Classification and reporting of constitutional copy-number variants (CNVs) in the clinical setting can be challenging and may result in discrepancies in interpretation for various reasons. In 2019, the ACMG and ClinGen published technical standards (2019 CNV guidelines) to guide the evaluation of constitutional CNVs. To improve consistency and accuracy in classification and reporting practices at ARUP Laboratories, we began using the 2019 CNV guidelines for genomic microarray testing beginning in 2020, including the adoption of the 5-tier variant classification system, incorporation of the scoring metrics into our CNV investigation workflow, and changing our reporting practices for CNVs involving reduced penetrance or autosomal recessive (AR) disease risk.

Reevaluation and reanalysis of previously performed genomic tests is becoming more commonplace, through laboratory-initiated processes, clinician and even patient requests. According to recent ACMG policy statements, laboratories share the duty to recontact when variants or gene-disease relationships are reclassified. Establishing processes for reanalysis and recontact represents a significant challenge for clinical laboratories, particularly cytogenetics laboratories with microarray and interpretive data which often predate implementation of more sophisticated databases. We share our experiences from a first attempt at establishing a process for reevaluation of copy number variant (CNV) classifications in an academic reference laboratory, where access to clinical information and clinician-patient recontact represent notable obstacles. Approximately 4000 CNVs identified through microarray testing were reviewed, including CNVs classified as variants of uncertain significance (VUS) or likely benign (LB) and overlapping genes and regions with established haploinsufficiency/triplosensitivity by ClinGen Dosage Sensitivity Map curations (DS Score=3, DS3). Of 45 VUS/LB CNVs overlapping DS3 genes, 10 were reclassified to pathogenic (P) or likely pathogenic (LP) based on new evidence in the literature. Amended reports and verbal communication of the updated result was provided in all cases. In some, updated clinical history obtained during reevaluation aided in re-interpretation of the variant. Additionally, 12/45 CNVs involving DS3 genes remained classified as VUS, 9 of which only partially overlap the gene of interest, the functional effect of which is difficult to predict by microarray. We also identified 44 multiply-encountered CNVs (close-match: ≥99% overlap/size similarity) involving 244 cases with discordant classifications, many represented recurrent CNVs with reduced penetrance or deletions overlapping recessive-disease genes. Such discordances illustrate the need for better standards of classification of CNVs, which likely require refinement beyond the current 5-tier classification system. Although challenges remain, this CNV reevaluation process has informed our evolving curation and data sharing processes, with the overarching goal of improved patient care.

The recognition that organisms mount physiological responses to DNA damage initially came from work on Escherichia coli and was surprising to many scientists. This chapter first traces the intellectual development of the present model for SOS regulation in prokaryotes and the identification of genes under SOS control. It considers various molecular mechanisms that are used to fine-tune the expression of individual SOS genes and summarizes our present understanding of the physiology of the SOS responses. The genetic studies of recA, lexA, recA mutants, and lexA mutants indicate the existence of the SOS system. Then, it presents essential elements of SOS transcriptional regulation. It also presents identifying SOS genes by the use of fusions, searching for potential lexA-binding sites and expression microarray analysis. A variety of experiments now support the unifying view that the ultimate signal for SOS induction in vivo is the generation of regions of ssDNA within the cell, which in turn results in the formation of sufficient RecA nucleoprotein filaments to mediate LexA cleavage. Next, the chapter briefly discusses additional subtleties in transcriptional regulation of the SOS responses, and known and putative SOS responses of E. coli from a physiological perspective. Other covered topics are SOS responses in pathogenesis, toxicology, and other bacteria.

This chapter focuses solely on the human as an experimental organism. The data discussed are derived from unique experimental approaches. There is substantial evidence that cancer segregates in many additional families, albeit at a reduced frequency compared with that for the more severe syndromes. There is also epidemiological evidence for significant variation in DNA repair capacity among individuals in the population and evidence that those with mildly reduced capacity may be more likely to exhibit a cancer predisposition. A great many publications have proposed associations between specific genetic variants (polymorphisms) in DNA repair and/or damage response genes and a cancer predisposition. Evidence documenting the impact of a polymorphism on protein function is generally lacking. Thus, the appreciation of a specific role for the variant proteins in disease, while logical in theory, remains an important aspect of DNA repair and mutagenesis that is still under development.

