Newborn screening for X-linked adrenoleukodystrophy in New York State: diagnostic protocol, surveillance protocol and treatment guidelines.

PURPOSE: To describe a diagnostic protocol, surveillance and treatment guidelines, genetic counseling considerations and long-term follow-up data elements developed in preparation for X-linked adrenoleukodystrophy (X-ALD) newborn screening in New York State. METHODS: A group including the director from each regional NYS inherited metabolic disorder center, personnel from the NYS Newborn Screening Program, and others prepared a follow-up plan for X-ALD NBS. Over the months preceding the start of screening, a series of conference calls took place to develop and refine a complete newborn screening system from initial positive screen results to long-term follow-up. RESULTS: A diagnostic protocol was developed to determine for each newborn with a positive screen whether the final diagnosis is X-ALD, carrier of X-ALD, Zellweger spectrum disorder, acyl CoA oxidase deficiency or D-bifunctional protein deficiency. For asymptomatic males with X-ALD, surveillance protocols were developed for use at the time of diagnosis, during childhood and during adulthood. Considerations for timing of treatment of adrenal and cerebral disease were developed. CONCLUSION: Because New York was the first newborn screening laboratory to include X-ALD on its panel, and symptoms may not develop for years, long-term follow-up is needed to evaluate the presented guidelines.

Decreased rates of methionine synthesis by methylene tetrahydrofolate reductase-deficient fibroblasts and lymphoblasts.

Methionine synthesis from homocysteine was measured in intact human fibroblasts and lymphoblasts using a [14C]formate label. Seven fibroblast lines and two lymphoblast lines derived from patients with 5,10-methylene tetrahydrofolate reductase deficiency had rates of methionine synthesis that were from 4 to 43% of normal. When the patients were divided by clinical status into mildly (two patients), moderately (two patients), and severely (three patients) affected, methionine biosynthesis expressed as a percent of control values was 43 and 33%, 11 and 10%, and 7, 6, and 4%, respectively, in fibroblasts. Similar data for the two lymphoblast lines were 36 and 26% for a mildly and moderately affected patient, respectively. These data are to be contrasted with the measurement of residual enzyme activity in cell extracts which agrees less precisely with the clinical status of the patients. In the presence of normal methionine synthetase activity, the rate of synthesis of methionine from homocysteine is a function of the activity of the enzyme 5,10-methylene tetrahydrofolate reductase, and measurement of the methionine biosynthetic capacity of cells deficient in this enzyme accurately reflects the clinical status of the patient from whom the cells were derived.

Genetic prothrombotic mutations are common in neonates but are not associated with umbilical catheter-associated thrombosis.

OBJECTIVE: To evaluate the prevalence of hereditary prothrombotic mutations, and their effect on the incidence and severity of umbilical arterial or venous catheter (UAC or UVC)-associated thrombosis. STUDY DESIGN: All neonates with a UAC or UVC were studied prospectively for the presence, severity and timing of thrombosis with duplex Doppler ultrasound scan. Genetic testing for factor V Leiden (FVL), prothrombin mutation (PTm) and methylene-tetrahydrofolate reductase (MTHFR) mutations was performed using PCR and restriction fragment length polymorphism assays. RESULT: Umbilical catheter (UC)-associated thrombosis developed in 16/53 (31%) neonates; 23% of UACs and 22% of UVCs were associated with thrombosis. The prevalence of a significant prothrombotic mutation was present in 10/51 (20%) of infants: FVL (8%), MTHFR667 homozygosity (10%), MTHFR1298 homozygosity (2%) and PTm (0%). There was no increase in the risk of UC-associated thrombus in patients carrying these prothrombotic mutations; our study had the power to detect a 2.5-fold increased risk of thrombosis for any of these significant mutations. In addition, MTHFR667 heterozygosity was found in 41% of infants and MTHFR1298 heterozygosity in 52% and also were not associated with increased risk of UC-associated thrombus. The risk of MTHFR double heterozygosity (db het) was 14%, the risk of a significant or db het was 17/51 (33%) and the risk of any mutation was 90%. CONCLUSION: Prothrombotic genetic mutations are common in our Neonatal Intensive Care Unit population but do not appear to increase the risk of UC-associated thrombosis.

Regulation of methionine adenosyltransferase in normal diploid and simian virus 40-transformed human fibroblasts.

