A method is described enabling prenatal diagnosis of glycogenosis II (Pompe's disease) within 7—10 days after amniocentesis in the 14th—16th week of pregnancy. The procedure is based on amniotic fluid cell cultivation on thin plastic foil, freezing and freeze-drying of the cells and subsequent microdissection of pieces of plastic foil each containing 70-250 lyophilized cells. These are incubated in microliter volumes of substrate. The extinction or fluorescence values are measured in microcuvettes adapted to a normal spectrofluorometer or in microcapillaries using a microscope spectrofluoiometer design. The methods developed are compared with conventional biochemical analyses.

INTRODUCTION

Hers¹ delineated the basic defect in type II glycogenosis (Pompe's disease) as a deficient activity of the lysosomal acid a-i,4-glucosidase. In patients with Pompe's disease this enzyme deficiency has been demonstrated in various tissues and organs².³. Nitowsky and Gninfeld⁴ showed that the acid α-glucosidase deficiency is also ex­pressed in leucocytes and in cultivated skin fibroblasts. This not only facilitated the diagnosis of patients and heterozygous carriers but also formed the basis for prenatal detection of this autosomal recessive disease. The first examples of prenatal diag­nosis of Pompe's disease were based on a deficiency of α-glucosidase in amniotic fluid, in uncultivated and cultivated fluid cells⁵.⁶. Later, it became apparent that the use of amniotic fluid and uncultivated cells could result in erroneous interpretations (refs. 7, 8), because of the possibility of contamination with maternal components and because of the presence in amniotic fluid of an α-glucosidase with different properties than the lysosomal acid glucosidase which is deficient in Pompe's disease'.⁹.

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Summary. 1. A method for freezing uncultured amniotic fluid cells is presented, which allows their use as pregnancy-age matched controls in prenatal diagnosis of metabolic diseases. 2. Amniotic fluid cells were successfully cultured after up to 7 days in transport, which makes prenatal diagnosis available to parents living a long way from specialized centers.

Zusammenjassung. 1. Eine Einfriermethode fiir nichtkultivierte Fruchtwasserzellen wird beschrieben, wodurch man diese wie Kontrollmuster anwenden kann bei pranataler Diagnostik von Stoffwechselkrankhciten. 2. Fruchtwasserzellen wurden erfolgreich kultiviert nach lang-zeitigem Transport (7 Tage); dieser Befund bringt eine pranatale Diagnostik im Bereich ent-fernt von spezialisierten Zentren lebender Eltern.

An increasing number of inborn errors of metabolism nan he detected in early pregnancy (Milunsky et al., 1970; Milunsky and Littlofield, 1972). Until now metabolic defects have been determined by biochemical analysis of cell homo-genates obtained from large numbers (millions) of cultured amniotic-fluid cells. As a consequence the period of time required for prenatal diagnosis in most in­stances has been very long (4—10 weeks) (Brady et al., 1971 ; Fratantoni et al., 1969; Epstein et al., 1972; Cox et al., 1970).

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This thesis deals with several methodological and practical aspects of prenatal diagnosis.

A genetic metabolic disease can only be detected in utero if the enzymatic defect or storage of a specific metabolite is expressed in cultured cells from an affected individual. This should be demonstrated for each separate disease. An^- example of such a study is presented in appendix paper I. The data in­dicate, that the enzymatic defect in the haemolytic anaemia caused by pyru­vate kinase deficiency (an autosomal recessive disease) is not detectable in fibroblasts and cultured amniotic fluid cells, since these only produce the M-type of this enzyme, whereas the anaemia is related to a deficient activi­ty of the L-type. As a consequence, prenatal diagnosis of this disease is not possible.

The prenatal diagnosis of enzymatic defects requires analysis of a sufficient number of amniotic fluid cell cultures cultivated under identical conditions and for a comparable period as the diagnostic sample. Sufficient amniotic fluid samples from normal pregnancies will generally not be available at the very moment these are required. To solve this problem a method was develop-ped to store uncultured amniotic fluid cells without loss of cell viability. This method is described in appendix paper II and has enabled us to collect suffi­cient control material for later use in prenatal diagnosis of enzymatic defects. The possible detrimental effects of transport periods of up to a few days on the viability of amniotic fluid cells was tested simultaneously and the results indicate that transport during periods of up to seven days did not interfere with succesfull cell cultivation. This means that prenatal diagnosis can be offered to people living at long distances from the center performing the ana­lysis.

