- Email: [email protected]
HLA-A locus specificities identified by sequence specific PCR
56
Abstracts
IV-54
IV-53 Peter Krausa 1, Jonathan Moses 2. Walter Bodmer 1. Julia Bodmer 1,2, Michael
Mike Bunco, and Ken I. Welsh Clinical Transplantation Immunology, Nuffield Dept. of Surgery, Oxford Transplant Centre, Oxford, OX3 7L J, England
Browning l I Cancer Immunology
Laboratory.
Imperial Cancer Research Fund.
Institute
Rapid HLA-Cw typing using sequence-specific primers (PCR-SSP)
of Molocu|ar Medicine. John Radcliffe Hospital. Oxford OX3 9DU and 2Tissue Antigen Laboratory. Imperial Cancer Research Fund.
61 Lincoln's Inn Fields.
Loudo, WC2A 3PX
semlogicat HLA-Cw typing i$ generally unreliedle for the antigens HLA-Cw6, Cw7 and
HLA-A locus specificities identified by Sequence Specific PCR
Cw8 due to a lack of monosc~cific Cw reagents. In addition the Cw alleles Cw'1201, Cw'1202. Cw'1301, Cw*1401 and the Cw*15 group of alleles are all undetected by semlugicel methods even though they are expressed as products. We describe here a DNAJoaseOCw typing medxx:lwhch resolves the i~oblems of Cw detection by serology.
We have established a system for typing the HLA Class I 'A' locus from genomic DNA, by a one-step polymcrase chain reaction (PCR) based on ARMS (Amplification Refractory
Mutation System l). The ARMS principle allows
specificity to be determthed by the use of oligonucleotide primers in which a 3' single base mismatch inhibits the priming of non-specific reactions. Combinations
primer combination has been validated against DNA from ceils of known Primer combinations have been designed to distinguish HLA-A
locus specificities HLA-A], A2, A3, A9 (A23 & A24), A10 (A25 & A26), A1 I, A28 (A6801, A6802 & A69). and A29, A30, A31. A32 & A33. All reactions are run under a single set of conditions, and a standard
PCR-SSP takes two hours to perform and is therefore surtai~e for the genotyping of cadaveric donors. We have deszgned a set of pnrners, which m nine PCR reactions, will monol~ecifically amplity the HLA-Cw alleles corresponding to the semlogicatly defined series Cwl. Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8 and the rlon-semlogicatly defined
of such primers allow sequence-specific amplification of
target DNA, to yield PCR products of defined size. The spe.cificity of each tissue type,
Arnplificetion of DNA using sequence-sbecific primers (PCR-SSP) has proved a reliable and rapid moftmO for typing HLA-DRB1 (1) and DQBt (manuscnlX sul~mitted) genes.
internal control defines the
success of each reaction. The HLA-A locus tissue type is determined by the
Cw°1501. Cw'1502 and Cw'1503. Amplilication primers for the remaining HLA-Cw alleles, Cw°1201. Cw'1202. Cw'1301 and Cw'1401 are still being tested. Seventeen hornczygous cell lines and 18 individuals have been typed by the Cw-PCR-SSP technique and compared to serology. NO raise nagatlve or false positive amplihcations were recorded. Further tests are being done to verify the allate-speeific nature of the Cw a~ions in all Cw hatemzygote coatoinefions, Cw-PCR-SSP is non-isotope and can
presence or absence of appropriate sized bands on the gel.
take as little as 130 n~nctee tram start to finish, including DNA praparation. Our Cw-SSP typ=ng method is designed to be coml~ementaTyto the published DRB1-SSP and our
In practical terms, the method uses renewable reagents and standard
own DQB1-SSP in that the same 13gl reac~onvolumes and PCR parameters are used,
laboratory equipment, widening the scope of tissue typing for clinical or research puq~oses. A further advantage of the system is that it does not require viable ceils, and can be used to type material derived from cells that cannot be tissue typed
by conventional means, Extension of this approach
Ref. Olemp O, Zatterquist H: HI.A-DR tYl~ng by PCR amplifcation with sequencespaclic primers (PCR-SSP) in 2 hours: An alternative to semlngcal DR typing in clinical p ~ including Oonor-mcip*entmatctling in cadavenc translNantat=on.Tissue Aoflgens 39: 225, 1992.
should allow other HLA Class 1 loci to be typed, This method
holds significant implications for both
histocompatibility
testing for organ transplantation and HLA disease association. (1). New[on CR, Graham A, Heptins~li LEet aL (1989) Nucleic Acids Res 17. 2503-
IV-56
2516.
