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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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    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)
    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)
    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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    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.
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    Bioinformatics-Based Identification of Expanded Repeats: A Non-reference Intronic Pentamer Expansion in RFC1 Causes CANVAS
    Rafehi, H ; Szmulewicz, DJ ; Bennett, MF ; Sobreira, NLM ; Pope, K ; Smith, KR ; Gillies, G ; Diakumis, P ; Dolzhenko, E ; Eberle, MA ; Garcia Barcina, M ; Breen, DP ; Chancellor, AM ; Cremer, PD ; Delatycki, MB ; Fogel, BL ; Hackett, A ; Halmagyi, GM ; Kapetanovic, S ; Lang, A ; Mossman, S ; Mu, W ; Patrikios, P ; Perlman, SL ; Rosemergy, I ; Storey, E ; Watson, SRD ; Wilson, MA ; Zee, DS ; Valle, D ; Amor, DJ ; Bahlo, M ; Lockhart, PJ (CELL PRESS, 2019-07-03)
    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.
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    Expanding the clinical and radiological phenotypes of leukoencephalopathy due to biallelic HMBS mutations
    Stutterd, CA ; Kidd, A ; Florkowski, C ; Janus, E ; Fanjul, M ; Raizis, A ; Wu, TY ; Archer, J ; Leventer, RJ ; Amor, DJ ; Lukic, V ; Bahlo, M ; Gow, P ; Lockhart, PJ ; van Der Knaap, MS ; Delatycki, MB (WILEY, 2021-10)
    Pathogenic heterozygous variants in HMBS encoding the enzyme hydroxymethylbilane synthase (HMBS), also known as porphobilinogen deaminase, cause acute intermittent porphyria (AIP). Biallelic variants in HMBS have been reported in a small number of children with severe progressive neurological disease and in three adult siblings with a more slowly, progressive neurological disease and distinct leukoencephalopathy. We report three further adult individuals who share a distinct pattern of white matter abnormality on brain MRI in association with biallelic variants in HMBS, two individuals with homozygous variants, and one with compound-heterozygous variants. We present their clinical and radiological features and compare these with the three adult siblings previously described with leukoencephalopathy and biallelic HMBS variants. All six affected individuals presented with slowly progressive spasticity, ataxia, peripheral neuropathy, with or without mild cognitive impairment, and/or ocular disease with onset in childhood or adolescence. Their brain MRIs show mainly confluent signal abnormalities in the periventricular and deep white matter and bilateral thalami. This recognizable pattern of MRI abnormalities is seen in all six adults described here. Biallelic variants in HMBS cause a phenotype that is distinct from AIP. It is not known whether AIP treatments benefit individuals with HMBS-related leukoencephalopathy. One individual reported here had improved neurological function for 12 months following liver transplantation followed by decline and progression of disease.