Molecular characterisation of sickle cell disease and classification of major haplotypes associated with the ß-globin cluster (HBB gene) by means of SNP marker sequencing in a group of samples from Bolívar, Colombia.

BACKGROUND: Colombia has a mestizo population and the prevalence of haemoglobin variants varies according to each region, but heterozygous carriers can be found in all of them. AIM: To characterise sickle cell disease (SCD) haematologically, biochemically, and molecularly, and detect classic haplotypes by DNA sequencing in a group of samples from Bolívar, Colombia. SUBJECTS AND METHODS: Blood samples were collected after informed consent from volunteers from eight communities in the Bolívar department, plus samples from the Pacific region, Providencia Island, and Bogotá were included. Data were obtained from: (1) haematological analyses; (2) biochemical tests: dHPLC was used to determine haemoglobin (Hb); and (3) DNA sequencing data through five SNPs. RESULTS: 101 samples were identified by rs334 through Sanger's Sequencing, structural haemoglobinopathies HbAS (34.65%), HbSS (2.97%) and HbAC (1.98%) were found. When contrasting the Hb identification results between SNP rs334 Vs. dHPLC/Isoelectric Focusing (IEF), a coincidence was found in 39/43 samples analysed, therefore, when comparing these techniques, a significant correlation was found (Pearson's correlation coefficient r = 0.998). 26 samples previously analysed by rs334 were classified into classical haplotypes CAR (50.0%), BEN (30.76%), CAM (7.69%), SEN (3.84%), and ATP-I (7.69%). CONCLUSIONS: SCD characterisation and SNPs-based classification through Sanger's DNA sequencing have not been performed before in Colombia. The results of this work will make it possible to expand the data or records of carriers and those affected, which will benefit patients and their families.

Identification of a Novel Variant c.163delG in HBB Gene Resulting in a Beta Null Phenotype in a Proband with Thalassemia Intermedia.

A 21-year-old patient presented with a previous medical history of pallor, mild icterus, increased fatigue, low hemoglobin, and abnormal hemoglobin variant analysis with more than 70 transfusions. He was referred for genetic analysis to identify the pathogenic variations in the ß-globin gene. Sanger's sequencing of the proband and his family revealed the presence of a novel frame shift variant HBB:c.163delG in a compound heterozygous state with hemoglobin E (HbE) (HBB:c.79G > A) variant. The father and the sibling of the patient were found to be normal for the HBB gene. Mother was found to be heterozygous for HbE (HBB:c.79G > A) variant. In silico analysis by Mutalyzer predicted that c.163delG variant generated a premature stop codon after seven codons, leading to a truncated protein. FoldX protein stability analysis showed a positive ΔΔG value of 45.27 kcal/mol suggesting a decrease in protein stability. HBB:c.79G > A is a known variant coding for HbE variant, which results in the reduced synthesis of ß-globin chain and shows mild thalassemia. Combined effect of HBB:c.163delG and HBB:c.79G > A variants in the proband might have led to the reduced synthesis of ß-globin chains resulting in a thalassemia intermedia type of clinical manifestation.

Hb Mizuho Case Report; Early Genomic Testing Facilitates a Life Changing Diagnosis.

Unstable variant hemoglobinopathies are an uncommon cause of hemolysis in the pediatric patient and may cause a delay in diagnosis if there is not a high index of suspicion. Hemoglobin (Hb) Mizuho is a rare unstable hemoglobinopathy caused by a pathogenic variant of the HBB gene with a severe phenotype. Here we report on the first known case of Hb Mizuho in Australia, presenting with features of acute and chronic hemolysis. The morphological features on blood film review, in conjunction with biochemical findings and other clinical features, did not immediately suggest an alternative diagnosis and a Next Generation Sequencing gene analysis approach was taken to investigate genes associated with red blood cell disorders and atypical uremic syndrome. The HBB Mizuho variant was detected and established the diagnosis. This report highlights the challenge of diagnosing Hb Mizuho on conventional testing and the need for early genomic testing to clarify a diagnosis.

A Novel Frameshift Mutation of HBB Causing Dominant ß-Thalassemia in a Chinese Individual.

