Identification of a new familial case of 3q29 deletion syndrome associated with cleft lip and palate via whole-exome sequencing.

Many unbalanced large copy number variants reviewed in the paper are associated with syndromic orofacial clefts, including a 1.6 Mb deletion on chromosome 3q29. The current report presents a new family with this recurrent deletion identified via whole-exome sequencing and confirmed by array comparative genomic hybridization. The proband exhibited a more severe clinical phenotype than his affected mother, comprising right-sided cleft lip/alveolus and cleft palate, advanced dental caries, heart defect, hypospadias, psychomotor, and speech delay, and an intellectual disability. Data analysis from the 3q29 registry revealed that the 3q29 deletion increases the risk of clefting by nearly 30-fold. No additional rare and pathogenic nucleotide variants were identified that could explain the clefting phenotype and observed intrafamilial phenotypic heterogeneity. These data suggest that the 3q29 deletion may be the primary risk factor for clefting, with additional genomic variants located outside the coding sequences, methylation changes, or environmental exposure serving as modifiers of this risk. Additional studies, including whole-genome sequencing or methylation analyses, should be performed to identify genetic factors underlying the phenotypic variation associated with the recurrent 3q29 deletion.

Identification of novel susceptibility genes for non-syndromic cleft lip with or without cleft palate using NGS-based multigene panel testing.

For non-syndromic cleft lip with or without cleft palate (ns-CL/P), the proportion of heritability explained by the known risk loci is estimated to be about 30% and is captured mainly by common variants identified in genome-wide association studies. To contribute to the explanation of the "missing heritability" problem for orofacial clefts, a candidate gene approach was taken to investigate the potential role of rare and private variants in the ns-CL/P risk. Using the next-generation sequencing technology, the coding sequence of a set of 423 candidate genes was analysed in 135 patients from the Polish population. After stringent multistage filtering, 37 rare coding and splicing variants of 28 genes were identified. 35% of these genetic alternations that may play a role of genetic modifiers influencing an individual's risk were detected in genes not previously associated with the ns-CL/P susceptibility, including COL11A1, COL17A1, DLX1, EFTUD2, FGF4, FGF8, FLNB, JAG1, NOTCH2, SHH, WNT5A and WNT9A. Significant enrichment of rare alleles in ns-CL/P patients compared with controls was also demonstrated for ARHGAP29, CHD7, COL17A1, FGF12, GAD1 and SATB2. In addition, analysis of panoramic radiographs of patients with identified predisposing variants may support the hypothesis of a common genetic link between orofacial clefts and dental abnormalities. In conclusion, our study has confirmed that rare coding variants might contribute to the genetic architecture of ns-CL/P. Since only single predisposing variants were identified in novel cleft susceptibility genes, future research will be required to confirm and fully understand their role in the aetiology of ns-CL/P.

The Prevalence and Morphology of Supernumerary Teeth in Children With Nonsyndromic Cleft Lip and Palate.

INTRODUCTION: Cleft lip with or without cleft palate (CL/P) is congenital deformity associated with hyperdontia. OBJECTIVE: To determine the prevalence and characteristics of supernumerary teeth in patients with CL/P. DESIGN: Retrospective descriptive and correlation clinical study. PATIENTS: One hundred thirteen children with cleft (age ranged 9.3-19.2; 67 males and 46 females) treated in Clinic of Congenital Facial Deformities Medical University of Lublin were included in the study. METHODS: Records evaluation was conducted regarding age, gender, cleft type (Q36, Q37-International Classification of Diseases 10th revision), cleft side, and incidence of supernumeraries. In all supernumerary teeth, size, shape, and developmental degree were analyzed and correlation between the incidence of hyperdontia with different variables was checked. Correlations were detected using chi-square and the Yates correction. RESULTS: The majority of the examined group were males-59.29% with Q37 (67.26%) and the cleft on the left side (62.83%). Hyperdontia was noted in 26.55%. Only upper lateral incisors were affected. They usually had atypical shape (56.67%), reduced size (83.33%), and delayed development (56.67%). CONCLUSIONS: The prevalence of supernumerary permanent teeth in patients with cleft was higher than in the general population. Anomaly was more frequent in male patients and occurred mainly on the cleft side. The severity of the cleft did not influenced the frequency of supernumerary teeth, their shape, size, and developmental degree. Supernumerary teeth were characterized by reduced crown size, abnormal structure, incorrect inclination, and delayed development phase.

Paraoxonase 1 gene (PON1) variants concerning hepatitis C virus (HCV) spontaneous clearance in hemodialysis individuals: a case-control study.

