Non-invasive prenatal screening & diagnosis of ?-thalassaemia in an affected foetus

Background & objectives: Non-invasive prenatal testing (NIPT) of maternally inherited alleles of ?-thalassaemia (MIB) remains to be a challenge. Furthermore, current techniques are not available for use as routine tests. NIPT for ?-thalassaemia disease was developed by using a specific droplet digital polymerase chain reaction (ddPCR) assay to analyze the cell-free foetal DNA (cffDNA) derived from maternal plasma. Methods: Pregnant women and their spouses who are at risk of bearing an offspring with ?-thalassaemia disease from common MIB mutations (CD 41/42-TCTT, CD17A>T, IVS1-1G>T and CD26G>A) were enrolled. The ddPCR assay sets were constructed for each of the four mutations. All cell-free DNA samples were first screened for the paternally inherited ?-thalassaemia (PIB) mutation. The PIB-negative samples were considered as non-disease and were not further analyzed. For PIB-positive samples, DNA fragments of 50-300 base pairs in size were isolated and purified, and further analyzed for MIB mutation. The allelic ratio between the mutant and the wild-type was used to determine the presence of MIB in cffDNA. All cases underwent a prenatal diagnosis by amniocentesis for a definite diagnosis. Results: Forty two couples at risk were enrolled. Twenty two samples were positive for PIBs. Among these 22 samples, there were 10 cases with allelic ratio >1.0 (MIB positive). All foetuses with over-represented mutant alleles were further diagnosed with ?-thalassaemia disease; eight with compound heterozygous and two with homozygous mutations. The 20 PIB-negative and 12 MIB-negative foetuses were non-affected. Interpretation & conclusions: The results of this study suggest that NIPT utilizing the ddPCR assay can be effectively used for the screening and diagnosis of foetal ?-thalassaemia in at risk pregnancies.

A successful strategy for Preimplantation Genetic Diagnosis of beta-thalassemia and simultaneous detection of Down's syndrome using multiplex fluorescent PCR.

OBJECTIVES: Preimplantation Genetic Diagnosis (PGD) is an alternative to prenatal diagnosis providing couples the chance to start a pregnancy with an unaffected fetus. The objective of the present study was to develop and apply quick, sensitive and accurate single cell PCR protocols for PGD of beta-thalassemia and Down's syndrome detection. MATERIAL AND METHOD: Two couples carrying beta-thalassemia codon41-42 mutation underwent routine IVF procedures. Embryo biopsy was performed on Day-3 post-fertilisation and single cell multiplex fluorescent PCR was employed for mutation analysis, contamination detection and diagnosis of trisomy 21 cases. RESULTS: Seventeen embryos were tested in two clinical PGD cycles. This resulted in the first birth following PGD for a single gene disorder in Thailand and South East Asia, confirmed by prenatal testing. Two embryos were shown to be affected by Down's syndrome. CONCLUSION: Successful strategy for PGD of beta-thalassemia and Down's syndrome detection using multiplex fluorescent PCR was introduced.

Risk of a couple having a child with severe thalassemia syndrome, prevalence in lower northern Thailand.

Thalassemia screening in pregnant women and their spouses was performed at Buddhachinaraj Provincial Hospital and 8 community hospitals in Phitsanulok; lower northern Thailand. The prevalence of thalassemic carrier state was determined of 1,198 couples. Of these, 4.8% had heterozygous alpha thalassemia-1, 1.6% had heterozygous beta thalassemia, 12.4% had heterozygous hemoglobin (Hb) E, 2.7% had homozygous Hb E and 0.25% of others had abnormal Hb. Eighteen at risk couples (1.5%) were identified. Fifteen couples were at risk for compound heterozygous Hb E / beta thalassemia and the remaining 3 were at risk for homozygous alpha thalassemia-1. Prenatal diagnosis (cordocentesis) was performed in 4 couples at risk, but no fetuses with severe thalassemic disease were detected.

Hemoglobin E levels in double heterozygotes of hemoglobin E and SEA-type alpha-thalassemia.

Coinheritance of alpha-thalassemia and hemoglobin E (Hb E) is prevalent in Thailand, where the gene frequencies of thalassemia and hemoglobinopathies are high. Hb E carriers with, concomitant inheritance of alpha-thalassemia 1 are known to have a lower level of Hb E. In this study, we reviewed the Hb E levels in Hb E carriers, who either had or did not have Southeast Asian (SEA)-type alpha-thalassemia, in order to seek a Hb E level that may be used as a predictor for concomitant alpha-thalassemia carrier status. The Hb E levels as measured by microcolumn chromatography in 844 Hb E carriers detected during a prenatal screening program for severe thalassemia at Chiang Mai University Hospital were reviewed. Hb E levels ranged from 12.3-35.0% (23.3 +/- 3.1%) in 751 Hb E carriers without SEA-type alpha-thalassemia and from 11.6-32.0% (17.0 +/- 3.7%) in 93 concomitant Hb E and SEA-type alpha-thalassemia carriers. The difference was significant (p < 0.01). However, the absence of SEA-type alpha-thalassemia could not be predicted by the higher Hb E level alone, as 3% of double heterozygotes demonstrated a level of more than 25%. Our study confirms a lower Hb E level in double heterozygotes with Hb E and SEA-type alpha-thalassemia. Nevertheless, the difference does not provide sufficient discriminatory power for the reliable exclusion of alpha-thalassemia status.

Prevalence of thalassemia in pregnant women at Maharaj Nakorn Chiang Mai Hospital.