We reported a rare ß-thalassemia patient, a 41-year-old Chinese male with small cell hypopigmentation anemia, jaundice and splenomegaly as the main clinical symptoms. By using Next-Generation Sequencing (NGS), we identified a novel de novo HBB mutation(c.358_365dup, p.Phe123Alafs*39) which resulted in an abnormally prolonged ß-globin chain comprising 159 amino acid residues. The secondary and three-dimensional structures of the ß-globin predicted that the novel prolonged ß-globin chain has a considerable risk of instability in the hemoglobin, and leads to clinical phenotype. This study contributes to the enrichment of the genetic pathogenic mutation database for thalassemia and underscores the significance of NGS in the screening of mutations for thalassemia families.

lncRNA Neat1 regulates neuronal dysfunction post-sepsis via stabilization of hemoglobin subunit beta.

Sepsis-associated encephalopathy (SAE) is characterized by acute and diffuse brain dysfunction and correlates with long-term cognitive impairments with no targeted therapy. We used a mouse model of sepsis-related cognitive impairment to examine the role of lncRNA nuclear enriched abundant transcript 1 (Neat1) in SAE. We observed that Neat1 expression was increased in neuronal cells from septic mice and that it directly interacts with hemoglobin subunit beta (Hbb), preventing its degradation. The Neat1/Hbb axis suppressed postsynaptic density protein 95 (PSD-95) levels and decreased dendritic spine density. Neat1 knockout mice exhibited decreased Hbb levels, which resulted in increased PSD-95 levels, increased neuronal dendritic spine density, and decreased anxiety and memory impairment. Neat1 silencing via the antisense oligonucleotide GapmeR ameliorated anxiety-like behavior and cognitive impairment post-sepsis. In conclusion, we uncovered a previously unknown mechanism of the Neat1/Hbb axis in regulating neuronal dysfunction, which may lead to a novel treatment strategy for SAE.

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.

Mutation Spectrum of ß-Thalassemia in Some Ethnic Groups of North Maharashtra, India.

Beta-thalassemia is the most common inherited single-gene disorder in the world, caused by more than 200 known mutations in the HBB gene. In India, the average prevalence of ß-thalassemia carriers is 3-4%. Several ethnic groups have a much higher prevalence, about 8% in the tribal groups, according to the 2011 census. The study's main goal is to identify common ß-thalassemia mutations and the frequencies of different haplotypes in various communities in North Maharashtra. Nashik district had the highest prevalence of ß-thalassemia (34%), followed by Ahmednagar (29%), Jalgaon (16%), Dhule (14%), and Nandurbar (7.0%). Prevalence of ß-thalassemia was highest in the schedule caste community (SC) (48%), followed by (17%) in Muslims, (14%) in other backward classes (OBC), (13%) in Schedule Tribe (ST), and (8.0%) in the general population The six most common ß-thalassemia mutations detected in this study are IVS 1 > 5 (G→C), Cd 15(G→A), Cd 41/41 (-TCTT), Cd 8/9(+G), IVS 1 > 1(G→T) and Cap + 1(A > G). Among these mutations, IVS 1 > 5 (G > C) was the most common type of mutation found in ß-thalassemia patients in the North Maharashtra population. Type-I haplotype was the most prevalent among all communities. Nashik and Ahmednagar districts were highly affected by ß-thalassemia. Among different ethnic groups, the SC and Muslim communities were the worst affected with a higher proportion of ß-thalassemia and increased frequency of mutations.

Identification of the ß thalassemia allele ß-50 and analysis of the hematology data of carriers in a southern Chinese population.

