Prenatal diagnosis of galactosemia

Eur J Pediatr (1995) 154 [Suppl 2] : $33-$36 9 Springer-Verlag 1995

C. Jakobs W, J. Kleijer J. Allen J. B. Holton

C. Jakobs ([~) Department of Paediatrics, Free University Hospital, De Boelelaan 1117, NL-1081 HV Amsterdam, The Netherlands Tel.: ++31-20-444 2416 Fax: ++31-20-444 2422 W. J. Kleijer Department of Clinical Genetics, Erasmus University, Rotterdam, The Netherlands J. Allen Department of Clinical Chemistry, Southmead Hospital, Bristol, UK J. B. Holton Department of Child Health, Royal Hospital for Sick Children, Bristol, UK

Prenatal diagnosis of galactosemia

A b s t r a c t The experience from tlu'ee different European centres with the prenatal diagnosis o f galactose-lphosphate-uridyltransferase (GALT) deficiency is presented and the question whether or not there is a need for prenatal diagnosis of this disorder is discussed. Most prenatal diagnoses (n -- 50) have been performed by assay o f G A L T activity in cultured amniotic fluid cells. The assay used is reliable and clearly distinguishes hom o z y g o u s affected fetuses (n = 11; 0 % - 2 . 3 % of mean control e n z y m e activity) from non-(homozygous)-affected fetuses. The G A L T assay for cultured amniocytes was adapted to assay the e n z y m e directly in chorionic villi. The experience with chorionic villi comprises 23 cases with 5 affected fetuses ( 0 % - 4 . 2 % of mean control e n z y m e activity). In 36 cases galactitoI was determined in amniotic fluid supernatant by gas chro-

Introduction Although all disorders of galactose metabolism are amenable to prenatal diagnosis there is very little experience except with galactose-l-phosphate uridyltransferase ( G A I T ) deficiency (EC 2.7.7.10; McKusick 230400). It is possible to perform prenatal diagnosis of this disorder by the direct assay of GALT in chorionic villi or in cultured amniotic fluid cells, and by galactitol measurement in amniotic fluid supernatant [5]. Prenatal diagnosis is undertaken rarely at present. In this paper the experience of three major European centres is presented, and the question whether or not there

matography-mass spectrometry. This method also differentiated affected (n = 11; galactitol 5.9-10.6 gmol/1) and unaffected pregnancies (galactitol 0.23-1.6 gmol/1) clearly and has the advantage of providing a result within a day or two after amniocentesis. Prenatal diagnosis of galactosemia is undertaken rarely and sometimes for the wrong reasons, but it should perhaps be considered more seriously until better methods o f treatment are established.

Key w o r d s

Prenatal diagnosis. G A L T deficiency 9 Amniocytes 9 Chorionic villi - Amniotic fluid

A b b r e v i a t i o n s GALT galactose1-phosphate-uridyltransferase 9 GC gas chromatography 9 MS mass spectrometry 9 G/D galactosemia/ Duarte c o m p o u n d heterozygote G/N galactosemia heterozygote

is a need for prenatal diagnosis o f this disorder is discussed.

Materials and methods Amniocentesis was performed in all cases between 16-18 weeks of gestation. Amniotic fluid supematant was frozen at -20~ until analysis for galactitol and GALT activity in cultured amniotic fluid cells was measured according to Monk and Holton [12]. In chorionic villi, GALT was measured according to Kleijer et aL [9]. Galactitol in amniotic fluid supematant was determined by the Bristol group, first by gas chromatography (GC) with flame ionization detection according to Allen et al. [1, 2], and subsequently by GC and mass spectrometry (MS) (unpublished). The galaetitol

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Table 1 Prenatal diagnosis by measurement of GALT activitya in cultured amniocytes (nmol/h/mg protein)b. Affected cases have been confirmed either by determination of GALT activity in fetal fibroblasts or in the child after birth. Unaffected cases include N/N, G/N and probably G/D genotypes

Controls Mean _+SD Range n

Rotterdam group

Bristol group

47 + 14 29-82 15

75 _+ 13 58-116 31

Table 3 Prenatal diagnosis by measurement of the concentration of galactitol in second trimester amniotic fluid supernatant (btmol/ 1). Affected cases have been confirmed either by determination of GALT activity in fetal fibroblasts or in the child after birth. Unaffected cases include N/N, N/G and probably G/D genotypes Amsterdam group Bristol group Controls Mean _+SD Range n

0.80 • 0.15 ~ 0.44-1.2 30

1.38 -t- 0.53 b 0.6-2.2 20

0.72 _+0.14~ 0.50-0.95 20

At risk."

