American Journal of Hematology 75:220–224 (2004)
Accurate and Rapid Prenatal Diagnosis of the Most Frequent East Mediterranean b-Thalassemia Mutations R.P. Naja,1 H. Kaspar,2 H. Shbaklo,1 N. Chakar,1 N.J. Makhoul,1 and P.A. Zalloua1,2,3* 1 Genetics Research Laboratory, Chronic Care Center, Beirut, Lebanon Department of Obstetrics and Gynecology, American University of Beirut, Beirut, Lebanon 3 Program for Population Genetics, Harvard School of Public Health, Boston, Massachusetts 2
b-Thalassemia is the most common genetic disorder in the Lebanese population. Of the 200 different mutations in the b-globin gene that leads to thalassemia, the IVSI-110 (29.87%), IVSI-6 (20.74%), IVSI-1 (14.07%), IVSII-1 (9.13%), Cd29 (9.13%), and Cd30 (3.95%) mutations are the most frequent among Lebanese thalassemic patients. These mutations are also present at high frequencies in the East Mediterranean region. Due to this high prevalence of certain b-thalassemia mutations, a rapid technique for the prenatal diagnosis of these mutations was implemented. The technique used is based on Real-Time PCR quantification and melting curve analysis of the amplified fragment using the LightCycler. The DNA samples used for amplification were obtained from CVS or amniotic fluid. Six mutations were easily and efficiently detected using only 3 sets of probes. With this method, mutant genotypes can be easily distinguished from normal alleles. In prenatal diagnosis, the accuracy and the speed of testing are paramount. The method of prenatal b-thalassemia mutations detection described here is efficient and fast, with the entire procedure including DNA preparation taking less than half a workday. It is safe, does not involve radioactivity, and is accurate showing 100% concordance with conventional DNA sequencing methods. Am. J. Hematol. 75:220–224, 2004. ª 2004 Wiley-Liss, Inc. Key words: thalassemia; LightCycler; diagnosis; prenatal; melting curves
INTRODUCTION
Thalassemia belongs to a group of inherited autosomal recessive disorders of the blood collectively known as the hemoglobinopathies (1), characterized by the defective production of hemoglobin, which leads to anemia (2). Hemoglobinopathies are a major public health problem in Lebanon as well as in many Mediterranean countries (3). b-Thalassemia occurs most frequently in people of Mediterranean origin and it is the most common hemoglobin disorder in Lebanon (4). The gene frequency of b-thalassemia, in the Lebanese population, is estimated to be 2–3% (5). The b-globin gene is under regulatory control and consists of 3 coding sequences and 2 non-coding intervening sequences (IVS) (6). b-Thalassemia is caused by any one of numerous mutations in the b-globin gene and its regulatory sequences (7). In a study of the molecular basis of b-thalassemia in Lebanon, eight different mutations were identified (8). Six of these mutations (IVSI-6, ª 2004 Wiley-Liss, Inc.
IVSI-110, IVSII-1, IVSII-745, Cd8, and Cd39) are common to the Mediterranean region whereas the other two mutations (IVSI-5 and Cd29) are more specific to Lebanon (8). The mutational analysis of 500 Lebanese thalassemic patients showed that the IVSI-110 (29.87%), IVSI-6 (20.74%), IVSI-1 (14.07%), IVSII-1 (9.13%), Cd29 (9.13%), and Cd30 (3.95%) mutations were the most frequent (unpublished data). Due to the high prevalence of certain b-thalassemia mutations in Lebanon, a rapid and accurate technique for the prenatal diagnosis of the most frequent mutations was developed and implemented in our laboratory. *Correspondence to: Zalloua Pierre A, Ph.D., Genetics Research Laboratory, Chronic Care Center, Hazmieh, Lebanon. E-mail:
[email protected] Received for publication 18 March 2003; Accepted 17 October 2003 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ajh.20013
Technique: b-Thalassemia Mutations Using Real-Time PCR
This technique is based on the employment of RealTime PCR quantification and melting curve formation for the mutation detection. The method relies on the use of fluorescent-labeled oligonucleotide probes hybridizing to complementary sequences harboring the mutations, hence producing curves with melting peaks that clearly distinguish between the wild type and the mutant genotypes [9,10]. One individual hybridization probe was designed for each of the IVSI-110 and the IVSII-1 mutations and a third probe was designed to detect the remaining four mutations, namely, Cd29, Cd30, IVSI-6, and IVSI-1 mutations. This LightCycler method is rapid, cheap, accurate (showing 100% concordance with conventional sequencing methods), and can be used routinely in prenatal diagnosis. The technique is quite robust and easily interpreted and reproduced. The principle used here can also be applied to detect point mutations in other genetic diseases. MATERIALS AND METHODS Patients and Samples
