Absence of G1528C mutation in long-chain 3-hydroxyacyl-CoA dehydrogenase in four Indian patients with pregnancy-related liver disease

Sir, Acute fatty liver of pregnancy (AFLP) is a rare pregnancyrelated liver disorder manifesting in late pregnancy with acute liver failure [1], while the hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome is a microangiopathy affecting the liver and other organs in late pregnancy [2]. Long-chain 3-hydroxyacyl-coenzyme-A dehydrogenase (LCHAD) deficiency in the fetus has been associated with these hepatic disorders of the mother during pregnancy [3]. The commonest mutation is G1528C in exon 15 of the mitochondrial trifunctional protein (MTP) α subunit, which leads to a substitution of a glutamine for glutamate, which selectively decreases LCHAD activity [4]. Though the occurrence of the G1528C mutation has been studied in Western populations [4, 5], where it is common, the information on this from other populations is scarce. We studied the presence of the G1528C mutation in three patients with AFLP (all fulfilled “Swansea” criteria for diagnosis, none had liver biopsy, two had coexistent HELLP, and one had coexistent pre-eclampsia and partial HELLP) [6] and one patient with HELLP syndrome (coexistent preeclampsia). All patients (age: 23; 19–28 years, median; range) presented in third trimester (36; 32–38 weeks of gestation). Three were primigravida while one patient had a previous uneventful pregnancy. All patients were eventually discharged in stable condition, and the immediate fetal outcome was favorable in all. The placenta is known to carry out mitochondrial β oxidation, and its genetic make up is identical to the fetus [7], and hence, we used placental DNA for our genetic analysis by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) analysis. This study was approved by the Institutional Review Board, and consent was obtained from the patient for the study. As required by the criteria, all patients had an unyielding make up for the etiology of acute liver disease, including serology tests for acute viral hepatitis A, B, and E. Controls were placenta from normal deliveries of equal gestational age. The blood was drained, and the placental tissue was rinsed thrice with saline and stored at −80 °C. Baseline laboratory parameters in the four patients analyzed are summarized in Table 1. DNAwas isolated from 100 mg placenta and amplified using forward (5′-CCCTTGCCAGGTGATTGGC-3′) and reverse (5′-GTATAGAAGCCAGGTCCATCCTGCCAAG-3 ′) primers [8]. The PCR products were purified, digested with Pst1, and separated by agarose gel electrophoresis to identify the G1528C mutation. Purified PCR products were bidirectionally sequenced using the BigDye Terminator V3.1 kit in a 3130 genetic analyzer (Applied Biosystems, Carlsbad, USA) and analyzed using NovoSNP software (http://www.molgen. ua.ac.be/bioinfo/novosnp/). PCR was used to amplify the whole of exon 15 of the α subunit, which codes for LCHAD activity of the MTP (Fig. 1a, first lane on left). All healthy controls as well as AFLP and HELLP patients showed two characteristic fragments after Pst1 digestion (465 and 175 bp), indicating an absence of the G1528C mutation [8]. V. Raghupathy :K. R. Thangaraj :K. A. Balasubramanian : A. Ramachandran (*) The Wellcome Trust Research Laboratory, Division of Gastrointestinal Sciences, Christian Medical College, Vellore 632 004, India e-mail: anup@cmcvellore.ac.in