1998 Oxford University Press
Human Molecular Genetics, 1998, Vol. 7, No. 11 1825–1829
Segregation of a missense mutation in the microtubule-associated protein tau gene with familial frontotemporal dementia and parkinsonism Cécile Dumanchin1,+, Agnès Camuzat2,+, Dominique Campion1, Patrice Verpillat2, Didier Hannequin1, Bruno Dubois2,3, Pascale Saugier-Veber1, Cosette Martin1, Christiane Penet2, Françoise Charbonnier1, Yves Agid2,3, Thierry Frebourg1,* and Alexis Brice2,3,* 1Génétique
et Hématologie Moléculaires (JE 2006), Centre Hospitalo-Universitaire de Rouen, 76031 Rouen, France and IFRMP, 76821 Mont-Saint-Aignon, France, 2INSERM U289, Paris, France and 3Fédération de Neurologie, Hôpital de la Salpêtrière, 75013 Paris, France Received July 8, 1998; Revised and Accepted July 31, 1998
Frontotemporal dementia and parkinsonism (FTDP) is the second most common cause of neurodegenerative dementia after Alzheimer’s disease. Recently, several kindreds with an autosomal dominant form of FTDP have been reported and in some families the pathological locus was mapped to a 2 cM interval on 17q21–22. The MAPT gene, located on 17q21 and coding for the human microtubule-associated protein tau, is a strong candidate gene, since tau-positive neuronal inclusions have been observed in brains from some FTDP patients. Direct sequencing of the MAPT exonic sequences in 21 French FTDP families revealed in six index cases the same missense mutation in exon 10 resulting in a Pro→Leu change at amino acid 301. Co-segregation of this mutation with the disease was demonstrated by restriction fragment analysis in two families for which several affected relatives were available. The Pro301Leu mutation was not observed in either 50 unrelated French controls or in 11 patients with sporadic frontotemporal dementia. This mutation, which occurs in the second microtubule-binding domain of the MAPT protein, is likely to have a drastic functional consequence. The observation of this mutation in several FTDP families might suggest that disruption of binding of MAPT protein to the microtubule is a key event in the pathogenesis of FTDP. INTRODUCTION Recently, several families have been reported in which an insidious presenile form of dementia with prominent behavioural disturbance and motor manifestations segregates as an autosomal dominant trait. In some families, the pathological locus was mapped to a 2 cM
interval on 17q21–22 (1–11) and the corresponding condition was therefore designated frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) (12). However, genetic heterogeneity has been demonstrated, since another locus for frontotemporal dementia has been mapped on chromosome 3 in a single large pedigree (13). Despite descriptions that have emphasized one or another clinical or neuropathological feature, FTDP-17 kindreds share similar characteristics: gross neuropathological examination shows atrophy of the frontotemporal and basal ganglia contrasting with relative sparing of the hippocampus. In some, but not all, cases, microscopical examination of affected brains reveals tau-positive neuronal inclusions, without the characteristics of Pick bodies, and/or tau-positive glial inclusions (12). The gene coding for the human microtubule-associated protein tau (MAPT) is located on 17q21 and is therefore a strong candidate for FTDP-17. However, in three FTDP-17 families, analysis of the MAPT coding sequence had failed to detect any pathogenic mutation in affected subjects (4,8,9). We had previously identified 21 French pedigrees with FTDP and we report in this study identification of the same MAPT mutation in six families. RESULTS We studied 21 families with FTDP, in which age of onset ranged from 38 to 73 years (Table 1). For each family, we analysed the MAPT gene in one affected proband. Direct sequencing of the MAPT exons revealed in six families the same C→T substitution (CCG→CTG) in exon 10 (data not shown), resulting in a Pro→Leu change at amino acid 301 (Table 1). This substitution removes an MspI restriction site which allowed simple confirmation of the mutation in index cases and detection of the mutation in the relatives. Co-segregation of the Pro301Leu mutation with the disease was established in two families for which DNA from several relatives was available (Fig. 1). In other pedigrees, lack of living affected relatives precluded such an analysis. The Pro301Leu mutation was not observed in 50 unrelated controls and in 11 patients with
*To whom correspondence should be addressed. T.F.: Tel: +33 2 32 88 81 82; Fax: +33 2 32 88 80 80; Email:
[email protected]. A.B.: Tel: +33 1 42 16 21 82; Fax: +33 1 44 24 36 58; Email:
[email protected] +These authors contributed equally to this work
1826 Human Molecular Genetics, 1998, Vol. 7, No. 11
Figure 1. Segregation of the Pro301Leu MAPT mutation in the ROU 113 and SAL 525 pedigrees. Filled symbols, FTDP patients; open symbols, asymptomatic individuals; +/+, wild-type genotype; +/301, heterozygous Pro301Leu mutation. In the ROU 113 and SAL 525 families, nine individuals (four affected and five unaffected) and five individuals (two affected and three unaffected) were analysed, respectively. Under each pedigree, the detection of the mutation by restriction fragment analysis is shown. The fragment sizes (in bp) of the molecular weight marker (HinfI-digested ΦX174) are shown to the left of the gels. Lanes correspond to subjects shown in the corresponding pedigree. The wild-type PCR product (271 bp) after digestion generates 167 and 104 bp restriction fragments, whereas the mutant product containing the Pro301Leu substitution is not digested.
sporadic frontotemporal dementia from the French population. In addition, six other MAPT nucleotide variations were also identified in affected probands of the FTDP families (Table 2). DISCUSSION We have detected a missense mutation (Pro301Leu) of the MAPT gene in six French FTDP families. The human MAPT gene includes 11 coding exons (1–5, 7 and 9–13). In human brain, exons 2, 3 and 10 are alternatively spliced, thus producing six different isoforms, which range from 352 to 441 amino acids and differ from each other by the presence or absence of three inserts (14). Although we cannot exclude that the Pro301Leu substitution is in linkage disequilibrium with the causal mutation, this mutation, located in exon 10, is likely to be pathogenic. (i) This substitution, which is predicted to produce a drastic change in the conformation of the protein, occurs in the second of four repeats (Pro-Gly-Gly-Gly) located in the C-terminal half of the MAPT protein which are highly conserved among species. These repeats constitute the microtubule-binding domains, which suggests that the Pro301Leu mutation might alter binding to the microtubule. However, since exon 10 is alternatively spliced in human brain, this mutation will only affect the subset of MAPT isoforms containing the four microtubule-binding domains. (ii) The Pro301Leu was not found in 50 French controls, indicating that this variation is not a common polymorphism. (iii) Co-segregation analysis, performed in two families (Fig. 1) confirmed that the Pro301Leu mutation is probably involved in FTDP. In contrast, the six other MAPT sequence variations that we have detected correspond to polymorphisms, since they were observed
in unaffected controls (Table 2). Furthermore, five out of these six variations were observed in the proband of family ROU 100 harbouring the Pro301Leu mutation, were homozygous in some probands (Tables 1 and 2) and the three variations identified in exons 7 and 9 do not change the amino acid. In the six families in which we identified the Pro301Leu mutation, the age of onset ranged from 46 to 62 years (Table 1), suggesting that other genetic or non-genetic factors may interact with MAPT mutations to determine onset of the disease. In families with early-onset Alzheimer’s disease associated with APP mutations, such an interaction has been documented with the ApoE genotype and it has been reported that the ApoE4 isoform decreases the age of onset (15), possibly via an interaction between MAPT and ApoE (16). It has also been reported that in sporadic frontal lobe dementia, the age of onset decreases as the number of APOE ε4 allele increases (17). In this study, the 10 affected individuals harbouring the Pro301Leu mutation and corresponding to six distinct families (Table 1) had an ApoE ε3/ε3 genotype. This result is not surprising, since the ε3 allele frequency in the French population is ∼0.8. Considering the variation in age of onset observed in these families (Table 1), this result does not support the hypothesis that APOE is a modifier gene in FTDP-17 families. While this study was in progress, three groups have reported the occurrence of various MAPT mutations in FTDP-17 (18–20). The Pro301Leu mutation was also identified in two families originating from The Netherlands and from the USA (19). Strikingly, we detected this mutation in six out of 21 families of French origin. This high proportion might suggest that, in the French population, the Pro301Leu mutation could result from a founder effect.
