Dimitri G. Trembath

Current Developments in the Genetics of Autism : From Phenome to Genome

Despite compelling evidence from twin and family studies indicating a strong genetic involvement in the etiology of autism, the unequivocal detection of autism susceptibility genes remains an elusive goal. The purpose of this review is to evaluate the current state of autism genetics research, with attention focused on new techniques and analytic approaches. We first present a brief overview of evidence for the genetic basis of autism, followed by an appraisal of linkage and candidate gene study findings and consideration of new analytic approaches to the study of complex psychiatric conditions, namely, genome-wide association studies, assessment of structural variation within the genome, and the incorporation of endophenotypes in genetic analysis.

MERTK receptor tyrosine kinase is a therapeutic target in melanoma

Metastatic melanoma is one of the most aggressive forms of cutaneous cancers. Although recent therapeutic advances have prolonged patient survival, the prognosis remains dismal. C-MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase with oncogenic properties that is often overexpressed or activated in various malignancies. Using both protein immunohistochemistry and microarray analyses, we demonstrate that MERTK expression correlates with disease progression. MERTK expression was highest in metastatic melanomas, followed by primary melanomas, while the lowest expression was observed in nevi. Additionally, over half of melanoma cell lines overexpressed MERTK compared with normal human melanocytes; however, overexpression did not correlate with mutations in BRAF or RAS. Stimulation of melanoma cells with the MERTK ligand GAS6 resulted in the activation of several downstream signaling pathways including MAPK/ERK, PI3K/AKT, and JAK/STAT. MERTK inhibition via shRNA reduced MERTK-mediated downstream signaling, reduced colony formation by up to 59%, and diminished tumor volume by 60% in a human melanoma murine xenograft model. Treatment of melanoma cells with UNC1062, a novel MERTK-selective small-molecule tyrosine kinase inhibitor, reduced activation of MERTK-mediated downstream signaling, induced apoptosis in culture, reduced colony formation in soft agar, and inhibited invasion of melanoma cells. This work establishes MERTK as a therapeutic target in melanoma and provides a rationale for the continued development of MERTK-targeted therapies.

Detection of Dysplasia in Barrett's Esophagus With In Vivo Depth-Resolved Nuclear Morphology Measurements

BACKGROUND & AIMS Patients with Barretts esophagus (BE) show increased risk of developing esophageal adenocarcinoma and are routinely examined using upper endoscopy with biopsy to detect neoplastic changes. Angle-resolved low coherence interferometry (a/LCI) uses in vivo depth-resolved nuclear morphology measurements to detect dysplasia. We assessed the clinical utility of a/LCI in the endoscopic surveillance of patients with BE. METHODS Consecutive patients undergoing routine surveillance upper endoscopy for BE were recruited at 2 endoscopy centers. A novel, endoscope-compatible a/LCI system measured the mean diameter and refractive index of cell nuclei in esophageal epithelium at 172 biopsy sites in 46 patients. At each site, an a/LCI measurement was correlated with a concurrent endoscopic biopsy specimen. Each biopsy specimen was assessed histologically and classified as normal, nondysplastic BE, indeterminate for dysplasia, low-grade dysplasia (LGD), or high-grade dysplasia (HGD). The a/LCI data from multiple depths were analyzed to evaluate its ability to differentiate dysplastic from nondysplastic tissue. RESULTS Pathology characterized 5 of the scanned sites as HGD, 8 as LGD, 75 as nondysplastic BE, 70 as normal tissue types, and 14 as indeterminate for dysplasia. The a/LCI nuclear size measurements separated dysplastic from nondysplastic tissue at a statistically significant (P < .001) level for the tissue segment 200 to 300 μm beneath the surface with an accuracy of 86% (147/172). A receiver operator characteristic analysis indicated an area under the curve of 0.91, and an optimized decision point gave 100% (13/13) sensitivity and 84% (134/159) specificity. CONCLUSIONS These preliminary data suggest a/LCI is accurate in detecting dysplasia in vivo in patients with BE.

Studies of reduced folate carrier 1 (RFC1) A80G and 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms with neural tube and orofacial cleft defects.

