GENOMICS 5, 199-203 (1989)

Linked Markers Flanking the Gene for Multiple Endocrine Neoplasia Type 2A

Y. NAKAMURA,* C. G. P. MA-rtiEw,t H. SOSOL,$ D. F. EASTON,!~ H. TELENIUS,t T. BRAGG,* K. Ct-wt J. CLARK,t C. JONES,” G. M. LENOIR,* R. WHITE,* AND B. A. 1. Pommt

*Howard Hughes Medical Institute, University of Utah, Salt Lake City, Utah 84132; tSection of Human Cancer Genetics and §Section of Epidemiology, Institute of Cancer Research, Sutton, Surrey SM2 5rVG, United Kingdom; Writ of

Mechanisms of Carcinogenesis, International Agency for Research on Cancer, Lyon, France; and I’Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado 80262

Received November 7, 1988; revised February 9, 1989

The inherited cancer syndrome multiple endocrine neoplasia type 2A (MENBA) has recently been mapped to chromosome 10. We have typed 29 families with this disorder with DNA markers from the pericen- tromeric region of chromosome 10. Two markers, RBP3 and MCKS, were tightly linked to the MENBA gene at recombination fractions of less than 3%. Mul- tipoint analysis of the linkage data suggests that the gene is located within a ~-CM interval defined by the markers RBPS/MCKB on one side and TB14.34 on the other. No evidence for locus heterogeneity was de- tected in any of the 27 families from 14 countries who were informative for the markers tested. The data confirm and refine the original assignment and provide the basis for presymptomatic screening for this dis- order. IC) 1989 Academic Press, Inc.

INTRODUCTION

Multiple endocrine neoplasia type 2A (MENSA) is an inherited cancer syndrome characterized by med- ullary thyroid carcinoma, pheochromocytoma, and hy- perplasia of the parathyroid glands. The gene is trans- mitted as an autosomal dominant trait and although clinical penetrance is incomplete, most gene carriers can be detected by biochemical screening tests before the age of 40 (Gage1 et al., 1982). The predisposing gene for the disorder has recently been mapped to chromosome 10 by the detection of linkage between MENZA and both the retinol-binding protein gene RBP3 (Mathew et al., 1987; Simpson et al., 1987) and the DNA marker DlOS5 (Simpson et al., 1987). Since then, there has been considerable progress in the de- velopment of a genetic linkage map of chromosome 10, which has led to the isolation of four new markers closely linked to RBP3 (Lathrop et al., 1988; Nakamura

et al., 1988a). The linkage map of these markers is il- lustrated in Fig. 1.

We have analyzed a panel of 29 MENSA families with these markers to establish both the position of the MENBA gene on the linkage map and whether the syndrome might be caused by mutations at more than one genetic locus. Our data suggest that two closely linked markers near the centromere of chromosome 10 flank the MENBA gene and that the predisposing mu- tation lies within this region in all informative families in the panel.

MATERIALS AND METHODS

MEN2A Families

All individuals at risk were screened for C-cell hy- perplasia or medullary thyroid carcinoma by measure- ment of serum calcitonin after pentagastrin stimulation (Telenius-Berg et al., 1977). Individuals with normal test results were considered unaffected; all such indi- viduals used in this study who were potential gene car- riers had tested normally until at least the age of 15 years. The family panel consisted of 16 families from the Institute of Cancer Research (ICR), 11 families from the GETC/IARC (Groupement d’etude des Tu- meurs a Calcitonine/International Agency for Research on Cancer), and 2 families from the Howard Hughes Medical Institute at Salt Lake City.

DNA Probes and Restriction Fragment Length Polymorphisms (RFLPs)

The following DNA markers were used:

1. IRBP.H4/BglII/RBP3 (Liou et al., 1987)

2. IRBP.H4/Styl/RBP3 (Chin et al., 1988)

3. TBIRBPS/Taql/RBP3 (Nakamura et al., 1988a)

199 0888.7543/89 $3.00 Copyright 0 1989 by Academic Press, Inc.

All rights of reproduction in any form reserved.

NAKAMURA El’ AL. 200

4. MCK2/Mspl or Styl/DlOSl5 (Nakamura et al., 1988b)

5. TBlO.l63/A4spl/DlOS22 (Bragg et al.. 1988)

6. TBQlG/Mspl/DlOS30 (Nakamura et al., 1988a)

7. TB14.34/Tczql/DlOS34 (Nakamura et al., 1988a).

The ICR families were typed with all seven markers and the other families with markers 1, 2, 4, and 5. Linkage data with the marker IRBP.H4 have been published for a small subset of the ICR (Mathew et al., 1987) and GETC/IARC (Sob01 et al., 1988) families.

