Biallelic loss-of-function ZFYVE19 mutations are associated with congenital hepatic fibrosis, sclerosing cholangiopathy and high-GGT cholestasis.

BACKGROUND: For many children with intrahepatic cholestasis and high-serum gamma-glutamyl transferase (GGT) activity, a genetic aetiology of hepatobiliary disease remains undefined. We sought to identify novel genes mutated in children with idiopathic high-GGT intrahepatic cholestasis, with clinical, histopathological and functional correlations. METHODS: We assembled a cohort of 25 children with undiagnosed high-GGT cholestasis and without clinical features of biliary-tract infection or radiological features of choledochal malformation, sclerosing cholangitis or cholelithiasis. Mutations were identified through whole-exome sequencing and targeted Sanger sequencing. We reviewed histopathological findings and assessed phenotypical effects of ZFYVE19 deficiency in cultured cells by immunofluorescence microscopy. RESULTS: Nine Han Chinese children harboured biallelic, predictedly complete loss-of-function pathogenic mutations in ZFYVE19 (c.314C>G, p.S105X; c.379C>T, p.Q127X; c.514C>T, p.R172X; c.547C>T, p.R183X; c.226A>G, p.M76V). All had portal hypertension and, at liver biopsy, histopathological features of the ductal plate malformation (DPM)/congenital hepatic fibrosis (CHF). Four children required liver transplantation for recurrent gastrointestinal haemorrhage. DPM/CHF was confirmed at hepatectomy, with sclerosing small-duct cholangitis. Immunostaining for two primary-cilium axonemal proteins found expression that was deficient intraluminally and ectopic within cholangiocyte cytoplasm. ZFYVE19 depletion in cultured cells yielded abnormalities of centriole and axoneme. CONCLUSION: Biallelic ZFYVE19 mutations can lead to high-GGT cholestasis and DPM/CHF in vivo. In vitro, they can lead to centriolar and axonemal abnormalities. These observations indicate that mutation in ZFYVE19 results, through as yet undefined mechanisms, in a ciliopathy.

Low-GGT intrahepatic cholestasis associated with biallelic USP53 variants: Clinical, histological and ultrastructural characterization.

BACKGROUND & AIMS: In about 20% of children with cholestasis and normal or low serum gamma-glutamyltransferase (GGT) activity, no aetiology is identified. We sought new genes implicated in paediatric hepatobiliary disease. METHODS: We conducted whole-exome sequencing in 69 children evaluated at our centre from 2011 to 2018 who had low-GGT cholestasis and in whom homozygous/compound heterozygous predictedly pathogenic variants (PPVs) in ATP8B1, ABCB11, NR1H4, MYO5B or TJP2 were not found. Clinical records and findings on light microscopy and transmission electron microscopy of liver biopsy materials were reviewed. RESULTS: In seven patients from seven unrelated families, biallelic PPVs (10 in total) were found in USP53, recently associated with intrahepatic cholestasis. Seven variants were classified as pathogenic: one canonical splicing, c.569 + 2T > C, and six nonsense or frameshifting: c.169C > T (p.Arg57Ter), c.581delA (p.Arg195GlufsTer38), c.831_832insAG (p.Val279GlufsTer16), c.1012C > T (p.Arg338Ter), c.1426C > T (p.Arg476Ter) and c.1558C > T (p.Arg520Ter). Three were likely pathogenic: c.297G > T (p.Arg99Ser), c.395A > G (p.His132Arg) and c.878G > T (p.Gly293Val). In all patients, jaundice began at age <7 months. Cholestasis was transient, with documented resolution of hyperbilirubinaemia in all (oldest patient now aged 5 years) except one, who was lost to follow-up. Light microscopy identified intralobular cholestasis, giant-cell change of hepatocytes and perisinusoidal-perihepatocytic and portal-tract fibrosis. Ultrastructural study revealed elongated hepatocyte-hepatocyte tight junctions. One patient was deaf. CONCLUSION: USP53 interacts with the tight junction constituent TJP2. TJP2 mutation can cause low-GGT intrahepatic cholestasis, with elongated hepatocyte-hepatocyte tight junctions, as well as deafness. Our findings extend a preliminary report of USP53 disease and indicate that USP53 mutation may generate a partial phenocopy of TJP2 disease.

Molecular findings in children with inherited intrahepatic cholestasis.

