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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.

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: 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

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.

Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) is a relatively common birth defect often associated with additional congenital anomalies such as vertebral, anal, cardiovascular, renal and limb defects, the so-called VACTERL association. Yet, little i

Item does not contain fulltext, OBJECTIVE: To evaluate the clinical course, genetic etiology, and visual prognosis in patients with cone dystrophy (CD) and cone-rod dystrophy (CRD). DESIGN: Clinic-based, longitudinal, multicenter study. PARTICIPANTS: Consecutive probands with CD (N = 98), CRD (N = 83), and affected relatives (N = 41 and N = 17, respectively) from various ophthalmogenetic clinics in The Netherlands, Belgium, and the United Kingdom. METHODS: Data on best-corrected Snellen visual acuity, color vision, ophthalmoscopy, fundus photography, Goldmann perimetry, and full-field standard electroretinogram (ERG) from all patients were registered from medical charts over a mean follow-up of 19 years. The ABCA4, CNGB3, KCNV2, PDE6C, and RPGR genes were analyzed by direct sequencing in autosomal recessive (AR) and X-linked (XL), respectively. Genotyping was not undertaken for autosomal-dominant cases. MAIN OUTCOME MEASURES: The 10-year progression of all clinical parameters and cumulative lifetime risk of low vision and legal blindness were assessed. RESULTS: The mean age onset for CD was 16 years (standard deviation, 11), and of CRD 12 years (standard deviation, 11; P = 0.02). The pattern of inheritance was AR in 92% of CD and 90% of CRD. Ten years after diagnosis, 35% of CD and 51% of CRD had a bull's eye maculopathy; 70% of CRD showed absolute peripheral visual field defects and 37% of CD developed rod involvement on ERG. The mean age of legal blindness was 48 (standard error [SE], 3.1) years in CD, and 35 (SE, 1.1; P<0.001) years in CRD. ABCA4 mutations were found in 8 of 90 (9%) of AR-CD, and in 17 of 65 (26%) of AR-CRD. Other mutations were detected in CNGB3 (3/90; 3%), KCNV2 (4/90; 4%), and in PDE6C (1/90; 1%). The RPGR gene was mutated in the 2 XL-CD and in 4 of 5 (80%) of XL-CRD. ABCA4 mutations as well as age of onset <20 years were significantly associated with a faster progression to legal blindness (P<0.001). CONCLUSIONS: Although CD had a slightly more favorable clinical course tha

Item does not contain fulltext, OBJECTIVE: To evaluate the clinical course, genetic etiology, and visual prognosis in patients with cone dystrophy (CD) and cone-rod dystrophy (CRD). DESIGN: Clinic-based, longitudinal, multicenter study. PARTICIPANTS: Consecutive probands with CD (N = 98), CRD (N = 83), and affected relatives (N = 41 and N = 17, respectively) from various ophthalmogenetic clinics in The Netherlands, Belgium, and the United Kingdom. METHODS: Data on best-corrected Snellen visual acuity, color vision, ophthalmoscopy, fundus photography, Goldmann perimetry, and full-field standard electroretinogram (ERG) from all patients were registered from medical charts over a mean follow-up of 19 years. The ABCA4, CNGB3, KCNV2, PDE6C, and RPGR genes were analyzed by direct sequencing in autosomal recessive (AR) and X-linked (XL), respectively. Genotyping was not undertaken for autosomal-dominant cases. MAIN OUTCOME MEASURES: The 10-year progression of all clinical parameters and cumulative lifetime risk of low vision and legal blindness were assessed. RESULTS: The mean age onset for CD was 16 years (standard deviation, 11), and of CRD 12 years (standard deviation, 11; P = 0.02). The pattern of inheritance was AR in 92% of CD and 90% of CRD. Ten years after diagnosis, 35% of CD and 51% of CRD had a bull's eye maculopathy; 70% of CRD showed absolute peripheral visual field defects and 37% of CD developed rod involvement on ERG. The mean age of legal blindness was 48 (standard error [SE], 3.1) years in CD, and 35 (SE, 1.1; P<0.001) years in CRD. ABCA4 mutations were found in 8 of 90 (9%) of AR-CD, and in 17 of 65 (26%) of AR-CRD. Other mutations were detected in CNGB3 (3/90; 3%), KCNV2 (4/90; 4%), and in PDE6C (1/90; 1%). The RPGR gene was mutated in the 2 XL-CD and in 4 of 5 (80%) of XL-CRD. ABCA4 mutations as well as age of onset <20 years were significantly associated with a faster progression to legal blindness (P<0.001). CONCLUSIONS: Although CD had a slightly more favorable clinical course tha

