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1.  A Twin Study of Heritable and Shared Environmental Contributions to Autism 
The present study examined genetic and shared environment contributions to quantitatively-measured autism symptoms and categorically-defined ASD. Participants included 568 twins from the Interactive Autism Network. Autism symptoms were obtained using the Social Communication Questionnaire and Social Responsiveness Scale. Categorically-defined ASD was based on clinical diagnoses. DeFries-Fulker and liability threshold models examined etiologic influences. Very high heritability was observed for extreme autism symptom levels (h2g=.92-1.20). Extreme levels of social and repetitive behavior symptoms were strongly influenced by common genetic factors. Heritability of categorically-defined ASD diagnosis was comparatively low (.21, 95% CI=0.15-0.28). High heritability of extreme autism symptom levels confirms previous observations of strong genetic influences on autism. Future studies will require large, carefully ascertained family pedigrees and quantitative symptom measurements.
PMCID: PMC4104233  PMID: 24604525
autism; twins; genetic; heritability; environment; diagnosis
2.  Selective Roles of E2Fs for ErbB2- and Myc-mediated Mammary Tumorigenesis 
Oncogene  2013;34(1):119-128.
Previous studies have demonstrated that cyclin D1, an upstream regulator of the Rb/E2F pathway, is an essential component of the ErbB2/Ras (but not the Wnt/Myc) oncogenic pathway in the mammary epithelium. However, the role of specific E2fs for ErbB2/Ras-mediated mammary tumorigenesis remains unknown. Here we show that in the majority of mouse and human primary mammary carcinomas with ErbB2/HER2 over-expression, E2f3a is up-regulated, raising the possibility that E2F3a is a critical effector of the ErbB2 oncogenic signaling pathway in the mammary gland. We examined the consequence of ablating individual E2fs in mice on ErbB2-triggered mammary tumorigenesis in comparison to a comparable Myc-driven mammary tumor model. We found that loss of E2f1 or E2f3 led to a significant delay on tumor onset in both oncogenic models, whereas loss of E2f2 accelerated mammary tumorigenesis driven by Myc-over-expression. Furthermore, Southern blot analysis of final tumors derived from conditionally deleted E2f3−/loxP mammary glands revealed that there is a selection against E2f3−/− cells from developing mammary carcinomas, and that such selection pressure is higher in the presence of ErbB2 activation than in the presence of Myc activation. Taken together, our data suggest oncogenic activities of E2F1 and E2F3 in ErbB2- or Myc-triggered mammary tumorigenesis, and a tumor suppressor role of E2F2 in Myc-mediated mammary tumorigenesis.
PMCID: PMC4032808  PMID: 24276244
Myc; ErbB2; E2F; mammary tumorigenesis; oncogene
3.  Duration of Tamoxifen Use and the Risk of Contralateral Breast Cancer in BRCA1 and BRCA2 Mutation Carriers 
Women with a mutation in BRCA1 or BRCA2 face a lifetime risk of breast cancer of approximately 80%. Tamoxifen treatment of the first cancer has been associated with a reduction in the risk of a subsequent contralateral cancer.
We studied 1504 women with a known BRCA1 or BRCA2 mutation, 411 women with bilateral breast cancer (cases) and 1093 women with unilateral breast cancer (controls) in a matched case-control study. Control women were of similar age and had a similar age of diagnosis of first breast cancer as the cases. For each woman who used tamoxifen, the starting and stopping dates were abstracted and the duration of tamoxifen use was calculated.
331 women had used tamoxifen (22%); of these 84 (25%) had completed four or more years of tamoxifen, the remainder stopped prematurely or were current users. For women with up to one year of tamoxifen use, the odds ratio for contralateral breast cancer was 0.37 (95% CI: 0.20 – 0.69; p = 0.001) compared to women with no tamoxifen use. Among women with one to four years of tamoxifen use the odds ratio was 0.53 (95% CI: 0.32-0.87; p = 0.01). Among women with four or more years of tamoxifen use the odds ratio was 0.83 (95% CI: 0.44 -1.55; p = 0.55).
Short-term use of tamoxifen for chemoprevention in BRCA1 and BRCA2 mutation carriers may be as effective as a conventional five year course of treatment.
PMCID: PMC4131437  PMID: 24951267
Breast cancer; tamoxifen; oophorectomy; BRCA1; BRCA2
4.  Germline disruption of Pten localization causes enhanced sex-dependent social motivation and increased glial production 
Human Molecular Genetics  2014;23(12):3212-3227.
PTEN Hamartoma Tumor Syndrome (PHTS) is an autosomal-dominant genetic condition underlying a subset of autism spectrum disorder (ASD) with macrocephaly. Caused by germline mutations in PTEN, PHTS also causes increased risks of multiple cancers via dysregulation of the PI3K and MAPK signaling pathways. Conditional knockout models have shown that neural Pten regulates social behavior, proliferation and cell size. Although much is known about how the intracellular localization of PTEN regulates signaling in cancer cell lines, we know little of how PTEN localization influences normal brain physiology and behavior. To address this, we generated a germline knock-in mouse model of cytoplasm-predominant Pten and characterized its behavioral and cellular phenotypes. The homozygous Ptenm3m4 mice have decreased total Pten levels including a specific drop in nuclear Pten and exhibit region-specific increases in brain weight. The Ptenm3m4 model displays sex-specific increases in social motivation, poor balance and normal recognition memory—a profile reminiscent of some individuals with high functioning ASD. The cytoplasm-predominant protein caused cellular hypertrophy limited to the soma and led to increased NG2 cell proliferation and accumulation of glia. The animals also exhibit significant astrogliosis and microglial activation, indicating a neuroinflammatory phenotype. At the signaling level, Ptenm3m4 mice show brain region-specific differences in Akt activation. These results demonstrate that differing alterations to the same autism-linked gene can cause distinct behavioral profiles. The Ptenm3m4 model is the first murine model of inappropriately elevated social motivation in the context of normal cognition and may expand the range of autism-related behaviors replicated in animal models.
