Hugo A. Barrera-Saldaña

Landscape of genomic alterations in cervical carcinomas

Cervical cancer is responsible for 10–15% of cancer-related deaths in women worldwide. The aetiological role of infection with high-risk human papilloma viruses (HPVs) in cervical carcinomas is well established. Previous studies have also implicated somatic mutations in PIK3CA, PTEN, TP53, STK11 and KRAS as well as several copy-number alterations in the pathogenesis of cervical carcinomas. Here we report whole-exome sequencing analysis of 115 cervical carcinoma–normal paired samples, transcriptome sequencing of 79 cases and whole-genome sequencing of 14 tumour–normal pairs. Previously unknown somatic mutations in 79 primary squamous cell carcinomas include recurrent E322K substitutions in the MAPK1 gene (8%), inactivating mutations in the HLA-B gene (9%), and mutations in EP300 (16%), FBXW7 (15%), NFE2L2 (4%), TP53 (5%) and ERBB2 (6%). We also observe somatic ELF3 (13%) and CBFB (8%) mutations in 24 adenocarcinomas. Squamous cell carcinomas have higher frequencies of somatic nucleotide substitutions occurring at cytosines preceded by thymines (Tp*C sites) than adenocarcinomas. Gene expression levels at HPV integration sites were statistically significantly higher in tumours with HPV integration compared with expression of the same genes in tumours without viral integration at the same site. These data demonstrate several recurrent genomic alterations in cervical carcinomas that suggest new strategies to combat this disease.

Conserved Methylation Patterns of Human Papillomavirus Type 16 DNA in Asymptomatic Infection and Cervical Neoplasia

ABSTRACT DNA methylation contributes to the chromatin conformation that represses transcription of human papillomavirus type16 (HPV-16), which is prevalent in the etiology of cervical carcinoma. In an effort to clarify the role of this phenomenon in the regulation and carcinogenicity of HPV-16, 115 clinical samples were studied to establish the methylation patterns of the 19 CpG dinucleotides within the long control region and part of the L1 gene by bisulfite modification, PCR amplification, DNA cloning, and sequencing. We observed major heterogeneities between clones from different samples as well as between clones from individual samples. The methylation frequency of CpGs was measured at 14.5%. In addition, 0.21 and 0.23%, respectively, of the CpA and CpT sites, indicators of de novo methylation, were methylated. Methylation frequencies exceeded 30% in the CpGs overlapping with the L1 gene and were about 10% for most other positions. A CpG site located in the linker between two nucleosomes positioned over the enhancer and promoter of HPV-16 had minimal methylation. This region forms part of the HPV replication origin and is close to binding sites of master-regulators of transcription during epithelial differentiation. Methylation of most sites was highest in carcinomas, possibly due to tandem repetition and chromosomal integration of HPV-16 DNA. Methylation was lowest in dysplasia, likely reflecting the transcriptional activity in these infections. Our data document the efficient targeting of HPV genomes by the epithelial methylation machinery, possibly as a cellular defense mechanism, and suggest involvement of methylation in HPV oncogene expression and the early-late switch.

Folate levels and N(5),N(10)-methylenetetrahydrofolate reductase genotype (MTHFR) in mothers of offspring with neural tube defects: a case-control study.

BACKGROUND Neural tube defects (NTDs) have been associated with biochemical factors involved in the conversion of homocysteine to methionine as folate deficiency and the mutation 677T in the N(5),N(10)-methylenetetrahydrofolate reductase gene (MTHFR). METHODS A case-control study was performed to detect this mutation in 38 unrelated women with NTD deceased products and 31 mothers without antecedents of NTD offspring. All products were born in Nuevo León (northeastern Mexico) during 1997. Erythrocyte and plasmatic folate levels and the genotype of the 677 polymorphism at the MTHFR locus were analyzed in both groups. RESULTS Although no significant differences were found in mean blood folate levels, the percentage of women in the case group with erythrocyte folate levels <160 ng/mL was significantly higher than in the control group (75 vs. 51.2%, p <0.05). The proportion of women with plasma folate levels <3.5 ng/mL was higher in the case group (16.2 vs. 0%, p <0.01). Genotype analysis demonstrated a significantly higher proportion of 677T homozygous mothers with NTD products (39.6 vs. 9.1%, p <0.05). Allele frequencies for the 677T mutation were 0.55 and 0.36 for cases and controls, respectively. The odds ratio (OR) for having a NTD product was 6.1 (95%, CI 1.56-23.6) for homozygous 677T mothers vs. homozygous 677C and heterozygous mothers. Significantly low levels of erythrocyte folate were found in the 677C homozygous case group and in plasma folate in the 677C/677T heterozygous case mothers. CONCLUSIONS Our study suggests that folate deficiency and MTHFR unfavorable genotype in mothers are important risk factors for severe NTD phenotype in our population.