At risk:

Affected Mean Range n

0.7 0.0-1.1 3

0.13 0.0-0.5 8

Affected Mean Range n

7.6 a 5.9-9.2 4

15.9b 14.0-18.7 4

8.7~ 6.7-10.6 7

Unaffected Mean Range n

33 15-60 16

64 21-130 23

Unaffected Mean Range n

0.72 a 0.23-1.2 15

1.05b 0.5-1.8 11

0.8 lC 0.4-1.6 10

Monk and Holton [ 12] b Enzyme and protein assays were done on total cell homogenates in Rotterdam and on centrifuged homogenates in Bristol Table 2 Prenatal diagnosis by measurement of GALT activity [9] in chorionic villi (nmol/h/mg protein), a Affected cases have been confirmed in fetal fibroblasts. Unaffected cases include N/N, N/G and probably G/D genotypes Rotterdam group

Bristol group

74 + 25 38-130 15

205 + 45 122-267 18

Affected Mean Range n

0.0; 3.1 2

0.0; 0.0; 0.2 3

Unaffected Mean Range n

64 21-105 10

172 125-239 8

Controls Mean + SD Range n At risk:

a Enzyme and protein assays were performed on total cell homogenates in Rotterdam and on centrifuged homogenates in Bristol

determination in amniotic fluid supernatant by the Amsterdam group was based on stable isotope dilution GC/MS, as described by Jakobs et al. [7].

Results and discussion The results of the prenatal diagnoses for G A L T deficiency by m e a s u r e m e n t of e n z y m e activity in cultured amnio-

a Stable isotope dilution GC/MS; Jakobs et al. [7] b GC; Allen et al. [1]

~ GC/MS; unpublished The 2 cohorts are not identical cytes, chorionic villi and the galactitol determination in amniotic fluid are presented in Tables 1-3. Most prenatal diagnoses for classical galactosemia have been performed by the assay of G A L T in cultured amniotic fluid cells. The assay used [12] is reliable and can distinguish not only a h o m o z y g o u s affected from an unaffected fetus, but also a galactosemic heterozygote from a galactosemic/Duarte (G/D) c o m p o u n d heterozygote, the latter having about 1 0 % - 1 5 % of m e a n normal e n z y m e activity [5]. This degree of discrimination is important, since families where one parent is a G/D heterozygote and the other a galactosemia heterozygote (G/ N) are not u n c o m m o n . The offspring in such families could be true cases of classical galactosemia or G/D heterozygotes, and the significance with respect to terminating a p r e g n a n c y would be different in these two cases. More recently, the G A L T method was adapted to assay the enzyme directly in chorionic villus biopsies [9]. The experience with chorionic villi of the Rotterdam and Bristol groups together comprises 23 cases with 5 affected fetuses. In all cases the p r e g n a n c y was terminated and the results confirmed by determination of G A L T e n z y m e activity in fetal fibroblasts. As in cultured amniocytes the G A L T activity determination in chorionic villi m a y allow the discrimination b e t w e e n heterozygote activity and normal [5] but, due to lack of consistent information about the genotype of the n o n affected fetuses at risk' evidence for the discrimination between the heterozygous state and the h o m o z y g o u s n o r m a l state cannot be presented. A l l e n et al. [1, 2] first reported raised levels of galactitol in a m n i o t i c fluid of a g a l a c t o s e m i c fetus and sug-

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gested that this method would be useful for prenatal diagnosis of GALT deficiency. The group initially used GC with flame ionization detection but refined their method by the use of GC with MS detection. Later, Jakobs et al. [7] improved the GC/MS method by using stable isotope labelled galactitol as internal standard. The galactitol method has been used now in both laboratories in a total of 50 Cases (Table 3). It differentiated affected and unaffected pregnancies clearly and has the advantage of providing a result within a day or two after amniocentesis compared to 2-3 weeks by using cultured cells. Both control and affected at risk ranges of the Bristol (GC/MS) and the Amsterdam groups are comparable. The Bristol group, however, suggested that heterozygotes might be distinguished from normal [5], but this is not supported by the results of the Amsterdam group. The fear that this method becomes unreliable when the mother adheres to a lactosefree or lactose-restricted diet during pregnancy, which has previously been recommended for pregnancies at risk, proved unnecessary, as in two affected pregnancies galactirol accumulated in amniotic fluid despite dietary restriction [8]. This observation, together with the evidence presented elsewhere for in utero production of galactose-1phosphate [6], makes the benefit of this restriction questionable. Nevertheless, the prenatal treatment does not interfere with a reliable prenatal diagnosis based on measurement of galactitol in amniotic fluid. The Bristol group has measured the galactitol concentration in first trimester amniotic fluid supernatant of controls; there is no significant difference between these values and the established control range in the second trimester (unpublished observations). To date however, they have not had any amniotic fluid sample from a galactosemic fetus to validate the use of galactitol for prenatal diagnosis in the first trimester. At least 20 specific mutations have now been found in the gene [3, 10]. In all populations studied so far, 6 0 % 70% of the galactosemic alleles were due to a single mutation (Q188R). Other mutations occur with much lower frequency. If both parents at risk for producing a galactosaemic child carry Q188R or another known mutation, prenatal diagnosis will be possible by mutation analysis which to our knowledge, has until now not been performed. The indication for prenatal diagnosis of galactosemia is a difficult issue. The justification for aborting a galactosemic fetus is questioned by many, because it is seen as a treatable condition, although it has been known for a number of years that the intellectual outcome can be poor even with early and carefully controlled dietary treatment. Most concern is directed towards the long-term complica-

