Early Detection and Diagnosis
Narrowing of the regions of allelic losses of chromosome 1p36 in meningioma tissues by an improved SSCP analysis
Version of Record online: 18 DEC 2007
Copyright © 2007 Wiley-Liss, Inc.
International Journal of Cancer
Volume 122, Issue 8, pages 1820–1826, 15 April 2008
How to Cite
Guan, Y., Hata, N., Kuga, D., Yoshimoto, K., Mizoguchi, M., Shono, T., Suzuki, S. O., Tahira, T., Kukita, Y., Higasa, K., Yokoyama, N., Nagata, S., Iwaki, T., Sasaki, T. and Hayashi, K. (2008), Narrowing of the regions of allelic losses of chromosome 1p36 in meningioma tissues by an improved SSCP analysis. Int. J. Cancer, 122: 1820–1826. doi: 10.1002/ijc.23297
- Issue online: 19 FEB 2008
- Version of Record online: 18 DEC 2007
- Manuscript Accepted: 16 OCT 2007
- Manuscript Received: 1 AUG 2007
- Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. Grant Numbers: 17659450, 18390400
Mapping loss of heterozygosity (LOH) regions in the genomes of tumor tissues is a practical approach for identifying genes whose loss is related to tumorigenesis. Conventional LOH analyses using microsatellite or single nucleotide polymorphism (SNP) markers require the simultaneous examination of tumor- and matched normal-DNA. Here, we improved the previously developed SNP-based LOH assay using single strand conformation polymorphism (SSCP) analysis, so that LOH in tumor samples heavily contaminated with normal DNA can now be precisely estimated, even when matched normal DNA is not available. We demonstrate the reliability of the improved SSCP-based LOH detection method, called the LOH estimation by quantitative SSCP analysis using averaged control (LOQUS-AC), by comparing the results with those of the previous “LOH estimated by quantitative SSCP assay” (LOQUS) method. Using the LOQUS-AC assay, LOH was detected at a high consistency (98.1%) with the previous LOQUS method. We then applied this new method to characterize LOH profiles in 130 meningiomas, using 68 SNPs (i.e., a mean inter-SNP interval of 441 kbp) that are evenly distributed throughout chromosome 1p36. Benign, atypical and anaplastic meningiomas exhibited 1p36 LOH at frequencies of 48.39, 84.62 and 100.00%, respectively, using LOQUS-AC. Subsequently, we detected a candidate common LOH region on 1p36.11 that might harbor tumor suppressor genes related to malignant progression of meningioma. © 2007 Wiley-Liss, Inc.
Finding genetic alterations in tumor tissues is a basic approach to elucidate the mechanism of tumor formation or progression. Among the methods available, loss of heterozygosity (LOH) analysis is widely used for various tumor types, including meningioma, a common form of brain tumor arising from meningothelial cells. The majority (80–90%) of meningiomas have a benign character and are classified as World Health Organization grade I. The remaining meningiomas, however, which are categorized as grade II or III, are more aggressive and have a higher recurrence rate than grade I tumors.1–3 An LOH on 22q, which leads to the inactivation of the NF2 tumor suppressor gene located on 22q12.2, is detected in 40–70% of meningiomas.4, 5 This genetic alteration is an early event in the pathogenesis of meningiomas; mutations and/or deletions of this gene are frequently observed, even in grade I tumors.6, 7 On the other hand, the progression of meningiomas is thought to involve a multi-step process with the cumulative acquisition of genetic alterations.8 Little is known, however, about the actual genes associated with malignant progression. The investigation of LOH has revealed some chromosomal regions that are rarely affected in grade I meningiomas, but are frequently deleted in grade II or III meningiomas.9–11 Among them, chromosome 1p is considered to be one of the most probable regions in which a deletion is associated with the malignant progression of meningiomas and correlates with increased morbidity.12, 13 It has also been suggested that a putative area on chromosome 1p harbors tumor suppressor genes, which are yet to be identified; therefore, more detailed mapping of LOH regions is important to clarify the mechanism of meningioma progression.14–16
In a previous study, we established a new LOH-detection method, named LOQUS (LOH Estimation by Quantitative single-strand conformation polymorphism [SSCP] analysis), that can detect LOH at the single nucleotide polymorphism (SNP) level using a conventional capillary sequencer.17 The remarkable advantages of this system are (i) high sensitivity for LOH detection, which allows for the examination of highly heterogeneous tumor samples (e.g., samples contaminated with up to 80% noncancer cells), and (ii) flexibility of experimental design (i.e., optional high-density analyses can be performed simply by adding SNPs from public databases or other sources).
