Brief Communication
Two novel HLA-DQBl"O6 alleles reveal additional heterogeneity of HLA-DQw1 T.S. Fenske, L. A. Baxter-Lowe. Two novel HLA-DQB1*06 alleles reveal additional heterogeneity of HLA-DQwl. Tissue Antigens 1992: 40: 49-52.
Timothy S. Fenske and Lea Ann Baxter-lowe The Blood Center of Southeastern Wisconsin and the Medical College of Wisconsin, Milwaukee, Wisconsin, U.S.A.
Key words: HIA-DQ - nucleotide sequence oligonucleotide typing
-
Received 13 February, revised, accepted for pub lication 24 kbNaV 1992
The HLA-DQB1 locus possesses a high degree of heterogeneity which has recently been characterized using nucleotide sequencing, revealing polymorphism which has been implicated in transplantation and autoimmune disease (1-6). To date, 16 HLA-DQB 1 alleles encoding unique polypeptides have been reported (1, 2). Although these are not all resolved using routine serological typing, nucleic acid-based methods such as oligotyping can be designed to resolve all known alleles. Here we report nucleotide sequences demonstrating the existence of two additional HLA-DQB 1 alleles which are similar but not identical to DQB1*0603. In addition, the previously reported partial sequence of DQB1*0504 was confirmed and extended. Genomic DNA was isolated from peripheral blood leukocytes and enzymatically amplified using the polymerase chain reaction (7) under the conditions described by Molkentin et al. (8). The amplified DNA was ligated into pCR 1000 (Invitrogen, San Diego, CA), E. coli were transformed, and kanamycin-resistant clones were screened for the presence of inserts of the expected size. Nucleotide sequences of the inserts were determined using the Sanger dideoxy chain termination method (9) with standard vector primers and an internal HLADQBl primer. For each allele, at least four independent clones derived from amplified genomic DNA were sequenced to rule out experimental artifacts. The presence of the polymorphic sequences in the original sample was confirmed by sequencespecific hybridization to DNA which was amplified from genomic DNA templates.
One allele, designated DQB 1*06BRII, was found to differ from DQB1*0603 by a single nucleotide substitution at codon 57. GTT (valine) replaced GAT (aspartic acid), which is present in DQBl*0603. GTT is present at codon 57 of DQB1*0501, *0604, and *0605 (1,2). Another allele, designated DQB 1*06BRI2', was identical to DQBl*O603 except for a single nucleotide substitution at codon 70. DQBl *O603 encodes glycine (GGG) at position 70 and DQB1*06BRI2 encodes arginine (AGG). Codon 70 is AGG in several HLA-DQB 1 alleles: DQB 1*0601, *0604, *0605, *0201, *0301, *0302, *0303 and *0304 (1, 2, 10) An allele identical to DQB1*0504 was also sequenced, including 50 nucleotides which weje not previously reported. The nucleotide sequences of DQB 1*06BRI1, *06BRI2, and *0504 are aligned with all other reported HLA-DQB1 alleles in Fig. 1. The corresponding deduced amino acid sequences of these alleles are displayed in Fig. 2. An oligotyping system reported by Molkentin et al. (8) was designed to discriminate any combination of known DQB1*05 and *06 alleles. However, the recent report of DQB1*05032 (1, 2, l l) required the use of an additional oligonucleotide probe in order to allow continued resolution of all possible combinations of known alleles (Table 1). DQB1*05032 is a silent variant of DQB1*05031 (previously called DQBI *0503). The probe (5' CTC
' The names DQBlL06BRI1 and DQB1*06BRI2 are provisional names. These two sequences have been submitted to GenBank and assigned the accession numbers M87041 and M87042, respectively. 49
Fenske and Baxter-Lowe 10 20 30 40 R D S P E D F ~ ~ ~ F K G * C ~ F T ~ C T E R ~ R L ~ ~ R Y I ~ ~ R E E ~ A AtAWCTCTCCC~GWTTTCGlGTACUG~TTMCGGI%TGT~A~TU~CG~~~GCGT~GT~TGl~CUWTAUTCTATMCC~~~GTAC~~KTTC~I%G~ffil~ C. .................~bb.............. A ........r A......C A C...............................................
