detection of genetic alterations in hereditary colorectal cancer screening
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Mutation Research 693 (2010) 19–31
Contents lists available at ScienceDirect
Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis
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etection of genetic alterations in hereditary colorectal cancer screening�
arta Pinedaa,1, Sara Gonzáleza,1, Conxi Lázaroa, Ignacio Blancob, Gabriel Capelláa,∗
Translational Research Laboratory and Cancer Genetic Counseling Program, Av. Gran Via de l’Hospitalet 199-203, 08907 L’Hospitalet de Llobregat, Barcelona, SpainInstitut Català d’Oncologia-IDIBELL, Av. Gran Via de l’Hospitalet 199-203, 08907 L’Hospitalet de Llobregat, Barcelona, Spain
r t i c l e i n f o
rticle history:eceived 18 March 2009eceived in revised form 3 November 2009ccepted 10 November 2009vailable online 27 November 2009
a b s t r a c t
There are two major hereditary colorectal cancer syndromes: Adenomatous Polyposis, secondary to APCgermline alterations (FAP, Familial Adenomatous Polyposis) or secondary to MUTYH germline alterations(MAP, MUTYH associated Polyposis), and Lynch syndrome, associated with germline mutations in mis-match repair genes (MLH1, MSH2, MSH6 and PMS2). The elucidation of their genetic basis has depicted anincreasingly complex picture that has lead to the implementation of complex diagnostic algorithms thatinclude both tumor profiling and germline analyses. A variety of techniques at the DNA, RNA and protein
eywords:PC geneamilial Adenomatous Polyposisynch syndromeMR genes
level are used to screen for molecular alterations both in tumor biopsies (microsatellite instability anal-ysis, mismatch repair protein immunohistochemistry, BRAF-Val600Glu detection and MLH1 promoterhypermethylation analysis) and in the germline (point mutation screening, copy number assessment).Also functional tests are more often used to characterize variants of unknown significance. Method-
UTYHutation detectionUTYH associated polyposis
ological issues associated with the techniques analyzed, as well as the algorithms used, are discussed.
© 2009 Elsevier B.V. All rights reserved.
ontents
1. Molecular basis of colorectal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202. Molecular basis of hereditary colorectal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.1. Molecular basis of adenomatous polyposis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.1.1. Familial Adenomatous Polyposis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.1.2. MUTYH associated polyposis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2. Molecular basis of Lynch syndrome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213. Molecular profiling of tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.1. Analysis of microsatellite instability and immunohistochemistry of MMR proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233.2. Detection of p.Val600Glu BRAF mutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233.3. Detection of MLH1 promoter hypermethylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
4. Germline mutation detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234.1. Mutation analysis in adenomatous polyposis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244.2. Mutation analysis in Lynch syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244.3. Future directions in mutation detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
5. Germline epimutations detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256. Assessment of functional effects of unclassified variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
6.1. MMR genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
6.2. APC and MUTYH variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7. Final remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Conflict of interest statement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
� Grant support: This work was supported by grants SAF 06-6084 and 2009-07319 fromundació La Caixa, F05-01 from the Fundació Gastroenterologia Dr. Francisco Vilardell, 20RD/06/0020/1050 and 1051].∗ Corresponding author. Tel.: +34 932607952; fax: +34 932607466.
E-mail addresses: [email protected], [email protected] (G. Capellá).1 These authors contributed equally.
027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.mrfmmm.2009.11.002
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the Spanish Ministry of Education and Science, BM 04-107-0 from the09SGR290 from Generalitat de Catalunya and Spanish Networks RTICCC
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. Molecular basis of colorectal cancer
Colorectal cancer (CRC) is a major medical and public healthhallenge: with nearly 150,000 new cases and 50,000 deathsxpected in 2008, it is the third most common and second leadingause of cancer deaths in the United States [1]. CRC tumorigene-is is driven by the accumulation of a limited number of geneticlterations in oncogenes and tumor supressors genes [2,3], as wells epigenetics events [4]. Three major pathways have been postu-ated as main players in the colorectal carcinogenesis as a sourcef genetic instability: chromosomal instability (CIN), microsatellitenstability (MSI) and CpG island methylator pathway (CIMP).
CIN has been pointed as the responsible for the generation ofpproximately 85% of the sporadic CRCs. Tumors arising throughhis genetic instability pathway present with numerical and struc-ural chromosomic alterations affecting many different actors.
olecular events in the Adenomatous Polyposis Coli gene (APC)uch as point mutations, promoter hypermethylation or allelicoss, as well as other events in genes involved in the same signalransduction pathway (such as CTNNB1, TCF4 and AXIN2) are nec-ssary and sufficient for the development of an adenoma troughhe CIN pathway [5]. Mutations in KRAS, p16INK4a and TP53 havelso been found mutated in the majority of CIN positive tumors6–8].
Microsatellite instability (MSI) is the molecular fingerprint ofutations in mismatch repair (MMR) genes such as MLH1, MSH2,SH6 and PMS2 [9–11]. Approximately 8–15% of sporadic CRCs dis-
lay MSI. Genetic and epigenetic alterations of genes involved inhe MMR system produce an accelerated adenoma to carcinomaequence. MSI introduces genetic instability that is reflected by theumber of secondary mutations in oncogenes and tumor suppres-or genes containing coding and nor-coding microsatellite regions12,13]. Sporadic MSI tumors harbor more frequently mutations inRAF and less frequently in TP53 and KRAS compared to CIN positiveumors [14].