One of the limitations of LOQUS analysis is that it requires paired DNAs of tumor and normal tissues from the same individual. In the present study, we modified the LOQUS method and established an improved LOH detection system that does not require matched nontumor DNA. In this new method, LOH is evaluated based on the distribution of the signal ratio of the two alleles of heterozygotes, which are predetermined using a limited number of unrelated normal individuals. In addition, this method also has the advantage of performing higher throughput analysis than that described for LOQUS.
We analyzed the LOH for 68 SNP markers on chromosome 1p36 (average inter-SNP distance of 441 kbp) in 130 meningiomas and eventually identified a narrowed region of LOH hot spots at 1p36.11 that might harbor tumor suppressor genes associated with meningiomas.
Material and methods
Samples and DNA preparation
Meningioma samples were obtained from patients during surgery at Kyushu University Hospital. A part of the tumor tissue was saved for histopathologic examination, the rest was snap-frozen in liquid nitrogen and stored at −80°C. Tumors were histologically diagnosed by qualified neuropathologists (S.O.S. and T.I.) and graded according to WHO criteria.1 A total of 130 tumor tissue samples were collected from 122 patients with meningiomas, including 8 anaplastic meningiomas, 27 atypical meningiomas and 95 benign meningiomas (see Table I for details). Multiple samples obtained from different surgeries of the same patients were included if the histologic diagnosis of the recurrent tumor was different from that of the primary tumor. Of these samples, 30 were also used for LOH analysis, using microsatellite markers. Tumor DNA was isolated from the frozen blocks using a QIAamp DNA Mini Tissue Kit (Qiagen, Valencia, CA). Normal DNA was isolated from blood samples using a QIAamp DNA Blood Kit (Qiagen). The present study was approved by the Ethics Committee of Kyushu University.
|WHO grade and histologic subtype||LOH no./Total no.||LOH (%)|
Single nucleotide polymorphism
We chose 96 SNPs on the distal portion of chromosome 1p (from the telomeric end to 1p35.3) from the public database, International Hapmap Project (http://www.hapmap.org/index.html.en). SNPs with allele frequencies greater than 40% in the Japanese population were chosen from the database. The genomic sequences, including the chosen SNPs, were downloaded from NCBI (http://www.ncbi.nlm.nih.gov/), and the repetitive sequences were masked by use of Repeatmasker (http://www.repeatmasker.org). Polymerase chain reaction (PCR) primer-pairs for all SNPs were designed for nonredundant regions, using Primer3 software,18 to obtain a product of 80–120 bp and a standardized primer melting temperature (Tm) of 60°C. Oligonucleotide primer pairs (custom synthesized at SIGMA Genosys, Hokkaido, Japan) were made to carry either a 5′-ATT or 5′-GTT for postlabeling purposes, as described previously.19
Polymerase chain reaction
PCR was performed in a total volume of 5 μl containing 25 ng of template DNA, 0.25 μM of each primer, 0.2 mM of each nucleotide, 0.125 U of AmpliTaq® DNA polymerase (Applied Biosystems, Foster City, CA), 27.5 ng of TaqStart™ antibody (Clontech Laboratories, Palo Alto, CA), 2 mM MgCl2, 10 mM Tris-HCl, pH 8.3, 50 mM KCl and 5% DMSO. The thermal cycle profile was 1 min at 94°C for initial heating, followed by 40 cycles of 30 sec at 94°C, 30 sec at 60°C and 1 min at 72°C.
