BACKGROUND:Significant recent efforts have facilitated increased access to clinical genetics assessment and genomic sequencing for children with rare diseases in many centres, but there remains a service gap for adults. The Austin Health Adult Undiagnosed Disease Program (AHA-UDP) was designed to complement existing UDP programs that focus on paediatric rare diseases and address an area of unmet diagnostic need for adults with undiagnosed rare conditions in Victoria, Australia. It was conducted at a large Victorian hospital to demonstrate the benefits of bringing genomic techniques currently used predominantly in a research setting into hospital clinical practice, and identify the benefits of enrolling adults with undiagnosed rare diseases into a UDP program. The main objectives were to identify the causal mutation for a variety of diseases of individuals and families enrolled, and to discover novel disease genes. METHODS:Unsolved patients in whom standard genomic diagnostic techniques such as targeted gene panel, exome-wide next generation sequencing, and/or chromosomal microarray, had already been performed were recruited. Genome sequencing and enhanced genomic analysis from the research setting were applied to aid novel gene discovery. RESULTS:In total, 16/50 (32%) families/cases were solved. One or more candidate variants of uncertain significance were detected in 18/50 (36%) families. No candidate variants were identified in 16/50 (32%) families. Two novel disease genes (TOP3B, PRKACB) and two novel genotype-phenotype correlations (NARS, and KMT2C genes) were identified. Three out of eight patients with suspected mosaic tuberous sclerosis complex had their diagnosis confirmed which provided reproductive options for two patients. The utility of confirming diagnoses for patients with mosaic conditions (using high read depth sequencing and ddPCR) was not specifically envisaged at the onset of the project, but the flexibility to offer recruitment and analyses on an as-needed basis proved to be a strength of the AHA-UDP. CONCLUSION:AHA-UDP demonstrates the utility of a UDP approach applying genome sequencing approaches in diagnosing adults with rare diseases who have had uninformative conventional genetic analysis, informing clinical management, recurrence risk, and recommendations for relatives.

Tandem repeat (TR) catalogs are important components of repeat genotyping studies as they define the genomic coordinates and expected motifs of all TR loci being analyzed. In recent years, genome-wide studies have used catalogs ranging in size from fewer than 200,000 to over 7 million loci. Where these catalogs overlapped, they often disagreed on locus boundaries, hindering the comparison and reuse of results across studies. Now, with multiple groups developing public databases of TR variation in large population cohorts, there is a risk that, without sufficient consensus in the choice of locus definitions, the use of divergent repeat catalogs will lead to confusion, fragmentation, and incompatibility across future resources. In this paper, we compare existing TR catalogs and discuss desirable features of a comprehensive genome-wide catalog. We then present a new, richly annotated catalog designed for large-scale analyses and population databases. Our catalog stratifies TRs into two groups: 1) isolated TRs suitable for repeat copy number analysis using short read or long read data and 2) so-called variation clusters that contain TRs within wider polymorphic regions that are best studied through sequence-level analysis. To define variation clusters, we present a novel algorithm that leverages long-read HiFi sequencing data to group repeats with surrounding polymorphisms. We show that the human genome contains at least 25,000 complex variation clusters, most of which span over 120 bp and contain five or more TRs. Resolving the sequence of entire variation clusters instead of individually genotyping constituent TRs leads to a more accurate analysis of these regions and enables us to profile variation that would have been missed otherwise. ### Competing Interest Statement The authors have declared no competing interest.

Figures S1-S3 and Supplementary Methods

Hereditary cerebellar ataxias are a heterogenous group of progressive neurological disorders that are disproportionately caused by repeat expansions (REs) of short tandem repeats (STRs). Genetic diagnosis for RE disorders such as ataxias are difficult as the current gold standard for diagnosis is repeat-primed PCR assays or Southern blots, neither of which are scalable nor readily available for all STR loci. In the last five years, significant advances have been made in our ability to detect STRs and REs in short-read sequencing data, especially whole-genome sequencing. Given the increasing reliance of genomics in diagnosis of rare diseases, the use of established RE detection pipelines for RE disorders is now a highly feasible and practical first-step alternative to molecular testing methods. In addition, many new pathogenic REs have been discovered in recent years by utilising WGS data. Collectively, genomes are an important resource/platform for further advancements in both the discovery and diagnosis of REs that cause ataxia and will lead to much needed improvement in diagnostic rates for patients with hereditary ataxia.

PDF - 85K, Supplementary Figure S5. Induction of apoptotic cell death by CB-839.

Adult-onset cerebellar ataxias are a group of neurodegenerative conditions that challenge both genetic discovery and molecular diagnosis. In this study, we identified an intronic (GAA) repeat expansion in fibroblast growth factor 14 (FGF14). Genetic analysis of 95 Australian individuals with adult-onset ataxia identified four (4.2%) with (GAA)>300 and a further nine individuals with (GAA)>250. PCR and long-read sequence analysis revealed these were pure (GAA) repeats. In comparison, no control subjects had (GAA)>300 and only 2/311 control individuals (0.6%) had a pure (GAA)>250. In a German validation cohort, 9/104 (8.7%) of affected individuals had (GAA)>335 and a further six had (GAA)>250, whereas 10/190 (5.3%) control subjects had (GAA)>250 but none were (GAA)>335. The combined data suggest (GAA)>335 are disease causing and fully penetrant (p = 6.0 × 10−8, OR = 72 [95% CI = 4.3–1,227]), while (GAA)>250 is likely pathogenic with reduced penetrance. Affected individuals had an adult-onset, slowly progressive cerebellar ataxia with variable features including vestibular impairment, hyper-reflexia, and autonomic dysfunction. A negative correlation between age at onset and repeat length was observed (R2 = 0.44, p = 0.00045, slope = −0.12) and identification of a shared haplotype in a minority of individuals suggests that the expansion can be inherited or generated de novo during meiotic division. This study demonstrates the power of genome sequencing and advanced bioinformatic tools to identify novel repeat expansions via model-free, genome-wide analysis and identifies SCA27B/ATX-FGF14 as a frequent cause of adult-onset ataxia.

