Familial Cancer (2012) 11:307–311 DOI 10.1007/s10689-012-9509-7
SHORT COMMUNICATION
Prevalence of TP53 germ line mutations in young Pakistani breast cancer patients Muhammad U. Rashid • Sidra Gull • Kashif Asghar • Noor Muhammad • Asim Amin • Ute Hamann
Published online: 7 February 2012 Ó Springer Science+Business Media B.V. 2012
Abstract Women from Pakistan and India are more often diagnosed with early-onset breast cancer than Caucasian women. Given that only 12% of Pakistani women diagnosed with breast cancer at or before 30 years of age have previously been shown to harbor germ line mutations in the breast cancer susceptibility genes BRCA1 and BRCA2, the genetic causes of the majority of early-onset cases are unexplained. Since germ line mutations in the tumor suppressor gene TP53 predispose women to early-onset breast cancer, we assessed the prevalence of TP53 mutations in 105 early-onset breast cancer patients from Pakistan, who had previously been found to be negative for BRCA1 and BRCA2 germ line mutations. The patient group included 67 women diagnosed with early-onset breast cancer at or before age 30 with no family history of breast or ovarian cancer (EO30NFH group) and 38 women diagnosed with breast cancer at or before age 40 with one or more first- or second-degree relatives with breast or ovarian cancer (EO40FH group). Mutation analysis of the complete TP53 coding region was performed using denaturing high-performance liquid chromatography analysis, followed by DNA sequencing of variant fragments. One deleterious mutation, c.499-500delCA in exon 5, was identified in the M. U. Rashid S. Gull K. Asghar N. Muhammad Shaukat Khanum Memorial Cancer Hospital and Research Centre (SKMCH & RC), Lahore, Pakistan M. U. Rashid U. Hamann (&) Molecular Genetics of Breast Cancer, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany e-mail:
[email protected] A. Amin Levine Cancer Institute, Carolinas Medical Center, Charlotte, NC, USA
105 breast cancer patients (1%). This mutation is novel in the germ line and has not been described in other populations. It was detected in a 28-year-old patient with no family history of breast or ovarian cancer. This mutation is rare as it was not detected in additional 157 recently recruited non-BRCA1 and non-BRCA2-associated earlyonset breast cancer patients. Our findings show that TP53 mutations may account for a minimal portion of early-onset breast cancer in Pakistan. Keywords TP53 Germ line mutations Early-onset breast cancer Pakistan
Introduction In Pakistan breast cancer is the most frequent malignancy among women accounting for 34.6% of all female cancers [1]. A recent analysis of breast cancer diagnoses using data from the United States National Cancer Institute’s surveillance, epidemiology and end results (SEER) cancer program showed that Pakistani and Indian women in the US are more often diagnosed with breast cancer before age 40 than Caucasian women (16.2 vs. 6.2%) [2]. A similar finding was obtained in another study conducted among US-residing Pakistani/Indian immigrants that showed a higher proportion of breast cancers diagnoses before age 40 in Pakistani/Indian women compared to non-Hispanic White women (14.8 vs. 4.5%) [3]. Germ line mutations in the TP53 tumor suppressor gene predispose to several different malignancies including early-onset breast cancer. They are the underlying cause of Li-Fraumeni syndrome (LFS), a rare autosomal dominant disorder that predisposes to breast cancer, soft tissue sarcomas, osteosarcomas, brain tumors, leukemia,
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adrenocortical carcinomas and many rarer tumors [4, 5]. In this syndrome, most of breast cancers occur in young women, with approximately 30% being diagnosed before age 30 [6]. TP53 germ line mutations have been found in up to 83% of families/individuals with complete LFS features [4, 6–10] and in up to 30% of families or individuals with partial LFS features, the latter defined as Li-Fraumenilike (LFL) syndrome [6, 7, 11–13]. The life time risk of cancer in TP53 mutation carriers is high and more pronounced in women (nearly 100%) than in men (73%), primarily due to female breast cancer [12, 14]. Outside the context of LFS/LFL families, TP53 mutations have been shown to contribute to a small number of early-onset breast cancer cases. Prevalence of TP53 mutations ranging from less than 1 to 5% has been reported in studies conducted in Caucasian populations [15–24]. Few data exist on the contribution of TP53 to early-onset breast cancer in Non-Caucasian populations. In a recent study conducted in Chinese women including 76 with early-onset cases diagnosed before age 35, one TP53 mutation (1.3%) was identified [25]. In Pakistan 12% of women diagnosed with breast cancer at or before 30 years of age have previously been shown to harbor germ line mutations in the breast cancer susceptibility genes 1 and 2 (BRCA1 and BRCA2) leaving the genetic causes of the majority of early-onset cases unexplained [26]. In this study we assessed the prevalence of TP53 mutations in young Pakistani breast cancer patients. Our study included 105 early-onset breast cancer patients of Pakistani origin, who had previously been shown to be negative for BRCA1 and BRCA2 germ line mutations. Comprehensive mutation screening of the entire coding region of the TP53 gene was performed.
