Clinical Genetics 1979: 15: 245-251
Regional mapping of the HLA on the short arm of chromosome 6 R. BERGER~, A. BERNHEM, M.
G. HAUPTMANN3, J. M. FELMUSS
,%SPORTe@,
HORS4,
L. LEG-
AND
1 Laboratoire
de CytogBnCtique, Institut de Recherches sur les Maladies du Sang, HGpital Saint-Louis, Paris; 2 Unite de Recherche d’ImmunogtnCtique et de Transplantation humaine INSERM U93, HGpital Saint-Louis, Paris; 3 Centre de Transfusion de Strasbourg; 4 France-Transplant, HGpital Saint-Louis, Paris; 5 Laboratoire d’Immuno-Hematolopie, HGpital Saint-Louis, Paris, France A detailed gene marker study was performed on a partial 6p trisomic child resulting from a balanced maternal translocation t (2;6) (p 2505; p 2105). HLA typing and mixed lymphocyte reaction showed that the breakpoint on chromosome 6 was located within the HLA gene cluster, allowing an accurate location of the D determinants. Localization of the P blood group locus within the region 6 p 2105 to 6 p ter was excluded. Received 28 July, revised 3 October, accepted for publication 10 October 1978 Key words: Chromosome 6; complement; HLA; PI blood group; translocation mapping.
The location of the HLA gene cluster was first assigned to chromosome No. 6 by Jongsma et al. (1973) using human-Chinese hamster hybrid cells. The assignment of the major histocompatibility complex (MHC) gene cluster to the short arm of chromosome 6 was ascertained by Francke & Pellegrino (1977) and by Breuning et al. (1977). A biological study of a No. 6 short arm trisomic patient was performed in a child from whom clinical data have been reported in another paper (Bernheim et al. in preparation).
H L A typing. HLA determinants were typed by cytotoxicity using mono- or bispecific sera (Mittal et al. 1968): HLA-A, B, C on peripheral lymphocytes, cell lines and skin fibroblasts, and HLA-DR on B lymphocytes enriched by rosetting (70% pure). Additionally, complement fixation was used to detect HLA-A and B specificities on platelets (Colombani et al. 1971) and for DR determinant analysis of cell line and PHA-stimulated lymphocytes (Colombani et al. 1977).
Material and Methods
Mixed lymphocyte reaction ( M L R ) . Primary MLR were performed following the technique of Fradelizi et al. (1978).
Cytogenetics. Chromosome studies were performed on lymphocyte cultures using the GT, RH, RFA and CBG banding techniques.
Enzymes and proteins. Phosphoglucomutase 3 (PGM3) was studied using the technique of Spencer et al. (1964), red cell glyoxalase
0009-9163/79/030245-07 $02.50/0 0 1979 Munksgaard, Copenhagen
246
BERGER ET AL.
I (GLO I) using the technique of Kompf et al. (1975) and properdin factor Bf using the technique of Alper et al. (1972). Complement studies. Immunochemical titrations of C3, C4 and C5 were performed using commercial immunoplates (Hyland Laboratories, Costa Mesa, California, USA, for C3 and C4; Meloy Laboratories, Springfield, Virginia, for C5). C l q and factor Bf were estimated by radial immunodiffusion in 1% agarose, barbital 0.05 M and EDTANaz 0.01 M, p H 8.6 buffer using commer-
cial antisera and reference standards (Atlantic Antibodies, Westbrook, Maine). A C2-deficient serum (Hauptmann et al. 1977) was used for hemolytic titration of C2, according to Ngan et al. (1977). Except for C3 and C4, the titers of complement components were expressed as percentage activity or concentration of a pool of 40 normal sera stored in small aliquots at -70°C. Polymorphism of C2 was studied by analytical isoelectric focusing and hemolytic detection with C2-deficient serum, as described by Meo et al. (1977).
A
Flg. 1. Partial karyotypes. A (G-bands) and B (R-bands): maternal balanced translocation t(2;6). C (G-bands): partial 6p trisomy of 111-1.
REGIONAL M A P P I N G OF HLA
247
FAMILY 0..
I
II
m Flg. 2. Pedigree of the D family.
