Prenatal Diagnosis Of Beta-thalassemia Research Articles

Genotype-Phenotype Profile of Beta-thalassemia.

Beta thalassemias are extremely heterogenous hereditary monogenic blood disorders and preventable genetic hemolytic anemia caused by >200 mutations in HBB gene. In Nepal, it is more prevalent in Tharu tribe but it seen in other communities as well. Out of more than 200 mutations of beta globin gene, approximate 20 different alleles are responsible for >80% of the mutations. Mutations vary in different geographic population and are responsible for manifestation of different phenotypes. This study was done to find common mutations of HBB gene in Nepal which were responsible for different phenotypic manifestations and to know clinical severity according to the mutations. This was a descriptive, cross sectional study conducted in the pediatric and medicine department of Nepalgunj Medical College and Bheri Zonal Hospital, Nepalgunj from January 2020 to December 2020. The genotype and phenotype profiles of thalassemia cases were reported. The data was analyzed by SPSS 20. The results obtained showed that clinical presentation differed with different β-globin gene mutations present. Individuals with HBB:c.47G>A and HBB:c.20A>T/ c.79G>A mutations showed milder presentation than those with HBB:c.47G>A/-619del and HBB:c.20A>T/c.47G>A. Therefore, these findings can be used to predict clinical severity so that we can take appropriate measures by early genotype identification for prenatal diagnosis of beta thalassemia.

Incidental Detection of a Chromosomal Aberration by Array-CGH in an Early Prenatal Diagnosis for Monogenic Disease on Coelomic Fluid.

Turner syndrome is a rare genetic condition in which a female is partly or completely missing an X chromosome. Signs and symptoms vary among those affected. In fetuses that survive at birth and without congenital malformations, the prognosis is usually positive, but it has high lethality in utero, especially in the first trimester of pregnancy. We report a case of monosomy X detected during a prenatal diagnosis for beta thalassemia on coelomic fluid (CF) at the VIII week of gestation. Beta globin gene analysis, whole genome amplification (WGA), quantitative fluorescent PCR and array comparative genomic hybridization (array-CGH) were performed on DNA extracted from CF. A monoallelic pattern of all Short Tandem Repeats mapped on the X chromosome was found and array-CGH performed on WGA from a few fetal erythroblasts confirmed monosomy X. This report underlines the importance of an early prenatal diagnosis and the countless potentialities of array-CGH that could make definition of molecular karyotype possible from a few fetal cells, unlike conventional cytogenetic techniques that require a greater cellular content. This is the first report of a molecular karyotype obtained from two cells selected by micromanipulation of CF and defined at such an early gestational age.

Prevention programmes and prenatal diagnosis for beta thalassemia in Pakistan: A narrative review.

Beta thalassemia in Pakistan is a serious health concern with an estimated 5-8% carrier frequency and birth of 5000 major children every year in the country. The treatment of beta thalassemia major patients poses a great economic burden; hence, the ideal approach towards this disease should encompass effective prevention services. At present only one government funded project "Punjab Thalassemia Prevention Programme" existed in Punjab province, and providing free of cost services for beta thalassemia screening and prenatal diagnosis. Complete blood count and haemoglobin electrophoresis remains the preliminary test for screening, while chorionic villi sampling and amplification refractory mutation system method have been most widely used for molecular diagnosis of beta thalassemia. Modern molecular techniques, non-invasive prenatal diagnosis, and pre-implantation diagnosis are in trial phases. In this review we have discussed the available diagnostic facilities and status of prevention programmes for beta thalassemia in Pakistan as well as future perspectives.

Six-year experience of prenatal diagnosis for beta thalassemia in twin pregnancies and selective foetal reduction - A case series.

A retrospective study was conducted for prenatal diagnosis and selective foetal reduction, which included 41 beta thalassemia carrier couples with twin pregnancy. Chorionic villi sampling was carried out at 12-14 weeks of gestation. Thirty-three couples presented with dizygotic while eight had monozygotic twins. Molecular analysis for beta thalassemia revealed similar results in both the twins in 27 cases, while different results were obtained in 14 cases. Selective foetal reduction was offered to couples with discordant results. One miscarriage occurred due to the procedure. The results of prenatal molecular analysis were confirmed by postnatal molecular analysis. This series of trans-abdominal Chorionic villi sampling in twin pregnancies with the option of selective foetal reduction of the affected foetus was found to be useful as well as acceptable by the at-risk couples.

