Clinical and genetic study of ABCB4 gene-related cholestatic liver disease in China: children and adults.

BACKGROUND: ABCB4 gene-related cholestatic liver diseases have a wide spectrum of clinical and genetic variations. The correlation between genotype and clinical phenotype still unclear. This study retrospectively analyzed the clinical and pathological characteristics of 23 patients with ABCB4 gene-related cholestatic liver diseases. Next-generation sequencing was used to identify the genetic causes. RESULTS: The 23 included patients (15 children and 8 adults) were diagnosed as progressive familial intrahepatic cholestasis type 3 (PFIC3), drug-induced liver injury (DILI), cirrhosis cholestasis, cirrhosis, and mild liver fibrosis. Nineteen patients underwent liver pathological examination of the liver, exhibiting fibrosis, small bile duct hyperplasia, CK7(+), Cu(+), bile duct deletion, and cirrhosis. Thirty ABCB4 variants were identified, including 18 novel variants. CONCLUSION: ABCB4 gene-related cholestatic liver diseases have a wide spectrum of clinical and genetic variations. Biallelic ABCB4 mutation carriers tended to severe PFIC3, which mostly occurs in children; while ABCB4 non-biallelic variants can lead to milder ICP, LACP, DILI or overlapping, mostly in adults. Thus, the ABCB4 genotype has a specific correlation with the phenotype, but there are exceptions. Non-biallelic null mutations can cause severe diseases. The mechanisms underlying this genetic phenotype require further investigation.

NR1H4 disease: rapidly progressing neonatal intrahepatic cholestasis and early death.

BACKGROUND: Clinical studies on progressive familial intrahepatic cholestasis (PFIC) type 5 caused by mutations in NR1H4 are limited. METHODS: New patients with biallelic NR1H4 variants from our center and all patients from literature were retrospectively analyzed. RESULTS: Three new patients were identified to be carrying five new variants. Liver phenotypes of our patients manifests as low-γ-glutamyl transferase cholestasis, liver failure and related complications. One patient underwent liver transplantation (LT) and survived, and two other patients died without LT. Nine other patients were collected through literature review. Twelve out of 13 patients showed neonatal jaundice, with the median age of onset being 7 days after birth. Reported clinical manifestations included cholestasis (13/13, 100%), elevated AFP (11/11, 100%), coagulopathy (11/11, 100%), hypoglycemia (9/13, 69%), failure to thrive (8/13, 62%), splenomegaly (7/13, 54%), hyperammonemia (7/13, 54%), and hepatomegaly (6/13, 46%). Six of 13 patients received LT at a median age of 6.2 months, and only one patient died of acute infection at one year after LT. Other 7 patients had no LT and died with a median age of 5 months (range 1.2-8). There were 8 patients with homozygous genotype and 5 patients with compound heterozygous genotype. In total, 13 different variants were detected, and 5 out of 12 single or multiple nucleotides variants were located in exon 5. CONCLUSIONS: We identified three newly-diagnosed patients and five novel mutations. NR1H4-related PFIC typically cause progressive disease and early death. LT may be the only lifesaving therapy leading to cure.

[Analysis of clinical characteristic of children with progressive familial intrahepatic cholestasis type 3].

