Screening For Hemoglobinopathies Research Articles

Newborn Screening for Hemoglobinopathies and Red Cell Enzymopathies in Tripura State: A Malaria-Endemic State in Northeast India

Hemoglobinopathies are a group of inherited single gene disorders. There are reports on hemoglobin (Hb) variants identified in the tribal and non-tribal populations of Tripura State in northeastern India. This study aimed to determine the spectrum of hemoglobinopathies and enzymopathies by newborn screening in Tripura State and assess the extent of neonatal jaundice. A total of 2400 cord blood samples were collected and analyzed by high performance liquid chromatography (HPLC). Further confirmation of any abnormal HPLC was done by DNA analysis. The samples were also screened for deficiency of enzymopathies, glucose-6-phosphate dehydrogenase (G6PD) deficiency and pyruvate kinase. Of 2400 cord blood samples screened, 225 (9.3%) were Hb E (HBB: c.79G>A) heterozygotes, 80 (3.3%) were Hb E homozygotes and one carried Hb E-β-thalassemia (β-thal). Other Hb abnormalities were also detected including 15 Hb S (HBB: c.20A>T) heterozygotes, two Hb D-Punjab (HBB: c.364G>C) heterozygotes and two compound heterozygotes for Hb D-Punjab and Hb E. Of the 80 homozygous Hb E babies, four were non-tribal and 76 babies were tribal, and 225 patients carried Hb E trait, 141 were tribal, while 84 were non-tribal. Of 40 G6PD deficient babies identified, 13 had coinherited Hb E and two babies had pyruvate kinase deficiency. α Genotyping was performed in 162 affected babies, 50 of them carried α gene deletions. Newborn screening programs for Hb E, other hemoglobinopathies and G6PD deficiency must be encouraged in the malaria-endemic northeastern region of India. Drug-induced hemolysis can also be avoided by screening for G6PD deficiency at birth.

Antenatal carrier screening for thalassemia and related hemoglobinopathies: A hospital-based study

Background: Thalassemias are an important cause of morbidity and mortality worldwide. They create a huge emotional and financial deficit on the affected individuals and their families, society and the country. However, severely symptomatic disease can be prevented by various methods. Aims: The present study was undertaken with an aim to find the prevalence of carriers of thalassemia and other hemoglobinopathies in pregnant females and their husbands so as to identify the couples at risk of having severely affected children. Materials and Methods: The study included 2000 antenatal patients of microcytic hypochromic anemia detected on routine hematological examination. Various formulae used to differentiate between thalassemia and iron deficiency anemia were calculated. Serum ferritin levels were performed, and cases with normal or high serum ferritin were analyzed for hemoglobin variants by high-performance liquid chromatography using Bio-Rad variant II hemoglobin testing system-beta thalassemia short program. Results and Conclusion: Sixty-three of the 2000 women screened (3.15%) were identified as carriers of beta thalassemia trait and other hemoglobinopathies. Most of them that is 59 cases (2.95%) were beta thalassemia carriers while one each were carriers of HbE (0.05%), HbS (0.05%), HbD (0.05%), and double heterozygous for beta thalassemia and HbE. Among the various indices evaluated in the present study, Shine and Lal index could identify almost all cases of beta thalassemia carriers (98.3% sensitivity) whereas all the other indices could not be relied on in the identification of all the cases.

Experiences of a High-Risk Population with Prenatal Hemoglobinopathy Carrier Screening in a Primary Care Setting: a Qualitative Study

Carrier screening for hemoglobinopathies (HbPs; sickle cell disease and thalassemia) aims to facilitate autonomous reproductive decision-making. In the absence of a Dutch national HbP carrier screening program, some primary care midwives offer screening on an ad hoc basis. This qualitative descriptive study explores how pregnant women perceive an offer of HbP carrier screening by their midwife. Semi-structured interviews (n = 26) were conducted with pregnant women at risk of being a HbP carrier, and whom were offered screening at their booking appointment in one of two midwifery practices in Amsterdam. The results showed that half of the respondents were familiar with HbPs. Generally, women perceived the offer of HbP carrier screening as positive, and most women (n = 19) accepted screening. Seven declined, of whom two already knew their carrier status. Important reasons to accept screening were to obtain knowledge about their own carrier status and health of their unborn child, and the ease of the procedure. A multistep process of decision-making was observed, as many women did not give follow-up testing (e.g. partner, invasive diagnostics) much consideration while deciding on accepting or declining HbP screening. Women experienced information overload, and preferred receiving the information at a different moment (e.g. before the intake by a leaflet, or preconceptionally). In conclusion, while prenatal HbP carrier screening is perceived as positive, informed decision-making seems to be suboptimal, and both the content and timing of the information provided needs improvement.

