Prenatal diagnosis of pyruvate carboxylase deficiency

Cfzmcu Chimicu A&z,

119 (1982) 121-127

Elsevier Biomedical Press CCA 2034

Prenatal diagnosis of pyruvate carboxylase deficiency C.Marsac

p * , Ch. Augereaua, G. Feldman b, B. Wolf b, ‘T.L. HansenC and R. 13ergerd

a iJ7.5INSERM

Btochzmze Wdicufe, Faczdt& de M&deczne Necker-Enjunts-Muludes~ 156, rue de Vaugzrurd 7.5730 Purrs Cidex 15 {Francei, ’ Depurtment of Human Genetzcs and Pediutrzcs, Medzcuf College of Vzrginzu. Richmond VA 23298 (USA). ‘ Departmentof Cfzntcaf Genetzcs, Rzks~~~itulet, Unzuersr+ of Capenhlrgen (Denmark), * Depurtment of Pedzutrzcs, Lahorutozy of Developmentrtf Bzocfzemist~. State Unzversi!v of Groningen, Bfoemszngef IO (The Netherlands)

(Received July 13th: revision October 16th, 1981)

Deficient pyruvate carboxylase activity was found in the amniotic fluid cells obtained from a pregnancy at risk for pyruvate carboxylase deficiency. The results were corroborated by four independent laboratories. The diagnosis was confirmed by the demonstration that pyruvate carboxylase activity was undetectable in all the tissues examined from the aborted fetus. This represents the first prenatal diagnosis of pyruvate carboxylase deficiency.

Introduction Pyruvate carboxylase (PC), which catalyzes the conversion of pyruvate to oxaloacetate, is a key enzyme of the gluconeogenic pathway. Isolated deficiencies of this enzyme are rare, but represent a well-described clinical entity [l-4]. Individuals with this disorder are characterized clinically by neurological deterioration and growth retardation; biochemical findings include lactic acidosis, ketosis and hyperpyruvicemia. No efficient therapy is currently available and the course of the disease is usually fatal within the first year of life. PC activity can be demonstrated in all the tissues examined, including liver, kidney, brain, leukocytes and skin fibroblasts [l]. In recent reports Feldman and Wolf [S] and Hansen and Christensen [6,7] measured PC activity in amniotic fluid cells and predicted that PC deficiency could be prenatally diagnosed. We have had an opportunity to monitor the pregnancy of a couple whose first two children died

* To whom correspondence should be addressed. 0009-898 1/82/0000-OOOO,/$O2,75 0 i 982 Elsevier Biomedical Press

of PC deficiency [S]. This represents with PC deficiency.