BACKGROUND: To explore associations between PON1 rs854560, rs662, 705,379, HCV clearance, and interactions between tested PON1 single nucleotide variants (SNVs) and interferon-λ4 gene (IFNL4) rs368234815 variant in hemodialyzed individuals. METHODS: The study included 83 HD individuals who spontaneously resolved HCV infection (all had known IFNL4 rs368234815 variant) and 104 individuals with persistently positive blood tests for HCV RNA (102 were IFNL4 rs368234815 variant successfully genotyped). We genotyped PON1 by high-resolution melt analysis (rs662) or predesigned TaqMan SNV Genotyping Assay (rs854560, rs705379). We used a logistic regression model to assess the association between genetic data and HCV outcome while adjusting for clinical confounding variables. Epistatic interactions between tested PON1 SNVs and IFNL4 rs368234815 were analyzed by the multifactor dimensionality reduction method. RESULTS: In the recessive inheritance model, PON1 rs662 GG (OR 9.94, 95% CI 1.20-82.7, P = 0.022) and rs854560 TT (OR 4.31, 95% CI 1.62-11.5, P = 0.003) genotypes were associated with a higher probability for HCV clearance. The haplotype composed of rs662A_rs854560A_rs705379 was not associated with spontaneous HCV clearance. The IFNL4 rs368234815 TT/TT variant was equally distributed among individuals bearing different PON1 SNVs. The epistatic gene-gene analysis did not reveal the interaction between tested PON1 SNVs and IFNL4 rs368234815 (P = 0.094). Regression model, including the PON1 rs662 GG genotype, the PON1 rs854560 genotype, the IFNL4 rs368234815 TT/TT genotype, age at RRT onset, RRT duration, and chronic glomerulonephritis as possible explanatory variables for spontaneous HCV clearance, showed that significant predictors of spontaneous HCV clearance were the IFNL4 rs368234815 TT/TT genotype (OR 2.607, 95% CI 1.298-5.235, P = 0.007), PON1 rs854560 TT (OR 6.208, 1.962-19.644, P = 0.002), PON1 rs662 GG (OR 10.762, 1.222-94.796, P = 0.032), and RRT duration (OR 0.930, 95% CI 0.879-0.984, P = 0.011). CONCLUSION: In HD individuals, PON1 rs662 GG and rs854560 TT are associated with a higher frequency of spontaneous HCV clearance.

Polymorphism rs368234815 of interferon lambda 4 gene and spontaneous clearance of hepatitis C virus in haemodialysis patients: a case-control study.

BACKGROUND: In non-uremic subjects, IFNL4 rs368234815 predicts HCV clearance. We investigated whether rs368234815 is associated with spontaneous HCV clearance in haemodialysis patients and whether it is a stronger predictor of HCV resolution than the IFNL polymorphisms already associated with HCV clearance in dialysis subjects. We also evaluated an association of rs368234815 with patients` survival and alterations in transcription factor binding sites (TFBS) caused by IFNL polymorphisms. METHODS: Among 161 haemodialysis patients with positive anti-HCV antibodies, 68 (42.2%) spontaneously resolved HCV infection, whereas 93 remained HCV RNA positive. Patients were tested for near IFNL3 rs12980275, IFNL3 rs4803217, IFNL4 rs12979860, IFNL4 rs368234815, and near IFNL4 rs8099917. IFNL4 rs368234815 polymorphism (TT/TT, ΔG/TT, ΔG/ΔG) was genotyped by restriction fragment length polymorphism analysis; other IFNL polymorphisms - by high resolution melting curve analysis. We used the Kaplan-Meier method with the log-rank test for survival analysis. In silico analysis included the use of ENCODE TFBS ChIP-seq data, HOCOMOCO, JASPAR CORE, and CIS-BP databases, and FIMO software. RESULTS: The probability (OR, 95%CI, P) of spontaneous HCV clearance for rs368234815 TT/TT patients was higher than for the ΔG allele carriers (2.63, 1.38-5.04, 0.003). This probability for other major homozygotes varied between 2.80, 1.45-5.43, 0.002 for rs12980275 and 2.44, 1.27-4.69, 0.007 for rs12979860. In the additive model, rs368234815 TT/TT was the strongest predictor of HCV clearance (6.38, 1.69-24.2, 0.003). Survival analysis suggested an association of the ΔG allele with mortality due to neoplasms (log-rank P = 0.005). The rs368234815 ∆G allele caused TFBS removal for PLAGL1. CONCLUSIONS: In haemodialysis patients, the association of rs368234815 with the spontaneous HCV clearance is better than that documented for other IFNL3/IFNL4 polymorphisms only in the additive mode of inheritance. However, identifying the homozygosity in the variant ∆G allele of rs368234815 means a more potent prediction of persistent HCV infection in haemodialysis subjects that we observe in the case of the variant homozygosity of other tested IFNL3/IFNL4 polymorphisms. Removal of PLAGL1 TFBS in subjects harbouring the rs368234815 ∆G allele may contribute to cancer susceptibility. The association of rs368234815 with cancer-related mortality needs further studies in HCV-exposed subjects.

