Objective In this population-based study, we aimed to determine whether neuropsychiatric history, medication or family history of neuropsychiatric disorders predicted cognitive and/or behavioural impairment in motor neuron disease (MND). Methods People with MND (pwMND) on the Scottish Clinical, Audit, Research and Evaluation of MND (CARE-MND) register, diagnosed from January 2015 to January 2018, with cognitive and/or behavioural data measured using the Edinburgh Cognitive and Behavioural ALS Screen were included. Data were extracted on patient neuropsychiatric, medication and family history of neuropsychiatric disorders. We identified patients with cognitive impairment (motor neuron disease with cognitive impairment (MNDci)), behavioural impairment (motor neuron disease with behavioural impairment (MNDbi), both (motor neuron disease with cognitive and behavioural impairment (MNDcbi)) or motor neuron disease–frontotemporal dementia (MND-FTD). Results Data were available for 305 pwMND (mean age at diagnosis=62.26 years, SD=11.40), of which 60 (19.7%) had a neuropsychiatric disorder. A family history of neuropsychiatric disorders was present in 36/231 (15.58%) of patients. Patient premorbid mood disorders were associated with increased apathy (OR=2.78, 95% CI 1.083 to 7.169). A family history of any neuropsychiatric disorder was associated with poorer visuospatial scores, MNDbi (OR=3.14, 95% CI 1.09 to 8.99) and MND-FTD (OR=5.08, 95% CI 1.26 to 20.40). A family history of mood disorders was associated with poorer overall cognition (exp(b)=0.725, p=0.026), language, verbal fluency and visuospatial scores, and MND-FTD (OR=7.57, 95% CI 1.55 to 46.87). A family history of neurotic disorders was associated with poorer language (exp(b)=0.362, p<0.001), visuospatial scores (exp(b)=0.625, p<0.009) and MND-FTD (OR=13.75, 95% CI 1.71 to 110.86). Conclusion Neuropsychiatric disorders in patients and their families are associated with cognitive and behavioural changes post-MND diagnosis, with many occurring independently of MND-FTD and C9orf72 status. These findings support an overlap between MND, frontotemporal dementia and neuropsychiatric disorders, particularly mood disorders.

In addition to motor neurone degeneration, up to 50% of amyotrophic lateral sclerosis (ALS) patients present with cognitive decline. Understanding the neurobiological changes underlying these cognitive deficits is critical, as cognitively impaired patients exhibit a shorter survival time from symptom onset. Given the pathogenic role of synapse loss in other neurodegenerative diseases in which cognitive decline is apparent, such as Alzheimer’s disease, we aimed to assess synaptic integrity in the ALS brain. Here, we have applied a unique combination of high-resolution imaging of post-mortem tissue with neuropathology, genetic screening and cognitive profiling of ALS cases. Analyses of more than 1 million synapses using two complimentary high-resolution techniques (electron microscopy and array tomography) revealed a loss of synapses from the prefrontal cortex of ALS patients. Importantly, synapse loss was significantly greater in cognitively impaired cases and was not due to cortical atrophy, nor associated with dementia-associated neuropathology. Interestingly, we found a trend between pTDP-43 pathology and synapse loss in the frontal cortex and discovered pTDP-43 puncta at a subset of synapses in the ALS brains. From these data, we postulate that synapse loss in the prefrontal cortex represents an underlying neurobiological substrate of cognitive decline in ALS.

