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Thalassemia is a genetic disease that seriously affects the health of the fetus. At present, invasive prenatal diagnosis is the main method of thalassemia screening, but invasive prenatal diagnosis has the risk of fetal abortion. The discovery of cell-free fetal DNA (cffDNA) in the peripheral blood of pregnant women provides the possibility for non-invasive prenatal diagnosis (NIPD). Rapid and efficient capture of mutational information on cffDNA in maternal plasma can help prevent the birth of children with thalassemia major. Currently, strategies for cffDNA-based NIPD of thalassemia include the detection of paternal mutations in maternal plasma, detection of a proportion of wild and mutant alleles in maternal plasma, linkage disequilibrium single nucleotide polymorphism (SNP) based on pedigree probands, and prediction of fetal genotypes by bioinformatics combined with population information. Therefore, this paper will focus on the above aspects, in order to provide an essential reference to the prevention and treatment of thalassemia. [ABSTRACT FROM AUTHOR]

The discovery of circulating cell-free fetal DNA (cff-DNA) in maternal plasma has inspired the noninvasive prenatal testing (NIPT) approaches for various genetic fetal screening including rhesus D typing, sex determination, aneuploidies, and single-gene disorders. Noninvasive determination of paternally inherited beta-thalassemia mutations in maternal total cell-free DNA (cf-DNA) by using allele-specific amplification refractory mutation system (ARMS) real-time PCR (RT-PCR) in concordance with the conventional invasive method. An observational study was conducted at the Armed Forces Institute of Blood Transfusion in collaboration with the genetics resource center from March 2021 to August 2021. A total number of 26 couples were selected having a history of previously affected children with beta-thalassemia. A routine chorionic villus sampling (CVS) invasive procedure was carried out, and the mutation analysis was done using conventional PCR. To assess NIPT, a total cf-DNA was also extracted from maternal plasma and analyzed using allele-specific ARMS RT-PCR. Based on conventional PCR testing, 13 of 26 couples were found having beta-thalassemia carriers with homozygous mutation, and 13 couples were carriers with heterozygous mutations. Further to assess NIPT, the cf-DNA of 13 pregnant females among the couples with different mutational patterns was analyzed by allele-specific ARMS RT-PCR to detect paternally inherited mutations. In comparison with conventional PCR, 11 cases (84.6%) were matched successfully, while two cases (15.4%) had no concordance with conventional invasive prenatal testing (IPT). NIPT using maternal cf-DNA by allele-specific ARMS RT-PCR can be feasible to screen paternal inherited mutant alleles to rule out pregnant women from invasive procedures where the test would be negative for paternal inheritance. However, a low amount of fetal DNA in maternal plasma is a limiting factor and required further improvement to enrich fetal cf-DNA for complete concordance with conventional IPT. [ABSTRACT FROM AUTHOR]

β-Thalassemia (β-thal) is a monogenic disease characterized by mutations on the HBB gene, affecting the production of globin that results in hypochromic and microcytic anemia. The aim of this study was to determine the prevalence of six common β-thal mutations, and their frequency and inheritance pattern in affected populations of North Waziristan Agency, Pakistan. In this study, 130 blood samples from 37 unrelated β-thalassemic families having a minimum of one transfusion-dependent child with β-thal major (β-TM), were retrieved either from the Thalassaemia Centre for Women and Children Hospital Bannu or their home towns situated in Noth Waziristan Agency. All samples were analyzed by the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) using six allele-specific primers for the presence of the six β-thal mutations common in the Pakistani population. Of the six common mutations, our study demonstrated five HBB mutations comprising HBB: c.27_28insG, HBB: c.92+5G>C, HBB: c.126_129delCTTT, HBB: c.92+1G>T and HBB: c.17_18delCT from the families studied, while mutation HBB: c.47G>A [codon 15 (G>A)] was not detected in any of the studied families. Furthermore, the HBB: c.27_28insG and HBB: c.92+5G>C were noted to be the most common with frequencies of 42.85 and 31.42%, respectively. The findings of the present study may be useful in launching carrier screening and prenatal diagnosis (PND) programs by screening analyzed and other unanalyzed affected families for the possible presence of common mutations through the ARMS-PCR technique that will help to control the disease. [ABSTRACT FROM AUTHOR]

