Genetic heterogeneity in tuberous sclerosis. Study of a large collaborative dataset.

Tuberous sclerosis (TSC) is a multisystem autosomal dominant hamartosis whose genetics is complicated by reduced penetrance and widely varying clinical expression. Results of linkage analyses have variously suggested two different locations for a TSC gene. A collaborative dataset has been assembled to clarify the issue of genetic heterogeneity. We have now analyzed the data from a combined sample of 111 families. Using Ott's HOMOG programs, we completed three tests of homogeneity: (1) for chromosome 9q, (2) for chromosome 11q, and (3) for the combined 9q and 11q data. For test 1 the chi-square (1 df) was 21.54 (p less than 0.001), for test 2 the chi-square (1 df) was 0.13 (p greater than 0.35), and for test 3 the chi-square (2 df) was 37.61 (p less than 0.0001). Additionally, we examined the combined data for evidence that a third, as yet unlinked locus exists. Results of this last test were suggestive but not significant. Clearly loci for TSC are present on both chromosomes 9q and 11q. The maximum likelihood estimate of the proportion of chromosome 9q-linked families is 0.38, for chromosome 11q-linked families is 0.47, and for the unlinked type 0.15. Alternative explanations for these latter families include chance sampling of recombinants, nongenetic phenocopies, or misclassification.

Myotonic dystrophy: correlation of clinical symptoms with the size of the CTG trinucleotide repeat.

An unstable DNA sequence of a gene encoding a protein kinase has been identified as the molecular basis of myotonic dystrophy. The correlation between different symptoms of myotonic dystrophy and the size of this unstable base triplet (CTG)n repeat was investigated in 14 patients. DNA was prepared from whole blood by standard procedures. Detailed clinical, psychological, electrophysiological (quantified measurement of myotonia, electrocardiography) and other laboratory examinations (muscle biopsy in 4 patients, slit lamp examination) were performed. Triplet size correlated significantly with muscular disability and inversely with age at onset of the disease. A greater frequency of mental and gonadal dysfunction could be observed in patients with a larger repeat size. Other symptoms, however, such as cataract, myotonia, gastrointestinal dysfunction and cardiac abnormalities were not correlated with repeat size. Somatic mosaicism with different amplification rates in various tissues might be one possible explanation for the variable phenotypes. Furthermore, other factors such as different expression of the myotonic dystrophy gene might contribute to the clinical variability of the disease at a given triplet size.

The fragile-X phenotype. Computer assisted analysis of the dysmorphological features and discrimination from the Sotos phenotype.

Fragile-X and Sotos phenotypes may be difficult to distinguish. This is illustrated with a case report. Computer assisted phenotype analyses (MDDB), using the complete trait list of this patient, suggested the fragile-X diagnosis, which later was confirmed by molecular techniques. The results of corresponding phenotype analyses are summarized for 17 children with proven fragile-X, and 10 children with suggested Sotos syndrome.

Infratentorial meningioma in an 8-year-old child as first sign of neurofibromatosis type 2.

Meningiomas are rare intracranial tumors in pediatric patients. In contrast to meningiomas in adults, childhood ones have a poorer prognosis because of their high growth potential and tendency to recur. Meningiomas are often associated with neurofibromatosis type 2 (NF2) which is an autosomal-dominant disorder. In contrast to adults who primarily present with symptoms due to vestibular tumors, the initial symptoms in children with NF2 are subtle skin tumors, posterior capsular cataracts, or neurological signs secondary to cranial nerve(s) schwannoma excluding vestibular nerve, and/or brainstem or spinal cord compression. Here we report on the clinical, radiological, and histological findings in an 8-year-old boy who was diagnosed with an isolated infratentorial meningioma and a novel splice site mutation in the NF2 gene. The same mutation was detected in the boy's mother who suffered from hearing loss and tinnitus due to a bilateral vestibular schwannoma. Our patient demonstrates the need for molecular testing for NF2 gene mutations even in isolated childhood meningiomas although they do not fulfill the clinical criteria of NF2.

