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Whole exome sequencing: a state-of-the-art approach for defining (and exploring!) genetic landscapes in pediatric nephrology

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Abstract

The genesis of whole exome sequencing as a powerful tool for detailing the protein coding sequence of the human genome was conceptualized based on the availability of next-generation sequencing technology and knowledge of the human reference genome. The field of pediatric nephrology enriched with molecularly unsolved phenotypes is allowing the clinical and research application of whole exome sequencing to enable novel gene discovery and provide amendment of phenotypic misclassification. Recent studies in the field have informed us that newer high-throughput sequencing techniques are likely to be of high yield when applied in conjunction with conventional genomic approaches such as linkage analysis and other strategies used to focus subsequent analysis. They have also emphasized the need for the validation of novel genetic findings in large collaborative cohorts and the production of robust corroborative biological data. The well-structured application of comprehensive genomic testing in clinical and research arenas will hopefully continue to advance patient care and precision medicine, but does call for attention to be paid to its integrated challenges.

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Acknowledgements

American Society of Nephrology, Ben J. Lipps Program.

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Authors and Affiliations

  1. Division of Nephrology, Department of Pediatrics, Yale University School of Medicine, New Haven, CT, USA

    Ashima Gulati

  2. Departments of Internal Medicine and Genetics, Yale University School of Medicine, New Haven, CT, USA

    Stefan Somlo

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  1. Ashima Gulati
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Correspondence to Ashima Gulati.

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Glossary

Bioinformatics tools

Sophisticated computer programs enabling processing and analysis of large scale genomic data to generate meaningful information with speed and accuracy

Complex inheritance

More than one or many genes with small individual effects collectively contributing to a given phenotype

Coding variants

Genetic variants that lie within the protein-coding or exonic region of the genome

Coverage depth

Number of times a particular chromosomal position is sequenced. Greater coverage depth may increase the authenticity or the confidence of a variant call at that particular chromosomal position

Deleterious mutation

A mutation predicted to have an impact on protein function and thus likely to have a biological effect

De novo variant

New mutation arising in an embryo that is not carried by the somatic cells of either of the parents

DNA fragmentation

A step in the WES technique that employs physical, enzymatic or chemical methods to break DNA into smaller fragments so as to generate a nucleic acid sequence length that will be compatible for subsequent sequencing. Most modern next-generation sequencers can read up to 150 bp in length

Exome

All the exons in a genome collectively constitute the exome. The exome is the sum total of the transcribed portion of the genome, which thus includes all the protein coding genomic regions

Exome capture

A step in the WES technique that uses short oligonucleotide (DNA or RNA) sequences complementary to exon sequences in the genome to selectively bind to the exonic regions for subsequent sequencing, thus leaving behind the intervening nonprotein-coding introns. Most exome capture techniques use DNA or RNA sequences in a solution phase (solution-phase exome capture) for hybridization to the exonic regions of a sample of genomic DNA being subjected to WES

Genotyping

Testing a genome for a panel of known genetic variants

Genetic heterogeneity

Disease conditions where varied alleles of the same genes (allelic heterogeneity) or multiple genes at different chromosomal loci (locus heterogeneity) can account for similar phenotypic presentation

Gene modifiers

Modification of disease expression due to other genes (modifier genes) interacting with the primary disease-causing gene and modifying its effect. This genetic interaction may be a result of the involvement of common or intersecting biological pathways by different genes. The net biological effect may be different from that expected from simply additive properties of individual gene effects (epistasis)

Heritable variation

The proportion of phenotypic variation in a trait or disease condition accounted for by genetic factors

Hybridization baits

DNA or RNA sequences complementary to exonic regions in the genome that are used to hybridize and capture exons in the process of exome capture

Linkage analysis

Genes located in close proximity within a chromosomal location likely remain associated during random chromosomal crossover in meiosis and may thus be inherited together. Common inheritance of certain chromosomal regions in individuals with the same disease condition may thus provide information on the chromosomal location of the causative gene

Loss of function variants

These include truncation mutation, frame shift mutation, and canonical splice site variants, as these can most likely be predicted to result in loss of protein function

Minor allele frequency

Population frequency of the second most common allele at a particular chromosomal location

Mutation nomenclature (gene name) (longest mRNA transcript RefSeq database) (nucleotide change at exon location and coding sequence position “c”) (amino-acid change at protein position “p”)

Nonsynonymous (missense) mutation: e.g., PKD1: NM_001009944:exon2:c.T221A:p.V74D Human PKD1 with nucleotide change T>A at coding sequence position 221 results in amino-acid change at protein position 74 from a valine to aspartic acid Stop gain (truncating) mutation: PKD1: NM_001009944:exon5: c. G914A: p. W305X Introduction of a stop codon resulting in protein sequence termination at position 305 normally coding for a tryptophan residue Frameshift insertion: PKD1: NM_001009944:exon46: c.12627_12628insAG:p. P4210fs Two base-pair deletion at coding positions 12627 and 12628 causing frameshift (fs) at protein position 4210 Frameshift deletion: PKD1: NM_001009944:exon7: c.1426delG: p. V476fs One base-pair deletion at coding position 1426 causing frameshift at protein position 476 Splice site mutation: PKD1: NM_001009944:exon38: c.11016+1G>A Nucleotide change G to A at one base pair position downstream to the exon–intron junction causing alteration of the canonical exon–intron splicing

Nonsynonymous SNV

SNV that results in an amino-acid change in a protein sequence

Repetitive sequences

DNA sequences in the genome that share high homology or similarity with each other and hence may get mis-mapped to the reference genome, resulting in false variant calls

Single nucleotide variation (SNV)

Or a single base pair substitution, e.g., guanine (G) is replaced by adenine (A)

Structural variation

Large insertion/deletions or copy number variation

Trio

Proband and both biological parents

Variant annotation

Characterization of genetic variants, e.g., based on the type of mutation, the frequency in public databases, quality scoring parameters, bioinformatics predictions for effect on protein function

Variant filtering

Downsizing the number of variant calls in a WES analysis dataset based on various parameters such as population frequency, quality scores, coverage depth, predicted effect on protein function, relevance to the particular inheritance model being analyzed

Whole exome sequencing (WES)

Sequencing all the protein coding regions or exons in their entirety. The exon–intron boundaries are usually included in the sequencing, whereas the intervening intronic regions are not

Whole genome sequencing (WGS)

Sequencing the entire genome including all the exons or the protein-coding regions and the nonprotein-coding intronic regions

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Gulati, A., Somlo, S. Whole exome sequencing: a state-of-the-art approach for defining (and exploring!) genetic landscapes in pediatric nephrology. Pediatr Nephrol 33, 745–761 (2018). https://doi.org/10.1007/s00467-017-3698-0

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  • DOI: https://doi.org/10.1007/s00467-017-3698-0

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