Common Β-thalassemia Mutations Research Articles

Mutation Spectrum of β-Thalassemia in Some Ethnic Groups of North Maharashtra, India

Beta-thalassemia is the most common inherited single-gene disorder in the world, caused by more than 200 known mutations in the HBB gene. In India, the average prevalence of β-thalassemia carriers is 3-4%. Several ethnic groups have a much higher prevalence, about 8% in the tribal groups, according to the 2011 census. The study’s main goal is to identify common β-thalassemia mutations and the frequencies of different haplotypes in various communities in North Maharashtra. Nashik district had the highest prevalence of β-thalassemia (34%), followed by Ahmednagar (29%), Jalgaon (16%), Dhule (14%), and Nandurbar (7.0%). Prevalence of β-thalassemia was highest in the schedule caste community (SC) (48%), followed by (17%) in Muslims, (14%) in other backward classes (OBC), (13%) in Schedule Tribe (ST), and (8.0%) in the general population The six most common β-thalassemia mutations detected in this study are IVS 1 > 5 (G→C), Cd 15(G→A), Cd 41/41 (-TCTT), Cd 8/9(+G), IVS 1 > 1(G→T) and Cap + 1(A > G). Among these mutations, IVS 1 > 5 (G > C) was the most common type of mutation found in β-thalassemia patients in the North Maharashtra population. Type-I haplotype was the most prevalent among all communities. Nashik and Ahmednagar districts were highly affected by β-thalassemia. Among different ethnic groups, the SC and Muslim communities were the worst affected with a higher proportion of β-thalassemia and increased frequency of mutations.

Adenine base editor–mediated correction of the common and severe IVS1-110 (G>A) β-thalassemia mutation

Abstractβ-Thalassemia (BT) is one of the most common genetic diseases worldwide and is caused by mutations affecting β-globin production. The only curative treatment is allogenic hematopoietic stem/progenitor cells (HSPCs) transplantation, an approach limited by compatible donor availability and immunological complications. Therefore, transplantation of autologous, genetically-modified HSPCs is an attractive therapeutic option. However, current gene therapy strategies based on the use of lentiviral vectors are not equally effective in all patients and CRISPR/Cas9 nuclease-based strategies raise safety concerns. Thus, base editing strategies aiming to correct the genetic defect in patients’ HSPCs could provide safe and effective treatment. Here, we developed a strategy to correct one of the most prevalent BT mutations (IVS1-110 [G>A]) using the SpRY-ABE8e base editor. RNA delivery of the base editing system was safe and led to ∼80% of gene correction in the HSPCs of patients with BT without causing dangerous double-strand DNA breaks. In HSPC-derived erythroid populations, this strategy was able to restore β-globin production and correct inefficient erythropoiesis typically observed in BT both in vitro and in vivo. In conclusion, this proof-of-concept study paves the way for the development of a safe and effective autologous gene therapy approach for BT.

Genetic Background Studies of Eight Common Beta Thalassemia Mutations in Thailand Using β-Globin Gene Haplotype and Phylogenetic Analysis.

Single nucleotide polymorphisms are informative for haplotype analysis associated with genetic background and clinical linkage studies of β-thalassemia mutations. Hence, the aim of this study was to investigate five polymorphisms (codon 2 (C/T), IVS II-16 (C/G), IVS II-74 (G/T), IVS II-81 (C/T) and the Hinf I (T/A) polymorphism) on the β-globin gene, related to eight common β-thalassemia mutations in Thailand, including NT-28 (A > G), codon 17 (A > T), codon 19 (A > G), HbE (G > A), IVS I-1 (G > C), IVS I-5 (G > C), codon 41/42 (-TTCT) and IVS II-654 (C > T). The strongest LD (100%) between the β-thalassemia mutation allele and all five SNPs was found in NT-28 (A > G), codon 17 (A > T) and codon 19 (A > G). In the haplotype analysis, we found three haplotypes (H1, H2 and H7) related to Hb E, whereas we only found two haplotypes related to codon 41/42 (-TTCT) (H1, H3) and IVS I-1 (G > C) (H3, H4). Of interest is the finding relating to a single haplotype in the remaining β-thalassemia mutations. Furthermore, phylogenetic tree analysis revealed three clusters of these common β-thalassemia mutations in the Thai population and enabled us to determine the origin of these mutations. Here, we present the results of our study, including four intragenic polymorphisms and the finding that the Hinf I polymorphism could be informative in genetic background analysis, population studies and for predicting the severity of β-thalassemia in Thailand.

