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A novel locus for dementia with Lewy bodies: a clinically and genetically heterogeneous disorder

(CC)
Veerle Bogaerts, Sebastiaan Engelborghs, Samir Kumar-Singh, Dirk Goossens, Barbara Pickut, Julie van der Zee, Kristel Sleegers, Karin Peeters, Jean-Jacques Martin, Jurgen Del-Favero, Thomas Gasser, Dennis W. Dickson, Zbigniew K. Wszolek, Peter P. De Deyn, Jessie Theuns, Christine Van Broeckhoven
DOI: http://dx.doi.org/10.1093/brain/awm167 2277-2291 First published online: 6 August 2007

Summary

Dementia with Lewy bodies (DLB) represents the second most frequent type of neurodegenerative dementia in the elderly. Although most patients have sporadic DLB, a limited number of DLB families have been described, suggesting that genetic factors may contribute to DLB pathogenesis. Here, we describe a three-generation Belgian family with prominent dementia and parkinsonism, consistent with a diagnosis of DLB, that was autopsy confirmed for the index patient. In a genome-wide scan and subsequent finemapping of candidate loci we obtained significant linkage to 2q35-q36 (Z = 3.01 at D2S1242). Segregation analysis defined a candidate region of 9.2 Mb between D2S433 and chr2q36.3-8, adjacent to the previously reported PARK11 locus. In addition, haplotype sharing studies in another DLB family of close geographical origin with similar clinical and neuropathological features highlighted the specificity of a 2q35-q36 haplotype harbouring a pathogenic mutation that causes DLB in the Belgian family. So far, extensive sequence analysis of five candidate genes within the 2q35-q36 region has not revealed a disease-causing mutation. Together, our data re-emphasize the genetic heterogeneity of DLB, and strongly support the existence of a gene for familial DLB on 2q35-q36. Once identified this will be the first novel causal gene for DLB and can be expected to open new avenues for biological studies of the disease process.

  • dementia with Lewy bodies
  • autosomal dominant inheritance
  • linkage analysis
  • genetic heterogeneity
  • 2q35-q36

Introduction

Dementia with Lewy bodies (DLB) accounts for the second-largest neuropathological subgroup of neurodegenerative dementia after Alzheimer's disease (AD) (McKeith et al., 1996). DLB has an estimated prevalence of up to 30.5% among dementia patients (reviewed by Zaccai et al., 2005), and an incidence of 0.1% a year in the general population (Miech et al., 2002). DLB patients present with progressive dementia, accompanied by core clinical characteristics as fluctuating cognition, recurrent visual hallucinations and parkinsonism. Features suggestive for DLB are REM sleep behaviour disorder, severe neuroleptic sensitivity and low dopamine reuptake in basal ganglia upon functional imaging, whereas additional features including repeated falls, transient loss of consciousness, severe autonomic dysfunction and depression are supportive for a clinical diagnosis of DLB (McKeith et al., 2005). Neuropathologically, DLB is classified into three types (McKeith et al., 1996) depending on the distribution and density of Lewy bodies (LB): the brainstem-predominant type, the limbic (transitional) type and the neocortical type [also known as diffuse Lewy body disease (DLBD) (Kosaka et al., 1984)]. In addition, DLB patients often have accompanying AD neuropathology in various degrees (Marui et al., 2004), and according to the presence or absence of co-occurring AD pathology, DLB is further classified into respectively a common form and a pure form (Kosaka, 1990). LB are also the neuropathological hallmark of Parkinson's disease (PD), moreover DLB and PD have clinical features in common. Since 25–30% of PD patients develop dementia (Parkinson's disease with dementia, PDD) (Aarsland et al., 2005) and most DLB patients have parkinsonism (Burn et al., 2003) it has been hypothesized that PD, PDD and DLB are not distinct nosological entities, but may exist along the spectrum of Lewy body diseases (LBD). A DLB/PDD Working Group recently proposed the use of LBD as an umbrella term for studying the biology of these disorders (PD, PDD and DLB), but suggested to maintain the three individual terms for clinical needs (Lippa et al., 2007).

DLB is generally a sporadic disorder, and it must be considered as a complex disorder resulting from the interaction between genetic and environmental risk factors. Nevertheless, a number of studies reported DLB families segregating the disease as an autosomal dominant (Golbe et al., 1990; Waters and Miller 1994; Denson et al., 1997; Wakabayashi et al., 1998; Galvin et al., 2002; Tsuang et al., 2002; Bonner et al., 2003) or recessive trait (Ohara et al., 1999).

Molecular genetic studies identified several mutations associated with DLB, suggesting a large genetic heterogeneity in the development of DLB. A triplication of α-synuclein (SNCA) was shown to cause autosomal dominant PD in a large kindred with mixed phenotype ranging from typical PD to DLB (Singleton et al., 2003). Later, evidence illustrated that multiplication mutations of SNCA are a rare cause of PD and DLB (Johnson et al., 2004). Zarranz et al. reported the identification of the SNCA p.Glu46Lys mutation segregating in a Spanish family with autosomal dominant parkinsonism, dementia and visual hallucinations. The neuropathological findings were consistent with DLB with a high density of LB in cortical and subcortical areas (Zarranz et al., 2004). SNCA p.Ala53Thr was reported in an elderly Greek man presenting with progressive memory impairment, parkinsonism, recurrent visual hallucinations and fluctuating attention and alertness (Morfis and Cordato, 2006). The patient had a strong family history of PD, but neither co-segregation data nor post-mortem studies were available. β-Synuclein (SNCB) mutations (p.Val70Met, p.Pro123His) were identified in two unrelated patients with DLB though co-segregation with the disease could not be established unambiguously (Ohtake et al., 2004). A 3-bp deletion in exon 12 (p.▵Thr440) of presenilin 1 (PSEN1) was reported in a patient with familial parkinsonism and dementia, neuropathologically characterized by widespread occurrence of LB and cotton wool plaques, but again co-segregation was not tested (Ishikawa et al., 2005). Finally, Koide et al. described a heterozygous p.Met232Arg mutation in the prion protein gene (PRNP) in a patient that was clinically diagnosed with Creutzfeldt–Jacob disease (Koide et al., 2002). However, at autopsy the patient was diagnosed with DLB, based on the presence of many LB in the substantia nigra and cerebral cortex (Koide et al., 2002).

