Computational protocol: Detailed investigations of proximal tubular function in Imerslund-Gräsbeck syndrome

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Protocol publication

[…] Nucleotides are numbered according to GenBank accession numbers [NM_001081.3] (CUBN) and [NM_030943.3] (AMN) with +1 corresponding to the A of the ATG translation initiation codon and the initiation codon corresponding to codon 1. CUBN and AMN exons, with flanking intronic regions, were amplified using standard PCR procedures with sequence specific primers (primer sequences are available upon request) and a polymerase (HOT FIREPol® DNA polymerase; Solis Biodyne, Estonia). AMN exons were amplified in 7 fragments with addition of the PCR additive S-Solution for amplification of GC-rich templates. Amplified products were enzymatically purified (ExoSAP-IT; USB Corporation, Cleveland, Ohio, USA) and used as template in sequencing reactions (Big Dye® Terminator v1.1 Cycle Sequencing Kit; Applied Biosystems, Naerum, Denmark). Sequencing products were purified using pre-soaked Sephadex G-50 (GE Healthcare Orsay, France) 96-well multiscreen filter plates (Millipore, Molsheim, France). Purified products were analysed on an automated 48-capillary sequencer (ABI 3730 Genetic analyser; Applied Biosystems, Courtaboeuf, France) and the results interpreted using the SeqScape® software (Applied Biosystems). Novel sequence variants were compared to commercially available control alleles (Human random control panels; Health Protection Agency Culture Collections, Salisbury, United Kingdom) to exclude commonly occurring polymorphisms. In silico splicing prediction analyses was performed using the NNSPLICE server (0.9 version) ( No additional patient material was available for analyses of AMN splicing. […]

Pipeline specifications

Software tools SeqScape, NNSplice
Applications WGS analysis, Sanger sequencing
Organisms Homo sapiens
Diseases Adrenoleukodystrophy, Anemia, Genetic Diseases, Inborn
Chemicals Vitamin B 12, Vitamin D