Objective: To analyze the clinical manifestations, pathology, and gene variant characteristics in children with progressive familial intrahepatic cholestasis type 3 (PFIC3). Methods: This retrospective study assessed the clinical manifestations, pathological features, gene variants, and prognosis data of 11 children with PFIC3 hospitalized in the Department of Hepatology, Fifth Medical Center, PLA General Hospital, from January 2015 to December 2022. Panel or whole exome sequencing was performed on the probands, followed by Sanger sequencing for verification within the family. Detected pathogenic variants were compared with known disease databases. Additionally, the new variants were predicted the deleteriousness and protein structure using relevant software to evaluate their pathogenicity. Results: Among the 11 PFIC3 children, 8 were boys and 3 were girls. The age of onset was 3.1 (0.2, 15.6) years. The main complaint of onset was different in the 11 patients;5 of them were abnormal liver function, 3 of them were liver and spleen enlargement, 2 of them were abdominal distension, and 1 of them was jaundice. Alanine aminotransferase, asparate aminotransferase and γ-glutamyltransferase increased in all the patients, which were(113±40), (150±44) and (270±156) U/L respectively. Moreover, direct bilirubin increased in 9 patients, and cholestasis was showed in 8 patients. All patients showed liver fibrosis on imaging, and 8 patients had cirrhosis. The pathological features of 8 cases by liver biopsy were as follows: 8 cases of fibrosis in the portal area, 7 cases of small bile duct hyperplasia, 4 cases of positive copper staining, and 5 cases of cirrhosis. A total of 17 ABCB4 gene variants were detected, including 9 new variants: c.589C>T(p.Q197X), c.1230+1G>A(Splicing), c.2914G>A(P.D972N), c.1058G>A(p.C353Y), c.956G>T(p.G319V), c.473T>A(p.L158Q), c.164T>C(p.L55S), c.2493G>C(p.R831S), and c.1150G>C(p.G384R). All 11 patients were treated with ursodeoxycholic acid and followed up for 5.1(0.6, 7.4) years. Among them, 4 cases of cirrhosis progressed continuously, 3 cases had liver transplantations, and the remaining 4 cases were stable after medical treatment. Conclusions: Children with PFIC3 have early onset, diverse clinical manifestations, rapid progression of fibrotic and cholestasis, as well as poor prognosis. Genetic testing helps to confirm the diagnosis.

Genome-wide characterization of circulating metabolic biomarkers.

Genome-wide association analyses using high-throughput metabolomics platforms have led to novel insights into the biology of human metabolism1-7. This detailed knowledge of the genetic determinants of systemic metabolism has been pivotal for uncovering how genetic pathways influence biological mechanisms and complex diseases8-11. Here we present a genome-wide association study for 233 circulating metabolic traits quantified by nuclear magnetic resonance spectroscopy in up to 136,016 participants from 33 cohorts. We identify more than 400 independent loci and assign probable causal genes at two-thirds of these using manual curation of plausible biological candidates. We highlight the importance of sample and participant characteristics that can have significant effects on genetic associations. We use detailed metabolic profiling of lipoprotein- and lipid-associated variants to better characterize how known lipid loci and novel loci affect lipoprotein metabolism at a granular level. We demonstrate the translational utility of comprehensively phenotyped molecular data, characterizing the metabolic associations of intrahepatic cholestasis of pregnancy. Finally, we observe substantial genetic pleiotropy for multiple metabolic pathways and illustrate the importance of careful instrument selection in Mendelian randomization analysis, revealing a putative causal relationship between acetone and hypertension. Our publicly available results provide a foundational resource for the community to examine the role of metabolism across diverse diseases.

Exosomes from Intrahepatic Cholestasis of Pregnancy Induce Cell Apoptosis Through the miRNA-6891-5p/YWHAE Pathway.

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is associated with adverse pregnancy outcomes; however, the underlying mechanisms are not fully understood. AIMS: This study aimed to determine the role of exosomal miR-6891-5p in placental trophoblast dysfunction in ICP and identify new biomarkers for ICP diagnosis. METHODS: Serum samples were collected from ICP patients and healthy pregnant women, and serum exosomes were extracted and identified. Fluorescent dye labeling of exosomes and cell-verified cell phagocytosis were performed. In vitro experiments were conducted by adding taurocholic acid to simulate the ICP environment. Cell proliferation and apoptosis levels were detected using flow cytometry and the cell counting kit-8 assay. Mimics were constructed to overexpress miR-6891-5p in cells, and the binding site between miR-6891-5p and YWHAE was verified using luciferase reporter genes. RESULTS: miR-6891-5p expression was significantly decreased in serum exosomes of ICP patients. Co-culturing with exosomes derived from ICP patients' serum (ICP-Exos) decreased HTR-8/SVeno cell proliferation and increased apoptosis levels. miR-6891-5p upregulation in HTR-8/SVeno cells significantly increased cell viability and reduced cell apoptosis levels, as determined by the cell counting kit-8 assay and flow cytometry. A double luciferase assay confirmed that miR-6891-5p affected the expression of the downstream YWHAE protein. CONCLUSIONS: This study indicates that serum exosomes from ICP patients can impact the apoptosis of placental trophoblast HTR-8/SVeno cells through the miR-6891-5P/YWHAE pathway and can serve as specific molecular markers for ICP diagnosis.

