Genetic and clinical features of patients with intrahepatic cholestasis caused by citrin deficiency

Introduction

Citrin deficiency (CD) was first identified in Japan by Kobayashi [1] with the mutation of the SLC25A13 gene on chromosome 7q21.3 and had been reported frequently in several countries [2]. CD should be confirmed by genetic testing. All the 54 different genetic mutations of SLC25A13 had been detected, including nonsense (12/54), missense (17/54), insertion (5/54), deletion (5/54), and splicing (14/54), among which c.852_855del and IVS16ins3kb mutations could be almost identified in patients with CD in the East Asia [3, 4].

CD is associated with various clinical features from infancy to adulthood, including intrahepatic cholestatic hepatitis, fatty liver, hepatic fibrosis, hypoalbuminemia, coagulopathy, hypoglycemia, and elevated alpha-fetoprotein (AFP) levels [5]. Levels of amino acidemias, such as citrulline, arginine, threonine, methionine, phenylalanine, and tyrosine are elevated in patients with CD [6]. The clinical manifestations of CD are diverse. There are no specific markers to differentiate CD from intrahepatic cholestasis.

Dysregulation of bile acid synthesis and metabolism is a primary indicator of liver dysfunction. It has been shown that plasma bile acid level is a sensitive indicator of hepatobiliary diseases, and it can be used as one of the reliable indicators of liver function [7]. It has been reported that bile acid levels could be applicable to diagnose cholestatic hepatobiliary diseases [8].

The present study aimed to analyse genetic mutations and clinical characteristics of a series of patients with CD. We collected data on plasma bile acid profiles in CD patients and idiopathic cholestasis (IC) group, hoping to find a potential biomarker for non-invasive early diagnosis of CD.

Patients and methods

A total of 14 patients (12 males and 2 females, age 1–18 months, mean 3.6 months) diagnosed with CD were admitted to the Maternal and Child Hospital of Hubei Province (Wuhan, China) from May 2015 to December 2021. The patients’ demographic and clinical data, including age, sex, birth weight, biochemical characteristics, clinical symptoms, and molecular findings were collected.

In addition, 30 age-matched patients (15 males and 15 females, age 1–20 months, mean 3.8 months) with idiopathic cholestasis (IC) were assigned to the control group for evaluation of plasma bile acid profiles. According to the guidelines of the North American Society for Paediatric Gastroenterology, Hepatology and Nutrition [9], IC was defined as a group of disorders of unknown origin that are associated with cholestasis in the newborn and young infant.

LC-MS/MS has a short analysis time, can separate of isomers, and has a simple sample preparation method and process that does not require derivatisation. It has high accuracy, with lower limits of quantification (LLOQ) typically reaching 0.1 ng/mL, and can accurately isolate and quantify different types of BA. The ultra-performance liquid chromatography (UPLC) method provides a shorter elution time and higher separation efficiency. UPLC coupled to MS is the mainstream method for the simultaneous detection of mixed bile acid profiles; it can improve the separation of isomers with the same ion fragments [10]. In this study, plasma bile acid levels in the CD and IC groups were measured using the UPLC-MS/MS method. Detection was performed using an AB Sciex 4,500 mass spectrometer equipped with a Waters Acquisity UPLC I-Class liquid phase system. The mass spectrometer source gas parameters including curtain gas were set at 25 psi, the ion spray voltage was −4,500 V and the temperature was 600 °C. The liquid chromatography column temperature was maintained at 50 °C. Mobile phase A consisted of double-distilled water, 10 mmol/L ammonium acetate and 0.1 % ammonium hydroxide. Mobile phase B consisted of acetonitrile (70 %), methanol (30 %), 10 mmol/L ammonium acetate, and 0.3 % ammonium hydroxide. A gradient mode was used at a flow rate of 0.5 mL/min. The sample temperature was 8 °C.

Plasma samples were prepared using the protein precipitation method. The protein precipitation method, which is simple, fast and inexpensive, has been widely used as the main preparation method, using methanol as the precipitant. The internal standards, calibrators and quality control samples must be centrifuged at a low speed of 2,000 rpm for 1 min, and the calibrators were diluted to different levels. The diluted internal standards (150 μL) were added to the calibration and quality control on the template, sealed and vortexed for 1 min. After centrifugation for 30 min, 80 μL supernatant was removed and 80 μL purified water was added, the membrane was sealed and shaken for 2 min for LC-MS/MS analysis.

