Pseudodeficiency Allele Research Articles

Arylsulfatase a psedodeficiency: A common polymorphism which is associated with a unique haplotype

The allele for pseudodeficiency (PD) of the lysosomal enzyme arylsulfatase A (ARSA) is a common polymorphism in all populations. The PD allele frequency in different Israeli ethnic groups was found to range from 9.2-22.7%. The PD allele includes two different mutations PD(1) and PD(2) in an approximately 1 Kb interval. In this study we confirmed that while PD(1) may be found alone as a polymorphism, PD(2) is always associated with the PD allele (660 alleles screened). Analysis of three ARSA intragenic polymorphisms showed a complete linkage disequilibrium between the PD allele and an haplotype defined by the three polymorphic restriction sites. The results suggest that the origin of the PD polymorphism may be a common founder, or recurrent mutations which are occurring in a unique haplotype.

Molecular genetics of metachromatic leukodystrophy.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulphatase A. The disease is characterized by a progressive demyelination that causes a variety of neurological symptoms. Patients die within a few years after the age of onset. Clinically the disease is heterogeneous and according to the age of onset three different forms can be distinguished. The gene of arylsulphatase A has been cloned and several mutations causing metachromatic leukodystrophy have been characterized. The distribution of these alleles among patients with different clinical forms of the disease has revealed a genotype-phenotype correlation. A major determinant of the clinical phenotype is the residual enzyme activity that it associated with a particular genotype. Homozygosity for alleles that do not allow the synthesis of arylsulphatase A polypeptides causes the most severe form of disease, whereas homozygosity for alleles that encode arylsulphatase A with low residual enzyme activity is found in the mild late-onset forms of disease. A substantial arylsulphatase A deficiency can also be found in healthy individuals at high frequency. This phenomenon has been termed pseudodeficiency. It is often difficult to distinguish whether an arylsulphatase A deficiency is due to metachromatic leukodystrophy or harmless pseudodeficiency. The characterization of the mutations causing pseudodeficiency has allowed the detection of the pseudodeficiency allele in the DNA of probands and has thus improved the diagnosis and genetic counselling for metachromatic leukodystrophy.

Searching for mutations in the arylsulphatase A gene.

Metachromatic leukodystrophy (MLD, McKusick 250100), a lysosomal storage disorder, is caused by deficiency of arylsulphatase A (ASA) (EC 3.1.3.6) activity. The pseudodeficiency (PD) allele, which causes diminished ASA activity in clinically healthy individuals, is characterized by two A→G transitions, of which only the one destroying the polyadenylation signal is the cause of the lowered enzyme activity (Gieselmann et al 1989). Discrimination of MLD and pseudodeficiency based on ASA activity determination is difficult and unreliable. This is a serious problem in carrier detection and prenatal diagnosis in MLD families who also carry a PD allele

Metachromatic leukodystrophy: a nonsense mutation (Q486X) in the arylsulphatase A (ARSA) gene

Metachromatic Leukodystrophy (MLD) is an autosomal recessive lysosomal storage disorder characterised by defective catabolism of myelin and the accumulation of cerebroside sulphate in neural tissue. The disorder is caused by a deficiency of the lysosomal hydrolase, arylsulphatase A (ARSA). MLD patients present with a broad spectrum of clinical phenotypes, the disorder has therefore been divided into three broad subtypes based on the age of onset of clinical symptoms. Late-infantile MLD is typically diagnosed before the age of 2 years, juvenile between ages 3 and 16 and adult MLD which is diagnosed after puberty (1). Biochemical diagnosis of MLD is complicated by the existence of an ARSA pseudodeficiency allele, which causes the loss of the major mRNA species (2.1 kb) of ARSA and a concomitant 90% reduction in the level of ARSA activity. Approximately 1% of the normal population is homozygous for this allele with no apparent clinical sequelae (2). The 507 amino acid ARSA polypeptide is encoded by a compact 3.2 kb gene containing 8 exons (3). Each of the ARSA exons and intron boundaries were PCR amplified and subjected to single-stranded conformation polymorphism analysis (SSCP) to detect sequence alterations (4). Exons exhibiting alterations in electrophoretic mobility on SSCP gels were sequenced using a modified linear based PCR sequencing protocol (5). SSCP analysis of the ARSA gene from a late-infantile MLD patient revealed a shift in the mobility of a band derived from exon v m (Figure 1). Sequence analysis of exon 8 revealed a heterozygous C to T transition at nucleotide number 1458 of the ARSA cDNA resulting in the alteration of a glutamine codon (CAG) at position 486 to a stop codon (TAG), (Q486X). No other sequence changes have yet been observed in this patient. Allele specific oligonucleotides were designed to detect the normal (5'-CGCCCTGCAGATCTGCTG-3') and mutant (5'-CGCCCTGTAGATCTGCTG-3') sequences Of Q486X (Figure 2) on Southern blotted filters containing exon 8 PCR products. After hybridisation filters were washed twice in 4xSSC for 10 minutes and once at 60°C in 2 xSSC, 0.1 % SDS (4). The patient's mother was found to be heterozygous for Q ggX (her father was unavailable for testing), however the mutation was not found in 27 other MLD patients, 21 ARSA pseudodeficient or 42 normal individuals.