the first prenatal

diagnosis

of a fetus affected

Materials and methods Chemicals “C-labelled barium carbonate was obtained from CEA (Commissariat a I’Energie Atomique). NaHt4C0, was prepared by displacing Ba14C0, with 2.5 mol/l H,SO, and collecting 14C0, in 1 mol/l NaHCO,. Malate dehydrogenase was obtained from Boehringer-Mannheim (France). Other biochemicals were purchased from Sigma Chemical Co (Saint Louis, MO, USA). Fibroblast and amniotic fluid cell cultures Human skin fibroblasts were grown in RPM1 1640 (Eurobio, France) medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 pg/ml streptomycin, 10 pg/ml terramycin and 500 pg/ml Lincocin. The cells were fed twice weekly, and harvested 5 to 7 days after confluency by treating them with 0.25% trypsin 2X (Choay Chimie) in phosphate buffered saline (Ca’+- and Mg’+ -free Dulbecco’s “A” solution; PBS). The cells were collected by low speed centrifugation (2000 X g), washed twice with PBS, and the pellets frozen at - 80°C. Fibroblast cultures were derived from various unaffected controls, an affected sibling (index case), his parents and the aborted fetus. Amniotic fluid cells were obtained from unaffected controls (amniocentesis performed for other indications) and the fetus at 17-weeks gestation. The cell cultures were free from mycoplasma contamination [9]. Skin fibroblasts, liver, kidney and brain tissues were obtained from the fetus immediately following abortion. Post-mortem liver, kidney and brain tissues were obtained from the index case and another unpublished patient with PC deficiency (two patients with congenital lactic acidosis). Enzyme assays Frozen cells were suspended in buffer containing 0.2 mol/l sucrose, 20 mmol/l triethanolamine-HCl buffer, pH 7.4, and 1 mmol/l EDTA. The cells were disrupted by sonication (1 X 5 s burst at setting 30 on an Artek Ultrasonic Dismembrator). PC, propionyl CoA carboxylase (PCC), P-methylcrotonyl CoA carboxylase (PMCC) and phosphoenolpyruvate carboxykinase (PEPCK) were assayed by determining the enzyme-dependent incorporation of 14C-bicarbonate into the non-volatile product following procedures [ 10,111. The standard reaction mixture for PC in a total volume of 0.1 ml was: 50 mmol/l Tris-HCl, pH 7.5, 10 mmol/l MgCl,, 2 mmol/l neut.ralized sodium ATP, 50 mmol/l NaH14C0, (40 pCi/mmol), 0.36 U malate dehydrogenase, 0.1% Triton X- 100, 5 mmol/l sodium pyruvate, 0.5 mmol/l acetyl CoA, 50 mmol/l KCl. Blanks were prepared by omitting acetyl CoA. For the PCC and PMCC assays, the substrates 3 mmol/l propionyl CoA or 3 mmol/l P-methylcrotonyl CoA, respectively, were added in place of pyruvate and acetyl CoA; malate dehydrogenase was omitted. Blanks were prepared by omitting the specific substrate.

1516ell7 (n=22)

Mean normal activity (n = number studied)

* * ** ***

1863~127 (n= 18)

2335 1530

2066 **

Standard deviation Two different controls simultaneously processed. Control simultaneously processed. Two different assays of the same cells.

Mother Brother

*** ***

990 * 85 (n= 14)

1336

I Ill

**

2908*336 or= 13)

3180

7602 120 (n=9)

401-802 195 412 IO

*

(II= 14)

5292212

517-1718 425 390 (13

PC

PEPCK

PC

MCC

PC

PCC

Feldman

’ protein) Hansen

(pmol.min-“mg

Marsac

IN FIBROBLASTS

K K

ACTIVITIES

1196 ** 121-154 616-790 15

I

Controls Father K

ENZYME

TABLE

*

040 *

863% 102 (,1=8)

723-l 1617 I 602 1192

PCC

and Wolf

700

1000 **

PC

Berger

E

For PEPCK activity assay the reaction mixture contained in a total volume of 0.1 ml: 60 mmol/l Hepes buffer, pH 6.6; 1.2 mmol/l MnCl,; 6 mmol/l MgCl,; 0.1% Triton X-100; 1 mmol/l NADH; 50 mmol/l NaH14C0, (40 pCi/mmol); 1.2 mmol/l IDP; 0.36 U malate dehydrogenase and 3 mmol/l phosphoenolpyruvate. Blanks were prepared by omitting phosphoenolpyruvate. The reactions were initiated by the addition of 40 ~1 cell sonicate and assay performed in duplicate. All four enzyme assays were linear for O-30 min and from 20 to 200 pg extract protein per assay tube. The reactions were stopped by adding 10 ~1 50% trichloracetic acid. The tubes were centrifuged at 2000 X g for 1 min and 50 ~1 of the supernatant was transferred to a scintillation vial. The contents of the vial were evaporated for 10 min to remove unincorporated 14C0,. Two ml Unisolve scintillation fluid (Koch-Light Laboratories Ltd.) were added to each vial and counted in a Packard-Tricarb scintillation counter. Protein concentrations were determined by Spector’s method utilizing Coomassie Blue [ 121. Feldman and Wolf measured PC and PCC activities by the procedure of Feldman and Wolf [ 131 and Wolf et al [ 141, respectively. PC activities were measured by Hansen as described [7] by a method modified as described by Utter and Keech [ 151 and Berger used the method described by Atkin et al [ 11. Results . Enzyme activities of PC, PCC, MCC, PEPCK in fibroblasts from the parents of the fetus and his previously affected sibling are shown in Table I. Except for PC, the activities of all enzymes were normal. PC activity in the fibroblasts of the index case was 0 to 2% of that in control cells. All laboratories obtained PC activities

