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Palmoplantar Keratoderma in Association with Carcinoma of the Esophagus Maps to Chromosome 17q Distal to the Keratin Gene Cluster
SHORT COMMUNICATION Palmoplantar Keratoderma in Association with Carcinoma of the Esophagus Maps to Chromosome 17q Distal to the Keratin Gene Cluster HANS-CHRISTIAN HENNIES,* MANFRED HAGEDORN,†
AND
ANDRE´ REIS*,1
*Institute of Human Genetics, Virchow-Klinikum, Humboldt-Universita¨t zu Berlin, D-13353 Berlin, Germany; and †Department of Dermatology, Municipal Hospital, Darmstadt, Germany Received March 17, 1995; accepted July 17, 1995
Palmoplantar keratoderma is a group of hereditary disorders of keratinization involving hyperkeratosis of palms and soles. Two different forms of palmoplantar keratoderma have recently been shown to be caused by mutations in the body site-specific keratin 9 gene and in the keratin 1 gene, respectively. Now we have analyzed a large German family with autosomal dominantly inherited palmoplantar keratoderma in association with carcinoma of the esophagus. Linkage to both the type I keratin gene cluster on chromosome 17q and the type II keratin gene cluster on chromosome 12q could be excluded. In contrast, we mapped palmoplantar keratoderma in this family to chromosome 17q distal to the type I keratin genes. Two-point linkage data at D17S801 gave a lod score Zmax Å 5.1 at u Å 0.00. Therefore, palmoplantar keratoderma is shown to be heterogeneous clinically as well as genetically and may be caused by mutations in keratins as well as in nonkeratins. q 1995 Academic Press, Inc.
Palmoplantar keratoderma (PPK) is a common hereditary cutaneous disorder characterized by marked hyperkeratosis on the surface of palms and soles. Various clinically and histopathologically different types of PPK can be distinguished by the shape of the palmar and plantar keratosis, the age of onset of the disease, and the occurrence of any associated features. Two forms of PPK have recently been mapped to the type I and the type II keratin gene cluster on chromosome 17 and chromosome 12, respectively. We and others have identified various mutations in the gene for the body site-specific type I keratin 9 in epidermolytic PPK (2, 3, 10, 11, 18, 21), whereas a mutation in the type II keratin 1 gene was identified in a nonepidermolytic form of PPK (15). Here we present a large German family with a clinically distinct form of PPK associated with carcinoma of the esophagus (MIM (17) No. 1 To whom correspondence should be addressed at the Institute of Human Genetics, Virchow-Klinikum, Humboldt-Universita¨t zu Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany. Telephone: /49-30-450 66113. Fax: /49-30-450-66904.
148500). To investigate whether the underlying mutation for the type of PPK in this family could also be in a keratin gene, we analyzed polymorphisms within a number of keratins as well as microsatellites at anonymous loci in the chromosomal regions of keratin genes on chromosomes 12q and 17q. After excluding both type I and type II keratin genes as candidate genes, we identified linkage of PPK associated with esophageal cancer in this family to a region on chromosome 17q distal to the type I keratin gene cluster. This localization confirms a recent report of linkage in one large English family from Liverpool with tylosis and esophageal cancer to chromosome 17q23–qter (19). The family is a four-generation family from a formerly isolated Black Forest valley with 43 members; 19 of these are affected by PPK. Twenty-four family members, 12 of them affected, were investigated in this study (Fig. 1). The patients show a diffuse hyperkeratosis of palms and soles with a late onset. Plantar hyperkeratosis became evident between the ages of 8 and 15 years, whereas manifestation of palmar hyperkeratosis was later, between 14 and 25 years of age. Histopathological investigations show marked orthokeratotic hyperkeratosis and acanthosis but no signs of keratolysis. Five patients from the family have died; two of these, individuals II,2 and III,4 (Fig. 1), died from esophageal cancer. The carcinoma was first diagnosed at 56 and 48 years of age, respectively. Further features of the syndrome in this family are congenital oral and esophageal leukoplakia (Fig. 1) and a follicular keratosis, especially on the upper arms and thighs (7). One member of the family, proband III,4, was first described in 1978 (8). The disease is inherited autosomal dominantly with complete penetrance. Polymorphisms within the following keratin genes were analyzed to assess them directly for linkage: polymorphisms in the genes for type II keratins 2e and 4 and type I keratin 10 were investigated by PCR and subsequent SSCP analysis as described elsewhere (12). Microsatellite polymorphisms were investigated as described (13) but either with radioactive end-labeling of one primer using [g-32P]ATP and T4 polynucleotide GENOMICS
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FIG. 1. Pedigree of the family with palmoplantar keratoderma associated with esophageal cancer that was investigated and likely haplotypes at microsatellite loci on chromosome 17q between D17S948 and D17S802. Individuals II,2 and III,4 died from esophageal cancer. Symbols are as follows: filled, affected proband with PPK and esophageal cancer; one-quarter open, affected proband with PPK and leukoplakia; half open, affected proband with PPK; open symbol, unaffected. In probands IV,7 and IV,8, the phase at D17S939 in parents is unknown, and haplotypes were deduced from the results from other family members. The boxed haplotype completely cosegregates with the PPK phenotype.
kinase (20) or using one fluorescein-labeled primer. Radioactive PCR products were separated on 6% polyacrylamide sequencing gels; fluorescent PCR products were loaded on 6.6% polyacrylamide gels and analyzed in an automated DNA sequencer (A.L.F., Pharmacia). Microsatellites at D12S59, D12S64 (4), D12S96 (23), and D17S802 (6) were amplified with an initial denaturation step for 7 min at 947C followed by 27 cycles of 10 s denaturation at 947C, 10 s annealing at 577C (D12S59, D12S64), 537C (D12S96), or 597C (D17S802), and 10 s elongation at 727C and a final extension for 10
min at 727C in a thermal cycler 9600 (Perkin–Elmer). Microsatellites at D17S250 (22), D17S579 (9), D17S784, D17S785, D17S801, D17S807, D17S929, D17S939, and D17S949 (6) were amplified for 27 cycles of 1 min at 947C, 1 min at the annealing temperature, and 1.5 min at 727C in a thermal cycler 480 (Perkin– Elmer). The annealing temperatures were as follows: D17S250, 537C; D17S579, 557C; D17S784, 547C; D17S801, 577C; D17S785 and D17S807, 527C; D17S929, 587C; D17S939, 597C; D17S949, 507C. Two-point linkage data between each marker locus
TABLE 1 Pairwise Lod Scores Using Two-Point Linkage Data between Markers in the Chromosomal Regions of Keratin Genes on Chromosomes 12 and 17 and PPK Associated with Esophageal Cancer in One Family from Germany Pairwise lod scores at u of Locus
0.00
0.01
0.05
0.10
0.20
0.30
0.40
KRT2E KRT4 D12S59 D12S96 D12S64
0` 0` 0` 0` 0`
04.06 01.43 010.85 09.63 07.06
01.99 00.70 05.47 04.91 03.74
01.15 00.40 03.32 03.01 02.45
00.43 00.14 01.49 01.36 01.34
00.13 00.05 00.69 00.61 00.76
00.01 00.01 00.28 00.23 00.35
KRT10 D17S250 D17S579
0` 0` 0`
02.69 02.71 04.23
01.28 01.27 01.49
00.72 00.69 00.47
00.28 00.25 /0.17
00.12 00.12 /0.18
00.06 00.06 0.00
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FIG. 2. Genetic map of markers on part of chromosome 17q (1, 6). The approximate genetic distance between markers is given in cM (numbers below the line). A locus for palmoplantar keratoderma associated with carcinoma of the esophagus maps to the interval between D17S929 and D17S802 approximately 45 cM distal to the keratin gene cluster.
