Newborn screening for mucopolysaccharidoses: a pilot study of measurement of glycosaminoglycans by tandem mass spectrometry

Background

Mucopolysaccharidoses (MPS) are a group of inborn errors of metabolism that are progressive and usually result in irreversible skeletal, visceral, and/or brain damage, highlighting a need for early diagnosis.

Methods

This pilot study analyzed 2862 dried blood spots (DBS) from newborns and 14 DBS from newborn patients with MPS (MPS I, n?=?7; MPS II, n?=?2; MPS III, n?=?5). Disaccharides were produced from polymer GAGs by digestion with chondroitinase B, heparitinase, and keratanase II. Heparan sulfate (0S, NS), dermatan sulfate (DS) and mono- and di-sulfated KS were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS). Median absolute deviation (MAD) was used to determine cutoffs to distinguish patients from controls. Cutoffs were defined as median?+?7× MAD from general newborns.

Results

The cutoffs were as follows: HS-0S?>?90 ng/mL; HS-NS?>?23 ng/mL, DS?>?88 ng/mL; mono-sulfated KS?>?445 ng/mL; di-sulfated KS?>?89 ng/mL and ratio di-KS in total KS?>?32 %. All MPS I and II samples were above the cutoffs for HS-0S, HS-NS, and DS, and all MPS III samples were above cutoffs for HS-0S and HS-NS. The rate of false positives for MPS I and II was 0.03 % based on a combination of HS-0S, HS-NS, and DS, and for MPS III was 0.9 % based upon a combination of HS-0S and HS-NS.

Conclusions

Combination of levels of two or more different GAGs improves separation of MPS patients from unaffected controls, indicating that GAG measurements are potentially valuable biomarkers for newborn screening for MPS.

     

Experience with artificial liver support in 16 living related liver transplant recipients.

This study was a retrospective investigation about the indication and efficacy of artifical liver support for liver transplant recipients. Apheresis was performed in 16 of 41 patients subjected to living related liver transplantation (LRLTx) as articial liver support, including plasmapheresis (PP) in 13 cases, continuous hemodiafiltration (CHDF) in 7 cases, and plasma adsorption (PA) in 2 cases. One patient with cryptogenic liver cirrhosis was subjected to PP before the LRLTx, and the result was satisfactory. On the contrary, the results of PP and CHDF for graft, respiratory, or cardiac failure were not acceptable. Only 1 patient survived despite multiple organ failure. Both PP and PA for patients with hyperbilirubinemia were effective and improved their critical conditions. We conclude that apheresis for liver transplant patients is effective to treat hyperbilirubinemia, but it is not indicated for respiratory and cardiac failure nor for hepatic failure.     

Analysis of the inflammatory reaction induced by the catfish (Cathorops spixii) venoms.

Cathorops spixii is one of the most abundant venomous fish of the southeastern coast of the State of S?o Paulo, and consequently causes a great part of the accidents seen there. The accidents affect mainly fishermen, swimmers and tourists and are characterized by punctiform or wide wounds, erythema, edema, pain, sudoresis, indisposition, fever, nausea, vomiting and secondary infection. The objective of this work was to characterize the inflammatory response induced in mice by both venoms (mucus and sting) of the catfish C. spixii. Our results demonstrated that both venoms induced a great number of rolling and adherent leukocytes in the post-capillary venules of cremaster muscle of mice, and an increase in the vascular permeability in peritoneal cavity. Mucus induced the recruitment of neutrophils immediately after injection followed later by macrophage infiltration. In contrast, the cellular infiltration elicited by sting venom was rapidly resolved. The peritonitis reaction provoked by venoms was characterized by cytokine (IL-6), chemokines (MCP-1 and KC) or lipid mediator (LTB4) production in the peritoneal cavity. The macrophages from 7-day mucus venom-induced exudates upon in vitro mucus venom stimulation, expressed CD11c x MHC class II and release bioactive IL-12p70. On the other hand, sting venom-elicited peritoneal macrophages lost the ability to differentiate into dendritic cells, following re-stimulation in vitro with sting venom, they do not express CD11c, nor do they exhibit sufficient levels of MHC class II. In conclusion, both types of venoms (mucus or sting) promote inflammatory reaction with different profiles, and the inflammatory reaction induced by the first was characterized by antigen persistence in peritoneal cavity that allowed the activation of phagocytic cells with capacity of antigenic presentation.     