Our genetics research laboratory is situated in the Chronic Care Center in Lebanon. This center harbors the country’s only professional unit for the prevention and treatment of b-thalassemia. Currently 580 thalassemic children are being treated at the center. Couples at-risk of bearing a child with a hemoglobin defect come to our laboratory after having consulted their own physician or the center’s hematologist. The couples are counseled by a geneticist, and are informed of the risks involved in bearing a thalassemic child. If a couple decides to undergo prenatal diagnosis, a trained social worker and a nurse read and explain a test consent form to both parents. After both parents sign the consent form, the test is then performed. DNA Extraction
The prenatal diagnostic assay requires 5 ml of amniotic fluid that is free of maternal blood or a pinhead sized clean chorionic villus sample (CVS). A 500-ml blood sample is required from each parent to confirm the diagnosis, preferably before the amniocentesis or the CVS is performed. DNA from blood: 500 ml of each sample are lysed using 1 ml red cell lysis buffer (RCLB): 0.144 M NH4Cl–1 mM KHCO3 in repeating steps of incubation at 37 C and centrifugation at 13,000 rpm for one minute. The cell pellet is resuspended in 700 ml of white cell lysis buffer (WCLB): 10 mM Tris-Cl pH 8.0 – 5 mM ethylenediaminetetra-acetic acid (EDTA) – 400 mM NaCl), 15 ml of 10% sodium dodecyl sulfate (SDS) and 7 ml of proteinase K (20mg/ml) and incubated at 55 C for 2 hr. After incubation, 240 ml of 5.5 M NaCl is
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added and the sample is well vortexed and centrifuged for 15 min at 13,000 rpm. The supernatant was then transferred into a fresh tube containing 600 ml of 99% ethanol. The tube is gently mixed then again centrifuged for 10 min. at 13,000 rpm. Ethanol is discarded, and the pellet is washed by 200 ml of 70% ethanol; the tube was then centrifuged for 5 min. at 13,000 rpm, Ethanol aspirated from the tube, and the pellet air dried and resuspended in 20 ml of Tris-EDTA buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA). DNA from CVS: clear white villi are isolated under a dissecting microscope and washed in 150 ml of 1 PBS. One-hundred fifty ml of WCLB was added to the villi, and further processing was followed using the same procedure described above for the blood samples after adjusting the relative volumes. DNA from amniotic fluid: 5 ml of amniotic fluid are spun down at 10,000 rpm for 5 minutes. The pellet is subsequently washed with 150 ml of 1 PBS. One hundred fifty ml of WCLB are added to the pellet and further processing follows using the same procedure described above for the blood samples after adjusting the relative volumes. Design of Oligonucleotide Primers and Probes
Forward primer BetaG(F) 50 -TAGCAACCTCAAACAGACACC-30 and Reverse primer BetaG(R) 50 -TCACAGTGCAGCTCACTCAGT-30 annealing in the bglobin at positions (Accession number L26463) 843–863 and 1298–1278 respectively are used to amplify the 457 bp region spanning the IVSI-110 (G1065A), IVSI-1 (G956A), IVSI-6 (T961C), Cd29 (C953T), and Cd30 (G955C) mutations. The 137 bp region spanning the IVSII-1 mutation is amplified using primers IVS2 (Forward) 50 -CCACACTGAGTGAGCTGCA-30 and IVS2 (Reverse) 50 -GTTACTTCTCCCCTTCC-TATGACA-30 annealing at positions (Accession number L26463) 1253–1271 and 1389–1366 respectively. Probes
Two sequence-specific fluorescent-labeled Hbb1 and Hbb2 probes (Table I) were designed in collaboration with Tib MolBiol (Berlin, Germany) to span the IVSI1, IVSI-6, Cd29, and Cd30 mutations (Fig. 1). The probe sequences were based on the wild type or the mutant sequence depending on the mutation. This is to maximize the difference in the probe melting temperature between the wild type and the mutant genotype. For example, Hbb1 probe was based on the wild type sequence and the difference in the probe melting temperature between the wild type (64.6 C) and the Cd29 mutant genotype (68.71 C) is 4.11 C. Probe Hbb1 (anchor) is labeled with fluorescein at the 30 end and probe Hbb2 (sensor) is labeled with LightCycler-
