Methotrexate (MTX) embryopathy was described nearly 50 years ago, when this agent began to be used as a cancer treatment and abortifacient. In this report we describe a case with typical features of MTX syndrome together with new features to expand the phenotype.
A 29-year-old woman decided to terminate her unwanted pregnancy because of ill health, as she had conceived soon after her last delivery by cesarian section. At 6 weeks of gestation, she took 2.5 mg of MTX 3 times a day for 7 days. The pregnancy termination failed, and the pregnancy was carried to term. A female infant was delivered who was growth retarded and had characteristic features of MTX embryopathy in addition to holoprosencephaly and other brain malformations, facial hypertrichosis, and long eyelashes—features that have not hitherto been described.
Methotrexate (MTX; amethopterin), a methyl derivative of aminopterin, is an antimetabolite that was first described in 1947 (Seger et al.,1947). It blocks the enzyme dihydrofolate reductase and thus inhibits the production of thymidine, which is required for DNA synthesis. MTX interferes with cell growth, specifically with rapidly dividing cells. MTX primarily affects the cytotrophoblast and inhibits the implantation process. MTX and aminopterin have been known to be teratogenic in humans since the 1950s (Thiersch,1952; Milunsky et al.,1968), and many malformations have been reported since then. The critical period for teratogenicity of these drugs is 6–8 weeks after conception (Feldkamp and Carey,1993). We present the characteristic features of MTX embryopathy together with features that have not previously been described.
The proband, a 3-month-old girl, was delivered at term by elective cesarian section. The mother was 29 years old. This was her fourth pregnancy, and the other 3 children were normal. The parents were cousins. The mother conceived soon after her last delivery by cesarian section and did not feel well. She had no relevant illnesses during her pregnancy. The couple decided to terminate this pregnancy because of the mother's health, without seeking medical advice. She took 2.5 mg of MTX 3 times a day for 7 days, for a total of 52.5 mg starting on the sixth week of gestation. She was told that the pregnancy would terminate after 7 days, and stopped the medication. No folic acid or medication (other than MTX) was taken during the pregnancy. The pregnancy failed to terminate and was carried to term. An antenatal ultrasound examination at 28 weeks of gestation revealed intrauterine growth retardation, short limbs, an abnormally shaped head, and 18-mm dilated lateral ventricles.
The baby was a female with a birth weight of 1310 gm, length of 41 cm, and head circumference of 27.5 cm (all below the 10th centile). She had multiple dysmorphic features (Fig. 1) consisting of prominent eyes, long eyelashes, hypertelorism, epicanthic folds, shallow orbits and supraorbital ridges, a beaked nose, a long philtrum, a cleft palate, micrognathia, low-set malformed ears, a small narrow forehead, closed metopic and coronal sutures and wide posterior fontanel, a low hairline covering the whole forehead, posteriorly combed hair appearance, short forearms (mesomelia), and hypoplastic first toes and nails. She had no cardiac involvement and no organomegaly. She was fed via gavage because she was unable to suck. Chromosome studies showed a normal female karyotype. The renal ultrasound findings were normal. Magnetic resonance imaging studies of the brain (Fig. 2) showed agenesis of the corpus callosum, a single ventricle, fused thalami, absent cavum septum pellucidum and falx, and a hypoplastic cerebellum with prominent cisterna magna. These findings were in keeping with alobar holoprosencephaly (HPE). However, the facial phenotypic features did not look like those of HPE. The baby was discharged to her home at 2 months of age, and the family was counseled.
The first suggestions that folic acid antagonists were teratogenic in humans were based on reports of failed pregnancy termination in mothers exposed to aminopterin in the first trimester. Reports of abnormalities included neural tube and skull and limb defects with a gestational age of exposure of 4–12 weeks. The constellation of these abnormalities was called “aminopterin syndrome” (Warkany,1978). Thiersch (1952) first noted abnormal morphogenesis in 3 abortuses and 1 full-term offspring of mothers who received aminopterin 4–9 weeks after conception. Subsequently, other cases have been published, including an account of teratogenesis secondary to MTX exposure.
In a comprehensive review of the effects of MTX on pregnancy, fertility, and lactation, Lloyd et al. (1999) listed all reported cases of MTX exposure during pregnancies with known outcomes. There were 71 cases of exposed fetuses, 42 of which were exposed during the first trimester. Of these 42 cases, 12 abnormal cases were reported (10 involved physical abnormalities, 9 involved skull abnormalities, and 6 involved peripheral skeletal problems). All cases of definite physical abnormality occurred following exposure in the first trimester. It has been suggested that the critical period for exposure to MTX is 6–8 weeks after conception, and that a maternal MTX dosage of >10 mg/week is necessary to produce defects in the fetus (Feldkamp and Carey,1993). Donnenfield (1994)reported 4 infants who were exposed during the first trimester between 0 and 6 weeks. All of these infants were normal. In addition, almost all infants who were exposed after the 12th week were normal.
In this case report we describe a baby with characteristic features of aminopterin-MTX syndrome, as previously described (Table 1). The fetus was exposed to MTX (a total of 52.5 mg was taken by the mother in an attempt to induce pregnancy termination from the 6th to the 7th week after conception). In addition to the previously described features of MTX embryopathy, to the best of our knowledge, our case is the first to be reported with brain malformations in the form of HPE, cerebellar hypoplasia, and absent corpus callosum. In a review by Lloyd et al. (1999), of 71 fetuses exposed to MTX during pregnancy, only 1 case of central nervous system (CNS) involvement with hydrocephalus was reported (Diniz et al.,1978). CNS involvement was reported more often with aminopterin exposure. Previous studies reported meningocele and hydrocephalus (Thiersch,1952), anencephaly (Thiersch,1956), and hydrocephalus (Reich et al.,1978). Other CNS abnormalities within the aminopterin-MTX syndrome include spina bifida and mental retardation. No case of HPE was reported with either MTX or aminopterin in the cases reviewed by Lloyd et al. (1999). The other unique features in our case are the striking long eyelashes and abundant facial hair with a hair distribution pattern similar to that reported by Shaw and Steinbach (1968), which they described as a “back-combed” appearance.