Few studies have investigated genetic risk factors for DLB. The ε4 allele of the apolipoprotein E gene (APOE), the major genetic risk factor for AD, also appeared to be a risk factor for DLB (Arai et al., 1994; Benjamin et al., 1994; Galasko et al., 1994; Lippa et al., 1995), as well as the *4 allele of debrisoquine 4-hydroxylase (CYP2D6) (Saitoh et al., 1995;Tanaka et al., 1998), although some studies did not detect significant differences in CYP2D6*4 allele frequencies between DLB patients and control individuals (Atkinson et al., 1999; Furuno et al., 2001; Huckvale et al., 2003). Moreover, a glucocerebrosidase (GBA) mutation analysis recently detected a GBA mutation rate of 23% in pathologically confirmed DLB patients (Goker-Alpan et al., 2006), which was markedly higher compared to control individuals. GBA mutations may thus contribute to DLB susceptibility, but replication studies are required.

Despite the aforementioned findings, little is known about the aetiology of DLB. Therefore, additional linkage and association studies in patients with DLB are necessary as the identification of novel causal and susceptibility genes in DLB patients will improve our understanding of this disorder situated along the spectrum of LBD.

Few families with pathologically confirmed DLB have been reported. Here, we describe a three-generation Belgian family, family DR246, with a mixed phenotype of dementia and parkinsonism, that was clinically consistent with a diagnosis of DLB, and of whom the index patient had neuropathological confirmed DLB. We performed a genome-wide scan, linkage and mutation analyses. Furthermore, we analysed a previously reported family, family G, whose index patient also demonstrated neocortical LB pathology on autopsy (Denson et al., 1997), for haplotype sharing with family DR246, and emphasize the clinical and genetic heterogeneity associated with DLB.

Material and methods

Families and control individuals

The index patient III.17 of the Belgian family DR246 (Fig. 1), diagnosed with DLB, was contacted for molecular genetic studies by research nurses under the supervision of a physician experienced in clinical and molecular genetic studies of neurodegenerative disorders. Through interviews, detailed information on family history was obtained and additional patients and unaffected family members, living throughout the province of Antwerpen in Flanders, the Dutch-speaking region of Belgium, were asked to participate in molecular genetic studies. Diagnoses of patients were verified through extensive review of medical records. All 22 participating family members, including eight patients, signed an informed consent form approved by the medical ethical committee of the University of Antwerp and venous blood samples were taken for DNA extraction using standard procedures.

Fig. 1

Pedigrees of DR246 (A) and family G (B) Left-filled symbols represent patients with dementia, right-filled symbols represent patients with parkinsonism. Patients with dementia and parkinsonism are indicated with filled symbols. Open symbols represent unaffected individuals or at-risk individuals with unknown phenotype; question marks indicate obligate carriers or deceased individuals with unknown phenotype. The Roman numbers to the left of the pedigree specify generations. The arrows indicate index patients III.17 of DR246 and III.3 of family G with pathologically confirmed DLB. For DR246, numbers below the symbols denote age at onset for patients and either age at last examination or age at death for unaffected individuals, at-risk individuals with unknown phenotype or obligate carriers. An asterisk (*) indicates individuals that were included in the genome-wide scan.

For family G (Denson et al., 1997), originating from the province Friesland in the northern part of The Netherlands (Fig. 2) and also having Dutch as native language, 20 DNA samples were available for genetic studies. These DNAs were an extended collection of samples of family G, and included DNA of five patients with dementia and/or parkinsonism. Diagnoses of these patients were established by clinical examination and review of medical records.

Fig. 2

Site map of Europe showing the province of Antwerpen in Flanders (Belgium), where family DR246 lives, as well as the province Friesland in The Netherlands, where family G originated from. Dutch is spoken in both regions. B = Belgium; CH = Switzerland; D = Germany; F = France; I = Italy; N = The Netherlands.

In this study, we provide neuropathological data for the index patient of family DR246, and we give an update of the previously reported neuropathology of the index patient of family G (Denson et al., 1997).

Unrelated control individuals (n = 363, M/F: 0.77, mean age at inclusion: 61.5 years (46–77 years)) were recruited in Flanders and had no clinical evidence or history of PD or any other disorder involving the central nervous system.

Neuropathological and immunohistochemical analysis

A post-mortem neuropathological study was performed on patient III.17 of family DR246. The right cerebral hemisphere was fixed by immersion in 10% formalin and embedded in paraffin. Sections 5 µm thick were taken from cortical area 6, superior temporal gyrus, area parietalis superior, area striata and cuneus, hippocampus, parahippocampal and lateral occipitotemporal gyri, neostriatum, basal ganglia, thalamus, amygdaloid nucleus, midbrain at the level of the substantia nigra, pons, medulla oblongata and cerebellum. Sections were examined by routine histopathological methods, including haematoxylin and eosin and cresyl violet, as well as Thioflavin S staining. Following antibodies were used for immunostaining: anti-ubiquitin (Dako, Glostrup, Denmark), anti-α-synuclein (Chemicon, Temecula, CA, USA), AT8 against hyperphosphorylated tau (TAU, Innogenetics, Zwijnaarde, Belgium) and 4G8 against residues 17–24 of amyloid β (Aβ, Signet, Dedham, MA, USA). Antigen retrieval was performed by treating sections with 98% formic acid for 5 min at room temperature (4G8, anti-α-synuclein), by boiling in citrate buffer (pH6) (anti-ubiquitin) or without pre-treatment (AT8). All dilutions were made in 0.1 mol/l of phosphate-buffered saline with 0.1% bovine serum albumin. Staining was performed with appropriate secondary antibodies and streptavidin-biotin-horseradish peroxidase using chromogen 3’,3’diaminobenzidine (DAB, Roche, New Jersey, NJ, USA), as described previously (Kumar-Singh et al., 2002; Pirici et al., 2006).