PAX7 nucleotide variants and the risk of non-syndromic orofacial clefts in the Polish population.

OBJECTIVE: The etiology of non-syndromic cleft lip with or without cleft palate (nsCL/P) is multifactorial, heterogeneous, and still not completely understood. The aim of the present study was to examine the associations between common and rare PAX7 nucleotide variants and the risk of this common congenital anomaly in a Polish population. SUBJECTS AND METHODS: Eight top nsCL/P-associated PAX7 variants identified in our cleft genome-wide association study (GWAS) were selected for replication analysis in an independent group of patients and controls (n = 247 and n = 445, respectively). In addition, mutation screening of the PAX7 protein-coding region was conducted. RESULTS: Analysis of the pooled data from the GWAS and replication study confirmed that common PAX7 nucleotide variants are significantly associated with the increased risk of nsCL/P. The strongest individual variant was rs1339062 (c.586 + 15617T > C) with a p-value = 2.47E-05 (OR = 1.4, 95%CI: 1.20-1.64). Sequencing analysis identified a novel synonymous PAX7 substitution (c.87G > A, p.Val29Val) in a single patient with nsCLP. This transition located in the early exonic position was predicted to disrupt potential splice enhancer elements. CONCLUSION: Our study confirmed that PAX7 is a strong candidate gene for nsCL/P. Nucleotide variants of this gene contribute to the etiology of nsCL/P in the homogenous Polish population.

Calcium-sensing receptor gene (CASR) polymorphisms and CASR transcript level concerning dyslipidemia in hemodialysis patients: a cross-sectional study.

BACKGROUND: There is scarce data on CASR associations with dyslipidemia. We investigated in hemodialysis (HD) patients whether CASR single nucleotide polymorphisms (SNPs) rs7652589 and rs1801725 have associations with dyslipidemia and show epistatic interactions with SNPs of the energy homeostasis-associated gene (ENHO), retinoid X receptor α gene (RXRA), and liver X receptor α gene (LXRA). METHODS: The study included 1208 HD subjects. For diagnosis of dyslipidemia, both K/DOQI criteria and atherogenic index ≥3.8 were used. CASR rs1801725 was genotyped by TaqMan SNP Genotyping Assay, other SNPs - by high-resolution melting curve analysis or polymerase chain reaction-restriction fragment length polymorphism, as appropriate. Relative transcript levels of CASR, ENHO, RXRA, and LXRA were measured in peripheral blood mononuclear cells. The occurrence of dyslipidemic phenotypes concerning tested polymorphisms was compared using models of inheritance. Haplotypes were estimated using the Haploview 4.2 software. Epistatic interactions between tested SNPs were analyzed using the logistic regression and epistasis option in the PLINK software. RESULTS: Rs7652589 indicated a greater probability of atherogenic dyslipidemia in the dominant inheritance model (OR 1.4, 95%CI 1.0-2.0, P = 0.026), principally because of increased triglyceride (TG) levels. The rs1801725 variant allele was associated with a decreased probability of dyslipidemia characterized by non-HDL-cholesterol ≥130 mg/dL and TG ≥200 mg/dL (OR 0.6, 0.4-0.9, P = 0.012). There were no epistatic interactions between CASR and RXRA, LXRA, and ENHO regarding dyslipidemia. Both rs7652589 and rs1801725 SNPs were not in linkage disequilibrium (D' = 0.091, r2 = 0.003 for the entire HD group) and their haplotypes did not correlate with dyslipidemia. Relative CASR transcript was lower at a borderline significance level in patients harboring the rs1801725 variant allele compared with homozygotes of the major allele (0.20, 0.06-7.80 vs. 0.43, 0.04-5.06, P = 0.058). CASR transcript correlated positively with RXRA transcript (adjusted P = 0.001), LXRA transcript (adjusted P = 0.0009), ENHO transcript (borderline significance, adjusted P = 0.055), dry body weight (adjusted P = 0.035), and renal replacement therapy duration (adjusted P = 0.013). CONCLUSIONS: CASR polymorphisms (rs7652589, rs1801725) are associated with dyslipidemia in HD patients. CASR correlates with RXRA, LXRA, and ENHO at the transcript level. Further investigations may elucidate whether other CASR SNPs contribute to associations shown in this study.

