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Printed in Singapore. All rights reserved Journal compilation # 2006 Blackwell Munksgaard Mutation analysis of BRCA1 and BRCA2from 793 Korean patients with sporadicbreast cancer Han S-H, Lee K-R, Lee D-G, Kim B-Y, Lee K-E, Chung W-S. Mutation analysis of BRCA1 and BRCA2 from 793 Korean patients with sporadic Clin Genet 2006: 70: 496–501. # Blackwell Munksgaard, 2006 To investigate the role of BRCA1 and BRCA2 mutations in Korean patients with sporadic breast cancer, 793 breast cancer patients were analyzed by Clinical Laboratories, Seoul, Korea,bDivision of Hematology and Oncology, denaturing high performance liquid chromatography and direct sequencing.
The 793 breast cancer patients enrolled in this study had no family history of affected first- or second-degree relatives with breast and/or ovarian cancer.
Seventy-nine different sequence variations were identified, of which 34 were novel. Fifteen deleterious mutations were detected in 20 out of 793 patients (2.5%): 11 frameshift mutations and 4 nonsense mutations (seven in BRCA1and eight in BRCA2), and no recurrent or founder mutations were observedin BRCA mutation screening. However, three mutations (K467X, 3972delTGAG, and R2494X in BRCA2) were identified in other studies of the Korean population. Of 793 patients, the clinicopathological information was obtained in 135 patients, who included 20 deleterious mutation-positive and 115 deleterious mutation-negative groups. The median age at diagnosis, histologic type, histologic grade and T stage did not show statistically significant difference between these two groups. BRCA-mutation-associated tumors showed lower estrogen receptor, progesterone receptor, and HER-2/neu but higher p53 expression. Although poor prognostic features were noted in BRCA-associated tumors, we did not find statistically fax: 182 2 2650 2719;e-mail: significant differences. The present study will be helpful in the evaluationof the need for the genetic screening of germline BRCA mutations and reliable genetic counseling for sporadic breast cancer patients.
accepted for publication 27 September2006 The incidence of breast cancer in Korea has been mutations result in truncation of the encoded increasing in recent years. Although this cancer is protein and malfunction of protein activation (6).
still much less prevalent in Korea than Europe Up to date, more than 900 deleterious BRCA and the United States, it became the most mutations, polymorphisms, and sequence varia- tions with unclassified significance have been (1). Remarkably, the peak age of Korean women described in the Breast Cancer Information Core diagnosed with breast cancer is in the 40s, which (BIC database;
is 15–20 years younger than that in the United Many researchers from different countries have States. Breast cancer is typically diagnosed at reported hundreds of disease-associated BRCA young age in Asian countries including Korea, mutations. Among those reported mutations, which suggests the important role of racial several founder mutations have been identified differences and genetic variations (2).
in specific ethnic groups such as Icelanders, Ashkenazi Jews, Russians, and Israelis. Due to genes account for genetic predisposition and founder effects including environmental and geo- increased risk of breast and ovarian cancer in the graphical factors, the prevalence of BRCA1 and majority of affected families (3–5). Most BRCA BRCA2 mutations is variable among different Mutation analysis of BRCA1/2 from 793 Korean sporadic breast cancer patients populations (7, 8). There have been few data on instrumentation equipped with a DNASep col- the contribution of germline BRCA mutations to umn (Transgenomic Inc.). The column is packed with proprietary, 2-mm, nonporous, alkylated poly (styrene divinylbenzene) particles (10, 11).
mutations were screened in 793 samples of breast An alternative phase consisting of 3.5-mm-wide cancer patients with no family history of breast pore, alkylated silica beads (Eclipse dsDNA cancer and/or ovarian cancer as well as in 167 Analysis Column, San Jose, CA) is available com- samples of normal controls with negative mam- mercially from Hewlett Packard (Palo Alto, CA).
mography to investigate the presence of BRCA1 The mobile phase was 0.1 M triethylammonium acetate buffer, pH 7.0 (PE Biosystems, San Jose, breast cancer patients in Korea. The mutational CA), containing 0.1 mM tetrasodium ethylenedi- analysis was performed by denaturing high amine-tetraacetic acid (Sigma, St Louis, MO).
