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 hyperbiliru-binaemia 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 homo-zygous 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 com-prehensive 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 N ICCD 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 bio- chemical indices from BA or INC. TBA excretion in NICCD appeared to be more severely disturbed than that of bilirubin and cholesterol.

Usefulness of a scoring system in the interpretation of histology in neonatal cholestasis

AIM:To ascertain the usefulness of a histological scoring system devised to assist in the interpretation of liver histology in neonatal cholestasis(NC) .METHODS:Liver biopsy specimens obtained from infants with NC referred to a tertiary pediatric unit in Malaysia were prospectively studied.The first author,blinded to the final diagnosis,devised the histological diagnosis based on a 7-feature(portal ductal proliferation,bile plugs in portal ductules,portoportal bridging,lymphocytic infiltration in portal region,multinucleated hepatocytes,neutrophilic infiltration,hepatocellular swelling) ,15-point(0 to 15) scoring system.The author classified the histological diagnosis as either biliary atresia(BA) or neonatal hepatitis(NH,all other diagnoses) ,and subsequently compared the author's diagnosis with the final diagnosis.RESULTS:Eighty-four biopsy specimens obtained from 78 patients were reviewed.Without the scoring system,BA was correctly diagnosed by the author histologically in 30 cases,labelled as NH in 3.For other diagnoses,BA was excluded correctly in 33 cases and mislabeled as BA in 2 cases.The overall sensitivity for BA was 91%,specificity 86% and accuracy 88%.With the scoring system,a score of ≥ 7 had the best diagnostic utility to differentiate BA from other intrahepatic cholestasis histologically(sensitivity 88%,specificity 94%,accuracy 92%) .Four patients with a score < 7 had BA,and 3 patients with a score ≥ 7 had NH.CONCLUSION:A 7-feature,15-point histological scoring system had good diagnostic accuracy in the interpretation of liver histology in neonatal cholestasis.

Neonatal cholestasis can be the first symptom of McCune–Albright syndrome:A case report

BACKGROUND McCune–Albright syndrome(MAS)is caused by postzygotic somatic mutations of the GNAS gene.It is characterized by the clinical triad of fibrous dysplasia,caféau-lait skin spots,and endocrinological dysfunction.Myriad complications in MAS,including hepatobiliary manifestations,are also reported.CASE SUMMARY This is a case of a 4-year-old boy who presented with MAS with neonatal cholestasis.He was suspected to have Alagille syndrome due to neonatal cholestasis with intrahepatic bile duct paucity in liver biopsy,peripheral pulmonary artery stenosis,and renal tubular dysfunction.By the age of 2 years,his cholestatic liver injury gradually improved,but he had repeated left femoral fractures.He did not exhibit endocrinological abnormality or café-au-lait skin spots.However,MAS was suspected due to fibrous dysplasia at the age of 4 years.No mutation was identified in the GNAS gene in the DNA isolated from the peripheral blood,but an activating point mutation(c.601C>T,p.Arg201Cys)was observed in the DNA extracted from the affected bone tissue and that extracted from the formalin-fixed paraffin-embedded liver tissue,which was obtained at the age of 1 mo.CONCLUSION MAS should be considered as a differential diagnosis for transient cholestasis in infancy.

Clinical Assessment of Differential Diagnostic Methods in Infants with Cholestasis due to Biliary Atresia or Non-Biliary Atresia

The different methods in differentiating biliary atresia(BA)from non-BA-related cholestasis were evaluated in order to provide a practical basis for a rapid,early and accurate differential diagnosis of the diseases.396 infants with cholestatic jaundice were studied prospectively during the period of May 2007 to June 2011.The liver function in all subjects was tested.All cases underwent abdominal ultrasonography and duodenal fluid examination.Most cases were subjected to hepatobiliary scintigraphy,magnetic resonance cholangiopancreatography(MRCP)and a percutaneous liver biopsy.The diagnosis of BA was finally made by cholangiography or histopathologic examination.The accuracy,sensitivity,specificity and predictive values of these various methods were compared.178 patients(108 males and 70 females with a mean age of 58±30 days)were diagnosed as having BA.218 patients(136 males and 82 females with a mean age of 61±24 days)were diagnosed as having non-BA etiologies of cholestasis jaundice during the follow-up period in which jaundice faded after treatment with medical therapy.For diagnosis of BA,clinical evaluation,hepatomegaly,stool color,serum gamma-glutamyltranspeptidase(GGT),duodenal juice color,bile acid in duodenal juice,ultrasonography(gallbladder),ultrasonography(griangular cord or strip-apparent hyperechoic foci),hepatobiliary scintigraphy,MRCP,liver biopsy had an accuracy of 76.0%,51.8%,84.3%,70.0%,92.4%,98.0%,90.4%,67.2%,85.3%,83.2%and 96.6%,a sensitivity of 83.1%,87.6%,96.1%,73.7%,90.4%,100%,92.7%,27.5%,100%,89.0%and 97.4%,a specificity of 70.2%,77.5%,74.8%,67.0%,94.0%,96.3%,88.5%,99.5%,73.3%,75.4%and 94.3%,a positive predictive value of 69.0%,72.6%,75.7%,64.6%,92.5%,95.7%,86.8%,98.0%,75.4%,82.6%and 98.0%,and a negative predictive value of 83.6%,8.5%,95.9%,75.7%,92.3%,100%,84.2%,93.7%,100%,84.0%and 92.6%,respectively.It was concluded that all the differential diagnosis methods are useful.The test for duodenal drainage and elements is fast and accurate.It is helpful in the differential diagnosis of BA and non-BA etiologies of cholestasis.It shows good practical value clinically.

