Rapid detection of common variants and deletions of CYP21A2 using MALDI-TOF MS.

Newborn screening (NBS) for congenital adrenal hyperplasia (CAH) based on hormonal testing is successfully implemented in many countries. However, this method cannot detect non-classic CAH and has high false positive rates. We have developed a novel MALDI-TOF MS assay that can identify common variants and deletions of CYP21A2 in the Chinese population. Thirty-seven clinical patients with CAH confirmed by Sanger sequencing and MLPA analysis were detected by MALDI-TOF MS assay. Two CYP21A2 variants were detected in 30 patients and one CYP21A2 variant was detected in 7 patients. The MALDI-TOF MS assay detected 67 mutant alleles in 37 patients with a detection rate of 90.5%. Sanger sequencing revealed that three variants in seven patients were not included in the designed panel. Eleven distinct CYP21A2 variants were identified, including five missense variants, two nonsense variants, two large gene deletions, one splice variant, and one frameshift variant. The most frequent variant was c.293-13C > G (37.84%), followed by c.518T > A (21.62%) and exon 1-7 deletion (17.57%). The high-throughput MALDI-TOF MS assay that can simultaneously detect common variants and deletions of CYP21A2. This assay can be used for population-based genetic screening and rapid detection of suspected patients, and is expected to be a valuable complement to biochemical-based testing for the detection of CAH.

Validation of a clinical breast cancer risk assessment tool combining a polygenic score for all ancestries with traditional risk factors.

PURPOSE: We previously described a combined risk score (CRS) that integrates a multiple-ancestry polygenic risk score (MA-PRS) with the Tyrer-Cuzick (TC) model to assess breast cancer (BC) risk. Here, we present a longitudinal validation of CRS in a real-world cohort. METHODS: This study included 130,058 patients referred for hereditary cancer genetic testing and negative for germline pathogenic variants in BC-associated genes. Data were obtained by linking genetic test results to medical claims (median follow-up 12.1 months). CRS calibration was evaluated by the ratio of observed to expected BCs. RESULTS: Three hundred forty BCs were observed over 148,349 patient-years. CRS was well-calibrated and demonstrated superior calibration compared with TC in high-risk deciles. MA-PRS alone had greater discriminatory accuracy than TC, and CRS had approximately 2-fold greater discriminatory accuracy than MA-PRS or TC. Among those classified as high risk by TC, 32.6% were low risk by CRS, and of those classified as low risk by TC, 4.3% were high risk by CRS. In cases where CRS and TC classifications disagreed, CRS was more accurate in predicting incident BC. CONCLUSION: CRS was well-calibrated and significantly improved BC risk stratification. Short-term follow-up suggests that clinical implementation of CRS should improve outcomes for patients of all ancestries through personalized risk-based screening and prevention.

Joint ABS-UKCGG-CanGene-CanVar consensus regarding the use of CanRisk in clinical practice.

BACKGROUND: The CanRisk tool, which operationalises the Breast and Ovarian Analysis of Disease Incidence and Carrier Estimation Algorithm (BOADICEA) is used by Clinical Geneticists, Genetic Counsellors, Breast Oncologists, Surgeons and Family History Nurses for breast cancer risk assessments both nationally and internationally. There are currently no guidelines with respect to the day-to-day clinical application of CanRisk and differing inputs to the model can result in different recommendations for practice. METHODS: To address this gap, the UK Cancer Genetics Group in collaboration with the Association of Breast Surgery and the CanGene-CanVar programme held a workshop on 16th of May 2023, with the aim of establishing best practice guidelines. RESULTS: Using a pre-workshop survey followed by structured discussion and in-meeting polling, we achieved consensus for UK best practice in use of CanRisk in making recommendations for breast cancer surveillance, eligibility for genetic testing and the input of available information to undertake an individualised risk assessment. CONCLUSIONS: Whilst consensus recommendations were achieved, the meeting highlighted some of the barriers limiting the use of CanRisk in clinical practice and identified areas that require further work and collaboration with relevant national bodies and policy makers to incorporate wider use of CanRisk into routine breast cancer risk assessments.

