing on a MiSeq instrument. With an innovative pooling strategy, high flexibility, scalability, and cost-efficiency were obtained, independent of the availability of commercial kits. The approach was validated for ∼250 genes associated with monogenic disorders. An overall sensitivity (>99%) similar to Sanger sequencing was observed in combination with a positive predictive value of >98%. The distribution of coverage was highly uniform, guaranteeing a minimal number of gaps to be filled with alternative methods. ISO15189-accreditation was obtained for the workflow. A major asset of the singleplex PCR-based enrichment is that new targets can be easily implemented. Diagnostic laboratories have validated assays available ensuring that the proposed workflow can easily be adopted. Although our platform was optimized for constitutional variant detection of monogenic disease genes, it is now also used as a model for somatic mutation detection in acquired diseases., (© 2014 WILEY PERIODICALS, INC.)

Autosomal-recessive Stargardt disease (STGD1) is hallmarked by a large proportion of patients with a single heterozygous causative variant in the disease gene ABCA4. Braun et al. () reported deep intronic variants of ABCA4 in STGD1 patients with one coding variant, prompting us to perform an augmented screen in 131 Belgian STGD1 patients with one or no ABCA4 variant to uncover deep intronic causal ABCA4 variants. This revealed a second variant in 28.6% of cases. Twenty-six percent of these carry the same causal variant c.4539+2001G>A (V4). Haplotyping in V4 carriers showed a common region of 63 kb, suggestive of a founder mutation. Genotype-phenotype correlations suggest a moderate-to-severe impact of V4 on the STGD1 phenotype. In conclusion, V4 occurs in a high fraction of Belgian STGD1 patients and represents the first deep intronic founder mutation in ABCA4. This emphasizes the importance of augmented molecular genetic testing of ABCA4 in Belgian STGD1., (© 2014 WILEY PERIODICALS, INC.)

This study was undertaken to investigate the prevalence of sequence variants in LCA5 in patients with Leber congenital amaurosis (LCA), early-onset retinal dystrophy (EORD), and autosomal recessive retinitis pigmentosa (arRP); to delineate the ocular phenotypes; and to provide an overview of all published LCA5 variants in an online database. Patients underwent standard ophthalmic evaluations after providing informed consent. In selected patients, optical coherence tomography (OCT) and fundus autofluorescence imaging were possible. DNA samples from 797 unrelated patients with LCA and 211 with the various types of retinitis pigmentosa (RP) were screened by Sanger sequence analysis of all LCA5 exons and intron/exon junctions. Some LCA patients were prescreened by APEX technology or selected based on homozygosity mapping. In silico analyses were performed to assess the pathogenicity of the variants. Segregation analysis was performed where possible. Published and novel LCA5 variants were collected, amended for their correct nomenclature, and listed in a Leiden Open Variation Database (LOVD). Sequence analysis identified 18 new probands with 19 different LCA5 variants. Seventeen of the 19 LCA5 variants were novel. Except for two missense variants and one splice site variant, all variants were protein-truncating mutations. Most patients expressed a severe phenotype, typical of LCA. However, some LCA subjects had better vision and intact inner segment/outer segment (IS/OS) junctions on OCT imaging. In two families with LCA5 variants, the phenotype was more compatible with EORD with affected individuals displaying preserved islands of retinal pigment epithelium. One of the families with a milder phenotype harbored a homozygous splice site mutation; a second family was found to have a combination of a stop mutation and a missense mutation. This is the largest LCA5 study to date. We sequenced 1,008 patients (797 with LCA, 211 with arRP) and identified 18 probands with LCA5 mutations. Mutations in LCA5 are a rare cause of childhood retinal dystrophy accounting for ∼2% of disease in this cohort, and the majority of LCA5 mutations are likely null. The LCA5 protein truncating mutations are predominantly associated with LCA. However, in two families with the milder EORD, the LCA5 gene analysis revealed a homozygous splice site mutation in one and a stop mutation in combination with a missense mutation in a second family, suggesting that this milder phenotype is due to residual function of lebercilin and expanding the currently known phenotypic spectrum to include the milder early onset RP. Some patients have remaining foveal cone structures (intact IS/OS junctions on OCT imaging) and remaining visual acuities, which may bode well for upcoming treatment trials., (© 2013 WILEY PERIODICALS, INC.)

