Kazuhiko Nakabayashi

Absence of a Paternally Inherited FOXP2 Gene in Developmental Verbal Dyspraxia

Mutations in FOXP2 cause developmental verbal dyspraxia (DVD), but only a few cases have been described. We characterize 13 patients with DVD--5 with hemizygous paternal deletions spanning the FOXP2 gene, 1 with a translocation interrupting FOXP2, and the remaining 7 with maternal uniparental disomy of chromosome 7 (UPD7), who were also given a diagnosis of Silver-Russell Syndrome (SRS). Of these individuals with DVD, all 12 for whom parental DNA was available showed absence of a paternal copy of FOXP2. Five other individuals with deletions of paternally inherited FOXP2 but with incomplete clinical information or phenotypes too complex to properly assess are also described. Four of the patients with DVD also meet criteria for autism spectrum disorder. Individuals with paternal UPD7 or with partial maternal UPD7 or deletion starting downstream of FOXP2 do not have DVD. Using quantitative real-time polymerase chain reaction, we show the maternally inherited FOXP2 to be comparatively underexpressed. Our results indicate that absence of paternal FOXP2 is the cause of DVD in patients with SRS with maternal UPD7. The data also point to a role for differential parent-of-origin expression of FOXP2 in human speech development.

Molecular and genomic studies of IMMP2L and mutation screening in autism and Tourette syndrome

We recently reported the disruption of the inner mitochondrial membrane peptidase 2-like (IMMP2L) gene by a chromosomal breakpoint in a patient with Gilles de la Tourette syndrome (GTS). In the present study we sought to identify genetic variation in IMMP2L, which, through alteration of protein function or level of expression might contribute to the manifestation of GTS. We screened 39 GTS patients, and, due to the localization of IMMP2L in the critical region for the autistic disorder (AD) locus on chromosome 7q (AUTS1), 95 multiplex AD families; however, no coding mutations were found in either GTS or AD patients. In addition, no parental-specific expression of IMMP2L was detected in somatic cell hybrids containing human chromosome 7 and human cell lines carrying a maternal uniparental disomy for chromosome 7 (mUPD7). Despite the fact that no deleterious mutations in IMMPL2 (other than the inverted duplication identified previously) were identified in either GTS or AD, this gene cannot be excluded as a possible rare cause of either disorder.

ZFAT expression in B and T lymphocytes and identification of ZFAT-regulated genes.

The human ZFAT gene encodes a 1243-amino-acid protein containing one AT hook and 18 C2H2 zinc finger domains, which are highly conserved among ZFAT orthologues from fish to mammalian species. Consistent with the presence of multiple predicted nuclear localization signals, endogenous ZFAT protein was found to be localized to the nucleus. In the mouse tissues examined by Western blotting, ZFAT was found to be expressed in thymus, spleen, and lymph nodes, but not in other tissues, including bone marrow. Furthermore, ZFAT protein was found to be up-regulated during the transition from CD4(-)CD8(-) to CD4(+)CD8(+) thymocytes and to be expressed only in B and T lymphocytes in peripheral lymphoid tissues. Expression array analyses demonstrated that genes that are down-regulated upon ZFAT overexpression in mouse Ba/F3 cells are significantly enriched for those functionally related to immune responses. These results suggest that ZFAT functions as a critical transcriptional regulator in B and T lymphocytes.

Heterogeneity of the 7q36 Breakpoints in the t(7;12) Involving ETV6 in Infant Leukemia

The t(7;12)(q36;p13) is a recurrent chromosome abnormality in infant leukemia. In these cases, the involvement of ETV6, with disruption of the gene consistently at its 5′ end, has been reported by several groups. A fusion transcript between ETV6 and HLXB9 has been detected in some, but not all, reported cases of t(7;12). We report here a study based on fluorescence in situ hybridization (FISH) mapping of the translocation breakpoints in seven patients and detailed molecular studies using Southern blotting on two of these patients. The FISH studies have shown a cluster of breakpoints within a cosmid contig proximal to the HLXB9 gene. Southern blotting analysis enabled us to define two distinct breakpoints within the area covered by the cosmid contig in two patients. The analysis of an unusual case of t(7;12)(q22;p13) [full karyotype: 46,XX,der(7)t(7;12)(q22;p13)del(7)(q22q36)] also revealed a break in 7q36, although in a region proximal to the overlapping cosmids. 5′ RACE PCR in one patient has shown a rearrangement involving the ETV6 allele not involved in the t(7;12), suggesting that no functional ETV6 allele might be present in this case. These data show some heterogeneity in the distribution of breakpoints in 7q36, indicating that the generation of a fusion gene might not be the mechanism responsible for leukemogenesis in the t(7;12), at least in some cases.

Analysis of KRAP expression and localization, and genes regulated by KRAP in a human colon cancer cell line

AbstractWe previously identified the human KRAP (Ki-ras-induced actin-interacting protein) gene from the cDNA library of human colon cancer HCT116 cells as one of the genes whose expression levels were up-regulated by activated Ki-ras. Although the KRAP gene is structurally conserved from fish to mammalian species, the expression pattern and function of KRAP still remain to be elucidated. Here, we have generated a specific polyclonal antibody for KRAP and characterized the histological expression of KRAP in mouse tissues. KRAP was ubiquitously expressed in mouse tissues, with high levels in pancreas, liver, and brown adipose tissues, and KRAP was co-localized with filamentous actin along the apical membranes in both pancreas and liver tissues. A subfractionation study revealed that KRAP is a cytoplasmic protein and that the majority is associated with the cytoskeleton. Furthermore, microarray gene expression profile by inhibiting KRAP expression in HCT116 cells showed that several receptors and signal molecules frequently deregulated in cancers were differentially expressed in the KRAP-knockdown cells. All of these results suggested that KRAP might be a cytoskeleton-associated protein involving the structural integrity and/or signal transductions in human cancers.

Functional and chromosomal clustering of genes responsive to 5-bromodeoxyuridine in human cells

5-Bromodeoxyuridine immediately and dramatically induces senescence-associated genes in human cells. We examined changes in gene expression in HeLa cells using cDNA microarrays containing ca. 39,000 human genes or ESTs. Addition of 5-bromodeoxyuridine for 4 days changed expression of 2.6% of them twice more (1.5%) or less (1.1%) than control levels. We functionally categorized 191 genes that showed greater than four times increase or decrease, and found that they have a various function. These genes were assigned to various human chromosomes, and half of them seemed to cluster at a few regions on individual chromosomes. These results suggest that multiple genes collectively act to induce cellular senescence and chromatin structure has a role in expression of the genes.

Expression of RAB27B is up-regulated in senescent human cells

Immortal SVts8 cells that express thermolabile SV40 T antigen exhibit a senescence-like phenomenon upon inactivation of the T antigen. By using a cDNA subtractive hybridization technique, RAB27B, a member of the RAB GTPase family, was found to be up-regulated in senescent SVts8 cells. The up-regulation of RAB27B depends on the p53 gene. Enhanced expression was also observed in replicative senescence in normal human fibroblasts.