Prospective Screening for Pediatric Mitochondrial Trifunctional Protein Defects in Pregnancies Complicated by Liver Disease FREE

Acute fatty liver of pregnancy (AFLP) is a devastating disorder of the third trimester that carries significant perinatal and maternal morbidity and mortality. Hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome is also a maternal illness of the third trimester that is a complication of severe preeclampsia and has a better prognosis than AFLP. The prevalence of AFLP and HELLP syndrome is approximately 1 in 13 000 and 5 in 1000 pregnancies, respectively.^1- 2 Recent evidence has linked these maternal disorders, which have long been considered mysterious syndromes of unknown etiology, to an inherited fetal disorder of fatty acid oxidation.

Throughout the past decade, several case reports have noted fetal deficiency of long-chain 3-hydroxyacyl coenzyme A dehydrogenase (LCHAD) in the offspring of women who had developed AFLP, HELLP syndrome, or both during gestation.^3- 8 Long-chain 3-hydroxyacyl coenzyme A dehydrogenase is part of a mitochondrial trifunctional protein (MTP) complex that catalyzes the last 3 steps in long-chain fatty acid oxidation and is a hetero-octamer of 4-α and 4-β subunits.^9- 10 Long-chain 3-enoyl coenzyme A hydratase and LCHAD activity reside in the MTP α subunit. The β subunit has the long-chain 3-ketoacyl coenzyme A thiolase activity. The human complementary DNA and genes encoding both subunits have been isolated and characterized.^6,11- 13

Human defects in the MTP complex are recessively inherited and cause either LCHAD deficiency with normal or partially reduced thiolase and hydratase activity or complete MTP deficiency with markedly reduced activity of all 3 enzymes.^12,14- 17 Recently, we reported the α subunit molecular defects and phenotypes in 24 patients with documented LCHAD deficiency or complete MTP deficiency.¹⁷ Patients with the more common LCHAD deficiency present predominantly at a few months of age with hypoglycemia and liver dysfunction and carry a prevalent mutation that changes glutamate to glutamine at position 474 (E474Q) in the mature MTP α subunit, whereas patients with complete MTP deficiency present predominantly with cardiomyopathy or neuromyopathy and carry mutations other than the E474Q mutation.¹⁷ The estimated prevalence of LCHAD deficiency in the United States is 1 in 62 000 pregnancies.¹⁷

The initial case reports that linked the maternal liver syndromes to the fetal LCHAD deficiency used biochemical or enzymatic assays as the diagnostic criteria in the pediatric patients.^3- 5 Recently we correlated the pediatric genotype to pregnancy maternal history in 24 families with documented pediatric MTP defects.¹⁷ In 19 heterozygous mothers who carried fetuses with LCHAD deficiency, 15 (79%) developed AFLP or HELLP syndrome. All fetuses in these affected pregnancies were either homozygous for the E474Q mutation or compound heterozygous with one mutation as the E474Q and the other mutation producing a stop codon or altering a splice site. These studies suggest that fetal LCHAD deficiency is unique among fatty acid oxidation disorders in its common association with maternal liver disease. It is rather striking that only 1 case of maternal liver disease has been reported in association with fetal medium-chain acyl coenzyme A dehydrogenase deficiency, the most common fatty acid oxidation disorder.¹⁸ Two other single-case reports have linked maternal complications to other fatty acid oxidation disorders.^19- 20 There seems to be a mechanism peculiar to LCHAD deficiency that causes this fetal-maternal interaction and may involve generation by the placenta or fetus of 3-hydroxy fatty acid metabolites toxic to the maternal liver.¹⁷

Although the association between liver disease in pregnancy and fetal LCHAD deficiency is well documented, the proportion of women who carry LCHAD-deficient fetuses in all pregnancies complicated by maternal AFLP or HELLP syndrome is unknown, which is relevant to the critical issue of whether newborns in all pregnancies complicated by AFLP or HELLP syndrome should undergo screening for LCHAD deficiency. Identifying LCHAD-deficient offspring can be lifesaving because this inborn error of metabolism is potentially treatable through diet modification.

From January 1997 to December 2001, 108 consecutive blood samples obtained from women who developed AFLP or HELLP syndrome, from their offspring, or from their partners were referred to our laboratory for molecular screening for MTP mutations. The sole criterion for conducting the molecular analysis was the maternal history of AFLP or HELLP syndrome. Records of the maternal obstetric histories were reviewed. Published criteria^1- 2,21 were used to classify patients as having HELLP syndrome or AFLP.

The obstetric history revealed a course compatible with AFLP in 27 women, and this diagnosis was proven by liver biopsy in 6 women. The remaining 81 women had clinical and biochemical abnormalities consistent with HELLP syndrome. Forty-eight cases were referred from North Carolina (7 AFLP and 41 HELLP syndrome); the remaining 60 cases (20 AFLP and 40 HELLP syndrome) were referred from 16 other states (California, Connecticut, Georgia, Illinois, Indiana, Kansas, Maine, Maryland, Missouri, New Jersey, New York, Ohio, Pennsylvania, Tennessee, Texas, and Virginia).

The institutional review board of Wake Forest University School of Medicine approved this study, and informed consent was obtained from the parents.

We used single-strand conformation variance analysis to detect mutations in the MTP α subunit, as described previously.^17,22 Nucleotide sequences of exons with abnormal single-strand conformation variance were determined with either the standard dideoxy chain termination method or an automated sequencer and the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Perkin Elmer, Wellesley, Mass).

