Genetic variation in the chicken genome: insights in selection

The chicken currently provides more than a quarter of the meat and nearly all eggs produced worldwide. For future improvements in production traits and animal welfare as well as to address future consumer demands it is necessary to understand the etiology and biology underlying production traits and diseases. The primary aim of the research described in this thesis was to investigate the utility of several molecular approaches to identify causative variants underlying a variety of traits in the chicken. The general introduction in chapter 1 provides an overview of the domestication history of the chicken - with a particular focus on commercial chicken breeds - and describes the importance to identify causative variants underlying production traits and diseases. Furthermore, several different molecular techniques and methods are introduced that are being used to detect causative variants underlying monogenic and polygenic traits. Linkage maps are essential for linkage analysis, important to study recombination rates and recombination hotspots within the genome and can assist in the sequence assembly of genomes. In chapter 2 we describe the construction of a new high-resolution linkage map of the chicken genome based on two chicken populations with a total of 1619 individuals. The two populations used are a purebred broiler line and a broiler x broiler cross. This high resolution allowed accurate identification of recombination hotspots in the chicken genome, including sex specific recombination. Furthermore, to improve the current reference genome (WASHUC2), 613 unmapped markers were included in the genome-wide assay that included a total of 17,790 SNPs. The resulting linkage map comprises 13,340 SNPs, of which 360 had not been assigned to a known chromosome on chicken genome build WASHUC2. The resulting linkage map is composed of 31 linkage groups, with a total length of 3,054 cM for the sex-average map of the combined population. Regional differences in recombination hotspots between the two mapping populations were observed for several chromosomes near the telomere of the p arm. The sex-specific analysis revealed that these regional differences were mainly caused by female-specific recombination hotspots in the broiler × broiler cross. In chapter 3 we describe the molecular characterization of the locus causing the late feathering phenotype; a monogenic trait in chicken that results in a delayed emergence of flight feathers at hatch. The late feathering phenotype is beneficial to breeders as it can be used for sex typing at hatch. The locus has, therefore, been extensively used in diverse commercial chicken breeds. However, a retrovirus closely linked to the late feathering allele causes a negative pleiotropic effect on egg production and causes viral infections. Within this chapter we describe the identification of a 180 kb tandem duplication in the late feathering allele using a quantitative PCR approach. The tandem duplication results in the partial duplication of two genes; the prolactin receptor and the gene encoding sperm flagellar protein 2. Sequence analysis revealed that the duplication is identical in broiler, white egg-layer, and brown egg-layer lines. This information was also used to design a molecular test to detect this duplication, particularly in heterozygous individuals. The recent advances in massive parallel sequencing technologies have enabled rapid and cost-effective detection of all genetic variants within genomes. The detection of all genetic variants within a genome has further increased our ability to identify causative variants underlying quantitative trait loci (QTL). In chapter 4, we combined a genome-wide association study with whole-genome resequencing to identify causative variants underlying the pulmonary hypertension syndrome (PHS), a polygenic trait in chicken. PHS is a metabolic disease that has been linked to intense selection on growth rate and feed conversion ratio of modern broilers (meat-type chicken). PHS has become one of the most frequent causes of mortality within the broiler industry and leads to substantial economic losses and reduced animal welfare. In total, 18 QTL regions were identified in the genome-wide association study. In order to detect causative variants underlying these QTL regions, we sequenced the genomes of twelve individuals. To maximize the detection of causative variants we selected the individuals based on extreme phenotypes for PHS. Within 8 QTL regions we identified a total of 10 genes that contain at least one variant that is predicted to affect protein function. Moreover, 7.62 million SNPs were detected within the twelve animals compared to the reference genome. These markers can be used in the development of future genome-wide assays. Genomic regions that have undergone selection should contain loci that influence important phenotypic traits and will, therefore, include causative variant(s) that could aid in further future improvement of production traits and disease resistance. In chapter 5, we applied hitch-hiking mapping to make a broad assessment of the effects of selection histories in domesticated chicken. Towards this end, we sampled commercial chickens representing all major breeding goals from multiple breeding companies. In addition, we sampled non-commercial chicken diversity by sampling almost all recognized traditional Dutch breeds and a representative sample of breeds from China. The broad sample of 67 commercial and non-commercial breeds were assessed for signatures of selection in the genome using information of 57,636 SNPs that were genotyped on pooled DNA samples. Our approach demonstrates the strength of including many different populations with similar, and breed groups with different selection histories to reduce stochastic effects based on single populations. The detection of regions of putative selection resulted in the identification of several candidate genes that could aid in further improvement of production traits and disease resistance. Finally, the general discussion in chapter 6 describes the main findings of this thesis. In this chapter recommendations are given for the best strategies to detect causative variants underlying monogenic or polygenic traits. All strategies can benefit substantially from the recent developments in massive parallel sequencing, although the high costs of this method currently prevent large scale studies. In order to perform powerful and cost-effective studies, several strategies are discussed that combine massive parallel sequencing with other existing methods and techniques. Furthermore, the limitations of the different strategies are addressed, as well as the improvements needed in the near future to identify causative variants underlying a variety of traits in, but not limited to, the chicken.   </p