TABLE

II

ENZYME

ACTIVITIES

Assay

Cell source

I

Control I Control 2 Control 3 Fetus K

IN AMNIOTIC

FLUID

CELLS

Marsac

Feldman

and Wolf

PC

PC

PCC

2178 1033 I459 IO

3 009

4887

806 788 802 70-33

441 317 129 0

I219 I459 1415 2187

645 341 IO-12

631 428 I3

620 I626 659

588-t217 (n= IO)

46lt222 (!I= 14)

I084 IO

2 100

3

Control Control

I021 594 IO

6 776 4768 4768

968 _C98 (n=20)

2 908 - 366 (n= 13)

Fetus K

Hansen PEPCK

Control I Control 2 Fetus K

Mean normal activity ( n = number studied)

-’ protein)

PC

2

I 2

(pmol.mn-‘.mg

Berger PC

1000 100

III

ACTIVITIES

1043 1213 10

Control 3 ** Fetus K **

1 **

child *

I 103

778

749

854 631 340 5

390

2300 40 40

g -’

tissue)

(nmol.min-‘-

protein)

ms - ’

269 348

318

PCC

* OR FETUSES

(pm01 min- ‘.

MCC

PC

PCC

CHILDREN

PC

FROM Liver

TISSUES

Skin fibroblasts

IN POST-MORTEM

2 **

Control Control

Control * Brother IL * Another PC-deficient

ENZYME

TABLE **

105 53

239

6200 9ooo 6000

PEPCK

66 0

50

1300 20 20

g -’

mintissue)

(nmol

PC

Kidney

‘.

0.045

0.96

0.023 0.029

I

mg-’

‘. protein)

(nmol . min-

PC

Brain

1.82

1.4

1.4 3.6

PCC

intermediate between normal and deficient values in the fibroblasts of both parents. PC activity m amniotic fluid cells from the fetus was found to be deficient (0 to 10% of control PC activity) in all four laboratories (Table II). PEPCK and PCC activities were normal, the latter excluding the possibility of multiple carboxylase deficiency. Based on the above findings the pregnancy was terminated and the fetal tissues were obtained for enzyme analyses (Table III). PC activity was undetectable in the fibroblasts, liver, kidney, and brain of the fetus, whereas PCC, PMCC. and PEPCK were normal in those tissues examined. These results concur with those in postmortem tissues of the index case and in another unpublished case of PC deficiency. Discussion PC deficiency was confirmed in the index patient by the absence of PC activity in his cultured skin fibroblasts and in post-mortem tissues. The parents each had 40-60% of normal PC activity in their fibroblasts, consistent with the conclusion of Atkin [16] that PC deficiency is inherited as an autosomal recessive trait. Because PC deficiency is characterized by severe developmental delay and ultimately death, and is not amenable to any known form of treatment, there is an obvious need for prenatal diagnosis of the disorder. The finding of less than 10% of PC activity in amniotic fluid cells from the at-risk fetus as well as normal activity of the various other enzymes, as determined by all our laboratories, indicated that the fetus was affected with isolated PC deficiency. This diagnosis was confirmed by failure to detect PC activity in four different tissues from the aborted fetus. These results are consistent with similar enzyme findings in post-mortem tissues of the index case and an unpublished case of PC deficiency. As suggested by Feldman and Wolf [5] and Hansen and Christensen [6,7], our results indicate that PC deficiency can be diagnosed prenatally. Furthermore, the prenatal diagnosis of a metabolic disorder for the first time can most confidently be demonstrated by having more than one competent laboratory perform the analyses. PC deficiency can now be added to the growing list of prenatally diagnosable inherited metabolic disorders. Acknowledgements This work was supported by NIH grants AM25675 and AM26127. G.L. Feldman was supported by an NIH predoctoral training grant (GM07492). B. Wolf is a recipient of an NIH Research Career Development Award (AM00677). The authors thank A. Moncion for her technical assistance and Dr. Saudubray and Dr. Vidailhet who referred their patients to us. They also thank Mr. and Mrs. BouC for their help. The financial support of the “Conseil Scientifique” of the “Faculte de Medecine Necker-Enfants-Malades” is gratefully acknowledged.