A 19% reduction in the birth prevalence of neural tube defects (NTDs) occurred following folic acid fortification of the US food supply [Honein et al., 2001] and a 31% reduction occurred in Chile after a similar fortification [Castilla et al., 2003]. The same change inprevalence hasnot beendetected for orofacial clefts (OFC) [Ray et al., 2003] even though there is a suggestion from experimental animal data [Paros and Beck, 1999; Bienengraber et al., 2001] and human case-control studies [Shaw et al., 1995; Itikala et al., 2001; Loffredo et al., 2001] that folic acid and multivitamin exposure decreases the risk for OFC. Therefore, genes involved in the folate pathway are of interest for studies aiming to unravel the genetic basis of these birth defects. One mechanism associated with the transport of folate molecules from the circulation to peripheral cells involves the internalization and transportation of these molecules through the reduced folate carrier 1 (RFC1) protein. RFC1 also regulates the delivery of 5,10-methylenetetrahydrofolate reductase (MTHFR) from the cell’s endocytotic vesicle into the cytoplasm [Kamen et al., 1988; Dixon et al., 1994; Hresko et al., 1994; Brigle et al., 1995; Chango et al., 2000]. MTHFR catalyzes the conversion of 5,10 methylenetetrahydrofolate into 5-methyltetrahydrofolate, which will provide the carbon moiety that is used to convert for example homocysteine into methionine. Recent evidence indicates that the A80G RFC1 polymorphism is associated with higher risk of spina bifida [De Marco et al., 2001; Shaw et al., 2002] and conotruncal heart defects but not with risk for OFC [Shaw et al., 2003]. It is unknown whether the RFC1 A80G polymorphism alters the transport function of RFC1. However, lower plasma folate levels and homocysteinemia were observed in individuals presenting with two copies of the rareRFC1 A80G variant compared with individuals presenting with no copies [Chango et al., 2000].NTD have also been associated with the mutation C677T in the MTHFR locus [van der Putt et al., 1995; Ou et al., 1996; Trembath et al., 1999] and this samemutation has been investigated for association with OFC with many conflicting results [Gaspar et al., 1999; Mills et al., 1999; Martinelli et al., 2001; Blanton et al., 2002; Prescott et al., 2002; Jugessur et al., 2003; Pezzetti et al., 2004; Shi et al., 2004]. We present the studies with the RFC1 A80G and MTHFR C677T polymorphisms in two populations affected by NTD and OFC. DNA samples from a total of 388 families in which the proband was affected with myelomeningocele were analyzed. These samples were gathered from affected individuals and their immediate family members from several U.S. clinics. Table I describes the type of NTD. These families came from 4 different sites (Nebraska—9; Minnesota—47; Iowa—126; Duke—206) and include samples on 350 affected individuals, 328 mothers, 245 fathers, and 167 unaffected siblings. In addition, DNA samples from 217 case-mother pairs with OFC from ECLAMC (Latin American Collaborative Study of Congenital Malformations) were studied (Table I). ECLAMC is a hospital-based birth defects registry study that includes sites in Argentina, Bolivia, Brazil, Chile, Ecuador, Paraguay, Uruguay, and Venezuela. This study population has been previously described in detail [Vieira et al., 2002, 2003]. Family-based approaches were previously employed in this NTD study population and found no association between the

The distribution of cerebrovascular amyloid in Alzheimer’s disease varies with ApoE genotype

We performed a comparative study to assess cerebral amyloid angiopathy and ApoE genotype in cases of Alzheimer’s disease (AD). Ten ApoE 3,3 and ten ApoE 4,4 AD brains, as well as ten normal control brains, were selected after matching for age, sex, and duration of disease. Sections of middle frontal and inferior parietal cortex including white matter sections were stained with an antibody against amyloid beta (Aβ), and extensive analysis of arteriolar Aβ deposition was performed using digital image analysis. Quantification of the staining revealed a larger cross-section of arteriolar walls occupied by Aβ in ApoE 4,4 and ApoE 3,3 AD subjects compared to controls. Our results show Aβ deposition in gray matter and white matter arterioles was predominantly found in ApoE 4,4 brains and, overall, Aβ deposition was greatest in these cases. This observation implies that there is greater vascular amyloid deposition (particularly in the white matter arterioles) in ApoE 4,4 AD individuals compared to ApoE 3,3 AD. These observations may give insight into the etiology behind the increased risk for AD associated with the ApoE-ε4 allele and the pathogenesis of vascular Aβ deposition.

MicroRNAs Classify Different Disease Behavior Phenotypes of Crohn's Disease and May Have Prognostic Utility.

