Purpose: Smith-Magenis syndrome (SMS) is a complex disorder that includes mental retardation, craniofacial and skeletal anomalies, and behavioral abnormalities. We report the molecular and genotype-phenotype analyses of 31 patients with SMS who carry 17p11.2 deletions or mutations in the RAI1 gene.

Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation disorder characterized by distinct craniofacial features and neurobehavioral abnormalities usually associated with an interstitial deletion in 17p11.2. Heterozygous point mutations in the retinoic acid induced 1 gene (RAI1) have been reported in nine SMS patients without a deletion detectable by fluorescent in situ hybridization (FISH), implicating RAI1 haploinsufficiency as the cause of the major clinical features in SMS. All of the reported point mutations are unique and de novo. RAI1 contains a polymorphic CAG repeat and encodes a plant homeo domain (PHD) zinc finger-containing transcriptional regulator. We report a novel RAI1 frameshift mutation, c.3103delC, in a non-deletion patient with many SMS features. The deletion of a single cytosine occurs in a heptameric C-tract (CCCCCCC), the longest mononucleotide repeat in the RAI1 coding region. Interestingly, we had previously reported a frameshift mutation, c.3103insC, in the same mononucleotide repeat. Furthermore, all five single base frameshift mutations preferentially occurred in polyC but not polyG tracts. We also investigated the distribution of the polymorphic CAG repeats in both the normal population and the SMS patients as one potential molecular mechanism for variability of clinical expression. In this limited data set, there was no significant association between the length of CAG repeats and the SMS phenotype. However, we identified a 5-year-old girl with an apparent SMS phenotype who was a compound heterozygote for an RAI1 missense mutation inherited from her father and a polyglutamine repeat of 18 copies, representing the largest known CAG repeat in this gene, inherited from her mother.

IntroductionThe etiologic agent of Kaposi sarcoma (KS), KS-associated herpesvirus (KSHV), 1 infects a number of cell types within KS lesions, including endothelial cells, monocyte-derived cells, and characteristic "spindle cells" that help define these tumors. 2,3 Within infected individuals, a variety of circulating bone marrowderived cells also can harbor KSHV DNA. Some of these cells can transform to spindle-shaped cells displaying morphology and cell surface characteristics similar to KS spindle cells. [4][5][6] Recent observations further implicate KSHV infection of bone marrow cells as a potential cause of pancytopenia, marrow engraftment failure, and hemophagocytic lymphohistiocytosis. [7][8][9][10] However, the precise identity of the infected marrow cells and whether they may serve as reservoirs for subsequent viral dissemination and the clinical manifestations of KSHV infection remain unknown.Human adult and fetal bone marrow contain a rare (0.01%-0.001%) but critical population of stroma-based cells-mesenchymal stem cells (MSCs)-that regulate the differentiation and proliferation of hematopoietic cells within bone marrow. 11,12 Although the mechanisms by which MSCs exert these effects are unclear, in vitro and in vivo data indicate that certain cell lineages that they influence also can be targets for KSHV infection in human patients, including CD34 ϩ progenitor cells. 12 In this study, we provide the first report of direct KSHV infection of primary human bone marrow cells, demonstrating susceptibility of fetal-derived MSCs to KSHV infection in culture. Our results raise the possibility that MSCs may play a role in the development of KSHV-related pathology and suggest the potential of this cell culture system for studying the long-term effects of KSHV infection on relevant human hematopoietic precursor cells. Study design Isolation of mesenchymal stem cellsIsolation and enrichment of adherent stem cells from human fetal bone marrow was undertaken with commercially available reagents (RosetteSep and MesenCult enrichment media, StemCell Technologies, Vancouver, BC, Canada), following the manufacturer's recommendations. MSCs were identified by flow cytometry (FACS Calibur) using a panel of fluorochromeconjugated monoclonal cell surface antibodies ( Figure 1) from Caltag (Burlingame, CA). KSHV infection of MSCsThe KSHV-infected primary effusion lymphoma (PEL) cell line, BCBL-1, was chemically induced to support lytic KSHV replication, as described previously. 13 MSCs were incubated for 2 hours with KSHV concentrated from BCBL-1 supernatants, or for 16 hours with induced BCBL-1 cells. 14 KSHV-infected MSCs were identified with an immunofluorescence assay that detects expression of the KSHV latency-associated nuclear antigen (LANA) in the nuclei of fixed cells. 14 Quantification of input KSHVThe concentration of encapsidated viral genomes from each viral preparation was determined using a fluorescently labeled KSHV DNA probe complementary to ORF73. 14 Unless otherwise stated, all cell-free infections were c...

Overexpression of the transcriptional coregulators C-terminal binding proteins 1 and 2 (CtBP) occurs in many human solid tumors and is associated with poor prognosis. CtBP modulates oncogenic gene expression programs and is an emerging drug target, but its oncogenic role is unclear. Consistent with oncogenic potential, exogenous CtBP2 transformed primary mouse and human cells to anchorage independence similarly to mutant H-Ras. To investigate CtBP’s contribution to in vivo tumorigenesis, Apcmin/+ mice, which succumb to massive intestinal polyposis, were bred to Ctbp2+/− mice. CtBP interacts with Adenomatous Polyposis Coli (APC) protein, and is stabilized in both APC-mutated human colon cancers and Apcmin/+ intestinal polyps. Ctbp2 heterozygosity increased the median survival of Apcmin/+ mice from 21 to 48 weeks, and reduced polyp formation by 90%, with Ctbp2+/− polyps exhibiting reduced levels of β-catenin and its oncogenic transcriptional target, cyclin D1. Ctbp’s potential as a therapeutic target was studied by treating Apcmin/+ mice with the CtBP small molecule inhibitors 4-methlythio-2-oxobutyric acid and 2-hydroxy-imino phenylpyruvic acid, both of which reduced polyposis by more than half compared with vehicle treatment. Phenocopying Ctbp2 deletion, both Ctbp inhibitors caused substantial decreases in the protein level of Ctbp2, as well its oncogenic partner β-catenin, and the effects of the inhibitors on CtBP and β-catenin levels could be modeled in an APC mutated human colon cancer cell line. CtBP2 is thus a druggable transforming oncoprotein critical for the evolution of neoplasia driven by Apc mutation.

Duplications of 17(p11.2p11.2) have been associated with various behavioral manifestations including attention deficits, obsessive-compulsive symptoms, autistic traits, and language delay. We are conducting a genetic study of autism and are screening all cases for submicroscopic chromosomal abnormalities, in addition to standard karyotyping, and fragile X testing. Using array-based comparative genomic hybridization analysis of data from the Affymetrix GeneChip(R) Human Mapping Array set, we detected a duplication of approximately 3.3 Mb on chromosome 17p11.2 in a male child with autism and severe expressive language delay. The duplication was confirmed by measuring the copy number of genomic DNA using quantitative polymerase chain reaction. Gene expression analyses revealed increased expression of three candidate genes for the Smith-Magenis neurobehavioral phenotype, RAI1, DRG2, and RASD1, in transformed lymphocytes from Case 81A, suggesting gene dosage effects. Our results add to a growing body of evidence suggesting that duplications of 17(p11.2p11.2) result in language delay as well as autism and related phenotypes. As Smith-Magenis syndrome is also associated with language delay, a gene involved in acquisition of language may lie within this interval. Whether a parent of origin effect, gender of the case, the presence of allelic variation, or changes in expression of genes outside the breakpoints influence the resultant phenotype remains to be determined.