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    Unexpected diagnosis of myotonic dystrophy type 2 repeat expansion by genome sequencing
    Rafehi, H ; Green, C ; Bozaoglu, K ; Gillies, G ; Delatycki, MB ; Lockhart, PJ ; Scheffer, IE ; Bahlo, M (SPRINGERNATURE, 2022-08-09)
    Several neurological disorders, such as myotonic dystrophy are caused by expansions of short tandem repeats (STRs) which can be difficult to detect by molecular tools. Methodological advances have made repeat expansion (RE) detection with whole genome sequencing (WGS) feasible. We recruited a multi-generational family (family A) ascertained for genetic studies of autism spectrum disorder. WGS was performed on seven children from four nuclear families from family A and analyzed for REs of STRs known to cause neurological disorders. We detected an expansion of a heterozygous intronic CCTG STR in CNBP in two siblings. This STR causes myotonic dystrophy type 2 (DM2). The expansion did not segregate with the ASD phenotype. Repeat-primed PCR showed that the DM2 CCTG motif was expanded above the pathogenic threshold in both children and their mother. On subsequent examination, the mother had mild features of DM2. We show that screening of STRs in WGS datasets has diagnostic utility, both in the clinical and research domain, with potential management and genetic counseling implications.
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    Sporadic hypothalamic hamartoma is a ciliopathy with somatic and bi-allelic contributions
    Green, TE ; Motelow, JE ; Bennett, MF ; Ye, Z ; Bennett, CA ; Griffin, NG ; Damiano, JA ; Leventer, RJ ; Freeman, JL ; Harvey, AS ; Lockhart, PJ ; Sadleir, LG ; Boys, A ; Scheffer, IE ; Major, H ; Darbro, BW ; Bahlo, M ; Goldstein, DB ; Kerrigan, JF ; Heinzen, EL ; Berkovic, SF ; Hildebrand, MS (OXFORD UNIV PRESS, 2022-02-08)
    Hypothalamic hamartoma with gelastic seizures is a well-established cause of drug-resistant epilepsy in early life. The development of novel surgical techniques has permitted the genomic interrogation of hypothalamic hamartoma tissue. This has revealed causative mosaic variants within GLI3, OFD1 and other key regulators of the sonic-hedgehog pathway in a minority of cases. Sonic-hedgehog signalling proteins localize to the cellular organelle primary cilia. We therefore explored the hypothesis that cilia gene variants may underlie hitherto unsolved cases of sporadic hypothalamic hamartoma. We performed high-depth exome sequencing and chromosomal microarray on surgically resected hypothalamic hamartoma tissue and paired leukocyte-derived DNA from 27 patients. We searched for both germline and somatic variants under both dominant and bi-allelic genetic models. In hamartoma-derived DNA of seven patients we identified bi-allelic (one germline, one somatic) variants within one of four cilia genes-DYNC2I1, DYNC2H1, IFT140 or SMO. In eight patients, we identified single somatic variants in the previously established hypothalamic hamartoma disease genes GLI3 or OFD1. Overall, we established a plausible molecular cause for 15/27 (56%) patients. Here, we expand the genetic architecture beyond single variants within dominant disease genes that cause sporadic hypothalamic hamartoma to bi-allelic (one germline/one somatic) variants, implicate three novel cilia genes and reconceptualize the disorder as a ciliopathy.
