Gap-polymerase Chain Reaction Research Articles

Analysis of Cut-off Value in Screening of Thalassemia by Capillary Hemoglobin Electrophoresis for Pregnant Women from Shenzhen Region of China

To investigate the cut-off value in screening of thalassemia in pregnant women from Shenzhen region by capillary hemoglobin electrophoresis. The data of capillary hemoglobin electrophoresis and genetic diagnosis of thalassemia from 2122 examined prenatal women were retrospectively analyzed. Capillary hemoglobin electrophoresis and α-, β- genetic diagnosis of thalassemia were carried out for every woman. Hemoglobin electrophoresis was performed using Capillarys 2 full-automated electrophoresis instrument. Gap polymerase chain reaction and reverse dot blot were used for genetic diagnosis of thalassemia genotyping test. The cut-off value in screening of thalassemia was determined by receiver operating characteristic curve and next to analyze the value of HbA2 and HbF in screening of thalassemia using the decided cut-off value. The areas under the curve (AUC(Roc)) of HbA2 for diagnosis of α-, β- thalassemia were 0.75 and 0.981 respectively, and the AUC(Roc) of HbF for diagnosis of β-thalassemia was 0.787. When HbA2 ≤ 2.55 was taken as the cut-off value of HbA2 for diagnosis of α-thalassemia, the sensitivity, specificity, positive likelihood ratio (LR(+)) and negative likelihood ratio (LR(-)) were 89.5%, 54.8%, 1.98, 0.19 respectively. When HbA2 ≥3.9 was taken as the cut off value of HbA2 for diagnosis of β-thalassemia, the sensitivity, specificity, LR(+) and LR(-) were 96.1%, 99.8% 480.5, 0.04 respectively. When HbF ≥0.75 was taken as the cut off value of HbF for diagnosis of β-thalassemia, the sensitivity, specificity, LR(+) and LR(-) were 83.6%, 61.8% respectively. The cut-off value in screening of thalassemia by capillarys 2 full automated electrophoresis instrument is different from that of the traditional method of hemoglobin electrophoresis, such as cellulose acetate membrane electrophoresis and agarose gel electrophoresis. Each laboratory should establish their own respective cut off value.

The Natural History of Hb S/Hereditary Persistence of Fetal Hemoglobin in 13 Children from the State of Minas Gerais, Brazil

Children with Hb S (HBB: c.20A > T)/hereditary persistence of fetal hemoglobin (Hb S/HPFH) have a mild clinical phenotype, but some complications have been reported. The natural history of Hb S/HPFH in children from the State of Minas Gerais, Brazil newborn cohort is described. Clinical and hematological data regarding participants’ phenotypes were retrieved from medical records. The HPFH-1, HPFH-2, and HPFH-3 and α-thalassemia (α-thal) deletions were detected by gap-polymerase chain reaction (gap-PCR). Thirteen children were included, nine (69.2%) had the Hb S/HPFH-2 deletion, and four (30.8%) had Hb S/HPFH-1 deletion; 11 children (84.6%) had αα/αα, and two (15.4%) carried the αα/−α3.7 (rightward) deletion. The mean concentration of total hemoglobin (Hb) and Hb F was 12.52 ± 0.56 g/dL and 42.31% ± 1.97%, respectively. Mild microcytosis and hypochromia were observed. We found acute clinical manifestations of sickle cell disease, such as acute chest syndrome (ACS) and acute pain crisis in four children; nine (69.2%) children were completely asymptomatic during the follow-up period. All children were classified as having low-risk transcranial Doppler (TDC). In conclusion, children with Hb S/HPFH have a mild clinical phenotype of sickle cell disease, although acute clinical manifestations may occur. High Hb F levels and absence of anemia are common hematological characteristics.

