Experience of a multidisciplinary task force with exome sequencing for Mendelian disorders.

BACKGROUND: In order to optimally integrate the use of high-throughput sequencing (HTS) as a tool in clinical diagnostics of likely monogenic disorders, we have created a multidisciplinary "Genome Clinic Task Force" at the University Hospitals of Geneva, which is composed of clinical and molecular geneticists, bioinformaticians, technicians, bioethicists, and a coordinator. METHODS AND RESULTS: We have implemented whole exome sequencing (WES) with subsequent targeted bioinformatics analysis of gene lists for specific disorders. Clinical cases of heterogeneous Mendelian disorders that could potentially benefit from HTS are presented and discussed during the sessions of the task force. Debate concerning the interpretation of identified variants and the content of the final report constitutes a major part of the task force's work. Furthermore, issues related to bioethics, genetic counseling, quality control, and reimbursement are also addressed. CONCLUSIONS: This multidisciplinary task force has enabled us to create a platform for regular exchanges between all involved experts in order to deal with the multiple complex issues related to HTS in clinical practice and to continuously improve the diagnostic use of HTS. In addition, this task force was instrumental to formally approve the reimbursement of HTS for molecular diagnosis of Mendelian disorders in Switzerland.

DNA repair in mammalian cells : Nucleotide excision repair: variations on versatility.

Nucleotide excision repair (NER) is one of the most versatile DNA repair systems. It can be subdivided into several, differentially regulated, subpathways: global genome repair (GGR), transcription-coupled repair (TCR), and transcription domain-associated repair (DAR). This review begins with a brief overview of the numerous types of DNA lesions handled by NER, and proceeds to describe in detail the molecular mechanisms of NER. It then addresses heterogeneities in NER activity in physiological situations (e. g. in differentiated cells) and explores the underlying regulatory mechanism. It then reviews several inherited diseases associated with NER deficiencies: xeroderma pigmentosum, Cockayne syndrome, trichothiodystrophy, UV-sensitive syndrome. It concludes by discussing several currently unresolved issues, relating either to the cause of the above diseases or to the mechanistic details of the various NER subpathways and of their regulation. (Part of a Multi-author Review).

DNA repair in differentiated cells: some new answers to old questions.

Terminally differentiated cells need never replicate their genomes and may therefore dispense with the daunting task of maintaining several repair systems to constantly scan their entire complement of DNA. Obviously, transcribed genes need to be repaired, so that cells can carry out their specialized functions, but dedicated mechanisms such as transcription-coupled repair and differentiation-associated repair can ensure the maintenance of those transcriptionally active domains. Many groups have studied DNA repair in differentiated cells, often with divergent results, possibly because there are distinct classes of differentiated cells, with unique properties. Thus neurons ought to last for a lifetime, whereas myocytes are backed by precursor cells, while white blood cells like macrophages are constantly being replaced. More importantly, different DNA repair systems can vary in their response to cellular differentiation, possibly depending on whether they can be coupled to transcription. Nucleotide excision repair (NER) is probably the most versatile DNA repair system and is coupled to transcription. NER was shown to be attenuated by differentiation in several cell types, including neurons. The attenuation occurs only at the global genome level, with transcribed genes still being efficiently repaired. We have determined that this attenuation results from the lack of ubiquitination of a NER factor, most likely owing to differences in phosphorylation of the ubiquitin-activating enzyme E1. Because there is only one E1 in human cells, it is likely that other metabolic pathways are similarly affected, depending on whether they rely on an E2 enzyme which is sensitive to the state of E1 phosphorylation.

Terminally differentiated human neurons repair transcribed genes but display attenuated global DNA repair and modulation of repair gene expression.

