Jana Djakow1, Lenka Kramná1, Lenka Dušátková1, Jiří Uhlík2, Juha-Pekka Pursiheimo3, Tamara Svobodová1, Petr Pohunek1, Ondřej Cinek1. 1. Department of Paediatrics, 2nd Faculty of Medicine, Charles University in Prague and Motol University Hospital, Czech Republic. 2. Department of Histology and Embryology, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic. 3. Department of Medical Biochemistry and Genetics, University of Turku, Turku, Finland.
Abstract
BACKGROUND: Primary ciliary dyskinesia (PCD) is a multigenic autosomal recessive condition affecting respiratory tract and other organs where ciliary motility is required. The extent of its genetic heterogeneity is remarkable. The aim of the study was to develop a cost-effective pipeline for genetic diagnostics using a combination of Sanger and next generation sequencing (NGS). MATERIALS AND METHODS: Data and samples of 33 families with 38 affected subjects with PCD diagnosed in childhood were collected over the territory of the Czech Republic. A panel of 18 PCD causative or candidate genes was implemented into an Illumina TruSeq Custom Amplicon NGS assay, and three ancestral mutations in SPAG1 were screened by conventional Sanger sequencing, which was also used for the confirmation of the NGS results and for the analysis of familial segregation. RESULTS: The causative gene was DNAH5 in 11/33 (33%) probands, SPAG1 in 8/33 (24%), and DNAI1, CCDC40, LRRC6 in one family each. If the high proportion of subjects with bi-allelic ancestral mutations in SPAG1 is corroborated in other Caucasian populations, a simple Sanger sequencing test for these three mutations may serve as an effective pre-screening step, being followed by an NGS panel for other, much larger, PCD genes. CONCLUSIONS: We present a combination of Sanger sequencing with an NGS panel for known and candidate PCD genes, implemented in a moderate-size national collection of patients. This strategy has proven to be cost-effective, rapid and reliable, and was able to detect the causative gene in two thirds of our PCD patients.
BACKGROUND:Primary ciliary dyskinesia (PCD) is a multigenic autosomal recessive condition affecting respiratory tract and other organs where ciliary motility is required. The extent of its genetic heterogeneity is remarkable. The aim of the study was to develop a cost-effective pipeline for genetic diagnostics using a combination of Sanger and next generation sequencing (NGS). MATERIALS AND METHODS: Data and samples of 33 families with 38 affected subjects with PCD diagnosed in childhood were collected over the territory of the Czech Republic. A panel of 18 PCD causative or candidate genes was implemented into an Illumina TruSeq Custom Amplicon NGS assay, and three ancestral mutations in SPAG1 were screened by conventional Sanger sequencing, which was also used for the confirmation of the NGS results and for the analysis of familial segregation. RESULTS: The causative gene was DNAH5 in 11/33 (33%) probands, SPAG1 in 8/33 (24%), and DNAI1, CCDC40, LRRC6 in one family each. If the high proportion of subjects with bi-allelic ancestral mutations in SPAG1 is corroborated in other Caucasian populations, a simple Sanger sequencing test for these three mutations may serve as an effective pre-screening step, being followed by an NGS panel for other, much larger, PCD genes. CONCLUSIONS: We present a combination of Sanger sequencing with an NGS panel for known and candidate PCD genes, implemented in a moderate-size national collection of patients. This strategy has proven to be cost-effective, rapid and reliable, and was able to detect the causative gene in two thirds of our PCDpatients.
Authors: Adam J Shapiro; Stephanie D Davis; Deepika Polineni; Michele Manion; Margaret Rosenfeld; Sharon D Dell; Mark A Chilvers; Thomas W Ferkol; Maimoona A Zariwala; Scott D Sagel; Maureen Josephson; Lucy Morgan; Ozge Yilmaz; Kenneth N Olivier; Carlos Milla; Jessica E Pittman; M Leigh Anne Daniels; Marcus Herbert Jones; Ibrahim A Janahi; Stephanie M Ware; Sam J Daniel; Matthew L Cooper; Lawrence M Nogee; Billy Anton; Tori Eastvold; Lynn Ehrne; Elena Guadagno; Michael R Knowles; Margaret W Leigh; Valery Lavergne Journal: Am J Respir Crit Care Med Date: 2018-06-15 Impact factor: 21.405
Authors: Alexander Ing; Alissa Wlodaver; Dawn Kirschmann; Erica Toledo; Christopher McCabe; Sabah Kadri; Mary Kate McIntyre; Joanne Salazar; Kristina Firestein; Joel Charrow; Victoria Sanders; Theresa Laguna; Kai Lee Yap Journal: Cold Spring Harb Mol Case Stud Date: 2021-02-19
Authors: Dinu Antony; Elif Gulec Yilmaz; Alper Gezdirici; Lennart Slagter; Zeineb Bakey; Helen Bornaun; Ibrahim Cansaran Tanidir; Tran Van Dinh; Han G Brunner; Peter Walentek; Sebastian J Arnold; Rolf Backofen; Miriam Schmidts Journal: Front Genet Date: 2022-04-13 Impact factor: 4.772