Genetic predictions of life expectancy in southern Thai patients with β0-thalassemia/Hb E.

The types of β-thalassemia mutations, α-thalassemia interactions, and Hb F-associated SNPs have been described in association with variable disease phenotypes. This study aimed to determine the updated spectrum of β-thalassemia mutations and evaluate the contribution of primary and secondary genetic modifiers and SNPs to disease severity, age at onset, and predicted life expectancy in southern Thai β-thalassemia patients. A total of 181 β-thalassemia patients were enrolled and 135 β0-thalassemia/Hb E patients without α-thalassemia interactions were divided into three categories according to disease severity, age at onset, and predicted life expectancy. A total of 16 β-thalassemia mutations were identified in this study, and the three most common β-thalassemia mutations accounted for 61.4% of all mutations. It was also found that the XmnI polymorphism and rs2071348 were associated with age at onset and the predicted life expectancy. More than 82% of β0-thalassemia/Hb E patients with CC genotype (XmnI) were 3 years old or younger at onset. Additionally, >90% of the higher predicted life expectancy in β0-thalassemia/Hb E patients had the T allele of XmnI. Therefore, genetic prediction for age at onset and life expectancy is beneficial and practical during prenatal diagnosis or newborn screening for better genetic counseling and optimal management.

Multiplex Quantitative Real-Time Polymerase Chain Reaction and High-Resolution Melting Analysis for Identification of a Couple At-Risk of Having a Newborn with Severe Thalassemia

Many polymerase chain reaction (PCR)-based techniques have been used for routine diagnosis of α- and β-thalassemias. However, most require a multi step of post-PCR processes that are time-consuming and labor-intensive procedures. This study reported the successful use of multiplex quantitative real-time PCR (qPCR), with high-resolution melting (HRM) analysis for diagnosis of two common deletional α0-thalassemia (α0-thal) and 15 common β-thalassemia (β-thal) mutations, in order to identify a couple at-risk of having a newborn with severe thalassemia in the northern region of Thailand. With this approach, 22 (7.2%) of 306 couples were diagnosed as being at-risk for having a child with severe thalassemia, including three homozygous α0-thal, five homozygous β-thal and 14 Hb E (HBB: c.79G>A)/β0-thal disease. Our findings indicated that multiplex qPCR with HRM is applicable for routine molecular diagnosis in order to identify a couple at-risk of having a newborn with severe thalassemia, especially in an endemic region.

Direct Amplification of Whole Blood and Amniotic Fluid Specimens for Prenatal and Postnatal Diagnosis of Hb E-β 0-Thalassemia Diseases.

Prenatal and postnatal diagnosis of hemoglobin E-β 0-thalassemia can be made using polymerase chain reaction (PCR) analysis mostly on purified DNA. We have establihed a direct amplification method without DNA extraction on whole blood (WB) and amniotic fluid (AF) specimens to diagnose the disease. Three reactions of WB PCR assays and 7 reactions of AF PCR tests were developed for postnatal and prenatal diagnosis, respectively. Assays were validated against routine tests in a blinded trial. The results showed 100% concordance with routine DNA PCR assays. Among 309 β-thalassemia carriers, 191 patients (61.8%) carried common β-thalassemia mutations. Among 448 AF specimens, 116 (25.9%) fetuses were found to be affected, 247 (55.1%) fetuses were carriers, and 85 (19%) fetuses were unaffected. We found that WB and AF PCR assays are simple, rapid, and reliable. The developed techniques could be applicable in routine settings.

