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`# 2002 Oxford University Press
`
`Human Molecular Genetics, 2002, Vol. 11, No. 23
`
`2961–2967
`
`Biallelic germline mutations in MYH predispose
`to multiple colorectal adenoma and somatic
`G:C!T:A mutations
`
`Siaˆ n Jones1, Paul Emmerson1, Julie Maynard1, Jacqueline M. Best2, Sheila Jordan1,
`Geraint T. Williams2, Julian R. Sampson1 and Jeremy P. Cheadle1,*
`
`1Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK and
`2Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK.
`
`Received July 26, 2002; Revised and Accepted September 3, 2002
`
`We have recently demonstrated that inherited defects of the base excision repair gene MYH predispose to
`multiple colorectal adenomas and carcinoma. Three affected siblings from a single British family were
`identified as Y165C/G382D compound heterozygotes and both missense mutations were shown to be
`functionally compromised. Here, we report the identification of seven further unrelated patients with >100
`colorectal adenomas (six with colorectal cancer) and biallelic germline mutations in MYH: four were
`homozygous for truncating mutations, two were homozygous for Y165C and one was a Y165C/G382D
`compound heterozygote. As predicted from studies of the bacterial and yeast orthologues of MYH, colorectal
`tumours from affected individuals displayed a significant excess of somatic G:C!T:A mutations in APC, as
`2¼ 242.96, P < 10
`2¼ 194.85, P < 10
`20) or FAP-associated ( v
`20) colorectal tumours.
`compared to sporadic ( v
`the somatic G:C!T:A mutations was predominantly AA,
`The sequence immediately downstream of
`irrespective of the nature of the germline MYH mutations. These findings confirm the role of MYH in
`colorectal adenoma and carcinoma predisposition.
`
`INTRODUCTION
`
`Inherited factors are thought to play a major role in at least 15%
`of colorectal cancers (CRC), but established CRC predisposi-
`tion genes account for only a minority of these (1). Familial
`adenomatous polyposis (FAP) (MIM 175100) is an autosomal
`dominant disorder
`associated with the development of
`hundreds or thousands of colorectal adenomas, some of which
`progress to cancer. It is caused by inherited mutations in the
`adenomatous polyposis coli
`(APC ) gene that acts as a
`gatekeeper
`regulating proliferation of colonic cells
`(2).
`Attenuated FAP (AFAP) is associated with smaller numbers
`of adenomas and is caused by mutations in the extreme 50 or 30
`ends of APC or in the alternatively spliced region of exon 9 (2).
`Hereditary non-polyposis CRC (HNPCC; MIM 114500) is a
`distinct autosomal dominant disorder characterized by a family
`history of early-onset CRC and other cancers in the absence of
`florid polyposis. It is caused by inherited deficiencies in the
`mismatch repair (MMR) pathway (3).
`Until recently,
`inherited deficiencies in the base excision
`repair (BER) pathway had not been causally linked with any
`human genetic disorder. The BER pathway plays a significant
`role in the repair of mutations caused by reactive oxygen
`
`species that are generated during aerobic metabolism (4).
`8-oxo-7,8-dihydro20deoxyguanosine (8-oxoG)
`is
`the most
`stable product of oxidative DNA damage (5) and readily
`mispairs with A residues (6), leading to G:C!T:A mutations
`in repair-deficient bacteria and yeast (7–10). In Escherichia
`coli, three enzymes help protect cells against the mutagenic
`effects of guanine oxidation (8). MutM Glycosylase removes
`the oxidized base from 8-oxoG:C base pairs in duplex DNA,
`MutY glycosylase excises A residues misincorporated oppo-
`site unrepaired 8-oxoG during replication, and MutT, an
`8-oxo-dGTPase, prevents the incorporation of 8-oxo-dGMP
`into nascent DNA. Homologues of mutM, MutY and mutT have
`been identified in human cells and termed OGG1 (11),
`MYH (12) and MTH1 (13), respectively. MYH interacts with
`proteins involved in long-patch BER (14) and is associated
`with the replication foci, suggesting a role in replication-
`coupled repair (15).
