doi:10.1093/brain/awy184 BRAIN 2018: Page 1 of 4 | e1

LETTER TO THE EDITOR

A novel MCM3AP mutation in a Lebanese family with recessive Charcot-Marie-Tooth neuropathy

Marina L. Kennerson,1,2,3 Alastair C. Corbett,2,4 Melina Ellis,1 Gonzalo Perez-Siles1,2 and Garth A. Nicholson1,2,3

1 Northcott Neuroscience Laboratory, ANZAC Research Institute, Sydney, Australia 2 Sydney Medical School, University of Sydney, Sydney, Australia 3 Molecular Medicine Laboratory, Concord Hospital, Sydney, Australia 4 Department of Neurology, Concord Hospital, Sydney, Australia Correspondence to: Marina L. Kennerson Northcott Neuroscience Laboratory ANZAC Research Institute Gate 3 Hospital Road Concord NSW, 2139 Australia E-mail: [email protected]

Sir, progressive polyneuropathy and ptosis (Schuurs-Hoeijmakers The article recently published by Ylikallio et al. (2017) et al.,2013). highlights mutations in the mini chromosome maintenance We would like to report the use of whole exome sequen- complex component 3 associated protein gene (MCM3AP; cing to identify a novel missense mutation (NM_003906.3 OMIM 603294) as a new cause of autosomal recessive c.2609T4C p.Leu870Ser; chr21:g.47690334) in exon 9 of Charcot-Marie-Tooth (CMT) neuropathy associated with the MCM3AP gene in three siblings (two sisters and one intellectual disability (Ylikallio et al.,2017). The authors brother) of Lebanese ethnicity. The onset of slowly progres- identified four compound heterozygous mutations sive distal muscle weakness involving wrists, hand intrinsic (p.Pro148Leufs*48/p.Val1272Met; p.Tyr889*/p.Ala867Asp; muscles, feet and ankles occurred between ages 6 and 10 p.Gln619=/p.Asn1317fs*18; p.Ser1478*/p.Glu1577Lys) and years. Early motor milestones had been normal but by early a homozygous missense mutation (p.Met762Thr) in five teens all had developed foot drop, wrist and finger drop families of different ethnicity. Three of the mutant alleles and were significantly disabled. The parents were clinically were frameshift nonsense mutations in which one was de normal and distantly related. One additional brother had novo (p.Tyr889*). One of the mutant alleles was a synon- no neurological deficits and was unavailable for testing. ymous mutation (p.Gln619=) located in the splice donor posi- The three affected siblings were homozygous for the muta- tion of exon 5 and predicted to create an exonic splicing tion and both parents were heterozygous carriers for the silencer site. Since the article of Ylikallio et al., two additional p.Leu870Ser mutant allele (Fig. 1A). homozygous mutations have been reported in Kurdish The clinical features of the affected family members are (p.Arg878His) and Iranian (p.Ser951Pro) consanguineous shown in Table 1. When first reviewed at the Concord families and a compound heterozygous mutation Hospital Adult Neuromuscular Clinic aged in their early (Gln1804*/Ser145_Leu1459dup) in a non-consanguineous 20 s, the affected siblings all had marked wasting of hand Iranian family (Karakaya et al.,2017). Prior to the report intrinsic and forearm muscles and wasting of the foot of this gene for recessive CMT, studies of families with intel- intrinsic and leg anterior and posterior compartment mus- lectual disability described two siblings with a homozygous cles. Proximal muscle bulk was normal. All had grade 1–2 variant in the MCM3AP gene (p.Glu915Lys). Interestingly, power in hand intrinsic muscles and finger extensors with this gene was selected as a candidate gene in which the sib- grade 4 strength in long finger flexors and wrist flexors. lings presented with borderline to mild intellectual disability, Proximal upper limb power remained normal. In the legs