performance liquid chromatography (DHPLC) Crude polymerase chain reaction (PCR) prod- analysis through the entire coding exons and ucts, subjected to an additional 95°C denaturing exon–intron boundaries of the BRCA1 and step for 5 min followed by gradual reannealing from 95°C to 23°C over a period of 40 min priorto analysis, were eluted with a linear acetonitrile(J.T. Baker, Phillipsburg, NJ) gradient at a flowrate of 0.9 ml/min. The start- and end-points of the gradient were adjusted according to the size of the PCR products using an algorithm pro- A total of 793 pathologically confirmed breast cancer patients were enrolled in this study.
software. Generally, an analysis took 5–10 min, Family histories were examined by interviewing including column regeneration and re-equilibra- patients, and only cases without a family history tion to the starting condition. The temperature of affected first- or second-degree relatives with required for successful resolution of heteroduplex breast and/or ovarian cancer were selected for this study. Additionally, 167 normal control melting algorithm. Details of the temperature conditions and the algorithm can be obtained lyzed, who had no family history of breast and/or ovarian cancer. Among the 793 breast cancer DHPLC gradients and temperatures were deter- patients, the clinicopathological information mined by WAVE Maker System software.
could be acquired in 135 patients by medical Heterozygous profiles were identified by visual record review, which was divided into deleterious inspection of the chromatograms on the basis of mutation-positive (n ¼ 20) and deleterious additional earlier eluting peaks compared with mutation-negative group (n ¼ 115) for compar- one peak in corresponding homozygous profiles.
ison between them. The clinicopathologicalcharacteristics such as the age at diagnosis, family history, histological subtype, histologic Once subset of PCR products with an aberrant grade, T stage, and estrogen receptor (ER), DHPLC pattern had been identified, its PCR progesterone receptor (PR), p53, and HER-2/ products were amplified using the same primers neu expression were obtained. This study was as used for DHPLC, and DNA was sequenced on approved by an institutional board, and in- a ABI 3100 sequencer (Perkin Elmer Applied formed consent was obtained form all subjects.
Biosystems, Foster City, CA) in both directions.
The sequence was compared with the Breast Denaturing high performance chromatography Cancer Information Core (BIC database; http:// Genomic DNA was extracted from peripheral, Human Genome whole-blood samples, using standard techniques Mutation Database ( with the Gentra kit Systems (Gentra Inc., ac/index.php) and the National Center for Bio- Minneapolis, MN). All samples were tested for technology Information database (http://www.
small deletion, insertion, or point mutations on all exons of the BRCA1 and BRCA2 gene using DHPLC (Transgenomic Inc., San Jose, CA).
DHPLC analysis was performed on a WAVE Clinicopathologic characteristics and BRCA Maker System as previously described (9).
mutation results were analyzed using SPSS (Statistical Package for the Social Sciences, Chicago, IL) 12.0 for Windows. Differences in mutations (1047C.T, 2167delA, 2478delG, and categorical variables between deleterious muta- 5616del10 in BRCA1; 2487delT, 8662delGGA, tion-positive and deleterious mutation-negative and 10378C.T in BRCA2) were novel. The muta- group were compared using chi-square analysis tional spectrum is summarized in Tables 1 and 2.
or Fisher’s exact test. p value of ,0.05 was con- (1047C.T, 2167delA, 2478delG, 2552delC,3746insA, 4184delTCAA, and 5616del10) pro-duced a truncated protein signal at codons 310, 700, 791, 811, 1218, 1364, and 1839, respectively.
