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The detection of copy-number variations (CNVs) from NGS data is underexploited as chip-based or targeted techniques are still commonly used. We assessed the performances of a workflow centered on CANOES, a bioinformatics tool based on read depth information. We applied our workflow to gene panel (GP) and whole-exome sequencing (WES) data, and compared CNV calls to quantitative multiplex PCR of short fluorescent fragments (QMSPF) or array comparative genomic hybridization (aCGH) results. From GP data of 3776 samples, we reached an overall positive predictive value (PPV) of 87.8%. This dataset included a complete comprehensive QMPSF comparison of four genes (60 exons) on which we obtained 100% sensitivity and specificity. From WES data, we first compared 137 samples with aCGH and filtered comparable events (exonic CNVs encompassing enough aCGH probes) and obtained an 87.25% sensitivity. The overall PPV was 86.4% following the targeted confirmation of candidate CNVs from 1056 additional WES. In addition, our CANOES-centered workflow on WES data allowed the detection of CNVs with a resolution of single exons, allowing the detection of CNVs that were missed by aCGH. Overall, switching to an NGS-only approach should be cost-effective as it allows a reduction in overall costs together with likely stable diagnostic yields. Our bioinformatics pipeline is available at: https://gitlab.bioinfo-diag.fr/nc4gpm/canoes-centered-workflow .

Fifty years after the recognition of the Li-Fraumeni syndrome (LFS), our perception of cancers related to germline alterations of TP53 has drastically changed: (i) germline TP53 alterations are often identified among children with cancers, in particular soft-tissue sarcomas, adrenocortical carcinomas, central nervous system tumours, or among adult females with early breast cancers, without familial history. This justifies the expansion of the LFS concept to a wider cancer predisposition syndrome designated heritable TP53-related cancer (hTP53rc) syndrome; (ii) the interpretation of germline TP53 variants remains challenging and should integrate epidemiological, phenotypical, bioinformatics prediction, and functional data; (iii) the penetrance of germline disease-causing TP53 variants is variable, depending both on the type of variant (dominant-negative variants being associated with a higher cancer risk) and on modifying factors; (iv) whole-body MRI (WBMRI) allows early detection of tumours in variant carriers and (v) in cancer patients with germline disease-causing TP53 variants, radiotherapy, and conventional genotoxic chemotherapy contribute to the development of subsequent primary tumours. It is critical to perform TP53 testing before the initiation of treatment in order to avoid in carriers, if possible, radiotherapy and genotoxic chemotherapies. In children, the recommendations are to perform clinical examination and abdominal ultrasound every 6 months, annual WBMRI and brain MRI from the first year of life, if the TP53 variant is known to be associated with childhood cancers. In adults, the surveillance should include every year clinical examination, WBMRI, breast MRI in females from 20 until 65 years and brain MRI until 50 years.

In oncology, the expanding use of multi-gene panels to explore familial cancer predisposition and tumor genome analysis has led to increased secondary findings discoveries (SFs) and has given rise to important medical, ethical, and legal issues. The American College of Medical Genetics and Genomics published a policy statement for managing SFs for a list of genes, including 25 cancer-related genes. Currently, there are few recommendations in Europe. From June 2016 to May 2017, the French Society of Predictive and Personalized Medicine (SFMPP) established a working group of 47 experts to elaborate guidelines for managing information given on the SFs for genes related to cancers. A subgroup of ethicists, lawyers, patients' representatives, and psychologists provided ethical reflection, information guidelines, and materials (written consent form and video). A subgroup with medical expertise, including oncologists and clinical and molecular geneticists, provided independent evaluation and classification of 60 genes. The main criteria were the "actionability" of the genes (available screening or prevention strategies), the risk evaluation (severity, penetrance, and age of disease onset), and the level of evidence from published data. Genes were divided into three classes: for class 1 genes (n = 36), delivering the information on SFs was recommended; for class 2 genes (n = 5), delivering the information remained questionable because of insufficient data from the literature and/or level of evidence; and for class 3 genes (n = 19), delivering the information on SFs was not recommended. These guidelines for managing SFs for cancer-predisposing genes provide new insights for clinicians and laboratories to standardize clinical practices.