Progressive familial intrahepatic cholestasis—farnesoid X receptor deficiency due to NR1H4 mutation:A case report

BACKGROUND Functioning farnesoid X receptor(FXR;encoded by NR1H4)is key to normal bile acid homeostasis.Biallelic mutations in NR1H4 are reported in a few children with intrahepatic cholestasis.We describe a boy with progressive familial intrahepatic cholestasis and homozygous mutation in NR1H4.CASE SUMMARY A boy had severe neonatal cholestasis with moderate hypercholanemia and persistently elevated alpha-fetoprotein.Despite medical treatment,coagulopathy was uncontrollable,prompting liver transplantation at age 8 mo with incidental splenectomy.The patient experienced catch-up growth with good liver function and did not develop allograft steatosis.However,1 year after transplant,he died from an acute infection,considered secondary to immunosuppression and asplenia.A homozygous protein-truncating mutation,c.547C>T,p.(Arg183Ter),was subsequently identified in NR1H4,and both parents were shown to be heterozygous carriers.Absence of FXR and of bile salt export pump expression was confirmed by immunostaining of explanted liver.CONCLUSION Severe cholestasis with persistently high alpha-fetoprotein and modest elevation of serum bile acid levels may suggest FXR deficiency.Some patients with FXR deficiency may not develop allograft steatosis and may respond well to liver transplantation.

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 RTPCR was 100%.CONCLUSION:RT-PCR can detect the mutation 851del4 in infants with intrahepatic cholestasis with an accuracy of 100%.

SOX9 in biliary atresia: New insight for fibrosis progression

Background:Liver fibrosis is a hallmark determinant of morbidity in biliary atresia(BA)even in successfully operated cases.Responsible factors for this rapid progression of fibrosis are not completely defined.Aberrant expression of the transcription factor SOX9 and hepatic progenitor cells(HPCs)proliferation have roles in fibrogenesis in cholestatic disorders.However,they were not investigated sufficiently in BA.We aimed to delineate the relation of SOX9 and HPCs to fibrosis and its progression in BA.Methods:Forty-eight patients with BA who underwent an initial diagnostic liver biopsy(LB)and consequent intraoperative LB were recruited and compared to 28 cases with non-BA cholestasis that had an LB in their diagnostic workup.Liver fibrosis,tissue SOX9 and HPC expressions were studied in both BA and non-BA-cholestasis cases.Liver fibrosis,SOX9,and HPCs’dynamic changes in BA cases were assessed.Relation of fibrosis and its progression to SOX9 and HPCs in BA was assessed.Results:SOX9 and HPCs in ductular reaction(DR)form were expressed in 100%of BA and their grades increased significantly in the second biopsy.The rapidly progressive fibrosis in BA,represented by fibrosis grade of the intraoperative LB,correlated significantly to SOX9-DR and HPC-DR at the diagnostic(r=0.420,P=0.003 and r=0.405,P=0.004,respectively)and the intraoperative(r=0.460,P=0.001 and r=0.467,P=0.001,respectively)biopsy.On the other hand,fibrosis,SOX9-DR,and HPC-DR were significantly lower in non-BA cases at a comparable age(P<0.001,P=0.006,and P=0.014,respectively).Conclusions:Fibrosis in BA is rapidly progressive within a short time and is significantly correlated to SOX9 and HPCs.Assessment of targeting SOX9 and HPCs on fibrosis progression is warranted.

Biliary atresia: Where do we stand now?

The pathway from clinical suspicion to establishing the diagnosis of biliary atresia in a child with jaundice is a daunting task. However, investigations available help to point towards the correct diagnosis in reasonable time frame. Imaging by Sonography has identified several parameters which can be of utility in the diagnostic work up. Comparison of Sonography with imaging by Nuclear medicine can bring out the significant differences and also help in appropriate imaging. The battery of Biochemical tests, available currently, enable better understanding of the line-up of investigations in a given child with neonatal cholestasis. Management protocols enable standardized care with optimal outcome. The place of surgical management in biliary atresia is undisputed, although Kasai procedure and primary liver transplantation have been pitted against each other. This article functions as a platform to bring forth the various dimensions of biliary atresia.

Novel Mutations in the 3β-hydroxy-A5-C27-steroid Dehydrogenase Gene （HSD3B7）in a Patient with Neonatal Cholestasis

Bile acid synthetic defect （BASD） is a rare category of genetic disorders that are responsible for approximately 2% of persistent cholestasis in infants. Until date, four enzynles responsible for congenital defects of bile acid synthesis （CBAS） have been identified. 313-hydroxy-A5-C27-steroid dehydrogenase （3β-HSD）, the deficiency of which can cause CBAS 1 （OMIM No. 607765）, is encoded by the gene HSD3B7 and works in the second step of transforming the steroid into primary bile acids.

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.