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Many patients with neuropathies, ataxias, dystonias and other neurological diseases remain without a genetic diagnosis following gold standard diagnostic testing. Lack of a diagnosis could be because new disease genes need to be found, or because diagnostic methods fail to detect challenging variants in known disease genes. It could also be because research into adult-onset genetic diseases, which many of these diseases are, has been relatively neglected compared to early-onset diseases. Our trans-Australia collaboration between Perth, Sydney, Melbourne, Brisbane and Adelaide is analysing DNA from patients with peripheral motor and sensory neuropathies, ataxias, dystonia, hereditary spastic paraplegia etc. who have not received a molecular diagnosis following testing in Australian or overseas diagnostic laboratories, with the aim of identifying the molecular diagnosis. We are using short-read and long-read genome sequencing and optical genome mapping allied to new bioinformatic tools including the T2T and pangenome genomes. Our collaborating laboratories have multiple model systems in which to model candidate variants. To date we have demonstrated that long-read sequencing can overcome issues with pseudogenes (SORD) and clarify pathogenic structural variants. We have identified deep intronic variants causing peripheral neuropathy (MME), participated in the identification of FGF14 STR ataxia (SCA27B), further clarified the genetics of RFC1 ataxia and peripheral neuropathy, and developed new tools for targeted long-read sequencing and short-tandem repeat detection. We have used C. elegans modelling to analyse riboflavin transport deficiency and are modelling additional disorders in zebrafish. We have completed two reviews: one proposing methods to identify the remaining hidden genetics of inherited peripheral neuropathy and one on the use of iPSCs in analysing neurogenic disorders. Together, our collaboration has led to numerous diagnoses for Australian patients. [ABSTRACT FROM AUTHOR]

Perilipins are a family of proteins that coat the surface of lipid droplets and regulate lipid turnover. Perilipin 4, encoded by PLIN4 on chr 9, is highly expressed in skeletal muscle where it mainly localizes to the subsarcolemmal regions of type 1 myofibres. An expansion of the perilipin 4 amphipathic domain (40 x 99 repetitive sequences in exon 3) was recently identified in a family suffering from an autosomal (AD) distal vacuolar myopathy. We aim to describe two unrelated Spanish families suffering from a highly similar distal myopathy caused by two different size repeat expansions in PLIN4 , respectively. Affected patients in both families presented around the age of 30 with distal weakness in the anterior compartment of legs eventually progressing to involve proximal and distal muscles of four limbs, with loss of ambulation and respiratory insufficiency later in the course of the disease. Muscle biopsies showed unique pathological features characterized by large numbers of rimmed vacuoles, located under the sarcolemma and within the cytoplasm. Under EM these autophagic vacuoles were often located at the periphery of myofibres. There were large numbers of membrane-bound lysosomes often in continuity with the extracellular space indicating exocytosis. The basal lamina showed abundant duplications and multiple folds. In addition, there were collections of tubulofilaments within the sarcoplasm. Immunohistochemical analysis showed prominent p62 co-localizing with PLIN4 at the sarcolemma and within the cytoplasm. WB analysis for PLIN4 revealed a band of higher molecular weight corresponding to mutated PLIN4 when compared to controls. Targeted ONT sequencing of affected individuals revealed a single 'normal' PLIN4 allele of 29 x 99bp repeats and a single expanded allele of 39 x 99bp repeats in the proband of family 1, and an expanded allele of 37 x 99bp repeats in affected individuals of family two. Unaffected members of both families harboured two copies of the 'normal' PLIN4 allele. We report two new size expansions of the perilipin 4 amphipathic domain in two unrelated families suffering from an AD distal myopathy. Subsarcolemmal p62 expression seems to be a unique marker of this disease. No correlation between the size of the expansion and clinical severity could be established. PLIN4 expansions should be considered in the diagnosis of distal vacuolar myopathies when conventional NGS technologies fail to identify. [ABSTRACT FROM AUTHOR]