Mutations in mitochondrial transfer ribonucleic acid genes in preeclampsia

Mutations in mitochondrial transfer ribonucleic acid genes in preeclampsia Terese Folger0, MD, ~ Norunn Storbakk, MSc," Torberg Torbergsen, MD, PhD," and P~I Oian, MD, Phi) ~ TromsO, Norway OBJECTIVE: We investigated whether maternally inherited mitochondrial deoxyribonucleic acid mutations could be associated with preeclampsia because mendelian models fail to explain all the aspects of inheritance in preeclampsia. STUDY DESIGN: In two families with a high occurrence of preeclampsia and eclampsia the 22 mitochondrial transfer ribonucleic acid genes were sequenced in eight and three women, respectively. RESULTS: An A-to-G mutation in transfer ribonucleic acid~ at nucleotide 3243 was found in one family, and in the other there was an A-to-G mutation at nucleotide 12308 in transfer ribonucleic acid~u[CUN]. Mutations of mitochondrial transfer ribonucleic acid genes are generally considered to have systemic consequences, which might explain the multiorgan involvement seen in preeclampsia. CONCLUSION: We report for the first time mutations in mitochondrial transfer ribonucleic acid genes in two families with a high occurrence of preeclampsia and eclampsia. Mitochondrial dysfunction caused by point mutations of mitochondrial deoxyribonucleic acid is maternally inherited, but in the case of mutations of nuclear genes mitochondrial dysfunction can be inherited as an autosomal recessive or dominant trait. (AM J OBSTETGYNECOL1996;174:1626-30.)

Key words: Preeclampsia, genetics, mitochondrial dysfunction, mitochondrial transfer ribonucleic acid mutations

Preeclampsia seems to be strongly heritable, and differs ent genetic models have been discussed. A simple recessive model with the genes acting in the mother and a model of a dominant gene with 50% penetrance also acting in the mother have been suggested) -3 Mitochondrial inheritance has not been excluded and could explain aspects of inheritance not accounted for by other models. Mitochondria harbor the only extranuclear deoxyribonucleic acid (DNA) in the cell. The mitochondrial DNA (mtDNA) codes for 13 suhunits of the enzyme complexes essential for oxidative phosphorylation. Nuclear coded subunits are transported into mitochondria and assembled with mitochondrial coded subunits. The mtDNA also encodes two ribosomal ribonucleic acids (RNA) and 22 transfer RNAs (tRNAs) necessary for mitochondrial protein synthesis.4 The mtDNAs are inherited exclusively from the mother. The ovum contains thousands of mitochondria but the sperm only a few. Even if the midpiece of the

bu the Departments of Obstetrics and Gynecologf and Neurology,~ University of Tromsr Received for publication May 25, 1995; revised September 18, 1995; accepted September28, 1995. Reprint requests: Terese Folgerr MD, Department of Obstetrics and Gynecology, University of TromsO,N-9038 Troms#, Norway. Copyright 9 1996 by Mosby-YearBook, Inc. 0002-9378/96 ~5.00+ 0 6/1/69617 1626

spermatozoon should penetrate the ovum, sperm mtDNAs might be eliminated by several mechanisms. Diseases caused by mtDNA nmtations are therefore maternally inherited. The mother transmit the mutation to all daughters and sons, but only daughters pass it to succeeding generations) In oxidative phosphorylation, which takes place in the mitochondria, adenosine triphosphate (ATP) is formed. ATP is essential for cell structure and function, and 95% of the total ATP requirements of human cells is supplied by the mitochondria. ~ Reduced mitochondrial ATP production results in symptoms from various tissues, depending on metabolic demands. 5 A woman with mitochondrial dysfunction might have sufficient ATP production in the nonpregnant state, but it might be insufficient during pregnancy when metabolic demands increase. Redman 7 in 1993 described preeclampsia as a two-stage disease. The first stage comprises processes affecting the maternal spiral arteries, which result in a deficient maternal blood supply to the placenta. The second stage encompasses the effect of the ensuing placental ischemia on both mother and fetus. Stage one is preclinical without symptoms and stage two is clinical. A mitochondrial dysfunction may explain both stages. We report here mutations in mitochondrial tRNA genes in two familes with a high occurrence of preeclampsia and eclampsia.

