Electron microscopy as a means for carrier detection and genetic counselling in families at risk of tuberous sclerosis

Summary In otherwise asymptomatic parents of two unrelated children severely affected with tuberous sclerosis (TS), the ultrastructure of hypomelanotic skin lesions was investigated. In the hypomelanotic macules of both mothers of the two families, the population density of melanocytes was normal, melanization, however, was markedly suppressed. Therefore, these macules represent typical white leaf-shaped macules characteristic of tuberous sclerosis. Especially by applying the dihydroxyphenylalanine (DOPA) reaction to the ultrastructural level, it was possible to separate the TS-macules from the vitiliginous lesions of one father with a decrease of functional melanocytes as well as from other congenital circumscribed hypomelanoses. Thus electron microscopy in combination with the DOPA reaction may be helpful in recognizing a forme fruste of the dominantly inherited tuberous sclerosis (i.e. identification of white leaf-shaped macules) to enable genetic counselling and family planning by marking carriers of the mutant TS gene amongst the relatives of a patient and to exclude a sporadic mutation.     

Differential ultrastructural aberrations of collagen fibrils in Ehlers-Danlos syndrome types I–IV as a means of diagnostics and classification

Among the different subtypes of Ehlers-Danlos syndrome (EDS), the dominant types I–III have, so far, been uninformative biochemically and molecular genetically, and diagnostic problems with subgroup boundaries often arise. We have investigated the ultrastructural pattern of connective tissue macromolecules in skin biopsy specimens of some 85 patients aged 4 months-54 years who exhibit clinical symptoms or the suspicion of EDS I–IV. Based on the differential features of collagen fibrils and ground substance material, four distinct groups could be established. Group I (clinically EDS type I) showed disorganized collagen bundles and dense aggregations of collagen fibrils with bizarre shapes. Group II (clinically varying from EDS types I–III) revealed collagen bundles that regularly contained numerous “composite collagen fibrils” with enlarged “flower-like” cross-sections and rope-like longitudinal sections, often associated with increased amounts of matrix substances in the form of electron-dense irregular strands and filaments in a branched network. Group III (clinically EDS types II–III) presented smaller isolated collagen flowers and ropes associated with excessive filamentous ground substance material and flocculent material. Group IV (with clinical symptoms of EDS type IV) had a dermis thinned to one third of the normal and a reduced number of collagen bundles with small diameter fibrils. In 13 patients, the abnormal ultrastructural dermal architecture did not coincide with any of these four groups or with the pattern of any other inherited connective tissue disorder. In 16 additional patients with mostly mild clinical symptoms, such as muscle weakness and small joint hyperlaxity, no ultrastructural aberrations could be found. Even though the primary defects underlying the respective aberration of the collagen fibrils are still unknown, the differential ultrastructural changes of the collagen fibrils together with clinical symptoms should, as in other heterogeneous genetic disorders, facilitate the (provisional?) classification of EDS and permit the diagnosis of individual cases.     

A new Schizosaccharomyces pombe base excision repair mutant,nth1, reveals overlapping pathways for repair of DNA base damage

Endonuclease III (Nth) enzyme from Escherichia coli is involved in base excision repair of oxidised pyrimidine residues in DNA. The Schizosaccharomyces pombe Nth1 protein is a sequence and functional homologue of E. coli Nth, possessing both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activity. Here, we report the construction and characterization of the S. pombe nth1 mutant. The nth1 mutant exhibited no enhanced sensitivity to oxidising agents, UV or gamma-irradiation, but was hypersensitive to the alkylating agent methyl methanesulphonate (MMS). Analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea showed that the purified Nth1 enzyme did not remove alkylated bases such as 3-methyladenine and 7-methylguanine whereas methyl-formamidopyrimidine was excised efficiently. The repair of AP sites in S. pombe has previously been shown to be independent of Apn1-like AP endonuclease activity, and the main reason for the MMS sensitivity of nth1 cells appears to be their lack of AP lyase activity. The nth1 mutant also exhibited elevated frequencies of spontaneous mitotic intrachromosomal recombination, which is a phenotype shared by the MMS-hypersensitive DNA repair mutants rad2, rhp55 and NER repair mutants rad16, rhp14, rad13 and swi10. Epistasis analyses of nth1 and these DNA repair mutants suggest that several DNA damage repair/tolerance pathways participate in the processing of alkylation and spontaneous DNA damage in S. pombe.     