"~)ufoss~ F.,'" Loiceau P, ""Ragu~n~s O, "Cracco P, "'Charron D. " ' F 6 r e c C, "'*Mercer B.
IV-55 M. F a l c o * , L. Delfino*, A. Colonna*#, a n d G.B. Ferrara*&
Lonqo*,
A.
Morabito*,
M.
* Laboratory of Immunogenetica. Iatituto Nazionale per la Rioarca aul Cancro, 16132 Geneva, Italy # D e p a r t m e n t of S i o c h e m e a t r y and M o l e c u l a r Biology, Harvard University, Cambrldqa, MA 02138, U S A & Centre Ricarche Immunoematologicha, A.V.I.S., Bergamo, Italy ANALYSIS O F MI,%-C POLYMORPHISM e Y P C R A N D HLA-C polylorphiam
has in
seroloqical restricted
met.hods. the
by alloantlbod£ea
been shown to comparison with However, limited
eso
display a limited MLA-A and HLA-B by
theaa studies availability
hava been of human
specific for HLA-C, due to low antiqanicity in v i v o of H L A - C molecules, p o s s i b l y resulting from a very low expression on cell surface. Thus, the MLA-C polylRorphllm was analysed at the OMA level by polymeraae chain reaction (PCR) and hybridization with sequence specific oligonucleotidea (SSO) in a panel inoludlng 56 homozygoue cell lines of the 10 IHW and 40 random individuals free the Italian p o p u l a t i o n . All the s a m p l e s were also e e r o l o q l c a l l y
typed. Two p r i m e r s p a i r s w e r e c h o s e n for the s p e c i f i c amplification of the second axon of HLA-C. A third primers pair amplified the third oxen of HLA-C and taw a l l e l e s of MLA-B. T h e p o l y m o r p h l l m of the a m p l l f i a d fragmenEe was analysed b y hybridization with 2 7 probes. Specific hybridization patterns were identified CorEelpondinq to MLA-CVl, -Cw2, -Cw3, -Cv4, -C~d5, -C~d6, -Cw7, -Cw8 serological epeclflcitiee. Moreover we were able to split the C w6 a11ele= in HLA-C*0601 and Cw6w, the Cv7 in MLA-C*0701 and HLA-C*0702, and the Cw8 in HLAC*0801 and HLA-C*0802. A d d i t i o n a l M L A - C a l l e l e s p r e v i o u s l y d e f i n e d by D N A sequencing were also identified, including HLA-C blanks a l l e l e s , such as H L A - C * I 4 0 1 , BaWo, HLA-C*I201, HLAC*I202, HLA-C*I301, CX, and C1.10.
C e n t r e R~gional de Transfusion Sanguine, 21 r u e C. Gu~rin - 59012 LILLE "" H61)ital Saint Louis, Laboretoire crlmmunologie et d'Histocomoatibilit6 - 1 avenue C. Vellelaux - 75010 PARIS *'* Centre de Blogen~tique - C D.T.S - BP 454 - 29275 BREST •
HLA D R TYPING BY PCR-SSP : Advantages and Irmonvenlenoel alter Ida months of routine use in three laboretorml
The HLA DR locus is known to be the most polymorpmc locus coding tor the HLA class II molecules. During the last years different typing methods have been developed : serology, RFLP, PCR-SSO and PCR-RFLP. The serological typing error rate is up to 25%. The molecular biology techniques were time ¢onsummg as five days were necessary for the RFLP and one clay for the PCR-SSO and PCR-RFLP. The besonption in 1992 of the PCR-SSP, a/low*ng an HLA DR typing in lesl than three hours, renders possible the molecular biology typing in a time coml~tib~ with trans!~antafion. The exact determination of the a/leads can then be performed by direct digestion of the apeofic aml~ifisd fragments. The PCR-SSP is routinely used since Judy 1992 by our three laboratories. Here we report the results obtained and the advantages and inconvenisnta of this method wiU be dmousesO. Biistly more than 1000 cell= have been routinely typed. No false po~tive was obeen,od. A retrosl:ectve study on 155 organ's donors showed that this method allowed the identification of 98% of the antigens. Moreover this method is now routmely used for the t y i n g of organ's donors, and 13 typings have been pedonned in emergency in the last 5 months. This method is a/so used in one laboratory for the HLA DRB1 atlolic typing of bone marrow donors. We conclube that this method allowing a rapid HLA DR typing can be routirla/y used. This routinely use o1 this method and the development of exchange of organs between the tranplantatlons centers could allow a better matching between donor and recipient of organ transplantation,
Abstracts
IV-54
IV-53 Peter Krausa 1, Jonathan Moses 2. Walter Bodmer 1. Julia Bodmer 1,2, Michael
Mike Bunco, and Ken I. Welsh Clinical Transplantation Immunology, Nuffield Dept. of Surgery, Oxford Transplant Centre, Oxford, OX3 7L J, England
Browning l I Cancer Immunology
Laboratory.
