Ontogeny of fetal liver glucose-6-phosphatase activity

A reliable and sensitive microassay for the measurement of liver glucose-6-phosphatase is described. Human fetal liver was assayed for glucose-6-phosphatase activity from 7.5 to 24 weeks of gestation and was found to have a mean activity of 2.11 nmol per min per mg of protein. This was approximately 30 per cent of the postnatal controls assayed by the same method, but there was no evidence of a change in activity during the gestational period examined. If fetal liver tissue can be reliably obtained, it may be possible to determine a deficiency of glucose-6-phosphatase in fetuses who are at risk.     

Fetal intestinal microvilli in human amniotic fluid

The intestinal microvilli of fetal origin in human amniotic fluid were purified by Ca²⁺ precipitation of contaminating organelles followed by differential centrifugation of microvillar membranes. In the purified preparation, the specific activity of the microvillar marker-enzymes maltase and sucrase increased about 77-fold over that in cell-free amniotic fluid. Significant contamination of the purified preparation by endoplasmic reticulum (microsomes) and lysosomes was ruled out on the basis of a low content of the marker enzymes glucose-6-phosphatase (microsomes) and acid phosphatase (lysosomes). Amniotic fluid microvilli contain typical enzymes of the fetal intestine including maltase, sucrase, trehalase, alkaline phosphatase and γ-glutamyltransferase, and their morphology by electron microscopy resembles that of vesiculated intestinal microvilli. Prenatal detection of genetic diseases due to a deficiency of a protein expressed in these membranes or associated to abnormal microvilli seems feasible.     

Possibility of prenatal diagnosis of hereditary triose phosphate isomerase deficiency

Prenatal diagnosis has been performed on umbilical cord blood of an 18 weeks fetus of heterozygous triosephosphate isomerase (TPI) deficient parents. After excluding maternal blood contamination, TPI activity was measured and found to be 60 per cent of the normal mean whereas the value of glucose-6-phosphate dehydrogenase activity was in the normal range of fetal blood. In addition, the analysis of the characteristics of fetal TPI, i.e. K_m measurements for glyceraldehyde-3-phosphate, heat stability tests and electrophoretic studies, did not show any evidence of a special form of TPI in fetal blood. These results were consistent with the heterozygous state and were confirmed at birth.     

Fetal presentation of morquio disease type A

A fetus with mucopolysaccharidosis type IV A (Morquio type A) is described. The family had one affected child exhibiting symptoms of classical Morquio A disease, and late in the subsequent pregnancy prenatal diagnosis was requested. At 23 weeks' gestation, moderate ascites was detected by detailed ultrasound scan and keratan sulphate was found in the amniotic fluid. The pregnancy was terminated by prostaglandin induction and the diagnosis of mucopolysaccharidosis type IV A was confirmed by demonstration of a deficiency of N-acetylgalactosamine-6-sulphate (GalNac-6-S) sulphatase in cultured amniotic cells and in post-mortem fibroblast cultures. The activities of β-galactosidase and arylsulphatase A were normal, ruling out Morquio disease type B and multiple sulphatase deficiency. These results indicate that mucopolysaccharidosis IV A (a disease that predominantly affects the skeletal system) may produce ascites in the fetus to such an extent that it can be detected by ultrasound.     

Prenatal diagnosis of molybdenum cofactor deficiency and isolated sulfite oxidase deficiency

Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency are autosomal recessive inborn errors of metabolism with severe neurological symptoms resulting from a lack of sulfite oxidase activity. The deficiencies can be diagnosed prenatally by monitoring sulfite oxidase activity in chorionic villus sampling (CVS) tissue. In those families in which the specific defects have been identified, diagnosis can be achieved by mutation analysis or linkage studies directed at affected genes. These include MOCS1, MOCS2 or GEPH, in cases of molybdenum cofactor deficiency, or SUOX in patients with isolated sulfite oxidase deficiency. Copyright © 2002 John Wiley & Sons, Ltd.     