Results

Mutation spectrum and clinical features of patients with citrin deficiency

In this study, we identified eight different SLC25A13 mutations, and detailed SLC25A13 variant profiles in the CD group were shown in Table 1. Genetic testing revealed that 7 out of 14 patients were homozygous for the c.852_855del(p.M285Pfs*2), and the rest were compound heterozygous. The other variants included IVS16ins3kb(5/28), c.1638_1660dup(p.A554Gfs*17) (2/28), c.955C>T(p.R319X) (1/28), and c.615+5G>A(p.A206Vfs*7) (1/28). Three novel variants of SLC25A13 were identified: ① c.1043C>T (p.P348L) (n=1) in exon11 (Figure 1A). Nucleotide 1,043 in the coding region was changed from cytosine to thymine, resulting in a change of amino acid 348 changed from proline to leucine, which was a missense mutation. Sanger sequencing result of the novel mutation showed that it was inherited from his father. ② c.1216dupG(p.A406Gfs*13) (n=1) in exon12 (Figure 1B), a guanine was inserted in the coding region 1,216, resulting in an amino acid change that was a frameshift mutation. Pedigree validation analysis showed that the patient’s father was heterozygous for this mutation. ③ c.135G>C (p.L45F) (n=1) in exon3 (Figure 1C). Nucleotide 135 in the coding region was changed from guanine to cytosine, resulting in a change of amino acid 45 from leucine to phenylalanine, which was a missense mutation and inherited from his mother.

Table 1:

Demographic data and mutations of SLC25A13 in 14 patients with CD.

Patients	Gender	Birth weight, g	Gestational age	Age at presentation	Mutations
1	Male	3,500	39w1d	2m5d	c.955C>T(p.R319X)
					c.852_855del(p.M285Pfs*2)
2	Male	4,000	40w	2m	c.1043C>T (p.P348L) (novel)
					IVS16ins3kb
3	Male	3,450	39w	1m	c.1216dupG(p.A406 Gfs*13) (novel)
					IVS16ins3kb
4	Male	3,000	39w	5m	c.852_855del (p.M285Pfs*2)
					c.852_855del (p.M285Pfs*2)
5	Male	3,000	39w5d	4m23d	c.852_855del (p.M285Pfs*2)
					IVS16ins3kb
6	Male	3,600	40w	1m11d	c.852_855del (p.M285Pfs*2)
					c.852_855del (p.M285Pfs*2)
7	Female	3,000	40w2d	2m6d	c.1638_1660dup(p.A554Gfs*17)
					IVS16ins3kb
8	Male	2,380	37w	1m5d	c.852_855del (p.M285Pfs*2)
					c.852_855del (p.M285Pfs*2)
9	Male	3,250	38w2d	1m	c.852_855del (p.M285Pfs*2)
					c.852_855del (p.M285Pfs*2)
10	Male	2,600	37w5d	2m	c.852_855del (p.M285Pfs*2)
					c.852_855del (p.M285Pfs*2)
11	Female	2,750	38w1d	8m	c.1638_1660dup(p.A554Gfs*17)
					IVS16ins3kb
12	Male	3,200	39w3d	1m25d	c.852_855del (p.M285Pfs*2)
					c.852_855del (p.M285Pfs*2)
13	Male	3,000	40w	18m	c.852_855del (p.M285Pfs*2)
					c.852_855del (p.M285Pfs*2)
14	Male	3,250	39w	1m	c.615+5G>A (p.A206Vfs*7)
					c.135G>C (p.L45F) (novel)

1. CD, citrin deficiency.

Figure 1:

The three novel variants of SLC25A13 gene and Sanger sequencing confirmed the mutations. (A) Patient 1: the c.1043C>T (p.P348L) in exon11,which was a missense mutation and inherited from his father. (B) Patient 2: the c.1216dupG(p.A406 Gfs*13) in exon12, which was a frameshift mutation and inherited from his father. (C) Patient 3: the c.135G>C (p.L45F) in exon3, which was a missense mutation and inherited from his mother.

The phenotype and clinical features of the mutation carriers were highly consistent with the disease caused by CD. None of these novel mutations were observed in the Human Gene Mutation Database (HGMD) or the Genome Aggregation Database (gnomAD) or the 1000 Genomes Project.

In addition, all the 14 patients in the CD group had prolonged neonatal jaundice. Biochemical analysis showed elevated total bilirubin (TBil) (14/14), direct bilirubin (DBil) (14/14), indirect bilirubin (Ibil) (14/14), and total bile acids (TBA) (14/14), accompanying with significantly higher levels of alphafetoprotein (AFP) (13/14), hyperlactatemia (12/14), hypoglycemia (10/14), hypertriglyceridemia (4/14), hypercholesterolaemia (2/14) and aspartate transaminase (AST)/alanine transaminase (ALT) ratio greater than 1 (12/14), and decreased albumin (9/14). Half of the patients had significantly elevated citrulline, arginine, methionine, and threonine while 3 of 14 had large amounts of 4-hydroxyphenyl lactic acid and 4-hydroxyphenyl-pyruvic acid on urine chromatography-mass spectrometry analysis (shown in Table 2).

