12-Year Trends of Orofacial Clefts in the United States: Highlighting Racial/Ethnic Differences in Prevalence of Cleft Lip and Cleft Palate.

OBJECTIVE: Discrepancies in prevalence among infants with orofacial clefts are public health research priorities. Our objective was to calculate updated estimated prevalence of orofacial clefts in the United States, with sub-analyses by racial/ethnic group. DESIGN: The National Birth Defect Prevention Network database was used to evaluate trends in cases with orofacial cleft in the United States from 2006 to 2018. Cases with cleft lip with and without cleft palate (CL ± P) and cleft palate (CP) alone were sub-stratified by racial/ethnic category. Estimated prevalence was calculated using the total live births reported in each maternal racial/ethnic group. The odds ratio (OR) was calculated to measure the strength of association between racial/ethnic group and risk of orofacial clefts. RESULTS: Estimated prevalence rates show that maternally-reported Native American/Alaskan Native individuals were 43.8% (p < 0.0001) and 36.0% (p < 0.0001) more likely to have CL ± P and CP alone, respectively, compared to maternally-reported non-Hispanic White individuals. Estimated prevalence of CL ± P in maternally-reported non-Hispanic Black individuals (OR = 0.64) and maternally-reported Asians/Pacific Islander individuals were significantly lower than in maternally-reported non-Hispanic White individuals (OR = 0.63, p < 0.0001). Estimated prevalence of CP alone was significantly lower in maternally-reported non-Hispanic Black individuals (OR = 0.64, p < 0.0001), maternally-reported Asians/Pacific Islander individuals (OR = 0.69, p < 0.0001), and maternally-reported Hispanic individuals (OR = 0.81, p < 0.0001). CONCLUSIONS: Across the total population, there was no significant change in estimated orofacial cleft prevalence. However, there were significant disproportions in estimated orofacial cleft prevalence across racial/ethnic groups, which may guide further discussion among craniofacial health care providers and centers and their patients regarding differences in cleft risk factors.

Acellular Dermal Matrix Cover Improves Skin Growth during Tissue Expansion by Affecting Distribution of Mechanical Forces.

BACKGROUND: Biological cover over tissue expander prostheses has been introduced to provide soft-tissue support for tissue expanders during breast reconstruction. However, its impact on mechanically induced skin growth remains unknown. This study investigates the hypothesis that covering the tissue expander with acellular dermal matrix (ADM) affects mechanotransduction without compromising the efficacy of tissue expansion. METHODS: Tissue expansion, with and without use of ADM, was performed on a porcine model. The tissue expanders were inflated twice with 45 mL of saline, and the full-thickness skin biopsy specimens were harvested from expanded and control unexpanded skin 1 week and 8 weeks after the final inflation. Histologic evaluation, immunohistochemistry staining, and gene expression analysis were performed. Skin growth and total deformation were evaluated using isogeometric analysis. RESULTS: The authors' results demonstrate that use of ADM as a biological cover during tissue expansion does not impede mechanotransduction that leads to skin growth and blood vessel formation. Isogeometric analysis revealed similar total deformation and growth of expanded skin with and without a biological cover, confirming that its use does not inhibit mechanically induced skin growth. In addition, the authors found that use of an ADM cover results in more uniform distribution of mechanical forces applied by the tissue expander. CONCLUSIONS: These results suggest that ADM improves mechanically induced skin growth during tissue expansion by facilitating a more uniform distribution of mechanical forces applied by the tissue expander. Therefore, the use of a biological cover has potential to improve outcomes in tissue expansion-based reconstruction.

Drop-off in axonal regeneration along the length of a cross-face nerve graft: An experimental study in rats.

