A LINE-1 mediated deletion resulting in germline retinoblastoma predisposition.

Retinoblastoma is an ocular cancer associated with genomic variation in the RB1 gene. In individuals with bilateral retinoblastoma, a germline variant in RB1 is identified in virtually all cases. We describe herein an individual with bilateral retinoblastoma for whom multiple clinical lab assays performed by outside commercial laboratories failed to identify a germline RB1 variant. Paired tumor/normal exome sequencing, long-read whole genome sequencing, and long-read isoform sequencing was performed on a translational research basis ultimately identified a germline likely de novo Long Interspersed Nuclear Element (LINE)-1 mediated deletion resulting in a premature stop of translation of RB1 as the underlying genetic cause of retinoblastoma in this individual. Based on these research findings, the LINE-1 mediated deletion was confirmed via Sanger sequencing in our clinical laboratory, and results were reported in the patient's medical record to allow for appropriate genetic counseling.

Noninvasive prenatal screening (NIPS) for fetal chromosome abnormalities in a general-risk population: An evidence-based clinical guideline of the American College of Medical Genetics and Genomics (ACMG).

PURPOSE: This workgroup aimed to develop an evidence-based clinical practice guideline for the use of noninvasive prenatal screening (NIPS) for pregnant individuals at general risk for fetal trisomy 21, trisomy 18, or trisomy 13 and to evaluate the utility of NIPS for other chromosomal disorders. METHODS: The NIPS Evidence-Based Guideline Work Group (n = 7) relied on the results from the recent American College of Medical Genetics and Genomics (ACMG) systematic review to form the evidentiary basis of this guideline. Workgroup members used the Grading of Recommendations Assessment, Development, and Evaluation Evidence to Decision framework to draft recommendations. The guideline underwent extensive internal and external peer review with a public comment period before approval by the ACMG Board of Directors. RESULTS: Evidence consistently demonstrated improved accuracy of NIPS compared with traditional screening methods for trisomies 21, 18, and 13 in singleton and twin gestations. Identification of rare autosomal trisomies and other microdeletion syndromes with NIPS is an emerging area of interest. CONCLUSION: ACMG strongly recommends NIPS over traditional screening methods for all pregnant patients with singleton and twin gestations for fetal trisomies 21, 18, and 13 and strongly recommends NIPS be offered to patients to screen for fetal sex chromosome aneuploidy.

Characterization of unusual iAMP21 B-lymphoblastic leukemia (iAMP21-ALL) from the Mayo Clinic and Children's Oncology Group.

Acute lymphoblastic leukemia (B-ALL) with intrachromosomal amplification of chromosome 21 (iAMP21-ALL) represents a recurrent high-risk cytogenetic abnormality and accurate identification is critical for appropriate clinical management. Identification of iAMP21-ALL has historically relied on fluorescence in situ hybridization (FISH) using a RUNX1 probe. Current classification requires ≥ five copies of RUNX1 per cell and ≥ three additional copies of RUNX1 on a single abnormal iAMP21-chromosome. We sought to evaluate the performance of the RUNX1 probe in the identification of iAMP21-ALL. This study was a retrospective evaluation of iAMP21-ALL in the Mayo Clinic and Children's Oncology Group cohorts. Of 207 cases of iAMP21-ALL, 188 (91%) were classified as "typical" iAMP21-ALL, while 19 (9%) cases were classified as "unusual" iAMP21-ALL. The "unusual" iAMP21 cases did not meet the current definition of iAMP21 by FISH but were confirmed to have iAMP21 by chromosomal microarray. Half of the "unusual" iAMP21-ALL cases had less than five RUNX1 signals, while the remainder had ≥ five RUNX1 signals with some located apart from the abnormal iAMP21-chromosome. Nine percent of iAMP21-ALL cases fail to meet the FISH definition of iAMP21-ALL demonstrating that laboratories are at risk of misidentification of iAMP21-ALL when relying only on the RUNX1 FISH probe. Incorporation of chromosomal microarray testing circumvents these risks.

