Skeletal ciliopathies are a group of disorders caused by dysfunction of the cilium, a small signaling organelle present on nearly every vertebrate cell. This group of disorders is marked by genetic ...and clinical heterogeneity, which complicates accurate diagnosis. In this study, we developed a robust, standardized immunofluorescence approach to accurately diagnose a subset of these disorders. Hereto we determined and compared the cilium phenotype of healthy individuals to patients from three different ciliopathy subgroups, using skin-derived fibroblasts. The cilium phenotype assay consists of three parameters; (1) ciliogenesis, based on the presence or absence of cilium markers, (2) cilium length, measured by the combined signal of an axonemal and a cilium membrane marker, and (3) retrograde intraflagellar transport (IFT), quantified by the area of the ciliary tip. Analysis of the cilium phenotypic data yielded comparable and reproducible results and in addition, displayed identifiable clusters for healthy individuals and two ciliopathy subgroups, i.e. ATD and CED. Our results illustrate that standardized analysis of the cilium phenotype can be used to discriminate between ciliopathy subgroups. Therefore, we believe that standardization of functional assays analyzing cilium phenotypic data can provide additional proof for conclusive diagnosis of ciliopathies, which is essential for routine diagnostic care.
Copy-number variation is a common source of genomic variation and an important genetic cause of disease. Microarray-based analysis of copy-number variants (CNVs) has become a first-tier diagnostic ...test for patients with neurodevelopmental disorders, with a diagnostic yield of 10-20%. However, for most other genetic disorders, the role of CNVs is less clear and most diagnostic genetic studies are generally limited to the study of single-nucleotide variants (SNVs) and other small variants. With the introduction of exome and genome sequencing, it is now possible to detect both SNVs and CNVs using an exome- or genome-wide approach with a single test.
We performed exome-based read-depth CNV screening on data from 2,603 patients affected by a range of genetic disorders for which exome sequencing was performed in a diagnostic setting.
In total, 123 clinically relevant CNVs ranging in size from 727 bp to 15.3 Mb were detected, which resulted in 51 conclusive diagnoses and an overall increase in diagnostic yield of ~2% (ranging from 0 to -5.8% per disorder).
This study shows that CNVs play an important role in a broad range of genetic disorders and that detection via exome-based CNV profiling results in an increase in the diagnostic yield without additional testing, bringing us closer to single-test genomics.Genet Med advance online publication 27 October 2016.
Loss-of-function mutations of the
MECP2 gene at Xq28 are associated with Rett syndrome in females and with syndromic and nonsyndromic forms of mental retardation (MR) in males. By array comparative ...genomic hybridization (array-CGH), we identified a small duplication at Xq28 in a large family with a severe form of MR associated with progressive spasticity. Screening by real-time quantitation of 17 additional patients with MR who have similar phenotypes revealed three more duplications. The duplications in the four patients vary in size from 0.4 to 0.8 Mb and harbor several genes, which, for each duplication, include the MR-related
L1CAM and
MECP2 genes. The proximal breakpoints are located within a 250-kb region centromeric of
L1CAM, whereas the distal breakpoints are located in a 300-kb interval telomeric of
MECP2. The precise size and location of each duplication is different in the four patients. The duplications segregate with the disease in the families, and asymptomatic carrier females show complete skewing of X inactivation. Comparison of the clinical features in these patients and in a previously reported patient enables refinement of the genotype-phenotype correlation and strongly suggests that increased dosage of
MECP2 results in the MR phenotype. Our findings demonstrate that, in humans, not only impaired or abolished gene function but also increased MeCP2 dosage causes a distinct phenotype. Moreover, duplication of the
MECP2 region occurs frequently in male patients with a severe form of MR, which justifies quantitative screening of
MECP2 in this group of patients.
