Pharmacogenetic selection of volunteers increases stringency of bioequivalence studies; the case of clopidogrel.

Clinical response to clopidogrel varies widely due to under-dosing, drug interactions and intrinsic interindividual differences resulting from genetic polymorphisms. Cytochrome P450-2C19 is the principal enzyme involved in the activation of the prodrug and loss-of-function alleles have been described. Upon expiration of the pharmaceutical patent of clopidogrel, generic manufacturers have started to subject interchangeable formulations to bioequivalence studies. The purpose of the current investigation was to study the effect of selection of volunteers homozygous for the CYP2C19*1 haplotype on the bioavailability of clopidogrel. A regular 2×2 bioequivalence study between two formulations of clopidogrel was performed in volunteers selected and unselected for relevant CYP2C19 haplotypes for the Mexican population. It was found that selection of volunteers homozygous for the CYP2C19*1 haplotype, increased the stringency of bioequivalence statistics and resulted in bioinequivalence of a generic clopidogrel compound that otherwise proved equivalent when tested in an open unselected population. Augmentation of bioequivalence strictness is expected to result from pharmacogenetic selection of volunteers.

Treatment of vitiligo with a chimeric monoclonal antibody to CD20: a pilot study.

Five patients with active disseminated vitiligo were given 1g of a chimeric (murine/human) monoclonal antibody to CD20 in a single intravenous infusion and followed-up for 6 months. Three of the patients showed an overt clinical and histological improvement of the disease, one presented slight improvement and the remaining patient showed no changes. Improvement was neither associated with changes in laboratory parameters nor to a specific human leucocyte antigen D-related (HLA-DR) phenotype. We believe that these preliminary results are encouraging, and further clinical trials should be undertaken. An important aim should be the finding of a marker with a good response to this therapeutic approach.

Molecular monitoring of the treatment of patients with BCR/ABL (+) chronic myelogenous leukemia.

BACKGROUND: The molecular follow-up of patients with chronic myelogenous leukemia (CML) has been described as useful in other countries, but there are not data reported in Mexico. METHODS: All patients studied at Laboratories Clínicos de Puebla/Centro de Hematología y Medicina Interna de Puebla in which the BCR-ABL hybrid gene was identified by means of polymerase chain reaction were analyzed. In 22 individuals the molecular marker of the disease was studied at diagnosis and in different instances afterwards; these patients were treated with chemotherapy, interferon, autologous or allogeneic bone marrow transplantation. RESULTS: Only the six patients that were allografted from HLA-identical siblings cleared the molecular marker of the disease; the rest of them did not achieve molecular remissions. The median survival (SV) of the whole group has not been reached, whereas the 53-month SV is 68%. One of the allografted patients died as a result of complications of graft versus-host disease. CONCLUSIONS: We have found useful the molecular monitoring of the treatment of patients with CML. Using this approach, we found that molecular remissions can be accomplished only with allografting; however, other therapeutic approaches may also result in long-lasting hematologic remissions.

Heterozygosity for the H63D mutation in the hereditary hemochromatosis (HFE) gene may lead into severe iron overload in beta-thalassemia minor: observations in a thalassemic kindred.

Heterozygosity for beta-thalassemia (minor) by itself does not lead into iron overload; however, when it is inherited together with a homozygous state for either the H63D or the C282Y mutations of the hereditary hemochromatosis gene (HFE gene), iron overload may ensue. We describe here a kindred in which the propositus, being heterozygote for beta-thalassemia and the H63D mutation of the HFE gene, developed severe iron overload and in turn, chronic liver failure with portal hypertension. Other members of the family with either beta-thalassemia or heterozygous for the H63D gene mutation did not develop iron overload. The interaction between beta-thalassemia and hereditary hemochromatosis is briefly discussed and speculations about other possible genetic mutations leading into familial iron loading are done.

Primary thrombophilia in Mexico. II. Factor V G1691A (Leiden), prothrombin G20210A, and methylenetetrahydrofolate reductase C677T polymorphism in thrombophilic Mexican mestizos.

We have shown that in Mexican mestizo patients with clinical features of primary thrombophilia, 39% have activated protein C resistance phenotype, 5% protein C deficiency, and 2% protein S deficiency. In the present study, in a group of 37 thrombophilic Mexicans and 50 normal controls, we assessed the factor V G1691A (Leiden), the prothrombin G20210A, and the methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphisms. Four patients were found to be heterozygous for factor V Leiden, 5 heterozygous for the prothrombin 20210, 16 heterozygous, and 6 homozygous for the MTHFR 677. There were four individuals with co-segregation of alleles: two heterozygotes for the factor V Leiden/prothrombin 20210, one heterozygote for prothrombin 20210/MTHFR 677, and one heterozygote for prothrombin 20210/homozygote for MTHFR 677. For factor V Leiden, prothrombin 20210, and MTHFR 677 mutations, the allele frequencies were respectively 1% (+/-0.2%, alpha = 0.05), <1% and 51% (+/-5%, alpha = 0.05), with calculated relative risks for thrombosis of 5.94 (P = 0.08), >7.66 (P < 0.05), and 0.44 (P NS), respectively. In Mexican mestizo thrombophilic patients, the low prevalence of the factor V Leiden mutation (10.8%) and the high prevalence of the prothrombin 20210 mutation (13.5%) contrast with those identified in Caucasian thrombophilic patients (21% and 6%, respectively; P < 0.01). On the other hand, the high prevalence of the MTHFR 677 mutation gene both in normal controls (78%) and thrombophilic patients (61%) does not support a role of this mutation in the thrombogenesis of Mexican mestizo patients.