Table 1. Reported Features of Fetal Aminopterin-Methotrexate Syndrome*
Of interest, our patient does not have the typical face of children with HPE. Ming and Muenke (2002) suggested that in some cases HPE may be due to teratogens interacting with genes that are known to be responsible for this defect. The zinc finger protein of cerebellum 2 (ZIC2) is a gene that is responsible for HPE and is generally associated with a normal face. HPE is associated with a diagnostic facial pattern ∼80% of the time. Barr and Cohen (2002) reported 3 siblings with autosomal-recessive alobar HPE and essentially normal-appearing faces. A similar family was reported by Khan et al. (1970). Alobar HPE with essentially normal faces has also been observed in infants of diabetic mothers (Barr et al.,1983).
In animal studies of pregnant rabbits, exposure to MTX during early gestation led to universal litter resorption, whereas administration during midgestation caused abnormalities, including hydrocephalus, micrognathia, cleft lip and palate, and dysplastic vertebrae, and administration later in pregnancy caused mainly distal limb dysplasia (Jordan et al.,1977). In a study of rats, malformations were largely confined to the caudal vertebrae in up to 75% of fetuses exposed to MTX (Wilson et al.,1979). No case of HPE was found in these reports or in other species exposed to MTX. Animal models demonstrated that HPE can be induced with ethanol exposure during gastrulation (Sulik and Johnston,1982), which may be due to interference with prechordal plate function (Blader and Strahle,1998). The HPE spectrum in humans has also been noted in association with prenatal exposure to ethanol (Ronen and Andrews,1991; Croen et al.,2000; Cohen and Shiota,2002). HPE in humans has also been reported in association with prenatal exposure to retinoic acid (Lammer et al.,1985; Cohen and Shiota,2002). Wallis and Muenke (2000) reviewed gene mutations associated with HPE. They indicated that at least 12 different loci have been associated with HPE and several distinct genes have been identified, including Sonic Hedgehog (SHH), (ZIC2), (HPE 5), sine oculis homeobox (SIX), and transforming growth factor β-induced factor (TGIF). Chromosome aberrations involving chromosomes 2, 3, 7, 13, 18, and 21 were also associated with HPE. In a review article, Ming and Muenke (2002) hypothesized that for some conditions, specifically those that originate during early embryogenesis, alterations in modifier genes or interactions with environmental factors are required for full expression of the disease (i.e., the “multiple-hit” hypothesis). They focused particularly on HPE. Although it appears likely that multiple genes contribute to the phenotype of human HPE, evidence from both human studies and animal models implicates environmental factors in the pathogenesis of HPE. The protective role of folic acid against neural tube defects is well established. In another review, Ramsey-Goldman and Schilling (1997) suggested that women exposed to MTX should continue supplementation throughout pregnancy. In support of this, none of the 3 cases found in the review by Lloyd et al. (1999) in which folic acid supplementation was given with low-dose MTX resulted in fetal abnormality. Because dihydrofolate reductase has a far greater affinity for MTX than for folic acid, complete reversal of the antifolate effects of MTX requires the administration of folinic acid. When given soon after MTX exposure in pregnant rabbits, leucovorin (a close structural analog of folinic acid) virtually eliminates teratogenic effects (DeSesso and Goeringer,1991). Folinic acid administration at the equivalent MTX dose is recommended for at least 4 months following cessation of MTX administration in women who wish to continue their pregnancy (Lloyd et al.,1999).
The results of the chromosome analysis of our case were normal; however, we need to perform a molecular genetic analysis, which may reveal a novel gene mutation or the ZIC2 gene. Such a finding would support the multiple-hit hypothesis of Ming and Muenke (2002), as our patient had been exposed to a known teratogen (MTX) at a critical period of gestation (6–8 weeks) without the protection of folic acid. In a previous study, of 42 fetuses exposed to MTX in the first trimester (some without maternal folic acid supplementation during pregnancy), 30 were normal (Lloyd et al.,1999). In addition, some fetuses exposed to extremely large amounts of MTX within the first trimester were normal, but an abnormal fetus was born after exposure to a low dose of 7.5 mg. These reports give support to the multiple-hit theory and the notion that genetic differences in placental and fetal response to toxins may explain the variable expression of the outcome in fetuses exposed to teratogenic agents. Ming and Muenke (2002) posed the hypothesis that even relatively low dose of teratogens, which may not be sufficient to cause HPE or any other clinical abnormality, may act in concert with other environmental or genetic variables to generate the HPE phenotype. This is a valid hypothesis in the light of the available data.
Our case is the first case of MTX embryopathy to be reported in association with HPE and hypoplastic cerebellum. Other features include long eyelashes and peculiar hair distribution. Both physicians and the public should be aware of the effects of MTX on the fetus, especially during the critical period of organogenesis and throughout pregnancy.
The authors thank Mr. Mandy Sarmiento and Mr. Abdullah Palma from the Department of Education and Training for the medical photography, and Mrs. Bing Borromeo for her secretarial assistance.