Study of the genotypic and hematological feature of hemoglobin H disease in West Guangxi area

To analyze the incidence, genotype and hematological feature of hemoglobin H (HbH) disease in West Guangxi region. A total of 1246 patients diagnosed with HbH disease from January 2013 to December 2018 in our hospital were enrolled. Red blood cell parameters, hemoglobin electrophoresis, Gap-polymerase china reaction (Gap-PCR) and polymerase chain reaction-reverse dot blot (PCR-RDB) techniques were used to detect the 6 common α-thalassemia mutations and 17 common β-thalassemia mutations. The results were compared with those of other regions. The detection rate for HbH disease was 5.66%. Among the 1246 patients, 614 (49.28%) had deletion-type HbH disease, including -α 3.7/--SEA (35.32%),-α 4.2/--SEA(13.72%) and -a 3.7/--THAI(0.24%), 632(50.72%) had non-deleted HbH disease, mainly α CS α /--SEA (44.86%), followed by α WS α/--SEA (4.33%), α QS α /--SEA (1.45%) and α CS α/--THAI(0.08%). Co-committent HbH disease and β-thalassemia were detected in 54 cases (4.33%). Most patients with HbH disease showed mild to moderate anemia. Very few had severe anemia. Among these, patients with HbH-CS had the most severe anemia, and HbH-WS were the mildest. Hb levels in patients with HbH disease alone were lower than those with co-committent HbH and β-thalassemia. Compared with other regions, the incidence and genotype of HbH disease of West Guangxi are different. The prevalence of HbH disease is high in West Guangxi region, and the main genotypeis non-deletion. α CS α /--SEA is the most common, and most of them had moderate anemia. Compared with the deletion-type HbH disease, non-deleted HbH patients were more severe. When HbH disease co-committed with β-thalassemia, the severity of anemia is reduced. The difference between West Guangxi and other regions may account for the variance of clinical manifestations and incidence of HbH disease in this region.

Development of DNA controls for detection of β-thalassemia mutations commonly found in Asian.

Several DNA-based approaches including a reverse dot-blot hybridization (RDB) have been established for detection of β-thalassemia genotypes to provide accurate genetic counseling and prenatal diagnosis for prevention and control of severe β-thalassemia. However, one of major concerns of these techniques is a risk of misdiagnosis due to a lack of DNA controls. Here, we constructed positive DNA controls for β-thalassemia genotyping in order to ensure that all steps in the analysis are performed properly. Four recombinant β-globin plasmids, including a normal sequence and three different mutant panels covering 10 common β-thalassemia mutations in Asia, were constructed by a conventional cloning method followed by sequential rounds of site-directed mutagenesis. These positive DNA controls were further validated by RDB analysis. We demonstrated the applicability of established positive DNA controls for β-thalassemia genotyping in terms of accuracy and reproducibility by RDB analysis. We further combined three mutant β-globin plasmids into a single positive control, which showed positive signals for both normal and mutant probes of all tested mutations. Therefore, only two positive DNA controls, normal and combined mutant β-globin plasmids, are required for detecting 10 common β-thalassemia mutations by RDB, reducing the cost, time, and efforts in the routine diagnosis. The β-globin DNA controls established here provide efficient alternatives to a conventional DNA source from peripheral blood, which is more difficult to obtain. They also provide a platform for future development of β-globin plasmid controls with other mutations, which can also be suitable for other DNA-based approaches.

