Prenatal diagnosis for haemophilia: the Thai experience.

BACKGROUND: Haemophilia is a lifelong X-linked recessive inherited bleeding disorder. Since the haemophilia management in economically less-developed countries is inadequately provided, prevention of new cases of haemophilia is essentially required. SUBJECTS AND METHODS: A total of 42 pregnancies in 37 women at risk for severe and moderate haemophilia (A = 33, B = 4) were enrolled. The prenatal diagnostic (PND) procedure was performed in 32 women, while 10 women refused further PND procedure after knowing their foetuses were female (n = 8) and male (n = 2). The foetal specimen was obtained through chorionic villus sampling (n = 14), amniocentesis (n = 1) and cordocentesis (n = 17). The status of haemophilia was determined using informative RFLP markers and inversion of intron 22 of the F8 gene, and/or foetal FVIII:C or FIX:C. RESULTS: The final diagnosis revealed normal males (n = 18), haemophilia A males (n = 9), normal females (n = 3) and haemophilia A carrier females (n = 2). All women with affected haemophilia sons requested to terminate their pregnancies except one woman. One of 32 pregnancies (3.1%) had spontaneous abortion. At follow-up after birth, the PND was accurately confirmed in one haemophilia A male, three normal females and two carrier females by laboratory testing, and 18 unaffected normal males by history taking of no bleeding manifestations. However, 10 women who continued their pregnancies after knowing foetal sex turned out to have two haemophilia A males, one normal female, one haemophilia A carrier female and six normal or carrier females. CONCLUSION: The PND of haemophilia could be accurately determined but it was not well accepted by all couples at risk.

Detection of known haemophilia B mutations and carrier testing by microarray.

The molecular basis of haemophilia B is heterogeneous and many mutations of the Factor IX (FIX) gene have been characterised. Using the allele-specific arrayed primer extension (AS-APEX) technology, we have designed a FIX array to simultaneously analyse 69 mutations found in British, Thai and Chinese patients. This technology overcomes the problem of multiple reverse dot-blot analysis and has a 100% accuracy in the detection of both affected subjects and carriers in families with known mutations. In seven unknown mutations from Thailand, the array could detect the specific mutation in five and in the remainders the normal primer at specific spots failed to extend due to a mutation a few nucleotides upstream, thus allowing their identification. Hence this FIX array can detect 53% of the 2891 mutation entries in the FIX database. Each of the microarray slide can be used for three different test samples and would be useful for carrier testing for common mutations and prenatal diagnosis. It is simpler and more cost effective than genome sequencing and would be particularly useful in laboratories with limited technical capabilities.

A comparison of the allelic frequencies of ten DNA polymorphisms associated with factor VIII and factor IX genes in Thai and Western European populations.

The frequency of five factor VIII gene intragenic and linked DNA polymorphisms and five factor IX gene intragenic polymorphisms was studied in Thai females. The polymorphisms in the FVIII gene were detected by restriction enzymes BclI, XbaI, BglI and at linked loci DX13 (DXS15) and St14 (DXS52) by BglII and TaqI, respectively, and in the FIX gene by MseI, DdeI, XmnI, TaqI and HhaI. With the exception of the BglI restriction fragment length polymorphism (RFLP), which is absent in Thais, factor VIII polymorphism frequencies were similar in Thais and Caucasians. Combined use of XbaI and TaqI/St14 resulted in a heterozygosity rate of greater than 90% in Thai females. For FIX, the recently described MseI RFLP in the 5' flanking region was the most informative polymorphism in Thais, 43% of females being heterozygous. The other four polymorphisms added little to the overall heterozygosity rate. The appropriate polymorphisms were used to track defective factor VIII and IX genes through 22 Thai pedigrees with haemophilia to enable carrier status to be assigned to female family members. The information obtained during this study will form the basis for carrier detection and prenatal diagnosis of haemophilia A and B by DNA polymorphism analysis in Thailand.

Hemogram in normal newborn babies with special reference to platelet count.

