Inherited predisposition to colorectal adenomas caused by multiple rare alleles of MUTYH but not OGG1, NUDT1, NTH1 or NEIL 1, 2 or 3.

BACKGROUND: MUTYH-associated polyposis (MAP) is a recessive trait characterised by multiple colorectal adenomas and a high risk of colorectal cancer. MUTYH functions in the DNA base excision repair pathway and has a key role in the repair of oxidative DNA damage. OBJECTIVES: To assess the contribution of inherited variants in genes involved in base excision repair and oxidative DNA damage including MUTYH, OGG1, NEIL1, NEIL2, NEIL3, NUDT1 and NTH1 to the multiple colorectal adenoma phenotype. METHODS: Inherited variants of MUTYH, OGG1, NEIL1, NEIL2, NEIL3, NUDT1 and NTH1 were sought in 167 unrelated patients with multiple colorectal adenomas whose family histories were consistent with recessive inheritance. These variants were also characterised in approximately 300 population controls. RESULTS: Thirty-three patients (20%) and no controls were MUTYH homozygotes or compound heterozygotes (ie, carried two mutations) and therefore had MAP. Eight different pathogenic MUTYH mutations were identified, of which four were novel. MAP cases had significantly more adenomas than non-MAP cases (p = 0.0009; exact test for trends in proportions) and presented earlier (p = 0.013; analysis of variance). Twenty-four protein-altering variants were identified upon screening of OGG1, NEIL1, NEIL2, NEIL3, NUDT1 and NTH1. However, all combinations of two (or more) variants that were identified at an individual locus in patients were also seen in controls, and no variants were significantly over-represented (or under-represented) in cases. CONCLUSION: Multiple rare alleles of MUTYH are associated with autosomal recessive MAP, while OGG1, NEIL1, NEIL2, NEIL3, NUDT1 and NTH1 do not contribute significantly to autosomal recessive polyposis.

MutYH (MYH) and colorectal cancer.

MAP (MutYH-associated polyposis) is a recently described colorectal adenoma and carcinoma predisposition syndrome that is associated with biallelic-inherited mutations of the human MutY homologue gene, MutYH. MutYH is often also termed MYH. MAP tumours display a mutational signature of somatic guanine-to-thymine transversion mutations in the adenomatous polyposis coli and K-ras genes, reflecting the normal role of MutYH in the base excision repair of adenines misincorporated opposite 7,8-dihydro-8-oxoguanine, a prevalent and stable product of oxidative damage to DNA. However, the full genetic pathway of MAP tumorigenesis has not been elucidated.

Increased frequency of the k-ras G12C mutation in MYH polyposis colorectal adenomas.

Colorectal tumours from MYH polyposis patients display an excess of somatic G : C --> T : A transversions in the adenomatous polyposis coli gene. Here, we identify k-ras mutations in nine out of 54 (16.7%) MYH polyposis tumours. Their presence was associated with increased dysplasia and tubulovillous morphology (P=0.005). G : C --> T : A transversions in k-ras were significantly more frequent in MYH polyposis adenomas than in sporadic or familial adenomatous polyposis-associated tumours (P

Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin.

Critical functions of hamartin and tuberin, encoded by the TSC1 and TSC2 genes, are likely to be closely linked. The proteins interact directly with one another and mutations affecting either gene result in the tuberous sclerosis phenotype. However, the regions of hamartin and tuberin that interact have not been well defined, and the relationship between their interaction and the pathogenesis of tuberous sclerosis has not been explored. To address these issues a series of hamartin and tuberin constructs were used to assay for interaction in the yeast two-hybrid system. Hamartin (amino acids 302-430) and tuberin (amino acids 1-418) interacted strongly with one another. A region of tuberin encoding a putative coiled-coil (amino acids 346-371) was necessary but not sufficient to mediate the interaction with hamartin, as more N-terminal residues were also required. A region of hamartin (amino acids 719-998) predicted to encode coiled-coils was capable of oligermerization but was not important for the interaction with tuberin. Subtle, non-truncating mutations identified in patients with tuberous sclerosis and located within the putative binding regions of hamartin (N198_F199delinsI;593-595delACT) or tuberin (G294E and I365del), abolished or dramatically reduced interaction of the proteins as assessed by yeast two-hybrid assays and by co-immunoprecipitation of the full-length proteins from Cos7 cells. In contrast, three non-pathogenic missense polymorphisms of tuberin (R261W, M286V, R367Q) in the same region as the disease-causing TSC2 mutations did not. These results indicate a requirement for interaction in critical growth suppressing functions of hamartin and tuberin.

Analysis of the TSC1 and TSC2 genes in sporadic renal cell carcinomas.

