Phenotypic screen of sixty-eight colorectal cancer cell lines identifies CEACAM6 and CEACAM5 as markers of acid resistance.

Elevated cancer metabolism releases lactic acid and CO2 into the under-perfused tumor microenvironment, resulting in extracellular acidosis. The surviving cancer cells must adapt to this selection pressure; thus, targeting tumor acidosis is a rational therapeutic strategy to manage tumor growth. However, none of the major approved treatments are based explicitly on disrupting acid handling, signaling, or adaptations, possibly because the distinction between acid-sensitive and acid-resistant phenotypes is not clear. Here, we report pH-related phenotypes of sixty-eight colorectal cancer (CRC) cell lines by measuring i) extracellular acidification as a readout of acid production by fermentative metabolism and ii) growth of cell biomass over a range of extracellular pH (pHe) levels as a measure of the acid sensitivity of proliferation. Based on these measurements, CRC cell lines were grouped along two dimensions as "acid-sensitive"/"acid-resistant" versus "low metabolic acid production"/"high metabolic acid production." Strikingly, acid resistance was associated with the expression of CEACAM6 and CEACAM5 genes coding for two related cell-adhesion molecules, and among pH-regulating genes, of CA12. CEACAM5/6 protein levels were strongly induced by acidity, with a further induction under hypoxia in a subset of CRC lines. Lack of CEACAM6 (but not of CEACAM5) reduced cell growth and their ability to differentiate. Finally, CEACAM6 levels were strongly increased in human colorectal cancers from stage II and III patients, compared to matched samples from adjacent normal tissues. Thus, CEACAM6 is a marker of acid-resistant clones in colorectal cancer and a potential motif for targeting therapies to acidic regions within the tumors.

Adult stem cell activity in naked mole rats for long-term tissue maintenance.

The naked mole rat (NMR), Heterocephalus glaber, the longest-living rodent, provides a unique opportunity to explore how evolution has shaped adult stem cell (ASC) activity and tissue function with increasing lifespan. Using cumulative BrdU labelling and a quantitative imaging approach to track intestinal ASCs (Lgr5+) in their native in vivo state, we find an expanded pool of Lgr5+ cells in NMRs, and these cells specifically at the crypt base (Lgr5+CBC) exhibit slower division rates compared to those in short-lived mice but have a similar turnover as human LGR5+CBC cells. Instead of entering quiescence (G0), NMR Lgr5+CBC cells reduce their division rates by prolonging arrest in the G1 and/or G2 phases of the cell cycle. Moreover, we also observe a higher proportion of differentiated cells in NMRs that confer enhanced protection and function to the intestinal mucosa which is able to detect any chemical imbalance in the luminal environment efficiently, triggering a robust pro-apoptotic, anti-proliferative response within the stem/progenitor cell zone.

A historical perspective on HLA.

The discovery of the history of the HLA system is reviewed from the earliest attempts at cancer transfers between mice, through the discovery of the mouse H-2 system on mouse red blood cells, the discovery of HLA class II and II antigens by use of sera from multiparous women, to the resolution of the HLA and H-2 functions explained by the attachment of intra cellular peptides to the HLA antigen grooves on the cell surface. The study of the associations between HLA types and diseases forms the basis for the subsequent extensive study of the genetics of human complex disease and phenotypes by GWAS (Genome Wide Association Studies).

Acid-adapted cancer cells alkalinize their cytoplasm by degrading the acid-loading membrane transporter anion exchanger 2, SLC4A2.

Acidic environments reduce the intracellular pH (pHi) of most cells to levels that are sub-optimal for growth and cellular functions. Yet, cancers maintain an alkaline cytoplasm despite low extracellular pH (pHe). Raised pHi is thought to be beneficial for tumor progression and invasiveness. However, the transport mechanisms underpinning this adaptation have not been studied systematically. Here, we characterize the pHe-pHi relationship in 66 colorectal cancer cell lines and identify the acid-loading anion exchanger 2 (AE2, SLC4A2) as a regulator of resting pHi. Cells adapt to chronic extracellular acidosis by degrading AE2 protein, which raises pHi and reduces acid sensitivity of growth. Acidity inhibits mTOR signaling, which stimulates lysosomal function and AE2 degradation, a process reversed by bafilomycin A1. We identify AE2 degradation as a mechanism for maintaining a conducive pHi in tumors. As an adaptive mechanism, inhibiting lysosomal degradation of AE2 is a potential therapeutic target.

mRNA-Lipid Nanoparticle (LNP) Delivery of Humanized EpCAM-CD3 Bispecific Antibody Significantly Blocks Colorectal Cancer Tumor Growth.

