Philip P. Dembure

A prevalent mutation for galactosemia among black Americans

OBJECTIVE To define the mutation causing galactosemia in patients of black American origin who have no galactose-1-phosphate uridyltransferase (GALT) activity in erythrocytes but good clinical outcome. METHODS We discovered a mutation caused by a C-->T transition at base-pair 1158 of the GALT gene that results in a serine-to-leucine substitution at codon 135 (S135L). We developed a method with which to screen populations for its prevalence. We compared galactose-1-phosphate uridyltransferase among erythrocytes, leukocytes, and transformed lymphoblasts, as well as total body oxidation of D-(13C)-galactose to 13CO2 among three genotypes for GALT (S135L/S135L, Q188R/Q188R, and Normal/Normal). RESULTS We found a 48% prevalence of the S135L mutation among 17 black American patients with classic galactosemia and a 1% prevalence in a population of 50 black Americans without galactosemia. The S135L mutation was not found in 84 white patients with G/G galactosemia nor in 87 white control subjects without galactosemia. We found normal whole body oxidation of D-(13C)-galactose by the patient homozygous for S135L and various degrees of enzyme impairment among different tissues. CONCLUSIONS The S135L mutation in the GALT gene is a prevalent cause of galactosemia among black patients. Because GALT activity varies in different tissues of patients homozygous for S135L, they may have a better clinical outcome than patients who are homozygous for Q188R when both are treated from infancy.

Neural-tube defects are associated with low concentrations of cobalamin (vitamin B12) in amniotic fluid.

While folate supplementation reduces the risk of recurrent neural‐tube defects (NTD), both folate and cobalamin deficiencies may be independent risk‐factors for neural‐tube defects. Folate‐dependence and impaired remethylation of homocysteine are implicated as mechanisms for NTD. There are few references reported for folate, cobalamin, homocysteine and methionine in the fetal compartment. This case‐controlled pilot study of amniotic fluid (AF) samples derived from 16 NTD pregnancies and 64 age‐matched controls quantitates total homocysteine (tHcy), total cysteine (tCys), folate, cobalamin (B12), and methionine. Only decreased AF B12 concentrations were found (150 pg/ml versus 540 pg/ml, P<0·02). Since cobalamin, folate and homocysteine participate in the remethylation of homocysteine, via methyl transfer from 5‐methyltetrahydrofolate to B12, to methionine, we compared ratios of these methionine synthase (EC 2.1.1.13) ‐related intermediates. The ratio of B12/folate for NTD versus controls was 48 (34–90) versus 126 (123–182), P<0·001. The ratio of methionine/(folate×tHcy) was 1·4 (1·2–2·2) versus 2·7 (2·4–3·3), P<0·001. We conclude that AF from pregnancies with NTD have lower B12 concentrations, and that ratios of product to substrate(s) of homocysteine remethylation suggest impaired methionine synthase in the fetal compartment through the early second trimester.

Defective urinary carnitine transport in heterozygotes for primary carnitine deficiency

Purpose: Primary carnitine deficiency is an autosomal recessive disorder caused by defective carnitine transport and manifests as nonketotic hypoglycemia or skeletal or heart myopathy.Methods: To define the mechanisms producing partially reduced plasma carnitine levels in the parents of affected patients, we examined carnitine transport in vivo and in the fibroblasts of a new patient and his heterozygous parents.Results: Kinetic analysis of carnitine transport in fibroblasts revealed an absence of saturable carnitine transport in the probands cells and a partially impaired carnitine transport in fibroblasts from both parents, whose cells retained normal Km values toward carnitine (6–9 μM) but reduced Vmax. At steady state, normal fibroblasts accumulated carnitine to a concentration that was up to 80 times the extracellular value (0.5 μM). By contrast, cells from the proband had minimal carnitine accumulation, and cells from both parents had intermediate values of carnitine accumulation. Plasma carnitine levels were slightly below normal in both heterozygous, yet clinically normal, parents and in the paternal grandfather and the maternal grandmother. To define the mechanism producing partially decreased carnitine levels, we studied urinary carnitine losses in heterozygous parents compared with controls. Urinary losses increased linearly (P < 0.05) with plasma carnitine levels in normal controls. When urinary carnitine losses were normalized to plasma carnitine levels, a significant difference was observed between controls and heterozygous individuals (P < 0.01).Conclusions: These results indicate that fibroblasts from heterozygotes for primary carnitine deficiency have a decreased capacity to accumulate carnitine and that heterozygotes have increased urinary losses, which may contribute to their reduced plasma carnitine levels.