The genetic events involved in the aetiology of non-clear-cell renal cell carcinoma (RCC) and a proportion of clear cell RCC remain to be defined. Germline mutations of the TSC1 and TSC2 genes cause tuberous sclerosis (TSC), a multi-system hamartoma syndrome that is also associated with RCC. We assessed 17 sporadic clear cell RCCs with a previously identified VHL mutation, 15 clear-cell RCCs without an identified VHL mutation and 15 non-clear-cell RCCs for loss of heterozygosity (LOH) at chromosomes 9q34 and 16p13.3, the chromosomal locations of TSC1 and TSC2. LOH was detected in 4/9, 1/11 and 3/13 cases informative at both loci. SSCP analysis of the whole coding region of the retained allele did not reveal any cases with a detectable intragenic second somatic mutation. Furthermore, RT-PCR analysis of TSC1 and TSC2 on total RNA from 8 clear-cell RCC cell lines confirmed expression of both TSC genes. These data indicate that biallelic inactivation of TSC1 or TSC2 is not frequent in sporadic RCC and suggests that the molecular mechanisms of renal carcinogenesis in TSC are likely to be distinct.

Tuberous sclerosis causing mutants of the TSC2 gene product affect proliferation and p27 expression.

The autosomal dominant disease tuberous sclerosis (TSC) is caused by mutations in either TSC1 on chromosome 9q34, encoding hamartin, or TSC2 on chromosome 16p13.3, encoding tuberin. TSC is characterized by hamartomas that occur in many organs of affected patients and these have been considered to likely result from defects in proliferation control. Although the true biochemical functions of the two TSC proteins have not been clarified, a series of independent investigations demonstrated that modulated hamartin or tuberin expression cause deregulation of proliferation/cell cycle in human, rodent and Drosophila cells. In support of tuberin acting as a tumor suppressor, ectopic overexpression of TSC2 has been shown to decrease proliferation rates of mammalian cells. Furthermore, overexpression of TSC2 has been demonstrated to trigger upregulation of the cyclin-dependent kinase inhibitor p27. We report that three different naturally occurring and TSC causing mutations within the TSC2 gene eliminate neither the anti-proliferative capacity of tuberin nor tuberin's effects on p27 expression. For the first time these data provide strong evidence that deregulation of proliferation and/or upregulation of p27 are not likely to be the primary/only mechanisms of hamartoma development in TSC. These results demand reassessment of previous hypotheses of the pathogenesis of TSC.

LD-PCR coupled to long-read direct sequencing: an approach for mutation detection in genes with compact genomic structures.

A number of techniques have been developed as primary screens to scan for DNA sequence variants, including denaturing gradient gel electrophoresis, denaturing high-performance liquid chromatography, single-strand conformation polymorphism and heteroduplex analysis. Variant alleles detected by these assays are subsequently characterised by DNA sequencing. Sequencing itself is rarely used as a primary screen because of labour intensity, cost, and, upon automation, occasional inaccuracy in identifying heterozygous sites. We have previously developed an approach based on coupling long-distance PCR (LD-PCR) to long-read direct sequencing to allow the detection of mutations in the approximately 1.1 kb exon 3 of MECP2. Our use of dye-labelled primers generated high-quality bi-directional sequence runs > 650 bp and allowed easy discrimination of heterozygous bases. We now describe the application of this approach to the detection of mutations in a considerably larger 6.35 kb LD-PCR fragment spanning 10 exons (exons 32-41) of the structurally complex, but genomically compact, TSC2 gene. In a blind analysis, 15/15 previously characterised mutations were successfully identified using seven overlapping bi-directional sequencing reactions. Our approach of long-read sequencing of long-distance PCR products may allow rapid sequencing of multiple exons of compact genes and may be appropriate as a highly sensitive primary screen for mutations.

Molecular analysis of the TSC1 and TSC2 tumour suppressor genes in sporadic glial and glioneuronal tumours.

Reduced expression of the TSC2 tumour suppressor gene product, tuberin, has been reported in sporadic astrocytomas, suggesting that the TSC genes may play a role in formation of sporadic glial or glioneuronal tumours. We studied paired constitutional and tumour DNA samples from 100 patients with sporadic glial and glioneuronal tumours for loss of heterozygosity (LOH) at the TSC1 and TSC2 loci using a combination of seven previously reported and seven novel polymorphic markers. LOH was seen in 1/16 astrocytomas, 3/15 ependymomas, 5/16 gangliogliomas, 2/14 glioblastoma multiforme, 0/7 oligodendrogliomas, 0/7 tumours of mixed oligodendrocytic/astrocytic histology, 2/11 pilocytic astrocytomas and 0/1 subependymal giant cell astrocytomas informative at both loci. However, SSCP screening of all coding exons of the TSC1 or TSC2 genes in the tumours displaying LOH, and of both genes in 21 gangliogliomas, revealed no intragenic mutations. The lack of demonstrable inactivation of both alleles of either TSC gene in any of the tumours investigated suggests that they do not play a frequent role in the aetiology of sporadic glial or glioneuronal tumours.