During a routine test, we identified a 38-year-old man who had a positive hematology screening result but was negative for hot spot variants of his thalassemia gene. Further analysis identified ß-50 (HBB: c.-100G>A). It was first suggested that ß-50 was a ß+ -thal allele, and some research groups suggested this allele was a silent ß-thal allele. To fully understand the hematological phenotype of the ß-50 allele, we screened for individuals carrying ß-50 in the general population and performed hematology analysis on these carriers. A real-time PCR detection system was designed to verify samples carrying ß-50 . Twenty-one thousand samples and 43 pedigree samples were screened, and 86 ß-50 carriers were detected. We performed hematological analysis on 65 individuals older than 3 years who had normal serum ferritin and analyzed the data. A total of 34.62% of the ß-50 /ßN individuals had mean cellular volume (MCV) or mean cellular hemoglobin (MCH) values slightly lower than the positive cutoff value of screening; the ß-50 carriers' Hb A2 value was slightly elevated. According to the test results, ß-50 carriers have slight changes in hematology parameters, including slight decreases in MCV and MCH and slight increases in Hb A2 ; however, these effects do not reach the degree of traditional ß+ alleles. Females with genotype ß-50 /ß0 show a degree of decline in hematological indicators during pregnancy. Therefore, we should describe ß-50 as a ß++ thalassemia allele, and identification of ß-50 can explain slight changes in hematological indicators in some carriers.

Role of hemoglobin alpha and hemoglobin beta in non-small-cell lung cancer based on bioinformatics analysis.

The differentially expressed genes (DEGs) were identified and screened differentially in non-small-cell lung cancer (NSCLC) using information from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases, and the correlation of DEGs in protein interaction, function, and pathway enrichment were analyzed to search for new biomarkers and potential therapeutic targets for NSCLC. Protein-protein interaction network (PPI) analysis showed that CDK1 and GNGT1 were the most significantly upregulated hub nodes, while FPR2 was the most significantly downregulated. Gene Ontology enrichment analysis showed that upregulated DEGs were significantly enriched in protein heterodimerization activity and other functions, while downregulated DEGs were enriched in functions such as heparin-binding. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that upregulation of DEGs were significantly associated with neuroactive ligand-receptor interaction pathways, while downregulation of DEGs were significantly associated with malaria pathways. According to the analysis results, we identified hemoglobin alpha (HBA) and hemoglobin beta (HBB) as the genes of interest for further study. Through tissue level and cell level experiments, we found that the expressions of HBA and HBB in NSCLC tissues were significantly lower than those in paracancerous tissues, and downregulation of HBA and HBB could significantly affect the proliferation ability of NSCLC cells. In addition, we also found that changes in HBA and HBB may affect NSCLC cells through the p38/MAPK pathway and JNK pathway, and ultimately affect the occurrence and development of NSCLC.

First report of successful treatment for hemoglobin Bristol-Alesha by hemopoietic stem cell transplantation.

HBB gene mutations lead to many kinds of diseases, of which, except for the two most common diseases of thalassemia and sickle cell anemia, rare kinds of hemolytic anemia, such as hemoglobin Bristol-Alesha, are rarely reported, no ideal treatment in clinic. A child suffered from chronic recurrent hemolytic attacks and the related genes of hereditary hemolytic anemia were detected on her. Hematopoietic stem cell transplantation was conducted in the treatment of the patient. The patient was diagnosed as hemoglobin Bristol-Alesha and achieved complete recovery after hematopoietic stem cell transplantation. For Bristol-Alesha, without characteristic clinical manifestation and specific biochemical examination, diagnosis is dependent on the gene mutation detection and hematopoietic stem cell transplantation is an effective and curable method.

Highly expressed genes in multiple myeloma cells - what can they tell us about the disease?

Cancer cells can convert proto-oncoproteins into oncoproteins by increasing the expression of genes that are oncogenic when expressed at high levels. Such genes can promote oncogenesis without being mutated. To find overexpressed genes in cancer cells from patients with multiple myeloma, we retrieved mRNA expression data from the CoMMpass database and ranked genes by their expression levels. We grouped the most highly expressed genes based on a set of criteria and we discuss the role a selection of them can play in the disease pathophysiology. The list was highly concordant with a similar list based on mRNA expression data from the PADIMAC study. Many well-known "myeloma genes" such as MCL1, CXCR4, TNFRSF17, SDC1, SLAMF7, PTP4A3, and XBP1 were identified as highly expressed, and we believe that hitherto unrecognized key players in myeloma pathogenesis are also enriched on the list. Highly expressed genes in malignant plasma cells that were absent or expressed at only a low level in healthy plasma cells included IFI6, IFITM1, PTP4A3, SIK1, ALDOA, ATP5MF, ATP5ME, and PSMB4. The ambition of this article is not to validate the role of each gene but to serve as a guide for studies aiming at identifying promising treatment targets.

