Detection of a novel gross deletion in the UNC13D gene ends the diagnostic odyssey for a family with familial hemophagocytic lymphohistiocytosis 3.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis (FHL) is an immunological disorder characterized by overactivation of macrophages and T lymphocytes. This autosomal recessive condition has been characterized into multiple types depending on the genetic etiology. FHL type 3 is associated with bi-allelic pathogenic variants in the UNC13D gene. CASE PRESENTATION: We present a 12-year diagnostic odyssey for a family with FHL that signifies the advances of FHL genetic testing in a clinical genetic diagnostic laboratory setting. We describe the first case of a large UNC13D gross deletion in trans to a nonsense variant in a family with FHL3, which may have been mediated by Alu elements within introns 12 and 25 of the UNC13D gene. CONCLUSIONS: This case highlights the importance of re-evaluating past genetic testing for a patient and family as test technology evolves in order to end a diagnostic odyssey.

Assortative mating and parental genetic relatedness contribute to the pathogenicity of variably expressive variants.

We examined more than 97,000 families from four neurodevelopmental disease cohorts and the UK Biobank to identify phenotypic and genetic patterns in parents contributing to neurodevelopmental disease risk in children. We identified within- and cross-disorder correlations between six phenotypes in parents and children, such as obsessive-compulsive disorder (R = 0.32-0.38, p < 10-126). We also found that measures of sub-clinical autism features in parents are associated with several autism severity measures in children, including biparental mean Social Responsiveness Scale scores and proband Repetitive Behaviors Scale scores (regression coefficient = 0.14, p = 3.38 × 10-4). We further describe patterns of phenotypic similarity between spouses, where spouses show correlations for six neurological and psychiatric phenotypes, including a within-disorder correlation for depression (R = 0.24-0.68, p < 0.001) and a cross-disorder correlation between anxiety and bipolar disorder (R = 0.09-0.22, p < 10-92). Using a simulated population, we also found that assortative mating can lead to increases in disease liability over generations and the appearance of "genetic anticipation" in families carrying rare variants. We identified several families in a neurodevelopmental disease cohort where the proband inherited multiple rare variants in disease-associated genes from each of their affected parents. We further identified parental relatedness as a risk factor for neurodevelopmental disorders through its inverse relationship with variant pathogenicity and propose that parental relatedness modulates disease risk by increasing genome-wide homozygosity in children (R = 0.05-0.26, p < 0.05). Our results highlight the utility of assessing parent phenotypes and genotypes toward predicting features in children who carry rare variably expressive variants and implicate assortative mating as a risk factor for increased disease severity in these families.

Assortative mating and parental genetic relatedness drive the pathogenicity of variably expressive variants.

We examined more than 38,000 spouse pairs from four neurodevelopmental disease cohorts and the UK Biobank to identify phenotypic and genetic patterns in parents associated with neurodevelopmental disease risk in children. We identified correlations between six phenotypes in parents and children, including correlations of clinical diagnoses such as obsessive-compulsive disorder (R=0.31-0.49, p<0.001), and two measures of sub-clinical autism features in parents affecting several autism severity measures in children, such as bi-parental mean Social Responsiveness Scale (SRS) scores affecting proband SRS scores (regression coefficient=0.11, p=0.003). We further describe patterns of phenotypic and genetic similarity between spouses, where spouses show both within- and cross-disorder correlations for seven neurological and psychiatric phenotypes, including a within-disorder correlation for depression (R=0.25-0.72, p<0.001) and a cross-disorder correlation between schizophrenia and personality disorder (R=0.20-0.57, p<0.001). Further, these spouses with similar phenotypes were significantly correlated for rare variant burden (R=0.07-0.57, p<0.0001). We propose that assortative mating on these features may drive the increases in genetic risk over generations and the appearance of "genetic anticipation" associated with many variably expressive variants. We further identified parental relatedness as a risk factor for neurodevelopmental disorders through its inverse correlations with burden and pathogenicity of rare variants and propose that parental relatedness drives disease risk by increasing genome-wide homozygosity in children (R=0.09-0.30, p<0.001). Our results highlight the utility of assessing parent phenotypes and genotypes in predicting features in children carrying variably expressive variants and counseling families carrying these variants.

Genetic Testing in Patients with Neurodevelopmental Disorders: Experience of 511 Patients at Cincinnati Children's Hospital Medical Center.