Myotonic dystrophy type 1 (DM1) is a multisystem disorder, caused by expansion of a CTG trinucleotide repeat in the 3′-untranslated region of the DMPK gene. The repeat expansion is somatically unstable and tends to increase in length with time, contributing to disease progression. In some individuals, the repeat array is interrupted by variant repeats such as CCG and CGG, stabilising the expansion and often leading to milder symptoms. We have characterised three families, each including one person with variant repeats that had arisen de novo on paternal transmission of the repeat expansion. Two individuals were identified for screening due to an unusual result in the laboratory diagnostic test, and the third due to exceptionally mild symptoms. The presence of variant repeats in all three expanded alleles was confirmed by restriction digestion of small pool PCR products, and allele structures were determined by PacBio sequencing. Each was different, but all contained CCG repeats close to the 3′-end of the repeat expansion. All other family members had inherited pure CTG repeats. The variant repeat-containing alleles were more stable in the blood than pure alleles of similar length, which may in part account for the mild symptoms observed in all three individuals. This emphasises the importance of somatic instability as a disease mechanism in DM1. Further, since patients with variant repeats may have unusually mild symptoms, identification of these individuals has important implications for genetic counselling and for patient stratification in DM1 clinical trials.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by an expansion of a polyglutamine tract within the ATXN1 gene. Normal alleles have been reported to range from 6 to 35 repeats, intermediate alleles from 36 to 38 repeats and fully penetrant pathogenic alleles have at least 39 repeats. This distribution was based on relatively few samples and the narrow intermediate range makes the accuracy of the repeat sizing crucial for interpreting and reporting diagnostic tests, which can vary between laboratories. Here, we examine the distribution of 6378 SCA1 chromosomes and identify a very late onset SCA1 family with a fully penetrant uninterrupted pathogenic allele containing 38 repeats. This finding supports the theory that polyQ toxicity is related to the increase of the length of the inherited tracts and not as previously hypothesized to the structural transition occurring above a specific threshold. In addition, the threshold of toxicity shifts to a shorter polyQ length with the increase of the lifespan in SCA1. Furthermore, we show that SCA1 intermediate alleles have a different behavior compared to the other polyglutamine disorders as they do not show reduced penetrance when uninterrupted. Therefore, the pathogenic mechanism in SCA1 is distinct from other cytosineadenineguanine (CAG) repeat disorders. Accurately sizing repeats is paramount in precision medicine and can be challenging particularly with borderline alleles. We examined plasmids containing cloned CAG repeat tracts alongside a triplet repeat primed polymerase chain reaction (TP PCR) CAG repeat ladder to improve accuracy in repeat sizing by fragment analysis. This method accurately sizes the repeats irrespective of repeat composition or length. We also improved the model for calculating repeat length from fragment analysis sizing by fragment analyzing 100 cloned repeats of known size. Therefore, we recommend these methods for accurately sizing repeat lengths and restriction enzyme digestion to identify interruptions for interpretation of a given allele's pathogenicity.

Genetic understanding of motor neuron disease (MND) has evolved greatly in the past 10 years, including the recent identification of association between MND and variants in TBK1 and NEK1. Our aim was to determine the frequency of pathogenic variants in known MND genes and to assess whether variants in TBK1 and NEK1 contribute to the burden of MND in the Scottish population. SOD1, TARDBP, OPTN, TBK1, and NEK1 were sequenced in 441 cases and 400 controls. In addition to 44 cases known to carry a C9orf72 hexanucleotide repeat expansion, we identified 31 cases and 2 controls that carried a loss-of-function or pathogenic variant. Loss-of-function variants were found in TBK1 in 3 cases and no controls and, separately, in NEK1 in 3 cases and no controls. This study provides an accurate description of the genetic epidemiology of MND in Scotland and provides support for the contribution of both TBK1 and NEK1 to MND susceptibility in the Scottish population.

