Missense Mutations of MADH4: Characterization of the Mutational Hot Spot and Functional Consequences in Human Tumors

Vol. 10, 1597–1604, March 1, 2004 Clinical Cancer Research 1597

Featured Article Missense Mutations of MADH4: Characterization of the Mutational Hot Spot and Functional Consequences in Human Tumors

Christine A. Iacobuzio-Donahue,1 Jason Song,5 Introduction Giovanni Parmiagiani,4 Charles J. Yeo,2,3 Human pancreatic ductal adenocarcinomas inactivate the Ralph H. Hruban,1,2 and Scott E. Kern2 tumor suppressor gene MADH4 (DPC4, SMAD4) with a high frequency (1). This inactivation occurs most commonly by Departments of 1Pathology, 2Oncology, 3Surgery, and 4Public Health, The Johns Hopkins Medical Institutions, Baltimore, Maryland, and homozygous deletion (HD), but some tumors may also inacti- 5Temple University School of Medicine, Philadelphia, Pennsylvania vate the gene by loss of heterozygosity (LOH) coupled with a mutation in the remaining allele. Inactivation by nonsense mutation may cause the loss of protein expression by enhanced Abstract proteosomal degradation (2, 3). Even when expressed as protein, Purpose and Experimental Design: The mutational spec- missense mutations may result in loss of a specific function of trum of MADH4 (DPC4/SMAD4) opens valuable insights the Madh4 protein such as DNA binding or Smad protein into the functions of this protein that confer its tumor- interactions (2–9). Thus, the location of these mutations can suppressive nature in human tumors. We present the provide clues to key structural features that mediate the tumor- MADH4 genetic status determined on a new set of pancre- suppressive function of MADH4. atic, biliary, and duodenal cancers with comparison to the Members of the Smad protein family, including Madh4, mutational data reported for various tumor types. have two evolutionarily conserved regions termed the MH1

Results: Homozygous deletion, followed by inactivating and MH2 domains (Mad homology 1 and 2). The NH2-termi- nonsense or frameshift mutations, is the predominant form nal MH1 domain (codons 1 through 142) is responsible for of MADH4 inactivation in pancreatic cancers. Among the sequence-specific DNA binding (5–7, 10–12), whereas hetero- naturally occurring MADH4 missense mutations, the MH2 merization and transactivation functions have been largely at- domain is the most frequent target (77%) of missense mu- tributed to the MH2 domain [codons 319–552 (6, 8, 13)]. In tations in human tumors. A mutational hot spot resides addition, the MH2 region has been shown to partially interfere within the MH2 domain corresponding to codons 330 to 370, with the DNA-binding function of the MH1 region (5, 7, 8, 12, termed the mutation cluster region (MCR). A relationship 14). These domains are separated by a linker region that con- was found between the locations of the missense mutations tains a 48-amino acid segment called the Smad4 activation (the MH1 domain, the MH2-MCR, and the MH2 outside of domain required for the activation of Smad4-dependent signal- the MCR) and the tumor types, suggesting environmental or ing responses (15). selective influences in the development of MADH4 muta- In the seminal study by Hahn (1), six pancreatic carcino- tions. Immunohistochemical studies for Madh4 protein in mas were found to have intragenic mutations of the MADH4 nine archival cancers (six pancreatic cancers, two duodenal gene, only one of which was a missense mutation. Schutte et al. cancers, and one biliary cancer) with known missense mu- (16) reported additional missense mutations in a pancreatic tations indicated that all mutations within the MH1 or MH2 cancer cell line and an ovarian carcinoma. Missense mutations domain COOH-terminal to the MCR (seven of nine cases) have since been reported in biliary cancers (17), neuroendocrine had negative or weak labeling, whereas two cancers with tumors (18), colorectal cancer (19–23), juvenile polyposis syn- mutations within the MCR had strong positive nuclear la- drome (24–28), ovarian cancer (29), and lung cancers (30). beling for Madh4 protein. Thus, the mapping of these mutations to the known domains of Conclusions: These findings have important implica- the MADH4 gene and their presumed effect on the function of tions for in vitro functional studies, suggesting that the ma- the Dpc4 protein are in need of update. We present new data jority of missense mutations inactivate Madh4 by protein characterizing the mutations of the MADH4 gene in a large degradation in contrast to those that occur within the MCR. series of additional pancreatic cancer xenografts and cell lines. These combined data help to better define the spectrum of mutations in the MADH4 gene with respect to clustering and potential effects on tumor-suppressive function in pancreatic Received 7/8/03; revised 12/8/03; accepted 12/8/03. cancers. We compare our findings with those reported for other Grant support: Supported by the NIH Specialized Programs of Re- human tumor types in an effort to consider the possible associ- search Excellence in Gastrointestinal Cancer Grant CA 62924 and Grant ation between the mutation location and tumor types. CA 68228. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Materials and Methods advertisement in accordance with 18 U.S.C. Section 1734 solely to Generation of Xenografts and Cell Lines. The genera- indicate this fact. tion of xenografted tumors derived from pancreatic, biliary, and Requests for reprints: Scott E. Kern, Department of Oncology, Room 461, Cancer Research Building, 1650 Orleans Street, The Johns Hop- other tumor types has been described in detail previously (31). kins University School of Medicine, Baltimore, Maryland 21231. Human pancreatic cancer cell lines Panc 9.06, Panc 8.13, Panc Phone: (410) 614-3316; Fax: (410) 614-9705; E-mail: [email protected]. 3.27, Panc 2.8, and PL45 are low-passage pancreatic carcinoma Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2004 American Association for Cancer Research. 1598 MADH4 Missense Mutations in Human Tumors

