Coucke, Paul

Cueto-González, AM

De Paepe, Anne

Guillemyn, Brecht

Kayserlili, H

Malfait, Fransiska

Menten, Björn

Rosseel, Toon

Symoens, Sofie

Syx, Delfien

Van Maldergem, L

English

Ghent University Academic Bibliography

10x 10xImplementation of an in-house designed skeletal dysplasia gene panel as a first screening step to diagnose unsolved osteogenesis imperfecta (–like) patientsThe pace of disease-related gene discovery hasaccelerated phenomenally in recent years due toadvances of next generation sequencing (NGS)technologies, such as whole exome sequencing(WES). In the field of skeletal dysplasias, particularlyfor osteogenesis imperfecta (OI), NGS added aremarkable number of disease loci, therebyexpanding the brittle bone disease to apredominantly collagen-related disorder. However,for a small subset of OI patients the genetic causeremains elusive. We designed a skeletal dysplasiagene panel and evaluated the detection rate in acohort of unsolved OI (-like) patients.Twenty-one OI-like patients, in who no type Icollagen defect was detected, were selectedbased on clinical severity (most patients hadmultiple fractures) and positive familial anamnesis(patients with a consanguineous background wereprioritized during the selection procedure). WESsample prep was performed using the AgilentSureSelect XT target enrichment system.Figure 1: Whole exome sequencing, facts and figures.1,2Guillemyn B.1, Syx D.1, Rosseel T.1, Kayserili H.2, Cueto-González AM.3, Van Maldergem L4, Menten B1, De Paepe A.1, Coucke P.1, Malfait F.1, Symoens S.1Targeted sequencingWES is perhaps the most widely used targeted sequencing methodExome vs genomeThe exome, the protein-coding region of the human genome, accounts for less than 2% of the genome but contains ~85% of known disease-related variantsSuccess rateIn clinical setting, reported detection rates for deleterious mutations range from 25 to 50% WES libraries were sequenced on Illumina platforms, data wereanalysed by SeqPlorer (an in-house developed pipeline, whichintegrates population database information/qualityscores/coverage data and in silico prediction algorithms).The generated data was mapped against a newly designedskeletal dysplasia gene panel, including 566 genes. This genepanel includes known loci for heritable skeletal disorders, butalso candidate genes for bone fragility are included (which arethe result of GWAS and animal studies linking aberrant BMDscores to candidate genes for osteoporosis).3,4,5,6,7Figure 2: Methods used in this study.Clinical phenotypeLEPRE1 – case (male, 2y)Six fractures in first 2 years, short stature, triangular face, brachiocephaly, flat occiput, psychomotor retardationALPL – case (female, 1y)No fractures, short stature, blue sclerae, dimples, bowed humeriCTSK – case (female, 16y)Multiple fractures, osteopetrosis, mild short stature, long palpebral fissure, pinched nose with long columella, blue sclerae, dentinogenesis imperfectaProtein changeClinical diagnosisVariant classification DEOGEN2 8,9p.[(Leu149Arg)];[(Leu149Arg)] p.[(His482Asn)];[(His482Asn)] p.[(Arg241*)];[(Arg241*)]autosomal recessive (AR) OI AR hypophosphatasia AR osteopetrosisDEOGEN2 is not applicable for nonsense variantsCLASS 3 CLASS 3 CLASS 5 Figure 3: Overview of the 3 solved cases.0X 10X20X30X40X50X60X70X80X90X100X100%  98%    96%    92%    85%   78%    70%    62%    53%    46%   38%Figure 4: Averageskeletal dysplasia genepanel coverage of the21 exomes included inthis study. An exome isconsidered as ‘wellsequenced’ if morethan 95% of the bases is20x covered. Error bars,SD.Prolyl-3-hydroxylase-1 (P3H1) is required for proper collagen biosynthesis, folding, and assemblyCathepsin K is a cysteine protease which digests bone matrix proteins during bone resorption.Liver / bone / kidney alkaline phosphatases are membrane-bound glycoproteins, playing a role in bone mineralization.Protein functionReferences1 Van Dijk EL et al, Trends Genet, 2014: Ten years of next-generation sequencing technology.2 Atwal PS et al, Genet Med, 2014: Clinical whole-exome sequencing: are we there yet?3 Bonafe L et al, Am J Med Genet Part A, 2015: Nosology and classification of genetic skeletal disorders: 2015 revision4 Rocha-Braz M.G.M. et al, Arch Endocrinol Metab., 2016: Genetics of osteoporosis: searching for candidate genes for bone fragility5 Brent Richards J. et al, Nat Rev Genet, 2012: Genetics of osteoporosis from genome-wide association studies: advances and challenges6 Urano T et al, J Clin Endocrinol Metab., 2012: GPR98/Gpr98 gene is involved in the regulation of human and mouse bone mineral density.7 JP Kemp et al, Nat Genet, 2017: Identification of 153 new loci associated with heel bone mineral density and functional involvement of GPC6 in osteoporosis8 Raimondi et al, Nucleic Acids Research, 2017: DEOGEN2: prediction and interactive visualization of single amino acid variant deleteriousness in human proteins.9 Tanyalcin I et al, Computing in Science & Engineering, 2018: Lexicon Visualization Library and JavaScript for Scientific Data Visualization.Author affiliations1 Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium2 University of Istanbul, Medical Genetics, Millet Cad. Capa, 34390, Istanbul, Turkey3 Vall D’Hebron Hospital Materno Infantil, Clinical Genetics, 08035 Barcelona, Spain4 Centre Hospitalier Universitaire de Besancon – Génétique, 25030 Besancon Cedex, FranceContact informationBrecht.Guillemyn@UGent.beSofie.Symoens@UGent.beSearching for the needle in the haystackAn average of 41636 variants were generated using the GRCh38/hg38 build across the 21 exome samples included in this studyApplicationsExome sequencing can efficiently identify coding variants across a wide range of applications, including population genetics, genetic disease, and cancer studiesConclusions and future perspectivesOur findings show that the implementation of this skeletal dysplasia gene panel is a powerful firstscreening step in molecularly diagnosing OI(-like) patients, especially for those patients whom aredifficult to diagnose due to phenotypically overlapping clinical symptoms.With regards to the 18 unsolved exome cases: with the use of SeqPlorer, we were able to generatea shortlist of 71 variants in total, of which further prioritization, confirmational sanger sequencing,segregational analysis and functional studies will have to aid in the elucidation of these cases.Besides the used seqplorer pipeline, we are currently testing tools in order to pick up copy numbervariations from the same exome datasets (such as cn.MOPS, CoNIFER and ExomeDepth).

Implementation of an in-house designed skeletal dysplasia gene panel as a first screening step to diagnose unsolved osteogenesis imperfecta(-like) patients

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Implementation of an in-house designed skeletal dysplasia gene panel as a first screening step to diagnose unsolved osteogenesis imperfecta(-like) patients

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