RESUMEN
Hb H disease is generally associated with moderate to severe anemia but rarely requires regular blood transfusion. We recently studied two apparently unrelated patients with transfusion-dependent Hb H disease. Hemoglobin studies demonstrated Hb H and Hb Bart's without other detectable abnormal globin species. Extensive molecular analyses of the alpha globin genes and their regulatory sequence (HS-40) revealed that both patients are compound heterozygotes for alpha0 thalassemia (--(SEA)) and a novel point mutation, a thymidine insertion after codon 131 of the alpha1 gene. The resulting frameshift gives rise to a highly unstable alpha globin chain, which we refer to as "Hb Pak Num Po," containing an additional 34 amino acids. This unusual alpha1 globin variant clearly causes alpha thalassemia, but the unexpectedly severe phenotype suggests that this mutation may have additional effects on red cell physiology. A PCR-based (ARMS) assay was developed for rapid detection of this novel mutation, and this might be useful to study the prevalence of this novel mutation which poses potentially significant clinical consequences in populations of Southeast Asia. Detecting carriers of this mutation using the molecular diagnostic procedures described will provide the means to screen and prevent a potentially severe form of alpha thalassemia in Thailand.
Asunto(s)
Transfusión Sanguínea , Hemoglobinas Anormales/genética , Mutación Puntual/genética , Talasemia alfa/genética , Talasemia alfa/terapia , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Preescolar , Codón/genética , Análisis Mutacional de ADN , Femenino , Globinas/genética , Haplotipos , Heterocigoto , Humanos , Hidropesía Fetal/sangre , Hidropesía Fetal/genética , Masculino , Datos de Secuencia Molecular , Timidina/genética , Talasemia alfa/sangre , Talasemia alfa/patologíaRESUMEN
Shortened red cell life span and excess iron cause functional and physiological abnormalities in various organ systems in thalassemia patients. In an earlier study, we showed that beta-thalassemia patients have a high prevalence of renal tubular abnormalities. The severity correlated with the degree of anemia, being least severe in patients on hypertransfusion and iron chelation therapy, suggesting that the damage might be caused by the anemia and increased oxidation induced by excess iron deposits. This study was designed to define the renal abnormalities associated with alpha-thalassemia and to correlate the renal findings with clinical parameters. Thirty-four pediatric patients (mean age 8.2+/-2.8 years) with Hb H disease or Hb H/Hb CS were studied. Ten patients (group 1) were splenectomized, with a mean duration post splenectomy of 3.5+/-1.4 years; 24 patients (group 2) had intact spleens. The results were compared with 15 normal children. Significantly higher levels of urine N-acetyl-beta- d-glycosaminidase, malondialdehyde (MDA), and beta(2)-microglobulin were found in both groups compared with normal children. An elevated urine protein/creatinine ratio was recorded in 60% of group 1 and 29% of group 2. Two patients (5.9%), 1 in each group, had generalized aminoaciduria. We found proximal tubular abnormalities in alpha-thalassemia patients. Increased oxidative stress, possibly iron induced, may play an important role, since urine MDA levels were significantly increased in both groups of patients.
Asunto(s)
Enfermedades Renales/etiología , Enfermedades Renales/fisiopatología , Túbulos Renales/fisiopatología , Talasemia alfa/complicaciones , Talasemia alfa/fisiopatología , Acetilglucosaminidasa/orina , Adolescente , Nitrógeno de la Urea Sanguínea , Niño , Preescolar , Creatinina/sangre , Humanos , Malondialdehído/orina , Estrés Oxidativo , Aminoacidurias Renales/etiología , Aminoacidurias Renales/fisiopatología , Bazo , Esplenectomía , Microglobulina beta-2/orinaRESUMEN
DNA linkage analysis was performed in Thai hemophilia A families to evaluate its value for carrier detection. Both intragenic and extragenic polymorphic DNA regions of the factor VIII gene, including Bcl I-RFLP in intron 18, microsatellites (CA repeats) in introns 13 and 22, and extragenic Stl4 (DXS 52) VNTR, were amplified by polymerase chain reaction (PCR) before analyses by appropriate electrophoretic procedures. A total of 80 Thai hemophilia A families (48 with a family history and 32 with a sporadic case), containing 349 DNA samples from 90 hemophilia A patients, 143 parents, and 116 relatives, were analyzed. Heterozygosities in the patients' mothers from both families with a family history and with a sporadic case were observed in 71 out of 80 families (88.75%) for all polymorphic DNA markers analyzed. The carrier status could be identified in 36 females and excluded in 44 females. This result indicates that the DNA linkage analysis can be used for carrier detection or exclusion in the majority of Thai hemophilia A families. It should also be useful for prenatal diagnosis in families at risk of hemophilia A, which is part of the prevention and control of this disease.
Asunto(s)
Ligamiento Genético , Hemofilia A/genética , Heterocigoto , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo Genético , Adulto , Secuencia de Bases , Niño , Preescolar , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Tamización de Portadores Genéticos , Hemofilia A/epidemiología , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Polimorfismo de Longitud del Fragmento de Restricción , Estudios Retrospectivos , TailandiaRESUMEN
BACKGROUND AND OBJECTIVES: Hemoglobin Constant Spring (Hb CS), caused by a termination codon mutation (TAA-->CAA) in the a2 gene, is the most common non-deletional type of a thalassemia in Southeast Asia. This mutation can most easily be detected by loss of an MseI-restriction site (T/TAA) spanning the termination codon. Recently, we sequenced the a globin genes from patients with a thalassemia in whom this MseI site was absent. This revealed, a previously described termination codon mutation (TAA-->TAT) associated with Hb Paksé. This prompted us to re-evaluate the molecular basis of a thalassaemia in other Thai patients with non-deletional types of Hb H disease. DESIGN AND METHODS: DNA samples from 30 patients, previously diagnosed as having Hb H-CS disease, were characterized by direct genomic sequencing and by using a mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Clinical and hematologic data were assessed. RESULTS: Hemoglobin electrophoresis in almost all 30 unrelated patients with non-deletional a thalassemia revealed a slow migrating band resembling Hb CS. Five of these patients were found to have Hb H-Paksé disease and the remainder had Hb H-CS disease. Comparing the hematology in patients with these two genotypes, no significant differences were found except that the proportion of Hb H was higher in patients with Hb H-Paksé disease. INTERPRETATION AND CONCLUSIONS: These results suggest that termination codon mutations may have been previously misidentified in many cases of non-deletional Hb H disease. Findings from six unrelated families described in this study suggest that the proportion of patients with the Hb Paksé mutation might be underestimated and that this mutation could be prevalent in Southeast Asia. Analysis of mismatched-PCR-RFLP, described here, was shown to provide an unequivocal diagnosis and will be applicable in population screening programs.