The Spectrum of HBB Mutations among 2315 Beta Thalassemia Patients of a Reference Clinic in Tehran-Iran.

Beta Thalassemia is the most prevalent and well-studied single gene disorder in Iran. Here, we investigated the spectrum of HBB gene mutations, identified among 2315 patients, referred to a reference thalassemia clinic in Tehran, on the basis of suspicion to thalassemia major or intermedia. The patients were homozygous or compound heterozygous for HBB mutations, and were referred from various Iranian provinces, during 15 years (2001- 2016). The HBB mutations were classified based on their frequency, and the result was compared to a meta-analysis of 14,293 beta thalassemia cases in the Iranian population, within the same time period. The mutation spectrum in this study contained 43 HBB mutations, compared to the 90, presented by the meta-analysis. Similar to the meta-analysis, IVSII-1 (G > A) and IVSI-5 (G > C) were the most common mutations in this study. These two comprised 62.40% of the total HBB mutant alleles in the studied population, comparable to 51.92% of that in the meta-analysis. IVSII-1 (G > A) and IVSI-5 (G > C), followed by 17 other mutations that had frequencies ranging from 0.15% to 5.44%, were among the 20 common HBB mutations in Iran and neighboring countries, according to the meta-analysis. This study provided further evidence to support the spectrum of the most common HBB mutations in the Iranian population.

Novel mutations in mitochondrial carrier family gene SLC25A38, causing congenital sideroblastic anemia in Iranian families, identified by whole exome sequencing.

Sideroblastic anemias are heterogeneous rare hematological disorders, representing diverse phenotypes. In this study, the genetic cause of congenital, transfusion dependent anemia in four unrelated families consisting of eighteen individuals, with one affected member was investigated. Probands were suspected to rare anemias, including sideroblastic anemia. Whole exome sequencing in probands followed by segregation analysis in families was performed. Two novel frame shift mutations and one previously reported missense mutation in SLC25A38 gene was identified in these families. Mutations and their recessive mood of inheritance in each family were confirmed by PCR and Sanger sequencing. These findings suggest that sideroblastic anemia must be considered a possible etiology in cases with unexplained hemolytic anemia. Furthermore, mutations in SLC25A38 gene could be a prevalent cause of congenital sideroblastic anemia (CSA) in the Iranian population. Considering that parents of all affected individuals had consanguineous marriage and belong to sub populations, where consanguineous marriage is prevalent, it is important to perform carrier screening and genetic counseling in these families and their close relatives as prevention strategy in populations at risk.

Electromobility Shift Assay Reveals Evidence in Favor of Allele-Specific Binding of RUNX1 to the 5' Hypersensitive Site 4-Locus Control Region.

In our previous studies on the Iranian ß-thalassemia (ß-thal) patients, we identified an association between the severity of the ß-thal phenotype and the polymorphic palindromic site at the 5' hypersensitive site 4-locus control region (5'HS4-LCR) of the ß-globin gene cluster. Furthermore, a linkage disequilibrium was observed between this region and XmnI-HBG2 in the patient population. Based on this data, it was suggested that the well-recognized phenotype-ameliorating role assigned to positive XmnI could be associated with its linked elements in the LCR. To investigate the functional significance of polymorphisms at the 5'HS4-LCR, we studied its influence on binding of transcription factors. Web-based predictions of transcription factor binding revealed a binding site for runt-related transcription factor 1 (RUNX1), when the allele at the center of the palindrome (TGGGG(A/G)CCCCA) was A but not when it was G. Furthermore, electromobility shift assay (EMSA) presented evidence in support of allele-specific binding of RUNX1 to 5'HS4. Considering that RUNX1 is a well-known regulator of hematopoiesis, these preliminary data suggest the importance of further studies to confirm this interaction and consequently investigate its functional and phenotypical relevance. These studies could help us to understand the molecular mechanism behind the phenotype modifying role of the 5'HS4-LCR polymorphic palindromic region (rs16912979), which has been observed in previous studies.

The Impact of XmnI-HBG2, BCL11A and HBS1L-MYB Single Nucleotide Polymorphisms on Hb F Variation of Hematologically Normal Iranian Individuals.

