Mutation analysis of SMAD2, SMAD3, and SMAD4 genes in hereditary non-polyposis colorectal.
- SourceAvailable from: A. Syed Sameer[show abstract] [hide abstract]
ABSTRACT: The development and progression of colorectal cancer has been extensively studied and the genes responsible have been well characterized. However the correlation between the SMAD4 gene mutations with KRAS mutant status has not been explored by many studies so far. Here, in this study we aimed to investigate the role of SMAD4 gene aberrations in the pathogenesis of CRC in Kashmir valley and to correlate it with various clinicopathological variables and KRAS mutant genotype. We examined the paired tumor and normal tissue specimens of 86 CRC patients for the occurrence of aberrations in MCR region of SMAD4 and exon 1 of KRAS by PCR-SSCP and/or PCR-Direct sequencing. The overall mutation rate of mutation cluster region (MCR) region of SMAD4 gene among 86 patients was 18.6% (16 of 86). 68.75% (11/16) of the SMAD4 gene mutants were found to have mutations in KRAS gene as well. The association between the KRAS mutant genotype with SMAD4 mutants was found to be significant (P = or < 0.05). Further more, we found a significant association of tumor location, tumor grade, node status, occupational exposure to pesticides and bleeding PR/Constipation with the mutation status of the SMAD4 gene (P = or < 0.05). Our study suggests that SMAD4 gene aberrations are the common event in CRC development but play a differential role in the progression of CRC in higher tumor grade (C+D) and its association with the KRAS mutant status suggest that these two molecules together are responsible for the progression of the tumor to higher/advanced stage.BMC Cancer 01/2010; 10:300. · 3.33 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The human gene, human giant larvae (Hugl-1/Llg1/Lgl1) has significant homology to the Drosophila tumour suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that binds Myosin II and is involved in maintaining cell polarity and epithelial integrity. The human protein, Hugl-1 contains several conserved functional domains found in Lgl, suggesting that these proteins may have closely related functions. Whether loss of Hugl expression plays a role in human tumorigenesis has so far not been extensively investigated. Thus, we evaluated tumour tissues from 94 patients undergoing surgery for colorectal cancer (CRC) for loss of Hugl-1 transcription and compared our findings with the clinical data from each of these patients. We found that Hugl-1 was lost in 75% of tumour samples and these losses were associated with advanced stage and particularly with lymph node metastases. Reduced Hugl-1 expression during the adenoma-carcinoma sequence occurring as early as in colorectal adenomas was detected by both immunohistochemical and reverse transcription-polymerase chain reaction analysis. Functional assays with ecdysone-inducible cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Our studies thus indicate that downregulation of Hugl-1 contributes to CRC progression.Oncogene 05/2005; 24(19):3100-9. · 7.36 Impact Factor
whether there was an association between the presence of
the diVuse ring and breast cancer. There were 49 samples
from women with breast cancer and 59 from unaVected
women. A ?2value of 0.86 was obtained. For 1%
significance, a value of 6.63 and for 5% significance, a
value of 3.84 is required. Thus, it can be concluded that
there is no measurable association between the diVuse ring
and breast cancer. The trace element (Zn, Cu, Fe, and S)
analysis of intact hair showed no correlation with the ring
structure in the diVraction pattern or with the subjects’
group. The women in the normal population group whose
hair had shown the diVuse ring were examined and shown
not to have breast cancer.
Our x ray diVraction data do not support the recent
claim that hair from breast cancer patients or those at high
risk (BRCA1/BRCA2 mutation carriers) show a distinct
diVuse ring. This conclusion for breast cancer diagnosis
was also reached on a much smaller study of head hair
only.6In our study, diVraction patterns from 75% (37 of
49) of the breast cancer patients do not show this ring.
