PGxome - Whole Exome Sequencing

PGxome Diagnostic (Test Code #5000) Requisition Form

Background

PGxome is PreventionGenetics' whole exome sequencing (WES) test. The PGxome assesses almost all genes from the human genome including coding regions and adjacent introns. This test is intended for health care providers who are looking for a genetic diagnosis when the clinical phenotype is unclear and/or previous test results have been uninformative. This is important as more than 50% of patients with genetic diseases are not given a specific diagnosis even after repeat clinical examinations and tests (Shashi et al. 2014). The standard clinical practice often involves examinations for specific phenotypes, imaging, biochemical testing for inborn errors of metabolism, genomic tests such as karyotyping or microarrays, and single gene or panel tests (Iglesias et al. 2014). However, patients remain without a genetic diagnosis, and patients and health care providers are caught in a long term diagnosis search, known as a diagnostic odyssey. This can lead to failures in identifying potential treatments, and unknown recurrence and prognosis risks (Yang et al. 2013).

Reported diagnostic rates from commercial and academic laboratories have found that WES assays have a ~20-40% positive diagnostic rate, with higher rates being reported from trio analysis (i.e. proband and parents) compared to singleton analysis (Atwal et al. 2014; Iglesias et al. 2014; Farwell et al. 2015). Notably, ~5-7% of individuals who have WES have had dual diagnoses (i.e. two non-overlapping clinical presentations) (Yang et al. 2014; Farwell et al. 2015). One study reported that 30% of genetics diagnoses have only been recently resolved due to new literature reports, highlighting the fast pace of gene-disease discovery and the need of genetic testing laboratories to be current of the medical literature (Yang et al. 2014). The use of a whole exome sequencing test may aid in altering clinical management, predict recurrence and prognosis risks, and reduce costs of additional testing, and may offer advantages over traditional molecular tests in certain patients (Valencia et al. 2015).

The PGxome is ideal for individuals with:

  • Unresolved genetic testing such as normal karyotyping or microarray analysis, and negative single gene or gene panel sequencing results
  • Disorders with significant genetic heterogeneity
  • Global developmental delay/intellectual disability, with or without dysmorphic features
  • Dysmorphic features, multiple congenital anomalies, or birth defects

Test Ordering Details

Trio Pricing: $4,490

Duo Pricing: $3,490

Individual Pricing: $2,490

Sequencing cost to additional family members beyond trio: $990 (no report)

If report is needed for any additional family members, add $990

Test Requisition Form & Provider Statement

Clinical History: Clinical notes and pedigree must be included

Test Code: #5000

CPT Code: 81415 (Proband), 81416 (for each family member)

Turnaround Time: Maximum of 6 weeks.

Proband and Parental Specimen Requirements

See test requisition form for full specimen and shipping details.

Specimen Type: Blood

Specimen requirements: Draw blood in EDTA (purple-top) or ACD (yellow-top) tubes

Infants <2 years old: 1mL-2mL blood in EDTA or ACD tubes

Adults and children >2yrs: 3mL-5mL blood in EDTA or ACD tubes

Specimen Type: DNA

Specimen requirements: Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 ng/µl.

Specimen Type: Tissue (Fresh frozen)

Specimen requirements: Fresh frozen tissue accepted. Please contact us for details. Fixed, paraffin embedded samples are not accepted.

Cultured Cells

Specimen requirements: Ship at least two T25 flasks of confluent cells.

Storage and shipping: T25 flasks need to be shipped in appropriate complete culture medium filled to the brim. Ship T25 flasks at room temperature in an insulated container once 90% confluency is reached and by overnight delivery.

Specimen Type: Saliva

OrageneTM Saliva Collection kit used according to manufacturer instructions.

Test Methods

For the PGxome we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced using Illumina's Reversible Dye Terminator (RDT) platform NextSeq 500 using 150 by 100 bp paired end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. Data analysis and interpretation is performed by the internally developed software Titanium-Exome. In brief, the output data from the NextSeq 500 is converted to fastqs by Illumina Bcl2Fastq 1.8.4, and mapped by BWA. Variant calls are made by the GATK Haplotype caller and annotated using in house software and SnpEff. Variants are filtered and annotated using VarSeq (www.goldenhelix.com). Common benign, likely benign, and low quality variants are filtered from analysis. All reported pathogenic, likely pathogenic, and variants of uncertain significance are confirmed by Sanger sequencing.

