Identification of 2 Loci at Chromosomes 9 and 14 in a Multiplex Family With Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis FREE

Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are 2 severe neurodegenerative disorders. Frontotemporal lobar degeneration has a prevalence close to that of Alzheimer disease in individuals younger than 65 years,¹ whereas ALS is the most common motor neuron disorder.² Frontotemporal lobar degeneration and ALS belong to an overlapping clinicopathological spectrum of disorders that might have common genetic etiologies.³ Clinically, patients with FTLD present with behavioral, personality, and language disturbances, while memory impairment and other cognitive dysfunctions occur in a later stage. Signs of ALS or parkinsonism may also occur.⁴ Conversely, a minority of patients with ALS develop dementia and up to 50% show disturbances in executive functions.^5- 6 Frontotemporal lobar degeneration is caused by degeneration of neurons in frontal and/or temporal lobes, while ALS results from motor neuron degeneration in the motor cortex, brainstem, and spinal cord. Ubiquitinated transactivation response DNA-binding protein 43 (TDP-43) is a major component of pathological inclusions in both tau-negative FTLD (FTLD-TDP) and ALS.^7- 8 A positive family history is observed in up to 50% of FTLD patients⁹ and in 5% to 10% of ALS patients,^10- 13 indicating a significant genetic contribution to disease etiology. Mutations causing FTLD were observed in genes that encode the microtubule-associated protein tau (MAPT),¹⁴ granulin (GRN),^15- 16 the valosin-containing protein (VCP),¹⁷ and the charged multivesicular body protein 2B (CHMP2B),¹⁸ while in ALS patients, mutations in the genes encoding superoxide dismutase 1 (SOD1)¹⁹ and TDP-43 (TARDBP)^20- 24 and FUS^25- 26 were found, among other genes. Furthermore, several families segregating both FTLD and ALS are linked to the ALSFTD2 locus at chromosome 9p13-21, but the causal gene remains elusive.^27- 32 Mutations in these genes explain about 35% of familial patients, indicating that other genes exist. Herein, we performed a genome-wide linkage scan and mutation analyses in an FTLD-ALS family with TDP-43 pathology but without a GRN mutation.

The proband, patient III-12 (Figure 1 and Figure 2), who had FTLD (subtype frontotemporal dementia [FTD]), was contacted for molecular genetic studies. Detailed information on family history was obtained and family members were asked to participate in the study. Clinical information was collected through family informants and medical records from treating physicians. Informed consent was obtained from all 29 participating family members (aged ≥18 years). The research was approved by the local medical ethics committee of the University of Antwerp, University Hospital Antwerp, and ZNA Middelheim. Blood samples were collected for genomic DNA (gDNA) extractions and Epstein-Barr virus transformations.

A postmortem neuropathological study was performed on the brain of the proband (Figure 1 and Figure 2). After 3 months' fixation in 10% buffered formalin, the brain was embedded in paraffin. Sections of 5 μm were prepared from 3 frontocortical regions and Brodmann area 9, the olfactory bulb and tracts, cingulate gyrus, corpus callosum, superior temporal gyrus, hippocampus, parahippocampal gyrus, lateral occipitotemporal gyrus, occipital cortex with area striata, cerebellum, midbrain, and pons with locus coeruleus. The sections were examined using routine histopathological methods and immunostained using antibody AT8 against hyperphosphorylated tau (Innogenetics, Zwijnaarde, Belgium), 4G8 against residues 17 through 24 of amyloid β (Signet, Dedham, Massachusetts), 3F4 against prion protein (Signet), antiglial fibrillary acidic protein (Dako, Glostrup, Denmark), CD68 (Dako), antiubiquitin (Dako), anti-p62 (Progen Biotechnik), rabbit TDP-43 antisera (Proteintech Group Inc, Chicago, Illinois), and phospho-TDP-43 against 409/410 serines of TDP-43.⁷ Antigen retrieval and staining were performed as described.³³