Objectives: The high-resolution melting (HRM) technique is fast, effective and successful method for mutation detection. The aim of this study was to determine the sensitivity and specificity of the HRM method for detection of a paternally inherited mutation in a fetus as a noninvasive prenatal diagnosis of β-thalassemia.Methods: Genomic DNAs were prepared from 50 β-thalassemia minor couples whose pregnancy was at risk for homozygous β-thalassemia. Ten milliliters of the maternal blood from each pregnant woman were collected and after separating plasma stored at -80 °C until analysis. The extracted DNAs were analyzed by HRM real-time PCR for detection of IVS-II-I (G-A) as a paternally inherited mutation. The gold standard was the result of a chorionic villus sampling by a standard reverse dot blotting test.Results: The sensitivity and specificity of HRM real-time PCR were 92.6% and 82.6%, respectively. Also, the positive and negative predictive values were 86.2% and 90.47%, respectively.Conclusions: HRM real-time PCR was a sensitive and specific method for determining the paternally inherited mutation in the fetus at risk with thalassemia major. [ABSTRACT FROM AUTHOR]

Objective: To design and evaluate a noninvasive protocol for prenatal diagnosis (PND) of β-thalassemia, using cell free fetal DNA (cff-DNA) in maternal circulation. Traditional current PND which is mainly based on chorionic villous sampling (CVS), amplification refractory mutation system and sequencing holds as gold standard. Methods: Ten thalassemia trait couples with distinct mutations for the husband and wife were included in this study. The mutations in carrier fathers were IVSI-1, IVSI-5, FR8/9 and CD44. After maternal plasma isolation and free DNA extraction, all samples subjected to designed protocol including DNA size separation on agarose gel, elution of DNA from the gel slices using a simple and efficient manual purification method, with or without whole genome amplification and the detection method was allele-specific real-time PCR. Results: Presence or absence of the paternal mutant allele was correctly determined in all of cases and the accuracy of designed protocol was determined 100%. Conclusions: The protocol described here is very simple, inexpensive and easy to perform, but with satisfactory accuracy in detection of paternal mutations in cff-DNA. Due to the risk of fetal loss with current invasive sampling for PND, a noninvasive alternative is highly demanded in clinical setting. [ABSTRACT FROM AUTHOR]

α-Thalassemia (α-thal) is one of the most common inherited hemoglobin (Hb) disorders in the world. In addition to large deletions, over 50 different α-thal point mutations were detected around the world, thus, patients showed different phenotypes with regard to genotype. This study evaluated the genetic frequency of α-thal in Khuzestan Province, Southwest Iran, to help implement premarital and prenatal screening programs. The study was conducted on couples proposing to get married and parents who were referred to the genetic center of Shafa Hospital, Ahvaz, Iran, for prenatal diagnosis (PND) in 2012. Genomic DNA was purified by the salting-out method and tested using multiplex gap-polymerase chain reaction (gap-PCR), amplification refractory mutation system-PCR (ARMS-PCR), reverse hybridization test strips and DNA sequencing. Overall, 11 mutations were found on the α-globin genes. Based on gene frequency, the most common mutant allele was –α3.7 (rightward) (71.3%) followed by the two gene deletion – –MED (9.7%). Other common mutations were αcodon 19α (GCG>GC–, α2) (8.4%), the polyadenylation (polyA1) site αpolyA1α (AATAAA>AATAAG) (2.8%), and α–5 ntα (–TGAGG) (2.0%). In addition, an extremely rare mutation at αcodon 21α [Hb Fontainebleau, HBA2: c.64G > C (or HBA1)] was also found. The results of this study are critical for correct diagnosis of α-thal carriers, premarriage counseling and PND. This study suggests that the distribution of mutations on the α-globin genes differs among the ethnic groups in Khuzestan Province as well as in other provinces. [ABSTRACT FROM AUTHOR]