Cell cultures. Fibroblast cell cultures were established from skin biopsies of the proposita and from her mother. Lymphoblastoid cell lines were established from peripheral blood lymphocytes of the proposita and her mother. Blood groups. The following blood group typings were performed: ABO, MNS, PI, K, K,, CDE, Fy, Jk and Lu.
Results Cytogenetics. The proband, S.D., had a karyotype 46,XX,2p and the mother had a balanced reciprocal translocation t(2;6)(p25;p21). The father and maternal grandparents had normal karyotypes. The translocation was thus considered to be de novo in the child’s mother. A careful analysis of the chromosomes
+,
prepared using the G and R banding techniques was made to ascertain the breakpoints. Due to the fact that the mother was a balanced reciprocal translocation carrier, no chromosomal material was expected to have been lost. The extreme distal part of 2p (telomeric region) was thus translocated onto the deleted 6p. But the banding patterns of these regions of the chromosomes made it impossible to decide whether the exact breakpoint was at 2p 2500 or 2p 2509, or between these two subregions. Nevertheless, the breakpoints were most probably at 2p 2505 and 6p 2105. The child was thus trisomic for bands 6p 2105 to 6 pter and monosomic for a part of or almost the totality of band 2p 25 (Fig. 1). HLA typing. The proposita was found to have three haplotypes for the HLA, A and B loci: A3 B12 inherited from her father
248
BERGER ET AL.
Table 1 C h r o m o s o m e 6 g e n e m a r k e r s in t h e D family Subject
Relationship
Haplotype designation
HLA-A
I-1
Grandfather
a b
A11 A2
d
1-2
11-1 11-2
Grandmother Father Mother
HLA-C
HLA-B
Bf
HLA-DR
Cw4
Bw35 Bw21
S
-
DRwG DRw4
A3 643)
Cw4 Cve
Bw35 El8
DRw7 DRwG
e f
A3 A1
-
-
812 837
-
a d
A11 A3
Cve
A2 A3
-
-
C
A2 A3
e a d
A3 A11 A3
-
C
I13
Uncle
b
II 4
Aunt
b
C
I11-1
Patient
Cw4
Cw4 Cw4
GLO
PGM3
DRwl
2-1
1
Bw35 B18
DRwG
1
1
Bw21 Bw35
DRw4 DRw7
Bw21 Bw35
DRw4 DRw7 1
1
812 Bw35 El8
Cw4 Cve
S S S
DRwl DRwG
-
and A3 Cve B18 and A l l Cw4 Bw35 inherited from her mother. Only two determinants, however, have been identified for DR: DRwl transmitted from her father and DRw6 transmitted from her mother. The second maternally transmitted allele could, as shown by the family study, be DRw6 (Fig. 2, Tables 1 and 2).
not only gave, as expected, a positive MLR with the father’s cells and cells from unrelated controls, but also with the mother’s cells (Table 3), even though the child shared two HLA haplotypes (a, d) with the mother. Enzymes and proteins. PGM3 and GLO phenotypes were not informative (Table 1).
MLR. A primary MLR was performed within the family. The child as responder
Complement. The complement-component levels of C2, C4 and Bf were elevated in
Table 2 HLA markers in t h e patient
Cytotoxicity: Lymphocytes Lyrnphoblastoid cell line Skin fibroblasts Complement fixation: Platelets Lymphoblastoid cell line PHA-stimulated lymphocytes
A3
A11
Bw35
+ + +
+ + +
+
+
+
+ +
+
NT
NT
B18
Cw4
DRw3
+ +
+
+ +
+ +
-
+
+
NT NT
NT NT
NT NT
NT N
NT T NT
+
t f
+ NT
812
+
NT +
+ NT
N NT
+
NT T NT
-
-
DRw4
DRwG
NT +
+
HLA markers have been tested by two techniques (cytotoxicity and complement fixation) on 6 different NT=not tested. sources of antigen.
