Genome-wide association study of thyroid-stimulating hormone highlights new genes, pathways and associations with thyroid disease.

Thyroid hormones play a critical role in regulation of multiple physiological functions and thyroid dysfunction is associated with substantial morbidity. Here, we use electronic health records to undertake a genome-wide association study of thyroid-stimulating hormone (TSH) levels, with a total sample size of 247,107. We identify 158 novel genetic associations, more than doubling the number of known associations with TSH, and implicate 112 putative causal genes, of which 76 are not previously implicated. A polygenic score for TSH is associated with TSH levels in African, South Asian, East Asian, Middle Eastern and admixed American ancestries, and associated with hypothyroidism and other thyroid disease in South Asians. In Europeans, the TSH polygenic score is associated with thyroid disease, including thyroid cancer and age-of-onset of hypothyroidism and hyperthyroidism. We develop pathway-specific genetic risk scores for TSH levels and use these in phenome-wide association studies to identify potential consequences of pathway perturbation. Together, these findings demonstrate the potential utility of genetic associations to inform future therapeutics and risk prediction for thyroid diseases.

Molecular radiobiology and the origins of the base excision repair pathway: an historical perspective.

PURPOSE: To demonstrate how the search by the Molecular Radiobiologists for enzymes that could recognize and remove DNA damage produced by ionizing radiation was intertwined with the development of the Base Excision Repair pathway. CONCLUSION: The Base Excision Repair pathway repairs the vast majority of radiation-induced DNA damages including base damages, alkali labile lesions, and single strand breaks. It turns out that Base Excision Repair actually evolved to repair some thirty to forty thousand endogenous lesions formed in each of our cells every day. Thus, this process is extremely efficient and accordingly, at relatively low doses of radiation, the single lesions repaired by base excision repair result in few lethal or mutagenic events. This efficiency is a double-edged sword since ionizing radiation-induced hydroxyl radicals produced along the radiation track form both bistranded and tandem clustered lesions in DNA. These damages are recognized by the efficient Base Excision Repair enzymes, which, during attempted repair, lead to double strand breaks and/or multiple lesions that can collapse replication forks. Double strand breaks and other complex or clustered lesions formed by ionizing radiation present distinct challenges to DNA repair systems compared to the relative ease and efficiency by which isolated lesions are repaired.

Consequences and repair of radiation-induced DNA damage: fifty years of fun questions and answers.

PURPOSE: To summarize succinctly the 50 years of research undertaken in my laboratory and to provide an overview of my career in science. It is certainly a privilege to have been asked by Carmel Mothersill and Penny Jeggo to contribute to this special issue of the International Journal of Radiation Biology focusing on the work of women in the radiation sciences. CONCLUSION: My students, post-docs and I identified and characterized a number of the enzymes that recognize and remove radiation-damaged DNA bases, the DNA glycosylases, which are the first enzymes in the Base Excision Repair (BER) pathway. Although this pathway actually evolved to repair oxidative and other endogenous DNA damages, it is also responsible for removing the vast majority of radiation-induced DNA damages including base damages, alkali-labile lesions and single strand breaks. However, because of its high efficiency, attempted BER of clustered lesions produced by ionizing radiation, can have disastrous effects on cellular DNA. We also evaluated the potential biological consequences of many of the radiation-induced DNA lesions. In addition, with collaborators, we employed computational techniques, x-ray crystallography and single molecule approaches to answer many questions at the molecular level.

Mouse Embryonic Fibroblasts Isolated From Nthl1 D227Y Knockin Mice Exhibit Defective DNA Repair and Increased Genome Instability.

Oxidative DNA damage as a result of normal cellular metabolism, inflammation, or exposure to exogenous DNA damaging agents if left unrepaired, can result in genomic instability, a precursor to cancer and other diseases. Nth-like DNA glycosylase 1 (NTHL1) is an evolutionarily conserved bifunctional DNA glycosylase that primarily removes oxidized pyrimidine lesions. NTHL1 D239Y is a germline variant identified in both heterozygous and homozygous state in the human population. Here, we have generated a knockin mouse model carrying Nthl1 D227Y (mouse homologue of D239Y) using CRISPR-cas9 genome editing technology and investigated the cellular effects of the variant in the heterozygous (Y/+) and homozygous (Y/Y) state using murine embryonic fibroblasts. We identified a significant increase in double stranded breaks, genomic instability, replication stress and impaired proliferation in both the Nthl1 D227Y heterozygous Y/+ and homozygous mutant Y/Y MEFs. Importantly, we identified that the presence of the D227Y variant interferes with repair by the WT protein, possibly by binding and shielding the lesions. The cellular phenotypes observed in D227Y mutant MEFs suggest that both the heterozygous and homozygous carriers of this NTHL1 germline mutation may be at increased risk for the development of DNA damage-associated diseases, including cancer.