Our institution developed and continuously improved a Neurodevelopmental Reflex (NDR) algorithm to help physicians with genetic test ordering for neurodevelopmental disorders (NDDs). To assess its performance, we performed a retrospective study of 511 patients tested through NDR from 2018 to 2019. SNP Microarray identified pathogenic/likely pathogenic copy number variations in 27/511 cases (5.28%). Among the 484 patients tested for Fragile X FMR1 CGG repeats, a diagnosis (0.20%) was established for one male mosaic for a full mutation, a premutation, and a one-CGG allele. Within the 101 normocephalic female patients tested for MECP2, two patients were found to carry pathogenic variants (1.98%). This retrospective study suggested the NDR algorithm effectively established diagnoses for patients with NDDs with a yield of 5.87%.

A rare case of postnatal mosaic trisomy 12 with severe congenital heart disease and literature review.

Trisomy 12 is a rare autosomal aneuploidy. All postnatally diagnosed individuals with trisomy 12 have been mosaic for this chromosome abnormality. We herein report an infant girl presented at 2 weeks of age with severe congenital heart defect, tracheobronchomalacia, and dysmorphic features. All of the dysmorphic features of this patient fit into the known phenotype spectrum of mosaic trisomy 12, although this patient uniquely presented with macrocephaly. Tracheo-bronchomalacia has been described once previously but had a significant impact on this patient's clinical course. The patient passed away at 2-month-old due to cardiac and respiratory complications. Chromosomal single nucleotide polymorphism (SNP) microarray analysis on a peripheral blood sample from the patient revealed trisomy 12 in approximately 50% of cells. Concurrent fluorescence in situ hybridization analysis of uncultured blood cells detected a comparable level of trisomy 12 mosaicism. Compared to conventional cytogenetics, SNP microarray examines all nucleated cells without sampling bias, has an increased power to estimate mosaicism level, and can provide a quick assessment of the underlying mechanism. Here we demonstrate the utilization of SNP microarray in the clinical diagnosis of those once considered rare disorders but might have been missed by conventional cytogenetic techniques.

3q29 microduplication syndrome: Clinical and molecular description of eleven new cases.

Interstitial duplications of 3q29 have recently been described in association with a new genetic syndrome characterized by a neurodevelopmental phenotype. A total of 16 individuals with the 3q29 duplication have been reported in the literature with clinical features that include intellectual disability, language delay, epilepsy, structural brain anomalies, micro/macrocephaly, generalized obesity, ocular abnormalities, distinctive facial features, cleft palate, and musculoskeletal anomalies. In this paper, we summarize the current literature and present eleven additional cases from nine families with the 3q29 microduplication identified by microarray analysis at the Cincinnati Children's Hospital Medical Center Laboratory of Genetics and Genomics. Three diagnoses were made prenatally, making this the first case series to describe 3q29 duplications incidentally identified during the prenatal period. We further delineate the minimal region of overlap previously described in the literature and explore the modifying effects of "second hit" genetic aberrations, which were frequent in our cohort.

Diffuse Intrinsic Pontine Glioma Cells Are Vulnerable to Mitotic Abnormalities Associated with BMI-1 Modulation.

Diffuse intrinsic pontine glioma (DIPG) is a poor-prognosis pediatric brain tumor with a median survival of less than 1 year. No effective therapy is currently available, and no therapeutic advances have been made in several decades. We have previously identified BMI-1 as a potential therapeutic target in DIPG and have shown that BMI-1 is highly expressed in DIPG tumors regardless of histone 3 subtype. In the present study, we show that the modulation of BMI-1 leads to DNA damage, M phase cell-cycle arrest, chromosome scattering, and cell death. Interestingly, EZH2 inhibition did not alter these effects. Furthermore, modulation of BMI-1 sensitizes DIPG patient-derived stem-like cells to ionizing radiation (IR). Treatment of DIPG stem-like cells with PTC596, a BMI-1 modulator, and IR impairs the kinetics of DNA damage response (DDR). Both DDR foci formation and resolution were delayed, resulting in further reduction in cell viability compared with either treatment alone. In vivo, treatment of mice bearing DIPG xenografts with PTC596 leads to decreased tumor volume and growth kinetics, increased intratumoral apoptosis, and sustained animal survival benefit. Gene expression analysis indicates that BMI-1 expression correlates positively with DIPG stemness and BMI-1 signature. At the single-cell level, the analysis reveals that BMI-1 pathway is upregulated in undifferentiated cells and positively correlates with stemness in DIPG tumors. IMPLICATIONS: Together, our findings indicate that BMI-1 modulation is associated with mitotic abnormalities, impaired DDR, and cell death, supporting the combination of BMI-1 modulation and radiation as a promising novel therapy for children with DIPG.

