Fetal hepatic alpha-fetoprotein mRNA expression in fetuses with trisomy 21 and 18 at 12–15 weeks gestation

Fetal hepatic alpha-fetoprotein mRNA expression in fetuses with trisomy 21 and 18 at 12- 15 weeks gestation Maria L. Brizota, Andrew T. Mckieb, Constantin S. von Kaisenberg”, Farzin Farzanehb, Kypros H. Nicolaides* a aHawis Birthright Research Centre for Fetal Afe&ine, King’s College Hospital Medical School, Demnark HI& London, SE.5 8RX. UK bDepartment of Molecular Medicine. K&g’s College Hwpital Me&cal School, Denmark Hilt, Lot&n, SE5 8RX, UK Received

4 July 1995; revision

received

24 July 1995; accepted

26 July 1995

-AM

In thisstudyweexaminedalpha-fetoprotein(AFP) mRNA expression in fetal liver at 12- 15 weeksof gestationin trisomy 21 (n = 13), thorny 18(n = 5) and control fehues(n = 24). No significant ditkence was found in the steady-state level of fetal liver AFP mRNA levels in

either of the two trisomy groups studied. These findinga suggest that the d4weasein maternal serumAFP concentration found in trisomic pregnancies is unlikely to be the ConseQuenceof impaired transcription of the AFP geneby the fetal liver. Keyuotdr: Alpha-fctoprotein (AFP); Trisomkq Geneexpression;Fetal liver; Pregnancy

1. Iaaolpctloa

Human alpha-fetoproteiu (AFT) gene is located on the long arm of chromosome 4 and has a high degrez of sequence homology with albumin VI. It is initially synthesized by the yolk sac and later predominantly by the fetal liver [6]. In pregaa&es with fetal trisomy 21 or 18 the maternal serum AFP concentration is decreased [2,9] and this was postulated to be the consequence of impaired fetal l

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hepatic synthesis[lo]. The aim of the presentstudy was to investigatethis hypothesis by determining fetal hepatic AFP mRNA levels in normal and trisomic fetusesat 12-15 weeks of gestation. 2. Materials and methads Specimensof liver were collected from 13 trisomy 21 fetuses, five trisomy 18 fetusesand 24 normal fetusesafter surgical termination of pregnancy at 12- 15weeks of gestation. The diagnosis of trisomy was made by chorion villus sampling in singleton pregnancies referred to our centre because of increased fetal nuchal translucencythicknessdetectedat routine ultrasound examination [ 13,141.Liver tissue was obtained immediately after the procedure, snap-frozen in liquid nitrogen and stored at -70°C. In all cases,gestational age was determined by ultrasound measurementsof fetal crown rump length (CRL). The study was approved by the hospital ResearchEthics Committee and tissuecollection was made in accordance with the Polkinghorne guidelines [16] on the researchuse of fetal material. 2.1. RNA extraction and blotting Total RNA was extracted from 200 mg of frozen liver using a modified version of the singlestepprotocol of Chomcxynskiand Sacchi[3]. For Northern blot analysis,samplescontaining 30 pg of total RNA were loaded onto a denaturing gel containing 1% agarose, 6 ml of 37% formaldehyde and transferred onto nylon membranes following the manufacturers instructions (Gene screen, Dupont, Stevenage,UK). RNA slot blots were also performed using a slot manifold (HybriSlotm Manifold, BRL-Bethesda Research Laboratories, USA), to facilitate comparison of hybridization signals by densitometric measurements. 2.2. DNA probes The probe for #-actin usedas a control for loading and transfer, was a 1.1-kb pst1 fragment of plasmid pAL41 as described by Alonso et al. [l]. The AFP probe was obtained as a plasmid (pHAF2) and this was kindly donated by Professor T. Tamaoki. This was cut out as a > 900 bp (base pair) Pst I fragment [11,12]. This fragment was excisedfrom a low melting point gel, purified using Wizard PCR preps (Promega, Southampton, UK) and labelled with [“P]dCTP according to the hexanucleotide priming method of Feinburg and Vogelstein [4]. Membranes were prehybridii with a solution of 0.2% BSA, 0.2% polyvinylpyrrolidone (m.w. 40 000), 0.2% ficoll (m.w. 400 000), 50 mM Tris-HCI (pH 7.9, 0.15% sodium pyrophosphate, 1.0% SDS, 10% dextram sulphate, 50% formamide, 1.0 M NaCl and denatured salmon sperm DNA (0.1 mg/ml) for at least2 h. The labelkd probes were then added and the hybridisation continued overnight. Prior to autoradiography, membranes were washed stringently with a solution of 0.2 x SSC, 0.2 x SSC + 0.1% SDS at 65°C for 1 h or more and then placed in contact with films (Kodak RX) in cassettescontaining intensifying screensand left exposed at -70°C for 2-20 h. Autoradiographies were led using a densitometer (GDS 2000, Mitsubishi) and the densitometric scoresof AFP were normal&d to &a&in mRNA.