249
REGIONAL MAPPING OF HLA Table 3
Primary MLR in t h e D family. Responders Father: F Mother: M Child: C Control: T1 T2
y
F 200’ 55,008 18,453 49,129 56,116
irradiated stimulators M C T1 22,764 4.348 2,687 23,175 11,672 356 39,600 13,195 53.779 27,963
~~
14,449 34,773 23,721 2,232 73,562
T2 26.548 46,446 18,228 56,927 1,165
~
* Median value of triplicate cpm.
the proposita, contrasting with the normal levels of the C3, Clq and C5 components. The polymorphism study of C2 and Bf was not informative (Tables 3 and 4). Blood group typing. The child was P2, the mother P1 and the father P2. It can be concluded that the mother was a PUP2 heterozygote and transmitted only the P2 allele to her daughter. Discussion
The proposita was partially trisomic for the short arm of chromosome No. 6. She had three haplotypes for HLA-A and B and received two HLA-A, B and C haplotypes from her mother. These loci are thus located in this chromosomal region. Within the D region, only two HLA-DR specificities were serologically detected: DRwl
inherited paternally and DRw6 maternally. The MLR gave unexpected results. The most likely explanation is that the mother possessed an antigen which was not present in the child. From serological family data, the mother could be homozygous for DRw6, but the standard serological techniques do not allow the assessment of homozygosity for the DRw6 specificity. One possibility is that the breakpoint on the chromosome 6 interacts with the D region to produce a modified HLA-DR specificity undetectable by serological techniques. Due to the meiotic segregation, the proposita did not receive the 6p- chromosome on which the hypothetical modified determinant could be present. Experiments are now in progress to investigate other possibilities. It can be concluded that the breakpoint on the rearranged chromosome 6 was within the HLA gene cluster very near or inside the D locus, or at least between HLA-B and HLA-D, since the sequence of genes from centromere to telomere is D, B, C, A. These results are thus a direct proof of the location of HLA genes on chromosome 6p. The exact localization of the complex is the 6p 2105 region. This is in good agreement with previous data : localization “a little distal to the midpoint of either the short arm or the long arm of chromosome No. 6” was claimed from the study of a familial pericentric inversion of
Table 4 C o m p l e m e n t c o m p o n e n t s in t h e D family
111-2 11-2 11-1 Normal range
t 5
Clq R.I.’ %§
c4
c2
R.I.’ mgidl
H.T.7
91 86 86 67-135
53 24 28.5 15-45
c5
%§
c3 R.I.’ mgldl
R.I.’ mgldl
Bf R.I.’ mgldl
270 74 64 6 5 1 35
122 139 139 1ocL190
20 11.5 12.5 &25
81 35.5 37.5 21-50
R.I.: radial immunodiffusion. H.T.: hemolytic titration. %: percent of mean normal level (pool of 40 normal sera stored at -7OOC)
250
BERGER ET AL.
this chromosome (Lamm et al. 1974) : localization to 6p 22 from the study of a familial t(6;21) translocation (Borgaonkar & Bias 1974); localization to the 6p 21 band close to the transition to 6p 22 calculated from a t(6;20)(p21;p13) translocation in a large Dutch family (Breuning et al. 1977); localization proximal to 6p 22 estimated from interspecific hydrid human fibroblasts, having a balanced reciprocal translocation t(1;6) (p 3200 p 2100) x Chinese hamster cell studies (Francke & Pellegrino 1977). The map of chromosome No. 6 has been intensively studied by various methods, and some genes have been localized to the short arm of this chromosome (Bodmer 1976). Studies of phosphoglucomutase 3, PGM3 (Jongsma et al. 1973), as would be expected, were not informative in our family since the locus of the gene responsible has been estimated to be at a distance of 17 cM from the centromere (Ott et al. 1976) and the gene for PGM3 assigned to band 6p 12 (Francke & Pellegrino 1977). The red cell glyoxalase I (GLO I) study was also uninformative. The study of complementcomponent C2, C4 and Bf showed that the three loci are located in the region 6p 21 to 6p ter. The Bf locus was localized between HLAB and HLA-D with very close linkage to HLA-B. The genes for complement factors C2 and C4 have been claimed to be linked to the HLA gene cluster (review in Svejgaard et al. 1976), the C4 gene being on the side of the HLA-B (Teisberg et al. 1976, Ochs et al. 1977). The P1 blood group was tentatively assigned to chromosome No. 6 (Fellow et al. 1973, Lamm et al. 1975, Bijnen et al. 1976). Our results allow exclusion of the P1 blood group locus from the region 6p 2105 to 6p ter. Other considerations can be stressed from the study of the above reported family. First. no Dosition effects for either the
