@article {792, title = {Quantitative Assessment of Parental Somatic Mosaicism for Copy-Number Variant (CNV) Deletions.}, journal = {Curr Protoc Hum Genet}, volume = {106}, year = {2020}, month = {2020 06}, pages = {e99}, abstract = {

As genome sequencing methodologies have become more sensitive in detecting low-frequency rare-variant events, the link between post-zygotic mutagenesis and somatic mosaicism in the etiology of several human genetic conditions other than cancers has become more clear. Given that current clinical-genomics diagnostic methods have limited detection sensitivity for mosaic events, a copy-number variant (CNV) deletion inherited from a parent with low-level (<10\%) mosaicism can be erroneously interpreted in the proband to represent a de novo germline event. Here, we describe three sensitive, precise, and cost-efficient methods that can quantitatively assess the potential degree of parental somatic mosaicism levels for CNV deletions: droplet digital PCR (ddPCR), PCR amplicon-based next-generation sequencing (NGS), and quantitative PCR. ddPCR using the EvaGreen fluorescent dye protocol can specifically quantify the deleted or non-deleted alleles by analyzing the number of droplets positive for a fluorescent signal for each event. PCR amplicon-based NGS assesses the allele frequencies of a heterozygous single-nucleotide polymorphism within a deletion region. The difference in number of reads between the two genotypes indicates the level of somatic mosaicism for the CNV deletion. Quantitative PCR can be applied where the relative quantity of the deletion junction-specific product represents the level of mosaicism. Clinical implementation of these quantitative variant-detection methods enables potentially more accurate assessment of disease recurrence risk in family-based genetic counseling, allowing couples to engage in more informed family planning. {\textcopyright} 2020 by John Wiley \& Sons, Inc. Basic Protocol: Droplet digital PCR (ddPCR) Alternate Protocol 1: PCR amplicon-based next-generation sequencing Alternate Protocol 2: Quantitative real-time PCR (qPCR).

}, keywords = {DNA Copy Number Variations, Genetic Diseases, Inborn, High-Throughput Nucleotide Sequencing, Humans, Mosaicism, Parents, Polymorphism, Single Nucleotide, Real-Time Polymerase Chain Reaction}, issn = {1934-8258}, doi = {10.1002/cphg.99}, author = {Liu, Qian and Grochowski, Christopher M and Bi, Weimin and Lupski, James R and Stankiewicz, Pawe{\l}} } @article {339, title = {Resolution of Disease Phenotypes Resulting from Multilocus Genomic Variation.}, journal = {N Engl J Med}, volume = {376}, year = {2017}, month = {2017 01 05}, pages = {21-31}, abstract = {

BACKGROUND: Whole-exome sequencing can provide insight into the relationship between observed clinical phenotypes and underlying genotypes.

METHODS: We conducted a retrospective analysis of data from a series of 7374 consecutive unrelated patients who had been referred to a clinical diagnostic laboratory for whole-exome sequencing; our goal was to determine the frequency and clinical characteristics of patients for whom more than one molecular diagnosis was reported. The phenotypic similarity between molecularly diagnosed pairs of diseases was calculated with the use of terms from the Human Phenotype Ontology.

RESULTS: A molecular diagnosis was rendered for 2076 of 7374 patients (28.2\%); among these patients, 101 (4.9\%) had diagnoses that involved two or more disease loci. We also analyzed parental samples, when available, and found that de novo variants accounted for 67.8\% (61 of 90) of pathogenic variants in autosomal dominant disease genes and 51.7\% (15 of 29) of pathogenic variants in X-linked disease genes; both variants were de novo in 44.7\% (17 of 38) of patients with two monoallelic variants. Causal copy-number variants were found in 12 patients (11.9\%) with multiple diagnoses. Phenotypic similarity scores were significantly lower among patients in whom the phenotype resulted from two distinct mendelian disorders that affected different organ systems (50 patients) than among patients with disorders that had overlapping phenotypic features (30 patients) (median score, 0.21 vs. 0.36; P=1.77{\texttimes}10).

CONCLUSIONS: In our study, we found multiple molecular diagnoses in 4.9\% of cases in which whole-exome sequencing was informative. Our results show that structured clinical ontologies can be used to determine the degree of overlap between two mendelian diseases in the same patient; the diseases can be distinct or overlapping. Distinct disease phenotypes affect different organ systems, whereas overlapping disease phenotypes are more likely to be caused by two genes encoding proteins that interact within the same pathway. (Funded by the National Institutes of Health and the Ting Tsung and Wei Fong Chao Foundation.).

}, keywords = {Exome, Genetic Diseases, Inborn, Genetic Variation, Genotyping Techniques, High-Throughput Nucleotide Sequencing, Humans, Phenotype, Retrospective Studies, Sequence Analysis, DNA}, issn = {1533-4406}, doi = {10.1056/NEJMoa1516767}, author = {Posey, Jennifer E and Harel, Tamar and Liu, Pengfei and Rosenfeld, Jill A and James, Regis A and Coban Akdemir, Zeynep H and Walkiewicz, Magdalena and Bi, Weimin and Xiao, Rui and Ding, Yan and Xia, Fan and Beaudet, Arthur L and Muzny, Donna M and Gibbs, Richard A and Boerwinkle, Eric and Eng, Christine M and Sutton, V Reid and Shaw, Chad A and Plon, Sharon E and Yang, Yaping and Lupski, James R} }