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Patients and methods Family A. In this three-generation family with mitochondrial dysfunction 8 2 of 5 sisters in generation I had eclampsia. Another had severe preeclampsia with intrauterine death in 6 of her 10 pregnancies. There was a remarkably high incidence of a b n o r m a l pregnancies (abortions, preterm births, stillbirths, and pregnancy-induced hypertension) in generation II. Patient 11, the mother of generation III, had 2 spontaneous early abortions and 4 childbirths. In the first and third pregnancy she had preeclampsia with blood pressures of 150/100 and 170/120 mmHg. In generation III patient 16 had severe preeclampsia in 2 pregnancies and pregnancyinduced hypertension in another. One sister (patient 17) had preeclampsia and patient 18 had eclampsia. The youngest sister (patient 19) had a normal pregnancy until the thirty-seventh week, when she had a spontaneous delivery (Fig. 1). Family B. There were no signs of mitochondrial dysfunction in the nonpregnant state. Patient 1 had severe preeclampsia in both her pregnancies. Her daughters (patients 2 and 3) had eclampsia in their first pregnancies. Their second pregnancies were normotensive (Fig. 1). Controls. Control subjects were seven healthy women who had normotensive pregnancies and who had maternal relatives with normotensive pregnancies. Methods. Blood samples for analysis were accessible from patients 10, 11, 12, 14, 15, 16, 17, 18, and 19. The mtDNA was purified from blood platelets as described by Wrischnik et al. 9 from patients 10, 11, 14, 15, 16, 17, 18, and 19, all descending from patient 5 in family A; mtDNA was also purified from seven controls. Total DNA was isolated from blood from patients 10, 11, 12, 15, 17, and 18 in family A and from patients 1 to 3 in family B. a~ Informed consent was obtained from all patients and controls. The 22 tRNA genes in mitochondrial DNA were sequenced in patients 10, 12, and 16 in family A; in patients 1 to 3 in family B; and in seven controls. Appropriate primers were used, with one biotinylated primer in each pair, and the 22 tRNA genes were amplified by polymerase chain reaction with deoxyribonucleoside triphosphate set (Pharmacia, Uppsala) and TaqDNApolymerase (Promega, Madison, Wis., or Gibco, Bethesda, Md.). Polymerase chain reaction products were immobilized by Dynabeads M-280 streptavidin as described by the manufacturer (Dynal A.S., Oslo). DNA sequencing was performed with appropriate sequencing primers with a con> mercial T v sequencing kit (Pharmacia). In patients 11 and 14 to 19 in family A the 909 nucleofide region flanking nucleotide 32434 was amplified with polymerase chain reaction. The primers used were nucleotides 2834 to 2856 and 3726 to 3746, and mtDNA was amplified with the Gene Amp PCR reagent kit (PerkinElmer Cetus, Norwalk, Conn.). The polymerase chain

Folgero et al. 1627

reaction product was digested with the restriction enzyme

ApaI (United States Biochemical) and the fragments were separated on a I% agarose get.

Results An A-to-G mutation in tRNAI~u[UUR] (U=uracil, R = adenine [A] or guanine [G]) at nucleotide 3243 was found in family A but not in family B nor in controls. The mutation was heteroplasmic, which means that both mutant and wild-type (normal) mitochondrial DNA were present (Fig. 2). Other mutations were not found in the 22 tRNA genes. The mutation at nucleotide 3243 creates a new restriction site for the enzyme Apa I (GGGCC'C). Apa I digestion of the 909 nucleotide polymerase chain reaction fragment flanking nucleotide 3243 resulted in three bands in patients 10, 11, and 14 to 19--909, 500, and 409 nucleotide--which is consistent with a heteroplasmic mutation. In family B an A-to-G mutation at nucleotide 12308 in tRNA~"u[CUN] ( C= cytosine, N = A , C, G, or U) was found but not in family A or controls. In family B the mutation was homoplasmic (i:e., only mutant mtDNA was present) (Fig. 3). This was the only tRNA mutation in the family.