PMCID: PMC4030776  PMID: 24470394
5.  Second Malignant Neoplasms in Patients With Cowden Syndrome With Underlying Germline PTEN Mutations 
Journal of Clinical Oncology  2014;32(17):1818-1824.
Patients with Cowden syndrome (CS) with underlying germline PTEN mutations are at increased risk of breast, thyroid, endometrial, and renal cancers. To our knowledge, risk of subsequent cancers in these patients has not been previously explored or quantified.
Patients and Methods
We conducted a 7-year multicenter prospective study (2005 to 2012) of patients with CS or CS-like disease, all of whom underwent comprehensive PTEN mutational analysis. Second malignant neoplasms (SMNs) were ascertained by medical records and confirmed by pathology reports. Standardized incidence ratios (SIRs) for all SMNs combined and for breast, thyroid, endometrial, and renal cancers were calculated.
Of the 2,912 adult patients included in our analysis, 2,024 had an invasive cancer history. Germline pathogenic PTEN mutations (PTEN mutation positive) were identified in 114 patients (5.6%). Of these 114 patients, 46 (40%) had an SMN. Median age of SMN diagnosis was 50 years (range, 21 to 71 years). Median interval between primary cancer and SMN was 5 years (range, < 1 to 35 years). Of the 51 PTEN mutation–positive patients who presented with primary breast cancer, 11 (22%) had a subsequent new primary breast cancer and 10-year second breast cancer cumulative risk of 29% (95% CI, 15.3 to 43.7). Risk of SMNs compared with that of the general population was significantly elevated for all cancers (SIR, 7.74; 95% CI, 5.84 to 10.07), specifically for breast (SIR, 8.92; 95% CI, 5.85 to 13.07), thyroid (SIR, 5.83; 95% CI, 3.01 to 10.18), and endometrial SMNs (SIR, 14.08.07; 95% CI, 7.10 to 27.21).
Patients with CS with germline PTEN mutations are at higher risk for SMNs compared with the general population. Prophylactic mastectomy should be considered on an individual basis given the significant risk of subsequent breast cancer.
PMCID: PMC4039869  PMID: 24778394
6.  Germline and Somatic SDHx Alterations in Apparently Sporadic Differentiated Thyroid Cancer 
Endocrine-related cancer  2015;22(2):121-130.
Along with breast and endometrial cancers, thyroid cancer is a major component cancer in Cowden syndrome (CS). Germline variants in SDHB/C/D (SDHx) genes account for subsets of CS/CS-like cases, conferring a higher risk of breast and thyroid cancers over those with only germline PTEN mutations. To investigate if SDHx alterations at both germline and somatic levels occur in apparently sporadic breast cancer and differentiated thyroid cancer (DTC), we analyzed SDHx genes in following 4 groups: 1) 48 individuals with sporadic invasive breast adenocarcinoma for germline mutation; 2) 48 (expanded to 241) DTC for germline mutation; 3) 37 pairs DTC tumor-normal tissues for germline and somatic mutation and mRNA expression levels; and 4) 476 TCGA thyroid carcinoma dataset for validation. No germline SDHx variant was found in a pilot series of 48 breast cancer cases. Because germline SDHx variants were found in our pilot of 48 thyroid cancer cases, we expanded to 3 series of DTC comprising a total 754 cases, and found 48 (6%) with germline SDHx variants (p<0.001 compared to 0/350 controls). In 513 tumors, we found 27 (5%) with large somatic duplications within chromosome 1 encompassing SDHC. Both papillary and follicular thyroid tumors showed consistent loss of SDHC/D gene expression (p<0.001), which is associated with earlier disease onset and higher pathological-TNM stage. Therefore we conclude that both germline and somatic SDHx mutations/variants occur in sporadic DTC but are very rare in sporadic breast cancer, and overall loss of SDHx gene expression is a signature of DTC.
PMCID: PMC4335266  PMID: 25694510
SDHx; Variant; Gene Expression; Papillary Thyroid Cancer
7.  Emergence, Involution, and Progression to Carcinoma of Mutant Clones in Normal Endometrial Tissues 
Cancer research  2014;74(10):2796-2802.
Sporadic somatic inactivation of genes such as PTEN within histologically normal endometrium (latent precancers) is an early step in endometrial carcinogenesis. We have used clone-specific mutations of PTEN to determine the fate of latent precancers over time in women who do (high risk) and do not (low risk) develop endometrial neoplasia.
