To our knowledge, so far this is the largest BKTD cohort studied in China. The large number of screened newborns in this study provided a more comprehensive perspective on the incidence of BKTD detected via NBS in China. Biochemical, clinical, and molecular features of Chinese BKTD patients were summarized, contributing to NBS, early diagnosis and timely treatment of this rare disease.
Few studies regarding the incidence of BKTD have been reported. The incidence is estimated at 1:190,000 in northern Vietnam , 1:313,000 in North Carolina and 1:232,000 in Minnesota in the United States [5, 17]. Our study investigated for the first time more than 16 million newborns with 14 identified BKTD patients for an incidence of approximately 1:1,000,000 in China. The incidence is lower than those reported in other studies [5, 10, 17], possibly due to differences in ethnic backgrounds, screening experience and awareness of the disease. It is noteworthy that the actual incidence of BKTD may be higher in China because some early neonatal deaths and patients who suffered mild episodes may not be identified.
BKTD is an ideal disease for NBS from a clinical perspective. However, false-negatives (FN) has been reported in several NBS programs [5, 6, 18]. Although both C5OH and C5:1 acylcarnitines are well-known markers for BKTD screening, these two markers are not necessary high during NBS and even at acute metabolic crisis. In our study, two patients (No. 4 and 11) had increased C4OH only but not C5OH and C5:1, even in the period of acute decompensation. However, we also found one patient (No. 10) showed increased levels of C5OH and C5:1 but no increase in C4OH. Notably, C4OH level in patient No. 4 was significantly high during NBS but returned to normal when recalled, then C4OH increased again at 11 months old after injection of meningococcal vaccine, indicating that C4OH is sometimes variable and could be normal in a stable condition. Beta-ketothiolase not only acts in ketone body utilization (ketolysis) by catalyzing thiolytic cleavage of acetoacetyl-coenzyme A to produce 2 molecules of acetyl CoA in extrahepatic tissues, but also catalyzes conversion of 2-methylacetoacetyl-coenzyme A in isoleucine catabolism. D-3-hydroxybutyrate ketone body can be converted into D-3-hydroxybutyrylcarnitine (C4OH) in vivo and in vitro . It is therefore reasonable that there is not only an increase in C5OH and C5:1 but also an increase in C4OH in BKTD patients. Taking together, we speculate that some BKTD patients may be missed if either one of the three acylcarnitines (C5OH, C5:1 and C4OH) is used independently as a screening marker. Conversely, the FN results can be reduced by using several markers and/or their combinations. Our study therefore strongly suggests that C4OH is a very useful and powerful marker for the detection of BKTD. The performance of BKTD NBS may be improved by adding C4OH to C5OH and C5:1 combination in NBS.
In this study, despite almost all patients (except patient No. 4) exhibited the characteristic increase of urinary 2M3HB and TIG, both urinary markers were slightly elevated or undetectable even during acute crisis. Sometimes the analysis of urinary organic acid profiles is not typical, as observed in patient No. 4. Thus the diagnosis of BKTD should be confirmed by genetic test. In addition, elevated excretion of urinary 2M3HB and TIG indicates not only BKTD but also HSD10 mitochondrial disease (HSD10MD, OMIM #300438), which is a rare X-linked recessive disorder caused by a hemizygous or heterozygous mutation in the HSD17B10 gene . Fukao et al. reported a 6-year-old Japanese boy who was initially diagnosed with BKTD based on metabolic profiles, however, enzyme activity assays and mutation analysis finally confirmed the patient had HSD10MD . Grunert et al. recently described two patients who may actually have HSD10MD but were misdiagnosed as BKTD in earlier reports . Thus the diagnosis of BKTD cannot be based solely on metabolite data. Given the confusing blood acylcarnitine and urinary organic acid profiles between the two disorders, enzyme activity assays or mutation analysis are essential for differential diagnosis.
Most symptomatic patients in this cohort presented with acute metabolic decompensations or displayed neurologic impairment. Similar to previous study, neonatal presentation was very rare in this cohort and only two patients with neonatal onset . About two thirds of patients have a favorable outcome. However, three clinical patients who did not undergo NBS presented with acute metabolic decompensations and died early, highlighting the importance of NBS for BKTD. NBS may be the only method for early detection of BKTD, and severe metabolic crises could be avoided if patients are properly managed. It is well known that acute episodes of most BKTD patients were associated with infections. Consistent with previous studies, six of our patients were triggered by respiration tract infections or diarrhea . Notably, this study reported two patients developed severe metabolic crises triggered by vaccination and one had died. The patient who died experienced acute metabolic crises at 8 months that were triggered by febrile reaction to inactivated Japanese encephalitis vaccine, indicating that the risk of metabolic decompensation should be considered and special caution should be taken for BKTD patients before and after the injection of the vaccine.
At least 105 ACAT1 variants associated with BKTD have been described so far . Most are private variants, only four variants have been identified in more than six families. The most frequent variant, c.622C > T (p.R208*), was found in 28 families and most of them are Vietnamese origin [22–24]. This variant was detected in 6 families and 10 individuals in our patient cohort, and counted for 17.2% of all the mutant alleles identified in Chinese patients. This is consistent with previous studies that it is the most common variant. The second most common variant was c.1006-1G > C, a splice site variant associated with exon 11 skipping, that was detected in 13 families and most of them are also Vietnamese [22, 25]. This variant was identified in 4 families in this cohort and as reported in previous literature was the second common variant in China. Two other common variants, c.578T > G (p.M193R) and c.455G > C (p.G152A), however, were not observed in Chinese patients [8, 26]. Notably, another second common variant in our cohort was c.1124A > G (p.N375S), but it was rarely identified in other populations. This variant has been proven to activate a cryptic splice donor site and cause aberrant splicing [8, 22, 27]. Thus the ACAT1 mutational spectrum appears to vary among different ethnic groups. Our study identified five previously unreported variants. They are c.1119dup (p.V374Sfs*86), c.631C > A (p.Q211K), c.1154A > T (p.H385L), c.401T > C (p.M134T), and c.481T > C (p.Y161H). And all novel variants were predicted to be pathogenic by in silico analysis, expanding the molecular profiles of ACAT1.
To summarize, this study for the first time revealed that the incidence of BKTD in China was about 1 per 1 million newborns. Most patients have a favorable outcome, but severe metabolic decompensation and even death may occur. NBS is an effective method to identify BKTD early and prevent severe metabolic crises. C4OH is a potential screening marker, the performance of BKTD NBS may be improved and FN results can be reduced by adding C4OH to C5OH and C5:1 for the combination in NBS. The mutational spectrum of ACAT1 in Chinese population was established. Previously unreported five novel variants were identified, expanding the molecular profiles of ACAT1.