The epithelial cell adhesion molecule (EpCAM) is often overexpressed in many types of tumors, including colorectal cancer. We sequenced and humanized an EpCAM mouse antibody and used it to develop bispecific EpCAM-CD3 antibodies. Three different designs were used to generate bispecific antibodies such as EpCAM-CD3 CrossMab knob-in-hole, EpCAM ScFv-CD3 ScFv (BITE), and EpCAM ScFv-CD3 ScFv-human Fc designs. These antibody designs showed strong and specific binding to the EpCAM-positive Lovo cell line and T cells, specifically killed EpCAM-positive Lovo cells and not EpCAM-negative Colo741 cells in the presence of T cells, and increased T cells' IFN-gamma secretion in a dose-dependent manner. In addition, transfection of HEK-293 cells with EpCAM ScFv-CD3 ScFv human Fc mRNA-LNPs resulted in antibody secretion that killed Lovo cells and did not kill EpCAM-negative Colo741 cells. The antibody increased IFN-gamma secretion against Lovo target cells and did not increase it against Colo741 target cells. EpCAM-CD3 hFc mRNA-LNP transfection of several cancer cell lines (A1847, C30, OVCAR-5) also demonstrated functional bispecific antibody secretion. In addition, intratumoral delivery of the EpCAM-CD3 human Fc mRNA-LNPs into OVCAR-5 tumor xenografts combined with intravenous injection of T cells significantly blocked xenograft tumor growth. Thus, EpCAM-CD3 hFc mRNA-LNP delivery to tumor cells shows strong potential for future clinical studies.

Cancer Immunotherapy: Where Next?

The fundamental problem of dealing with cancer is that cancer cells are so like normal cells that it is very hard to find differences that can be a basis for treatment without severe side effects. The key to successful cancer immunotherapy will be based on a very careful choice of cancer targets that are sufficiently cancer specific not to cause serious side effects. There are two fundamentally different ways to deploy the immune system for such cancer treatments. One is to increase the efficacy of the cancer patient's own immune system so that it attacks these differences. This has been achieved by "checkpoint blocking" which is very successful but only with a relatively small proportion of cancers. Secondly, one can produce antibodies, or T cells, whose specificity is directed against proteins expressed differentially in cancers. CART cell treatments have proved very effective for some blood cancers but not so far for common solid tumours. Humanised, unmodified monoclonal antibodies have been used extensively for the treatment of certain adenocarcinomas with modest success. However, using antibodies together with the body's own immune system to treat cancers by engineering monoclonal antibodies that are directed at both a target antigen on the cancer cell surface and also against T cells shows promise for the development of novel immunotherapies. Genes can be found which are expressed highly in some cancers but with a low or absent expression on normal tissues and so are good novel targets. It is so far, only immune-based killing that can kill bystander target negative cells, which is essential for successful treatment since hardly ever will all the cells in a cancer express any desired target. We conclude that, while there still may be many hurdles in the way, engineered bispecific T cell attracting monoclonal antibody-mediated killing of cancer cells may be the most promising approach for achieving novel effective cancer immunotherapies.

The Newfoundland and Labrador mosaic founder population descends from an Irish and British diaspora from 300 years ago.

The founder population of Newfoundland and Labrador (NL) is a unique genetic resource, in part due to its geographic and cultural isolation, where historical records describe a migration of European settlers, primarily from Ireland and England, to NL in the 18th and 19th centuries. Whilst its historical isolation, and increased prevalence of certain monogenic disorders are well appreciated, details of the fine-scale genetic structure and ancestry of the population are lacking. Understanding the genetic origins and background of functional, disease causing, genetic variants would aid genetic mapping efforts in the Province. Here, we leverage dense genome-wide SNP data on 1,807 NL individuals to reveal fine-scale genetic structure in NL that is clustered around coastal communities and correlated with Christian denomination. We show that the majority of NL European ancestry can be traced back to the south-east and south-west of Ireland and England, respectively. We date a substantial population size bottleneck approximately 10-15 generations ago in NL, associated with increased haplotype sharing and autozygosity. Our results reveal insights into the population history of NL and demonstrate evidence of a population conducive to further genetic studies and biomarker discovery.