Molecular Pathology Education: A Suggested Framework for Primary Care Resident Training in Genomic Medicine: A Report of the Association for Molecular Pathology Training and Education Committee.

Developments in genomics are profoundly influencing medical practice. With increasing use of genetic and genomic testing across every aspect of the health care continuum, patients and their families are increasingly turning to primary care physicians (PCPs) for discussion and advice regarding tests, implications, and results. Yet, with the rapid growth of information, technology, and applications, PCPs are finding it challenging to fill the gaps in knowledge and support the growing needs of their patients. A critical component in expanding PCP genomic literacy lies in the education of physicians in training and in practice. Although a framework for developing physician competencies in genomics has already been developed, the Association for Molecular Pathology is uniquely situated to actively utilize the skills of its members to engage and support PCPs in this effort. This report provides an overview and a suggested basic teaching framework, which can be used by molecular professionals in their individual institutions as a starting point for educational outreach.

Standards for the classification of pathogenicity of somatic variants in cancer (oncogenicity): Joint recommendations of Clinical Genome Resource (ClinGen), Cancer Genomics Consortium (CGC), and Variant Interpretation for Cancer Consortium (VICC).

PURPOSE: Several professional societies have published guidelines for the clinical interpretation of somatic variants, which specifically address diagnostic, prognostic, and therapeutic implications. Although these guidelines for the clinical interpretation of variants include data types that may be used to determine the oncogenicity of a variant (eg, population frequency, functional, and in silico data or somatic frequency), they do not provide a direct, systematic, and comprehensive set of standards and rules to classify the oncogenicity of a somatic variant. This insufficient guidance leads to inconsistent classification of rare somatic variants in cancer, generates variability in their clinical interpretation, and, importantly, affects patient care. Therefore, it is essential to address this unmet need. METHODS: Clinical Genome Resource (ClinGen) Somatic Cancer Clinical Domain Working Group and ClinGen Germline/Somatic Variant Subcommittee, the Cancer Genomics Consortium, and the Variant Interpretation for Cancer Consortium used a consensus approach to develop a standard operating procedure (SOP) for the classification of oncogenicity of somatic variants. RESULTS: This comprehensive SOP has been developed to improve consistency in somatic variant classification and has been validated on 94 somatic variants in 10 common cancer-related genes. CONCLUSION: The comprehensive SOP is now available for classification of oncogenicity of somatic variants.

Guiding the global evolution of cytogenetic testing for hematologic malignancies.

Cytogenetics has long represented a critical component in the clinical evaluation of hematologic malignancies. Chromosome banding studies provide a simultaneous snapshot of genome-wide copy number and structural variation, which have been shown to drive tumorigenesis, define diseases, and guide treatment. Technological innovations in sequencing have ushered in our present-day clinical genomics era. With recent publications highlighting novel sequencing technologies as alternatives to conventional cytogenetic approaches, we, an international consortium of laboratory geneticists, pathologists, and oncologists, describe herein the advantages and limitations of both conventional chromosome banding and novel sequencing technologies and share our considerations on crucial next steps to implement these novel technologies in the global clinical setting for a more accurate cytogenetic evaluation, which may provide improved diagnosis and treatment management. Considering the clinical, logistic, technical, and financial implications, we provide points to consider for the global evolution of cytogenetic testing.

Conventional Cytogenetic Analysis of Hematologic Neoplasms: A 20-Year Review of Proficiency Test Results From the College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee.