Analysis of HFE-codon 63/282 (H63D/C282Y) gene variants in mexican mestizos. Blood donors and patients with hereditary hemochromatosis.

BACKGROUND: The prevalence of hereditary hemochromatosis (HH) (H63D/C282Y) gene variants in Mexico is unknown. METHODS: Using amplification refractory mutation system polymerase chain reaction, an analysis of HFE-codon 63/282 (H63D/C282Y) gene variants was performed in a group of 153 Mexican mestizo blood donors and six individuals with familial iron overload. RESULTS: In normal blood donors, three heterozygotes for the C282Y mutation (2.0%) were found, whereas 18 heterozygotes and one homozygote for the H63D mutation (11.8% and 0.6%, respectively) were identified; there was one compound heterozygote for the C282Y/H63D mutation. These data resulted in allele frequencies of 0.013 (+/-0. 2%, alpha = 0.05) and 0.062 (+/-0.9%, alpha = 0.05), respectively, for these two mutations, results similar to those found in whites. In the six patients with the HH phenotype, two were found to be heterozygous for C282Y and one heterozygous for H63D; three individuals with HH had no gene mutations. Two heterozygous HH individuals were found to have iron overload associated with other conditions: one heterozygous for C282Y infected with HIV, and another heterozygous for H63D with heterozygous beta-thalassemia. CONCLUSIONS: The prevalence of C282Y and H63D HFE gene mutations in Mexican mestizos is similar to that found in other populations. In addition, other gene mutations responsible for HH in the Mexican mestizo population should be investigated, because, in three of six individuals with the HH phenotype, neither of the two mutations was recorded.

Assessment of residual disease in acute leukemia by means of polymerase chain reaction.

Along a 5-year period in a single institution, specific molecular markers were prospectively looked for in consecutive patients with acute leukemia, by means of polymerase chain reaction (PCR): In patients with acute lymphoblastic leukemia (ALL), the BCR/ABL and TEL-AML1 fusion transcripts as well as clonotypic immunoglobulin gene rearrangements were investigated, whereas in patients with acute myelogenous leukemia (AML) the PML-RAR alpha, AML1-ETO and CBF beta-MYH11 fusion proteins were assessed. Specific molecular markers were identified in 15/75 patients: Four with ALL (three with clonotypic IgG rearrangements and one with BCR/ABL) and 11 with AML (nine with the PML/RAR alpha fusion protein--M3 AML-, and two with the AML1/ETO fusion protein--M2 AML-). During follow-up periods ranging from 1 to 60 months, seven patients cleared the residual disease assessed by PCR (RD-PCR), whereas eight patients had either persistence of RD-PCR or a molecular relapse. For patients without or with RD-PCR, the 30-month survival (SV) was 86% and 14%, respectively, whereas median SV was > 60 and two months, also respectively (p < 0.01). Six of eight patients with detectable RD-PCR died, all of them within three months after the detection of the RD-PCR, whereas two of the patients that relapsed were rescued with treatment and entered a second molecular remission. Two of the three molecular relapses were detected without an overt morphological relapse. It is concluded that PCR is a valuable method for assessing residual disease and that early diagnosis of relapses may lead into effective salvage treatment in some instances.

Primary thrombophilia in Mexico: a prospective study.

A group of 102 Mexican Mestizo patients with appropriate clinical features suggestive of primary thrombophilia was prospectively studied. Thirty-nine percent of them had activated protein C resistance, but only four patients displayed the factor V Leiden mutation. Five percent of the individuals were found to be protein C deficient, whereas 2% had protein S deficiency. No cases of abnormalities in antithrombin III, plasminogen, tissue-type plasminogen activator or plasminogen activator inhibitor were found. The low prevalence of the activated protein C resistance genotype, probably stemming from the genetic admixture of the Mexican Mestizo group is noteworthy.

The G20210A polymorphism in the 3'-untranslated region of the prothrombin gene in Mexican mestizo patients with primary antiphospholipid syndrome.

