Nondeletional Mutations Research Articles

SNPscan Combined With CNVplex as a High-Performance Diagnostic Method for Thalassemia.

Thalassemia is a Mendelian-inherited blood disorder with severe consequences, including disability and mortality, making it a significant public health concern. Therefore, there is an urgent need for precise diagnostic technologies. We introduce two innovative diagnostic techniques for thalassemia, SNPscan and CNVplex, designed to enhance molecular diagnostics of thalassemia. The SNPscan and CNVplex assays utilize variations in PCR product length and fluorescence to identify multiple mutations. In the SNPscan method, we designed three probes per locus: two 5' and one 3', and incorporated allele identification link sequences into one of the 5' probes to distinguish the alleles. The detection system was designed for 67 previously reported loci in the Chinese population for a specific genetic condition. CNVplex identifies deletion types by analyzing the specific positions of probes within the globin gene. This innovative approach enables the detection of six distinct deletional mutations, enhancing the precision of thalassemia diagnostics. We evaluated and refined the methodologies in a training cohort of 100 individuals with confirmed HBA and HBB genotypes. The validation cohort, consisting of 1647 thalassemia patients and 100 healthy controls, underwent a double-blind study. Traditional diagnostic techniques served as the control methods. In the training set of 100 samples, 10 mutations (Hb QS, Hb CS, Hb Westmead, CD17, CD26, CD41-42, IVS-II-654, --SEA, -α3.7 and -α4.2) were identified, consistent with those identified by traditional methods. The validation study showed that SNPscan/CNVplex offered superior molecular diagnostic capabilities for thalassemia, with 100% accuracy compared to 99.43% for traditional methods. Notably, the assay identified three previously undetected mutations in 10 cases, including two deletion mutations (Chinese Gγ(Aγδβ)0 del and SEA-HPFH), and one non-deletion mutation (Hb Q-Thailand). The SNPscan/CNVplex assay is a cost-effective and user-friendly tool for diagnosing thalassemia, demonstrating high accuracy and reliability, and showing great potential as a primary diagnostic method in clinical practice.

Disease burden, management strategies, and unmet needs in α-thalassemia due to hemoglobin H disease.

Alpha-thalassemia is an inherited blood disorder caused by impaired α-globin chain production, leading to anemia and other complications. Hemoglobin H (HbH) disease is caused by a combination of mutations generally affecting the expression of three of four α-globin alleles; disease severity is highly heterogeneous, largely driven by genotype. Notably, non-deletional mutations cause a greater degree of ineffective erythropoiesis and hemolysis, higher transfusion burden, and increased complication risks versus deletional mutations. There are limited treatment options for HbH disease, and effective therapies are needed. This review discusses the pathophysiology of HbH disease, current management strategies, unmet needs, and emerging treatment options.

A rare hemoglobinopathy duo: Hb Adana×Hb SEA in a 1-year-old patient – a case report and a brief literature review

Introduction and importance: Alpha thalassemia, resulting from nondeletional mutations, typically presents a more severe clinical manifestation compared to deletional mutations. Severe outcomes, such as hydrops fetalis, are associated with two specific nondeletional mutations. Therefore, DNA-based investigation is crucial for suspected carriers exhibiting subtle hematological abnormalities to facilitate proper diagnosis and effective family counseling. Case presentation: In this report, the authors describe a phenotypically normal 1-year-old girl with a rare and unique alpha-thalassemia genotype due to the presence of Hb Adana, a nondeletional alpha-chain mutation compounded with Hb SEA, an alpha-globin gene deletion. Clinical discussion: Mutations determine the clinical manifestations of alpha-thalassemia. DNA testing is recommended for suspected carriers with relatively small hematological abnormalities, for precise diagnosis and family counseling. To provide clinicians with a reference for diagnostic assessment, the authors established a genotype-phenotype correlations based on reported cases of Hb Adana following an exhaustive literature review. Being interested in determining which ethnicities and genotypes are associated with a higher risk of complications, including hydrops fetalis and transfusion dependence, the authors formalized a diagnostic evaluation guide and a guide for early screening to improve outcomes. Conclusion: Precise genetic evaluation is important for the diagnosis of alpha thalassemia. Hematologists play a critical role in managing these disorders, understanding genotype-phenotype correlations, and highlighting the significance of genetic counseling for high-risk patients. Extensive studies on these various genophenotypes are required to improve the diagnosis and prognosis of such medical conditions and advocate preventative strategies.