Moreover, the ?2test shows no association between the
diVuse ring and breast cancer and, as such, the claim that x
ray diVraction of pubic hair can be used as a screening
method for breast cancer or breast cancer predisposition is
J GÜNTER GROSSMANN†
KAN C CHEUNG†‡
D GARETH R EVANS§
S SAMAR HASNAIN†‡
*CRC Department of Medical Oncology,Christie Hospital NHS Trust,
Wilmslow Road,Manchester M20 4BX,UK
†CLRC Daresbury Laboratory,Warrington WA4 4AD,Cheshire,UK
‡Faculty of Applied Sciences,DeMontfort University,Leicester LE1 9BH,UK
§Department of Medical Genetics,St Mary’s Hospital,Manchester
Correspondence to: Dr Evans, Gevans@central.cmht.nwest.nhs.uk
1 Wiesener W, Gorner W, Niese S, Grund W, Hennig M, Mende T. Neutron
activation analytical determination of trace elements in human hair.
2 Astbury WT, Woods HJ. The X-ray interpretation of the structure and elas-
tic properties of hair keratin. Nature 1930;126:913-14.
3 James VJ, Yue DK, McLennan SV. Changes in the molecular structure of
human hair in insulin-dependant diabetes. Biochem Biophys Res Commun
4 James V, Kearsley J, Irving T, Amemiya Y, Cookson D. Using hair to screen
for breast cancer. Nature 1999;398:33-4.
5 Fraser RDB, MacRea TP, Rogers GE, Filshie BK. Lipids in keratinised tis-
sues. J Mol Biol 1963;7:90-1.
6 Briki F, Busson B, Salicru B, Esteve F, Doucet J. Breast-cancer diagnosis
using hair. Nature 1999;400:226.
J Med Genet 2000;37:298–300
Mutation analysis of SMAD2, SMAD3,
and SMAD4 genes in hereditary
non-polyposis colorectal cancer
EDITOR—Transforming growth factor-? (TGF-?) family
members are known to be involved in the regulation of cell
TGF-? family include TGF-?s, activins, and bone morpho-
genetic proteins (BMPs). Their signals are mediated to the
cell nucleus by a network of transmembrane serine/
threonine kinase receptors and their downstream eVectors,
the SMAD proteins.2SMAD proteins play a key role in
intracellular TGF-? signalling and inactivating mutations of
SMADs, such as SMAD2, SMAD3, and SMAD4, provide
resistance of cells to TGF-? induced growth inhibition.
To date, eight human SMADs have been identified. Two
of them, SMAD2 and SMAD4, have been reported to be
mutated in a subset of colorectal carcinomas.3–6Germline
mutations of SMAD4 have been found in patients with
juvenile polyposis, a condition predisposing to colorectal
SMAD3 mutations have not been reported in human
cancers.In a recent study by Arai et al,11SMAD3 mutations
were analysed in 35 sporadic colorectal and 15 HNPCC
cancers and no mutations were found. Targeted disruption
of the SMAD3 gene in mice has been reported to lead to
development of colorectal cancer,12though other studies
have not detected a clear association.13 14No genetic altera-
tions in other SMADs have been reported in malignancy.
Hereditary non-polyposis colorectal cancer (HNPCC) is
an autosomal dominantly inherited cancer susceptibility
syndrome,associated with germline mutations in five DNA
mismatch repair genes: MLH1, PMS1,PMS2,MSH2, and
MSH6.15–19Inactivation of both alleles of a mismatch repair
gene results in microsatellite instability (MSI) that is a
hallmark of HNPCC tumours.20–23The genes responsible
for microsatellite stable (MSS) HNPCC are still unknown.