Exome sequencing revealed a homozygous missense mutation (c.317C>G p.Arg106Pro) in POC1B, encoding POC1 centriolar protein B, in three siblings with autosomal-recessive cone dystrophy or cone-rod ...dystrophy and compound-heterozygous POC1B mutations (c.199_201del p.Gln67del and c.810+1G>T) in an unrelated person with cone-rod dystrophy. Upon overexpression of POC1B in human TERT-immortalized retinal pigment epithelium 1 cells, the encoded wild-type protein localized to the basal body of the primary cilium, whereas this localization was lost for p.Arg106Pro and p.Gln67del variant forms of POC1B. Morpholino-oligonucleotide-induced knockdown of poc1b translation in zebrafish resulted in a dose-dependent small-eye phenotype, impaired optokinetic responses, and decreased length of photoreceptor outer segments. These ocular phenotypes could partially be rescued by wild-type human POC1B mRNA, but not by c.199_201del and c.317C>G mutant human POC1B mRNAs. Yeast two-hybrid screening of a human retinal cDNA library revealed FAM161A as a binary interaction partner of POC1B. This was confirmed in coimmunoprecipitation and colocalization assays, which both showed loss of FAM161A interaction with p.Arg106Pro and p.Gln67del variant forms of POC1B. FAM161A was previously implicated in autosomal-recessive retinitis pigmentosa and shown to be located at the base of the photoreceptor connecting cilium, where it interacts with several other ciliopathy-associated proteins. Altogether, this study demonstrates that POC1B mutations result in a defect of the photoreceptor sensory cilium and thus affect cone and rod photoreceptors.
Pathogenic variants in the OPN1LW/OPN1MW gene cluster are causal for a range of mild to severe visual impairments with color deficiencies. The widely utilized short-read next-generation sequencing ...(NGS) is inappropriate for the analysis of the OPN1LW/OPN1MW gene cluster and many patients with pathogenic variants stay underdiagnosed. A diagnostic genetic assay was developed for the OPN1LW/OPN1MW gene cluster, consisting of copy number analysis via multiplex ligation-dependent probe amplification and sequence analysis via long-read circular consensus sequencing. Performance was determined on 50 clinical samples referred for genetic confirmation of the clinical diagnosis (n = 43) or carrier status analysis (n = 7). A broad range of pathogenic haplotypes were detected, including deletions, hybrid genes, single variants and combinations of variants. The developed genetic assay for the OPN1LW/OPN1MW gene cluster is a diagnostic test that can detect both structural and nucleotide variants with a straightforward analysis, improving diagnostic care of patients with visual impairment.
The Joubert-Meckel syndrome spectrum is a continuum of recessive ciliopathy conditions caused by primary cilium dysfunction. The primary cilium is a microtubule-based, antenna-like organelle that ...projects from the surface of most human cell types, allowing them to respond to extracellular signals. The cilium is partitioned from the cell body by the transition zone, a known hotspot for ciliopathy-related proteins. Despite years of Joubert syndrome (JBTS) gene discovery, the genetic cause cannot be identified in up to 30% of individuals with JBTS, depending on the cohort, sequencing method, and criteria for pathogenic variants. Using exome and targeted sequencing of 655 families with JBTS, we identified three individuals from two families harboring biallelic, rare, predicted-deleterious missense TMEM218 variants. Via MatchMaker Exchange, we identified biallelic TMEM218 variants in four additional families with ciliopathy phenotypes. Of note, four of the six families carry missense variants affecting the same highly conserved amino acid position 115. Clinical features included the molar tooth sign (N = 2), occipital encephalocele (N = 5, all fetuses), retinal dystrophy (N = 4, all living individuals), polycystic kidneys (N = 2), and polydactyly (N = 2), without liver involvement. Combined with existing functional data linking TMEM218 to ciliary transition zone function, our human genetic data make a strong case for TMEM218 dysfunction as a cause of ciliopathy phenotypes including JBTS with retinal dystrophy and Meckel syndrome. Identifying all genetic causes of the Joubert-Meckel spectrum enables diagnostic testing, prognostic and recurrence risk counseling, and medical monitoring, as well as work to delineate the underlying biological mechanisms and identify targets for future therapies.
