Multipoint Linkage Analysis in Menkes Disease T

Am. J. Hum. Genet. 50:1012-1017, 1992

Multipoint Linkage Analysis in Menkes Disease T. T0nnesen,* A. Petterson,* T. A. KruseT A.-M. Gerdest and N. Horn*

*John F. Kennedy Institute, Glostrup, Denmark; TInstitute of Human Genetics, University of Aarhus, Aarhus, Denmark; and *Department of Clinical Chemistry, Odense Sygehus, Odense, Denmark

Summary

Linkage analyses were performed in 11 families with X-linked Menkes disease. In each family more than one affected patient had been diagnosed. Forty informative meioses were tested using 11 polymorphic DNA markers. From two-point linkage analyses high lod scores are seen for DXS146 (pTAK-8; maximal lod score 3.16 at recombination fraction [0] = .0), for DXS1 (p-8; maximal lod score 3.44 at 0 = .0), for PGK1 (maximal lod score 2.48 at 0 = .0), and for DXS3 (p19-2; maximal lod score 2.90 at 0 = .0). This indicates linkage to the pericentromeric region. Multilocus linkage analyses of the same data revealed a peak for the location score between DXS146(pTAK-8) and DXYSlX(pDP34). The most likely location is between DXS159 (cpX289) and DXYS1X(pDP34). Odds for this location relative to the second-best-supported region, between DXS146(pTAK-8) and DXS159 (cpX289), are better than 74:1. Visualization of individual recombinant X chromosomes in two of the Menkes families showed the Menkes locus to be situated between DXS159(cpX289) and DXS94(pXG-12). Combination of the present results with the reported absence of Menkes symptoms in male patients with deletions in Xq21 leads to the conclusion that the Menkes locus is proximal to DXSYlX(pDP34) and located in the region Xql2 to Xql3.3.

Introduction (Wienker et al. 1983). Use of a centromeric chromo- Menkes disease (McKusick 30940) is an inherited dis- somal marker in five Danish families with Menkes as an X-linked disease suggested a close linkage between the Menkes order of copper metabolism transmitted locus and the centromeric region (Horn et al. 1984). recessive trait. Patients show specific copper-defi- man and as lack keratinization and By comparative gene mapping between ciency symptoms, such of mouse, a localization close to the human PGK1 locus pigmentation ofhair, degenerative changes ofthe elas- et al. In a three- in blood and scorbutic at Xql3 was suggested (Horn 1984). tic tissue the aorta and vessels, the Menkes locus was tenta- bone changes (Danks et al. 1972). In addition, pro- point linkage analysis, retardation with seizures and tively localized on the long arm of the X chromosome gressive psychomotor proximal to DXYS1X (Tonnesen et al. 1986, p. 627). temperature instability are seen, and the affected males of Menkes disease more than 3 The incidence is A girl having clinical symptoms rarely survive for years. a translocation with the on the to be live births et al. and having breakpoint estimated 1:300,000 (Tonnesen active X chromosome in Xql3.2-Xql3.3 was recently 1991). found (Kapur et al. 1987; Verga et al. 1991). The The exact localization of the Menkes locus has not have now been extended to 11 A tentative localization has been linkage analyses yet been established. Menkes families, and the results on 40 informative proposed at Xpl 1.2, defined by the locus DXS7 of these data meioses in these families indicate a localization of the (Wieacker et al. 1983). Further analyses Menkes on the part of Xq. defined the Menkes locus to a region distal to DXS7 gene proximal

Received October 23, 1991; revision received December 26, Subjects and Methods 1991. Address for correspondence and reprints: Dr. T. T0nnesen, John Subjects F. Kennedy Institute, GI. Landevej 7, DK-2600 Glostrup, Den- Four German, three American, two British, one mark. i 1992 by The American Society of Human Genetics. All rights reserved. Danish, and one Argentinian family were used in this 0002-9297/92/S005-0015$02.00 study. For each of these 11 investigated families, more

