Single-Tube Multiplex-PCR Screen for Anti-3.7 - Clinical Chemistry

Clinical Chemistry 49, No. 10, 2003

A. Andreoli (Divisione di Gastroenterologia, Ospedale N. Regina Margherita, Roma); C. Papi and L. Capurso (Gastroenterologia, S. Filippo Neri, Roma); R. D’Inca´ and G. Sturniolo (Cattedra di Gastroenterologia, Universita´ di Padova); M. Oliva (Gastroenterologia, Ospedale “Cervello”, Palermo); G. Lombardi, S. Fiorella, G. Corritore, and T. Latiano (Gastroenterologia, Ospedale CSSIRCSS, San Giovanni Rotondo); M. Rizzetto, M. Astegiano, and F. Bresso (Dipartimento di Gastroenterologia, Ospedale Molinette, Torino); and A. Pera and M.T. Fiorentini (Divisione di Gastroenterologia, Ospedale Mauriziano, Torino). This work was supported by grants from the Center for Translational Medicine at Thomas Jefferson University (to P.F.), the Gastroenterology Research Unit at IRCCS Casa Sollievo della Sofferenza (to V.A. and A.L.), and the Italian Ministry of Health (RC2003, to B.D.).

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19. Ronaghi M. Pyrosequencing sheds light on DNA sequencing [Review]. Genome Res 2001;11:3–11. 20. Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn’s disease. Nature 2001;411:599 – 603. 21. Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, et al. A frameshift mutation in NOD2 associated with susceptibility to Crohn’s disease. Nature 2001;411:603– 6. 22. Hampe J, Cuthbert A, Croucher PJ, Mirza MM, Mascheretti S, Fisher S, et al. Association between insertion mutation in NOD2 gene and Crohn’s disease in German and British populations. Lancet 2001;357:1925– 8. 23. Fakhrai-Rad H, Pourmand N, Ronaghi M. Pyrosequencing: an accurate detection platform for single nucleotide polymorphisms [Review]. Hum Mutat 2002;19:479 – 85. 24. Ferraris A, Rappaport E, Santacroce R, Pollak E, Krantz I, Toth S, et al. Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss. Hum Mutat 2002;20:312–20. 25. Nordfors L, Jansson M, Sandberg G, Lavebratt C, Sengul S, Schalling M, et al. Large-scale genotyping of single nucleotide polymorphisms by pyrosequencing and validation against the 5⬘ nuclease (Taqman) assay. Hum Mutat 2002;19:395– 401.

