TTC4, a Novel Candidate Tumor Suppressor Gene at 1P31 Is Often Mutated in Malignant Melanoma of the Skin

Oncogene (2000) 19, 5817 ± 5820 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc SHORT REPORT TTC4, a novel candidate tumor suppressor gene at 1p31 is often mutated in malignant melanoma of the skin Micaela Poetsch*,1, Thomas Dittberner2, John K Cowell4 and Christian Woenckhaus3 1Institute of Forensic Medicine, University of Greifswald, Germany; 2Department of Dermatology, University of Greifswald, Germany; 3Institute of Pathology, University of Greifswald, Germany; 4Center for Molecular Genetics, Cleveland Clinic Foundation, Ohio, USA A novel candidate tumor suppressor gene, TTC4,on (TPR) has been mapped in this part of the short arm chromosome 1p31 has been described recently. Since of chromosome 1 and named TTC4 (Su et al., 1999). A aberrations in this region have been detected in variety of other genes belong to this family and malignant melanoma, we investigated DNA of paran- dierent functions have been assigned to genes with embedded sections from 16 typical naevi, 19 atypical TPR motifs (Blatch and LaÈ ssle, 1999). The members of naevi, 32 primary melanomas (15 super®cial spreading the human TPR motif gene family include co- melanomas, 17 nodular melanomas) and 25 metastases chaperones like IEFSSP3521 (Honore et al., 1992) or and DNA from four melanoma cell lines by PCR and FKBPRr38 (Lam et al., 1995), genes with protein direct sequencing analysis for mutations in all exons of transport functions like PXR1 (Fransen et al., 1995), TTC4. Tumors comprised a wide range of thickness genes with importance in phosphate turnover like PP5 (Breslow index) and Clark levels. No mutations could be (Chen et al., 1994), and a gene with cell cycle control detected in typical or atypical naevi, but we found seven functions (Schrick et al., 1995). Very recently, addi- dierent point mutations in the tumor samples, six of tional genes with TPR motifs have been found which them causing an amino acid change. Ten melanoma demonstrate interactions with heat shock proteins or samples belonging to nine patients showed one or more are necessary for the formation of the phagocyte of these mutations. In detail, in six of 25 metastases, in NADPH oxidase (Ballinger et al., 1999; Koga et al., two of 17 nodular melanomas and in two of 15 1999; Zhang and Grishin, 1999). Evidence for an super®cial spreading melanomas point mutations could involvement of TPR motif genes in neoplasia is more be detected. In two cell lines, a loss of a whole exon dicult. Another member of the TPR motif family could be demonstrated and in one cell line we found a (Tg737) seems to be involved in liver tumorigenesis point mutation. In addition, three polymorphisms were (Isfort et al., 1997). Tg737 showed deletions or found. Our ®ndings indicate that TTC4 may participate rearrangements in 40% of carcinogen-induced liver in the pathogenesis of malignant melanomas of the skin. tumors in rats and Isfort et al. (1997) were able to Oncogene (2000) 19, 5817 ± 5820. detect similar changes in human liver, kidney and pancreas tumors. In addition, they demonstrated a Keywords: TTC4; malignant melanoma; sequencing suppression of the tumor forming ability in nude mice analysis by overexpressing Tg737 in cells otherwise lacking this gene. These facts and the localization of TTC4 within a region of interest in certain cancer initiated our study of DNA from paran embedded tissue and corre- Deletions of the short arm of chromosome 1 are sponding constitutional DNA from 16 typical naevi, 19 among the most frequent aberrations in many solid atypical naevi, 15 super®cial spreading melanomas, 17 tumors including neuroblastoma, breast cancer and nodular malignant melanomas and 25 metastatic malignant melanoma of the skin (Heim and Mitelman, melanomas from 48 non-selected melanoma patients 1995). Although Dracopoli et al. (1989) demonstrated a comprising a wide range of thickness (Breslow index) deletion especially in the subterminal region of 1p by and Clark levels which were staged and classi®ed as loss of heterocygosity analysis (LOH), cytogenetic stated elsewhere (Poetsch et al., 1998). Six patients had analysis and comparative genomic hybridization ana- multiple metastatic lesions. DNA isolation from lysis (CGH) found chromosomal loss in all parts of 1p paran embedded tissue was performed as described in malignant melanoma (Thompson et al., 1995; before (Poetsch et al., 2000). In addition, the four Wiltshire et al., 1995). In a recent ¯uorescence in situ melanoma cell lines M19, DX3 (Albino et al., 1981), hybridization (FISH) study with YAC DNA probes, MEWO (Siracki et al., 1982), and LT5.1 have been we were able to demonstrate deletions not only in included in this study. Using PCR and direct 1p36, but also in 1p31 (Poetsch et al., 