Multiplex Gap-polymerase Chain Reaction Research Articles

Hematological Profile of Hb Adana Among High School Students in Northeast Peninsular Malaysia.

Background Hb Adana is a non-deletional alpha (α)-thalassaemia variant resulting from mutations in α1- or α2-globin codon 59 (αCD59), leading to the production of unstable α-globin. Clinical manifestations can vary from silent carrier status to dependence on blood transfusions, hepatosplenomegaly, skeletal deformities, and spinal cord compression. Despite the significance of Hb Adana inheritance, studying this variant poses challenges due to the scarcity of molecular tests and the potential for routine diagnoses to be overlooked. This study aims to investigate the prevalence of Hb Adana among local high school students and assess the hematological parameters and hemoglobin analysis of Hb Adana in Malaysia. Methodology This retrospective study analyzed 13,721 blood samples collected from high school students participating in Malaysia's National Thalassaemia Screening Program at Hospital Raja Perempuan Zainab II (HRPZ II). Deletional α-thalassaemia was detected using multiplex gap-polymerase chain reaction (PCR), while common non-deletional α-thalassaemia was identified using multiplex amplification refractory mutation system (ARMS) PCR. Data were extracted from the HRPZ II database for analysis. Results Among the participants, 2327 individuals were found to have either common deletional (n=1037, 44.6%) or non-deletional (n=1290, 55.4%) α-thalassaemia. Hb Constant Spring was the most prevalent non-deletional α-thalassaemia, accounting for 53.03% of cases. Thirty-one participants (1.33%) exhibited αCD59α/αα, and one (0.04%) had αCD59α/-α3.7. Among the 32 subjects with Hb Adana, 87.5% were Malay, and 12.5% were Orang Asli. Additionally, seven cases of HbE/Hb Adana co-inheritance were identified. Hemoglobin levels in heterozygous Hb Adana individuals ranged from mild anemia to normal, between 95 g/L and 153 g/L. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were approximately 73 fL and 23 pg, respectively. Conclusion This study delineates the distribution ofα-thalassaemia mutation patterns among high school students in Kelantan, Northeast Peninsular Malaysia. Our findings indicate that Hb Adana is rare in our region and co-inheritance with an α-gene deletion results in α+-thalassaemia and with HbE, α0-thalassaemia. All heterozygous Hb Adana individuals exhibited low MCVs and MCHs.

Hb Q-Thailand heterozygosity unlinked with the (–α4.2/) α+-thalassemia deletion allele identified by long-read SMRT sequencing: hematological and molecular analyses

ABSTRACTObjectiveIn the present study, two unrelated cases of Hb Q-Thailand heterozygosity unlinked with the (–α4.2/) α+-thalassemia deletion allele were identified by long-read single molecule real-time (SMRT) sequencing in southern China. The aim of this study was to report the hematological and molecular features as well as diagnostic aspects of the rare manifestation.MethodsHematological parameters and hemoglobin analysis results were recorded. A suspension array system for routine thalassemia genetic analysis and long-read SMRT sequencing were applied in parallel for thalassemia genotyping. Traditional methods, including Sanger sequencing, multiplex gap-polymerase chain reaction (gap-PCR) and multiplex ligation-dependent probe amplification (MLPA), were used together to confirm the thalassemia variants.ResultsLong-read SMRT sequencing was used to diagnose two Hb Q-Thailand heterozygous patients for whom the hemoglobin variant was unlinked to the (–α4.2/) allele for the first time. The hitherto undescribed genotypes were verified by traditional methods. Hematological parameters were compared with those of Hb Q-Thailand heterozygosity linked with the (–α4.2/) deletion allele in our study. For the positive control samples, long-read SMRT sequencing revealed a linkage relationship between the Hb Q-Thailand allele and the (–α4.2/) deletion allele.ConclusionsIdentification of the two patients confirms that the linkage relationship between the Hb Q-Thailand allele and the (−α4.2/) deletion allele is a common possibility but not a certainty. Remarkably, as it is superior to traditional methods, SMRT technology may eventually serve as a more comprehensive and precise method that holds promising prospects in clinical practice, especially for rare variants.