Table 2:

Biochemical characteristics of 14 patients with CD.

Parameters (reference range)	Mean ± SD	Positive numbers, n (%)
ALT (7–81 U/L)	80.9 ± 76.4	5 (36)
AST (21–80 U/L)	142.5 ± 87.9	10 (71)
r-GGT (9–150 U/L)	195.9 ± 85.2	11 (78)
ALP (98–532 U/L)	880.6 ± 416.8	11 (78)
TBIL (5–21 umol/L)	155.8 ± 58.4	14 (100)
DBIL (0–3.4 umol/L)	74.7 ± 36.2	14 (100)
TBA (0–15 umol/L)	250.2 ± 125.7	14 (100)
ALB (35–50 g/L)	32.8 ± 6.5	9 (64)
AST/ALT>1	3.03 ± 2.12	12 (85)
AFP (0–28 ng/mL)	>3,000	13 (92)
Lac (0.5–1.0 mmol/L)	2.5 ± 0.7	12 (85)
BS (3.9–6.1 mmol/L)	2.82 ± 1.43	10 (71)
TG (0–1.7 mmol/L)	1.74 ± 0.92	4 (28)
CHOL (3–5.7 mmol/L)	4.65 ± 1.01	2 (14)
LDL (1.89–4.21 mmol/L)	2.17 ± 1.10	7 (50)
citrulline (7–35 umol/L)	284 ± 212.3	7 (50)
arginine (11–69 umol/L)	124 ± 65	7 (50)
methionine (5–34 umol/L)	225 ± 124	7 (50)
threonine (40–211 umol/L)	523 ± 226	7 (50)
4-HPL (8.6–73.2 umol/L)	123 ± 76.5	3 (21)
4-HPP (0–70 umol/L)	218 ± 128.7	3 (21)

1. CD, citrin deficiency; ALT, alanine aminotransferase; AST, aspartate aminotransferase; γ-GGT, γ-glutamyl transpeptidase; ALP, alkaline phosphatase; TBIL, total bilirubin; DBIL, direct bilirubin; IBIL, indirect bilirubin; TBA, total bile acids; ALB, albumin; AFP, alpha-feto protein; Lac, lactic acid; BS, blood sugar; TG, triglyceride; CHOL, cholesterol; LDL, low density lipoprotein; 4-HPL, 4-hydroxyphenyl lactate; 4-HPP, 4-hydroxyphenyl pyruvate.

A lactose-free, medium-chain triacylglycerol (MCT) enriched therapeutic formula and oral ursodeoxycholic acid was started immediately. The majority of patients were self-limited. Only one patient was diagnosed with hepatic fibrosis and liver failure and eventually died at the age of 1 year due to uncorrectable abnormal coagulation function.

Discussion

In the present study, three novel mutation sites, c.1043C>T (p.P348L) in exon11, c.1216dupG (p.A406 Gfs*13) in exon12 and c.135G>C (p.L45F) in exon3, which had not been previously reported in HGMD or gnomAD and the 1000 Genomes Project, were found for the first time in patients with CD. The deleterious effects were supported by computational evidence. The phenotype or family history of the heterocarrier was highly consistent with the disease caused by the mutation of the gene SLC25A13 gene and finally diagnosed with CD. These three novel variants could provide a molecular genetic reference. The other pathogenic variants included c.955C>T, c.852_855del, c.1638_1660dup, c.615+5G>A, and IVS16ins3kb, which had been reported previously [4, 11, 12], and the mutations, such as c.852_855del, and IVS16ins3kb were more frequently identified in China [4]. The pool of gene mutations was increased in many reports [13, 14].

The main clinical manifestations caused by CD in the present study were neonatal cholestatic jaundice, higher levels of γ-GGT, ALP, and AFP. Other biochemical findings, including AST/ALT ratio >1, dyslipidemia, and hypoglycemia were relatively common in CD patients. The protein of citrin is localised in the liver-type aspartate/glutamate carrier isoform 2 (AGC2) in the mitochondrial membrane [15]. This protein imports glutamate together with H+ from the cytosol into the mitochondrial matrix and exports aspartate in the opposite direction, playing a crucial role in the urea cycle. CD could exacerbate liver cell damage, creating a vicious circle. Mitochondrial dysfunction and severe damage could cause AST to rise more than ALT and other clinical manifestations.