We have developed and validated for the diagnosis of inherited colorectal cancer (CRC) a massive parallel sequencing strategy based on: (i) fast capture of exonic and intronic sequences from ten genes involved in Mendelian forms of CRC (MLH1, MSH2, MSH6, PMS2, APC, MUTYH, STK11, SMAD4, BMPR1A and PTEN); (ii) sequencing on MiSeq and NextSeq 500 Illumina platforms; (iii) a bioinformatic pipeline that includes BWA-Picard-GATK (Broad Institute) and CASAVA (Illumina) in parallel for mapping and variant calling, Alamut Batch (Interactive BioSoftware) for annotation, CANOES for CNV detection and finally, chimeric reads analysis for the detection of other types of structural variants (SVs). Analysis of 1644 new index cases allowed the identification of 323 patients with class 4 or 5 variants, corresponding to a 20% disease-causing variant detection rate. This rate reached 37% in patients with Lynch syndrome, suspected on the basis of tumour analyses. Thanks to this strategy, we detected overlapping phenotypes (e.g., MUTYH biallelic mutations mimicking Lynch syndrome), mosaic alterations and complex SVs such as a genomic deletion involving the last BMPR1A exons and PTEN, an Alu insertion within MSH2 exon 8 and a mosaic deletion of STK11 exons 3-10. This strategy allows, in a single step, detection of all types of CRC gene alterations including SVs and provides a high disease-causing variant detection rate, thus optimizing the diagnosis of inherited CRC.

Primary familial brain calcification (PFBC) is a rare cerebral microvascular calcifying disorder with a wide spectrum of motor, cognitive, and neuropsychiatric symptoms. It is typically inherited as an autosomal-dominant trait with four causative genes identified so far: SLC20A2, PDGFRB, PDGFB, and XPR1. Our study aimed at screening the coding regions of these genes in a series of 177 unrelated probands that fulfilled the diagnostic criteria for primary brain calcification regardless of their family history. Sequence variants were classified as pathogenic, likely pathogenic, or of uncertain significance (VUS), based on the ACMG-AMP recommendations. We identified 45 probands (25.4%) carrying either pathogenic or likely pathogenic variants (n = 34, 19.2%) or VUS (n = 11, 6.2%). SLC20A2 provided the highest contribution (16.9%), followed by XPR1 and PDGFB (3.4% each), and PDGFRB (1.7%). A total of 81.5% of carriers were symptomatic and the most recurrent symptoms were parkinsonism, cognitive impairment, and psychiatric disturbances (52.3%, 40.9%, and 38.6% of symptomatic individuals, respectively), with a wide range of age at onset (from childhood to 81 years). While the pathogenic and likely pathogenic variants identified in this study can be used for genetic counseling, the VUS will require additional evidence, such as recurrence in unrelated patients, in order to be classified as pathogenic.

Interpretation of variants of unknown significance (VUS) is a major challenge for laboratories performing molecular diagnosis of hereditary breast and ovarian cancer (HBOC), especially considering that many genes are now known to be involved in this syndrome. One important way these VUS can have a functional impact is through their effects on RNA splicing. Here we present a custom RNA-Seq assay plus bioinformatics and biostatistics pipeline to analyse specifically alternative and abnormal splicing junctions in 11 targeted HBOC genes. Our pipeline identified 14 new alternative splices in BRCA1 and BRCA2 in addition to detecting the majority of known alternative spliced transcripts therein. We provide here the first global splicing pattern analysis for the other nine genes, which will enable a comprehensive interpretation of splicing defects caused by VUS in HBOC. Previously known splicing alterations were consistently detected, occasionally with a more complex splicing pattern than expected. We also found that splicing in the 11 genes is similar in blood and breast tissue, supporting the utility and simplicity of blood splicing assays. Our pipeline is ready to be integrated into standard molecular diagnosis for HBOC, but it could equally be adapted for an integrative analysis of any multigene disorder.