HLA-A, B and C determinants or for the complement-components due to the translocated HLA genes were found in the mothers of the proposita. This is the reason why the lack of one maternal D locus in the proposita can be considered as significant. Second, the distance in map units from the centromere of chromosome No. 6 to the HLA gene cluster was estimated to be about 32 cM. This distance is approximately equivalent to that of the region 6p 1 following the Paris Conference nomenclature. A more detailed correspondence between factorial and chromosomal maps cannot be established accurately at the present time, since insufficient data on the non-uniformity of crossing over are available in man. Acknowledgments
We gratefully acknowledge the help of Prof. J. Colombani in HLA typing, Dr. Nguyen Van Cong for PGM3-typing and Miss M. L. North for GLO-typing. References
Alper, C. A,, T. Boenisch & L. Watson (1972). Genetic polymorphism in human glycin-rich0-glycoprotein. J . exp. Med. 135, 68-80. Bernheim, A., R. Berger, G. Vaugier, J. C. Thieffry & Y . Matet (1978). Trisomy 6p. In preparation. Bijnen, A. B., I. Schreuder, P. Meera Khan, F. H. Allen, G. M. Giles, W. R. 1. Los, W. S. Volkers & J. J. Van Rood (1976). Linkage relationships of the loci of the major histocompatibility complex in a family with a recombinant in the HLA region. J . Immunogenet. 3, 171-183. Bodmer, W. F. (1976). Report of the Committee on the genetic constitution of chromosome 6. Baltimore Conference (1975). 3rd International Workshop on Human Gene Mapping. Birth Defects: Original Article Series, Vol. X I , No. 3, 24-30. Borgaonkar, D. S. & W. B. Bias (1974). HL-A loci and chromosome 6. New Haven Conference (1973). 1st International Conference on Human Gene Mapping. _ _ - Birth Defects: Original Artcle Series, Vol. X , No. 3, 67-68.
R E G I O N A L MAPPING OF H L A
Breuning, M. H., E. M. Van den Berg-Loonen, L. F. Bernini, J . B. Bijlsma, E. Van Loghem, P. M. Khan & L. E. Nijenhuis (1977). Localization of HLA on the short arm of chromosome 6. Hum. Genet. 37, 131-139. Colombani, J., J . d’Amaro, B. Gabb, G. Smith & A. Svejgaard (1971). International agreem-nt on a microtechnique of platelet complement fixation PI. c Fixi. Transpl. Proc. 3, 121-126. Colombani, J., M. Colombani, H. Pastot, M. Reboul & L. Degos (1977). Detection of human B alloantigens by complement fixation. Transplantation 24, 230-234. Fellous, M., P. Couillin, C. Neauport-Sautts, J. FrBzal, C. Billardon & J. Dausset (1973). Studies of human alloantigens on man-mouse hybrids: possible syntheny between HL-A and P systems. Europ. J . Immunol. 3, 543-548. Fradelizi, D., A. Nunez-Roldan & M. Sasportes (1978). Human Ia-like DRw lymphocyte antigens stimulating activity in primary mixed lymphocyte reaction. Europ. J . Imrnunol. 8, 88-93. Francke, U. & M. A. Pellegrino (1977). Assignment of the major histocompatibility complex t o a region of the short arm of human chromosome 6. Proc. nut. Acad. Sci. (Wash.) 74, 1147-1150. Hauptmann, G., M. M. Tongio, H. GrosseWilde & S. Mayer (1977). Linkage between C2 deficiency and the HLA-AIO, BlS, Dw2/ BfS haplotype in a French family. Immunogenetics 4, 736-741. Jongsma, A., H. Van Someren, A. Westerveld, A Hagemeijer & P. Pearsori (1973). Localization of genes on human chromosomes by studies of human-Chinese hamster somatic cell hybrids. Assignment of PGM3 to chromosome C6 and regional mapping of the PGD, PGMl and Pep-C genes on chromosome Al. Hum. Genet. 20, 195-202. Kompf, J., S. Bissbort, S. Gussmann & H. Ritter (1975). Polymorphism of red cell glyoxalase 1 (E, C : 4. 4. 1. 5 ) A new genetic marker in man. Hum. Genet. 27, 141-143. Lamm, L. U., U. Friedrich, C. B. Petersen, J. Jorgensen, J. Nielsen, A. J. Therkelsen & F. Kissmeyer-Nielsen (1974). Assignment of the major histocompatibility complex to chromosome No. 6 in a family with a pericentric inversion. Hum. Hered. 24, 273-284. Lamm, L. U., I. Thorsen, G. Bruun-Petersen, J. Jorgensen, K. Henningsen, B. Bech & F.