R
............... ....... ........................................................ ............................................... ........................................................ ...... ....................................... CG.l ........................................c...c.c.................................. ................................. Gbb ..........c ........................ T ...................... ..................................... ............................... .w;G.. ........c.. .................... ..t.. .................... ................ .......... c........................T ...................... .............................................. A .......T...................... ...... C. ................ CT ...........C .................. T........ ..........TA ............................... C...T ...................... ......................... T ............................................................................................................. ................................................................................ ......C...... .................................... ................................................................................ ....................................... C ........................................ A.................. .................................... .............................................................. A.. ................. G....AG...............A...AT..T...................... ........................ C ..................................... ................................................................................................................. ..................... .............................................................. C.................................................. AA..................... ................................................................................................................. ..................... A. ..................................... C ..................................... TA....................................A................... ......................... .................................... c....T ......GGG .......................................................... ......................... .................................... ............*........c....c AGA~CICTCCCGhG~TllCGTGTACUGTlT~~TClGC~A~T~C~CGG~ffi~~GCGlGCGT~l~~CUWTAU~CTATMC~tAG~~~~~Tl~U~ GGG
A A......C...............................................
DPBlW201
TA....................................A...................
1.
A
1 1
CONSENSUS
5' ACTC7CC~GG4llTCGTG-~
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................................. ........................... ..... ..... ......... ........ C.......................... T....................................A....................................... ........ ......................... TT............................................................................ .......... .A., ..... .............TT.. ................................... ..G. .......1C.G ......G .......................... ......T.C... ........ ..... ................................ .AG... .................................... .G.. ......TC.G.. ......W.. ...................... .C.C.C. A. .A... .....T.C... ..... T., ................................................................... t... .....TC.G... .W.. ....................... .A ........T.C... ........ ..... ............................. T................................ .... A.W .......TC.C...... G. ................................. .......................... .I%U ......... ........ ..... T ...........C ............................................ A .......A ........................................C... ........T ............. ...... ..cc.......................... .......................... T7 ..................................................................................... ..................................................................................... c ...........T ............. ........ c........... 1............. ................................. .TT.. .................................. .............................................. 6. .A.. ......1 .C. .......... ................................. .TT.. ................................. .A.. ............................................. .G. .A.. ......T.C. .......... .A..T ..............T..T .....T .....C ....................... ....a.......A . . M .......C.G ......t ...................C..T .....C.....AC..........C..C.. ........................................... ...................... T.....C A. ........................................ .C. .C. . ....-1................T .....C.. ...C. .................................. ...................................... .C.. .T... ....C...C. .... ...AC.. ....... .... .T._.. ............T.. ...C.. ........................................ .A.. ...................................... AC... .......C.X.. .A.. ...................................... .C. .T.. ...C.. ..AC.. ........C. S.. .... .T.. ............. .T.. ...C. ........................................ ..... .................................... A ......................................... ...................... T.....C ............... C..T .....C .....AC......C.C. ..... 1................T ........T ..................T ...........I%.........A.W .......1C.G ....... .....T ................ i....~.........~......u.....~.tA....~c.~....c.~........c.r...c........c.~.
G T G T A C C ~ C G G t W C G C ~ G G ~ C T ~ G U ~ G T A ~ ~ U ~ U ~ ~ T ~ G t A G ~ ~ C ~ C ~ G l l ~ ~ C G G T G l G U ~ ~ T AG U.... A...TC. C. ....G C
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Figure 1. Alignment of DQB1*06BRIl, *06BRI2, and *0504 nucleotide sequences with published DQBl sequences (DQB1*0304, reference 10; other sequences summarized in reference 1). The consensus sequence reflects the most common nucleotide for a given
position. The bars show the location of amplification primers.
GGC ATC AGG C 3') has been named PDAE.57.2 and has successfully been used for accurate determination of HLA-DQB 1 oligotypes. However, the
CONSENSUS DQB1'0504 DQB1*06BRIl DQB1*06BRIZ DQB1*050 1 DQB1*0502 DQB1*05031 DQB1*05032 DQB 1* 0601 DQB1'0602 DQB1'0603 DQB1'0604 DQBl * 0605 DQBl*OZOl DQB1*0301 DQB1*0302 DQB1*03031 DQB1*03032 DQB1*0304 DQBl'O401 DQB1*0402 CONSENSUS
discovery of DQB 1*06BRI1 and DQBl*06BRI2 introduces new ambiguities into all oligotyping strategies published to date (8, 12). Specifically, an
10 20 30 40 50 60 70 80 90 RDSPEDF'VYQFKGMCYFTNGTERVRLVTRYSYNREEYARFDSDVGVYRA'ITPPGRPDAEYWNSQKEVLERTRAELDTVCRMYEVELRGTLQRR
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10
20
30
40
50
60
70
80
90
Figure 2. Alignment of deduced second exon amino acid sequences coded by a11 HLA-DQBI alleles, including DQB1*06BR11, DQBl'06BRI2, and DQB1*0504.