Comparison of the ABC and ACMG systems for variant classification.

The ABC and ACMG variant classification systems were compared by asking mainly European clinical laboratories to classify variants in 10 challenging cases using both systems, and to state if the variant in question would be reported as a relevant result or not as a measure of clinical utility. In contrast to the ABC system, the ACMG system was not made to guide variant reporting but to determine the likelihood of pathogenicity. Nevertheless, this comparison is justified since the ACMG class determines variant reporting in many laboratories. Forty-three laboratories participated in the survey. In seven cases, the classification system used did not influence the reporting likelihood when variants labeled as "maybe report" after ACMG-based classification were included. In three cases of population frequent but disease-associated variants, there was a difference in favor of reporting after ABC classification. A possible reason is that ABC step C (standard variant comments) allows a variant to be reported in one clinical setting but not another, e.g., based on Bayesian-based likelihood calculation of clinical relevance. Finally, the selection of ACMG criteria was compared between 36 laboratories. When excluding criteria used by less than four laboratories (<10%), the average concordance rate was 46%. Taken together, ABC-based classification is more clear-cut than ACMG-based classification since molecular and clinical information is handled separately, and variant reporting can be adapted to the clinical question and phenotype. Furthermore, variants do not get a clinically inappropriate label, like pathogenic when not pathogenic in a clinical context, or variant of unknown significance when the significance is known.

Laboratory testing for preconception/prenatal carrier screening: A technical standard of the American College of Medical Genetics and Genomics (ACMG).

Carrier screening has historically assessed a relatively small number of autosomal recessive and X-linked conditions selected based on frequency in a specific subpopulation and association with severe morbidity or mortality. Advances in genomic technologies enable simultaneous screening of individuals for several conditions. The American College of Medical Genetics and Genomics recently published a clinical practice resource that presents a framework when offering screening for autosomal recessive and X-linked conditions during pregnancy and preconception and recommends a tier-based approach when considering the number of conditions to screen for and their frequency within the US population in general. This laboratory technical standard aims to complement the practice resource and to put forth considerations for clinical laboratories and clinicians who offer preconception/prenatal carrier screening.

Harmonization of homologous recombination deficiency testing in ovarian cancer: Results from the MITO16A/MaNGO-OV2 trial.

BACKGROUND: Homologous Recombination Deficiency (HRD) status predicts response to treatment with poly(ADP-ribose) polymerase inhibitors in Ovarian Cancer (OC) patients. The Myriad myChoiceCDx Assay is approved by Food and Drug Agency for the HRD assessment. Here we compared the HRD status obtained by three commercial panels with the results from Myriad reference test. METHODS: The HRD analysis was performed on DNA from formalin-fixed and paraffin-embedded tumor samples of 100 untreated OC patients for which Myriad assay results were available, using TruSight Oncology 500 HRD assay (Illumina), Oncomine Comprehensive Assay Plus (Thermo Fisher Scientific) and SOPHiA DDM HRD solution panel (SOPHiA Genetics). RESULTS: A good overall concordance with the reference method was demonstrated at three different levels: BRCA mutational status (from 94.4 % to 97.7 %), the genomic instability value (from 88.2 % to 95.3 %) and for the HRD status (from 90.4 % to 97.6 %). Moreover, a trend in favour of HRD positive patients for response rate, progression-free survival and overall survival similar to Myriad was observed for all three tests. DISCUSSION: Our data suggest the feasibility of commercial testing for assessing HRD status, with a good concordance with the reference method and association with clinical outcome.

Results of a Pilot External Quality Assessment Scheme for Genetic Testing of Newborns with Spinal Muscular Atrophy.