Prenatal diagnosis and neonatal monitoring of a fetus with glutaric aciduria type II due to electron transfer flavoprotein (beta-subunit) deficiency.

The prenatal diagnosis of a male fetus with glutaric aciduria type II and the time course of metabolite urinary excretion, starting immediately after birth, are described. Prenatal diagnosis was undertaken at the 17th wk of gestation by immunoblot analysis and pulse labeling experiments of amniocytes and, retrospectively, by stable isotope dilution analysis of six metabolites in amniotic fluid. The results were as follows: 1) The immunochemical analysis on cultured amniocytes showed that the fetus, as the previous index case in this family, was affected with a deficiency of the beta-subunit of electron transfer flavoprotein. 2) Glutarate concentration was significantly increased in the cell-free supernatant of the amniotic fluid. In the postnatal period, most of the organic acids and acylglycines characteristic of the disorder appeared in urine within a week, although an increased excretion of hexanoylglycine was the only biochemical abnormality detectable in the first urine sample collected at 9 h after birth. Growth and development of this infant were normal during the following 6 mo of life, when he was receiving oral supplementation with L-carnitine and riboflavin. It should be underscored that transient abnormalities in routine blood tests (glutamic oxaloacetic transaminase, lactate dehydrogenase, and creatine phosphokinase) were present soon after birth, despite his asymptomatic clinical course. Early detection and aggressive treatment could be effective in such a form of glutaric aciduria type II.     

Heterogeneity of defects in mitochondrial acetoacetyl-CoA thiolase biosynthesis in fibroblasts from four patients with 3-ketothiolase deficiency

Deficient mitochondrial acetoacetyl-CoA thiolase in fibroblasts from four patients with 3-ketothiolase deficiency was studied using immunochemical methods. We also examined fibroblasts from two heterozygotes, the mother and the brother of the case 1 patient, identified on the basis of the results of the enzyme activity measurements, using 2-methylacetoacetyl-CoA as substrate. The results were as follows: 1) in fibroblasts from all four patients, the thiolase activity using acetoacetyl-CoA was not activated by K+, although that of the controls and the heterozygotes was activated about 2-fold. 2) by immunoblot analyses, mitochondrial acetoacetyl-CoA thiolase was not detectable in fibroblasts from cases 2 and 3, although a very faint band was seen in tissues from cases 1 and 4. However, the band of mitochondrial 3-ketoacyl-CoA thiolase was clearly detected in all patients to the same extent as in the controls. 3) mitochondrial acetoacetyl-CoA thiolase was observed after pulse labeling for 1-h and a 72-h chase period in three cell lines (cases 1, 2, and 4), but was fainter compared to the controls. In another cell line (case 3), a fluorographic band at the same position was detected following a 1-h pulse, but disappeared following a 6-h chase. These results demonstrate heterogeneity in the enzyme defect resulting in a deficiency of mitochondrial acetoacetyl-CoA thiolase in fibroblasts from patients with 3-ketothiolase deficiency.     

Cord blood lymphocyte responses to food antigens for the prediction of allergic disorders.

