Ana Rebeca Jaloma-Cruz

Allele frequency distributions of six amp-FLPS (D1S80, APO-B, VWA, TH01, CSF1PO and HPRTB) in a Mexican population

Six amplified fragment length polymorphisms or Amp-FLPs, two VNTRs (D1S80 and APO-B) and four STRs (VWA, TH01, CSF1PO and HPRTB), were typed in a Mexican population of the Jalisco state by means of non-denaturing polyacrylamide gel electrophoresis (native PAGE) in standard gel units and silver staining. Genotype distribution was in agreement with Hardy-Weinberg expectations (HWE) for all six markers. Heterozygosity ranged from 70.6 to 83.5%, the cumulated chance of exclusion (CE) and power of discrimination (PD) were 99.4 and 99.99%, respectively. STRs and D1S80 allele frequency distributions (AFD) were similar (P > 0.05) to U.S. Hispanics, but different to U.S. Caucasians and African-Americans. APO-B exhibited similarities with White Brazilians, Spaniards, but differences (P < 0.05) with Amerindian and Black Brazilians.

Y-Chromosome Haplotypes for Six Short Tandem Repeats (STRs) in a Mexican Population

BACKGROUND Short tandem repeats (STRs) on the non-pseudoautosomal region of the Y-chromosome are DNA polymorphic markers able to solve special cases in legal medicine, for instance in paternity testing where the alleged father is not available, and in forensic situations, such as rape cases, where mixtures of male/female DNA are present. METHODS Six STR polymorphisms from the Y-chromosome (DYS19, DYS385, DYS389/I, DYS390, DYS391, and DYS393) were PCR-typed in 120 males from the northwest region of Mexico by means of native polyacrylamide gel electrophoresis and silver staining. RESULTS Allele frequencies were estimated for each STR. Their gene diversity ranged from 51.4% for DYS393 to 92.5% for DYS385. Mexican Y-STR allele distributions displayed similarity (p >0.05) with previously reported U.S. Hispanics for DYS19, DYS389/I, DYS390, DYS391, and DYS393. Although Mexicans showed the same modal allele for DYS385 (11/14; 24.4%) with regard to most European populations, differences in allele distributions were observed (p <0.01). The haplotype diversity and the male discriminatory capacity of this six-locus system were 99.3 and 84.1%, respectively. CONCLUSIONS This knowledge permits the effective use of these six Y-chromosome markers in legal medicine casework in the studied population. This STR-system offers a great potential to identify males and male-lineages, and can be used confidentially in paternity testing and forensic analysis in the Mexican population.

Nine independent F9 mutations in the Mexican hemophilia B population: nonrandom recurrences of point mutation events in the human germline.

The factor IX gene (F9) is a valuable model for studying germ‐line mutations. Nine mutations were detected in nine Mexican patients with hemophilia B by direct sequencing using genomic amplification with transcript sequencing (GAWTS): six single base changes, one micro‐deletion, and two large deletions. Germline origins of mutations were found in three of six families with sporadic cases. Curiously, the four independent single base substitutions which were not at CpG dinucleotides occurred at only two different nucleotide positions (17,678 and 17,747) one transition and one transversion at each. The two remaining substitutions were identical changes at a CpG dinucleotide, but were determined to be independent by germline origin analysis. A statistical analysis suggests that the independent recurrence of mutations at these locations may reflect an unusual aspect of F9 mutagenesis in the Mexican population. These data raise the possibility of population‐specific differences in human germline mutations. Hum Mutat 15:116–117, 2000.

Novel hotspot detector software reveals a non-CpG hotspot of germline mutation in the factor IX gene (F9) in Latin Americans.

Two‐base substitutions at each of two nucleotides in the factor IX gene (F9), but not part of CpG dinucleotides, were recently reported in a small population sample collected in Mexico, a significant observation of recurrent sites (“hotspots”) of mutation (P=0.00005). When these new data were combined with previously collected mutation data into two progressively larger and inclusive Latin American samples, additional mutations were observed at one recurrent site, nucleotide 17747, and an additional recurrent nucleotide was observed such that the recurrent nucleotides in these larger samples were also significant (P=0.0003 and 0.0003). In contrast, in three non‐Latin American control samples, there was at most only one nucleotide that recurred only once, most likely a chance recurrence (P≥0.5). When the significance of substitutions was analyzed at each recurrent nucleotide individually, nucleotide 17747 was shown to be a significant recurrent nucleotide by itself in all the Latin American population samples (P≤0.02). Furthermore, a standard statistical comparison of mutation frequencies in the previously collected data alone confirmed that the frequency of mutation at nucleotide 17747 is significantly higher in Latin Americans than in all other populations combined (P=0.01). Thus, nucleotide 17747 is a germline mutation hotspot in F9 specific to Latin American populations. This may be the first evidence for population‐specific effects on germline mutation that causes human genetic disease. The significance of the observed recurrent sites was analyzed using new software called Hotspot Detector which is capable of detecting significant recurrent sites in small samples, extending the sensitivity of F9 as a human germline mutagen test. Hotspot Detector uses a Monte‐Carlo simulation method that was validated by comparing its results with those from an exact probability formula derived from statistical theory. Hum Mutat 16:203–210, 2000.

