Adapting SureSelect enrichment protocol to the Ion Torrent S5 platform in molecular diagnostics of craniosynostosis

Abstract

Obtaining reliable and high fidelity next-generation sequencing (NGS) data requires to choose a suitable sequencing platform and a library preparation approach, which both have their inherent assay-specific limitations. Here, we present the results of successful adaptation of SureSelect hybridisation-based target enrichment protocol for the sequencing on the Ion Torrent S5 platform, which is designed to work preferably with amplicon-based panels. In our study, we applied a custom NGS panel to screen a cohort of 16 unrelated patients affected by premature fusion of the cranial sutures, i.e. craniosynostosis (CS). CS occurs either as an isolated malformation or in a syndromic form, representing a genetically heterogeneous and clinically variable group of disorders. The approach presented here allowed us to achieve high quality NGS data and confirmed molecular diagnosis in 19% of cases, reaching the diagnostic yield similar to some of the published research reports. In conclusion, we demonstrated that an alternative enrichment strategy for library preparations can be successfully applied prior to sequencing on the Ion Torrent S5 platform. Also, we proved that the custom NGS panel designed by us represents a useful and effective tool in the molecular diagnostics of patients with CS.

Figure 1

Comparison of per-base coverage depth for all samples. Additional horizontal line indicates 95% of total bases in panel target regions.

Figure 2

Clinical characteristics at the age of 12 months (a,b) as well as molecular results of Patient 1 (c,d). Patient 1, in addition to sagittal craniosynostosis, maxillary hypoplasia, high palate and proptosis, presented with broad halluces and skin syndactyly of 2^nd and 3^rd toes (a). X-ray of the feet showed small hypoplastic middle phalanges of all toes, relative widening of 1^st metatarsals and broadening of phalangeal bones forming halluces, and no bone syndactyly of 2^nd and 3^rd toes (b) Representation of the heterozygous FGFR2 deleterious variant c.868T > G p.Trp290Gly detected in Patient 1 by means of targeted next-generation sequencing (c) and validation studies of the proband and parental testing of the FGFR2 gene with the use of Sanger sequencing (d). Pathogenic variant c.868T > G p.Trp290Gly was confirmed in the index case and excluded in his unaffected parents, clearly indicating a de novo occurrence.

Figure 3

Clinical characteristics at the age of 2 years (a) and 6 years (b,c) as well as molecular results of Patient 7 (d,e). Patient 7 presented with complex craniosynostosis involving sagittal and bilateral coronal synostosis, dolichocephaly, macrocephaly, prominent forehead (a), flat face, proptosis (full facial picture not shown),brachydactyly and broad halluces (b,c). Representation of the heterozygous FGFR2 deleterious variant c.1694A > G p.Glu565Gly unraveled in Patient 7 by means of targeted next-generation sequencing (d) and validation studies of the proband and parental testing of the FGFR2 gene with the use of Sanger sequencing (e). Pathogenic variant c.1694A > G p.Glu565Gly was confirmed in the index case and excluded in his unaffected parents, clearly indicating a de novo occurrence.

Figure 4

Clinical characteristics of Patient 15 at the age of 11 (a–d) and 9.5 years (e,f) as well as molecular results of the patient (g-j). Patient 15 presented with complex craniosynostosis composed of bilateral coronal synostosis (complete right-sided and partial left-sided) as well as partial left-sided lambdoid synostosis shown in 3D modelling of the skull (a–c). CT scan of the head (d). Coronal sutures are prematurely fused. The right coronal suture is completely fused (a), while the left one is only partially fused (b); consequently, there is marked enlargement of the anterior fontanelle and widening of the sagittal suture, (a,c). Asymmetry of the skull and brain, including lateral ventricles, and enlargement of left subarachnoid space seen on horizontal section (d). Limb defect clinically recognized as bilateral split foot malformation with syndactyly of the remaining toes, extremely short and hypoplastic thumbs and 5^th fingers, short 5^th metacarpals and valgus deformity of the right 2^nd finger (e,f). Representation of the compound heterozygous RECQL4 deleterious variants c.308C > T p.Pro103Leu and 3062G > A p.Arg1021Gln detected in Patient 15 by means of targeted next-generation sequencing (g,i). Both pathogenic variants were confirmed with the use of Sanger sequencing (h,j).