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Introduction Molecular analysis in haemophilia is currently used in the diagnosis, treatment and prognosis of this disease. Hispanic populations in Latin America have been of interest to researchers due to the reportedly high prevalence of inhibitors in these patients. Aim To perform next-generation sequencing (NGS) in a cohort of Mexican patients with HA and HB and correlate with clinical phenotypes. Methods Patients with Haemophilia A (HA) or haemophilia B (HB), were evaluated using NGS with an Ion AmpliSeq Custom Panel. Odds ratios (ORs) for associations between F8 variants and inhibitors were obtained. Results A total of 85 patients (60 with HA and 25 with HB) were included. Pathogenic variants in F8 were found in 93.3% of HA patients and in F9 in 96% of HB patients. Twelve novel potentially pathogenic variants were found. Inhibitors were observed in 20% of patients with severe HA. Four patients clinically diagnosed with HA were negative for F8 variants. Conclusion Overall detection rate of pathogenic variants in F8 and F9 genes was 94.6%. We identified 12 non previously reported variants and pathogenic variants in other coagulation related genes. Molecular diagnosis of HA and HB permits better options for management, assessment and genetic counseling.

Background The pathophysiology of hemophilic arthropathy is complex and not completely understood. In this study, we aimed to identify biomarkers that can affect the hemophilic arthropathy severity. Methods Fifty patients were analyzed for biomarker frequencies; in 37 patients, articular symptoms were evaluated based on the physical joint examination score, and in 18, it was based on magnetic resonance imaging. Eight polymorphisms, namely FV 1691G>A, FII 20210G>A, MTHFR 677C>T and 1298A>C, TNFα‐308G>A and ‐238G>A, ACAN VNTR, and IL1RN*2‐VNTR were identified. Results Patients with the MTHFR 677TT genotype showed a higher number of affected joints (1.83 ± 0.9 vs. 0.55 ± 0.7 for CC; p = .023), whereas those with the MTHFR 1298AC genotype exhibited higher effusion according to two radiologists (0.90 ± 0.31/1.20 ± 0.63 vs. 0.38 ± 0.52/0.50 ± 0.53 for AA genotype; p = .043/0.036, respectively). In addition, patients with the TNFα‐308GA genotype had more subchondral cysts (0.75 ± 0.95 vs. 0.07 ± 0.26 for GG genotype; p = .041). Conclusions The distribution of risk genotypes for MTHFR and TNFα‐308GA suggests their association with clinical parameters of hemophilic arthropathy. Cohort studies are essential to verify these associations.

Idiopathic recurrent pregnancy loss (IRPL) is defined by three or more consecutive miscarriages occurring before the twentieth week of gestation as a result of unidentified etiological factors. The results of previous studies have indicated that prothrombotic factors play a pathogenic role in early and late pregnancy. This study aimed to identify inherited prothrombotic and hypofibrinolytic risk factors in Mexican-Mestizo patients with IRPL. Fifty-six women with IRPL and 50 control women with at least two full-term pregnancies and no history of RPL were included in this case-control study. Four prothrombotic (F5 G1691A, F2 G20210A, MTHFR C677T-A1298C) and one hypofibrinolytic (PAI1 4G/5G) restricted fragment length polymorphisms were subjected to molecular analysis. In the case of hypofibrinolytic ACE Ins/Del (I/D), identification was performed by direct PCR. The independent risk correlated with the presence of polymorphisms in IRPL patients was estimated using odds ratio (OR) with a 95% confidence interval (CI). MTHFR 677TT was the most frequent prothrombotic factor in the IRPL group (23%), followed by the compound-heterozygous C677T-A1298C (16%) and heterozygous F2 20210GA (3.6%). The heterozygous ACE I/D (62%) was the main hypofibrinolytic risk factor of IRPL, followed by the homozygote PAI1 4G/4G (18%). The ACE I/D polymorphism was the only significantly different factor among the cases and controls. The dominant genetic model D/D+I/D vs I/I showed an OR (95%CI) of 2.89 (1.22-6.89) and P = 0.019 in Mexican-Mestizo women. The results of this study support an association between the ACE I/D polymorphism and IRPL risk in a Mexican population.

Detection of minimal residual disease (MRD) in patients with B-cell acute lymphoblastic leukemia (B-ALL) has been achieved using several radioactive labelling methodologies; however, limited information exists about the use of chemiluminescent labelling. Although many malignant disorders are related to cytogenetic alterations, there is not a consistent chromosomal translocation that could serve as a tumour marker for the monitoring of MRD. ALL are derived from B-lymphocytes in 80% of cases. In the early stages of their maturation, the immunoglobulin heavy chain genes (IgH) undergo rearrangements among their V, D, and J segments, giving rise to the Complementary Determining Regions (CDR). Among these, CDR3 is considered unique for each lymphocyte and used as a tumour-specific marker in B-ALL patients. In this study, the CDR3 was labelled with digoxigenin and used as a patient-specific probe to test its sensitivity for further detection of MRD. Fourteen pretreatment samples of bone marrow (BM) or peripheral blood (PB) from B-ALL patients were included. Tumour-specific probes were designed from each clonal product by elimination of the consensus sequences. Ten digoxigenin-labelled probes were hybridized with a mixture of their respective clonal DNA and the polyclonal product from a normal healthy donor, in serial dilutions from 1:1 up to 1:10(7). A sensitivity range of 1:10(3)-1:10(6) was obtained, with an average of 1:10(5). Crossed tests performed in four patients, showed right probe specificity in all cases. We propose that the design of allele-specific probes with chemiluminescent labelling, represents a reliable, sure and sensitive alternative methodology for MRD detection in patients with B-cell lymphoproliferative disorders.

