Associated with Gerstmann-Sträussler-Scheinker

OBSERVATION A New PRNP Mutation (G131V) Associated With Gerstmann-Stra¨ussler-Scheinker Disease

Peter K. Panegyres, PhD, FRACP; Kathrine Toufexis, BSc (Hons); B. A. Kakulas, MD, FRCPA, FRACP; Larisa Cernevakova, MD; P. Brown, MD; B. Ghetti, MD; P. Piccardo, MD; S. R. Dlouhy, PhD

Background: Gerstmann-Stra¨ussler-Scheinker dis- Results: Genetic analysis of the prion protein gene ease is a rare form of prion disease. showed a novel mutation at codon 131 that caused a valine-for-glycine substitution (G131V) and homozygos- Objective: To determine the prion mutation in a 51-year- ity at codon 129 (129M). Proteinase K–resistant prion old man without a family history of neurologic disease who protein was detected by Western blot analysis. died from Gerstmann-Stra¨ussler-Scheinker disease. Conclusions: This is the first mutation described in the Patient and Methods: The patient was a 51-year-old short, antiparallel ␤-sheet domain of the prion protein. man who died after a 9-year illness characterized by de- This report highlights the importance of genetic analy- mentia and eventually ataxia. Neuropathologic studies sis of patients with atypical dementia even in the ab- were performed, the results of which revealed abundant sence of a family history. prion protein–immunopositive amyloid plaques in the cerebellum without spongiform degeneration. Arch Neurol. 2001;58:1899-1902

ERSTMANN- Stra¨ussler- tection of the PrP deposits, antibodies rec- Scheinker disease (GSS) ognizing the N-terminus (PrP 23-40), mid- is an autosomal domi- region (PrP 95-108, 3F4), and C-terminus nant neurodegenerative (PrP 220-231) of PrP were used (Figure 1). disease caused by muta- The PrP immunoreactivity was intense in Gtions in the prion protein gene (PRNP).1 the molecular layer of the cerebellum. In ad- Clinically, it is characterized by progres- dition, PrP deposition was found in the fol- sive ataxia and dementia; pathologically, lowing brain regions: Ammon horn; fron- it is characterized by prion protein (PrP) tal, temporal, and parietal cortices; and amyloid plaques and neuronal loss. corpus striatum and thalamus. In addi- Mutations in PRNP have been found tion, PrP was not detected in the midbrain, to be associated with GSS, Creutzfeldt- pons, medulla, and mamillary bodies. There From the Department of Jakob disease (CJD), and fatal familial in- was no spongiform degeneration. Neuropathology, Royal Perth somnia. The GSS phenotype has been as- By modified Bielschowsky stain, oc- Hospital, Perth, Western sociated with point and insertional casional neurons with neurofibrillary Australia (Drs Panegyres and mutations.2-5 Some of these PRNP muta- tangles (NFTs) were seen in the Ammon Kakulas and Ms Toufexis); tions have been used to establish genetic horn and entorhinal cortex. However, Jerome H. Holland Laboratory, linkage between the mutation and the dis- American Red Cross, Rockville, NFTs were not found in any other area of Md (Dr Cernevakova); ease phenotype. Herein, we report a novel the central nervous system. Laboratory of Central Nervous PRNP mutation in a patient with an un- System Studies, National usual phenotype. GENETIC ANALYSIS Institute of Neurological Disorders and Stroke, National RESULTS DNA was extracted from the cerebellum, Institutes of Health, Bethesda, and polymerase chain reaction was used Md (Dr Brown); and NEUROPATHOLOGIC FINDINGS to amplify the open reading frame of the Departments of Pathology and PRNP gene using the following human Laboratory Medicine (Drs Ghetti and Piccardo) and On autopsy, the brain weighed 1243 g. PRNP primers (Genbank submission 6 Ј Ј Medical and Molecular There was mild cerebral and cerebellar at- M13899). For forward 5 -3 , region 1, po- Genetics (Dr Dlouhy), Indiana rophy. Microscopically, numerous con- sition 31 to 51, 21-mer CCG AGG CAG University School of Medicine, gophilic amyloid plaques were found in the AGC AGT CAT TAT; region 2, position Indianapolis. cerebellum. For immunohistochemical de- 311 to 329, 19-mer GGC TGG GGT CAA

