Distinct Mechanisms of Nuclease-Directed DNA-Structure-Induced Genetic Instability in Cancer Genomes.

Sequences with the capacity to adopt alternative DNA structures have been implicated in cancer etiology; however, the mechanisms are unclear. For example, H-DNA-forming sequences within oncogenes have been shown to stimulate genetic instability in mammals. Here, we report that H-DNA-forming sequences are enriched at translocation breakpoints in human cancer genomes, further implicating them in cancer etiology. H-DNA-induced mutations were suppressed in human cells deficient in the nucleotide excision repair nucleases, ERCC1-XPF and XPG, but were stimulated in cells deficient in FEN1, a replication-related endonuclease. Further, we found that these nucleases cleaved H-DNA conformations, and the interactions of modeled H-DNA with ERCC1-XPF, XPG, and FEN1 proteins were explored at the sub-molecular level. The results suggest mechanisms of genetic instability triggered by H-DNA through distinct structure-specific, cleavage-based replication-independent and replication-dependent pathways, providing critical evidence for a role of the DNA structure itself in the etiology of cancer and other human diseases.

Recombination regulator PRDM9 influences the instability of its own coding sequence in humans.

PRDM9 plays a key role in specifying meiotic recombination hotspot locations in humans and mice via recognition of hotspot sequence motifs by a variable tandem-repeat zinc finger domain in the protein. We now explore germ-line instability of this domain in humans. We show that repeat turnover is driven by mitotic and meiotic mutation pathways, the latter frequently resulting in substantial remodeling of zinc fingers. Turnover dynamics predict frequent allele switches in populations with correspondingly fast changes of the recombination landscape, fully consistent with the known rapid evolution of hotspot locations. We found variation in meiotic instability between men that correlated with PRDM9 status. One particular "destabilizer" variant caused hyperinstability not only of itself but also of otherwise-stable alleles in heterozygotes. PRDM9 protein thus appears to regulate the instability of its own coding sequence. However, destabilizer variants are strongly self-limiting in populations and probably have little impact on the evolution of the recombination landscape.

Proteasome inhibition rescues clinically significant unstable variants of the mismatch repair protein Msh2.

MSH2 is required for DNA mismatch repair recognition in eukaryotes. Deleterious mutations in human MSH2 account for approximately half of the alleles associated with a common hereditary cancer syndrome. Previously, we characterized clinically identified MSH2 missense mutations, using yeast as a model system, and found that the most common cause of defective DNA mismatch repair was low levels of the variant Msh2 proteins. Here, we show that increased protein turnover is responsible for the reduced cellular levels. Increasing gene dosage of more than half of the missense alleles fully restored function. A titration experiment revealed that raising the expression level of one variant to less than wild-type levels restored mismatch repair, suggesting that overexpression is not always required to regain function. We found that the ubiquitin-mediated proteasome degradation pathway is the major mechanism for increased turnover of the Msh2 variants and identified the primary ubiquitin ligase as San1. Deletion of San1 restored protein levels for all but one variant, but did not elevate wild-type Msh2 levels. The unstable variants interacted with San1, whereas wild-type Msh2 did not. Additionally, san1Δ suppressed the mismatch repair defect of unstable variants. Of medical significance, the clinically approved drug Bortezomib partially restored protein levels and mismatch repair function for low-level variants and reversed the resistance to cisplatin, a common chemotherapeutic. Our results provide the foundation for an innovative therapeutic regime for certain mismatch-repair-defective cancers that are refractory to conventional chemotherapies.

Características fenotípicas e histológicas de los pacientes con melanoma cutáneo en función de los polimorfismos del MC1R / Phenotypic and Histologic Characteristics of Cutaneous Melanoma in Patients With Melanocortin-1 Receptor Polymorphisms

