Demographic and clinical information on gTSE cases is given in Table . A definite neuropathological diagnosis was available in 52% of the cases, ranging from more than 87% of FFI to 22% of GSS patients, while in 47% of gCJD autopsy was not performed. Data on the polymorphic codon 129 of PRNP were available in 86% of all cases. For some patients the only available information was about PRNP with no information on codon 129 genotype. The majority of gTSE patients were either homozygous for methionine (MM, 58%) or heterozygous (MV, 36%), only a few were homozygous for valine (6%). In a few cases, where the PrPSc typing was available, it was invariable PrPSc type I pattern.
Summary of clinical features of patients in the study
Quantitative levels of brain-derived proteins
The CSF concentrations of tau protein ranged from 70.0 to 35,440.0 pg/ml (median 6,255.5 pg/ml) in the gCJD group, from 75.0 to 20,370.0 pg/ml (median 2,354.0 pg/ml) in the insert gTSE group, from 296.0 to 1,698.0 pg/ml (median 675.0 pg/ml) in the GSS group, and from 97.0 to 4,126.0 pg/ml (median 464.5 pg/ml) in the FFI group (Fig. a). CSF levels of tau protein significantly differ in the four groups of gTSEs (p < 0.001, Kruskal–Wallis test), but did not vary within the gCJD group of patients (p = 0.13).
Fig. 1 a Box plots of CSF markers: total CSF tau protein levels in gCJD (n = 52), GSS (n = 6), FFI (n = 18), and insert gTSE subjects (n = 17). b Box plots of CSF S100b protein levels in gCJD ( (more ...)
The median CSF concentrations of S100b protein were above the cut-off level in all groups but one (FFI), ranging from 1.0 to 16.0 ng/ml in gCJD (median 9.0 ng/ml), from 3.0 to 17.0 ng/ml in insert gTSE (median 11.0 ng/ml), and from 1.0 to 6.0 ng/ml in FFI (median 3.0 ng/ml), Fig. b. The only GSS patient tested for S100b protein had a CSF concentration of 23.0 ng/ml. Statistical analysis showed a significant difference among the three groups (p = 0.002, Kruskal–Wallis test).
The CSF concentrations of NSE protein ranged from 15.0 to 140.0 ng/ml (median 38.5 ng/ml) in only 10 gCJD patients where it was tested, from 10.0 to 190.0 ng/ml (median 17.0 ng/ml) in insert gTSE (n = 5), and from 9.0 to 16.0 ng/ml (median 12.5 ng/ml) in FFI (n = 2). The GSS patient tested for NSE protein had a CSF concentration of 13.0 ng/ml. Difference in CSF levels of NSE protein within the four groups of gTSE patients did not reach significance (p = 0.08, Kruskal–Wallis test) (Fig. c).
Abnormal cerebrospinal fluid findings in gTSEs
There was a statistically significant difference in the rate of elevated 14-3-3 levels among the four groups of gTSEs, (p < 0.001), see Table . A 14-3-3 positive test was present in the majority of gCJD patients, irrespective of the mutation they carried (R208H, V203I, E211Q, T188K, and R148H). Thus, no significant difference in percentage of abnormal tests for 14-3-3 was found between E200K (81%) andV210I (85%) or versus other forms of gCJD (72%). Insert gTSE patients had also a high proportion of positive tests (69%), while the 14-3-3 test was positive in only 10% of GSS and in 13% of FFI cases.
Sensitivity (positive/total) of CSF investigations by forms of gTSE
A high rate of tau levels was found in gCJD and insert gTSE groups, while in GSS only 40% of cases had tau levels above the cut-off level of 1,300 pg. Only a single FFI patient had abnormal tau levels in CSF (Table . The levels of tau protein in the CSF were statistically different in the four groups (p < 0.001).
A similar distribution was observed for S100b values. Elevated S100b levels were found in 87% of gCJD and 77.8% of insert gTSE patients. These values were lower in GSS and FFI patients. The differences in the gTSE groups were statistically significant, (p = 0.002).
Only 64.3% of gCJD patients and 50% of insert gTSE patients had NSE values above the cut-off level, while NSE values were normal in GSS and FFI patients.
Effect of octapeptide repeats
The number of octapeptide repeats was inversely correlated with the values of tau (Spearman ρ −0.51 (p = 0.036)); patients with high number of repeats (5 × 24) had low tau levels. A similar trend was found for the 14-3-3 test; patients with high number of repeats had low percentage of abnormal tests : 1 × 24 (1/1); 3 × 24 (1/1); 4 × 24 (5/5); 5 × 24 (5/10) Fisher’s p value = 0.22.
Effects of patient characteristics on abnormal findings
The effect of clinical characteristics on percentage of abnormal findings in gCJD patients was the age at disease onset (p = 0.014) and the PRNP codon 129 genotype (VV vs. MV p = 0.043). Sensitivity of 14-3-3 was higher in old patients compared to young ones (25.0% in patients younger than 40 years, 83.9 in patients aged 40–60 years, 86.5% in 60–80 years and 100% in patients older than 80 years).
Regarding the percentage of abnormal findings in the 14-3-3 test in relation to the PRNP codon 129 genotype, this was lower in gCJD patients homozygous for valine than in heterozygous patients (VV vs. MV p = 0.043). However, when we adjusted by type of mutation, this statistical significance was lost. Although the 14-3-3 test performed worse in homozygous for methionine than in heterozygous gCJD patients, this difference was not significant (χ2 test, p = 0.08) (Table ). In E200K and V210I patients, the 14-3-3 sensitivity was higher in heterozygous (88.2 and 100%) than in methionine homozygous patients (78.1 and 77.8%), but these differences did not reach any significance (Table ).
Sensitivity of tests in gCJD
Disease duration had no influence on 14-3-3 test results (84.6% abnormal findings in patients with disease duration below the median, and 87.3% in patients with disease duration above the median survival time, Table ). In gCJD patients, there was no significant influence of the disease stage at the time of lumbar puncture in relation to 14-3-3 results (p
= 0.4) (100% in te first third of disease duration, 89% in the second third and 82% in the last third, Table , as reported in sporadic CJD [21
Regarding the other CSF tests, there was a lower percentage of abnormal findings for the S100b test in gCJD patients homozygous for methionine than in heterozygous patients, while the opposite was found for tau and NSE (Table ), though the differences were not statistically significant (p = 0.06 for tau, p = 0.92 for S100b, and p = 0.24 for NSE, χ2 test).
Multivariate analysis, performed on all gTSE patients, included as variables disease duration, age at onset, gender, type of mutation and 129 genotype, and as covariates gender and country of residence. This analysis revealed that the type of mutation is the only variable that significantly influenced 14-3-3 test (p < 0.004). Particularly, in patients carrying the D178N-Met mutation (FFI), the percentage of abnormal findings of 14-3-3 decreased significantly in comparison to gCJD (p < 0.007) and insert gTSE (p < 0.001). Interestingly, regarding the 129 codon polymorphism, 14-3-3 had lower percentage of abnormal tests in patients homozygous for valine than in patients homozygous for methionine or heterozygous, though no significant differences were found (crude p value = 0.5; adjusted p value = 0.76). Table .
CSF biomarker in genetic TSE (reports in the literature)
Multivariate analysis did not revealed any variables that influenced tau, NSE or S100b levels in gTSE patients.
In all gTSE there was a statistically significant negative correlation between tau levels and disease duration (Spearman ρ2 between duration and tau levels = −0.52 (p < 0.001). However, when the data were stratified by diagnostic groups, numbers become too low and were not significant (only borderline for gCJD: r2 = −0.32, p = 0.057).