Susanna Bunge

GENOTYPE-PHENOTYPE CORRELATIONS IN MUCOPOLYSACCHARIDOSIS TYPE I USING ENZYME KINETICS, IMMUNOQUANTIFICATION AND IN VITRO TURNOVER STUDIES

Fibroblasts from 16 patients with known alpha-L-iduronidase gene mutations and different clinical phenotypes of mucopolysaccharidosis type I (MPS I) were investigated in order to establish genotype/phenotype correlations. Enzyme kinetic studies were performed using the specific alpha-L-iduronidase substrate iduronosyl anhydro[1-3H]mannitol-6-sulfate. Specific residual enzyme activities were estimated using the kinetic parameters and an immunoquantification assay which determines levels of alpha-L-iduronidase protein. Cells were cultured in the presence of [35S]sulfate and the in vivo degradation of accumulated labelled glycosaminoglycans measured after different chase times. Residual enzyme activity and different amounts of residual enzyme protein were present in extracts from 9 of 16 cell lines covering a wide spectrum of clinical severity. Catalytic capacity, calculated as the product of kcat/Km and ng iduronidase protein per mg cell protein, was shown in most cases to be directly related to the severity of clinical phenotype, with up to 7% of normal values for patients with the attenuated form of MPS I (Scheie) and less than 0.13% for severely affected patients (Hurler) In vitro turnover studies allowed further refinement of correlations between genotype and phenotype. Scheie disease compared to Hurler disease patients were shown to accumulate smaller amounts of glycosaminoglycans that were also turned over faster. A combination of turnover and residual enzyme data established a correlation between the genotype, the biochemical phenotype and the clinical course of this lysosomal storage disorder.

Identification of 31 novel mutations in the N‐acetylgalactosamine‐6‐sulfatase gene reveals excessive allelic heterogeneity among patients with Morquio A syndrome

Mutation analysis of the N‐acetylgalactosamine‐6‐sulfate sulfatase gene was performed in a group of 35 patients with mucopolysaccharidosis type IVA from 33 families, mainly of European origin. By nonradioactive SSCP screening, 35 different gene mutations were identified, 31 of them novel. Together they account for 88.6% of the disease alleles of the patients investigated. The vast majority of the gene alterations proved to be point mutations, 23 missense, 2 nonsense, and 3 affecting splicing. Six small deletions (1–27 bp) and one insertion were also characterized. In a Polish family, two mildly affected siblings were compound heterozygotes for R94G and R259Q. Their mother was homozygous for the latter point mutation, leading to enzyme deficiency and a borderline disease phenotype. Hum Mutat 10:223–232, 1997.

Identification of 16 Sulfamidase Gene Mutations Including the Common R74C in Patients With Mucopolysaccharidosis type IIIA (Sanfilippo A)

Mucopolysaccharidosis type IIIA (MPS IIIA or Sanfilippo A disease) is a storage disorder caused by deficiency of the lysosomal enzyme sulfamidase. Mutation screening, using SSCP/heteroduplex analyses on cDNA and genomic DNA fragments, was performed in a group of 42 European patients. Sixteen of the 17 different gene mutations characterized have not been previously described. The spectrum of gene lesions consists of two 1‐bp deletions (1091delC, 1093delG), an 18‐bp duplication (421ins18), a splice site mutation (IVS2‐2A→G), and 13 different missense point mutations. As in other lysosomal storage disorders, the phenotypic heterogeneity is associated with a considerable genetic heterogeneity. The missense mutation R74C, which alters an evolutionary conserved amino acid in the active site of the enzyme, was found on 56% of alleles of 16 Polish patients, whereas it was less frequent among German patients (21% of disease alleles). R245H, a previously reported common mutation, represents 35% of disease alleles in German patients, but only 3% in Polish patients. As the combined frequency of the common mutations (R74C and R245H) in German and Polish populations exceeds 55%, screening for these two mutations will assist molecular genetic diagnosis of MPS IIIA and allow heterozygote testing in these populations. Hum Mutat 10:479–485, 1997.

Mucopolysaccharidosis type IIIB (Sanfilippo B): identification of 18 novel α-N-acetylglucosaminidase gene mutations

Mucopolysaccharidosis type IIIB (MPS IIIB or Sanfilippo B disease) is an autosomal recessive storage disorder caused by deficiency of the lysosomal enzyme α-N-acetylglucosaminidase. Mutation screening was performed on a group of 22 patients using a combination of SSCP/heteroduplex analysis of amplified genomic fragments and direct sequencing of cDNA fragments. Twenty-one different mutations were identified, 18 of them novel. Together they account for 82% of the disease alleles. The mutation spectrum consists of two small insertions, two small deletions, three nonsense mutations, and 14 different missense mutations, one of them (M1L) affecting the initiation codon. The vast genetic heterogeneity seen in this disorder is reflected by the fact that only three of the mutations were identified in more than one patient.

