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Distinct mutations in MLH1 and MSH2 genes in Hereditary
Non-polyposis Colorectal Cancer (HNPCC) families from
China
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Wenqian Wei , Fangqi Liu , Lei Liu , Zuofeng Li , Xiaoyan Zhang , Fan Jiang , Qu Shi , Xiaoyan Zhou , Weiqi
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Sheng , Sanjun Cai , Xuan Li *, Ye Xu * & Peng Nan *
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Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University,
Shanghai 200433, 2Colorectal Surgery, Shanghai Cancer Hospital of Fudan University, Shanghai 20032, 3Shanghai Center for
Bioinformation Technology, Shanghai, 200235, 4School of Life Science and Technology, Tongji University, Shanghai 200092, China
Hereditary non-polyposis Colorectal Cancer (HNPCC) is an autosomal dominant inheritance syndrome. HNPCC is the most
common hereditary variant of colorectal cancer (CRC), which
accounts for 2-5% CRCs, mainly due to hMLH1 and hMSH2
mutations that impair DNA repair functions. Our study aimed
to identify the patterns of hMSH2 and hMLH1 mutations in
Chinese HNPCC patients. Ninety-eight unrelated families from
China meeting Amsterdam or Bethesda criteria were included
in our study. Germline mutations in MLH1 and MSH2 genes,
located in the exons and the splice-site junctions, were
screened in the 98 probands by direct sequencing. Eleven mutations were found in ten patients (11%), with six in MLH1
(54.5%) and five in MSH2 (45.5%) genes. One patient had
mutations in both MLH1 and MSH2 genes. Three novel mutations in MLH1 gene (c.157_160delGAGG, c.2157dupT and
c.-64G＞T) were found for the first time, and one suspected
hotspot in MSH2 (c.1168C＞T) was revealed. [BMB reports
2011; 44(5): 317-322]
INTRODUCTION
Hereditary nonpolyposis colorectal cancer (HNPCC) (MIM#120435), also known as Lynch syndrome, is the most common
known form of hereditary colorectal cancer (CRC) (1). It accounts for approximately 2-5% of all the colorectal cancer cas-
*Corresponding author. Peng Nan, Tel: 86-21-65642957; Fax: 8621-65642468; E-mail: [email protected], Ye Xu, Tel: 86-2164175590 Ext1106; Fax: 86-21-64035387; E-mail: xu.shirley021@
gmail.com, Xuan Li, Tel: 86-21-54924305; Fax: 54924015; E-mail:
[email protected]
#
These authors contributed equally to this work.
DOI 10.5483/BMBRep.2011.44.5.317
Received 4 January 2011 , Accepted 22 February 2011
Keywords: HNPCC, MLH1, MSH2, Mutations
http://bmbreports.org
es (2). It is an autosomal dominant inheritance syndrome associated with heritable defects in DNA mismatch repair genes
(MMR). HNPCC is characterized by earlier onset of colorectal
cancer, proximal colonic cancer predominance, and an increased incidence of cancer in extracolonic organs such as endometrial, stomach, ovary, small bowel, hepatobiliary tract, renal pelvis, ureter, skin, and brain (3, 4). Germline mutations in
at least 5 DNA mismatch-repair genes (MMR) cause HNPCC:
MLH1 (3p21-23) (MIM*120436), MSH2 (2p21) (MIM*609309),
MSH6 (2p21) (MIM*600678), PMS1 (2q31- q33) (MIM*600258),
and PMS2 (7p22) (MIM*600259). Most (90%) of the mutations
were found in two MMR genes: MLH1 (50%) and MSH2
(40%), whereas 7% mutations occurred in MSH6 and 3% for
the remaining genes (5).