ENHO, RXRA, and LXRA polymorphisms and dyslipidaemia, related comorbidities and survival in haemodialysis patients.

BACKGROUND: The energy homeostasis-associated gene (ENHO), retinoid X receptor alpha gene (RXRA), and liver X receptor alpha gene (LXRA) are involved in adipogenic/lipogenic regulation. We investigated whether single-nucleotide polymorphisms in these genes (ENHO rs2281997, rs72735260; RXRA rs749759, rs10776909, rs10881578; LXRA rs2279238, rs7120118, rs11039155) are associated with dyslipidaemia, related comorbidities and survival of haemodialysis (HD) patients also tested for T-helper (Th) cell interleukin genes (IL). METHODS: The study was carried out in 873 HD patients. Dyslipidaemia was diagnosed by the recommendations of the Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines (2003); atherogenic dyslipidaemia was referred to if the TG/HDL cholesterol ratio was equal to or higher than 3.8. Genotyping of ENHO SNPs, LXRA SNPs, and IL12A rs568408 was carried out using HRM analysis. RXRA SNPs, IL12B rs3212227, and IL18 rs360719 were genotyped using PCR-RFLP analysis. The circulating adropin concentration was determined in 126 patients by enzyme-linked immunosorbent assay. Survival probability was analysed using the Kaplan-Meier method in 440 patients followed through 7.5 years. RESULTS: Dyslipidaemia by K/DOQI was diagnosed in 459 patients (91% revealed hyper-LDL- cholesterolaemia), atherogenic dyslipidaemia was diagnosed in 454 patients, and 231 patients were free of dyslipidaemia by both criteria. The variant allele (T) of ENHO rs2281997 was associated with the hyper-LDL cholesterolaemic pattern of dyslipidaemia by K/DOQI. The frequency of atherogenic dyslipidaemia was lower in T-allele bearers than in CC-genotype patients. The rs2281997 T allele was associated with lower cardiovascular mortality in HD patients showing atherogenic dyslipidaemia. ENHO, RXRA, and LXRA showed epistatic interactions in dyslipidaemia. Circulating adropin was lower in atherogenic dyslipidaemia than in non-atherogenic conditions. RXRA rs10776909 was associated with myocardial infarction. Bearers of LXRA rs2279238, rs7120118 or rs11039155 minor alleles showed higher mortality. ENHO SNP positions fell within the same DNase 1 hypersensitivity site expressed in the Th1 cell line. Epistatic interactions occurred between rs2281997 and Th1 IL SNPs (rs360719, rs568408). CONCLUSIONS: Atherogenic dyslipidaemia occurs in HD patients in whom ENHO encodes less adropin. ENHO, RXRA, and LXRA SNPs, separately or jointly, are associated with dyslipidaemia, myocardial infarction, and survival in HD patients. Differences in the availability of transcription binding sites may contribute to these associations.

Association of CDKAL1 nucleotide variants with the risk of non-syndromic cleft lip with or without cleft palate.

Although the aetiology of non-syndromic cleft lip with or without cleft palate (nsCL/P) has been studied extensively, knowledge regarding the role of genetic factors in the pathogenesis of this common craniofacial anomaly is still limited. We conducted a follow-up association study to confirm that CDKAL1 nucleotide variants identified in our genome-wide association study (GWAS) for nsCL/P are associated with the risk of this anomaly. In addition, we performed a sequence analysis of the selected CDKAL1 exons. A mega-analysis of the pooled individual data from the GWAS and a replication study revealed that six out of thirteen CDKAL1 variants were positively replicated and reached the threshold of statistical significance (Ptrend < 3.85E-03). They represented a single association signal and were located within the fifth intron of CDKAL1. The strongest individual variant was rs9356746 with a Ptrend value = 5.71E-06 (odds ratio (OR) = 1.60, 95% confidence interval (CI): 1.30-1.97). Sequencing analysis did not reveal any pathogenic mutations of this gene. This study provides the first evidence that chromosomal region 6p22.3 is a novel susceptibility locus for nsCL/P. The location of the risk variants within the CDKAL1 intronic sequence containing enhancer elements predicted to regulate the SOX4 transcription may suggest that SOX4, rather than CDKAL1, is a potential candidate gene for this craniofacial anomaly.

The Calcium-Sensing Receptor Gene Polymorphism rs1801725 and Calcium-Related Phenotypes in Hemodialysis Patients.