Prenatal ultrasonic diagnosis of obstructive bowel disease: A retrospective analysis

Fetal obstructive bowel disease was diagnosed in 29 patients at 22–37 weeks (median 32 weeks) of gestation, seven (24 per cent) of whom also displayed other anomalies. Polyhydramnios was present in 20/29 cases (69 per cent). An abnormal karyotype existed in 7/29 cases (24 per cent), of which six were diagnosed prenatally (trisomy 21, n = 5; 69, XXX, n = 1) and one postnatally (trisomy 21). There was always an association with the ultrasonic ‘double bubble’ sign. Obstructive bowel disease was confirmed postnatally in 20/29 (69 per cent) cases, i.e., oesophageal atresia (n = 1), duodenal obstruction (n = 12), and small bowel obstruction (n = 7). Other anomalies existed in 6/29 (21 per cent) cases, i.e., multicystic kidney (n = 1) and multiple congenital anomalies (n = 5). The perinatal mortality rate was 35 per cent (7/20).     

拟柱孢藻生长及碱性磷酸酶活性对不同磷浓度和磷形态响应的株系间差异

拟柱孢藻(Cylindrospermopsis raciborskii)是热带地区普遍存在的蓝藻种类,近年来广泛扩张到温带地区水体,耐受低磷环境和多生态型的存在被认为是该藻能成功入侵的重要原因.为进一步了解不同藻株对磷波动的生理响应是否存在差异,本研究以广东省镇海水库分离的4株拟柱孢藻(N1、 N8、 N9和N10)为材料,观测它们的生长和碱性磷酸酶(ALP)活性在不同无机磷(Pi)浓度(HP=7.13 mg·L~(-1)、 MP=0.64 mg·L~(-1)、 LP=0.03 mg·L~(-1))和磷形态[磷酸氢二钾(K_2HPO_4)、焦磷酸钾(K_4P_2O_7)、三聚磷酸钾(K_5P_3O_(10))、 D-葡萄糖-6-磷酸(D-G-6-P)、三磷酸腺苷(ATP)、环磷酸腺苷(cAMP)]的变化.结果表明, 4株拟柱孢藻的生长对Pi浓度变化的响应基本一致,即它们的生物量都随Pi浓度的升高而增加,而ALP活性则反之;无论在LP、MP或HP条件下,N8藻株的ALP活性均显著低于其他3藻株,表明该藻株更能适应环境中的磷波动.在不同磷源培养下,拟柱孢藻N8和N9在3种无机磷下的生物量显著高于3种有机磷组,两者的比生长速率在K_2HPO_4最高,在ATP中最低,这表明拟柱孢藻偏好无机磷,但也能利用有机磷生长;在有机磷源条件下,N8藻株在ATP中的ALP活性显著高于其他2种有机磷,而N9藻株的ALP活性在3种有机磷培养下无显著差异,这表明N8藻株对无机磷缺乏的响应较N9藻株更为敏感.本结果表明来源于同一水库的拟柱孢藻藻株间存在显著差异,其中N8藻株对磷浓度变化的适应及响应能力均高于其它藻株.株系差异性的存在有利于拟柱孢藻适应各种环境变化,增强自身的竞争优势.     

贵州碘缺乏病区环境中碘的特征研究

为探讨贵州碘缺乏病与环境的关系,以及环境中岩石、土壤和水对碘缺乏病分布的影响因素,对贵州碘缺乏病的重病区、轻病区和非病区进行了一定量的岩石、土壤、水及粮食作物(大米和玉米)样品采集,测试碘背景含量,并进行对比分析。结果显示,环境中岩、土及水碘背景与贵州碘缺乏病分布有一定的关系。重病区主要属岩石、土壤和水碘背景含量低的分布区,其岩石类型主要为碳酸盐岩;非病区主要属岩石、土壤和水碘背景含量相对较高的分布区,其岩石类型主要为煤系;轻病区分布的岩石、土壤和水碘背景含量则介于重病区和非病区之间,其岩石类型主要为浅变质岩和碎屑岩。      

Prenatal diagnosis of multiple acyl-CoA dehydrogenase deficiency: association with elevated α-fetoprotein and cystic renal changes

