Publications by authors named "Neal Sondheimer"

56 Publications

Nuclear genome-wide associations with mitochondrial heteroplasmy.

Sci Adv 2021 Mar 17;7(12). Epub 2021 Mar 17.

Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada.

The role of the nuclear genome in maintaining the stability of the mitochondrial genome (mtDNA) is incompletely known. mtDNA sequence variants can exist in a state of heteroplasmy, which denotes the coexistence of organellar genomes with different sequences. Heteroplasmic variants that impair mitochondrial capacity cause disease, and the state of heteroplasmy itself is deleterious. However, mitochondrial heteroplasmy may provide an intermediate state in the emergence of novel mitochondrial haplogroups. We used genome-wide genotyping data from 982,072 European ancestry individuals to evaluate variation in mitochondrial heteroplasmy and to identify the regions of the nuclear genome that affect it. Age, sex, and mitochondrial haplogroup were associated with the extent of heteroplasmy. GWAS identified 20 loci for heteroplasmy that exceeded genome-wide significance. This included a region overlapping mitochondrial transcription factor A (), which has multiple roles in mtDNA packaging, replication, and transcription. These results show that mitochondrial heteroplasmy has a heritable nuclear component.
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http://dx.doi.org/10.1126/sciadv.abe7520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7968846PMC
March 2021

Characterization of mitochondrial health from human peripheral blood mononuclear cells to cerebral organoids derived from induced pluripotent stem cells.

Sci Rep 2021 Feb 25;11(1):4523. Epub 2021 Feb 25.

Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, M5S 1A8, Canada.

Mitochondrial health plays a crucial role in human brain development and diseases. However, the evaluation of mitochondrial health in the brain is not incorporated into clinical practice due to ethical and logistical concerns. As a result, the development of targeted mitochondrial therapeutics remains a significant challenge due to the lack of appropriate patient-derived brain tissues. To address these unmet needs, we developed cerebral organoids (COs) from induced pluripotent stem cells (iPSCs) derived from human peripheral blood mononuclear cells (PBMCs) and monitored mitochondrial health from the primary, reprogrammed and differentiated stages. Our results show preserved mitochondrial genetics, function and treatment responses across PBMCs to iPSCs to COs, and measurable neuronal activity in the COs. We expect our approach will serve as a model for more widespread evaluation of mitochondrial health relevant to a wide range of human diseases using readily accessible patient peripheral (PBMCs) and stem-cell derived brain tissue samples.
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http://dx.doi.org/10.1038/s41598-021-84071-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907388PMC
February 2021

A pathogenic UFSP2 variant in an autosomal recessive form of pediatric neurodevelopmental anomalies and epilepsy.

Genet Med 2021 Jan 20. Epub 2021 Jan 20.

Children's Medical Center Research Institute, UT Southwestern Medical Center, Dallas, TX, USA.

Purpose: Neurodevelopmental disabilities are common and genetically heterogeneous. We identified a homozygous variant in the gene encoding UFM1-specific peptidase 2 (UFSP2), which participates in the UFMylation pathway of protein modification. UFSP2 variants are implicated in autosomal dominant skeletal dysplasias, but not neurodevelopmental disorders. Homozygosity for the variant occurred in eight children from four South Asian families with neurodevelopmental delay and epilepsy. We describe the clinical consequences of this variant and its effect on UFMylation.

Methods: Exome sequencing was used to detect potentially pathogenic variants and identify shared regions of homozygosity. Immunoblotting assessed protein expression and post-translational modifications in patient-derived fibroblasts.

Results: The variant (c.344T>A; p.V115E) is rare and alters a conserved residue in UFSP2. Immunoblotting in patient-derived fibroblasts revealed reduced UFSP2 abundance and increased abundance of UFMylated targets, indicating the variant may impair de-UFMylation rather than UFMylation. Reconstituting patient-derived fibroblasts with wild-type UFSP2 reduced UFMylation marks. Analysis of UFSP2's structure indicated that variants observed in skeletal disorders localize to the catalytic domain, whereas V115 resides in an N-terminal domain possibly involved in substrate binding.

Conclusion: Different UFSP2 variants cause markedly different diseases, with homozygosity for V115E causing a severe syndrome of neurodevelopmental disability and epilepsy.
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http://dx.doi.org/10.1038/s41436-020-01071-zDOI Listing
January 2021

The mitochondrial genome of .

Mitochondrial DNA B Resour 2020 May 22;5(3):2147-2148. Epub 2020 May 22.

Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Canada.

is a wild guinea pig found throughout South America. The previously published mitochondrial sequence for was highly divergent from the sequence and contained stop codons within open reading frames. Here we resequenced the mitochondrial genomes of and . Both sequences reflect gene organization typical for mammalian mitochondrial DNA. Our mtDNA sequence shows that all of the open reading frames are intact, but confirms the strikingly low level of sequence identity (92.7%) with the closely related mtDNA.
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http://dx.doi.org/10.1080/23802359.2020.1768918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782685PMC
May 2020

Specifications of the ACMG/AMP standards and guidelines for mitochondrial DNA variant interpretation.

Hum Mutat 2020 Dec 10;41(12):2028-2057. Epub 2020 Nov 10.

Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.