Detection of Non-Deletional Αlpha Thalassaemia in Hospital Tengku Ampuan Rahimah, Klang, Malaysia

Background: To date, more than 70 variants of non-deletional mutations associated with α-thalassaemia have been recognized and recorded, showcasing the diverse genetic manifes-tations of the condition. Objective: This study is to provide data on the non-deletional alpha (α) thalassaemia cases at Hospital Tengku Ampuan Rahimah (HTAR), Klang, Selangor and to compare the hematological parameters (Hb, RBC, MCH, MCV, MCHC) of non-deletional α thalassaemia with the deletional variant. Methods: This was a cross-sectional study involving data extraction from the HTAR hematology laboratory registry on confirmed thalassaemia cases via DNA analysis from January 2017 to December 2019. Hematological parameters were obtained from the laboratory database. Results: A total of 479 α thalassaemia cases were recruited, 98 (20.5%) were non-deletional type. Among these, 75 (76.5%) were hetero-zygous Hb CS, 17 (17.3%) heterozygous Hb Adana, 3 (3.1%) heterozygous Hb Quong Sze (QS) and 1 (1.0%) case each of compound heterozygous Hb Constant Spring (CS) and Hb Adana (αCSα/αCD59α), compound heterozygous Hb CS and -3.7kb deletion (αCSα /-α3.7) and compound heterozygous of Hb CS and SEA deletion (αCSα/--SEA). In the single and double gene mutated groups, there was no significant difference of hematological parameters be-tween the deletional and non-deletional groups. Among the non-deletional variants, there was a significant association between Hb, MCH and MCHC parameters and the number of mu-tated α gene. Conclusion: The result can update the data of non-deletional α thalassaemia.

Detection of α-thalassemia mutations by Multiplex ligation-dependent probe amplification

Vietnam is a country with a high rate of people carrying α-globin gene mutations, therefore, it is highly important to screen for α-thalassemia carriers, especially in couples with one diagnosed α-carrier, in order to prevent and avoid having α-thalassemia major offspring. α-thalassemia is an inherited autosomal recessive disease, of which the phenotype depends on the degree of α-globin chain deficiency. Research samples were collected and analysed gene mutations at the Gene - Protein Research Center, Hanoi Medical University. With the objective of screening for deletional and nondeletional α-globin gene by using Multiplex ligation-dependent probe amplification (MLPA) method on eighty five people, whose spouses were α-thalassemia carriers, the results of this study have successfully identified 38/85 carriers including thirty five deletional carriers (26 --SEA, 5 -α3.7, 1 -α4.2, 1 --THAI, 1 whole α-globin deletion, and 1 POLR3K-ITFG3 gene deletion) and 3 cases of non-deletional mutation (2 -αHbCs, 1 anti3.7). Deletional mutations account for 92.1% while non-deletional mutations account for only 7.9%.

