TITLE: A recurrent de novo nonsense variant in ZSWIM6 results in severe intellectual disability without frontonasal or limb malformations.

AUTHOR LIST: AUTHOR LIST: Elizabeth E. Palmer1,2,3$, Raman Kumar4 $

Christopher T. Gordon 5,6 $, Marie Shaw4, Laurence Hubert5,6, Renee Carroll4, Marlène

Rio8, Lucinda Murray1, Melanie Leffler1, Tracy Dudding-Byth1, Myriam Oufadem4,5,

Seema Lalani9, Andrea M. Lewis9, Fan Xia9, Pawel Stankiewicz9, Sau Wai Cheung9,

Allison Tam9, Richard Webster10, Susan Brammah12 , Francesca Filippini5,6 John

Pollard12, Judy Spies13, Andre Minoche3, Mark C. Cowley3, Sarah Risen14, Nina N.

Powell-Hamilton15, Jessica E. Tusi15, LaDonna Immken16, Honey Nagakura16,

Christine Bole-Feysot6,17, Patrick Nitschké6,18, Alexandrine Garrigue6,19, Geneviève de

Saint Basile6,19,20, Emma Kivuva21, DDD Study22,Richard Scott23,24, Augusto

Rendon23,25, Arnold Munnich6,7,8, William Newman,26,27*, Bronwyn Kerr26,27*, Charles

Schwartz28, Claude Besmond6,7, Jill Rosenfeld9, Jeanne Amiel5,6,8# Michael Field1#,

Jozef Gecz4,29#

AFFILIATIONS:

1. Genetics of Learning Disability Service, Hunter Genetics, Waratah, NSW

2298, Australia

2. School of Women and Children’s Health, University of New South Wales,

Randwick, NSW 2031, Australia

3. The Kinghorn Centre for Clinical Genomics, The Garvan Institute, Darlinghurst

NSW,NSW 2010, Australia

4. Adelaide Health and Medical School and the Robinson Research Institute,

The University of Adelaide, North Adelaide, SA 5006, Australia

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5. Laboratory of Embryology and Genetics of Human Malformations, Institut

National de la Santé et de la Recherche Médicale (INSERM) UMR 1163,

Institut Imagine, 75015 Paris, France

6. Paris Descartes-Sorbonne Paris Cité University, Institut Imagine, 75015 Paris,

France

7. Translational Genetics, INSERM UMR 1163, Institut Imagine, 75015 Paris,

France

8. Service de Génétique, Hôpital Necker-Enfants Malades, Assistance Publique

- Hôpitaux de Paris (APHP), 75015 Paris, France

9. Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas,

77030, USA

10.Children’s Hospital at Westmead, Westmead, NSW 2145, Australia

11.Electron Microscope Unit, Anatomical Pathology, Concord NSW 2139,

Australia

12. Brain Mind Research Institute, The University of Sydney, Camperdown, NSW

2050, Australia

13.Department of Neurology, Royal Prince Alfred Hospital. Camperdown, NSW

2050, Australia

14.Meyer Centre for Developmental Pediatrics, Texas Children’s Hospital Autism

Center, Houston, Texas 77054, USA

15.Medical Genetics, Nemours/Alfred I. duPont Hospital for Children, Wilmington,

Delaware 19803, USA

16.Dell Children's Medical Center of Central Texas, Texas 78723, USA

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17.Genomic Platform, INSERM UMR 1163, Institut Imagine, 75015 Paris,

France.

18.Bioinformatic Platform, INSERM UMR 1163, Institut Imagine, 75015 Paris,

France

19.Laboratory of Normal and Pathological Homeostasis of the Immune System,

INSERM UMR 1163, Institut Imagine, 75015 Paris, France

20.Centre d’Etudes des Déficits Immunitaires, Hôpital Necker-Enfants Malades,

APHP, 75015 Paris, France

21.Peninsula Clinical Genetics, Royal Devon and Exeter NHS Foundation Trust,

Exeter, EX1 2ED, UK

22.Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK

23.Genomics England, William Harvey Research Institute, Queen Mary

University of London, Charterhouse Square, London EC1M 6BQ, UK

24.Great Ormond Street Hospital, Great Ormond St, London WC1N 3JH, UK

25.Department of Haematology, University of Cambridge, Long Road,

Cambridge, CB2 0PT, UK

26.Manchester Centre for Genomic Medicine, St Mary’s Hospital, Central

Manchester University Hospitals NHS Foundation Trust, Manchester M13

9PL, UK

27.Division of Evolution and Genomic Sciences School of Biological Sciences,

University of Manchester, Manchester M13 9PL, UK

28.The Greenwood Center, Greenwood, SC 29646, USA

29.Healthy Mothers and Babies, South Australian Health and Medical Research

Institute, Adelaide, SA 5000, Australia

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CORRESPONDENCE EMAILS(S): jozef.gecz@adelaide.edu.au*;