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Figure 1 The p.Leu870Ser MCM3AP mutation in a Lebanese family localizes to the Sac3 domain of GANP. (A) The pedigree shows segregation of the mutation that has been confirmed by (B) Sanger sequencing analysis traces. Circles denote females and squares denote males. The asterisk denotes the nucleotide position at which a homozygous T to C transition is observed in all affected siblings and both parents are heterozygous for the T to C transition. A sequence trace of a neurologically normal unrelated individual shows a homozygous T allele. (C) Alignment analysis for the p.Leu870Ser mutation for MCM3AP orthologues in different species. Amino acid position 870 is boxed in red. (D) GANP protein showing the different domains and location of mutations for recessive CMT. Previously reported mutations (below) and the novel mutation in this study (above) are shown. (E) Structure of the TREX2 complex including the GANP protein, showing CMT causing mutations within the Sac3 domain. Diagram was drawn using the crystal structure of the S. cerevisiae homologue (PDB: 3t5v) (Ellisdon et al., 2012). Sac3 residues are shown in blue, in complex with Thp1 (human homologue PCID2) in red and Sem1 (human homologue DSS1) in yellow. CMT mutations are shown as grey sticks and the newly identified p.Leu870Ser mutation in orange. Box area is enlarged to show CMT mutations enriched around the Sac3/Thp1 interaction interface (Sac3 interacting residues highlighted in green).

power was graded as ankle dorsiflexion 2, peronei 2, tibia- developed obstructive sleep apnoea but no dysfunction of lis posterior 4, plantar flexion 4 + with normal power bulbar musculature. proximally. Upper limb and knee tendon reflexes were Two of the affected siblings have had one or more nerve normal and ankle jerks could just be elicited. All three conduction studies. At age 26 years the youngest daughter had reduced sensation to pain and temperature to the (Patient II:3) had nerve conduction studies demonstrating mid forearm and mid-thigh. All had intact vibration and normal median, ulnar and sural sensory nerve action poten- proprioception. The affected siblings were unsteady but tials. The motor compound action potentials were absent independently mobile with bilateral foot drop and frequent or markedly reduced in the upper limbs and reduced but falls. All married in their early 20 s and have managed to less affected in the legs. Motor conduction velocities were maintain independent gait and continued to manage their reduced but sensory velocities normal. For the oldest sibling activities of daily living with assistance from their spouses (Patient II:1) at age 24 years, sensory nerve action poten- and parents. Each has had more than one child all of tials could be elicited but the amplitudes were reduced. whom remain normal. All of the affected siblings have Motor compound action potentials were reduced or become increasing obese and two have required treatment absent, terminal motor latencies normal and motor conduc- for diabetes mellitus type 2 with oral agents. All three have tion velocities reduced. By age 28 there was a significant

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Table 1 Summary of clinical features of the patients

Patient II:1 II:2 II:3 Gender F M F First evaluation age 23 25 21 UL weakness proximal/distal À/+ + + À/+++ À/+ + + LL weakness proximal/distal À/+ + + À/+++ À/+ + + Bilateral foot drop + + + + + + + + + Sensory loss Distally to pain Distally to pain Distally to pain Nerve conduction studies age 24 years 26 years Median motor, mV/m/s Absent Absent Ulnar motor, mV/m/s 0.6/38.4 Not recorded 0.3 Tibial motor, mV/m/s 2.4 Not recorded 4.2/43 Common peroneal motor, mV/m/s Absent Absent Median sensory, mV/m/s 7.3/49.8 Not recorded 6.9/48 Ulnar, mV/m/s 3.7/52.6 Not recorded 4/52.4 Sural, mV/m/s Not recorded Not recorded 5.1/48 Posterior tibial evoked potentials Normal Not recorded Normal Brain MRI Middle fossa arachnoid cysts; Normal otherwise normal Spinal MRI Normal Additional medical problems Obesity, DM type 2, psychosis Obesity Obesity, epigastric hernia repair, DM type 2

DM = diabetes mellitus; F = female; LL = lower limb; M = male; UL = upper limb. À = normal power; + + + = less than antigravity power.

