Multiplex gap-PCR Research Articles

Diagnosis of Challenging Spinal Muscular Atrophy Cases with Long-Read Sequencing

Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disorder primarily caused by the deletion or mutation of the Survival Motor Neuron 1 (SMN1) gene. This study assesses the diagnostic potential of Long-Read Sequencing (LRS) in three SMA patients. For Patient 1, who has a heterozygous SMN1 deletion, LRS unveiled a missense mutation in SMN1 exon 5. In Patient 2, an Alu/Alu-mediated rearrangement covering the SMN1 promoter and exon 1 was identified through a blend of Multiplex Ligation-Dependent Probe Amplification (MLPA), LRS, and Gap-PCR. The third patient, born to a consanguineous family, bore four copies of hybrid SMN genes. LRS determined the genomic structures, indicating two distinct hybrids of SMN2 exon 7-SMN1 exon 8. However, a discrepancy was found between the SMN1-SMN2 ratio interpretations by LRS (0:2) and MLPA (0:4), which suggested a limitation of LRS in SMA diagnosis. In conclusion, this newly adapted long PCR-based third-generation sequencing introduces an additional avenue for SMA diagnosis.

‘Mind the gap’ in using multiplex gap-PCR for the diagnosis of alpha thalassaemia in Polynesian, Melanesian and Aboriginal populations

‘Mind the gap’ in using multiplex gap-PCR for the diagnosis of alpha thalassaemia in Polynesian, Melanesian and Aboriginal populations

A large cohort of deletional high hemoglobin F determinants in Thailand: A molecular revisited and identification of a novel mutation

Background and aimsHigh hemoglobin F determinants can be classified into hereditary persistence of fetal hemoglobin (HPFH) and δβ-thalassemia with different phenotype. We report the molecular basis and hematological features in a large cohort of deletional high Hb F determinants in Thailand. Materials and methodsSubjects (n = 28,177) encountered during 2015–2022 were reviewed, and those with phenotypically suspected of having high Hb F determinants were selected. Combined PCR, multiplex ligation-dependent probe amplification, next-generation sequencing, and DNA sequencing were used to identify the mutations. ResultsAmong 28,177 subjects investigated, 300 (1.06 %) were found to carry deletional high Hb F determinants in a total of 302 alleles, including heterozygote, compound heterozygote with β-hemoglobinopathies, and homozygote. DNA analysis identified eight different DNA deletions, including δβ0-thalassemia (12.6 kb deletion) (73.8 %), HPFH-6 (14.9 %), Indian deletion-inversion Aγδβ0-thalassemia (3.6 %), Thai deletion-inversion-insertion Aγδβ0-thalassemia (3.0 %), SEA-HPFH (3.0 %), Chinese Aγδβ0-thalassemia (1.0 %), Thai δβ0-thalassemia (11.3 kb deletion) (0.3 %), and a novel δβ0-thalassemia (137.1 kb deletion) (0.3 %). In addition, three novel genetic interactions, including Chinese Aγδβ0-thalassemia/Hb E, δβ0-thalassemia/Indian deletion-inversion Aγδβ0-thalassemia, and homozygous δβ0-thalassemia were found. Hematological features and Hb analysis results of 20 different genotypes were recorded. Multiplex gap-PCR assays for detection of these genetic determinants were described. ConclusionsDeletional high Hb F determinants are common and heterogeneous in Thailand. Data on the prevalence, molecular spectrum, phenotypic expression, and complex interactions of these genetic determinants should prove useful in the study and a prevention and control program of hemoglobinopathies in the region.

Clinical and haematological characteristics of 38 individuals with Hb G-Makassar in Malaysia.