Two contrasting papers stimulate a commentary on the origins of tumour immunology, current cancer immunotherapies, and the future potential for cancer immunotherapy.

Two early papers expressing conflicting views on the occurrence of effective immune attack against cancers stimulate an analysis of the gradual development of an understanding of tumour biology. This understanding has led to the development of the strikingly effective check point blocking and CART anti-cancer immunotherapies, and the promise of more widely applicable therapies based on T cell attracting genetically engineered monoclonal antibodies.

GMMchi: gene expression clustering using Gaussian mixture modeling.

BACKGROUND: Cancer evolution consists of a stepwise acquisition of genetic and epigenetic changes, which alter the gene expression profiles of cells in a particular tissue and result in phenotypic alterations acted upon by natural selection. The recurrent appearance of specific genetic lesions across individual cancers and cancer types suggests the existence of certain "driver mutations," which likely make up the major contribution to tumors' selective advantages over surrounding normal tissue and as such are responsible for the most consequential aspects of the cancer cells' gene expression patterns and phenotypes. We hypothesize that such mutations are likely to cluster with specific dichotomous shifts in the expression of the genes they most closely control, and propose GMMchi, a Python package that leverages Gaussian Mixture Modeling to detect and characterize bimodal gene expression patterns across cancer samples, as a tool to analyze such correlations using 2 × 2 contingency table statistics. RESULTS: Using well-defined simulated data, we were able to confirm the robust performance of GMMchi, reaching 85% accuracy with a sample size of n = 90. We were also able to demonstrate a few examples of the application of GMMchi with respect to its capacity to characterize background florescent signals in microarray data, filter out uninformative background probe sets, as well as uncover novel genetic interrelationships and tumor characteristics. Our approach to analysing gene expression analysis in cancers provides an additional lens to supplement traditional continuous-valued statistical analysis by maximizing the information that can be gathered from bulk gene expression data. CONCLUSIONS: We confirm that GMMchi robustly and reliably extracts bimodal patterns from both colorectal cancer (CRC) cell line-derived microarray and tumor-derived RNA-Seq data and verify previously reported gene expression correlates of some well-characterized CRC phenotypes. AVAILABILITY: The Python package GMMchi and our cell line microarray data used in this paper is available for downloading on GitHub at https://github.com/jeffliu6068/GMMchi .

Solute exchange through gap junctions lessens the adverse effects of inactivating mutations in metabolite-handling genes.

Growth of cancer cells in vitro can be attenuated by genetically inactivating selected metabolic pathways. However, loss-of-function mutations in metabolic pathways are not negatively selected in human cancers, indicating that these genes are not essential in vivo. We hypothesize that spontaneous mutations in 'metabolic genes' will not necessarily produce functional defects because mutation-bearing cells may be rescued by metabolite exchange with neighboring wild-type cells via gap junctions. Using fluorescent substances to probe intercellular diffusion, we show that colorectal cancer (CRC) cells are coupled by gap junctions assembled from connexins, particularly Cx26. Cells with genetically inactivated components of pH regulation (SLC9A1), glycolysis (ALDOA), or mitochondrial respiration (NDUFS1) could be rescued through access to functional proteins in co-cultured wild-type cells. The effect of diffusive coupling was also observed in co-culture xenografts. Rescue was largely dependent on solute exchange via Cx26 channels, a uniformly and constitutively expressed isoform in CRCs. Due to diffusive coupling, the emergent phenotype is less heterogenous than its genotype, and thus an individual cell should not be considered as the unit under selection, at least for metabolite-handling processes. Our findings can explain why certain loss-of-function mutations in genes ascribed as 'essential' do not influence the growth of human cancers.

Critical roles for EGFR and EGFR-HER2 clusters in EGF binding of SW620 human carcinoma cells.

