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Cited by 61Metabolism and Genetic DisordersLysosomal Storage Disorders ResearchHereditary Neurological Disorders

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

Open access
Rattay, Tim W., Rautenberg, Maren, Söhn, Anne S., Hengel, Holger, Traschütz, Andreas, Röben, Benjamin, Hayer, Stefanie N., Schüle, Rebecca +7 more·2020·Scientific Reports
DOI 10.1038/s41598-020-71248-8PMC PMC7494896
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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.

AI snapshot

Written from the paper’s full text.

TL;DR

In genetically confirmed X-ALD and X-ALD-like Non-X-ALD patients, ROC analysis identified optimized serum VLCFA cutoffs, especially C24:0/C22:0 < 1.0 and C26:0/C22:0 < 0.02, that improve sensitivity over prior in-house cutoffs. Serum VLCFA ratios, interpreted with lipid profiles, are proposed as economical screening biomarkers to guide ABCD1 genetic testing.

Key findings

  • Among 153 phenotypically similar cases (34 genetically confirmed X-ALD, 15 ABCD1-negative with elevated VLCFA, 104 phenotypic controls), the best differentiation was C24:0/C22:0 < 1.0: sensitivity 97%, specificity 94.1%, PPV 83.8%; C26:0/C22:0 < 0.02 gave sensitivity 90.9%, specificity 95.0%, PPV 80.6%.
  • C26:0 < 1.2 µmol/l had sensitivity 81.8%, specificity 94.1%, PPV 82.1%; C24:0 < 92 µmol/l had sensitivity 24.2%, specificity 94.1%, PPV 50.0%; C22:0 < 105 µmol/l had sensitivity 0%, specificity 92.4%, PPV 0%. C26:0 was superior to C24:0 for detection of X-ALD.
  • New cutoffs increased sensitivity versus previous in-house cutoffs, e.g. C26:0 +15.1%, C24:0/C22:0 +6.1%, C26:0/C22:0 +24.2%, but increased false-positive findings by n = 3 (total n = 18) when all VLCFA values were considered.
  • Total cholesterol and LDL correlated with absolute VLCFA levels (LDL: C22:0 rs = 0.521, C24:0 rs = 0.548, C26:0 rs = 0.415; all p < 0.001), but associations were lost when values were analyzed as ratios; mutation type and VLCFA levels did not correlate.

Methods

  • Retrospective single-center study at University Hospital Tübingen: 1,953 serum VLCFA tests from 01/2008–01/2020 in patients where X-ALD was a differential diagnosis; 45 had at least one positive VLCFA result and were genetically tested, yielding 30 ABCD1-mutation-positive and 15 ABCD1-negative cases. Four additional X-ALD patients with VLCFA below old cutoffs were added, totaling 34 X-ALD cases; 104 phenotypically matched Non-X-ALD controls (63 HSP, 41 other neurodegenerative) were used for ROC analysis.
  • VLCFA measured by gas chromatography after Lepage/Roy transesterification; routine lipid analysis was performed for n = 90 cases. ROC analysis optimized cutoffs; correlations used Spearman/Pearson methods with Bonferroni-corrected significance at p < 0.002.

Limitations

  • Stated by the paper: even optimized cutoffs did not achieve 100% sensitivity and specificity; one female with c.1816T>C, p.S606P would have been missed using the adjusted ratio cutoffs; VLCFA can detect only about 85% of heterozygous female carriers; cutoffs are laboratory-, method-, and assay-specific; spontaneous fasted and unfasted samples were used, and fasted lipid status plus serial testing are recommended for uncertain results.
  • Stated/evident: the study did not include neurologically healthy controls, instead using phenotypically similar patients as controls; the X-ALD group was small (n = 34), with only 15 ABCD1-negative elevated-VLCFA cases, limiting precision and generalizability.
  • Evident from the text: single-center retrospective design and use of clinical/stored samples may introduce selection and preanalytical variability.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41598-020-71248-8

Cites

29 of 29 references verified0 citation errors · 0 not checkable

Statistics

5 tests recomputed · 0 with the reported and recomputed p on opposite sides of .05

Claims the paper makes

5 of 26 cited sources checked · 1 supported · 0 flagged · accuracy not yet measured

  • Interpretationreasoning

    The number of false-positives from reference ranges is too large to allow screening for X-ALD in neurologically affected patients with an incidence at birth of approximately 1:17,000.