Haemoglobin (Hb) G-Makassar is a rare Hb variant. It presents a diagnostic challenge as it imitates sickle Hb (Hb S) in standard electrophoresis and high-performance liquid chromatography assays requiring DNA analysis to confirm diagnosis. Both have point mutations in codon 6, exon 1 in the β-globin (HBB) gene with different pathogenicities. This study describes the clinical phenotype, haematology and genotype of Hb G-Makassar. Clinical and laboratory data of 38 cases of Hb G-Makassar over 8 years were analysed. Hb G-Makassar was confirmed by a direct sequencing of HBB gene and co-inheritance of α-thalassaemia determined through multiplex gap-PCR and multiplex Amplification Refractory Mutation System polymerase chain reaction. All cases were Malays, predominantly from Terengganu (n=20, 52.6%). There were 14 (36.8%) males and 24 (63.2%) females with median age of 25 years. Majority (n=33, 86.8%) had features of thalassaemia trait with mean±SD for Hb, mean cell volume (MCV) and mean cell haemoglobin (MCH) as 13.21g/dL±1.69, 73.06±4.48fL and 24.71±1.82pg, respectively. None had evidence of haemolysis or thromboembolic complications. Six genotypes were identified; ßG-Makassar/ß,αα/αα (n=19, 50.0%), ßG-Makassar/ßE,αα/αα (n=4, 10.5%), ßG-Makassar/ßNewYork,αα/αα (n=1, 2.6%), ßG-Makassar/ß,αα/-α (n=11, 28.9%), ßG-Makassar/ß,αα/αAdanaα (n=2, 5.3%) and ßG-Makassar/ß,αα/-SEA (n=1, 2.6%). The ßG-Makassar/ß,αα/αα showed that features of thalassaemia trait with mean±SD for Hb, MCV and MCH were 13.74g/dL±2.40, 76.18±6.02fL and 25.79±2.41pg, respectively. This is the largest study reporting a significant number of Hb G-Makassar in Malaysia. Although the mutation is similar to Hb S, the phenotype is benign.

Revisiting and updating molecular epidemiology of α-thalassemia mutations in Thailand using MLPA and new multiplex gap-PCR for nine α-thalassemia deletion

α-thalassemia is an inherited blood disorder that is most frequently found in Southeast Asian populations. In Thailand, molecular characterization can diagnose most patients with α-thalassemia; however, several atypical patients are also observed in routine analyses. Here, we characterized α-thalassemia mutations among 137 Hemoglobin H (Hb H) disease patients and three fetuses of Hb Bart’s hydrops, a fatal clinical phenotype of α-thalassemia. Specifically, we performed multiplex ligation-dependent probe amplification (MLPA) followed by direct DNA sequencing. We noticed common genotypes in 129 patients and eight patients had rare Hb H disease caused by compound heterozygous α0-thalassemia (--CR or --SA deletion) with α+-thalassemia (-α3.7/-α4.2/αConstant Springα). Furthermore, two affected fetuses had the --SA/--SEA and one had the --CR/--SEA genotypes. Next, we developed and validated a new multiplex gap-PCR and applied this method to 844 subjects with microcytic red blood cells (RBCs) from various parts of Thailand. The frequency of heterozygous α0-thalassemia was dominated by --SEA 363/844 (43%), followed by --THAI 3/844 (0.4%), --SA 2/844 (0.2%), and --CR 2/844 (0.2%) mutations. These findings suggest that aforementioned four mutations should be routinely applied to increase the effectiveness of diagnosis and genetic counseling in this region.

Diagnosis of α-thalassaemia by colorimetric gap loop mediated isothermal amplification

α-Thalassaemia is an inherited haemoglobin disorder that results from the defective synthesis of α-globin protein. Couples whom both carry the α-thalassaemia 1 gene are at risk of having a foetus with the most severe thalassaemia, Hb Bart’s hydrops fetalis, with a risk of maternal mortality. However, haematological parameters alone cannot distinguish between a α-thalassaemia 1 carrier and a homozygous α-thalassaemia 2, in which one α-globin gene has been deleted on each chromosome. A rapid and accurate molecular detection assay is essential for prevention of the disease in populations where α-thalassaemia 1 is common. Multiplex Gap-PCR analysis is widely used for diagnosis of α-thalassaemia. However, the technique requires a thermocycler and post-amplification processing, which limits its application in primary care or in rural areas in developing countries. Loop mediated isothermal amplification (LAMP) amplifies target DNA at a constant temperature and does not require a thermocycler. This study developed a colorimetric Gap-LAMP using malachite green to allow naked eye visualization of two deletional α-thalassaemia 1 commonly found in Asian populations, the Southeast Asian type (--SEA) and the Thai type (--THAI) deletions. The Gap-LAMP was performed on DNA samples from 410 individuals carrying various α-thalassaemia gene defects with 100% concordance with conventional Gap-PCR analysis. This method eliminates post-amplification processing or the use of expensive sophisticated equipment and allows screening large populations for the prevention and control of α-thalassaemia.