The Amsterdam criteria were established by the International Collaborative Group on Hereditary Nonpolyposis Colorectal Cancer (ICG-HNPCC) in 1990. As the widely accepted
criteria for diagnosis of HNPCC, it has some limitations. It is
too restrictive and difficult to identify HNPCC cases in small
family (2). Thus, some other guidelines, such as Bethesda criteria, were also formed to identify suspected patients (6). The
golden standard for diagnostic of HNPCC is germline mutation
in MMR genes. At present, direct sequencing is the key procedure in recognizing MMR genes defects. As most studies of
HNPCC are limited in race and sample sizes, several MMR
gene mutation databases were established to share data
around the world. The most authoritative one is InSiGHT
(http://www.insight-group.org/mutations/), set up by the International Society of Gastrointestinal Hereditary Tumors.
InSiGHT collected a large amount of data on MLH1 and
MSH2 genes. The total number of variants for MLH1 was
6079, and for MSH2 was 4428 (accessed in May 2010). Also
there is a database with unclassified variants (www.mmruv.info)
supporting the InSiGHT.
Our previous studies based on InSiGHT suggested there
were differences in MLH1 and MSH2 mutations between
Caucasian and Asian populations (7). In the meantime, Sheng
et al. indicated that the incidence of early onset CRC in Hong
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Mutations in MLH1 & MSH2 genes in HNPCC families from China
Wenqian Wei, et al.
Kong, was more than Western countries (8). A predominance
of distal tumors (9) and primary extracolonic tumors in stomach (10) in Asian HNPCC patients were reported, which were
also different from those in Western countries. Kim et al. found
the three novel germline mutations in MLH1 and MSH2 in
Korea (11). The study was to analyze MLH1 and MSH2 mutations in 98 unrelated probands of the suspected HNPCC families, which is one of the large scale studies in Chinese
population. In addition to those reported, several novel MLH1
and MSH2 mutations and a suspected hotspot in the group of
Chinese HNPCC patients were identified.
RESULTS
A total of eleven mutations were found in ten patients (11%)
from the 98 probands, with six in MLH1 (54.5%) and five in
MSH2 (45.5%) genes (Table 1). Four families who met the
Amsterdam criteria harbored mutations, and six families who
met Bethesda criteria had mutations.
Three novel mutations which had never been reported were
founded in patients H76 (MLH1 c.157_160delGAGG), H86
(MLH1 c.-64G＞T) and H224 (MLH1 c.2157dupT). There
were five mutations previously reported, including one known
polymorphism in MLH1 c.655A＞G (dsSNP1799977). Interestingly, c.1168C＞T of MSH2, a missense mutation, was found
in four unrelated probands (H88, H224, H231 and H245).
Furthermore, one patient (H224) was a compound heterozygote for a novel pathogenic frameshift mutation in MLH1
and a missense mutation in MSH2.
Patient H86
Sequence analysis of MLH1 revealed a novel G＞T substitution at nucleotide -64 (Fig. 1a), which is in the promoter
area. It may result in alteration or loss of MLH1 gene function.
No mutation was identified in MSH2.
Patient H76
Sequence analysis of MLH1 coding area revealed the deletion
of four nucleotides GAGG at 157_160 in exon 2 (Fig. 1b). This
deletion caused a frameshift mutation, results in the replacement of a Gly with an Ala at codon 54, and early termination
at two amino acids after. Comprehensive sequence analysis of
MLH1 and MSH2 genes revealed no other mutations.
Patient H224
An insertion (c.2157dupT, Fig. 1c) in MLH1 was found in patient H224, which resulted in a frameshift mutation and replacement of Val with Cys at codon 720, and ended in three
amino acids. Meanwhile, a C＞T substitution at nucleotide
1168, resulted in the replacement of a phenylalanine-encoding
codon with a leucine-encoding codon at codon 390 in MSH2
gene.
DISCUSSION
In this study, eleven germline mutations were found in ten
probands. Among them were eight distinct mutations, all of
which were heterozygote. Although some researchers found
bi-allelic mutations in HNPCC patients, most alterations are
mono-allelic. Among the eight germline mutations we identified, four are missense mutations, two are frameshift mutations, one is aberrant splicing, and one is in promoter area.
