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Braz J Med Biol Res 36(11) 2003 Alpha-globin gene analysis
Screening for mutations in human
alpha-globin genes by nonradioactive
single-strand conformation polymorphism
Departamentos de 1Patologia Clínica and 2Clínica Médica,
Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, SP, Brasil
S.B. Jorge1, M.B. Melo2, F.F. Costa2 and M.F. Sonati1
Abstract
Point mutations and small insertions or deletions in the human α-globin genes may produce α-chain structural variants and α-thalas-semia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns), the α-globin genes are duplicate (α2 and α1) and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electro-phoresis in order to detect mutations in these genes employing nonra-dioactive single-strand conformation polymorphism (SSCP). Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here com-bined and the resultant fragments (6 new besides 6 original per α-gene) submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions includ-ing two electrophoretic apparatus (PhastSystem™ and GenePhor™, Amersham Biosciences), different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem™ and 20% gels at 15oC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repeti-tions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.
Correspondence
M.F. Sonati
Departamento de Patologia Clínica FCM, UNICAMP Caixa Postal 6111 13083-970 Campinas, SP Brasil Fax: +55-19-3788-9434 E-mail: [email protected] Research supported by FAPESP (Nos. 1997/11725-1 and 1999/ 11121-4) and CNPq. Received October 15, 2002 Accepted July 31, 2003 Key words •Alpha-globin genes •Nonradioactive SSCP •Alpha-globin structural variants •Hemoglobinopathies •Mutation screening
Mutations in the human α-globin genes
have not been as extensively investigated as ß-globin gene alterations in the Brazilian population, although it has been demonstrated that they are prevalent, frequently in associa-tion with other hemoglobinopathies and sometimes resulting in important clinical and hematological manifestations (1-4).
Because the α-globin genes are G-C rich,
artifacts in the sequencing reaction are com-mon. Thus, the use of mutation screening methods is interesting, especially for labora-tories which do not have an automated se-quencer available.
There are two main proposals in the lit-erature for the screening of mutations in the
Brazilian Journal of Medical and Biological Research (2003) 36: 1471-1474 ISSN 0100-879X Short Communication
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Braz J Med Biol Res 36(11) 2003
S.B. Jorge et al.
α-genes: Ayala et al. (5), used the single-strand conformation polymorphism (SSCP) technique described by Orita et al. (6), and Harteveld et al. (7) used SSCP in combina-tion with denaturating gradient gel
electro-phoresis (DGGE) (8). These two methods, however, present some disadvantages be-cause the former is a radioactive SSCP and the latter combines two techniques and two different types of equipment, with the DGGE Figure 1. A, Schematic
represen-tation of fragment and primer origin. B, Schematic representa-tion of the α-globin gene muta-tions screened by nonradioac-tive single-strand conformation polymorphism in the present study.
