[Phacomatoses: structural substare of epilepsy]. / Fakomatozy--strukturalne podloze padaczki.

AIM: Evaluation of frequency and characteristics of seizures in the most frequent phacomatoses and assessment of relationship between fits and structural changes in CNS. MATERIAL AND METHODS: 135 children with tuberous sclerosis (TS), 73 with NF-1 and 30 with Sturge-Weber syndrome took part in the study. Except for careful anamnesis in all patients with fits were done brain CT or MR studies. RESULTS: Seizures were reported in 128 of 135 (95%) patients with TS, usually, between 3rd and 6th month of life. Their early presentation was related to developmental delay. Cortical and subcortical lesions detected in neuroimaging studies were responsible for drug-resistant epilepsy in the children. 13 of 73 (18%) children with NF-1 had seizures. In 9 of them CNS lesions were detected on neuroimaging. In Sturge-Weber syndrome inherited meningo-encephalic lesions correlated with hemilateral seizures, even in first months of life. Most children did not show apparent developmental delay. CONCLUSIONS: Epileptic seizures in phacomatoses had their own specificity. They were correlated with structural lesions in CNS.

[Tests for loss of heterozygosity in tuberous sclerosis]. / Badania nad utrata heterozygotycznosci w stwardnieniu guzowatym.

The authors assessed loss of heterozygosity in TSC1 and TSC2 genes in three patients undergoing resection of TSC hamartomas: two sub-ependymal giant cell astrocytomas and one kidney angiomyolipoma. Loss with heterozygosity was found only in the patient with kidney angiomyolipoma. A germline mutation, TSC2 E35 645 > A 1549Y > N missense mutation was identified by DHPLC and direct genome sequencing in the blood and loss of the normal allele was demonstrated in the tumor. In one of the patients with brain tumors no germline mutation was identified in the blood, but a heterozygous TSC2 E14 1516C > T 505R > X nonsense mutation was found in the SEGA. Another patient demonstrated germline mutation in TSC2 E38 5116C > T 1706R > C, but tumor DNA indicated that there was retention of heterozygosity for this mutation. The presence of LOH in internal organ tumors is consistent with the Knudson's two-hit model in TS. The frequency of LOH depends on the type of tumor and type of mutation (TSC1 or TSC2).

Superiority of denaturing high performance liquid chromatography over single-stranded conformation and conformation-sensitive gel electrophoresis for mutation detection in TSC2.

We evaluated denaturing high pressure liquid chromatography (DHPLC) as a scanning method for mutation detection in TSC2, and compared it to conformation-sensitive gel electrophoresis (CSGE) and single-stranded conformation polymorphism analysis (SSCP). The first 20 exons of TSC2 were amplified from 84 TSC patients and screened initially by CSGE and then by DHPLC. Optimization of DHPLC analysis of each exon was carried out by design of primers with minimum variation in the melting temperature of the amplicon, and titration of both elution gradient and temperature. CSGE analysis identified 40 shifts (21 unique) in the 84 patients and 20 exons. All of these variants were detected by DHPLC, and an additional 27 changes (14 unique) were identified. Overall 15 of 28 (54%) unique single base substitutions were detected by CSGE; all were detected by DHPLC. 25 definite or probable mutations were found in these 84 patients (30%) in exons 1-20 of TSC2. In a subsequent blinded analysis of 15 samples with 18 distinct TSC2 sequence variants originally detected by SSCP in another centre, all variants were detected by DHPLC except one where the variation occurred within the primer. Ten other (7 unique) sequence variants were detected in these samples which had not been detected by SSCP. Overall, 11 of 16 (69%) unique single base substitutions were detected by SSCP; all were detected by DHPLC. We conclude that DHPLC is superior to both CSGE and SSCP for detection of DNA sequence variation in TSC2, particularly for single base substitution mutations.

Mutational analysis in a cohort of 224 tuberous sclerosis patients indicates increased severity of TSC2, compared with TSC1, disease in multiple organs.

Tuberous sclerosis (TSC) is a relatively common hamartoma syndrome caused by mutations in either of two genes, TSC1 and TSC2. Here we report comprehensive mutation analysis in 224 index patients with TSC and correlate mutation findings with clinical features. Denaturing high-performance liquid chromatography, long-range polymerase chain reaction (PCR), and quantitative PCR were used for mutation detection. Mutations were identified in 186 (83%) of 224 of cases, comprising 138 small TSC2 mutations, 20 large TSC2 mutations, and 28 small TSC1 mutations. A standardized clinical assessment instrument covering 16 TSC manifestations was used. Sporadic patients with TSC1 mutations had, on average, milder disease in comparison with patients with TSC2 mutations, despite being of similar age. They had a lower frequency of seizures and moderate-to-severe mental retardation, fewer subependymal nodules and cortical tubers, less-severe kidney involvement, no retinal hamartomas, and less-severe facial angiofibroma. Patients in whom no mutation was found also had disease that was milder, on average, than that in patients with TSC2 mutations and was somewhat distinct from patients with TSC1 mutations. Although there was overlap in the spectrum of many clinical features of patients with TSC1 versus TSC2 mutations, some features (grade 2-4 kidney cysts or angiomyolipomas, forehead plaques, retinal hamartomas, and liver angiomyolipomas) were very rare or not seen at all in TSC1 patients. Thus both germline and somatic mutations appear to be less common in TSC1 than in TSC2. The reduced severity of disease in patients without defined mutations suggests that many of these patients are mosaic for a TSC2 mutation and/or have TSC because of mutations in an as-yet-unidentified locus with a relatively mild clinical phenotype.

Comprehensive mutational analysis of the TSC1 gene: observations on frequency of mutation, associated features, and nonpenetrance.

We performed a comprehensive analysis for mutations in the TSC1 gene using Southern blot analysis, and SSCP and heteroduplex analysis of amplified exons in 13 families with genetic linkage to the TSC1 region, 22 small families without linkage information, and 126 sporadic patients. 17 unique mutations were identified in 21 patients. Mutations were found in 7/13 (54%) TSC1-linked families, 1/22 (5%) small families without linkage, and 13 of 126 (10%) sporadic cases. The mutations were all chain-terminating, with 14 small deletions, 1 small insertion, and 6 nonsense mutations. In families with mutations, all individuals carrying a mutation met formal diagnostic criteria for TSC, apart from a 3-year-old girl who had inherited a deletion mutation, and who had no seizures, normal intelligence, normal abdominal ultrasound, and hypomelanotic macules only on physical exam. We assessed the incidence and severity of mental retardation in the 13 sporadic patients with TSC1 mutations versus the entire sporadic cohort, and found no significant difference. The observations indicate that TSC1 mutations are all inactivating, suggest that TSC1 disease occurs in only 15-20% of the sporadic TSC population, and demonstrate that presymptomatic TSC does occur.