Hemoglobinopathies in the Çukurova Region and Neighboring Provinces

To contribute to the creation of a mutation map of the region, we aimed to determine the mutation spectrum of thalassemias and abnormal hemoglobins (Hbs) in the Çukurova region and surrounding provinces. In this study, a total of 8135 samples from Adana, Hatay, Mersin, Konya and Kayseri provinces between 1993 and 2014 were analyzed. Complete blood cell (CBC) counts and Hb typing were carried out using automatic cell counters, cellulose acetate membrane electrophoresis and high performance liquid chromatography (HPLC), respectively. For the molecular analyses, genomic DNA was extracted using both manual and automated DNA extraction devices. Determination of Hb mutations were done by microarray, restriction fragment length polymorphism (RFLP), amplification refractory mutation system (ARMS) and gap-polymerase chain reaction (gap-PCR) methodologies. Samples were analyzed for abnormal Hb and thalassemia mutations. Out of 8135 samples, 1382 were observed to be carrying Hb mutations. It was identified that 826 mutation carriers included abnormal Hbs with a frequency of 59.7%, 416 carriers included β-thalassemia (β-thal) mutations with a frequency of 30.7% and 136 carriers included α-thalassemia (α-thal) mutations with a frequency of 9.9%. In this study, the most frequently observed abnormal Hb in the region was Hb S [β6(A3)Glu→Val (GTG > GAG), HBB: c.20T > A], whereas the most commonly observed mutations were the IVS-I-110 (G > A) (HBB: c.93–21G > A) point mutation in β-thal and the 3.7 kb deletion in α-thal.

Evaluation of α-Globin Gene Mutations Among Different Ethnic Groups in Khuzestan Province, Southwest Iran

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

Impact of null genotypes of GSTT1 and GSTM1 with uterine leiomyoma risk in Iranian population.

Few studies have investigated the role of the GSTM1 and GSTT1 genes in uterine leiomyoma. Therefore, in the current study the distribution of these genotypes in Iranian women and susceptibility to uterine leiomyoma was investigated. Blood samples of 50 patients with uterine leiomyoma and 50 healthy individual controls were collected in this cross-sectional study. Genomic DNA was extracted, and subsequently GSTM1 and GSTT1 null genotypes were detected by the Gap-polymerase chain reaction method. A total of 42% of patients appeared to lack GSTM1 enzyme activity due to the presence of an extended deletion (GSTM1 0/0 genotype), compared with 18% in a control group (odds ratio [OR], 3.56; 95% confidence interval [CI], 1.35-9.37; P < 0.010). In addition, the prevalence of the GSTT1 null genotype in patients was higher than that in the control group (42% to 14%, P < 0.009). Also, it was shown that individuals with both null genotypes (-/-) had a 19.23-fold higher risk of developing the disease in comparison to people who showed both present genotypes (+/+). (P = 0.007; 95%CI, 2.20-167.41). Besides, it was observed that at least one null genotype increases the risk of myoma to 2.6 compared to the both present genotype (P-value < 0.03, 95%CI, 1.05-6.82). To our knowledge, this is first significant correlation between risk of uterine leiomyoma and null GSTM1 and GSTT1 genotypes among Iranian patients. Our data support the involvement of GSTM1 and GSTT1 in uterine leiomyoma liability, and especially its role as a genetic factor in the occurrence of this disease.

Analysis of α1 and α2 globin genes among patients with hemoglobin Adana in Malaysia.

Hemoglobin (Hb) Adana [HBA2: c179G>A (or HBA1); p.Gly60Asp] is a non-deletional α-thalassemia variant found in Malaysia. An improvement in the molecular techniques in recent years has made identification of Hb Adana much easier. For this study, a total of 26 Hb Adana α-thalassemia intermedia and 10 Hb Adana trait blood samples were collected from patients. Common deletional and non-deletional α-thalassemia genotypes were determined using multiplex gap polymerase chain reaction (PCR) and multiplex ARMS PCR techniques. Identification of the Hb Adana location on the α-globin gene was carried out using genomic sequencing and the location of the mutation was confirmed via restriction fragment length polymorphism-PCR. Among the 36 samples, 24 (66.7%) had the -α(3.7)/α(Cd59)α mutation, while the -α(3.7)/α(Cd59)α mutation accounted for 2 samples (5.6%) and the remaining 10 (27.8%) samples were α/α(Cd59)α. All 36 samples were found to have the Hb Adana mutation on the α2-globin gene. The position of the α-globin gene mutation found in our cases was similar to that reported in Indonesia (16%) but not to that in Turkey (0.6%). Our results showed that the Hb Adana mutation was preferentially present in the α2-globin genes in Malays compared to the other ethnicities in Malaysia. Thus, the Malays might have similar ancestry based on the similarities in the Hb Adana position.

