title: A labor and cost effective next generation sequencing of PKHD1 in autosomal recessive polycystic kidney disease patients.
abstract: The Sanger sequencing of patients with recessive polycystic kidney disease is challenging due to the length and heterogeneous mutational spectrum of the PKHD1 gene. Next generation sequencing (NGS) might thus be of special interest to search for PKHD1 mutations. The study involved a total of 22 patients with autosomal recessive polycystic kidney disease (ARPKD) and 8 parents of non-available ARPKD patients. Five pools of 6 samples each were sequenced with the Personal Genome Machine (PGM, Ion Torrent). For each DNA pool, a total of 109 fragments that covered the entire PKHD1 coding sequence were amplified in only two tubes followed by library preparation and NGS with the PGM. To validate the technique, each pool contained the DNA of at least one patient with known mutation. The putative mutations identified in each pool were confirmed and assigned to specific individuals through Sanger sequencing. All but one of the 109 amplicons were successfully read, and we identified the two PKHD1 mutations in 11 of the ARPKD cases, one mutation in 9 patients, and no mutation in only 2 patients. Six of the 8 parents from non-available patients were mutation carriers. The reported procedure would facilitate the large scale analysis of PKHD1 with a significant reduction in cost and labor.
authors.affiliation: Genética-Laboratorio de Medicina, Hospital Universitario Central Asturias, Oviedo, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Tavira
authors.affiliation: Genética-Laboratorio de Medicina, Hospital Universitario Central Asturias, Oviedo, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Gómez
authors.affiliation: Pediatría, Hospital Universitario Central Asturias, Oviedo, Spain; Departamento de Medicina, Universidad de Oviedo, Oviedo, Spain.
authors.forename: Serafín
authors.initials: S
authors.lastname: Málaga
authors.affiliation: Pediatría, Hospital Universitario Central Asturias, Oviedo, Spain; Departamento de Medicina, Universidad de Oviedo, Oviedo, Spain.
authors.forename: Fernando
authors.initials: F
authors.lastname: Santos
authors.affiliation: Genética-Laboratorio de Medicina, Hospital Universitario Central Asturias, Oviedo, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Fernández-Aracama
authors.affiliation: Genética-Laboratorio de Medicina, Hospital Universitario Central Asturias, Oviedo, Spain.
authors.forename: Belén
authors.initials: B
authors.lastname: Alonso
authors.affiliation: Genética-Laboratorio de Medicina, Hospital Universitario Central Asturias, Oviedo, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Iglesias
authors.affiliation: Genética-Laboratorio de Medicina, Hospital Universitario Central Asturias, Oviedo, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Benavides
authors.affiliation: Genética-Laboratorio de Medicina, Hospital Universitario Central Asturias, Oviedo, Spain.
authors.forename: Inés
authors.initials: I
authors.lastname: Hernando
authors.affiliation: Genética-Laboratorio de Medicina, Hospital Universitario Central Asturias, Oviedo, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Plasencia
authors.affiliation: Genética-Laboratorio de Medicina, Hospital Universitario Central Asturias, Oviedo, Spain.
authors.forename: Victoria
authors.initials: V
authors.lastname: Alvarez
authors.affiliation: Genética-Laboratorio de Medicina, Hospital Universitario Central Asturias, Oviedo, Spain; Departamento de Medicina, Universidad de Oviedo, Oviedo, Spain; Red de Investigación Renal (REDINREN), Madrid, Spain; Fundación Renal I. Alvarez de Toledo, Madrid, Spain. Electronic address: eliecer.coto@sespa.princast.es.
authors.forename: Eliecer
authors.initials: E
authors.lastname: Coto
mesh_terms: D001483:Base Sequence; D005260:Female; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007668:Kidney / Q000473:pathology; D008297:Male; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D007690:Polycystic Kidney Diseases / Q000235:genetics*; D011956:Receptors, Cell Surface / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: C451750:PKHD1 protein, human; D011956:Receptors, Cell Surface
keywords: Genetic counseling; Ion Torrent sequencing; Next generation sequencing; PKHD1 gene; Polycystic kidney disease