Prime Editing of the α-Thalassemia Hb Constant Spring Mutation

Hemoglobin Constant Spring (HbCS) is the most common non-deletional α-thalassemic mutation and is an important cause of HbH-like disease in Southeast Asia. Although the α-globin is expressed from two copies of homologous genes ( HBA2 and HBA1), the constant spring (CS) mutation CD142 (UAA > CAA) occurs only in HBA2 gene that leads to the instability of both mRNA and protein in erythroid cells. The newly developed gene editing technology prime editors (PEs) can generate all types of substitutions, providing therapeutic potential in different types of diseases. However, the efficiency of primer editor in primary hematopoietic stem cells is still a big challenge and lack of study in the HbCS mutation. We first created the endogenous HbCS mutation in human umbilical cord blood-derived erythroid progenitor-2 (HUDEP-2) cells, providing as a model for prime editing optimization. The protein of α cs chain (a larger protein with extra 31 amino acids) was detected in the model cells with HBA2 bi-allelic CS mutation and normal HBA1, which exhibited the delay of expansion, higher apoptosis rate and altered erythroid maturation markers after induced differentiation. To correct the CS mutation for converting HbCS to HbA, we performed ex vivo delivery of mRNA encoding the prime editor together with a pegRNA and a nick guide RNA into the Hb CS HUDEP2 model cells and achieved the successful in-situ correction with an efficiency of 39.883±9.720% of CS alleles without enrichment, while no measurable editing in the same loci of HBA1 as evaluated by both Sanger sequencing and NGS. More importantly, the correction resulted in a significant decrease of α cs chain, thereby alleviated erythroid phenotype after induced maturation with reducing the apoptosis rate. After that, we collected the primary Hb H-CS patients derived CD34 + hematopoietic stem/progenitor cells (HSPCs) and confirmed that the defect on erythroid expansion, differentiation in Hb H-CS patients derived CD34 + HSPCs compared to the normal donor, especially demonstrated by lower enucleation rate and higher apoptosis. Moreover, the protein of α cs chain was detected in the erythroblast from Hb H-CS CD34+ HSPCs. We further successfully corrected the CS mutation in Hb H-CS patient derived CD34 + HSPCs with an efficiency of 23.68% by mRNA electroporation. At the same time, the editing efficiency was achieved with minimal generation of insertions and deletions and a low level of Cas9-dependent DNA off-target editing. Importantly, we observed an ~23% decrease of α cs chain protein and significant restoration of the enucleation defect from the differentiated erythroblast after PE correction in CD34 + HSPCs. In summary, these results suggests that the delivery of PE mRNA together with synthesized pegRNA and nick sgRNA can successfully achieve the in-situ correction in Hb HbCS mutation site in the model cells and the primary Hb H-CS patient derived CD34 + HSPCs. Our results provide proof of principle for prime editing for its therapeutic potential in directly and precisely correction of the Hb-CS mutation.

Combined approaches for increasing fetal hemoglobin (HbF) and de novo production of adult hemoglobin (HbA) in erythroid cells from β-thalassemia patients: treatment with HbF inducers and CRISPR-Cas9 based genome editing.

Genome editing (GE) is one of the most efficient and useful molecular approaches to correct the effects of gene mutations in hereditary monogenetic diseases, including β-thalassemia. CRISPR-Cas9 gene editing has been proposed for effective correction of the β-thalassemia mutation, obtaining high-level "de novo" production of adult hemoglobin (HbA). In addition to the correction of the primary gene mutations causing β-thalassemia, several reports demonstrate that gene editing can be employed to increase fetal hemoglobin (HbF), obtaining important clinical benefits in treated β-thalassemia patients. This important objective can be achieved through CRISPR-Cas9 disruption of genes encoding transcriptional repressors of γ-globin gene expression (such as BCL11A, SOX6, KLF-1) or their binding sites in the HBG promoter, mimicking non-deletional and deletional HPFH mutations. These two approaches (β-globin gene correction and genome editing of the genes encoding repressors of γ-globin gene transcription) can be, at least in theory, combined. However, since multiplex CRISPR-Cas9 gene editing is associated with documented evidence concerning possible genotoxicity, this review is focused on the possibility to combine pharmacologically-mediated HbF induction protocols with the "de novo" production of HbA using CRISPR-Cas9 gene editing.