Epidermal growth factor (EGF) signalling regulates normal epithelial and other cell growth, with EGF receptor (EGFR) overexpression reported in many cancers. However, the role of EGFR clusters in cancer and their dependence on EGF binding is unclear. We present novel single-molecule total internal reflection fluorescence microscopy of (i) EGF and EGFR in living cancer cells, (ii) the action of anti-cancer drugs that separately target EGFR and human EGFR2 (HER2) on these cells and (iii) EGFR-HER2 interactions. We selected human epithelial SW620 carcinoma cells for their low level of native EGFR expression, for stable transfection with fluorescent protein labelled EGFR, and imaged these using single-molecule localization microscopy to quantify receptor architectures and dynamics upon EGF binding. Prior to EGF binding, we observe pre-formed EGFR clusters. Unexpectedly, clusters likely contain both EGFR and HER2, consistent with co-diffusion of EGFR and HER2 observed in a different model CHO-K1 cell line, whose stoichiometry increases following EGF binding. We observe a mean EGFR : EGF stoichiometry of approximately 4 : 1 for plasma membrane-colocalized EGFR-EGF that we can explain using novel time-dependent kinetics modelling, indicating preferential ligand binding to monomers. Our results may inform future cancer drug developments.

Sequencing of autosomal, mitochondrial and Y-chromosomal forensic markers in the People of the British Isles cohort detects population structure dominated by patrilineages.

Short tandem repeat (STR) polymorphisms are traditionally assessed by measuring allele lengths via capillary electrophoresis (CE). Massively parallel sequencing (MPS) reveals differences among alleles of the same length, thus improving discrimination, but also identifying groups of alleles likely related by descent. These may have relatively restricted geographical distributions and thus MPS could detect population structure more effectively than CE-based analysis. We addressed this question by applying an MPS multiplex, the Promega PowerSeq™ Auto/Mito/Y System prototype, to 362 individuals chosen to represent a wide geographical spread from the People of the British Isles (PoBI) cohort, which represents at least three generations of local rural ancestry. As well as 22 autosomal STRs (aSTRs; equivalent to PowerPlex Fusion loci) the system sequences 23 Y-STRs (the PowerPlexY 23 loci) and the control region (CR) of mitochondrial DNA (mtDNA), allowing population structure to be compared across biparentally and uniparentally inherited segments of the genome. For all loci, FST-based tests of population structure were done based on historical, linguistic, and geographical partitions, and for aSTRs the clustering algorithm STRUCTURE was also applied. STRs were considered using both length and sequence. Sequencing increased aSTR allele diversity by 87.5% compared to CE-based designations, reducing random match probability to 1.25E-30, compared to a CE-based 6.72E-27. Significant population structure was detectable in just one pairwise comparison (Central/South East England compared to the rest), and for sequence-based alleles only. The 362 samples carried 308 distinct mtDNA CR haplotypes corresponding to 13 broad haplogroups, representing a haplotype diversity of 0.9985 ( ± 0.0005), and a haplotype match probability of 0.0043. No significant population structure was observed. Y-STR haplotypes belonged to ten broad predicted Y-haplogroups. Allele diversity increased by 33% when considered at the sequence rather than length level, although haplotype diversity was unchanged at 0.999969 ( ± 0.000001); haplotype match probability was 2.79E-03. In contrast to the biparentally and maternally inherited loci, Y-STR haplotypes showed significant population structure at several levels, but most markedly in a comparison of regions subject to Anglo-Saxon influence in the east with the rest of the sample. This was evident for both length- and sequence-based allele designations, with no systematic difference between the two. We conclude that MPS analysis of aSTRs or Y-STRs does not generally reveal stronger population structure than length-based analysis, that UK maternal lineages are not significantly structured, and that Y-STR haplotypes reveal significant population structure that may reflect the Anglo-Saxon migrations to Britain in the 6th century.

CRISPR-Cas9 screen identifies oxidative phosphorylation as essential for cancer cell survival at low extracellular pH.

Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumors. Genes that facilitate survival under acid stress are therefore potential targets for cancer therapies. We performed a genome-wide CRISPR-Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1-/- cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy that lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option.

Genomic landscape of colorectal carcinogenesis.

