Germline variation in BRCA1/2 is highly ethnic-specific: Evidence from over 30,000 Chinese hereditary breast and ovarian cancer patients.

BRCA1 and BRCA2 play essential roles in maintaining the genome stability. Pathogenic germline mutations in these two genes disrupt their function, lead to genome instability and increase the risk of developing breast and ovarian cancers. BRCA mutations have been extensively screened in Caucasian populations, and the resulting information are used globally as the standard reference in clinical diagnosis, treatment and prevention of BRCA-related cancers. Recent studies suggest that BRCA mutations can be ethnic-specific, raising the question whether a Caucasian-based BRCA mutation information can be used as a universal standard worldwide, or whether an ethnicity-based BRCA mutation information system need to be developed for the corresponding ethnic populations. In this study, we used Chinese population as a model to test ethnicity-specific BRCA mutations considering that China has one of the latest numbers of breast cancer patients therefore BRCA mutation carriers. Through comprehensive data mining, standardization and annotation, we collected 1,088 distinct BRCA variants derived from over 30,000 Chinese individuals, one of the largest BRCA data set from a non-Caucasian population covering nearly all known BRCA variants in the Chinese population (https://dbBRCA-Chinese.fhs.umac.mo). Using this data, we performed multi-layered analyses to determine the similarities and differences of BRCA variation between Chinese and non-Chinese ethnic populations. The results show the substantial differences of BRCA data between Chinese and non-Chinese ethnicities. Our study indicates that the current Caucasian population-based BRCA data is not adequate to represent the BRCA status in non-Caucasian populations. Therefore, ethnic-based BRCA standards need to be established to serve for the non-Caucasian populations.

Transforming Growth Factor ß Mediates Drug Resistance by Regulating the Expression of Pyruvate Dehydrogenase Kinase 4 in Colorectal Cancer.

Drug resistance is one of the main causes of colon cancer recurrence. However, our understanding of the underlying mechanisms and availability of therapeutic options remains limited. Here we show that expression of pyruvate dehydrogenase kinase 4 (PDK4) is positively correlated with drug resistance of colon cancer cells and induced by 5-fluorouracil (5-FU) treatment in drug-resistant but not drug-sensitive cells. Knockdown of PDK4 expression sensitizes colon cancer cells to 5-FU or oxaliplatin-induced apoptosis in vitro and increases the effectiveness of 5-FU in the inhibition of tumor growth in a mouse xenograft model in vivo In addition, we demonstrate for the first time that TGFß mediates drug resistance by regulating PDK4 expression and that 5-FU induces PDK4 expression in a TGFß signaling-dependent manner. Mechanistically, knockdown or inhibition of PDK4 significantly increases the inhibitory effect of 5-FU on expression of the anti-apoptotic factors Bcl-2 and survivin. Importantly, studies of patient samples indicate that expression of PDK4 and phosphorylation of Smad2, an indicator of TGFß pathway activation, show a strong correlation and that both positively associate with chemoresistance in colorectal cancer. These findings indicate that the TGFß/PDK4 signaling axis plays an important role in the response of colorectal cancer to chemotherapy. A major implication of our studies is that inhibition of PDK4 may have considerable therapeutic potential to overcome drug resistance in colorectal cancer patients, which warrants the development of PDK4-specific inhibitors.

Two PALB2 germline mutations found in both BRCA1+ and BRCAx familial breast cancer.

Partner and localizer of BRCA2 (PALB2), plays an important functional role in DNA damage repair. Recent studies indicate that germline mutations in PALB2 predispose individuals to a high risk of developing familial breast cancer. Therefore, comprehensive identification of PALB2 germline mutations is potentially important for understanding their roles in tumorigenesis and for testing their potential utility as clinical targets. Most of the previous studies of PALB2 have focused on familial breast cancer cases with normal/wild-type BRCA1 and BRCA2 (BRCAx). We hypothesize that PALB2 genetic mutations also exist in individuals with BRCA mutations (BRCA+). To test this hypothesis, PALB2 germline mutations were screened in 107 exome data sets collected from familial breast cancer families who were either BRCA1+ or BRCAx. Two novel heterozygous mutations predicted to alter the function of PALB2 were identified (c.2014G>C, p.E672Q and c.2993G>A, p.G998E). Notably, both of these mutations co-existed in BRCA1+ and BRCA1x families. These studies show that mutations in PALB2 can occur independent of the status of BRCA1 mutations, and they highlight the importance to include BRCA1+ families in PALB2 mutation screens.