Challenges in prenatal diagnosis of beta thalassaemia: couples with normal HbA2 in one partner.

To undertake β-genotyping in couples having normal/borderline HbA2 levels in one partner to offer the possibility of prenatal diagnosis of thalassaemia. A total of 967 couples were screened for β-thalassaemia. Haematological analysis was carried out on a Sysmex K-1000 analyser. The HbA2 and HbF levels were measured by High Performance Liquid Chromatography-Variant II analyser (Bio-Rad, USA). β-globin gene analysis was done by reverse dot blot hybridization, amplification refractory mutation system or DNA sequencing. Alpha globin gene triplication was determined by Multiplex PCR. In 33 of 967 couples, one partner had a normal/borderline HbA2 level (1-3.5%); however a β-thalassaemia mutation could be identified in 24 of these individuals. Molecular analysis of the β-globin gene revealed the presence of the capsite +1 (A → C) [HBB: c.-50 A → C] mutation in 15 cases (60%), Poly A(T → C) [HBB: c.*110 T → C] mutation in two cases (8%), IVS 1-5 (G → C) [HBB: c. 92 + 5 (G → C)] mutation in four cases (17%) and the CD 15 (G → A) [HBB: c. 47 G → A] mutation, CD 16 (-C) [HBB: c. 51 del C] mutation and CD 30 (G → C) [HBB: c. 93 G → C] mutation in one case each (4%). Alpha gene triplication was found in five cases, while four cases remained uncharacterized. β-genotyping should always be done in a couple if one partner is a β-thalassaemia carrier irrespective of the RBC indices and HbA2 levels of the other partner.

Correlation of beta-thalassemia mutations with alpha-thalassemia: an experience of the southwestern region of Iran

Background and aims: Thalassemias are the commonest monogenic diseases worldwide. Mutations in genes coding for the globin proteins that alter protein output, produce the thalassemia syndromes. This study aimed to present the cases of alpha-thalassemia in beta-thalassemic patients from Khuzestan province in Iran.Materials and methods: A total of 227 subjects (10–45 years, mean age of 14·8 years) including thalassemia patients (n=43), and their parents and siblings (n=184) from unrelated Khuzestani families were included in this study. The standard diagnostic marker for beta-thalassemia is elevation of the hemoglobin A2 (HbA2) level (>3·5%), while low mean corpuscular volume (MCV<80) and mean cell hemoglobin (MCH<27) with a normal HbA2 level indicate alpha-thalassemia carrier.Results: The amplification refractory mutation system polymerase chain reaction confirmed that among 227 subjects, 147 (64·75%) were heterozygous beta-thalassemia patients and 80 (35·25%) were identified either normal or with unknown mutations. Out of these 147 patients, nine cases were diagnosed as compound heterozygous. The IVS11-1(G-C) mutation was found in majority of chromosomes (40·08%). Out of nine beta/alpha-thalassemia patients, four showed −α3·7/αα (1·76%), two had −α3·7/−α3·7 (0·88%), one had −α4·2/αα (0·44%) and one had – −MED/αα (0·44%) genotypes.Conclusion: The present study suggests the need to establish a program for genetic counseling and prenatal diagnosis of beta-thalassemia for affected families and for initiating a control program by prospective screening of pregnant women. The application of the knowledge about mutation pattern was found to be beneficial since the mutations could be screened in the order in which they are present in our population. Hence, it will not only help to reduce the screening cost but also to promote early genetic counseling and prevention of an affected child.