Introducción: El receptor de la melanocortina-1 (MC1R) es un importante determinante del riesgo de melanoma debido a su función en la producción de melanina en respuesta a la exposición solar. Objetivos: Analizar las características fenotípicas e histológicas de los pacientes con melanoma cutáneo portadores de mutaciones del MC1R asociadas a riesgo de melanoma y la influencia de la exposición solar en la aparición del melanoma. Material y métodos: Se incluyeron 224 pacientes diagnosticados de melanoma atendidos en el Servicio de Dermatología del Hospital General Universitario Gregorio Marañón (septiembre de 2004 -diciembre de 2009). Se realizó la secuenciación genómica del ADN del MC1R mediante PCR. Resultados: El 58% presentaba al menos una de las siguientes variantes de MC1R (V60L, V92M, I155T, R160W, D294H, R163Q). Estos pacientes presentaban antecedentes de quemaduras solares (p=0,018), melanomas localizados en áreas de exposición solar intermitente (p=0,019), con predominio del tipo histológico de extensión superficial. Estas asociaciones fueron especialmente significativas en los portadores de las variantes R160W y D294H. Los portadores de R160W presentaron además melanomas asociados a nevus melanocíticos (p=0,028). Conclusión: Los resultados obtenidos sugieren que puede existir una relación entre la expresión de determinadas variantes de MC1R y los hábitos de exposición solar, antecedentes de quemadura y tipo de piel del paciente, así como una mayor frecuencia de melanomas de extensión superficial y melanomas asociados a nevus en portadores de ciertas mutaciones de MC1R (AU)

Background: The melanocortin-1 receptor (MC1R) is an important risk factor for melanoma due to its role in the production of melanin in response to sun exposure. Objectives: To analyze the phenotypic and histologic characteristics of cutaneous melanoma in patients carrying mutations in MC1R and assess the influence of sun exposure on the occurrence of melanoma. Material and methods: A total of 224 patients with a diagnosis of melanoma seen in the Department of Dermatology at Hospital General Universitario Gregorio Marañón in Madrid, Spain between September 2004 and December 2009 were included in the study. The genomic sequence of MC1R was analyzed by polymerase chain reaction. Results: At least one of the following MC1R variants was present in 58% of the patients: V60L, V92M, I155T, R160W, D294H, and R163Q. Carriers of those variants had a history of sunburn (P=.018) and melanomas located on areas with intermittent sun exposure (P=0.019), and the majority had a diagnosis of superficial spreading melanoma. These associations were especially significant in patients with the R160W and D294H variants. Carriers of R160W also had melanomas associated with melanocytic nevi (P=0.028). Conclusions: The results of our study suggest that there may be a relationship between the expression of certain MC1R variants and sun exposure, history of sunburn, and skin type. They also indicate a higher frequency of superficial spreading melanomas and melanomas associated with melanocytic nevi in patients carrying certain mutations in MC1R (AU)

Sperm rates of 7q11.23, 15q11q13 and 22q11.2 deletions and duplications: a FISH approach.

Genomic disorders are human diseases caused by meiotic chromosomal rearrangements of unstable regions flanked by Low Copy Repeats (LCRs). LCRs act as substrates for Non-Allelic Homologous Recombination (NAHR) leading to deletions and duplications. The aim of this study was to assess the basal frequency of deletions and duplications of the 7q11.23, 15q11-q13 and 22q11.2 regions in spermatozoa from control donors to check differences in the susceptibility to generate anomalies and to assess the contribution of intra- and inter-chromatid NAHR events. Semen samples from ten control donors were processed by FISH. A customized combination of probes was used to discriminate among normal, deleted and duplicated sperm genotypes. A minimum of 10,000 sperm were assessed per sample and region. There were no differences in the mean frequency of deletions and duplications (del + dup) among the 7q11.23, 15q11-q13 and 22q11.2 regions (frequency ± SEM, 0.37 ± 0.02; 0.46 ± 0.07 and 0.27 ± 0.07%, respectively) (P = 0.122). Nevertheless, hierarchical cluster analysis reveals interindividual differences suggesting that particular haplotypes could be the main source of variability in NAHR rates. The mean frequency of deletions was not different from the mean frequency of duplications in the 7q11.23 (P = 0.202) and 15q11-q13 (P = 0.609) regions, indicating a predominant inter-chromatid NAHR. By contrast, in the 22q11.2 region the frequency of deletions slightly exceed duplications (P = 0.032), although at the individual level any donor showed differences. Altogether, our results support the inter-chromatid NAHR as the predominant mechanism involved in the generation of sperm deletions and duplications.

The myotonic dystrophies: diagnosis and management.