BACKGROUND: Genetic defects account for a substantial proportion of pediatric cholestasis. This study explored the molecular findings in a large cohort of Chinese patients with inherited cholestasis. METHODS: Between January 2012 and June 2016, 809 Chinese pediatric patients with suspected inherited intrahepatic cholestasis were evaluated by Sanger sequencing and/or panel sequencing. RESULTS: Of the 809 patients, 273 (33.7%) obtained a genetic diagnosis. The rate of positive genetic diagnosis in patients with disease onset at 0-3 month of age was higher than that in patients with disease onset at 4 month of age or later. There were 17 distinct genetic defects diagnosed. The top 4 resulted from mutations in SLC25A13 (44.3%), JAG1 (24.5%), ABCB11 (11.0%), and ATP8B1 (5.9%). All 17 genetic disorders were diagnosed in patients with disease onset at 0-3 months of age; but only 5 were diagnosed in patients with disease onset beyond 4 months of age. A total of 217 distinct pathogenic variants, including 41 novel variants, were identified. Ten recurrent mutations were detected in SLC25A13, ATP8B1, and CYP27A1. They accounted for 48.2% of the total 477 mutant alleles. CONCLUSIONS: There were 17 distinct genetic disorders diagnosed in Chinese pediatric patients with inherited cholestasis.

Novel missense mutation in VPS33B is associated with isolated low gamma-glutamyltransferase cholestasis: Attenuated, incomplete phenotype of arthrogryposis, renal dysfunction, and cholestasis syndrome.

The typical phenotype of arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome involves three cardinal symptoms as the name describes, harboring biallelic mutations on VPS33B or VIPAS39. Except for ARC syndrome, low gamma-glutamyltransferase (GGT) cholestasis often implies hereditary hepatopathy of different severity; however, some remain undiagnosed. Several monogenic defects typically with multiorgan manifestations may only present liver dysfunction at times, such as DGUOK defect and AGL defect. Previously, four VPS33B mutated cases were reported without arthrogryposis, or with less severe symptoms and longer lifespan, indicating the possibility of incomplete ARC phenotype of isolated hepatopathy. So we retrospectively reviewed all patients with confirmed VPS33B/VIPARS39 defect in our center and identified three presenting isolated low-GGT cholestasis with intractable pruritus. Distinguished from others with typical ARC phenotype, these patients did not suffer the other two typical characteristics, survived much longer, and shared a novel missense VPS33B variation c.1726T>C, p.Cys576Arg, causing declined protein expression and abolished interaction with VIPAS39 in-vitro. Serum bile acid profiles of our VPS33B/VIPAS39 mutated patients revealed similar changes to primary defect of bile salt export pump, among which those with isolated cholestasis phenotype had a higher level of total secondary bile acids than that with typical ARC phenotype, indicating the partial residual function of VPS33B.

Defects in myosin VB are associated with a spectrum of previously undiagnosed low γ-glutamyltransferase cholestasis.

Hereditary cholestasis in childhood and infancy with normal serum gamma-glutamyltransferase (GGT) activity is linked to several genes. Many patients, however, remain genetically undiagnosed. Defects in myosin VB (MYO5B; encoded by MYO5B) cause microvillus inclusion disease (MVID; MIM251850) with recurrent watery diarrhea. Cholestasis, reported as an atypical presentation in MVID, has been considered a side effect of parenteral alimentation. Here, however, we report on 10 patients who experienced cholestasis associated with biallelic, or suspected biallelic, mutations in MYO5B and who had neither recurrent diarrhea nor received parenteral alimentation. Seven of them are from two study cohorts, together comprising 31 undiagnosed low-GGT cholestasis patients; 3 are sporadic. Cholestasis in 2 patients was progressive, in 3 recurrent, in 2 transient, and in 3 uncategorized because of insufficient follow-up. Liver biopsy specimens revealed giant-cell change of hepatocytes and intralobular cholestasis with abnormal distribution of bile salt export pump (BSEP) at canaliculi, as well as coarse granular dislocation of MYO5B. Mass spectrometry of plasma demonstrated increased total bile acids, primary bile acids, and conjugated bile acids, with decreased free bile acids, similar to changes in BSEP-deficient patients. Literature review revealed that patients with biallelic mutations predicted to eliminate MYO5B expression were more frequent in typical MVID than in isolated-cholestasis patients (11 of 38 vs. 0 of 13). CONCLUSION: MYO5B deficiency may underlie 20% of previously undiagnosed low-GGT cholestasis. MYO5B deficiency appears to impair targeting of BSEP to the canalicular membrane with hampered bile acid excretion, resulting in a spectrum of cholestasis without diarrhea. (Hepatology 2017;65:1655-1669).