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    Germline variants in tumor suppressor FBXW7 lead to impaired ubiquitination and a neurodevelopmental syndrome
    Stephenson, SEM ; Costain, G ; Blok, LER ; Silk, MA ; Nguyen, TB ; Dong, X ; Alhuzaimi, DE ; Dowling, JJ ; Walker, S ; Amburgey, K ; Hayeems, RZ ; Rodan, LH ; Schwartz, MA ; Picker, J ; Lynch, SA ; Gupta, A ; Rasmussen, KJ ; Schimmenti, LA ; Klee, EW ; Niu, Z ; Agre, KE ; Chilton, I ; Chung, WK ; Revah-Politi, A ; Au, PYB ; Griffith, C ; Racobaldo, M ; Raas-Rothschild, A ; Ben Zeev, B ; Barel, O ; Moutton, S ; Morice-Picard, F ; Carmignac, V ; Cornaton, J ; Marle, N ; Devinsky, O ; Stimach, C ; Wechsler, SB ; Hainline, BE ; Sapp, K ; Willems, M ; Bruel, A ; Dias, K-R ; Evans, C-A ; Roscioli, T ; Sachdev, R ; Temple, SEL ; Zhu, Y ; Baker, JJ ; Scheffer, IE ; Gardiner, FJ ; Schneider, AL ; Muir, AM ; Mefford, HC ; Crunk, A ; Heise, EM ; Millan, F ; Monaghan, KG ; Person, R ; Rhodes, L ; Richards, S ; Wentzensen, IM ; Cogne, B ; Isidor, B ; Nizon, M ; Vincent, M ; Besnard, T ; Piton, A ; Marcelis, C ; Kato, K ; Koyama, N ; Ogi, T ; Goh, ES-Y ; Richmond, C ; Amor, DJ ; Boyce, JO ; Morgan, AT ; Hildebrand, MS ; Kaspi, A ; Bahlo, M ; Fridriksdottir, R ; Katrinardottir, H ; Sulem, P ; Stefansson, K ; Bjornsson, HT ; Mandelstam, S ; Morleo, M ; Mariani, M ; Scala, M ; Accogli, A ; Torella, A ; Capra, V ; Wallis, M ; Jansen, S ; Waisfisz, Q ; de Haan, H ; Sadedin, S ; Lim, SC ; White, SM ; Ascher, DB ; Schenck, A ; Lockhart, PJ ; Christodoulou, J ; Tan, TY (CELL PRESS, 2022-04-07)
    Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.
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    Pathogenic Variants in GPC4 Cause Keipert Syndrome
    Amor, DJ ; Stephenson, SEM ; Mustapha, M ; Mensah, MA ; Ockeloen, CW ; Lee, WS ; Tankard, RM ; Phelan, DG ; Shinawi, M ; de Brouwer, APM ; Pfundt, R ; Dowling, C ; Toler, TL ; Sutton, VR ; Agolini, E ; Rinelli, M ; Capolino, R ; Martinelli, D ; Zampino, G ; Dumic, M ; Reardon, W ; Shaw-Smith, C ; Leventer, RJ ; Delatycki, MB ; Kleefstra, T ; Mundlos, S ; Mortier, G ; Bahlo, M ; Allen, NJ ; Lockhart, PJ (CELL PRESS, 2019-05-02)
    Glypicans are a family of cell-surface heparan sulfate proteoglycans that regulate growth-factor signaling during development and are thought to play a role in the regulation of morphogenesis. Whole-exome sequencing of the Australian family that defined Keipert syndrome (nasodigitoacoustic syndrome) identified a hemizygous truncating variant in the gene encoding glypican 4 (GPC4). This variant, located in the final exon of GPC4, results in premature termination of the protein 51 amino acid residues prior to the stop codon, and in concomitant loss of functionally important N-linked glycosylation (Asn514) and glycosylphosphatidylinositol (GPI) anchor (Ser529) sites. We subsequently identified seven affected males from five additional kindreds with novel and predicted pathogenic variants in GPC4. Segregation analysis and X-inactivation studies in carrier females provided supportive evidence that the GPC4 variants caused the condition. Furthermore, functional studies of recombinant protein suggested that the truncated proteins p.Gln506∗ and p.Glu496∗ were less stable than the wild type. Clinical features of Keipert syndrome included a prominent forehead, a flat midface, hypertelorism, a broad nose, downturned corners of mouth, and digital abnormalities, whereas cognitive impairment and deafness were variable features. Studies of Gpc4 knockout mice showed evidence of the two primary features of Keipert syndrome: craniofacial abnormalities and digital abnormalities. Phylogenetic analysis demonstrated that GPC4 is most closely related to GPC6, which is associated with a bone dysplasia that has a phenotypic overlap with Keipert syndrome. Overall, we have shown that pathogenic variants in GPC4 cause a loss of function that results in Keipert syndrome, making GPC4 the third human glypican to be linked to a genetic syndrome.