Methods of DNA electrophoresis, blotting, and hy- bridization were as described previously (Mathew et al., 1987). Families in whom recombinant5 were de- tected were typed with the multiallelic locus-specific minisatellite probes XMSl and XMS31 (Wong et al.. 1987) to check family relationships.

Linkage Analysis

Likelihood computations were carried out using the LINKAGE program (Lathrop et al., 1985). The age- specific penetrance of the MEN2A gene was based on previously derived estimates (Easton et al., 1988) and a gene frequency of 2 X 10e5 was assumed (Carter et al., 1987). The multipoint analysis of MENBA relative to the DNA markers was based primarily on the chro- mosome 10 linkage map of Lathrop et al. (1988) as revised by Nakamura et al. (1988a). Since RBP3 and

MCK2 are very tightly linked, a definitive order of these markers relative to TBQl6 has yet to emerge. We have therefore considered both map orders RBP3-MCKB- TBQ16 and MCK2-RBP3-TBQ16 here. One recom- binant family was excluded from both the pairwise and the multipoint analysis because DNA fingerprint,ing showed that six offspring were the product of one mother and three different fathers, which rendered the matings uninformative. In another family, two indi- viduals in a sibship of five who had been diagnosed as gene carriers on the basis of the stimulated calcitonin assay were recombinant with six informative markers between pMHZ15 (which is 30 CM proximal to TB14.34, Lathrop et al., 1988) and TB10.163. This suggests either that the two siblings represent examples of a false-positive screening assay or that two cross- overs occurred within a small interval in both individ- uals. The family was excluded from the pairwise anal- ysis. Inclusion of this family in the multipoint analysis did not affect the preferred order (see Results).

RESULTS

Pairwise Analysis

The lod scores for the pairwise analysis of MEN2A with the chromosome 10 markers are shown in Table 1. The probe TBIRBPS is a genomic fragment adjacent to the RBP3 gene and is considered as the same locus in this analysis. No recombinants between IRBP.H4

TABLE 1

Pairwise and Maximum Lod Scores for Linkage between MEN2A and Chromosome 10 Markers

Recombination fraction H (8, = or)”

Marker 0.0 0.001 0.005 0.01 0.05 0.1 0.2 0.3 0.4

Pairwise lod scores

TB14.34 -2.03 0.75 2.05 2.64 3.56 3.55 2.83 1.79 0.69 RBP3 6.79 13.62 17.39 17.83 17.73 16.14 11.85 7.05 2.62 MCKP 14.83 18.83 19.38 19.48 18.53 16.67 12.38 7.13 3.19 TBQl6 0.67 4.58 5.22 5.43 5.55 5.09 3.79 2.36 0.98 TB10.163 -33.32 -8.03 ~2.56 -0.25 4.42 5.43 4.72 2.10 1.04

Marker 2

TB14.34 3.61 RBP3 18.10 MCK2 19.48 TBQl6 5.63 TB10.163 5.48

em = 8fb

Maximum lod scores (2)

8 i

0.07 3.89 0.024 18.79 0.011 19.73 0.029 5.81 0.12 6.13

8, f Bf’

& 8 “1

0.089 0.0 0.048 0.0 0.02 0.0 0.044 0.0 0.159 0.035

’ 0, and Br refer to male and female recombination fractions respectively. b Assuming no sex differences in recombination. ’ Sex-specific recombination rates.

LINKED MARKERS FOR MENBA 201

and TBIRBPS have been detected (Nakamura et al., 1988a). As can be seen from Table 1, the markers RBP3, MCKB, and TBQl6 are all closely linked to MENBA. RBP3 has been mapped to lOp11.2-lOq11.2 by in situ hybridization (Liou et al., 1987) and to 1Oq by somatic cell hybrid analysis (Lathrop et al., 1988). On the basis of the pairwise analysis and the linkage map of the pericentromeric markers (Fig. I), it might be thought that the MEN2A gene is located on the proximal long arm of chromosome 10. However, the confidence limits for the values of 0 are wide. For ex- ample, the 95% confidence interval for RBP3-MENBA is 0.004-0.07. We have therefore carried out a multi- point linkage analysis of the data to order MENSA relative to the markers.

