Genomic technologies such as next-generation sequencing (NGS) are revolutionizing molecular diagnostics and clinical medicine. However, these approaches have proven inefficient at identifying pathogenic repeat expansions. Here, we apply a collection of bioinformatics tools that can be utilized to identify either known or novel expanded repeat sequences in NGS data. We performed genetic studies of a cohort of 35 individuals from 22 families with a clinical diagnosis of cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS). Analysis of whole-genome sequence (WGS) data with five independent algorithms identified a recessively inherited intronic repeat expansion [(AAGGG) exp ] in the gene encoding Replication Factor C1 (RFC1). This motif, not reported in the reference sequence, localized to an Alu element and replaced the reference (AAAAG) 11 short tandem repeat. Genetic analyses confirmed the pathogenic expansion in 18 of 22 CANVAS-affected families and identified a core ancestral haplotype, estimated to have arisen in Europe more than twenty-five thousand years ago. WGS of the four RFC1-negative CANVAS-affected families identified plausible variants in three, with genomic re-diagnosis of SCA3, spastic ataxia of the Charlevoix-Saguenay type, and SCA45. This study identified the genetic basis of CANVAS and demonstrated that these improved bioinformatics tools increase the diagnostic utility of WGS to determine the genetic basis of a heterogeneous group of clinically overlapping neurogenetic disorders.

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

Complex I deficiency is the most common biochemical phenotype observed in individuals with mitochondrial disease. With 44 structural subunits and over 10 assembly factors, it is unsurprising that complex I deficiency is associated with clinical and genetic heterogeneity. Massively parallel sequencing (MPS) technologies including custom, targeted gene panels or unbiased whole-exome sequencing (WES) are hugely powerful in identifying the underlying genetic defect in a clinical diagnostic setting, yet many individuals remain without a genetic diagnosis. These individuals might harbor mutations in poorly understood or uncharacterized genes, and their diagnosis relies upon characterization of these orphan genes. Complexome profiling recently identified TMEM126B as a component of the mitochondrial complex I assembly complex alongside proteins ACAD9, ECSIT, NDUFAF1, and TIMMDC1. Here, we describe the clinical, biochemical, and molecular findings in six cases of mitochondrial disease from four unrelated families affected by biallelic (c.635G>T [p.Gly212Val] and/or c.401delA [p.Asn134Ilefs(∗)2]) TMEM126B variants. We provide functional evidence to support the pathogenicity of these TMEM126B variants, including evidence of founder effects for both variants, and establish defects within this gene as a cause of complex I deficiency in association with either pure myopathy in adulthood or, in one individual, a severe multisystem presentation (chronic renal failure and cardiomyopathy) in infancy. Functional experimentation including viral rescue and complexome profiling of subject cell lines has confirmed TMEM126B as the tenth complex I assembly factor associated with human disease and validates the importance of both genome-wide sequencing and proteomic approaches in characterizing disease-associated genes whose physiological roles have been previously undetermined.

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Objective: To characterize the clinical features and neuropathology associated with recessive VAC14 mutations. Methods: Whole-exome sequencing was used to identify the genetic etiology of a rapidly progressive neurological disease presenting in early childhood in two deceased siblings with distinct neuropathological features on post mortem examination. Results: We identified compound heterozygous variants in VAC14 in two deceased siblings with early childhood onset of severe, progressive dystonia, and neurodegeneration. Their clinical phenotype is consistent with the VAC14-related childhood-onset, striatonigral degeneration recently described in two unrelated children. Post mortem examination demonstrated prominent vacuolation associated with degenerating neurons in the caudate nucleus, putamen, and globus pallidus, similar to previously reported ex vivo vacuoles seen in the late-endosome/lysosome of VAC14-deficient neurons. We identified upregulation of ubiquitinated granules within the cell cytoplasm and lysosomal-associated membrane protein (LAMP2) around the vacuole edge to suggest a process of vacuolation of lysosomal structures associated with active autophagocytic-associated neuronal degeneration. Interpretation: Our findings reveal a distinct clinicopathological phenotype associated with recessive VAC14 mutations.