HbAdrian (α1:c.251del, p.Leu84Argfs*19)—A Novel Pathogenic Variant in the α1-Globin Gene Associated with Microcytosis from the North of Iran

Background: Alpha thalassemia is one of the most common human genetic abnormalities. More than 400 different variations of the α-globin protein have been introduced, most of which are not associated with noticeable clinical manifestations. The identification of all variants of Hb in different regions helps in acquiring comprehensive knowledge concerning thalassemia disease, and it can be used in preventive programs as well as prenatal diagnosis (PND). Aims: In the present study, we describe a new α1 gene mutation that leads to a frameshift after codon 83. Methods: As a plan for a national screening program of thalassemia, routine cell blood count (CBC) and Hb capillary electrophoresis tests were applied. After taking written informed consent, genomic DNA was extracted, and, for identifying common Mediterranean α-Globin gene deletion, multiplex Gap-PCR was performed; for detecting other mutations on α- and β-Globin genes, a DNA sequencing method was used. Results: The results of CBC and capillary electrophoresis tests showed microcytosis in a female subject. The sequencing of the α-Globin gene showed that the case is heterozygote for a single-nucleotide deletion at codon 83 of the α1-Globin Gene. We named this mutation Hb Adrian (α1: c.251–T), which is a novel mutation. The mentioned mutation was also detected in the subject’s mother. Conclusions: The introduced mutation (Hb Adrian) leads to a frameshift change that produces a protein with 100 amino acids, which in comparison to a normal α-chain is shorter, and its amino acids are altered after codon 83. This hemoglobin is undetectable via the use of electrophoresis. Although no major hematological abnormalities were observed in the carriers, Hb Adrian should be considered in screening programs to help prevent Hb H disease in high-risk couples.

Molecular and hematological studies in a cohort of beta zero South East Asia deletion (β°-thal SEA) from Malaysian perspective.

We report the haematological parameters and molecular characterization of beta zero (β°) South East Asia (SEA) deletion in the HBB gene cluster with unusually high levels of Hb F compared to a classical heterozygous beta zero (β°)-thalassaemia. Retrospective study on 17 cases of (β°) South East Asia (SEA) deletion from 2016 to 2019 referred to Institute for Medical Research were conducted. The clinical information and haematological profiles were evaluated. The mutation was analyzed, and the results were compared with other β°-thalassaemia groups. For HBB gene genotyping, all the cases were subjected for multiplex gap-PCR, 5 cases were subjected for HBB gene sequencing for exclusion of compound heterozygous with other beta variants. Co-inheritance of α-thalassaemia were determined using multiplex gap-PCR and multiplex ARMS-PCR. Seventeen cases were positive for β°-thal SEA deletion. Fifteen cases were heterozygous and two were compound heterozygous for β°-thal SEA deletion. The results were compared with 182 cases of various heterozygous β° deletions and mutations. The mean Hb for heterozygous β°-thal SEA deletion (13.44 ± 1.45 g/dl) was normal and significantly higher than heterozygous IVS 1-1 and Codon 41/42 (post hoc test, p < 0.05). The medians for the MCV and MCH of β°-thal SEA deletion were significantly higher than for all heterozygote β°-thalassaemia traits (Mann Whitney test, p < 0.05). Patients with β°-thal SEA deletion had elevated levels of Hb A2 consistent with β-thalassaemia traits, with Hb F levels consistent with HPFH or δβ-thalassaemia carriers. The median for Hb A2 was 4.00 + 1.00%, similar to that observed in other β°-thalassaemia groups except for IVS 1-1 mutation (median 5.30 + 0.45%) and β°-Filipino (∼45 kb deletion) deletion (median 6.00 + 0.58). Interestingly, we found that Hb F levels for β°-thal SEA deletion were statistically higher than other β°-thalassaemia mutations (median 19.00 + 5.50%, p < 0.05), except for the β°-thal 3.5 kb deletion group. We conclude that β°-thal SEA deletion has a unique haematological parameters of beta zero thalassaemia trait. We affirm toclassifying this deletion as SEA-HPFH based on previous studiesconsidering the phenotype features rather than the molecular defect of β°-thal SEA deletion, as this will make it easier to offer genetic counselling to affected individuals.