Bone marrow (BM) is accepted as the tissue of choice for the detection of monoclonal populations in leukemias and lymphomas; however, obtaining BM can be painful and traumatic for the patients. Although it is possible to detect clonality in peripheral blood (PB) samples, there are no reports comparing the results observed from BM with those from PB. Lymphoblastic leukemias and lymphomas are derived from B-lymphocytes in 80% of cases. In the early stages of their maturation, the immunoglobulin heavy chain genes (IgH) undergo rearrangements among their V, D, and J segments, giving rise to the Complementarity Determining Regions (CDR). Of these, CDR3 is unique for each lymphocyte and therefore it can be used as a tumour-specific marker in these malignant disorders. Among the 104 patients from whom we obtained pre-treatment paired samples of PB and BM, 94 (90.4%) showed concordant results. Similarly, at the end of treatment, 40 of 44 patients (90.9%) showed this concordance. During treatment only 24 patients were monitored and monoclones disappeared in 12 patients; in the other half, they persisted either partial or totally. We demonstrate that the detection and monitoring of monoclonal populations in the PB, in comparison with BM, was achieved with a statistical sensitivity of 90% and specificity of 92%. Copyright © 2003 John Wiley & Sons, Ltd.

Summary In Mexico, 15% of haemophilia A (HA) patients develop inhibitory alloantibodies in response to replacement therapy with factor VIII (FVIII), requiring bypass therapy such as activated prothrombin complex concentrate (APCC). Because bypass therapy has not been broadly available in Mexico even in recent years, this study aimed to evaluate the thrombin generation assay (TGA) in assessing the response to FVIII or APCC treatment in patients with severe HA positive to inhibitors. We studied 189 patients with severe HA. Clinical severity was verified by one-stage APTT-based clotting assay. Inhibitors to FVIII were investigated by the Nijmegen–Bethesda (N–B) method, and type of inhibition was assessed through serial plasma dilutions. Thrombin generation was measured with the calibrated automated thrombogram in inhibitor-positive plasmas previously spiked and incubated with FVIII or APCC. Data were analysed using anova, Student or Fisher's exact tests. We detected 47 (24.9%) subjects with high-titre (5–1700 N–B U mL−1) and 25 (13.2%) subjects with low-titre inhibitor antibodies (0.6–4.7 N–B U mL−1). We found an association between kinetic behaviour and clinical response to FVIII (P = 0.0049) or vs. FVIII response evaluated with TGA (P = 0.0007). Global concordance between clinical and in vitro response was 70%. By evaluating the capacity of thrombin formation in a plasma sample, TGA predicts the response to FVIII or APCC therapy and allows individual optimization of resources in patients with severe HA and high-titre inhibitors. The inhibition pattern of the antibodies to FVIII:C correlated with the TGA parameters and showed an association with the clinical response to FVIII.

The clinical phenotype of patients with haemophilia A (HA) often differs between individuals with the same factor VIII (FVIII) gene defect (e.g. within the same family) or the same coagulant activity of FVIII (FVIII:C). We proposed that because the thrombin generation assay in platelet-poor plasma of HA patients provides more information [peak thrombin concentration, endogenous thrombin potential (ETP), rate of thrombin generation and lag-time] than a clot-based FVIII assay it might provide insight into these differences. We therefore investigated the relation between the results of the thrombin generation assay and the clinical severity in nine families with HA (23 patients with different phenotypes). We also examined the contribution of prothrombotic risk factors: (FV Leiden G1691A and prothrombin G20210A), the coagulant activity of FVIII and tissue factor (5'UTR) polymorphisms. Our data detect marked differences between individuals but these did not correlate with the reported clinical phenotype. These differences were also reflected in a marked difference in response to the therapeutic amounts of FVIII. This might account for differences in amounts of treatment consumption. Reduced peak and possibly rate of thrombin generation, rather than FVIII:C or ETP appear to represent the critical defects in FVIII-deficient plasma. We suggest that the analysis of parameters in thrombin generation is a useful tool to detect bleeding tendency in HA but not to predict the modulation of the haemorrhagic tendency in patients within families. However the presence of the other factors such as vessel wall components, protein C and platelets might need to be incorporated into this system.

Detection of minimal residual disease (MRD) in patients with B-cell acute lymphoblastic leukemia (B-ALL) has been achieved using several radioactive labelling methodologies; however, limited information exists about the use of chemiluminescent labelling. Although many malignant disorders are related to cytogenetic alterations, there is not a consistent chromosomal translocation that could serve as a tumour marker for the monitoring of MRD. ALL are derived from B-lymphocytes in 80% of cases. In the early stages of their maturation, the immunoglobulin heavy chain genes (IgH) undergo rearrangements among their V, D, and J segments, giving rise to the Complementary Determining Regions (CDR). Among these, CDR3 is considered unique for each lymphocyte and used as a tumour-specific marker in B-ALL patients. In this study, the CDR3 was labelled with digoxigenin and used as a patient-specific probe to test its sensitivity for further detection of MRD. Fourteen pretreatment samples of bone marrow (BM) or peripheral blood (PB) from B-ALL patients were included. Tumour-specific probes were designed from each clonal product by elimination of the consensus sequences. Ten digoxigenin-labelled probes were hybridized with a mixture of their respective clonal DNA and the polyclonal product from a normal healthy donor, in serial dilutions from 1:1 up to 1:10(7). A sensitivity range of 1:10(3)-1:10(6) was obtained, with an average of 1:10(5). Crossed tests performed in four patients, showed right probe specificity in all cases. We propose that the design of allele-specific probes with chemiluminescent labelling, represents a reliable, sure and sensitive alternative methodology for MRD detection in patients with B-cell lymphoproliferative disorders.

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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.

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.