Breast cancer 1-associated protein 1 (BAP1) is a nuclear deubiquitinase that regulates gene expression, transcription, DNA repair, and more. Several findings underscore the apparent driver role of ...BAP1 in malignant mesothelioma (MM). However, the reported frequency of somatic BAP1 mutations in MM varies considerably, a discrepancy that appeared related to either methodological or ethnical differences across various studies.
To address this discrepancy, we carried out comprehensive genomic and immunohistochemical (IHC) analyses to detect somatic BAP1 gene alterations in 22 frozen MM biopsies from U.S. MM patients.
By combining Sanger sequencing, multiplex ligation-dependent probe amplification, copy number analysis, and cDNA sequencing, we found alteration of BAP1 in 14 of 22 biopsies (63.6%). No changes in methylation were observed. IHC revealed normal nuclear BAP1 staining in the eight MM containing wild-type BAP1, whereas no nuclear staining was detected in the 14 MM biopsies containing tumor cells with mutated BAP1. Thus, IHC results were in agreement with those obtained by genomic analyses. We then extended IHC analysis to an independent cohort of 70 MM biopsies, of which there was insufficient material to perform molecular studies. IHC revealed loss of BAP1 nuclear staining in 47 of these 70 MM biopsies (67.1%).
Our findings conclusively establish BAP1 as the most commonly mutated gene in MM, regardless of ethnic background or other clinical characteristics. Our data point to IHC as the most accessible and reliable technique to detect BAP1 status in MM biopsies.
Malignant mesothelioma is associated with the exposure to asbestos fibers. Recent discovery of the
BAP1
cancer syndrome, a Mendelian disorder with high-penetrance autosomal dominant inheritance ...fostered the genotyping for nucleotide-level or larger structural alteration of germline DNA. Inherited heterozygous mutations of the
BAP1
gene increase the susceptibility to carcinogenic fibers, leading to a concept of gene x environment interaction (GxE) as a pathogenetic mechanism of mesothelioma. Several studies on cohorts of unselected patients with mesothelioma or on familial/early-onset cohorts of mesothelioma cases converged on
BAP1
as the more frequent germline mutated gene, followed by other genes involved in DNA repair and homologous recombination. Evidence has been emerging that patients with mesothelioma carrying germline mutations of
BAP1
and of other genes, such as those involved in DNA repair and tumor suppressor genes, have better prognosis and higher chemosensitivity when compared with patients with germline wildtype Bap1. We report here a germline genomic analysis targeted 22 genes in a cohort of 101 Japanese patients irrespective of asbestos exposure, age at diagnosis, or personal or family history of cancer. By comparing the results with the Human Genetic Variation Database (HGVD) and the Genome Aggregation Database (gnomAD) we selected rare germline variants with a Combined Annotation Dependent Depletion (CADD) >20. We show here that 31 of 101 subjects were carrying 25 rare variants in 14 genes, neither reported in the HGVD nor in the gnomAD database for 14/25 variants. Besides pathogenic variants of
BAP1
, rare missense variants were found in genes encoding lysine-specific histone methyltransferase
SETD2
and
SETDB1
and genes encoding subunits of the mSWI/SNF chromatin remodeling complex. The complete scenario of the genetic background consisting of pathogenic germline variants required for the predisposition and GxE for pathogenesis of mesothelioma appears complex, and further large-scale studies are warranted.
