Common variants in DLG1 locus are associated with non-syndromic cleft lip with or without cleft palate.

Non-syndromic cleft lip with or without cleft palate (nsCL/P) is a common craniofacial anomaly with a complex and heterogeneous aetiology. Knowledge regarding specific genetic factors underlying this birth defect is still not well understood. Therefore, we conducted an independent replication analysis for the top-associated variants located within the DLG1 locus at chromosome 3q29, which was identified as a novel cleft-susceptibility locus in our genome-wide association study (GWAS). Mega-analysis of the pooled individual data from the GWAS and replication study confirmed that common DLG1 variants are associated with the risk of nsCL/P. Two single nucleotide polymorphisms (SNPs), rs338217 and rs7649443, were statistically significant even at the genome-wide level (Ptrend = 9.70E-10 and Ptrend = 8.96E-09, respectively). Three other SNPs, rs9826379, rs6805920 and rs6583202, reached a suggestive genome-wide significance threshold (Ptrend < 1.00E-05). The location of the strongest individual SNP in the intronic sequence of the gene encoding DLG1 antisense RNA suggests that the true causal variant implicated in the risk of nsCL/P may affect the DLG1 gene expression level rather than structure of the encoded protein. In conclusion, we identified a novel cleft-susceptibility locus at chromosome 3q29 with a DLG1 as a novel candidate gene for this common craniofacial anomaly.

GREM2 nucleotide variants and the risk of tooth agenesis.

OBJECTIVE: The etiology of tooth agenesis (TA) is multifactorial and still not fully understood. The aim of the study was to test whether variants of GREM2, encoding a bone morphogenetic protein (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 TA. 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.

Nucleotide variants of genes encoding components of the Wnt signalling pathway and the risk of non-syndromic tooth agenesis.

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.

MiRNA-149 as a Candidate for Facial Clefting and Neural Crest Cell Migration.

Nonsyndromic cleft lip with or without palate (nsCL/P) ranks among the most common human birth defects and has a multifactorial etiology. Human neural crest cells (hNCC) make a substantial contribution to the formation of facial bone and cartilage and are a key cell type in terms of nsCL/P etiology. Based on increasing evidence for the role of noncoding regulatory mechanisms in nsCL/P, we investigated the role of hNCC-expressed microRNAs (miRNA) in cleft development. First, we conducted a systematic analysis of miRNAs expressed in human-induced pluripotent stem cell-derived hNCC using Affymetrix microarrays on cell lines established from 4 unaffected donors. These analyses identified 152 candidate miRNAs. Based on the hypothesis that candidate miRNA loci harbor genetic variation associated with nsCL/P risk, the genomic locations of these candidates were cross-referenced with data from a previous genome-wide association study of nsCL/P. Associated variants were reanalyzed in independent nsCL/P study populations. Jointly, the results suggest that miR-149 is implicated in nsCL/P etiology. Second, functional follow-up included in vitro overexpression and inhibition of miR-149 in hNCC and subsequent analyses at the molecular and phenotypic level. Using 3'RNA-Seq, we identified 604 differentially expressed (DE) genes in hNCC overexpressing miR-149 compared with untreated cells. These included TLR4 and JUNB, which are established targets of miR-149, and NOG, BMP4, and PAX6, which are reported nsCL/P candidate genes. Pathway analyses revealed that DE genes were enriched in pathways including regulation of cartilage development and NCC differentiation. At the cellular level, distinct hNCC migration patterns were observed in response to miR-149 overexpression. Our data suggest that miR-149 is involved in the etiology of nsCL/P via its role in hNCC migration.

The novel polymorphic variants within the paired box of the PAX9 gene are associated with selective tooth agenesis.

It has been reported that two genes MSX1 and PAX9, which encode transcription factors, are associated with selective tooth agenesis. Expression of these genes specifically marks the regions of the mesenchyme where the tooth buds are formed. A mutation in the MSX1 gene, detected in a single family, resulting in an Arg-->Pro substitution in the homeodomain of the protein product of this gene has previously been associated with the deficiency of second premolars and third molars. However, mutations of the MSX1 gene were excluded in the patients with agenesis of the other type of teeth. In a single family with the lack of first and second molars, a mutation in the PAX9 gene was found. In our group of patients with the deficiency of various teeth, in 20% of the patients and their relatives sequence analysis revealed a C-->T transition in the coding sequence of the PAX9 gene. However, this polymorphism does not alter amino acid sequence of the protein product of this gene.

