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 (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 (OI; MIM 166200, 166210, 610854, 259420, 166220, 610967, 610968, 610682, 610915, and 259440 for type I to IX of the disease) is a genetic disorder characterized by bone fragility and susceptibility to fractures after minimal trauma. Disease severity ranges from very mild forms without fractures to intrauterine fractures and perinatal lethality.1Rauch F. 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Mutations in all genes known to cause autosomal-dominant and -recessive OI (see above) had been excluded by direct Sanger sequencing of genomic DNA. We therefore decided to follow a genome-wide sequencing approach to identify the cause of the disease in this patient. The study was approved by the Medical Ethics Committee of the Radboud University Nijmegen Medical Centre. Written informed consent to participate in the study and to publish clinical data and radiographs was obtained for the index patient and for all other OI patients described in this paper. We sequenced the exome (∼18,600 genes) of the index patient by using the SureSelect human exome kit (Agilent, Santa Clara, CA, USA) and one-quarter of a SOLiD 4 system sequencing slide (Life Technologies, Carlsbad, CA, USA). We obtained 3.7 Gb of mappable sequence data. Color space reads were mapped to the hg18 reference genome with the SOLiD bioscope software version 1.2, which utilizes an iterative mapping approach. In total, 71% of bases came from the targeted exome, resulting in a mean coverage of 65-fold (Table S1, available online). Single nucleotide variants were subsequently called by the DiBayes algorithm with high call stringency. Ninety-two percent of the targeted exons were covered more than ten times. Small insertions and deletions were detected with the SOLiD Small InDel Tool. Called SNP variants and indels were then combined and annotated with a custom analysis pipeline. Initial quality filtering (>5 variant reads and >15% variant reads) resulted in the identification of 13,487 genetic variants, including 6,298 nonsynonymous changes, in the coding regions or the canonical dinucleotides of the splice sites (Table S2). We applied a prioritization scheme to identify the pathogenic mutation, similar to what was done in recent studies.27Gilissen C. Arts H.H. Hoischen A. Spruijt L. Mans D.A. Arts P. van Lier B. Steehouwer M. van Reeuwijk J. Kant S.G. et al.Exome sequencing identifies WDR35 variants involved in Sensenbrenner syndrome.Am. J. Hum. Genet. 2010; 87: 418-423Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 28Hoischen A. van Bon B.W. Gilissen C. Arts P. van Lier B. Steehouwer M. de Vries P. de Reuver R. Wieskamp N. Mortier G. et al.De novo mutations of SETBP1 cause Schinzel-Giedion syndrome.Nat. Genet. 2010; 42: 483-485Crossref PubMed Scopus (348) Google Scholar, 29Vissers L.E. de Ligt J. Gilissen C. Janssen I. Steehouwer M. de Vries P. van Lier B. Arts P. Wieskamp N. del Rosario M. et al.A de novo paradigm for mental retardation.Nat. Genet. 2010; 42: 1109-1112Crossref PubMed Scopus (590) Google Scholar For a recessive disease, it is possible that pathogenic mutations are listed as benign polymorphisms in dbSNP or in our internal variant database because individuals without the respective phenotype can be heterozygous carriers. However, given the rare incidence of autosomal-recessive OI, we considered such a scenario to be unlikely. We therefore excluded known dbSNP130 variants as well as variants from our in-house variant database, reducing the number of candidates by more than 98% to a total of 318 variants. For further analysis, we applied an autosomal-recessive disease model and assumed that the causal mutation was inherited from a common ancestor because of parental consanguinity. Under this assumption, only 17 autosomal candidate genes with homozygous variants in the index patient remained. To prioritize these candidate genes, we developed an algorithm for unbiased identification of homozygous