High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing

Halbritter, Jan; Diaz, Katrina A.; Chaki, Moumita; Porath, Jonathan D.; Tarrier, Brendan; Fu, Clementine; Innis, Jamie L.; Allen, Susan J.; Lyons, Robert H.; Stefanidis, Constantinos J.; Omran, Heymut; Soliman, Neveen A.; Otto, Edgar A.

doi:10.1136/jmedgenet-2012-100973

Cited by 109 publications

(144 citation statements)

References 45 publications

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“…As previously published, median coverage values .203 are sufficient to exclude false-negative results in high-throughput exon sequencing (5,6). In this study, only eight of 143 individuals (5.7%), and 37 of 518 amplicons (7.1%) had a median coverage ,203.…”

Section: Coverage Statisticssupporting

confidence: 57%

“…Mutation analysis was performed using a barcoded multiplex PCR-based approach, as previously described (5,6). We designed 518 target-specific primers for 381 coding exons and the adjacent splice sites of 30 genes that are known monogenic causes of NL/NC (defined by OMIM; www.ncbi.nlm.nih.gov/omim).…”

Section: Mutation Analysismentioning

confidence: 99%

“…Amplicon sizes were chosen to range from 250 to 300 bp (primer sequences are available from the authors). The use of barcoded multiplex PCR (Fluidigm 48.48-Access Arrays system) allowed parallel amplification of all 518 amplicons in 48 individuals at a time (5,6). Subsequently, the pooled libraries were sequenced on an Illumina MiSeq instrument using the v2 chemistry.…”

Section: Mutation Analysismentioning

confidence: 99%

“…However, through the availability of high-throughput multiplex PCR and next-generation sequencing, rapid mutation analysis of multiple genes in large cohorts has become an efficient and cost-effective screening method (5)(6)(7).…”

Section: Introductionmentioning

confidence: 99%

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Prevalence of Monogenic Causes in Pediatric Patients with Nephrolithiasis or Nephrocalcinosis

Braun¹,

Lawson²,

Gee

et al. 2016

Clinical Journal of the American Society of Nephrology

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108

110

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Background and objectives Nephrolithiasis is a prevalent condition that affects 10%-15% of adults in their lifetime. It is associated with high morbidity due to colicky pain, the necessity for surgical intervention, and sometimes progression to CKD. In recent years, multiple monogenic causes of nephrolithiasis and nephrocalcinosis have been identified. However, the prevalence of each monogenic gene in a pediatric renal stone cohort has not yet been extensively studied.Design, setting, participants, & measurements To determine the percentage of cases that can be explained molecularly by mutations in one of 30 known nephrolithiasis/nephrocalcinosis genes, we conducted a high-throughput exon sequencing analysis in an international cohort of 143 individuals ,18 years of age, with nephrolithiasis (n=123) or isolated nephrocalcinosis (n=20). Over 7 months, all eligible individuals at three renal stone clinics in the United States and Europe were approached for study participation.Results We detected likely causative mutations in 14 of 30 analyzed genes, leading to a molecular diagnosis in 16.8% (24 of 143) of affected individuals; 12 of the 27 detected mutations were not previously described as disease causing (44.4%). We observed that in our cohort all individuals with infantile manifestation of nephrolithiasis or nephrocalcinosis had causative mutations in recessive rather than dominant monogenic genes. In individuals who manifested later in life, causative mutations in dominant genes were more frequent. ConclusionsWe present the first exclusively pediatric cohort examined for monogenic causes of nephrolithiasis/ nephrocalcinosis, and suggest that important therapeutic and preventative measures may result from mutational analysis in individuals with early manifestation of nephrolithiasis or nephrocalcinosis.

