Genetic heterogeneity in tuberous sclerosis

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by widespread hamartosis. Preliminary evidence of linkage between the TSC locus and markers on chromosome 9q34 was established, but subsequently disputed. More recently, a putative TSC locus on chromosome 11 has been suggested and genetic heterogeneity seems likely. Here we describe an approach combining multipoint linkage analysis and heterogeneity tests that has enabled us to obtain significant evidence for locus heterogeneity after studying a relatively small number of families. Our results support a model with two different loci independently causing the disease. One locus (TSC1) maps in the vicinity of the Abelson oncogene at 9q34 and a second locus (TSC2) maps in the region of the anonymous DNA marker Lam L7 and the dopamine D2 receptor gene at 11q23.     

Analysis of TSC1 truncations defines regions involved in TSC1 stability,aggregation and interaction

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, interact to form a protein complex that inhibits signal transduction to the downstream effectors of the target of rapamycin complex 1 (TORC1). Here we investigate TSC1 structure and function by analysing a series of truncated TSC1 proteins. We identify specific regions of the protein that are important for TSC1 stability, localisation, interactions and function.     

Localization of one gene for tuberous sclerosis within 9q32-9q34, and further evidence for heterogeneity.

Tuberous sclerosis (TSC) is an autosomal dominant disorder with both neurological and cutaneous manifestations often resulting in significant disability. Although it has been studied clinically and biochemically for many years, the underlying pathophysiology remains unknown. Genetic linkage analysis provides an alternative strategy for understanding the genetic etiology of this disease. Genetic linkage of a gene for TSC to loci in 9q32-9q34 has been reported but has not been a universal finding, since absence of linkage to 9q loci, as well as linkage to loci on 11q, have also been reported. We present here data on 22 families (21 previously unreported) segregating TSC. Our results strongly support a TSC locus in the 9q32-34 region for approximately one-third of families and provide significant evidence for genetic heterogeneity. Application of newly described highly polymorphic dinucleotide repeat marker loci in TSC greatly enhanced the informativeness of our pedigrees and was vital for detecting the heterogeneity. No clear evidence of linkage to chromosome 11q22 markers was found, suggesting that a still unidentified TSC locus elsewhere in the genome may account for the majority of TSC families.     

The tuberous sclerosis genes and regulation of the cyclin-dependent kinase inhibitor p27

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor syndrome that affects approximately 1 in 6000 individuals. It is characterized by the development of tumors, named hamartomas, in the kidneys, heart, skin and brain. The latter often cause seizures, mental retardation, and a variety of developmental disorders, including autism. This disease is caused by mutations within the tumor suppressor gene TSC1 on chromosome 9q34 encoding hamartin or within TSC2 on chromosome 16p13.3 encoding tuberin. TSC patients carry a mutant TSC1 or TSC2 gene in each of their somatic cells, and loss of heterozygosity has been documented in a wide variety of TSC tumors. Recent data suggest that functional inactivation of TSC proteins might also be involved in the development of other diseases not associated with TSC, such as sporadic bladder cancer, breast cancer, ovarian carcinoma, gall bladder carcinoma, non-small-cell carcinoma of the lung, and Alzheimer's disease. Tuberin and hamartin form a heterodimer, suggesting they might affect the same processes. Tuberin is assumed to be the functional component of the complex and has been implicated in the regulation of different cellular functions. The TSC proteins regulate cell size control due to their involvement in the insulin signalling pathway. Furthermore, they are potent positive regulators of the cyclin-dependent kinase inhibitor p27, a major regulator of the mammalian cell cycle. Here we review the current knowledge on how mutations within the TSC genes could trigger deregulation of stability and localization of the tumor suppressor p27.     