Phospholipid-Sensitive Ca2+-Dependent Protein Kinase Preferentially Phosphorylates Serine-115 of Bovine Myelin Basic Protein

Abstract: Phospholipid‐sensitive Ca2+‐dependent protein kinase (PL‐Ca‐PK) and cyclic AMP‐dependent protein kinase (A‐PK) both preferentially phosphorylated serine residues of bovine myelin basic protein (MBP). Tryptic peptide maps of MBP phosphorylated by PL‐Ca‐PK or A‐PK, however, revealed different phosphopeptides, suggesting a difference in the intramolecular substrate specificity for the two enzymes. Serine‐115 of MBP, in the sequence (‐Arg‐Phe‐Ser(115)‐Trp‐), was found to be a preferred and probably major phosphorylation site for PL‐Ca‐PK. Because serine‐115 of bovine MBP corresponds to serine‐113 of rabbit MBP, an in vivo phosphorylation site reported by Martenson et al. (1983), and PL‐Ca‐PK is present at a very high level in brain and myelin, it is suggested that the enzyme may be responsible for the in vivo phosphorylation of this and other sites in MBP.

A molecular approach to galactosemia

Classical galactosemia (G/G) is caused by the lack of galactose-1-phosphate uridyltransferase (GALT) activity. A more common clinical variant, Duarte/Classical (D/G) produces partial enzymatic impairment. Although neonatal death due to G/G galactosemia has been largely eliminated by populationbased screening and intervention, long-term outcome in some is associated with impaired growth, ovarian failure, dyspraxic speech and neurologic deficits. At least 32 variants in the nucleotide sequence of the GALT gene have been identified and 9 have transferred impaired GALT activity to transformed cells in transfection experiments. We here define the prevalence and biochemical phenotype of two mutations. An A to G transition in exon 6 of the GALT gene converts a predicted glutamine at codon 188 to an arginine (Q188R), and introduces a new HpaII cut site into the gene which enables population screening by polymerase chain reaction. An A to G transition in exon 10 in the GALT gene produces a codon change converting an asparagine to aspartic acid at codon 314 (N314D) and adds an AVA II cut site. We screened a large population for the Q188R and N314D sequence changes to investigate the prevalence of Q188R in G/G galactosemia, the effect of homozygosity for Q188R on outcome, and the prevalence and biochemical phenotype of the N314D sequence change. We found that the Q188R mutation has a prevalence of 62% in a predominately Caucasian population of 107 patients with G/G galactosemia. homozygosity for Q188R was associated with a poor clinical outcome in a subgroup of these patients. The N314D mutation is associated with the Duarte biochemical phenotype with extraordinary concordance.

Phenylalanine Alters the Mean Power Frequency of Electroencephalograms and Plasma L-DOPA in Treated Patients with Phenylketonuria

ABSTRACT: Phenylketonuria is a human model for the study of the effects of phenylalanine on brain function. We found previously a correlation between high blood phenylalanine, prolonged performance times on neuropsychological tests of higher integrative function, and decreased urinary dopamine in 10 patients. In this protocol we examine changes in triplicate of plasma dihydroxyphenylalanine (L-DOPA) and the mean power frequency of the electroencephalogram in eight additional older patients with phenylketonuria using longer intervals in a blinded, cross-over design. Mean power frequency was obtained by Fourier transform of the power spectrum from traditional eight channel electroencephalograms. Plasma L-DOPA was quantitated by radioenzymatic methods. In all patients statistically significant decreases were found in the mean power frequency of the electroencephalogram and in plasma L-DOPA when plasma L-phenylalanine increased. These findings were reversible and correlated in the reverse direction when plasma L-phenylalanine was reduced. Thus changes in the mean power frequency of electroencephalograms and circulating L-DOPA offer sensitive parameters of human brain function in vivo. These findings indicate reversible effects of elevated plasma phenylalanine on electrical function of the brain which may be mediated in part through inhibition of catecholamine synthesis.

The Duarte allele impairs biostability of galactose‐1‐phosphate uridyltransferase in human lymphoblasts