`We previously studied a British Caucasian family with three
`affected siblings with multiple colorectal adenomas and
`carcinoma and excluded an inherited defect of the APC or
`MMR genes (16). We showed that the siblings were compound
`heterozygotes for
`the functionally compromised missense
`mutations Y165C and G382D in MYH. Colorectal tumours
`
`*To whom correspondence should be addressed at: Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff,
`CF14 4XN, United Kingdom. Tel: þ44 2920742652; Fax: þ44 2920746551; Email: cheadlejp@cardiff.ac.uk or sampson@cardiff.ac.uk
`
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`Human Molecular Genetics, 2002, Vol. 11, No. 23
`
`from these individuals exhibited a preponderance of somatic
`G:C!T:A transversions. We now describe the identification of
`seven further unrelated patients with multiple colorectal
`adenomas (six with colorectal carcinoma) and biallelic germ-
`line MYH mutations, including four cases homozygous for
`truncating mutations. Colorectal tumours from these indivi-
`duals exhibit a significant excess of somatic G:C!T:A
`mutations, as compared to sporadic and FAP-associated
`tumours, confirming that biallelic mutations in MYH pre-
`dispose to colorectal adenomas and carcinoma.
`
`RESULTS
`
`Biallelic germline mutations in MYH
`
`We sequenced the entire open reading frame (ORF) of MYH in
`twenty-one unrelated patients with multiple colorectal adeno-
`mas with or without carcinoma. We identified seven patients
`with biallelic mutations (Figure 1, Table 1), six of whom were
`presumed to be homozygous for MYH variants since no wild
`type allele could be detected upon sequence analysis. One
`Pakistani case (MA27) was homozygous for
`the exon 3
`nonsense mutation Y90X (270 C!A); two British Caucasian
`cases (MA22 and MA34) were homozygous for the exon 7
`missense mutation Y165C (494 A!G); one British Caucasian
`case (MA25) was compound heterozygous for Y165C/G382D
`(1145 G!A); and three cases from different unrelated Indian
`families (MA20, MA24 and MA26) were homozygous for the
`exon 14 nonsense mutation E466X (1396 G!T). All but one
`of the cases were sporadic, with no history of colorectal
`adenomas or carcinoma in first degree relatives. MA24 had two
`siblings affected by multiple colorectal adenomas, one of
`whom had CRC, but
`their samples were unavailable for
`analysis. No patients carried single mutant MYH alleles.
`The recurrent MYH variants Y90X (2 mutations, patient
`MA27) and Y165C (5 mutations, patients MA22, MA34 and
`MA25) were found in association with the G allele of the exon
`12 polymorphism 972 C!G (H324Q) and E466X (6
`mutations, patients MA20, MA24 and MA26) was always
`found in association with the C allele. Sequencing of the entire
`ORFs of OGG1 and MTH1 in the fourteen MYH negative
`cases, did not identify any likely pathogenic changes.
`
`Phenotypes of patients with MYH mutations
`
`All seven cases presented with symptoms and signs attributable
`to CRC or colorectal polyposis (diarrhoea, bleeding, anaemia,
`weight loss or abdominal pain) between 36 and 65 years of age.
`Six had over 100 separate macroscopic polyps (one had >400)
`and another had a cancer and 25 adenomas in only 22 cm of
`resected bowel. None had extracolonic signs of FAP or a
`history of other extracolonic tumours.
`
`Identification of somatic G:C!T:A mutations in
`colorectal tumours
`
`Using denaturing high performance liquid chromatography
`(dHPLC) analysis, we tested for somatic mutations in the APC
`gene in 108 colorectal tumours from the seven patients with
`
`biallelic germline mutations of MYH (Table 2). We screened a
`region of APC spanning codons 653–1589 which encompassed
`the mutation cluster region (MCR, codons 1286–1513, ref. 17),
`a known hotspot for somatic mutations (2). In total, 50 somatic
`mutations were identified of which 49 (98%) were G:C!T:A
`transversions creating nonsense codons (Figure 2, Table 2).