M. U. Rashid et al.
For this study, two groups of early-onset breast cancer patients were included: (1) women diagnosed with breast cancer at or before age 30 and no family history of breast or ovarian cancer (EO30NFH group), and (2) Women diagnosed with breast cancer at or before age 40 with one or more first- or second-degree relatives with breast or ovarian cancer (EO40FH group). In addition, we tested 157 recently recruited early-onset cancer patients (EO30NFH group: n = 97 and EO40FH group: n = 59), who were also found to be negative for BRCA1 and BRCA2 germ line mutations (Muhammad U. Rashid, unpublished data) and 112 healthy female controls. Participants were included only after they had given informed written consent per institutional policy. The study was approved by the Institutional Review Board of the SKMCH & RC. Mutation screening Genomic DNA was extracted as previously described [26]. The entire coding region and exon–intron junctions of the TP53 (Genbank accession number U94788) gene were screened using DHPLC analysis. DHPLC analysis was carried out with the WAVE system (Transgenomics, Omaha, NE, USA). PCR-primer pairs for exon 4–9, set-up of PCR reactions, cycling conditions and DHPLC running conditions were selected from the IARC TP53 database (http://wwwp53.iarc.fr/p53sequencing.html). PCR primer pairs and DHPLC running conditions for exon 2, 3, and 11 were as published previously [27] and exon 10 was as described elsewhere [21]. When available, a mutation positive control for each exon was included in the DHPLC analyses. DNA sequence analysis
Materials and methods Study subjects One hundred and five Pakistani early-onset breast cancer patients were selected from a database and DNA repository housed at the SKMCH & RC and the DKFZ, respectively. These patients had been offered genetic testing for BRCA1 and BRCA2 germ line mutations as part of a research protocol. The complete coding sequence of the BRCA1 and BRCA2 genes was screened to detect small-range alterations using single strand conformational polymorphism analysis, denaturing high performance liquid chromatography (DHPLC) analysis and the protein truncation assay, followed by bi-directional genomic DNA sequencing of variant fragments. Patients/families, histopathological characteristics and BRCA1 and BRCA2 mutation analyses have been described previously [26].
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Each sample revealing variants identified by DHPLC analysis was sequenced using an automated DNA CEQ 8000 sequencer (Beckman, Hilden, Germany) according to the manufacturers’ instructions. Bi-directional genomic DNA sequencing was performed to verify the presence of a mutation.
Results We studied 105 unrelated female breast cancer patients from Pakistan, all of whom have previously been tested and found to be negative for BRCA1 and BRCA2 germ line mutations. Among these were 67 women in the EO30NFH group (mean age of diagnosis 27 years, range 22–30) and 38 women in the EO40FH group (mean age of diagnosis 32 years, range 22–40). Fourteen of 105 women were diagnosed with bilateral breast cancer. No family history was consistent with LFS/LFL.