Impact of age-dependent red blood cell parameters on α-globin gene genotyping in children.

When screening for α-thalassemia in children, adult cut-offs for mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) are generally applied to guide genetic evaluation. However, the normal ranges for MCV and MCH are lower in children than in adults, so we hypothesized that using age-matched cut-offs could lead to a more rational diagnostic strategy. The aim of this study was to evaluate if age-matched cut-offs could be applied advantageously. Data on children referred to a hemoglobin fractionation at the Department of Clinical Biochemistry, Aarhus University Hospital between 2016-2021 were identified in the laboratory information system. α-globin gene (HBA1/HBA2) genotyping was performed using multiplex gap-polymerase chain reaction. A total of 387 children were identified. HBA1/HBA2-genotyping was performed in 207 children (53%), and α-thalassemia was diagnosed in 47 children (23%) with -α3.7/αα being the predominant genotype (13%). We found that 23 children had MCV and MCH levels in the normal age-matched range, and two of these children (9%) were α+ thalassemia carriers with three functional α-globin genes. Using age-specific cut-off levels resulted in a reduction of 23 (11%) genotypes performed. In conclusion, applying age-matched cut-offs for MCV and MCH when screening children for α-thalassemia lead to 11% fewer genotypes performed while 9% carriers of α+ thalassemia (of the medically innocuous genotype -α3.7/αα) would have been overlooked.

The Effect of Alpha Thalassemia, HbF and HbC on Haematological Parameters of Sickle Cell Disease Patients in Ibadan, Nigeria.

BackgroundSickle cell disease is a protean disease with limited data on Nigeria’s phenotypic and genetic variants. This study was conducted to provide baseline data on these variants by characterising the existing forms of sickle cell disease and correlating these with basic haematological parameters.MethodsAdult and paediatric patients with SCD were recruited from a tertiary health centre in Nigeria. Patients were age and sex-matched with healthy controls. Blood samples were obtained for Full Blood Count, phenotyping by High-Performance Liquid Chromatography, and genotyping for alpha thalassemia by multiplex Gap-polymerase chain reaction. Data analysis was done using IBM SPSS statistics version 23.ResultsA total of 130 patients with sickle cell disease and 117 controls were studied. Alpha thalassemia in the study population was due to a 3.7kb deletion in the alpha-globin gene cluster at a prevalence of 45.4% in the patients and 47% in the controls. The prevalence of the various existing forms of SCD genotype was: Homozygous S without alpha gene deletion (HbSS)- 39.2%; HbSC - 10.8%; HbSSα+1- 35.4%; HbSSα+2 - 6.9% and HbSF- 7.7%. In the control population, HbAA without alpha gene deletion had a prevalence of 42.7%, HbAAα+1 was 25.6%, HbAA α+2 was 6%, HbAS- 7.7%, HbAS α+1 – 11.1%, HbAS α+2 - 2.6%, HbAC – 2.6% and HbAC α+1 – 1.7%. HbA2 was significantly elevated in HbSS individuals with two alpha gene deletions but reduced in normal controls (HbAA) with alpha gene deletions. HbF and HbA2 were negatively correlated with each other (r= −0.587, p < 0.001). Individuals with the HbSC genotype followed by HbSSα+2 had the best haematological parameters.ConclusionsHaematological parameters vary with haemoglobin genotype. The C haemoglobin and homozygous alpha-thalassemia deletion had a better ameliorating effect on SCD haematological parameters than the F haemoglobin in this population. The effect of alpha thalassemia on some haematological parameters in SCD patients are reversed in normal controls.

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.

