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1.  Biochemical characteristics of neonatal cholestasis induced by citrin deficiency 
AIM: To explore differences in biochemical indices between neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) and that with other etiologies.
METHODS: Patients under 6 mo of age who were referred for investigation of conjugated hyperbilirubinaemia from June 2003 to December 2010 were eligible for this study. After excluding diseases affecting the extrahepatic biliary system, all patients were screened for the two most common SLC25A13 mutations; the coding exons of the entire SLC25A13 gene was sequenced and Western blotting of citrin protein performed in selected cases. Patients in whom homozygous or compound heterozygous SLC25A13 mutation and/or absence of normal citrin protein was detected were defined as having NICCD. Cases in which no specific etiological factor could be ascertained after a comprehensive conjugated hyperbilirubinaemia work-up were defined as idiopathic neonatal cholestasis (INC). Thirty-two NICCD patients, 250 INC patients, and 39 infants with cholangiography-confirmed biliary atresia (BA) were enrolled. Laboratory values at their first visit were abstracted from medical files and compared.
RESULTS: Compared with BA and INC patients, the NICCD patients had significantly higher levels of total bile acid (TBA) [all measures are expressed as median (inter-quartile range): 178.0 (111.2-236.4) μmol/L in NICCD vs 112.0 (84.9-153.9) μmol/L in BA and 103.0 (70.9-135.3) μmol/L in INC, P = 0.0001]. The NICCD patients had significantly lower direct bilirubin [D-Bil 59.6 (43.1-90.9) μmol/L in NICCD vs 134.0 (115.9-151.2) μmol/L in BA and 87.3 (63.0-123.6) μmol/L in INC, P = 0.0001]; alanine aminotransferase [ALT 34.0 (23.0-55.0) U/L in NICCD vs 108.0 (62.0-199.0) U/L in BA and 84.5 (46.0-166.0) U/L in INC, P = 0.0001]; aspartate aminotransferase [AST 74.0 (53.5-150.0) U/L in NICCD vs 153.0 (115.0-239.0) U/L in BA and 130.5 (81.0-223.0) U/L in INC, P = 0.0006]; albumin [34.9 (30.7-38.2) g/L in NICCD vs 38.4 (36.3-42.2) g/L in BA and 39.9 (37.0-42.3) g/L in INC, P = 0.0001]; glucose [3.2 (2.0-4.4) mmol/L in NICCD vs 4.1 (3.4-5.1) mmol/L in BA and 4.0 (3.4-4.6) mmol/L in INC, P = 0.0014] and total cholesterol [TCH 3.33 (2.97-4.00) mmol/L in NICCD vs 4.57 (3.81-5.26) mmol/L in BA and 4.00 (3.24-4.74) mmol/L in INC, P = 0.0155] levels. The D-Bil to total bilirubin (T-Bil) ratio was significantly lower in NICCD patients [all measures are expressed as median (inter-quartile range): 0.54 (0.40-0.74)] than that in BA patients [0.77 (0.72-0.81), P = 0.001] and that in INC patients [0.74 (0.59-0.80), P = 0.0045]. A much higher AST/ALT ratio was found in NICCD patients [2.46 (1.95-3.63)] compared to BA patients [1.38 (0.94-1.97), P = 0.0001] and INC patients [1.48 (1.10-2.26), P = 0.0001]. NICCD patients had significantly higher TBA/D-Bil ratio [3.36 (1.98-4.43) vs 0.85 (0.72-1.09) in BA patients and 1.04 (0.92-1.14) in INC patients, P = 0.0001], and TBA/TCH ratio [60.7 (32.4-70.9) vs 24.7 (19.8-30.2) in BA patients and 24.2 (21.4-26.9) in INC patients, P = 0.0001] compared to the BA and INC groups.
CONCLUSION: NICCD has significantly different biochemical indices from BA or INC. TBA excretion in NICCD appeared to be more severely disturbed than that of bilirubin and cholesterol.
PMCID: PMC3482648  PMID: 23112554
Cholestasis; Biliary atresia; Infants; Idiopathic neonatal cholestasis; SLC25A13
2.  Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency in Korean Infants 
Journal of Korean Medical Science  2007;22(6):952-956.
Citrin is a liver-type mitochondrial aspartate-glutamate carrier encoded by the SLC25A13 gene, and its deficiency causes adult-onset type II citrullinemia and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). Here, the authors investigated clinical findings in Korean infants with NICCD and performed mutation analysis on the SLC25A13 gene. Of 47 patients with neonatal cholestasis, three infants had multiple aminoacidemia (involving citrulline, methionine, and arginine) and galactosemia, and thus were diagnosed as having NICCD. Two of these three showed failure to thrive. The laboratory findings showed hypoproteinemia and hyperammonemia, and liver biopsies revealed micro-macrovesicular fatty liver and cholestasis. The three patients each harbored compound heterozygous 1,638-1,660 dup/ S225X mutation, compound heterozygous 851del4/S225X mutation, and heterozygous 1,638-1,660 dup mutation, respectively. With nutritional manipulation, liver functions were normalized and catch-up growth was achieved. NICCD should be considered in the differential diagnosis of cholestatic jaundice in Korean infants.
PMCID: PMC2694627  PMID: 18162705
Cholestasis; Citrin; Citrullinemia; SLC25A13; Mutation
3.  SLC25A13 Gene Analysis in Citrin Deficiency: Sixteen Novel Mutations in East Asian Patients, and the Mutation Distribution in a Large Pediatric Cohort in China 
PLoS ONE  2013;8(9):e74544.
