Molecular analysis of genomic DNA allows rapid, and accurate, prenatal diagnosis of peroxisomal D-bifunctional protein deficiency.

Prenatal diagnosis was requested for a couple with a previous child affected by the peroxisomal disorder D-bifunctional protein deficiency. Prior analysis of the D-bifunctional protein cDNA sequence from the propositus had shown that it was missing 22 bp. This was subsequently attributed to a point mutation in the intron 5 donor site (IVS5 + 1G>C) of the D-bifunctional protein gene. Consistent with parental consanguinity, the patient was shown to be homozygous for this mutation, which is associated with loss of a Hph 1 restriction site in the genomic sequence. Prenatal testing of the fetus using genomic DNA isolated from uncultured amniocytes indicated that both alleles of the D-bifunctional protein had the IVS5 + 1G>C substitution. The peroxisomal defect was later confirmed biochemically using cultured amniocytes, which were found to have elevated levels of very long chain fatty acids (VLCFA). This is the first report of prenatal diagnosis of D-bifunctional protein deficiency using molecular analysis of genomic DNA.

A common PEX1 frameshift mutation in patients with disorders of peroxisome biogenesis correlates with the severe Zellweger syndrome phenotype.

Peroxisome biogenesis disorders are a heterogeneous group of human neurodegenerative diseases caused by peroxisomal metabolic dysfunction. At the molecular level, these disorders arise from mutations in PEX genes that encode proteins required for the import of proteins into the peroxisomal lumen. The Zellweger syndrome spectrum of diseases is a major sub-set of these disorders and represents a clinical continuum from Zellweger syndrome (the most severe) through neonatal adrenoleukodystrophy to infantile Refsum disease. The PEX1 gene, which encodes a cytoplasmic AAA ATPase, is the responsible gene in more than half of the Zellweger syndrome spectrum patients, and mutations in PEX1 can account for the full spectrum of phenotypes seen in these patients. In these studies, we have undertaken mutation analysis of PEX1 in skin fibroblast cell lines from Australasian Zellweger syndrome spectrum patients. A previously reported common PEX1 mutation that gives rise to a G843D substitution and correlates with the less severe disease phenotypes has been found to be present at high frequency in our patient cohort. We also report a novel PEX1 mutation that occurs at high frequency in Zellweger syndrome spectrum patients. This mutation produces a frameshift in exon 13, a change that leads to the premature truncation of the PEX1 protein. A Zellweger syndrome patient who was homozygous for this mutation and who survived for less than two months from birth had undetectable levels of PEX1 mRNA. This new common mutation therefore correlates with a severe disease phenotype. We have adopted procedures for the detection of this mutation for successful prenatal diagnosis.

Prenatal diagnosis of mucolipidosis II--electron microscopy and biochemical evaluation.

Prenatal diagnosis of mucolipidosis type II (I-cell disease) can be performed quickly and reliably by electron microscopy of chorionic villus tissue. This study reports the results of studies in three prenatal assessments (two families) where the pregnancy was at one in four risk of the disorder. In all three cases, electron microscopy showed marked vacuolation in chorionic villus cells, consistent with the fetus being affected by the disorder. Further studies in cultured chorionic villus cells showed a marked deficiency of a number of lysosomal enzymes. All pregnancies were terminated. Follow-up studies in fetal tissue (where available) confirmed the prenatal diagnosis as correct.

Mutations among Italian mucopolysaccharidosis type I patients.

A group of 27 Italian patients was screened for alpha-L-iduronidase mucopolysaccharidosis type I mutations. Mutations were found in 18 patients, with 28 alleles identified. The two most common mutations in northern Europeans (W402X and Q70X) accounted for 11% and 13% of the alleles, respectively. The R89Q mutation, uncommon in Europeans, was found only in one patient, accounting for 1 of 54 alleles (1.9%). The other mutations, P533R, A327P and G51D, accounted for 11%, 5.6% and 9.3% of the total alleles, respectively. Interestingly, the high frequency of the P533R mutation seems to be confined to Sicily and is higher than the 3% reported in a British/Australian study.

Pitfalls in the prenatal diagnosis of peroxisomal beta-oxidation defects by chorionic villus sampling.