BACKGROUND: This study aims to evaluate the ability of laboratories to perform spinal muscular atrophy (SMA) genetic testing in newborns based on dried blood spot (DBS) samples, and to provide reference data and advance preparation for establishing the pilot external quality assessment (EQA) scheme for SMA genetic testing of newborns in China. METHODS: The pilot EQA scheme contents and evaluation principles of this project were designed by National Center for Clinical Laboratories (NCCL), National Health Commission. Two surveys were carried out in 2022, and 5 batches of blood spots were submitted to the participating laboratory each time. All participating laboratories conducted testing upon receiving samples, and test results were submitted to NCCL within the specified date. RESULTS: The return rates were 75.0% (21/28) and 95.2% (20/21) in the first and second surveys, respectively. The total return rate of the two examinations was 83.7% (41/49). Nineteen laboratories (19/21, 90.5%) had a full score passing on the first survey, while in the second survey twenty laboratories (20/20, 100%) scored full. CONCLUSIONS: This pilot EQA survey provides a preliminary understanding of the capability of SMA genetic testing for newborns across laboratories in China. A few laboratories had technical or operational problems in testing. It is, therefore, of importance to strengthen laboratory management and to improve testing capacity for the establishment of a national EQA scheme for newborn SMA genetic testing.

A new genetic diagnosis strategy for paroxysmal kinesigenic dyskinesia: Targeted high-throughput detection of PRRT2 gene c.649 locus.

BACKGROUND: Paroxysmal kinesigenic dyskinesia (PKD) is the most prevalent kind type of paroxysmal Dyskinesia, characterized by recurrent and transient episodes of involuntary movements. Most PKD cases were attributed to the proline-rich transmembrane protein 2 (PRRT2) gene, in which the c.649 region is a hotspot for known mutations. Even though some patients with PKD have been genetically diagnosed using whole-exome sequencing (WES) and Sanger sequencing, there are still cases of missed diagnoses due to the limitations of sequencing technology and analytic methods on throughput. METHODS: Patients meeting the diagnosis criteria of PKD with negative results of PRRT2-Sanger sequencing and WES were included in this study. Mutation screening and targeted high-throughput sequencing were performed to analyze and verify the sequencing results of the potential mutations. RESULTS: Six patients with PKD with high mutation ratios of c.649dupC were screened using our targeted high-throughput sequencing from 26 PKD patients with negative results of PRRT2-Sanger sequencing and WES (frequency = 23.1%), which compensated for the comparatively shallow sequencing depth and statistical flaws in this region. Compared with the local normal population and other patients with PKD, the mutation ratios of c.649dupC of these six patients with PKD were much higher and also had truncated protein structures and differentially altered mRNA expression. CONCLUSION: Based on the above studies, we emphasize the routine targeted high-throughput sequencing of the c.649 site in the PRRT2 gene in so-called genetic-testing-negative patients with PKD, and manually calculate the deletion and duplication mutations depth and ratios to lower the rate of clinical misdiagnosis.

Establishing national reference materials for genetic testing of cytochrome P450.

OBJECTIVES: Reference materials for in-vitro diagnostic reagents play a critical role in determining the quality of reagents and ensuring the accuracy of clinical test results. This study aimed to establish a national reference material (NRM) for detecting cytochrome P450 (CYP) genes related to drug metabolism by screening databases on the Chinese population to identify CYP gene polymorphism characteristics. METHODS: To prepare the NRM, we used DNA extracted from healthy human immortalized B lymphoblastoid cell lines as the raw material. Samples of these cell lines were obtained from the Chinese Population PGx Gene Polymorphism Biobank. Further, we used Sanger sequencing, next-generation sequencing, and commercial assay kits to validate the polymorphic genotypes. RESULTS: Among the CYP superfamily genes, we confirmed 24 riboswitch loci related to drug metabolism, with evidence levels of 1A, 2A, 3, and 4. We confirmed the polymorphic loci and validated their genotypes using various sequencing techniques. Our results were consistent with the polymorphism information of samples obtained from the biobank, thus demonstrating high precision and stability of the established NRM. CONCLUSION: An NRM (360 056-202 201) for CYP genetic testing covering 24 loci related to drug metabolism was established and approved to assess in-vitro diagnostic reagents containing CYP family gene polymorphisms and perform clinical inter-room quality evaluations.

A framework for the evaluation and reporting of incidental findings in clinical genomic testing.