INTRODUCTION: The average nerve graft length utilized in cross-face nerve grafting for reconstruction of facial nerve palsy is 20-22 cm. While the graft length is thought to be one of the greatest determinants of muscle strength, the mechanism through which this happens remains unknown. We studied changes in axonal regeneration along the length of a 2 cm cross-face nerve graft in a rat model. The hypothesis was that axon count would decrease along the length of the graft. METHODS: A 2 cm nerve graft (sciatic nerve) was used as a cross-face nerve graft in 16 adult female, 210-250 g, Sprague Dawley rats. Thirteen weeks later, 5 mm nerve biopsies were taken at four sites: the facial nerve trunk (control), proximal graft, midpoint of graft (1 cm distal to coaptation) and distal graft (2 cm distal to coaptation). Retrograde nerve labeling with FluoroGold was performed at the biopsied nerve site and the facial motor nucleus was taken 1 week later. Microscopic imaging and manual counting of axons and labeled motor nuclei was performed. RESULTS: Retrograde-labeled motor neuron counts were decreased at the midway point of the graft compared to the facial trunk (1517 ± 335 axons, Δ% = 92.5, p = .01) and even further decreased at the distal end of the graft (269 ± 293 axons, Δ% = 175.5, p = .006). Analysis of the nerve biopsies demonstrated no significant differences in myelinated axon count between the nerve trunk and over the length of the nerve graft (range 6207-7179 axons, Δ% = 14.5, p = .07). CONCLUSION: In a rat model, the number of regenerating motor neurons drops off along the length of the graft and axon count is preserved due to axon sprouting. How this pattern correlates to ultimate muscle strength remains unknown, but this study provides insight into why shorter grafts may afford better outcomes.

Biological Cover Mitigates Disruption of the Dermal Structure in Mechanically Expanded Skin in a Porcine Model.

Tissue expansion is an integral procedure of the vast majority of breast reconstruction and has a significant impact on the final clinical outcomes. Therefore, technological advances leading to a fewer number of unfavorable outcomes and a decrease in complication rates are imperative. In this study, using a porcine model, we investigated an effect of acellular dermal matrix (ADM) used as a tissue expander cover on the dermal changes induced by mechanical forces during tissue expansion. After 14 days of expansion, skin samples were collected from one animal, while the second animal underwent radiation, and tissue was collected 8 weeks later. Tissue expanded without the use of ADM and unexpanded skin served as the controls. Collected skin biopsies were used for histological and immunohistochemical evaluation, and for gene expression analysis. We revealed that the biological cover incorporation into host tissue is facilitated by macrophages without inducing a broad inflammatory response. The utilization of ADM mitigated disruption in the dermal structure, excessive collagen deposition, and capsule formation in non-irradiated expanded skin. The protective effect was not fully maintained in irradiated skin. These results demonstrate that tissue expansion might be improved by using the tissue expander cover.

Langerhans cells and SFRP2/Wnt/beta-catenin signalling control adaptation of skin epidermis to mechanical stretching.

Skin can be mechanically stimulated to grow through a clinical procedure called tissue expansion (TE). Using a porcine TE model, we determined that expansion promptly activates transcription of SFRP2 in skin and we revealed that in the epidermis, this protein is secreted by Langerhans cells (LCs). Similar to well-known mechanosensitive genes, the increase in SFRP2 expression was proportional to the magnitude of tension, showing a spike at the apex of the expanded skin. This implies that SFRP2 might be a newly discovered effector of mechanotransduction pathways. In addition, we found that acute stretching induces accumulation of b-catenin in the nuclei of basal keratinocytes (KCs) and LCs, indicating Wnt signalling activation, followed by cell proliferation. Moreover, TE-activated LCs proliferate and migrate into the suprabasal layer of skin, suggesting that LCs rebuild their steady network within the growing epidermis. We demonstrated that in vitro hrSFRP2 treatment on KCs inhibits Wnt/b-catenin signalling and stimulates KC differentiation. In parallel, we observed an accumulation of KRT10 in vivo in the expanded skin, pointing to TE-induced, SFRP2-augmented KC maturation. Overall, our results reveal that a network of LCs delivers SFRP2 across the epidermis to fine-tune Wnt/b-catenin signalling to restore epidermal homeostasis disrupted by TE.

Bayesian calibration of a computational model of tissue expansion based on a porcine animal model.

