- Email: [email protected]
Characterization of DRB5*0105 by PCR-SSP and direct sequencing
116
Abstracts
p630
p631
CHARACTERIZATION OF AN ELUSNE IlLA-A*6803 ALLELE Ma ry Ell e xs on 1• Marie Lau 2 • Pa ul Terasaki - , and Willi am Hilde hra nd l 1 University of Okl ahom a Health Sciences Cente r. Okla homa City. OK. USA; 2University of California, Los Angeles. CA. USA. We have characte rized a novel A'6803 allele from an Hispanic individual which most resembles A'680 12. This new A'68 allele was cloned and sequenced from a cell run on the Intern ation al Ce ll Exchange (I CE) which wa s se ro logic ally typed by 169 laboratories and DNA typed (at the HLA-A locus) by 14 laboratories. A'6803 differs from A'68012 by only one nucle otid e at positi on 28 2 whi c h tran slat es to a co ding difference at positi on 70 of the a I domain situated atop the a helix of the liL A class 1 molecule . With only one amioo acid difference from A'680 l , A' 6803 was di fficult to type when run on the ICE; A*6803 went undetected when typed by serology whil e only tw o of the fo urte en labor ator ies usi ng high resolution DNA typi ng methods detected an A'68 variant. The undete cted A*680 1/A* 6803 polymorphism at amino acid 70 is pos iti oned to af fec t the stereochem istr y of bot h B and C speci fici ty pockets, so that unde tected polym orph ism s at re sidue 70 will likely provo ke a llorcac ti ve res pon ses. Considering th at new HLA class I pol ymorph isms continue to be det ected, DNA sequence analysis will remai n a valuable mean s for characte rizing novel class ] mol ecules.
DNA-SEQUENCING OF NESTED PeR PRODUCTS AS A HLA-B27 SUBTYPING METHOD BV ANCVWSING SPONDVLITIS R. Frank, S. Kastel~ C. Seid1., E. Seifried, Institulefor Transfusion Medicine and Imrnunhaematology, Red Cross Donor Service Hessen, Frankfurt, Germany
The ankyIosing spondylitis (AS) is highly associated with HLA-B27 and detectable in 96% of Caucasian AS patients. Therefore the detennination of HLA-B27 is used to confinn the diagnosis of the disease. In molecularbiological HLA typing HLA B27 represent a group of nine different alleles HLA B·2701·B 2709. We have established a HLA-B27 subtyping strategic
where groupspecific amplification is combined with automatic DNA sequencing. The amplificlitilm was carrying out by nested PeR with primer sequences whichare located in exon 2 and 3 of the gene. Only alleles of the HLA-B27 group were amplified in this approach. After amplification and seperation of single stranded DNA with magnetic beads the sequence analysis was conducted on an AD! 373 DNA sequencer using Sequenase and fluorescence coupledddNTP's. We used this method to subtype 64 HLA B27 positivsamples from 3 I AS patients and 33 heahhy people (control samples). Only two different subtypes HLA B"2702 and HLA B·2705 were observed. In case of 7 (2 1%) control and 5 (16%) patient samples the allele HLA-B·2702 was found. Altogether 26 (79"10) control and 26 (84%) patient samples were HLA B·2705 positive. There was no significant difference in the appearance of HLA-B27 alleles betweenAS patient and controlsamples.
p632
p633
A mRNA BASED METHOD FOR TYPING THE HLA-C LO CUS GENE BV AUTOMATED SEQUENC ING BASED TYPING
HIGH RESOLUTION DRS TYPING BY SSP·PCR AND AUTOMATED DNA SEQUENCING Malco1m D. McGinnis, CandiaL. Brown, David M IOVlIDIli.sci, MorganP. Conrad and Mel Kronick. Applied Biosysterns Divisiooof Pertin Elmer.FoslerCity, CA, USA The genetic complexity of theORBregionof the hwnanMIIC has required deve1q>menl of molec:ular typing tEchniques with increasingly higher levels of resolution. Melhods such as PCR-SSOP ... SSP-PeR are not alwaysable 10 discriminate amoog the ...,.., Ihan130recognized alle1
Day Sarinder, Ross Joe, UK Transplant Support Service Authority, Bristol, UK We describe here a sequencing based typing (SBT) method for HLA-e locus using mRNA as the starting material, where exon 2 and exon 3 are sequenced together in one reaction. Messenger RNA was extracted from B Iymphoblastoid cell lines using the ' mRNA direct kit' (Dynal). Solid phase eDNA synthesis was carried out using M-MuLV reverse transcriptase, C-locus sequences including exon 2 and exon 3 were amplified with C-lueus specific primers (Santamaria et aI, 1993, Human Immunology 37:39) tailed with -2 1 ~f1 3 universal sequencing primer. Sequencing reactions were carried out using the Dye Primer Cycle Sequencing Ready Reaction -21 M13 kit with AmpliTaq DNA Polymerase FS (ABO). Sequencing reaction products were analysed on the ABD 373 sequencer. Each sample was sequenced in both forward and reverse orientations to resolve any ambiguous nucleotide positions and to confirm the HLA type. lILA types were assigned using published C locus sequence data for exon 2 and exon 3. This approach only requires two reactions to sequence in both directions. Furthermore the use of cycle sequencing with AmpliTaq FS eliminates the need for a single strand preparation step whilst producing even signal peak height, for accurate heterozygote base calling.