Methionine adenosyltransferase activity in normal diploid and simian virus 40 (SV40)-transformed human fibroblasts increased severalfold when cell monolayers were cultured in medium deficient in L-methionine. This increase in methionine adenosyltransferase activity required RNA and protein syntheses and probably represented a derepression of the enzyme's biosynthesis. Furthermore, studies with RNA synthesis inhibitors suggested that the regulation of this enzyme activity in human fibroblasts involved posttranscriptional mechanisms. The inclusion of homocysteine thiolactone, a metabolic precursor of methionine, in the methionine-deficient medium inhibited the derepression in normal human fibroblasts but augmented the derepression in fully transformed fibroblasts. These differences in derepression patterns thus appeared related to altered metabolism of homocysteine and/or methionine in SV40-transformed human fibroblasts and as such may serve as a transformation marker in SV40-transformed cells.

Transfer of tumor cells between cell aggregates as a model for adhesive changes in metastasis.

A model for cell detachment which may influence metastasis in vivo is described involving the transfer of hamster melanoma cells from aggregates of those tumor cells to hamster fibroblast aggregates. The aggregates were made by the spontaneous association of cultured cells. Tumor and fibroblast aggregates were then incubated together but separated by a nylon net that allowed only single cells to pass. Melanoma cells rapidly separated from the tumor aggregates, crossed the net, and attached to the fibroblast aggregates, but fibroblast cells did not. This model of metastasis reflects the postulated role of cell-to-cell adhesion in metastasis and will allow further study of the role of cell attachments in metastasis.

Folate metabolism in cells from fragile X syndrome patients and carriers.

The in vitro folate sensitivity of the fragile site at Xq27 and the claims of a beneficial response of patients given folic acid prompted us to examine the folate metabolism in cells cultured from fragile X syndrome patients and carriers. Using Epstein-Barr virus we established permanent lymphoblastoid lines from 4 fragile X syndrome males and 3 carriers from 7 families. All these lines expressed the fragile site when 0.1 microM 5-fluorodeoxyuridine (FUdR) was added to the cultures 24 hr prior to harvest; thus, the lines seemed suitable for seeking an intrinsic defect. Fragile X syndrome patient and carrier lines and normal control cell lines did not differ in regard to folate requirement for growth, the ability to use homocysteine in place of methionine, the ability to utilize reduced folates as the sole folate source, or methotrexate sensitivity. These results suggest that no intrinsic defect in folate metabolism is present in fragile X syndrome cells.

Antifolate-induced misincorporation of deoxyuridine monophosphate into DNA by cells from patients with the fragile X syndrome.

The fragile site at Xq27 is expressed in vitro under conditions that lead to decreased intracellular thymidine triphosphate concentration, a condition which has also been shown to promote the misincorporation into DNA of deoxyuridine monophosphate (dUMP) in place of thymidine. We tested for increased whole-cell misincorporation of dUMP as a possible molecular mechanism for the expression of the fragile X abnormality. Neither deoxyuridine triphosphatase nor uracil-DNA-glycosylase, the two enzymes that normally prevent the accumulation of dUMP in DNA, was deficient in fragile X syndrome cells. Misincorporation of dUMP occurred in comparably low levels in both normal and fragile X syndrome lymphoblasts. Although these results provide strong evidence against generalized misincorporation of dUMP in fragile X syndrome cells, a substantial real difference present at Xq27 might not be detected in these studies of whole cells containing the diploid chromosome complement.

Vitamin B12 responsive homocystinuria and megaloblastic anemia: heterogeneity in methylcobalamin deficiency.

A male infant with methyl-B12 deficiency (cblE) presented at age 6 weeks with lethargy, staring spells, and vomiting. He later became hypotonic and unresponsive to stimuli and required intubation and ventilation. He had homocystinuria and hypomethioninemia with megaloblastic anemia but normal serum folate and vitamin B12 concentrations. No methylmalonic aciduria was detected. Fibroblasts, cultured from the patient, were unable to grow in medium in which homocysteine replaced methionine and incorporated abnormally small amounts of [14C]-methyl-tetrahydrofolate but normal amounts of [14C]-propionate into protein. Methyl-B12 content of fibroblasts was low, while the adenosyl-B12 content was normal. Methionine synthase activity was decreased when the assay was performed under both optimal and suboptimal reducing conditions, suggesting heterogeneity in the cblE disease. The patient responded dramatically to hydroxocobalamin treatment. Homocystinuria disappeared after 10 days of therapy, and methionine was normalized after 3 weeks. Psychometric testing at age 15 months showed a developmental age of 9 months.

