Mouse Dspp frameshift model of human dentinogenesis imperfecta.

Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5' mutations affecting an N-terminal targeting sequence and 3' mutations that shift translation into the - 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp-1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a DsppP19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. DsppP19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. DsppP19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp-1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp+/+ and DsppP19L dentin. The Dspp-1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5' and 3' Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.

Odontogenesis-associated phosphoprotein truncation blocks ameloblast transition into maturation in OdaphC41*/C41* mice.

Mutations of Odontogenesis-Associated Phosphoprotein (ODAPH, OMIM *614829) cause autosomal recessive amelogenesis imperfecta, however, the function of ODAPH during amelogenesis is unknown. Here we characterized normal Odaph expression by in situ hybridization, generated Odaph truncation mice using CRISPR/Cas9 to replace the TGC codon encoding Cys41 into a TGA translation termination codon, and characterized and compared molar and incisor tooth formation in Odaph+/+, Odaph+/C41*, and OdaphC41*/C41* mice. We also searched genomes to determine when Odaph first appeared phylogenetically. We determined that tooth development in Odaph+/+ and Odaph+/C41* mice was indistinguishable in all respects, so the condition in mice is inherited in a recessive pattern, as it is in humans. Odaph is specifically expressed by ameloblasts starting with the onset of post-secretory transition and continues until mid-maturation. Based upon histological and ultrastructural analyses, we determined that the secretory stage of amelogenesis is not affected in OdaphC41*/C41* mice. The enamel layer achieves a normal shape and contour, normal thickness, and normal rod decussation. The fundamental problem in OdaphC41*/C41* mice starts during post-secretory transition, which fails to generate maturation stage ameloblasts. At the onset of what should be enamel maturation, a cyst forms that separates flattened ameloblasts from the enamel surface. The maturation stage fails completely.

Mutations in RELT cause autosomal recessive amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a collection of isolated (non-syndromic) inherited diseases affecting dental enamel formation or a clinical phenotype in syndromic conditions. We characterized three consanguineous AI families with generalized irregular hypoplastic enamel with rapid attrition that perfectly segregated with homozygous defects in a novel gene: RELT that is a member of the tumor necrosis factor receptor superfamily (TNFRSF). RNAscope in situ hybridization of wild-type mouse molars and incisors showed specific Relt mRNA expression by secretory stage ameloblasts and by odontoblasts. Relt-/- mice generated by CRISPR/Cas9 exhibited incisor and molar enamel malformations. Relt-/- enamel had a rough surface and underwent rapid attrition. Normally unmineralized spaces in the deep enamel near the dentino-enamel junction (DEJ) were as highly mineralized as the adjacent enamel, which likely altered the mechanical properties of the DEJ. Phylogenetic analyses showed the existence of selective pressure on RELT gene outside of tooth development, indicating that the human condition may be syndromic, which possibly explains the history of small stature and severe childhood infections in two of the probands. Knowing a TNFRSF member is critical during the secretory stage of enamel formation advances our understanding of amelogenesis and improves our ability to diagnose human conditions featuring enamel malformations.

Symptom-based network classification identifies distinct clinical subgroups of liver diseases with common molecular pathways.