The human SLC25A13 gene encodes citrin, the liver-type mitochondrial aspartate/glutamate carrier isoform 2 (AGC2), and SLC25A13 mutations cause citrin deficiency (CD), a disease entity that encompasses different age-dependant clinical phenotypes such as Adult-onset Citrullinemia Type II (CTLN2) and Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD). The analyses of SLC25A13 gene and its protein/mRNA products remain reliable tools for the definitive diagnoses of CD patients, and so far, the SLC25A13 mutation spectrum in Chinese CD patients has not been well-characterized yet.
Methods and Results
By means of direct DNA sequencing, cDNA cloning and SNP analyses, 16 novel pathogenic mutations, including 9 missense, 4 nonsense, 1 splice-site, 1 deletion and 1 large transposal insertion IVS4ins6kb (GenBank accession number KF425758), were identified in CTLN2 or NICCD patients from China, Japan and Malaysia, respectively, making the SLC25A13 variations worldwide reach the total number of 81. A large NICCD cohort of 116 Chinese cases was also established, and the 4 high-frequency mutations contributed a much larger proportion of the mutated alleles in the patients from south China than in those from the north (χ2 = 14.93, P<0.01), with the latitude of 30°N as the geographic dividing line in mainland China.
This paper further enriched the SLC25A13 variation spectrum worldwide, and formed a substantial contribution to the in-depth understanding of the genotypic feature of Chinese CD patients.
PMCID: PMC3777997  PMID: 24069319
4.  Treatment with Lactose (Galactose)-Restricted and Medium-Chain Triglyceride-Supplemented Formula for Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency 
JIMD Reports  2011;2:37-44.
Citrin plays a role in the transfer of NADH-reducing equivalent from cytosol to mitochondria as part of the malate–aspartate shuttle in liver. Citrin deficiency may cause an impairment of glycolysis due to an increase in the cytosolic NADH/NAD ratio leading to an energy shortage in the liver. Mutations of the SLC25A13 gene are responsible for neonatal intrahepatic cholestasis (NICCD) and adult-onset type II citrullinemia (CTLN2). Most patients with NICCD show a resolution of symptoms within the first year of life, but some patients present with severe symptoms and require liver transplantation. We treated four patients including three siblings with NICCD by lactose (galactose)-restricted and medium-chain triglyceride (MCT)-supplemented formula. This formula rapidly improved the clinical condition and laboratory findings. Early treatment was more effective and did not require long-term administration. Lactose (galactose)-restriction can avoid further increase in the cytosolic NADH/NAD ratio in the liver and MCT supplementation can provide energy to hepatic cells by producing an excess of acetyl-CoA in mitochondria. Early treatment with lactose (galactose)-restricted and MCT-supplemented formula is recommended for patients with NICCD and possibly for patients with CTLN2.
PMCID: PMC3509838  PMID: 23430852
5.  Inspissated bile syndrome in an infant with citrin deficiency and congenital anomalies of the biliary tract and esophagus: Identification and pathogenicity analysis of a novel SLC25A13 mutation with incomplete penetrance 
Biallelic mutations of the SLC25A13 gene result in citrin deficiency (CD) in humans. Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is the major CD phenotype in pediatrics; however, knowledge on its genotypic and phenotypic characteristics remains limited. The present study aimed to explore novel molecular and clinical characteristics of CD. An infant suspected to have NICCD as well as her parents were enrolled as the research subjects. SLC25A13 mutations were investigated using various methods, including cDNA cloning and sequencing. The pathogenicity of a novel mutation was analyzed bioinformatically and functionally with a yeast model. Both the infant and her father were heterozygous for c.2T>C and c.790G>A, while the mother was only a c.2T>C carrier. The novel c.790G>A mutation proved bioinformatically and functionally pathogenic. The infant had esophageal atresia and an accessory hepatic duct, along with bile plug formation confirmed by laparoscopic surgery. However, the father seemed to be healthy thus far. The findings of the present study enrich the genotypic and phenotypic characteristics of CD patients, and provided clinical and molecular evidence suggesting the possible non-penetrance of SLC25A13 mutations and the likely involvement of this gene in primitive foregut development during early embryonic life.
PMCID: PMC4199400  PMID: 25216257
citrin deficiency; inspissated bile syndrome; esophageal atresia; functional analysis
6.  Screening of SLC25A13 mutation in the Thai population 
AIM: To determine the prevalence of SLC25A13 mutations in the Thai population.
METHODS: A total of 1537 subjects representing the Thai population were screened for a novel pathologic allele p.Met1? (c.2T > C) and six previously known common SLC25A13 mutations: [I] (c.851_854delGTAT), [II] (g.IVS11 + 1G > A), [III] (c.1638_1660dup), [IV] (p.S225X), [V] (IVS13 + 1G > A), and [XIX] (g.IVS16ins3kb) using a newly developed TaqMan and established HybProbe assay, respectively. Sanger sequencing was employed for specimens showing an aberrant peak to confirm the targeted mutation as well as the unknown aberrant peaks detected. Frequencies of the mutations identified were compared in each region. Carrier frequency and disease prevalence of citrin deficiency caused by SCL25A13 mutations were estimated.