mike.field@health.nsw.gov.au**

ADDITIONAL FOOTNOTES $ # These authors contributed equally to this work; *On

behalf of the 100,000 Genomes Project and Genomics England

ABSTRACT

A recurrent de novo missense variant within the C-terminal Sin3-like domain of

ZSWIM6 has been previously reported to cause acromelic frontonasal dyostosis

(AFND), an autosomal dominant severe frontonasal and limb malformation

syndrome, associated with neurocognitive and motor delay, via a proposed gain of

function effect. We present detailed phenotypic information on seven unrelated

individuals with a recurrent de novo nonsense variant (p.Arg913Ter) in the

penultimate exon of ZSWIM6 who have severe-profound intellectual disability and

additional central and peripheral nervous system symptoms but an absence of

frontonasal or limb malformations. We show that the p.Arg913Ter variant does not

trigger nonsense-mediated decay of the ZSWIM6 mRNA in affected individual-

derived cells. This supports the existence of a truncated ZSWIM6 protein lacking the

Sin3-like domain, which may have a dominant negative effect. This study builds

support for a key role for ZSWIM6 in neuronal development and function, in addition

to its putative roles in limb and craniofacial development, and provides a striking

example of different variants in the same gene leading to distinct phenotypes.

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MAIN TEXT

Advances in high-throughput DNA sequencing combined with databases that allow

the sharing of clinical and genotypic information among clinicians and researchers,

such as those in the Matchmaker Exchange hub, has changed the landscape of

research collaborations leading to accelerated disease variant discovery and

validation for neurocognitive disorders during recent years1;2. This has led to an

explosion in the diagnostic rate for neurocognitive disorders1, with increasing

appreciation of the complexity of clinical presentations of variants even in the same

gene2. In a multicentre study, we discovered a recurrent protein-truncating variant in

the ZSWIM6 gene (MIM: 615951) in a cohort of affected individuals with overlapping

neurocognitive phenotypes. A recurrent de novo nonsynonymous ZSWIM6 variant

(p.Arg1163Trp) was reported to cause the rare Mendelian disorder acromelic

frontonasal dysostosis (AFND)3; 4 characterized by severe frontonasal dysplasia,

tibial hemimelia, pre-axial polydactyly, brain malformations and severe

neurocognitive and motor delay3; 4. The p.Arg1163Trp variant was postulated to

perturb the function of the highly conserved Sin3-like domain at the C-terminus of the

protein, with three-dimensional modelling suggesting disruption of an interaction

surface4. A gain of function mechanism was proposed, based on the observation that

affected individuals with chromosomal deletions spanning ZSWIM65 lack the

craniofacial and limb abnormalities seen in AFND4.

Via Matchmaker Exchange6 and contact with individual diagnostic laboratories, we

identified seven unrelated individuals (four female; three male) with a recurrent

single nucleotide variant in ZSWIM6 that introduced a premature termination codon

(PTC) in exon 13 (Chr5[GRCh37]: g60837744C>T; NM_020928.1: c.2737C>T). All

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had a severe-profound intellectual disability (ID)/developmental delay (DD) and

additional neurological features but lacked the frontonasal or limb malformations

reported in individuals with the p.Arg1163Trp variant. None of the affected

individuals had any other plausible causative variants identified by exome/genome

sequencing or prior diagnostic testing. An overview of the clinical data is provided in

Table 1, and further clinical details in Table S2.

Genetic studies were approved by local ethics committees and written informed

consent was obtained for molecular genetic analysis, functional studies on affected

individual-derived cells, the publication of clinical and radiological data and

photographs from participants or their legal guardians. In all cases the p.Arg913Ter

variant was confirmed by Sanger sequencing using standard methodology, and

segregation analysis was consistent with the variant being de novo for individuals

two to seven. For individual one the variant was not present in his unaffected mother

or either of his two unaffected brothers, while his unaffected father’s DNA was

unavailable for testing.

All affected individuals had a severe-to-profound ID and required early

developmental interventions or a special school. The two oldest individuals

(individual six and seven) live in fully-supported independent accommodation. A

particular deficit in verbal communication was noted for all individuals. Five of the

affected individuals (71%) met diagnostic criteria for Autism or Autism Spectrum

Disorder: this diagnosis was based on features of repetitive movements and limited

play and communication. All affected individuals were noted to be content and

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socially responsive, showing affection to and interest in their families. Hyperactivity

was noted in four of the seven (57%).

