Kazuo Sengoku

Promoter Hypermethylation Contributes to Frequent Inactivation of a Putative Conditional Tumor Suppressor Gene Connective Tissue Growth Factor in Ovarian Cancer

Connective tissue growth factor (CTGF) is a secreted protein belonging to the CCN family, members of which are implicated in various biological processes. We identified a homozygous loss of CTGF (6q23.2) in the course of screening a panel of ovarian cancer cell lines for genomic copy number aberrations using in-house array-based comparative genomic hybridization. CTGF mRNA expression was observed in normal ovarian tissue and immortalized ovarian epithelial cells but was reduced in many ovarian cancer cell lines without its homozygous deletion (12 of 23 lines) and restored after treatment with 5-aza 2-deoxycytidine. The methylation status around the CTGF CpG island correlated inversely with the expression, and a putative target region for methylation showed promoter activity. CTGF methylation was frequently observed in primary ovarian cancer tissues (39 of 66, 59%) and inversely correlated with CTGF mRNA expression. In an immunohistochemical analysis of primary ovarian cancers, CTGF protein expression was frequently reduced (84 of 103 cases, 82%). Ovarian cancer tended to lack CTGF expression more frequently in the earlier stages (stages I and II) than the advanced stages (stages III and IV). CTGF protein was also differentially expressed among histologic subtypes. Exogenous restoration of CTGF expression or treatment with recombinant CTGF inhibited the growth of ovarian cancer cells lacking its expression, whereas knockdown of endogenous CTGF accelerated growth of ovarian cancer cells with expression of this gene. These results suggest that epigenetic silencing by hypermethylation of the CTGF promoter leads to a loss of CTGF function, which may be a factor in the carcinogenesis of ovarian cancer in a stage-dependent and/or histologic subtype-dependent manner.

Frequent Inactivation of a Putative Tumor Suppressor, Angiopoietin-Like Protein 2, in Ovarian Cancer

Angiopoietin-like protein 2 (ANGPTL2) is a secreted protein belonging to the angiopoietin family, the members of which are implicated in various biological processes, although its receptor remains unknown. We identified a homozygous loss of ANGPTL2 (9q33.3) in the course of screening a panel of ovarian cancer (OC) cell lines for genomic copy-number aberrations using in-house array-based comparative genomic hybridization. ANGPTL2 mRNA expression was observed in normal ovarian tissue and immortalized normal ovarian epithelial cells, but was reduced in some OC lines without its homozygous deletion (18 of 23 lines) and restored after treatment with 5-aza 2-deoxycytidine. The methylation status of sequences around the ANGPTL2 CpG-island with clear promoter activity inversely correlated with expression. ANGPTL2 methylation was frequently observed in primary OC tissues as well. In an immunohistochemical analysis of primary OCs, ANGPTL2 expression was frequently reduced (51 of 100 cases), and inversely correlated with methylation status. Patients with OC showing reduced ANGPTL2 immunoreactivity had significantly worse survival in the earlier stages (stages I and II), but better survival in advanced stages (stages III and IV). The restoration of ANGPTL2 expression or treatment with conditioned medium containing ANGPTL2 inhibited the growth of OC cells originally lacking the expression of this gene, whereas the knockdown of endogenous ANGPTL2 accelerated the growth of OC cells with the expression of ANGPTL2. These results suggest that, at least partly, epigenetic silencing by hypermethylation of the ANGPTL2 promoter leads to a loss of ANGPTL2 function, which may be a factor in the carcinogenesis of OC in a stage-dependent manner.

Male infertility and its genetic causes.

Infertility is a serious social problem in advanced nations, with male factor infertility accounting for approximately half of all cases of infertility. Here, we aim to discuss our laboratory results in the context of recent literature on critical genes residing on the Y chromosome or autosomes that play important roles in human spermatogenesis.

Single-nucleotide polymorphisms in the SEPTIN12 gene may be a genetic risk factor for Japanese patients with Sertoli cell-only syndrome.

Genetic mechanisms have been implicated as a cause of some cases of male infertility. Recently, 10 novel genes involved in human spermatogenesis, including human SEPTIN12, were identified by expression microarray analysis of human testicular tissue. Septin12 is a member of the septin family of conserved cytoskeletal GTPases that form heteropolymeric filamentous structures in interphase cells. It is expressed specifically in the testis. Therefore, we hypothesized that mutation or polymorphisms of SEPTIN12 participate in male infertility, especially Sertoli cell-only syndrome (SCOS). To investigate whether SEPTIN12 gene defects are associated with azoospermia caused by SCOS, mutational analysis was performed in 100 Japanese patients by direct sequencing of coding regions. Statistical analysis was performed in patients with SCOS and in 140 healthy control men. No mutations were found in SEPTIN12 ; however, 8 coding single-nucleotide polymorphisms (SNP1-SNP8) could be detected in the patients with SCOS. The genotype and allele frequencies in SNP3, SNP4, and SNP6 were notably higher in the SCOS group than in the control group (P < .001). These results suggest that SEPTIN12 might play a critical role in human spermatogenesis.