PURPOSE: The molecular pathogenesis of solid tumour was first assessed in colorectal cancer (CRC). To date, ≤ 100 genes with somatic alterations have been found to inter-connectively promote neoplastic transformation through specific pathways. The process of colorectal carcinogenesis via genome landscape is reviewed on the basis of an adenoma-to-carcinoma sequence, as shown by serial histological and epidemiological observations. METHODS: The relevant literatures from PubMed (1980-2021) have been reviewed for this article. RESULTS: The major routes of CRC development, chromosomal instability (CIN), microsatellite instability (MSI), and the serrated route either via CIN or MSI, proceed through the respective molecular pathway of colorectal carcinogenesis. Particular aspects of CRC carcinogenesis can also be determined by evaluating familial CRCs (FCRC) and genotype-phenotype correlations. Specific causative gene alterations still leave to be identified in several FCRCs. Otherwise, recently verified FCRC can be particularly notable, for example, EPCAM-associated Lynch syndrome, polymerase proofreading-associated polyposis, RNF43-associated polyposis syndrome or NTHL1 tumour syndrome, and hereditary mixed polyposis syndrome. The oncogenic landscape is described, including representative pathway genes, the three routes of carcinogenesis, familial CRCs, genotype-phenotype correlations, the identification of causative genes, and consensus molecular subtypes (CMS). CONCLUSION: Whole genome research using multi-gene panels (MGPs) has facilitated high through-put detection of previously unidentified genes involved in colorectal carcinogenesis. New approaches designed to identify rare variants are recommended to consider their alterations implicated in the molecular pathogenesis.

Genotypic and Phenotypic Characteristics of Hereditary Colorectal Cancer.

The genomic causes and clinical manifestations of hereditary colorectal cancer (HCRC) might be stratified into 2 groups, namely, familial (FCRC) and a limited sense of HCRC, respectively. Otherwise, FCRC is canonically classified into 2 major categories; Lynch syndrome (LS) or associated spectra and inherited polyposis syndrome. By contrast, despite an increasing body of genotypic and phenotypic traits, some FCRC cannot be clearly differentiated as definitively single type, and the situation has become more complex as additional causative genes have been discovered. This review provides an overview of HCRC, including 6 LS or associated spectra and 8 inherited polyposis syndromes, according to molecular pathogenesis. Variants and newly-identified FCRC are particularly emphasized, including MUTYH (or MYH)-associated polyposis, Muir-Torre syndrome, constitutional mismatch repair deficiency, EPCAM-associated LS, polymerase proofreading-associated polyposis, RNF43- or NTHL1-associated serrated polyposis syndrome, PTEN hamartoma tumor syndrome, and hereditary mixed polyposis syndrome. We also comment on the clinical utility of multigene panel tests, focusing on comprehensive cancer panels that include HCRC. Finally, HCRC surveillance strategies are recommended, based on revised or notable concepts underpinned by competent validation and clinical implications, and favoring major guidelines. As hereditary syndromes are mainly attributable to genomic constitutions of distinctive ancestral groups, an integrative national HCRC registry and guideline is an urgent priority.

A novel xenonucleic acid-mediated molecular clamping technology for early colorectal cancer screening.

BACKGROUND: Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Early detection is critical to reduce CRC morbidity and mortality. In order to meet this need, we developed a molecular clamping assay called the ColoScape TM assay for early colorectal cancer diagnostics. METHODS: Nineteen mutations in four genes (APC, KRAS, BRAF and CTNNB1) associated with early events in CRC pathogenesis are targeted in the ColoScapeTM assay. Xenonucleic Acid (XNA)-mediated qPCR clamping technology was applied to minimize the wild-type background amplification in order to improve assay sensitivity of CRC mutation detection. The assay analytical performance was verified and validated, cfDNA and FFPE CRC patient samples were evaluated, and an ROC curve was applied to evaluate its performance. RESULTS: The data showed that the assay analytical sensitivity was 0.5% Variant Allele Frequency, corresponding to ~7-8 copies of mutant DNA with 5 ng total DNA input per test. This assay is highly reproducible with intra-assay CV of <3% and inter-assay CV of <5%. We have investigated 380 clinical samples including plasma cfDNA and FFPE samples from patients with precancerous and different stages of CRC. The preliminary assay clinical specificity and sensitivity for CRC cfDNA were: 100% (95% CI, 80.3-97.5%) and 92.2% (95% CI, 94.7-100%), respectively, with AUC of 0.96; 96% specificity (95% CI, 77.6-99.7%) and 92% sensitivity (95% CI, 86.1-95.6%) with AUC of 0.94 for CRC FFPE; 95% specificity (95% CI, 82.5%-99.1%) and 62.5% sensitivity (95% CI, 35.8%-83.7%) with AUC of 0.79 for precancerous lesions cfDNA. CONCLUSIONS: The XNA-mediated molecular clamping assay is a rapid, precise, and sensitive assay for the detection of precancerous lesions cfDNA and CRC cfDNA or FFPE samples.

