Research ArticleNIEMANN-PICK TYPE C DISEASE

Development of a bile acid–based newborn screen for Niemann-Pick disease type C

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Science Translational Medicine  04 May 2016:
Vol. 8, Issue 337, pp. 337ra63
DOI: 10.1126/scitranslmed.aaf2326
  • Fig. 1. NPC1 marker screening.

    (A) Three-tier targeted metabolomic strategy for identification of bile acid markers. First-tier screen includes 49 MRM transitions (17-min run time). Second-tier screen includes 10 MRM transitions (7.5-min run time) to characterize peaks with signal-to-noise ratio greater than five. Third-tier screen (6-min run time) quantifies unknown bile acid peaks (A, B, and C) that are elevated in NPC1 compared to control. (B) Comparison of bile acid concentration in NPC1 (n = 12) versus control (n = 11) samples obtained from second-tier profiling. Data are presented as mean fold change + SD normalized to control. *P = 0.0005, 0.0003, and 0.0007 for bile acids A, B, and C in NPC1 versus controls, respectively. CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; GCA, glycocholic acid; GDCA, glycodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; GLCA, glycolithocholic acid; TCA, taurocholic acid; TDCA, taurodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TLCA, taurolithocholic acid; BA-A, bile acid A; BA-B, bile acid B; BA-C, bile acid C. (C) Comparison of bile acids A, B, and C in NPC1 (n = 12) and control (n = 11) plasma samples obtained from third-tier profiling. Data are presented as means ± 95% confidence interval (CI) peak area. P < 0.0001 for bile acids A and B in NPC1 versus controls. P = 0.0005 for bile acid C in NPC1 versus controls. (D) Correlation between bile acids A and B in NPC plasma samples, r2 = 0.83, P < 0.0001; correlation between bile acids A and C in NPC plasma samples, r2 = 0.39, P = 0.0008.

  • Fig. 2. Identification and confirmation of structure of unknown bile acids.

    (A) Strategy for identification of bile acid structures. HRMS, high-resolution MS; HDXMS, hydrogen/deuterium exchange MS; HRMS/MS, high-resolution MS/MS. (B and C) High-resolution mass spectra are shown for bile acid A (B) and bile acid B (C). mmu, milli-mass unit. (D and E) H/D exchange mass spectra are shown for bile acid A (D) and bile acid B (E). (F and G) HCD mass spectra are shown for bile acid A (F) and bile acid B (G).

  • Fig. 3. Biosynthesis of bile acid A from cholestane-3β,5α,6β-triol.

    (A) Synthetic bile acid A detected by MRM transition m/z 407→407. cps, counts per second. (B) Bile acid A generated from cholestane-3β,5α,6β-triol in Hep G2 cells and detected by MRM transition m/z 407→407. (C) d4-Bile acid A generated from d4-cholestane-3β,5α,6β-triol in Hep G2 cells and detected by MRM transition m/z 411→411.

  • Fig. 4. Detection of bile acid markers in dried blood spots.

    (A) Bile acid A in NPC1 (n = 10) and control (n = 16) dried blood spot samples. Data are presented as means ± 95% CI peak area. P < 0.0001 for NPC1 versus controls. (B) Bile acid B in NPC1 (n = 10) and control (n = 16) dried blood spots sample. Data are presented as means ± 95% CI peak area. P < 0.0001 for NPC1 versus controls.

  • Fig. 5. Two-tier newborn screen for NPC1 disease.

    (A) Chromatograms of bile acid B in dried blood spots from a newborn control, adult NPC1 carrier, and NPC1 patient, as resolved with short LC (first-tier assay) and long LC conditions (second-tier assay). Bile acid B was eluted at 1.7 and 4.05 min under short and long LC conditions, respectively. There are two interferences that eluted close to bile acid B. An interference peak that presents in most newborn dried blood spots was baseline-resolved from bile acid B under both short (1.63 min) and long LC (3.85 min) conditions. The dried blood spots from NPC1 subjects and carriers showed an interference peak that was co-eluted with bile acid B at 1.7 min under the short LC condition, but baseline-separated from bile acid B under the long LC condition at 4.23 min. (B) Algorithm for two-tier newborn screening of NPC1 disease.

  • Fig. 6. Establishment and validation of cutoff for NPC1 newborn screen.

