Diagnostic significance and source of lactate dehydrogenase and its isoenzymes in cerebrospinal fluid of children with a variety of neurological disorders.

Two hundred and thirty-four cerebrospinal fluid (CSF) specimens from 183 different children were analysed for total lactate dehydrogenase (LD) activity and LD isoenzyme distribution. The LD activities were elevated in the CSF of patients with meningitis, especially with bacterial infections, and with central nervous system (CNS) leukaemia. The CSF LD isoenzyme patterns of both groups generally reflected the number and distribution of lymphocytes and granulocytes in the CSF. Increases in CSF LD levels also occurred in patients with other neurological disorders, such as hydrocephalus, raised intracranial pressure, and epileptic seizures. However, no significant increases in CSF LD activity nor abnormality of the isoenzyme distribution were noted in children who had had a non-specific febrile convulsion.

Diagnosis of Tay-Sachs disease using [3H]N-acetylneuraminic acid labelled GM2 ganglioside as substrate.

GM2 ganglioside labelled with tritium in the N-acetylneuraminic acid moiety was prepared and used to measure beta-hexosaminidase A activity in cultured humans skin fibroblasts extracts. The latter convert this substrate to the correspondingly labelled GM3 ganglioside which can easily be separated from the substrate by thin-layer chromatography. No cleavage of the N-acetylneuraminic acid group was observed under our conditions. Two methods are described for the determination of GM2-beta-hexosaminidase A activity in fibroblasts. The application of these methods to the diagnosis of Tay-Sachs disease is discussed.

A transient variant of serum lactate dehydrogenase isoenzymes.

An unusual variant of the serum lactate dehydrogenase (LDH) isoenzyme pattern is described in an apparently healthy young woman. The abnormal pattern consisted of a single zone of LDH activity, having the same mobility as, but more diffuse than, normal LDH-4. The molecular weight of the abnormal LDH complex is approximately 280 000, but the nature of the additional component remains unknown, as the isoenzyme pattern spontaneously reverted to normal six weeks after it was first noticed.

A micro-radiochemical assay for alpha-1,4-glucosidase and its use in the assessment of type II glycogenosis (Pompe's disease).

An assay for alpha-1,4-glucosidase (acid maltase) activity which is deficient in Pompe's disease is described. The assay can be used to measure the enzyme in cultured skin fibroblasts, cultured amniotic cells and peripheral blood leucocytes. [U-14 C]Maltose is used as the substrate in a total assay volume of 8 microliter. The product, [U-14C]glucose, is separated from the substrate by cellulose thin-layer chromatography. The procedure permits replicate assays from 400 microliter whole blood and from amniotic cells in primary culture. Discrimination of the heterozygous Pompe state appears to be facilitated.

Type 2 Gaucher disease: the collodion baby phenotype revisited.

The association of Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase (EC 3.2.1.45), and congenital ichthyosis was first noted a decade ago. Subsequently, a null allele type 2 Gaucher mouse was generated that also exhibited ichthyotic skin, confirming that the skin disorder and enzyme deficiency were directly related. This paper details the clinical and molecular characterisation of 6 cases of type 2 Gaucher disease presenting with the collodion baby phenotype. The identified mutant glucocerebrosidase alleles include two novel mutations (S196P and R131L) and two rare point mutations (R120W and R257Q), as well as alleles resulting from recombination with the nearby glucocerebrosidase pseudogene. There is significant genotypic heterogeneity in this rare subset of patients with type 2 Gaucher disease. Gaucher disease should be considered in the differential diagnosis of congenital ichthyosis in the newborn period.

Adult-onset exercise intolerance due to phosphorylase b kinase deficiency.

Muscle-specific phosphorylase b kinase deficiency is an unusual form of glycogen storage disorder. The majority of patients are male with an age at diagnosis between 15 to 36 years. Clinical features include exercise intolerance, myalgia and muscle weakness. A forearm ischaemic exercise test is usually normal and histochemical staining for myophosphorylase positive. The demonstration of reduced muscle phosphorylase b kinase activity by biochemical assay confirms the diagnosis. We report a 36 year old male with phosphorylase b kinase deficiency and symptom onset in adult life.

Neonatal screening for cystic fibrosis using immunoreactive trypsinogen and direct gene analysis: four years' experience.

