@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 {515, title = {Clinical exome sequencing reveals locus heterogeneity and phenotypic variability of cohesinopathies.}, journal = {Genet Med}, volume = {21}, year = {2019}, month = {2019 03}, pages = {663-675}, abstract = {

PURPOSE: Defects in the cohesin pathway are associated with cohesinopathies, notably Cornelia de Lange syndrome (CdLS). We aimed to delineate pathogenic variants~in known and candidate cohesinopathy genes from a clinical exome perspective.

METHODS: We retrospectively studied patients referred for clinical exome sequencing (CES, N = 10,698). Patients with causative variants in novel or recently described cohesinopathy genes were enrolled for phenotypic characterization.

RESULTS: Pathogenic or likely pathogenic single-nucleotide and insertion/deletion variants (SNVs/indels) were identified in established disease genes including NIPBL (N = 5), SMC1A (N = 14), SMC3 (N = 4), RAD21 (N = 2), and HDAC8 (N = 8). The phenotypes in this genetically defined cohort skew towards the mild end of CdLS spectrum as compared with phenotype-driven cohorts. Candidate or recently reported cohesinopathy genes were supported by de novo SNVs/indels in STAG1 (N = 3), STAG2 (N = 5), PDS5A (N = 1), and WAPL (N = 1), and one inherited SNV in PDS5A. We also identified copy-number deletions affecting STAG1 (two de novo, one of unknown inheritance) and STAG2 (one of unknown inheritance). Patients with STAG1 and STAG2 variants presented with overlapping features yet without characteristic facial features of CdLS.

CONCLUSION: CES effectively identified disease-causing alleles at the mild end of the cohensinopathy spectrum and enabled characterization of candidate disease genes.

}, keywords = {Adolescent, Alleles, Antigens, Nuclear, Biological Variation, Population, Carrier Proteins, Cell Cycle Proteins, Child, Child, Preschool, Chromosomal Proteins, Non-Histone, Cohort Studies, De Lange Syndrome, Exome, Female, Gene Frequency, Genetic Heterogeneity, Humans, INDEL Mutation, Male, Mutation, Nuclear Proteins, Phenotype, Polymorphism, Single Nucleotide, Proto-Oncogene Proteins, Retrospective Studies, Whole Exome Sequencing}, issn = {1530-0366}, doi = {10.1038/s41436-018-0085-6}, author = {Yuan, Bo and Neira, Juanita and Pehlivan, Davut and Santiago-Sim, Teresa and Song, Xiaofei and Rosenfeld, Jill and Posey, Jennifer E and Patel, Vipulkumar and Jin, Weihong and Adam, Margaret P and Baple, Emma L and Dean, John and Fong, Chin-To and Hickey, Scott E and Hudgins, Louanne and Leon, Eyby and Madan-Khetarpal, Suneeta and Rawlins, Lettie and Rustad, Cecilie F and Stray-Pedersen, Asbj{\o}rg and Tveten, Kristian and Wenger, Olivia and Diaz, Jullianne and Jenkins, Laura and Martin, Laura and McGuire, Marianne and Pietryga, Marguerite and Ramsdell, Linda and Slattery, Leah and Abid, Farida and Bertuch, Alison A and Grange, Dorothy and Immken, LaDonna and Schaaf, Christian P and Van Esch, Hilde and Bi, Weimin and Cheung, Sau Wai and Breman, Amy M and Smith, Janice L and Shaw, Chad and Crosby, Andrew H and Eng, Christine and Yang, Yaping and Lupski, James R and Xiao, Rui and Liu, Pengfei} } @article {335, title = {De Novo Disruption of the Proteasome Regulatory Subunit PSMD12 Causes a Syndromic Neurodevelopmental Disorder.}, journal = {Am J Hum Genet}, volume = {100}, year = {2017}, month = {2017 Feb 02}, pages = {352-363}, abstract = {

Degradation of proteins by the ubiquitin-proteasome system (UPS) is an essential biological process in the development of eukaryotic organisms. Dysregulation of this mechanism leads to numerous human neurodegenerative or neurodevelopmental disorders. Through a multi-center collaboration, we identified six de novo genomic deletions and four de novo point mutations involving PSMD12, encoding the non-ATPase subunit PSMD12 (aka RPN5) of the 19S regulator of 26S proteasome complex, in unrelated individuals with intellectual disability, congenital malformations, ophthalmologic anomalies, feeding difficulties, deafness, and subtle dysmorphic facial features. We observed reduced PSMD12 levels and an accumulation of ubiquitinated proteins without any impairment of proteasome catalytic activity. Our PSMD12 loss-of-function zebrafish CRISPR/Cas9 model exhibited microcephaly, decreased convolution of the renal tubules, and abnormal craniofacial morphology. Our data support the biological importance of PSMD12 as a scaffolding subunit in proteasome function during development and neurogenesis in particular; they enable the definition of a neurodevelopmental disorder due to PSMD12 variants, expanding the phenotypic spectrum of UPS-dependent disorders.

