Non-concordance of CVS and liver glycine cleavage enzyme in three families with non-ketotic hyperglycinaemia (NKH) leading to false negative prenatal diagnoses.

We report three false negative prenatal diagnostic results, using direct measurement of glycine cleavage enzyme activity in uncultured chorionic villus tissue from 290 pregnancies at risk for non-ketotic hyperglycinaemia (NKH). Testing was done by two centres: Vancouver, Canada and Lyon, France. One false negative result had activity near the lower limit of the normal range but two samples gave completely normal results well within the control range. All three pregnancies continued and the three children were born affected with NKH. Because of the first result, we now counsel that there is a grey zone of uninterpretable activity where affected and normal enzyme values overlap. Because of the other two results we now counsel that there is an approximately 1% chance of a pregnancy with a normal CVS activity resulting in an affected child. The clinical and biochemical findings in the three families are discussed.

Molecular fragile X screening in normal populations.

In December, 1993, we initiated a pilot project in which DNA fragile X (fraX) testing was offered during routine prenatal or genetic counseling to all pregnant women seen at the Genetics & IVF Institute, most of whom were referred for the indication of advanced maternal age. A brochure on fragile X syndrome was sent to each patient prior to her appointment and was reviewed by a counselor or physician during the counseling session. As of June 1995, 3,345 patients were offered testing; 474 women with no identified family history of mental retardation or learning disability and 214 women with a positive family history accepted the test on a self-pay basis. The second population screened was 271 potential donors in our anonymous egg donor program. DNA from blood was tested by Southern blot using EcoRI/EagI and StB12.3. If an expansion was detected, CGG repeat number was determined by PCR-based analysis. Among the 474 patients with unremarkable family histories, three fraX carriers were identified (repeat sizes = 60+), whereas none were found in the 214 patients with a positive family history. Among the potential egg donors, two high borderline patients were identified (repeat sizes = between 50 and 59). Our ongoing study indicates that screening of pregnant or preconceptual populations for fraX carrier status using DNA testing is accepted by many patients and is an important addition to current medical practice.

Prenatal diagnosis of uniparental disomy 15 following trisomy 15 mosaicism.

Maternal uniparental disomy 15 (UPD15), responsible for approximately 25 per cent of Prader-Willi syndrome cases, is usually caused by maternal meiosis I non-disjunction associated with advanced maternal age. These cases may initially be detected as mosaic trisomy 15 during routine prenatal diagnostic studies. In such cases, PCR (polymerase chain reaction) microsatellite analysis of uncultured cells makes prospective prenatal diagnosis for UPD15 possible with results available in 2-4 days. We have performed molecular analyses on a series of seven cases of mosaic trisomy 15 identified in amniotic fluid (AF, n = 3) or chorionic villus samples (CVS, n = 4) from patients initially referred for advanced maternal age or abnormal triple screen. In all cases, the maternal ages were > or = 35 years and maternal meiosis I non-disjunction was documented as the cause of the trisomy in all informative cases (n = 5). Of the three case with mosaic trisomy 15 at amniocentesis, two showed the presence of the trisomy in the fetus. Molecular analysis showed one case with maternal UPD15 in the euploid cell line and one case with biparental inheritance. Both of these families elected to terminate the pregnancies based on the presence of true fetal mosaicism. In the third case, low-level trisomy 15 mosaicism in the amniotic fluid was not confirmed in a follow-up amniotic fluid sample and molecular analysis indicated biparental inheritance in the fetus. For the four trisomy 15 mosaics detected at CVS, molecular analysis was performed on direct amniotic fluid cell lysates for prospective diagnosis of UPD at 14-16 weeks' gestation. Follow-up cytogenetic analysis of the amniotic fluid in all four cases was normal, indicating confined placental mosaicism. Molecular analysis showed one of these four cases to have maternal heterodisomy 15. Based on the likelihood of Prader-Willi syndrome due to maternal UPD15, the couple chose to terminate the pregnancy. The total of two of seven cases of trisomy 15 mosaicism resulting in UPD15 is consistent with the theoretical expectation of one-third and indicates a high risk of UPD in such pregnancies. Therefore, UPD testing should be offered in all cases of mosaic trisomy 15 encountered in CVS or amniocentesis.

Preimplantation genetic diagnosis.

Preimplantation genetic diagnosis now represents an alternative reproductive option for parents at high risk of having offspring affected with certain genetic diseases. Progress in the past year has included increasing reliability in embryo sexing by both polymerase chain reaction and fluorescent in situ hybridization techniques; delivery of babies free of specific diseases such as cystic fibrosis, Lesch-Nyhan syndrome, and Tay-Sachs disease; and successful development of molecular techniques for detecting common diseases such as fragile-X syndrome. In addition, sperm separation in combination with preimplantation genetic diagnosis appears to be an exciting advance in yielding more in vitro fertilization female embryos for transfer and subsequent pregnancy in families at risk for X-linked diseases. Accumulated world experience can now be reviewed to provide couples considering preimplantation genetic diagnosis with observed pregnancy rates and accuracy of diagnosis.

