Sujin Kanokpongsakdi

Prenatal diagnosis of β-thalassaemia by reverse dot-blot hybridization

Thalassaemia is the most common genetic disease and is a public health problem of Thailand. Prevention and control of β‐thalassaemia diseases need accurate diagnosis of carriers and proper genetic counselling. Prenatal diagnosis is needed to prevent birth of the thalassaemic offspring in the couple at risk. This can be performed in the first trimester of pregnancy by DNA analysis using the polymerase chain reaction (PCR). Since there are more than 20 mutations causing β‐thalassaemia in Thailand, the point mutation detection by reverse dot‐blot allele‐specific oligonucleotide (ASO) hybridization was developed using two sets of ASO probes. The first battery of ASO probes has been designed to detect 10 common β‐globin gene mutations including codon 26, G→A (Hb E); codons 41/42, ‐TCTT; codon 17, A→T; IVS 2 nt 654, C→T; IVS 1 nt 1, G→T; IVS 1 nt 5, G→C; codon 19, A→G (Hb Malay); codon 35, C→A; codons 71/72, +A and ‐28 ATA, A→G. The second set of ASO probes detect 14 uncommon β‐thalassaemia mutations.

Detection of α-thalassemia-1 (Southeast Asian type) and its application for prenatal diagnosis

A simple non‐radioactive method based on the polymerase chain reaction was used to detect the Southeast Asian type of α‐thalassemia 1 (–). Three oligonucleotide primers, one of which was adjacent to the breakpoint of the α‐thalassemia‐1 allele, were used to amplify the 570 and 194 bp DNA fragments. The 570 bp product was specific to the α‐thalassemia‐1 determinant and the 194 bp fragment was amplified from either the α‐thalassemia‐2 (‐α) or normal α‐globin (αα) determinants. In Hb Barts hydrops fetalis (–/–), only the 570 bp fragment was obtained, whereas the 194 bp fragment was amplified in normal individual (αα/αα) and α‐thalassemia‐2 trait (‐α/αα). Both 570 and 194 bp fragments were detected in α‐thalassemia‐1 trait (–/αα) and Hb H patients (–/‐α). This procedure is useful for the rapid screening of α‐thalassemia‐1 trait and prenatal diagnosis of Hb Barts hydrops fetalis in populations with a high frequency of the Southeast Asian Type of α‐thalassemia‐1.

Noninvasive Prenatal Diagnosis for Hemoglobin Bart’s Hydrops Fetalis

Hemoglobin (Hb) Bart’s hydrops fetalis, the most severe thalassemic disease, occurs from homozygosity of α-thalassemia 1. Deletion of all 4 α-globin genes (- -/- -) in this condition results in the absence of α-globin chains, and the physiologic dysfunction of Hb Bart’s (γ4) leads to lethality, either in utero or soon after birth. The best strategy for prevention and control of the disease is prenatal diagnosis in the mothers at risk. However, conventional prenatal diagnosis involves invasive procedures that may result in infection or abortion. In this study, a simple technique was developed to identify the presence or absence of α-globin chains in fetal nucleated red blood cells (NRBCs) enriched from maternal blood. Mononuclear cells including fetal NRBCs were isolated from maternal blood at 10 to 26 weeks of pregnancy by density-gradient centrifugation. Immunomagnetic separation with anti-CD71 antibody was employed to enrich fetal NRBCs, whose numbers increase with increasing gestational age. For the unaffected fetus, fetal NRBCs were detected by immunofluorescence microscopy after staining with rabbit antihuman α-globin antibody. In contrast, fetal red blood cells homozygous for α-thalassemia 1, which were identified from their size and morphology, did not stain for a-globin antibody. In this study, 3 affected fetuses were detected from 10 pregnancies at risk of Hb Bart’s hydrops fetalis, and the results were confirmed by DNA analysis. In the remaining cases, all fetal NRBCs were positive for immunofluorescence staining. DNA analysis revealed that 2 cases were normal, 1 was heterozygous for α-thalassemia 2, and the other 4 cases were heterozygous for α-thalassemia 1.

Reverse Twin-Twin Transfusion Syndrome after Fetoscopic Laser Photocoagulation of Chorionic Anastomoses: A Case Report

A case of severe twin-twin transfusion syndrome (TTTS) which developed at menstrual age of 17 weeks underwent a fetoscopic laser ablation of the anastomosing vessels. The vascular equator of the anastomoses was noticed to be deviated due to marginal cord insertion of the recipient fetus. The procedure was accomplished uneventfully. However, the recipient fetus died 6 h after the procedure. After the pregnancy was terminated, the donor was found to be counterintuitively plethoric, and the recipient was pale. Similar appearances were noted on the placental territories of each fetus. This is consistent with reverse TTTS. Dye injection study and microscopic examination revealed a residual deep vein-vein anastomosis. This subchorionic vascular connection is still a challenge to coagulate with current placental surgery techniques. To the best of our knowledge, this is the first confirmed case of reverse TTTS as a cause of fetal loss following laser photocoagulation. This report also discusses the technical considerations in the complicated case of in utero placental surgery for TTTS.

Predicting fetal anemia by using reference centile charts for liver length, spleen perimeter and umbilical vein maximum flow velocity in Thai fetuses throughout gestation

Aim: To create reference centile charts for liver length, spleen perimeter and umbilical vein maximum flow velocity (UVVmax) in Thai fetuses in order to predict fetal anemia in Thai fetuses.