RNA-Based Mutation Screening in Hereditary Nonpolyposis

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1 RNA-Based Mutation Screening in Hereditary Nonpolyposis Colorectal Cancer Maija Kohonen-Corish,' Veronica L. Ross,' William F. Doe,' Deborah A. KooI,2 Edward Edkins,2 Ian Faragher,3 Juul Wijnen,5 P. Meera Khan,5 Finlay Macrae,4 and D. James B. St John4 Am. J. Hum. Genet. 59: , Division of Molecular Medicine, John Curtin School of Medical Research, Australian National University, Canberra; 2Genetic Services of Western Australia, Princess Margaret Hospital for Children, Perth; 3Ludwig Institute for Cancer Research and 4Department of Gastroenterology, The Royal Melbourne Hospital, Melbourne; and 5MGC-Department of Human Genetics, Sylvius Laboratory, Leiden University, Leiden Summary Hereditary nonpolyposis colorectal cancer (HNPCC) is a cancer syndrome inherited in an autosomal dominant fashion. Four susceptibility genes are known, which code for DNA mismatch repair enzymes. The purpose of this study was to identify the HNPCC gene defects in a cohort of Australian HNPCC families and to evaluate the use of RNA-based screening methods. Six mutations were identified, four in the hmlh1 gene and two in hmsh2, by using a combination of DNA-based and RNA-based methods. One of the hmlh1 defects was a missense mutation, and the other five mutations would be expected to result in a shortened protein. These included a rare type of mrna splicing mutation in hmlh1 exon 17. By use of reverse-transcriptase (RT) PCR, defective transcripts were detectable for three of the hmlh1 mutations but not for the fourth one, which was predicted to cause skipping of exon 15. Furthermore, many more alternative transcripts for the hmlh1 gene were found than previously described, and these were more abundant in the RNA samples prepared from whole blood than from lymphoblastoid cell lines. This confounded RNA-based screening for HNPCC mutations, because it was difficult to determine which aberrant RT-PCR fragment was the real hereditary defect. One of the splice-site mutations reported here causes skipping of exons 9 and 10, which also occurs as an alternative transcript. When the protein-truncation test was used, the results were indistinguishable between the patients in this family and controls. Other aberrant transcripts were also observed that varied in size between individuals but were unrelated to the hereditary defects. This study has important implications for the design of reliable diagnostic tests for HNPCC gene defects. Received March 25, 1996; accepted for publication July 3, Address for correspondence and reprints: Dr. Maija Kohonen-Corish, Division of Molecular Medicine, John Curtin School of Medical Research, GPO Box 334, Canberra, ACT 2601, Australia. maija.corish@anu.edu.au X 1996 by The American Society of Human Genetics. All rights reserved /96/ $ Introduction Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common of the recognized inherited colorectal cancer (CRC) syndromes, comprising at least 2%-6% of all CRCs. The recent identification of the human homologues of the yeast mismatch repair genes hmsh2 (Fishel et al. 1993; Leach et al. 1993), hmlh1 (Bronner et al. 1994; Papadopoulos et al. 1994), hpms1, hpms2, (Nicolaides et al. 1994) and the G/T binding protein (GTBP, Palombo et al. 1995) offered the prospect of genetic screening leading to an extensive search for mutations in HNPCC families. The majority of defects have been detected in hmsh2 and hmlh1. To date, only three mutations have been published for hpms1 and hpms2 (Nicolaides et al. 1994; Hamilton et al. 1995). No germ-line defects have yet been detected for GTBP, a gene closely linked to hmsh2, although mutations have been found in three colon cancer cell lines (Papadopoulos et al. 1995b). Both DNA-based and RNA-based methods have been used in mutation screening in HNPCC. The DNA-based methods have involved PCR amplification of individual exons and subsequent analysis by single-strand conformational polymorphism (Han et al. 1995), denaturing gradient-gel electrophoresis (DGGE; Wijnen et al. 1995, 1996), and sequencing (Kolodner et al. 1994, 1995). The advantages are that genomic DNA is more stable than RNA, both alleles are present in equal numbers, and sequencing represents direct and definitive detection. But the distribution of mutations across the entire length of the gene requires the screening of large numbers of exons, and sequencing is tedious and expensive. Genomic deletions and mutations involving the primerrecognition site or those outside the amplified region, however, may result in amplification of the normal allele only. This problem is largely overcome by the RNAbased methods, in which the whole coding region of the gene is amplified from cdna in two or more overlapping fragments (Liu et al. 1994, 1995; Papadopoulos et al. 1994). Alleles with genomic deletions spanning intron-exon junctions are seen as shortened fragments.