Additional neurological features were described in all affected individuals. Low

truncal tone, delayed motor milestones in the first year of life, and delayed onset of

walking (age range to achieve independent ambulation two to five years) was

universal. When ambulant, all have a wide-based, unsteady gait. Progressive

neurological features are described: individuals one, four and six have progressive

spasticity, individual one lost independent ambulation in mid-childhood and

individuals four and six require a wheelchair or stroller for mobility outside of the

home. In addition, individual one has a progressive neuropathy and has lost strength

and reflexes in his distal lower limbs. No sequence or structural candidate variants in

known neuropathy genes were identified on genome analysis for this individual

(Table S2). Nerve conduction studies for individual one demonstrated mixed axonal

and demyelinating features, and light and electron microscopy of a sural nerve

biopsy (Figure 2B) showed a reduction in myelinated fiber density, reduction in the

thickness of myelination in many myelinated axons and evidence of abnormal

accumulation of neurofilaments (Figure 2B). Individuals three and seven have low

sensitivity to pain, leading to self-sustained injuries. No other individuals in the cohort

have had nerve conduction or nerve biopsy studies, indeed detailed neurological

assessments to appropriately screen for neuropathies were not possible in the

majority of individuals due to the severity of their neurocognitive disabilities or young

age. Unusual movements have also been described in six of the seven (86%)

affected individuals: four have paroxysmal tongue movements (tongue thrusting or

tremor), two have stereotypical hand movements, one has head tics and two have

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episodes of whole body hypertonicity which was not consistent with a seizure but

which raised the question of a possible dystonia. Four (57%) had strabismus and

individual one had limitation of the extremes of gaze, although none had a confirmed

visual or hearing impairment. Three of the individuals developed a seizure disorder

which could be controlled on medication, while individual seven had a suspected

seizure disorder which has never required antiepileptic medication. Brain MRI,

performed in all individuals, was reported as normal in five; mild cortical atrophy was

reported consistently only for individual one (Figure S3).

Many of the extra-neurocognitive features in the cohort are commonly seen in

children with ID/DD due to other causes. None had structural congenital anomalies.

Feeding difficulties, failure to thrive, or gastro-esophageal reflux were reported as

significant for six of the seven, with two individuals requiring

fundoplication/gastrostomy. Growth parameters were generally within the normal

range; however, three individuals had progressive microcephaly whereas one

(individual six) had progressive macrocephaly. When assessed as a group, some

similarity in facial features were apparent, particularly at younger ages (Figure 2A).

None of the affected individuals with the p.Arg913Ter variant had the midline clefting,

parietal foramina or extreme hypertelorism characteristic of affected individuals with

the p.Arg1163Trp variant3; 4. Two of the three older male individuals (individuals one

and six) had progressive coarsening of their facial features, and in both individuals a

differential diagnosis of Coffin-Lowry syndrome had been considered (based on

facial and neurological features without the characteristic hand and foot features),

but no pathogenic variants in RPS6KA3 had been identified. In view of this, we

screened a cohort of 90 RPS6KA3 mutation-negative affected individuals with facial

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features suggestive of Coffin-Lowry syndrome, for the recurrent p.Arg913Ter variant

using a PCR and HphI restriction site digestion screen (see supplemental methods).

However, none of these 90 individuals carried the p.Arg913Ter variant. We also

used PCR and HphI restriction site digestion approach for screening this variant in

an additional cohort of 672 individuals who were recruited from South Australia with

a diagnosis of developmental delay, ID or autism, and who had normal fragile X and

chromosomal microarray but did not identify any further individuals carrying the

variant. The nonsense variant in individuals three-five was identified by exome

sequencing of 6,100 cases with a neurological phenotype at Baylor Genetics, and

one out of X cases with a neurological phenotype at INSERM, suggesting this variant

has a frequency of 4.9 x10-4 -X in individuals with neurological disorders.

The severe neurodevelopmental phenotype in individuals harbouring the

p.Arg913Ter variant is consistent with several lines of evidence showing

neurodevelopmental roles for ZSWIM6. ZSWIM6 is expressed in a localized and

developmentally regulated manner in the brain. In zebrafish larvae, zswim6 is

relatively highly expressed in regions of the telencephalon, midbrain, hindbrain and

retina4. In the embryonic mouse, Zswim6 is initially expressed in the ganglionic

eminences and subsequently also in the cortical plate, developing amygdala and

portions of the thalamus and hypothalamus. Postnatally, telencephalic expression

becomes more restricted to the striatum7. A similar distribution has been noted

during human brain development7. Zswim6 knockout mice have abnormal

neocortical and striatal development with reduced cortical and striatal volumes, and

alternations in the number and structure of medium spiny neurons in the striatum7.