Spectrum of HBB gene mutations among 696 ß-thalassemia patients and carriers in Southern Vietnam.

BACKGROUND: Thalassemias are common inherited blood disorders that have been extensively studied in Asia. Thus far, data on mutations of the HBB gene in Vietnamese patients with ß-thalassemia are limited to small studies. METHODS: We recruited 696 ß-thalassemia patients and carriers in southern Vietnam and analyzed for the HBB gene mutations using Sanger sequencing technology. RESULTS: We documented 27 types of known mutations and 10 types of novel variants on 737 alleles out of 1392 surveyed alleles. The three most common mutations, which account for more than ¾ of all mutant alleles, were c.79G > A (HbE), c.124_127delTTCT, and c.52A > T. The novel variants were mainly located in 5' untranslated region (c.-92delC and c.-67A > G) and 3' untranslated region (c.*4C > T, c.*116_*117insA, c.*142 T > C, c.*156G > C, c.*176_*177insA, and c.*247 T > C), except for one in intron 2 (c.316-99 T > G) and one in exon 3 (c.385delG). CONCLUSION: We provide here a comprehensive mutation spectrum of the HBB gene in Southern Vietnam, which is crucial for carrier screening and prenatal diagnosis in the future.

Identification of a Novel Mutation in the 3' Untranslated Region of the ß-Globin Gene (HBB:c.*132C>G) in a Chinese Family.

We describe a new ß-globin mutation causing silent ß-thalassemia (ß-thal). The proband was a 5-year-old boy who presented with the phenotype of thalassemia intermedia. Molecular diagnoses revealed a genomic alteration at position 1606 of the HBB gene (HBB:c.*132C>G) in combination with a common ß0-thal mutation (HBB:c.126_129delCTTT). The 3'-untranslated region (UTR) mutation was inherited from his father who showed a normal mean corpuscular volume (MCV) and Hb A2 level. The discovery of rare mutations provides important information related to both genetic counseling for families involved.

Consideration of Splenectomy in Unstable Hemoglobinopathy: A Case Report of Hb Hammersmith (HBB: c.128T>C).

A 5-year-old female has been diagnosed with Hb Hammersmith (HBB: c.128T>C) and has required three blood transfusions thus far, with hemoglobin (Hb) levels dropping as low as 5.4 g/dL. An elective splenectomy is now being considered in order to reduce hemolysis and the need for transfusions. Of 18 previously reported cases of Hb Hammersmith, eight patients have reportedly undergone splenectomy, with only four of those studies reporting clinical improvement. Therefore, the role of splenectomy in unstable hemoglobinopathies remains unclear, but seems to be a promising option in Hb Hammersmith.

Multi-Omics Analysis in ß-Thalassemia Using an HBB Gene-Knockout Human Erythroid Progenitor Cell Model.

ß-thalassemia is a hematologic disease that may be associated with significant morbidity and mortality. Increased expression of HBG1/2 can ameliorate the severity of ß-thalassemia. Compared to the unaffected population, some ß-thalassemia patients display elevated HBG1/2 expression levels in their red blood cells. However, the magnitude of up-regulation does not reach the threshold of self-healing, and thus, the molecular mechanism underlying HBG1/2 expression in the context of HBB-deficiency requires further elucidation. Here, we performed a multi-omics study examining chromatin accessibility, transcriptome, proteome, and phosphorylation patterns in the HBB homozygous knockout of the HUDEP2 cell line (HBB-KO). We found that up-regulation of HBG1/2 in HBB-KO cells was not induced by the H3K4me3-mediated genetic compensation response. Deletion of HBB in human erythroid progenitor cells resulted in increased ROS levels and production of oxidative stress, which led to an increased rate of apoptosis. Furthermore, in response to oxidative stress, slower cell cycle progression and proliferation were observed. In addition, stress erythropoiesis was initiated leading to increased intracellular HBG1/2 expression. This molecular model was also validated in the single-cell transcriptome of hematopoietic stem cells from ß-hemoglobinopathy patients. These findings further the understanding of HBG1/2 gene regulatory networks and provide novel clinical insights into ß-thalassemia phenotypic diversity.