p634 DRBI BIGB RESOLUTION SEQUENCING BASED TYPING INDIVIDUALS; COMPARISON WITH PCR -SSP
or
30 llNRELATED
Da i e y de Br u yn, Pa ul Savel koul and El la van d e n Be rg-Loo nen
Tiss ue Typ ing Laboratory , Ma as t r ic ht, The Nethe r lands
Unive r s i ty
Hos p ita l
Maastri cht,
Ac c urate al lele as signment for HLA- ORBI al l e l es i s rather c omplex . S in ce t he number o f d i ff ere nt al le l es r ecog ni zed i s e xpand i n g qu i ckly , mo r e al lele comb i n at i o n s wil l b e f ound that r e sult in ident ical sequences. I n high re solut ion typing met hods as SS T thi s wi ll lead t o ambiguou s typi ng r e s u l t s , for Pe R-SSP t h i s wil l l e ad t o the ne c e s s i t y o f unaccep tab l e numbe r s of sSP react ions.
e
PCR:~S; ~;~nds:;u~K ~e ;:;:~~t j8 u%~\~~e~eBi;~~vf~~:t~~A~l ~::~tfil~f h::Pia;:~e~~~t~-i~~s~ ~:rthle~r·~~r c~:i~:~ i~~:
used . With our cu r r e nt LR/ HR SSP t yp i ng t.a c h n Lqu e 79 different DRBl al le l e s can be di s t i ngUi s h e d . t or saT the sequen ce an alys is was c onducted b y solid phaae d irect DNA sequencing on a Pharmacia. Alfexpreas, tyPing wa s performed with t he SBTyper sof tw are. No dfeccepenckee between SST an d PCR-S SP were no t ice d . 2 1 out o f 3 0 s anl.plea c o u l d be direc t l y t yped by a n a l ys i u o f the heteroz ygo us sequenc es . Fo r 9 sample s al lele epec Lf Ic ampli fi c ation was ne ce~aary to ob t a i n an unamb iguou s
~~Pi~fieref~~~ · £~a~net~:mP~~S;~~l~es~tiel:ma~g~i~t~~~usw~~: amp lified by the same pr i mer group.