    “This number of false-positives is too large to allow screening for X-ALD in neurologically affected patients with an incidence at the birth of ~ 1:17,0002.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [2] Engelen, M. et al. X-linked adrenoleukodystrophy (X-ALD): clinical presentation and guidelines for diagnosis, follow-up and management. Orphanet J. Rare Dis. 7, 51. https://doi.org/10.1186/1750-1172-7-51 (2012). source

      The Epidemiology section gives an estimated birth incidence of 1 in 17,000, and Differential diagnosis notes that elevated plasma VLCFA can yield false positives. Neither passage says that false positives from reference ranges are too numerous to allow screening neurologically affected patients. The Biochemical and molecular diagnosis section instead describes testing males with neurological symptoms when X-ALD is suspected. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Premisecitation

    VLCFA are elevated in all male X-ALD patients regardless of age, disease duration, metabolic status, or clinical symptoms.

    “VLCFA are elevated in all male X-ALD patients regardless of age, disease duration, metabolic status, or clinical symptoms15.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [15] Wiesinger, C., Eichler, F. S. & Berger, J. The genetic landscape of X-linked adrenoleukodystrophy: inheritance, mutations, modifier genes, and diagnosis. Appl. Clin. Genet. 8, 109. https://doi.org/10.2147/TACG.S49590 (2015). source
  • Premisecitation

    The mutation c.1816 T > C, p.S606P is shown to be pathogenic in at least seven patients according to the ALD mutation database.

    “Two of the three cases which were not recognized by a cutoff ≥ 0.02 for the ratio of C26:0/C22:0 presented with the mutation c.1816 T > C, p.S606P (both females), shown to be pathogenic in at least seven patients according to the ALD mutation database (http://adrenoleukodystrophy.info18–21.”
    Quote in the paperNot a substantial claim1 of 4 sources checked
    • cites [18] Wang, Y. et al. X-linked adrenoleukodystrophy: ABCD1 de novo mutations and mosaicism. Mol. Genet. Metab. 104, 160–166. https://doi.org/10.1016/j.ymgme.2011.05.016 (2011). source
    • cites [19] Feigenbaum, V. et al. Mutational and protein analysis of patients and heterozygous women with X-linked adrenoleukodystrophy. Am. J. Hum. Genet. 58, 1135–1144 (1996).
    • cites [20] Shukla, P. et al. Molecular analysis of ABCD1 gene in Indian patients with X-linked Adrenoleukodystrophy. Clin. Chim. Acta 412, 2289–2295. https://doi.org/10.1016/j.cca.2011.08.026 (2011). source
    • cites [21] Pereira, F. d. S. et al. Mutations, clinical findings and survival estimates in South American patients with X-linked adrenoleukodystrophy. PLOS ONE 7, e34195. https://doi.org/10.1371/journal.pone.0034195 (2012). source

      Table 1 in Results > Molecular results lists c.1816T>C (p.Ser606Pro) in a male patient with cerebral X-linked adrenoleukodystrophy, supporting its association with disease. Our copy does not say the ALD mutation database classifies this variant as pathogenic in at least seven patients; the table documents one patient in this series. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Findingown data

    There was no positive correlation between VLCFA levels and mutation type (missense vs. frameshift/deletion/nonsense) for C22:0, C24:0, C26:0, and the ratios C24:0/C22:0 and C26:0/C22:0.

    “There was no positive correlation using a point-biserial coefficient between VLCFA levels and mutation type (missense vs. frameshift/deletion/nonsense) C22:0 (r(32) = − 0.10, p = 0.592) and C24:0 (r(32) = − 0.06, p = 0.975), C26:0 (r(32) = − 0.06, p = 0.973) nor the ratios C24:0/C22:0 (r(33) = 0.11, p = 0.537) and C26:0/C22:0 (r(33) = 0.05, p = 0.766).”
    Quote in the paperNot a substantial claim
  • Findingown data

    In 15 cases, no ABCD1 gene mutation or another underlying disease was identified.

    “In 15 cases, no ABCD1 gene mutation or another underlying disease was identified.”
    Quote in the paperNot a substantial claim
  • Classified as probably not a substantial claim. 24
  • May combine two claims. 20
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The AMN phenotype is an important differential diagnosis to hereditary spastic paraplegias (HSP).

