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DOI 10.1007/s12311-014-0643-7
LETTER TO THE EDITOR
Clinical and Neuropathological Features of Spastic Ataxia
in a Spanish Family with Novel Compound Heterozygous
Mutations 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 heterogeneous neurodegenerative disorders. Although many ataxia
genes have been identified, about 50 % of cases await the
identification of the genetic cause [1]. High-throughput sequencing, namely whole-exome sequencing (WES), has
changed the paradigm of genetic analysis of rare heterogeneous Mendelian disorders [2], and allowed the identification
of STUB1 mutations as the cause of autosomal recessive
ataxia syndromes [3–7]. We report on clinical and pathological 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. Bettencourt
Department of Molecular Neuroscience, UCL Institute of Neurology,
London, UK
C. Bettencourt
Institute for Molecular and Cell Biology (IBMC), University of
Porto, Porto, Portugal
C. Bettencourt (*)
Center of Research in Natural Resources (CIRN) and Department of
Biology, University of the Azores, Ponta Delgada, Portugal
e-mail: [email protected]
J. G. de Yébenes : J. L. López-Sendón : R. Ros
Department of Neurology, Hospital Ramón y Cajal, Madrid, Spain
B. Quintáns : M.<J. Sobrido
Fundación Pública Galega de Medicina Xenómica and Health
Research Institute of Santiago de Compostela (IDIS), Clinical
Hospital of Santiago de Compostela-SERGAS, Santiago de
Compostela, Spain
M. R. Bevova
European Research Institute for the Biology of Ageing,
Groningen, The Netherlands
S. Jain : P. Heutink : P. Rizzu
German Center for Neurodegenerative Diseases, Tübingen, Germany
O. Shomroni : I. M. C. Bakker : S. Heetveld : M. R. Bevova :
S. Jain : P. Heutink : P. Rizzu
Department of Clinical Genetics, Neuroscience Campus Amsterdam,
VU University Medical Center, Amsterdam, The Netherlands
M. Bugiani
Department of Pediatrics, Child Neurology, Neuroscience Campus
Amsterdam, VU University Medical Center,
Amsterdam, The Netherlands
X. Zhang : S.<B. Qian
Division of Nutritional Sciences, Cornell University, Ithaca,
NY, USA
M. Bugiani
Department of Pathology, VU University Medical Center,
Amsterdam, The Netherlands
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mutations identified by WES. The study had ethics
committee approval, and all subjects provided written
informed consent.
Two out of three siblings born to nonconsanguineous healthy
Spanish parents were affected (Fig. 1), suggesting autosomal
recessive inheritance. The older sibling (proband) was a woman who presented progressive incoordination of movement
and unsteadiness since her 20s. Initial examination revealed
upper and lower limb dysmetria, dysdiadochokinesia, cerebellar dysarthria, and saccadic ocular pursuit with increased
tendon reflexes in the four limbs. Assessment of other neurological systems was initially unremarkable. Ataxia and pyramidal tract dysfunction became more prominent, and unassisted walking became impossible in her late 30s. She later
presented with cognitive impairment that progressed to
dementia, mild generalized choreatric movements, and spontaneous myoclonus. At age 52, she was bedridden and suffered severe neurogenic dysphagia, which led to aspiration
pneumonia and death. Her brother was diagnosed with ataxia
with retained reflexes at age 22. Subsequent neurological
examinations revealed a more generalized cerebellar deficit
causing dysarthria, limb ataxia, wide-base gait, and hyperactive tendon reflexes with lower limb spasticity. In his 40s, he
exhibited progressive neurological deterioration with generalized ataxia, spasticity, myoclonus, and cognitive deterioration
that led to dementia. He died at age 46 due to neurological
complications. Metabolic and endocrine studies as well as
electromyogram and nerve conduction velocities were normal
in both patients. MRI brain scans revealed progressive midline
and hemispheric cerebellar atrophy. We did not find any signs
Fig. 1 a Pedigree of a Spanish ataxia family showing the STUB1
mutations identified in each family member. The arrow points to the
proband. WT wild-type; M1: heterozygous for the c.633G>A, p.Met211Ile
mutation; 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 between species at those positions. c CHIP protein domain structures (tetratricopeptide repeat domains—TPR and U-Box domain) with all STUB1
mutations described in the literature depicted (in red are highlighted the
mutations found in our family)
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of hypogonadism in any of the patients. The parents (in their
80s), the younger sibling, and the two daughters of the older
patient remain unaffected.
Previous classical genetic studies excluded major
ataxia-causing mutations (Appendix e–1). WES revealed
compound heterozygous variants in STUB1 gene
(c.633G>A, p.Met211Ile and c.712G>T, p.Glu238Ter) to
be 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 score
by 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 disease
causing (>0.99) by Mutation Taster [8], and are not
present in ∼560 population-matched control chromosomes
Fig. 2 Neuropathological
examination of an ataxia patient
with compound heterozygous
mutations in the STUB1 gene
(p.Met211Ile and p.Glu238Ter).
a, b Hematoxylin-eosin staining
showing severe decrease of
Purkinje cells and neurons of the
granular layer in the cerebellum
of the patient (b) as compared to
the control brain (a). c, d CHIPlabeling showing cytoplasmic
staining in neurons of the
paraventricular nuclei of the
hypothalamus (c) and extending
to the apical part of axons in
neurons of the temporal cortex
(d). e Varicosities labeled with
CHIP antibody in the nerve
processes of the frontal cortex. f
Predominant nuclear staining
with CHIP antibody in the
pyramidal neurons of the
medio-entorhinal cortex
or in public databases. By screening a Spanish ataxia cohort
(N=185), no homozygous or compound heterozygous variants were found. STUB1 (OMIM# 607207) encodes the Cterminal of Hsp70-interacting protein (CHIP), a dimeric protein involved in the metabolism of protein misfolding. The p.
