Introduction

Frontotemporal lobar degeneration (FTLD) is a neurodegenerative disease characterized by alterations in behavior and speech (Scarpini et al. 2006). The majority of FTLD cases are sporadic, and only a minor fraction (5–10%) is monogenic due to alterations in the microtubule-associated protein tau (MAPT, OMIM +157140), granulin (GRN, OMIM *138945), valosin-containing protein (VCP, OMIM *601023) or the chromatin-modifying protein 2B (CHMP2B, OMIM +609512) (Cruts et al. 2006; Scarpini et al. 2006).

The current consensus criteria identify three clinical FTLD subtypes: progressive nonfluent aphasia (PA), semantic dementia (SD) and frontotemporal dementia (FTD) (Neary et al. 1998). Few genes have been considered as genetic susceptibility factors for sporadic FTLD, including MAPT itself (Fenoglio et al. 2007). The association between FTLD and APOE-ε4 (OMIM +107741) in the Italian population is controversial (Bernardi et al. 2006; Verpillat et al. 2002).

An impairment of serotonin (5-HT) transmission in FTLD has been reported (Yang and Schmitt 2001). The 5-HT transporter is encoded by the single gene SLC6A4 (OMIM *182138) (17q11.1-q12), and it modulates 5-HT reuptake (Lesch et al. 1994). A functional polymorphism in the promoter region of SLC6A4, called the 5-HT transporter gene-linked polymorphic region (rs4795541), was reported by Heils et al. (1996). The rs4795541 consists of a 43-bp deletion (short variant S) or insertion (long variant L), the S-allele reducing SLC6A4 transcription two times (Collier et al. 1996). The SLC6A4 promoter region contains other functional polymorphic sites, such as rs25531 (A→G) (Wendland et al. 2008).

We performed a case-control association study in the Italian population to evaluate rs4795541, rs25531 and APOE-ε4 as genetic risk factors for FTLD. Moreover, we performed a dHPLC screening of our population, searching for rare coding variants.

Materials and methods

Patient recruitment

Two hundred and twenty-five independent subjects were recruited from the following clinical centers: “Fondazione Ospedale Maggiore Policlinico” (Milan, Italy) and “Luigi Sacco” Hospital (Milan, Italy). All patients underwent screening laboratory tests, neurocognitive evaluation, brain magnetic resonance imaging (MRI) or computed tomography (CT scan). Cognitive dysfunctions were assessed by the Clinical Dementia Rating (CDR), the Mini Mental State Examination (MMSE), the Frontal Assessment Battery (FAB), the Wisconsin Card Sorting Test (WCST) and the Tower of London.

Seventy-four subjects were diagnosed as FTLD, according to Neary et al. and subsequent revision by McKhann et al. (Neary et al. 1998; McKhann et al. 2001). An accurate follow-up was done to confirm the diagnosis. Age-matched healthy controls (CT) had no memory and psychobehavioral complaints. All subjects (or their relatives) gave their informed consent to participation in the study, whose protocol was approved by the human investigation scientific committee of the Italian Ministry of Health.

Blood samples collection, rs4795541, rs25531 and APOE genotyping

Genomic DNA (gDNA) was isolated from whole blood using a commercial Flexigene kit (Qiagen, Hildren, Gemany).

To assess the rs4795541 genotype, 50 ng of gDNA was amplified by polymerase chain reaction (PCR) using the following primers: for-5′ggcgttgccgctctgaatgc-3′ and rev-5′ gagggactgagctggacaacca-3′ (Nakamura et al. 2000).

The rs25531 genotype was assessed by allele-specific PCR using the following primers: common rev: 5′tggagtccgcgcgggattctggtgccacct-3′; for-allele A specific: 5′-acccctcgcggcatcccccctgcacccaca-3′; for-allele G specific: 5′-acccctcgcggcatcccccctgcacccacg-3′.

APOE genotype was determined by restriction fragment length polymorphism (RFLP) using the following primers: for-5′ tcggccgcagggcgctgatgg-3′; rev-5′ctcgcgggcccccggccccggcctggta-3′ and CfoI as restriction enzyme (Roche, Basel, Switzerland).

dHPLC screening

SLC6A4 (NM_001045) exons 1–15 and the relative 5′- and 3′-intronic flanking regions (50 bp) were screened by dHPLC (Wave 3500, Transgenomic, Omaha, NE) using the experimental conditions set up by the instrument’s software (Navigator, Transgenomic, Omaha, NE). The presence of an heterozygous nucleotidic substitution was confirmed by double-stand DNA sequencing.

Statistical analysis

Frequency distributions were compared using χ2 test or Fisher’s exact test. The odds ratios (OR) were calculated by a 2 × 2 contingency table at 95% confidence interval (CI). The statistical significance limit was set at P = 0.05. Analyses were performed using StatView program version 5.0 or RxC program (http://www.marksgeneticsoftware.net). The program HaploView 4.1 was used to evaluate linkage disequilibrium (LD).