ML. B&of et al. /Early Human Development 44 (19%) 155-159 CONTROL

rRlSOMY

21

AFP

ACTIN

Fig. 1. Representative Northern blot analysis of liver RNA hybridized with an AFP specific DNA probe and &actin probe in 9 trisomy 21 fetuses (T21) and normal controls (C) matched for gestational age. The bands represent the 2.4-kb AFP gene and 1.9-Lb &tctin gene.

2.3. Statistical anaiysis

The median was calculated for liver AFP mRNA levels in normal controls and trisomic fetusesfor eachgestational age. Mann-Whitney test was used to determine the significanceof differences between the two groups. 3. Resdts

A representative Northern blot analysis of liver AFP mRNA from trisomy 21 fetusesand normal controls is shown in Fig. 1. Slot blot RNA was also performed and laser densitometric determination of relative abundance of fetal liver AFP mRNA from normal and control fetuseswas ana@&. In the normal fetuses,the median normalised densitometric scorefor fetal liver AFP mRNA was relatively high at 13 weeksof gestation (3.24) compared to median scoresat 12, 14 and 15 weeks’ gestation(1.29,0.75, and 0.79, respectively,Fig. 2). A similar pattern of change with gestationwas observedin pregnancieswith trisomy 21 or trisomy 18fm; mediin scoreswere not significantly different from those in matched controls (P = 0.27 and P = 0.48, respectively). 4. DlsTcudon

The fmdmgs of this study suggestthat in normal fetusesat 12-15 weeksof gestation, the highest level of hepatic AFP mRNA expressianis at 13weeks.Thesefmdings are campatible with the results of previous reports that maximum fetal platnrta cancentration of AFP is reachedat 13weeksof gestationand declinesexpo~~tiahy there&w [s]. This decreasehas beenattributed to decry& synt$asisand the diiutional e%ct of expanding plasma volume. In maternal blood, AFP leveis rise throughout the fast and second trimesters and decline only after 32 weeks. This

158

ML. Brizot et al. /Early Human Development 44 (1996) 155-159

AFP (NDS) 10

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Fig. 2. Normdial den&WC scores (NDg) of liver mRNA encoding alpha-fetoprotein (AFP) in controls 0, trisomy 21 (A) and t&my 18 (U) fetuaee pbtted against gestation in vi&a.

increaseis thought to be due to increasedplacental permeability to fetal plasmaproteins with advancing gestation [a]. In trisomy 21 and trisomy 18 fetusesat 12- 15 weeks of gestation, hepatic AFP mRNA expressionis not significantly different from normal. This is compatible with the fmdings of a previous study of trisomy 21 fetusesat 17-22 weeks which also reported no significant Merence from normal in AFP mRNA levels [8]. Thesefindings suggestthat the decrease in maternal serum AFP concentration found in txisomicpregnanciesis unlikely to be the consequenceof impaired transcription of the AFP geneby the fetal liver. Consequently,the low levels of maternal serum AFP in trisomy 21and 18pregnanciesmay be due to post-tmnscriptional alterations such

ML. Brizot et al. /Early Human Development 44 (19%) 155-159

1%

as mRNA degradation, protein stability, impaired protein transport across the placenta, modified serum stability of AFP or increased degradation in maternal serum of the released protein. Similarly, studies that measured fetal serum AFP concentrations from at&&d pregnancies reported normal levels and suggested that low AFP levels in amniotic fluid and maternal serum may be due to impaired fetal kidney function and impaired placental transport, respectively [ 15,17,18). Aeknowkdgememts

M.L. Brizot was supported by a grant from CNPq (Conselho National Pesquisa e Desenvolvimento Cientifico e Tecnologico - Brazil).