Fragile sites and high-resolution chromosome studies in multiple endocrine neoplasia type 2A.

Affected individuals from four kindreds with multiple endocrine neoplasia type 2A syndrome (MEN-2A), were studied for the possible existence of a specific fragile site that might be associated with the MEN-2A gene. The chromosomes were also examined with high-resolution banding with particular emphasis on those chromosomes (#1, 10, 20, and 22) that have been implicated by previous studies from several laboratories as being associated with this disease. There was no evidence for a unique fragile site or a unique high-resolution banding pattern in subjects with MEN-2A. These findings, in combination with all previous cytogenetic studies, indicate that it is unlikely that current techniques will be useful in developing a simple cytogenetic test for this disease.

Effects of lead, cadmium and mercury on brain adenylate cyclase.

The effects of lead, cadmium and mercury ions on adenylate cyclase activity of rat cerebrum, cerebellum and brain stem were studied in vitro and in vivo. Adenylate cyclase activity in homogenates of cerebellum as well as cerebrum and brain stem was found to be inhibited by micromolar concentrations of these heavy metal ions in vitro. Administration of lead acetate trihydrate (25 mg/kg body wt i.v.) produced an initial increase of adenylate cyclase activity in the cerebellum and brain stem 1 h after injection, followed by a significant decrease of enzyme activity in cerebrum and cerebellum 4 h after the injection. Chronic lead treatment achieved by feeding lead containing diets, which generated blood lead levels of 31.3 +/- 3.8, 68.8 +/- 1.5 and 121.5 +/- 8.6 microgram Pb/100 g blood resp., produced a significant increase of brain lead levels and a 10-30% reduction of adenylate cyclase activity in cerebrum, cerebellum and brain stem. Phosphodiesterase activity was reduced under these conditions in the range of 10-20% in cerebellum and brain stem, but not in cerebrum.

Thymidylate metabolism in fragile X syndrome cells.

The observation that decreased thymidylate supply in vitro induces the expression of the Xq27 chromosome fragile site prompted us to examine cellular thymidylate metabolism. Using a sensitive enzyme assay for deoxyribonucleotide triphosphates, we found that the total cellular thymidine triphosphate pools in cell lines from fragile X patients and carriers do not differ from normal controls under either basal or folate-deficient conditions. This agrees with our earlier observation that the thymidylate synthase enzyme activities in crude cell extracts of five fragile X syndrome lymphoblast lines do not differ from those in normal controls under standard assay conditions. Although a difference in the amount of thymidine triphosphate available at the replication fork for DNA synthesis remains a possibility, our results indicate that a readily demonstrable defect in thymidylate metabolism is not present in fragile X syndrome cells.

High in vivo rates of methionine biosynthesis in transformed human and malignant rat cells auxotrophic for methionine.

Unlike normal cells, malignant rat and two simian virus 40-transformed human cell lines can neither grow nor survive in B12-and folate-supplemented media in which methionine is replaced by homocysteine. Yet three lines of evidence indicate that the malignant and transformed cells synthesize large amounts of methionine endogenously through the reaction catalyzed by 5-methyltetrahydropteroyl-L-glutamate; L-homocysteine S-methyltransferase (EC 2.1.1.13). (1) The activities of this methyltransferase were comparable in extracts of malignant and normal cells. (2) The uptake of radioactive label from [5-14C]methyltetrahydropteroyl-L-glutamic acid (5-Me-H4PteGlu) was at least as great in the malignant cells as in the normals and was nearly totally dependent on the addition of homocysteine, the methyl acceptor; furthermore, 59-84% of the label incorporated by cells was recovered as methionine.

Decreased purine synthesis during amino acid starvation of human lymphoblasts.

Normal human lymphoblasts starved for each of several essential, but not essential, amino acids had decreased DNA and RNA synthesis but no change in free intracellular purine nucleotides. The rates of purine nucleotide synthesis via the de novo and salvage pathways were measured by incorporating [14C]formate and [14C]hypoxanthine labels, respectively, into lymphoblasts starved for an amino acid or treated with a protein synthesis inhibitor. After 3 h of starvation, purine synthesis via the de novo pathway decreased 90% and via the salvage pathway decreased 60%. Cycloheximide and puromycin each reduced de novo synthesis by 96% and salvage synthesis by 72%. The decrease in purine synthesis de novo after removal of the amino acid was of first order kinetics and was fully and rapidly reversed by reconstitution with the amino acid. The synthesis of alpha-N-formylglycinamide ribonucleotide declined 97% after amino acid starvation; the synthesis of purines from 5-aminoimidazole-4-carboxamide riboside decreased 41%. The synthesis of guanylates decreased more than the synthesis of adenylates during amino acid starvation.