PTEN immunohistochemistry was performed on 45 occurrences of endometrial neoplasia and their paired antecedent benign biopsies, along with age matched sample pairs from 167 patients who did not develop a neoplasm. When PTEN deficient cells were present at both timepoints, DNA sequencing was performed to determine if they were single, or multiple independent, events. Loss of PTEN protein in isolated glands was common in the initial normal biopsies of high and low risk groups (42% and 27% respectively, p=0.066). Protein deficient glands have a tendency to disappear over time in low risk women (p=0.047), and even when “persistent” are infrequently (19%, 3/16) confirmed to be the same clone. Similarly, only a small proportion (6.7%, 1/15) of latent precancers seen in high risk women are the direct progenitors of subsequent neoplasia.
There is a high rate of latent precancer turnover in both low and high risk patients, with rare long-term persistence of unique clones which may or may not progress to a histologic lesion. The temporal dynamics of clonal emergence, persistence, and involution are sufficiently complex that in the individual patient the presence of a latent precancer has an unknown contribution to long term cancer risk.
PMCID: PMC4058864  PMID: 24662919
Endometrium; PTEN; latent precancer; carcinoma
8.  Increased prevalence of eosinophilic gastrointestinal disorders (EGID) in pediatric PTEN hamartoma tumor syndromes (PHTS) 
The PTEN hamartoma tumor syndromes (PHTS) are a collection of disorders caused by germline mutations of the tumor suppressor gene PTEN. Eosinophilic gastrointestinal disorders (EGID) are rare diseases characterized by food-induced, eosinophil-dominant inflammation in various segments of the gastrointestinal tract. On the basis of our clinical observations of several patients with EGID-PHTS, we investigated whether there is an association between these two disorders.
Cincinnati Children’s Hospital Medical Center (CCHMC) Informatics for Integrating Biology & the Bedside (i2b2) warehouse was queried for the years 2007-2012 using ICD 9 codes for PTEN-related diseases; the results were cross-referenced with participants enrolled in the Cincinnati Center for Eosinophilic Disorder’s EGID database to identify patients with both disorders. Similarly, the Cleveland Clinic Genomic Medicine Institute PTEN database was queried for cases between 2005 and 2012. Inclusion criteria were age ≥18 years, history of PHTS, and an esophagogastroduodenoscopy (EGD) and/or colonoscopy with at least one histologic EGID diagnosis confirmed by a CCHMC pathologist. Pearson’s Chi-square was used to determine the odds of EGID enrichment in PHTS.
Of the 1,058,260 CCHMC distinct patients identified by the i2b2 search, 53 had clinical diagnoses suggestive of PHTS. Thirteen of the 53 had PTEN mutations, with 8/13 (62%) having had an EGD and/or colonoscopy. Five of the 8 had confirmed EGID. At the Cleveland Clinic, 3/75 patients with PHTS had confirmed EGID. CCHMC i2b2 query data showed a substantial enrichment of EGID in PHTS (OR=272; CI 89-831, p<0.0001). An EGID prevalence estimate from the i2b2 query supported a marked enrichment of EGID in PHTS in the Cleveland Clinic database (p<0.0001). Among the 8 subjects with EGID and PHTS, the age at EGID and PHTS diagnosis was 7.6 ± 3.2 and 7.9 ± 5.8 years, respectively. Patients with EGID-PHTS had excess eosinophils in biopsies of the esophagus (75%), stomach (38%), and colon (13%) with a notable presence of eosinophil-rich gastrointestinal polyposis (88%).
EGID is a previously unrecognized comorbid disease in pediatric patients with PHTS. These data suggest a potential role of PTEN in contributing to EGID susceptibility.
PMCID: PMC4129455  PMID: 24345843
9.  Demographic and clinical correlates of autism symptom domains and autism spectrum diagnosis 
Demographic and clinical factors may influence assessment of autism symptoms. This study evaluated these correlates and also examined whether social communication and interaction and restricted/repetitive behavior provided unique prediction of autism spectrum disorder diagnosis. We analyzed data from 7352 siblings included in the Interactive Autism Network registry. Social communication and interaction and restricted/repetitive behavior symptoms were obtained using caregiver-reports on the Social Responsiveness Scale. Demographic and clinical correlates were covariates in regression models predicting social communication and interaction and restricted/repetitive behavior symptoms. Logistic regression and receiver operating characteristic curve analyses evaluated the incremental validity of social communication and interaction and restricted/repetitive behavior domains over and above global autism symptoms. Autism spectrum disorder diagnosis was the strongest correlate of caregiver-reported social communication and interaction and restricted/repetitive behavior symptoms. The presence of comorbid diagnoses also increased symptom levels. Social communication and interaction and restricted/repetitive behavior symptoms provided significant, but modest, incremental validity in predicting diagnosis beyond global autism symptoms. These findings suggest that autism spectrum disorder diagnosis is by far the largest determinant of quantitatively measured autism symptoms. Externalizing (attention deficit hyperactivity disorder) and internalizing (anxiety) behavior, low cognitive ability, and demographic factors may confound caregiver-report of autism symptoms, potentially necessitating a continuous norming approach to the revision of symptom measures. Social communication and interaction and restricted/repetitive behavior symptoms may provide incremental validity in the diagnosis of autism spectrum disorder.
PMCID: PMC4269571  PMID: 24104512
autism spectrum disorder; autism symptoms; diagnosis; prediction
10.  Germline and somatic SDHx alterations in apparently sporadic differentiated thyroid cancer 
Endocrine-Related Cancer  2015;22(2):121-130.