Comment We have found two different tRNN e= mutations in mitochondrial DNA in two families with a high occurrence of preeclampsia and eclampsia, tRNA molecules have an essential function in protein synthesis. They read the information of the messenger RNA and transport the right amino acid to the proper position in the polypeptide chain. Structural RNA molecules, like the tRNAs, fold into compact three-dimensional forms. The folding is determined by tertiary interactions among different parts of the molecule and consists primarily of hydrogen bonds between conserved base pairs and conserved unpaired residues. Mutational changes in these base triplets reduce the function of structural RNAs. 1I Both the wild-type A at nucleotide 3243 in the dihydrouridine loop of tRNN "u[UUR] and the wild-type A at nucleotide 12308 in the variable loop of tRNN"u[CUN] are highly conserved between species during evolution J 2' i3 It is therefore likely that mutations of these bases affect a proper folding and thereby the function of the two tRNA molecules. The 3243 mutation also affects the transcriptional terminator and may alter the ratio of mitochondrial ribosomal and messenger RNA transcripts? Leucine is the most frequent amino acid in proteins coded for by mtDNA in humans?4 A reduced function of one of the two tRNA leu molecules therefore affects a large part of the amino acids of proteins synthesized in the mitochondria and is likely to have biologic consequences.

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FAMILY A

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Pre-eclampsia ill Eclampsia II

Fig. 1. Pedigrees of two families with high occurrence of preeclampsia a n d eclampsia.

In 1989 the obstetric history of family A was presented, and the question whether preeclampsia is a mitochondrial disorder was raised. 1~The molecular defect had n o t been identified at that time. Later there were reports on ultrastructural changes in preeclampsia, suggesting a systemic metabolic disorder involving m i t o c h o n d r i a ) < ~7 Most h u m a n cells have h u n d r e d s of m i t o c h o n d r i a and thousands of mtDNAs, s The tRNA~eu[UUR] mutation in family A is heteroplasmic. This means that wild-type (normal) and mutated mitochondrial DNA coexist in the same cell. As the cells u n d e r g o divisions, the mitochondrial genotype can drift toward either pure mutant or qcl t,q~ rntI3~XTAS ( h n m o n l a s m v ] T h u s cells n r individu-

als with identical nuclear genotypes, such as monozygotic twins, can have different cytoplasmic genotypes and therefore different phenotypes2 A n o t h e r feature of mitochondrial genetics is threshold expression, which may explain the differences i n symptoms within a family. In patients with mitochondrial mutations the phenotype is d e p e n d e n t on two i m p o r t a n t factors: the severity of the defect in oxidative phosphorylation (i.e., the nature of the mutation and percentage of mutant mtDNAs) and the relative reliance of each organ system on mitochondrial energy production. ~ A w o m a n with a mitochondrial tRNA mutation might have sufficient mitochondrial ATP production in the n o n p r e g n a n t

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IT C G AIT C G A~T C GAIt C G Air C a Ai Fig. 2. Sequences of mtDNA including heteroplasmic mutation in mitochondrial tRNA~""[UUR] in three patients in family A (sequences 1 to 3). Corresponding sequences from two control subjects (sequences 4 to 5) are shown. Sequencing was performed of the DNA strand complementary to one commonly referred to (4), and base substitution ofT to C in our patients corresponds to A-to-G mutation at nucleotide 3243.

state. When metabolic demands increase during pregnancy, mitochondrial ATP production could be insufficient. The cell energy crisis resulting from mitochondrial dysfunction may lead to an accumulation of adenosine diphosphate, adenosine monophosphate, or inosine monophosphate. These nucleotides are degraded to inosine and hypoxanthine and further metabolized to xanthine and uric acid. ~*This may explain some characteristic findings in preeclampsia. Hyperuricemia is regularly seen, and adenosine diphosphate is a potent vasoconstrictor and platelet aggregator. Generally, mutations of mitochondrial tRNA genes are considered to have systemic phenotypic consequences.; It is very likely that reduced mitochondrial ATP production in pregnancy affects most cells and organs and might explain the multiorgan involvement in preeclampsia. The central nervous system is most dependent on mitochondrial energy, followed by skeletal muscle, heart, kidney, and liver) There might be symptoms from the central nervous system in severe preeclampsia and obviously in eclampsia. Ultrastructural changes of mitochondria in myocardium have recently been shown in a woman with preeclampsia. ~7The liver and kidney are often affected in preeclampsia. Glomerular endotheli0sis is considered characteristic, and ultrastructural findings include endothelial cytoplasmic foamy alterations and vacuoles containing lipid, m These are ultrastructural changes often found in skeletal muscle in mitochondrial dysfunctions.'-'" Endothelial cell dysfunction seems to be important in preeclampsia, 2~ and ultrastructural changes of mitochondria in endothelial cells have been found. ''~We have also observed ultrastrucmral changes of mitochondria in skeletal muscle from preeclamptic women (unpublished data). The placenta is important in the pathogenesis of preeclampsia and eclampsia. There is a failure ofcytotrophoblast invasion of spiral arteries. The second wave of trophoblast migration into the myometrial segments of the spiral arteries does not occur. 7 Trophoblast invasion is