    “The AMN phenotype is an important differential diagnosis to hereditary spastic paraplegias (HSP)6,7, which are clinically and genetically very heterogeneous (80+ genes are described—see8).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [6] Chen, Y.-J. et al. Chinese patients with adrenoleukodystrophy and Zellweger spectrum disorder presenting with hereditary spastic paraplegia. Parkinsonism Related Disord. 65, 256–260. https://doi.org/10.1016/j.parkreldis.2019.06.008 (2019). source
    • cites [7] D’Amore, A. et al. Next generation molecular diagnosis of hereditary spastic paraplegias: an italian cross-sectional study. Front. Neurol. https://doi.org/10.3389/fneur.2018.00981 (2018). source

      The Results (Molecular Findings; page unavailable) report ABCD1 variants in two patients with an X-linked HSP diagnosis, providing some evidence of diagnostic overlap. The paper does not identify those patients as having an AMN phenotype or state that AMN is an important differential diagnosis for HSP. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Hereditary spastic paraplegias are clinically and genetically very heterogeneous, with more than 80 genes described.

    “The AMN phenotype is an important differential diagnosis to hereditary spastic paraplegias (HSP)6,7, which are clinically and genetically very heterogeneous (80+ genes are described—see8).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [8] Bis-Brewer, D. M. & Zuchner, S. Perspectives on the Genomics of HSP Beyond Mendelian Inheritance. Front. Neurol. 9, 958. https://doi.org/10.3389/fneur.2018.00958 (2018). source

      The Introduction describes HSPs as genetically heterogeneous and distinguishes pure and complicated forms with different clinical features. It reports over 76 genomic loci but 58 corresponding genes, so it does not support the claim that more than 80 genes had been described. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Pearson correlation indicated a significant positive association between total blood cholesterol and VLCFA values.

    “Pearson correlation indicated a significant positive association between total blood cholesterol and VLCFA values.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In Non-X-ALD patients, false-positive elevations occurred for C22:0 (n=9), C24:0 (n=7), C26:0 (n=5), ratio C24:0/C22:0 (n=6), and ratio C26:0/C22:0 (n=6).

    “On an individual level, the following VLCFA values were elevated in Non-X-ALD patients resulting in false-positive findings with the possibility of more than one increased VLCFA value per patient: C22:0 n = 9, C24:0 n = 7, C26:0 n = 5, and the ratios C24:0/C22:0 n = 6 and C26:0/C22:0 n = 6.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Using the new cutoff values, sensitivity increased substantially for C26:0 (+15.1%), ratio C24:0/C22:0 (+6.1%), and ratio C26:0/C22:0 (+24.2%).

    “We found substantially increased sensitivity values particularly for C26:0 (+ 15.1%) and the ratios C24:0/C22:0 (+ 6.1%) and C26:0/C22:0 (+ 24.2%) (see Table 5) using the new cutoff values but also an increase by n = 3 false-positive findings (total n = 18) when taking all VLCFA values into account.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Findingown data

    In addition to the 45 positive VLCFA cases (30 X-ALD and 15 Non-X-ALD), four further X-ALD patients with VLCFA values below the previously used cutoffs were added, resulting in 34 genetically confirmed X-ALD cases.

    “In addition to the 45 positive VLCFA cases (30 X-ALD and 15 Non-X-ALD (see Table 2 and Fig. 1), four further X-ALD patients were added to the cohort with VLCFA values below the previously used cutoffs. Resulting in 34 genetically confirmed X-ALD cases (four minors; all mutations are specified in Supplementary Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    When using ratios relating to C22:0, not one single C26:0/C22:0 ratio and only three C24:0/C22:0 ratios remained elevated.

    “However, when using the ratios (relating to C22:0), not one single C26:0/C22:0 ratio and only three C24:0/C22:0 ratios remained elevated.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    There was no correlation between VLCFA levels and the ratios (C24:0/C22:0 and C26:0/C22:0).

    “There was no correlation between VLCFA levels and the ratios (C24:0/C22:0 and C26:0/C22:0).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims. The quote only partly states this claim.

    Findingown data

    The phenotypical control group (n = 104) consisted of patients with the clinical diagnosis of hereditary spastic paraplegia (n = 63) and other matched neurodegenerative cases (n = 41).