Results

Case-control study to assess rs4795541 genotype distribution

Table 1 summarizes the population demographics. Seventy patients were classified as FTD (95%), while four patients (5%) were diagnosed as PA. For subsequent analysis, FTD and PA genotypes were considered aggregated. Sex distribution was balanced (male-to-female ratio 0.85). In controls, females were slightly over-represented (male-to-female ratio 0.7).

Table 1 Demographics of the population sample recruited for the case-control studies
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The genotypic frequencies of rs4795541 for CT and FTLD respected Hardy-Weinberg equilibrium (data not shown), and we found a significant difference between CT and FTLD (P = 0.046, χ2-test) (Table 2). The S-allele in the FTLD group was increased (47.3 vs. 35.8% in controls; P = 0.019, χ2-test). The calculated OR with 95% CI interval for the rs4795541 (S/L + S/S) genotypes was 2.1, with an associated P-value of 0.018. Sample stratification by gender gave no difference.

Table 2 Genotypic and allelic frequencies of SLC6A4 and APOE polymorphisms
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Case-control study to assess rs25531 and apolipoprotein E (APOE) genotypes

We genotyped our sample for rs25531 (Table 2). The results did not evidence a difference between CT and FTLD, either at the genotypic or allelic level. A similar picture was found for the APOE-ε4 allele (Table 2). An FTD subject was excluded from APOE analyses due to an ambiguous genotyping result. APOE distribution did not differ between CT and FTLD, both for genotypic and allelic frequencies.

Once the genotypic distribution of rs4795541, rs25531 and APOE had been assessed separately, we verified a possible interaction among these polymorphisms. The APOE-ε4(−) group had n = 187 subjects, and by comparing the rs4795541 allelic frequency between CT and FTLD, an almost significant increase of the S-allele in FTLD was detectable (P = 0.068, χ2-test). The same was true for the APOE-ε4(+) group (sample size n = 37; FTLD S-allele frequency 46.4 vs. 26.0% in CT; P = 0.073, χ2-test). No significant or almost-significant difference was detected between APOE-ε4(−) and APOE-ε4(+) subjects for rs25531 genotypic or allelic distribution.

We linked rs4795541, rs25531 and APOE-ε4 genotypes to FTLD age at onset. No correlation came to light (data not shown).

dHPLC screening of SLC6A4

We performed a dHPLC screening of the SLC6A4 gene in FTLD and CT searching for coding or splicing variants. In 6 independent patients and 12 CTs, we found the nucleotidic change (A→C, nt121 of NM_001045) that mapped within the 5′-untranslated region (5′-UTR), had already been described in databank (rs6354) and was then genotyped by direct sequencing in all FTLDs and CTs. The polymorphism respected Hardy-Weinberg equilibrium (data not shown), and no significant difference was found (Table 2).

Discussion

We aimed at evaluating rs4795541, rs25531 and APOE-ε4 as risk factors for FTLD in Italy. For rs4795541, we found a significant difference between CT and FTLD, the presence of at least one S-allele associating to an increased FTLD risk of 2.1 times. Interestingly, a 5-HT deficit in FTLD was reported by autopsy, imaging and biochemical studies that evaluated the CSF content of 5-hydroxyindoleacetic acid (5-HIAA, a 5-HT metabolite) (Huey et al. 2006).

We were unable to link rs25531 to FTLD susceptibility. The dHPLC screening found a polymorphism in SLC6A4 5′-UTR (rs6354) whose frequency did not differ between FTLD and CT. Our dHPLC analysis suggests a low frequency of SLC6A4 coding mutations. We also evaluated LD among rs4795541, rs25531 and rs6354, but no LD was found (Fig. 1).

Fig. 1
figure 1

Linkage disequilibrium coefficients D′ and r 2 calculated by HalpoView among the SLC6A4 polymorphisms rs4795541, rs25531 and rs6354 (1, 2 and 3, respectively, in the graphs)

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Our data about APOE-ε4 are not in agreement with a paper finding a positive association between APOE-ε4 and FTLD in the Italian population (Bernardi et al. 2006). However, the FTLD sample was composed not only by sporadic (n = 54), but also by familial (n = 46) FTLD subjects (even if monogenic FTLD cases were excluded), thus making a rigorous comparison difficult. As for rs4795541, rs25531 or APOE-ε4 status and FTLD age at onset, our failure in detecting any relation underlines the need for a more comprehensive approach to perform this kind of analysis (Borroni et al. 2008).

In conclusion, our data highlight rs4795541 as possible genetic modulator of FTLD susceptibility in Italy to be confirmed in larger association studies.