Caught in motion: human NTHL1 undergoes interdomain rearrangement necessary for catalysis.

Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and ß-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.

Adding dynamic consent to a longitudinal cohort study: A qualitative study of EXCEED participant perspectives.

BACKGROUND: Dynamic consent has been proposed as a process through which participants and patients can gain more control over how their data and samples, donated for biomedical research, are used, resulting in greater trust in researchers. It is also a way to respond to evolving data protection frameworks and new legislation. Others argue that the broad consent currently used in biobank research is ethically robust. Little empirical research with cohort study participants has been published. This research investigated the participants' opinions of adding a dynamic consent interface to their existing study. METHODS: Adult participants in the Extended Cohort for E-health, Environment and DNA (EXCEED) longitudinal cohort study who are members of the EXCEED Public and Participant Engagement Group were recruited. Four focus groups were conducted and analysed for thematic content. Discussion topics were derived from a review of the current literature on dynamic consent. RESULTS: Participants were in favour of many aspects of a dynamic consent interface, such as being able to update their information, add additional data to their records and choose withdrawal options. They were supportive provided it was simple to use and not intrusive. Participants expressed a markedly high level of trust in the study and its investigators and were unanimously happy with their current participation. No strong support was found for adding a dynamic consent interface to EXCEED. CONCLUSIONS: Trust in the study researchers was the strongest theme found. Openness and good data security were needed to retain their trust. While happy to discuss dynamic consent, participants were satisfied with the current study arrangements. There were indications that changing the study might unnecessarily disturb their trust. This raised the question of whether there are contexts where dynamic consent is more appropriate than others. This study was limited by the small number of participants who were committed to the study and biased towards it. More research is needed to fully understand the potential impact of adding a dynamic consent interface to an existing cohort study.

Intussusception in 2 Children With Severe Acute Respiratory Syndrome Coronavirus-2 Infection.

We note that intussusception was likely associated with severe acute respiratory syndrome coronavirus-2 infection in 2 infants in Wuhan and London. The intussusception was reduced by enemas in Wuhan; the outcome was fatal. The intussusception was not reduced by enemas in London and required surgery; the outcome was favorable.

How Can We Not Waste Legacy Genomic Research Data?

Enabling genomic and biomedical data to be shared for secondary research purposes is not always straightforward for existing "legacy" data sets. Researchers may not know whether their data meet ethical and regulatory requirements for sharing. As a result, these data, collected using public funds and the good will and efforts of the donors and investigators, may not be used beyond their original purpose. Single-use plastics are now being banned in many countries; single-use research should be avoided if possible. This paper describes a filter developed through the driver projects of the Global Alliance for Genomics and Health that can be used by researchers to help them determine the extent of sharing possible for their legacy data and actions to be taken to enable further sharing.

Unhooking of an interstrand cross-link at DNA fork structures by the DNA glycosylase NEIL3.

Interstrand DNA-DNA cross-links (ICLs) are generated by endogenous processes, drugs, and environmental toxins. Understanding the cellular pathways by which various ICLs are repaired is critical to understanding their biological effects. Recent studies showed that replication-dependent repair of an ICL derived from the reaction of an abasic (AP) site with an adenine residue (dA) on the opposing strand of duplex DNA proceeds via a novel mechanism in which the DNA glycosylase NEIL3 unhooks the ICL. Here we examined the ability of the glycosylase domain of murine NEIL3 (MmuNEIL3-GD) to unhook dA-AP ICLs. The enzyme selectively unhooks the dA-AP ICL located at the duplex/single-strand junction of splayed duplexes that model the strand-separated DNA at the leading edge of a replication fork. We show that the ability to unhook the dA-AP ICL is a specialized function of NEIL3 as this activity is not observed in other BER enzymes. Importantly, NEIL3 only unhooks the dA-AP ICL when the AP residue is located on what would be the leading template strand of a model replication fork. The same specificity for the leading template strand was observed with a 5,6-dihydrothymine monoadduct, demonstrating that this preference is a general feature of the glycosylase and independent of the type of DNA damage. Overall, the results show that the glycosylase domain of NEIL3, lacking the C-terminal NPL4 and GRF zinc finger motifs, is competent to unhook the dA-AP ICL in splayed substrates and independently enforces important substrate preferences on the repair process.

