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Technical Briefs |
1
Medizinische Klinik II, Klinikum der Johann Wolfgang Goethe-Universität and
2
Blutspendedienst Hessen, D-60590 Frankfurt a.M., Germany;
a address correspondence to this author at: Medizinische Klinik II, Zentrum der Inneren Medizin, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt a.M., Germany
Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominantly inherited disease caused by loss of function of DNA mismatch repair genes. Defects in MLH1 and MSH2 account for ~98% of the mutations in HNPCC families (1). Identification of gene carriers within these families is of great importance because surveillance may be restricted to genetically affected relatives. Identification of mutations by direct sequencing is time-consuming and not feasible in a large-scale clinical setting. Molecular screening strategies, including single-strand conformation polymorphism analysis (2), denaturing gradient-gel electrophoresis (3), constant denaturant gel electrophoresis (4), or in vitro transcription/translation assays (5), have been described and may facilitate the detection of mutations. However, these techniques often have low sensitivity with mutation detection rates of only 35–70% (6), or they are highly accurate but are technically difficult to perform (3)(4)(5). In the present study, we developed and evaluated a modified base excision sequence scanning (BESS) protocol (7) for the detection of MLH1 and MSH2 germline mutations. This simple method is based on the incorporation of dUTP into the PCR products. Subsequent digestion with uracil N-glycosylase, which releases uracil from both single-stranded and double-stranded DNA and thus creates apyrimidinic sites, and endonuclease IV, which cleaves the phosphodiester bond at these sites, generates a defined series of fragments (7)(8).
Lymphocytes were prepared from whole blood of patients with HNPCC and
healthy subjects using Vacutainer cell preparation tubes (Becton
Dickinson). After extraction of total RNA (Tri-Star-Kit; AGS),
complementary DNA synthesis was performed with reverse transcriptase
(Superscript; Life Technologies) and random hexamer oligonucleotides or
2.5 µmol/L reverse primers (Table 1
). The PCR amplification was carried out in a Perkin-Elmer 9700
PCR system in a total volume of 50 µL containing 5 U of AmpliTaq Gold
polymerase (Perkin-Elmer); 60 mmol/L Tris-HCl, pH 8.5; 15 mmol/L
(NH4)2SO4;
3.5 mmol/L MgCl2; 200 µmol/L dATP, dTTP, dGTP,
and dCTP; 16 µmol/L dUTP (Biozym Diagnostik); and 2.5 µmol/L
forward and reverse primers (Table 1
). Either the forward or the
reverse primer was labeled with 6-carboxy-fluorescein.
Amplification conditions were optimized and applied as follows: 10 min
at 95 °C; 50 cycles of 30 s at 95 °C and 30 s at 50,
55, or 65 °C, depending on the amplified fragments (see Table 1
),
and 1 min at 72 °C; and final extension 10 min at 72 °C. PCR
products were purified on 1.5% agarose gels using the Qiaquick Gel
purification kit (Qiagen). The eluted DNA was digested in 20 µL of a
solution with 2 µL of excision enzyme mixture containing uracil
N-glycosylase and endonuclease IV (Biozym Diagnostik), 50
mmol/L Tris-HCl (pH 9.0), 20 mmol/L
(NH4)2SO4,
and 10 mmol/L EDTA at 37 °C for 45 min. The fragments of the
digested PCR products, ranging in size from 22 to 480 bp, were mixed
with 11.5 µL of formamide and 1.5 µL of TAMRA size marker
(N,N,N',N'-tetramethyl-6-carboxyrhodamine; Perkin-Elmer) and
electrophoresed for 40 min on an automated ABI 310 DNA sequencer with
laser scanning and linear detection characteristics (Perkin-Elmer). The
peak pattern represented fragments ending with dUTP and was comparable
with the "T" lane of a conventional sequencing reaction. BESS
analyses of the MLH1 and MSH2 genes were
performed on samples from four patients with known sequence-confirmed
mutations and subsequently prospectively on samples from four patients
who fulfilled the Amsterdam criteria. The appearance, disappearance, or
change in intensity of a peak in comparison with a control, which
indicated the presence of a mutation, was assessed by an investigator
who was unaware of the direct sequence data. All patients consented to
participate in the study, which was approved by the Ethics Committee
for Medical Research in Frankfurt a.M. in accordance with the
Declaration of Helsinki.