Mitochondrial DNA (mtDNA) variant pathogenicity interpretation has special considerations given unique features of the mtDNA genome, including maternal inheritance, variant heteroplasmy, threshold effect, absence of splicing, and contextual effects of haplogroups. Currently, there are insufficient standardized criteria for mtDNA variant assessment, which leads to inconsistencies in clinical variant pathogenicity reporting. An international working group of mtDNA experts was assembled within the Mitochondrial Disease Sequence Data Resource Consortium and obtained Expert Panel status from ClinGen. This group reviewed the 2015 American College of Medical Genetics and Association of Molecular Pathology standards and guidelines that are widely used for clinical interpretation of DNA sequence variants and provided further specifications for additional and specific guidance related to mtDNA variant classification. These Expert Panel consensus specifications allow for consistent consideration of the unique aspects of the mtDNA genome that directly influence variant assessment, including addressing mtDNA genome composition and structure, haplogroups and phylogeny, maternal inheritance, heteroplasmy, and functional analyses unique to mtDNA, as well as specifications for utilization of mtDNA genomic databases and computational algorithms.
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http://dx.doi.org/10.1002/humu.24107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717623PMC
December 2020

Homozygous GLUL deletion is embryonically viable and leads to glutamine synthetase deficiency.

Clin Genet 2020 Dec 1;98(6):613-619. Epub 2020 Oct 1.

Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, University of Toronto, Toronto, Canada.

Glutamine synthetase (GS) is the enzyme responsible for the biosynthesis of glutamine, providing the only source of endogenous glutamine necessary for several critical metabolic and developmental pathways. GS deficiency, caused by pathogenic variants in the glutamate-ammonia ligase (GLUL) gene, is a rare autosomal recessive inborn error of metabolism characterized by systemic glutamine deficiency, persistent moderate hyperammonemia, and clinically devastating seizures and multi-organ failure shortly after birth. The four cases reported thus far were caused by homozygous GLUL missense variants. We report a case of GS deficiency caused by homozygous GLUL gene deletion, diagnosed prenatally and likely representing the most severe end of the spectrum. We expand the known phenotype of this rare condition with novel dysmorphic, radiographic and neuropathologic features identified on post-mortem examination. The biallelic deletion identified in this case also included the RNASEL gene and was associated with immune dysfunction in the fetus. This case demonstrates that total absence of the GLUL gene in humans is viable beyond the embryonic period, despite the early embryonic lethality found in GLUL animal models.
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http://dx.doi.org/10.1111/cge.13844DOI Listing
December 2020

Rod bipolar cell dysfunction in POLG retinopathy.

Doc Ophthalmol 2021 Feb 21;142(1):111-118. Epub 2020 Jun 21.

Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Canada.

Objective: To report the clinical and novel electrophysiological features in a child with POLG-related sensory ataxic neuropathy, dysarthria and ophthalmoparesis (SANDO).

Methods: The proband, a male child of Indian descent, underwent serial systemic and ophthalmological evaluations from birth until 14 years of age. Eye examinations included visual acuity and extraocular movement assessments, fundus photography, spectral domain optical coherence tomography and full-field electroretinography (ERG). Detailed genetic testing was also performed.

Results: The child carried a homozygous mutation in POLG (c.911T > G/p.Leu304Arg) and manifested systemic features such as seizures, headaches, areflexia, hypotonia, myopathy and vomiting. The child's distance visual acuity was 0.50 and 0.40 LogMAR in the right and left eyes, respectively. Bilateral ophthalmoplegia and ptosis were observed at 5 years of age. The dark-adapted (DA) ERG responses to 2.29 cd s m and 7.6 cd s m stimuli showed a markedly reduced b/a ratio; an electronegative configuration was noted to a DA 7.6 ERG.

Conclusion: This is the first documented case of an electronegative ERG in a POLG-related disorder consistent with generalized rod ON-bipolar dysfunction. The rest of the proband's systemic and ophthalmological features were consistent with SANDO but some features overlapped with other POLG-related disorders such as Alpers-Huttenlocher syndrome and autosomal dominant progressive external ophthalmoplegia demonstrating the wide phenotypic overlap expected due to POLG mutations.
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http://dx.doi.org/10.1007/s10633-020-09777-wDOI Listing
February 2021

Utility of metabolic screening in neurological presentations of infancy.

Ann Clin Transl Neurol 2020 07 4;7(7):1132-1140. Epub 2020 Jun 4.

Division of Neurology, Hospital for Sick Children, Toronto, ON, Canada.

Background: The first-line use of specialized metabolic screening laboratories in the investigation of hypotonia and/or developmental delay remains a standard practice despite lack of supporting evidence. Our study aimed to address the utility of such testing by determining the proportion of patients whose diagnosis was directly supported by metabolic screening.

Methods: We performed a retrospective chart review study of 164 patients under age one who had screening metabolic laboratory testing done within the time period of one calendar year.

Results: Of patients screened, 9/164 (5.5%) had diagnoses supported by metabolic testing (two with nonketotic hyperglycinemia, three with ornithine transcarbamylase deficiency, one with propionic acidemia, one with a congenital disorder of glycosylation, one with D-bifunctional protein deficiency, and one with GM1 Gangliosidosis). Of patients specifically evaluated for hypotonia and/or developmental delay, 5/79 (6.3%) were diagnosed with the aid of metabolic testing. All patients with positive screens presented with acute decompensation. Outside of this subgroup of high-risk patients, no patients were diagnosed using metabolic testing. Screening laboratories were also ineffective in an outpatient setting, identifying only one of the seven outpatients who was ultimately diagnosed with an inborn error of metabolism.

Conclusions: These findings demonstrate that the yield of specialized metabolic screening testing is extremely low in infants with hypotonia and/or developmental delay, approaching zero outside of the specific setting of clinical decompensation or multi-system involvement. Furthermore, many outpatient cases of IEM are not identified by screening studies. This information will help guide the diagnostic evaluation of hypotonia and/or global developmental delay.
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http://dx.doi.org/10.1002/acn3.51076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359104PMC
July 2020

Liver transplantation for Gaucher disease presenting as neonatal cholestasis: Case report and literature review.

Pediatr Transplant 2020 06 23;24(4):e13718. Epub 2020 Apr 23.

