A novel mutation of the SLC25A13 gene in a Chinese patient with citrin deficiency detected by target next-generation sequencing

Type II citrullinaemia, also known as citrin deficiency, is an autosomal recessive metabolic disorder, which is caused by pathogenic mutations in the SLC25A13 gene on chromosome 7q21.3. One of the clinical manifestations of type II citrullinaemia is neonatal intrahepatic cholestatic hepatitis caused by citrin deficiency (NICCD, OMIM# 605814). In this study, a 5-month-old female Chinese neonate diagnosed with type II citrullinaemia was examined. The diagnosis was based on biochemical and clinical findings, including organic acid profiling using a gas chromatography mass spectrometry (GC/MS), and the patient's parents were unaffected. Approximately 14 kb of the exon sequences of the SLC25A13 and two relative genes (ASS1 and FAH) from the proband and 100 case-unrelated controls were captured by array-based capture method followed by high-throughput next-generation sequencing. Two single-nucleotide mutations were detected in the proband, including the previous reported c.1177+1G>A mutation and a novel c.754G>A mutation in the SLC25A13 gene. Sanger sequence results showed that the patient was a compound heterozygote for the two mutations. The novel mutation (c.754G>A), which is predicted to affect the normal structure and function of citrin, is a candidate pathogenic mutation. Target sequence capture combined with high-throughput next-generation sequencing technologies is proven to be an effective method for molecular genetic testing of type II citrullinaemia.     

Identification of two novel mutations in the SLC25A13 gene and detection of seven mutations in 102 patients with adult-onset type II citrullinemia

Adult-onset type II citrullinemia (CTLN2) is characterized by a liver-specific deficiency of argininosuccinate synthetase (ASS) protein. We have recently identified the gene responsible for CTLN2, viz., SLC25A13, which encodes a calcium-binding mitochondrial carrier protein, designated citrin, and found five mutations of the SLC25A13 gene in CTLN2 patients. In the present study, we have identified two novel mutations, 1800ins1 and R605X, in SLC25A13 mRNA and the SLC25A13 gene. Diagnostic analysis for the seven mutations in 103 CTLN2 patients diagnosed by biochemical and enzymatic studies has revealed that 102 patients had one or two of the seven mutations and 93 patients were homozygotes or compound heterozygotes. These results indicate that CTLN2 is caused by an abnormality in the SLC25A13 gene, and that our criteria for CTLN2 before DNA diagnosis are correct. Five of 22 patients from consanguineous unions have been shown to be compound heterozygotes, suggesting a high frequency of the mutated genes. The frequency of homozygotes is calculated to be more than 1 in 20,000 from carrier detection (6 in 400 individuals tested) in the Japanese population. We have detected no cross-reactive immune materials in the liver of CTLN2 patients with any of the seven mutations by Western blot analysis with anti-human citrin antibody. From these findings, we hypothesize that CTLN2 is caused by a complete deletion of citrin, although the mechanism of ASS deficiency is still unknown.     

Novel SLC20A2 mutations identified in southern Chinese patients with idiopathic basal ganglia calcification

Idiopathic basal ganglia calcification (IBGC) is a rare neuropsychiatric disorder characterized by bilateral and symmetric cerebral calcifications. Recently, SLC20A2 was identified as a causative gene for familial IBGC, and three mutations were reported in a northern Chinese population. Here, we aimed to explore the mutation spectrum of SLC20A2 in a southern Chinese population. Sanger sequencing was employed to screen mutations within SLC20A2 in two IBGC families and 14 sporadic IBGC cases from a southern Han Chinese population. Four novel mutations (c.82G > A p.D28N, c.185T > C p.L62P, c.1470_1478delGCAGGTCCT p.Q491_L493del and c.935-1G > A) were identified in two families and two sporadic cases, respectively; none were detected in 200 unrelated controls. No mutation was found in the remaining 12 patients. Different mutations may result in varied phenotypes, including brain calcification and clinical manifestations. Our study supports the hypothesis that SLC20A2 is a causative gene of IBGC and expands the mutation spectrum of SLC20A2, which facilitates the understanding of the genotype–phenotype correlation of IBGC.     

Downregulation of citrin, a mitochondrial AGC, is associated with apoptosis of hepatocytes

Citrin is a mitochondrial aspartate–glutamate carrier primarily expressed in liver. Adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin, and patients with this condition do not express citrin. We found apoptotic hepatocytes in one such patient. This finding prompted us to investigate the role of citrin in hepatocyte survival. Knockdown of citrin by a vector-based short-hairpin RNA technique reduced cell viability and induced apoptosis of a hepatocellular carcinoma cell line, Hep3B cells. Caspase-3/7 and caspase-9 were activated, and PARP was cleaved. Citrin knockdown also increased the expression of Bax and Bak, and reduced expression of Bcl-xL and Bcl-2. These alterations resulted in the release of cytochrome c from the mitochondria. Our results indicated that citrin downregulation induces apoptosis of hepatocytes through the mitochondrial death pathway, highlighting the importance of citrin in survival of hepatocytes and maintenance of liver function.     

