Clinical characterization of NTCP deficiency in paediatric patients : A case-control study based on SLC10A1 genotyping analysis.

Na+ -taurocholate cotransporting polypeptide deficiency (NTCPD) is a newly described disorder arising from biallelic mutations of the SLC10A1 gene. As a result of a lack of compelling evidence from case-control studies, its genotypic and phenotypic features remain open for in-depth investigation. This study aimed to explore the genotypic and clinical phenotypic characteristics of paediatric patients with NTCPD. The SLC10A1 genotypes of all NTCPD patients were confirmed by screening for the prevalent variant c.800C>T and Sanger sequencing when necessary. The clinical presentations and laboratory changes were collected, reviewed and analysed, and then qualitatively and quantitatively compared with the relevant controls. A total of 113 paediatric NTCPD patients were diagnosed while c.374dupG and c.682_683delCT were detected as two novel pathogenic mutations. Hypercholanemia was observed in 99.12% of the patients. Indirect hyperbilirubinemia in affected neonates exhibited higher positive rates in comparison to controls. Moreover, transient cholestatic jaundice, elevated liver enzymes and 25-hydroxyvitamin D (Vit D) deficiency during early infancy were more commonly observed in patients than in controls. All NTCPD patients exhibited favourable clinical outcomes as a result of symptomatic and supportive treatment. The findings enriched the SLC10A1 mutation spectrum and provided comprehensive insights into the phenotypic characteristics of NTCPD. NTCPD should be considered and SLC10A1 gene should be analysed in patients with above age-dependent clinical features. Furthermore, over investigation and intervention should be avoided in the management of NTCPD patients.

Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency: In vivo and in vitro studies of the aberrant transcription arising from two novel splice-site variants in SLC25A13.

Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD) is an autosomal recessive disease resulting from biallelic SLC25A13 mutations, and its diagnosis relies on genetic analysis. This study aimed to characterize the pathogenicity of 2 novel splice-site variants of SLC25A13 gene. Two patients (C0476 and C0556) suspected to have NICCD, their family members and 9 healthy volunteers were recruited as the research subjects. The SLC25A13 genotypes NG_012247.2(NM_014251.3): c.[852_855del]; [69+5G > A] in patient C0476 and c.[1453-1G > A]; [1751-5_1751-4ins (2684)] in patient C0556 were identified by means of polymerase chain reaction, long and accurate polymerase chain reaction, as well as Sanger sequencing. The 2 splice-site variants were absent in control databases and predicted to be pathogenic by computational analysis. The alternative splice variants in monocyte-derived macrophages from patient C0476 demonstrated exon 2 skipping [r.16_69del; p.(Val6_Lys23del)] in vivo, while minigene analysis revealed both exon 2-skipping and retained products from c.69+5G > A in vitro. In the patient C0556, an aberrant transcript [r.1453del; p.(Gly485Valfs*22)] resulting from c.1453-1G > A was detected on minigene splicing study. Thus, c.69+5G > A and c.1453-1G > A were both proved to be pathogenic. The 2 novel splice-site variants expanded the SLC25A13 mutation spectrum and provided reliable molecular markers for the definite diagnosis and genetic counseling of NICCD in the affected families.

Clinical and molecular study of a pediatric patient with sodium taurocholate cotransporting polypeptide deficiency.

The human solute carrier family 10 member 1 (SLC10A1) gene encodes sodium taurocholate cotransporting polypeptide (NTCP), the principal transporter of conjugated bile salts from the plasma into hepatocytes. Although the function of NTCP has been studied extensively and a number of SLC10A1 variations have been identified in humans, information regarding NTCP deficiency is limited. To date, only one patient with NTCP deficiency has been described; however, in the present study a pediatric patient who experienced intractable and striking hypercholanemia is presented. Analysis of the SLC10A1 gene in the patient revealed a homozygous p.Ser267Phe (c.800C>T) variation, which proved to be a single-nucleotide polymorphism (SNP) in the allele frequency of 4.7% of healthy controls. This variation involved a conserved amino acid residue on the orthologous alignment that was predicted to be 'disease-causing' by functional analysis using a number of bioinformatic tools. Next generation sequencing was performed; however, no other genetic causes were identified that would affect the bile acid homeostasis in the patient. Moreover, an adult, with the same genotype as the pediatric patient, was identified for the first time as experiencing mild hypercholanemia. The molecular and clinical findings in the present study suggest, for the first time, that there is an association between p.Ser267Phe SNP and hypercholanemia, and this information may be used to clinically identify NTCP deficiency worldwide.

Inspissated bile syndrome in an infant with citrin deficiency and congenital anomalies of the biliary tract and esophagus: identification and pathogenicity analysis of a novel SLC25A13 mutation with incomplete penetrance.

