Peripheral myelin protein-22 gene maps in the duplication in chromosome 17p11.2 associated with Charcot-Marie-Tooth 1A.

Charcot-Marie-Tooth disease 1A (CMT1A) is a hereditary demyelinating peripheral neuropathy, associated with a DNA duplication on chromosome 17p11.2. A related disorder in the mouse, trembler (Tr), maps to mouse chromosome 11 which has syntenic homology to human chromosome 17p. Recently, the peripheral myelin protein-22 (pmp-22) gene was identified as the likely Tr locus. We have constructed a partial yeast artificial chromosome contig spanning the CMT1A gene region and mapped the PMP-22 gene to the duplicated region. These observations further implicate PMP-22 as a candidate gene for CMT1A, and suggest that over-expression of this gene may be one mechanism that produces the CMT1A phenotype.

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.

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.

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.

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.

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.

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.

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.

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.

Analysis of the DNA duplication 17p11.2 in Charcot-Marie-Tooth neuropathy type 1 pedigrees: additional evidence for a third autosomal CMT1 locus.

We have restudied two clinically typical Charcot-Marie-Tooth neuropathy type 1 (CMT1; also known as hereditary motor and sensory neuropathy 1) pedigrees that were previously reported to be unlinked to the regions of proximal chromosome 1q and chromosome 17p by multipoint linkage analyses. In these two pedigrees, there is no evidence for linkage to additional DNA markers that flank and span the CMT1A locus on chromosome 17p11.2, and a duplication associated with CMT1A is not present in these pedigrees. These findings confirm that the CMT1 locus in these two pedigrees does not map to chromosome 17p11.2 or 1q, and provide further evidence for the existence of a third autosomal locus for CMT1.

Trisomy 17p associated with Charcot-Marie-Tooth neuropathy type 1A phenotype: evidence for gene dosage as a mechanism in CMT1A.

Charcot-Marie-Tooth neuropathy type 1A (CMT1A) is associated with a DNA duplication on chromosome 17, band p11.2, resulting in partial trisomy for this region in CMT1A patients. The 17p11.2 duplication may lead to the CMT1A phenotype either through disruption of a gene at the duplication breakpoint junction or by trisomic dosage and overexpression of a gene within the duplication. To test the latter model, we evaluated a patient with complete translocation trisomy 17p for signs of CMT1A. In addition to the dysmorphic features seen in trisomy 17p, a neurologic examination and electrophysiologic studies detected a demyelinating neuropathy, compatible with CMT1A. A karyotype on the patient's father found a balanced translocation [t(14;17)] with breakpoints on chromosome 17 in either band p11.1 or proximal p11.2. An analysis of the patient's DNA confirmed trisomy 17p and mapped the translocation breakpoint to a region in 17p11.2, proximal to the duplication breakpoint in CMT1A. Our observations in this patient with trisomy 17p are relevant to an understanding of the genetic mechanism in CMT1A and provide strong evidence that gene dosage through segmental trisomy for 17p11.2 results in the CMT1A phenotype.

Biochemical and immunological characterization of the DNA binding protein (RBP-J kappa) to mouse J kappa recombination signal sequence.

We have investigated whether J kappa recombination signal sequence (RS) binding protein (RBP-J kappa) has any partial catalytic activities involved in the VDJ recombination reaction, such as cleavage, ligation, and bending of DNA. Murine RBP-J kappa protein purified by J kappa-RS affinity chromatography did not show DNA cleavage activities but contained a strong DNA ligase activity. To obtain a large amount of purified RBP-J kappa protein, recombinant RBP-J kappa was synthesized in Escherichia coli as a fusion protein and also in silkworm cells. Although recombinant RBP-J kappa produced in silkworm cells could bind J kappa-RS, it failed to show either ligase or DNA bending activity. Since the DNA affinity-purified RBP-J kappa has the ligase activity, the RBP-J kappa protein may form a complex with a ligase in vivo. We have raised monoclonal antibodies against the RBP-J kappa fusion protein which was synthesized in E. coli and unable to bind J kappa-RS. Using the anti-RBP-J kappa monoclonal antibody we have shown that the RBP-J kappa protein is expressed ubiquitously in mammalian tissues. The ubiquitous expression of the RBP-J kappa protein is consistent with the hypothesis that the RBP-J kappa protein may have dual function [Furukawa et al. (1991) J. Biol. Chem. 266, 23334-23340].