Using exome and targeted sequencing, we identified biallelic pathogenic TMEM218 variants in two families with Joubert and Meckel syndrome. Via MatchMaker Exchange, we identified four additional families with ciliopathy phenotypes. Combined with prior work implicating TMEM218 in ciliary transition zone function, we establish that TMEM218 dysfunction causes ciliopathies along the Joubert-Meckel spectrum.
Duplications in Xq28 involving the methyl CpG binding protein 2 gene ( MECP2 ) have been described in male patients with severe mental disability, delayed milestones, absence of language, hypotonia ...replaced by spasticity and retractions, and recurrent and often severe infections. In a study involving five patients in two families, multiplex ligation-dependent probe amplification was used to screen the Xq28 region that includes MECP2 , focusing on the presence of gene duplications. Some manifestations of the disease observed in these patients may occur less regularly than the classical abnormalities. Epilepsy with frequent seizures of the myoclonic-astatic type was observed in these patients and was associated with a slowing of the background electroencephalographic activity, rather than the generalized spike-waves or polyspike-waves usually observed in this type of seizure. In addition, cerebral abnormalities were observed with magnetic resonance imaging that were inconstant and nonspecific but that could nonetheless assist in diagnosis of this genetic pathology.
Camptodactyly-arthropathy-coxa vara-pericarditis (CACP, OMIM: #208250) syndrome is a rare autosomal recessive disease that can be difficult to recognise not only because of its wide clinical ...variability but also because of its clinical resemblance to juvenile idiopathic arthritis (JIA). PRG4 is the only gene so far known to be associated with CACP syndrome. Children with CACP syndrome lack the glycoprotein lubricin due to recessive mutations in PRG4. Lubricin serves as a lubricant in joints, tendons and visceral cavities (pleural cavity, pericardium) and inhibits synovial proliferation. Children with CACP syndrome suffer from congenital camptodactyly, arthropathy, coxa vara and sometimes pericarditis. This report concerns a child with CACP syndrome complicated by protein-losing enteropathy (PLE), caused by constrictive pericarditis and so contributes to knowledge of the presentation of CACP syndrome.
A 10- year-old girl with consanguineous parents suffered from congenital camptodactyly and progressive swollen and painful joints. Her father and his sister had similar childhood-onset joint complaints. Laboratory tests showed no signs of inflammation but showed persistent low protein- and IgG- levels, indicating a secondary immunodeficiency. Increased alpha antitrypsin clearance confirmed PLE. Abdominal ultrasound with Doppler showed hepatomegaly and portal hypertension. Echocardiography suggested constrictive pericarditis. However, heart catheterization could not confirm this. Ultrasound and X-ray examination of the joints combined with a puncture of the synovial fluid were performed. These results, combined with the clinical presentation and the consanguinity, suggested CACP syndrome. Due to excessive enteral protein losses, the patient was treated with Cotrimoxazol prophylaxis and immunoglobulin supplements. These supplements were inadequate to achieve normal IgG values. As constrictive pericarditis with subsequent PLE was the best explanation for the excessive IgG losses, pericardiectomy was performed with good results. Genetic testing in our patient was complicated but revealed a pathogenic mutation within the repeat sequence in exon 7 of the PRG4 gene.
PLE resulting from constrictive pericarditis can be a complication of CACP syndrome. As serious complications can arise from the resulting secondary immunodeficiency, we recommend regular evaluation of clinical symptoms of constrictive pericarditis and PLE in children with CACP syndrome.
Stargardt disease is caused by variants in the
gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, ...small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells. We therefore devised a novel strategy in which a bacterial artificial chromosome was employed to generate midigenes, splice vectors of varying lengths (up to 11.7 kb) covering almost the entire
gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on
pre-mRNA splicing. Intriguingly, multi-exon skipping events were observed, as well as exon elongation and intron retention. The analysis of all reported NCSS variants in
allowed us to reveal the nature of aberrant splicing events and to classify the severity of these mutations based on the residual fraction of wild-type mRNA. Our strategy to generate large overlapping splice vectors carrying multiple exons, creating a toolbox for robust and high-throughput analysis of splice variants, can be applied to all human genes.
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