    “The phenotypical control group (n = 104, compare Fig. 2A) consisted of: a) Patients with the clinical diagnosis of hereditary spastic paraplegia (n = 63).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A cutoff of less than 0.02 for the ratio C26:0/C22:0 leads to a sensitivity of 90.9%, a specificity of 95.0%, and a positive predictive value of 80.6%.

    “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%.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For C26:0 (< 1.9 μmol/l), the sensitivity was 66.7% with 99.2% specificity and a PPV of 92% in genetically confirmed X-ALD cases.

    “For C24:0 (< 85 µmol/l) the sensitivity was 36.4% with 88.2% specificity and a PPV of 48%, and for C26:0 (< 1.9 µmol/l)13 the sensitivity was 66.7% with 99.2% specificity and a PPV of 92%.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [13] Morell, B. K., Teichler, J., Budak, K., Vollenweider, J. & Pavlicek, V. X-linked adrenoleukodystrophy presenting as Addison’s disease. Case Rep. 2010, bcr1120092419 (2010). source

  • May combine two claims.

    Findingown data

    The sensitivity increased with the addition of 37 samples from consecutive tests: C24:0 by 14.4% to 36.6%; C26:0 by 3.9% to 85.7%, while the sensitivity for the ratios C24:0/C22:0 and C26:0/C22:0 remained virtually unchanged.

    “The sensitivity increased with the addition of 37 samples from consecutive tests (see Supplementary Material): C24:0 by 14.4% to 36.6%; C26:0 by 3.9% to 85.7%, while the sensitivity for the ratios C24:0/C22:0 and C26:0/C22:0 remained virtually unchanged.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    Of the hereditary spastic paraplegia patients, 73% (n = 49) had a genetically confirmed HSP diagnosis and 14 patients remained genetically unsolved despite exome analysis.

    “Thereof, 73% (n = 49) had a genetically confirmed HSP diagnosis (one minor) and 14 patients remained genetically unsolved despite exome analysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    A total of 1,953 VLCFA samples were tested for elevated VLCFA at the University Hospital Tübingen since 01/2008.

    “In a first step, a total of n = 1,953 VLCFA samples were tested for elevated VLCFA in the Institute for Clinical Chemistry and Pathobiochemistry of the University Hospital Tübingen since 01/2008 (Fig. 1).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    There was a significant positive correlation between total serum cholesterol (driven by serum LDL levels) and VLCFA values.

    “Spearman’s coefficient indicated that there was a significant positive correlation between total serum cholesterol (driven by serum LDL levels) and VLCFA values.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    For the ratio C24:0/C22:0 (< 1.11), the sensitivity was 90.9% with 96.6% specificity and a PPV of 91% in genetically confirmed X-ALD cases.

    “For the ratio C24:0/C22:0 (< 1.11)13 the sensitivity was 90.9% with 96.6% specificity and a PPV of 91% for C26:0/C22:0 (< 0.04) the sensitivity was 66.7% with 100% specificity and a PPV of 96%.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [13] Morell, B. K., Teichler, J., Budak, K., Vollenweider, J. & Pavlicek, V. X-linked adrenoleukodystrophy presenting as Addison’s disease. Case Rep. 2010, bcr1120092419 (2010). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    The coefficients of variation were 2.25%, 2.8% and 9.4% for C22:0, C24:0 and C26:0 in QC 1, and 4.96%, 3.87% and 5.29% for C22:0, C24:0 and C26:0 in control 2.

    “The coefficient of variations was 2.25, 2.8 and 9.4% for C22:0, C24:0, C26:0 (QC 1) and 4.96, 3.87 and 5.29% for C22:0, C24:0, C26:0 for control 2.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    An increasingly important alternative for determination of VLCFA is the use of 26:0-lyso-PC in dried blood spots, which is sensitive for females.

    “An increasingly important alternative for the determination of VLCFA is the use of 1-hexacosanosyl-2-lyso-sn-3-glycero-phosphatidylcholine (26:0-lyso-PC) in dried blood spots (Hubbard WC et al., 2009), being sensitive for females (Huffnagel et al. 2017) as well.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Hubbard WC et al., 2009

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Huffnagel et al. 2017

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The coefficient of variation from day to day was below 5% for all lipid analytes.