and the PPK phenotype were used for calculation of lod scores with the LINKAGE (version 5.10) computer programs (16). Based on the segregation patterns found in this family (Fig. 1) we assumed autosomal dominant inheritance of the PPK with full penetrance. In addition, the observation of esophageal cancer in two consecutive generations and of oral and esophageal leukoplakia in the second and third generation argues against the possibility of a phenocopy. Linkage analyses at the loci of keratin genes KRT2E, KRT4, and KRT10 showed recombination events at each locus clearly excluding these genes as candidate genes for the disease (Table 1). Moreover, analysis of microsatellites flanking the keratin gene loci revealed an exclusion of the whole chromosomal regions containing the type I and the type II keratin genes (Table 1). All of the known keratin genes map to the keratin gene clusters on chromosomes 12 and 17. Therefore, by direct and indirect analysis, these results show that the defect in the family presented here does not reside in a keratin. In contrast, positive lod scores were obtained testing further microsatellite markers on chromosome 17q distal to the keratin gene cluster (Fig. 2 and Table 2). A maximum lod score of Zmax Å 5.06 at u Å 0.00 was calculated at D17S801 located 45 cM distal to KRT10 (1, 6). No recombination was observed at loci D17S801, D17S785, and D17S939 (Table 2). The genotypes at several chromosome 17 markers were
aligned to likely haplotypes (Fig. 1). Recombination events in probands III,8, III,10, and III,12 clearly define a 7-cM interval between D17S929 and D17S802 (6) containing a gene that is responsible for the manifestation of PPK with esophageal cancer (Figs. 1 and 2). An association of PPK with carcinoma of the esophagus was first published by Howel-Evans et al. in 1958 (14). One of the families from Liverpool described there has recently been reviewed (5), and linkage of the disease to chromosome 17q23–qter was identified in that family with a maximum lod score at D17S515 (Zmax Å 6.2 at u Å 0.10) and D17S785 (Zmax Å 6.0 at u Å 0.02) (19). PPK in the Liverpool families also shows late manifestation, i.e., at puberty, but the mean age of onset of esophageal cancer is 45 years as reported by Howel-Evans et al. In contrast, the carcinoma was first diagnosed in the German family described here at 48 and 56 years of age, respectively. Only 2 of 10 patients over 50 years of age have developed esophageal cancer in the German family compared to 21 from 32 deceased probands in the one from Liverpool. However, our linkage data are in accordance with those in the Liverpool family, indicating that the same gene is probably affected in both families. A different mutation in that gene might be responsible for the slightly divergent clinical severity of the disease. Several disorders of keratinization have now been shown to be caused by mutations in keratin genes. Par-
TABLE 2 Pairwise Lod Scores Using Two-Point Linkage Data between Markers on Chromosome 17q and PPK Associated with Esophageal Cancer in One Family from Germany Pairwise lod scores at u of Locus
0.00
0.01
0.05
0.10
0.20
0.30
0.40
D17S807 D17S949 D17S929 D17S801 D17S785 D17S939 D17S802 D17S784
0` 0` 0` /5.06 /4.35 /3.18 0` 0`
00.49 /1.41 /1.74 /5.03 /4.28 /3.20 00.19 04.85
/0.80 /1.85 /2.13 /4.81 /3.98 /3.20 /1.01 02.17
/1.20 /1.82 /2.02 /4.40 /3.59 /3.04 /1.34 01.13
/1.31 /1.46 /1.46 /3.38 /2.76 /2.48 /1.36 00.27
/1.06 /0.94 /0.75 /2.16 /1.86 /1.70 /1.07 /0.05
/0.61 /0.39 /0.11 /0.84 /0.91 /0.78 /0.61 /0.11
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ticularly, different forms of PPK have been mapped to the keratin gene clusters, and mutations in keratins have been identified (2, 3, 10, 11, 15, 18, 21). However, our results give further evidence that PPK is heterogeneous clinically as well as on the molecular level and might also be caused by mutations in nonkeratins. A candidate gene for PPK with esophageal cancer must be expressed in both cornifying and wet stratified epithelia in contrast to other genes involved in PPK identified so far. To our knowledge, no obvious candidate gene has yet been mapped to the region in question on chromosome 17q. Besides, it remains puzzling why this PPK manifests only relatively late in life in comparison with other PPK forms. Recently, we have identified linkage of the striated form of PPK to a region on chromosome 18q12 containing a desmosomal cadherin gene cluster (12). The identification of linkage to chromosome 17q distal to the keratin gene cluster in a family with PPK associated with carcinoma of the esophagus now reveals that mutations in quite a few different genes might cause hyperkeratosis of palms and soles. Therefore, various forms of PPK that can be distinguished clinically and histopathologically actually exhibit distinct, nonallelic, cutaneous diseases.