`We compared the proportion of somatic G:C!T:A trans-
`version mutations in APC that were detected in colorectal
`tumours from patients with biallelic mutations of MYH, to a
`database of 503 reported somatic APC mutations from sporadic
`colorectal adenomas and carcinomas and 308 somatic muta-
`tions from FAP associated colorectal tumours (16). The excess
`of somatic G:C!T:A transversions in patients with biallelic
`MYH mutations was highly significant (49/50 versus 49/503,
`w2¼ 242.96, P < 10
`20; and 49/50 versus 30/308, w2¼ 194.85,
`20, respectively).
`P < 10
`
`Sequence surrounding the somatic
`G:C!T:A mutations
`
`Examination of the sequence context surrounding the 49
`somatic G:C!T:A mutations revealed that
`the two bases
`immediately 30
`to the mutated G were almost always AA,
`irrespective of the nature of the germline MYH mutations
`(Table 2). The preponderance of G:C!T:A mutations at GAA
`sequences is significant, since other sequences that could
`undergo G:C!T:A mutation to stop codons are highly
`prevalent in the region of APC assayed for somatic mutations
`(83 GAA sites versus 67 non-GAA sites, w2¼ 20.07,
`P¼ 7.5 10
`6).
`
`DISCUSSION
`
`in a single family,
`We have previously demonstrated that,
`compound heterozygosity for the missense mutations Y165C
`and G382D in MYH was associated with multiple colorectal
`adenoma and carcinoma. Functional analysis of the equivalent
`mutations
`in E. coli MYH showed that
`these changes
`significantly compromised adenine glycosylase activities with
`both 8-oxoG:A and G:A substrates (16). In this study, we
`identified another patient compound heterozygous for Y165C/
`G382D and two patients homozygous for Y165C. More
`significantly, we also report the identification of four unrelated
`patients homozygous for nonsense mutations in MYH. The
`absence of any history of colorectal adenomas or carcinoma in
`the fourteen obligate heterozygote parents and the occurrence
`of similar phenotypes in two siblings of one index case, is
`consistent with transmission of colorectal polyposis due to
`MYH mutation as an autosomal recessive trait. Together with
`the highly significant excess of somatic G:C!T:A mutations
`in tumours from these patients,
`this data unequivocally
`confirms that biallelic inactivation of MYH predisposes to
`colorectal adenoma and carcinoma.
`The recurrent mutations Y90X, Y165C and E466X cannot be
`readily explained in terms of the well characterized mecha-
`nisms of hypermutagenesis and are associated with specific
`(and different) alleles of the polymorphism 972 C!G in exon
`12 of MYH. We therefore speculate that these mutations are not
`independent mutational events, but are likely to be derived
`from the same ancestral chromosomes. In total, we have
`
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`Table 1. Biallelic germline mutations of MYH in patients with multiple