PLACE-SSCP and data analysis
Post-PCR labeling was performed, followed by removal of the residual fluorescent nucleotides by gel filtration, as described previously.20 After post-PCR labeling, 2 μl/well of the product was added to the prepared loading plate containing 28 μl/well of a premix solution (27.75 μl/well of 0.5 mM EDTA and 0.25 μl/well of homemade single-strand size marker). The samples were heated at 90°C for 3 min. Electrophoresis was performed at 27°C under SSCP conditions using a 36 cm capillary in the ABI Prism® 3100 Genetic Analyzer (Applied Biosystems). The samples were loaded by electrokinetic injection at 2 kV for 10 sec and separated at 15 kV for 30 min at 27°C, using the sieving matrix of 10% linear polydimethylacrylamide in 2 × TME (60 mM Tris, 70 mM MES and 2 mM Na2EDTA), as described previously.21 Output data were converted to ASCII format and then imported to Quantitative Interpretation of SSCP in Capillary Array (QUISCA) software for analysis.22
Tumor and nontumor DNA was evaluated by a PCR-based LOH assay using 26 microsatellite markers located on chromosome 1p, as shown in Supplemental Table S1. PCR and fluorescence labeling were performed according to the method described previously.23 Capillary electrophoresis was performed with a 3730 Prism Genetic Analyzer (Applied Biosystems). Alleles were identified and peak heights were measured using GeneMapper software (Applied Biosystems, Version 3.0). Allelic status was assessed by the criteria established in a previous study.23
Microsatellite analysis on entire chromosome 1p
To examine the overall LOH status on chromosome 1p in meningiomas, we first performed the LOH analysis using 26 microsatellite markers distributed throughout the entire short arm of this chromosome. For this analysis, we used a subset30 of the tumor samples obtained from patients whose normal DNA samples were available. As shown in Figure 1, LOH was detected most frequently on 1p36, which is in agreement with several previous studies.14, 24 We then attempted to narrow down the region by a SNP-based LOH assay.
Selection of SNP markers
We first chose 96 SNP markers, distributed over the distal portion of chromosome 1p (from the telomeric end to 1p35.3), to cover the entire 1p36, as described in the Materials and Methods. PCR reactions on nontumor DNA from 8 individuals were performed for each sequence-tagged site containing a SNP. Subsequently, PCR products were divided and used for sequencing and PLACE-SSCP analyses, to confirm the SNPs and to select SNPs suitable for precise quantitative analysis of alleles by SSCP. A total of 68 SNPs were used for subsequent LOQUS analyses (see Supplemental Table S2 for details).
Averaged control method
In the LOQUS assay, paired DNAs of tumor and normal samples are repeatedly analyzed by PLACE-SSCP to assess the variability of the allele signal ratio. LOH was statistically determined based on the distribution of the ratios of normal samples. Unfortunately, this method has the disadvantage of not being able to test tumor samples when the matched normal DNA is unavailable. Therefore, we exploited the possibility of detecting LOH using the signal ratio of alleles in heterozygous samples from multiple individuals as a control, instead of using matched nontumor DNA from the same patient. Here, we addressed the surgical tumor tissues including normal cells at the detective level so that LOH of tumors without matched normal DNA could be distinguished from homozygosity.
To test the possibility mentioned above, we first assessed the variability of the peak height ratios of alleles of all examined SNPs in many heterozygotes. We picked up 55 meningioma samples in which the corresponding nontumor DNAs were available. PLACE-SSCP analyses of all 68 SNPs were then performed using these samples. These 55 tumor DNAs were separately arranged to 2 μl plates and nontumor DNA samples from 24 different individuals were also included for the analysis by adding to each plate. We then determined the following values for all SNPs.
