A Molecular Characterization of the Allelic Expression of the BRCA1 Founder Δ9-12 Pathogenic Variant and Its Potential Clinical Relevance in Hereditary Cancer.

Hereditary breast and ovarian cancer (HBOC) syndrome is a genetic condition that increases the risk of breast cancer by 80% and that of ovarian cancer by 40%. The most common pathogenic variants (PVs) causing HBOC occur in the BRCA1 gene, with more than 3850 reported mutations in the gene sequence. The prevalence of specific PVs in BRCA1 has increased across populations due to the effect of founder mutations. Therefore, when a founder mutation is identified, it becomes key to improving cancer risk characterization and effective screening protocols. The only founder mutation described in the Mexican population is the deletion of exons 9 to 12 of BRCA1 (BRCA1Δ9-12), and its description focuses on the gene sequence, but no transcription profiles have been generated for individuals who carry this gene. In this study, we describe the transcription profiles of cancer patients and healthy individuals who were heterozygous for PV BRCA1Δ9-12 by analyzing the differential expression of both alleles compared with the homozygous BRCA1 control group using RT-qPCR, and we describe the isoforms produced by the BRCA1 wild-type and BRCA1Δ9-12 alleles using nanopore long-sequencing. Using the Kruskal-Wallis test, our results showed a similar transcript expression of the wild-type allele between the healthy heterozygous group and the homozygous BRCA1 control group. An association between the recurrence and increased expression of both alleles in HBOC patients was also observed. An analysis of the sequences indicated four wild-type isoforms with diagnostic potential for discerning individuals who carry the PV BRCA1Δ9-12 and identifying which of them has developed cancer.

A Pathogenic Variant Reclassified to the Pseudogene PMS2P1 in a Patient with Suspected Hereditary Cancer.

The PMS2 gene is involved in DNA repair by the mismatch repair pathway. Deficiencies in this mechanism have been associated with Lynch Syndrome (LS), which is characterized by a high risk for colorectal, endometrial, ovarian, breast, and other cancers. Germinal pathogenic variants of PMS2 are associated with up to 5% of all cases of LS. The prevalence is overestimated for the existence of multiple homologous pseudogenes. We report the case of a 44-year-old woman diagnosed with breast cancer at 34 years without a relevant cancer family history. The presence of pathogenic variant NM_000535.7:c.1A > T, (p.Met1Leu) in PMS2 was determined by next-generation sequencing analysis with a panel of 322 cancer-associated genes and confirmed by capillary sequencing in the patient. The variant was determined in six family members (brothers, sisters, and a son) and seven non-cancerous unrelated individuals. Analysis of the amplified region showed high homology of PMS2 with five of its pseudogenes. We determined that the variant is associated with the PMS2P1 pseudogene following sequence alignment analysis. We propose considering the variant c.1A > T, (p.Met1Leu) in PMS2 for reclassification as not hereditary cancer-related, given the impact on the diagnosis and treatment of cancer patients and families carrying this variant.

A Previously Unrecognized Molecular Landscape of Lynch Syndrome in the Mexican Population.

Lynch syndrome (LS) is the main hereditary colorectal cancer syndrome. There have been few reports regarding the clinical and molecular characteristics of LS patients in Latin America; this is particularly true in the Mexican population, where no information is available. The present study aims to describe the clinical and molecular spectrum of variants in a cohort of patients diagnosed with LS in Mexico. We present a retrospective analysis of 412 patients with suspected LS, whose main site of cancer diagnosis was the colon (58.25%), followed by the endometrium (18.93%). Next-generation sequencing analysis, with an extensive multigene panel, showed that 27.1% (112/414) had a variant in one of the genes of the mismatch repair pathway (MMR); 30.4% (126/414) had a variant in non-MMR genes such as CHEK2, APC, MUTYH, BRCA1, and BRCA2; and 42.5% (176/414) had no genetic variants. Most of the variants were found in MLH1. Pathogenic variants (PVs) in MMR genes were identified in 65.7% (96/146) of the total PVs, and 34.24% (45/146) were in non-MMR genes. Molecular and clinical characterization of patients with LS in specific populations allowed personalized follow-up, with the option for targeted treatment with immune checkpoint inhibitors and the development of public health policies. Moreover, such characterization allows for family cascade testing and consequent prevention strategies.

