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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.

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.

Objective The etiology of tooth agenesis is multifactorial and still not fully understood. The aim of the study was to test whether variants of GREM2, encoding a BMP antagonist, are associated with the risk of this common dental anomaly in a Polish population. Subjects and Methods Direct sequencing of the GREM2 coding sequence including exon/intron boundaries was performed in 95 patients with both hypodontia and oligodontia. All identified GREM2 variants were then further tested in an independent group of patients (n=163) and controls (n=184). Results The previously described, functional GREM2 mutation (c.226C>G, p.Gln76Glu) was identified in two patients with hypodontia and associated dental anomalies, including taurodontism and microdontia. This mutation generating an allele with increased inhibitory activity was not detected in the control group. The second identified GREM2 variant, c.-1-21C>T (rs11806449), was not associated with the risk tooth agenesis. The polymorphism allele frequency in both patients and controls was 0.21 (OR=1.0, 95%CI: 0.76–1.46). The rs11806449 did not correlate either with the overall TA phenotype or hypodontia/oligodontia phenotypes. Conclusion Our study confirmed that GREM2 is a candidate gene for tooth agenesis, which mutations can explain, however, only a small fraction of the genetic contribution to the pathogenesis of this anomaly. This article is protected by copyright. All rights reserved.

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 ptrend values were identified for rs16969681, rs16969816, and rs1258763 (ptrend 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. Birth Defects Research (Part A), 2015. © 2015 Wiley Periodicals, Inc.

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.

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. Birth Defects Research (Part A), 100:670–678, 2014. © 2014 Wiley Periodicals, Inc.

Tooth agenesis is one of the most common dental anomalies, with a complex and not yet fully elucidated aetiology. Given the crucial role of the Wnt signalling pathway during tooth development, the purpose of this study was to determine whether nucleotide variants of genes encoding components of this signalling pathway might be associated with hypodontia and oligodontia in the Polish population. A set of 34 single nucleotide polymorphism (SNPs) in 13 WNT and WNT-related genes were analyzed in a group of 157 patients with tooth agenesis and a properly matched control group (n = 430). In addition, direct sequencing was performed to detect mutations in the MSX1, PAX9 and WNT10A genes. Both single-marker and haplotype analyses showed highly significant association between SNPs in the WNT10A gene and the risk for tooth agenesis. Moreover, nine pathogenic mutations within the coding region of the WNT10A gene were identified in 26 out of 42 (62%) tested patients. One novel heterozygous mutation was identified in the PAX9 gene. Borderline association with the risk of non-syndromic tooth agenesis was also observed for the APC, CTNNB1, DVL2 and WNT11 polymorphisms. In conclusion, nucleotide variants of genes encoding important components of the Wnt signalling pathway might influence the risk of tooth agenesis.

Among different groups of Poles in Brussels there are an estimated twenty thousand Polish migrants performing undocumented work. The presence of homeless Poles in Brussels indicates the vulnerability of some of the European labour migrants. The article is based on fieldwork conducted amongst Polish people sleeping rough in Brussels in 2008 and 2009. Most of the homeless informants were construction workers, who lost their living quarters due to seasonal unemployment, alcohol problems, illness or other incidents. In the article I analyse their narratives using Julian Wolpert's concept of “place utility” to confront the way they talk about their adaptation to the environment with the risks and opportunities they attach to staying in Brussels and their possible return migration to Poland. I present four types of homeless migrants and their different situations and survival strategies. The analysis includes their perception of life in Brussels and Poland. The narratives of most of them seem to share the perception of Poland's lower “anticipated place utility” in comparison with Brussels. The decision not to return to Poland minimizes the perceived risks and uncertainty. It avoids psychological strains, such as feelings of shame, frustration and confronting their families and friends. Living on the streets of Brussels seems “optimal” to them, under the circumstances. This example shows that economically unsuccessful migrations cannot be easily terminated; that the risks and profits are not equally distributed across family members; and that the different rationalities of all the actors and their self-limitations should also be taken into account. Further studies of homelessness among working immigrants may contribute to a better understanding of some of the migration phenomena.