Mutation analysis

Genomic DNA (gDNA) of III.17 was screened for mutations in the coding exons 3 to 12 of PSEN1 and PSEN2, coding exons 16 and 17 of the amyloid precursor protein gene (APP), coding exon 2 of PRNP, exons 1 and 9 to 13 of the microtubule associated protein tau gene (MAPT), all exons of parkin (PARK2), coding exons 2 to 6 of SNCA, all 7 exons of DJ1 (PARK7) and exons 24–25, 29–31, 35 and 41 of leucine-rich repeat kinase 2 (LRRK2) by direct sequence analysis (primer sequences available upon request). 20 nanogram gDNA was PCR amplified in 20 µl reactions using empirically defined reaction conditions. PCR products were purified with 10 U exonuclease I (USB Corporation, Cleveland, OH, USA) and 2 U shrimp alkaline phosphatase (USB Corporation). After purification, amplification products were sequenced in both directions using the BigDye Terminator Cycle Sequencing kit v3.1 (Applied Biosystems, Foster City, CA, USA) and analysed on an ABI3730 DNA Analyzer (Applied Biosystems).

SNCA dosage was quantified using a TaqMan® MGB Real-Time PCR assay (Applied Biosystems). Primers and TaqMan® MGB probes for SNCA exon 2 and β-2-microglobulin (B2M) exon 2 were designed using the Primer Express Software, version 2.0 (Applied Biosystems) and sequences are available upon request. Real-time PCR was performed using the universal amplification protocol on the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). Duplex amplification reactions (30 µl) were carried out in triplicate on 20 ng of gDNA, 1x qPCR™ Mastermix Plus without uracil-N-glycosylase (UNG) (Eurogentec, Seraing, Belgium), 100 nM of SNCA exon 2 primers, 83 nM of TaqMan® MGB SNCA probe, 150 nM of B2M exon 2 primers and 125 nM of TaqMan MGB B2M probe. SNCA copy number was interpolated based on the normalized standard curve of serial dilutions (0.3, 3 and 30 ng/µl) of a calibrator sample and serial dilutions (1/10, 1/100) of three control samples with respectively two normal copies of SNCA, a SNCA duplication and SNCA triplication.

Parkin was screened for exonic copy number variations by use of an in-house developed technique for Multiplex Amplicon Quantification (MAQ) (www.vibgeneticservicefacility.be/) and SYBR® Green and TaqMan® MGB real-time PCR. The MAQ assay comprises a multiplex PCR (mPCR) amplification, that enables simultaneous amplification of several amplicons in one reaction by use of different primer sets (Brouwers et al., 2006). mPCR amplification on 50 ng gDNA of two pools of fluorescently labelled target (parkin exons 2–8 and 10–11) and six reference amplicons randomly located on different chromosomes (primer mixes are available upon request), was followed by fragment analysis on an ABI3730 DNA Analyzer (Applied Biosystems). The comparison of normalized peak areas between the index patient and control individuals resulted in amplicon dosage quotients, calculated using an in-house developed MAQ software (MAQs) package, indicating the copy number of the parkin exons. SYBR® Green and TaqMan® MGB real-time PCR assays for all parkin exons were designed using the Primer Express Software, version 2.0 (Applied Biosystems) and sequences are available upon request. Real-time PCR was performed on an ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) in a 30 µl reaction containing 1x qPCR™ Mastermix Plus for SYBR® Green I (without UNG) (Eurogentec, Seraing, Belgium) or TaqMan® Universal PCR Master Mix (Applied Biosystems), 20 ng gDNA and 400 nM of the respective primers. Amplification was done according to the manufacturer's recommendations. The parkin amplicons were normalized for the reference genes B2M and ubiquitin C (UBC) and dosage quotients were calculated and compared to 100 healthy individuals.

Statistical genetics

The statistical power to obtain genome-wide significant linkage was assessed by use of the SLINK program (Ott, 1989) (Weeks et al., 1990). DLB was treated as a dichotomous, autosomal dominant trait with age-dependent penetrance. A cumulative risk curve was calculated and seven penetrance classes were defined using mean age at onset (AAO) in the family and assuming a maximum penetrance of 90%. Phenocopy rates were age-dependent and based on dementia prevalence in Western Europe (Ferri et al., 2005), with DLB accounting for 15% of all dementia patients (McKeith et al., 2004; Zaccai et al., 2005). Assuming a hypothetical marker with four equifrequent alleles and a disease allele frequency of 0.1%, simulation analyses for family DR246 indicated an average maximum logarithm of odds (LOD) score of 2.96 at recombination fraction (θ) 0.04. The estimated average maximum false positive LOD score was 2.16 (θ = 0.5), and under the same assumption of no linkage, there was a 0.2% possibility for a suggestive LOD score (LOD > 1.9). Simulation calculations for family G indicated an average maximum LOD score of 0.57 at θ = 0.0 and an estimated average maximum false positive LOD score of 0.47 at θ = 0.5.

Genome-wide scan

An 8 cM density genome-wide scan was performed for family DR246, using 425 autosomal short tandem repeat (STR) markers, with an average heterozygosity of 0.73, of an in-house developed human genome mapping panel consisting of 30 mPCR reactions (Deprez et al., 2007). gDNA was amplified in 20 µl mPCR reactions using standard optimized conditions and fluorescently labelled primers. PCR products were separated and analysed on an ABI3730 DNA Analyzer (Applied Biosystems) and genotypes were assigned using in-house developed TracI genotyping software (http://www.vibgeneticservicefacility.be/). PedCheck (O'Connell and Weeks, 1998) was used to check Mendelian inconsistencies.

Linkage analysis

Two-point and multi-point parametric linkage analyses were performed using MLINK and LINKMAP of the Linkage program, version 5.2 (Lathrop et al., 1985) and LOD scores > 1.0 were regarded as requiring further investigation. All patients of family DR246 with dementia and/or parkinsonism were considered affected in the linkage analysis, and the phenotype of individual II.2 was considered unknown. Two-point and multi-point LOD scores were calculated with the same parameters as in the simulation study, and marker allele frequencies were set equal. For comparison, we also calculated LOD scores assuming a maximal penetrance of 60%.