Molecular genetic advances in tuberous sclerosis.

Over the past decade, there has been considerable progress in understanding the molecular genetics of tuberous sclerosis, a disorder characterised by hamartomatous growths in numerous organs. We review this progress, from cloning and characterising TSC1 and TSC2, the genes responsible for the disorder, through to gaining insights into the functions of their protein products hamartin and tuberin, and the identification and engineering of animal models. We also present the first comprehensive compilation and analysis of all reported TSC1 and TSC2 mutations, consider their diagnostic implications and review genotype/phenotype relationships.

Application and evaluation of denaturing HPLC for molecular genetic analysis in tuberous sclerosis.

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in multiple tissues and organs. TSC exhibits locus heterogeneity with genes at 9q34 (TSC1) and 16p13.3 (TSC2) that have 21 and 41 coding exons, respectively. The mutational spectrum at both loci is wide and previous studies have shown that 60%-70% of cases are sporadic and represent new mutations. We have formatted denaturing high performance liquid chromatography (DHPLC) for rapid screening of all coding exons of TSC1 and TSC2. DHPLC analysis detected likely disease-causing mutations in 103 of 150 unrelated cases (68%), compared with 92/150 (61%) and 87/150 (58%) for single-strand conformation polymorphism analysis (SSCP) and conventional heteroduplex analysis (HA), respectively. Capital, consumable and labour costs were determined for each exon screening procedure. Estimated costs per patient sample depended on throughput, particularly for DHPLC, where a high proportion of costs are fixed, and were pounds sterling 257, pound sterling 216 and pound sterling 242 for DHPLC, SSCP and HA, respectively, assuming a throughput of 252 samples per year, or pound sterling 354, pound sterling 233 and pound sterling 259, assuming a throughput of 126 samples per year. DHPLC had the advantages of increased sensitivity and reduced labour costs when compared with more traditional approaches to exon screening but, unless expensive DHPLC equipment is being efficiently utilised for a very high proportion of the time available, overall costs are slightly higher.

Long-read sequence analysis of the MECP2 gene in Rett syndrome patients: correlation of disease severity with mutation type and location.

Mutations in the methyl-CpG-binding protein gene MECP2 at Xq28 cause Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder characterized by a period of stagnation followed by regression in the development of young girls. Mutations were sought in MECP2 in 48 females with classical sporadic RTT, seven families with possible familial RTT and five sporadic females with features suggestive, but not diagnostic of RTT. Long distance PCR coupled with long-read direct sequencing was employed to sequence the entire MECP2 gene coding region in all cases. Mutations were identified in 44/55 (80%) unrelated classical sporadic and familial RTT patients, but only 1/5 (20%) sporadic cases with suggestive but non-diagnostic features of RTT. Twenty-one different mutations were identified (12 missense, four nonsense and five frame-shift mutations); 14 of these were novel. All missense mutations were located either in the methyl-CpG-binding domain or in the transcription repression domain. Nine recurrent mutations were characterized in a total of 33 unrelated cases (73% of all cases with MECP2 mutations). Significantly milder disease was noted in patients carrying missense mutations as compared with those with truncating mutations ( P = 0. 0023), and milder disease was associated with late as compared with early truncating mutations ( P = 0.0190).

The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination.

We report here that overexpression of the tuberous sclerosis-1 (TSC1) gene product hamartin results in the inhibition of growth, as well as changes in cell morphology. Growth inhibition was associated with an increase in the endogenous level of the product of the tuberous sclerosis-2 (TSC2) gene, tuberin. As overexpression of tuberin inhibits cell growth, and hamartin is known to bind tuberin, these results suggested that hamartin stabilizes tuberin and this contributes to the inhibition of cell growth. Indeed, transient transfection of TSC1 increased the endogenous level of tuberin, and transient co-transfection of TSC1 with TSC2 resulted in higher tuberin levels. The stabilization was explained by the finding that tuberin is highly ubiquitinated in cells, while the fraction of tuberin that is bound to hamartin is not ubiquitinated. Co-expression of tuberin stabilized hamartin, which is weakly ubiquitinated, in transiently transfected cells. The amino-terminal two-thirds of tuberin was responsible for its ubiquitination and for stabilization of hamartin. A mutant of tuberin from a patient missense mutation of TSC2 was also highly ubiquitinated, and was unable to stabilize hamartin. We conclude that hamartin is a growth inhibitory protein whose biological effect is likely dependent on its interaction with tuberin.