1827 Human Genetics, 1998, 7, No. NucleicMolecular Acids Research, 1994, Vol. Vol. 22, No. 1 11 1827 Table 1. Characteristics of the 21 FTDP families screened for MAPT mutations Families
Number of affected individuals
Age of onset (range)
Mutationa
ROU 113 ROU 100 SAL 501 SAL 525 SAL 526 AL 097 AL 092 ROU 001 ROU 098 AL 093 SAL 522 AL 036 ROU 280 SAL 513 AL 055 AL 045 SAL 521 SAL 535 SAL 539 SAL 504 ROU 288
8 6 11 5 3 4 3 2 2 4 2 2 2 4 3 4 3 2 3 2 2
50–60 52–62 41–62 46–61 51–55 51–54 50–58 44–55 59–67 50–60 53–55 45–60 52–58 38–50 55–73 51–64 45–63 40–45 45–52 68–69 68–70
Pro301Leub Pro301Leu Pro301Leu Pro301Leuc Pro301Leu Pro301Leu –d – – – – – – – – – – – – – –
aNumbered
according to the longest MAPT isoform. in four affected individuals (Fig. 1). cDetected in two affected individuals (Fig. 1). dNo pathogenic mutation was detected. bDetected
Table 2. MAPT variants identified in FTDP families Location
Variant
Identified in
Allele frequencya
Exon 7, codon 176b
CCG→CCA Pro→Pro
ROU 100c, AL 092d, ROU 001d, ROU 098d, AL 093d
0.42e
Exon 9, codon 227b
GCA→GCG Ala→Ala
ROU 100c, AL 092d, ROU 001d, ROU 098dAL 093d, AL 045d, SAL504d
Exon 9, codon
255b,g
Intron 2, splice donor site +18
100c,
001d, ROU
098d,
ROU
C→T
ROU 100c, AL 092d, ROU 001d, ROU 098d, AL 093d AL
AL
093d,
AAT→AAC Asn→Asn
092d,
AL
092d, ROU
AL
045d, SAL504c
093d
Intron 11, splice donor site +34
G→A
AL
3′ UTR, stop codon +29
T→C
ROU 100c, AL 092d, ROU 001d, ROU 098d, AL 093d, SAL504c
0.32f 0.33f 0.29f 0.33f 0.30f
aFrequency
of the allele corresponding to the variant. according to the longest MAPT isoform. cHomozygous substitution. dHeterozygous substitution. eEstimated on 12 controls. fEstimated on 35 controls. gPreviously reported (18). bNumbered
In 15 FTDP families, we did not detect any pathogenic mutation in the MAPT coding region. Since we had no linkage data for these families, we cannot exclude that these negative results might be explained by genetic heterogeneity. The alternative hypothesis is that, at least in some families, MAPT mutations might fall outside the coding region, such as in the promoter or the introns. From a biological point of view, identification of the Pro301Leu mutation in eight FTDP families (this study; 19) may be highly informative. Since this mutation is located in one of the microtubule-binding domains of the MAPT protein, it could be
speculated that disruption of binding of the MAPT protein to the microtubule is a key event in the pathogenesis of FTDP. MATERIALS AND METHODS Patients Twenty one FTDP families with autosomal dominant inheritance were selected for this study. All affected subjects fulfilled the Lund and Manchester criteria for frontotemporal dementia (21): they presented with similar clinical features, including behaviou-
1828 Human Molecular Genetics, 1998, Vol. 7, No. 11 ral disturbances (disinhibition, stereotypic behaviour and apathy), memory impairment, relative preservation of spatial orientation and speech disturbance with reduced speech output progressing to muteness. CT scans showed frontotemporal atrophy and functional imaging anterior hypoperfusion. As shown in Table 1, ages of onset were mainly in the fifth or sixth decade. Eleven patients with sporadic frontotemporal dementia were selected using the same clinical criteria. After informed consent was obtained, peripheral blood lymphocytes were collected and genomic DNA was extracted using a standard procedure.