Variability in the level of expression of very long chain fatty acids (VLCFAs) is documented in cultured chorionic villus (CV) cells derived from two fetuses, one at risk for an unusual peroxisomal fatty acid beta-oxidation defect, and the other at risk for the X-linked form of adrenoleucodystrophy (ALD). Cells from early subcultures of chorionic cells from both cases gave normal values for VLCFA ratios. The results for the fetus at risk for the beta-oxidation defect were interpreted to indicate that the fetus was not affected; however, at birth, the infant was clinically and biochemically affected. In the case of the fetus at risk for X-linked ALD, although VLCFAs were normal in subculture 1, the levels of these fatty acids increased dramatically in subculture 3, suggesting an abnormal fetus. Termination of the pregnancy and subsequent biochemical and morphological follow-up confirmed that the fetus was indeed affected by ALD.

Neonatal screening for cystic fibrosis using immunoreactive trypsinogen and direct gene analysis: four years' experience.

OBJECTIVE: To assess the performance and impact of a two tier neonatal screening programme for cystic fibrosis based on an initial estimation of immunoreactive trypsinogen followed by direct gene analysis. DESIGN: Four year prospective study of two tier screening strategy. First tier: immunoreactive trypsinogen measured in dried blood spot samples from neonates aged 3-5 days. Second tier: direct gene analysis of cystic fibrosis mutations (delta F508, delta I506, G551D, G542X, and R553X) in samples with immunoreactive trypsinogen concentrations in highest 1% and in all neonates with meconium ileus or family history of cystic fibrosis. SETTING: South Australian Neonatal Screening Programme, Adelaide. SUBJECTS: All 88,752 neonates born in South Australia between December 1989 and December 1993. INTERVENTIONS: Neonates with two identifiable mutations were referred directly for clinical assessment and confirmatory sweat test; infants with only one identifiable mutation were recalled for sweat test at age 3-4 weeks. Parents of neonates identified as carriers of cystic fibrosis mutation were counselled and offered genetic testing. MAIN OUTCOME MEASURES: Identification of all children with cystic fibrosis in the screened population. RESULTS: Of 1004 (1.13%) neonates with immunoreactive trypsinogen > or = 99th centile, 912 (90.8%) had no identifiable mutation. 23 neonates were homozygotes or compound heterozygotes; 69 carried one identifiable mutation, of whom six had positive sweat tests. Median age at clinical assessment for the 29 neonates with cystic fibrosis was 3 weeks; six had meconium ileus and two had affected siblings. 63 neonates were identified as carriers of a cystic fibrosis mutation. Extra laboratory costs for measuring immunoreactive trypsinogen and direct gene analysis were $A1.50 per neonate screened. CONCLUSION: This strategy results in early and accurate diagnosis of cystic fibrosis and performs better than screening strategies based on immunoreactive trypsinogen measurement alone.

Mutation analysis of 19 North American mucopolysaccharidosis type I patients: identification of two additional frequent mutations.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive genetic disorder caused by deficiency of the lysosomal glycosidase alpha-L-iduronidase. Patients with this disorder present with varied clinical phenotypes ranging from early severe onset of disease and death in early childhood to mild manifestations compatible with adult life. An understanding of the molecular basis of iduronidase deficiency and its correlation to clinical phenotype will improve prognostic prediction at diagnosis, aid in genetic counselling of families, and provide a framework to more accurately assess experimental treatment protocols. We have used the approach of single-strand conformational polymorphism analysis and direct sequencing of the alpha-L-iduronidase gene in an attempt to define the molecular basis of iduronidase deficiency in affected individuals. An initial series of 19 patients representing 35 independently segregating mutant alleles were studied. In addition to five previously identified mutations (W402X, Q70X, E274X, H82P, and P533R) two novel mutations (A75T and 474-2a-->g) were found. These seven mutations account for 71% of the mutant alleles and 53% of the genotypes in this group of patients. Analysis of a larger independently ascertained group of 103 MPS I patients, mainly of Northern European origin, revealed that together the two novel mutations account for 7% of mutant alleles and are associated with severe clinical phenotypes. These mutations are the most frequent MPS I mutations detected so far after W402X and Q70X. With the definition of these two mutations, a clear picture of the molecular heterogeneity of MPS I is emerging.

Mutation analysis of 28 Gaucher disease patients: the Australasian experience.