This chapter discusses the molecular mechanism of nucleotide excision repair (NER) in eukaryotes, with emphasis on the reaction mechanism in mammalian cells and in the budding yeast, Saccharomyces cerevisiae. The NER mechanism is highly conserved in eukaryotes, and most components and features of the reaction mechanism are very similar in these two organisms. A cell-free system that reflects NER in mammalian cells was developed in the late 1980s and was followed by experimental approaches that measure repair synthesis or the excision of damage-containing oligonucleotide fragments in extracts from mammalian cells and yeast. These techniques have served to identify and track proteins required for NER and have provided specific assays for the purification of NER proteins. The results of genetic studies and the biochemical systems have facilitated the reconstitution of the mammalian and yeast NER machinery with purified protein components and DNA molecules containing single lesions placed at specific sites. To set the stage for the discussion to follow, it is useful to first summarize the reconstitution results. The incision step of NER in the yeast S.cerevisiae has been reconstituted with UV-irradiated DNA and a set of proteins comprising Rad14, Rad4-Rad23, RPA, TFIIH, and the nucleases Rad2 and Rad1-Rad10. The chapter also discusses mechanism of assembly and action of the NER machinery, modulation and regulation of NER in eukaryotes, and evolution of the eukaryotic NER system.

Background: While microarray testing can identify chromosomal abnormalities missed by karyotyping, its prenatal use is often avoided in low-risk pregnancies due to the possible identification of variants of uncertain significance (VOUS). Methods: We tested 2,970 prenatal samples of all referral indications using a rapid BACs-on-Beads-based assay with probes for sex chromosomes, common autosomal aneuploidies, and 20 microdeletion/microduplication syndromes, designed as an alternative to microarray in low-risk pregnancies and an alternative to rapid aneuploidy testing in pregnancies also undergoing microarray analysis. Results: Interpretable results were obtained in 2,940 cases (99.0%), with 89% receiving results in 1 day. Aneuploidies were detected in 7.3% and partial chromosome abnormalities in 0.45% (n = 13), including 5 referred for maternal age, abnormal maternal serum screen, or isolated ultrasound markers. The added detection above karyotype was 1 in 745 in lower-risk cases with normal ultrasounds or isolated ultrasound markers/increased nuchal measurements and 1 in 165 for fetuses with structural/growth abnormalities. Neither false negatives nor false positives were found within test limitations. Female polyploidy could not be detected, while polyploidies with Y chromosomes were suspected and confirmed through additional analysis. Conclusion: When combined with karyotyping, this assay provides increased interrogation of specific chromosomal regions, while limiting VOUS identification.

Cytogenetic alterations are strong outcome prognosticators in uveal melanoma (UVM). Monosomy 3 (-3) and MYC amplification at 8q24 are commonly tested by fluorescence in situ hybridization (FISH). Alternatively, microarray analysis provides whole genome data, detecting partial chromosome loss, loss of heterozygosity (LOH), or abnormalities unrepresented by FISH probes. Nonfixed frozen tissue is conventionally used for microarray analysis but may not always be available. We assessed the feasibility of genomic microarray analysis for high resolution interrogation of UVM using formalin-fixed paraffin-embedded tissue (FFPET) as an alternative to frozen tissue (FZT). Enucleations from 44 patients (clinical trial NCT00952939) yielded sufficient DNA from FFPET (n = 34) and/or frozen tissue (n = 41) for comparative genomic hybridization and select single nucleotide polymorphism analysis (CGH/SNP) on Roche-NimbleGen OncoChip arrays. CEP3 FISH analysis was performed on matched cytology ThinPrep material. CGH/SNP analysis was successful in 30 of 34 FFPET and 41 of 41 FZT samples. Of 27 paired FFPET/FZT samples, 26 (96.3%) were concordant for at least four of six major recurrent abnormalities (-3, +8q, -1p, +6p, -6q, -8p), and 25 of 27 (92.6%) were concordant for -3. Results of CGH/SNP were concordant with the CEP3 FISH results in 27 of 30 (90%) FFPET and 38 of 41 (92.6%) FZT cases; partial -3q was detected in two CEP3 FISH-negative cases and whole chromosome 3, 4, and 6 SNP-LOH in one case. CGH detection of -3, +8q, -8p on FFPET and FZT showed significant correlation with the clinical outcome measures (metastasis development, time to progression, survival). Results of the UVM genotyping by CGH/SNP on FFPET are highly concordant with those of the FZT analysis and with those of the CEP3 FISH analysis, and therefore CGH/SNP is a practical method for UVM prognostication. Genome-wide coverage provides additional data with potential relevance to UVM biology, diagnosis, and prognosis.