We are thankful to all of the family members who participated in this investigation. We thank Ralf Eckhardt, Fabienne Trotier, and Silke Woweries for excellent technical assistance. Dr. Dietmar Mischke kindly performed the analysis of keratin gene polymorphisms. This work was supported by the Deutsche Forschungsgemeinschaft and the Maria Sonnenfeld-Geda¨chtnis-Stiftung.
9.
10.
11.
12.
13.
14.
16.
17. 18.
REFERENCES 1. Anderson, L. A., Friedman, L., Osborne Lawrence, S., Lynch, E., Weissenbach, J., Bowcock, A., and King, M. C. (1993). Highdensity genetic map of the BRCA1 region of chromosome 17q12–q21. Genomics 17: 618–623. 2. Bonifas, J. M., Matsumura, K., Chen, M. A., Berth-Jones, J., Hutchinson, P. E., Zloczower, M., Fritsch, P. O., and Epstein, E. H. (1994). Mutations of keratin 9 in two families with palmoplantar epidermolytic hyperkeratosis. J. Invest. Dermatol. 103: 474–477. 3. Bowden, P. E., Watts, C. E., and Marks, R. (1994). Mutations in adjacent codons of human keratin 9 (HK9) gene found in EPPK patients. Br. J. Dermatol. 131: 425. 4. Dawson, E., Shaikh, S., Weber, J. L., Wang, Z., Weissenbach, J., Powell, J. F., and Gill, M. (1993). A continuous linkage map of 22 short tandem repeat polymorphisms on human chromosome 12. Genomics 17: 245–248. 5. Ellis, A., Field, J. K., Field, E. A., Friedmann, P. S., Fryer, A., Howard, P., Leigh, I. M., Risk, J., Shaw, J. M., and Whittaker, J. (1994). Tylosis associated with carcinoma of the oesophagus and oral leukoplakia in a large Liverpool family—A review of six generations. Eur. J. Cancer Oral Oncol. 30B: 102–112. 6. Gyapay, G., Morissette, J., Vignal, A., Dib, C., Fizames, C., Millasseau, P., Marc, S., Bernardi, G., Lathrop, M., and Weis-
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ACKNOWLEDGMENTS
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senbach, J. (1994). The 1993–94 Ge´ne´thon human genetic linkage map. Nature Genet. 7: 246–339. Hagedorn, M. (1983). Angeborene Tylosis palmaris et plantaris. Hautarzt 34: 81–83. Hagedorn, M., and Thomas, C. (1978). Tylosis Palmaris et Plantaris. Akt. Dermatol. 4: 245–248. Hall, J. M., Friedman, L., Guenther, C., Lee, M. K., Weber, J. L., Black, D. M., and King, M. C. (1992). Closing in on a breast cancer gene on chromosome 17q. Am. J. Hum. Genet. 50: 1235–1242. Hennies, H. C., Zehender, D., Kunze, J., Ku¨ster, W., and Reis, A. (1994). Keratin 9 gene mutational heterogeneity in patients with epidermolytic palmoplantar keratoderma. Hum. Genet. 93: 649–654. Hennies, H. C., Ku¨ster, W., Mischke, D., Hagedorn, M., Patton, M. A., and Reis, A. (1995). Genetic heterogeneity and mutational variation in palmoplantar keratoderma (PPK). Arch. Dermatol. Res., in press. Hennies, H. C., Ku¨ster, W., Mischke, D., and Reis, A. (1995). Localization of a locus for the striated form of palmoplantar keratoderma to chromosome 18q near the desmosomal cadherin gene cluster. Hum. Mol. Genet. 4: 1015–1020. Hennies, H. C., and Reis, A. (1993). Three dinucleotide microsatellite polymorphisms on human chromosome 13. Hum. Mol. Genet. 2: 87. Howel-Evans, W., McConnell, R. B., Clarke, C. A., and Sheppard, P. M. (1958). Carcinoma of the oesophagus with keratosis palmaris et plantaris (tylosis): A study of two families. Quart. J. Med. 27: 413–429. Kimonis, V., DiGiovanna, J. J., Yang, J.