We report the occurrence of multiple acyl-CoA dehydrogenase deficiency (MADD) in two consecutive pregnancies in a young, Caucasian, non-consanguineous couple. In the first pregnancy, the maternal serum α-fetoprotein was elevated. A sonogram showed growth delay, cystic renal disease, and oligohydramnios; the parents decided to terminate the pregnancy. Postmortem examination confirmed the cystic renal disease and showed hepatic steatosis, raising the suspicion of a metabolic disorder. The diagnosis of MADD was made by immunoblot studies on cultured fibroblasts. In the subsequent pregnancy, a sonogram at 15 weeks' gestation showed an early growth delay but normal kidneys. The maternal serum and amniotic fluid concentrations of α-fetoprotein were elevated, and the amniotic fluid acylcarnitine profile was consistent with MADD. In vitro metabolic studies on cultured amniocytes confirmed the diagnosis. A follow-up sonogram showed cystic renal changes. These cases provide additional information regarding the evolution of renal changes in affected fetuses and show a relationship with elevated α-fetoprotein, which may be useful in counseling the couple at risk. MADD should be considered in the differential diagnosis of elevated α-fetoprotein and cystic renal disease. Early growth delay may be an additional feature. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal diagnosis of respiratory chain deficiency by direct mutation screening

Respiratory chain deficiency (RCD) is responsible for a clinically heterogeneous group of early-onset untreatable disorders. Enzymological prenatal diagnosis (PD) can only be offered to a fraction of families. Moreover, due to the two-fold genetic origin of the respiratory chain (nuclear and mitochondrial DNA) and owing to the large number of nuclear genes involved in the respiratory chain assembly, maintenance and functioning, the identification of the disease causing gene in a given family remains challenging. Here, we report on PD of RCD by direct screening of NDUFV1, SDH-Fp, SCO1 and SURF1 mutations in five unrelated families with complex I, II and IV deficiency, respectively. The identification of the disease-causing gene in a given family with RCD is a major issue to provide both adequate genetic counselling and early, reliable PD. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal diagnosis for arginase deficiency by second-trimester fetal erythrocyte arginase assay and first-trimester ARG1 mutation analysis

Hyperargininemia is a progressive neurometabolic disorder caused by deficiency of hepatic cytosolic arginase I, resulting from mutations in the ARG1 gene. We diagnosed arginase deficiency in a three-year-old male child of first-cousin Palestinian Arab parents. Prenatal diagnosis of an unaffected fetus was achieved in the second trimester of a subsequent pregnancy by cordocentesis and analysis of arginase activity in fetal erythrocytes. ARG1 mutation analysis in the proband revealed homozygosity for a deletion of 10 753 bp extending from the first intron to beyond the poly (A) site of the gene. This is the first gross deletion in the ARG1 gene to be identified and the first mutation to be described in an arginase-deficient patient of this ethnic origin. The identification of the ARG1 deletion in this family enabled first-trimester prenatal diagnosis in a subsequent pregnancy by multiplex PCR analysis performed on chorionic villous DNA. Copyright © 2004 John Wiley & Sons, Ltd.     

Activity and multiple forms of α-L-fucosidase and hexosaminidase in chorion biopsy specimens and some fetal organs

It was shown that activities and isoenzyme patterns of x-L-fucosidase and hexosaminidase were similar in biopsy specimens of chorion obtained immediately before induced abortion in normal pregnancy and in chorion tissue itself. A comparative study of isoenzyme patterns of these glycosidases showed their similarity in chorion and human fetal kidney, liver and lungs. The data obtained may be applied to the investigation of the multiple forms of α-L-fucosidase and hexosaminidase in chorion biopsy specimens for the prenatal diagnosis of fucosidosis (α-L-fucosidase deficiency). Tay-Sachs diseases (hexosaminidase A deficiency) and Sandhoff disease (deficiency of hexosaminidase A and B).     

Prenatal diagnosis of ornithine transcarbamylase deficiency: Results in SPFASH mice

Ornithine transcarbamylase (OTC) deficiency is a human X-chromosome-linked disease (McKusick 31125). The presence of OTC activity in the human placenta encouraged us to examine the possible diagnosis of the disease in an animal model (Spf^ash mice) by enzymatic assay on placental samples. A significant positive correlation (P< 0·02) was found between placental and hepatic activities; the pH dependence of OTC was similar in the placenta and liver when compared within normal or homozygous mutant mice. The apparent K_m (ornithine) and K_m (carbamoylphosphate) values of the enzyme did not show any significant differences when compared in both placentae and livers of normal fetuses. The use of OTC assay in the placenta for prenatal diagnosis of OTC deficiency in mouse fetuses obtained by the crossbreeding of Spf^ash/+ with +/Y has shown that our method has good diagnostic value. We made the diagnosis of OTC deficiency in male fetuses with a sensitivity and a specificity of 1·0. The + gene from the father in Spf^ash/ + animals is preferentially inactivated in extraembryonic tissues, explaining why very low placental OTC activity was observed in 12 of the 18 female fetuses studied. Because these 12 females have variable OTC activity in their livers, it is not possible to appreciate the true residual activity in their livers by measuring this activity in the placenta.     