Department of Pediatrics, Kingston Health Sciences Center, Queen's University, Kingston, ON, Canada.

Background: We present a rare case of neonatal cholestasis in a female infant with Gaucher Disease (GD), who received liver transplantation. We review the relevant literature on similar disease presentations.

Methods: A chart review of the index case was performed. PubMed and Medline databases were searched to identify other cases.

Results: A 4-day-old female was referred with conjugated hyperbilirubinemia. Physical examination revealed icterus with hepatosplenomegaly and normal neurologic examination. The diagnosis of GD was confirmed through liver biopsy, low glucocerebrosidase enzyme activity, and two pathogenic mutations in GBA gene. Despite early initiation of ERT, the patient had worsening of her liver failure and underwent a left lateral segment liver transplant from a living donor at 7 months of age. She experienced improvement of her liver enzymes and coagulation, but passed away at 8 months due to the late onset of neurologic involvement. Nine other cases of GD presenting with neonatal cholestasis have been reported. Forty-four percent (4/9) of cases received ERT and none were considered for transplant. Overall, the literature suggests a poor prognosis with death reported in 77% (7/9) cases.

Conclusions: Neonatal presentation of GD represents a poor prognosis despite early initiation of treatment. Diagnosis remains a challenge as the presentation is rare and multiple tests such as BM biopsy, liver biopsy with both light and electron microscopy, enzymology, and genetic testing may need to be completed to reach a diagnosis. Neurological sequelae may manifest later making the decision to proceed with liver transplantation a difficult one.
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http://dx.doi.org/10.1111/petr.13718DOI Listing
June 2020

Evaluation of the quality of clinical data collection for a pan-Canadian cohort of children affected by inherited metabolic diseases: lessons learned from the Canadian Inherited Metabolic Diseases Research Network.

Orphanet J Rare Dis 2020 04 10;15(1):89. Epub 2020 Apr 10.

Health Sciences Centre Winnipeg, University of Manitoba, Winnipeg, Manitoba, Canada.

Background: The Canadian Inherited Metabolic Diseases Research Network (CIMDRN) is a pan-Canadian practice-based research network of 14 Hereditary Metabolic Disease Treatment Centres and over 50 investigators. CIMDRN aims to develop evidence to improve health outcomes for children with inherited metabolic diseases (IMD). We describe the development of our clinical data collection platform, discuss our data quality management plan, and present the findings to date from our data quality assessment, highlighting key lessons that can serve as a resource for future clinical research initiatives relating to rare diseases.

Methods: At participating centres, children born from 2006 to 2015 who were diagnosed with one of 31 targeted IMD were eligible to participate in CIMDRN's clinical research stream. For all participants, we collected a minimum data set that includes information about demographics and diagnosis. For children with five prioritized IMD, we collected longitudinal data including interventions, clinical outcomes, and indicators of disease management. The data quality management plan included: design of user-friendly and intuitive clinical data collection forms; validation measures at point of data entry, designed to minimize data entry errors; regular communications with each CIMDRN site; and routine review of aggregate data.

Results: As of June 2019, CIMDRN has enrolled 798 participants of whom 764 (96%) have complete minimum data set information. Results from our data quality assessment revealed that potential data quality issues were related to interpretation of definitions of some variables, participants who transferred care across institutions, and the organization of information within the patient charts (e.g., neuropsychological test results). Little information was missing regarding disease ascertainment and diagnosis (e.g., ascertainment method - 0% missing).

Discussion: Using several data quality management strategies, we have established a comprehensive clinical database that provides information about care and outcomes for Canadian children affected by IMD. We describe quality issues and lessons for consideration in future clinical research initiatives for rare diseases, including accurately accommodating different clinic workflows and balancing comprehensiveness of data collection with available resources. Integrating data collection within clinical care, leveraging electronic medical records, and implementing core outcome sets will be essential for achieving sustainability.
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http://dx.doi.org/10.1186/s13023-020-01358-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149838PMC
April 2020

Higher Order Organization of the mtDNA: Beyond Mitochondrial Transcription Factor A.

Front Genet 2019 20;10:1285. Epub 2019 Dec 20.

Institute of Medical Sciences and the Department of Paediatrics, The University of Toronto, Toronto, ON, Canada.

The higher order organization of eukaryotic and prokaryotic genomes is pivotal in the regulation of gene expression. Specifically, chromatin accessibility in eukaryotes and nucleoid accessibility in bacteria are regulated by a cohort of proteins to alter gene expression in response to diverse physiological conditions. By contrast, prior studies have suggested that the mitochondrial genome (mtDNA) is coated solely by mitochondrial transcription factor A (TFAM), whose increased cellular concentration was proposed to be the major determinant of mtDNA packaging in the mitochondrial nucleoid. Nevertheless, recent analysis of DNase-seq and ATAC-seq experiments from multiple human and mouse samples suggest gradual increase in mtDNA occupancy during the course of embryonic development to generate a conserved footprinting pattern which correlate with sites that have low TFAM occupancy (ChIP-seq) and tend to adopt G-quadruplex structures. These findings, along with recent identification of mtDNA binding by known modulators of chromatin accessibility such as MOF, suggest that mtDNA higher order organization is generated by cross talk with the nuclear regulatory system, may have a role in mtDNA regulation, and is more complex than once thought.
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http://dx.doi.org/10.3389/fgene.2019.01285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961661PMC
December 2019

Whole-exome sequencing identifies a homozygous pathogenic variant in in a girl with palmoplantar keratoderma.

Mol Genet Metab Rep 2019 Dec 22;21:100534. Epub 2019 Nov 22.

Clinical Genetics, Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Canada.