Prevalence and Genetic Analysis of α- and β-Thalassemia and Sickle Cell Anemia in Southwest Iran

This prospective study assessed the prevalence and genetic analysis of α- and β-thalassemia and sickle cell anemia (SCA) in Southwest Iran. Hematological indices were measured in 17,581 couples living in Khuzestan Province, Southwest Iran. Individuals with mean corpuscular volume <80, mean corpuscular hemoglobin <27, hemoglobin A2 ≥3/5 were considered as β-thalassemia traits. Prevalence of minor β-thalassemia, α-thalassemia, SCA, iron deficiency anemia, and silent thalassemia were respectively identified in 995 (5.6%), 1169 (6.65%), 1240 (7.05%), 911 (5.18%), and 1134 (6.45%) individuals using a multiplex amplification refractory mutation system, and direct DNA sequencing of globin genes. Three codons IVS-II-1 (G → A; 26%; n = 13), IVS-I-1 (G → T; 16%; n = 8), and IVS-I-110 (G → A; 14%; n = 7) were the most frequent mutants and IVS-II-1 was the most common β-thalassemia mutation. Also, based on a gap-polymerase chain reaction assay, genotype frequencies of α -globin mutations were –α3.7kb (50%; n = 25), Med/ααthal (12%; n = 6), and –α 4.2/αα (10%; n = 5), which were the most frequent deletion mutants (72% in total). The most common deletion (50%) was –αα3.7kb. Our data suggest that the population of Southwest Iran is at high risk of α- and β-thalassemia caused by these deletion mutants and SCA. Our findings will be useful for developing an efficient control program and genetic counseling.

Haplotype Analysis of Three Common β-Thalassemia Mutations in Syrian Patients

β-Globin haplotypes were used to investigate the origin of three common β-globin mutations, IVS-I-110 (G>A); HBB: c.93-21G>A, IVS-I-1 (G>A); HBB: c.92 + 1G>A and codon 39 (C>T); HBB: c.118C > T in Syrian patients. Haplotype analysis was done for 49 unrelated patients with β-thalassemia (β-thal) and 20 unrelated healthy subjects by polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) for the β-globin gene cluster of the following polymorphic restriction sites: HincII 5' to ε, HindIII 5' to Gγ, HindIII 5' to Aγ, HincII in ψβ, HincII 3' to ψβ, AvaII in β, and HinfI 3' to β. The IVS-I-110 mutation was associated with three haplotypes: I [+ – – – – + +] (79.4%), V [+ – – – – + –] (5.9%) and VII [+ – – – – – +] (14.7%), while, the two mutations IVS-I-1 and codon 39 were be linked to a single haplotype V (100.0%) and II [– + + – + + +] (100.0%), respectively. The normal chromosomes (βA/βA) were associated with four haplotypes, I (50.0%), II (7.5%), V (32.5%) and VII (10.0%). In the Syrian population, the IVS-I-110 mutation was associated with multi haplotypes, whereas the IVS-I-1 and codon 39 mutations have a single origin. More studies for the other mutations will be very useful for genetic epidemiological studies in Syria.

Non-invasive prenatal testing for fetal inheritance of maternal β-thalassaemia mutations using targeted sequencing and relative mutation dosage: a feasibility study.

To evaluate whether targeted sequencing and relative mutation dosage can be used to diagnose correctly inheritance of maternal β-thalassaemia mutations in cell-free DNA. Feasibility study using samples collected in a prenatal clinic. South East Asia. Couples where both partners were known to be carriers of one of four common β-thalassaemia mutations or an HbE mutation, and therefore at risk of carrying a fetus affected with β-thalassaemia. 49 samples previously identified as having inherited a paternal β-thalassaemia mutation were amplified using nested polymerase chain reaction (PCR), and then sequencing. Relative mutation dosage was used to classify the fetus as having inherited the wild-type or mutant maternal allele. Classification of the fetus as 'unaffected' (if the maternal wild-type allele was inherited) or 'affected' with β-thalassaemia (if the maternal mutant allele was inherited). A classification for inheritance of maternal allele was obtained in 48/49 samples (98.0%). A concordant call was made in 44/48 cases (91.7%): one false-positive and three false-negatives were obtained. Thus, we had an overall sensitivity of 87.5% [95% confidence interval (CI) 67.6-97.3%] and a specificity of 95.8% (95% CI 78.9-99.9%) for inheritance of maternal genotype. RMD for detection of inheritance of maternal β-thalassaemia mutations has potential for clinical use. Our sequential approach could be applied to other single-gene disorders. NIPT for β-thalassaemia achieved using nested-PCR followed by relative mutation dosage.

