LETTER TO THE EDITOR

Clinical and Neuropathological Features of Spastic Ataxiain a Spanish Family with Novel Compound HeterozygousMutations in STUB1

Conceição Bettencourt & Justo García de Yébenes & José Luis López-Sendón &

Orr Shomroni & Xingqian Zhang & Shu-Bing Qian & Ingrid M. C. Bakker &

Sasja Heetveld & Raquel Ros & Beatriz Quintáns & María-Jesús Sobrido &

Marianna R. Bevova & Shushant Jain & Marianna Bugiani & Peter Heutink &

Patrizia Rizzu

# Springer Science+Business Media New York 2015

Hereditary ataxias are clinically and genetically heteroge-neous neurodegenerative disorders. Although many ataxiagenes have been identified, about 50 % of cases await theidentification of the genetic cause [1]. High-throughput se-quencing, namely whole-exome sequencing (WES), has

changed the paradigm of genetic analysis of rare heteroge-neous Mendelian disorders [2], and allowed the identificationof STUB1 mutations as the cause of autosomal recessiveataxia syndromes [3–7]. We report on clinical and pathologi-cal features of a family with spastic ataxia due to novel STUB1

Electronic supplementary material The online version of this article(doi:10.1007/s12311-014-0643-7) contains supplementary material,which is available to authorized users.

C. BettencourtDepartment ofMolecular Neuroscience, UCL Institute of Neurology,London, UK

C. BettencourtInstitute for Molecular and Cell Biology (IBMC), University ofPorto, Porto, Portugal

C. Bettencourt (*)Center of Research in Natural Resources (CIRN) and Department ofBiology, University of the Azores, Ponta Delgada, Portugale-mail: c.bettencourt@ucl.ac.uk

J. G. de Yébenes : J. L. López-Sendón : R. RosDepartment of Neurology, Hospital Ramón y Cajal, Madrid, Spain

O. Shomroni : I. M. C. Bakker : S. Heetveld :M. R. Bevova :S. Jain : P. Heutink : P. RizzuDepartment of Clinical Genetics, Neuroscience Campus Amsterdam,VU University Medical Center, Amsterdam, The Netherlands

X. Zhang : S.<B. QianDivision of Nutritional Sciences, Cornell University, Ithaca,NY, USA

B. Quintáns :M.<J. SobridoFundación Pública Galega de Medicina Xenómica and HealthResearch Institute of Santiago de Compostela (IDIS), ClinicalHospital of Santiago de Compostela-SERGAS, Santiago deCompostela, Spain

M. R. BevovaEuropean Research Institute for the Biology of Ageing,Groningen, The Netherlands

S. Jain : P. Heutink : P. RizzuGermanCenter for NeurodegenerativeDiseases, Tübingen, Germany

M. BugianiDepartment of Pediatrics, Child Neurology, Neuroscience CampusAmsterdam, VU University Medical Center,Amsterdam, The Netherlands

M. BugianiDepartment of Pathology, VU University Medical Center,Amsterdam, The Netherlands

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mutations identified by WES. The study had ethicscommittee approval, and all subjects provided writteninformed consent.Two out of three siblings born to nonconsanguineous healthySpanish parents were affected (Fig. 1), suggesting autosomalrecessive inheritance. The older sibling (proband) was a wom-an who presented progressive incoordination of movementand unsteadiness since her 20s. Initial examination revealedupper and lower limb dysmetria, dysdiadochokinesia, cerebel-lar dysarthria, and saccadic ocular pursuit with increasedtendon reflexes in the four limbs. Assessment of other neuro-logical systems was initially unremarkable. Ataxia and pyra-midal tract dysfunction became more prominent, and unas-sisted walking became impossible in her late 30s. She laterpresented with cognitive impairment that progressed to

dementia, mild generalized choreatric movements, and spon-taneous myoclonus. At age 52, she was bedridden and suf-fered severe neurogenic dysphagia, which led to aspirationpneumonia and death. Her brother was diagnosed with ataxiawith retained reflexes at age 22. Subsequent neurologicalexaminations revealed a more generalized cerebellar deficitcausing dysarthria, limb ataxia, wide-base gait, and hyperac-tive tendon reflexes with lower limb spasticity. In his 40s, heexhibited progressive neurological deterioration with general-ized ataxia, spasticity, myoclonus, and cognitive deteriorationthat led to dementia. He died at age 46 due to neurologicalcomplications. Metabolic and endocrine studies as well aselectromyogram and nerve conduction velocities were normalin both patients. MRI brain scans revealed progressive midlineand hemispheric cerebellar atrophy. We did not find any signs

Fig. 1 a Pedigree of a Spanish ataxia family showing the STUB1mutations identified in each family member. The arrow points to theproband.WTwild-type;M1: heterozygous for the c.633G>A, p.Met211Ilemutation; M2: heterozygous for the c.712G>T, p.Glu238Ter mutation*Whole-exome sequencing was performed for these individuals. b STUB1

mutations found in the ataxia family, highlighting the conservation be-tween species at those positions. c CHIP protein domain structures (tetra-tricopeptide repeat domains—TPR and U-Box domain) with all STUB1mutations described in the literature depicted (in red are highlighted themutations found in our family)

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of hypogonadism in any of the patients. The parents (in their80s), the younger sibling, and the two daughters of the olderpatient remain unaffected.