Brief communications Table 1. Oligotyping scheme"
OQB1'01 Allele
Oligonucleotide probes
VFQF.09
TRHI.30
+ +
0501 0502 05031 05032 0504 0601 0602 0603 0604
+
+
+
+
+ + + +
+
06BRll 06BR12
+ + + +
0501/0603 05032106BR12 0501/06BR11 05032/0604
+
0603/0604 06BR11/06BR12
+ +
+
+ +
+
+
+
0605
~~
TRYI.30 AELCl74 PDAE57.1 PDAE57.2C PSAE.57 PVAE.57 QK01.66 EGAR71 EGTR.71 LERT.71 ASVD74
+
r +
+ +
+
+
4.
+ + +
+ +
+ +
+ +
+
+
+
+ +
+
+ + + + + +
+
+ + +
+
+
+ + +
+ + + + + + +
+
+ +
+
+
+ +
+
+
+
+
+ + +
+
+ + + +
+ + +
~~~
DQB1-05" and 06- high-resolution detection, as described in Molkentin et al. (8). The probe named PDAE57 in Molkentin et al. (8) has been renamed PDAE57.1 to prevent confusion. Oligonucleotide probe POAE57.2 has been added to the panel described by Molkentin et al. (8). PDAE57.2 is necessary to distinguish OQB1'0501. *0.502, and '05031 homozygous individuals from 0501/05032, 0502/05032, and 05031105032 heterozygous individuals, respectively. This probe also resolves the following ambiguities: 050110604 from 05032/0604, 0501/0605 from 05032/0605. 0502/0504 from 0503210504, 05031I0601 from 05032/0601, and 0501/06BR12 from 05032/06BR12.
individual possessing DQB 1*0501 and DQB 1*0603 would display a hybridization pattern identical to that of a DQBl*05032/*06BRI2 heterozygote. This would also be the case for DQB 1*050 1/ *06BRI1 and DQB 1*05032/*0604 heterozygotes as well as DQB 1*0603/ *0604 and DQBl*06BRIl/*06BRI2. Table 1 depicts these situations within the context of the oligotyping scheme of Molkentin et al. (8). Allele-specific amplification can be used to resolve these ambiguous heterozygote combinations. However, as the sequence information of the HLADQBI locus increases, HLA typing methods will need to be modified if resolution of all potentially ambiguous oligotypes is desired. An oligotyping system which would distinguish all combinations of HLA-DQB1 alleles reported to date would require several amplifications, some demanding very selective conditions to specifically amplify alleles by means of one base pair mismatches. Future reports of new alleles will only add complexity to oligotyping schemes, making alternative techniques based on DNA sequencing increasingly appealing for HLA typing. Alternatively, understanding the relationship between HLA structure and function may demonstrate that HLA typing can be limited to a small number of polymorphic sequences, rather than resolving all known alleles.
Note added in proof
Official names for the sequences reported in this paper were assigned by the WHO Nomenclature Committee in February 1992. The sequence DQB 1*0607 was assigned DQB 1*06BRI1. The sequence DQB 1*0608 was assigned DQB 1*06BRI2. Acknowledgments
We would like to thank Dave Majewski for excellent technical assistance as well as thoughtful discussions. We are also grateful to the members of the Histocompatibility and Immunogenetics Laboratory and the DNA Diagnostics Laboratory of the Blood Center of Southeastern Wisconsin for their assistance in this study. This work was supported by the National Institutes of Health grant #R01 A128034 and The Blood Center of Southeastem Wisconsin. References 1. Marsh SGE, Bodmer JG. HLA class I1 nucleotide sequences, 1991. Human Immunology 1991: 31: 207-27. 2. Bodmer JG, Marsh SGE, Albert ED, et al. Nomenclature for factors of the HLA system, 1990. Tissue Antigens 1991: 37:97-104.
