Mahasandana C

Circulating Anticoagulants in the Newborn: Relation to Hypercoagulability and the Idiopathic Respiratory Distress Syndrome

Extract: Cord blood from 106 high risk and 68 normal deliveries was studied for hypercoagulability and levels of the circulating anticoagulants, antithrombin III (AT-III) and antiactivated factor X (anti-Xa). Table I shows that normal, term, appropriate for gestational age (AGA) infants showed a deficiency at AT-III when compared with normal children and adults (P < 0.001). Preterm (Pr) AGA infants had a lower level of AT-III than term (T) AGA infants (P < 0.001). The lowest levels of AT-III were seen in infants who developed idiopathic respiratory distress syndrome (IRDS); 66% of all infants with an AT-III time of 18 sec or less (mean ± sem AT-III value for T-AGA infants, 37 ± 2.8) developed IRDS. All except three of the infants (Fig. 1) with IRDS and/or low AT-III levels were offspring of mothers in the high risk category of premature labor or third trimester bleeding. When the thrombelastogram was used as a measurement of whole blood coagulability, T-AGA infants showed hypercoagulability when compared with adults and children (P = 0.025). The infants with the most hypercoagulability were offspring of the groups of severe erythroblastosis fetalis, third trimester bleeders, and premature labor.As seen in Table I and Figure 2, anti-Xa activity is slightly lower in the Pr-AGA group than in adults (P < 0.01), but the level of anti-Xa does not correlate well with the development of IRDS.Speculation: The susceptibility of the ill newborn infant to thrombotic, hemorrhagic, and necrotic pathologic lesions may be related to a deficiency of a potent, naturally occurring anticoagulant, antithrombin III. This AT-III, like α-1-antitrypsin, is a proteinase inhibitor. Both of these substances are severely deficient in infants with IRDS. The therapeutic implications of replacement of these proteins in groups of infants at risk to develop IRDS and hemorrhagic-thrombotic lesions remains to be explored.

Malignant lymphoma in Thailand: changes in the frequency of malignant lymphoma determined from a histopathologic and immunophenotypic analysis of 425 cases at Siriraj Hospital

BACKGROUND Analysis of malignant lymphoma in a single institution at different periods of time can determine the changing status of the disease in the region. METHODS To compare with the large series of 1095 lymphoma cases reported between 1957-1971 at Siriraj Hospital, the largest hospital in Thailand, a similar study was performed through histopathologic evaluation of 425 lymphoma cases diagnosed consecutively at the same institution between August 1993 and October 1995. Phenotypic analysis was performed by paraffin section-immunoperoxidase studies. RESULTS A striking increase in lymphoma cases was noted from 73 cases/year in the first series to 189 cases/year in the second series (an increase of 158.9%). Lymphoma occurred in all age groups, with a peak incidence at the seventh decade of life. The male to female ratio decreased from 2:1 in 1957-1971 to 1.3:1 in the more recent series. The incidence of Hodgkins disease (HD) was found to have decreased from 28.9% to 8.5%. There were 36 cases (8.5%) of HD and 389 cases (91.5%) of non-Hodgkins lymphoma (NHL) reported in the second series. The subtypes of HD included 16 cases of mixed cellularity, 13 cases of nodular sclerosis, 6 cases of lymphocyte depletion, and 1 case of lymphocyte predominance. According to the Working Formulation, the 389 NHL cases included low grade (14.1%), intermediate grade (57.3%), high grade (11.3%), and miscellaneous groups (17.2%). They were classified as small lymphocytic (9.5%), follicular (11.1%), diffuse (50.9%), immunoblastic (4.1%), small noncleaved (4.4%), lymphoblastic (2.8%), anaplastic large cell (9.0%), mycosis fungoides (1.8%), hairy cell leukemia (0.3%), true histiocytic (0.5%), and extramedullary plasmacytoma (1.0%). The immunophenotypes of the 359 NHL cases available for paraffin section-immunoperoxidase studies were B-cell (71.0%), T-cell (24.5%), histiocyte (0.6%), and undetermined phenotypes (3.9%). CONCLUSIONS The incidence of malignant lymphoma is increasing in Thailand, with a high frequency of intermediate to high grade NHL of B-cell phenotype reported.

Congenital afibrinogenaemia caused by uniparental isodisomy of chromosome 4 containing a novel 15-kb deletion involving fibrinogen Aα-chain gene

Among rare inherited deficiencies of coagulation factors, congenital afibrinogenaemia is characterised by the lack of fibrinogen in plasma. In the last few years, several genetic defects underlying afibrinogenaemia (mostly point mutations) have been described in the fibrinogen gene cluster. In this study, the molecular basis responsible for afibrinogenaemia in a Thai proband was defined. Point mutation screening was accomplished by directly sequencing the three fibrinogen genes. The impossibility to amplify fibrinogen Aα-chain gene (FGA) exons 5 and 6 suggested the presence of a homozygous deletion. A specific long-range PCR assay enabled the identification of a novel 15-kb deletion, representing the largest afibrinogenaemia-causing deletion described so far. Direct sequencing of the deletion junction allowed mapping of the breakpoints in FGA intron 4 and in the intergenic region between Aα- and Bβ-chain genes. Since the mutation was inherited only from the mother and nonpaternity was ruled out, a maternal uniparental disomy (UPD) was hypothesised. UPD test, carried out with markers covering the whole chromosome 4, revealed that maternal isodisomy was responsible for homozygosity of the 15-kb deletion in the proband. The apparently normal phenotype of the proband, except for afibrinogenaemia, suggests that UPD for chromosome 4 is clinically silent. This represents the first case of a documented complete isodisomy of chromosome 4 causing the phenotypic expression of a recessive disorder. In silico analyses of the regions surrounding the breakpoints suggested that the 15-kb deletion might have originated from an inappropriate repair of a double-strand break by the nonhomologous end joining mechanism.