Finally, 10–20% of CRCs present high proportion of hyperme-hylation in several gene promoters such as p16INK4a, p14ARF, APC,GMT, LKB1, MLH1, RASSF1A, CRBP1 y RARB2 [15]. This fact has
ed to propose the existence of a CpG island methylator fenotypeCIMP). This pathway is the third major mechanism introducingenetic instability in CRCs. These tumors are associated with theresence of MSI in sporadic CRC and absence of KRAS mutations14,16].
. Molecular basis of hereditary colorectal cancer
It is assumed that most tumors (85–90%) are secondary to envi-onmental exposure while in 5–10% heredity plays a significantole [17]. There are two main familial colorectal cancer syndromes:
amilial Adenomatous Polyposis (FAP) and Lynch syndrome [18].ere we aim to review genetic diagnostic strategies used in thevaluation of these patients. After a brief introduction to the molec-lar basis of these diseases we will focus on the pros and pitfalls ofhe methodologies used in the molecular testing.able 1ereditary CRC genes.
Gene Chromosomal location N◦ exons
MLH1 3p21.3 19MSH2 2p22-p21 16MSH6 2p16 10PMS2 7p22.2 15APC 5q21-q22 15MUTYH 1p32-1p34 16
earch 693 (2010) 19–31
2.1. Molecular basis of adenomatous polyposis
2.1.1. Familial Adenomatous PolyposisFamilial Adenomatous Polyposis (FAP) is an autosomal domi-
nant hereditary syndrome that accounts for less than 1% of all CRCs[18]. Its classical form is characterized by the presence of 100 ormore adenomatous polyps in the colon and rectum [19] that areusually diagnosed at age 20–30, that leads inevitably to developcolorectal carcinoma by the age of 40–50 years. Various extra-colonic manifestations are also observed, mainly desmoid tumors,osteomas, and upper gastrointestinal polyps. In the last years muchattention has been paid to the attenuated form (AFAP) associatedwith a lower number of polyps, a less aggressive evolution, a laterage of onset and a decreased incidence of extracolonic manifesta-tions [20,21].
APC (Adenomatous Polyposis Coli) tumor suppressor gene islocated at 5q21-q22 (Table 1). The most abundant transcript is8532 bp long divided in 15 exons, where exon 15 accounts for77% of the coding region. APC codifies a multifunctional proteinwith distinct domains [22] that regulates several cellular processes,including transcription, cell cycle control, migration, differentia-tion and apoptosis. The APC protein plays an integral role in theWnt signaling pathway, as it binds and down-regulates �-catenin[23]. When Wnt, a secreted glycoprotein, binds the Frizzled recep-tor a transduction signal is generated that allows �-catenin nuclearaccumulation where it acts as a Tcf4 transactivator. MYC and cyclinD1 have been identified as prominent transcriptional targets of thispathway [24,25]. APC-�-catenin binding also influences cell-cellinteractions by interacting with E-cadherin.
In more than 80% of patients with typical FAP, a mutationcan be identified in the APC gene that constitutively activatesthe Wnt pathway. Pathogenic mutations are scattered through-out the whole coding region although there are two mutationalhotspots (codons 1061 and 1309) accounting for approximately17% and 11% of all germline APC mutations, respectively. A list ofrecurrent and founder mutations in APC gene is shown in Table 2[26–32]. The yield of APC gene mutations is much lower in patientswith AFAP (∼25%; [21]). In contrast with other colorectal hered-itary syndromes no germline epimutations have been identifiedin FAP cases otherwise negative for APC and/or MUTYH alterations[33].
2.1.2. MUTYH associated polyposisA minority of classical and up to 30% attenuated FAP
are secondary to biallelic germline alterations in the MUTYHgene. The human glycosylase MUTYH, together with OGG1 andMTH1, play a major role in the excision of damaged basesfrom DNA (BER, Base Excision Repair). MTH1 hydrolyzes 8-oxo-7,8-dihydroxy-2′-deoxyguanosine triphosphate to 8-oxo-7,8-dihydroxy-2′-deoxyguanosine monophosphate, OGG1 removes
8-oxoG from 8-oxoG·C base pairs, and MUTYH removes the inap-propriate A from 8-oxoG·A base pairs [34]. The identification ofthese mutations lead to the concept of MUTYH associated polyposis(MAP) [35]. The phenotype of MAP is similar to attenuated familialadenomatous polyposis with upper gastrointestinal lesions beingSize open reading frame (bp) N◦ amino acids
2524 7563145 9344320 13602836 8628532 28431888 535
M. Pineda et al. / Mutation Research 693 (2010) 19–31 21
Table 2Recurrent/founder mutations identified in hereditary colorectal cancer genes.