Rapid Targeted Next-Generation Sequencing Platform for Molecular Screening and Clinical Genotyping in Subjects with Hemoglobinopathies

Hemoglobinopathies are among the most common autosomal-recessive disorders worldwide. A comprehensive next-generation sequencing (NGS) test would greatly facilitate screening and diagnosis of these disorders. An NGS panel targeting the coding regions of hemoglobin genes and four modifier genes was designed. We validated the assay by using 2522 subjects affected with hemoglobinopathies and applied it to carrier testing in a cohort of 10,111 couples who were also screened through traditional methods. In the clinical genotyping analysis of 1182 β-thalassemia subjects, we identified a group of additional variants that can be used for accurate diagnosis. In the molecular screening analysis of the 10,111 couples, we detected 4180 individuals in total who carried 4840 mutant alleles, and identified 186 couples at risk of having affected offspring. 12.1% of the pathogenic or likely pathogenic variants identified by our NGS assay, which were undetectable by traditional methods. Compared with the traditional methods, our assay identified an additional at-risk 35 couples. We describe a comprehensive NGS-based test that offers advantages over the traditional screening/molecular testing methods. To our knowledge, this is among the first large-scale population study to systematically evaluate the application of an NGS technique in carrier screening and molecular diagnosis of hemoglobinopathies.

Abstracts of Presentations Scheduled for the 10th ISNS-Asia Pacific Regional Meeting, Ulaanbataar, Mongolia, 24–26 August 2017

The International Society for Neonatal Screening (ISNS) recognises six different geographical regions [...]

English

English

Carrier Screening for the Haemoglobinopathies: Past, Present and Future

Carrier screening for the haemoglobinopathies has undergone many technological improvements in haematological and molecular diagnostic techniques since the first prenatal diagnoses by DNA analysis in the 1970s by Southern blot analysis enabled the implementation of effective successful prevention programmes for beta thalassaemia involving public education, carrier screening, genetic counselling and prenatal diagnosis in Mediterranean countries. The application of a wide variety of PCR-based molecular diagnostic techniques which allows the detection of the complete range of haemoglobinopathy mutations has led to the establishment of comprehensive national prevention programmes in many developing countries and also in countries such as those in Northern Europe in which the prevalence and heterogeneity of the haemoglobinopathies has been significantly increased by population immigration. Despite the great technological advances in mutation detection, the screening of haemoglobinopathies still requires the combined use of haematological and molecular techniques to arrive at an accurate diagnosis, and requires specialist knowledge of genotype/phenotype relationships because of the multitude of complex phenotypes which result from interactions between genotypes and co-inherited globin gene disorders relationships. The latest technological advances in mutation analysis techniques and the application of some of these for the noninvasive approach of analysis of fetal DNA in maternal blood are anticipated to improve haemoglobinopathy prevention programmes in the future.

Evaluation of four hemoglobin separation analyzers for hemoglobinopathy diagnosis.

Four automated hemoglobin separation devices are compared in their ability to detect hemoglobinopathies, both in HbA1c and in hemoglobinopathy mode. Quality control material and 58 samples, including one heterozygous α-thalassemia sample, six heterozygote β-thalassemia samples and 32 samples with a known hemoglobin variant, were used to assess imprecision of HbF and HbA2 measurements, correlation with the gold standard and sensitivity for detecting β-thalassemia and Hb variants on D-100 (Bio-Rad Laboratories), HA 8180T (Menarini), HLC-723G8 (Tosoh Bioscience) and Capillarys 2 Flex Piercing (Sebia). Imprecision was <10% for both HbF and HbA2 in all modes of all analyzers. Correlation studies for HbF and HbA2 demonstrated statistically significant but small biases when compared to the gold standard. All six β-thalassemia samples but one were detected on all analyzers using a HbA2 cut-off value of 3.5%. D-100, HA8180T and the Hb-pathy mode of the HLC-723G8 and the Capillarys are able to detect the most common important Hb variants (Hb C, D, E and S), but more seldom variants can be missed as they co-elute with HbA0. The HbA1c mode of the Capillarys correctly detected all measured hemoglobin variants and can therefore be used as a hemoglobinopathy screening device. This was also the case for the most common important Hb variants on the HbA1c mode of the HLC-723G8, but two rare variants were not detected. This study stresses the importance for individual laboratories to know the advantages and drawbacks of their hemoglobin separation analyzer and its different modes in the diagnosis of hemoglobinopathies.