51
Fenske and Baxter-Lowe 3. Park MS, Terasaki PI, Barbetti A, Han H, Cecka JM. Significance of the HLA molecular structure to transplantation. Clinical Transplants 1988: 301-28. 4. Gregersen PK. HLA class I1 polymorphism: implications for genetic susceptibility to autoimmune disease. Lab Invest 1989: 61: 5-19. 5. Thorsby E, Lundin KEA, Ronningen KS, Sollid LM, Vartdal F. Molecular basis and functional importance of some disease-associated HLA polymorphism. Tissue Antigens 1990: 34: 39-49. 6. Erlich HA. HLA class I1 polymorphism and genetic susceptibility to insulin-dependent diabetes mellitus. Curr Top Microbiol Immunol 1990 164: 41-55. 7. Saiki RK, Gelfand DH, Stoffel S , et al. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988: 239 487-91. 8. Molkentin J, Gorski J, Baxter-Lowe LA. Dktection of 14 HLA-DQB 1 alleles by oligotyping. Human Immunology 1991: 31: 114-22. 9. Sanger F, Nicklen S, Coulson A. DNA sequencing with
52
chain terminating inhibitors. Proc Natl Acad Sci USA 1977: 74: 5463-7. 10. Fenske TS, Baxter-Lowe LA. Characterization of a novel DQB I allele associated with HLA-DQw3: Implications for oligotyping. Human Immunology (in press). 11. Scharf SJ, Freidmann A, Steinman L, Brautbar C, Erlich HA. Specific HLA-DQB and HLA-DRBI alleles confer susceptibility to pemphigus vulgaris. Proc Natl Acad Sci USA 1989: 86: 6215-9. 12. Bugawan TL, Erlich HA. Rapid typing of HLA-DQBI DNA polymorphism using non-radioactive oligonucleotide probes and amplified DNA. Immunogenetics 1991: 33: 163-70. Address: Dr. Lee Ann Baxter-Lowe The Blood Center of Southeastern Wisconsin 1701 W. Wisconsin Ave., Milwaukee WI 53233, U.S.A. Phone (414) 937-6143
Two novel HLA-DQBl"O6 alleles reveal additional heterogeneity of HLA-DQw1 T.S. Fenske, L. A. Baxter-Lowe. Two novel HLA-DQB1*06 alleles reveal additional heterogeneity of HLA-DQwl. Tissue Antigens 1992: 40: 49-52.
Timothy S. Fenske and Lea Ann Baxter-lowe The Blood Center of Southeastern Wisconsin and the Medical College of Wisconsin, Milwaukee, Wisconsin, U.S.A.
Key words: HIA-DQ - nucleotide sequence oligonucleotide typing
-
Received 13 February, revised, accepted for pub lication 24 kbNaV 1992
The HLA-DQB1 locus possesses a high degree of heterogeneity which has recently been characterized using nucleotide sequencing, revealing polymorphism which has been implicated in transplantation and autoimmune disease (1-6). To date, 16 HLA-DQB 1 alleles encoding unique polypeptides have been reported (1, 2). Although these are not all resolved using routine serological typing, nucleic acid-based methods such as oligotyping can be designed to resolve all known alleles. Here we report nucleotide sequences demonstrating the existence of two additional HLA-DQB 1 alleles which are similar but not identical to DQB1*0603. In addition, the previously reported partial sequence of DQB1*0504 was confirmed and extended. Genomic DNA was isolated from peripheral blood leukocytes and enzymatically amplified using the polymerase chain reaction (7) under the conditions described by Molkentin et al. (8). The amplified DNA was ligated into pCR 1000 (Invitrogen, San Diego, CA), E. coli were transformed, and kanamycin-resistant clones were screened for the presence of inserts of the expected size. Nucleotide sequences of the inserts were determined using the Sanger dideoxy chain termination method (9) with standard vector primers and an internal HLADQBl primer. For each allele, at least four independent clones derived from amplified genomic DNA were sequenced to rule out experimental artifacts. The presence of the polymorphic sequences in the original sample was confirmed by sequencespecific hybridization to DNA which was amplified from genomic DNA templates.
