Mónica Alejandra Rosales-Reynoso

Gene expression profiling identifies WNT7A as a possible candidate gene for decreased cancer risk in fragile X syndrome patients.

BACKGROUND AND AIMS Although sporadic cases of cancer in patients with fragile X syndrome (FXS) have been reported, extensive studies carried out in Denmark and Finland concluded that cancer incidence in these patients is lower than in the general population. On the other hand, the FMR1 protein, which is involved in the translation process, is absent in FXS patients. Hence, it is reasonable to assume that these patients exhibit an abnormal expression of some proteins involved in regulating tumor suppressor genes and/or oncogenes, thus explaining its decreased cancer frequency. We undertook this study to analyze the expression of oncogenes and tumor suppressor genes in fragile X syndrome patients. METHODS Molecular analysis of the FMR1 gene was achieved in 10 male patients and controls. Total RNA from peripheral blood was used to evaluate expression of oncogenes and tumor suppressor genes included in a 10,000 gene microarray library. Quantitative real-time PCR was utilized to confirm genes with differential expression. RESULTS Among 27 genes showing increased expression in FXS patients, only eight genes exhibited upregulation in at least 50% of them. Among these, ARMCX2 and PPP2R5C genes are tumor suppressor related. Likewise, 23/65 genes showed decreased expression in >50% of patients. Among them, WNT7A gene is a ligand of the beta-catenin pathway, which is widely related to oncogenic processes. Decreased expression of WNT7A was confirmed by quantitative RT-PCR. Expression of c-Myc, c-Jun, cyclin-D and PPARdelta genes, as target of the beta-catenin pathway, was moderately reduced in FXS patients. CONCLUSIONS Results suggest that this diminished expression of the WNT7A gene may be related to a supposed protection of FXS patients to develop cancer.

Peripheral T-lymphocytes express WNT7A and its restoration in leukemia-derived lymphoblasts inhibits cell proliferation

BackgroundWNT7a, a member of the Wnt ligand family implicated in several developmental processes, has also been reported to be dysregulated in some types of tumors; however, its function and implication in oncogenesis is poorly understood. Moreover, the expression of this gene and the role that it plays in the biology of blood cells remains unclear. In addition to determining the expression of the WNT7A gene in blood cells, in leukemia-derived cell lines, and in samples of patients with leukemia, the aim of this study was to seek the effect of this gene in proliferation.MethodsWe analyzed peripheral blood mononuclear cells, sorted CD3 and CD19 cells, four leukemia-derived cell lines, and blood samples from 14 patients with Acute lymphoblastic leukemia (ALL), and 19 clinically healthy subjects. Reverse transcription followed by quantitative Real-time Polymerase chain reaction (qRT-PCR) analysis were performed to determine relative WNT7A expression. Restoration of WNT7a was done employing a lentiviral system and by using a recombinant human protein. Cell proliferation was measured by addition of WST-1 to cell cultures.ResultsWNT7a is mainly produced by CD3 T-lymphocytes, its expression decreases upon activation, and it is severely reduced in leukemia-derived cell lines, as well as in the blood samples of patients with ALL when compared with healthy controls (p ≤0.001). By restoring WNT7A expression in leukemia-derived cells, we were able to demonstrate that WNT7a inhibits cell growth. A similar effect was observed when a recombinant human WNT7a protein was used. Interestingly, restoration of WNT7A expression in Jurkat cells did not activate the canonical Wnt/β-catenin pathway.ConclusionsTo our knowledge, this is the first report evidencing quantitatively decreased WNT7A levels in leukemia-derived cells and that WNT7A restoration in T-lymphocytes inhibits cell proliferation. In addition, our results also support the possible function of WNT7A as a tumor suppressor gene as well as a therapeutic tool.

DNA repair/replication transcripts are down regulated in patients with Fragile X Syndrome

BackgroundFragile X Syndrome (FXS) and its associated disorders are caused by the expansion of the CGG repeat in the 5’ untranslated region of the fragile X mental retardation 1 (FMR1) gene, with disease classification based on the number of CGG repeats. The mechanisms of repeat expansion are dependent on the presence of cis elements and the absence of trans factors both of which are not mutually exclusive and contribute to repeat instability. Expansions associated with trans factors are due to the haploinsuffient or reduced expression of several DNA repair/metabolizing proteins. The reduction of expression in trans factors has been primarily conducted in animal models without substantial examination of many of these expansion mechanisms and trans factors in humans.ResultsTo understand the trans factors and pathways associated with trinucleotide repeat expansion we have analyzed two microarray datasets which characterized the transcript expression in patients with FXS and in controls.ConclusionWe observed significant down regulation of DNA damage/repair pathway transcripts. This observation was consistent in both datasets, which used different populations. Within these datasets, several transcripts overlapped in the direction of association and fold change. Further characterization of these genes will be critical to understand their role in trinucleotide repeat instability in FXS.