Background:There is a dire need for reliable prognostic markers that can guide effective therapeutic intervention in Crohns disease (CD). We examined whether different phenotypes in CD can be classified based on colonic microRNA (miRNA) expression and whether miRNAs have prognostic utility for CD. Methods:High-throughput sequencing of small and total RNA isolated from colon tissue from patients with CD and controls without Inflammatory Bowel Disease (non-IBD) was performed. To identify miRNAs associated with specific phenotypes of CD, patients were stratified according to disease behavior (nonstricturing, nonpenetrating; stricturing; penetrating), and miRNA profiles in each subset were compared with those of the non-IBD group. Validation assays were performed using quantitative reverse transcription polymerase chain reaction. These miRNAs were further evaluated by quantitative reverse transcriptase polymerase chain reaction on formalin-fixed, paraffin-embedded tissue (index biopsies) of patients with nonpenetrating CD at the time of diagnosis that either retained the nonpenetrating phenotype or progressed to penetrating/fistulizing CD. Results:We found a suite of miRNAs, including miR-31-5p, miR-215, miR-223-3p, miR-196b-5p, and miR-203 that stratify patients with CD according to disease behavior independent of the effect of inflammation. Furthermore, we also demonstrated that expression levels of miR-215 in index biopsies of patients with CD might predict the likelihood of progression to penetrating/fistulizing CD. Finally, using a novel statistical simulation approach applied to colonic RNA-sequencing data for patients with CD and non-IBD controls, we identified miR-31-5p and miR-203 as candidate master regulators of gene expression profiles associated with CD. Conclusions:miRNAs may serve as clinically useful prognostic markers guiding initial therapy and identifying patients who would benefit most from effective intervention.

Gray zones in brain tumor classification: evolving concepts.

The World Health Organization recently updated its classification of central nervous system tumors, adding 8 entities, as well as defining new variants and morphologic patterns of existing entities. Despite the continued refinement of brain tumor histologic classification and grading, there remain some diagnostic “gray zones” that challenge general surgical pathologists and neuropathologists alike. These include the presence of oligodendroglial features in (mixed) oligoastrocytomas and glioblastomas (GBMs), GBM variants (such as small cell GBM), meningioma classification and grading, medulloblastoma variants, ependymoma grading, the presence of “neuronal features” in otherwise morphologically classic gliomas, and low-grade gliomas with high Ki-67 labeling indices. In the current review, we discuss these issues and offer some practical guidelines for dealing with problematic cases.

A novel small molecule that selectively inhibits glioblastoma cells expressing EGFRvIII

BackgroundMutations of the epidermal growth factor receptor (EGFR) are a possible molecular target for cancer therapy. EGFR is frequently amplified in glioblastomas and 30 to 40% of glioblastomas also express the deletion mutation EGFRvIII. This frequent oncogenic mutation provides an opportunity for identifying new anti-glioblastoma therapies. In this study, we sought small molecule inhibitors specific for cancer cells expressing EGFRvIII, using isogenic parental cells without EGFRvIII as a control.ResultsA screen of the NCI small molecule diversity set identified one compound, NSC-154829, which consistently inhibited growth of different human glioblastoma cells expressing EGFRvIII, but permitted normal growth of matched control cells. NSC-154829 had no previously established medicinal use, but has a purine-like structural component. Further experiments showed this compound increased apoptosis in cells with EGFRvIII, and moderately affected the expression of p21, independent of any changes in p53 levels or in Akt phosphorylation.ConclusionThese initial results suggest that NSC-154829 or a closely related structure might be further investigated for its potential as an anti-glioblastoma drug, although its precise molecular mechanism is still undefined.

Cytopathology of mesenchymal chondrosarcomas: a report and comparison of four patients.

Mesenchymal chondrosarcoma (MC) is an infrequent neoplasm, representing approximately 1% of all chondrosarcomas. Cytologic descriptions of MCs have been confined to rare case reports. In the current report, the authors describe their experience with the cytologic features of four MCs: two primary tumors and two metastatic lesions.

Phospho-ERK and AKT status, but not KRAS mutation status, are associated with outcomes in rectal cancer treated with chemoradiotherapy

BackgroundKRAS mutations may predict poor response to radiotherapy. Downstream events from KRAS, such as activation of BRAF, AKT and ERK, may also confer prognostic information but have not been tested in rectal cancer (RC). Our objective was to explore the relationships of KRAS and BRAF mutation status with p-AKT and p-ERK and outcomes in RC.MethodsPre-radiotherapy RC tumor biopsies were evaluated. KRAS and BRAF mutations were assessed by pyrosequencing; p-AKT and p-ERK expression by immunohistochemistry.ResultsOf 70 patients, mean age was 58; 36% stage II, 56% stage III, and 9% stage IV. Responses to neoadjuvant chemoradiotherapy: 64% limited, 19% major, and 17% pathologic complete response. 64% were KRAS WT, 95% were BRAF WT. High p-ERK levels were associated with improved OS but not for p-AKT. High levels of p-AKT and p-ERK expression were associated with better responses. KRAS WT correlated with lower p-AKT expression but not p-ERK expression. No differences in OS, residual disease, or tumor downstaging were detected by KRAS status.ConclusionsKRAS mutation was not associated with lesser response to chemoradiotherapy or worse OS. High p-ERK expression was associated with better OS and response. Higher p-AKT expression was correlated with better response but not OS.