References 1. Sandler RS. Epidemiology of inflammatory bowel disease. In: Targan SR, Shanahan F, eds. Inflammatory bowel disease. From bench to bedside. Baltimore: Williams & Wilkins, 1994:5–31. 2. Satsangi J, Jewell DP, Bell JI. The genetics of inflammatory bowel disease. Gut 1997;40:572– 4. 3. Ahmad T, Armuzzi A, Bunce M, Mulcahy-Hawes K, Marshall SE, Orchard TR, et al. The molecular classification of the clinical manifestations of Crohn’s disease. Gastroenterology 2002;122:854 – 66. 4. Farrell RJ, Peppercorn MA. Ulcerative colitis. Lancet 2002;359:331– 40. 5. Shanahan F. Crohn’s disease. Lancet 2002;359:62–9. 6. Cuthbert AP, Fisher SA, Mirza MM, King K, Hampe J, Croucher PJ, et al. The contribution of NOD2 gene mutations to the risk and site of disease in inflammatory bowel disease. Gastroenterology 2002;122:867–74. 7. Lesage S, Zouali H, Cezard JP, Colombel JF, Belaiche J, Almer S, et al. CARD15/NOD2 mutational analysis and genotype-phenotype correlation in 612 patients with inflammatory bowel disease. Am J Hum Genet 2002;70: 845–57. 8. Hampe J, Grebe J, Nikolaus S, Solberg C, Croucher PJP, Mascheretti S, et al. Association of NOD2 (CARD15) genotype with clinical course of Crohn’s disease: a cohort study. Lancet 2002;359:1661–5. 9. Vermeire S, Wild G, Kocher K, Cousineau J, Dufresne L, Bitton A, et al. CARD15 genetic variation in a Quebec population: prevalence, genotypephenotype relationship, and haplotype structure. Am J Hum Genet 2002; 71:74 – 83. 10. Bonen DK, Cho JH. The genetics of inflammatory bowel disease [Review]. Gastroenterology 2003;124:521–36. 11. Hugot JP, Laurent-Puig P, Gower-Rousseau C, Olson JM, Lee JC, Beaugerie L, et al. Mapping of a susceptibility locus for Crohn’s disease on chromosome 16. Nature 1996;379:821–3. 12. Mirza MM, Lee J, Teare D, Hugot JP, Laurent-Puig P, Colombel JF, et al. Evidence of linkage of the inflammatory bowel disease susceptibility locus on chromosome 16 (IBD1) to ulcerative colitis. J Med Genet 1998;35:218 – 21. 13. Duerr RH, Barmada MM, Zhang L, Davis S, Preston RA, Chensny LJ, et al. Linkage and association between inflammatory bowel disease and a locus on chromosome 12. Am J Hum Genet 1998;63:95–100. 14. Cho JH, Nicolae DL, Gold LH, Fields CT, LaBuda MC, Rohal PM, et al. Identification of novel susceptibility loci for inflammatory bowel disease on chromosomes 1p, 3q, and 4q: evidence for epistasis between 1p and IBD1. Proc Natl Acad Sci U S A 1998 23;95:7502–7. 15. Annese V, Latiano A, Bovio P, Forabosco P, Piepoli A, Lombardi G, et al. Genetic analysis in Italian families with inflammatory bowel disease supports linkage to the IBD1 locus. A GISC study. Eur J Hum Genet 1999;7: 567–73. 16. Forabosco P, Collins A, Latiano A, Annese V, Clementi M, Andriulli A, et al. Combined segregation and linkage analysis of inflammatory bowel disease in the IBD1 region using severity to characterise Crohn’s disease and ulcerative colitis. Eur J Hum Genet 2000;8:846 –52. 17. Cavanaugh J, IBD International Genetics Consortium. International collaboration provides convincing linkage replication in complex disease through analysis of a large pooled data set: Crohn disease and chromosome 16. Am J Hum Genet 2001;68:1165–71. 18. van Heel DA, McGovern DP, Cardon LR, Dechairo BM, Lench NJ, Carey AH, et al. Fine mapping of the IBD1 locus did not identify Crohn diseaseassociated NOD2 variants: implications for complex disease genetics. Am J Med Genet 2002;111:253–9.

Single-Tube Multiplex-PCR Screen for Anti-3.7 and Anti-4.2 ␣-Globin Gene Triplications, Wen Wang,1 Edmond S.K. Ma,5 Amy Y.Y. Chan,5 John Prior,7 Wendy N. Erber,7 Li C. Chan,5 David H.K. Chui,6 and Samuel S. Chong1– 4* (Departments of 1 Pediatrics and 2 Obstetrics & Gynecology, National University of Singapore, Singapore 119074, Singapore; 3 The Children’s Medical Institute and Molecular Diagnosis Center, Department of Laboratory Medicine, National University Hospital, Singapore 119074, Singapore; 4 Departments of Pediatrics and Gynecology & Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, MD 21287; 5 Division of Hematology, Department of Pathology, The University of Hong Kong and Queen Mary Hospital, Hong Kong, People’s Republic of China; 6 Departments of Medicine and Pathology, Boston University School of Medicine, Boston, MA 02118; 7 The Western Australian Centre for Pathology and Medical Research, Nedlands, WA 6009, Australia; * address correspondence to this author at: Department of Pediatrics, National University of Singapore, Level 4, National University Hospital, 5 Lower Kent Ridge Road, Singapore 119074, Singapore; fax 65-6779-7486, e-mail [email protected]) The coexistence of ␣-globin gene triplication (␣␣␣) is an important modulator of the severity of ␤-thalassemia trait or ␤-thalassemia intermedia, exacerbating the phenotypic severity of ␤-thalassemia by causing more globin chain imbalance (1, 2 ). Typically, the inheritance of a single ␤-thalassemia allele is associated with mild anemia and hypochromic microcytic red cells. Compared with simple ␤-heterozygotes, co-inheritance of triplicated or quadruplicated ␣-globin genes in ␤-heterozygotes often leads to more significant anemia, splenomegaly, more pronounced red cell abnormalities, the presence of circulating normoblasts, higher hemoglobin F concentrations, and even the presence of inclusion bodies in erythroblasts (3, 4 ). Because the ␣- and ␤-globin gene clusters are physically unlinked and segregate independently,

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Technical Briefs

Fig. 1. Formation and molecular detection of ␣-globin gene triplications. (A), misalignment and unequal crossover at the ␣-globin gene cluster generates a single gene deletion and reciprocal gene triplication. Crossovers between misaligned Z boxes give rise to ⫺␣3.7 and ␣␣␣anti3.7 chromosomes (rightward crossover). Crossovers between misaligned X boxes give rise to ⫺␣4.2 and ␣␣␣anti4.2 chromosomes (leftward crossover). (B), determination of ␣-globin genotype by a combination of seven-deletion multiplex-PCR (top gel) and anti-3.7/4.2 multiplex-PCR (bottom gel). Lane M, 1-kb DNA ladder (Fermentas); lane AL, allelic ladder. (C), expected hybridizing bands of various unequal crossover derivative alleles (left) and genotypes (middle), and actual autoradiogram of three DNA samples (right), after Southern hybridization of BamHI and BglII digests with [32P]dATP-labeled ␣-globin gene probe.