Rij(N): The peak-height ratio of alleles (fast to slow) of ith SNP in jth heterozygous nontumor sample.
Rik(T): The peak-height ratio of the alleles (fast to slow) of ith SNP in kth heterozygous tumor sample.
Ai(N): The average of Rij(N).
Vik(T): min [Rik(T)/Ai(N), Ai(N)/Rik(T)].
Some values were not determined because the individuals were homozygous for the particular SNPs. However, 5–19 (average 11.6) individuals were heterozygous for the SNPs examined. As a negative control, we replaced the data of tumor DNAs above to those of nontumor DNAs obtained from the same patients and performed the same analyses, then Rik(C) and Vik(C), which are the control data of Rik(T) and Vik(T) respectively, were calculated.
As shown in Figure 2a, the distribution of Vik(T) (1701 determinations) revealed a bimodal pattern, presumably representing a group of SNPs in a LOH region and another group representing those in a ROH region. Consistent with this interpretation, the distribution of the second group closely resembled that of Vik(C) (Fig. 2b). We named this procedure the LOH estimation by quantitative SSCP analysis using averaged control: LOQUS-AC.
In conclusion, the signal ratio of alleles in heterozygous samples from multiple individuals were used as an alternative control in the LOQUS assay, and based on the bimodal distribution pattern above, the appropriate threshold value of LOH was estimated to be between 0.65 and 0.85.
One would expect that the results analyzed by LOQUS-AC using the most appropriate threshold value, should be consistent with the conventional LOQUS method. Therefore, to infer the most appropriate threshold value of LOH, we tested an LOH estimation on 1701 loci analyzed above, based on a variable threshold value from 0.65 to 0.85. We also performed the conventional LOQUS assay using the data of paired DNAs within the same dataset. Subsequently, the total concordance rate, defined as the fraction of the loci showing the consistent determinations (LOH or ROH) in both assays, was calculated for each threshold value tested. In addition, specificity and sensitivity were also calculated, assuming that the LOQUS method provides an accurate answer. As shown in Figure 3, the maximum concordance rate of 98.1% was reached at the threshold value of 0.78, where the specificity is almost 100% (99.9%) and there is only a slight loss in the sensitivity (97.0%; 97.6% at maximum). Accordingly, we inferred that 0.78 should be the appropriate threshold value for detecting LOH by LOQUS-AC.
To further support this threshold value, we estimated the sample-to-sample variability of the allele signal ratio using the data set of Rij(N) and Ai(N) above. We determined the overall variability of Rij(N) (734 determinations) by calculating Vij(N), which is Rij(N)/Ai(N), and the variability of Vij(N) can be approximated by the normal distribution, judged by the Shapiro-Wilk W test using JMP software (SAS Institute, Cary, NC; Version 5.0.1J, 2002. Available at: http://www.sas.com/) (Supplementary Fig. 1).25 The mean and SD of Vij(N) were 1.000 and 0.072, respectively, comparable to the value of run-to-run variability of the peak-height ratios for normal samples in the previous study.17 The data set of another 24-individuals for all SNPs showed that the mean and SD of Vij(N) were highly reproducible. Accordingly, when we adopt three times the standard deviation as the threshold for determining the LOH, the threshold value becomes 0.784 (1–3 × 0.072), which is close to the inferred threshold value of Vij(T) for the LOH determination earlier. We thus conclude that a Vik(T) of less than 0.78 (∼3 times the SD from the mean) can be regarded as an LOH at a confidence level of 99.7% (according to the probability density of the Gaussian distribution). We adopted this criterion as an indication of LOH in the LOQUS-AC assay described later.