Tuberous sclerosis in two sibs of normal parents.

We report on a family with two sibs suffering from tuberous sclerosis. The parents were normal in all clinical tests including Wood's light examination of the skin, ophthalmoscopy, X-ray computerized tomography of brain, liver, and kidneys, cardiac echography and MR imaging of the brain. The most likely explanation is a germinal cell mosaic in one of the parents. A recurrence risk of 20 to 37% seems appropriate. The implications for risk assessment of sporadic cases are emphasized.

Tuberous sclerosis in a child with de novo translocation t(3;12) (p26.3;q23.3).

We report on an 8-year-old boy with severe mental retardation, epileptic seizures, autistic behaviour, and X-ray CT findings of the skull characteristics for tuberous sclerosis. At the age of 9 years, first signs of adenoma sebaceum developed. Chromosomal analysis revealed a translocation t(3;12)(p26.3;q23.3). The parents were both healthy and had normal karyotypes. As non-random association of a chromosomal abnormality and tuberous sclerosis is hypothesized, a third locus for this disorder on 3p26 or 12q23 has to be taken in account.

A third gene locus for tuberous sclerosis is closely linked to the phenylalanine hydroxylase gene locus.

Following the observation of a patient suffering from tuberous sclerosis (TSC) with a de novo reciprocal translocation t(3;12) (p26.3;q23.3), we have undertaken a linkage study in 15 TSC families using polymorphic DNA markers neighbouring the chromosome breakpoints. Significant lod scores have been obtained for markers D12S7 (Zmax = 2.34, theta = 0.14) and PAH (phenylalanine hydroxylase (Zmax = 4.34, theta = 0.0). In multipoint linkage analysis, the peak lod score was 4.56 at the PAH gene locus. These data suggest the existence of a third gene locus for TSC (TSC3) on chromosome 12q22-24.1. The regions that have been found to be linked to TSC in different families map to the positions of three enzymes, phenylalanine hydroxylase (12q22-24), tyrosinase (11q14-22), and dopamine-beta-hydroxylase (9q34), all of which are involved in the conversion of phenylalanine to catecholamine neurotransmitters or melanin. Disorders of these biochemical pathways might be involved in the pathogenesis of TSC.

A novel mutation L1425P in the GAP-region of the NF1 gene detected by temperature gradient gel electrophoresis (TGGE). Mutation in brief no. 230. Online.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with an incidence of between 1: 3000 and 1: 4000. Common clinical signs include more than six café-au-lait spots, multiple cutaneous neurofibromas and iris Lisch nodules. Rarer are skeletal anomalies, learning disabilities and an increased risk of malignancy. The NF1 gene contains at least 60 exons with intron sizes ranging from 60 bp to more than 40 kb. Despite using different techniques including PTT, SSCP heteroduplex analyses and direct sequencing, only a relatively small number of mutations have been reported world-wide. Using the more sensitive technique of temperature gradient gel electrophoresis (TGGE), we analysed a part of the NF1-GAP-region, namely exon 25, in DNA samples from 131 unrelated patients. We have identified a novel mutation L1425P in exon 25 of the NF1 gene in a 12-year-old boy (clinically diagnosed with NF1 at the age of 7). In contrast to those cases diagnosed with having both GAP-region mutations and malignant tumours, neither the proband nor four clinically affected family members with this mutation showed any evidence of malignancies.

Familial reciprocal translocation t(17;19) (q11.2;q13.2) associated with neurofibromatosis type 1, including one patient with non-Hodgkin lymphoma and an additional t(14;20) in B lymphocytes.

The cosegregation of a reciprocal translocation t(17;19) (q11.2;13.2) with neurofibromatosis type 1 in three generations suggested that the breakpoint on chromosome 17 involved the NF1 gene. In order to map the breakpoint, we analysed DNAs of patients using parts of the NF1 gene as probes. Southern analysis revealed that the chromosome 17 breakpoint lies within intron 23 of the NF1 gene. One of the patients of the family developed a non-Hodgkin lymphoma. An additional translocation t(14;20) (q32;13.1) in his B lymphocytes points to a gene on chromosome 20 that is juxtaposed to the IGH locus on 14q32, and that may be of relevance for the development of this tumor type.