    “The coefficient of variation from day to day was below 5% for all analytes.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Both patient samples and controls were cross-checked with a reference laboratory at least yearly to verify the accuracy of the analysis.

    “Both patient samples and controls were cross-checked with a reference laboratory at least yearly to verify the accuracy of the analysis.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    VLCFA values are partially influenced by current diet, including ketogenic or vegetarian diet, with vegetarians potentially showing elevated values.

    “Preanalytical issue need to be considered e.g. VLCFA values are partially influenced by current diet e.g. by ketogenic (shown by Moser et al.10 and others) or vegetarian diet, with vegetarians potentially showing elevated values (Table 1).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Moser et al. [10] Moser, A. B. et al. Plasma very long chain fatty acids in 3,000 peroxisome disease patients and 29,000 controls. Ann. Neurol. 45, 100–110 (1999). source
    • cites Table 1

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Both quality controls were analyzed every run together with patients' samples.

    “Both quality controls were analyzed every run together with patients' samples.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    All patient samples with concentrations of C26:0 greater than 0.5 μmol/l were analyzed a second time independently, not to miss an elevated VLCFA.

    “All patient samples with concentrations of C26:0 > 0.5 µmol/l were analyzed a second time independently, not to miss an elevated VLCFA.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Moser et al. introduced a discriminant function based on measurement of C26:0 and the ratios C26/22 and C24/22 to enhance the distinction between X-ALD males and control males and between X-ALD females and control females.

    “Moser et al.10 introduced a discriminant function based on the measurement of C26:0 and the two ratios C26/22 and C24/22 to enhance the distinction between values obtained for X-ALD males and control males and X-ALD females and control females.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Moser, A. B. et al. Plasma very long chain fatty acids in 3,000 peroxisome disease patients and 29,000 controls. Ann. Neurol. 45, 100–110 (1999). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Repeated analysis yielded results differing by less than 8% from the first analysis.

    “In any case, we found that the repeated analysis yielded results differing by less than 8% from the first analysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The study was carried out in compliance with the Helsinki Declaration and approved by the Institutional Review Board of the University of Tübingen, reference number (690/2011BO1).

    “This study was carried out in compliance with the Helsinki Declaration and approved by the Institutional Review Board of the University of Tübingen, reference number (690/2011BO1).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    X-linked Adrenoleukodystrophy (X-ALD) is caused by mutations in the X-chromosomal ABCD1 gene.

    “X-linked Adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder worldwide1,2 and caused by mutations in the X-chromosomal ABCD1 gene3.”
    • Also stated in: § Abstract · plain
      “X-linked Adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene resulting in the accumulation of very long chain fatty acids (VLCFA).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [3] Mosser, J. et al. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with Abc transporters. Nature 361, 726–730. https://doi.org/10.1038/361726a0 (1993). source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Written informed consent was obtained from all patients or caregivers/legal guardians if applicable.

    “Written informed consent was obtained from all patients or caregivers/legal guardians if applicable.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    2.5% out of 17,000 healthy individuals equals 425 who will show elevated VLCFA, which is far too many false-positive values.

    “Instead of using reference ranges, the establishment of a threshold or cutoff is more helpful for the clinician in the case of X-ALD: 2.5% out of 17,000 healthy individuals = 425 will show elevated VLCFA, i.e. far too many false-positive values will be obtained.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Only genetically tested positive ABCD1 mutation carriers were considered to be X-ALD cases.

    “Additionally, only genetically tested positive ABCD1 mutation carriers were considered to be X-ALD cases.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Our observations that both total cholesterol and LDL-cholesterol levels, but not triglycerides levels, show a significant correlation to VLCFA C24:0 and C26:0 levels indicate that low-density lipoprotein contains lipids carrying VLCFA.

    “Our observations that both total cholesterol and LDL-cholesterol levels, but not triglycerides levels, show a significant correlation to VLCFA C24:0 and C26:0 levels (Table 4) indicate that low-density lipoprotein contains lipids carrying VLCFA.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: LDL is composed of approximately 50% cholesterol, most of it esterified with fatty acids, and 20% phospholipids containing fatty acids.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Vegetarian diet in healthy individuals seems to influence VLCFA levels shifting values towards those found in X-ALD patients.