About one third of patients with rhabdoid tumors (RT) harbor a heterozygous germline variant in SMARCB1. Molecular diagnosis therefore keeps a crucial place in the diagnosis of RT, and genetic counseling should be systematically recommended. However, immunohistochemistry has progressively replaced molecular tools to assess the status of SMARCB1 in tumors; the necessity of analyzing SMARCB1 status in the tumor may thus be less considered by neuropathologists and pediatric neuro-oncologists. In the present manuscript as aforementioned, we report on two patients with bifocal RT in the first month of life and in whom no germline variant was initially found in the SMARCB1 coding sequence. Careful analysis of SMARCB1 status in the tumors revealed that only one of the two inactivating hits was found in the coding sequence. By sequencing the tumor cells RNA, we were able to detect an insertion with an abnormal sequence, due to the same intronic variant of SMARCB1, which led to the exonisation of the first intron. This cryptic variant was absent in the germline DNA of both patients. Of note, we previously reported one patient with the same deep intronic variant in the germline in a soft tissue RT. To our mind, this additional report on two patients clearly demonstrates that this intronic variant is a new hotspot that should now be systematically added to the germline screening of SMARCB1. We therefore recommend searching for and cautiously interpreting germline analysis if SMARCB1 has not been extensively studied in the tumor.

Primary brain calcification (PBC) is a dominantly inherited calcifying disorder of the brain. SLC20A2 loss-of-function variants account for the majority of families. Only one genomic deletion encompassing SLC20A2 and six other genes has been reported. We performed whole-exome sequencing (WES) in 24 unrelated French patients with PBC, negatively screened for sequence variant in the known genes SLC20A2, PDGFB, PDGFRB and XPR1. We used the CANOES tool to detect copy number variations (CNVs). We detected two deletions of exon 2 of SLC20A2 in two unrelated patients, which segregated with PBC in one family. We then reanalyzed the same series using a QMPSF assay including one amplicon in each exon of SLC20A2 and detected two supplemental partial deletions in two patients: one deletion of exon 4 and one deletion of exons 4 and 5. These deletions were missed by the first screening step of CANOES but could finally be detected after readjustment of bioinformatic parameters and use of a genotyping step of CANOES. This study reports the first partial deletions of SLC20A2 and strengthens its position as the major PBC-causative gene. It is possible to detect short CNVs from WES data, although the sensitivity of such tools should be evaluated in comparison with other methods.

Causative variants in APP, PSEN1 or PSEN2 account for a majority of cases of autosomal dominant early-onset Alzheimer disease (ADEOAD, onset before 65 years). Variant detection rates in other EOAD patients, that is, with family history of late-onset AD (LOAD) (and no incidence of EOAD) and sporadic cases might be much lower. We analyzed the genomes from 264 patients using whole-exome sequencing (WES) with high depth of coverage: 90 EOAD patients with family history of LOAD and no incidence of EOAD in the family and 174 patients with sporadic AD starting between 51 and 65 years. We found three PSEN1 and one PSEN2 causative, probably or possibly causative variants in four patients (1.5%). Given the absence of PSEN1, PSEN2 and APP causative variants, we investigated whether these 260 patients might be burdened with protein-modifying variants in 20 genes that were previously shown to cause other types of dementia when mutated. For this analysis, we included an additional set of 160 patients who were previously shown to be free of causative variants in PSEN1, PSEN2 and APP: 107 ADEOAD patients and 53 sporadic EOAD patients with an age of onset before 51 years. In these 420 patients, we detected no variant that might modify the function of the 20 dementia-causing genes. We conclude that EOAD patients with family history of LOAD and no incidence of EOAD in the family or patients with sporadic AD starting between 51 and 65 years have a low variant-detection rate in AD genes.