Multipoint Analysis

Two families were particularly informative in this analysis. One family included an individual in whom there had been recombination between MENBA and the markers RBP3 and TB10.163, but who was not recombinant with TB14.34. The other family contained two individuals who were recombinant with TB14.34 but not with RBP3 and MCKB. All three individuals in whom the recombinant events were observed had histologically proven medullary thyroid carcinoma. Nonpaternity was excluded by fingerprinting using minisatellite probes. The odds for various marker and gene orders are shown in Table 2. The order that places MEN2A between TB14.34 and RBP3 is preferred by more than 60-fold over other possible orders. The lod score for various positions of MENSA relative to the markers is plotted in Fig. 2. Likelihood ratios for the various orders were also calculated including the two

15 -1

i

em=1

14 I 13 1 I

12

F

T814.34

11.2 I1 0.03

TB10.163

24

25

E 26 u

10

FIG. 1. Linkage map of markers in the pericentromeric region of chromosome 10 (adapted from Ref. (15)). The order RBP3/MCKP is uncertain. Bm = f: sex-averaged recombination fraction.

TABLE 2

Likelihood Ratios for Various Gene Orders as Determined by Multipoint Analysis”

Order NO. Order Hati

1 MEN2A-TB14.34-RBP3-MCK2-TBQl6-TBlo.l63 1 2 TB14.34-MENZA-RBP3-MCKZ-TBQX-TB10.163 52,602 3 TBi4.34-RBP3-MENZA-MCKZ-TBQlfi-TBlO.lfi3 402 4 TB14.34-RBP3-MCK2-MENZA-TBQl6-TBlO.163 21 5 TB14.34-RBP3-MCK2--TBQl6-MEN2A-TBlO.l63 0.0091 6 TB14.34-RBP3-MCK2-TBQ16-TBlO.l63MEN2A 1 x 1or9 7 MEN’LA-TB14.34-MCKP-RBP3-TBQIG-TB10.163 0.0091 8 TB14.34-MENZA-MCKZ-RBPS-TBQlG-TB10.163 785

9 TB14.34-MCKZ-MENZA-RBP3-TBQ16-TB10.163 419 10 TB14.34&MCK2-RBP3-MEN2A-TBQl6-TB10.163 41 11 TB14.34-MCK2-RBP3-TBQl6-MEN2A-TBlO.163 0.013 12 TB14.34-MCKZ-RBP3-TBQ16TB10.163-MEN2A 1.3 x 10-y

’ Likelihoods are expressed as a ratio of that obtained for order 1. The likelihoods for orders 7 to 12 are based on the alternative marker order with RBP3 and MCKY reversed and include a factor off for the relative likelihood of this marker order compared with the first (15).

individuals with C-cell hyperplasia who were recom- binant with the string of markers between pMHZ15 and TB10.163 (see Methods). The two most preferred orders were unchanged, with order 2 in Table 2 having a likelihood ratio of 44,055 and order 8 having one of 415.

Locus Heterogeneity

A total of 29 families originating from Sweden, Den- mark, England, Holland, Germany, Belgium, Spain, South Africa, Tunisia, Morocco, Italy, Switzerland, France, and the United States were informative for linkage analysis with one or more of the pericentro- merit chromosome 10 markers. Segregation of the markers was consistent with linkage to MENSA in all cases where two or more meioses could be scored (data not shown, but available on request). Lod scores of greater than 3, which is considered strong evidence for linkage (Morton, 1955), were observed in four different families.

DISCUSSION

The lod scores obtained for the pairwise linkage analysis of the chromosome 10 markers and MENSA (Table 1) provide overwhelming support for the original assignment of this gene to chromosome 10 (Mathew et al., 1987; Simpson et al., 1987). The data suggest that both RBP3 and MCK2 are within 3 CM of MENBA, which is similar to our earlier estimates of 4 CM for RBP3-MENBA (Mathew et al., 1987), but smaller than the 12CM estimate of Simpson et al. (1987). The reason for this difference is not yet clear, but it may result from chance factors such as the proportion of families with a female affected parent in the two sample sets.

202 NAKAMURA ET AL.

171

\ I

17:

,sl I -/ I - 1 I 1 -i 15' L 1 I 1 \ 1

-20 -15 -10 -5 0 5 10 15 20 25 30 35 -20 -15 -10 -5 0 5 IO 15 20 25 30 35

GENETIC LOCATION (CENTIMORGANS) GENETIC LOCATION (CENTIMORGANS)

FIG. 2. Lod scores for various positions of MENZA relative to the DNA markers. The locations are given relative to that of TB14.34, with genetic distances corresponding to those of the female map. (a) Marker positions for the most likely order determined by Nakamura et al. (15). (b) The positions of RBP3 and MCK2 have been reversed. This order is less likely than (a) by odds of 7:l (15). All other possible marker orders have been strongly excluded. The dashed line (- - -) is drawn 1 LOD interval below the maximum likelihood ratio. Regions of the curve lying above this line approximate the 95% confidence region for the position of the MENZA gene.