Family-specific, novel, deleterious germline variants provide a rich resource to identify genetic predispositions for BRCAx familial breast cancer.

BACKGROUND: Genetic predisposition is the primary risk factor for familial breast cancer. For the majority of familial breast cancer, however, the genetic predispositions remain unknown. All newly identified predispositions occur rarely in disease population, and the unknown genetic predispositions are estimated to reach up to total thousands. Family unit is the basic structure of genetics. Because it is an autosomal dominant disease, individuals with a history of familial breast cancer must carry the same genetic predisposition across generations. Therefore, focusing on the cases in lineages of familial breast cancer, rather than pooled cases in disease population, is expected to provide high probability to identify the genetic predisposition for each family. METHODS: In this study, we tested genetic predispositions by analyzing the family-specific variants in familial breast cancer. Using exome sequencing, we analyzed three families and 22 probands with BRCAx (BRCA-negative) familial breast cancer. RESULTS: We observed the presence of family-specific, novel, deleterious germline variants in each family. Of the germline variants identified, many were shared between the disease-affected family members of the same family but not found in different families, which have their own specific variants. Certain variants are putative deleterious genetic predispositions damaging functionally important genes involved in DNA replication and damaging repair, tumor suppression, signal transduction, and phosphorylation. CONCLUSIONS: Our study demonstrates that the predispositions for many BRCAx familial breast cancer families can lie in each disease family. The application of a family-focused approach has the potential to detect many new predispositions.

Genome instability in blood cells of a BRCA1+ breast cancer family.

BACKGROUND: BRCA1 plays an essential role in maintaining genome stability. Inherited BRCA1 germline mutation (BRCA1+) is a determined genetic predisposition leading to high risk of breast cancer. While BRCA1+ induces breast cancer by causing genome instability, most of the knowledge is known about somatic genome instability in breast cancer cells but not germline genome instability. METHODS: Using the exome-sequencing method, we analyzed the genomes of blood cells in a typical BRCA1+ breast cancer family with an exon 13-duplicated founder mutation, including six breast cancer-affected and two breast cancer unaffected members. RESULTS: We identified 23 deleterious mutations in the breast cancer-affected family members, which are absent in the unaffected members. Multiple mutations damaged functionally important and breast cancer-related genes, including transcriptional factor BPTF and FOXP1, ubiquitin ligase CUL4B, phosphorylase kinase PHKG2, and nuclear receptor activator SRA1. Analysis of the mutations between the mothers and daughters shows that most mutations were germline mutation inherited from the ancestor(s) while only a few were somatic mutation generated de novo. CONCLUSION: Our study indicates that BRCA1+ can cause genome instability with both germline and somatic mutations in non-breast cells.

Can unknown predisposition in familial breast cancer be family-specific?

Genetic predisposition plays a key role in the development of familial breast cancer. In spite of strong familial clustering of the disease and extensive efforts made during the past decade; however, progress has been slow in identifying genetic predisposition for the majority of familial breast cancer families. The question arises therefore as to whether current approaches are adequate in identifying the unknown genetic predisposition. We analyzed eight members of a BRCA1-, BRCA2-, p53-, and PTEN-negative breast cancer family, of which five had breast cancer, one is an obligate gene carrier, and two were unaffected. We sequenced the entire coding region of the genome for each member using exome sequencing to identify nonsynonymous variants. We identified 55 nonsynonymous germline variants affecting 49 genes in multiple members of the family, of which 22 are predicted to have damaging effects. We validated 20 of the 22 selected variants in the family by Sanger sequencing. Two variants in KAT6B, an acetal transferase gene, were identified in six family members of which five were affected with breast cancer and one is the unaffected obligate carrier. We further examined the presence of the identified variants in a cohort of 40 additional breast cancer cases from 22 familial breast cancer families, but none of the 22 variants was detected in these cases. Sequencing the entire coding exons in KAT6B detects no variants in these cases. Our results show that genetic predisposition for familial breast cancer can be rich in an affected family, but the predisposition can be family-specific. As such, it will be difficult to detect them by applying population-based approach. Our study supports the concept that focusing on each affected family will be required to determine the genetic predisposition for many familial breast cancer families whose genetic dispositions remain unknown.