Palmoplantar keratoderma (PPK) is a defect in cornification that is characterized by progressive hyperkeratosis of palms and soles. Many phenotypes are linked with PPK, making exome-based diagnosis increasingly efficient. In this report, we identified tyrosinemia type II on whole-exome sequencing in a 7-year-old Syrian refugee that presented with PPK. Dietary therapy helped improve her overall symptoms.
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http://dx.doi.org/10.1016/j.ymgmr.2019.100534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881597PMC
December 2019

Homozygous pathogenic variant in associated with nonprogressive cerebellar ataxia.

Neurol Genet 2019 Oct 4;5(5):e359. Epub 2019 Sep 4.

Division of Neurology (A.M., G.Y.), Department of Paediatrics, University of Toronto, The Hospital for Sick Children, Canada; Department of Genome Dynamics (Z.C., K.W.C., H.H.), Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic; Faculty of Science (Z.C.), Charles University in Prague, Czech Republic; Department of Neuroscience (M.T.), Université de Montréal, CHUM, Montréal, Québec, Canada; Department of Paediatric Laboratory Medicine (L.M.), Hospital for Sick Children; Department of Lab Medicine and Pathobiology (L.M.), University of Toronto, Ontario, Canada; Program in Genetics and Genome Biology (N.S.), SickKids Research Institute, Toronto, Ontario, Canada; Division of Clinical and Metabolic Genetics (N.S., G.Y.), Department of Paediatrics, University of Toronto, The Hospital for Sick Children, Toronto, Canada; and Genome Damage and Stability Centre (K.W.C., H.H.), School of Life Sciences, University of Sussex, Falmer, Brighton, UK.

Objective: To investigate the pathogenicity of a novel homozygous variant in 2 siblings with nonprogressive cerebellar ataxia (NPCA) through functional studies on primary and immortalized patient cell lines.

Methods: BRAT1 protein levels and ataxia-telangiectasia mutated (ATM) kinase activity in patient-derived and control cell lines were assessed by Western blotting. The impact of the novel variants on mitochondrial function was also assessed, by comparing patient and control cell lines for rates of oxygen consumption and for phosphorylation (S293) of the E1⍺ subunit of pyruvate dehydrogenase (PDH).

Results: Two male siblings with NPCA, mild intellectual disability, and isolated cerebellar atrophy were found to be homozygous for a c.185T>A (p.Val62Glu) variant in by whole exome sequencing. Western blotting revealed markedly decreased BRAT1 protein levels in lymphocytes and/or fibroblast cells from both affected siblings compared to control cell lines. There were no differences between the patient and control cells in ATM kinase activation, following ionizing radiation. Mitochondrial studies were initially suggestive of a defect in regulation of PDH activity, but there was no evidence of increased phosphorylation of the E1⍺ subunit of the PDH complex. Measurement of oxygen consumption rates similarly failed to identify differences between patient and control cells.

Conclusions: Biallelic pathogenic variants in can be associated with NPCA, a phenotype considerably milder than previously reported. Surprisingly, despite the molecular role currently proposed for BRAT1 in ATM regulation, this disorder is unlikely to result from defective ATM kinase or mitochondrial dysfunction.
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http://dx.doi.org/10.1212/NXG.0000000000000359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773431PMC
October 2019

Contribution of a mitochondrial tyrosyl-tRNA synthetase mutation to the phenotypic expression of the deafness-associated tRNA 7511A>G mutation.

J Biol Chem 2019 12 4;294(50):19292-19305. Epub 2019 Nov 4.

Division of Medical Genetics and Genomics, Children's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China

Nuclear modifier genes have been proposed to modify the phenotypic expression of mitochondrial DNA mutations. Using a targeted exome-sequencing approach, here we found that the p.191Gly>Val mutation in mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) interacts with the tRNA 7511A>G mutation in causing deafness. Strikingly, members of a Chinese family bearing both the YARS2 p.191Gly>Val and m.7511A>G mutations displayed much higher penetrance of deafness than those pedigrees carrying only the m.7511A>G mutation. The m.7511A>G mutation changed the A4:U69 base-pairing to G4:U69 pairing at the aminoacyl acceptor stem of tRNA and perturbed tRNA structure and function, including an increased melting temperature, altered conformation, instability, and aberrant aminoacylation of mutant tRNA. Using lymphoblastoid cell lines derived from symptomatic and asymptomatic members of these Chinese families and control subjects, we show that cell lines harboring only the m.7511A>G or p.191Gly>Val mutation revealed relatively mild defects in tRNA or tRNA metabolism, respectively. However, cell lines harboring both m.7511A>G and p.191Gly>Val mutations displayed more severe defective aminoacylations and lower tRNA and tRNA levels, aberrant aminoacylation, and lower levels of other tRNAs, including tRNA, tRNA, tRNA, and tRNA, than those in the cell lines carrying only the m.7511A>G or p.191Gly>Val mutation. Furthermore, mutant cell lines harboring both m.7511A>G and p.191Gly>Val mutations exhibited greater decreases in the levels of mitochondrial translation, respiration, and mitochondrial ATP and membrane potentials, along with increased production of reactive oxygen species. Our findings provide molecular-level insights into the pathophysiology of maternally transmitted deafness arising from the synergy between tRNA and mitochondrial YARS mutations.
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http://dx.doi.org/10.1074/jbc.RA119.010598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916496PMC
December 2019

Heteroplasmy Shifting as Therapy for Mitochondrial Disorders.

Adv Exp Med Biol 2019 ;1158:257-267

Institute of Medical Science, The University of Toronto, Toronto, ON, Canada.