Clinical symptoms, biochemistry, and liver histology during the native liver period of progressive familial intrahepatic cholestasis type 2.

BACKGROUND: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is an ultra-rare disease caused by mutations in the ABCB11 gene. This study aimed to understand the course of PFIC2 during the native liver period. METHODS: From November 2014 to October 2015, a survey to identify PFIC2 patients was conducted in 207 hospitals registered with the Japanese Society of Pediatric Gastroenterology, Hepatology, and Nutrition. Investigators retrospectively collected clinical data at each facility in November 2018 using pre-specified forms. RESULTS: Based on the biallelic pathogenic variants in ABCB11 and/or no hepatic immunohistochemical detection of BSEP, 14 Japanese PFIC2 patients were enrolled at seven facilities. The median follow-up was 63.2 [47.7-123.3] months. The median age of disease onset was 2.5 [1-4] months. Twelve patients underwent living donor liver transplantation (LDLT), with a median age at LDLT of 9 [4-57] months. Two other patients received sodium 4-phenylbutyrate (NaPB) therapy and survived over 60 months with the native liver. No patients received biliary diversion. The cases that resulted in LDLT had gradually deteriorated growth retardation, biochemical tests, and liver histology since the initial visit. In the other two patients, jaundice, growth retardation, and most of the biochemical tests improved after NaPB therapy was started, but pruritus and liver fibrosis did not. CONCLUSIONS: Japanese PFIC2 patients had gradually worsening clinical findings since the initial visit, resulting in LDLT during infancy. NaPB therapy improved jaundice and growth retardation but was insufficient to treat pruritus and liver fibrosis.

Clinical outcomes of ABCB4 heterozygosity in infants and children with cholestatic liver disease.

OBJECTIVES: Biallelic variants in the adenosine triphosphate binding cassette subfamily B member 4 (ABCB4) gene which encodes the multidrug resistance 3 protein (MDR3) leads to progressive familiar intrahepatic cholestasis type 3. However, monoallelic variants are increasingly recognized as contributing to liver disease in adults. Our aim was to describe the clinical characteristics of MDR3 heterozygous variants in a large cohort of infants and children with cholestatic liver disease. METHODS: The clinical and genotypic data on pediatric patients seen at King's College Hospital, London, between 2004 and 2022 and found to harbour heterozygous variants in ABCB4 were reviewed. RESULTS: Ninety-two patients amongst 1568 tested were identified with a monoallelic variant (5.9%). The most common presenting problem was conjugated hyperbilirubinemia (n = 46; 50%) followed by cholelithiasis (n = 12; 13%) and cholestatic hepatitis (n = 10; 11%). The median values of liver biochemistry at presentation were: GGT 105 IU/L and total bilirubin 86 µmol/L. Thirty-two genetic variants were identified including 22 missense (69%), 4 deletions (13%), 5 splice site (16%) and 1 termination (3%). At a median follow up of 1 year there was resolution of liver disease. CONCLUSIONS: Rare variants in ABCB4 were found amongst infants and children with cholestatic liver disease. The presenting problems were variable and abnormalities tended to normalize over time. Those with severe mutations could develop liver disease later in life when exposed to further insult and should be counseled appropriately.

The role of ERp29/FOS/EMT pathway in excessive apoptosis of placental trophoblast cells in intrahepatic cholestasis of pregnancy.