2 Kohonen-Corish et al.: RNA-Based Mutation Screening in HNPCC Similarly, skipped exons caused by splice-site or branchpoint mutations that may be missed by amplifying the individual exon from genomic DNA are detected by amplifying cdna. The major disadvantage of the RNA-based methods, however, is that mutation detection is indirect. Defective alleles may be only weakly expressed or not expressed at all (Liu et al. 1994; Papadopoulos et al. 1995a). In addition, alternative transcripts of the mismatch repair genes (Charbonnier et al. 1995) may confuse the analysis, because they may be indistinguishable from skipped exons caused by inherited germ-line mutations. In this paper, we describe the use of reverse-transcriptase (RT) PCR and the protein-truncation test (PTT), also known as in vitro synthesized protein assay (Liu et al. 1994, 1995), to screen for mutations in hmlh1 and hmsh2 and describe six mutations. Amplification of some cdna samples produced fragments resulting in truncated proteins that did not represent the inherited genetic defect. These were particularly evident in cdna derived from whole-blood total RNA collected from both HNPCC-affected individuals and from normal control subjects. Our results suggest that while PTT is useful as the first screening of mutations in HNPCC it is not sufficient for diagnostic purposes, especially when whole-blood RNA is used. Patients, Material, and Methods Patients The HNPCC families were ascertained in three centers, The Royal Melbourne Hospital, The Princess Margaret Hospital for Children, and the John Curtin School of Medical Research. Family Oz-4 (formerly 238) has been described elsewhere (Kohonen-Corish et al. 1995). All families fulfilled the criteria for HNPCC (Vasen et al. 1991; Lynch et al. 1993). After informed consent was obtained, blood samples were collected from at least two cancer patients per family. Lymphoblastoid cell lines were established from selected patients. Preparation of DNA and cdna Genomic DNA was prepared from whole-blood buffy-coat cells by using standard methods. Total RNA was extracted from buffy-coat cells and lymphoblastoid cell lines as described by Chomczynski and Sacchi (1987) or by use of the RNeasy Total RNA kit (Qiagen). cdna was prepared using AMV RT (Promega) or Superscript II (Life Technologies). DNA Amplification and the PTT The hmlh1 cdna was amplified in two overlapping fragments, A and B. including codons and , respectively, by using primers described by Papadopoulos et al. (1994). For half-nested PCR of fragment A, the nesting reverse primer was 5'- GGAATCTGT- ACGAACCATCTGG-3'. PCR was carried out at 940C (30 s), 570C (30 s), and 700C (60 s), for 35 cycles, after an initial denaturation step of 3 min. Otherwise, PCR conditions were as described by Kohonen-Corish et al. (1995). The PTT was carried out with the primers described by Liu et al. (1995), except for the hmlh1 cdna fragment A that was amplified as shown above but with the T7 forward primer in the second-round PCR. The TNT Coupled Reticulocyte system (Promega) was used for the analysis. The full-length hmsh2 cdna was amplified using a 1:1 mixture of Taq Extender (Stratagene) and Tth plus (Biotech International) with primers 5'-GGGTCGCGC ATTTTCTTCAAC-3' and 5'-CCTTCATTCCATTAC- TGGGATTTT-3'. PCR was carried out at 94 C (1 min), 50 C (1 min), and 720C (3 min), for 35 cycles, with 6 min initial denaturation and 9 min final elongation. Fragment A, including codons 1-548, was then reamplified using nesting primers 5'-GGATCCTAATACGAC- TCACTATAGGGAGACCACCATGGCGGTGCA- GCCGAA-3' (includes the T7 sequence for transcription and translation) and 5'-CACCATTCTTCTGGATAT- CTACAG-3'. PCR was carried out at 94 C (30 s), 55 C (1 min), and 72 C (2 min), for 30 cycles, with 5 min initial denaturation and 9 min final elongation. Alternative transcripts involving a deletion of hmlh1 exon 9 in the cdna were detected by using a forward primer 5 '-GGAAATGCTGTTAGTCGATC-3' designed from the end sequence of exon 8 and the beginning of exon 10, together with the fragment A reverse primer. Exon 9 and 10 deletion was detected in the same way by using 5'-GGAAATGCTGTTAGTCGTT-3' as the forward primer. The genomic hmlh1 exons 9 and 15 were amplified with primers described by Han et al. (1995) and Wijnen et al. (1996), respectively. Primers to amplify other exons from genomic DNA were designed from published sequences. DGGE and Sequence Analysis DGGE and sequencing of hmlh1 exon 15 were performed as described by Wijnen et al. (1996). Other hmlh1 exons and cdna fragments A and B were either cloned for sequencing (pgem-t, Promega) or sequenced directly from PCR products by using PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) and an ABI 373A DNA Sequencer. The hmsh2 exons and cdna were sequenced manually from PCR products using Exo- Pfu (Stratagene). Results 819 RT-PCR of the hmlh1 gene was initially carried out for 12 HNPCC families, and the overlapping fragments A and B were visualized on an agarose gel. In two families, Oz-2 and Oz-3, first-round RT-PCR showed a