Mitochondrial disease can arise due to pathogenic sequence variants in the mitochondrial DNA (mtDNA) that prevent cells from meeting their energy demands. Mitochondrial diseases are often fatal and currently there are no treatments directed towards the underlying cause of disease. Pathogenic variants in mtDNA often exist in a state of heteroplasmy, with coexistence of pathogenic and wild type mtDNA. The load of heteroplasmy, defined as the relative amount of pathogenic mtDNA to wild type mtDNA, corresponds to timing and symptom severity. Thus, changing the heteroplasmy load may lead to a shift in disease onset and symptom severity. Here we review techniques aimed at preventing inheritance of pathogenic mtDNA via mitochondrial replacement therapy (MRT) and strategies geared toward shifting of heteroplasmy in individuals with active mitochondrial disease. MRT strategies seek to create embryos with the nuclear genetic makeup of the intended parents and wild type mtDNA from a donor in order to avoid known maternal pathogenic variants. Heteroplasmy shift approaches in patients are of two categories: nuclease dependent and nuclease independent strategies. Despite initial success in mouse models and patient cells, these techniques have not reached clinical use. Translational attempts in this area are urgently needed to improve therapies for a currently untreatable set of disorders.
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http://dx.doi.org/10.1007/978-981-13-8367-0_14DOI Listing
September 2019

Health Care for Mitochondrial Disorders in Canada: A Survey of Physicians.

Can J Neurol Sci 2019 11;46(6):717-726

School of Epidemiology & Public Health, University of Ottawa, Ottawa, Ontario, Canada.

Background: An improved understanding of diagnostic and treatment practices for patients with rare primary mitochondrial disorders can support benchmarking against guidelines and establish priorities for evaluative research. We aimed to describe physician care for patients with mitochondrial diseases in Canada, including variation in care.

Methods: We conducted a cross-sectional survey of Canadian physicians involved in the diagnosis and/or ongoing care of patients with mitochondrial diseases. We used snowball sampling to identify potentially eligible participants, who were contacted by mail up to five times and invited to complete a questionnaire by mail or internet. The questionnaire addressed: personal experience in providing care for mitochondrial disorders; diagnostic and treatment practices; challenges in accessing tests or treatments; and views regarding research priorities.

Results: We received 58 survey responses (52% response rate). Most respondents (83%) reported spending 20% or less of their clinical practice time caring for patients with mitochondrial disorders. We identified important variation in diagnostic care, although assessments frequently reported as diagnostically helpful (e.g., brain magnetic resonance imaging, MRI/MR spectroscopy) were also recommended in published guidelines. Approximately half (49%) of participants would recommend "mitochondrial cocktails" for all or most patients, but we identified variation in responses regarding specific vitamins and cofactors. A majority of physicians recommended studies on the development of effective therapies as the top research priority.

Conclusions: While Canadian physicians' views about diagnostic care and disease management are aligned with published recommendations, important variations in care reflect persistent areas of uncertainty and a need for empirical evidence to support and update standard protocols.
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http://dx.doi.org/10.1017/cjn.2019.240DOI Listing
November 2019

G-quadruplex-mediated reduction of a pathogenic mitochondrial heteroplasmy.

Hum Mol Genet 2019 10;28(19):3163-3174

Institute of Medical Science.

Disease-associated variants in mitochondrial DNA (mtDNA) are frequently heteroplasmic, a state of co-existence with the wild-type genome. Because heteroplasmy correlates with the severity and penetrance of disease, improvement in the ratio between these genomes in favor of the wild-type, known as heteroplasmy shifting, is potentially therapeutic. We evaluated known pathogenic mtDNA variants and identified those with the potential for allele-specific differences in the formation of non-Watson-Crick G-quadruplex (GQ) structures. We found that the Leigh syndrome (LS)-associated m.10191C variant promotes GQ formation within local sequence in vitro. Interaction of this sequence with a small molecule GQ-binding agent, berberine hydrochloride, further increased GQ stability. The GQ formed at m.10191C differentially impeded the processivity of the mitochondrial DNA polymerase gamma (Pol γ) in vitro, providing a potential means to favor replication of the wild-type allele. We tested the potential for shifting heteroplasmy through the cyclical application of two different mitochondria-targeted GQ binding compounds in primary fibroblasts from patients with m.10191T>C heteroplasmy. Treatment induced alternating mtDNA depletion and repopulation and was effective in shifting heteroplasmy towards the non-pathogenic allele. Similar treatment of pathogenic heteroplasmies that do not affect GQ formation did not induce heteroplasmy shift. Following treatment, heteroplasmic m.10191T>C cells had persistent improvements and heteroplasmy and a corresponding increase in maximal mitochondrial oxygen consumption. This study demonstrates the potential for using small-molecule GQ-binding agents to induce genetic and functional improvements in m.10191T>C heteroplasmy.
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http://dx.doi.org/10.1093/hmg/ddz153DOI Listing
October 2019

Pyruvate carboxylase deficiency type A and type C: Characterization of five novel pathogenic variants in PC and analysis of the genotype-phenotype correlation.

Hum Mutat 2019 06 13;40(6):816-827. Epub 2019 Apr 13.

Department of Pediatrics, Klinikum Reutlingen, Reutlingen, Germany.

Pyruvate carboxylase deficiency (PCD) is caused by biallelic mutations of the PC gene. The reported clinical spectrum includes a neonatal form with early death (type B), an infantile fatal form (type A), and a late-onset form with isolated mild intellectual delay (type C). Apart from homozygous stop-codon mutations leading to type B PCD, a genotype-phenotype correlation has not otherwise been discernible. Indeed, patients harboring biallelic heterozygous variants leading to PC activity near zero can present either with a fatal infantile type A or with a benign late onset type C form. In this study, we analyzed six novel patients with type A (three) and type C (three) PCD, and compared them with previously reported cases. First, we observed that type C PCD is not associated to homozygous variants in PC. In silico modeling was used to map former and novel variants associated to type A and C PCD, and to predict their potential effects on the enzyme structure and function. We found that variants lead to type A or type C phenotype based on the destabilization between the two major enzyme conformers. In general, our study on novel and previously reported patients improves the overall understanding on type A and C PCD.
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http://dx.doi.org/10.1002/humu.23742DOI Listing
June 2019

An Infant Refugee with Anemia and Low Serum Vitamin B.