Currently, there are no widely accepted recommendations in the genomics field guiding the return of incidental findings (IFs), defined here as unexpected results that are unrelated to the indication for testing. Consequently, reporting policies for IFs among laboratories offering genomic testing are variable and may lack transparency. Herein we describe a framework developed to guide the evaluation and return of IFs encountered in probands undergoing clinical genome sequencing (cGS). The framework prioritizes clinical significance and actionability of IFs and follows a stepwise approach with stopping points at which IFs may be recommended for return or not. Over 18 months, implementation of the framework in a clinical laboratory facilitated the return of actionable IFs in 37 of 720 (5.1%) individuals referred for cGS, which is reduced to 3.1% if glucose-6-phosphate dehydrogenase (G6PD) deficiency is excluded. This framework can serve as a model to standardize reporting of IFs identified during genomic testing.

Temporal trends and regional variability in BRAF and KRAS genetic testing in Denmark (2010-2022): Implications for precision medicine.

OBJECTIVE: This study aims to evaluate the developments in the testing of Kirsten Rat Sarcoma viral oncogene homolog (KRAS) and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations across different cancer types and regions in Denmark from 2010 to 2022. STUDY DESIGN AND SETTING: Using comprehensive data from the Danish health registries, we linked molecular test results from the Danish Pathology Registry with cancer diagnoses from the Danish National Patient Registry between 2010 and 2022. We assessed the frequency and distribution of KRAS and BRAF mutations across all cancer types, years of testing, and the five Danish regions. RESULTS: The study included records of KRAS testing for 30 671 patients and BRAF testing for 30 860 patients. Most KRAS testing was performed in colorectal (78%) and lung cancer (18%), and BRAF testing in malignant melanoma (13%), colorectal cancer (67%), and lung cancer (12%). Testing rates and documentation mutational subtypes increased over time. Reporting of wildtype results varied between lung and colorectal cancer, with underreporting in lung cancer. Regional variations in testing and reporting were observed. CONCLUSION: Our study highlights substantial progress in KRAS and BRAF testing in Denmark from 2010 to 2022, evidenced by increased and more specific reporting of mutational test results, thereby improving the precision of cancer diagnosis and treatment. However, persistent regional variations and limited testing for cancer types beyond melanoma, colorectal, and lung cancer highlight the necessity for a nationwide assessment of the optimal testing approach.

Best practice guidelines on genetic diagnostics of facioscapulohumeral muscular dystrophy: Update of the 2012 guidelines.

The gold standard for facioscapulohumeral muscular dystrophy (FSHD) genetic diagnostic procedures was published in 2012. With the increasing complexity of the genetics of FSHD1 and 2, the increase of genetic testing centers, and the start of clinical trials for FSHD, it is crucial to provide an update on our knowledge of the genetic features of the FSHD loci and renew the international consensus on the molecular testing recommendations. To this end, members of the FSHD European Trial Network summarized the evidence presented during the 2022 ENMC meeting on Genetic diagnosis, clinical outcome measures, and biomarkers. The working group additionally invited genetic and clinical experts from the USA, India, Japan, Australia, South-Africa, and Brazil to provide a global perspective. Six virtual meetings were organized to reach consensus on the minimal requirements for genetic confirmation of FSHD1 and FSHD2. Here, we present the clinical and genetic features of FSHD, specific features of FSHD1 and FSHD2, pros and cons of established and new technologies (Southern blot in combination with either linear or pulsed-field gel electrophoresis, molecular combing, optical genome mapping, FSHD2 methylation analysis and FSHD2 genotyping), the possibilities and challenges of prenatal testing, including pre-implantation genetic testing, and the minimal requirements and recommendations for genetic confirmation of FSHD1 and FSHD2. This consensus is expected to contribute to current clinical management and trial-readiness for FSHD.

Embryology with precision: effective quality control in the in vitro fertilization laboratory.