Molecular and Haematological Characteristics of alpha-Thalassemia Deletions in Yogyakarta Special Region, Indonesia.

alpha-Thalassemia is caused primarily by deletions of one to two alpha-globin genes and is characterized by absent or deficient production of alpha-globin protein. The South-East Asia (SEA) deletion, 3.7-kb and 4.2-kb deletions are the most common causes. The present study aimed to observe the molecular characteristics of this common alpha-Thalassemia deletions and analyse its haematological parameter. Blood samples from 173 healthy volunteers from thalassemia carrier screening in Yogyakarta Special Region were used. Haematological parameters were analysed and used to predict the carrier subjects. Genotype of suspected carriers was determined using multiplex gap-polymerase chain reaction and its haematological parameters were compared. The boundary site of each deletion was determined by analysing the DNA sequences. Seventeen (9.8%) of the volunteers were confirmed to have alpha-Thalassemia trait. Of these, four genotypes were identified namely -α3.7/αα (58.8%), -α4.2/αα (5.9%), -α3.7/-α4.2 (5.9%) and - -SEA/αα (29.4%). The 5' and 3' breakpoints of SEA deletion were located at nt165396 and nt184700 of chromosome 16, respectively. The breakpoint regions of 3.7-kb deletion were 176-bp long, whereas for 4.2-kb deletion were 321-bp long. The haematological comparison between normal and those with alpha-Thalassemia trait genotype indicated a significant difference in mean corpuscular volume (MCV) (p< 0.001) and mean corpuscular haemoglobin (MCH) (p< 0.001). As for identifying the number of defective genes, MCH parameter was more reliable (p= 0.003). The resultant molecular and haematological features provide insight and direction for future thalassemia screening program in the region.

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.

Molecular Genetic Analysis of α-Thalassemia in Hamadan Province, West Iran

Identifying couples who are carriers of thalassemia-causing mutations, followed by prenatal diagnosis (PND), is undoubtedly an effective way to prevent the birth of children with the disease. Our aim in this study was to report for the first time the spectrum of α-globin gene mutations in the population living in Hamadan Province, West Iran. Multiplex gap-polymerase chain reaction (gap-PCR), amplification refractory mutation system (ARMS)-PCR, and direct DNA sequencing of HBA1 and HBA2 genes were used to identify the α-thalassemia (α-thal)-causing mutations in a cohort of 389 individuals including 328 α-thal carriers and 61 normal subjects. A total of 17 different mutations and 25 different genotypes were detected. The –α3.7 (rightward) deletion (NG_000006.1: g.34164_37967del3804) was the most frequent mutation, accounting for more than half of all mutations (61.04%). This study revealed that there is a variety of α-thal mutations and α-thal genotypes in Hamadan Province, West Iran. This observation is probably due to the complexity of the Hamadan Province population that is composed of Persians (Fars), Turks, Kurds, and Lurs/Laks. In conclusion, our results demonstrated the spectrum of mutations in α-globin genes in Iran and increased our understanding of their distribution in this country.

Rare β-Globin Gene Mutations including a de novo Mutation of Hemoglobin Hammersmith in Southern Thailand

Objective: The aim of this study was to characterize unknown β-globin gene mutations in individuals who attended Songklanagarind Hospital for thalassemia screening and genetic counseling.Material and Methods: β-thalassemia mutations in individuals with hemoglobin (Hb) A2 levels &gt;3.5% originating from various provinces in southern Thailand were characterized by reverse dot blot hybridization (RDB) and multiplex gappolymerase chain reaction using a panel of 30 allele-specific probes for point mutations and 6 sets of specific primers for large deletions. Mutations which could not be identified by these two methods were further analyzed by direct deoxyribonucleic acid (DNA) sequencing.Results: Nineteen subjects found to have uncharacterized β-globin gene mutations were analyzed by direct DNA sequencing. Nine different rare mutations were identified, four of which have not been to date described in Thailand: -30 (T&gt;C), codon 5 (-CT), Hb Monroe (codon 30, G&gt;C) and Hb Hammersmith (codon 42, T&gt;C). An Hb Hammersmith mutation detected in one subject appeared to be a spontaneous mutation, unrelated to family history. The other five mutations have been reported previously within Thailand, but here they were identified in the southern part of Thailand for the first time: -31 (A&gt;G), codon 15 (-T), codon 35 (C&gt;A), codon 95 (+A) and Hb Dhonburi (codon 126, T&gt;G). The presence of the mutations was confirmed by RDB.Conclusion: In addition to the already reported β-globin gene mutations, 9 other different types of mutations were identified. This information should be useful for planning genetic counseling and prenatal diagnosis programs for prevention and control of thalassemia diseases.