Exon 1 Exon 1 Exon 1 Exon 1
Exon 1 = D65+C69 (5’- ACT- CCC- CTg- Cgg- TCC- gg -3’+++++ 5’- gAA- ggA- CAg- gAA- CAT- CCT -3’) Exon 2
Exon 2 Exon 2 Exon 2
Exon 2 = D68+C72 (5’- ACA- ggC- CAC- CCT- CAA- CCg- T -3’ + + + + 5’- CAg- gCC- CgC- AgC- CgC- CgC- CT -3’)+ Exon 3
Exon 3Exon 3 Exon 3
Exon 3(((((α11111) ) ) ) ) = D71+C113 (5’- AgA- ggA- TCA- CgC- ggg- TTg- C -3’ +++++ 5’- AAA- ACT- CAg- gCA- CAC- ACA- gg -3’)
Exon 3( Exon 3(Exon 3( Exon 3(
Exon 3(α22222) ) ) ) ) = D71+C110 (5’- AgA- ggA- TCA- CgC- ggg- TTg- C -3’ +++++ 5’- ATT- CCg- ggA- CAg- AgA- gAA- CC -3’)
Fragment I Fragment I Fragment I Fragment I
Fragment I = S1+S12 (5’- CAC- AgA- CTC- AgA- gAg- AAC- C -3’ + + + + + 5’- ggA- Agg- ACA- ggA- ACA- TCC- Tg -3’) Fragment II
Fragment II Fragment II Fragment II
Fragment II = S3+S6 (5’- ggC- Aag- Aag- gTg- gCC- gAC -3’ +++++ 5’- TCC- ATT- gTT- ggC- ACA- TTC- C -3’) Fragment III
Fragment III Fragment III Fragment III
Fragment III = S7+S18 (5’- Tgg- Agg- gTg- gAg- ACg- TCC- Tg -3’ + + + + + 5’- CgT- Tgg- gCA- TgT- CgT- CCA- C -3’) Fragment IV
Fragment IV Fragment IV Fragment IV
Fragment IV = S3+S8 (5’- ggC- Aag- Aag- gTg- gCC- gAC -3’ +++++ 5’- CTg- gCA- CgT- TTC- TgA- ggg- AA -3’) Fragment A
Fragment A Fragment A Fragment A
Fragment A = D65+S12 (5’- ACT- CCC- CTg- Cgg- TCC- gg -3’ + + + + + 5’- ggA- Agg- ACA- ggA- ACA- TCC- Tg -3’) Fragment B
Fragment B Fragment B Fragment B
Fragment B = D65+C10seq (5’- ACT- CCC- CTg- Cgg- TCC- gg -3’+++++ 5’- ACg- gTT- gAg- ggT- ggC- CTg- T -3’) Fragment C (
Fragment C (Fragment C ( Fragment C (
Fragment C (α11111) ) ) = D71+S8 (5’- AgA- ggA- TCA- CgC- ggg- TTg- C -3’+) ) ++++5’- CTg- gCA- CgT- TTC- TgA- ggg- AA -3’)
Fragment C ( Fragment C (Fragment C ( Fragment C (
Fragment C (α22222) ) ) = D71+S6 (5’- AgA- ggA- TCA- CgC- ggg- TTg- C -3’ +) ) ++++5’- TCC- ATT- gTT- ggC- ACA- TTC- C -3’)
Fragment D Fragment D Fragment D Fragment D
Fragment D = S3+C72 (5’- ggC- Aag- Aag- gTg- gCC- gAC -3’ + + + + + 5’- CAg- gCC- CgC- AgC- CgC- CgC- CT -3’) Fragment E
Fragment E Fragment E Fragment E
Fragment E = D68+S18 (5’- ACA- ggC- CAC- CCT- CAA- CCg- T -3’ +++++5’- CgT- Tgg- gCA- TgT- CgT- CCA- C -3’) Fragment F
Fragment F Fragment F Fragment F
Fragment F = S1+S18 (5’- CAC- AgA- CTC- AgA- gAg- AAC- C -3’ ++++5’- CgT- Tgg- gCA- TgT- CgT- CCA- C -3’)+
A AA A A B BB BB Gene α2 Gene α1 Hph I Hph I Nco I Nco I
Dde I Apa I Dde I Nla I
Nla III Hind III Nla III Hind III
Dde I Hph I Dde I Dde I Hph I Dde I
Mbo I Mbo I Apa I Hph I Hinc II Apa I
Hph I
Hind III Hind III
Apa I Nco I
Nco I Nla III
Exon 1 Exon 1Exon 1 Exon 1Exon 1 Exon 2 Exon 2Exon 2 Exon 2 Exon 2 Exon 1 Exon 1 Exon 1 Exon 1 Exon 1 Exon 2 Exon 2Exon 2 Exon 2 Exon 2 Exon 3 Exon 3Exon 3 Exon 3Exon 3 Exon 3 Exon 3 Exon 3 Exon 3 Exon 3 Fragment I Fragment I Fragment I Fragment I
Fragment I Fragment IIFragment IIFragment IIFragment IIFragment II Fragment IFragment IFragment IFragment IFragment I