One allele, designated DQB 1*06BRII, was found to differ from DQB1*0603 by a single nucleotide substitution at codon 57. GTT (valine) replaced GAT (aspartic acid), which is present in DQBl*0603. GTT is present at codon 57 of DQB1*0501, *0604, and *0605 (1,2). Another allele, designated DQB 1*06BRI2', was identical to DQBl*O603 except for a single nucleotide substitution at codon 70. DQBl *O603 encodes glycine (GGG) at position 70 and DQB1*06BRI2 encodes arginine (AGG). Codon 70 is AGG in several HLA-DQB 1 alleles: DQB 1*0601, *0604, *0605, *0201, *0301, *0302, *0303 and *0304 (1, 2, 10) An allele identical to DQB1*0504 was also sequenced, including 50 nucleotides which weje not previously reported. The nucleotide sequences of DQB 1*06BRI1, *06BRI2, and *0504 are aligned with all other reported HLA-DQB1 alleles in Fig. 1. The corresponding deduced amino acid sequences of these alleles are displayed in Fig. 2. An oligotyping system reported by Molkentin et al. (8) was designed to discriminate any combination of known DQB1*05 and *06 alleles. However, the recent report of DQB1*05032 (1, 2, l l) required the use of an additional oligonucleotide probe in order to allow continued resolution of all possible combinations of known alleles (Table 1). DQB1*05032 is a silent variant of DQB1*05031 (previously called DQBI *0503). The probe (5' CTC
' The names DQBlL06BRI1 and DQB1*06BRI2 are provisional names. These two sequences have been submitted to GenBank and assigned the accession numbers M87041 and M87042, respectively. 49
Fenske and Baxter-Lowe 10 20 30 40 R D S P E D F ~ ~ ~ F K G * C ~ F T ~ C T E R ~ R L ~ ~ R Y I ~ ~ R E E ~ A AtAWCTCTCCC~GWTTTCGlGTACUG~TTMCGGI%TGT~A~TU~CG~~~GCGT~GT~TGl~CUWTAUTCTATMCC~~~GTAC~~KTTC~I%G~ffil~ C. .................~bb.............. A ........r A......C A C...............................................
R
............... ....... ........................................................ ............................................... ........................................................ ...... ....................................... CG.l ........................................c...c.c.................................. ................................. Gbb ..........c ........................ T ...................... ..................................... ............................... .w;G.. ........c.. .................... ..t.. .................... ................ .......... c........................T ...................... .............................................. A .......T...................... ...... C. ................ CT ...........C .................. T........ ..........TA ............................... C...T ...................... ......................... T ............................................................................................................. ................................................................................ ......C...... .................................... ................................................................................ ....................................... C ........................................ A.................. .................................... .............................................................. A.. ................. G....AG...............A...AT..T...................... ........................ C ..................................... ................................................................................................................. ..................... .............................................................. C.................................................. AA..................... ................................................................................................................. ..................... A. ..................................... C ..................................... TA....................................A................... ......................... .................................... c....T ......GGG .......................................................... ......................... .................................... ............*........c....c AGA~CICTCCCGhG~TllCGTGTACUGTlT~~TClGC~A~T~C~CGG~ffi~~GCGlGCGT~l~~CUWTAU~CTATMC~tAG~~~~~Tl~U~ GGG
A A......C...............................................
DPBlW201
TA....................................A...................
1.
A
1 1
CONSENSUS
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Y
R
m
a
50
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A
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90
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................................. ........................... ..... ..... ......... ........ C.......................... T....................................A....................................... ........ ......................... TT............................................................................ .......... .A., ..... .............TT.. ................................... ..G. .......1C.G ......G .......................... ......T.C... ........ ..... ................................ .AG... .................................... .G.. ......TC.G.. ......W.. ...................... .C.C.C. A. .A... .....T.C... ..... T., ................................................................... t... .....TC.G... .W.. ....................... .A ........T.C... ........ ..... ............................. T................................ .... A.W .......TC.C...... G. ................................. .......................... .I%U ......... ........ ..... T ...........C ............................................ A .......A ........................................C... ........T ............. ...... ..cc.......................... .......................... T7 ..................................................................................... ..................................................................................... c ...........T ............. ........ c........... 1............. ................................. .TT.. .................................. .............................................. 6. .A.. ......1 .C. .......... ................................. .TT.. ................................. .A.. ............................................. .G. .A.. ......T.C. .......... .A..T ..............T..T .....T .....C ....................... ....a.......A . . M .......C.G ......t ...................C..T .....C.....AC..........C..C.. ........................................... ...................... T.....C A. ........................................ .C. .C. . ....-1................T .....C.. ...C. .................................. ...................................... .C.. .T... ....C...C. .... ...AC.. ....... .... .T._.. ............T.. ...C.. ........................................ .A.. ...................................... AC... .......C.X.. .A.. ...................................... .C. .T.. ...C.. ..AC.. ........C. S.. .... .T.. ............. .T.. ...C. ........................................ ..... .................................... A ......................................... ...................... T.....C ............... C..T .....C .....AC......C.C. ..... 1................T ........T ..................T ...........I%.........A.W .......1C.G ....... .....T ................ i....~.........~......u.....~.tA....~c.~....c.~........c.r...c........c.~.