Clinical and haematological features in a compound heterozygote (HBB:c.92 + 5G &gt; C/HBB:c.93‐2A &gt; C) case of thalassaemia major

An Indian Muslim boy was diagnosed with thalassaemia major at 3 months of age. His blood investigations revealed haemoglobin: 5.3 gm%, MCV: 68 fl, MCH 26.6 pg, MCHC: 39%, haemoglobin variant analysis: HbA(2): 2.8%, HbF: 20.3% and HbA: 75.2% (post-transfusion). His fathers' haemoglobin was 10.2 gm%, MCV: 68 fl, MCH: 23.9 pg, MCHC: 35% HbA(2): 4.7%, HbF: 0.7% and HbA: 85.2% and his mothers' haemoglobin was 10.9 gm%, MCV: 67.4 fl, MCH 22.6 pg, MCHC: 33.5%, HbA(2): 5.3%, HbF: 0% and HbA: 85.4%. The boy was found to be compound heterozygote for beta globin gene mutations (HBB:c.92 + 5G > C/HBB:c.93-2A > C). The mutation HBB:c.93-2A > C was inherited from his father. This report confirms the presence of HBB:c.93-2A > C in the Indian subcontinent and has important implications for screening and prenatal diagnosis of beta thalassaemia. This report also supports inclusion of this mutation in the beta globin gene mutation database.

Molecular diagnostics for haemoglobinopathies

The globin gene mutations have been used as a prototype for the development of many techniques of mutation detection for > 30 years, and consequently there have been numerous PCR-based methods described that can be used for the molecular diagnosis of the haemoglobinopathies. This review describes the most commonly used current methods for the detection of each of the different types of thalassaemia mutation and abnormal haemoglobin, and reviews the most promising newer technology. In addition, it outlines the various diagnostic strategies commonly used for prenatal diagnosis by fetal DNA analysis and discusses the state of progress of the molecular analysis of cell-free DNA for non-invasive prenatal diagnosis. An understanding of which are the best current techniques for carrier detection and prenatal diagnosis of the full range of haemoglobinopathy mutations, and which of the newer, more complex technologies are being applied to the emerging approach of non-invasive prenatal diagnosis of beta thalassaemia and sickle cell disease. The haemoglobinopathies are a very heterogeneous group of genetic disorders with > 1300 different mutations described, from point mutations to large deletions and complex gene rearrangements. Despite the complexity of the disorders and the plethora of high-tech diagnostic methods developed, mutation screening and prenatal diagnosis are still carried out in many countries by simple and cheap in-house methods developed many years ago; however, the more advanced technologies may well play an essential role in the various approaches to non-invasive prenatal diagnosis haemoglobinopathies in the future.

Simple, Efficient, and Cost-Effective Multiplex Genotyping with Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Hemoglobin Beta Gene Mutations

A number of common mutations in the hemoglobin beta (HBB) gene cause beta-thalassemia, a monogenic disease with high prevalence in certain ethnic groups. As there are 30 HBB variants that cover more than 99.5% of HBB mutant alleles in the Thai population, an efficient and cost-effective screening method is required. Three panels of multiplex primer extensions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were developed. The first panel simultaneously detected 21 of the most common HBB mutations, while the second panel screened nine additional mutations, plus seven of the first panel for confirmation; the third panel was used to confirm three HBB mutations, yielding a 9-Da mass difference that could not be clearly distinguished by the previous two panels. The protocol was both standardized using 40 samples of known genotypes and subsequently validated in 162 blind samples with 27 different genotypes (including a normal control), comprising heterozygous, compound heterozygous, and homozygous beta-thalassemia. Results were in complete agreement with those from the genotyping results, conducted using three different methods overall. The method developed here permitted the detection of mutations missed using a single genotyping procedure. The procedure should serve as the method of choice for HBB genotyping due to its accuracy, sensitivity, and cost-effectiveness, and can be applied to studies of other gene variants that are potential disease biomarkers.

Thalassaemia‐like carriers not linked to the β‐globin gene cluster

This study describes the largest series reported to date, of individuals belonging to unrelated families carrying a beta-thalassaemia-like phenotype in whom the beta-globin gene was found to be structurally intact by sequence analysis. This genetic determinant appears haematologically heterogeneous, displaying either a silent beta-thalassaemia-like phenotype or a typical beta-thalassaemia carrier-like phenotype in different families. Compound heterozygosity for both beta-thalassaemia-like determinant and typical beta-thalassaemia allele resulted either in thalassaemia intermedia or thalassaemia major. By linkage analysis both the silent and the typical beta-like determinants were found not to be linked to the beta-globin cluster. Sequence analysis of the hypersensitive site cores of locus control region and of the genes coding for the transcription factors erythroid Kruppel-like factor and nuclear factor (erythroid-derived 2) were normal. beta-globin mRNA levels determined by real-time polymerase chain reaction were reduced in both types of beta-like carriers. These results indicate the existence of causative genetic determinants not yet molecularly defined, but most likely, resulting from either the reduction or loss of function of a gene coding for unknown transcriptional regulator(s) of the beta-globin gene. The knowledge of these rare beta-thalassaemia-like determinants have implications for clinical and, especially, prenatal diagnosis of beta-thalassaemia.