Fragment III Fragment IIIFragment III Fragment III
Fragment III Fragment IIIFragment IIIFragment IIIFragment IIIFragment III
Fragment IV Fragment IV Fragment IV Fragment IV Fragment IV Fragment A Fragment A Fragment A Fragment A Fragment A Fragment B Fragment BFragment B Fragment B Fragment B Fragment C Fragment C Fragment C Fragment C Fragment C Fragment D Fragment D Fragment D Fragment D Fragment D Fragment E Fragment E Fragment E Fragment E Fragment E Fragment F Fragment F Fragment F Fragment F Fragment F Fragment A Fragment A Fragment A Fragment A Fragment A Fragment B Fragment BFragment B Fragment BFragment B Fragment D Fragment DFragment D Fragment DFragment D Fragment E Fragment EFragment E Fragment EFragment E Fragment F Fragment F Fragment F Fragment F Fragment F Fragment C Fragment C Fragment C Fragment C Fragment C Combined primers Harteveld et al. (7) Ayala et al. (5) α1 Exon 2 Exon 2 Exon 2 Exon 2
Exon 2 Exon 3Exon 3Exon 3Exon 3Exon 3 Exon 1Exon 1Exon 1Exon 1Exon 1 Exon 2Exon 2Exon 2Exon 2Exon 2 Exon 3Exon 3Exon 3Exon 3Exon 3
IVS IVS IVS IVS IVS IIIII IVS IVS IVS IVS IVS IIIII IVS IVS IVS IVS IVS II IIII II II IVS IVS IVS IVS IVS II II II II II Exon 1 Exon 1 Exon 1 Exon 1 Exon 1 Gene α2 Gene α1 Exon 2 Exon 2 Exon 2 Exon 2
Exon 2 Exon 3Exon 3Exon 3Exon 3Exon 3 Exon 1Exon 1Exon 1Exon 1Exon 1 Exon 2Exon 2Exon 2Exon 2Exon 2 Exon 3Exon 3Exon 3Exon 3Exon 3
IVS IVSIVS IVSIVS IIIII IVS IVSIVS IVS IVS IIIII IVS IVS IVS IVS IVS II II II II II IVS IVS IVS IVS IVS II II II II II Exon 1 Exon 1 Exon 1 Exon 1 Exon 1
→ Hb CampinasHb CampinasHb CampinasHb CampinasHb Campinas → Hb J-RovigoHb J-RovigoHb J-RovigoHb J-RovigoHb J-Rovigo → Hb Daneshgah-TehranHb Daneshgah-TehranHb Daneshgah-TehranHb Daneshgah-TehranHb Daneshgah-Tehran → αααααHph IHph IHph IHph IHph Iααααα
→ Hb G-Pest Hb G-Pest Hb G-Pest Hb G-Pest Hb G-Pest
→ Hb WestmeadHb WestmeadHb WestmeadHb WestmeadHb Westmead
→ Hb West OneHb West OneHb West OneHb West OneHb West One →→→→→ Hb KurosakiHb KurosakiHb KurosakiHb KurosakiHb Kurosaki
→ Hb Hasharon (Hb Hasharon (Hb Hasharon (Hb Hasharon (Hb Hasharon (ααααα22222ααααα11111))))) → Hb Stanleyville-II (Hb Stanleyville-II (Hb Stanleyville-II (Hb Stanleyville-II (Hb Stanleyville-II (ααααα22222ααααα11111)))))
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Braz J Med Biol Res 36(11) 2003 Alpha-globin gene analysis
equipment being specially manufactured for α-gene analysis.
Using the primers described by the cited
investigators and the C10seq primer described
by Hall et al. (9) for sequencing, in combina-tions to produce six new fragments (named A to F), we optimized the conditions for the
screening of mutations in the α-genes by
conventional silver staining SSCP (10). Fig-ure 1A shows a schematic representation of all amplified fragments and the primer pairs used.