These morphological changes are accompanied by alterations in motor control and

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behavior, including impaired learning on the accelerating rotarod, hyperactivity, and

an increase in stereotypical repetitive movements7. Finally, genome-wide association

(GWA) studies provide further support for a role for ZSWIM6 in brain development

and function. ZSWIM6 has been reported as one of the top 15 candidate genes

implicated most consistently across various GWA analyses with variation in

educational attainment8. ZSWIM6 was also included within a chromosomal region

that reached genome-wide significance for schizophrenia9; 10.

ZSWIM6 is a 14-exon gene at 5q12.1 and encodes a 133.5 kDa protein belonging to

a recently described group of proteins characterised by a zinc finger ‘SWIM’ (found

in SWI2/SNF2 and MuDR proteins) domain11 (Figure 1A). SWIM domains are

present in diverse archaeal, bacterial and eukaryotic proteins, including bacterial

ATPases of the SWI2/SNF2 family and vertebrate MEK kinase-1 and have putative

roles in DNA and protein binding 11. In addition to the SWIM domain, ZSWIM6

(NP_065979.1) contains a BC-box, Cul2-box, a Cut8/STS1-like domain and a C-

terminal Sin-3 like domain (amino acids 1148-1215)4 (Figure 1A). The position of the

p.Arg913Ter variant in the ZSWIM6 gene and protein is shown in Figure 1A. The

variant lies within the penultimate exon 13, 48 base pairs (bp) upstream from the last

exon/exon junction, suggesting that the ZSWIM6 PTC-encoding mRNA may escape

nonsense mediated decay (NMD). We therefore sought to confirm this in cells from

the affected individuals. Lymphoblastoid cell lines (LCLs) from affected individual one

and two and controls were generated using standard methods (see supplemental

data). Sanger sequencing of reverse transcribed ZSWIM6 mRNAs (cDNA) from

p.Arg913Ter LCLs (in affected individual one) cultured in the presence or absence of

a translational inhibitor cycloheximide which would suppress protein translation and

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as such NMD, showed that the p.Arg913Ter transcript is not subjected to NMD

(Figure 1B). This was consistent with the ZSWIM6 expression, as assayed by real-

time quantitative PCR (RT-qPCR) that showed no significant difference between the

affected individual and his unaffected brother and four unrelated male control LCLs

(Figure 1C). Sequencing of cDNA generated from peripheral blood of individual 2

also suggested that the variant does not result in NMD (Fig S1). We could not detect

the full length (in LCLs of unaffected brother of individual 1) or truncated (in affected

individual 1) ZSWIM6 protein using an anti-ZSWIM6 antibody (Sigma HPA035938)

raised against 634-696 aa of the ZSWIM6 protein. As an alternative commercial

antibody against the N-terminal region of ZSWIM6 protein is unavailable, we could

not determine the presence of a truncated ZSWIM6 protein, which is assumed to be

produced from the mRNA encoding the PTC.

This case series indicates that the recurrent de novo nonsense variant p.Arg913Ter

in ZSWIM6 should be considered causal of severe-profound autosomal dominant ID

associated with a wide-based ataxic gait, limited communication, happy disposition,

significant gastrointestinal symptoms, repetitive behaviors on the autistic spectrum,

distinctive facial features, progressive spasticity, weakness and strabismus. In one

older individual, a debilitating progressive peripheral neuropathy is a striking feature,

and we would recommend close surveillance of all affected individuals for this

potential complication, to determine how frequently this is part of the clinical

condition.

This report expands the phenotypes previously associated with pathogenic variants

in ZSWIM6. Previously, the recurrent de novo p.Arg1163Trp variant located within

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the C-terminal Sin3-like domain has been implicated in AFND3; 4. Although the

p.Arg913Ter cohort shares the severe-profound ID of affected individuals with

AFND, all lack the characteristic facial clefting, significant hypertelorism,

interhemispheric lipomas and limb anomalies. Moreover, although phenotype

expansions are recurrently described in the age of genomic sequencing12 and

challenge the traditional ‘one gene, one phenotype’ concept13 such exquisite

genotype-phenotype correlations are still relatively rare. Once such example is

FGFR1-related disorders where loss and gain of function mutations in FGFR1 cause

the very different syndromes of Kallman and Pfeiffer syndromes respectively14.