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Open defects of the neural tube (anencephaly and meningomyelocele) are re­latively frequent congenital malformations of multifactorial origin. The recur­rence risk after one affected child is about 5% and after two affected child­ren it increases to + 10% (Bonaiti-Pellie & Smith, 1974). The malformation in the subsequent child may be either identical or of a different type. An affected parent has a 3 - 4% risk for an affected child (Carter, 1974). Prenatal detection of open neural tube defects is possible by analysis of the concentration of alpha-fetoprotein in the amniotic fluid (Brock & Sutcliffe, 1972; Allan et aL, 1973); closed defects, some of which may have milder clinical manifestations, cannot be detected with this method (Laurence et al., 1973) and the parents should be informed accordingly.

This recent development will be a quite common indication for prenatal moni­toring, and in most cases this will be for parents with an affected child. Elevated levels of amniotic fluid alpha-fetoprotein can in some cases also be detected in maternal serum (Brock et al., 1973). In a number of studies the value of this maternal indicator for fetal neural tube defects was explored (Harris et al., 1974; Seller et al., 1974; Wald et al., 1974). The possibili­ties for large-scale screening so far are not very promising.

Future developments in prenatal diagnosis

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About 50 severe recessive autosomal or X-linked diseases can now be detected in utero by biochemical assays of cultured amniotic fluid cells (for reviews, see: Milunsky et al., 1970; Milunsky & Littlefield, 1972; Burton et al.,1974). In autosomal recessive diseases the diagnosis of a first affected child usually is the first indication that both parents are carriers of the same gene mutation. The recurrence risk is 25%. Prenatal diagnosis in future pregnancies of these parents will only be reliable if cultured cells of the affected child are a-vailable. The prenatal diagnosis will be based on a comparison of the bio­chemical assays on amniotic fluid cells from the pregnancy at risk, control cells from a normal pregnancy cultured under identical conditions and fibro- blasts from the patient with the metabolic defect.

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When the mother is a carrier of an X-linked disease, there is a 50% chance for her son to be affected. Prenatal sex determination by chromosome analysis may help these parents to limit their families to unaffected daughters (who will be carriers in 50% of the cases). The limitations of this approach are ob­vious, but it is the only possibility for these parents to have their own child­ren, without being forced to accept a high risk for a severely affected child. This approach applies to those X-linked disorders where the basic defect is un­known, (e.g. Duchenne's muscular dystrophy, X-linked mental retardation, etc.) or is not yet detectable in amniotic fluid or cultured amniotic fluid cells (e.g. hemophilia). In a few X-linked diseases the biochemical defect is expressed in cultured amniotic fluid cells and this allows prenatal identifica­tion of an affected male fetus (Fabry's disease; Hunter's disease; the Lesch-Nyhan syndrome; for references: see Burton et al., 1974).

The most frequent examples are the recurrence risk of trisomy 21 (Down's syn­drome) and the risk for chromosomal aneuploidies at advanced maternal age (38 years and older).

The recurrence risk for trisomy 21 is 1% as calculated either from retrospective data (Pfeiffer et al., 1973) or from prospective series of prenatal diagnoses for this indication (Milunsky, 1973). The recurrence risk of other numerical aberra­tions is less well known. This is related to the question of the presence in man of a familial tendency to non-disjunction. Two types of evidence for this phenomenon are present:

a. A number of sibships have been described with aneuploidies for different chro­mosomes (Girardet et al.,1972; Bell & Cripps, 1974; Holmgren & AWhn, 1971; Hamerton, 1971).

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4 - 6% of the liveboms are affected with some form of congenital disease (Warkany, 1971; Trimble & Doughty, 1974). Accordingly, in The Netherlands each year 8000 - 12.000 children are born with a more or less severe handi­cap. In some cases morphological and/or functional abnormalities are apparent shortly after birth, as is the case in anencephaly, meningomyelocele, conge­nital heart defects, several chromosomal aberrations and inborn errors of meta­bolism. In other congenital diseases the onset of clinical manifestations occurs in early or late childhood and some sex-chromosomal aberrations will not be detected before puberty.

Finally, a number of these diseases is not manifest before adulthood, like Huntington's chorea and a number of variants of inherited metabolic disorders. The three main categories of congenital diseases are:

1 . Chromosomal aberrations, with an incidence of 1 : 200 liveborns (Jacobs et al., 1974); about 1000 patients per year in The Netherlands.

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