The impact of Hb F on severity of sickle cell disease and ß-thalassemia (ß-thal) is well documented. The XmnI-HBG2, BCL11A and HBS1L-MYB single nucleotide polymorphisms (SNPs) have been introduced as the most important factors causing variation in fetal hemoglobin (Hb F) levels in different population studies. However, the extent of their effect could be population-specific. In this study, multivariate linear regression analysis was used to evaluate the association of Hb F with age, sex, and eight SNPs, including XmnI-HBG2, four BCL11A, two HBS1L-MYB SNPs and the polymorphic palindromic 5' hypersensitive 4-locus control region (5'HS4-LCR). One hundred and twenty-two hematologically normal individuals, from a previous study cohort, constituted our study population. In multivariate regression analyses, no association of Hb F was observed with age or sex of the individuals and SNPs in this study. We conducted a univariate regression analysis to further investigate the results, which among all the factors only detected XmnI-HBG2 and 5'HS4 SNPs as significant modifiers of Hb F. The significance of these two factors disappeared in a bivariate analysis. These results suggest that either XmnI-HBG2 or 5'HS4-LCR have a stronger contribution in Hb F variations of the Iranian population than BCL11A and HBS1L-MYB SNPs. Furthermore, the effect of low population size and technical limitations on obtained results could not be ruled out.

The influence of the BCL11A polymorphism on the phenotype of patients with beta thalassemia could be affected by the beta globin locus control region and/or the Xmn1-HBG2 genotypic background.

To study the influence of the ß globin locus control region (LCR) genotypic background on the phenotype modifying role of BCL11A polymorphisms, 100 cases of thalassemia, 48 homozygous for the A allele and 52 homozygous for the G allele at the 5'HS4-LCR palindromic polymorphic site were genotyped for two BCL11A single nucleotide polymorphisms (rs11886868 and rs766432) in the intronic region of this gene. The effect of these polymorphisms on HbF variation was also examined in 122 normal individuals. The 5'HS4-LCR had the most significant role in determining the phenotype of these thalassemia patients. BCL11A polymorphisms showed a significant role in determining the phenotype of patients homozygous for the G allele at 5'HS4-LCR. However, the majority of patients homozygous for the A allele at 5'HS4-LCR, showed a severe phenotype, regardless of the BCL11A genotype. These results, without undermining the strength of BCL11A as a silencer of the γ globin gene, suggest that the LCR background, by governing the state of BCL11A binding to this region, plays a more significant role in determining the thalassemia phenotype than the level of BCL11A protein expression, that might be influenced by single nucleotide polymorphisms in intronic regions of the BCL11A gene. Functional studies to confirm the interactions between BCL11A and LCR could be useful in designing pharmacogenetic strategies for the treatment of beta thalassemia major.

The modifying effect of Xmn1-HBG2 on thalassemic phenotype is associated with its linked elements in the beta globin locus control region, including the palindromic site at 5'HS4.

The core sequence of 5'HS4-beta globin locus control region and Xmn1-HBG2 site were analyzed and compared among 86 thalassemia patients with homozygous or compound heterozygous beta globin gene mutations and 101 normal individuals. Frequency of the G allele in the polymorphic palindromic sequence of 5'HS4 (TGGGG A/G CCCCA) and positive Xmn1-HBG2 profile was significantly higher in thalassemia patients compared to the normal population. Linkage disequilibrium was observed between the G allele and positive Xmn1-HBG2 profile in patient population. Furthermore, dominance of IVSII-1 in the mutation spectrum of the patients enabled us to identify linkage disequilibrium relationships between IVSII-1, positive Xmn1-HBG2 and the G allele at 5'HS4. The frequency of milder clinical phenotype was significantly higher in patients with GG/++ than cases with AA/-- genotypic pattern in 5'HS4/Xmn1-HBG2 loci. These data together with biochemical evidence suggesting a role for the A/G polymorphism at 5'HS4 palindromic site on modifying chromatin structure and in the absence of any evidence from functional studies relating the Xmn1-HBG2 site to the increased gamma chain expression, suggest that the phenotype modifying role long time assigned to Xmn1-HBG2 is possibly played by more functionally potent elements linked to it in LCR.

The XmnI and BCL11A single nucleotide polymorphisms may help predict hydroxyurea response in Iranian ß-thalassemia patients.

Hydroxyurea (HU), a drug which can reactivate fetal hemoglobin (Hb F) production, is frequently prescribed to ß-thalassemia (ß-thal) patients. However, transfusion requirements of only a subset of patients are reduced upon HU treatment. Because of its potential side-effects, targeted prescription of HU is imperative. To identify genetic markers that correlate with drug response, we have carried out a retrospective association study of single nucleotide polymorphisms (SNPs) in three Hb F quantitative trait loci (QTLs): the XmnI polymorphism, BCL11A, and the HBS1L-MYB intergenic region, with the response to HU in a cohort of 81 transfusion-dependent Iranian ß-thal patients. An increase in blood transfusion intervals post-therapy was used to measure drug response. Our results suggest that presence of the XmnI T/T genotype or the BCL11A rs766432 C allele correlates strongly with response to HU (p <0.001). Accordingly, these markers may be used to accurately predict the HU response of Iranian ß-thal patients.

