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Newborn Screening Method Uses Mass Spec to Test for Niemann-Pick Disease Type C

NEW YORK (GenomeWeb) – A Washington University School of Medicine-led research team has developed a mass spectrometry-based screen for the cholesterol storage disorder Niemann-Pick disease type C in newborns.

The team led by WUSTL's Daniel Ory identified bile acids that are present at increased levels in NPC patients as compared to controls, and devised a mass spec-based method to test for these acids in dried blood spots, such as those obtained from newborn heel pricks. As the researchers reported in Science Translational Medicine today, this test could distinguish NPC patients from controls with high sensitivity and specificity.

"This dried blood spot-based assay provides the basis for a screen for NPC that is ready for piloting in newborn screening programs," Ory and his colleagues wrote in their paper.

Currently, there is often a delay in NPC diagnosis as the initial symptoms are nonspecific, and a test such as this could, the researchers argued, enable treatment before the onset of neurological symptoms, delay disease progression, and perhaps extend life.

Ory and his colleagues focused their analyses on bile acids because they are known to be affected by sterol metabolism changes, there have been reports of unusual bile acids in NPC patients' urine, and they can be detected using mass spec.

Using a three-tier targeted metabolomic approach relying on LC-MS/MS operating in multiple reaction monitoring mode, the researchers homed in on three candidate bile acid species that were elevated in the plasma of NPC1 patients, but not in controls. Bile acids A and B were elevated 41- and 144-fold, respectively, in NPC1 plasma and could discriminate between NPC1 and control plasma samples, they reported. Bile acid C was elevated six-fold in NPC1 patients, but overlapped with control samples.

By combining high-resolution MS, hydrogen/deuterium exchange MS, and high-resolution MS/MS, the researchers sought to resolve the structures of these unknown bile acids. Based on their mass/charge ratios, hydrogen/deuterium exchange, and likely hydroxyl group positions, they proposed that the bile acids they dubbed 'A' and 'B' were 5 α-cholanic acid-3β,5α,6β-triol and 5 α-cholanic acid-3β,5α,6β-triol N-(carboxymethyl)-amide, respectively.

They then confirmed their identities by synthesizing them and performing a comparative LC-MS/MS of the synthesized and endogenous compounds.

These bile acids are metabolites of cholestane-3β,5α,6β-triol, an oxysterol that is elevated in NPC.

After confirming that these bile acids could be measured and distinguished in dried blood spots from a small number of NPC patients and controls, Ory and his colleagues set about developing an assay to detect bile acid B in dried blood spots.

They adopted a two-tier strategy in which the first tier involves a short, 2.2-minute LC condition and the second tier involves a long, 7-minute LC condition. With this approach, the researchers said some 500 samples could be analyzed each day using the first-tier method, with only ones that can't be resolved undergoing second-tier analysis.

To establish their cut-off values, the researchers analyzed dried blood spot samples from 1,013 controls, 130 NPC1 carriers, and 25 NPC1 patients to yield control reference ranges for each group. Using a cut-off of 13.5 ng/ml yielded 100 percent sensitivity and specificity, they reported, and only one normal sample had to undergo second-tier testing. This, they added, should minimize false positives.

This screen, Ory and his colleagues wrote, is ready for pilot testing.

"Broad implementation of newborn screening for NPC would eliminate the diagnostic delay and shift diagnosis of the disease to the newborn period before the onset of neurological symptoms," they added. "Drug intervention and non-pharmacological supportive care during this asymptomatic period have the potential to markedly delay disease progression and extend life."