STR genotyping

Twenty fluorescently labelled STR markers spanning the 23.2 cM region between D2S2382 and D2S427 were used for finemapping of the candidate region. Sixteen STR markers were selected from the Marshfield gender-averaged genetic map (http://research.marshfieldclinic.org/genetics/) and four novel STR markers (chr2q35-10, chr2q35-18, chr2q36.3-8 and chr2q36.3-9) were identified using the Tandem Repeat Finder program (Benson, 1999). These STR markers were genotyped in all 22 individuals of family DR246 that were included in the genome-wide scan. Amplification reactions and subsequent analyses of these STR markers were done as described for the genome-wide scan.

The same twenty STR markers, and two additional STR markers from the genome-wide scan (D2S126 and D2S1363), were also genotyped in the available family members of family G. Using the genotypes of these 22 STR markers, haplotypes were reconstructed across the genotyped chromosome 2 region.

Candidate gene analysis

The genes located in the candidate region were determined using NCBI Map Viewer, based on Homo sapiens Genome Build 36.2 (http://www.ncbi.nlm.nih.gov/mapview/). Candidate genes were prioritized according to their expression in brain and biologically relevant function, considering a possible role in the pathogenesis of DLB. Selected candidate genes were analysed for mutations by direct sequencing of all exons, as well as exon–intron boundaries and 5' and 3' regulatory regions. Direct sequencing of selected candidate genes was done in four patients (II.8, III.1, III.3 and III.17) and three control individuals (III.9, III.10 and III.26) of family DR246. Intronic primers flanking the coding exons were developed using Primer3 (Rozen and Skaletsky, 2000). Primers for 5' regulatory regions were designed using Primer Express Software, version 2.0 (Applied Biosystems). Twenty nanogram gDNA was amplified in 20 µl PCR reactions using empirically defined reaction conditions. PCR products were purified with 10 U exonuclease I (USB Corporation) and 2 U shrimp alkaline phosphatase (USB Corporation). Purified amplification products were sequenced in both directions using the BigDye® Terminator Cycle Sequencing kit v3.1 (Applied Biosystems) and analysed on an ABI3730 DNA Analyzer (Applied Biosystems). Variations segregating on the disease haplotype of family DR246 were tested in 46 control chromosomes. Three hundred and forty additional control individuals were available for genotyping of variations absent in the initially tested 46 control chromosomes. For screening of these variations in control individuals, either direct sequencing or Pyrosequencing™ (Biotage, Uppsala, Sweden) were used.

Results

Belgian DLB family

Family DR246 was ascertained through its index patient III.17 who had been referred to our Molecular Diagnostic Unit for genetic testing for early onset dementia. A total of eight patients and 14 at-risk individuals cooperated in the research project and blood samples were collected for genetic linkage studies (Fig. 1). III.17 presented with gait changes, slight resting tremor and hypomimia at the age of 62 years. At that time, structural imaging with computed tomography showed moderate cerebral atrophy, mainly frontally. Neurological examination 1 year later showed a confused patient afflicted with slowly progressive parkinsonism with prominent hypomimia, postural instability and difficulty in rising from a chair and reduced arm swing. Three years after disease onset the index patient was admitted to a psychiatric hospital due to severe nocturnal episodes of confusion, paranoid delusions and visual hallucinations leading to pronounced agitation and verbal and physical aggression. Magnetic resonance imaging at age 65 years showed enlarged ventricles and cortical sulci, associated with enlarged Virchow–Robin spaces. Mini-mental state examination (MMSE) score at that time was 18/30 and the index patient was clinically diagnosed with probable DLB. III.17 died at age 72 years and was pathologically diagnosed with DLBD.

In total, nine individuals in two generations were affected in this family, including one patient (II.4) without offspring who had deceased prior to the start of our study. Table 1 summarizes the clinical characteristics of all nine affected family members. The disease phenotype of the kindred was highly variable. The index patient's mother (II.8) and an uncle (II.10) presented with dementia, while two individuals in the youngest generation displayed only PD features (III.1, III.18). The remaining five patients suffered from a combination of dementia and parkinsonism. The mean AAO, based on the age at which either features of dementia or parkinsonism were first noted, was 70.7 ± 6.3 years (age range: 62–80 years) with a mean duration of disease of 11.5 ± 5.2 years (range of duration: 4–19 years). Using direct sequence analysis, we excluded mutations in previously reported AD genes (PSEN1, PSEN2, APP), as well as in PRNP, MAPT and the PD genes SNCA, parkin, DJ1 and LRRK2. We also excluded the presence of gene or exon dosage variations in SNCA and parkin.

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Table 1

Clinical characteristics of patients DR246 and family G

IndividualM/FAge at onseta (years)Age at death+ or current age (years)Clinical involvement cStructural imaging
DRBRTPIOther
A. DR246
II.4bFP 77 D 9096++NKNKNKNK
II.7FD 67 P 8082+++Hypomimia, Fluctuating cognition, impaired gait
II.8FD 7591++MMSE: 7/30, Concentration and memory disturbances, apraxia, disoriented in time/spaceCortico-subcortical atrophy (CT)
II.9FD 72 P 7376+++++DysarthriaCortico-subcortical atrophy (CT)
II.10MD 8086++
III.1FP 7383+Gait instabilityCortico-subcortical and cerebellar atrophy (MRI)
III.3MP 63 D 7376++++++Repeated falls, hallucinationsSlight atrophy (CT)
III.17MP 62 D 6372++++++Hypomimia, hallucinationsSortico-subcortical atrophy (CT and MRI)
III.18FP 6767++++Bipolar disorder
B. Family G
II.2FNK92++
II.3MNK77+NK+++
III.1MD 78 P 8082++++Postural tremor
III.2MD 7080++
III.3MP 48 D 7076+++++Incontinence
III.4MP 74 D 8190++++++
III.5MP 51 D 7881++++++Incontinence, Frontal lobe release signs
III.6FP 7687+++Postural tremor
III.7MP 52 D 6468++++++Orthostatic hypotension
  • aAge at onset is given for dementia (D) and parkinsonism (P), in order of manifestation. bFor individual DR246 II.4 diagnosis was obtained through a family informant. cFor clinical involvement the presence or absence of dementia (D), rigidity (R), bradykinesia (B), resting tremor (RT) and postural instability (PI) is indicated. M = male; F = female; NK = not known; CT = computerized tomography; MRI = magnetic resonance imaging.