25 1
Kissmeyer-Nielsen (1975). Data on the HLA linkage group. Ann. hum. Genet. 38, 383389. Meo, T., J. P. Atkinson, M. Bernoco & R. Ceppellini (1977). Structural heterogeneity of C2 complement protein and its genetic variants in man: a new polymorphism of the HLA region. Proc. nat. Acad. Sci. (Wash.) 74, 1672-1675. Mittal, K. K., M. R. Mickey, D. P. Singal & P. I. Terasaki (1968). Serotyping for homotransplantation. 18. Refinement of microdroplet lymphocyte cytotoxicity test. Transplantation 6, 9 13-927. Ngan, B. Y . , E. W. Gelfand & J. 0. Minta (1977). A simple one-step hemolytic assay for C2 with C2-deficient human serum. J . Immunol. 118, 736-741. Ochs, H. D., S. I. Rosenfeld, S. Thomas, E. R. Giblett, C. A. Alper, B. Dupont, J. G. Schaller, B. C . Gilliland, J. A. Hanse & R. J. Wesgwood (1977). Linkage between the gene (or genes) controlling synthesis of the fourth component of complement and the major histocompatibility complex. N e w Engl. J . Med. 296, 470-475. Ott, J., D. Linder, B. Kaiser McCaw, E. W. Lovrien & F. Hecht (1976). Estimating distances from the centromere by means of benign ovarian teratomas in man. Ann. hum. Genet. 40, 191-196. Spencer, N., D. A. Hopkinson & H. Harris (1964). Phosphoglucomutase polymorphism in man. Nature 204, 742-745. Svejgaard, A., M. Hauge, C. Jersilid, P. Platz, L. P. Ryder, L. Staub-Nielsen & M. Thomsen (1976). The HLA system. An introductory survey. Monograph in Human Genetics, No. 7, Series ed. Beckman, L. & M. Hauge. Basel, Karger. Teisberg, P., I. Akesson, B. Olaisen, T. GeddeDahl, Jr. & E. Thorsby (1976). Genetic polymorphism of C4 in man and localisation of a structural C4 locus to the HLA gene complex of chromosome 6. Nature 264, 253-254. Address : R . Berger, M . D . Laboratoire d e Cytogknnktique Institut de Recherche sur les Maladies du Sang HBpital Saint-Louis, 2 place du D r Fournier 75475 Paris Cedex 10 France
Regional mapping of the HLA on the short arm of chromosome 6 R. BERGER~, A. BERNHEM, M.
G. HAUPTMANN3, J. M. FELMUSS
,%SPORTe@,
HORS4,
L. LEG-
AND
1 Laboratoire
de CytogBnCtique, Institut de Recherches sur les Maladies du Sang, HGpital Saint-Louis, Paris; 2 Unite de Recherche d’ImmunogtnCtique et de Transplantation humaine INSERM U93, HGpital Saint-Louis, Paris; 3 Centre de Transfusion de Strasbourg; 4 France-Transplant, HGpital Saint-Louis, Paris; 5 Laboratoire d’Immuno-Hematolopie, HGpital Saint-Louis, Paris, France A detailed gene marker study was performed on a partial 6p trisomic child resulting from a balanced maternal translocation t (2;6) (p 2505; p 2105). HLA typing and mixed lymphocyte reaction showed that the breakpoint on chromosome 6 was located within the HLA gene cluster, allowing an accurate location of the D determinants. Localization of the P blood group locus within the region 6 p 2105 to 6 p ter was excluded. Received 28 July, revised 3 October, accepted for publication 10 October 1978 Key words: Chromosome 6; complement; HLA; PI blood group; translocation mapping.