3 820 Oz bp 1219 _ 1012 Frag. A Frag. B Figure 1 Amplification of the hmlh1 cdna from affected members of HNPCC families (overlapping fragments A and B). Total RNA was extracted from lymphoblastoid cell lines, except for family Oz-3, where blood RNA was used. In family Oz-2, exons 9 and 10 are deleted from fragment A, yielding a shorter fragment of 1,012 bp. In Oz-3, exon 17 is deleted from fragment B, producing a 1,331-bp fragment. shorter fragment, in addition to the expected normal length fragment, although it was faint in Oz-2 (fig. 1). Sequence analysis revealed a deletion of hmlh1 exon 17 in Oz-3 and exons 9 and 10 in Oz-2. The mutations causing splicing out of exons were confirmed from genomic DNA in at least two affected members per family. The exon 17 skipping is apparently caused by a 2-nt deletion in codon 659 (positions -14 and -15 of the splice-donor site), which leads to a frameshift and a new stop codon in exon 17. Splicing out of both exons 9 and 10 appears to be caused by a mutation in the exon 9 splice-donor site (table 1). In families Oz-1 and Oz-4, the amplification of cdna from the lymphoblastoid cell lines produced the same fragments as in the controls. Linkage analysis implicated hmlh1 in these two families (data not shown), and therefore the cdna was sequenced. In Oz-1, a T-to-G substitution was found in hmlh1 codon 185 in exon 7, which caused an amino-acid change from valine to Am. J. Hum. Genet. 59: , 1996 glycine. This mutation was confirmed from genomic DNA in 3 affected members and was not found in 16 healthy members tested and in 70 unrelated controls by making use of the disruption of the RsaI site in exon 7 (data not shown). For family Oz-4, no mutations were found by sequencing cdna. RNA was available from three affected family members. By use of the PTT, a truncated protein was detected in one affected member only (fig. 2), but this was reproducible. In one PTT experiment, another faint truncated protein (-30 kd) was also detected, but this was difficult to confirm in further PTT experiments. Selected exons were subsequently amplified from genomic DNA. DGGE analysis of hmlh1 exon 15 showed an aberrant pattern in five affected members (data not shown). Exon 15 was sequenced and a G-to-A substitution was found in the last nucleotide of the exon. This was predicted to cause splicing out of exon 15, making the transcript unstable and the truncated protein (27 kd) difficult to detect. In the remaining eight families, the PTT screening detected no hereditary defects for the hmlh1 gene, and the analysis is continuing for hmsh2. A further series of six HNPCC families were first screened for hmsh2 mutations. In family Oz-5, an 865- bp cdna deletion involving hmsh2 exons 2-6 was found in two affected members tested. Only DNA-based screening was used for family Oz-6, and a stop codon was found in hmsh2 exon 12. A summary of the mutations and tumor spectrum in each family is shown in table 1. In the other four families, no hereditary defects were detected using the PTT for the hmsh2 gene, and the analysis is continuing for hmlh1. In addition to sequencing, PTT was also carried out for the Oz-1, Oz-2, and Oz-3 families. Figure 2 shows a representative result for hmlh1 fragments A and B. The deletion of exons 9 and 10 in Oz-2 is clearly evident as a truncated protein when derived from both blood Table 1 Pathogenic Mutations in the HNPCC Families Family Gene cdna Change Genomic DNA Mutation Tumor Spectruma Oz-1 hmlh1 GTA-+GGA GTA-GGA C (7); C, GB; Val-Gly Exon 7/codon 185 C, SI, BD; 0; UN Oz-2 hmlh1 Del exons 9+10 Exon 9 splice donor site: TCAACCgtaagt-+TCAACCgttaagt C (12); K; B (2) Oz-3 hmlh1 Del exon 17 Exon 17: CTTCGACTAGCCACTGA-CTFACTAGCCACTGA C (4); C, B; E; mrp Oz-4 hmlh1 Del exon 15 Exon 15 splice donor site: TTATC~gta-+TTATCAgta C (5); C, 0, E (2); C, E; UN (2) Oz-5 hmsh2 Del exons 2-6 not identified C (3); C, E; B Oz-6 hmsh2 TAC--TAG TAC-TAG C (4); C, U Tyr-+stop codon Exon 12/codon 656 a Tumors observed in individual patients are separated by semicolons. Numbers in parentheses indicate the number of members with similar tumors or tumor sets. B = brain; BD = bile duct; C = colon; E = endometrium; GB = gall bladder; K = kidney; mrp = malignant rectal polyp; 0 = ovary; SI = small intestine; U = uterus; and UN = unknown primary site of cancer. Only those tumor sites are listed that have been previously considered part of the HNPCC tumor spectrum, but not all the cancer patients were tested for the presence of the mutation. In family Oz-6, the presence of other cancers was not ascertained.

