Diagnosing X-linked Ichthyosis by Monoplast Single-round Duplex PCR

13 4 Vol13 No4 2009 8 Life Science Research Aug 2009 2009 PCR X- *,,,,,,,, 410078 : X- (X-linked ichthyosis, XLI), XLI, XLI (preimplantation genetic diagnosis, PGD) PCR amelogenin (Amel), 958% 909%, 15%; Amel-Y 919% 927%, 14% 0; ADO 62% PCR XLI, XLI PGD : PCR; X- (XLI); ; amelogenin ; (PGD) : R3943 :A :1007-7847(2009)04-0332-05 Diagnosing X-linked Ichthyosis by Monoplast Single-round Duplex PCR ZHONG Chang-gao LI Lu-yun LU Chang-fu GONG Fei LIAO Hong-qing ZHANG Zhan GAO Bai-di ZHANG Shuo-ping LU Guang-xiu * Institute of Reproduction and Stem Cell Engineering Central South University Reproductive & Genetic Hospital CITIC-XIANGYA Changsha 410078 Hunan China Abstract X-linked ichthyosis XLI is a sort of sex linked recessive inherited diseases It is one of the most common congenital metabolic diseases of the mankind Diagnosing XLI on monoplast level using monoplast PCR that can provide a basis for preimplantation genetic diagnosis PGD of XLI The gene and the amelogenin Amel gene were amplified by single-round duplex PCR with single lymphocytes from the patient the normal female and single blastomere from the normal person The amplification success rates were 958% and 909% on gene in the groups of single lymphocytes and single blastomere from the normal person respectively The false positive rate was 15% on gene in the groups of single lymphocytes from the patient The amplification success rates were 919% and 927% on the gene loci of and Amel-Y respectively The contamination rates were 14% and 0 on the above 2 gene loci respectively The allele dropout rate ADO was 62% on the Amel loci To some extent the protocol of monoblast single-round duplex PCR for diagnosing XLI is accurate specific and reliable It will help us further develop the PGD of XLI : 2009-04-24 : 2009-05-31 : 1013-8 03JJY3120 : 1968- * 1939- Tel 0731-84497661 E-mail lgxdirector@sinacom

4 : PCR X- 333 Key words monoblast single-round duplex PCR X-linked ichthyosis XLI gene amelogenin gene preimplantation genetic diagnosis PGD Life Science Research 2009 13 4 332~336 X XLI HEPES 1 IVC-HTF HEPES 02 μl 25 μl 200 μl 02 μl [1] XLI steroid sulfatase 113 PCR X Xp2232 164 3 kb 10 85% ~90% -F 5 - GCTTCAGCTGTGCTGTCCTT-3 -R 5 -CCCAAGCCTTCAGGTGATAA-3 PCR 223 bp [5] [2] PGD amelogenin Amel-F 5 -ATCAGAG 1989 Handyside CTTAAACTGGGAAGCT-3 Amel-R 5 -CCTGGG PGD CTCTGTAAAGAATAG-3 PCR X [3] PGD 105 bp Y 111 bp 12 [4] 121 PGD PCR 20 μl 65 X 10 min -70 PCR 122 PCR 1 11 111 X Sigma 1 PCR Buffer 03 mmol / L dntp 0625 U HotStarTaq Ivitrogen PCR IVC-HTF HEPES 95 12 min 30 s 96 2 min In Vitro Care International 96 25 s 65 30 s 05 72 30 s 10 94 25 s 60 [4] 02 μl 25 μl 25 s 72 25 s 49 72 5 min 200 μl 4 02 μl 123 PCR 112 25 μl 200 mmol/l Tris Gly [6] Tricine ph 473 Sigma 25 μl 02 μmol / L 008 μmol/l Amel 2 mmol/l MgCl 2 01% 1 μl 1 loading buffer 10% 29 1 500 V acrylamide gel electrophoresis PAGE 2 h Tyrode 1 min IVC-HTF

334 2009 2 21 PCR PCR 68 PCR 1 223 bp Amel 15% 1 / 68 Amel X Y 105 bp X 63 927% 63 / 68 111 bp Y 3 X Y ADO 105 bp Amel 2 Amel ADO 62% 4/65 105 bp 111 bp 37 1 Amel X allele dropout 27% 1 / 37 1 ADO Table 1 1 Amplification results of single lymphocytes and blastomeres Cell types No Noof blank control Amplified gene Amplification success rate False positive rate Single lymphocyte of patient of deletion type Single lymphocyte of normal female Blastomere of normal person 68 48 44 37 23 13 Amel-Y Amel-Y 15% 1 / 68 927% 63 / 68 927% 63 / 68 958% 46 / 48 917% 44 / 48 909% 40 / 44 909% 40 / 44 546% 24 / 44 15% 1 / 68 27% 1 / 37 0 0 / 37 0 0 / 23 0 0 / 23 bp 331 242 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 190 147 111 110 1 PCR M: DNA ; 1~4: PCR ; 5~9: PCR ; 10~14: PCR ; 15 16: gdna ; 17~20: HEPES Fig1 The amplification results of monoplast with single-round duplex PCR M puc Mix Marker 8 molecular weight marker Lanes 1~4 the PCR products of single lymphocyte from the patient Lanes 5~9 the PCR products of single lymphocyte from the normal female Lanes 10~14 the PCR products of single blastomere from the normal person 15 16 gdna positive control of the male and the female respectively Lane 17~20 blank control with washing solution of single lymphocyte from the patient single lymphocyte from normal female single blastomere from the normal person and blank control with HEPES culture medium respectively 22 PCR PCR 46 958% 46 / 48 Amel X 48 44 917% 44 / 48