The location of the HLA gene cluster was first assigned to chromosome No. 6 by Jongsma et al. (1973) using human-Chinese hamster hybrid cells. The assignment of the major histocompatibility complex (MHC) gene cluster to the short arm of chromosome 6 was ascertained by Francke & Pellegrino (1977) and by Breuning et al. (1977). A biological study of a No. 6 short arm trisomic patient was performed in a child from whom clinical data have been reported in another paper (Bernheim et al. in preparation).
H L A typing. HLA determinants were typed by cytotoxicity using mono- or bispecific sera (Mittal et al. 1968): HLA-A, B, C on peripheral lymphocytes, cell lines and skin fibroblasts, and HLA-DR on B lymphocytes enriched by rosetting (70% pure). Additionally, complement fixation was used to detect HLA-A and B specificities on platelets (Colombani et al. 1971) and for DR determinant analysis of cell line and PHA-stimulated lymphocytes (Colombani et al. 1977).
Material and Methods
Mixed lymphocyte reaction ( M L R ) . Primary MLR were performed following the technique of Fradelizi et al. (1978).
Cytogenetics. Chromosome studies were performed on lymphocyte cultures using the GT, RH, RFA and CBG banding techniques.
Enzymes and proteins. Phosphoglucomutase 3 (PGM3) was studied using the technique of Spencer et al. (1964), red cell glyoxalase
0009-9163/79/030245-07 $02.50/0 0 1979 Munksgaard, Copenhagen
246
BERGER ET AL.
I (GLO I) using the technique of Kompf et al. (1975) and properdin factor Bf using the technique of Alper et al. (1972). Complement studies. Immunochemical titrations of C3, C4 and C5 were performed using commercial immunoplates (Hyland Laboratories, Costa Mesa, California, USA, for C3 and C4; Meloy Laboratories, Springfield, Virginia, for C5). C l q and factor Bf were estimated by radial immunodiffusion in 1% agarose, barbital 0.05 M and EDTANaz 0.01 M, p H 8.6 buffer using commer-
cial antisera and reference standards (Atlantic Antibodies, Westbrook, Maine). A C2-deficient serum (Hauptmann et al. 1977) was used for hemolytic titration of C2, according to Ngan et al. (1977). Except for C3 and C4, the titers of complement components were expressed as percentage activity or concentration of a pool of 40 normal sera stored in small aliquots at -70°C. Polymorphism of C2 was studied by analytical isoelectric focusing and hemolytic detection with C2-deficient serum, as described by Meo et al. (1977).
A
Flg. 1. Partial karyotypes. A (G-bands) and B (R-bands): maternal balanced translocation t(2;6). C (G-bands): partial 6p trisomy of 111-1.
REGIONAL M A P P I N G OF HLA
247
FAMILY 0..
I
II
m Flg. 2. Pedigree of the D family.
Cell cultures. Fibroblast cell cultures were established from skin biopsies of the proposita and from her mother. Lymphoblastoid cell lines were established from peripheral blood lymphocytes of the proposita and her mother. Blood groups. The following blood group typings were performed: ABO, MNS, PI, K, K,, CDE, Fy, Jk and Lu.
Results Cytogenetics. The proband, S.D., had a karyotype 46,XX,2p and the mother had a balanced reciprocal translocation t(2;6)(p25;p21). The father and maternal grandparents had normal karyotypes. The translocation was thus considered to be de novo in the child’s mother. A careful analysis of the chromosomes
+,
prepared using the G and R banding techniques was made to ascertain the breakpoints. Due to the fact that the mother was a balanced reciprocal translocation carrier, no chromosomal material was expected to have been lost. The extreme distal part of 2p (telomeric region) was thus translocated onto the deleted 6p. But the banding patterns of these regions of the chromosomes made it impossible to decide whether the exact breakpoint was at 2p 2500 or 2p 2509, or between these two subregions. Nevertheless, the breakpoints were most probably at 2p 2505 and 6p 2105. The child was thus trisomic for bands 6p 2105 to 6 pter and monosomic for a part of or almost the totality of band 2p 25 (Fig. 1). HLA typing. The proposita was found to have three haplotypes for the HLA, A and B loci: A3 B12 inherited from her father
248
BERGER ET AL.