Comprehensive bile acid profiling in hereditary intrahepatic cholestasis: Genetic and clinical correlations.

BACKGROUND & AIMS: Genetic defects causing dysfunction in bile salt export pump (BSEP/ABCB11) lead to liver diseases. ABCB11 mutations alter the bile acid metabolome. We asked whether profiling plasma bile acids could reveal compensatory mechanisms and track genetic and clinical status. METHODS: We compared plasma bile acids in 17 ABCB11-mutated patients, 35 healthy controls and 12 genetically undiagnosed cholestasis patients by ultra-high-performance liquid chromatography/multiple-reaction monitoring-mass spectrometry (UPLC/MRM-MS). We developed an index to rank bile acid hydrophobicity, and thus toxicity, based on LC retention times. We recruited 42 genetically diagnosed hereditary cholestasis patients, of whom 12 were presumed to have impaired BSEP function but carried mutations in genes other than ABCB11, and 8 healthy controls, for further verification. RESULTS: The overall hydrophobicity indices of total bile acids in both the ABCB11-mutated group (11.89 ± 1.07 min) and the undiagnosed cholestasis group (11.46 ± 1.07 min) were lower than those of healthy controls (13.69 ± 0.77 min) (both p < 0.005). This was owing to increased bile acid modifications. Secondary bile acids were detected in patients without BSEP expression, suggesting biliary bile acid secretion through alternative routes. A diagnostic panel comprising lithocholic acid (LCA), tauro-LCA, glyco-LCA and hyocholic acid was identified that could differentiate the ABCB11-mutated cohort from healthy controls and undiagnosed cholestasis patients (AUC=0.946, p < 0.0001) and, in non-ABCB11-mutated cholestasis patients, could distinguish BSEP dysfunction from normal BSEP function (9/12 vs 0/38, p < 0.0000001). CONCLUSIONS: Profiling of plasma bile acids has provided insights into cholestasis alleviation and may be useful for the clinical management of cholestatic diseases.

Splicing analysis of rare/novel synonymous or intronic variants identified in ABCB11 heterozygotes presenting as progressive intrahepatic cholestasis with low γ-glutamyltransferase.

AIM: The aim of this study was to analyze the pathogenicity of rare/novel synonymous or intronic variants identified in ABCB11 heterozygotes presenting as progressive intrahepatic cholestasis with low γ-glutamyltransferase. METHODS: The enrolled variants were identified in ABCB11 between October 2009 and June 2016. The effects on pre-RNA splicing were analyzed by in silico tools and minigene splicing assay. RESULTS: There were three intronic (c.908 + 5G > A, c.2815-8A > G, and c.612-15_-6del10bp) and two synonymous (c.1809G > A, p.K603 K and c.2418C > T, p.G806G) variants with unknown significance identified in ABCB11 of five ABCB11 heterozygotes. Parental studies were carried out for four patients, and revealed that the variants with unknown significance were compound heterozygous with other pathogenic variants. The five variants with unknown significance had minor allele frequency <0.1% or were absent from controls, and had positive prediction results by in silico tools. The effects on pre-RNA splicing were further confirmed by minigene splicing assay. c.908 + 5A caused abnormal splicing in at least 78.5 ± 3.8% of products using a cryptic splice site (ss) 22 nucleotides (nt) upstream of the wild-type (WT) 5'ss. Seven nucleotides of intron 22 upstream of the WT 3'ss was retained for all products from c.2815-8G. c.612-15_-6del caused exon 8 skipping in 24.8 ± 7.7% of products, and 55 nt of exon 8 downstream of the WT 3'ss removal in remaining products. c.1809A led to exon 15 skipping. c.2418 T removed exon 20 and 62 nt of exon 21 downstream of the WT 3'ss by using a cryptic ss. CONCLUSIONS: We successfully identified five pathogenic synonymous or intronic variants with some common features. These features might help to choose the right variant for further functional assay.

Novel NBAS mutations and fever-related recurrent acute liver failure in Chinese children: a retrospective study.

