Prenatal diagnosis of the fragile X syndrome using fetal blood and amniotic fluid

Nineteen pregnancies at risk for the Martin–Bell syndrome have been monitored during the second trimester for the presence of the fragile Xq27. Of the 19 potential carrier mothers, 14 showed the presence of the fragile X in their lymphocytes at a level of 4 per cent or above. As one was a twin pregnancy, fetal blood was obtained at fetoscopy from 20 fetuses and amniotic fluid obtained simultaneously from 19 of them. Of the 20 fetuses, 18 were males (including both of the twins) and two were females. Of these 18 males, seven were found to carry the fragile Xq27 in lymphocytes and subsequently six of the seven were terminated. The diagnosis was confirmed in five of the six terminated fetuses (the sixth case was a patient whose pregnancy was terminated abroad) and also in a full-term male baby. Five of the seven males without the marker X who came to term had their karyotypes confirmed post natally. Of the two female fetuses one was found to be a carrier of the fragile X and the other was not. Both babies had full-term deliveries and both had their karyotypes confirmed post natally. In some cases the diagnosis made in fetal lymphocytes was confirmed later in amniocytes.     

Early prenatal diagnosis of the fragile site AT Xq27.3 associated with Martin-Bell syndrome

Early prenatal diagnosis of the fragile X was attempted in 44 pregnancies, including one twin pregnancy at risk of Martin-Bell (MB) syndrome. The sex ratio was 24M:21F. The fragile site was reproducibly demonstrated in cultured chorionic villus (CV) cells in eight male and five female fetuses. Six of the male and three of the female fetuses were terminated. Simultaneous RFLP analysis provided confirmative data with flanking DNA markers in 3 of 13 analysed cases. Recombination and/or non-informativeness at available distal and/or proximal loci were found in nine cases. In one male fetus, discordance between the haplotype and cyto-genetics (fragile-X-negative) suggested the presence of a normal male transmitter, a double meiotic cross-over within the region, or a false-negative cytogenetic diagnosis. However, discordance between prenatal and post-termination/postnatal cytogenetic findings was not observed in this series. The use of excess thymidine for induction of the fragile X in cultured CV cells provided in the majority of cases a safe and rapid method for cytogenetic diagnosis, with options for early induced termination in fragile-X-positive pregnancies, for simultaneous RFLP analysis, and for subsequent second-trimester analysis of fetal blood in complicated cases.     

Prenatal diagnosis of fragile X in a heterozygous female fetus and postnatal follow-up

An apparently normal female infant was born after the prenatal diagnosis of fragile Xq27×28 present in about 4 per cent of amniocytes. The mildly retarded mother had been found in early pregnancy to be heterozygous for fragile X. The child, now 9 months old. showed about the same level of fragile X expression as her mother. Variations in the proportion of cells with fragile X appeared to be related to cell type and laboratory techniques. The infant's growth and development have been normal. Different techniques to induce or increase the expression of fragile X are discussed.     

Prenatal diagnosis of the fragile X using thymidine induction

Eleven pregnancies in ten patients at risk for the fragile X were monitored by amniocentesis or chorion villus biopsy and induction of the fragile site using thymidine, methotrexate and FUdR. Three female fetuses and one male fetus were found to have the fragile X. The results obtained using thymidine induction were superior to those using methotrexate induction and probably better than those obtained using FUdR induction.     

Evaluation of transcervical chorionic villus sampling with a completed follow-up of 1550 consecutive pregnancies

Data from 1550 consecutive pregnancies after first-trimester prenatal diagnosis by transcervical chorionic villus sampling (TC-CVS) are presented. The sampling efficacy was 97.8 per cent; the mean amount of collected villus tissue was 23 mg (range 5–100 mg). There were 97 affected fetuses, mainly (73.2 per cent) with a chromosomal abnormality or a male karyotype in carriers of X-linked disease. Pregnancy termination in these and four other women for social reasons resulted in 1449 continuing pregnancies. In these pregnancies, the fetal loss rate up to 28 weeks of gestation was 5.1 per cent with the highest loss rate (3.9 per cent) before 16 weeks. When relating this fetal loss rate to maternal age, this was 6.1 per cent in the advanced maternal age group (⩾36 years) against 3.1 per cent in the younger age group. In 1376 pregnancies continuing beyond 28 weeks, the perinatal mortality rate was 1.1 per cent; the percentage of non-genetic congenital anomalies was 0.9 per cent. The reproductive pattern of women at high genetic risk after CVS followed by pregnancy termination was evaluated. Within 12 months after the first CVS followed by pregnancy termination, 70 percent of women again requested CVS in a subsequent pregnancy.     