}, keywords = {Adolescent, Animals, Child, Child, Preschool, Disease Models, Animal, DNA Copy Number Variations, Down-Regulation, Female, Gene Deletion, Humans, Infant, Intellectual Disability, Male, Microcephaly, Neurodevelopmental Disorders, Polymorphism, Single Nucleotide, Proteasome Endopeptidase Complex, Zebrafish}, issn = {1537-6605}, doi = {10.1016/j.ajhg.2017.01.003}, author = {K{\"u}ry, S{\'e}bastien and Besnard, Thomas and Ebstein, Fr{\'e}d{\'e}ric and Khan, Tahir N and Gambin, Tomasz and Douglas, Jessica and Bacino, Carlos A and Craigen, William J and Sanders, Stephan J and Lehmann, Andrea and Latypova, X{\'e}nia and Khan, Kamal and Pacault, Mathilde and Sacharow, Stephanie and Glaser, Kimberly and Bieth, Eric and Perrin-Sabourin, Laurence and Jacquemont, Marie-Line and Cho, Megan T and Roeder, Elizabeth and Denomm{\'e}-Pichon, Anne-Sophie and Monaghan, Kristin G and Yuan, Bo and Xia, Fan and Simon, Sylvain and Bonneau, Dominique and Parent, Philippe and Gilbert-Dussardier, Brigitte and Odent, Sylvie and Toutain, Annick and Pasquier, Laurent and Barbouth, Deborah and Shaw, Chad A and Patel, Ankita and Smith, Janice L and Bi, Weimin and Schmitt, S{\'e}bastien and Deb, Wallid and Nizon, Mathilde and Mercier, Sandra and Vincent, Marie and Rooryck, Caroline and Malan, Val{\'e}rie and Brice{\~n}o, Ignacio and G{\'o}mez, Alberto and Nugent, Kimberly M and Gibson, James B and Cogn{\'e}, Benjamin and Lupski, James R and Stessman, Holly A F and Eichler, Evan E and Retterer, Kyle and Yang, Yaping and Redon, Richard and Katsanis, Nicholas and Rosenfeld, Jill A and Kloetzel, Peter-Michael and Golzio, Christelle and B{\'e}zieau, St{\'e}phane and Stankiewicz, Pawe{\l} and Isidor, Bertrand} } @article {357, title = {Lessons learned from additional research analyses of unsolved clinical exome cases.}, journal = {Genome Med}, volume = {9}, year = {2017}, month = {2017 03 21}, pages = {26}, abstract = {

BACKGROUND: Given the rarity of most single-gene Mendelian disorders, concerted efforts of data exchange between clinical and scientific communities are critical to optimize molecular diagnosis and novel disease gene discovery.

METHODS: We designed and implemented protocols for the study of cases for which a plausible molecular diagnosis was not achieved in a clinical genomics diagnostic laboratory (i.e. unsolved clinical exomes). Such cases were recruited to a research laboratory for further analyses, in order to potentially: (1) accelerate novel disease gene discovery; (2) increase the molecular diagnostic yield of whole exome sequencing (WES); and (3) gain insight into the genetic mechanisms of disease. Pilot project data included 74 families, consisting mostly of parent-offspring trios. Analyses performed on a research basis employed both WES from additional family members and complementary bioinformatics approaches and protocols.

RESULTS: Analysis of all possible modes of Mendelian inheritance, focusing on both single nucleotide variants (SNV) and copy number variant (CNV) alleles, yielded a likely contributory variant in 36\% (27/74) of cases. If one includes candidate genes with variants identified within a single family, a potential contributory variant was identified in a total of ~51\% (38/74) of cases enrolled in this pilot study. The molecular diagnosis was achieved in 30/63 trios (47.6\%). Besides this, the analysis workflow yielded evidence for pathogenic variants in disease-associated genes in 4/6 singleton cases (66.6\%), 1/1 multiplex family involving three affected siblings, and 3/4 (75\%) quartet families. Both the analytical pipeline and the collaborative efforts between the diagnostic and research laboratories provided insights that allowed recent disease gene discoveries (PURA, TANGO2, EMC1, GNB5, ATAD3A, and MIPEP) and increased the number of novel genes, defined in this study as genes identified in more than one family (DHX30 and EBF3).

CONCLUSION: An efficient genomics pipeline in which clinical sequencing in a diagnostic laboratory is followed by the detailed reanalysis of unsolved cases in a research environment, supplemented with WES data from additional family members, and subject to adjuvant bioinformatics analyses including relaxed variant filtering parameters in informatics pipelines, can enhance the molecular diagnostic yield and provide mechanistic insights into Mendelian disorders. Implementing these approaches requires collaborative clinical molecular diagnostic and research efforts.

}, keywords = {Adenosine Triphosphatases, ATPases Associated with Diverse Cellular Activities, Computational Biology, DNA Copy Number Variations, DNA-Binding Proteins, Exome, Female, Genetic Diseases, Inborn, Genomics, GTP-Binding Protein beta Subunits, Humans, Male, Membrane Proteins, Metalloendopeptidases, Mitochondrial Proteins, Pilot Projects, Polymorphism, Single Nucleotide, Sequence Analysis, DNA, Transcription Factors}, issn = {1756-994X}, doi = {10.1186/s13073-017-0412-6}, author = {Eldomery, Mohammad K and Coban-Akdemir, Zeynep and Harel, Tamar and Rosenfeld, Jill A and Gambin, Tomasz and Stray-Pedersen, Asbj{\o}rg and K{\"u}ry, S{\'e}bastien and Mercier, Sandra and Lessel, Davor and Denecke, Jonas and Wiszniewski, Wojciech and Penney, Samantha and Liu, Pengfei and Bi, Weimin and Lalani, Seema R and Schaaf, Christian P and Wangler, Michael F and Bacino, Carlos A and Lewis, Richard Alan and Potocki, Lorraine and Graham, Brett H and Belmont, John W and Scaglia, Fernando and Orange, Jordan S and Jhangiani, Shalini N and Chiang, Theodore and Doddapaneni, Harsha and Hu, Jianhong and Muzny, Donna M and Xia, Fan and Beaudet, Arthur L and Boerwinkle, Eric and Eng, Christine M and Plon, Sharon E and Sutton, V Reid and Gibbs, Richard A and Posey, Jennifer E and Yang, Yaping and Lupski, James R} }