This chapter discusses repair pathways of DNA such as alternative excision repair (AER). An AER pathway that has been most extensively defined by studies of E. coli involves a specific endonuclease called endonuclease V. Endonuclease V-mediated incision of DNA is presumably followed by other biochemical events that complete the excision repair of deaminated bases and other substrates. Early studies demonstrated enzymatic activity primarily on DNA containing deoxyinosine, a product of the deamination of deoxyadenosine. This activity was thus designated deoxyinosine 3' endonuclease of E. coli. A series of genetic studies indicate that endonuclease V of E. coli also incises DNA containing the purine analog N-6-hydroxylaminopurine (HAP). Then, the chapter talks about AER mediated by other endonucleases. It is evident from the examples provided in the chapter that nature has been versatile in evolving mechanisms that afford the repair of various types of spontaneous and environmentally generated base damage in DNA. In summary, the chapter provides a cogent reminder that DNA damage is a pervasive phenomenon in living cells and it is obvious that there is no dearth of strategies adopted by nature to mitigate the lethal and mutagenic effects of such damage.

This chapter begins with a brief review of the biochemistry of RecQ helicases and is followed by a discussion of biological data obtained from the study of less complex organisms such as Escherichia coli and yeast. The genomes of these organisms bear single recQ gene homologs, and isolated mutants have provided insights into the general function of this family of proteins. The chapter then talks about human diseases that are characterized by defects in the human RECQ homologs. The gene defective in Bloom syndrome (BS) (the BLM gene) was the first of the disease genes to be identified as a member of this human gene family. This was followed by identification of the gene mutated in Werner syndrome (WS) (the WRN gene). Analysis of two additional homologs, RECQL4 and RECQL5, revealed RECQL4 gene mutations in some (but not all) patients with the cancer-prone human disorder Rothmund-Thomson syndrome (RTS) and in patients with RAPADILINO syndrome. In contrast to xeroderma pigmentosum, where mutations in any one of a number of different genes that function in nucleotide excision repair yield the same clinical phenotypes, the disorders described in the chapter exhibit distinct clinical and cellular features, suggesting that these similar proteins function at different stages of DNA metabolism. Each of the disorders is considered separately in the chapter, with a concluding summary of key similarities and differences.

Sources and Consequences of DNA Damage Errol C. Friedberg, Errol C. Friedberg Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TexasSearch for more papers by this authorGraham C. Walker, Graham C. Walker Department of Biology, Massachusetts Institute of Technology, Cambridge, MassachusettsSearch for more papers by this authorWolfram Siede, Wolfram Siede Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TexasSearch for more papers by this authorRichard D. Wood, Richard D. Wood Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PennsylvaniaSearch for more papers by this authorRoger A. Schultz, Roger A. Schultz Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TexasSearch for more papers by this authorTom Ellenberger, Tom Ellenberger Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MassachusettsSearch for more papers by this author Book Author(s):Errol C. Friedberg, Errol C. Friedberg Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TexasSearch for more papers by this authorGraham C. Walker, Graham C. Walker Department of Biology, Massachusetts Institute of Technology, Cambridge, MassachusettsSearch for more papers by this authorWolfram Siede, Wolfram Siede Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TexasSearch for more papers by this authorRichard D. Wood, Richard D. Wood Hillman Cancer Center, University of Pittsburgh, Pittsburgh, PennsylvaniaSearch for more papers by this authorRoger A. Schultz, Roger A. Schultz Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TexasSearch for more papers by this authorTom Ellenberger, Tom Ellenberger Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MassachusettsSearch for more papers by this author First published: 22 November 2005 https://doi.org/10.1128/9781555816704.part1 AboutPDFPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShareShare a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. DNA Repair and Mutagenesis, Second Edition RelatedInformation

This chapter examines two genetic disorders, each exhibiting locus heterogeneity and each representing the biological consequences of mutations in different genes in a specific DNA repair pathway. Both of these disorders present with a significant cancer predisposition and have associated cellular characteristics reflective of inherent chromosome instability. However, experimental approaches to the identification and characterization of the underlying genetics have taken strikingly different paths. The first of these disorders, hereditary nonpolyposis colon cancer (HNPCC), results from mutations in different genes associated with DNA mismatch repair (MMR). Given that MMR had been extensively studied before its connection to HNPCC was established, more recent studies have focused on evaluating additional MMR candidate genes for mutations in patients with HNPCC and elucidation of the biological basis for the organ-specific nature of the disorder. In contrast, the second disease considered in this chapter, is a disorder represented by multiple complementation groups that all exhibit defects in cross-link repair and responses to oxidative damage. These are not repair functions that are well defined or representative of established biochemical pathways. Therefore, experimental approaches have focused on the identification of the different genes responsible for FA and characterization of how the proteins involved define a unique biological pathway.

