Cibele Masotti

Region 8q24 is a susceptibility locus for nonsyndromic oral clefting in Brazil

BACKGROUND Nonsyndromic cleft lip with or without cleft palate is a relatively common craniofacial defect with multifactorial inheritance. The association of the rs987525 single nucleotide variant, located in a gene desert at 8q24.21 region, has been consistently replicated in European populations. We performed a structured association approach combined with transcriptional analysis of the MYC gene to dissect the role of rs987525 in oral clefting susceptibility in the ethnically admixed Brazilian population. METHODS We performed the association study conditioned on the individual ancestry proportions in a sample of 563 patients and 336 controls, and in an independent sample of 221 patients and 261 controls. The correlation between rs987525 genotypes and MYC transcriptional levels in orbicularis oris muscle mesenchymal stem cells was also investigated in 42 patients and 4 controls. RESULTS We found a significant association in the larger sample (p = 0.0016; OR = 1.80 [95% confidence interval {CI}, 1.21-2.69], for heterozygous genotype, and 2.71 [95% CI, 1.47-4.96] for homozygous genotype). We did not find a significant correlation between rs987525 genotypes and MYC transcriptional levels (p = 0.14; r = -0.22, Spearman Correlation). CONCLUSIONS We present a positive association of rs987525 in the Brazilian population for the first time, and it is likely that the European contribution to our population is driving this association. We also cannot discard a role of rs987515 in MYC regulation, because this locus behaves as an expression quantitative locus of MYC in another tissue.

IRF6 is a risk factor for nonsyndromic cleft lip in the Brazilian population

Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a complex disorder with a worldwide incidence estimated at 1:700. Among the putative susceptibility loci, the IRF6 gene and a region at 8q24.21 have been corroborated in different populations. To test the role of IRF6 in NSCL/P predisposition in the Brazilian population, we conducted a structured association study with the SNPs rs642961 and rs590223, respectively, located at 5′ and 3′ of the IRF6 gene and not in strong linkage disequilibrium (LD), in patients from five different Brazilian locations. We also evaluated the effect of these SNPs in IRF6 expression in mesenchymal stem cells (MSC). We observed association between rs642961 and cleft lip only (CLO) (P = 0.009; odds ratio (OR) for AA genotype = 1.83 [95% Confidence interval (CI), 0.64–5.31]; OR for AG genotype = 1.72 [95% CI, 1.03–2.84]). This association seems to be driven by the affected patients from Barbalha, a location which presents the highest heritability estimate (H2 = 0.85), and the A allele at rs642961 is acting through a dominant model. No association was detected for the SNP rs590223. We did not find any correlation between expression levels and genotypes of the two loci, and it is possible that these SNPs have a functional role in some specific period of embryogenesis.

Human Stem Cell Cultures from Cleft Lip/Palate Patients Show Enrichment of Transcripts Involved in Extracellular Matrix Modeling By Comparison to Controls

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies.

Auriculo-condylar syndrome: mapping of a first locus and evidence for genetic heterogeneity

Auriculo-condylar syndrome (ACS), an autosomal dominant disorder of first and second pharyngeal arches, is characterized by malformed ears (‘question mark ears’), prominent cheeks, microstomia, abnormal temporomandibular joint, and mandibular condyle hypoplasia. Penetrance seems to be complete, but there is high inter- and intra-familial phenotypic variation, with no evidence of genetic heterogeneity. We herein describe a new multigeneration family with 11 affected individuals (F1), in whom we confirm intra-familial clinical variability. Facial asymmetry, a clinical feature not highlighted in other ACS reports, was highly prevalent among the patients reported here. The gene responsible for ACS is still unknown and its identification will certainly contribute to the understanding of human craniofacial development. No chromosomal rearrangements have been associated with ACS, thus mapping and positional cloning is the best approach to identify this disease gene. To map the ACS gene, we conducted linkage analysis in two large ACS families, F1 and F2 (F2; reported elsewhere). Through segregation analysis, we first excluded three known loci associated with disorders of first and second pharyngeal arches (Treacher Collins syndrome, oculo-auriculo-vertebral spectrum, and Townes–Brocks syndrome). Next, we performed a wide genome search and we observed evidence of linkage to 1p21.1–q23.3 in F2 (LOD max 3.01 at θ=0). Interestingly, this locus was not linked to the phenotype segregating in F1. Therefore, our results led to the mapping of a first locus of ACS (ACS1) and also showed evidence for genetic heterogeneity, suggesting that there are at least two loci responsible for this phenotype.