Rapid detection of trisomy 21 by quantitative PCR.

Chromosomal aneuploidy is a major cause of fetal loss and genetic disease. We have devised a polymerase chain reaction (PCR)-based test that allows prenatal detection of trisomy 21 in as few as 15 fetal cells within 1 day. A pair of fluorescein-tagged primers directs amplification of a 216-bp fragment of the human S100B gene on chromosome 21. Primers that direct amplification of a 165-bp fragment of the IGF1 gene on chromosome 12 are included to generate an internal standard for quantitation. After 31 cycles of PCR, the amounts of S100B and IGF1 amplification products are determined on an Automated Laser Fluorescent DNA Sequencer. In trisomic cells, the relative amount of the S100B product is approximately 1.5-fold higher than that from normal cells. The test may be useful for non-invasive prenatal diagnosis performed on fetal cells isolated from maternal blood.

Nearby stop codons in exons of the neurofibromatosis type 1 gene are disparate splice effectors.

Stop mutations are known to disrupt gene function in different ways. They both give rise to truncated polypeptides because of the premature-termination codons (PTCs) and frequently affect the metabolism of the corresponding mRNAs. The analysis of neurofibromin transcripts from different neurofibromatosis type 1 (NF1) patients revealed the skipping of exons containing PTCs. The phenomenon of exon skipping induced by nonsense mutations has been described for other disease genes, including the CFTR (cystic fibrosis transmembrance conductance regulator) gene and the fibrillin gene. We characterized several stop mutations localized within a few base pairs in exons 7 and 37 and noticed complete skipping of either exon in some cases. Because skipping of exon 7 and of exon 37 does not lead to a frameshift, PTCs are avoided in that way. Nuclear-scanning mechanisms for PTCs have been postulated to trigger the removal of the affected exons from the transcript. However, other stop mutations that we found in either NF1 exon did not lead to a skip, although they were localized within the same region. Calculations of minimum-free-energy structures of the respective regions suggest that both changes in the secondary structure of the mRNA and creation or disruption of exonic sequences relevant for the splicing process might in fact cause these different splice phenomena observed in the NF1 gene.

Mutational analysis and expression studies of the neurofibromatosis type 2 (NF2) gene in a patient with a ring chromosome 22 and NF2.

The case of a seriously disabled and retarded female patient with neurofibromatosis type 2 (NF2) is reported. She suffered from bilateral vestibular schwannomas, multiple intracranial meningiomas and neurinomas. The constitutional karyotype of the patient was 46, XX, r(22)/45,XX,-22. A constitutional G to A transition in the proximal 3' untranslated region of isoforms 1 and 2 was identified in the patient's NF2 gene and shown not to affect differential splicing or mRNA stability. The instability of the ring chromosome 22 with the associated loss of tumor suppressor genes on chromosome 22, in particular the loss of the NF2 gene, are assumed to have caused multiple tumorigenesis in this patient.

Analysis of the NF1 gene by temperature gradient gel electrophoresis reveals a high incidence of mutations in exon 4b.

A total of 196 unrelated patients with neurofibromatosis type 1 (NF1) was screened for mutations in exons 4a-c of the NF1 gene by temperature gradient gel electrophoresis (TGGE) of polymerase chain reaction (PCR)-amplified genomic DNA fragments using intron-based primers. DNA samples with abnormal TGGE band patterns were subjected to sequence analysis. Sequence alterations were identified in ten patients (5.1%): 496delGT (1), 499delTGTT (4), T528A = D176E (2), T539A = L180X (1), 540insA (1), C574T = R192X (1). Thus, a total of six different mutations was identified in exon 4b but none in exons 4a and 4c. Only the missense mutation D176E, which we assume to be a nonpathogenic polymorphism, and the 4-base pair (bp) deletion 499delTGTT have been described before. The reason for the high incidence of mutations in exon 4b is obviously a tetranucleotide tandem repeat comprising nucleotides 495-502 (TGTTTGTT) that may give rise to slipped mispairing and subsequent deletion of one repeat unit during replication. Additionally, the recurrent 4 bp deletion was found as a second hit in a malignant schwannoma of a further NF1 patient, suggesting that microlesions may be as frequent among somatic as among germline mutations. This is the first report of a systematic study of NF1 exons 4a-c in a large group of NF1 patients.

