Exome Sequencing Identifies Truncating Mutations in Human SERPINF1 in Autosomal-Recessive Osteogenesis Imperfecta

Abstract

Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder characterized by bone fragility and susceptibility to fractures after minimal trauma. After mutations in all known OI genes had been excluded by Sanger sequencing, we applied next-generation sequencing to analyze the exome of a single individual who has a severe form of the disease and whose parents are second cousins. A total of 26,922 variations from the human reference genome sequence were subjected to several filtering steps. In addition, we extracted the genotypes of all dbSNP130-annotated SNPs from the exome sequencing data and used these 299,494 genotypes as markers for the genome-wide identification of homozygous regions. A single homozygous truncating mutation, affecting SERPINF1 on chromosome 17p13.3, that was embedded into a homozygous stretch of 2.99 Mb remained. The mutation was also homozygous in the affected brother of the index patient. Subsequently, we identified homozygosity for two different truncating SERPINF1 mutations in two unrelated patients with OI and parental consanguinity. All four individuals with SERPINF1 mutations have severe OI. Fractures of long bones and severe vertebral compression fractures with resulting deformities were observed as early as the first year of life in these individuals. Collagen analyses with cultured dermal fibroblasts displayed no evidence for impaired collagen folding, posttranslational modification, or secretion. SERPINF1 encodes pigment epithelium-derived factor (PEDF), a secreted glycoprotein of the serpin superfamily. PEDF is a multifunctional protein and one of the strongest inhibitors of angiogenesis currently known in humans. Our data provide genetic evidence for PEDF involvement in human bone homeostasis.

Osteogenesis imperfecta

“…Osteogenesis imperfecta type VI is caused by reces sive null mutations in SERPINF1, which encodes pigment epithelium derived factor (PEDF). PEDF is an anti angiogenic factor that also interacts with recep tor activator of nuclear factor κβ ligand (RANKL; also known as TNFSF11) pathway, thereby increasing the activity of osteoclasts [83][84][85] (FIG. 4a).…”

Disease gene identification strategies for exome sequencing

“…After this, typically between 150 and 500 private non-synonymous or splicesite variants are prioritized as potential pathogenic variants (see Figures 1 and 2). 4,[28][29][30][31][32][33][34] It is important to emphasize that prioritization may discard the pathogenic variant. A variant that is present at low frequency in a heterozygous state in the normal population may be removed even though it causes disease if present in a homozygous state.…”

“…Homozygous variants can therefore be prioritized by their presence in large homozygous regions of the patient's genome. These regions can be identified by SNP microarrays and used during the prioritization process, 35 but Becker et al 31 recently showed that exome data itself may contain sufficient numbers of informative SNPs to allow reliable homozygosity mapping. In this study, 17 of the 318 private non-synonymous variants observed in the index patient were autosomal homozygous variants, but only three were located in large homozygous regions and the causative mutation was located in the largest of these three.…”

“…Yamaguchi et al 56 Glazov et al 57 Puente et al 58 Gunay-Aygun et al 59 Weedon et al 60 Homozygosity (Figure 3b Becker et al 31 Walsh et al 35 Wang et al 51a Bolze et al 61 Caliskan et al 62 Bilguvar et al 63 O'Sullivan et al 64 Barak et al 65 Hanson et al 66 Shaheen et al 67 Doi et el Only a single experiment is required.…”

Effect of high-dose vitamin D supplementation on bone density in youth with osteogenesis imperfecta: A randomized controlled trial

“…SERPINF1 mutations have been shown to cause premature termination codons and subsequent loss of PEDF expression [46]. How this leads to under mineralized bone in extracellular matrix has yet to be explained.…”