Gerstmann-Sträussler-Scheinker disease - prion diseases - prions
doença de Gerstmann-Sträussler-Scheinker - doenças de prion - príons
Human spongiform encephalopathies, or prion diseases, are rare fatal neurodegenerative
conditions associated with conversion of prion protein (PrPC) to its misfolded pathogenic
isoform (PrPsc)[1]. According to their etiology, prion diseases can be classified as sporadic, genetic
or acquired by infection and their estimated annual incidence is approximately one
per million individuals[2],[3],[4].
Genetic forms of prion diseases are inherited as an autosomal dominant trait and associated
with mutations in the prion protein gene (PRNP), accounting for around 15% of cases[5]. The PRNP mutations lead to three major phenotypes: Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker
(GSS) disease and familial fatal insomnia[4].
Gerstmann-Sträussler-Scheinker disease was first reported in an Austrian family in
1936 and its main clinical features are slowly progressive cerebellar ataxia associated
with cognitive decline[6]. The molecular basis of GSS was identified in 1989, when the substitution of a proline
at position 102 for leucine (p.Pro102Leu) in the PRNP gene was recognized in two families[7]; this same variant was subsequently identified in the original Austrian family[8]. A number of other mutations associated with GSS were also reported, predominantly
missense and nonsense pathogenic variants located at codons 105, 117, 131, 187, 198,
202, 212, 217, and 232, or copy number variation in octapeptide repeats[9]. The common variant in the PRNP gene, p.Met129Val, might act as a disease modifier in prion disease, but alone is
not solely responsible for this condition. Clinical and pathological phenotypic variability
among individuals with the same mutation had previously been reported worldwide[10],[11],[12],[13],[14]. Several factors may contribute to this variability, such as the genotype for the
variant p.Met129Val and differences in molecular prion strains[14],[15],[16]. A study conducted in the largest reported GSS disease kindred confirmed marked
phenotypic variability and suggested that the p.Met129Val variant genotype was associated
with it[14].
In the present study, we report two unrelated Brazilian families with GSS disease
due to a p.Pro102Leu mutation. Clinical, molecular and/or pathological data of five
cases in one family and two cases in another family are reported. Age at onset ranged
from 25 to 66 years old and disease duration ranged from eight months to 15 years.
METHODS
Patients
Two individuals from Brazilian families with clinical symptoms suggestive of prion
disease were first evaluated, and GSS disease confirmed based on the finding of the
variant p.Pro102Leu in one allele of the PRNP gene. Based on this diagnosis, data from other family members were collected and
prospective follow-up performed. Where possible, family members were interviewed,
patients examined and blood samples taken for molecular analysis after informed consent.
Additional investigations, such as brain MRI, protein 14.3.3 in cerebrospinal fluid
(CSF) and an electroencephalogram were performed when possible. This study was approved
by the Institutional Ethics Committee and participating families signed the informed
consent form.
Genetic analysis
The DNA was extracted according to standard protocols using the Wizard Genomic DNA
Purification Kit®. The PRNP coding region amplification and sequencing were performed adopting previously published
procedures[17]. In order to investigate the segregation of the variants p.Pro102Leu and p.Met129Val,
PCR products of the complete coding region of PRNP were cloned using pGEM®-T Easy Vector Systems (Promega) and then sequenced. For apolipoprotein E (apoE) gene
analysis, allele-specific real-time PCR was performed[18].
Brain tissue analyses
Formalin-fixed fragments of the cerebral cortex, basal ganglia, hippocampus and cerebellum
were immersed in 95% formic acid, and then processed for paraffin embedding. Histological
sections were stained with H&E and Congo Red. Antigen retrieval was performed after
tissue exposure to 10mM citrate buffer, pH6.0 for 40 minutes at 96oC. Immunohistochemical studies were performed using anti-PrPC monoclonal antibody (clone PrP3F4;DAKO) followed by Polymer Secondary System (Novolink,
Leica). Unfortunately, there was no availability of frozen unfixed brain tissue for
western blot analysis of protease-resistant prion protein.
RESULTS
Clinical descriptions
Family A
In this family, there were five affected individuals (pedigree shown in [Figure 1]). Clinical and/or pathological data of five patients over two generations are given
in [Table 1]. In the first generation, the father of three affected individuals died after the
seventh decade of life while the mother died in her forties, without symptoms of GSS
disease. Three siblings from the second generation, aged from 61 to 69 years old,
have no symptoms to-date. Fifteen relatives of the third generation, aged from 27
to 51 years old, are also asymptomatic.