Table 1 C h r o m o s o m e 6 g e n e m a r k e r s in t h e D family Subject
Relationship
Haplotype designation
HLA-A
I-1
Grandfather
a b
A11 A2
d
1-2
11-1 11-2
Grandmother Father Mother
HLA-C
HLA-B
Bf
HLA-DR
Cw4
Bw35 Bw21
S
-
DRwG DRw4
A3 643)
Cw4 Cve
Bw35 El8
DRw7 DRwG
e f
A3 A1
-
-
812 837
-
a d
A11 A3
Cve
A2 A3
-
-
C
A2 A3
e a d
A3 A11 A3
-
C
I13
Uncle
b
II 4
Aunt
b
C
I11-1
Patient
Cw4
Cw4 Cw4
GLO
PGM3
DRwl
2-1
1
Bw35 B18
DRwG
1
1
Bw21 Bw35
DRw4 DRw7
Bw21 Bw35
DRw4 DRw7 1
1
812 Bw35 El8
Cw4 Cve
S S S
DRwl DRwG
-
and A3 Cve B18 and A l l Cw4 Bw35 inherited from her mother. Only two determinants, however, have been identified for DR: DRwl transmitted from her father and DRw6 transmitted from her mother. The second maternally transmitted allele could, as shown by the family study, be DRw6 (Fig. 2, Tables 1 and 2).
not only gave, as expected, a positive MLR with the father’s cells and cells from unrelated controls, but also with the mother’s cells (Table 3), even though the child shared two HLA haplotypes (a, d) with the mother. Enzymes and proteins. PGM3 and GLO phenotypes were not informative (Table 1).
MLR. A primary MLR was performed within the family. The child as responder
Complement. The complement-component levels of C2, C4 and Bf were elevated in
Table 2 HLA markers in t h e patient
Cytotoxicity: Lymphocytes Lyrnphoblastoid cell line Skin fibroblasts Complement fixation: Platelets Lymphoblastoid cell line PHA-stimulated lymphocytes
A3
A11
Bw35
+ + +
+ + +
+
+
+
+ +
+
NT
NT
B18
Cw4
DRw3
+ +
+
+ +
+ +
-
+
+
NT NT
NT NT
NT NT
NT N
NT T NT
+
t f
+ NT
812
+
NT +
+ NT
N NT
+
NT T NT
-
-
DRw4
DRwG
NT +
+
HLA markers have been tested by two techniques (cytotoxicity and complement fixation) on 6 different NT=not tested. sources of antigen.
249
REGIONAL MAPPING OF HLA Table 3
Primary MLR in t h e D family. Responders Father: F Mother: M Child: C Control: T1 T2
y
F 200’ 55,008 18,453 49,129 56,116
irradiated stimulators M C T1 22,764 4.348 2,687 23,175 11,672 356 39,600 13,195 53.779 27,963
~~
14,449 34,773 23,721 2,232 73,562
T2 26.548 46,446 18,228 56,927 1,165
~
* Median value of triplicate cpm.