Thalassaemia is the commonest monogenic disease in Sri Lanka, affecting over 3500 children and half-a-million thalassaemia carriers. This is a review of 82 amniocenteses performed from 2006 to 2016, in the largest prenatal diagnoses study for thalassaemia carried out in Sri Lanka. Amniocenteses were performed between 11 and 12 weeks of ultrasonically confirmed gestation, on mothers with previous thalassaemia major children pregnant for the second time and nulliparous thalassaemia trait women married to trait partners. The Consultant Radiologist, using local analgesia, under ultrasound cover, performed these as an outpatient procedure, at the Teaching Hospital Kandy & Suwasevana Hospital Kandy. The amniotic fluid was analysed by the team of Senior Geneticists, at the Genetech Molecular Diagnostics and School of Gene Technology, Colombo, via the polymerase-chain-reaction based ARMS (Amplification Refractory Mutation Systems) assay. The genetic results indicated the presence of 21% thalassaemia major foetuses, 53% thalassaemia traits and 26% foetuses without thalassaemia mutations. The predominance of the IVS1-5(G-C) mutation in the Sri Lankan population is exemplified, with a low prevalence of HbE thalassaemia. Impact statement Thalassaemia is the commonest monogenic disease in Sri Lanka affecting over 3500 children and half-a-million thalassaemia carriers. Although pre-natal diagnosis by amniocentesis was practised universally for many years, this could not be performed in Sri Lanka as genetic diagnostic facilities were not available until 2005. Therefore, parents with a thalassaemia major child limited their families to one child, by choice or by termination. The results of this study point to a 21% probability of thalassaemia major in the next child, giving the parents a guarded optimism to conceive another child without thalassaemia disease. With siblings being the highest HLA compatibility for Bone Marrow Transplant, that is now being established in Sri Lanka as a permanent cure for thalassaemia, this will bring a ray of hope for these desperate parents to finally cure their previous sick child. Although, 95% of the Sri Lankan mutated genetic sites for thalassaemia are known, more research will be needed to identify the other rare sites. The publication of this paper, with its novelty for pre-natal diagnosis, would encourage clinicians to practice it in other centres and to extend it to families with other genetic disorders. [ABSTRACT FROM AUTHOR]

β-Thalassemia major (β-TM) is an inherited disease and efforts have been made in several countries to reduce the number of affected births. In the present study, we aimed to evaluate the Iranian thalassemia prevention program, considered to be an important program in the region. The time period of the present study ranges from 2007-2009, during which new thalassemic births and the relevant causes were evaluated throughout the country. A cross-sectional analytical study was conducted at the Iranian Blood Transfusion Organization (IBTO), Tehran, Iran. A questionnaire was forwarded to all blood centers of the IBTO so as to obtain information about the new cases of thalassemia and the causes of these thalassemic births. Provincial thalassemia societies also received the questionnaires so that screening and prenatal diagnosis (PND) errors would be recorded. The results showed that 755 new thalassemia cases were born during 2007-2009 with the average fall in affected thalassemia births of 80.82%. The main cause of the new births was attributed to unregistered 'timeless religious marriages' based on the conventions of the Sunni community which accounted for 43.17% of all new cases mainly having occurred in Sistan & Baluchestan Province. Not using PND was evaluated to be another main cause. Although the prevention program has led to a great reduction in thalassemic births, new measures are required, including research on how to make the program compatible with social and economic conventions and norms of Sistan & Baluchestan Province. The province of Kohgiluyeh Boyer Ahmad also needs to be revisited in terms of the program efficacy. [ABSTRACT FROM AUTHOR]