Does the Novel KLF1 Gene Mutation Lead to a Delay in Fetal Hemoglobin Switch?

The Kruppel-like factor 1 (KLF1) gene is an essential transcription factor that is required for the proper maturation of the erythroid cells. Recent studies have reported that KLF1 variations are associated with increased fetal hemoglobin (HbF) levels. Here we report a novel KLF1 gene variation codon 211 A→G (c.632 A>G) in a family who was referred for hemoglobinopathy screening. Both parents were classical β-thalassemia trait (mother: HbA2 4.1%, HbF 8.6%; father: HbA2 5.5%, HbF 0.6%) codon 15 G→A heterozygous, and the child was β-thalassemia homozygous. Because the mother showed a high HbF level, the genetic determinants for raised HbF were screened. We detected a novel KLF1 gene variant in the mother and the child in the heterozygous state. The co-inheritance of this novel KLF1 variant might have increased the HbF levels in the mother and may have ameliorated the clinical manifestations of the 6-year-old untransfused β-thalassemia homozygous child. Identification of KLF1 gene variants may act as a novel target for increasing HbF levels in patients with β-hemoglobinopathies.

Care for Haemoglobinopathy Patients in Slovakia.

The paper presents the results od 22-year study of screening and follow-up of haemoglobinopathies in Slovakia, an overview of genetic mutations, the coincidence with hereditary haemochromatosis mutations, and the procedure in genetic councelling. Between 1993-2015, in three centres in Bratislava and in one centre in Kosice, carriers of beta-thalassaemic genes or other haemoglobinopathies were searched for. Diagnosis was performed by haematologists, whereby the family history was evaluated, together with the overall clinical condition, blood count and blood smear, iron and haemolysis parameters, mutations of hereditary haemochromatosis, and haemoglobin electrophoresis testing. In the last years the haemoglobin division also examined by high performance liquid chromatography (HPLC). A clinical suspicion of the heterozygous form of beta-thalassaemia or other haemoglobinopathies was documented in 554 patients. Of them 32 (5.8%) were foreigners. 213 (38.45%) patients were genetically examined. In 190 (33.93%) of them heterozygote beta-thalassaemia was confirmed. The most frequent mutations were IVS 1.110 (33.15%), IVS 2.1 (33.15%), and IVS 1.6 (14.7%). Evidence of haemoglobin S (heterozygote sickle cell anaemia) was also notable in two non-relative children, whose fathers were of African origin, and one patient from Ghana. One female patient was followed up for haemoglobin Santa Ana (non-stabile haemoglobin previously diagnosed as mutation de novo). In our group, we took care of pregnant patients with haemoglobinopathies. The study showed that there is a higher number of heterozygotes for beta-thalassaemia and rarely haemoglobinopathies in Slovakia. Over the past years, we have recorded an increase number of foreigners coming to our country. It is necessary to continue in search of pathological gene carriers to avoid serious forms of haemoglobinopathies.

Next-generation sequencing improves thalassemia carrier screening among premarital adults in a high prevalence population: the Dai nationality, China.

Thalassemia is one of the most common monogenic diseases in southwestern China, especially among the Dai ethnic group. Here, we explore the feasibility of a next-generation sequencing (NGS) screening method specifically for the Dai people. Blood samples were obtained from Dai people for premarital screening. Double-blind, parallel hemoglobinopathy screening was conducted using both traditional hematological methods (red cell indexes and hemoglobin electrophoresis, then DNA sequencing) and an NGS approach. Among 951 tested individuals, we found a thalassemia carrier rate of 49.5% (471/951) using the NGS screen, in contrast to 22.0% (209/951) found using traditional methods. Almost 74.8% (217/290) of α-thalassemia carriers and 30.5% (25/82) of composite α- and β-thalassemia carriers were missed by traditional screens. The proportion of such α- and β-thalassemia carriers among the Dai people is 8.6% (82/951). For β-thalassemia carriers, the high ratio (66/99) of CD26 mutations may suggest a correlation between CD26 and the environmental adaption of the Dai people. Methodological comparisons demonstrate the superiority of NGS for both sensitivity and specificity, provide a comprehensive assessment of thalassemia screening strategies, and indicate that NGS is a competitive screening method, especially among populations with a high prevalence of disease.Genet Med advance online publication 26 January 2017.