Plasma amino acid analysis in newborn screening (NBS) revealed moderately elevated plasma levels of citrulline, arginine, and threonine, and mildly elevated levels of methionine. However, in our study, only half of the NBS showed typical profiles of multiple aminoacidemia, suggesting that not all the patients with CD had obvious clinical manifestations of the disease and abnormal results of the NBS [16]. Several studies had showed that normal plasma amino acid levels might lead to missed diagnosis of CD [6, 17]. There are no specific markers to differentiate CD from intrahepatic cholestasis. We also expected that more laboratory markers might be specific for the diagnosis of early-stage CD.

To date, a large number of studies have found the bile acid spectrum to be an indicator for the diagnosis of liver disease [18]. The bile acid spectrum of patients with progressive familial intrahepatic cholestasis(PFIC) showed 2–7 times higher ratios of taurine: glycine conjugated primary bile acids, and unchanged levels of secondary bile acids compared to healthy controls [19]. Patients with biliary atresia had a distinct bile acid profile characterised by higher levels of taurochenodeoxycholic acid (TCDCA) and lower levels of chenodeoxycholic acid (CDCA). The ratio of the plasma TCDCA to CDCA was significantly higher in patients with biliary atresia [20].

The classical pathway of bile acid synthesis from cholesterol is catalysed by cholesterol 7 alpha-hydroxylase (CYP7A1). These primary bile acids synthesised by the liver, including cholic acid (CA) and chenodeoxycholic acid (CDCA), are dehydroxylated by microorganisms in the intestine to form secondary bile acids, such as deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) [21]. The farnesoid X receptor (FXR) is highly expressed in hepatocytes and intestine and could be activated by bile acid. CDCA is a hydrophobic bile acid that is the most potent ligand of FXR [22]. Activation of FXR could inhibit bile acid synthesis in the liver to prevent the accumulation of bile acid in liver cells [23]. In the present study, it was revealed that the concentrations of GCDCA, TCA, and TCDCA were found to be significantly higher in the CD group than those in the IC group. Fu et al. collected the samples from patients with neonatal intrahepatic cholestasis caused by citrin deficiency(NICCD) to test bile acid profiles, and showed that elevated plasma total bile acid concentrations correlated with elevated conjugated primary bile acids in patients with NICCD [8]. Yang et al. also found that plasma total bile acid concentrations, including primary or secondary bile acids, were higher in infants with CD than in healthy controls [7]. Contrary to Yang’s valuable report, TLCA was not significantly different between the two groups in our study. CD may cause damage to liver function, which increases the amount of bile acid that enters the blood through the hepatic sinusoid. To maintain the normal enterohepatic circulation of bile acids, the number of enterohepatic circulations must be increased. FXR activity is inhibited in patients and CYP7A1 inhibition is reduced, in addition it also leads to changes in bile acid receptors and transporters. The abundance and quantity of intestinal flora also changed, and taurine metabolising bacteria increased. These changes could lead to changes in total bile acid levels. Furthermore, LCA is a monohydroxy and toxic bile acid. With the progression of the disease caused by CD, the spectrum of bile acids would be different, and this could be the reason for the difference from previous research results.

Previous studies of CD have mainly shown a benign condition, with the majority of patients were self-limiting after a period of compensating for their high protein, high fat, and low carbohydrate diet between the ages of 6 and 12 months [24, 25]. However, although rare, some cases eventually developed chronic liver failure and required liver transplantation to survive [26, 27]. In addition, about twenty cases of CD with infection and bleeding associated with coagulopathy in the setting of liver failure had been previously reported and they eventually died [17]. In the present study, only one patient was diagnosed with hepatic fibrosis and liver failure, who eventually died from uncorrectable abnormal coagulation function. Therefore, it is important to remain vigilant and avoid irreversible liver failure in the course of CD.

Our study had several limitations. First, plasma bile acid profiles were influenced by diet, and it was difficult to distinguish the influencing factors. Secondly, we did not collect data on bile acid profiles after treatment. Finally, the sample size was extremely small and no normal control group was established. Further studies are needed to address the above mentioned limitations and to verify the results of this study.

Conclusions

This study described the clinical characteristics and gene mutations of 14 patients with CD, and identified three novel mutations in the SLC25A13 gene. Compared with the IC group, plasma bile acid profiles, such as GCDCA, TCA, and TCDCA could be diagnostic markers for CD. In addition, not all patients with CD had benign outcomes, and we should seriously counsel patients’ family members to avoid misdiagnosis.