Anneke Seller

Clinical and Molecular Genetic Analysis of 19 Wolfram Syndrome Kindreds Demonstrating a Wide Spectrum of Mutations in WFS1

Wolfram syndrome is an autosomal recessive neurodegenerative disorder characterized by juvenile-onset diabetes mellitus and progressive optic atrophy. mtDNA deletions have been described, and a gene (WFS1) recently has been identified, on chromosome 4p16, encoding a predicted 890 amino acid transmembrane protein. Direct DNA sequencing was done to screen the entire coding region of the WFS1 gene in 30 patients from 19 British kindreds with Wolfram syndrome. DNA was also screened for structural rearrangements (deletions and duplications) and point mutations in mtDNA. No pathogenic mtDNA mutations were found in our cohort. We identified 24 mutations in the WFS1 gene: 8 nonsense mutations, 8 missense mutations, 3 in-frame deletions, 1 in-frame insertion, and 4 frameshift mutations. Of these, 23 were novel mutations, and most occurred in exon 8. The majority of patients were compound heterozygotes for two mutations, and there was no common founder mutation. The data were also analyzed for genotype-phenotype relationships. Although some interesting cases were noted, consideration of the small sample size and frequency of each mutation indicated no clear-cut correlations between any of the observed mutations and disease severity. There were no obvious mutation hot spots or clusters. Hence, molecular screening for Wolfram syndrome in affected families and for Wolfram syndrome-carrier status in subjects with psychiatric disorders or diabetes mellitus will require complete analysis of exon 8 and upstream exons.

Reassessment of Mendelian gene pathogenicity using 7,855 cardiomyopathy cases and 60,706 reference samples

Purpose:The accurate interpretation of variation in Mendelian disease genes has lagged behind data generation as sequencing has become increasingly accessible. Ongoing large sequencing efforts present huge interpretive challenges, but they also provide an invaluable opportunity to characterize the spectrum and importance of rare variation.Methods:We analyzed sequence data from 7,855 clinical cardiomyopathy cases and 60,706 Exome Aggregation Consortium (ExAC) reference samples to obtain a better understanding of genetic variation in a representative autosomal dominant disorder.Results:We found that in some genes previously reported as important causes of a given cardiomyopathy, rare variation is not clinically informative because there is an unacceptably high likelihood of false-positive interpretation. By contrast, in other genes, we find that diagnostic laboratories may be overly conservative when assessing variant pathogenicity.Conclusions:We outline improved analytical approaches that evaluate which genes and variant classes are interpretable and propose that these will increase the clinical utility of testing across a range of Mendelian diseases.Genet Med 19 2, 192–203.

DNA testing for hypertrophic cardiomyopathy: a cost-effectiveness model

Aims To explore the cost-effectiveness of alternative methods of screening family members for hypertrophic cardiomyopathy (HCM), the most common monogenic cardiac disorder and the most frequent cause of sudden cardiac death (SCD) in young people. Methods and results Economic decision model comparing cascade screening by genetic, as opposed to clinical methods. The incremental cost per life year saved was 14,397 euro for the cascade genetic compared with the cascade clinical approach. Genetic diagnostic strategies are more likely to be cost-effective than clinical tests alone. The costs for cascade molecular genetic testing were slightly higher than clinical testing in the short run, but this was largely because the genetic approach is more effective and identifies more individuals at risk. Conclusion The use of molecular genetic information in the diagnosis and management of HCM is a cost-effective approach to the primary prevention of SCD in these patients.

Next-generation sequencing (NGS) as a diagnostic tool for retinal degeneration reveals a much higher detection rate in early-onset disease.

Inherited retinal degeneration (IRD) is a common cause of visual impairment (prevalence ∼1/3500). There is considerable phenotype and genotype heterogeneity, making a specific diagnosis very difficult without molecular testing. We investigated targeted capture combined with next-generation sequencing using Nimblegen 12plex arrays and the Roche 454 sequencing platform to explore its potential for clinical diagnostics in two common types of IRD, retinitis pigmentosa and cone-rod dystrophy. 50 patients (36 unknowns and 14 positive controls) were screened, and pathogenic mutations were identified in 25% of patients in the unknown, with 53% in the early-onset cases. All patients with new mutations detected had an age of onset <21 years and 44% had a family history. Thirty-one percent of mutations detected were novel. A de novo mutation in rhodopsin was identified in one early-onset case without a family history. Bioinformatic pipelines were developed to identify likely pathogenic mutations and stringent criteria were used for assignment of pathogenicity. Analysis of sequencing metrics revealed significant variability in capture efficiency and depth of coverage. We conclude that targeted capture and next-generation sequencing are likely to be very useful in a diagnostic setting, but patients with earlier onset of disease are more likely to benefit from using this strategy. The mutation-detection rate suggests that many patients are likely to have mutations in novel genes.