OBJECTIVE: To determine the prevalence of thalassemia including alpha-thalassemia-1 trait (SEA type), beta-thalassemia trait, hemoglobin E (HbE) trait, homozygous HbE, the combination of alpha-thalassemia-1 (SEA type) and beta-thalassemia trait, alpha-thalassemia-1 (SEA type) and hemoglobin E trait, and beta-thalassemia hemoglobin E disease in pregnant women. METHOD: A cross-sectional descriptive study was conducted on pregnant women who attended the antenatal clinic at Maharaj Nakorn Chiang Mai Hospital, from 1 August to 31 October 2001. All subjects had blood taken for diagnosis of thalassemia trait or diseases, based on quantitative electrophoresis, and PCR (polymerase chain reaction) technique RESULTS: 516 pregnant women were recruited 81.0% resided in Chiang Mai province, and the remainder were in other northern provinces of Thailand. The mean (+/-SD) age was 27.7+/-6.3 years old. 5.6% of cases had anemia. Overall prevalence of thalassemia trait was 25.4% which were classified as follows: alpha-thalassemia-1 (SEA type) trait 6.6%, beta-thalassemia trait 3.7%, hemoglobin E trait 11.6%, homozygous hemoglobin E 0.8%, the combination of alpha-thalassemia-1 (SEA type) and beta-thalassemia trait 1.2% and the combination of alpha-thalassemia-1 (SEA type) and hemoglobin E trait 1.5%. Additionally, the authors also found beta-thalassemia hemoglobin/E disease 0.2%. CONCLUSION: The prevalence of thalassemia carriers among pregnant women at Maharaj Nakorn Chiang Mai hospital was high, indicating the necessity of a screening thalassemia program aimed at prevention and control of this disease.

Homogeneity of beta(0)-thalassemia codon 17 (A-->T) alleles in Northern Thailand using a direct DNA sequencing method.

The aim of this study was to characterize beta-globin gene micro-haplotype polymorphisms (frameworks) associated with a beta-thalassemia mutations common in Northern Thailand using a direct DNA sequencing method. A total of 11 beta-thalassemia major patients homozygous for the codon 17 (A-->T) mutation admitted to Chiang Mai University Hospital were examined. All 22 alleles were found to contain the Asian framework 3A. The homogeneity of the framework associated with the codon 17 (A-->T) mutation indicates a relatively recent origin of the codon 17 (A-->T) mutation. Similar studies in other East Asian populations may provide information concerning the origin and the migrational spread of this beta-thalassemia mutation.

Hematological and molecular characterization of beta-thalassemia/Hb Tak compound heterozygote.

We report a case of beta-thalassemia/Hb Tak compound heterozygote. The 7 year-old Thai boy presented with plethora since birth. Hemoglobin electrophoresis showed a major band between Hb A2 and Hb F and absent Hb A. DNA sequencing study demonstrated an AC insertion at the terminal codon of the beta-globin gene. The clinical feature of polycythemia reflected a high oxygen affinity of Hb Tak.

Prenatal diagnosis of alpha-thalassemia-1 (SEA type) by chorionic villus sampling.

OBJECTIVE: To describe the experience of prenatal diagnosis for Hb Bart's disease, by chorionic villus sampling (CVS) with DNA analysis. DESIGN: Descriptive study SETTINGS: Department of Obstetrics and Gynecology, Faculty of Medicine, Chiang Mai University. SUBJECTS: Sixteen high risk pregnancies at risk of Hb Bart's disease who were eligible for CVS criteria between 1 January, 1999 and May 31, 2000. MATERIAL AND METHOD: Fetal villi were obtained by either transcervical (TC) or transabdominal (TA) CVS route to extract DNA and detect for alpha-thal-1 gene deletion (SEA type) with modified Chang's method. The CVS results were confirmed by either serial ultrasound or cordocentesis or diagnosis after pregnancy termination. MAIN OUTCOME MEASURES: The efficacy, safety and pregnancy outcomes. RESULTS: CVS was successfully done in all of 16 cases (5 with TC and 11 with TA), The mean gestational age was 13.25 +/- 2.9 weeks. The procedure time for TA was shorter than that of TC (4.64 +/- 5.4 vs 10.4 +/- 11.3 min). The CVS result showed as follows: 3 normal fetuses, 7 alpha-thal-1 carriers, 4 fetal Hb Bart's, 1 misdiagnosis and 1 failure to diagnosis due to technical error. The sensitivity and specificity were 100 per cent (4/4) and 90.91 per cent (10/11), respectively. One case of Hb Bart's misdiagnosis and one failure case were later confirmed for alpha-thal-1 trait and alpha-thal-1/ Hb E trait by cordocentesis, respectively. The pregnancy outcomes included 11 livebirths, 4 terminated cases and 1 fetal loss of continuing pregnancies. No serious complications occurred. CONCLUSION: This preliminary experience suggests that CVS is an effective method for early prenatal diagnosis of fetal Hb Bart's.

Psychosocial problems in children with thalassemia and their siblings.

Beta-thalassemia is a chronic illness causing serious symptoms to children and a burden to families. The purpose of this study was to evaluate psychosocial problems in children with thalassemia and their siblings by using a semi-structured interview and the Pediatric Symptom Checklist (PSC). The study sample included 82 children with thalassemia, 20 siblings, and 50 control children without a chronic illness. With children and families demographically controlled, psychosocial problems were significantly more common in children with thalassemia than in those without chronic illness, 28.05 per cent vs 4 per cent (p=0.001), but there was no difference between siblings and the controls, 5 per cent vs 4 per cent (p=0.64). The mean PSC score in children with thalassemia was higher than that in the sibling and control group (18.34 vs 10.95 and 10.28, respectively; p<0.001). These findings suggest an increased risk of psychosocial problems in children with thalassemia that psychosocial intervention may be required to prevent major psychiatric disorders.