A total of 79 sequence variations in BRCA1 and Four of these seven BRCA1 deleterious mutations BRCA2 were identified in 793 patients, which (1047C.T, 2167delA, 2478delG, and 5616del10) consisted of 38 missense, 26 polymorphism, and were novel. In BRCA2, four frameshift muta- 15 deleterious (11 frameshift and 4 nonsense) mu- tions (1537delAAAG, 2487delT, 3423delTAAT, tations, by mutation type. The novel sequence varia- tions were identified in 34 out of 79 sequence (8662delGGA), and three nonsense mutations variations (43%). Out of 79 sequence variations, (1627A.T, 7708C.T, and 10378C.T) were iden- 15 deleterious mutations were detected in 20 out tified. Among the eight deleterious mutations in of 793 patients (2.5%), which were not recurrent or founder mutation. Seven out of 15 deleterious 8662delGGA, and 10378C.T) were identified.
Table 1. Sequence alterations detected in BRCA1 Exon Nucleotide change Amino acid change Mutation type Patients (na ¼ 793) Normal controls (na ¼ 167) BIC entries BIC, the Breast Cancer Information Core (last modified: Tuesday, 17 January 2006); FS, frameshift; MS, missense; NS,nonsense; P, polymorphism.
cMutations show a high frequency in both patients and normal controls.
Mutation analysis of BRCA1/2 from 793 Korean sporadic breast cancer patients Table 2. Sequence alterations detected in BRCA2 Exon Nucleotide change Amino acid change Mutation type Patients (na ¼ 793) Normal controls (na ¼ 167) BIC entries BIC, the Breast Cancer Information Core (last modified: Tuesday, 17 January 2006); FS, frameshift; IFD, in frame deletion; MS,missense; NS, nonsense; P, polymorphism; UTR, untranslated region.
bMutations identified independently in previous studies on Korean population.
dMutations show a high frequency in both patients and normal controls.
Three out of eight BRCA2 deleterious mutations tified in previous studies on Korean population (12–15). A family study was not performed for detected in 7 of 20 patients, which were also iden- patients with deleterious mutations.
Out of 38 missense mutations in BRCA1 and novel mutation in this study (47%) was similar to three previous studies in Korean populations remaining 11 were spread at exons 5, 16, and (54.5–66.7%) (12–14). The spectrum of BRCA1 22 in BRCA1 and exons 2, 3, 10, 14 15, 17, and and BRCA2 mutations in the Korean population 27 in BRCA2. Among the 38 missense mutations, appears different from those of Ashkenazi Jew 3 novel BRCA1 and 10 novel BRCA2 mutations and other populations. None of three founder mutations identified in Ashkenazi Jewish descent A total of 30 sequence variations were identi- fied in 167 normal controls, which consisted of 18 6174delT for BRCA2) or 999del5 mutation for missense and 12 polymorphisms by mutation type.
BRCA2 common to the Icelandic population was No deleterious mutations were detected in normal detected in this study. This study did not show controls. Among the 13 novel missense mutations, 12 (2 in BRCA1 and 10 in BRCA2) mutations in Korean breast cancer patients. However, the were not found in 167 normal controls (334 chromosomes). Seven BRCA1 sequence variations (2201T, 2430C, 2731T, 3232G, 3667G, 4427C, of 20 patients was found in this study, as well as and 4956G) and nine BRCA2 sequence varia- in other previous studies (12–15). Although more population-based study is required, these find- ings suggest that the above three mutations at a high frequency in 167 normal controls. They might be a recurrent mutation and have impor- also showed similar frequency in 793 breast can- tant implication for screening strategies for breast cancer among Korean population.
The clinicopathological information was ob- The prevalence of germline deleterious muta- tained in 135 patients, who included 20 deleteri- tions in BRCA1 and BRCA2 was estimated to be ous mutation-positive and 115 deleterious 5.1 and 6.7% in previous studies, and phenotypes mutation-negative patients. The median age at diag- were variable by ethnicity (2, 16). This study nosis of both the BRCA deleterious mutation- showed a lower prevalence of BRCA mutations positive and deleterious mutation-negative group (2.5%), which was similar to previous studies on was similar, 43 and 48 years, respectively. Infil- trating ductal carcinoma was the predominant patients with sporadic breast cancer (2.8–3.1%) histologic subtype in both groups. The cases with (14, 15). Further studies on large numbers of deleterious BRCA mutations showed lower ER, cases may give a more accurate estimation of PR, and HER-2/neu and higher p53 expression prevalence and variations between Asian pop- compared with mutation-negative cases, of which ulations. The true contribution of BRCA genes difference was not statistically significant.
to sporadic breast cancer remains controversialfor a number of reasons. First, missense muta-tions with an unknown significance could havea pathogenic effect. Second, in addition to missense mutations, silent polymorphisms and In this study, we aimed to evaluate the role of intronic variants may affect splicing mechanisms.