Evidence for an iduronate-sulfatase pseudogene near the functional Hunter syndrome gene in Xq27.3-q28

We are currently characterizing mutations of the iduronate-2-sulfatase (IDS) gene in patients with Hunter syndrome (mucopolysaccharidosis type II). Surprisingly, all 17 patients with a mutation in exon III of the IDS gene identified by us were found to carry both the mutant and wild-type sequences in polymerase chain reaction (PCR) products amplified from genomic DNA. Similarly, two unaffected male controls showed a heterozygous pattern for two different point mutations in exon III. Collectively, the data suggest that at least intron 2, exon III, and the 3′-half of exon II of the functional IDS gene are present in the human genome as (part of) a non-expressed IDS gene. Deletion mapping further suggests that the pseudogene is in distal Xq in physical proximity to the functional IDS gene. The high degree of sequence homology observed between the functional IDS gene and pseudogene results in permanent co-amplification in PCR-based screening methods and makes mutation analysis at the genomic DNA level difficult.

[3]Simple and nonisotopic methods to detect unknown gene mutations in nucleic acids

Publisher Summary This chapter discusses simple and nonisotopic methods to detect unknown gene mutations in nucleic acids. Regardless of the method used to identify a gene, the demonstration of mutations that cosegregate with the disease phenotype is both an important step and an essential condition toward proving the involvement of the gene (product) in the pathological process. The principle of the method of single-strand conformation polymorphism (SSCP) analysis is that the electrophoretic mobility of single-stranded nucleic acid fragments under nondenaturing conditions depends largely on their conformation. The chapter presents a simple and nonisotopic method that can be carried out in any laboratory without special equipment and combines the SSCP and heteroduplex analyses. This method has been successfully used for mutation screening in 15 different genes responsible for inherited diseases. Because of the invention of highly selective DNA amplification by a thermostable DNA polymerase and repeated cycles of denaturation, primer annealing, and chain elongation, the polymerase chain reaction (PCR) has become one of the most widely used techniques in molecular genetics. The PCR provides fast and highly effective synthesis of any DNA (or cDNA) fragment specified by a pair of primers from a complex mixture of nucleic acids without time-consuming cloning procedures.

Common CFTR mutations are not likely to predispose to chronic bronchitis in northern Germany.

The frequency of six common mutations in the cystic fibrosis transmembrane conductance regulator gene was studied in 100 patients hospitalized with chronic bronchitis. Only one patient with chronic bronchitis and diffuse bronchiectasis was heterozygous for the common ΔF508 mutation. R553X, G542X, G551D, N1303K and 621+1G→T were not detected. This result is not significantly different from the frequency of cystic fibrosis carriers in Northern Europe. Predisposition of heterozygotes for chronic bronchitis is therefore unlikely.

Clinical, biochemical and molecular findings in a two-generation Morquio A family

Mucopolysaccharidosis type IVA (Morquio A) is caused by a deficiency of N‐acetylgalactosamine‐6‐sulfatase (GALNS), an enzyme capable of cleaving the sulfate group from both N‐acetylgalactosamine‐6‐sulfate and galactose‐6‐sulfate. We describe here a two‐generation Morquio A family with two distinct clinical phenotypes. The two probands from the second generation showed intermediate signs of the disease whereas their affected mother, aunt and two uncles had only very mild symptoms. Galactose‐6‐sulfatase (GALS) activity in leukocytes and fibroblasts of the affected family members was clearly deficient. Molecular genetic analysis of the GALNS gene revealed that two different point mutations segregate in the family, which correlated well with the clinical phenotype. The probands with intermediate symptoms were compound heterozygotes for the mutations R259Q and R94G, the latter one being inherited from the unaffected father. The mother and her affected siblings with the unusually mild phenotype were proven to be homozygous for the novel missense point mutation R259Q.

EXTENSIVE GENETIC HETEROGENEITY IN AUTOSOMAL DOMINANT RETINITIS PIGMENTOSA

The most prevalent group of genetically determined progressive retinopathies, currently affecting approximately 1.5 million people, is collectively termed retinitis pigmentosa (RP). RP describes a heterogeneous group of disorders primarily involving photoreceptor degeneration. The rapid development of highly informative DNA polymorphisms as genetic markers throughout the human genome has facilitated the localisation of genes responsible for many human disorders such as RP. Furthermore, techniques for rapid identification of sequence variation have provided an effective means of investigating genes that are considered to be ‘candidates’ for a particular disease in a given patient population. Such techniques have been successfully applied to the study of RP. In this paper, we will deal mainly with the recent developments in the autosomal dominantly inherited forms of RP, in particular highlighting the extensive genetic heterogeneity which we now know to be inherent in this group of diseases.

Phenotypes of Carboxyl-Terminal Rhodopsin Mutations in Autosomal Dominant Retinitis Pigmentosa

We compared the ocular findings in 13 patients from 4 families with autosomal dominant retinitis pigmentosa and 4 different mutations in the carboxyl-terminal sequence of rhodopsin. Phenotypic similarities were found among patients with point mutations predicting the amino acid changes valine-345-methionine, proline-347-serine or proline-347-leucine in the rhodopin molecule. These patients had no measurable rod function and an early impairment of cone function, which was most profound in the proline-347-serine genotype. One patient with a valine-345-methionine mutation showed a regional predilection of fundus abnormalities and cone sensitivity loss. A different phenotype with relatively mild disease expression could be observed in a family with a deletion of 8 base pairs (codons 341–343). One 34 year-old member showed regionally varying rod sensitivity loss, which was less severe in the peripheral visual field, and well maintained cone function, as measured by electroretinography and psychophysical tests.