4 Kohonen-Corish et al.: RNA-Based Mutation Screening in HNPCC A. Oz-I Oz-2 Oz-4 P N P P P P2 P B. Oz-3 Oz-4 N PP1 p ~ BL BL BL BL Figure 2 PTT of hmlh1 cdna in two overlapping fragments A and B. Truncated proteins (asterisks [*]) are caused by either alternative transcripts (lanes Al, A2, and A9) in blood (BL) RNA or by a genomic mutation that results in splicing out of exons 9 and 10 (lanes A4 and AS) or exon 17 (B2). In family Oz-4, the truncated protein (27 kd) caused by the defect in exon 15 is not visible. The nature of the truncated proteins in lanes A8 and B3 are under investigation. P = CRC patient; and N = healthy member with normal hmlh1 alleles. Molecular weights of the marker proteins are given in kilodaltons. and cell-line RNA. The same truncated protein was, however, also evident in many other families when the RNA was derived from blood, including both healthy and affected members of family Oz-1, in which the point mutation in exon 7 is not expected to cause a shortened protein. To investigate this further, RT-PCR was used to compare samples of blood RNA with lymphoblastoid cell-line RNA from the same subjects. As shown in figure 3, several fragments visible in the samples obtained from blood RNA were not seen in the cell-line RNA when a half-nested PCR procedure was used. A PCR probe, containing the purified normal-length hmlh1 fragment A, hybridized with all the fragments, whereas a probe specific for exon 9 failed to hybridize with many of the shorter fragments (fig. 3). These findings suggest the presence of some alternative transcripts of hmlh1, many of which involve deletion of exon 9. Some alternative transcripts are also present in the cell-line RNA, although they were less abundant. We designed PCR primers that would amplify cdna derived from transcripts that have a deletion of exon 9 or both exons 9 and 10. As shown in figure 4, both transcripts were detected in cell-line RNA. Moreover, when we cloned and sequenced the hmlh1 fragment A derived from Oz-1 cell-line RNA, one clone was obtained that included a deletion of exon 9, in addition to the normal length clones. For this family, we subsequently amplified and sequenced exon 9 from genomic DNA, but no splice-site mutations were found. A similar but less extensive analysis was also carried out for hmlh1 fragment B. Extra fragments were found in some samples but there was no increased frequency in the bloodderived samples (data not shown) and fragment size varied between the samples. Again, when we cloned the hmlh1 fragment B, derived from family Oz-4 cell-line RNA, one clone was obtained that included a deletion of exon 17. Subsequent sequence analysis of exon 17 from genomic DNA did not reveal any splice-site mutations. Discussion Mutation detection in HNPCC is still problematic. In the large study carried out by Liu et al. (1996) HNPCC mutations were not detected in 14 of 48 families, although, in some HNPCC kindreds, genetic linkage to hmlh1 or bmsh2 could be demonstrated. Our results support their interpretation that, because of the nature of some HNPCC mutations, current RNA-based screening methods are not adequate. Not only are some mutations undetectable with standard techniques (Papadopoulos et al. 1995a), but also unstable mutant transcripts can easily be missed. Moreover, as shown in this paper, the presence of alternative transcripts can seriously confound mutation screening, including the PTT, a problem not discussed by Liu et al. (1996). Few laboratories have reported the use of the PTT in HNPCC. Liu et al. (1996) found that 71% of the 34 HNPCC mutations detected were identifiable by PTT, giving a success rate of 50% for mutation screening by PTT in the whole series. Luce et al. (1995) detected truncated proteins for either hmsh2 or hmlh1 in 6 of 12 HNPCC kindreds but reported the nature of the underlying genomic DNA defect for only 2 of them.