Along with breast and endometrial cancers, thyroid cancer is a major component cancer in Cowden syndrome (CS). Germline variants in SDHB/C/D (SDHx) genes account for subsets of CS/CS-like cases, conferring a higher risk of breast and thyroid cancers over those with only germline PTEN mutations. To investigate whether SDHx alterations at both germline and somatic levels occur in apparently sporadic breast cancer and differentiated thyroid cancer (DTC), we analyzed SDHx genes in the following four groups: i) 48 individuals with sporadic invasive breast adenocarcinoma for germline mutation; ii) 48 (expanded to 241) DTC for germline mutation; iii) 37 pairs DTC tumor-normal tissues for germline and somatic mutation and mRNA expression levels; and iv) data from 476 patients in the Cancer Genome Atlas thyroid carcinoma dataset for validation. No germline SDHx variant was found in a pilot series of 48 breast cancer cases. As germline SDHx variants were found in our pilot of 48 thyroid cancer cases, we expanded to three series of DTC comprising a total 754 cases, and found 48 (6%) with germline SDHx variants (P<0.001 compared with 0/350 controls). In 513 tumors, we found 27 (5%) with large somatic duplications within chromosome 1 encompassing SDHC. Both papillary and follicular thyroid tumors showed consistent loss of SDHC/D gene expression (P<0.001), which is associated with earlier disease onset and higher pathological-TNM stage. Therefore, we conclude that both germline and somatic SDHx mutations/variants occur in sporadic DTC but are very rare in sporadic breast cancer, and overall loss of SDHx gene expression is a signature of DTC.
PMCID: PMC4335266  PMID: 25694510
SDHx; variant; gene expression; papillary thyroid cancer
11.  A Unified Nomenclature and Amino Acid Numbering for Human PTEN 
Science signaling  2014;7(332):pe15.
The tumor suppressor PTEN is a major brake for cell transformation, mainly due to its phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] phosphatase activity that directly counteracts the oncogenicity of phosphoinositide 3-kinase (PI3K). PTEN mutations are frequent in tumors and in the germ line of patients with tumor predisposition or with neurological or cognitive disorders, which makes the PTEN gene and protein a major focus of interest in current biomedical research. After almost two decades of intense investigation on the 403-residue-long PTEN protein, a previously uncharacterized form of PTEN has been discovered that contains 173 amino-terminal extra amino acids, as a result of an alternate translation initiation site. To facilitate research in the field and to avoid ambiguities in the naming and identification of PTEN amino acids from publications and databases, we propose here a unifying nomenclature and amino acid numbering for this longer form of PTEN.
PMCID: PMC4367864  PMID: 24985344
12.  The Development of a Clinical Screening Instrument For Tumour Predisposition Syndromes In Childhood Cancer Patients 
Identification of tumour predisposition syndromes in patients who have cancer in childhood is paramount for optimal care. A screening instrument that can help to identify such patients will facilitate physicians caring for children with cancer. The complete screening instrument should consist of a standardized series of pictures and a screening form for manifestations not visible in the pictures. Here we describe the development of such a screening form based on an international two-stage Delphi process and an initial validation of the complete instrument.
Patients and Methods
We identified manifestations that may contribute to the diagnosis of a tumour predisposition syndrome through the Winter-Baraitser Dysmorphology Database and the textbook “Gorlin's Syndromes of the Head and Neck”. In a two-round Delphi process, eight international content-experts scored the contribution of each of these manifestations. We performed a clinical validation of the instrument in a selected cohort of ten paediatric cancer patients from another centre.
In total, 49 manifestations were found to contribute to the diagnosis of a tumour predisposition syndrome and were included in the screening form. The pilot validation study showed that patients suspect for having a tumour predisposition syndrome were recognized. Excellent correlation for indication for referral of a patient between the screening instrument and the reference standard (personal evaluation by an experienced clinical geneticist) was found.).
The Delphi process performed by international specialists with a function as opinion leaders in their field of expertise has led to a screening form and instrument with which those childhood cancer patients can be identified who may have a tumour predisposition syndrome and thus have an indication to be referred for further genetic analysis.
PMCID: PMC4277698  PMID: 23855994
Paediatric oncology; childhood cancer; screening instrument; tumour predisposition syndromes; Delphi process; questionnaires
13.  Pulmonary arterial hypertension in a patient with Cowden syndrome and the PTEN mutation 
Pulmonary Circulation  2014;4(4):728-731.
The pathogenesis of pulmonary arterial hypertension (PAH) exhibits many neoplastic-like features. Cowden syndrome is a difficult-to-recognize heritable cancer syndrome caused by a germline mutation in the phosphatase-and-tensin homolog deleted on the chromosome 10 (PTEN) gene. PTEN regulation has been implicated in cancer development and, more recently, PAH pathogenesis. Here we report a case of PAH in a patient with Cowden syndrome and the response to pulmonary vasodilators.
