Multivariate characterization of white matter heterogeneity in autism spectrum disorder.

The complexity and heterogeneity of neuroimaging findings in individuals with autism spectrum disorder has suggested that many of the underlying alterations are subtle and involve many brain regions and networks. The ability to account for multivariate brain features and identify neuroimaging measures that can be used to characterize individual variation have thus become increasingly important for interpreting and understanding the neurobiological mechanisms of autism. In the present study, we utilize the Mahalanobis distance, a multidimensional counterpart of the Euclidean distance, as an informative index to characterize individual brain variation and deviation in autism. Longitudinal diffusion tensor imaging data from 149 participants (92 diagnosed with autism spectrum disorder and 57 typically developing controls) between 3.1 and 36.83 years of age were acquired over a roughly 10-year period and used to construct the Mahalanobis distance from regional measures of white matter microstructure. Mahalanobis distances were significantly greater and more variable in the autistic individuals as compared to control participants, demonstrating increased atypicalities and variation in the group of individuals diagnosed with autism spectrum disorder. Distributions of multivariate measures were also found to provide greater discrimination and more sensitive delineation between autistic and typically developing individuals than conventional univariate measures, while also being significantly associated with observed traits of the autism group. These results help substantiate autism as a truly heterogeneous neurodevelopmental disorder, while also suggesting that collectively considering neuroimaging measures from multiple brain regions provides improved insight into the diversity of brain measures in autism that is not observed when considering the same regions separately. Distinguishing multidimensional brain relationships may thus be informative for identifying neuroimaging-based phenotypes, as well as help elucidate underlying neural mechanisms of brain variation in autism spectrum disorders.

A phase II study of metronomic paclitaxel/cyclophosphamide/capecitabine followed by 5-fluorouracil/epirubicin/cyclophosphamide as preoperative chemotherapy for triple-negative or low hormone receptor expressing/HER2-negative primary breast cancer.

PURPOSE: Better treatments for triple-negative breast cancer (TNBC) are needed. To address this need, we studied the effects of preoperative metronomic paclitaxel/cyclophosphamide/capecitabine (mPCX) followed by 5-fluorouracil (FU)/epirubicin/cyclophosphamide (FEC) as preoperative chemotherapy in TNBC patients. METHODS: Forty primary TNBC patients received four cycles of metronomic paclitaxel (80 mg/m(2) on Days 1, 8, and 15), cyclophosphamide (50 mg/body daily), and capecitabine (1,200 mg/m(2) daily), followed by four cycles of 5-FU (500 mg/m(2)), epirubicin (100 mg/m(2)), and cyclophosphamide (500 mg/m(2)) every 3 weeks. The primary end point was the pathological complete response (pCR) rate. RESULTS: Forty patients formed the intent-to-treat population. The median dose intensities of paclitaxel, cyclophosphamide, and capecitabine were 89.7, 92.1, and 89.8%, respectively. Five patients discontinued mPCX and two discontinued FEC, primarily because of adverse events, resulting in a per-protocol population (PPS) of 33 patients. The pCR (ypT0/Tis ypN0) rate was 47.5% (19/40) in the intent-to-treat population and 54.5% (18/33) in the PPS. The clinical response rates were 36/40 (90.0%) and 31/33 (93.9%) in the intent-to-treat and PPS, respectively. The breast conservation rate was 72.7% (24/33), and 5/13 patients underwent partial resection instead of pre-planned total mastectomy. Grade 3-4 adverse events included neutropenia (35%), leukopenia (25%), and hand-foot syndrome (8%). CONCLUSIONS: Metronomic PCX followed by FEC chemotherapy was associated with a high pCR rate and low toxicity in TNBC patients. Further studies of this regimen in larger numbers of patients are warranted.

A high-density SNP genome-wide linkage scan in a large autism extended pedigree.

