Ann. Hum. Genet., Lolad. (1977), 41, 53

Printed in Great Britain 53

Family studies with the chromosome 9 markers ABO, AK,, ACON, and 9qh

BY E. B. ROBSON, P. J. L. COOK M.R.C. Human Biochemical Genetics Unit , The Galton Laboratory, University College London

AND K. E. BUCKTON M.R.C. Clinical and Population Cytogenetics Unit, Western General Hospital, Edinburgh

The ABO blood group locus (ABO) was shown to be linked to that for the Nail-patella syndrome ( N p ) by Renwick & Lawler in 1955. No additions were made to this linkage group until 1967 when Rapley et aZ. demonstrated linkage between A B O and the locus determining the polymorphism of the red cell enzyme adenylate kinase (AK,). Both of these claims have been confirmed by further work by these and other authors. N p must be very close to AK, because no recombinants have been observed out of 61 reported opportunities (Sobel, Tiger & Gerald, 1971 ; Schleutermann et al. 1969).

Since 1974 several laboratories have assigned AK, to chromosome 9 by the use of human- rodent somatic cell hybrids (Westerveld et al. 1976; Povey, et al. 1976; Grzeschik, 1976; Nguyen Van Cong et al. 1976). There is also evidence from hybrid studies that another adenylate kinase locus (AK,), and the locus for soluble aconitase (ACON,) may also be on chromosome 9 (Povey et al. 1976). Electrophoretic variants of ACON, have been described in family studies (Slaughter, Hopkinson & Harris, 1975) but no variants of AK, have yet been found (Wilson, Povey & Harris, 1976). Family studies on chromosome 9 have shown that conventional linkage studies are practicable since the heterochromatic region near the centromere (9qh) shows a remarkable degree of variation in size and position, whilst usually remaining a constant individual characteristic (Robinson et al. 1976).

In this paper we will present data on the segregation of ABO, AK,, ACON, and 9qh analysed to measure the recombination fraction between each pair of loci. We will also show that some unassigned loci cannot lie close to these markers. In a subsequent paper an attempt will be made to assign the loci ABO, AK, and ACON, to particular regions of the chromosome using families with abnormalities involving chromosome 9. A preliminary report of some of this work has already been published (Cook, Buckton & Spowart, 1976).

MATERIALS AND METHODS

Informative families were selected from the records of the MRC Human Biochemical Genetics Unit. They include the families used by Rapley et al. (1967), and Kindred T (de la Chapelle et al. 1974) which is also MRC 1905. Several other families have been published for a variety of unrelated reasons, but all with an MRC code number. Some of the families informa- tive for ACON, have been reported by Slaughter et al. (1975). Families segregating for 9qh were originally tested in the course of a family study of chromosome polymorphisms (Robinson et al. 1976), but subsequently families informative for ACON, or AK, were selected for 9qh typing.

ABO blood groups were determined by Miss J. E. Noades using a tube method. All A and AB

Pai

rs o

f m

arke

rs

AB

O : A

K,

AB

O :g

qh

AB

O :

AC

ON

,

AK

,:gq

h

AR

, : A

CO

N,

gqh:

AC

ON

,

Tab

le 1

. Lin

kuge

dat

a on

AB

O, A

K,,

AC

ON

, an

d 9q

h 3 -

gene

rati

on

Sex

of

No.

of

No.

of

dat

a*

lod

scor

es fo

r re

com

bina

tion

fra

ctio

ns o

f:

hete

ro-

hete

ro-

off-

(-A-,

zygo

te z

ygot

es s

prin

g rc

nr

c 3.

05

0'1

0

0.15

0

'20

0.

25

0.30

0.

35

2-

and

3-g

ener

atio

n d

ata:

A

M

62

174

2

I3

+15.

82

+16

.35

+15

.18

+13

.27

+10

.79

+8.

33

+5.