PMCID: PMC4278632  PMID: 25610608
pulmonary hypertension; PTEN mutation; Cowden syndrome
14.  The impact of pregnancy on breast cancer survival in women who carry a BRCA1 or BRCA2 mutation 
Physicians are often approached by young women with a BRCA mutation and a recent history of breast cancer who wish to have a baby. They wish to know if pregnancy impacts upon their future risks of cancer recurrence and survival. To date, there is little information on the survival experience of women who carry a mutation in one of the BRCA genes and who become pregnant. From an international multi-center cohort study of 12,084 women with a BRCA1 or BRCA2 mutation, we identified 128 case subjects who were diagnosed with breast cancer while pregnant or who became pregnant after a diagnosis of breast cancer. These women were age-matched to 269 mutation carriers with breast cancer who did not become pregnant (controls). Subjects were followed from the date of breast cancer diagnosis until the date of last follow-up or death from breast cancer. The Kaplan–Meier method was used to estimate 15-year survival rates. The hazard ratio for survival associated with pregnancy was calculated using a left-truncated Cox proportional hazard model, adjusting for other prognostic factors. Among women who were diagnosed with breast cancer when pregnant or who became pregnant thereafter, the 15-year survival rate was 91.5 %, compared to a survival of 88.6 % for women who did not become pregnant (adjusted hazard ratio = 0.76; 95 % CI 0.31–1.91; p = 0.56). Pregnancy concurrent with or after a diagnosis of breast cancer does not appear to adversely affect survival among BRCA1/2 mutation carriers.
PMCID: PMC3940343  PMID: 24136669
BRCA1; BRCA2; Breast cancer; Pregnancy; Survival
15.  A Novel Germline Mutation in BAP1 Predisposes to Familial Clear-Cell Renal Cell Carcinoma 
Molecular cancer research : MCR  2013;11(9):1061-1071.
Renal cell carcinoma (RCC) clusters in some families. Familial RCC arises from mutations in several genes, including VHL, which is also mutated in sporadic RCC. However, a significant percentage of familial RCC remains unexplained. Recently, we discovered that the BAP1 gene is mutated in sporadic RCC. BAP1, which encodes a nuclear deubiquitinase, is a two-hit tumor suppressor gene. Somatic BAP1 mutations are associated with high-grade ccRCC and poor patient outcomes. To determine whether BAP1 predisposes to familial RCC, we sequenced the BAP1 gene in 83 unrelated probands with unexplained familial RCC. We identified a novel variant (c.41T>A; p.L14H), which cosegregated with the RCC phenotype. The p.L14H variant disrupts a highly conserved residue in the catalytic domain, a domain frequently targeted by missense mutations. The family with the BAP1 variant was characterized by early-onset clear cell RCC, occasionally of high Fuhrman grade, and lacked other features that characterize von Hippel-Lindau syndrome. These findings suggest that BAP1 is a familial RCC predisposing gene.
PMCID: PMC4211292  PMID: 23709298
renal cell carcinoma; BAP1; cancer; inherited RCC; predisposition; tumor suppressor
16.  PTEN Germline Mutations in Patients Initially Tested for Other Hereditary Cancer Syndromes: Would Use of Risk Assessment Tools Reduce Genetic Testing? 
The Oncologist  2013;18(10):1083-1090.
PTEN Hamartoma Tumor syndrome (PHTS) is a hereditary condition causing increased risk for neoplasia types seen in persons with Hereditary Breast and Ovarian Cancer, Lynch, and Adenomatous Polyposis syndromes. We reviewed our series of patients with PHTS to determine how often testing criteria for these syndromes were met and how often other-gene testing was ordered before testing PTEN.
Learning Objectives
Cite the risk assessment tools available for several hereditary cancer predisposition syndromes.Describe ways in which use of these risk assessment tools can lead to cost savings and decreased time to correct diagnosis.Explain the impact of correct genetic diagnosis on the patient's medical management and on predictive testing for family members.
PTEN Hamartoma Tumor syndrome (PHTS) includes patients with Cowden syndrome or other syndromes with germline mutation of the PTEN tumor suppressor gene. The risk for breast, colorectal, and endometrial cancer and polyposis is increased, creating clinical overlap with hereditary breast and ovarian cancer (HBOC), Lynch syndrome (LS), and adenomatous polyposis syndromes (APS). We reviewed our series of patients with PHTS to determine how often testing criteria for these syndromes were met and how often other-gene testing was ordered before testing PTEN.
Patients and Methods.
Patients were prospectively recruited by relaxed International Cowden Consortium criteria or presence of known germline PTEN mutation. Mutations were identified by mutation scanning/multiplex ligation-dependent probe amplification analysis and confirmed by sequencing/quantitative polymerase chain reaction. Patients were excluded if they were adopted, were <18 years of age, or if they were diagnosed with Cowden syndrome before 1998. Standard risk-assessment models were applied to determine whether patients met HBOC testing criteria, LS-relevant Amsterdam II/Bethesda 2004 criteria, or had adenomatous polyps. Prior probability of PTEN mutation was estimated with the Cleveland Clinic PTEN risk calculator.
Of 137 PTEN mutation-positive adult probands, 59 (43.1%) met testing criteria for HBOC or LS. Of these, 45 (32.8%) were first offered HBOC, LS, or APS testing. Of those who underwent APS testing, none of the six patients met criteria. Initial risk assessment by a genetics specialist was significantly associated with immediate PTEN testing in patients also meeting HBOC testing criteria. Using this PTEN risk assessment tool could have spared gene testing for 22 unlikely syndromes, at a total cost of $66,080.
PHTS is an important differential diagnosis for patients referred for HBOC, LS, or APS. Risk assessment tools may help focus genetic analysis and aid in the interpretation of multiplex testing.