However, these variants cannot be classified as Korean patients with sporadic breast cancer.
associated with disease in the absence of a good Whereas the majority of studies on BRCA gene functional assay system for BRCA gene. Once mutations have focused on the western popula- a functional assay becomes available, it could be tion with a family history or ovarian cancer, only important to elucidate the relevance of such a relatively small number of investigations on the variations with currently unknown significance.
role of the BRCA genes have been undertaken in Moreover, it should be noted that no currently Asian sporadic breast cancer populations (2).
available technique can guarantee detection of all In this study, BRCA mutation analysis of 793 disease-associated mutations in the BRCA genes.
breast cancer patients showed 34 novel variants In this study, the BRCA mutation analysis for 167 out of 79 sequence variations, which may be of normal controls with normal mammography was Korean-specific origin. Fifteen deleterious muta- performed to compare the frequency of BRCA tions (11 frameshift and 4 nonsense mutations) in mutation with the breast cancer patient group.
Deleterious mutations were not found in normal termination producing shortened proteins were controls. Also, 12 novel missense mutations detected. Among the 15 mutations, 7 (47%) had detected in this study were not found in normal not been published before. The incidence of controls (334 control chromosomes), which Mutation analysis of BRCA1/2 from 793 Korean sporadic breast cancer patients could have a pathogenic effect. The sequence present study allows a better evaluation of the variations listed in Results showed high fre- need for the genetic screening of germline BRCA quency in normal controls. They also showed mutations and reliable genetic counseling for similar frequency in 793 breast cancer patients.
It is speculated that they are not likely to bedisease-associated mutations in the BRCA1 andBRCA2 genes. Although haplotype analysis was not performed, the above BRCA mutations with 1. Korean Breast Cancer Society. Clinical Characteristics of high allele frequency might have high possibility Breast Cancer Patients in Korea in 2000. Arch Surg 2004: In this study, scanning for the presence of 2. De Leon Matsuda ML, Liede A, Kwan E. BRCA1 and BRCA2 mutations among breast cancer patients from the sequence variations was performed by mutational Philippines. Int J Cancer 2002: 98: 596–603.
analysis through the entire coding exons and exon– 3. Easton DF, Bishop DT, Ford D, Crockford GP. Genetic linkage analysis in familial breast and ovarian cancer genes using DHPLC analysis. DHPLC offers the results from 214 families. The Breast Cancer Linkage advantage of distinct elution profiles that differ not Consortium. Am J Hum Genet 1993: 52: 678–701.
only between different sequence variants but also 4. Frank TS, Manley SA, Olopade OI et al. Sequence analysis of BRCA1 and BRCA2: correlation of mutations with between combinations of them. In this study, to family history and ovarian cancer risk. J Clin Oncol 1998: verify the sensitivity of DHPLC screening, 960 fragments were tested in a blind manner by direct 5. Hall JM, Lee MK, Newman B et al. Linkage of early-onset DNA sequencing, and no discrepancies were found familial breast cancer to chromosome 17q21. Science 1990: (data not shown). As shown in Tables 1 and 2, 6. Wooster R, Bignell G, Lancaster J et al. Identification of many sequence variations including novel variants the breast cancer susceptibility gene BRCA2. Nature 1995: and single substitutions were detected using this system, which also demonstrates the efficiency of 7. Loman N, Johannsson O, Kristoffersson U, Olsson H, Borg A. Family history of breast and ovarian cancers and BRCA1 and BRCA2 mutations in a population-based series of early- clinicopathological information about 20 delete- onset breast cancer. J Natl Cancer Inst 2001: 93: 1215–1223.
