A novel germline 1.8-kb deletion of hMLH1 mimicking alternative splicing: a founder mutation in the Chinese population

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We have previously reported that there is a high incidence of microsatellite instability (MSI) and germline mismatch repair gene mutation in colorectal cancer arising from young Hong Kong Chinese. Most of the germline mutations involve hMSH2, which is different from the mutation spectrum in the Western population. It is well known that alternative splicing is common in hMLH1, which complicates RNA based mutation detection methods. In contrast, large deletions in hMLH1, commonly observed in some ethnic groups, tend to escape detection by exon-by-exon direct DNA sequencing. Here we report the detection of a novel germline 1.8 kb deletion involving exon 11 of hMLH1 in a local hereditary non-polyposis colorectal cancer family. This mutation generates a mRNA transcript with deletion of exons 10–11, which is indistinguishable from one of the most common and predominant hMLH1 splice variants. A diagnostic test based on PCR of the breakpoint region led to the identification of an additional young colorectal cancer patient with this mutation. Haplotype analysis suggests that they may share a common ancestral mutation. Our results caution investigators in the interpretation of alternative splicing and have important implications for the design of hMLH1 mutation detection strategy in the Chinese population.


Microsatellite instability (MSI) is the phenotypic manifestation of mismatch repair deficiency. It is present in the majority of tumors arising from Hereditary Non-polyposis Colorectal Cancer (HNPCC) syndrome, which is the most common form of inherited colorectal cancer (Aaltonen et al., 1993; Ionov et al., 1993; Thibodeau et al., 1993). We and others have also reported the presence of MSI in 50–60% of early-onset colorectal cancer (Liu et al., 1995; Farrington et al., 1998; Chan et al., 1999; Ho et al., 2000). Germline mutations in one of the DNA mismatch repair (MMR) genes are implicated as the cause in these patients. It is known that mutation detection for the MMR genes are technically difficult for several reasons. Firstly, germline mutations in five mismatch repair genes, including hMSH2, hMLH1, hMSH6, hPMS1 and hPMS2, have been detected in HNPCC kindreds, and each of these genes are very big in size (Fishel et al., 1993; Leach et al., 1993; Papadopoulos et al., 1994; Bronner et al., 1994; Nicolaides et al., 1994; Palombo et al., 1995; Drummond et al., 1995; Akiyama et al., 1997; Miyaki et al., 1997). Secondly, there are no mutational hotspots, and no single mutational detection strategy can identify all types of mutation (Peltomaki and Vasen, 1997). So far, germline mutation, mostly involving hMSH2 or hMLH1, can be identified in around 60% of HNPCC kindreds (Wijnen et al., 1995, 1996; Buerstedde et al., 1995; Miyaki et al., 1995; Moslein et al., 1996; Liu et al., 1996; Nystrom-Lahti et al., 1996; Froggatt et al., 1996; Weber et al., 1997; Viel et al., 1997; Lamberti et al., 1999; Bapat et al., 1999) and a similar percentage of young patients with MSI colorectal cancer (Liu et al., 1995; Farrington et al., 1998; Chan et al., 1999). The mechanisms underlying the mismatch repair deficit in the remaining cases are poorly understood.

Finding mutation in hMLH1 poses a particularly complicated problem because alternative splicing is common in this gene, which may generate transcripts that code for truncated protein (Charbonnier et al., 1995; Kohonen-Corish et al., 1996; Palmirotta et al., 1998). Some of the commonly observed splice variants include those with skipping of exons 10 and 11, exons 9 and 10, exons 9–11, exons 15 and 16. These may mimic pathogenic mutations when RNA-based mutation detection strategies are utilized. In contrast, in the literature there are rare examples of large genomic deletions involving complete exons of hMLH1 (Nystrom-Lahti et al., 1995; Mauillon et al., 1996). These mutations are not demonstrable by PCR-based exon-by-exon genomic sequencing and hence reverse transcription-polymerase chain reaction based methodologies are essential for their detection. Here, we report an unusual situation where a novel large genomic deletion of hMLH1 results in generation of a mRNA transcript identical to one of the most prominent alternative splice variants. This mutation is found in two unrelated Chinese patients. Haplotype analysis suggests that they may originate from the same founder mutation. Taken together with the high incidence of early-onset colorectal cancer in Hong Kong Chinese but a low incidence of hMLH1 germline mutation from our previous studies (Yuen et al., 1997; Chan et al., 1999), the results have important implications for the design of mutation detection strategy especially for HNPCC patients of Chinese origin.

