Nextera rapid enrichment kit

Informed consent for molecular genetic analysis was obtained for each patient and the investigated family members. To extract genomic DNA from EDTA blood, we used standard procedures and the Blood and Cell Kit by Qiagen (Qiagen GmbH, Hilden, Germany). For index patients panel sequencing (95 genes including all 11 HPS genes; Supplementary Material S1 gene list) was performed using a custom-designed Nextera Rapid Enrichment Kit (Illumina) followed by sequencing on a MiSeq (Illumina). The average sequencing depth overall genes for the 3 patients investigated was 98% for 20x and 91% for 100x, respectively. SeqPilot (JSI medical systems) was used for data analyses. The variants were exported and filtered by allele frequency and serious consequences. We utilized supporting software ALAMUT^®(v.2.15), pathogenicity prediction (SIFT, MutTaster, PolyPhen2, and CADD), occurrence in population and disease databases (HGMD public version, Huizing HPS Mutation update (Huizing et al., 2020 (link))) in order to classify the variants. These analyses were performed in accordance with the ACMG (American College of Medical Genetics) guidelines (Richards et al., 2015 (link)). For segregation analysis Sanger sequencing was conducted.

To extract genomic DNA from EDTA-blood, we applied standard procedures and the Blood and Cell Kit by Qiagen (Qiagen GmbH, Hilden, Germany). Panel sequencing (95 genes including all 11 HPS-genes) was performed in both patients using a custom designed Nextera Rapid Enrichment Kit (Illumina, San Diego, California/USA) followed by sequencing on a MiSeq (Illumina). SeqPilot (JSI medical systems) was used for data analyses. The variants were exported and filtered by allele frequency and serious consequences. We used supporting software ALAMUT^® (v.2.15), pathogenicity prediction (CADD; Combined Annotation Dependent Depletion), occurrence in population and disease databases (HGMD public version, Huizing HPS Mutation update [18 (link)]) to classify the variants. These analyses were conducted following the ACMG (American College of Medical Genetics) guidelines [41 (link)]. Sanger sequencing was performed for confirmation. BLOC1S5 exon 2 and intronic boundaries were amplified using the following primers (F, forward; R, reverse): F- 5′- TCT CTT AGT GGG GAA GGG AGA GAG T-3′ and R- 5′-CCC TAG AGC AGG CAC CAG AAC T-3′. Array-CGH (comparative genomic hybridization) analyses were performed using the SNP array Infinium^® CytoSNP-850K (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions.