Development of a rapid multiplex PCR assay for identification of the three common Hemoglobin‐Lepore variants (Boston‐Washington, Baltimore, and Hollandia) and identification of a new Lepore variant
Authors: Roberto H. Nussenzveig, Derek L. Vanhille, Dottie Hussey, Archana M. Agarwal
Journal: American Journal of Hematology (2012)

Hemoglobin (Hb) Lepore is a β+-thalassemic variant composed of two α-globin chains and two δβ-globin hybrid chains. Three common Hb Lepore variants have been identified based on their deletion breakpoints: Hb Lepore-Hollandia, Baltimore, and Boston/Washington. Although standard diagnostic methods may suffice for identification of many hemoglobinopathies, they cannot be used for routine detection and confirmation of Hb Lepore. We report the development of a rapid multiplex Gap-PCR assay for identification of the three common variants in a single tube format and identification of a novel breakpoint variant. Nineteen patient samples with suspected Hb Lepore, 1 known case of Hb Lepore-Baltimore, and 10 wild-type samples were tested and subsequently sequenced to confirm breakpoints. Fourteen Boston-Washington, 3 Baltimore, and 2 Hollandia samples were identified. One sample suspected of harboring an Hb Lepore by HPLC was negative by both multiplex Gap-PCR and sequencing. Breakpoints for the other 19 positive samples were confirmed by sequencing. The breakpoint for one sample, initially identified as Hb Lepore-Baltimore by our Gap-PCR assay, was that of a novel Hb Lepore (δ31Leu/β51Thr), which we have named Hb Lepore-ARUP. The single reaction tube format, high throughput, and rapid turnaround make our assay highly useful in the clinical laboratory. Hemoglobin (Hb) Lepore is a β+-thalassemia-producing hemoglobin variant composed of two α-globin chains and two δβ-globin hybrid chains. Hemoglobin (Hb) Lepore was first described by Gerald and Diamond [1], however Baglioni [2] was the first to recognize the molecular cause for this abnormal hemoglobin. Because of the significant homology between the δ and β globin genes frequent recombination events resulting in unequal crossover between both genes can lead to deletion of a 7.4 kb DNA fragment. The result of these events is an in frame fusion that includes the 5′ half of the δ gene with the 3′ half of the β gene. In addition, decreased transcription efficiency of the δβ hybrid chain is noted [3] since transcription from the δ gene promoter is less efficient than the β gene promoter. Due to decreased quantity of the δβ hybrid chain, a β-thalassemia minor phenotype with mild hypochromic, microcytic anemia is noted. Homozygosity or compound heterozygosity of Hb Lepore and β-thalassemia can result in thalassemia major or intermedia phenotypes [3]. Hb Lepore occurs worldwide and in many ethnic groups, however it is seen most commonly in southern Europeans [4-6]. It is one of the most common abnormal Hbs in Caucasians of Central Portugal [4]. In contrast, it is rare in African Americans with few cases having been reported [7, 8]. Three common Hb Lepore variants have been identified based on the primary sequence of the fusion proteins: Hb Lepore-Hollandia (δ 1-22/β 50-146), Hb Lepore-Baltimore (δ 1-50/β 86-146), and Hb Lepore-Boston/Washington (δ 1-87/β 116-146) (Supporting Information Fig. S1 A) [3, 9]. Hemoglobin Lepore-Boston-Washington is by far the most common variant reported worldwide [10]. Hb Lepore-Hollandia is extremely rare and restricted mostly to Bangladesh and New-Guinea [10]. In heterozygous individuals, the relative percentage of Hb Lepore ranges between 6 and 15% with normal or reduced Hb A2 and increased Hb F levels [3, 5]. The presence of Hb Lepore is often suggested by hemoglobin electrophoresis or cation exchange high performance liquid chromatography (HPLC). Hb Lepore separates from HbA and A2 by alkaline gel electrophoresis with a mobility similar to that of Hb S; in contrast, on acid gel electrophoresis Hb Lepore comigrates with HbA and A2 [5]. Hb Lepore can also be identified by globin chain analysis using reversed phase HPLC [5]. However, molecular methods are required for confirmation of a HPLC result, to determine the specific variant, and may also be useful for patient management, genetic counseling, and prenatal diagnosis. A total of 30 samples were tested by a novel multiplex Gap-PCR assay: 19 patient samples with suspected Hb Lepore, 1 previously confirmed Hb Lepore Baltimore (Coriell, Camden, NJ), and 10 wild-type control samples. A diagram of Hb Lepore variants and primer locations is depicted in Supporting Information Figure S1 B. Patient's age ranged from 1 to 89 years with an average age of 38 years. Patient sample testing was positive for 14 Hb Lepore-Boston/Washington, 3 Hb Lepore-Baltimore, 2 Hb Lepore-Hollandia, and 1 patient sample was Hb Lepore negative (wild type). Experimental results are summarized in Supporting Information Table SI. Hematological data was available for nine patient samples (6 Boston-Washington, 2 Hb Lepore-Hollandia, and 1 Hb Lepore- Baltimore). Patients hemoglobin ranged from 9.3 to 14.3 g/dL and the mean corpuscular volume (MCV) ranged from 62.5 to 82.7 fL. Hematological findings are summarized in Supporting Information Table S-II. No difference in hematological data was observed between the three different Hb Lepore variants. One patient identified as homozygous Lepore-Boston-Washington in our multiplex assay was determined to be hemizygous with deleted δ and β globin genes on one allele by MLPA (data not shown) and Hb Lepore-Boston-Washington on the other. Representative results for the multiplex Gap-PCR for the three Hb Lepore variants and a wild-type sample are shown in Supporting Information Figure S2 (A and B, QIAxcel system and agarose gel respectively). No Hb Lepore amplicon was detected in the wild-type control samples by multiplex Gap-PCR (data not shown). The amplification and