We performed a high-density, single nucleotide polymorphism (SNP), genome-wide scan on a six-generation pedigree from Utah with seven affected males, diagnosed with autism spectrum disorder. Using a two-stage linkage design, we first performed a nonparametric analysis on the entire genome using a 10K SNP chip to identify potential regions of interest. To confirm potentially interesting regions, we eliminated SNPs in high linkage disequilibrium (LD) using a principal components analysis (PCA) method and repeated the linkage results. Three regions met genome-wide significance criteria after controlling for LD: 3q13.2-q13.31 (nonparametric linkage (NPL), 5.58), 3q26.31-q27.3 (NPL, 4.85) and 20q11.21-q13.12 (NPL, 5.56). Two regions met suggestive criteria for significance 7p14.1-p11.22 (NPL, 3.18) and 9p24.3 (NPL, 3.44). All five chromosomal regions are consistent with other published findings. Haplotype sharing results showed that five of the affected subjects shared more than a single chromosomal region of interest with other affected subjects. Although no common autism susceptibility genes were found for all seven autism cases, these results suggest that multiple genetic loci within these regions may contribute to the autism phenotype in this family, and further follow-up of these chromosomal regions is warranted.

Detailed genetic characterization of the interleukin-23 receptor in psoriasis.

Using a multi-tiered, case-control association design, scanning 25 215 gene-centric SNPs, we previously identified two psoriasis susceptibility genes: IL12B and IL23R. These results have recently been confirmed. To better characterize the IL23R psoriasis-association, we used a fine mapping strategy to identify 59 additional IL23R-linked SNPs, which were genotyped in our three independent, white North American sample sets (>2800 individuals in toto). A sliding window of haplotype association demonstrates colocalization of psoriasis susceptibility effects within the boundaries of IL23R across all sample sets, thereby decreasing the likelihood that neighboring genes, particularly IL12RB2, are driving the association at this region. Additional haplotype work identified two 5-SNP haplotypes with strong protective effects, consistent across our three sample sets (OR(common)=0.67; P(comb)=4.32E-07). Importantly, heterogeneity of effect was extremely low between sample sets for these haplotypes (P(Het)=0.961). Together, these protective haplotypes attain a frequency of 16% in controls, declining to 11% in cases. The characterization of association patterns within IL23R to specific predisposing/protective variants will play an important role in the elucidation of psoriasis etiology and other related phenotypes. Further, this work is essential to lay the foundation for the role of IL23R genetics in response to pharmaceutical therapy and dosage.

Variants in the 5q31 cytokine gene cluster are associated with psoriasis.

A multitiered genetic association study of 25 215 single-nucleotide polymorphisms (SNPs) in three case-control sample sets (1446 patients and 1432 controls) identified three IL13-linked SNPs (rs1800925, rs20541 and rs848) associated with psoriasis. Although the susceptibility effects at these SNPs were modest (joint allelic odds ratios (ORs): 0.76 to 0.78; P(comb): 1.3E-03 to 2.50E-04), the association patterns were consistent across the sample sets, with the minor alleles being protective. Haplotype analyses identified one common, susceptible haplotype CCG (joint allelic OR=1.27; P(comb)=1.88E-04) and a less common, protective haplotype TTT (joint allelic OR=0.74; P(comb)=7.05E-04). In combination with the other known genetic risk factors, HLA-C, IL12B and IL23R, the variants reported here generate an 11-fold psoriasis-risk differential. Residing in the 5q31 cytokine gene cluster, IL13 encodes an important T-cell-derived cytokine that regulates cell-mediated immunity. These results provide the foundation for additional studies required to fully dissect the associations within this cytokine-rich genomic region, as polymorphisms in closely linked candidate genes, such as IRF1, IL5 or IL4, may be driving these results through linkage disequilibrium.

Siah-1 mediates a novel beta-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein.