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TABLE I. Probe Sequences and Their Positions Within the b Globin Gene (Accession Number L26463) Name
Target DNA
Type
Position
Nucleotide sequence (50 –30 )a
Hbb1 Hbb2 Hbb3 Hbb4 Hbb5 Hbb6
Hbb IVSI-1, IVSI-6, Cd29, Cd30
Anchor Sensor Anchor Sensor Anchor Sensor
1007–976 974–947 1080–1103 105II–1077 1300–1278 1324–1301
50 -TGCCCAGTTTCTATTGGTCTCCTTAAACCTGT-30 50 -TTGTAACCTTGATACCAACCTACCCAGG-30 50 -CCTTAGGCTGCTGGTGGTCTACCC-30 50 -CTCTCTGCCTATTAGTCTATTTTCCC-30 50 -TCTCAGGATCCACGTGCAGCTTG-30 50 -GGTCCCATAGACTCACCCTGAAG-30
Hbb IVSI-110 Hbb IVSII-1
a
Underlined nucleotides indicate modifications made from the reference sequence to allow mutation detection.
Red 705 (LC-Red 705) at the 50 -end. Upon hybridization, the two probes come in close proximity resulting in fluorescence resonance energy transfer (FRET). During FRET, the Hbb1 fluorescein is excited by the LC light source donating part of the excitation energy to the acceptor fluorophore LC-Red of probe Hbb2 which then emits energy that is measured by the optical unit of the LightCycler instrument. Hbb2, spanning the mutation sites, is designed to hybridize at a three base distance from the anchor probe Hbb1. Based on the same concept, IVSI-110 mutation is detected using
Fig. 1. Mutations in the b-globin gene (accession number L26463) are indicated by vertical arrows and the hybridization probes (sensor) are shown below the partial b-globin DNA sequence. (a) Bases 1048–1077 showing the IVSI-110 mutation (G to A) at position 1065 and the sequence of the Hbb3 probe. (b) Bases 1299–1328 showing the IVSII-1 mutation (G to A) at position 1309 and Hbb5 (sensor) hybridization probe sequence. (c) Bases 944–977 showing the Cd29 (C to T), Cd30 (G to C), IVSI-1 (G to A), and IVSI-6 (T to C) mutations at positions 953, 955, 956, and 961, respectively, and Hbb2 (sensor) hybridization probe sequence.
probes Hbb3/Hbb4 (separated by two bases) and IVSII-1 mutation is detected using probes Hbb3/ Hbb4 (separated by one base) (Fig. 1). The reference sequence of the b-globin gene used had the Gene bank database accession number L26463. LightCycler-PCR (LC-PCR) Protocol
LC-PCR is performed using 60 ng of genomic DNA, 4 mM MgCl2, 0.5 units of Uracil N-Glycosylase (UNG), and 0.2 mM of the fluorescein-labeled and the LC-Red hybridization probes with the LightCyclerFaststart DNA Master Hybridization Probes Kit (Roche Biodiagnostics, Mannheim, Germany) according to the manufacturer’s recommendations in a final reaction volume of 20 ml using disposable glass capillaries. Cycling was started after a 5-min incubation period at room temperature to enable UNG activity. Forty-five amplification cycles are performed as follows: Denaturation and AmpliTaqGold activation (95 C for 10 min), annealing (50 C for 10 sec), and extension (72 C for 10 sec). The ramp rates are programmed at 20 C/sec from denaturation to annealing, 5 C/sec from annealing to extension and 20 C/sec from extension to denaturation. At the end of the amplification, a cycle for melting temperature (TM) analysis is added as follows: Heating to 95 C (20 C/sec), without hold, cooling to 40 C (20 C/sec), a 10-sec hold at 40 C, heating slowly at 0.2 C/sec up to 85 C and a final recooling to 40 C (20 C/sec). Genotyping is decided after melting curve analyses is done by comparing the melting peaks of the suspected patient with that of individuals with known genotypes. RESULTS
After melting curve analysis, the presence or absence of a mutation can be recognized by a noticeable shift in the melting peak as compared to the wild-type or the known genotype control. IVSI-110 Mutation
A melting peak from the Hbb3/4 probes is observed at 66.9 C indicating the IVSI-110 mutation,
Technique: b-Thalassemia Mutations Using Real-Time PCR
distinctly different from the melting peak of the wildtype allele observed at 60.5 C (Fig. 2a). An IVSI-110 homozygous individual shows a single peak at 66.9 C as compared to a single peak at 60.5 C for a homozygous normal individual. A heterozygous individual displays two peaks, one at 66.9 C and one at 60.5 C.