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In families with HNPCC, 214 different germline mutations have been
described to date by direct sequence analysis: 127 mutations were
located in the MLH1 gene and 81 in the MSH2 gene,
whereas only 6 mutations were detected in the MSH6, the
PMS1, or the PMS2 gene (9). To
establish and evaluate BESS as a screening method, three representative
mutations and one polymorphism of MLH1 and MSH2
were analyzed. Initially, we investigated a heterozygous ACG
ATG
missense mutation in codon 117, exon 4 of the MLH1 gene
leading to a change from threonine to methionine (MLH1,
Thr117Met). BESS analysis identified this mutation by an additional
peak at nucleotide position 350 (Fig. 1
A). In addition, we performed BESS analysis on a patient with a
heterozygous TGTCGT missense mutation in codon 697, exon 13 of the
MSH2 gene (MSH2, Cys697Arg). As shown in Fig. 1B
, this mutation was detectable by a 50% reduction of the T peak
at nucleotide position 2089. As a third representative mutation, we
investigated an as yet unidentified 1-bp deletion within codon 782,
exon 14 of the MSH2 gene (MSH2, DEL782FS). This
frameshift mutation produced a complex fragment pattern in the BESS
analysis, which was caused by the superimposed band pattern of the
wild-type and mutant alleles. When compared with the BESS pattern of a
healthy control subject, the deletion at nucleotide position 2345 was
clearly identified (Fig. 1C
). However, in a patient with a known
GGCGGG polymorphism in codon 713 of the MSH2 gene
(MSH2, Gly713Gly), a BESS pattern identical to those of
healthy control subjects (n = 7) was observed (data not shown).
Subsequently, we investigated the MLH1 and MSH2
genes of patients fulfilling the Amsterdam criteria prospectively
by the BESS protocol. In two patients, missense mutations in the
MSH2 gene, one in codon 322 (MSH2, Gly965Asp) and
one as yet unidentified mutation in codon 388 (MSH2,
Pro1165Leu), were detected. In the third patient, BESS analysis showed
another as yet unidentified missense mutation in codon 618 of the
MLH1 gene (MLH1, Glu1853Asp). All mutations
detected by the BESS protocol were confirmed by direct sequencing. In
the remaining patient, BESS analysis and direct sequencing revealed no
mutation in MLH1 and MSH2.
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RNA-based techniques provide an interesting approach for mutation
screening because 81% of these genetic defects are located in exons
and the remaining 19% are relevant intronic mutations that lead to
detectable splicing variants. BESS analysis identifies missense
mutations, deletions, insertions, repeat expansions, and frameshift
mutations at sites involving dTTP, which account for 96% of known
MLH1 and MSH2 mutations
(1)(9). To date, only 8 of 208 MLH1
and MSH2 mutations have been identified as GC or CG
missense mutations (9), which cannot be detected by the BESS
method. Thus, the sensitivity of BESS analysis (7) for
detecting MLH1 and MSH2 mutations theoretically
should be higher compared with in vitro transcription/translation
(62%) (5) and single-strand conformation polymorphism
techniques (35–70%) (6). However, for the definite
determination of accuracy of the BESS method, a large prospective
evaluation is required. In healthy controls, we did not observe
aberrant BESS peak patterns, indicating high specificity.
BESS analysis of MLH1 and MSH2 can be performed on an automated sequencer in less than 24 h with a hands-on time of 6 h. In addition, a potential mutation must be confirmed by sequence analysis. Nevertheless, the costs of BESS analysis are <50% compared with complete genomic sequence analysis. BESS is considerably less labor-intensive than genomic sequencing and several other screening methods, such as constant denaturant gel electrophoresis, denaturing gradient-gel electrophoresis, and in vitro transcription/translation assays (3)(4)(5). With the described BESS method, we could analyze fragments of up to 500 bp. To further accelerate mutation screening, we attempted to analyze PCR fragments of up to 750 bp. Despite extension of the electrophoresis time, we were not able to resolve distinct fragment peaks; this problem might be overcome by the use of longer capillaries or an optimized polymer.
In summary, the described modified BESS method allows rapid, efficient, and simple detection of MLH1 and MSH2 germline mutations in HNPCC. Its application can improve the genetic diagnosis of hereditary cancer susceptibility syndromes caused by germline mutations of large genes without mutation hotspots.
Footnotes
fax 49-69-6301-4807, e-mail Zeuzem{at}em.uni-frankfurt.de
References
The following articles in journals at HighWire Press have cited this article:
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  H. Yamasaki, M. Nakao, Y. Sako, K. Nakaya, M. O. Sato, W. Mamuti, M. Okamoto, and A. Ito DNA Differential Diagnosis of Human Taeniid Cestodes by Base Excision Sequence Scanning Thymine-Base Reader Analysis with Mitochondrial Genes J. Clin. Microbiol., October 1, 2002; 40(10): 3818 - 3821. [Abstract] [Full Text] [PDF]
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 J. Raedle, J. Trojan, A. Brieger, N. Weber, D. Schafer, G. Plotz, E. Staib-Sebler, S. Kriener, M. Lorenz, and S. Zeuzem Bethesda Guidelines: Relation to Microsatellite Instability and MLH1 Promoter Methylation in Patients with Colorectal Cancer Ann Intern Med, October 16, 2001; 135(8_Part_1): 566 - 576. [Abstract] [Full Text] [PDF]
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 R. B Chadwick, R. E Pyatt, T. H Niemann, S. K Richards, C. K Johnson, M. W Stevens, J. E Meek, H. Hampel, T. W Prior, and A. de la Chapelle Hereditary and somatic DNA mismatch repair gene mutations in sporadic endometrial carcinoma J. Med. Genet., July 1, 2001; 38(7): 461 - 466. [Full Text]
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