Loss of growth inhibition by TGF-? is an important step
in colon tumorigenesis and in HNPCC tumours with MSI
this is mainly the result of frameshift mutations within a
polyadenine sequence repeat in the TGF-? type II receptor
(TGF?RII) gene.24It has been proposed that mutations in
TGF?RII could underlie the cancer predisposition in MSS
HNPCC,25and also that other genes involved in the
TGF-? pathway are candidates for MSS HNPCC.26
Chromosomal deletions are common genetic alterations
in cancer and they are targeted at tumour suppressor
loci.27 28Previous studies have shown that one copy of
chromosome 18q is lost in over 70% of sporadic colorectal
cancers.29–32The DCC (deleted in colorectal cancer) gene
has been suggested as a candidate target gene in this region
and loss of expression of DCC has also been reported in
colorectal cancers.33However, mutations in the coding
region of DCC seem to be rare34and the position of DCC
as a candidate tumour suppressor is not clear. Two other
candidate genes, SMAD4 and SMAD2, have recently been
identified at the same 18q region3 35emphasising the possi-
ble role of the SMAD genes in colorectal tumorigenesis.
The aim of the present study was to investigate whether
germline mutations in SMAD2, SMAD3, and SMAD4
underlie microsatellite stable HNPCC.
Mutation screening was performed in 14 Finnish
HNPCC kindreds from which lymphoblastoid cell lines
were available. Based on genealogical evidence the families
are unrelated, though the existence of early common
ancestors cannot be excluded. One aVected subject per
family was included in the study. Of the kindreds, six
fulfilled the Amsterdam criteria for HNPCC.36Other
patients represent familial HNPCC-like colorectal cancer
(CRC); the number of patients with CRC or endometrial
cancer ranged from two to six per family (average three)
(table 1). All kindreds selected for this study have
previously been shown to be MLH1 and MSH2 mutation
negative.37In three kindreds,DNA from tumour tissue had
not been available. From 10 families one and in one family
two colorectal cancer samples were available and no
evidence of MSI had been detected (table 1). The study
was approved by the ethical committee of the Department
of Medical Genetics, University of Helsinki.
Total cellular RNA was extracted from lymphoblasts by
RNA extraction kit (QIAGEN). The SMAD2, SMAD3,
and SMAD4 genes were amplified from RNA using an
RT-PCR procedure. First, 20 µl cDNA was created from
0.8 µg of RNA using standard random priming methods
with Mu-MLV reverse transcriptase (Promega) and
RNAse inhibitor (Promega). The cDNA sequences for
SMAD2, SMAD3, and SMAD4 were derived from
GenBank database (accession numbers U65019, U76622,
and U44378, respectively). PCR primers for cDNA ampli-
fication were designed using the Primer3 server (http://
Each gene was divided into five fragments, covering the
whole coding region of the gene. The forward (F) and
reverse (R) primers and size of each PCR product are listed
in table 2.
The PCR reactions were carried out in a 50 µl reaction
volume including 2 µl of cDNA, 1 × PCR reaction buVer
(Perkin Elmer Applied Biosystems Division),200 µmol/l of
each dNTP (Finnzymes), 0.8 µmol/l of each primer, and 2
units of AmpliTaq GOLD polymerase (PE/ABI). The
MgCl2concentration was 1.5 mmol/l for SMAD2 frag-
ments 1 and 2, SMAD3 fragment 3, and all SMAD4 frag-
ments. For all other fragments the MgCl2concentration
was 2.5 mmol/l.SMAD3 fragment 2 reaction also included
10% of DMSO. The PCR conditions are available upon
After PCR, 5 µl of the PCR product was run on a 3%
agarose (NuSieve) gel to verify the specificity of the PCR
reaction. The rest of the PCR product was purified using
QIAquick PCR purification Kit (QIAGEN). Direct
sequencing of the PCR products was performed using the
ABI PRISM Dye Terminator or ABI PRISM dRhodamine
cycle sequencing kits (PE/ABI). Cycle sequencing prod-
ucts were electrophoresed on 6% Long Ranger gels (FMC
Bioproducts) and analysed on an Applied Biosystems
model 373A or 377 DNA sequencer (PE/ABI).
To screen for the presence of a base substitution in
SMAD3 in controls, restriction enzyme digestion was per-
formed. HgaI (New England BioLabs) digestion was used
to detect A to G change in SMAD3 exon 3 at codon 170.