BACKGROUND/AIMS: The calcium-sensing receptor gene (CASR) rs1801725 variant is responsible for a non-conservative amino-acid change (A986S) in the calcium-sensing receptor cytoplasmic tail. We hypothesized that rs1801725 polymorphism might be helpful in understanding Ca-related abnormalities in HD patients. METHODS: In 1215 subjects (245 on cinacalcet), we determined the associations of rs1801725 with secondary hyperparathyroidism (sHPT)-related laboratory parameters, PTH-decreasing effect of cinacalcet hydrochloride, coronary artery disease (CAD), myocardial infarction (MI), nephrolithiasis-related ESRD, and mortality. CASR rs7652589(AT) haplotypes and rs1801725 epistatic interactions with vitamin D signaling pathway genes were examined for associations with selected phenotypes. RESULTS: The rs1801725 variant allele showed an increasing independent effect on plasma PTH (Pcorrected = 0.009). CASR rs7652589_rs1801725 AT haplotype was associated with 1.7-fold higher frequency of PTH levels over 437 pg/mL than the reference haplotype GG (P = 0.001). CASR rs7652589_rs1801725 AG haplotype was 1.5-fold more frequent in nephrolithiasis-related ESRD than the GG haplotype (P = 0.004). There were no significant associations between rs1801725, CAD, MI, and response to cinacalcet. Variant homozygosity of rs1801725 correlated independently with higher infection-related mortality compared with heterozygosity (HR 7.95, 95%CI 2.15 - 29.37, P = 0.003) and major homozygosity (HR 5.89, 95%CI 1.69 - 20.55, P = 0.040). CASR rs1801725 did not show epistatic interactions with vitamin D signaling pathway genes concerning tested associations. CONCLUSION: The variant allele of CASR rs1801725 solely and together with the variant allele of rs7652589 increases risk of more advanced sHPT. Homozygosity of the rs1801725 variant allele contributes to infection-related mortality in HD patients.

The assessment of GWAS - identified polymorphisms associated with infertility risk in Polish women with endometriosis.

OBJECTIVES: Genome-wide association studies in patients with endometriosis revealed ten significant single nucleo-tide polymorphisms (SNPs) in the Caucasian population, which include rs12700667 near NFE2L3, rs12037376 in WNT4, rs7521902 near WNT4, rs13394619 in GREB1, rs10859871 near VEZT, rs1537377 near CDKN2B-AS1, rs4141819 near ETAA1, rs7739264 near ID4, rs1519761 near RND3 and rs6542095 near IL1A. MATERIAL AND METHODS: We replicated ten polymorphisms among infertile women with endometriosis (n = 315) and healthy fertile women (n = 406) in the Polish Caucasian population. Genotyping was conducted either by high-resolution melting curve analysis or by a pre-designed TaqMan probe. RESULTS: For all infertile women with endometriosis, the p values of the Cochran-Armitage trend test for the rs12700667 SNP was ptrend = 0.038 and the odds ratio (OR) for the risk allele frequency (RAF) of rs12700667 was 1.304 (95% CI = 1.009-1.685; p = 0.042). In patients with endometriosis with severity stages III/IV, ptrend for rs12700667 SNP was 0.036 and OR for the RAF was 1.394 (95% CI = 1.010-1.923; p = 0.043). In infertile women with endometriosis with severity stages III/IV for rs4141819 SNP, we observed ptrend = 0.026 and for RAF the OR = 1.350 (95% CI = 1.032-1.766; p = 0.029). CONCLUSIONS: Our results demonstrate association of RAF of rs12700667 and rs4141819 SNPs with infertility in Polish women with advanced endometriosis.

Polymorphisms of T helper cell cytokine-associated genes and survival of hemodialysis patients - a prospective study.

BACKGROUND: Circulating pro-inflammatory cytokines were associated with increased relative mortality risk, while immune parameters reflecting improved T-cell function were predictors of survival in hemodialysis (HD) patients. We evaluated in the prospective study whether variants in T helper cell cytokine-associated genes are determinants of mortality in HD patients. METHODS: The study was carried out in 532 prevalent HD subjects who were followed-up for 7 years. HRM analysis was used for IFNL3, IL12A, IL13, and IL4R genotyping. CCL2, IL12B, and IL18 were genotyped using PCR-RFLP analysis. Survival analyses were conducted using the Kaplan-Meier method and the Cox proportional hazard model. RESULTS: In univariate analyses, IFNL3 rs8099917 was associated with all-cause mortality in recessive model of inheritance (log-rank test P = 0.044), IL12A rs568408 - in dominant model (log-rank test P = 0.029). Minor homozygotes (the genotype GG) in IFNL3 rs8099917 showed shorter survival during the study (3.6, 1.0-7.0 years vs 4.7, 0.1-7.0 years, P = 0.009) than the major allele (T) bearers. The rs8099917 GG patients demonstrated higher risk of death than the remaining patients (GT + TT) (OR 1.94, 95%CI 1.11-3.40, P = 0.020). Major homozygosity (the genotype GG) in IL12A rs568408 was associated with higher mortality than that shown in bearers of the minor allele (AA + AG) (HR 1.31, 95%CI 1.02-1.69, P = 0.035). In multivariate analyses, however, the mentioned polymorphisms were not independent predictors of survival. CONCLUSIONS: Polymorphisms of IFNL3 rs8099917 and IL12A rs568408 contribute to survival of HD patients, but not as independent factors.