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type allele observed at 64.5 C (Fig. 2b). An IVSII-1 homozygous individual shows one peak at 60.0 C compared to a single peak at 64.5 C for a homozygous normal individual. A heterozygous individual displays two peaks, one at 60.0 C and one at 64.5 C. Cd29, Cd30, IVSI-1, and IVSI-6 Mutations
IVSII-1 Mutation
The melting peak from the Hbb5/6 probes is observed at 60.0 C indicating the IVSII-1 mutation, distinctly different from the melting peak of the wild-
Fig. 2. Real-time genotyping of the b-globin gene by hybridization probe melting curve analysis. Analysis is presented by plotting the first negative derivative of the fluorescence (F ) with respect to temperature [– (dF/dT) versus T ]. (a) Probes Hbb3/4 detecting an IVSI-110 homozygous mutant (—) and a normal genotype (– – –) at a TM of 66.9 and 60.5 C, respectively. Also shown is the heterozygous IVSI-110/normal genotype (– – –). (b) Probes Hbb5/6 detecting an IVSII-1 homozygous mutant (—) and a normal genotype (– – –) at TM of 60.0 and 64.5 C, respectively. Also shown the heterozygous IVSII-1/normal genotype (– – –).
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Because of the close proximity of the Cd29, Cd30, IVSI-1, and IVSI-6 mutations, a single pair of probes was designed to detect all the mutated alleles simultaneously. Probes Hbb1/2 distinguish four melting peaks at 68.71 C, 59.28 C, 58.48 C, and 59.88 C for the Cd29, Cd30, IVSI-1, and IVSI-6 mutations respectively, in addition to a distinctive peak at 64.6 C for the normal genotype (Fig. 3). A homozygous mutant for any of the four mutations showed one peak at the mentioned temperature compared to a single distinctive peak at 64.6 C for a homozygous normal individual. Heterozygotes display two peaks, one at the TM of the
Fig. 3. Multiplex genotyping of the IVSI-1, Cd30, IVSI-6, and Cd29 mutations of the human b-globin gene. (a) Probe Hbb1 is labeled with fluorescein at its 30 end and serves as an anchor probe for Hbb2. Hbb2, the sensor probe, is labeled with LC-Red 705 at its 50 end and spans the DNA region having the four mutations. (b) Probe melting curve analysis of homozygous mutants of IVSI-1, Cd30, IVSI-6, and Cd29 show distinctive peaks at TM of 58.48, 59.28, 59.88, and 68.71 C, respectively. The probes also detect a normal genotype having a melting peak at a TM 64.6 C. (Genotypes are indicated on top of each peak.)