(REPRINTED) ARCH NEUROL / VOL 58, NOV 2001 WWW.ARCHNEUROL.COM 1899

A white man, born in 1945, resigned in 1987 from his position as a teacher following complaints from colleagues regarding his inability to discipline his class. His driving was erratic and he was unable to find his way on the roads. There was impairment of his short- term memory, reduced learning capacity, persever- ance, and emotional immaturity. His family reported aloofness, anxiety, and increasing anger. The family, including the patient’s 2 siblings, who are in their 50s, and his teenage son, has no history of neurologic disease. Figure 1. Anti–prion protein (PrP) immunohistochemistry. The molecular In 1987, during a neurologic examination, the layer of the cerebellar cortex shows large PrP deposits (hematoxylin man appeared to have no insight into his condition. counterstain, original magnification ϫ400). He was apraxic and had a tremor of the limbs and gegenhalten. Visuospatial skills and spatial orienta- tion were impaired and he was dyscalculic. The deep- 421 ccttggcggc ta catg ctg g ga agtgccat gagcaggccc atcatacatt tcggcagtga tendon reflexes were pathologically brisk; the cra- X96/323 F ccttggcggc ta catg ctg g Ta agtgccat gagcaggccc atcatacatt tcggcagtga nial nerve examination results and peripheral motor D94-483 F systems were otherwise normal. There was no ataxia. D92-307 F A neuropsychological assessment revealed a D98-414 F Mini-Mental State Examination score of 22/30 and a 129 131

verbal IQ of 88, a performance IQ of 67, and a full- 481 ctatgaggac cgttactatc gtgaaaacat gcaccgttac cccaaccaag tgtactacag scale IQ of 78. Results of block design and object as- X96/323 F sembly subtests from the Wechsler Intelligence Scale D94-483 F were abnormal. He was grossly impaired on tests of D92-307 F verbal short-term memory, reasoning, comprehen- D98-414 F sion of visual information, and visuospatial organi- zation. The computed tomography and magnetic reso- nance images showed cerebral and cerebellar atrophy. 541 gcccatggat gagtacagca accagaacaa ctttgtgcac gac tgcgtca atatcacaat Results of examination of the cerebrospinal fluid were X96/323 F normal, except for a slightly elevated protein level con- D94-483 F ctttgtgcac Aac tgcgtca D92-307 F centration of 0.046 g/dL (reference range, 0.015- D98-414 F 0.045 g/dL). The electroencephalogram did not show 178 periodic sharp waves. His dementia progressed and ataxia developed. 601 caagcagcac acggtcacca caaccaccaa gggggagaac ttcacc gag a ccgacgttaa He eventually required institutionalization in a locked X96/323 F ward because of uncontrollable aggressive behav- D94-483 F ior. He died by choking at the age of 51 years, 9 years D92-307 F gggggagaac ttcacc Aag a ccgacgttaa after the onset of symptoms. D98-414 F 200

Figure 2. Sequence data of the open reading from the prion protein (PRNP) gene. The nucleotide number is shown in the upper left of each row and the GGA GCT G; and region 3, position 582 to 603, normal sequence at the top of each row; slashes represent homology to the wild-type sequence and the codon number is under each rectangular shaded 22-mer ACT GCG TCA ATA TCA CAA TCA A. For re- box. The sequence of our patient X96/323 is shown under the wild-type verse 5Ј-3Ј, region 1, position 400 to 417, 18-mer CCC sequence. A point mutation at g → T is shown at codon 131 and methionine ACC ACT GCC CCA GCT; region 2, position 673 to 693, homozygosity at codon 129. The controls were run concurrently. D98-414 is a healthy patient; D92-307, familial Creutzfeldt-Jakob disease with codon 21-mer ATA CAC ATC TGC TCA ACG ACG; and re- 200 mutation; and D94-483, familial fatal insomnia with codon 178 mutation. gion 3, position 945 to 963, 19-mer AGT GCC CAT CAG Identical results were obtained on 3 separate runs with sequencing TGC CAA G. performed in a forward (F) and reverse direction. An automated DNA sequencer (ABI 377; Applied Bio- systems, Inc, Foster City, Calif) was used. The sequenc- PrP ANALYSIS ing was performed in a forward and reverse direction on 3 separate occasions. A G→T transversion mutation at codon Protein was extracted from the cerebellum and processed 131 was found, resulting in a substitution of valine (V, GTA) as previously described.7 The presence of protease- for glycine (G, GGA) at that position (G131V). In addi- resistant PrP was determined by digestion with proteinase tion, codon 129 was homozygous for methionine K (PK) and fractionation in sodium dodecyl sulfate– (Figure 2). The mutation was confirmed in subsequent polyacrylamide gel electrophoresis. The gels were probed experiments in the laboratories of the American Red Cross, with the monoclonal antibody 3F4 (1:50000), which Rockville, Md, and Indiana University, Indianapolis, and recognizes positions 109 to 112 of human PrP. The im- has not been seen in the DNA from other patients with prion munoblots were visualized using electrochemilumines- diseases or healthy controls (N=50). cence. Non–PK-treated samples contained PrP species of

(REPRINTED) ARCH NEUROL / VOL 58, NOV 2001 WWW.ARCHNEUROL.COM 1900

©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 approximately 18, 26 to 27, and 31 to 36 kd. In addition, AB C D PK digestion demonstrated evidence of proteinase- resistant PrP with bands at approximately 8, 18, 26, and kd 31 kd (Figure 3).

