Lynn D. Fleisher

Cystathionine synthase deficiency: Heterozygote detection using cultured skin fibroblasta

Summary An assay for cystathionine synthase in cultured fibroblasts, of high sensitivity and not rate-limited for substrate, is described. Enzymatic activity was found to be highest in controls (mean ± SEM = 20.97 ± 1.81 nmole cystathionine/mg protein/hour), intermediate in obligate heterozygotes for synthase deficiency (4.40 ± 0.92), and lowest in patients (0.77 ± 0.42), with no overlap between controls and heterozygotes. One clinically and biochemically atypical patient had a synthase activity at the low end of the heterozygote range. Thus, this method is effective for the detection of heterozygotes for cystathionine synthase deficiency.

Cystathionine |[beta]|-Synthase Deficiency: Differences in Thermostability between Normal and Abnormal Enzyme from Cultured Human Cells

Summary: The thermostability of cystathionine β-synthase and the effect of pyridoxal phosphate (PLP) and other B6 vitamers on this thermostability were studied in extracts of cultured skin fibro-blasts from normal subjects, from heterozygotes for synthase deficiency, and from patients with homocystinuria due to synthase deficiency.Incubation of crude extracts of normal fibroblasts at 55° (preincubation) for short periods prior to assay consistently resulted in an increase of cystathionine synthase activity (activation) that reached a maximum at 2 min (Fig. 1). Further preincubation resulted in inactivation. Addition of 0.4 mM PLP to the preincubation mixture resulted in greater activation that reached a maximum at 3–5 min. The subsequent inactivation phase proceeded at a slower rate than had occurred in the absence of PLP, resulting in a doubling of the half-life of thermal inactivation at 55°. PLP was the only one of the B6 vitamers to have a protective effect.No significant activation of synthase was observed when extracts of fibroblasts from synthase-deficient patients were preincubated at 55°. Cells from eight patients were studied: extracts from those of four patients demonstrated activity too low to permit measurement of thermostability; extracts of cells from two patients were less thermostable than those from control subjects and showed a protective effect of PLP; extracts of cells from two other patients (sibs) showed very high heat stability in the absence of PLP, but significant inactivation of synthase in the presence of the coenzye (Fig. 2, A and A).Three patterns of response to heating were observed with extracts of fibroblasts from seven obligate hétérozygotes (Fig. 3): 1) no activation; 2) activation with and without prior addition of PLP to the preincubation mixture; and 3) activation only in the presence of PLP. The half-life of inactivation at 55° of the extracts from heterozygotes was similar to that seen in extracts from control subjects.Synthase from cultured long term lymphoid cells also showed activation followed by inactivation when preincubated at 55°, as well as protection by PLP.Speculation: Heat-induced activation of cystathionine synthase is a physical characteristic of the normal enzyme molecule which is altered in enzyme from patients and from some heterozygotes for synthase deficiency. Although in vivo vitamin B6 therapy restores the ability of hepatic synthase from patients to be activated, this restoration appears not to be the result of a direct stabilizing effect of the coenzyme PLP on the apoenzyme molecule. Furthermore, since PLP does not consistently protect enzyme from patients against heat inactivation in vitro, the increase of hepatic synthase activity observed in some patients receiving megavitamin therapy may not be mediated by a protective or stabilizing effect of the coenzyme.

Carnosinase deficiency: a new variant with high residual activity.

Summary: Plasma carnosinase deficiency was discovered in a 12-yr-old male with profound mental retardation, severe athetoid spastic quadriparesis, optic atrophy, sensory peripheral neuropathy, and suprabulbar signs. Amino acid analysis revealed persistent carno-sinuria but no detectable carnosinemia. After ingestion of L-carnosine (100 mg/kg), the patient had carnosine in his plasma and excreted 28% of the administered load as carnosine (an agematched control excreted 1.3% as carnosine). Urinary 1-methyl-histidine was measurable in the patient and increased greatly during a high anserine diet. Plasma carnosinase activity in the patient was 0.28 μmoles per ml plasma per hr (control mean, 2.00; range, 1.10-2.85), his parents had activity of 1.36 and 1.30, and 2 sibs had activities of 1.10 and 1.86. Carnosinase activity in liver from the patient was 43% of control liver. We have demonstrated that carnosinase activity is present in human nerve and that sural nerve from the patient had activity that was 46% of control nerve. Histopathologic examination of the patients nerve showed axonal degeneration. Histidine levels in the patients liver and nerve were normal, and neither β-alanine nor carnosine was detectable. The unusually high residual carnosinase activity in plasma and tissues from this patient may explain his apparent ability to metabolize anserine and would suggest that this represents a new variant form of carnosinase deficiency.Speculation: Carnosinuria due to plasma carnosinase deficiency may be merely associated with the striking neurologic findings that have been reported rather than causally related.

Homocystinuria: investigations of cystathionine synthase in cultured fetal cells and the prenatal determination of genetic status.