There are currently two clinically and molecularly defined forms of myotonic dystrophy: (1) myotonic dystrophy type 1 (DM1), also known as 'Steinert's disease'; and (2) myotonic dystrophy type 2 (DM2), also known as proximal myotonic myopathy. DM1 and DM2 are progressive multisystem genetic disorders with several clinical and genetic features in common. DM1 is the most common form of adult onset muscular dystrophy whereas DM2 tends to have a milder phenotype with later onset of symptoms and is rarer than DM1. This review will focus on the clinical features, diagnosis and management of DM1 and DM2 and will briefly discuss the recent advances in the understanding of the molecular pathogenesis of these diseases with particular reference to new treatments using gene therapy.

SnoN expression is differently regulated in microsatellite unstable compared with microsatellite stable colorectal cancers.

BACKGROUND: SnoN is an important regulator of the transforming growth factor beta (TGFbeta) signalling pathway and has been shown to exhibit both tumour promotion and suppression activity. METHODS: To further explore the role of this complex molecule in colorectal tumorigenesis, we examined 52 paired normal and tumour colorectal specimens stratified by level of microsatellite instability; 18 with high-level microsatellite instability (MSI-H) and 34 microsatellite stable (MSS). SnoN transcript expression was quantitated by real-time PCR and analysed with respect to clinical indicators of prognosis. RESULTS: Within the MSI-H subgroup, SnoN was commonly either up-regulated (6/18, 33%) or down-regulated (7/18, 39%). A significantly different distribution of SnoN expression was observed in MSS cancers compared with MSI-H (P < or = 0.001). Whilst 17/34 (50%) of MSS tumours demonstrated up-regulation, none showed down-regulated expression. Within the MSI-H subgroup, up-regulation was significantly correlated with lack of repeat tract mutation in the TGFbetaRII gene (P < or = 0.025), suggesting that SnoN is more frequently up-regulated in the presence of functional TGFbeta signalling. CONCLUSION: Together these data support the notion that SnoN has both oncogenic and tumour suppressive properties depending on other genetic changes within the tumour, and that the MSI-H pathway of colorectal tumorigenesis presents an excellent model for the study of these opposing functions.

Variability and inequity in testing of somatic tissue for hereditary cancer: a survey of UK clinical practice.

A growing body of literature demonstrates the benefits of molecular pathological investigations of tumour material in the identification of individuals with hereditary non-polyposis colorectal cancer and debates the best detection strategies. This testing is novel as it is the first widespread use of somatic tissue testing to inform genetic analysis and requires the co-ordination of both histopathology and molecular genetics laboratories. However, the clinical use and experience of microsatellite instability (MSI) testing and immunohistochemical analysis have not been reported. A respondent from every cancer genetics centre in the UK (n= 24, response rate 100%) and laboratory performing MSI testing (n= 5, response rate 100%) was interviewed by telephone to ascertain test availability, testing methods, eligibility criteria and post-test management. Twenty centres (83%) offer eligible clients at least one form of tumour testing, and all use tumour testing to determine who should have access to germ line genetic testing. However, no two laboratories used the same testing methods, seven different testing strategies were applied and there was considerable variation in eligibility criteria. The implications of these variations are considered, and recommendations for the development of a consistent service for testing of somatic tissue offered.

Microsatellite instability (MSI) increases with age in normal somatic cells.

Small pool PCR (SP-PCR) is a sensitive method for the detection and quantification of microsatellite instability (MSI) in somatic cells. Here we propose that mutant microsatellite fragments accumulate with age in normal somatic cells and that this increase in MSI can be quantified by SP-PCR. MSI at 6 microsatellite loci was determined by SP-PCR in PBL DNA from 17 "normal" blood bank donors. These individuals varied in age from 20 to 67 y/o. MSI phenotypes were plotted against age in a regression analyses. A positive slope indicated a correlation between age and MSI phenotype (p=0.0006). The mean weighted average mutant frequencies across all loci for all individuals in the age groups (0.009 for 20-30 y/o; 0.019 for 35-50 y/o; 0.034 for 60-70 y/o) were also significantly different from each other (p<0.01). A baseline for increases of MSI with age in human somatic cells was therefore begun and the effectiveness of SP-PCR to evaluate low, but significant, levels of MSI, established.

