Infants with biliary atresia exhibit an altered amino acid profile in their newborn screening

Abstract

Introduction: Biliary atresia (BA) is a rare progressive neonatal cholangiopathy with unknown pathophysiology and time of onset. Newborn Screening (NBS) in Germany is routinely performed in the first days of life to identify rare congenital diseases utilizing dried blood spot (DBS) card analyses. Infants with biliary atresia (BA) are known to have altered amino acid profiles (AAP) at the time point of diagnosis, but it is unclear whether these alterations are present at the time point of NBS.

Objectives: We aimed to analyze amino acid profiles in NBS-DBS of infants with Biliary Atresia.

Methods: Original NBS-DBS cards of 41 infants who were later on diagnosed with BA were retrospectively obtained. NBS-DBS cards from healthy newborns (n = 40) served as controls. In some BA infants (n = 14) a second DBS card was obtained at time of Kasai surgery. AAP in DBS cards were analyzed by targeted metabolomics.

Results: DBS metabolomics in the NBS of at that time point seemingly healthy infants later diagnosed with BA revealed significantly higher levels of Methionine (14.6 ± 8.6 μmol/l), Histidine (23.5 ± 50.3 μmol/l), Threonine (123.9 ± 72.8 μmol/l) and Arginine (14.1 ± 11.8 μmol/l) compared to healthy controls (Met: 8.1 ± 2.6 μmol/l, His: 18.6 ± 10.1 μmol/l, Thr: 98.1 ± 34.3 μmol/l, Arg: 9.3 ± 6.6 μmol/l). Methionine, Arginine and Histidine showed a further increase at time point of Kasai procedure. No correlation between amino acid levels and clinical course was observed.

Conclusion: Our data demonstrate that BA patients exhibit an altered AAP within 72 h after birth, long before the infants become symptomatic. This supports the theory of a prenatal onset of the disease and, thus, the possibility of developing a sensitive and specific NBS. Methionine might be particularly relevant due to its involvement in glutathione metabolism. Further investigation of AAP in BA may help in understanding the underlying pathophysiology.

Keywords: Dried blood spot analysis; Hypermethionemia; Pediatric hepatology; Targeted metabolomics.

Fig. 1

Clinical outcome of BA patients with acquired NBS card during a mean follow-up period of 45 (± 20) months (n = 35; JFNL = jaundice free native liver survival, JNL = jaundice native liver survival, LTX = Liver transplantation)

Fig. 2

Stability of amino acid levels in NBS–DBS cards during exposure to room temperature over time. (AA: amino acids, DBS: dried blood spots, HC: healthy controls, BA: Biliary Atresia)

Fig. 3

a The levels of Methionine, Histidine, Arginine, and Threonine in the NBS DBS in infants with BA are increased in newborns later clinically diagnosed with BA (n = 41) compared to healthy controls (n = 40). (unpaired t-test; ****p < 0.0001, **p < 0.01, *p < 0.05). b Changes in levels of the four significantly altered amino acids from birth to the time point of KPE. Each connected value belongs to the same BA patient at birth and at KPE. (n = 14, mean age at KPE 57 ± 18 days). (paired t-test; ****p < 0.0001, **p < 0.01, *p < 0.05, ns = not significant). (HC: healthy controls, BA: Biliary Atresia, BA-NBS: Biliary Atresia Newborn Screening, KPE: Kasai Portoenterostomy)

Fig. 4

Other analyzed amino acids did not show significant differences between BA patients (n = 41) and healthy newborns (n = 40) at time of NBS (unpaired t-test; ns = not significant) (HC: healthy controls, BA: Biliary Atresia-Newborn Screening)