To the Editor: In suspected autosomal recessive disease without a genetic diagnosis, the risk to future pregnancies is generally reported as 25%, assuming inheritance of one causative allele from each parent. However, it is also possible that a de novo mutation (DNM) could contribute to disease in these cases. The contribution of DNMs to autosomal dominant developmental disorders is widely recognized, but their frequency in autosomal recessive disease is thought to be low, as found in a recent Genetics in Medicine article by Retterer et al.,1.Retterer K, Juusola J, Cho MT, et al. Clinical application of whole-exome sequencing across clinical indications. Genet Med; e-pub ahead of print 3 December 2015.Google Scholar titled “Clinical Application of Whole-Exome Sequencing Across Clinical Indications,” and other studies.2.Yang Y. Muzny D.M. Xia F. Molecular findings among patients referred for clinical whole-exome sequencing.1:CAS:528:DC%2BC2MXisFWqsb4%3D10.1001/jama.2014.14601JAMA. 2014; 312: 1870-1879Google Scholar,3.Deciphering Developmental Disorders Study. Large-scale discovery of novel genetic causes of developmental disorders.10.1038/nature14135Nature. 2015; 519: 223-228Google Scholar The genetic diagnosis of such conditions is important because it allows precise understanding of disease etiology and accurate reporting of recurrence risk. This enables families to make informed decisions about future reproduction and provides the opportunity for earlier genetic screening in future pregnancies. Without a genetic diagnosis, recurrence risk is estimated on the basis of the clinical phenotype alone. In a series of nine families in which severe fetal malformation led to the termination of a pregnancy, we used trio-based exome sequencing to identify two unrelated cases in which an autosomal recessive disorder was caused by the combination of one de novo and one inherited variant. Compound heterozygous loss-of-function variants in EVC2 were identified in the first fetus. Variants in this gene have been shown to cause Ellis-van Creveld syndrome, which was consistent with the phenotype of the fetus. The first variant was a 1-bp deletion in exon 16 (ENST00000344408.5:c.2746delA) that results in a frameshift generating a premature stop codon (ENSP00000342144.5: p.Ser916AlafsTer6). This was inherited from the mother. The second was a de novo nonsense variant in exon 18 (ENST00000344408.5:c.3141G>A; ENSP00000342144.5:p.Trp1047Ter), which allele-specific polymerase chain reaction confirmed had arisen on the paternal haplotype. In the second family, compound heterozygous loss-of-function variants in FRAS1 were identified in the fetus; these mutations cause Fraser syndrome, which was consistent with the phenotype. The first variant was a 20-bp deletion in exon 41 (ENST00000264895.6:c.5664_5665+19delinsT), leading to the loss of two bases of coding sequence and a donor splice site, which is predicted to lead to skipping of exon 41 and a frameshift in the open reading frame, generating a premature stop codon (ENST00000264895.6:p.Asp1845ThrfsTer10). This variant was inherited from the mother. The second variant was a de novo 1-bp deletion in exon 66 (ENST00000264895.6:c.10287delC), resulting in a frameshift mutation and a premature stop codon (ENSP00000264895.6:p.Tyr3429Ter). Allele-specific polymerase chain reaction confirmed that the DNM had arisen on the paternal chromosome. The variants were validated by Sanger sequencing, and the biological relationships within both trios were confirmed, showing that these were true DNMs. For these two fetuses, the clinical phenotypes were suggestive of autosomal recessive syndromes with a reported recurrence risk of 25%. In both cases, because one of the causative variants arose de novo, the recurrence risk is markedly reduced, with significant implications for future reproductive decisions. A small finite risk remains, however, owing to the possibility of germ-line mosaicism in the father in both families. Three recently published trio-based exome sequencing studies, each including several thousand cases, a proportion of which presented as congenital malformations, did not report a single case of recessive disorder in which one of the causative alleles was a DNM.1.Retterer K, Juusola J, Cho MT, et al. Clinical application of whole-exome sequencing across clinical indications. Genet Med; e-pub ahead of print 3 December 2015.Google Scholar,2.Yang Y. Muzny D.M. Xia F. Molecular findings among patients referred for clinical whole-exome sequencing.1:CAS:528:DC%2BC2MXisFWqsb4%3D10.1001/jama.2014.14601JAMA. 2014; 312: 1870-1879Google Scholar,3.Deciphering Developmental Disorders Study. Large-scale discovery of novel genetic causes of developmental disorders.10.1038/nature14135Nature. 2015; 519: 223-228Google Scholar Retterer et al.1.Retterer K, Juusola J, Cho MT, et al. Clinical application of whole-exome sequencing across clinical indications. Genet Med; e-pub ahead of print 3 December 2015.Google Scholar reported exome sequencing in more than 3,000 cases and Yang et al.2.Yang Y. Muzny D.M. Xia F. Molecular findings among patients referred for clinical whole-exome sequencing.1:CAS:528:DC%2BC2MXisFWqsb4%3D10.1001/jama.2014.14601JAMA. 2014; 312: 1870-1879Google Scholar in more than 2,000 cases; together these studies report more than 400 autosomal recessive molecular diagnoses, without a single case of a DNM contributing to the genetic etiology. The Deciphering Developmental Disorders project did not identify enrichment for any functional class of DNMs in autosomal recessive developmental disorder–linked genes.3.Deciphering Developmental Disorders Study. Large-scale discovery of novel genetic causes of developmental disorders.10.1038/nature14135Nature. 2015; 519: 223-228Google Scholar Although our study size is small, it suggests that DNMs may have a more important role in autosomal recessive congenital malformation than has previously been considered. Given that the identification of a causative DNM in recessive disease leads to a low predicted recurrence risk but has hitherto been infrequently reported, we recommend that the frequency of this mode of inheritance be analyzed systematically in a larger series of cases, particularly in patients presenting with a congenital malformation. Future studies of recessive disease should specifically report whether this mechanism contributes to any of their cases. For accurate clinical reporting, this mode of inheritance should always be considered a possible contributor to the cause of autosomal recessive congenital malformation. Clinical confirmation of parental carrier status will help to ensure accurate reporting of recurrence risk in autosomal recessive disease. T.J.A. is in receipt of speaker honoraria from Illumina and consultancy fees from AstraZeneca. The other authors declare no conflict of interest. T.J.A. acknowledges an award of translational funding from the Medical Research Council.