Imperial Cancer Research Fund.
Institute
Rapid HLA-Cw typing using sequence-specific primers (PCR-SSP)
of Molocu|ar Medicine. John Radcliffe Hospital. Oxford OX3 9DU and 2Tissue Antigen Laboratory. Imperial Cancer Research Fund.
61 Lincoln's Inn Fields.
Loudo, WC2A 3PX
semlogicat HLA-Cw typing i$ generally unreliedle for the antigens HLA-Cw6, Cw7 and
HLA-A locus specificities identified by Sequence Specific PCR
Cw8 due to a lack of monosc~cific Cw reagents. In addition the Cw alleles Cw'1201, Cw'1202. Cw'1301, Cw*1401 and the Cw*15 group of alleles are all undetected by semlugicel methods even though they are expressed as products. We describe here a DNAJoaseOCw typing medxx:lwhch resolves the i~oblems of Cw detection by serology.
We have established a system for typing the HLA Class I 'A' locus from genomic DNA, by a one-step polymcrase chain reaction (PCR) based on ARMS (Amplification Refractory
Mutation System l). The ARMS principle allows
specificity to be determthed by the use of oligonucleotide primers in which a 3' single base mismatch inhibits the priming of non-specific reactions. Combinations
primer combination has been validated against DNA from ceils of known Primer combinations have been designed to distinguish HLA-A
locus specificities HLA-A], A2, A3, A9 (A23 & A24), A10 (A25 & A26), A1 I, A28 (A6801, A6802 & A69). and A29, A30, A31. A32 & A33. All reactions are run under a single set of conditions, and a standard
PCR-SSP takes two hours to perform and is therefore surtai~e for the genotyping of cadaveric donors. We have deszgned a set of pnrners, which m nine PCR reactions, will monol~ecifically amplity the HLA-Cw alleles corresponding to the semlogicatly defined series Cwl. Cw2, Cw3, Cw4, Cw5, Cw6, Cw7, Cw8 and the rlon-semlogicatly defined
of such primers allow sequence-specific amplification of
target DNA, to yield PCR products of defined size. The spe.cificity of each tissue type,
Arnplificetion of DNA using sequence-sbecific primers (PCR-SSP) has proved a reliable and rapid moftmO for typing HLA-DRB1 (1) and DQBt (manuscnlX sul~mitted) genes.
internal control defines the
success of each reaction. The HLA-A locus tissue type is determined by the
Cw°1501. Cw'1502 and Cw'1503. Amplilication primers for the remaining HLA-Cw alleles, Cw°1201. Cw'1202. Cw'1301 and Cw'1401 are still being tested. Seventeen hornczygous cell lines and 18 individuals have been typed by the Cw-PCR-SSP technique and compared to serology. NO raise nagatlve or false positive amplihcations were recorded. Further tests are being done to verify the allate-speeific nature of the Cw a~ions in all Cw hatemzygote coatoinefions, Cw-PCR-SSP is non-isotope and can
presence or absence of appropriate sized bands on the gel.
take as little as 130 n~nctee tram start to finish, including DNA praparation. Our Cw-SSP typ=ng method is designed to be coml~ementaTyto the published DRB1-SSP and our
In practical terms, the method uses renewable reagents and standard
own DQB1-SSP in that the same 13gl reac~onvolumes and PCR parameters are used,
laboratory equipment, widening the scope of tissue typing for clinical or research puq~oses. A further advantage of the system is that it does not require viable ceils, and can be used to type material derived from cells that cannot be tissue typed
by conventional means, Extension of this approach
Ref. Olemp O, Zatterquist H: HI.A-DR tYl~ng by PCR amplifcation with sequencespaclic primers (PCR-SSP) in 2 hours: An alternative to semlngcal DR typing in clinical p ~ including Oonor-mcip*entmatctling in cadavenc translNantat=on.Tissue Aoflgens 39: 225, 1992.
should allow other HLA Class 1 loci to be typed, This method
holds significant implications for both
histocompatibility
testing for organ transplantation and HLA disease association. (1). New[on CR, Graham A, Heptins~li LEet aL (1989) Nucleic Acids Res 17. 2503-
IV-56
2516.