A Fetus with Hb Bart’s Disease Due to Maternal Uniparental Disomy for Chromosome 16

We here report an unusual case of Hb Bart’s (γ4) disease. Thalassemia screening of a couple showed that the wife was an α0-thalassemia (α0-thal) carrier and her husband’s mean corpuscular volume (MCV) was normal. Chorionic villus sampling (CVS) was performed at 13 weeks’ gestation for positive Down syndrome screening and chromosomal study of the cultured CVS showed a normal karyotype. Ultrasound examination at 22 weeks’ gestation showed fetal cardiomegaly and raised middle cerebral artery peak systolic velocity. Cordocentesis confirmed fetal anemia and showed Hb Bart’s disease. Multiplex gap-polymerase chain reaction (gap-PCR) for α-thal deletions on DNA extracted from the CVS showed the presence of a homozygous α0-thal − −SEA (Southeast Asian) deletion. The husband was found to be a carrier of the α+-thal −α3.7 (rightward) deletion. Non paternity was excluded by fluorescent PCR using short tandem repeat (STR) markers on chromosomes 13, 18 and 21. A de novo terminal deletion of chromosome 16 was excluded by array comparative genomic hybridization (aCGH). Detection of uniparental disomy (UPD), using STR markers on chromosome 16 showed maternal uniparental isodisomy from 16pter to 16p13.2, and uniparental heterodisomy from 16p13.13 to 16qter.

Application of multiplex ligation-dependent probe amplification technique in prenatal diagnosis of α-thalassemia

OBJECTIVE To assess the application value of multiplex ligation-dependent probe amplification (MLPA) for the detection of gene deletion and prenatal diagnosis of α-thalassemia. METHODS MLPA was applied for 2 cases with α-thalassemia phenotype by whole blood cell counting and hemoglobin component detection but were ruled out by regular molecular diagnosis. Potential gene deletions and point mutations of α-thalassemia gene were detected with regular Gap-polymerase chain reaction (Gap-PCR) and reverse dot blotting (RDB) in 89 cases where one or both partners were carriers of α-thalassemia mutations. Meanwhile, MLPA was used for detecting α-globin gene deletion among the 89 samples. RESULTS For the 2 cases with α-thalassemia phenotype, no α globin gene deletion was detected by MLPA, but were subsequently confirmed as iron-deficiency anemia. The results of MLPA and Gap-PCR detection for the 88 cases were consistent, except for 1 fetal sample (chorionic villi) which could not be diagnosed by Gap-PCR and was confirmed to be - SEA/αα by MLPA. CONCLUSION MLPA can be applied to prenatal diagnosis of α-thalassemia as an effective supplement to Gap-PCR to reduce both misdiagnosis and missed diagnosis and improve the accuracy of prenatal diagnosis.

A novel gap-PCR with high resolution melting analysis for the detection of α-thalassaemia Southeast Asian and Filipino β°-thalassaemia deletion.

Homozygosity for the α-thalassaemia Southeast Asian (α-SEA) and Filipino β0-thalassaemia (β-FIL) deletions can cause serious complications leading to foetal death or life-long blood transfusions. A rapid and accurate molecular detection assay is essential in populations where the deletions are common. In this study, gap-polymerase chain reaction (PCR) with high resolution melting (HRM) analysis was developed to detect both the large deletions. Melting curves at 86.9 ± 0.1 °C were generated by normal individuals without the α-SEA deletion, 84.7 ± 0.1 °C by homozygous α-SEA deletion individuals and two melting curves at 84.7 ± 0.1 °C and 86.9 ± 0.1 °C by α-SEA deletion carriers. Normal individuals without the β-FIL deletion produce amplicons with a melting temperature (Tm) at 74.6 ± 0.1 °C, homozygous β-FIL individuals produce amplicons with Tm at 73.6 ± 0.1 °C and heterozygous β-FIL individuals generate two amplicons with Tm at 73.6 ± 0.1 °C and 74.6 ± 0.1 °C. Evaluation using blinded tests on 220 DNA samples showed 100% sensitivity and specificity. The developed assays are sensitive and specific for rapid molecular and prenatal diagnosis for the α-SEA and β-FIL deletions.