Reduced transcription of TCOF1 in adult cells of Treacher Collins syndrome patients

BackgroundTreacher Collins syndrome (TCS) is an autosomal dominant craniofacial disorder caused by frameshift deletions or duplications in the TCOF1 gene. These mutations cause premature termination codons, which are predicted to lead to mRNA degradation by nonsense mediated mRNA decay (NMD). Haploinsufficiency of the gene product (treacle) during embryonic development is the proposed molecular mechanism underlying TCS. However, it is still unknown if TCOF1 expression levels are decreased in post-embryonic human cells.MethodsWe have estimated TCOF1 transcript levels through real time PCR in mRNA obtained from leucocytes and mesenchymal cells of TCS patients (n = 23) and controls (n = 18). Mutational screening and analysis of NMD were performed by direct sequencing of gDNA and cDNA, respectively.ResultsAll the 23 patients had typical clinical features of the syndrome and pathogenic mutations were detected in 19 of them. We demonstrated that the expression level of TCOF1 is 18-31% lower in patients than in controls (p < 0.05), even if we exclude the patients in whom we did not detect the pathogenic mutation. We also observed that the mutant allele is usually less abundant than the wild type one in mesenchymal cells.ConclusionsThis is the first study to report decreased expression levels of TCOF1 in TCS adult human cells, but it is still unknown if this finding is associated to any phenotype in adulthood. In addition, as we demonstrated that alleles harboring the pathogenic mutations have lower expression, we herein corroborate the current hypothesis of NMD of the mutant transcript as the explanation for diminished levels of TCOF1 expression. Further, considering that TCOF1 deficiency in adult cells could be associated to pathologic clinical findings, it will be important to verify if TCS patients have an impairment in adult stem cell properties, as this can reduce the efficiency of plastic surgery results during rehabilitation of these patients.

Does germ-line deletion of the PIP gene constitute a widespread risk for cancer?

