Francisco Barros

Naturalistic pharmacogenetic study of treatment resistance to typical neuroleptics in European-Brazilian schizophrenics

Objectives This study aimed to explore the influence of variation in DRD2, DRD3, CYP2D6, CYP3A4, and CYP3A5 genes on treatment resistance to typical neuroleptics in a Brazilian sample of patients with schizophrenia. Methods One polymorphism at DRD2 gene, five at DRD3, 24 at CYP2D6, nine at CYP3A4 gene, and one at CYP3A5 gene were genotyped in a sample of 186 patients with schizophrenia. Results From the nine studied CYP3A4 single nucleotide polymorphisms, only the −392A>G was polymorphic, and significant associations were observed between this single nucleotide polymorphism and efficacy of neuroleptic treatment. Homozygous individuals for the −392A variant [P=0.014, odds ratio (OR)=3.32] were more frequent in the treatment-resistant group, compared with carriers of one copy of the −392G variant. The CYP3A5 low expressor genotype (CYP3A5*3/CYP3A5*3) was found to be associated with refractoriness to neuroleptic treatment (P=0.003, OR=3.16). Among the haplotypes observed in DRD3 gene, the T/A/G/A/C haplotype showed an association with refractoriness to neuroleptics (χ2=5.342, P=0.021, OR=1.75). This association showed that carriers of one copy of this haplotype presented intermediate values between noncarriers and homozygous individuals for the haplotype. No association was observed with polymorphisms in DRD2 and CYP2D6 genes. Multiple logistic regression analyses showed that the number of copies of DRD3 T/A/G/A/C haplotype and CYP3A5 low expressor genotype were predictors of refractoriness to neuroleptic after controlling for selected risk factors. CYP3A5*3 individuals carrying at least one copy of the T/A/G/A/C haplotype showed a higher risk to be refractory to neuroleptics than CYP3A5*3 homozygotes+non-T/A/G/A/C carriers (χ2=5.533, P=0.019, OR=2.32, 95% confidence interval=1.08–5.02). No significant associations were observed with DRD2 and CYP2D6 polymorphisms. Conclusion Our results suggest a role for CYP3A5 and DRD3 gene variants on refractoriness to neuroleptic treatment in Brazilians with schizophrenia.

Pharmacogenetic analysis in neoadjuvant chemoradiation for rectal cancer: high incidence of somatic mutations and their relation with response.

AIMS The identification of predictive markers of response to chemoradiotherapy treatment remains a promising approach for patient management in order to obtain the best response with minor side effects. Initially, we investigated whether the analysis of several markers previously studied and others not yet evaluated could predict response to 5-fluorouracil- and capecitabine-based neoadjuvant treatment in locally advanced rectal cancer. METHODS & MATERIALS We studied germline and tumoral samples of 65 stage II/III rectal patients. A panel of pharmacogenetic markers was genotyped in paired peripheral blood samples and rectal cancer tumors. RESULTS Our results seem to confirm the previously described association of thymidylate synthase and the prediction of chemoradiotherapy response in rectal cancer. However, it failed to confirm the clinical utility proposed for XRCC1, ERCC1, ERCC2, MTHFR and EGFR polymorphisms in blood/germline samples. Subsequently, with the aim of improving prediction of individual response and assessing the role of studied polymorphisms in response to treatment, we determined if changes in tumor response to these markers could predict clinical outcome. We found a high degree of changes between germline and tumor samples, mainly somatic mutations without microsatellite instability, and a minor frequency of loss-of-heterozygosity events. In tumoral samples, XRCC1 appeared to be significantly associated (p = 0.006) with downstaging of the tumor (odds ratio: 7.93; 95% CI: 1.03-60.83), but the increasing of TYMS low-expression alleles contradict the previous results observed in germline samples. CONCLUSION The detection of somatic mutations in rectal cancer tumors led us to re-evaluate the utility of the tests performed in blood samples for these polymorphisms in rectal cancer. Furthermore, studies aimed at assessing the influence of pharmacogenetic markers in treatment response performed in blood samples should take into account the particular pattern of hypermutability present in each tumor type. We hypothesize that different patterns of hypermutability present in each tumor type would be related to the different results in association studies related to response to the treatment.

