William F. Carey

Population frequency of the arylsulphatase A pseudo-deficiency allele

SummaryThe 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%.

Late-onset visceral presentation with cardiomyopathy and without neurological symptoms of adult Sanfilippo A syndrome

Sanfilippo A syndrome, mucopolysaccharidosis type IIIA, is caused by a deficiency of heparan sulphamidase activity, and usually presents in childhood with neurodegeneration leading to death in teenage years. Visceral symptoms are limited to coarsening and diarrhea. We now describe an adult patient who presented with cardiomyopathy. At age 45 years she had hypertension, and the next year she developed a progressively worsening cardiomyopathy with prominent apical hypertrophy and atrial fibrillation. At age 53, she had severe concentric hypertrophic nonobstructive cardiomyopathy in both ventricles. There was no coarsening of features. Neurologic function, skeleton, cornea, liver, and spleen were normal. Percutaneous endomyocardial biopsy showed ballooned cardiomyocytes with storage vacuoles, containing acid mucopolysaccharides. Leucocytes, uterus, and brain biopsy did not show this storage material. There was a slight increase in total urine mucopolysaccharides, with an increased proportion of heparan sulfates. Heparan sulphamidase activity was deficient in leukocytes and heparan sulphamidase protein and activity were reduced in cultured fibroblasts. No mutations were identified after sequencing of the heparan sulphamidase gene at the cDNA and the genomic level. This new clinical presentation expands the clinical spectrum of Sanfilippo A syndrome to include a primary visceral presentation of cardiomyopathy without neurologic symptoms in the adult. The late onset may be related to the residual heparan sulphamidase activity. The genetic basis of this new variant is still unclear. Physicians evaluating adults must remain aware of possible new adult presentations of storage conditions.

Adult-onset exercise intolerance due to phosphorylase b kinase deficiency.

Muscle-specific phosphorylase b kinase deficiency is an unusual form of glycogen storage disorder. The majority of patients are male with an age at diagnosis between 15 to 36 years. Clinical features include exercise intolerance, myalgia and muscle weakness. A forearm ischaemic exercise test is usually normal and histochemical staining for myophosphorylase positive. The demonstration of reduced muscle phosphorylase b kinase activity by biochemical assay confirms the diagnosis. We report a 36 year old male with phosphorylase b kinase deficiency and symptom onset in adult life.

Frequency of intron 8 CFTR polythymidine sequence variant in neonatal blood specimens

2 analysis. The genotype (ac/ac) was found to be significantly associated with AD. The other heterozygous genotypes (ac/at) and (ac/gc) were not associated with AD. Therefore, the influence of the homozygous genotype (ac/ac) would be recessively inherited, whereas the other responsible or risk genes are dominantly inherited. The genotypes were examined in the context of the presence or absence of the apolipoprotein E (APOE) e4 allele, 3

Isolated Glycerol Kinase Deficiency in a Neonate

Glycerol kinase deficiency occurs either as a relatively benign isolated enzyme deficiency, or as part of a syndrome resulting from a microdeletion in the p21 region of the X chromosome associated with congenital adrenal hypoplasia and/or Duchenne muscular dystrophy. Developmental delay is a consistent feature of the microdeletion syndrome but not of the isolated enzyme defect. We report a case of isolated glycerol kinase deficiency in a neonate presenting with hypotonia, apnea, mild developmental delay, and glyceroluria, without evidence of adrenal insufficiency or myopathy. A mild communicating hydrocephalus was noted on magnetic resonance imaging brain scan. It is important, therefore, to exclude glyceroluria in infants being investigated for apnea and hypotonia. (J Child Neurol 1994;9:70-73).

Identification of a cystic fibros is mutation: deletion of isoleucine506

SummaryThe recent isolation of the cystic fibrosis (CF) gene has resulted in the identification of a common mutation (AF508) that is found on about 70% of CF chromosomes and that comprises a deletion of 3 bp and results in the omission of Phe508 from within a putative ATP-binding domain of the predicted gene product. We describe a CF mutation that involves the deletion of 3 bp encoding Ile508 or Ile507. This is a rare mutation found in less than 1% of CF chromosomes and could be mistaken for ΔAF508 using the current methods for the molecular diagnosis of CF.

An arylsulphatase A (ARSA) frameshift mutation (289insG) in metachromatic leukodystrophy (MLD)

Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disorder caused by a deficiency of arylsulphatase A (ARSA) (EC 3.1.6.8). MLD involves progressive demyelination, resulting in a variety of neurological symptoms varying in severity (Kolodny and Fluharty, 1995). The ARSA gene consists of eight exons encoding the 507 amino acid enzyme (Stein et al, 1989). It is transcribed into three mRNA species, a major species of 2.1 kb, and two minor species of 3.7 and 4.8 kb. Over 90 largely missense mutations and polymorphisms have been identified in the ARSA gene (Human Gene Mutation Database). Some healthy individuals, referred to as having pseudodeficiency of arylsulphatase A (ARSA-PD), have very low ARSA activity. The molecular defect responsible for ARSA-PD is characterised by two A to G transitions (Gieselmann et al, 1989), the first of which causes the substitution of a glycosylated asparagine with a serine residue at position 350 (N350S), resulting in about 50% of enzyme being mistargeted. The second results in a non-functional polyadenylylation signal and a deficiency of the major 2.1 kb mRNA species, leading to a 70% reduction of polyadenylylated mRNA (Harvey et al, 1998). As a result of the combined effect of these variants, ARSA-PD homozygotes have only about 10% of normal enzyme activity but this is sufficient to prevent the development of MLD symptoms. The individual described in this study showed features of an MLD-like neuropathy and exhibited ARSA activity in the pseudodeficiency range. He was found to be negative for all known MLD mutations, but heterozygous for the ARSA-PD allele. To detect possible additional sequence alterations, all eight exons and their intron boundaries were PCR amplified and subjected to single stranded conformational polymorphism (SSCP) analysis as previously described (Harvey et al, 1993). The exon 2 PCR product revealed that he was heterozygous for a change in this exon (Figure 1A). Direct sequence analysis revealed an insertion of a single G nucleotide in a run of 7 Gs starting at ARSA cDNA position 289, resulting in a frameshift mutation (289insG; Figure 1B). Figure 1 (A) SSCP analysis of exon 2 genomic PCR products. Tracks 2-4 are controls, track 1 is of the individual described in this study, and contains a band of altered conformation as indicated. (B) Reverse automated sequence analysis of the altered conformation ... This frameshift mutation results in normal translation for 99 amino acids followed by 31 aberrant amino acids prior to premature termination of the protein. Consequently, this mutation is likely to totally disrupt ARSA activity and can be considered a new null allele for MLD. The identity of the mutation was confirmed by allele specific oligonucleotide (ASO) analysis of 100 normal alleles, 31 MLD alleles and 33 ARSA-PD alleles (using the following ASO oligonucleotides: 289n, 5′-CCGGGGGGGCCTGC-3′; 289insG, 5′-CCGGGGGGGGCCTG-3′). 289insG was not present on any of these alleles except in the index case described in this paper, who was heterozygous. Further ASO analysis revealed that his mother carried 289insG and his father carried the ARSA-PD allele (Figure 2), accounting for the low enzyme activity observed in both parents. Figure 2 Duplicate filters containing exon 2 products were probed as indicated with the normal and mutant oligonucleotides for 289insG. A control normal sample was included. The pedigree is as follows; grey represents the ARSA-PD allele, black; the 289insG mutation. ...

Arylsulfatase A lysosomal enzyme Map position 22q13.3

Metachromat ic l eukodys t rophy is an au tosomal recessive d i sorder of myel in metabol i sm caused by a deficiency of the lysosomal enzyme arylsul fa tase A (ARSA) [1]. The ARSA gene has p rev ious ly been local ized to chromosome 22 between 22q13 and qter us ing h u m a n rodent hybr id clones [2]. The human ARSA cDNA probe conta ined the whole coding region and consis ted of a 2.0-kb insert in pBluescript [3]. Fluorescence in situ hybr id iza t ion was as descr ibed in [4], except that no prereassocia t ion was necessary and chromosomes were s ta ined before analysis with both p r o p i d i u m iod ide (as counters ta in) and DAPI (for chromosome identification). Twenty metaphases from a normal male all showed specific label l ing of one or both chromat ids of chromosome 22 in the region ex tending from dis ta l 22q13.31 to 22q13.33. Two non-specific backg round dots were observed. A similar result was shown in a second normal male (15 h igh-resolu t ion metaphases) , conf irming the distal 22q13.3 posi t ion. 1. Gieselmann Vet al. (1995) Hum Mutat 4: 233-242. 2. Geurts van Kissel AHM et al. (1980) Cytogenet Cell Ganet 28: 169-!72. 3. Stein C et al. (1989) J Biol Chem 264: 1252-1259. 4. Callen DF et al. (1990) Ann Gfnft 33: 219-221.Metachromat ic l eukodys t rophy is an au tosomal recessive d i sorder of myel in metabol i sm caused by a deficiency of the lysosomal enzyme arylsul fa tase A (ARSA) [1]. The ARSA gene has p rev ious ly been local ized to chromosome 22 between 22q13 and qter us ing h u m a n rodent hybr id clones [2]. The human ARSA cDNA probe conta ined the whole coding region and consis ted of a 2.0-kb insert in pBluescript [3]. Fluorescence in situ hybr id iza t ion was as descr ibed in [4], except that no prereassocia t ion was necessary and chromosomes were s ta ined before analysis with both p r o p i d i u m iod ide (as counters ta in) and DAPI (for chromosome identification). Twenty metaphases from a normal male all showed specific label l ing of one or both chromat ids of chromosome 22 in the region ex tending from dis ta l 22q13.31 to 22q13.33. Two non-specific backg round dots were observed. A similar result was shown in a second normal male (15 h igh-resolu t ion metaphases) , conf irming the distal 22q13.3 posi t ion. 1. Gieselmann Vet al. (1995) Hum Mutat 4: 233-242. 2. Geurts van Kissel AHM et al. (1980) Cytogenet Cell Ganet 28: 169-!72. 3. Stein C et al. (1989) J Biol Chem 264: 1252-1259. 4. Callen DF et al. (1990) Ann Gfnft 33: 219-221.