Gene Mutation Founder (F)Recurrent (R)
Estimated age (years) Population Reference
MLH1 c.454-1G > A F 125–525 Finnish [123,124]c.545 + 3A > G F Italian [130]c.1667 + 2 1667 + 8del7ins4 F Danish [121]c.1757 1758insC (p.Met587fs) F Korean [126]c.1831delAT (p.Ile611fs) F Italian-Quebec [130]c.1381A > T (p.Lys461X) F North-American [128]c.2142G > A (p.Trp714X) F >200 Swiss [120]c.2269 2270insT F Italian (north) [111]Exon 11 deletion F Chinese [114]Exon 16 deletion F 400–1075 Finnish [123,124]
MSH2 c.942 + 3A > T F >300 Canadian (Newfoundland) [119]c.1452 1455delAATG (p.Asn486fs) F 550–2575 Chinese (Guangdong) [113]c.1788 1790delAAT (p.Asn596del) F Danish [125]c.1906G > C (p.Ala636Pro) F 200–500 Ashkenazi Jewish [118,129]c.2063T > G (p.Met688Arg) F Spanish (Tenerife Island) [122]Exon 1–6 deletion F ≈500 North-American [116,132]Exon 1–6 deletion F Italian (northeast) [127]c.942 + 3A > T R [117]
MSH6 c.1346T > C (p.Leu449Pro) F Swiss [112]c.2931C > G (p.Trp977X) F Swiss [112]c.2983G > T (p.Glu995X) F Finnish [131]
PMS2 c.736 741del6ins11 (p.Pro246fs) F ≈1625 English/Swiss [116]
APC c.423-1G > A F 200 Newfoundlander [26]c.426 427delAT (p.Leu143fs) F 400 American from England [27]c.3183 3187delACAAA (p.Gln1062fs) F Spanish (Balearic Island) [28]c.3183 3187delACAAA (p.Gln1062fs) R [29]c.3927 3931delAAAGA (p.Lys1310fs) R [30]c.3920T > A (p.Ile1307Lys) F and R Ashkenazi Jews [30–32]
MUTYH p.Tyr90X R Pakistani [105]c.494A > G (p.Tyr165Cys) R Caucasian [34]c.892-2A > G R Japanese [109]
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c.1145G > A (p.Gly382Asp) Rc.1186 1187insGG (p.Glu396fs) Rc.1395 1397delGGA (p.Glu466del) R
etected. However, MAP shows a recessive pattern of inheritancend a lesser number of extraintestinal manifestations.
MUTYH is located at chromosome 1p32-34, has a 1888 bp openeading frame, and 16 exons, encoding a protein of 535 residuesTable 1). Two mutational hot spots were identified in the MUTYHene, p.Tyr165Cys and p.Gly382Asp [34]. These variants seem toe the most common mutations found in Caucasians of northernuropean origin [34] (Table 2). In addition to these variants, approx-mately 82 germline mutations have been found in the MUTYH ofatients with colorectal adenomas and carcinomas [36].
.2. Molecular basis of Lynch syndrome
Lynch syndrome is the most common of the known CRCredisposing syndromes. It was also referred to as Hereditary Non-olyposis Colorectal Cancer (HNPCC). Clarification of the geneticasis and full phenotypic expression of this disease mandates aore clinically useful name that clarifies the consideration of non-
olonic cancers in a family history, and unifies the diagnosis aroundhe germline mutation in a DNA mismatch repair (MMR) gene37,38]. The term ‘Lynch syndrome’ is proposed for the autosomalominant disease caused by a germline mutation in a DNA MMRene: MLH1, MSH2, MSH6 or PMS2 [39,40] (Table 1).
Clinically, Lynch syndrome is an autosomal dominant inher-
ted disease characterized by an early-onset colorectal cancernd an increased risk of other cancers, including cancers of thendometrium, stomach, ovary, upper urinary tract, hepatobiliaryract, pancreas, small bowel, skin and rarely the brain [39]. Ams-erdam II and revised Bethesda criteria (Table 3) are currently usedCaucasian [34]Portuguese [107]Southern European caucasian and Indian [105,106,108]
to select patients at risk of Lynch syndrome [41,42] (Fig. 1). Otherpredictors, i.e. PREMM, MMRpro and Barnetson models, can pro-vide also an estimate of the likelihood of finding MMR germlinemutations [43–45] but no guidelines have endorsed their use yet.Families that meet Amsterdam criteria (Table 3) but do not haveevidence for MMR deficiency should be referred to as having ‘Famil-ial CRC’ [40], also called ‘Hereditary Colorectal Cancer Type-X’ [46].These families have lower incidence of colorectal cancer, later ageof onset and no evidence of increased risk of extracolonic tumors[46].
The human MMR proteins are involved in detection and repairof errors that occur during the DNA replication. MSH2 and MSH6work as heterodimers preferentially recognizing and binding singlebase pair mismatches or short insertion-deletion loops. Throughan energy-requiring process, they interact with DNA at the siteof a mismatch, exchange ADP for ATP, and form a sliding clamparound the DNA. The MLH1–PMS2 heterodimer interacts with theMSH2–MSH6–DNA complex, and together with Exo1 and otherenzymes required, the newly synthesized strand containing theerror is excised and resynthesis is done. Whereas MSH2 and MLH1are essential MMR components, other proteins apart from MSH6and PMS2 can dimerize with them, playing partially redundantroles in MMR (reviewed in [47]).
In carriers of germline MMR mutations, a somatic inactivation
of the MMR wild-type allele is required for tumor development,leaving the cell with a defective MMR system (reviewed in [48]).The multiple errors in repetitive DNA sequences (microsatellites)result from a failure of the DNA mismatch repair system to editthem [10,49]. This mutator phenotype can be revealed by evalua-
22 M. Pineda et al. / Mutation Research 693 (2010) 19–31
Table 3Amsterdam II criteria and revised Bethesda guidelines.
Amsterdam criteria IIThere should be at least three relatives with colorectal cancer (CRC) or with a Lynch syndrome-associated cancer: cancer of the endometrium, small bowel,
ovary, ureter or renal pelvis, brain, hepatobiliary tract and skin (sebaceous tumors).One relative should be a first-degree relative of the other two.At least two successive generations should be affected.At least one tumor should be diagnosed before the age of 50 years.FAP should be excluded in any CRC cases and tumors should be verified by histopathological examination.