Impact of human papillomavirus coinfections on the risk of high-grade squamous intraepithelial lesion and cervical cancer.

OBJECTIVE: The molecular and epidemiologic effect of human papillomavirus (HPV) coinfections in the risk of developing cervical cancer is yet unclear. The aim of this study was to determine the frequency HPV coinfections at different stages of cervical lesions in the development of cervical cancer and the impact of HPV specific type interactions on high-grade squamous intraepithelial lesions (HSIL) and invasive cervical cancer (ICC) risk. METHODS: HPV testing was performed in 931 cervical samples diagnosed as: negative for intraepithelial lesion or malignancy (NILM); low-grade squamous intraepithelial lesion (LSIL); HSIL; and ICC. For HPV detection and typing two sets of primers from the L1 region were used in the polymerase chain reaction method (PCR) (MY09/MY11/HMB01 and L1C1/L1C2.1/L1C2.2) and HPV type was determined by PCR product sequence. To look for multiple HPV infections, the E6 nested multiplex PCR method was performed in all DNA samples. Odds ratios were calculated as indexes of the strength of the association between the sample category (LSIL/NILM or ICC/HSIL) and the presence of a given viral combination. RESULTS: In HPV positive samples, coinfections are as common in ICC/HSIL as in LSIL/NILM (47.12% and 40.17%, respectively). There is an increased risk to ICC/HSIL when multiple high-risk HPV types are present. The coinfection of HPV68 with HPV16 increases the risk of ICC/HSIL (OR=14.54, P=0.012, after multivariate adjustment), related to the presence of HPV16 or HPV68 alone. CONCLUSIONS: These results sustain that specific HPV coinfections confer an increased risk to develop ICC/HSIL.

Molecular changes in adipocyte-derived stem cells during their interplay with cervical cancer cells.

PURPOSE: Obesity is as an important risk factor and has been associated with a worse prognosis in at least 13 distinct tumor types. This is partially due to intercellular communication between tumor cells and adipose tissue-derived stem cells (ADSCs), which are increased in obese individuals. As yet, however, little is known about the molecular changes occurring in ADSCs in these conditions. Cervical cancer has a high incidence and mortality rate in women from developing countries, particularly in those with a high body mass index (BMI). METHODS: We analyzed the expression profile of ADSCs co-cultured with cervical cancer cells through massive RNA sequencing followed by evaluation of various functional alterations resulting from the modified transcriptome. RESULTS: A total of 761 coding and non-coding dysregulated RNAs were identified in ADSCs after co-culture with HeLa cells (validation in CaSki and SiHA cells). Subsequent network analysis showed that these changes were correlated with migration, stemness, DNA repair and cytokine production. Functional experiments revealed a larger ALDHhigh subpopulation and a higher migrative capacity of ADSCs after co-culture with HeLa cells. Interestingly, CXCL3 and its intragenic long-noncoding RNA, lnc-CXCL3, were found to be co-regulated during co-culture. A loss-of-function assay revealed that lnc-CXCL3 acts as a key regulator of CXCL3 expression. CONCLUSIONS: Our results suggest that intercellular communication between ADSCs and cervical cancer cells modifies the RNA expression profile in the former, including that of lncRNAs, which in turn can regulate the expression of diverse chemokines that favor malignancy-associated capacities such as migration.

Familial erythrocytosis 2 and von Hippel-Lindau disease in the same pediatric patient.

BACKGROUND: Patients with familial erythrocytosis type 2 have no increased risk of von Hippel-Lindau-associated tumors, although mutations in the VHL gene cause both pathologies. CASE REPORT: We present a case of a compound heterozygote patient with von Hippel-Lindau disease and familial erythrocytosis type 2. One of the mutations found in our patient, c.416C>G (p.Ser139Cys) of the VHL gene, has not been previously reported. This case is the second one reported where von Hippel-Lindau disease and familial erythrocytosis type 2 coexist in the same individual. CONCLUSIONS: Despite the low frequency of familial erythrocytosis type 2 in patients with von Hippel-Lindau disease, the possibility of this diagnosis should be considered to avoid unnecessary invasive studies to explain the polyglobulia in these patients and guarantee an adequate follow-up and vigilance of both diseases.