-M., Doyle, S. Z., Bale, S. J., and Compton, J. G. (1994). A mutation in the V1 end domain of keratin 1 in non-epidermolytic palmar–plantar keratoderma. J. Invest. Dermatol. 103: 764–769. Lathrop, G. M., Lalouel, J. M., Julier, C., and Ott, J. (1984). Strategies for multilocus linkage analysis in humans. Proc. Natl. Acad. Sci. USA 81: 3443–3446. McKusick, V. A. (1994). ‘‘Mendelian Inheritance in Man,’’ 11th ed., Johns Hopkins Univ. Press, Baltimore. Reis, A., Hennies, H. C., Langbein, L., Digweed, M., Mischke, D., Drechsler, M., Schro¨ck, E., Royer-Pokora, B., Franke, W. W., Sperling, K., and Ku¨ster, W. (1994). Keratin 9 gene mutations in epidermolytic palmoplantar keratoderma (EPPK). Nature Genet. 6: 174–179. Risk, J. M., Field, E. A., Field, J. K., Whittaker, J., Fryer, A., Ellis, A., Shaw, J. M., Friedman, P. S., Bishop, D. T., Bodmer, J., and Leigh, I. M. (1994). Tylosis oesophageal cancer mapped. Nature Genet. 8: 319–321. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). ‘‘Molecular cloning: A Laboratory Manual,’’ 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Torchard, D., Blanchet Bardon, C., Serova, O., Langbein, L., Narod, S., Janin, N., Goguel, A. F., Bernheim, A., Franke, W. W., Lenoir, G. M., and Feunteun, J. (1994). Epidermolytic palmoplantar keratoderma cosegregates with a keratin 9 mutation in a pedigree with breast and ovarian cancer. Nature Genet. 6: 106–110. Weber, J. L., Kwitek, A. E., May, P. E., Wallace, M. R., Collins, F. S., and Ledbetter, D. H. (1990). Dinucleotide repeat polymorphisms at the D17S250 and D17S261 loci. Nucleic Acids Res. 18: 4640. Weissenbach, J., Gyapay, G., Dib, C., Vignal, A., Morissette, J., Millasseau, P., Vaysseix, G., and Lathrop, M. (1992). A secondgeneration linkage map of the human genome. Nature 359: 794–801.
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ANDRE´ REIS*,1
*Institute of Human Genetics, Virchow-Klinikum, Humboldt-Universita¨t zu Berlin, D-13353 Berlin, Germany; and †Department of Dermatology, Municipal Hospital, Darmstadt, Germany Received March 17, 1995; accepted July 17, 1995
Palmoplantar keratoderma is a group of hereditary disorders of keratinization involving hyperkeratosis of palms and soles. Two different forms of palmoplantar keratoderma have recently been shown to be caused by mutations in the body site-specific keratin 9 gene and in the keratin 1 gene, respectively. Now we have analyzed a large German family with autosomal dominantly inherited palmoplantar keratoderma in association with carcinoma of the esophagus. Linkage to both the type I keratin gene cluster on chromosome 17q and the type II keratin gene cluster on chromosome 12q could be excluded. In contrast, we mapped palmoplantar keratoderma in this family to chromosome 17q distal to the type I keratin genes. Two-point linkage data at D17S801 gave a lod score Zmax Å 5.1 at u Å 0.00. Therefore, palmoplantar keratoderma is shown to be heterogeneous clinically as well as genetically and may be caused by mutations in keratins as well as in nonkeratins. q 1995 Academic Press, Inc.
