Coinheritance of Sicilian (뫧)0-Thalassemia and Two Rare Hemoglobin Variants: A Complex Case of Hemoglobinopathy.

α-Thalassemia (α-thal) is considered as the most common inherited hemoglobin disorder worldwide. The present study describes the first observation of a combination of rare α-chain variants, and ß-globin gene cluster deletion. A 21-year-old woman with thalassemia trait, marked microcytosis, mild anemia, and normal range of Hb F was referred to Amirkola genetic center in the North of Iran for routine molecular test of thalassemia in the context of carrier detection and prevention of thalassemia major birth. Nucleotide sequencing revealed a novel compound heterozygosity status for two non-deletional mutations on HBA2, Hb O Indonesia (α116(GH4)Glu â†’ Lys), and Hb Matsue-Oki (α75 (EF4) Asp â†’ Asn), together with heterozygosity for the sicilian (δß)0-thal mutation. This finding highlights the necessity of deep molecular investigation of thalassemia in regions where thalassemia is abundant, and present highly heterogeneous population.

A Comprehensive Molecular Investigation of α-Thalassemia in an Iranian Cohort from Different Provinces of North Iran.

α-Thalassemia (α-thal) is the most common monogenic disease that is caused by the absence or reduced expression of α-globin genes. The aim of this study was to investigate common α-globin mutations and their associated haplotypes in four northern provinces of Iran (Gilan, Mazandaran, Golestan, Khorasan). One thousand, one hundred and ninety-one persons were tested for α-thal mutations by gap-polymerase chain reaction (PCR), reverse dot-blot hybridization, restriction fragment length polymorphism (RFLP) analysis and sequencing. Of the nine different mutations found, the most frequent were -α3.7 (rightward deletion) (45.6%), polyadenylation site (αp°lyA2α) (α2) (AATAAA>AATGAA; HBA2: c.*92 A>G) (15.27%), - -MED (Mediterranean deletion) (6.86%), -α4.2 (leftward deletion), (6.17%), αCSα [Hb Constant Spring (Hb CS) (HBA2: c.427 T>C)] (4.62%), -α-5 nt (HBA2: c.95+2_95+6delTGAGG) (3.70%). All chromosomes bearing an α-globin point mutation [αp°lyA2α, -α-5 ntα, αCSα, αp°lyA1α (AATAAA> AATAAG; HBA2: c.*94 A>G)] showed only one haplotype that was present in most normal chromosomes, while the -α3.7 deletion was associated with three distinct haplotypes. Our results indicate that α-thal mutations are heterogeneous and -α3.7 and αp°lyA2α are the most prevalent mutations in this region. The presence of -α3.7 with three different haplotypes suggests an older history for this mutation. The high prevalence of αp°lyA2α in Mazandaran Province, Iran compared to other parts of the country and the world, suggests a founder effect. Altogether, we here provide further data confirming the heterogeneity of the northern population of Iran. These data may contribute to the establishment of a national mutation database, more accurate genetic counseling and prenatal diagnosis (PND).

Non-invasive prenatal diagnosis of ß-thalassemia by detection of the cell-free fetal DNA in maternal circulation: a systematic review and meta-analysis.

The discovery of fetal DNA (f-DNA) opens the possibility of early non-invasive procedure for detection of paternally inherited mutation of beta-thalassemia. Since 2002, some studies have examined the sensitivity and specificity of this method for detection of paternally inherited mutation of thalassemia in pregnant women at risk of having affected babies. We conducted a systematic review of published articles that evaluated using this method for early detection of paternally inherited mutation in maternal plasma. A sensitive search of multiple databases was done in which nine studies met our inclusion criteria. The sensitivity and specificity was 99 and 99 %, respectively. The current study found that detection of paternally inherited mutation of thalassemia using analysis of cell-free fetal DNA is highly accurate. This method could replace conventional and invasive methods.

Identification of a Rare ß(0)-Thalassemia Mutation, Codon 54 (-T) (HBB: c.165delT) in an Iranian Family.

ß-Thalassemia (ß-thal) is the most widespread autosomal recessive disorder worldwide. The present study describes a very rare ß-globin gene mutation, codon 54 (-T) (HBB: c.165delT), in a family from northern Iran. Nucleotide sequencing of amplified DNA obtained from a 28-year-old man revealed a deletion (-T) at codon 54 of the ß-globin gene that results in a nonsense sequence at codon 60 and inphase termination at codon 59. Moreover, the haplotype combination of six different restriction enzyme sites in the ß-globin cluster was determined for this mutation. To the best of our knowledge, this is the second article reporting the codon 54 mutation worldwide and the first report of this mutation in the Iranian population, emphasizing the high heterogeneity of this population.

