Search

Background\ud The severity of Tuberous Sclerosis Complex (TSC) can vary among affected individuals. Complications of TSC can be life threatening, with significant impact on patients’ quality of life. Management may vary dependent on treating physician, local and national policies, and funding. There are no current UK guidelines. We conducted a Delphi consensus process to reach agreed guidance for the management of patients with TSC in the UK.\ud \ud Methods\ud We performed a literature search and reviewed the 2012/13 international guideline for TSC management. Based on these, a Delphi questionnaire was formed. We invited 86 clinicians and medical researchers to complete an online survey in two rounds. All the people surveyed were based in the UK. Clinicians were identified through the regional TSC clinics, and researchers were identified through publications. In round one, 55 questions were asked. In round two, 18 questions were asked in order to obtain consensus on the outstanding points that had been contentious in round one. The data was analysed by a core committee and subcommittees, which consisted of UK experts in different aspects of TSC. The Tuberous Sclerosis Association was consulted.\ud \ud Results\ud 51 TSC experts took part in this survey. Two rounds were required to achieve consensus. The responders were neurologists, nephrologists, psychiatrist, psychologists, oncologists, general paediatricians, dermatologist, urologists, radiologists, clinical geneticists, neurosurgeons, respiratory and neurodisability clinicians.\ud \ud Conclusions\ud These new UK guidelines for the management and surveillance of TSC patients provide consensus guidance for delivery of best clinical care to individuals with TSC in the UK.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in either the TSC1 or the TSC2 genes and characterized by the development of benign hamartomatous growths in multiple organ systems. We have inactivated Tsc1 in the mouse germ line by gene targeting in ES cells and confirmed that the mutant allele (Tsc1-) has a recessive embryonic lethal phenotype. We found that a significant number (approximately 27%) of heterozygous (Tsc1+/-) mice on the C57BL/6 background died before weaning (P = 0.014) and show that these mice die in the post-natal period (P = 0.033), normally at 1-2 days, from unknown causes. Forty-four percent (7/16) of Tsc1+/- mice on a C3H background developed macroscopically visible renal lesions as early as 3-6 months, increasing to 95% (37/39) by 15-18 months. Renal lesions progressed from cysts through cystadenomas to solid carcinomas. Eighty percent (16/20) of Tsc1+/- mice on a Balb/c background exhibited solid renal cell carcinomas (RCC) by 15-18 months and in 41%, RCCs wereor = 5 mm, resulting in grossly deformed kidneys. Some RCCs had a sarcomatoid morphology of spindle cells in whorled patterns and metastasized to the lungs. We detected loss of the wild-type Tsc1 allele and elevated levels of p-mTOR and p-S6 in lesions from Tsc1+/- mice. This new murine model of hamartin deficiency exhibits a more severe phenotype than existing models.

The base excision repair DNA glycosylase MutY homolog (MYH) is responsible for removing adenines misincorporated into DNA opposite guanine or 7,8-dihydro-8-oxo-guanine (8-oxoG), thereby preventing G:C to T:A mutations. Biallelic germline mutations in the human MYH gene predispose individuals to multiple colorectal adenomas and carcinoma. We have recently demonstrated that hMYH interacts with the mismatch repair protein hMSH6, and that the hMSH2/hMSH6 (hMutSalpha) heterodimer stimulates hMYH activity. Here, we characterize the functional effect of two missense mutations (R227W and V232F) associated with hMYH polyposis that lie within, or adjacent to, the putative hMSH6 binding domain. Neither missense mutation affects the physical interaction between hMYH and hMSH6. However, hMYH(R227W) has a severe defect in A/8-oxoG binding and glycosylase activities, while hMYH(V232F) has reduced A/8-oxoG binding and glycosylase activities. The glycosylase activity of the V232F mutant can be partially stimulated by hMutSalpha but cannot be restored to the wild-type level. Both mutants also fail to complement mutY-deficiency in Escherichia coli. These data define the pathogenic mechanisms underlying two further hMYH polyposis-associated mutations.

Base excision repair (BER) protects against damage to DNA from reactive oxygen species, methylation, deamination, hydroxylation and other by-products of cellular metabolism. Until last year, inherited deficiencies in the BER pathway had not been causally linked with any human genetic disorder. An apparent explanation was functional redundancy between proteins in this and other pathways. However, it was recently discovered that biallelic mutations in the BER DNA glycosylase MYH lead to an autosomal recessive syndrome of adenomatous colorectal polyposis and very high colorectal cancer risk. We review the molecular mechanism of tumourigenesis in MYH polyposis, the preliminary delineation of the MYH polyposis phenotype and the functional overlap of MYH with other repair proteins.