cell lines kindly provided by Dr. Elizabeth Jaffee (32) or de- the MH2] in relation to the primary tumor type was determined scribed previously (33). All were recently made available by ␹2 analysis. Values of Յ0.05 were considered significant. through the American Type Culture Collection (Manassas, VA). All cell lines were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics (100 units/ml penicillin and Results 100 ␮g/ml streptomycin). Cells were incubated at 37°Cina MADH4 Gene Inactivation in Pancreatic Cancer. We

humidified atmosphere of 5% CO2 in air. analyzed the MADH4 gene in 63 new cases representing 54 Determination of LOH. Previous work demonstrated a pancreatic cancer xenografts, 2 duodenal cancer xenografts, 2 high frequency of LOH at 18q21.1 in these pancreatic cancer biliary cancer xenografts, and 5 pancreatic cancer cell lines. xenografts by use of dinucleotide markers, described previously LOH at 18q21 was determined to be present for all but one of in detail (34). the duodenal cancers. Homozygous deletion was found in 13 Amplification and Sequencing of Exons 0–11 of cases (10 xenografts and 3 cell lines of pancreatic cancer). Ten MADH4. PCR amplification of exons 0–11 from genomic of these HDs involved the entire coding region of MADH4. In DNA was performed as described previously (16, 35). PCR- three additional cases, only a portion of the coding region was amplified products were purified using QIAquick (Qiagen) and homozygously deleted, corresponding to exons 7–11 in cell line studied by automated sequencing using nested primers and an Panc 8.13, exons 5–11 for xenograft PX191, and exons 1–4 for ABI Prism model 3700 (Applied Biosystems, Foster City, CA). xenograft PX194. In all 13 cases, the presence of a HD was Sequence analysis used Sequencher version 4.0.5 software confirmed by the failure of PCR to amplify contiguous DNA (Gene Codes, Ann Arbor, MI). Verification of the mutation was segments in the presence of appropriate positive controls. accomplished by sequencing of a second PCR product derived For the 50 cases in which no HD was found, exons 0–11 of independently from the original template. the MADH4 gene were PCR amplified and sequenced. Eighteen Immunohistochemistry. Unstained 5-␮m sections were different intragenic mutations in 17 cases were identified, cor- cut from the archival paraffin blocks of eight pancreatic, biliary, responding to 13 pancreatic cancer xenografts, two duodenal or duodenal cancers with tumorigenic missense mutations of the cancer xenografts, one biliary cancer xenograft, and one pan- MADH4 gene. Paraffin blocks corresponding to one of the creatic cancer cell line (Table 1). In four pancreatic cancer duodenal carcinomas having a missense mutation were unavail- able. For this case, samples of the normal duodenal mucosa and xenografts and the cancer cell line, an insertion/deletion muta- xenografted tumor were fixed overnight in 10% buffered for- tion was found, and each was predicted to cause a frameshift in malin and embedded in paraffin, and sections were cut. Slides the coding sequence. In three additional pancreatic cancer xe- were deparaffinized by routine techniques followed by incuba- nografts, a nonsense mutation was found, and in nine cases (six tion in 1ϫ sodium citrate buffer (diluted from 10ϫ heat-induced pancreatic cancer xenografts, two duodenal cancer xenografts, epitope retrieval buffer; Ventana-Bio Tek Solutions, Tucson, and one biliary cancer xenograft), a missense mutation was AZ) before steaming for 20 min at 80°C. Slides were cooled for identified in the retained MADH4 allele. One of the missense 5 min and incubated with a 1:100 dilution of monoclonal anti- mutations in a pancreatic cancer xenograft