Development of a comprehensive approach to adult hereditary cancer testing in Ontario.

BACKGROUND: Genetic testing for hereditary cancer susceptibility has advanced over time due to the discovery of new risk genes, improved technology and decreased cost. In the province of Ontario, testing eligibility criteria were initially developed to include hereditary breast, ovarian and colorectal cancer syndromes. The rapid evolution of genetic technologies has facilitated the ability to interrogate a large number of genes concurrently. This, coupled with new knowledge about risk genes, necessitated a coordinated approach to expanding the scope of genes and indications tested and synchronisation of access and test utilisation across the province as required in a publicly funded universal healthcare system. METHODS: Ontario Health-Cancer Care Ontario convened expert working groups to develop a standardised and comprehensive cancer gene list for adults and accompanying hereditary cancer testing (HCT) criteria using an evidence-based framework and broad laboratory and clinical genetics engagement. RESULTS: A standardised 76-cancer-gene panel, organised into 13 larger disease site panels and 25 single/small gene panels, was developed and endorsed by the working groups. Provincial genetic testing eligibility criteria were updated to align with the new panels and to guide clinical decision-making. In the first year following the implementation of these changes, 10 564 HCT panels were performed with an overall mutation detection rate of 12.2%. CONCLUSION: Using an evidence framework and broad clinical engagement to develop and endorse an updated guidance document, cancer genetic testing for adults in Ontario is now standardised and coordinated across the province.

Practice guidelines for BRCA1/2 tumour testing in ovarian cancer.

The purpose of this document is to provide pre-analytical, analytical and post-analytical considerations and recommendations to Canadian clinical laboratories developing, validating and offering next-generation sequencing (NGS)-based BRCA1 and BRCA2 (BRCA1/2) tumour testing in ovarian cancers. This document was drafted by the members of the Canadian College of Medical Geneticists (CCMG) somatic BRCA Ad Hoc Working Group, and representatives from the Canadian Association of Pathologists. The document was circulated to the CCMG members for comment. Following incorporation of feedback, this document has been approved by the CCMG board of directors. The CCMG is a Canadian organisation responsible for certifying medical geneticists and clinical laboratory geneticists, and for establishing professional and ethical standards for clinical genetics services in Canada. The current CCMG Practice Guidelines were developed as a resource for clinical laboratories in Canada; however, they are not inclusive of all information laboratories should consider in the validation and use of NGS for BRCA1/2 tumour testing in ovarian cancers.

Data sharing to improve concordance in variant interpretation across laboratories: results from the Canadian Open Genetics Repository.

BACKGROUND: This study aimed to identify and resolve discordant variant interpretations across clinical molecular genetic laboratories through the Canadian Open Genetics Repository (COGR), an online collaborative effort for variant sharing and interpretation. METHODS: Laboratories uploaded variant data to the Franklin Genoox platform. Reports were issued to each laboratory, summarising variants where conflicting classifications with another laboratory were noted. Laboratories could then reassess variants to resolve discordances. Discordance was calculated using a five-tier model (pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), benign (B)), a three-tier model (LP/P are positive, VUS are inconclusive, LB/B are negative) and a two-tier model (LP/P are clinically actionable, VUS/LB/B are not). We compared the COGR classifications to automated classifications generated by Franklin. RESULTS: Twelve laboratories submitted classifications for 44 510 unique variants. 2419 variants (5.4%) were classified by two or more laboratories. From baseline to after reassessment, the number of discordant variants decreased from 833 (34.4% of variants reported by two or more laboratories) to 723 (29.9%) based on the five-tier model, 403 (16.7%) to 279 (11.5%) based on the three-tier model and 77 (3.2%) to 37 (1.5%) based on the two-tier model. Compared with the COGR classification, the automated Franklin classifications had 94.5% sensitivity and 96.6% specificity for identifying actionable (P or LP) variants. CONCLUSIONS: The COGR provides a standardised mechanism for laboratories to identify discordant variant interpretations and reduce discordance in genetic test result delivery. Such quality assurance programmes are important as genetic testing is implemented more widely in clinical care.

Alpha-1-antitrypsin molecular testing in Canada: A seven year, multi-centre comparison.