    (A) Bile acid B concentrations in dried blood spots from newborn control, control at other age (>1 month old), NPC1 carrier, and NPC1 patients in a training set. Bile acid B concentrations below the LLOQ (5 ng/ml) were quantifiable, although the percent coefficient of variance (%CV) and percent relative error (%RE) for these samples were above acceptance criteria for the validated assay. Data presented on semilog plots are shown as means ± 95% CI. Samples with no detectable bile acid B peak were assigned as 0.1 ng/ml for purposes of plotting. P < 0.0001 for NPC1 versus controls and NPC1 carriers. (B) Determination of bile acid B concentrations and application of the 13.5 ng/ml cutoff to a test sample set consisting of newborn control, NPC1 carrier, and NPC1 dried blood spots. All newborn control and NPC1 samples are new samples, and carriers denoted by pink symbols are new samples. The NPC1 carriers in blue were analyzed in training set and reanalyzed in validation set. Samples were coded and randomized, and the operator was blinded to group assignment, thus reducing bias and noise/variance in the results and enabling unbiased statistical analysis of the data. Data are presented on semilog plots and are shown as means ± 95% CI. Samples with no detectable bile acid B peak were assigned as 0.1 ng/ml for purposes of plotting. P < 0.0001 for NPC1 versus controls and NPC1 carriers. (C) Application of cutoff value of 13.5 ng/ml yields sensitivity and specificity of 100%, and ROC area under the curve of 1.0. (D) Bile acid B concentrations in SLOS, ASMD, CTX, and LALD dried blood spots from cutoff validation sample set. Data are presented on semilog plots and are shown as means ± 95% CI. Samples with no detectable bile acid B peak were assigned as 0.1 ng/ml for purposes of plotting. P < 0.0001 for ASMD versus controls.

  • Fig. 7. Comparison of bile acid B in dried blood spots with cholestane-3β,5α,6β-triol in plasma.

    (A) Correlation of bile acid B in dried blood spots with cholestane-3β,5α,6β-triol in plasma (n = 47), r = 0.59, P < 0.0001. x, bile acid cutoff for NPC1 carriers and controls; y, cholestane-3β,5α,6β-triol cutoff for NPC1 carriers; z, cholestane-3β,5α,6β-triol cutoff for controls. (B) The inset bounded by the dotted purple line in (A) has been expanded to more clearly compare assay specificity.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/337/337ra63/DC1

    Materials and Methods

    Fig. S1. Structures of bile acids and analogs used to study fragmentation patterns of the AMPP derivatives.

    Fig. S2. HCD mass spectra of AMPP-derivatized bile acids 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), 6 (F), 7 (G), 8 (H), 9 (I), and 10 (J).

    Fig. S3. Proposed fragmentation pathway to fragments A to H in AMPP derivatives of unconjugated bile acids 1 to 7.

    Fig. S4. Proposed fragmentation pathway to fragments I to K in AMPP derivatives of unconjugated bile acids 1 to 7.

    Fig. S5. Proposed fragmentation pathway to fragments L (L′) to M (M′) in AMPP derivatives of unconjugated bile acids 1 to 7.

    Fig. S6. Proposed fragmentation pathway to fragments N to P in AMPP derivatives of unconjugated bile acids 1 to 6.

    Fig. S7. Proposed fragmentation pathway to fragments Q to T in AMPP derivatives of unconjugated bile acids 1 to 7.

    Fig. S8. Proposed fragmentation pathway to fragments GA to GE and GH in AMPP derivatives of glycine-conjugated bile acids 8 to 10.

    Fig. S9. Proposed fragmentation pathway to fragments GF, GG, and GI in AMPP derivatives of glycine-conjugated bile acids 8 to 10.

    Fig. S10. Proposed fragmentation pathway to fragments GJ to GM in AMPP derivatives of glycine-conjugated bile acids 8 to 10.

    Fig. S11. Proposed fragmentation pathway to fragments GN to GW in AMPP derivatives of glycine-conjugated bile acids 8 to 10.

    Fig. S12. Proposed fragmentation pathway to fragments GX to GZ and GAA to GAB in glycine-conjugated bile acid AMPP derivatives.

    Fig. S13. Comparison of chromatograms and HCD mass spectra of AMPP derivatives of bile acids A and B in NPC plasma and solutions of synthetic compounds.

    Fig. S14. Structures of bile acid markers for NPC.

    Fig. S15. Comparison of chromatograms of bile acids A and B in NPC1 plasma and solutions of synthetic compounds.

    Fig. S16. Correlation of bile acid B concentrations with patient parameters.

    Table S1. Accurate masses and calculated elemental composition of fragment ions of 21,26,27-trinorcholestan-25-oic acid-3β,5α,6β-triol and bile acid A AMPP derivatives.

    Table S2. Accurate masses and calculated elemental composition of fragment ions of bile acid B AMPP derivative.

    Table S3. Accurate masses and calculated elemental composition of fragment ions of DCA, CDCA, and 5β-cholanic acid-3α,4β,7α-triol AMPP derivatives.