Early prenatal diagnosis by celocentesis.

Celocentesis is the ultrasound-guided aspiration of fluid from the extra-amniotic cavity at 7-8 weeks of gestation. This paper reports on the clinical application of celocentesis for early prenatal diagnosis. Celocentesis was successfully performed in nine pregnancies and 1-2 mL of fluid were obtained after one needle insertion. The indications were prenatal diagnosis of beta-thalassemia or sickle cell disease (n = 6), Marfan syndrome (n = 1) and paternity testing (n = 2). Molecular biological techniques were used to analyze the celomic fluid and this was successfully carried out in all cases. In two cases pregnancy termination was performed at the request of the mother because in one case the fetus was found to have sickle cell anemia and in the second case paternity testing demonstrated that the father was not the woman's husband. In both cases the results were confirmed using the placental samples collected after pregnancy termination. In six of the seven pregnancies with desirable results, amniocentesis was performed at 16 weeks and the results were concordant with those obtained from celocentesis. All pregnancies were uneventful and resulted in the delivery of healthy and appropriately grown babies. Celocentesis may be a viable alternative to the currently used tests of chorionic villus sampling and amniocentesis.

Prenatal exclusion of β thalassaemia major by examination of maternal plasma

The discovery of the presence of fetal DNA in maternal plasma has provided a new approach for non-invasive prenatal diagnosis. At present, the prenatal diagnosis of beta thalassaemia relies on invasive methods. We designed allele-specific primers and a fluorescent probe for detection of the codon 41/42 (-CTTT) mutation in the beta globin gene from maternal plasma by real-time PCR. The specificity and sensitivity of the allele-specific assay was confirmed by subjecting plasma, buffy coat, and amniotic fluid samples from 100 pregnancies to screening for the mutation. Subsequently, the assay was applied to the prenatal testing of eight fetuses at risk of beta thalassaemia major, the aim being to exclude fetal inheritance of paternally transmitted codon 41/42 mutation. The fetal genotype was completely concordant with conventional analysis and beta thalassaemia major was excluded in two of the pregnancies non-invasively.

Usefulness of automated chromatography for rapid fetal blood analysis for second trimester prenatal diagnosis of beta-thalassemia.

Prenatal diagnosis of beta-thalassemia is now ideally done in the first trimester of pregnancy by chorionic villus tissue DNA analysis. Nevertheless, fetal blood analysis in the second trimester is required either when the mutation in both parents cannot be characterised or when the couple comes late for investigations. We evaluated the usefulness of analysis of fetal blood on the Biorad Variant Hemoglobin Testing System using the beta-thalassemia short programme in comparison with the conventional globin biosynthesis in 58 pregnancies. The beta/alpha biosynthesis ratios in 13 homozygous fetuses ranged from 0 to 0.03 and the adult hemoglobin (HbA) levels by automated chromatography varied from 0% to 0.4%. The normal or heterozygous fetuses had beta/alpha ratios of >0.04 and HbA levels ranging from 2.1% to 10.6%. In 17 fetuses we also correlated the beta gene mutations with the predicted genotypes using automated high-performance liquid chromatography (HPLC). Follow-up of 18 unaffected fetuses using the Variant System at birth showed a significant increase in HbA levels.

Beta-thalassaemia in Cubans: novel allele increases the genetic diversity at the HBB locus in the Caribbean.