Frequency of the arylsulphatase A pseudodeficiency allele in the Spanish population.

Pseudodeficiency in arylsulphatase A (ASA) is a relatively frequent condition in healthy individuals. It produces a reduction in enzyme activity similar to that found in metachromatic leukodystrophy (MLD). Unambiguous discrimination between the two conditions cannot be achieved through conventional enzyme activity assays. A PCR method has been developed which detects the pseudodeficiency (pd) allele using a single pair of primers encompassing the mutation site and hair follicles as the source of DNA. The frequency of this allele in the Spanish population has been evaluated and correlations between different genotypes and ASA activity levels have been established. Ten out of 55 individuals were heterozygous for the ASA pd allele, while two were homozygous. The allele frequency was thus 12.7%.

The molecular detection of the pseudodeficiency allele for arylsulphatase A in patients with neurological symptoms and low arylsulphatase A activity.

Journal of Inherited Metabolic DiseaseVolume 16, Issue 6 p. 1048-1049 Short Report The molecular detection of the pseudodeficiency allele for arylsulphatase A in patients with neurological symptoms and low arylsulphatase A activity S. Goldenfum, S. Goldenfum Division of Biochemistry & Genetics, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UKSearch for more papers by this authorS. Malcolm, S. Malcolm Division of Biochemistry & Genetics, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UKSearch for more papers by this authorE. Young, E. Young Division of Biochemistry & Genetics, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UKSearch for more papers by this authorB. Winchester, B. Winchester Division of Biochemistry & Genetics, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UKSearch for more papers by this author S. Goldenfum, S. Goldenfum Division of Biochemistry & Genetics, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UKSearch for more papers by this authorS. Malcolm, S. Malcolm Division of Biochemistry & Genetics, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UKSearch for more papers by this authorE. Young, E. Young Division of Biochemistry & Genetics, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UKSearch for more papers by this authorB. Winchester, B. Winchester Division of Biochemistry & Genetics, Institute of Child Health, 30 Guilford Street, London, WC1N 1EH UKSearch for more papers by this author First published: 01 November 1993 https://doi.org/10.1007/BF00711527Citations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume16, Issue6November 1993Pages 1048-1049 RelatedInformation

Metachromatic leukodystrophy: Multiple nonfunctional and pseudodeficiency alleles in a pedigree: Problems with diagnosis and counseling

Metachromatic leukodystrophy is due to deficient activity of arylsulfatase A, an enzyme important in myelin catabolism. The deficiency can be caused by different point mutations in the gene coding for arylsulfatase A (nonfunctional alleles). In addition, certain mutations result in low levels of enzyme activity detectable with artificial substrates in vitro but no clinical dysfunction (pseudodeficiency alleles). The described family has various combinations of normal, nonfunctional, and pseudodeficiency alleles that presented diagnostic and counseling dilemmas which were resolved at the genomic level. We find no evidence that compound heterozygote individuals have subclinical involvement of the nervous system. We report the clinical, pathological, electrophysiological, imaging, biochemical, and genetic data of this family and discuss the difficulties in analyzing such pedigrees.

Medical genetics: advances in brief: A pseudodeficiency allele common in non-Jewish Tay-Sachs carriers: implications for carrier screening

Medical genetics: advances in brief: A pseudodeficiency allele common in non-Jewish Tay-Sachs carriers: implications for carrier screening

Diagnosis of arylsulfatase A deficiency.