Palmoplantar keratoderma (PPK) is a common hereditary cutaneous disorder characterized by marked hyperkeratosis on the surface of palms and soles. Various clinically and histopathologically different types of PPK can be distinguished by the shape of the palmar and plantar keratosis, the age of onset of the disease, and the occurrence of any associated features. Two forms of PPK have recently been mapped to the type I and the type II keratin gene cluster on chromosome 17 and chromosome 12, respectively. We and others have identified various mutations in the gene for the body site-specific type I keratin 9 in epidermolytic PPK (2, 3, 10, 11, 18, 21), whereas a mutation in the type II keratin 1 gene was identified in a nonepidermolytic form of PPK (15). Here we present a large German family with a clinically distinct form of PPK associated with carcinoma of the esophagus (MIM (17) No. 1 To whom correspondence should be addressed at the Institute of Human Genetics, Virchow-Klinikum, Humboldt-Universita¨t zu Berlin, Augustenburger Platz 1, D-13353 Berlin, Germany. Telephone: /49-30-450 66113. Fax: /49-30-450-66904.
148500). To investigate whether the underlying mutation for the type of PPK in this family could also be in a keratin gene, we analyzed polymorphisms within a number of keratins as well as microsatellites at anonymous loci in the chromosomal regions of keratin genes on chromosomes 12q and 17q. After excluding both type I and type II keratin genes as candidate genes, we identified linkage of PPK associated with esophageal cancer in this family to a region on chromosome 17q distal to the type I keratin gene cluster. This localization confirms a recent report of linkage in one large English family from Liverpool with tylosis and esophageal cancer to chromosome 17q23–qter (19). The family is a four-generation family from a formerly isolated Black Forest valley with 43 members; 19 of these are affected by PPK. Twenty-four family members, 12 of them affected, were investigated in this study (Fig. 1). The patients show a diffuse hyperkeratosis of palms and soles with a late onset. Plantar hyperkeratosis became evident between the ages of 8 and 15 years, whereas manifestation of palmar hyperkeratosis was later, between 14 and 25 years of age. Histopathological investigations show marked orthokeratotic hyperkeratosis and acanthosis but no signs of keratolysis. Five patients from the family have died; two of these, individuals II,2 and III,4 (Fig. 1), died from esophageal cancer. The carcinoma was first diagnosed at 56 and 48 years of age, respectively. Further features of the syndrome in this family are congenital oral and esophageal leukoplakia (Fig. 1) and a follicular keratosis, especially on the upper arms and thighs (7). One member of the family, proband III,4, was first described in 1978 (8). The disease is inherited autosomal dominantly with complete penetrance. Polymorphisms within the following keratin genes were analyzed to assess them directly for linkage: polymorphisms in the genes for type II keratins 2e and 4 and type I keratin 10 were investigated by PCR and subsequent SSCP analysis as described elsewhere (12). Microsatellite polymorphisms were investigated as described (13) but either with radioactive end-labeling of one primer using [g-32P]ATP and T4 polynucleotide GENOMICS
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0888-7543/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Pedigree of the family with palmoplantar keratoderma associated with esophageal cancer that was investigated and likely haplotypes at microsatellite loci on chromosome 17q between D17S948 and D17S802. Individuals II,2 and III,4 died from esophageal cancer. Symbols are as follows: filled, affected proband with PPK and esophageal cancer; one-quarter open, affected proband with PPK and leukoplakia; half open, affected proband with PPK; open symbol, unaffected. In probands IV,7 and IV,8, the phase at D17S939 in parents is unknown, and haplotypes were deduced from the results from other family members. The boxed haplotype completely cosegregates with the PPK phenotype.