The transcriptome of human CD34+ hematopoietic stem-progenitor cells.

Studying gene expression at different hematopoietic stages provides insights for understanding the genetic basis of hematopoiesis. We analyzed gene expression in human CD34(+) hematopoietic cells that represent the stem-progenitor population (CD34(+) cells). We collected >459,000 transcript signatures from CD34(+) cells, including the de novo-generated 3' ESTs and the existing sequences of full-length cDNAs, ESTs, and serial analysis of gene expression (SAGE) tags, and performed an extensive annotation on this large set of CD34(+) transcript sequences. We determined the genes expressed in CD34(+) cells, verified the known genes and identified the new genes of different functional categories involved in hematopoiesis, dissected the alternative gene expression including alternative transcription initiation, splicing, and adenylation, identified the antisense and noncoding transcripts, determined the CD34(+) cell-specific gene expression signature, and developed the CD34(+) cell-transcription map in the human genome. Our study provides a current view on gene expression in human CD34(+) cells and reveals that early hematopoiesis is an orchestrated process with the involvement of over half of the human genes distributed in various functions. The data generated from our study provide a comprehensive and uniform resource for studying hematopoiesis and stem cell biology.

Gene transfer efficiency and genome-wide integration profiling of Sleeping Beauty, Tol2, and piggyBac transposons in human primary T cells.

In this study, we compared the genomic integration efficiencies and transposition site preferences of Sleeping Beauty (SB or SB11), Tol2, and piggyBac (PB) transposon systems in primary T cells derived from peripheral blood lymphocytes (PBL) and umbilical cord blood (UCB). We found that PB demonstrated the highest efficiency of stable gene transfer in PBL-derived T cells, whereas SB11 and Tol2 mediated intermediate and lowest efficiencies, respectively. Southern hybridization analysis demonstrated that PB generated the highest number of integrants when compared to SB and Tol2 in both PBL and UCB T cells. Tol2 and PB appeared more likely to promote clonal expansion than SB, which may be in part due to the dysregulated expression of cancer-related genes near the insertion sites. Genome-wide integration analysis demonstrated that SB, Tol2, and PB integrations occurred in all the chromosomes without preference. Additionally, Tol2 and PB integration sites were mainly localized near transcriptional start sites (TSSs), CpG islands and DNaseI hypersensitive sites, whereas SB integrations were randomly distributed. These results suggest that SB may be a preferential choice of the delivery vector in T cells due to its random integration site preference and relatively high efficiency, and support continuing development of SB-mediated T-cell phase I trials.

Common genetic variants contribute to incomplete penetrance: evidence from cancer-free BRCA1 mutation carriers.

PURPOSE: The presence of pathogenic germline mutation in BRCA1 gene is considered as the most penetrant genetic predisposition for breast cancer. However, a portion of BRCA1 mutation carriers never develops breast cancer throughout their lifetime. This phenomenon is called incomplete penetrance. Genetic factor is proposed to contribute to this phenomenon, but the details regarding the genetic factor remain elusive. BRCA1 mutations were inherited from the ancestors of the mutation carrier families during human evolution, and their presence is a consistent threat to the survival of the mutation carrier population. In the present study, we hypothesize that evolution could positively select genetic components in the mutation carrier population to suppress the oncogenesis imposed by the predisposition. EXPERIMENTAL DESIGN: To test our hypothesis, we used whole exome sequencing to compare germline variation of all genes in pairs of breast cancer-unaffected and breast cancer-affected BRCA1 mutation carriers, each pair was from the same family carrying the same BRCA1 mutation. RESULTS: We identified a group of 'beneficial' variants enriched in the breast cancer-unaffected carrier group. These were the common variants in human population distributed in multiple genes involved in multiple functionally important pathways. We found a single-nucleotide polymorphism, rs3735400 located in ANLN gene, which plays an essential role in controlling cytokinesis and is often found to be overexpressed in cancer. The carriers of this variant had lower cumulative risk of developing breast cancer; overexpression of the variant-containing ANLN decreased ANLN nuclear localization suppressed expression of the variant-containing ANLN, and decreased the cellular proliferation respectively. CONCLUSION: Our findings support our hypothesis that common genetic variants can be evolutionarily selected in BRCA1 mutation carrier population to counterpart the oncogenic effects imposed by mutation predisposition in BRCA1, contributing to the incomplete penetrance.