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Clinical Chemistry 49, No. 10, 2003

Table 1. Anti-3.7/4.2 multiplex-PCR primers and expected amplicon sizes (top), and results of blinded analysis of 31 samples (bottom). Primer

LIS1-2.5F LIS1-2.5R AT3.7-F AT3.7-R AT4.2-F AT4.2-R

5ⴕ33ⴕ sequence

GTC GAT CCA CAG CCT GAA

GTC TCC CCT CGG TGC CTG

ACT AGG CCA GCA ACC GCT

GGC TTG TTC GGA GGC GAA

AGC TAG TCC GGA CCT AGG

GTA ACG AAC ACG TCC GAT

GAT C GAC TG CAC G TG GCA G

GenBank no.: nucleotides

Concentration, ␮M

HSLIS10: 4073428 HSLIS10: 290932887 HSGG1: 36371336391 HSGG1: 34498334480 HSGG1: 34525334544 HSGG1: 31978331957

0.5 0.5 0.3 0.3 0.4 0.4

Anti-3.7/4.2 multiplex-PCR: presence/absence (ⴙ/ⴚ) of amplicon

Amplicon (size)

LIS1 3⬘UTRa (2503 bp) Anti-3.7 (1932/1939 bp) Anti-4.2 (1711 bp)

Seven-deletion multiplex-PCR: presence/absence (ⴙ/ⴚ) of ampliconb

Southern blot genotype

No. of samples

LIS1 (2.5 kb)

Anti-3.7 (1.9 kb)

Anti-4.2 (1.7 kb)

LIS1 (2.3 kb)

ⴚ␣3.7 (2.0 kb)

␣2 (1.8 kb)

ⴚ␣4.2 (1.6 kb)

ⴚ ⴚSEA (1.3 kb)

␣␣/␣␣␣anti3.7 ␣␣/␣␣␣anti4.2 ␣␣␣anti3.7/⫺␣4.2 ␣␣␣anti4.2/⫺␣4.2 ␣␣␣anti4.2/⫺␣3.7 ␣␣␣anti3.7/⫺ ⫺SEA ␣␣␣anti4.2/⫺ ⫺SEA ␣␣/␣␣

7 7 2 2 1 1 1 10

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺

⫺ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺

a b

UTR, untranslated region. ⫺ ⫺THAI (1.2 kb), ⫺(␣)20.5 (1.0 kb), ⫺ ⫺MED (0.8 kb), and ⫺ ⫺FIL (0.5 kb) amplicons were also screened for but were negative in these samples.

␤-thalassemia carriers who also carry triplicated or quadruplicated ␣-globin genes have a 25% risk of having a similarly affected offspring, although their partners may be entirely normal. Triplicated ␣-globin genes appear to be ubiquitous and have been found in most populations (2 ). They result from misalignment and unequal crossover between the homologous X-, Y-, and Z-box segments of the ␣-globin gene cluster during meiosis (Fig. 1A). Generally, two types of triplicated alleles can be generated from an unequal crossover, ␣␣␣anti3.7 and ␣␣␣anti4.2. If the crossover occurs between the homologous Z2 and Z1 boxes, also referred to as a “rightward crossover”, this produces a ⫺␣3.7 single-gene deletion allele and the reciprocal ␣␣␣anti3.7 triplicated allele. However, if the crossover occurs between the X2 and X1 boxes (a “leftward crossover”), a ⫺␣4.2 single-gene deletion allele and the reciprocal ␣␣␣anti4.2 triplicated allele are generated (5 ). A Sri Lankan study of individuals with severe to moderate ␤-thalassemia revealed a 2% frequency of ␣-globin gene triplications (6 ), whereas a preliminary study in Hong Kong suggests that the frequency of ␣-globin gene triplication carriers among individuals with ␤-thalassemia and iron deficiency is ⬃3.3% (E.S.K. Ma, A.Y.Y. Chan, L.C. Chan, unpublished observation). The true prevalence of ␣-globin gene triplications is not well defined in other populations. Because the ␣- and ␤-globin gene clusters are unlinked, it is likely that the percentages derived from the study of ␤-thalassemia cohorts are a reasonable indicator of the general population frequency of ␣-globin gene triplication carriers. Current PCR methods can detect the anti-3.7 but not the anti-4.2 triplication (7, 8 ), and Southern blotting is re-