Prenatal exclusion of X-linked hydrocephalus-stenosis of the aqueduct of sylvius sequence using closely linked DNA markers

X-linked hydrocephalus-stenosis of the aqueduct of Sylvius sequence (H-SAS, MIM number 307 000) is a rare genetic disorder characterized by hydrocephalus, macrocephaly, adducted thumbs, spasticity, mental retardation, and cerebral malformations. This regularly lethal condition is usually diagnosed at birth or prenatally by ultrasound, but hydrocephalus may be moderate or even undetectable on fetal ultrasound examination. Moreover, since heterozygous women are asymptomatic, carrier detection is at present impossible before the birth of an affected son. Therefore, mapping the H-SAS locus to distal Xq (Xq28) was of primary importance for genetic counselling and prenatal diagnosis. Here, we report prenatal exclusion of H-SAS with a probability of 97.6 per cent in two male fetuses with a 50 per cent a priori risk of being affected using closely linked Xq28 DNA markers.     

Fetal blood sampling and cytogenetic abnormalities

From September 1984 to April 1991, we performed cytogenetic analysis on fetal blood samples from 214 second-and third-trimester pregnancies. One hundred and thirty-four cases were referred to consider the possibility of chromosomal mosaicism following amniocyte studies. The confirmation rate of mosaicism is at 0 per cent (0/9), 1·4 per cent (1/70), and 40 per cent (22/55) for cases of level I, level II, and level III mosaicism, respectively. Four out of 17 cases were positive for the diagnosis of fragile X syndrome. Of 63 cases with abnormal ultrasound findings, blood disorders, or other genetically related clinical conditions, 11 were found to have a chromosome abnormality. Fetal blood sampling is a valuable adjunct to other methods in the prenatal diagnosis of chromosomal mosaicism or pseudomosaicism. It is also useful when rapid cytogenetic diagnosis is desired because of malformations detected in pregnancies at a late gestational age.     

Transabdominal chorionic villus sampling for fetal genetic diagnosis. Technical and obstetrical evaluation of 100 cases

First trimester fetal diagnosis was established in 100 pregnancies at risk by transabdominal chorionic villus sampling (TA-CVS). Forty-eight per cent of the women were 35 years or more at the time of sampling. Using the double needle technique, both the aspiration and the diagnostic success rate were 100 per cent. The mean amount of villi aspirated was 28·2 mg (10–50 mg). The mean needle time was 3 min. Vaginal spotting appeared in 2 per cent of the women. Four women had therapeutic abortion due to abnormal findings and one for social reasons. Three fetuses with normal karyotypes were lost. Excluding the therapeutic abortions, the fetal loss rate was 3±2 per cent. The fetal loss rate in the amniocentesis control group (n = 200) was 3±6 per cent. The cytogenetic diagnosis was carried out by the direct preparation technique as well as by chorion villus cultivation. All karyotypes were confirmed by lymphocyte cultures from umbilical cord blood or heel blood from the newborn or from aborted fetal tissue. Transabdominal CVS is deemed a safe and easy tool for achieving chorionic villi in the first trimester.     