Refined localization of TSC1 by combined analysis of 9q34 and 16pl3 data in 14 tuberous sclerosis families

Tuberous sclerosis (TSC) is a heterogeneous trait. Since 1990, linkage studies have yielded putative TSC loci on chromosomes 9, 11, 12 and 16. Our current analysis, performed on 14 Dutch and British families, reveals only evidence for loci on chromosome 9q34 (TSC1) and chromosome 16p13 (TSC2). We have found no indication for a third locus for TSC, linked or unlinked to either of these chromosomal regions. The majority of our families shows linkage to chromosome 9. We have refined the candidate region for TSC1 to a region of approximately 5 cM between ABL and ABO.     

Evidence for genetic heterogeneity in tuberous sclerosis: one locus on chromosome 9 and at least one locus elsewhere.

Linkage of tuberous sclerosis complex (TSC), an autosomal dominant disorder, to markers on chromosome 9 was reported first in 1987. This assignment was confirmed by an international collaborative study that suggested more than one locus may be responsible for the phenotype. We studied 14 multigenerational TSC families (13 previously unreported) with markers for nine loci in the linked region of chromosome 9q32-q34. Our results confirm the previous reports that the genetic locus in one-third to one-half of families maps to chromosome 9. Comparison of clinical findings in the chromosome 9-linked families with those in the chromosome 9-unlinked families reveals only a higher incidence of ungual fibromata in the chromosome 9-linked families.     

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

Summary 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 (z _max=2.34, =0.14) and PAH (phenylalanine hydroxylase) (z _max=4.34, =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.     

The TOR1A (DYT1) gene family and its role in early onset torsion dystonia

Most cases of early onset torsion dystonia are caused by a 3-bp deletion (GAG) in the coding region of the TOR1A gene (alias DYT1, DQ2), resulting in loss of a glutamic acid in the carboxy terminal of the encoded protein, torsin A. TOR1A and its homologue TOR1B (alias DQ1) are located adjacent to each other on human chromosome 9q34. Both genes comprise five similar exons; each gene spans a 10-kb region. Mutational analysis of most of the coding region and splice junctions of TOR1A and TOR1B did not reveal additional mutations in typical early onset cases lacking the GAG deletion (N = 17), in dystonic individuals with apparent homozygosity in the 9q34 chromosomal region (N = 5), or in a representative Ashkenazic Jewish individual with late onset dystonia, who shared a common haplotype in the 9q34 region with other late onset individuals in this ethnic group. A database search revealed a family of nine related genes (50-70% similarity) and their orthologues in species including human, mouse, rat, pig, zebrafish, fruitfly, and nematode. At least four of these genes occur in the human genome. Proteins encoded by this gene family share functional domains with the AAA/HSP/Clp-ATPase superfamily of chaperone-like proteins, but appear to represent a distinct evolutionary branch.     

Evidence that lymphangiomyomatosis is caused by TSC2 mutations: chromosome 16p13 loss of heterozygosity in angiomyolipomas and lymph nodes from women with lymphangiomyomatosis.

Lymphangiomyomatosis (LAM) is a rare disease, of unknown etiology, affecting women almost exclusively. Lung transplantation is the only consistently effective therapy for LAM. Microscopically, LAM consists of a diffuse proliferation of smooth muscle cells. LAM can occur without evidence of other disease (referred to as "sporadic LAM") or in association with tuberous sclerosis complex (TSC). TSC is an autosomal dominant tumor suppressor gene syndrome characterized by seizures, mental retardation, and tumors in the brain, heart, skin, and kidney. Renal angiomyolipomas occur in approximately 50% of sporadic LAM patients and in 70% of TSC patients. Loss of heterozygosity (LOH) in the chromosomal region for the TSC2 gene occurs in 60% of TSC-associated angiomyolipomas. Because of the similar pulmonary and renal manifestations of TSC and sporadic LAM, we hypothesized that LAM and TSC have a common genetic basis. We analyzed renal angiomyolipomas, from 13 women with sporadic LAM, for LOH in the regions of the TSC1 (chromosome 9q34) and TSC2 (chromosome 16p13) genes. TSC2 LOH was detected in seven (54%) of the angiomyolipomas. We also found TSC2 LOH in four lymph nodes from a woman with retroperitoneal LAM. No TSC1 LOH was found. Our findings indicate that the TSC2 gene may be involved in the pathogenesis of sporadic LAM. However, genetic transmission of LAM has not been reported. Women with LAM may have low-penetrance germ-line TSC2 mutations, or they may be mosaic, with TSC2 mutations in the lung and the kidney but not in other organs.     