4 : PCR X- 335 23 1 PCR PCR 23 PCR PCR PCR DNA 44 40 PCR 909% 40/44 Amel X 40 909% 40 / 44 44 Amel Y 24 546% 24 / 44 13 1 HotstarTaq 10 Touchdown PCR 3 59 DNA XLI 1 6 000 1 2 000 931% 149 / 160 PCR XLI PCR 1 PCR 50% 50% 50% PCR PCR XLI 85%~90% XLI 3 PGD PGD XLI X- XLI PGD SRY Amel SRY PGD SRY Amel PGD Amel X / Y 1 X PCR Y PCR PCR 6 105 bp [5,8] 1 105 bp 111 bp PCR Genebank Amel X Y PCR PCR 105 bp 111 bp [5] 106 bp 112 bp Amel SRY primer extension ADO preamplification PEP Y PCR Zhang Amel X Wells [4,7] PCR 10 [9,10] PEP 78% ~90% 96 ADO 65 Amel PGD PCR 938% 61/65 ADO 62% 4/65

336 2009 [11] 5%~20% 2005 31 3 186-188 958% 909% Amel-Y 919% 147/160 927% [10] 90% PCR DNA 68 Yan-Lin LI Qi PAN Qian et al 1 15% South Univ Med Sci 2002 27 6 DNA 73 1 Amel- X 14% 1 / 73 hybridization[j] Nucleic Acids Res 1999 Amel-Y PCR XLI XLI PGD (References): Xue-jun YANG Sen Progress of molecular pathogenesis on X-linked ichthyosis[j] F Med Sci Sect Dermatol Venereol [3] GOOSSENS V HARTON G MOUTOU C et al ESHRE PGD Consortium data collection VIII cycles from January to December 2005 with pregnancy follow-up to October 2006[J] Hum Reprod 2008 23 12 2629-2645 [4] ZHANG L CUI X F SCHMITT K et al Whole genome amplification from a single cell Implications for genetic analysis[j] Proc Nat Acad Sci USA 1992 89 5847-5851 [5] [J] MA Single cell PCR for sex determination in preimplantation genetic diagnosis[j] J Cent 487-490 [6] De RYCKE M GEORGIOU I SERMON K et al PGD for autosomal dominant polycystic kidney disease type 1[J] Mol Hum Reprod 2005 11 65-71 [7] WELLS D SHERLOCK J K HANDYSIDE A H et al Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic 274 1214-1218 [8] NAKAHORI Y HAMANO K IWAYA M et al Sex identification by polymerase chain reaction using X-Y homologous primer[j] Am J Med Genet 1991 396 472-473 [9] RAY P F HANDSIDE A H Increasing the denaturation temperature during the first cycles of amplification reduces allele drop out from single cells for preimplantation diagnosis [J] Mol Hum Reprod 1996 2 213-218 [10] PCR [J] ZHONG Chang- [1] [J] gao LI Lu-yun LU Chang-fu et al Diagnosing achondroplasia LU Guo-fang ZHANG Ying Progress by single cell nested-pcr [J] Chin J Med Genet of pathogenesis on genetic ichthyosis[j] Chinese Journal of Bir-th Health and Heredity 2007 15 12 120-121 124 [11] 2003 20 3 228-231 HARPER J C WELLS D Recent advances and future [2] developments in PGD[J] Prenat Diagn 1999 19 1193- [J] XIAO Feng-li ZHANG 1199 CA BIOSIS (BA) Medical Index AJ NCBI CABI 20 2005 2006-2009 B 2007 Journal of Genetics and Genomics 2008 SCI-E ISSN 1673-8527 CN11-5450/R 12 2-819 M63 2010 50 600 http://wwwjgenetgenomicsorg/ ISSN 0253-9772 CN11-1913/R 12 2-810 M62 2010 50 600 http:// wwwchinagenecn 5, : 1 :100101 010-64807669 010-64807786 : E-mail:ybxue@geneticsaccn : E-mail:swli@geneticsaccn