Recommendations for laboratory workflow that better support centralised amalgamation of genomic variant data: findings from CanVIG-UK national molecular laboratory survey.

BACKGROUND: National and international amalgamation of genomic data offers opportunity for research and audit, including analyses enabling improved classification of variants of uncertain significance. Review of individual-level data from National Health Service (NHS) testing of cancer susceptibility genes (2002-2023) submitted to the National Disease Registration Service revealed heterogeneity across participating laboratories regarding (1) the structure, quality and completeness of submitted data, and (2) the ease with which that data could be assembled locally for submission. METHODS: In May 2023, we undertook a closed online survey of 51 clinical scientists who provided consensus responses representing all 17 of 17 NHS molecular genetic laboratories in England and Wales which undertake NHS diagnostic analyses of cancer susceptibility genes. The survey included 18 questions relating to 'next-generation sequencing workflow' (11), 'variant classification' (3) and 'phenotypical context' (4). RESULTS: Widely differing processes were reported for transfer of variant data into their local LIMS (Laboratory Information Management System), for the formatting in which the variants are stored in the LIMS and which classes of variants are retained in the local LIMS. Differing local provisions and workflow for variant classifications were also reported, including the resources provided and the mechanisms by which classifications are stored. CONCLUSION: The survey responses illustrate heterogeneous laboratory workflow for preparation of genomic variant data from local LIMS for centralised submission. Workflow is often labour-intensive and inefficient, involving multiple manual steps which introduce opportunities for error. These survey findings and adoption of the concomitant recommendations may support improvement in laboratory dataflows, better facilitating submission of data for central amalgamation.

Germline mismatch repair (MMR) gene analyses from English NHS regional molecular genomics laboratories 1996-2020: development of a national resource of patient-level genomics laboratory records.

OBJECTIVE: To describe national patterns of National Health Service (NHS) analysis of mismatch repair (MMR) genes in England using individual-level data submitted to the National Disease Registration Service (NDRS) by the NHS regional molecular genetics laboratories. DESIGN: Laboratories submitted individual-level patient data to NDRS against a prescribed data model, including (1) patient identifiers, (2) test episode data, (3) per-gene results and (4) detected sequence variants. Individualised per-laboratory algorithms were designed and applied in NDRS to extract and map the data to the common data model. Laboratory-level MMR activity audit data from the Clinical Molecular Genetics Society/Association of Clinical Genomic Science were used to assess early years' missing data. RESULTS: Individual-level data from patients undergoing NHS MMR germline genetic testing were submitted from all 13 English laboratories performing MMR analyses, comprising in total 16 722 patients (9649 full-gene, 7073 targeted), with the earliest submission from 2000. The NDRS dataset is estimated to comprise >60% of NHS MMR analyses performed since inception of NHS MMR analysis, with complete national data for full-gene analyses for 2016 onwards. Out of 9649 full-gene tests, 2724 had an abnormal result, approximately 70% of which were (likely) pathogenic. Data linkage to the National Cancer Registry demonstrated colorectal cancer was the most frequent cancer type in which full-gene analysis was performed. CONCLUSION: The NDRS MMR dataset is a unique national pan-laboratory amalgamation of individual-level clinical and genomic patient data with pseudonymised identifiers enabling linkage to other national datasets. This growing resource will enable longitudinal research and can form the basis of a live national genomic disease registry.

Identification of people with Lynch syndrome from those presenting with colorectal cancer in England: baseline analysis of the diagnostic pathway.