Neuropathology

On macroscopic examination, the brain of III.17 showed general cortical atrophy, ventricular dilatation and severe degeneration of the substantia nigra (Table 2). Microscopic inspection demonstrated neuronal loss and microspongiosis in the hippocampus, and severe neuronal loss in the substantia nigra pars compacta and locus coeruleus associated with release of neuromelanin granules in glial cells and macrophages. Immunohistochemical studies for ubiquitin and α-synuclein detected the omnipresent and abundant occurrence of LB throughout cortex, limbic system and brainstem (Fig. 3). The frontal and temporal gyri were the most affected regions with densities of LB from 1.5 to 25/mm2, while lesser amounts of LB were found in parietal cortex and nearly none in the area striata. Up to 3 LB were detected in a single neuronal cell body. Numerous LB were also found in the gyrus cinguli, the amygdaloid nucleus, the ento- and transentorhinal zone (10–15 LB/mm2) of the gyrus parahippocampalis. The remaining neurons of the substantia nigra pars compacta contained many LB, and some perikarya contained multiple LB. Abundant LB, ubiquitin immunoreactive dystrophic neurites, and some Lewy neurites were present in the locus coeruleus. Neurons belonging to the reticular formation of the brainstem contained LB at the levels of the mesencephalon, pons and medulla oblongata. Large amounts of LB (15/mm2) and Lewy neurites were also present in the dorsal motor nucleus of the vagus nerve (X). AT8-positive neurofibrillary tangles (NFT), dystrophic neurites and neuropil threads as well as ghost tangles were present in transentorhinal and entorhinal cortex, subiculum and parasubiculum and in the pyramidal cells of hippocampal CA1, CA2 and CA4. Rare NFT were also present in midbrain and superior temporal gyrus consistent with a Braak staging of II/III. Staining with 4G8 and Thioflavin S revealed moderate numbers of diffuse and dense-core plaques in the motor cortex, superior temporal gyrus, occipital lobe and hippocampus, compatible with a moderate CERAD senile plaque rating (Mirra et al., 1991). Some of the dense-core plaques were also neuritic as demonstrated by ubiquitin, AT8, and α-synuclein staining of the surrounding neurites. A mild degree of amyloid angiopathy was present in the leptomeningeal arteries. The neuropathological diagnosis of the index patient was consistent with DLBD (Kosaka et al., 1984; McKeith et al., 2005) with concurrent low-to-intermediate AD (Ball et al., 1997).

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Table 2

Summary of main clinical and neuropathological features of families DR246 and G [based on (Denson et al., 1997) and with recent update]

DR246Family G
Clinical phenotypeDLB/PDD (5), dementia only (2), parkinsonism only (2)DLB/PDD (5), dementia only (2), parkinsonism only (4)
Mean age at onset (range)70.7 ± 6.3 years (62–80 years)62.2 ± 13.3 years (48–78 years)
Mean duration of disease (range)11.5 ± 5.2 years (4–19 years)20 years (10–30 years)
Comparison of index patients
DR246 (III.17)Family G (III.3)
    Age at death72 years76 years
    Disease duration10 years28 years
    Neuropathological diagnosisDLBDlimbic (transitional) form of LBD
    Pathological DLB criteria(McKeith et al., 2005)(McKeith et al., 2005)
Macroscopy
Cortical atrophyMild to moderateMild
Ventricles dilatationMild to moderatePresent
Basal gangliaCaudate nucleus mildly atrophiedCaudate nucleus mildly atrophied
Substantia nigraDepigmentedSeverely depigmented
Locus coeruleusSeverely dipigmentedNK
Microscopy
Lewy-related pathology
Fig. 3Fig. 4
Neocortical regionsFrontal42
Temporal42
Parietal31
Basal forebrain/limbicCingulated43
    regionsTransentorhinal/parahippocampal gyri43
Amygdale43
Nucleus basalis of MeynertNK3
BrainstemSubstantia nigra3; up to 3 LB per residual neuron3
Locus coeruleus3+
Dorsal vagal nucleus4+
AD-type pathology
PlaquesNumerous plaques in motor cortex, occipital lobe, superior temporal gyrus and hippocampusNo plaques in hippocampus, some in entorhinal and superficial cortex, and amygdala
Neurofibrillary tangles (NFT)Very rare NFT in midbrain and superior temporal gyrus, several NFT, dystrophic neurites and neuropil threads in the hippocampal formation (Braak stage II/III)No NFT in neocortex, rare in hippocampus and several in entorhinal cortex (Braak stage II)
  • DLB = dementia with Lewy bodies; DLBD = diffuse Lewy body disease; LB = Lewy body; PDD = Parkinson's disease with dementia,+ = present; 1 = mild; 2 = moderate; 3 = severe; 4 = very severe [according to (McKeith et al., 2005)]; NK = not known.

Fig. 3

Ubiquitin (left panel) and α-synuclein (right panel) staining of the index patient of family DR246 in regions of substantia nigra (A, B), locus coeruleus (C, D), cingulate gyrus (E, F), superior temporal gyrus (G, H), superior parietal lobe (I, J) and hippocampus (K, L) reveal numerous Lewy body inclusions. In (B), an inset shows three intracellular inclusions in tegmentum. Scale bar represents 20 μm.

Fig. 4

Haematoxylin and eosin staining (A, D, G) and α-synuclein immunohistochemistry (B, C, E, F, H) of the index patient of family G showing brainstem type Lewy bodies (A, B) and glial α-synuclein-positive inclusions (C) in dorsal raphe, cortical type Lewy bodies (D, E) and Lewy neurites (F) in amygdala, and brainstem type Lewy bodies in nucleus basalis of Meynert (G, H). Original magnification (A–F) × 400 and (G–H) × 200.