BACKGROUND: Laboratory confirmation of alpha-1-antitrypsin (A1AT) deficiency may be achieved by multiple methods. Here, we compare the relative comprehensiveness and efficiency of pathogenic variant (PV) detection of four different protocols utilized at different diagnostic centres in Canada. METHODS: Diagnostic results from 2011 to 2018 at clinical laboratories in British Columbia (BC), Alberta (AB), Ontario (ON), and Québec (QC) were reviewed. The four labs utilize the following protocols: BC-CGID (serum A1AT Concentration/Genotyping/Isoelectric focussing (IEF)/SERPINA1 DNA sequencing), AB-CID (serum A1AT Concentration/IEF/DNA sequencing), ON-CD (serum A1AT Concentration/DNA sequencing), and QC-G (Genotyping). As the respective catchment areas varied in size and ethnic composition, the comprehensiveness of PV detection was assessed by comparing the frequency of individual genotypes to the ZZ genotype, which is clearly identified by all protocols. RESULTS: Collectively 5399 index patients were tested identifying 396 ZZ genotypes. Serum A1AT concentration as a determinant of further testing efficiently identified PV. ON-CD had the highest detection rate for PV; genotypes with at least one PV, other than S, Z or F, were identified at 0.67/ZZ as compared to <0.2/ZZ (all others). However, ON-CD had the highest rates of undefined molecular variants (UMV) (0.16/ZZ) or likely benign variants (LBV) (0.08/ZZ), compared to all others (<0.12/ZZ and < 0.06/ZZ, respectively). The F variant was identified at 0.10/ZZ, only in the ON-CD and the AB-CID protocols. Collectively, MMalton was the next most common variant, identified as a compound heterozygous genotype at 0.04/ZZ, only in the ON-CD and BC-CGID protocols. CONCLUSION: Strategies which readily detect variants across the full coding sequence of SERPINA1 detect more PV as well as more UMV and LBV.

Microdeletions of ELP4 Are Associated with Language Impairment, Autism Spectrum Disorder, and Mental Retardation.

Copy-number variations (CNVs) are important in the aetiology of neurodevelopmental disorders and show broad phenotypic manifestations. We compared the presence of small CNVs disrupting the ELP4-PAX6 locus in 4,092 UK individuals with a range of neurodevelopmental conditions, clinically referred for array comparative genomic hybridization, with WTCCC controls (n = 4,783). The phenotypic analysis was then extended using the DECIPHER database. We followed up association using an autism patient cohort (n = 3,143) compared with six additional control groups (n = 6,469). In the clinical discovery series, we identified eight cases with ELP4 deletions, and one with a partial duplication of ELP4 and PAX6. These cases were referred for neurological phenotypes including language impairment, developmental delay, autism, and epilepsy. Six further cases with a primary diagnosis of autism spectrum disorder (ASD) and similar secondary phenotypes were identified with ELP4 deletions, as well as another six (out of nine) with neurodevelopmental phenotypes from DECIPHER. CNVs at ELP4 were only present in 1/11,252 controls. We found a significant excess of CNVs in discovery cases compared with controls, P = 7.5 × 10(-3) , as well as for autism, P = 2.7 × 10(-3) . Our results suggest that ELP4 deletions are highly likely to be pathogenic, predisposing to a range of neurodevelopmental phenotypes from ASD to language impairment and epilepsy.

Absent CNKSR2 causes seizures and intellectual, attention, and language deficits.

Synaptic function is central to brain function. Understanding the synapse is aided by studies of patients lacking individual synaptic proteins. Common neurological diseases are genetically complex. Their understanding is likewise simplified by studies of less common monogenic forms. We detail the disease caused by absence of the synaptic protein CNKSR2 in 8 patients ranging from 6 to 62 years old. The disease is characterized by intellectual disability, attention problems, and abrupt lifelong language loss following a brief early childhood epilepsy with continuous spike-waves in sleep. This study describes the phenotype of CNKSR2 deficiency and its involvement in systems underlying common neurological disorders.

CHD2 haploinsufficiency is associated with developmental delay, intellectual disability, epilepsy and neurobehavioural problems.

