Hideaki Sawai

Statin treatment rescues FGFR3 skeletal dysplasia phenotypes

Gain-of-function mutations in the fibroblast growth factor receptor 3 gene (FGFR3) result in skeletal dysplasias, such as thanatophoric dysplasia and achondroplasia (ACH). The lack of disease models using human cells has hampered the identification of a clinically effective treatment for these diseases. Here we show that statin treatment can rescue patient-specific induced pluripotent stem cell (iPSC) models and a mouse model of FGFR3 skeletal dysplasia. We converted fibroblasts from thanatophoric dysplasia type I (TD1) and ACH patients into iPSCs. The chondrogenic differentiation of TD1 iPSCs and ACH iPSCs resulted in the formation of degraded cartilage. We found that statins could correct the degraded cartilage in both chondrogenically differentiated TD1 and ACH iPSCs. Treatment of ACH model mice with statin led to a significant recovery of bone growth. These results suggest that statins could represent a medical treatment for infants and children with TD1 and ACH.

First-line intraperitoneal carboplatin-based chemotherapy for 165 patients with epithelial ovarian carcinoma: results of long-term follow-up.

OBJECTIVE Currently, no long-term follow-up data are available on intraperitoneal (IP) carboplatin-based chemotherapy for ovarian carcinoma. In this study we evaluated retrospectively the survival and recurrence of a retrospective cohort of patients with epithelial ovarian cancer treated with first-line IP carboplatin-based therapy. METHODS Records were reviewed of 174 patients with epithelial ovarian cancer who received IP carboplatin-based therapy between 1990 and 2000. All patients underwent surgical staging, and implantable port systems were placed regardless of residual tumor size. The pathological slides were submitted and reviewed, and then nine patients were excluded because of borderline malignancies (n = 8), and wrong histology (n = 1). Therefore, the records of 165 patients were analyzed for survival. Tumor grade was determined by the Universal grading system. Statistical analysis included tests for association between potential prognostic factors, and between prognostic factors and survival. Survival probabilities were estimated by Kaplan-Meier methods, and prognostic factors for survival were evaluated by a Cox regression model. RESULTS The mean age of the patients was 53.7 years (range 21-83). The median follow-up was 41 months. The distribution by stage and histology was as follows: high risk (grade 2/3, clear cell, capsule rupture) stage I, 54; II, 21; III, 72; IV, 18; and serous, 75; clear cell, 30; mucinous, 27; endometrioid, 20; others, 13. The chemotherapy regimen was either carboplatin alone (n = 22) or in combination with cyclophosphamide (n = 116) or paclitaxel (n = 27). Catheter-related complications occurred in 16 (9.7%) cases. The chemotherapeutic response in 54 patients with measurable disease was 66.4%. The 5-year survival was 94.4% for stage I, and 87.9% for stage II. The median survival for optimal and suboptimal stage III/IV patients was 51 months and 34 months, respectively. The median survival of patients with stage III/IV disease was 51 months with carboplatin doses of 400 mg/m(2) or more, but it was only 25 months with carboplatin doses smaller than 400 mg/m(2). Poor prognostic factors, determined by Cox regression multivariate analysis, were clear cell histology (P < 0.001) and a carboplatin dose smaller than 400 mg/m(2) (P = 0.002). CONCLUSIONS Survival of patients who underwent carboplatin-based IP chemotherapy was excellent when the dose of carboplatin was higher than 400 mg/m(2). A prospective evaluation of IP carboplatin therapy with modern combination is warranted.

Direct Production of the Fab Fragment Derived From the Sperm Immobilizing Antibody Using Polymerase Chain Reaction and cDNA Expression Vectors

PROBLEM: Sperm immobilizing antibodies cause infertility mainly through complement dependent sperm immobilization. To analyze any effect of sperm immobilizing antibody on fertilization, we had already established cell lines that secrete IgM monoclonal antibody (MAb H6‐3C4) and IgG monoclonal antibody (MAb EnBCMGS). The latter was a class‐switched recombinant IgG antibody that shares the same variable region as MAb H6‐3C4. The biological effects of the IgG antibody were also reported previously to eliminate sperm immobilizing or sperm agglutinating activities. However, the method of chemical digestion of IgG had some disadvantage to prepare the purified Fab fragment stably and in large quantities. This time we report a unique method to obtain the recombinant Fab fragments (Fab EnBCMGS) using polymerase chain reaction (PCR) and cDNA expression vectors.