In order to establish the molecular basis of beta-thalassaemia in Cubans, a total of 75 unrelated individuals, with beta-thalassaemia major (7), Hb S-beta-thalassaemia (28), Hb C-beta-thalassaemia (1), and beta-thalassaemia trait (39) yielding 82 beta-thalassaemia alleles, were analyzed. Seventeen different point mutations were identified accounting for 93% of the beta-thalassaemia alleles studied, revealing a high genetic heterogeneity at the HBB locus in this population. The more prevalent mutations, namely, CD 39 (C --> T) (30.5%), -29 (A --> G) (13.4%), IVS-I-110 (G --> A) (8.5%), and IVS-II-1 (G --> A) (8.5%), reflect the Mediterranean and African predominant ancestry of the extant Cuban population. We also report the identification of a novel allele, IVS-I-108 (T --> C), that possibly activates a cryptic branch site, in a beta-thalassaemia carrier with no other molecular defect within the beta-globin gene and its proximal promoter. This study shows that prenatal diagnosis of beta-thalassaemia should be feasible in about 60% of at-risk pregnancies by direct detection of selected point mutations. However, due to the wide spectrum of mutations, and in order to offer fully informative prenatal diagnosis to more than 87% of at-risk couples, the screening for beta-thalassaemia mutations in Cubans should be performed by using a general point mutation detection method, such as DGGE (denaturing gradient gel electrophoresis).

Prenatal diagnosis of beta-thalassaemia by coelocentesis.

Coelomic fluid and placental tissue were obtained from four women undergoing termination of pregnancy at 7-9 weeks gestation for psychological reasons. All four women and their partners were known carriers of beta-thalassaemia and DNA analysis in their blood identified the mutation carried by each of them. Allelespecific polymerase chain reaction and denaturing gradient gel electrophoresis techniques were used to detect and identify the mutations in the DNA extracted from the coelomic cells and placental tissue. Three fetuses were found to be carriers of either the paternal or maternal mutation, while one was found to be affected by beta-thalassaemia. There was concordance in the results obtained from the chorionic villi and coelomic cells. Amplification of the apolipoprotein B gene variable number tandem repeats (VNTR), in the DNA of the coelomic cells showed normal sagregation of alleles in the fetuses, thus excluding maternal contamination. The results of this study demonstrate that coelocentesis may be a reliable alternative technique for the diagnosis of beta-thalassaemia from as early as 7 weeks gestation.

Regional distribution of beta-thalassemia mutations in India.

We have characterized the mutations in 1050 carriers of the beta-thalassemia gene and analyzed their regional distribution in India. The majority of beta-thalassemia carriers were migrants from Pakistan and their pattern of mutations differed from the rest. The frequency of the 619-bp deletion was 33.3% among the migrants from Pakistan, 8-17% in the northern states, and less than 5% in the other states. Among non-migrant subjects, the predominant mutation was IVS-I-5 (G-->C), varying from 85% in the southern states and 66-70% in the eastern states to 47-60% in the northern states. The mutation IVS-I-1 (G-->T) was observed at high frequency among the migrants from Pakistan (26.2%), but with very low/zero frequency in the other states. Mutations at codons 8/9 (+G) and codons 41/42 (-CTTT) were distributed in all regions of India with a frequency varying from 3% to 15%. Only eight of 12 published rare mutations were observed in subjects from different parts of India. Mutations of codon 5 (-CT) and codons 47/48 (+ATCT) were found exclusively in migrants from Pakistan, and mutation -88 (C-->T) was detected only in subjects from Punjab, Haryana, and Uttar Pradesh. Using the amplification refractory mutation system technique, mutations were successfully identified in 98.2% of subjects. Overall, 91.8% of the subjects had one of the five commonest mutations [IVS-I-5 (G-->C), 34.1%; 619-bp deletion, 21.0%; IVS-I-1 (G-->T) 15.8%; codons 8/9 (+G), 12.1%, and codons 41/42 (-CTTT), 8.7%], 5.9% of the subjects had a less common mutation, while 1.8% of the carriers remained uncharacterized. The application of this knowledge has helped to successfully establish a program of genetic counselling and prenatal diagnosis of beta-thalassemia in order to reduce the burden of this disease in India.

Application of natural and amplification-created restriction sites in prenatal and preimplantation diagnosis of beta-thalassemia.

