Molecular Psychiatry https://doi.org/10.1038/s41380-018-0020-x ARTICLE A set of regulatory genes co-expressed in embryonic human brain is implicated in disrupted speech development 1 1 1 2 3 Else Eising ● Amaia Carrion-Castillo ● Arianna Vino ● Edythe A. Strand ● Kathy J. Jakielski ● 4,5 6 7 8 9 Thomas S. Scerri ● Michael S. Hildebrand ● Richard Webster ● Alan Ma ● Bernard Mazoyer ● 1,10 4,5 6,11 6,12 13 Clyde Francks ● Melanie Bahlo ● Ingrid E. Scheffer ● Angela T. Morgan ● Lawrence D. Shriberg ● Simon E. Fisher 1,10 Received: 22 September 2017 / Revised: 3 December 2017 / Accepted: 2 January 2018 © The Author(s) 2018. This article is published with open access Abstract Genetic investigations of people with impaired development of spoken language provide windows into key aspects of human biology. Over 15 years after FOXP2 was identified, most speech and language impairments remain unexplained at the molecular level. We sequenced whole genomes of nineteen unrelated individuals diagnosed with childhood apraxia of speech, a rare disorder enriched for causative mutations of large effect. Where DNA was available from unaffected parents, CHD3 SETD1A WDR5 fi 1234567890();,: we discovered de novo mutations, implicating genes, including , and . In other probands, we identi ed novel loss-of-function variants affecting KAT6A, SETBP1, ZFHX4, TNRC6B and MKL2, regulatory genes with links to neurodevelopment. Several of the new candidates interact with each other or with known speech-related genes. Moreover, they show significant clustering within a single co-expression module of genes highly expressed during early human brain development. This study highlights gene regulatory pathways in the developing brain that may contribute to acquisition of proficient speech. Introduction The capacity to acquire complex spoken language appears to be unique to humans [1]. The majority of children, when exposed to linguistic input in their environment, develop skills to understand others, to convert thoughts into spoken Electronic supplementary material The online version of this article utterances and to produce intelligible speech. Remarkably, (https://doi.org/10.1038/s41380-018-0020-x) contains supplementary material, which is available to authorized users. this sophisticated suite of abilities emerges rapidly within * Simon E. Fisher 7 Department of Neurology and Neurosurgery, Children’s Hospital [email protected] Westmead, Sydney, NSW, Australia 1 Language and Genetics Department, Max Planck Institute for 8 Department of Clinical Genetics, Children’s Hospital Westmead, Psycholinguistics, Nijmegen 6525 XD, The Netherlands Sydney, NSW, Australia 2 Department of Neurology, Mayo Clinic, Rochester, MN 55905, 9 University of Bordeaux, IMN, UMR 5293, Bordeaux, France USA 10 Donders Institute for Brain, Cognition and Behaviour, Radboud 3 Department of Communication Sciences and Disorders, University, Nijmegen 6500 HE, The Netherlands Augustana College, Rock Island, IL 61201, USA 11 Austin Health and Royal Children’s Hospital, Melbourne 3052, 4 Population Health and Immunity Division, Walter and Eliza Hall Australia Institute of Medical Research, Melbourne 3052, Australia 12 Neuroscience of Speech, Murdoch Childrens Research Institute, 5 Department of Medical Biology, University of Melbourne, Melbourne 3052, Australia Melbourne 3010, Australia 13 Waisman Center, University of Wisconsin-Madison, Madison, WI 6 Faculty of Medicine, Dentistry and Health Sciences, University of 53705, USA Melbourne, Melbourne 3010, Australia E. Eising et al. the first few years of life without the need for formal implicated in a neurodevelopmental syndrome that includes teaching or special effort. Several complementary lines of language delays, although a diagnosis of speech apraxia biological research suggest that there are strong genetic was not specifically reported in these cases [10]. Still, the underpinnings for such skills, ranging from evidence of majority of speech apraxia cases do not seem to carry causal significant heritability from twin and family studies to mutations in FOXP2, BCL11A or ERC1 [6, 11, 12]. observations of gene associations in molecular studies of The present study aimed to take advantage of cutting- relevant traits [2]. Identification of the responsible genes not edge genomic strategies to identify novel genes implicated only sheds novel light on the pathways underlying the in CAS, and thereby move the field significantly beyond disorders, but can also greatly enhance our fundamental FOXP2. In particular, we applied whole-genome sequen- knowledge about the neurobiological mechanisms enabling cing (WGS) in 19 probands with a diagnosis of CAS, to humans to acquire language [3]. identify single nucleotide variants (SNVs) and small inser- Most cases of