Molecular Epidemiological Investigation of Thalassemia in the Chengdu Region, Sichuan Province, Southwest China

Thalassemia is the most common inherited disease in southern China. However, this disorder is usually ignored by the health system in the Sichuan Province due to the lack of epidemiological data. To provide basic epidemiological data for thalassemia screening, genetic counseling, and prenatal diagnosis (PND) in the Chengdu region, a total of 3262 healthy subjects were assessed by complete blood count (CBC), reverse dot-blot gene chip, gap-polymerase chain reaction (gap-PCR), and PCR-DNA sequencing. A frequency of heterozygous thalassemia of 3.43% (112/3262) was found, of which 2.21% (72/3262) patients carried α-thalassemia (α-thal), 1.19% (39/3262) β-thalassemia (β-thal) and 0.3% (1/3262) hereditary persistence of fetal hemoglobin (Hb) (HPFH)/δβ-thalassemia (δβ-thal). Four types of α-thal mutations were found, the most prevalent being – –SEA (68.06%), followed by −α3.7 (rightward deletion, 25.0%), Hb Quong Sze (Hb QS; HBA2: c.377 T > C) (4.17%), and −α4.2 (leftward deletion, 2.78%). The seven β-thal mutations included: codons 41/42 (−TTCT), HBB: c.126_129delCTTT (13/39, 33.33%); codon 17 (A > T), HBB: c.52 A > T (11/39, 28.95%); IVS-II-654 (C > T), HBB: c.316-197 C > T (9/39, 23.68%); −28 (A > G), HBB: c.–78 A > G (3/39, 7.69%); −29 (A > G), HBB: c.–79 A > G (1/39, 2.56%); codons 27/28 (+C), HBB: c.84_85insC (1/39, 2.56%), and the rare IVS-II-850 (G > T), HBB: c.316-1 G > T (1/39, 2.56%). Only one case of the Southeast Asian HPFH deletion was found. This is the first detailed molecular epidemiological survey of thalassemia in the Chengdu region, Sichuan Province, People’s Republic of China (PRC).

Frequencies of HKαα and anti-HKαα Alleles in Chinese Carriers of Silent Deletional α-Thalassemia

The HKαα (HongKongαα) allele is an unusual rearrangement of the α-globin gene cluster containing both the −α3.7 (rightward) and αααanti 4.2 crossover deletion/duplication. The anti-HKαα (anti-HongKongαα) allele is the reciprocal product containing both the −α4.2 (leftward) and αααanti 3.7 unequal crossover deletion/duplication. In clinical practice of thalassemia screening, gap-polymerase chain reaction (gap-PCR) approaches are used to detect the common −α3.7 and −α4.2 deletions of α-thalassemia (α-thal). Because the HKαα and anti-HKαα alleles also contain the single α-globin gene deletion, individuals with these alleles would be misdiagnosed as −α3.7 or −α4.2 carriers. This would likely produce misleading or incorrect information in genetic counseling. In this study, we investigated the HKαα and anti-HKαα alleles in Chinese carriers of silent deletional α-thal, and reported their frequencies to be 2.27 and 0.35% in −α3.7 and −α4.2 carriers, respectively. Given the rarity of the HKαα and anti-HKαα alleles, a routine screening for these two rearrangements are unlikely to be necessary on most occasions.

A Novel Single Gene Deletion (−αMAL3.5) Giving Rise to Silent α Thalassemia Carrier Removing the Entire HBA2 Gene Observed in Two Chinese Patients with Hb H Disease: Case Report of Two Probands

We report a novel deletion at the HBA2 presented with Hb H disease in two Malaysian- Chinese patients. The two unrelated probands were diagnosed with Hb H disease in a primary hematological screening for thalassemia. Results from routine molecular analysis with gap-polymerase chain reaction (PCR) method revealed a genotype asynchrony with the observed clinical presentation. Subsequent DNA analysis using a battery of molecular methods such as gap-PCR, multiplex ligation dependent probe amplification, DNA sequencing, confirmed the presence of a novel deletion in both the index cases removing the entire α2 globin gene. We have designated the deletion as (−αMAL3.5). Hematological indices and clinical findings suggest that the deletion has an α+ phenotype. The molecular process of this deletion is the result from misalignment and unequal crossover event between the duplicated homologous Y-boxes within the α globin gene cluster. Uncharacterized deletions, single nucleotide polymorphism and other nucleotide indels at the primer binding sites may impede the optimum condition for its annealing and extension and therefore may invalidate the gap-PCR obscuring the real genotype.