at 50–60C and 40 s at 72C, preceded by 2 min at 95C and followed by 5 min at 72C. PCR products were purified by electrophoresis on low melting point agarose gels and directly sequenced on both strands using the PRISM AmpliTaqFS Ready Reaction Dye Terminators or Rhodamine Dye Terminators sequencing kits (Applied Biosystems, Perkin Elmer-Cetus) and an Applied Biosystems model 373A or 377 automated sequencer. Nucleotide sequences obtained in patients were compared with that obtained in a neurologically normal subject using the SeqEd 675 DNA Sequence Editor Software (Applied Biosystems, Perkin Elmer-Cetus).
Table 3. Primers used for PCR amplification of the MAPT gene
Restriction fragment analysis Primera
Sequence
1F
5′-CCCAACACTCCTCAGAACTT-3′b
1R
5′-CAGTGATCTGGGCCTGCTGT-3′b
2F
5′-AGCTCCACAGGACACTGCTC -3′
2R
5′-ACCGAAGGAGTGAGCACATC-3′b
3F
5′-ACTTCAGGGCTGCTTTCTGG-3′b
3R
5′-AATCCACCCATGTTCCTGCC-3′b
4F
5′-GGTACAGAGAGCTTGGTTTC-3′b
4R
5′-GTCCAAATGATCTTTTCAGG-3′
5F
5′-GGCTTTCTGTGAACAGTGAA-3′b
5R
5′-GCTTCTCTGTAAACTTGACC-3′b
7F
5′-GGTGGCAGTAACTTTTCCCA-3′
7R
5′-GAGAGCTTCAGCTTCCTCTA-3′
9F
5′-CCCCAGGGCCTTTTCTGAC-3′b
9R
5′-ATGCACAGTCCACGACTCCA-3′b
10Fc
5′-GGAGGCGTCCTTGCGAGCAAG-3′
10Rd
5′-GTGTACGACTCACACCACTTC-3′
11F
5′-TTGCTCATTCTCTCTCCTCC-3′b
11R
5′-CTCTTCTGAAGTCTGGAGC-3′
12F
5′-CCACAGAACCACAGAAGATGA-3′b
12R
5′-AAGGCAGCATCCAACCCACC-3′
13F
5′-TCTCTGGCACTTCATCTCAC-3′b
13R
5′-CACTCTCTCATTCTCTCCTC-3′b
14F
5′-TGACCTTGATGTCTTGAGAGC-3′b
14R
5′-GAATTCGGGACATTGTGACG-3′b
aNumbers
correspond to the amplified exon; F, sense primer; R, antisense primer. bAccording to Froelich et al. (9). cContaining an M13R additional site: CAG GAA ACA GCT ATG ACC. dContaining an M13-21 additional site: TGT AAA ACG ACG GCC AGT.
Sequencing analysis Exons 1–5, 7 and 9–14 of the MAPT gene and the corresponding flanking intronic sequences were PCR amplified using the primers described by Froelich et al. (9) and primers described in Table 3. PCR reactions were performed in a final volume of 50 µl containing 0.5 µM each primer and 1 U Taq DNA polymerase (Eurobio). The PCR consisted of 40 cycles of 15 s at 94C, 15 s
Screening for the Pro301Leu mutation was performed on genomic DNA by restriction fragment analysis. A 271 bp fragment was amplified with the 10F and 10R primers (Table 3) and one third of the PCR reaction (15 µl) was digested with 15 U MspI restriction enzyme (New England Biolabs) for 2 h in a final volume of 20 µl and resolved on a 3% agarose gel. ApoE genotypes were determined according to Hixson and Vernier (22). ACKNOWLEDGEMENTS We thank Y. Pothin and J. Bou for their excellent technical assistance and A. Michon for referring patients. This work was supported by a grant from the Conseil Régional de HauteNormandie (to C.D.).
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