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Cerebellar Ataxia, Neuropathy and Vestibular Areflexia Syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing (WGS) data from nearly 10,000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n=6 from 5 families), AAGGC (n=2 from 1 family), AGAGG (n=1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, here we show a pathogenic role for large AAAGG repeat configuration expansions (n=5). Long read sequencing was used to fully characterise the entire repeat sequence and revealed a pure AGGGC expansion in six patients, whereas the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs seem to have arisen from a common haplotype and are predicted to form highly stable G quadruplexes, which have been previously demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions, since the AAAGG motif when very large (>500 repeats) or in the presence of AAGGG interruptions. Accurate sizing and full sequencing of the satellite repeat with long read is recommended in clinically selected cases, in order to achieve an accurate molecular diagnosis and counsel patients and their families.

Introduction:Cerebellar Ataxia, Neuropathy and Vestibular Areflexia Syndrome (CANVAS) is an autosomal recessive neurodegenerative disease characterized by adult onset and slowly progressive sensory neuropathy, cerebellar dysfunction, and vestibular impairment. In most cases, the disease is caused by biallelic (AAGGG)nrepeat expansions in the second intron of the Replication Factor Complex subunit 1 (RFC1). However, a small number of cases with typical CANVAS do not carry the common biallelic repeat expansion. The objective of this study was to expands the genotypic spectrum of CANVAS by identifying point mutations inRFC1coding region associated with this condition.Methods:Fifteen individuals diagnosed with CANVAS and carrying only one heterozygous (AAGGG)nexpansion inRFC1underwent WGS or WES to test for the presence of a second variant inRFC1or other unrelated gene. To assess the impact of truncating variants onRFC1expression we tested the level of RFC1 transcript and protein on patients’ derived cell lines.Results:We identified seven patients from five unrelated families with clinically defined CANVAS carrying a heterozygous (AAGGG)nexpansion together with a second truncating variantin transinRFC1, which included: c.1267C>T (p.Arg423Ter), c.1739_1740del (p.Lys580SerfsTer9), c.2191del (p.Gly731GlufsTer6) and c.2876del (p.Pro959GlnfsTer24). Patient fibroblasts containing the c.1267C>T (p.Arg423Ter) or c.2876del (p.Pro959GlnfsTer24) variants demonstrated nonsense-mediated mRNA decay and reduced RFC1 transcript and protein.Discussion:Our report expands the genotype spectrum of RFC1 disease. FullRFC1sequencing is recommended in cases affected by typical CANVAS and carrying monoallelic (AAGGG)nexpansions. Also, it sheds further light on the pathogenesis of RFC1 CANVAS as it supports the existence of a loss of function mechanism underlying this complex neurodegenerative condition.

Radiometric calibration methods are described that enable long-term deployment of uncooled microbolometer infrared imagers without on-board calibration sources. These methods involve tracking the focal-plane-array and/or camera-body temperatures and compensating for the changing camera response. The compensation is derived from laboratory measurements with the camera viewing a blackbody source while the camera temperature is varied in a thermal chamber. Results demonstrate absolute temperature uncertainty of ≤0.35 °C in a 24-hour period, with more than half of the uncertainty inherent in the blackbody source to which the data are compared. Although this work was driven by environmental remote sensing applications, the methods are relevant to a wide range of infrared imaging applications.

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New Compounds 2745 added. The mixture was heated for fifteen minutes on a steam-bath and then diluted with 300 ml. of water. The precipitation and purification of the base was as described in method A.

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