Analysis of Common β-Thalassemia Mutations in North Vietnam

Available and flexible choice of methods for screening and detecting β-thalassemia (β-thal) can promote control of thalassemia in developing countries. In this study, two methods, the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) and reverse dot-blot hybridization assays were developed to detect common β-thal mutations in 244 thalassemia patients and 152 healthy people in North Vietnam. The most common mutation was codon 26 (G>A), also known as Hb E (HBB: c.79G>A), accounting for 26.4% of the total studied chromosomes, followed by codons 41/42 (–TCTT) (HBB: c.126_129delCTTT) and codon 17 (A>T) (HBB: c.c.52A>T), accounting for 19.4 and 16.4%, respectively. In addition, codon 95 (+A) (HBB: c.c.287_288insA) that is known as the Vietnamese mutation, accounted for 0.6%. Moreover, the heterozygous state of the four mutations was also found in healthy people, of which Hb E was again the most common mutation with a frequency 3.0%. The results of this study provide available methods and indicative data for preventive and control strategies concerning the genetic diagnosis of thalassemia.

Primary study on application of cell-free fetal DNA (cffDNA) in non-invasive prenatal testing of β-thalassemia

β-thalassemia is a common autosomal recessive disorders, caused by mutations in the β-globin gene. It severely influences the physical and mental development of affected children and place an immense burden of care and treatment on the family and society. Therefore, the prenatal screening of β-thalassemia to control its increase in the community is anessential part of preventive genetics. Currently, besides traditional prenatal diagnostic tests that requires invasive sampling techniques such as amniocentesis or chorionic villous sampling (CVS), the use of cffDNA for non-invasive prenatal testing of β-thalassemia is a new, safer, and more effective direction. In this study, we extracted and enriched the percent of cffDNA in the maternal peripheral blood as well as optimized AS-PCR to detect three most common β-thalassemia mutations CD17, CD26, and CD41/42. Presence or absence of the paternal mutant allele was then correctly determined in all of 7 cases compared to fetal genotyping that determined with amniocentesis.

A multiplex ARMS PCR approach to detection of common β-globin gene mutations

Backgroundβ-thalassaemia is a group of inherited single-gene disorders worldwide. Each ethnic population has its own common mutations. Heterogeneity of β-thalassaemia mutations in multi-ethnic population of Surat, makes molecular diagnosis expensive and time consuming. MethodsSpecific primers were used to differentiate four common mutations, IVS I-5 (G→C), Codon 41/42 (- TCTT), 619-bp deletion and FS 8/9 (+G), by a simple PCR involving a multiplex amplification refractory mutation system. ResultsSeveral high prevalence β-Thalassemia trait groups constituted by Muslims, Patels, Sindhis, ModhBanias, and Mahayavanshi. Four most common mutations detected in them are IVS I-5 (G→C), Codon 41/42 (- TCTT), 619-bp deletion and FS 8/9 (+G). We identified each of these β-thalassemia mutations in multiplexed ARMS from positive control samples. Our multiplex-ARMS-PCR system was first standardized on positive DNA samples with above known four most common β-thalassemia mutations, and these positive samples had been diagnosed with β-thalassemia and also all these samples belonged to Surat ethnic groups. The system was subsequently tested on 110 blood samples from different ethnic backgrounds with unknown β-thalassemia mutations which were in all specimens. ConclusionThe ARMS multiplex system was found reliable, cost effective, fast and most applicable for mutation screening of Thalassemia in Surat populations.

Postnatal and non-invasive prenatal detection of β-thalassemia mutations based on Taqman genotyping assays.

