The landscape of reported VUS in multi-gene panel and genomic testing: Time for a change.

PURPOSE: Variants of uncertain significance (VUS) are a common result of diagnostic genetic testing and can be difficult to manage with potential misinterpretation and downstream costs, including time investment by clinicians. We investigated the rate of VUS reported on diagnostic testing via multi-gene panels (MGPs) and exome and genome sequencing (ES/GS) to measure the magnitude of uncertain results and explore ways to reduce their potentially detrimental impact. METHODS: Rates of inconclusive results due to VUS were collected from over 1.5 million sequencing test results from 19 clinical laboratories in North America from 2020 to 2021. RESULTS: We found a lower rate of inconclusive test results due to VUSs from ES/GS (22.5%) compared with MGPs (32.6%; P < .0001). For MGPs, the rate of inconclusive results correlated with panel size. The use of trios reduced inconclusive rates (18.9% vs 27.6%; P < .0001), whereas the use of GS compared with ES had no impact (22.2% vs 22.6%; P = ns). CONCLUSION: The high rate of VUS observed in diagnostic MGP testing warrants examining current variant reporting practices. We propose several approaches to reduce reported VUS rates, while directing clinician resources toward important VUS follow-up.

Stankiewicz-Isidor syndrome: expanding the clinical and molecular phenotype.

PURPOSE: Haploinsufficiency of PSMD12 has been reported in individuals with neurodevelopmental phenotypes, including developmental delay/intellectual disability (DD/ID), facial dysmorphism, and congenital malformations, defined as Stankiewicz-Isidor syndrome (STISS). Investigations showed that pathogenic variants in PSMD12 perturb intracellular protein homeostasis. Our objective was to further explore the clinical and molecular phenotypic spectrum of STISS. METHODS: We report 24 additional unrelated patients with STISS with various truncating single nucleotide variants or copy-number variant deletions involving PSMD12. We explore disease etiology by assessing patient cells and CRISPR/Cas9-engineered cell clones for various cellular pathways and inflammatory status. RESULTS: The expressivity of most clinical features in STISS is highly variable. In addition to previously reported DD/ID, speech delay, cardiac and renal anomalies, we also confirmed preaxial hand abnormalities as a feature of this syndrome. Of note, 2 patients also showed chilblains resembling signs observed in interferonopathy. Remarkably, our data show that STISS patient cells exhibit a profound remodeling of the mTORC1 and mitophagy pathways with an induction of type I interferon-stimulated genes. CONCLUSION: We refine the phenotype of STISS and show that it can be clinically recognizable and biochemically diagnosed by a type I interferon gene signature.

Exome and genome sequencing for pediatric patients with congenital anomalies or intellectual disability: an evidence-based clinical guideline of the American College of Medical Genetics and Genomics (ACMG).

PURPOSE: To develop an evidence-based clinical practice guideline for the use of exome and genome sequencing (ES/GS) in the care of pediatric patients with one or more congenital anomalies (CA) with onset prior to age 1 year or developmental delay (DD) or intellectual disability (ID) with onset prior to age 18 years. METHODS: The Pediatric Exome/Genome Sequencing Evidence-Based Guideline Work Group (n = 10) used the Grading of Recommendations Assessment, Development and Evaluation (GRADE) evidence to decision (EtD) framework based on the recent American College of Medical Genetics and Genomics (ACMG) systematic review, and an Ontario Health Technology Assessment to develop and present evidence summaries and health-care recommendations. The document underwent extensive internal and external peer review, and public comment, before approval by the ACMG Board of Directors. RESULTS: The literature supports the clinical utility and desirable effects of ES/GS on active and long-term clinical management of patients with CA/DD/ID, and on family-focused and reproductive outcomes with relatively few harms. Compared with standard genetic testing, ES/GS has a higher diagnostic yield and may be more cost-effective when ordered early in the diagnostic evaluation. CONCLUSION: We strongly recommend that ES/GS be considered as a first- or second-tier test for patients with CA/DD/ID.

Limited diagnostic impact of duplications <1 Mb of uncertain clinical significance: a 10-year retrospective analysis of reporting practices at the Mayo Clinic.