Confirmatory testing illustrates additional risks for structural sex chromosome abnormalities in fetuses with a non-invasive prenatal screen positive for monosomy X.

More and more women rely on non-invasive prenatal screening (NIPS) to detect fetal sex and risk for aneuploidy. The testing applies massively parallel sequencing or single nucleotide polymorphism (SNP) microarray to circulating cell-free DNA to determine relative copy number. In addition to trisomies 13, 18, and 21, some labs offer screening for sex chromosome abnormalities as part of their test. In this study, an index neonate screened positive for monosomy X and had discordant postnatal chromosomes indicating an X;autosome translocation. This patient prompted a retrospective chart review for similar cases at a large NIPS testing center. The review found 28 patients with an abnormal NIPS for monosomy X who were eventually diagnosed with additional discrepant structural sex chromosome abnormalities including translocations, isochromosomes, deletions, rings, markers, and uniparental disomy. The majority of these were mosaic with monosomy X, but in seven cases, there was no evidence of mosaicism on confirmatory testing. The identification of multiple sex chromosome aneuploidies in these cases supports the need for additional genetic counseling prior to NIPS testing and following abnormal NIPS results that are positive for monosomy X. This finding broadens our knowledge about the variable outcomes of positive monosomy X NIPS results and emphasizes the importance of confirmatory testing and clinical follow up for these patients.

Assessing Genomic Copy Number Alterations as Best Practice for Renal Cell Neoplasia: An Evidence-Based Review from the Cancer Genomics Consortium Workgroup.

Renal cell neoplasia are heterogeneous with diverse histology, genetic alterations, and clinical behavior that are diagnosed mostly on morphologic features. The Renal Cell Neoplasia Workgroup of the Cancer Genomics Consortium systematically evaluated peer-reviewed literature on genomic studies of renal cell carcinoma (RCC), including clear cell RCC, papillary RCC, chromophobe RCC, and the translocation RCC involving TFE3, TFEB and MITF rearrangements, as well as benign oncocytoma, which together comprise about 95% of all renal cell neoplasia. The Workgroup curated recurrent copy number alterations (CNAs), copy-neutral loss-of-heterozygosity (cnLOH), rearrangements, and mutations, found in each subtype and assigned clinical relevance according to established criteria. In clear cell RCC, loss of 3p has a disease-initiating role and most likely also in progression with mutations detected in VHL and other genes mapped to this arm, and loss of 9p and/or 14q has well-substantiated prognostic utility. Gain of chromosomes 7 and 17 are hallmark CNAs of papillary RCC, but patterns of other CNAs as detected by chromosomal microarray analysis (CMA) afford sub-classification into Type 1 and 2 with prognostic value, and for further sub-stratification of Type 2. Inherent chromosome loss in chromophobe RCC as detected by CMA is useful for distinguishing the eosinophilic variant from benign oncocytoma which in contrast exhibits few CNAs or rearranged CCND1, but share mitochondrial DNA mutations. In morphologically atypical RCCs, rearrangement of TFE3 and TFEB should be considered in the differential diagnosis, portending an aggressive RCC subtype. Overall, this evidence-based review provides a validated role for assessment of CNAs in renal cell neoplasia in the clinical setting to assist in renal cell neoplasm diagnosis and sub-classification within subtypes that is integral to the management of patients, from small incidentally found renal masses to larger surgically resected specimens, and simultaneously identify the presence of key alterations portending outcome in malignant RCC subtypes.

The classification of pediatric and young adult renal cell carcinomas registered on the children's oncology group (COG) protocol AREN03B2 after focused genetic testing.