New PCR primers for genomic exon 3 amplification were
designed using the Primer3 server. The primers were:
5'-ATCGACACTGAGCCACCTCT (forward) and 5'-
CCCACGTGCCTACCTCTG (reverse). The PCR reac-
tions were carried out in a 50 µl reaction volume including
100 ng genomic DNA, 1 × PCR reaction buVer (PE/ABI),
200 µmol/l of each dNTP (Finnzymes), 0.8 µmol/l of each
primer, 2 units of AmpliTaq GOLD polymerase (PE/ABI),
and 1.5 mmol/l of MgCl2.The following PCR cycles were
used for amplification: 10 minutes at 95°C, 40 cycles of 45
seconds at 95°C, 45 seconds at 56°C, one minute at 72°C,
and final extension for 10 minutes at 72°C. HgaI cuts the
PCR fragment (187 bp) that contains the substitution into
two fragments (134 bp and 53 bp in size) whereas the wild
type fragment lacks the restriction site and is not digested.
The digestion was performed in 1 × NEBuVer (New Eng-
land BioLabs) at 37°C overnight. After digestion, the PCR
products were electrophoresed through a 3% agarose gel.
In this work we analysed SMAD2,SMAD3,and SMAD4
mutations in 14 familial colon cancer kindreds, 11 of these
displaying at least one MSS tumour. The microsatellite
analysis data derived typically from one single tumour per
family suggest that these kindreds do not segregate DNA
mismatch repair gene mutations, but does not exclude this
possibility. Previous studies had evaluated MLH1 and
the traditional Amsterdam criteria,eight patients represent familial
colorectal cancer.All except three kindreds displayed microsatellite stable
tumours;in F33,F65,and F74 the MSI/MSS status was unknown
(tumour sample not available).Sites of cancer and age at diagnosis (in
parentheses) in proband and first degree relatives are presented
Features of the families studied.Six out of 14 families fulfilled
Sites of cancers and age of diagnosis
in proband and first degree relatives
MSS CRC (38, 39, 48, 62) small
CRC (34, 40, 44, 50), cervix
(40), liver (71)
CRC (61, 67), stomach (56),
CRC (33, 53)
CRC (54), cervix (?)
CRC (24, 43, 51, 52, 76)
F 33Not known
F 42Familial CRC MSS
F 65Not knownCRC (32, 41, 54), liver (?)
CRC (66, 68)
CRC (64, 67)
CRC (41, 45)
CRC (47, 67), melanoma (70)
F 84MSS CRC (43, 56, 59), breast (44)
CRC = colorectal cancer.
Primer3 server.Each gene was divided into five fragments,covering the
whole coding region of the gene.The forward (F) and reverse (R) primers
and size of the each PCR product are listed below
PCR primers for cDNA amplification were designed using the
Gene/fragment Primer sequence (5'→3')
changed to guanine (marked by an arrow),which is predicted to convert
isoleucine to valine at amino acid 170.
SMAD3 polymorphism identified by sequencing.Adenine has
MSH2 mutations in the series with negative results.37 38
SMAD gene mutation analysis was performed by auto-
mated sequencing covering the translated region of the
genes. Genetic alterations were not detected in SMAD2 or
SMAD4 in any of these patients. In SMAD3, three
discrepancies were detected between GenBank sequence
(U76622) and sequences from our patients, firstly the A to
G change at the third position of codon 103 (exon 2).
Homozygous A to G change was seen in 11 of our 14
patients and in three of them the substitution was hetero-
zygous. This discrepancy has been reported earlier and the
variant does not cause any amino acid change.9 11A second,
silent change detected was a C to T transition at nucleotide
907 (exon 6). This change was homozygous and it was
present in one of our 14 HNPCC patients (in family 31).