RESULTS: p.Met1? was identified in the heterozygous state in 85 individuals, giving a carrier frequency of 1/18, which suggests possible selective advantage of this variant. The question of p.Met1? homozygote lethality remains unanswered which may serve as an explanation as to why this homozygote has yet to be identified in patients/controls even with high allele frequency. The p.Met1? mutation has rarely been studied in populations other than Thai and Chinese; therefore, may have been overlooked. Development of the TaqMan assay in the present study would allow a simple, rapid, and cost-effective method for mass screening. Heterozygous mutations: [XIX] and [I] were identified in 17 individuals, giving a carrier rate of 1/90 and a calculated homozygote rate of 1/33000. Two novel variants, g.IVS11 + 17C > G and c.1311C > T, of unknown clinical significance were identified at low frequency.
CONCLUSION: This study highlighted the current underestimation of citrin deficiency and suggests the possible selective advantage of the p.Met1? allele.
PMCID: PMC3837273  PMID: 24282362
Aspartate-glutamate carrier; Isoform 2; Citrin deficiency; Type II citrullinemia; Neonatal intrahepatic cholestasis caused by citrin deficiency; SLC25A13
7.  Slc25a13-Knockout Mice Harbor Metabolic Deficits but Fail To Display Hallmarks of Adult-Onset Type II Citrullinemia 
Molecular and Cellular Biology  2004;24(2):527-536.
Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn−/− mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn−/− mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.
PMCID: PMC343808  PMID: 14701727
8.  Most common SLC25A13 mutation in 400 Chinese infants with intrahepatic cholestasis 
AIM: To establish the real time fluorescence polymerase chain reaction (RT-PCR) with dual labeled probes for fast detection of SLC25A13 gene mutation 851del4.
METHODS: Four hundred infants (< 1 year of age) with unexplained intrahepatic cholestasis from 18 provinces or municipalities in China were enrolled in this study for detecting their SLC25A13 gene mutation 851del4. Suitable primers and fluorescence-labeled probes for detecting SLC25A13 gene mutation 841del4 were designed. Normal and mutant sequences were detected by PCR with two fluorescence-labeled probes. After a single RT-PCR, results were obtained by analyzing the take-off curves. Twenty-four positive and 14 negative samples were retested by direct sequencing.
RESULTS: Eight homozygous and 30 heterozygous mutations were detected in 46 mutant alleles with a 851del4 mutation rate of 5.8% (46/800). Twenty-six and 20 mutant alleles were observed respectively, in 474 and 242 alleles from the intermediate and southern areas of China. No mutant allele was detected in 84 alleles from northern China. Twenty-four positive samples including 4 homozygous and 20 heterozygous mutations, and 14 negative samples were retested by direct sequencing, which confirmed that the accuracy of RT-PCR was 100%.
CONCLUSION: RT-PCR can detect the mutation 851del4 in infants with intrahepatic cholestasis with an accuracy of 100%.
PMCID: PMC2868222  PMID: 20458766
851del4 mutation; Neonatal intrahepatic cholestasis; Real-time fluorescent polymerase chain reaction; SLC25A13 gene
9.  Different regional distribution of SLC25A13 mutations in Chinese patients with neonatal intrahepatic cholestasis 
AIM: To investigate the differences in the mutation spectra of the SLC25A13 gene mutations from specific regions of China.
METHODS: Genetic analyses of SLC25A13 mutations were performed in 535 patients with neonatal intrahepatic cholestasis from our center over eight years. Unrelated infants with at least one mutant allele were enrolled to calculate the proportion of SLC25A13 mutations in different regions of China. The boundary between northern and southern China was drawn at the historical border of the Yangtze River.
RESULTS: A total of 63 unrelated patients (about 11% of cases with intrahepatic cholestasis) from 16 provinces or municipalities in China had mutations in the SLC25A13 gene, of these 16 (25%) were homozygotes, 28 (44%) were compound heterozygotes and 19 (30%) were heterozygotes. In addition to four well described common mutations (c.851_854del, c.1638_1660dup23, c.615+5G>A and c.1750+72_1751-4dup17insNM_138459.3:2667 also known as IVS16ins3kb), 13 other mutation types were identified, including three novel mutations: c.985_986insT, c.287T>C and c.1349A>G. According to the geographical division criteria, 60 mutant alleles were identified in patients from the southern areas of China, 43 alleles were identified in patients from the border, and 4 alleles were identified in patients from the northern areas of China. The proportion of four common mutations was higher in south region (56/60, 93%) than that in the border region (34/43, 79%, χ2 = 4.621, P = 0.032) and the northern region (2/4, 50%, χ2 = 8.288, P = 0.041).
CONCLUSION: The SLC25A13 mutation spectra among the three regions of China were different, providing a basis for the improvement of diagnostic strategies and interpretation of genetic diagnosis.
PMCID: PMC3725380  PMID: 23901231
Citrin deficiency; Mutation spectrum; Intrahepatic cholestasis; SLC25A13
10.  Clinical, Molecular and Functional Investigation on an Infant with Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency (NICCD) 
PLoS ONE  2014;9(2):e89267.
Background and Objective
SLC25A13 analysis has provided reliable evidences for the definitive diagnosis of citrin deficiency (CD) in the past decade. Meanwhile, these studies generated some issues yet to be resolved, including the pathogenicity of SLC25A13 missense mutations and the mRNA product from the mutation c.615+5G>A. This study aims to investigate the effect of a novel missense mutation on the aspartate/glutamate carrier (AGC) function of citrin protein, and to explore the aberrant transcript from c.615+5G>A in the same CD infant.