In the course of examination for germline hMLH1 mutation using RT–PCR and cDNA sequencing in young patients with high level MSI colorectal cancer, one patient showed an abnormal splicing pattern when amplified by primers flanking exon 6 to exon 12. In normal individuals, the amplification products from blood leucocytes showed, apart from the wild-type amplicon, three splice variants corresponding to the skipping of exon 9 (Δ9 transcript), exons 10 and 11 (Δ10–11 transcript), and exons 9–11 (Δ9–11 transcript). In patient K89, the Δ10–11 transcript was particularly prominent (Figure 1a). We then examined his microdissected colorectal cancer tissue and found that the Δ10–11 transcript was the predominant splice variant, there being a corresponding loss of the wild-type transcript (Figure 1b). Direct sequencing of this shorter amplicon confirmed that nucleotides 791–1038 were absent, corresponding to skipping of exons 10 and 11. Sequencing of the PCR amplified genomic DNA, however, did not reveal any mutation in both coding regions and intron-exon boundaries of the corresponding exons.

Figure 1

RT–PCR analysis for hMLH1 fragment 2 encompassing exons 6–12. Transcripts generated by alternative splicing are labeled as follows: I, wild-type transcript; II, δ9 transcript; III, δ10–11; IV, δ9–11 transcript. (a) Blood leucocytes (lanes 1–5) and normal colonic tissue (lanes 6–11) from 11 young patients with MSI colorectal cancer; lane 2 is from patient K89; lane 11 is from patient K116; lane M, Bluescript/MspI DNA marker. Transcript III is the predominant spliced variant in patient K89. Patient K116 shows only a mild prominence of transcript III, which is not distinguishable from the remaining patients who were subsequently confirmed not harboring the specific deletion mutation. (b) The microdissected colorectal cancer tissue of patient K89 expresses mostly transcript III with a loss of the wild-type transcript. mT, microdissected colorectal cancer tissue. Samples were from young colorectal cancer patients referred to our Hereditary Gastrointestinal Cancer Registry based at Queen Mary Hospital, Hong Kong. Venous blood was drawn, with informed consent from the patients. This study was approved by the Ethics Committees of Queen Mary Hospital and the University of Hong Kong. Total RNA was extracted from peripheral blood leucocytes, frozen normal colonic tissue or microdissected colorectal cancer tissue. An aliquot (2 μg) was reverse-transcribed using random hexamer. The complete open reading frame of hMLH1 cDNA was amplified and sequenced in four overlapping fragments (1–4) using primers and conditions as described previously (Chan et al., 1999). In brief, 5 μl of RT–PCR product was treated with 2 μl of presequencing enzyme (Amersham Pharmacia). Sequencing was performed using the DYEnamic ET Terminator Cycle Sequencing Kits (Amersham Pharmacia) and analysed by the Applied Biosystems 377 automated sequencer. Both forward and reverse RT–PCR primers were used in sequencing of each fragment. To search for splice site mutations, exons 10 and 11 including the intron–exon boundaries were amplified from genomic DNA and sequenced. The primer sequences for RT–PCR and genomic sequencing are available on request

To define the mutation on the genomic level, Southern-blot analysis was performed using DNA extracted from his peripheral blood leucocytes. An aberrant restriction fragment around 0.8 kb shorter than the constitutional band was detected in the ApaLI genomic digest when hybridized with hMLH1 cDNA probe encompassing exons 9–12. This abnormal band did not show a signal when hybridized with hMLH1 cDNA probe spanning exons 10–11 (Figure 2a). Presence of a deletion involving either exon 10, exon 11 or both was thus suspected.

