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BackgroundClinical exome sequencing typically achieves diagnostic yields of 30%–57.5% in individuals with monogenic rare diseases. Undiagnosed diseases programmes implement strategies to improve diagnostic outcomes for these individuals.AimWe share the lessons learnt from the first 3 years of the Undiagnosed Diseases Program-Victoria, an Australian programme embedded within a clinical genetics service in the state of Victoria with a focus on paediatric rare diseases.MethodsWe enrolled families who remained without a diagnosis after clinical genomic (panel, exome or genome) sequencing between 2016 and 2018. We used family-based exome sequencing (family ES), family-based genome sequencing (family GS), RNA sequencing (RNA-seq) and high-resolution chromosomal microarray (CMA) with research-based analysis.ResultsIn 150 families, we achieved a diagnosis or strong candidate in 64 (42.7%) (37 in known genes with a consistent phenotype, 3 in known genes with a novel phenotype and 24 in novel disease genes). Fifty-four diagnoses or strong candidates were made by family ES, six by family GS with RNA-seq, two by high-resolution CMA and two by data reanalysis.ConclusionWe share our lessons learnt from the programme. Flexible implementation of multiple strategies allowed for scalability and response to the availability of new technologies. Broad implementation of family ES with research-based analysis showed promising yields post a negative clinical singleton ES. RNA-seq offered multiple benefits in family ES-negative populations. International data sharing strategies were critical in facilitating collaborations to establish novel disease–gene associations. Finally, the integrated approach of a multiskilled, multidisciplinary team was fundamental to having diverse perspectives and strategic decision-making.

Ornithine transcarbamylase deficiency (OTCD) is an X-chromosome linked urea cycle disorder (UCD) that causes hyperammonemic episodes leading to white matter injury and impairments in executive functioning, working memory, and motor planning. This study aims to investigate differences in functional connectivity of two resting-state networks--default mode and set-maintenance--between OTCD patients and healthy controls. Sixteen patients with partial OTCD and twenty-two control participants underwent a resting-state scan using 3T fMRI. Combining independent component analysis (ICA) and region-of-interest (ROI) analyses, we identified the nodes that comprised each network in each group, and assessed internodal connectivity. Group comparisons revealed reduced functional connectivity in the default mode network (DMN) of OTCD patients, particularly between the anterior cingulate cortex/medial prefrontal cortex (ACC/mPFC) node and bilateral inferior parietal lobule (IPL), as well as between the ACC/mPFC node and the posterior cingulate cortex (PCC) node. Patients also showed reduced connectivity in the set-maintenance network, especially between right anterior insula/frontal operculum (aI/fO) node and bilateral superior frontal gyrus (SFG), as well as between the right aI/fO and ACC and between the ACC and right SFG. Internodal functional connectivity in the DMN and set-maintenance network is reduced in patients with partial OTCD compared to controls, most likely due to hyperammonemia-related white matter damage. Because several of the affected areas are involved in executive functioning, it is postulated that this reduced connectivity is an underlying cause of the deficits OTCD patients display in this cognitive domain.

Patients with the Aicardi–Goutières syndrome, an autosomal recessive disorder that affects the central nervous system, immune system, and skin, have severe neurodevelopmental abnormalities and abnormal signaling in the interferon pathway. In patients in this study, Janus kinase inhibition decreased skin manifestations and improved neurologic function.