Tissue expansion is a technique used clinically to grow skin in situ to correct large defects. Despite its enormous potential, lack of fundamental knowledge of skin adaptation to mechanical cues, and lack of predictive computational models limit the broader adoption and efficacy of tissue expansion. In our previous work, we introduced a finite element model of tissue expansion that predicted key patterns of strain and growth which were then confirmed by our porcine animal model. Here we use the data from a new set of experiments to calibrate the computational model within a Bayesian framework. Four 10×10cm2 patches were tattooed in the dorsal skin of four 12 weeks-old minipigs and a total of six patches underwent successful tissue expander placement and inflation to 60cc for expansion times ranging from 1 h to 7 days. Six patches that did not have expanders implanted served as controls for the analysis. We find that growth can be explained based on the elastic deformation. The predicted area growth rate is k∈[0.02,0.08] [h-1]. Growth is anisotropic and reflects the anisotropic mechanical behavior of porcine dorsal skin. The rostral-caudal axis shows greater deformation than the transverse axis, and the time scale of growth in the rostral-caudal direction is given by rate parameters k1∈[0.04,0.1] [h-1] compared to k2∈[0.01,0.05] [h-1] in the transverse direction. Moreover, the calibration results underscore the high variability in biological systems, and the need to create probabilistic computational models to predict tissue adaptation in realistic settings. STATEMENT OF SIGNIFICANCE: Tissue expansion is a widely used technique in reconstructive surgery because it triggers growth of skin for the correction of large skin lesions and for breast reconstruction after mastectomy. Despite of its potential, complications and undesired outcomes persist due to our incomplete understanding of skin mechanobiology. Here we quantify the deformation and growth fields induced by an expander over 7 days in a porcine animal model and use these data to calibrate a computational model of skin growth using finite element simulations and a Bayesian framework. The calibrated model is a leap forward in our understanding skin growth, we now have quantitative understanding of this process: area growth is anisotropic and it is proportional to stretch with a characteristic rate constant of k∈[0.02,0.08] [h-1].

Transcriptomic analysis reveals dynamic molecular changes in skin induced by mechanical forces secondary to tissue expansion.

Tissue expansion procedures (TE) utilize mechanical forces to induce skin growth and regeneration. While the impact of quick mechanical stimulation on molecular changes in cells has been studied extensively, there is a clear gap in knowledge about sequential biological processes activated during long-term stimulation of skin in vivo. Here, we present the first genome-wide study of transcriptional changes in skin during TE, starting from 1 h to 7 days of expansion. Our results indicate that mechanical forces from a tissue expander induce broad molecular changes in gene expression, and that these changes are time-dependent. We revealed hierarchical changes in skin cell biology, including activation of an immune response, a switch in cell metabolism and processes related to muscle contraction and cytoskeleton organization. In addition to known mechanoresponsive genes (TNC, MMPs), we have identified novel candidate genes (SFRP2, SPP1, CCR1, C2, MSR1, C4A, PLA2G2F, HBB), which might play crucial roles in stretched-induced skin growth. Understanding which biological processes are affected by mechanical forces in TE is important for the development of skin treatments to maximize the efficacy and minimize the risk of complications during expansion procedures.

Modeling Tissue Expansion with Isogeometric Analysis: Skin Growth and Tissue Level Changes in the Porcine Model.

BACKGROUND: Tissue expansion relies on the ability of skin to grow in response to sustained mechanical strain. This study focuses on correlation of cellular and histologic changes with skin growth and deformation during tissue expansion. METHODS: Tissue expanders were placed underneath the skin of five Yucatan minipigs and inflated with one fill of 60 cc of saline 1 hour, 24 hours, 3 days, and 7 days before the animals were killed, or two fills of either 30 cc or 60 cc at 10 and 3 days or 14 and 7 days before the animals were killed. Skin biopsy specimens and three-dimensional photographs were used to calculate skin growth and stretch according to the authors' novel finite element analysis model. RESULTS: The mitotic index of keratinocytes in the basal layer increased 1 hour after stimulus was applied (4 percent) (p = 0.022), peaked at approximately day 3 (26 percent) (p < 0.0001), and tapered by day 7 (12.5 percent) (p = 0.012) after tissue expansion. The authors demonstrated that it is the volume per fill rather than the total volume in the expander that scales the magnitude of response. Lastly, the authors demonstrated that the ratio of deformation attributable to growth versus stretch (Fgrowth/Fstretch) after 60 cc of tissue expansion fill was 1.03 at 1 hour, 0.82 at 1 day, 0.85 at day 3, and 0.95 at 7 days. CONCLUSIONS: Peak cell proliferation occurred 3 days after tissue expansion fill and is scaled in response to stimulus magnitude. The growth component of deformation equilibrates to the stretch component at day 7, as cell proliferation has started to translate to skin growth.

Mechanical stretching stimulates growth of the basal layer and rete ridges in the epidermis.

We have developed four experimental models of mechanical stimulation applied to the back skin using tissue expansion (TE) procedure performed on minipigs. The technique is used by plastic surgeons for decades, to amend the congenital or accidental skin defects, though underlying changes in epidermis are not well understood. We found that the initial stretching increased proliferation of basal keratinocytes leading to elongation of the basal layer, and increased cellular density. The increased number of the rete ridges, suggests that they absorbed the impact of excessive proliferation, preserving layered organization of epidermis. We found ß1 integrin to be a very sensitive responder to stimulation instigated by TE procedure, able to dynamically relocate to adjust the basal cell against external force. Repeated mechanical stimulation with a seven-day interval generated healthy tissue without detrimental effects. Given the similarities between the structure of the porcine and human epidermis, we speculate that a similar mechanism functions in human skin. A better understanding of the underlying process could help improve medical care and outcomes for patients undergoing surgical reconstruction.