Neonatal presentation of adult-onset type II citrullinemia

Adult-onset type II citrullinemia (CTLN2) is characterized by a liver-specific argininosuccinate synthetase deficiency caused by a deficiency of the citrin protein encoded by the SLC25A13 gene. Until now, however, no SLC25A13 mutations have been reported in children with liver diseases. We described three infants who presented as neonates with intrahepatic cholestasis associated with hypermethioninemia or hypergalactosemia detected by neonatal mass screening. DNA analyses of SLC25A13 revealed that one patient was a compound heterozygote for the 851de14 and IVS11+IG-->A mutations and two patients (siblings) were homozygotes for the IVS11+lG-->A mutation. These results suggested that there may be a variety of liver diseases related to CTLN2 in children.     

Characterization of seven novel mutations on the HEXB gene in French Sandhoff patients

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by mutations in the HEXB gene encoding the beta subunit of hexosaminidases A and B, two enzymes involved in GM2 ganglioside degradation. Eleven French Sandhoff patients with infantile or juvenile forms of the disease were completely characterized using sequencing of the HEXB gene. A specific procedure was developed to facilitate the detection of the common 5′-end 16 kb deletion which was frequent (36% of the alleles) in our study. Eleven other disease-causing mutations were found, among which four have previously been reported (c.850C>T, c.793T>G, c.115del and c.800_817del). Seven mutations were completely novel and were analyzed using molecular modelling. Two deletions (c.176del and c.1058_1060del), a duplication (c.1485_1487dup) and a nonsense mutation (c.552T>G) were predicted to strongly alter the enzyme spatial organization. The splice mutation c.558+5G>A affecting the intron 4 consensus splice site led to a skipping of exon 4 and to a truncated protein (p.191X). Two missense mutations were found among the patients studied. The c.448A>C mutation was probably a severe mutation as it was present in association with the known c.793T>G in an infantile form of Sandhoff disease and as it significantly modified the N-terminal domain structure of the protein. The c.171G>C mutation resulting in a p.W57C amino acid substitution in the N-terminal region is probably less drastic than the other abnormalities as it was present in a juvenile patient in association with the c.176del. Finally, this study reports a rapid detection of the Sandhoff disease-causing alleles facilitating genetic counselling and prenatal diagnosis in at-risk families.     

Identification of a novel ZNF469 mutation in a large family with Ehlers–Danlos phenotype

Brittle cornea syndrome (BCS) is a genetically heterogeneous disorder characterized by extreme corneal fragility and thinning, which may lead to spontaneous or trauma-induced corneal rupture. BCS-1 and BCS-2 are caused by recessive mutations in ZNF469 and PRDM5, respectively. Both genes play a role in the regulatory pathway of corneal development and maintenance. We report a consanguineous family with five patients affected with the cardinal ocular features of BCS and significant musculoskeletal findings primarily in the form of joint hypermobility and severe kyphoscoliosis. The patients had thin velvety skin, hallux valgus, variable sensorineural hearing loss and arachnodactyly. Interestingly, one of the patients additionally had phenylketonuria and showed a milder ophthalmological and musculoskeletal phenotype than his affected siblings. The urinary pyridinoline and deoxypyridinoline concentrations and their ratios were mildly elevated indicating increased bone-collagen turnover. A novel homozygous 14 bp duplication in exon 2 of ZNF469 (c.8817_8830dup) was uncovered by direct sequencing. This family highlights the phenotypic overlap between BCS and Ehlers–Danlos syndrome.     

The mitochondrial solute carrier SLC25A5 at Xq24 is a novel candidate gene for non-syndromic intellectual disability

Loss-of-function mutations in several different neuronal pathways have been related to intellectual disability (ID). Such mutations often are found on the X chromosome in males since they result in functional null alleles. So far, microdeletions at Xq24 reported in males always have been associated with a syndromic form of ID due to the loss of UBE2A. Here, we report on overlapping microdeletions at Xq24 that do not include UBE2A or affect its expression, in patients with non-syndromic ID plus some additional features from three unrelated families. The smallest region of overlap, confirmed by junction sequencing, harbors two members of the mitochondrial solute carrier family 25, SLC25A5 and SLC25A43. However, identification of an intragenic microdeletion including SLC25A43 but not SLC25A5 in a healthy boy excluded a role for SLC25A43 in cognition. Therefore, our findings point to SLC25A5 as a novel gene for non-syndromic ID. This highly conserved gene is expressed ubiquitously with high levels in cortex and hippocampus, and a presumed role in mitochondrial exchange of ADP/ATP. Our data indicate that SLC25A5 is involved in memory formation or establishment, which could add mitochondrial processes to the wide array of pathways that regulate normal cognitive functions.     

Two novel RAD21 mutations in patients with mild Cornelia de Lange syndrome-like presentation and report of the first familial case

Cornelia de Lange syndrome (CdLS) is a developmental disorder characterized by limb reduction defects, characteristic facial features and impaired cognitive development. Mutations in the NIPBL gene predominate; however, mutations in other cohesin complex genes have also been implicated, particularly in atypical and mild CdLS cases. Missense mutations and whole gene deletions in RAD21 have been identified in children with growth retardation, minor skeletal anomalies and facial features that overlap findings in individuals with CdLS. We report the first intragenic deletion and frameshift mutations identified in RAD21 in two patients presenting with atypical CdLS. One patient had an in-frame deletion of exon 13, while the second patient had a c.592_593dup frameshift mutation. The first patient presented with developmental delay, hypospadias, inguinal hernia and dysmorphic features while, the second patient presented with developmental delay, characteristic facial features, hirsutism, and hand and feet anomalies, with the first patient being milder than the second. The in-frame deletion mutation was found to be inherited from the mother who had a history of melanoma and other unspecified medical problems.