Addition of galactose-1-phosphate measurement enhances newborn screening for classical galactosemia.

Galactosemia is an inborn disorder of carbohydrate metabolism of which early detection can prevent severe illness. Although the assay for galactose-1-phosphate uridyltransferase (GALT) enzyme activity has been available since the 1960s, many issues prevented it from becoming universal. In order to develop the Israeli newborn screening pilot algorithm for galactosemia, flow injection analysis tandem mass spectrometry measurement of galactose-1-phosphate in archived dried blood spots from newborns with classical galactosemia, galactosemia variants, epimerase deficiency, and normal controls, was conducted. Out of 431 330 newborns screened during the pilot study (30 months), two with classical galactosemia and four with epimerase deficiency were identified and confirmed. Five false positives and no false negatives were recorded. Following this pilot study, the Israeli final and routine newborn screening algorithm, as recommended by the Advisory Committee to the National Newborn Screening Program, now consists of galactose-1-phosphate measurement integrated into the routine tandem mass spectrometry panel as the first-tier screening test, and GALT enzyme activity as the second-tier performed to identify only newborns suspected to be at risk for classical galactosemia. The GALT enzyme activity cut-off used in the final algorithm was lowered in order to avoid false positives.

The role of orotic acid measurement in routine newborn screening for urea cycle disorders.

Urea cycle disorders (UCDs), including OTC deficiency (OTCD), are life-threatening diseases with a broad clinical spectrum. Early diagnosis and initiation of treatment based on a newborn screening (NBS) test for OTCD with high specificity and sensitivity may contribute to reduction of the significant complications and high mortality. The efficacy of incorporating orotic acid determination into routine NBS was evaluated. Combined measurement of orotic acid and citrulline in archived dried blood spots from newborns with urea cycle disorders and normal controls was used to develop an algorithm for routine NBS for OTCD in Israel. Clinical information and genetic confirmation results were obtained from the follow-up care providers. About 1147986 newborns underwent routine NBS including orotic acid determination, 25 of whom were ultimately diagnosed with a UCD. Of 11 newborns with OTCD, orotate was elevated in seven but normal in two males with early-onset and two males with late-onset disease. Orotate was also elevated in archived dried blood spots of all seven retrospectively tested historical OTCD patients, only three of whom had originally been identified by NBS with low citrulline and elevated glutamine. Among the other UCDs emerge, three CPS1D cases and additional three retrospective CPS1D cases otherwise reported as a very rare condition. Combined levels of orotic acid and citrulline in routine NBS can enhance the detection of UCD, especially increasing the screening sensitivity for OTCD and differentiate it from CPS1D. Our data and the negligible extra cost for orotic acid determination might contribute to the discussion on screening for proximal UCDs in routine NBS.

Calpastatin is upregulated in non-immune neuronal cells via toll-like receptor 2 (TLR2) pathways by lipid-containing agonists.

Calpain (intracellular Ca(2+)-dependent protease) and calpastatin (calpain specific endogenous inhibitor) are widely distributed in biological systems, and have been implicated in many cellular physiological and pathological processes. Calpastatin level is of central importance to the control of calpain activity. We demonstrated for the first time that calpastatin is overexpressed in mycoplasma-contaminated cultured cells (SH-SY5Y cells that are infected by a strain of Mycoplasma hyorhinis (NDMh)). We have found that the calpastatin-upregulating activity resides in the mycoplasmal membrane lipoproteins, and is associated with NF-κB activation. Calpain-promoted proteolysis is attenuated in the NDMh lipoprotein-treated cells. Here we show that the NDMh lipoproteins promoted an increase in calpastatin in SH-SY5Y cells via the TLR2/TAK1/NF-κB pathway. The synthetic mycoplasmal lipopeptide MALP-2 and the bacterial lipopeptide PAM3CSK4 (TLR2 agonists) also promoted calpastatin upregulation. LPS (TLR4 agonist) activated NF-κB without calpastatin increase in the cell. In contrast, lipoteichoic acid (TLR2 agonist) upregulated calpastatin not via NF-κB activation, but via the MEK1/ELK1 pathway. Zymosan and peptidoglycan, TLR2 agonists that lack lipids, did not induce calpastatin upregulation. Cell treatment with a calpastatin-upregulating agonist (lipoteichoic acid) led to the attenuation of Ca(2+)-promoted calpain activity, whereas agonists that do not upregulate calpastatin (LPS, Zymosan) were ineffective. Overall, the results indicate that in these non-immune cells, calpastatin is upregulated by TLR2-agonists containing lipids, with more than one downstream pathway involved. Such agonists may be useful for studying mechanisms and factors involved in calpastatin regulation. In addition, suitable TLR2 agonists may be of interest in devising treatments for pathological processes involving excessive calpain activation.