Zika virus propagation and release in human fetal astrocytes can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869.

Zika virus (ZIKV) is a neurotrophic flavivirus that is capable of infecting humans, leading to brain abnormalities during fetal development. The ZIKV infectivity in neural target cells remains poorly understood. Here, we found that ZIKV specifically infected glial fibrillary acidic protein- and S100B-positive primary human astrocytes derived from fetal brains. In contrast, neuron-specific Class III ß-tubulin (TuJ1)-positive neurons in the astrocyte cultures and SOX2-positive neural progenitor cells derived from the fetal brains were less susceptible to ZIKV infection compared with astrocytes. The infected astrocytes released competent viral particles and manifested programmed cell death with a progressive cytopathic effect. Interestingly, ZIKV infection in human fetal astrocytes induced a significant increase of extracellular vesicles (EVs). Treatment with GW4869, a specific inhibitor of neutral sphingomyelinase-2, decreased EV levels, suppressed ZIKV propagation, and reduced the release of infectious virions in astrocytes. Therefore, ZIKV infects primary human fetal astrocytes and the infection can be suppressed by neutral sphingomyelinase-2 inhibitor GW4869. Further investigation into sphingomyelin metabolism and EVs may provide insights to the therapeutic treatment of ZIKV infection.

Unique Features of Germline Variation in Five Egyptian Familial Breast Cancer Families Revealed by Exome Sequencing.

Genetic predisposition increases the risk of familial breast cancer. Recent studies indicate that genetic predisposition for familial breast cancer can be ethnic-specific. However, current knowledge of genetic predisposition for the disease is predominantly derived from Western populations. Using this existing information as the sole reference to judge the predisposition in non-Western populations is not adequate and can potentially lead to misdiagnosis. Efforts are required to collect genetic predisposition from non-Western populations. The Egyptian population has high genetic variations in reflecting its divergent ethnic origins, and incident rate of familial breast cancer in Egypt is also higher than the rate in many other populations. Using whole exome sequencing, we investigated genetic predisposition in five Egyptian familial breast cancer families. No pathogenic variants in BRCA1, BRCA2 and other classical breast cancer-predisposition genes were present in these five families. Comparison of the genetic variants with those in Caucasian familial breast cancer showed that variants in the Egyptian families were more variable and heterogeneous than the variants in Caucasian families. Multiple damaging variants in genes of different functional categories were identified either in a single family or shared between families. Our study demonstrates that genetic predisposition in Egyptian breast cancer families may differ from those in other disease populations, and supports a comprehensive screening of local disease families to determine the genetic predisposition in Egyptian familial breast cancer.

Ccdc3: A New P63 Target Involved in Regulation Of Liver Lipid Metabolism.

TAp63, a member of the p53 family, has been shown to regulate energy metabolism. Here, we report coiled coil domain-containing 3 (CCDC3) as a new TAp63 target. TAp63, but not ΔNp63, p53 or p73, upregulates CCDC3 expression by directly binding to its enhancer region. The CCDC3 expression is markedly reduced in TAp63-null mouse embryonic fibroblasts and brown adipose tissues and by tumor necrosis factor alpha that reduces p63 transcriptional activity, but induced by metformin, an anti-diabetic drug that activates p63. Also, the expression of CCDC3 is positively correlated with TAp63 levels, but conversely with ΔNp63 levels, during adipocyte differentiation. Interestingly, CCDC3, as a secreted protein, targets liver cancer cells and increases long chain polyunsaturated fatty acids, but decreases ceramide in the cells. CCDC3 alleviates glucose intolerance, insulin resistance and steatosis formation in transgenic CCDC3 mice on high-fat diet (HFD) by reducing the expression of hepatic PPARγ and its target gene CIDEA as well as other genes involved in de novo lipogenesis. Similar results are reproduced by hepatic expression of ectopic CCDC3 in mice on HFD. Altogether, these results demonstrate that CCDC3 modulates liver lipid metabolism by inhibiting liver de novo lipogenesis as a downstream player of the p63 network.