The Spectrum of α-Thalassemia Mutations in Kurdistan Province, West Iran

In order to identify the α-thalassemia (α-thal) mutation spectrum in Kurdistan Province, West Iran, a total of 217 individuals, including 154 α-thal carriers and 63 normal subjects were investigated in this study. Molecular analysis of α1- and α2-globin genes using multiplex gap-polymerase chain reaction (gap-PCR), amplification refractory mutation system (ARMS)-PCR or direct DNA sequencing, showed 11 different α-globin variants. The –α3.7 (rightward) deletion (NG_000006.1: g.34164_37967del3804) (70.32%), polyadenylation signal (polyA2) site (AATAAA>AATGAA) (αpolyA2α) (HBA2: c.*92A>G) (7.74%), –α4.2 (leftward) deletion (6.45%) and codon 59 (or Hb Adana) (G>A) (ααcodon 59) (HBA1: c.179G>A) (4.52%) were the most frequent mutations in the present study. In conclusion, the spectrum of α-thal mutations in Kurdistan Province is closest to that in western provinces of Iran (Kurdish and Laki populations). In addition, it was revealed that the codon 59 mutation is common in the Kurdish population. On the other hand, despite the same ethnic background of Kurds in Iran and Iraq, the – –MED I double gene deletion and polyA2 point mutation have different distributions in these two populations. Therefore, further studies are needed to identify the cause of these differences.

α-Thalassemia Mutations in Ilam Province, West Iran

Despite several studies performed in different provinces of Iran to identify the spectrum of α-globin gene mutations, no such study has so far been carried out in Ilam Province. A total of 274 individuals, including 201 α-thalassemia (α-thal) carriers and 73 normal subjects, originating from the northern counties of Ilam Province, participated in this study. Analysis of α-globin defects was performed using multiplex gap-polymerase chain reaction (gap-PCR), amplification refractory mutation system (ARMS)-PCR and direct sequencing, which revealed a total of 11 different mutations and 22 different genotypes. The –α3.7 (rightward) (NG_000006.1: g.34164_37967del3804), α−5 ntα (HBA2: c.95 + 2_95 + 6delTGAGG), and –α4.2 (leftward) deletions were the most prevalent mutations identified in our study, with frequencies of 66.23, 10.09 and 8.33%, respectively. In conclusion, the present study showed that the α-thal mutation spectrum in Ilam Province, at least in the northern part of the province, is different from that in other geographical regions of Iran. These results increase our knowledge about the spectrum and distribution of α-globin gene mutations in Iran.

Genetic and phenotypic analysis of a rare asymptomatic case of a homozygous Chinese Gγ+(Aγδβ)0-thalassemia deletion in a Chinese family