G T G T A C C ~ C G G t W C G C ~ G G ~ C T ~ G U ~ G T A ~ ~ U ~ U ~ ~ T ~ G t A G ~ ~ C ~ C ~ G l l ~ ~ C G G T G l G U ~ ~ T AG U.... A...TC. C. ....G C
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Figure 1. Alignment of DQB1*06BRIl, *06BRI2, and *0504 nucleotide sequences with published DQBl sequences (DQB1*0304, reference 10; other sequences summarized in reference 1). The consensus sequence reflects the most common nucleotide for a given
position. The bars show the location of amplification primers.
GGC ATC AGG C 3') has been named PDAE.57.2 and has successfully been used for accurate determination of HLA-DQB 1 oligotypes. However, the
CONSENSUS DQB1'0504 DQB1*06BRIl DQB1*06BRIZ DQB1*050 1 DQB1*0502 DQB1*05031 DQB1*05032 DQB 1* 0601 DQB1'0602 DQB1'0603 DQB1'0604 DQBl * 0605 DQBl*OZOl DQB1*0301 DQB1*0302 DQB1*03031 DQB1*03032 DQB1*0304 DQBl'O401 DQB1*0402 CONSENSUS
discovery of DQB 1*06BRI1 and DQBl*06BRI2 introduces new ambiguities into all oligotyping strategies published to date (8, 12). Specifically, an
10 20 30 40 50 60 70 80 90 RDSPEDF'VYQFKGMCYFTNGTERVRLVTRYSYNREEYARFDSDVGVYRA'ITPPGRPDAEYWNSQKEVLERTRAELDTVCRMYEVELRGTLQRR
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10
20
30
40
50
60
70
80
90
Figure 2. Alignment of deduced second exon amino acid sequences coded by a11 HLA-DQBI alleles, including DQB1*06BR11, DQBl'06BRI2, and DQB1*0504.
Brief communications Table 1. Oligotyping scheme"
OQB1'01 Allele
Oligonucleotide probes
VFQF.09
TRHI.30
+ +
0501 0502 05031 05032 0504 0601 0602 0603 0604
+
+
+
+
+ + + +
+
06BRll 06BR12
+ + + +
0501/0603 05032106BR12 0501/06BR11 05032/0604
+
0603/0604 06BR11/06BR12
+ +
+
+ +
+
+
+
0605
~~
TRYI.30 AELCl74 PDAE57.1 PDAE57.2C PSAE.57 PVAE.57 QK01.66 EGAR71 EGTR.71 LERT.71 ASVD74
+
r +
+ +
+
+
4.
+ + +
+ +
+ +
+ +
+
+
+
+ +
+
+ + + + + +
+
+ + +
+
+
+ + +
+ + + + + + +
+
+ +
+
+
+ +
+
+
+
+
+ + +
+
+ + + +
+ + +
~~~
DQB1-05" and 06- high-resolution detection, as described in Molkentin et al. (8). The probe named PDAE57 in Molkentin et al. (8) has been renamed PDAE57.1 to prevent confusion. Oligonucleotide probe POAE57.2 has been added to the panel described by Molkentin et al. (8). PDAE57.2 is necessary to distinguish OQB1'0501. *0.502, and '05031 homozygous individuals from 0501/05032, 0502/05032, and 05031105032 heterozygous individuals, respectively. This probe also resolves the following ambiguities: 050110604 from 05032/0604, 0501/0605 from 05032/0605. 0502/0504 from 0503210504, 05031I0601 from 05032/0601, and 0501/06BR12 from 05032/06BR12.