We performed a genome-wide scan using 425 short tandem repeat (STR) markers, with a mean intermarker distance of 8 cM as described.³⁴ For fine mapping of regions with a multipoint logarithm of odds (LOD) score above 1.9, additional STR markers were selected from the Marshfield genetic map (http://research.marshfieldclinic.org/genetics) or from the Microsatellites track in the University of California–Santa Cruz Human Genome Browser (http://genome.ucsc.edu), based on Tandem Repeats Finder.³⁵ Primers were designed with an algorithm implemented in a polymerase chain reaction (PCR) multiplexer program (primers are available from the authors upon request). Polymerase chain reaction amplification reactions and analyses were performed as described.³⁴

Genes were selected for mutation analysis on gDNA or complementary DNA (cDNA) of 2 patients (III-9 and III-12) (Figure 1 and Figure 2) and 2 married-in control individuals. Epstein-Barr virus–transformed lymphoblasts were treated with 100-μg/mL cycloheximide or with dimethyl sulfoxide for 4 hours prior to RNA isolation. Total RNA was isolated using the Ribopure Kit (Ambion; Applied Biosystems, Foster City, California) and first-strand cDNA was synthesized using the SuperScript III First-Strand Synthesis System for reverse transcriptase–PCR kit (Invitrogen, Carlsbad, California). Primers were designed using Primer 3 (available from the authors upon request).³⁶ Standard PCRs on gDNA amplified exons and exon-intron boundaries with optimized conditions. Amplified cDNA of the entire open reading frame was analyzed for aberrantly sized transcripts by agarose gel electrophoresis. Amplicons of both gDNA and cDNA were purified, sequenced in both directions using BigDye Terminator Cycle Sequencing kit, version 3.1 (Applied Biosystems), and analyzed on an ABI3730 DNA Analyzer (Applied Biosystems). Sequences were analyzed using novoSNP.³⁷ Variations segregating with the disease were tested in at least 90 neurologically healthy control individuals.

Oligonucleotide-based array comparative genomic hybridization (CGH) was performed in the proband and a control individual not carrying the disease haplotype using 385K Tiling arrays (Nimblegen, Madison, Wisconsin) of chromosomes 9 and 14, with a mean probe spacing of 255 base pairs (bp) and 200 bp, respectively. Data were analyzed with SignalMap (Nimblegen). Copy number variations (CNVs) were validated by quantitative real-time PCR using SYBR Green assays on the ABI Prism 7900HT Sequence Detection System (Applied Biosystems) as described previously.³⁸ Confirmed CNVs were tested for segregation in family DR14 using quantitative PCR.

The statistical power of family DR14 to obtain genome-wide significant linkage was assessed using SLINK,^39- 40 assuming autosomal dominant inheritance with age-dependent penetrance and phenocopy rates. A cumulative risk curve was calculated based on the mean age at onset (58.1 years), and 9 liability classes for disease penetrance were defined with a maximum penetrance of 90%.

Two-point and multipoint parametric linkage analyses were performed using MLINK and LINKMAP from the Linkage package, version 5.2,⁴¹ or Simwalk, version 2.91,⁴² for genome-wide multipoint parametric linkage analysis. All patients with FTLD, dementia, and/or ALS were considered affected, and the phenotypes of individuals I-1, I-2, II-6, and II-7 were considered unknown (Figure 1 and Figure 2). Parameters were the same as in the simulation study and marker allele frequencies were set equally.

At age 57 years, patient III-12 presented with marked behavioral disturbances and personality changes, including agitation, irritability, and compulsive behavior, and experienced forgetfulness. His brain computed tomographic scan showed moderate frontal atrophy. One year later, the patient's personality disturbances had progressively worsened and the patient showed mild impairment of short- and long-term memory, impaired concentration, and impaired verbal fluency. His Mini-Mental State Examination score was 28 of 30. Brain-perfusion single-photon emission computed tomography showed hypoperfusion in the frontotemporal region at both sides, and on brain magnetic resonance imaging severe frontotemporal atrophy was observed (eFigure). At age 59 years, the patient presented with severe wandering (away from home), and his cognitive state had severely declined, as illustrated by a Mini-Mental State Examination score of 3 of 30. He did not show muscle weakness, fasciculations, or hyperactive reflexes. The patient received a diagnosis of familial FTLD (subtype FTD)⁴ and died at age 60 years.