Haematological and Molecular Characterisation of Haemoglobin E with Deletional Alpha-Thalassaemia among Kelantan Subjects

Heterozygous haemoglobin E (Hb E) with deletional alpha (α)-thalassaemia is commonly seen in Southeast Asia including Kelantan, a Northeastern state of Malaysia. Studies in Malaysia showed that Hb E is the commonest among Malay Kelantan, Penang and Senoi group of Orang Asli [1-5].&#x0D; &#x0D; This study aimed to compare the haematological parameters (Hb, RBC, MCV, MCH, RDW and Hb E) among the heterozygous Hb E with deletional α-thalassaemia subjects. This study also intended to ascertain a cut-off value for heterozygous Hb E with deletional α-thalassaemia using automated capillary electrophoresis method. &#x0D; &#x0D; A cross-sectional study was conducted involving secondary data collection of 219 samples of heterozygous Hb E with possible α-thalassaemia from all districts of Kelantan. Full blood count (FBC) was analysed using Sysmex XN 3000 automated blood cell analyser (Kobe, Japan). Hemoglobin (Hb) analysis was performed using automated CE system (Capillarys2; Sebia, France). Molecular characterisation was performed using multiplex gap-PCR and ARMS-PCR to detect both deletional and non-deletional α-thalassaemia, respectively. Qualitative data were expressed as frequency and percentage while quantitative data were expressed as mean, ± SEM and median. One-way ANOVA was used to test for mean Hb level, RBC count, MCV level, MCH level, and RDW level differences among six groups of heterozygous Hb E with concurrent deletional alpha thalassaemia in this study. The ROC curve was used to determine the cut-off point for Hb E level.&#x0D; &#x0D; Eighty-nine samples of heterozygous Hb E were confirmed to have concurrent deletional α-thalassaemia. Heterozygous Hb E with heterozygous α3.7 gene deletion was the most common deletional α-thalassaemia observed in 56 samples (62.9%) and followed by heterozygous SEA gene deletion which were detected in 21 samples (23.6%). The haematological parameters showed there was a significant difference between groups. The cut-off point for Hb E level for predicting heterozygous Hb E with deletional α-thalassaemia from CE method was 24.7% (Table 1). The diagnostic performance for the cut-off point for Hb E level showed sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 100% for all. &#x0D; This cut-off value can be used in assisting the selection of heterozygous Hb E individual to proceed with DNA analysis for α-thalassaemia. &#x0D;

Detection of BCL11A, HMIP, and XmnI Polymorphisms among Anemic Pregnant Women in Hospital Universiti Sains Malaysia

BACKGROUND: Anemia is one of the most common conditions in women during pregnancy. Fetal hemoglobin (HbF) levels are usually &lt;1.0%. There are several genetic loci that have a significant influence on HbF levels. AIMS AND OBJECTIVES: The aim of this study is to determine the association of HbF level and DNA polymorphism at BCL11A rs1186868, HMIP rs9376090, and XmnI rs7482144 in anemic pregnant women. MATERIALS AND METHODS: Blood samples were collected from 164 anemic pregnant women at Obstetrics and Gynecology Clinic, Hospital Universiti Sains Malaysia. High-performance liquid chromatography was used to determine the HbF and HbA2 levels. RESULTS: Multiplex amplification-refractory mutation system-polymerase chain reaction (PCR) and gap-PCR were performed for 44 samples with a high HbA2 level (&gt;3.2%) and normal HbA2 level (≤3.2%) to detect mutation at b-globin gene cluster. Allelic discrimination for rs1186868, rs9376090, and rs7482144 was performed using the real-time PCR technique for samples with no mutation detected. The mean age of patients was 31 ± 5.9 years. The HbF and Hb levels showed a strong positive correlation (r = 0.61). Out of 22 samples, 15 mutations at the b-globin gene and no mutation at the δβ-globin gene were detected. The mean difference of HbF level of patients with and without b-globin gene cluster was 1.07 ± 0.6 and 1.79 ± 3.5, respectively. The minor allele frequency (MAF) for the studied single nucleotide polymorphisms as follows: rs1186868 (MAF = 0%), rs9376090 (MAF = 19%), and rs7482144 (MAF = 32.8%). CONCLUSION: In conclusion, the baseline HbF levels were unaltered by BCL11A, HMIP, and XmnI genetic polymorphisms.