Missense mutations are also the common mutation type in
Chinese population. The missense mutations (MLH1
c.655A＞G, MLH1 c.2042C＞T, MSH2 c.23C＞T and MSH2
c.1168C＞T) were identified as “missense mutations of unknown significance”, functions of which were controversial.
Four probands had one same mutation - MSH2 c.1168C＞T
(p.L390P). We searched for MSH2 c.1168C＞T in InSiGHT database and the original papers, and found the mutations (about
80%) frequently occurred in East Asia populations, including
Table 1. Germline MLH1 and MSH2 mutations in all patients
Family ID
H9
H76
H86
H88
H166
H167
H224
H231
H236
H245
Mutation
Gene
Exon
MSH2
MLH1
MLH1
MSH2
MLH1
MLH1
MLH1
MSH2
MSH2
MLH1
MSH2
1
2
1
7
8
17
19
7
7
18
7
Nucleotide
c.23C＞T
c.157_160del GAGG
c.-64G＞T
c.1168C＞T
c.655A＞G
c.1989G＞A
c.2157dupT
c.1168C＞T
c.1168C＞T
c.2042C＞T
c.1168C＞T
Consequence
p.Thr8Met
p.Gly54Alafs*2
p.Phe390Leu
p.Ile219Val
p.Val720Cysfs*3
p.Phe390Leu
p.Phe390Leu
p.Ala681Val
p.Phe390Leu
Type
Missense
Frameshift
Uncertain
Missense
Missense
Aberrant splicing
Frameshift
Missense
Missense
Missense
Missense
Clinical criteria
Amsterdam
Amsterdam
Bethesda
Bethesda
Amsterdam
Amsterdam
Bethesda
Bethesda
Bethesda
Bethesda
Having been
reported
(times)#
Yes (5)
No
No
Yes (34)
Yes (224)
Yes (2)
No
Yes (34)
Yes (34)
Yes (1)
Yes (34)
#
According to the InSiGHT database (http://www.insight-group.org/mutations/), InSiGHT database is the largest and most authoritative database in
HNPCC studies, which integrated the data from MMR Gene Unclassified Variants Database (www.mmruv.info), Mismatch Repair Genes Variants
Database (www.med.mun.ca/MMRvariants), and the Human Gene Mutation Database (www.hgmd.cf.ac.uk/ac). By May 2010, the InSiGHT database lists a total number of 6079 and 4428 variants and 1079 and 924 unique DNA mutations for MLH1 and MSH2, respectively.
318 BMB reports
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Mutations in MLH1 & MSH2 genes in HNPCC families from China
Wenqian Wei, et al.
Fig. 2. Reported MSH2 c.1168C＞T variants. The percentage of
the patients’ number in MSH2 c.1168 C＞T based on the InSiGHT
database and the original papers.
Fig. 1. Three novel mutations found in patients H86, H76 and
H224. (a) A single nucleotide substitution (G＞T) at position -64
in the promoter region of the MLH1 gene in patient H86. (b)
Comparison of sequence pattern of exon 2 in MLH1 both normal
and patient H76, deletion of four nucleotides (GAGG) in patient
H76. (c) Sequence analysis of exon 19 in MLH1, insertion of one
nucleotide (T) in patient H224.
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Chinese Han, Japanese, Korean and Singaporean (Fig. 2).
However, in a study of 1721 German suspected HNPCC families, Mangold detected no instance of MSH2 c.1168C＞T (12).
