Precision Newborn Screening for Lysosomal Disorders

Purpose: The implementation of newborn screening for lysosomal disorders has uncovered overall poor specificity, psychosocial harm experienced by caregivers, and costly follow-up testing of false-positive cases. We report an informatics solution proven to minimize these issues. Methods: The Kentucky Department for Public Health outsourced testing for mucopolysaccharidosis type I (MPS I) and Pompe disease, conditions recently added to the recommended uniform screening panel, plus Krabbe disease, which was added by legislative mandate. A total of 55,161 specimens were collected from infants born over 1 year starting from February 2016. Testing by tandem mass spectrometry was integrated with multivariate pattern recognition software (Collaborative Laboratory Integrated Reports), which is freely available to newborn screening programs for selection of cases for which a biochemical second-tier test is needed. Results: Of five presumptive positive cases, one was affected with infantile Krabbe disease, two with Pompe disease, and one with MPS I. The remaining case was a heterozygote for the latter condition. The false-positive rate was 0.0018% and the positive predictive value was 80%. Conclusion: Postanalytical interpretive tools can drastically reduce false-positive outcomes, with preliminary evidence of no greater risk of false-negative events, still to be verified by long-term surveillance

The autophagic protein p62 is a target of reactive aldehydes in human and murine cholestatic liver disease.

Inflammatory cholestatic liver diseases, including Primary Sclerosing Cholangitis (PSC), are characterized by periportal inflammation with progression to cirrhosis. The objective of this study was to examine interactions between oxidative stress and autophagy in cholestasis. Using hepatic tissue from male acute cholestatic (bile duct ligated) as well as chronic cholestatic (Mdr2KO) mice, localization of oxidative stress, the antioxidant response and induction of autophagy were analyzed and compared to human PSC liver. Concurrently, the ability of reactive aldehydes to post-translationally modify the autophagosome marker p62 was assessed in PSC liver tissue and in cell culture. Expression of autophagy markers was upregulated in human and mouse cholestatic liver. Whereas mRNA expression of Atg12, Lamp1, Sqstm1 and Map1lc3 was increased in acute cholestasis in mice, it was either suppressed or not significantly changed in chronic cholestasis. In human and murine cholestasis, periportal hepatocytes showed increased IHC staining of ubiquitin, 4-HNE, p62, and selected antioxidant proteins. Increased p62 staining colocalized with accumulation of 4-HNE-modified proteins in periportal parenchymal cells as well as with periportal macrophages in both human and mouse liver. Mechanistically, p62 was identified as a direct target of lipid aldehyde adduction in PSC hepatic tissue and in vitro cell culture. In vitro LS-MS/MS analysis of 4-HNE treated recombinant p62 identified carbonylation of His123, Cys128, His174, His181, Lys238, Cys290, His340, Lys341 and His385. These data indicate that dysregulation of autophagy and oxidative stress/protein damage are present in the same periportal hepatocyte compartment of both human and murine cholestasis. Thus, our results suggest that both increased expression as well as ineffective autophagic degradation of oxidatively-modified proteins contributes to injury in periportal parenchymal cells and that direct modification of p62 by reactive aldehydes may contribute to autophagic dysfunction

Fig 8 -

Colocalization of p62 and 4-HNE in liver in cholestasis A. Control and PSC liver (200X). B. Sham and BDL cholestatic liver (100X) C. WT and Mdr2KO liver (100X). Arrows indicate periportal hepatocytes of increased staining/colocalization. n = 4/group, Blue-Dapi, Green-4-HNE, Cyan-p62. CV-central vein, PT-portal triad.</p

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Quantification of necrosis, fibrosis, H2Ax positive nuclei and the ductular reaction (CK7), in liver tissue from C57BL/6 sham and 3-day BDL mice.

A. Percent necrotic injury. B. Quantification of Picrosirius red staining (PSR). C. Percent cytokeratin 7 (CK7) positive cells. D. qPCR analysis of mRNA for fibrogenic genes Timp1 and Col1a1 in liver tissue from control and 3D BDL mouse liver (N = 4/group). E. H2Ax positive nuclei/100X field. Data were statistically analyzed using Student’s t-test and are presented as Mean ± STDEV. ****p (TIF)</p

Increased expression of autophagic proteins in acute murine cholestasis.

A. qPCR analysis of mRNA for autophagic proteins in liver tissue isolated from 3-day sham and BDL adult mice (N = 5/group). Expression was normalized against HPRT. B. Western analysis of autophagic proteins in liver tissue isolated from BDL mice (N = 5/group). Blots were normalized to GAPDH expression. C. qPCR analysis of mRNA for autophagic proteins in liver tissue isolated from 10-week old Mdr2KO mice (N = 4/group). D. Western analysis of autophagic proteins in liver tissue isolated from 10 week on Mdr2KO mice (N = 6/group). Blots were normalized to GAPDH expression Data are presented as Mean ± STDEV and were statistically analyzed using a Student’s t-test, *p<0.05, **p<0.01, ***P<0.001, ****p<0.0001, N = 6/group.</p

Serum biochemical analysis of liver injury in WT and BDL treated mice.

Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, total bilirubin and total serum bile acid concentrations. Data were analyzed statistically using a Student’s t-test and are presented as Mean ± STDEV, ***p (TIF)</p

Histochemical assessment of liver injury and ductular proliferation in C57BL/6 sham and 3-day BDL mice.

A. Hematoxylin and Eosin (H&E) 200X. B. Picrosirius Red (PSR) 100X. C. Cytokeratin 7 (CK7) 200X. CV-central vein, PT-portal triad, NEC-necrosis, n = 4/group. (TIF)</p