`colorectal adenomas
`
`Table 2. Somatic APC mutations in colorectal tumours from patients with
`germline MYH mutations
`
`Human Molecular Genetics, 2002, Vol. 11, No. 23
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`Sequence contextb
`
`TGAAGAG*
`TGAAGAG*
`TGAAGAG*
`TGAATGT*
`AGAAGAT
`TGAAGAG*
`TGAAGAG*
`TGAAATA
`AGAAGAT
`AGAACAG
`TGAAGAG*
`TGAAAAG
`TGAAATA
`AGAAAAA
`TGAAAAG
`AGAAGAG
`TGAAGAG*
`NA
`AGAATTA
`TGGAATG
`TGAATTT*
`TGAAGAG*
`TGAAGAA*
`TGAAAAC
`TGAATGT*
`TGAAAAG
`TGAAATA
`TGAATGT*
`TGAAATA
`TGAATGT*
`TGAATGT*
`AGAACAG
`AGAATCA
`AGAATAC
`TGAAAAG
`TGAACAC
`AGAAGAT
`AGAATCA
`TGAAGAT
`TGAACAC
`TGAAGAT
`TGAAAAG
`TGAAGAG*
`TGAAAAG
`AGAAGAA
`TGAAAAC
`TGAAGAT
`AGAAGAG
`TGAAATA
`TGAAAAT*
`
`S836X
`S836X
`S836X
`S932X
`E1265X
`S836X
`S836X
`E1286X
`E1265X
`E1151X
`S836X
`E1560X
`E1059X
`E1554X
`E1461X
`E1156X
`S836X
`R1450X
`E763X
`G1412X
`S943X
`S836X
`S1356X
`E1547X
`S932X
`E1461X
`E1286X
`S932X
`E1059X
`S932X
`S932X
`E1151X
`E1345X
`E955X
`E1560X
`E1374X
`E1265X
`E1544X
`E1317X
`E1374X
`E1284X
`E1560X
`S836X
`E1560X
`E1155X
`E1547X
`E988X
`E1156X
`E1573X
`S1196X
`
`Nucleotide change Amino acid change
`2507 C!A
`2507 C!A
`2507 C!A
`2795 C!A
`3793 G!T
`MA27_18A 2507 C!A
`2507 C!A
`MA27_18B
`3856 G!T
`MA27_34A 3793 G!T
`3451 G!T
`MA27_40
`2507 C!A
`MA22_3
`4678 G!T
`3175 G!T
`4660 G!T
`4381 G!T
`MA22_7A
`MA22_10A 3466 G!T
`2507 C!A
`MA22_10B
`4348 C!T
`MA22_12A 2287 G!T
`4234 G!T
`2828 C!A
`MA22_12B
`MA22_13A 2507 C!A
`4067 C!A
`MA22_13B
`4639 G!T
`MA22_15A 2795 C!A
`4381 G!T
`3856 G!T
`2795 C!A
`3175 G!T
`2795 C!A
`2795 C!A
`3451 G!T
`4033 G!T
`2863 G!T
`4678 G!T
`4120 G!T
`MA20_6
`3793 G!T
`MA20_7
`4630 G!T
`MA20_9A
`3949 G!T
`MA20_10
`MA20_11A 4120 G!T
`3850 G!T
`MA20_12B
`4678 G!T
`MA24_1B
`2507 C!A
`MA24_15C
`4678 G!T
`3463 G!T
`4639 G!T
`2962 G!T
`3466 G!T
`4717 G!T
`3587 C!A
`
`Samplea
`
`MA27_1
`MA27_12
`MA27_17c
`
`MA22_5
`
`MA22_15B
`MA22_16
`MA22_23
`MA34_2D
`MA25_20C
`MA20_4
`
`MA20_5A
`
`MA26_2
`
`MA26_3
`MA26_6
`
`MA26_9
`
`aWe analysed 26 adenomas from MA27 (mutations identified in MA27_1, 12,
`17, 18A, 18B, 34A and 40), 18 adenomas from MA22 (mutations identified in
`MA22_3, 5, 7A, 10A, 10B, 12A, 12B, 13A, 13B, 15A, 15B, 16 and 23), 6
`adenomas from MA34 (mutation identified in MA34_2D), 25 adenomas from
`MA25 (mutation identified in MA25_20C), 15 adenomas in MA20 (mutations
`identified in MA20_4, 5A, 6, 7, 9A, 10, 11A and 12B), 9 adenomas from
`MA24 (mutations identified in MA24_1B and 15C), and 9 adenomas from
`MA26 (mutations identified in MA26_2, 3, 6 and 9).
`bSequence context surrounding the G:C!T:A mutations (italicised) (sequence of
`the non-transcribed strand is shown except for those marked with *).
`cThree somatic mutations were identified in MA27_17; this is likely to reflect a
`mixed population of tumour cells. NA, not applicable.
