RESUMEN
Our 25 years of experience in carrier diagnosis of hemophilia A (HA) and B (HB) in Mexican population comprises linkage analysis of intragenic F8/F9 neutral variants along with, in severe HA (SHA), detection of F8 int22h and int1h inversions. In symptomatic carriers (SCs) we explored Lyonization to explain their symtomatology. From a DNA-Bank of 3,000 samples, intragenic restriction fragment length (RFLPs) and short tandem repeats (STRs) of F8/F9 genes were assessed by PCR-PAGE and GeneScan. In SHA patients, F8 inversions were detected by inverse shifting-PCR/diagnostic and complementary tests. In SCs, we evaluated hemorrhagic symptoms, clotting FVIII/FIX and X-chromosome inactivation (XCI) patterns were assessed by HUMARA assay and the search of XIST promoter pathogenic variants. Informativeness of linkage analysis for HA carrier diagnosis with RFLP's/STR's increased to 74% and reached 80% with five RFLPs for HB. Combined Inv22/Inv1 diagnosed 113 possible carriers, three de novo Inv22-1, and confirmed 45 mothers as obligate or sporadic carriers. Among 21 SCs, four showed extreme skewed XCI pattern (~80:20) but had normal karyotype and no C43G pathogenic variant in XIST promoter. Clotting FVIII/FIX correlated with the active X in leukocytes. Our data integrate the largest comprehensive research worldwide on the molecular diagnosis of HA and HB carriers in terms of the number of studied and diagnosed cases, in addition to the genetic analysis in SCs. Intragenic RFLPs and STRs of F8/F9 genes along with F8 int22h/int1h inversions in SHA emerge as optimal variants for molecular diagnosis in Mexican population. In counseling SCs, inheritance of skewed X-inactivation should be considered.
Asunto(s)
Hemofilia A , Inversión Cromosómica , Factor VIII/genética , Pruebas Genéticas , Hemofilia A/diagnóstico , Hemofilia A/genética , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
X chromosome inactivation is a stochastic event that occurs early in female embryo development to achieve dosage compensation with males. Certain genetic mechanisms affect the normal process causing a skewed X inactivation pattern which has clinical relevance in female carriers of X-linked recessive disorders, like haemophilia. The most commonly used assay to evaluate the X inactivation pattern is the PCR amplification of the human androgen receptor gene (HUMARA). The use of this technique in bleeding carriers and women with haemophilia allows identifying if their hemorrhagic symptoms are due to an unfavourable lyonization. Furthermore, these studies are important for understanding the X chromosome inactivation process in humans.
Asunto(s)
Hemofilia A/genética , Heterocigoto , Inactivación del Cromosoma X , Femenino , Marcadores Genéticos , Humanos , Receptores Androgénicos/genéticaRESUMEN
La inactivación del cromosoma X es un fenómeno estocástico que ocurre en la embriogénesis temprana femenina para lograr una compensación de dosis génica respecto a los varones. Ciertos mecanismos genéticos afectan el proceso normal, propiciando una inactivación sesgada con efectos clínicos relevantes en portadoras de trastornos recesivos ligados al cromosoma X, como la hemofilia. La herramienta molecular mayormente utilizada para la evaluación del patrón de inactivación del cromosoma X es la amplificación por PCR del gen del receptor de andrógenos humano (HUMARA). El empleo de esta técnica en portadoras sintomáticas y mujeres con hemofilia permite esclarecer si las manifestaciones de la enfermedad se deben a una lyonización desfavorable. Estos estudios, además, son importantes para la comprensión del proceso de inactivación del cromosoma X en humanos.
X chromosome inactivation is a stochastic event that occurs early in female embryo development to achieve dosage compensation with males. Certain genetic mechanisms affect the normal process causing a skewed X inactivation pattern which has clinical relevance in female carriers of X-linked recessive disorders, like haemophilia. The most commonly used assay to evaluate the X inactivation pattern is the PCR amplification of the human androgen receptor gene (HUMARA). The use of this technique in bleeding carriers and women with haemophilia allows identifying if their hemorrhagic symptoms are due to an unfavourable lyonization. Furthermore, these studies are important for understanding the X chromosome inactivation process in humans.