Cooperation of the NEIL3 and Fanconi anemia/BRCA pathways in interstrand crosslink repair.

The NEIL3 DNA glycosylase is a base excision repair enzyme that excises bulky base lesions from DNA. Although NEIL3 has been shown to unhook interstrand crosslinks (ICL) in Xenopus extracts, how NEIL3 participants in ICL repair in human cells and its corporation with the canonical Fanconi anemia (FA)/BRCA pathway remain unclear. Here we show that the NEIL3 and the FA/BRCA pathways are non-epistatic in psoralen-ICL repair. The NEIL3 pathway is the major pathway for repairing psoralen-ICL, and the FA/BRCA pathway is only activated when NEIL3 is not present. Mechanistically, NEIL3 is recruited to psoralen-ICL in a rapid, PARP-dependent manner. Importantly, the NEIL3 pathway repairs psoralen-ICLs without generating double-strand breaks (DSBs), unlike the FA/BRCA pathway. In addition, we found that the RUVBL1/2 complex physically interact with NEIL3 and function within the NEIL3 pathway in psoralen-ICL repair. Moreover, TRAIP is important for the recruitment of NEIL3 but not FANCD2, and knockdown of TRAIP promotes FA/BRCA pathway activation. Interestingly, TRAIP is non-epistatic with both NEIL3 and FA pathways in psoralen-ICL repair, suggesting that TRAIP may function upstream of the two pathways. Taken together, the NEIL3 pathway is the major pathway to repair psoralen-ICL through a unique DSB-free mechanism in human cells.

Single molecule glycosylase studies with engineered 8-oxoguanine DNA damage sites show functional defects of a MUTYH polyposis variant.

Proper repair of oxidatively damaged DNA bases is essential to maintain genome stability. 8-Oxoguanine (7,8-dihydro-8-oxoguanine, 8-oxoG) is a dangerous DNA lesion because it can mispair with adenine (A) during replication resulting in guanine to thymine transversion mutations. MUTYH DNA glycosylase is responsible for recognizing and removing the adenine from 8-oxoG:adenine (8-oxoG:A) sites. Biallelic mutations in the MUTYH gene predispose individuals to MUTYH-associated polyposis (MAP), and the most commonly observed mutation in some MAP populations is Y165C. Tyr165 is a 'wedge' residue that intercalates into the DNA duplex in the lesion bound state. Here, we utilize single molecule fluorescence microscopy to visualize the real-time search behavior of Escherichia coli and Mus musculus MUTYH WT and wedge variant orthologs on DNA tightropes that contain 8-oxoG:A, 8-oxoG:cytosine, or apurinic product analog sites. We observe that MUTYH WT is able to efficiently find 8-oxoG:A damage and form highly stable bound complexes. In contrast, MUTYH Y150C shows decreased binding lifetimes on undamaged DNA and fails to form a stable lesion recognition complex at damage sites. These findings suggest that MUTYH does not rely upon the wedge residue for damage site recognition, but this residue stabilizes the lesion recognition complex.

The lyase activity of bifunctional DNA glycosylases and the 3'-diesterase activity of APE1 contribute to the repair of oxidized bases in nucleosomes.

The vast majority of oxidized bases that form in DNA are subject to base excision repair (BER). The DNA intermediates generated during successive steps in BER may prove mutagenic or lethal, making it critical that they be 'handed' from one BER enzyme to the next in a coordinated fashion. Here, we report that the handoff of BER intermediates that occurs during the repair of naked DNA substrates differs significantly from that in nucleosomes. During BER of oxidized bases in naked DNA, products generated by the DNA glycosylase NTHL1 were efficiently processed by the downstream enzyme, AP-endonuclease (APE1). In nucleosomes, however, NTHL1-generated products accumulated to significant levels and persisted for some time. During BER of naked DNA substrates, APE1 completely bypasses the inefficient lyase activity of NTHL1. In nucleosomes, the NTHL1-associated lyase contributes to BER, even in the presence of APE1. Moreover, in nucleosomes but not in naked DNA, APE1 was able to process NTHL1 lyase-generated substrates just as efficiently as it processed abasic sites. Thus, the lyase activity of hNTHL1, and the 3' diesterase activity of APE1, which had been seen as relatively dispensable, may have been preserved during evolution to enhance BER in chromatin.

