Prace oryginalne Endokrynologia, Diabetologia i Choroby Przemiany Materii Wieku Rozwojowego 2007, 13, 4, 183-186 ISSN 1234-625X
Multiplex minisequencing applied in detection of human functional CYP21 gene mutations Zastosowanie metody multipleksowego minisekwencjonowania (multiplex minisequencing) do wykrywania mutacji w ludzkim genie CYP21. Ma³gorzata Tokarska1, Ewa Barg2, Beata Wikiera2, Tadeusz Dobosz3, Magdalena ¯o³êdziewska3, Katarzyna Brzeziñska4, Anna Jonkisz3, Barbara Kosowska4 Research Institute, Polish Academy of Sciences, Bia³owie¿a, Poland Zak³ad Badania Ssaków Polskiej Akademii Nauk w Bia³owie¿y, Polska 2 Department of Endocrinology for Children and Adolescents, Wroclaw Medical University, Poland Katedra i Klinika Endokrynologii i Diabetologii Wieku Rozwojowego AM we Wroc³awiu, Polska 3 Department of Forensic Medicine, Wroc³aw Medical University, Poland Katedra Medycyny S¹dowej AM we Wroc³awiu, Polska 4 Department of Genetics and Animal Breeding, Wroc³aw University of Environmental And Life Sciences, Poland Katedra Genetyki i Ogólnej Hodowli Zwierz¹t Uniwersytetu Przyrodniczego we Wroc³awiu, Polska
1 Mammal
Abstract We analyzed seven most common mutations within the CYP21B gene, responsible for congenital adrenal hyperplasia (CAH), using the minisequencing method. Functional CYP21B gene sequences were amplified with the pair of specific primers that pevented amplification of pseudogene CYP21P or pseudogene CYP21P/active CYP21 hybrids. Multiplex minisequencing (SNaPShot PCR) assay was performed with fluorescent dideoxynucleotides ([F]ddNTPs) and originally designed primers, claiming seven most common mutation sites responsible for the CAH symptoms. Using the method we detected five novel substitutions of unknown effect on the CAH course in five out of seven analyzed mutation sites. Compared to classic SNPs analyzing methods, especially single SNP detection, multiplex minisequencing is the same highly specific and sensitive but much faster one. The method is recommended for any population screened for known mutations.
Key words: SNaPShot PCR, CYP21 analyses Streszczenie Poddaliœmy analizie siedem najczêœciej wystêpuj¹cych mutacji genu CYP21B, odpowiedzialnych za wrodzony przerost kory nadnerczy (WPN, ang. CAH), wykorzystuj¹c metodê multipleksowego minisekwencjonowania. Sekwencje funkcjonalnego genu CYP21B namno¿one zosta³y przy u¿yciu pary primerów, zapobiegaj¹cych amplifikacji pseudogenu CYP21P lub hybryd pseudogenu CYP21B i genu CYP21P. Minisekwencjonowanie multipleksowe (SNaPShot PCR) wykonano z u¿yciem fluorescencyjnie znakowanych dideoksynukleotydów ([F]ddNTPs) i oryginalnie zaprojektowanych primerów, komplementarnych do miejsc wystêpowania siedmiu najczêstszych mutacji, odpowiedzialnych za objawy WPN. W piêciu sposród siedmiu analizowanych miejsc mutacji stwierdziliœmy piêæ nieopisanych dot¹d substytucji o nieznanym wp³ywie na przebieg choroby. W porównaniu do klasycznych metod analizy polimorfizmu pojedynczych podstawieñ, multiplex minisequencing jest metod¹ równie specyficzn¹ i czu³¹, ale znacznie szybsz¹. Metoda jest szczególnie polecana do populacyjnej, przesiewowej analizy znanych miejsc mutacji.
S³owa kluczowe: SNaPShot PCR, gen CYP21
Introduction The big interest in SNP (single nucleotide polymorphism) applications arises from their versatility, they may serve as genetic markers for forensic identification, tissue typing, genetic population, evolutionary studies and disease genetics diaAdres do korespondencji: Dr Ma³gorzata Tokarska Zak³ad Badania Ssaków, Polska Akademia Nauk ul. Waszkiewicza 1, 17-230 Bia³owie¿a, Poland tel. (+48 85) 682 77 61, tel./fax (+48 85) 682 77 52 e-mail:
[email protected]
gnostics. Deficiency of 21-steroid hydroxylase, which is responsible for more than 90% of congenital adrenal hyperplasia (CAH) cases in humans, is conditioned by numerous mutations in the gene coding 21-hydroxylase – CYP21 (1, 2). SNP type mutations in human active CYP21 gene are of a unique scientific value since most of them code the disorder and may serve as markers of 21-steroid hydroxylase deficiency. Minisequencing is a highly specific electrophoretical assay able to interrogate up to ten single nucleotide polymorphisms (SNPs) of known mutation sites in one tube (3, 4). The method has become a valuable qualitative diagnostic tool, also in the 21-hydroxylase deficiency syndrome (5).