Genetic amniocentesis: 505 cases performed before the sixteenth week of gestation

Between May 1987 and November 1988, 505 early amniocentesis within the 15th week of gestation were performed at the First Department of Obstetrics and Gynaecology,‘L. Mangiagalli’ of the University of Milan and at the Department of Obstetrics and Gynaecology of ‘Gaslini’ hospital in Genoa. A total number often abnormal fetal karyotypes were diagnosed. In addition, one case of pseudomosaicism (not confirmed on fetal blood) and one case of osteogenesis imperfecta type II (observed at ultrasound examination) were also detected. Eleven pregnancies were therefore terminated because of an abnormal fetus. Out of 494 pregnancies (excluding terminated pregnancies) there were 16 fetal losses within the 28th week; ten of these occurred in the 2 weeks following the procedure. There were 475 live-births, of which 447 were term deliveries and the other 28 deliveries occurred before the 37th week of gestation.     

Amniotic fluid disaccharidases in the prenatal detection of cystic fibrosis

Intestinal disaccharidases in amniotic fluid were studied in 41 pregnancies with a recurrence risk for cystic fibrosis (CF). In 11 out of 13 pregnancies with CF fetuses the maltase and sucrase activities were either below the control range (8 cases) or below the 10th percentile of control values (3 cases). Trehalase and lactase were slightly less informative indicators of CF. Of the other 28 pregnancies 3 had low amniotic fluid activities of several intestinal enzymes and were terminated, 12 resulted in the birth of a healthy child and 13 are continuing. The findings in fetal CF suggest an impairment of the defaecation of intestinal contents into the amniotic fluid. Reduced or low amniotic fluid disaccharidase activities were also found in other fetal disorders with demonstrated or presumed intestinal anomalies: e.g. anal atresia (2 cases), anencephaly (3 our of the 7 cases), trisomy 13 (5 cases), trisomy 18 (3 of the 5 cases) and trisomy 21 (19 of the 22 cases). Reduced amniotic fluid disaccharidase activities, although not specific for CF, are highly informative in pregnancies at high risk for CF. Using the 10th percentile of the normal range for amniotic fluid disaccharidase activities as an action line, the sensitivity of CF detection is estimated at 80 to 90 per cent, which could in high risk pregnancies reduce the risk of having another affected child from 1 in 4 to 1 in 20.     

Multicenter clinical experience with non-invasive cell-free DNA screening for monosomy X and related X-chromosome variants

Objective

We aimed to investigate how the presence of fetal anomalies and different X chromosome variants influences Cell-free DNA (cfDNA) screening results for monosomy X.

Methods

From a multicenter retrospective survey on 673 pregnancies with prenatally suspected or confirmed Turner syndrome, we analyzed the subgroup for which prenatal cfDNA screening and karyotype results were available. A cfDNA screening result was defined as true positive (TP) when confirmatory testing showed 45,X or an X-chromosome variant.

Results

We had cfDNA results, karyotype, and phenotype data for 55 pregnancies. cfDNA results were high risk for monosomy X in 48/55, of which 23 were TP and 25 were false positive (FP). 32/48 high-risk cfDNA cases did not show fetal anomalies. Of these, 7 were TP. All were X-chromosome variants. All 16 fetuses with high-risk cfDNA result and ultrasound anomalies were TP. Of fetuses with abnormalities, those with 45,X more often had fetal hydrops/cystic hygroma, whereas those with “variant” karyotypes had different anomalies.

Conclusion

Both, 45,X or X-chromosome variants can be detected after a high-risk cfDNA result for monosomy X. When there are fetal anomalies, the result is more likely a TP. In the absence of fetal anomalies, it is most often an FP or X-chromosome variant.     

Prenatal diagnosis of fragile x syndrome: (cgg)n expansion and methylation of chorionic villus samples

Fragile X syndrome is the most common form of inherited mental retardation, due to an expansion of the (CGG)_n trinucleotide repeat in the FMR-1 gene and hypermethylation of its 5′ upstream CpG island. Two major problems remain to be resolved for fragile X prenatal diagnosis: the abnormal methylation patterns of chorionic villus samples (CVS) and the inability to predict the mental status of females with the full mutation. We present here the results of ten prenatal diagnoses of fragile X syndrome using Southern blotting and polymerase chain reaction (PCR) amplification, and the analysis of 50 further CVS to test the methylation status of the CpG island of the FMR-1 gene. In the ten ‘at-risk’ CVS, eight normal (five males and three females) and two affected male fetuses were detected. Absence of methylation in the CVS was observed in two cases, which was not found upon subsequent examination of the newborn or of fetal tissues. In the 50 CVS not ‘at risk’ for fragile X syndrome, abnormal fragment patterns for probe StB12.3 were detected in 32 per cent for female and 24 per cent for male fetuses. This abnormal pattern could be due to absent or partial methylation of the CpG island of the FMR-1 gene in chorionic villus tissues.     