Genetic white matter disorders have heterogeneous etiologies and overlapping clinical presentations. We performed a study of the diagnostic efficacy of genome sequencing in 41 unsolved cases with prior exome sequencing, resolving an additional 14 from an historical cohort (n = 191). Reanalysis in the context of novel disease‐associated genes and improved variant curation and annotation resolved 64% of cases. The remaining diagnoses were directly attributable to genome sequencing, including cases with small and large copy number variants (CNVs) and variants in deep intronic and technically difficult regions. Genome sequencing, in combination with other methodologies, achieved a diagnostic yield of 85% in this retrospective cohort.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses threats to individuals with rare disease, in part because so little is known about the impact of COVID-19 infection and vaccination safety in rare disease populations. Of particular concern, given the overlap in disease manifestations and interferon dysregulation, are a group of heritable autoinflammatory conditions called type I interferonopathies. The most common of these, Aicardi Goutières Syndrome (AGS), is caused by altered nucleic acid metabolism and sensing, resulting in additional concerns surrounding the use of mRNA vaccination approaches. To determine whether mRNA vaccines induce an interferon response in AGS, we applied mRNA SARS-CoV-2 vaccines to whole blood samples and assessed internalization and interferon signaling gene expression responses to the mRNA. In all cases (11 AGS and 11 control samples), interferon signatures did not significantly increase from baseline, regardless of baricitinib treatment status in the AGS subjects, and were even decreased, when using codon optimized SARS-CoV-2 di-proline modified spike sequence (S2P). Internalization of S2P in human dendritic cells was verified by Western Blot, and in control and AGS blood cells was verified by Luciferase activity. Although numbers of tested samples in this rare disease are small, based on these findings, we suggest that COVID vaccination is unlikely to directly stimulate the interferon signaling gene expression in AGS patients via response to mRNA internalization. The in vitro nature of this study cannot exclude an exaggerated interferon response to spike protein production at a systemic level in individuals with a primary heritable interferonopathy. In the context of continued SARS-CoV-2 spread in the community, we do not recommend withholding vaccination in this rare disease group. However, we recommend that vaccinations for AGS patients are provided in a controlled setting with appropriate observation and used with caution in individuals with prior vaccine associated adverse events.

Alexander disease results from gain of function mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed in astrocytes. At least eight GFAP isoforms have been described, however, the predominant alpha isoform accounts for approximately 90% of GFAP protein in the central nervous system. Here we describe exonic variants identified in three unrelated families with Type II Alexander disease that alter the splicing of GFAP pre-mRNA and result in upregulation of a previously uncharacterized GFAP lambda isoform (NM 001363846.1). Affected members of Family 1 and Family 2 shared the same missense variant, NM 001363846.1:c.1289G>A;p.(Arg430His) while in Family 3 we identified a synonymous variant in the adjacent nucleotide, NM 001363846.1:c.1290C>A;p.(Arg430Arg). Using RNA and protein analysis of brain autopsy samples, and a mini-gene splicing reporter assay, we demonstrate both variants result in upregulation of the lambda isoform. We assessed other GFAP variants in the ClinVar database for predicted aberrant splicing and using the same assay demonstrated significant changes to splicing for two selected variants. Our approach demonstrates the importance of characterizing the effect of GFAP variants on mRNA splicing in order to inform future pathophysiologic and therapeutic study for Alexander disease.

Mechanically activated (MA) ion channels convert physical forces into electrical signals. Despite the importance of this function, the involvement of mechanosensitive ion channels in human disease is poorly understood. Here we report heterozygous missense mutations in the gene encoding the MA ion channel TMEM63A that result in an infantile disorder resembling a hypomyelinating leukodystrophy. Four unrelated individuals presented with congenital nystagmus, motor delay, and deficient myelination on serial scans in infancy, prompting the diagnosis of Pelizaeus-Merzbacher (like) disease. Genomic sequencing revealed all four individuals carry heterozygous missense variants in the pore-forming domain of TMEM63A. These variants were confirmed to have arisen de novo in three of the four individuals. While the physiological role of TMEM63A is incompletely understood, it is highly expressed in oligodendrocytes and it has recently been shown to be a mechanically activated (MA) ion channel. Using patch clamp electrophysiology, we demonstrated that each of the modelled variants result in strongly attenuated stretch-activated currents when expressed in naïve cells. Unexpectedly, the clinical evolution of all four individuals has been surprisingly favorable, with substantial improvements in neurological signs and developmental progression. In the three individuals with follow-up scans after four years of age, the myelin deficit had almost completely resolved. Our results suggest a previously unappreciated role for mechanosensitive ion channels in myelin development.