PMCID: PMC3805149  PMID: 24037976
Risk assessment; Genetic testing; PTEN hamartoma tumor syndrome; Cowden syndrome; Hereditary cancer syndromes
17.  Cowden syndrome-associated germline SDHD variants alter PTEN nuclear translocation through SRC-induced PTEN oxidation 
Human Molecular Genetics  2014;24(1):142-153.
Germline mutations in the PTEN tumor-suppressor gene and germline variations in succinate dehydrogenase subunit D gene (SDHD-G12S, SDHD-H50R) are associated with a subset of Cowden syndrome and Cowden syndrome-like individuals (CS/CSL) and confer high risk of breast, thyroid and other cancers. However, very little is known about the underlying crosstalk between SDHD and PTEN in CS-associated thyroid cancer. Here, we show SDHD-G12S and SDHD-H50R lead to impaired PTEN function through alteration of its subcellular localization accompanied by resistance to apoptosis and induction of migration in both papillary and follicular thyroid carcinoma cell lines. Other studies have shown elevated proto-oncogene tyrosine kinase (SRC) activity in invasive thyroid cancer cells; so, we explore bosutinib, a specific inhibitor for SRC, to explore SRC as a mediator of SDH-PTEN crosstalk in this context. We show that SRC inhibition could rescue SDHD dysfunction-induced cellular phenotype and tumorigenesis only when wild-type PTEN is expressed, in thyroid cancer lines. Patient lymphoblast cells carrying either SDHD-G12S or SDHD-H50R also show increased nuclear PTEN and more oxidized PTEN after hydrogen peroxide treatment. Like in thyroid cells, bosutinib decreases oxidative PTEN in patient lymphoblast cells carrying SDHD variants, but not in patients carrying both SDHD variants and PTEN truncating mutations. In summary, our data suggest a novel mechanism whereby SDHD germline variants SDHD-G12S or SDHD-H50R induce thyroid tumorigenesis mediated by PTEN accumulation in the nucleus and may shed light on potential treatment with SRC inhibitors like bosutinib in PTEN-wild-type SDHD-variant/mutation positive CS/CSL patients and sporadic thyroid neoplasias.
PMCID: PMC4262496  PMID: 25149476
18.  Germline and somatic KLLN alterations in breast cancer dysregulate G2 arrest 
Human Molecular Genetics  2013;22(12):2451-2461.
PTEN is a well-described predisposition gene for Cowden syndrome (CS), a familial cancer syndrome characterized by a high risk of breast and other cancers. KLLN, which shares a bidirectional promoter with PTEN, causes cell cycle arrest and apoptosis. We previously identified germline hypermethylation of the KLLN promoter in 37% of PTEN mutation-negative CS/CS-like (CSL) patients. Patients with germline KLLN hypermethylation have an increased prevalence of breast and renal cancers when compared with PTEN mutation carriers. We have consequently sought to identify and characterize germline KLLN variants/mutations in CS/CSL and in apparently sporadic breast cancer patients. KLLN variants in CS/CSL patients are rare (1 of 136, 0.007%). Interestingly, among 438 breast cancer patients, 13 (3%) have germline KLLN variants when compared with none in 128 controls (P = 0.049). Patients with KLLN variants have a family history of breast cancer when compared with those without (P = 0.02). We demonstrate that germline KLLN variants dysregulate the cell cycle at G2. Of 24 breast carcinomas analyzed, 3 (13%) have somatic KLLN hemizygous deletions, with somatic loss of the wild-type allele in a patient with germline KLLN p.Leu119Leu. Of 452 breast carcinomas in The Cancer Genome Atlas project, 93 (21%) have KLLN hemizygous or homozygous deletions. This is the first study to associate germline KLLN variants with sporadic breast cancer and to recognize somatic KLLN deletions in breast carcinomas. Our observations suggest that KLLN may be a low penetrance susceptibility factor for apparently sporadic breast cancer.
PMCID: PMC3858115  PMID: 23446638
19.  Prevalence of Germline PTEN, BMPR1A, SMAD4, STK11, and ENG Mutations in Patients with Moderate-Load Colorectal Polyps 
Gastroenterology  2013;144(7):1402-1409.e5.
Gastrointestinal polyposis is a common clinical problem, yet there is no consensus on how to best manage patients with moderate-load polyposis. Identifying genetic features of this disorder could improve management, and especially surveillance, of these patients. We sought to determine the prevalence of hamartomatous polyposis associated mutations in the susceptibility genes PTEN, BMPR1A, SMAD4, ENG, and STK11 in individuals with 5 or more gastrointestinal polyps, including at least 1 hamartomatous or hyperplastic/serrated polyp.
We performed a prospective, referral-based study of 603 patients (median age 51 y; range, 2–89 y), enrolled from June 2006 through January 2012. Genomic DNA was extracted from peripheral lymphocytes and analyzed for specific mutations and large rearrangements in PTEN, BMPR1A, SMAD4, and STK11, as well as mutations in ENG. Recursive partitioning analysis was used to determine cutoffs for continuous variables. The prevalence of mutations was compared using Fischer’s exact test. Logistic regression analyses were used to determine univariate and multivariate risk factors.