    Table S4. Accurate masses and calculated elemental composition of fragment ions of CA, α-muricholic acid, and β-muricholic acid AMPP derivatives.

    Table S5. Accurate masses and calculated elemental composition of fragment ions of GCA, GCDCA, and GDCA AMPP derivatives.

    Table S6. MRM transitions and MS parameters for bile acids in the first-tier marker screening.

    Table S7. MRM transitions and MS parameters for bile acids in the second-tier marker screening.

    Table S8. Accuracy and precision of QC samples.

    References (4042)

  • Supplementary Material for:

    Development of a bile acid–based newborn screen for Niemann-Pick disease type C

    Xuntian Jiang, Rohini Sidhu, Laurel Mydock-McGrane, Fong-Fu Hsu, Douglas F. Covey, David E. Scherrer, Brian Earley, Sarah E. Gale, Nicole Y. Farhat, Forbes D. Porter, Dennis J. Dietzen, Joseph J. Orsini, Elizabeth Berry-Kravis, Xiaokui Zhang, Janice Reunert, Thorsten Marquardt, Heiko Runz, Roberto Giugliani, Jean E. Schaffer, Daniel S. Ory*

    *Corresponding author. Email: dory{at}wustl.edu

    Published 4 May 2016, Sci. Transl. Med. 8, 337ra63 (2016)
    DOI: 10.1126/scitranslmed.aaf2326

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Structures of bile acids and analogs used to study fragmentation patterns of the AMPP derivatives.
    • Fig. S2. HCD mass spectra of AMPP-derivatized bile acids 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), 6 (F), 7 (G), 8 (H), 9 (I), and 10 (J).
    • Fig. S3. Proposed fragmentation pathway to fragments A to H in AMPP derivatives of unconjugated bile acids 1 to 7.
    • Fig. S4. Proposed fragmentation pathway to fragments I to K in AMPP derivatives of unconjugated bile acids 1 to 7.
    • Fig. S5. Proposed fragmentation pathway to fragments L (L′) to M (M′) in AMPP derivatives of unconjugated bile acids 1 to 7.
    • Fig. S6. Proposed fragmentation pathway to fragments N to P in AMPP derivatives of unconjugated bile acids 1 to 6.
    • Fig. S7. Proposed fragmentation pathway to fragments Q to T in AMPP derivatives of unconjugated bile acids 1 to 7.
    • Fig. S8. Proposed fragmentation pathway to fragments GA to GE and GH in AMPP derivatives of glycine-conjugated bile acids 8 to 10.
    • Fig. S9. Proposed fragmentation pathway to fragments GF, GG, and GI in AMPP derivatives of glycine-conjugated bile acids 8 to 10.
    • Fig. S10. Proposed fragmentation pathway to fragments GJ to GM in AMPP derivatives of glycine-conjugated bile acids 8 to 10.
    • Fig. S11. Proposed fragmentation pathway to fragments GN to GW in AMPP derivatives of glycine-conjugated bile acids 8 to 10.
    • Fig. S12. Proposed fragmentation pathway to fragments GX to GZ and GAA to GAB in glycine-conjugated bile acid AMPP derivatives.
    • Fig. S13. Comparison of chromatograms and HCD mass spectra of AMPP derivatives of bile acids A and B in NPC plasma and solutions of synthetic compounds.
    • Fig. S14. Structures of bile acid markers for NPC.
    • Fig. S15. Comparison of chromatograms of bile acids A and B in NPC1 plasma and solutions of synthetic compounds.
    • Fig. S16. Correlation of bile acid B concentrations with patient parameters.
    • Table S1. Accurate masses and calculated elemental composition of fragment ions of 21,26,27-trinorcholestan-25-oic acid-3β,5α,6β-triol and bile acid A AMPP derivatives.
    • Table S2. Accurate masses and calculated elemental composition of fragment ions of bile acid B AMPP derivative.
    • Table S3. Accurate masses and calculated elemental composition of fragment ions of DCA, CDCA, and 5β-cholanic acid-3α,4β,7α-triol AMPP derivatives.
    • Table S4. Accurate masses and calculated elemental composition of fragment ions of CA, α-muricholic acid, and β-muricholic acid AMPP derivatives.
    • Table S5. Accurate masses and calculated elemental composition of fragment ions of GCA, GCDCA, and GDCA AMPP derivatives.
    • Table S6. MRM transitions and MS parameters for bile acids in the first-tier marker screening.
    • Table S7. MRM transitions and MS parameters for bile acids in the second-tier marker screening.
    • Table S8. Accuracy and precision of QC samples.
    • References (4042)

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