Prime Editor 3 Mediated Beta-Thalassemia Mutations of the HBB Gene in Human Erythroid Progenitor Cells.

Recently developed Prime Editor 3 (PE3) has been implemented to induce genome editing in various cell types but has not been proven in human hematopoietic stem and progenitor cells. Using PE3, we successfully installed the beta-thalassemia (beta-thal) mutations in the HBB gene in the erythroid progenitor cell line HUDEP-2. We inserted the mCherry reporter gene cassette into editing plasmids, each including the prime editing guide RNA (pegRNA) and nick sgRNA. The plasmids were electroporated into HUDEP-2 cells, and the PE3 modified cells were identified by mCherry expression and collected using fluorescence-activated cell sorting (FACS). Sanger sequencing of the positive cells confirmed that PE3 induced precise beta-thal mutations with editing ratios from 4.55 to 100%. Furthermore, an off-target analysis showed no unintentional edits occurred in the cells. The editing ratios and parameters of pegRNA and nick sgRNA were also analyzed and summarized and will contribute to enhanced PE3 design in future studies. The characterization of the HUDEP-2 beta-thal cells showed typical thalassemia phenotypes, involving ineffective erythropoiesis, abnormal erythroid differentiation, high apoptosis rate, defective alpha-globin colocalization, cell viability deterioration, and ROS resisting deficiency. These HUDEP-2 beta-thal cells could provide ideal models for future beta-thal gene therapy studies.

Haematological abnormalities in children with sickle cell disease and non-severe malaria infection in western Kenya.

BACKGROUND: In Plasmodium falciparum infection, clinical conditions such as anaemia, thrombocytopenia and leukocytosis are common. Mutation in haemoglobin sub-unit beta gene (HBB) may be a genetic factor responsible for these haematological changes during infection. However, the contributions of the carriage of different HBB genotypes on these changes remain largely unknown. METHODOLOGY: In this cross-sectional study, we evaluated haematological abnormalities in P. falciparum-infected children (n = 217, aged 1-192 months) with different haemoglobin sub-unit beta (HBB) genotypes (HbAA, HbAS and HbSS). Children with acute febrile conditions were recruited at Jaramogi Oginga Odinga Teaching and Referral Hospital at the outpatient clinic. Haematological parameters were determined using Beckman Coulter counter ACTdiff2™ while HBB genotyping was done using TaqMan® SNP genotyping assay. Chi-square (χ2) was used to determine differences between proportions. Differences in haematological parameters were compared across groups using Kruskal Wallis test and between groups using Mann Whitney U test. Partial correlation test was used to determine correlation between haematological parameters and sickle cell genotypes while controlling for age and sex. RESULTS: Haemoglobin (Hb), [median (IQR); 7.3 (1.3), P = 0.001], haematocrit (HCT), [median (IQR); 26.4 (4.4), P = 0.009], red blood cells (RBC), [median (IQR); 3.2 (1.7), P = 0.048] were markedly reduced in HbSS, however, red cell distribution with (RDW) [median (IQR); 14.9 (3.3), P = 0.030] was increased in malaria infected children with HbSS. Severe anaemia was highest in HbSS (23.1%) followed by HbAA (8.6%) and HbAS (7.1%). There were no differences in platelet count (P = 0.399) hence no severe thrombocytopeania across the genotypes. Leukocytosis was highest in HbSS (69.2%), 42% in HbAS and 31% in HbAA. The RBC, HCT and Hb had negative correlation with RDW in HbSS in malarial-infected children (r = - 0.725, P = 0.008), (r = - 0.718, P = 0.009) and (r = - 0.792, P = 0.002), respectively. CONCLUSION: Our study reveals that anaemia is the most common abnormality in malaria-infected children with carriage of HbSS. The RBC, HCT and Hb concentration decrease with increase in RDW levels in infected children with carriage of HbSS compared to other HBB genotypes. Therefore, carriage of HbSS genotype is correlated with severity of haematological abnormalities.

Noninvasive prenatal diagnosis for pregnancies at risk for ß-thalassaemia: a retrospective study.