Exome-based Variant Detection in Core Promoters.

Core promoter controls the initiation of transcription. Core promoter sequence change can disrupt transcriptional regulation, lead to impairment of gene expression and ultimately diseases. Therefore, comprehensive characterization of core promoters is essential to understand normal and abnormal gene expression in biomedical studies. Here we report the development of EVDC (Exome-based Variant Detection in Core promoters) method for genome-scale analysis of core-promoter sequence variation. This method is based on the fact that exome sequences contain the sequences not only from coding exons but also from non-coding region including core promoters generated by random fragmentation in exome sequencing process. Using exome data from three cell types of CD4+ T cells, CD19+ B cells and neutrophils of a single individual, we characterized the features of core promoter-mapped exome sequences, and analysed core-promoter variation in this individual genome. We also compared the core promoters between YRI (Yoruba in Ibadan, Nigeria) and the CEU (Utah residents of European decedent) populations using the exome data generated by the 1000 Genome project, and observed much higher variation in YRI population than in CEU population. Our study demonstrates that the EVDC method provides a simple but powerful means for genome-wile de novo characterization of core promoter sequence variation.

Prevalence and spectrum of BRCA germline variants in mainland Chinese familial breast and ovarian cancer patients.

Germline mutations in BRCA1 and BRCA2 are the most penetrating genetic predispositions for breast and ovarian cancer, and their presence is largely ethnic-specific. Comprehensive information about the prevalence and spectrum of BRCA mutations has been collected in European and North American populations. However, similar information is lacking in other populations, including the mainland Chinese population despite its large size of 1.4 billion accounting for one fifth of the world's population. Herein, we performed an extensive literature analysis to collect BRCA variants identified from mainland Chinese familial breast and ovarian cancer patients. We observed 137 distinct BRCA1 variants in 409 of 3,844 and 80 distinct BRCA2 variants in 157 of 3,024 mainland Chinese patients, with an estimated prevalence of 10.6% for BRCA1 and 5.2% for BRCA2. Of these variants, only 40.3% in BRCA1 and 42.5% in BRCA2 are listed in current Breast Cancer Information Core database. We observed higher frequent variation in BRCA1 exons 11A, 11C, 11D, and 24 and BRCA2 exon 10 in Chinese patients than in the patients of other populations. The most common pathogenic variant in BRCA1 wasc.981_982delAT in exon 11A, and in BRCA2 c.3195_3198delTAAT in exon 11B and c.5576_5579delTTAA in exon 11E; the most common novel variant in BRCA1 was c.919A>G in exon 10A, and in BRCA2 c.7142delC in exon 14. None of the variants overlap with the founder mutations in other populations. Our analysis indicates that the prevalence of BRCA variation in mainland Chinese familial breast and ovarian cancer patients is at a level similar to but the spectrum is substantially different from the ones of other populations.

Differences of Variable Number Tandem Repeats in XRCC5 Promoter Are Associated with Increased or Decreased Risk of Breast Cancer in BRCA Gene Mutation Carriers.