Compound Heterozygosity for HKαα and an in Cis Deletion of Double α Genes Presents as α-Thalassemia Trait

The HKαα (Hong Kongαα) allele is an unusual rearrangement of the α-globin gene cluster containing both the –α3.7 (rightward) and αααanti 4.2 crossover deletion/insertion. During our thalassemia screening program, we identified 10 adult individuals and two newborns who were confirmed to be compound heterozygotes for HKαα and the Southeast Asian deletion (– –SEA). Their hematological data showed a typical α-thalassemia (α-thal) trait. The routine gap-polymerase chain reaction (gap-PCR) based assay revealed the presence of –α3.7, – –SEA and normal α2 alleles in the α-globin gene clusters. These confusing findings indicated the existence of more complex derivative alleles produced possibly by repeated unequal crossover of recombinant alleles between α-globin gene clusters. A two-round nested PCR strategy confirmed the diagnosis of HKαα. Considering the large-scale population screening in the thalassemia-prevalent regions in China, the current diagnostic strategy might need to be modified accordingly. The detection of HKαα would improve accuracy in genetic counseling, especially in couples where one partner was a – –SEA carrier and the other carries a –α3.7 deletion identified by routine gap-PCR methods.

Medium-based noninvasive preimplantation genetic diagnosis for human α-thalassemias-SEA.

To develop a noninvasive medium-based preimplantation genetic diagnosis (PGD) test for α-thalassemias-SEA.The embryos of α-thalassemia-SEA carriers undergoing in vitro fertilization (IVF) were cultured. Single cells were biopsied from blastomeres and subjected to fluorescent gap polymerase chain reaction (PCR) analysis; the spent culture media that contained embryo genomic DNA and corresponding blastocysts as verification were subjected to quantitative-PCR (Q-PCR) detection of α-thalassemia-SEA. The diagnosis efficiency and allele dropout (ADO) ratio were calculated, and the cell-free DNA concentration was quantitatively assessed in the culture medium.The diagnosis efficiency of medium-based α-thalassemias–SEA detection significantly increased compared with that of biopsy-based fluorescent gap PCR analysis (88.6% vs 82.1%, P < 0.05). There is no significant difference regarding ADO ratio between them. The optimal time for medium-based α-thalassemias–SEA detection is Day 5 (D5) following IVF.Medium-based α-thalassemias–SEA detection could represent a novel, quick, and noninvasive approach for carriers to undergo IVF and PGD.

Molecular analysis of beta-globin gene mutations among Thai beta-thalassemia children: results from a single center study

BackgroundBeta-thalassemia is one of the most common genetic disorders in Thailand. Clinical phenotype ranges from silent carrier to clinically manifested conditions including severe beta-thalassemia major and mild beta-thalassemia intermedia.ObjectiveThis study aimed to characterize the spectrum of beta-globin gene mutations in pediatric patients who were followed-up in Phramongkutklao Hospital.Patients and methodsEighty unrelated beta-thalassemia patients were enrolled in this study including 57 with beta-thalassemia/hemoglobin E, eight with homozygous beta-thalassemia, and 15 with heterozygous beta-thalassemia. Mutation analysis was performed by multiplex amplification refractory mutation system (M-ARMS), direct DNA sequencing of beta-globin gene, and gap polymerase chain reaction for 3.4 kb deletion detection, respectively.ResultsA total of 13 different beta-thalassemia mutations were identified among 88 alleles. The most common mutation was codon 41/42 (-TCTT) (37.5%), followed by codon 17 (A>T) (26.1%), IVS-I-5 (G>C) (8%), IVS-II-654 (C>T) (6.8%), IVS-I-1 (G>T) (4.5%), and codon 71/72 (+A) (2.3%), and all these six common mutations (85.2%) were detected by M-ARMS. Six uncommon mutations (10.2%) were identified by DNA sequencing including 4.5% for codon 35 (C>A) and 1.1% initiation codon mutation (ATG>AGG), codon 15 (G>A), codon 19 (A>G), codon 27/28 (+C), and codon 123/124/125 (-ACCCCACC), respectively. The 3.4 kb deletion was detected at 4.5%. The most common genotype of beta-thalassemia major patients was codon 41/42 (-TCTT)/codon 26 (G>A) or betaE accounting for 40%.ConclusionAll of the beta-thalassemia alleles have been characterized by a combination of techniques including M-ARMS, DNA sequencing, and gap polymerase chain reaction for 3.4 kb deletion detection. Thirteen mutations account for 100% of the beta-thalassemia genes among the pediatric patients in our study.