BACKGROUND: Underlying causes in Chinese children with recurrent acute liver failure (RALF), including liver crises less than full acute liver failure, are incompletely understood. We sought to address this by searching for genes mutated in such children. METHODS: Five unrelated Chinese boys presenting between 2012 and 2015 with RALF of unexplained etiology were studied. Results of whole exome sequencing were screened for mutations in candidate genes. Mutations were verified in patients and their family members by Sanger sequencing. All 5 boys underwent liver biopsy. RESULTS: NBAS was the only candidate gene mutated in more than one patient (biallelic mutations, 3 of 5 patients; 5 separate mutations). All NBAS mutations were novel and predictedly pathogenic (frameshift insertion mutation c.6611_6612insCA, missense mutations c.2407G > A and c.3596G > A, nonsense mutation c.586C > T, and splicing-site mutation c.5389 + 1G > T). Of these mutations, 3 lay in distal (C-terminal) regions of NBAS, a novel distribution. Unlike the 2 patients without NBAS mutations, the 3 patients with confirmed NBAS mutations all suffered from a febrile illness before each episode of liver crisis (fever-related RALF), with markedly elevated alanine aminotransferase and aspartate aminotransferase activities 24-72 h after elevation of body temperature, succeeded by severe coagulopathy and mild to moderate jaundice. CONCLUSIONS: As in other countries, so too in China; NBAS disease is a major cause of fever-related RALF in children. The mutation spectrum of NBAS in Chinese children seems different from that described in other populations.

Severe Neonatal Cholestasis in Cerebrotendinous Xanthomatosis: Genetics, Immunostaining, Mass Spectrometry.

OBJECTIVES: Cerebrotendinous xanthomatosis (CTX) is caused by defects in sterol 27-hydroxylase (CYP27A1, encoded by CYP27A1), a key enzyme in the bile acid synthesis pathway. CTX usually presents as neurologic disease in adults or older children. The rare reports of CTX manifest as neonatal cholestasis assess the cholestasis as transient, with patient survival. Our experience differs. METHODS: Homozygous or compound heterozygous CYP27A1 mutations were detected in 8 neonatal cholestasis patients by whole exome sequencing, panel sequencing, or Sanger sequencing. Their clinical and biochemical data were retrospectively reviewed. Immunostaining for CYP27A1 was conducted in liver of 4 patients. Mass spectrometry was used to analyze patients' urine samples. RESULTS: All 8 infants had severe cholestasis. Five died from, or were transplanted for, liver failure; 3 cleared their jaundice eventually. Marking for CYP27A1 was weak or absent in 3 of the 4 patient specimens. Mass spectrometry of urine revealed a predominance of sulfated and doubly conjugated (sulfated-glucuronidated) bile alcohols. No patient harbored a putatively pathogenic mutation in genes other than CYP27A1 that have been implicated in cholestatic liver disease. CONCLUSIONS: CTX manifest as neonatal cholestasis has a bile acid profile different from CTX manifest in later life, and thus may be overlooked. Immunostaining, mass spectrometry of urine, and genetic studies can support one another in making the diagnosis. A substantial proportion of CTX patients with severe neonatal cholestasis may die or need liver transplantation. CTX manifest in infancy as severe cholestasis warrants further investigation of biochemical diagnostic criteria and best management.

Fibrinogen storage disease in a Chinese boy with de novo fibrinogen Aguadilla mutation: Incomplete response to carbamazepine and ursodeoxycholic acid.

BACKGROUND: Fibrinogen storage disease (FSD) is a rare autosomal-dominant disorder caused by mutation in FGG, encoding the fibrinogen gamma chain. Here we report the first Han Chinese patient with FSD, caused by de novo fibrinogen Aguadilla mutation, and his response to pharmacologic management. CASE PRESENTATION: Epistaxis and persistent clinical-biochemistry test-result abnormalities prompted liver biopsy in a boy, with molecular study of FGG in him and his parents. He was treated with the autophagy enhancer carbamazepine, reportedly effective in FSD, and with ursodeoxycholic acid thereafter. Inclusion bodies in hepatocellular cytoplasm stained immune-histochemically for fibrinogen. Selective analysis of FGG found the heterozygous mutation c.1201C > T (p.Arg401Trp), absent in both parents. Over more than one year's follow-up, transaminase and gamma-glutamyl transpeptidase activities have lessened but not normalized. CONCLUSION: This report expands the epidemiology of FSD and demonstrates idiosyncrasy in response to oral carbamazepine and/or ursodeoxycholic acid in FSD.

