Cholestasis is defined as a conjugated bilirubin level >1 mg/dL(17.1 μmol/L)if total serum bilirubin is ≤5 mg/dL(85.5 μmol/L),or conjugated bilirubin fraction >20％of total bilirubin when the total bilirubin is >5 mg/dL(85.5 μmol/L).In the recent years,the diagnosis and management of genetic cholestasis have caused considerable attention in the pediatric world,in pace with the development,maturation,and clinical application of the theories and techniques in genomics as well as molecular genetics.With a diversity of causative genes,genetic cholestasis usually demonstrates nonpathognomonic clinical manifestations.The etiology diagnosis such a disease relies on genetic tests,the treatment is often difficult,and the prognosis varies disparately,usually causing tremendous pain and burden on the affected patient and the family as well.Taking citrin deficiency,mitochondrial DNA depletion syndrome,microvi-llus inclusion disease and sodium taurocholate cotransporting polypeptide deficiency as samples,the recent advances in the diagnosis and treatment of genetic cholestasis are addressed.

Objective To develope both qualitative and quantitative analytic method of the four urinary markers,i.e.lactate,uracil,orotate and hippurate,from ornithine transcarbamylase deficiency(OTCD) by Gas Chromatography-Mass Spectrometry(GC-MS).Methods Urea in urine samples was decomposed with urease,and heptadecanoiate was added as internal standard,then protein was denatured with ethanol and removed by centrifugation.After evaporation,the residue was derivatized trimethylsilylly by BSTFA/TMCS,and analyzed by GC-MS.ResultsThe markers can be separated in total ion current profiles,with indentifications confirmed by mass spectra.The significantly elevated levels of lactate,uracil and orotate in urine from OTCD patient were droped to normal or subnormal levels,together with large amount of hippurate excretion in the urine,after clinical therapeutic measures,including introduction of benzoic acid,were performed.Conclusion GC-MS analysis of the urinary markers is a valuable tool for the diagnosis and evaluation of the therapeutic outcome of OTCD.

The elevated NADH/ NAD + ratio in the plasma of the hepatocyte is a key pathophysiologic altera-tion for the development of neonatal intrahepatic cholestasis caused by Citrin deficiency (NICCD). The elevated ratio results in energy shortage in hepatocytes and impairs the function of the ATP - dependent canalicular transporters,in-cluding BSEP,MDR3,FIC1,Sterolin 1 / 2 and MRP2,leading to intrahepatic cholestasis in NICCD patients. On the other hand,the increased NADH/ NAD + ratio inhibits galactose metabolism and thus gives rise to secondary galactosemia, damaging the hepatocyte as well as extrahepatic organs including the lens of the eyes. The lactose - free and medium -chain triglyceride - enriched formulas can rapidly correct the energy shortage of the hepatocyte and alleviate secondary galactosemia,hence improving the clinical presentations effectively in NICCD patients.

Sodium-taurocholate cotransporting polypeptide (NTCP) deficiency is a new hereditary bile acid metabolic disease due to biallelic mutations of the SLC10A1 gene and is not rare in China. Marked and persistent hypercholanemia in childhood is the major clinical feature of NTCP deficiency, and this condition might be involved in the development of neonatal hyperbilirubinemia, cholestasis in early infancy, and cholestasis in pregnancy. At present, there lack specific therapies for NTCP deficiency, but such patients tend to have good prognosis. SLC10A1 gene detection may facilitate the timely and definite diagnosis of this disease and thus avoid unnecessary examinations and interventions.

OBJECTIVETo explore the clinical and genetic characteristics of a family affected by genetic cholestasis.

METHODSClinical data of the patient was collected. Targeted exome sequencing was carried out to detect the pathogenic mutations. The results were confirmed by Sanger sequencing.

RESULTSThe patient, a 5-year-old boy, presented with severe cholestatic cirrhosis. Genetic analysis revealed that he has carried compound heterozygous mutations c.1006-2A>G and c.3580C>T (p.R1194X) of the ABCB4 gene, which were inherited from his father and mother, respectively. By structural prediction, the mutation c.3580C>T can give rise to a truncated multi-drug resistance protein 3 (MDR3).