Ultrasound evaluation of pregnancies at risk for homozygous α-thalassaemia-1

Twenty-six pregnant Chinese women who were at risk of giving birth to a fetus affected with homozygous α-thalassaemia-1 were examined serially by ultrasound. Six of these 26 pregnancies were affected. In one third of the affected pregnancies progressive fetal ascites appeared before 24 weeks gestation and these pregnancies were terminated. In the remaining two thirds abnormal estimated fetal weight-placental volume (EFW-PV) ratio and fetal growth retardation as evidenced by a falling biparietal diameter (BPD), femur length (FL) but a normal abdominal circumference (AC) was apparent by 28 weeks gestation. Increased transverse cardiac (TC) diameter was another consistent finding but appeared late. All these features appeared before the onset of fetal ascites. A normal EFW-PV ratio and fetal growth until 28 weeks gestation was a reassuring sign of normality. Abnormal EFW-PV ratio was the earliest sign to appear in affected pregnancies and a normal ratio until 28 weeks gestation had a 100 per cent predictive value.     

Prenatal diagnosis of fragile X syndrome: Management of the male fetus with a premutation

Direct detection of the fragile X mutation by DNA analysis has greatly simplified prenatal diagnosis of this disease. However, women carrying a fragile X premutation may pass their expanded trinucleotide repeat to sons without expansion to a full mutation. Such sons are predicted to be intellectually normal. In this situation, the accuracy with which the fetal status can be inferred from analysis of chorionic villus sample (CVS) DNA is unclear. We describe such a case, in which it was felt necessary to proceed to fetal blood sampling despite technically unambiguous DNA results from the CVS. The lack of prospective data means that this dilemma may be expected to recur over the next few years when performing prenatal diagnosis on fragile X premutation carriers.     

Prenatal diagnosis of the hurler syndrome: Report on 40 pregnancies at risk

In 40 pregnancies at risk for the Hurler syndrome 13 affected fetuses were detected by the demonstration of an α-L -iduronidase deficiency and an increased level of ³⁵S-sulphate incorporation. The diagnoses were confirmed by the analysis of fetal tissues and/or cultured fetal skin fibroblasts. Microassays for α-L -iduronidase, using phenyl α-L -iduronide and more recently 4–methyl-umbelliferyl α-L -iduronide, enabled a reliable diagnosis to be made within 15 to 18 days after amniocentesis. ³⁵S-sulphate incorporation has been a valuable adjunct in cases with a low (heterozygote) enzyme activity.     

Creatine kinase estimation in pure fetal blood samples for the prenatal diagnosis of duchenne muscular dystrophy

This paper compares the results of a survey of plasma creatine kinase (CK) activity measured in fetuses at-risk for Duchenne muscular dystrophy (DMD) with a reliable control series. Only pure fetal blood samples obtained by fetoscopy at between 17–24 weeks gestational age were used. Of the at-risk group 19 male pregnancies, mostly at low risk for DMD, proceeded to term with a normal outcome; there was no significant difference between their fetal plasma CK activities and the control group. Another 21 male pregnancies were terminated. This group included the highest risk mothers and hence was expected to contain a significant proportion of affected fetuses. The fetal plasma CK activity range was overlapping but significantly higher than the control group. No grossly elevated CK value was obtained. We conclude that, on average, DMD fetuses at this gestational age have higher plasma CK activity than controls. The problems of applying this finding to the prenatal diagnosis of DMD are discussed.     

