Patients and controls

We studied peripheral blood samples from 20 TCS patients (TCS1 to 20, ranging from 3 to 41 years old) referred to our center for genetic counseling and 12 controls (ranging from 20 to 50 years old). We also obtained tissue samples from TCS16, from three other patients (TCS21 to 23), and from six controls submitted to reconstructive plastic surgery at University of São Paulo Medical School. The study was approved by the ethical committee of our Institution and informed consent was obtained from both patients and control subjects or from their legal tutors. In total, 23 TCS patients were studied.

Isolation of cells from periosteum of TCS patients

During corrective surgery, overlying periosteum from the face of four TCS patients (two males and two females aged from 6 to 29 years) was meticulously dissected away from surrounding tissues to isolate intact periosteal flaps. Control periosteum was obtained using the same procedure from facial region of six subjects (four males and two females aged from 11 months to 20 years) with no evidence of bone disease during corrective surgery.

Mesenchymal cells were isolated as described by previous reports from our group [18, 19]. The periosteal flaps were thoroughly washed with sterile phosphate-buffered saline (PBS) supplemented with 4% antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin; Invitrogen), and digested with trypsin solution (TrypLe; Invitrogen) for 1 h at 37°C. Once digested, the tissue was transferred with minimal dissection into 35 mm Petri dishes (Corning, NY) containing Dulbecco's modified Eagle's medium (DMEM; GIBCO) with 10% fetal bovine serum (FBS; GIBCO), 100 units/mL penicillin, and 100 mg/mL streptomycin (Invitrogen). After two weeks, cells were washed with PBS, then dissociated in trypsin solution and seeded at 10⁴ cells per 25 cm² for the first passage. For expansion, cells were cultured in monolayer in growth medium at 37°C in a humidified atmosphere of 5% CO2. The medium was replaced every 3 days. In order to prevent cell differentiation, cultures were maintained semiconfiuent and subcultured every 4-5 days with daily medium changes.

Flow cytometry

Cells were harvested with TrypLe (Invitrogen), washed with PBS, and incubated at 4°C for 30 min with the following anti-human antibodies: CD29-PE CY5, CD90 (Thy-1), CD45-FITC, CD73, CD105, CD117, CD 31-PE (Becton Dickinson) and SH3 (Case Western Reserve University). After the wash, unconjugated primary antibodies were incubated with anti-mouse-PE secondary antibody (Guava Technologies) for additional 15 min at 4°C. Finally, the cell suspension was washed with PBS, and 10⁴ labeled cells were acquired with an EasyCyte flow cytometer (Guava Technologies). Control samples were incubated with PBS instead of primary antibody, followed by incubation with anti-mouse-PE secondary antibody. All the generated plots were analyzed in Guava ExpressPlus software (Guava Technologies).

DNA and RNA isolation and cDNA synthesis

Genomic DNA from peripheral blood samples was obtained according to reference [20] and genomic DNA from culture cells was extracted using NucleoSpin Tissue extraction kits (Macherey-Nagel). Total RNA was isolated from leucocytes and mesenchymal cells using TRIzol^® (Gibco BRL), treated with DNAse (Promega) and submitted to reverse transcription using Superscript II Reverse Transcriptase (Gibco BRL), according to manufacturer's protocol. Aliquots from DNAse treated RNAs were used for amplification of an intronic region of MLH1 gene as a control for DNA contamination (primers sequences on request).

Mutation screening

Real Time PCR

Real time quantitative PCR reactions were performed in duplicates with a final volume of 20 μl, using 12.5 ng of cDNA, 1× SYBR Green PCR Master Mix (Applied Biosystems) and 200 or 400 nM of each primer. We used ABI Prism 7700 Sequence Detection System (Applied Biosystems) with standard temperature protocol. Primers were designed with Primer Express software V.2.0 (Applied Biosystems; primers sequence in Additional file 1) and the amplification efficiency (E) of each primer was calculated according to the equation E = 10^(-1/slope). The expression data of TCOF1 transcripts was determined by relative quantification in comparison to a pool of RNA from 10 controls. Four endogenous controls genes (GAPDH, BCRP, HPRT1, and HMBS), were used and their stability was verified through geNorm VBA applet designed for Microsoft Excel. This tool calculates the most stable reference genes from a set of tested candidate reference genes in a given sample panel, and calculates the gene expression normalization factor (NF) for each target sample based on the geometric mean of a user-defined number of housekeeping genes [22]. We calculated the NF for each sample based on the four endogenous controls and the expression data was calculated according to reference [23].

Molecular characterization of the TCS patients

Pathogenic mutations were identified in 13 of the novel 15 screened individuals: five had already been reported as pathogenic [9, 21, 24] and eight are herein described for the first time. These new mutations were considered pathogenic because they are a nonsense mutation (c.1609C>T), disrupt splicing (c.639+1G>A) or alter the reading frame (c.3853delC, c.1298delC, c.218_222insAACC, c.4344dupA, c.431delC). Mutation c.4375_4377delAAG (TCS18) causes an in phase deletion and excludes a Lysine residue from the protein C-terminal nucleolar localization signal [25]. In Table 1, mutation and tissue availability of each patient are depicted.

RT-PCR in leucocytes

We first verified the range of TCOF1 transcript levels in leucocytes from 20 patients (Table 1) and 12 controls by real-time PCR, using as reference a pool of RNA from 10 controls. We observed a wide variation in TCOF1 expression in leucocytes from normal individuals (Figure 1; SD = 1.24). This wide variation of gene expression was also observed among TCS patients (Table 1; SD = 1.46), and was not correlated to age (p = 0.39; Two-tailed Pearson correlation regression).

Comparing patients (mean ± SEM = 6.846 ± 0.3278; N = 20) and controls (mean ± SEM = 8.093 ± 0.3587; N = 12), we observed that TCOF1 expression levels were significantly lower in patients than in controls (p = 0.0164; Two-tailed unpaired t test with Welch's correction; Figure 1), and the variances were not significantly different between these two groups (p = 0.5828, F test). TCS patients had approximately 18% less TCOF1 transcript levels than normal individuals. Excluding the four patients without an identified pathogenic mutation, we obtained similar results (~18.5%, p = 0.0039).

RT-PCR in mesenchymal cells

We also examined whether TCOF1 expression levels were decreased in mesenchymal cells. Flow cytometry results showed that most of the cells were positive for mesenchymal cell markers (>95%) and negative for endothelial and hematopoietic markers (see Additional files 2 and 3) in primary cell cultures from four patients and four controls.

We also observed a great variance in TCOF1 expression among mesenchymal cells from TCS patients (n = 4) and controls (n = 6), and the variances in these samples were also not significantly different between the groups (p = 0.90, F test). Although the number of tested individuals was smaller, we also detected a significantly lower expression of TCOF1 in TCS patients (~31%; Figure 2; patients mean ± SEM = 0.9687 ± 0.1122; controls mean ± SEM = 1.414 ± 0.09010; p = 0.0213; Two-tailed unpaired t test with Welch's correction). For this analysis, we also used as reference a pool of RNAs.

Mutant allele detection

In order to confirm the existence of NMD mechanism, we evaluated the expression of mutant alleles. We compared peak heights obtained through sequencing analysis of the nucleotides flanking the pathogenic mutation of the gDNA versus cDNA (Figure 2). We observed the expression of the mutant alleles in two patients (TCS16 and 23), but in patients TCS21 and 22 we could only detect the wild-type allele (Figure 2).