Macroscopic examination of the autopsied brain demonstrated a generalized cortical atrophy, which was most prominent in the frontal lobe resulting in dilatation of frontal (and temporal) horns of the lateral ventricles. On microscopic examination, marked neuronal loss was observed mostly in cortical layers I and II of the frontal cortical regions, including the prefrontal cortex (Figure 3A), but it was also observed in the cortex of the gyrus temporalis superior and the CA1 segment of the hippocampus. Mild microspongiosis and astrocytic gliosis were present in the cortical layers I and II of the prefrontal cortex and the deep cortical layers, respectively. Except for some very rare neurofibrillary tangles in the hippocampus, AT8 staining did not reveal neurofibrillary tangles or Pick bodies in any other region analyzed (Figure 3D), and in most regions 4G8 staining showed some focal accumulations of diffuse or senile amyloid β plaques (Figure 3E), but amyloid angiopathy was absent. These observations, accompanied by corpora amylacea in these regions, most likely represent age-dependent phenomena. Immunoreactivity with the antiprion antibody was negative.

Ubiquitin staining frequently demonstrated a small number of rim-shaped, granular, or occasionally large globular ubiquitin-positive neuronal cytoplasmic inclusions in the superficial cortical layers, especially of the frontal neocortex (Figures 3B and C). In addition, small ubiquitin-positive neuritic profiles were observed in the affected neocortical layers. Immunoreactivity of p62 was observed in a subset of neuronal cytoplasmic inclusions but never in the nucleus or neurites (Figure 3F). Immunostaining with TDP-43 and phospho-TDP-43 antibodies also showed a strong presence of different neuronal cytoplasmic inclusions in the superficial cortical layers with clearing of nuclei of inclusion-bearing neurons as well as short neuritic profiles or dot-like structures (Figures 3G and H). Although the observed FTLD-TDP pathology did not completely fit into one of the described FTLD-TDP subtypes,^43- 44 the presence of neuronal cytoplasmic inclusions without neuronal intranuclear inclusions was more typical for type III.⁴³

Family DR14 was ascertained through its proband, individual III-12, who initially had been referred to our Molecular Diagnostic Unit for genetic testing of FTLD genes. No mutations were observed in MAPT, GRN, VCP, or CHMP2B. Informed consent was obtained for further genetic testing, and mutations were also excluded in the presenilin genes PSEN1 and PSEN2; exons 16 and 17 of the amyloid precursor protein gene (APP); the coding exon 2 of PRNP; TARDBP; and the intraflagellar transport 74 homologue gene (IFT74).²⁹

Genealogical analysis identified a 4-generation family that showed indications of autosomal dominant transmission of dementia (Figure 1 and Figure 2). In total, we collected DNA from 29 family members, including 3 patients in generation III and 11 at-risk individuals in both generation III and IV. The mean age at onset was 58.1 years (SD, 4.2 years [n = 9]; range, 51-65 years) and mean disease duration was 6.4 years (SD, 4.9 years; range 1-17 years). Two patients (III-2 and III-12) (Figure 1 and Figure 2) had dementia specified as FTLD (subtype FTD), while patient III-10 had a diagnosis of ALS. The clinical characteristics of the 9 patients are summarized in Table 1. Of individuals I-1, I-2, II-6, and II-7, no or very limited clinical information was available. In individuals II-6 and II-7, some suggestive symptoms pointed toward an (unconfirmed) ALS phenotype, including swallowing problems and loss of muscle strength.