Characterization of a Kinesin-Related Gene ATSV, within the Tuberous Sclerosis Locus (TSC1) Candidate Region on Chromosome 9Q34

In the search for candidate genes for the tuberous sclerosis (TSC1) disease locus on chromosome 9q34, we have isolated an overlapping series of 22 plasmid and phage cDNA clones covering nearly 7 kb and with an open reading frame of 5070 bp encoding a protein of 1690 amino acids. The putative protein product is a member of the kinesin superfamily and is homologous to the mouse KIF1A and theCaenorhabditas elegansunc-104 genes. Both KIF1A and unc-104 function in the anterograde axonal transport of synaptic vesicles. The human homolog is therefore termed H-ATSV (axonal transporter of synaptic vesicles, HGMW-approved nomenclature ATSV) Screening of DNA from 107 tuberous sclerosis patients and 80 unaffected individuals with H-ATSV cDNA probes by pulsed-field gel electrophoresis/Southern blotting following digestion by rare-cutting methylation-sensitive restriction enzymes showed variant banding patterns in three patients with tuberous sclerosis. However, further analysis indicated that these variant fragments represent a rare polymorphism probably associated with methylation of clustered restriction sites. There is no evidence to support H-ATSV as a candidate gene for TSC1.     

Allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions.

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation, and hamartomatous lesions. Although hamartomas can occur in almost any organ, they are most common in the brain, kidney, heart, and skin. Allelic loss or loss of heterozygosity (LOH) in TSC lesions has previously been reported on chromosomes 16p13 and 9q34, the locations of the TSC2 and TSC1 genes, respectively, suggesting that the TSC genes act as tumor-suppressor genes. In our study, 87 lesions from 47 TSC patients were analyzed for LOH in the TSC1 and TSC2 chromosomal regions. Three findings resulted from this analysis. First, we confirmed that the TSC1 critical region is distal to D9S149. Second, we found LOH more frequently on chromosome 16p13 than on 9q34. Of the 28 patients with angiomyolipomas or rhabdomyomas, 16p13 LOH was detected in lesions from 12 (57%) of 21 informative patients, while 9q34 LOH was detected in lesions from only 1 patient (4%). This could indicate that TSC2 tumors are more likely than TSC1 tumors to require surgical resection or that TSC2 is more common than TSC1 in our patient population. It is also possible that small regions of 9q34 LOH were missed. Lastly, LOH was found in 56% of renal angiomyolipomas and cardiac rhabdomyormas but in only 4% of TSC brain lesions. This suggests that brain lesions can result from different pathogenic mechanisms than kidney and heart lesions.     

A radiation-reduced hybrid cell line containing 5 Mb/17 cM of human DNA from 9q34.

Disease gene loci for tuberous sclerosis (TSC1), idiopathic torsion dystonia (DYT1), and nail-patella syndrome (NPS1) have been mapped by genetic linkage analysis to human chromosome 9q band 34. To create a resource for physical mapping and manipulation of this region of the genome, we have created a radiation-reduced hybrid cell line containing DNA from human 9q34 as its only human component. This cell line, E6B, has been characterized by Southern blot and PCR analysis using a panel of 9q markers and fluorescent in situ hybridization. We estimate that it contains 5 Mb of human DNA, equal to 17 cM of genetic distance, extending from AK1 to ABO on 9q34.     

Molecular analysis of chromosome 9q deletions in two Gorlin syndrome patients.