The Promising Role of miR-21 as a Cancer Biomarker and Its Importance in RNA-Based Therapeutics.

MicroRNAs are small noncoding transcripts that posttranscriptionally regulate gene expression via base-pairing complementarity. Their role in cancer can be related to tumor suppression or oncogenic function. Moreover, they have been linked to processes recognized as hallmarks of cancer, such as apoptosis, invasion, metastasis, and proliferation. Particularly, one of the first oncomiRs found upregulated in a variety of cancers, such as gliomas, breast cancer, and colorectal cancer, was microRNA-21 (miR-21). Some of its target genes associated with cancer are PTEN (phosphatase and tensin homolog), PDCD4 (programmed cell death protein 4), RECK (reversion-inducing cysteine-rich protein with Kazal motifs), and STAT3 (signal transducer activator of transcription 3). As a result, miR-21 has been proposed as a plausible diagnostic and prognostic biomarker, as well as a therapeutic target for several types of cancer. Currently, research and clinical trials to inhibit miR-21 through anti-miR-21 oligonucleotides and ADM-21 are being conducted. As all of the evidence suggests, miR-21 is involved in carcinogenic processes; therefore, inhibiting it could have effects on more than one type of cancer. However, whether miR-21 can be used as a tissue-specific biomarker should be analyzed with caution. Consequently, the purpose of this review is to outline the available information and recent advances regarding miR-21 as a potential biomarker in the clinical setting and as a therapeutic target in cancer to highlight its importance in the era of precision medicine.

Clinical Evaluation of BRCA1/2 Mutation in Mexican Ovarian Cancer Patients.

Ovarian cancer (OC) is an important cause of gynecologic cancer-related deaths. In Mexico, around 4700 new cases of OC are diagnosed per year and it represents the second cause of gynecological cancer mortality with more than 2700 deaths. Germline mutations in BRCA1/2 genes are present in 13-18% of OC cases. Few studies have evaluated the presence of mutations in BRCA genes in a population of OC Mexican patients and their relationship with clinical response and survival rates. A total of 179 OC patients were studied by molecular testing for BRCA1/2 through next-generation sequencing and multiplex ligation-dependent probe amplification. Recurrence-free survival (RFS) was estimated by the Kaplan-Meier method. BRCA mutation was detected in 33% of patients. A percentage of 66.1% were BRCA1 mutated and 33.9% were BRCA2 mutated. BRCA1 mutation carriers had a worst RFS compared with BRCA2 mutation carriers (37.6 [29-46.2] vs 72.7 [38.4-107.2]; P = 0.030). The most common mutation for BRCA1 was ex9-12del (28.2%) (Mexican founder mutation). The Mexican founder mutation had a better RFS than other BRCA1 mutations (86.1 [37.2-135.1] vs 34.5 [20.7-48.2]; P = 0.033). The presence of BRCA2 mutations in the ovarian cancer cluster region (OCCR) had a significantly better RFS than mutations in breast cancer cluster regions (BCCR) and not-related risk region (NRR) (NR vs 72.8 [39-106.6] vs 25.8 [8.3-43.2]; P = 0.013). These results demonstrate that the prevalence of BRCA1/2 positive patients in OC Mexican patients are the highest reported. Patients with mutations in BRCA2 have a better prognosis than those mutated in BRCA1. The Mexican founder mutation has an important role in clinical outcomes. These results highlight the importance to test all the HGSP (high-grade serous papillary) OC patients with or without cancer family history (CFH) in Mexican population.

MicroRNAs in Tumor Cell Metabolism: Roles and Therapeutic Opportunities.