Revised Bethesda guidelines1. CRC diagnosed in a patient aged <50 years.2. Presence of synchronous, metachronous colorectal or other Lynch syndrome-related tumors*, regardless of age.3. CRC with MSI-H histologya diagnosed in a patient aged <60 years.4. Patient with CRC and a first-degree relative with a Lynch syndrome-related tumor, with one of the cancers diagnosed at age <50 years.5. Patient with CRC with two or more first-degree or second degree relatives with a Lynch syndrome-related tumor, regardless of age.
n, pana
ction
tn
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* Lynch syndrome-related tumors include colorectal, endometrial, stomach, ovariand keratoacanthomas, and carcinoma of the small bowel.
a Presence of tumor-infiltrating lymphocytes, Crohn disease-like lymphocytic rea
ion of length changes in the microsatellite sequences, a phenotypeamed microsatellite instability (MSI) (see below).
Mutations in MLH1 and MSH2 are the most frequently iden-ified with similar prevalence: 34% and 42%, respectively in aomprehensive study of Amsterdam I families [46] and 44%nd 38%, respectively in the Leiden Open Variation Databasehttp://chromium.liacs.nl/LOVD2/colon cancer/home.php). MSH6
utations are responsible for a smaller fraction of Lynch syndrome,nd tend to be more strongly associated with endometrial can-er and a lower degree of microsatellite instability (reviewed in47]). Mutations in PMS2 are a rare cause of Lynch syndrome [50]lthough they might be underestimated given the technical dif-culty of identifying them [50,51]. Mutations identified in MMRenes are widespreadly distributed within the genes. Recently
LH1 germline epimutations have been reported to be a new causef Lynch syndrome [52–59]. Nonetheless, the heritability of methy-ation is still under investigation. MSH2 germline epimutationsave been also identified that are linked to deletions in the closely
ocated TACSTD1 gene [60,61].
Fig. 1. Strategy for identification of patients with hereditary colorectal cancer. IHC, imm
creas, ureter, renal pelvis, biliary tract and brain tumors, sebaceous gland adenomas
, mucinous/signet-ring differentiation, or medullary growth pattern.
It is noteworthy that patients with biallelic germline muta-tions in one of the MMR genes suffer from a condition that ischaracterized by the development of childhood cancers, mainlyhaematological malignancies and/or brain tumors, as well asearly-onset colorectal cancers. This condition has been termed as“constitutional mismatch repair-deficiency syndrome” (reviewedin [62]).
3. Molecular profiling of tumors
Since most (90%) of Lynch syndrome tumors show microsatelliteinstability (MSI), it is clinically used as a useful screening markerfor identifying those patients at increased risk (a proposed diagnos-
tic algorithm for hereditary colorectal cancer is shown in Fig. 1).However, as mentioned in Section 1, MSI is also involved in thegenesis of 8–15% of sporadic CRC, where it strongly associates withsomatic CpG island hypermethylation of the MLH1 promoter andp.Val600Glu BRAF mutation [63,64].unohistochemistry; MSI, microsatellite instability; MSS, microsatellite stability.
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.1. Analysis of microsatellite instability andmmunohistochemistry of MMR proteins
The standard testing procedure recommended by the Nationalancer Institute/International Collaborative Group on HNPCC forSI testing is the analysis of the tumor and corresponding nor-al tissue by using five microsatellite repeat markers [42]. These 5arkers include 2 quasimonomorphic markers, BAT25 and BAT26,
nd 3 dinucleotide markers, D2S123, D5S346, and D17S250. MSI isefined by the presence of two or more of these markers as mutated,
.e. when new alleles, usually of shorter size, are evident. When onearker is mutated the panel should be expanded to a minimum of
0 markers. MSI is categorized in three groups: MSI-High (MSI-H)hose with ≥30–40% of the markers showing instability; MSI-LowMSI-L) those with <30–40% of the markers showing instability and
S-Stable (MSS) those showing no instability. The existence of aSI-L phenotype is a matter of controversy [65,66].The analysis of BAT26 is sufficient for detecting MSI phenotype
n most, but not all cases, mainly when the two MSH2 alleles areost in the tumor [67]. BAT26 and also BAT25 can be used withouthe corresponding normal tissue, however short alleles of these
arkers can be found, leading to false-positive results [68]. Moreecently, a pentaplex panel of five mononucleotide repeats (BAT26,AT25, NR21, NR22, and NR24) proved to be more sensitive forSI-H identification than other microsatellite markers [68]. The
vailability of a marketed test for this panel amenable for its useith capillary electrophoresis has promoted its use in a wide range
f laboratories although it has not been officially endorsed.Relevant methodological issues in MSI analysis include: (i) the
eed that tumor cell content should be at least 50–70%; (ii) thewareness that, in spite of its long-time clinical use, still someack of definition is present regarding the existence of MSI-L andhe number and type of markers to be assessed; (iii) the limitedsefulness of MSI for MSH6 carrier identification; (iv) the differ-nt information provided by adenomas (less likely to be MSI thanarcinomas) in Lynch syndrome patients; (v) to avoid reportingSI when loss of heterozygosity is present or suspected (vi) to be
autious when a larger size allele is detected; (vii) the usefulnessf duplicate independent scoring of results; and (viii) the need toarticipate, when available, in proficiency testing programs [69].