Improving tissue expansion protocols through computational modeling.

Tissue expansion is a common technique in reconstructive surgery used to grow skin in vivo for correction of large defects. Despite its popularity, there is a lack of quantitative understanding of how stretch leads to growth of new skin. This has resulted in several arbitrary expansion protocols that rely on the surgeon's personal training and experience rather than on accurate predictive models. For example, choosing between slow or rapid expansion, or small or large inflation volumes remains controversial. Here we explore four tissue expansion protocols by systematically varying the inflation volume and the protocol duration in a porcine model. The quantitative analysis combines three-dimensional photography, isogeometric kinematics, and finite growth theory. Strikingly, all four protocols generate similar peak stretches, but different growth patterns: Smaller filling volumes of 30 ml per inflation did not result in notable expander-induced growth neither for the short nor for the long protocol; larger filling volumes of 60 ml per inflation trigger skin adaptation, with larger expander-induced growth in regions of larger stretch, and more expander-induced growth for the 14-day compared to the 10-day expansion protocol. Our results suggest that expander-induced growth is not triggered by the local stretch alone. While stretch is clearly a driver for growth, the local stretch at a given point is not enough to predict the expander-induced growth at that location. From a clinical perspective, our study suggests that longer expansion protocols are needed to ensure sufficient growth of sizable skin patches.

The expression of fgfr3 in the zebrafish head.

Fibroblast growth factor (FGF) signaling is essential for many developmental processes and plays a pivotal role in skeletal homeostasis, regeneration and wound healing. FGF signals through one of five tyrosine kinase receptors: Fgfr1a, -1b, -2, -3, -4. To characterize the expression of zebrafish fgfr3 from the larval stage to adulthood, we used RNAscope in situ hybridization on paraffin sections of the zebrafish head. Our study revealed spatial and temporal distribution of fgfr3 transcript in chondrocytes of the head cartilages, osteoblasts involved in bone formation, ventricular zone of the brain, undifferentiated mesenchymal cells of the skin, and lens epithelium of the eye. In general, the expression pattern of zebrafish fgfr3 is similar to the expression observed in higher vertebrates.

Digital analysis yields more reliable and accurate measures of dermal and epidermal thickness in histologically processed specimens compared to traditional methods.

Changes in the thickness of the dermis and epidermis have been described in the scenario of tissue expansion as well as inflammatory skin processes (psoriasis, contact hypersensitivity and so on). These changes have previously been quantified using ocular micrometers to obtain and then average a limited number of spot measurements, leading to suboptimal accuracy. We describe a rapid method of using freely available ImageJ software to analyze digitized images of fixed skin specimens. By determining the cross-sectional area and surface length of a skin layer, a simple calculation produces more accurate and reproducible measurements of its thickness compared to historical methods, with excellent inter-rater reliability.

The role of prospero homeobox 1 (PROX1) expression in follicular thyroid carcinoma cells.

The prospero homeobox 1 (Prox1) transcription factor is a key player during embryogenesis and lymphangiogenesis. Altered Prox1 expression has been found in a variety of human cancers, including papillary thyroid carcinoma (PTC). Interestingly, Prox1 may exert tumor suppressive or tumor promoting effect, depending on the tissue context. In this study, we have analyzed Prox1 expression in normal and malignant human thyroid carcinoma cell lines. Moreover, we determined the effect of Prox1 silencing and overexpression on the cellular processes associated with the metastatic potential of tumor cells: proliferation, migration, invasion, apoptosis and anchorage-independent growth, in the follicular thyroid carcinoma (FTC) FTC-133 cell line. We found that Prox1 expression was significantly higher in FTC-derived cells than in PTC-derived cells and normal thyroid, and it was associated with the PI3K/Akt signaling pathway. In the FTC-133 cells, it was associated with cell invasive potential, motility and wound closure capacities, but not with proliferation or apoptosis. Modifying Prox1 expression also induced substantial changes in the cytoskeleton structure and cell morphology. In conclusion, we have shown that Prox1 plays an important role in the development of FTC and that its suppression prevents, whereas its overexpression promotes, the malignant behavior of thyroid follicular cancer cells.