Genetic heterogeneity of thalassemia major patients in Rembang Regency, Central Java, Indonesia

Link of Video Abstract: https://www.youtube.com/watch?v=RG95aJkUndg Background: Thalassemia and hemoglobinopathies are caused by mutations in the globin genes that reduce haemoglobin synthesis. In many Asian countries, including Indonesia, thalassemia is a common inherited disorder. Thalassemia has specific characteristics due to the wide variability of the mutations from various populations. Therefore, screening protocol should be designed on specific populations’ regional gene mutation patterns. This study aims to investigate the mutational diversity of the globin genes in thalassemia major patients in Rembang Regency, Central Java, Indonesia. Methods: Genetic mutations were carried out in all major thalassemia patients who were recorded and underwent routine blood transfusions at the Rembang District Hospital from 2018 – 2020. Mutation identification was carried out using a PCR- RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) based method, the ACRS (Amplified Created Restriction Site) method, and genome sequencing. Results: The most common genotypes are double heterozygous HbE and Cd 35 mutation (-C) (42.87%), HbE and IVS1-nt.5 mutations (G> C) (28.57%), HbE and IVS1- nt.2 mutations (T> C), HbE and codon 8/9 (insertion + G), codon 8/9 (insertion + G) and Hb Malay, as well as Hb Adana and Hb Constant Spring (1.74%). Conclusion: The β-thalassemia and α-thalassemia mutations most commonly found in Rembang Regency are Cd 35 (-C) and non-deletional mutations.

Molecular spectrum of α- and β-thalassemia among individuals of reproductive age in the Zhuhai region of southern China.

Thalassemia is the most common monogenic disease in South and Southeast Asia. An accurate assessment of the relative frequency and composition of thalassemia mutations is important for the design of appropriate strategies to prevent the disease. In this study, we aimed to decode the molecular characterization of thalassemia mutations in Zhuhai region of southern China. A total of 8048 individuals who were potential thalassemia carriers were enrolled. Gap-polymerase chain reaction (Gap-PCR) and reverse dot-blot (RDB) hybridization methods were employed to detect common deletional and non-deletional thalassemia mutations. Multiplex ligation dependent probe amplification (MLPA) and Sanger sequencing were used to analyze and verify rare and complex mutations. We diagnosed 3433 individuals as thalassemia carriers or patients. Of these, 2395 (69.76%) individuals with α-thalassemia harbored 13 α-globin gene mutations. The three most common α-thalassemia mutations were --SEA (60.08%), -α3.7 (20.62%) and -α4.2 (9.25%). We diagnosed 903 (26.30%) individuals with β-thalassemia and identified 20 β-globin gene mutations, of which the three most frequent were CD41/42 (-TCTT) (38.10%), IVS-II-654 (C>T) (23.69%) and TATAbox-28 (A>G) (15.18%). In addition, we identified 15 rare thalassemia variants. We also summarized the association between the thalassemia genotype and hematological parameters, which demonstrated the broad phenotypic heterogeneity caused by globin gene mutations. This is the first survey of thalassemia molecular epidemiology and hematological phenotype in Zhuhai region. It uncovered a high prevalence and complex molecular spectrum of thalassemia. These findings can be used as a basis for thalassemia diagnosis, counseling and prevention management.

Gene Mutation Spectrum among Alpha-Thalassaemia Patients in Northeast Peninsular Malaysia.