Simultaneous detection of Hb constant spring (α142, TAA>CAA, α2) and the α2 IVS-I donor site (-TGAGG) deletion by a simple polymerase chain reaction-based method in Iran.

Hb Constant Spring (Hb CS, codon 142, TAA>CAA, α2) (HBA2:c.427T>C) and α2 IVS-I donor site (GAGGTGAGG>GAGG - - - - -) (HBA2:c.95+2_95+6delTGAGG) are nondeletional α-thalassemia (α-thal) mutations found all over the world. Identification of α-thal genotypes in at-risk couples for severe anemia or in highly heterogeneous populations requires rapid, accurate and cost-effective genotyping methods. In this study, a pair of primers were used to specifically amplify an 883 bp fragment from the α2-globin gene in order to simultaneously identify these two mutations by a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method. We determined the genotypic frequencies of Hb CS and the α2 IVS-I donor site mutations after amplification and enzymatic digestion with Tru9I in 238 northern Iranian samples referred for α-thal testing. Hb CS and the α2 IVS-I donor site mutations accounted for 21 (8.8%) and 29 (12.2%) of the nondeletional cases. This genotyping assay has proven to be a rapid, reliable and useful diagnostic tool for simultaneous detection of these two anomalies for genetic counseling or further prenatal diagnosis.

A comprehensive molecular characterization of beta thalassemia in a highly heterogeneous population.

In Iran, the prevalence of beta-thalassemia trait is approximately 4-8% in most areas, and in Mazandaran province 10% of the population are carriers. Twenty four beta-globin gene mutations were identified in 1635 persons with beta-thalassemia trait using reverse dot blot and restriction fragment length polymorphism analysis. The predominant mutations included IVSII-1 (G-A) (61%), codon 30 (G-C) (7.5%), codon 22 (-7bp) (6.2%), codon 8 (-AA) (5.4%) and IVSI-5 (G-C) (3.6%). These mutations were in different haplotypes, with IVSII-1 being the most heterogeneous. Other less frequent mutations included IVS-II-745 (C-G), codon 44 (-C), codon 39 (C-T), codon 5 (-CT), IVS I-110 (G-A), IVSI-130 (G-C), Fr8/9 (+G), IVSI-1 (G-A), and IVSI (-25bp). All rare mutations except IVSI-130 were encountered in a unique haplotype. The diversity of these mutations reflects the historical admixture of genes in the region. The high prevalence of IVSII-1 (G-A) compared to other parts of the country and the world suggests a founder effect. Our data provide a basis for genetic counseling and prenatal diagnosis.

High-resolution melting analysis for noninvasive prenatal diagnosis of IVS-II-I (G-A) fetal DNA in minor beta-thalassemia mothers.

OBJECTIVES: The high-resolution melting (HRM) technique is fast, effective and successful method for mutation detection. The aim of this study was to determine the sensitivity and specificity of the HRM method for detection of a paternally inherited mutation in a fetus as a noninvasive prenatal diagnosis of ß-thalassemia. METHODS: Genomic DNAs were prepared from 50 ß-thalassemia minor couples whose pregnancy was at risk for homozygous ß-thalassemia. Ten milliliters of the maternal blood from each pregnant woman were collected and after separating plasma stored at -80 °C until analysis. The extracted DNAs were analyzed by HRM real-time PCR for detection of IVS-II-I (G-A) as a paternally inherited mutation. The gold standard was the result of a chorionic villus sampling by a standard reverse dot blotting test. RESULTS: The sensitivity and specificity of HRM real-time PCR were 92.6% and 82.6%, respectively. Also, the positive and negative predictive values were 86.2% and 90.47%, respectively. CONCLUSIONS: HRM real-time PCR was a sensitive and specific method for determining the paternally inherited mutation in the fetus at risk with thalassemia major.

A bimetallic nanocomposite modified genosensor for recognition and determination of thalassemia gene.