the proposita, contrasting with the normal levels of the C3, Clq and C5 components. The polymorphism study of C2 and Bf was not informative (Tables 3 and 4). Blood group typing. The child was P2, the mother P1 and the father P2. It can be concluded that the mother was a PUP2 heterozygote and transmitted only the P2 allele to her daughter. Discussion
The proposita was partially trisomic for the short arm of chromosome No. 6. She had three haplotypes for HLA-A and B and received two HLA-A, B and C haplotypes from her mother. These loci are thus located in this chromosomal region. Within the D region, only two HLA-DR specificities were serologically detected: DRwl
inherited paternally and DRw6 maternally. The MLR gave unexpected results. The most likely explanation is that the mother possessed an antigen which was not present in the child. From serological family data, the mother could be homozygous for DRw6, but the standard serological techniques do not allow the assessment of homozygosity for the DRw6 specificity. One possibility is that the breakpoint on the chromosome 6 interacts with the D region to produce a modified HLA-DR specificity undetectable by serological techniques. Due to the meiotic segregation, the proposita did not receive the 6p- chromosome on which the hypothetical modified determinant could be present. Experiments are now in progress to investigate other possibilities. It can be concluded that the breakpoint on the rearranged chromosome 6 was within the HLA gene cluster very near or inside the D locus, or at least between HLA-B and HLA-D, since the sequence of genes from centromere to telomere is D, B, C, A. These results are thus a direct proof of the location of HLA genes on chromosome 6p. The exact localization of the complex is the 6p 2105 region. This is in good agreement with previous data : localization “a little distal to the midpoint of either the short arm or the long arm of chromosome No. 6” was claimed from the study of a familial pericentric inversion of
Table 4 C o m p l e m e n t c o m p o n e n t s in t h e D family
111-2 11-2 11-1 Normal range
t 5
Clq R.I.’ %§
c4
c2
R.I.’ mgidl
H.T.7
91 86 86 67-135
53 24 28.5 15-45
c5
%§
c3 R.I.’ mgldl
R.I.’ mgldl
Bf R.I.’ mgldl
270 74 64 6 5 1 35
122 139 139 1ocL190
20 11.5 12.5 &25
81 35.5 37.5 21-50
R.I.: radial immunodiffusion. H.T.: hemolytic titration. %: percent of mean normal level (pool of 40 normal sera stored at -7OOC)
250
BERGER ET AL.
this chromosome (Lamm et al. 1974) : localization to 6p 22 from the study of a familial t(6;21) translocation (Borgaonkar & Bias 1974); localization to the 6p 21 band close to the transition to 6p 22 calculated from a t(6;20)(p21;p13) translocation in a large Dutch family (Breuning et al. 1977); localization proximal to 6p 22 estimated from interspecific hydrid human fibroblasts, having a balanced reciprocal translocation t(1;6) (p 3200 p 2100) x Chinese hamster cell studies (Francke & Pellegrino 1977). The map of chromosome No. 6 has been intensively studied by various methods, and some genes have been localized to the short arm of this chromosome (Bodmer 1976). Studies of phosphoglucomutase 3, PGM3 (Jongsma et al. 1973), as would be expected, were not informative in our family since the locus of the gene responsible has been estimated to be at a distance of 17 cM from the centromere (Ott et al. 1976) and the gene for PGM3 assigned to band 6p 12 (Francke & Pellegrino 1977). The red cell glyoxalase I (GLO I) study was also uninformative. The study of complementcomponent C2, C4 and Bf showed that the three loci are located in the region 6p 21 to 6p ter. The Bf locus was localized between HLAB and HLA-D with very close linkage to HLA-B. The genes for complement factors C2 and C4 have been claimed to be linked to the HLA gene cluster (review in Svejgaard et al. 1976), the C4 gene being on the side of the HLA-B (Teisberg et al. 1976, Ochs et al. 1977). The P1 blood group was tentatively assigned to chromosome No. 6 (Fellow et al. 1973, Lamm et al. 1975, Bijnen et al. 1976). Our results allow exclusion of the P1 blood group locus from the region 6p 2105 to 6p ter. Other considerations can be stressed from the study of the above reported family. First. no Dosition effects for either the
HLA-A, B and C determinants or for the complement-components due to the translocated HLA genes were found in the mothers of the proposita. This is the reason why the lack of one maternal D locus in the proposita can be considered as significant. Second, the distance in map units from the centromere of chromosome No. 6 to the HLA gene cluster was estimated to be about 32 cM. This distance is approximately equivalent to that of the region 6p 1 following the Paris Conference nomenclature. A more detailed correspondence between factorial and chromosomal maps cannot be established accurately at the present time, since insufficient data on the non-uniformity of crossing over are available in man. Acknowledgments
We gratefully acknowledge the help of Prof. J. Colombani in HLA typing, Dr. Nguyen Van Cong for PGM3-typing and Miss M. L. North for GLO-typing. References
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