BACKGROUND AND OBJECTIVE: Liver disease is a multifactorial complex disease with high global prevalence and poor long-term clinical efficacy and liver disease patients with different comorbidities often incorporate multiple phenotypes in the clinic. Thus, there is a pressing need to improve understanding of the complexity of clinical liver population to help gain more accurate disease subtypes for personalized treatment. METHODS: Individualized treatment of the traditional Chinese medicine (TCM) provides a theoretical basis to the study of personalized classification of complex diseases. Utilizing the TCM clinical electronic medical records (EMRs) of 6475 liver inpatient cases, we built a liver disease comorbidity network (LDCN) to show the complicated associations between liver diseases and their comorbidities, and then constructed a patient similarity network with shared symptoms (PSN). Finally, we identified liver patient subgroups using community detection methods and performed enrichment analyses to find both distinct clinical and molecular characteristics (with the phenotype-genotype associations and interactome networks) of these patient subgroups. RESULTS: From the comorbidity network, we found that clinical liver patients have a wide range of disease comorbidities, in which the basic liver diseases (e.g. hepatitis b, decompensated liver cirrhosis), and the common chronic diseases (e.g. hypertension, type 2 diabetes), have high degree of disease comorbidities. In addition, we identified 303 patient modules (representing the liver patient subgroups) from the PSN, in which the top 6 modules with large number of cases include 51.68% of the whole cases and 251 modules contain only 10 or fewer cases, which indicates the manifestation diversity of liver diseases. Finally, we found that the patient subgroups actually have distinct symptom phenotypes, disease comorbidity characteristics and their underlying molecular pathways, which could be used for understanding the novel disease subtypes of liver conditions. For example, three patient subgroups, namely Module 6 (M6, n = 638), M2 (n = 623) and M1 (n = 488) were associated to common chronic liver disease conditions (hepatitis, cirrhosis, hepatocellular carcinoma). Meanwhile, patient subgroups of M30 (n = 36) and M36 (n = 37) were mostly related to acute gastroenteritis and upper respiratory infection, respectively, which reflected the individual comorbidity characteristics of liver subgroups. Furthermore, we identified the distinct genes and pathways of patient subgroups and the basic liver diseases (hepatitis b and cirrhosis), respectively. The high degree of overlapping pathways between them (e.g. M36 with 93.33% shared enriched pathways) indicates the underlying molecular network mechanisms of each patient subgroup. CONCLUSIONS: Our results demonstrate the utility and comprehensiveness of disease classification study based on community detection of patient network using shared TCM symptom phenotypes and it can be used to other more complex diseases.

DNA Methylation of the hTERT Gene in Breast Cancer Revisited: Diagnostic and Clinical Implications.

BACKGROUND: It is documented that in tumor cell lines, the hTERT gene exhibits prominent methylation at a CpG island rich region about -600 bp upstream of the transcription start site, but mixed or allelically absent around ±150 bp region. Given the potential clinical implications of the findings in breast cancer diagnostics, we set out to investigate if such findings are reproducible on primary surgically resected invasive breast carcinomas. METHODS: The cohort consisted of 50 cases of freshly sampled and formalin-fixed paraffin embedded (FFPE) invasive breast cancers and normal tissue. A modified quasi-quantitative methylation specific polymerase chain reaction was used to determine methylation status in cancer relative to normal tissue at the 2 CpG island-rich regions. RESULTS: A global hypermethylation is evident at the -600 bp region in both fresh and parallel FFPE breast cancers as compared to normal tissue. In contrast, most of the tumor and normal tissue remains unmethylated around the ±150 bp region. CONCLUSIONS: Our results support further development of hTERT hypermethylation in the -600 bp region as a biomarker for breast cancer diagnostics. The unmethylated status of ±150 bp region in normal breast tissue does not support the suggestion that unmethylation is required for hTERT gene expression.

The potential of identification of a malignancy-associated biomarker in breast cancer diagnosis and research: hTERT gene DNA methylation.

