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Item does not contain fulltext, Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies. Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases. Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.

Contains fulltext : 282710.pdf (Publisher’s version ) (Open Access), In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results.

Contains fulltext : 246089.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 225504.pdf (Publisher’s version ) (Closed access), PURPOSE: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. METHODS: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. RESULTS: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. CONCLUSION: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

We present key points from the updated Dutch-Flemish guideline on comprehensive diagnostics in disorders/differences of sex development (DSD) that have not been widely addressed in the current (inter)national literature. These points are of interest to physicians working in DSD (expert) centres and to professionals who come across persons with a DSD but have no (or limited) experience in this area. The Dutch-Flemish guideline is based on internationally accepted principles. Recent initiatives striving for uniform high-quality care across Europe, and beyond, such as the completed COST action 1303 and the European Reference Network for rare endocrine conditions (EndoERN), have generated several excellent papers covering nearly all aspects of DSD. The Dutch-Flemish guideline follows these international consensus papers and covers a number of other topics relevant to daily practice. For instance, although next-generation sequencing (NGS)-based molecular diagnostics are becoming the gold standard for genetic evaluation, it can be difficult to prove variant causality or relate the genotype to the clinical presentation. Network formation and centralisation are essential to promote functional studies that assess the effects of genetic variants and to the correct histological assessment of gonadal material from DSD patients, as well as allowing for maximisation of expertise and possible cost reductions. The Dutch-Flemish guidelines uniquely address three aspects of DSD. First, we propose an algorithm for counselling and diagnostic evaluation when a DSD is suspected prenatally, a clinical situation that is becoming more common. Referral to ultrasound sonographers and obstetricians who are part of a DSD team is increasingly important here. Second, we pay special attention to healthcare professionals not working within a DSD centre as they are often the first to diagnose or suspect a DSD, but are not regularly exposed to DSDs and may have limited experience. Their thoughtful co

Lysine acetyltransferase 6A (KAT6A) and its paralog KAT6B form stoichiometric complexes with bromodomain- and PHD finger-containing protein 1 (BRPF1) for acetylation of histone H3 at lysine 23 (H3K23). We report that these complexes also catalyze H3K23 propionylatio

Purpose: Using exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability. Methods: Sequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects. Results: In 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects. Conclusion: Our data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.

X-linked cone dysfunction disorders such as Blue Cone Monochromacy and X-linked Cone Dystrophy are characterized by complete loss (of) or reduced L- and M- cone function due to defects in the OPN1LW/OPN1MW gene cluster. Here we investigated 24 affected males from 16 families with either a structurally intact gene cluster or at least one intact single (hybrid) gene but harbouring rare combinations of common SNPs in exon 3 in single or multiple OPN1LW and OPN1MW gene copies. We assessed twelve different OPN1LW/MW exon 3 haplotypes by semi-quantitative minigene splicing assay. Nine haplotypes resulted in aberrant splicing of >=20% of transcripts including the known pathogenic haplotypes (i.e. 'LIAVA', 'LVAVA') with absent or minute amounts of correctly spliced transcripts, respectively. De novo formation of the 'LIAVA' haplotype derived from an ancestral less deleterious 'LIAVS' haplotype was observed in one family with strikingly different phenotypes among affected family members. We could establish intrachromosomal gene conversion in the male germline as underlying mechanism. Gene conversion in the OPN1LW/OPN1MW genes has been postulated, however, we are first to demonstrate a de novo gene conversion within the lineage of a pedigree.