BACKGROUND: The chromodomain helicase DNA binding domain (CHD) proteins modulate gene expression via their ability to remodel chromatin structure and influence histone acetylation. Recent studies have shown that CHD2 protein plays a critical role in embryonic development, tumor suppression and survival. Like other genes encoding members of the CHD family, pathogenic mutations in the CHD2 gene are expected to be implicated in human disease. In fact, there is emerging evidence suggesting that CHD2 might contribute to a broad spectrum of neurodevelopmental disorders. Despite growing evidence, a description of the full phenotypic spectrum of this condition is lacking. METHODS: We conducted a multicentre study to identify and characterise the clinical features associated with haploinsufficiency of CHD2. Patients with deletions of this gene were identified from among broadly ascertained clinical cohorts undergoing genomic microarray analysis for developmental delay, congenital anomalies and/or autism spectrum disorder. RESULTS: Detailed clinical assessments by clinical geneticists showed recurrent clinical symptoms, including developmental delay, intellectual disability, epilepsy, behavioural problems and autism-like features without characteristic facial gestalt or brain malformations observed on magnetic resonance imaging scans. Parental analysis showed that the deletions affecting CHD2 were de novo in all four patients, and analysis of high-resolution microarray data derived from 26,826 unaffected controls showed no deletions of this gene. CONCLUSIONS: The results of this study, in addition to our review of the literature, support a causative role of CHD2 haploinsufficiency in developmental delay, intellectual disability, epilepsy and behavioural problems, with phenotypic variability between individuals.

Ectopic TLX1 expression accelerates malignancies in mice deficient in DNA-PK.

The noncluster homeobox gene HOX11/TLX1 (TLX1) is detected at the breakpoint of the t(10;14)(q24;q11) chromosome translocation in patients with T cell acute lymphoblastic leukemia (T-ALL). This translocation results in the inappropriate expression of TLX1 in T cells. The oncogenic potential of TLX1 was demonstrated in IgHµ-TLX1(Tg) mice which develop mature B cell lymphoma after a long latency period, suggesting the requirement of additional mutations to initiate malignancy. To determine whether dysregulation of genes involved in the DNA damage response contributed to tumor progression, we crossed IgHµ-TLX1(Tg) mice with mice deficient in the DNA repair enzyme DNA-PK (Prkdc(Scid/Scid) mice). IgHµ-TLX1(Tg)Prkdc(Scid/Scid) mice developed T-ALL and acute myeloid leukemia (AML) with reduced latency relative to control Prkdc(Scid/Scid) mice. Further analysis of thymi from premalignant mice revealed greater thymic cellularity concomitant with increased thymocyte proliferation and decreased apoptotic index. Moreover, premalignant and malignant thymocytes exhibited impaired spindle checkpoint function, in association with aneuploid karyotypes. Gene expression profiling of premalignant IgHµ-TLX1(Tg)Prkdc(Scid/Scid) thymocytes revealed dysregulated expression of cell cycle, apoptotic and mitotic spindle checkpoint genes in double negative 2 (DN2) and DN3 stage thymocytes. Collectively, these findings reveal a novel synergy between TLX1 and impaired DNA repair pathway in leukemogenesis.

Disruption of the ASTN2/TRIM32 locus at 9q33.1 is a risk factor in males for autism spectrum disorders, ADHD and other neurodevelopmental phenotypes.

Rare copy number variants (CNVs) disrupting ASTN2 or both ASTN2 and TRIM32 have been reported at 9q33.1 by genome-wide studies in a few individuals with neurodevelopmental disorders (NDDs). The vertebrate-specific astrotactins, ASTN2 and its paralog ASTN1, have key roles in glial-guided neuronal migration during brain development. To determine the prevalence of astrotactin mutations and delineate their associated phenotypic spectrum, we screened ASTN2/TRIM32 and ASTN1 (1q25.2) for exonic CNVs in clinical microarray data from 89 985 individuals across 10 sites, including 64 114 NDD subjects. In this clinical dataset, we identified 46 deletions and 12 duplications affecting ASTN2. Deletions of ASTN1 were much rarer. Deletions near the 3' terminus of ASTN2, which would disrupt all transcript isoforms (a subset of these deletions also included TRIM32), were significantly enriched in the NDD subjects (P = 0.002) compared with 44 085 population-based controls. Frequent phenotypes observed in individuals with such deletions include autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), speech delay, anxiety and obsessive compulsive disorder (OCD). The 3'-terminal ASTN2 deletions were significantly enriched compared with controls in males with NDDs, but not in females. Upon quantifying ASTN2 human brain RNA, we observed shorter isoforms expressed from an alternative transcription start site of recent evolutionary origin near the 3' end. Spatiotemporal expression profiling in the human brain revealed consistently high ASTN1 expression while ASTN2 expression peaked in the early embryonic neocortex and postnatal cerebellar cortex. Our findings shed new light on the role of the astrotactins in psychopathology and their interplay in human neurodevelopment.

eNOS overexpressing bone marrow cells are safe and effective in a porcine model of myocardial regeneration following acute myocardial infarction.