Thrombin generation and international normalized ratio in inherited thrombophilia patients receiving thromboprophylactic therapy

BACKGROUND Thrombin generation assay (TGA) is useful as a global functional test for assessing bleeding or thrombotic risk and its modification with therapy. We investigated TGA to assess anticoagulation status compared with the international normalized ratio (INR) system in patients with primary thrombophilia receiving and not undergoing thromboprophylaxis. MATERIALS AND METHODS We studied 50 patients with at least one thrombotic event and a confirmed diagnosis of inherited thrombophilia. Thrombin generation was measured in platelet-poor plasma by calibrated automated thrombography (CAT). RESULTS Patients in optimal anticoagulation (INR: 2.0-3.0) showed an endogenous thrombin potential (ETP) of 14-56% of normal and a peak of 18-55% of normal. A significant inverse relationship between INR and thrombin generation parameters (ETP, peak and velocity index) and a linear correlation for lag time was found in patients treated with vitamin-K antagonists (VKA). Receiver-operating characteristics (ROC) analysis showed that the optimal cutoff for ETP was 1600.2 nM · min (111.6% of normal, with a sensitivity of 96.6% and a specificity of 92.9%) and for the peak was 298.3 nM (112.1% of normal, with a sensitivity of 96.4% and a specificity of 100%). According to this analysis, ETP was able to identify patients with increased thrombotic and hemorrhagic risk, correlating with severe clinical complications. CONCLUSION TGA showed excellent sensitivity and specificity for assessing anticoagulation status in patients with primary thrombophilia receiving VKA, with significant advantages with regard to INR. Clinical data strongly support ETP as a valuable indicator of thrombotic or hemorrhagic risk in patients receiving or not receiving thromboprophylaxis.

Phenotypic variability in a family with haemophilia B and prothrombin G20210A

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F8 inversions of introns 22 and 1 confer a moderate risk of inhibitors in Mexican patients with severe hemophilia A. Concordance analysis and literature review

Intron-22 (Inv22) and intron-1 (Inv1) inversions account for approximately one half of all severe cases of hemophilia A (SHA) worldwide. Inhibitor development against exogenous factor VIII (FVIII) represents a major complication in HA. The causative F8 mutation is considered the most decisive factor conditioning inhibitor development. We aimed to investigate prevalence of Inv22 and Inv1 mutations, and its association as risk factors for developing inhibitors to FVIII. We investigated Inv22 and Inv1 in 255 SHA Mexican patients from 193 unrelated families using the inverse shifting-polymerase chain reaction (IS-PCR). We analyzed the association between inversions and inhibitor development via logistic regression introducing as covariates the populations, the inversions, F8-haplotypes and the age of patients at enrollment. Inv22 was found in 91/193 (47.2%: 38.9% exhibited Inv22-1 and 8.3% Inv22-2), and Inv1 in 2/193 (1.0%) independent families. Absolute inhibitor prevalence (IP) for Inv22 in unrelated patients was 15% (10-19). The cohorts and age of patients were independent predictors of inhibitor risk, but not inversions or haplotypes. Inversions presence in our population was associated to a moderate risk of developing inhibitors. Inv1 was found for the first time in two Mexican families. A relevant genetic component was observed by the strong concordance among brother-pairs.

Genotype-Phenotype Interaction Analyses in Hemophilia

As with monogenic diseases, hemophilia A and hemophilia B have a direct relationship between factor VIII and factor IX gene mutations, respectively, and their causative effect on protein deficiency either in function or reduced antigen level in plasma. These aspects are related to, but do not totally explain, a more complex clinical phenotype such as the age of initial symptom onset, bleeding tendency, inhibitor development, arthropathy tendency, carrier bleeding symptoms, etc., which currently are critical complications under study in various clinical protocols in order to improve medical care in patients with hemophilia. The complex relationship between clinical behavior and genetics of hemophilia is changing the approach to diagnosis and research methods, expanding the scope of analysis to other related genes (bleeding tendency, immune system, regulatory genes of X-chromosome expression, etc.). In addition, novel functional approaches can provide prognostic parameters of clinical behavior such as gene expression assays and biochemical analyses including kinetics of inhibitors to factor VIII or thrombin generation assay by the standardized method of calibrated automated thrombography. This method describes the overall clotting capacity of patients’ plasma in vitro and ex vivo. This chapter will focus on certain studies regarding genotype-phenotype interactions in hemophilia that have been applied in Mexican hemophilia families for molecular diagnosis and genetic counseling. These studies have also been used to determine prognostic factors for clinical behavior and treatment response in hemophilia patients in order to improve hematological management as well as to optimize the use of therapeutic resources, an important consideration in developing countries such as Mexico.