Genetic Diagnosis of Pyruvate Kinase Deficiency in Undiagnosed Iranian Patients with Severe Hemolytic Anemia, using Whole Exome Sequencing.

BACKGROUND: After ruling out the most common causes of severe hemolytic anemia by routine diagnostic tests, certain patients remain without a diagnosis. The aim of this study was to elucidate the genetic cause of the disease in these patients using next generation sequencing (NGS). METHODS: Four unrelated Iranian families including six blood transfusion dependent cases and their parents were referred to us from a specialist center in Tehran. There was no previous history of anemia in the families and the parents had no abnormal hematological presentations. All probands presented severe congenital hemolytic anemia, neonatal jaundice and splenomegaly. Common causes of hemolytic anemia were ruled out prior to this investigation in these patients and they had no diagnosis. Whole exome sequencing (WES) was performed in the probands and the results were confirmed by Sanger sequencing and subsequent family studies. RESULTS: We identified five variants in the PKLR gene, including a novel unpublished frameshift in these families. These variants were predicted as pathogenic according to the ACMG guidelines by Intervar and/or Varsome prediction tools. Subsequent family studies by Sanger sequencing supported the diagnosis of pyruvate kinase deficiency (PKD) in six affected individuals and the carrier status of disease in their parents. CONCLUSION: These findings show that PKD is among the rare blood disorders that could remain undiagnosed or even ruled out in Iranian population without performing NGS. This could be due to pitfalls in clinical, hematological or biochemical approaches in diagnosing PKD. Furthermore, genotyping PKD patients in Iran could reveal novel mutations in the PKLR gene.

Analyzing 5'HS3 and 5'HS4 LCR core regions and NF-E2 in Iranian thalassemia intermedia patients with normal or carrier status for beta-globin mutations.

Our data on 114 Iranian individuals with thalassemia intermedia phenotype revealed homozygous or compound heterozygous beta-globin mutations to be the predominant disease factor in 86.2% of cases. However, 8.2% of these individuals were found to be heterozygous or wild type for beta-globin mutations. In search for determinants outside of the beta-globin gene, which could be responsible for the unexpected thalassemia intermedia phenotype in these subjects, we screened the alpha-globin genes, the 5'HS3 and 5'HS4 regions of the beta-globin LCR, and the NF-E2 transcription factor for sequence variations in selected individuals. The -3.7 deletion was the only alpha-globin mutation detected, and no alterations were found in 5'HS3 and NF-E2. Sequence analysis of the 5'HS4 LCR core region identified three known SNPs in a single patient, who required irregular blood transfusions. The A/G polymorphism in the 5'HS4 palindromic region was also observed to be variable. Family studies were carried out on a female G/G homozygous patient, who received irregular blood transfusions. Her father, who had the same heterozygous IVSII-1 beta-globin mutation but the A/G genotype at the 5'HS4 palindromic site, presented with mild anemia and no requirement for blood transfusions. This suggests an impact of SNPs in the 5'HS4 LCR core region on the thalassemia phenotype and offers an interesting subject for further investigations in the Iranian population.

Frequency of positive XmnIGgamma polymorphism and coinheritance of common alpha thalassemia mutations do not show statistically significant difference between thalassemia major and intermedia cases with homozygous IVSII-1 mutation.

From 362 thalassemia cases referred to adult thalassemia clinic of the Iranian blood transfusion organization (IBTO) for genotyping, 103 cases (28.5%) had a common primary disease factor, IVSII-1 mutation in homozygous state. 61 (59.2%) of these individuals represented thalassemia major and 42 (40.8%) thalassemia intermedia clinical phenotype. To re-evaluate our current diagnostic criteria, XmnI(G)gamma polymorphism and coexistence of alpha thalassemia mutations, frequently recalled as important factors modifying the clinical phenotype of homozygous beta zero thalassemia cases in our country, were examined in both groups. No statistically significant difference in the frequency of positive XmnI(G)gamma polymorphism was observed between thalassemia intermedia and thalassemia major patients. Double gene deletion --(Med) was observed in only one thalassemia major case, while -a(3.7) in heterozygous state (-a(3.7)/aa) was identified in 6 (9.8%) of thalassemia major and 8 (19%) of thalassemia intermedia patients. -a(4.2) was observed in only one thalassemia major case. No statistically significant difference in the frequency of coinheritance of alpha thalassemia was observed between the two groups. These results imply that other interacting mechanisms which modify the phenotype of thalassemia patients is still in the dark in our current diagnostic criteria of thalassemia.