Obesity increases histone H3 lysine 9 and 18 acetylation at Tnfa and Ccl2 genes in mouse liver.

Obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD), which is characterized by the upregulated expression of two key inflammatory mediators: tumor necrosis factor (Tnfa) and monocyte chemotactic protein 1 (Mcp1; also known as Ccl2). However, the chromatin make-up at these genes in the liver in obese individuals has not been explored. In this study, to identify obesity-mediated epigenetic changes at Tnfa and Ccl2, we used a murine model of obesity induced by a high-fat diet (HFD) and hyperphagic (ob/ob) mice. Chromatin immunoprecipitation (ChIP) assay was used to determine the abundance of permissive histone marks, namely histone H3 lysine 9 and 18 acetylation (H3K9/K18Ac), H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 36 trimethylation (H3K36me3), in conjunction with polymerase 2 RNA (Pol2) and nuclear factor (Nf)-κB recruitment in the liver. Additionally, to correlate the liver tissue-derived ChIP measurements with a robust in vitro transcriptional response at the Tnfa and Ccl2 genes, we used lipopolysaccharide (LPS) treatment to induce an inflammatory response in Hepa1-6 cells, a cell line derived from murine hepatocytes. ChIP revealed increased H3K9/K18Ac at Tnfa and Ccl2 in the obese mice, although the differences were only statistically significant for Tnfa (p<0.05). Unexpectedly, the levels of H3K4me3 and H3K36me3 marks, as well as Pol2 and Nf-κB recruitment, did not correspond with the increased expression of these two genes in the obese mice. By contrast, the acute treatment of Hepa1-6 cells with LPS significantly increased the H3K9/K18Ac marks, as well as Pol2 and Nf-κB recruitment at both genes, while the levels of H3K4me3 and H3K36me3 marks remained unaltered. These results demonstrate that increased Tnfa and Ccl2 expression in fatty liver at the chromatin level corresponds to changes in the level of histone H3 acetylation.

Extracellular matrix and cytochrome P450 gene expression can distinguish steatohepatitis from steatosis in mice.

One of the main questions regarding nonalcoholic fatty liver disease is the molecular background of the transition from simple steatosis (SS) to the inflammatory and fibrogenic condition of steatohepatitis (NASH). We examined the gene expression changes during progression from histologically normal liver to SS and NASH in models of obesity caused by hyperphagia or a high-fat diet. Microarray-based analysis revealed that the expression of 1445 and 264 probe sets was changed exclusively in SS and NASH samples, respectively, and 1577 probe sets were commonly altered in SS and NASH samples. Functional annotations indicated that transcriptome alterations that were common for NASH and SS, as well as exclusive for NASH, involved extracellular matrix (ECM)-related processes, although they differed in the type of matrix structure change. The expression of 80 genes was significantly changed in all three comparisons: SS versus control, NASH versus control and NASH versus SS. Of these genes, epithelial membrane protein 1, IKBKB interacting protein and decorin were progressively up-regulated in both SS and NASH compared to normal tissue. The molecular context of interactions of encoded 80 proteins revealed that they are highly interconnected and significantly enriched for processes involving metabolism by cytochrome P450. Validation of 10 selected mRNAs encoding genes related to ECM and cytochrome P450 with quantitative RT-PCR analysis showed consistent changes in their expression during NASH development. The expression profile of these genes has the potential to distinguish NASH from SS and normal tissue and may possibly be beneficial in the clinical diagnosis of NASH.

Mitochondrial-related proteomic changes during obesity and fasting in mice are greater in the liver than skeletal muscles.

Although mitochondrial dysfunction is implicated in the pathogenesis of obesity, the molecular mechanisms underlying obesity-related metabolic abnormalities are not well established. We performed mitochondrial quantitative proteomic and whole transcriptome analysis followed by functional annotations within liver and skeletal muscles, using fasted and non-fasted 16- and 48-week-old high-fat diet (HFD)-fed and normal diet-fed (control group) wild-type C56BL/6J mice, and hyperphagic ob/ob and db/db obese mice. Our study identified 1,675 and 704 mitochondria-associated proteins with at least two peptides in liver and muscle, respectively. Of these, 221 liver and 44 muscle proteins were differentially expressed (adjusted p values ≤ 0.05) between control and all obese mice, while overnight fasting altered expression of 107 liver and 35 muscle proteins. In the liver, we distinguished a network of 27 proteins exhibiting opposite direction of expression changes in HFD-fed and hyperphagic mice when compared to control. The network centered on cytochromes P450 3a11 (Cyp3a11) and 4a14 (Cyp4a14), and fructose-bisphosphate aldolase B (Aldob) proteins which bridged proteins cluster involved in Metabolism of xenobiotics with proteins engaged in Fatty acid metabolism and PPAR signaling pathways. Functional annotations revealed that most of the hepatic molecular alterations, which characterized both obesity and fasting, related to different aspects of energy metabolism (such as Fatty acid metabolism, Peroxisome, and PPAR signaling); however, only a limited number of functional annotations could be selected from skeletal muscle data sets. Thus, our comprehensive molecular overview revealed that both obesity and fasting states induce more pronounced mitochondrial proteome changes in the liver than in the muscles.