α-Thalassemia (α-thal) encompasses a spectrum of mutations including deletion and point mutations on the α-globin chains that is characterized by a reduction or complete absence of α-globin genes. Most of the α-thal cases are deletions involving one (α+) or both (α0) α-globin genes, although point mutations (αTα or ααT) are found as well. In this study, 314 individuals with low hematological values, normal Hb A2 who were not affected with β-thal or iron deficiency, were investigated for the presence of α-thal mutations. The most common deletion was −α3.7 (rightward) with a frequency of 70.7%, followed by α−5 nt (-TGAGG) (8.7%), −α4.2 (leftward) (4.7%), the polyadenylation signal (polyA2) site (AAT AAA > AAT GAA) (4.2%), −(α)20.5 (3.8%), Hb Constant Spring [Hb CS, α142, Stop→Gln; HBA2: c.427T > C] (2.9%), polyA1 (AATAA A > AATAA G) and αcodon 19 (GCG > GC-, α2) (16%), and - -MED (0.9%). The results of this study may be valuable for designing a plan for carrier screening, premarital genetic counseling, prenatal diagnosis (PND) and reducing excessive health care costs to an affordable level in Isfahan Province, Iran. [ABSTRACT FROM AUTHOR]

Our study aimed to determine the number of couples with normal hemoglobin (Hb) electrophoresis and low-borderline hematological values, which may come up with a clinically critical status in their offspring. The number of couples at risk for severe α-thalassemia (α-thal) needed to be estimated before recommending genetic counseling and prenatal diagnosis (PND). During the past 14 years, from at least 7000 referrals, 754 couples were investigated for α-thal by direct mutation detection methods followed by reverse strip assay and α-globin gene sequencing for inconclusive cases. Detection of silent β-thalassemia (β-thal) mutations was done in suspected cases by complete β-globin gene sequencing. We were able to provide a molecular diagnosis in 87.3% (658/754) of couples. A total of 9.1% (60/658) may have a clinically significant hemoglobinopathy in their offspring. Significant conditions included hydrops fetalis (20.0%; 12/60), certain Hb H (β4) genotypes (78.3%; 47/60) and β-thal intermedia (β-TI) (1.7%; 1/60). The diagnostic flowchart for couples with microcytic hypochromic anemia in countries with a high prevalence of hemoglobinopathies should include α and β gene sequencing. As our results indicate, every nine out of 100 of these couples will face significant hemoglobinopathies and every two out of 100 can carry Hb Bart's (γ4) hydrops fetalis in their pregnancies. For such cases, PND should be utilized to allow the carrier couples to decide whether or not to abort the fetus. [ABSTRACT FROM AUTHOR]

Hb Hammersmith [β42(CD1)Phe → Ser; HBB: c.128T > C] is a rare, unstable hemoglobin (Hb) variant. In this case report, we describe another male case of Hb Hammersmith. A 39-year-old male had hemolytic anemia, cyanosis and splenomegaly since 6 months after birth. He passed the disease allele to his daughter, a 3-year-old girl, who also had hemolytic anemia and splenomegaly. This mutation was not identified in the parents and two brothers of the father. Early prenatal diagnosis was performed in the second pregnancy in this family. This is the first case of Hb Hammersmith in an adult male patient. [ABSTRACT FROM AUTHOR]

The molecular basis of β-thalassemia (β-thal) syndromes have been well documented, while the spectrum of mutations causing δ-thalassemia (δ-thal) has not been well characterized. δ-Thalassemia has no clinical symptoms but its coinheritance with heterozygous β-thal may cause misdiagnosis, especially in countries with a high prevalence of β-thal where prevention programs have been implemented. The coinheritance of β- and δ-globin mutations in India is not common. This association may interfere with correct diagnosis and genetic counseling of β-thal in screening programs. Here we report two families showing borderline Hb A2 levels belonging to the Koli Community, indigenous to the Saurashtra Province of Gujarat, India. They were referred to us for thalassemia molecular screening as they had children clinically presenting before 2 years of age and requiring regular blood transfusions. Interestingly, both families carried a novel δ-globin gene mutation at codon 100 (C>T) linked to a polyadenylation (polyA) site [AATAAA>A(-AATAA)] 5 bp deletional β-thal mutation, never before reported in the Indian population. This report highlights the importance of considering δ-globin gene analysis during β-thal screening to avoid false-negative results in the detection of at-risk couples. It also highlights how incomplete diagnosis of a borderline or normal Hb A2 level may lead to the probable birth of a β-thal major (β-TM) child. This has important implications in prenatal diagnosis. [ABSTRACT FROM AUTHOR]