Prenatal diagnosis of thanatophoric dysplasia by mutational analysis of the fibroblast growth factor receptor 3 gene and a proposed correction of previously published PCR results

Thanatophoric dysplasia (TD) is the most frequent form of neonatal lethal skeletal dysplasia. Recently, mutations in the fibroblast growth factor receptor 3 (FGFR3) gene that cause two subtypes of this disorder, type I (TDI) and type II (TDII), have been identified. This discovery has now made it possible to make a definite diagnosis of TD by molecular methods. To date, prenatal diagnosis of TD has been accomplished by ultrasonography in the second trimester. However, it is not always possible to distinguish TD fetuses in utero from the other osteochondrodysplasias by ultrasonography or radiography. We report on the prenatal diagnosis of a TD fetus, showing severe shortness of limbs and polyhydramnios, by identification of a mutation in the FGFR3 gene. Genomic DNA was isolated from the amniotic fluid and then subjected to PCR amplification. The common TDI mutation, C→T transition at nucleotide 742 in the FGFR3 gene, was identified using restriction enzyme analysis. This information was critical in obstetric management decisions later in pregnancy. However, although the mutation responsible for TDI was detected previously, we noticed some inconsistencies in the published PCR results and have proposed a correction. Copyright

Prevalence of c.1559delT in ALPL, a common mutation resulting in the perinatal (lethal) form of hypophosphatasia in Japanese and effects of the mutation on heterozygous carriers

Hypophosphatasia (HPP) is an inherited disorder caused by mutations in ALPL that encodes an isozyme of alkaline phosphatase (ALP), TNSALP. One of the most frequent ALPL mutations is c.1559delT, which causes the most severe HPP, the perinatal (lethal) form (pl-HPP). c.1559delT has been found only in Japanese and its prevalence is suspected to be high; however, the allele frequency of c.1559delT in Japanese remains unknown. We designed a screening system for the mutation based on high-resolution melting curve analysis, and examined the frequency of c.1559delT. We found that the c.1559delT carrier frequency is 1/480 (95% confidence interval, 1/1562–1/284). This indicates that ∼1 in 900 000 individuals to have pl-HPP caused by a homozygous c.1559delT mutation. In our analysis, the majority of c.1559delT carriers had normal values of HPP biochemical markers, such as serum ALP and urine phosphoethanolamine. Our results indicate that the only way to reliably detect whether individuals are pl-HPP carriers is to perform the ALPL mutation analysis.

Low prevalence of genetic prenatal diagnosis in Japan.

Aiko Sasaki1, Hideaki Sawai2, Hideaki Masuzaki3, Fumiki Hirahara4 and Haruhiko Sago1* 1Department of Maternal-Fetal and Neonatal Medicine, National Center for Child Health and Development, Tokyo, Japan 2Department of Obstetrics and Gynecology, Hyogo College of Medicine, Nishinomiya, Japan 3Department of Obstetrics and Gynecology, Nagasaki University, Nagasaki, Japan 4Department of Obstetrics and Gynecology, Yokohama City University, Yokohama, Japan

Live births from isolated primary/early secondary follicles following a multistep culture without organ culture in mice

Although the ovary has a large store of germ cells, most of them do not reach mature stages. If a culture system could be developed from early growing follicles to mature oocytes, it would be useful for biological research as well as for reproductive medicine. This study was conducted to establish a multistep culture system from isolated early growing follicles to mature oocytes using a mouse model. Early growing follicles with diameters of 60-95 μm corresponding to primary and early secondary follicles were isolated from 6-day-old mice and classified into three groups by diameter. These follicles contained oocytes with diameters of ~45 μm and one or a few layered granulosa cells on the basal lamina. Embedding in collagen gel was followed by first-step culture. After 9-day culture, the growing follicles were transferred onto collagen-coated membrane in the second step. At day 17 of the culture series, the oocyte-granulosa cell complexes were subjected to in vitro maturation. Around 90% of the oocytes in follicles surviving at day 17 resumed second meiosis (metaphase II oocytes: 49.0-58.7%), regardless of the size when the follicle culture started. To assess developmental competence to live birth, the eggs were used for IVF and implantation in pseudopregnant mice. We successfully obtained two live offspring that produced next generations after puberty. We thus conclude that the culture system reported here was able to induce the growth of small follicles and the resultant mature oocytes were able to develop into normal mice.