1012 Localization of the Menkes Locus 1013

Table I Results Names and Regional Assignments of Probes Two-Point Linkage Analyses Informative in Present Study A number ofX chromosome-specific DNA markers HMG 10.5 Probe Regional have been used in our search for linkage to the Menkes Nomenclature Designation Assignment locus (table 1). Two-point linkage analyses on the data DXS84 ...... L754 Xp21.1 obtained from 40 informative meioses in 11 Menkes DXS146 ...... pTAK-8 Xpl1.22 families are shown in table 2. For some of the DNA DXS14 ...... p58-1 Xpl 1.21 markers (pTAK-8, p-8, pSPT19-1, and pl9-2) high DXS1 ...... p8 Xqll.2-ql2 lod scores are seen, which indicates linkage between DXS159...... cpX289 Xql2 the Menkes locus and the pericentromeric region. DXS106...... cpX203 Xql2 PGK1 ...... pSPT19.1 Xql3 Multilocus Linkage Analyses DXYS1X...... pDP34 Xq21.31 DXS3 ...... p19-2 Xq21.3 When the data are subjected to multilocus linkage DXS94...... pXG-12 Xq22 analyses, the following picture emerges (fig. 1). The DXS91 ...... pXG-17 Xq24-q26 location score shows a peak between pTAK-8 and DXS52 ...... Stl4-1 Xq28 pDP34. But the most likely localization is between cpX289 and pDP34, with a maximal location score of than one affected Menkes patient has been diagnosed. 26.3, equivalent to a multipoint lod score of5.7. Odds Informative individuals are persons for whom pedi- for this location relative to the second-best-supported gree information alone is sufficient to establish the region (between pTAK-8 and cpX289) are greater genotype. A total of 40 informative meioses have been than 74:1. The present results give no indication of investigated. linkage heterogeneity. DNA Analyses Haplotype Analyses The DNA probes applied for this investigation are We next looked in more details on individual recom- listed in table 1. For hybridization, 0.05-0.10 gg puri- binant X chromosomes (fig. 2). In the left-hand panel fied human insert DNA was used as a template in of figure 2 are shown the results of the RFLP analyses oligonucleotide-primed synthesis of 32P-labeled DNA in one Menkes family. It is evident that the healthy (Feinberg and Vogelstein 1983). Subsequent to the boy (III-2) has received the complete X chromosome labeling, the DNA probes were hybridized to nylon of his maternal grandfather. The Menkes boy (111-3) filters onto which 5-10 gg of human genomic DNA has received the alternative X chromosome from his had been Southern blotted, after restriction-endo- mother. This X chromosome must thus carry the nuclease digestion and subsequent separation of the Menkes mutation, as the mother is an obligate carrier. DNA fragments by agarose gel electrophoresis. The Both 111-4 and III-6 have recombinant X chromo- filters were autoradiographed by exposure to X-ray somes, as they carry parts of the Menkes X chromo- films for 2-10 d. some and parts of the healthy X chromosome. From these analyses we may conclude that the Menkes locus Linkage Analyses is on Xq, distal to cpX289. Genotypic data were obtained using the probes For a second Menkes family the results of the RFLP listed in table 1. Pairwise and multilocus (nine-point) analyses are shown in the right-hand panel of figure 2. linkage analyses were performed using the LINKAGE In this Menkes family we know that 111-3 suffered from 4.7 package of computer programs (Lathrop and La- Menkes disease. 111-4 is one ofthe very few girls known louel 1984; Lathrop et al. 1984). Gene order was ex- to express the Menkes gene (Gerdes et al. 1990). In amined by multilocus likelihood calculations using the this family the X chromosome carrying the Menkes ILINK and LINKMAP programs. Genetic distances mutation in the obligate carrier mother (11-3) is derived were calculated using Haldane's mapping function, from her father, probably as a new mutation during which converts recombination fractions to genetic dis- spermatogenesis. II-3 has donated the main part ofher tances in centiMorgans. The frequency of the Menkes Menkes X chromosome to her Menkes boy. Further- gene in the general population was taken to be .0001, more, she has given most of the Menkes X chromo- and the published RFLP allele frequencies were used. some to her Menkes daughter (III-4). The daughter 1014 Tonnesen et al. Table 2 Lod Score Results of Pairwise Linkage Analyses for I I Different Markers. LOD SCORE AT RECOMBINATION FRACTION OF MAXIMUM MAXIMUM 95% DNA LOD RECOMBINATION CONFIDENCE MARKER .00 .001 .01 .05 .1 .2 .3 .4 SCORE FRACTION LIMITS L754 ...... - 2.27 -1.28 -.62 -.36 -.15 -.05 -.01 .00 .50 .02-.50 pTAK-8 ..... 3.16 3.15 3.10 2.87 2.56 1.91 1.20 .49 3.16 .00 .00-.16 p58-1 . . - 3.65 - 1.67 -.39 .06 .32 .30 .17 .33 .24 .03-.50 p-8 ...... 3.44 3.44 3.38 3.12 2.78 2.07 1.33 .61 3.44 .00 .00-.15 cpX289 ...... -.95 .05 .71 .88 .79 .51 .21 .89 .12 .01-.50 cpX203 ...... - 2.15 - 1.16 -.50 -.26 -.08 -.02 -.01 .00 .50 .02-.50 pSPT19-1 ... 2.48 2.48 2.44 2.26 2.03 1.53 .98 .42 2.48 .00 .00-.21 pDP34 ...... - - 7.63 - 3.80 - 1.21 -.28 .31 .34 .15 .36 .25 .08-.50 p19-2...... 2.90 2.90 2.85 2.62 2.32 1.68 .99 .33 2.90 .00 .00-.17 pXG-12 ...... - 4.59 - 1.90 -.06 .53 .79 .62 .29 .79 .20 .04-.50 pXG-17 ...... - 6.45 - 3.48 - 1.51 -.78 -.22 -.05 -.01 .00 .50 .08-50