Involvement of 17ß-hydroxysteroid dehydrogenase type gene 1 937 A>G polymorphism in infertility in Polish Caucasian women with endometriosis.

PURPOSE: Endometriosis is considered to be an estrogen-related chronic inflammatory disease. The 17ß-hydroxysteroid dehydrogenase 1 (HSD17B1) converts estrone to 17ß estradiol. The role of HSD17B1 937 A>G (rs605059) single nucleotide polymorphism (SNP) in development of endometriosis is still disputable. This study evaluated the association of the HSD17B1 937 A>G (rs605059) SNP with infertile women affected by endometriosis from Polish Caucasian population. METHODS: The genotyping of cases (n = 290) and fertile women (n = 410) was conducted by high-resolution melting curve analysis. RESULTS: Statistical analysis demonstrated that the HSD17B1 937 A>G SNP is associated with endometriosis in stages I and II. The p trend and p allelic values calculated for the HSD17B1 937 A>G polymorphism were statistically significant and were equal to 0.001 and 0.0009, respectively. There was a significant association for the dominant model: (AG + GG vs AA) OR = 1.973 (95% CI = 1.178-3.304), p = 0.009, and for the recessive model: (GG vs AG + AA) OR = 1.806 (95% CI = 1.178-2.770), p = 0.006. However, we did not find statistical association of HSD17B1 937 A>G polymorphism with all infertile women with endometriosis or infertile women with endometriosis in stages III and IV. CONCLUSION: Our genetic study demonstrated HSD17B1 937 G variant as a risk factor for infertility in women with stage I and II endometriosis in Polish Caucasian patients.

Association between polymorphisms at the GREM1 locus and the risk of nonsyndromic cleft lip with or without cleft palate in the Polish population.

BACKGROUND: The locus on chromosome 15q13.3 containing GREM1 is correlated with the risk of nonsyndromic cleft lip with or without cleft palate (NSCL/P). The aim of the present study was to find the GREM1 functional variants implicated in the aetiology of this common developmental anomaly in the Polish population. METHODS: Eight polymorphisms were genotyped in 334 NSCL/P patients and 955 controls. In addition, the GREM1 protein-coding region was sequenced in 96 NSCL/P patients. RESULTS: Significant association with a risk of oral clefts was found for 5 tested polymorphisms. The lowest p(trend) values were identified for rs16969681, rs16969816, and rs1258763 (p(trend) 4.09E-05, 3.35E-05, and 0.0002, respectively). The putative functional variant rs16969681, located in a region that has enhancer activity, was associated with a 2.6-fold lower risk for NSCL/P (odds ratio [OR] = 0.38; 95% confidence interval [CI], 0.24-0.61, p = 2.37E-05). The previously reported association of rs1258763 with NSCL/P was replicated (OR = 0.57; 95% CI, 0.44-0.73; p = 1.10E-05). For all tested GREM1 variants, no significant sex-by-genotype interaction effects were observed. The sequencing analysis did not detect any rare variants implicated in the development of oral clefts. CONCLUSION: Our results might suggest that variants influencing GREM1 expression levels, rather than variants affecting the function of the encoded protein, are significant factors in NSCL/P etiology.

WNT10A coding variants and maxillary lateral incisor agenesis with associated dental anomalies.

Congenital maxillary lateral incisor agenesis (MLIA) is one of the most common subtypes of dental agenesis. Because little is known with regard to the aetiology of this anomaly, the aim of the study was to determine the contribution of nucleotide variants in wingless-type MMTV integration site family, member 10A (WNT10A), msh homeobox 1 (MSX1), and paired box 9 (PAX9) to the risk of MLIA in a Polish population. Coding regions of the selected genes were analysed by direct sequencing in a group of 20 individuals with unilateral and bilateral MLIA, associated or not with other dental anomalies. The frequencies of the identified nucleotide variants were assessed in an additional cohort of patients with isolated dental agenesis (n = 147) and in 178 controls. Mutation screening showed four non-synonymous substitutions located in the highly conserved coding sequence of WNT10A in five (25%) of the 20 patients. Analysis of genotyping results revealed that three of these variants--p.Arg113Cys, p.Phe228Ile, and the newly identified p.Arg171Leu--may represent aetiological mutations underlying MLIA with associated dental anomalies. No mutations that were potentially aetiologic were identified in MSX1 and PAX9. In conclusion, this is the first report implicating coding variants in the WNT10A gene in the aetiology of MLIA. These results will require further confirmation using larger-scale studies.