Utility of screening golf ball inclusions in hemoglobin H disease and its clinico-hematological profile

BACKGROUND: In India, hemoglobinopathy screening programs focus on reducing the burden of beta-thalassemia by screening the antenatal females and young adolescents. Although alpha (α)-thalassemia is common in the Mediterranean countries, alpha (α)-thalassemia screening and diagnosis are performed in a few centers in India. HbH disease is an uncommon clinical phenotype of α-thalassemia with variable clinical presentation. The hallmark feature of HbH is the presence of golf ball inclusions in the red cells demonstrated with supravital staining. PATIENTS AND METHODS: Here, we discuss an algorithmic approach to screen hemograms for thalassemic indices, followed by evaluation of reticulocyte preparation after 40 minutes (supravital stain preparation for golf ball inclusions). If golf ball inclusions were positive, Hb analysis on Hb-capillary zone electrophoresis (Hb-CZE)was done. RESULTS: We received 28,000 samples for complete hemogram and blood film examination, over a period of 8 months. Reticulocyte count was required in 11,600 samples out of which 13 cases were positive for golf ball inclusions. Of these, nine were identified as HbH disease on Hb-CZE. These patients presented with pallor and generalized weakness. On examination, jaundice (3/9 patients) and hepatosplenomegaly (4/9 patients) were relatively uncommon. The majority of them (6/9) required infrequent blood transfusions. All patients had microcytic hypochromic red cell indices with median HbA of 89% (range: 68.1%–96.9%), HbA2 of 1.4% (range: 0.8%–1.7%), and fast-moving band in zone 15 (HbH) of 5.6% (range: 1.4%–30.3%) on Hb-CZE. CONCLUSION: We emphasize a systematic screening of hemograms and peripheral smears along with a simple and cost-effective screening test for golf ball inclusions to diagnose HbH disease that often poses a clinical dilemma due to its variable clinical presentation.

Hemoglobin variants identified in the Uganda Sickle Surveillance Study

AbstractThe Uganda Sickle Surveillance Study analyzed dried blood spots that were collected from almost 100 000 infants and young children from all 10 regions and 112 districts in the Republic of Uganda, with the primary objective of determining the prevalence of sickle cell trait and disease. An overall prevalence of 13.3% sickle cell trait and 0.7% sickle cell disease was recently reported. The isoelectric focusing electrophoresis technique coincidentally revealed numerous hemoglobin (Hb) variants (defined as an electrophoresis band that was not Hb A, Hb F, Hb S, or Hb C) with an overall country-wide prevalence of 0.5%, but with considerable geographic variability, being highest in the northwest regions and districts. To elucidate these Hb variants, the original isoelectric focusing (IEF) gels were reviewed to identify and locate the variant samples; corresponding dried blood spots were retrieved for further testing. Subsequent DNA-based investigation of 5 predominant isoelectric focusing patterns identified 2 α-globin variants (Hb Stanleyville II, Asn78Lys; Hb G-Pest, Asp74Asn), 1 β-globin variant (Hb O-Arab, Glu121Lys), and 2 fusion globin variants (Hb P-Nilotic, β31-δ50; Hb Kenya, Aγ81Leu-β86Ala). Compound heterozygotes containing an Hb variant plus Hb S were also identified, including both Hb S/O-Arab and HbS/Kenya. Regional differences in the types and prevalence of these hemoglobin variants likely reflect tribal ancestries and migration patterns. Algorithms are proposed to characterize these Hb variants, which will be helpful for emerging neonatal hemoglobinopathy screening programs that are under way in sub-Saharan Africa.