OBJECTIVE: To evaluate the clinical feasibility of noninvasive prenatal diagnosis (NIPD) for ß-thalassaemia using circulating single molecule amplification and re-sequencing technology (cSMART). DESIGN: Through carrier screening, 102 pregnant Chinese couples carrying pathogenic HBB gene variants were recruited to the study. Pregnancies were managed using traditional invasive prenatal diagnosis (IPD). Retrospectively, we evaluated the archived pregnancy plasma DNA by NIPD to evaluate the performance of our cSMART assay for fetal genotyping. SETTING: Chinese prenatal diagnostic centres specialising in thalassaemia testing. POPULATION: Chinese carrier couples at high genetic risk for ß-thalassaemia. METHODS: Fetal cell sampling was performed by amniocentesis and HBB genotypes were determined by reverse dot blot. NIPD was performed by a newly designed HBB cSMART assay and fetal genotypes were called by measuring the allelic ratios in the maternal cell-free DNA. MAIN OUTCOME MEASURES: Concordance of HBB fetal genotyping between IPD and NIPD and the sensitivity and specificity of NIPD. RESULTS: Invasive prenatal diagnosis identified 29 affected homozygotes or compound heterozygotes, 54 heterozygotes and 19 normal homozygotes. Compared with IPD results, 99 of 102 fetuses (97%) were correctly genotyped by our NIPD assay. Two of three discordant samples were false positives and the other sample involved an incorrect call of a heterozygote carrier as a homozygote normal. Overall, the sensitivity and specificity of our NIPD assay was 100% (95% CI 88.06-100.00%) and 97.26% (95% CI 90.45-99.67%), respectively. CONCLUSIONS: This study demonstrates that our cSMART-based NIPD assay for ß-thalassaemia has potential clinical utility as an alternative to IPD for pregnant HBB carrier couples. TWEETABLE ABSTRACT: A new noninvasive test for pregnancies at risk for ß-thalassaemia.

Report of Two Novel Thalassemia Variants, HBB: c.181delG and HBA1: c.121_126delAAGACC, in Chinese Individuals.

In this study, we report two novel thalassemia variants detected in Chinese individuals using targeted NGS technology. We detected a novel frameshift variant, HBB: c.181delG, in a 32-year-old Chinese individual. This novel variant [a single nucleotide deletion at nucleotide 181 of codon 60 (-G)], was detected by targeted next generation sequencing (NGS), resulting in a stop codon at codon 60 in exon 2 of the HBB gene. The impact of this novel variant was further analyzed by an in vitro model. We also identified a novel in-frame variant, HBA1: c.121_126delAAGACC [codons 40/41 (-AAGACC)], in another Chinese individual in this study. We named these two novel variants, HBB: c.181delG and HBA1: c.121_126delAAGACC according to the Human Genome Variation Society (HGVS), which were detected by the first author. These two novel variants have expanded the mutation spectrum of thalassemia and it would be beneficial for carrier screening, genetic counseling and prenatal diagnosis (PND) of thalassemia.

Elevated Hb A2 is Not Always Indicative of ß-Thalassemia.

Hb A2 levels are usually high in carriers of ß-thalassemia (ß-thal). These levels also provide a sensitive marker for the identification of hemoglobin (Hb) variants. In this study, we aimed to examine two patients from two Chinese families who showed elevated Hb A2 levels but did not show any signs of ß-thal. The HBB variants were analyzed using direct sequencing of HBB and in silico prediction analysis. Moreover, the family's genetic history was investigated. We examined two probands from different Chinese families with elevated Hb A2 levels who were not afflicted with ß-thal, although several nucleotide changes were found at codon 81 (CTC>CTA) (HBB: c.246C>A) in Family 1 and a compound heterozygosity for codon 40 (AGG>AAG) (HBB: c.122G>A) and IVS-II-478 (C>A) (HBB: c.316-373C>A) in Family 2. After investigating the genetic history of both families including the ß-thal aspect, we found that these mutations were not responsible for the elevated Hb A2 levels. It is rarely reported that high Hb A2 level is not indicative of ß-thal. In contrast, low or normal Hb A2 level is always found with ß-thal due to other molecular defects that mask their ß-thal genotype. Our results highlight the importance of considering the genetic factors related and unrelated to ß-thal to improve the accuracy of future genetic counseling.