Whole-exome sequencing and molecular analyses

For Family 1, genomic DNA was obtained from the parents’ peripheral blood and the patients’ fibroblasts. Whole-exome sequencing of both patients and their parents were performed in the National Centre of Genomic Analysis (CNAG; Barcelona, Spain) using the Illumina HiSeq-2000 platform. Exome capture was performed with Agilent SureSelect v5 (Agilent, CA, USA). The filtering criteria were as in Urreizti et al. [20]. Sequencing data are available on demand.

The mean coverage was 156.84, 151.36, 217.18, and 143.30 reads for patient 1, patient 2, father, and mother, respectively, and a minimum of 99.2% of the target region was covered with at least 10 reads (C10). Six variants were selected for validation by Sanger sequencing (Supplementary Table 2). Primer sequences and PCR conditions are available on request. PCR reaction, purification, and sequencing were performed as described previously [20].

For Family 2, genomic DNA was obtained from peripheral blood. Whole-exome sequencing was performed on the proband (Patient 3) and the proband’s father at Genewiz (South Plainfield, NJ, USA). An Agilent SureSelect Exome kit (v6) was used for library preparation, and sequencing was performed on an Illumina HiSeq 2500 instrument (Illumina, San Diego, CA, USA) with 100-bp, paired-end reads. Alignment and variant calling was completed with an in-house GATK-based pipeline. A total of 122,433,475 reads were generated for the proband’s sample and 117,272,439 reads were generated for the father’s sample. 93.2% and 94.3% of the target had ≥ 30 x coverage for the proband’s and father’s samples, respectively. Variants were filtered with Ingenuity Variant Analysis (Qiagen, Redwood City) based on confidence (call quality ≥ 20), frequency (variants excluded if frequency was at least 0.5% in the 1000 Genomes Project, NHLBI ESP exomes, ExAC, or gnomAD databases), predicted deleteriousness (frameshift, in-frame indel, or start/stop codon change, missense change, splice site loss up to six bases into intron or as predicted by MaxEntScan, CADD score > 15, disease-associated variant according to computed ACMG guidelines classification criteria of pathogenic or likely pathogenic, or listed in HGMD or ClinVar included). The identified DPH1 variant was confirmed by PCR and Sanger sequencing.