With the expanded availability of next generation sequencing (NGS)-based clinical genetic tests, clinicians seeking to test patients with Mendelian diseases must weigh the superior coverage of ...targeted gene panels with the greater number of genes included in whole exome sequencing (WES) when considering their first-tier testing approach. Here, we use an in silico analysis to predict the analytic sensitivity of WES using pathogenic variants identified on targeted NGS panels as a reference.
Corresponding nucleotide positions for 1533 different alterations classified as pathogenic or likely pathogenic identified on targeted NGS multi-gene panel tests in our laboratory were interrogated in data from 100 randomly-selected clinical WES samples to quantify the sequence coverage at each position. Pathogenic variants represented 91 genes implicated in hereditary cancer, X-linked intellectual disability, primary ciliary dyskinesia, Marfan syndrome/aortic aneurysms, cardiomyopathies and arrhythmias.
When assessing coverage among 100 individual WES samples for each pathogenic variant (153,300 individual assessments), 99.7% (n = 152,798) would likely have been detected on WES. All pathogenic variants had at least some coverage on exome sequencing, with a total of 97.3% (n = 1491) detectable across all 100 individuals. For the remaining 42 pathogenic variants, the number of WES samples with adequate coverage ranged from 35 to 99. Factors such as location in GC-rich, repetitive, or homologous regions likely explain why some of these alterations were not detected across all samples. To validate study findings, a similar analysis was performed against coverage data from 60,706 exomes available through the Exome Aggregation Consortium (ExAC). Results from this validation confirmed that 98.6% (91,743,296/93,062,298) of pathogenic variants demonstrated adequate depth for detection.
Results from this in silico analysis suggest that exome sequencing may achieve a diagnostic yield similar to panel-based testing for Mendelian diseases.
Exome sequencing of a single individual for a clinical indication may result in the identification of incidental deleterious variants unrelated to the indication for testing (secondary findings). ...Given the recent availability of clinical exome testing, there is a limited knowledge regarding the disclosure preferences and impact of secondary findings in a clinical diagnostic setting. In this article, we provide preliminary data regarding the preferences for secondary findings results disclosure based on the first 200 families referred to Ambry Genetics for diagnostic exome sequencing.
Secondary findings were categorized into four groups in the diagnostic exome sequencing consent: carrier status of recessive disorders, predisposition to later-onset disease, predisposition to increased cancer risk, and early-onset disease. In this study, we performed a retrospective analysis of patient responses regarding the preferences for secondary findings disclosure.
The majority of patients (187/200; 93.5%) chose to receive secondary results for one or more available categories. Adult probands were more likely than children to opt for blinding of secondary data (16 vs. 4%, respectively). Among responses for blinding, preferences were evenly scattered among categories.
These data represent the unprecedented results of a large reference laboratory providing clinical exome sequencing. We report, for the first time, the preferences of patients and families for the receipt of secondary findings based on clinical genome sequencing. Overwhelmingly, families undergoing exome sequencing opt for the disclosure of secondary findings. The data may have implications regarding the development of guidelines for secondary findings reporting among patients with severe and/or life-threatening disease undergoing clinical genomic sequencing.
Background
Clinical diagnostic whole‐exome sequencing (WES) is a powerful tool for patients with undiagnosed genetic disorders. To demonstrate the clinical utility, we surveyed healthcare providers ...(HCP) about changes in medical management and treatment, diagnostic testing, reproductive planning, and use of educational services subsequent to WES testing.
Methods
For a period of 18 months, an 18‐question survey was sent to HCPs attached to the WES reports. We analyzed the molecular diagnosis, patient clinical features, and the medical management changes reported in the returned surveys.
Results
A total of 62 (2.2% of 2,876) surveys were returned, consisting of 37.1% patients with a positive or likely positive pathogenic alteration, 51.6% negative results, 9.7% uncertain findings, and 1 patient (1.6%) with a novel candidate finding. Overall, 100% of the HCPs of patients with positive or likely positive WES results (n = 23) and HCPs of patients with uncertain WES results (n = 6) responded positively to one of the 18 queries. Of note, 37.5% of the HCPs of patients with negative WES results (n = 32) responded positively to at least one query.
Conclusion
Overall, these data clearly demonstrate the clinical utility of WES by demonstrating the impact on medical management irrespective of the exome result.
To demonstrate the clinical utility of whole‐exome sequencing (WES), we surveyed healthcare providers (HCP) about changes in medical management and treatment, diagnostic testing, reproductive planning, and use of educational services subsequent to WES testing. Overall, 100% of the HCPs of patients with positive or likely positive WES results (n = 23) and HCPs of patients with uncertain WES results (n = 6) responded positively to one of the 18 queries. Of note, 37.5% of the HCPs of patients with negative WES results (n = 32) responded positively to at least one query.
Diagnostic exome sequencing was immediately successful in diagnosing patients in whom traditional technologies were uninformative. Herein, we provide the results from the first 500 probands referred ...to a clinical laboratory for diagnostic exome sequencing.