Establishment and characterization of a granulocyte-macrophage colony-stimulating factor-dependent human myeloid cell line.

A new human myeloid cell line has been established recently from the bone marrow cells of a patient with chronic myelogenous leukemia in blast crisis. The active proliferation and survival of the cells in RPMI 1640 medium containing fetal calf serum are clearly dependent on the presence of either natural or recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Despite permanent culturing in rhGM-CSF (100 U/mL), the cells do not differentiate and bear the myelomonocytic surface markers CD34, CD13, CD36, as well as HLA-DR, but not CD3, CD7, CD10, CD11b, CD14, CD20, or CD42b. The predominant karyotype, apart from tetraploidy in several cells, is 45, XX, -9, -17, -19, -22, 7p-, 9q+ (der t[9;22]), der (13q), with three additional marker chromosomes, from which one was observed in the patient's leukemic cells. On BglII-digested DNA, Southern blot analysis with bcr 5' as the probe detected two additional hybridizing restriction fragments of 8.6 and 11.0 kilobase pairs.

Spinal neurofibromatosis without café-au-lait macules in two families with null mutations of the NF1 gene.

Spinal neurofibromatosis (SNF) is considered to be an alternative form of neurofibromatosis, showing multiple spinal tumors and café-au-lait macules. Involvement of the neurofibromatosis type 1 (NF1) locus has been demonstrated, by linkage analysis, for three families with SNF. In one of them, a cosegregating frameshift mutation in exon 46 of the NF1 gene was identified. In the present study, we report four individuals from two families who carry NF1 null mutations that would be expected to cause NF1. Three patients have multiple spinal tumors and no café-au-lait macules, and the fourth has no clinical signs of NF1. In the first family, a missense mutation (Leu2067Pro) in NF1 exon 33 was found, and, in the second, a splice-site mutation (IVS31-5A-->G) enlarging exon 32 by 4 bp at the 5' end was found. The latter mutation has also been observed in an unrelated patient with classical NF1. Both NF1 mutations cause a reduction in neurofibromin of approximately 50%, with no truncated protein present in the cells. This demonstrates that typical NF1 null mutations can result in a phenotype that is distinct from classical NF1, showing only a small spectrum of the NF1 symptoms, such as multiple spinal tumors, but not completely fitting the current clinical criteria for SNF. We speculate that this phenotype is caused by an unknown modifying gene that compensates for some, but not all, of the effects caused by neurofibromin deficiency.

New markers for the neurofibromatosis-2 region generated by microdissection of chromosome 22.

To identify new DNA markers around the neurofibromatosis-2 gene on human chromosome 22, the critical region (22q12-q13.1) was microdissected and microcloned from GTG-banded metaphase chromosomes. Eighteen thousand recombinant clones were obtained. Twenty-seven of 55 clones tested (50%) detected single-copy DNA sequences. Nine of nine clones analyzed in detail were found to map to chromosome 22. Interestingly one clone (EAN04) is part of the leukemia inhibitory factor gene which has previously been mapped to 22q11.2-q13.1. Four clones (EAN01, EAN47, EAN57, and EAN68) detect DNA polymorphisms. These probes were used to compare constitutional and tumor genotypes of 41 patients with acoustic neurinoma. Loss of constitutional heterozygosity was identified in 17 of 31 informative cases (55%). From our data we conclude that the microdissection library is a valuable resource for physical and genetic mapping studies in neurofibromatosis-2.

Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain.

More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, <20% represent C-->T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles played by exons 12a and 15 and part of exon 16.