Cone dystrophy with supernormal rod response (CDSRR) is considered to be a very rare autosomal recessive retinal disorder. CDSRR is associated with mutations in KCNV2, a gene that encodes a modulatory subunit (Kv8.2) of a voltage-gated potassium channel. In this study, we found that KCNV2 mutations are present in a substantial fraction (2.2-4.3%) of a sample of 367 independent patients with a variety of initial clinical diagnoses of cone malfunction, indicating that CDSRR is underdiagnosed and more common than previously thought. In total, we identified 20 different KCNV2 mutations; 15 of them are novel. A new finding of this study is the substantial proportion of large deletions at the KCNV2 locus that accounts for 15.5% of the mutant alleles in our sample. We determined the breakpoints and size of all five different deletions, which ranged between 10.9 and 236.8 kb. Two deletions encompass the entire KCNV2 gene and one also includes the adjacent VLDLR gene. Furthermore, we investigated N-terminal amino acid substitution mutations for its effect on interaction with Kv2.1 using yeast two-hybrid technology. We found that these mutations dramatically reduce or abolish this interaction suggesting a lack of assembly of heteromeric Kv channels as one underlying pathomechanism of CDSRR., (© 2011 Wiley-Liss, Inc.)

Ciliopathies are an emerging group of disorders, caused by mutations in ciliary genes. One of the most intriguing disease genes associated with ciliopathies is CEP290, in which mutations cause a wide variety of distinct phenotypes, ranging from isolated blindness over Senior-Loken syndrome (SLS), nephronophthisis (NPHP), Joubert syndrome (related disorders) (JS[RD]), Bardet-Biedl syndrome (BBS), to the lethal Meckel-Grüber syndrome (MKS). Despite the identification of over 100 unique CEP290 mutations, no clear genotype-phenotype correlations could yet be established, and consequently the predictive power of a CEP290-related genotype remains limited. One of the challenges is a better understanding of second-site modifiers. In this respect, there is a growing interest in the potential modifying effects of variations in genes encoding other members of the ciliary proteome that interact with CEP290. Here, we provide an overview of all CEP290 mutations identified so far, with their associated phenotypes. To this end, we developed CEP290base, a locus-specific mutation database that links mutations with patients and their phenotypes (medgen.ugent.be/cep290base)., (Hum Mutat 31:1097-1108, 2010. © 2010 Wiley-Liss, Inc.)

Leber Congenital Amaurosis (LCA), the most severe inherited retinal dystrophy, is genetically heterogeneous, with 14 genes accounting for 70% of patients. Here, 91 LCA probands underwent LCA chip analysis and subsequent sequencing of 6 genes (CEP290, CRB1, RPE65, GUCY2D, AIPL1and CRX), revealing mutations in 69% of the cohort, with major involvement of CEP290 (30%). In addition, 11 patients with early-onset retinal dystrophy (EORD) and 13 patients with Senior-Loken syndrome (SLS), LCA-Joubert syndrome (LCA-JS) or cerebello-oculo-renal syndrome (CORS) were included. Exhaustive re-inspection of the overall phenotypes in our LCA cohort revealed novel insights mainly regarding the CEP290-related phenotype. The AHI1 gene was screened as a candidate modifier gene in three patients with the same CEP290 genotype but different neurological involvement. Interestingly, a heterozygous novel AHI1 mutation, p.Asn811Lys, was found in the most severely affected patient. Moreover, AHI1 screening in five other patients with CEP290-related disease and neurological involvement revealed a second novel missense variant, p.His758Pro, in one LCA patient with mild mental retardation and autism. These two AHI1 mutations might thus represent neurological modifiers of CEP290-related disease., (© 2010 Wiley-Liss, Inc.)

Blepharophimosis Syndrome (BPES) is an autosomal dominant developmental disorder of the eyelids with or without ovarian dysfunction caused by FOXL2 mutations. Overall, FOXL2deletions represent 12% of all genetic defects in BPES. Here, we have identified and characterized 16 new and one known FOXL2 deletion combining multiplex ligation-dependent probe amplification (MLPA), custom-made quantitative PCR (qPCR) and/or microarray-based copy number screening. The deletion breakpoints could be localized for 13 out of 17 deletions. The deletion size is highly variable (29.8 kb - 11.5 Mb), indicating absence of a recombination hotspot. Although the heterogeneity of their size and breakpoints is not reflected in the uniform BPES phenotype, there is considerable phenotypic variability regarding associated clinical findings including psychomotor retardation (8/17), microcephaly (6/17), and subtle skeletal features (2/17). In addition, in all females in whom ovarian function could be assessed, FOXL2 deletions proved to be associated with variable degrees of ovarian dysfunction. In conclusion, we present the largest series of BPES patients with FOXL2 deletions and standardized phenotyping reported so far. Our genotype-phenotype data can be useful for providing a prognosis (i.e. occurrence of associated features) in newborns with BPES carrying a FOXL2 deletion., ((c) 2010 Wiley-Liss, Inc.)