Methods and Results
By means of screening for prevalent SLC25A13 mutations and exons sequencing, the patient proved a compound heterozygote of c.615+5G>A and a novel c.1064G>A (p.Arg355Gln) mutation. An aberrant transcript with retention of the entire intron 6, r.[615+1_615+1789ins; 615+5 g>a] (GenBank accession number KJ128074), which was resulted from c.615+5G>A, was detected by RT-PCR and cDNA sequencing. After bioinformatic analyses of the novel missense mutation c.1064G>A, the growth abilities of three agc1Δ yeast strains were tested, which had been transformed with recombinant or empty vectors, respectively. Besides the bioinformatically pathogenic evidences, the growth ability of the agc1Δ strains transformed with mutant recombinant was the same as with empty vector, but significantly lower than that with normal control in functional analysis.
A CD infant was definitely diagnosed in this paper by a genetic, transcriptional and functional analysis of SLC25A13 gene. This study provided direct laboratory evidences supporting the splice-site nature of the c.615+5G>A mutation, and the novel c.1064G>A variation, which proved a pathogenic mutation bioinformatically and functionally, enriched the SLC25A13 mutation spectrum.
PMCID: PMC3931723  PMID: 24586645
11.  Diversity of Disorders Causing Neonatal Cholestasis – The Experience of a Tertiary Pediatric Center in Germany 
Background and Objective: Rapidly establishing the cause of neonatal cholestasis is an urgent matter. The aim of this study was to report on the prevalence and mortality of the diverse disorders causing neonatal cholestasis in an academic center in Germany.
Methods: Clinical chemistry and cause of disease were retrospectively analyzed in 82 infants (male n = 42, 51%) that had presented with neonatal cholestasis to a tertiary medical center from January 2009 to April 2013.
Results: Altogether, 19 disorders causing neonatal cholestasis were identified. Biliary atresia was the most common diagnosis (41%), followed by idiopathic cases (13%), progressive familial intrahepatic cholestasis (PFIC, 10%), cholestasis in preterm infants (10%), α1AT deficiency, Alagille syndrome, portocaval shunts, mitochondriopathy, biliary sludge (all 2%), and others. Infants with biliary atresia were diagnosed with a mean age of 62 days, they underwent Kasai portoenterostomy ~66 days after birth. The majority of these children (~70%) received surgery within 10 weeks of age and 27% before 60 days. The 2-year survival with their native liver after Kasai procedure was 12%. The time span between Kasai surgery and liver transplantation was 176 ± 73 days. Six children (7%), of whom three patients had a syndromic and one a non-syndromic biliary atresia, died prior to liver transplantation. The pre- and post-transplant mortality rate for children with biliary atresia was ~12 and ~17%, respectively.
Conclusion: Neonatal cholestasis is a severe threat associated with a high risk of complications in infancy and it therefore requires urgent investigation in order to initiate life saving therapy. Although in the last 20 years new causes such as the PFICs have been identified and newer diagnostic tools have been introduced into the clinical routine biliary atresia still represents the major cause.
PMCID: PMC4066316  PMID: 25003101
neonatal cholestasis; neonatal jaundice; biliary atresia; Kasai procedure; ERCP
12.  Cholestatic jaundice in infancy. The importance of familial and genetic factors in aetiology and prognosis. 
Archives of Disease in Childhood  1981;56(8):622-627.
One hundred and twenty-four infants admitted to hospitals in Norway between 1955 and 1974 during the first 3 months of life with cholestatic jaundice were studied retrospectively. Sixty-four infants had had extrahepatic atresia of the biliary tree and 60 had had intrahepatic cholestasis. This gives an incidence of about 1:9000 live births for cholestasis. In 4 of the 64 infants with extra-hepatic atresia a bile duct-to-bowel anastomosis had been performed but this was successful in only 2. Sixty of these infants had died by their 2nd birthday. Twenty-six of the infants with intrahepatic cholestasis had died by 1978 and the most common causes of death were cholestasis complicated by infection, bleeding, or hepatoma. The survivors aged between 4 and 23 years were followed up in 1978. In about two-thirds of them aetiological factors--such as alpha-1-antitrypsin deficiency, arteriohepatic dysplasia, cholestasis with lymphoedema--and other familial or genetic factors, or infections were found. Four of the 34 survivors are known to have cirrhosis. Twenty patients had biochemical abnormalities, and 12 had normal liver function tests. Two patients could not be examined. Of the 19 patients with familial or genetic aetiological factors, 4 had cirrhosis, 14 had biochemical abnormalities, and only 5 had normal liver function tests. Of 11 survivors with idiopathic disease or septicaemia, none had cirrhosis and only 4 had abnormal liver function tests.
PMCID: PMC1627279  PMID: 7271301
13.  Novel ABCB11 mutations in a Thai infant with progressive familial intrahepatic cholestasis 
Progressive familial intrahepatic cholestasis (PFIC) type 2 is caused by mutations in ABCB11, which encodes bile salt export pump (BSEP). We report a Thai female infant who presented with progressive cholestatic jaundice since 1 mo of age, with normal serum γ-glutamyltransferase. Immunohistochemical staining of the liver did not demonstrate BSEP along the canaliculi, while multidrug resistance protein 3 was expressed adequately. Novel mutations in ABCB11, a four-nucleotide deletion in exon 3, c.90_93delGAAA, and a single-nucleotide insertion in exon 5, c.249_250insT, were identified, with confirmation in her parents. These mutations were predicted to lead to synthesis of truncated forms of BSEP. Immunostaining and mutation analysis thus established the diagnosis of PFIC type 2.