The adenomatous polyposis coli (APC) tumor-suppressor protein, together with Axin and GSK3beta, forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of beta-catenin by the proteasome. Siah-1, the human homolog of Drosophila seven in absentia, is a p53-inducible mediator of cell cycle arrest, tumor suppression, and apoptosis. We have now found that Siah-1 interacts with the carboxyl terminus of APC and promotes degradation of beta-catenin in mammalian cells. The ability of Siah-1 to downregulate beta-catenin signaling was also demonstrated by hypodorsalization of Xenopus embryos. Unexpectedly, degradation of beta-catenin by Siah-1 was independent of GSK3beta-mediated phosphorylation and did not require the F box protein beta-TrCP. These results indicate that APC and Siah-1 mediate a novel beta-catenin degradation pathway linking p53 activation to cell cycle control.

TGF-beta1 inhibits BRCA1 expression through a pathway that requires pRb.

TGF-beta1 inhibits BRCA1 expression, which contradicts the model that TGF-beta1 prevents carcinogenesis by activating tumor suppressor genes. To resolve this apparent contradiction, we examined BRCA1 expression in Mv1Lu cells, a well-established model system for studying the TGF-beta1 tumor suppressor pathway. We found that inactivation of pRb by the papillomavirus type 16 E7 protein increased BRCA1 expression and abolished the ability of TGF-beta1 to inhibit BRCA1 expression. We conclude that TGF-beta1 inhibits BRCA1 expression through a pathway that requires pRb. We propose a model to explain the inhibition of BRCA1 as a target in the TGF-beta1 tumor suppressor signaling pathway. Our results suggest that the tumor suppressor functions of BRCA1 are initiated by the inactivation of pRb, and therefore that the activation of pRb by TGF-beta1 might alleviate the requirement for BRCA1 function.

Identification of high-risk breast cancer patients from genetic changes of their tumors.

To identify the genetic prognostic markers for breast cancer, we analyzed loss of heterozygosity (LOH) at 11p, 16q, 17p, 17q, and 18q, as well as amplification of the ERBB2, INT2, and MYC genes, in 131 patients with breast carcinoma, 49 of whom had lymph node involvement, but none of whom had distant metastases. Among the several chromosome arms tested, LOH at 17q was correlated with lymph node metastasis. Amplification of the ERBB2, MYC, and INT2 genes was found more frequently in tumors from patients with lymph node metastases than in tumors from those without lymph node metastases. Univariate analysis demonstrated that LOH at 17q and INT2 amplification were factors influencing disease-free survival (DFS). A multivariate analysis was performed on 89 tumors that were able to be evaluated for both LOH at 17q and INT2 amplification, and the results showed that patients who had tumors with these genetic changes were more likely to have a poor prognosis. The findings of this study suggest that investigating genetic changes, in addition to conventional clinicopathologic factors, may contribute to defining groups of breast cancer patients with differences in prognosis.

Malignant phyllodes tumor with liposarcomatous components: a case report with cytological presentation.

Cytological and histological findings of a rare case of a malignant phyllodes tumor (with liposarcomatous components) of the breast are presented. The atypia of the stromal cells and naked nuclei in a lesion considered benign clinically and on imaging alerted us to the possibility of a phyllodes tumor, despite the low cellularity of the preoperative fine-needle aspiration smears. The excisional biopsy was histologically diagnosed as malignant phyllodes tumor with liposarcomatous components. Peculiar atypical cells with large, pale, transparent cytoplasm, or with intranuclear chromatolytic areas similar to intranuclear cytoplasmic inclusions, in addition to atypical lipoblasts revealed in imprint smears from the excised tumor, may be important for cytopathologists to achieve a definitive cytological diagnosis, and also to observe patients over long periods for recurrences and metastasis after surgery for this rare breast tumor.

Inhibition of topoisomerase IIalpha expression by transforming growth factor-beta1 is abrogated by the papillomavirus E7 protein.