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    Rapid Diagnosis of Spinocerebellar Ataxia 36 in a three-Generation Family Using Short-Read Whole-Genome Sequencing Data
    Rafehi, H ; Szmulewicz, DJ ; Pope, K ; Wallis, M ; Christodoulou, J ; White, SM ; Delatycki, MB ; Lockhart, PJ ; Bahlo, M (WILEY, 2020-05-14)
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    Cerebrospinal fluid liquid biopsy for detecting somatic mosaicism in brain
    Ye, Z ; Chatterton, Z ; Pflueger, J ; Damiano, JA ; McQuillan, L ; Harvey, AS ; Malone, S ; Do, H ; Maixner, W ; Schneider, A ; Nolan, B ; Wood, M ; Lee, WS ; Gillies, G ; Pope, K ; Wilson, M ; Lockhart, PJ ; Dobrovic, A ; Scheffer, IE ; Bahlo, M ; Leventer, RJ ; Lister, R ; Berkovic, SF ; Hildebrand, MS (OXFORD UNIV PRESS, 2021-01-28)
    Brain somatic mutations are an increasingly recognized cause of epilepsy, brain malformations and autism spectrum disorders and may be a hidden cause of other neurodevelopmental and neurodegenerative disorders. At present, brain mosaicism can be detected only in the rare situations of autopsy or brain biopsy. Liquid biopsy using cell-free DNA derived from cerebrospinal fluid has detected somatic mutations in malignant brain tumours. Here, we asked if cerebrospinal fluid liquid biopsy can be used to detect somatic mosaicism in non-malignant brain diseases. First, we reliably quantified cerebrospinal fluid cell-free DNA in 28 patients with focal epilepsy and 28 controls using droplet digital PCR. Then, in three patients we identified somatic mutations in cerebrospinal fluid: in one patient with subcortical band heterotopia the LIS1 p. Lys64* variant at 9.4% frequency; in a second patient with focal cortical dysplasia the TSC1 p. Phe581His*6 variant at 7.8% frequency; and in a third patient with ganglioglioma the BRAF p. Val600Glu variant at 3.2% frequency. To determine if cerebrospinal fluid cell-free DNA was brain-derived, whole-genome bisulphite sequencing was performed and brain-specific DNA methylation patterns were found to be significantly enriched (P = 0.03). Our proof of principle study shows that cerebrospinal fluid liquid biopsy is valuable in investigating mosaic neurological disorders where brain tissue is unavailable.
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    Reducing the exome search space for Mendelian diseases using genetic linkage analysis of exome genotypes
    Smith, KR ; Bromhead, CJ ; Hildebrand, MS ; Shearer, AE ; Lockhart, PJ ; Najmabadi, H ; Leventer, RJ ; McGillivray, G ; Amor, DJ ; Smith, RJ ; Bahlo, M (BIOMED CENTRAL LTD, 2011-01-01)
    Many exome sequencing studies of Mendelian disorders fail to optimally exploit family information. Classical genetic linkage analysis is an effective method for eliminating a large fraction of the candidate causal variants discovered, even in small families that lack a unique linkage peak. We demonstrate that accurate genetic linkage mapping can be performed using SNP genotypes extracted from exome data, removing the need for separate array-based genotyping. We provide software to facilitate such analyses.
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    Neuropathology of childhood-onset basal ganglia degeneration caused by mutation of VAC14
    Stutterd, C ; Diakumis, P ; Bahlo, M ; Fernandez, MF ; Leventer, RJ ; Delatycki, M ; Amor, D ; Chow, CW ; Stephenson, S ; Meisler, MH ; Mclean, C ; Lockhart, PJ (WILEY, 2017-12-01)
    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.