PMCID: PMC2744194  PMID: 19750581
ABCB11; Bile salt export pump; Progressive familial intrahepatic cholestasis
14.  Evaluation of cholestasis in Iranian infants less than three months of age 
Aim: The aim of this study is to find-out the possible etiologies in Iranian infants less than three months in Shiraz, South of Iran.
Background: Cholestatic jaundice most probably occurs due to a pathological condition and the most frequent causes in early infancy are neonatal hepatitis and biliary atresia. Early diagnosis and treatment of infantile cholestasis can improve prognosis of liver diseases by prevention of the complications of these disorders.
Patients and methods: In this retrospective study, 122 infants under 3 months of age with cholestasis were studied in Nemazee Hospital (affiliated to Shiraz University of Medical Sciences) during the years 2001-2011. Demographic data, duration of jaundice, liver biopsy and the causes of cholestasis were recorded.
Results: There were 76 males (62.3%) and 46 females (37.7%) with a mean age of 54.4 ± 23.7 days. The most common clinical finding was jaundice that was seen in all patients (100%).The onset of jaundice was the first day to the fifty two days of age, with an average age of 15.6 ± 16.1 days. Other findings included hepatomegaly in 92 patients (76.4%), clay-color stool in 54 (44.3%), and splenomegaly in 29 patients (23.8%). In this study, the most common causes of cholestasis were biliary atresia (30=24.6%), idiopathic neonatal hepatitis (30= 24.6%) and bile ducts paucity (16=10.3).
Conclusion: The results of this study showed that biliary atresia and neonatal hepatitis are the most common causes of infantile cholestasis in this area. It is recommended that biliary atresia should be discriminated from other forms of neonatal cholestasis.
PMCID: PMC4285931  PMID: 25584175
Cholestasis; Biliary atresia; Neonatal jaundice
15.  Biopsy-driven diagnosis in infants with cholestatic jaundice in Iran 
AIM: To determine the frequencies of diagnoses confirmed by liver biopsy in infants with cholestasis in an Iranian pediatric hospital.
METHODS: This was a retrospective study conducted in a tertiary referral children’s hospital in Iran. We retrieved all pathology reports of liver biopsies from children less than two years of age who had presented for evaluation of cholestatic jaundice from March 2001 to March 2011. Additional specimen samples obtained from archived pathology blocks were reviewed by a pathologist blinded to the final diagnosis. These results were compared with the pathology reports from chart records to ensure consensus and eliminate any inconsistencies in final diagnoses. A structured checklist was used to gather information on multiple variables including age, sex, gestational age at birth, birth weight, age at which hyperbilirubinemia manifested, presence and identification of associated anomalies, clinical manifestations, and histological findings from liver biopsies. The baseline data are reported using descriptive statistics, and differences between groups were assessed by Fisher’s exact test and Student’s t test when indicated.
RESULTS: Fifty-five cases (28 females; 27 males) of infantile cholestasis (IC) were included in this study. The mean serum total bilirubin and direct bilirubin at presentation were 13.6 ± 5.9 and 7.3 ± 3.4, respectively. Forty cases (72.7%) were the product of term pregnancies. Common associated clinical findings were acholic stool in 33 cases (60.0%), hepatomegaly in 30 cases (54.5%), and dark-colored urine in 21 cases (38.2%). Biliary atresia (BA) was the most frequent diagnosis, found in 32 cases (58.2%), followed by intrahepatic bile duct paucity found in 6 cases (10.9%), metabolic disease in 6 cases (10.9%), idiopathic neonatal hepatitis in 5 cases (9.1%), choledochal cyst in 2 cases (3.6%), liver cirrhosis in 2 cases (3.6%), and progressive familial intrahepatic cholestasis and portal fibrosis each in 1 case (1.8%). The mean times for jaundice onset and liver biopsy were 43.8 and 102.0 d, respectively. In BA, the mean age at jaundice presentation was 21 d and for liver biopsy was 87.5 d, representing a mean delay of 66.5 d.
CONCLUSION: A significant delay was found between IC presentation and liver biopsy, which is detrimental in conditions that can cause irreversible liver damage, such as BA.
PMCID: PMC3921528  PMID: 24574777
Cholestasis; Neonate; Hepatitis; Biliary atresia; Neonatal hepatitis; Infant; Conjugated hyperbilirubinemia; Liver biopsy
16.  Screening of newborn infants for cholestatic hepatobiliary disease with tandem mass spectrometry 
BMJ : British Medical Journal  1999;319(7208):471-477.
To assess the feasibility of screening for cholestatic hepatobiliary disease and extrahepatic biliary atresia by using tandem mass spectrometry to measure conjugated bile acids in dried blood spots obtained from newborn infants at 7-10 days of age for the Guthrie test.
Three tertiary referral clinics and regional neonatal screening laboratories.
Unused blood spots from the Guthrie test were retrieved for infants presenting with cholestatic hepatobiliary disease and from the two cards stored on either side of each card from an index child. Concentrations of conjugated bile acids measured by tandem mass spectrometry in the two groups were compared.
Main outcome measures
Concentrations of glycodihydroxycholanoates, glycotrihydroxycholanoates, taurodihydroxycholanoates, and taurotrihydroxycholanoates. Receiver operator curves were plotted to determine which parameter (or combination of parameters) would best predict the cases of cholestatic hepatobiliary disease and extrahepatic biliary atresia. The sensitivity and specificity at a selection of cut off values for each bile acid species and for total bile acid concentrations for the detection of the two conditions were calculated.
218 children with cholestatic hepatobiliary disease were eligible for inclusion in the study. Two children without a final diagnosis and five who presented at <14 days of age were excluded. Usable blood spots were obtained from 177 index children and 708 comparison children. Mean concentrations of all four bile acid species were significantly raised in children with cholestatic hepatobiliary disease and extrahepatic biliary atresia compared with the unaffected children (P<0.0001). Of 177 children with cholestatic hepatobiliary disease, 104 (59%) had a total bile acid concentration >33 μmol/l (97.5th centile value for comparison group). Of the 61 with extrahepatic biliary atresia, 47 (77%) had total bile acid concentrations >33 μmol/l. Taurotrihydroxycholanoate and total bile acid concentrations were the best predictors of both conditions. For all cholestatic hepatobiliary disease, a cut off level of total bile acid concentration of 30 μmol/l gave a sensitivity of 62% and a specificity of 96%, while the corresponding values for extrahepatic biliary atresia were 79% and 96%.
Most children who present with extrahepatic biliary atresia and other forms of cholestatic hepatobiliary disease have significantly raised concentrations of conjugated bile acids as measured by tandem mass spectrometry at the time when samples are taken for the Guthrie test. Unfortunately the separation between the concentrations in these infants and those in the general population is not sufficient to make mass screening for cholestatic hepatobiliary disease a feasible option with this method alone.
Key messagesThe prognosis of cholestatic hepatobiliary disease in infancy, in particular biliary atresia, is improved by early detectionInfants destined to present with cholestatic jaundice in the first few months of life have raised concentrations of bile acids in the blood spots obtained at 7-10 days for current neonatal screening programmesTandem mass spectrometry can be used to detect this marker of neonatal cholestasisUnfortunately there is too much overlap between bile acid concentrations in infants with cholestasis and those in control infants for this to be used as a single screening test for cholestatic hepatobiliary disease in general and biliary atresiaTandem mass spectrometry is a powerful tool for neonatal screening but every potential application must be carefully assessed
PMCID: PMC28198  PMID: 10454398
17.  Neonatal cholestasis as the presenting feature in cystic fibrosis. 
Between 1960 and 1994 cystic fibrosis was found in nine out of 1474 infants investigated for neonatal cholestasis. Four had delay in passing meconium. In all patients cholestatic jaundice was present during the first 48 hours and in three patients cholestasis was complete, mimicking biliary atresia. Serum cholesterol concentrations were normal in all but two children. Sweat chloride was repeatedly above 95 mmol/l in all instances. Three children had another condition enhancing the risk of cholestasis (alpha1-antitrypsin deficiency, hypopituitarism, perinatal asphyxia, and total parenteral nutrition). Liver histology displayed portal fibrosis and inflammation with bile duct proliferation; mucous plugs in bile ducts were observed in only one patient. Only one child died from cirrhosis. These results indicate that cystic fibrosis is not a major cause of neonatal cholestasis. However early signs of intestinal obstruction and low concentrations of serum cholesterol may indicate cystic fibrosis, regardless of liver histology. Neonatal cholestasis has no prognostic value concerning evolution to cirrhosis.
PMCID: PMC1511658  PMID: 8813874
18.  Bile salts and liver disease in childhood 
Postgraduate Medical Journal  1974;50(584):354-360.
Recognition of neonatal liver disease has been heavily dependent on the occurrence of jaundice. In most instances the jaundice is related to specific disturbances in bilirubin transport and other tests of liver function are normal. In contrast, hepatitis and other liver diseases not specifically affecting bilirubin transport often go undetected unless jaundice occurs.
The development of practical methods for the estimation of bile acids in serum has permitted an evaluation of hepatic excretory function in neonates and children independent of bilirubin excretion. Since bile acid excretion by the liver each day greatly exceeds bilirubin excretion it was not surprising to find that elevations in serum bile acids occur regularly in anicteric hepatitis. Because the excretion of bile acids generates canalicular bile flow a reduction in the capacity to excrete bile acids intimates the presence of cholestasis. Early cholestasis is not associated with hyperbilirubinaemia but as cholestasis becomes more severe, jaundice occurs and the possibility of biliary atresia arises in neonates. Serum bile acid patterns in neonates being evaluated for biliary atresia indicate two distinctive patterns. Those infants with severe cholestasis and patent bile ducts usually have pre-dominantly cholic acid in serum. This observation is consistent with the bile acid patterns found in intrahepatic and extrahepatic cholestasis occurring in adult life. Infants found to have extrahepatic biliary atresia have marked elevations in the proportion of chenodeoxycholate in serum. Since elevations of chenodeoxycholate in serum are associated with hepatitis, the findings are consistent with the view that extrahepatic atresia is a rare sequalae of hepatitis.
Studies of bile acid metabolism and excretion give promise of providing further insight on the pathogenesis of cholestatic liver disease.
PMCID: PMC2495736  PMID: 4464509
Gastroenterology  2013;144(5):945-e15.
The final step in bile acid synthesis involves conjugation with glycine and taurine, which promotes a high intraluminal micellar concentration to facilitate lipid absorption. We investigated the clinical, biochemical, molecular, and morphologic features of a genetic defect in bile acid conjugation in 10 pediatric patients with fat-soluble vitamin deficiency, some with growth failure or transient neonatal cholestatic hepatitis.