BACKGROUND: DNA hypermethylation has been documented to be prominent at a CpG island rich region about 600 bp upstream the transcription start site of the hTERT gene using qualitative methylation specific PCR on DNA isolated from tumor cell lines. In order to assess the potential significance of this biomarker in breast cancer research and diagnosis, we explored if such findings are reproducible on surgically resected fresh breast tumor cells. METHODS: Using quantitative pyrosequencing technology, we investigated and present methylation status of four CpG islands of this region in a cohort of 77 invasive breast carcinomas using normal breast tissue as controls. RESULTS: Globally, a significant hypermethylation in tumor cells was observed in the four CpG islands as a sum, in comparison to methylation of the normal breast tissue. Individually, certain CpG islands displayed methylation greater than 50% in about 3/4 of the 77 breast cancers, but in none of the normal breast tissue. Our results highlight the value of DNA hypermethylation in the -600 bp region of the hTERT gene as a potential marker for breast cancer diagnosis. CONCLUSIONS: We believe that integration of this novel, malignancy-associated molecular testing with morphology is of significant value in the accurate interpretation of small tumor sample size obtained via fine needle aspiration biopsy, ductal lavage, and nipple fluid aspirates both in clinical practice and in breast cancer research. Diagn. Cytopathol. 2016;44:670-675. © 2016 Wiley Periodicals, Inc.

Characterization of KRAS Mutation in Acinar and Langerhans Islet Cells of Patients With Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: KRAS mutations are frequent in pancreatic ductal adenocarcinoma, chronic pancreatitis, and mucinous neoplasms. In animal studies, KRAS mutations in acinar and Langerhans islets are associated with pancreatic intraepithelial neoplasia. Clinically, KRAS mutation is sometimes requested on cytology/biopsy specimens and negative results are helpful to rule out pancreatic ductal adenocarcinoma. This study set out to further elucidate these issues. METHODS: Surgical specimens with pancreatic ductal adenocarcinoma, premalignant lesions, and chronic pancreatitis were reviewed. Tissue microdissections on 53 such areas of 21 cases were performed followed by polymerase chain reaction and pyrosequencing. RESULTS: KRAS codon 12 mutations were detected in 100% pancreatic ductal adenocarcinomas. No KRAS codon 12 and 13 mutations were detected in benign acinar and Langerhans islets that lie adjacent to or away from the tumor. Variable mutation frequencies were seen in premalignant lesions. CONCLUSIONS: The results support such clinical practice that negative KRAS mutation helps rule out pancreatic ductal adenocarcinomas on small cytology/biopsy specimens. Negative KRAS mutations, however, cannot rule out pancreatic premalignant lesions. Additionally, the results that benign pancreas are negative for KRAS mutations complement the findings of other relevant study that KRAS mutation-associated premalignant lesions do not appear to arise from acinar cells or Langerhans islets.

Progressive loss of malignant behavior in telomerase-negative tumorigenic adrenocortical cells and restoration of tumorigenicity by human telomerase reverse transcriptase.

Replicative senescence/crisis is thought to act as a tumor suppressor mechanism. Although recent data indicate that normal human cells cannot be converted into cancer cells without telomerase, the original concept of senescence as a tumor suppressor mechanism is that senescence/crisis would act to limit the growth of telomerase-negative tumors. We show here that this concept is valid when oncogene-expressing human and bovine cells are introduced into immunodeficient mice using tissue reconstruction techniques, as opposed to conventional subcutaneous injection. Primary human and bovine adrenocortical cells were transduced with retroviruses encoding Ha-Ras(G12V) and SV40 large T antigen and transplanted in immunodeficient mice using tissue reconstruction techniques. Transduced cells were fully malignant (invasive and metastatic) in this model. They had negligible telomerase activity both before transplantation and when recovered from tumors. When serially transplanted, tumors showed progressively slower growth, decreased invasion and metastasis, shortened telomeres, and morphological features of crisis. Whereas telomerase was not essential for malignant behavior, expression of human telomerase reverse transcriptase enabled cells from serially transplanted tumors that had ceased growth to reacquire tumorigenicity. Moreover, telomerase-negative oncogene-expressing cells were tumorigenic only when transplanted using tissue reconstruction techniques; human telomerase reverse transcriptase was required for cells to form tumors when cells were injected subcutaneously. This work provides a new model to study crisis in an in vivo setting and its effects on malignancy; despite having invasive and metastatic properties, cells are eventually driven into crisis by proliferation in the absence of a telomere maintenance mechanism.