BACKGROUND: Abnormal bile acid metabolism leading to changes in placental function during pregnancy. To determine whether endoplasmic reticulum protein 29 (ERp29) can mediate the pregnancy effects of cholestasis by altering the level of trophoblast cell apoptosis. METHODS: ERp29 in serum of 66 intrahepatic cholestasis of pregnancy (ICP) pregnant women and 74 healthy were detected by ELISA. Subcutaneous injection of ethinyl estradiol (E2) was used to induce ICP in pregnant rats. Taurocholic acid (TCA) was used to simulate the ICP environment, and TGF-ß1 was added to induce the epithelial mesenchymal transformation (EMT) process. The scratch, migration, and invasion test were used to detect the EMT process. ERp29 overexpression/knockdown vector were constructed and transfected to verify the role of ERp29 in the EMT process. Downstream gene was obtained through RNA-seq. RESULTS: Compared with the healthy pregnant women, the expression levels of ERp29 in serum of ICP pregnancy women were significantly increased (P < 0.001). ERp29 in the placenta tissue of the ICP pregnant rats increased significantly, and the level of apoptosis increased. The placental tissues of the ICP had high expression of E-cadherin and low expression of N-cadherin, snail1, vimentin. After HTR-8/SVneo cells were induced by TCA, EMT was inhibited, while the ERp29 increased. Cell and animal experiments showed that, knockdown of ERp29 reduced the inhibition of EMT, the ICP progress was alleviated. Overexpression of FOS salvaged the inhibitory effects of ERp29 on cell EMT. DISCUSSION: The high level of ERp29 in placental trophoblast cells reduced FOS mRNA levels, inhibited the EMT process and aggravated the occurrence and development of ICP.

Diagnostic algorithm for neonatal intrahepatic cholestasis integrating single-gene testing and next-generation sequencing in East Asia.

BACKGROUND AND AIM: Advances in molecular genetics have uncovered causative genes responsible for neonatal cholestasis. Panel-based next-generation sequencing has been used clinically in infants with neonatal cholestasis. We aimed to evaluate the clinical application of single-gene testing and next-generation sequencing and to develop a diagnostic algorithm for neonatal intrahepatic cholestasis. METHODS: From January 2010 to July 2021, patients suspected of having neonatal intrahepatic cholestasis were tested at the Seoul National University Hospital. If there was a clinically suspected disease, single-gene testing was performed. Alternatively, if it was clinically difficult to differentiate, a neonatal cholestasis gene panel test containing 34 genes was performed. RESULTS: Of the total 148 patients examined, 49 (33.1%) were received a confirmed genetic diagnosis, including 14 with Alagille syndrome, 14 with neonatal intrahepatic cholestasis caused by citrin deficiency, 7 with Dubin-Johnson syndrome, 5 with arthrogryposis-renal dysfunction-cholestasis syndrome, 5 with progressive familial intrahepatic cholestasis type II, 1 with Rotor syndrome, 1 with Niemann-Pick disease type C, 1 with Kabuki syndrome, and 1 with Phenylalanyl-tRNA synthetase subunit alpha mutation. Sixteen novel pathogenic or likely pathogenic variants of neonatal cholestasis were observed in this study. Based on the clinical characteristics and laboratory findings, we developed a diagnostic algorithm for neonatal intrahepatic cholestasis by integrating single-gene testing and next-generation sequencing. CONCLUSIONS: Alagille syndrome and neonatal intrahepatic cholestasis caused by citrin deficiency were the most common diseases associated with genetic neonatal cholestasis. Single-gene testing and next-generation sequencing are important and complementary tools for the diagnosis of genetic neonatal cholestasis.