OBJECTIVE: To assess the performance and impact of a two tier neonatal screening programme for cystic fibrosis based on an initial estimation of immunoreactive trypsinogen followed by direct gene analysis. DESIGN: Four year prospective study of two tier screening strategy. First tier: immunoreactive trypsinogen measured in dried blood spot samples from neonates aged 3-5 days. Second tier: direct gene analysis of cystic fibrosis mutations (delta F508, delta I506, G551D, G542X, and R553X) in samples with immunoreactive trypsinogen concentrations in highest 1% and in all neonates with meconium ileus or family history of cystic fibrosis. SETTING: South Australian Neonatal Screening Programme, Adelaide. SUBJECTS: All 88,752 neonates born in South Australia between December 1989 and December 1993. INTERVENTIONS: Neonates with two identifiable mutations were referred directly for clinical assessment and confirmatory sweat test; infants with only one identifiable mutation were recalled for sweat test at age 3-4 weeks. Parents of neonates identified as carriers of cystic fibrosis mutation were counselled and offered genetic testing. MAIN OUTCOME MEASURES: Identification of all children with cystic fibrosis in the screened population. RESULTS: Of 1004 (1.13%) neonates with immunoreactive trypsinogen > or = 99th centile, 912 (90.8%) had no identifiable mutation. 23 neonates were homozygotes or compound heterozygotes; 69 carried one identifiable mutation, of whom six had positive sweat tests. Median age at clinical assessment for the 29 neonates with cystic fibrosis was 3 weeks; six had meconium ileus and two had affected siblings. 63 neonates were identified as carriers of a cystic fibrosis mutation. Extra laboratory costs for measuring immunoreactive trypsinogen and direct gene analysis were $A1.50 per neonate screened. CONCLUSION: This strategy results in early and accurate diagnosis of cystic fibrosis and performs better than screening strategies based on immunoreactive trypsinogen measurement alone.

Neonatal screening strategy for cystic fibrosis using immunoreactive trypsinogen and direct gene analysis.

OBJECTIVE: To assess the effectiveness of a two tier neonatal screening strategy for cystic fibrosis, which combines estimation of immunoreactive trypsinogen followed by direct gene analysis in dried blood spot samples collected at age 5 days. DESIGN: Prospective study of two tier screening strategy. The first tier of testing immunoreactive trypsinogen concentration was measured in dried blood spot samples from neonates aged 4-5 days. In the second tier direct gene analysis to detect cystic fibrosis mutations deltaF508 and deltaI506 was performed in those blood spot samples which produced the highest 1% of immunoreactive trypsinogen values. Direct gene analysis was also performed on blood spot samples from infants with suspected or confirmed meconium ileus, regardless of the immunoreactive trypsinogen value. SETTING: The South Australian Neonatal Screening Programme, operating from the department of chemical pathology at Adelaide Children's Hospital. Subjects--All 12,056 neonates born in South Australia between December 1989 and June 1990. No selection criteria were applied. INTERVENTIONS: All infants found to have two recognised cystic fibrosis mutations on direct gene analysis were referred directly for clinical management, and those with one recognised cystic fibrosis mutation were recalled for a sweat test; their families were given genetic counselling. MAIN OUTCOME MEASURES: Direct or exclusion of cystic fibrosis by sweat testing of neonates identified as being at high risk of cystic fibrosis on screening and of those at minimum risk but whose subsequent clinical history raised suspicion about the disease. RESULTS: Of the 12,056 infants screened, 11,907 (98.8%) were reported as "cystic fibrosis not indicated" on the basis of low immunoreactive trypsinogen values. Of the 148 (1.23%) infants with raised immunoreactive trypsinogen values and one (0.008%) with meconium ileus, 132 (1.09%) were reported as cystic fibrosis not indicated, four (0.033%) were identified as having cystic fibrosis, and 13 (0.108%) were recalled for sweat testing after direct gene analysis for the presence of the deltaF508 and deltaI506 cystic fibrosis mutations. No cases of affected infants are known to have been missed to date. CONCLUSION: The strategy of measurement of immunoreactive trypsinogen followed by direct gene analysis is a highly specific neonatal screen for cystic fibrosis, requiring only 2.8 families to be contacted for every case of cystic fibrosis diagnosed.

Variability of fibroblast lysosomal acid hydrolases with reference to the detection of enzyme deficiencies.