With an assumed hypothetical marker with 4 equifrequent alleles and a disease allele frequency of 0.01%, simulation analyses indicated an average maximum LOD score of 2.45 at recombination fraction (θ) 0.04. The mean maximum false-positive LOD score was 2.26 (θ = 0.5).

Multipoint LOD scores were calculated for markers of the genome-wide linkage scan (Figure 4). At 3 loci, we obtained a LOD score above 1.9, which is compatible with suggestive linkage⁴⁵: one at chromosome 9 with a maximal LOD score of 2.71 between D9S1121 and D9S270, one at chromosome 14 with the highest LOD score of 2.61 between D14S302 and D14S611, and one at chromosome 11 with a maximal LOD score of 1.99 between D11S4464 and D11S4410.

At chromosome 9, 31 additional STR markers were analyzed, in an interval of 80.7 cM (85.9 Mb) between D9S2169 and D9S253 (eTable 1). A maximal 2-point LOD score of 2.82 was obtained with D9S171 (θ = 0.0), and a maximal multipoint LOD score of 3.38 was calculated in the region between D9S1833 and D9S1121 (Table 2). Haplotypes were reconstructed for all individuals, and segregation analysis defined a candidate region of 64.6 cM (74.7 Mb) between D9S235 and D9S257 at 9p23-q21. Obligate meiotic recombinants defining the minimal interval were observed in patient III-9 (Figure 1).

At chromosome 14, 17 additional STR markers were genotyped in an interval of 41.8 cM (17.9 Mb) between D14S1005 and the 14q telomere (deTable 2). The marker D14S302 reached the highest 2-point LOD score of 2.45, and a maximal multipoint LOD score of 2.79 was obtained between D14S1030 and D14S987 (Table 3). Based on an obligate meiotic recombination in patient II-5 between D14S1015 and D14S973, the disease haplotype was delineated to a region of a maximal length of 33.2 cM (14.6 Mb) at 14q31-q32 between D14S1015 and the 14q telomere (Figure 2). Individual II-7 carried only part of the haplotype owing to a telomeric recombinant between D14S985 and D14S1051. However, his disease status was unclear.

At chromosome 11, 11 additional STR markers were analyzed in an interval of 42.0 cM (21.0 Mb) between D11S897 and D11S4112. MFD316 reached the maximal 2-point LOD score of 1.73, and the highest multipoint LOD score of 2.18 was obtained in the interval between MFD316 and D11S4464 (Table 4). Haplotype analysis showed a disease haplotype with a maximal length of 11.0 cM (7.0 Mb) at 11q23-q25 between D11S1998 and D11S933, defined by obligate meiotic recombinants in patients II-5 and III-3.

Because of the significant or nearly significant linkage at chromosomes 9p23-q21 and 14q31-q32, we prioritized genes in these 2 loci. Positional candidate genes were selected from the National Center for Biotechnology Information Reference Sequence (RefSeq) database of protein-coding genes. The chromosome 9p23-q21 locus in DR14 harbored 265 genes. Because this region did not reduce the known ALSFTD2 locus at 9p13-p21, we based the selection of genes on published segregation data in chromosome 9–linked FTLD-ALS families with a multipoint LOD score above 3.0.^{27- 28,30- 32} We defined a minimal region of 6.96 Mb between D9S169²⁸ and D9S1805³¹ that segregated in all these families and contained 27 protein-coding genes. Based on functions, protein homology and interactions with known FTLD genes, we analyzed the 17 best candidate genes by sequencing of the complete coding sequence of cDNA from lymphoblasts treated with or without cycloheximide, allowing the detection of putatively degraded aberrant transcripts. These 17 genes included MOBKL2B, C9orf72, ACO1, DDX58, TOPORS, NDUFB6, DNAJA1, SMU1, B4GALT1, BAG1, CHMP5, AQP3, NOL6, UBE2R2, UBAP2, WDR40A, and UBAP1. We did not identify novel variants segregating on the disease haplotype.