The main roles of DNA in the cells are to maintain and properly express genetic information. It is important to have analytical methods capable of fast and sensitive detection of DNA damage. DNA hybridization sensors are well suited for diagnostics and other purposes, including determination of bacteria and viruses. Beta thalassemias (ßth) are due to mutations in the ß-globin gene. In this study, an electrochemical biosensor which detects the sequences related to the ß-globin gene issued from real samples amplified by polymerase chain reaction (PCR) is described for the first time. The biosensor relies on the immobilization of 20-mer single stranded oligonucleotide (probe) related to ßth sequence on the carbon paste electrode (CPE) modified by 15% silver (Ag) and platinum (Pt) nanoparticles to prepare the bimetallic nanocomposite electrode and hybridization of this oligonucleotide with its complementary sequence (target). The extent of hybridization between the probe and target sequences was shown by using linear sweep voltammetry (LSV) with methylene blue (MB) as hybridization indicator. The selectivity of sensor was investigated using PCR samples containing non-complementary oligonucleotides. The detection limit of biosensor was calculated about 470.0pg/µL.

Hb Knossos: HBB:c.82G>T Associated with HBB:c.315+1G>A Beta Zero Mutation Causes Thalassemia Intermedia.

ß-thalassemia is the most common single gene disorder worldwide and in Iran. In the present study we report for the first time a rare variant of hemoglobin HBB:c.82G>T; Codon 27 GCC→TCC (Ala→Ser), Hb Knossos, using sequencing and reverse dot blot hybridization, in members of a family from North Iran. The family has a 16 years-old compound heterozygous thalassemia intermedia male child presenting this variant together with HBB:c.315+1G>A (IVSII-I) mutation. The father, heterozygous for Hb Knossos, showed borderline hematological indices. To our knowledge, this is the first report of Hb Knossos in trans with the ß(O) IVSII-I allele leading to thalassemia intermedia. Our data also highlight the necessity of deep molecular characterization of subjects presenting normal HbA2 level associated with abnormal or borderline red cell indices.

Beta globin frameworks in thalassemia major patients from north iran.

OBJECTIVE: Four combinations of five neutral sequence changes at rs713040, rs10768683, rs7480526, rs7946748, and rs1609812 occurring in the human beta globin gene defined as frameworks have been reported in beta globin gene. Here we report for the frequency of these frameworks in thalassemia major patients of North Iran. METHODS: Beta globin gene frameworks of 46 thalassemia major patients of North Iran were determined using Denaturing Gradient Gel Electrophoresis. FINDINGS: All these frameworks called framework 1, 2, 3, 3a were present at the frequency of 23.9%, 45.7%, 6.5% and 23.9% respectively. CONCLUSION: These frameworks may be used for tracking mutant alleles in prenatal diagnosis programs.

Hematologic features of alpha thalassemia carriers.

Alpha thalassemia (α-thal) is relatively common worldwide. Most carriers are defective in either one or two alpha globin genes out of four functional ones, with deletions being more common than point mutations. The hematologic features are very important for the selection of the appropriate molecular tests while determining the genotype. The aim of this study was to compare hematologic features of patients with various types of α globin mutations. Hematological indices including red blood cells (RBC), hemoglobin concentration (Hb), mean cell volume (MCV), mean cell hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC) and percentage of Hemoglobin (HBA1, HBA2 and HBF) of seven-hundred and twenty two patients presenting ten different α-thal genotypes were considered. All patients showed reduced MCV and/or MCH values.Moreover, MCV and MCH were lower in patients with two functional alpha globin genes in comparison to patients with one mutated alpha globin gene (P value<0.001). In conclusion, MCV and MCH valuescan be helpful for the selection of the appropriate molecular tests to determine the genotype of alphathalassemia carriers.

IVSII-666 of human beta-globin gene: a polymorphic marker linked to codon 8(-AA) mutation.

AIMS: IVSII-666 (C-T) is one of the polymorphic sites located in the second intron of the ß-globin gene. Its polymorphism rate and relationship to a specific mutation are studied for the first time on 211 DNA samples of thalassemia trait patients living in Mazandaran province in North Iran using Ssp1 restriction enzyme. ß-Globin haplotype determination at XmnI/(G)γ, HincII/3'Ψß, HinfI/5'ß, RsaI/5'ß, and SspI/ß sites was also performed by analysis of family members. RESULTS: Nineteen different haplotypes were encountered in 211 unrelated thalassemia trait patients. One hundred twenty-seven patients (60.2%) were homozygous (+/+), 81 (38.4%) were heterozygous (+/-), and 3 (1.4%) were homozygous (-/-) for Ssp1 polymorphism. Of 24 mutant chromosomes negative for SspI, 16 were linked to mutation in codon 8(-AA). All codon 8(-AA) mutations were linked to the SspI-negative site. CONCLUSION: The SspI site can be used as a marker for tracking either normal ß-globin gene (11.9%) or mutant alleles at codon 8 during prenatal diagnosis.