80Genomic technologies such as Next Generation Sequencing (NGS) are revolutionizing 81 molecular diagnostics and clinical medicine. However, these approaches have proven 82 inefficient at identifying pathogenic repeat expansions. Here, we apply a collection of 83 bioinformatics tools that can be utilized to identify either known or novel expanded repeat 84 sequences in NGS data. We performed genetic studies of a cohort of 35 individuals from 22 85 families with a clinical diagnosis of cerebellar ataxia with neuropathy and bilateral vestibular 86 areflexia syndrome (CANVAS). Analysis of whole genome sequence (WGS) data with five 87 independent algorithms identified a recessively inherited intronic repeat expansion 88 [(AAGGG) exp ] in the gene encoding Replication Factor C1 (RFC1). This motif, not reported 89 in the reference sequence, localized to an Alu element and replaced the reference (AAAAG) 11 90 short tandem repeat. Genetic analyses confirmed the pathogenic expansion in 18 of 22 91 CANVAS families and identified a core ancestral haplotype, estimated to have arisen in 92 Europe over twenty-five thousand years ago. WGS of the four RFC1 negative CANVAS 93 families identified plausible variants in three, with genomic re-diagnosis of SCA3, spastic 94 ataxia of the Charlevoix-Saguenay type and SCA45. This study identified the genetic basis of 95 CANVAS and demonstrated that these improved bioinformatics tools increase the diagnostic 96 utility of WGS to determine the genetic basis of a heterogeneous group of clinically 97 overlapping neurogenetic disorders. 98 99 126 specific neurophysiological protocol. 18 A characteristic radiological pattern of cerebellar 127 atrophy has also been described and verified on post-mortem pathology. 11 The characteristic 128 oculomotor abnormality seen in combined cerebellar and vestibular impairment is the 129 visually-enhanced vestibulo-ocular reflex (VVOR), and this can now be evaluated using a 130 commercially available instrumented assessment tool. 19-21 Altogether, these advances have 131 allowed the formulation of diagnostic criteria to aid identification of CANVAS, contributing 132 both research and clinical benefits including improved prognostication and targeted 133 157 A number of bioinformatics tools now exist that allow screening of short-read 158 sequencing data for expanded STRs. 25 Initially, STR detection tools, such as lobSTR and 159 hipSTR, were limited to short STRs that were encompassed by a single sequencing read. 160 However, in the last two years, multiple methods have been released that can screen WES 161 and WGS datasets for REs without being limited by read length. These include 162 ExpansionHunter (EH) 28 , exSTRa 8 , TREDPARSE 29 , STRetch 30 and GangSTR. 31 These are 163 all reference based methods -i.e. they rely on a catalogue of STR loci and motifs and are 164 therefore limited to detecting expansion of previously defined STRs, such as those catalogued 165 in the UCSC track. Moreover, the normal variability in STR length and repeat composition ...

Conventional single cell RNA-seq methods are destructive, such that a given cell cannot also then be tested for fate and function, without a time machine. Here, we develop a clonal method SIS-seq, whereby single cells are allowed to divide, and progeny cells are assayed separately in SISter conditions; some for fate, others by RNA-seq. By cross-correlating progenitor gene expression with mature cell fate within a clone, and doing this for many clones, we can identify the earliest gene expression signatures of dendritic cell subset development. SIS-seq could be used to study other populations harboring clonal heterogeneity, including stem, reprogrammed and cancer cells to reveal the transcriptional origins of fate decisions. Main textSingle cell analyses including flow cytometry, microscopy, colony assays, clonal lineage tracing, and most recently single cell genomics methods, have revolutionised our understanding of biological systems and their heterogeneity 1 . As demonstrated by clonal assays, haematopoietic stem and progenitor cells (HSPCs) are particularly heterogeneous in their fate 2 , and the molecular programs governing these are gradually being characterised 3-7 .One challenge for connecting transcriptional signatures with functional heterogeneity is that these properties can rarely be measured on the same single cell i.e. single cell RNA-seq is destructive, so the same cell cannot then be tested for fate and, vice versa, a single cell tested for fate divides and differentiates such that the founder cell cannot be tested for its molecular profile. A time machine could conceivably allow one to first ascertain one feature of a given cell, then go back in time and re-test the same cell for the other feature, allowing cross-

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