PURPOSE. Anterior segment dysgenesis (ASD) comprises a heterogeneous group of developmental abnormalities that affect several structures of the anterior segment of the eye. The main purpose of this study was to assess the proportion of FOXC1 and PITX2 mutations and copy number changes in 80 probands with ASD. METHODS. The patients were examined for FOXC1 and PITX2 copy number changes and mutations using MLPA (multiplex ligation-dependent probe amplification) and direct sequencing. Subsequently, the identified copy number changes were fine-mapped using high-resolution microarrays. In the remaining mutation-negative patients, sequencing of the FOXC1 and- PITX2 3″untranslated regions (UTRs) and three other candidate genes (P32, PDP2, and FOXC2) was performed. RESULTS. Thirteen FOXC1 and eight PITX2 mutations were identified, accounting for 26% (21/80) of the cases. In addition, six FOXC1 and five PITX2 deletions were found, explaining 14% (11/80) of the cases. The smallest FOXC1 and PITX2deletions were 5.4 and 1.6 kb in size, respectively. Six patients carrying FOXC1 deletions presented with variable extraocular phenotypic features such as hearing defects (in 4/6) and mental retardation (in 2/6). No further genetic defects were found in the remaining mutation-negative patients. CONCLUSIONS. FOXC1 and PITX2 genetic defects explain 40% of our large ASD cohort. The current spectrum of intragenic FOXC1 and PITX2 mutations was extended considerably, the identified copy number changes were fine mapped, the smallest FOXC1 and PITX2 deletions reported so far were identified, and the need for dedicated copy number screening of the FOXC1 and PITX2 genomic landscape was emphasized. This study is unique in that sequence and copy number changes were screened simultaneously in both genes.

Blepharophimosis Syndrome (BPES) is an autosomal dominant developmental disorder of the eyelids with or without ovarian dysfunction caused by FOXL2 mutations. Overall, FOXL2 deletions 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.

Context: Short stature has an incidence of three in 100 in children. Reliable molecular genetic testing may be crucial in the context of beneficial disease management. Deletions spanning or surrounding the SHOX gene account for a significant proportion of patients with idiopathic short stature (ISS) and allied disorders, such as Leri-Weill dyschondrosteosis. Objective: Several shortcomings of current strategies for copy number profiling of the SHOX region prompted us to develop an improved test for molecular diagnostics of the SHOX region. Design and Results:Weintroduced a quantitative PCR (qPCR)-based copy number profiling test, consisting of 11 amplicons targeting clinically relevant regions, i.e. the SHOX gene and regulatory regions. To ensure an optimal sensitivity and specificity, this test was validated in 32 controls and 18 probands with previously identified copy number changes. In addition, 152 probands with SHOX-associated phenotypes were screened, revealing 10 novel copy number changes. Conclusion: This highly validated qPCR test supersedes other approaches for copy number screening of the SHOX region in terms of reliability, accuracy, and cost efficiency. In addition, another strong point is the fact that it can be easily implemented in any standard equipped molecular laboratory. Our qPCR-based test is highly recommended for molecular diagnostics of idiopathic short stature and allied disorders. Copyright