Proliferative responses of cord blood lymphocytes (CBLs) to food antigens and cord blood IgE concentrations were measured in 37 full term newborn infants for the prediction of allergic disorders. In these 37 infants who were followed up for two years, allergic history of the family was found in four (sensitivity 57.1%) and cord blood IgE concentrations were greater than 0.5 IU/ml in three (sensitivity 42.9%) of seven infants who developed allergic disorders. When CBLs were stimulated twice by ovalbumin or bovine serum albumin, the value of the stimulation index in proliferative responses of CBLs to ovalbumin or bovine serum albumin was greater than 1.5 in six (sensitivity 85.7%) of seven infants who developed allergic disorders. The specificity of the responses of CBLs in the prediction of the development of allergic disorders was 93.3%. The proliferative responses of CBLs to food antigens were useful in the prediction of not only development of allergic disorders but also offending allergens. These observations provide further evidence that sensitisation is occurring in utero. This would appear to be increasingly important in the genesis of early atopic problems. As our follow up is only two years, in utero sensitisation is a prediction for the early development of atopic disease but only longer follow up will show whether this holds good for allergic disorders at any age.     

Restoration of biochemical function of the peroxisome in the temperature-sensitive mild forms of peroxisome biogenesis disorder in humans

We have found that peroxisome assembly is temperature-sensitive (ts) in mild forms of peroxisome biogenesis disorders (PBDs), that is all infantile Refsum disease (IRD) patients and a few neonatal adrenoleukodystrophy patients of several complementation groups. The number of peroxisomes increased daily in incubation at 30 degrees C in the ts cells. Oxidation of very long-chain fatty acids, processing of acyl-CoA oxidase and dihydroxyacetonephosphate acyltransferase activity also improved after 8 days incubation at 30 degrees C in the IRD fibroblasts. These biochemical functions of the peroxisome did not change at 30 degrees C in Zellweger fibroblasts. Number of peroxisomes gradually decreased after 4 days when the temperature shifted from 30 to 37 degrees C in the ts cells. These results indicate that the biochemical functions of peroxisome are also restored by incubation at 30 degrees C in the mild and ts phenotype of PBDs, and the results will aid to predict the severity and the prognosis of affected children.     

Significance of measurement of pre-S2 antigen for the prevention of vertical transmission of hepatitis B virus in infants born to HBsAg carrier mothers

Terazawa S, Kondo N, Orii T. Significance of measurement of pre-S2 antigen for the prevention of vertical transmission of hepatitis B virus in infants born to HBsAg carrier mothers. Acta Pædiatr 1994;83:30–4. Stockholm. ISSN 0803–5253
The significance of pre-S2 antigen (pre-S2 Ag) as a marker of hepatitis B virus (HBV) infection, especially in infants born to HBsAg carrier mothers who are HBeAg-negative or HBeAg-positive, was evaluated. Pre-S2 Ag was measured by enzyme immunoassay. HBsAg carrier mothers who were HBeAg-negative and HBeAb-positive were divided into two groups: group A, mothers whose infants were not infected with HBV ( n = 10) and group B, mothers whose infants were infected with HBV ( n = 13). Absorption rates of pre-S2 Ag in group A and B were 0.09 k 0.04 and 1.36 ± 0.95, respectively. The values for pre-S2 Ag in group B were significantly higher than those in group A. Values for pre-S2 Ag among HBsAg carrier mothers who were HBeAg-positive and HBeAb-negative were also measured by reversc passive hemagglutination. In the same way, HBsAg carrier mothers who were HBeAg-positive and HBeAb-negativc were divided into two groups: group C, mothers whose infants did not become HBsAg carriers ( n = 15) and group D, mothers whose infants became HBsAg carriers (n = 11). The titers of pre-S2 Ag (reverse passive hemagglutination) in group C and D were 2^{5.75 ± 1.68} and 2^10.45±^1.69, respectively. The values for pre-S2 Ag in group D were significantly higher than those in group C. The values for pre-S2 Ag as markers of infectivity became higher with increasing amounts of HBV-DNA. Therefore, our results show that measurement of pre-S2 Ag in HBsAg carrier mothers who are HBeAg or HBeAb-positive is useful in the detection of high-risk groups of vertical transmission of HBV.