CONTEXT.­: One goal of the joint College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee is to ensure the accurate detection and description of chromosomal abnormalities in both constitutional and neoplastic specimens, including hematologic neoplasms. OBJECTIVE.­: To report a 20-year performance summary (1999-2018) of conventional chromosome challenges focusing on hematologic neoplasms. DESIGN.­: A retrospective review was performed from 1999 through 2018 to identify karyotype challenges specifically addressing hematologic neoplasms. The overall performance of participants was examined to identify potential recurring errors of clinical significance. RESULTS.­: Of 288 total conventional chromosome challenges from 1999-2018, 87 (30.2%) were presented in the context of a hematologic neoplasm, based on the provided clinical history, specimen type, and/or chromosomal abnormalities. For these 87 hematologic neoplasm challenges, 91 individual cases were provided and graded on the basis of abnormality recognition and karyotype nomenclature (ISCN, International System for Human Cytogenomic [previously Cytogenetic] Nomenclature). Of the 91 cases, 89 (97.8%) and 87 (95.6%) exceeded the required 80% consensus for grading of abnormality recognition and correct karyotype nomenclature, respectively. The 2 cases (2 of 91; 2.2%) that failed to meet the 80% consensus for abnormality recognition had complex karyotypes. The 4 cases (4 of 91; 4.4%) that failed to meet the 80% consensus for correct karyotype nomenclature were the result of incorrect abnormality recognition (2 cases), missing brackets in the karyotype (1 case), and incorrect breakpoint designation (1 case). CONCLUSIONS.­: This 20-year review demonstrates clinical cytogenetics laboratories have been and continue to be highly proficient in the detection and description of chromosomal abnormalities associated with hematologic neoplasms.

Evidence-based review of genomic aberrations in B-lymphoblastic leukemia/lymphoma: Report from the cancer genomics consortium working group for lymphoblastic leukemia.

Clinical management and risk stratification of B-lymphoblastic leukemia/ lymphoma (B-ALL/LBL) depend largely on identification of chromosomal abnormalities obtained using conventional cytogenetics and Fluorescence In Situ Hybridization (FISH) testing. In the last few decades, testing algorithms have been implemented to support an optimal risk-oriented therapy, leading to a large improvement in overall survival. In addition, large scale genomic studies have identified multiple aberrations of prognostic significance that are not routinely tested by existing modalities. However, as chromosomal microarray analysis (CMA) and next-generation sequencing (NGS) technologies are increasingly used in clinical management of hematologic malignancies, these abnormalities may be more readily detected. In this article, we have compiled a comprehensive, evidence-based review of the current B-ALL literature, focusing on known and published subtypes described to date. More specifically, we describe the role of various testing modalities in the diagnosis, prognosis, and therapeutic relevance. In addition, we propose a testing algorithm aimed at assisting laboratories in the most effective detection of the underlying genomic abnormalities.

GGC Repeat Expansion and Exon 1 Methylation of XYLT1 Is a Common Pathogenic Variant in Baratela-Scott Syndrome.

Baratela-Scott syndrome (BSS) is a rare, autosomal-recessive disorder characterized by short stature, facial dysmorphisms, developmental delay, and skeletal dysplasia caused by pathogenic variants in XYLT1. We report clinical and molecular investigation of 10 families (12 individuals) with BSS. Standard sequencing methods identified biallelic pathogenic variants in XYLT1 in only two families. Of the remaining cohort, two probands had no variants and six probands had only a single variant, including four with a heterozygous 3.1 Mb 16p13 deletion encompassing XYLT1 and two with a heterozygous truncating variant. Bisulfite sequencing revealed aberrant hypermethylation in exon 1 of XYLT1, always in trans with the sequence variant or deletion when present; both alleles were methylated in those with no identified variant. Expression of the methylated XYLT1 allele was severely reduced in fibroblasts from two probands. Southern blot studies combined with repeat expansion analysis of genome sequence data showed that the hypermethylation is associated with expansion of a GGC repeat in the XYLT1 promoter region that is not present in the reference genome, confirming that BSS is a trinucleotide repeat expansion disorder. The hypermethylated allele accounts for 50% of disease alleles in our cohort and is not present in 130 control subjects. Our study highlights the importance of investigating non-sequence-based alterations, including epigenetic changes, to identify the missing heritability in genetic disorders.

Diagnostic cytogenetic testing following positive noninvasive prenatal screening results: a clinical laboratory practice resource of the American College of Medical Genetics and Genomics (ACMG).