kinase (20) or using one fluorescein-labeled primer. Radioactive PCR products were separated on 6% polyacrylamide sequencing gels; fluorescent PCR products were loaded on 6.6% polyacrylamide gels and analyzed in an automated DNA sequencer (A.L.F., Pharmacia). Microsatellites at D12S59, D12S64 (4), D12S96 (23), and D17S802 (6) were amplified with an initial denaturation step for 7 min at 947C followed by 27 cycles of 10 s denaturation at 947C, 10 s annealing at 577C (D12S59, D12S64), 537C (D12S96), or 597C (D17S802), and 10 s elongation at 727C and a final extension for 10
min at 727C in a thermal cycler 9600 (Perkin–Elmer). Microsatellites at D17S250 (22), D17S579 (9), D17S784, D17S785, D17S801, D17S807, D17S929, D17S939, and D17S949 (6) were amplified for 27 cycles of 1 min at 947C, 1 min at the annealing temperature, and 1.5 min at 727C in a thermal cycler 480 (Perkin– Elmer). The annealing temperatures were as follows: D17S250, 537C; D17S579, 557C; D17S784, 547C; D17S801, 577C; D17S785 and D17S807, 527C; D17S929, 587C; D17S939, 597C; D17S949, 507C. Two-point linkage data between each marker locus
TABLE 1 Pairwise Lod Scores Using Two-Point Linkage Data between Markers in the Chromosomal Regions of Keratin Genes on Chromosomes 12 and 17 and PPK Associated with Esophageal Cancer in One Family from Germany Pairwise lod scores at u of Locus
0.00
0.01
0.05
0.10
0.20
0.30
0.40
KRT2E KRT4 D12S59 D12S96 D12S64
0` 0` 0` 0` 0`
04.06 01.43 010.85 09.63 07.06
01.99 00.70 05.47 04.91 03.74
01.15 00.40 03.32 03.01 02.45
00.43 00.14 01.49 01.36 01.34
00.13 00.05 00.69 00.61 00.76
00.01 00.01 00.28 00.23 00.35
KRT10 D17S250 D17S579
0` 0` 0`
02.69 02.71 04.23
01.28 01.27 01.49
00.72 00.69 00.47
00.28 00.25 /0.17
00.12 00.12 /0.18
00.06 00.06 0.00
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FIG. 2. Genetic map of markers on part of chromosome 17q (1, 6). The approximate genetic distance between markers is given in cM (numbers below the line). A locus for palmoplantar keratoderma associated with carcinoma of the esophagus maps to the interval between D17S929 and D17S802 approximately 45 cM distal to the keratin gene cluster.
and the PPK phenotype were used for calculation of lod scores with the LINKAGE (version 5.10) computer programs (16). Based on the segregation patterns found in this family (Fig. 1) we assumed autosomal dominant inheritance of the PPK with full penetrance. In addition, the observation of esophageal cancer in two consecutive generations and of oral and esophageal leukoplakia in the second and third generation argues against the possibility of a phenocopy. Linkage analyses at the loci of keratin genes KRT2E, KRT4, and KRT10 showed recombination events at each locus clearly excluding these genes as candidate genes for the disease (Table 1). Moreover, analysis of microsatellites flanking the keratin gene loci revealed an exclusion of the whole chromosomal regions containing the type I and the type II keratin genes (Table 1). All of the known keratin genes map to the keratin gene clusters on chromosomes 12 and 17. Therefore, by direct and indirect analysis, these results show that the defect in the family presented here does not reside in a keratin. In contrast, positive lod scores were obtained testing further microsatellite markers on chromosome 17q distal to the keratin gene cluster (Fig. 2 and Table 2). A maximum lod score of Zmax Å 5.06 at u Å 0.00 was calculated at D17S801 located 45 cM distal to KRT10 (1, 6). No recombination was observed at loci D17S801, D17S785, and D17S939 (Table 2). The genotypes at several chromosome 17 markers were