BACKGROUND: Renal cell carcinomas (RCCs) are rare in young patients. Knowledge of their pathologic and molecular spectrum remains limited, and no prospective studies have been performed to date in this population. This study analyzes patients diagnosed with RCC who were prospectively enrolled in the AREN03B2 Children's Oncology Group (COG). The objective was to classify these tumors with the aid of focused genetic testing and to characterize their features. METHODS: All tumors registered as RCC by central review were retrospectively re-reviewed and underwent additional ancillary studies. Tumors were classified according to the 2016 World Health Organization classification system when possible. RESULTS: In total, 212 tumors were identified, and these were classified as microphthalmia transcription factor (MiT) translocation RCC (MiT-RCC) (41.5%), papillary RCC (16.5%), renal medullary carcinoma (12.3%), chromophobe RCC (6.6%), clear cell RCC (3.3%), fumarate hydratase-deficient RCC (1.4%), and succinate dehydrogenase-deficient RCC (0.5%). Other subtypes included tuberous sclerosis-associated RCC (4.2%), anaplastic lymphoma kinase (ALK)-rearranged RCC (3.8%), thyroid-like RCC (1.4%), myoepithelial carcinoma (0.9%), and unclassified (7.5%). MiT-RCCs were classified as either transcription factor E3 (TFE3) (93.2%) or EB (TFEB) (6.8%) translocations, and characterization of fusion partners was possible in most tumors. CONCLUSIONS: The current study delineates the frequency of distinct RCC subtypes in a large prospective series of young patients and contributes knowledge to the diagnostic, clinical, and genetic features of MiT-RCC, the most common subtype among this age group. The identification of rare subtypes expands the spectrum of RCC in young patients, supporting the need for a thorough diagnostic workup. These studies may aid in the introduction of specific therapies for different RCC subtypes in the future. Cancer 2018. © 2018 American Cancer Society.

Placental Histomorphology in a Case of Double Trisomy 48,XXX,+18.

BACKGROUND: Approximately 50% of early spontaneous abortions are found to have chromosomal abnormalities. In these cases, certain histopathologic abnormalities are suggestive of, although not diagnostic for, the presence of chromosomal abnormalities. However, placental histomorphology in cases of complex chromosomal abnormalities, including double trisomies, is virtually unknown. CASE REPORT: We present the case of a 27-year-old G3P22002 female presenting at 19 weeks and 1 day of gestation by last menstrual period for scheduled prenatal visit. Ultrasound revealed a single fetus without heart tones and adequate amniotic fluid. Limited fetal measurements were consistent with estimated gestational age of 17 weeks. Labor was induced with misoprostol due to fetal demise. Autopsy revealed an immature female fetus with grade 1-2 maceration. The ears were low-set and posteriorly rotated. The fingers were short bilaterally, and the right foot showed absence of the second and third digits. Evaluation of the organs showed predominantly marked autolysis consistent with retained stillbirth. Placental examination revealed multiple findings, including focal pseudovillous papilliform trophoblastic proliferation of the undersurface of the chorionic plate and clustering of perpendicularly oriented sclerotic chorionic villi in the chorion laeve, which have not been previously reported in cases of chromosomal abnormalities. Karyotype of placental tissue revealed a 48,XXX,+18 karyotype and the same double trisomy of fetal thymic tissue by FISH. CONCLUSION: In addition to convoluted outlines of chorionic villi, villous trophoblastic pseudoinclusions, and clusters of villous cytotrophoblasts, the previously unreported focal pseudovillous papilliform trophoblastic proliferation of the undersurface of the chorionic plate and clustering of perpendicularly oriented sclerotic chorionic villi in the chorion laeve were observed in this double trisomy case. More cases have to be examined to show if the histology is specific for this double trisomy.

Alu element insertion in PKLR gene as a novel cause of pyruvate kinase deficiency in Middle Eastern patients.

Pyruvate kinase deficiency (PKD) is the most frequent red blood cell enzyme abnormality of the glycolytic pathway and the most common cause of hereditary nonspherocytic hemolytic anemia. Over 250 PKLR-gene mutations have been described, including missense/nonsense, splicing and regulatory mutations, small insertions, small and gross deletions, causing PKD and hemolytic anemia of variable severity. Alu retrotransposons are the most abundant mobile DNA sequences in the human genome, contributing to almost 11% of its mass. Alu insertions have been associated with a number of human diseases either by disrupting a coding region or a splice signal. Here, we report on two unrelated Middle Eastern patients, both born from consanguineous parents, with transfusion-dependent hemolytic anemia, where sequence analysis revealed a homozygous insertion of AluYb9 within exon 6 of the PKLR gene, causing precipitous decrease of PKLR RNA levels. This Alu element insertion consists a previously unrecognized mechanism underlying pathogenesis of PKD.