PURPOSE. The purpose of this study was to investigate the phenotype and long-term clinical course of female carriers of RPGR mutations. METHODS. This was a retrospective cohort study of 125 heterozygous RPGR mutation carriers from 49 families. RESULTS. Eighty-three heterozygotes were from retinitis pigmentosa (RP) pedigrees, 37 were from cone-/cone-rod dystrophy (COD/CORD) pedigrees, and 5 heterozygotes were from pedigrees with mixed RP/CORD or unknown diagnosis. Mutations were located in exon 1-14 and in ORF15 in 42 of 125 (34%) and 83 of 125 (66%) subjects, respectively. The mean age at the first examination was 34.4 years (range, 2.1 to 86.0 years). The median follow-up time in heterozygotes with longitudinal data (n = 62) was 12.2 years (range, 1.1 to 52.2 years). Retinal pigmentary changes were present in 73 (58%) individuals. Visual symptoms were reported in 51 (40%) cases. Subjects with both symptoms and pigmentary fundus changes were older than the other heterozygotes (P = 0.01) and had thinner foveal outer retinas (P = 0.006). Complete expression of the RP or CORD phenotype was observed in 29 (23%) heterozygotes, although usually in milder forms than in affected male relatives. Best-corrected visual acuity (BCVA) was <20/40 and <20/400 in at least one eye in 45 of 116 (39%) and 11 of 116 (9%) heterozygotes, respectively. Myopia was observed in 74 of 101 (73%) subjects and was associated with lower BCVA (P = 0.006). Increasing age was associated with lower BCVA (P = 0.002) and decreasing visual field size (P = 0.012; I4e isopter). CONCLUSIONS. RPGR mutations lead to a phenotypic spectrum in female carriers, with myopia as a significantly aggravating factor. Complete disease expression is observed in some individuals, who may benefit from future (gene) therapeutic options.

Purpose: We aimed to identify the genetic cause in a cohort of 11 unrelated cases and two sisters with 46,XX SRY-negative (ovo)testicular disorders of sex development (DSD). Methods: Whole-exome sequencing (n = 9), targeted resequencing (n = 4), and haplotyping were performed. Immunohistochemistry of sex-specific markers was performed on patients' gonads. The consequences of

Contains fulltext : 162145.pdf (publisher's version ) (Closed access), Butterfly-shaped pigment dystrophy is an eye disease characterized by lesions in the macula that can resemble the wings of a butterfly. Here we report the identification of heterozygous missense mutations in the CTNNA1 gene (encoding alpha-catenin 1) in three families with butterfly-shaped pigment dystrophy. In addition, we identified a Ctnna1 missense mutation in a chemically induced mouse mutant, tvrm5. Parallel clinical phenotypes were observed in the retinal pigment epithelium (RPE) of individuals with butterfly-shaped pigment dystrophy and in tvrm5 mice, including pigmentary abnormalities, focal thickening and elevated lesions, and decreased light-activated responses. Morphological studies in tvrm5 mice demonstrated increased cell shedding and the presence of large multinucleated RPE cells, suggesting defects in intercellular adhesion and cytokinesis. This study identifies CTNNA1 gene variants as a cause of macular dystrophy, indicates that CTNNA1 is involved in maintaining RPE integrity and suggests that other components that participate in intercellular adhesion may be implicated in macular disease.