111-1 has received the intact Menkes X chromosome 3), the most likely localization of the Menkes locus is from her mother and should be a carrier of this trait. between cpX289 and pXG-12. For III-2 we observe two recombinational events, giv- ing her two regions ofher mother's Menkes X chromo- Discussion some. From these analyses we may conclude that the Menkes locus is proximal to pXG-12. When the re- Our two-point linkage analyses indicate linkage be- sults obtained for these two families are combined (fig. tween the Menkes locus and the pericentromeric re-

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Figure I LINKMAP analysis of the location of the Menkes locus. The location scores were calculated for different locations of the Menkes locus relative to a fixed map of eight markers. Localization of the Menkes Locus 1015 n1

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5 1 2 3 4 Figure 2 Left, Danish Menkes family presented by Horn et al. (1984; family 14). The prenatal diagnosis performed on 111-6 has been reported elsewhere (Tonnesen et al. 1989). From top to bottom, the informative probes used for this family are L754, p58-1, cpX289, p19-2, and Stl4-1. For all the two-allele probes the largest allele is given the number "1", and the smallest allele is given the number "2." For the probe Stl4-1 the HMG 10 allele system is used. Right, Menkes family from Germany. The clinically affected Menkes girl (III-4) recently has been reported elsewhere (Gerdes et al. 1990). From top to bottom, the informative probes used for this family are pTAK-8, cpX203, pSPT19-1, p19-2, pXG-12, and Stl4-1. Descriptions of the allele identifications for all the probes are as in the left-hand panel.

gion (table 2). The results obtained from our The Menkes gene is thus localized on the proximal multipoint analyses indicate that the Menkes locus is part of Xq, in the region Xql2-Xq2l.3. localized between cpX289 and pDP34. This localiza- Male patients with deletions of the region Xq21.1- tion is further illustrated from the haplotype analyses Xq21.33 and with no clinical Menkes symptoms have of individual crossover events (figs. 2 and 3), where a been reported (Cremers et al. 1988, 1989; Schwartz localization between cpX289 and pXG-12 is found. et al. 1988; Merry et al. 1989; Yang et al. 1990). If we assume that Menkes disease is caused by a loss of function, we can narrow the region of interest to Xql2-Xql3.3. x x Quite recently the localization of the breakpoint in a girl with Menkes symptoms and an X,autosomal 22 [ translocation has been confined to the region Xql3.2-