Prevalence of TP53 germ line mutations
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The combined approach of DHPLC and DNA sequencing analysis revealed that of the 105 cases, 1 (1%, 95% confidence interval 0.02–5.2) carried a deleterious TP53 frame shift mutation in exon 5, c.499-500delCA. This germ line mutation was previously reported as a somatic mutation in four other cancer types [28–31], but it is the first incidence of it occurring in the germ line (Table 1). It was found in a 28-year-old early-onset breast cancer patient of Punjabi ethnicity with no family history of breast or ovarian cancer. The mutation carrier presented with a grade 3, lymph node status positive, invasive ductal, carcinoma, which was negative for expression of estrogen receptor, progesterone receptor, but positive for epidermal growth factor receptor 2 (data not shown). Two of her paternal uncles presented with pancreatic and renal cell cancer, respectively, both at age 70. The mutation is deemed causative as it is predicted to result in premature termination codon at position 179. It was not found in 157 additional recently recruited early-onset breast cancer patients, who were also tested negative for BRCA1 and BRCA2 germ line mutations (Muhammad U. Rashid, unpublished data). In addition to the deleterious mutation already described, nine distinct TP53 sequence variants were detected (Table 1). These include seven variants previously classified as polymorphisms in IARC TP53 database and two novel variants. The silent c.903A[G mutation was previously found in three somatic tumors other than breast [32–34] and now it was detected for the first time in the germ line. It was identified in a patient of Punjabi origin, who was diagnosed with breast cancer at 27 years of age, but not in 112 healthy controls. The effect of the silent
c.903A[G was evaluated for the pattern of pre-mRNA splicing using exonic splicing enhancers (ESE) finder which compares the wild type and mutant sequence using the four Ser/Arg-rich protein (SR protein) motif-scoring matrices (http://rulai.cshl.edu/tools/ESE/). Using a threshold value of 3.0 the c.903A[G reduced the SF2/ASF (IgM-BRCA1) high-score motif and eliminated the SC35 and SRp40 motifs. The alignment of TP53 exon 8 from human, chimpanzee, mouse and rat showed that this nucleotide is identical, indicating high conservation during evolution and might have a functional importance in splicing. Since synonymous variants alter the RNA processing and post-transcriptional regulation, the relative synonymous codon usage (DRSCU) of c.903A[G was also measured as described recently [35]. The DRSCU value of -0.66 revealed that this change initiates a rarer codon that may be linked with a slower local rate of translation elongation compared with the wild type. The variant is deemed likely to have an effect on the protein. The second novel variant was a G to A change at nucleotide 144 in the 30 untranslated region (30 UTR). It was detected in a patient of Punjabi background diagnosed with breast cancer at 27 years of age and was also not found in 112 healthy controls.
Discussion In the present study we estimated the contribution of TP53 germ line mutations to early-onset breast cancer in Pakistan including 67 women in the EO30NFH group and 38 women in the EO40FH group. We screened the entire coding sequence of the TP53 gene using DHPLC analyses
Table 1 TP53 germ line mutations and nucleotide changes in Pakistani early-onset breast cancer patients Location
Coding (c.) DNA sequencea
Effect
SNP linkb
Classificationc P
Exon 4
c.108G[A (P36P)
Silent
rs1800370
Exon 4
c.215C[G (R72P)
Missense
rs1042522
P
Exon 5
c.499_500delCA
Frame shift
–
M
Exon 8
c.903A>G (P301P)
Silent
–
Md
Intron 2
c.74?38C[G
Intronic
rs1642785
P
Intron 3
c.97-29C[A
Intronic
rs17883323
P
Intron 3
c.96?41_96?56del16
Intronic
rs17878362
P
Intron 6
c.673-36G[C
Intronic
rs17880604
P
Intron 10
c.1100?30A[T
Intronic
rs17880847
P
30 UTR
c.*144G>A
Noncoding (30 UTR)
–
VUS
Novel germ line mutations and nucleotide changes are marked in bold P polymorphism, M mutation, VUS variant of unknown significance a
Nomenclature follows human genome variation society (HGVS) (http://www.hgvs.org). Numbering start at the first A of the first coding ATG (located in exon 2) of NCBI reference sequence NC_000017.9
b
Link to NCBI SNP database (http://ncbi.nlm.nih.gov/projects/SNP/)
c
Classification is based on information from the IARC TP53 database (http://www-p53.iarc.fr)
d
The mutation is likely to be deleterious based on in silico analyses