Copy number variation (CNV) is a polymorphism in the human genome involving DNA fragments larger than 1 kb. Copy number variation sites provide hotspots of somatic alterations in cancers. Herein, we ...examined somatic alterations at sites of CNV in DNA from 20 invasive breast cancers using a Comparative Genomic Hybridization array specifically designed to detect the genome‐wide CNV status of approximately 412 000 sites. Somatic copy number alterations (CNAs) were detected in 39.9% of the CNV probes examined. The most frequently altered regions were gains of 1q21‐22 (90%), 8q21‐24 (85%), 1q44 (85%), and 3q11 (85%) or losses of 16q22‐24 (80%). Gene ontology analyses of genes within the CNA fragments revealed that cascades related to transcription and RNA metabolism correlated significantly with human epidermal growth factor receptor 2 positivity and menopausal status. Thirteen of 20 tumors showed CNAs in more than 35% of sites examined and a high prevalence of CNAs correlated significantly with estrogen receptor (ER) negativity, higher nuclear grade (NG), and higher Ki‐67 labeling index. Finally, when CNA fragments were categorized according to their size, CNAs smaller than 10 kb correlated significantly with ER positivity and lower NG, whereas CNAs exceeding 10 Mb correlated with higher NG, ER negativity, and a higher Ki‐67 labeling index. Most of these findings were confirmed or supported by quantitative PCR of representative DNA fragments in 72 additional breast cancers. These results suggest that most CNAs are caused by gain or loss of large chromosomal fragments and correlate with NG and several malignant features, whereas solitary CNAs of less than 10 kb could be involved in ER‐positive breast carcinogenesis.
Short‐length somatic alterations at the site of copy number variation were involved in a portion of breast cancer with a lower grade malignancy.
Precise quantification of copy‐number alterations (CNAs) in a tumor genome is difficult. We have applied a comprehensive copy‐number analysis method, digital multiplex ligation‐dependent probe ...amplification (digitalMLPA), for targeted gene copy‐number analysis in clear cell renal cell carcinoma (ccRCC). Copy‐number status of all chromosomal arms and 11 genes was determined in 60 ccRCC samples. Chromosome 3p loss and 5q gain, known as early changes in ccRCC development, as well as losses at 9p and 14q were detected in 56/60 (93.3%), 31/60 (51.7%), 11/60 (18.3%), and 33/60 (55%), respectively. Through gene expression analysis, a significant positive correlation was detected in terms of 14q loss determined using digitalMLPA and downregulation of mRNA expression ratios with HIF1A and L2HGDH (P = .0253 and .0117, respectively). Patients with early metastasis (<1 y) (n = 18) showed CNAs in 6 arms (in median), whereas metastasis‐free patients (n = 34) showed those in significantly less arms (3 arms in median) (P = .0289). In particular, biallelic deletion of CDKN2A/2B was associated with multiple CNAs (≥7 arms) in 3 tumors. Together with sequence‐level mutations in genes VHL, PBRM1, SETD2, and BAP1, we performed multiple correspondence analysis, which identified the association of 9p loss and 4q loss with early metastasis (both P < .05). This analysis indicated the association of 4p loss and 1p loss with poor survival (both, P < .05). These findings suggest that CNAs have essential roles in aggressiveness of ccRCC. We showed that our approach of measuring CNA through digitalMLPA will facilitate the selection of patients who may develop metastasis.
Our digitalMLPA approach of measuring copy‐number alterations for all chromosomal arms can distinguish patients with early metastasis from metastasis‐free patients, and will facilitate the selection of patients who may develop metastasis.