Prenatal diagnosis of morquio's disease type a (n-acetylgalactosamine 6-sulphate sulphatase deficiency)

Prenatal monitoring of three pregnancies at risk of Morquio's disease type A by determination of N-acetylgalactosamine 6-sulphate sulphatase activity in cultured amniotic cells is reported. In one pregnancy prenatal diagnosis of one affected fetus was made. Enzyme determinations in tissues of the aborted fetus confirmed the prenatal diagnosis.     

First trimester diagnosis of Gaucher disease in a fetus with trisomy 21

A 38-year-old lady, who had a previous infant with type 2 Gaucher disease, underwent prenatal diagnosis by chorionic villus sampling at 9 weeks' gestation. Results on the fresh villus revealed a 47,XY,+21 karyotype and a marked deficiency (2 per cent of control) of β-glucosidase activity. Following termination, villus material was cultured which initially revealed only a partial enzyme deficiency and a normal female karyotype, i.e., maternal cells. A subsequent culture contained 47,XY, + 21 cells which were deficient in β-glucosidase activity, thus confirming the diagnosis. The results in this interesting case illustrate the potential dangers of maternal cell contamination in cultured villus cells.     

Prenatal diagnosis of carnitine palmitoyltransferase 2 deficiency in chorionic villi: a novel approach

Carnitine palmitoyltransferase 2 (CPT2) deficiency, the most common autosomal recessive inherited disease of the mitochondrial long-chain fatty acid (LCFA) β-oxidation, may result in three distinct clinical phenotypes, namely, a mild adult muscular form, a severe infantile hepatocardiomuscular disease, and a neonatal form, which includes dysmorphic features in addition to hepatocardiomuscular symptoms. Both the latter forms are life-threatening diseases, and prenatal diagnosis (PND) can be offered to couples at a one-fourth risk of having an affected child. PND of CPT2 deficiency hitherto relied mostly on mutation detection from fresh chorionic villi (10 weeks' gestation), since CPT2 activity could be assayed on cultured amniocytes only (16–17 weeks' gestation). We devised a CPT2 activity assay from 10 mg of chorionic villi sampling (CVS). Combining this enzymatic assay to haplotype study using polymorphic markers linked to the CPT2 gene, we were able to carry out within 2 days, CPT2 deficiency PND, in two unrelated families, using a CVS performed at the 11th week of gestation. Copyright © 2003 John Wiley & Sons, Ltd.     

Evaluation of a fetus at risk for dihydropteridine reductase deficiency by direct mutation analysis using denaturing gradient gel electrophoresis

Dihydropteridine reductase (DHPR) is an enzyme involved in the recycling of tetrahydrobiopterin (BH₄), which is an obligate co-factor of the aromatic amino acid hydroxylases. DHPR deficiency is a rare, autosomal recessive disorder caused by mutations in the QDPR gene. DHPR-deficient patients are diagnosed by a lack of response to a low phenylalanine diet and by severe neurological symptoms. Final diagnosis is made by measurements of neurotransmitters and pterin metabolites in cerebrospinal fluid (CSF) and urine, in addition to DHPR enzyme activity, which can be assessed in whole red blood cells. Treatment of DHPR deficiency can be difficult and the outcome is not always satisfying, even if all treatment strategies are followed. Therefore prenatal diagnosis is of great importance in affected families. Prenatal diagnosis is possible by measuring DHPR activity in different cell types but this is time consuming. More than 25 different mutations have to date been identified in the QDPR gene and direct identification of a mutation in a fetus would be easy and rapid. We have developed a method based on denaturing gradient gel electrophoresis (DGGE) for the analysis of the QDPR gene. The method is useful for rapid and simultaneous scanning of all exons and flanking intronic sequences of the QDPR gene. We describe the first prenatal diagnosis conducted using this method. Copyright © 2001 John Wiley & Sons, Ltd.     