The marginal zones of the Caspian Sea and the Persian Gulf have a higher prevalence of thalassemia compared to other regions of Iran. This disease has disabled many people and resulted in increasing health care costs. The aim of this study was to assess the incidence of β-thalassemia (β-thal) and to evaluate the outcome of applied preventive strategies over a 14-year period in Fars Province, Southern Iran. This cross-sectional study comprised all new cases of β-thal recorded during 1997-2011. The data were obtained from the Non-Communicable Diseases Surveillance Department of Shiraz University of Medical Sciences, Shiraz, Iran, and are presented as mean ± standard deviation (SD). The Fars Health Network System screened 840 686 males and females applying for marriage certificates. Among the carriers, 50.5% cancelled their marriages, 42.5% married, and 7.0% did not show up at the clinics. The rate of cancelled marriages has reduced since 2000, when marriage candidates were given the option of prenatal diagnosis. From 2000 to 2011, a total of 3539 married couples were referred for prenatal diagnosis. Of these, 806 fetuses were found to carry thalassemia and 800 aborted. It is impressive to note that while 101 cases of thalassemia were recorded in 1997, this figure was reduced to two cases by 2011. This study has established that an integrated primary health care approach, with good infrastructure for implementing successful strategies, can significantly reduce the incidence of β-thal. [ABSTRACT FROM AUTHOR]

β-Thalassemia (β-thal) is a public health problem in the Gaza Strip, Palestine, where about 320 patients are currently managed through blood transfusions and iron chelation. Within the restrictive environment of the Gaza Strip, no advanced molecular analysis [sequencing, real-time polymerase chain reaction (real-time PCR)] technology is currently available for developing a premarital screening protocol and providing couples at risk with prenatal diagnosis. Therefore, genetic identification of samples with indicators of β-thal is delayed for weeks before the samples can be sequenced outside the country. As nine causative mutations have been identified in the majority of β-thal cases in the Gaza Strip, a basic genetic screening strategy was designed to improve timeliness in mutation identification and reduce costs to the Palestinian health system. In the present study, we developed a reliable method for the detection of nine Mediterranean β-thal mutations common to the Palestinian population using a panel of restriction enzyme digests. This strategy utilizes standard instrumentation (thermocycler and agarose gel electrophoresis) that would be available in any basic molecular genetics or biochemical laboratory and provides a reliable method of genetic screening and counseling for patients at risk for β-thal. [ABSTRACT FROM AUTHOR]

Thalassemia is the most common monogenic inherited disease worldwide and it affects most countries to various extents. This review summarizes the current approaches to phenotypic and genotypic diagnosis of thalassemia in clinical practice. Prevention strategies that encompass carrier screening, genetic counseling and prenatal diagnosis are discussed. The importance of public education and an awareness of a changing perception regarding this group of diseases are emphasized. It also addresses the impact of the rapidly increasing knowledge in disease severity modification by hemoglobin F (Hb F). [ABSTRACT FROM AUTHOR]

Introduction: The carriers of the same autosomal recessive disorder are usually unaware of onset of the genetic diseases in the children even if screenings are available for many of these disorders. In this paper, we report the experience of the Prenatal Diagnosis Center of AOU Federico II and we discuss the role of the screening for beta-thalassemia (BT), cystic fibrosis (CF) and for other rare genetic disorders. Materials and Methods: We analyzed retrospectively the indication for Prenatal Diagnosis (PD) of all the couples referred to our center from January 1993 to May 2013. We divided our sample into three groups: couples at high risk for BT, for CF and for other rare genetic disorders. Results: From January 1993 to May 2013, we performed 1269 PD for genetic disorders. There are still couples who discovered to be carriers of BT by screening after the birth of the affected child ( n = 51 (11,3%)); the majority of the people were screened for CF carrier after the birth of an affected child ( n = 155 (80,7%)) or through the cascade screening ( n = 28 (14,6%)). Large-scale screenings for rare genetic conditions are not available and people were screened only if they have a positive familial history. Conclusion: Parental screening is available for many severe and rare diseases whose genetic origin is known. The proportion of patients referred for very high-risk indications increased over time with an higher demand for rare disease. An adequate counseling is fundamental to identify women at risk for having affected child. Screening, counseling and PD of genetic diseases is a complex matter and needs for a continuous update. [ABSTRACT FROM AUTHOR]

