The mutation spectrum of SLC25A13 gene in citrin deficiency: identification of novel mutations in Vietnamese pediatric cohort with neonatal intrahepatic cholestasis.

BACKGROUND: Citrin deficiency (CD), a disorder caused by mutations in the SLC25A13 gene, may result in neonatal intrahepatic cholestasis. This study was purposely to explore the mutation spectrum of SLC25A13 gene in Vietnamese CD patients. METHODS: The 292 unrelated CD patients were first screened for four high-frequency mutations by PCR/PCR-RFLP. Then, Sanger sequencing was performed directly for heterozygous or undetected patients. Novel mutations identified would need to be confirmed by their parents. RESULTS: 12 pathogenic SLC25A13 mutations were identified in all probands, including three deletions c.851_854del (p.R284Rfs*3), c.70-63_133del (p.Y24_72Ifs*10), and c.[1956C>A;1962del] (p.[N652K;F654Lfs*45]), two splice-site mutations (IVS6+5G>A and IVS11+1G>A), one nonsense mutations c.1399C>T (p.R467*), one duplication mutation c.1638_1660dup (p.A554fs*570), one insertion IVSl6ins3kb (p.A584fs*585), and four missense mutation c.2T>C (p.M1T), c.1231G>A (p.V411M), c.1763G>A (p.R588Q), and c.135G>C (p.L45F). Among them, c.851_854del (mut I) was the most identified mutant allele (91.78%) with a total of 247 homozygous and 42 heterozygous genotypes of carriers. Interestingly, two novel mutations were identified: c.70-63_133del (p.Y24_72Ifs*10) and c.[1956C>A;1962del] (p.[N652K;F654Lfs*45]). CONCLUSION: The SLC25A13 mutation spectrum related to intrahepatic cholestasis infants in Vietnam revealed a quite similar pattern to Asian countries' reports. This finding supports the use of targeted SLC25A13 mutation for CD screening in Vietnam and contributed to the SLC25A13 mutation spectra worldwide. It also helps emphasize the role of DNA analysis in treatment, genetic counseling, and prenatal diagnosis.

Clinical and genetic features of congenital bile acid synthesis defect with a novel mutation in AKR1D1 gene sequencing: Case reports.

RATIONALE: Congenital bile acid synthesis defect (BASD) is a rare disease caused by mutations in the aldo-keto reductase 1D1 gene, which encodes the primary Δ4-3-oxosteroid 5ß-reductase enzyme. Early disease diagnosis is critical for early treatment with bile acid replacement therapy, with an excellent chance for recovery. In contrast, protracted diagnosis and treatment may lead to poor outcomes, including decompensated hepatic cirrhosis, liver transplant, and even death. PATIENT CONCERNS: Three clinical congenital bile acid synthesis defect cases in the Vietnamese population are herein reported. These pediatric patients presented with symptoms of prolonged postpartum jaundice and abnormal loose stool (mucus, lipids, and white). The clinical examinations showed hepatosplenomegaly. Urinalysis showed a very low fraction of primary bile acids and atypical 3-oxo-Δ4- bile acids in all three patients. DIAGNOSES: The patients were diagnosed with primary Δ4-3-oxosteroid 5ß-reductase deficiency. Next-generation gene sequencing revealed two homozygous mutations in the aldo-keto reductase family 1 member D1 gene. The first is a documented variant, c.797G>A (p.Arg266Gln), and the second is a novel mutation at c.155T>C (p.Ile52Thr). INTERVENTIONS: Immediately after diagnosis, patients were treated with oral chenodeoxycholate 5 mg/kg/d. OUTCOMES: The patients' symptoms, signs, and primary bile acids levels improved significantly. LESSONS: Clinicians should consider genetic disorders related to cholestasis for effective and life-saving treatment. A prompt genetic analysis by next-generation gene sequencing enables patients to access bile acid replacement therapy earlier, significantly improving short- and long-term outcomes.

Identification of a wide spectrum of ciliary gene mutations in nonsyndromic biliary atresia patients implicates ciliary dysfunction as a novel disease mechanism.

BACKGROUND: Biliary atresia (BA) is the most common obstructive cholangiopathy in neonates, often progressing to end-stage cirrhosis. BA pathogenesis is believed to be multifactorial, but the genetic contribution, especially for nonsyndromic BA (common form: > 85%) remains poorly defined. METHODS: We conducted whole exome sequencing on 89 nonsyndromic BA trios to identify rare variants contributing to BA etiology. Functional evaluation using patients' liver biopsies, human cell and zebrafish models were performed. Clinical impact on respiratory system was assessed with clinical evaluation, nasal nitric oxide (nNO), high speed video analysis and transmission electron microscopy. FINDINGS: We detected rare, deleterious de novo or biallelic variants in liver-expressed ciliary genes in 31.5% (28/89) of the BA patients. Burden test revealed 2.6-fold (odds ratio (OR) [95% confidence intervals (CI)]= 2.58 [1.15-6.07], adjusted p = 0.034) over-representation of rare, deleterious mutations in liver-expressed ciliary gene set in patients compared to controls. Functional analyses further demonstrated absence of cilia in the BA livers with KIF3B and TTC17 mutations, and knockdown of PCNT, KIF3B and TTC17 in human control fibroblasts and cholangiocytes resulted in reduced number of cilia. Additionally, CRISPR/Cas9-engineered zebrafish knockouts of KIF3B, PCNT and TTC17 displayed reduced biliary flow. Abnormally low level of nNO was detected in 80% (8/10) of BA patients carrying deleterious ciliary mutations, implicating the intrinsic ciliary defects. INTERPRETATION: Our findings support strong genetic susceptibility for nonsyndromic BA. Ciliary gene mutations leading to cholangiocyte cilia malformation and dysfunction could be a key biological mechanism in BA pathogenesis. FUNDING: The study is supported by General Research Fund, HMRF Commissioned Paediatric Research at HKCH and Li Ka Shing Faculty of Medicine Enhanced New Staff Start-up Fund.