Use of a comprehensive panel of biomarkers to predict response to a fluorouracil–oxaliplatin regimen in patients with metastatic colorectal cancer

AIM Polymorphisms in the metabolism, detoxification or DNA repair pathways have been proposed as potential predictors of response to 5-fluorouracil and oxaliplatin. We have studied the predictive value of a set of germline genetic polymorphisms in metastatic colorectal cancer patients treated with mFolfox-6. MATERIALS & METHODS A total of 72 patients, comprising 50 men (69.4%) and 22 women (30.6%), were included after the signing of an informed consent form. Median age was 65.5 years (range: 32-80). All participants received mFolfox-6. DNA was extracted from peripheral blood samples and genotyped by direct sequencing, SnapShot(®) and multiplex PCR techniques. Eight polymorphisms within six genes were investigated: TS 5´-UTR (variable number tandem repeat + G/C), TS 3´-UTR (TS1494del6); MTHFR C677T and A1298C; GSTP1 I105V; ERCC1 C118T; XPD Lys751Gln and XRCC1 Arg399Gln. Association was evaluated by univariate analysis, and Cox regression and Kaplan-Meier assessed survival. The local ethics committee approved the pharmacogenetic study protocol and all subjects signed an informed consent before participating in the study. RESULTS The sample was in Hardy-Weinberg equilibrium. Only XPD Lys751Gln was found to be significantly associated with a favorable progression-free survival (PFS). Median PFS for XPD Lys751Gln patients (n = 33) was 16 months (95% CI: 9.2-22.7), 10 months (95% CI: 6.1-13.9) for Gln/Gln (n = 11) and 8 months (95% CI: 5.8-10.2) for Lys/Lys (n = 28), p = 0.019. The increased risk of progression was: 1.93 (95% CI: 1.13-13.30; p = 0.017) for Lys/Lys and 2.1 (95% CI: 1.01-4.22; p = 0.047) for Gln/Gln. Patients with one or two Val alleles of GSTP1 tended to a lower risk of progression compared with Ile/Ile homozygotes, p = 0.067. When XPD Lys751Gln and GSTP1 were analyzed jointly, patients who carried one or two favorable genotypes, XPD Lys751Gln and Val, had a longer median PFS: 11 months (95% CI: 7.4-14.6) compared with six (95% CI: 4.6-7.4) with unfavorable genotypes, p < 0.001. CONCLUSION In metastatic colorectal cancer patients treated with mFolfox-6, the combination of haplotype XPD Lys751Gln-GSTP1 105Val seems to predict the risk of progression.

Sequence variation of a hypervariable short tandem repeat at the D12S391 locus.

A short tandem repeat (STR) in the D12S391 locus was sequenced in more than 40 individuals. Twenty different alleles were found and these could be grouped into 12 allelic classes in accordance with the total number of repeats. This is a compound STR consisting of blocks of (AGAT) and (AGAC) repeats with basic sequence structure (AGAT)8-17(AGAC)6-10(AGAT)0-1. Whereas smaller alleles (15-18) have variation limited to the (AGAT) unit, in larger alleles the complexity is greater with variation in the number of tandem arrays in the two motifs (AGAT) and (AGAC). Population data showed that this is a highly polymorphic STR with a heterozygosity of more than 0.9. This fact, together with its simple structure, makes this STR an interesting DNA polymorphism for forensic and genetic purposes.

Analysis of BRCA1 and BRCA2 in breast and breast/ovarian cancer families shows population substructure in the Iberian peninsula.

An estimated 5–10% of all breast and ovarian cancers are due to an inherited predisposition, representing a rather large number of patients. In Spain 1/13–1/14 women will be diagnosed with breast cancer during their lifetime. Two major breast cancer genes, BRCA1 and BRCA2, have been identified. To date, several hundred pathogenic mutations in these two genes have been published or reported to the Breast Cancer Information Core, BIC database (http://www.nhgri.nih.gov/Intramural_research_Labtransfer/Bic/index.html). In the present study, 30 Spanish breast and breast/ovarian cancer families (29 from Galicia, NW Spain, and 1 from Catalonia, NE Spain) were screened for mutations in the BRCA1 and BRCA2 genes. The analysis of these genes was carried out by SSCP for shorter exons and direct sequencing in the case of longer ones. Mutations were found in 8 of the 30 families studied (26.66%). It is important to note that all mutations were detected within the BRCA1 gene: 330 A>G, 910_913delGTTC, 2121 C>T, 3958_3962delCTCAGinsAGGC, and 5530 T>A. The BRCA1 330 A>G mutation was found in four unrelated families and accounted for 50% of all identified mutations.