occurred in a location body to Madh4 protein (clone B8; Santa Cruz Biotechnology, predicted to cause a splice site alteration at exon 8. One of the Santa Cruz, CA) using the Bio Tek-Mate 1000 automated duodenal cancer xenografts (PX255) contained two different stainer (Ventana-Bio Tek Solutions). This antibody has previ- mutations, a missense mutation of codon 361 resulting in re- ously been shown to be a sensitive and specific marker of placement of an Arg with a Trp residue at that site, and a MADH4 genetic status in pancreatic cancers (36). Anti-Madh4 nonsense mutation of codon 445 resulting in replacement of an antibody was detected by secondary antibody, followed by Arg residue with a termination signal. This latter xenograft was avidin-biotin complex and 3,3Ј-diaminobenzidine chromagen. the one tumor lacking LOH. In all cases, intragenic mutations Sections were counterstained with hematoxylin and evaluated were confirmed by sequencing a second PCR product independ- by two of the authors (C. A. I-D. and S. E. K.), with agreement ently generated from the original template DNA. in all cases. Thus, genetic inactivation of MADH4 was found in 27 of Protein Structure Analysis. The crystal structures of the 59 pancreatic cancers analyzed (46%), in addition to two duo- Smad3 MH1 domain bound to the Smad binding element, and denal xenografts and one biliary cancer xenograft. the Smad4 MH2 domains were obtained from the National Mapping of Mutations and Identification of the Muta- Center for Biotechnology Information Structure database.6 tional Hot Spots of MADH4. Forty-two pancreatic cancer Cn3D software version 4.1, also available from this web site,6 xenografts and 13 pancreatic cancer cell lines have previously was used to visualize the protein domains and to identify the been analyzed for genetic alterations of MADH4 and reported in positions corresponding to missense mutations in these struc- detail (1, 16). Together with the current data, MADH4 inactiva- tures. Statistical Analysis. The location of missense mutations tion was found in a total of 60 of 114 pancreatic cancer xe- [MH1, mutation cluster region (MCR), or COOH-terminus of nografts or cell lines (53%), of which 38 (63%) are due to HD, 14 (23%) are due to nonsense or frameshift mutation, and 8 (13%) are due to a missense mutation. Table 2 summarizes the missense mutations within MADH4 in human neoplasms and syndromes reported in the 6 www.ncbi.nlm.nih.gov/Structure. National Center for Biotechnology Information PubMed data-

Table 1 Genetic alterations of the MADH4 gene identified in the current study Case Type of mutation Codon Sequence change Predicted effect PL9 1-bp deletion 121 TTA to TTAA Frameshift PX93 2-bp deletion 259 ACT to A Frameshift PX108 Nonsense 328 TAC to TAA Tyr to Stop PX133 Missense 118 GCG to GTG Ala to Val PX139 1-bp insertion 121 TTA to TTAA Frameshift PX141 Missense 127 GTC to GCC Val to Ala PX197 Nonsense 245 CAG to TAG Gln to Stop PX226 Nonsense 245 CAG to TAG Gln to Stop PX240 Missense 86 GCT to GTT Ala to Val PX248 1-bp insertion 106 AAA to AAAA Frameshift PX255a Missense and nonsenseb 361 CGG to TGG Arg to Trp 445 CGA to TGA Arg to Stop PX260 Missense 99 TGG to CGG Trp to Arg PX264a Missense 353 TAC to TGC Tyr to Cys PX281 Missense 499 TGC to TAC Cys to Tyr PX350 Splice site acceptor change Exon 8 AGT to AAT Aberrant splicing PX359 2-bp insertion 410 CAG to CACAG Frameshift MX82c Missense 492 GTT to TTT Val to Phe a These two xenografts were derived from primary adenocarcinomas of the duodenum. b This xenograft contained a nonsense mutation in exon 8 and a missense mutation in exon 10. All other mutations were homozygous. c This xenograft was derived from a primary biliary carcinoma.