The ranges of variability of eight lysosomal acid hydrolases were examined in 47 cultured human skin fibroblast lines through 5 successive subcultures. No overall trend in activity values was found for any of the enzymes tested between the first and final subcultures. Similarly, no significant differences could be found between the overall ranges of fibroblast enzyme activity between 20 clinically normal children and 27 individuals being investigated for non-specific metabolic diseases. It is concluded that for diagnostic reference purposes, wide ranges of normal enzyme values must be established. Such ranges can be validly drawn from a continuation of both clinical groups irrespective of the passage number (up to 5) of the cells at the time of isolation. It is noted that in certain cell lines derived from Fabry and Gaucher patients the 'residual' activity of the expectedly deficient enzyme could not easily be distinguished from the lowest values observed in normals, whereas, as expected, the activity of cells derived from patients with GM1 and GM2 gangliosidoses was markedly reduced.

A method for enrichment of hybrid somatic cells: complementation studies in certain lysosomal enzymopathies.

An improved method, which combined a number of published techniques, is described for the polyethylene-glycol-induced fusion of mononuclear human skin fibroblasts in the presence of phytohaemagglutinin-P and for the subsequent isolation of polynuclear cells by Ficoll gradient sedimentation. Enriched cultures contain between 60 and 75% multinucleated cells and may be maintained in culture without fetal calf serum for up to 14 days without significant overgrowth by the few contaminating mononuclear parental cells. Complementation appears not to occur between GM1 gangliosidosis and mucopolysaccharidosis, type VI B (Morquio) cell strains; this experimental observation provides support for the earlier hypothesis that the mutations for these conditions are allelic. Earlier observations that complementation does not occur between selected phenotypic variants (viz., neuronopathic forms and those without neurological involvement) of sphingomyelin storage (Niemann-Pick) disease or Gaucher's disease are confirmed.

Microvillar enzymes in amniotic fluid. Considerations for the prenatal diagnosis of cystic fibrosis.

The activity of gamma-glutamyl transpeptidase (GGT) and the proportion of alkaline phosphatase (ALP) activity that remains in cell-free amniotic fluid (AF) after inhibition by amino acids ("residual ALP activity") have been evaluated as possible prenatal diagnostic tests for cystic fibrosis. A total of 1511 AFs were examined. In 1435 "reference" AFs (excluding those from pregnancies with a known fetal abnormality and those with a known one in four risk of cystic fibrosis) at 14-24 weeks' gestation, the mean residual ALP activity in the presence of 2.5 mmol/L L-phenylalanine was 32% (median, 28%) and in 10.0 mmol/L L-homoarginine it was 70% (median, 72%). AFs were arbitrarily considered to be "abnormal" if the residual activity was greater than 50% in L-phenylalanine and less than 50% in L-homoarginine. An abnormal residual ALP activity pattern was found in nine of 27 pregnancies which resulted in a neural tube (or other developmental) defect and in five of the 10 pregnancies with a chromosomal abnormality; a further 23 (1.6%) abnormal patterns occurred in the 1435 reference AFs from pregnancies that were believed to have had a normal outcome. However, of an additional 23 AFs that were sampled at the 25th week of pregnancy or later, 13 had an abnormal residual activity pattern; the outcome at term in each case was normal. The residual activity of ALP in the presence of either inhibitor did not change with increasing gestational age. However, the absolute amount of ALP that was inhibited by L-phenylalanine (the so-called "phe-inhibitable activity") was greatest at 18 weeks. GGT activity decreased with increasing length of gestation. Of the AF samples from 16 pregnancies at one in four risk for cystic fibrosis that were obtained at 18 weeks' gestation, 11 AFs had a normal residual ALP activity pattern, normal GGT and normal phe-inhibitable ALP activity. Of these 11, five have come to term and the infants are not affected by cystic fibrosis. Three of the 16 AFs had an abnormal residual ALP activity pattern, low GGT and low phe-inhibitable ALP activity; these pregnancies were assessed to be affected by cystic fibrosis and termination was chosen in each case. The two remaining AFs had borderline, paradoxical GGT and residual ALP activity, and initially could not be classified clearly into either of the two groups; however, phe-inhibitable ALP activity was low in each. These pregnancies are continuing.

Inhibition of Tamm-Horsfall glycoprotein induction of alkaline phosphatase in cystic fibrosis fibroblasts by medium conditioned by normal cells.

It is confirmed that the level of alkaline phosphatase in fibroblasts derived from cystic fibrosis patients can be induced many-fold by growing the cells in the presence of Tamm-Horsfall glycoprotein. It is further shown that normal fibroblasts produce a "CF corrective factor" which markedly inhibits this phenomenon. These observations support a previous hypothesis on the nature of the metabolic defect in cystic fibrosis.

Improved method for determination of chlorothalonil and related residues in cranberries.