Updated neuropathological data of the previously described family G (Denson et al., 1997) are included in Table 2 and Fig. 4.

Genetic studies

In an 8 cM density genome-wide scan, we obtained two-point LOD scores ≥ 1.0 for seven STR markers in five different chromosomal regions (Table 3 and Supplementary Material 1). The highest LOD score of 2.60 at θ = 0.0 was obtained with D2S126 located at 2q36, and approaches the simulated average maximum LOD score. The distal marker flanking D2S126 also gave a positive LOD score of 1.09 at θ = 0.0.

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Table 3

Summary of genome-wide scan genetic data

Chromosomal regionSTR markerLOD score (θ)
1q24ATA38A051.01 (0.0)
2q36D2S1262.60 (0.0)
D2S13631.09 (0.0)
3q13D3S30451.42 (0.1)
D3S35261.20 (0.1)
8q21D8S11261.33 (0.0)
22q11D22S4201.44 (0.0)
  • DLB was treated as a dichotomous, autosomal dominant trait with age-dependent penetrance. A cumulative risk curve was calculated and seven penetrance classes were defined using mean age at onset in the family and assuming a maximum penetrance of 90%. Phenocopy rates were age-dependent and based on dementia prevalence in Western Europe (Ferri et al., 2005), with DLB accounting for 15% of all dementia patients (McKeith et al., 2004; Zaccai et al., 2005). Marker allele frequencies were set equal. A full overview of the two-point linkage data of the genome-wide scan can be found in Supplementary Material 1.

We analysed 20 additional STR markers in the 23.2 cM interval flanked by D2S2382 and D2S427, and obtained a maximum two-point LOD score of 3.01 for D2S1242 in the absence of recombinants (Table 4). A maximum multipoint LOD score of 3.01 was obtained in the interval chr2q35-18–D2S126. Based on genotype data of the 22 STR markers, between D2S2382 and D2S427, we reconstructed haplotypes for all individuals in the linkage pedigree. All patients segregated the same disease haplotype at 2q35-q36 (Fig. 5A) and obligate recombinants defined a candidate region of 13–15 cM (9.2 Mb) between D2S433 (II.8) and chr2q36.3-8 (II.9).

View this table:
Table 4

Two-point LOD scores for 2q35-q36 STR markers in DR246

MarkeraGenetic distanceb (cM)Physical distancec (Mb)HeterozygosityAge-dependent penetrance of maximum 90% LOD at θ =Age-dependent penetrance of maximum 60% LOD at θ = 
0.000.010.050.100.200.300.400.00
D2S2382213.5216.80.81−3.39−2.13−1.02−0.54−0.16−0.04−0.01−2.64
D2S2249215.8218.50.77−1.82−0.64−0.030.180.250.170.05−1.29
D2S433216.3219.60.65−0.770.340.850.930.770.450.13−0.65
chr2q35-18219.81.781.741.571.360.910.470.131.47
chr2q35-10220.01.991.951.791.591.140.650.191.78
D2S163218.5220.60.802.902.852.632.351.751.070.362.56
D2S2359218.5220.80.711.061.040.960.850.610.360.121.03
D2S1242218.5221.00.883.012.962.742.451.821.130.392.63
D2S377220.6221.60.710.790.780.710.620.440.240.070.75
D2S126221.1221.80.822.602.552.362.101.550.940.312.27
D2S2372221.1221.90.682.822.772.562.281.681.020.342.51
D2S339221.1222.50.772.162.111.931.681.170.640.171.89
D2S130222.7223.60.750.790.810.850.820.650.390.121.06
D2S1363227.0226.90.791.091.070.980.870.640.380.131.04
D2S2308227.0227.10.842.052.031.941.781.360.840.272.26
D2S2354227.5227.20.802.362.322.131.891.360.790.242.10
D2S2389228.6227.50.520.120.120.090.070.040.020.010.19
D2S1349228.0227.60.571.341.321.201.050.740.420.131.18
D2S159228.6227.60.770.070.070.050.040.030.020.010.16
D2S2158229.1227.90.771.751.741.651.491.090.610.171.96
chr2q36.3-8228.8−1.18−0.230.380.570.570.360.11−0.83
chr2q36.3-9228.9−1.61−0.550.020.190.230.140.04−1.36
D2S2297232.4230.20.87−1.96−0.080.560.740.700.450.14−1.21
D2S427236.7231.90.76−1.78−0.510.070.240.270.170.05−1.47
  • aMarkers from the 8 cM density genome-wide scan are in bold. bGenetic distances were obtained from the Marshfield gender-averaged genetic map, cM = centimorgans and cphysical positions were deduced from the UCSC Human Genome Browser, Mb = megabase.

Fig. 5

Linkage pedigrees of DR246 (A) and family G (B). Left-filled symbols represent patients with dementia, right-filled symbols represent patients with parkinsonism and filled symbols indicate patients with dementia and parkinsonism. Haplotypes are based on a selection of 12 informative STR markers on 2q35-q36. Haplotypes for deceased individuals were inferred from genotype data of offspring (shown between brackets). For DR246 the disease haplotype was arbitrarily set for I.1, and numbers in diamonds indicate the number of genotyped at-risk individuals. For family G the haplotype segregating with parkinsonism was arbitrarily set for II.3 and II.4, and numbers in diamonds indicate the number of non-genotyped at-risk individuals in generation III, or the number of genotyped at-risk individuals in generation IV. For confidentiality reasons haplotypes are only shown for patients and obligate carriers.

Genotyping of the same 22 STR markers spanning the 9.2 Mb disease locus in all available family members of family G enabled the reconstruction of haplotypes at 2q35-q36 (Fig. 5B). There was no obvious allele sharing between the Belgian family DR246 and the Dutch family G. Linkage of family G to 2q35-q36 could not be excluded based on two-point LOD score calculations, under the same assumptions as used for family DR246.

