A 68-year-old male was diagnosed with severe HEMA in early childhood, with less than 0.001% factor VIII activity. The brother of the proband also suffered from severe HEMA, thus the mother must have been carrier of the causative mutation. The children of the proband were male, and consequently, in this part of the family the mutation has not been passed on. Due to the distant past of the diagnosis, no genetic tests had been performed to identify the causative mutation. Around 30% of patients with severe HEMA develop inhibitors during their treatment with factor VIII, especially patients with large deletions and intron inversions. Thus, genetic factors can influence inhibitor development, and different treatment approaches are chosen according to risk of inhibitor development [
7]. However, the proband never developed factor VIII inhibitors, possibly suggesting a smaller and less frequent mutation in
F8 than the large intron inversion. Following blood transfusion, the proband was tested positive for HIV-1 and hepatitis C virus in the late 1980s and early 1990s, respectively. The patient was cured for his Hepatitis C infection, but never received any treatment for his HIV-1 infection, since he remained with normal CD4 T cell count over time and was considered an HIV long-term non-progressor (LTNP).
To identify the HEMA causative mutation (as well as possible mutations explanatory for his HIV LTNP phenotype), a blood sample was drawn in EDTA tubes (FLUKA), and peripheral blood mononuclear cells (PBMCs) were isolated over ficoll gradient (GE-healthcare). Integrating HIV DNA in CD4 T cells might result in false positive (somatic mosaic) mutations, or disturb the quality of sequencing. Therefore, CD4 T cells were depleted by magnetic purification (miltenyi biotec). DNA from non-CD4 T cells was purified using allprep DNA/RNA mini kit (Qiagen). Whole exome sequencing (WES) was performed employing Kapa HTP Library preparation and Nimblegen SeqCap EZ MedExome Plus kit and analysed using Nextseq v2 chemistry (2 × 150 bp). SNPs were called relative to hg19. Variant call files (VCF) were uploaded to Ingenuity Variant Analysis (IVA, Qiagen) and variants were compared to population frequencies of variants in the Allele Frequency Community (AFC) database and to frequencies in the 1000 Geneomes project. One hundred thirty thousand six hundred eighty-seven variants were identified in 16,957 genes in the patient, of which seven were located in the
F8 gene. Two variants did not pass quality control, thus five variants could be possibly causative (see Table
1). Four of the remaining variants had an allele frequency much higher than the disease frequency and were therefore judged as being irrelevant. Therefore, one variant (c.5411_5412delTCT, p.F1804del) remained a potential cause of disease (Fig.
1b). The mutation was verified in the raw BAM file (Additional file
1: Figure S1). Ingenuity did not provide any dbSNP ID or frequency for this variant, which is thus denoted as novel. Moreover, the variant was not reported in Coagulation Factor Variant Databases EAHAD.CFDB (
https://databases.lovd.nl/shared/variants/F8), which provides all 5418 known transcript variants in the
F8 gene, confirming that the c.5411_5413delTCT, p.F1804del must indeed be novel.
Table 1
Identified genetic variants passing quality control
c.6115 + 103 T > C | Intronic | SNV | normal | Benign | | < 10 | 4,074,307 | 22.442 | 44.079 |
c.5998 + 91 T > A | Intronic | SNV | normal | Benign | | < 10 | 4,898,352 | 22.653 | 44.132 |
c.5411_5413delTCT | p.F1804del | Exonic | Deletion | loss | Likely Pathogenic | in-frame | * | | | |
c.3780C > G | p.D1260E | Exonic | SNV | gain | Benign | missense | < 10 | 1,800,291 | 18.924 | 25.642 |
c.1010-27G > A | Intronic | SNV | normal | Benign | | < 10 | 7,058,826 | 11.328 | 7.735 |