Clin Chem 2018 11;64(11):1567-1570

Division of Clinical and Biochemical Genetics, The Hospital for Sick Children, Toronto, Canada;

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http://dx.doi.org/10.1373/clinchem.2017.283283DOI Listing
November 2018

DNAJC12-associated developmental delay, movement disorder, and mild hyperphenylalaninemia identified by whole-exome sequencing re-analysis.

Eur J Hum Genet 2018 12 23;26(12):1867-1870. Epub 2018 Aug 23.

Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada.

Hyperphenylalaninemia, movement disorder, and intellectual disability due to variants in DNAJC12 is a recently reported inherited neurotransmitter disorder. We report two new patients with this new genetic disorder. Patient 1 is a 6-year-11-month-old boy with mild hyperphenylalaninemia and global developmental delay (GDD). Seventeen-year-old male sibling of patient 1 had GDD from the first year of life. He had mild hyperphenylalaninemia at 11.5 years of age following his younger brother's diagnosis. He had low levels of homovanillic acid and 5-hydroxyindolacetic acid in the cerebrospinal fluid. Whole-exome sequencing (WES) was normal in 2016. After the first description of DNAJC12-associated hyperphenylalaninemia, dystonia, and intellectual disability in 2017, WES re-analysis identified a homozygous c.58_59delGG (p.(Gly20Metfs*2)) variant in DNAJC12. His younger brother was homozygous for the same variant, confirming the diagnosis of DNAJC12-associated hyperphenylalaninemia, movement disorder, and intellectual disability. Mild hyperphenylalaninemia and GDD should warrant targeted DNAJC12 genetic testing for the early diagnosis of DNAJC12-associated hyperphenylalaninemia, movement disorder, and intellectual disability.
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http://dx.doi.org/10.1038/s41431-018-0237-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6244406PMC
December 2018

Reply.

Environ Mol Mutagen 2019 06 14;60(5):465. Epub 2018 Mar 14.

Department of Psychiatry, Division of Behavioral Medicine, Columbia University Medical Center, New York, NY, 10032.

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http://dx.doi.org/10.1002/em.22181DOI Listing
June 2019

Periodic reanalysis of whole-genome sequencing data enhances the diagnostic advantage over standard clinical genetic testing.

Eur J Hum Genet 2018 05 16;26(5):740-744. Epub 2018 Feb 16.

Genome Diagnostics, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, ON, Canada.

Whole-genome sequencing (WGS) as a first-tier diagnostic test could transform medical genetic assessments, but there are limited data regarding its clinical use. We previously showed that WGS could feasibly be deployed as a single molecular test capable of a higher diagnostic rate than current practices, in a prospectively recruited cohort of 100 children meeting criteria for chromosomal microarray analysis. In this study, we report on the added diagnostic yield with re-annotation and reanalysis of these WGS data ~2 years later. Explanatory variants have been discovered in seven (10.9%) of 64 previously undiagnosed cases, in emerging disease genes like HMGA2. No new genetic diagnoses were made by any other method in the interval period as part of ongoing clinical care. The results increase the cumulative diagnostic yield of WGS in the study cohort to 41%. This represents a greater than 5-fold increase over the chromosomal microarrays, and a greater than 3-fold increase over all the clinical genetic testing ordered in practice. These findings highlight periodic reanalysis as yet another advantage of genomic sequencing in heterogeneous disorders. We recommend reanalysis of an individual's genome-wide sequencing data every 1-2 years until diagnosis, or sooner if their phenotype evolves.
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http://dx.doi.org/10.1038/s41431-018-0114-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945683PMC
May 2018

The Personal Genome Project Canada: findings from whole genome sequences of the inaugural 56 participants.

CMAJ 2018 02;190(5):E126-E136

The Centre for Applied Genomics (Reuter, Walker, Thiruvahindrapuram, Whitney, Yuen, Trost, Paton, Pereira, Herbrick, Wintle, Merico, Howe, MacDonald, Lu, Nalpathamkalam, Sung, Wang, Patel, Pellecchia, J. Wei, Strug, Bell, Kellam, Mahtani, Hosseini, Fiume, Marshall, Buchanan, Scherer); Divisions of Clinical Pharmacology and Toxicology (I. Cohn), or Clinical, and Metabolic Genetics (Sondheimer, Weksberg, Shuman, Bowdin, Meyn, Monfared), The Hospital for Sick Children; Departments of Paediatrics (Sondheimer, R. Cohn) and Molecular Genetics (Yuen, Weksberg, Shuman, R. Cohn, Ellis, Meyn), University of Toronto; Deep Genomics Inc. (Merico); Department of Psychiatry (Bassett), University Health Network and Centre for Addiction and Mental Health, University of Toronto; Li Ka Shing Knowledge Institute (Bombard), St. Michael's Hospital; Institute of Health Policy, Management and Evaluation (Bombard), University of Toronto; Centre for Genetic Medicine (Stavropoulos, Bowdin, Ray, Monfared); Molecular Genetics Laboratory (Stavropoulos, Ray, Marshall), Division of Genome Diagnostics, Paediatric Laboratory Medicine; Developmental and Stem Cell Biology (Hildebrandt, W. Wei, Romm, Pasceri, Ellis); Ted Rogers Cardiac Genome Clinic (Hosseini); Cytogenetics Laboratory (Joseph-George), Division of Genome Diagnostics, Paediatric Laboratory Medicine, The Hospital for Sick Children; Departments of Biochemistry and Laboratory Medicine, and Pathobiology (Keeley), University of Toronto; DNAstack (Cook, Fiume); McLaughlin Centre (Lee, Scherer), University of Toronto; Medcan Health Management Inc. (Davies, Hazell); Dalla Lana School of Public Health (Szego), Department of Family and Community Medicine, and The Joint Centre for Bioethics, University of Toronto; Centre for Clinical Ethics (Szego), St. Joseph's Health Centre, Toronto, Ont.

