Project description:We previously established the contribution of de novo damaging sequence variants to Tourette disorder (TD) through whole-exome sequencing of 511 trios. Here, we sequence an additional 291 TD trios and analyze the combined set of 802 trios. We observe an overrepresentation of de novo damaging variants in simplex, but not multiplex, families; we identify a high-confidence TD risk gene, CELSR3 (cadherin EGF LAG seven-pass G-type receptor 3); we find that the genes mutated in TD patients are enriched for those related to cell polarity, suggesting a common pathway underlying pathobiology; and we confirm a statistically significant excess of de novo copy number variants in TD. Finally, we identify significant overlap of de novo sequence variants between TD and obsessive-compulsive disorder and de novo copy number variants between TD and autism spectrum disorder, consistent with shared genetic risk.
Project description:Decreased blood-brain barrier (BBB) efflux function of the P-glycoprotein (P-gp) transport system could facilitate the accumulation of toxic compounds in the brain, increasing the risk of neurodegenerative pathology such as Parkinson's disease (PD). This study investigated in vivo BBB P-gp function in patients with parkinsonian neurodegenerative syndromes, using [11C]-verapamil PET in PD, PSP and MSA patients. Regional differences in distribution volume were studied using SPM with higher uptake interpreted as reduced P-gp function. Advanced PD patients and PSP patients had increased [11C]-verapamil uptake in frontal white matter regions compared to controls; while de novo PD patients showed lower uptake in midbrain and frontal regions. PSP and MSA patients had increased uptake in the basal ganglia. Decreased BBB P-gp function seems a late event in neurodegenerative disorders, and could enhance continuous neurodegeneration. Lower [11C]-verapamil uptake in midbrain and frontal regions of de novo PD patients could indicate a regional up-regulation of P-gp function.
Project description:BACKGROUND:Intensification of systemic therapy for high-risk neuroblastoma (HRNB) has resulted in improved local control and overall survival (OS) leaving potential for de-escalation of primary site radiotherapy. The utility of primary site de-escalation should be evaluated in the context of potential for successful local-regional salvage. We evaluated salvage strategies and outcomes in patients with HRNB with local-regional recurrence as a component of first failure. METHODS:Twenty of 89 patients with HRNB experienced local-regional recurrence as a component of first relapse after chemotherapy, radiotherapy, surgery, and stem cell transplant from 1997 to 2013. We reviewed salvage therapy strategies and disease control, and report on the impact of local therapy as salvage for local-regional relapse. RESULTS:Six of 20 patients with local-regional failure (LRF) were alive after a median follow-up of 13 years (range, 0.9-25.2 years). Median OS was 4.6 years (95% CI, 0.6 to not reached) versus 0.6 years (95% CI, 0.05-2.6) after LRF with and without distant failure, respectively (P = 0.03). OS in patients receiving salvage radiotherapy was comparable to those receiving initial adjuvant but no salvage radiotherapy. Time to first failure and death was significantly impacted by the intensity of frontline systemic therapy (P = 0.03). Salvage radiotherapy reduced the hazard for subsequent LRF (hazard ratio 0.3, 95% CI 0.1-0.9, P = 0.04) but not OS (P = 0.07). CONCLUSIONS:Our study highlights the potential of local control strategies at first failure in patients with LRF when primary site radiotherapy was initially omitted, and delineates potential selection factors which may further improve the therapeutic ratio.
Project description:Tandem segmental duplications (SDs) greater than 10 kb are widespread in complex genomes. They provide material for gene divergence and evolutionary adaptation, while formation of specific de novo SDs is a hallmark of cancer and some human diseases. Most SDs map to distinct genomic regions termed 'duplication blocks'. SDs organization within these blocks is often poorly characterized as they are mosaics of ancestral duplicons juxtaposed with younger duplicons arising from more recent duplication events. Structural and functional analysis of SDs is further hampered as long repetitive DNA structures are underrepresented in existing BAC and YAC libraries. We applied Transformation-Associated Recombination (TAR) cloning, a versatile technique for large DNA manipulation, to selectively isolate the coronary artery disease (CAD) interval sequence within the 9p21.3 chromosome locus from a patient with coronary artery disease and normal individuals. Four tandem head-to-tail duplicons, each ?50 kb long, were recovered in the patient but not in normal individuals. Sequence analysis revealed that the repeats varied by 10-15 SNPs between each other and by 82 SNPs between the human genome sequence (version hg19). SNPs polymorphism within the junctions between repeats allowed two junction types to be distinguished, Type 1 and Type 2, which were found at a 2:1 ratio. The junction sequences contained an Alu element, a sequence previously shown to play a role in duplication. Knowledge of structural variation in the CAD interval from more patients could help link this locus to cardiovascular diseases susceptibility, and maybe relevant to other cases of regional amplification, including cancer.