Dysregulated metabolism is a common feature of cancer cells and is considered a hallmark of cancer. Altered tumor-metabolism confers an adaptive advantage to cancer cells to fulfill the high energetic requirements for the maintenance of high proliferation rates, similarly, reprogramming metabolism confers the ability to grow at low oxygen concentrations and to use alternative carbon sources. These phenomena result from the dysregulated expression of diverse genes, including those encoding microRNAs (miRNAs) which are involved in several metabolic and tumorigenic pathways through its post-transcriptional-regulatory activity. Further, the identification of key actionable altered miRNA has allowed to propose novel targeted therapies to modulated tumor-metabolism. In this review, we discussed the different roles of miRNAs in cancer cell metabolism and novel miRNA-based strategies designed to target the metabolic machinery in human cancer.

Mexican BRCA1 founder mutation: Shortening the gap in genetic assessment for hereditary breast and ovarian cancer patients.

The deletion of exons 9 to 12 of BRCA1 (9-12 del BRCA1) is considered a founder mutation in the Mexican population. We evaluate the usefulness of the target detection of 9-12 del BRCA1 as the first molecular diagnostic strategy in patients with Hereditary Breast and Ovarian Cancer (HBOC). We performed the genetic assessment of 637 patients with suspected HBOC. The region corresponding to the breakpoints for the 9-12 del BRCA1 was amplified by polymerase chain reaction (PCR). An analysis of the clinical data of the carriers and non-carriers was done, searching for characteristics that correlated with the deletion. The 9-12 del BRCA1 was detected in 5% of patients with suspected HBOC (30/637). In patients diagnosed with ovarian cancer, 13 of 30 were 9-12 del BRCA1 carriers, which represents 43%. We found a significant association between the 9-12 del BRCA1 carriers with triple negative breast cancer and high-grade papillary serous ovarian cancer. We concluded that the detection of the 9-12 del BRCA1 is useful as a first molecular diagnostic strategy in the Mexican population. In particular, it shortens the gap in genetic assessment in patients with triple negative breast cancer and ovarian cancer.

Comprehensive Analysis of Germline Variants in Mexican Patients with Hereditary Breast and Ovarian Cancer Susceptibility.

Hereditary breast and ovarian cancer syndrome (HBOC) represents 5⁻10% of all patients with breast cancer and is associated with high-risk pathogenic alleles in BRCA1/2 genes, but only for 25% of cases. We aimed to find new pathogenic alleles in a panel of 143 cancer-predisposing genes in 300 Mexican cancer patients with suspicion of HBOC and 27 high-risk patients with a severe family history of cancer, using massive parallel sequencing. We found pathogenic variants in 23 genes, including BRCA1/2. In the group of cancer patients 15% (46/300) had a pathogenic variant; 11% (33/300) harbored variants with unknown clinical significance (VUS) and 74% (221/300) were negative. The high-risk group had 22% (6/27) of patients with pathogenic variants, 4% (1/27) had VUS and 74% (20/27) were negative. The most recurrent mutations were the Mexican founder deletion of exons 9-12 and the variant p.G228fs in BRCA1, each found in 5 of 17 patients with alterations in this gene. Rare VUS with potential impact at the protein level were found in 21 genes. Our results show for the first time in the Mexican population a higher contribution of pathogenic alleles in other susceptibility cancer genes (54%) than in BRCA1/2 (46%), highlighting the high locus heterogeneity of HBOC and the necessity of expanding genetic tests for this disease to include broader gene panels.

Locus on chromosome 6p linked to elevated HDL cholesterol serum levels and to protection against premature atherosclerosis in a kindred with familial hypercholesterolemia.

Heterozygous familial hypercholesterolemia (FH) is a highly atherogenic genetic disorder leading to premature coronary heart disease (CHD), usually before 60 years of age. We studied an extended multigenerational kindred with FH linked to chromosome 1p32 in which atherosclerotic complications were either delayed or prevented in individuals with elevated HDL cholesterol (HDL-C) levels or hyperalphalipoproteinemia (HA). Premature CHD was observed in FH individuals without HA. The study of this family established that the HA trait in the family also followed an autosomal dominant mode of inheritance with a pattern of segregation independent from FH. We identified a locus on chromosome 6 linked to elevated HDL-C levels (HA) in this family. Haplotype analysis refined the localization to a 7.32-cM interval (73 to 80 cM from pter) flanked by markers D6S1280 and D6S1275. Parametric 2-point and multipoint analyses yielded maximum LOD scores of 3.05 and 3.17, respectively. This finding was confirmed with a nonparametric multipoint score of 3.78 (P=0.0009). We propose that this locus, linked to elevated HDL-C levels, confers protection against premature CHD within an FH context.