Background: The Personal Genome Project Canada is a comprehensive public data resource that integrates whole genome sequencing data and health information. We describe genomic variation identified in the initial recruitment cohort of 56 volunteers.

Methods: Volunteers were screened for eligibility and provided informed consent for open data sharing. Using blood DNA, we performed whole genome sequencing and identified all possible classes of DNA variants. A genetic counsellor explained the implication of the results to each participant.

Results: Whole genome sequencing of the first 56 participants identified 207 662 805 sequence variants and 27 494 copy number variations. We analyzed a prioritized disease-associated data set ( = 1606 variants) according to standardized guidelines, and interpreted 19 variants in 14 participants (25%) as having obvious health implications. Six of these variants (e.g., in or mosaic loss of an X chromosome) were pathogenic or likely pathogenic. Seven were risk factors for cancer, cardiovascular or neurobehavioural conditions. Four other variants - associated with cancer, cardiac or neurodegenerative phenotypes - remained of uncertain significance because of discrepancies among databases. We also identified a large structural chromosome aberration and a likely pathogenic mitochondrial variant. There were 172 recessive disease alleles (e.g., 5 individuals carried mutations for cystic fibrosis). Pharmacogenomics analyses revealed another 3.9 potentially relevant genotypes per individual.

Interpretation: Our analyses identified a spectrum of genetic variants with potential health impact in 25% of participants. When also considering recessive alleles and variants with potential pharmacologic relevance, all 56 participants had medically relevant findings. Although access is mostly limited to research, whole genome sequencing can provide specific and novel information with the potential of major impact for health care.
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http://dx.doi.org/10.1503/cmaj.171151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798982PMC
February 2018

Mitochondrial DNA, nuclear context, and the risk for carcinogenesis.

Environ Mol Mutagen 2019 06 14;60(5):455-462. Epub 2018 Jan 14.

Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, Ontario, M5G1X8, Canada.

The inheritance of mitochondrial DNA (mtDNA) from mother to child is complicated by differences in the stability of the mitochondrial genome. Although the germ line mtDNA is protected through the minimization of replication between generations, sequence variation can occur either through mutation or due to changes in the ratio between distinct genomes that are present in the mother (known as heteroplasmy). Thus, the unpredictability in transgenerational inheritance of mtDNA may cause the emergence of pathogenic mitochondrial and cellular phenotypes in offspring. Studies of the role of mitochondrial metabolism in cancer have a long and rich history, but recent evidence strongly suggests that changes in mitochondrial genotype and phenotype play a significant role in the initiation, progression and treatment of cancer. At the intersection of these two fields lies the potential for emerging mtDNA mutations to drive carcinogenesis in the offspring. In this review, we suggest that this facet of transgenerational carcinogenesis remains underexplored and is a potentially important contributor to cancer. Environ. Mol. Mutagen. 60:455-462, 2019. © 2018 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/em.22169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045969PMC
June 2019

Divergent Patterns of Mitochondrial and Nuclear Ancestry Are Associated with the Risk for Preterm Birth.

J Pediatr 2018 03 14;194:40-46.e4. Epub 2017 Dec 14.

Department of Genetics, The University of Pennsylvania, Philadelphia, PA; Department of Pediatrics, The University of Toronto, Toronto, Ontario, Canada. Electronic address:

Objective: To examine linkages between mitochondrial genetics and preterm birth by assessing the risk for preterm birth associated with the inheritance of nuclear haplotypes that are ancestrally distinct from mitochondrial haplogroup.

Study Design: Genome-wide genotyping studies of cohorts of preterm and term individuals were evaluated. We determined the mitochondrial haplogroup and nuclear ancestry for individuals and developed a scoring for the degree to which mitochondrial ancestry is divergent from nuclear ancestry.

Results: Infants with higher degrees of divergent mitochondrial ancestry were at increased risk for preterm birth (0.124 for preterm vs 0.105 for term infants; P< .05). This finding was validated in 1 of 2 replication cohorts. We also observed that greater degrees of divergent ancestry correlated with earlier delivery within the primary study population, but this finding was not replicated in secondary cohorts born preterm.

Conclusions: Individuals with divergent patterns of mitochondrial and nuclear ancestry are at increased risk for preterm birth. These findings may in part explain the higher rates of preterm birth in African Americans and in individuals with a matrilineal family history of preterm birth.
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http://dx.doi.org/10.1016/j.jpeds.2017.10.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987530PMC
March 2018

Predicting the pathogenicity of novel variants in mitochondrial tRNA with MitoTIP.

PLoS Comput Biol 2017 12 11;13(12):e1005867. Epub 2017 Dec 11.

Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, Ontario, Canada.