deterioration in both motor and sensory parameters with with homozygous mutations all present with debilitating neuro- sural sensory potentials no longer recordable. Needle EMG pathy with two mutations (p.Met762Thr and p.Glu915Lys) demonstrated active and chronic neurogenic changes. associated with intellectual disability. Posterior tibial nerve somatosensory evoked potentials The MCM3AP gene encodes the germinal centre asso- recorded in the early 20 s for Patients II:1 and II:3 demon- ciated nuclear protein (GANP) and is ubiquitously strated normal lumbar and cerebral potential waveforms expressed (Abe et al., 2000). The nuclear phase of the and latencies. None of the siblings experienced significant gene expression pathway is the export of mature mRNAs pain, sensory symptoms or cramping. All were able to walk to the cytoplasm through nuclear pore complexes. GANP is independently and have used ankle foot orthoses intermit- a subunit of TREX-2, which is an important mRNA export tently. Because of severe hand weakness they were not able complex (Jani et al., 2012). All the homozygous mutations to use their hands to hold walking aids. The eldest sibling reported to date cluster in the Sac3 domain (Fig. 1D). (Patient II:1) developed psychosis with catatonia requiring Modelling the mutations in the crystal structure of the treatment with electroconvulsive therapy subsequently com- Saccharomyces cerevisiae homologue (Ellisdon et al., pletely controlled. 2012) reveals that they lie in close proximity to the Sac3- The p.Leu870Ser mutation we report was absent from ThpI (human PCID2) binding interface (Fig. 1E). public databases including dbSNP 150, gnomAD, 1000 Maintenance of this interface is needed for mRNA export genomes and the Greater Middle East Variome Project through the nuclear pore complexes. It is therefore possible (GME Variome). The variant was also excluded from 200 that our mutation and others reported within this domain neurologically normal chromosomes (n = 100 in-house con- could affect mRNA export by disturbing the Sac3-PCID2 trols). The amino acid is highly conserved and a suite of interface; however, further functional studies will be prediction tools suggest the mutation is disease causing required to confirm this hypothesis. Mutations in the MCM3AP gene highlight the importance of understanding (Fig. 1C and Supplementary Table 1). As reported for the mRNA export in the pathogenic process of axonal degen- other MCM3AP homozygous mutations, the CADD score eration that underlies CMT. for p.Leu870Ser (29.0) was 420 indicating the substitution is in the top 1% most deleterious changes to occur in the genome (Kircher et al., 2014). As noted in previous studies, the genotype–phenotype corre- Web resources lation of patients with MCM3AP mutations is unclear. With The URLs used in this study: the addition of the p.Leu870Ser mutation, there are now five Online Mendelian Inheritance in Man; https://www.omim. homozygous mutations reported. Interestingly, the homozygous org/ mutations are all located in the suppressor of actin 3 (Sac3) UCSC Genome Browser; https://genome.ucsc.edu/ domain (amino acid 366–990) of GANP. Patients presenting Combined Annotation Dependent Depletion (CADD; http://

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cadd.gs.washington.edu/home Genome Aggregation Database gnomAD; http://gnomad. References broadinstitute.org/ Abe E, Kuwahara K, Yoshida M, Suzuki M, Terasaki H, Matsuo Y, 1000 Genomes Browser; http://www.internationalgenome. et al. Structure, expression, and chromosomal localization of the org/1000-genomes-browsers/ human gene encoding a germinal center-associated nuclear protein The Greater Middle East Variome Project GME; http://igm. (GANP) that associates with MCM3 involved in the initiation of DNA replication. Gene 2000; 255: 219–27. ucsd.edu/gme/ Ellisdon AM, Dimitrova L, Hurt E, Stewart M. Structural basis Mutation Taster; http://www.mutationtaster.org/ for the assembly and nucleic acid binding of the TREX-2 Polyphen; http://genetics.bwh.harvard.edu/pph2/ transcription-export complex. Nat Struct Mol Biol 2012; 19: SIFT and Provean; http://sift.jcvi.org/ 328–36. Clustal Omega; https://www.ebi.ac.uk/Tools/msa/clustalo/ Jani D, Lutz S, Hurt E, Laskey RA, Stewart M, Wickramasinghe VO. Functional and structural characterization of the mammalian TREX- 2 complex that links transcription with nuclear messenger RNA export. Nucleic Acids Res 2012; 40: 4562–73. Acknowledgements Karakaya M, Mazaheri N, Polat I, Bharucha-Goebel D, Donkervoort S, Maroofian R, et al. Biallelic MCM3AP mutations cause Charcot- We would like to thank the family members for participat- Marie-Tooth neuropathy with variable clinical presentation. Brain ing in this study. 2017; 140: e65. Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet 2014; 46: 310–15. Funding Schuurs-Hoeijmakers JH, Vulto-van Silfhout AT, Vissers LE, van This work was supported by the Australian National de Vondervoort II, van Bon BW, de Ligt J, et al. Identification of pathogenic gene variants in small families with intellectually dis- Health and Medical Research Council project grant abled siblings by exome sequencing. J Med Genet 2013; 50: (APP1046690) awarded to M.L.K and G.A.N. 802–11. Ylikallio E, Woldegebriel R, Tumiati M, Isohanni P, Ryan MM, Stark Z, et al. MCM3AP in recessive Charcot-Marie-Tooth neur- opathy and mild intellectual disability. Brain 2017; 148: Supplementary material 2093–103. Supplementary material is available at Brain online.

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