Novel Point Mutation of the α2-Globin Gene (HBA2) and a Rare 2.4 kb Deletion of the α1-Globin Gene (HBA1), Identified in Two Chinese Patients with Hb H Disease

Two Chinese patients with mild and moderate Hb H disease were investigated for rare mutations on the α-globin genes (HBA1, HBA2) in addition to the – –SEA deletion. One patient was a 41-year old man with mild anemia (Hb 11.3 g/dL). Multiplex ligation-dependent probe amplification (MLPA) revealed a rare 2392 bp deletion involving the entire HBA1 gene. Mapping by gap-polymerase chain reaction (gap-PCR) defined the exact breakpoints of this deletion (HBA1: g36859_39252del2392) and confirmed its identity with a recently reported HBA1 deletion found in a Southern Chinese. The other patient was a 53-year old man with moderate anemia (Hb 9.5 g/dL). Automated direct nucleotide (nt) sequencing identified a novel single nt deletion at codon 40 (HBA2: c.123delG). This leads to a frameshift that modifies the C-terminal sequence to (40)Lys-Pro-Thr-Ser-Arg-Thr-Ser-Thr(47)COOH and the introduction of a stop codon TGA 23 nts downstream. These two cases demonstrate the power of MLPA and direct nt sequencing to detect and characterize rare and novel mutations. They also highlight the differential effect of HBA1 and HBA2 gene mutations on an α-thalassemia (α-thal) phenotype due to their different transcriptional activity.

The Value of Red Blood Cells (Rbc) Indices and Osmotic Fragility Test as Screening Tests in Malay Pregnant Women with Alpha Thalassaemia

Red blood cell (RBC) parameters obtained from hematology analyser has been very useful as the first line screening for thalassaemia. The changes are prominent in most type of β-thalassaemia but milder in α-thalassaemia. RBC parameters could have been normalized especially during pregnancy and in such situation screening these patients would require a different protocol. Blood samples from two hundred (200) Malay pregnant women attended obstetric clinic at Hospital Universiti Sains Malaysia (HUSM) were collected to screen for double α-gene deletion (-SEA, and –THAI), and the two single α-globin gene deletion (–α3.7 and -α4.2). Standard hematological analyses including red blood cell count and indices, and hemoglobin quantitation were performed on the blood samples. Of these, sixteen were excluded as they had HbA2 levels more than 4% and were diagnosed as HbE or β-thalassaemia trait. Then, multiplex GAP polymerase chain reaction (PCR) analyses of α-globin gene were performed on the remaining 184 blood samples. Total of 17 from 184 subjects confirmed to have α-thalassaemia (-α3.7/αα and --SEA/ αα genotype). The RBC indices were compared between those with α-thalassaemia and normal pregnant women and they were significantly different. -α3.7 kb single gene deletion (8.1%) was the commonest type followed by double gene South East Asia (--SEA) deletion (1.1%). We conclude that mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) with the cut off value of less than 86.3fl and 27.4 pg respectively are two useful RBC indices which can be used for screening of α-thalassaemia in pregnant women.

Thalassemia and hemoglobin e in southern thai blood donors.