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    Recent advances in the detection of repeat expansions with short-read next-generation sequencing.
    Bahlo, M ; Bennett, MF ; Degorski, P ; Tankard, RM ; Delatycki, MB ; Lockhart, PJ (F1000 Research Ltd, 2018)
    Short tandem repeats (STRs), also known as microsatellites, are commonly defined as consisting of tandemly repeated nucleotide motifs of 2-6 base pairs in length. STRs appear throughout the human genome, and about 239,000 are documented in the Simple Repeats Track available from the UCSC (University of California, Santa Cruz) genome browser. STRs vary in size, producing highly polymorphic markers commonly used as genetic markers. A small fraction of STRs (about 30 loci) have been associated with human disease whereby one or both alleles exceed an STR-specific threshold in size, leading to disease. Detection of repeat expansions is currently performed with polymerase chain reaction-based assays or with Southern blots for large expansions. The tests are expensive and time-consuming and are not always conclusive, leading to lengthy diagnostic journeys for patients, potentially including missed diagnoses. The advent of whole exome and whole genome sequencing has identified the genetic cause of many genetic disorders; however, analysis pipelines are focused primarily on the detection of short nucleotide variations and short insertions and deletions (indels). Until recently, repeat expansions, with the exception of the smallest expansion (SCA6), were not detectable in next-generation short-read sequencing datasets and would have been ignored in most analyses. In the last two years, four analysis methods with accompanying software (ExpansionHunter, exSTRa, STRetch, and TREDPARSE) have been released. Although a comprehensive comparative analysis of the performance of these methods across all known repeat expansions is still lacking, it is clear that these methods are a valuable addition to any existing analysis pipeline. Here, we detail how to assess short-read data for evidence of expansions, reviewing all four methods and outlining their strengths and weaknesses. Implementation of these methods should lead to increased diagnostic yield of repeat expansion disorders for known STR loci and has the potential to detect novel repeat expansions.
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    Functional analysis of a hypomorphic allele shows that MMP14 catalytic activity is the prime determinant of the Winchester syndrome phenotype
    de Vos, IJHM ; Tao, EY ; Ong, SLM ; Goggi, JL ; Scerri, T ; Wilson, GR ; Low, CGM ; Wong, ASW ; Grussu, D ; Stegmann, APA ; van Geel, M ; Janssen, R ; Amor, DJ ; Bahlo, M ; Dunn, NR ; Carney, TJ ; Lockhart, PJ ; Coull, BJ ; van Steensel, MAM (OXFORD UNIV PRESS, 2018-08-15)
    Winchester syndrome (WS, MIM #277950) is an extremely rare autosomal recessive skeletal dysplasia characterized by progressive joint destruction and osteolysis. To date, only one missense mutation in MMP14, encoding the membrane-bound matrix metalloprotease 14, has been reported in WS patients. Here, we report a novel hypomorphic MMP14 p.Arg111His (R111H) allele, associated with a mitigated form of WS. Functional analysis demonstrated that this mutation, in contrast to previously reported human and murine MMP14 mutations, does not affect MMP14's transport to the cell membrane. Instead, it partially impairs MMP14's proteolytic activity. This residual activity likely accounts for the mitigated phenotype observed in our patients. Based on our observations as well as previously published data, we hypothesize that MMP14's catalytic activity is the prime determinant of disease severity. Given the limitations of our in vitro assays in addressing the consequences of MMP14 dysfunction, we generated a novel mmp14a/b knockout zebrafish model. The fish accurately reflected key aspects of the WS phenotype including craniofacial malformations, kyphosis, short-stature and reduced bone density owing to defective collagen remodeling. Notably, the zebrafish model will be a valuable tool for developing novel therapeutic approaches to a devastating bone disorder.