LOQUS-AC assay of meningioma tissue samples
To detect the critical LOH region on 1p in meningiomas, we analyzed LOH of the 68 SNPs on 1p36 in 130 meningiomas. PCR, subsequent post-PCR labeling, and electrophoresis for each SNP were performed with two 96-well microtiter plates, both of which contained 24 normal DNA samples. Figure 4 shows the summary of the assay. For example, Figure 4a reveals a complete 1p36 loss as the frequent pattern of benign meningiomas, which is in agreement with the results from the microsatellite analysis (see Fig. 1). The LOH on 1p36.12-35.3 in an atypical meningioma (M-160) and terminal LOH on 1p36.33-36.32 in a benign meningioma (M-50a) are shown in Figures 4b–4c, respectively.
When tumor tissues are heavily contaminated with normal cells, the accuracy of detecting LOH is often reduced. Not surprisingly, we found 3 such tumor samples (M-007, M-035 and M-145) in the present study. In these samples, the majority of the Vik(T) values were near 0.78, the threshold value of LOH detection in the present method, but none were in the range of 0.9–1.0, where most of the Vik(T) of the SNPs in retention of heterozygosity (ROH) regions are clustered (Supplemental Fig. 2). We thus excluded these three samples from the following data analyses.
LOH profiles of 1p36 in meningiomas
The LOH profiles of the 127 meningiomas studied here are summarized in Figure 5. The tumor frequencies with LOH in 1p36 were 48.39% (45/93 cases) for Grade I, 84.62% (22/26 cases) for Grade II and 100.00% (8/8 cases) for Grade III, suggesting the progressive nature of LOH, in accordance with the grade of malignancy (See Table I for detail). These results were consistent with previous reports about genetic alterations of meningiomas.8, 26, 27 We then estimated the correlation between the LOH fraction and the grade of tumor malignancy. Statistical analysis showed significant differences between Grade I and Grade II (p = 0.001; Fisher's exact test), as well as Grade I and Grade III (p = 0.004; Fisher's exact test), but there was no significant difference between Grade II and Grade III (p = 0.322; Fisher's exact test), suggesting that 1p36 LOH is associated with the malignancy progression from benign meningiomas to atypical and/or anaplastic meningiomas. The LOH fractions were also compared among the histologic subtypes of Grade I meningiomas and there were no significant differences.
We also attempted to narrow down the common LOH regions that might harbor tumor suppressor genes related with the meningioma progression on 1p36. In total, 75 tumors with LOH were assembled (Fig. 6), and among them, all of the Grade III and the majority of Grade II meningiomas showed complete LOH of 1p36. We also noted that a small fraction of meningiomas showed a partial terminal LOH (M-050a and M-050b), interstitial LOH (M-003a and M-160), and complex LOH pattern (two or more discontinuous LOH, as observed for M-048, M-152 and M-049). Furthermore, based on the overlapping continuous LOH region in M-048, M-049, M-152 and M-160, the region that spans 2.02 Mbp, from rs6671571 to rs9438620, might be a novel candidate locus, which harbors tumor suppressor genes associated with meningiomas. The genes located in these regions and their possible involvement in meningioma progression are discussed later.
In the present study, we improved the LOQUS assay that we previously proposed for LOH-detection17 by developing a new approach called LOQUS-AC, which has the advantage of detecting LOH in tumor samples for which matched normal DNA samples are unavailable. The present method is also more efficient because of less repetition in the measurement, compared with the previous method. Conventional LOH analysis using microsatellite markers always requires paired DNA from the same patient because of the fact that the polymorphism could show many genotype patterns in individuals because they are multi-allelic. The recently reported strategy of microsatellite analysis based on real-time quantitative PCR, called Quantitative Microsatellite Analysis, also allows for LOH detection without using paired nontumor DNA, but this method requires tumor tissue containing at least 70% cancerous cells.28
On the other hand, normally only a single pattern of a heterozygous genotype can exist on SNPs; theoretically, this means LOH analyses using SNPs could be performed as long as at least one control DNA sample displaying the heterozygous genotype was available. We evaluated the sample-to-sample variability of the peak-height ratio of SNP alleles (Rh) in heterozygous individuals with high reproducibility. This result indicates that DNAs from unspecified individuals can be used as a control for the estimation of Rh. We also compared the LOH status using both methods and demonstrated that the consistency rate of LOH determination was 98.1%, demonstrating the high reliability of this new method.