Aβ-related cerebral amyloid angiopathy (CAA) is a major cause of primary non-traumatic brain hemorrhage. In families with an early onset of the disease, CAA can be due to amyloid precursor protein (APP) pathogenic variants or duplications. APP duplications lead to a ~1.5-fold increased APP expression, resulting in Aβ overproduction and deposition in the walls of leptomeningeal vessels. We hypothesized that rare variants in the 3'untranslated region (UTR) of APP might lead to APP overexpression in patients with CAA and no APP pathogenic variant or duplication. We performed direct sequencing of the whole APP 3'UTR in 90 patients with CAA and explored the functional consequences of one previously unreported variant. We identified three sequence variants in four patients, of which a two-base pair deletion (c.*331_*332del) was previously unannotated and absent from 175 controls of same ethnicity. This latter variant was associated with increased APP expression in vivo and in vitro. Bioinformatics and functional assays showed that the APP c.*331_*332del variant affected APP messenger RNA (mRNA) structure and binding of two microRNAs (miR-582-3p and miR-892b), providing a mechanism for the observed effects on APP expression. These results identify APP 3'UTR sequence variants as genetic determinants of Aβ-CAA.

To determine if the at-risk single-nucleotide polymorphism (SNP) alleles for colorectal cancer (CRC) could contribute to clinical situations suggestive of an increased genetic risk for CRC, we performed a prospective national case-control study based on highly selected patients (CRC in two first-degree relatives, one before 61 years of age; or CRC diagnosed before 51 years of age; or multiple primary CRCs, the first before 61 years of age; exclusion of Lynch syndrome and polyposes) and controls without personal or familial history of CRC. SNPs were genotyped using SNaPshot, and statistical analyses were performed using Pearson's χ(2) test, Cochran-Armitage test of trend and logistic regression. We included 1029 patients and 350 controls. We confirmed the association of CRC risk with four SNPs, with odds ratio (OR) higher than previously reported: rs16892766 on 8q23.3 (OR: 1.88, 95% confidence interval (CI): 1.30-2.72; P=0.0007); rs4779584 on 15q13.3 (OR: 1.42, CI: 1.11-1.83; P=0.0061) and rs4939827 and rs58920878/Novel 1 on 18q21.1 (OR: 1.49, CI: 1.13-1.98; P=0.007 and OR: 1.49, CI: 1.14-1.95; P=0.0035). We found a significant (P<0.0001) cumulative effect of the at-risk alleles or genotypes with OR at 1.62 (CI: 1.10-2.37), 2.09 (CI: 1.43-3.07), 2.87 (CI: 1.76-4.70) and 3.88 (CI: 1.72-8.76) for 1, 2, 3 and at least 4 at-risk alleles, respectively, and OR at 1.71 (CI: 1.18-2.46), 2.29 (CI: 1.55-3.38) and 6.21 (CI: 2.67-14.42) for 1, 2 and 3 at-risk genotypes, respectively. Combination of SNPs may therefore explain a fraction of clinical situations suggestive of an increased risk for CRC.

Fanconi anaemia (FA) is characterized by progressive bone marrow failure, congenital anomalies, and predisposition to malignancy. In a minority of cases, FA results from biallelic FANCD1/BRCA2 mutations that are associated with early-onset leukaemia and solid tumours. Here, we describe the clinical and molecular features of a remarkable family presenting with multiple primary colorectal cancers (CRCs) without detectable mutations in genes involved in the Mendelian predisposition to CRCs. We unexpectedly identified, despite the absence of clinical cardinal features of FA, a biallelic mutation of the FANCD1/BRCA2 corresponding to a frameshift alteration (c.1845&#95;1846delCT, p.Asn615Lysfs*6) and a missense mutation (c.7802A>G, p.Tyr2601Cys). The diagnosis of FA was confirmed by the chromosomal analysis of lymphocytes. Reverse transcriptase (RT)-PCR analysis revealed that the c.7802A>G BRCA2 variation was in fact a splicing mutation that creates an aberrant splicing donor site and results partly into an aberrant transcript encoding a truncated protein (p.Tyr2601Trpfs*46). The atypical FA phenotype observed within this family was probably explained by the residual amount of BRCA2 with the point mutation c.7802A>G in the patients harbouring the biallelic FANCD1/BRCA2 mutations. Although this report is based in a single family, it suggests that CRCs may be part of the tumour spectrum associated with FANCD1/BRCA2 biallelic mutations and that the presence of such mutations should be considered in families with CRCs, even in the absence of cardinal features of FA.