Previous classical genetic studies excluded majorataxia-causing mutations (Appendix e–1). WES revealedcompound heterozygous variants in STUB1 gene(c.633G>A, p.Met211Ile and c.712G>T, p.Glu238Ter) tobe segregating with the phenotype (Fig. 1, Appendix e–1).These variants are located in highly conserved residues(Fig. 1b), with a 100 vertebrates basewise conservation scoreby PhyloP (available at www.genome.ucsc.edu) of 8.8 and 9.7 for p. Met211Ile and p.Glu238Ter, respectively.Furthermore, both variants are predicted as diseasecausing (>0.99) by Mutation Taster [8], and are notpresent in ∼560 population-matched control chromosomes

or in public databases. By screening a Spanish ataxia cohort(N=185), no homozygous or compound heterozygous vari-ants were found. STUB1 (OMIM# 607207) encodes the C-terminal of Hsp70-interacting protein (CHIP), a dimeric pro-tein involved in the metabolism of protein misfolding. The p.Glu238Ter mutation causes a premature stop codon leading tothe truncation of the protein and/or to nonsense-mediateddecay, while p.Met211Ile is located in a conserved residue,essential for CHIP dimerization and function. Both HEK293cells expressing these mutants (Supplementary Fig. e–1) andproband’s fibroblasts (reported elsewhere [9]) show decreasedlevels of CHIP when compared with controls, supporting aloss-of-function mechanism.

Neuropathological analysis of the proband’s brotherbrain revealed dramatic cerebellar cortical cell loss, with

Fig. 2 Neuropathologicalexamination of an ataxia patientwith compound heterozygousmutations in the STUB1 gene(p.Met211Ile and p.Glu238Ter).a, b Hematoxylin-eosin stainingshowing severe decrease ofPurkinje cells and neurons of thegranular layer in the cerebellumof the patient (b) as compared tothe control brain (a). c, d CHIP-labeling showing cytoplasmicstaining in neurons of theparaventricular nuclei of thehypothalamus (c) and extendingto the apical part of axons inneurons of the temporal cortex(d). e Varicosities labeled withCHIP antibody in the nerveprocesses of the frontal cortex. fPredominant nuclear stainingwith CHIP antibody in thepyramidal neurons of themedio-entorhinal cortex

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severe loss of Purkinje cells and neurons of the granularlayer, accompanied by reactive Bergmann gliosis(Fig. 2a, b). Staining with CHIP antibody showed main-ly cytoplasmic diffuse reactivity in neurons, often ex-tending to dendrites and axons (Fig. 2c, d), and also inastrocytes. Some swollen axonal processes in the frontalwhite matter stained as well (Fig. 2e). Remarkably,intense nuclear staining in neurons of the deep layersof the frontal cortex and in the pyramidal stellate neu-rons of medio-entorhinal cortex, with only mild cyto-plasmic staining, was observed in this patient (Fig. 2f).Staining with HSP70-antibody showed an overlap withCHIP in all investigated brain tissues. P62-antibodystained a few neuronal intranuclear inclusions in smallneurons of frontal cortex and caudate, and seldom ap-parently free-lying bodies in the cerebellum and putamen(data not shown).

Mutations in STUB1 have been recently described inGordon Holmes syndrome and in additional autosomalrecessive forms of ataxia [3–7]. The phenotype of ourpatients is similar to most of the previous reports,including an ataxic syndrome with onset in the mid-20s, progressive pyramidal tract dysfunction, and myoc-lonus. We also observed an increasing impairment ofmemory, language, and executive function leading todementia in the later stages of the disease, likely relatedto a delayed involvement of frontal and temporal cortexas revealed by the neuropathological findings. In addi-tion, our patients presented acute and partially reversibleepisodes of sudden neurological deterioration, unreport-ed in other cases with mutations in the STUB1 gene.All previous reports suggest a classical loss-of-functionmechanism of the protein—CHIP, a chaperone widelyexpressed in the nervous system. In our family, thepresence of compound heterozygous mutations and apedigree compatible with an autosomal recessive inher-itance is in line with those reports. Our study addspathological information and expands the mutationalspectrum of STUB1-associated ataxias. This study fur-ther reinforces the utility of clinical genomics in thediagnosis of heterogeneous neurodegenerative disorders.

Acknowledgments The authors thank all the patients who participatein this study and their families, as well as people involved in the collectionof samples; the Netherlands Brain Bank (Amsterdam, the Netherlands)for providing brain tissue of the controls; and A. Ingrassia and Dr W.D.van de Berg for technical support in the immunohistochemistry experi-ments. This study was supported by the “CIBERNED,” the “Ministeriode Economía y Competitividad—“Gobierno de España” (AIB2010PT-00182) and “Instituto de Salud Carlos III (PI12/00742),” and the “Xuntade Galicia, Dirección Xeral de Investigación, Desenvolvemento eInnovación” (10 PXIB 9101 280 PR). CB was recipient of a postdoctoralfellowship from the “Fundação para a Ciência e a Tecnologia”—FCT[SFRH/BPD/63121/2009] until January 2013, and is presently supportedby the Medical Research Council (UK).

Conflict of Interest The authors declare no conflict of interest.

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