Repair of UV-induced DNA lesions in terminally differentiated human hNT neurons was compared to that in their repair-proficient precursor NT2 cells. Global genome repair of (6-4)pyrimidine-pyrimidone photoproducts was significantly slower in hNT neurons than in the precursor cells, and repair of cyclobutane pyrimidine dimers (CPDs) was not detected in the hNT neurons. This deficiency in global genome repair did not appear to be due to denser chromatin structure in hNT neurons. By contrast, CPDs were removed efficiently from both strands of transcribed genes in hNT neurons, with the nontranscribed strand being repaired unexpectedly well. Correlated with these changes in repair during neuronal differentiation were modifications in the expression of several repair genes, in particular an up-regulation of the two structure-specific nucleases XPG and XPF/ERCC1. These results have implications for neuronal dysfunction and aging.

Complementation of transformed fibroblasts from patients with combined xeroderma pigmentosum-Cockayne syndrome.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are human hereditary disorders characterized at the cellular level by an inability to repair certain types of DNA damage. Usually, XP and CS are clinically and genetically distinct. However, in rare cases, CS patients have been shown to have mutations in genes that were previously linked to the development of XP. The linkage between XP and CS has been difficult to study because few permanent cell lines have been established from XP/CS patients. To generate permanent cell lines, primary fibroblast cultures from two patients, displaying characteristics associated with CS and belonging to XP complementation group G, were transformed with anorigin-of-replication-deficient simian virus 40 (SV40). The new cell lines, summation operatorXPCS1LVo- and summation operatorXPCS1ROo-,were characterized phenotypically and genotypically to verify that properties of the primary cells are preserved after transformation. The cell lines exhibited rapid growth in culture and were shown, by immunostaining, to express the SV40 T antigen. The summation operatorXPCS1LVo- and summation operatorXPCS1ROo- cell lines were hypersensitive to UV light and had an impaired ability to reactivate a UV-irradiated reporter gene. Using polymerase chain reaction (PCR) amplification and restriction enzyme cleavage, the summation operatorXPCS1ROo- cells were shown to retain the homozygous T deletion at XPG position 2972. This mutation also characterizes the parental primary cells and was evident in the XPG RNA. Finally, to characterize the XPG DNA repair deficiency in these cell lines, an episomal expression vector containing wild-type XPG cDNA was used to correct UV-induced damage in a beta-galactosidase reporter gene.

A common mutational pattern in Cockayne syndrome patients from xeroderma pigmentosum group G: implications for a second XPG function.

Xeroderma pigmentosum (XP) patients have defects in nucleotide excision repair (NER), the versatile repair pathway that removes UV-induced damage and other bulky DNA adducts. Patients with Cockayne syndrome (CS), another rare sun-sensitive disorder, are specifically defective in the preferential removal of damage from the transcribed strand of active genes, a process known as transcription-coupled repair. These two disorders are usually clinically and genetically distinct, but complementation analyses have assigned a few CS patients to the rare XP groups B, D, or G. The XPG gene encodes a structure-specific endonuclease that nicks damaged DNA 3' to the lesion during NER. Here we show that three XPG/CS patients had mutations that would produce severely truncated XPG proteins. In contrast, two sibling XPG patients without CS are able to make full-length XPG, but with a missense mutation that inactivates its function in NER. These results suggest that XPG/CS mutations abolish interactions required for a second important XPG function and that it is the loss of this second function that leads to the CS clinical phenotype.

Defective transcription-coupled repair of oxidative base damage in Cockayne syndrome patients from XP group G.

In normal human cells, damage due to ultraviolet light is preferentially removed from active genes by nucleotide excision repair (NER) in a transcription-coupled repair (TCR) process that requires the gene products defective in Cockayne syndrome (CS). Oxidative damage, including thymine glycols, is shown to be removed by TCR in cells from normal individuals and from xeroderma pigmentosum (XP)-A, XP-F, and XP-G patients who have NER defects but not from XP-G patients who have severe CS. Thus, TCR of oxidative damage requires an XPG function distinct from its NER endonuclease activity. These results raise the possibility that defective TCR of oxidative damage contributes to the developmental defects associated with CS.