We report the problems in diagnosis faced by two families referred for prenatal diagnosis of thalassemia where cordocentesis and fetal blood analysis by high performance liquid chromatography (HPLC) had to be done. The Hb A levels of the fetal blood measured by HPLC on the VARIANT™ Hemoglobin Testing System were 1.2 and 6.7%%, respectively, suggestive of a heterozygous β-thalassemia (β-thal) fetus in the first case and a normal fetus in the second case. In one family, one of the parents had a borderline Hb A2 level and in the other, one parent had normal RBC indices. However, DNA sequencing, done later, showed that in the first case the fetus was a compound heterozygote for the IVS-I-5 (G>C) and the polyadenylation signal site [poly A (T>C)] mutation, while in the second case, the fetus was homozygous for the poly A mutation. This emphasizes that characterization of β-thal mutations must be done whenever one of the parents has a borderline Hb A2 level or normal RBC indices, and one should not rely on fetal blood analysis by HPLC for prenatal diagnosis of β-thal so as to avoid misdiagnosis. [ABSTRACT FROM AUTHOR]

We report a novel mutation at codon 24 of the α2-globin gene ( HBA2: c.75T > A) found in a Sundanese family. This novel mutation was detected during prenatal diagnosis. The couple already had a 7-year-old boy who exhibited clinically severe α-thalassemia intermedia (α-TI), and he was found to be a compound heterozygote for the novel mutation at codon 24 and the previously described Hb Adana ( HBA2: c.179G > A) at codon 59 of the α2-globin gene. The father was a carrier of the novel point mutation and showed normal hemoglobin (Hb) and a low mean corpuscular volume (MCV) and mean corpuscular Hb (MCH) value. [ABSTRACT FROM AUTHOR]

Prenatal diagnosis (PND) of β-thalassemia is offered by DNA based mutation analysis at 10–12 weeks gestation using chorionic villous biopsy specimens. Most centers offering PND search for five common mutations prevalent in India, which cover more than 90% of patients. However in those families where the mutation cannot be identified in the parents, PND becomes difficult by mutation detection. The technique thus followed is estimation of globin chain synthesis ratio in second trimester cord blood samples. However, this technique is cumbersome and not available in most of the centres. We evaluated 112 cord blood samples (74 were informative) for analyzing hemoglobin A (HbA) levels by cation-exchange high performance liquid chromatography (CE-HPLC) using the BioRad β-thal short program between 18 and 22 weeks of gestation. A normal range for the population was calculated by running 14 cord blood samples of non-thalassemic abortuses of the same gestation. A 6 month post-natal follow-up was possible in 18 cases. All but one of these corroborated their PND. [ABSTRACT FROM AUTHOR]

While knowledge of HBB gene mutations is necessary for offering prenatal diagnosis (PND) of β-thalassemia (β-thal), a genotype-phenotype correlation may not always be available for rare variants. We present for the first time, genotype-phenotype correlation for a compound heterozygous status with IVS-I-5 (G>C) (HBB: c.92+5G>C) and HBB: c.407C>T (Hb Alperton) mutations on the HBB gene in an Indian family. Hb Alperton is a very rare hemoglobin (Hb) variant with scant published information about its clinical presentation, especially when accompanied with another HBB gene mutation. Here we provide biochemical as well as clinical details of this variant. [ABSTRACT FROM AUTHOR]