AIM: Cell therapy has been shown to be effective in improving LV function postmyocardial infarction (MI). We hypothesized that eNOS-transfected bone marrow cells (BMCs) are safe in a swine model of myocardial infarction (MI). We also hypothesized that endothelial nitric oxide synthase (eNOS) transfection would enhance cell function, as assessed by myocardial functional recovery post-MI. METHODS: Fifteen female Yorkshire pigs underwent bone marrow aspiration and creation of MI. Bone marrow cells were cultured for 7 days, and each pig received either autologous BMCs transiently transfected with eNOS plasmid (eNOS-BMC, n = 5), nontransfected BMCs (nt-BMC, n = 4), or phosphate-buffered saline (PBS) control (n = 6). Cardiac MRI was performed at baseline (1 week post-MI) and 6 weeks post-MI. RESULTS: There was no difference in safety outcomes between groups. Absolute left ventricular ejection fraction (LVEF) at 6 weeks showed a trend toward improvement in both cell therapy groups compared with baseline but worsened in the PBS control group. The absolute improvement in LVEF was significantly greater in both cell therapy groups compared with PBS control. Infarct mass was significantly lower in the eNOS-BMC group between baseline and 6 weeks, but the absolute change in infarct mass was not different between groups. Finally, there was a trend toward reduced LV mass in the eNOS-BMC group. CONCLUSIONS: Bone marrow cell delivery, with and without eNOS overexpression, is safe and leads to improvement in LVEF when administered in the coronary circulation 7 days following acute MI in swine. Transfection of healthy BMCs with eNOS resulted in some improvement in left ventricular remodeling. Further study is warranted in a preclinical model that approximates the impact of cardiovascular risk factors on BMC function.

Rare exonic deletions implicate the synaptic organizer Gephyrin (GPHN) in risk for autism, schizophrenia and seizures.

The GPHN gene codes for gephyrin, a key scaffolding protein in the neuronal postsynaptic membrane, responsible for the clustering and localization of glycine and GABA receptors at inhibitory synapses. Gephyrin has well-established functional links with several synaptic proteins that have been implicated in genetic risk for neurodevelopmental disorders such as autism spectrum disorder (ASD), schizophrenia and epilepsy including the neuroligins (NLGN2, NLGN4), the neurexins (NRXN1, NRXN2, NRXN3) and collybistin (ARHGEF9). Moreover, temporal lobe epilepsy has been linked to abnormally spliced GPHN mRNA lacking exons encoding the G-domain of the gephyrin protein, potentially arising due to cellular stress associated with epileptogenesis such as temperature and alkalosis. Here, we present clinical and genomic characterization of six unrelated subjects, with a range of neurodevelopmental diagnoses including ASD, schizophrenia or seizures, who possess rare de novo or inherited hemizygous microdeletions overlapping exons of GPHN at chromosome 14q23.3. The region of common overlap across the deletions encompasses exons 3-5, corresponding to the G-domain of the gephyrin protein. These findings, together with previous reports of homozygous GPHN mutations in connection with autosomal recessive molybdenum cofactor deficiency, will aid in clinical genetic interpretation of the GPHN mutation spectrum. Our data also add to the accumulating evidence implicating neuronal synaptic gene products as key molecular factors underlying the etiologies of a diverse range of neurodevelopmental conditions.

Deletions in 16q24.2 are associated with autism spectrum disorder, intellectual disability and congenital renal malformation.