Genetic variation of the FMR1 gene among four Mexican populations: Mestizo, Huichol, Purepecha, and Tarahumara.

Fragile X syndrome is the most common cause of inherited mental retardation; it is caused by expansion of CGG repeats in the first exon of the FMR1 gene. The number of CGG repeats varies between 6 and 50 triplets in normal individuals; the most common alleles have 29 or 30 repeats. Allelic patterns in the global populations are similar; however; some reports show statistical differences among several populations. In Mexico, except by a single report on a western Mestizo population, the allelic frequencies of the FMR1 gene are unknown. In this study, we analyze 207, 140, 138, and 40 chromosomes from Mestizos, Tarahumaras, Huichols, and Purepechas respectively. After PCR amplification on DNA modified by sodium bisulfite treatment, molecular analysis of the FMR1 gene showed 30 different alleles among the 525 chromosomes evaluated. Trinucleotide repeat number in the different Mexican populations varied from 15 to 87, with modal numbers of 32 and 30 in Mestizos and Tarahumaras, 29 and 32 in Purepechas and 30 among Huichols. Together, these allelic patterns differ significantly from those reported for Caucasian, Chinese, African, Indonesian, Brazilian, and Chilean populations. The increased number of the unusual allele of 32 repeats observed in the Mexican mestizo population can be explained from its frequency in at least two Mexican native populations. Am. J. Hum. Biol., 2008.

FMR1 Protein Expression in Blood Smears for Fragile X Syndrome Diagnosis in a Mexican Population Sample

Molecular diagnosis of fragile X syndrome (FXS) is carried out by Southern blot or polymerase chain reaction-Southern analysis; however, these procedures are expensive and time consuming, making it impractical for mass screening programs. Willemsen et al. developed and tested the diagnostic potential of a rapid antibody test on blood smears, based on the presence of fragile X mental retardation protein (FMRP) in peripheral lymphocytes from normal individuals and its absence in male patients with FXS. The diagnostic power of this antibody test is perfect for men, whereas the results are less specific for women. Validation of this procedure has been achieved mainly in the Caucasian population, but no reports including Latin American individuals have been published. To test this procedure, expression of FMRP in peripheral lymphocytes was achieved both in Mexican FXS patients and normal men and was compared with the molecular analysis of the CGG repetitive sequences of the FMR1 gene. The results of the antibody test, which measure the FMRP expression, entirely correlated with the molecular tests using polymerase chain reaction on DNA modified. Sensitivity and specificity of the test and the positive and negative predictive values were 100%. This noninvasive test requires one or two blood drops; it is rapid, simple, and inexpensive, making this procedure an ideal choice for screening large groups of male patients with mental retardation.

AXIN2 Polymorphisms and Their Association with Colorectal Cancer in Mexican Patients

AIM The aim of this study was to investigate the association of the rs2240308 and rs1133683 polymorphisms in the AXIN2 gene with colorectal cancer (CRC) in Mexican patients. MATERIALS AND METHODS Genomic DNAs from 201 CRC patients and 100 healthy blood donors were analyzed for AXIN2 gene polymorphisms by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Statistical associations were calculated using the odds ratio (OR) test. RESULTS The genotype distribution of the rs1133683 polymorphism C > T showed a statistical difference between the two study groups (p = 0.0019). Moreover, OR analyses demonstrated that individuals with either the C/T or T/T genotype have a decreased risk for CRC compared with individuals with the C/C genotype (OR = 0.47, 95% confidence interval [CI] = 0.25-0.86, p = 0.0134 and OR = 0.24, 95% CI = 0.10-0.57, p = 0.005, respectively). This association was also evident in a stratified analysis based on tumor-node-metastasis (TNM) stage. For the rs2240308 polymorphism C > T, the OR analysis showed a significantly increased risk for carriers of the T/T genotype (OR = 2.64, 95% CI = 1.12-6.24, p = 0.0236) and this association was also evident in the stratified analysis by TNM stage. CONCLUSION Our results indicate the possibility that variations in the AXIN2 gene may play a significant role in promoting or preventing CRC development.