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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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    Neonatal BCG vaccination is associated with a long-term DNA methylation signature in circulating monocytes
    Bannister, S ; Kim, B ; Dominguez-Andres, J ; Kilic, G ; Ansell, BRE ; Neeland, MR ; Moorlag, SJCFM ; Matzaraki, V ; Vlahos, A ; Shepherd, R ; Germano, S ; Bahlo, M ; Messina, NL ; Saffery, R ; Netea, MG ; Curtis, N ; Novakovic, B (AMER ASSOC ADVANCEMENT SCIENCE, 2022-08-05)
    Trained immunity describes the capacity of innate immune cells to develop heterologous memory in response to certain exogenous exposures. This phenomenon mediates, at least in part, the beneficial off-target effects of the BCG vaccine. Using an in vitro model of trained immunity, we show that BCG exposure induces a persistent change in active histone modifications, DNA methylation, transcription, and adenosine-to-inosine RNA modification in human monocytes. By profiling DNA methylation of circulating monocytes from infants in the MIS BAIR clinical trial, we identify a BCG-associated DNA methylation signature that persisted more than 12 months after neonatal BCG vaccination. Genes associated with this epigenetic signature are involved in viral response pathways, consistent with the reported off-target protection against viral infections in neonates, adults, and the elderly. Our findings indicate that the off-target effects of BCG in infants are accompanied by epigenetic remodeling of circulating monocytes that lasts more than 1 year.
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    Detection of repeat expansions in large next generation DNA and RNA sequencing data without alignment
    Fearnley, LG ; Bennett, MF ; Bahlo, M (NATURE PORTFOLIO, 2022-07-30)
    Bioinformatic methods for detecting short tandem repeat expansions in short-read sequencing have identified new repeat expansions in humans, but require alignment information to identify repetitive motif enrichment at genomic locations. We present superSTR, an ultrafast method that does not require alignment. superSTR is used to process whole-genome and whole-exome sequencing data, and perform the first STR analysis of the UK Biobank, efficiently screening and identifying known and potential disease-associated STRs in the exomes of 49,953 biobank participants. We demonstrate the first bioinformatic screening of RNA sequencing data to detect repeat expansions in humans and mouse models of ataxia and dystrophy.
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    Cutting edge approaches to detecting brain mosaicism associated with common focal epilepsies: implications for diagnosis and potential therapies
    Ye, Z ; Bennett, MF ; Bahlo, M ; Scheffer, IE ; Berkovic, SF ; Perucca, P ; Hildebrand, MS (TAYLOR & FRANCIS LTD, 2021-09-24)
    INTRODUCTION: Mosaic variants arising in brain tissue are increasingly being recognized as a hidden cause of focal epilepsy. This knowledge gain has been driven by new, highly sensitive genetic technologies and genome-wide analysis of brain tissue from surgical resection or autopsy in a small proportion of patients with focal epilepsy. Recently reported novel strategies to detect mosaic variants limited to brain have exploited trace brain DNA obtained from cerebrospinal fluid liquid biopsies or stereo-electroencephalography electrodes. AREAS COVERED: The authors review the data on these innovative approaches published in PubMed before 12 June 2021, discuss the challenges associated with their application, and describe how they are likely to improve detection of mosaic variants to provide new molecular diagnoses and therapeutic targets for focal epilepsy, with potential utility in other nonmalignant neurological disorders. EXPERT OPINION: These cutting-edge approaches may reveal the hidden genetic etiology of focal epilepsies and provide guidance for precision medicine.
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    Genetic aetiologies for childhood speech disorder: novel pathways co-expressed during brain development
    Kaspi, A ; Hildebrand, MS ; Jackson, VE ; Braden, R ; van Reyk, O ; Howell, T ; Debono, S ; Lauretta, M ; Morison, L ; Coleman, MJ ; Webster, R ; Coman, D ; Goel, H ; Wallis, M ; Dabscheck, G ; Downie, L ; Baker, EK ; Parry-Fielder, B ; Ballard, K ; Harrold, E ; Ziegenfusz, S ; Bennett, MF ; Robertson, E ; Wang, L ; Boys, A ; Fisher, SE ; Amor, DJ ; Scheffer, IE ; Bahlo, M ; Morgan, AT (SPRINGERNATURE, 2022-09-18)
    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.