8. Neuhausen SL. Ethnic differences in cancer risk resulting rious mutation-positive and 115 deleterious from genetic variation. Cancer 1999: 86: 2575–2582.
mutation-negative groups are similar to that of 9. Gross E, Amold N, Pfeifer K, Bandick K, Kiechle M.
the other studies: BRCA-associated tumors Identification of specific BRCA1 and BRCA 2 variants by DHPLC. Hum Mutat 2000: 16: 345–353.
higher p53 expression, findings in accordance 10. Huber CG, Oefner PJ, Bonn GK. High-resolution liquid with previous studies. Although poor prognostic chromatography of oligonucleotides on nonporous alky-lated styrene-divinylbenzene copolymers. Anal Biochem features were noted in BRCA-associated tumors, the differences were not statistically significant.
11. Huber CG, Oefer PJ, Bonn GK. Rapid and accurate sizing These data suggest that neither hormone recep- of DNA fragments by ion-pair chromatography on tors nor HER-2/neu stimulation seems to be alkylated nonporous poly (styrene-divinylbenzene) par- important in the pathogenesis of cancers arising ticles. Anal Chem 1995: 67: 578–585.
in mutation carriers. A reasonable explanation 12. Kang HC, Kim IJ, Park JH et al. Germline mutations of BRCA1 and BRCA2 in Korean breast and/or ovarian for these results is that p53 loss rescues the cancer families. Hum Mutat 2002: 20: 235–239.
proliferative deficiency of BRCA mutant cells at 13. Choi DH, Lee MH, Bale AE, Carter D, Haffty BG. Inci- the expense of genetic instability (17).
dence of BRCA1 and BRCA2 mutations in young Korean In conclusion, a total of 793 Korean patients breast cancer patients. J Clin Oncol 2004: 22: 1638–1645.
with sporadic breast cancer were analyzed for 14. Seo JH, Cho DY, Ahn SH et al. BRCA1 and BRCA2 germline mutations in Korean patients with sporadic breast germline mutations in the BRCA1 and BRCA2 cancer. Hum Mutat 2004: 24: 350–356.
genes, using a combination of DHPLC and direct 15. Ahn SH, Hwang UK, Kwak BS et al. Prevalence of sequencing. The present study revealed 79 BRCA1 and BRCA2 mutations in Korean breast cancer different sequence variations including 15 dele- patients. J Korean Med Sci 2004: 19: 269–274.
terious mutations, 34 of which have not been 16. Liede A, Malik IA, Aziz Z et al. Contribution of BRCA1 reported previously and may be of Korean- and BRCA2 mutations to breast and ovarian cancer inPakistan. Am J Hum Genet 2002: 71: 595–606.
specific origin. The genetic testing of BRCA 17. Xu X, Qiao W, Linke SP et al. Genetic interactions between mutations remains too expensive and laborious tumor suppressors Brca1 and p53 in apoptosis, cell cycle and thus is restricted to high-risk families. The and tumorigenesis. Nat Genet 2001: 28: 266–271.


ORIGINAL CONTRIBUTION Osteopathic Manipulative Treatment in the Emergency Department for Patients With Acute Ankle Injuries Anita W. Eisenhart, DOTheodore J. Gaeta, DO, MPHDavid P. Yens, PhD Study Objective : The purpose of this study was to evaluate pression dressings, elevation of the affected ankle, analgesia the efficacy of osteopathic manipulative treatment (OMT) (specifically

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Letteratura moderna e contemporanea AA.VV., L'altro visto dall'altro - Letteratura araba ed ebraica a confronto , Milano, Cortina, 1992 AA.VV., L'altro Mediterraneo - Antologia di scrittori arabi del Novecento , a c. V. Colombo), Milano, Oscar Mondadori, 2004 AA.VV., Silenzi , a cura di I. Camera d'Afflitto, ed. Avagliano 1999 Albert ABOU ABDALLAH, La poesia araba moderna e i

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