It is believed that >95% of people with Lynch syndrome (LS) remain undiagnosed. Within the National Health Service (NHS) in England, formal guidelines issued in 2017 state that all colorectal cancers (CRC) should be tested for DNA Mismatch Repair deficiency (dMMR). We used a comprehensive population-level national dataset to analyse implementation of the agreed diagnostic pathway at a baseline point 2 years post-publication of official guidelines. Using real-world data collected and curated by the National Cancer Registration and Analysis Service (NCRAS), we retrospectively followed up all people diagnosed with CRC in England in 2019. Nationwide laboratory diagnostic data incorporated somatic (tumour) testing for dMMR (via immunohistochemistry or microsatellite instability), somatic testing for MLH1 promoter methylation and BRAF status, and constitutional (germline) testing of MMR genes. Only 44% of CRCs were screened for dMMR; these figures varied over four-fold with respect to geography. Of those CRCs identified as dMMR, only 51% underwent subsequent diagnostic testing. Overall, only 1.3% of patients with colorectal cancer had a germline MMR genetic test performed; up to 37% of these tests occurred outside of NICE guidelines. The low rates of molecular diagnostic testing in CRC support the premise that Lynch syndrome is underdiagnosed, with significant attrition at all stages of the testing pathway. Applying our methodology to subsequent years' data will allow ongoing monitoring and analysis of the impact of recent investment. If the diagnostic guidelines were fully implemented, we estimate that up to 700 additional people with LS could be identified each year.

Exposure profiling of reactive compounds in complex mixtures.

Humans are constantly exposed to mixtures, such as tobacco smoke, exhaust from diesel, gasoline or new bio-fuels, containing several 1000 compounds, including many known human carcinogens. Covalent binding of reactive compounds or their metabolites to DNA and formation of stable adducts is believed to be the causal link between exposure and carcinogenesis. DNA and protein adducts are well established biomarkers for the internal dose of reactive compounds or their metabolites and are an integral part of science-based risk assessment. However, technical limitations have prevented comprehensive detection of a broad spectrum of adducts simultaneously. Therefore, most studies have focused on measurement of abundant individual adducts. These studies have produced valuable insight into the metabolism of individual carcinogens, but they are insufficient for risk assessment of exposure to complex mixtures. To overcome this limitation, we present herein proof-of-principle for comprehensive exposure assessment, using N-terminal valine adduct profiles as a biomarker. The reported method is based on our previously established immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with modification to enrich all N-terminal valine alkylated peptides. The method was evaluated using alkylated peptide standards and globin reacted in vitro with alkylating agents (1,2-epoxy-3-butene, 1,2:3,4-diepoxybutane, propylene oxide, styrene oxide, N-ethyl-N-nitrosourea and methyl methanesulfonate), known to form N-terminal valine adducts. To demonstrate proof-of-principle, the method was successfully applied to globin from mice treated with four model compounds. The results suggest that this novel approach might be suitable for in vivo biomonitoring.

In silico identification of carboxylate clamp type tetratricopeptide repeat proteins in Arabidopsis and rice as putative co-chaperones of Hsp90/Hsp70.

The essential eukaryotic molecular chaperone Hsp90 operates with the help of different co-chaperones, which regulate its ATPase activity and serve as adaptors to recruit client proteins and other molecular chaperones, such as Hsp70, to the Hsp90 complex. Several Hsp90 and Hsp70 co-chaperones contain the tetratricopeptide repeat (TPR) domain, which interacts with the highly conserved EEVD motif at the C-terminal ends of Hsp90 and Hsp70. The acidic side chains in EEVD interact with a subset of basic residues in the TPR binding pocket called a 'carboxylate clamp'. Since the carboxylate clamp residues are conserved in the TPR domains of known Hsp90/Hsp70 co-chaperones, we carried out an in silico search for TPR proteins in Arabidopsis and rice comprising of at least one three-motif TPR domain with conserved amino acid residues required for Hsp90/Hsp70 binding. This approach identified in Arabidopsis a total of 36 carboxylate clamp (CC)-TPR proteins, including 24 novel proteins, with potential to interact with Hsp90/Hsp70. The newly identified CC-TPR proteins in Arabidopsis and rice contain additional protein domains such as ankyrin, SET, octicosapeptide/Phox/Bem1p (Phox/PB1), DnaJ-like, thioredoxin, FBD and F-box, and protein kinase and U-box, indicating varied functions for these proteins. To provide proof-of-concept of the newly identified CC-TPR proteins for interaction with Hsp90, we demonstrated interaction of AtTPR1 and AtTPR2 with AtHsp90 in yeast two-hybrid and in vitro pull down assays. These findings indicate that the in silico approach used here successfully identified in a genome-wide context CC-TPR proteins with potential to interact with Hsp90/Hsp70, and further suggest that the Hsp90/Hsp70 system relies on TPR co-chaperones more than it was previously realized.