Mutations in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome.

Next-generation RNA sequence analysis of platelets from an individual with autosomal recessive gray platelet syndrome (GPS, MIM139090) detected abnormal transcript reads, including intron retention, mapping to NBEAL2 (encoding neurobeachin-like 2). Genomic DNA sequencing confirmed mutations in NBEAL2 as the genetic cause of GPS. NBEAL2 encodes a protein containing a BEACH domain that is predicted to be involved in vesicular trafficking and may be critical for the development of platelet α-granules.

Homozygosity mapping with SNP arrays confirms 3p21 as a recessive locus for gray platelet syndrome and narrows the interval significantly.

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by thrombocytopenia and the absence of α-granules in platelets. Patients with GPS present with mild to moderate bleeding and many develop myelofibrosis. The genetic cause of GPS is unknown. We present 2 Native American families with a total of 5 affected persons and a single affected patient of Pakistani origin in which GPS appears to be inherited in an autosomal recessive manner. Homozygosity mapping using the Affymetrix 6.0 chips demonstrates that all 6 GPS-affected persons studied are homozygous for a 1.7-Mb region in 3p21. Linkage analysis confirmed the region with a logarithm of the odds score of 2.7. Data from our families enabled us to significantly decrease the size of the critical region for GPS from the previously reported 9.4-Mb region at 3p21.

Gene expression analysis of human otosclerotic stapedial footplates.

Otosclerosis is a complex disease that results in a common form of conductive hearing loss due to impaired mobility of the stapes. Stapedial motion becomes compromised secondary to invasion of otosclerotic foci into the stapedio-vestibular joint. Although environmental factors and genetic causes have been implicated in this process, the pathogenesis of otosclerosis remains poorly understood. To identify molecular contributors to otosclerosis we completed a microarray study of otosclerotic stapedial footplates. Stapes footplate samples from otosclerosis and control patients were used in the analysis. One-hundred-and-ten genes were found to be differentially expressed in otosclerosis samples. Ontological analysis of differentially expressed genes in otosclerosis provides evidence for the involvement of a number of pathways in the disease process that include interleukin signaling, inflammation and signal transduction, suggesting that aberrant regulation of these pathways leads to abnormal bone remodeling. Functional analyses of genes from this study will enhance our understanding of the pathogenesis of this disease.

Transcriptional profiling of lymphoblast lines from subjects with panic disorder.

In attempts to isolate genetic vulnerability factors for panic disorder (PD), a number of investigators have used genome-wide linkage or association analyses. But these attempts have been only modestly successful which suggests that alternative approaches may be needed to define the biology of PD. Therefore, using recently developed genome-wide gene expression profiling, we explored whether transcriptional signatures associated with PD are present in lymphoblast cell line. The expression of 2,469 transcripts in lymphoblast cell lines from 16 subjects was arithmetically increased in every line and significantly increased overall and 354 transcripts was arithmetically decreased in every cell line and significantly decreased overall as compared to those lymphoblast lines from 17 subjects without a history of behavioral illness. Further sex specific analyses showed that in those 10 lines derived from female probands, the expression of a further 67 transcripts was arithmetically increased in every line and significantly increased overall and a further 332 transcripts was arithmetically decreased in every cell line and significantly decreased. Conversely, in cell lines from the six male probands, the expression of an additional 212 was arithmetically increased in every line and significantly increased overall and a further 332 transcripts was arithmetically decreased in every cell line. We conclude that lymphoblast cell lines derived from subjects with PD have significant, partially sex dependent changes in gene transcription. Further studies are necessary to correlate these changes in these hemopoetically derived cells with those changes postulated to occur in the CNS in association with PD.

Transcriptional profiling of subjects from the Iowa adoption studies.

Transcriptional profiling has been used to identify gene expression patterns indicative of general medical illnesses such as atherosclerosis. However, whether these methods can identify common psychiatric disorders has not been established. To answer this question with respect to nicotine use, we used genome-wide expression profiling lymphoblast cell lines from six actively smoking Iowa Adoption Studies (IAS) subjects and nine "clean" control subjects, followed by real-time PCR (RT-PCR) of gene expression patterns in lymphoblast derived RNA from 94 subjects in the IAS. As compared to those from controls without a history of smoking (n = 9), the expression levels of 579 of 29,098 genes were significantly up-regulated and expression levels of 584 of 29,098 genes were significantly down-regulated in lymphoblast lines from currently smoking subjects (n = 6). RT-PCR confirmation of four select RNA levels confirmed the validity of the overall profile and revealed highly significant relationships between the expression of some of these transcripts and (1) major depression, (2) antisocial personality, (3) nicotine dependence, and (4) cannabis dependence. We conclude that the use of expression profiling may contribute significant insights into the biology of complex behavioral disorders.