regions in the genome: we extracted the genotypes of all dbSNP130-annotated autosomal SNPs that had a read coverage of at least 15-fold from the exome sequencing data of the index patient. This resulted in a total of 299,494 genotypes, which increased the number of potential genetic markers for the identification of homozygous regions in the genome more than 17-fold. Variants displaying an identical SNP allele in ≥95% of all reads were considered to be homozygous, SNPs with 30%–70% variation reads were considered to be heterozygous, and SNPs with <30% or with 70%–95% variation reads were considered ambiguous. A genomic region was identified as a homozygous stretch if at least 500 consecutive SNP markers were called as homozygous; there was a maximum tolerance of two heterozygous SNPs per 500 markers (to account for possible sequencing or mapping errors). In the dataset of the index patient, this algorithm identified seven homozygous regions on chromosomes 1, 6, 11, and 17 (Table S3 and Figure S1). Only three of the 17 homozygous variants in the index patient's exome were located in one of these homozygous regions: two missense variants on chromosome 1 (p.Gln361Arg [c.1082A>G] in LPHN2 [RefSeq accession number NM_012302.2] and p.Thr1799Met [c.5396C>T] in INADL [MIM 603199; NM_176877.2]) and a stop mutation (p.Tyr232X [c.696C>G]) affecting SERPINF1 (MIM 172860; NM_002615.4) on chromosome 17p13.3 (Table S4). The stop mutation was embedded into a homozygous stretch spanning 2.99 Mb (Figure 1D ). We regarded SERPINF1 as the best candidate for further evaluation. Validation by Sanger sequencing confirmed that the c.696C>G (p.Tyr232X) mutation was indeed present in a homozygous state in the index patient (Figure 1C). We also tested DNA samples of the patient's parents, his affected brother (patient 2), and his two healthy sisters. Only the affected brother carried the mutation in a homozygous state, whereas both parents and both sisters were heterozygous carriers, compatible with autosomal-recessive inheritance of the disorder in the family (Figure S2A). The mutation was not detected on 460 control chromosomes from individuals with the same regional ancestry as the index patient. These results confirmed that the identified mutation is not a rare polymorphism in the United Arab Emirates population. No other SERPINF1 nonsense variant was observed in more than 120 in-house exomes, indicating the rare nature of loss-of-function mutations in this gene. As a next step, we performed Sanger sequencing of all seven coding SERPINF1 exons on genomic DNA samples obtained from two unrelated Turkish patients (patients 3 and 4), an Iraqi patient, and an Italian patient, all clinically diagnosed with OI. These four patients had no detectable COL1A1 or COL1A2 mutations. The Italian patient had moderate OI (type IV in the Sillence classification23Sillence D.O. Senn A. Danks D.M. Genetic heterogeneity in osteogenesis imperfecta.J. Med. Genet. 1979; 16: 101-116Crossref PubMed Scopus (1470) Google Scholar), whereas the other patients suffered from a severe form of OI, comparable to the course of the disease in the Arabian index family. Only the Turkish patients were born to consanguineous parents. In these two patients, we identified truncating SERPINF1 mutations: patient 3 was homozygous for the insertion c.324_325dupCT (p.Tyr109SerfsX5) in exon 4, resulting in a frameshift with a premature stop codon (Figure 2 and Figure S3A). Patient 4 carried the homozygous stop mutation c.1132C>T (p.Gln378X) in exon 8 (Figure 2 and Figure S3B). The patients' parents (as obligate carriers) and two healthy sisters of patient 3 were heterozygous for the respective mutation (Figures S2B and S2C). The insertion mutation c.324_325dupCT was excluded in 460 Turkish control chromosomes, and the stop mutation c.1132C>T was not present on 272 Turkish control alleles, demonstrating that neither of these two sequence changes is a rare polymorphism. The Iraqi and the Italian patient were negative for mutations in the coding region of