Expression of miR-let7b and miR-19b in progressive familial intrahepatic cholestasis (PFIC) children / Expresión de miR-let7b y miR-19b en niños con colestasis intrahepática familiar progresiva (PFIC)

Background MicroRNAs (miRNAs) are a group of small non-coding RNAs that bind to the target mRNA and regulate gene expression. Recently circulating microRNAs were investigated as markers of diseases and therapeutic targets. Although various studies analyze the miRNA expression in liver disease, these studies on PFIC are few. Progressive familial intrahepatic cholestasis (PFIC) is a rare liver disease with autosomal recessive inheritance. Most children with PFIC progress to cirrhosis and liver failure and consequently need to have a liver transplant. The aim of this study is the investigation of the miR-19b and miR-let7b expression levels in Iranian PFIC children. Methods 25 PFIC patients, 25 healthy children and 25 Biliary Atresia patients were considered as case and two control groups respectively. Blood samples were obtained and Liver function tests (LFTs) were measured. After RNA extraction and cDNA synthesis, quantitative PCR was performed using specific primers for miR-19b and miR-let7b. The U6 gene is used as an internal control. Results qPCR on PFIC patients’ samples demonstrated that the miR-19b and the miR-let7b expression were significantly decreased in patients compared to the control groups, with a p-value<0.0001 and p-value=0.0006 receptively. Conclusion In conclusion, circulating micro-RNA like miR-19b and miR-let7b have a potential opportunity to be a non-invasive diagnostic marker or therapeutic target for PFIC in the future (AU)

Antecedentes Los microRNA (miRNA) son un grupo de pequeños RNA no codificantes que se unen al ARNm diana y regulan la expresión génica. Recientemente se han investigado los microRNA circulantes como marcadores de enfermedades y dianas terapéuticas. Aunque varios estudios analizan la expresión de miRNA en enfermedades hepáticas, estos estudios sobre PFIC son escasos. La colestasis intrahepática familiar progresiva (PFIC) es una enfermedad hepática rara con herencia autosómica recesiva. La mayoría de los niños con PFIC progresan a cirrosis e insuficiencia hepática y, en consecuencia, requieren de un trasplante de hígado. El objetivo de este trabajo es la investigación de los niveles de expresión de miR-19b y miR-17b en niños iraníes con PFIC. Métodos Se consideraron 25 pacientes con PFIC, 25 niños sanos y 25 pacientes con atresia biliar como grupos de casos y controles. Se obtuvieron muestras de sangre y se midieron las pruebas de función hepática (LFT). Después de la extracción de RNA y la síntesis de cDNA, se realizó PCR cuantitativa usando cebadores específicos para miR-19b y miR-17b. El gen U6 se utiliza como control interno. Resultados La qPCR en muestras de pacientes con PFIC demostró que la expresión de miR-19b y miR-17b disminuyó significativamente en los pacientes en comparación con dos grupos de control, con un valor de p<0,0001 y un valor de p=0,0006, receptivamente. Conclusión En conclusión, los micro-RNA circulantes, como miR-19b y miR-let7b, tienen una oportunidad potencial de ser un marcador de diagnóstico no invasivo o un objetivo terapéutico para PFIC en el futuro (AU)

Expression of miR-let7b and miR-19b in progressive familial intrahepatic cholestasis (PFIC) children.

BACKGROUND: MicroRNAs (miRNAs) are a group of small non-coding RNAs that bind to the target mRNA and regulate gene expression. Recently circulating microRNAs were investigated as markers of diseases and therapeutic targets. Although various studies analyze the miRNA expression in liver disease, these studies on PFIC are few. Progressive familial intrahepatic cholestasis (PFIC) is a rare liver disease with autosomal recessive inheritance. Most children with PFIC progress to cirrhosis and liver failure and consequently need to have a liver transplant. The aim of this study is the investigation of the miR-19b and miR-let7b expression levels in Iranian PFIC children. METHODS: 25 PFIC patients, 25 healthy children and 25 Biliary Atresia patients were considered as case and two control groups respectively. Blood samples were obtained and Liver function tests (LFTs) were measured. After RNA extraction and cDNA synthesis, quantitative PCR was performed using specific primers for miR-19b and miR-let7b. The U6 gene is used as an internal control. RESULTS: qPCR on PFIC patients' samples demonstrated that the miR-19b and the miR-let7b expression were significantly decreased in patients compared to the control groups, with a p-value<0.0001 and p-value=0.0006 receptively. CONCLUSION: In conclusion, circulating micro-RNA like miR-19b and miR-let7b have a potential opportunity to be a non-invasive diagnostic marker or therapeutic target for PFIC in the future.