Germline alterations of the tumour suppressor TP53 gene are detected approximately in 25% of the families suggestive of Li-Fraumeni syndrome (LFS), characterised by a genetic predisposition to a wide tumour spectrum, including soft-tissue sarcomas, osteosarcomas, premenopausal breast cancers, brain tumours, adrenocortical tumours, plexus choroid tumours, leukaemia and lung cancer. The aim of this study was to determine the contribution of germline copy number variations (CNVs) to LFS in families without detectable TP53 mutation. Using a custom-designed high-resolution array CGH, we evaluated the presence of rare germline CNVs in 64 patients fulfilling the Chompret criteria for LFS, but without any detectable TP53 alteration. In 15 unrelated patients, we detected 20 new CNVs absent in 600 controls. Remarkably, in four patients who had developed each brain tumour, the detected CNV overlap the KDM1A, MTA3, TRRAP or SIRT3 genes encoding p53 partners involved in histone methylation or acetylation. Focused analysis of SIRT3 showed that the CNV encompassing SIRT3 leads to SIRT3 overexpression, and that in vitro SIRT3 overexpression prevents apoptosis, increases G2/M and results in a hypermethylation of numerous genes. This study supports the causal role of germline alterations of genes involved in chromatin remodelling in genetic predisposition to cancer and, in particular, to brain tumours.

Several studies have reported that, in Lynch syndrome resulting from mutations of the mismatch repair (MMR) genes, a CA repeat ≤17 within the IGF1 promoter, SNPs within the xenobiotic metabolizing enzyme gene CYP1A1 and SNPs on 8q23.3 and 11q23.1 modify colorectal cancer (CRC) risk in MMR mutation carriers. We analysed the impact of these polymorphisms on CRC risk in 748 French MMR mutation carriers derived from 359 families. We also analysed the effect of the Novel 1 SNP (18q21), which has recently been shown to increase CRC risk in the general population. We observed a significant difference in the CRC-free survival time between males and females, between MSH2 and MSH6 mutation carriers and between MLH1 and MSH6, indicating that this series is representative of Lynch syndrome. In contrast, the univariate log-rank test, as well as multivariate Cox model analysis controlling for familial aggregation and mutated MMR gene, year of birth and gender showed that the polymorphic alleles tested were not associated with a significant CRC risk increase, neither on the entire sample nor among males and females. This discrepancy with previous reports might be explained both by the genetic heterogeneity between the different populations analysed and the allelic heterogeneity of the MMR mutations. We conclude that genotyping of these polymorphisms is not useful to evaluate CRC risk in MMR mutation carriers and to optimize their clinical follow-up.

Neuroblastoma, a tumour derived from the peripheral sympathetic nervous system, is one of the most frequent solid tumours in childhood. It usually occurs sporadically but familial cases are observed, with a subset of cases occurring in association with congenital malformations of the neural crest being linked to germline mutations of the PHOX2B gene. Here we conducted genome-wide comparative genomic hybridization analysis on a large series of neuroblastomas. Copy number increase at the locus encoding the anaplastic lymphoma kinase (ALK) tyrosine kinase receptor was observed recurrently. One particularly informative case presented a high-level gene amplification that was strictly limited to ALK, indicating that this gene may contribute on its own to neuroblastoma development. Through subsequent direct sequencing of cell lines and primary tumour DNAs we identified somatic mutations of the ALK kinase domain that mainly clustered in two hotspots. Germline mutations were observed in two neuroblastoma families, indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were overexpressed, hyperphosphorylated and showed constitutive kinase activity. The knockdown of ALK expression in ALK-mutated cells, but also in cell lines overexpressing a wild-type ALK, led to a marked decrease of cell proliferation. Altogether, these data identify ALK as a critical player in neuroblastoma development that may hence represent a very attractive therapeutic target in this disease that is still frequently fatal with current treatments.

