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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, 2022-07-21)
    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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    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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    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)
    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)
    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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    De novo and inherited private variants in MAP1B in periventricular nodular heterotopia
    Heinzen, EL ; O'Neill, AC ; Zhu, X ; Allen, AS ; Bahlo, M ; Chelly, J ; Chen, MH ; Dobyns, WB ; Freytag, S ; Guerrini, R ; Leventer, RJ ; Poduri, A ; Robertson, SP ; Walsh, CA ; Zhang, M ; Hallmayer, J (PUBLIC LIBRARY SCIENCE, 2018-05)
    Periventricular nodular heterotopia (PVNH) is a malformation of cortical development commonly associated with epilepsy. We exome sequenced 202 individuals with sporadic PVNH to identify novel genetic risk loci. We first performed a trio-based analysis and identified 219 de novo variants. Although no novel genes were implicated in this initial analysis, PVNH cases were found overall to have a significant excess of nonsynonymous de novo variants in intolerant genes (p = 3.27x10-7), suggesting a role for rare new alleles in genes yet to be associated with the condition. Using a gene-level collapsing analysis comparing cases and controls, we identified a genome-wide significant signal driven by four ultra-rare loss-of-function heterozygous variants in MAP1B, including one de novo variant. In at least one instance, the MAP1B variant was inherited from a parent with previously undiagnosed PVNH. The PVNH was frontally predominant and associated with perisylvian polymicrogyria. These results implicate MAP1B in PVNH. More broadly, our findings suggest that detrimental mutations likely arising in immediately preceding generations with incomplete penetrance may also be responsible for some apparently sporadic diseases.
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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.