ATM is required for SOD2 expression and homeostasis within the mammary gland.

PURPOSE: ATM activates the NF-κB transcriptional complex in response to genotoxic and oxidative stress. The purpose of this study was to examine if the NF-κB target gene and critical antioxidant SOD2 (MnSOD) in cultured mammary epithelium is also ATM-dependent, and what phenotypes arise from deletion of ATM and SOD2 within the mammary gland. METHODS: SOD2 expression was studied in human mammary epithelial cells and MCF10A using RNAi to knockdown ATM or the NF-κB subunit RelA. To study ATM and SOD2 function in mammary glands, mouse lines containing Atm or Sod2 genes containing LoxP sites were mated with mice harboring Cre recombinase under the control of the whey acidic protein promoter. Quantitative PCR was used to measure gene expression, and mammary gland structure was studied using histology. RESULTS: SOD2 expression is ATM- and RelA-dependent, ATM knockdown renders cells sensitive to pro-oxidant exposure, and SOD mimetics partially rescue this sensitivity. Mice with germline deletion of Atm fail to develop mature mammary glands, but using a conditional knockout approach, we determined that Atm deletion significantly diminished the expression of Sod2. We also observed that these mice (termed AtmΔ/Δ) displayed a progressive lactation defect as judged by reduced pup growth rate, aberrant lobulo-alveolar structure, diminished milk protein gene expression, and increased apoptosis within lactating glands. This phenotype appears to be linked to dysregulated Sod2 expression as mammary gland-specific deletion of Sod2 phenocopies defects observed in AtmΔ/Δ dams. CONCLUSIONS: We conclude that ATM is required to promote expression of SOD2 within the mammary epithelium, and that both ATM and SOD2 play a crucial role in mammary gland homeostasis.

Home electroacupuncture for persistent postsurgical pain: a patient's report.

A 30-year-woman presented with iatrogenic myofascial facial pain of 12 months' duration after surgical treatment for bilateral temporomandibular joint dysfunction. Limited response to manual acupuncture was followed by a trial of electroacupuncture. Response to electroacupuncture was limited in duration and one option was to teach her partner to perform electroacupuncture at home. The patient and partner, trained in beauty therapy, embraced this idea and it proved equally effective as therapist-provided acupuncture. We describe this case report from the perspective of the patient, caregiver or acupuncture partner and therapist. Home electroacupuncture seems to be safe, acceptable and practicable as a maintenance treatment for patients with persistent postsurgical pain of myofascial origin.

DNA methyltransferase inhibitor, zebularine, delays tumor growth and induces apoptosis in a genetically engineered mouse model of breast cancer.

Zebularine is a novel potent inhibitor of both cytidine deaminase and DNA methylation. We examined the effect of zebularine on mammary tumor growth in genetically engineered MMTV-PyMT transgenic mice that develop mammary tumors at 60 days of age with 100% penetrance. The MMTV-PyMT transgenic mice were randomized at 46 days of age into control (n = 25) and zebularine (n = 25) treatment groups and monitored for parameters of tumor growth. Zebularine was administered at 5 mg/mL in drinking water. We observed a significant delay in the growth of mammary tumors in zebularine-treated mice with a statistically significant reduction (P = 0.0135) in total tumor burden at 94 days of age when the mice were sacrificed. After 48 days of zebularine treatment, the tumors were predominantly necrotic compared with untreated animals. In addition, a high apoptotic index by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was observed as early as 13 days following treatment. Immunoblot analysis showed depletion of DNMT1 and partial depletion of DNMT3b after zebularine treatment. Microarray analyses of global gene expression identified upregulation of twelve methylation-regulated genes as well as a set of candidate cancer genes that participate in cell growth and apoptosis. In summary, zebularine inhibits the growth of spontaneous mammary tumors and causes early onset of tumor cell necrosis and apoptosis in a genetically engineered mouse model of breast cancer. Defining the parameters of zebularine-mediated tumor inhibition may advance the future development of DNA methyltransferase inhibitors as an effective cancer treatment.

WIF1 is a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix.