Differences in treatment of stage I colorectal cancers:A population-based study of colorectal cancers detected within and outside of a screening program

Background:Screen-detected colorectal cancers (CRCs) are often treated less invasively than stage-matched nonscreen-detected CRCs, but the reasons for this are not fully understood. This study evaluated the treatment of stage I CRCs detected within and outside of the screening program in the Netherlands. Methods:Data from the Netherlands Cancer Registry for all stage I CRCs diagnosed between January 1, 2008 and December 31, 2020 were analyzed, comparing patient, tumor, and treatment characteristics of screen-detected and nonscreen-detected stage I CRCs. Multivariable logistic regression was used to assess the association between treatment (local excision only vs. surgical oncologic resection) and patient and tumor characteristics, stratified for T stage and tumor location. Results:Screen-detected stage I CRCs were relatively more often T1 than T2 compared with non-screen-detected stage I CRCs (66.9 % vs. 53.3 %; P 0.001). When only T1 tumors were considered, both screen-detected colon and rectal cancers were more often treated with local excision only than non-screen-detected T1 cancers (odds ratio [OR] 2.19, 95%CI 1.93 2.49; and OR 1.29, 95 %CI 1.05 1.59, respectively), adjusted for sex, tumor location, lymphovascular invasion (LVI) status, and tumor differentiation. Conclusions:Less invasive treatment of screen-detected stage I CRC is partly explained by the higher rate of T1 cancers compared with non-screen-detected stage I CRCs. T1 stage I screen-detected CRCs were also more likely to undergo less invasive treatment than non-screen-detected CRCs, adjusted for risk factors such as LVI and tumor differentiation. Future research should investigate whether the choice of local excision was related to unidentified cancerrelated factors or the expertise of the endoscopists.</p

Trends in incidence, treatment, and relative survival of colorectal cancer in the Netherlands between 2000 and 2021

Background: The epidemiology of colorectal cancer (CRC) has changed rapidly over the years. The aim of this study was to assess the trends in incidence, treatment, and relative survival (RS) of patients diagnosed with CRC in the Netherlands between 2000 and 2021. Patients and methods: 2 75667 patients diagnosed with CRC between 2000 and 2021 were included from the Netherlands Cancer Registry. Analyses were stratified for disease extent (localised: T1-3N0M0; regional: T4N0M0/T1-4N1-2M0; distant: T1-4N0-2M1) and localisation (colon; rectum). Trends were assessed with joinpoint regression. Results: CRC incidence increased until the mid-2010s but decreased strongly thereafter to rates comparable with the early 2000s. Amongst other trend changes, local excision rates increased for patients with localised colon (2021: 13.6 %) and rectal cancer (2021: 34.9 %). Moreover, primary tumour resection became less common in patients with distant colon (2000–2021: 60.9–12.5 %) or rectal cancer (2000–2021: 47.8–6.9 %), while local treatment of metastases rates increased. Five-year RS improved continuously for localised and regional colon (97.7 % and 72.0 % in 2017, respectively) and rectal cancer (95.2 % and 76.3 % in 2017, respectively). The rate of anti-cancer treatments decreased in distant colon (2010–2021: 80.3 % to 67.2 %; p &lt; 0.001) and rectal cancer (2011–2021: 86.0 % to 77.0 %; p &lt; 0.001). The improvement of five-year RS stagnated for distant colon (2010–2017: 11.2 % to 11.9 %; average percentage of change [APC]: 2.1, 95 % confidence interval [CI]: −7.6, 4.7) and rectal cancer (2009–2017: 12.7 % to 15.6 %; APC: 1.4, 95 % CI: −19.1, 5.5). Conclusions: Major changes in the incidence and treatment of CRC between 2000 and 2021 were identified and quantified. Five-year RS increased continuously for patients with localised and regional CRC, but stagnated for patients with distant CRC, likely caused by decreased rates of anti-cancer treatment in this group.</p

Trends in incidence, treatment, and relative survival of colorectal cancer in the Netherlands between 2000 and 2021

Background: The epidemiology of colorectal cancer (CRC) has changed rapidly over the years. The aim of this study was to assess the trends in incidence, treatment, and relative survival (RS) of patients diagnosed with CRC in the Netherlands between 2000 and 2021. Patients and methods: 2 75667 patients diagnosed with CRC between 2000 and 2021 were included from the Netherlands Cancer Registry. Analyses were stratified for disease extent (localised: T1-3N0M0; regional: T4N0M0/T1-4N1-2M0; distant: T1-4N0-2M1) and localisation (colon; rectum). Trends were assessed with joinpoint regression. Results: CRC incidence increased until the mid-2010s but decreased strongly thereafter to rates comparable with the early 2000s. Amongst other trend changes, local excision rates increased for patients with localised colon (2021: 13.6 %) and rectal cancer (2021: 34.9 %). Moreover, primary tumour resection became less common in patients with distant colon (2000–2021: 60.9–12.5 %) or rectal cancer (2000–2021: 47.8–6.9 %), while local treatment of metastases rates increased. Five-year RS improved continuously for localised and regional colon (97.7 % and 72.0 % in 2017, respectively) and rectal cancer (95.2 % and 76.3 % in 2017, respectively). The rate of anti-cancer treatments decreased in distant colon (2010–2021: 80.3 % to 67.2 %; p &lt; 0.001) and rectal cancer (2011–2021: 86.0 % to 77.0 %; p &lt; 0.001). The improvement of five-year RS stagnated for distant colon (2010–2017: 11.2 % to 11.9 %; average percentage of change [APC]: 2.1, 95 % confidence interval [CI]: −7.6, 4.7) and rectal cancer (2009–2017: 12.7 % to 15.6 %; APC: 1.4, 95 % CI: −19.1, 5.5). Conclusions: Major changes in the incidence and treatment of CRC between 2000 and 2021 were identified and quantified. Five-year RS increased continuously for patients with localised and regional CRC, but stagnated for patients with distant CRC, likely caused by decreased rates of anti-cancer treatment in this group.</p