The molecular and cellular mechanisms to explain how the two ZSWIM6 variants

cause distinct phenotypes remain incompletely understood. Hypothesizing that the

recurrent p.Arg913Ter mutation is not a simple haploinsufficiency allele, we

examined the frequency of ZSWIM6 LoF variants or chromosomal deletions in case

and control populations. In the Database of Genomic Variants and the Copy Number

Variation Morbidity map available through the UCSC genome browser, the only

deletions affecting coding sequence correspond to a small, highly GC-rich region

containing exon 1 and are therefore likely false positives (Figure S2A and B). The

only LoF variants listed in the gnomAD database15 fall within a region of very poor

coverage in a highly GC-rich and repetitive region of exon 1 (Figure S2C); all are

indels and visual inspection suggests these are also artefactual. No individuals in our

in-house cohorts, DECIPHER or the literature have chromosomal deletions confined

to ZSWIM6. The shortest deletion involving ZSWIM6 in DECIPHER (affected

individual 267601) is a 2.91Mb deletion that also contains six other protein coding

genes including the OMIM morbid listed gene KIF2A, in which monoallelic missense

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variants have been reported to cause malformations of cortical development16. The

phenotype in this affected individual is mild ID in a healthy, sociable girl with short

stature and microcephaly (D. Fitzpatrick, personal communication). ZSWIM6 is one

of the 12 genes in the 2.63Mb shortest region of overlap of microdeletions at 5q12.1,

which are characterised by neurocognitive disorders5. Given the number of genes in

the above two intervals, the contribution of ZSWIM6 haploinsufficiency to the

phenotype remains uncertain. Analysis of over 10,000 whole exomes performed at

the Institut Imagine indicated one further LoF variant in ZSWIM6, p.Gln874Ter

(Figure 1A), in a proband with an immunodeficiency phenotype caused by a

homozygous recessive mutation in IFNGR2 (MIM 147569, NM_005534.3, c.679G>A,

p.Gly227Arg). Sanger sequencing indicated that this heterozygous ZSWIM6 variant

was inherited from the proband’s mother; neither individual had significant

neurological deficits. p.Gln874Ter, falling within exon 12, is predicted to result in

NMD and therefore suggests that ZSWIM6 haploinsufficiency does not cause ID. As

the p.Arg913Ter ZSWIM6 variant mRNA escapes NMD, cellular consequences may

be due to a dominant negative effect caused by the presence of truncated ZSWIM6

protein. Several human diseases are known to be caused by clustered C-terminal

truncating mutations17; 18. Although the variant encoding p.Arg913Ter falls on a CpG

dinucleotide, and therefore the recurrence of this variant may be influenced by

nucleotide composition, it is striking that no other truncating mutations in the final two

exons were identified. An intriguing possibility is that truncation at Arg913 results in a

highly specific alteration in protein structure not recapitulated by slightly more C-

terminal truncations. In any case, p.Arg913Ter would result in a ZSWIM6 protein that

lacks the C-terminal Sin3-like domain (Figure 1A). In comparison, the p.Arg1163Trp

variant that causes AFND lies within the Sin3-like domain, and is postulated to have

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a gain of function effect4. The Sin3-like domain has similarity to the four paired

amphipathic alpha-helix (PAH) motifs that are a prominent feature of the protein

encoded by the yeast Sin3 gene and its mammalian paralogues SIN3A and SIN3B 19

that code for part of the key SIN3-HDAC-MeCP2 transcriptional co-repressor

complex involved in gene regulation in neuronal progenitors20. The biochemical

function of the ZSWIM6 Sin3-like domain, and the divergent molecular

consequences of its truncation (p.Arg913Ter) or local surface alteration

(p.Arg1163Trp) will be an important area for future investigations.

Additional and or progressive neuronal features were described in several affected

individuals in the cohort, including progressive spasticity, movement disorders,

seizure disorders and abnormal pain threshold. A striking finding for individual one

was his prominent, progressive peripheral neuropathy. Sural nerve biopsy (Figure

2B) demonstrated neuropathological findings reminiscent of those seen in disorders

of ubiquitination such as giant axonal neuropathy 1 (GAN1) and also in certain toxic

neuropathies such as secondary to exposure to n-Hexane and acrylamide. GAN1

(MIM 256850) is an autosomal recessive progressive neurodegenerative disorder

characterised by a combination of peripheral motor and sensory neuropathy and

central nervous system impairment and caused by biallelic pathogenic variants in

GAN which encodes gigaxonin, a subunit of an E3 ubiquitin ligase21. This similarity in

pathology between a known disorder of ubiquitination and ZSWIM6 p.Arg913Ter-

related neuropathy is intriguing given a postulated role of ZSWIM6 in the ubiquitin

pathway7. ZSWIM6 has been shown by mass spectrometry to interact with the HECT

type ubiquitin E3 ligase HECW2, also known as NEDL2, in HEK293T cells 22. De

novo missense variants in HECW2 have recently been shown to cause

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neurocognitive disorders 23; 24. ZSWIM6 has a paralogue, ZSWIM8, the function of

whose orthologue, EBAX-1, has been studied in C. elegans. EBAX-1 encodes a

substrate recognition subunit in the Elongin BC-containing Cullin-RING ubiquitin E3

Ligase (CRL), and has been shown to play a role in the control of neuronal axon

pathfinding, and in collaboration with Hsp90, to regulate general protein quality within

axons25. Given these observations, we speculate that ZSWIM6 may have a role in a

protein ubiquitination pathway in neurons.