A report of 8 cases with hemoglobin H disease in an Iranian family.

alpha-Thalassemia is a common genetic disorder in Iran. However, no comprehensive data on epidemiology of severe forms of alpha-thalassemia, including hemoglobin H (HbH) or hydrops fetalis, is available in this population. This is a first case report of an Iranian family with large number of HbH individuals. The proband is a 48-year-old woman, referred to our center with anemia and no history of previous blood transfusions. Similar clinical phenotype has been observed in all of her 5 siblings, 2 of her 4 children, and her granddaughter, whose parents are first cousins. A reverse hybridization assay covering 21 alpha globin mutations was performed to determine the genotype in 11 members of this family and a fetus. HbH genotype was identified in 9 individuals, representing 3 generations, including a fetus. The high prevalence of alpha-thalassemia carriers together with the high rate of consanguineous marriages could lead to a large number of individuals with HbH or even hydrops fetalis in Iranian families. Therefore, to avoid the risk of having affected offspring, carrier detection, genetic counseling, and prenatal diagnosis would be of vital importance for individuals with low red blood cell (RBC) indices, normal iron status, and normal HbA(2) level, who are suspected to be alpha-thalassemia carriers.

Molecular mechanisms underlying thalassemia intermedia in Iran.

To improve the differentiation of thalassemia intermedia from other hemoglobinopathies in Iran, four known genetic mechanisms-XmnI (G)gamma polymorphism, inheritance of mild and silent beta-thalassemia alleles, delta beta deletion, and coinheritance of alpha- and beta-thalassemia-were investigated in 52 Iranian individuals suspected to have thalassemia intermedia based on clinical and hematological characteristics. Beta-globin mutations were studied using a reverse-hybridization assay and sequencing of the total beta-globin gene. The XmnI (G)gamma polymorphism, the Sicilian delta beta deletion, and four alpha-globin mutations (-a(3.7), -a(4.2), -(MED), aaa(anti-3.7)) were studied using PCR-based techniques. The inheritance of the XmnI (G)gamma polymorphism with severe beta-thalassemia alleles in the homozygous or compound heterozygous state was the predominant mechanism observed in 27 individuals (55.3%). In five cases, this status overlapped with the -a(3.7)/aa genotype. The second most frequent cause for thalassemia intermedia (14.8%) was the inheritance of mild beta-thalassemia alleles, including IVS-I-6 (T > C), -88 (C > A), and + 113 (A > G). In three subjects (4.3%) the Sicilian delta beta deletion was identified. HbS in association with beta-zero-thalassemia was found in three patients with thalassemia intermedia phenotype. In 11 cases (21.3%) no causative genetic alteration could be identified. Our results reflect the diversity underlying thalassemia intermedia, and the limitations of the applied clinical, hematological, and molecular approaches for correct diagnosis. Some of the unresolved cases will offer an opportunity to discover additional molecular mechanisms leading to thalassemia intermedia.

The Iranian Human Mutation Gene Bank: a data and sample resource for worldwide collaborative genetics research.

As Human Genome Project exploration continues, the necessity of having a broader spectrum of genomic DNA material from different nationalities to study various aspects of hereditary disease becomes more obvious. The existence of high genetic polymorphism within and between different communities in the world makes it necessary for the gene hunters to investigate many different populations. Iran, a large country with close to 66 million people, is a land of different nationalities, tribes, and religions that offers a highly heterogeneous gene pool to the genetics researcher. The purity of many different races in this country has been highly conserved by geographical borders and by an ancient culture that has always encouraged intrafamilial marriages. All these have created a population that is remarkably heterogeneous yet high in consanguinity rate. During the last five years of investigation we have established a DNA bank, the Iranian Human Mutation Gene Bank (www.IHMGB.com), which contains all genetic diseases studied in Iran that have the Mendelian mode of inheritance. Some of the samples are assigned to common or novel mutations and others belong to patients with clinical profiles associated with particular genetic diseases but undefined mutation. This bank stores samples of DNA from the patient and his/her first-degree relatives together with a comprehensive pedigree and clinical profile for each sample. To facilitate collaboration with other scientists around the world with the same interests, we decided to present our experimental projects online. This DNA bank provides opportunities for us to collaborate with scientists outside Iran. It offers a sample resource to research scientists around the world, at no charge, for the purpose of investigating the various aspects of genetic disorders from prenatal diagnosis to gene structure and function. It is strongly stressed that no commercial benefit is involved in the establishment of this DNA bank and the DNA samples are free of charge. However, to meet our goals and to respect ethical values, DNA samples can only be used under certain conditions stated in the User Consent Form.

alpha-globin gene deletion and point mutation analysis among in Iranian patients with microcytic hypochromic anemia.

We tested 67 Iranian individuals, presenting with low mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) levels, normal hemoglobin electrophoresis and iron status, for the presence of twelve common alpha-thalassemia gene deletions and point mutations. Five different mutations (-alpha(3.7), -alpha(4.2), --MED, -(alpha)20.5, Hb Constant Spring) were identified in a total of 43 cases