Biallelic mutations of the SLC25A13 gene result in citrin deficiency (CD) in humans. Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is the major CD phenotype in pediatrics; however, knowledge on its genotypic and phenotypic characteristics remains limited. The present study aimed to explore novel molecular and clinical characteristics of CD. An infant suspected to have NICCD as well as her parents were enrolled as the research subjects. SLC25A13 mutations were investigated using various methods, including cDNA cloning and sequencing. The pathogenicity of a novel mutation was analyzed bioinformatically and functionally with a yeast model. Both the infant and her father were heterozygous for c.2T>C and c.790G>A, while the mother was only a c.2T>C carrier. The novel c.790G>A mutation proved bioinformatically and functionally pathogenic. The infant had esophageal atresia and an accessory hepatic duct, along with bile plug formation confirmed by laparoscopic surgery. However, the father seemed to be healthy thus far. The findings of the present study enrich the genotypic and phenotypic characteristics of CD patients, and provided clinical and molecular evidence suggesting the possible non-penetrance of SLC25A13 mutations and the likely involvement of this gene in primitive foregut development during early embryonic life.

Clinical, molecular and functional investigation on an infant with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD).

BACKGROUND AND OBJECTIVE: SLC25A13 analysis has provided reliable evidences for the definitive diagnosis of citrin deficiency (CD) in the past decade. Meanwhile, these studies generated some issues yet to be resolved, including the pathogenicity of SLC25A13 missense mutations and the mRNA product from the mutation c.615+5G>A. This study aims to investigate the effect of a novel missense mutation on the aspartate/glutamate carrier (AGC) function of citrin protein, and to explore the aberrant transcript from c.615+5G>A in the same CD infant. METHODS AND RESULTS: By means of screening for prevalent SLC25A13 mutations and exons sequencing, the patient proved a compound heterozygote of c.615+5G>A and a novel c.1064G>A (p.Arg355Gln) mutation. An aberrant transcript with retention of the entire intron 6, r.[615+1_615+1789ins; 615+5 g>a] (GenBank accession number KJ128074), which was resulted from c.615+5G>A, was detected by RT-PCR and cDNA sequencing. After bioinformatic analyses of the novel missense mutation c.1064G>A, the growth abilities of three agc1Δ yeast strains were tested, which had been transformed with recombinant or empty vectors, respectively. Besides the bioinformatically pathogenic evidences, the growth ability of the agc1Δ strains transformed with mutant recombinant was the same as with empty vector, but significantly lower than that with normal control in functional analysis. CONCLUSIONS: A CD infant was definitely diagnosed in this paper by a genetic, transcriptional and functional analysis of SLC25A13 gene. This study provided direct laboratory evidences supporting the splice-site nature of the c.615+5G>A mutation, and the novel c.1064G>A variation, which proved a pathogenic mutation bioinformatically and functionally, enriched the SLC25A13 mutation spectrum.

[Detection of urine DD3/PSA mRNA ratio by duplex TaqMan RT-PCR assay and evolution of its primary application].

OBJECTIVE: To establish a duplex TaqMan RT-PCR assay for urine DD3/PSA mRNA ratio, and to evaluate its effect in diagnosis of prostate cancer (PCa). METHODS: Urine samples were obtained from 34 patients with PCa and 44 patients with benign prostate hypertrophy (BPH) by prostate massage. A duplex TaqMan RT-PCR assay was developed: PCR primers were designed to amplify the fragments between the exon1 and exon2 in the PSA mRNA and between the exon1 and exon3 in the DD3 mRNA, and the PCR TaqMan-MGB probes were labeled with HEX and FAM respectively in 5' for PSA mRNA. LNCaP cells were used as template. DD3/PSA mRNA ratio was measured. Receiver operating characteristic curve (ROC) was drawn so as to evaluate its diagnostic efficacy. RESULTS: Sequencing showed that the PCR products were specific for PSA mRNA and DD3 mRNA. The minimum detection level was approximately 0.6 cells/reaction for PSA mRNA and was 60 cells/reaction for DD3 mRNA in the LNCaP cell cDNA. The intra- and inter-assay coefficients of variation of DD3/PSA mRNA were 3.8%-4.7% and 4.1%-4.9% respectively. Urine DD3/PSA mRNA ratio in PCa group was significantly higher than the BPH group (P < 0.01). When the cutoff value was defined as 0.254, the area under curve (AUC) of DD3/PSA mRNA ratio was 0.746 (95% CI: 0.630-0.862), and the sensitivity and specificity were 64.7% and 77.3% respectively. The urine DD3/PSA mRNA ratio positive rate was not correlated with clinical and pathological parameters. CONCLUSION: A duplex TaqMan RT-PCR assay for urine DD3/PSA mRNA ratio has been established with an excellent clinical performance and specificity for PCa, saving time and reducing costs. It may be a promising method in the early diagnosis of PCa.