Association of Retinoid X Receptor Alpha Gene Polymorphism with Clinical Course of Chronic Glomerulonephritis.

BACKGROUND Vitamin D (VD), VD binding protein, VD receptor (VDR), and retinoids are involved in pathogenesis of chronic glomerulonephritis (ChGN). We aimed to compare distribution of VD pathway gene polymorphisms in ChGN patients showing glomerular filtration rate (GFR) category 1-3, GFR category 5D, and healthy controls in order to elucidate the role of VD-related polymorphisms in the course of ChGN. MATERIAL AND METHODS GFR category 1-3 ChGN patients (n=195), GFR category 5D ChGN patients (n=178), and controls (n=751) underwent testing for polymorphisms of genes encoding VD binding protein (GC, rs2298849, rs7041, rs1155563), VDR (VDR, rs2228570, rs1544410), and retinoid X receptor alpha (RXRA, rs10776909, rs10881578, rs749759). RESULTS Among GFR 1-3 subjects possessing TT genotype of RXRA rs10776909, 75% of patients had nephrotic syndrome, and 37.5% had glomerular hyperfiltration defined as GFR >140 ml/min/1.73 m2, and, consequently, serum creatinine was lower in these patients compared to the remaining subjects (0.67±0.26 vs. 0.94±0.34, P=0.014). In GFR category 5D ChGN patients, frequencies of RXRA rs10776909 allele T (25% vs. 19%) and CT+TT (46% vs. 34%) were higher compared to frequencies of respective variants in controls (Ptrend=0.004, Pgenotype=0.008). CONCLUSIONS RXRA rs10776909 allele T is specifically involved in the pathogenesis of ChGN. This risk allele may be also associated with worse clinical course of ChGN.

Genetic variants in BRIP1 (BACH1) contribute to risk of nonsyndromic cleft lip with or without cleft palate.

BACKGROUND: The etiology of nonsyndromic cleft lip with or without cleft palate (NSCL/P) is very complex and still not well elucidated. Given the critical role of DNA damage repair in the embryonic development, we decided to test the hypothesis that polymorphisms of selected DNA repair genes might contribute to the risk of NSCL/P in the Polish population. METHODS: Analysis of 36 polymorphisms in 12 DNA damage repair genes (ATM, BLM, BRCA1, BRIP1, E2F1, MLH1, MRE11A, MSH2, MSH6, NBN, RAD50, and RAD51) was conducted using TaqMan assays in a group of 263 NSCL/P patients and matched control group (n = 526). RESULTS: Statistical analysis of genotyping results revealed that nucleotide variants in the BRIP1 (BACH1) gene were associated with the risk of NSCL/P. Under assumption of a dominant model, the calculated odds ratios (ORs) for BRIP1 rs8075370 and rs9897121 were 1.689 (95% confidence interval [CI], 1.249-2.282; p = 0.0006) and 1.621 (95% CI, 1.200-2.191; p = 0.0016), respectively. These results were statistically significant even after applying multiple testing correction. Additional evidence for a causative role of BRIP1 in NSCL/P etiology was provided by haplotype analysis. Borderline association with a decreased risk of this anomaly was also observed for BLM rs401549 (ORrecessive = 0.406; 95% CI, 0.223-1.739; p = 0.002) and E2F1 rs2071054 (ORdominant = 0.632; 95% CI, 0.469-0.852; p = 0.003). CONCLUSION: Our study suggests that polymorphic variants of DNA damage repair genes play a role in the susceptibility to NSCL/P. BRIP1 might be novel candidate gene for this common developmental anomaly.

Vitamin D receptor gene BsmI, FokI, ApaI and TaqI polymorphisms and the risk of systemic lupus erythematosus.

