Calvarial doughnut lesions with bone fragility in a French-Canadian family; case report and review of the literature.

Calvarial Doughnut Lesions with Bone Fragility (CDL) is an autosomal dominant genetic disease, characterized by low bone mineral density, multiple fractures starting in childhood, and sclerotic doughnut-shaped lesions in the cranial bones. Aubé and colleagues described in 1988 a French-Canadian family of 12 affected members who had a clinical diagnosis of doughnut lesions of the skull, with pathological fractures, osteopenia, "bone in bone" in the vertebral bodies and squaring of metatarsal and metacarpal bones. Herein we study new members of this family. Sequential genetic testing identified a nonsense variant c.148C>T, p. Arg50⁎ in SGMS2 previously reported in other families. SGMS2 encodes Sphingomyelin Synthase 2, which produces Sphingomyelin (SM), a major lipid component of the plasma membrane that plays a role in bone mineralization. The nonsense variant is associated with milder phenotype. The proband presents with bone in bone vertebral appearance that had been defined uniquely in the first cases described in the same family. The proband's son was identified to carry the same variant, which makes him the sixth generation with the diagnosis of CDL. We also report that the same pathogenic variant was identified in another previously described family, from France. These reports further confirm the genetic basis of CDL, the recurrence of the same variant (p.Arg50*) in individuals of the same ancestry, and the variable penetrance of some of the clinical findings.

Three-Dimensional Nuclear Telomere Profiling as a Biomarker for Recurrence in Oligodendrogliomas: A Pilot Study.

Mechanisms of recurrence in oligodendrogliomas are poorly understood. Recurrence might be driven by telomere dysfunction-mediated genomic instability. In a pilot study, we investigated ten patients with oligodendrogliomas at the time of diagnosis (first surgery) and after recurrence (second surgery) using three-dimensional nuclear telomere analysis performed with quantitative software TeloView® (Telo Genomics Corp, Toronto, Ontario, Canada). 1p/19q deletion status of each patient was determined by fluorescent in situ hybridization on touch preparation slides. We found that a very specific 3D telomeric profile was associated with two pathways of recurrence in oligodendrogliomas independent of their 1p/19q status: a first group of 8 patients displayed significantly different 3D telomere profiles between both surgeries (p < 0.0001). Their recurrence happened at a mean of 231.375 ± 117.42 days and a median time to progression (TTP) of 239 days, a period defined as short-term recurrence; and a second group of three patients displayed identical 3D telomere profiles between both surgery samples (p > 0.05). Their recurrence happened at a mean of 960.666 ± 86.19 days and a median TTP of 930 days, a period defined as long-term recurrence. Our results suggest a potential link between nuclear telomere architecture and telomere dysfunction with time to recurrence in oligodendrogliomas, independently of the 1p/19q status.

Study of Telomere Dysfunction in TP53 Mutant LoVo Cell Lines as a Model for Genomic Instability.

Telomere restriction fragment, 3D quantitative FISH on nuclei, and quantitative FISH on metaphases are complementary approaches that explore telomere dysfunction genomically, cellularly, and chromosomally, respectively. We used these approaches to study association between telomere dysfunction and degree of genomic instability related to TP53 mutations in LoVo isogenic cell lines. We found a strong correlation between degree of genomic instability, telomere dysfunction, and specific mutations of TP53. The use of complementary approaches to study telomere biology is essential to have a comprehensive understanding of telomere involvement in genomic instability.

Expression of Genes Associated with Telomere Homeostasis in TP53 Mutant LoVo Cell Lines as a Model for Genomic Instability.

We describe a method that assesses the impact of specific mutations of TP53 and genomic instability on gene expression of the most important genes involved in telomere length and structure homeostasis. The approaches consist of using a reverse transcriptase method and a quantitative PCR that were applied to isogenic cell lines from a colon cancer.

Different TP53 mutations are associated with specific chromosomal rearrangements, telomere length changes, and remodeling of the nuclear architecture of telomeres.