We attempted the strategy of natural and amplification-created restriction sites for early prenatal and preimplantation diagnosis of Chinese beta-thalassemia. Mutagenesis primers were designed for 11 mutations reported for the Chinese population. The diagnosis was established after polymerase chain reaction and digestion of products by specific enzymes. The results were confirmed by direct sequencing of enzymatically amplified double-stranded DNA. The beta-globin gene was amplified from triploid embryos and isolated blastomeres using mismatched primers. The mutant and normal alleles can be distinguished clearly by this new method. Early prenatal diagnosis was successfully achieved in 9 cases. The beta-globin gene was successfully amplified from single blastomeres and tripronuclear embryos with mismatched primers. Natural and amplification-created restriction sites are a reliable method for rapid prenatal diagnosis of beta-thalassemia. Furthermore, the strategy provides a possible approach for the preimplantation diagnosis of beta-thalassemia.

Beta-thalassemia mutations in Singapore--a strategy for prenatal diagnosis.

The strategy for early prenatal diagnosis of beta-thalassemia in Singapore by direct detection of the mutant beta-globin gene requires the spectrum of mutations producing the disorder in this population to be characterized. We analyzed 134 beta-thalassemia alleles from Singapore by specific oligonucleotide hybridization after DNA amplification, using a nonradioactive enhanced chemiluminescence detection system. The mutations were identified in 90% of the alleles using five oligonucleotide probes for the following mutations: codons 41/42 (deletion-TCTT), IVS II nt 654 (C-->T), codon 17 (A-->T), IVS I nt 5 (G-->C), and -28 TATA box (A-->G). Together with the strategy of direct sequencing, a total of 97% of the mutations were identified. In the Chinese subpopulation, 97% of the mutations were detected by the oligonucleotide probes. Using just four oligonucleotide probes would identify 96% of the mutations, and 76% of the mutations were accounted for by codon 41/42 (-TCTT) and IVS II nt 654 (C-->T) mutations. Thus in this subpopulation early prenatal diagnosis would be possible in virtually all the affected families.

BamH1 polymorphism in the Chinese, Malays, and Indians in Singapore and its application in the prenatal diagnosis of beta-thalassemia.

The distribution of restriction fragment length polymorphism (RFLP) at the BamH1 site of the beta-globin gene was investigated in the Chinese, Indian, and Malay race in Singapore. The sample comprised of 183 normal individuals and 35 beta-thalassemia carriers in which 13 were couples with at least one beta-major child. The results from this study indicate that BamH1 polymorphism will be informative in 22% of pregnancies at risk for beta-thalassemia major in Chinese, 19% in Malays and 7% in Indians. In prenatal diagnosis using BamH1 polymorphism for one beta-major affected family, the fetus was diagnosed to be normal or beta-carrier. The validity of BamH1 polymorphism in the exclusion of beta-thalassemia major was subsequently confirmed at birth by globin chain biosynthesis.

Nonradioactive Quantification of Low Concentrations of Hemoglobin a by HPLC for Midtrimester Prenatal Diagnosis of β-Thalassemia

The usual methods for prenatal diagnosis of beta-thalassemia and other hemoglobinopathies by assay of fetal blood erythrocytes are either complex (analysis of globin chains synthesis by carboxymethylcellulose chromatography) or only semiquantitative [isoelectric focusing of hemoglobin (Hb)]. To further simplify the diagnostic procedure and to obtain quantitative data, we measured the small concentrations of Hb A in fetal erythrocytes by using a high-pressure liquid chromatography (HPLC) instrument (DIAMAT-TM; Bio-Rad) equipped with the new column proposed for measuring Hb A2. We analyzed 212 uncontaminated fetal blood samples obtained by cordocentesis between the 18th and 22nd weeks of pregnancy, using the HPLC procedure, and compared the results with those obtained by the above-named methods. The Hb A values obtained ranged between 0% and 8.5%; they were less than or equal to 1.8% in 44 fetuses affected by homozygous beta-thalassemia and greater than 2.5% in 168 unaffected fetuses. The method was simple, rapid, and reproducible (CV 3.2%) and there was good correlation between Hb A concentrations determined by HPLC and the beta/gamma ratio determined by carboxymethylcellulose chromatography (r = 0.7687; P less than 0.0001). No false-negative or false-positive results were observed, and there was no overlap of values between affected and unaffected fetuses.