65

F

50

144

7 13

-7

.73

-1.9

0 +I.OO

+2

'11

+2

.41]

+

2.19

+

1.69

M

29

76

9 8

- 1

9.40

-1

1.37

-7

.24

-4.6

6 -2

-92

-1-7

5 -0

.99

2

o

-22'0

0

-1.3

9 -1

.05

-0.8

0 -0

.60

-0

.4

-0.

31

M

2

I +

0.46

$0

.43

+0.

40

+0.

36

+0.

31

+0

26

+

o.zo

F

2

2

0

M

8 25

0

2

-4.6

8 -2

.58

- 1.

48

-0.8

2 -0.40

-0.1

4 +0.04

F

6 27

0

I -2

.60

-0.9

4 -0

.17

+o

.zz

+o-

40

+0.

43

+0.

36

F

33

92

I4

I5

-18.

52

-10'

73

-6.6

7 -4.16

-2.5

1

-1'4

3 -0

.71

I

M

2

5 3

0

-3'7

2 -2

'54

-1.86

-1'3

9 -1

'03

-0'7

4 -0

.51

3 7

0 z

-0.88

-0.38

-0.1

2

+o.o

z +

O.I

O

+o.

14

+O

.I~

M

F

I

30

o to

's3

+

0.46

t-

0.39

1

0.3

2

+0

24

s0

.17

1-

0.10

* rc

= r

ecom

bina

nt,

nrc

= n

on-r

ecom

bina

nt.

0.40

+ 3.

19

+ 1.05

- 0

.43

- 0.

27

-0.1

9

+ 0.0

6 + 0

.24

-0.3

1

+O

.IZ

+ 0.05

s0.1

4

7

0.45

+ 1.16

+ 0.44

- 0.

04

- 0.

09

+ 0.0

6 + 0

.09

-0.1

4

-0.1

2

+ 0.0

7

+ 0.0

7 + 0

'01

9

0 w w c3

0 z

Family studies with chromosome 9 markers 55

Table 2. Linkage data on ABO and AK, in relation to chromosomes I and 15

Markers

ABO :rqh

Ferguson-Smith et al* (1975)

AK, :rqh

ABO : Fy

AK,:Fy

A BO : 15p

Sex of No. of No. of hetero- hetero- off- zygote zygotes spring

M 23 66 F 20 5 0

M + F 10 -

M 3 I 1

F 2 '4 M 62 180 F 21 64 M 34 96 F 24 78 M I8 66 F 22 61 M F

- - - -

M + F I I -

M 4 I4 F 6 18

3 -generation data*

7 4

rc

6 5 0

I 0

16

5 5 5 3

I 0

- - 0

0

0

nrc

3 6 0

I

0

8

5 3 8

I 2

I 0 - - 0

0

3

2- and 3-generation data: lod scores for recombination

fractions of 7- - 0'1

- 9.6 - 5.6 + 3.0 - 1.6 - 0.8 - 19.7 - 6.9 - 8.4 - 9.6 - 3.6 - 4.0 - 3.1 -0'1

- 6.6

- 3'3 + 1'2

0'2

-4.1 - 2'2 -

- 0.6

- 7'3

- 3'1 - 4 2 - 0.4 - 0.7 - 0.3 + 0.8

- 0'1

- 2'2

-

- 1.7 + 1'2

0.3 0'4 -1.7 -0.6 -0.7 -0.1 + 1.3 -

-0 .2 -0.1 +O.I 0

-2.5 -0.7 -0.4 +O.I

-1.0 -00'2 -1.7 -0 .5 +0.4 +0.5 +0.3 +0.4 +0.5 +0.6 +0.9 +0.5 - 1.3 -

-0.9 -0.3 +0.7 +0.3

* rc = recombinants. nrc = non-recombinants.

samples were classified as A,, A,, A,B or A,B on a tile using Dolichos or a similar seed extract. Dr T. E. Cleghorn determined the blood groups of some early families, and Dr R. Sanger supplied the results on some others. AK, types were determined on red cell haemolysates by starch-gel electrophoresis (Harris & Hopkinson, 1976), and ACON, phenotypes were determined on extracts of white blood cells or placentae (Slaughter et al. 1975). Other markers (see Tables 2 and 3) were determined by members of the MRC Human Biochemical Genetics Unit using standard blood grouping or enzyme techniques (Harris & Hopkinson, 1976). The size and position of the 9qh region were assessed visually after C-banding by members of the MRC Clinical and Population Cytogenetics Unit where other chromosomal polymorphisms were also scored (Robinson et al. 1976).