ObjectiveThe clinical and hematologic features of thalassemia are due to different factors, and patients with identical genotypes may regularly exhibit variable severity. In the present work, one homozygous Chinese Gγ+(Aγδβ)0-thalassemia case with an asymptomatic phenotype, which is contrary to traditional views, was identified. Analysis of the underlying causes of this rare clinical phenotype involved accurate genetic diagnosis and detection of several genetic modifications. MethodsSix members of the proband’s family were enrolled in the study. Hematological parameters and hemoglobin analysis results were recorded. A suspension-array system, multiplex gap-polymerase chain reaction (gap-PCR) and multiplex ligation-dependent probe amplification (MLPA) were used together to characterize genotypes. Sanger sequencing was utilized to examine the KLF1 gene and four primary fetal hemoglobin (Hb F)-associated single-nucleotide polymorphisms (SNPs). ResultsFour family members carried the Chinese Gγ+(Aγδβ)0-thalassemia mutation, and a homozygous state was ultimately diagnosed for the proband. All of the Chinese Gγ+(Aγδβ)0 mutation-positive cases were coinherited with the Southern Asian α-thalassemia deletion (– – SEA/αα). Two SNP variants, rs7776054 and rs9399137, in the HBS1L-MYB locus were detected in the proband. ConclusionsThus far, this is the first study to describe the molecular characterization of a homozygous Chinese Gγ+(Aγδβ)0-thalassemia patient who exhibits no clinical symptoms. Our findings suggest that coinheritance of α-thalassemia or HBS1L-MYB locus variants may affect the clinical severity of Chinese Gγ+(Aγδβ)0-thalassemia. We conclude that the molecular examination of genetic determinants known to be associated with clinical outcomes in Chinese Gγ+(Aγδβ)0-thalassemia should be emphasized.

Identification of a Novel α-Thalassemia Deletion: –(α)4.9 from a Current Multiplex Gap-Polymerase Chain Reaction

α-Thalassemia (α-thal) is a genetic disorder caused by a reduced production of α-globin chain and commonly results from a large deletion involving the α-globin gene. This study aimed to investigate...

Iranian patients with hemoglobin H disease: genotype-phenotype correlation.

Thalassemia is one of the most common monogenic hereditary disorders. Despite noticeable advances made in prevention strategies, it is still highly prevalent in the Iranian population. A key approach to management and early diagnosis of the disease is through revealing the regions with high prevalence and determining common genetic and phenotypic diversity. In the current study Hemoglobin H (HbH) disease patients were analyzed as the most common form of thalassemia intermedia in Iran. A total of 80 patients suspected of being thalassemic according to their mild to moderate anemia, microcytosis and normal iron levels were included in this study at the hemoglobinopathy and thalassemia center of Ahvaz University of Medical Science. Patients were analyzed for hematological parameters and HbH mutations using Multiplex Gap Polymerase Chain Reaction and Multiplex Amplification Refractory Mutation System. Twelve mutations were detected in the studied population. The most common genotype was -α3.7/--MED (45%) followed by Homozygote αPoly A2 (17.5%). A total of ten different alpha-globin (α-globin) mutations were observed in patients which --MED, being the most common mutation (26.27%), followed by -α3.7 (24.37%) and αpolyA2(A>G) (18.12%). Hematological parameters such as Hb, MCV, MCH and HbH were assessed and results showed that they varied significantly among genotypes, adjusted to age and gender. This study reveals a highly diverse range of HbH patients different from what was thought in terms of both genotype and phenotype in the Khuzestan region of Iran. These findings could contribute to improve the thalassemia managing policies in this province.

The Spectrum of α-Thalassemia Mutations in the Lak Population of Iran

α-Thalassemia (α-thal) is one of the most common genetic disorders worldwide. The aim of this study was to investigate for the first time the α-thal mutation spectrum in the Lak population living in Lorestan Province, Iran. One hundred and seventy-six α-thal carriers participated in the study. Multiplex gap-polymerase chain reaction (gap-PCR), amplification refractory mutation system (ARMS)-PCR and direct sequencing were used for the detection of different mutations on the α-globin (HBA1 and HBA2) genes. A total of 11 different mutations was identified. The –α3.7 (rightward; NG_000006.1: g.34164_37967del3804) deletion was observed most frequently (56.35%), followed by α−5 ntα (HBA2: c.95+2_95+6delTGAGG), αpolyA2α (HBA2: c.*92A>G) and – –MED I (NG_000006.1: g.24664_41064del16401), with frequencies of 15.47, 9.39, and 6.08%, respectively. These four mutations accounted for more than 87.0% of the total mutated alleles. Moreover, 19 different genotypes were identified. The types and distribution pattern of the mutations identified in this study, in comparison with other studies conducted in Iran, was most similar to the Kurdish population of Kermanshah Province, Iran. Due to the lack of information on α-thal in Lorestan Province, it was not possible to compare the mutation spectrum in the Lur and Lak populations. In conclusion, our results may help in setting up a strategy for an α-thal screening program and genetic counseling in the Lak people.