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Methods In this attempt we used the Multiplex SNaPshot® (ABI Prism) minisequencing kit to genotype seven most common mutation sites, related with CAH symptoms in humans. Genotyping the most common point mutations and deletions in the CYP21 gene is expected to detect 94 to 99% of deleterious alleles associated with CAH in any Middle European population (6). DNA samples of 15 patients with clinically diagnosed CAH and their families (n = 50) were isolated using QIAamp DNA Blood Kit (Qiagen), with final DNA concentrations of approx. 30 ng/µl. The analysis was performed in two steps. CYP21 is an active gene while CYP21P, located closely, is its inactive mutated copy. Due to their obvious structural similarities, both gene forms may converse and create a chimeric CYP21P/CYP21 gene, responsible for a large part of CAH cases (2). In the first step of PCR we used specific pri-
mers, which bound only to the functional form of the CYP21 gene to avoid amplification of the pseudogene or their chimeric form (gene/pseudogene). Reverse primer (5’-atc act ggc tgt ggg ccg agg gga ggc-3’) is complementary to both active CYP21 gene and CYP21P pseudogene. Forward primer (5’-aga ggg ccc agg tgg ggg cgg aca cta-3’) allows for specific amplification of coding CYP21, but not of a pseudogene (after Lee et al. – modified fig. 1A). Amplified CYP21B gene fragment (1750 nt) includes all the analysed mutation sites. In the second step, seven (P30L, Intron2”g”, G110∆8nt, I172N, I236N, V237E and M239K) out of over forty known mutations responsible for 21-hydroxylase deficiency symptoms were minisequenced. Out of seven original primers used, four primers were forward (F) and three reversely (R) orientated (tab. I) Each of the minisequencing primers was designed to bind its last 3’ base to the premutation site nucleotide. Since the primers were of similar length, each was added a poly deoxy adenine triphosphate (dATP)
Table I: Multiplex SNaPshot primers designed and used for the study Tabela I: Primery do multipleksu SnaPshot zaprojektowane i wykorzystane w badaniach Mutation location and name Miejsce i nazwa mutacji
5'-3' primer sequence Sekwencja primera w kierunku 5'-3'
Primer position (5'-3')* Pozycja primera (5'-3')*
dATP tail length D³ugoœæ ogonka dATP
gca aga agc ccg ggg caa gag gc
1790 - 1768 nt
-
intron II Intron2"g"
cag ttc cca ccc tcc agc ccc ca
2310 - 2333 nt
6 (dATP)
exon III G110∆8nt
ttg tgg gct ttc cag agc agg ga
2415 - 2393 nt
9 (dATP)
exon IV I172N
tgt ctc cga agg tga ggt aac ag
2700 - 2677 nt
15 (dATP)
exon VI I236N
ggc cat aga gaa gag gga tca ca
3035 - 3057 nt
21 (dATP)
exon VI V237E
cat aga gaa gag gga tca cat cg
3038 - 3060 nt
27(dATP)
exon VI M239K
gaa gag gga tca cat cgt gga ga
3044 - 3066 nt
33 (dATP)
exon I P30L
* nucleotide positions are given in accordance to GeneBank accession numbers M12792 and M23280/pozycje nukleotydów s¹ zgodne ze zg³oszeniami do GeneBank nr M12792 i M23280
Table II: Novel substitutions found in five CAH mutation sites using multiplex minisequencing Tabela II: Nowe substytucje wykryte przy u¿yciu multipleksowego minisekwencjonowania w piêciu miejscach mutacji zwi¹znych z wystêpowaniem WPN (exon - ekson, intron - intron, deletion - delecja) Mutation location and name Miejsce i nazwa mutacji
Mutation type and position* Typ i pozycja mutacji
Novel substitutions Nowe substytucje
C → T (1767 nt)
G
intron II Intron2"g"
A, C → G(2333 nt):
T
exon III G110∆8nt
8 nt deletion C → G(2385-2392 nt)
A
exon IV I172N
T → C (2677 nt)
C
exon VI I236N
T → A (3058 nt)
G
exon I P30L
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* nucleotide positions are given in accordance to GeneBank accession numbers M12792 and M23280 pozycje nukleotydów s¹ zgodne ze zg³oszeniami do GeneBank nr M12792 i M23280
tail of different length at its 5’ end to differentiate their lengths and thus enable their precise identification and readout after completing ABI electrophoresis run. Since multiplex SNaPshot reaction mix contained exclusively fluorescently labeled dideoxynucleotides ([F]ddNTPs), annealing of the SNaP primer was restricted to only one nucleotide. Each of four [F]ddNTP kinds was labeled with different dye, allowing for acute identification of the fluorescent signal (fig. 1B). CYP21B amplification (first step PCR) was performed in total volume of 20 µl, including 2.5 µl of 10XPCR Buffer (Qiagen), 3.0 µl of dNTP mix (DNA Gdansk), 0.5 µl (25 pmol/µl) of each primer (Bionovo), 1.5 µl MgCl2 and 12 µl of deionized water. PCR thermal conditions for gene amplifying were: 2 minutes at 96°C followed by 30 cycles of 1 min at 96°C, 3 min at 74°C and elongation at 74°C for 7 min. PCR products were cleaned up using Sap and Exo I (Applied Biosystems), according to producer’s protocol. Minisequencing PCR was performed in total volume of 5 µl, including 1.5 µl of SNaPshot mix (Applied Biosystems), 1.6 µl of primer mix (tab. II), 0.5 µl of cleaned up PCR product and 1.35 µl of deionized water. Concentrations of distinct primers in a final reaction volume were