Immunohistochemical (IHC) analysis in tumor tissue using anti-odies against the MMR-proteins involved in Lynch syndrome mayetect their loss of expression. Since the mismatch repair pro-eins form heterodimeric complexes, the distinct staining patternsbtained can be suggestive of the underlying gene defect. This hasead to promote MMR IHC as an alternative to MSI testing. Among itsdvantages are: (i) its more widespread availability; (ii) the lowerost; and (iii) the potential ability to direct the following muta-ion analysis that is especially relevant for MSH6 staining. HoweverHC has also some limitations including: (i) the low sensitivityf MLH1 staining; (ii) the difficulty in interpreting some patternsuch as focal staining, lack of positive internal control and cyto-lasmic staining; and (iii) the intrinsic limitation associated withhe fact that not all pathogenic mutations result in loss of proteiny IHC [69,70]. In general IHC and MSI testing may be viewed asomplementary in the selection of patients candidate for germlineutation analysis with some differences in what technique is to
e used first depending upon the selection criteria used (Fig. 1). Ofote, scarce evidence is available on the analytical validity of bothethods in the clinical setting [71–73].
.2. Detection of p.Val600Glu BRAF mutation
BRAF, a RAS effector, is a member of the RAF family of proteininases (reviewed by [74]). The most common BRAF mutation cor-espond to a T > A transversion at position 1799, resulting in the
earch 693 (2010) 19–31 23
substitution of Valine by Glutamate at position 600 of the protein(p.Val600Glu, formerly known as p.Val599Glu). This mutation isthought to mimic phosphorylation of Thr 599 and Ser 602, render-ing BRAF constitutively active [14,75].
BRAF somatic mutations have been reported in about 10% of spo-radic colorectal tumors [14,75]. The frequency of the p.Val600GluBRAF mutation is higher in sporadic CRC with MSI and is associatedwith somatic MLH1 promoter hypermethylation [76]. Interestingly,p.Val600Glu BRAF mutation has not been detected in tumors fromindividuals with a germline mutation in MLH1, MSH2 or MSH6 sofar [76,77]. However, it has been recently identified in 3 individu-als with monoallelic PMS2 pathogenic mutations [50]. While it hasbeen proposed the use of p.Val600Glu BRAF screening to excludeLynch Syndrome in MSI tumors [78] (Fig. 1), it may not be applicablein the case of PMS2 mutation carriers.
Detection of p.Val600Glu BRAF mutation has been achievedby several methods. Among methods for scanning BRAF gene,direct sequencing (dideoxy sequencing [75,76] and pyrosequenc-ing [79]) and screening methods in combination with sequencing(Single Strand Conformation Polymorphism [80], heteroduplexanalysis [75] and High-Resolution Melting Analysis [81,82]) arecommonly used. In addition, methods used for specific detectionof p.Val600Glu BRAF mutation includes: Allele-specific PCR [83],Restriction Fragment Length Polymorphism [84] and real-time PCRallelic discrimination [83]. Whichever the method must work effi-ciently in paraffin-embedded tissues, analytical sensitivity shouldbe known prior to its use and adequate positive controls should beincluded in every run.
3.3. Detection of MLH1 promoter hypermethylation
MSI, when associated with Lynch syndrome, is caused bygermline MMR gene mutations rather than promoter hypermethy-lation. Thus, the examination of MLH1 promoter hypermethylationhas been proposed as a tool for the discrimination between spo-radic and hereditary colon cancers similar to BRAF p.Val600Glu[72,85]. However, this subject is currently under intense discussion[86,87]. It has been demonstrated that silencing of MLH1 correlatedwith methylation of the proximal but not the distal region of theMLH1 promoter [88]. However, a high proportion of the publishedstudies have analyzed nonspecific regions [89], being difficult todraw definitive conclusions. A partial discussion on the pros andlimitations of hypermethylation analysis is included below in thegermline epimutation section.
4. Germline mutation detection
Irrespective of the availability of clinical and molecular work-flows to select patients for germline mutation analysis of a givenhereditary colorectal gene (Fig. 1), study of the whole coding regionplus regulatory sequences of the gene is mandatory (Table 1). Com-plete sequencing of these regions would be the gold standard formutation scanning although it is still an expensive technique anddepending on the structure and size of the gene could not beaffordable for all laboratories. In this sense, a variety of screeningmethods could be used as adjuncts to sequencing. Most screen-ing methods are based on properties of heteroduplex (i.e.: DGGE,Denaturing Gradient Gel Electrophoresis [90]; CSCE, ConformationSensitive Capillary Electrophoresis [91]; dHPLC, Denaturing HighPerformance Liquid Chromatography [92]) or properties of single-
stranded DNA (i.e. SSCP, Single Strand Conformation Polymorphism[93]). Recent publications demonstrate the utility of real-timeinstruments to scan for mutations using the High-Resolution Melt-ing approach (HRM) based on the ability to record and evaluatefluorescence intensities as a function of the melting (dissociation)
24 M. Pineda et al. / Mutation Research 693 (2010) 19–31
Table 4Different approaches to screen for known mutations or SNPs.
Approach Instrument Company Assay N◦ SNPs
RFLP Electrophoresis system – RFLP (Restriction Fragment Length Polymorphism) 1TaqMan Real-Time Thermocycler Applied Biosystems 5′exonuclease/PCR 1Melting curve analysis LightCycler Roche PCR-FRET probes (Fluorescence Resonance Energy Transfer) 1–2SNPstream Scanner Beckman Coulter SBE (Single Base Extension) 1–12iPLEX Mass Spectrometer Sequenom ASPE (Allele Specific Primer Extension) 12–36
tcpu
omstudOspr4dcsfnpvetaa
pfadotc
4
rpbdbrhi
pmtrm
different approaches (i.e. long-range PCR, cDNA sequencing) to
SNPlex Capillary Sequencer Applied BiosystemsGoldenGate Scanner High Resolution IlluminaGeneChip Scanner High Resolution AffymetrixInfinium II Scanner High Resolution Illumina
emperature of PCR products [94]. Additionally, the Protein Trun-ation Test (PTT), a method designed to detect truncated proteinsroduced by frameshift or nonsense DNA mutations has also beensed in the analysis of several CRC tumor suppressor genes [95].