Figure 1 Pedigree: A) Family A; B) Family B.
Table 1
Clinical data and ancillary tests from seven patients with Gerstmann-Sträussler-Scheinker.
Variable
|
Family A
|
Family B
|
Patients
|
IIIa -1
|
IIa - 1
|
IIa – 2
|
IIIa – 2
|
IIa-3
|
IIIb -1
|
IIIb - 2
|
Sex
|
F
|
M
|
F
|
F
|
F
|
F
|
M
|
Age at onset (y)
|
27
|
53
|
54
|
38
|
66
|
35
|
25
|
Onset symptoms
|
Dementia
|
+
|
+
|
-
|
-
|
-
|
+
|
-
|
Ataxia
|
-
|
-
|
+
|
+
|
-
|
-
|
+
|
Paresthesias
|
-
|
-
|
-
|
+
|
+
|
-
|
-
|
Social withdrawal
|
-
|
-
|
-
|
-
|
+
|
-
|
-
|
Age at death (y)
|
42
|
53
|
58
|
45
|
Living
|
Living
|
Living
|
Disease duration (y)
|
15
|
08/12
|
4
|
7
|
2
|
4
|
8
|
Dementia
|
+
|
+
|
+
|
+
|
+
|
+
|
+
|
Pyramidal syndrome
|
+
|
-
|
+
|
+
|
-
|
+
|
+
|
Parkinsonian syndrome
|
+
|
+
|
+
|
-
|
-
|
-
|
-
|
Cerebellar syndrome
|
-
|
-
|
+
|
+
|
+
|
+
|
+
|
Myoclonus
|
+
|
+
|
+
|
-
|
-
|
-
|
-
|
Epilepsy
|
-
|
-
|
+
|
+
|
-
|
-
|
-
|
Brain MRI
|
Cerebellar and cerebral atrophy
|
Frontal atrophy
|
Cerebral atrophy, DWI frontal and parietal cortex hyperintensities
|
Cerebellar and cerebral atrophy
|
Leukoaraiosis
|
Cerebellar and cerebral atrophy
|
Cerebellar and cerebral atrophy
|
Frontal hypoperfusion
|
Cerebellar vermis hypoperfusion
|
Brain SPECT
|
EEG PSD
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
CSF 14.3.3 protein
|
-
|
ND
|
ND
|
ND
|
ND
|
-
|
ND
|
PRNP gene analysis
|
Codon 102
|
Pro/Leu
|
ND
|
Pro/Leu
|
Pro/Leu
|
Pro/Leu
|
Pro/Leu
|
Pro/Leu
|
Codon 129
|
Met/Val
|
ND
|
Met/Met
|
Met/Val
|
Met/Met
|
Met/Val
|
Met/Val
|
Leu102 in cis with
|
Met129
|
ND
|
Met129
|
Met129
|
Met129
|
Val129
|
Val129
|
ApoE gene analysis
|
e2/e3
|
ND
|
e2/e3
|
e2/e3
|
e2/e3
|
e3/e3
|
e3/e3
|
Neuropathological data
|
+
|
+
|
ND
|
+
|
ND
|
ND
|
ND
|
(-) absent; (+) present; ND: not done; PSD: periodic synchronous discharge; DWI: diffusion
weighted imaging; MRI: magnetic resonance imaging; SPECT: single photon emission computed
tomography; M: methionine; V: valine. EEG: electroencephalogram ; CSF: cerebrospinal
fluid; PRNP: prion protein gene .
Patient IIIa - 1 (index)
Disease onset occurred at the age of 27 years with rapid cognitive decline progressing
to severe dementia in two years. Gait instability and dysarthria emerged after five
years. Neurologic examination seven years after onset showed severe dementia, grasping
reflex, parkinsonian gait with bilateral support, hyperactive tendon reflexes with
Babinski sign, and plastic rigidity in the upper limbs. Myoclonus occurred eight months
later. She died 15 years after the first symptoms.
Family B
This Brazilian family is from Portuguese descent and had six probably-affected individuals
(pedigree shown in [Figure 1]). The first known case was the paternal grandfather of the proband (Ib - 1). He
died before the age of 40 with a disease that rendered him dependent for several years.
In the next generation, there were reports of at least three individuals who died
at around the fourth decade of life, with similar features. The father of the proband
(IIb - 1) died at age 33 after eight years of disease, with gait and speech impairment
and cognitive decline. In the third generation, two patients were evaluated. Clinical
data for these two patients are given in [Table 1].