In the present study, we analyzed genomic alterations of BRCA1‐associated protein 1 (BAP1) in 23 malignant mesotheliomas (MMs), 16 epithelioid and seven non‐epithelioid, consisting of 18 clinical ...specimens and five established cell lines. In examining these samples for homozygous deletions and sequence‐level mutations, we found biallelic BAP1 gene alterations in 14 of 23 MMs (61%). Seven of these 14 MMs had homozygous deletions of the partial or entire BAP1 gene, another five had sequence‐level mutations, including small deletions, a nonsense mutation, and missense mutations with additional monoallelic deletions, and the remaining two had homozygous mutations without allelic loss. All but one of the 14 BAP1 gene mutations were found in the epithelioid‐type MMs; BAP1 mutations were found in 13 of 16 epithelioid‐type MMs, but in only one of seven non‐epithelioid‐type MMs (13/16 vs 1/7; P = 0.005). There was no BAP1 mRNA expression in MMs with biallelic deletion and repressed expression was confirmed in MM specimens with deletion/mutation as compared with Met5a, SV40‐transformed normal mesothelial cells. Western blot showed that seven of eight epithelioid MMs analyzed were BAP1 negative. Immunostaining with anti‐BAP1 antibody in normal lung tissues revealed clear nuclear staining of normal mesothelial cells. No nuclear staining was observed among BAP1 mutation‐positive MM tumors, whereas nuclear staining was observed among BAP1 mutation‐negative MM tumors. These results suggest that the lack of the tumor suppressor BAP1 may be more specifically involved in the pathogenesis of epithelioid MM rather than non‐epithelioid MM, and would be useful for diagnosis of epithelioid‐type MM. (Cancer Sci 2012; 103: 868–874)
We recently discovered an inherited cancer syndrome caused by BRCA1-Associated Protein 1 (BAP1) germline mutations, with high incidence of mesothelioma, uveal melanoma and other cancers and very high ...penetrance by age 55. To identify families with the BAP1 cancer syndrome, we screened patients with family histories of multiple mesotheliomas and melanomas and/or multiple cancers. We identified four families that shared an identical BAP1 mutation: they lived across the US and did not appear to be related. By combining family histories, molecular genetics, and genealogical approaches, we uncovered a BAP1 cancer syndrome kindred of ~80,000 descendants with a core of 106 individuals, whose members descend from a couple born in Germany in the early 1700s who immigrated to North America. Their descendants spread throughout the country with mutation carriers affected by multiple malignancies. Our data show that, once a proband is identified, extended analyses of these kindreds, using genomic and genealogical studies to identify the most recent common ancestor, allow investigators to uncover additional branches of the family that may carry BAP1 mutations. Using this knowledge, we have identified new branches of this family carrying BAP1 mutations. We have also implemented early-detection strategies that help identify cancers at early-stage, when they can be cured (melanomas) or are more susceptible to therapy (MM and other malignancies).
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
BRCA1‐associated protein 1 (BAP1) is a deubiquitinating enzyme that is involved in the regulation of cell growth. Recently, many somatic and germline mutations of BAP1 have been reported in a broad ...spectrum of tumors. In this study, we identified a novel somatic non‐synonymous BAP1 mutation, a phenylalanine‐to‐isoleucine substitution at codon 170 (F170I), in 1 of 49 patients with esophageal squamous cell carcinoma (ESCC). Multiplex ligation‐dependent probe amplification (MLPA) of BAP1 gene in this ESCC tumor disclosed monoallelic deletion (LOH), suggesting BAP1 alterations on both alleles in this tumor. The deubiquitinase activity and the auto‐deubiquitinase activity of F170I‐mutant BAP1 were markedly suppressed compared with wild‐type BAP1. In addition, wild‐type BAP1 mostly localizes to the nucleus, whereas the F170I mutant preferentially localized in the cytoplasm. Microarray analysis revealed that expression of the F170I mutant drastically altered gene expression profiles compared with expressed wild‐type BAP1. Gene‐ontology analyses indicated that the F170I mutation altered the expression of genes involved in oncogenic pathways. We found that one candidate, TCEAL7, previously reported as a putative tumor suppressor gene, was significantly induced by wild‐type BAP1 as compared to F170I mutant BAP1. Furthermore, we found that the level of BAP1 expression in the nucleus was reduced in 44% of ESCC examined by immunohistochemistry (IHC). Because the nuclear localization of BAP1 is important for its tumor suppressor function, BAP1 may be functionally inactivated in a substantial portion of ESCC. Taken together, BAP1 is likely to function as a tumor suppressor in at least a part of ESCC.
The deubiquitinase activity of F170I‐mutant BAP1 found in ESCC was markedly suppressed compared with wild‐type. In addition, wild‐type BAP1 mostly localizes to the nucleus, whereas the F170I mutant preferentially localized in the cytoplasm. Furthermore, the level of BAP1 expression in the nucleus was reduced in more than 40% of ESCCs examined by IHC, and some of the nuclear BAP1‐depleted cases showed strong cytosolic expression of BAP1.