Mutation analysis of selected candidate genes

Seventy-seven genes are located within the 9.2 Mb candidate region comprising 22 reviewed, nine validated, 22 provisional and six predicted RefSeq genes supported by existence of cDNA clones, expressed sequence tags, or protein homologies, as well as two microRNA coding sequences. We extensively screened five genes for mutations i.e. DnaJ (Hsp40) homolog, subfamily B, member 2 (DNAJB2), EPH receptor A4 (EPHA4), obscurin-like 1 (OBSL1), serine threonine kinase 16 (STK16) and sphingosine-1-phosphate phosphotase 2 (SGPP2), and herein we identified 74 variations in total. These 74 variations comprised six missense mutations, 14 silent mutations, 29 intronic variations and 25 variations in either 5' or 3' regulatory regions (see Supplementary Material 2 for a detailed overview). Four variations in two genes segregated with the disease haplotype in family DR246 (Table 5). Analysis in control individuals indicated that all four variations were common (DNAJB2) or rare (OBSL1 p.Ala1477Thr) polymorphisms. None of the selected genes contained mutations, in exons (coding and untranslated), exon–intron boundaries or regulatory regions, which could be causative.

View this table:
Table 5

Variations segregating with DLB in the Belgian family DR246

Genea, variation (numbering relative to NT_ 005403.16)Relative position within gene, predicted amino acid substitutionLinked alleleFrequencyb in control individuals (%)
DNAJB2 (+)
g.70353826G>C (rs908197)IVS1-110G>CC20.5c
g.70354702C>T (rs2276638)IVS2-14C>TT19.6c
g.70357148C>T (rs2276639)IVS6 + 81C>TT19.6c
OBSL1 (−)
g.70630338C>Tp.Ala1477ThrdT0.9e
  • aDNAJB2: DnaJ (Hsp40) homologue, subfamily B, member 2 situated on plus (+) strand; OBSL1: obscurin-like 1 situated on reverse (−) strand. bAllele frequencies estimated in 46 control chromosomes. For OBSL1 p.Ala1477Thr over 600 additional control chromosomes were tested. c Tested by direct sequencing. dOBSL1 amino acid numbering according to GenBank Accession EF063638 (Geisler et al., 2007). e Tested by Pyrosequencing™ (forward: 5′-bio- TGTGCGGGCAGAGGAAGG-3′, reverse: 5′- GCCATGG ACAGGCGAGAG-3′, sequencing: 5'- GCGCACGGCCCCCGC TG-3'). An overview of all variations identified during the sequencing of the five candidate genes can be found in Supplementary Material 2.

Discussion

Examination of large families has led to the identification of multiple causal genes for neurodegenerative brain diseases such as AD and PD. Several reports have identified kindreds segregating both dementia and parkinsonism (Denson and Wszolek, 1995; Kurz et al., 2006). However, most described patients in which parkinsonism preceded the onset of dementia by many years. In contrast, only few families were described in which dementia precedes the onset of parkinsonism (Tsuang et al., 2004), and those families potentially represent examples of familial DLB. So far, genetic investigations of familial DLB have been very limited and with little result. In this study, we ascertained a three-generation Belgian family, DR246, through its index patient who fulfilled both clinical and pathological criteria for DLB. The family history suggested an autosomal dominant inheritance pattern with a mixed phenotype of dementia and parkinsonism in nine individuals across two generations. The clinical symptoms of five patients were compatible with DLB or PDD, two patients only exhibited parkinsonism, whereas the two remaining patients suffered from dementia, including the mother of our index patient. A clinical diagnosis of DLB could not be assessed retrospectively for these patients, but the absence of parkinsonism (after 16 years of disease) in the mother of the index patient might underscore that parkinsonism is a common but not absolute feature of DLB (McKeith et al., 1996; McKeith et al., 2005). Brain pathology was obtained for the index patient and the ubiquitous presence of LB throughout the cerebral cortex, limbic system and brainstem supported the pathological DLBD diagnosis (Table 2).

In a genome-wide scan with an average intermarker spacing of 8 cM, we observed significant linkage to chromosome 2q35-q36 (multi-point LOD score of 3.01). Obligate recombinants between D2S433 and chr2q36.3-8 defined a candidate region of 9.2 Mb. All patients of family DR246 segregated the same disease haplotype at 2q35-q36, related to the same genetic defect. We thus suggest that the 2q35-q36 locus is specific to a DLB-causing mutation segregating in family DR246. At least in family DR246 this mutation appears to have a high penetrance, which is also supported by the fact that all asymptomatic carriers of the disease haplotype are currently still 1 SD (6.3 years) below the mean AAO (70.7 years, age range: 62–80 years) (data not shown for reasons of confidentiality).

The 9.2 Mb candidate region contains 77 genes, and an extensive sequence analysis of all exons, exon–intron boundaries, 5' and 3' regulatory regions of five of these candidate genes (DNAJB2, EPHA4, OBSL1, SGPP2 and STK16) did not reveal a disease-causing mutation. However, the presence of mutations other than single base pair substitutions or small insertions and deletions within these five genes remains possible. Moreover, the 2q35-q36 locus contains other interesting functional candidate genes as well, and these have been prioritized for mutation analysis.

