High-throughput DNA sequencing technology has transformed genetic research and is starting to make an impact on clinical practice. However, analyzing high-throughput sequencing data remains challenging, particularly in clinical settings where accuracy and turnaround times are critical. We present a new approach to this problem, implemented in a software package called Platypus. Platypus achieves high sensitivity and specificity for SNPs, indels and complex polymorphisms by using local de novo assembly to generate candidate variants, followed by local realignment and probabilistic haplotype estimation. It is an order of magnitude faster than existing tools and generates calls from raw aligned read data without preprocessing. We demonstrate the performance of Platypus in clinically relevant experimental designs by comparing with SAMtools and GATK on whole-genome and exome-capture data, by identifying de novo variation in 15 parent-offspring trios with high sensitivity and specificity, and by estimating human leukocyte antigen genotypes directly from variant calls.
The Mediator is an evolutionarily conserved, multi-subunit complex that regulates multiple steps of transcription. Mediator activity is regulated by the reversible association of a four-subunit module comprising CDK8 or CDK19 kinases, together with cyclin C, MED12 or MED12L, and MED13 or MED13L. Mutations in
MED12
,
MED13,
and
MED13L
were previously identified in syndromic developmental disorders with overlapping phenotypes. Here, we report
CDK8
mutations (located at 13q12.13) that cause a phenotypically related disorder. Using whole-exome or whole-genome sequencing, and by international collaboration, we identified eight different heterozygous missense
CDK8
substitutions, including 10 shown to have arisen
de novo
, in 12 unrelated subjects; a recurrent mutation, c.185C>T (p.Ser62Leu), was present in five individuals. All predicted substitutions localize to the ATP-binding pocket of the kinase domain. Affected individuals have overlapping phenotypes characterized by hypotonia, mild to moderate intellectual disability, behavioral disorders, and variable facial dysmorphism. Congenital heart disease occurred in six subjects; additional features present in multiple individuals included agenesis of the corpus callosum, ano-rectal malformations, seizures, and hearing or visual impairments. To evaluate the functional impact of the mutations, we measured phosphorylation at STAT1-Ser727, a known CDK8 substrate, in a
CDK8
and
CDK19
CRISPR double-knockout cell line transfected with wild-type (WT) or mutant
CDK8
constructs. These experiments demonstrated a reduction in STAT1 phosphorylation by all mutants, in most cases to a similar extent as in a kinase-dead control. We conclude that missense mutations in
CDK8
cause a developmental disorder that has phenotypic similarity to syndromes associated with mutations in other subunits of the Mediator kinase module, indicating probable overlap in pathogenic mechanisms.
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