Josephine C. Dorsman

Fanconi anemia is associated with a defect in the BRCA2 partner PALB2

The Fanconi anemia and BRCA networks are considered interconnected, as BRCA2 gene defects have been discovered in individuals with Fanconi anemia subtype D1. Here we show that a defect in the BRCA2-interacting protein PALB2 is associated with Fanconi anemia in an individual with a new subtype. PALB2-deficient cells showed hypersensitivity to cross-linking agents and lacked chromatin-bound BRCA2; these defects were corrected upon ectopic expression of PALB2 or by spontaneous reversion.

RNA-Seq of Tumor-Educated Platelets Enables Blood-Based Pan-Cancer, Multiclass, and Molecular Pathway Cancer Diagnostics

Summary Tumor-educated blood platelets (TEPs) are implicated as central players in the systemic and local responses to tumor growth, thereby altering their RNA profile. We determined the diagnostic potential of TEPs by mRNA sequencing of 283 platelet samples. We distinguished 228 patients with localized and metastasized tumors from 55 healthy individuals with 96% accuracy. Across six different tumor types, the location of the primary tumor was correctly identified with 71% accuracy. Also, MET or HER2-positive, and mutant KRAS, EGFR, or PIK3CA tumors were accurately distinguished using surrogate TEP mRNA profiles. Our results indicate that blood platelets provide a valuable platform for pan-cancer, multiclass cancer, and companion diagnostics, possibly enabling clinical advances in blood-based “liquid biopsies”.

Characterisation of retinoblastomas without RB1 mutations: genomic, gene expression, and clinical studies.

BACKGROUND Retinoblastoma is the childhood retinal cancer that defined tumour-suppressor genes. Previous work shows that mutation of both alleles of the RB1 retinoblastoma suppressor gene initiates disease. We aimed to characterise non-familial retinoblastoma tumours with no detectable RB1 mutations. METHODS Of 1068 unilateral non-familial retinoblastoma tumours, we compared those with no evidence of RB1 mutations (RB1(+/+)) with tumours carrying a mutation in both alleles (RB1(-/-)). We analysed genomic copy number, RB1 gene expression and protein function, retinal gene expression, histological features, and clinical data. FINDINGS No RB1 mutations (RB1(+/+)) were reported in 29 (2·7%) of 1068 unilateral retinoblastoma tumours. 15 of the 29 RB1(+/+) tumours had high-level MYCN oncogene amplification (28-121 copies; RB1(+/+)MYCN(A)), whereas none of 93 RB1(-/-) primary tumours tested showed MYCN amplification (p<0·0001). RB1(+/+)MYCN(A) tumours expressed functional RB1 protein, had fewer overall genomic copy-number changes in genes characteristic of retinoblastoma than did RB1(-/-) tumours, and showed distinct aggressive histological features. MYCN amplification was the sole copy-number change in one RB1(+/+)MYCN(A) retinoblastoma. One additional MYCN(A) tumour was discovered after the initial frequencies were determined, and this is included in further analyses. Median age at diagnosis of the 17 children with RB1(+/+)MYCN(A) tumours was 4·5 months (IQR 3·5-10), compared with 24 months (15-37) for 79 children with non-familial unilateral RB1(-/-) retinoblastoma. INTERPRETATION Amplification of the MYCN oncogene might initiate retinoblastoma in the presence of non-mutated RB1 genes. These unilateral RB1(+/+)MYCN(A) retinoblastomas are characterised by distinct histological features, only a few of the genomic copy-number changes that are characteristic of retinoblastoma, and very early age of diagnosis. FUNDING National Cancer Institute-National Institutes of Health, Canadian Institutes of Health Research, German Research Foundation, Canadian Retinoblastoma Society, Hyland Foundation, Toronto Netralaya and Doctors Lions Clubs, Ontario Ministry of Health and Long Term Care, UK-Essen, and Foundations Avanti-STR and KiKa.

Identification of the Fanconi anemia complementation group I gene, FANCI

To identify the gene underlying Fanconi anemia (FA) complementation group I we studied informative FA-I families by a genome-wide linkage analysis, which resulted in 4 candidate regions together encompassing 351 genes. Candidates were selected via bioinformatics and data mining on the basis of their resemblance to other FA genes/proteins acting in the FA pathway, such as: degree of evolutionary conservation, presence of nuclear localization signals and pattern of tissue-dependent expression. We found a candidate, KIAA1794 on chromosome 15q25-26, to be mutated in 8 affected individuals previously assigned to complementation group I. Western blots of endogenous FANCI indicated that functionally active KIAA1794 protein is lacking in FA-I individuals. Knock-down of KIAA1794 expression by siRNA in HeLa cells caused excessive chromosomal breakage induced by mitomycin C, a hallmark of FA cells. Furthermore, phenotypic reversion of a patient-derived cell line was associated with a secondary genetic alteration at the KIAA1794 locus. These data add up to two conclusions. First, KIAA1794 is a FA gene. Second, this gene is identical to FANCI, since the patient cell lines found mutated in this study included the reference cell line for group I, EUFA592.