Item does not contain fulltext, PURPOSE: To elucidate the functional effect of the ABCA4 variant c.5461-10T-->C, one of the most frequent variants associated with Stargardt disease (STGD1). DESIGN: Case series. PARTICIPANTS: Seventeen persons with STGD1 carrying ABCA4 variants and 1 control participant. METHODS: Haplotype analysis of 4 homozygotes and 11 heterozygotes for c.5461-10T-->C and sequence analysis of the ABCA4 gene for a homozygous proband. Fibroblasts were reprogrammed from 3 persons with STGD1 into induced pluripotent stem cells, which were differentiated into photoreceptor progenitor cells (PPCs). The effect of the c.5461-10T-->C variant on RNA splicing by reverse-transcription polymerase chain reaction was analyzed using PPC mRNA. In vitro assays were performed with minigene constructs containing ABCA4 exon 39. We analyzed the natural history and ophthalmologic characteristics of 4 persons homozygous for c.5461-10T-->C. MAIN OUTCOME MEASURES: Haplotype and rare variant data for ABCA4, RNA splice defects, age at diagnosis, visual acuity, fundus appearance, visual field, electroretinography (ERG) results, fluorescein angiography results, and fundus autofluorescence findings. RESULTS: The frequent ABCA4 variant c.5461-10T-->C has a subtle effect on splicing based on prediction programs. A founder haplotype containing c.5461-10T-->C was found to span approximately 96 kb of ABCA4 and did not contain other rare sequence variants. Patient-derived PPCs showed skipping of exon 39 or exons 39 and 40 in the mRNA. HEK293T cell transduction with minigenes carrying exon 39 showed that the splice defects were the result of the c.5461-10T-->C variant. All 4 subjects carrying the c.5461-10T-->C variant in a homozygous state showed a young age of STGD1 onset, with low visual acuity at presentation and abnormal cone ERG results. All 4 demonstrated severe cone-rod dystrophy before 20 years of age and were legally blind by 25 years of age. CONCLUSIONS: The ABCA4 variant c.5461-10T-->C is located on a fo

Contains fulltext : 168023.pdf (publisher's version ) (Closed access), Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV.

Item does not contain fulltext, PURPOSE: USH2A mutations are an important cause of retinitis pigmentosa (RP) with or without congenital sensorineural hearing impairment. We studied genotype-phenotype correlations and compared visual prognosis in Usher syndrome type IIa and nonsyndromic RP. DESIGN: Clinic-based, longitudinal, multicenter study. PARTICIPANTS: Consecutive patients with Usher syndrome type IIa (n = 152) and nonsyndromic RP (n = 73) resulting from USH2A mutations from ophthalmogenetic clinics in the Netherlands and Belgium. METHODS: Data on clinical characteristics, visual acuity, visual field measurements, retinal imaging, and electrophysiologic features were extracted from medical charts over a mean follow-up of 9 years. Cumulative lifetime risks of low vision and blindness were estimated using Kaplan-Meier survival analysis. MAIN OUTCOME MEASURES: Low vision and blindness. RESULTS: Participant groups had similar distributions of gender (48% vs. 45% males in Usher syndrome type IIa vs. nonsydromic RP; P = 0.8), ethnicity (97% vs. 99% European; P = 0.3), and median follow-up time (6.5 years vs. 3 years; P = 0.3). Usher syndrome type IIa patients demonstrated symptoms at a younger age (median age, 15 years vs. 25 years; P < 0.001), were diagnosed earlier (median age, 26 years vs. 36.5 years; P < 0.001), and became visually impaired 13 years earlier (median age, 41 years vs. 54 years; P < 0.001) based on VF and 18 years earlier based on VA (median age, 54 years vs. 72 years; P < 0.001) than nonsyndromic RP patients. The presence of 2 truncating mutations in USH2A was associated mostly with the syndromic phenotype, whereas other combinations were present in both groups. We found novel variants in Usher syndrome type IIa (25%) and nonsyndromic RP (19%): 29 missense mutations, 10 indels, 14 nonsense mutations, 9 frameshift mutations, and 5 splice-site mutations. CONCLUSIONS: Most patients with USH2A-associated RP have severe visual impairment by age 50. However, those with Usher syndrome t

X-linked cone dysfunction disorders such as Blue Cone Monochromacy and X-linked Cone Dystrophy are characterized by complete loss (of) or reduced L- and M- cone function due to defects in the OPN1LW/OPN1MW gene cluster. Here we investigated 24 affected males from 16 families with either a structurally intact gene cluster or at least one intact single (hybrid) gene but harbouring rare combinations of common SNPs in exon 3 in single or multiple OPN1LW and OPN1MW gene copies. We assessed twelve different OPN1LW/MW exon 3 haplotypes by semi-quantitative minigene splicing assay. Nine haplotypes resulted in aberrant splicing of ≥20% of transcripts including the known pathogenic haplotypes (i.e. 'LIAVA', 'LVAVA') with absent or minute amounts of correctly spliced transcripts, respectively. De novo formation of the 'LIAVA' haplotype derived from an ancestral less deleterious 'LIAVS' haplotype was observed in one family with strikingly different phenotypes among affected family members. We could establish intrachromosomal gene conversion in the male germline as underlying mechanism. Gene conversion in the OPN1LW/OPN1MW genes has been postulated, however, we are first to demonstrate a de novo gene conversion within the lineage of a pedigree.