Fetal cell-free DNA fraction in maternal plasma is affected by fetal trisomy.

The purpose of this noninvasive prenatal testing (NIPT) study was to compare the fetal fraction of singleton gestations by gestational age, maternal characteristics and chromosome-specific aneuploidies as indicated by z-scores. This study was a multicenter prospective cohort study. Test data were collected from women who underwent NIPT by the massively parallel sequencing method. We used sequencing-based fetal fraction calculations in which we estimated fetal DNA fraction by simply counting the number of reads aligned within specific autosomal regions and applying a weighting scheme derived from a multivariate model. Relationships between fetal fractions and gestational age, maternal weight and height, and z-scores for chromosomes 21, 18 and 13 were assessed. A total of 7740 pregnant women enrolled in the study, of which 6993 met the study criteria. As expected, fetal fraction was inversely correlated with maternal weight (P<0.001). The median fetal fraction of samples with euploid result (n=6850) and trisomy 21 (n=70) were 13.7% and 13.6%, respectively. In contrast, the median fetal fraction values for samples with trisomies 18 (n=35) and 13 (n=9) were 11.0% and 8.0%, respectively. The fetal fraction of samples with trisomy 21 NIPT result is comparable to that of samples with euploid result. However, the fetal fractions of samples with trisomies 13 and 18 are significantly lower compared with that of euploid result. We conclude that it may make detecting these two trisomies more challenging.

Molecular analysis of the Y chromosome AZFc region in Japanese infertile males with spermatogenic defects

Cytogenetic and molecular studies of azoospermic and oligozoospermic males have suggested the presence of azoospermia factors (AZF) in the human Y chromosome. Deletion in three Y chromosomal regions--AZFa, AZFb and AZFc--has been reported to disrupt spermatogenesis and cause infertility. Several candidate genes responsible for spermatogenesis have been identified in these regions and some of them are thought to be functional in human spermatogenesis. Here we report on clinical and molecular studies of Y chromosome micro-deletions in Japanese. In these studies the data from 157 infertile Japanese men with azoospermia and oligozoospermia was analyzed and divided into 5 categories based on spermatozoa count. Sixteen sets of primers were used for polymerase chain reaction (PCR) to amplify sequence tagged site markers. One common deletion in the AZFc region was identified in infertile men. On the other hand, no deletions around the AZFc region were identified in fertile men. Japanese infertile men in our study had a common deletion in the AZFc region of the Y chromosome. A genomic clone was obtained by PCR screening of the P1 phage artificial chromosome (PAC) library. This clone was analyzed by Southern blotting using a PCR amplified probe of sY240. Our analysis of the genomic sequence of the clone suggests that this locus may contain specific genes for spermatogenesis.

Prenatal diagnosis of short-rib polydactyly syndrome type 3 (Verma-Naumoff type) by three-dimensional helical computed tomography

We present a case of short‐rib polydactyly syndrome (SRPs) type 3 in which accurate prenatal diagnosis was feasible using both ultrasonography and 3D‐CT. SRP encompass a heterogeneous group of lethal skeletal dysplasias. However, the phenotypes overlap with those of nonlethal skeletal dysplasias (i.e. Ellis‐van Creveld syndrome and Jeune syndrome). As accurate prenatal diagnosis of SRP is helpful for parents, we used 3D‐CT in the early third trimester to examine a fetus suggested to have phenotypes of ‘short‐rib dysplasia group’ on ultrasonography. 3D‐CT showed mild modification of the vertebral bodies, small ilia with horizontal acetabula and triangular partial ossification defects, and subtle metaphyseal irregularities of the femora. These CT findings and an extensive literature search regarding the phenotypes of various diseases categorized as short‐rib dysplasia group led to a correct prenatal diagnosis of SRP type 3. This case exemplified the usefulness of 3D‐CT for the precise prenatal diagnosis of skeletal dysplasias.