Mostowska A, Hozyasz KK, Biedziak B, Wojcicka P, Lianeri M, Jagodzinski PP. Genotype and haplotype analysis of WNT genes in non-syndromic cleft lip with or without cleft palate. Eur J Oral Sci 2012; 120: 1–8. © 2012 Eur J Oral Sci The wingless-type MMTV integration site family (Wnt) signalling pathway plays a crucial role in craniofacial development. Recently, nucleotide variants in WNT genes have been shown to be associated with oral congenital anomalies, including facial clefts. Therefore, in the current study we decided to assay the association of nucleotide variants in selected WNT genes with the risk of non-syndromic cleft lip with or without cleft palate (NCL/P) in the Polish population. Fourteen polymorphisms in WNT3, WNT3A, WNT5A, WNT8A, WNT9B, and WNT11 were tested in a group of 210 patients with NCL/P and in a properly matched control group. The most significant results were found for the WNT3 rs3809857 variant, which, under the assumption of a recessive model, was associated with a two-fold decrease in the risk of NCL/P (ORTT vs. GT + GG = 0.492, 95% CI: 0.276–0.879, P = 0.015). Moreover, haplotype analysis revealed that WNT3 is significantly correlated with NCL/P. The global P-values for haplotypes of rs12452064_rs7207916 and rs3809857_rs12452064_rs7207916 were 0.0034 and 0.0014, respectively, and these results were statistically significant, even after the permutation test correction. In conclusion, our study confirmed the involvement of polymorphisms in the WNT3 gene in NCL/P aetiology in the tested population.

BACKGROUND Non-syndromic cleft lip and palate (CLP) is one of the most common birth defects. Recent genome-wide association studies (GWAS) have identified several novel risk loci associated with this craniofacial anomaly. Therefore, the objective of this report was to investigate the contribution of the top seven polymorphisms reaching genome-wide statistical significance in GWAS analyses in the Polish population. METHODS and RESULTS Nucleotide variants located at chromosomal regions 1p22.1, 10q25.3, 17q22, and 20q12 were tested in a group of 206 patients with nonsyndromic CLP and a properly matched control group. Significant results, which persisted even after Bonferroni correction (p < 0.0071), were observed for polymorphisms located at 10q25.3 (rs7078160 and rs4752028) and 17q22 (rs227731). Under a recessive model, both rs7078160 and rs4752028 were associated with a greater than fourfold increase in the risk of CLP (odds ratio [OR] = 4.536; 95% confidence interval [CI], 1.678–12.265; p = 0.0012 and OR = 4.573; 95% CI, 1.817–11.512; p = 0.0004, respectively). Polymorphism rs227731 increased the risk of CLP when analyzed under a dominant model (OR = 1.732; 95% CI, 0.184–2.253; p = 0.0044). Borderline association was alsoidentified for the 1p22.1 locus (rs481931). Moreover, 10q25.3 haplotypes were significantly associated with a susceptibility to CLP. CONCLUSION Our evaluation study confirmed a substantial association of polymorphisms located at chromosomal regions 10q25.3 and 17q22 with nonsyndromic CLP in the Polish population. Birth Defects Research (Part A), 2012. © 2011 Wiley Periodicals, Inc.

Mostowska A, Hozyasz KK, Biedziak B, Misiak J, Jagodzinski PP. Polymorphisms located in the region containingBHMTandBHMT2genes as maternal protective factors for orofacial clefts. Eur J Oral Sci 2010; 118: 325–332. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci Nonsyndromic cleft lip with or without cleft palate (NCL/P) is one of the most common craniofacial malformations; however, its aetiology is still unclear. Because the effects of maternal nutrition on fetal development are well known, we decided to pursue the question of whether polymorphic variants of genes encoding enzymes involved in choline metabolism might be associated with the maternal risk of having a baby with NCL/P. Analysis of 18 single nucleotide polymorphisms (SNPs) of betaine-homocysteine methyltransferase (BHMT), betaine-homocysteine methyltransferase-2 (BHMT2), choline dehydrogenase (CHDH), choline kinase (CHKA), dimethylglycine dehydrogenase (DMGDH), choline-phosphate cytidylyltransferase A (PCYT1A), and phosphatidylethanolamine N-methyltransferase (PEMT) provided evidence that polymorphisms located in the region containing BHMT and BHMT2 were protective factors against NCL/P affected pregnancies in our population. The strongest signal was found for the SNP located in the intronic sequence of BHMT2. Women carrying two copies of the rs625879 T allele had a significantly decreased risk of having offspring with orofacial clefts. These results were significant, even after correction for multiple comparisons. Moreover, the gene–gene interaction analysis revealed a significant epistatic interaction of BHMT2 (rs673752), PEMT (rs12325817), and PCYT1A (rs712012) with maternal NCL/P susceptibility. Altogether, our study identified a novel gene, the nucleotide variants of which were be associated with a decreased risk of having a baby with NCL/P.