Of 603 patients, 119 (20%) had a personal history of colorectal cancer and most (461; 76%) had fewer than 30 polyps. Seventy-seven patients (13%) were found to have polyposis-associated mutations, comprising 11 in ENG (1.8%), 13 in PTEN (2.2%), 13 in STK11 (2.2%), 20 in BMPR1A (3.3%), and 21 in SMAD4 (3.5%). Univariate clinical predictors for risk of having these mutations included age at presentation less than 40 years (19% vs 10%; P=.008), a polyp burden of 30 or more (19% vs 11%; P=0.014), and male sex (16% vs 10%; P=.03). Patients who had 1 or more ganglioneuromas (29% vs 2%; P<.001) or presented with polyps of 3 or more histologic types (20% vs 2%; P=.003) were more likely to have germline mutations in PTEN.
Age less than 40 years, male sex, and specific polyp histologies are significantly associated with risk of germline mutations in hamartomatous-polyposis associated genes. These associations could guide clinical decision making and further investigations.
PMCID: PMC3969031  PMID: 23399955
Hamartomatous polyposis; Juvenile Polyposis Syndrome; Peutz-Jeghers syndrome; Cowden syndrome
20.  Formative Evaluation of Clinician Experience with Integrating Family History-Based Clinical Decision Support into Clinical Practice 
Journal of Personalized Medicine  2014;4(2):115-136.
Family health history is a leading predictor of disease risk. Nonetheless, it is underutilized to guide care and, therefore, is ripe for health information technology intervention. To fill the family health history practice gap, Cleveland Clinic has developed a family health history collection and clinical decision support tool, MyFamily. This report describes the impact and process of implementing MyFamily into primary care, cancer survivorship and cancer genetics clinics. Ten providers participated in semi-structured interviews that were analyzed to identify opportunities for process improvement. Participants universally noted positive effects on patient care, including increases in quality, personalization of care and patient engagement. The impact on clinical workflow varied by practice setting, with differences observed in the ease of integration and the use of specific report elements. Tension between the length of the report and desired detail was appreciated. Barriers and facilitators to the process of implementation were noted, dominated by the theme of increased integration with the electronic medical record. These results fed real-time improvement cycles to reinforce clinician use. This model will be applied in future institutional efforts to integrate clinical genomic applications into practice and may be useful for other institutions considering the implementation of tools for personalizing medical management.
PMCID: PMC4263968  PMID: 25563219
family health history; clinical decision support; implementation; risk stratification; barriers; facilitators
21.  Specific kinesin expression profiles associated with taxane resistance in breast cancer 
Breast cancer is a genetically heterogenous disease with subtypes differing in prognosis and chemosensitivity. The basal-like breast cancer (BLBC) molecular subtype is associated with poorer outcomes, but is more responsive to taxane-based chemotherapy, which is used in multiple settings of breast cancer. Kinesins are intracellular transport proteins that interact with microtubules, which are also the mechanistic target for taxanes. We investigated the relationship between taxane resistance in BLBC and kinesins using both expression and functional studies.
Kinesin (KIF) expression was evaluated in three settings in relation to taxane resistance: (i) the NCI-60 cancer cell lines, (ii) pre-treatment samples from four BLBC patient cohorts receiving neoadjuvant chemotherapy regimens with and without taxanes, and (iii) post-treatment samples from residual breast cancer following neoadjuvant taxane-containing chemotherapy. We used a novel functional approach to gene modification, validation-based insertional mutagenesis, to select kinesin-overexpressing clones of BLBC cells for evaluation of related mechanisms of taxane resistance.
In the NCI-60 cell line dataset, overexpression of the kinesin KIFC3 is significantly correlated with resistance to both docetaxel (p<0.001) and paclitaxel (p<0.001), but not to platinum-based chemotherapy, including carboplatin (p=0.49) and cisplatin (p=0.10). Overexpression of KIFC3 and KIF5A in pre-chemotherapy samples similarly predicted resistance to paclitaxel in the MDACC cohorts (p=0.01); no KIF predicted resistance to fluorouracil-epirubicin-cyclophosphamide or cisplatin in BLBC patient cohorts treated without taxanes. KIF12 is the most overexpressed KIF gene in post-chemotherapy taxane-resistant residual breast cancers (2.8 fold-change). Functional studies established that overexpression of KIFC3, KIF5A and KIF12 were specific in mediating resistance to docetaxel and not vincristine or doxorubicin. We demonstrated that mutation of the ATP-binding domain of a kinesin abolishes its ability to mediate docetaxel resistance.
We show that kinesin overexpression correlates with specific taxane resistance in BLBC cell lines and tissue. Our results suggest a novel approach to overcoming taxane resistance in breast cancer through concurrent or sequential use of kinesin inhibitors, highlighting the ATP-binding domain as a drug development target.
PMCID: PMC4038085  PMID: 21479552
22.  Cowden syndrome-related mutations in PTEN associate with enhanced proteasome activity 
Cancer research  2013;73(10):3029-3040.
Germline mutations in PTEN have been described in a spectrum of syndromes that are collectively known as PTEN hamartoma tumor syndrome (PHTS). In addition to being mutated in the germline in PHTS, somatic loss-of-function PTEN mutations are seen in a wide range of sporadic human tumors. Here, we show evidence of upregulated proteasome activity in PHTS-derived lymphoblasts, Pten knock-in mice and cell lines expressing missense and nonsense PTEN mutations. Notably, elevated nuclear proteasome activity occurred in cells expressing the nuclear mislocalized PTEN-K62R mutant, whereas elevated cytosolic proteasome activity was observed in cells expressing the cytosolic-predominant mutant PTEN (M3M4 and C136R). Treatment with proteasome inhibitor MG-132 was able to restore both nonsense and missense mutant PTEN protein levels in vitro. PHTS patients with destabilizing PTEN mutations and proteasome hyperactivity are more susceptible to develop neurological symptoms such as mental retardation and autism than mutation-positive patients with normal proteasome activity. A detailed molecular and functional analysis shows that PTEN mutants most likely cause proteasome hyperactivity via two different mechanisms, namely, induction of proteotoxic stress and loss of protein phosphatase activity. These results provide novel insights into the cellular functions of PTEN and reveal molecular mechanisms whereby PTEN mutations increase proteasome activity and lead to neurological phenotypes.