BACKGROUND: The contribution of copy-number variation (CNV) to disease has been highlighted with the widespread adoption of array-based comparative genomic hybridisation (aCGH) and microarray technology. Contiguous gene deletions involving ANKRD11 in 16q24.3 are associated with autism spectrum disorder (ASD) and intellectual disability (ID), while 16q24.1 deletions affecting FOXF1 are associated with congenital renal malformations, alveolar capillary dysplasia, and various other abnormalities. The disease associations of deletions in the intervening region, 16q24.2, have only been defined to a limited extent. AIM: To determine whether deletions affecting 16q24.2 are correlated with congenital anomalies. METHODS: 35 individuals, each having a deletion in 16q24.2, were characterised clinically and by aCGH and/or SNP-genotyping microarray. RESULTS: Several of the 35 16q24.2 deletions identified here closely abut or overlap the coding regions of FOXF1 and ANKRD11, two genes that have been previously associated with the disease. 25 patients were reported to have ASD/ID, and three were found to have bilateral hydronephrosis. 14 of the deletions associated with ASD/ID overlap the coding regions of FBXO31 and MAP1LC3B. These same genes and two others, C16orf95 and ZCCHC14, are also included in the area of minimal overlap of the three deletions associated with hydronephrosis. CONCLUSIONS: Our data highlight 16q24.2 as a region of interest for ASD, ID and congenital renal malformations. These conditions are associated, albeit without complete penetrance, with deletions affecting C16orf95, ZCCHC14, MAP1LC3B and FBXO31. The function of each gene in development and disease warrants further investigation.

Rare deletions at the neurexin 3 locus in autism spectrum disorder.

The three members of the human neurexin gene family, neurexin 1 (NRXN1), neurexin 2 (NRXN2), and neurexin 3 (NRXN3), encode neuronal adhesion proteins that have important roles in synapse development and function. In autism spectrum disorder (ASD), as well as in other neurodevelopmental conditions, rare exonic copy-number variants and/or point mutations have been identified in the NRXN1 and NRXN2 loci. We present clinical characterization of four index cases who have been diagnosed with ASD and who possess rare inherited or de novo microdeletions at 14q24.3-31.1, a region that overlaps exons of the alpha and/or beta isoforms of NRXN3. NRXN3 deletions were found in one father with subclinical autism and in a carrier mother and father without formal ASD diagnoses, indicating issues of penetrance and expressivity at this locus. Notwithstanding these clinical complexities, this report on ASD-affected individuals who harbor NRXN3 exonic deletions advances the understanding of the genetic etiology of autism, further enabling molecular diagnoses.

Migration of cells from the yolk sac to hematopoietic tissues after in utero transplantation of early and mid gestation canine fetuses.

BACKGROUND: In utero hematopoietic cell transplantation offers a means of early intervention for the treatment of diseases before birth. Delivery of cells to the yolk sac is a minimally invasive approach that results in low levels of chimerism. However, there is little information on the optimal doses, timing of delivery, and migration of transplanted cells from the yolk sac into the fetus. METHODS: Varying cell doses of mesenchymal stromal cells or bone marrow mononuclear cells labeled with fluorescent supraparamagnetic iron oxide nanoparticles and a fluorescent intracellular dye, 5- and 6-([(4-chloromethyl)benzoyl]-amino) tetramethylrhodamine, were transplanted under ultrasound guidance to the yolk sacs of day 25 or day 35 canine fetuses. Ex vivo whole body fluorescence imaging and microscopy of tissue sections were correlated with the presence of iron oxide in injected and control fetuses. RESULTS: Day 25 and day 35 recipients showed similar survival rates after injection of cells into yolk sacs, although increased fetal morality was associated with cell doses greater than 10 cells/kg to day 25 fetuses. The fluorescence and iron oxide signals were predominantly localized to the abdominal regions, with no fluorescence visible in yolk sacs. Microscopy of tissues revealed colocalization of fluorophore with iron oxide in donor cells detected in the fetal livers and bone marrow of recipients 7 and 17 days after receiving mesenchymal stromal cells or bone marrow mononuclear cells. CONCLUSIONS: These studies demonstrated that cells injected into the yolk sacs of early gestation canine fetuses migrate to recipient hematopoietic tissues. Thus, yolk sac injection offers a safe and effective approach for engraftment of cells to fetal hematopoietic tissues.

Long-term tracking of bone marrow progenitor cells following intracoronary injection post-myocardial infarction in swine using MRI.