Recently, several approaches were established to study LOH using SNPs. Among them, SNP-mass spectrometry-genotyping technology has been applied to detect LOH at high-resolution.29 In this study, a rapid source of high-throughput method was provided and thus a high-resolution investigation of LOH for given chromosomal regions was carried out. However, this method detects LOH by comparing the genotype of tumor DNA to that of the matched normal DNA and therefore requires the examination of both DNAs simultaneously. In addition, LOH can be detected precisely only when the population of normal cell contamination is less than 10%. On the other hand, current generations of SNP arrays provide high-density markers, making it possible to estimate LOH regions with extremely high resolution. In some of these methods, LOH can be inferred by the probability of the appearance of consecutive homozygous genotypes.30, 31 In recent studies, hidden Markov model-based interpretation of the data obtained from SNP array or SNP-beads, allowed for an accurate LOH detection for most of the examined SNP markers. Compared with their previous study using paired nontumor DNA as control,30 a significant loss in tolerance of normal DNA contamination was observed for this method which requires samples of tumor cell lines or tumor tissues that are almost free of normal DNA, an unrealistic event in the field of clinical testing.32, 33 In contrast to these past methods, our current method can be applied to samples without paired nontumor DNAs and also maintains tolerance of tumor DNA that is highly contaminated with normal DNA. We thus suggest our method would be appropriate for clinical application because clinically obtained tumor samples should include nontumoral cells to some extent, derived from blood or connective tissues, and because paired normal DNAs may not be obtained from some patients because of various clinical issues. It is true that LOH is not detectable by this method in the tumor tissues containing very little amount of normal cells because their LOH is hardly distinguishable from homozygosity, but such cases are extremely rare in surgically obtained samples.
Allelic loss of 1p, one of the most frequent chromosomal abnormalities in meningioma, is predominant in atypical and anaplastic histologic types, and is therefore recognized as a progression-associated genetic aberration in meningioma.8, 34 Several studies have succeeded in narrowing down the critical regions on 1p that relate to tumorigenesis of meningiomas.12–14, 35, 36 Several candidate genes on 1p have been discussed with regard to their relationship with meningioma tumorigenesis or progression, including TP73, CDKN2C (encoding p18INK4c), RAD54L and ALPL.37–40 The roles of these genes, however, are still controversial.
In the present study, we successfully applied the improved LOQUS method, LOQUS-AC, to detect the allelic status of chromosome 1p36 in various stages of meningiomas at a higher resolution compared with previous reports.12–14 We finally defined a candidate common LOH region which might associate with meningioma. This region, located at 1p36.11, was considerably narrowed down when compared with a 19.3- Mbp candidate locus, R2, located at 1p36.21-p34.1 by Buckley et al.16 Genes located at this region are shown in Supplemental Table S3. Among them, interesting candidate genes include RUNX3 and EXTL1. RUNX3 is frequently deleted or transcriptionally silenced in various types of cancer.41–44EXTL1 is a member of the human tumor suppressor EXT gene family.45 Further analyses are needed to identify tumor suppressor genes from these candidate genes. Finally, LOQUS-AC might also be applicable to detect LOH in other tumor types.
- 1World Health Organization classification of tumors. Tumors of the nervous system. Lyon: IARC Press, 2000; 29–39., .
- 40Loss of alkaline phosphatase activity in meningiomas: a rapid histochemical technique in indicating progression-associated deletion of a putative tumor suppressor gene on the distal part of the short arm of chromosome 1. J Neuropathol Exp Neurol 1997; 56: 879–86., , , , , .
This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0020-7136/suppmat .
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