Prevalence of CYP2C9 and VKORC1 alleles in the Argentine population and implications for prescribing dosages of anticoagulants.

Dicumarinic oral anticoagulants have a narrow therapeutic range and a great individual variability in response, which makes calculation of the correct dose difficult and critical. Genetic factors involved in this variability include polymorphisms of genes that encode the metabolic enzyme CYP2C9 and the target enzyme vitamin K epoxide reductase complex 1 (VKORC1); these polymorphisms can be associated with reduced enzymatic expression. We examined the frequency of the most relevant variants encoding CYP2C9 (alleles *1, *2 and *3) and VKORC1 (SNP -1639A>G) in the Argentinian population. Molecular typing was performed by PCR-RFLP on a randomly selected sample of 101 healthy volunteers from the Hospital Italiano de Buenos Aires gene bank. Fifty-seven subjects were identified as homozygous for CYP2C9*1 and 14 for *2, while 24 and 5 were heterozygous for *2 and *3 alleles; one individual was a composite heterozygote (*2/*3). When we examined VKORC1, 21 subjects were AA homozygous, 60 were AG heterozygotes and 20 were GG homozygotes. This is the first analysis of genotypic frequencies for CYP2C9 and VKORC1 performed in an Argentinian population. These allele prevalences are similar to what is known for Caucasian population, reflecting the European ancestor of our patient population, coming mostly from Buenos Aires city and surroundings. Knowledge of this prevalence information is instrumental for cost-effective pharmacogenomic testing in patients undergoing oral anticoagulation treatment.

CD24 as a genetic modifier of disease progression in multiple sclerosis in Argentinean patients.

INTRODUCTION: Previous reports have shown that CD24 gene polymorphisms have an important role in the risk of development and progression of multiple sclerosis (MS). OBJECTIVE: To investigate the association between P226 polymorphisms (T/C), P1056 (A/G), P1527 (TG/del) and P1626 (A/G) of the CD24 gene and MS, comparing allele and genotype frequencies of patients versus controls. MATERIALS AND METHODS: We analyzed DNA samples from 102 MS patients and from 205 unrelated healthy individuals. DNA was extracted from peripheral blood and polymorphic regions were amplified by nested PCR. Genotyping was performed by restriction fragments length polymorphisms. Time from disease onset to reach EDSS 6 and time to conversion to secondary progressive phase (SP) were used as variables of survival as well as percentage of patients that reached those endpoints. We used the log Rank test for data comparison (significant p≤0.05). RESULTS: We found no differences between cases and controls in frequency of polymorphisms at the CD24 gene. 44.6% of patients with the AA genotype (P1626) reached an EDSS 6 vs 16% of patients with other genotypes (p<0.001, HR 3.2, 95% CI 1.4 to 7.4). 45.8% of patients with the AA genotype reached SPMS vs 16.7% without this genotype (p<0.001, HR 3.4, 95% CI 1.5 to 7.8). CONCLUSIONS: This study showed a strong association between the presence of AA genotype in the 1626 polymorphism of the CD24 gene and the risk of disease progression in MS patients.

Pharmacogenetics of immunosuppressant polymorphism of CYP3A5 in renal transplant recipients.

The tacrolimus is metabolized primarily by CYP3A5, a member of the single nucleotide polymorphism family. It shows cytochrome P450 (SNP) in intron 3, which consists of a change of base, G for A, producing a stop codon. The result is a nonfunctional protein (allele *3). Allele *1 is the wild type. The patients that show the allelic variant *3 in homozygosis (G/G) are slow metabolizers of the immunosuppressant, increasing its concentration in blood. In contrast, heterozygote A/G alleles *1/*3 are intermediate metabolizers, whereas those of allele *1 in homozygosis (A/A) are normal metabolizers. The aim of this study was to determine CYP 3A5 polymorphism among adult renal transplant recipients and the general Argentinean population. We analyzed 21 recipients and 36 healthy controls. All subjects gave written informed consent approved by the local committee. To determine the polymorphism, we extracted DNA from peripheral blood and used polymerase chain reaction (PCR) to amplify intron 3 of the CYP 3A5. The presence of variant was confirmed by direct sequencing. Among the controls the CYP3A5 genotype *3/*3 (G/G) was detected in 32 individuals, 4 showed *1/*3 (A/G), and none had *1/*1 (A/A); among the recipients, the results were as follows: 18, 2, and 1, respectively. The frequencies of polymorphism in both groups were similar, although they differed from those published for other populations. These results are the basis for the development of a pharmacogenomic program applied to organ transplantation. The genetic polymorphisms can determine responses to drugs. The molecular diagnosis must be transferred to clinical practice so as to guide selection of medicine and drug doses to be optimal for each individual.

HLA-DRB1 and multiple sclerosis in Argentina.

