Genome-wide detection of tandem DNA repeats that are expanded in autism.

Tandem DNA repeats vary in the size and sequence of each unit (motif). When expanded, these tandem DNA repeats have been associated with more than 40 monogenic disorders1. Their involvement in disorders with complex genetics is largely unknown, as is the extent of their heterogeneity. Here we investigated the genome-wide characteristics of tandem repeats that had motifs with a length of 2-20 base pairs in 17,231 genomes of families containing individuals with autism spectrum disorder (ASD)2,3 and population control individuals4. We found extensive polymorphism in the size and sequence of motifs. Many of the tandem repeat loci that we detected correlated with cytogenetic fragile sites. At 2,588 loci, gene-associated expansions of tandem repeats that were rare among population control individuals were significantly more prevalent among individuals with ASD than their siblings without ASD, particularly in exons and near splice junctions, and in genes related to the development of the nervous system and cardiovascular system or muscle. Rare tandem repeat expansions had a prevalence of 23.3% in children with ASD compared with 20.7% in children without ASD, which suggests that tandem repeat expansions make a collective contribution to the risk of ASD of 2.6%. These rare tandem repeat expansions included previously undescribed ASD-linked expansions in DMPK and FXN, which are associated with neuromuscular conditions, and in previously unknown loci such as FGF14 and CACNB1. Rare tandem repeat expansions were associated with lower IQ and adaptive ability. Our results show that tandem DNA repeat expansions contribute strongly to the genetic aetiology and phenotypic complexity of ASD.

Rare tandem repeat expansions associate with genes involved in synaptic and neuronal signaling functions in schizophrenia.

Tandem repeat expansions (TREs) are associated with over 60 monogenic disorders and have recently been implicated in complex disorders such as cancer and autism spectrum disorder. The role of TREs in schizophrenia is now emerging. In this study, we have performed a genome-wide investigation of TREs in schizophrenia. Using genome sequence data from 1154 Swedish schizophrenia cases and 934 ancestry-matched population controls, we have detected genome-wide rare (<0.1% population frequency) TREs that have motifs with a length of 2-20 base pairs. We find that the proportion of individuals carrying rare TREs is significantly higher in the schizophrenia group. There is a significantly higher burden of rare TREs in schizophrenia cases than in controls in genic regions, particularly in postsynaptic genes, in genes overlapping brain expression quantitative trait loci, and in brain-expressed genes that are differentially expressed between schizophrenia cases and controls. We demonstrate that TRE-associated genes are more constrained and primarily impact synaptic and neuronal signaling functions. These results have been replicated in an independent Canadian sample that consisted of 252 schizophrenia cases of European ancestry and 222 ancestry-matched controls. Our results support the involvement of rare TREs in schizophrenia etiology.

Genome-wide tandem repeat expansions contribute to schizophrenia risk.

Tandem repeat expansions (TREs) can cause neurological diseases but their impact in schizophrenia is unclear. Here we analyzed genome sequences of adults with schizophrenia and found that they have a higher burden of TREs that are near exons and rare in the general population, compared with non-psychiatric controls. These TREs are disproportionately found at loci known to be associated with schizophrenia from genome-wide association studies, in individuals with clinically-relevant genetic variants at other schizophrenia loci, and in families where multiple individuals have schizophrenia. We showed that rare TREs in schizophrenia may impact synaptic functions by disrupting the splicing process of their associated genes in a loss-of-function manner. Our findings support the involvement of genome-wide rare TREs in the polygenic nature of schizophrenia.

Genome-wide enhancer-associated tandem repeats are expanded in cardiomyopathy.

