Coincidence of two novel arylsulfatase A alleles and mutation 459+1G>A within a family with metachromatic leukodystrophy: molecular basis of phenotypic heterogeneity.

In a family with three siblings, one developed classical late infantile metachromatic leukodystrophy (MLD), fatal at age 5 years, with deficient arylsulfatase A (ARSA) activity and increased galactosylsulfatide (GS) excretion. The two other siblings, apparently healthy at 12(1/2) and 15 years, respectively, and their father, apparently healthy as well, presented ARSA and GS values within the range of MLD patients. Mutation screening and sequence analysis disclosed the involvement of three different ARSA mutations being the molecular basis of intrafamilial phenotypic heterogeneity. The late infantile patient inherited from his mother the frequent 0-type mutation 459+1G>A, and from his father a novel, single basepair microdeletion of guanine at nucleotide 7 in exon 1 (7delG). The two clinically unaffected siblings carried the maternal mutation 459+1G>A and, on their paternal allele, a novel cytosine to thymidine transition at nucleotide 2435 in exon 8, resulting in substitution of alanine 464 by valine (A464V). The fathers genotype thus was 7delG/A464V. Mutation A464V was not found in 18 unrelated MLD patients and 50 controls. A464V, although clearly modifying ARSA and GS levels, apparently bears little significance for clinical manifestation of MLD, mimicking the frequent ARSA pseudodeficiency allele. Our results demonstrate that in certain genetic conditions MLD-like ARSA and GS values need not be paralleled by clinical disease, a finding with serious diagnostic and prognostic implications. Moreover, further ARSA alleles functionally similar to A464V might exist which, together with 0-type mutations, may cause pathological ARSA and GS levels, but not clinical outbreak of the disease.

A new polymorphism of arylsulfatase A within the coding region.

A 10-year-old boy with juvenile metachromatic leukodystrophy (MLD) presented with the 459 + 1G-->A arylsulfatase A (ASA) mutation on one allele. To detect his complete genotype, the other ASA allele was sequenced and a T-to-C transition at nucleotide 376 in exon 2 was identified. This missense mutation results in a substitution of leucine 76 by proline. Of 20 MLD unrelated controls, 18 carried the L/P76 mutation either in the homozygous (n = 6) or heterozygous (n = 12) state. The presence or absence of L/P76 did not influence leukocyte ASA activity or urinary sulfatide excretion. Apparently, the substitution of leucine 76 by proline is a common ASA polymorphism, neither being related to MLD nor creating ASA pseudodeficiency. However, because of its frequency and location, L/P76 may be of particular importance in genetic studies requiring the differentiation of the ASA alleles within a kindred. Further studies are directed to the as yet unresolved genotype of the index case with juvenile MLD.

Human rhinovirus 2A proteinase mutant and its second-site revertants.

The 2A proteinases of human rhinoviruses are cysteine proteinases with marked similarities to serine proteinases. In the absence of a three-dimensional structure, we developed a genetical screening system for proteolytic activity and identified Phe-130 as a key residue. The mutation Phe-130-->Tyr almost completely inhibited enzyme activity at 37 degrees C; activity was, however, partially restored by the following exchanges: Ser-27-->Pro, His-135-->Arg or His-137-->Arg. To investigate this phenotypic reversion, 2A proteinases with the mutations Phe-130-->Tyr, Phe-130-->Tyr/His-135-->Arg, Phe-130-->Tyr/His-137-->Arg, His-135-->Arg or His-137-->Arg were expressed in Escherichia coli and purified. None of these mutations affected the affinity of the enzyme for a peptide substrate. However, the temperature-dependence of enzyme activity, as assayed by cleavage of a peptide substrate and by monitoring the toxicity of the proteinases towards the E. coli strain BL21(DE3), and the structural stability, as monitored by 8-anilino-I-naphthalenesulphonic acid fluorescence and CD spectrometry, were affected. The thermal transition temperatures for both the activity and the stability of the Phe-130-->Tyr 2A proteinase were reduced by about 17 degrees C compared with the wild-type enzyme. The presence of the additional mutations His-135-->Arg or His-137-->Arg in the Phe-130-->Tyr mutant increased temperature stability by 3 degrees C and 6 degrees C respectively. Thus essential interactions exist within the C-terminal domain of human rhinoviral 2A proteinases which contribute to the overall stability and integrity of the enzyme.