The recurrent p.Arg913Ter variant was found in 3/6,100 cases with a neurological

phenotype who had exome sequencing through Baylor Genetics but was not present

in any of the 60,706 unrelated individuals included in the ExAC database, which is

depleted of individuals with severe pediatric disease. Our screening of a cohort of

672 individuals with neurocognitive disease did not reveal any further individuals

carrying the variant. Although this variant is rare, we postulate that it is likely to be

present in more affected individuals who have already had diagnostic genomic

sequencing, as the lack of the distinctive AFND phenotype reported for the separate

recurrent p.Arg1163Trp may have meant that the variant was reported as a variant of

uncertain significance.

In summary we have described a new syndromic neurocognitive disorder due to a

recurrent de novo ZSWIM6 p.Arg913Ter variant which is distinct from the multiple

congenital anomaly disorder AFND, caused by the recurrent p.Arg1163Trp variant

within the C-terminal Sin3-like domain. The distinct clinical phenotypes likely reflect

differing molecular mechanisms: the p.Arg913Ter variant may result in a dominant

negative effect due to production of a truncated ZSWIM6 protein that lacks the Sin-3-

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like domain whereas the p.Arg1163Trp variant that may result in a gain of function.

Such exquisite genotype-phenotype relationships are still rare in clinical genetics,

however, this may be due to a bias in exome/genome data interpretation. Our study

further highlights genotype-phenotype complexity2 and serves as a cautionary tale to

clinicians and pathologists analysing high-throughput sequencing data not to

discount the possibility that a new clinical phenotype may result from novel variants

within a Mendelian gene. Our study also highlights the huge benefits of platforms

such as Matchmaker Exchange that can connect clinicians and genomicists and

result in the rapid building of cohorts of affected individuals to delineate the natural

history of novel genetic conditions2. The consistency of a severe ID phenotype

across both p.Arg913Ter and p.Arg1163Trp cohorts builds on accumulating

evidence that ZSWIM6 has critical roles in neuronal development and function, and

that this gene is a valid target for ongoing basic and clinical neuroscientific research.

APPENDICES

SUPPLEMENTAL DATA DESCRIPTION

The supplemental data contains four figures, supplemental methods, and two

supplemental tables.

CONFLICTS OF INTEREST

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The Department of Molecular and Human Genetics at Baylor College of Medicine

receives revenue from clinical genetic testing done at Baylor Genetics Laboratory.

ACKNOWLEDGEMENTS

The authors thank the affected individuals and their families for participation in this

study. JG was supported by NHMRC Program Grant 1091593 and Senior Research

Fellowship 1041920 and Channel 7 Children’s Research Foundation. The DDD

Study presents independent research commissioned by the Health Innovation

Challenge Fund (grant number HICF-1009-003), a parallel funding partnership

between the Wellcome Trust and the Department of Health, and the Wellcome Trust

Sanger Institute (grant number WT098051). The views expressed in this publication

are those of the authors and not necessarily those of the Wellcome Trust or the

Department of Health. The study has UK Research Ethics Committee approval

(10/H0305/83, granted by the Cambridge South REC, and GEN/284/12 granted by

the Republic of Ireland REC). We acknowledge the support of the National Institute

for Health Research, through the Comprehensive Clinical Research Network. CTG

and JA were supported by funding from the Agence Nationale de la Recherche

(ANR-10-IAHU-01, CranioRespiro). CB received support from the Fondation

maladies rares.