Transforming growth factor-beta (TGF-beta) protects normal cells from etoposide-induced cell death, yet the mechanism has remained speculative. Studies have shown that etoposide modifies the activity of the topoisomerase IIalpha (topo IIalpha) enzyme, thereby causing DNA damage and inducing cell death. Expression of topo IIalpha is necessary for etoposide-induced cell death, and peak expression of topo IIalpha normally occurs during the G2 phase of the cell cycle. We predicted that by arresting growth in the G1 phase, TGF-beta1 would prevent the induction of topo IIalpha expression that normally occurs subsequent to the G1-S transition, thereby protecting cells from etoposide-induced cell death. Accordingly, we hypothesized that the inhibition of topo IIalpha expression by TGF-beta1 would be dependent on the ability of TGF-beta1 to arrest cell cycle progression in G1. Using mink lung epithelial cells (MvlLu), we found that TGF-beta1 decreases topo IIalpha mRNA expression, and the decrease occurs as cells begin to accumulate in the G1 phase of the cell cycle. Topo IIalpha protein expression decreases subsequent to the fall in mRNA expression. In contrast, topo IIalpha expression is not affected by TGF-beta1 in cells that fail to undergo G1 arrest because of inactivation of the retinoblastoma tumor suppressor protein (pRb) by the papillomavirus type 16 E7 protein. Our studies suggest that inhibition of topo IIalpha by TGF-beta1 is the principal mechanism that protects mink lung epithelial cells (Mv1Lu) from etoposide-induced toxicity. Furthermore, the inhibition of topo IIalpha protein expression by TGF-beta1 is dependent on pRb-mediated cell cycle arrest, suggesting that TGF-beta1 will not reduce the sensitivity of pRb-deficient cancers to etoposide.

Persistent transgene expression and normal differentiation of immortalized human keratinocytes in vivo.

Cells transduced ex vivo with transgenes encoded on retroviruses have constant and prolonged expression in vitro; however, in vivo expression is quickly lost. Much attention has been directed at methods to circumvent this problem. We have shown that loss of transgene expression does not occur when transduced immortalized 3T3 cells are transplanted to the in vivo setting of athymic mice. Ease of acquisition and potential for clinical application led us to assess the potential of using immortalized human keratinocytes for expression of transgenes in vivo. Human keratinocytes were immortalized with a HPV16-E6/E7 retrovirus, transduced with a lacZ retrovirus, cloned by limiting dilution, seeded onto a physiologic dermal substrate, and transplanted to athymic mice. Six weeks after transplantation, the immortalized transgene expressing keratinocytes had formed an epidermis that was indistinguishable from one formed by nonimmortalized keratinocytes; furthermore, there was no loss of expression of the lacZ gene. These observations show that methods to extend cell survival are an alternative approach to achieving stable and prolonged expression of transgenes in vivo and that HPV16-E6/ E7 immortalized keratinocytes generate an epidermis with normal morphology.

E7-transduced human breast epithelial cells show partial differentiation in three-dimensional culture.

Disruption of the retinoblastoma (RB) tumor suppressor pathway is a common and important event in breast carcinogenesis. To examine the role of the retinoblastoma protein (pRB) in this process, we created human mammary epithelial cells (HMEC) deficient for pRB by infecting primary outgrowth from breast organoids with the human papillomavirus type 16 (HPV16) E7 gene. HPV16 E7 binds to and inactivates pRB and also causes a significant down-regulation of the protein. Culturing normal HMEC in a reconstituted basement membrane (rBM) provides a correct environment and signaling cues for the formation of differentiated, acini-like structures. When cultured in this rBM, HMEC+E7 were found to respond morphologically as normal HMEC and form acinar structures. In contrast to normal HMEC, many of the cells within the HMEC+E7 structures were not growth arrested, as determined by a 5-bromo-2'-deoxyuridine incorporation assay. pRB deficiency did not affect polarization of these structures, as indicated by the normal localization of the cell-cell adhesion marker E-cadherin and the basal deposition of a collagen IV membrane. However, in HMEC+E7 acini, we were unable to detect by immunofluorescence microscopy the milk protein lactoferrin or cytokeratin 19, both markers of differentiation expressed in the normal HMEC structures. These data suggest that loss of RB in vivo would compromise differentiation, predisposing these cells to future tumor-promoting actions.

Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect.

Evidence from epidemiological studies, clinical trials, and animal experiments indicates that inhibitors of prostaglandin synthesis lower the risk of colon cancer. We tested the hypothesis that abnormal expression of prostaglandin H synthase 2 (PHS-2), which can be induced by oncogenes and tumor promoters, occurs during colon carcinogenesis by examining its level in colon tumors. Human colon cancers were found to have an increased expression of PHS-2 mRNA compared with normal colon specimens from the same patient (n = 5). In situ hybridization showed that the neoplastic colonocytes had increased expression of PHS-2 (n = 4). Additionally, five colon cancer cell lines were shown to express high levels of PHS-2 mRNA even in the absence of a known inducer of PHS-2. To study the basis for this increased gene expression, we transfected a colon cancer cell line, HCT-116, with a reporter gene containing 2.0 kb of the 5' regulatory sequence of the PHS-2 gene. Constitutive transcription of the reporter gene was observed, whereas normal control cell lines transcribed the reporter only in response to an exogenous agonist. We conclude that PHS-2 is transcribed abnormally in human colon cancers and that this may be one mechanism by which prostaglandins or related compounds that support carcinogenesis are generated.

The tetranucleotide repeat polymorphism D21S1245 demonstrates hypermutability in germline and somatic cells.

Six novel polymorphic short sequence repeats were identified and localized on the linkage map of human chromosome 21 by genotyping the CEPH reference pedigrees. One of these markers, the tetrameric (AAAG)n repeat D21S1245, was found to be hypermutable. In the DNAs from lymphoblastoid cell lines of members of the 40 CEPH families a total of 18 new alleles were detected. These new alleles, sometimes appearing in mosaic forms, arose equally in paternal and maternal DNAs, and could be equally larger or smaller than the alleles from which they were derived. The larger alleles of D21S1245 are more prone to be converted to new alleles. None of the new alleles with mosaicism were present in the corresponding genomic blood DNA, and therefore originated during or after the establishment of the lymphoblastoid cell lines; half of the new alleles without mosaicism were also found in genomic blood DNA of the appropriate CEPH individuals. The range of germline mutation rate observed in the 716 meioses examined was 0.56-1.4 x 10(-2); the range of somatic mutations observed in the 405 cell lines examined was 1.96-3.46 x 10(-2). This is one of the most hypermutable microsatellite repeat polymorphism in the human genome detected to date. D21S1245, is highly polymorphic (heterozygosity of 0.96) and maps between D21S231 and D21S198.

Physical and genetic mapping of human chromosome 3 loci containing microsatellite repeats.

One hundred and six microsatellite repeat-containing loci, including 59 CA-containing repeats from the CEPH/Genethon collection, were regionally assigned on human chromosome 3 using a somatic cell hybrid mapping panel, diving the chromosome into 14 intervals. The others were dinucleotide and tetranucleotide repeat-containing loci newly developed for human chromosome 3, of which 26 were also localized by means of genetic linkage analysis against selected CEPH microsatellites. The regional assignment of these two marker sets in a common mapping panel facilitates their integration. Incorporation of these highly polymorphic loci into the developing physical and genetic maps should provide useful information for studies of various diseases involving chromosome 3.

Demonstration of promoter activity and alternative splicing in the region 5' to exon 1 of the APC gene.