We also used restriction fragment length polymorphism analysis to detect the E474Q mutation in exon 15 of the MTP α subunit, as described previously.⁶ This method allows rapid diagnosis for homozygosity and heterozygosity for this mutation.

Table 1 summarizes the demographics and some clinical and laboratory characteristics of the women who developed AFLP or HELLP syndrome. All pregnancies resulted in live births, except 3 that were associated with stillbirths (2 complicated by maternal AFLP and 1 by HELLP syndrome). No DNA could be obtained from the 3 stillborn fetuses, but molecular analysis was conducted on both parents to evaluate for MTP mutations.

Pregnancies Complicated by AFLP. Mutations in the MTP α subunit that cause LCHAD deficiency were identified in 5 of the 27 families (19%; 95% confidence interval, 9%-54%) with a history of maternal AFLP. All these families were white and of European origin. The pediatric and maternal genotypes and phenotypes in these 5 families are shown in Table 2. In the first family, the mother developed AFLP at 37 weeks of gestation, with complete recovery following cesarean delivery. Blood was obtained for genetic testing 9 weeks after delivery because of the maternal history. As mutational analysis was under way, the infant became ill with a metabolic crisis associated with cardiogenic shock. Molecular analysis revealed compound heterozygosity of the infant for mutations in exons 4 and 15.¹⁷ The currently 5-year-old child is receiving dietary treatment (a low-fat, high-carbohydrate diet) and doing well. The mothers in families 2 and 3 developed AFLP at 32 and 35 weeks of gestation, respectively. Both recovered completely after prompt delivery. The LCHAD-deficient newborns were clinically asymptomatic at molecular diagnosis (2 and 3 months after birth, respectively). Single-strand conformation variance analysis and nucleotide sequencing revealed homozygosity of both infants for the E474Q mutation. Both infants have remained asymptomatic while receiving treatment with frequent feedings of a low-fat diet in which the fats were medium-chain triglycerides (current age of infants, 24 and 16 months [from families 2 and 3, respectively]). The mother in family 4 developed severe AFLP at 25 weeks of gestation but recovered completely 1 week after delivery. The newborn was homozygous for the E474Q mutation. He died because of severe prematurity. The maternal AFLP in family 5 was associated with intrauterine fetal demise at 36 weeks of gestation. The mother was heterozygous for the common exon 15 mutation; the father was heterozygous for an exon 12 donor-site splice mutation (T to C transversion at +2 position). Unfortunately, there was no tissue available from the presumably affected fetus to test for these mutations.

Pregnancies Complicated by HELLP Syndrome. Heterozygosity for the E474Q mutation was detected in only 1 woman with HELLP syndrome. She had HELLP syndrome at 38 weeks, with full recovery after induced vaginal delivery. No MTP mutations were detected in the newborn, who is now aged 2 years.

Our results document that the association between AFLP and the recessively inherited fetal LCHAD deficiency is significant: 19% of AFLP cases were associated with fetal MTP mutations that cause LCHAD deficiency. In our study, none of the children born to mothers diagnosed as having HELLP syndrome carried MTP mutations.

Three previous reports assessing women with a history of AFLP or HELLP syndrome had contradictory results. In the first report, Treem and coworkers²³ reported enzymatic activity levels consistent with LCHAD deficiency in the offspring of 7 of 12 women diagnosed as having AFLP or HELLP syndrome. In the second report,²⁴ no E474Q mutation was detected in 14 women diagnosed as having AFLP. In the third report, 113 Dutch women with a history of HELLP syndrome were screened for the E474Q mutation, and only 1 was heterozygous for it.²⁵ Molecular genetic studies were not universally done on the mothers and their offspring in these studies.

Our results justify screening newborns prospectively for the E474Q mutation at birth in all pregnancies complicated by AFLP. Initial screening for the E474Q mutation is crucial because all LCHAD-deficient fetuses were either homozygous for this mutation or compound heterozygous with the E474Q mutation on 1 allele. Homozygosity and heterozygosity for this mutation can be detected rapidly by restriction fragment length polymorphism or single-strand conformation variance.^6,17 Treatment with a low-fat diet in which long-chain fatty acids are replaced by medium-chain fatty acids and avoidance of prolonged fasting should be implemented promptly if the E474Q mutation is detected. If the newborn is only heterozygous for the E474Q mutation, further analysis is necessary to search for another mutation on the other allele.

It is important for pediatricians, perinatologists, obstetricians, and gastroenterologists to be informed about the association between pediatric LCHAD deficiency and AFLP. Newborn screening for LCHAD deficiency and other fatty acid oxidation disorders is a public health issue that is being debated in the United States and has already been implemented in 9 states.²⁶ To our knowledge, our results demonstrate for the first time that prospective screening of newborns for the E474Q mutation in pregnancies complicated by AFLP is useful and can be lifesaving because it allows early diagnosis and dietary treatment in affected infants before clinical manifestations. In addition, identification of heterozygous women with risk of recurrence of maternal complications enables genetic counseling and molecular prenatal diagnosis in subsequent pregnancies by using chorionic villous sampling.²⁷

In contrast, our results do not justify screening newborns in pregnancies complicated by HELLP syndrome. However, screening might be considered in cases of recurrent HELLP syndrome and families with a history of sudden unexplained infant death.