5 822 In this paper, a variety of techniques were used. In four of five cases, the mutations were detectable from RNA after the defective gene was first identified by linkage or a deletion was obvious using RT-PCR. For the rest of the families, linkage data were not available, and the RNA-based analysis is continuing. In family Oz-4, it was concluded that the truncated protein (fig. 2) found in only one member did not represent the hereditary mutation and that the predicted skipping of hmlh1 exon 15 caused an unstable transcript. This has been confirmed in another HNPCC family who had a mutation in the intronic splice-donor site of exon 15 (Wijnen et al. 1996). In family Oz-3, a frameshift mutation introduced a new stop codon at the end of exon 17, leading to exon skipping as described in Marfan syndrome and gyrate athropy (Dietz et al. 1993). It is interesting to note that in this case the mutant transcript amplified as strongly with RT-PCR as did the normal allele. A. bp 1200 _ B. Co Figure 3 A, Half-nested amplification of hmlhl cdna fragment A, showing the difference between whole blood (lanes 1-4) and cell line-derived (lanes 5-8) RNA samples. In addition to the normal length fragment (1,200 bp), several other fragments are visible in the blood-derived samples, one of which is the same molecular weight as the fragment containing a deletion of exons 9 and 10 (993 bp) in family Oz-2 (lanes 2 and 6). Other lanes show results from family Oz-1 (lanes 1 and 5), Oz-4 (lanes 3 and 7), and a healthy control (lanes 4 and 8). Panels B and C show hybridization results of the same gel. B, PCR probe, coding for hmlh1 exon 9, which fails to hybridize to the shorter fragments. C, PCR probe, coding for hmlh1 codons 1-388, which hybridizes with all the fragments. Am. J. Hum. Genet. 59: , bp Figure 4 Detection of hmlh1 alternative transcripts by RT- PCR from lymphoblastoid cell lines from patients Oz-1, Oz-2, Oz-4, and a healthy control (lanes 2-5 and 7-10). The 391-bp fragment contains a deletion of exon 9 (lanes 1-5). The 297-bp fragment contains a deletion of exons 9 and 10 (lanes 6-10). Lanes 1 and 6 show amplification from cloned cdna containing a deletion of exon 9 or both exons 9 and 10, respectively. Some amplification of the normal length transcript (504 bp) is observed in lanes 2-5, and similarly the exon 9 deleted transcript is evident in lanes Difficulties were encountered in analyzing hmlh1 cdna derived from blood RNA, because multiple bands were commonly detected, particularly for fragment A, which covered exons Our results are in agreement with those of Charbonnier et al. (1995), who described three alternative hmlh1 transcripts from blood lymphocytes, one involving deletion of exons 9 and 10, one of exons 10 and 11, and one of exons 9, 10, and 11. In our material, however, more than three extra bands were observed with varying intensities, possibly because of the amplification of a longer stretch of the cdna. We also commonly observed other shortened RT-PCR fragments from both blood and cell-line RNA, which were particularly evident after reamplification and varied in size between individuals but appeared unrelated to the hereditary defect. Even when we cloned seemingly pure first-round PCR fragments amplified from cell-line RNA, some clones were obtained that had exons deleted. It has been reported that families with APC gene mutations, involving those exons of the gene that are alternatively spliced, have later onset of CRC (van der Luijt et al. 1995) or have the attenuated form of APC (Samowitz et al. 1995). In this study, family Oz-2 was found to have a germ-line mutation that resulted in splicing out of hmlh1 exons 9 and 10. Two living affected members who were distant relatives shared the same mutation and had been diagnosed with CRC at the ages of 46 and 33 years. However, only one of the two had first-degree relatives diagnosed with CRC. The mother of the second affected member was tested and was found to have the same mutation, but she remains healthy at the age of 62 years. Therefore, it is possible that this mutation has lower penetrance. On the other hand, the hmlh1 exon 17 mutation is associated with a very severe clinical CRC phenotype in family Oz-3, although our results suggest that the splicing out of this exon may also occur as an alternative transcript. One member was also diagnosed with the type of brain tumor, glioblas-

6 Kohonen-Corish et al.: RNA-Based Mutation Screening in HNPCC 823 toma multiforme, that has been described to be part of the HNPCC tumor spectrum by Hamilton et al. (1995), who suggest that these rare families could be classified as Turcot syndrome. In conclusion, our study shows that in HNPCC the results of RT-PCR and the PTT should be interpreted with caution. The mutation should be confirmed in at least two patients per family, or from genomic DNA, before other, as-yet unaffected, members are tested. Also, the use of whole-blood RNA for screening of HNPCC mutations can be problematic. Cell-line RNA is safer, but in a routine diagnostic laboratory it will not be possible to raise lymphoblastoid cell lines for all specimens. In our hands, RNA-based tests not only produced false-positive results caused by alternative transcripts but also at least one false negative. 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