Mutations in genes encoding the glycine cleavage system predispose to neural tube defects in mice and humans.

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt(-/-) mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.

Prenatal molecular diagnosis of a severe type of L1 syndrome (X-linked hydrocephalus).

OBJECT: The aim of this study was to evaluate the feasibility of prenatal L1CAM gene testing for X-linked hydrocephalus (XLH). METHODS: In a nationwide study conducted in Japan between 1999 and 2009, the authors identified 51 different L1CAM gene mutations in 56 families with XLH. Of these 56 families, 9 obligate carriers requested prenatal gene mutation analysis for the fetal L1CAM gene in 14 pregnancies. RESULTS: In 2004, new clinical guidelines for genetic testing were established by 10 Japanese genetic medicine-related societies. These guidelines stated that the genetic testing of carriers should be done only with their consent and with genetic counseling. Therefore, because females are carriers, since 2004, L1CAM gene analysis has not been performed for female fetuses. The authors report on 7 fetal genetic analyses that were performed at the request of families carrying L1CAM mutations, involving 3 female (prior to 2004) and 4 male fetuses. Of the 7 fetuses, 3 (1 male and 2 female) carried L1CAM mutations. Of these 3, 1 pregnancy (the male fetus) was terminated; in the other cases, the pregnancies continued, and 3 female and 3 male babies without the XLH phenotype were born. CONCLUSIONS: Prenatal L1CAM gene testing combined with genetic counseling was beneficial for families carrying L1CAM mutations.

Searching for genes for cleft lip and/or palate based on breakpoint analysis of a balanced translocation t(9;17)(q32;q12).

OBJECTIVE: Identification of the breakpoints of disease-associated chromosome rearrangements can provide informative clues to a positional cloning approach for genes responsible for inherited diseases. Recently, we found a three-generation Japanese family segregating balanced chromosome translocation t(9;17)(q32;q12). One of the subjects had cleft lip and palate. We examined whether regions near the breakpoint could be associated with cleft lip and/or palate. METHODS: We determined the breakpoints involved in the translocation by fluorescence in situ hybridization analysis and subsequent long-range polymerase chain reaction. In order to study the role of these disrupted regions in nonsyndromic cleft lip and/or palate, we performed mutation analysis and a haplotype-based transmission disequilibrium test using tagging single-nucleotide polymorphisms in the flanking regions of the breakpoints in white and Filipino nonsyndromic cleft lip and/or palate populations. RESULTS: Sequence analysis demonstrated that two genes, SLC31A1 (solute carrier family 31 member 1) on chromosome 9 and CCL2 (chemokine ligand 2) on chromosome 17, were rearranged with the breaks occurring within their introns. It is interesting that SLC31A1 lies closed to BSPRY (B-box and SPRY domain), which is a candidate for involvement with cleft lip and/or palate. Some of the variants in BSPRY and CCL2 showed significant p values in the cleft lip and/or palate population compared with the control population. There was also statistically significant evidence of transmission distortion for haplotypes on both chromosomes 9 and 17. CONCLUSIONS: The data support previous reports that genes on chromosomal regions of 9q and 17q play an important role in facial development.

Establishment of a polymerase chain reaction method for detection of Escherichia coli in amniotic fluid in patients with chorioamnionitis.

OBJECTIVE: We investigated whether Escherichia coli could be detected by E. coli of reference (ECOR) grouping and virulence factors (VFs) in amniotic fluid using polymerase chain reaction (PCR). METHOD: From 18 patients with clinical symptoms, such as cyclic uterine contraction, genital bleeding and cervical ripening, who subsequently developed abortion or labor before term, and from 40 normal pregnant women undergoing diagnostic amniocentesis, amniotic fluid was obtained. All samples were negative for standard culture. Six patients with symptoms were classified into the ECOR group, and with VFs, E. coli was detected in 6 patients. Thus, 4 patients were positive for both tests. CONCLUSION: We could establish a detection method for E. coli in amniotic fluid using both ECOR grouping and VFs with PCR.

Sibling risk of pervasive developmental disorder estimated by means of an epidemiologic survey in Nagoya, Japan.