TP53 mutations are the most common mutations in human cancers, and TP53-R175H and TP53-R273H are the most frequent. The impact of these mutations on genomic instability after tumor initiation is still uncovered. To gain insight into this, we studied the effects of three specific TP53 mutants (TP53-V143A, TP53-R175H, and TP53-R273H) on genomic instability using four isogenic lines of LoVo cells. Multicolor fluorescence in situ hybridization (FISH), three-dimensional (3D) quantitative FISH (Q-FISH) on interphase and Q-FISH on metaphases were used to investigate genomic instability. We found that LoVo cells expressing mutant TP53-R175H displayed the highest level of chromosomal instability among the LoVo cell lines. Furthermore, we observed that mutant TP53-R175H and TP53-V143A showed more alterations in their 3D nuclear architecture of telomeres than the mutant TP53-R273H and the wild type. Moreover, we noted an association between some chromosomal abnormalities and telomere elongation in the mutant TP53-R175H. Taken together, our results indicate that the mutation TP53-R175H is more likely to cause higher levels of genomic instability than the other TP53 mutations. We proposed that the type of TP53 mutations and the genetic background of a cancer cell are major determinants of the TP53-dependent genomic instability.

A new der(1;7)(q10;p10) leading to a singular 1p loss in a case of glioblastoma with oligodendroglioma component.

The combined 1p-/19q- deletions in oligodendrogliomas originate from translocation between both chromosomes. In the few cases of oligoastrocytomas and glioblastomas with an oligodendroglioma component (GBMO) where only 1p deletion was described, the origin remains unknown. We report the first case of GBMO, in which a single 1p deletion was detected and was linked to a translocation between chromosomes 1 and 7. Fresh surgical specimens were collected during surgery and the samples were used for cell culture, touch preparation smear slides (TP slides) and DNA extraction. Peripheral venous blood was also collected from the patient. G-banding using Trypsin and stained with Giemsa (GTG) banding and karyotyping were performed and 1p-/19q-, TP53, PTEN and c-MYC were analyzed by fluorescent in situ hybridization (FISH). Multicolor FISH (mFISH) and microsatellites analyses were also performed to complete the investigation. Three-dimensional quantitative FISH (3D-QFISH) of telomeres was performed on nuclei from TP slides and analyzed using TeloView(TM) to determine whether the 3D telomere profile as an assessment of telomere dysfunction and a characterization of genomic instability could predict the disease aggressiveness. An unbalanced chromosomal translocation was found in all metaphases and confirmed by mFISH. The karyotype of the case is: 50∼99,XXX, +der(1;7)(q10;p10),inc[47] The derivative chromosome was found in all 47 analyzed cells, but the number of derivatives varied from one to four. There was neither imbalance in copy number for genes TP53 and PTEN, nor amplification of c-MYC gene. We did not find loss of heterozygosity with analysis of microsatellite markers for chromosomes 1p and 19q in tumor cells. The 3D-telomere profile predicted a very poor prognostic and short-term survival of the patient and highlights the potential clinical power of telomere signatures as a solid biomarker of GBMO. Furthermore, this translocation between chromosomes 1 and 7 led to a singular 1p deletion in this GBMO and may generate the 1p and 7q deletions.

Cytosine containing dipyrimidine sites can be hotspots of cyclobutane pyrimidine dimer formation after UVB exposure.