SERPINF1. SERPINF1 encodes pigment-epithelium-derived factor (PEDF), a 50 kDa secreted glycoprotein of the serpin superfamily. The p.Tyr232X and p.Tyr109SerfsX5 mutations can be predicted to cause null alleles because of nonsense-mediated mRNA decay (NMD). We found experimental evidence for NMD of the mutated transcript in peripheral blood by direct sequencing of a PCR-amplified cDNA fragment encompassing the frameshift mutation p.Tyr109SerfsX5 (Figure S4). In the father of patient 3, the cDNA sequence of the mutated allele was only detectable as a faint background signal behind the wild-type sequence, whereas Sanger sequencing on genomic DNA derived from this obligate mutation carrier displayed equally strong signals for the mutated and the wild-type allele. This observation suggests that the number of mutated transcripts serving as templates for the preceding RT-PCR reaction is strongly reduced in comparison to the number of transcripts with the wild-type sequence. The c.1132C>T (p.Gln378X) mutation is located in the last coding exon of SERPINF1, 123 bp upstream of the regular stop codon (Figure 2). The corresponding transcript should therefore escape the NMD machinery.30Nagy E. Maquat L.E. A rule for termination-codon position within intron-containing genes: When nonsense affects RNA abundance.Trends Biochem. Sci. 1998; 23: 198-199Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar However, the C terminus of PEDF (amino acids 384–415) represents one of the four highly conserved regions of the protein.31Tombran-Tink J. Aparicio S. Xu X. Tink A.R. Lara N. Sawant S. Barnstable C.J. Zhang S.S. PEDF and the serpins: Phylogeny, sequence conservation, and functional domains.J. Struct. Biol. 2005; 151: 130-150Crossref PubMed Scopus (34) Google Scholar Extensive mutagenesis studies with cDNA expression vectors of human PEDF revealed that any deletion of the C terminus, which encompasses more than the four C-terminal amino acids (amino acids 415–418), abolishes secretion of the protein by transiently transfected mammalian cells.32Shao H. Schvartz I. Shaltiel S. Secretion of pigment epithelium-derived factor. Mutagenic study.Eur. J. Biochem. 2003; 270: 822-831Crossref PubMed Scopus (18) Google Scholar Moreover, the premature stop codon identified in patient 3 resides within the reactive center loop (RCL) of PEDF (Figure 2). An in vitro deletion of a segment of the RCL (Pro373–Ala380) as well as substitutions of Gly376 and Leu377 by alanine resulted in abrogation of PEDF secretion, suggesting that the RCL is important for the interaction of PEDF with the secretory apparatus.32Shao H. Schvartz I. Shaltiel S. Secretion of pigment epithelium-derived factor. Mutagenic study.Eur. J. Biochem. 2003; 270: 822-831Crossref PubMed Scopus (18) Google Scholar The p.Gln378X mutation therefore most likely represents a functional null allele. This interpretation is supported by the observation that the phenotypic consequences of the p.Gln378X mutation are indistinguishable from those of the p.Tyr232X and p.Tyr109SerfsX5 mutations (see below). The four affected children with SERPINF1 mutations (referred to as patients 1–4) exhibited similar clinical symptoms and disease severity, currently classified as OI type III. There were no intrauterine fractures reported, and birth length and weight were normal. Dentinogenesis imperfecta was not present. Formal hearing tests were not performed, but there were no clinical signs of hearing impairment. The sclerae were grayish. No ophthalmological problems were seen. The serum levels showed moderately elevated levels for alkaline phosphatase and procollagen-1-C-peptide and moderately increased values for the osteoclast marker deoxypyridinoline (Table 1). Compared to other OI patients, these biochemical findings are not uncommon and can also be found in patients with COL1A1 or COL1A2 mutations. All four patients had fractures of long bones and severe vertebral compression fractures, and resulting deformities were seen as early as in their first year of life. Some of the