Allele .ss ignment of H~ genes by QQqu e nc e an a lys i s improves the reeolution of HLA typing results. SaT proved a powerful tool in high resolution HLA ORB1-typi ng . The determination of the ac tu a l nucleotide sequence facilita.tes the d iscovery of new a lle1e8. The expanding number of r e cogn ized DRBl alleles will eventually lead to considerable time g ain of SBT over ssp and other DNA typing techniques. J
P635
CJlARACTERIZA'1ION OF DRBS· Ol0S BY' PeR-SSP AND DIREC'1 S EQUENCI NG
Fr a nc e-sea pc j L, Pa ol a Bi a nc h i, Lo re tt a Crespi a t ico , Ell a van d e n ae rc - t.ocnen " . Olle o l.e r up - anc Gi r olamo s t r c n t e Cent ro Tr a s f u s i on a l e e d i Irnrnun o l ogi a d e i Trapian t i an d Di v i s i one di Ema t .ol oqf a Ospe d e I e Ma ggi o r e Poli cli ni c o , v i a Fran ce s c o sr or ae 35, 20 1 22 Mi la n o - Ita l y 1 Cha i r raan a n d co-chai rman of AHS#ll We de sc ribe a nove l DRBS" 01 seque nc e f ou nd in an Ita li an s ubject ( 557 0), t ype d as, HLA-A 2 . 9 , 83 5 . X, DRSI' ll. 1 501, DQ.',! 'O l 02 . 0501 ; 0081 "0 602 , 030 1. Th i s n ew a ll e l e wa s r ecogni z ed by a n unusua l p a tte r n o f a:npl it i catl on ob ta in ed wi th PCR- SS P . Sample 5570 did amp lify ·.... i t h t h e p r I rre r mi x wh i c h i d e nti fi es HLA - OR8S· 0 101/ 0102 / 02 03 a ll el e s an d wi t h t hat wh ich i dent ifies a ll s eque nc ed DRB5 a ll eles exc e p t 020 1. but fa iled t o arap l Lf y wi t h p r ime r s spec i fi c f o r 0101. 0 102, 02011 020 2 / 02 03, 0 201 / 020 2 a nd 0201 res p e ct i vely . Wi t h o li g o typing , u s i ng pr ime rs a nd p r ob es of t h e 12t. h Hist oc orr:pat ibil i t y Workshop , the a e mp ke resul ted ORBS" 01 01. be i ng po a tr. tve wi t h p r ob e 280 5 wh i c h i dent i fies DR8 ' · 01 0t·. Th e e x on 2 nuc leot i de sequ ence o f ORBS new v a r i a n t wa s perf ormed bY di rect s equ e n c i ng 4 t imes i n b o th d ire c t i on s, wi t h p roducts o f 4 d if fere nt PeR r e a c ti on s. Th e sequence is id en t i c a l to t h a t o f' DRBS -O lO l exc ept for co don J B. where t h e triplet t e s eque nc e i s ~TG ins tead o f ,ITG . Thi s results i n an ami noa c i d substi t u tion f r om Leu to Va l . PCR- SSOP wa s n ot a ble to i d e n t ify this new v ariant s in c e th e probes u s ed f o r DR85 t y pi ng were l oca l i ze d in r e g i o n s d i f f ere n t from whe r e t h e nu cle ot i d e substituti on h ad occu rr ed .
This a lle l e ha s been offi c i a lly name d ORBS· Ol 0S .
Abstracts
p630
p631
CHARACTERIZATION OF AN ELUSNE IlLA-A*6803 ALLELE Ma ry Ell e xs on 1• Marie Lau 2 • Pa ul Terasaki - , and Willi am Hilde hra nd l 1 University of Okl ahom a Health Sciences Cente r. Okla homa City. OK. USA; 2University of California, Los Angeles. CA. USA. We have characte rized a novel A'6803 allele from an Hispanic individual which most resembles A'680 12. This new A'68 allele was cloned and sequenced from a cell run on the Intern ation al Ce ll Exchange (I CE) which wa s se ro logic ally typed by 169 laboratories and DNA typed (at the HLA-A locus) by 14 laboratories. A'6803 differs from A'68012 by only one nucle otid e at positi on 28 2 whi c h tran slat es to a co ding difference at positi on 70 of the a I domain situated atop the a helix of the liL A class 1 molecule . With only one amioo acid difference from A'680 l , A' 6803 was di fficult to type when run on the ICE; A*6803 went undetected when typed by serology whil e only tw o of the fo urte en labor ator ies usi ng high resolution DNA typi ng methods detected an A'68 variant. The undete cted A*680 1/A* 6803 polymorphism at amino acid 70 is pos iti oned to af fec t the stereochem istr y of bot h B and C speci fici ty pockets, so that unde tected polym orph ism s at re sidue 70 will likely provo ke a llorcac ti ve res pon ses. Considering th at new HLA class I pol ymorph isms continue to be det ected, DNA sequence analysis will remai n a valuable mean s for characte rizing novel class ] mol ecules.