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followed by direct DNA sequencing of variant fragments and found one pathogenic germ line TP53 mutation. All breast cancer patients were originally selected for genetic testing for BRCA1 and BRCA2 mutations as part of another research study, and were not selected on the basis of a family history suggestive of LFS/LFL. Among the study subjects, none met LFS/LFL criteria. Data on the frequency of TP53 germ line mutations to early-onset/familial breast cancer in Asian populations are scarce. One study conducted in China including 76 women diagnosed with breast cancer before age 35 reported a mutation frequency of 1.3% [25]. This mutation frequency is similar to that found in the present study (1/105, 1%). However, these frequencies may have been underestimated, because in both studies screening for regulatory mutations outside the coding region, which may affect transcription, and for large genomic deletions and rearrangements, which account for approximately 5–10% of all TP53 mutations [36], were not performed. Studies conducted in Caucasian populations to evaluate prevalence of TP53 mutations have shown a range from less than 1 to 7%. One mutation was detected in a study including 167 unselected breast cancer patients (\1%), one of 40 patients diagnosed before age 35 (2.5%), but not in 30 patients with at least one-first degree relative with breast cancer [15]. Both carrier cases met LFS criteria. Another study reported one mutation of unclear functional significance among 126 patients diagnosed before age 40 [24], while no mutations were detected in another study on five early-onset breast cancer families [19]. One mutation was identified in 14 women with non-familial early-onset breast cancer diagnosed before age 30 (7%) and in none of 15 cases between ages 30 and 49 [16]. Recent studies using population-based series of early-onset breast cancer cases yielded higher mutation frequencies. A frequency of 4% was reported in a population-based series of 100 early-onset breast cancer cases from the United Kingdom diagnosed at or before age 30 and unselected for family history including 37 cases with a family history of breast or ovarian cancer and 63 cases without a family history. Two case carriers met LFS/LFL criteria and two did not [18]. A similar frequency of 5% was found in an Australian population-based series of 94 early-onset breast cancer cases including 52 cases diagnosed before age 30 and unselected for family history and 42 cases diagnosed between the ages of 30 and 39 and a family history of breast or ovarian cancer [23]. Two of five case carriers met LFS/LFL criteria. In our study, the c.499-500delCA frame shift mutation was identified in a young Pakistani patient of Punjabi origin with no family history of breast cancer or ovarian cancer. Since DNA samples of the parents of the index patient were not available, the pattern of transmission could not be studied. This mutation would be considered deleterious
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because it is predicted to result in premature termination at codon 179, is located in a highly conserved region of exon 5, a region that corresponds mainly to the DNA binding domain, and is absent in 112 healthy female controls. Its prevalence is low as it was not detected in 157 recently recruited BRCA1- and BRCA2-negative early-onset breast cancer patients. This is the first report of this mutation being identified in the germ line. Previously, it was reported as a somatic mutation in four patients with other cancer types: a women from Kashmir valley (Indian part) diagnosed with esophageal squamous cell carcinoma at age 50 [31], a Japanese male diagnosed with adenocarcinoma of the lung at age 60 [28], a German women diagnosed with squamous cell carcinoma of larynx at age 46 [29] and a male patient from the US diagnosed with adenocarcinoma of the descending/sigmoid colon at 61 years of age [30]. Our study included women from only one ethnic group providing uniformity of subjects, adding to the robustness of data. All patients had been selected for BRCA1 and BRCA2 testing by virtue of having an early-onset of disease or a family history suggestive of hereditary breast/ovarian cancer [26]. Carriers of TP53 mutations have a poor prognosis [37, 38], therefore it is possible that patients who passed away early after diagnosis could have been excluded. However, this is unlikely because blood samples from the majority (85%) of patients were drawn within the first 12 months after diagnosis. Detailed comprehensive family history data over three to four generations was collected from all study participants allowing a correct categorization of pedigrees according to LFS/LFL criteria. In this first study on the prevalence of TP53 germ line mutations in 105 early-onset breast cancer patients from Pakistan only one deleterious mutation was identified. Our finding suggests that TP53 germ line mutations play a minor role in early-onset breast cancer in this population and imply that TP53 mutation screening of early-onset breast cancer patients with or without a family history of breast or ovarian cancer would not be warranted. Acknowledgments We are grateful to all family members for their participation in this study. We thank Pierre Hainaut and Bert Vogelstein for DNA samples of TP53 mutation controls and Antje Seidel-Renkert for expert technical assistance. We thank Magali Olivier for critical reading of the manuscript. This study was supported by the Deutsches Krebsforschungszentrum and the Shaukat Khanum Memorial Cancer Hospital & Research Centre. Conflict of interest conflict of interest.
We, the authors of this study don’t have any
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