Infantile Progressive Intrahepatic Cholestasis And Mutations In Bile Salt Export Pump Gene

Infantile cholestatic diseases are common in children in Chinese Mainland,largepart of the disease have good prognosis.Little part of the patients with infantilecholestasis progressed to liver cirrhosis in the first decade of life.In America livertransplantation in children share 12.5% of all liver transplantation.In China infantilecholestatic diseases had become a very popular topic in recent years.Recent molecular and genetic studies have allowed the precise identification ofgenes responsible for at least three subtypes of PFICs, with all of them exhibitingmutations in genes that code proteins relevant for hepatobiliary transport. Here wewill focus on PFIC subtype 2, which is caused by mutations in BSEP. According torecent reports, the BSEP gene is located in 2q24 and encodes a liver-specificATP-binding cassette transporter that is a sister of P-glycoprotein (SPGP)BSEPprotein is involved in ATP-dependent bile acid transportation from hepatocytes tobiliary canaliculi. Many studies have been performed in patients in certain ethnicpopulations, including Amish, European, Arabic patients and kinds of missense,premature termination, frame shift, and splicing junction mutations associated withthe BSEP gene has been revealed. To elucidate the role of BSEP in chronicintrahepatic cholestasis in Chinese Mainland, we present 19 Chinese PFIC patientswith genetic mutations of BSEP gene.Part one: Progonostic value of serum GGT in Infants withIntrahepatic CholestasisObjective:Most infants with intrahepatic cholestasis will recover completely, only a smallproportion of them will progress to cirrhosis or death. Some author found that normalor low serum GGT in the infants with idiopathic neonatal hepatitis is an indicator ofpoor prognosis. The aim of this study is to investigate the prognostic value of serumGGT in Chinese infants with intrahepatic cholestasis.Methods:A retrospective study was conducted in 38 cases of infantile hepatitis, with whom the extrahepatic bile atresia or other bile abnormality had been excluded. Children withdeath, liver transplantation or waiting for liver transplantation, persistent or recurrentjaundice over 1 year of age were regarded as poor prognosis. According to theserumGGT level at presentation, the 38 patients were divided into two groups:≤50U/L and＞50U/L. The prognoses were compared between different groups.Results:1. Patients with GGT≤50U/L at presentation have poorer prognosis than those withGGT＞50U/L (P=0.001). Poor prognoses were observed in 8 patients. The serumGGTlevel was normal in 5 of them during the whole follow up period. 2 of 5 patients diedbefore 1 year old. Jaundice persistent or repeated with persistent obvious pruritis wasfound in the other 3.2. The other 3 of 8 patients had elevated GGT at presentation, but the GGT leveldropped to normal while the clinical conditions deteriorated. In 72.4% of the caseswith benign prognoses, AST declined with jaundice when they recovered. However,the peak of GGT was appeared during the process of jaundice remission in 79.3%patients with benign prognosis.Brief summary:1.GGT is also a prognostic parameter in Chinese intrahepatic cholestasis infants.2.The level of GGT drop to normal in infants with persistent jaundice often indicatedthe failure of liver function.3.Pregressive familial intrahepatic cholestasis may also existed in Chinese infants.Part two: Mutations of BSEP gene in 19 Chinese patientswith progressive intrahepatie eholestasisObjective:To elucidate the role of BSEP in chronic intrahepatic cholestasis with lowgama-glutamyltranspeptidase (GGT) levels in Chinese Mainland, we present 19 PFICpatients with genetic mutations of BSEP gene.Methods:Polymerase chain reaction was performed using 27 primer sets designed foramplification of the bile salt export pump DNA. Direct sequencing was undertaken,and identified sequences were compared with the sequence for bile salt export pumpgene registered with GenBank. In addition, BSEP expression was determined in liver biopsies by immunohistochymistry.Results:1. 19 patients were diagnosed as progressive intrahepatic cholestasis.All of themwere not older than 5 years.5 of them lost of their lives during following period.2. Genetic analysis revealed 9 gene mutations in BSEP, including 6 missensemutations,1 nonsense mutation and 2 Splicing site mutations. Heterozygous formissense mutation G188W combined with Splicing site mutation IVS22+3 A＞Tof bile salt exportpump protein were detected in patient 5. Results of gene analysisin case 11 revealed 2 heterozygous missense mutations: 1496G＞A(G499E) and2606A＞C (Q869P). 2 heterozygous missense mutation were happened in case15: 2023G＞C(D675H) and 3569C＞T(Al190E),of the 2 mutations,2023G＞C(D675H) was also observed in patient 18. An early stop codon atposition 702 (Q702X) was detected in of patient 3,and a Splicing site mutationIVS25-6 was in case 14.3. Secondary structure of the bile salt export pump displayed that A167T located inABC transporter transmembrane region, the other 5 missense mutations inintracellular region. G188W were in the forward ABC-type multidrug transportsystem and in the first intracellular loop. Q869P were in the latter ABC-typemultidrug transport system and in the forth intracellular loop.4. 4 of 6 missense mutations were displayed in high conservative regions comparinghomo with several other species such as Pan troglodytes, Rattus norvegicus andMus musculus.5. Immunohistochemestry dyeing of BSEP in liver sections of patients 5,11,15 and18 showed no expression in canalicular membrane,all of the four patients wererevealed with gene mutations. Immunohistochemestry dyeing of BSEP in cases 8and 16 showed normal expression and there were no gene mutation in both ofthem.Brief summary1. Novel BSEP mutations were found in Chinese patients with chronic intrahepaticcholestasis with low GGT levels.2. Gene mutatios of BSEP were observed in exon 7, 14,17,18,21and 26, including 8missense mutations, 1 nonsense mutation and 2 Splicing site mutations.3. Secondary structure of the bile salt export pump displayed that G188W were inthe forward ABC-type multidrug transport system and in the first intracellular loop. Q869P were in the latter ABC-type multidrug transport system and in theforth intracellular loop.There was no mutations in other nations happened in firstor forth intracellular loop.4. 4of 6 missense mutations were displayed in high conservative regions.5. Mutations in other nations reported in Gene Bank were not observed in ourpatients,mutations revealed in our patients were not found in Gene Bank,whichshowed the difference in heredity background.6. Immunohistochemestry dyeing of BSEP in liver sections of patients 5,11,15 and18 showed no expression in canalicular membrane,all of the four patients wererevealed with gene mutations. Immunohistochemestry dyeing of BSEP in cases 8and 16 showed normal expression and there were no gene mutation in both ofthem. All of those showed that gene mutation influenced on expression of BSEP.Conclusions:1. GGT is also a prognostic parameter in Chinese intrahepatic cholestasis infants.2. Progressive familial intrahepatic cholestasis may also existed in Chinese infants.3. Novel BSEP mutations were found in Chinese patients with chronic intrahepaticcholestasis with low GGT levels.