DNA-SEQUENCING OF NESTED PeR PRODUCTS AS A HLA-B27 SUBTYPING METHOD BV ANCVWSING SPONDVLITIS R. Frank, S. Kastel~ C. Seid1., E. Seifried, Institulefor Transfusion Medicine and Imrnunhaematology, Red Cross Donor Service Hessen, Frankfurt, Germany
The ankyIosing spondylitis (AS) is highly associated with HLA-B27 and detectable in 96% of Caucasian AS patients. Therefore the detennination of HLA-B27 is used to confinn the diagnosis of the disease. In molecularbiological HLA typing HLA B27 represent a group of nine different alleles HLA B·2701·B 2709. We have established a HLA-B27 subtyping strategic
where groupspecific amplification is combined with automatic DNA sequencing. The amplificlitilm was carrying out by nested PeR with primer sequences whichare located in exon 2 and 3 of the gene. Only alleles of the HLA-B27 group were amplified in this approach. After amplification and seperation of single stranded DNA with magnetic beads the sequence analysis was conducted on an AD! 373 DNA sequencer using Sequenase and fluorescence coupledddNTP's. We used this method to subtype 64 HLA B27 positivsamples from 3 I AS patients and 33 heahhy people (control samples). Only two different subtypes HLA B"2702 and HLA B·2705 were observed. In case of 7 (2 1%) control and 5 (16%) patient samples the allele HLA-B·2702 was found. Altogether 26 (79"10) control and 26 (84%) patient samples were HLA B·2705 positive. There was no significant difference in the appearance of HLA-B27 alleles betweenAS patient and controlsamples.
p632
p633
A mRNA BASED METHOD FOR TYPING THE HLA-C LO CUS GENE BV AUTOMATED SEQUENC ING BASED TYPING
HIGH RESOLUTION DRS TYPING BY SSP·PCR AND AUTOMATED DNA SEQUENCING Malco1m D. McGinnis, CandiaL. Brown, David M IOVlIDIli.sci, MorganP. Conrad and Mel Kronick. Applied Biosysterns Divisiooof Pertin Elmer.FoslerCity, CA, USA The genetic complexity of theORBregionof the hwnanMIIC has required deve1q>menl of molec:ular typing tEchniques with increasingly higher levels of resolution. Melhods such as PCR-SSOP ... SSP-PeR are not alwaysable 10 discriminate amoog the ...,.., Ihan130recognized alle1
Day Sarinder, Ross Joe, UK Transplant Support Service Authority, Bristol, UK We describe here a sequencing based typing (SBT) method for HLA-e locus using mRNA as the starting material, where exon 2 and exon 3 are sequenced together in one reaction. Messenger RNA was extracted from B Iymphoblastoid cell lines using the ' mRNA direct kit' (Dynal). Solid phase eDNA synthesis was carried out using M-MuLV reverse transcriptase, C-locus sequences including exon 2 and exon 3 were amplified with C-lueus specific primers (Santamaria et aI, 1993, Human Immunology 37:39) tailed with -2 1 ~f1 3 universal sequencing primer. Sequencing reactions were carried out using the Dye Primer Cycle Sequencing Ready Reaction -21 M13 kit with AmpliTaq DNA Polymerase FS (ABO). Sequencing reaction products were analysed on the ABD 373 sequencer. Each sample was sequenced in both forward and reverse orientations to resolve any ambiguous nucleotide positions and to confirm the HLA type. lILA types were assigned using published C locus sequence data for exon 2 and exon 3. This approach only requires two reactions to sequence in both directions. Furthermore the use of cycle sequencing with AmpliTaq FS eliminates the need for a single strand preparation step whilst producing even signal peak height, for accurate heterozygote base calling.
p634 DRBI BIGB RESOLUTION SEQUENCING BASED TYPING INDIVIDUALS; COMPARISON WITH PCR -SSP
or
30 llNRELATED
Da i e y de Br u yn, Pa ul Savel koul and El la van d e n Be rg-Loo nen
Tiss ue Typ ing Laboratory , Ma as t r ic ht, The Nethe r lands
Unive r s i ty
Hos p ita l
Maastri cht,
Ac c urate al lele as signment for HLA- ORBI al l e l es i s rather c omplex . S in ce t he number o f d i ff ere nt al le l es r ecog ni zed i s e xpand i n g qu i ckly , mo r e al lele comb i n at i o n s wil l b e f ound that r e sult in ident ical sequences. I n high re solut ion typing met hods as SS T thi s wi ll lead t o ambiguou s typi ng r e s u l t s , for Pe R-SSP t h i s wil l l e ad t o the ne c e s s i t y o f unaccep tab l e numbe r s of sSP react ions.