correct genotyping of each variant was determined by consistent production of amplicons of appropriate size. Breakpoints for 18/19 samples (∼95%), including the Coriell positive control, were confirmed by sequencing (Supporting Information Table SI). The breakpoint for one patient sample, initially identified as Hb Lepore-Baltimore by our Gap-PCR assay, was not confirmed by sequencing; instead, a novel Hb Lepore breakpoint between δ31Leu and β50Thr was identified, which we have named Hb Lepore-ARUP (Supporting Information Fig. S3 A and B). One of the drawbacks of our assay is the possibility for misdiagnosis of compound heterozygosity of deletional δβ0-thalassemia and Hb Lepore as homozygous Hb Lepore. However, this is an inherent problem with Gap-PCR deletional assays and requires high degree of clinical suspicion and correlation for proper diagnosis. One sample suspected to harbor an Hb Lepore mutation based on HPLC and gel electrophoresis was found to be negative (wild-type) by our multiplex PCR assay and sequencing control reaction, thus emphasizing the importance of our novel multiplex PCR test for confirmation of HPLC results. Molecular testing for Hb Lepore is useful for confirmation of HPLC hemoglobinopathy screening, as well as breakpoint identification for the purpose of genetic counseling. In conclusion, we developed an accurate, fast, sensitive, and specific assay for the molecular identification/confirmation of the three most common Hb Lepore variants. While standard diagnostic methods may suffice for identification of many hemoglobinopathies, they cannot be solely relied on for routine detection and confirmation of Hb Lepore. Moreover, with increasing migrant population, complex patterns of Hb Lepore in combination with β-thalassemic mutations such as Hb E/Hb Lepore are not uncommon. Besides causing thalassemia intermedia, these complex patterns can also lead to a diagnostic conundrum [11, 12]. Our novel multiplex Gap-PCR assay can be used to confirm an Hb Lepore variant as well as which of the three common variants is present. The single reaction tube format, ease of setup, high throughput, and rapid turnaround time make our assay highly useful in the clinical laboratory. The discovery of the new variant (Hb Lepore-ARUP) provides additional information regarding the variability seen in the β-globin gene cluster, especially in relation to hybrid globins. A total of 19 peripheral blood samples were collected using an approved Institutional Review Board protocol. One sample with confirmed Hb Lepore-Baltimore was obtained from Coriell laboratories (Coriell, Camden, NJ). Nineteen samples, submitted to our laboratory for hemoglobin evaluation, were suspected of harboring an Hb Lepore variant based on HPLC testing (Variant I HPLC system, Bio-Rad, Hercules, CA). Ten samples from normal healthy control volunteers with no known hemoglobin dysfunction were used as wild-type controls. Samples were extracted using the MAGna Pure Compact system (Roche, Indianapolis, IN). PCR master-mix included Platinum Taq polymerase (Invitrogen, Grand Island, NY) and EpiCentre Premix-D (EpiCentre, Madison, WI). Cycling conditions: 96°C for 5 min, (96°C for 20 sec, 55°C for 30 sec, 72°C for 45 sec) x35 cycles, and 72°C for 5 min. PCR primers were purchased from IDT, (Integrated DNA Technologies, Coralville, IA). PCR amplification of the three common Hb Lepore variants was performed using a common forward primer and a variant specific reverse primer. Primer sequences can be found in Supporting Information Table S-III. Resulting PCR amplicons were separated on the QIAxcel system (Qiagen, Valencia, CA) or by 2% agarose gel electrophoresis. On the basis of the diagram of Hb Lepore variants and primer locations (Supporting Information Fig. S1 B), it should be clear that amplification from the variant specific Hb Lepore-Hollandia reverse primer (smallest variant amplicon) will also result in amplification from the other two variant specific reverse primers. Conversely, amplification from the variant specific Hb Lepore-Boston/Washington reverse primer (largest variant amplicon) will not amplify the two other variants since their reverse specific primers are located within the deleted region. Amplification products for each Hb Lepore variant are as follows: Boston/Washington will have a single variant specific 616 bp band; Baltimore will have 2 bands (a 552 bp variant specific band, and the Boston/Washington band); and Hollandia will have 3 bands (a 359 bp variant specific band and two other bands for Boston/Washington and Baltimore). PCR controls include primers within the common Hb Lepore deleted region (wild-type allele) and primers specific to the 3′UTR of the LIS1 gene (amplification control) that generate PCR amplicons of 439 base pairs and 197 base pairs respectively. A generic primer set, flanking all three Hb Lepore variants, was used to generate a ∼700 bp amplicon from genomic DNA of the multiplex Gap-PCR positive Hb Lepore patient samples. PCR products were treated with ExoSAP-IT (GE Life Sciences, Piscataway, NJ) prior to sequencing. Sequencing was performed using BigDye Terminator v1.1 (Applied Biosystems, Foster City, CA), and 0.8 μM of the appropriate primer in a total volume of 10 μL. Immediately following thermocycling unincorporated terminator dyes and salts were removed by gel filtration, and the eluate was loaded and separated on a 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). Sequencing results were analyzed with the Mutation Surveyor software (SoftGenetics, State College, PA). Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Roberto H. Nussenzveig*, Derek L. Vanhille*, Dottie Hussey*, Archana M. Agarwal* , * Department of Pathology/ARUP Laboratories, Associated Regional University Pathologists (ARUP) Laboratories, Salt Lake City, Utah, Department of Pathology, University of Utah, Salt Lake City, Utah.