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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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    Atypical development of Broca's area in a large family with inherited stuttering
    Thompson-Lake, DGY ; Scerri, TS ; Block, S ; Turner, SJ ; Reilly, S ; Kefalianos, E ; Bonthrone, AF ; Helbig, I ; Bahlo, M ; Scheffer, IE ; Hildebrand, MS ; Liegeois, FJ ; Morgan, AT (OXFORD UNIV PRESS, 2022-03-14)
    Developmental stuttering is a condition of speech dysfluency, characterized by pauses, blocks, prolongations and sound or syllable repetitions. It affects around 1% of the population, with potential detrimental effects on mental health and long-term employment. Accumulating evidence points to a genetic aetiology, yet gene-brain associations remain poorly understood due to a lack of MRI studies in affected families. Here we report the first neuroimaging study of developmental stuttering in a family with autosomal dominant inheritance of persistent stuttering. We studied a four-generation family, 16 family members were included in genotyping analysis. T1-weighted and diffusion-weighted MRI scans were conducted on seven family members (six male; aged 9-63 years) with two age and sex matched controls without stuttering (n = 14). Using Freesurfer, we analysed cortical morphology (cortical thickness, surface area and local gyrification index) and basal ganglia volumes. White matter integrity in key speech and language tracts (i.e. frontal aslant tract and arcuate fasciculus) was also analysed using MRtrix and probabilistic tractography. We identified a significant age by group interaction effect for cortical thickness in the left hemisphere pars opercularis (Broca's area). In affected family members this region failed to follow the typical trajectory of age-related thinning observed in controls. Surface area analysis revealed the middle frontal gyrus region was reduced bilaterally in the family (all cortical morphometry significance levels set at a vertex-wise threshold of P < 0.01, corrected for multiple comparisons). Both the left and right globus pallidus were larger in the family than in the control group (left P = 0.017; right P = 0.037), and a larger right globus pallidus was associated with more severe stuttering (rho = 0.86, P = 0.01). No white matter differences were identified. Genotyping identified novel loci on chromosomes 1 and 4 that map with the stuttering phenotype. Our findings denote disruption within the cortico-basal ganglia-thalamo-cortical network. The lack of typical development of these structures reflects the anatomical basis of the abnormal inhibitory control network between Broca's area and the striatum underpinning stuttering in these individuals. This is the first evidence of a neural phenotype in a family with an autosomal dominantly inherited stuttering.
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    Self-reported impact of developmental stuttering across the lifespan
    Boyce, JO ; Jackson, VE ; van Reyk, O ; Parker, R ; Vogel, AP ; Eising, E ; Horton, SE ; Gillespie, NA ; Scheffer, IE ; Amor, DJ ; Hildebrand, MS ; Fisher, SE ; Martin, NG ; Reilly, S ; Bahlo, M ; Morgan, AT (WILEY, 2022-03-21)
    AIM: To examine the phenomenology of stuttering across the lifespan in the largest prospective cohort to date. METHOD: Participants aged 7 years and older with a history of developmental stuttering were recruited. Self-reported phenotypic data were collected online including stuttering symptomatology, co-occurring phenotypes, genetic predisposition, factors associated with stuttering severity, and impact on anxiety, education, and employment. RESULTS: A total of 987 participants (852 adults: 590 males, 262 females, mean age 49 years [SD = 17 years 10 months; range = 18-93 years] and 135 children: 97 males, 38 females, mean age 11 years 4 months [SD = 3 years; range = 7-17 years]) were recruited. Stuttering onset occurred at age 3 to 6 years in 64.0%. Blocking (73.2%) was the most frequent phenotype; 75.9% had sought stuttering therapy and 15.5% identified as having recovered. Half (49.9%) reported a family history. There was a significant negative correlation with age for both stuttering frequency and severity in adults. Most were anxious due to stuttering (90.4%) and perceived stuttering as a barrier to education and employment outcomes (80.7%). INTERPRETATION: The frequent persistence of stuttering and the high proportion with a family history suggest that stuttering is a complex trait that does not often resolve, even with therapy. These data provide new insights into the phenotype and prognosis of stuttering, information that is critically needed to encourage the development of more effective speech therapies. WHAT THIS PAPER ADDS: Half of the study cohort had a family history of stuttering. While 75.9% of participants had sought stuttering therapy, only 15.5% identified as having recovered. There was a significant negative correlation between age and stuttering frequency and severity in adults.
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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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    Defective lipid signalling caused by mutations in PIK3C2B underlies focal epilepsy
    Gozzelino, L ; Kochlamazashvili, G ; Baldassari, S ; Mackintosh, AI ; Licchetta, L ; Iovino, E ; Liu, YC ; Bennett, CA ; Bennett, MF ; Damiano, JA ; Zsurka, G ; Marconi, C ; Giangregorio, T ; Magini, P ; Kuijpers, M ; Maritzen, T ; Norata, GD ; Baulac, S ; Canafoglia, L ; Seri, M ; Tinuper, P ; Scheffer, IE ; Bahlo, M ; Berkovic, SF ; Hildebrand, MS ; Kunz, WS ; Giordano, L ; Bisulli, F ; Martini, M ; Haucke, V ; Hirsch, E ; Pippucci, T (OXFORD UNIV PRESS, 2022-07-04)
    Epilepsy is one of the most frequent neurological diseases, with focal epilepsy accounting for the largest number of cases. The genetic alterations involved in focal epilepsy are far from being fully elucidated. Here, we show that defective lipid signalling caused by heterozygous ultra-rare variants in PIK3C2B, encoding for the class II phosphatidylinositol 3-kinase PI3K-C2β, underlie focal epilepsy in humans. We demonstrate that patients' variants act as loss-of-function alleles, leading to impaired synthesis of the rare signalling lipid phosphatidylinositol 3,4-bisphosphate, resulting in mTORC1 hyperactivation. In vivo, mutant Pik3c2b alleles caused dose-dependent neuronal hyperexcitability and increased seizure susceptibility, indicating haploinsufficiency as a key driver of disease. Moreover, acute mTORC1 inhibition in mutant mice prevented experimentally induced seizures, providing a potential therapeutic option for a selective group of patients with focal epilepsy. Our findings reveal an unexpected role for class II PI3K-mediated lipid signalling in regulating mTORC1-dependent neuronal excitability in mice and humans.