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Reporting

Reports will consist of six different sections:

  1. Variants in genes known to be associated with phenotype
  2. Variants in genes possibly associated with phenotype
  3. Medically actionable variants from the ACMG recommended list of genes (if requested) (Kalia et al. 2016)
  4. Variants in genes not associated with phenotype but result in a Mendelian disorder (if requested)
  5. Carrier status for variants that are causative for recessive disease (if requested)
  6. Pharmacogenetic variants that may influence drug response (if requested) (currently limited to 15 genes; see list below under “Limitations and Other Test Notes” for gene list and associated drugs)

All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories (Pathogenic, Likely Pathogenic, Variant of Uncertain Significance, Likely Benign and Benign) per ACMG Guidelines (Richards et al. 2015). Pathogenic, Likely Pathogenic and Variants of Uncertain Significance will be reported in the first and second sections (1 & 2). For sections three through five (3-5), only definitive pathogenic and likely pathogenic variants will be reported. Rare and undocumented synonymous variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing. Additionally, only actionable pharmacogenetic variants will be reported (section 6). The interpretation of and terminology used for identified pharmacogenetic findings are based on CPIC guidelines, PharmGKB, workgroup recommendations, and peer-reviewed literature (Caudle et al. 2016; Kalman et al. 2015). Variants with unknown or uncertain significance in drug response will not be reported. Likely benign and benign variants are not included in the reports. However, a full list of all sequence variants will be provided to the ordering physician upon request.

Nomenclature for sequence variants comes from Human Genome Variation Society (HGVS) (http://www.hgvs.org).

Limitations and Other Test Notes

Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When sequencing does not reveal any heterozygous differences from the reference sequence, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not capture or amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele. Our Sanger and NGS tests (including PGxome) are generally not capable of detecting Copy Number Variants (CNVs).

For technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of sequence variants relevant to the patient's health will not be detected.

We sequence coding exons for most given transcripts, plus ~10 bp of flanking non-coding DNA for each exon. Unless specifically indicated, test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions, uncharacterized alternative exons, chromosomal rearrangements, repeat expansions, epigenetic effects, and mitochondrial genome variants.

In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient's nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during amplification.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes if taken from whole blood). Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the specimen arrives at PreventionGenetics.

A negative finding does not rule out a genetic diagnosis.

Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended. Results of PGxome testing can be used for both diagnostic and scientific research purposes.

Important Pharmacogenetic Variants

Footnotes:

"Important Pharmacogenetic Variants" refers to common pharmacogenetic variants that have been reported with sufficient evidence. PreventionGenetics will identify and report these common variants along with other rare and/or novel variants identified in the coding regions of genes analyzed that are suspected to affect drug response. Pharmacogenetic information based on US FDA drug labels: (1) genetic testing required, (2) genetic testing recommended, (3) actionable PGx. (a) To increase clinical sensitivity, in the genes where loss of function (LOF) is a known mechanism of abnormal drug response, rare or previously undiscovered LOF variants (e.g., null sense, splicing and frameshift insertions/deletions) will also be considered. (b) Due to X-linked chromosome inactivation in females, it is impossible to predict the G6PD activity in heterozygous females based on genotype alone. Therefore, an enzyme activity test is needed to determine G6PD phenotype. (c) The variant rs887829 (c.-364C>T; UGT1A1*80) is not covered by this test due to technical limitations. Actionable pharmacogenetic variants are reported with the level of evidence: strong, where a CPIC guideline is available; moderate, in which the strength of the evidence is lower but still sufficient to determine effects; and weak, in which the data is limited by the quality and consistency of the studies.

Contacts

Genetic Counselors: GC Team - clinicaldnatesting@preventiongenetics.com

Geneticist: Jerry Machado, PhD, DABMG, FCCMG - jerry.machado@preventiongenetics.com

References

Atwal P.S. et al. 2014. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 16: 717-9. PubMed ID: 24525916

Caudle et al. 2016. Genetics in Medicine. PubMed ID: 27441996

Farwell K.D. et al. 2015. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 17: 578-86. PubMed ID: 25356970

Iglesias A. et al. 2014. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 16: 922-31. PubMed ID: 24901346

Kalia S.S. et al. 2016. Genetics in Medicine: Official Journal of the American College of Medical Genetics. Advance online publication. doi:10.1038/gim.2016.190. PubMed ID: 27854360

Kalman et al. 2015. Clin Pharmacol Ther. 99(2): 172-85. PubMed ID: 26479518

Richards S et al. 2015. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 17: 405-24. PubMed ID: 25741868

Shashi V. et al. 2014. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 16: 176-82. PubMed ID: 23928913

Valencia C.A. et al. 2015. Frontiers in Pediatrics. 3: 67. PubMed ID: 26284228

Yang Y. et al. 2013. The New England Journal of Medicine. 369: 1502-11. PubMed ID: 24088041

Yang Y. et al. 2014. JAMA. 312: 1870-9. PubMed ID: 25326635

PGxome Health Screen (Test Code #4000) Requisition Form

Background

PGxome is PreventionGenetics' whole exome sequencing (WES) assay. The PGxome assesses almost all genes from the human genome including coding regions and adjacent introns. The PGxome Health Screen is intended for patients who are basically healthy, but who want to learn their carrier status for recessive disease and/or their susceptibility to adult onset disorders, and/or their response to certain prescription medications.