Figure 2

(a) Mapping of hMLH1 deletion. ApaLI genomic digest from patient K89 and control (lane C) hybridized with hMLH1 cDNA probe spanning exons 9–12 (I). An aberrant fragment (arrowhead) around 0.8 kb shorter than the constitutional band is detected in patient K89. This aberrant fragment does not show a signal when hybridized with hMLH1 cDNA probe spanning exons 10 and 11 (II). (The aberrant fragment is subsequently found to result from a 1.8 kb genomic deletion removing an ApaLI site in exon 11). Ten micrograms of control and genomic DNA from patient K89 were digested by ApaLI and subjected to electrophoresis in 0.8% agarose gel. The DNA was transferred to hybond N membrane and hybridized with hMLH1 cDNA probes spanning exons 9–12 or exons 10 and 11. Hybridization and autoradiography were carried out according to conventional methods. (b) Long PCR products containing exons 9–12 and their intervening introns, amplified from the genomic DNA of patient K89 compared with control (lane C). A wild-type amplicon of 11.8 kb is detected in control DNA while DNA from patient K89 shows an additional shorter amplicon of 10 kb (arrow). Lane M, 1 kb DNA ladder. Control and genomic DNA from the patient were amplified using primers 5′-AATGGACAGGCACAGGAGGACC-3′ from intron 8 (161 bp proximal to exon 9) and 5′-TCTCCTCATCTTGCTGCCTAGCC-3′ from exon 12 by the Expand Long Template PCR kit (Boehringer Mannheim, Roche) according to the manufacturer's protocol. Four sets of primers flanking exons 10 and 11, each of around 400 bp apart, were then used to fine map the breakpoint. These primer sequences are available on request. The amplification products were directly sequenced

We next constructed long PCR products extending from intron 8 to exon 12. While specific wild-type amplicon (11.8 kb) was amplified successfully from the normal individual, a unique shortened product (10 kb) was amplified from patient K89 (Figure 2b). We then narrowed the analysis to regions flanking exons 10 and 11. Four different sets of primers, about 400 bp apart from each other, were designed to clone the breakpoint. The deletion was finally mapped to the region encompassing exon 11 but sparing exon 10. An amplicon with size 1.8 kb shorter than the expected wild-type product was observed with different sets of primers flanking the same region. The break points were confirmed by direct DNA sequencing of the PCR product and was located at 597 bp upstream and 1120 bp downstream of exon 11 (Figure 3b). It is interesting that this deletion involves exon 11 only, but eventually leads to splicing out of exon 10 as well, resulting in a mRNA identical to one of the commonest splice variants.

Figure 3

(a) Schematic representation of the proposed mechanism of recombination. The proximal 2.1 kb of intron 10 is joined to the distal 4.2 kb of intron 11 at a 3-bp homologous region (TAT) where the recombination has occurred. It is possible that Alu no. 1 paired with Alu no. 3 to form a hair-pin-like structure. This may facilitate the pairing between introns 10 and 11 where a 47% homology can be detected in region I and a 45% homology in region II. (b) Direct DNA sequencing of the PCR product amplified across the breakpoint region

As this is a novel mutation which is easily missed by RT–PCR as well as direct DNA sequencing, we designed a duplex PCR reaction using primers around the breakpoint region, to simultaneously amplify fragments specific for the normal and mutated allele. We screened the genomic DNA from 13 patients whose colorectal cancers were both MSI and had lost the hMLH1 protein as detected by immunohistochemistry. An identical mutant allele was found in one of the patients (patient K116) (Figure 4). This mutated PCR fragment was subsequently sequenced and confirmed to contain the same breakpoint fusion product as patient K89. Amplification of the hMLH1 cDNA spanning exons 6–12 from the normal colonic tissue of this patient showed only a mild increase in intensity of the Δ10–11 splice variant, which was indistinguishable from the case-to-case variation in intensity of this splice variant in normal individuals (Figure 1a). However, when we examined the RNA extracted from his microdissected tumour tissue, again the wild-type transcript was absent. The genomic deletion was further confirmed by Southern-blot and long PCR.