Numerous reports have highlighted the contribution of MSH2 and MLH1 genomic deletions to hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch's syndrome, but genomic duplications of these genes have been rarely reported. Using quantitative multiplex PCR of short fluorescent fragments (QMPSF), 962 and 611 index cases were, respectively, screened for MSH2 and MLH1 genomic rearrangements. This allowed us to detect, in 11 families, seven MSH2 duplications affecting exons 1-2-3, exons 4-5-6, exon 7, exons 7-8, exons 9-10, exon 11, and exon 15, and three MLH1 duplications affecting exons 2-3, exon 4 and exons 6-7-8. All duplications were confirmed by an independent method. The contribution of genomic duplications of MSH2 and MLH1 to HNPCC can therefore be estimated approximately to 1% of the HNPCC cases. Although this frequency is much lower than that of genomic deletions, the presence of MSH2 or MLH1 genomic duplications should be considered in HNPCC families without detectable point mutations.

Mutations and polymorphisms in the SPINK1 gene, which encodes trypsin's physiological inhibitor, pancreatic secretory trypsin inhibitor, have been found to be associated with chronic pancreatitis. However, to date, all currently reported SPINK1 variants are either single-nucleotide substitutions or microinsertions/deletions. It is possible that large genomic rearrangements at this locus may underlie certain cases of chronic pancreatitis. However, such events, if indeed they exist, may have been overlooked by conventional PCR-based techniques. Here we attempted to screen all four exons as well as the promoter region of the SPINK1 gene for large genomic deletions by means of quantitative high-performance liquid chromatography analysis. Of the 47 pancreatitis families (not carrying any known PRSS1, SPINK1 and CFTR variants/mutations after screening the coding regions by our previously established denaturing high-performance liquid chromatography methods), one family was suggested to carry a large genomic deletion in the SPINK1 gene. The aberrant chromosomal junction was encapsulated by long-range PCR and the breakpoints were determined by direct sequencing of the rearranged fragment. A 2-bp short direct repeat was present at the deletion breakpoints; this simple deletion (c.1-320&#95;c.55+961del1336 bp) can thus in principle be explained by replication slippage. Identification of this lesion has not only expanded the SPINK1 mutational spectrum but also served to identify a novel mutational mechanism causing chronic pancreatitis.

Splenomegaly with sea-blue histiocytes, thrombocytopenia and hypertriglyceridemia is a very rare association that has been described in only one report to date. The molecular defect in the two reported patients consists in a deletion of a leucine at position 149 in the receptor-binding region of the apoE molecule. Here, we report on another family in whom the proband and his brother were diagnosed with splenomegaly, thrombocytopenia and hypertriglyceridemia. An apoE p.Leu149del mutation was found in both subjects. A large beta band in the VLDL fraction and elevated VLDL cholesterol-to-plasma triglyceride ratio was observed in the proband only. Their mother, presenting with isolated hypertriglyceridemia, also carried the same p.Leu149del mutation. The coexistence of factors facilitating the development of hypertriglyceridemia and/or low HDL-cholesterol level could explain why the proband and his brother developed a splenomegaly with thrombocytopenia, whereas the mother did not. Moreover, the presence of an apoE2 allele in the proband likely explains the more severe phenotype we observed in this subject. In conclusion, the apoE p.Leu149del mutation results in a very striking phenotype including one or all symptoms among splenomegaly, thrombocytopenia and hypertriglyceridemia, and should be considered as a differential diagnosis of storage disorders in the causes of splenomegaly with sea-blue histiocytes.