Expression of the human argininosuccinate synthetase gene in hamster transferents.

The structural gene for human argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5] was transferred to argininosuccinate synthetase-deficient Chinese hamster cells via metaphase chromosomes isolated from human lymphoblast line MGL8D1, a constitutive overproducer of argininosuccinate synthetase, and from its repressible parent, MGL8B2. Argininosuccinate synthetase expression was selected for in citrulline-containing medium, and the human origin of the argininosuccinate synthetase expressed by seven transferents was identified by isoelectric focusing. Stable transferents expressing MGL8D1 argininosuccinate synthetase fell into two classes: (i) those whose argininosuccinate synthetase activity was reduced to 10-50% by arginine, similar to the repression of argininosuccinate synthetase synthesis observed in normal human lymphoblasts, and (ii) those that constitutively expressed argininosuccinate synthetase when grown in the presence of arginine or citrulline. Two transferents from the MGL8B2 donor constitutively expressed human arginonosuccinate synthetase. Three hamster revertants were isolated that constitutively expressed hamster argininosuccinate synthetase. Transferents and revertants exhibited growth-dependent changes in argininosuccinate synthetase activity, in contrast to the constant synthetase activity levels in donor lymphoblasts during growth. The isolation of stable transferents that constitutively or repressibly express argininosuccinate synthetase makes possible the analysis of regulatory signals influencing expression of the argininosuccinate synthetase gene.

Decreased methylation rates of DNA in SV40-transformed human fibroblasts.

The rates of methylation of total cellular DNA and newly synthesized DNA were measured in four unrelated SV40-transformed human fibroblast lines and in four control parent fibroblast lines. Rates of methylation of total cellular DNA were decreased by a factor of 1.8-2.3 in the transformed cells relative to control cells. Methylation was largely (75%-87%) restricted to newly synthesized DNA in control and transformed fibroblasts, and methylation rates of newly synthesized DNA were diminished in transformed cells by 12- to 19-fold relative to control cells. Growth rates were similar in the normal and transformed cells. The cellular uptake of methionine and conversion to S-adenosylmethionine were similar in the normal and transformed cells, suggesting no major differences between the normal and transformed cells in the cellular transport of methionine, methionine S-adenosyltransferase activity, or the intracellular concentrations of methionine and S-adenosylmethionine. The diminished rates of DNA methylation that we have observed suggest a possible mechanism for altered gene expression and growth control in transformed cells.

Prevention of fetal damage through dietary control of maternal hyperphenylalaninemia.

Maternal phenylketonuria is a new entity in obstetrics. If unrecognized and for this or other reasons untreated, it produces a substantial risk for fetal damage. Our knowledge of the pathophysiology of the fetal complications in maternal PKU is very limited, but the degree of maternal hyperphenylalaninemia seems to be important. The management differs from the other high-risk pregnancies in the need for a special diet beginning before conception. An effective program of dietary therapy designed in collaboration with a PKU clinic will reduce the likelihood of fetal damage and improve pregnancy outcome.

Lysyl-protocollagen hydroxylase deficiency in fibroblasts from siblings with hydroxylysine-deficient collagen.

Cell culture studies were performed on members of a family in which two sisters, ages 9 and 12, have a similar disorder characterized clinically by severe scoliosis, joint laxity and recurrent dislocations, hyperextensible skin, and thin scars. The skin collagen from the sisters was markedly deficient in hydroxylysine, but other amino acids were present in normal amounts. Hydroxylysine in collagen from fascia and bone was reduced to a lesser extent. Since the most likely explanation for the hydroxylysine deficiency was a reduction in enzymatic hydroxylation of lysine residues in protocollagen, we measured the activity of lysyl-protocollagen hydroxylase in crude lysates of cultured skin fibroblasts. Enzyme activities in the two affected children were 14 and 10% of controls, whereas the activity was about 60% of normal in the mother, a pattern most consistent with autosomal recessive inheritance. The mutant enzyme demonstrated the same cofactor requirements as that from normal cells. Deficiency of lysyl-protocollagen hydroxylase is the first inborn error of human collagen metabolism to be defined at the biochemical level.