Clinical dividends from the molecular genetic diagnosis of craniosynostosis

A dozen years have passed since the first genetic lesion was identified in a family with craniosynostosis, the premature fusion of the cranial sutures. Subsequently, mutations in the FGFR2, FGFR3, TWIST1, and EFNB1 genes have been shown to account for ∼25% of craniosynostosis, whilst several additional genes make minor contributions. Using specific examples, we show how these discoveries have enabled refinement of information on diagnosis, recurrence risk, prognosis for mental development, and surgical planning. However, phenotypic variability can present a significant challenge to the clinical interpretation of molecular genetic tests. In particular, the difficulty of analyzing the complex interaction of genetic background and prenatal environment in determining clinical features, limits the value of identifying low penetrance mutations.

Clinical, biochemical, cellular and molecular characterization of mitochondrial DNA depletion syndrome due to novel mutations in the MPV17 gene

Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are severe autosomal recessive disorders associated with decreased mtDNA copy number in clinically affected tissues. The hepatocerebral form (mtDNA depletion in liver and brain) has been associated with mutations in the POLG, PEO1 (Twinkle), DGUOK and MPV17 genes, the latter encoding a mitochondrial inner membrane protein of unknown function. The aims of this study were to clarify further the clinical, biochemical, cellular and molecular genetic features associated with MDS due to MPV17 gene mutations. We identified 12 pathogenic mutations in the MPV17 gene, of which 11 are novel, in 17 patients from 12 families. All patients manifested liver disease. Poor feeding, hypoglycaemia, raised serum lactate, hypotonia and faltering growth were common presenting features. mtDNA depletion in liver was demonstrated in all seven cases where liver tissue was available. Mosaic mtDNA depletion was found in primary fibroblasts by PicoGreen staining. These results confirm that MPV17 mutations are an important cause of hepatocerebral mtDNA depletion syndrome, and provide the first demonstration of mosaic mtDNA depletion in human MPV17 mutant fibroblast cultures. We found that a severe clinical phenotype was associated with profound tissue-specific mtDNA depletion in liver, and, in some cases, mosaic mtDNA depletion in fibroblasts.

Gross deletions in TCOF1 are a cause of Treacher–Collins–Franceschetti syndrome

Treacher–Collins–Franceschetti syndrome (TCS) is an autosomal dominant craniofacial disorder characterised by midface hypoplasia, micrognathia, downslanting palpebral fissures, eyelid colobomata, and ear deformities that often lead to conductive deafness. A total of 182 patients with signs consistent with a diagnosis of TCS were screened by DNA sequence and dosage analysis of the TCOF1 gene. In all, 92 cases were found to have a pathogenic mutation by sequencing and 5 to have a partial gene deletion. A further case had a novel in-frame deletion in the alternatively spliced exon 6A of uncertain pathogenicity. The majority of the pathogenic sequence changes were found to predict premature protein termination, however, four novel missense changes in the LIS1 homology motif at the 5′ end of the gene were identified. The partial gene deletions of different sizes represent ∼5.2% of all the pathogenic TCOF1 mutations identified, indicating that gene rearrangements account for a significant proportion of TCS cases. This is the first report of gene rearrangements resulting in TCS. These findings expand the TCOF1 mutation spectrum indicating that dosage analysis should be performed together with sequence analysis, a strategy that is predicted to have a sensitivity of 71% for patients in whom TCS is strongly suspected.

Variable phenotype and discrete alterations of immune phenotypes in CTP synthase 1 deficiency: Report of 2 siblings