In the linkage analysis the lod score method as tabulated by Maynard-Smith, Penrose & Smith (1961) was used. Families were scored only when both parents were tested and where at least one parent was a double heterozygote. Where a parent was of the phenotype A or B the family was only scored for ABO when there was evidence from a relative that they were hetero- zygous. Because of their rarity ACONs families were scored even when both parents were not fully tested by weighting the scores for each possible genotype by the probability of the parents being of that genotype given their race and the phenotypes of their relatives.

56 E. B. ROBSON, P. J. L. COOK AND K. E. BUCKTON

Table 3. Minimum male recombination fraction between chromosome 9 markers and various unassigned markers (P > 500: 1)

Marker

Tf

MNSs Do* Gc

Gm Pi Jk Km C6t GPT P Kell Lu* sec co* Le

Marfan’s syndrome

El

Hba

c 3 t E,

Minimum distance from r--

ABO A K , 9rlh

27 22

24 31 26

5 30 19

26 16 29 18

14 19

5 3

14

I 2

21

I 1

I 2

21

15 25 27 22 2

23 19 I 2

20 I 0

23 16 15 1 2 2 1

15 5 4 6 5

Excluded from being

iiiside i l B O : AZi,

Ires Yes Yes Yes Yes Yes Yes Yes Yes YeR Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes

* The Do, Lu and Co results were supplied by Dr R. Sanger and many of the Do families have been

The complement typing was done by Dr M. Hobart and the C6 results have been published in detail by published by Tippett et al (1972).

Hobart, Cook & Lachmann (1977).

RESULTS

The lod scores between the markers ABO, AK,, ACON, and 9qh are given in Table 1, classified by the sex of the double heterozygote. They include the three generation data which are also tabulated separately as recombinants and non-recombinants. Table 2 gives the lod scores between ABO and AK, and selected markers not on chromosome 9, because of various hints or claims of linkage made in the past. Table 3 gives lod scores between chromosome 9 markers and disease loci or markers which had not been unambiguously assigned to any chromosome by September 1975 (Baltimore Conference, 1976).

DISCUSSION ABO: Np: A K ,

This is a well-established linkage group. Renwick (1968) added some of his own data to the original data of Rapley et al. (1967) and estimated the recombination fraction between ABO and A K , to be 8 yo in the male and 24 % in the female. Weitkamp et al. (1969), Wille &, Ritter (1969), Wendt et al. (1971) and Fenger & S~rensen (1975) all agree that these loci are linked and that the recombination fraction lies between 6 yo and 25 %. Only Weitkamp and Fenger et al. give their results separately for males and females and neither could confirm a sex difference in recombination fraction, although Weitkamp comments that he had relatively little data on

Family studies with chromosome 9 maikem 57

males. Our current results (Table 1) which include those of Rapley et al. (1967) still show a marked sex effect and give maximum likelihood estimates of 8 % for males and 24 yo for females. These estimates fit reasonably well with Schleutermann et aZ.’s estimate of the N p : ABO distance of 10 % for males and 15 yo for females, if the N p : AK, distance is 0 %. The formal independence of N p and AK, has not been demonstrated since no recombinant has been directly observed, but since the Npl allele has been observed in coupling with AKI1 on 31 occasions and with AK12 on two occasions (Sobel et al. 1971) we have assumed that N p and AK, are separate loci. On the basis of the female recombination fractions the order AK, : N p : ABO has been assumed for the present :