    “Vegetarian diet in healthy individuals seems to influence VLCFA levels shifting values towards those found in X-ALD patients16.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Martinez, M., Mougan, I., Roig, M. & Ballabriga, A. Blood polyunsaturated fatty acids in patients with peroxisomal disorders. A multicenter study. Lipids 29, 273–280. https://doi.org/10.1007/BF02536332 (1994). source

  • May combine two claims.

    Interpretationown data

    Elevated LDL levels influence VLCFA levels but only barely C24:0/C22:0 and C26:0/C22:0 ratios.

    “Thus, elevated LDL levels influence VLCFA levels but only barely C24:0/C22:0 and C26:0/C22:0 ratios.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationnone given

    Serum VLCFA are economical and established biomarkers suitable for guiding genetic testing that matches the X-ALD phenotype.

    “Usage of serum VLCFA are economical and established biomarkers suitable for the guidance of genetic testing matching the X-ALD phenotype.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    VLCFA tests are only performed in neurological patients if X-ALD is considered a relevant differential diagnosis.

    “VLCFA tests are only performed in neurological patients if X-ALD is considered a relevant differential diagnosis (e.g. in our specialty clinic for hereditary spastic paraplegias17, where VLCFA are routinely tested).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [17] Schüle, R. et al. Hereditary spastic paraplegia: Clinicogenetic lessons from 608 patients. Ann. Neurol. 79, 646–658. https://doi.org/10.1002/ana.24611 (2016). source

  • May combine two claims.

    Study design and conductreasoning

    In contrast to previous studies, this study did not include a large number of healthy controls but focused on phenotypical controls mimicking the X-ALD phenotype.

    “In contrast to previous studies10, this study did, therefore, not include a large number of healthy controls but focused on phenotypical controls mimicking the X-ALD phenotype.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Moser, A. B. et al. Plasma very long chain fatty acids in 3,000 peroxisome disease patients and 29,000 controls. Ann. Neurol. 45, 100–110 (1999). source
36 of 36

  • The paper relies on this without support.

    Impaired peroxisomal beta-oxidation leads to accumulation of very long chain fatty acids (>22 carbon atoms) in blood and a variety of tissues.

    “Impaired peroxisomal beta-oxidation leads to the accumulation of very long chain fatty acids (VLCFA > 22 carbon atoms) in blood and a variety of tissues (e.g. brain white matter, spinal cord, and adrenal cortex).”

  • The paper relies on this without support.

    Adults with X-ALD usually develop an adrenomyeloneuropathy phenotype characterized by progressive spastic paraparesis with bladder disturbance, sensory ataxia with impaired vibration sense, leg pain, and adrenal failure in male patients.

    “Adults usually develop an adrenomyeloneuropathy (AMN) phenotype characterized by progressive spastic paraparesis with bladder disturbance, sensory ataxia with impaired vibration sense, leg pain, and adrenal failure in male patients.”

  • The paper relies on this without support.

    Up-to-date assessment of VLCFA cutoffs in patients with a mimicking phenotype as well as in genetically confirmed X-ALD cases is missing.

    “Up to date assessment of VLCFA cutoffs in patients with a mimicking phenotype as well as in genetically confirmed X-ALD cases is missing.”

  • The paper relies on this without support.

    LDL is composed of approximately 50% cholesterol, most of it esterified with fatty acids, and 20% phospholipids containing fatty acids.

    “LDL is composed of approximately 50% cholesterol, most of it is esterified with fatty acids and 20% phospholipids containing fatty acids.”
4 of 4

Statistics that don’t add up

Each reported p-value recomputed from its test statistic and degrees of freedom.

  • Inconsistent p-value: r(32) = − 0.06, p = .973

    Minor

    The paper reports p = .973 here; recomputing from the test statistic and its degrees of freedom gives p = .736 (two-tailed). The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .973 · recomputed p = .736

  • Inconsistent p-value: r(32) = − 0.06, p = .975

    Minor

    The paper reports p = .975 here; recomputing from the test statistic and its degrees of freedom gives p = .736 (two-tailed). The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .975 · recomputed p = .736

2 of 2

Statistics recomputed

Every reported test result, recomputed from its test statistic and degrees of freedom. A result shows as not adding up only once an independent check has confirmed it.

