Semiquantitative reverse transcription-polymerase chain reaction with the Agilent 2100 Bioanalyzer.

We have applied a method to monitor mRNA expression in a semiquantitative fashion on the Agilent 2100 Bioanalyzer. The method was originally described in 1994 by Wong et al. and referred to as the "primer-dropping" method. This polymerase chain reaction (PCR) technique uses multiple sets of primer pairs in a coamplification reaction that amplifies the target of interest within a predetermined range specific for each target. Separation, detection and quantification of PCR products were accomplished using the Agilent 2100 Bioanalyzer in conjunction with the DNA 500 and the DNA 1000 Lab-Chip kits for the detection of DNA fragments with a maximum size of 500 and 1000 bp, respectively. Using primers specific for the inducible form of hsp72 and primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal standard we were able to rapidly monitor and quantify inducible hsp72-mRNA expression.

Characterization of four arylsulfatase A missense mutations G86D, Y201C, D255H, and E312D causing metachromatic leukodystrophy.

Metachromatic leukodystrophy is a lysosomal storage disease caused by the deficiency of arylsulfatase A. Here we describe a hitherto unknown arylsulfatase A allele carrying a E312D missense mutation and characterize the effects of this and three previously described missense mutations, G86D, Y201C, and D255H, on arylsulfatase A. In transfection experiments no enzyme activity can be expressed from arylsulfatase A cDNAs coding for the D255H substituted enzyme, whereas Y201C and E312D mutations were associated with low amounts of residual enzyme activity. All amino acid substitutions lead to a decreased stability of the mutant enzyme, and metabolic labeling experiments indicated that except for the E312D substitution the mutations cause arrest of the mutant arylsulfatase A polypeptides in a prelysosomal compartment.

Translational control of arylsulfatase A expression in mouse testis.

Arylsulfatase A is a lysosomal enzyme that is involved in the degradation of sulfated glycolipids. High levels of arylsulfatase A mRNA are found in germ cells of mouse testis. In late pachytene and secondary spermatocytes the level of arylsulfatase A mRNA is increased 20-fold when compared with other tissues. These high levels of arylsulfatase A mRNA are maintained in round spermatids and decrease in late elongating spermatids. The increase of arylsulfatase A mRNA levels is not accompanied by a similar increase in enzyme activity or polypeptides. Subcellular fractionation revealed that the majority of arylsulfatase A mRNA is not associated with polysomes but is found in fractions of lower buoyancy. The failure to become translated is ascribed to the association of arylsulfatase A mRNA with nonpolysomal ribonucleoproteins. This translational repression may be due to proteins that bind to arylsulfatase A mRNA and prevent its translation. Within the 639-nucleotide 5'-untranslated region and the 700-nucleotide 3'-untranslated region of the arylsulfatase A mRNA, we identified two regions that specifically bind proteins present in extracts prepared from testicular cells. These RNA binding proteins were absent from extracts prepared from liver or brain.

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.

Structure of the mouse arylsulfatase A gene and cDNA.

The murine arylsulfatase A (ARSA) gene and cDNA have been cloned and sequenced. The gene is 3.8 kb long and contains eight exons. All intron/exon splice junctions conform to the GT/AG consensus sequence. The genomic structure is similar to that of the human gene. One major RNA species of 3.2 kb is transcribed. This RNA species has a 5' untranslated region of 638 nucleotides and terminates in a region around nucleotide 700 downstream of the termination codon. In addition, a rare mRNA species terminating at a polyadenylation signal 135 nucleotides downstream of the termination codon has been found. A larger transcript of 4 kb can be detected in liver. The size difference is due to initiation of transcription 5' of the cap site of the 3.2-kb mRNA species. The entire ARSA cDNA has been cloned by PCR from reverse-transcribed RNA. The coding sequence has 1518 nucleotides and predicts a protein of 506 amino acids. The nucleotide as well as the amino acid sequence is highly conserved among humans and mice.

Molecular basis of different forms of metachromatic leukodystrophy.

BACKGROUND: Metachromatic leukodystrophy is an autosomal recessive inherited lysosomal storage disorder caused by a deficiency of arylsulfatase A. Three forms of the disease can be distinguished according to severity and the age at onset: late infantile (1 to 2 years), juvenile (3 to 16), and adult (greater than 16). METHODS AND RESULTS: To understand the molecular basis of the different forms of the disease, we analyzed arylsulfatase A alleles associated with metachromatic leukodystrophy. Two alleles (termed I and A) were identified and accounted for about half of all arylsulfatase A alleles among 68 patients with metachromatic leukodystrophy whom we examined. Sufficient information was available for 66 of the patients to allow classification of their disease. Of the six instances of homozygosity for allele I, all were associated with the late-infantile form of the disease; of the eight instances of homozygosity for allele A, five were associated with the adult form and three with the juvenile form. When both alleles were present, the juvenile form resulted (seven of seven instances). Heterozygosity for allele I (with the other allele unknown) is usually associated with late-infantile disease, and heterozygosity for allele A with a later onset of the disease. The clinical variability can be explained by the different levels of residual arylsulfatase A activity associated with these genotypes. CONCLUSIONS: Like many lysosomal storage disorders, metachromatic leukodystrophy shows clinical heterogeneity that seems to reflect genetic heterogeneity. One of the known alleles (allele I) is associated with earlier and more severe disease than the other (allele A).

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.

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.