Aims: The Schubert-Bornschein type of complete congenital stationary night blindness (CSNB) is a genetically heterogeneous retinal disorder. It is characterized by a non-progressive disease course, often associated with high myopia and nystagmus. So far mutations in two genes, NYX (nyctalopin) and GRM6 (metabotropic glutamate receptor 6) have been associated with this form of CSNB. The purpose of this study was to identify the genetic defect in affected male patients from Flemish families with complete CSNB. Methods: Probands with CSNB from 3 large Flemish families underwent ophthalmologic examination. DNA was extracted from peripheral blood and the coding region of NYX along with parts of the 5;UTR and 3;UTR and intronic regions covering the splice sites were PCR amplified and sequenced. Results: In the affected individuals of 3 Flemish families with the complete form of CSNB a novel NYX mutation, c.855delG was identified. This deletion is predicted to lead to a frameshift mutation, p.Asp286ThrfsX62 causing a premature stop codon. Conclusion: Previously, both single families with different mutations in NYX as well as different families with an identical mutation, suggestive of a founder mutation, have been described. The c.855delG deletion in NYX seems to be a common mutation associated with CSNB in the Flemish population from Belgium. Thus we suggest performing diagnostic testing for CSNB in the Flemish population initially directed towards the identification of this mutation. Subsequent screening for other mutations in NYX or GRM6 could be performed as a second step.

PURPOSE. Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disease. Although electroretinographic (ERG) measurements can discriminate clinical subgroups, the identification of the underlying genetic defects has been complicated for CSNB because ofgenetic heterogeneity, the uncertainty about the mode of inheritance, and time-consuming and costly mutation scanning and direct sequencing approaches. METHODS. To overcome these challenges and to generate a time- and cost-efficient mutation screening tool, the authors developed a CSNB genotyping microarray with arrayed primer extension (APEX) technology. To cover as many mutations as possible, a comprehensive literature search was performed, and DNA samples from a cohort of patients with CSNB were first sequenced directly in known CSNB genes. Subsequently, oligonucleotides were designed representing 126 sequence variations in RHO, CABP4, CACNA1F, CACNA2D4, GNAT1,GRM6, NYX, PDE6B, and SAG and spotted on the chip. RESULTS. Direct sequencing of genes known to be associated with CSNB in the study cohort revealed 21 mutations (12 novel and 9 previously reported). The resultant microarray containing oligonucleotides, which allow to detect 126 known and novel mutations, was 100% effective in determining the expected sequence changes in all known samples assessed. In addition, investigation of 34 patients with CSNB who were previously not genotyped revealed sequence variants in 18%, of which 15% are thought to be disease-causing mutations. CONCLUSIONS. This relatively inexpensive first-pass genetic testing device for patients with a diagnosis of CSNB will improve molecular diagnostics and genetic counseling of patients and their families and gives the opportunity to analyze whether, for example, more progressive disorders such as cone or cone–rod dystrophies underlie the same gene defects.

Mutations in FOXL2, a forkhead transcription factor gene, have recently been shown to cause blepharo-phimosis-ptosis-epicanthus inversus syndrome (BPES) types I and II, a rare genetic disorder. In BPES type I a complex eyelid malformation is associated with premature ovarian failure (POF), whereas in BPES type II the eyelid defect occurs as an isolated entity. In this study, we describe the identification of novel mutations in the FOXL2 gene in BPES types I and II families, in sporadic BPES patients, and in BPES families where the type could not be established. In 67% of the patients studied, we identified a mutation in the FOXL2 gene. In total, 21 mutations (17 of which are novel) and one microdeletion were identified. Thirteen of these FOXL2 mutations are unique. In this study, we demonstrate that there is a genotype-phenotype correlation for either types of BPES by the finding that mutations predicted to result in a truncated protein either lacking or containing the forkhead domain lead to BPES type I. In contrast, duplications within or downstream of the forkhead domain, and a frameshift downstream of them, all predicted to result in an extended protein, cause BPES type II. In addition, in 30 unrelated patients with isolated POF no causal mutations were identified in FOXL2. Our study provides further evidence that FOXL2 haploinsufficiency may cause BPES types I and III by the effect of a null allele and a hypomorphic allele, respectively. Furthermore, we propose that in a fraction of the BPES patients the genetic defect does not reside within the coding region of the FOXL2 gene and may be caused by a position effect.