PMCID: PMC3655114  PMID: 23475934
Cowden syndrome; PHTS; PTEN instability; localization; germline mutation
23.  Elevated Plasma Succinate Among PTEN, SDHB and SDHD Mutation Positive Individuals 
Cowden Syndrome (CS) results from germline mutations in phosphatase and tensin homologue deleted on chromosome ten (PTEN) and from variants in succinate dehydrogenase (SDH) B and D subunits. We hypothesized that succinate accumulation may be common among individuals with SDH variants/mutations and those with PTEN mutations.
Urine and blood were collected from individuals meeting full or partial CS diagnostic criteria, those with paraganglioma or a known susceptibility PGL-associated gene mutation and succinate was measured. PTEN, SDHB, SDHC, and SDHD-genes were sequenced from genomic DNA.
Elevated plasma succinate was observed in 13/21 (62%) individuals with germline PTEN, SDHB or SDHD mutations compared to 5/32 (16%) controls (p<0.001); 10/15 (67%) individuals with pathogenic PTEN mutations, but in <20% of mutation negative individuals meeting identical criteria; and among individuals with mutations in SDHB (1/1, 100%) and SDHD (2/5, 40%).
Our data suggest that mutations in PTEN, SDHB and SDHD reduce catalytic activity of SDH and result in succinate accumulation and identify a common biochemical alteration between these two patient populations. Plasma organic acid analysis may provide an effective and inexpensive screening method to determine when more expensive gene sequencing of PTEN and SDH-genes is warranted.
PMCID: PMC4019996  PMID: 22261759
PTEN; SDH; Succinate; Cowden Syndrome
24.  Hypomethylation of Noncoding DNA Regions and Overexpression of the Long Noncoding RNA, AFAP1-AS1, in Barrett’s Esophagus and Esophageal Adenocarcinoma 
Gastroenterology  2013;144(5):956-966.e4.
Alterations in methylation of protein-coding genes are associated with Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC). Dys-regulation of noncoding RNAs occurs during carcinogen-esis but has never been studied in BE or EAC. We applied high-resolution methylome analysis to identify changes at genomic regions that encode noncoding RNAs in BE and EAC.
We analyzed methylation of 1.8 million CpG sites using massively parallel sequencing-based HELP tagging in matched EAC, BE, and normal esophageal tissues. We also analyzed human EAC (OE33, SKGT4, and FLO-1) and normal (HEEpic) esophageal cells.
BE and EAC exhibited genome-wide hypomethylation, significantly affecting intragenic and repetitive genomic elements as well as noncoding regions. These methylation changes targeted small and long noncoding regions, discriminating normal from matched BE or EAC tissues. One long noncoding RNA, AFAP1-AS1, was extremely hypomethylated and overexpressed in BE and EAC tissues and EAC cells. Its silencing by small interfering RNA inhibited proliferation and colony-forming ability, induced apoptosis, and reduced EAC cell migration and invasion without altering the expression of its protein-coding counterpart, AFAP1.
BE and EAC exhibit reduced methylation that includes noncoding regions. Methylation of the long noncoding RNA AFAP1-AS1 is reduced in BE and EAC, and its expression inhibits cancer-related biologic functions of EAC cells.
PMCID: PMC3739703  PMID: 23333711
Esophageal Cancer Progression; Tumor Development; Gene Regulation; Noncoding RNA
25.  The Cognitive Characteristics of PTEN Hamartoma Tumor Syndromes 
To characterize cognition in individuals with germline PTEN mutations (N=23) as well as in PTEN mutation-negative individuals with classic Cowden Syndrome or Bannayan-Riley-Ruvalcaba Syndrome (N=2).
Twenty-five individuals completed a comprehensive neuropsychological evaluation. One sample t-tests and effect sizes were used to examine differences in participant test scores compared with normal controls. Composite scores were created for each patient within each of the cognitive domains assessed and classified as above average, average, or below average according to normative standards. Chi-square analyses compared these classifications to expected proportions in normal control samples.
The mean IQ was in the average range, and there was a wide range of intellectual functioning (extremely low to very superior). However, scores were lower than expected on measures of motor functioning, executive functioning, and memory recall suggesting a pattern of frontal lobe dysfunction in a large subset of participants.
This is the first study to characterize cognition in individuals with PTEN mutations and associated syndromes using a comprehensive neuropsychological battery. Contrary to previous association with intellectual disability, mean IQ was average, and there was a broad range of intellectual abilities. Specific evidence of frontal lobe dysfunction may have implications for treatment compliance and cancer surveillance and warrants further investigation.
PMCID: PMC3956109  PMID: 23470840
PTEN; PTEN Hamartoma Tumor Syndromes; Cowden Syndrome; Bannayan-Riley-Ruvalcaba Syndrome; Cognition; Neuropsychology

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