Characterization of β-Globin Gene Cluster Deletions Using Multiplex-Gap Polymerase Chain Reaction (PCR) and Multiplex Ligation-Dependent Probe Amplification (MLPA)

Background : Deletions in the β-globin gene cluster are usually rare, problematic to detect, and subsequently possess a challenge in many diagnostic laboratories. They are normally related to the heterozygous of the delta beta (δβ) thalassemia, hereditary persistence of fetalhemoglobin (HPFH) and some of the hemoglobin variants. These disorders are typically presented by elevated levels of hemoglobin F (Hb F), but with low to normal hemoglobin A2 (Hb A2). However, despite their existence, there is still a limited number of studies focusing on this area, hence no definitive diagnosis could be conclusively established. Therefore, this pilot study was carried out to fill this knowledge gap.&#x0D; Methods: In this study, screening of the selected deletional mutations in the β-globin gene cluster among patients with Hb F (&gt;1%) and Hb A2 (&lt;4%) were performed using multiplex Gap-PCR and multiplex ligation-dependent probe amplification (MLPA).&#x0D; Results: The results showed that out of 54 samples tested using multiplex Gap-PCR against four target deletions; Thai (δβ)°-thalassemia, HPFH-6, Siriraj J and HbLepore, one sample was found positive with Thai (δβ)°-thalassemia. Further findings from the MLPA screening on 12 randomly selected samples revealed that another patient was positive with double deletions within the region of the β-globin gene cluster. These deletions occur at the gamma-globin gene 1 (HBG1) and gamma-globin gene 2 (HBG2) in exon 3.&#x0D; Conclusions: In conclusion, this study highlighted the importance of these deletions’ characterization using multiplex Gap-PCR and MLPA which helps in establishing a definitive diagnosis among this selected group of patients.&#x0D; Bangladesh Journal of Medical Science Vol.20(3) 2021 p.618-624

β-globin gene cluster mutations and large deletions among anaemic patients with elevated HbF level in a tertiary teaching hospital in Kelantan, Malaysia

Mutations in the β-globin gene cluster can lead to β-thalassaemia, δβ-thalassaemia, hereditary persistence of foetal haemoglobin (HPFH) and some of the haemoglobin variants. The clinical and haematological spectrum of thalassaemia range from benign to pathogenic conditions including severe transfusion dependent thalassaemia. Therefore, it is important to accurately diagnose β-globin gene cluster mutations to prevent thalassaemia major or intermedia offspring. The objective of this study is to detect β-globin gene cluster mutations and large deletions among anaemic patients with elevated HbF level in a tertiary teaching hospital in Kelantan, Malaysia. This study involved 144 anaemic patients with HbF level &gt;1.0%. High-Performance Liquid Chromatography (HPLC) was used to determine the HbF and HbA2 levels. Multiplex-ARMS (ARMS)-PCR and gap-PCR were performed for those patients with high HbA2 level (&gt;3.2%) and normal HbA2 level (≤3.2%) to detect β-globin gene cluster mutations and large deletions respectively. The majority of patients were Malays (99.3%) with mean age of 19.99 ± 1.64 years and female 61.1% predominance. Out of 87 samples tested using multiplex ARMS-PCR against eight targeted single mutation; Cd 41/42, IVS 1–5, Cd 26, Cd 17, Cd 71/72, IVS 1–1, Cd 8/9 and -28 mutations, 65 (74.7%) patients were detected which comprises of Cd 26 (56.3%), Cd 41/42 (11.5%), compound Cd 26 and Cd 41/42 (3.4%) and IVS 1–1 (3.4%). Meanwhile, for multiplex gap-PCR which detect four types of large deletions; Thai (δβ)o-thalassaemia, HPFH-6, Siriraj J and Hb Lepore, one out of 57 patients (1.8%) was found positive with Thai (δβ)o-thalassaemia. There was a significant difference between the mean of HbF level, MCV level as well as MCH level of patients with and without β-globin gene mutations and large deletions (P&lt;0.05). This study highlighted the presence of various types of β-globin gene cluster mutations detection in establishing a definitive diagnosis among this selected group of patients for the large-scale screening of the thalassaemia gene.