Compound Heterozygote for a Novel Elongated C-Terminal β-Globin Variant (HBB: c.364delG) and Hb E (HBB: c.79G>A) with Heterozygous α-Thalassemia-2

This study reports the case of 2-year-old Northeastern Thai girl with β-thalassemia (β-thal) disease who has required regular blood transfusions since she was 8 months old. Hemoglobin (Hb) analysis by high performance liquid chromatography (HPLC) separated Hb A2/E (16.5%), Hb F (22.7%), Hb A (51.8%) and an abnormal peak (Hb X) found at a retention time (RT) of 5.05 min. (C-window) with 2.8%. Multiplex gap-polymerase chain reaction (gap-PCR) revealed heterozygous α-thalassemia-2 (α-thal-2) (–α3.7/αα; NG_000006.1: g.34164_37967 del3804). This patient was suspected of having a β-globin chain variant and Hb E (HBB: c.79G>A) according to the high Hb F level and disease presentations. Surprisingly, Hb Mahasarakham (the geographic origin of the proband), a novel single nucleotide deletion (–G) at the first nucleotide of codon 121 (HBB: c.364delG), was identified by direct DNA sequencing and secondary confirmation by PCR-restriction fragment length polymorphism (PCR-RFLP). This novel mutation causes a frameshift mutation and added 10 more residues to the β-globin chain that was elongated to 156 amino acids. Molecular basis of this novel mutation in the heterozygous state is required to confirm the mode of inheritance.

First Report of a Novel Deletion Due to εγδβ-Thalassemia in a Chinese Family

A fetus of Chinese descent presented with ultrasound features of anemia at 20 weeks’ gestation. Father had low a mean corpuscular volume (MCV) level. Multiplex gap-polymerase chain reaction (gap-PCR) excluded common α-thalassemia (α-thal) deletions and mutations and PCR sequencing of the α1- and α2-globin genes were negative. The fetus had a normal karyotype. Array comparative genomic hybridization (aCGH) showed a single copy loss of 189.87 kb in chromosome 11p15.4, involving the whole β-globin gene cluster, inherited from the father. Multiplex ligation-dependent probe amplification (MLPA) confirmed the deletion included the ε-globin gene, confirming the diagnosis of heterozygous (εγδβ)0-thalassemia [(εγδβ)0-thal], also inherited from the father. The fetus had a worsening anemic condition in utero and required a transfusion at 26 weeks’ gestation, raising the hemoglobin (Hb) level from 5.3 to 12.6g/dL. A cesarean-section was subsequently performed at 32 weeks’ gestation because of reduced fetal movements, and a 1650g baby girl with good Apgar scores was delivered. Hemoglobin at birth was 12.8g/dL, gradually dropping to 6.8 g/dL, requiring three neonatal transfusions. Her condition gradually stabilized after 2 months with Hb stable at 8.0 g/dL. Family screening by MLPA showed that the paternal grandmother carried the same deletion. The deletion in this case is distinct and is the reported first case. The deletion transmitted across three successive generations with great phenotypic variation. The final adult phenotype of (εγδβ)0-thal is usually mild, therefore, with accurate prenatal diagnosis this condition is salvageable by in utero and early neonatal transfusions, preventing adverse pregnancy and neonatal outcomes.

