Utility of whole-exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care.

An accurate diagnosis is an integral component of patient care for children with rare genetic disease. Recent advances in sequencing, in particular whole-exome sequencing (WES), are identifying the genetic basis of disease for 25-40% of patients. The diagnostic rate is probably influenced by when in the diagnostic process WES is used. The Finding Of Rare Disease GEnes (FORGE) Canada project was a nation-wide effort to identify mutations for childhood-onset disorders using WES. Most children enrolled in the FORGE project were toward the end of the diagnostic odyssey. The two primary outcomes of FORGE were novel gene discovery and the identification of mutations in genes known to cause disease. In the latter instance, WES identified mutations in known disease genes for 105 of 362 families studied (29%), thereby informing the impact of WES in the setting of the diagnostic odyssey. Our analysis of this dataset showed that these known disease genes were not identified prior to WES enrollment for two key reasons: genetic heterogeneity associated with a clinical diagnosis and atypical presentation of known, clinically recognized diseases. What is becoming increasingly clear is that WES will be paradigm altering for patients and families with rare genetic diseases.

Hypercontractile properties of cardiac muscle fibers in a knock-in mouse model of cardiac myosin-binding protein-C.

Myosin-binding protein-C (MyBP-C) is a component of all striated-muscle sarcomeres, with a well established structural role and a possible function for force regulation. Multiple mutations within the gene for cardiac MyBP-C, one of three known isoforms, have been linked to familial hypertrophic cardiomyopathy. Here we generated a knock-in mouse model that carries N-terminal-shortened cardiac MyBP-C. The mutant protein was designed to have a similar size as the skeletal MyBP-C isoforms, whereas known myosin and titin binding sites as well as the phosphorylatable MyBP-C motif were not altered. We have shown that mutant cardiac MyBP-C is readily incorporated into the sarcomeres of both heterozygous and homozygous animals and can still be phosphorylated by cAMP-dependent protein kinase. Although histological characterization of wild-type and mutant hearts did not reveal obvious differences in phenotype, left ventricular fibers from homozygous mutant mice exhibited an increased Ca(2+) sensitivity of force development, particularly at lower Ca(2+) concentrations, whereas maximal active force levels remained unchanged. The results allow us to propose a model of how cMyBP-C may affect myosin-head mobility and to rationalize why N-terminal mutations of the protein in some cases of familial hypertrophic cardiomyopathy could lead to a hypercontractile state.

A rapid protocol for cardiac troponin T gene mutation detection in familial hypertrophic cardiomyopathy.

Mutations in the human cardiac troponin T gene (TNNT2) are associated with familial hypertrophic cardiomyopathy (FHC) linked to chromosome 1q3 (CMH2). Mutation analyses of TNNT2 have been restricted to RNA-based screening methods because only the TNNT2 cDNA sequence was known. We characterized the genomic structure of 15 TNNT2 exons spliced into the adult isoform. A protocol for rapid mutation detection based on direct sequencing of large PCR-amplified genomic DNA fragments revealed a known TNNT2 mutation (Phe110Ile) in one of 30 FHC probands. Three polymorphic short tandem repeat elements (D1S477, D1S2622, and D1S1723), useful for FHC pedigree analyses at CMH2, were shown to be physically tightly linked to TNNT2.