TP63 gene mutations have recently been shown to be disease causing in EEC and SHFM. Two other overlapping syndromes with ectrodactyly as a major feature, have been mapped to chromosome 3q27 close by the TP63 locus, namely the LMS and ADULT syndromes. Here, we report on a missense TP63 gene mutation in an isolated ADULT syndrome case. This finding widens the spectrum of abnormalities to be ascribed to TP63 gene in human and emphasise on the variable roles of the different Tp63 isotypes.

The human p53 protein acts mainly as a stress inducible transcription factor transactivating several genes involved in cell cycle arrest (e.g. p21) or apoptosis (e.g. Bax, PIG3). Roughly half of all human tumours contains p53 missense mutations. Virtually all tumour-derived p53 mutants are unable to activate Bax transcription but some retain the ability to activate p21 transcription. Identification of these mutants may have valuable clinical implications. We have determined the transactivation ability of 77 p53 mutants using reporter yeast strains containing a p53-regulated ADE2 gene whose promoter is regulated by p53 responsive elements derived from the regulatory region of the p21, Bax and PIG3 genes. We also assessed the influence of temperature on transactivation. Our results indicate that a significant proportion of mutants [16/77 (21%); 10/64 (16%) considering only tumour-derived mutants] are transcriptionally active, especially with the p21 promoter. Discriminant mutants preferentially affect less conserved (P<0.04, Fisher's exact test), more rarely mutated (P<0.006, Fisher's exact test) amino acids. Temperature sensitivity is frequently observed, but is more common among discriminant than non-discriminant mutants (P<0.003, Fisher's exact test). Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours that surprisingly were indistinguishable from wild type in standard transcription, growth suppression and apoptosis assays in human cells, but showed gain of function in transformation assays. The incidence of transcriptionally active mutations among this group was significantly higher than in the panel of mutants studied previously (P<0.001, Fisher's exact test). Since it is not possible to predict the behaviour of a mutant from first principles, we propose that the yeast assay be used to compile a functional p53 database and fill the gap between the biophysical, pharmacological and clinical fields.

Many p53 mutants found in human cancer have an altered ability to bind DNA and transactivate gene expression. Re-expression of functional p53 in cells in which the endogenous TP53 gene is inactivated has been demonstrated to restore a non-tumorigenic phenotype. Pharmacological modulation of p53 mutant conformation may therefore represent a mechanism to reactivate p53 function and consequently improve response to radio- and chemotherapy. We have recently reported that the radio- and chemoprotector Amifostine (WR2721, Ethyol) activates wild-type p53 in cultured mammalian cells. In the present study, we have used a yeast functional assay to investigate the effect of WR2721 on the transcriptional activity of p53. WR2721 restored this activity in a temperature-sensitive mutant V272M (valine to methionine at codon 272) expressed at the non-permissive temperature and it also partially restored the transcriptional activity of several other conformationally flexible p53 mutants. The results indicate that the yeast functional assay may be used to identify compounds that modulate p53 activity, with potential therapeutic implications.

The apolipoprotein E (APOE, gene; apoE, protein) isoforms are associated with differential risk of Alzheimer's disease (AD). An additional involvement of APOE promoter polymorphisms in AD risk has recently been suggested by several studies. Indeed, three polymorphisms of the APOE regulatory region (-219 G/T, -427 C/T and -491 A/T) have been found associated with AD even after adjustment on the apoE status. We analysed these three promoter region polymorphisms in a large French case-control study (388 AD cases and 386 controls). We found that the -427 T and -491 A alleles were associated with an increased risk of developing AD, but not the -219 G/T alleles. However, a strong linkage disequilibrium was observed between the alleles of these promoter region polymorphisms and the APOE coding region alleles. We therefore retested association after adjustment on apoE status and found that the sole association which remained significant was the association with the -427 T allele. The alpha level was equal to 0.03 (0.09 after Bonferroni correction for multiple comparisons). Analysis of promoter haplotypes also yielded non-significant results. Thus our study does not reinforce the hypothesis of an independent involvement of the APOE promoter region polymorphisms in AD risk.