Mutations that disable the DNA repair gene XPG in a xeroderma pigmentosum group G patient.

The human XPG (ERCC5) gene encodes a large acidic protein that corrects the ultraviolet light sensitivity of cells from both xeroderma pigmentosum complementation group G and rodent ERCC group 5. Here we characterize five XPG sequence alterations and a minor splicing defect in XP-G patient XP125LO. Three of these changes are polymorphic variants whereas the remaining two, one in each XPG allele, inactivate complementation in vivo. These single point mutations provide formal proof that defects in XPG give rise to the group G form of xeroderma pigmentosum, and their locations suggest ways in which this may occur.

Complementation of the DNA repair defect in xeroderma pigmentosum group G cells by a human cDNA related to yeast RAD2.

Defects in human DNA repair proteins can give rise to the autosomal recessive disorders xeroderma pigmentosum (XP) and Cockayne's syndrome (CS), sometimes even together. Seven XP and three CS complementation groups have been identified that are thought to be due to mutations in genes from the nucleotide excision repair pathway. Here we isolate frog and human complementary DNAs that encode proteins resembling RAD2, a protein involved in this pathway in yeast. Alignment of these three polypeptides, together with two other RAD2 related proteins, reveals that their conserved sequences are largely confined to two regions. Expression of the human cDNA in vivo restores to normal the sensitivity to ultraviolet light and unscheduled DNA synthesis of lymphoblastoid cells from XP group G, but not CS group A. The XP-G correcting protein XPGC is generated from a messenger RNA of approximately 4 kilobases that is present in normal amounts in the XP-G cell line.

Insulin signalling and regulation of glucokinase gene expression in cultured hepatocytes.

In cultured rat hepatocytes, transcription of the glucokinase gene is turned on by insulin and turned off by glucagon/cAMP, the latter being the dominant effector system. It is thus possible that in the absence of hormones the gene is maintained in a repressed state by the basal level of cAMP and that insulin turns on transcription by relieving cAMP repression, for instance via activation of a cyclic-nucleotide phosphodiesterase. Three inhibitors of this class of enzymes were tested for their effect on the insulin-dependent induction of the glucokinase gene in hepatocytes. Isobutyl methylxanthine, the prototype inhibitor, abrogated the gene response to insulin, as shown by run-on transcription assay. Among the drugs investigated, Ly186126, a preferential inhibitor of type-III phosphodiesterase, proved the most potent in inhibiting insulin-induced accumulation of glucokinase mRNA. Type-III phosphodiesterase is inhibited by cGMP. Induction of glucokinase mRNA was prevented in hepatocytes challenged with insulin in presence of 8-bromoguanosine-3',5'-phosphate. These results are consistent with the involvement of type-III phosphodiesterase in transduction of the insulin signal to the glucokinase gene. However, we were unable to detect significant decreases in total cellular cAMP level or cAMP-dependent-protein-kinase ratio after the addition of insulin to hepatocytes. Many effects of glucagon are mediated via cAMP-dependent protein-kinase phosphorylation of regulatory proteins and, conversely, insulin effects are often accompanied by protein dephosphorylation. A specific inhibitor of protein phosphatases PP1 and PP2A, okadaic acid, was shown to abolish the transcriptional response of the glucokinase gene to insulin. Thus, interference of insulin with the cAMP signal transduction pathway at several steps may be a critical aspect of insulin action on hepatic glucokinase gene expression. In addition, insulin induction of glucokinase mRNA was suppressed by inhibitors of protein synthesis. The underlying mechanism was a severe inhibition of the transcriptional effect of insulin, rather than mRNA destabilization, as demonstrated by run-on transcription assays with nuclei from cycloheximide-treated or pactamycin-treated cells. Transcription of the glucokinase gene may therefore depend on de novo synthesis of the product of an early-response gene induced by insulin, or may require a short-lived trans-acting or accessory factor of transcription. Alternatively, insulin signalling may be compromised in hepatocytes by a mechanism indirectly related to the arrest of protein synthesis.