The β-thalassemias are genetic disorder caused by more than 200 mutations in the β-globin gene, resulting in a total (β0) or partial (β+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for β-thalassemia are: β039, β+IVSI-110, β+IVSI-6 and β0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human β-globin gene. Four TaqMan® genotyping assays for the most common β-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 β-thalassemia patients. In addition, 15 specimens at late gestation (21–39 gestational weeks) and 11 at early gestation (5–18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations β039, β+IVSI-110, β+IVSI-6, β0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of β-globin gene mutations, starting from the 9th gestational week.

Genotype-Phenotype Correlations of β-Thalassemia Mutations in an Azerbaijani Population.

β-Thalassemia is the most common inherited disorder in Azerbaijan. The aim of our study was to reveal genotype-to-phenotype correlations of the most common β-thalassemia mutations in an Azerbaijani population. Patients with codon 8 (-AA), IVS-I-6 (T>C), and IVS-II-1 (G>A) mutations, which are reportedly the most common β-globin gene mutations among the local population, were tested for hematologic parameters. Fifty-five previously tested patients with known genotypes were included in the study. Hematologic indices and hemoglobin fractions were tested in order to reveal the phenotypic manifestation of the mutations. The results obtained indicate that clinical presentation varies between different β-globin gene mutations: individuals with IVS-I-6 (T>C) mutations showed milder presentation than those with codon 8 (-AA) and IVS-II-1 (G>A), which is associated with the molecular basis of the mutations. These data can be of assistance to predict clinical presentation and select the best possible therapeutic approach via early genotype identification.

Molecular patterns of β-thalassemia mutations of Saudi patients referred to King Faisal Specialist Hospital and Research Center

Background: Beta thalassemias are a group of hereditary blood disorders that are characterized by reduction or complete absence of the β-globin chain synthesis due to mutations, affecting critical areas of the β-globin gene on the chromosome 11. The disease is inherited in an autosomal recessive manner, with severity ranging from asymptomatic individuals to transfusion dependent anemia, according to the nature of the mutation. The prevalence of the β-globin gene in various areas of Saudi Arabia, previously reported as 0.01 to 0.15% in general population. Materials And Methods: A cohort of 131 samples, which were submitted for β-globin gene mutation analysis in King Faisal Specialist Hospital and Research Center during 2008 to 2013, were tested for the entire genomic region (3-Exons and 2-Introns) of the HBB gene by direct Sanger sequencing technique. Results: Out of the total population tested, 28 (21%) were undetectable cases and 103 (79%) were detectable for β-globin chain mutations. Nineteen different mutations of the HBB gene were identified in all detectable cases (103 patients). Among these mutations c.315+1G>A, c.118C>T, and c.92+5G>C were detected in the majority of cases (66%) with five novel mutations (c.410G>A, c.-151C>T, c.68_74delAAGTTGG, c.316-3C>A, and c.-31C>T) that are reported for the first time in Saudi population. Discussion: The result of this retrospective study confirms the previously reported common β-thalassemia mutations among Saudi population.