FIG 1. Pneumococcal serotype-specific antibody concentrations before and after booster vaccination at age 35 months. Pn, Pneumococcal serotype. Arrows indicate age at vaccination with the 13-valent pneumococcal conjugate vaccine (Prev(e)nar 13, Pfizer Inc); the dashed line specifies a serotype-specific IgG concentration of 0.35 mg/mL. To the Editor: Loss-of-function homozygous mutations in the CTP synthase 1 (CTPS1) gene in humans have only recently been discovered and reveal the role of this gene in lymphocyte proliferation. To date a single report including 8 individuals describes a severe clinical phenotype including early onset of severe chronic viral infections, recurrent encapsulated bacterial infections, and EBV-related B-cell non-Hodgkin lymphoma. We report 2 further sibling cases of CTSP1 deficiency identified through whole-exome sequencing (WES), further illustrating the phenotype of CTPS1 deficiency and demonstrating the value of WES for rapid diagnosis of primary immunodeficiency even for conditions whose phenotype is not well recognized. The 2 siblings (boy, now 6 years old; girl, now 3 years old) described in this report are the only children of nonconsanguineous and healthy white parents with no personal or family history of immunodeficiency, autoimmunity, or EBV-related disorders. Both children had no dysmorphic features and normal growth. The boy was born at term and was noted to have a significant burden of infections from early infancy. He was admitted to hospital at age 2 months with fever, lethargy, and poor feeding of unknown cause, responding rapidly to 48 hours of intravenous antibiotics. At age 7 months, he had an episode of invasive serotype 19A pneumococcal disease despite prior vaccination with the 13-valent pneumococcal conjugate vaccine (Prev(e)nar 13, Pfizer Inc, New York, NY) at age 2 and 4 months. At age 16months,while on holiday inTurkey, hewas admitted to hospital with severe tonsillitis and dehydration.At the age of 34months, he developed an acute EBV infection with persistent viremia (see Fig E1 in this article’s Online Repository at www.jacionline.org) but no features of hemophagocytic lymphohistiocytosis. In addition to these hospital admissions, he suffered from recurrent otitis media and lower respiratory tract infections, and had moderate to severe eczema from the neonatal period. He developed chronic diarrhea of unknown etiology lasting for approximately 8 weeks when he was 3.5 years old. An upper and lower gastrointestinal endoscopic biopsy showed reactive lymphoid aggregates in the duodenum and colon without signs of active inflammation, ulceration, or infectious organisms. The immunologic workup was largely normal (see Table E1 and the Methods section in this article’s Online Repository at www.jacionline.org). However, he had only moderate responses to a 13-valent pneumococcal conjugate vaccine booster with poor persistence of vaccine antibodies (Fig 1). He also showed rapid waning of antibodies against H influenzae type b polysaccharide and tetanus toxoid protein. His younger sister first presented at the age of 8 months with a likely viral illness involving fever, possible meningitis (mild pleocytosis) and unresponsive episodes along with vomiting, and prolonged diarrhea. She also suffered from eczema and recurrent upper and lower respiratory tract infections from age 12 months. However, she did not have frequent ear infections, had never suffered from invasive bacterial diseases, and remains EBV negative by both PCR and serology. Initial immunological testing was normal (Table E1) apart from a poor response to a booster dose of pneumococcal conjugate vaccine and undetectable antiH influenzae type b IgG concentration despite booster vaccination. Detailed analysis of cytokine production in both children showed a normal TH1 axis and normal TLR responses. However, IFN-g production in response to T-cell agonists was significantly reduced, pointing to T-cell impairment (see Fig E2 in this article’s Online Repository at www.jacionline.org). Following negative results upon initial investigation for causes of immunodeficiency, targeted WES was performed on the affected boy when he was 4 years old. A homozygous variant within the final nucleotide in intron 17 of the CTPS1 gene (NM_001905.2:c.1692-1G>C) was thought to be causative (see the Methods section). Flow cytometric analysis of lymphocytes at the age of 59 and 25 months, respectively, revealed lymphopenia and marked deficiency of B cells in the boy (Fig 2, A andB), possibly resulting from chronic EBV infection (Fig E1). Both children showed a relative predominance of transitional B cells and a deficiency of naive B cells (Fig 2, C). The distribution of major T cells (Fig 2,D) andCD4T-cell subpopulationswas largely unaffected (Fig 2, E), whereas major disturbances were seen within the CD8 T-cell compartment (Fig 2, F). The identification of the CTPS1 mutation has significantly altered the clinical management of the 2 affected siblings, emphasizing the clinical use of next-generation sequencing in children with unknown immunodeficiencies. The index case has already

Detection rate of pathogenic mutations in ABCA4 using direct sequencing: clinical and research implications.

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Pulsed Field Gel Electrophoresis for Detection of Gene Rearrangements in Duchenne Muscular Dystrophy

In diseases with a high new mutation rate, such as Duchenne and Becker muscular dystrophy (DMD, BMD), linkage analysis often produces highly unsatisfactory results for carrier diagnosis compared to methods that rely on the direct detection of the mutation. The size of the dystrophin gene and the nature of mutations at this locus that give rise to DMD/BMD make pulsed field gel electrophoresis (PFGE) an appropriate and powerful technique for detection of mutations and hence accurate carrier diagnosis in these diseases.