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References [I] Alonso, S., Minty. A., Bourlet, Y. and Buckingham, M. (1986): Compsrison of three a&n-coding sequences in the mouse; evolutionary relationships between the actin genes of warm-blooded vertebrates. J. Mol. Evol., 23, 1l-22. [2] &nick, J.A., Palomaki, G.E. and Gsathanondh, R. (1990): Prenatal screening for ttisomy 18 in the second trimester. PrenataJ Diagn., 10,546-548. [3] Chomcaynski. P. and Sac&i, N. (1987): Single-step method of RNA isolation by acid guanidium thi~phenol-chloroform extraction. Anal. Biocbem., 162, 156-159. [4] Feinburg, A.P. and Vogelstein, G. (1983): A tcchniquc for radiolabelhng DNA restriction endonucka8e fragments to a high spc6ifii activity. Anal. Biochem., 132 6-13. [J] Gitlin, D., Perricelli. A. and Gitlin. GM. (1972): Synthesis of alpha-fetoprotein by liver, yolk sac and gastromtestinal tract of the human conceptus. Cancer Rcs., 32, 979-982. [6] Gitliu. D. (1975): Normal biology of alpha-fetoprotein. Ann. New York Acad. sci., 259, 7-15. [7] Harper, ME. and Dugakzyk, A. (1983): Linkage of the evolutionarily-related serum albumin and a-fetoprotein genes within ql l-22 of human chromosome 4. Am. J. Hum. Genet., 35, 565-572. [8] Kronquist, K., Dreaxen, E., Keener, S., Nicholas, T. and Crandall, B. (1990): Reduced fetal hepatic alpha-fetoprotein levels in Down’s syndrome. Prenatal Diagn., 10, 739-751. [9] Merkatx, LR., Nitowsky, H.M., Macri, J.N. and Johnson, W.E. (1984): An association between low maternal serum alpha-fetoprotein and fetal chromosomai abnormalities. Am. J. Obstet. Gyuccol., 148.886-894. [lo] Mihmsky. A. (1992): The USCof biochemical markers in maternal serum screening for chromosome defects. In: Genetic Disorders and the Fetus, 3rd edn., pp. 565-592. Editor: A. Mibmsky. John Hopkins Press, Baltimore, MD. [ll] Mormaga, T., Sakai, M., Wegmann, T.G. and Tamaoki, T. (1982): Alphafetoprotein messenger RNA in human embryonal carcinoma grown in nude mice, and cloning of it8 complementary DNA. Oncodcv. Biol. Med., 3, 301-313. 1121 Morinaga, T., Sakai, M.. Wegmann. T.G. and Tamaoki, T. (1983): Primary structure of human afetoprowin and its mRNA. Biochemistry, 80,~4608. 1131 Nicolaides, K.H., Brixot, ML., Patel, F. and Snijders, R. (1994): Comparison ofchorion villus sampling and amniocentesis for fetal karyotyping at 10-13 weeks gestation. Lancet, 344,435-439. [14] Niilaides, K.H., Briaot, M.L. and Snijders, R. (1994): Fetal m&al translucency: ultrasound screen@ for fetal trisomy in the fmt trimester of pregnancy. Br. J. Obstet. Gynaccol., 101. 782-786. [15] Nicohni, U., Hubinont, C., Santolaya, J., Fisk, N.M., Rode& C.H. and Johnson, R.D. (1988): FetaJ serum alpha-fetoprotein in fetuam with chromoromnt abnormahties. Lancc& ii, 1316-1317. [16] PoJkinghome, 1. (1989): Review of the Gu&nce on the Research Use of Fetuses and Fetal Material, Cm762. HMSO, London. [17] Sdkr, M.J. (1990): Alpha-fetoprotein in midtrimester D0wn’S ayndromc fetal serum. J. Med. Ganet., 27, 240-243. 1181 van L&h, J.M.M., Beekhuis, J.R., van Loon, A-J., Mantis@, A., de Wolf, B.T.H. and Breed, S.P.M. (1991): Alpha-fetoprotein in fetal serum, amniotic fluid, and maternal serum. Prenatal nira.m 11 1;7<-rem