Whole-Genome Sequencing Reveals Large ATP8B1 Deletion/Duplications as Second Mutations Missed by Exome-Based Sequencing.

Progressive familial intrahepatic cholestasis type 1 (PFIC1) results from biallelic pathogenic variants in ATP8B1. This study sought second pathogenic variants in ATP8B1 by whole-genome sequencing (WGS) in four unrelated low γ-glutamyl transpeptidase cholestasis patients in whom clinical suspicion of PFIC1 was high and gene-panel or Sanger sequencing had identified only one pathogenic variant in ATP8B1. Sanger sequencing confirmed WGS findings and determined the origin of each variant. Novel nonrecurrent structural variants in three patients (patient 1 to patient 3) were identified in trans: g.55396652_55403080del (6427-bp deletion), g.55335906_55346620dup (10,715-bp duplication), and g.55362063_55364293dup (2231-bp duplication). One synonymous variant in patient 4 was recognized in trans (c.1029G>A, p. Thr343Thr) and demonstrated as deleterious. In conclusion, WGS improves genetic diagnostic yield in PFIC1. These findings expand the gene-variant spectrum associated with familiar intrahepatic cholestasis 1 (FIC1) disease and for the first time report tandem duplication in ATP8B1 associated with cholestasis.

Recurrent acute liver failure associated with novel SCYL1 mutation: A case report.

BACKGROUND: Pediatric recurrent acute liver failure (RALF) with recovery between episodes is rare. Causes include autoimmune disease, which may flare and subside; intermittent exposure to toxins, as with ingestions; and metabolic disorders, among them the fever-associated crises ascribed to biallelic mutations in SCYL1, with RALF beginning in infancy. SCYL1 disease manifest with RALF, as known to date, includes central and peripheral neurologic and muscular morbidity (hepatocerebellar neuropathy syndrome). Primary ventilatory and skeletal diseases also have been noted in some reports. CASE SUMMARY: We describe a Han Chinese boy in whom fever-associated RALF began at age 14 mo. Bilateral femoral head abnormalities and mild impairment of neurologic function were first noted aged 8 years 6 mo. Liver biopsy after the third RALF episode (7 years) and during resolution of the fourth RALF episode (8 years 6 mo) found abnormal architecture and hepatic fibrosis, respectively. Whole-exome sequencing revealed homozygosity for the novel frameshift mutation c.92_93insGGGCCCT, p.(H32Gfs*20) in SCYL1 (parental heterozygosity confirmed). CONCLUSION: Our findings expand the mutational and clinical spectrum of SCYL1 disease. In our patient a substantial neurologic component was lacking and skeletal disease was identified relatively late.

Δ4-3-oxosteroid-5ß-reductase deficiency: Responses to oral bile acid therapy and long-term outcomes.

BACKGROUND: Disorders of primary bile acid synthesis may be life-threatening if undiagnosed, or not treated with primary bile acid replacement therapy. To date, there are few reports on the management and follow-up of patients with Δ4-3-oxosteroid 5ß-reductase (AKR1D1) deficiency. We hypothesized that a retrospective analysis of the responses to oral bile acid replacement therapy with chenodeoxycholic acid (CDCA) in patients with this bile acid synthesis disorder will increase our understanding of the disease progression and permit evaluation of this treatment regimen as an alternative to the Food and Drug Administration (FDA) approved drug cholic acid, which is currently unavailable in China. AIM: To evaluate the therapeutic responses of patients with AKR1D1 deficiency to oral bile acid therapy, specifically CDCA. METHODS: Twelve patients with AKR1D1 deficiency, confirmed by fast atom bombardment ionization-mass spectrometry analysis of urine and by gene sequencing for mutations in AKR1D1, were treated with differing doses of CDCA or ursodeoxycholic acid (UDCA). The clinical and biochemical responses to therapy were monitored over a period ranging 0.5-6.4 years. Dose adjustment, to optimize the therapeutic dose, was based on changes in serum biochemistry parameters, notably liver function tests, and suppression of the urinary levels of atypical hepatotoxic 3-oxo-Δ4-bile acids measured by mass spectrometry. RESULTS: Physical examination, serum biochemistry parameters, and sonographic findings improved in all 12 patients during bile acid therapy, except one who underwent liver transplantation. Urine bile acid analysis confirmed a significant reduction in atypical hepatotoxic 3-oxo-Δ4 bile acids concomitant with clinical and biochemical improvements in those patients treated with CDCA. UDCA was ineffective in down-regulating endogenous bile acid synthesis as evidenced from the inability to suppress the urinary excretion of atypical 3-oxo-Δ4-bile acids. The dose of CDCA required for optimal clinical and biochemical responses varied from 5.5-10 mg/kg per day among patients based on maximum suppression of the atypical bile acids and improvement in serum biochemistry parameters, and careful titration of the dose was necessary to avoid side effects from CDCA. CONCLUSION: The primary bile acid CDCA is effective in treating AKR1D1 deficiency but the therapeutic dose requires individualized optimization. UDCA is not recommended for long-term management.

