Article

Genetic Mapping and Exome Sequencing Identify Variants Associated with Five Novel Diseases

Innsbruck Medical University, Austria
PLoS ONE (Impact Factor: 3.53). 01/2012; 7(1):e28936. DOI: 10.1371/journal.pone.0028936
Source: PubMed

ABSTRACT The Clinic for Special Children (CSC) has integrated biochemical and molecular methods into a rural pediatric practice serving Old Order Amish and Mennonite (Plain) children. Among the Plain people, we have used single nucleotide polymorphism (SNP) microarrays to genetically map recessive disorders to large autozygous haplotype blocks (mean = 4.4 Mb) that contain many genes (mean = 79). For some, uninformative mapping or large gene lists preclude disease-gene identification by Sanger sequencing. Seven such conditions were selected for exome sequencing at the Broad Institute; all had been previously mapped at the CSC using low density SNP microarrays coupled with autozygosity and linkage analyses. Using between 1 and 5 patient samples per disorder, we identified sequence variants in the known disease-causing genes SLC6A3 and FLVCR1, and present evidence to strongly support the pathogenicity of variants identified in TUBGCP6, BRAT1, SNIP1, CRADD, and HARS. Our results reveal the power of coupling new genotyping technologies to population-specific genetic knowledge and robust clinical data.

0 Followers
 · 
315 Views
  • Source
    • "For Cases 4–8, mutations were identified by direct Sanger sequencing of SLC6A3. The mutations in Cases 4 and 5 were briefly reported previously (Puffenberger et al., 2012). Novel previously unreported mutations were identified in all others. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Dopamine transporter deficiency syndrome due to SLC6A3 mutations is the first inherited dopamine 'transportopathy' to be described, with a classical presentation of early infantile-onset progressive parkinsonism dystonia. In this study we have identified a new cohort of patients with dopamine transporter deficiency syndrome, including, most significantly, atypical presentation later in childhood with a milder disease course. We report the detailed clinical features, molecular genetic findings and in vitro functional investigations undertaken for adult and paediatric cases. Patients presenting with parkinsonism dystonia or a neurotransmitter profile characteristic of dopamine transporter deficiency syndrome were recruited for study. SLC6A3 mutational analysis was undertaken in all patients. The functional consequences of missense variants on the dopamine transporter were evaluated by determining the effect of mutant dopamine transporter on dopamine uptake, protein expression and amphetamine-mediated dopamine efflux using an in vitro cellular heterologous expression system. We identified eight new patients from five unrelated families with dopamine transporter deficiency syndrome. The median age at diagnosis was 13 years (range 1.5-34 years). Most significantly, the case series included three adolescent males with atypical dopamine transporter deficiency syndrome of juvenile onset (outside infancy) and progressive parkinsonism dystonia. The other five patients in the cohort presented with classical infantile-onset parkinsonism dystonia, with one surviving into adulthood (currently aged 34 years) and labelled as having 'juvenile parkinsonism'. All eight patients harboured homozygous or compound heterozygous mutations in SLC6A3, of which the majority are previously unreported variants. In vitro studies of mutant dopamine transporter demonstrated multifaceted loss of dopamine transporter function. Impaired dopamine uptake was universally present, and more severely impacted in dopamine transporter mutants causing infantile-onset rather than juvenile-onset disease. Dopamine transporter mutants also showed diminished dopamine binding affinity, reduced cell surface transporter, loss of post-translational dopamine transporter glycosylation and failure of amphetamine-mediated dopamine efflux. Our data series expands the clinical phenotypic continuum of dopamine transporter deficiency syndrome and indicates that there is a phenotypic spectrum from infancy (early onset, rapidly progressive disease) to childhood/adolescence and adulthood (later onset, slower disease progression). Genotype-phenotype analysis in this cohort suggests that higher residual dopamine transporter activity is likely to contribute to postponing disease presentation in these later-onset adult cases. Dopamine transporter deficiency syndrome remains under-recognized and our data highlights that dopamine transporter deficiency syndrome should be considered as a differential diagnosis for both infantile- and juvenile-onset movement disorders, including cerebral palsy and juvenile parkinsonism.
    Brain 03/2014; 137(4). DOI:10.1093/brain/awu022 · 10.23 Impact Factor
  • Source
    • "Among the three identified USH2 genes, USH2A (USH2A), GPR98 (USH2C), and DFNB31 (USH2D), USH2A mutations have been shown to be responsible for 70–80% of USH2 cases (Besnard et al. 2012; Le Quesne Stabej et al. 2012). Until recently, CLRN1 (USH3A) was the only gene known responsible for USH3 (Joensuu et al. 2001), but the HARS gene was recently proposed as a novel USH3 gene (Puffenberger et al. 2012). In addition, a twelfth gene, PDZD7, contributes to USH2 as a modifier of the retinal phenotype on a USH2A background or in digenic inheritance with GPR98 (Ebermann et al. 2010). "
    [Show abstract] [Hide abstract]
    ABSTRACT: We show that massively parallel targeted sequencing of 19 genes provides a new and reliable strategy for molecular diagnosis of Usher syndrome (USH) and nonsyndromic deafness, particularly appropriate for these disorders characterized by a high clinical and genetic heterogeneity and a complex structure of several of the genes involved. A series of 71 patients including Usher patients previously screened by Sanger sequencing plus newly referred patients was studied. Ninety-eight percent of the variants previously identified by Sanger sequencing were found by next-generation sequencing (NGS). NGS proved to be efficient as it offers analysis of all relevant genes which is laborious to reach with Sanger sequencing. Among the 13 newly referred Usher patients, both mutations in the same gene were identified in 77% of cases (10 patients) and one candidate pathogenic variant in two additional patients. This work can be considered as pilot for implementing NGS for genetically heterogeneous diseases in clinical service.
    01/2014; 2(1):30-43. DOI:10.1002/mgg3.25
  • [Show abstract] [Hide abstract]
    ABSTRACT: There has been an academic "gold rush" with researchers mining the deep seams of whole-exome and whole-genome sequencing since 2008. Although undoubtedly a major advance initially for identifying new disease-associated genes for rare monogenetic disorders-more recently, common and complex conditions have been successfully studied using these techniques. With great power comes great responsibility, however, and we must not forget that next generation sequencing produces unique ethical conundrums and validation challenges. We review the progression of published papers using whole-exome sequencing from a clinical and technical viewpoint before then reflecting on the key arguments that need to be fully understood before these tools can become a routine part of clinical practice and we ask what may be the role for the biomedical scientists?
    01/2012; 89:27-63. DOI:10.1016/B978-0-12-394287-6.00002-1
Show more