Optimal temperature selection for mutation detection by denaturing HPLC and comparison to single-stranded conformation polymorphism and heteroduplex analysis.

BACKGROUND: Denaturing HPLC (DHPLC) is a semi-automated method for detecting unknown DNA sequence variants. The sensitivity of the method is dependent on the temperature at which the analysis is undertaken, the selection of which is dependent on operator experience. To circumvent this, software has been developed for predicting the optimal temperature for DHPLC analysis. We examined the utility of this software. METHODS: To maximize the relevance of our data for other investigators, we have screened 42 different amplimers from CFTR, TSC1, and TSC2. The samples consisted of 103 unique sequence heterozygotes and 126 wild-type homozygous controls. RESULTS: At the temperature recommended by the software, 96% (99 of 103) of heterozygotes and all of the wild-type controls were correctly classified. This compares favorably with sensitivities of 85% for single-stranded conformation polymorphism and 82% for gel-based heteroduplex analyses of the same fragments. CONCLUSIONS: Software-optimized DHPLC is a highly sensitive method for mutation detection. However, where sensitivity >96% is required, our data suggest that in addition to the recommended temperature, fragments should also be run at the recommended temperature plus 2 degrees C.

Comprehensive mutation analysis of TSC1 and TSC2-and phenotypic correlations in 150 families with tuberous sclerosis.

Tuberous sclerosis (TSC [MIM 191090 and MIM 191100]) is an autosomal dominant disorder characterized by hamartomas in many organs. Two thirds of cases are sporadic and are thought to represent new mutations. TSC is caused by mutations affecting either of the presumed tumor-suppressor genes, TSC1 and TSC2. Both appear to function as tumor suppressors, because somatic loss or intragenic mutation of the corresponding wild-type allele is seen in the associated hamartomas. Here we report the first comprehensive mutation analysis of TSC1 and TSC2 in a cohort of 150 unrelated TSC patients and their families, using heteroduplex and SSCP analysis of all coding exons and using pulsed-field gel electrophoresis and conventional Southern blot analysis and long PCR to screen for large rearrangements. Mutations were characterized in 120 (80%) of the 150 cases, affecting TSC1 in 22 cases and TSC2 in 98 cases. TSC1 mutations were significantly underrepresented in sporadic cases (P=. 000185). Twenty-two patients had TSC2 missense mutations that were found predominantly in the GAP-related domain (eight cases) and in a small region encoded in exons 16 and 17, between nucleotides 1849 and 1859 (eight cases), consistent with the presence of residues performing key functions at these sites. In contrast, all TSC1 mutations were predicted to be truncating, consistent with a structural or adapter role for the encoded protein. Intellectual disability was significantly more frequent in TSC2 sporadic cases than in TSC1 sporadic cases (P=.0145). These data provide the first representative picture of the distribution and spectrum of mutations across the TSC1 and TSC2 loci in clinically ascertained TSC and support a difference in severity of TSC1- and TSC2-associated disease.

Molecular genetic and phenotypic analysis reveals differences between TSC1 and TSC2 associated familial and sporadic tuberous sclerosis.

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterised by the development of hamartomatous growths in many organs. Sixty to seventy percent of cases are sporadic and appear to represent new mutations. TSC exhibits locus heterogeneity: the TSC2 gene is located at 16p13.3 whilst the TSC1 gene, predicted to encode a novel protein termed hamartin, has recently been cloned from 9q34. With the exception of a contiguous gene deletion syndrome involving TSC2 and PKD1 , TSC1 and TSC2 phenotypes have been considered identical. We have now comprehensively defined the TSC1 mutational spectrum in 171 sequentially ascertained, unrelated TSC patients by single strand conformation polymorphism and heteroduplex analysis of all 21 coding exons, and by assaying a restriction fragment spanning the whole locus. Mutations were identified in 9/24 familial cases, but in only 13/147 sporadic cases. In contrast, a limited screen revealed TSC2 mutations in two of the familial cases and in 45 of the sporadic cases. Thus TSC1 mutations were significantly under-represented among sporadic cases (Fisher's exact p -value = 3.12 x 10(-4)). Both large deletions and missense mutations were common at the TSC2 locus whereas most TSC1 mutations were small truncating lesions. Mental retardation was significantly less frequent among carriers of TSC1 than TSC2 mutations (odds ratio 5.54 for sporadic cases only, 6.78 +/- 1.54 when a single randomly selected patient per multigeneration family was also included). No correlation between mental retardation and the type of mutation was found. We conclude that there is a reduced risk of mental retardation in TSC1 as opposed to TSC2 disease and that consequent ascertainment bias, at least in part, explains the relative paucity of TSC1 mutations in sporadic TSC.