(1) Background: Alpha (α)-thalassaemia is a genetic disorder that affects 5% of the world population. Deletional or nondeletional mutations of one or both HBA1 and HBA2 on chromosome 16 will result in reduced production of α-globin chains, a component of haemoglobin (Hb) that is required for the formation of red blood cells (RBCs). This study aimed to determine the prevalence, haematological and molecular characterisations of α-thalassaemia. (2) Method: The parameters were based on full blood count, high-performance liquid chromatography and capillary electrophoresis. The molecular analysis involved gap-polymerase chain reaction (PCR), multiplex amplification refractory mutation system-PCR, multiplex ligation-dependent probe amplification and Sanger sequencing. (3) Results: With a total cohort of 131 patients, the prevalence of α-thalassaemia was 48.9%, leaving the remaining 51.1% with potentially undetected α gene mutations. The following genotypes were detected: -α3.7/αα (15.4%), -α4.2/αα (3.7%), --SEA/αα (7.4%), αCSα/αα (10.3%), αAdanaα/αα (0.7%), αQuong Szeα/αα (1.5%), -α3.7/-α3.7 (0.7%), αCSα/αCSα (0.7%), -α4.2/αCSα (0.7%), -SEA/αCSα (1.5%), -SEA/αQuong Szeα (0.7%), -α3.7/αAdanaα (0.7%), --SEA/-α3.7 (2.2%) and αCSα/αAdanaα (0.7%). Indicators such as Hb (p = 0.022), mean corpuscular volume (p = 0.009), mean corpuscular haemoglobin (p = 0.017), RBC (p = 0.038) and haematocrit (p = 0.058) showed significant changes among patients with deletional mutations, but not between patients with nondeletional mutations. (4) Conclusions: A wide range of haematological parameters was observed among patients, including those with the same genotype. Thus, a combination of molecular technologies and haematological parameters is necessary for the accurate detection of α-globin chain mutations.

Alpha-Thalassemia: Diversity of Clinical Phenotypes and Update on the Treatment

One of the more common single-gene disorders worldwide is α-thalassemia, carriers of which are found at variable frequencies (>1%) across all tropical and subtropical countries. Two linked α-globin genes on each allele of chromosome 16 regulate α-globin chain production. Deletion of one or more α-globin genes is the most frequent molecular defect found in α-thalassemia, whereas non-deletional mutations also occur, leading to unstable α-globin chains. HbH is the most common clinically important α-thalassemia disease and occurs when three α-globin genes are deleted/mutated, leaving only one copy of the gene intact. HbH can be divided into deletional (--/-α) and non-deletional genotypes (--/αTα). Whereas clinical phenotypes of the former are usually homogenously mild to moderate, those of the latter can be diverse. As HbH disease is particularly prevalent in Southeast Asia and some parts of the Mediterranean region, where β-thalassemia is also prevalent, affected patients are sometimes left undertreated. Therefore, hematologists and general physicians need to be educated to provide optimal disease monitoring and early identification of those with more severe phenotypes. Some issues regarding transfusion and iron chelation management differ from those of β-thalassemia, and these need to be recognized. Hb Bart’s hydrops fetalis syndrome (BHFS) is the most severe form of α-thalassemia; affected patients lack production of α-globin chains. Recent advances in fetal medicine and neonatal intensive care have made it possible for BHFS to no longer constitute a universally fatal disorder. Transfusion and chelation strategies for rare survivors are distinct and require updating.

Clinical and Haematological Parameters of Commonly Reported Non-deletional α-thalassaemia Mutations in Southeast Asia: A Review

Alpha (α)-thalassaemia is a common genetic disorder worldwide caused by the deletion and rarely non-deletional mutations of the α-globin gene. Nearly 70 types of non-deletional mutations have been reported worldwide, and this review focuses on the common ones affecting α-thalassaemia patients. The common mutations are initiation codon mutation, codon 30, haemoglobin (Hb) Constant Spring, Hb Quang Sze, Hb Adana and Hb Evora. The haematological parameters of non-deletional mutations usually show mild changes. However, a severe reduction in haemoglobin level, mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), and mean corpuscular haemoglobin count (MCHC) has been observed among compound heterozygous HbH disease, involving both deletional and non-deletional mutations. Although non-deletional mutations are rarely reported, it requires the study of more cases to understand the clinical phenotypes that lead to severe clinical manifestations.

One-step genotyping of α-thalassaemia by multiplex symmetric PCR melting curve

AimsAlpha-thalassaemia is one of the most common monogenic disorders worldwide. Due to high guanine-cytosine (GC) content and high mutation diversity in α-globin gene cluster, deletional and non-deletional mutations were usually...

Molecular Spectrum, Ethnic and Geographical Distribution of Thalassemia in the Southern Area of Hainan, China.

