Multi-omics identifies large mitoribosomal subunit instability caused by pathogenic MRPL39 variants as a cause of pediatric onset mitochondrial disease.

MRPL39 encodes one of 52 proteins comprising the large subunit of the mitochondrial ribosome (mitoribosome). In conjunction with 30 proteins in the small subunit, the mitoribosome synthesizes the 13 subunits of the mitochondrial oxidative phosphorylation (OXPHOS) system encoded by mitochondrial Deoxyribonucleic acid (DNA). We used multi-omics and gene matching to identify three unrelated individuals with biallelic variants in MRPL39 presenting with multisystem diseases with severity ranging from lethal, infantile-onset (Leigh syndrome spectrum) to milder with survival into adulthood. Clinical exome sequencing of known disease genes failed to diagnose these patients; however quantitative proteomics identified a specific decrease in the abundance of large but not small mitoribosomal subunits in fibroblasts from the two patients with severe phenotype. Re-analysis of exome sequencing led to the identification of candidate single heterozygous variants in mitoribosomal genes MRPL39 (both patients) and MRPL15. Genome sequencing identified a shared deep intronic MRPL39 variant predicted to generate a cryptic exon, with transcriptomics and targeted studies providing further functional evidence for causation. The patient with the milder disease was homozygous for a missense variant identified through trio exome sequencing. Our study highlights the utility of quantitative proteomics in detecting protein signatures and in characterizing gene-disease associations in exome-unsolved patients. We describe Relative Complex Abundance analysis of proteomics data, a sensitive method that can identify defects in OXPHOS disorders to a similar or greater sensitivity to the traditional enzymology. Relative Complex Abundance has potential utility for functional validation or prioritization in many hundreds of inherited rare diseases where protein complex assembly is disrupted.

Comparing saliva and blood for the detection of mosaic genomic abnormalities that cause syndromic intellectual disability.

We aimed to determine whether SNP-microarray genomic testing of saliva had a greater diagnostic yield than blood for pathogenic copy number variants (CNVs). We selected patients who underwent CMA testing of both blood and saliva from 23,289 blood and 21,857 saliva samples. Our cohort comprised 370 individuals who had testing of both, 224 with syndromic intellectual disability (ID) and 146 with isolated ID. Mosaic pathogenic CNVs or aneuploidy were detected in saliva but not in blood in 20/370 (4.4%). All 20 individuals had syndromic ID, accounting for 9.1% of the syndromic ID sub-cohort. Pathogenic CNVs were large in size (median of 46 Mb), and terminal in nature, with median mosaicism of 27.5% (not exceeding 40%). By contrast, non-mosaic pathogenic CNVs were 100% concordant between blood and saliva, considerably smaller in size (median of 0.65 Mb), and predominantly interstitial in location. Given that salivary microarray testing has increased diagnostic utility over blood in individuals with syndromic ID, we recommend it as a first-tier testing in this group.

The clinical utility of exome sequencing and extended bioinformatic analyses in adolescents and adults with a broad range of neurological phenotypes: an Australian perspective.

Currently there is no secured ongoing funding in Australia for next generation sequencing (NGS) such as exome sequencing (ES) for adult neurological disorders. Studies have focused on paediatric populations in research or highly specialised settings, utilised standard NGS pipelines focusing only on small insertions, deletions and single nucleotide variants, and not explored impacts on management in detail. This prospective multi-site study performed ES and an extended bioinformatics repeat expansion analysis pipeline, on patients with broad phenotypes (ataxia, dementia, dystonia, spastic paraparesis, motor neuron disease, Parkinson's disease and complex/not-otherwise-specified), with symptom onset between 2 and 60 years. Genomic data analysis was phenotype-driven, using virtual gene panels, reported according to American College of Medical Genetics and Genomics guidelines. One-hundred-and-sixty patients (51% female) were included, median age 52 years (range 14-79) and median 9 years of symptoms. 34/160 (21%) patients received a genetic diagnosis. Highest diagnostic rates were in spastic paraparesis (10/25, 40%), complex/not-otherwise-specified (10/38, 26%) and ataxia (7/28, 25%) groups. Findings were considered 'possible/uncertain' in 21/160 patients. Repeat expansion detection identified an unexpected diagnosis of Huntington disease in an ataxic patient with negative ES. Impacts on management, such as more precise and tailored care, were seen in most diagnosed patients (23/34, 68%). ES and a novel bioinformatics analysis pipepline had a substantial diagnostic yield (21%) and management impacts for most diagnosed patients, in heterogeneous, complex, mainly adult-onset neurological disorders in real-world settings in Australia, providing evidence for NGS and complementary multiple, new technologies as valuable diagnostic tools.

Rapid exome sequencing and adjunct RNA studies confirm the pathogenicity of a novel homozygous ASNS splicing variant in a critically ill neonate.