A report of Hb Fontainebleau [α21 (B2) Ala > Pro] as a result of founder effect phenomenon

ObjectiveTo date, more than 400 alpha chain variants or point mutations in the α-globin genes that lead to single amino acid substitutions have been described. Here, we state a rare alpha-globin gene variant Hb Fontainebleau [a21 (B2) Ala>Pro] identified in heterozygote state in two families from the north of Iran during premarital thalassemia screening program. MaterialTwo first cousin couples with reduced hematological indices compatible with α-thalassemia were referred to Lab for premarital screening of thalassemia. At first CBC and capillary electrophoresis tests were applied. For molecular investigation genomic DNA was isolated from peripheral blood samples. Then, multiplex-Gap PCR and Sanger sequencing techniques were applied to discover common α- globin deletions and probable point mutations, respectively. ResultsIn the capillary electrophoresis results, one unknown peak was observed to the left of Hb A. Sanger sequencing results revealed that all cases are heterozygote for Hb Fontainebleau (HBA2: c.64G4C, p.Ala21Pro). Family history analysis has shown that all presented cases have belonged to the families which are originally from India. ConclusionThe presence of Hb Fontainebleau in Mazandaran province and in subjects with Indian origin may indicate the founder effect phenomenon. DNA analysis of subjects with reduced hematological indices is recommended for finding common and rare mutations to predict childbirth with thalassemia in premarital screening programs.

Molecular prevalence of thalassemia and hemoglobinopathies among the Lao Loum Group in the Lao People's Democratic Republic.

Thalassemias and hemoglobinopathies are the most prevalent inherited anemias detected in South East Asians. These disorders represent not only a clinical health problem but also a socioeconomic problem for this region. Regarding the prevention and control of thalassemias and hemoglobinopathies in the Lao PDR, screening and diagnostic strategies should be strongly considered. The knowledge about the prevalence and molecular genotyping of thalassemias and hemoglobinopathies among the Lao Loum group, which includes the majority of Lao people, is now limited, making the prevention and control of thalassemias difficult. This study aimed to determine the prevalence of thalassemia among Lao Loum subjects of reproductive age. Multiplex gap PCR and direct sequencing were used to investigate the mutations of α-globin and β-globin genes. Thalassemias and hemoglobinopathies were detected in 154 of 354 (43.50%) patients, and 22 different genotypes were identified in this cohort. Remarkably, high frequencies of hemoglobin E, α0 -thalassemia (--SEA ), and α+ -thalassemia (-α3.7 ) were noted. A variety of hematologic features was observed, including co-inheritance of heterozygous HbE and heterozygous α-thalassemia, which was associated with significantly lower levels of MCV and MCH values than those observed in typical HbE heterozygotes. Female participants who were heterozygous for β0 or co-inheritance of heterozygous βE with heterozygous α-thalassemia exhibited mild anemia. Our data show that thalassemias and hemoglobinopathies have become health problems imposing a serious burden in the Lao PDR. Prevention programs aimed at decreasing the incidence of severe thalassemia diseases should be designed and initiated.

Influence of alpha thalassemia on clinical and laboratory parameters among nigerian children with sickle cell anemia.

There is paucity of data on the influence of alpha thalassemia on the clinical and laboratory parameters among Nigerian sickle cell anemia (SCA) patients. This study aimed to determine the prevalence of alpha thalassemia and the influence of alpha thalassemia on laboratory parameters and clinical manifestations in a group of young Nigerian SCA patients. This was a cross-sectional retrospective study conducted on 100 patients with SCA and 63 controls. The diagnosis of SCA was confirmed by DNA studies. Alpha thalassemia genotyping was performed by multiplex gap-PCR method. Laboratory parameters including complete blood count, hemoglobin quantitation, serum lactate dehydrogenase (LDH), and bilirubin were determined with standard techniques. Alpha thalassemia was found in 41 (41.0%) patients compared to 24 (38.1%) controls (P=0.744), and all were due to the 3.7κb α-globin gene deletions. Alpha thalassemia was associated with more frequent bone pain crisis, higher hemoglobin concentration, red blood cell count, and HbA2 level among the patients. On the contrary, patients with alpha thalassemia had lower mean corpuscular volume, mean corpuscular hemoglobin, and white blood cell count (WBC) (P˂0.05). There were 6 (6.0%) patients with leg ulcers, and none of them had alpha thalassemia, P=0.04. This study confirms that coexistence of alpha thalassemia with SCA significantly influences both the clinical and laboratory manifestations of young Nigerian SCA patients. The coexistence of this genetic modifier is associated with increased bone pain crisis and protects against sickle leg ulcers among the patients.