  • Doesn’t add upr(32) = -0.06reported p = .975 · recomputed p = .736

    “r(32) = − 0.06, p = .975”

  • Doesn’t add upr(32) = -0.06reported p = .973 · recomputed p = .736

    “r(32) = − 0.06, p = .973”

  • Consistentr(32) = -0.1reported p = .592 · recomputed p = .574

    “r(32) = − 0.10, p = .592”

  • Consistentr(33) = 0.11reported p = .537 · recomputed p = .529

    “r(33) = 0.11, p = .537”

  • Consistentr(33) = 0.05reported p = .766 · recomputed p = .775

    “r(33) = 0.05, p = .766”

5 of 5

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Perfect specificity conflicts with a PPV below 100%

    Major

    For a test with detected positive cases, 100% specificity means there are no false positives, so its positive predictive value must also be 100%, not 96%.

  • HSP percentage does not match the stated count

    Minor

    The phenotypical control group includes 63 HSP patients, of whom 49 had a genetically confirmed diagnosis. That is 77.8%, not 73%.

    Reported 73 · recomputed 77.777778

  • Reported cholesterol correlation method changes

    Minor

    The abstract attributes the significant association between total cholesterol and VLCFA values to Pearson correlation, but the results and Table 4 attribute it to Spearman correlation.

  • Cholesterol correlation includes more cases than the stated lipid-analysis cohort

    Minor

    The methods state that routine lipid analysis was performed for 90 cases, but every total-cholesterol correlation in Table 4 reports 92 cases.

4 of 4

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Moser, H. W., Mahmood, A. & Raymond, G. V. X-linked adrenoleukodystrophy. Nat. Rev. Neurol. 3, 140 (2007). · DOI · Paper page

Cited by

Sorted by citations

Related

Authors
Rattay, T. W., Rautenberg, M., Söhn, A. S., Hengel, H., Traschütz, A., Röben, B., Hayer, S. N., Schüle, R., Wiethoff, S., Zeltner, L., Haack, T. B., Cegan, A., Schöls, L., Schleicher, E., & Peter, A.
Affiliations
  • German Center for Neurodegenerative Diseases (DE)
  • University of Tübingen (DE)
  • Hertie Institute for Clinical Brain Research (DE)
  • Helmholtz Munich (DE)
  • German Center for Diabetes Research (DE)
Corresponding author
Schleicher, Erwin
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2020-09-15
Volume
10
Issue
1
Pages
15093-15093
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-020-71248-8
PMC
PMC7494896
OpenAlex
W3084746432
Semantic Scholar
8b1edf77329bbb7b584466cdb202c7a5ef4a85ab
Cited by
61
References
29
Citations by year
2026: 5 · 2025: 6 · 2024: 3 · 2023: 3 · 2022: 5 · 2021: 33 · 2020: 6
FWCI
6.19
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • Digitální knihovna Univerzity Pardubice (Univerzity Pardubice) (submittedVersion, cc-by)
  • Site cant be reached (submittedVersion, cc-by)
  • PuSH - Publication Server of Helmholtz Zentrum München (submittedVersion, cc-by)
Funders
  • Medizinischen Fakultät, Eberhard Karls Universität Tübingen (Clinician Scientist Program Grant: #386-0-0, fortüne program (#2554-0-0)., Clinician Scientist Program Grant: #434-0-0)
  • Deutsche Forschungsgemeinschaft (Open Access Publishing Fund of University of Tübingen, 418081722)
  • European Reference Network for Rare Neurological Diseases (Project ID No 739510)
  • Ministry of Science, Research and the Arts of Baden-Württemberg (31-7635)
  • European Social Fund (41/67/1)
  • Projekt DEAL
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Metabolism and Genetic Disorders
  • Health Sciences › Medicine › Physiology › Lysosomal Storage Disorders Research
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Hereditary Neurological Disorders
Keywords
X-linked adrenoleukodystrophy, very long chain fatty acids, diagnostic thresholds, ABCD1 mutations, peroxisomal disorders, diagnostic biomarkers, spastic paraparesis, adrenal insufficiency, serum cholesterol, genetic testing
MeSH terms
Adrenoleukodystrophy, Adult, Aged, Astrocytes, Fatty Acids, Female, Humans, Male, Middle Aged, Mutation, Sensitivity and Specificity, Biomarkers, ATP-Binding Cassette Transporters

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-20.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-20.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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