The safety of fetoscopy: (I). Effect of umbilical vessel puncture on the fetal heart rate and cord histology

The fetal heart rate (FHR) was continuously monitored during 42 umbilical vessel punctures performed at the placental insertion of the cord in 24 diagnostic fetoscopies in which pure fetal blood was obtained. In only one patient did a deceleration first appear during puncture and aspiration of fetal blood. In two patients decelerations preceded fetoscopy and in two others they began during the fetoscopy but before puncture of an umbilical vessel. In 19 patients, the FHR did not change at all during the procedure. Fetal haemorrhage after sampling was either absent or minimal. Six pregnancies were terminated because a positive diagnosis had been made and 18 healthy babies were born. Umbilical cords were examined after 7 terminations of pregnancy and after 6 deliveries. In the former group the puncture could just be seen with the naked eye and the needle track was demonstrated histologically in 6. No traces of the puncture or other abnormalities were found in the cords after delivery. Fetal blood sampling from umbilical cord vessels, particularly at the placental insertion of the cord, is the technique of choice since pure fetal blood can be obtained without increasing the risk of fetoscopy.     

Prenatal diagnosis of hemoglobinopathies in twin pregnancies by double simultaneous fetoscopy

A new technique for sampling fetal blood in twin pregnancies using two fetoscopes simultaneously is described. Two fetoscopes were inserted, one after the other, into both amniotic cavities and fetal blood samples were obtained from either the chorionic plate vessels or the umbilical cord insertion area. The observation of the bright tip of the second fetoscope behind the septum using the first fetoscope assured the successful entry of the two fetoscopes into the two different amniotic sacs. This technique was performed on 15 out of 17 patients. In all patients the fetuses were at risk of β-thalassemia major. Sampling was successful in all cases. Double simultaneous fetoscopy seems to be a safe and accurate technique without technical problems or complications. The simultaneous use of two fetoscopes opens new possibilities in intrauterine fetal surgery and research.     

Fetal chromosome analysis: Screening for chromosome disease?

The aim of the study was to investigate the rationale of the current indications for fetal chromosome analysis. 5372 women had 5423 amniocenteses performed, this group constituting a consecutive sample at the chromosome laboratory, Rigshospitalet, Copenhagen from March 1973 to September 1980 (Group A + B). Pregnant women 35 years of age, women who previously had a chromosomally abnormal child, families with translocation carriers or other heritable chromosomal disease, families where the father was 50 years or more and women in families with a history of Down's syndrome (group A), were compared to women having amniocentesis, although considered not to have any increased risk of fetal chromosome abnormality (1390 pregnancies, group B). They were also compared with 750 consecutive pregnancies in women 25–34 years of age, in whom all heritable diseases were excluded (group Q. The risk of unbalanced chromosome abnormality in group A (women with elevated risk) is significantly higher than in group B + C (women without elevated risk) (relative risk 2–4). Women with a known familial translocation and women 40 years or more have a relative risk of 5–7 of having an unbalanced chromosome abnormality compared with women without elevated risk. Spontaneous abortion rate and prematurity rate did not differ from rates expected without amniocentesis. It is concluded that current indications may be characterized as a mixture of evident high risk factors and factors with only a minor influence on risk. Indications for amniocentesis should therefore be reconsidered. Because it must be considered impractical and ethically wrong to limit amniocentesis to the two mentioned real high risk groups, and illogical to continue the present policy, which is not based on clearcut evidence, the possibility of offering amniocentesis to all who want it, is discussed. Screening for chromosome disease in all pregnancies is not without problems, but may be reasonable in some localities.     

Short communications normal fetal growth despite persistent amniotic fluid leakage after genetic amniocentesis

Two women not only lost relatively large amounts of amniotic fluid immediately following genetic amniocentesis, but continued to lose fluid for the remainder of their pregnancies. Periodic ultrasonographic assessment confirmed normal fetal growth and presence of some amniotic fluid. Both women were delivered at term of normal offspring who showed no evidence of fetal deformations. Although amnionitis is a risk, cautious surveillance may permit continuation of pregnancies complicated by copious or persistent amniotic fluid leakage following genetic amniocentesis.