Glu238Ter mutation causes a premature stop codon leading to
the truncation of the protein and/or to nonsense-mediated
decay, while p.Met211Ile is located in a conserved residue,
essential for CHIP dimerization and function. Both HEK293
cells expressing these mutants (Supplementary Fig. e–1) and
proband’s fibroblasts (reported elsewhere [9]) show decreased
levels of CHIP when compared with controls, supporting a
loss-of-function mechanism.
Neuropathological analysis of the proband’s brother
brain revealed dramatic cerebellar cortical cell loss, with
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severe loss of Purkinje cells and neurons of the granular
layer, accompanied by reactive Bergmann gliosis
(Fig. 2a, b). Staining with CHIP antibody showed mainly cytoplasmic diffuse reactivity in neurons, often extending to dendrites and axons (Fig. 2c, d), and also in
astrocytes. Some swollen axonal processes in the frontal
white matter stained as well (Fig. 2e). Remarkably,
intense nuclear staining in neurons of the deep layers
of the frontal cortex and in the pyramidal stellate neurons of medio-entorhinal cortex, with only mild cytoplasmic staining, was observed in this patient (Fig. 2f).
Staining with HSP70-antibody showed an overlap with
CHIP in all investigated brain tissues. P62-antibody
stained a few neuronal intranuclear inclusions in small
neurons of frontal cortex and caudate, and seldom apparently free-lying bodies in the cerebellum and putamen
(data not shown).
Mutations in STUB1 have been recently described in
Gordon Holmes syndrome and in additional autosomal
recessive forms of ataxia [3–7]. The phenotype of our
patients is similar to most of the previous reports,
including an ataxic syndrome with onset in the mid20s, progressive pyramidal tract dysfunction, and myoclonus. We also observed an increasing impairment of
memory, language, and executive function leading to
dementia in the later stages of the disease, likely related
to a delayed involvement of frontal and temporal cortex
as revealed by the neuropathological findings. In addition, our patients presented acute and partially reversible
episodes of sudden neurological deterioration, unreported in other cases with mutations in the STUB1 gene.
All previous reports suggest a classical loss-of-function
mechanism of the protein—CHIP, a chaperone widely
expressed in the nervous system. In our family, the
presence of compound heterozygous mutations and a
pedigree compatible with an autosomal recessive inheritance is in line with those reports. Our study adds
pathological information and expands the mutational
spectrum of STUB1-associated ataxias. This study further reinforces the utility of clinical genomics in the
diagnosis of heterogeneous neurodegenerative disorders.
Acknowledgments The authors thank all the patients who participate
in this study and their families, as well as people involved in the collection
of 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 experiments. This study was supported by the “CIBERNED,” the “Ministerio
de Economía y Competitividad—“Gobierno de España” (AIB2010PT00182) and “Instituto de Salud Carlos III (PI12/00742),” and the “Xunta
de Galicia, Dirección Xeral de Investigación, Desenvolvemento e
Innovación” (10 PXIB 9101 280 PR). CB was recipient of a postdoctoral
fellowship from the “Fundação para a Ciência e a Tecnologia”—FCT
[SFRH/BPD/63121/2009] until January 2013, and is presently supported
by the Medical Research Council (UK).
Conflict of Interest The authors declare no conflict of interest.
References
1. Hersheson J, Haworth A, Houlden H. The inherited ataxias: genetic
heterogeneity, mutation databases, and future directions in research
and clinical diagnostics. Hum Mutat. 2012;33(9):1324–32.
2. Bettencourt C, Lopez-Sendon J, Garcia-Caldentey J, Rizzu P, Bakker
I, Shomroni O, et al. Exome sequencing is a useful diagnostic tool for
complicated forms of hereditary spastic paraplegia. Clinical genetics.
2014;85(2):154–8.
3. Shi CH. Schisler JC. Tan S, Song B, McDonough H, et al. Ataxia and
hypogonadism caused by the loss of ubiquitin ligase activity of the U
box protein CHIP. Human molecular genetics. 2014;23(4):1013–24.
4. Shi Y, Wang J, Li JD, Ren H, Guan W, He M, et al. Identification of
CHIP as a novel causative gene for autosomal recessive cerebellar
ataxia. PLoS One. 2013;8(12):e81884.
5. Depondt C, Donatello S, Simonis N, Rai M, van Heurck R,
Abramowicz M, et al. Autosomal recessive cerebellar ataxia of adult
onset due to STUB1 mutations. Neurology. 2014;82(19):1749–50.
6. Cordoba M, Rodriguez-Quiroga S, Gatto EM, Alurralde A, Kauffman
MA. Ataxia plus myoclonus in a 23-year-old patient due to STUB1
mutations. Neurology. 2014;83(3):287–8.
7. Synofzik M, Schule R, Schulze M, Gburek-Augustat J, Schweizer R,
Schirmacher A, et al. Phenotype and frequency of STUB1 mutations:
next-generation screenings in Caucasian ataxia and spastic paraplegia
cohorts. Orphanet j rare dis. 2014;9(1):57.
8. Schwarz JM, Cooper DN, Schuelke M, Seelow D. MutationTaster2:
mutation prediction for the deep-sequencing age. Nat Methods.
2014;11(4):361–2.
9. Casarejos MJ, Perucho J, Lopez-Sendon JL. Garcia de Yebenes J,
Bettencourt C, Gomez A, et al. Trehalose Improves Human
Fibroblast Deficits in a New CHIP-Mutation Related Ataxia. PLoS
One. 2014;9(9):e106931.