Distal duplication of chromosome 16q22.1q23.1 in a Vietnamese patient with midface hypoplasia and intellectual disability.

The clinical presentation of distal duplications of the long arm of chromosome (chr) 16 is currently not well described. Only one case of microduplication of chr16q22.1 and another involving the chr16q22.1q23.1 region have been reported so far. Here, using array comparative genomic hybridization, we identified a second case of chr16q22.1q23.1 duplication in a Vietnamese boy, who shares significant clinical phenotype with the previously described case. Aside from developmental delay, intellectual disability and midface hypoplasia, our patient also displays a forked tongue, visual impairment and external ptosis. Our report further expands the clinical spectrum associated with duplication of this region.

A novel homozygous mutation IVS6+5G>T in CYP11B1 gene in a Vietnamese patient with 11ß-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease which is characterized by a deficiency of one of the enzymes involved in the synthesis of cortisol from cholesterol by the adrenal cortex. CAH cases arising from impaired 11ß-hydroxylase are the second most common form. Mutations in the CYP11B1 gene are the cause of 11ß-hydroxylase deficiency. This study was performed on a patient with congenital adrenal hyperplasia and with premature development such as enlarged penis, muscle development, high blood pressure, and bone age equivalent of 5 years old at 2 years of chronological age. Biochemical tests for steroids confirmed the diagnosis of CAH. We used PCR and sequencing to screen for mutations in CYP11B1 gene. Results showed that the patient has a novel homozygous mutation of guanine (G) to thymine (T) in intron 6 (IVS6+5G>T). The analysis of this mutation by MaxEntScan boundary software indicated that this mutant could affect the gene splicing during transcription.

Targeted next-generation sequencing on Hirschsprung disease: a pilot study exploits DNA pooling.

To adopt an efficient approach of identifying rare variants possibly related to Hirschsprung disease (HSCR), a pilot study was set up to evaluate the performance of a newly designed protocol for next generation targeted resquencing. In total, 20 Chinese HSCR patients and 20 Chinese sex-matched individuals with no HSCR were included, for which coding sequences (CDS) of 62 genes known to be in signaling pathways relevant to enteric nervous system development were selected for capture and sequencing. Blood DNAs from eight pools of five cases or controls were enriched by PCR-based RainDance technology (RDT) and then sequenced on a 454 FLX platform. As technical validation, five patients from case Pool-3 were also independently enriched by RDT, indexed with barcode and sequenced with sufficient coverage. Assessment for CDS single nucleotide variants showed DNA pooling performed well (specificity/sensitivity at 98.4%/83.7%) at the common variant level; but relatively worse (specificity/sensitivity at 65.5%/61.3%) at the rare variant level. Further Sanger sequencing only validated five out of 12 rare damaging variants likely involved in HSCR. Hence more improvement at variant detection and sequencing technology is needed to realize the potential of DNA pooling for large-scale resequencing projects.

Screening of the RET gene of Vietnamese Hirschsprung patients identifies 2 novel missense mutations.

BACKGROUND/PURPOSE: Hirschsprung disease (HSCR; megacolon, congenital aganglionosis) is a congenital disorder characterized by the absence of ganglion cells along variable segments of the gut. Both rare (RV) and common variants of the RET gene are associated with HSCR. The aim of this study is to assess, for the first time, the variation in the RET gene of Vietnamese HSCR patients. METHODS: We used Sanger sequencing to screen the coding sequence of the RET gene of 97 Vietnamese HSCR patients of Southern Chinese ancestry. The healthy population consisted of 250 Southern Chinese individuals with no diagnosis of HSCR. RESULTS: We detected 8 heterozygous RVs distributed among 13 patients (13.40%) and that were not present in healthy individuals. Among those variants, there were 2 novel and deleterious (R133C [c.397 C>T]; R144C [c.430 C>T]) missense amino acid substitutions, 2 novel silent variants (P667P [c.2001 A>T]; Y809Y [c.2427 C>T]), and 4 previously described missense substitutions (R114H [c.341 G>A]; V292M [c.874 G>A]; G533S [c.1597 G>A]; R982C [c.2944 C>T]). As expected, the common RET coding sequence variants rs1800858 (A45A [c.135 G>A]) and rs1800861 (L769L [c.2307 T>G]) were highly associated with the disease. CONCLUSIONS: The identification of novel deleterious variants together with the fact RET RVs are virtually unique to HSCR patients indicates that the RET gene is a target for mutations among Hirschsprung patients of any population.