BACKGROUND: Cardiomyopathy is a clinically and genetically heterogeneous heart condition that can lead to heart failure and sudden cardiac death in childhood. While it has a strong genetic basis, the genetic aetiology for over 50% of cardiomyopathy cases remains unknown. METHODS: In this study, we analyse the characteristics of tandem repeats from genome sequence data of unrelated individuals diagnosed with cardiomyopathy from Canada and the United Kingdom (n = 1216) and compare them to those found in the general population. We perform burden analysis to identify genomic and epigenomic features that are impacted by rare tandem repeat expansions (TREs), and enrichment analysis to identify functional pathways that are involved in the TRE-associated genes in cardiomyopathy. We use Oxford Nanopore targeted long-read sequencing to validate repeat size and methylation status of one of the most recurrent TREs. We also compare the TRE-associated genes to those that are dysregulated in the heart tissues of individuals with cardiomyopathy. FINDINGS: We demonstrate that tandem repeats that are rarely expanded in the general population are predominantly expanded in cardiomyopathy. We find that rare TREs are disproportionately present in constrained genes near transcriptional start sites, have high GC content, and frequently overlap active enhancer H3K27ac marks, where expansion-related DNA methylation may reduce gene expression. We demonstrate the gene silencing effect of expanded CGG tandem repeats in DIP2B through promoter hypermethylation. We show that the enhancer-associated loci are found in genes that are highly expressed in human cardiomyocytes and are differentially expressed in the left ventricle of the heart in individuals with cardiomyopathy. INTERPRETATION: Our findings highlight the underrecognized contribution of rare tandem repeat expansions to the risk of cardiomyopathy and suggest that rare TREs contribute to ∼4% of cardiomyopathy risk. FUNDING: Government of Ontario (RKCY), The Canadian Institutes of Health Research PJT 175329 (RKCY), The Azrieli Foundation (RKCY), SickKids Catalyst Scholar in Genetics (RKCY), The University of Toronto McLaughlin Centre (RKCY, SM), Ted Rogers Centre for Heart Research (SM), Data Sciences Institute at the University of Toronto (SM), The Canadian Institutes of Health Research PJT 175034 (SM), The Canadian Institutes of Health Research ENP 161429 under the frame of ERA PerMed (SM, RL), Heart and Stroke Foundation of Ontario & Robert M Freedom Chair in Cardiovascular Science (SM), Bitove Family Professorship of Adult Congenital Heart Disease (EO), Canada Foundation for Innovation (SWS, JR), Canada Research Chair (PS), Genome Canada (PS, JR), The Canadian Institutes of Health Research (PS).

Genome sequencing broadens the range of contributing variants with clinical implications in schizophrenia.

The range of genetic variation with potential clinical implications in schizophrenia, beyond rare copy number variants (CNVs), remains uncertain. We therefore analyzed genome sequencing data for 259 unrelated adults with schizophrenia from a well-characterized community-based cohort previously examined with chromosomal microarray for CNVs (none with 22q11.2 deletions). We analyzed these genomes for rare high-impact variants considered causal for neurodevelopmental disorders, including single-nucleotide variants (SNVs) and small insertions/deletions (indels), for potential clinical relevance based on findings for neurodevelopmental disorders. Also, we investigated a novel variant type, tandem repeat expansions (TREs), in 45 loci known to be associated with monogenic neurological diseases. We found several of these variants in this schizophrenia population suggesting that these variants have a wider clinical spectrum than previously thought. In addition to known pathogenic CNVs, we identified 11 (4.3%) individuals with clinically relevant SNVs/indels in genes converging on schizophrenia-relevant pathways. Clinical yield was significantly enriched in females and in those with broadly defined learning/intellectual disabilities. Genome analyses also identified variants with potential clinical implications, including TREs (one in DMPK; two in ATXN8OS) and ultra-rare loss-of-function SNVs in ZMYM2 (a novel candidate gene for schizophrenia). Of the 233 individuals with no pathogenic CNVs, we identified rare high-impact variants (i.e., clinically relevant or with potential clinical implications) for 14 individuals (6.0%); some had multiple rare high-impact variants. Mean schizophrenia polygenic risk score was similar between individuals with and without clinically relevant rare genetic variation; common variants were not sufficient for clinical application. These findings broaden the individual and global picture of clinically relevant genetic risk in schizophrenia, and suggest the potential translational value of genome sequencing as a single genetic technology for schizophrenia.

Genome sequencing identifies rare tandem repeat expansions and copy number variants in Lennox-Gastaut syndrome.