A prospective study was performed to verify the hemogram of 318 healthy fullterm newborn babies aged one hour to thirty days. The mean hemoglobin, hematocrit and reticulocyte count were between 17.6-17.9 g/dl, 52.2-53.4% and 5.7-6.7% in the first 72 hours of life. After that, they were decreased gradually approaching adult level at the later half of one month old. The white blood count at 12 to 24 hours was 18,482 +/- 6,600/microliters, gradually decreased to 9,817 +/- 2,496/microliters during 14 to 30 days. The ratio of immature to total neutrophils between 1-12 hours was 0.07 and 12 hour to 30 day was 0.04-0.05. The mean platelet counts in the first 72 hours was between (280 +/- 69) x 10(3)-(285 +/- 93) x 10(3)/microliters, it increased thereafter until 30 days old with a peak of 402 x 10(3)/microliters during 7-14 days. The platelet counts below 150 x 10(3)/microliters in the newborn period should be considered as thrombocytopenia.

Zinc and copper status of thalassemic children.

We investigated the amount of both zinc and copper in plasma, erythrocytes and hair in 11 patients with hemoglobin H disease, 59 patients with beta-thalassemia/HbE disease and 20 patients with homozygous beta-thalassemia. Plasma and hair zinc levels were found to be much lower, but erythrocyte zinc levels were higher, in thalassemic patients than in controls. The levels of copper in both plasma and erythrocytes were higher in the patients than in the controls. The mechanism with respect to the increase of the amount of both zinc and copper in erythrocytes was not clear; this result may reflect the impairment of zinc and copper utilization in tissues in the pathogenesis of these thalassemic patients.

Platelet counts in thalassemic children before and after splenectomy.

Even though thrombotic risks in thalassemia patients from standpoints of platelet dysfunction and coagulation factors are controversial, they are in favor of thrombosis due to thrombocytosis. From the study of 74 cases of thalassemia in children, marked thrombocytosis occurred during day 8 to 4 months during which one should be aware of the thrombosis. However, none of thalassemia children had acute thrombosis even at platelet counts of 1.6 million/microliters.

DNA polymorphisms for carrier detection of hemophilia in Thailand.

The assessment of carrier state based on the pedigree and laboratory testing in 55 females from 34 Thai hemophilia families (24 affected by hemophilia A, 10 by hemophilia B) was studied. The laboratory testing included phenotypic analysis (FVIII:C/vWF: Ag ratio, FIX:C) and two types of DNA polymorphisms, restriction fragment length polymorphisms (RFLP) and variable number tandem repeats (VNTR) in/and close to the factor VIII genes (Bcl I, Xba I RFLP, St 14 VNTR) and factor IX genes (Mse I, Dde I RFLP). Fifteen out of seventeen (88%) obligate hemophilia A carriers and one out of five (20%) obligate hemophilia B carriers were diagnosed by phenotypic analysis. All hemophilia A carriers were informative for at least one polymorphism (Bcl I, Xba I or St 14) while 42% of hemophilia B carriers were informative for Mse I RFLP only. DNA polymorphism analysis has advantage over phenotypic analysis since it generally gives an absolute diagnosis when informative. Most DNA polymorphism analyses are performed by PCR technique which is a simple, inexpensive and quick procedure. However, it is limited by non-informativeness and high incidence of new mutations.

DNA extraction and amplification of 10-day, room-temperature blood samples.

DNA was serially studied in 20 samples of buffy coat stored at room temperature. Each sample was divided into 5 equal volumes, namely D0, D3, D5, D7 and D10. DNA extraction was performed on days 0, 3, 5, 7 and 10 after blood collection. The mean ratio of OD260/OD280 of the DNA obtained from D0 to D10 ranged from 1.77 to 1.79, and the mean amounts of the DNA obtained from D0 to D10 ranged from 602 to 740 ng/ul. There were no significant differences in the mean ratio and amounts of DNA obtained among these samples (p > 0.05). Subsequently, amplification was successfully performed from this template DNA to yield products of 1.4 kb and 142 bp at the sites associated with beta globin and factor VIII genes, respectively. These findings suggest the possibility of sending blood samples for DNA analysis by mail, or no ice is required during transportation.

Vitamin E status, glutathione peroxidase activity and the effect of vitamin E supplementation in children with thalassemia.

Vitamin E and selenium statuses were studied in thalassemic children in comparison with 16 normal controls. Twelve Hb H disease, 46 beta-thal/Hb E and 7 beta-thal major patients had lower plasma vitamin E level than controls but plasma vitamin E/total lipids ratio of Hb H disease subjects was not different from normal. Twelve Hb H disease and 33 beta-thal/Hb E patients had normal RBC Se but increased RBC GSH-Px activity. Ten vitamin E-deficient thalassemic subjects had been supplemented with 200 mg of oral vitamin E for 4-8 weeks. After supplementation, their plasma vitamin E increased and H2O2 hemolysis decreased to normal values. Their RBC GSH-Px activity also decreased but hematocrit did not change significantly. The results demonstrate that some types of thalassemic patients have vitamin E deficiency and support that vitamin E and selenium have related functions in the prevention of RBC oxidation. Vitamin E supplementation increased RBC resistance to oxidative damage.

