Mutation surveyor v4.08

PCRs were performed using AmpliTaq Gold DNA polymerase (Applied Biosystems by Life Technologies). Products were run on an agarose gel and purified using the A’SAP PCR cleanup kit (Arctic Zymes) and Sanger sequenced at FIMM. Sequences were analyzed manually and with Mutation Surveyor (v4.08) (Softgenetics). PCRs from archival samples were performed in at least three replicates. For cDNA synthesis, RNA was reverse transcribed using random primers and Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega). The primers are listed in Supplementary Material, Table S9 and were designed with Primer3web (v4.0.0) (34 (link),35 (link)).

All of the genetic investigations performed in the proband and his family members were performed after informed consent.
A peripheral blood sample was used as the source of DNA for testing, and DNA isolation was performed using the A&A DNA isolation kit (Blood Mini kit, A&A Biotechnology, Gdynia, Poland). A sequencing analysis of the whole coding region of the APOB gene, including 25 bp flanking intronic regions, was performed using the NGS method and the HaloPlex Design Panel (Agilent, Santa Clara, CA, USA), which also included LDL receptor (LDLR) gene enrichment probes. Sequencing was performed using the MiSeq Reagent Kit v2 (500 cycles) on a MiSeq^TM sequencer (Illumina, San Diego, CA, USA).
For the APOB gene, the mean coverage corresponded to 842.68-fold; 98.79% of the bases were covered >50-fold, 0.71% of the bases were covered 10–49-fold, and 0.5% of the bases were covered 1–10-fold. There were no bases without any coverage.
The presence of all variants was confirmed by direct DNA sequencing. Mutation Surveyor V 4.0.8 (Softgenetics, State College, PA, USA) was employed to analyze fluorograms. To describe variants, reference sequences according to HGMD^® Professional [13 (link)], gene names according to HUGO Gene Nomenclature Committee (HGNC), and variant names according to HGVS v15.11 were applied.