The candidate region at chromosome 14q31-q32 harbored 121 protein-coding RefSeq genes. Sixty-six genes located in the region between D14S1015 and D14S1051defined in individual II-7 (Figure 2) were prioritized for exon-based genomic sequencing. We identified 9 novel variants segregating on the disease haplotype; however, all were present in at least 1 control individual (Table 5). The Gly191Ala variant in YY1 and the IVS5-43G>C variant in SERPINA9 were found in only 1 of 300 and 1 of 360 control individuals with age at last examination of 83 and 77 years, respectively; no positive familial history of dementia was described.

We performed high-density array CGH at chromosomes 9 and 14 that covered the complete candidate regions. In the chromosome 9 locus, we identified 2 large CNVs (chromosome 9: 24492891-24508926 and chromosome 9: 29082732-29087816) covered by 64 and 20 CGH probes, respectively. Only the deletion of the second region was confirmed in the proband by 6 quantitative PCR fragments that demonstrated a deleted region of at least 5273 bp (chromosome 9: 29082677-29087949) (data not shown). This deletion did not segregate with disease in DR14 and represented a polymorphism, because it was also present in individuals not carrying the disease haplotype and a frequent CNV had previously been reported at this position (chromosome 9: 29082445-29088195).⁴⁶ The chromosome 14 locus did not show the presence of a large CNV except for a known CNV region in the immunoglobulin heavy chain gene family cluster near the 14q telomere.

We identified a multigenerational autosomal dominant FTLD-ALS family, family DR14, of whom the proband presented clinically with FTD and neuropathologically with FTLD-TDP. We showed significant linkage to chromosome 9p23-q21 overlapping with the known ALSFTD2 locus and nearly significant linkage to a novel locus at chromosome 14q31-q32. At both loci, the disease haplotype segregated in all patients (Figure 1 and Figure 2). Also, an asymptomatic individual in generation III, aged 76 years at last examination, carried the complete chromosome 9 disease haplotype, possibly suggesting reduced penetrance of the mutation. In addition, the current ages of asymptomatic at-risk individuals were within or below the wide age at onset range (individual data not shown for confidentiality). Therefore, recombinants in these individuals were not accounted for to define the candidate regions. Alternatively, because the asymptomatic carrier of the chromosome 9 haplotype did not carry the chromosome 14 disease haplotype, the chromosome 9 mutation is possibly insufficient to cause the disease, implicating a digenic effect by which the disease will only be fully expressed if mutant genes at both loci are present in the same individual. The latter is supported by the observation that all affected individuals carry both the chromosome 9 and 14 disease haplotypes. Another potential explanation could be that a mutation in a gene in the one locus has a modifying effect on the disease-causing mutation at the other. Alternatively, 1 of the 2 loci might be a false-positive finding due to coincidental segregation even if both loci reached a near equal level of genetic significance. The slight difference in the maximum LOD scores is not essential, because they largely depend on the allele frequencies in the control population. Owing to accumulating evidence for an FTLD-ALS locus at 9p23-q21 and because family DR14 has a similar clinicopathological phenotype than other chromosome 9–linked families,^27- 32 one might reason that the chromosome 14 linkage is more likely false.^27- 32,47

At chromosome 9, in a minimal region shared between chromosome 9–linked families,^{27- 28,30- 32} we excluded in 17 genes the presence of simple mutations, exonic insertions or deletions, and detectable altered splicing by transcript analysis. Currently, all 27 RefSeq genes located in this minimal region have been analyzed for mutations on gDNA and/or cDNA in 1 or more families.^{28- 29,31- 32} In addition, we excluded large CNVs in the complete DR14 candidate region. However, mutations might still have been missed because most candidate genes were either exclusively screened on gDNA or in families not conclusively linked to chromosome 9.²⁹ Also, a noncoding mutation in 1 of the screened RefSeq genes or a mutation in a non-RefSeq gene or outside an annotated gene cannot be excluded. Furthermore, the potential of phenocopies within these families and subsequent false meiotic recombinants makes it possible that the disease gene is actually located outside the delineated minimal candidate region. Alternatively, although the increasing number of families with similar phenotypes linked to chromosome 9p supported the idea that 1 common gene might be responsible for the occurrence of both FTLD and ALS, the possibility remains that the FTLD and ALS symptoms are the result of 1 larger genomic rearrangement affecting 2 different disease genes. The array CGH applied here could only detect CNVs of at least 1 kb, while inversions or small insertions or deletions could not be detected at all. Performing other techniques, including macrorestriction mapping, would provide more information on the presence of more complex rearrangements.