The DNA-instability test as a specific marker of malignancy and its application to detect cancer clones in borderline malignancy.

Recent progress in cytogenetic and biochemical mutation assay technologies has enabled us to detect single gene alterations and gross chromosomal rearrangements, and it became clear that all cancer cells are genetically unstable. In order to detect the genome-wide instability of cancer cells, a new simple method, the DNA-instability test, was developed. The methods to detect genomic instability so far reported have only demonstrated the presence of qualitative and quantitative alterations in certain specific genomic loci. In contrast to these commonly used methods to reveal the genomic instability at certain specific DNA regions, the newly introduced DNA-instability test revealed the presence of physical DNA-instability in the entire DNA molecule of a cancer cell nucleus as revealed by increased liability to denature upon HCl hydrolysis or formamide exposure. When this test was applied to borderline malignancies, cancer clones were detected in all cases at an early-stage of cancer progression. We proposed a new concept of "procancer" clones to define those cancer clones with "functional atypia" showing positivities for various cancer markers, as well as DNA-instability testing, but showing no remarkable ordinary "morphological atypia" which is commonly used as the basis of histopathological diagnosis of malignancy.

Asociación entre la inestabilidad de microsatélites y las características clínicas y anatomopatológicas en pacientes con cáncer de colon esporádico / Association between microsatellite instability and clinico-pathological characteristics in sporadic colon cancer

FUNDAMENTO Y OBJETIVO: La inestabilidad de microsatélites derivada del fallo en la reparación delos falsos emparejamientos del ADN es la alteración característica de los tumores de la vía mutadorao inestables (MSI). Tales casos parecen presentar diferencias desde el punto de vista clinicopatológicocon los tumores de la vía supresora o estables (MSS). Los tumores con alto gradode inestabilidad (MSI-H) parecen constituir una nueva entidad de tumores con diferencias endeterminadas características anatomopatológicas y clínicas con respecto a los tumores estables(MSS) e inestables de bajo grado (MSI-L). En el presente estudio se valora la posible asociaciónentre el alto grado de inestabilidad de microsatélites con la localización, contenido mucinoso,grado de diferenciación, estadio, así como el intervalo libre de enfermedad y supervivencia.PACIENTES Y MÉTODO: Se clasifica a 117 pacientes con cáncer de colon esporádico en las poblacionesMSS/MSI-L y MSI-H (siguiendo las recomendaciones del National Cancer Institute) mediantereacción en cadena de la polimerasa y electroforesis de 7 microsatélites.RESULTADOS: Los tumores MSI-H tendieron a localizarse en el colon derecho (p = 0,022) y a presentarcontenido mucinoso (p = 0,04). El conjunto de pacientes MSI-H de estadios II y III no presentóintervalos libres de enfermedad ni períodos de supervivencia más prolongados (p = 0,54, p= 0,37, respectivamente). Los tumores MSI-H de estadio II presentaron períodos de supervivenciamás prolongados que los tumores MSS/MSI-L (p = 0,027). No observamos diferencias en la respuestaa quimioterapia con 5-fluorouracilo y leucovorín entre los grupos MSS/MSI-L y MSI-H (p =0,38).CONCLUSIONES: El alto grado de inestabilidad de microsatélites se asocia con determinadas característicaspatológicas, así como con períodos de supervivencia más prolongados para los tumoresde estadios II