Acquired bleeding disorders: the impact of health problems in the developing world

Summary.  Several acquired bleeding disorders in the developing world have impacts on health, including late vitamin K deficiency bleeding (VKDB) in infants, dengue haemorrhagic fever (DHF), and malaria. This paper describes their clinical manifestations, mechanisms involved, and treatment.

Frameshift mutations with severe and moderate clinical phenotypes in Thai hemophilia A patients

Six frameshift mutations in exon 14 of the factor VIII gene were identified in Thai hemophilia A patients. Although all these mutations created premature stop codons and expected to cause severe disease, the molecular defects and clinical severity were in discrepancy in some patients. Four mutations (delT3490, delACAC3618‐21, delGA4429‐30, and delA4658) were found in the patients with the severe clinical phenotype while two (delA3629‐37 and insA4372‐9) were observed in the patients who had moderate severity, with FVIII:C of 4.2 and 2.8%. The frameshift mutations in these two patients were due to deletion and insertion of an ‘A’ nucleotide in the stretches of 9As and 8As in codons 1191‐4 and 1439‐41, respectively. This indicates that deletion or insertion in the stretches of poly A nucleotides in exon 14 of the factor VIII gene is a likely cause of the moderate clinical severity in some cases of Thai hemophilia A patients. Hum Mutat 16:530–531, 2000.

Mutations of the factor VIII gene in Thai hemophilia A patients

Hemophilia A is a common X‐linked bleeding disorder caused by mutations in the coagulation factor VIII gene. The entire coding and essential sequences of the factor VIII gene were generated by a combination of genomic DNA amplification and long reverse transcription‐polymerase chain reaction (long RT‐PCR) using factor VIII transcripts prepared from lymphocytes. Mutations were then screened by non‐radioactive single strand conformation polymorphism (SSCP) analysis and characterized by DNA sequencing. We have identified six potentially pathogenic mutations in the factor VIII gene in Thai hemophilia A patients, including two nonsense mutations (R‐5X and R1966X), three missense mutations (D542Y, G1850V, and G2325C), and a 4‐bp insertion (ACTA) at codon 2245. Three of these mutations (D542Y, G2325C, and 4‐bp insertion) have never been previously reported, and the ins2245 is the first example of such insertion probably causing factor VIII elongation. R1966X, D542Y, G1850V, and 4‐bp insertion were associated with a severe hemophiliac phenotype whereas R‐5X and G2325C were observed in moderately affected patients. Mutations in the factor VIII gene in Thai hemophilia A patients are likely to be heterogeneous. This study represents the first attempt to further the understanding of the molecular basis of hemophilia A in Thai. Hum Mutat 15:177–118, 2000.

Genotype and phenotype of haemophilia A in Thai patients

Summary.  To study genotype and phenotype correlation of haemophilia A in Thai patients, molecular defects of the factor VIII (FVIII) gene were examined and their correlation with clinical phenotypes were evaluated. The molecular pathologies of FVIII in Thai patients were found to be heterogeneous. The most common mutation was FVIII intron 22 inversion accounting for about 30% of the severe cases while gene deletion was rare. Sixteen point mutations were identified, comprising two nonsense mutations (R‐5X and R1966X), five missense mutations (T233I, D542Y, G1850V, W2229S and G2325C), five nucleotide deletions (1145delT, 1187–8delACAC, 1191–4delA, 1458delGA and 1534delA), three nucleotide insertions (1439–41insA, 1934insTA and 2245insACTA) and one splicing defect (IVS15+1G>T). Nine mutations (T233I, D542Y, 1145delT, 1458delGA, 1534delA, 1934insTA, W2229S, 2245insACTA and G2325C) were novel, firstly identified in Thai patients. The genotypes were found to correlate with clinical phenotypes in a majority of cases. However, in five patients the molecular defects did not correlate with clinical severity and FVIII:C level. Cellular and molecular mechanisms were proposed to be responsible in amelioration of clinical severity caused by deleterious mutations. Carrier detection by direct mutation analysis was also demonstrated.

Mutation causing exon 15 skipping and partial exon 16 deletion in factor VIII transcript, and a method for direct mutation detection

A splicing defect with 201 nucleotide deletion in the factor VIII transcript due to IVS15 + 1G > T mutation inactivating this donor splice site and activating a cryptic acceptor splice site in exon 16 was identified in a severe haemophilia A patient. Allele specific amplification (ASA) method was successfully developed for direct detection of this mutation.

Determination of haemophilia A carrier status by mutation analysis

A reliable method for determination of carrier status and genetic counselling is required for effective control of haemophilia. Linkage analysis is currently the most widely used method for this purpose; however, in cases where there is no prior family history and/or unavailability of informative polymorphic markers it is less applicable. Detection of a mutation characterized in each family may be an alternative method for determination of the carrier status. In this study, linkage analysis using four polymorphic DNA markers, and direct mutation analysis were compared to determine the carrier status in six unrelated Thai haemophilia A families, two with a family history and four without. In the two families with a family history of haemophilia A, the carrier and noncarrier statuses could readily be determined in eight females by either linkage or direct mutation analysis. In the four families without a family history, the polymorphic DNA markers for linkage analysis were informative in two families and uninformative in the other two. The carrier status could be excluded in all four female siblings of the patients in the former. However, the specific FVIII gene mutation was not observed in the mother of one patient, who should have carried the mutation. In the remaining two families with uninformative polymorphic DNA markers, the carrier and noncarrier statuses of four female members could only be determined by direct mutation analysis. Therefore, direct mutation analysis could circumvent the limitations of linkage analysis in the determination of haemophilia A carrier status in families without a previous history or informative polymorphic markers.