Subdividing Y-chromosome haplogroup R1a1 reveals Norse Viking dispersal lineages in Britain.

The influence of Viking-Age migrants to the British Isles is obvious in archaeological and place-names evidence, but their demographic impact has been unclear. Autosomal genetic analyses support Norse Viking contributions to parts of Britain, but show no signal corresponding to the Danelaw, the region under Scandinavian administrative control from the ninth to eleventh centuries. Y-chromosome haplogroup R1a1 has been considered as a possible marker for Viking migrations because of its high frequency in peninsular Scandinavia (Norway and Sweden). Here we select ten Y-SNPs to discriminate informatively among hg R1a1 sub-haplogroups in Europe, analyse these in 619 hg R1a1 Y chromosomes including 163 from the British Isles, and also type 23 short-tandem repeats (Y-STRs) to assess internal diversity. We find three specifically Western-European sub-haplogroups, two of which predominate in Norway and Sweden, and are also found in Britain; star-like features in the STR networks of these lineages indicate histories of expansion. We ask whether geographical distributions of hg R1a1 overall, and of the two sub-lineages in particular, correlate with regions of Scandinavian influence within Britain. Neither shows any frequency difference between regions that have higher (≥10%) or lower autosomal contributions from Norway and Sweden, but both are significantly overrepresented in the region corresponding to the Danelaw. These differences between autosomal and Y-chromosomal histories suggest either male-specific contribution, or the influence of patrilocality. Comparison of modern DNA with recently available ancient DNA data supports the interpretation that two sub-lineages of hg R1a1 spread with the Vikings from peninsular Scandinavia.

In Vitro Analyses of Interactions Between Colonic Myofibroblasts and Colorectal Cancer Cells for Anticancer Study.

BACKGROUND/AIM: Interactions between colorectal cancer (CRC) cells and myofibroblasts govern many processes such as cell growth, migration, invasion and differentiation, and contribute to CRC progression. Robust experimental tests are needed to investigate the nature of these interactions for future anticancer studies. The purpose of the study was to design and validate in vitro assays for studying the communication between myofibroblasts and CRC epithelial cell lines. MATERIALS AND METHODS: The influence of co-culture of myofibroblasts and CRC cell lines is discussed using various in vitro assays including direct co-culture, transwell assays, Matrigel-based differentiation and cell invasion experiments. RESULTS: The results from these in vitro assays clearly demonstrated various aspects of the crosstalk between myofibroblasts and CRC cell lines, which include cell growth, differentiation, migration and invasion. CONCLUSION: The reported in vitro assays provide a basis for investigating the factors that control the myofibroblast-epithelial cell interactions in CRC in vivo.

Polygenic inheritance, GWAS, polygenic risk scores, and the search for functional variants.

The reconciliation between Mendelian inheritance of discrete traits and the genetically based correlation between relatives for quantitative traits was Fisher's infinitesimal model of a large number of genetic variants, each with very small effects, whose causal effects could not be individually identified. The development of genome-wide genetic association studies (GWAS) raised the hope that it would be possible to identify single polymorphic variants with identifiable functional effects on complex traits. It soon became clear that, with larger and larger GWAS on more and more complex traits, most of the significant associations had such small effects, that identifying their individual functional effects was essentially hopeless. Polygenic risk scores that provide an overall estimate of the genetic propensity to a trait at the individual level have been developed using GWAS data. These provide useful identification of groups of individuals with substantially increased risks, which can lead to recommendations of medical treatments or behavioral modifications to reduce risks. However, each such claim will require extensive investigation to justify its practical application. The challenge now is to use limited genetic association studies to find individually identifiable variants of significant functional effect that can help to understand the molecular basis of complex diseases and traits, and so lead to improved disease prevention and treatment. This can best be achieved by 1) the study of rare variants, often chosen by careful candidate assessment, and 2) the careful choice of phenotypes, often extremes of a quantitative variable, or traits with relatively high heritability.