We wish to draw the attention of cancer geneticists to a particular genetic variant of the Prolactin-Induced Protein (PIP) gene that may be an important predisposing factor to cancer because of its high frequency and significant association with cancer, as determined in this study. In an initial copy number variation (CNV) screen for germ-line deletions in 123 Brazilian cancer patients selected as high risk either because of early age of onset (pediatric cancer) or a positive family history (TP53-negative Li–Fraumeni and APC/MUTYH-negative Familial Adenomatous Polyposis patients), a previously undescribed microdeletion was discovered in four patients. The deletion carried in these patients was apparently identical (Figure 1), with a size of 69 kb and similar base-pair position in chromosome region 7q34. All four deletions were validated by qPCR and found to harbor only the PIP and TAS2R39 (taste receptor type 2 member 39) genes. TAS2R39 is a member of ∼30 TAS2R bitter taste receptors,1 several of which are known to exhibit variation in copy number. It is unlikely that TAS2R39 has a role in tumorigenesis, and will not be considered further in this submission. We here refer to the deletion encompassing the PIP gene as PIP-Sao Paulo, following the convention of adding the place of discovery to the gene name. This deletion was not detected in a control group of 260 non-related individuals from the Sao Paulo urban area that had attended our genetic service for reasons other than cancer (normal relatives of patients with intellectual disabilities). The difference in deletion frequency between patients and controls was significant at the 0.01 level (Fishers exact test). Another example of the PIP-Sao Paulo deletion was serendipitously detected in a TP53-mutated patient who was not part of these cohorts, but had been investigated by aCGH for presenting with an atypically severe course of cancer that commenced at 4 years of age with the diagnosis of a rhabdomyosarcoma, followed by choroid plexus tumor (7 years of age), liposarcoma and osteochondroma (10 years of age), and finally passed away at 11 years. This patient was not included in the statistics of the patient–control comparison; however, a possible interaction between the TP53 mutation and PIP-Sao Paulo deletion may have contributed to the severity of the cancer progression. Figure 1 PIP-Sao Paulo microdeletion at 7q34, detected by array-CGH in cancer patients. Array-CGH profile of a chromosome region at 7q34 (microarray platform Agilent 180K—Agilent Technologies, Santa Clara, CA, USA), showing heterozygous losses ... Following our initial observations, we interrogated the 1000 Genomes Project database and found a comparable deletion to be present in 13 out of 1097 individuals: 9 of European and 4 of Latin-American origin (see Supplementary Table 1). Manual review of the sequence data in this region indicated that 7 out of the 13 individuals had sufficient sequence depth to determine that they shared identical breakpoints. From this information, we designed PCR primers and were able to amplify across the breakpoints in all the five Brazilian deletion cases. Subsequent sequencing of the PCR fragments (see Supplementary Figure 1) demonstrated that all identified PIP-Sao Paulo deletions were identical to the deletions present in the 1000 Genomes Project data set. In a replicate study, we determined by breakpoint PCR the frequency of PIP deletions in an independent Brazilian cancer group (219 individuals) that had either presented with more than one primary cancer prior to 60 years of age (166 individuals) or were probands of hereditary melanoma families (53 individuals with no CDKN2A or CDK4 mutations). This identified a further 6 individuals, who had had cancer and carried the PIP deletion (2.6%), as opposed to 10 out of 847 individuals (1.2%) from a second control group. As in the initial study, the deletion frequency in the replicate study in cancer patients was higher than that in controls, although this was not significant at the 5% level (P=0.11). However, when cancer and control samples from both studies were pooled, statistical significance was attained (P=0.04, Fishers exact test). Importantly, heterogeneity tests demonstrated that no significant differences existed at the 0.05 level between the two control and two patient samples, respectively. We conclude that the relatively frequent germ-line deletion of the PIP gene has more than a two-fold increase in frequency in the Brazilian cancer patients compared with the Brazilian controls. The clinical phenotype, mutation status, type of tumor and age of diagnosis of all patients carrying the PIP-Sao Paulo deletion are summarized in Table 1, demonstrating that several types of cancer are involved in this study and that no single type or group of cancers predominates. Presence of the PIP-Sao Paulo deletion was investigated in paraffin blocks from several different primary tumors taken from four of the deleted patients. In all samples, except for one from patient 7, the PIP-Sao Paulo deletion was detected (Supplementary Figure 1); additionally, at least one exon of the PIP gene was amplified from each of the tested samples, indicating that one allele was retained, similar to blood. Unfortunately, DNA quality did not allow sequence screening for point mutations or other deletions that may have occurred in the remaining allele. We conclude that if the PIP-Sao Paulo deletion is directly responsible for tumor induction, this has not occurred by induction of homozygosity of the deletion itself in the cases studied. Table 1 Characteristics of the PIP-Sao Paulo deletion patients: mutation status, clinical phenotype, type of tumor and age at diagnosis To date, several publications have reported association between PIP expression and tumor progression, particularly for prostate and breast cancers.2, 3 The gene has been implicated in multiple functions, including apoptosis, cell proliferation and migration,4 but mutations have only been studied in tumor cell lines and never investigated as germ-line mutations. According to the TCGA database (http://cancergenome.nih.gov/), PIP gains in tumors are more frequent than deletions; however, data in different type of tumors are very heterogeneous and difficult to interpret. We have no specific insights into the mechanisms through which the PIP-Sao Paulo deletion could be oncogenic. It is evident from the foregoing that the PIP-Sao Paulo deletion is not specific to Brazil. Inspection of the HapMap3 database shows that the haplotype adjacent to the deletion (Table 2) is either absent or extremely rare in African populations. Interestingly, although about ∼25% of the 1094 individuals sequenced in data set 1 of the 1000 Genome Project were of African origin, none of the 13 individuals carrying the deletion were African and, except for one Colombian and two Mexican individuals from Los Angeles, CA, USA, all others were of European origin. Ethnic certainty of Brazilian cancer patients and controls is obscured by miscegenation and ethnic diversity; however, they are most likely to be mainly Caucasians, given the relatively high Caucasian composition of Sao Paulo, and through hospital attendance preferences and other socioeconomic differences. It is probable that PIP-Sao Paulo arose in an European population as a founder mutation that was subsequently exported to the New World. Table 2 SNPs, genomic positions (Hg19) and the common haplotype deduced from the proximal region of the patients with 7q34 deletion The crucial question of whether the PIP-Sao Paulo deletion constitutes a widespread cancer risk can only be answered by similar investigations in other countries, which we hope will be stimulated by this report. However, maintenance of a deletion variant associated with cancer at such high frequency is surprising and probably implies that, if the cancer association is confirmed by independent studies, PIP-Sao Paulo appears to have a relatively low cancer penetrance rate so that many carriers either do not manifest cancer or do so at later age, following transmission of the deletion to their offspring, similar to the well-documented Brazilian TP53 variant R337H4. Whereas R337H has an estimated population frequency of ∼0.3% in Southern Brazil and exhibits an ∼8% penetrance rate in families with adrenocortical cancer,5 PIP-Sao Paulo is practically an order of magnitude more frequent (estimated at ∼2% in European populations from the 1000 Genome Project data set) and appears as a very frequent factor associated with increased cancer risk. Further, if the PIP-Sao Paulo deletion frequency is at least two-fold higher in European cancer patients than in controls, as in the Sao Paulo study, then even allowing for a low penetrance rate, there would be an increased cancer risk for Europeans. In the case of PIP-Sao Paulo, it is the high frequency and not the penetrance that constitutes the main risk parameter. This completely unanticipated aspect deserves a rapid, coordinated response to confirm whether indeed such an increased risk exists, and if so, to establish its magnitude and specificity. Rapid and cost-effective detection of the deletion can be performed using either PCR across the breakpoints, or copy number estimates by RT-PCR, MLPA and so on. The exact genome location of PIP-Sao Paulo is chr7: 142824847-142893913 (Genome build GRCh37/hg19).

Analysis of allelic differential expression in the human genome using allele-specific serial analysis of gene expression tags

Recent reports have demonstrated that a significant proportion of human genes display allelic differential expression (ADE). ADE is associated with phenotypic variability and may contribute to complex genetic diseases. Here, we present a computational analysis of ADE using allele-specific serial analysis of gene expression (SAGE) tags representing 1295 human genes. We identified 472 genes for which unequal representation (>3-fold) of allele-specific SAGE tags was observed in at least one SAGE library, suggesting the occurrence of ADE. For 235 out of these 472 genes, the difference in the expression level between both allele-specific SAGE tags was statistically significant (p < 0.05). Eleven candidate genes were then subjected to experimental validation and ADE was confirmed for 8 out of these 11 genes. Our results suggest that at least 25% of the human genes display ADE and that allele-specific SAGE tags can be efficiently used for the identification of such genes.