Neither, sequencing strategies nor classical screening meth-ds are designed to identify deletions or duplications of single orultiple consecutive exons. To overcome this technical limitation
everal quantitative approaches have been developed. Quantita-ive real-time PCR of the region of interest has demonstratedseful in some instances [96]. In this sense, Multiplex Ligation-ependent Probe Amplification (MLPA) uses the specificity of theLA (Oligonucelotide Ligation Assay) to increase the accuracy of
imple quantitative PCR and is it very popular due to its sim-licity, relatively low cost, possibility of high-throughput andobustness [97]. MLPA is a multiplex technology testing up to5 sequences simultaneously for copy number variations. Probeesigning is time consuming and need expertise but commer-ial probe sets are available for the hereditary colorectal genes. Aimilar approach, Quantitative Multiplex PCR of Short Fluorescentragments (QMPSF), has demonstrated to be useful to detect copyumber variations in CRC genes as well [98,99]. For diagnostic pur-oses, when possible, it is desirable to confirm any copy numberariation with a different methodology and, if possible, search thexact molecular nature of the deletion. Other techniques as conven-ional Southern blotting, Fluorescence In Situ Hybridization (FISH),rray-comparative genomic hybridization (aCGH) or SNP-arraysre used to confirm and detect copy number variations.
Once the mutational pathology of a given gene is known it isossible to design specific cost-effective strategies to screen forounder or recurrent mutations. More than hundred PCR-basedpproaches are currently available. The election of the methodepends on the project and should take into account: the numberf samples to screen, the number and molecular nature of muta-ions, as well as the facilities of the laboratory. Several of the mostommon methods used are summarized in Table 4.
.1. Mutation analysis in adenomatous polyposis
Full gene sequencing of APC coding region is the most accu-ate test available to detect APC mutations [100] (Fig. 1) althoughrotein truncation testing has demonstrated to be useful [101],ecause nearly 94% of APC germline mutations involve the intro-uction of a premature stop codon. For copy number analysesoth MLPA and QMPSF techniques have been used with compa-able results [99]. In some specific areas where founder mutationsave been detected (Table 2) [26–28] molecular analyses could be
nitiated with their study.Most germline APC mutations involve the introduction of a
remature stop codon, either by a nonsense (30%) or frameshiftutation (68%) or gross deletion (5%) [99], leading to truncation of
he protein product in the C-terminal region [102]. The number ofeported characterized APC splice-site mutations is low. Missenseutations can also be detected albeit at low frequency [103,104].
OLA (Oligonucleotide Ligation Assay) 24–48ASPE/OLA 96–1536ASH (Allele-Specific Hybridization) 105–9 × 106
SBE 3.7 × 105–106
Recurrent mutations in MUTYH gene have been identified indistinct populations (Table 2) [34,105–109] allowing the designof sequence specific analysis adapted to geographic areas. Somelaboratories initiate their analysis by screening of local recurrentmutations whereas others prefer to perform complete sequenc-ing instead. When using a screening approach, the identificationof a recurrent mutation in heterozygosis should be followed by acomprehensive sequencing analysis (Fig. 1). An important part ofMUTYH mutations are missense mutations with unknown signifi-cance, presenting a diagnostic challenge.
4.2. Mutation analysis in Lynch syndrome
Over the past years different screening approaches have beendeveloped to identify MMR gene mutations (reviewed in [110]).Several laboratories sequence directly the whole coding region ofinterest due to several facts including: the fact that one can iden-tify the specific gene of interest in a given patient/family on thebasis of immunohistochemistry patterns, the relative medium sizeof these genes (Table 1) and the random distribution of mutationsthroughout the genes, together with the lower prices of sequenc-ing reactions. However, it must be noted that in a recent systematicreview it was concluded that there is very little information avail-able about the analytical validity of tests used in the diagnosis ofHNPCC [71].
A number of recurrent/founder mutations have been identi-fied in MMR genes (Table 2) [111–132]. Mutations usually resultin truncated proteins but large deletions are relatively commonin MSH2 (about 10%), less common in MLH1 and rare in MSH6and PMS2 (Leiden Open Variation Database). Missense mutationsare especially common in MLH1, MSH6 and PMS2 although theyare often also detected in the MSH2 gene (Leiden Open Varia-tion Database). Concerning their biological significance, up to 30%of mutations identified in MMR genes are variants of unknownsignificance.
MLPA screening is one of the most popular approaches todetect copy number alterations of single or multiple exons for itssimplicity and robustness. Results from several groups indicatethat prevalence of copy number alterations varies among genes(reviewed in [133,134]). Confirmation of the identified genomicrearrangements with another technique is a safe strategy to avoidfalse-positive results.
Technical difficulties in the analysis of PMS2 hampered theassessment of the contribution of PMS2 mutations to Lynch syn-drome. PMS2 is located in a complex genomic region with severalpseudogenes on the same chromosome making it difficult toperform mutation analysis. However, recent reports developed
overcome this problem [50,116,135] and conclude than PMS2mutations contribute significantly to Lynch syndrome.