127

References I Atkin BM, Utter MF, Weinberg MB. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase activity in leukocytes and fibroblasts from a patient with pyruvate carboxylase defictency. Pedtatr Res 1979; 13: 38-43. 2 Saudubray .JM, Marsac C, Charpentier C, Cathelineau L, Besson-Leaud M, Leroux JP. Neonatal congenital lactic acidosis with pyruvate carboxylase deficiency m two siblings Acta Paedtatr Stand 1976; 65: 717-724. 3 Van Biervliet JPGM, Brumvis L, Van der Hetden C, Ketting D, Wadman SK, Willems JL, Monnens JLLAH. Report of a patient wtth severe, chronic lacttc acidaemia and pyruvate carboxylase deficiency. Develop Med Child Neural 1977; 19: 392-401. 4 De Vivo DC, Haymond MW, Leckre MP, Bussman YL, McGougal DB, Paghara AS. The climcal and biochemtcal implication of pyruvate carboxylase deficiency. J Clm Endocrinol Metab 1977: 45: 1281-1296. 5 Feldman CL, Wolf B. Measurement of pyruvate carboxylase activity in amniottc fluid cells. Pedtatr Res 1979; 14: 153 6 Hansen TL, Christensen E. Studies on pyruvate carboxylase from human diploid fibroblasts and amniotic fluid cells. The Society for the Study of Inborn Errors of Metabolism. 16th annual meeting, Bristol, England: 1978. 7 Hansen TL, Christensen E. Studies on pyruvate carboxylase from cultured human fibroblasts and amniotic fluid cells. J Inher Metab Dis 1979; 2: 23-28. 8 Marsac C, Augereau Ch, Boue J, Vidailhet M. Antenatal diagnosis of pyruvate carboxylase deftctency. Lancet 1981; I: 675. 9 Hamet M, Bonissol C, Cartier P. Enzymatic activities in purine and pyrimidine metabolism in tune mycoplasma species contaminating cell cultures. Clin Chim Acta 1980; 103: 15-22. IO Hsia YE, Scully KJ. Propionic acidemia: diagnosis by enzyme assay m frozen leukocytes. J Pediatr 1973; 83: 625-628. I I Ballard FJ. Hanson RW. Phosphoenolpyruvate carboxykinase and pyruvate carboxylase m developing rat liver. Biochem J 1967; 104: 8666871. 12 Spector T. Refinement of the Coomassie Blue method of protein quantitation. Analyt Btochem 1978: 86: 142-146. 13 Feldman GL, Wolf B. Evidence for two genetic complementatton groups in pyruvate carboxylasedeficient human fibroblast cell lines. Biochem Genet 1980; 18: 6 17-624. 14 Wolf B, Hsia YE, Rosenberg LE. Biochemical differences between mutant propionyl CoA carboxylases from two complementation groups. Am J Hum Genet 1978; 30: 455-464. 15 Utter MF, Keech DB. Pyruvate carboxylase. I. Nature of the reaction. J Biol Chem 1963: 23X: 2603-2608. 16 Atkin BM. Carrier detection of pyruvate carboxylase deficiency in fibroblasts and lymphocytes. Pediatr Res 1979; 13: 1101-l 104.