Broad-spectrum autism, referred to as pervasive developmental disorder (PDD), may be associated with genetic factors. We examined 241 siblings in 269 Japanese families with affected children. The sibling incidence of PDD was 10.0% whereas the prevalence of PDD in the general population in the same geographic region was 2.1%. Both of these rates are higher than those reported previously, probably because of the expanded clinical criteria applied. The prevalence in males of the general population was 3.3% and that in females was 0.82%. The sibling incidences were 7.7 and 20.0% for families in which the probands were male and female, respectively. Because the reversed sex ratios correspond to the general rule for a multifactorial threshold model, we suggest that most PDD cases result from the cumulative effects of multiple factors (mostly genetic). The sibling incidences were 0 and 10.9% for families in which the proband had low and normal birth-weight, respectively, suggesting the risk is lower in families with low-birth-weight probands.

Evaluation levels of cytokines in amniotic fluid of women with intrauterine infection in the early second trimester.

Amniotic fluid was obtained from 180 patients by amniocentesis at 16-22 weeks of gestation and assayed for the levels of interleukin (IL)-6, IL-8, leukocyte elastase (LE), and glucose. Ten of cases had clinical symptoms, such as uterine contraction, genital bleeding, and cervical ripening, and the other 170 were assessed for fetal chromosomal features. Four of the ten cases with uterine contraction developed abortion, while 10 of those screened had findings of fetal chromosomal anomalies, and 7 cases then underwent induced abortion artificially. In the cases of abortion, levels of IL-6, IL-8 and LE were higher than in the samples from the 160 pregnant women without clinical symptoms and a normal karyotype, while glucose in amniotic fluid was lower. Of 6 cases with clinical symptoms, but not developing abortion, 4 developed preterm labor, and in these IL-6 and IL-8 also were significantly elevated, with LE being slight high compared to normal. The results suggest that IL-6, IL-8, LE, and glucose in amniotic fluid at early second trimester can be used as markers of severe infection in the uterus, and with the first two being particularly sensitive.

Elevated aldosterone in amniotic fluid and maternal blood has diagnostic potential in pregnancies complicated with a fetus of Bartter syndrome.

Pregnancies with fetuses affected with the Bartter syndrome, an autosomal recessive disorder of hyperreninism and hyperaldosteronism, are complicated by early onset of polyhydramnios which results in preterm deliveries. We have assessed biochemical changes in amniotic fluid and the mother's blood with a view to early diagnosis. Aldosterone levels of both amniotic fluid and the mother's blood were found to be increased at 27 weeks of gestation, while electrolyte levels did not differ significantly from those reported earlier for controls. After birth the baby suffered from polyuria with hyponatremia, hypomagnesemia and hypercalciuria which could be controlled by treatment with sodium chloride and magnesium. Elevated aldosterone thus might be a useful marker for early diagnostic purposes.

Paternal uniparental disomy of chromosome 14 and unique exchange of chromosome 7 in cases of spontaneous abortion.

To investigate the involvement of uniparental disomies (UPDs) in spontaneous abortion, the polymorphic patterns of microsatellites on each chromosome were analyzed in 164 cases of abortion. Eighty-three of the 164 cases had chromosomal abnormalities. In 79 of the remaining 81 cases with normal karyotypes, the microsatellite analysis revealed that biparental patterns were present in the informative microsatellites in all chromosomes. In one of the remaining two cases, however, the polymorphic patterns of chromosome 14 appeared to be both of paternal origin. The patterns of the distal of the long arm were homozygous, and those of the remaining region were heterozygous. That is, this fetus had paternal UPD 14, originating from meiosis I nondisjunction. In the other case, the polymorphic patterns of the distal one third of the long arm of chromosome 7 were uniparental (maternal) in origin whereas those of the remaining region of this chromosome were biparental. These findings thus suggested that this chromosome might have originated from chromatid exchange between the long arms of paternal and maternal chromosome 7 at the first mitotic division. Microsatellite analysis, however, produced no evidence of duplication or deletion of any segments. The findings also suggest the possibility that some UPDs may cause spontaneous abortion.

Parental origin and cell stage of non-disjunction of double trisomy in spontaneous abortion.