Figure 4

(a) Schematic representation of the primers used for detecting the exon 11 deletion mutation in hMLH1. (b) Lanes 1–13, amplification products from genomic DNA of patients with hMLH1 protein-negative MSI colorectal cancer. Methods and criteria used for detection of MSI and immunohistochemical staining for hMLH1 protein were previously described (Leung et al., 1998, 1999). Lane 4 is from patient K116 who shows the mutated allele (119 kb) in addition to the wild-type allele (140 bp). DNA from K89 is used as positive control; lane M, Bluescript/MspI marker. Duplex PCR was designed to simultaneously amplify the wild-type and the novel junctional fragment. Forward primer (1) 5′-GCCCAAGGTCACACAAAGAA-3′ is located in intron 10 (677 bp proximal to exon 11), forward primer (2) 5′-GGAGGAAGAAGTTCTGTCTGGA-3′ in intron 11 (1019 bp distal to exon 11) and reverse primer (3) 5′-TGTGTGGCCTCATCTCATCC-3′ in intron 11 (1159 bp distal to exon 11). Primers (2) and (3) amplified a 140 bp fragment specific for the normal allele. Primers (1) and (3) amplified a 119 bp fragment specific for the mutated allele

Patient K89 had a family history of colorectal cancer satisfying the Amsterdam criteria. He developed colorectal cancer at the age of 33. His father and his paternal aunt had colorectal cancer at the age of 62 and 45 respectively, and both died of the disease. Patient K116 did not have a family history of cancer but he developed colorectal cancer at the age of 38. They were descended from two unrelated families. Haplotype analysis was then performed on these patients and their relatives using five microsatellite markers spanning 0.2–7.2 cM around hMLH1 (Figure 5). As patient K89 was homozygous in four of the five markers, the phase in the one remaining mapped marker (D3S3564) could not be known with certainty, but by comparison with one unaffected sib, the most likely disease haplotype was reconstructed. This haplotype was absent in screening of 15 child–parent pairs from local familial adenomatous polyposis/HNPCC families without this mutation. Thus, we can conclude that the haplotype associated with this deletion is uncommon in Hong Kong Chinese. Although reconstruction of the disease haplotype was not possible in patient K116 because of the unavailability of a family member, all disease alleles of patient K89 were present in patient K116. Moreover, this constellation of alleles was not detected in the 15 child–parent pairs without the mutation. Thus, it is highly likely that patients K89 and K116 shared a common ancestral mutation.

Figure 5

Reconstructed disease haplotypes of two patients with germline hMLH1 exon 11 deletion. Marker loci are shown from telomere to centromere. Patient K89 shows one identical haplotype with his sib who does not harbour the mutation, so the remaining haplotype 33353 is the most likely disease haplotype. This disease haplotype is not seen in 45 control haplotyes from the same population. Patient K116 contains all alleles in this disease haplotype. Five polymorphic microsatellite markers (D3S3718, D3S1612, D3S1611, D3S1298, D3S3564) located within and flanking the hMLH1 gene were used. The distances between these marker loci and their most likely order were calculated based on the Genethon Map obtained from the UniSTS database of the National Center for Biotechnology Information, NIH. All forward primers were tagged with M13(-21). PCR were performed in the presence of fluorescent dye-labelled M13(-21) primer and analysed using the Applied Biosystems automated sequencer 377 using method as described (Schuelke, 2000). Alleles were numbered consecutively according to increasing size

Alu elements have been repeatedly implicated in human gene rearrangement, including two previously reported large genomic deletions of hMLH1 (Nystrom-Lahti et al., 1995; Mauillon et al., 1996). Whilst the exact mechanism leading to deletion of exon 11 in our patients is not certain, intron 10 is rich in Alu repetitive elements and the breakpoint in intron 10 was located 31 bp upstream of the third Alu (Figure 3a). The first and third Alu repeats in intron 10, which share an 85% homology, are arranged in opposite orientation. We speculate that they may pair up to form a 2 kb long hair-pin loop which brings the two breakpoints in introns 10 and 11 close together and helps in the recombination event. Also there are regions around the breakpoint where a 45–47% homology can be detected.