X-Ray Cross-Complementing Group 1 and Thymidylate Synthase Polymorphisms Might Predict Response to Chemoradiotherapy in Rectal Cancer Patients

PURPOSE 5-Fluorouracil-based chemoradiotherapy before total mesorectal excision is currently the standard treatment of Stage II and III rectal cancer patients. We used known predictive pharmacogenetic biomarkers to identify the responders to preoperative chemoradiotherapy in our series. METHODS AND MATERIALS A total of 93 Stage II-III rectal cancer patients were genotyped using peripheral blood samples. The genes analyzed were X-ray cross-complementing group 1 (XRCC1), ERCC1, MTHFR, EGFR, DPYD, and TYMS. The patients were treated with 225 mg/m(2)/d continuous infusion of 5-fluorouracil concomitantly with radiotherapy (50.4 Gy) followed by total mesorectal excision. The outcomes were measured by tumor regression grade (TRG) as a major response (TRG 1 and TRG 2) or as a poor response (TRG3, TRG4, and TRG5). RESULTS The major histopathologic response rate was 47.3%. XRCC1 G/G carriers had a greater probability of response than G/A carriers (odds ratio, 4.18; 95% confidence interval, 1.62-10.74, p = .003) Patients with polymorphisms associated with high expression of thymidylate synthase (2R/3G, 3C/3G, and 3G/3G) showed a greater pathologic response rate compared with carriers of low expression (odds ratio, 2.65; 95% confidence interval, 1.10-6.39, p = .02) No significant differences were seen in the response according to EGFR, ERCC1, MTHFR_C677 and MTHFR_A1298 expression. CONCLUSIONS XRCC1 G/G and thymidylate synthase (2R/3G, 3C/3G, and 3G/3G) are independent factors of a major response. Germline thymidylate synthase and XRCC1 polymorphisms might be useful as predictive markers of rectal tumor response to neoadjuvant chemoradiotherapy with 5-fluorouracil.

Molecular diversity at the CYP2D6 locus in healthy and schizophrenic southern Brazilians

AIMS The delineation of allele distribution and frequency is required to effectively translate pharmacogenetics to the clinic and given the paucity of CYP2D6 data in the Brazilian population, the purpose of this research was to characterize CYP2D6 alleles and genotype frequencies in Brazilians of European and African ancestries. Moreover, since it is suggested in the literature that CYP2D6 poor metabolism might be involved with susceptibility to schizophrenia, we included data from Brazilian schizophrenic patients to verify if CYP2D6 poor metabolism phenotypes are associated with susceptibility to schizophrenia. MATERIALS & METHODS We investigated 24 CYP2D6 polymorphisms, gene deletions and gene multiplications in 179 healthy individuals from Brazil, 92 of European descent and 87 African Brazilians. CYP2D6 gene polymorphisms were genotyped by a MassARRAY SNP genotyping system. RESULTS A total of 19 different alleles and five allele duplications were identified in African and European Brazilians. No significant differences in CYP2D6 allele function or poor metabolizer predicted phenotype frequencies were observed between healthy controls and schizophrenic patients, but the predicted metabolic phenotype distribution showed a significant higher frequency of intermediate metabolizers in African Brazilians than in European Brazilians (p = 0.001). CONCLUSIONS CYP2D6 poor metabolizer genotype seems not to be a determining factor of schizophrenia susceptibility in Brazilians. The characterization of CYP2D6 variability will be very useful for future pharmacogenetic studies in the Brazilian population.