BACKGROUND AND OBJECTIVE: Currently, colon cancer is a leading cause of cancer death worldwide.It progresses according to three molecular pathways, named suppressor, mutador andmethylator. Microsatellite instability is a hallmark of the lack of reparation, of DNA mismatchesand it characterizes a subset of colon tumors (unstable tumors, MSI). MSI-H patients (high degreeof microsatellite instability) seem to share clinico-pathological differences with MSS (microsatellitestable) and MSI-L (low degree of microsatellite instability) patients. In this study,associations between high degree of microsatellite instability and pathological (location, mucinouscontent, differentiation grade, stages T3N0, stages II and III) and clinical features (responseto chemotherapy, disease-free survival and overall survival) were evaluated.PATIENTS AND METHOD: 117 patients with sporadic colon cancer were classified into two populations(MSS/MSI-L and MSI-H) by using PCR and electrophoresis of seven microsatellites, accordingto the National Cancer Institute recommendations.RESULTS: MSI-H tumors tended to be located in the right colon (p = 0.022) and were of mucinoushistologic type (p = 0.04). No differences in disease-free survival and overall survival betweengroup of stage II and III patients with MSS/ MSI-L and corresponding ones with MSI-Hcolon cancer were found (p = 0.54, p = 0.37, respectively). Conversely, MSI-H patients withstage II colon cancer had a favourable prognosis (p = 0.027). Nevertheless, response to 5-fluorouracil(5-FU) and leucovorin was similar in MSS/ MSI-L and MSI-H groups (p = 0.38).CONCLUSIONS: MSI-H patients are characterized by certain pathological features; those MSI-Hpatients with a stage II seem to have a better prognosis than MSS/ MSI-L patients

Evolution and functional classification of vertebrate gene deserts.

Large tracts of the human genome, known as gene deserts, are devoid of protein-coding genes. Dichotomy in their level of conservation with chicken separates these regions into two distinct categories, stable and variable. The separation is not caused by differences in rates of neutral evolution but instead appears to be related to different biological functions of stable and variable gene deserts in the human genome. Gene Ontology categories of the adjacent genes are strongly biased toward transcriptional regulation and development for the stable gene deserts, and toward distinctively different functions for the variable gene deserts. Stable gene deserts resist chromosomal rearrangements and appear to harbor multiple distant regulatory elements physically linked to their neighboring genes, with the linearity of conservation invariant throughout vertebrate evolution.

Mitochondrial DNA instability in malignant melanoma of the skin is mostly restricted to nodular and metastatic stages.

Ultraviolet (UV) radiation is thought to be a major contributor to the development of sporadic malignant melanoma of the skin. It may induce alterations in genomic or mitochondrial DNA (mtDNA), especially C to T or CC to TT changes. Mutations or other alterations in mtDNA have been reported in a variety of human cancers and may be due to different mechanisms. In this study, we have attempted to elucidate whether aberrations in the mtDNA of melanoma are due to UV radiation or other factors by investigating two parts of the mitochondrial D-loop and two mitochondrial genes, as well as looking for the delta4977 mtDNA deletion and mtDNA duplications, in 61 primary malignant melanomas and neighbouring normal skin tissue (in 70% of primary tumours; otherwise, corresponding blood samples). Point mutations were a rare feature, occurring in only seven tumour samples and never as a C to T change, whereas mtDNA instability in the D-loop (mtMSI) was found in 13% of primary nodular tumours and 20% of metastases. A de novo delta4977 mtDNA deletion was demonstrated in 10% of melanomas; in 20% of patients, mtDNA duplications and/or the delta4977 mtDNA deletion was detectable. Our data indicate that mtDNA alterations in malignant melanoma are not induced by UV radiation. In addition, point mutations and mtMSI were mostly a feature of nodular and metastatic melanoma samples.

Relationship between mitochondrial DNA instability, mitochondrial DNA large deletions, and nuclear microsatellite instability in head and neck squamous cell carcinomas.

Mitochondrial DNA (mtDNA) mutations in coding and noncoding regions have been reported in a variety of human cancers. Despite a greater number of studies, the relationship between such alterations and nuclear microsatellite instability (nMSI) of the tumor cells remains controversial. To contribute new data to this discussion, we investigated head and neck squamous cell carcinomas (HNSCC) for mutations and mitochondrial microsatellite instability (mtMSI) in 2 parts of the mitochondrial D-loop as well as mutations in 2 mitochondrial genes and for the delta4977 mtDNA deletion. These results were compared with data of an analysis for microsatellite instability at IGFIIR, hMSH3, hMSH6, and 5 dinucleotide repeats. We found mtMSI, low nMSI, and high nMSI in 42%, 36%, and 13% of HNSCC primary tumors, respectively. A de novo delta4977 mtDNA deletion could be demonstrated in 25% of HNSCCs. A correlation between mtMSI and nMSI or between a de novo occurrence of the delta4977 mtDNA deletion and nMSI could not be detected in our HNSCC samples (P values 0.527 and 0.078, respectively). Nevertheless, the high rate of mtMSI suggests an involvement of mtDNA alterations in the tumorigenesis of this head and neck cancer and supports the proposal that this aberration may be a new tumor marker.