Supplementary Figure 2 from Antitumor Activity of PR-171, a Novel Irreversible Inhibitor of the Proteasome

PDF - 126K, Supplementary Figure S3. TNBC primary tumors and cell lines have elevated glutaminase (GAC) mRNA levels.

ABSTRACT We describe FoundHaplo, a novel identity-by-descent algorithm designed to identify individuals with known, untyped, disease-causing variants using only SNP array data. FoundHaplo leverages knowledge of shared disease haplotypes for inherited disease-causing variants to identify individuals who share the disease haplotype and are, therefore, likely to carry the rare (MAF<0.01) variant. We performed a simulation study to evaluate the performance of FoundHaplo across 33 known disease-harbouring loci. We demonstrated the ability of FoundHaplo to infer the presence of two rare (MAF<0.01) pathogenic variants, SCN1B c.363C>G (p.Cys121Trp) and WWOX c.49G>A (p.E17K), which can cause mild dominant and severe recessive epilepsy respectively, in two large cohorts including 1,573 individuals with epilepsy from the Epi25 cohort and 468,481 individuals from the UK Biobank. We demonstrate that FoundHaplo performs substantially better at inferring the presence of these variants than existing genome-wide imputation approaches. FoundHaplo is a valuable, low-cost screening tool that can be applied to search SNP genotyping array data for disease-causing variants with known founder effects based on shared disease haplotypes. FoundHaplo is available at https://github.com/bahlolab/FoundHaplo .

ABSTRACT Adult-onset cerebellar ataxias are a group of neurodegenerative conditions that challenge both genetic discovery and molecular diagnosis. In this study, we identified a novel intronic GAA repeat expansion in the gene encoding Fibroblast Growth Factor 14 ( FGF14 ). Genetic analysis identified 4/95 previously unresolved Australian affected individuals (4.2%) with (GAA) >335 and a further nine individuals with (GAA) >250 . Notably, PCR and long-read sequence analysis revealed these were pure GAA repeats. In comparison, no controls had (GAA) >300 and only 2/311 control individuals (0.6%) encoded a pure (GAA) >250 . In a German validation cohort 9/104 (8.7%) of affected individuals had (GAA) >335 and a further six had (GAA) >250 . In comparison no controls had (GAA) >335 and 10/190 (5.3%) encoded (GAA) >250 . The combined data suggests (GAA) >335 are disease-causing and fully penetrant [P-value 6.0×10 −8 , OR 72 (95% CI=4.3-1227)], while (GAA) >250 is likely pathogenic, albeit with reduced penetrance. Affected individuals had an adult-onset, slowly progressive cerebellar ataxia with a clinical phenotype that may include vestibular impairment, hyper-reflexia and autonomic dysfunction. A negative correlation between age at onset and repeat length was observed (R2=0.44 p=0.00045, slope = -0.12). This study demonstrates the power of genome sequencing and advanced bioinformatic tools to identify novel repeat expansion loci via model free, genome-wide analysis and identifies SCA50/ATX-FGF14 is a frequent cause of adult-onset ataxia.

PDF - 189K, Supplementary Figure S2. CB-839 has potent anti-proliferative activity in TNBC cells that is associated with selective impairment of glutamine utilization.

Supplementary Figures S1-S4; Supplementary Tables S1-S2.

PDF - 246K, Supplementary Materials and Methods.

PDF - 125K, Supplementary Figure S4. Markers of glutamine utilization are increased in TNBC cell lines relative to receptor-positive cell lines.

Childhood apraxia of speech (CAS), the prototypic severe childhood speech disorder, is characterized by motor programming and planning deficits. Genetic factors make substantive contributions to CAS aetiology, with a monogenic pathogenic variant identified in a third of cases, implicating around 20 single genes to date. Here we aimed to identify molecular causation in 70 unrelated probands ascertained with CAS. We performed trio genome sequencing. Our bioinformatic analysis examined single nucleotide, indel, copy number, structural and short tandem repeat variants. We prioritised appropriate variants arising de novo or inherited that were expected to be damaging based on in silico predictions. We identified high confidence variants in 18/70 (26%) probands, almost doubling the current number of candidate genes for CAS. Three of the 18 variants affected SETBP1, SETD1A and DDX3X, thus confirming their roles in CAS, while the remaining 15 occurred in genes not previously associated with this disorder. Fifteen variants arose de novo and three were inherited. We provide further novel insights into the biology of child speech disorder, highlighting the roles of chromatin organization and gene regulation in CAS, and confirm that genes involved in CAS are co-expressed during brain development. Our findings confirm a diagnostic yield comparable to, or even higher, than other neurodevelopmental disorders with substantial de novo variant burden. Data also support the increasingly recognised overlaps between genes conferring risk for a range of neurodevelopmental disorders. Understanding the aetiological basis of CAS is critical to end the diagnostic odyssey and ensure affected individuals are poised for precision medicine trials.

PDF - 113K, Supplementary Figure S7. Oral dosing of CB-839 results in high exposure in mouse plasma and is well tolerated in immunocompromised mice.

PDf - 136K, Supplementary Figure S1. CB-839 is a non-competitive inhibitor of rHu-GAC.

Supplementary Figure 2 from Antitumor Activity of PR-171, a Novel Irreversible Inhibitor of the Proteasome