BackgroundThalassemia is one of the most common genetic diseases in southern China. Accurate population frequency data regarding the occurrence and distribution of thalassemia are important for designing appropriate prevention strategies for thalassemia. This study aims to reveal the molecular spectrum, ethnic and geographical distribution of thalassemia in the southern area of Hainan Province, China.MethodsA total of 9813 suspected carriers of thalassemia were screened for genetic analysis by using the PCR-reverse dot blot hybridization method targeting three known deletions of α-thalassemias (--SEA, -α3.7, and -α4.2), three nondeletional mutations of α-thalassaemias (αCS, αQS, and αWS) and the 17 most common mutations of β-thalassaemias in the Chinese population.ResultsApproximately 6,924 subjects were genetically diagnosed as thalassemia carriers or patients, including 5812 cases of α-thalassemia (83.9%), 369 cases of β-thalassemia (5.3%), and 743 cases of α-composite β-thalassemia (10.7%). A total of 21 distinct genotypes were identified among the 5,812 α-thalassemia carriers, -α4.2/αα, -α3.7/αα, and -α3.7/-α4.2 were the most common α-thalassemia genotypes. The most frequent β-thalassemia genotype was βCD41−42/βN, with a notable proportion of 69.6%, followed by the β−28M/βN, βIVS−II−654/βN, βCD71−72/βN, βE/βN, and βCD17/βN genotypes. In addition, 37 genotypes were detected among the 743 cases of both α- and β-thalassemia mutations. The α-thalassemia genotypes were most commonly found in the Li people, who accounted for 73.5% of α-thalassemia carriers. The β-thalassemia genotypes were most commonly identified in the Han people, who accounted for 59.4% of β-thalassemia carriers. Among the subjects carrying both α- and β-thalassemia variations, only three ethnic minorities were identified, including the Li, Han, and Miao people, accounting for 82.0, 17.4, and 0.7%, respectively.ConclusionsOur study indicates that there is high genetic heterogeneity, geographical and ethnic differences in thalassemia in populations in the southern area of Hainan Province. These findings will be helpful in guiding genetic counseling and prenatal diagnosis of thalassemia in Hainan Province.

Genotype\u2013phenotype correlation in patients with deletional and nondeletional mutations of Hb H disease in Southwest of Iran

We studied the alpha-globin gene genotypes, hematologic values, and transfusion-dependence of patients with Hb H disease. Molecular characterization of alpha-thalassemia was performed. We identified 120 patients with Hb H disease. Of these patients, 35 (29.16%) had deletional form of Hb H disease, and 85 (70.83%) had different form of non-deletional Hb H disease. The most frequently observed Hb H genotypes were --Med/–α3.7 in 33 patients (27.5%), αCD19(-G) α/αCD19(-G) α in 25 cases (20.83%), αpolyA2α/αpolyA2α in 15 (12.5%), and αpolyA1α/αpolyA1α in 13 (10.83%) respectively. The probability of receiving at least one transfusion blood in deletional form was observed in 3 of 35 (8.57%) patients which just seen in 3 of 33 (9%) patients with --Med/–α3.7 genotype. This form was also observed in 8 of 85 (9.4%) patients in non-deletional Hb H diseases which five of them had Med deletion in compound with alpha globin point mutations. Nondeletional Hb H disease was more severe than deletional Hb H disease requiring more blood transfusions. We can recommend that Med deletion in compound with alpha-globin point mutations, polyA1 and constant spring in homozygous form needs to be taken into consideration when offering counseling to high-risk couples.

