Computational protocol: Microdeletions in 9q33.3-q34.11 in five patients with intellectual disability, microcephaly, and seizures of incomplete penetrance: is STXBP1 not the only causative gene?

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Protocol publication

[…] Mutational screening of RALGPS1 and GARNL3 by Sanger sequencing was performed on 192 individuals seen at the University Medical Genetics Clinics in Bonn, 96 of whom had mild ID/DD and 96 of whom had moderate to severe ID/DD. This study group consisted of 66 females (34 %) and 126 males (66 %). A total of 82 individuals showed no or only negligible dysmorphisms (43 %), while 109 had dysmorphisms (57 %); of the latter group, 40 patients also showed organ malformations (21 %). Standard evaluation consisted of detailed clinical investigation, conventional karyotyping, and the exclusion of clinically recognizable syndromes with known etiology. The presence of Fragile X syndrome had been excluded in almost all patients. Previous molecular karyotyping with Illumina microarrays (550, 610, 660 K or Omni1-Quad) had identified no pathogenic aberrations. The study was approved by the Institutional Review Board of the University Hospital of Bonn, and informed consent was obtained for all participants.To target the coding regions of RALGPS1 and GARNL3 and flanking intronic sequence (at least 15 bp), primers were designed using the online program Primer3 (http://primer3.ut.ee/). All known protein-coding RefSeq isoforms were analyzed: NM_014636, NM_001190728, NM_001190729 and NM_001190730 for RALGPS1, NM_032293 and NM_001286779 for GARNL3. Sequencing was performed with either genomic or whole genome amplified DNA (REPLI-g WGA kit, Qiagen, Hilden, Germany) of the 192 patients with ID/DD using standard procedures. Amplicons were sequenced bi-directionally using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA). The fluorescently labeled fragments were analyzed on a capillary sequencing system (3130XL Genetic Analyzer, Applied Biosystems). Sequences were analyzed using SeqMan II software (DNAStar, Madison, WI, USA). All primer sequences as well as PCR conditions are available upon request. The frequency in control cohorts was analyzed for all detected variants using dbSNP build 142 and the NHLBI GO-ESP cohort (Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), http://evs.gs.washington.edu/EVS/; July 2015). The in silico tool CADD (Combined Annotation Dependent Depletion, http://cadd.gs.washington.edu/, []) was employed to predict pathogenicity. […]

Pipeline specifications

Software tools Primer3, CADD
Databases dbSNP Exome Variant Server
Applications WGS analysis, WES analysis, qPCR
Organisms Homo sapiens
Diseases Epilepsy, Microcephaly, Movement Disorders