Nine structural mutations and one 5-nucleotide thalassemic deletion were tested:
[Hbs Kurosaki (α7(A5) Lys→Glu - nt22
A→G), Campinas (α26(B7) Ala→Val - nt80
C→T), Hasharon (α47(CE5) Asp→His
-nt259 G→C), J-Rovigo (α53(E2) Ala→Asp
- nt278 C→A), Daneshgah-Tehran (α72(EF1)
His→Arg - nt335 A→G), G-Pest(α74(EF3)
Asp→Asn - nt340 G→A), Stanleyville-II
(α78(EF7) Asn→Lys - nt354 C→G),
Westmead (α122(H5) His→Gln - nt628
C→G), West One (α126(H9) Asp→Gly
-nt639 A→G), and αHphIα (-5nt-IVS-I - nts
134-138-TGAGG)]. They were detected in our laboratory by manual direct sequencing
of the entire genes and represent all the
α-globin structural variants we encountered in our population up to now. Figure 1B shows
the location of these mutations in the α-genes.
Selective amplification of the α1- and α2
-globin genes was carried out by PCR as described by Dodé et al. (11), and the prod-uct was used as template for a second PCR round, resulting in 24 subfragments (see Fig-ure 1A). The general SSCP conditions were
as follows: the second round of PCR prod-ucts was mixed with a formamide solution
(95% formamide, 20 mM Na2EDTA,
0.05% bromophenol blue and 0.05% xy-lene cyanol) in equal volumes (1:1), heated at 95ºC for 5 min and then cooled on ice. The mixture was then loaded onto a ho-mogeneous polyacrylamide gel. Electro-phoresis was performed in both PhastSys-tem™ and GenePhor™ apparatus (Amer-sham Biosciences, Uppsala, Sweden). Af-ter a pre-run at 400 V, 100 Vh, 15ºC, differ-ent conditions were tested: for Phast-System™, 12.5 and 20% polyacrylamide gels (PhastGels™) and run temperatures of 4º and 15ºC, at 400 V; for GenePhor™, only 12.5% gels (GeneGels™) and run tem-peratures of 5º and 15ºC, at 600 V. Run time was about 3 h for both systems. Gels were stained with silver (10).
A second denaturing solution, 10% low ionic strength (LIS) (12), was also tested, as well as complete and shortened fragments (cut with restriction enzymes, see Figure 1A). The fragments and conditions that showed the best quality of electrophoretic separation and allowed 100% mutation detection are presented in Table 1. No difference was observed between LIS and formamide dena-turing solutions. The PhastSystem™ appa-ratus was the best system, probably because 20% gels could be used, which resulted in better electrophoretic performance. Reduc-ing fragment length, in this case, did not improve the sensitivity of mutation detec-tion.
The basic composition of the fragments Table 1. Recommended single-strand conformation polymorphism primer combinations and conditions.
Fragments Primer pairs* PCR annealing PhastGelTM PhastSystemTM Run temperature Detection
(ºC) concentration (%) (Vh) (ºC) (%)
A D65 + S12 58 20 600 15 100
C D71 + S6 61 20 890 15 100
D S3 + C72 61 20 400 15 100
E D68 + S18 61 20 400 15 100
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Braz J Med Biol Res 36(11) 2003
S.B. Jorge et al.
rather than their length was the most impor-tant parameter examined and was dependent on the primer pair choice, since each frag-ment produces one main stable conforma-tion which is determined by the intramolecu-lar interactions of the primary sequence (13). Therefore, after optimization, nonradio-active SSCP proved to be a sensitive and
efficient method for the screening of
muta-tions in the α-globin genes. This strategy
may be important for laboratories that wish to work on these genes and do not have an automated DNA sequencer. It will speed up the procedures, will reduce the costs in-volved in frequent sequencing repetitions, and will increase the reliability of the results.
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