quired to detect both the anti-3.7 and anti-4.2 triplication and quadruplication alleles (9 ). Although highly sensitive and specific, Southern blotting is labor-intensive and time-consuming, and is thus not an ideal screening tool. We have developed and validated a simple, rapid, and reliable single-tube multiplex-PCR assay to screen for the presence of the anti-3.7 and anti-4.2 ␣-globin gene triplications. Genomic DNA samples carrying either an ␣␣␣anti3.7 or ␣␣␣anti4.2 triplication allele, as determined by Southern blot analysis, were used in assay optimization. Final validation of the optimized assay was accomplished by blinded analysis of 31 samples that were either positive (n ⫽ 21) or negative (n ⫽ 10) for triplicated ␣-globin genes. Oligonucleotide primers were designed to specifically amplify the unequal crossover region(s) within the anti3.7 or anti-4.2 chromosomes. Primers AT3.7-F and AT3.7-R were designed to anneal to the unique stretches between the Y1 and Z1 homology boxes and between the Z2 and X1 boxes, respectively, to amplify an ⬃1.9-kb fragment only from the hybrid Z1Z2 box of the anti-3.7 allele (Fig. 1A). Similarly, primers AT4.2-F and AT4.2-R anneal to the unique regions between the Z2 and X1 boxes and between the X2 and Y2 boxes, respectively, to amplify an ⬃1.7-kb fragment only from the hybrid X1X2 box of the anti-4.2 allele. Additionally, amplification of a large fragment (⬃2.5 kb) of the 3⬘ untranslated region of the LIS1 gene was included as a control for amplification success, using primers LIS1-2.5F and LIS1-2.5R. Each 50 ␮L of anti-3.7/4.2 multiplex-PCR reaction contained 200 ␮M each of the deoxynucleotide triphosphates,

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1.5 mM MgCl2, 1⫻ Q-solution (Qiagen), 2 U of HotStarTaq DNA polymerase in supplied reaction buffer (Qiagen), 100 ng of genomic DNA, and the six primers at different concentrations (Table 1). Thermal cycling was performed in a T3 instrument (Biometra) under conditions identical to those for the seven-deletion multiplexPCR assay (10 ). We analyzed 10 ␮L of each anti-3.7/4.2 multiplex-PCR product by electrophoresis through a 1% agarose gel in 1⫻ Tris-borate-EDTA buffer at 15 V/cm for 1 h. The seven-deletion multiplex-PCR assay was performed and analyzed exactly as described previously and was used to detect the seven common ␣-thalassemia deletions (10 ). To validate the anti-3.7/4.2 multiplex-PCR assay, we performed a double-blind analysis of 21 DNA samples harboring either an anti-3.7 or an anti-4.2 ␣-globin gene triplication, together with 10 negative control samples (␣␣/␣␣). The ␣-globin genotypes of these samples had been determined previously by Southern blot analysis. The anti-3.7/4.2 multiplex-PCR was assessed in combination with the ␣-thalassemia seven-deletion multiplex-PCR assay (10 ) to determine their ␣-globin genotype. Therefore, for each DNA sample tested, two amplification reactions were performed. The seven-deletion multiplexPCR assay screens for the seven common ␣-thalassemia deletions. In this assay, a control ␣2-globin gene fragment is amplified to indicate heterozygosity for a nondeleted allele when a deletion is also present (Fig. 1B); it cannot, however, distinguish between a wild-type nondeleted allele (␣␣) and a triplicated (␣␣␣) allele. We used the anti-3.7/4.2 multiplex-PCR assay to detect the presence of the triplicated allele in these samples (Fig. 1B). In the blinded analysis, the anti-3.7/4.2 multiplex-PCR assay detected the presence of the correct triplicated allele (␣␣␣anti3.7 or ␣␣␣anti4.2) in all 21 triplication-positive DNA samples (Table 1). No anti-3.7 or anti-4.2 junction fragment was detected in any of the 10 negative control DNA samples. In combination with the seven-deletion multiplex-PCR assay, the ␣-globin genotype of all 31 DNA samples was correctly determined (Table 1). The anti-3.7/ 4.2 multiplex-PCR assay thus serves as a useful rapid screen for the presence of the anti-3.7 and/or the anti-4.2 types of triplication. For the majority of individuals negative for a triplication by this rapid PCR assay (⬃96.5– 98.0% based on data extrapolation), no further analysis is required. Southern blot analysis is required only in the presumptive 2.0 –3.5% of samples that are PCR-positive, to distinguish between the heterozygous and homozygous states (Fig. 1C). It is likely that the result of our anti-3.7/4.2 multiplexPCR assay will also be abnormal with the rarer ␣␣␣␣anti3.7 and ␣␣␣␣anti4.2 quadruplicated alleles, which have two copies of the crossover junction, one copy more than their triplicated counterparts. To distinguish between the triplicated and quadruplicated alleles, however, will require Southern blotting because both triplicated and quadruplicated anti-3.7 and anti-4.2 alleles should yield essentially