Project description:Worldwide, nontuberculous mycobacteria (NTM) have become emergent pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an estimated prevalence ranging from 5 to 20%. This work investigated the presence of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to longitudinal clinical supervision at a regional reference center in Rio de Janeiro, Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10 (7.75%) out of 129 children were obtained. Molecular identification of NTM was performed by using PCR restriction analysis targeting the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene, and susceptibility tests were performed that followed Clinical and Laboratory Standards Institute recommendations. For evaluating the genotypic diversity, pulsed-field gel electrophoresis (PFGE) and/or enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) was performed. The species identified were Mycobacterium abscessus subsp. bolletii (n = 24), M. abscessus subsp. abscessus (n = 6), Mycobacterium fortuitum (n = 3), Mycobacterium marseillense (n = 2), and Mycobacterium timonense (n = 1). Most of the isolates presented resistance to five or more of the antimicrobials tested. Typing profiles were mainly patient specific. The PFGE profiles indicated the presence of two clonal groups for M. abscessus subsp. abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one clone detected in two patients. Given the observed multidrug resistance patterns and the possibility of transmission between patients, we suggest the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease.
Project description:Autism is a common and often severe neurodevelopmental disorder for which diverse pathophysiological processes have been proposed. Recent gene expression data comparing autistic and control brains suggest that the normal differential gene expression between frontal and temporal cortex is attenuated in autistic brains. It is unknown if regional de-differentiation occurs elsewhere in autistic brain. Using high resolution, genome-wide RNA expression microarrays and brain specimens meeting stringent selection criteria we evaluated gene expression data of two other regions: Brodmann area 19 (occipital cortex) and cerebellar cortex. In contrast to frontal/temporal cortical data, our data do not indicate an attenuation of regional specialization between occipital and cerebellar cortical regions in autistic brains.
Project description:De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We investigated global patterns of germline mutation by whole-genome sequencing of monozygotic twins concordant for ASD and their parents. Mutation rates varied widely throughout the genome (by 100-fold) and could be explained by intrinsic characteristics of DNA sequence and chromatin structure. Dense clusters of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing data sets. Our findings suggest that regional hypermutation is a significant factor shaping patterns of genetic variation and disease risk in humans.
Project description:Alterations in DNA methylation have been associated with genome-wide hypomethylation and regional de novo methylation in numerous cancers. De novo methylation is mediated by the de novo methyltransferases Dnmt3a and 3b, but only Dnmt3b has been implicated in promoting cancer by silencing of tumor-suppressor genes. In this study, we have analyzed the role of Dnmt3a in lung cancer by using a conditional mouse tumor model. We show that Dnmt3a deficiency significantly promotes tumor growth and progression but not initiation. Changes in gene expression show that Dnmt3a deficiency affects key steps in cancer progression, such as angiogenesis, cell adhesion, and cell motion, consistent with accelerated and more malignant growth. Our results suggest that Dnmt3a may act like a tumor-suppressor gene in lung tumor progression and may be a critical determinant of lung cancer malignancy.
Project description:Activating mutations in tyrosine kinase (TK) genes (eg, FLT3 and KIT) are found in more than 30% of patients with de novo acute myeloid leukemia (AML); many groups have speculated that mutations in other TK genes may be present in the remaining 70%. We performed high-throughput resequencing of the kinase domains of 26 TK genes (11 receptor TK; 15 cytoplasmic TK) expressed in most AML patients using genomic DNA from the bone marrow (tumor) and matched skin biopsy samples ("germline") from 94 patients with de novo AML; sequence variants were validated in an additional 94 AML tumor samples (14.3 million base pairs of sequence were obtained and analyzed). We identified known somatic mutations in FLT3, KIT, and JAK2 TK genes at the expected frequencies and found 4 novel somatic mutations, JAK1(V623A), JAK1(T478S), DDR1(A803V), and NTRK1(S677N), once each in 4 respective patients of 188 tested. We also identified novel germline sequence changes encoding amino acid substitutions (ie, nonsynonymous changes) in 14 TK genes, including TYK2, which had the largest number of nonsynonymous sequence variants (11 total detected). Additional studies will be required to define the roles that these somatic and germline TK gene variants play in AML pathogenesis.
Project description:Activating mutations in tyrosine kinase (TK) genes (e.g. FLT3 and KIT) are found in more than 30% of patients with de novo acute myeloid leukemia (AML); many groups have speculated that mutations in other TK genes may be present in the remaining 70%. We performed high-throughput re-sequencing of the kinase domains of 26 TK genes (11 receptor TK and 15 cytoplasmic TK) that are expressed in most AML patients, using genomic DNA from the bone marrow (tumor) and matched skin biopsy samples (germline) from 94 patients with de novo AML; sequence variants were validated in an additional 94 AML tumor samples (14.3 million base pairs of sequence were obtained and analyzed). We identified known somatic mutations in FLT3, KIT, and JAK2 TK genes at the expected frequencies, and found four novel somatic mutations, JAK1V623A, JAK1T478S, DDR1A803V and NTRK1S677N, once each in four respective patients out of 188 tested. We also identified novel germline sequence changes encoding amino acid substitutions (i.e. non-synonymous changes) in 14 TK genes, including TYK2, which had the largest number of non-synonymous sequence variants (11 total detected). Additional studies will be required to define the roles that these somatic and germline TK gene variants play in AML pathogenesis. Experiment Overall Design: 188 patient samples analysed