WEB RESOURCES

The URLs for data presented herein are as follows:

UCSC Genome Browser, http://genome.ucsc.edu

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DECIPHER, https://decipher.sanger.ac.uk/

Matchmaker Exchange, http://www.matchmakerexchange.org/

gnomAD database, http://gnomad.broadinstitute.org

ExAC database, http://exac.broadinstitute.org

CADD, http://cadd.gs.washington.edu

PROVEAN, http://provean.jcvi.org

Mendelian Inheritance in Man, http://www.omim.org

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FIGURE TITLES AND LEGENDS

Figure 1:

ZSWIM6 domain structure and variants and molecular data on mRNA encoding

the p.Arg913Ter variant

(A) Diagram showing predicted ZSWIM6 protein domains (lower) relative to exonic

structure (upper) and positions of the p.Arg913Ter and p.Gln874Ter variants

reported here (in red and green, respectively) and the previously reported

p.Arg1163Trp variant (information adapted from Tischfeld et al., 2017). The recurrent

missense variant p.Arg1163Trp is located within the C-terminal Sin3-like domain. For

clarity, the correspondence between the exon boundaries and the protein sequence

is depicted only for the final three exons. Introns not drawn to scale. (B) The mRNA

encoding p.Arg913Ter is not degraded by nonsense-mediated decay (NMD) in LCLs.

Sequence chromatograms showing normal and p.Arg913Ter-encoding reverse

transcribed mRNAs (cDNA) from LCLs cultured in the presence or absence of the

NMD inhibitor cycloheximide (100 g/6hr; CHX) and genomic DNA (gDNA) of the

affected individual and his unaffected brother. Presence of the variant mRNA in the

affected individual’s LCLs cultured without CHX indicates absence of NMD.

Normal/variant nucleotides are boxed. (C) ZSWIM6 expression levels do not vary

between affected individual 1 and his unaffected brother or four unrelated male

controls. ZSWIM6 expression was determined by real-time quantitative PCR and

normalised to the housekeeping gene HRPT1.

Figure 2:

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Facial and neurological features of individuals with the p.Arg913Ter ZSWIM6

variant. (A) facial features of individuals one to seven. In early childhood frequent

features include fine, arched eyebrows, short nose with depressed bridge and blunt

tip, broad columella, thick everted lower lip vermillion, widely-spaced teeth,

downturned corners to mouth, mouth often held open and esotropia. Individuals one

and six were noted with age to develop prominent forehead and supraorbital ridges,

thick eyebrows and thicker everted lower lip vermillion, reminiscent of the facial

appearance of older individuals with Coffin Lowry syndrome. This was not the case

for individual 6. (B) Sural nerve biopsy from individual one demonstrating

demyelination and abnormal accumulation of neurofilaments within numerous

myelinated axons (a) Low power electron micrograph showing three fibres containing

proliferated neurofilaments (arrows), amongst thinly myelinated fibres which

demonstrate the normal density of axonal filaments. (b) Accumulated neurofilaments

[n] in an axon with thick and folded myelin. (c) Demyelinated axon [d] packed with

neurofilaments that displace other axonal organelles. (d) High magnification of (c)

showing mitochondria and other organelles in septa between neurofilaments. (e)

Demyelinated axon with proliferated neurofilaments in distinct whorled bundles. (f)

High magnification of proliferated neurofilament bundle. The individual filaments

measure 9-15nm in diameter.

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TABLE TITLE AND LEGENDS

Table 1: Comparison of clinical features of affected individuals with the

p.Arg913Ter or p.Arg1163Trp ZSWIM6 variants.

Abbreviations: AFO, ankle foot orthoses; ASD, autism or autism spectrum disorder,

DN, de novo; GM, gross motor; GERD, gastro-esophageal reflux disease; GTC,

generalised tonic-clonic; ID, intellectual disability; IS, infantile spasms; mo, months;

MRI, magnetic resonance imaging; ND, not done; NT, not tested; OFC, occipital

frontal circumference; PECS, picture exchange communication system; yr, year.

#not inherited from mother, not present in either of two unaffected brothers, father not

available for testing; * mildly affected mother with mosaicism and fetus terminated at

20 weeks excluded.

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p.Arg913Ter affected

individual 1

p.Arg913Ter affected

individual 2

p.Arg913Ter affected

individual 3

p.Arg913Ter affected

individual 4

p.Arg913Ter affected

individual 5

p.Arg913Ter affected

individual 6

p.Arg913Ter affected individual 7

Summary p.Arg913Ter cohort (n=7)

Summary postnatal non-mosaic* p.Arg1163Trp cohort (n=6)

Inheritance not maternal# DN DN DN DN DN DN 6/7 DN; 1/7 unknown 4/6 DN; 1 NT; 1/6 inherited from mosaic

parentAge (yr) 16 7 4 5 3 29 29 3-29 yr unknown

Gender M F F F F M M 3/7 male (43%) 4/6 male (67%)

Level of ID severe-profound

severe severe severe severe profound severe 7/7 severe- profound (100%)

6/6 severe (100%)

OFC normocephaly progressive microcephaly

(-2S.D.)

progressive microcephaly

normocephaly progressive microcephal

y

progressive macrocephaly to 90-97th

centile

normocephaly

progressive microcephaly 3/7 (43%);

progressive macrocephaly 1/7 (14%)

NR

Infantile hypotonia / delayed GM milestones?