Disclaimer: ACMG Clinical Laboratory Practice Resources are developed primarily as an educational tool for clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to these practice resources is voluntary and does not necessarily assure a successful medical outcome. This Clinical Laboratory Practice Resource should not be considered inclusive of all proper procedures and tests or exclusive of other procedures and tests that are reasonably directed to obtaining the same results. In determining the propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own professional judgment to the specific circumstances presented by the individual patient or specimen. Clinical laboratory geneticists are encouraged to document in the patient's record the rationale for the use of a particular procedure or test, whether or not it is in conformance with this Clinical Laboratory Practice Resource. They also are advised to take notice of the date any particular guideline was adopted, and to consider other relevant medical and scientific information that becomes available after that date. It also would be prudent to consider whether intellectual property interests may restrict the performance of certain tests and other procedures.Noninvasive prenatal screening (NIPS) using cell-free DNA has been rapidly adopted into prenatal care. Since NIPS is a screening test, diagnostic testing is recommended to confirm all cases of screen-positive NIPS results. For cytogenetics laboratories performing confirmatory testing on prenatal diagnostic samples, a standardized testing algorithm is needed to ensure that the appropriate testing takes place. This algorithm includes diagnostic testing by either chorionic villi sampling or amniocentesis samples and encompasses chromosome analysis, fluorescence in situ hybridization, and chromosomal microarray.

CRELD2: gene mapping, alternate splicing, and comparative genomic identification of the promoter region.

CRELD2 is the second member of the CRELD family of proteins. The only other CRELD family member, encoded by CRELD1, is also known as the AVSD2 gene as mutations in CRELD1 are associated with cardiac atrioventricular septal defects (AVSD). Like CRELD1, CRELD2 is ubiquitously expressed during development and by mature tissues. Recently, a specific CRELD2 isoform (CRELD2beta) was implicated as a regulator of alpha4beta2 nicotinic acetylcholine receptor expression, suggesting that the CRELD family has widely diverse biological roles in both developmental events and subsequent cell function. Here we report additional characterization of CRELD2, which was undertaken to further our understanding of this important family. Mapping of CRELD2 by FISH shows that it maps to 22q13 rather than the GenBank reported locus of 22p13. Comparative genomic analysis of upstream sequences shows a discrete region that is highly conserved among diverse species with hallmark features indicative of a promoter region. Functional analysis demonstrates that this region has promoter activity. Consistent with widespread expression of CRELD2, this region is GC-rich and lacks a TATA box. Overall, the highest levels of CRELD2 expression occur in adult endocrine tissues. However, alternative splicing of CRELD2 is extensive with positive identification of several splice variants expressed by most normal fetal and adult tissues. Confirmed splice variants encode 5 different CRELD2 isoforms that differ significantly in composition indicating that CRELD2 function is varied and as yet poorly understood.

Interstrand crosslink-induced radials form between non-homologous chromosomes, but are absent in sex chromosomes.

Fanconi anemia (FA) and cells lacking functional BRCA1 and BRCA2 proteins are hypersensitive to interstrand crosslinking (ICL) agents and show increased numbers of chromosomal breaks and radials. Although radial formation has been used to diagnose FA for more than 30 years, there has been little analysis of these characteristic formations. In this study, radials were analyzed from FA-A and FA-G fibroblasts as well as normal and retrovirally-corrected FA-A fibroblasts treated with high doses of ICLs. Radials were found to only involve non-homologous chromosome interactions and to be distributed nearly randomly along the length of chromosomes. Sites on chromosomes that did show increased frequency of radial involvement did not correlate with known fragile sites or pericentric regions. Hybrid radials were observed between mouse and human chromosomes in human-mouse hybrid cells produced by microcell-mediated chromosome transfer of mouse chromosomes into human FA-A fibroblasts. Both X and Y chromosomes were notably not involved in radials. These observations suggest that ICL repair may involve short stretches of homology, resulting in aberrant radial formation in the absence of FA proteins.