Low or absent unconjugated estriol in pregnancy: an indicator for steroid sulfatase deficiency detectable by fluorescence in situ hybridization and biochemical analysis

It has been previously reported that a low or absent maternal serum unconjugated estriol (uE3) level is associated with placental steroid sulfatase (STS) deficiency. Here we report a correlation between patients who present with a very low or absent maternal serum uE3 and a deletion of the STS gene as assessed by fluorescence in situ hybridization (FISH). We studied nine prenatal cases that presented to the clinical laboratory with an abnormal triple screen, specifically low or absent maternal serum uE3 and a 46,XY karyotype. FISH analysis showed complete deletion of a probe containing the STS gene in six cases and one case had a partial deletion (reduced but not absent signal). The remaining two cases were not deleted for the STS probe. All mothers tested whose fetus showed a deletion were shown to be STS deletion carriers using FISH. Biochemical analysis was performed on 7/9 prenatal specimens. All fetuses deleted for the STS probe were also found to be deficient for STS by biochemical analysis of cultured amniotic fluid (5/5). Of the two fetuses not deleted for the STS probe, one was deficient for STS activity, while the other had a normal result. The abnormal result of enzyme deficiency by biochemical analysis in a non-deletion case likely represents a mutation in the STS gene, not detectable by this FISH assay. Postnatal FISH confirmation of the STS deletion was performed in 1/7 cases. Clinical follow-up was available for 4/9 cases following birth. Copyright © 2002 John Wiley & Sons, Ltd.     

Second-trimester maternal serum screening using alpha-fetoprotein,human chorionic gonadotrophin,and unconjugated oestriol: Experience of a regional programme

Over a 2-year period from January 1991 to December 1992, second-trimester maternal serum screening for Down's syndrome using alpha-fetoprotein (aFP), human chorionic gonadotrophin (hCG), and unconjugated oestriol (uE₃) was made available to five health districts in East Anglia, with a total population of 1·2 million. Amniocentesis was offered when the risk of Down's syndrome at term was 1:200 or greater. 25359 singleton pregnancies were screened, representing an uptake of 77 per cent. The recall rate for the 24 per cent of women who had not had a dating scan prior to the test was 9·4 per cent compared with 3·9 per cent for those who had been scanned (P<0·0005). Seventy-five per cent (36/48) of Down's syndrome pregnancies were detected for a false-positive rate of 4·0 per cent. Twenty-five out of 36 of detected Down's syndrome pregnancies were dated by scan prior to sampling, and in the 11 remaining cases, the dates were confirmed by scan after a high-risk result was obtained. The exclusion of uE₃ from the screening protocol would have reduced the detection rate to 52 per cent (25/48) for the same false-positive rate. Eighty-five per cent of women identified at high risk accepted the offer of an amniocentesis. Other fetal abnormalities detected were trisomy 18 (3), trisomy 13 (2), 45,X (6), 69,XXX (5), other chromosome abnormalities (9), open neural tube defects (26), hydrocephalus (7), abdominal wall defects (4), and steroid sulphatase deficiency (6).     

Molecular analysis of genomic DNA allows rapid,and accurate,prenatal diagnosis of peroxisomal D-bifunctional protein deficiency

Prenatal diagnosis was requested for a couple with a previous child affected by the peroxisomal disorder D-bifunctional protein deficiency. Prior analysis of the D-bifunctional protein cDNA sequence from the propositus had shown that it was missing 22 bp. This was subsequently attributed to a point mutation in the intron 5 donor site (IVS5+1G>C) of the D-bifunctional protein gene. Consistent with parental consanguinity, the patient was shown to be homozygous for this mutation, which is associated with loss of a Hph 1 restriction site in the genomic sequence. Prenatal testing of the fetus using genomic DNA isolated from uncultured amniocytes indicated that both alleles of the D-bifunctional protein had the IVS5+1G>C substitution. The peroxisomal defect was later confirmed biochemically using cultured amniocytes, which were found to have elevated levels of very long chain fatty acids (VLCFA). This is the first report of prenatal diagnosis of D-bifunctional protein deficiency using molecular analysis of genomic DNA. Copyright © 2002 John Wiley & Sons, Ltd.