Common low-penetrance risk variants associated with breast cancer in Polish women.

BACKGROUND: Breast cancer is the most common type of cancer and the second leading cause of cancer-death among women in Poland. The known high-risk mutations account for 25% of familial aggregation cases and 5% of total breast cancer predisposition. Genome-wide association studies have identified a number of common low-penetrance genetic variants, but their contribution to disease risk differs between populations. METHODS: To verify selected associations with breast cancer susceptibility among Polish women, the replication study was performed, included 1424 women with breast cancer and 1788 healthy persons. Sixteen single-nucleotide polymorphisms (SNPs) were analyzed using TaqMan SNP Genotyping Assays. Allele frequency differences were tested using chi2-test implemented in PLINK v1.07 and Cochran-Armitage trend test was performed using R software. RESULTS: Significant differences (Bonferroni corrected p-valuecor ≤ 0.0197) in the frequency of alleles distribution between all cancer and control subjects were observed for four (rs2736098, rs13281615, rs1219648, rs2981582) out of 16 SNPs. The same result was obtained for group of patients without high-risk BRCA1/2 mutations. The rs1219648 (p-valuecor ≤ 6.73E-03) and rs2981582 (p-valuecor ≤ 6.48E-03) SNPs showed significant association with both familial and sporadic cancers. Additionally, rs2736098 (p-valuecor ≤ 0.0234) was associated with only sporadic cancers; also in group without carriers of high-risk mutation. All these associations revealed their significance also in Cochran-Armitage trend test. Opposite to other SNPs, rs2736098 was associated with a decreased risk of breast cancer. CONCLUSION: The association of four known susceptibility SNPs, representing three individual loci, with breast cancer risk in Polish women was confirmed. One of them (rs2736098) seems to be specific for the Polish population. Due to the population differences in allele frequencies, identification of general genetic risk factors requires sets of association studies conducted on different populations.

Pooled sample-based GWAS: a cost-effective alternative for identifying colorectal and prostate cancer risk variants in the Polish population.

BACKGROUND: Prostate cancer (PCa) and colorectal cancer (CRC) are the most commonly diagnosed cancers and cancer-related causes of death in Poland. To date, numerous single nucleotide polymorphisms (SNPs) associated with susceptibility to both cancer types have been identified, but their effect on disease risk may differ among populations. METHODS: To identify new SNPs associated with PCa and CRC in the Polish population, a genome-wide association study (GWAS) was performed using DNA sample pools on Affymetrix Genome-Wide Human SNP 6.0 arrays. A total of 135 PCa patients and 270 healthy men (PCa sub-study) and 525 patients with adenoma (AD), 630 patients with CRC and 690 controls (AD/CRC sub-study) were included in the analysis. Allele frequency distributions were compared with t-tests and χ(2)-tests. Only those significantly associated SNPs with a proxy SNP (p<0.001; distance of 100 kb; r(2)>0.7) were selected. GWAS marker selection was conducted using PLINK. The study was replicated using extended cohorts of patients and controls. The association with previously reported PCa and CRC susceptibility variants was also examined. Individual patients were genotyped using TaqMan SNP Genotyping Assays. RESULTS: The GWAS selected six and 24 new candidate SNPs associated with PCa and CRC susceptibility, respectively. In the replication study, 17 of these associations were confirmed as significant in additive model of inheritance. Seven of them remained significant after correction for multiple hypothesis testing. Additionally, 17 previously reported risk variants have been identified, five of which remained significant after correction. CONCLUSION: Pooled-DNA GWAS enabled the identification of new susceptibility loci for CRC in the Polish population. Previously reported CRC and PCa predisposition variants were also identified, validating the global nature of their associations. Further independent replication studies are required to confirm significance of the newly uncovered candidate susceptibility loci.