Contains fulltext : 151962.pdf (publisher's version ) (Open Access), PURPOSE: To identify mutations in FAM161A underlying autosomal recessive retinitis pigmentosa (arRP) in the Dutch and Belgian populations and to investigate whether common FAM161A-associated phenotypic features could be identified. METHODS: Homozygosity mapping, amplification-refractory mutation system (ARMS) analysis, and Sanger sequencing were performed to identify mutations in FAM161A. Microsatellite and SNP markers were genotyped for haplotype analysis. Patients with biallelic mutations underwent detailed ophthalmologic examinations, including measuring best-corrected visual acuity, extensive fundus photography with reflectance and autofluorescence imaging, and optical coherence tomography. RESULTS: Homozygosity mapping in 230 Dutch individuals with suspected arRP yielded five individuals with a homozygous region harboring FAM161A. Sanger sequencing revealed a homozygous nonsense mutation (c.1309A>T; p.[Arg437*]) in one individual. Subsequent ARMS analysis and Sanger sequencing in Dutch and Belgian arRP patients resulted in the identification of seven additional individuals carrying the p.(Arg437*) mutation, either homozygously or compound heterozygously with another mutation. Haplotype analysis identified a shared haplotype block of 409 kb surrounding the p.(Arg437*) mutation in all patients, suggesting a founder effect. Although the age of onset was variable among patients, all eight developed pronounced outer retinal loss with severe visual field defects and a bull's eye-like maculopathy, followed by loss of central vision within 2 decades after the initial diagnosis in five subjects. CONCLUSIONS: A founder mutation in FAM161A p.(Arg437*) underlies approximately 2% of arRP cases in the Dutch and Belgian populations. The age of onset of the retinal dystrophy appears variable, but progression can be steep, with almost complete loss of central vision later in life.

Contains fulltext : 153526.pdf (publisher's version ) (Open Access), Bietti's crystalline dystrophy (BCD) is a rare, autosomal recessive retinal degenerative disease associated with mutations in CYP4V2. In this study, we describe the genetic and clinical findings in 19 unrelated BCD patients recruited from five international retinal dystrophy clinics. Patients underwent ophthalmic examinations and were screened for CYP4V2 mutations by Sanger sequencing and quantitative polymerase chain reaction (qPCR) copy number variation screening. Eight CYP4V2 mutations were found in 10/19 patients, including three patients in whom only monoallelic mutations were detected. Four novel mutations were identified: c.604G>A; p.(Glu202Lys), c.242C>G; p.(Thr81Arg), c.604+4A>G; p.(?), and c.1249dup; p.(Thr417Asnfs*2). In addition, we identified a heterozygous paternally inherited genomic deletion of at least 3.8 Mb, encompassing the complete CYP4V2 gene and several other genes, which is novel. Clinically, patients demonstrated phenotypic variability, predominantly showing choroidal sclerosis, attenuated vessels, and crystalline deposits of varying degrees of severity. To our knowledge, our study reports the first heterozygous CYP4V2 deletion and hence a novel mutational mechanism underlying BCD. Our results emphasize the importance of copy number screening in BCD. Finally, the identification of CYP4V2-negative patients with indistinguishable phenotypes from CYP4V2-positive patients might suggest the presence of mutations outside the coding regions of CYP4V2, or locus heterogeneity, which is unreported so far.