Exposure to the UV component of sunlight is the principal factor leading to skin cancer development. Cyclobutane pyrimidine dimers (CPD) are considered to be the most important pre-mutagenic type of DNA damage involved in skin carcinogenesis. To better understand the biological mechanisms of UV carcinogenesis, it is critical to understand the CPD distribution between the four types of dipyrimidine sites. Most of our knowledge regarding CPD distribution comes from in vitro studies or from investigations using UVC, even though we are not naturally exposed to these UV wavelengths. We exposed normal human fibroblasts and purified DNA to UVB. Using ligation-mediated PCR, we quantified the CPD formation at 952 dipyrimidine sites among the PGK1 (phosphoglycerate kinase 1), JUN, HRAS, KRAS, NRAS and TP53 genes. In cellulo, we found a CPD distribution of 27 : 27 : 25 : 21 for TT : CC : TC : CT. This distribution is similar to that observed in vitro. In the analysed genes, we observed some extremely frequently damaged dipyrimidine sites and many of these occurred at potentially frequently mutated sites, i.e. at dipyrimidine sites containing cytosine. Also, most of the frequently damaged dipyrimidine sites in cellulo that are not frequently damaged in vitro are found on TP53 and NRAS. This indicates that many of the frequently damaged dipyrimidine sites in cellulo are on genes frequently mutated in skin cancer. All these results support the view that CPD are the main UVB-induced mutagenic photoproducts and provide evidence of the importance of CPD formation at sites containing cytosine.

Update of PAX2 mutations in renal coloboma syndrome and establishment of a locus-specific database.

Renal coloboma syndrome, also known as papillorenal syndrome is an autosomal-dominant disorder characterized by ocular and renal malformations. Mutations in the paired-box gene, PAX2, have been identified in approximately half of individuals with classic findings of renal hypoplasia/dysplasia and abnormalities of the optic nerve. Prior to 2011, there was no actively maintained locus-specific database (LSDB) cataloguing the extent of genetic variation in the PAX2 gene and phenotypic variation in individuals with renal coloboma syndrome. Review of published cases and the collective diagnostic experience of three laboratories in the United States, France, and New Zealand identified 55 unique mutations in 173 individuals from 86 families. The three clinical laboratories participating in this collaboration contributed 28 novel variations in 68 individuals in 33 families, which represent a 50% increase in the number of variations, patients, and families published in the medical literature. An LSDB was created using the Leiden Open Variation Database platform: www.lovd.nl/PAX2. The most common findings reported in this series were abnormal renal structure or function (92% of individuals), ophthalmological abnormalities (77% of individuals), and hearing loss (7% of individuals). Additional clinical findings and genetic counseling implications are discussed.

A survey of APC mutations in Quebec.

This is an 11-year survey of molecular analysis of APC germline mutations for the province of Quebec done at the Molecular Pathology Unit of the Jewish General Hospital which offers genetic testing for hereditary forms of colorectal cancer for the whole of Quebec province. We report on 47 unique mutations seen in 66 families affected with familial adenomatous polyposis. Of these unique mutations, 60% are short indels, 28% are point mutations, and 6% are whole exon deletions. The absence of founder mutations and the variety of mutations encountered reinforce the value of RNA-based testing and the need for gene dosage techniques such as multiplex ligation-dependent probe amplification.

Neurofibromatosis type 1 and the "elephant man's" disease: the confusion persists: an ethnographic study.

BACKGROUND: In 1986, two Canadian geneticists had demonstrated that Joseph Merrick, better known as the Elephant Man, suffered from the Proteus syndrome and not from neurofibromatosis type 1 (NF1), as was alleged by dermatologist Parkes in 1909. Despite this and although the two diseases differ at several levels: prevalence, diagnostic criteria, clinical manifestations and transmission, the confusion between NF1 and the "elephant man's" disease continues in medical and social representations by current linguistic usage, and in some media reports. With this article, we want to 1) document the persistence and extent of this fallacy, 2) identify certain critical factors that contribute to its persistence, and 3) evaluate its impact on the health and well being of patients with NF1 and their family members. METHODOLOGY: Participant observation in the course of an ethnographic study on intergenerational dialogue between individuals with neurofibromatosis and their parents - Analysis of the scientific literature and of pinpoint articles in the print and online news media. FINDINGS: Our findings show that because physicians have little knowledge about NF1, several print and online news media and a lot of physicians continue to make the confusion between NF1 and the disease the "elephant man". This misconception contributes to misinformation about the disease, feeding prejudices against affected patients, exacerbating the negative impacts of the disease on their quality of life, their cognitive development, their reproductive choices, as well as depriving them of proper care and appropriate genetic counseling. CONCLUSION: If family physicians and pediatricians were properly informed about the disease, they could refer their patients with NF1 to NF clinics and to specialists. Thus, patients and their family members would benefit from better-tailored clinical management of their cases, perhaps even optimal management. [corrected]

Neurofibromatosis type 1: persisting misidentification of the "elephant man" disease.