Screening of fetal brain and fetal retina complementary DNA (cDNA) libraries and exon-connection experiments using brain cDNA have identified three exons 5' to exon 1 of the adenomatous polyposis coli gene. The exons are termed (from 5'-3') 0.3, 0.1, and 0.2; exons 0.1 and 0.2 are contiguous genomically. Library screening revealed alternatively spliced cDNAs containing the following combinations of 5'-exons: 0.3 + 1 + 2, 0.3 + 2, 0.1 + 0.2 + 1 + 2, and 0.1 + 1 + 2. Exon-connection experiments also identified these four forms in mRNAs from tissues and cultured cell lines, along with two additional forms, 0.1 + 0.2 + 2 and 0.1 + 2. The multiple splice forms may lead to proteins of differing activity; for example, products derived from cDNAs without exon 1 will lack most of a heptad-repeat domain that supports formation of homodimers. No mRNA species combining 0.3 with either 0.1 or 0.2 were identified. The existence of two apparently separate 5'-ends of APC suggests the possibility of two independent promoters. The genomic sequence adjacent to exon 0.3 confers promotor activity when cloned in a chloramphenicol acetyltransferase expression vector and transfected into a colon cancer cell line.

A radiation hybrid map of the BRCA1 region.

A locus on chromosome 17q, designated "BRCA1," has been identified as a predisposition gene for breast cancer. A panel of chromosome 17-specific radiation-reduced somatic cell hybrid clones has been assembled for high-resolution mapping of chromosome 17. A series of 35 markers, known to span the BRCA1 locus, were tested against this hybrid panel by PCR assays. Statistical analysis of these data yields a BRCA1 radiation hybrid map at a density sufficient to initiate YAC cloning and pulsed-field gel electrophoretic mapping of the candidate region. In addition, many of the markers reveal genetic polymorphisms and may be tested in breast cancer families and in loss-of-heterozygosity studies of sporadic breast cancers to better define the BRCA1 gene candidate region.

Recognition sequence of a highly conserved DNA binding protein RBP-J kappa.

DNA binding specificity of the RBP-J kappa protein was extensively examined. The mouse RBP-J kappa protein was originally isolated as a nuclear protein binding to the J kappa type V(D)J recombination signal sequence which consisted of the conserved heptamer (CACTGTG) and nonamer (GGTTTTTGT) sequences separated by a 23-base pair spacer. Electrophoretic mobility shift assay using DNA probes with mutations in various parts of the J kappa recombination signal sequence showed that the RBP-J kappa protein recognized the sequence outside the recombination signal in addition to the heptamer but did not recognize the nonamer sequence and the spacer length at all. Database search identified the best naturally occurring binding motif (CACTGTGGGAACGG) for the RBP-J kappa protein in the promoter region of the m8 gene in the Enhancer of split gene cluster of Drosophila. The binding assay with a series of m8 motif mutants indicated that the protein recognized mostly the GTGGGAA sequence and also interacted weakly with ACT and CG sequences flanking this hepta-nucleotide. Oligonucleotides binding to the RBP-J kappa protein were enriched from a pool of synthetic oligonucleotides containing 20-base random sequences by the repeated electrophoretic mobility shift assay. The enriched oligomer shared a common sequence of CGTGGGAA. All these data indicate that the RBP-J kappa protein recognizes a unique core sequence of CGTGGGAA and does not bind to the V(D)J recombination signal without the flanking sequence.

A somatic cell hybrid map of human chromosome 13.

We have constructed a chromosome 13 somatic cell hybrid map using seven cell lines: PGMEA6, a hybrid containing the entire chromosome 13, and six hybrids containing various deletions of chromosome 13 (BARF7, PPF22, KBF11, KSF39, CF25, and CF27). We have mapped 80 markers that define 10 regions of chromosome 13 with respect to 10 breakpoints in the mapping panel; these regions range in size from 4 to 24 Mb, with an average size of 8 Mb. The 80 markers sublocalized on our mapping panel include 10 Alu-PCR clones, 6 of which were converted to sequence-tagged sites; 40 (CA)n repeat-containing clones, 27 of which are microsatellite PCR markers; 8 (AAAG)n repeat-containing PCR markers, 1 two-allele PCR marker, 4 genes or expressed sequences, and 17 anonymous DNA probes. This low-resolution physical map can be used as a backbone map for more refined physical mapping using radiation hybrids or yeast artificial chromosomes.