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1.  A combination of mutations in AKR1D1 and SKIV2L in a family with severe infantile liver disease 
Infantile cholestatic diseases can be caused by mutations in a number of genes involved in different hepatocyte molecular pathways. Whilst some of the essential pathways have a well understood function, such as bile biosynthesis and transport, the role of the others is not known. Here we report the findings of a clinical, biochemical and molecular study of a family with three patients affected with a severe infantile cholestatic disease. A novel homozygous frameshift germline mutation (c.587delG) in the AKR1D1 gene; which encodes the enzyme Δ 4-3-oxosteroid 5β–reductase that is required for synthesis of primary bile acids and is crucial for establishment of normal bile flow, was found in all 3 patients. Although the initial bile acid analysis was inconclusive, subsequent testing confirmed the diagnosis of a bile acid biogenesis disorder. An additional novel homozygous frameshift mutation (c.3391delC) was detected in SKIV2L in one of the patients. SKIV2L encodes a homologue of a yeast ski2 protein proposed to be involved in RNA processing and mutations in SKIV2L were recently described in patients with Tricohepatoenteric syndrome (THES). A combination of autozygosity mapping and whole-exome-sequencing allowed the identification of causal mutations in this family with a complex liver phenotype. Although the initial 2 affected cousins died in the first year of life, accurate diagnosis and management of the youngest patient led to successful treatment of the liver disease and disease-free survival.
doi:10.1186/1750-1172-8-74
PMCID: PMC3659031  PMID: 23679950
Bile acid metabolism; Diarrhoea; Gene mutation; Whole exome sequencing; Paediatric liver disease
2.  Mutation detection in cholestatic patients using microarray resequencing of ATP8B1 and ABCB11  
F1000Research  2013;2:32.
Background : Neonatal cholestasis is a common presentation of childhood liver diseases and can be a feature of various conditions including disorders of bile acid biogenesis and transport, various inborn errors of metabolism and perinatal infections. Some inherited metabolic diseases can be easily screened using biochemical assays, however many can only be accurately diagnosed by DNA sequencing. Fluorescent capillary Sanger sequencing (FS) is the gold standard method used by clinical laboratories for genetic diagnosis of many inherited conditions; however, it does have limitations. Recently microarray resequencing (MR) has been introduced into research and clinical practice as an alternative method for genetic diagnosis of heterogeneous conditions. In this report we compared the accuracy of mutation detection for MR with FS in a group of patients with ‘low-normal’ gamma glutamyl transpeptidase (gGT) cholestasis without known molecular diagnoses.
Methods : 29 patient DNA samples were tested for mutations in the ATP8B1 and ABCB11 genes using both FS and MR. Other known causes of “low gGT cholestasis” such as ARC syndrome and bile acid biosynthesis disorders were excluded.
Results : Mutations were identified in 13/29 samples. In 3/29 samples FS and MR gave discordant results: MR had a false positive rate of 3.4% and a false negative rate of 7%.
Conclusions : The major advantage of MR over FS is that multiple genes can be screened in one experiment, allowing rapid and cost-effective diagnoses.  However, we have demonstrated that MR technology is limited in sensitivity. We therefore recommend that MR be used as an initial evaluation, with FS deployed when genetic and clinical or histopathological findings are discordant.
doi:10.12688/f1000research.2-32.v2
PMCID: PMC3907151  PMID: 24627769
3.  Mutation detection in cholestatic patients using microarray resequencing of ATP8B1 and ABCB11  
F1000Research  2013;2:32.
Background : Neonatal cholestasis is a common presentation of childhood liver diseases and can be a feature of various conditions including disorders of bile acid biogenesis and transport, various inborn errors of metabolism and perinatal infections. Some inherited metabolic diseases can be easily screened using biochemical assays, however many can only be accurately diagnosed by DNA sequencing. Fluorescent capillary Sanger sequencing (FS) is the gold standard method used by clinical laboratories for genetic diagnosis of many inherited conditions; however, it does have limitations. Recently microarray resequencing (MR) has been introduced into research and clinical practice as an alternative method for genetic diagnosis of heterogeneous conditions. In this report we compared the accuracy of mutation detection for MR with FS in a group of patients with ‘low-normal’ gamma glutamyl transpeptidase (gGT) cholestasis without known molecular diagnoses.