PURPOSE: Copy-number variants (CNVs) of uncertain clinical significance are routinely reported in a clinical setting only when exceeding predetermined reporting thresholds, typically based on CNV size. Given that very few genes are associated with triplosensitive phenotypes, it is not surprising that many interstitial duplications <1 Mb are found to be inherited and anticipated to be of limited or no clinical significance. METHODS: In an effort to further refine our reporting criteria to maximize diagnostic yield while minimizing the return of uncertain variants, we performed a retrospective analysis of all clinical microarray cases reported in a 10-year window. A total of 1112 reported duplications had parental follow-up, and these were compared by size, RefSeq gene content, and inheritance pattern. De novo origin was used as a rough proxy for pathogenicity. RESULTS: Approximately 6% of duplications 500 kb-1 Mb were de novo observations, compared with approximately 14% for 1-2 Mb duplications (p = 0.0005). On average, de novo duplications had higher gene counts than inherited duplications. CONCLUSION: Our data reveal limited diagnostic utility for duplications of uncertain significance <1 Mb. Considerations for revised reporting criteria are discussed and are applicable to CNVs detected by any genome-wide exploratory methodology, including exome/genome sequencing.

Most noninvasive prenatal screens failing due to inadequate fetal cell free DNA are negative for trisomy when repeated.

OBJECTIVE: We aimed to test for an association between the amount of circulating fetal cell-free DNA and trisomy, and whether NIPS failure due to low fetal fraction indicates trisomy risk. METHOD: Maternal BMI, maternal age, fetal sex, gestational age, fetal cfDNA fraction, and NIPS results was collected on 2374 pregnancies. Additional clinical information was available for 1180 research consented patients. We investigated associations between fetal fraction and available variables and determined the success rate of repeat NIPS testing. RESULTS: Fetal trisomy was marginally associated with decreased fetal fraction (P = .067). However, the proportions of trisomy events were not significantly increased in women who had failed NIPS due to low fetal fraction (<4%) (OR = 1.37 [0.3-7.4]; P = .714). 66% of repeated NIPS after a second blood draw were successful. CONCLUSION: Failure to meet the clinical cutoff of 4% fetal fraction established for NIPS accuracy did not suggest increased risk for trisomy in our cohort. Because repeat testing was successful in the majority of cases and most failures were explained by high BMI and low gestational age, a redraw may be an appropriate next step before invasive screening due to concerns for trisomic pregnancies.

Biallelic variants in CTU2 cause DREAM-PL syndrome and impair thiolation of tRNA wobble U34.

The wobble position in the anticodon loop of transfer ribonucleic acid (tRNA) is subject to numerous posttranscriptional modifications. In particular, thiolation of the wobble uridine has been shown to play an important role in codon-anticodon interactions. This modification is catalyzed by a highly conserved CTU1/CTU2 complex, disruption of which has been shown to cause abnormal phenotypes in yeast, worms, and plants. We have previously suggested that a single founder splicing variant in human CTU2 causes a novel multiple congenital anomalies syndrome consisting of dysmorphic facies, renal agenesis, ambiguous genitalia, microcephaly, polydactyly, and lissencephaly (DREAM-PL). In this study, we describe five new patients with DREAM-PL phenotype and whose molecular analysis expands the allelic heterogeneity of the syndrome to five different alleles; four of which predict protein truncation. Functional characterization using patient-derived cells for each of these alleles, as well as the original founder allele; revealed a specific impairment of wobble uridine thiolation in all known thiol-containing tRNAs. Our data establish a recognizable CTU2-linked autosomal recessive syndrome in humans characterized by defective thiolation of the wobble uridine. The potential deleterious consequences for the translational efficiency and fidelity during development as a mechanism for pathogenicity represent an attractive target of future investigations.

Mate pair sequencing improves detection of genomic abnormalities in acute myeloid leukemia.