Acute promyelocytic leukemia (APL) is characterized by promyelocytic leukemia (PML)-retinoic acid receptor, alpha (RARA) fusion gene as a result of t(15;17)(q24.1;q21.2).1 Current advances in APL therapy have dramatically improved patient outcome.2,3 However, patients with APL are at risk of potentially lethal coagulopathy if appropriate therapy is delayed.4

Various microarray platforms, including BAC, oligonucleotide, and SNP arrays, have been shown to -provide clinically useful diagnostic and prognostic information for patients with myelodysplastic syndromes (MDS). Clinically useful arrays are designed with specific purposes in mind and with attention to genomic content and probe density. All array types have been shown to detect genomic copy gains and losses, with SNP arrays having the added advantage of detecting copy neutral loss of heterozygosity (CNLOH). The finding of CNLOH has led to the identification of certain disease genes implicated in the initiation or progression of myeloid diseases. In addition, SNP karyotyping alone, or in conjunction with routine cytogenetics, can affect the outcome prediction and improve prognostic stratification of patients with MDS. Patients who were reclassified after array testing as having adverse-risk chromosomal findings correlated with poor survival. Results of over 25 published studies support the use of arrays in MDS testing. Because few balanced translocations are found in MDS, this disease is particularly amenable to microarray testing, and studies have shown better disease classification, identification of cryptic changes, and prognostication in this heterogeneous group of disorders. Novel genomic alterations identified by array testing may lead to better targeted therapies for treating patients with MDS.

Molecular cytogenetic alterations, specifically monosomy 3, are strongly correlated with metastases and death in uveal melanoma (UVM). Although FISH can be used for the identification of monosomy 3, a subset of UVM exhibit only partial loss of chromosome 3 potentially missed with chromosome enumeration probes. Moreover, often limited material available can compromise detection of additional alterations with potential clinical relevance. Microarray analysis is an alternative method for the analysis of such specimens affording whole genome interrogation. In the current study we performed microarray to detect genomic changes in DNA from FFPET and FZT UVM samples (clinical trial NCT00952939). Of the available cases, 23.5% yielded DNA of sufficient quantity and quality to obtain interpretable microarray results, representing 28 patients. All but one case demonstrated significant abnormalities. Gains of 8q, consistent with an apparent i(8)(q10) karyotype, were the most common finding, seen in 21 patients. Monosomy 3 was detected by microarray in 15 cases. A single case showed partial loss of 3 (3q11.2q25.31), the only clinically significant abnormality detected in that case. All cases lacking chromosome 3 abnormalities showed a copy gain of 6p sharing a small 28.18 Mb region of overlap at 6p25.2p21.33. Additional findings included TERT and NEDD9 amplifications, and CDKN2A/B and LUM gene deletions. SNP analysis revealed three cases with unique regions of copy neutral LOH involving 5p15.33q35.3, 15q11.2q21.1 and one case with whole chromosome LOH for chromosomes 3, 4 and 6. The latter was detected in a FZT specimen, while the paired FFPET sample lacked evidence of LOH and showed monosomy 3, trisomy 4 and whole arm gains and losses of 6p and 6q, respectively, suggesting the presence of tumor heterogeneity. Microarray analysis has identified new recurrent abnormalities associated with UVM with potential relevance to UVM biology, diagnosis and prognosis.