Patient IIIb – 1
Disease onset occurred at age 35 with cognitive decline and balance deficit. Severe
dementia and abasia occurred three years later. Neurologic examination three years
after onset revealed severe dementia with frontal syndrome and global cerebellar ataxia.
Spasticity, brisk tendon reflexes in all four limbs and Babinski sign were also present.
The age of onset in the two reported families ranged from 27 to 66 years old. The
duration of the disease ranged from eight months to 15 years. The clinical diagnosis
of possible CJD was initially proposed in two patients. Typical GSS features were
observed in four patients (IIa-2, IIa-3, IIIa-2 and IIIb-2).
Molecular data
The p.Pro102Leu mutation was found in all six patients (IIa-2, IIa-3, IIIa-1, IIIa-2,
IIIb-1 and IIIb-2) who underwent genetic analyses. This investigation was not possible
in one patient (IIa-1). Four patients were heterozygote at codon 129 (methionine/valine,
Met129Val) and two were homozygote for methionine. All patients from family A were
e3/e3 and all patients from family B were e2/e3 for apoE gene ([Table 1]).
Neuropathological data
Neuropathological study was performed in three of the five patients from family A.
Spongiform encephalopathy was found on frontal lobe biopsy of patient IIa-1. At the
time immunohistochemistry for PrPSc was not available, and the slides and blocks could not be retrieved for further studies.
The study of patient IIIa-1 showed that focal neuronal loss, spongiosis and gliosis
were minimal, both in the cerebral and the cerebellar cortex. However, there were
multicentric plaques in the molecular layer of the cerebellum ([Figure 2]) and deep regions of the temporal cortex, including regions of the hippocampus,
adjacent to pyramidal cells and granular cells of the dentate fascia ([Figure 3]). The basal ganglia, thalami, midbrain, pons, and medulla were normal.
Figure 2 Patient IIIa-1. Multicentric plaques in the molecular layer of the cerebellum.
Figure 3 Patient IIIa-1. Multicentric plaque adjacent to granular cells of the dentate fascia
of the hippocampus.
In patient IIIa-2, there was severe diffuse cerebral and cerebellar cortical atrophy,
in addition to extensive involvement of the subjacent white matter, which was rarefied
and collapsed, in some areas containing small cavities ([Figure 4]). The basal ganglia and thalami were also extremely reduced in size. There was extensive
neuronal loss, reactive gliosis and spongiosis, both in the cerebral and cerebellar
cortex, with extensive Purkinje and granular cell loss. The subjacent white matter
was rarefied, with hardly any myelin ([Figures 5] and [6]). Multicentric plaques, immunopositive for PrPSc ([Figure 7]), some of them containing amyloid material, confirmed with Congo Red staining, were
observed in the cerebral and cerebellar cortex.
Figure 4 Patient IIIa-2. Severe diffuse cerebral (a) and cerebellar (b) cortical atrophy,
in addition to extensive involvement of the subjacent white matter.
Figure 5 Patient IIIa-2. Cerebral white matter rarefied, with hardly any myelin.
Figure 6 (A, B and C) Patient IIIa-2. Multicentric plaques, immunopositive for PrPSc observed
in the cerebral cortex.
Figure 7 Patient IIIa-2. Cerebellar white matter was rarefied (A) and the cortex contained
multicentric plaques (B).
DISCUSSION
The p.Pro102Leu variant is the most frequent mutation associated with GSS and has
been reported in a number of kindreds from Austria, Germany, China, Japan, UK, Italy,
Argentina and USA families[14],[19],[20].
Classically, GSS with the p.Pro102Leu mutation is characterized by slowly progressive
cerebellar ataxia with symptom onset in the fifth or sixth decade of life. During
the course of the disease, pyramidal signs and cognitive decline are common. Movement
disorders, typically myoclonus and athetosis, may occur. Seizures, deafness and cranial
nerve palsies number among the less common features[9]. Mean survival of GSS is from five to seven years, longer than seen in other prion
diseases. Of the seven individuals from two families reported herein, four died, within
eight months, four, seven and 15 years of disease onset, respectively. Survival of
longer than 10 years is uncommon and the longest reported disease duration, so far,
was 13 years[9].
The clinical phenotype of GSS disease ranges from ataxia followed by dementia to primary
cognitive decline, while the disease progression rate also varies considerably. Phenotypic
variability has been reported in other series and appears to be unrelated to the p.Met129Val
variant genotype[21]. Patients IIIa - 1 and IIIb - 1 had cognitive decline as the first symptom and exhibited
very early onset, which is unusual but consistent with some other previous reports[13]. One patient presented with clinical features of CJD (patient IIa - 1), while another
had rapidly-progressively dementia with a long survival (patient IIIa - 1). Many studies
support the existence of three dementia phenotypes associated with the p.Pro102Leu
variant: rapid global dementia resembling CJD[21],[22], slowly progressive cognitive decline[23] and frontotemporal type dementia[24].