Vitamin K prophylaxis in the neonates by oral route with different dosages.

This paper evaluated the effect of VKP in the neonates by oral route with different dosages compared to the standard parenteral route giving a single dose at birth. Two hundred and thirty-six healthy, breast-fed infants were divided into 4 groups receiving vitamin K1 1 mg intramuscularly and 2, 3, 5 mg orally during 2-4 hours after birth. The vitamin K dependent clotting factors were measured by the thrombotest at the age of 2 weeks and 4-6 weeks. The result showed no statistical differences among these 4 groups regarding the mean prothrombin complex level and the number of PC deficient subjects. Vitamin K prophylaxis in the newborn babies by 2 mg oral route would be benefit and can be applied routinely as well as 1 mg parenteral route to prevent both HDN and APCD syndrome particularly in breast fed infants. The routine practice of giving vitamin K1 prophylaxis 2 mg orally or 0.5-1 mg intramuscularly should be recommended to all newborn infants. Giving VKP by oral route is practical for developing countries because of simple way of administration, low cost, low toxicity, as well as high efficacy.

Neonatal jaundice and glucose-6-phosphate dehydrogenase deficiency.

Ninety-six newborn aged 0-7 days with serum bilirubin of more than 15 mg/dl were studied for the G6PD status using semiquantitative and the assay method. It was found that the result of the 2 methods corresponded. The prevalence of G6PD deficiency was 12.4 per cent (there were 11 boys and one girl). Among the G6PD deficiency and the normal group, there was no difference in the age of the patient, onset of jaundice, bilirubin level, hematocrit status and the reticulocyte count. The semiquantitative method is a reliable as the assay method.

The usefulness of X-linked polymorphic loci as gene markers to track X allele and chimerism in a post-allogeneic peripheral blood stem cell transplant patient with Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS), an X-linked recessive disorder, is characterized by progressive T-cell immunodeficiency. Laboratory findings generally demonstrate reduced response to T-cell mitogens, markedly decreased serum concentration of IgM, and thrombocytopenia with small platelet volume. Allogeneic HLA-matched sibling bone marrow transplantation (BMT) can correct this disorder. We report the usefulness of X-linked polymorphic loci to detect X-allele gene tracking among WAS siblings and chimerism between a pre- and post-allogeneic matched sibling peripheral blood stem cell transplantation (PBSCT). A 3 1/2 year old boy with clinical and laboratory findings consistent with WAS underwent allogeneic matched sibling PBSCT. We used BclI restriction fragment length polymorphism (RFLP) of intron 18 of factor VII gene and MseI RFLP of the 5' flanking region of factor IX gene to detect X-allele gene tracking among siblings and family members and chimerism in patients between pre-and post-allogeneic matched sibling PBSCT. We were able to demonstrate that determination of BclI and MseI RFLP can be employed to recognize the difference in X-allele genes between the recipient and donor for allogeneic matched sibling PBSCT. The authors also were able to demonstrate that these polymorphic loci can detect full chimerism of donor hematopoietic cells in recipient blood after allogeneic PBSCT. This finding was correlated with improvement of post-PBSCT clinical and laboratory findings. BclI and MseI RFLP associated with X-chromosome can effectively track X-allele, detect carrier state, and demonstrate the different X-allele among male siblings, and chimerism of hematopoietic cells between donors and recipients in a setting of allogeneic matched sibling BMT or PBSCT for X-linked hereditary diseases such as Wiskott-Aldrich syndrome.

Prevalence of factor V Leiden in Thai blood donors.

Factor V Leiden was recently found to be the most common cause of familial venous thrombosis in the European population. We have studied the prevalence of factor V Leiden by DNA analysis among 500 Thai blood donors (male 285, female 215). Their ages ranged from 18 to 60 years with a mean of 33 years and 2 months. All of them were healthy voluntary blood-donors who met the standard criteria of the American Association of Blood Banks. No history of thrombosis was found. The results revealed that factor V Leiden was not present among 1,000 chromosomes from Thai blood donors. This suggests that factor V Leiden is not the common genetic predisposing factor of venous thrombosis in the Thai population as compared to the European population.