Aspects of programmed cell death during early senescence of barley leaves: possible role of nitric oxide.

Leaf senescence is a highly coordinated process which involves programmed cell death (PCD). Early stages of leaf senescence occurring during normal leaf ontogenesis, but not triggered by stress factors, are less well known. In this study, we correlated condensation of chromatin and nuclear DNA (nDNA) fragmentation, two main features of PCD during early senescence in barley leaves, with the appearance of nitric oxide (NO) within leaf tissue. With the help of the alkaline version of the comet assay, together with measurements of nDNA fluorescence intensity, we performed a detailed analysis of the degree of nDNA fragmentation. We localised NO in vivo and in situ within the leaf and photometrically measured its concentration with the NO-specific fluorochrome 4-amino-5-methylamino-2',7'-difluorofluorescein. We found that both nDNA fragmentation and chromatin condensation occurred quite early during barley leaf senescence and always in the same order: first nDNA fragmentation, in leaves of 6-day-old seedlings, and later chromatin condensation, in the apical part of leaves from 10-day-old seedlings. PCD did not start simultaneously even in neighbouring cells and probably did not proceed at the same rate. NO was localised in vivo and in situ within the cytoplasm, mainly in mitochondria, in leaves at the same stage as those in which chromatin condensation was observed. Localisation of NO in vascular tissue and in a large number of mesophyll cells during the senescence process might imply its transport to other parts of the leaf and its involvement in signalling between cells. The fact that the highest concentration of NO was found in the cytoplasm of mesophyll cells in the earliest stage of senescence and lower concentrations were found during later stages might suggest that NO plays an inductive role in PCD.

Maternal MTR genotype contributes to the risk of non-syndromic cleft lip and palate in the Polish population.

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-formyltetrahydrofolate 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.

Monitoring the mitochondrial transmembrane potential with the JC-1 fluorochrome in programmed cell death during mesophyll leaf senescence.

Analysis of the mitochondrial transmembrane potential (Delta Psi(m)) with the help of the JC-1 fluorochrome (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide) during mesophyll leaf senescence was performed in order to determine whether a reduction of Delta Psi(m) takes place during mesophyll senescence and whether plant mitochondria, like mammalian ones, might be involved in the induction of programmed cell death. Fluorescence analysis of mesophyll protoplasts of Pisum sativum in a confocal microscope, fluorescent spectra analysis and time dependence of fluorescence intensity of monomers and of J-aggregates revealed that JC-1 is incorporated and accumulated specifically in plant mitochondria. Analysis of Delta Psi(m) during mesophyll protoplast senescence revealed that two subpopulations of mitochondria which differ in Delta Psi(m) exist in all analyzed stages of leaf senescence. The first subpopulation contains mitochondria with red fluorescence of J-aggregates due to an unperturbed high Delta Psi(m). The second subpopulation comprises mitochondria with green fluorescence of monomers due to a low Delta Psi(m), proving total depolarization of mitochondrial membranes. Fluorescence analysis demonstrated that even in the latest analyzed stages of leaf senescence, mitochondria with a high Delta Psi(m) still exist. Fluorometric measurements revealed that the fluorescence intensity of J-aggregates decreases with the age of plants, which indicates that a reduction of Delta Psi(m) during the mesophyll senescence process takes place; however, it does not take place within the whole population of mitochondria of the same protoplast. The reason of this can be due to a dramatic reorganization of mitochondria in mesophyll cells and the appearance of large mitochondria with local heterogeneity of Delta Psi(m) in the oldest analyzed stages. All mitochondria in every stage of senescence maintained their membrane organization even when their size, distribution, and spatial organization in protoplasts changed dramatically. We stated that the reduction of Delta Psi(m) does not directly induce programmed cell death in mesophyll cells, as opposed to animal apoptosis.