BACKGROUND: during informal interviews in the course of an ethnographic study on intergenerational dialogue between individuals with neurofibromatosis and their parents, many members of Canadian neurofibromatosis associations have stated that they continue to be told the condition that afflicts them or their children is "elephant man's disease." Today, even though well-established clinical criteria make it possible to diagnose and differentiate the 2 diseases, the confusion between neurofibromatosis type 1 (NF1) and elephant man's disease persists in both the media's and physicians' representations. METHODS: this was an ethnographic study in medical anthropology. DISCUSSION: some reference sources and print and online news media have all contributed to the persistence of the association between NF1 and elephant man's disease. Our observations suggest that confusing NF1 with the Elephant Man's condition harms the interests of those with NF1 and thus increases the burden of the disease. CONCLUSION: changes of attitude regarding medical teaching and the media could dispel the confusion among physicians and journalists.

Formation of stress-specific p53 binding patterns is influenced by chromatin but not by modulation of p53 binding affinity to response elements.

The p53 protein is crucial for adapting programs of gene expression in response to stress. Recently, we revealed that this occurs partly through the formation of stress-specific p53 binding patterns. However, the mechanisms that generate these binding patterns remain largely unknown. It is not established whether the selective binding of p53 is achieved through modulation of its binding affinity to certain response elements (REs) or via a chromatin-dependent mechanism. To shed light on this issue, we used a microsphere assay for protein-DNA binding to measure p53 binding patterns on naked DNA. In parallel, we measured p53 binding patterns within chromatin using chromatin immunoprecipitation and DNase I coupled to ligation-mediated polymerase chain reaction footprinting. Through this experimental approach, we revealed that UVB and Nutlin-3 doses, which lead to different cellular outcomes, induce similar p53 binding patterns on naked DNA. Conversely, the same treatments lead to stress-specific p53 binding patterns on chromatin. We show further that altering chromatin remodeling using an histone acetyltransferase inhibitor reduces p53 binding to REs. Altogether, our results reveal that the formation of p53 binding patterns is not due to the modulation of sequence-specific p53 binding affinity. Rather, we propose that chromatin and chromatin remodeling are required in this process.

Sizing the ends: normal length of human telomeres.

The ends of human chromosomes are constituted of telomeres, a nucleoprotein complex. They are mainly formed by the entanglement of repeat DNA and telomeric and non-telomeric proteins. Telomeric sequences are lost in each cell division and this loss happens in vitro as well as in vivo. The diminution of telomere length over the cell cycle has led to the consideration of telomeres as a 'mitotic clock'. Telomere lengths are heterogeneous because they differ among tissues, cells, and chromosome arms. Cell proliferation capacity, cellular environment, and epigenetic factors are some elements that affect this telomere heterogeneity. Also, genetic and environmental factors modulate the difference in telomere lengths between individuals. Telomere length is regulated by telomere structure, telomerase, the enzyme that elongates the 3'-end of telomeres, and alternative lengthening of telomeres (ALT) used exclusively in immortalized and cancer cells. The understanding of telomere length dynamic in the normal population is essential to develop a deeper insight into the role of telomere function in pathological settings.

p53 functions and cell lines: have we learned the lessons from the past?

p53 has a determinant role in cancer prevention and is among the most studied proteins in the world. The majority of studies devoted to this protein are carried out in cell lines because they are easy to use and have naturally emerged as the main research tool in laboratories. However, the p53 pathway is commonly deregulated in cancer cells, from which the experimental cell lines are generally derived. The fact that the pathway is deregulated challenges the relevance of using cancer-derived cell lines to study wild-type p53 activities, or, in a broader sense, to study any normal cellular process. In the present article, we identify and discuss a number of limitations of cell lines using examples related to p53. Finally, we point out the general limitations of cell lines and propose solutions as alternatives to these cells.