OBJECTIVE: Acute myeloid leukemia (AML) can be subtyped based on recurrent cytogenetic and molecular genetic abnormalities with diagnostic and prognostic significance. Although cytogenetic characterization classically involves conventional chromosome and/or fluorescence in situ hybridization (FISH) assays, limitations of these techniques include poor resolution and the inability to precisely identify breakpoints. METHOD: We evaluated whether an NGS-based methodology that detects structural abnormalities and copy number changes using mate pair sequencing (MPseq) can enhance the diagnostic yield for patients with AML. RESULTS: Using 68 known abnormal and 20 karyotypically normal AML samples, each recurrent primary AML-specific abnormality previously identified in the abnormal samples was confirmed using MPseq. Importantly, in eight cases with abnormalities that could not be resolved by conventional cytogenetic studies, MPseq was utilized to molecularly define eight recurrent AML-fusion events. In addition, MPseq uncovered two cryptic abnormalities that were missed by conventional cytogenetic studies. Thus, MPseq improved the diagnostic yield in the detection of AML-specific structural rearrangements in 10/88 (11%) of cases analyzed. CONCLUSION: Utilization of MPseq represents a precise, molecular-based technique that can be used as an alternative to conventional cytogenetic studies for newly diagnosed AML patients with the potential to revolutionize the diagnosis of hematologic malignancies.

Copy number variant analysis using genome-wide mate-pair sequencing.

Copy number variation (CNV) is a common form of structural variation detected in human genomes, occurring as both constitutional and somatic events. Cytogenetic techniques like chromosomal microarray (CMA) are widely used in analyzing CNVs. However, CMA techniques cannot resolve the full nature of these structural variations (i.e. the orientation and location of associated breakpoint junctions) and must be combined with other cytogenetic techniques, such as karyotyping or FISH, to do so. This makes the development of a next-generation sequencing (NGS) approach capable of resolving both CNVs and breakpoint junctions desirable. Mate-pair sequencing (MPseq) is a NGS technology designed to find large structural rearrangements across the entire genome. Here we present an algorithm capable of performing copy number analysis from mate-pair sequencing data. The algorithm uses a step-wise procedure involving normalization, segmentation, and classification of the sequencing data. The segmentation technique combines both read depth and discordant mate-pair reads to increase the sensitivity and resolution of CNV calls. The method is particularly suited to MPseq, which is designed to detect breakpoint junctions at high resolution. This allows for the classification step to accurately calculate copy number levels at the relatively low read depth of MPseq. Here we compare results for a series of hematological cancer samples that were tested with CMA and MPseq. We demonstrate comparable sensitivity to the state-of-the-art CMA technology, with the benefit of improved breakpoint resolution. The algorithm provides a powerful analytical tool for the analysis of MPseq results in cancer.

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.

American College of Medical Genetics and Genomics: standards and guidelines for documenting suspected consanguinity as an incidental finding of genomic testing.

Genomic testing, including single-nucleotide polymorphism-based microarrays and whole-genome sequencing, can detect long stretches of the genome that display homozygosity. The presence of these segments, when distributed across multiple chromosomes, can indicate a familial relationship between the proband's parents. This article describes the detection of possible consanguinity by genomic testing and the factors confounding the inference of a specific p-arental relationship. It is designed to guide the documentation of suspected consanguinity by clinical laboratory professionals and to alert laboratories to the need to establish a reporting policy in conjunction with their ethics review committee and legal counsel.

ACMG Standards and Guidelines for constitutional cytogenomic microarray analysis, including postnatal and prenatal applications: revision 2013.

Microarray methodologies, including array comparative genomic hybridization and single-nucleotide polymorphism-detecting arrays, are accepted as an appropriate first-tier test for the evaluation of imbalances associated with intellectual disability, autism, and multiple congenital anomalies. This technology also has applicability in prenatal specimens. To assist clinical laboratories in validation of microarray methodologies for constitutional applications, the American College of Medical Genetics and Genomics has produced the following revised professional standards and guidelines.

Alström syndrome caused by maternal uniparental disomy.

Purpose: To describe a case of Alström syndrome arising from maternal uniparental disomy. Observations: A 13-month-old boy with poor vision and nystagmus was diagnosed with Alström syndrome based on genetic testing that identified a homozygous pathogenic variant, ALMS1 c.2141_2141del (p.Ser714Tyrfs*6), that was only found in his mother and not his father. In contrast to the usual autosomal recessive inheritance pattern in which a child inherits a variant from each parent, multi-step genetic testing of the child and both parents confirmed uniparental disomy as the mechanism of inheritance. Conclusions and Importance: Confirmation of uniparental disomy in autosomal recessive disorders allows for parental assurance that future offspring will be unaffected.

Clinical Validation of Tagmentation-Based Genome Sequencing for Germline Disorders.