In an effort to identify alleles associated with an increased risk of venous thrombosis in patients with primary antiphospholipid syndrome, we studied the G20210A polymorphism (the G-->A mutation at nucleotide position 20210) in the 3'-untranslated region of the prothrombin gene in a group of 14 patients with primary antiphospholipid syndrome. We did not find any patient with the mutated gene. Since the prothrombin mutation is more prevalent in white populations, this finding may be related with the genetic composition of the Mexican mestizos, in whom the white component is low. The polymorphism of the prothrombin gene in Mexican mestizo patients with antiphospholipid syndrome does not seem to be related to the thrombophilia observed in these patients.

Molecular follow-up of patients with promyelocytic leukaemia treated with all-trans retinoic acid.

Six consecutive patients with promyelocytic leukaemia displaying the PML/RAR-alpha fusion messenger ribonucleic acid (mRNA)--the molecular marker of the disease--were prospectively treated with all-trans retinoic acid: Haematological complete remission was achieved in all of them. However, in three of them the PML/RAR-alpha mRNA persisted. Subsequent treatment with ablative chemotherapy rendered the molecular remission in all cases. Two of the patients that did not achieve the molecular remission with ATRA were autografted with peripheral blood stem cells after clearing the PML/RAR-alpha fusion mRNA. One patient with tretinoin-induced molecular remission relapsed two years after diagnosis and died; the remaining five survive in complete haematological and molecular remission for periods ranging from 405 to 1695 days. Additional studies are needed to clarify the significance of the molecular follow-up of patients with PML.

Tuberculosis-associated fatal hemophagocytic syndrome in a patient with lymphoma treated with fludarabine.

A patient with a stage IV high-grade non-Hodgkin's lymphoma who developed a fatal hemophagocytic syndrome is presented: When the patient had achieved complete remission and receiving fludarabine and chlorambucil/prednisone, she developed miliary tuberculosis, the CD4+ T-cell count then being 50/microL; the hemophagocytic syndrome ensuing at this point was fatal. Speculations about the predisposing factors that could have led to this complication are discussed focusing on the severe cellular immunosuppression which developed probably related to the use of fludafabine: it could be useful in the future to use anti-tuberculous prophylaxis in selected patients treated with this purine nucleoside analog.

Inherited activated protein C resistance in a patient with familial primary antiphospholipid syndrome.

The mechanism of thrombophilia in patients with antiphospholipid antibodies (aPL) is not clearly understood. A number of contributing factors have been described, but more than one may be operative. It was recently found that aPL may cause the acquired activated protein C resistance phenotype, whereas in familial thrombophilia, activated protein C resistance frequently results from a point mutation in the factor V gene (replacing arginine 506 with a glutamine) that leads into the (R-506-Q), the so-called Leiden mutation, that produces a mutated factor V, resistant to the catalytic action of activated protein C, otherwise normal in its procoagulant properties. We describe one patient heterozygous for the activated protein C resistance genotype with a familial form of primary antiphospholipid syndrome, who had a nephew who died with this disease. In individuals who are heterozygous for the factor V Leiden mutation, the presence of aPL may cause further activated protein C resistance, resulting in increased thrombophilia.

Activated protein C resistance phenotype and genotype in patients with primary antiphospholipid syndrome.

We studied both inherited and acquired activated protein C (APC) resistance in a group of 22 patients with primary antiphospholipid syndrome (APS). The APC resistance genotype was assessed using a PCR-based analysis for the factor V R506Q (Leiden) mutation. One patient with primary APS was found to be heterozygous for the factor V Leiden mutation. He and other family members were affected by severe thrombophilia and had a familial form of primary APS. The APC resistance phenotype was assessed by measuring the prolongation of the activated partial thromboplastin clotting time in response to APC. It was found in five out of six patients with APS, in one of them transiently. We have found that the APC resistance phenotype is more frequent than the genotype in primary APS. It would seem that patients with thrombophilia should be investigated for APC resistance even if found to have antiphospholipid antibodies and/or lupus anticoagulants.

Nodal radiotherapy in refractory tuberculosis in an AIDS patient.

A 25-year-old female with transfusion-associated acquired immunodeficiency syndrome (AIDS) treated with zidovudine (AZT) developed cervical lymph node enlargement. The histological study disclosed granulomas and the culture revealed M. tuberculosis. The patient was treated with isoniazid, rifampicin and pirazynamide and the lymphadenomegaly resolved. Five years later, with a CD4 cell count of 245, the lymph node enlargement reappeared, the biopsy and special studies confirming tuberculosis (TB). She was then given ciprofloxacin, azithromycin, ethambutol, amikacin and pirazynamide without success. In two instances the enlarged nodes were surgically removed. Facing progressive obstruction of both the airway and the esophagus, localized radiotherapy (1800 cGy in nine fractions) to the right aspect of the neck was delivered with dramatic resolution of the node enlargement; however, dissemination of the infection leading to a severe lung infiltration and respiratory failure ended the life of the patient.