Acetylsalicylic acid inhibits hepatitis C virus RNA and protein expression through cyclooxygenase 2 signaling pathways

It has been reported that salicylates (sodium salicylate and aspirin) inhibit the replication of flaviviruses, such as Japanese encephalitis virus and dengue virus. Therefore, we considered it important to test whether acetylsalicylic acid (ASA) had anti–hepatitis C virus (HCV) activity. To this end, we examined the effects of ASA on viral replication and protein expression, using an HCV subgenomic replicon cell culture system. We incubated Huh7 replicon cells with 2‐8 mM ASA for different times and measured HCV‐RNA and protein levels by northern blot, real‐time polymerase chain reaction, and western analysis, respectively. We found that ASA had a suppressive effect on HCV‐RNA and protein levels (nearly 58%). ASA‐dependent inhibition of HCV expression was not mediated by the 5′‐internal ribosome entry site or 3′‐untranslated regions, as determined by transfection assays using bicistronic constructs containing these regulatory regions. However, we found that HCV‐induced cyclooxygenase 2 (COX‐2) messenger RNA and protein levels and activity and these effects were down‐regulated by ASA, possibly by a nuclear factor kappa B–independent mechanism. We also observed that the ASA‐dependent inhibition of viral replication was due in part to inhibition of COX‐2 and activation of p38 and mitogen‐activated protein kinase/extracellular signal‐regulated kinase kinase 1/2 (MEK1/2) mitogen‐activated protein kinases (MAPKs). Inhibition of these kinases by SB203580 and U0126, respectively, and by short interfering RNA silencing of p38 and MEK1 MAPK prevented the antiviral effect of ASA. Taken together, our findings suggest that the anti‐HCV effect of ASA in the Huh7 replicon cells is due to its inhibitory effect on COX‐2 expression, which is mediated in part by the activation of MEK1/2/p38 MAPK. Conclusion: These findings suggest the possibility that ASA could be an excellent adjuvant in the treatment of chronic HCV infection. (HEPATOLOGY 2008.)

Papillomavirus Subtypes Are Natural and Old Taxa: Phylogeny of Human Papillomavirus Types 44 and 55 and 68a and -b

ABSTRACT A human papillomavirus (HPV) type is defined as an HPV isolate whose L1 gene sequence is at least 10% different from that of any other type, while a subtype is 2 to 10% different from any HPV type. In order to analyze the phylogeny behind the subtype definition, we compared 49 isolates of HPV type 44 (HPV-44) and its subtype HPV-55, previously misclassified as a separate type, and 41 isolates of the subtype pair HPV-68a and -b, sampled from cohorts in four continents. The subtypes of each pair are separated by deep dichotomic branching, and three of the four subtypes have evolved large phylogenetic clusters of genomic variants forming a “star” phylogeny, with some branches specific for ethnically defined cohorts. We conclude that subtypes of HPV types are natural and old taxa, equivalent to types, which either diverged more recently than types or evolved more slowly.

Absence of human placental lactogen and placental growth hormone (HGH-V) during pregnancy: PCR analysis of the deletion.