DNA profiling of primary serous ovarian and Fallopian tube carcinomas with array comparative genomic hybridization and multiplex ligation-dependent probe amplification†

Primary serous ovarian carcinoma (OVCA) and serous Fallopian tube carcinoma (FTC), both belonging to the BRCA‐linked tumour spectrum, share many properties and are treated similarly. However, a detailed molecular comparison has been lacking. We hypothesized that comparative genomic studies of serous OVCAs and FTCs should point to gene regions critically involved in their tumorigenesis. Array comparative genomic hybridization (array CGH) analysis indicated that serous OVCAs and serous FTCs displayed common but also more distinctive patterns of recurrent changes. Targeted gene identification using a dedicated multiplex ligation‐dependent probe amplification (MLPA) probe set directly identified EIF2C2 on 8q as a potentially important driver gene. Other previously unappreciated gained/amplified genes included PSMB4 on 1q, MTSS1 on 8q, TEAD4 and TSPAN9 on 12p, and BCAS4 on 20q. SPINT2 and ACTN4 on 19q were predominantly found in FTCs. Gains/amplifications of CCNE1 and MYC, often in conjunction with changes in genes of the AKT pathway, EVI1 and PTK2, seemed to be involved at earlier stages, whereas changes of ERBB2 were associated with advanced stages. The only BRCA1‐mutated FTC shared common denominators with the sporadic tumours. In conclusion, the data suggest that serous OVCAs and FTCs, although related, exhibit differences in genomic profiles. In addition to known pathways, new genes/pathways are likely to be involved, with changes in an miRNA‐associated gene, EIF2C2, as one important new feature. Dedicated MLPA sets constitute potentially important tools for differential diagnosis and may provide footholds for tailored therapy. Copyright

Genome-wide-array-based comparative genomic hybridization reveals genetic homogeneity and frequent copy number increases encompassing CCNE1 in Fallopian tube carcinoma

Fallopian tube carcinoma (FTC) is a rare, poorly studied and aggressive cancer, associated with poor survival. Since tumorigenesis is related to the acquisition of genetic changes, we used genome-wide array comparative genomic hybridization to analyse copy number aberrations occurring in FTC in order to obtain a better understanding of FTC carcinogenesis and to identify prognostic events and targets for therapy. We used arrays of 2464 genomic clones, providing ∼1.4 Mb resolution across the genome to map genomic DNA copy number aberrations quantitatively from 14 FTC onto the human genome sequence. All tumors showed a high frequency of copy number aberrations with recurrent gains on 3q, 6p, 7q, 8q, 12p, 17q, 19 and 20q, and losses involving chromosomes 4, 5q, 8p, 16q, 17p, 18q and X. Recurrent regions of amplification included 1p34, 8p11–q11, 8q24, 12p, 17p13, 17q12–q21, 19p13, 19q12–q13 and 19q13. Candidate, known oncogenes mapping to these amplicons included CMYC (8q24), CCNE1 (19q12–q21) and AKT2 (19q13), whereas PIK3CA and KRAS, previously suggested to be candidate driver genes for amplification, mapped outside copy number maxima on 3q and 12p, respectively. The FTC were remarkably homogeneous, with some recurrent aberrations occurring in more than 70% of samples, which suggests a stereotyped pattern of tumor evolution.

Mutant huntingtin activates Nrf2-responsive genes and impairs dopamine synthesis in a PC12 model of Huntington's disease

BackgroundHuntingtons disease is a progressive autosomal dominant neurodegenerative disorder that is caused by a CAG repeat expansion in the HD or Huntingtons disease gene. Although micro array studies on patient and animal tissue provide valuable information, the primary effect of mutant huntingtin will inevitably be masked by secondary processes in advanced stages of the disease. Thus, cell models are instrumental to study early, direct effects of mutant huntingtin. mRNA changes were studied in an inducible PC12 model of Huntingtons disease, before and after aggregates became visible, to identify groups of genes that could play a role in the early pathology of Huntingtons disease.ResultsBefore aggregation, up-regulation of gene expression predominated, while after aggregates became visible, down-regulation and up-regulation occurred to the same extent. After aggregates became visible there was a down-regulation of dopamine biosynthesis genes accompanied by down-regulation of dopamine levels in culture, indicating the utility of this model to identify functionally relevant pathways. Furthermore, genes of the anti-oxidant Nrf2-ARE pathway were up-regulated, possibly as a protective mechanism. In parallel, we discovered alterations in genes which may result in increased oxidative stress and damage.ConclusionUp-regulation of gene expression may be more important in HD pathology than previously appreciated. In addition, given the pathogenic impact of oxidative stress and neuroinflammation, the Nrf2-ARE signaling pathway constitutes a new attractive therapeutic target for HD.