Methods : 29 patient DNA samples were tested for mutations in the ATP8B1 and ABCB11 genes using both FS and MR. Other known causes of “low gGT cholestasis” such as ARC syndrome and bile acid biosynthesis disorders were excluded.
Results : Mutations were identified in 13/29 samples. In 3/29 samples FS and MR gave discordant results: MR had a false positive rate of 3.4% and a false negative rate of 7%.
Conclusions : The major advantage of MR over FS is that multiple genes can be screened in one experiment, allowing rapid and cost-effective diagnoses.  However, we have demonstrated that MR technology is limited in sensitivity. We therefore recommend that MR be used as an initial evaluation, with FS deployed when genetic and clinical or histopathological findings are discordant.
doi:10.12688/f1000research.2-32.v1
PMCID: PMC3907151  PMID: 24627769
4.  MUTATIONS IN TTC37 CAUSE TRICHOHEPATOENTERIC SYNDROME (PHENOTYPIC DIARRHOEA OF INFANCY) 
Gastroenterology  2010;138(7):2388-2398.e2.
Background
Trichohepatoenteric syndrome (THES) is an autosomal recessive disorder characterised by life-threatening diarrhoea in infancy, immunodeficiency, liver disease, trichorrhexis nodosa, facial dysmorphism, hypopigmentation and cardiac defects. We attempted to characterise the phenotype and elucidate the molecular basis of THES.
Methods
Twelve patients with classical THES from 11 families had detailed phenotyping. Autozygosity mapping was undertaken in 8 patients from consanguineous families using 250k single nucleotide polymorphism (SNP) arrays and linked regions evaluated using microsatellite markers. Linkage was confirmed to one region from which candidate genes were analysed. The effect of mutations on protein production and/or localisation in hepatocytes and intestinal epithelial cells from affected patients was characterised by immunohistochemistry.
Results
Previously unrecognised platelet abnormalities (reduced platelet α-granules, unusual stimulated alpha granule content release, abnormal lipid inclusions, abnormal platelet canalicular system and reduced number of microtubules) were identified. The THES locus was mapped to 5q14.3 – 5q21.2. Sequencing of candidate genes demonstrated mutations in TTC37, which encodes the uncharacterised tetratricopeptide repeat protein, thespin. Bioinformatic analysis suggested thespin to be involved in protein-protein interactions or chaperone. Preliminary studies of enterocyte brush-border ion transporter proteins (NHE2, NHE3, Aquaporin 7, Na/I symporter and H / K ATPase) showed reduced expression or mislocalisation in all THES patients with different profiles for each. In contrast the basolateral localisation of Na/K ATPase was not altered.
Conclusion
THES is caused by mutations in TTC37. TTC37 mutations have a multisystem effect which may be due to abnormal stability and / or intracellular localisation of TTC37 target proteins.
doi:10.1053/j.gastro.2010.02.010
PMCID: PMC3166659  PMID: 20176027
phenotypic diarrhoea of infancy; platelet alpha granules; ion transporter proteins; thespin
5.  TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum 
Nature genetics  2011;43(3):189-196.
Ciliary dysfunction leads to a broad range of overlapping phenotypes, termed collectively as ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifying alleles to clinically distinct disorders. Here we show that mutations in TTC21B/IFT139, encoding a retrograde intraflagellar transport (IFT) protein, cause both isolated nephronophthisis (NPHP) and syndromic Jeune Asphyxiating Thoracic Dystrophy (JATD). Moreover, although systematic medical resequencing of a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations unmasked a significant enrichment of pathogenic alleles in cases, suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy patients. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies, as well as interact in trans with other disease-causing genes, and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of disorders.
doi:10.1038/ng.756
PMCID: PMC3071301  PMID: 21258341

Results 1-5 (5)