Conversion of diploidy to haploidy by creating somatic cellhybrids has been demonstrated useful to identify cryptic mutationsin MMR genes undetectable by standard methods [136,137].
n Res
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.3. Future directions in mutation detection
Conventional Sanger method technology for sequencing analy-is is starting to be partially supplanted by several next-generationequencing (NGS) technologies with a more cost-effective through-ut. The first three NGS platforms in the market include the54 GS20/FLX system (Roche Applied Science: www.roche-pplied-science.com) [138], the Solexa 1G analyzer (Illumina, Inc:ww.illumina.com) [139] and the SOLiD instrument (Appliediosystems: www.appliedbiosystems.com) [140]. These platformsse different sequencing approaches with variable read lengths.he ultimate goal of these platforms is reduce costs in order toequence a complete genome at an affordable price (ideally, auman genome per 1000 USD).
Although the development of new sequencing methodologiesas brought a huge capacity for DNA sequencing at a very lowost per base, the usefulness and cost-effectiveness of these newechniques for genetic diagnosis remains to be established. NGSpproaches were conceived for sequencing complete genomes; inrder to use them in a genetic diagnosis setting, several techni-al developments are required. First, new sequencing techniqueshould be adapted to sequence one or a discrete number of genes.n these sense several strategies are currently used as multi-lex PCR or custom capture arrays, among others (reviewed in141]). Second, in order to take advantage of sequencing capac-ty and lower the cost per sample, it is important to develop andptimize protocols that allow the inclusion of multiple patientsn the same sequencing reaction as it is the case of the use of
IDs (Multiplex Identifiers) for 454 GS20/FLX system, for exam-le.
In summary, NGS is opening a new era on our understanding of
he human genome and especially it will improve our knowledgebout the main molecular alterations in specific cancer genomes.owever, the use of these new platforms in molecular diagno-is laboratories is still challenging and its implementation shoulde accompanied by a conceptual change, not only in the experi-
able 5eb pages of interest in the assessment of pathogenicity of hereditary colorectal cancer
Database of hereditary colorectal cancer gene variantshttp://www.mmrmissense.org/default.aspx; http://www.mmruv.info It cont
interphttp://www.med.mun.ca/mmrvariants/search.aspx The W
varianhttp://www.insight-group.org/mutations/ The In
has a sMSH2Datab
http://chromium.liacs.nl/LOVD2/colon cancer/home.php In TheMmrm
http://www.hgmd.cf.ac.uk/ac/index.php The H(publi
Impact on splicinghttp://www.fruitfly.org/seq tools/splice.html Splice
and scESEfinder: http://exon.cshl.org/ESE; RESCUE-ESE:
http://genes.mit.edu/burgelab/rescue-eseThese(exon
Impact on the function and structure of the proteinhttp://mappmmr.blueankh.com/ A mul
bioinfmisse
http://genetics.bwh.harvard.edu/pph/ PolyPhaminostraigh
http://blocks.fhcrc.org/sift/SIFT.html SIFT psequenaturamisse
earch 693 (2010) 19–31 25
mental design, but in data analysis and results interpretation aswell.
5. Germline epimutations detection
As previously mentioned, MLH1 germline epimutations havebeen reported to be a new cause of Lynch syndrome [52–59].Germline epimutation provides a mechanism for phenocopyingof genetic disease. The mosaicism and nonmendelian inheritancethat are characteristic of epigenetic states could produce pat-terns of disease risk that resemble those of polygenic or complextraits. These research reports have used a number of techniquesto study this issue, all of them based on bisulphite pre-treatmentof DNA, that converts unmethylated cytosines to uracils, whilemethylated cytosines do not react [142]. All bisulphite-dependentdetection techniques face the challenge of assay robustness basedon their dependence on complete bisulphite conversion. Thehighly sensitive Methylation-Specific PCR (MSP) is probably themost commonly used technique but relies on the analysis ofCpGs residues present in the primer sequence, that are thosemore commonly methylated in tumor cells. COBRA (COmbinedBisulphite-Restriction Analysis) provides semiquantitative databased on the creation of new restriction site after bisulphitetreatment. Both techniques are extremely sensitive to incompleteconversion. In a more recent report MS-MLPA has been used[57]. Here, conventional MLPA results are compared with thoseobtained after bisulphite treatment providing a semiquantitativeanalysis of the methylation status of the region analyzed. In allcases, sequencing of cloned PCR products after bisulphite treat-ment is needed to verify the results [143]. While MLH1 epimutation
may be used in the near future in the clinical setting no MSH2epimutation analyses is envisioned since it has been linked todeletion of the neighbouring gene TACSTD1, opening the doorto the use of MLPA with specific TACSTD1 probes in selectedcases.gene variants.