I

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Ay A~ IT CG A[TC G AIT CG A I Fig. 3. Sequences of mtDNA including homoplasmic mutation in mitochondrial tRNAk'*'[CUN] in three patients in family B. probably an ATP-dependent process. There is also a high oxidative metabolism in the placenta. A mitochondrial disease will probably cause placental dysfunction and thereby intrauterine growth restriction. In a recent study decreased levels of one of the mitochondrial enzyme complexes were found in preeclamptic placentas. "2~This supports the significance of mutations in mtDNA in preeclamptic women. Preeclampsia is considered a complication of the first pregnancy. This was true for the two daughters in family B but not for the mother. In family A the obstetric histories varied. The first pregnancy might introduce a p e r m a n e n t change in the metabolism favorable for subsequent pregnancies, for example, proliferation of mitochondria as a response to increased energy requirements. Metabolic adjustments in the mother do not explain better placental function in later pregnancies unless the first pregnancy results in permanent changes in the spiral arteries. The incidence of preeclampsia in mothers, daughters, sisters, and granddaughters is two to five times higher than in mothers-in-law, daughters-in-law, and "control populations. "~ The restriction of the phenotype to one sex and to pregnancy makes genetic analysis more complicated than for any ordinary phenotype under genetic control. Different genetic models have been proposed.'-'

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N o n e of these explain all aspects of inheritance, and n o n e offer a satisfactory explanation for the lack of concordance shown by monozygous twins2 ~A heteroplasmic mtDNA mutation with different proportions of wild-type and mutated mtDNA in the two twins could result in different cytoplasmic phenotypes and differences in mitochondrial ATP production. It is likely that mutations of different genes result in preeclampsia. Mitochondrial dysfunction might be one of several metabolic disorders causing preeclampsia. Different nuclear and mitochondrial DNA mutations cause mitochondrial dysfunction. Autosomal recessive inheritance is c o m m o n and includes defects of fatty acid oxidation, pyruvate carboxylase deficiency, defects of the Krebs cycle, and several defects involving oxidative phosphorylation. A nuclear gene defect can predispose to m t D N A mutations and might be inherited as an autosomal dominant trait. 5 Thus mitochondrial dysfunction with reduced ATP production can be inherited as an autosomal recessive or d o m i n a n t trait or could be maternally inherited, as in the two families studied here. Mutations of nuclear genes not affecting mitochondria may cause preeclampsia in others. Recently, Ward et al. -~4 found a molecular variant of angiotensinogen in preeclampsia. T r e a t m e n t with succinate, vitamins and coenzyme Q has been tried in mitochondrial dysfunctions, with symptomatic i m p r o v e m e n t in some patients. 5 A characterization of the genetic defect in preeclampsia opens for identifying susceptible w o m e n and trying treatment or prophylaxis. Apart from the clinical presentation of our family A, ~ there has to our knowledge b e e n only one case report on preeclampsia in a patient with mitochondrial myopathy. ~ In pedigrees with mitochondrial disorders, most attention has b e e n paid to neuromuscular symptoms, and the obstetric histories in these families have probably n o t b e e n explored .... In conclusion, we report here for the first time mutations in mitochondrial tRNA genes in families with a high occurrence of preeclampsia and eclampsia. These mutations most likely affect the function of the tRNAs and result in impaired mitochondrial protein synthesis, which leads to r e d u c e d ATP production. This might explain the multiorgan involvement in preeclampsia. Maternally inherited mitochondrial DNA mutations should be considered, especially in families with a high occurrence of preeclampsia and eclampsia. REFERENCES

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