Epilepsies are a group of common neurological disorders with a substantial genetic basis. Despite this, the molecular diagnosis of epilepsies remains challenging due to its heterogeneity. Studies utilizing whole-genome sequencing may provide additional insights into genetic causes of epilepsies of unknown aetiology. Whole-genome sequencing was used to evaluate a cohort of adults with unexplained developmental and epileptic encephalopathies (n = 30), for whom prior genetic tests, including whole-exome sequencing in some cases, were negative or inconclusive. Rare single nucleotide variants, insertions/deletions, copy number variants and tandem repeat expansions were analysed. Seven pathogenic or likely pathogenic single nucleotide variants, and two pathogenic deleterious copy number variants were identified in nine patients (32.1% of the cohort). One of the copy number variants, identified in a patient with Lennox-Gastaut syndrome, was too small to be detected by chromosomal microarray techniques. We also identified two tandem repeat expansions with clinical implications in two other patients with Lennox-Gastaut syndrome: a CGG repeat expansion in the 5'untranslated region of DIP2B, and a CTG expansion in ATXN8OS (previously implicated in spinocerebellar ataxia type 8). Three patients had KCNA2 pathogenic variants. One of them died of sudden unexpected death in epilepsy. The other two patients had, in addition to a KCNA2 variant, a second de novo variant impacting potential epilepsy-relevant genes (KCNIP4 and UBR5). Overall, whole-genome sequencing provided a genetic explanation in 32.1% of the total cohort. This is also the first report of coding and non-coding tandem repeat expansions identified in patients with Lennox-Gastaut syndrome. This study demonstrates that using whole-genome sequencing, the examination of multiple types of rare genetic variation, including those found in the non-coding region of the genome, can help resolve unexplained epilepsies.

In Vivo Validation of PAPSS1 (3'-phosphoadenosine 5'-phosphosulfate synthase 1) as a Cisplatin-sensitizing Therapeutic Target.

Purpose: Our previous screening efforts found that inhibition of PAPSS1 increases the potency of DNA-damaging agents in non-small cell lung cancer (NSCLC) cell lines. Here, we explored the clinical relevance of PAPSS1 and further investigated it as a therapeutic target in preclinical model systems.Experimental Design: PAPSS1 expression and cisplatin IC50 values were assessed in 52 lung adenocarcinoma cell lines. Effects of PAPSS1 inhibition on A549 cisplatin sensitivity under hypoxic and starvation conditions, in 3D spheroids, as well as in zebrafish and mouse xenografts, were evaluated. Finally, the association between PAPSS1 expression levels and survival in patients treated with standard chemotherapy was assessed.Results: Our results show a positive correlation between low PAPSS1 expression and increased cisplatin sensitivity in lung adenocarcinoma. In vitro, the potentiation effect was greatest when A549 cells were serum-starved under hypoxic conditions. When treated with low-dose cisplatin, PAPSS1-deficient A549 spheroids showed a 58% reduction in size compared with control cells. In vivo, PAPSS1 suppression and low-dose cisplatin treatment inhibited proliferation of lung tumor cells in zebrafish xenografts and significantly delayed development of subcutaneous tumors in mice. Clinical data suggest that NSCLC and ovarian cancer patients with low PAPSS1 expression survive longer following platinum-based chemotherapy.Conclusions: These results suggest that PAPSS1 inhibition enhances cisplatin activity in multiple preclinical model systems and that low PAPSS1 expression may serve as a biomarker for platin sensitivity in cancer patients. Developing strategies to target PAPSS1 activity in conjunction with platinum-based chemotherapy may offer an approach to improving treatment outcomes. Clin Cancer Res; 23(21); 6555-66. ©2017 AACR.

Sulfonation, an underexploited area: from skeletal development to infectious diseases and cancer.

Sulfonation is one of the most abundant cellular reactions modifying a wide range of xenobiotics as well as endogenous molecules which regulate important biological processes including blood clotting, formation of connective tissues, and functionality of secreted proteins, hormones, and signaling molecules. Sulfonation is ubiquitous in all tissues and widespread in nature (plants, animals, and microorganisms). Although sulfoconjugates were discovered over a century ago when, in 1875, Baumann isolated phenyl sulfate in the urine of a patient given phenol as an antiseptic, the significance of sulfonation and its roles in human diseases have been underappreciated until recent years. Here, we provide a current overview of the significance of sulfonation reactions in a variety of biological functions and medical conditions (with emphasis on cancer). We also discuss research areas that warrant further attention if we are to fully understand how deficiencies in sulfonation could impact human health which, in turn, could help define treatments to effect improvements in health.

Nanoscale Reaction Vessels Designed for Synthesis of Copper-Drug Complexes Suitable for Preclinical Development.