Novel or rare variants in mitochondrial tRNA sequences may be observed after mitochondrial DNA analysis. Determining whether these variants are pathogenic is critical, but confirmation of the effect of a variant on mitochondrial function can be challenging. We have used available databases of benign and pathogenic variants, alignment between diverse tRNAs, structural information and comparative genomics to predict the impact of all possible single-base variants and deletions. The Mitochondrial tRNA Informatics Predictor (MitoTIP) is available through MITOMAP at www.mitomap.org. The source code for MitoTIP is available at www.github.com/sonneysa/MitoTIP.
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http://dx.doi.org/10.1371/journal.pcbi.1005867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5739504PMC
December 2017

DNM1L Variant Alters Baseline Mitochondrial Function and Response to Stress in a Patient with Severe Neurological Dysfunction.

Biochem Genet 2018 Apr 6;56(1-2):56-77. Epub 2017 Nov 6.

Department of Anesthesia and Pain Medicine, The Hospital for Sick Children, 555 University Avenue, Toronto, M5G 1X8, Canada.

Mitochondria play vital roles in brain development and neuronal activity, and mitochondrial dynamics (fission and fusion) maintain organelle function through the removal of damaged components. Dynamin-like protein-1 (DRP-1), encoded by DNM1L, is an evolutionarily conserved GTPase that mediates mitochondrial fission by surrounding the scission site in concentric ring-like structures via self-oligomerization, followed by GTPase-dependant constriction. Here, we describe the clinical characteristics and cellular phenotype of a patient with severe neurological dysfunction, possessing a homozygous DNM1L variant c.305C>T (p.T115M) in the GTPase domain. For comparative analysis, we also describe a previously identified heterozygous variant demonstrating a rapidly fatal neurocognitive phenotype (c.261dup/c.385:386del, p.W88M*9/E129K*6). Using patient-generated fibroblasts, we demonstrated both DNM1L variants undergo adverse alterations to mitochondrial structure and function, including impaired mitochondrial fission, reduced membrane potential, and lower oxidative capacity including an increased cellular level of reactive oxygen species (ROS) and dsDNA breaks. Mutation of DNM1L was also associated with impaired responses to oxidative stress, as treatment with hydrogen peroxide dramatically increased cellular ROS, with minimal exacerbation of already impaired mitochondrial function. Taken together, our observations indicate that homozygous p.T115M variant of DNM1L produces a neurological and neurodevelopmental phenotype, consistent with impaired mitochondrial architecture and function, through a diminished ability to oligomerize, which was most prevalent under oxidative stress.
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http://dx.doi.org/10.1007/s10528-017-9829-2DOI Listing
April 2018

Red Blood Cells Homeostatically Bind Mitochondrial DNA through TLR9 to Maintain Quiescence and to Prevent Lung Injury.

Am J Respir Crit Care Med 2018 02;197(4):470-480

1 Pulmonary, Allergy and Critical Care Division and.

Rationale: Potentially hazardous CpG-containing cell-free mitochondrial DNA (cf-mtDNA) is routinely released into the circulation and is associated with morbidity and mortality in critically ill patients. How the body avoids inappropriate innate immune activation by cf-mtDNA remains unknown. Because red blood cells (RBCs) modulate innate immune responses by scavenging chemokines, we hypothesized that RBCs may attenuate CpG-induced lung inflammation through direct scavenging of CpG-containing DNA.

Objectives: To determine the mechanisms of CpG-DNA binding to RBCs and the effects of RBC-mediated DNA scavenging on lung inflammation.

Methods: mtDNA on murine RBCs was measured under basal conditions and after systemic inflammation. mtDNA content on human RBCs from healthy control subjects and trauma patients was measured. Toll-like receptor 9 (TLR9) expression on RBCs and TLR9-dependent binding of CpG-DNA to RBCs were determined. A murine model of RBC transfusion after CpG-DNA-induced lung injury was used to investigate the role of RBC-mediated DNA scavenging in mitigating lung injury in vivo.

Measurements And Main Results: Under basal conditions, RBCs bind CpG-DNA. The plasma-to-RBC mtDNA ratio is low in naive mice and in healthy volunteers but increases after systemic inflammation, demonstrating that the majority of cf-mtDNA is RBC-bound under homeostatic conditions and that the unbound fraction increases during inflammation. RBCs express TLR9 and bind CpG-DNA through TLR9. Loss of TLR9-dependent RBC-mediated CpG-DNA scavenging increased lung injury in vivo.

Conclusions: RBCs homeostatically bind mtDNA, and RBC-mediated DNA scavenging is essential in mitigating lung injury after CpG-DNA. Our data suggest a role for RBCs in regulating lung inflammation during disease states where cf-mtDNA is elevated, such as sepsis and trauma.
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http://dx.doi.org/10.1164/rccm.201706-1161OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821907PMC
February 2018

Topological requirements of the mitochondrial heavy-strand promoters.

Transcription 2017 25;8(5):307-312. Epub 2017 Aug 25.

b The Hospital for Sick Children , Toronto , ON , Canada.

In vitro studies of mitochondrial transcription often use linear templates that fail to replicate key features of transcription on a circular genome. We developed a plasmid-based system for the analysis of heavy-strand promoters that recapitulates key features of native mtDNA to study topological and protein requirements of promoter activation. The heavy-strand promoters (HSP1 and HSP2) are simultaneously active on a circular template. HSP2 requires supercoiling for maximal activation. Increasing TFAM concentrations suppress HSP2 at levels that result in HSP1 stimulation. This study shows distinct modes of promoter activation, providing opportunities for the regulation of mitochondrial gene expression by promoter selection.
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http://dx.doi.org/10.1080/21541264.2017.1331156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703240PMC
March 2018

Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test.

Genet Med 2018 04 3;20(4):435-443. Epub 2017 Aug 3.

Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, Ontario, Canada.

PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.MethodsWe prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing.ResultsWGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24%; P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A.ConclusionWGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.
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http://dx.doi.org/10.1038/gim.2017.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895460PMC
April 2018