Hemoglobinopatias em Portugal e a intervenção do médico de família

Hemoglobinopathies are among the most common inherited diseases. Early diagnosis can reduce morbidity and mortality with prompt implementation of preventive and therapeutic measures. The family physician can perform screening tests for hemoglobinopathies to help achieve this. This article reviews the clinical and epidemiological features of the major hemoglobinopathies. Based on these characteristics, an approach is proposed for primary health care services to identify and manage the complications related to these conditions. In order to promote critical reflection on this topic, we have collected the literature on the epidemiology of these conditions in Portugal. There is only one Portuguese guideline on hemoglobinopathies. This was published in 2004 but there have been no epidemiological studies published in Portugal since the 1980s. Given the patterns of external and internal migration of the population since that time, health professionals should reflect on the need for targeted screening for hemoglobinopathies in the risk areas identified previously. We conclude that is urgent to sensitize and train family physician to address these conditions. A new study of the national epidemiology of hemoglobinopathies and possible regional variations is necessary in order to make decisions based on scientific evidence and focused on the specific needs of the population.

The New -474(C→T) Substitution Discovered in the HBG2 Promoter of a Sardinian δβ-Thalassemia Carrier

During a screening for hemoglobinopathies, we found a carrier of the Sardinian δβ-thalassemia condition. The proband's hematology and hemoglobin (Hb) profile agreed with those of the other carriers previously identified during our diagnostic program except for the fetal Hb (HbF) composition, which consisted of both α₂^Aγ₂ and α₂^Gγ₂ instead of nearly 100% α₂^Aγ₂. In order to explain the unusual γ-chain ratio, sequencing of the ^Gγ promoter was carried out and revealed two nucleotide substitutions in cis: C→T at position -474 and A→G at position -309 from the Cap site. The latter had previously been observed in subjects with raised HbF levels, although it has not yet been evaluated at functional level. We used the luciferase assay to determine whether the two mutations modify the transcriptional activity of the ^Gγ promoter. Results indicated that the observed in vivo ^Gγ-globin production cannot be translated into increased in vitro promoter function, suggesting that the assessed mutations cannot be considered as functional single nucleotide polymorphisms per se; instead, a more complex regulatory mechanism might be involved.

Rapid phenotype hemoglobin screening by high-resolution mass spectrometry on intact proteins

BackgroundGiven the excellent performance of modern mass spectrometers, their clinical application for the analysis of macromolecules is a growing field of interest. This principle is explored by hemoglobin analysis, which is a representative example by its molecular weight and clinical relevance in e.g. screening programs for thalassemia and hemoglobin variants. Considering its abundance and cellular containment, pre-analysis is significantly reduced allowing for essential rapid acquisitions. MethodsBy parallel analysis of routine diagnostics for hemoglobin variants and thalassemia, we acquired samples of adults who were consented for hemoglobinopathy screening in our clinical laboratory. The pre-analytical process comprised of red cell lysis only; without further digestion and purification steps, the samples were directly injected in an electrospray ionization quadrupole time-of-flight setup and the intact proteins were analyzed by flow injection analysis. After optimization of process parameters, the deconvoluted mass spectra revealed the presence of α- and β-globulins. The reference ranges for the average mass of both globulins and their intensity ratio (α/β-ratio) were deduced from a disease-free subgroup and patients with a hemoglobinopathy were compared. ResultsThe α/β-ratio is a poor marker for thalassemia patients, yet deviant α/β-ratios are found for patients with a hemoglobin variant. Mass deviations down to 1Da can be resolved; even if the patient suffers from a heterozygotic disorder, the average mass is found outside the established reference interval. ConclusionsAlthough subjects with mild thalassemia were not detected, all patients with a hemoglobin variant were resolved by top-down mass spectrometry using the average globulin mass and the α/β-ratio as screening parameters.

Newborn screening for sickling and other haemoglobin disorders using tandem mass spectrometry: A pilot study of methodology in laboratories in England.

To determine (i) if electrospray mass spectrometry-mass spectrometry with the SpOtOn Diagnostics Ltd reagent kit for sickle cell screening could be integrated into the English newborn screening programme, under routine screening conditions, and provide mass spectrometry-mass spectrometry results which match existing methods, and (ii) if common action values could be set for all manufacturers in the study, for all assessed haemoglobins, to indicate which samples require further investigation. Anonymised residual blood spots were analysed using the SpOtOn reagent kit as per manufacturer's instructions, in parallel with existing techniques at four laboratories. Mass spectrometry-mass spectrometry instrumentation at Laboratories A and B was AB Sciex (Warrington, UK) AP4000, and at Laboratories C and D, Waters Micromass (Manchester, UK), Xevo TQMS and Premier, respectively. There were 23,898 results accepted from the four laboratories. Excellent specificity at 100% sensitivity was observed for haemoglobin S, haemoglobin C, haemoglobin E and haemoglobin OArab. A common action value was not possible for Hb C, but action values were set by manufacturer. The two haemoglobin DPunjab cases at Laboratory D were not detected using the common action value. Conversely, false-positive results with haemoglobin DPunjab were a problem at the remaining three laboratories. This multicentre study demonstrates that it is possible to implement mass spectrometry-mass spectrometry into an established screening programme while maintaining consistency with existing methods for haemoglobinopathy screening. However, one of the instruments investigated cannot be recommended for use with this application.