developmental language impairments are tions and deletions (indels) against a genome-wide back- likely to involve genetic complexity, resulting from the ground. Crucially, for half the WGS cohort of the present inheritance of multiple risk factors with small individual study (9 probands), we could also sequence the entire effect sizes [2]. Nonetheless, it has been established that genomes of nuclear family members without CAS (both disorders of speech and language sometimes occur in parents, and in one family a sibling as well), allowing us to monogenic form. One relevant disorder that may be enri- directly pinpoint de novo variants, which are known to have ched for damaging gene variants of large effect size is an increased likelihood of being causal [13]. Moreover, childhood apraxia of speech (CAS), also known as devel- given the established evidence that genes involved in opmental verbal dyspraxia. CAS is a rare severe develop- speech and language disruptions cluster in related functional mental disorder characterized by difficulties with pathways [14, 15], we coupled our WGS findings to co- automatically and accurately sequencing speech sounds into expression network analysis, identifying correlated expres- syllables, syllables into words, and words into sentences sion patterns across developing human brain tissue samples. with the correct prosody [4]. In CAS, it is thought that We further validated findings by comparison to WGS data impairments in the neural planning and/or programming of from an independent set of healthy controls, analyzed using spatiotemporal parameters of movement sequences result in the same procedures as the CAS cohort. Our work uncov- errors in speech sound production and prosody. Diagnostic ered a neural co-expression module of functionally-related features of CAS include inconsistency in the types of genes, which brings together ERC1 and BCL11A with speech errors that are made, and greater problems as the newly implicated candidate genes in CAS susceptibility, complexity and length of the utterance increase. several of which have also been connected with neurode- In 2001, studies of a large multigenerational pedigree velopment through studies of other disorders. named the KE family, along with an unrelated case with a chromosomal translocation, led to the discovery that dis- rupting one copy of the FOXP2 gene (on chromosome Patients and Methods 7q31) is sufficient to cause CAS [5]. Multiple different cases of FOXP2 disruption have since been identified, Ethics including missense and nonsense mutations, insertion/ deletions and chromosomal rearrangements of various The research in this study was approved by the appropriate kinds; CAS is the most consistent phenotypic consequence review boards: the Social and Behavioral Sciences Institu- in the affected people [2, 6]. More than 15 years after the tional Review Board of the University of Wisconsin- identification of FOXP2, progress in identifying additional Madison [Protocol 2013-0438], the Augustana College genetic risk factors has been slow, and mainly driven by Institutional Review Board, and the Mayo Clinic Institu- studies of chromosomal rearrangements. For example, tional Review Board [Protocol PR08-002372] (primary deletion of a ~600-kb region in human chromosomal band CAS cohort); the Melbourne Human Research Ethics 16p11.2, encompassing >25 genes, significantly increases Committee [project 27053] (Australian case); and the risk of CAS, amongst other phenotypic consequences [7]. Basse-Normandie local ethics committee [reference CPP- The BCL11A and ERC1 genes have been found to be dis- 2006-16] (control data set). rupted by deletions at 2p16.1 and 12p13.33, respectively in children with CAS or with broader problems that also Participants involve intellectual disability, motor difficulties, develop- mental problems (for BCL11A deletions) and psychiatric The primary data set comprised 19 probands who were manifestations (for ERC1 deletions) [8, 9]. Recently, point ascertained based on a formal clinical diagnosis of CAS. mutations that disrupt BCL11A function have been Participants were recruited for a study of pediatric motor Regulatory genes co-expressed in embryonic human brain... speech disorders at two collaborative sites, as described [20], were used as healthy controls to test for potential previously [11, 12]. All probands were evaluated using the enrichment bias that might result from variant filtering Madison Speech Assessment Protocol and the Speech procedures (see below). Informed consent was obtained Disorders Classification System to identify and classify from all participants. BGI (Hong Kong/Shenzhen) per- speakers’ speech status at assessment, including CAS formed WGS on this data set using Illumina’s HiSeq Xten (Supplementary