AN OBSERVATIONAL STUDY OF THE EFFECT OF HEMOGLOBINOPATHY, ALPHA THALASSEMIA AND HEMOGLOBIN E ON P. VIVAX PARASITEMIA

BackgroundThe protective effect of α-thalassemia, a common hematological disorder in Southeast Asia, against Plasmodium falciparum malaria has been well established. However, there is much less understanding of the effect of α-thalassemia against P. vivax. Here, we aimed to investigate the proportion of α-thalassemia including the impact of α-thalassemia and HbE on the parasitemia of P. vivax in Southeast Asian malaria patients in Thailand.MethodsA total of 210 malaria patients, admitted to the Hospital for Tropical Diseases, Thailand during 2011–2012, consisting of 159 Myanmeses, 13 Karens, 26 Thais, 3 Mons, 3 Laotians, and 6 Cambodians were recruited. Plasmodium spp. and parasite densities were determined. Group of deletion mutation (--SEA, −α3.7, −α4.2deletion) and substitution mutation (HbCS and HbE) were genotyped using multiplex gap-PCR and PCR-RFLP, respectively.ResultsIn our malaria patients, 17/210 homozygous and 74/210 heterozygous −α3.7 deletion were found. Only 3/210 heterozygous −α4.2 and 2/210 heterozygous--SEA deletion were detected. HbE is frequently found with 6/210 homozygotes and 35/210 heterozygotes. The most common thalassemia allele frequencies in Myanmar population were −α3.7 deletion (0.282), followed by HbE (0.101), HbCS (0.013), −α4.2 deletion (0.009), and --SEA deletion (0.003). Only density of P. vivax in α-thalassemia trait patients (−α3.7/−α3.7, --SEA/αα, −α3.7/−α4.2) but not in silent α-thalassemia (−α3.7/αα, −α4.2/αα, ααCS/αα) were significantly higher compared with non-α-thalassemia patients (p=0.027). HbE did not affect P. vivax parasitemia. The density of P. falciparum significantly increased in heterozygous HbE patients (p=0.046).ConclusionsAlpha-thalassemia trait is associated with high levels of P. vivax parasitemia in malaria patients in Southeast Asia.

Red Cell Exchange Transfusion in Sickle Cell Anaemia Patients at the University College Hospital, Ibadan, Nigeria

Level of HbA2 more than 4% is the reliable parameter to identify β- thalassemia carrier. However, in some cases the level is not typically increased hence leading to diagnostic dilemma. Thus the objectives of this study were to evaluate the existence of β- thalassemia among borderline HbA2 sample. Out of 11,790 samples received for thalassemia screening, 405 (3.4%) were found to have borderline HbA2 level. Out of this, 117(28.9%) samples were selected by simple random sampling for PCR. Multiplex ARMS-PCR was used to detect β-globin gene mutation and multiplex gap PCR for α-globin genes deletion. The result revealed 36 (30.8%) had β-globin gene mutations, 8 (6.8%) had α-globin gene deletions and 1 (0.9%) had coexistence of α and β-globin gene defects. The commonest gene mutation detected were CD 19 (A-G), detected from 17 (45.9%) samples, followed by 9 (24.3%) with IVS 1-1 (G-A) mutation, 5 (13.5%) with Poly A mutation and 1 (2.7%) showed CAP +1 (A-C) mutation. Two samples (5.4%) showed mutations of Poly A and CD19, 2 (5.4%) showed mutation of IVS 1-1 and CD 19 while 1 (2.7%) showed IVS 1-5 and CD 19 mutations. This study showed 10 (27.0%) positive molecular results even though the HbA2 level was at only 3.0%. There was significant number of people with HbA2 between 3.0% and 3.9% and confirmed as thalassemia carriers by PCR. This data may suggest the level of HbA2 need to be revised to ensure the success of thalassemia screening programme in our population.