Due to the GC-rich, repetitive nature of C9orf72 hexanucleotide repeat expansions, PCR based detection methods are challenging. Several limitations of PCR have been reported and overcoming these could help to define the pathogenic range. There is also a need to develop improved repeat-primed PCR assays which allow detection even in the presence of genomic variation around the repeat region. We have optimised PCR conditions for the C9orf72 hexanucleotide repeat expansion, using betaine as a co-solvent and specific cycling conditions, including slow ramping and a high denaturation temperature. We have developed a flanking assay, and repeat-primed PCR assays for both 3' and 5' ends of the repeat expansion, which when used together provide a robust strategy for detecting the presence or absence of expansions greater than ∼100 repeats, even in the presence of genomic variability at the 3' end of the repeat. Using our assays, we have detected repeat expansions in 47/442 Scottish ALS patients. Furthermore, we recommend the combined use of these assays in a clinical diagnostic setting.

Paget's disease of bone (PDB) is a common disorder with a strong genetic component characterized by increased but disorganized bone remodelling. Previous genome-wide association studies identified a locus on chromosome 14q32 tagged by rs10498635 which was significantly associated with susceptibility to PDB in several European populations. Here we conducted fine-mapping and targeted sequencing of the candidate locus to identify possible functional variants. Imputation in 741 PDB patients and 2699 controls confirmed that the association was confined to a 60 kb region in the RIN3 gene and conditional analysis adjusting for rs10498635 identified no new independent signals. Sequencing of the RIN3 gene identified a common missense variant (p.R279C) that was strongly associated with the disease (OR = 0.64; P = 1.4 × 10(-9)), and was in strong linkage disequilibrium with rs10498635. A further 13 rare missense variants were identified, seven of which were novel and detected only in PDB cases. When combined, these rare variants were over-represented in cases compared with controls (OR = 3.72; P = 8.9 × 10(-10)). Most rare variants were located in a region that encodes a proline-rich, intrinsically disordered domain of the protein and many were predicted to be pathogenic. RIN3 was expressed in bone tissue and its expression level was ∼10-fold higher in osteoclasts compared with osteoblasts. We conclude that susceptibility to PDB at the 14q32 locus is mediated by a combination of common and rare coding variants in RIN3 and suggest that RIN3 may contribute to PDB susceptibility by affecting osteoclast function.

