{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,5]],"date-time":"2026-10-05T00:14:06Z","timestamp":1791159246085,"version":"4.1.0"},"reference-count":44,"publisher":"BMJ","issue":"12","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J Med Genet"],"accepted":{"date-parts":[[2012,10,1]]},"published-print":{"date-parts":[[2012,12]]},"abstract":"<jats:sec>\n                  <jats:title>Objective<\/jats:title>\n                  <jats:p>To identify disease-causing mutations within coding regions of 11 known NPHP genes (<jats:italic>NPHP1-NPHP11<\/jats:italic>) in a cohort of 192 patients diagnosed with a nephronophthisis-associated ciliopathy, at low cost.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>Mutation analysis was carried out using PCR-based 48.48 Access Array microfluidic technology (Fluidigm) with consecutive next-generation sequencing. We applied a 10-fold primer multiplexing approach allowing PCR-based amplification of 475 amplicons (251 exons) for 48 DNA samples simultaneously. After four rounds of amplification followed by indexing all of 192 patient-derived products with different barcodes in a subsequent PCR, 2\u00d7100 paired-end sequencing was performed on one lane of a HiSeq2000 instrument (Illumina). Bioinformatics analysis was performed using \u2018CLC Genomics Workbench\u2019 software. Potential mutations were confirmed by Sanger sequencing and shown to segregate.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>Bioinformatics analysis revealed sufficient coverage of 30\u00d7for 168\/192 (87.5%) DNA samples (median 449\u00d7) and of 234 out of 251 targeted coding exons (sensitivity: 93.2%). For proof-of-principle, we analysed 20 known mutations and identified 18 of them in the correct zygosity state (90%). Likewise, we identified pathogenic mutations in 34\/192 patients (18%) and discovered 23 novel mutations in the genes <jats:italic>NPHP3<\/jats:italic> (7), <jats:italic>NPHP4<\/jats:italic> (3), <jats:italic>IQCB1<\/jats:italic> (4), <jats:italic>CEP290<\/jats:italic> (7), <jats:italic>RPGRIP1L<\/jats:italic> (1), and <jats:italic>TMEM67<\/jats:italic> (1). Additionally, we found 40 different single heterozygous missense variants of unknown significance.<\/jats:p>\n               <\/jats:sec>\n               <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>We conclude that the combined approach of array-based multiplexed PCR-amplification on a Fluidigm Access Array platform followed by next-generation sequencing is highly cost-efficient and strongly facilitates diagnostic mutation analysis in broadly heterogeneous Mendelian disorders.<\/jats:p>\n               <\/jats:sec>","DOI":"10.1136\/jmedgenet-2012-100973","type":"journal-article","created":{"date-parts":[[2012,11,27]],"date-time":"2012-11-27T05:08:35Z","timestamp":1353992915000},"page":"756-767","source":"Crossref","is-referenced-by-count":105,"title":["High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing"],"prefix":"10.1136","volume":"49","author":[{"given":"Jan","family":"Halbritter","sequence":"first","affiliation":[{"name":"Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katrina","family":"Diaz","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Moumita","family":"Chaki","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan D","family":"Porath","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brendan","family":"Tarrier","sequence":"additional","affiliation":[{"name":"DNA Sequencing Core, University of Michigan, Ann Arbor, Michigan, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clementine","family":"Fu","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jamie L","family":"Innis","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susan J","family":"Allen","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Robert H","family":"Lyons","sequence":"additional","affiliation":[{"name":"DNA Sequencing Core, University of Michigan, Ann Arbor, Michigan, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Constantinos J","family":"Stefanidis","sequence":"additional","affiliation":[{"name":"Pediatric Nephrology, \u2018A. and P. 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