Thalassemia and hemoglobin E (Hb E) are common in Thailand. Individuals with thalassemia trait usually have a normal hemoglobin concentration or mild anemia. Therefore, thalassemic individuals who have minimum acceptable Hb level may be accepted as blood donors. This study was aimed at determining the frequency of α-thalassemia 1 trait, β-thalassemia trait, and Hb E-related syndromes in Southern Thai blood donors. One hundred and sixteen voluntary blood donors, Southern Thailand origin, were recruited for thalassemia and Hb E screening by red blood cell indices/dichlorophenolindophenol precipitation test. β-Thalassemia and Hb E were then identified by high performance liquid chromatography and 4 common α-thalassemia deletions were characterized by a single tube-multiplex gap-polymerase chain reaction. Overall frequency of hemoglobinopathies was 12.9%, classified as follows: homozygous α-thalassemia 2 (1.7%), heterozygous α-thalassemia 1 (1.7%), heterozygous β-thalassemia without α-thalassemia (0.9%), heterozygous Hb E without α-thalassemia (5.2%), double heterozygotes for Hb E/α-thalassemia 1 (1.7%), homozygous Hb E without α-thalassemia (0.9%), and homozygous Hb E with heterozygous α-thalassemia 2 (0.9%). The usefulness of thalassemia screening is not only for receiving highly effective red blood cells in the recipients but also for encouraging the control and prevention program of thalassemia in blood donors.

Mitochondrial DNA 4977-bp deletion in endometriosis

Endometriosis is a multifactorial gynecological condition characterized by the presence of ectopic endometrial and stromal tissue outside the uterus. Free radicals and Oxidative stress have been proposed to be involved in the pathogenesis of the endometriosis. It has been shown that mitochondrial DNA (mtDNA) is particularly susceptible to oxidative damage and mutations due to the high rate of reactive oxygen species production and limited DNA repair capacity in mitochondria. While a number of deletions can occur, the most commonly studied in human is a 4977-bp deletion that removes all or parts of the genes for NADH dehydrogenase subunits 3, 4, 4L and 5, cytochrome C oxidase subunit III and ATP synthase subunits 6 and 8.” We evaluated whether mtDNA common deletion is related with the susceptibility to endometriosis in northern Iran. In this study 80 endometriosis cases and 100 controls were enrolled. Total DNA was extracted from endometrial tissue samples. The mitochondrial common deletion was determined by Gap- polymerase chain reaction (Gap-PCR). It was found that the mitochondrial common deletion was more likely to be present in patients with endometriosis. Assessing indicate that 60 % of patients and 8 % of controls show mtDNA 4977-bp deletion (Odds Ratio [OR] = 17.25, P < 0.0001, confidence interval [CI] = 5.18–57.36). The mtDNA 4977 deletion may play a role in endometriosis. Further studies with larger numbers of patients are required for further evaluation and confirmation of our finding.

Variable Presentation of HB H Disease Due to Homozygosity for the Rare Polyadenylation Signal A TIndian (AATAAA>AATA– –) Mutation in Four Indian Families

The aim of this study was to identify the molecular defects leading to the variable clinical and hematological presentation of four patients with Hb H disease. Investigations included a complete blood count, high performance liquid chromatography (HPLC) analyses, cellulose acetate electrophoresis (pH 8.9), heat stability test, α genotyping by multiplex gap polymerase chain reaction (gap-PCR) to screen for the eight common α-globin gene deletions and DNA sequencing to detect the other deletional and nondeletional α-globin gene mutations. Two patients aged 15 and 5.5 years had a mild clinical presentation. The first patient aged 3 years had a severe presentation requiring regular transfusions. This patient also had an enlarged spleen and had to undergo splenectomy. The third patient, aged 5 years, also had severe anemia, had been transfused once and had a spleen of 4.5 cms. The hemoglobin (Hb) levels in the four patients ranged from 4.2 to 8.2 g/dL and they all had reticulocytosis (10.0 to 31.0%). Cellulose acetate electrophoresis at pH 8.9 showed a fast moving band that ranged from 18.0 to 25.9%. All the four patients were homozygous for the polyadenylation signal A (polyA) TIndian (AATAAA>AATA–) mutation. This mutation has been seen in Eastern India but not from Maharashtra and Uttar Pradesh where our patients originated.