Clinical validation of a single-tube PCR and reverse dot blot assay for detection of common α-thalassaemia and β-thalassaemia in Chinese

ObjectiveTo evaluate a novel reverse dot blot assay for the simultaneous detection six types of common α-thalassaemia alleles (three deletional and three common non-deletional mutations) and 19 types of common β-thalassaemia alleles in a Chinese population.MethodsGenomic DNA samples were collected from three hospitals in southern China. The novel thalassaemia gene assay involved one multiplex polymerase chain reaction amplification system and one round of hybridization. Each of the clinically validated DNA samples was re-tested using the new multiplex polymerase chain reaction/reverse dot blot assay II (M-PCR/RDB II) assay in a double-blind manner.ResultsA total of 1060 unrelated study participants, including 829 patients with thalassaemia and 231 healthy control subjects, were analysed. The whole PCR and RDB procedures were completed in 260 min. All the samples, including heterozygous thalassaemia, homozygous thalassaemia and compound heterozygous thalassaemia, were correctly genotyped, yielding 100% concordance with the reference assays. HKαα/--SEA and HKαα/−α4.2, which were not included in the detection panel, yielded a contradictory result with this new assay.ConclusionThe novel M-PCR/RDB II assay was simple, rapid and accurate, suggesting that it could be used for the genetic screening and clinical diagnosis of common α-thalassaemia and β-thalassaemia variants in Chinese populations.

Clinical and genetic characteristics of hemoglobin H disease in Iran

Hemoglobin H (Hb H) disease is a subtype of α-thalassemia caused by deletional and/or non-deletional mutations in three alpha-globin genes in which the various genotypes determine the disease severity. This study was aimed to investigate the frequency of alpha gene mutations and genotypes and their correlation with hematological and clinical characteristics in Iran. Among 202 patients diagnosed with Hb H disease through a national study in Iran according to standard methods, we had access to the hematologic and clinical findings and genetic data of 101 patients in whom genetic study was performed. Genomic DNA from peripheral blood was extracted and analyzed for identification of α-globin gene mutations using Multiplex Gap Polymerase Chain Reaction, Reverse Hybridization Assay, and finally Direct DNA Sequencing method. Twenty-one different mutations and thirty genotypes were detected in 101 patients with Hb H disease. In total, 39 patients (38.6%) were deletional and 62 patients (61.4%) were non-deletional type of the disease. The -- MED mutation was highly prevalent in almost half of the patients (56.4%). Among various genotypes, –MED/-a3.7 (29.7%) and -α20 .5/-α5NT (6.9%) were the most prevalent genotypes found in the studied group. Patients with non-deletional type presented with more severe hematological and clinical findings. Hb H percentage and serum ferritin levels were significantly higher in non-deletional patients in comparison to the deletional group (p < 0.05). 12 (11.9%) and 40 (39.6%) out of 101 patients were on regular and occasional transfusions, respectively. 83% of those with regular transfusion belonged to the non-deletional group. Among transfusion-dependent patients, –MED/αCSα and α20 .5/-α5NT were the most common genotypes. In this study, two patients with -α20 .5/αCSα and –MED/α−5NT genotypes experienced thrombotic events. This study indicated that although non-deletional genotypes of Hb H disease were responsible for more clinical severity of the disease, due to the presence of severe phenotypes even in deletional types, no definite correlation was found between genotype and phenotype.

Analysis of Gene Deficiency Types of Thalassemia in Lingui District of Guilin City