BAC microarray analysis of 15q11-q13 rearrangements and the impact of segmental duplications.

Chromosome 15q11-q13 is one of the most variable regions of the human genome, with numerous clinical rearrangements involving a dosage imbalance. Multiple clusters of segmental duplications are found in the pericentromeric region of 15q and at the breakpoints of proximal 15q rearrangements. Using sequence maps and previous global analyses of segmental duplications in the human genome, a targeted microarray was developed to detect a wide range of dosage imbalances in clinical samples. Clones were also chosen to assess the effect of paralogous sequences in the array format. In 19 patients analysed, the array data correlated with microsatellite and FISH characterisation. The data showed a linear response with respect to dosage, ranging from one to six copies of the region. Paralogous sequences in arrayed clones appear to respond to the total genomic copy number, and results with such clones may seem aberrant unless the sequence context of the arrayed sequence is well understood. The array CGH method offers exquisite resolution and sensitivity for detecting large scale dosage imbalances. These results indicate that the duplication composition of BAC substrates may affect the sensitivity for detecting dosage variation. They have important implications for effective microarray design, as well as for the detection of segmental aneusomy within the human population.

Matrix attachment region (MAR) properties and abnormal expansion of AT island minisatellites in FRA16B fragile sites in leukemic CEM cells.

AT-rich minisatellites (AT islands) are sites of genomic instability in cancer cells and targets for extremely lethal AT-specific drugs, such as bizelesin. Here we investigated the AT islands in the FRA16B fragile site region for their possible roles in the organization of DNA on the nuclear matrix. The FRA16B AT island nominally spans approximately 3 kb of mostly >90% A/T DNA. In silico analysis indicates that this domain exhibits characteristics of nuclear matrix attachment regions (MARs): an exceptionally intense computed 'MAR potential' and profound duplex destabilization and flexibility. FRA16B repeats specifically bind to isolated nuclear matrices, which indicates their in vitro MAR function. This binding is several-fold greater than that of a known MAR in the c-myc gene. AT islands in fragile sites FRA16B and FRA16D are significantly more abundant in CEM cells that are hypersensitive to bizelesin compared to normal WI-38 cells. FRA16B overabundance in CEM is due to an approximately 10-fold expansion of FRA16B repeats. The expanded FRA16B minisatellites in CEM cells preferentially localize to the nuclear matrix-associated DNA indicating their in vivo MAR function. The unexpanded repeats in WI-38 cells localize to the loop DNA. The c-myc MAR is also matrix-associated in CEM cells while localizing to loop DNA in WI-38 cells. These results are the first to demonstrate that AT islands in fragile sites can function as MARs both in vitro and in vivo. The ability of FRA16B-mediated MAR sites to rearrange depending on the repeat expansion status could be relevant to both genomic instability of cancer cells and their sensitivity to AT-island targeting drugs.

Insertional DNA and spontaneous mutation at the white locus in Drosophila simulans.

A large body of data on molecular analyses of several multiallelic loci in Drosophila melanogaster has demonstrated a high incidence of mobile DNA element insertions among spontaneous mutations. In the sibling species D. simulans, the dispersed, middle repetitive, nomadic sequences are reduced to about one-seventh that of its sibling species (Dowsett and Young 1982). Does this reduced amount of middle repetitive DNA (or mobile DNA sequences) mean that in D. simulans the occurrence of insertion mutants will be rare compared with that of D. melanogaster? To test this possibility, we collected seven different spontaneous white mutants of D. simulans and studied their molecular gene structures. Five out of seven mutants had insertion sequences which varied in length from 0.4 kb to 16 kb. One bore a deletion spanning the w region and another showed no gross structural alteration. Thus the proportion of insertional mutations at the white locus in D. simulans is equivalent to that observed in D. melanogaster. Among the five insertional mutants, one, wmky, showed genetic instability; the other four were stable. wmky was found to mutate at a frequency of 2.1 x 10(-5) in meiotic cells and may also be unstable in somatic cells.