Familial erythrocytosis 2 and von Hippel-Lindau disease in the same pediatric patient / Eritrocitosis familiar 2 y enfermedad de von Hippel-Lindau en el mismo paciente

Abstract Background: Patients with familial erythrocytosis type 2 have no increased risk of von Hippel-Lindau-associated tumors, although mutations in the VHL gene cause both pathologies. Case report: We present a case of a compound heterozygote patient with von Hippel-Lindau disease and familial erythrocytosis type 2. One of the mutations found in our patient, c.416C>G (p.Ser139Cys) of the VHL gene, has not been previously reported. This case is the second one reported where von Hippel-Lindau disease and familial erythrocytosis type 2 coexist in the same individual. Conclusions: Despite the low frequency of familial erythrocytosis type 2 in patients with von Hippel-Lindau disease, the possibility of this diagnosis should be considered to avoid unnecessary invasive studies to explain the polyglobulia in these patients and guarantee an adequate follow-up and vigilance of both diseases.

Resumen Introducción: Los pacientes con eritrocitosis familiar tipo 2 no muestran un riesgo incrementado de desarrollar tumores asociados con la enfermedad de von Hippel-Lindau, a pesar de que ambas afecciones están causadas por variantes patogénicas en el gen VHL. Caso clínico: Se presenta el caso de un paciente heterocigoto compuesto con enfermedad de von Hippel-Lindau y eritrocitosis familiar tipo 2. Una de las variantes patogénicas en el paciente, VHL c.416C>G (p.Ser139Cys), no ha sido previamente reportada. Este es el segundo reporte de caso en que la enfermedad de von Hippel-Lindau y la eritrocitosis familiar tipo 2 coexisten en el mismo individuo. Conclusiones: A pesar de la baja frecuencia de la eritrocitosis familiar tipo 2 en pacientes con enfermedad de von Hippel-Lindau, la posibilidad del diagnóstico debe considerarse con el fin de evitar estudios invasivos innecesarios para explicar la presencia de poliglobulia en estos pacientes y para garantizar un adecuado seguimiento y una correcta vigilancia de ambas enfermedades.

Mutations in MODY genes are not common cause of early-onset type 2 diabetes in Mexican families.

CONTEXT: Maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes mellitus characterized by autosomal dominant inheritance, early age of onset and a primary insulin secretion defect. Certain MODY gene sequence variants may be involved in polygenic forms of type 2 diabetes. OBJECTIVE: We assessed the contribution of MODY genes to the etiology of type 2 early-onset diabetes in 23 Mexican families, including five with apparently autosomal dominant inheritance. PATIENTS: Twenty-three unrelated Mexican families with early-onset type 2 diabetes previously screened for the presence of glucokinase mutations, were studied. DESIGN: We screened MODY genes for sequence variants by PCR-SSCP analysis and automated sequencing. We performed a functional analysis of the HNF-1alpha P379H recombinant protein in vitro in both HeLa and RINm5f beta-cell lines. MAIN OUTCOME MEASURES: MODY gene mutation screening and P379H mutant protein transactivation assay. RESULTS: No mutations were detected in the HNF-4alpha, IPF-1, NEUROD1 or HNF-1beta genes in any of the families studied. A new mutation (P379H) of the HNF-1alpha gene was identified in one MODY family. RINm5f and HeLa cell transfection assays revealed decreased transactivation activity of the mutant protein on the human insulin promoter. CONCLUSIONS: All known MODY genes were screened for abnormalities in this cohort of early-onset diabetes families which included 5 MODY pedigrees. We identified a new HNF-1alpha MODY mutation (P379H) and demonstrated that it reduces the transactivation potential of the mutant protein on the human insulin promoter. No other mutation was identified in this cohort indicating that abnormalities in MODY genes are generally not a common cause of early-onset diabetes and this includes MODY families in Mexico.