Low frequency of replication errors in primary nervous system tumours

OBJECTIVES Automated DNA technology was used to analyze the incidence of microsatellite instability (MIN) among the most frequent types of adult primary CNS tumours and to determine its relation with clinicopathological characteristics. METHODS Fifty six gliomas, 32 meningiomas and 11 schwannomas were screened for size changes at eight microsatellite loci using fluorescent polymerase chain reaction (PCR) followed by fragment analysis in an automated sequencer. A tumour was considered as MIN+ when a different electrophoretic pattern between constitutional and tumour DNA was evidenced in one or more microsatellite markers and as replication error positive (RER+) when at least 25% of the markers analyzed (2/8) showed instability. The MIN phenotype was correlated with relevant clinical and pathological parameters. RESULTS Globally, instability was found in 19/767 analyses (2.47%), with a higher rate among tetranuceotide than dinucleotide repeats (χ2 test, p=0.018). Ten gliomas (17.9%), two meningiomas (6.3%), and two schwannomas (18.2%) were MIN+, whereas one glioma (1.8%), two meningiomas (6.3%), and one schwannoma (9.1%) were classified as RER+. A possible association between microsatellite instability and a shorter duration of clinical course was found in meningiomas. The MIN+ phenotype was more frequent in spinal than intracranial schwannomas (Fishers exact test, p=0.018). No other significant association with clinical or histological features was detected. CONCLUSIONS Although microsatellite instability can be demonstrated at a low rate in some primary CNS tumours, a true replication error phenotype (revealed by widespread microsatellite instability at numerous loci) is uncommon and unlikely to play an important part in the pathogenesis of these neoplasms. This form of instability was more frequent in tetranucleotide than in dinucleotide repeats. To our knowledge, this is the first report of MIN in schwannomas, where it was associated with the spinal localisation of the tumour.

A novel stop mutation in the vascular endothelial growth factor-C gene (VEGFC) results in Milroy-like disease

Background Milroy and Milroy-like disease are rare disorders characterised by congenital lymphoedema caused by dysfunctional lymphatic vessel formation. Loss of extracellular response mediated by vascular endothelial growth factor receptor 3 (VEGFR-3) is associated with Milroy disease, and VEGFR-3 gene is mutated in around 70% of the cases diagnosed. The only genetic alteration known to be associated with Milroy-like disease was recently identified in a family with a frameshift mutation in vascular endothelial growth factor C (VEGFC) gene, which encodes a VEGFR3 ligand. Methods and results We report a newborn patient with an external phenotype consistent with Milroy disease and a truncating mutation (p.R210X) in the VEGFC gene detected by exome sequence analysis. Subsequent analysis, by lymphoscintigraphic scan, performed for research purposes, allowed us to correct the diagnosis, confirming patient’s disease as Milroy-like. The mutation segregates with the phenotype in the family according to a dominant model with full penetrance. Conclusions The clinical presentation, similar to Milroy disease, indicates an overlapping of the external phenotype of both diseases, suggesting that genetic analysis of VEGFC would be useful in diagnosing patients that present with Milroy features but have no mutation in VEGFR-3. Establishing a well-defined genetic pattern would help with differential diagnosis.

The value of genetic polymorphisms to predict toxicity in metastatic colorectal patients with irinotecan-based regimens

PURPOSE We are trying to identify predictive factors of high risk of toxicity by analyzing candidate genes in the irinotecan pathways in order to identify useful tools to improve mCRC patient management under real practice conditions. METHODS Genomic DNA was genotyped for UGT1A1 (*28, *60 and *93) from all 101 patients, and irinotecan dose was 180 mg/m(2) every second week. Clinical data were obtained by retrospective chart review. The primary endpoint is to find out whether the pharmacogenetic test in the clinical practice may predict toxicity. RESULTS Grade 3/4 diarrhea occurred in twelve patients and required dose reduction in six patients, and neutropenia reached grade 3/4 in 19 patients (only one patient with *28/*28 genotype). The UGT1A1*93 seemed to relate with grade 3/4 neutropenia but only in the heterozygote state (G/A), p = 0.071, and UGT1A*60 showed no association with neutropenia. Twenty-eight percentage of patients required the use of G-CSF; 64.3% of them harbored *1/*28 or *28/*28 genotypes, p = 0.003. Thirty-seven (36.6%) patients required dose reduction of irinotecan and/or 5-FU owing to toxicity, mainly neutropenia and diarrhea. No significant association was detected between *28, *60 and *93 UGT1A variants and severe irinotecan-associated hematologic or GI toxicity. CONCLUSION The impact of increased risk of toxicity attributed to the UGT1A variants may be offset by irinotecan in clinical practice by dose reduction or the use of colony-stimulating factor.