Next generation sequencing
Coding genomic regions were enriched with a SureSelectXT Human All Exon Kit V7 (Agilent technologies, Santa Clara, California) for subsequent sequencing on a NovaSeq 6000 (Illumina, San Diego, California).
Case presentations
Patient 1
The patient is the second child of non-consanguineous parents. The father is of Vietnamese origin, the mother of Romanian ancestry. The 2-year-old sister is healthy. The girl was born at 38 gestational weeks after an uneventful pregnancy. Her birth weight was 2770g, birth length 48 cm, and head circumference 32 cm, Apgar 10/10. The family was discharged from hospital on day 4, but the girl presented again on day 8 with neonatal icterus (total bilirubin 21.2 mg/dl, direct bilirubin 0.8 mg/dl) requiring phototherapy. Apart from hyperbilirubinemia the clinical condition was very good, and the girl was fully breastfed. Phototherapy could be terminated after 23 hours and bilirubin levels remained stable thereafter.
At the age of 8 weeks the child presented to the local pediatrician with a mild febrile airway infection. The girl was severely icteric but otherwise still in good clinical condition. The mother reported that the child required feeding every other hour, also at night. No stool or urine abnormalities were observed. She was admitted to the hospital for further diagnostic work-up. Laboratory testing revealed hepatopathy with the following parameters: prothrombin time 40%, INR 1.64, partial thromboplastin time 41 sec., fibrinogen 63 mg/dL (normal 130-330 mg/dL), antithrombin 35%, AST 86 U/L (normal 10-35 U/L) , ALT 28 U/L (normal 10-35 U/L), alkaline phosphatase 1367 U/L (normal < 449 U/L), gamma-GT 149 U/L (8-178 U/L), total bilirubin 10.2 mg/dL (normal < 1 mg/dL), direct bilirubin 4.9 mg/dL (normal < 0.3 mg/dL), total protein 4.0 g/dL (normal 4.4-7.6 g/dL), albumin 2.7 g/dL (normal 3.8-5.4 g/dL), alpha-fetoprotein >12,000 ng/mL (normal < 77 ng/mL), and ferritin 1402 ng/mL (normal 15-150 ng/mL). The blood count was normal, and there were no signs of infection (CRP 3.4 mg/dL). Blood gas analysis yielded an elevated lactate concentration of 6.1 mmol/L. Ammonia concentration was 123 µmol/L (normal < 70 µmol/L).
Abdominal ultrasound showed liver size at the upper normal limit with enhanced echogenicity. No splenomegaly was detected, and the gall bladder as well as the bile ducts were normal. Polymerase chain reaction (PCR) analyses for CMV, enterovirus and parechovirus were negative, and toxoplasmosis was excluded serologically. Immunological investigations were not suggestive for immunological causes of the hepatopathy. Elastase concentration in stool was normal and not suggestive of cystic fibrosis. Ultrasound of the brain, electrocardiogram and echocardiography were unremarkable.
During the following days the child displayed recurrent episodes of asymptomatic severe hypoglycemia with minimal glucose concentrations of 1.3 mmol/L despite frequent feeding (every 2 hours). Lactate concentrations ranged from 0.9 to 6.1 mmol/L.
Metabolic investigations were interpreted as not suggestive of a specific disorder. Organic acids in urine showed massive tyrosyluria, well compatible with hepatopathy. Acylcarnitine analysis in dried blood spots yielded an elevated concentration of free carnitine with unspecifically elevated levels of several acylcarnitines. Amino acid analysis showed elevated concentrations of several amino acids including citrulline (331 µmol/L, normal < 35 µmol/L). The sialotransferrin pattern (screening for congenital disorders of glycosylation) was normal.
Due to the trias of hepatopathy/impaired hepatic function, lactic acidosis and recurrent hypoglycemia a hepatic form of a mitochondrial depletion syndrome was initially suspected, also due to the elevated concentrations of alpha fetoprotein and ferritin. Clinically, no neurologic involvement was observed, and the brain MRI showed unremarkable results. To prevent hypoglycemia breast milk was supplemented with MCT oil and maltodextrin. After 2 weeks, the child had to be transferred to the intensive care unit due to severe recurrent hypoglycemia. In the end, normal blood glucose levels could only be achieved by continuous i.v. glucose supplementation or continuous oral feeding. Therefore, a percutaneous endoscopic gastrostomy (PEG) tube was inserted to enable normoglycemia and discharge from the hospital. Due to major PEG tube complications and a suspected intestinal perforation followed by a systemic infection inpatient treatment was prolonged. Lactate levels during this episode increased to 17 mmol/L. In the meanwhile, results of trio exome sequencing became available and revealed two mutations in the SLC25A13 gene, one frameshift variant c.852_855del, p.Met285Profs*2 and a novel deletion c.(69+1_70-1)_(212+1_231-1)del, p.?. The father was found heterozygous for the frameshift variant by Sanger sequencing, whereas the mother is a carrier of the deletion, confirmed by qPCR.
Since several attempts to implement tube-feeding with tea or small amounts of breast milk resulted in severe deterioration of abdominal symptoms, the PEG tube was removed after 3 weeks. Explorative laparotomy revealed extensive adhesions of the small intestine but no perforation. After removal of the PEG tube, the clinical condition stabilised and oral feeding could successfully be reintroduced. The child received a galactose-free, carbohydrate reduced, MCT-enriched diet consisting of Pregomin Proexpert (Milupa) and Basic–ch (Nutricia) (composition of nutrients displayed in Figure 1). Under this regimen blood glucose levels remained stable with feeding intervals of 4 hours. Hepatopathy and cholestasis resolved apart from a persistent mild elevation of transaminase activities. Lactate levels also normalised. At the age of 4 months the patient could be discharged from hospital in good clinical condition. Blood glucose monitoring at home revealed no further hypoglycemia.
At the age of six months, supplementary foods were started without any complications. The diet was still galactose-free, carbohydrate-reduced and rich in protein and MCT. The girl is now 2 years old and shows normal psychomotor development. The only pathological findings are slightly elevated transaminase activities. Relevant laboratory parameters during the course of the disease are shown in Figure 2.
Patient 2
One year later her younger brother was born. Genetic testing was initiated immediately after birth. Until the results became available, a diet consisting of 50% breast milk and 50% Pregomin Proexpert (lactose-free, 50% MCT fat) was recommended. On day 12, CD was genetically confirmed by Sanger sequencing for the paternal mutation and qPCR for the maternal mutation. A lactose-free, MCT-enriched diet was started on the same day. He clinically remained asymptomatic with no signs of cholestasis or icterus. Laboratory monitoring was performed at age 4, 7, 13 and 18 weeks. Results of liver enzymes, bilirubin and total protein concentrations are displayed in Figure 3.
Newborn screening
CD is not a target disease of newborn screening programs in Germany. Nevertheless, citrulline levels as well as the levels of other diagnostic amino acids are measured in dried blood spots. Results of the amino acids measured by tandem mass spectrometry in the dried blood spots of the newborn screening samples of our two patients are displayed in Table 1.