Intrahepatic cholestasis of pregnancy: insights into pathogenesis and advances in omics studies.

Intrahepatic cholestasis of pregnancy (ICP) is the most common pregnancy-specific liver disease. It is characterized by pruritus, abnormal liver function and elevated total bile acid (TBA) levels, increasing the risk of maternal and fetal adverse outcomes. Its etiology remains poorly elucidated. Over the years, various omics techniques, including metabolomics, microbiome, genomics, etc., have emerged with the advancement of bioinformatics, providing a new direction for exploring the pathogenesis, diagnosis and treatment of ICP. In this review, we first summarize the role of bile acids and related components in the pathogenesis of ICP and then further illustrate the results of omics studies.

Diagnostic yield and novel candidate genes by next generation sequencing in 166 children with intrahepatic cholestasis.

BACKGROUND AND AIMS: Cholestatic liver disease is a leading referral to pediatric liver transplant centers. Inherited disorders are the second most frequent cause of cholestasis in the first month of life. METHODS: We retrospectively characterized the genotype and phenotype of 166 participants with intrahepatic cholestasis, and re-analyzed phenotype and whole-exome sequencing (WES) data from patients with previously undetermined genetic etiology for newly published genes and novel candidates. Functional validations of selected variants were conducted in cultured cells. RESULTS: Overall, we identified disease-causing variants in 31% (52/166) of our study participants. Of the 52 individuals, 18 (35%) had metabolic liver diseases, 9 (17%) had syndromic cholestasis, 9 (17%) had progressive familial intrahepatic cholestasis, 3 (6%) had bile acid synthesis defects, 3(6%) had infantile liver failure and 10 (19%) had a phenocopy of intrahepatic cholestasis. By reverse phenotyping, we identified a de novo variant c.1883G > A in FAM111B of a case with high glutamyl transpeptidase (GGT) cholestasis. By re-analyzing WES data, two patients were newly solved, who had compound heterozygous variants in recently published genes KIF12 and USP53, respectively. Our additional search for novel candidates in unsolved WES families revealed four potential novel candidate genes (NCOA6, CCDC88B, USP24 and ATP11C), among which the patients with variants in NCOA6 and ATP11C recapitulate the cholestasis phenotype in mice models. CONCLUSIONS: In a single-center pediatric cohort, we identified monogenic variants in 22 known human intrahepatic cholestasis or phenocopy genes, explaining up to 31% of the intrahepatic cholestasis patients. Our findings suggest that re-evaluating existing WES data from well-phenotyped patients on a regular basis can increase the diagnostic yield for cholestatic liver disease in children.

Associations between ALDOB polymorphisms and intrahepatic cholestasis of pregnancy susceptibility in the Chinese Han population.

OBJECTIVES: To research the associations between fructose-bisphosphate aldolase B (ALDOB) gene polymorphisms and intrahepatic cholestasis of pregnancy (ICP) risk. MATERIAL AND METHODS: Whole-genome sequencing (WGS) was performed to detect ALDOB polymorphisms. Five web-available tools were employed to predict the effect of the site variant on the protein. Protein structure comparisons between the reference and ALDOB-modified samples were performed by SWISS-MODEL and Chimera 1.14rc, respectively. RESULTS: We identified 28 genetic variants in the ALDOB gene. When the cut-off value of minor allele frequency (MAF) of loci was 0.001 in four databases, five missense variants, including rs747604233, rs759204107, rs758242037, rs371526091 and rs77718928, were reserved for subsequent analysis. These variants were absent from the 1029 control individuals. The influence of all five variants on protein function was predicted to be damaging by the abovementioned five prediction software programs. Bioinformatics analysis demonstrated that these five missense variants were highly conserved among vertebrates. Compared to the wild-type protein structure, all five mutated protein structures showed a slight change in the chemical bond lengths of the enzyme activity domains. The combined clinical data indicate that the variant group had a significantly older age (p = 0.038), a higher level of indirect bilirubin (IDBIL, p = 0.033), and lower counts of white blood cells (WBCs, p = 7.38E-05) and platelets (PLTs, p = 0.018) than the wild-type group. CONCLUSIONS: This is the first study to examine the associations between ALDOB polymorphisms and ICP disease in 249 Chinese patients with ICP. Our present study expands the understanding of the pathogenesis of ICP.