Gaucher disease is the most common lysosomal storage disease. It is an autosomal recessive disorder that results from a deficiency of beta-glucocerebrosidase. Three clinical phenotypes have been described: non-neuronopathic, acute neuronopathic, and subacute neuronopathic. Genomic DNA from 28 Australasian patients of diverse ethnic origin with Gaucher disease was screened for 3 common mutations (1226G, 1448C and 84GG) using the amplification refractory mutation system (ARMS), and one uncommon mutation (1504T) by restriction enzyme digestion. Thirty-eight of the 56 independent alleles in these patients were characterized, with 1448C present in 42% and 1226G in 28% of the alleles. The 1226G mutation was associated only with the non-neuronopathic phenotype and 7 of the 15 patients who carried the 1448C mutation developed neuronopathic disease. Three infants who died in the neonatal period following a rapidly progressive neurodegenerative course carried no identifiable mutations. The 84GG mutation was carried by 2 Jewish patients and 1504T was present in one patient. It is now possible to rapidly identify the common Gaucher mutations using ARMS and restriction enzyme digestion, and our findings confirm the heterogeneity of mutations in Gaucher disease. It is also possible to predict in part the phenotypic outcome when screening patients for these mutations. We consider mutation analysis to be of most use in prenatal diagnosis and for carrier detection within affected families.

Identification of mutations in the alpha-L-iduronidase gene (IDUA) that cause Hurler and Scheie syndromes.

Mucopolysaccharidosis type I (MPS-I) is an autosomal recessive genetic disease caused by a deficiency of the lysosomal glycosidase alpha-L-iduronidase. Hurler (severe), Scheie (mild), and Hurler/Scheie (intermediate) syndromes are clinical subtypes of MPS-I, but it is difficult to distinguish between these subtypes by biochemical measurements. Mutation analysis was undertaken to provide a molecular explanation for the clinical variation seen in MPS-I. Using chemical cleavage and direct PCR sequencing, we have defined four previously undescribed mutations for MPS-I (delG1702, 1060 + 2t-->c, R89Q, and 678-7g-->a). R89Q and 678-7g-->a were found to be present in 40% of Scheie syndrome alleles. Expression of R89Q demonstrated reduced stability and activity of the mutant protein. The deleterious effect of R89Q may be potentiated by a polymorphism (A361T) to produce an intermediate phenotype. 678-7g-->a was found to be a mild mutation, since it was present in an index Scheie syndrome patient in combination with a severe allele (W402X). This mutation appears to allow a very small amount of normal mRNA to be produced from the allele which is likely to be responsible for the mild clinical phenotype observed. Both the 5' and 3' splice site mutations (1060 + 2t-->c and 678-7g-->a, respectively) result in high proportions of mature mRNAs containing introns, which has not been observed for other splicing mutations. The frameshift mutation (delG1702) and the 5' splice site mutation (1060 + 2t-->c) are both thought to be associated with severe MPS-I. The identification of these MPS-I mutations begins to document the expected genetic heterogeneity in MPS-I and provides the first molecular explanations for the broad range of clinical phenotypes observed.

An arylsulfatase A (ARSA) missense mutation (T274M) causing late-infantile metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disorder caused by a deficiency of arylsulfatase A (ARSA; EC 3.1.6.8). The 8 ARSA exons and adjacent intron boundaries from a patient with late-infantile metachromatic leukodystrophy were polymerase chain reaction (PCR) amplified in seven discrete reactions. Amplified ARSA exons were analysed for the presence of sequence alterations by single-strand conformation polymorphism analysis, followed by direct sequencing of PCR products. The patient was found to be homozygous for a C-->T transition in exon IV that results in the substitution of a highly conserved threonine residue at amino acid 274 with a methionine (T274M). Analysis of a further 29 MLD patients revealed the presence of five additional homozygotes for T274M. All 6 T274M homozygotes (representing four families) were of Lebanese descent, and all were known to be the result of consanguineous marriages. The altered amino acid is rigidly conserved among 10 sulfatases from Escherichia coli to humans; therefore, it is most likely that the resultant mutant protein will have little or no enzyme activity. This is consistent with the very low ARSA activity measured in these patients and their uniformly severe clinical presentation.

Mucopolysaccharidosis type I (Hurler syndrome): linkage disequilibrium indicates the presence of a major allele.

Two polymorphisms exist in the alpha-L-iduronidase (IDUA) gene, the gene that is defective in mucopolysaccharidosis type I (MPS I), viz. a KpnI polymorphism and a variable number of tandem repeats (VNTR) polymorphism with three common alleles. The analysis of allele and haplotype frequencies for these two polymorphisms in the normal population and in MPS I patients revealed the presence of linkage disequilibrium. The frequency of the 2,2 (VNTR, KpnI) allele in MPS I patients was 57% compared with only 37% in the normal population. The implications for the presence of a major MPS I allele and the ability to predict patient phenotype are discussed.