8 Male I

0 I

10 n t 1

Female 24

99h The overwhelming evidence for linkage between ABO and A K , combined with the evidence

assigning AK, to chromosome 9 from at least four different laboratories leaves little doubt that the AK,: N p : ABO linkage group is on chromosome 9. Earlier hints of the assignment of ABO to other chromosomes by family studies were not convincing. Yoder et al. (1974) found a hint of linkage between ABO and a large satellite on the short arms of chromosome 15. Our own results do not contradict this, but Ferguson-Smith et al. (1975) found evidence to the contrary (Table 2). The possibility that ABO was on chromosome 1 was raised by Ferguson-Smith’s data. The data in Table 2 make this claim untenable, especially when it is accepted that 1py is within 15 yo of lqh.

Although the heterochromatic region 9qh is officially designated as band 9q12 (Paris Con- ference, 1971) many families have been described where this region appears to show pericentric inversion to band 9pl l (Robinson et al. 1976; de la Chapelle et al. 1974). In this paper we have treated variants in the size of the 9q12 region and inv(9)(pllql3) as a single ‘locus’, 9qh. From the lods in Table 1 we can show that ABO is a t least 25% away from 9qh in males and that AK, is a t least 9 % away, accepting odds of 500: 1 against linkage (lod < - 2.7).

We can show that AK, is actually further away from 9qh by combining the male and female wores, and the 9qh : ABO and 9qh : A K , scores, assuming that the female recombination value is twice the male in this region and that the ABO : AK, distance is 8 % in males and 24 % in females. We must exclude from ABO:9qh the few families which also score for AK1:9qh (2 non- recombinants, two z, 11 scores and a z2 2100 in the male : 2 recombinants and 1 non-recombinant in the female). For example, at the AK1:9qh male recombination fraction of 10% the lod is - 2.58, and a t the female recombination fraction of 20 yo it is + 0.22. The ABO: 9qh recombi- nation fraction in the male would necessarily be 10yo+Syo, since the other alternative, 10 % - 8 yo is excluded by the ABO: 9qh data. The lod a t 18 yo is - 5.26 in the male, and a t 24Y0+2070 in the female the lod is -0.03. Hence the combined odds against the male

68 E. B. ROBSON, P. J. L. COOK AND K. E. BUCKTON

A K , : 9qh recombination fraction being 10 yo or less are over 40 000 000 : 1. Taking trial values in this manner we can say that A K , must be more than 20 yo from 9qh in the male, with odds of 600: 1. Similarly we can calculate that ABO must be more than 26% from 9qh in the male.

ACON, Only three of our six pedigrees of ACON, variants give linkage information. They exclude

very close linkage between ACON, and both ABO and A K , and show that ACON, cannot lie between them. In the family MRC 1962 (Cook et aZ. 1976) consideration of the gene frequencies, the phenotypes of the relatives and the known 8 yo recombination fraction between A K , and ABO shows that the three scorable children for ACON, and A K , are recombinants [P = 0,9991 and that the two scorable children for ACON, and ABO are recombinants [P = 0.9991. There are only four sibships informative for ACON, and 9qh and the lod scores do not rule out the possibility that this distance might just be measurable by family studies.

Unassigned markers We have a considerable body of negative data which suggests that several as yet unassigned

loci are not linked to the chromosome 9 markers. This is summarized in terms of minimum possible recombination fractions in Table 3. The calculations follow the arguments set out in the 9qh section. Many of these markers are also excluded from lying between ABO and AK,.

Inevitably there are some small positive lod scores at the larger recombination fractions. The only ones which exceed + 0.3 in the male are between 9qh and MNB, at 35 yo ( + 0.95), between A K , and C6 a t 29 yo ( + 0-36), between A K , and KeZZ at 25 % ( + 0.35) and between A K , and J k at 28 % ( + 0.36). We do not consider these scores to be of any significance at present.