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.

[Changes in granulocyte elastase caused by the use of ulinastatin during anesthesia for renal transplantation].

In patients with renal failure, releases of chemical mediators having inhibitory influences on organs and circulatory system have been observed. Therefore, in long-term dialysis patients, it is necessary to consider the biocompatibilities of the equipment for dialysis. On 13 cases of renal transplantations, we measured the pre- and post-operative changes of granulocyte elastase (GE) and evaluated the effect of ulinastatin. The renal transplanted patients receiving a long-term dialysis showed higher GE values than that of normal patients even before the operation. During the anesthesia, GE increased gradually and further increased after the operation. In patients given ulinastatin during anesthesia, the increase in GE during the operation was suppressed but an increase after the operation was observed. The renal transplanted patients are easily infectible. Therefore, it is useful to measure GE pre- and post-operatively for earlier diagnosis of the postoperative infection and septicemia. The administration of ulinastatin can inhibit the increase of GE. It seems that ulinastatin increases renal blood flow and improves renal function.

[Clinical evaluation of estramustine phosphate in the treatment of patients with advanced breast cancers].

Estramustine phosphate, an anti-prostatic cancer agent, was investigated on eleven patients to evaluate the efficacy in a treatment of advanced breast cancers. The daily dose of medication was 840 mg. According to criteria of Japan Society for Cancer Therapy, none was assessed as CR, three as PR, four as NC and PD. The response rate was 27.3%. There was no differences in response rates among estrogen receptor status. A favourable response was observed in postmenopausal patients but no response in premenopausal, as well as a good response in lesions of soft tissue and lung, a poor response in lesions of liver and bone. As to toxicity of estramustine phosphate, gastrointestinal disorders such as nausea, vomiting and diarrhea were noted frequently during the treatment, and a long term administration was not able to perform in premenopausal patients because of vaginal bleeding and discharge, and pain in breast. The estramustine phosphate therapy for advanced breast cancers was regarded as one of modalities for a treatment of postmenopausal patients as a second line therapy. This is the first report in Japan discussing the efficacy of estramustine phosphate for a treatment of breast cancer.

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.

[Diagnosis of minimal breast cancer by diagnostic hormonal treatment].

After diagnostic hormonal treatment of 129 patients, 11 (8.5%) were revealed to have breast cancer. The diagnostic accuracy in the case of a lesion of less than 1.0 cm in diameter was 11.8% for palpation, 18.2% for mammography, 33.3% for ultrasonography; the accuracy of this method is 66.6%. Minimal breast cancer was present in eight out of nine cases. Thus, diagnostic hormonal treatment was found to be useful in the diagnosis of minimal breast cancer that coexists with mastopathy.

Purification and characterization of a protein that binds to the recombination signal sequence of the immunoglobulin J kappa segment.

A protein that binds to the recombination signal sequence (RS) of the immunoglobulin J kappa segment was purified almost to homogeneity from the nuclear extract of a murine pre-B cell line 38B9. A similar binding protein was found in lymphoid cell lines but not in non-lymphoid cell lines. The binding activity was associated with a polypeptide with a molecular weight of 60,000. DNase I footprinting analysis demonstrated that this binding protein interacted with the heptamer and several 3' bases close to the heptamer. The Kd value of the J kappa RS binding protein to the J kappa RS was 1 nM. One base substitution in the heptamer of the J kappa RS greatly reduced the affinity of the J kappa RS binding protein. The high specificity of the binding site of the J kappa RS binding protein suggests that this protein may be involved in V-J recombination.