This would influence the (3 observed since the female: male recombination rate is about 2:l in this region of the genome (Lathrop et al., 1988). This sex difference is also reflected in our pairwise analysis (Table l), which shows that most recombinants were from af- fected female parents. Recently, Myers et al. (1988) have calculated the MENSA-RBP3 recombination fraction to be 0.07 by combining data from Mathew et al. (1987), Simpson et al. (1987), and Sobol et al. (1988).

The multipoint analysis of this data set provides strong evidence that the MENBA gene lies within the TBl4.34-RBP3/MCK2-TBQ16 linkage group and suggests that TB14.34 and RBP3/MCK2 represent flanking markers for the MEN2A gene (Table 2). Since these markers are linked at a sex-averaged distance of about 3 CM, this result defines a relatively small chro- mosomal interval within which the MENBA gene must reside. This is an important development in the search for the gene because new long-range mapping and cloning techniques such as pulsed-field gel electropho- resis (Schwartz and Cantor, 1984) and jumping libraries (Collins and Weissman, 1984; Poustka and Lehrach, 1986) can now be used to generate new markers and a physical map of the region. It is not yet clear whether the MEN2A gene lies on the proximal short arm or the long arm of chromosome 10, but it must be located very close to the centromere. Linkage studies with a polymorphic a-satellite DNA sequence that hybridizes specifically to the centromere of chromosome 10 (De- vilee et al., 1988) are in progress to resolve this point.

The fact that linkage of the gene to the chromosome 10 markers was detected in an extensive panel of fam- ilies from 14 countries suggests that this syndrome is unlikely to be caused by mutations at more than one genetic locus. However, the possibility that in a small fraction of families or an as-yet-untested ethnic pop-

ulation a different locus is involved cannot be excluded. There are also variants of the MEN2 syndrome that include families with medullary thyroid carcinoma only or with the more aggressive MEN2B. Preliminary ev- idence suggests that these syndromes arise from mu- tations at the same locus as that of MEN2A (Jackson et al., 1988; No11 et al., 1988).

The linkage data presented in this report have im- portant implications for the future diagnosis of gene carriers of MENBA. The tight linkage of the markers RBP3 and MCK2 to the gene implies that these mark- ers could be used for presymptomatic screening in the young offspring of affected individuals in these families. The penetrance of the gene on screening with the stimulated calcitonin assay is not complete until the age of about 30 (Gage1 et al., 1982; Easton et al., 1988), so that many gene carriers are not detected until adult- hood. The addition of DNA linkage analysis to the screening program of these families will be of great value in their clinical management. The existence of flanking markers will permit the detection of rare re- combination events at the locus in families informative for both TB14.34 and either RBP3 or MCK2 and thus significantly increase the accuracy of carrier detection. It will also lead to a more highly directed search for the MEN2A gene.

ACKNOWLEDGMENTS

The minisatellite probes XMSl and XMS31 were provided by Alec Jeffreys. We thank the following people, who provided us with clinical material for this work: T. Cummings, M. Telenius-Berg, M. Kaplan, H. Haak, S. Schifter, H. Hansen, S. Reichlin, S. Jansen, R. Carmena, C. Mahler, G. Benker, A. Frilling, G. Reinwein, S. Baylin, S. Wells, and S. Narod. We also thank the following people from the GETC: A. Boneu, C. Calmettes, D. Chadenas, G. Charpentier, J. Chatal, J. Dupond, N. Delepine, M. Delisle, P. Gardet, G. Godefroy, P. Guil-

LINKED MARKERS FOR MEN2A 203

lausseau, C. Guillausseau-Scholer, C. Houdent, J. Lalau, S. Mace.

C. Parmentier, F. Soubrier, and J. Tourniaire. This work was sup-

ported by a program grant from the Cancer Research Campaign.

H.T. was supported by Swedish grants from the Cancer Society in

Stockholm; The Foundation Blanceflor Boncompagni-Ludovisi, nee

Bildt; the Gyllenstierna-Krapperup Foundation; the Mrs. Berta Kamprad Foundation; The Soderberg Foundation; and the Sward

Foundation. R. W. is an Investigator of the Howard Hughes Medical

Institute.

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