COMMENT

The transmissible spongiform encephalopathies represent a unique group of neurologic conditions in which 30 genetic disorders can lead to transmissible neurologic disease. Herein, we describe a de novo mutation in PRNP associated with a GSS phenotype. In the proband’s fam- 20 ily, there was no history of neurologic disorders, and his 2 siblings, in their 50s, were examined by one of the au- thors (P.K.P.) and were found to be free of neurologic

signs. They declined predictive gene testing despite coun- 6 seling. The absence of a family history might suggest that this is a new mutation; however, this is rare for PRNP mutations. Figure 3. Immunoblot of Creutzfeldt-Jakob disease E200K (A, B) and Gerstmann-Stra¨ussler-Scheinker disease G131V (C, D) showing the effect of More than 20 mutations in PRNP have been de- proteinase K treatment. Brain extracts were untreated (A, C) or treated with scribed that cause CJD, GSS, fatal familial insomnia, and proteinase K (B, D). The position of relative molecular weight standards PrP cerebral amyloid angiopathy with different clinical corresponding to molecular masses (from top to bottom) of 30, 20, and 6 kd and neuropathologic phenotypes. Clinically, GSS is char- are shown. Immunoblot probed with monoclonal antibody 3F4. acterized by ataxia, parkinsonian signs, and dementia. In the proband, ataxia and dementia were the main signs. forms (ie, 21- to 30-kd bands). This case is character- The most common mutation associated with GSS is ized by PrPres species that are similar to those de- P102L, but other point mutations at codons 105, 117, scribed in GSS F198S and Q217R, 2 GSS variants without 198, 202, 212, and 217 and insert mutations (6 to 9) be- spongiform degeneration. The mutation reported herein tween codons 51 and 91 have been described.4 The dis- is the first one to cause an amino acid substitution (G → orders caused by these mutations have their onset in the V) in a short, antiparallel ␤-sheet domain comprising resi- third to sixth decades of life and usually present with cer- dues 128 to 131.8 The impact of such mutation on PrP ebellar ataxia that evolves to dementia. Our patient had folding and processing remains undetermined.14 cognitive impairment before the onset of ataxia, with sur- In the proband, NFTs were observed in the hippo- vival of about 9 years. Cognitive impairment before the campus and entorhinal cortex. The NFTs have been onset of ataxia has also been documented in a family with previously described in GSS with mutations F198S and a mutation at codon 198.8 Phenotypic heterogeneity has Q217R.15,16 In these 2 GSS variants, tau-immun- been previously emphasized in GSS, and it has been re- opositive NFTs were widespread in the neocortex and cently demonstrated again in 2 studies of an English and subcortical nuclei and were associated with neuritic French family with the A117V mutation and an octapep- plaques. The occurrence of different degrees of tau patho- tide insertional mutation, respectively.4,9,10 The pres- logic findings in GSS, including the G131V variant, re- ence of neurologic and psychiatric symptoms in mem- quires further investigation to determine whether hy- bers of these 2 families further illustrates the phenotypic perphosphorylation of the microtubule-associated protein variability of these conditions.9,10 tau and the formation of paired helical filaments are sec- Our patient was homozygous for methionine at ondary events to the presence of specific conformations codon 129. Homozygosity for either methionine or va- of PrP or reflect a nonspecific phenomenon. line affects susceptibility to CJD and the phenotypic ex- The G131V mutation is another example of the wide pression of mutations.11 For example, in the case of the spectrum of phenotypes associated with GSS and PRNP D178N mutation, the haplotype 129M-D178N is associ- gene mutations and highlights the importance of the mo- ated with fatal familial insomnia and the haplotype 129V- lecular classification of prion disorders, even in the ab- D178N is associated with CJD.12 sence of an identifiable family history. The diagnostic Prion diseases share a similar pathogenetic mecha- workup of patients with early-onset dementia and the sus- nism that involves the conversion of a normal PrP (PrPc) picion of prion disease necessitates analysis of the PRNP into a pathologic form that is insoluble in detergents and gene, since neuropathologic features, typical of prion dis- PK resistant (PrPres). Previous studies6 have shown that eases, might not always be found.17 patients with GSS accumulate N- and C-truncated PrPres fragments of approximately 8 to 15 kd and variable Accepted for publication January 17, 2001. amounts of higher-molecular-weight isoforms. In addi- The work was supported by the National Health and tion, in the GSS variant P102L, PrPres species of approxi- Medical Research Council of Australia, Canberra; Royal mately 21 to 30 kd, identical to those described in CJD, Perth Hospital Research Foundation, Perth; the Medical are also present when this variant is associated with se- Research Fund of Western Australia, Perth; and grant PHS vere spongiform degeneration.6,13 The pattern of PrPres P30 AG 10133 from the US Public Health Service, Wash- bands in the proband does not present CJD-like iso- ington, DC (Dr Ghetti).

