Here we report whole exome sequencing (WES) on a cohort of 71 patients with persistently unresolved white matter abnormalities with a suspected diagnosis of leukodystrophy or genetic leukoencephalopathy. WES analyses were performed on trio, or greater, family groups. Diagnostic pathogenic variants were identified in 35% (25 of 71) of patients. Potentially pathogenic variants were identified in clinically relevant genes in a further 7% (5 of 71) of cases, giving a total yield of clinical diagnoses in 42% of individuals. These findings provide evidence that WES can substantially decrease the number of unresolved white matter cases.

Whole exome sequencing in patients with white matter abnormalities.

Leukodystrophies comprise a large group of rare genetic disorders primarily affecting CNS white matter. Historically, the diagnostic process was slow and patient prognosis regarded as poor because curative treatment was only available for very few leukodystrophies in early stages of the disease. Whole-exome sequencing has both greatly increased the number of known leukodystrophies and improved diagnosis. Whether MRI keeps its central place in diagnosis and what the role is of whole-exome sequencing are relevant questions for neurologists. Improved diagnosis has revealed the phenotypic variability of leukodystrophies, requiring adaptation of prognostication. Technological advance in molecular techniques and improved insight into the pathophysiology of individual leukodystrophies have led to therapeutic developments, including drug design and gene therapy. Despite this progress, therapies are only beneficial early in the disease course, emphasising the need for a speedy diagnosis and for research on regenerative approaches to repair the damage already present.

Diagnosis, prognosis, and treatment of leukodystrophies

We aimed to develop an efficient, flexible and scalable approach to diagnostic genome-wide sequence analysis of genetically heterogeneous clinical presentations. Here we present G2P (
 www.ebi.ac.uk/gene2phenotype
 ) as an online system to establish, curate and distribute datasets for diagnostic variant filtering via association of allelic requirement and mutational consequence at a defined locus with phenotypic terms, confidence level and evidence links. An extension to Ensembl Variant Effect Predictor (VEP), VEP-G2P was used to filter both disease-associated and control whole exome sequence (WES) with Developmental Disorders G2P (G2PDD; 2044 entries). VEP-G2PDD shows a sensitivity/precision of 97.3%/33% for de novo and 81.6%/22.7% for inherited pathogenic genotypes respectively. Many of the missing genotypes are likely false-positive pathogenic assignments. The expected number and discriminative features of background genotypes are defined using control WES. Using only human genetic data VEP-G2P performs well compared to other freely-available diagnostic systems and future phenotypic matching capabilities should further enhance performance. Diagnostic filtering is an important step to analyze the functional and clinical significance of the large number of genetic variants identified from next-generation genome sequencing data. Here, the authors develop a flexible and scalable system for diagnostic filtering of genetic variants using G2P with Ensembl VEP.

/pdf/flexible-and-scalable-diagnostic-filtering-of-genomic-2vwciv3eu4.pdf

Flexible and scalable diagnostic filtering of genomic variants using G2P with Ensembl VEP.

Importance Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a frequent cause of adult-onset leukodystrophy known to be caused by autosomal dominant mutations in the CSF1R (colony-stimulating factor 1) gene. The discovery that CSF1R mutations cause ALSP led to more accurate prognosis and genetic counseling for these patients in addition to increased interest in microglia as a target in neurodegeneration. However, it has been known since the discovery of the CSF1R gene that there are patients with typical clinical and radiologic evidence of ALSP who do not carry pathogenic CSF1R mutations. These patients include those in whom the pathognomonic features of axonal spheroids and pigmented microglia have been found. Achieving a genetic diagnosis in these patients is important to our understanding of this disorder. Objective To genetically characterize a group of patients with typical features of ALSP who do not carry CSF1R mutations. Design, Settings, and Participants In this case series study, 5 patients from 4 families were identified with clinical, radiologic, or pathologic features of ALSP in whom CSF1R mutations had been excluded previously by sequencing. Data were collected between May 2014 and September 2015 and analyzed between September 2015 and February 2016. Main Outcomes and Measures Focused exome sequencing was used to identify candidate variants. Family studies, long-range polymerase chain reaction with cloning, and complementary DNA sequencing were used to confirm pathogenicity. Results Of these 5 patients, 4 were men (80%); mean age at onset of ALSP was 29 years (range, 15-44 years). Biallelic mutations in the alanyl-transfer (t)RNA synthetase 2 ( AARS2 ) gene were found in all 5 patients. Frameshifting and splice site mutations were common, found in 4 of 5 patients, and sequencing of complementary DNA from affected patients confirmed that the variants were loss of function. All patients presented in adulthood with prominent cognitive, neuropsychiatric, and upper motor neuron signs. Magnetic resonance imaging in all patients demonstrated a symmetric leukoencephalopathy with punctate regions of restricted diffusion, typical of ALSP. In 1 patient, brain biopsy demonstrated axonal spheroids and pigmented microglia, which are the pathognomonic signs of ALSP. Conclusions and Relevance This work indicates that mutations in the tRNA synthetase AARS2 gene cause a recessive form of ALSP. The CSF1R and AARS2 proteins have different cellular functions but overlap in a final common pathway of neurodegeneration. This work points to novel targets for research and will lead to improved diagnostic rates in patients with adult-onset leukoencephalopathy.

/pdf/analysis-of-mutations-in-aars2-in-a-series-of-csf1r-negative-1r18nma8i2.pdf

Analysis of Mutations in AARS2 in a Series of CSF1R-Negative Patients With Adult-Onset Leukoencephalopathy With Axonal Spheroids and Pigmented Glia.

Leukodystrophies and genetic leukoencephalopathies are a rare group of disorders leading to progressive degeneration of cerebral white matter. They are associated with a spectrum of clinical phenotypes dominated by dementia, psychiatric changes, movement disorders and upper motor neuron signs. Mutations in at least 60 genes can lead to leukoencephalopathy with often overlapping clinical and radiological presentations. For these reasons, patients with genetic leukoencephalopathies often endure a long diagnostic odyssey before receiving a definitive diagnosis or may receive no diagnosis at all. In this study, we used focused and whole exome sequencing to evaluate a cohort of undiagnosed adult patients referred to a specialist leukoencephalopathy service. In total, 100 patients were evaluated using focused exome sequencing of 6100 genes. We detected pathogenic or likely pathogenic variants in 26 cases. The most frequently mutated genes were NOTCH3, EIF2B5, AARS2 and CSF1R. We then carried out whole exome sequencing on the remaining negative cases including four family trios, but could not identify any further potentially disease-causing mutations, confirming the equivalence of focused and whole exome sequencing in the diagnosis of genetic leukoencephalopathies. Here we provide an overview of the clinical and genetic features of these disorders in adults.

/pdf/clinical-and-genetic-characterization-of-lrix98908d.pdf

Clinical and genetic characterization of leukoencephalopathies in adults.

https://espace.library.uq.edu.au/view/UQ:356542/UQ356542_OA.pdf

Marc C. Patterson

Papers

A clinical approach to the diagnosis of patients with leukodystrophies and genetic leukoencephelopathies