Schizosaccharomyces pombe encodes a mutated AP endonuclease 1

Mutagenic and cytotoxic apurinic/apyrimidinic (AP) sites are among the most frequent lesions in DNA. Repair of AP sites is initiated by AP endonucleases and most organisms possess two or more of these enzymes. Saccharomyces cerevisiae has AP endonuclease 1 (Apn1) as the major enzymatic activity with AP endonuclease 2 (Apn2) being an important backup. Schizosaccharomyces pombe also encodes two potential AP endonucleases, and Apn2 has been found to be the main repair activity, while Apn1 has no, or only a limited role in AP site repair. Here we have identified a new 5' exon (exon 1) in the apn1 gene and show that the inactivity of S. pombe Apn1 is due to a nonsense mutation in the fifth codon of this new exon. Reversion of this mutation restored the AP endonuclease activity of S. pombe Apn1. Interestingly, the apn1 nonsense mutation was only found in laboratory strains derived from L972 h(-) and not in unrelated isolates of S. pombe. Since all S. pombe laboratory strains originate from L972 h(-), it appears that all experiments involving S. pombe have been conducted in an apn1(-) mutant strain with a corresponding DNA repair deficiency. These observations have implications both for future research in S. pombe and for the interpretation of previously conducted epistatis analysis.     

Problems in prenatal diagnosis of the ichthyosis congenita group

Summary The late onset of normal keratinization after week 24 menstrual age (MA) of fetal life is the cause of considerable problems with the prenatal diagnosis of congenital ichthyosis. This paper summarizes the experiences with prenatal diagnosis in nine pregnancies at risk of congenital ichthyosis and one at risk of chondrodysplasia punctata, rhizomelic type. An important prerequisite—and the main problem—is the manifestation of the mutant genes early enough in fetal life to allow a safe exclusion. Continuous precocious keratinization of the interfollicular epidermis, hyperkeratosis, and/or specific markers of congenital ichthyosis such as various types of lipid inclusions had been expected. With a normal ultrastructure and development of fetal epidermis no evidence of ichthyosis was present in eight cases; all eight children were born healthy. Regional variations of the onset of keratinization of the interfollicular epidermis, observed in one of these eight fetuses as well as in one fetus at risk (but normal for) recessive dystrophic epidermolysis bullosa, posed considerable problems and might lead to a false-positive diagnosis. Examination after birth allowed one to localize these regions to areas close to the mamillae. Regional variations in addition to the well-known cranio-caudal gradient thus are normal findings: both children have normal skin. One fetus at risk of nonbullous congenital ichthyosiform erythroderma (type II) was involved without prenatal manifestation of interfollicular keratinization, specific markers, or increased numbers of cornified cells in the pilosebaceous follicles at 20 weeks MA. A slightly more irregular pattern of the horn cell contents was not regarded as sufficient evidence alone to indicate congenital ichthyosis. A severely affected boy was born in week 34 MA. Similarly the fetus at risk of chondrodysplasia punctata showed no skin abnormalities, neither at fetoscopy (week 22 MA) nor after abortion (week 24 MA) although based on other clinical features it was clearly affected. Thus, this genodermatosis cannot be diagnosed prenatally by its keratinization disturbances. In future cases, precocious keratinization and hyperkeratosis cannot be expected to be expressed before week 24 MA, and minor signs, such as irregularities of horn cell contents, have to be taken as an indication of involvement. Multiple biopsies are required, and a safe exclusion may be impossible before week 22 MA.     

Mutations in the COL5A1 gene are causal in the Ehlers-Danlos syndromes I and II.