aligned to likely haplotypes (Fig. 1). Recombination events in probands III,8, III,10, and III,12 clearly define a 7-cM interval between D17S929 and D17S802 (6) containing a gene that is responsible for the manifestation of PPK with esophageal cancer (Figs. 1 and 2). An association of PPK with carcinoma of the esophagus was first published by Howel-Evans et al. in 1958 (14). One of the families from Liverpool described there has recently been reviewed (5), and linkage of the disease to chromosome 17q23–qter was identified in that family with a maximum lod score at D17S515 (Zmax Å 6.2 at u Å 0.10) and D17S785 (Zmax Å 6.0 at u Å 0.02) (19). PPK in the Liverpool families also shows late manifestation, i.e., at puberty, but the mean age of onset of esophageal cancer is 45 years as reported by Howel-Evans et al. In contrast, the carcinoma was first diagnosed in the German family described here at 48 and 56 years of age, respectively. Only 2 of 10 patients over 50 years of age have developed esophageal cancer in the German family compared to 21 from 32 deceased probands in the one from Liverpool. However, our linkage data are in accordance with those in the Liverpool family, indicating that the same gene is probably affected in both families. A different mutation in that gene might be responsible for the slightly divergent clinical severity of the disease. Several disorders of keratinization have now been shown to be caused by mutations in keratin genes. Par-
TABLE 2 Pairwise Lod Scores Using Two-Point Linkage Data between Markers on Chromosome 17q and PPK Associated with Esophageal Cancer in One Family from Germany Pairwise lod scores at u of Locus
0.00
0.01
0.05
0.10
0.20
0.30
0.40
D17S807 D17S949 D17S929 D17S801 D17S785 D17S939 D17S802 D17S784
0` 0` 0` /5.06 /4.35 /3.18 0` 0`
00.49 /1.41 /1.74 /5.03 /4.28 /3.20 00.19 04.85
/0.80 /1.85 /2.13 /4.81 /3.98 /3.20 /1.01 02.17
/1.20 /1.82 /2.02 /4.40 /3.59 /3.04 /1.34 01.13
/1.31 /1.46 /1.46 /3.38 /2.76 /2.48 /1.36 00.27
/1.06 /0.94 /0.75 /2.16 /1.86 /1.70 /1.07 /0.05
/0.61 /0.39 /0.11 /0.84 /0.91 /0.78 /0.61 /0.11
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ticularly, different forms of PPK have been mapped to the keratin gene clusters, and mutations in keratins have been identified (2, 3, 10, 11, 15, 18, 21). However, our results give further evidence that PPK is heterogeneous clinically as well as on the molecular level and might also be caused by mutations in nonkeratins. A candidate gene for PPK with esophageal cancer must be expressed in both cornifying and wet stratified epithelia in contrast to other genes involved in PPK identified so far. To our knowledge, no obvious candidate gene has yet been mapped to the region in question on chromosome 17q. Besides, it remains puzzling why this PPK manifests only relatively late in life in comparison with other PPK forms. Recently, we have identified linkage of the striated form of PPK to a region on chromosome 18q12 containing a desmosomal cadherin gene cluster (12). The identification of linkage to chromosome 17q distal to the keratin gene cluster in a family with PPK associated with carcinoma of the esophagus now reveals that mutations in quite a few different genes might cause hyperkeratosis of palms and soles. Therefore, various forms of PPK that can be distinguished clinically and histopathologically actually exhibit distinct, nonallelic, cutaneous diseases.
We are thankful to all of the family members who participated in this investigation. We thank Ralf Eckhardt, Fabienne Trotier, and Silke Woweries for excellent technical assistance. Dr. Dietmar Mischke kindly performed the analysis of keratin gene polymorphisms. This work was supported by the Deutsche Forschungsgemeinschaft and the Maria Sonnenfeld-Geda¨chtnis-Stiftung.
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