To analyze the gene defect types and distribution characteristics of α- and β-thalassemia in Lingui District of Guilin City, Guangxi, so as to provide scientific basis for genetic consultation and prevention measures. A total of 6 496 suspected cases for screening the thalassemia during physical examination, premarital examination, pregnancy examination and hospitalization in the Second Affiliated Hospital of Guilin Medical University from May 2016 to October 2019 were analyzed. Gap-PCR, PCR-RDB and DNA sequencing techniques were used to detect the types and constituent ratios of gene defects in α- and β-thalassemia positive cases. Among 6 496 suspected patients, 1 363 were thalassemia carriers, the total positive rate was 20.98%. There were 677 cases of single-gene deletion and 26 cases of double-gene detetion on the deletional α-thalassemia, 115 cases of non-deletion α-thalassemia mutation and 4 cases of deletion plus mutation. The positive rate of α-thalassemia was 12.66%. There were 11 gene abnormalities for α-thalassemia, of which --SEA/αα (50.36%) was the most common, followed by -α3.7/αα (23.84%); the main α-gene mutation was ααCS (6.93%). There were 514 β-thalassemia gene carriers, with a positive rate of 7.93%. In 12 types of β-gene mutations, CD41-42 (-TTCT) (55.64%) was the most common, followed by CD17 (A→T) (20.23%). There were 25 cases of double heterozygous α and β thalassemia (0.39%), of which -α3.7/βCD17 (24%) and --SEA/β41-42 (16%) were numerically dominant. Two of rare thalassemia genotypes were identified by sequencing, which were heterozygous mutations of Chinese Hong Kong type α thalassemia (HKαα/αα or HKαα/-α3.7) and β gene mutations IVS-I (-2) or codon30 (A→G) β0, respectively. Lingui district of Guilin city is a high incidence area of thalassemia. The mutation rate of α-thalassemia --SEA/αα type deletion is relatively high, followed by that of the right deletion type (-α3.7/αα). CD41-42 (-TTCT) has the highest mutation rate in β-thalassemia, followed by CD17(A→T). The results of this study provide reference data for the regional screening, diagnosis and treatment of thalassemia and eugenics.

Molecular Detection of Alpha Thalassemia: A Review of Prevalent Techniques.

Alpha thalassemia (α-thalassemia) is an autosomal recessive disorder due to the reduction or absence of α globin chain production. Laboratory diagnosis of α-thalassemia requires molecular analysis for the confirmatory diagnosis. A screening test, comprising complete blood count, blood smear and hemoglobin quantification by high performance liquid chromatography and capillary electrophoresis, may not possibly detect all the thalassemia diseases. This review focused on the molecular techniques used to detect α-thalassemia, and the advantages and disadvantages of each technique were highlighted. Multiplex gap-polymerase chain reaction, single-tube multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, and loop-mediated isothermal amplification were used to detect common deletion of α-thalassemia. Furthermore, the reverse dot blot analysis and a single tube multiplex polymerase chain reaction could detect non-deletion mutation of the α-globin gene. Sanger sequencing is widely used to detect non-deletion mutations of α-thalassemia. Recently, next-generation sequencing was introduced in the diagnosis of both deletion and point mutations of α-thalassemia. Despite the advantages and disadvantages of different techniques, the routine method employed in the laboratory should be based on the facility, expertise, available equipment, and economic conditions.

Genotype-phenotype correlation of HbH disease in northern Iraq

BackgroundHbH disease results from dysfunction of three, less commonly two, α-globin genes through various combinations of deletion and non-deletion mutations. Characterization of the mutations and the underlying genotypes is fundamental for proper screening and prevention of thalassaemia in any region. The aim of this study was to explore the genetic arrangements of HbH disease and to correlate the genotypes with the clinical phenotypes.MethodsA total of 44 HbH disease patients were enrolled in this study. They were clinically and haematologically assessed. The patients were tested for 21 common α-globin gene mutations using multiplex PCR and reverse hybridization. According to the genotype, the patients were categorized into two separate sub-groups, deletion and non-deletion types HbH disease.ResultsWithin the studied HbH disease patients, eight different α-globin gene mutations were detected in nine different genetic arrangements. The --MED and -α3.7 deletions were the two most frequently encountered mutations (37.5 and 35.2% respectively). Patients with deletion genotypes constituted 70.4%. The most common detected genotype was --MED/−α3.7 (59.1%), followed by αpoly-A1α/αpoly-A1α (13.6%). For the first time, coinheritance of two relatively mild mutations (−α3.7/ααAdana) was unpredictably detected in a 1.5 year-old child with Hb of 7.1 g/dL.ConclusionThe HbH disease patients’ clinical characteristics were variable with no ample difference between the deletion and non-deletion types. These results can be of benefit for the screening and management of thalassaemia in this region.