Metachromatic leukodystrophy (MLD) is a neurologically devastating autosomal recessive disorder in humans associated with deficient arylsulfatase A activity. However, clinically normal individuals described as being pseudo-arylsulfatase-A deficient also demonstrate the same deficiency. Genotypically, they may be homozygous for the pseudodeficiency mutation (associated with 2 A-->G transitions in the cDNA of arylsulfatase A) or heterozygous with one pseudodeficiency and one MLD allele. Using as examples 2 families in which the pseudo deficiency condition occurs either independently or together with MLD, we demonstrate the utility of a proposed diagnostic protocol to provide complete genotype identification of individuals suffering from arylsulfatase A deficiency. Patient fibroblasts are extracted for DNA and a cytoplasmic fraction, which is used for arylsulfatase A enzyme assay. This will identify an arylsulfatase A-deficient group, which is further analyzed electrophoretically. Cells from the clinically affected patients with MLD are completely deficient in arylsulfatase A activity, whereas those from the pseudodeficient individuals demonstrate a characteristic residual arylsulfatase A activity detectable only after electrophoresis. Within this pseudodeficient group, gene amplification of DNA specific for the A-->G mutations will distinguish between those who are homozygous for the pseudodeficiency allele and those who are compound heterozygous for the pseudodeficiency and MLD alleles. This protocol of complete genotype identification requires only about 10(6) fibroblasts (1 x 100 mm dish) and 2 days to complete. Such variant-specific genotype identification increases accuracy and prognostic value of the diagnosis. It will likely become the preferred choice for diagnosis of genetic disease in the future as more variant-specific mutations are identified at the molecular level.

Genotype-phenotype relationship in various degrees of arylsulfatase A deficiency.

Arylsulfatase A (ASA) is a lysosomal enzyme that hydrolyzes sulfatide. Absence of ASA activity leads to metachromatic leukodystrophy (MLD). The clinical outcome resulting from ASA deficiency is highly variable with respect to age of onset and symptoms. So far the causes for the variability are poorly understood. We have studied the relationship between the ASA genotype and the clinical phenotype. Fibroblasts from a total of 34 subjects with low ASA activity were examined with immunoblotting, a sensitive ASA assay, and the sulfatide loading test in order to characterize low ASA activity further. By these methods, three different classes of ASA deficiency can be defined: homozygosity for the pseudodeficiency allele (ASAp), compound heterozygosity for the ASAp and MLD (ASA-) alleles, and ASA-/ASA- genotypes. These genotypes exhibit different levels of ASA residual activity. Only ASA-/ASA- genotypes are associated with MLD. For diagnostic purposes, however, the differentiation of the various ASA genotypes is essential.

An assay for the rapid detection of the arylsulfatase A pseudodeficiency allele facilitates diagnosis and genetic counseling for metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulfatase A (ASA). A substantial ASA deficiency has also been described in clinically healthy persons, a condition for which the term pseudodeficiency was introduced. The discrimination of both kinds of deficiencies based on ASA activity determination is difficult and unreliable. This creates a serious problem in the genetic counseling and diagnosis of MLD. The mutations characteristic for the pseudodeficiency (PD) allele have recently been identified. A non-radioactive assay based on the polymerase chain reaction is described, which allows the rapid detection of the ASA pd allele. The assay utilizes pairs of primers that allow either the amplification of the ASA PD allele or of other ASA alleles, since their 3' residues match either the ASA PD allele or other ASA alleles.

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%.

Molecular Genetics of Metachromatic Leukodystrophy

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by the deficiency of arylsulfatase A (ASA). The ASA cDNA as well as the gene has been cloned. The gene is about 3 kb long and consists of 8 exons. The two most frequent alleles causing MLD have been characterized and the distribution of these alleles among patients with different clinical forms of MLD has revealed a simple genotype-phenotype correlation. Some individuals have low ASA activities but are healthy. This condition has been called ASA pseudodeficiency. These individuals are homozygous for the ASA pseudodeficiency allele which only encodes 5-10% of the ASA activity compared to the normal allele. The mutations in the PD allele have been characterized. Based on the knowledge of these mutations diagnostic assays have been developed to differentiate ASA deficiencies associated with PD or MLD.

Phenotypic Consequences of Low Arylsulfatase A Genotypes (ASAp/ASAp and ASA<sup>–</sup>/ASAp): Does There Exist an Association with Multiple Sclerosis?