individual possessing DQB 1*0501 and DQB 1*0603 would display a hybridization pattern identical to that of a DQBl*05032/*06BRI2 heterozygote. This would also be the case for DQB 1*050 1/ *06BRI1 and DQB 1*05032/*0604 heterozygotes as well as DQB 1*0603/ *0604 and DQBl*06BRIl/*06BRI2. Table 1 depicts these situations within the context of the oligotyping scheme of Molkentin et al. (8). Allele-specific amplification can be used to resolve these ambiguous heterozygote combinations. However, as the sequence information of the HLADQBI locus increases, HLA typing methods will need to be modified if resolution of all potentially ambiguous oligotypes is desired. An oligotyping system which would distinguish all combinations of HLA-DQB1 alleles reported to date would require several amplifications, some demanding very selective conditions to specifically amplify alleles by means of one base pair mismatches. Future reports of new alleles will only add complexity to oligotyping schemes, making alternative techniques based on DNA sequencing increasingly appealing for HLA typing. Alternatively, understanding the relationship between HLA structure and function may demonstrate that HLA typing can be limited to a small number of polymorphic sequences, rather than resolving all known alleles.
Note added in proof
Official names for the sequences reported in this paper were assigned by the WHO Nomenclature Committee in February 1992. The sequence DQB 1*0607 was assigned DQB 1*06BRI1. The sequence DQB 1*0608 was assigned DQB 1*06BRI2. Acknowledgments
We would like to thank Dave Majewski for excellent technical assistance as well as thoughtful discussions. We are also grateful to the members of the Histocompatibility and Immunogenetics Laboratory and the DNA Diagnostics Laboratory of the Blood Center of Southeastern Wisconsin for their assistance in this study. This work was supported by the National Institutes of Health grant #R01 A128034 and The Blood Center of Southeastem Wisconsin. References 1. Marsh SGE, Bodmer JG. HLA class I1 nucleotide sequences, 1991. Human Immunology 1991: 31: 207-27. 2. Bodmer JG, Marsh SGE, Albert ED, et al. Nomenclature for factors of the HLA system, 1990. Tissue Antigens 1991: 37:97-104.
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Fenske and Baxter-Lowe 3. Park MS, Terasaki PI, Barbetti A, Han H, Cecka JM. Significance of the HLA molecular structure to transplantation. Clinical Transplants 1988: 301-28. 4. Gregersen PK. HLA class I1 polymorphism: implications for genetic susceptibility to autoimmune disease. Lab Invest 1989: 61: 5-19. 5. Thorsby E, Lundin KEA, Ronningen KS, Sollid LM, Vartdal F. Molecular basis and functional importance of some disease-associated HLA polymorphism. Tissue Antigens 1990: 34: 39-49. 6. Erlich HA. HLA class I1 polymorphism and genetic susceptibility to insulin-dependent diabetes mellitus. Curr Top Microbiol Immunol 1990 164: 41-55. 7. Saiki RK, Gelfand DH, Stoffel S , et al. Primerdirected enzymatic amplification of DNA with a thermostable DNA polymerase. Science 1988: 239 487-91. 8. Molkentin J, Gorski J, Baxter-Lowe LA. Dktection of 14 HLA-DQB 1 alleles by oligotyping. Human Immunology 1991: 31: 114-22. 9. Sanger F, Nicklen S, Coulson A. DNA sequencing with
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chain terminating inhibitors. Proc Natl Acad Sci USA 1977: 74: 5463-7. 10. Fenske TS, Baxter-Lowe LA. Characterization of a novel DQB I allele associated with HLA-DQw3: Implications for oligotyping. Human Immunology (in press). 11. Scharf SJ, Freidmann A, Steinman L, Brautbar C, Erlich HA. Specific HLA-DQB and HLA-DRBI alleles confer susceptibility to pemphigus vulgaris. Proc Natl Acad Sci USA 1989: 86: 6215-9. 12. Bugawan TL, Erlich HA. Rapid typing of HLA-DQBI DNA polymorphism using non-radioactive oligonucleotide probes and amplified DNA. Immunogenetics 1991: 33: 163-70. Address: Dr. Lee Ann Baxter-Lowe The Blood Center of Southeastern Wisconsin 1701 W. Wisconsin Ave., Milwaukee WI 53233, U.S.A. Phone (414) 937-6143