base7 to date. A total of 70 reported missense mutations includ- nance of mutations mapped to the MH2 domain, we mapped the ing this current study affect 51 unique codons. Missense muta- location of these mutations in relation to the MADH4 coding tions affecting eight codons in particular have each been sequence (Fig. 1). A distinct mutational hot spot within the MH2 reported in more than one study. These comprised codon 118, domain was identified, termed the MCR. This MCR corre- reported in this study in a pancreatic cancer xenograft (PX133) sponded to codons 330–370 and contained 28 (53%) of the total and in a colorectal cancer (21), codon 350 in a small intestinal 53 missense mutations reported within the MH2 domain. The cancer (37) and a colorectal cancer (21), codon 386 in syn- remainder of the missense mutations was distributed throughout dromic juvenile polyps (28) and in an ovarian cancer (29), the MH2 domain. codon 433 in a biliary cancer (17) and a colorectal cancer (22), The location of validated missense mutations (as defined in and codon 523 in a biliary cancer (17) and a pancreatic cancer Fig. 1 and Table 2) was also mapped to the MH1 and MH2 (38). Missense mutations affecting codons 330 and 355 were structural domains of the MadH4 protein (6, 43). As shown in each reported in three different studies. All three codon 330 Fig. 2, missense mutations occurring within the MH1 spanned a mutations were in a colorectal cancer (21, 22, 39), and codon variety of regions corresponding to the basic helix (codon 43), 355 mutations occurred in a pancreatic cancer cell line (40) and the ␤-hairpin (codon 86), and the L4 loop (codons 100 and 102), in two colorectal cancers (20, 39). Mutations affecting codon as well as within amino acids in proximity to these domains 351 were reported in five independent studies representing two (codons 95 and 99) or at the COOH end of the MH1 domain colorectal cancers (19, 23), one small bowel cancer (37), and (codons 118, 127, 129, 130, and 132). Within the MCR of the two ovarian cancers (16, 29). Missense mutations affecting MH2, missense mutations corresponded to the ␤-sheet or to the codon 361 were reported in three colorectal cancers (22, 41, 42), L2 loop of the loop-helix region thought to be critical for syndromic juvenile polyps (25–27), and a small intestinal cancer intermolecular interactions [codons 332, 350, 351, 353, 361, and (37) in addition to a duodenal cancer xenograft reported in the 365 (6)]. MH2 missense mutations outside of the MCR most current study (PX255). commonly localized to structural residues, such as the ␤-sheet Missense mutations are presumably selected for during (codons 401, 406, 433, and 523) or the three-helix bundle tumorigenesis due to their structural effects on a specific func- (codons 445, 492, 493, 497, 502, 537, and 540). tion or domain. Therefore, we mapped all 51 codons reported as Correlation of Mutation Distribution and Human Tu- affected by missense mutations to the coding sequence of the mor Type. The range of naturally occurring missense muta- MADH4 gene (Fig. 1). Missense mutations in MADH4 predom- tions in different human tumor types was also evaluated. Our inantly occurred within the coding region corresponding to the data indicate that MADH4 missense mutations tend to occur in MH2 domain. Overall, 53 of 70 missense mutations (76%) were distinct regions of the coding sequence of MADH4 in relation to located within the MH2 domain, 13 (18%) were located within the tumor type of origin. For example, missense mutations the MH1 domain, and only 4 mutations (6%) were located identified within pancreatic cancers were typically found out- within the linker region. Due to the overwhelming predomi- side of the MCR within the DNA-binding region of the MH1 domain or the COOH terminus of the MH2. In contrast, missense mutations identified within cancers derived from gastrointestinal mucosa (small bowel or colorectum) were most pre- 7 http://www.ncbi.nlm.nih.gov/PubMed. dominant within the MCR of the MH2 domain. A ␹2 test applied