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 2 (TSC2) gene is thought to function as a growth suppressor in sporadic and TSC-associated hamartomas and tumors. Clusters of dysplastic glial cells are a common feature of cortical tubers and subependymal nodules in tuberous sclerosis patients. In an effort to identify TSC2 gene alterations in sporadic gliomas, we detected a novel polymorphism adjacent to the 3′splice site of intron 4. We evaluated the distribution of this variant allele in a series of 244 patients with glial tumors, including 55 gangliogliomas, 31 pilocytic astrocytomas (WHO grade I), 50 astrocytomas (WHO grades II and III), and 108 glioblastomas (WHO grade IV). The allelic distribution in the general population was estimated by examining 381 healthy blood donors. This rare allele appeared in the control population and in the patients with astrocytic gliomas with a virtually identical frequency (8.14%, and 8.20%, respectively). The frequency of the rare allele in gangliogliomas, however, was significantly higher (15.5%; p = 0.024). The fact that both gangliogliomas and cortical tubers in tuberous sclerosis contain neuronal and astrocytic elements and may resemble each other histologically suggests that the TSC2 gene may be involved in the development of these tumors. The rare allele of the TSC2 gene emerges as a candidate for a predisposing factor for the formation of sporadic gangliogliomas.

Germ-line mutations of the TSC2 tumour suppressor gene have been identified in humans with tuberous sclerosis and in the Eker rat. Tuberin, the human TSC2 gene product, has a small region of homology with rap1GAP and stimulates rap1 GTPase activity in vitro, suggesting that one of its cellular roles is to function as a GTPase activating protein (GAP). We have undertaken a comparative analysis of the TSC2 gene in human and the pufferfish, Fugu rubripes. In addition to the GAP domain, three other regions of the proteins are highly conserved (peptide sequence similarity >80%). These regions are likely to represent further functional domains. To facilitate analysis of mutations within these domains we have determined the genomic structure of the human TSC2 gene. It comprises 41 exons, including exon 31 which was absent from the originally described spliceoform of the human TSC2 transcript and was identified following exon prediction from Fugu genomic sequence. These findings support the proposal of the Fugu genome as a tool for human gene analysis.

Renal involvement is common in tuberous sclerosis. Angiomyolipomas and cysts are found in approximately 50 and 30% of patients respectively, and often occur together. Tuberous sclerosis also appears to be associated with a small but increased risk of renal cell carcinoma. Recent studies have begun to elucidate the molecular genetic mechanisms underlying the renal manifestations of this systemic autosomal dominant disorder.

As a clinical geneticist, I am not too surprised that many public health practitioners appear to have ignored the wellpublicized advances in genome science. Their response is understandable since Mendelian and chromosomal disorders are generally rare, while for common, complex diseases, the progressive elucidation of genetic variation has yet to impact significantly upon prevention, diagnosis or treatment. But, like Zimmern, 1 I am concerned that the public health community needs to act now if it is to contribute to the ‘responsible and effective translation of genome-based knowledge and technologies for the benefit of population health’. Opportunities for the judicious application of genomics are emerging across a wide range of health settings, but the potential costs of irresponsible, ineffective or inappropriate application are high. Public health will simply not be able to contribute optimally to improving health in the twenty-first century unless it factors genomic science into its practice.

In a recent Commentary in Carcinogenesis, Hazra and colleagues described the discovery of a new family of mammalian enzymes for the repair of oxidatively damaged DNA and suggested that `the multiplicity of DNA glycosylases with overlapping substrate ranges could explain why . . . deficiency of the base excision enzymes examined so far has not been linked to susceptibility to cancer or other pathophysiological states' (1). However, we have recently shown that inherited defects in the base excision repair (BER) DNA glycosylase MYH cause multiple colorectal adenoma and carcinoma (2,3). 8-Oxo-7,8-dihydro-20-deoxyguanosine (8-oxoG) is the most stable product of oxidative DNA damage (4) and readily mispairs with A residues (5), leading to G:C!T:A mutations in repair-deficient bacteria and yeast (6±9). In Escherichia coli, three enzymes act synergistically to help protect cells against the mutagenic effects of guanine oxidation (7). MutM DNA glycosylase removes the oxidized base from 8-oxoG:C base pairs in duplex DNA, MutY DNA glycosylase excises A residues misincorporated opposite unrepaired 8-oxoG during replication and MutT, an 8-oxo-dGTPase, prevents the incorporation of 8-oxo-dGMP into nascent DNA. Homologues of mutM, mutY and mutT have been identified in human cells and termed OGG1 (10), MYH (11) and MTH1 (12), respectively. MYH DNA glycosylase interacts with AP endonuclease, PCNA and RPA, suggesting a role in long patch BER (13), and is associated with the replication foci, indicating a role in replication-coupled repair (14). Last year, we studied a British Caucasian family with three affected siblings with multiple colorectal adenomas and carcinoma and showed that these siblings were compound heterozygotes for the missense mutations Y165C and G382D in MYH (2). Analysis of the equivalent mutations in E.coli MutY showed that both variants significantly compromised adenine glycosylase activities (2). In a follow-up study, we identified seven further unrelated patients with 4100 colorectal adenomas (six with colorectal cancer) and biallelic germline mutations in MYH, including four patients homozygous for truncating mutations (3). As predicted from inactivation studies of the bacterial and yeast orthologues of MYH, colorectal tumours from affected individuals displayed a significant excess of somatic G:C!T:A mutations in APC, as compared with sporadic or familial adenomatous polyposis-associated colorectal tumours (2,3). More recently, Sieber et al. (15) identified a further 14 unrelated patients with multiple colorectal adenomas (six cases) or polyposis (eight cases) and biallelic germline variants in MYH. Taken together, these data unequivocally confirm that germline defects in the BER DNA glycosylase MYH cause a multiple colorectal adenoma and carcinoma phenotype.