Newborn screening for citrin deficiency and carnitine uptake defect using second-tier molecular tests

Tandem mass spectrometry (MS/MS) analysis is a powerful tool for newborn screening [1]. Many rare inborn errors of metabolism that were not covered by newborn screening are now screened by MS/MS. However, the sensitivity of MS/MS screening for the 20–30 diseases included in its screening panel varies among the individual diseases [2]. False negatives can also occur [3, 4], due to the nature of the specific diseases. Therefore, additional or second-tier testing may be required to improve screening for these diseases [5].

Citrin deficiency refers to two disease entities: adult-onset type II citrullinemia (CTLN2, OMIM#603471) and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD, OMIM#605814). Citrin is a mitochondrial membrane aspartate-glutamate carrier that functions as part of the malate-aspartate (MA) shuttle, transferring cytosolic NADH into the mitochondria of the liver [6]. Patients with NICCD often present with jaundice, hypoproteinemia, transient multiple aminoacidemia (citrulline, methionine, tyrosine, threonine), fatty liver, galactosemia, hypoglycemia, disturbed coagulation, and high α-fetoprotein [6, 7]. Citrin deficiency can be detected by newborn screening as an elevation of phenylalanine, methionine, or galactose levels, but the detection rate is only 50% [8]. It has been shown that 1 in 20 newborns affected with NICCD have normal dried blood spot (DBS) citrulline levels initially, but these levels increase at later time points [9].

Carnitine uptake defect (MIM #212140; CUD), also known as primary carnitine deficiency, is an autosomal recessive disorder of fatty acid oxidation caused by defects in OCTN2, a high-affinity carnitine transporter expressed at the plasma membrane [10, 11]. Carnitine is responsible for transporting fatty acids into the mitochondria, and defective carnitine uptake results in an intracellular carnitine deficiency, causing defects in the β-oxidation of fatty acids [12]. Patients with CUD can suffer from cardiomyopathy, muscle weakness, recurrent hypoketotic hypoglycemic coma, Reye-like syndrome, and premature death [13–15]. Because most symptoms are reversible, early treatment of the disease can result in a good patient outcome [12, 16]. CUD can be detected in newborns by measuring free carnitine levels in DBS [17]. However, because carnitine is transported through the placenta [18], carnitine can be supplemented by the mother, resulting in only half of fetuses with CUD being detected by newborn screening [19]. Additionally, a normal fetus can have low free carnitine levels due to carnitine deficiency in the mother [17].

The molecular defects associated with the aforementioned diseases have been elucidated. Citrin deficiency is caused by mutations in the SLC25A13 gene [20]. A study of 4 169 normal Chinese individuals revealed four SLC25A13 mutations: c.851_854del (851del4, p.M285PfsX2) (70%), c.1638_1660dup23 (1638ins23, p.A554GfsX17) (5%), IVS6+5 G>A (23%), and c.550C>T (p.R184X) (2%), and the total carrier rate was 1 in 65 [21]. The SLC22A5 gene encodes the carnitine transporter OCTN2 [10, 11]. A founder mutation, c.760C>T (p.R254X), in Chinese patients has a carrier rate of 1 in 125 [22]. To improve the newborn screening of citrin deficiency and CUD, we employed hotspot mutations analysis as the second-tier tests for these two diseases.

In this study, we sought to improve the performance of newborn screening for citrin deficiency and CUD. Newborn screening programs must determine cutoff values [2] to identify suspicious patients while minimizing false positives. However, patients with citrin deficiency may have nearly normal blood citrulline levels; similarly, patients with CUD may have normal carnitine levels due to placental transportation. Therefore, our strategy was to set a screening cutoff and a diagnostic cutoff, so we could test the inconclusive cases using a second-tier mutation analysis. We demonstrated that this strategy can improve the sensitivity of screening without increasing the false-positive rate.

In current practice, a successful molecular screening approach depends on the presence of founder or hotspot mutations. In citrin deficiency, the three mutations we screened (c.851_854del, c.1638_1660dup23, and IVS6+ 5 G>A) represent 95% of all mutations in Taiwanese patients [23] and 98% of all mutations in carrier screening [21]. In CUD, the p.R254X mutation accounts for 50% of all mutated alleles in clinical cases, but the prevalence of this mutation is lower in asymptomatic mothers and in screened newborns [17]. Therefore, to improve the performance of the screening program, we decided to subject these mutations to the second-tier testing.