Results of thalassemia screening and genetic diagnosis for 13 738 pregnant women

To report on the result of thalassemia screening and genetic diagnosis for pregnant women from Guiyang region. Prenatal screening for thalassemia was carried out based on erythrocyte parameters and hemoglobin electrophoresis. Single-tube multiplex GAP-PCR and PCR-reverse dot blot hybridization were performed on suspected cases to identify common alpha- and beta- thalassemia mutations, and direct sequencing was used for identifying rare mutations. Among 13 738 pregnant women, 1745 (12.70%) were suspected as thalassemia. In terms of native place, the provinces with highest screening-positive rates were Guangxi, Guangdong, Jiangxi and Guizhou. And the ethnic groups with highest screening-positive rates were Zhuang, Li, and Buyi. Among 801 women subjected to genetic testing, 457 (57.05%) were diagnosed with thalassemia. In total 9 genotypes of alpha- thalassemia were detected, with the most common genotypes being --SEA/alpha alpha (63.35%), - alpha3.7/alpha alpha (19.37%) and - alpha4.2/alpha alpha (8.90%). Eleven genotypes of beta- thalassemia were detected, with the most common genotypes being CD17/N (42.91%), CD41-42/N (32.46%) and IVS-II-654/N (11.94%). Two cases were detected with rare beta-thalassemia mutations (CD54-58/N and IVS-I-130/N). The screening-positive rate of thalassemia among pregnant women in Guiyang region is relatively high. The rates have shown substantial difference in terms of native place and ethnic group. Thalassemia-related mutations in Guizhou region have a diverse spectrum, which showed certain difference from those of other regions.

Design, Validation, and Clinical Implementation of a Gap-Polymerase Chain Reaction Method for α-Thalassemia Genotyping Using Capillary Electrophoresis

α-Thalassemia (α-thal) is genetically heterogeneous with most cases caused by variably sized deletions of the HBA1 and/or HBA2 loci. In this report, we describe the development, validation, and implementation of a novel gap-polymerase chain reaction (gap-PCR)/capillary electrophoresis (CE). method. This assay utilizes two multiplex reactions and CE to detect the following deletions: –α3.7 (rightward), –α4.2 (leftward), –(α)20.5, – –SEA (Southeast Asian), – –MED, – –FIL and – –THAI. Validation studies using 36 previously characterized patient samples and plasmid controls demonstrated 100.0% accuracy. Following clinical implementation, 423 patients were analyzed over 24 months. Two hundred and twenty-seven cases (46.0%) showed abnormal results including heterozygous –α3.7 (n = 114, 27.0%), homozygous –α3.7 (n = 96, 23.0%), heterozygous – –SEA (n = 9, 2.0%), heterozygous –α 4.2 (n = 5, 1.0%), heterozygous – –MED (n = 1, <1.0%), and compound heterozygous –α3.7/–α4.2 (n = 2, <1.0%) deletions. Correlation with red blood cell (RBC) parameters showed that patients with a deletion of two or more genes were associated with significantly lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin (Hb) (MCH) levels than patients with wild-type results. This novel multiplex gap-PCR protocol reliably detects the seven most common deletions giving rise to α-thal. Use of the fluorescently labeled CE method provides for a high throughput workflow suitable to a clinical diagnostic laboratory serving a multiethnic population.

The prevalence and genotypes of alpha-thalassemia in Adıyaman: two rare alpha variants

Abstract Introduction Alpha-thalassemia (α-thal) is one of the most common diseases in the world, and is more common in the Mediterranean countries, the Middle East, Southeast Asia, and Africa. The aim of this study was to determine the prevalence and genotypes of α-thal in high school students in Adıyaman province, Southeast Turkey. Methods In this study, a total of 289 people were screened for α-thal mutations by multiplex gap-PCR and ARMS. Results α-thal mutation frequency was determined to be 13.15%. Five different mutations were identified and the most common mutation was found to be −3.7 deletions with a frequency of 11.07%. We identified two heterozygous alpha hemoglobin variants. We described the second case of heterozygote Hb Fontainebleau of Turkish origin. Here, we also reported another heterozygote hemoglobin variant, Hb Q-Iran, previously reported in a few cases in another part of Turkey. Discussion and conclusion This study covers the frequency of α-thal disease, and the molecular analysis is the first study in the Southeastern Anatolia Region of Turkey. The prevalence of the α-thal trait is low in Adıyaman Province, compared to the other cities of Turkey.