Of interest is that the clinical phenotype of family DR246 is strikingly similar to that described previously for family G (Denson et al., 1997), with dementia being a prominent feature, in addition to parkinsonism, in several patients of both families (Table 2). The transmission pattern in family G is consistent with autosomal dominant inheritance and, not unimportantly, family G originated from Friesland, a province in the north of The Netherlands and thus in relative close geographical proximity to the Belgian province of Antwerpen, a Dutch-speaking province in the north of Belgium. Comparison of the neuropathology of the index patients of families DR246 and G supported a similar disease aetiology. At the macroscopic level, both brains showed cortical atrophy and depigmentation of the substantia nigra. Microscopic examination revealed neuronal loss and ubiquitin- and α-synuclein-positive LB in brainstem, limbic system and neocortex. Patient III.17 of DR246 was neuropathologically diagnosed with DLBD, whereas the index patient III.3 of family G was reported to have the limbic (transitional) form of LBD (McKeith et al., 1996; Denson et al., 1997). Both patients also had a Braak staging of at least II and presence of amyloid plaques consistent with mild AD pathology. Based on data of single autopsies it would be premature to draw any firm conclusions about the importance of plaque pathology to the pathological phenotype in both families. On the other hand, the concurrent neuropathological diagnosis of low-to-intermediate AD for patient III.17 of DR246 agrees with present neuropathological criteria for DLB (McKeith et al., 2005), in which combined neocortical LB pathology and AD pathology with low-to-intermediate severity have a high likelihood of being associated with a clinical diagnosis of DLB. Additionally, the symptomatic convergence between both families illustrates that DLBD and the LBD transitional form can hardly be differentiated clinically, and moreover the pathological border between the LBD transitional form and DLBD is unclear since these might represent consecutive pathological stages (Marui et al., 2004; Mori, 2005). Therefore, we concluded that families DR246 and G are neither clinically nor pathologically significantly different. Despite this, haplotype sharing analysis demonstrated that family G did not segregate the same 2q35-q36 disease haplotype as family DR246, and thus they are not distantly related. However, careful examination of the haplotype data indicated that we cannot exclude linkage of family G to the 2q35-q36 region. Allelic heterogeneity in families DR246 and G would not be uncommon in view of the fact that several different mutations have been identified in the same gene in other neurodegenerative brain diseases such as AD (http://www.molgen.ua.ac.be/ADMutations/) and PD (see Farrer, 2006 for a recent review).

A remarkable feature of both families is the prominent intrafamilial clinical heterogeneity. This has already been described earlier e.g. SNCA triplications were found to cause autosomal dominant LBD in two distinct families (Singleton et al., 2003; Farrer et al., 2004) with clinical phenotypes ranging from PD to DLB. The concept of a spectrum of LBD, encompassing PD and DLB, is attractive and presumes that these disorders would have pathophysiological and aetiological factors in common (O'Brien et al., 2006). A view that despite intensive research remains to be proven. The two patients alive of DR246, segregating the 2q35-q36 disease haplotype, are only afflicted with parkinsonism, a finding that is unusual for DLB. These patients may still develop dementia (PDD) later in their disease, as a result of the same neurodegenerative process as in DLB, but with different temporal and spatial courses (Papapetropoulos et al., 2006). The Belgian family DR246 therefore supports the concept of a spectrum of LBD, encompassing PD, PDD and DLB.

Currently it is unclear whether our 2q35-q36 locus and the PARK11 locus (Pankratz et al., 2002, 2003b) are pointing to a related genetic defect. Our candidate region overlaps at its telomeric end with the PARK11 locus, which was first detected in a genome-wide scan including 160 North American multiplex PD families (Pankratz et al., 2002). Follow-up studies in a partially overlapping sample using an autosomal dominant model of disease transmission generated significant LOD scores (Pankratz et al., 2003a, 2003b). In contrast, a replication study in 45 European PD pedigrees did not confirm linkage to PARK11 (Prestel et al., 2005). Also, the association of PD with rs10200894, identified in a high-resolution whole-genome association study of PD (Maraganore et al., 2005), and located within our candidate region, was not replicated (Elbaz et al., 2006; Farrer et al., 2006; Goris et al., 2006), except for one study (Li et al., 2006).

Powerful linkage analysis in our Belgian DLB family has led to the identification of a novel locus for familial DLB at 2q35-q36. This novel locus is of particular interest since the identification of the underlying genetic defect will reveal one of the first causal genes for DLB. The ultimate identification of the gene will have a major impact on our understanding of the molecular pathogenesis of DLB. This finding may also be pertinent for LBD and other neurodegenerative diseases with overlapping clinical and pathological features, and might help to differentiate DLB from AD and PD. Increased knowledge of DLB pathogenesis will hopefully contribute to a better identification and treatment of the disease in its earliest stages.

Electronic database information

Alzheimer Disease Mutation Database, http://www.molgen.ua.ac.be/ADMutations/

Marshfield Center for Medical Genetics, http://research.marshfieldclinic.org/genetics/

NCBI Map Viewer, http://www.ncbi.nlm.nih.gov/mapview/

UCSC Human Genome Browser, http://genome.ucsc.edu/cgi-bin/hgGateway

VIB Genetic Service Facility, http://www.vibgeneticservicefacility.be/

Supplementary material

Supplementary material is available at Brain online.

Acknowledgements

We are grateful to the participants of this study for their kind cooperation. We also acknowledge the personnel of the VIB Genetic Service Facility (http://www.vibgeneticservicefacility.be) and the Biobank of the Institute Born-Bunge. J. Searcy helped in obtaining additional data on family G. R. McComb provided hippocampus tissue of the index patient of family G. This study was supported by the Special Research Fund of the University of Antwerp, the Fund for Scientific Research Flanders (FWO-F), the Institute for Science and Technology – Flanders (IWT-F), the Interuniversity Attraction Poles program P5/19 and P6/43 of the Belgian Science Policy Office. The research was in part funded by EU contract LSHM-CT-2003-503330 (APOPIS). P.P.D.D. was funded by the Medical Research Foundation and Neurosearch Antwerp. T.G. was partly sponsored by the German Ministry for Education and Research and the Hertie-Institute for Clinical Brain Research, Tübingen, Germany. The work of D.W.D. and Z.K.W. is funded by the Morris K. Udall Parkinson's Disease Center of Excellence grant P50 NS40256. V.B. holds a PhD fellowship and S.E., K.S. and J.T. a postdoctoral fellowship of the FWO-F. J.V.D.Z. is holder of a PhD fellowship of the IWT-F. Funding to pay the Open Access publication charges for this article was provided by the Special Research Fund of the University of Antwerp.

Footnotes

  • Abbreviations:
    Abbreviations:
    AAO
    age at onset
    AD
    Alzheimer's disease
    DLB
    dementia with Lewy bodies
    DLBD
    diffuse Lewy body disease
    LB
    Lewy body
    LBD
    Lewy body disease
    LOD
    logarithm of odds
    MAQ
    multiplex amplicon quantification
    MMSE
    mini-mental state examination
    mPCR
    multiplex PCR
    NFT
    neurofibrillary tangles
    PD
    Parkinson's disease
    PDD
    Parkinson's disease with dementia
    STR
    short tandem repeat

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References

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