Revealing the Molecular Portrait of Triple Negative Breast Tumors in an Understudied Population through Omics Analysis of Formalin-Fixed and Paraffin-Embedded Tissues.

Triple negative breast cancer (TNBC), defined by the lack of expression of the estrogen receptor, progesterone receptor and human epidermal receptor 2, is an aggressive form of breast cancer that is more prevalent in certain populations, in particular in low- and middle-income regions. The detailed molecular features of TNBC in these regions remain unexplored as samples are mostly accessible as formalin-fixed paraffin embedded (FFPE) archived tissues, a challenging material for advanced genomic and transcriptomic studies. Using dedicated reagents and analysis pipelines, we performed whole exome sequencing and miRNA and mRNA profiling of 12 FFPE tumor tissues collected from pathological archives in Mexico. Sequencing analyses of the tumor tissues and their blood pairs identified TP53 and RB1 genes as the most frequently mutated genes, with a somatic mutation load of 1.7 mutations/exome Mb on average. Transcriptional analyses revealed an overexpression of growth-promoting signals (EGFR, PDGFR, VEGF, PIK3CA, FOXM1), a repression of cell cycle control pathways (TP53, RB1), a deregulation of DNA-repair pathways, and alterations in epigenetic modifiers through miRNA:mRNA network de-regulation. The molecular programs identified were typical of those described in basal-like tumors in other populations. This work demonstrates the feasibility of using archived clinical samples for advanced integrated genomics analyses. It thus opens up opportunities for investigating molecular features of tumors from regions where only FFPE tissues are available, allowing retrospective studies on the search for treatment strategies or on the exploration of the geographic diversity of breast cancer.

Gene expression profiles induced by E6 from non-European HPV18 variants reveals a differential activation on cellular processes driving to carcinogenesis.

Cervical cancer in developed countries remains as a major concern on public health policies due to incidence and mortality rates. Persistent infection with high risk human papillomavirus is a necessary etiological agent in the progression to invasive cervical carcinoma. A proposed hypothesis is the association between more aggressive HPV variants and the risk to develop cervical cancer. In order to have a global perspective in terms of cellular transcripts and molecular pathways affected by HPV18 E6 intratype variants; we conducted a genome wide analysis of gene expression. Our results show that E6 derived from non-European variants are able to up-regulate cellular transcripts associated to the hallmarks of cancer; such as cell cycle, migration, Wnt pathway and mTor signaling. Moreover, we were able to show that HPV18 E6 from African variant had a major effect on cellular processes such as cell cycle and migration as confirmed by functional studies.

Full-exon pyrosequencing screening of BRCA germline mutations in Mexican women with inherited breast and ovarian cancer.

Hereditary breast cancer comprises 10% of all breast cancers. The most prevalent genes causing this pathology are BRCA1 and BRCA2 (breast cancer early onset 1 and 2), which also predispose to other cancers. Despite the outstanding relevance of genetic screening of BRCA deleterious variants in patients with a history of familial cancer, this practice is not common in Latin American public institutions. In this work we assessed mutations in the entire exonic and splice-site regions of BRCA in 39 patients with breast and ovarian cancer and with familial history of breast cancer or with clinical features suggestive for BRCA mutations by massive parallel pyrosequencing. First we evaluated the method with controls and found 41-485 reads per sequence in BRCA pathogenic mutations. Negative controls did not show deleterious variants, confirming the suitability of the approach. In patients diagnosed with cancer we found 4 novel deleterious mutations (c.2805_2808delAGAT and c.3124_3133delAGCAATATTA in BRCA1; c.2639_2640delTG and c.5114_5117delTAAA in BRCA2). The prevalence of BRCA mutations in these patients was 10.2%. Moreover, we discovered 16 variants with unknown clinical significance (11 in exons and 5 in introns); 4 were predicted as possibly pathogenic by in silico analyses, and 3 have not been described previously. This study illustrates how massive pyrosequencing technology can be applied to screen for BRCA mutations in the whole exonic and splice regions in patients with suspected BRCA-related cancers. This is the first effort to analyse the mutational status of BRCA genes on a Mexican-mestizo population by means of pyrosequencing.