Tetrachloroisophthalonitrile (chlorothalonil) was applied under controlled conditions in 1985 to a cranberry bog for fungus control and for residue studies. Randomly selected samples of cranberries were analyzed for residues of chlorothalonil, its metabolite 4-hydroxy-2,5,6-trichloroisophthalonitrile, hexachlorobenzene, and pentachlorobenzonitrile by extraction, methylation, Florisil column cleanup, and electron capture gas chromatography. Because of interferences in the GC determinative step, previously reported methodology was modified. The total residues found in the test samples were well below the permissible limit for the parent and related compounds.

Considerations in the prenatal assessment of lysosomal enzyme deficiency in eight cases at risk.

Some of the factors which are to be considered in the antenatal diagnostic evaluation of lysosomal enzyme levels in cultured amniotic fluid cells are discussed in the light of eight consecutive cases in which the foetuses had a 1 in 4 chance of being homozygous for a lysosomal storage disease. There were 2 possible cases of GM1 gangliosidosis, 2 of neuropathic Gaucher's disease, 1 of Krabbe's disease and 2 of Pompe's disease. Each infant was predicted to be unaffected; the assessments were confirmed to be correct postnatally--7 by enzyme studies. Using a micro technique, 5 of the assessments were available in 28 days or less following amniocentesis. It is concluded that in certain circumstances skin fibroblasts may be used as valid controls and that the variability of assay results strongly militates against the confident assignment of heterozygous status and may cause difficulties in the identification of the homozygote in cases where 'residual' enzyme activity is high; concomitant family studies assist in the resolution of such problems.

Identification of a cystic fibrosis mutation: deletion of isoleucine506.

The recent isolation of the cystic fibrosis (CF) gene has resulted in the identification of a common mutation (delta F508) that is found on about 70% of CF chromosomes and that comprises a deletion of 3 bp and results in the omission of Phe508 from within a putative ATP-binding domain of the predicted gene product. We describe a CF mutation that involves the deletion of 3 bp encoding Ile506 or Ile507. This is a rare mutation found in less than 1% of CF chromosomes and could be mistaken for delta F508 using the current methods for the molecular diagnosis of CF.

Population frequency of the arylsulphatase A pseudo-deficiency allele.

The enzymatic diagnosis of metachromatic leukodystrophy is complicated by the frequent occurrence of the pseudo-deficiency of arylsulphatase A (ASA) enzyme activity. An A to G nucleotide transition in the first polyadenylation signal of the ASA gene results in the loss of its major mRNA species and a greatly reduced level of enzyme activity. This nucleotide change (nucleotide 1620 of the ASA cDNA) is the cause of ASA pseudo-deficiency and is closely linked to another A to G transition (nucleotide 1049), within the ASA gene, which changes Asn350 to serine but which does not affect ASA activity. The distribution of these 2 nucleotide changes has been investigated in 73 unrelated individuals from the Australian population. The two transitions were found together on 14 (9.6%) out of 146 chromosomes. The transition at nucleotide 1620 was not found alone; however, the other transition was found alone on 7 (4.8%) out of the 146 chromosomes. The carrier frequency of the ASA pseudo-deficiency mutation in Australia is thus estimated to be about 20%.

Importance of the glycosylation and polyadenylation variants in metachromatic leukodystrophy pseudodeficiency phenotype.

Metachromatic leukodystrophy (MLD) is an inborn error of myelin metabolism caused by a deficiency of the lysosomal hydrolase, arylsulfatase A (ASA). About 1% of the normal population have ASA activity levels approximating those of MLD patients. This non-pathogenic reduction in ASA activity is caused by homozygosity for the ASA pseudodeficiency allele (ASA-PD). Although this allele contains two sequence alterations, a polyadenylation defect and an amino acid substitution (N350S), the reduction in ASA activity previously has been attributed to the polyadenylation defect which reduces the amount of ASA mRNA and hence ASA protein by approximately 90%. The identification of MLD patients who are homozygous for the ASA-PD allele has brought about the need to re-evaluate the allele in light of the possible role that it may play in the development and progression of disease. Ribonuclease protection assay analysis of ASA mRNA transcripts and an investigation into the activity and lysosomal localization of protein expressed by an ASA expression construct containing the N350S variant indicated that both the N350S and polyadenylation defects play a role in biochemically defining the ASA-PD phenotype. The combined effect of the reduction in ASA mRNA due to the polyadenylation defect and the lowering of ASA activity and aberrant targeting of the expressed N350S ASA protein to the lysosome is estimated to reduce ASA activity in pseudodeficiency homozygotes to approximately 8% of normal.