CONCLUSIONThe patient was diagnosed with progressive familial intrahepatic cholestasis type 3 (PFIC-3) based on clinical and molecular findings. Detection of novel mutations of the ABCB4 gene has provided valuable clues for the diagnosis and genetic counseling.

OBJECTIVETo detect potential mutation of the AGL gene in two siblings affected with glycogen storage disease type IIIa.

METHODSClinical data of the two siblings was collected and analyzed. Genomic DNA was extracted from peripheral venous blood samples from the patients and their parents. All exons and their flanking sequences of the AGL gene were subjected to PCR amplification and Sanger sequencing. Suspected mutation was verified in 75 healthy controls.

RESULTSThe main clinical features of the two siblings included hypoglycemia and hepatomegaly, along with markedly elevated liver and myocardial enzymes. Genetic analysis revealed that both siblings harbored compound heterozygous mutations c.1735+1G>T and c.959-1G>C of the AGL gene. Among these, the splicing mutation c.959-1G>C was a novel one with an allele frequency of <1%.

CONCLUSIONBased on their clinical features and genetic analysis, the siblings were diagnosed with glycogen storage disease type IIIa. The c.959-1G>C has enriched the spectrum of AGL gene mutations.

Adolescent ; Amino Acid Sequence ; Female ; Glycogen Debranching Enzyme System ; genetics ; Glycogen Storage Disease Type III ; genetics ; Humans ; Infant ; Male ; Mutation ; genetics ; Siblings

OBJECTIVE: To explore the clinical and genetic features of an infant with citrin deficiency (CD). METHODS: Clinical data of the patient was collected and analyzed. Genomic DNA was extracted from peripheral blood samples collected from the patient and her parents. Targeted exome sequencing was performed to explore the genetic cause, and Sanger sequencing was used to confirm the detected variants. SLC25A13 mRNA was extracted from peripheral blood lymphocytes of the infant. The effect of novel mutation of SLC25A13 was analyzed by reverse transcription-PCR, cDNA cloning and Sanger sequencing. RESULTS: The SLC25A13 genotype of the patient was determined as c.845_c.848+1delG/c.1841+3_1841+4delAA, with the latter having not been reported. The mutation has affected the splicing of the SLC25A13 mRNA, giving rise to an aberrant transcript [r.1841_1842ins1841+1_1841+67; 1841+3_c.1841+4del]. CONCLUSION A novel SLC25A13 mutation c.1841+3_1841+4delAA and the resultant abnormal splicing variant were discovered by combined DNA sequencing and cDNA cloning. The finding has enabled definite diagnosis of CD and enriched the spectrum of SLC25A13 mutations.
Base Sequence ; Citrullinemia ; Female ; Humans ; Mitochondrial Membrane Transport Proteins ; genetics ; Mutation ; Pedigree

