Clinical utility of exome sequencing in individuals with large homozygous regions detected by chromosomal microarray analysis.

BACKGROUND: Chromosomal microarray analysis (CMA) is recommended as the first-tier clinical diagnostic test for individuals with developmental disabilities. In addition to detecting copy number variations, CMA platforms with single nucleotide polymorphism probes can detect large homozygous regions within the genome, which represent potential risk for recessively inherited disorders. METHODS: To determine the frequency in which pathogenic or likely pathogenic variants can be detected in these regions of homozygosity, we performed whole exome sequencing (WES) in 53 individuals where homozygosity was detected by CMA. These patients were referred to our clinical laboratory for a variety of neurodevelopmental conditions including autism spectrum disorder, developmental delay, epilepsy, intellectual disability and microcephaly. RESULTS: In 11.3% (6/53) of cases, the analysis of homozygous variants revealed pathogenic or likely pathogenic variants in GJB2, TPP1, SLC25A15, TYR, PCCB, and NDUFV2 which are implicated in a variety of diseases. The evaluation of heterozygous variants with autosomal dominant inheritance, compound heterozygotes and variants with X-linked inheritance revealed pathogenic or likely pathogenic variants in PNPLA4, CADM1, HBB, SOS1, SFTPC, OTC and ASMT in 15.1% (8/53) of cases. Two of these patients harbored both homozygous and heterozygous variants relevant to their phenotypes (TPP1 and OTC; GJB2 and ASMT). CONCLUSIONS: Our study highlights the clinical utility of WES in individuals whose CMA uncovers homozygosity. Importantly, we show that when the phenotype is complex and homozygosity levels are high, WES can identify a significant number of relevant variants that explain neurodevelopmental phenotypes, and these mutations may lie outside of the regions of homozygosity, suggesting that the appropriate follow up test is WES rather than targeted sequencing.

A novel SH2 recognition mechanism recruits Spt6 to the doubly phosphorylated RNA polymerase II linker at sites of transcription.

We determined that the tandem SH2 domain of S. cerevisiae Spt6 binds the linker region of the RNA polymerase II subunit Rpb1 rather than the expected sites in its heptad repeat domain. The 4 nM binding affinity requires phosphorylation at Rpb1 S1493 and either T1471 or Y1473. Crystal structures showed that pT1471 binds the canonical SH2 pY site while pS1493 binds an unanticipated pocket 70 Å distant. Remarkably, the pT1471 phosphate occupies the phosphate-binding site of a canonical pY complex, while Y1473 occupies the position of a canonical pY side chain, with the combination of pT and Y mimicking a pY moiety. Biochemical data and modeling indicate that pY1473 can form an equivalent interaction, and we find that pT1471/pS1493 and pY1473/pS1493 combinations occur in vivo. ChIP-seq and genetic analyses demonstrate the importance of these interactions for recruitment of Spt6 to sites of transcription and for the maintenance of repressive chromatin.

Crystal structures of the S. cerevisiae Spt6 core and C-terminal tandem SH2 domain.

The conserved and essential eukaryotic protein Spt6 functions in transcription elongation, chromatin maintenance, and RNA processing. Spt6 has three characterized functions. It is a histone chaperone capable of reassembling nucleosomes, a central component of transcription elongation complexes, and is required for recruitment of RNA processing factors to elongating RNA polymerase II (RNAPII). Here, we report multiple crystal structures of the 168-kDa Spt6 protein from Saccharomyces cerevisiae that together represent essentially all of the ordered sequence. Our two structures of the ∼900-residue core region reveal a series of putative nucleic acid and protein-protein interaction domains that fold into an elongated form that resembles the bacterial protein Tex. The similarity to a bacterial transcription factor suggests that the core domain performs nucleosome-independent activities, and as with Tex, we find that Spt6 binds DNA. Unlike Tex, however, the Spt6 S1 domain does not contribute to this activity. Crystal structures of the Spt6 C-terminal region reveal a tandem SH2 domain structure composed of two closely associated SH2 folds. One of these SH2 folds is cryptic, while the other shares striking structural similarity with metazoan SH2 domains and possesses structural features associated with the ability to bind phosphorylated substrates including phosphotyrosine. Binding studies with phosphopeptides that mimic the RNAPII C-terminal domain revealed affinities typical of other RNAPII C-terminal domain-binding proteins but did not indicate a specific interaction. Overall, these findings provide a structural foundation for understanding how Spt6 encodes several distinct functions within a single polypeptide chain.