[Mapping DNA damage to understand somatic mutagenesis]. / Comprendre la mutagenèse somatique grâce à la cartographie des dommages à l'ADN.

Somatic mutation theory explains how DNA damage can lead to the malignant transformation of cells. It therefore elucidates the connection between genotoxic agents and cancers. Mutational spectra, which tend to be characteristic of a cancer type, are available for certain genes like p53 which is frequently mutated in tumors. A mutational spectrum could therefore be the signature of the genotoxic agent(s) at the origin of the malignant transformation. Ligation-mediated PCR (LMPCR) is a genomic sequencing method that can be used for the mapping of DNA damage at nucleotide resolution. Such a mapping can then be compared to a mutational spectrum to test the hypothesis that implies one agent can cause mutations into one cancer type. LMPCR has been used this way to map DNA damage generated by different UV wavelengths. The frequently damaged sites following UVB irradiation correlate with the mutational spectrum of p53 in skin cancer. Similarly, BPDE, the activated form of the benzo[a]pyrene present in tobacco smoke, generates frequent adducts at sites corresponding to mutation hotspots of p53 in lung cancers. Still, the correlation between BPDE damage sites and p53 mutations is not perfect and this suggests a role of other genotoxic substances that are also present in tobacco smoke, such as the nitrosamine NNK. Finally, and beyond this objective of better understanding somatic mutagenesis, LMPCR is commonly used whenever DNA damage frequency and/or repair is to be investigated.

Three-dimensional nuclear telomere architecture is associated with differential time to progression and overall survival in glioblastoma patients.

The absence of biological markers allowing for the assessment of the evolution and prognosis of glioblastoma (GBM) is a major impediment to the clinical management of GBM patients. The observed variability in patients' treatment responses and in outcomes implies biological heterogeneity and the existence of unidentified patient categories. Here, we define for the first time three GBM patient categories with distinct and clinically predictive three-dimensional nuclear-telomeric architecture defined by telomere number, size, and frequency of telomeric aggregates. GBM patient samples were examined by three-dimensional fluorescent in situ hybridization of telomeres using two independent three-dimensional telomere-measurement tools (TeloView program [P(1)] and SpotScan system [P(2)]). These measurements identified three patients categories (categories 1-3), displaying significant differences in telomere numbers/nucleus (P(1) = .0275; P(2)

Fetal-maternal exchange of multipotent stem/progenitor cells: microchimerism in diagnosis and disease.

The biological concept of microchimerism, the bidirectional trafficking and stable long-term persistence of small numbers of allogeneic (fetal and maternal) cells in a genetically different organ, has gained considerable attention. Microchimerism is a common phenomenon in many species, including humans, and microchimeric cells can modify immunological recognition or tolerance, affect the course and outcome of various diseases and demonstrate stem cell-like or regenerative potential. Here, we review current knowledge of the biology of microchimerism and show how long-term allogeneic co-existence within an organism can impact on existing paradigms in chronic disease, cancer biology, regenerative medicine and fetal-maternal immunology. We discuss diagnostic challenges, clinical applications and future research directions in this exciting and rapidly emerging field of allogeneic fetal-maternal cell exchange.

p53 Pre- and post-binding event theories revisited: stresses reveal specific and dynamic p53-binding patterns on the p21 gene promoter.