`
`Patient
`
`MA27
`
`MA22
`
`MA34
`
`MA25
`
`MA20
`
`MA24
`
`MA26
`
`Exon
`
`3
`3
`
`7
`7
`
`7
`7
`
`7
`13
`
`14
`14
`
`14
`14
`
`14
`14
`
`Nucleotide change
`270 C!A
`270 C!A
`494 A!G
`494 A!G
`494 A!G
`494 A!G
`494 A!G
`1145 G!A
`1396 G!T
`1396 G!T
`1396 G!T
`1396 G!T
`1396 G!T
`1396 G!T
`
`Amino acid change
`
`Y90X
`Y90X
`
`Y165C
`Y165C
`
`Y165C
`Y165C
`
`Y165C
`G382D
`
`E466X
`E466X
`
`E466X
`E466X
`
`E466X
`E466X
`
`identified four British families that are either homozygous for
`Y165C or compound heterozygous for Y165C/G382D, three
`Indian families that are homozygous for E466X and a single
`Pakistani family that is homozygous for Y90X. Specific muta-
`tions in MYH are likely to be identified in different ethnic
`populations, consistent with founder effects and diagnostic
`screening strategies will have to be optimized accordingly. A
`question still remains as to how frequently MYH mutations
`contribute to the phenotype of apparently sporadic AFAP/FAP
`and further analyses of patients from distinct geographical and
`ethnic populations will help to resolve this issue.
`In a previous study of colorectal
`tumours from Y165C/
`G382D compound heterozygotes, we found that all somatic
`APC coding region G:C!T:A mutations were followed by two
`adenine bases (16). In this study, we have confirmed that the
`sequence immediately downstream of somatic G:C!T:A
`transversions
`is predominantly AA,
`irrespective of
`the
`nature of the germline mutations in MYH. Additional studies
`are therefore warranted to determine the basis of
`this
`sequence specificity, which may reflect
`susceptibility to
`guanine oxidation or defective recognition and/or repair by
`mutated MYH.
`likely
`As in our previous study (16), we did not detect
`pathogenic variants in the BER genes OGG1 or MTH1, in cases
`with multiple colorectal adenomas and carcinoma. It is possible
`that these genes are less frequently mutated than MYH, but
`cause a similar phenotype (as is seen with the MSH6, MSH3
`and MSH2 genes in HNPCC; ref. 3), or,
`the phenotype
`associated with inactivation of OGG1 or MTH1 may be unlike
`MYH-deficiency. It is also possible that mutations in OGG1 or
`MTH1 do not predispose to tumours in humans due to
`functional redundancy. Mouse models have provided only
`limited clues as to the function of these genes since Mth1-
`deficient mice display greater numbers of tumours in the lungs,
`liver and stomach compared to their wild-type littermates (18)
`and Ogg1-deficient mice do not exhibit an excess of tumours
`(19,20). Further studies in humans are therefore necessary to
`determine whether OGG1 or MTH1 play a role in CRC
`predisposition.
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`Figure 1. Identification of biallelic germline mutations in MYH in patients with multiple colorectal adenoma and carcinoma. Normal sequences are shown on the
`left with corresponding mutant sequences shown on the right. Sequences are shown in the forward direction except for (A), and arrows indicate the positions of the
`mutations. (A) Patient MA27 was homozygous for Y90X (270 C!A) in exon 3; (B) Patient MA22 was homozygous for Y165C (494 A!G) in exon 7; and
`(C) Patient MA26 was homozygous for E466X (1396 G!T) in exon 14.
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`Human Molecular Genetics, 2002, Vol. 11, No. 23
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`2965
`
`Figure 2. Identification of somatic G:C!T:A mutations in APC in colorectal tumours from patients with biallelic germline MYH mutations. dHPLC elution pro-
`files are shown on the left (vertical hashed lines indicate collection boundaries) and corresponding sequences are shown on the right. Arrows indicate the positions
`of the mutant peaks on the dHPLC and sequencing traces (shown in the forward direction). Although the majority of somatic mutations could be clearly resolved by
`direct sequencing of the unfractionated PCR products (for example (A) 4678 G!T in MA20_5A and (B) 4120 G!T in MA20_6), the resolution of some changes
`was substantially enhanced by isolating and sequencing dHPLC separated heteroduplexes (for example (C) 4067 C!A in MA22_13B).