fractured bones required surgical corrections of deformities and insertions of telescopic rods. Figure 3, Figure 4 show a selection of radiographs of patients 3 and 4. The radiological findings were typical for patients with severe forms of OI. There were no distinctive radiological abnormalities that would allow a further diagnostic subclassification. In Figure S5, we present whole-body DXA scans taken of patients 2–4 during their last bone-mineral-density measurement. These DXA scans give an impression of the degree of skeletal deformity.Table 1Clinical Features of Patients with Recessive OI and Mutations in SERPINF1FindingsPatient 1Patient 2Patient 3Patient 4OI typeaAccording to the Sillence classification.23IIIIIIIIIIIIAge at first visit (years)1 8/1211 4/126/121 1/12Age at last visit (years)3 2/1216 3/128 7/125 11/12Age at start of bisphosphonate treatment1 8/1211 4/127/121 1/12Birth length and birth weightnormalnormalnormalnormalConfirmed prenatal fracturesnonononoAge at first fracture (months)4unknown66Color of scleragrayishgrayishgrayishgrayishDentinogenesis imperfectanonononoHypermobility of jointsnonononoHearing impairmentnonononoOld fractures of extremitiesbAt first presentation.yesyesyesyesVertebral fracturesbAt first presentation.multiplemultiplemultiplemultipleBowing of upper extremitiesbAt first presentation.moderateseveremildmoderateBowing of lower extremitiesbAt first presentation.severeseveremoderatemoderateShortening of upper extremitiesbAt first presentation.moderateseveremildmildShortening of lower extremitiesbAt first presentation.moderateseveremoderatemildWeight at first visit kg/BMI (SD)9/−0.540/+3.27.5/−1.411.9/+1.8Weight at last visit kg/BMI (SD)9.9/1.145/+3.517/−1.414/−1.5Length at first visit cm/(SD)not done98/−8.867/0.278.3/−0.7Length at last visit cm/(SD)76/−5.6101/−10.2110/−3.8101.5/−2.8Retarded gross motor functionsyesyesyesyesMobility at first visit (BAMFcBrief assessment of motor function.60)2104Mobility at last visit (BAMFcBrief assessment of motor function.60)4266IntelligencenormalnormalnormalnormalCalcium levelbAt first presentation.normalnormalnormalnormalAlkaline phosphatase at first visit [U/l]353283295348Alkaline phosphatase at last visit [U/l]328154269344Procollagen-1-C-peptide (marker for osteoblastic activity) [μg/l]bAt first presentation.720430200375Deoxypyridinoline/creatinine (marker for osteoclastic activity) [nM/mM] at first visitnot done86.5118.9not doneDeoxypyridinoline/creatinine (marker for osteoclastic activity) [nM/mM] at last visit64.960.163.165.3First available bone density, DXA whole-body measurement (Z score)−6.1 (lumbar spine)−3.7−2.7−2.0a According to the Sillence classification.23Sillence D.O. Senn A. Danks D.M. Genetic heterogeneity in osteogenesis imperfecta.J. Med. Genet. 1979; 16: 101-116Crossref PubMed Scopus (1470) Google Scholarb At first presentation.c Brief assessment of motor function.60Cintas H.L. Siegel K.L. Furst G.P. Gerber L.H. Brief assessment of motor function: Reliability and concurrent validity of the Gross Motor Scale.Am. J. Phys. Med. Rehabil. 2003; 82: 33-41Crossref PubMed Scopus (25) Google Scholar Open table in a new tab Figure 4Radiological Features of Patient 4Show full caption(A) Hand radiogram at the age of 6 years shows no signs of fractures or skeletal dysplasia and only a mild delay of bone age.(B) The right arm at the age of 5.4 years shows severe deformities of the humerus with multiple old fractures and callus formation in the diaphysis.(C) Left hemithorax at the age of 4.3 years displays no deformities or fractures of rips.(D) Right leg at the age of 2.9 years after surgical rodding with an intramedullar telescopic rod because of a fracture.(E) Fractured right leg 3 months earlier. Also visible are the epiphyseal lines resulting from cyclic intravenous bisphosphonate treatment.(F) Left leg with a fracture after minimal trauma at the age of 2.2 years.(G) Left leg a few weeks later after surgical treatment with an intramedullar telescopic rod.View Large Image Figure ViewerDownload Hi-res