Gorlin syndrome is an autosomal dominant disorder characterized by multiple basal cell carcinomas, medulloblastomas, ovarian fibromas, and a variety of developmental defects. All affected individuals share certain key features, but there is significant phenotypic variability within and among kindreds with respect to malformations. The gene (NBCCS) maps to chromosome 9q22, and allelic loss at this location is common in tumors from Gorlin syndrome patients. Two recessive cancer-predisposition syndromes, xeroderma pigmentosum group A (XPAC) and Fanconi anemia group C (FACC), map to the NBCCS region; and unusual, dominant mutations in these genes have been proposed as the cause of Gorlin syndrome. This study presents cytogenetic and molecular characterization of germ-line deletions in one patient with a chromosome 9q22 deletion and in a second patient with a deletion of 9q22-q3l. Both have typical features of Gorlin syndrome plus additional findings, including mental retardation, conductive hearing loss, and failure to thrive. That Gorlin syndrome can be caused by null mutations (deletions) rather than by activating mutations has several implications. First, in conjunction with previous analyses of allelic loss in tumors, this study provides evidence that associated neoplasms arise with homozygous inactivation of the gene. In addition, dominant mutations of the XPAC and FACC1 genes can be ruled out as the cause of Gorlin syndrome, since the two patients described have null mutations. Finally, phenotypic features that show variable expression must be influenced by genetic background, epigenetic effects, somatic mutations, or environmental factors, since these two patients with identical alterations (deletions) of the Gorlin syndrome gene have somewhat different manifestations of Gorlin syndrome.     

A DNA methylation imprint, determined by the sex of the parent, distinguishes the angelman and Prader-Willi syndromes

The Angelman (AS) and Prader-Willi (PWS) syndromes are two clinically distinct disorders that are caused by a differential parental origin of chromosome 15q11-q13 deletions. Both also can result from uniparental disomy (the inheritance of both copies of chromosome 15 from only one parent). Loss of the paternal copy of 15q11-q13, whether by deletion or maternal uniparental disomy, leads to PWS, whereas a maternal deletion or paternal uniparental disomy leads to AS. The differential modification in expression of certain mammalian genes dependent upon parental origin is known as genomic imprinting, and AS and PWS represent the best examples of this phenomenon in humans. Although the molecular mechanisms of genomic imprinting are unknown, DNA methylation has been postulated to play a role in the imprinting process. Using restriction digests with the methyl-sensitive enzymes HpaII and HhaI and probing Southern blots with several genomic and cDNA probes, we have systematically scanned segments of 15q11-q13 for DNA methylation differences between patients with PWS (20 deletion, 20 uniparental disomy) and those with AS (26 deletion, 1 uniparental disomy). The highly evolutionarily conserved cDNA, DN34, identifies distinct differences in DNA methylation of the parental alleles at the D15S9 locus. Thus, DNA methylation may be used as a reliable, postnatal diagnostic tool in these syndromes. Furthermore, our findings demonstrate the first known epigenetic event, dependent on the sex of the parent, for a locus within 15q11-q13. We propose that expression of the gene detected by DN34 is regulated by genomic imprinting and, therefore, that it is a candidate gene for PWS and/or AS.     

Karyotypic abnormality of the X chromosome is rare in mutant HPRT-lymphocyte clones

Lymphocyte clones mutated at the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) locus on the X chromosome were studied by synchronization and G banding to determine the proportion of mutant clones having visible karyotypic change. 47 spontaneously mutant clones, 17 mutant clones induced by X-irradiation and 33 wild-type clones were studied. All clones were karyotypically normal except for 1 clone induced by X-irradiation in which an interstitial deletion of the short arm of the X chromosome had been inserted into the long arm of the same chromosome between q23 and q24; this change may have been coincidental or may have resulted in a position effect mutation. It was concluded that the great majority of mutations were not associated with a visible chromosome abnormality. This conclusion complements molecular studies which suggest that gene changes at the HPRT locus in HPRT- mutants generally extend over segments of DNA too small to be resolved by karyotypic analysis.     