1999). Lately, a sequencing analysis we screened for mutations in exons novel human gene with the tetratricopeptide repeat 1 through 10 of TTC4. In the constitutional DNA of all melanoma patients, in all 35 naevi and in all 57 investigated melanoma samples the 10 exons of TTC4 could be ampli®ed; this is, we did not ®nd any *Correspondence: M Poetsch, Institute of Forensic Medicine, Ernst homozygous deletions of the complete gene of a whole Moritz Arndt-University, Kuhstrasse 30, D-17489 Greifswald, Germany exon in these samples, but in the cell line LT5.1 exon 1 Received 1 April 2000; revised 21 September 2000; accepted 25 and in M19 exon 3 could not be ampli®ed, while the September 2000 remaining exons are intact. TTC4 mutations in melanoma M Poetsch et al 5818 No mutations could be found in any of the typical or paran embedded material which besides having atypical naevi, but seven point mutations could be dierently ®xed comprised archive material ± up to 10 detected among our tumor samples (Table 1), which years old ± where RNA is mostly degraded. In did not appear in the constitutional DNA isolated addition, no antibody for the TTC4 protein was from the blood of the patients. Therefore, these available for us thus restricting our analysis to the aberrations prove to be real mutations and not rare DNA of this gene. polymorphisms. Six of these mutations led to an amino Two of our seven mutations may have greater acid change. They were distributed over the exons 1 ± 6 importance than the others: the A?C change in codon and appeared with one exception only once. An A?C 77 occurring in ®ve tumor samples of four unrelated change in exon 3 could be demonstrated in ®ve patients results in an amino acid change from glutamic dierent tumor samples belonging to four unrelated acid to alanine (Figure 1a). It lies in the near patients (Figure 1a). In addition, in the cell line neighborhood of the ®rst potential TPR repeat motif MEWO a point mutation in exon 6 (codon 201) could and its appearance in three metastases, one SSM, and be detected, which resulted in a change from glutamine one NM hints at a possible signi®cance of this to alanine. mutation in melanoma. Since the chemical dierence The mutations appeared in six metastases (24%), two between glutamic acid and alanine is rather great, this nodular melanomas (12%), and two super®cial spread- mutation will possibly lead to a conformation change ing melanomas (13%), with one metastasis showing two of the protein implicating a reduction or loss of protein dierent mutations. In all metastases and in one nodular function. The potential importance of a second melanoma (NM47) only the mutated allele was present, mutation ± the C?T change in codon 135 ± derived indicating loss of heterozygosity in this region. Two of from its location in the gene: it resides in the middle of these tumor samples (MM11, MM14) belonging to one the second potential TRP repeat (Figure 1b). This patient displayed a deletion in 1p31 in our prior FISH mutation results in the amino acid phenylalanine study (Poetsch et al., 1999), in two melanomas no such instead of serine and occurred only in the metastasis deletion could be detected, the other six tumor samples MM11. Phenylalanine with its hydrophobic structure have not been included in the FISH study. At the in comparison to the hydrophilic serine may disturb moment we can not show a correlation between Breslow folding of the TRP repeat thus preventing the active index or Clark level and the mutations. In addition, no centrum of the protein from right function. relation between the occurrence of multiple metastatic In addition to the above mentioned mutations we lesions or survival after diagnosis and a mutation in found a variety of alterations referring to the published TTC4 could be demonstrated which might be due to the sequence (Su et al., 1999) in tumor DNA and the limited case number. However, the mutations occurred corresponding constitutional DNA. Three of them more often in metastasis and nodular melanomas, could be clearly de®ned as polymorphism (in exon 2, known to harbor a worse prognosis. We could not exon 5, and intron 8), since they occurred in normal analyse the expression of TTC4 mRNA in our tumor controls, in the naevi, and in the melanoma patients samples due to the fact that we investigated samples of with dierent heterozygosity rates (Table 2). The most Table 1 TTC4 mutations in SSM, NM and metastases Clark level Breslow index only primary Tumor sample Mutationa Exon/intron Codon Predicted effect del(1)(p31)b tumors Survival months after diagnosis SSM32c 230A?C exon 3 77 Glu?Ala n.d. I 0.5 mm 40, NEDd SSM55 469G?A exon 5 157 Gly?Asp n.d. II 0.7 mm 10, NED NM42 230A?C exon 3 77 Glu?Ala n.d. IV 5 mm 79, NED NM47 125T?A exon 2 42 Val?Asp n.d.

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