We identified the genetic defect that causes this disorder using mass spectrometry analysis of urine, bile, and serum samples, and sequence analysis of the genes encoding bile acid-CoA:amino acid N-acyltransferase (BAAT) and bile acid-Co A ligase (SLC27A5).
Levels of urinary bile acids were increased (432±248 μmol/L) and predominantly excreted in unconjugated forms (79.4%±3.9%), and as sulfates and glucuronides. Glycine or taurine conjugates were absent in the urine, bile and serum. Unconjugated bile acids accounted for 95.7%±5.8% of the bile acids in duodenal bile, with cholic acid accounting for 82.4%±5.5% of total. Duodenal bile acid concentrations were 12.1±5.9 mmol/L—a concentration too low for efficient lipid absorption. The biochemical profile was consistent with defective bile acid amidation. Molecular analysis of BAAT confirmed 4 different homozygous mutations in 8 patients tested.
Based on a study of 10 pediatric patients, genetic defects that disrupt bile acid amidation cause fat-soluble vitamin deficiency and growth failure, indicating the importance of bile acid conjugation in lipid absorption. Some patients developed liver disease with features of a cholangiopathy. These findings indicate that patients with idiopathic neonatal cholestasis or later onset of unexplained fat-soluble vitamin deficiency should be screened for defects in bile acid conjugation.
PMCID: PMC4175397  PMID: 23415802
Chronic Liver Disease; Hepatic; Inherited; Nutrient; Bile acid conjugation; Bile acid-CoA amino acid N-acyltransferase; BAAT; Glycine; Taurine; Mass Spectrometry; Cholestasis; Fat-soluble vitamin deficiency
20.  Early diagnosis of neonatal cholestatic jaundice 
Canadian Family Physician  2009;55(12):1184-1192.
To review best practices for early recognition and treatment of conditions resulting in neonatal cholestasis, in order to improve long-term outcomes for affected infants.
Studies, review articles, and meta-analyses pertaining to neonatal-onset cholestasis were sought via electronic databases. Reference lists of studies and review articles supplemented the electronic search. Studies were included if they examined the importance of early diagnosis and intervention for cholestatic jaundice of any cause, and mainly comprised Level II and Level III evidence.
Review of the relevant literature supports the recommendation that infants with jaundice at 2 weeks of age should be tested for cholestasis by quantifying the direct reacting bilirubin levels in their blood. Subsequent rapid investigation using a diagnostic algorithm enables early diagnosis of the specific cause and facilitates timely intervention for conditions whose outcomes are improved by early treatment.
Universal screening for neonatal cholestasis might help with early identification of cases and improve outcomes, although further study is required in the North American setting.
PMCID: PMC2793221  PMID: 20008595
21.  Atypical causes of cholestasis 
Cholestatic liver disease consists of a variety of disorders. Primary sclerosing cholangitis and primary biliary cirrhosis are the most commonly recognized cholestatic liver disease in the adult population, while biliary atresia and Alagille syndrome are commonly recognized in the pediatric population. In infants, the causes are usually congenital or inherited. Even though jaundice is a hallmark of cholestasis, it is not always seen in adult patients with chronic liver disease. Patients can have “silent” progressive cholestatic liver disease for years prior to development of symptoms such as jaundice and pruritus. In this review, we will discuss some of the atypical causes of cholestatic liver disease such as benign recurrent intrahepatic cholestasis, progressive familial intrahepatic cholestasis, Alagille Syndrome, biliary atresia, total parenteral nutrition induced cholestasis and cholestasis secondary to drug induced liver injury.
PMCID: PMC4110573  PMID: 25071336
Cholestasis; Benign recurrent intrahepatic cholestasis; Progressive familial intrahepatic cholestasis; Alagille syndrome; Biliary atresia; Total parenteral nutrition; Drug induced liver injury
22.  Screening for inborn errors of metabolism in high-risk children: a 3-year pilot study in Zhejiang Province, China 
BMC Pediatrics  2012;12:18.
Tandem mass spectrometry (MS/MS) has been available in China for 8 years. This technique makes it possible to screen for a wide range of previously unscreened inborn errors of metabolism (IEM) using a single test. This 3-year pilot study investigated the screening, diagnosis, treatment and outcomes of IEM in symptomatic infants and children.
All children encountered in the Newborn Screening Center of Zhejiang Province during a 3-year period with symptoms suspicious for IEM were screened for metabolic diseases. Dried blood spots were collected and analyzed by tandem mass spectrometry. The diagnoses were further confirmed by clinical symptoms and biochemical analysis. Neonatal intrahepatic cholestasis caused by citrin deficiency, ornithine transcarbamylase deficiency and primary carnitine deficiency were confirmed by DNA analysis.
A total of 11,060 symptomatic patients (6,720 boys, 4,340 girls) with a median age of 28.8 months (range: 0.04-168.2 months) were screened. Among these, 62 were diagnosed with IEM, with a detection rate of 0.56%. Thirty-five were males and 27 females and the median age was 3.55 months (range 0.07-143.9 months). Of the 62 patients, 27 (43.5%) had aminoacidemias, 26 (41.9%) had organic acidemias and nine (14.5%) had fatty acid oxidation disorders.
Because most symptomatic patients are diagnosed at an older age, mental retardation and motor delay are difficult to reverse. Additionally, poor medication compliance reduces the efficacy of treatment. More extensive newborn screening is thus imperative for ensuring early diagnosis and enhancing the treatment efficacy of IEM.