Using polymorphic analysis of microsatellites, we investigated the parental origin and mechanism of double trisomies seen in cases of spontaneous abortion. We obtained chorionic villi from spontaneous abortions, and peripheral blood from females who experienced abortion and their spouses. Chromosomal analysis of 170 cases revealed four cases with double trisomy. The karyotypes of these cases are 48,XX,+16,+22, 48,XXY,+18, 48,XX,+15,+21 and 48,XX,+2,+5. In the present study, the incidence of double trisomy was 2.4% of spontaneous abortions. Polymorphic analysis of microsatellites indicated that extra chromosomes were all of maternal origin in the four cases of double trisomy. The predominance of maternal origin in cases of double trisomy is similar to cases of single trisomy. The result also indicated that both extra chromosomes in two cases occurred by non-disjunction at the first meiotic division, and extra chromosomes in the other two cases occurred by non-disjunction at the first mitotic division. The mean maternal age in cases of double trisomy was significantly higher than that in cases of single trisomy. These findings suggest the possibility that abnormal separation of two or more chromosomes may occur simultaneously in oogonia, and that this phenomenon may increase in relation to the increase in age of women.

Reduction of pleural effusion by OK-432 in a fetus complicated with congenital hydrothorax.

A 29-year-old, primiparous woman was referred to our hospital at 21 weeks of gestation because of right pleural effusion in the fetus shown by routine ultrasonographic examination. Cytology revealed abundant lymphocytes, suggesting chylothorax. We removed the pleural effusion and injected OK-432 into the chest cavity at 24 and 25 weeks of gestation. Pleural effusion declined and an adhesion between the lung surface and the pleural membrane seemed to form. At 33 weeks of gestation, a female infant was born by cesarean section (1,090 g and Apgar score 6/8). Although she demonstrated slight retraction and tachypnea, management could be achieved by administration of oxygen alone without mechanical ventilation. Later, the baby was diagnosed as suffering from the Cornelia de Lange syndrome with characteristic features. OK-432 injections could thus prevent complications of chylothorax and hypoplastic lungs, without injury to either the baby or the mother.

Maternal uniparental disomy of chromosome 16 in a case of spontaneous abortion.

To investigate the involvement of uniparental disomies (UPDs) in spontaneous abortions, we analyzed in detail the polymorphism of microsatellites on each chromosome in cases of abortion. Of the 52 spontaneous abortions investigated, 25 had a normal karyotype. The polymorphic analysis of these cases revealed that, in the villi from 24 of the 25 cases, biparental patterns were present in informative microsatellites in all autosomes. In the remaining case with a 46,XX karyotype (case 18), however, the informative patterns of the microsatellites of chromosome 16 appeared to be both of maternal origin. The results also showed that the region from the distal end of the short arm to near the middle point of the long arm of chromosome 16 (pter to D16S3107) were heterozygous, and those of the remaining region of the long arm (D16S3018 to qter) were homozygous. That is, this fetus had maternal isodisomy and heterodisomy of chromosome 16, originating from a maternal, meiosis I non-disjunction of dyad 16 that accompanied a crossover at near the middle point of the long arm. The present finding suggests that some UPDs may become a cause for spontaneous abortions.

Antenatal evaluation of an encephalocele in a dizygotic twin pregnancy using fast magnetic resonance imaging.

We report a case of an encephalocele in a dizygotic twin pregnancy, following ovulatory induction. In the involved fetus, an abnormal shadow like an encapsulated-solid tumor located on the occiput was found by routine maternal transabdominal ultrasonography at 17 weeks of gestation. The parents did not accept induced abortion because of the presence of another fetus with no abnormality on ultrasonography. At 35 weeks of gestation, transabdominal ultrasound examination showed a large occipital cyst, composed of protrusive fetal brain and cerebrospinal fluid. Fast-scanning magnetic resonance imaging delineated more clearly the inside of the abnormal lesion and thus allowed confirmation of the putative diagnosis of fetal encephalocele during pregnancy. Surgical report was possible in this case, and the patient had no severe physical or neurological abnormalities 10 months after birth. Since the prognosis appears to depend primarily on how prominent the brain tissue is inside the herniated sac, this approach had benefit for clinical decision making.