Report on Ten Years’ Experience of Premarital Hemoglobinopathy Screening at a Center in Antalya, Southern Turkey

Thalassemia and hemoglobinopathies are a major public health problem in Turkey. Hemoglobinopathy prevention programs (HPPs) were started in 33 provinces situated in Thrace, Marmara, Aegean, Mediterranean and South Eastern regions of Turkey in 2003. A premarital hemoglobinopathy test is mandatory and free of charge in this program. According to the Ministry of Health reports, 46 first level hemoglobinopathy diagnostic centers were established for premarital tests. Within the last 10 years, approximately 79.0% of married individuals per year were screened by the centers. While the percentage of premarital screening tests was 30.0% of all couples in 2003, it reached 86.0% in 2013. The number of newborn with thalassemia and hemoglobinopathies were 272 in 2002 and dropped to 25 in 2013. There has been a 90.0% reduction in affected births. Our hemoglobinopathy diagnostic center was established in 2003 and licensed by the Ministry of Health in 2004. We studied a total of 89,981 blood samples from premarital tests for 10 years and the incidence of β- and α-thalassemia (β- and α-thal) trait was found to be 6.57 and 3.56%, respectively. The distribution of the most common abnormal hemoglobins (Hbs) was: Hb S (HBB: c.20A > T) (0.31%), Hb D-Los Angeles (HBB: c.364G > C) (0.15%), Hb G-Coushatta (HBB: c.68A > C) (0.06%) and Hb E (HBB: c.79G > A) (0.02%). A total of 60 couples, both carrying β-thal trait, were directed to the prenatal diagnosis (PND) center in 10 years. The premarital hemoglobinopathy screening program is running successfully at our center and other centers in Turkey.

Antenatal haemoglobinopathy screening in Australia.

Haemoglobinopathy screening should be performed in women with microcytic indices, women from high risk ethnic populations and those with unexplained anaemia. Early testing of women and their partners expedites appropriate management prior to and during pregnancy. Haemoglobinopathy screening is a multistep process beginning with a full blood count, ferritin assay, screening tests for haemoglobinopathies (ie, haemoglobin electrophoresis, high performance liquid chromatography, capillary electrophoresis) and assessment of clinical risk. Iron deficiency may obscure the diagnosis of β-thalassaemia trait. If possible, haemoglobinopathy testing should be performed when the woman is iron-replete. Genetic testing can be offered on the basis of the combined risk of the couple; but turnaround times are lengthy at present, hence the emphasis on early pregnancy or pre-conception screening. Screening processes vary between states and local health districts; a uniform approach to screening and genetic testing with a national registry to record results would improve management of this growing problem.

The Problem of Borderline Hemoglobin A2 Levels in the Screening for β-Thalassemia Carriers in Sardinia

Background: The increase in HbA2 is the most important parameter for the identification of thalassemia carriers. However, in routine screening for hemoglobinopathies, some cases are difficult to classify because the level of HbA2 is not typically elevated. In this work, we report the results of a molecular investigation on a cohort of subjects with borderline HbA2. Methods: All subjects with a β-thalassemia carrier partner and a borderline percentage level of HbA2 were investigated for the presence of a pathological mutation in the β-globin gene. All negative subjects were screened for both the KLF1 mutation and the presence of ααα/ or αααα/ alleles. The subjects with reduced MCV and/or MCH were also screened for deletional and nondeletional α-globin gene defects. Results: Various β-globin mutations and KLF1 gene defects are the most common genetic determinants responsible for this phenotype in our population. Conclusion: KLF1 mutations are important in a screening program for hemoglobinopathies. An increase in HbF in association with borderline HbA2 levels is a useful but not exclusive marker that suggests the investigation of this gene. On the basis of our findings, we are able to suggest the molecular procedure to use in a population characterized by a high prevalence of thalassemia carriers.