The Ehlers-Danlos syndrome (EDS) is a heterogeneous connective-tissue disorder of which at least nine subtypes are recognized. Considerable clinical overlap exists between the EDS I and II subtypes, suggesting that both are allelic disorders. Recent evidence based on linkage and transgenic mice studies suggest that collagen V is causally involved in human EDS. Collagen V forms heterotypic fibrils with collagen I in many tissues and plays an important role in collagen I fibrillogenesis. We have identified a mutation in COL5A1, the gene encoding the pro(alpha)1(V) collagen chain, segregating with EDS I in a four-generation family. The mutation causes the substitution of the most 5' cysteine residue by a serine within a highly conserved sequence of the pro(alpha)1(V) C-propeptide domain and causes reduction of collagen V by preventing incorporation of the mutant pro(alpha)1(V) chains in the collagen V trimers. In addition, we have detected splicing defects in the COL5A1 gene in a patient with EDS I and in a family with EDS II. These findings confirm the causal role of collagen V in at least a subgroup of EDS I, prove that EDS I and II are allelic conditions, and represent a, so far, unique example of a human collagen disorder caused by substitution of a highly conserved cysteine residue in the C-propeptide domain of a fibrillar collagen.     

The Streptomyces lividans 66 chromosome contains a 1 MB deletogenic region flanked by two amplifiable regions

Genetic instability in Streptomyces species often involves large deletions sometimes accompanied by DNA amplification. Two such systems in Streptomyces lividans 66 involve the production of mutants sensitive to chloramphenicol and the production of mutants resistant to the galactose analogue 2-deoxygalactose, respectively. Overlapping cosmids were isolated that span the ca. 1 Mb region between the two amplifiable regions. The structure of the region was confirmed by restriction mapping using the rarely cutting enzymes AseI, BfrI and DraI and pulsed-field gel electrophoresis. The region contains a non-clonable gap flanked by inverted repeats; the structure is consistent with the presence of a physical gap, i.e. a linear chromosome.     

Biochemical characterization and DNA repair pathway interactions of Mag1-mediated base excision repair in Schizosaccharomyces pombe

The Schizosaccharomyces pombe mag1 gene encodes a DNA repair enzyme with sequence similarity to the AlkA family of DNA glycosylases, which are essential for the removal of cytotoxic alkylation products, the premutagenic deamination product hypoxanthine and certain cyclic ethenoadducts such as ethenoadenine. In this paper, we have purified the Mag1 protein and characterized its substrate specificity. It appears that the substrate range of Mag1 is limited to the major alkylation products, such as 3-mA, 3-mG and 7-mG, whereas no significant activity was found towards deamination products, ethenoadducts or oxidation products. The efficiency of 3-mA and 3-mG removal was 5–10 times slower for Mag1 than for Escherichia coli AlkA whereas the rate of 7-mG removal was similar to the two enzymes. The relatively low efficiency for the removal of cytotoxic 3-methylpurines is consistent with the moderate sensitivity of the mag1 mutant to methylating agents. Furthermore, we studied the initial steps of Mag1-dependent base excision repair (BER) and genetic interactions with other repair pathways by mutant analysis. The double mutants mag1 nth1, mag1 apn2 and mag1 rad2 displayed increased resistance to methyl methanesulfonate (MMS) compared with the single mutants nth1, apn2 and rad2, respectively, indicating that Mag1 initiates both short-patch (Nth1-dependent) and long-patch (Rad2-dependent) BER of MMS-induced damage. Spontaneous intrachromosomal recombination frequencies increased 3-fold in the mag1 mutant suggesting that Mag1 and recombinational repair (RR) are both involved in repair of alkylated bases. Finally, we show that the deletion of mag1 in the background of rad16, nth1 and rad2 single mutants reduced the total recombination frequencies of all three double mutants, indicating that abasic sites formed as a result of Mag1 removal of spontaneous base lesions are substrates for nucleotide excision repair, long- and short-patch BER and RR.