IVA is known as a relatively rare, potentially life-threatening inborn error of leucine metabolism. Even with rapid diagnosis and effective treatment, patients with IVA are still at risk of metabolic decompensation, no matter what the form is. The disappointing outcome will increase the families economic and spiritual burden. Thus, prenatal diagnosis is an essential strategy for the family with IVA probands to prevent the recurrence of IVA. In this study, we shared our experiences with prenatal diagnosis of IVA in eight pregnancies.
The measurement of metabolite levels in AF by mass spectrometry has been increasingly used for the prenatal diagnosis of numerous inherited metabolic disorders, yet few reported in IVA. In a notable example, Shigematsu et al. reported that metabolite analysis of acylcarnitines by MS/MS and organic acids by GC/MS in AF allowed a rapid and reliable diagnosis, while it was based on a single case. In addition, it has been reported that IVG possibly present below the lowest levels, which remains a risk of false-negative results. Therefore, systematic application and evaluation of these biochemical methods are critical. Given that genetic analysis was generally recognized as the golden standard for prenatal diagnosis of IVA, in this study, we retrospectively reviewed prenatal diagnostic data from eight at-risk pregnancies and first analyzed the reliability of the biochemical approach for prenatal diagnosis of IVA by comparing the biochemical results with the molecular results.
Among these eight cases, the levels of C5 and C5/C2 were consistent with genetic results in both affected fetuses and in five of six unaffected fetuses, these two biochemical markers were also completely consistent with genetic results. Only the level of C5 in F6 was found to show a slight discrepancy with genetic results. One of the probable reasons might be associated with the selection of the cutoff value. It has been suggested that a reference range based exclusively on normal population might lead to many false positive results. Therefore, the reference range of prenatal metabolite levels need to be adjusted in response to the overlap between normal population and disorder range. However, it is difficult to establish more reasonable ranges on the basis of the small sample size in our study. The IVG assay was also performed for the prenatal diagnosis of IVA, which yet was found not to be valuable, since the AFs from all eight at-risk pregnancies contained no measurable IVG, whether they carried an affected or unaffected fetus. This is inconsistent with that described in the previous reports where the IVG levels in affected fetuses were notably 30-60 times higher than that in unaffected fetuses[16, 18]. The false negative results might be due to the low presentation of this metabolite in AFs and adsorption losses. According to our data and comparisons of the metabolite results and genetic results, the sensitivities of C5 and C5/C2 were both 100%, and the specificities of C5 and C5/C2 were 83.3% and 100%, respectively. Therefore, C5/C2 appeared to be the most reliable of these three tested biochemical markers. Due to the limitation of sample size in this study, further data are needed to determine the reliability of these three biochemical markers in the prenatal diagnosis of IVA.
Mutation analysis is generally recognized as the most reliable strategy for prenatal diagnosis of IVA, which yet may potentially lead to false negative results due to maternal cell contamination. Another limitation of this method is that it depends on the availability of known familial mutations. Luckily, all the probands in our center had found two causative variants and no samples had maternal cell contamination. However, in some IVA families, not more than one causative mutation was found in the proband, or genetic testing was not performed, especially in some remote areas, this can hinder a precise diagnosis by genetic analysis alone. In this situation, metabolite analysis by mass spectrometry could be optional which is able to provide reliable results using only a small amount of sample. Additionally, the biochemical results can be achieved within three days, which is much earlier than that of genetic analysis taking at least two weeks. This enables IVA families to make a timely decision concerning the pregnancies. This advantage was observed in our previous reports on prenatal metabolite analysis in methylmalonic acidemia, propionic acidemia and glutaric acidemia-Ⅰ[21–23]. Therefore, despite the accuracy of genetic testing, biochemical analysis appeared to be a reliable method in the prenatal diagnosis of IVA families, especially when genetic results were inconclusive, and this method could also allow a more precise diagnosis in conjunction with mutation analysis. However, the reliability of biochemical approach in the prenatal diagnosis of IVA is warranted to be further verified by expanding the sample size.