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characteristic mutation and one at 64.6 C for the normal gene. In addition, compound heterozygotes for any of the four mutations are clearly and successfully detected with this probe pair (data not shown). Prenatal Diagnosis Cases
Forty-eight b-thalassemia prenatal diagnoses were performed using the LightCycler method we developed. DNA samples from both parents, from the CVS or amniotic fluid as well from an affected child (when a sample was available) were used. Thirty cases were completely resolved by the LightCycler protocol, of these two compound heterozygous fetuses were identified. Twelve cases had one of the two mutant alleles detected. The remaining 6 cases were normal for the 6 tested mutations and needed further analysis. All obtained results were verified by conventional DNA sequencing and showed 100% concordance. DISCUSSION
In the past two decades, the molecular bases of b-thalassemia have been well characterized with over three hundred mutations reported in the b-globin gene that produce the b-thalassemia phenotype. These mutations differ among different ethnic groups and geographical areas. In Lebanon and the East Mediterranean, the IVSI-1, IVSI-6, Cd29, Cd30, IVSI-110, and IVSII-1 mutations are the most common [11–13]. The prevention of b-thalassemia and many other genetic diseases requires prenatal diagnosis and the termination of pregnancy in case of an affected fetus [14]. In this paper, we describe a practical method for carrier genotype determination, affected genotype determination, and prenatal diagnosis in DNA samples from Mediterranean patients with b-thalassemia. The development of PCR created a revolution within the DNA diagnostic field that facilitated the development of new DNA methods to identify mutations within the b-globin gene. Conventional methods such as ARMS-PCR (15) and DNA sequencing of the suspected DNA fragment can be highly accurate, but expensive and time-consuming. The advent of the melting curve analysis using Real-Time PCR in recent years introduced various applications in molecular genetic typing. Discrimination based on hybridization temperatures is a powerful tool to distinguish different sequences (9,10). Real-Time PCR has several advantages over conventional PCR regarding contamination and duration factors rendering it more applicable in cases of delicate prenatal diagnostic testing. Given the fact that the melting temperature of the probe is dependent on the stability of the probe/target duplex and that the LC-Red labeling can be provided in two detectable
forms, multiplexing can be done without the fear of primer–dimer misinterpretations. The cycling conditions of the LightCycler take no more than 35 min, giving our method the advantage over other methodologies, with the whole procedure including DNA preparation to take no more than half a working day to provide an accurate diagnosis. The custom designed Hbb1/2, Hbb3/4, and Hbb5/6 probes detect the six most common Lebanese mutations with probes Hbb1/2 detecting any of the IVSI-1, IVSI-6, Cd29, and Cd30 mutations saving time and reducing cost to the possible minimum. The method is safe, does not involve radioactivity, and is highly accurate showing 100% concordance with conventional DNA sequencing methods. It is a reliable technique, combining both PCR and fluorescent hybridization, which will replace the conventional methods in prenatal diagnosis. Finally, this method can be applied for the diagnosis of any given monogenic disorder. REFERENCES 1. Weatherall DJ, Clegg JB. Genetic disorders of hemoglobin. Semin Hematol 1999;36:24–37. 2. Perrimond H. [b-Thalassemia: clinical manifestations]. Bull Soc Pathol Exot 2001;94:92–94. 3. Angastiniotis M, Modell B. Global epidemiology of hemoglobin disorders. Ann NY Acad Sci 1998;850:251–269. 4. Hamamy H, Alwan A. Hereditary disorders in the Eastern Mediterranean Region. Bull World Health Org 1994;72:145–154. 5. Cabannes R, Taleb N, Ghorra F, Schmitt-Beurrier A. [Studies of hemoglobin types in the population of Lebanon]. Nouv Rev Fr Hematol 1965;5:851–856. 6. Forget BG. Structure and organization of the human globin genes. Tex Rep Biol Med 1980;40:77–86. 7. Thein SL, Hesketh C, Taylor P, et al. Molecular basis for dominantly inherited inclusion body b-thalassemia. Proc Natl Acad Sci U S A 1990;87:3924–3928. 8. Chehab FF, Der Kaloustian V, Khouri FP, Deeb SS, Kan YW. The molecular basis of b-thalassemia in Lebanon: application to prenatal diagnosis. Blood 1987;69:1141–1145. 9. Lyon E. Mutation detection using fluorescent hybridization probes and melting curve analysis. Expert Rev Mol Diagn 2001;1:92–101. 10. Pals G, Young C, Mao HS, Worsham MJ. Detection of a single base substitution in a single cell using the LightCycler. J Biochem Biophys Methods 2001;47:121–129. 11. Khlat M, Halabi S, Khudr A, Der Kaloustian VM. Perception of consanguineous marriages and their genetic effects among a sample of couples from Beirut. Am J Med Genet 1986;25:299–306. 12. Kattamis C, Kattamis AC. Genotypes and phenotypes of b-thalassemia in Mediterranean populations. Pediatr Hematol Oncol 1997;14:vii–ix. 13. Zahed L, Talhouk R, Saleh M, Abou-Jaoudeh R, Fisher C, Old J. The spectrum of b-thalassaemia mutations in the Lebanon. Hum Hered 1997;47:241–249. 14. Kazazian HH Jr. Prenatal diagnosis of b-thalassemia. Semin Perinatol 1991;15:15–24. 15. Bravo M, Salazar R, Arends A, Alvarez M, Velazquez D, Guevara JM, Castillo O. [Detection of b-thalassemia by the technique of refractory amplification of mutation systems (ARMS-PCR)]. Invest Clin 1999;40:203–213.