CONCLUSIONS A N D SUMMARY

We have failed to measure the recombination fraction between 9qh and the ABO:AK, linkage group. Since the total male map distance along chromosome 9 is likely to be a t least 120 cM, calculated from male chiasmata counts (Hulth, 1974) they may well not be within measurable distance of each other. ACON, does not lie between ABO and AK,, but there is so

t

I

=. 0.26 4 > 0.20 4

I

I

I I

> 0.01 I > 0.09

I

1 > 0.005

+ - - - 1--,+ ACON, 9qh ACON, AKlNp A60

/ \ \ /

Fig. 1. Tentative map of chromosome 9 based on studies of families with normal karyotypes. Estimates of the minimum male recombination fractions are derivod from the total data.

Family studies with chromosome 9 markers 59 little information on this marker from family studies that it could lie almost anywhere else on chromosome 9 and might be within measurable distance of 9qh.

The map of chromosome 9 based on studies on families with normal karyotypes is sum- marized in Fig. 1. We hope to improve this map in a subsequent paper based on observations on families with abnormal karyotypes.

REFERENCES

BALTIMORE CONFERENCE 1975 (1976). Third International Workshop on Human Gene Afappirzg. Birth Defects: Original Articlo Series, XII, 7, The National Foundation, New York.

COOK, P. J. L., BUCKTON, K. E. & SPOWART, 0. (1976). Family studies on chromosome 9. TILirtE Irbternational Workshop on Human Gene Mapping, Baltimore, 1975. Birth Defects: Original Article Series, XII, 7, p. 284. The National Foundation, New York.

DE LA CHAPELLE, A., SCKRODER, J., STENSTRAND, I<., FELLMAN, J., HERVA, R., SAARNI, M., ANTTOLAINEN, I., TALLILA, I., TERVISLA, L., HUSA, L., TALLQUIST, G., ROBSON, E. B., COOK, P. J. L. & SANGER, R. (1974). Pericentric inversions of human chromosomes 9 and 10. Am. J . H u m . Genet. 26, 746.

FENGER, K. & SBRENSEN, S. A. (1975). Evaliiation of a possiblo sex difference in rccnmbination for the ABO-AK linkage. Am. J . H u m . Genet 27, 784,

FERGUSON-SMITH, M. A., ELLJS, P. M., MUTCHINICK, O., GLEN, K. P., C ~ T E , G. 13. & EDWARDS, J. H. (1975). Centromeric linkage. Second Internationd Workshop on Human Gene Mapping, Rotterdam, 1974. Birth Defects: Original Article Series, XII, 3, p. 130. The National Foundation, Now York.

GRZESCIIIK, K.-H. ( 1976). Assignment of human genes : /3-glucuronidase to chromosome 7, adenylate kiriaso to 9, a second enzyme uith enolase activity to 12, and mitochondria1 IDH to 15. Third International Workshop on Human Gene Mopping, Baltimore, 1975. Birth Defects: Original Article Series, XII, 7, p. 142. The National Foundation, New York.

HARRIS, H. & HOPKINSON, D. A. (1976). Handbook of E m y m e Electrophoresis in Human Genetics. Amsterdam: North Holland.

HOBART, M. J., COOK, P. J. L. & LACHMANN, P. J. (1977). Linkage studios with CF. J . Immunogenetics 4, (in the press).

H U L T ~ N , M. (1974). Chiasma distribution a t diakiiiesis in the normal human mnlc. Hereditus 76, 55. MAYNARD-SMITH, S., PENROSE, L. 9. & SMITH, C. A. B. (1961). Mathematical Tables f o r Research Workers i n

Human Genetics. London : Churchill. NGUYEN VAN CONG, WEIL, D., FINAZ, C.. COCIIET, C., REBOURCET, R., GROUCHY, J. DE & F R ~ Z A L , J. (1976).

Assigmnent of tho ABO-Np-AK, linkage group to chromosome 9 in man-hamster Iiybrids. Third Inter- national Workshop o n H,uman Gene Mapping, Baltimore, 1975. Birth Defects : Original Article Series, XII, 7, p. 241. The National Foundation, New York.