A novel heterozygous deletion in ABCB4 gene in a Chinese family with intrahepatic cholestasis of pregnancy, neonatal hyperbilirubinemia, and cholelithiasis: Case reports and literature review.

BACKGROUND: ABCB4 gene (OMIM *171060) variant is associated with a wide clinical spectrum of hepatobiliary diseases, including familial intrahepatic cholestasis of pregnancy (ICP), progressive familial intrahepatic cholestasis type 3 (PFIC3), and neonatal hyperbilirubinemia due to impaired protection of the bile duct. The majority of reported cases, however, were missense or nonsense variants, with few deletion variant findings in the Chinese population. METHOD: We performed whole genome sequencing and confirmed it with Sanger sequencing of the proband infant and his families. Clinical courses and laboratory results were documented and collected from the proband infant and his mother. We also reviewed other published cases related to genetic variants in ABCB4 in the Chinese population. RESULTS: A 26-year-old Chinese female (II.2) who had recurrent intrahepatic cholestasis of pregnancy and her 49-day-old son (III.4) who had hyperbilirubinemia, both presented with extremely elevated total bile acid, cholestatic dominant pattern liver function abnormalities. They were able to stay relatively stable with mild pruritus on ursodeoxycholic acid treatment. After ruling out other possibilities, genetic sequencing revealed a diagnosis of heterozygous deletion variant NM_018849.3:c.1452_1454del (NP_061337.1:p.Thr485del) in ABCB4, which was not reported before, in the symptomatic mother (II.2), index patient (III.4), and the symptomatic grandmother (I.2). This variant resulted in clinical spectrums of ICP, neonatal hyperbilirubinemia, and cholelithiasis in our pedigree. CONCLUSION: We reported a novel heterozygous deletion variant of the ABCB4 gene in a Chinese family, as well as a literature review of ABCB4-related disorders. We aim to facilitate healthcare professionals to better understand genetic factors as an uncommon cause of hepatobiliary diseases, as well as improve therapeutic strategies in challenging clinical situations such as pregnancy and neonatal care.

Clinical and genetic analysis of 26 Chinese patients with neonatal intrahepatic cholestasis due to citrin deficiency.

BACKGROUND: Neonatal intrahepatic cholestasis due to citrin deficiency (NICCD) is an autosomal recessive disorder caused by SLC25A13 genetic mutations. We retrospectively analyzed 26 Chinese infants with NICCD (years 2014-2022) in Quanzhou City. METHODS: The plasma citrulline (CIT) concentration analyzed by tandem mass spectrometry (MS/MS), biochemical parameters and molecular analysis results are presented. RESULTS: Twelve genotypes were discovered. The relationship between the CIT concentration and genotype is uncertain. In total, 8 mutations were detected, with 4 variations, c.851_854delGTAT, c.615 + 5G > A, c.1638_1660dup and IVS16ins3kb, constituting the high-frequency mutations. Specifically, we demonstrated 2 patients with NICCD combined with another inborn errors of metabolism (IEM). Patient No. 22 possessed compound heterozygous mutations of c.615 + 5G > A and c.790G > A in the SLC25A13 gene accompanied by compound heterozygous variations of c.C259T and c.A155G in the PTS gene. Additionally, Patient No. 26 carried c.51C > G and c.760C > T in the SLC22A5 gene as well as c.615 + 5G > A and IVS16ins3kb in the SLC25A13 gene. CONCLUSIONS: We report a case of the simultaneous occurrence of primary carnitine deficiency (PCD) and NICCD.

Molecular and computational characterization of ABCB11 and ABCG5 variants in Tunisian patients with neonatal/infantile low-GGT intrahepatic cholestasis: Genetic diagnosis and genotype-phenotype correlation assessment.