Genome sequencing (GS) is a powerful clinical tool used for the comprehensive diagnosis of germline disorders. GS library preparation typically involves mechanical DNA fragmentation, end repair, and bead-based library size selection followed by adapter ligation, which can require a large amount of input genomic DNA. Tagmentation using bead-linked transposomes can simplify the library preparation process and reduce the DNA input requirement. Here we describe the clinical validation of tagmentation-based PCR-free GS as a clinical test for rare germline disorders. Compared with the Genome-in-a-Bottle Consortium benchmark variant sets, GS had a recall >99.7% and a precision of 99.8% for single nucleotide variants and small insertion-deletions. GS also exhibited 100% sensitivity for clinically reported sequence variants and the copy number variants examined. Furthermore, GS detected mitochondrial sequence variants above 5% heteroplasmy and showed reliable detection of disease-relevant repeat expansions and SMN1 homozygous loss. Our results indicate that while lowering DNA input requirements and reducing library preparation time, GS enables uniform coverage across the genome as well as robust detection of various types of genetic alterations. With the advantage of comprehensive profiling of multiple types of genetic alterations, GS is positioned as an ideal first-tier diagnostic test for germline disorders.

Best practices for the interpretation and reporting of clinical whole genome sequencing.

Whole genome sequencing (WGS) shows promise as a first-tier diagnostic test for patients with rare genetic disorders. However, standards addressing the definition and deployment practice of a best-in-class test are lacking. To address these gaps, the Medical Genome Initiative, a consortium of leading health care and research organizations in the US and Canada, was formed to expand access to high quality clinical WGS by convening experts and publishing best practices. Here, we present best practice recommendations for the interpretation and reporting of clinical diagnostic WGS, including discussion of challenges and emerging approaches that will be critical to harness the full potential of this comprehensive test.

American College of Medical Genetics standards and guidelines for interpretation and reporting of postnatal constitutional copy number variants.

Genomic microarrays used to assess DNA copy number are now recommended as first-tier tests for the postnatal evaluation of individuals with intellectual disability, autism spectrum disorders, and/or multiple congenital anomalies. Application of this technology has resulted in the discovery of widespread copy number variation in the human genome, both polymorphic variation in healthy individuals and novel pathogenic copy number imbalances. To assist clinical laboratories in the evaluation of copy number variants and to promote consistency in interpretation and reporting of genomic microarray results, the American College of Medical Genetics has developed the following professional guidelines for the interpretation and reporting of copy number variation. These guidelines apply primarily to evaluation of constitutional copy number variants detected in the postnatal setting.

American College of Medical Genetics recommendations for the design and performance expectations for clinical genomic copy number microarrays intended for use in the postnatal setting for detection of constitutional abnormalities.

Genomic copy number microarrays have significantly increased the diagnostic yield over a karyotype for clinically significant imbalances in individuals with developmental delay, intellectual disability, multiple congenital anomalies, and autism, and they are now accepted as a first tier diagnostic test for these indications. As it is not feasible to validate microarray technology that targets the entire genome in the same manner as an assay that targets a specific gene or syndromic region, a new paradigm of validation and regulation is needed to regulate this important diagnostic technology. We suggest that these microarray platforms be evaluated and manufacturers regulated for the ability to accurately measure copy number gains or losses in DNA (analytical validation) and that the subsequent interpretation of the findings and assignment of clinical significance be determined by medical professionals with appropriate training and certification. To this end, the American College of Medical Genetics, as the professional organization of board-certified clinical laboratory geneticists, herein outlines recommendations for the design and performance expectations for clinical genomic copy number microarrays and associated software intended for use in the postnatal setting for detection of constitutional abnormalities.

Section E9 of the American College of Medical Genetics technical standards and guidelines: fluorescence in situ hybridization.

This updated Section E9 has been incorporated into and supersedes the previous Section E9 in Section E: Clinical Cytogenetics of the 2008 Edition (Revised 02/2007) American College of Medical Genetics Standards and Guidelines for Clinical Genetics Laboratories. This section deals specifically with the standards and guidelines applicable to fluorescence in situ hybridization analysis.

Prenatal characterization of a novel inverted SMAD2 duplication by mate pair sequencing in a fetus with dextrocardia.

This case report underlines the importance of molecular characterization of genomic duplications and other structural variants in the prenatal setting to guide clinical interpretation, genetic counseling, and perinatal medical care.