The frequency of these variants in the normal population
was not analysed, as the changes were silent. The third
change was an adenine to guanine transition at nucleotide
545, which is predicted to convert isoleucine to valine at
amino acid 170 (fig 1). This change was detected in two
patients (in families 65 and 75).For this variant,110 Finn-
ish controls were analysed by restriction enzyme digestion
(HgaI). Seven out of 110 controls displayed the change
(6.4%).To compare further the frequency of this polymor-
phism in colon cancer patients and controls, 132 patients
were included in the analysis. Taken together, in the 14
HNPCC patients and 132 colon cancer patients the
frequency of this polymorphism was 8.9% (13/146). From
those 13 cancer patients who had valine instead of isoleu-
cine at codon 170, four turned out to be familial. Segrega-
tion of the polymorphism was analysed in two of these
families where DNA from multiple family members was
available and the polymorphism did not segregate with
cancer in these families.
SMAD2,SMAD3,or SMAD4 mutations were not found
in any of our patients using a cDNA based mutation analy-
sis.It should be noted that like all other mutation detection
methods, this method may miss a subset of mutations.
Also, the potential existence of founder mutations in the
Finnish population may have hampered our eVorts to
detect SMAD gene defects in HNPCC. However, it is
likely that defects of SMAD2,SMAD3, or SMAD4 are not
a common cause of familial colon cancer. Further work is
necessary to unravel the molecular background of MSS
We thank Siv Lindroos, Liisa Suksi, and Päivi Laiho for technical assistance.
This study was supported by grants from the Finnish Cancer Society, the Acad-
emy of Finland, Emil Aaltonen Foundation, Finnish Cultural Foundation,
Sigrid Juselius Foundation, and Biocentrum Helsinki.
L A AALTONEN*
*Department of Medical Genetics,Haartman Institute,PO Box 21,
(Haartmaninkatu 3),00014 University of Helsinki,Finland
†Human Cancer Genetics Program,Comprehensive Cancer Centre,Ohio
‡Second Department of Surgery,Helsinki University Central Hospital,
§Department of Surgery,Jyväskylä Central Hospital,Jyväskylä,Finland
Correspondence to: Dr Aaltonen, email@example.com
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Novel mutation in the MYOC gene in
primary open angle glaucoma patients
EDITOR—Glaucoma is the world’s leading cause of
irreversible blindness1and is characterised by progressive
optic disc cupping with corresponding visual field loss.
Both intraocular pressure (IOP) and positive family history
are risk factors for the development of the disease.2Juvenile
open angle glaucoma (JOAG) is a subtype of open angle
glaucoma characterised by an early onset (10 to 35 years of
age) and autosomal dominant inheritance with high
penetrance,3a characteristic which has led several authors
to investigate aVected families in an attempt to identify a
gene or genes associated with this condition.4–10With the
use of genetic linkage analysis in families with JOAG, a
genetic locus (GLC1A) was recognised on chromosome
1q21-q31.4The gene associated with GLC1A has been
identified and it codifies a 57 kDa protein named trabecu-
lar meshwork induced glucocorticoid response protein
(TIGR),10also known as myocilin (MYOC).11The MYOC
gene is composed of three exons of 604, 126, and 785 bp,
respectively.12During screening for mutations in the
MYOC gene in 25 unrelated Brazilian patients with JOAG,
an unreported mutation (Cys433Arg) was detected,
present in seven of them.
Patients were followed at the Glaucoma Service of the
State University of Campinas, Brazil. They underwent an
ocular examination, including gonioscopy by Posner lens,
applanation tonometry,slit lamp biomicroscopy,optic nerve
evaluation, and automated perimetry (Humphrey 630, pro-
gram 30-2).JOAG was defined as the presence of character-
istic bilateral optic nerve damage and visual field loss in the
presence of an open angle in subjects younger than 36 years
of age.Each patient included in this study came from diVer-
ent families according to interview data. The study was
approved by the Ethics Committee of the State University of
Campinas. At the time of the ocular examination, the mean
age of JOAG patients was 25.52 years (SD 6.99), ranging
from 10 to 35 years, and the mean IOP was 29.96 mm Hg
(SD 13.00). Thirteen patients (52%) were male and 12
patients (48%) were female;13 (52%) were white,11 (44%)
were black, and one (4%) was Asian. Some of the patients
had been taking antiglaucomatous medication or had
undergone surgical procedures for IOP control and showed
IOP levels lower than 22 mm Hg at the time of examination.