Family-based exome sequencing included whole-exome sequencing followed by family inheritance-based model filtering, comprehensive medical review, familial cosegregation analysis, and analysis of novel genes.
A positive or likely positive result in a characterized gene was identified in 30% of patients (152/500). A novel gene finding was identified in 7.5% of patients (31/416). The highest diagnostic rates were observed among patients with ataxia, multiple congenital anomalies, and epilepsy (44, 36, and 35%, respectively). Twenty-three percent of positive findings were within genes characterized within the past 2 years. The diagnostic rate was significantly higher among families undergoing a trio (37%) as compared with a singleton (21%) whole-exome testing strategy.
Overall, we present results from the largest clinical cohort of diagnostic exome sequencing cases to date. These data demonstrate the utility of family-based exome sequencing and analysis to obtain the highest reported detection rate in an unselected clinical cohort, illustrating the utility of diagnostic exome sequencing as a transformative technology for the molecular diagnosis of genetic disease.
Congenital generalized lipodystrophy (CGL) and pulmonary arterial hypertension (PAH) have recently been associated with mutations in the caveolin‐1 (
CAV1
) gene, which encodes the primary structural ...protein of caveolae. However, little is currently known about how these
CAV1
mutations impact caveolae formation or contribute to the development of disease. Here, we identify a heterozygous F160X
CAV1
mutation predicted to generate a C‐terminally truncated mutant protein in a patient with both PAH and CGL using whole exome sequencing, and characterize the properties of
CAV1
, caveolae‐associated proteins and caveolae in skin fibroblasts isolated from the patient. We show that morphologically defined caveolae are present in patient fibroblasts and that they function in mechanoprotection. However, they exhibited several notable defects, including enhanced accessibility of the C‐terminus of wild‐type CAV1 in caveolae, reduced colocalization of cavin‐1 with CAV1 and decreased stability of both 8S and 70S oligomeric CAV1 complexes that are necessary for caveolae formation. These results were verified independently in reconstituted CAV1
−/− mouse embryonic fibroblasts. These findings identify defects in caveolae that may serve as contributing factors to the development of PAH and CGL and broaden our knowledge of
CAV1 mutations associated with human disease.
We identify a heterozygous F160X mutation in the caveolin‐1 (CAV1) gene in a patient with both pulmonary arterial hypertension (PAH) and congenital generalized lipodystrophy (CGL) and examine its effect on caveolae assembly. We find that the mutant protein forms hybrid caveolae together with wild‐type CAV1 in patient skin fibroblasts and reconstituted CAV1−/− murine embryonic fibroblasts but that these caveolae are abnormal in several respects. These caveolaer defects may serve as contributing factors to the development of PAH and CGL.
Diagnostic exome sequencing (DES) is now a commonly ordered test for individuals with undiagnosed genetic disorders. In addition to providing a diagnosis for characterized diseases, exome sequencing ...has the capacity to uncover novel candidate genes for disease.
Family-based DES included analysis of both characterized and novel genetic etiologies. To evaluate candidate genes for disease in the clinical setting, we developed a systematic, rule-based classification schema.
Testing identified a candidate gene among 7.7% (72/934) of patients referred for DES; 37 (4.0%) and 35 (3.7%) of the genes received evidence scores of “candidate” and “suspected candidate,” respectively. A total of 71 independent candidate genes were reported among the 72 patients, and 38% (27/71) were subsequently corroborated in the peer-reviewed literature. This rate of corroboration increased to 51.9% (27/52) among patients whose gene was reported at least 12 months previously.
Herein, we provide transparent, comprehensive, and standardized scoring criteria for the clinical reporting of candidate genes. These results demonstrate that DES is an integral tool for genetic diagnosis, especially for elucidating the molecular basis for both characterized and novel candidate genetic etiologies. Gene discoveries also advance the understanding of normal human biology and more common diseases.
To identify the molecular basis for prenatally suspected cases of megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) (MIM 249210) in 3 independent families with clinical and ...radiographic evidence of MMIHS.
Whole-exome sequencing (WES) and Sanger sequencing of the ACTG2 gene.
We identified a novel heterozygous de novo missense variant in ACTG2 c.770G>A (p.Arg257His) encoding x03B3;-2 smooth muscle actin (ACTG2) in 2 siblings with MMIHS, suggesting gonadal mosaicism of one of the parents. Two additional de novo missense variants (p.Arg257Cys and p.Arg178His) in ACTG2 were identified in 2 additional MMHIS patients. All of our patients had evidence of fetal megacystis and a normal or slightly increased amniotic fluid volume. Additional findings included bilateral renal hydronephrosis, an enlarged fetal stomach, and transient dilated bowel loops. ACTG2 immunostaining of the intestinal tissue showed an altered muscularis propria, a markedly thinned longitudinal muscle layer, and a reduced amount and abnormal distribution of ACTG2.
Our study demonstrates that de novo mutations in ACTG2 are a cause of fetal megacystis in MMIHS and that gonadal mosaicism may be present in a subset of cases. These findings have implications for the counseling of families with a diagnosis of fetal megacystis with a preserved amniotic fluid volume and associated gastrointestinal findings.
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