BACKGROUND: The association of multiple sclerosis (MS) with HLA DR subtypes, and particularly human leukocyte antigen (HLA)-DRB1*15 has been a consistent finding across nearly all Caucasian MS populations. In South America, scarce data exist about this issue. As the complete characterization of the HLA association range around the world is important to understand the role of this locus in MS susceptibility, we analyzed the frequency of HLA-DRB1* allelic groups in an MS population in Argentina. METHODS: HLA-DRB1 locus was genotyped using PCR and sequence-specific primer amplification in 61 MS patients born in Buenos Aires, Argentina and 1216 healthy controls. Allele frequencies were compared between groups. RESULTS: The HLA-DRB1*15 allele frequency significantly differed between controls and patients (13.5% and 33.9% respectively, P < 0.001, OR 2.51, 95% CI: 1.7-3.0). The other allele frequencies did not show significant differences between patients and controls. CONCLUSIONS: The present HLA class II population study is in accordance with other Caucasian MS surveys which have found that HLA-DRB1*15 allele is strongly associated with MS disease. In Argentina, this is the first study performed to analyze the association of HLA-DRB1*15 and MS susceptibility in a Caucasian population and therefore contributes with new data to the immunogenomic global MS map.

Conversion from azathioprine [correction of azathioprina] to mycophenolate mofetil in pediatric renal transplant recipients with chronic rejection.

BACKGROUND: Chronic rejection is the leading cause of graft failure. Both nonimmunological and immunological mechanisms contribute to this pathology. METHODS: We studied changes in kidney function, mixed lymphocyte culture, cell-mediated lympholysis, serum HLA class I antigens, cytotoxic antibodies, and lymphocyte population before and after 6 months of follow-up in 22 pediatric renal transplanted patients. The immunosuppressive protocol used was: cyclosporine, azathioprine, and corticosteroids. Eight patients demonstrated chronic graft rejection (by biopsy), group I; and eight patients had no clinical evidence of chronic and/or acute rejection, group II. Substitution of mycophenolate mofetil (MMF) (600 mg/m2 bid for azathioprine was done in patients of groups I and II. Another six patients with chronic rejection, did not receive MMF, group III. RESULTS: Creatinine clearance increased in group I (44+/-5 vs. 51.1+/- ml/min/1.73 m2, P<0.03) but it decreased in group III (30+/-3 vs. 25+/-2, P<0.01). Urinary protein excretion decreased only in group I (0.3+/-0.03 to 0.06+/-0.03 g/24 hr, P<0.03). During MMF therapy antidonor mixed lymphocyte culture decreased 62 and 70% (P<0.05) in group I and II. Cell-mediated lympholysis against lymphocyte of the donor decreased 65% (P<0.05) in group I. Cell-mediated lympholysis toward control cells decreased 54% (P<0.01) in group II. Serum HLA class I antigens, only decreased from 0.7+/-0.1 to 0.5+/-0.1 microl/ml, P<0.05, in group I. CD19+ decreased from 7.9+/-1.1 to 5.6+/-0.8%, P<0.05, and 7.8+/-1.2 to 5.5+/-0.9%, P<0.05, in groups I and II, respectively. CD16+ increased from 5.7+/-1.1 to 8.6+/-1.3 (P<0.05) only in group I. CONCLUSIONS: Our data suggest that substituting MMF for azathioprine therapy leads to an improvement in the immunosuppression and renal function in children with on-going chronic rejection.

Microquimerismo y actividad bloqueante en mujeres con abortos espontáneos recurrentes (RSA) después de la aloinmunización con linfocitos del esposo / Microchimerism and blocking activity in women with recurrent spontaneous abortions after the allo immunization with their partner's leucocytes

Se han detectado células fetales en la circulación materna de seres humanos 4 semanas post-concepción (Thomas y colab. 1994). Se podría hipotetizar que el tráfico celular temprano a través de la placenta es importante y tal vez necesario para la inducción de tolerancia fetal. La localización de células fetales CD34+ en los órganos maternos linfoides puede ayudar a mantener la tolerancia fetal de una manera análoga al trasplante de órganos alogeneicos (Bianchi y colab. 1996). El reciente hallazgo de que células del dador circulan en el receptor de trasplante hasta 29 años post-trasplante, ha hecho pensar que éstas células quiméricas (Microquimerismo) pueden tener un rol en la inducción y perpetuación de la tolerancia. La terapia de aloinmunización con linfocitos del esposo podría ser efectiva para prevenir los abortos recurrentes de causa desconocida. Algunos investigadores establecieron que después de la aloinmunización se observa la presencia de factores bloqueantes (FB) en el suero de mujeres con embarazos exitosos en ensayos "in vitro" de cultivo mixto linfocitario (MLC). Nosotros hemos investigado la producción de FB en MLC antes y después de la aloinmunización y su posible relación con el desarrollo de microquimerismo (M). Antes del tratamiento estudiamos 14 parejas con 3 o más abortos quienes eran evaluadas clínicamente para descartar causas anatómicas, genéticas, estructurales, endócrinas, infecciosas y/o autoinmunes. El estudio de M fue hecho con la técnica llamada nested PCR-SSP para los alelos HLA-DR antes del tratamiento y después de 30 días de la última inmunización. Antes del tratamiento, solo 1 paciente tenía M positivo y ninguna tenía FB positivos con índice de inhibición (I.I) >50, solo 8 pacientes realizaron el tratamiento. Las pacientes recibieron entre 3 y 9 aloinmunizaciones (x=4.7). Después del tratamiento todas las pacientes tenían M positivo e I.I>50, 6 meses después de la última inmunización 4 pacientes tienen M positivo eII>50. En conclusión: la hipótesis propone que la aloinmunización establece un estado de microquimerismo que sería el estímulo alogénico necesario para la activación de células T y la inducción o mantenimiento de la tolerancia hacia el feto durante el embarazo