Searchable abstracts of presentations at key conferences on calcified tissues ISSN 2052-1219 (online)

OBJECTIVES:The objectives of this study were to: (i) establish whether the spinocerebellar ataxia type 8 (SCA 8) expansion is associated with ataxia in Scotland; (ii) test the hypothesis that SCA 8 is associated with neuropsychological impairment; and (iii) review neuroradiological findings in SCA 8.METHODS:The methods included: (i) measurement of SCA 8 expansion frequencies in ataxic patients and healthy controls; (ii) comprehensive neuropsychological assessment of patients with SCA 8 and matched controls, neuropsychiatric interview; and (iii) comparison of patient and matched control magnetic resonance imaging (MRI) scans.RESULTS:(i) 10/694 (1.4%) unrelated individuals with ataxia had combined CTA/CTG repeat expansions >100 compared to 1/1190 (0.08%) healthy controls (P < 0.0005); (ii) neuropsychological assessment revealed a dysexecutive syndrome among SCA 8 patients, not readily explained by motor or mood disturbance; neuropsychiatric symptoms occurred commonly; (iii) cerebellar atrophy was the only salient MRI abnormality in the patient group.CONCLUSIONS:The SCA 8 expansion is associated with ataxia in Scotland. The disorder is associated with a dysexecutive syndrome.

Identification of germline mutations in DNA mismatch repair genes in colorectal cancer probands without an extensive family history can be problematic when ascribing relevance to cancer causation. We undertook a structured assessment of the disease-causing potential of sequence variants identified in a prospective, population-based study of 932 colorectal cancer patients, diagnosed at <55 years of age. Patient samples were screened for germline mutations in MLH1, MSH2, and MSH6. Of 110 carriers, 74 (67%) had one of 33 rare variants of uncertain pathogenicity (12 MLH1, 11 MSH2, and 10 MSH6). Pathogenicity was assessed by determining segregation in families, allele frequency in large numbers of unaffected controls, effect on mRNA for putative splice-site mutations, effect on protein function by bioinformatic analysis and tumor microsatellite instability (MSI) status and DNA mismatch repair protein expression by immunohistochemistry. Because of the ambiguous nature of these variants and lack of concordance between functional assays and control allele frequency, we devised a scoring system to rank the degree of support for a pathogenic role. MLH1 c.200G>A p.G67E, MLH1 c.2041G>A p.A681T, and MSH2 c.2634+5G>C were categorized as pathogenic through assimilation of all available data, while 14 variants were categorized as benign (seven MLH1, three MSH2, and four MSH6). Interestingly, there is tentative evidence suggesting a possible protective effect of three variants (MLH1 c.2066A>G pQ689R, c.2146G>A p.V716M, and MSH2 c.965G>A p.G322D). These findings support a causal link with colorectal cancer for several DNA mismatch repair gene variants. However, the majority of missense changes are likely to be inconsequential polymorphisms.

1 Notermans NC, Wokke JHJ, Franssen H, van der Graaf Y, Vermeulen M, van der Berg L, Bar PR, Jennekens FG. Chronic idiopathic polyneuropathy presenting in middle or old age: a clinical and electrophysiological study of 75 patients. J Neurol Neurosurg Psychiaty 1993;56:1066-71. 2 Kaplan JG, Rosenberg R, Reinitz E, Buchbinder S, Schaumburg HH. Invited review: peripheral neuropathy in Sjogren's syndrome. Muscle Nerve 1990;13:570-9. 3 Mellgren SI, Conn DL, Stevens JC, Dyck PJ. Peripheral neuropathy in primary Sjogren's syndrome. Neurology 1989;39:390-4. 4 Griffin JW, Cornblath DR, Alexander E, Campbell J, Low PA, Bird S, Feldman EL. Ataxic sensory neuropathy and dorsal root ganglionitis associated with Sjogren's syndrome. Ann Neurol 1990;27:304-15. 5 Pierot L, Sauve C, Leger JM, Martin N, Koeger AC, Wechsler B, Chiras J. Asymptomatic cerebral involvement in Sjogren's syndrome. Neuroradiology 1993; 35:378-80.

responses by unpaired Student's t test. The time course of the effect was made from the data of several blocks of trials. The average time courses for all the patients were compared with the normal values described elsewhere2 by analysis of variance (ANOVA). Traces in the figure (A) are typical responses in a patient with Fisher's syndrome. The test magnetic stimulus elicited a response of above 1-6 mV in peak to peak amplitude (first trace). The conditioning electrical stimuli significantly (p < 0-01) reduced the size of EMG responses to the