Joubert syndrome and related disorders (JSRD) is a clinically and genetically heterogeneous condition with autosomal recessive or X-linked inheritance, which share a distinctive neuroradiological ...hallmark, the so-called molar tooth sign. JSRD is classified into six clinical subtypes based on associated variable multiorgan involvement. To date, 21 causative genes have been identified in JSRD, which makes genetic diagnosis difficult.
We report here a case of a 28-year-old Japanese woman diagnosed with JS with oculorenal defects with a novel compound heterozygous mutation (p.Ser219*/deletion) in the NPHP1 gene. Whole-exome sequencing (WES) of the patient identified the novel nonsense mutation in an apparently homozygous state. However, it was absent in her mother and heterozygous in her father. A read depth-based copy number variation (CNV) detection algorithm using WES data of the family predicted a large heterozygous deletion mutation in the patient and her mother, which was validated by digital polymerase chain reaction, indicating that the patient was compound heterozygous for the paternal nonsense mutation and the maternal deletion mutation spanning the site of the single nucleotide change.
It should be noted that analytical pipelines that focus purely on sequence information cannot distinguish homozygosity from hemizygosity because of its inability to detect large deletions. The ability to detect CNVs in addition to single nucleotide variants and small insertion/deletions makes WES an attractive diagnostic tool for genetically heterogeneous disorders.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
O6-methylguanine-DNA methyltransferase (MGMT) repairs the cytotoxic and mutagenic O6-alkylguanine produced by alkylating agents such as chemotherapeutic agents and mutagens. Recent studies have shown ...that in a subset of tumors, MGMT expression is inversely linked to hypermethylation of the CpG island in the promoter region; however, how the epigenetic silencing mechanism works, as it relates to hypermethylation, was still unclear. To understand the mechanism, we examined the detailed methylation status of the whole island with bisulfite-sequencing in 19 MGMT non-expressed cancer cell lines. We found two highly methylated regions in the island. One was upstream of exon 1, including minimal promoter, and the other was downstream, including enhancer. Reporter gene assay showed that methylation of both the upstream and downstream regions suppressed luciferase activity drastically. Chromatin immunoprecipitation assay revealed that histone H3 lysine 9 was hypermethylated throughout the island in the MGMT negative line, whereas acetylation on H3 and H4 and methylation on H3 lysine 4 were at significantly high levels outside the minimal promoter in the MGMT-expressed line. Furthermore, MeCP2 preferentially bound to the CpG-methylated island in the MGMT negative line. Given these results, we propose a model for gene silencing of MGMT that is dependent on the epigenetic state in cancer.
Malignant mesothelioma (MM) is an asbestos-related malignancy arising from surface serosal cells of pleural and peritoneal cavities. Somatic mutations of BRCA1 associated protein-1 (BAP1) gene were ...recently found in MM as well as in uveal melanoma and kidney cancer among the Caucasian and Japanese people. However, frequency of mutations varies among the reported studies, which might be due to presence of undetected gross rearrangements of BAP1 gene that might escape detection by sequencing strategy. We investigated the presence and frequency of gross genomic rearrangements in the BAP1 gene by multiplex ligation-dependent probe amplification (MLPA) in 17 Japanese cases of MM tumors. We found five tumors with partial deletion of BAP1 gene; each tumors displayed partial deletion of exons 1-4 (MM39), exons 1-5 (MM48), exons 11-17 (MM57), exons 1-15 (MM19) and exons 1-16 (MM21). Two tumors (MM34, MM14) had biallelic deletion and four tumors (MM29, MM35, MM45 and MM56) had monoallelic deletion of entire BAP1 gene. Therefore, MLPA analysis revealed large gene rearrangements of BAP1 gene in 65% of MM (11/17). Unusually high frequency of large deletions indicates that the 3p21 chromosomal region surrounding BAP1 gene is structurally unstable. MLPA was useful in characterizing both monoallelic and biallelic deletion of BAP1 gene precisely at exon level.
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