Aberrant activation of the Wnt/ß-catenin signaling axis is a prominent oncogenic mechanism in numerous cancers including cervical cancer. Wnt inhibitory factor-1 (WIF1) is a secreted protein that binds Wnt and antagonizes Wnt activity. While the WIF1 gene is characterized as a target for epigenetic silencing in some tumor types, WIF1 expression has not been examined in human cervical tissue and cervical cancer. Here, we show that WIF1 is unmethylated and its gene product is expressed in normal cervical epithelium and some cultured cervical tumor lines. In contrast, several cervical cancer lines contained dense CpG methylation within the WIF1 gene, and expression of both WIF1 transcript and protein was restored by culturing cells in the presence of the global DNA demethylating agent 5-aza-2'-deoxycytidine. Using single-molecule MAPit methylation footprinting, we observed differences in chromatin structure within the WIF1 promoter region between cell lines that express and those that do not express WIF1, consistent with transcriptional activity and repression, respectively. The WIF1 promoter was aberrantly methylated in ∼60% (10 of 17) high-grade highly undifferentiated squamous cell cervical tumors examined, whereas paired normal tissue showed significantly lower levels of CpG methylation. WIF1 protein was not detectable by immunohistochemistry in tumors with quantitatively high levels of WIF1 methylation. Of note, WIF1 protein was not detectable in two of the seven unmethylated cervical tumors examined, suggesting other mechanisms may contribute WIF1 repression. Our findings establish the WIF1 gene as a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix.

The transglutaminase 2 gene is aberrantly hypermethylated in glioma.

Transglutaminase 2 (TG2) is a ubiquitously expressed protein that catalyzes protein/protein crosslinking. Because extracellular TG2 crosslinks components of the extracellular matrix, TG2 is thought to function as a suppressor of cellular invasion. We have recently uncovered that the TG2 gene (TGM2) is a target for epigenetic silencing in breast cancer, highlighting a molecular mechanism that drives reduced TG2 expression, and this aberrant molecular event may contribute to invasiveness in this tumor type. Because tumor invasiveness is a primary determinant of brain tumor aggressiveness, we sought to determine if TGM2 is targeted for epigenetic silencing in glioma. Analysis of TGM2 gene methylation in a panel of cultured human glioma cells indicated that the 5' flanking region of the TGM2 gene is hypermethylated and that this feature is associated with reduced TG2 expression as judged by immunoblotting. Further, culturing glioma cells in the presence of the global DNA demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor Trichostatin A resulted in re-expression of TG2 in these lines. In primary brain tumors we observed that the TGM2 promoter is commonly hypermethylated and that this feature is a cancer-associated phenomenon. Using publically available databases, TG2 expression in gliomas was found to vary widely, with many tumors showing overexpression or underexpression of this gene. Since overexpression of TG2 leads to resistance to doxorubicin through the ectopic activation of NFκB, we sought to examine the effects of recombinant TG2 expression in glioma cells treated with commonly used brain tumor therapeutics. We observed that in addition to doxorubicin, TG2 expression drove resistance to CCNU; however, TG2 expression did not alter sensitivity to other drugs tested. Finally, a catalytically null mutant of TG2 was also able to support doxorubicin resistance in glioma cells indicating that transglutaminase activity is not necessary for the resistance phenotype.

Comparative epigenomics of human and mouse mammary tumors.

Gene silencing by aberrant epigenetic chromatin alteration is a well-recognized event contributing to tumorigenesis. Although genetically engineered tumor-prone mouse models have proven a powerful tool in understanding many aspects of carcinogenesis, to date few studies have focused on epigenetic alterations in mouse tumors. To uncover epigenetically silenced tumor suppressor genes (TSGs) in mouse mammary tumor cells, we conducted initial genome-wide screening by combining the treatment of cultured cells with the DNA demethylating drug 5-aza-2'-deoxycytidine (5-azadC) and the histone deacetylase inhibitor trichostatin A (TSA) with expression microarray. By conducting this initial screen on EMT6 cells and applying protein function and genomic structure criteria to genes identified as upregulated in response to 5-azadC/TSA, we were able to identify two characterized breast cancer TSGs (Timp3 and Rprm) and four putative TSGs (Atp1B2, Dusp2, FoxJ1 and Smpd3) silenced in this line. By testing a panel of 10 mouse mammary tumor lines, we determined that each of these genes is commonly hypermethylated, albeit with varying frequency. Furthermore, by examining a panel of human breast tumor lines and primary tumors we observed that the human orthologs of ATP1B2, FOXJ1 and SMPD3 are aberrantly hypermethylated in the human disease whereas DUSP2 was not hypermethylated in primary breast tumors. Finally, we examined hypermethylation of several genes targeted for epigenetic silencing in human breast tumors in our panel of 10 mouse mammary tumor lines. We observed that the orthologs of Cdh1, RarB, Gstp1, RassF1 genes were hypermethylated, whereas neither Dapk1 nor Wif1 were aberrantly methylated in this panel of mouse tumor lines. From this study, we conclude that there is significant, but not absolute, overlap in the epigenome of human and mouse mammary tumors.