Although this mutation was first reported by Konishi in 1996,
its function is still controversial (13). Banno et al. found this
mutation in two out of eleven (18%) endometrial cancer patients who had family histories of colorectal and endometrial
carcinoma. Later, a high occurrence rate (7%) of the mutation
was observed among 179 healthy Japanese individuals, so it
was reported to be a polymorphorism but not a causative mutation for CRC (14). Kim et al. also reported this mutation as
SNP in healthy Korean individuals, which showed no association with sporadic colorectal cancer (15), and was recently
identified as SNP (dsSNP17224367). However, Chen et al.
suggested this mutation might cause a polarity change at the
margin of the interaction region of MSH2 (16). Fan et al. analyzed molecular epidemiologic, structural, bioinformatic and
functional information of MSH2 c.1168C＞T. They found this
variant in 4.89% of CRC and only in one healthy individual
(0.89%). This variant resulted in a phenotype relevant to
young sporadic CRC or GC, which was suspected as predisposing factor to gastrointestinal cancer (17). In the same year,
Zhang et al. found significant difference in the frequency of
MSH2 c.1168C＞T between young-age patients and control
individuals, and considered this mutation associated with an
increased GC risk (18). In our study the incidence of MSH2
c.1168C＞T is up to 4% in 98 HNPCC probands. This result is
different from those Japanese and Korean studies. In Chinese
study, incidence of MSH2 c.1168C＞T is much higher in patients than in controls.
MLH1 c.655A＞G mutation was reported 224 times in
InSiGHT database. In our study, one patient who fulfilled the
Amsterdam criteria harbor MLH1 c.655A＞G mutation. The
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Mutations in MLH1 & MSH2 genes in HNPCC families from China
Wenqian Wei, et al.
significance of the sequence alteration is controversial. The
c.655A＞G variant is considered a polymorphism (dsSNP1799977)
and was reported in HapMap database (www.hapmap.org).
MLH1 c.655A＞G is in a conserved region of exon 8, and
both alleles result in non-polar and pH-neutral amino acids.
Functional analyses suggest that the variant has DNA repair efficiency (19, 20) and binding properties to PMS2 are similar to
the wild type (21). According to the result by SIFT (http://sift.jvci.org), this polymorphism may be tolerated by MLH1 protein.
But it is not an entirely benign mutation. Among sporadic colorectal cancer and young-onset lung cancer cases, c.655A＞G
was correlated with reduced MLH1 protein expression (15).
This variant may even compromise the MLH1 function due to
code change or change in the secondary structure of mRNA
(22). Recently Takahashi et al. provided evidence to support
this hypothesis by in vitro MMR assay (23). Campbell et al. reported association of the MLH1 c.655A＞G variant with colon
cancer risk for the people with a high Western diet pattern
(24). The association was extended to CRC patients by Nejda
et al., who showed patients with this variant displayed a better
outcome in sporadic colorectal cancer (25). This variant is not
only involved in colorectal cancer, but also in some other
types of cancers. MLH1 c.655A＞G was associated with an almost fivefold increased risk of ulcerative colitis (26), and 6- to
16-fold increased risk of acute lymphoblastic leukaemia when
combined with known genotypes of increased susceptibility to
leukeamia (27). The variant may be associated with the
young-onset of lung cancer, especially in histological squamous cell type (28), and may influence the disease onset of
prostate cancer (29). Our results suggest a possible role of
MLH1 c.655A＞G polymorphism in the Chinese HNPCC
group, and it remains to be further investigated with additional
samples.
We found three novel germline mutations. Two of these
novel mutations caused frameshift mutations in MLH1 gene
(H76 and H224) and made the coding terminate earlier. Both
of them were considered as pathogenic. The other one (H86)
was found in the promoter area of MLH1 (c.-64G＞T).
Previous researches found connection between mutations in
promoter area and diseases. The most frequent one in MLH1
gene is −93G＞A, While it is considered a SNP, some researchers noticed it was associated with CRC. Campbell et al.
and Raptis et al. found that −93G＞A was associated with an
increased risk of MSI-H CRC (24, 30). And in the study by
Allan et al., the risk in developing CRC increased to 3-fold,
particularly in patients with somatic loss of MLH1 protein expression (31). In addition, Zhong et al. found MLH1 −107C＞
G in three out of 163 CRC patients. They also found that this
variant reduced transcriptional activity by 51% and impeded
the promoter-binding capacity of nuclear proteins (32). Green
et al. demonstrated that the MLH1 promoter with −42C＞T
was less effective in binding nuclear proteins, and had only
37% of the activity of the wild-type promoter in driving the expression of a reporter gene in vivo (33). So this novel mutation
320 BMB reports
we found in MLH1 promoter area may impair MLH1 protein
function and may be associated with HNPCC.