Magnetic resonance imaging (MRI) can track progenitor cells following direct intramyocardial injection. However, in the vast majority of post-myocardial infarction (MI) clinical trials, cells are delivered by the intracoronary (IC) route, which results in far greater dispersion within the myocardium. Therefore, we assessed whether the more diffuse distribution of cells following IC delivery could be imaged longitudinally with MRI. In 11 pigs (7 active, 4 controls), MI was induced by 90-min balloon occlusion of the left anterior descending coronary artery. Seven (0) days [median (interquartile range)] following MI, bone marrow progenitor cells (BMCs) were colabeled with an iron-fluorophore and a cell viability marker and delivered to the left anterior descending coronary artery distal to an inflated over-the-wire percutaneous transluminal coronary angioplasty balloon. T2*-weighted images were used to assess the location of the magnetically labeled cells over a 6-wk period post-MI. Immediately following cell delivery, hypointensity characteristic of the magnetic label was observed in the infarct border rather than within the infarct itself. At 6 wk, the cell signal hypointensity persisted, albeit with significantly decreased intensity. BMC delivery resulted in significant improvement in infarct volume and ejection fraction (EF): infarct volume in cell-treated animals decreased from 7.1 +/- 1.5 to 4.9 +/- 1.0 ml (P < 0.01); infarct volume in controls was virtually unchanged at 4.64 +/- 2.1 to 4.39 +/- 2.1 ml (P = 0.7). EF in cell-treated animals went from 30.4 +/- 5.2% preinjection to 34.5 +/- 2.5% 6 wk postinjection (P = 0.013); EF in control animals went from 34.3 +/- 4.7 to 31.9 +/- 6.8% (P = 0.5). Immunohistochemical analysis revealed intracellular colocalization of the iron fluorophore and cell viability dye with the labeled cells continuing to express the same surface markers as at baseline. MRI can track the persistence and distribution of magnetically labeled BMCs over a 6-wk period following IC delivery. Signal hypointensity declines with time, particularly in the first week following delivery. These cells maintain their original phenotype during this time course. Delivery of these cells appears safe and results in improvement in infarct size and left ventricular ejection fraction.

Derivation and characterization of canine embryonic stem cell lines with in vitro and in vivo differentiation potential.

Embryonic stem cells (ESCs) represent permanent cell lines that can be maintained in an undifferentiated state. In an environment that induces differentiation, they form derivatives of the three embryonic germ layers: mesoderm, ectoderm, and endoderm. These characteristics give ESCs great potential for both basic research and clinical applications in the areas of regenerative medicine and tissue engineering. The establishment of ESCs from large animals that model human diseases is of significant importance. We describe the derivation of permanent canine cell lines from preimplantation-stage embryos. Similar to human ESCs, canine ESCs expressed OCT3/4, NANOG, SOX2, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase, whereas they expressed very low levels of SSEA-1. They maintained a normal karyotype and morphology typical of undifferentiated ESCs after multiple in vitro passages and rounds of cryopreservation. Plating cells in the absence of a feeder layer, either in attachment or suspension culture, resulted in the formation of embryoid bodies and their differentiation to multiple cell types. In vivo, canine ESCs gave rise to teratomas comprising cell types of all three embryonic germ layers. These cells represent the first pluripotent canine ESC lines with both in vitro and in vivo differentiation potential and offer the exciting possibility of testing the efficacy and safety of ESC-based therapies in large animal models of human disease.

HIV TAT variants differentially influence the production of glucocerebrosidase in Sf9 cells.

Gaucher disease, the most common lysosomal storage disorder, is currently treated with enzyme replacement therapy. This approach, however, is ineffective in altering the progression of neurodegeneration in type 2 and type 3 patients due to the difficulty of transferring the recombinant enzyme across the blood-brain barrier. Human immunodeficiency virus type 1 trans-activating transcriptional activator protein (HIV TAT) contains a protein transduction domain that can be added to a fusion protein partner to allow for transport of the partner across membranes. Consequently, we examined the creation, production, and secretion of fusion constructs containing glucocerebrosidase and either wild-type TAT or modified TAT in Sf9 cells. All three constructs exhibited successful expression, with wild-type TAT chimeras showing lower levels of expression than modified TAT chimeras.