p53 is a master transcription factor that prevents neoplasia and genomic instability. It is an important target for anticancer drug design. Understanding the molecular mechanisms behind its transcriptional activities in normal cells is a prerequisite to further understand the deregulation effected by mutant p53 in cancerous cells. Currently, how p53 coordinates transcription programs in response to stress remains unclear. One theory proposes that stresses induce pre-binding events that direct p53 to bind to specific response elements, whereas a second posits that, in response to stress, p53 binds most response elements and post-binding events then regulate transcription initiation. It is critical to establish the relevance of both theories and investigate whether stresses induce specific p53-binding patterns correlated with effector gene induction. Using unique in cellulo genomic footprinting experiments, we studied p53 binding to the five response elements of p21 in response to stresses and monitored p21 mRNA variant transcription. We show clear footprints of p53 bound to response elements in living cells and reveal that the binding of p53 to response elements is transient, subject to dynamic changes during stress responses, and influenced by response element pentamer orientations. We show further that stresses lead to specific p53-binding patterns correlated with particular p21 mRNA variant transcription profiles and that p53 binding is necessary but not sufficient to induce p21 transcription. Our results indicate that pre- and post-binding events act together to regulate adapted stress responses; this paves the way to the unification of pre- and post-binding event theories.

EGF receptor inhibitors in the treatment of glioblastoma multiform: old clinical allies and newly emerging therapeutic concepts.

Glioblastoma multiform (GBM) is the most common malignant brain tumour in adults. Despite decades of experimentation to improve the outcome of patients with GBM this highly aggressive tumour remains fatal. Primary GBM are often characterized by the over-expression of epidermal growth factor (EGF) receptor/HER1 and/or its mutational variants, with ligand-independent, constitutively active EGF receptor vIII variant most frequently observed in GBM. EGF receptor signalling can promote tumorigenesis by increasing cell proliferation, tissue invasion, neoangiogenesis, tumour cell chemoresistance, and by inhibiting apoptosis of cancer cells. EGF receptor was the first receptor to serve as target for cancer therapy of many solid tumours. After 2 decades of intensive targeting of EGF receptor for molecular therapy, several anti-EGF receptor inhibitors are now available in the clinic. Therapeutic strategies to target EGF receptor and EGF receptor mutant forms in GBM include humanized monoclonal antibodies, tyrosine kinase inhibitors, and RNAi compounds. However, despite the fact that most EGF receptor-directed glioma therapies to date have focused on single therapeutic agents, a multi-directional approach involving targeted inhibition of multiple signalling pathways has emerged as a more robust therapeutical approach. Furthermore, the emergence of the hypothesis of "brain cancer stem cells" in the bulb of GBM identifies this population of cells with self-renewal capacity as novel obligatory targets for efficient cure of GBM. Here we summarize current findings on the clinical role of these EGF receptor inhibitory therapeutic agents in the treatment of GBM.

Is the 1p/19q deletion a diagnostic marker of oligodendrogliomas?

The diagnosis and classification of diffusely infiltrative gliomas are based on their histopathological appearance; however, histopathological delineation of diffuse gliomas can be difficult because of vague and subjective histopathological criteria. Combined loss of chromosome arms 1p and 19q (denoted as 1p-/19q-) has proven to be a powerful predictor of chemotherapeutic response and survival in oligodendrogliomas. We undertook retrospective and prospective studies of brain tumor patients originally diagnosed as oligodendrogliomas or oligoastrocytomas patients followed at our institution using molecular genetic techniques. Fluorescence in situ hybridization using probes specific for chromosomes 1 and 19 was performed on 22 paraffin-embedded tissues retrospectively; 15 touch-preparation smear samples were studied prospectively; and loss of heterozygosity (LOH) screening was performed on 11 samples with microsatellite markers specific to chromosome 1 and chromosome 19. Of the 37 cases, 24 had 1p-/19q-, 1 case had 1p- only, 2 cases had 19q- only, and 10 cases had no deletion. The length of the largest deletion was mapped between markers D1S2795 (1p36.31 locus) and D1S2722 (1p34.2 locus) and between markers D19S416 (19q13.11 locus) and D19S397 (19q13.14 locus), using LOH. All of the pure oligodendrogliomas (n=7) harbored 1p-/19q-. In light of previous findings, the 1p-/19q- combination appears to be an objective diagnosis marker of classic oligodendrogliomas, one that can be used, in combination with histological examination, to improve the diagnosis of oligodendroglioma. Fluorescence in situ hybridization on touch preparations is a simple way to obtain information on 1p-/19q- in 24 hours.