Many inherited conditions cause hepatocellular cholestasis in infancy, including progressive familial intrahepatic cholestasis (PFIC), a heterogeneous group of diseases with highly overlapping symptoms. In our study, six unrelated Tunisian infants with PFIC suspicion were the subject of a panel-target sequencing followed by an exhaustive bioinformatic and modeling investigations. Results revealed five disease-causative variants including known ones: (the p.Asp482Gly and p.Tyr354 * in the ABCB11 gene and the p.Arg446 * in the ABCC2 gene), a novel p.Ala98Cys variant in the ATP-binding cassette subfamily G member 5 (ABCG5) gene and a first homozygous description of the p.Gln312His in the ABCB11 gene. The p.Gln312His disrupts the interaction pattern of the bile salt export pump as well as the flexibility of the second intracellular loop domain harboring this residue. As for the p.Ala98Cys, it modulates both the interactions within the first nucleotide-binding domain of the bile transporter and its accessibility. Two additional potentially modifier variants in cholestasis-associated genes were retained based on their pathogenicity (p.Gly758Val in the ABCC2 gene) and functionality (p.Asp19His in the ABCG8 gene). Molecular findings allowed a PFIC2 diagnosis in five patients and an unexpected diagnosis of sisterolemia in one case. The absence of genotype/phenotype correlation suggests the implication of environmental and epigenetic factors as well as modifier variants involved directly or indirectly in the bile composition, which could explain the cholestasis phenotypic variability.

Severe Neonatal Presentation of Progressive Familial Intrahepatic Cholestasis Type 4 in an Omani Infant.

Progressive familial intrahepatic cholestasis type 4 (PFIC4) is a relatively newly described autosomal recessive disorder caused by biallelic mutations in the gene encoding tight junction protein 2 (TJP2) which is located in chromosome 9q21. PFIC4 is characterised by cholestasis with or without other extrahepatic manifestations. Bleeding tendency due to vitamin k deficiency is a well-known complication of cholestasis. We present a neonate who presented to the Emergency Department at a tertiary care hospital in 2021 with cholestasis and multiple intracranial bleeds. He was found to have severe coagulopathy and his genetic work up revealed a homozygous variant mutation in TJP2 gene causing PFIC4. He had persistent cholestasis that necessitated an internal biliary diversion with some clinical improvement.

Features of liver injury in 138 Chinese patients with NICCD.

OBJECTIVES: To find biochemical and molecular markers can assist in identifying serious liver damage of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) patients. METHODS: 138 patients under 13 days to 1.1 year old diagnosed of NICCD in our center from 2004 to 2020. Base on the abnormal liver laboratory tests, we divided 138 patients into three groups: acute liver failure (ALF), liver dysfunction, and non-liver dysfunction groups, then compared their clinical, biochemical and, molecular data. RESULTS: 96 % of 138 patients had high levels of citrulline and high ratio of threonine to serine, which is the distinctive feature of plasma amino acid profile for NICCD. A total of 18.1 % of 138 patients had evidence of ALF who presented the most severity hepatic damage, 51.5 % had liver dysfunction, and the remaining 30.4 % presented mild clinical symptoms (non-liver dysfunction). In ALF group, the levels of citrulline, tyrosine, TBIL, ALP, and γ-GT was significantly elevated, and the level of ALB and Fisher ratio was pronounced low. Homozygous mutations of 1,638_1660dup, IVS6+5G.A, or IVS16ins3kb in SLC25A13 gene were only found in ALF and liver dysfunction groups. Supportive treatment including medium-chain triglyceride supplemented diet and fresh frozen plasma could be life-saving and might reverse ALF. CONCLUSIONS: High level of citrulline, tyrosine, TBIL, ALP, γ-GT, and ammonia, low level of albumin, and low Fisher ratio were predictors to suggest severe liver damage in NICCD patients who may go on to develop fatal metabolic disorder. Early identification and proper therapy is particularly important for these patients.