692. Genome Editing for Personalized Gene Therapy of IVSI-110 Beta-Thalassemia

Thalassemia is amongst the commonest single-gene disorders worldwide, caused by deficient production of α- or β-globin. Of particular clinical relevance is β-thalassemia, which as a severe monogenic disease of the hematopoietic system is an ideal target for gene therapy, either by gene addition or gene correction. Problems inherent to the gene-augmentation approach for β-thalassemia, including insertional mutagenesis or low expression of the therapeutic transgene, may be avoided using targeted gene correction of mutations with site-specific designer nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 or transcription activator-like effector nucleases (TALENs). Our study is focused on the development of a personalized gene-correction therapy of the common β-thalassemia mutation, HBBIVS1-110, which in most Mediterranean and many Western countries has a frequency of above 20% (with 80% on the island of Cyprus) amongst β-thalassemia carriers. HBBIVS1-110 creates an abnormal splice-acceptor site in intron I of the β-globin gene, leading to a pre-mature in-frame stop codon in the aberrantly spliced mRNA and to early transfusion dependence of homozygotes. We have developed and evaluated designer nucleases targeting the site of the HBBIVS1-110 β-thalassemia mutation, including direct comparison of targeted disruption efficiency and toxicity for CRISPR/Cas9 and TALEN tools in human embryonic kidney cells and in murine erythroleukemia (MEL) cells carrying a HBBIVS1-110mutant transgene based on the GLOBE (MA821) vector. Using the latter, we have assessed the therapeutic efficiency and cleavage properties of these designer nucleases. Analyses included immunoblots, absolute quantification of correctly and aberrantly spliced HBB mRNAs using multiplex RT-qPCR, sequencing and characterization of the resulting genome-editing events. We noted superior performance of several TALEN pairs compared to a single CRISPR/Cas9 nuclease suitable for the target site. Moreover, we demonstrate a significant increase of correct splicing and β-globin chain synthesis for genome-edited transgenic MEL MA821 HBBIVS1-110 cells. For the two most efficient TALEN pairs, β-globin protein levels in pools of edited cells reach over 20% of that detected in MEL cells harbouring a wild-type MA821 HBB control, up from 1% for the mock-treated MA821 HBBIVS1-110 control. Our findings validate our approach and indicate its suitability also for the correction of other intronic disease-causing mutations.

Rare Hemoglobin Variants: Hb G-Szuhu (HBB: c.243C>G), Hb G-Coushatta (HBB: c.68A>C) and Hb Mizuho (HBB: c.206T>C) in Sri Lankan Families

In this short communication, we describe the clinical presentation of unusual hemoglobin (Hb), variants in three Sri Lankan cases under study for β-thalassemia intermedia (β-TI). We believe this is the first report on their occurrence in Sri Lanka as well as from the Indian subcontinent. During a molecular study performed on β-TI patients, we identified three unusual Hb variants as Hb G-Szuhu (HBB: c.243C>G), Hb G-Coushatta (HBB: c.68A>C) and Hb Mizuho (HBB: c.206T>C) in three unrelated families. Hb G-Szuhu and Hb G-Coushatta were found in combination with the common β-thalassemia (β-thal) mutation, IVS-I-5 (G>C). Both probands had mild anemia with greatly reduced red cell indices and had non palpable livers and spleens, however, by ultrasound, both were observed to be enlarged. The final Hb variant, Hb Mizuho, was identified as a heterozygous mutation found in both proband and his mother. Both family members had severe anemia and were regularly transfused and had increased red cell parameters.

Non-invasive prenatal diagnostic testing for β-thalassaemia using cell-free fetal DNA and next generation sequencing.

To develop an accurate non-invasive prenatal test using next generation sequencing (NGS) for HbE and the four most common β-thalassaemia mutations found in South East Asia (namely -28A > G, CD17A > T, CD41/42(-TTCT) and IVS-II-654C > T). Cell-free DNA was extracted from maternal plasma from 83 families where both parents were carriers of the HbE mutation or one of four common β-thalassaemia mutations. Overlapping PCR amplicons covering each mutation were generated, pooled and sequenced using the Illumina MiSeq. Fastq files were analysed to detect inheritance of the paternal mutation. In two cases where the fathers were compound heterozygotes for HbE and -28A > G, the fetus was correctly diagnosed as having inherited one of the paternal mutations. In 35/85 cases, the paternal mutation was not detected, and in 50/85 cases, it was classified as inherited. Overall sensitivity for detection of paternal mutations was 100% (95% CI: 92.4-100%), and specificity was 92.1% (95% CI: 79.2-97.3%). We demonstrated that detection of paternal mutations using NGS can be readily achieved with high sensitivity and specificity, removing the need for an invasive test in 50% of pregnancies at risk of a thalassaemia in cases where the father and mother carry a different mutation. © 2014 John Wiley & Sons, Ltd.