Diagnosis of Fanconi Anemia: Mutation Analysis by Next-Generation Sequencing

Fanconi anemia (FA) is a rare genetic instability syndrome characterized by developmental defects, bone marrow failure, and a high cancer risk. Fifteen genetic subtypes have been distinguished. The majority of patients (≈85%) belong to the subtypes A (≈60%), C (≈15%) or G (≈10%), while a minority (≈15%) is distributed over the remaining 12 subtypes. All subtypes seem to fit within the “classical” FA phenotype, except for D1 and N patients, who have more severe clinical symptoms. Since FA patients need special clinical management, the diagnosis should be firmly established, to exclude conditions with overlapping phenotypes. A valid FA diagnosis requires the detection of pathogenic mutations in a FA gene and/or a positive result from a chromosomal breakage test. Identification of the pathogenic mutations is also important for adequate genetic counselling and to facilitate prenatal or preimplantation genetic diagnosis. Here we describe and validate a comprehensive protocol for the molecular diagnosis of FA, based on massively parallel sequencing. We used this approach to identify BRCA2, FANCD2, FANCI and FANCL mutations in novel unclassified FA patients.

Neuronal intranuclear and neuropil inclusions for pathological assessment of Huntington's disease

To evaluate the usefulness of neuronal intranuclear inclusions and neuropil inclusions for the pathological assessment of Huntington’s disease (HD), their presence in neocortex was assessed by ubiquitin and N‐terminal huntingtin immunohistochemistry in a consecutive series of 195 autopsy brains of individuals with a positive or tentative clinical diagnosis of, or at risk for, HD. The findings were correlated with striatal pathology (n = 190), CAG repeat length (n = 85) and original pathological diagnosis (n = 186). The antibodies detected both these inclusions in 181 patients with HD pathology ≥ Vonsattel et al’s grade I, five patients lacking striatal tissue for review, and two at‐risk individuals with grade 0 and grade I HD pathology, respectively. One patient with HD‐like pathology and two patients and four at‐risk individuals without HD pathology lacked HD inclusions. In the genetically analyzed cases, the inclusions were exclusively and consistently observed in association with repeat expansion [(CAG)n ≥ 39, n = 81]. Thirteen inclusion‐positive cases, including the grade 0 at‐risk individual, had a false negative original pathological diagnosis of HD and four had an unjustly questionable diagnosis. A false positive diagnosis was made in the inclusion‐negative case with HD‐like pathology. These results indicate that immunohistochemical analysis for HD inclusions facilitates the pathological evaluation of HD and enhances its accuracy.

RB1 mutation spectrum in a comprehensive nationwide cohort of retinoblastoma patients

Background Retinoblastoma (Rb) is a childhood cancer of the retina, commonly initiated by biallelic inactivation of the RB1 gene. Knowledge of the presence of a heritable RB1 mutation can help in risk management and reproductive decision making. We report here on RB1 mutation scanning in a unique nationwide cohort of Rb patients from the Netherlands. Methods From the 1173 Rb patients registered in the Dutch National Retinoblastoma Register until January 2013, 529 patients from 433 unrelated families could be included. RB1 mutation scanning was performed with different detection methods, depending on the time period. Results Our mutation detection methods revealed RB1 mutations in 92% of bilateral and/or familial Rb patients and in 10% of non-familial unilateral cases. Overall an RB1 germline mutation was detected in 187 (43%) of 433 Rb families, including 33 novel mutations. The distribution of the type of mutation was 37% nonsense, 20% frameshift, 21% splice, 9% large indel, 5% missense, 7% chromosomal deletions and 1% promoter. Ten per cent of patients were mosaic for the RB1 mutation. Six three-generation families with incomplete penetrance RB1 mutations were found. We found evidence that two variants, previously described as pathogenic RB1 mutations, are likely to be neutral variants. Conclusions The frequency of the type of mutations in the RB1 gene in our unbiased national cohort is the same as the mutation spectrum described worldwide. Furthermore, our RB1 mutation detection regimen achieves a high scanning sensitivity.