Relaxation of insulin-like growth factor-II gene imprinting in human gynecologic tumors

To test for the existence of genomic imprinting in human gynecologic tumors, we analyzed the allelic expression of human insulin-growth factor-II (IGF-II) genes. Genomic imprinting is the parental allele-specific expressions of genes, and recently imprinting of IGF-II gene has demonstrated that parental IGF-II was monoallelically expressed. To study whether IGF-II gene imprinting occurs in human gynecologic tumors, we examined allele-specific expression using an ApaI polymorphism in the 3 untranslated region of IGF-II gene exon 9. We used 19 gynecologic tumor cell lines, and 66 human gynecologic tumors. Four of 19 cell lines (21%) were informative, and three of these four cell lines (75%) revealed loss of imprinting (LOI). For gynecologic tumors, 24 of 66 were informative (36%), and 5 of the 24 (21%) had LOI. We have reported here that the IGF-II gene is expressed biallelically in some gynecologic tumors. We suggest that LOI of the IGF-II gene is involved in the development of some gynecologic tumors.

Clinicopathologic risk factors for recurrence of ovarian endometrioma following laparoscopic cystectomy

To identify epidemiologic risk factors and investigate whether the characteristics of removed ovarian tissue during surgery influence the recurrence of endometriomas.

First-trimester serum folate levels and subsequent risk of abortion and preterm birth among Japanese women with singleton pregnancies

ObjectivesTo determine whether a low serum folate level during the first trimester predicts subsequent late abortion, preterm birth, or fetal growth restriction (FGR).Study designA prospective cohort study involving 5,075 women whose serum folate levels were measured during the first trimester. The participants were informed of their serum folate levels.ResultsThe pregnancy duration, birthweight, rate of late abortion/preterm birth, and the rate of FGR did not differ significantly among the four groups classified according to folate status. The mean serum folate levels did not differ among quartiles classified according to the gestational week at the time of delivery. Nineteen of the 20 women with folate deficiency gave birth at term to infants with a birthweight of 3.132xa0±xa0321xa0g; only one infant had FGR.ConclusionLow serum folate levels during the first trimester were not associated with the risk of late abortion, preterm birth, or FGR.

Single nucleotide polymorphism in the UBR2 gene may be a genetic risk factor for Japanese patients with azoospermia by meiotic arrest

PurposeTo investigate the association between the UBR2 gene and the risk of azoospermia caused by meiotic arrest.MethodsMutational analysis of the UBR2 gene was performed using DNA from 30 patients with azoospermia by meiotic arrest to 80 normal controls.ResultsThe genotypic and allelic frequencies of c.1,066A>T variant were significantly higher in patient than control groups (pu2009<u20090.001).ConclusionThe c.1,066A>T variant in the UBR2 gene is associated with increased susceptibility to azoospermia caused by meiotic arrest.

A PLK4 mutation causing azoospermia in a man with Sertoli cell-only syndrome

About 15% of couples wishing to have children are infertile; approximately half these cases involve a male factor. Polo‐like kinase 4 (PLK‐4) is a member of the polo protein family and a key regulator of centriole duplication. Male mice with a point mutation in the Plk4 gene show azoospermia associated with germ cell loss. Mutational analysis of 81 patients with azoospermia and Sertoli cell‐only syndrome (SCOS) identified one man with a heterozygous 13‐bp deletion in the Ser/Thr kinase domain of PLK4. Division of centrioles occurred in wild‐type PLK4‐transfected cells, but was hampered in PLK‐4‐mutant transfectants, which also showed abnormal nuclei. Thus, this PLK4 mutation might be a cause of human SCOS and nonobstructive azoospermia.

Human male infertility and its genetic causes

Infertility affects about 15% of couples who wish to have children and half of these cases are associated with male factors. Genetic causes of azoospermia include chromosomal abnormalities, Y chromosome microdeletions, and specific mutations/deletions of several Y chromosome genes. Many researchers have analyzed genes in the AZF region on the Y chromosome; however, in 2003 the SYCP3 gene on chromosome 12 (12q23) was identified as causing azoospermia by meiotic arrest through a point mutation.