"~)ufoss~ F.,'" Loiceau P, ""Ragu~n~s O, "Cracco P, "'Charron D. " ' F 6 r e c C, "'*Mercer B.
IV-55 M. F a l c o * , L. Delfino*, A. Colonna*#, a n d G.B. Ferrara*&
Lonqo*,
A.
Morabito*,
M.
* Laboratory of Immunogenetica. Iatituto Nazionale per la Rioarca aul Cancro, 16132 Geneva, Italy # D e p a r t m e n t of S i o c h e m e a t r y and M o l e c u l a r Biology, Harvard University, Cambrldqa, MA 02138, U S A & Centre Ricarche Immunoematologicha, A.V.I.S., Bergamo, Italy ANALYSIS O F MI,%-C POLYMORPHISM e Y P C R A N D HLA-C polylorphiam
has in
seroloqical restricted
met.hods. the
by alloantlbod£ea
been shown to comparison with However, limited
eso
display a limited MLA-A and HLA-B by
theaa studies availability
hava been of human
specific for HLA-C, due to low antiqanicity in v i v o of H L A - C molecules, p o s s i b l y resulting from a very low expression on cell surface. Thus, the MLA-C polylRorphllm was analysed at the OMA level by polymeraae chain reaction (PCR) and hybridization with sequence specific oligonucleotidea (SSO) in a panel inoludlng 56 homozygoue cell lines of the 10 IHW and 40 random individuals free the Italian p o p u l a t i o n . All the s a m p l e s were also e e r o l o q l c a l l y
typed. Two p r i m e r s p a i r s w e r e c h o s e n for the s p e c i f i c amplification of the second axon of HLA-C. A third primers pair amplified the third oxen of HLA-C and taw a l l e l e s of MLA-B. T h e p o l y m o r p h l l m of the a m p l l f i a d fragmenEe was analysed b y hybridization with 2 7 probes. Specific hybridization patterns were identified CorEelpondinq to MLA-CVl, -Cw2, -Cw3, -Cv4, -C~d5, -C~d6, -Cw7, -Cw8 serological epeclflcitiee. Moreover we were able to split the C w6 a11ele= in HLA-C*0601 and Cw6w, the Cv7 in MLA-C*0701 and HLA-C*0702, and the Cw8 in HLAC*0801 and HLA-C*0802. A d d i t i o n a l M L A - C a l l e l e s p r e v i o u s l y d e f i n e d by D N A sequencing were also identified, including HLA-C blanks a l l e l e s , such as H L A - C * I 4 0 1 , BaWo, HLA-C*I201, HLAC*I202, HLA-C*I301, CX, and C1.10.
C e n t r e R~gional de Transfusion Sanguine, 21 r u e C. Gu~rin - 59012 LILLE "" H61)ital Saint Louis, Laboretoire crlmmunologie et d'Histocomoatibilit6 - 1 avenue C. Vellelaux - 75010 PARIS *'* Centre de Blogen~tique - C D.T.S - BP 454 - 29275 BREST •
HLA D R TYPING BY PCR-SSP : Advantages and Irmonvenlenoel alter Ida months of routine use in three laboretorml
The HLA DR locus is known to be the most polymorpmc locus coding tor the HLA class II molecules. During the last years different typing methods have been developed : serology, RFLP, PCR-SSO and PCR-RFLP. The serological typing error rate is up to 25%. The molecular biology techniques were time ¢onsummg as five days were necessary for the RFLP and one clay for the PCR-SSO and PCR-RFLP. The besonption in 1992 of the PCR-SSP, a/low*ng an HLA DR typing in lesl than three hours, renders possible the molecular biology typing in a time coml~tib~ with trans!~antafion. The exact determination of the a/leads can then be performed by direct digestion of the apeofic aml~ifisd fragments. The PCR-SSP is routinely used since Judy 1992 by our three laboratories. Here we report the results obtained and the advantages and inconvenisnta of this method wiU be dmousesO. Biistly more than 1000 cell= have been routinely typed. No false po~tive was obeen,od. A retrosl:ectve study on 155 organ's donors showed that this method allowed the identification of 98% of the antigens. Moreover this method is now routmely used for the t y i n g of organ's donors, and 13 typings have been pedonned in emergency in the last 5 months. This method is a/so used in one laboratory for the HLA DRB1 atlolic typing of bone marrow donors. We conclube that this method allowing a rapid HLA DR typing can be routirla/y used. This routinely use o1 this method and the development of exchange of organs between the tranplantatlons centers could allow a better matching between donor and recipient of organ transplantation,