Microchimerism and blocking activity in women with recurrent spontaneous abortion (RSA) after alloimmunization with the partner's lymphocytes.

Alloimmunization therapy using the partner's leukocytes has been reported to be effective in preventing the failure of pregnancy in patients who have suffered RSA of unknown cause. After alloimmunization, several investigators have reported the presence of blocking factors (BF) in women with successful pregnancies in in vitro assays of lymphocyte response. The recent discovery of small numbers of ubiquitous donor cells (microchimerism) in human transplants up to 29 years post-transplantation has raised questions about the migration of the chimeric cells and their role in the induction and perpetuation of tolerance. We have investigated the production of BF in the mixed lymphocyte reaction (MLR) before and in some patients after alloimmunization and its possible relation with the development of microchimerism (M). Before the treatment we studied 14 couples with three or more abortions who were evaluated clinically to rule-out genetic, structural, endocrine, infectious and autoimmune causes. The M study was done by nested PCR-SSP technique with HLA-DR alleles, before and after 30 days of the last immunization. Before the treatment only one patient was M positive and none were BF positive with inhibition effect (IE) > 50. Only eight underwent treatment. The patients had between three and nine alloimmunizations (x = 4.7). After treatment, all patients were M positive with IE > 50. Six months after the last immunization, four patients are M positive with IE > 50. In conclusion, the hypothesis proposes that alloimmunization establishes a state of microchimerism that would be the necessary allogeneic stimulus for T-cell activation, and the induction or maintenance of tolerance to the fetus during pregnancy. reserved.

Soluble HLA class I antigens in pediatric patients with renal transplants from related living donors without acute rejection and treated with triple therapy.

All HLA class I Ag-expressing cells may be the source of serum Ag sHLA I. T and B lymphocytes secrete considerable amounts of Ag sHLA I in a variety of in vitro and in vivo activation systems. The purpose of this study was to evaluate the level of Ag sHLA I in serum of children with kidney transplants from related living donors without acute rejection and with triple therapy. We studied 25 patients (2-21 years) with first kidney transplant, 19 individuals (10-20 years) undergoing hemodialysis without transplant, and 25 normal children (4-21 years). The levels of Ag sHLA in transplant patients was 0.2-3.2 micrograms/ml (mean = 1.04). The hemodialyzed patients was 0.48-4.5 micrograms/ml (mean = 2.09), and the normal control was 0.30-4.38 micrograms/ml (mean = 2.04). A statistically significant reduction was observed in transplant patients compared to normal control and hemodialyzed patients (p < 0.05 in both cases), whereas between normal and hemodialyzed patients no significant difference was seen (p > 0.05). The reduced levels of Ag sHLA I in blood could be an expression of adequate immunosuppressive treatment.

Human leukocyte antigen matching, radiographic score, and histologic findings in massive frozen bone allografts.

Forty-six patients receiving frozen bone allografts, preoperatively tissue typed for human leukocyte antigen and ABO antigens, were radiographically evaluated according to the Musculoskeletal Tumor Society scoring system at a mean followup of 55 months. Patients who matched for 1 or 2 Class I human leukocyte antigens with the donor scored higher than patients totally mismatched, but differences were not significant. Matching for Class II human leukocyte antigen and ABO antigens seemed not to influence radiographic outcome of allografts. In sixteen patients histologic specimens were obtained. Five of 16 patients who showed histologic parameters of an immune response scored significantly lower than those who did not. Processed frozen bone allografts, because of their lack of viable donor cells, most likely trigger an indirect pathway of alloantigen recognition in the recipient. This type of recognition may generate in the recipient either a chronic type of rejection or an immunologic state of tolerance to grafted antigens that cannot be measured with human leukocyte antigen blood tests. This may explain difficulties in correlating human leukocyte antigen mismatches between the donor and recipient with frozen bone allograft performances.