Prenatal diagnosis of Sjögren-Larsson syndrome using enzymatic methods.

Sjögren-Larsson syndrome (SLS) is an autosomal recessive disorder characterized by the presence of congenital ichthyosis, mental retardation, and spasticity. The primary biochemical defect in SLS has recently been identified to be a deficiency of fatty aldehyde dehydrogenase (FALDH), which is a component of fatty alcohol:NAD+ oxidoreductase (FAO). We monitored four pregnancies at risk for SLS by measuring FAO and FALDH in cultured amniocytes or cultured chorionic villus cells. The enzymatic results in one case using amniocytes obtained during the second trimester predicted an affected SLS fetus, which was confirmed at termination of the pregnancy. Another at-risk fetus was predicted to be affected with SLS using cultured chorionic villus cells obtained in the first trimester, and fetal skin fibroblasts confirmed a profound deficiency of FAO and FALDH. Two other fetuses were correctly predicted to be unaffected. These results demonstrate that SLS can be diagnosed prenatally using enzymatic methods.

Rare non-mosaic trisomies in chorionic villus tissue not confirmed at amniocentesis.

This paper reports eight cases of non-mosaic, rare, and typically lethal trisomies diagnosed in chorionic villi and not confirmed by amniocentesis. Four cases were 47,XX,+16; two cases were 47,XX,+2; one was 47,XX,+12; and one was 47,XY,+7. There have been no known complications in any of these gestations. These eight cases were found in a series of approximately 12,000 samples processed in our laboratory (0.07 per cent). We conclude that (1) rare non-mosaic trisomy not reflecting the fetal condition is an occasional source of diagnostic ambiguity in chorionic villus sampling; and (2) when encountered, a follow-up amniocentesis should be recommended to the patient to confirm or rule out the abnormality. We propose the term 'confined placental abnormality' to describe non-mosaic trisomies and other related abnormalities found only in chorionic tissue.

The risk and efficacy of chorionic villus sampling in multiple gestations.

Chorionic villus sampling (CVS) in the first trimester of pregnancy provides a safe and effective method for the early prenatal diagnosis of cytogenetic abnormalities in multiple gestations. In this multicentre study involving 126 twin and 2 triplet gestations primarily at risk because of advanced maternal age, the overall success rate of obtaining an adequate villus sample from each fetus was 99.2 per cent. For women of advanced maternal age, the rate of combined losses of chromosomally normal fetuses due to spontaneous abortion, stillbirths, and neonatal deaths was 5.0 per cent, compared with a 4.0 per cent total loss rate following CVS in singleton pregnancies derived from the same population (Rhoads et al., 1989). There was a 100 per cent success rate in obtaining a cytogenetic analysis; a cytogenetic abnormality was present in five of the multiple gestations (3.9 per cent) and involved seven fetuses (2.7 per cent). There were no diagnostic errors and no cases of normal cytogenetic diagnosis followed by the birth of a cytogenetically abnormal newborn. Based on cases of XX/XY admixture, cell contamination derived either from maternal decidua or the other twin occurred in 6 of 256 samples (2.3 per cent), giving an overall estimate of the frequency of cell contamination of 4.6 per cent; these cases did not present a diagnostic problem. However, there were two cases (0.8 per cent) in which the fetal sex was incorrect, due either to complete maternal cell contamination or to the possibility that in error one twin was sampled twice.

Confirmation of CVS mosaicism in term placentae and high frequency of intrauterine growth retardation association with confined placental mosaicism.

About 2 per cent of specimens from chorionic villus sampling (CVS) analysed either on direct preparation of cytotrophoblast cells or after culture of mesenchymal stroma reveal confined placental mosaicism (CPM), most commonly involving chromosomal trisomy. A significantly higher rate of prenatal loss (22 per cent) as well as the presence of intrauterine growth retardation (IUGR) has been reported among pregnancies with CPM. To evaluate more precisely the effect of these aneuploid cell lines confined to the placenta on intrauterine fetal growth and fetal survival, we have studied 34 term placentae from pregnancies with CPM diagnosed on CVS and confirmed identical mosaicism in 17 of these placentae. There was a direct correlation between a high number of aneuploid cells present at CVS and a high likelihood of their detection in term placenta. Also, the proportion of aneuploid cells in the mosaic term placentae correlated with that observed in CVS specimens. Among 17 gestations with confirmed CPM at delivery, there were six cases of IUGR identified, five in liveborns and one associated with intrauterine death.

Transvaginal chorionic villus sampling.

Chorionic villus sampling (CVS) with either transcervical catheters or transabdominal needles is a widely-accepted method for prenatal diagnosis. However, there exists a small subset of patients in whom sampling is difficult or impossible with either route because of individual anatomic variations. A new method of chorionic villus biopsy has been developed to circumvent these problems, utilizing transvaginal chorionic needle aspiration guided by an intravaginal ultrasound probe. This technique was performed successfully in 15 patients in whom villi could not be obtained by either of the conventional methods. This method now makes CVS possible in essentially all women regardless of their uterine anatomy or placental placement; it may also prove useful for very early chorionic sampling.