tions of the disease. Recent large scale, retrospective collaborative studies [13, 14] have emphasized the basic problems and have revealed that the number of patients with developmental delay and the degree to which they are affected are greater than earlier imagined. Specific speech impairment and ovarian dysfunction are now recognized as major problems. Growth retardation and neurological abnormalities occur, though less frequently [11, 13, 14]. There are no comprehensive data on the numbers performed, and the reasons for carrying out prenatal diagnoses for galactosemia, but it seems probable that only a small proportion is undertaken with a view to terminating affected pregnancies. This is consistent with the finding that relatively few diagnoses are done on chorionic villus biopsy because, although the technique has advantages, the increased risk of obtaining the specimen could only be justified if termination is being considered. Among the reasons why so few diagnoses are performed with possible abortion in mind, a lack of understanding, or of acceptance of the poor long-term outcome in galactosemia on the part of the counsellor must be important. It is also obvious that counselling the parents is particularly difficult when the index child in the family is alive and being enthusiastically treated. In this connection it is noteworthy that existing series of prenatal diagnoses for galactosemia contain a preponderance of families in which the index child succumbed in the neonatal period [4], although there is no inherent reason why prenatal diagnosis should be more necessary in this group. Other reasons encountered for prenatal diagnosis for galactosemia include: (1) the wish of parents to have foreknowledge of another affected child; (2) the relative ease with which a prenatal diagnosis can be made in comparison to a definitive postnatal diagnosis, with a view to very early treatment in positive cases; (3) to determine whether maternal galactose restriction is required during pregnancy. In our opinion, the first two of these reasons do not justify prenatal diagnosis. With regard to maternal galactose restriction available biochemical evidence is insufficient to suggest any advantage to the fetus [6, 8]. In addition, analysis of data from existing galactosemia surveys [13, 14] indicates that there may be little or no benefit to the long-term progress of the patient. Therefore, prenatal diagnosis for the intended prenatal treatment alone seems not justified. At present prenatal diagnosis is undertaken rarely and sometimes for the wrong reason, but it should perhaps be considered more seriously until better treatment modes are established.

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References 1. Allen JT, Gillett M, Holton JB, King GS, Pettit BR (1980) Evidence of galactosemia in utero. Lancet I: 603 2. Allen JT, Holton JB, Gillett M (1981) Gas liquid chromatographic determination of galactitol in amniotic fluid for possible use in prenatal diagnosis. Clin Chim Acta 110:59-63 3. Elsas LJ II, Langley S, Paulk EM, Hjelm LN, Dembure PP (1995) A molecular approach to galactosemia. Enr J Pediatr 154 [Suppl 2]: $21-$27 4. Holton JB, Raymond CM (1980) Prenatal screening for galactosaemia. In: Burman D, Holton JB, Pennock CA (eds) Inherited disorders of carbohydrate metabolism. MTP Press, Lancaster, UK: 141-147 5. Holton JB, Alien JT, Gitlett MG (t989) Prenatal diagnosis of disorders of galactose metabolism. J Inherited Metab Dis 12 [Suppl 1]: 202-206

6. Irons M, Levy HL, Piischel S, Castree K (1985) Accumulation of galactose- 1phosphate in the galactosemic fetus despite maternal milk avoidance. J Pediatr 107:261-263 7. Jakobs C, Warner TG, Sweetman L, Nyhan WL (1984) Stable isotope dilution analysis of galactitol in amniotic fluid: an accurate approach to the prenatal diagnosis of galactosemia. Pediatr Res 18:714-718 8. Jakobs C, Bakker HD, Gennip AH van, Przyrembel H, Kleijer WJ, Niermeijer MF (1988) Dietary restriction of maternal lactose intake does not prevent accumulation of galactitol in amniotic fluid of fetuses affected with galactosemia. Prenat Diagn 8:641-645 9. Kleijer W J, Janse HC, Diggelen OP van, Macek M, Hajek Z, Gillett M, Holton JB (1986) First-trimester diagnosis of galactosemia. Lancet I: 748

10. Leslie ND, Immerman ED, Flack JE, Flavez M, Friedovich-Keil JL, Elsas LJ (1992) The human galactose-l-phosphate uridyltransferase gene. Genomics 14:474480 l 1. Lo W, Packman S, Nash S, Schmidt RN, Ireland S, Diamond I, Ng WG, Donnell GN (1984) Curious neurologic sequelae in galactosemia. Pediatrics 73:309-312 12. Monk AM, Holton JB (1976) Galactose-1-phosphate uridyltransferase in cultured cells. Clin Chim Acta 73: 537-546 13. Schweitzer S, Shin YS, Jakobs C, Brodehl J (1993) Long-term outcome in 134 patients with galactosemia. Eur J Pediatr 152:36-43 14. Waggoner DD, Buist NRM, Donnell GN (1990) Long term prognosis in galactosaemia; results of a survey of 350 cases. J Inherited Metab Dis 13: 802-818