Objective: To explore the clinical and molecular genetic features of patients with Alagille syndrome (AS). Methods: The clinical data of eleven pediatric patients, who were suspected to have AS at the Department of Pediatrics in the First Affiliated Hospital of Jinan University from August 2010 to March 2017, were collected and analyzed. Genomic DNA was extracted from peripheral blood leukocytes of the patients and their parents. For 5 patients collected before March 2006, all JAG1 exons and their flanking sequences were directly sequenced. For the remaining 6 patients, high-throughput gene capture technology, chromosomal microarray analysis (CMA) and whole-genome copy-number variant(CNV) analysis were utilized, when necessary, to explore the genetic causes. Results: All patients had cholestasis. However, the γ-glutamyl transpeptidase (GGT) levels in one patient were normal. Nine patients had posterior embryotoxon and facial malformations. Eight patients displayed heart defects. Seven patients presented with vertebral anomalies and among them, 1 patient had sacralization of the cubitus and radius. The condition of nine patients tended to be stabilized on follow-up, but 1 patient died of liver failure in late infancy and 1 got worse. Seven JAG1 variants were detected in 9 out of the 11 AS patients, with c.1977G>A (p.Trp659*) and c.1106_1107delCC (p.Pro369fs) being two novel variants. Two heterozygous interstitial deletions of 3.0 Mb and 9.24 Mb in size, respectively, in chromosome 20 were discovered in the remaining 2 patients. Both deletions involved the entire JAG1 gene. De novo origin was unveiled for the detected variants in 7 patients and interstitial deletions in two. Although the mother of 2 patients carried the relevant variant, she did not demonstrate any clinical features of AS. Conclusions With cholestasis, posterior embryotoxon, facial malformations, heart defects and vertebral anomalies being the major manifestations, AS demonstrated variable clinical expressivities and incomplete penetrance. This study identified a total of 7 JAG1 variants as well as 2 interstitial deletions involving this gene, and among them, the variants c.1977G>A (p.Trp659* ) and c.1106_1107delCC (p.Pro369fs) as well as the 9.24 Mb chromosomal interstitial deletion had not been reported previously.

OBJECTIVE: To explore the clinical and genetic characteristics of a pediatric patient suspected for Autosomal Recessive Congenital Ichthyosis (ARCI). METHODS: Clinical data of the patient was analyzed. Peripheral blood samples were collected from the patient and his parents for the extraction of genomic DNA. Next-generation sequencing (NGS) was then carried out. Candidate variants were confirmed by Sanger sequencing. A variety of bioinformatic tools including Mutation Taster, PROVEAN, and PolyPhen2 were used to predict the pathogenicity of the variants based on guidelines from the American College of Medical Genetics and Genomics (ACMG). RESULTS: The patient, a 1-month-and-7-day-old male, had presented with cutaneous erythema and fine scaling of the whole body. NGS revealed that he has harbored compound heterozygous variants c.1579G>A (p.Val527Met) (paternal) and c.923T>C (p.Leu308Pro) (maternal) of the ALOX12B gene. The former was known to be likely pathogenic, while the latter was unreported previously and categorized as "likely pathogenic" based on the ACMG guidelines. Based on the clinical and genetic findings, the patient was diagnosed with ARCI. CONCLUSION The c.1579G>A and c.923T>C variants of the ALOX12B genes probably underlay the ARCI in this patient. Above finding has enriched the spectrum of ALOX12B mutations and enabled molecular diagnosis of the patient, based on which genetic counseling and prenatal diagnosis may be provided.
Arachidonate 12-Lipoxygenase/genetics* ; Child ; Female ; Genes, Recessive ; Genetic Testing ; High-Throughput Nucleotide Sequencing ; Humans ; Ichthyosis, Lamellar/genetics* ; Male ; Mutation ; Pregnancy

Professor Takeyori Saheki's team at Kagoshima University, Japan, published a paper in Nature Genetics in June 1999, pinpointing the pathogenic gene for adult-onset type Ⅱ citrullinemia as SLC25A13 and naming the protein product encoded by this gene as citrin. Over the past 25 years, the researches have made positive progress on the pathophysiological mechanism, clinical phenotype, molecular diagnosis, treatment, and prognosis of citrin deficiency (CD) as an autosomal recessive genetic disease. Currently, three age-dependent clinical phenotypes of CD have been found, namely neonatal intrahepatic cholestasis caused by citrin deficiency, failure to thrive and dyslipidemia caused by citrin deficiency, and adult-onset type Ⅱ citrullinemia. Although relevant internal medicine drugs are being researched and developed while liver transplantation has been used for the treatment of CD patients, scientific dietary therapy remains the foundation, core, and key for the management of this disease. Furthermore, CD management involves the full life cycle of patients, requiring the joint efforts of basic and clinical medicine as well as systematic articulation at multi-levels, such as the parents, family, and society. By full-cycle, multidisciplinary, and systematic management, it is an achievable goal for CD patients to learn, work, and live in a healthy manner.