+ + + + + + + 7/7 (100%) 6/6 (100%)

ASD? + - + + + - + 5/7 (71%) NR

Communication non verbal non verbal; limited

comprehension

gestures; non verbal

few words; PECS

babble and one word

(no); starting to use PECS

vocalizes, one sign

short sentences, articulation difficulties

6/7 non-verbal or only few words (86%)

2/2 non-verbal (100%)

Ambulation non ambulant (wheelchair);

previously ambulant at 2

1/2yr with wide

unsteady gait

ambulant (from 3 yr) with

wide based ataxic gait

ambulant with wide unsteady gait (from 2 yr)

ambulant with wide unsteady

gait, stroller used for

distances.

starting to cruise.

ambulant with wide

based gait; wheelchair

for distances.

ambulant with broad

based unsteady

gait;

5/7 ambulant (71%) with wide based gait

2/2 non ambulant (100%)

Temperament/behavior

happy and affectionate, interested in family and

TV; hyperactivity

happy disposition;

bursts of laughter;

hyperactivity

happy; hyperactivity;

repetitive behaviors;

pica

interactive and sociable

happy, affectionate; loves music and water

play.

happy, hyperactivity and attention deficit; history

of pica.

4/7 hyperactivity (57%) NR

Epilepsy + - (under - + + + 4/7 seizure / possible 1/6 (17%)

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(GTC and focal

dyscognitive)

investigation for starting

episodes and unusual

movements)

(IS: controlled

with medication)

(infrequent GTC and

absence from age 9)

possible absence

seizures (no medication)

seizure disorder (57%)

Progressive spasticity + - - + - + - 3/7 (43%) NR

Movement disorder? paroxysmal hypertonicity;

unusual tongue

movements

stereotypical hand

movements; midline tongue

protusion; ataxia

paroxysmal hypertonicity;

unusual tongue

movements; ataxia

body rocking ; ataxia

stereotypical hand

movements; ataxia

- ataxia, tongue

thrusting, head tics

6/7 (86%) NR

Ophthalmological features

impairment lateral gaze

strabismus - accommodative esotropia

right sided esotropia

- right sided esotropia

5/7 (71%) variable: cataract,

glaucoma, myopia, optic

nerve hypoplasia

Brain MRI cortical atrophy (14

yr)

normal normal normal (7 mo) normal mild cortical atrophy (22

mo) but later MRI

considered normal

normal cortical atrophy 1/7 (14%)

interhemispheric lipoma 6/6

(100%); abnormal

corpus callosum 3/6 (50%) other

abnormalities variable

Additional neurological features

mixed peripheral neuropathy

- truncal hypotonia; abnormally high pain threshold

truncal hypotonia

bilateral ankle

pronation and toe pointing (wears AFOs);

torticollis

- abnormally high pain threshold

5/7 (71%) NR

Gastro-intestinal symptoms

failure to thrive and

severe GERD requiring

fundoplication; constipation;

ulcerative colitis from mid-teens;

infantile cow's milk protein intolerance.

recurrent diarrhoea and vomiting from

18 mo improved on

gluten free diet

GERD severe GERD requiring

gastrostomy

GERD and failure to

thrive

- intermittent constipation

6/7 (86%) significant GI symptoms

NR

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Additional non- neurological features

marked equinovarus deformity of

feet; thoracolumba

r scoliosis

otitis media with effusions

(requiring tympanostomy

)

premature eruption of

teeth

facial asymmetry; premature eruption of

teeth: puberty at 12; male

pattern baldness by

age 17

severe bilateral

planovalgus; nocturnal eneuresis

5/6 (83%) limb abnormalities; 2/4 males (50%) cryptorchism ; 1/6 (17%) scoliosis; 2/6 (33%) hypopituitism

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Supplemental Data

Supplemental Figures

Figure S1: Sequencing of ZSWIM6 cDNA generated from peripheral blood of individual two and a control

Figure S2: ZSWIM6 loss of function single nucleotide and copy number variants in public databases

Figure S3: MRI Brain individual One.

Supplemental Methods

1. Whole exome sequencing (WES) and whole genome sequencing (WGS)

2. PCR and HphI restriction site digestion screen for the c.2737C>T (p.Arg913Ter) variant

3. RNA expression analysis from LCLs

4. Analysis of cDNA in peripheral blood

Supplemental Tables

Table S1 Primer sequences

Table S2 Additional clinical information regarding individuals one-seven with the recurrent p.Arg913Ter variant (presented

as Excel file).

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