Ku80 is a subunit of the Ku heterodimer that binds to DNA double-strand break ends as part of the non-homologous end joining (NHEJ) pathway. Ku80 is also involved in homologous recombination (HR) via its interaction with BRCA1. Ku80 is encoded by the XRCC5 gene that contains a variable number tandem repeat (VNTR) insertion in its promoter region. Different VNTR genotypes can alter XRCC5 expression and affect Ku80 production, thereby affecting NHEJ and HR pathways. VNTR polymorphism is associated with multiple types of sporadic cancer. In this study, we investigated its potential association with familial breast cancer at the germline level. Using PCR, PAGE, Sanger sequencing, and statistical analyses, we compared VNTR genotypes in the XRCC5 promoter between healthy individuals and three types of familial breast cancer cases: mutated BRCA1 (BRCA1 (+)), mutated BRCA2 (BRCA2 (+)), and wild-type BRCA1/BRCA2 (BRCAx). We observed significant differences of VNTR genotypes between control and BRCA1 (+) group (P < 0.0001) and BRCA2 (+) group (P = 0.0042) but not BRCAx group (P = 0.2185), and the differences were significant between control and cancer-affected BRCA1 (+) cases (P < 0.0001) and BRCA2 (+) cases (P = 0.0092) but not cancer-affected BRCAx cases (P = 0.4251). Further analysis indicated that 2R/2R (OR = 1.94, 95%CI = 1.26-2.95, P = 0.0096) and 2R/1R (OR = 1.58, 95%CI = 1.11-2.26, P = 0.0388) were associated with increased risk but 1R/1R (OR = 0.55, 95%CI = 0.35-0.84, P = 0.0196) and 1R/0R (OR = 0, 95%CI = 0-0.29, P = 0.0012) were associated with decreased risk in cancer-affected BRCA1 (+) group; 2R/1R (OR = 1.94, 95%CI = 1.14-3.32, P = 0.0242) was associated with increased risk in cancer-affected BRCA2 (+) group. No correlation was observed for the altered risk between cancer-affected or -unaffected carriers and between different age of cancer diagnosis in cancer-affected carriers. The frequently observed VNTR association with in BRCA1 (+) and BRCA2 (+) breast cancer group indicates that VNTR polymorphism in the XRCC5 promoter is associated with altered risk of breast cancer in BRCA1 (+) and BRCA2 (+) carriers.

2.45 GHz radiofrequency fields alter gene expression in cultured human cells.

The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the serial analysis of gene expression (SAGE) method to measure the RF effect on gene expression at the genome level. We observed that 221 genes altered their expression after a 2-h exposure. The number of affected genes increased to 759 after a 6-h exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the upregulated ones and the cell cycle genes among the downregulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.

Erk5 contributes to maintaining the balance of cellular nucleotide levels and erythropoiesis.

An adequate supply of nucleotides is essential for accurate DNA replication, and inappropriate deoxyribonucleotide triphosphate (dNTP) concentrations can lead to replication stress, a common source of DNA damage, genomic instability and tumourigenesis. Here, we provide evidence that Erk5 is necessary for correct nucleotide supply during erythroid development. Mice with Erk5 knockout in the haematopoietic lineage showed impaired erythroid development in bone marrow, accompanied by altered dNTP levels and increased DNA mutagenesis in erythroid progenitors as detected by exome sequencing. Moreover, Erk5-depleted leukemic Jurkat cells presented a marked sensitivity to thymidine-induced S phase stalling, as evidenced by increased H2AX phosphorylation and apoptosis. The increase in thymidine sensitivity correlated with a higher dTTP/dCTP ratio. These results indicate that Erk5 is necessary to maintain the balance of nucleotide levels, thus preventing dNTP misincorporation and DNA damage in proliferative erythroid progenitors and leukemic Jurkat T cells.

The genome of polymorphonuclear neutrophils maintains normal coding sequences.

Genetic studies often use genomic DNA from whole blood cells, of which the majority are the polymorphonuclear myeloid cells. Those cells undergo dramatic change of nuclear morphology following cellular differentiation. It remains elusive if the nuclear morphological change accompanies sequence alternations from the intact genome. If such event exists, it will cause a serious problem in using such type of genomic DNA for genetic study as the sequences will not represent the intact genome in the host individuals. Using exome sequencing, we compared the coding regions between neutrophil, which is the major type of polymorphonuclear cells, and CD4+ T cell, which has an intact genome, from the same individual. The results show that exon sequences between the two cell types are essentially the same. The minor differences represented by the missed exons and base changes between the two cell types were validated to be mainly caused by experimental errors. Our study concludes that genomic DNA from whole blood cells can be safely used for genetic studies.