We described one proband (H224) fulfilling Bethesda criteria with two different mutations in MLH1 and MSH2 - one
was a novel pathogenic frameshift mutation MLH1 c.2157
dupT, and the other one was a missense mutation MSH2
c.1168C＞T. Such rare case of double mutations was reported
before by others, in which patients were found to have two alterations in both MLH1 and MSH2 genes, and in most of the
cases, one was a deletion and the other one was a point mutation (34).
MATERIALS AND METHODS
Patients
Ninety-eight families with HNPCC or suspected HNPCC were
studied from 1994 to 2008 in Shanghai Cancer Hospital of
Fudan University. All were native Chinese without immigrant
ancestors. Fifteen families fulfilled Amsterdam II criteria and
were classified as Amsterdam group. The rest 83 families who
met the Bethesda criteria were classified as Bethesda group.
The study was approved by the Ethics Committee of Shanghai
Cancer Hospital of Fudan University, and the number of Ethics
Committee was 050432-4-1008A.
Extraction of DNA from blood
Blood samples were taken from 98 probands. Genomic DNA
was isolated from peripheral blood lymphocytes according to
the manufacturer’s instructions (TIANGEN BIOTECH, BEIJING).
o
The extracted genomic DNA was stored at −20 C for further
analysis.
Mutation of MLH1 and MSH2 genes
Exons 1-19 of MLH1 and exons 1-16 of MSH2, including the
splice-site junctions, were amplified by polymerase chain reaction (TaKaKa Biotechnology, Dalian). The PCR products
were purified by 1.5% agarose gel electrophoresis, following
the manufacturer’s instructions (TIANGEN BIOTECH, BEIJING).
All the purified products were sequenced by Shanghai Sunny
Biotechnology Co., Ltd with 3730XL of ABI. Each mutation
was amplified both the sense and antisense strand, and then
the experiment was repeated at least once. If the mutation was
exactly the same through at least three repeated experiments,
the mutation was confirmed. By comparing with the reference
sequences, missense and frameshift mutations were identified.
We submitted eleven mutations, six from MLH1 and five from
MSH2 gene from ten patients to InSiGHT database (http://
www.insight-group.org/mutations/). The IDs of the three novel
mutations are: MLH1-01495, MLH1-01496, and MLH1-01497.
Definition of pathogenic mutations
Sequence variants which would obviously impact the function
of MLH1 or MSH2 proteins, such as nonsense and frameshift
mutations, were considered pathogenic. Besides, the mutahttp://bmbreports.org
Mutations in MLH1 & MSH2 genes in HNPCC families from China
Wenqian Wei, et al.
tions at highly conserved splice sites were also considered
pathogenic. All the missense mutations were assessed for pathogenicity by searching against the InSiGHT database (http://
www.insight-group.org/mutations/), MMR Gene Unclassified
Variants Database (www.mmruv.info), Mismatch Repair Genes
Variants Database (www.med.mun.ca/MMRvariants), and the
Human Gene Mutation Database (www.hgmd.cf.ac.uk/ac). If
the missense mutations affected the promoter site, or have
been reported as pathogenic mutations in other studies, they
were also considered pathogenic. Those missense mutations
whose pathogenicity could not be confirmed were classified as
uncertain.
Acknowledgements
This study was supported by the grants from the 863 Hi-Tech
Program of China (2009AA02Z308, 2007AA02Z332, 2008AA02Z126), the Shanghai Project (07D19505, B111).
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