Table 2 Missense mutations of MADH4/DPC4/SMAD4 reported for all tumor types Tumor type Codon Nucleotide change Validateda Ref. no. Pancreatic cancer 43 TTG to TCG ϩ 50 100 AGG to ACG ϩ 1 355 GAC to GGC 40b 365 NAc ϩ 51 406 GCG to ACG ϩ 50 493 GAT to CAT ϩ 1 and 38 523 C to G 38 Biliary cancer 433 GCA to AGA ϩ 17b 497 CGC to CAC ϩ 17 502 AGG to GGG ϩ 17 523 TGC to TGG ϩ 17 Pancreatic neuroendocrine 369 AAT to GAT 18 tumors 457 GCA to TCA 18 Hepatocellular carcinoma 332 GAT to GGT ϩ 52 401 TGC to CGC ϩ 52 Small intestinal cancer 132 CAC to CTC ϩ 37 350 GTT to GAT ϩ 37 351 GAT to AAT ϩ 37 361 CGC to TGC ϩ 37 Colorectal cancer 64 GGA to GTA 19 95 TAT to AAT ϩ 20 115 TGT to CGT 42 118 GCG to GAG 21 129 AAT to AAG ϩ 20 130 ϩ 41 302 TGG to CGG 22 330 GAA to GCA ϩ 21, 22, and 39 340 AAG to GAG 21 350 GTT to GAT 21 351 GAT to CAT 19 and 23 355 GAC to GAA ϩ 20 and 39 361 CGC to AGC 22, 41, and 42 CGC to CAC 363 TGT to AGT 42 370 GTT to GAT 41 408 TTT to TCT 22 433 GCA to GTA 22 497 CGC to CAC 42 507 AAA to CAA 42 515 AGA to GGA 42 537 GAT to TAT ϩ 23 540 CTA to CGA ϩ 23 Juvenile polyposis 180 NAc ϩ 24 syndrome 361 CGC to TGC ϩ 25–27 386 NAc 28 390 GAA to AAA 27 445 NAc ϩ 26 448 NAc 28 Ovarian cancer 317 CAT to CGT 29 351 GAT to CAT ϩ 16 and 29 379 GCA to ACA 29 386 GGT to TGT 29 504 AGT to AGA 29 Cervical cancer 230 NAc 53 488 NAc 53 Lung cancer 420 CGT to CAT 30 441 CGT to CCT 30 Acute myelogenous 102 CCT to CTT ϩ 54 leukemia a Only those missense mutations that were confirmed by repeat sequencing of an independently derived PCR product, as indicated in “Materials and Methods” and/or “Results” of each study, are considered to be validated mutations. b Only exons 8–11 were analyzed in these studies. c Mutation sequence not provided in the report.

Fig. 1 Distribution of mutations reported for the MADH4 gene. The location of all missense mutations is indicated in reference to the MADH4 coding sequence. Arrow colors indicate the human tumor types from which the mutations are reported. Green, pancreatic cancer; pink, duodenal cancer; dark blue, sporadic colorectal cancer; light blue, juvenile polyposis syndrome; brown, small intestinal cancer; gray, acute myelogenous leukemia; yellow, biliary cancer; orange, hepatocellular cancer; red, ovarian cancer. Black arrows are mutations reported in MADH4 but not validated by sequencing of an independent amplification product. The coding regions corresponding to amino acids 330–370 are indicated by a bracket to illustrate the mutation cluster region of the MH2 domain. This distribution of mutations is significantly associated with tumor type (P Ͻ 0.001).

to the location of missense mutations (MH1, MCR, or the within the MH1 domain, and five contained a mutation within COOH terminus of MH2) in relation to the primary tumor type the MH2 domain, two of which corresponded to the MCR. indicated a statistically significant association (P Ͻ 0.001). The presence or absence of immunohistochemical labeling Relationship of Madh4 Protein Expression to Location for Madh4 protein was highly correlated to the location of the of Missense Mutation. Homozygous deletions and nonsense missense mutation within the coding sequence of MADH4.Of mutations of MADH4 are thought to result in loss of protein the four mutations located within the MH1 domain, three were expression or an unstable protein that may be targeted for negative, and one showed weak focal cytoplasmic labeling. All degradation (2, 36). We determined the status of Madh4 protein immunohistochemically analyzed mutations within the MH1 expression in nine cancers (six pancreatic cancers, two duodenal domain occurred in pancreatic cancers. Among the five mis- cancers, and one biliary cancer) in which MADH4 contained a sense mutations within the MH2 domain, two were strongly missense mutation (Fig. 3). Four cases contained a mutation positive, one was weakly positive, and two were negative for

Fig. 2 Distribution of missense mutations within functional domains of the highly similar Smad3 MH1 region or the Madh4 MH2 region. Only validated mutations are represented (as defined in Table 2). A, crystal structure of two molecules of the Smad3 MH1 protein domain bound to DNA.