21 [ Xq13.3 proximal to PKG1 (Verga et al. 1991). One E L754 likely explanation for the symptoms in this girl is a 11 pTAK- 8 destruction, by the translocation, of a functional CCp58 -1 11 _ Menkes gene on the active X chromosome. This result 12 E E CpX 203, cpX 289 is also in accordance with the present findings. 13 pSPT 19.1 [ The previous findings of a close linkage to the cen- 21[ tromeric region (Horn et al. 1984), as well as the ear- p19-2 q _I lier suggested close linkage to the PGK 22[ pXG -12 locus (Horn et 23 al. 1984) (a suggestion based on comparative gene 24 25 mapping between man and mouse), are also in 26 agreement with the present data. Our previous find- 27 ings of a localization on the proximal long arm of the 28 St 14-1 X chromosome, on the basis of a three-point linkage analysis in one Menkes family (T0nnesen et al. Figure 3 Combination of the results shown in the two panels 1986, of fig. 2. This presentation allows exclusion of certain regions of p. 627), are also in line with the present results. the X chromosome as candidate regions for the Menkes locus. From analyses of (a) a boy with Menkes disease and 1016 T0nnesen et al. a cisposition 46,XY,ins(X)(p1 1 .4q13.3q21.2) and Nussbaum RL, Schwartz M, Ropers H-H (1989) Physical (b) his mother, who has the same X chromosome in fine mapping of the choroideremia locus using Xq21 dele- a preferentially inactive state (Tumer et al., in press), tions associated with complex syndromes. Genomics 4: we can conclude that the Menkes locus is distal to 41-46 the X-inactivation center (XIC) localized in the Xql3 Cremers FPM, van de Pol TJR, Wieringa B, Hofker MH, region (Brown et al. 1991). When this result is com- Pearson PL, Pfeiffer RA, Mikkelsen M, et al (1988) Mo- lecular analysis of male-viable deletions and duplications bined with the results obtained by Verga et al. (1991 ), allows ordering of 52 DNA probes on proximal Xq. Am the Menkes locus should be located distal to XIC and J Hum Genet 43:452-461 proximal to PGK1. The present results support this Danks DM, Campbell PE, Stevens BJ, Mayne V, Cartwright conclusion. E (1972) Menke's kinky hair syndrome: an inherited de- The present results open the possibility of using fect in copper absorption with widespread effects. Pediat- DNA marker analysis for heterozygote detection and rics 50:188-201 prenatal diagnosis. Especially for carrier testing, such Feinberg AP, Vogelstein B (1983) A technique for radiola- an approach would be very valuable. Because of the beling DNA restriction endonuclease fragments to high random X inactivation, carriership cannot be ex- activity. Anal Biochem 132:6-13 cluded as a normal result of the biochemical analysis. Gerdes A-M, T0nnesen T, Horn N, Grisar T, Marg W, Muller A, Reinsch R, et al (1990) Clinical expression of Exclusion will, however, be possible on the basis of Menkes syndrome in females. Clin Genet 38:452-459 DNA analyses. If flanking DNA markers-such as Horn N, Stene J, M0llekxr A-M, Friedrich U (1984) Link- PGK1 and pX65H7(DXS72) on the distal side of the age studies in Menkes' disease: the Xg blood group system Menkes locus and cpX203, cpX289, and p8 on the and C-banding of the X-chromosome. Ann Hum Genet proximal side of the Menkes locus-are used, then 48:161-172 only double recombination events would lead to mis- Kapur S, Higgins JV, Delp K, Rogers B (1987) Menkes classification. As the markers mentioned above