Next-generation sequencing-based genome diagnostics across clinical genetics centers: Implementation choices and their effects

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care

Nationwide comprehensive gastro-intestinal cancer cohorts: the 3P initiative

Background: The increasing sub-classification of cancer patients due to more detailed molecular classification of tumors, and limitations of current trial designs, require innovative research designs. We present the design, governance and current standing of three comprehensive nationwide cohorts including pancreatic, esophageal/gastric, and colorectal cancer patients (NCT02070146). Multidisciplinary collection of clinical data, tumor tissue, blood samples, and patient-reported outcome (PRO) measures with a nationwide coverage, provides the infrastructure for future and novel trial designs and facilitates research to improve outcomes of gastrointestinal cancer patients. Material and methods: All patients aged ≥18 years with pancreatic, esophageal/gastric or colorectal cancer are eligible. Patients provide informed consent for: (1) reuse of clinical data; (2) biobanking of primary tumor tissue; (3) collection of blood samples; (4) to be informed about relevant newly identified genomic aberrations; (5) collection of longitudinal PROs; and (6) to receive information on new interventional studies and possible participation in cohort multiple randomized controlled trials (cmRCT) in the future. Results: In 2015, clinical data of 21,758 newly diagnosed patients were collected in the Netherlands Cancer Registry. Additional clinical data on the surgical procedures were registered in surgical audits for 13,845 patients. Within the first two years, tumor tissue and blood samples were obtained from 1507 patients; during this period, 1180 patients were included in the PRO registry. Response rate for PROs was 90%. The consent rate to receive information on new interventional studies and possible participation in cmRCTs in the future was >85%. The number of hospitals participating in the cohorts is steadily increasing. Conclusion: A comprehensive nationwide multidisciplinary gastrointestinal cancer cohort is feasible and surpasses the limitations of classical study designs. With this initiative, novel and innovative studies can be performed in an efficient, safe, and comprehensive setting

Origin and clinical relevance of chromosomal aberrations other than the common trisomies detected by genome-wide NIPS: Results of the TRIDENT study

PurposeNoninvasive prenatal screening (NIPS) using cell-free DNA in maternal blood is highly sensitive for detecting fetal trisomies 21, 18, and 13. Using a genome-wide approach, other chromosome anomalies can also be detected. We report on the origin

Nationwide comprehensive gastro-intestinal cancer cohorts: the 3P initiative

Background: The increasing sub-classification of cancer patients due to more detailed molecular classification of tumors, and limitations of current trial designs, require innovative research designs. We present the design, governance and current standing of three comprehensive nationwide cohorts including pancreatic, esophageal/gastric, and colorectal cancer patients (NCT02070146). Multidisciplinary collection of clinical data, tumor tissue, blood samples, and patient-reported outcome (PRO) measures with a nationwide coverage, provides the infrastructure for future and novel trial designs and facilitates research to improve outcomes of gastrointestinal cancer patients. Material and methods: All patients aged ≥18 years with pancreatic, esophageal/gastric or colorectal cancer are eligible. Patients provide informed consent for: (1) reuse of clinical data; (2) biobanking of primary tumor tissue; (3) collection of blood samples; (4) to be informed about relevant newly identified genomic aberrations; (5) collection of longitudinal PROs; and (6) to receive information on new interventional studies and possible participation in cohort multiple randomized controlled trials (cmRCT) in the future. Results: In 2015, clinical data of 21,758 newly diagnosed patients were collected in the Netherlands Cancer Registry. Additional clinical data on the surgical procedures were registered in surgical audits for 13,845 patients. Within the first two years, tumor tissue and blood samples were obtained from 1507 patients; during this period, 1180 patients were included in the PRO registry. Response rate for PROs was 90%. The consent rate to receive information on new interventional studies and possible participation in cmRCTs in the future was >85%. The number of hospitals participating in the cohorts is steadily increasing. Conclusion: A comprehensive nationwide multidisciplinary gastrointestinal cancer cohort is feasible and surpasses the limitations of classical study designs. With this initiative, novel and innovative studies can be performed in an efficient, safe, and comprehensive setting