Computational protocol: ALS5/SPG11/KIAA1840mutations cause autosomal recessive axonal Charcot–Marie–Tooth disease

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Protocol publication

[…] Genomic DNA was extracted from peripheral blood using the Promega Wizard® Genomic DNA isolation kit. Linkage study of the 28 families with ARCMT2 was performed using microsatellite markers flanking the ALS5/SPG11 locus, as previously described ( ). In the probands, all the coding exons of ALS5/SPG11/ KIAA1840 and at least 50 bp of flanking intronic sequence were PCR-amplified by primer pairs, as previously described ( ), and by Roche FastStart™ PCR Master Mix polymerase. All the PCR-amplified products were purified using a Qiagen PCR purification kit. Purified products were sequenced with respective forward and reverse primers by an Applied Biosystems 3130 Genetic Analyzer. Sequence analysis was performed using SeqScape software (v2.6; Applied Biosystems). Large genomic rearrangements of ALS5/SPG11/ KIAA1840 gene were investigated by multiplex ligation-dependent probe amplification method with the P306-B1 SPG11 probe mix, containing probes for each of the 40 KIAA1840 exons. Data were collected and analysed with GeneScan software (v.3.1.2; Applied Biosystems). For each sample, peak areas were calculated and compared with three wild-type controls, using Coffalyser software (v.7.0; MRC Holland). The segregation analysis within the family and the surveillance of mutations in the control population were performed using a PCR-restriction fragment length polymorphism method. Three hundred control chromosomes were obtained from healthy volunteers of mixed ethnic origins, including Caucasian, Brazilian and Japanese. An in silico pathogenicity prediction tool (Mutation Taster, ) was used to predict the effect of the novel mutations identified, as previously described ( ). In all probands, we performed direct sequencing of all exons and flanking introns of CMT2A2/HMSN2A2/ MFN2 (OMIM: #609260; ) , CMT2B1/ LMNA (OMIM: #605588; ), CMT2B2/ MED25 (OMIM: #605589; ), CMT2B5/ NEFL (OMIM: #607734; ), ARCMT2F/dHMN2B/ HSPB1 (OMIM: #608634; ), CMT2K/ GDAP1 (OMIM: #607831; ), CMT2P/ LRSAM1 (OMIM: #614436; ), CMT2R/ TRIM2 (OMIM: #615490; ), CMT2S/ IGHMBP2 (OMIM: #616155; ), CMT2T/ HSJ1 (OMIM: #616233; ), CMTRID/ COX6A1 (OMIM: #616039; ), ARAN-NM/ HINT (OMIM: #137200; ), GAN/ GAN (OMIM: #256850; ), SPG7/ PGN (OMIM: #607259; ), SPG15/ ZFYVE26 (OMIM: #270700; ), SPG21/ ACP33 (OMIM: #248900; ), SPG35/ FA2H (OMIM: #612319; ), SPG46/ GBA2 (OMIM: #614409; ; ), SPG55/ C12orf65 (OMIM: #615035; ), SPG56/ CYP2U1 (OMIM: #610670; ), and SLC12A6 (OMIM: #604878; ). Direct sequencing of POLG (OMIM: #174763; ) and TYMP (OMIM: #131222; ) genes was also carried out to exclude mitochondrial disorders related to CMT2 ( ). Furthermore, linkage to chromosome 8q13-21.1 was performed to investigate autosomal recessive CMT2H, as previously reported ( ). […]

Pipeline specifications

Software tools SeqScape, MutationTaster
Application Sanger sequencing
Organisms Homo sapiens