Anti-inflammatory and anti-arthritic properties of naringenin via attenuation of NF-κB and activation of the heme oxygenase ï´¾HOï´¿-1/related factor 2 pathway.

BACKGROUND: Naringenin, a bioflavonoid present in various species of citrus fruit, tomatoes and grapes, has been shown to have various pharmacological effects. We evaluated the anti-arthritic potential of naringenin in formaldehyde-induced inflammation and complete Freund's adjuvant (CFA)-induced arthritis. METHODS: For both evaluations, rats were divided into groups of six. Different doses of naringenin (5, 10 and 20mg/kg) were used in the models. Body weight and the arthritic index were assessed. Biochemical and antioxidant parameters were determined. Naringenin dose-dependently reduced joint inflammation, decreasing the joint diameter and inflammatory cell infiltration. RESULTS: Naringenin-treated rats showed an improvement in the synovium redox status (down-regulation of malondialdehyde and glutathione and up-regulation of Catalase (CAT) and superoxide dismutase levels). Naringenin significantly reduced the level of the inflammatory marker TNF-α. Naringenin increased Nrf-2/HO-1 and reduced NF-κB mRNA levels in CFA-treated animal joints. Additionally, naringenin treatment decreased the expression of extracellular matrix degrading enzymes, such as matrix metalloproteinase (MMP-3 and MMP-9), in CFA-induced arthritic rats. CONCLUSIONS: We conclude that naringenin exerts anti-arthritic effects by downregulating NF-κB and activating the Nrf-2/HO-1 pathway.

A Specially Designed Multi-Gene Panel Facilitates Genetic Diagnosis in Children with Intrahepatic Cholestasis: Simultaneous Test of Known Large Insertions/Deletions.

BACKGROUND AND AIMS: Large indels are commonly identified in patients but are not detectable by routine Sanger sequencing and panel sequencing. We specially designed a multi-gene panel that could simultaneously test known large indels in addition to ordinary variants, and reported the diagnostic yield in patients with intrahepatic cholestasis. METHODS: The panel contains 61 genes associated with cholestasis and 25 known recurrent large indels. The amplicon library was sequenced on Ion PGM system. Sequencing data were analyzed using a routine data analysis protocol and an internal program encoded for large indels test simultaneously. The validation phase was performed using 54 patients with known genetic diagnosis, including 5 with large insertions. At implement phase, 141 patients with intrahepatic cholestasis were evaluated. RESULTS: At validation phase, 99.6% of the variations identified by Sanger sequencing could be detected by panel sequencing. Following the routine protocol, 99.8% of false positives could be filtered and 98.8% of retained variations were true positives. Large insertions in the 5 patients with known genetic diagnosis could be correctly detected using the internal program. At implementation phase, 96.9% of the retained variations, following the routine protocol, were confirmed to be true. Twenty-nine patients received a potential genetic diagnosis when panel sequencing data were analyzed using the routine protocol. Two additional patients, who were found to harbor large insertions in SLC25A13, obtained a potential genetic diagnosis when sequencing data were further analyzed using the internal program. A total of 31 (22.0%) patients obtained a potential genetic diagnosis. Nine different genetic disorders were diagnosed, and citrin deficiency was the commonest. CONCLUSION: Specially designed multi-gene panel can correctly detect large indels simultaneously. By using it, we assigned a potential genetic diagnosis to 22.0% of patients with intrahepatic cholestasis.

Citrin deficiency presenting as acute liver failure in an eight-month-old infant.