Smad3 MH1 protein is shown in blue and purple, and DNA is shown in green and brown. The NH2 terminus of the structure is noted as well as the approximate locations of the double loop, basic helix, and ␤-hairpin motifs. Amino acid residues targeted by missense mutations in MADH4 are indicated in yellow. B and C, crystal structure of the MadH4 MH2 domain. The protein is shown in purple, and the amino acid residues targeted by missense mutations are indicated by yellow.InB, missense mutations corresponding to the mutation cluster region are shown, and in C, missense mutations corresponding to the COOH end of the MH2 are shown.

Fig. 3 Immunohistochemical detection of Madh4 protein in nine human cancers with known MADH4 missense mutations. Shown are four cancers with a missense mutation within the MH1 domain (top panels) and five cancers with a missense mutation within the MH2 domain (bottom panels). The codon affected by the mutation and the resultant amino acid changes are listed for each case. The presence or absence of Madh4 protein expression is seen to correlate with the location of the missense mutations. Within the MH1, mutations at codons 86, 99, and 127 result in loss of protein expression within the neoplastic epithelium, in contrast to the positive labeling of stromal cells. Madh4 protein is detectable in the case of mutation at codon 118, although labeling is weak and apparently confined to the cytoplasm. Mutations within the mutation cluster region of the MH2 domain (codons 353 and 361) have strong nuclear labeling in each case, whereas mutations outside of the cluster region at the 3Ј end of the gene weakly label (codon 492) or have complete loss of Madh4 protein (codons 493 and 499).

Madh4 protein. The two strongly positive cases, both duodenal tions occurring in the MH1 or COOH end of the MH2 domains. cancers, had missense mutations within the MCR and striking The decreased stability of missense mutations occurring in the nuclear localization of Madh4 protein. One weakly positive MH1 domain has been demonstrated in vitro (3, 5), where the case, a biliary cancer, had faint cytoplasmic labeling. The two protein is thought to be rapidly degraded in vivo by the ubiq- cases negative for Madh4 protein labeling corresponded to two uitin-proteasome pathway (44). Our data indicate that missense pancreatic cancers. mutations occurring COOH-terminal of the MH2 domain may also be targeted for rapid degradation because Madh4 protein Discussion expression is undetectable or at most weakly positive in pan- Homozygous deletion is the most common form of creatic cancers with missense mutations in this region. In the MADH4 inactivation. In these instances, the loss of tumor- prior studies by Wilentz et al. (36, 45) characterizing MadH4 suppressive function is clearly mediated by the loss of Madh4 protein expression in relation to MADH4 genetic status, no protein expression. A similar phenomenon may also occur in cancers with missense mutations were available for study. We human tumors where a nonsense, splice site, or frameshift are aware of only two other studies in which MADH4 missense mutation occurs; Madh4 protein is immunohistochemically un- mutations have been correlated to immunohistochemical label- detectable, a finding highly concordant with the MADH4 genetic ing for Madh4 (24, 38). Interestingly, both studies also reported status (36). In these examples, gross splicing or translation loss of Madh4 protein expression in tumors with missense errors are expected to preclude translation or result in an unsta- mutations COOH-terminal of the MCR and support the concept ble protein product that is targeted for degradation (2, 44). that this coding region is also exquisitely sensitive to structural In contrast to MADH4 inactivation caused by HD or non- changes introduced by single missense mutations. sense type mutations, the in vivo functional consequences of Our data also indicate that a MADH4 MCR exists for missense mutations have not been explored in detail. Our data naturally occurring missense mutations and that carcinomas indicate that the majority of missense mutations may also inac- with missense mutations within this MCR can retain Madh4 tivate MADH4 through enhanced degradation of an unstable protein expression. The MCR, spanning codons 330–370, cor- protein product. Specifically, we have noted that many missense responds to the loop/helix structure of the MH2 domain, sup- mutations target structural residues, in particular those muta- porting its functional significance proposed from structural stud-

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