In the largest published investigation involving 84 GSS individuals belonging to the
same extended family, methionine homozygosity at codon 129 was associated with early-onset
disease while other combinations were not associated with disease duration[14]. In contrast to this recent data, the earliest clinical onsets in the present study
occurred in four patients (IIIa - 1, IIIa - 2, IIIb - 1 and IIIb – 2) heterozygous
at codon 129, and the p.Pro102Leu mutation was located in the 129M allele for family
A and the 129V allele in family B. Besides the age at onset and duration of the disease,
other phenotypic differences were observed among the patients described.
Similar clinical contrasts, with different phenotypes within the same family, have
been reported worldwide. The causes of phenotypic variability within a family with
GSS mutation are unknown. The polymorphism at codon 129 alone is probably not the
only explanation for the pathologic and clinical heterogeneity in GSS (codon 102 mutation)[21]. However, they are of particular interest in the study of prion diseases since they
point to other, as yet unknown, factors that may contribute to the diverse phenotype
associated with the same mutation at PRNP. In family A, disease symptoms first manifested at 27 years old in the index case,
whereas, in her mother the symptoms were only reported at the age of 66.
Another genetic factor that could explain some clinical heterogeneity in prion diseases
is the apoE polymorphism. Conflicting results for the association of sporadic CJD
and the presence of apoE e4 have been published. Although apoE and PRNP may represent effect size of risk association in patients with sporadic CJD and e2
carriers have late onset disease[25], few data supports that apoE is not a risk factor for p.Pro102Leu GSS[26]. However, e4 carriers had delayed age of onset by 10 years in the largest GSS kindred
studied so far[14]. In our study, there were no e4 carriers and no difference in apoE polymorphism
among patients in the same family. Indeed, these polymorphisms could not explain the
clinical variability in the kindreds we are describing.
The presence of CSF 14-3-3 and the occurrence of periodic synchronous discharge is
unusual in GSS patients and may be confounding factors in some cases, leading to CJD
misdiagnosis[14]. In our sample, all the patients had an electroencephalogram, but none of them had
periodic synchronous discharge. Two patients had CSF analysis, but they were negative
for 14-3-3.
The pathognomonic neuropathological characteristic of GSS is the presence of multicentric
amyloid plaques with positive PrPc immunoreactivity. Other pathologic features that can be found in GSS include gliosis,
neuronal loss, neurofibrillary tangles and spongiform changes[27]. The degree of spongiform change is variable, and an absence of spongiosis in brain
tissue, which is uncommon in prion diseases, has been reported[11]. White matter degeneration, as observed in one of the present cases, may reflect
the degree of neuronal loss, rapidly progressive course and/or longer survival in
some cases and is described as the panencephalopathic form of prion disease[28], and has been previously reported in one patient with GSS[29].
There is evidence that, besides the mutant isoform, the protease-resistant wild-type
PrP may also be found in cerebral tissue of patients with GSS[16]. The frequency and spatial distribution of both PrPs could offer another explanation
for the variability in clinical and pathological findings between individuals in the
same family[16]. Atypical p.Pro102Leu phenotypes are associated with high levels of protease-resistant
wild-type PrP in few cases. In the present study, we could neither verify the distribution
of different forms of PrP, nor of the two abnormal fragments of ~21-30 kDa or 8 kDa.
In family A, third-generation patients presented with symptoms earlier than second-generation,
suggesting genetic anticipation. However, a recent report ruled out this genetic phenomenon
in prion diseases[30].
It is noteworthy that MRI findings also differ between GSS patients. Neuroimaging
studies are typically described as normal or with non-specific atrophy of cerebral
hemispheres and/or cerebellum[9]. There are, however, few reports of cortical and basal ganglia hyperintensities
on diffusion-weighted-MRI during the disease course[31],[32], as disclosed in patient IIa-2.
In conclusion, the two Brazilian families with GSS associated with p.Pro102Leu mutation
showed similar clinical heterogeneity to other cases described worldwide. However,
these results failed to corroborate the hypothesis that phenotype variation is attributable
to polymorphism at codon 129[14],[15]. Indeed, genomic and epigenetic variations within this family warrant further investigation.