Unimpaired effect of insulin on glucokinase gene expression in hepatocytes challenged with amylin.

Amylin appears to interfere with the action of insulin in muscle and possibly in liver. We have attempted to detect a direct antagonism between amylin and insulin in cultured rat hepatocytes. The stimulation of glucokinase gene expression was used as a marker of insulin action. Amylin proved ineffective in suppressing subsequent accumulation of glucokinase mRNA in response to maximal or submaximal doses of insulin. When applied to cells already induced by prior incubation with insulin alone, amylin failed to reverse induction, in contrast to the effectiveness of glucagon under the same conditions. Thus, amylin is not a physiological antagonist of insulin in the control of hepatic glucokinase gene expression.

Transcriptional induction of glucokinase gene by insulin in cultured liver cells and its repression by the glucagon-cAMP system.

Primary cultures of rat hepatocytes were used to investigate the regulation of glucokinase gene expression by insulin and glucagon. Insulin added in physiological concentrations to the culture medium causes de novo induction of glucokinase mRNA. The induced plateau is reached 4 to 8 h after insulin addition, and the mRNA level remains high as long as insulin is present. Comparison of the potencies of insulin, proinsulin, and insulin-like growth factor I in this system indicates that induction by insulin is mediated via the insulin receptor. The magnitude of the insulin effect is independent of the extracellular glucose concentration. Run-on transcription assays with isolated nuclei show that the mRNA build up depends primarily on a specific stimulation of glucokinase gene transcription. Glucagon added to hepatocytes together with a supramaximal concentration of insulin prevents induction of glucokinase mRNA in a dose-dependent manner. The inhibitory effect of glucagon is mimicked by 8-(4-chlorophenylthio)-cAMP. The effect of this agent has also been tested in hepatocytes first induced for maximal glucokinase gene transcription by culture with insulin alone for 12 h. The transcriptional activity of the gene as measured by run-on assay was completely turned off within 30 min after addition of the cyclic nucleotide. Under these conditions, glucokinase mRNA decays rapidly, with an apparent half-life of 45 min. The mRNA degradation rate was similarly rapid after insulin withdrawal from induced cells. Thus, a cAMP-mediated repression mechanism is a key aspect in the regulation of glucokinase gene transcription in the hepatocyte. Insulin may act by relieving the gene from repression.

Differential expression and regulation of the glucokinase gene in liver and islets of Langerhans.

Glucokinase, a key regulatory enzyme of glucose metabolism in mammals, provides an interesting model of tissue-specific gene expression. The single-copy gene is expressed principally in liver, where it gives rise to a 2.4-kilobase mRNA. The islets of Langerhans of the pancreas also contain glucokinase. Using a cDNA complementary to rat liver glucokinase mRNA, we show that normal pancreatic islets and tumoral islet cells contain a glucokinase mRNA species approximately 400 nucleotides longer than hepatic mRNA. Hybridization with synthetic oligonucleotides and primer-extension analysis show that the liver and islet glucokinase mRNAs differ in the 5' region. Glucokinase mRNA is absent from the livers of fasted rats and is strongly induced within hours by an oral glucose load. In contrast, islet glucokinase mRNA is expressed at a constant level during the fasting-refeeding cycle. The level of glucokinase protein in islets measured by immunoblotting is unaffected by fasting and refeeding, whereas a 3-fold increase in the amount of enzyme occurs in liver during the transition from fasting to refeeding. From these data, we conclude (i) that alternative splicing and/or the use of distinct tissue-specific promoters generate structurally distinct mRNA species in liver and islets of Langerhans and (ii) that tissue-specific transcription mechanisms result in inducible expression of the glucokinase gene in liver but not in islets during the fasting-refeeding transition.