Arylsulfatase A (ASA) pseudodeficiency does per definitionem not lead to metachromatic leukodystrophy. It is conceivable, however, that it may contribute to the susceptibility for more common, multifactorial disorders of the nervous system. In order to examine whether there is an association with multiple sclerosis (MS), the most common demyelinating disease, we screened 160 MS patients for ASA activity and looked for pseudodeficiency genotypes using polymerase chain reaction. Four homozygotes for the ASA pseudodeficiency allele were found among the MS patients, but only one in the control sample. Further studies are necessary to validate whether ASA pseudodeficiency is associated with MS.

Advances in the molecular genetics of metachromatic leukodystrophy

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulphatase A (EC 3.1.6.1). This results in the intralysosomal storage of cerebroside sulphate, which leads to a progressive demyelination of the nervous system. The patients usually die within a few years from the onset of symptoms. Clinically, there are different forms of the disease and the molecular basis for this heterogeneity is unknown. The gene for arylsulphatase A has recently been cloned and provides a necessary tool for the exact description of the molecular defects occurring in the different forms of metachromatic leukodystrophy. Metachromatic leukodystrophy can also be caused by the deficiency of an arylsulphatase A activator protein (sphingolipid activator protein B). The cDNA for the precursor of this protein has been isolated and a mutant cDNA of one patient has been analysed. A substantial arylsulphatase A deficiency can also occur in healthy individuals, a phenotype termed pseudodeficiency. Two concurrent mutations have been identified in this low arylsulphatase A activity allele. This permitted the development of a rapid assay which allows the detection of the pseudodeficiency allele. Bone marrow transplantation has been tried in several metachromatic leukodystrophy patients and there is evidence that this treatment might slow or even halt the progression of the disease. A final conclusion as to whether bone marrow transplantation is a suitable therapy for metachromatic leukodystrophy cannot be drawn yet.

Arylsulfatase A pseudodeficiency: loss of a polyadenylylation signal and N-glycosylation site.

Metachromatic leukodystrophy is a metabolic disorder caused by the deficiency of arylsulfatase A. Deficiency of this enzyme is also found in apparently healthy individuals, a condition for which the term pseudodeficiency was introduced. The arylsulfatase A (cerebroside-3-sulfate 3-sulfohydrolase; EC 3.1.6.8) (ASA) encoding gene was isolated from an individual homozygous for the ASA pseudodeficiency allele. Sequence analysis revealed two A----G transitions. One changes Arg-350 to serine, which leads to the loss of a utilized N-glycosylation site. This loss explains the smaller size of ASA in ASA pseudodeficiency fibroblasts. The introduction of Ser-350 into normal ASA cDNA does not affect the rate of synthesis, the stability, or the catalytic properties of ASA in stably transfected baby hamster kidney cells. Therefore, the loss of the N-linked oligosaccharide does not contribute to the reduction of ASA activity in ASA pseudodeficiency. The other A----G transition changes the first polyadenylylation signal downstream of the stop codon from AATAAC to AGTAAC. The latter causes a severe deficiency of a 2.1-kilobase (kb) mRNA species. The deficiency of the 2.1-kb RNA species provides an explanation for the diminished synthesis of ASA seen in pseudodeficiency fibroblasts. Amplification of genomic DNA and hybridization with allele-specific oligonucleotides detected both mutations in four unrelated individuals with ASA pseudodeficiency.

Pseudodeficiency of arylsulfatase A: a common genetic polymorphism with possible disease implications

At the locus for arylsulfatase A (ASA) at least four to five alleles exist: besides the normal ASA+ and at least two to three deficiency alleles (ASA-), a pseudodeficiency allele, ASAp, is known. On SDS-PAGE the ASAp enzyme migrates slightly faster than ASA+. Treatment of extracts from cells with ASA+/ASA+, ASAp/ASAp, or ASA+/ASAp genotypes with endoglycosidase F leads to the same deglycosylated subunit pattern. Presumably the degree of glycosylation is lower in ASAp than in ASA+. In a large-scale screening project we determined a gene frequency of 7.3% for ASAp. Thus, the ASA locus is polymorphic. In seven families, ASAp showed a codominant mode of inheritance with ASA+. Homozygosity for ASAp has no obvious clinical consequences. In subjects with the compound genotype ASA-/ASAp, the residual enzyme activity may fall below a critical threshold, so that the substrate can no longer be hydrolyzed sufficiently. Since these compounds are not so rare (estimated frequency 0.073%), this mechanism could be of importance in neuropsychiatric disorders with late onset.