Fourteen patients (56%) had a positive family history.
Genomic DNA from peripheral blood (withdrawn from
the antecubital vein) was prepared using DNAzolTM
Reagent according to the manufacturer’s instructions
(GIBCO/BRL, Gaithersburg, MD). Primers were de-
signed to amplify the three exons of the MYOC gene,
according to the reported sequence (GenBank accession
numbers U85257 and Z98750). Exon 3 was the first to be
analysed, since this is the region where the majority of the
mutations have been found.12SSCP analysis was per-
formed on a Pharmacia PhastSystem (Pharmacia, Upp-
sala, Sweden) using a 20% homogeneous polyacrylamide
gel.Samples with abnormal mobility shifts were sequenced
using Thermo Sequenase Cycle Sequencing Kit with ?-32P
ATP (Amersham Life Science Inc, Cleveland, Ohio),
according to the manufacturer’s instructions.
Seven patients (28%) showed a point mutation in exon 3,
a heterozygous T to C transition at nucleotide position
1374 leading to a cysteine (Cys) 433 for arginine (Arg)
substitution. The SSCP pattern shows two abnormal
mobility shifts (figs 1 and 2). Analysis through SSCP and
restriction digestion with FokI was performed in 130
control samples of diVerent ethnic origin in order to discard
the possibility of the new substitution being a polymor-
phism. Cleavage of the approximately 195 bp amplified
fragment with FokI generates three fragments of 13,50,and
132 bp in size in the absence of the mutation, and in its
presence the 132 bp fragment is cleaved into two additional
33 and 99 bp fragments. The mutation was not present in
any of the samples.The Cys433Arg mutation was present in
five (71%) white patients and two (29%) black patients. Six
patients (85%) of those with the mutation had a positive
familial history of glaucoma. The mean age of patients har-
bouring this mutation was 27.00 years (SD 6.02), ranging
from 15 to 35 years and the mean IOP was 39.13 mm Hg
(SD 12.62). In contrast, JOAG patients without the muta-
tion had a mean age of 24.02 (SD 7.46) and a mean IOP of
25.65 mm Hg (SD 11.68) (p>0.05). In another patient, a
previously reported mutation (Pro370Leu) was identified.13
One of the patients who showed the Cys433Arg
mutation had the family investigated for its presence. Nine
subjects were studied and four harboured the Cys433Arg
mutation (fig 3).Three of them had glaucoma and one had
ocular hypertension without optic nerve or visual field
damage. The other members who did not have the disease
were not aVected with the Cys433Arg mutation.
In order to discriminate between a founder eVect and a
de novo recurrence, haplotype analysis was performed in
the six patients with a positive family history who had the
new mutation and in one family using four microsatellite
markers, mapped at band 1q21-25, closely linked to the
NGA19. Polymerase chain reaction (PCR) was carried out
following standard procedures and primer sequences were
obtained from the Genome Data Bank. For allele scoring,
polyacrylamide-urea gel and autoradiographed.The analy-
sis of four microsatellite markers showed that the
Cys433Arg mutation is associated with a common
haplotype, suggesting that these patients inherited their
mutation from a common ancestor. A pedigree of the
family analysed can be seen in fig 3, depicting a potential
D1S2790, D1S1619, and
encompassing amino acids 412 to 476 of the MYOC gene.Lane 1:control
sample.Lanes 2-5:samples of JOAG patients.Samples 2,4,and 5 show
abnormal mobility shifts as compared with the control.
Silver stained SSCP gel showing the 195 bp PCR product