PMCID: PMC3306752  PMID: 22364411
Tandem mass spectrometry; Inborn errors of metabolism; Aminoacidemia; Fatty acid oxidation disorders; Organic acidemia
23.  Association between a Novel Mutation in SLC20A2 and Familial Idiopathic Basal Ganglia Calcification 
PLoS ONE  2013;8(2):e57060.
Familial idiopathic basal ganglia calcification (FIBGC) is a rare, autosomal dominant disorder involving bilateral calcification of the basal ganglia. To identify gene mutations related to a Chinese FIBGC lineage, we evaluated available individuals in the family using CT scans. DNA was extracted from the peripheral blood of available family members, and both exonic and flanking intronic sequences of the SLC20A2 gene were amplified by PCR and then sequenced. Non-denaturing polyacrylamide gel electrophoresis (PAGE) was used to confirm the presence of mutations. Allele imbalances of the SLC20A2 gene or relative quantity of SLC20A2 transcripts were evaluated using qRT-PCR. A novel heterozygous single base-pair deletion (c.510delA) within the SLC20A2 gene was identified. This deletion mutation was found to co-segregate with basal ganglia calcification in all of the affected family members but was not detected in unaffected individuals or in 167 unrelated Han Chinese controls. The mutation will cause a frameshift, producing a truncated SLC20A2 protein with a premature termination codon, most likely leading to the complete loss of function of the SLC20A2 protein. This mutation may also lead to a reduction in SLC20A2 mRNA expression by approximately 30% in cells from affected individuals. In conclusion, we identified a novel mutation in SLC20A2 that is linked to FIBGC. In addition to the loss of function at the protein level, decreasing the expression of SLC20A2 mRNA may be another mechanism that can regulate SLC20A2 function in IBGC individuals. We propose that the regional expression pattern of SLC20A1 and SLC20A2 might explain the unique calcification pattern observed in FIBGC patients.
PMCID: PMC3577762  PMID: 23437308
24.  Analysis of the histologic features in the differential diagnosis of intrahepatic neonatal cholestasis 
AIM: To compare the histologic features of the liver in intrahepatic neonatal cholestasis (IHNC) with infectious, genetic-endocrine-metabolic, and idiopathic etiologies.
METHODS: Liver biopsies from 86 infants with IHNC were evaluated. The inclusion criteria consisted of jaundice beginning at 3 mo of age and a hepatic biopsy during the 1st year of life. The following histologic features were evaluated: cholestasis, eosinophilia, giant cells, erythropoiesis, siderosis, portal fibrosis, and the presence of a septum.
RESULTS: Based on the diagnosis, patients were classified into three groups: group 1 (infectious; n = 18), group 2 (genetic-endocrine-metabolic; n = 18), and group 3 (idiopathic; n = 50). There were no significant differences with respect to the following variables: cholestasis, eosinophilia, giant cells, siderosis, portal fibrosis, and presence of a septum. A significant difference was observed with respect to erythropoiesis, which was more severe in group 1 (Fisher’s exact test, P = 0.016).
CONCLUSION: A significant difference was observed in IHNC of infectious etiology, in which erythropoiesis was more severe than that in genetic-endocrine-metabolic and idiopathic etiologies, whereas there were no significant differences among cholestasis, eosinophilia, giant cells, siderosis, portal fibrosis, and the presence of a septum.
PMCID: PMC2653371  PMID: 19152454
Intrahepatic cholestasis; Liver histopathology; Neonatal jaundice; Neonatal liver disease
25.  The multiple facets of ABCB4 (MDR3) deficiency 
Opinion Statement
ABCB4 (MDR3) is a lipid translocator that moves phosphatidylcholine from the inner to outer leaflet of the canalicular membrane for extraction into the lumen by bile salts. Genetic mutations of ABCB4 lead to 3 distinct but related hepatobiliary diseases. Progressive familial intrahepatic cholestasis (PFIC) type 3 is a chronic cholestatic syndrome characterized by a markedly elevated GGTP. Patients present later in life than other types of PFIC with jaundice, pruritis and hepatosplenomegaly. Peri-portal inflammation progresses to biliary cirrhosis and eventually causes portal hypertension. Ursodeoxycholic acid (UDCA), which alters biliary bile composition, normalizes liver function tests in approximately half of treated PFIC type 3 patients. Partial or non-responders will eventually require liver transplantation. Gallstone patients with ABCB4 mutations may have Low Phospholipid Associated Cholelithiasis Syndrome. This is characterized by cholesterol gallstones and intrahepatic microlithiasis, along with recurrent biliary symptoms, despite cholecystectomy. A distinct group of patients with ABCB4 mutations may also develop intrahepatic brown pigment stones. The current treatment for these two diseases is UDCA, which may improve biliary symptoms even before the dissolution of stones occurs. Additional therapies such as Farnesoid X receptor ligands/agonists and benzfibrates show promise as future therapeutic modalities. Intrahepatic cholestasis of pregnancy affects pregnant women with abnormal ABCB4 variants. These women suffer from disabling pruritis and may experience steatorrhea as well. Their fetuses are at high risk for prematurity and still births. The definitive treatment for this condition is delivery of the baby. In the interim, limited fat intake, fat soluble vitamin supplementation and UDCA, with or without S-adenosylmethionine can provide symptomatic relief. In the future, additional hepatobiliary diseases related to ABCB4 mutations are likely to be identified. It is likely that this will result in the discovery of additional therapies for PFIC Type 3, gallstones and intrahepatic cholestasis of pregnancy.
PMCID: PMC3888315  PMID: 18221610

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