Citrin deficiency typically presents as neonatal intrahepatic cholestasis and resolves in late infancy. Here we report a case of citrin deficiency that presented as acute liver failure in late infancy in an apparently healthy child. The full-term male infant weighed 3400 g at birth, and exhibited normal development for eight months, at which time he contracted bronchial pneumonia. The infant developed jaundice and laboratory tests indicated elevated bilirubin and ammonia levels and an abnormal coagulation profile. Plasma amino acid analysis showed elevated levels of tyrosine, methionine, citrulline, and arginine. Citrin deficiency was suspected, and genomic DNA analysis revealed a mutation (IVS16ins3kb) in SLC25A13, which encodes a mitochondrial aspartate-glutamate carrier protein. The infant was immediately put on a lactose-free, medium-chain-triglyceride-enriched formula with ursodeoxycholic acid and lipid-soluble vitamins. However, cholestasis and abnormal laboratory indices persisted, and the infant died at the age of 11.5 mo, two days before a scheduled liver transplantation. This case demonstrates that citrin deficiency can present in late infancy as acute liver failure triggered by infection, and may require liver transplantation.

Estrogen receptor-positive breast cancer molecular signatures and therapeutic potentials (Review).

In this review, the advances in the study of breast cancer molecular classifications and the molecular signatures of the luminal subtypes A and B of breast cancer were summarized. Effective clinical outcomes depend mainly on successful preclinical diagnosis and therapeutic decisions. Over the last few years, the ever-expanding investigations focusing on breast cancer diagnosis and the clinical trials have provided accumulating information on the molecular characteristics of breast cancer. Specifically, among the estrogen receptor (ER)-positive types of breast cancer, the luminal subtype A breast cancer has been shown to exhibit good clinical outcomes with endocrine therapy, whereas the luminal subtype B breast cancer represents the more complicated type, diagnostically as well as therapeutically. Furthermore, even in luminal subtype A breast cancer, the resistance to treatment has become the major limitation for endocrine-based therapy. Accumulating molecular data and further clinical trials may enable more accurate diagnostic and therapeutic decisions. The molecular signatures have emerged as a powerful tool for future diagnosis and therapeutic decisions, although currently available data are limited.

Antiproliferative effect of the Ginkgo biloba extract is associated with the enhancement of cytochrome P450 1B1 expression in estrogen receptor-negative breast cancer cells.

Ginkgo biloba is a dioecious tree and its extract is a complex mixture that has been used for thousands of years to treat a variety of ailments in traditional Chinese medicine. The aim of this study was to present our observations on the inhibitory effects of different Ginkgo biloba extracts on human breast cancer cell proliferation and growth. Our results demonstrated that treatment of MCF-7 and MDA-MB-231 human breast cancer cells with Ginkgo biloba leaves and ginkgo fruit extract inhibited cell proliferation. It was also observed that this inhibition was accompanied by the enhancement of cytochrome P450 (CYP) 1B1 expression in MDA-MB-231 cells. In addition, treatment with ginkgo fruit extract resulted in a higher CYP1B1 expression in MDA-MB-231 cells compared to treatment with the Ginkgo biloba leaves extract. Our results suggested that the inhibitory effects of the Ginkgo biloba extract on estrogen receptor-negative breast cancer proliferation and the induction of CYP1B1 expression may be exerted through an alternative pathway, independent of the estrogen receptor or the aryl hydrocarbon receptor pathway.

Lentivirus-mediated RNA interference targeting Bax inhibitor-1 suppresses ex vivo cell proliferation and in vivo tumor growth of nasopharyngeal carcinoma.

Bax inhibitor-1 (Bi-1), an anti-apoptotic protein that belongs to the Bcl-2 family, plays an important role in the mitochondrial apoptosis pathway to suppress Bax-induced apoptosis. In several human cancers, including nasopharyngeal carcinoma, its expression was found to be increased; however, up-regulated expression of this protein has been linked to increased cell proliferations. In this study, we down-regulated the gene expression of Bi-1 in nasopharyngeal carcinoma cells by using a lentivirus transfection system packed with short hairpin RNA targeting Bi-1 and used an in vivo model to assess its efficacy as a target in human gene therapy. The data indicated that human malignant nasopharyngeal carcinoma cells, CNE-1 and SUNE-1, transfected with lentiviral short hairpin RNA targeting Bi-1 grew more slowly and showed a higher degree of apoptosis. Moreover, the tumorigenicity of CNE-1 was significantly suppressed when inoculated mice were intratumorically injected with the same vector. Taken together, these data lead us to conclude that Bi-1 plays a crucial role in CNE-1 tumorigenesis and that Bi-1 may be a novel therapeutic target for nasopharyngeal carcinoma.