In the chromosome 14q31-q32, locus 66 genes were excluded for the presence of simple mutations in the coding exons and exon/intron boundaries. We selected these genes because of their localization within the candidate region delineated by a centromeric recombinant in patient II-5 and a telomeric recombinant in individual II-7 (Figure 2). In the latter patient, the disease status was uncertain, though it suggested ALS. Therefore, the remaining 55 RefSeq genes between the telomeric recombinant and the 14q telomere might still carry the mutation. Large CNVs were excluded in the complete candidate region by array CGH. Other types of mutations or mutations outside the screened sequences are still possible. Also at chromosome 11q23-q25, we observed suggestive linkage where we did not yet prioritize genes for mutation analysis because of its lower significance.

In conclusion, additional mutation analyses in both loci are needed to identify the underlying genetic etiology of the disease in family DR14. Although we did not reduce the known ALSFTD2 locus, this family provided supportive evidence for an important contribution of the chromosome 9 gene to FTLD and/or ALS and a novel disease-linked locus at chromosome 14q31-q32. We expect that the disease gene(s), once identified, will significantly contribute to understanding the neurodegenerative disease mechanisms in FTLD, ALS, and related diseases.

Correspondence: Marc Cruts, PhD, Neurodegenerative Brain Diseases Group, VIB–Department of Molecular Genetics, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium (marc.cruts@molgen.vib-ua.be).

Accepted for Publication: October 8, 2009.

Author Contributions: Dr Cruts had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Gijselinck, Van Broeckhoven, and Cruts. Acquisition of data: Gijselinck, Engelborghs, Maes, Cuijt, Peeters, Mattheijssens, Joris, Cras, Martin, De Deyn, Kumar-Singh, Van Broeckhoven, and Cruts. Analysis and interpretation of data: Gijselinck, Engelborghs, Maes, Cuijt, Joris, Cras, Martin, De Deyn, Kumar-Singh, Van Broeckhoven, and Cruts. Drafting of the manuscript: Gijselinck, Van Broeckhoven, and Cruts. Critical revision of the manuscript for important intellectual content: Gijselinck, Engelborghs, Maes, Cuijt, Peeters, Mattheijssens, Joris, Cras, Martin, De Deyn, Kumar-Singh, Van Broeckhoven, and Cruts. Obtained funding: Van Broeckhoven. Administrative, technical, and material support: Gijselinck, Engelborghs, Maes, Cuijt, Peeters, Mattheijssens, Joris, Cras, Martin, De Deyn, and Van Broeckhoven. Study supervision: Van Broeckhoven and Cruts.

Financial Disclosure: None reported.

Funding/Support: This research was funded in part by the Special Research Fund of the University of Antwerp, the Fund for Scientific Research Flanders, the Institute for Science and Technology–Flanders, the Methusalem Excellence Grant of the Flemish Government, the Interuniversity Attraction Poles program P6/43 of the Belgian Science Policy Office, the Stichting Alzheimer Onderzoek, and the Association for Frontotemporal Dementias. Drs Gijselinck and Engelborghs hold postdoctoral fellowships from the Fund for Scientific Research Flanders.

Additional Contributions: We are grateful to the DR14 family members for their cooperation. We further acknowledge the contribution of the personnel of the VIB Genetic Service Facility and the Biobank of the Institute Born-Bunge. We also thank Masato Hasegawa, PhD, for donating the phospho-TDP-43 antibody.