Whole-genome sequencing overcomes pseudogene homology to diagnose autosomal dominant polycystic kidney disease

Mallawaarachchi, Amali; Hort, Yvonne; Cowley, Mark J.; McCabe, Mark J.; Minoche, André E.; Dinger, Marcel E.; Shine, John; Furlong, Timothy J.

doi:10.1038/ejhg.2016.48

Cited by 72 publications

(79 citation statements)

References 31 publications

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“…It was argued that increasing the sequencing depth was insufficient to overcome the limitations and pitfalls of short‐read NGS approaches (Eisenberger et al., ; Qi et al., ). Similar to these short‐read NGS strategies (Eisenberger et al., ; Mallawaarachchi et al., ; Qi et al., ; Rossetti et al., ; Tan et al., ; Trujillano et al., ), our targeted approach combined with multiplexed sequencing can further accelerate ADPKD diagnostics, compared with the labor‐intensive Sanger sequencing (Rossetti et al., ; Tan et al., ). Despite the rather limited sample size, sufficient numbers were included in this study for a methodology evaluation.…”

Section: Discussionmentioning

confidence: 99%

“…In recent years, several attempts have been made to replace the standard ADPKD diagnostics by NGS approaches that would improve the screening of PKD1 gene (Eisenberger et al., ; Mallawaarachchi et al., ; Qi et al., ; Rossetti et al., ; Tan et al., ; Trujillano et al., ). These screenings were based on analyzing WGS or WES data (Mallawaarachchi et al., ; Qi et al., ), on the enrichment of PKD1 using LR‐PCR (Rossetti et al., ; Tan et al., ), or the hybridization capture of PKD1 (Eisenberger et al., ; Trujillano et al., ). Two of these studies were performed on short‐read NGS using targeted enrichment of PKD1 or PKD2 genes by LR‐PCR (Rossetti et al., ; Tan et al., ).…”

Section: Discussionmentioning

confidence: 99%

“…Several attempts have been made to improve the screening of PKD1 gene by using short‐read NGS approaches to replace the standard diagnostics based on Sanger sequencing and multiplex ligation‐dependent probe amplification (MLPA) assays (Eisenberger et al., ; Mallawaarachchi et al., ; Qi et al., ; Rossetti et al., ; Tan et al., ; Trujillano et al., ). These strategies provided a clear diagnosis with high sensitivity and specificity (97%–100%) for 115 out of 183 (Rossetti et al., ), 16 out of 25 (Tan et al., ), 10 out of 12 (Trujillano et al., ), 35 out of 55 (Eisenberger et al., ), and 24 out of 28 (Mallawaarachchi et al., ) screened ADPKD patients. Duplicated and high GC content genomic regions, such as that of PKD1 gene, can lead to ambiguous identification of variants when analyzed with short‐read NGS strategies (Lee & Schatz, ).…”

Section: Introductionmentioning

confidence: 99%

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DetectingPKD1variants in polycystic kidney disease patients by single-molecule long-read sequencing

et al. 2017

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A genetic diagnosis of autosomal‐dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC content, and homology of the PKD1 gene with six pseudogenes. Short‐read next‐generation sequencing approaches, such as whole‐genome sequencing and whole‐exome sequencing, often fail at reliably characterizing complex regions such as PKD1. However, long‐read single‐molecule sequencing has been shown to be an alternative strategy that could overcome PKD1 complexities and discriminate between homologous regions of PKD1 and its pseudogenes. In this study, we present the increased power of resolution for complex regions using long‐read sequencing to characterize a cohort of 19 patients with ADPKD. Our approach provided high sensitivity in identifying PKD1 pathogenic variants, diagnosing 94.7% of the patients. We show that reliable screening of ADPKD patients in a single test without interference of PKD1 homologous sequences, commonly introduced by residual amplification of PKD1 pseudogenes, by direct long‐read sequencing is now possible. This strategy can be implemented in diagnostics and is highly suitable to sequence and resolve complex genomic regions that are of clinical relevance.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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DetectingPKD1variants in polycystic kidney disease patients by single-molecule long-read sequencing

et al. 2017

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“…WGS was performed to 28–40× depth using Illumina HiSeq X at the Kinghorn Centre for Clinical Genomics, Australia, on the quartet, in 2016. Genomic data were processed according to the GATK best practices guidelines (Van der Auwera et al., ), using GATK (v3.3; (DePristo et al., ; McKenna et al., ), as previously described (Mallawaarachchi et al., ). Reads were aligned to the b37d5 (1000 genomes + decoy) reference genome using bwa mem (v0.7.10; (Li, )) followed by indel realignment and base quality recalibration.…”