The Duarte allele (D) is a missense mutation (N314D) that produces a characteristic isoform and partial impairment of galactose‐1‐phosphate uridyltransferase (GALT) in human erythrocytes, fibroblasts, and transformed lymphoblasts. The position of this amino acid is distant, however, from presumptive catalytic site(s) as deduced from a three‐dimensional model of crystallized Escherichia coli galT protein. To evaluate the mechanism(s) involved in the partial impairment of enzymatic activity, we compared the activity, abundance, biological stability, and mRNA of GALT in human lymphoblastoid cell lines cultured from individuals homozygous for wild‐type (WT/WT) and Duarte alleles (N314D/N314D). No other nucleotide differences were present in their GALT genes. The apparent Vmax was reduced in N314D/N314D cells to 31 ± 3.6 compared to WT/WT of 54 ± 6.5 nmole UDP‐galactose formed/g cell protein/hour. Both genotypes had similar apparent KMs for UDP‐glucose of 0.142 ± 0.057 mM and 0.133 ± 0.056 mM. This reduced Vmax was associated with a reduced abundance of the 86kD GALT dimer as determined by Western blots and densitometry. Using RNase protection assays, this reduced GALT protein in the N314D/N314D cell lines was not associated with reduced abundance of GALT mRNA. Using cycloheximide (3‐[2‐(3,5‐Dimethyl‐2‐oxocyclohexyl)‐2‐hydroxyethyl]glutarimide) inhibition of de novo protein synthesis, GALT enzyme activity, and its dimeric protein had a biological T1/2 of ˜24 hours in N314D/N314D cell lines as compared to 50 hours for WT/WT lymphoblasts. Upon exposure to 50°C for 15 minutes, N314D/N314D lymphoblasts retained 45% of GALT activity, whereas controls retained 77% activity. Reduced activity and thermal sensitivity caused by the N314D mutation reverted to control values when a lysine was substituted for a glutamic acid at amino acid 203 in cis (E203K). In summary, N314D/N314D lymphoblasts have reduced GALT enzyme capacity, dimeric protein abundance, biological, and thermal stability. We conclude that the substitution of aspartate for asparagine at amino acid 314 in the human GALT protein reduces the biostability of the active enzyme in human lymphoblasts. Hum Mutat 11:28–38, 1998.

The Treatment of Isovaleric Acidemia with Glycine Supplement

ABSTRACT: Although dietary leucine restriction and supplemental glycine are used to treat patients with isovaleric acidemia [deficient isovaleryl-CoA-dehydrogenase (E.C. 1.3.99.10)], little quantitative information is available regarding their optimum relationship. Herein we compare different glycine supplements and quantitate isovalerylgly cine produced in two patients with clinically different forms of isovaleric acidemia during restricted leucine intake and during oral leucine loading. We found that under stable conditions of leucine restriction, 150 mg glycine/kg/day is an optimum glycine supplement and that glycine supplements of more than 250 mg/kg/day may result in reduced isovalerylglycine production; that when isovaleric acid accumulation is increased, glycine supplements to 600 mg/kg/day will increase isovalerylglycine production; and that the phenotype of isovaleric acidemia is related not only to the extent of impaired isovaleryl-CoA dehydrogenase, but also the ability to detoxify accumulated isovaleryl CoA to isovalerylglycine.

Ascorbate regulation of collagen biosynthesis in Ehlers-Danlos syndrome, type VI

We studied two unrelated individuals with Ehlers-Danlos syndrome type VI, which is characterized by congenital hypotonia, lax joints, severe kyphoscoliosis, friable skin, and hemorrhagic hypotrophic scars. The diagnosis was confirmed by decreased hydroxylysine residues in dermal collagen and decreased collagen lysyl hydroxylase activities in their cultured skin fibroblasts. Despite the diminished hydroxylysine residues in dermal collagen from the probands, we found no differences in hydroxylysyl residues of collagen synthesized by fibroblasts in culture. When patient 1 was given oral sodium ascorbate (5 g/d) for 3 weeks, ascorbate concentrations increased two-fold in plasma and 300-fold in urine. Urinary excretion of hydroxylysine and hydroxyproline increased during ascorbate administration. After a 1-year interval, bleeding time, wound healing, and muscle strength improved. Ascorbate supplementation (50 micrograms/mL) to confluent fibroblasts cultured from the two patients and controls increased hydroxyprolyl and hydroxylysyl residues of fibroblasts four to seven and three to four-fold respectively. Total protein associated with the cell layer increased 14% to 32% without concomitant change in cellular DNA. Total soluble collagenous material recovered from culture media increased 61% to 103% with ascorbate supplementation. These studies demonstrate that ascorbate improves the clinical status of patients with impaired collagen lysyl hydroxylase activity by enhancing lysyl and prolyl hydroxylation and total collagen production.

Cystathionine-β-synthase deficiency: Detection of heterozygotes by the ratios of homocystein to cysteine and folate

Elevated total plasma homocysteine (tHcy) is recognized as an independent risk factor for occlusive vascular disease. However, it is not known how much of the observed hyperhomocysteinemia in patients with vascular disease is due to heterozygosity for cystathionine-beta-synthase (CbetaS) deficiency, because a clinically useful screening method is unavailable. To determine this, parents of children who are homozygous for CbetaS deficiency (affected with homocystinuria) and a control population were compared for tHcy, total plasma cysteine (tCys), plasma folate, and plasma vitamin B12. The group of obligate heterozygotes had increased tHcy (P < or = .01), decreased tCys (P < or = .01), and decreased plasma folate (P < or = .01). The calculated ratios of tHcy/tCys (P = .01) and tHcy/plasma folate (P = .003) were the best metabolic discriminants for genotype. These ratios are likely to prove useful in heterozygote screening for CPS deficiency and in the development of rational treatment strategies for patients with increased tHcy.