The aetiology of non-syndromic cleft lip with or without cleft palate (CL/P) is very complex. It has been shown that polymorphic variants of genes encoding key proteins of folate and methionine metabolism might be important maternal risk factors of having a child with this craniofacial anomaly. Therefore, in our study, mothers with CL/P children as well as control mothers were examined for prevalence of polymorphisms of genes that encode methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-formylte-trahydrofolate synthetase (MTHFD1) and reduced folate carrier 1 (RFC1). We observed that there were no statistical differences in allele and genotype frequencies of MTHFR c.677C>T, MTHFD1 c.1958G>A and RFC1 c.80G>A between mothers who had children with CL/P and control mothers. However, mothers with MTR c.2756AG or GG genotype displayed a 2.195-fold increased risk of having a child with CL/P (95% CI 1.189-4.050, p = 0.011). The mechanism by which polymorphic transition of MTR gene might increase the maternal risk of having CL/P progeny is unknown. Our observations are consistent with a significant role of the methyl cycle in the development of craniofacial structures in humans.

Tooth agenesis constitutes the most common anomalies of dental development in man. Despite this, little is known about the genetic defects responsible for this complex condition. To date, the only genes associated with the non-syndromic form of tooth agenesis are MSX1 and PAX9, which encode transcription factors that play a critical role during tooth development. This paper aims to review current literature about the molecular mechanisms responsible for selective tooth agenesis in humans.

Tooth development is regulated through a series of reciprocal interactions between the dental epithelium and mesenchyme and requires protein products of a number of genes. It has been reported that selective tooth agenesis is associated with mutations in human MSX and PAX9 genes. Mutational analysis of the two genes was performed in 25 individuals with familial or sporadic form of permanent tooth agenesis. Single-stranded conformational polymorphism analysis revealed no mutations in the entire coding sequence of the MSX1 gene. In PAX9, a novel, heterozygous G151A transition in the sequence encoding the paired domain of the PAX9 protein was detected in a patient with agenesis of third molars, second premolars and incisors, but not in her parents, the remaining patients or 162 individuals with normal dentition. This is the first de novo mutation described in PAX9. Our results support the view that mutations in PAX9 could constitute a causative factor of oligodontia. We hypothesize that the G151A transition in PAX9 might be responsible for the sporadic form of tooth agenesis in this patient.

A reduced rate of greening after continuous illumination was observed in dark-grown cress seedlings (Lepidium sativum L.) incubated with 5-aminolevulinate (ALA) or the complexing agents 2,2′-bipyridyl, 8-hydroxyquinoline or 1,10-phenanthroline. This effect cannot be explained merely by photodynamic damage caused by chlorophyll precursors which are accumulated in the dark under these conditions. Flash light experiments revealed that photoconversion of protochlorophyll(ide) to chlorophyllide was not influenced by chelator treatment. The next step in the chlorophyll pathway, the esterification of chlorophyllide, however, was inhibited. Simultaneously applicated ALA and complexing agents did not result in a synergistic reponse; on the contrary, ALA seemed to render cress plants less susceptible to the treatment with complexing agents upon subsequent irradiation. Ultrastructural studies demonstrated that grana formation in light was inhibited after pretreatment with ALA or complexing agents.

SUMMARY In microsporocytes of Stangeria, Impatiens and Tradescantia and in sporocytes of Equisetum all plastids and mitochondria aggregate for a short period at one side of the early prophase I nucleus. Later the organelles disaggregate and during metaphase I and anaphase I they organelles migrate towards the equator of the cell. In Impatiens, Clarkia, Lysimachia and Equisetum all these organelles aggregate in the equatorial plane, thus dividing a dyad into two parts. After meiosis II the equatorial aggregation of organelles is reshaped and divides a tetrad into four parts. Inside the aggregation of organelles, cell plates are simultaneously set up.