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Section: Coverage Statisticssupporting

confidence: 57%

Section: Mutation Analysismentioning

confidence: 99%

Section: Mutation Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Prevalence of Monogenic Causes in Pediatric Patients with Nephrolithiasis or Nephrocalcinosis

Braun¹,

Lawson²,

Gee

et al. 2016

Clinical Journal of the American Society of Nephrology

Self Cite

108

110

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“…Black line indicates the correlation of PCOLCE2 mRNA level with GFR. group in a rare disease cohort (Halbritter et al 2012;Gadegbeku et al 2013).…”

Section: Discussionmentioning

confidence: 99%

Defining cell-type specificity at the transcriptional level in human disease

et al. 2013

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Cell-lineage–specific transcripts are essential for differentiated tissue function, implicated in hereditary organ failure, and mediate acquired chronic diseases. However, experimental identification of cell-lineage–specific genes in a genome-scale manner is infeasible for most solid human tissues. We developed the first genome-scale method to identify genes with cell-lineage–specific expression, even in lineages not separable by experimental microdissection. Our machine-learning–based approach leverages high-throughput data from tissue homogenates in a novel iterative statistical framework. We applied this method to chronic kidney disease and identified transcripts specific to podocytes, key cells in the glomerular filter responsible for hereditary and most acquired glomerular kidney disease. In a systematic evaluation of our predictions by immunohistochemistry, our in silico approach was significantly more accurate (65% accuracy in human) than predictions based on direct measurement of in vivo fluorescence-tagged murine podocytes (23%). Our method identified genes implicated as causal in hereditary glomerular disease and involved in molecular pathways of acquired and chronic renal diseases. Furthermore, based on expression analysis of human kidney disease biopsies, we demonstrated that expression of the podocyte genes identified by our approach is significantly related to the degree of renal impairment in patients. Our approach is broadly applicable to define lineage specificity in both cell physiology and human disease contexts. We provide a user-friendly website that enables researchers to apply this method to any cell-lineage or tissue of interest. Identified cell-lineage–specific transcripts are expected to play essential tissue-specific roles in organogenesis and disease and can provide starting points for the development of organ-specific diagnostics and therapies.

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Renal‐hepatic‐pancreatic dysplasia: A sibship with skeletal and central nervous system anomalies and NPHP3 mutation

Copelovitch

O’Brien

Guttenberg

et al. 2013

American J of Med Genetics Pt A

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We report on five consecutive sibs three with fatal renal-hepatic-pancreatic dysplastic (RHPD) syndrome and two pregnancies ending in early abortion. Three of the fetuses reached term and two survived for 15 and 58 days. They had diffusely cystic kidneys with absence of the distal collecting tubules, hepatic fibrosis, bile duct paucity, and pancreatic fibrosis with irregularly dilated ducts. These findings correspond to many of those reported by Ivemark et al. [Ivemark et al. (1959); Acta Paediat Scand 48: 1-11] as part of the RHPD syndrome. There are several notable differences in this family: one patient had hypocalvaria and a choroid plexus cyst at the right foramen of Luschka, multiple bone abnormalities including widened growth plates and abnormal development of the trabeculae of the ribs, "handle-bar" clavicles, wedge defects of the inferior margin of several thoracic vertebrae; the second patient had hypocalvaria and abnormally developed brain with bilateral exposure of the insulae; and a third patient had anencephaly. Mutational analysis of the two who survived beyond post-delivery demonstrated compound heterozygous novel frameshift mutations in the nephronophthisis type 3 gene (NPHP3).

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High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed barcoded array-based PCR amplification and next-generation sequencing

Abstract: 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.

Cited by 109 publications

References 45 publications

Prevalence of Monogenic Causes in Pediatric Patients with Nephrolithiasis or Nephrocalcinosis

Prevalence of Monogenic Causes in Pediatric Patients with Nephrolithiasis or Nephrocalcinosis

Defining cell-type specificity at the transcriptional level in human disease

Renal‐hepatic‐pancreatic dysplasia: A sibship with skeletal and central nervous system anomalies and NPHP3 mutation

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