ains information from functional assays and other type of data to support theretation of MMR gene unclassified variantsoods database is a catalogue of known MMR gene variants including only thosets which have been published in peer-reviewed journalsternational Society for Gastrointestinal Hereditary Tumors (InSiGHT) web pageection containing a database of variants in the following genes: MLH1, MLH3,
, MSH6, PMS1 and PMS2 using the LOVD database style (Leiden Open Variationase)Leiden Open Variation Database entries from the Woods and InSiGHT andissense databases have been merged
uman Gene Mutation Database (HGMD) represents an attempt to collate knownshed) gene lesions responsible for human inherited disease
Site Prediction by Neural Network (SSPNN) is a program capable of recognizingoring potential donor/acceptor splice sitestwo programs are aimed to detect alteration in any putative ESE element
ic splice enhancer) that can alter the correct splicing of a given exon
tivariate analysis of protein polymorphisms-mismatch repair (MAPP-MMR) is aormatic algorithm that effectively classifies MLH1/MSH2 deleterious and neutralnse variantsen (=Polymorphism Phenotyping) is a tool which predicts possible impact of anacid substitution on the structure and function of a human protein usingtforward physical and comparative considerations
redicts whether an amino acid substitution affects protein function based onnce homology and the physical properties of amino acids. SIFT can be applied tolly occurring nonsynonymous polymorphisms and laboratory-induced
nse mutations
26 M. Pineda et al. / Mutation Research 693 (2010) 19–31
ction
6
6
gtlmmv
botaisis
Fig. 2. Proposed algorithm for the fun
. Assessment of functional effects of unclassified variants
.1. MMR genes
Most reported mutations in Lynch syndrome are detected in sin-le families and create truncating proteins. However, up to 30% ofhe aberrations found are missense variations with unclear bio-ogical significance [144] that might affect protein function and/or
RNA processing [145]. As the magnitude of the problem grows,ore attention is paid. A number of initiatives are ongoing to pro-
ide web-based updated information on variants (Table 5).Assessment of pathogenicity is a complex strategy that com-
ines several in silico and experimental procedures in differentrders (a proposed algorithm is shown in Fig. 2). It is of importanceo evaluate its putative effect on splicing influence using web-based
lgorithms that predict effects of variants on splice sites (reviewedn [146]; Table 5). This may occur by influencing consensus spliceites, forming cryptic splice sites, or altering binding sites for splic-ng enhancer proteins. cDNA analyses are advised to confirm theuspected impact on mRNA processing. Also, bioinformatics algo-al analysis of variants in MMR genes.
rithms are used to predict the functional impact on the nature of thechange and the evolutionary conservation of the affected residue(Table 5). Analysis of co-segregation within a family and frequencyin control populations is then of help when no prior information isavailable (Fig. 2).
If splice alterations are not suspected and the MMR gene vari-ant is not silent, a dysfunctional protein may or may not result.Some single amino acid substitutions can give rise to partly active,dominant negative, unstable, or nonfunctional proteins dependingon the nature of the individual mutation. It is not surprising that amultitude of assays in different experimental systems have beendeveloped to investigate their functional impact [147]. Cell-freeMMR assays [148–151] have been considered the gold standardfor assessing pathogenicity, despite the absence of their completevalidation. However, results indicating that a variant is deficient in
MMR can be considered strong evidence of pathogenicity with noclear recommendation about its clinical use. Importantly, a numberof variants may display normal activity in MMR in vitro assays. Inthis case, additional characterization may include detailed studiesof MMR protein stability, protein–protein interactions, and translo-
n Res
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M. Pineda et al. / Mutatio
ations in the cell (Fig. 2). These analyses are complex and must bearried out in specialized laboratories.
.2. APC and MUTYH variants
Functional assays have been occasionally used to assess theirathogenicity. In contrast with MMR genes where its transitiono the clinical setting is progressing, they should be considered a
atter of research. For APC variants assessment evaluation of theirmpact on �-catenin-mediated expression have been used alone103] or in combination with other techniques including �-cateninubcellular distribution and affinity binding experiments [152].
The most common MUTYH biallelic germlines mutations in Cau-asians (p.Tyr165Cys and p.Gly382Asp) and two additional variantsp.Arg227Trp and p.Val232Phe) associated with MAP have beeniochemically characterized [153–156]. The variants p.Tyr165Cys,.Gly382Asp, and p.ArgR227Trp expressed in E. coli are defective
n adenine glycosylase activity on A/GO mismatches and unableo complement the mutator phenotype of a mutY mutation. Onhe other hand MUTYH p.Val232Phe has reduced enzyme activityoth in vitro and in vivo. Of importance, functional characteriza-ion must also consider putative effect on splicing, nature of thehange and the evolutionary conservation of the affected residue,nalysis of co-segregation within a family and frequency in controlopulations.
. Final remarks
In the last 20 years a wealth of knowledge on the molecularasis of cancer, and of colorectal cancer in particular, has beenccumulated. The identification of the molecular basis of heredi-ary colorectal cancer has permitted the development of the geneticesting for the disease fostering the field of cancer genetic counsel-ng.
While we know more on the disease and its genetics a moreomplex picture is depicted. Initial expectations about the identi-cation of the underlying genetic defect in all familial cases haveot been met but an increasing proportion of familial cases benefits
rom molecular analysis. As more genes are identified and the spec-rum of aberrations expands, more techniques need to be set upnd more resources are to be allocated. Initially restricted to DNA-ased analyses, genetic testing is more often confronted to utilizeomplementary techniques analyzing mRNA, protein structure androtein function. As techniques spread from highly specialized labso routine settings (i.e. MSI testing, semiautomatic sequencing)ew techniques (i.e. DNA and RNA next-generation sequencing,onversion of diploidy to haploidy, functional assays) are makingts path from the research to the diagnostic setting in order toelp clinicians in charge of these patients. Diagnostic algorithmsre often created and immediately changed incorporating someew advance that is likely to improve the current strategy. Issuesuch as analytical and clinical validity become increasingly impor-ant while awareness about the limitations of the assays utilizeds of critical importance. The main challenge that face detectionf hereditary colorectal cancer alterations is that of the molecularedicine: to keep improving patient cancer care through a better
nowledge of the molecular basis of the disease.
onflict of interest statement
The authors declare that there are no conflicts of interest.
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