Analysis of CD46 mutations

Genomic DNA was extracted from peripheral blood leukocytes of patients and 50 healthy controls, as previously described [10]. Genetic analysis was done by polymerase chain reaction amplification of coding exons and splice sites using 0.5 µM of each primer (Table ​(Table1),1), 50–100 ng DNA, 1.25 mM MgCl₂, 0.25 mM of each dNTP (Invitrogen, Carlsbad, CA, USA) and 0.5 units of Taq polymerase (Invitrogen). The reactions were cycled on an ABI 9700 (thermocycler Applied Biosystems, Foster City, CA, USA) (Table ​(Table1).1). Amplified products were purified using Qiagen kits (Hilden, Germany) and sequenced on an ABI-3100 analyzer (Applied Biosystems). Nucleotide sequences were compared with the published cDNA sequences (GenBank ENSG00000117335).

Primer sequences used for CD46 screening

In silico prediction of pathogenicity of the variations was done using Human Splicing Finder (HSF) version 2.4.1 (http://www.umd.be/HSF/) to assess the effects of missense intronic and exonic variations on splicing [11]. PolyPhen-2 version 2.2.2 was also used to predict the impact of nonsynonymous exonic mutations on the structure and function of the protein [12]. Analysis was done using default threshold and cutoff values as mentioned in the software.