The purpose of this review is to address the critical need for standardization and clarity in the use of key performance indicators (KPIs) within the realm of in vitro fertilization (IVF), particularly emphasizing the integration of preimplantation genetic testing (PGT) processes. This review is timely and relevant given the persistently modest success rates of IVF treatments, which stand at approximately 30%, and the growing complexity of IVF procedures, including PGT practices. The review synthesizes recent findings across studies focusing on technical and clinical KPIs in embryology and genetic laboratories, identifying gaps in current research and practice, particularly the lack of standardized KPIs and terminology. Recent findings highlighted include the critical evaluation of technical KPIs such as Intracytoplasmic Sperm Injection (ICSI) fertilization rates, embryo development rates, and laboratory performance metrics, alongside clinical KPIs like the proportion of mature oocytes and clinical pregnancy rates. Notably, the review uncovers a significant gap in integrating and standardizing KPIs for PGT applications, which is essential for improving IVF outcomes and genetic diagnostic accuracy. The implications of these findings are profound for both clinical practice and research. For clinical practice, establishing a standardized set of KPIs, especially for PGT, could significantly enhance the success rates of IVF treatments by providing clearer benchmarks for quality and performance. For research, this review underscores the necessity for further studies to close the identified gaps, promoting a more integrated and standardized approach to KPIs in IVF and PGT processes. This comprehensive approach will not only aid in improving clinical outcomes but also in advancing the field of reproductive medicine.

Determining priority indicators of utility for genomic testing in rare disease: A Delphi study.

PURPOSE: Determining the value of genomic tests in rare disease necessitates a broader conceptualization of genomic utility beyond diagnostic yield. Despite widespread discussion, consensus toward which aspects of value to consider is lacking. This study aimed to use expert opinion to identify and refine priority indicators of utility in rare disease genomic testing. METHODS: We used 2 survey rounds following Delphi methodology to obtain consensus on indicators of utility among experts involved in policy, clinical, research, and consumer advocacy leadership in Australia. We analyzed quantitative and qualitative data to identify, define, and determine priority indicators. RESULTS: Twenty-five experts completed round 1 and 18 completed both rounds. Twenty indicators reached consensus as a priority in value assessment, including those relating to prognostic information, timeliness of results, practical and health care outcomes, clinical accreditation, and diagnostic yield. Whereas indicators pertaining to discovery research, disutility, and factors secondary to primary reason for testing were considered less of a priority and were removed. CONCLUSION: This study obtained expert consensus on different utility indicators that are considered a priority in determining the value of genomic testing in rare disease in Australia. Indicators may inform a standardized approach to evidence generation and assessment to guide future research, decision making, and implementation efforts.

Prenatal Genome-Wide Sequencing analysis (Exome or Genome) in detecting pathogenic Single Nucleotide Variants in fetal Central Nervous System Anomalies: systematic review and meta-analysis.

Prenatal Exome (pES) or Genome (pGS) Sequencing analysis showed a significant incremental diagnostic yield over karyotype and chromosomal microarray analysis (CMA) in fetal structural anomalies. Optimized indications and detection rates in different fetal anomalies are still under investigation. The aim of this study was to assess the incremental diagnostic yield in prenatally diagnosed Central Nervous System (CNS) anomalies. A systematic review on antenatal CNS anomalies was performed according to PRISMA guidelines, including n = 12 paper, accounting for 428 fetuses. Results were pooled in a meta-analysis fitting a logistic random mixed-effect model. The effect of interest was the incremental diagnostic rate of pES over karyotype/CMA in detecting likely pathogenic/pathogenic Single Nucleotide Variants (SNVs). A further meta-analysis adding the available pGS studies (including diagnostic coding SNVs only) and submeta-analysis on three CNS subcategories were also performed. The pooled incremental diagnostic yield estimate of pES studies was 38% (95% C.I.: [29%;47%]) and 36% (95% C.I.: [28%;45%]) when including diagnostic SNVs of pGS studies. The point estimate of the effect resulted 22% (95% C.I.: [15%;31%]) in apparently isolated anomalies, 33% (95% C.I.: [22%;46%]) in CNS-only related anomalies (≥1) and 46% (95% C.I.: [38%;55%]) in non-isolated anomalies (either ≥ 2 anomalies in CNS, or CNS and extra-CNS). Meta-analysis showed a substantial diagnostic improvement in performing Prenatal Genome-Wide Sequencing analysis (Exome or Genome) over karyotype and CMA in CNS anomalies.