The development of copper-drug complexes (CDCs) is hindered due to their very poor aqueous solubility. Diethyldithiocarbamate (DDC) is the primary metabolite of disulfiram, an approved drug for alcoholism that is being repurposed for cancer. The anticancer activity of DDC is dependent on complexation with copper to form copper bis-diethyldithiocarbamate (Cu(DDC)2), a highly insoluble complex that has not been possible to develop for indications requiring parenteral administration. We have resolved this issue by synthesizing Cu(DDC)2 inside liposomes. DDC crosses the liposomal lipid bilayer, reacting with the entrapped copper; a reaction that can be observed through a colour change as the solution goes from a light blue to dark brown. This method is successfully applied to other CDCs including the anti-parasitic drug clioquinol, the natural product quercetin and the novel targeted agent CX-5461. Our method provides a simple, transformative solution enabling, for the first time, the development of CDCs as viable candidate anticancer drugs; drugs that would represent a brand new class of therapeutics for cancer patients.

Impact of Commercial Strain Use on Saccharomyces cerevisiae Population Structure and Dynamics in Pinot Noir Vineyards and Spontaneous Fermentations of a Canadian Winery.

Wine is produced by one of two methods: inoculated fermentation, where a commercially-produced, single Saccharomyces cerevisiae (S. cerevisiae) yeast strain is used; or the traditional spontaneous fermentation, where yeast present on grape and winery surfaces carry out the fermentative process. Spontaneous fermentations are characterized by a diverse succession of yeast, ending with one or multiple strains of S. cerevisiae dominating the fermentation. In wineries using both fermentation methods, commercial strains may dominate spontaneous fermentations. We elucidate the impact of the winery environment and commercial strain use on S. cerevisiae population structure in spontaneous fermentations over two vintages by comparing S. cerevisiae populations in aseptically fermented grapes from a Canadian Pinot Noir vineyard to S. cerevisiae populations in winery-conducted fermentations of grapes from the same vineyard. We also characterize the vineyard-associated S. cerevisiae populations in two other geographically separate Pinot Noir vineyards farmed by the same winery. Winery fermentations were not dominated by commercial strains, but by a diverse number of strains with genotypes similar to commercial strains, suggesting that a population of S. cerevisiae derived from commercial strains is resident in the winery. Commercial and commercial-related yeast were also identified in the three vineyards examined, although at a lower frequency. There is low genetic differentiation and S. cerevisiae population structure between vineyards and between the vineyard and winery that persisted over both vintages, indicating commercial yeast are a driver of S. cerevisiae population structure. We also have evidence of distinct and persistent populations of winery and vineyard-associated S. cerevisiae populations unrelated to commercial strains. This study is the first to characterize S. cerevisiae populations in Canadian vineyards.

Combined Use of Gene Expression Modeling and siRNA Screening Identifies Genes and Pathways Which Enhance the Activity of Cisplatin When Added at No Effect Levels to Non-Small Cell Lung Cancer Cells In Vitro.

Platinum-based combination chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC). While cisplatin is effective, its use is not curative and resistance often emerges. As a consequence of microenvironmental heterogeneity, many tumour cells are exposed to sub-lethal doses of cisplatin. Further, genomic heterogeneity and unique tumor cell sub-populations with reduced sensitivities to cisplatin play a role in its effectiveness within a site of tumor growth. Being exposed to sub-lethal doses will induce changes in gene expression that contribute to the tumour cell's ability to survive and eventually contribute to the selective pressures leading to cisplatin resistance. Such changes in gene expression, therefore, may contribute to cytoprotective mechanisms. Here, we report on studies designed to uncover how tumour cells respond to sub-lethal doses of cisplatin. A microarray study revealed changes in gene expressions that occurred when A549 cells were exposed to a no-observed-effect level (NOEL) of cisplatin (e.g. the IC10). These data were integrated with results from a genome-wide siRNA screen looking for novel therapeutic targets that when inhibited transformed a NOEL of cisplatin into one that induced significant increases in lethality. Pathway analyses were performed to identify pathways that could be targeted to enhance cisplatin activity. We found that over 100 genes were differentially expressed when A549 cells were exposed to a NOEL of cisplatin. Pathways associated with apoptosis and DNA repair were activated. The siRNA screen revealed the importance of the hedgehog, cell cycle regulation, and insulin action pathways in A549 cell survival and response to cisplatin treatment. Results from both datasets suggest that RRM2B, CABYR, ALDH3A1, and FHL2 could be further explored as cisplatin-enhancing gene targets. Finally, pathways involved in repairing double-strand DNA breaks and INO80 chromatin remodeling were enriched in both datasets, warranting further research into combinations of cisplatin and therapeutics targeting these pathways.