American Journal of Medical Genetics Part AVolume 164, Issue 1 p. 259-263 Research Letter Refinement of the 8q22.1 microdeletion critical region associated with Nablus mask-like facial syndrome Justin Overhoff, Justin Overhoff Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, WashingtonSearch for more papers by this authorMarina M. Rabideau, Marina M. Rabideau Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, WashingtonSearch for more papers by this authorLynne M. Bird, Lynne M. Bird Department of Pediatrics, University of California San Diego and Rady Children's Hospital, San Diego, CaliforniaSearch for more papers by this authorDaniela N. Schweitzer, Daniela N. Schweitzer Department of Medical Genetics, Children's Hospital Los Angeles, Los Angeles, California Craniofacial and Cleft Center, Children's Hospital Los Angeles, Los Angeles, CaliforniaSearch for more papers by this authorKarla Haynes, Karla Haynes Craniofacial and Cleft Center, Children's Hospital Los Angeles, Los Angeles, CaliforniaSearch for more papers by this authorRoger A. Schultz, Roger A. Schultz Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, WashingtonSearch for more papers by this authorLisa G. Shaffer, Lisa G. Shaffer Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, WashingtonSearch for more papers by this authorJill A. Rosenfeld, Corresponding Author Jill A. Rosenfeld Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, Washington Correspondence to: Jill Rosenfeld, M.S., LCGC, Signature Genomic Laboratories, PerkinElmer, Inc., 2820 North Astor Street, Spokane, WA 99207. E-mail: jill.mokry@perkinelmer.comSearch for more papers by this authorJay W. Ellison, Jay W. Ellison Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, WashingtonSearch for more papers by this author Justin Overhoff, Justin Overhoff Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, WashingtonSearch for more papers by this authorMarina M. Rabideau, Marina M. Rabideau Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, WashingtonSearch for more papers by this authorLynne M. Bird, Lynne M. Bird Department of Pediatrics, University of California San Diego and Rady Children's Hospital, San Diego, CaliforniaSearch for more papers by this authorDaniela N. Schweitzer, Daniela N. Schweitzer Department of Medical Genetics, Children's Hospital Los Angeles, Los Angeles, California Craniofacial and Cleft Center, Children's Hospital Los Angeles, Los Angeles, CaliforniaSearch for more papers by this authorKarla Haynes, Karla Haynes Craniofacial and Cleft Center, Children's Hospital Los Angeles, Los Angeles, CaliforniaSearch for more papers by this authorRoger A. Schultz, Roger A. Schultz Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, WashingtonSearch for more papers by this authorLisa G. Shaffer, Lisa G. Shaffer Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, WashingtonSearch for more papers by this authorJill A. Rosenfeld, Corresponding Author Jill A. Rosenfeld Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, Washington Correspondence to: Jill Rosenfeld, M.S., LCGC, Signature Genomic Laboratories, PerkinElmer, Inc., 2820 North Astor Street, Spokane, WA 99207. E-mail: jill.mokry@perkinelmer.comSearch for more papers by this authorJay W. Ellison, Jay W. Ellison Signature Genomic Laboratories, PerkinElmer, Inc., Spokane, WashingtonSearch for more papers by this author First published: 20 November 2013 https://doi.org/10.1002/ajmg.a.36163Citations: 4 Conflict of interest: Justin Overhoff, Roger A. Schultz, and Jill A. Rosenfeld are employees of Signature Genomic Laboratories, a subsidiary of PerkinElmer, Inc. All other authors have no conflicts to declare. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume164, Issue1January 2014Pages 259-263 RelatedInformation

Acute promyelocytic leukemia (APL) is typically defined at the molecular level by a reciprocal translocation of the promyelocytic leukemia (PML) and retinoic acid receptor α (RARA) genes. An accurate diagnosis of APL is critical for appropriate choice of therapy and prognostic assessment. Cryptic and variant rearrangements in APL are discoverable by a variety of molecular methods including fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction, or gene sequencing. Rare reports of FISH-negative APL harboring cryptic rearrangements of PML-RARA detected by reverse transcriptase polymerase chain reaction or sequencing have been described. Here, we describe the detection of cryptic or variant PML-RARA rearrangements by translocation-based comparative genomic hybridization (tCGH), a recently described modification of traditional CGH technology that facilitates the detection of balanced translocations by means of the linear amplification of a potential translocation breakpoint region(s), in 2 unusual cases of APL. One tumor lacked detectable t(15;17) by karyotype and FISH, and the other tumor lacked the typical morphologic and immunophenotypic features of APL and had a variant 3-way translocation involving PML and RARA. PML-RARA translocations were identified by tCGH in both cases providing confirmation of the diagnosis of APL. These data emphasize the benefit of using complementary molecular methods including tCGH for detecting cryptic and variant PML-RARA translocations in unusual cases of APL.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, with the majority of cases being of precursor B-cell phenoltype. Conventional cytogenetic analysis plays an important role in the diagnosis of B-cell ALL, identifying characteristic chromosomal abnormalities associated with a given prognosis therein facilitating optimized treatment. The more recent introduction of microarray technology to the analysis of B-cell ALL has afforded both higher resolution for the detection of known abnormalities and an ability to identify novel copy number abnormalities (CNAs) with potential clinical relevance. In the current study, microarray analysis was performed on 20 cytogenetically abnormal B-cell ALL cases (10 pediatric and 10 adult), while a novel microarray-based balanced-translocation detection methodology (translocation CGH or tCGH) was applied to that subset of cases with a known or suspected recurrent balanced translocation. Standard microarray analysis identified that CNAs was not detected by previous conventional cytogenetics in 75% (15/20) cases. tCGH identified 9/9 (100%) balanced translocations defining BCR/ABL1 (x4), ETV6/RUNX1 (x3), and MLL/AFF1 (x2) breakpoints with high resolution. The results illustrate the improved molecular detail afforded by these technologies and a comparison of translocation breakpoints, CNAs and patient age offers new insights into tumor biology with potential prognostic significance.