Abstract Human placental lactogen (HPL) is produced in large amounts in normal pregnancies. We report a pregnancy with complete lack of HPL and the placental variant of the human growth hormone HGH-V. The pregnancy resulted in a severely growth-retarded but otherwise normal male baby. PCR analysis of DNA extracted from the placenta showed that the HPL encoding genes hPL-4 and hPL-3 were deleted along with the human growth hormone variant gene (hGH-V), which is located between these two active hPL genes and also expressed in the normal placenta. Of the five members of this multigene family, hGH-N, which is expressed in the pituitary gland, and hPL-1, a presumed pseudogene, were left intact. The latter (hPL-1) was expressed as RNA transcripts only at very low levels as is usually reported in normal pregnancies. Analysis of the parents’ DNA showed that both of them carried a different heterozygous deletion at the 3′ end of the hGH/hPL locus.

Detection and differentiation of the six Brucella species by polymerase chain reaction.

BackgroundBrucelosis is a severe acute febrile disease caused by bacteria of the genus Brucella. Its current diagnosis is based on clinical observations that may be complemented by serology and microbiological culture tests; however, the former is limited in sensitivity and specificity, the latter is time consuming. To improve brucelosis diagnosis we developed a test which is specific and sensitive and is capable of differentiating the six species of Brucella.Materials and MethodsFour primers were designed from B. abortus sequences at the well-conserved Omp2 locus that are able to amplify the DNAs of all six species of Brucella.ResultsOur test detected all six species of Brucella. Their differentiation resulted directly from differences in the amplification patterns or was achieved indirectly using a RFLP present in one of the PCR products. The sensitivity and specificity of the new test were then determined; it was applied successfully in confirming the diagnosis of a patient whose clinical history and serology indicated infection with Brucella.ConclusionsThe results make possible the use of a PCR test for Brucella detection and differentiation without relying on the measurement of the antibodies or microorganism culture. Our first results showed that the PCR test can confirm the presence of Brucella in blood samples of infected patients.

High-throughput profiling of the humoral immune responses against thirteen human papillomavirus types by proteome microarrays

We have developed microarrays with all eight proteins encoded by 13 different human papillomavirus types associated with anogenital cancer (HPV-16, -18, -31, -33, -35, -45, and -53), genital warts (HPV-6 and -11), or skin lesions (HPV-1, -2, -4, and -5). We analyzed the seroprevalence of antibodies in 546 patients, which had either cervical carcinomas, or precursor lesions, or which were asymptomatic. All patient groups contained sera ranging from high reactivity against multiple HPV proteins to low or no reactivity. Computational analyses showed the E7 proteins of carcinogenic HPV types as significantly more reactive in cancer patients compared to asymptomatic individuals and discriminating between cancer and HSIL or LSIL patients. Antibodies against E4 and E5 had the highest seroprevalence but did not exhibit differential reactivity relative to pathology. Our study introduces a new approach to future evaluation of the overall antigenicity of HPV proteins and cross-reaction between homologous proteins.

Growth hormone and placental lactogen: biology, medicine and biotechnology

Molecular cloning gave us access to the gene members of the human growth hormone and placental lactogen multigene family. Genomic sequencing provided clues for the understanding of the origin, functioning and regulation of this family. It has also allowed us to develop new diagnostic approaches for deficiencies of these hormones and to make new biotechnological contributions.

Additive effect of ethanol and HCV subgenomic replicon expression on COX‐2 protein levels and activity

Summary.  The mechanisms by which alcohol exacerbates liver injury in patients with hepatitis C are unknown. We used the hepatitis C virus (HCV) subgenomic replicon cell system to evaluate the effect of ethanol on HCV replication and viral protein synthesis. Our results demonstrate that alcohol stimulates HCV replicon expression at both HCV‐RNA and protein levels. Furthermore, we observed that ethanol treatment showed an additive effect in cyclooxygenase‐2 (COX‐2) protein expression and activity already induced by HCV viral proteins, and in turn increased HCV viral expression. Our results suggest that COX‐2 activity is involved in ethanol‐induced HCV‐RNA and NS5A protein expression, because acetylsalicylic acid (ASA), a COX‐1/2 inhibitor, blocked this induction and downregulated COX‐2 protein expression and activity. Therefore, we suggest that ethanol increases HCV replication expression, at least in part, by upregulating a key cellular regulator of oxidative stress pathway known as COX‐2 or its products.