The FOXL2 gene is one of 10 forkhead genes, the mutations of which lead to human developmental disorders, often with ocular manifestations. Mutations in FOXL2 are known to cause blepharophimosis syndrome (BPES), an autosomal dominant eyelid malformation associated (type I) or not (type II) with ovarian dysfunction, leading to premature ovarian failure (POF). In addition, a few mutations have been described in patients with isolated POF. Here, we review all currently described FOXL2 sequence variations and genomic rearrangements in BPES and POF. Using a combined mutation detection approach, it is possible to identify the underlying genetic defect in a major proportion (88%) of typical BPES patients. Of all genetic defects found in our BPES cohort, intragenic mutations represent 81%. They include missense changes, frameshift and nonsense mutations, in-frame deletions, and duplications, that are distributed along the single-exon gene. Genomic rearrangements comprising both deletions encompassing FOXL2 and deletions located outside its transcription unit, represent 12% and 5% of all genetic defects in our BPES cohort, respectively. One of the challenges of genetic testing in BPES is the establishment of genotype-phenotype correlations, mainly with respect to the ovarian phenotype. Genetic testing should be performed in the context of genetic counseling, however, and should be systematically complemented by a multidisciplinary clinical follow-up. Another challenge for health care professionals involved in BPES is the treatment of the eyelid phenotype and the prevention or treatment of POF., ((c) 2008 Wiley-Liss, Inc.)

Blepharophimosis syndrome (BPES) is caused by loss-of-function mutations in the single-exon forkhead transcription factor gene FOXL2 and by genomic rearrangements of the FOXL2 locus. Here, we focus on 92 new intragenic FOXL2 mutations, 34 of which are novel. Specifically, we found 10 nonsense mutations (11%), 13 missense mutations (14%), 40 deletions or insertions leading to a frameshift (43%), and 29 in-frame changes (32%), of which 28 (30%) lead to a polyalanine expansion. This study confirms the existence of two previously described mutational hotspots. Moreover, we gained novel insights in genotype-phenotype correlations, emphasizing the need to interpret genotype-phenotype correlations individually and always in the context of further clinical observations., ((c) 2008 Wiley-Liss, Inc.)

Researchers and clinicians ideally need instant access to all the variation in their gene/locus of interest to efficiently conduct their research and genetic healthcare to the highest standards. Currently much key data resides in the laboratory books or patient records around the world, as there are many impediments to submitting this data. It would be ideal therefore if a semiautomated pathway was available, with a minimum of effort, to make the deidentified data publicly available for others to use. The Human Variome Project (HVP) meeting listed 96 recommendations to work toward this situation. This article is planned to initiate a strategy to enhance the collection of phenotype and genotype data from the clinician/diagnostic laboratory nexus. Thus, the aim is to develop universally applicable forms that people can use when investigating patients for each inherited disease, to assist in satisfying many of the recommendations of the HVP Meeting [Cotton et al., 2007]. We call for comment and collaboration in this article., (Copyright 2007 Wiley-Liss, Inc.)

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES; MIM# 110100) is an autosomal dominant genetic condition in which an eyelid malformation is associated (type I) or not associated (type II) with premature ovarian failure (POF). In 2001, mutations in the FOXL2 gene, encoding a forkhead transcription factor, were shown to cause both BPES type I and II. Since then, a number of reports have appeared that describe intragenic FOXL2 mutations in BPES patients. In addition, a few FOXL2 variants have been reported in isolated POF patients and XX males. Previously, our group has described a large number of FOXL2 mutations, thereby demonstrating the existence of two mutational hotspots in FOXL2, intra- and interfamilial phenotypic variability in BPES families, and genotype-phenotype correlations for a number of mutations in BPES patients. Here we describe a locus-specific Human FOXL2 Mutation Database (http://medgen.ugent.be/foxl2/), created using the MuStaR software. Our database contains general information about the FOXL2 gene, as well as details about 135 intragenic mutations and variants of FOXL2, obtained from published papers, abstracts of meetings, and from unpublished data produced by our group. Not included in the current version of the database are variants residing outside the coding region of FOXL2 and molecular cytogenetic rearrangements of the FOXL2 locus. The Human FOXL2 Mutation Database was created to provide a unique publicly available online resource of information about human FOXL2 mutations/variants associated with BPES and POF. It allows remote users to submit new mutations to the database and to query the database using a web form. It will facilitate evaluation of the pathogenicity of a particular mutation, as it contains data about disease-causing mutations and polymorphisms in BPES and isolated POF patients, and a link to the known FOXL2 orthologs. Moreover, it will allow us to establish more accurate genotype-phenotype correlations, since clinical information is contained in the database., (Copyright 2004 Wiley-Liss, Inc.)