(REPRINTED) ARCH NEUROL / VOL 58, NOV 2001 WWW.ARCHNEUROL.COM 1901

©2001 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 We thank the staff of the Neuropathology Depart- Straüssler-Scheinker disease is associated with prion protein heterogeneity. ment, Royal Perth Hospital, for their help in the production J Neuropathol Exp Neurol. 1998;57:979-988. 8. Unverzagt FW, Farlow MR, Norton J, Dlouhy SR, Young K, Ghetti B. Neuro- of the manuscript. Rose Richardson, Albert William, and Yue psychological function in patients with Gerstmann-Straüssler-Scheinker dis- Feng provided valuable technical assistance. Bradley S. ease from the Indiana kindred (F198S). J Int Neuropsychol Soc. 1997;3:169- Glazier helped in the editing process. 178. Corresponding author and reprints: Peter K. Pan- 9. Mallucci GR, Campbell TA, Dickinson A, et al. Inherited prion disease with an egyres, PhD, Department of Neuropathology, Royal Perth alanine to valine mutation at codon 117 in the prion protein gene. Brain. 1999; 122:1823-1837. Hospital, Wellington Street, Perth, Western Australia 6000 10. Laplanche J-L, Hacimi KHE, Durieux I, et al. Prominent psychiatric features and (e-mail: [email protected]). early onset in an inherited prion disease with a new insertional mutation in the prior protein gene. Brain. 1999;122:2375-2386. 11. Collinge J, Palmer MS, Dryden AJ. Genetic predisposition to iatrogenic Creutzfeldt- REFERENCES Jacob disease. Lancet. 1991;337:1441-1442. 12. Goldfarb LG, Petersen RB, Tabaton M, et al. Fatal familial insomnia and familial 1. Prusiner SB. The prion diseases. Brain Pathol. 1998;8:499-513. Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymor- 2. Owen F, Poulter M, Lofthouse R, et al. Insertion in prion protein gene in familial phism. Science. 1992;258:806-808. Creutzfeldt-Jacob disease. Lancet. 1989;1:51-52. 13. Parchi P, Chen SG, Brown P, et al. Different patterns of truncated prion protein 3. Hsiao K, Baker HF, Crow TJ, et al. Linkage of a prion protein missense variant to fragments correlate with distinct phenotypes in P102L Gerstmann-Straüssler- Gerstmann-Straüssler Syndrome. Nature. 1989;338:342-345. Scheinker disease. Proc Natl Acad Sci U S A. 1998;95:8322-8327. 4. Gambetti P, Petersen RB, Parchi P, et al. Inherited prion diseases. In: Prusiner 14. Zahn R, Liu A, Luhrs T, et al. NMR solution structure of the human prion S, eds. Prion Biology and Diseases. New York, NY: Cold Spring Harbor Labora- protein. Proc Natl Acad Sci U S A. 2000;97:145-150. tory Press; 1999:509-583. 15. Ghetti B, Tagliavini F, Masters CL, et al. Gerstmann-Straüssler-Scheinker disease, II: 5. Boëllaard JW, Brown P, Tateishi J. Gerstmann-Straüssler-Scheinker disease: the neurofibrillary tangles and plaques with PrP amyloid coexist in an affected fam- dilemma of molecular and clinical correlations. Clin Neuropathol. 1999;18:271- ily. Neurology. 1989;39:1453-1461. 285. 16. Ghetti B, Dlouhy SR, Giaccone G, et al. Gerstmann-Straüssler-Scheinker dis- 6. Kretzschmar HA, Stowring LE, Westaway D, et al. Molecular cloning of a human ease and the Indiana kindred. Brain Pathol. 1995;5:61-75. prion protein cDNA. DNA. 1986;5:315-324. 17. Collinge J, Owen F, Poulter M, et al. Prion dementia without characteristic 7. Piccardo P, Dlouhy SR, Lievens PAJ, et al. Phenotypic variability of Gerstmann- pathology. Lancet. 1990;336:7-9.

(REPRINTED) ARCH NEUROL / VOL 58, NOV 2001 WWW.ARCHNEUROL.COM 1902