e
PCR:~S; ~;~nds:;u~K ~e ;:;:~~t j8 u%~\~~e~eBi;~~vf~~:t~~A~l ~::~tfil~f h::Pia;:~e~~~t~-i~~s~ ~:rthle~r·~~r c~:i~:~ i~~:
used . With our cu r r e nt LR/ HR SSP t yp i ng t.a c h n Lqu e 79 different DRBl al le l e s can be di s t i ngUi s h e d . t or saT the sequen ce an alys is was c onducted b y solid phaae d irect DNA sequencing on a Pharmacia. Alfexpreas, tyPing wa s performed with t he SBTyper sof tw are. No dfeccepenckee between SST an d PCR-S SP were no t ice d . 2 1 out o f 3 0 s anl.plea c o u l d be direc t l y t yped by a n a l ys i u o f the heteroz ygo us sequenc es . Fo r 9 sample s al lele epec Lf Ic ampli fi c ation was ne ce~aary to ob t a i n an unamb iguou s
~~Pi~fieref~~~ · £~a~net~:mP~~S;~~l~es~tiel:ma~g~i~t~~~usw~~: amp lified by the same pr i mer group.
Allele .ss ignment of H~ genes by QQqu e nc e an a lys i s improves the reeolution of HLA typing results. SaT proved a powerful tool in high resolution HLA ORB1-typi ng . The determination of the ac tu a l nucleotide sequence facilita.tes the d iscovery of new a lle1e8. The expanding number of r e cogn ized DRBl alleles will eventually lead to considerable time g ain of SBT over ssp and other DNA typing techniques. J
P635
CJlARACTERIZA'1ION OF DRBS· Ol0S BY' PeR-SSP AND DIREC'1 S EQUENCI NG
Fr a nc e-sea pc j L, Pa ol a Bi a nc h i, Lo re tt a Crespi a t ico , Ell a van d e n ae rc - t.ocnen " . Olle o l.e r up - anc Gi r olamo s t r c n t e Cent ro Tr a s f u s i on a l e e d i Irnrnun o l ogi a d e i Trapian t i an d Di v i s i one di Ema t .ol oqf a Ospe d e I e Ma ggi o r e Poli cli ni c o , v i a Fran ce s c o sr or ae 35, 20 1 22 Mi la n o - Ita l y 1 Cha i r raan a n d co-chai rman of AHS#ll We de sc ribe a nove l DRBS" 01 seque nc e f ou nd in an Ita li an s ubject ( 557 0), t ype d as, HLA-A 2 . 9 , 83 5 . X, DRSI' ll. 1 501, DQ.',! 'O l 02 . 0501 ; 0081 "0 602 , 030 1. Th i s n ew a ll e l e wa s r ecogni z ed by a n unusua l p a tte r n o f a:npl it i catl on ob ta in ed wi th PCR- SS P . Sample 5570 did amp lify ·.... i t h t h e p r I rre r mi x wh i c h i d e nti fi es HLA - OR8S· 0 101/ 0102 / 02 03 a ll el e s an d wi t h t hat wh ich i dent ifies a ll s eque nc ed DRB5 a ll eles exc e p t 020 1. but fa iled t o arap l Lf y wi t h p r ime r s spec i fi c f o r 0101. 0 102, 02011 020 2 / 02 03, 0 201 / 020 2 a nd 0201 res p e ct i vely . Wi t h o li g o typing , u s i ng pr ime rs a nd p r ob es of t h e 12t. h Hist oc orr:pat ibil i t y Workshop , the a e mp ke resul ted ORBS" 01 01. be i ng po a tr. tve wi t h p r ob e 280 5 wh i c h i dent i fies DR8 ' · 01 0t·. Th e e x on 2 nuc leot i de sequ ence o f ORBS new v a r i a n t wa s perf ormed bY di rect s equ e n c i ng 4 t imes i n b o th d ire c t i on s, wi t h p roducts o f 4 d if fere nt PeR r e a c ti on s. Th e sequence is id en t i c a l to t h a t o f' DRBS -O lO l exc ept for co don J B. where t h e triplet t e s eque nc e i s ~TG ins tead o f ,ITG . Thi s results i n an ami noa c i d substi t u tion f r om Leu to Va l . PCR- SSOP wa s n ot a ble to i d e n t ify this new v ariant s in c e th e probes u s ed f o r DR85 t y pi ng were l oca l i ze d in r e g i o n s d i f f ere n t from whe r e t h e nu cle ot i d e substituti on h ad occu rr ed .
This a lle l e ha s been offi c i a lly name d ORBS· Ol 0S .