A Case of ABL-BCR Negative, Philadelphia Positive CML with t(5;9)(q13;q34)

The Philadelphia chromosome is found in more than 90 percent of chronic myeloid leukemia (CML) patients. In most cases, it results from the reciprocal t(9;22)(q34;q11), with the ABL proto-oncogene from 9q34 fused to the breakpoint cluster region (BCR) locus on 22q11. In 5 to 10 percent of patients with CML, the Ph originates from variant translocations, involving various breakpoints in addition to 9q34 and 22q11. Here we report a rare case of a Philadelphia positive CML patient carrying t(5;9)(q13;q34) and deletion of ABL/BCR on der(9) as a separate event.     

Mutations in TPRN Cause a Progressive Form of Autosomal-Recessive Nonsyndromic Hearing Loss

We performed genome-wide homozygosity mapping in a large consanguineous family from Morocco and mapped the autosomal-recessive nonsyndromic hearing loss (ARNSHL) in this family to the DFNB79 locus on chromosome 9q34. By sequencing of 62 positional candidate genes of the critical region, we identified a causative homozygous 11 bp deletion, c.42_52del, in the TPRN gene in all seven affected individuals. The deletion is located in exon 1 and results in a frameshift and premature protein truncation (p.Gly15AlafsX150). Interestingly, the deleted sequence is part of a repetitive and CG-rich motive predicted to be prone to structural aberrations during crossover formation. We identified another family with progressive ARNSHL linked to this locus, whose affected members were shown to carry a causative 1 bp deletion (c.1347delG) in exon 1 of TPRN. The function of the encoded protein, taperin, is unknown; yet, partial homology to the actin-caping protein phostensin suggests a role in actin dynamics.     

Haploinsufficiency of <Emphasis Type="Italic">PAX9</Emphasis> is associated with autosomal dominant hypodontia

We recently identified a frame-shift mutation in the PAX9 gene as the underlying cause for hypodontia involving permanent molar teeth segregating in an autosomal dominant pattern in a single large family (Stockton et al. 2000). Here we report a small nuclear family in which a father and his daughter are affected with severe hypodontia, involving agenesis of all primary and permanent molars, evidently caused by deletion of the entire PAX9 gene. Hemizygosity at the PAX9 locus in the two affected individuals was initially discovered when an informative single nucleotide polymorphism, identified while sequencing the gene for mutations, appeared to demonstrate non-Mendelian inheritance. Fluorescence in situ hybridization (FISH) analysis with a cosmid containing the PAX9 gene yielded a signal on only one chromosome 14 homologue and confirmed the presence of a deletion encompassing the PAX9 locus. Analysis of microsatellite loci in the vicinity of PAX9 delineated one breakpoint of the deletion. These data, in concert with FISH analysis with cosmids encompassing a 199 kb region, indicated that the deletion is between approximately 44 kb and 100 kb. PAX9 is one of two genes, and the only odontogenic gene within the deletion interval, thus supporting the model of haploinsufficiency for PAX9 as the underlying basis for hypodontia.     

Mapping of epidermolysis bullosa simplex mutation to chromosome 12.

Epidermolysis bullosa simplex (EBS) is a dominantly inherited genodermatosis characterized by intraepidermal blister formation. Recent reports have suggested that EBS mutations may relate to keratin abnormalities. In this study, we conducted RFLP analyses to test the hypothesis that EBS is linked to one of the keratin gene clusters on chromosome 12 or chromosome 17. Although these keratin gene loci are not defined by RFLPs, several mapped RFLPs in the same chromosomal regions could be tested for linkage. A large EBS family with 14 affected and 12 unaffected individuals in three generations was analyzed for RFLP inheritance. Within this family there was no evidence for linkage of the EBS mutation to markers on chromosome 17q. However, there was evidence for close linkage to D12S17 located on chromosome 12q, with a maximum LOD score of 5.55 at theta = 0. Mapping of this mutation to chromosome 12 defines an EBS locus distinct from both EBS1 (Ogna) and EBS2 (Koebner), which are on chromosomes 8 and 1, respectively. Further mapping will determine whether this EBS locus on chromosome 12 resides within the keratin gene cluster at 12q11-q13.