Multiplex minisequencing applied in detection of human functional CYP21 gene mutations
Fig. 1A. Specificity of first step amplification primers to active gene sequence Ryc. 1A. Specyficznoœæ primerów pierwszego etapu amplifikacji do sekwencji genu (pseudogene – pseudogen, gene – gen, primer – primer, inactive CYP21P/CYP21 chimera gene – nieaktywny gen chimera CYP21P/CYP21)
Fig. 1B. SNP multiple minisequencing schema in the second step. [F]ddGTP, [F]ddCTP, [F]ddATP and [F]ddTTP stand for fluorescently labelled dideoxynucleotides. Blue, red, green or black fluorescent signals produced by the incorporated [F]ddNTP indicate presence or absence of deletorious substitution at studied mutation sites. Ryc. 1B. Schemat multipleksowego minisekwencjonowania SNP w drugim etapie. [F]ddGTP, [F]ddCTP, [F]ddATP i [F]ddTTP oznaczaj¹ fluorescencyjnie znakowane dideoksynukleotydy. Barwny sygna³ fluorescencyjne wysy³any przez ka¿dy do³¹czony do primera [F]ddNTP wskazuje na obecnoœæ lub brak niekorzystnej zmiany w badanym miejscu mutacji (amplified DNA strand – namno¿ona niæ DNA, fluorescent signal – sygna³ fluorescencyjny, unincorporated [F]ddNTPs – niedo³¹czone [F]ddNTPs, primer tail – niekomplementarny do badanej sekwencji ogonek poly(dATP) do³¹czony do primera)
diverse: from 0.3 to 2.0 pmol/µl. SNaPshot PCR thermal profile was 34 cycles of 96°C for 10 s, followed by 61°C for 5 s and 30 s at 72°C. Results of the SNaPshot PCR reaction were read out on ABIPrism 310 Genetic Analyser using GeneScan 2.1 software.
Results This study allowed to detect known CAH responsible mutations in 100% of patients. We also found novel substitutions of unknown effect at five out of seven mutations sites, in 8 patients and 3 family members (tab. II). The method enabled to point out potential large scale pseudogene-gene conversions resulting in no product of first step PCR. We observed that both extending the primers by using over 30
nt poly(dATP) tail as well as increasing the long primers concentration lead to am apparent decrease in the amplification yield of the primer’s product or even to inhibition of the whole reaction. The method thus requires very careful composition of primers concentration in the reaction mix. Nevertheless, we found a report on using as much as 13 multiplex primers with poly (dTTP) tails in SNP multiplex analysis (4). The possibility to analyse multiple mutation sites in one reaction tube significantly lowers costs and labour absorbing, while the accuracy of the examination may be compared with sequencing results. Nevertheless, the method is not appropriate in searching for novel mutations, it is restricted and recommended for fast detection of substitutions at known mutation sites. Praca wp³ynê³a do Redakcji: 02.04.2007. Zaakceptowano do druku: 19.09.2007
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References 1. White P.C., Speiser P.W.: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr. Rev., 2000, 21, 245-291. 2. Lee H.-H., Chang J.-G., Tsai C.-H. et al.: Analysis of the chimeric CYP21P/CYP21 gene in steroid 21-hydroxylase deficiency. Clin. Chem., 2000, 46, 606-611. 3. Pastinen T., Kurg A., Metspalu A. et al.: Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays. Genome Res., 1997, 7, 606614. 4. Krone N., Braun A., Weinert S., Peter M et al.: Multiplex minisequencing of the 21hydroxylase gene as a rapid strategy to confirm congenital adrenal hyperplasia. Clin. Chem., 2002, 48, 818-825.
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5. Keen-Kim D., Redman J.B., Alanes R.U. et al.: Validation and clinical application of a locus-specific polymerase chain reaction- and minisequencing-based assay for congenital adrenal hyperplasia (21-hydroxylase deficiency). J. Mol. Diagn., 2005, 7,. 236-246. 6. Dolzan V., Solyom J., Fekete G. et al.: Mutational spectrum of steroid 21-hydroxylase and the genotype-phenotype association in Middle European patients with congenital adrenal hyperplasia. Eur. J. Endocrinol., 2005, 153, 99-106.