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`Human Molecular Genetics, 2002, Vol. 11, No. 23
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`MATERIALS AND METHODS
`
`Samples
`
`We analysed twenty-one unrelated index cases with multiple
`(>10) colorectal adenomas with or without colorectal cancer.
`No patients harboured truncating mutations in exon 4 or the
`alternatively spliced region of exon 9 of APC (normally
`associated with AFAP). DNA was prepared from venous blood
`samples and from colorectal adenoma and carcinoma tissue
`that had been micro-dissected from paraffin blocks. The nature
`of all tissues was verified histologically.
`
`PCR amplification
`
`We amplified exons 1–16 of MYH, 1–8 of OGG1 and 2–5 of
`MTH1 as 16, 11 and 4 fragments, as previously described (16).
`We amplified a 2.8 kb segment of APC (between codons 653
`and 1589) which encompassed the somatic mutation cluster
`region, as eighteen overlapping fragments. Primer sequences
`are
`available
`at
`http://www.uwcm.ac.uk/study/medicine/
`medical_genetics/research/tmg/projects/hmyh.html
`
`Denaturing high performance liquid chromatography
`(dHPLC) analysis and fraction collection
`
`dHPLC was carried out using the 3500HT WAVE nucleic acid
`fragment analysis system (Transgenomic, Crewe, UK). To
`enhance the formation of heteroduplexes prior to analysis, the
`PCR products were denatured at 94C and reannealed by
`cooling to 50C at a rate of 1C per min. dHPLC was carried
`out at
`the melting temperatures predicted by Wavemaker
`(version 4.1) software (Transgenomic) with a 12% acetonitrile
`(ACN) gradient over 2.5 min (conditions are available at http://
`www.uwcm.ac.uk/study/medicine/medical_genetics/research/
`tmg/projects/myh2.html). Samples displaying aberrant dHPLC
`elution profiles were sequenced directly; those samples without
`clear sequence variations were reanalysed by isolating and
`sequencing dHPLC separated heteroduplexes. Fraction collec-
`tion of heteroduplexes was carried out using a Transgenomic
`FCW-200 in-line fragment collector and products were eluted
`in 8% ACN.
`
`Automated sequencing
`
`Amplification products were purified using the PCR purifica-
`tion kit (Qiagen, Crawley, W. Sussex, UK) and automated
`sequencing was carried out using the Big Dye Terminator
`Cycle Sequencing kit (Applied Biosystems [ABI], Warrington,
`Cheshire, UK) according to the manufacturer’s instructions.
`Sequencing reactions were purified by isopropanol precipita-
`tion and analysed on an ABI PRISM 3100 Genetic Analyser.
`Mutations were described according to the established
`nomenclature system (21).
`
`Assays for sequence variants
`
`All germline mutations in MYH and somatic mutations in APC
`were confirmed by sequencing at least two independent PCR
`
`products and/or dHPLC separated heteroduplexes, in forward
`and/or
`reverse directions. The germline mutations Y90X,
`G382D and E466X in MYH were further confirmed by
`restriction enzyme digestion (using RsaI, BglII and ApoI,
`respectively). The common polymorphism 972 C!G (H324Q)
`in exon 12 of MYH was assayed by sequencing.
`
`Somatic APC mutation database and statistical analysis
`
`We have previously compiled a database of 503 somatic
`mutations observed in sporadic colorectal tumours and 308
`somatic mutations observed in FAP and AFAP associated
`colorectal
`tumours (16). We carried out statistical analyses
`using the chi-squared test.
`
`ACKNOWLEDGEMENTS
`
`We thank I. Tomlinson, D.N. Cooper and N. Thomas for
`helpful discussions, R. Butler for use of a fraction collector and
`N. Al-Tassan, J. Myring, S. Palmer-Smith and M. McDonald
`for assistance with sample preparation. This work was
`supported by the Knowledge Exploitation Fund (ELWA) and
`a CETIC (Centres of Expertise in Technology and Industrial
`Collaboration) award for the W.O.A.
`
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