CYSTATHIONINE SYNTHASE DEFICIENCY (Fig. 1), which is inherited in an autosomal recessive manner , results in the clinical syndrome of homocystinuria. The full clinical picture is distinct 1 and may include dislocation of the optic lenses, progressive skeletal changes, severe t h r o m b o e m b o l i c disease , and, of ten, var iable degrees of mental retardation. Synthase activity, and its absence in affected individuals, has been demonstrated in liver and brain2,3,7; more recently it has been found in cultured skin fibroblasts 4, 6 and phytohemagglutininstimulated lymphocytes, s Heterozygotes can now be distinguished from patients and from normal individuals by determinat ion of synthase activity in cultured skin fibroblasts. 6 The utilization of cultured skin fibroblasts in the diagnosis of numerous enzymatic disorders often has been followed by attempts a t spec i f ic prenatal diagnosis by assay of cultured amniotic fluid cells. However, in order to establish proper control data, it is essential to determine optimal assay conditions for cultured amniotic

Fetal tissue amino acid concentrations in argininosuccinic aciduria and in “maternal homocystinuria”

Free amino acid concentrations have been measured in the tissues of a fetus at risk for argininosuccinic aciduria and of an obligate heterozygous fetus in a mother homozygous for homocystinuria. Argininosuccinic acid was detected in all tissues studied of the homozygous affected fetus from the heterozygous mother. Abnormal concentrations of methionine and cystathionine were observed in the tissues of the fetus who was an obligate heterozygote for homocystinuria. These abnormal free amino acid concentrations occur early in fetal development and may be related to later brain dysfunction.

ARGININOSUCCINIC ACIDURIA: PRENATAL DIAGNOSIS AND STUDIES OF AN AFFECTED FETUS

Argininoiuccinic acid (ASA) lyase deficiency, the basic defect in ASAuria, can be studied in cultured skin fibroblasts and cultured amniotic fluid (AF) cells. We diagnosed a fetus affected with ASAuria, after studying the obligate heterozygote parents and an affected child. The affected child had been thought to excrete cystathionine, which we showed to be an ASA anhydride. An increased ASA cone was found in AF from amniocenteses at 14, 15 and 17 weeks (fetus at risk = 0.3 μmoles/ml, norm i=0). ASA lyase activity in cultured AF cells from the fetus at risk, as measured by the ratio of 14C-cltrulline/3H leucine uptake into TCA-precipitable protein, was 0.4% of that in normal AF cells. The pregnancy was terminated, and the in utero diagnosis was confirmed by analysis of fetal liver for ASA lyase (1.8% of normal). It was demonstrated also that all 8 fetal tissues studied. Including brain, had significant accumulations of ASA (normal=0). These findings: demonstrate the possible pitfalls in the diagnosis of ASAuria by urinary amino acid analysis; confirm earlier suggestions that ASAuria may be diagnosed by determining ASA levels in amniotic fluid; cast doubt upon the value of “early” neonatal dietary therapy with arginine, as it appears that the enzymatic defect is already producing an accumulation of unmetabolized substrate in fetal tissues by the beginning of the second trimester; and provide evidence that the urea cycle is active prenatally.

714 EXPERIENCE IN THE PRENATAL DIAGNOSIS OF ARGININO-SUCCINIC ACID (ASA) URIA AND CITRULLINEMIA

Citrullinemia and ASA uria, which result from deficient activities of ASA synthetase and ASA lysase, respectively, are diagnosable in utero. Several laboratories have experienced difficulties with direct enzyme assay due to low activities of these enzymes in crude extracts of cultured amniotic fluid (AF) cells. Monitoring 4 at-risk pregnancies, with a microassay based on the incorporation of 14[C] citrulline and 3[H] leucine into protein in situ, has shown us that it is crucial for the proper control cell types to be used and grown in parallel with the at-risk cells. For example, control AF fibroblasts have a mean activity of 0.67 (14C/3H incorporated), whereas control AF epitheliod cells have a mean of only 0.09. The importance of this distinction was made clear when we recently monitored a pregnancy at-risk for citrullinemia. The first cells to grow out were epitheliod and had activity of 0.14. The culture later became fibroblastic and the activity rose to 0.7. Only when compared to their proper controls did both results indicate a normal fetus. We also have found that control and at-risk cells must be assayed at identical stages of confluence, since increasing activity is seen with increasing confluence. A 10-fold increase in the number of cells planted raised the activity in 2 of our control lines by 3 to 5-fold. Our experience thus indicates the critical need for precise control of tissue culture variables, and the advantages of the microassay system, for prenatal diagnosis of these disorders.

HOMOCYST INURIA DUE TO CYSTATHIONINE SYNTHASE (CS) DEFICIENCY: INVESTIGATIONS IN CULTURED LONG-TERM LYMPHOCYTES, FETAL SKIN FIBROBLASTS AND AMNIOTIC FLUID CELLS

Systematic delineation of optimal assay conditions for CS in adult skin fibroblasts (fib.) enabled us to differentiate obligate heterozygotes (het.) from affected individuals and controls (BBRC 55:38, 1973). We have now extended our studies to long-term lymphoid lines (LTL) and cultured fib. of fetal origin. We have detected CS activity in LTL from 12 normal donors (mean ± SEM=9.49 ± 0.98 nmoles/mg prot./h), its deficiency in a line from an affected individual (0.88), and intermediate activity in 3 obligate het. lines (3.21 ± 0.37), with no overlap. Activity in skin fib. from 5 control fetuses was 32.9 ± 5.06 and not different from control amniotic fluid (AF) cell cultures (40.7 ± 4.58) (.25>p>.01); both differed from adult skin fib. (21.0 ± 1.71) (p < .001). Activity in AF cells from an obligate het. mother was 73.8; prenatal diagnosis of a normal fetus was confirmed after birth by assay of skin fib. Thus: (1) LTL, which proliferate vigorously in apparently permanent cultures, are ideal for detection and study of the enzymatic defect in homozygotes and het. for CS deficiency. (2) CS values from adult skin fib. cannot be used as controls for AF cells. (3) Requirements for prenatal diagnosis of CS deficiency have now been met. (4) CS activity in het. LTL, as in liver and skin fib., was less than 50% of normal.