PARIS CONFERENCE ( 1971). Standardisation in Human Cytogenetics. Birth Dcfects : Original Article Scries VII, 7, 1972. The National Foundation, New York.

POVEY, S., SLAUGHTER, C. A., WILSON, D. E., GORMLEY, I. P., BUCKTON, I<. E., PERRY, P. & BOHROW, M. (1976). Evidence for the assignment of the loci AK,, Ah', and ACON, to cliromosomc 9 in man. Ann. Hum. Genet., Lond. 39, 413.

RAPLEY, S., ROBSON, E. B., HARRIS, H. & MAYNARD-SMITH, 8. (1967). Data on tho incidcnce, segregation arid linkage relations of the adenylate kinase (AK) polymorphism. Ann. Hum. Genet., Lond. 31, 237.

RENWICK, J. H. (1968). Ratios of female to male recombination fractions in man. Bull . Eur. SOC. H u m . Genet.

RENWICK, J. H. & LAWLER, S.D. (1955). Genetical linkagc bctween tho ABO and Nail-Patella loci. Ann.

ROBINSON, J. A., BUCKTON, K. E., SPOWART, G., NEWTON, M., JACOBS, P. A., EVANS, H. J. &HILL, R. (1976).

SCHLEUTERMANN, D. A., BIAS, W. B., MURDOCH, J. L. & MCKUSICK, V. A. (1969). Linkage of the loci for

SLAUGHTER, C. A., HOPKINSON, D. A. & HARRIS, H. (1975). Aconitase polymorphism in man. Ann. H u m .

SOBEL, R. S., TIGER, A. & GERALD, P. S. (1971). A second family with the nail-patolla allele and adcnylate

TIPPETT, P., GAVIN, J. & SANGER, R. (1972). The Dombrock system: linkage relations with othcr blood

WEITKAMP, L. R., SING, C. If'., SHREFFLER, D. C. & GUTTORMSEN, 8. A. (1969). The genetic linkage rolations

2, 7.

H u m . Genet., Lond. 19, 312.

The segregration of human chromosomal polymorphisms. Ann. Hum. Genet. Lond. 40, 113.

the Nail-Patella syndrome and adenylate kinase. Am. J . H u m . Genet. 21, 606.

Genet., Lond. 39, 193.

kinaso2 allele in coupling. Am. J . H u m . Genet. 23, 146.

group loci. J . Med. Genet. 9, 392.

of adenylate kinase: Further data on the ABO-AK linkage group. Am. J . H u m . Genet. 21, 600.

E. B. ROBSON, P. J. L. COOK AND K. E. BUCKTON WENDT, G. G., RITTER, H., ZILCH, I., TARIVERDIAN, G., KINDERMA", I. & KIRCHBERG, G. (1971). Genetics

and linkage analysis on adenylate kinase. Humntngenetik 13, 347. WESTERVELD, A., JONQSMA, A. P. M., MEERA KHAN, P., SOMEREN, H. VAN & BOOTSMA, D. (1976). Assign-

ment of the AK-1:Np:ABO linkage group to chromosome 9. PTOC. Nutn. Acad. Sci. U.S.A. 73, 895. W~LLE, R. 8: RITTER, H. (1969). Zur formalen Genetik der Adenylatkinasen; Hinweis auf Kopplung der

Loci fur AK und ABO. Humangenetik 7, 263. WILSON, D. E., POVEY, S. & HARRIS, H. (1976). Adenylate kinases in man: evidence far a third locus.

Ann. Hum. Genet., Lond. 39, 305. YODER, F. E., BIAS, W. B., BOROAONKAR, D. A., BAHR, G. F., YODER, I. A., YODER, 0. C. & GOLOMB,

H. M. (1974). Cytogenetic and linkage studies of a familial 15p+ variant. Am. J . Hum. Genet. 26, 535 .