Dickkopf-1 is an epigenetically silenced candidate tumor suppressor gene in medulloblastoma.

Medulloblastoma is a heterogeneous pediatric brain tumor with significant therapy-related morbidity, its five-year survival rates ranging from 30% to 70%. Improvement in diagnosis and therapy requires better understanding of medulloblastoma pathology. We used whole-genome microarray analysis to identify putative tumor suppressor genes silenced by epigenetic mechanisms in medulloblastoma. This analysis yielded 714 up-regulated genes in immortalized medulloblastoma cell line D283 on treatment with histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Dickkopf-1 (DKK1), a Wnt antagonist, was found to be up-regulated on HDAC inhibition. We examined DKK1 expression in primary medulloblastoma cells and patient samples by reverse transcriptase PCR and found it to be significantly down-regulated relative to normal cerebellum. Transfection of a DKK1 gene construct into D283 cell lines suppressed medulloblastoma tumor growth in colony focus assays by 60% (P < 0.001). In addition, adenoviral vector-mediated expression of DKK1 in medulloblastoma cells increased apoptosis fourfold (P < 0.001). These data reveal that inappropriate histone modifications might deregulate DKK1 expression in medulloblastoma tumorigenesis and block its tumor-suppressive activity.

Epigenetic regulation of maspin expression in the human placenta.

Maspin, a tumour suppressor gene, is differentially expressed in the human placenta. Decreased expression of maspin in the first trimester corresponds with the period of maximum trophoblast invasion, suggesting a role in cell invasion and motility. Although methylation of CpG islands regulates maspin expression in cancer cells, the mechanism of maspin regulation in the human placenta is unknown. Our objectives were to determine the role of epigenetic alterations in the regulation of maspin expression in the placenta. Placental samples obtained from 7 to 40 weeks' gestation were used for bisulphite sequencing and chromatin immunoprecipitation (ChIP) PCR. There was no significant change in the methylation indices in the promoter region of maspin throughout gestation. The levels of histone modifications associated with transcriptionally active chromatin were significantly different in placental tissues from second and third trimester relative to those from first trimester. Addition of trichostatin A (TSA) to placental explants increased the maspin mRNA expression (8- to 20-fold), whereas addition of 5-aza-cytidine (5-AzaC) had no effect on maspin expression. Our data suggest that maspin expression in the human placenta is regulated by changes in histone tail modifications. This is the first report of selective histone modifications associated with differential placental gene expression in human gestation.

Genome-wide analysis of epigenetic silencing identifies BEX1 and BEX2 as candidate tumor suppressor genes in malignant glioma.

Promoter hypermethylation and histone deacetylation are common epigenetic mechanisms implicated in the transcriptional silencing of tumor suppressor genes in human cancer. We treated two immortalized glioma cell lines, T98 and U87, and 10 patient-derived primary glioma cell lines with trichostatin A (TSA), a histone deacetylase inhibitor, or 5-aza-2'-deoxycytidine (5-AzaC), a DNA methyltransferase inhibitor, to comprehensively identify the cohort of genes reactivated through the pharmacologic reversal of these distinct but related epigenetic processes. Whole-genome microarray analysis identified genes induced by TSA (653) or 5-AzaC treatment (170). We selected a subset of reactivated genes that were markedly induced (greater than two-fold) after treatment with either TSA or 5-AzaC in a majority of glioma cell lines but not in cultured normal astrocytes. We then characterized the degree of promoter methylation and transcriptional silencing of selected genes in histologically confirmed human tumor and nontumor brain specimens. We identified two novel brain expressed genes, BEX1 and BEX2, which were silenced in all tumor specimens and exhibited extensive promoter hypermethylation. Viral-mediated reexpression of either BEX1 or BEX2 led to increased sensitivity to chemotherapy-induced apoptosis and potent tumor suppressor effects in vitro and in a xenograft mouse model. Using an integrated approach, we have established a novel platform for the genome-wide screening of epigenetically silenced genes in malignant glioma. This experimental paradigm provides a powerful new method for the identification of epigenetically silenced genes with potential function as tumor suppressors, biomarkers for disease diagnosis and detection, and therapeutically reversible modulators of critical regulatory pathways important in glioma pathogenesis.