Invasion suppressor cystatin E/M (CST6): high-level cell type-specific expression in normal brain and epigenetic silencing in gliomas.

DNA hypermethylation-mediated gene silencing is a frequent and early contributor to aberrant cell growth and invasion in cancer. Malignant gliomas are the most common primary brain tumors in adults and the second most common tumor in children. Morbidity and mortality are high in glioma patients because tumors are resistant to treatment and are highly invasive into surrounding brain tissue rendering complete surgical resection impossible. Invasiveness is regulated by the interplay between secreted proteases (eg, cathepsins) and their endogenous inhibitors (cystatins). In our previous studies we identified cystatin E/M (CST6) as a frequent target of epigenetic silencing in glioma. Cystatin E/M is a potent inhibitor of cathepsin B, which is frequently overexpressed in glioma. Here, we study the expression of cystatin E/M in normal brain and show that it is highly and moderately expressed in oligodendrocytes and astrocytes, respectively, but not in neurons. Consistent with this, the CST6 promoter is hypomethylated in all normal samples using methylation-specific PCR, bisulfite genomic sequencing, and pyrosequencing. In contrast, 78% of 28 primary brain tumors demonstrated reduced/absent cystatin E/M expression using a tissue microarray and this reduced expression correlated with CST6 promoter hypermethylation. Interestingly, CST6 was expressed in neural stem cells (NSC) and markedly induced upon differentiation, whereas a glioma tumor initiating cell (TIC) line was completely blocked for CST6 expression by promoter methylation. Analysis of primary pediatric brain tumor-derived lines also showed CST6 downregulation and methylation in nearly 100% of 12 cases. Finally, ectopic expression of cystatin E/M in glioma lines reduced cell motility and invasion. These results demonstrate that epigenetic silencing of CST6 is frequent in adult and pediatric brain tumors and occurs in TICs, which are thought to give rise to the tumor. CST6 methylation may therefore represent a novel prognostic marker and therapeutic target specifically altered in TICs.

The transglutaminase 2 gene (TGM2), a potential molecular marker for chemotherapeutic drug sensitivity, is epigenetically silenced in breast cancer.

Tissue transglutaminase (TG2) is a ubiquitously expressed enzyme capable of catalyzing protein cross-links. TG2-dependent cross-links are important in extracellular matrix integrity and it has been proposed that this TG2 activity establishes a barrier to tumor spread. Furthermore, TG2 controls sensitivity to the chemotherapeutic drug doxorubicin. Both doxorubicin sensitivity and TG2 expression are highly variable in cultured human breast cancer cell lines and inspection of the human gene (termed TGM2) determined that a canonical CpG island exists within its 5' flank. These features, when combined with its potential tumor suppressor activity, make TG2 an attractive candidate for epigenetic silencing. Consistent with this, we observed that culturing breast tumor cells with the DNA demethylating agent 5-aza-2'-deoxycytidine (5-azadC) resulted in a robust increase in TG2 expression. Analysis of DNA harvested from cultured lines and primary breast tumor samples indicated that TGM2 often displays aberrant hypermethylation and that there is a statistically significant correlation between gene methylation and reduced expression. Finally, we observed that doxorubicin-resistant MCF-7/ADR cells do not show TGM2 silencing but that doxorubicin-sensitive MCF-7 cells do and that culturing MCF-7 cells on 5-azadC and subsequently restoring TG2 expression reduced sensitivity to doxorubicin. This work indicates that the TGM2 gene is a target for epigenetic silencing in breast cancer and suggests that this aberrant molecular event is a potential marker for chemotherapeutic drug sensitivity.