Potential for diagnosis of infectious disease from the 100,000 Genomes Project Metagenomic Dataset: Recommendations for reporting results.

The identification of microbiological infection is usually a diagnostic investigation, a complex process that is firstly initiated by clinical suspicion. With the emergence of high-throughput sequencing (HTS) technologies, metagenomic analysis has unveiled the power to identify microbial DNA/RNA from a diverse range of clinical samples (1). Metagenomic analysis of whole human genomes at the clinical/research interface bypasses the steps of clinical scrutiny and targeted testing and has the potential to generate unexpected findings relating to infectious and sometimes transmissible disease. There is no doubt that microbial findings that may have a significant impact on a patient's treatment and their close contacts should be reported to those with clinical responsibility for the sample-donating patient. There are no clear recommendations on how such findings that are incidental, or outside the original investigation, should be handled. Here we aim to provide an informed protocol for the management of incidental microbial findings as part of the 100,000 Genomes Project which may have broader application in this emerging field. As with any other clinical information, we aim to prioritise the reporting of data that are most likely to be of benefit to the patient and their close contacts. We also set out to minimize risks, costs and potential anxiety associated with the reporting of results that are unlikely to be of clinical significance. Our recommendations aim to support the practice of microbial metagenomics by providing a simplified pathway that can be applied to reporting the identification of potential pathogens from metagenomic datasets. Given that the ambition for UK sequenced human genomes over the next 5 years has been set to reach 5 million and the field of metagenomics is rapidly evolving, the guidance will be regularly reviewed and will likely adapt over time as experience develops.

A functional assay-based procedure to classify mismatch repair gene variants in Lynch syndrome.

PURPOSE: To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cell-free in vitro MMR activity (CIMRA) assay. Here, we calibrate and validate the assay, enabling its integration with in silico and clinical data. METHODS: Two sets of previously classified MLH1 and MSH2 variants were selected from a curated MMR gene database, and their biochemical activity determined by the CIMRA assay. The assay was calibrated by regression analysis followed by symmetric cross-validation and Bayesian integration with in silico predictions of pathogenicity. CIMRA assay reproducibility was assessed in four laboratories. RESULTS: Concordance between the training runs met our prespecified validation criterion. The CIMRA assay alone correctly classified 65% of variants, with only 3% discordant classification. Bayesian integration with in silico predictions of pathogenicity increased the proportion of correctly classified variants to 87%, without changing the discordance rate. Interlaboratory results were highly reproducible. CONCLUSION: The CIMRA assay accurately predicts pathogenic and benign MMR gene variants. Quantitative combination of assay results with in silico analysis correctly classified the majority of variants. Using this calibration, CIMRA assay results can be integrated into the diagnostic algorithm for MMR gene variants.

The NEIL1 G83D germline DNA glycosylase variant induces genomic instability and cellular transformation.

Base excision repair (BER) is a key genome maintenance pathway. The NEIL1 DNA glycosylase recognizes oxidized bases, and likely removes damage in advance of the replication fork. The rs5745906 SNP of the NEIL1 gene is a rare human germline variant that encodes the NEIL1 G83D protein, which is devoid of DNA glycosylase activity. Here we show that expression of G83D NEIL1 in MCF10A immortalized but non-transformed mammary epithelial cells leads to replication fork stress. Upon treatment with hydrogen peroxide, we observe increased levels of stalled replication forks in cells expressing G83D NEIL1 versus cells expressing the wild-type (WT) protein. Double-strand breaks (DSBs) arise in G83D-expressing cells during the S and G2/M phases of the cell cycle. Interestingly, these breaks result in genomic instability in the form of high levels of chromosomal aberrations and micronuclei. Cells expressing G83D also grow in an anchorage independent manner, suggesting that the genomic instability results in a carcinogenic phenotype. Our results are consistent with the idea that an inability to remove oxidative damage in an efficient manner at the replication fork leads to genomic instability and mutagenesis. We suggest that individuals who harbor the G83D NEIL1 variant face an increased risk for human cancer.