Rapid genomic diagnosis programs are transforming rare disease diagnosis in acute pediatrics. A ventilated newborn with cerebellar hypoplasia underwent rapid exome sequencing (75 h), identifying a novel homozygous ASNS splice-site variant (NM_133436.3:c.1476+1G>A) of uncertain significance. Rapid ASNS splicing studies using blood-derived messenger RNA from the family trio confirmed a consistent pattern of abnormal splicing induced by the variant (cryptic 5' splice-site or exon 12 skipping) with absence of normal ASNS splicing in the proband. Splicing studies reported within 10 days led to reclassification of c.1476+1G>A as pathogenic at age 27 days. Intensive care was redirected toward palliation. Cost analyses for the neonate and his undiagnosed, similarly affected deceased sibling, demonstrate that early diagnosis reduced hospitalization costs by AU$100,828. We highlight the diagnostic benefits of adjunct RNA testing to confirm the pathogenicity of splicing variants identified via rapid genomic testing pipelines for precision and preventative medicine.

Feasibility of Ultra-Rapid Exome Sequencing in Critically Ill Infants and Children With Suspected Monogenic Conditions in the Australian Public Health Care System.

Importance: Widespread adoption of rapid genomic testing in pediatric critical care requires robust clinical and laboratory pathways that provide equitable and consistent service across health care systems. Objective: To prospectively evaluate the performance of a multicenter network for ultra-rapid genomic diagnosis in a public health care system. Design, Setting, and Participants: Descriptive feasibility study of critically ill pediatric patients with suspected monogenic conditions treated at 12 Australian hospitals between March 2018 and February 2019, with data collected to May 2019. A formal implementation strategy emphasizing communication and feedback, standardized processes, coordination, distributed leadership, and collective learning was used to facilitate adoption. Exposures: Ultra-rapid exome sequencing. Main Outcomes and Measures: The primary outcome was time from sample receipt to ultra-rapid exome sequencing report. The secondary outcomes were the molecular diagnostic yield, the change in clinical management after the ultra-rapid exome sequencing report, the time from hospital admission to the laboratory report, and the proportion of laboratory reports returned prior to death or hospital discharge. Results: The study population included 108 patients with a median age of 28 days (range, 0 days to 17 years); 34% were female; and 57% were from neonatal intensive care units, 33% were from pediatric intensive care units, and 9% were from other hospital wards. The mean time from sample receipt to ultra-rapid exome sequencing report was 3.3 days (95% CI, 3.2-3.5 days) and the median time was 3 days (range, 2-7 days). The mean time from hospital admission to ultra-rapid exome sequencing report was 17.5 days (95% CI, 14.6-21.1 days) and 93 reports (86%) were issued prior to death or hospital discharge. A molecular diagnosis was established in 55 patients (51%). Eleven diagnoses (20%) resulted from using the following approaches to augment standard exome sequencing analysis: mitochondrial genome sequencing analysis, exome sequencing-based copy number analysis, use of international databases to identify novel gene-disease associations, and additional phenotyping and RNA analysis. In 42 of 55 patients (76%) with a molecular diagnosis and 6 of 53 patients (11%) without a molecular diagnosis, the ultra-rapid exome sequencing result was considered as having influenced clinical management. Targeted treatments were initiated in 12 patients (11%), treatment was redirected toward palliative care in 14 patients (13%), and surveillance for specific complications was initiated in 19 patients (18%). Conclusions and Relevance: This study suggests feasibility of ultra-rapid genomic testing in critically ill pediatric patients with suspected monogenic conditions in the Australian public health care system. However, further research is needed to understand the clinical value of such testing, and the generalizability of the findings to other health care settings.

Early diagnosis of Pearson syndrome in neonatal intensive care following rapid mitochondrial genome sequencing in tandem with exome sequencing.

Rapid genomic testing is a valuable new diagnostic tool for acutely unwell infants, however exome sequencing does not deliver clinical-grade mitochondrial genome sequencing and may fail to diagnose mitochondrial disorders caused by mitochondrial DNA (mtDNA) variants. Rapid mitochondrial genome sequencing and analysis are not routinely available in rapid genomic diagnosis programmes. We present two critically ill neonates with transfusion-dependent anaemia and persistent lactic acidosis who underwent rapid mitochondrial genome sequencing in tandem with exome sequencing as part of an exome sequencing-based rapid genomic diagnosis programme. No diagnostic variants were identified on examination of the nuclear exome data for either infant. Mitochondrial genome sequencing identified a large mtDNA deletion in both infants, diagnosing Pearson syndrome within 74 and 55 h, respectively. Early diagnosis in the third week of life allowed the avoidance of a range of other investigations and appropriate treatment planning. Rapid mitochondrial genome analysis provides additional diagnostic and clinical utility and should be considered as an adjunct to exome sequencing in rapid genomic diagnosis programmes.