Recently, several studies have demonstrated the role of vitamin D receptor (VDR) polymorphisms in the development of systemic lupus erythematosus (SLE); however, these results are inconsistent between different cohorts. Therefore, we studied the prevalence of the VDR FokI (rs2228570), BsmI (rs1544410), ApaI (rs7975232) and TaqI (rs731236) genotypes and alleles in SLE patients (n = 258) and healthy individuals (n = 545) in a Polish population. We did not observe significant differences for either the VDR FokI, BsmI, ApaI and TaqI genotype and allele frequencies in patients with SLE and healthy individuals. However, the frequency of the VDR F/F and F/f genotypes of FokI was statistically different between patients with renal disease and patients without this symptom OR = 3.228 (1.534-6.792, p = 0.0014), p (corr) = 0.0476)]. There was no association of the studied VDR BsmI, ApaI and TaqI polymorphisms with clinical manifestations and laboratory profiles in patients with SLE. Our study indicates that the studied VDR FokI variant might increase the risk of some clinical presentations in patients with SLE.

DNMT1, DNMT3A and DNMT3B gene variants in relation to ovarian cancer risk in the Polish population.

Studies have demonstrated that changes in DNA methylation of cancer related genes can be an elementary process accounting for ovarian tumorigenesis. Therefore, we evaluated the possible association of single nucleotide polymorphisms (SNPs) of DNA methyltransferases (DNMTs) genes, including DNMT1, DNMT3B, and DNMT3A, with ovarian cancer development in the Polish population. Using PCR-RFLP and HRM analyses, we studied the prevalence of the DNMT1 rs8101626, rs2228611 and rs759920, DNMT3A rs2289195, 7590760, rs13401241, rs749131 and rs1550117, and DNMT3B rs1569686, rs2424913 and rs2424932 SNPs in patients with ovarian cancer (n=159) and controls (n=180). The lowest p values of the trend test were observed for the DNMT1 rs2228611 and rs759920 SNPs in patients with ovarian cancer (p trend=0.0118 and p trend=0.0173, respectively). Moreover, we observed, in the recessive inheritance model, that the DNMT1 rs2228611 and rs759920 SNPs are associated with an increased risk of ovarian cancer development [OR 1.836 (1.143-2.949), p=0.0114, p corr=0.0342, and OR 1.932 (1.185-3.152), p=0.0078, p cor=0.0234, respectively]. However, none of other nine studied SNPs displayed significant contribution to the development of ovarian cancer. Furthermore, haplotype and multifactor dimensionality reduction analysis of the studied DNMT1, DNMT3B, and DNMT3A polymorphisms did not reveal either SNP combinations or gene interactions to be associated with the risk of ovarian cancer development. Our results may suggest that DNMT1 variants may be risk factors of ovarian cancer.

Association of the interleukin-12 polymorphic variants with the development of antibodies to surface antigen of hepatitis B virus in hemodialysis patients in response to vaccination or infection.

Cytokines, involved in the T-helper 1 system, play a role in the regulation of hepatitis B virus (HBV) clearance and the immune response to HBV antigens during natural infection or planned vaccination. Our aim was to examine whether the polymorphic variants of IL-12 are equally associated with development of antibodies to HBV surface antigen (anti-HBs) in hemodialysis (HD) patients in the case of HBV vaccination or HBV infection. The IL-12A rs568408 and IL-12B rs3212227 polymorphisms were analyzed in relation to anti-HBs development in 602 HD patients with negative antibodies to HBV core antigen (anti-HBc) who were hepatitis B vaccinated (group I) as well as in 237 anti-HBc positive HD patients who were infected with HBV in the past (group II). In group I, 199 patients did not develop an anti-HBs titre >10 IU/L (subgroup Ia), whereas in group II, 55 patients did not develop an anti-HBs titre >10 IU/L (subgroup IIa). Patients of groups I and II that developed an anti-HBs >10 IU/L were included into subgroups Ib and IIb, respectively. In hepatitis B vaccinated HD patients, development of a protective anti-HBs titre was positively associated with vintage of renal replacement therapy (RRT), chronic glomerulonephritis as a cause of RRT, and GA rs 568408 IL-12A (OR 1.6, 95 % CI 1.0-2.5, P = 0.035), but a frequency distribution of this genotype between responders and non-responders was not significant when the Bonferroni correction was applied. In HBV infected HD patients, anti-HBs development was positively associated with AC rs3212227 IL-12B (OR 8.0, 95 % CI 2.6-24.9, P < 0.001), whereas HBsAg positivity, AA rs3212227 IL-12B (OR 0.3, 95 % CI 0.1-0.7, P = 0.007), and CC rs3212227 IL-12B (OR 0.1, 95 % CI 0.03-0.6, P = 0.011) were negative predictors of positive anti-HBs phenotype. When the Bonferroni correction was applied, if appropriate, these associations remained significant. In HD patients, the studied IL-12 polymorphic variants seem to be associated with the anti-HBs phenotype (a) with borderline significance for IL-12A in hepatitis B vaccinated patients, and (b) significantly for IL-12B in patients who underwent natural HBV infection.