Several studies have reported in schizophrenia a decrease of age of onset in successive family generations, and this observation is consistent with anticipation. Anticipation is known to result from expansion of CAG repeats in several neurodegenerative disorders. Longer alleles of the KCNN3 gene, which contains a highly polymorphic CAG repeat, and encodes a neuronal small conductance calcium-activated potassium channel, have recently been shown to be over-represented in sporadic cases of schizophrenia. In this report, we tested the hypothesis of an association between longer alleles of CAG repeat in the KCNN3 gene and schizophrenia in 20 families with clinical evidence for anticipation and in 151 unrelated schizophrenic cases. No significant difference in the distributions of allele frequencies was observed between familial cases of schizophrenia and controls, and between unrelated cases and controls. Furthermore, no intergenerational CAG repeat instability was detected in the 20 families. Our results do not support the involvement of the KCNN3 (hSKCa3) gene in the etiology of schizophrenia.

Recent studies have suggested that a rare class of p53 mutants found in tumours has a subtle transcriptional defect affecting bax induction but not p21 induction. We have therefore developed simple functional assays in yeast which can be used to identify these mutants. Analysis of 51 different mutations observed in human tumours showed that all mutants tested scored as mutant with the bax reporter strain but nine scored as wild-type with the p21 reporter strain. These results, which can be explained by the lower affinity of the p53 protein for the bax site, may suggest that p21 is not the key target of p53 mutations in tumours. Since p21 status has recently been shown to modulate the chemotherapeutic and radiotherapeutic sensitivities of cancerous cells, the functional assays described here may have important clinical implications.

Field cancerisation of the aerodigestive tract is caused by chronic exposure to alcohol and tobacco, but the nature of the genetic alterations preceding overt malignancy is unknown. To identify potential field changes we have used a functional assay which tests the transcriptional competence of human p53 expressed in yeast. To increase the sensitivity and reliability of the technique for samples containing under 20% mutant p53, the 5' and 3'-ends of the p53 cDNA were examined separately. With this split form of the assay the tissue p53 mRNA acts as its own control for RNA quality. Mutations were detected in 87% (46/53) of tumours, reflecting the high sensitivity of the technique. Multiple biopsies of histologically normal tissue from the upper aero-digestive tract were tested and clonal p53 mutations were identified in 76% (38/50) of biopsies from patients presenting with multiple tumours compared with 32% (38/117) of biopsies from patients presenting with single tumours (P<0.000001). All patients (16/16) presenting with multiple tumours had at least one positive biopsy, compared with only 53% (19/36) of patients presenting with single tumours (P <0.001). This defines expansion of multiple clones of mutant p53-containing cells as an important biological mechanism of field cancerisation, and provides a means to identify patients likely to benefit from intensive screening for the development of new head and neck tumours.

Alternative splicing affecting the p53 carboxy-terminus has previously been described in mouse but not in normal human cells. We report here the detection in normal human lymphocytes of an alternatively spliced form of human p53 mRNA containing an additional 133 bp exon derived from intron 9. This splice variant encodes a truncated protein of 341 amino-acids including 10 new amino-acids derived from the novel exon. The truncated protein, which lacks part of the p53 tetramerization domain, fails to bind DNA in vitro and has a transcriptional defect in vivo in both yeast and mammalian cells. Quantitative RT-PCR experiments suggest that the alternatively spliced form is only present in significant amounts in quiescent cells. Considering the numerous functions ascribed to the carboxy-terminus of the p53 protein, this splice variant may have important implications for the biological role of p53 in normal cells.