Section: Phenotypic Features Present In the Proband And Affected Siblingmentioning

confidence: 99%

Reanalysis and optimisation of bioinformatic pipelines is critical for mutation detection

et al. 2019

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Rapid advances in genomic technologies have facilitated the identification pathogenic variants causing human disease. We report siblings with developmental and epileptic encephalopathy due to a novel, shared heterozygous pathogenic 13 bp duplication in SYNGAP1 (c.435_447dup, p.(L150Vfs*6)) that was identified by whole genome sequencing (WGS). The pathogenic variant had escaped earlier detection via two methodologies: whole exome sequencing and high‐depth targeted sequencing. Both technologies had produced reads carrying the variant, however, they were either not aligned due to the size of the insertion or aligned to multiple major histocompatibility complex (MHC) regions in the hg19 reference genome, making the critical reads unavailable for variant calling. The WGS pipeline followed different protocols, including alignment of reads to the GRCh37 reference genome, which lacks the additional MHC contigs. Our findings highlight the benefit of using orthogonal clinical bioinformatic pipelines and all relevant inheritance patterns to re‐analyze genomic data in undiagnosed patients.

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“…When genetic testing for ADPKD is performed with next-generation sequencing (NGS), coverage of exon 1 of the PKD1 gene is generally low due to its high GC content [13]. To overcome this problem, we simultaneously performed conventional Sanger sequencing for exon 1 of PKD1.…”

Section: Mutation Analysis By Next Generation Sequencingmentioning

confidence: 99%

Genotype-Clinical Correlations in Polycystic Kidney Disease with No Apparent Family History

Sekine¹,

Fujimaru²,

Hoshino³

et al. 2019

Am J Nephrol

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Background: Genetic characteristics of polycystic kidney disease (PKD) patients without apparent family history were reported to be different from those with a positive family history. However, the clinical course of PKD patients with no apparent family history is not well documented in the literature. Methods: We evaluated the relationship between genotype and the clinical course of 62 PKD patients with no apparent family history. Results: The annual decline of renal function was faster in the patients with PKD1/PKD2 mutation (PKD1 truncating [–3.08; 95% CI –5.30 to –0.87, p = 0.007], PKD1 nontruncating [–2.10; –3.82 to –0.38, p = 0.02], and PKD2 [–2.31; –4.40 to –0.23, p = 0.03]) than in the other patients without PKD1/PKD2 mutation. Similar results were obtained after adjustment for gender, age, estimated glomerular filtration rate (eGFR), height-adjusted total kidney volume (TKV), and mean arterial pressure (MAP). There was no significant difference in the annual decline of renal function among the different PKD1/PKD2 groups, but Kaplan-Meier analysis showed that progression to eGFR < 15 mL/min/1.73 m² was significantly faster in PKD1 truncating group (p = 0.05). The annual rate of TKV increase was larger in the patients with PKD1/PKD2 mutation (PKD1 truncating [4.63; 95% CI 0.62–8.64, p = 0.03], PKD1 nontruncating [3.79; 0.55–7.03, p = 0.02], and PKD2 [2.11; –1.90 to 6.12, p = 0.29]) than in the other patients without PKD1/PKD2 mutation. Similar results were obtained after adjustment for gender, age, eGFR, and MAP. Conclusion: Detection of PKD1/PKD2 mutation, especially PKD1 truncating, is useful for predicting the renal outcome and rate of TKV increase in PKD patients with no apparent family history.

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Whole-genome sequencing overcomes pseudogene homology to diagnose autosomal dominant polycystic kidney disease

Cited by 72 publications

References 31 publications

DetectingPKD1variants in polycystic kidney disease patients by single-molecule long-read sequencing

DetectingPKD1variants in polycystic kidney disease patients by single-molecule long-read sequencing

Reanalysis and optimisation of bioinformatic pipelines is critical for mutation detection

Genotype-Clinical Correlations in Polycystic Kidney Disease with No Apparent Family History

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