Hematological and Molecular Characterization of a Novel Hb A2 Variant with Homozygous α-Thalassemia-2 in a Southern Thai Individual

We report here the hematological and molecular features of a novel δ-globin chain variant found in a Southern Thai woman. Her complete blood count was as follows: red blood cell (RBC) count 5.90 × 1012/L, hemoglobin concentration (Hb) 12.6 g/dL, packed cell volume (PCV) 0.41 L/L, mean corpuscular volume (MCV) 69.5 fL, mean corpuscular Hb (MCH) 21.4 pg, mean corpuscular Hb concentration (MCHC) 30.7 g/dL and RBC distribution width (RDW) 13.1%. The blood smear demonstrated microcytic hypochromic RBCs suggestive of thalassemia trait. Hemoglobin analysis identified Hb A2 + Hb A2-Kiriwong (2.4%) and Hb F (0.1%) on high performance liquid chromatography (HPLC). To characterize the α-thalassemia (α-thal) genotype, common α-thal-1 and α-thal-2 alleles were characterized by multiplex gap-polymerase chain reaction (gap-PCR). The results revealed homozygous α-thal-2 (–α3.7/–α3.7) in this case. DNA sequencing showed the presence of a novel δ-globin gene mutation [δ77(EF1)His→Arg; HBD: c.233A>G] that we named Hb A2-Kiriwong for the village from where the proband lived. In summary, the presence of microcytic hypochromic RBCs in this case was likely the result of the homozygous –α3.7 (rightward) deletion and was not affected by this Hb A2 variant.

Molecular Study of Deletional and Nondeletional Mutations on the α-Globin Locus in the Azeri Population of Northwestern Iran

The aim of this study was to determine the molecular spectrum and frequency of deletional and nondeletional α-thalassemia (α-thal) mutations and the genotype–phenotype correlation in common mutations in the Azeri population of Northwestern Iran. A total of 1256 potential carriers with microcytic and hypochromic anemia and normal Hb A2 levels (<3.5%) and without iron deficiency anemia plus three fetuses were identified. Multiplex gap-polymerase chain reaction (gap-PCR) and sequencing for α-thal mutations were carried out. In 606 individuals, the α-globin gene was normal, but in 650 persons (51.6%) and three fetuses, 10 different mutations were detected. The most frequent deletional genotypes were as follows: αα/–α3.7 (61.7%), –α3.7/–α3.7 (11.9%), αα/–α4.2 (4.6%), αα/– –MED (4.3%) and αα/–(α)20.5 (3.8%). The most frequent nondeletional genotypes were αα/αIVS-I (–5 nt)α (HBA2: c.95+2_95+6delTGAGG) and αα/αPoly A2α [polyadenylation signal (polyA2) (AATAAA>AATGAA); HBA2: c.*96G>A] with frequencies of 1.08% and 0.92%, respectively. Meanwhile, 7.71% of individuals with a proven β-thalassemia (β-thal) mutation were found to also carry an α-thal mutation. Persons having two functional α-globin genes showed lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (Hb) (MCH) values compared to those with one mutated α-globin gene, provided that they had normal β-globin genes. Overall, the incidence of α-thal was 2.7% in the Azeri population in Northwestern Iran. Our results showed that the variability of α-thal mutations are high in the Azeri population and that α-thal mutations are highly heterogeneous in both deletional and nondeletional genotype aspects.

The –(α)5.2 Deletion Detected in a Uruguayan Family: First Case Report in the Americas

In Uruguay, α-thalassemia (α-thal) mutations were introduced predominantly by Mediterranean European immigrant populations and by slave trade of African populations. A patient with anemia with hypochromia and microcytosis, refractory to iron treatment and with normal hemoglobin (Hb) electrophoresis was analyzed for α-thal mutations by multiplex gap-polymerase chain reaction (gap-PCR), automated sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses. Agarose gel electrophoresis of the multiplex gap-PCR showed a band of unexpected size (approximately 700 bp) in the samples from the proband and mother. Automated sequencing of the amplified fragment showed the presence of the –(α)5.2 deletion (NG_000006.1: g.32867_38062del5196) [an α-thal-1 deletion of 5196 nucleotides (nts)]. The MLPA analysis of the proband’s sample also showed the presence of the –(α)5.2 deletion in heterozygous state. We report here the presence of the –(α)5.2 deletion, for the first time in the Americas, in a Uruguayan family with Italian ancestry, detected with a previously described multiplex gap-PCR.