are syndrome in a girl with X-autosome translocation. Am J located within a region that is less than 20 cM, the Med Genet 26:503-510 risk of misclassification will be less than 1%. Even if Lathrop GM, Lalouel JM (1984) Easy calculations of lod -pDP34 is used as a distal marker, the error frequency scores and genetic risks on small computers. Am J Hum be less than 2%. Genet 36:460-465 would Lathrop GM, Lalouel JM, Julier JM, OttJ (1984) Strategies for multipoint linkage analysis in humans. Proc Natl Acad Sci USA 81:3443-3446 Acknowledgments Merry DE, Lesko JG, Sosnoski DM, Lewis RA, Lubinsky M, Trask B, van den Engh G, et al (1989) Choroideremia The authors are indebted to Drs. G. A. Bruns, P. Szabo, and deafness with stapes fixation: a contiguous gene dele- J. L. Mandel, B. Bakker, M. Hofker, and J. Singer-Sam for tion syndrome in Xq21. Am J Hum Genet 45:530-540 supplying DNA probes. We are furthermore indebted to Schwartz M, Yang H-M, Niebuhr E, Rosenberg T, Page DC the numerous colleagues who have referred their Menkes (1988) Regional localization of polymorphic DNA loci families to us. We are indebted to Danish BioBase for access on the proximal long arm of the X chromosome using to computing facilities. We thank J. Flindt and H. Heilstrup deletions associated with chroroideremia. Hum Genet 78: for technical assistance. This work was supported by Danish 156-160 Medical Research Council grants 12-7414, 5.17.4.2.53, Tonnesen T, Gerdes A-M, Damsgaard E, Miny P, Holz- 12-9292, and 12-9744), by Danish Biotechnological Re- greve W, Sondergaard F, Horn N (1989) First-trimester search and Developmental Program grant 5.18.03, and by diagnosis of Menkes disease: intermediate copper values the Foundation of 1870. Part of this work was presented at in chorionic villi from three affected male fetuses. J Prenat Human Gene Mapping 11, August 19-22,1991, in London. Diagn 9:159-165 Tonnesen T, Gerdes A-M, Horn N, Friedrich U, Grisar T, Muller A (1986) Localization of the Menkes gene to the References long arm of the X-chromosome. In: Proceedings of the Seventh International Congress on Human Genetics. Brown CJ, Lafreniere RG, Powers VE, Sebastio G, Ballabio Berlin A, Pettigrew AL, Ledbetter DH, et al (1991) Localization Tonnesen T, Kleijer WM, Horn N (1991) Incidence of of the X inactivation centre on the human X chromosome Menkes disease. Hum Genet 86:408-410 in Xq13. Nature 349:82-84 Tumer Z, Tommerup N, Tonnesen T, Kreuder J, Horn N. Cremers FPM, van de Pol TJR, Diergaarde Pj, Wieringa B, Mapping of the Menkes locus to Xql3.3 distal to the Localization of the Menkes Locus 1017

X-inactivation center by an intrachromosomal insertion Wienker TF, Wieacker P, Cooke HJ, Horn N, Ropers HH of the segment Xq13.3-21.2. Hum Genet (in press) (1983) Evidence that the Menkes locus maps on proximal Verga V, Hall BK, Wang S, Johnson S, Higgins JV, Glover Xp. Hum Genet 65:72-73 TW (1991) Localization of the translocation breakpoint Yang H-M, Lund T, Niebuhr E, Norby S, Schwartz M, Shen in a female with Menkes syndrome to Xql3.2-ql3.3 L (1990) Exclusion mapping of 12 X-linked disease loci proximal to PGK-1. Am J Hum Genet 48:1133-1138 and 10 DNA probes from the long arm of the X-chromo- Wieacker P, Horn N, Pearson P, Wienker TF, McKay E, some. Clin Genet 38:94-104 Ropers HH (1983) Menkes kinky hair disease: a search for closely linked restriction fragment length polymorphism. Hum Genet 64:139-142