Defining diagnostic cutoffs in neurological patients for serum very long chain fatty acids (VLCFA) in genetically confirmed X-Adrenoleukodystrophy

Abstract

X-linked Adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene resulting in the accumulation of very long chain fatty acids (VLCFA). X-ALD is the most common peroxisomal disorder with adult patients (male and female) presenting with progressive spastic paraparesis with bladder disturbance, sensory ataxia with impaired vibration sense, and leg pain. 80% of male X-ALD patients have an adrenal failure, while adrenal dysfunction is rare in women with X-ALD. The objective of this study was to define optimal serum VLCFA cutoff values in patients with X-ALD-like phenotypes for the differentiation of genetically confirmed X-ALD and Non-X-ALD individuals. Three groups were included into this study: a) X-ALD cases with confirmed ABCD1 mutations (n = 34) and two Non-X-ALD cohorts: b) Patients with abnormal serum VCLFA levels despite negative testing for ABCD1 mutations (n = 15) resulting from a total of 1,953 VLCFA tests c) Phenotypically matching patients as Non-X-ALD controls (n = 104). Receiver operating curve analysis was used to optimize VLCFA cutoff values, which differentiate patients with genetically confirmed X-ALD and Non-X-ALD individuals. The serum concentration of C26:0 was superior to C24:0 for the detection of X-ALD. The best differentiation of Non-X-ALD and X-ALD individuals was obtained with a cutoff value of < 1.0 for the C24:0/C22:0 ratio resulting in a sensitivity of 97%, a specificity of 94.1% and a positive predictive value (PPV) of 83.8% for true X-ALD. Our findings further suggested a cutoff of < 0.02 for the ratio C26:0/C22:0 leading to a sensitivity of 90.9%, a specificity of 95.0%, and a PPV of 80.6%. Pearson correlation indicated a significant positive association between total blood cholesterol and VLCFA values. Usage of serum VLCFA are economical and established biomarkers suitable for the guidance of genetic testing matching the X-ALD phenotype. We suggest using our new optimized cutoff values, especially the two ratios (C24:0/C22:0 and C26:0/C22:0), in combination with standard lipid profiles.

Figure 1

Flowchart of VLCFA testing. Since 01/2008, a total number of n = 1953 VLCFA tests were performed in the central laboratory of the University Hospital Tübingen in patients where X-ALD was considered an appropriate differential diagnosis. Thereof 45 patients had at least > 1 positive VLCFA results according to the cutoffs mentioned in the light blue box. By genetic testing, the 45 positive cases were grouped into two groups, the X-ALD positive cases (n = 30) and the X-ALD negative cases (n = 15). Cases with confirmed ABCD1-mutations (compare supplementary Table 1) were considered as X-ALD cases, and the Non-X-ALD cases consisted of cases without ABCD1-mutation or another genetically proven disorder (compare Table 2). Since, in many cases > 1 VLCFA result was positive, the specified findings in either the lighter green or light yellow box do not sum up to the number of cases specified above.

Figure 2

VLCFA evaluation in phenotypically similar patients. (A) Using the same dataset, the VLCFA values of phenotypically similar patients (black box—n = 153) were evaluated to redefine diagnostic cutoffs. The positive VLCFA cases (n = 45; 30 X-ALD and 15 Non-X-ALD) identified via the screening since 01/2008 (compare Fig. 1) were combined with four X-ALD patients with VLCFA values below the previously used cutoffs totaling 34 X-ALD cases. Phenotypical controls (n = 104) matching the Non-X-ALD cases phenotypically with elevated VLCFA (compare Table 3) were chosen from the same dataset. (B) ROC- analysis of X-ALD versus Non-X-ALD cases was performed using all five VLCFA measures (color coding see legend within the Figure) for the 153 phenotypically similar cases (as specified in the “Methods” section—see redefining diagnostic cutoffs). Sensitivity and specificity were highest for the C24:0/C22:0 ratio followed by the C26:0/C22:0 ratio and the C26:0 absolute value.

Figure 3

Correlation of LDL levels and VLCFA values. LDL levels in mg/dl are shown in scatter blots with the VLCFA values C22:0 (A), C24:0 (B), C26:0 (C) and the ratios C24:0/C22:0 (D) and C26:0/C22:0 (E). The phenotypical control cases are shown by white circles, the X-ALD cases by filled black circles with each circle representing a single patient. Spearman’s rank correlation coefficient indicated a significant correlation for LDL with C22:0 (r_s = 0.521; n = 89; p < 0.001), C24:0 (r_s = 0.548; n = 89; p < 0.001), and C26:0 (r_s = 0.415; n = 89; p < 0.001), but not for the ratios C24:0/C22:0 (r_s = 0.305; n = 89; p = 0.004), and C26:0/C22:0 (r_s = 0.262; n = 89; p = 0.013). A 2-tailed significance level of p < 0.002 was considered to be statistically significant (Bonferroni correction—see “Methods” section for details). Further correlations can be found in Table 4 together with the related p-values and the corresponding scatter blots for total cholesterol and VLCFA can be found in the supplementary material.