Altered lower extremity fracture characteristics in obese pediatric trauma patients.

OBJECTIVE: To determine whether there are differences in fracture patterns and femur fracture treatment choices in obese versus nonobese pediatric trauma patients. DESIGN: Prognostic study, retrospective chart review. SETTING: Two level I pediatric trauma centers. PATIENTS: The trauma registries of 2 pediatric hospitals were queried for patients with lower extremity long-bone fractures resulting from blunt trauma. 2858 alerts were examined, and 397 patients had lower extremity fractures. Three hundred thirty-one patients with a total of 394 femur or tibia fractures met the inclusion criteria, and 70 patients (21%) were obese. MAIN OUTCOME MEASUREMENTS: Weight for age >95th percentile was defined as obese. Radiographs were reviewed, and fractures were classified according the OTA/AO pediatric fracture classification system. Fracture patterns (OTA subsegment), severity, and choice of intervention for femur fractures were the primary outcomes. RESULTS: Overall, obese patients were twice as likely [risk ratio (RR), 2.20; 95% confidence interval (CI), 1.25-3.89] to have fractures involving the physis. Physeal fracture risk was greater for femur fractures (RR, 3.25; 95% CI, 1.35-7.78) than tibia fractures (RR, 1.58; 95% CI, 0.76-3.26). Severity did not differ between groups. Obese patients with femur fractures were more likely to be treated with locked nails. CONCLUSIONS: Obese pediatric trauma patients are more likely to sustain fractures involving the physis than nonobese patients. This could be related to intrinsic changes to the physis related to obesity or altered biomechanical forces. This is consistent with the observed relationships between obesity and other conditions affecting the physis including Blount disease and slipped capital femoral epiphysis. LEVEL OF EVIDENCE: Prognostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Pediatric obesity and traumatic lower-extremity long-bone fracture outcomes.

BACKGROUND: Pediatric obesity is associated with lower-extremity injuries and poor outcomes after blunt trauma. Our aim was to determine if obese pediatric patients with femur and tibia fractures have more severe injury patterns and worse outcomes compared with those of nonobese patients. METHODS: We performed a retrospective cohort study of obese and nonobese pediatric patients with femur or tibia fractures treated at two Level I trauma centers from 2004 to 2010. Patients weighing 95th percentile or greater for age and sex were classified as obese. Patients were compared regarding demographics, Injury Severity Score (ISS), as well as intra-abdominal and orthopedic injuries. Outcomes included fracture treatment, orthopedic complications, intensive care unit and hospital length of stay, ventilator days, and mortality. RESULTS: Of the 356 patients included in the study, 78 (21.9%) were obese and 278 (78.1%) were nonobese. Obese patients were older (mean [SD], 9.9 [3.7] years vs. 8.8 [3.9] years; p = 0.0162), had a higher ISS (20.8 [13.4] vs. 14.5 [10.8]; p = 0.0002), and sustained more intra-abdominal solid organ (24.4% vs.13.5%; p = 0.0200) and hollow viscus (3.9% vs. 0.0%; p = 0.0105) injuries. They had more pelvic fractures (15.4% vs. 6.9%; p = 0.0196), bilateral tibia fractures (8.0% vs. 0.0%; p = 0.0332), and operatively treated femur fractures (89.9% vs. 79.1%; p = 0.0484). Adjusting for age, obese patients were more likely to be admitted to the intensive care unit (relative risk, 1.68; 95% confidence interval, 1.10-2.55) and die in the hospital (relative risk, 3.45; 95% confidence interval, 1.14-10.41). Adjusting for ISS, these associations were nonsignificant. CONCLUSION: Obese patients with femur and tibia fractures have more severe injuries, which may predispose them to greater inpatient morbidity and mortality than do nonobese patients. LEVEL OF EVIDENCE: Epidemiologic study, level III.