the same ⬃1.9-kb and ⬃1.7-kb junction fragments, respectively, with this assay. Without an initial anti-3.7/4.2 multiplex-PCR screen, use of Southern analysis alone to detect triplications and quadruplications would require a minimum of two blots, each containing DNA digested by a different restriction enzyme, because no single enzyme digestion can provide full genotype information (9 ). For example, digestion with BamHI followed by hybridization with an ␣-globin gene probe enables detection of and differentiation between a triplication and a quadruplication, as well as determination of zygosity, but it cannot distinguish between the anti-3.7 and anti-4.2 types (Fig. 1C). Conversely, a BglII-digested DNA blot hybridized with the same probe enables the type of triplication or quadruplication to be determined (anti-3.7 or anti-4.2), but it does not provide information on zygosity. An initial anti-3.7/4.2 multiplex-PCR screen enables determination of the type of triplication (and potentially quadruplication) present in the patient (anti-3.7 or anti4.2), thus eliminating the need for a BglII-digested Southern blot. Therefore, only a BamHI-digested Southern blot hybridized with an ␣-globin probe is required to detect the presence of the rare quadruplicated and homozygous genotypes.

We thank A.S.C. Tan and G.H. Yeo for technical assistance. This work was supported by Grants NMRC/0365/ 1999 and NMRC/0732/2003 from the National Medical Research Council, Singapore (to S.S.C.).

References 1. Weatherall DJ. Pathophysiology of thalassaemia. Baillieres Clin Haematol 1998;11:127– 46. 2. Weatherall DJ. Phenotype-genotype relationships in monogenic disease: lessons from the thalassaemias. Nat Rev Genet 2001;2:245–55. 3. Ma SK, Au WY, Chan AY, Chan LC. Clinical phenotype of triplicated ␣-globin genes and heterozygosity for ␤0-thalassemia in Chinese subjects. Int J Mol Med 2001;8:171–5. 4. Beris P, Solenthaler M, Deutsch S, Darbellay R, Tobler A, Bochaton-Pialat ML, Gabbiani G. Severe inclusion body ␤-thalassaemia with haemolysis in a patient double heterozygous for ␤0-thalassaemia and quadruplicated ␣-globin gene arrangement of the anti-4.2 type. Br J Haematol 1999;105:1074 – 80. 5. Weatherall DJ. The thalassemias. In: Stamatoyannopoulos G, Majerus PW, Perlmutter RM, Varmus H, eds. The molecular basis of blood diseases, 3rd ed. Philadelphia: WB Saunders, 2001:183–226. 6. Fisher CA, Premawardhena A, de Silva S, Perera G, Rajapaksa S, Olivieri NA, et al. The molecular basis for the thalassaemias in Sri Lanka. Br J Haematol 2003;121:662–71. 7. Dode C, Krishnamoorthy R, Lamb J, Rochette J. Rapid analysis of ⫺␣ 3.7 thalassaemia and ␣␣␣ anti 3.7 triplication by enzymatic amplification analysis. Br J Haematol 1993;83:105–11. 8. Liu YT, Old JM, Miles K, Fisher CA, Weatherall DJ, Clegg JB. Rapid detection of ␣-thalassaemia deletions and ␣-globin gene triplication by multiplex polymerase chain reactions. Br J Haematol 2000;108:295–9. 9. Gu YC, Landman H, Huisman TH. Two different quadruplicated ␣-globin gene arrangements. Br J Haematol 1987;66:245–50. 10. Tan AS, Quah TC, Low PS, Chong SS. A rapid and reliable 7-deletion multiplex polymerase chain reaction assay for ␣-thalassemia. Blood 2001; 98:250 –1.