The most important conclusion of this study is the finding of a large genomic deletion in hMLH1 that can lead to generation of a transcript mimicking alternative splicing. As there is some variation in the intensity of the Δ10–11 splice variant even in normal individuals, this specific mutation would not be easily discernible in the RNA level using RT–PCR-based detection method. Neither would it be detectable using PCR-based genomic sequencing. In our cases, examination of the microdissected tumour RNA has provided substantial help. The 2nd hit in the tumour has led to loss of the wild-type allele, and thus unmasked the germline mutation. Interestingly, although deletion of exon 11 is the germline event, there is no observable transcript with deletion of exon 11 in the RT–PCR assay. The mechanism underlying generation of the full-length versus alternatively spliced variants remains poorly understood. It is possible that some sequences within the deleted introns 10 and 11 may play an important role in generation of the full-length transcript, without which the splicing machinery preferentially generates the Δ10–11 transcript. Alternatively, it is not unusual for deletion of one exon to lead to splicing out of the adjacent exons. A similar situation has been reported previously where deletion of exons 13–16 of hMLH1 results in splicing out of exon 17 in a proportion of transcripts (Mauillon et al., 1996).

Mutation detection remains a great challenge in HNPCC families. In our previous series of early-onset colorectal cancers with MSI, the majority of germline mutations were detected in hMSH2 (Chan et al., 1999). This differs from the mild preponderance of hMLH1 over hMSH2 mutations in most other populations (Peltomaki and Vasen, 1997). The results may suggest a differential involvement of these two genes in different populations, or alternatively, the hMLH1 mutations in the Chinese population may take forms that are not easily detectable using our current strategy. Most mutations in hMSH2 and hMLH1 reported to date are point mutations, small deletions or splice site mutations (Peltomaki and Vasen, 1997). Large deletions are relatively uncommon. This may be an artefact of the methodology commonly used, which is unable to detect this type of mutation. Indeed, a 3.5 kb genomic deletion including exon 16 constitutes one of the commonest mutations due to a founder effect in Finnish putative HNPCC kindreds (Nystrom-Lahti et al., 1995, 1996). More recently, there are increasing data on the presence of large genomic re-arrangements such as deletion or duplications affecting hMSH2 (Wijnen et al., 1998; Charbonnier et al., 2000). Moreover, there are some preliminary data that suggest segregation of specific alternative splicing pattern of hMLH1 with disease phenotypes in HNPCC families without an identifiable genomic mutation (Stella et al., 2000).

Although the exact incidence of this specific 1.8 kb deletion of hMLH1 in Chinese and other populations are not yet known, our findings broaden the mutational spectrum of hMLH1, and caution investigators in the interpretation of alternative splicing. As most current PCR-based mutation detection methods tend to miss this deletion, we advocate the addition of steps to screen for this specific alteration by our duplex-PCR method in putative HNPCC families.


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We thank Professor Andrew H Wyllie for useful discussions and comments. We also thank Professor John Wong, Professor JST Sham, Dr David Higgins and Dr RJ Collins for their support, Mr Ivan SH Ho for his technical assistance, and Dr N Trendell-Smith for his help in the preparation of this manuscript. This work was supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region (HKU 7330/00M), a donation from the Hong Kong Cancer Fund and a donation from the Hong Kong Society of Gastroenterology.

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Correspondence to Suet Yi Leung.

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  • hMLH1
  • germline mutation
  • intragenic deletion
  • alternative splicing
  • HNPCC syndrome

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