Adding a second-tier mutation test can improve screening performance. In citrin deficiency, the two cases that we identified would have been missed by our original screening cutoff (19.5 μM), but we identified these cases after lowering the screening cutoff. The incidence of citrin deficiency derived from the current study (1:23 350) is close to the incidence (1:16 900) calculated from a mutation carrier rate of 1:70 [24]. From our study, in the group of newborns with high citrulline, methionine, tyrosine, or galactose, the carrier rates (i.e., 1:80 for the first period and 1:24 for the second period) were not significantly different from those identified in the previous study [24] or from those found in another study that included 479 healthy controls from Southern Taiwan [25]. The carriers that we detected in this study with elevated citrulline levels might harbor another SLC25A13 mutation, or heterozygous carriers may even have slightly increased citrulline levels. Nevertheless, we would likely continue to miss some cases in which the subjects have normal citrulline levels at the newborn stage and only later present with cholestasis.

In CUD, the population carrier rate for p.R254X was shown to be 1:125, which suggests an incidence of 1:62 500 [22]. This mutation represented 50% of all mutations identified in clinically diagnosed patients and only 30% of mutations identified by newborn screening [17], which suggests that the actual incidence of CUD may be much higher. The CUD patient identified during this study was a compound heterozygote, which had an incidence of approximately 1 in 30 000 during this study period. Although this patient might have been identified without a second-tier mutation testing, not utilizing a second-tier mutation test approach would have required retesting 206 newborns (0.7%). Additionally, in the group of newborns exhibiting borderline low free carnitine, the carrier rate of the p.R254X mutation was 1:19 (11/206). Because 10 of the p.R254X heterozygous newborns were found to have normal free carnitine levels in the second screen, we speculate that those were true heterozygous carriers, not patients. This is a reasonable assumption because carriers of CUD may present with slightly low free carnitine, and our selection biased the carrier rate of this mutation. Although carnitine administration ameliorates all symptoms of CUD, we continue to screen patients using a second screen, but we do not apply this new algorithm using a second-tier test due to cost.

The major limitation of this study was our selection of patients based on an abnormal initial screening. If the levels of citrulline, galactose, methionine, tyrosine or carnitine were normal, the second-tier testing would not have been performed. A rapid genetic test for citrin deficiency performed by screening for 11 common mutations of SLC25A13 has been developed [26], but further validation using a larger scale of dried blood samples is necessary prior to the clinical application of this technique. In addition, the low incidence of these diseases makes it difficult to attain a case number of sufficient size for a statistically significant conclusion. Lastly, the cost of molecular testing may hinder the availability of genetic screening programs. However, if molecular screening for other diseases has previously been conducted, a second-tier molecular test may be easier to perform because the cost would be minimized by previous DNA extraction.

In Taiwan, we initiated newborn screening for citrin deficiency and CUD in 2001. Newborn screening for citrin deficiency has been shown to result in early treatment for newborns suffering from NICCD, or preventing erroneous management if CTLN2 develops in these patients. The plasma citrulline level is the current marker for newborn screening of NICCD, but this marker is not sensitive enough to detect all patients with NICCD: we have encountered patients with normal newborn screening results for whom a diagnosis could not be made before the occurrence of liver failure. Similarly, we have shown that newborn screening leads to early treatment for newborns suffering from CUD and that carnitine administration prevents the occurrence of symptoms in these patients. Plasma carnitine is the current marker for CUD, but both false-positive and false-negative results occur: we diagnosed one CUD patient who had recurrent hyperammonemia but whose newborn screening, performed by another screening center, was normal. Therefore, while it is very important to maintain current screening for citrin deficiency and CUD, there is an urgent need to find a method to further improve these screening tests.

We demonstrate the improvement of newborn screening for citrin deficiency and carnitine uptake defects using a second-tier mutation analysis. Further adjustments to improve the sensitivity of this test are warranted.