Although we sequence nearly all human genes, we analyze and report sequence variants only in genes that have been proven with high confidence to be involved in Mendelian (also called single gene) disorders (MacArthur et al. 2014). Our list of "clinically-relevant" genes currently includes about 4000 genes and is updated quarterly. We do not report variants in genes that for technical reasons cannot be accurately sequenced (primarily due to the presence of pseudogenes).

In addition, although we identify and interpret all sequence variants (differences between the patient's sequence and the reference sequence (build hg19)), we report only Pathogenic and Likely Pathogenic variants (Richards et al. 2015).

We have found through our exome sequencing at PreventionGenetics that the average person is a recessive disease carrier for less than five Pathogenic or Likely Pathogenic variants. Note, however, that the average person also carries approximately 75 variants of Uncertain significance and thousands of Benign variants.

For this test, patients also have the option of receiving results of Pathogenic and Likely Pathogenic variants in genes that predispose to or confirm a diagnosis of adult onset disorders such as cancer and heart disease (Kalia et al. 2016).

Additionally, patients have the option of receiving results of actionable variants within 15 genes known to influence prescription drug response. Reportable genes were selected based on evidence supporting gene-drug interactions (https://www.pharmgkb.org). Different from allele specific genotyping, our pharmacogenetics section is mainly based on full gene sequencing, which also detects rare or previously undiscovered loss of function variants (e.g., nonsense, splicing and frameshift insertions/deletions). The list of reported genes can be found below.

Important Pharmacogenetic Variants

Footnotes:

"Important Pharmacogenetic Variants" refers to common pharmacogenetic variants that have been reported with sufficient evidence. PreventionGenetics will identify and report these common variants along with other rare and/or novel variants identified in the coding regions of genes analyzed that are suspected to affect drug response. Pharmacogenetic information based on US FDA drug labels: (1) genetic testing required, (2) genetic testing recommended, (3) actionable PGx. (a) To increase clinical sensitivity, in the genes where loss of function (LOF) is a known mechanism of abnormal drug response, rare or previously undiscovered LOF variants (e.g., null sense, splicing and frameshift insertions/deletions) will also be considered. (b) Due to X-linked chromosome inactivation in females, it is impossible to predict the G6PD activity in heterozygous females based on genotype alone. Therefore, an enzyme activity test is needed to determine G6PD phenotype. (c) The variant rs887829 (c.-364C>T; UGT1A1*80) is not covered by this test due to technical limitations. Actionable pharmacogenetic variants are reported with the level of evidence: strong, where a CPIC guideline is available; moderate, in which the strength of the evidence is lower but still sufficient to determine effects; and weak, in which the data is limited by the quality and consistency of the studies.

Because new Mendelian disease genes are being continuously identified and because our ability to interpret sequence variants is steadily improving, it is important to consider reanalysis and reinterpretation of exome data in future. For this purpose, PreventionGenetics retains raw sequence data indefinitely from each exome test.

The PGxome test may also be ordered for diagnostic purposes (see Test #5000).

Test Ordering Details

Singleton Pricing (sequencing and report): $2,490

Couple Pricing (sequencing and report for each person): $4,490

PGxome Health Screen Test Requisition Form and Healthcare Provider Statement (Required)

Clinical History: Clinical notes and pedigree/family history information should be included when appropriate

Test Code: #4000

CPT Code: 81415 (x2 if couple)

Turnaround Time: Maximum of 6 weeks.

Specimen Requirements

See test requisition form for full specimen and shipping details.

Specimen Type: Blood

Specimen requirements: Draw 3mL-5mL blood in EDTA (purple-top) or ACD (yellow-top) tubes

Specimen Type: DNA

Specimen requirements: Send in screw cap tube at least 5µg-10µg of purified DNA at a concentration of at least 20 ng/µl.

Specimen Type: Tissue (Fresh frozen)

Specimen requirements: Fresh frozen tissue accepted. Please contact us for details. Fixed, paraffin embedded samples are not accepted.

Cultured Cells

Specimen requirements: Ship at least two T25 flasks of confluent cells.

Storage and shipping: T25 flasks need to be shipped in appropriate complete culture medium filled to the brim. Ship T25 flasks at room temperature in an insulated container once 90% confluency is reached and by overnight delivery.

Specimen Type: Saliva

OrageneTM Saliva Collection kit used according to manufacturer instructions.

Test Methods

For the PGxome we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced using Illumina's Reversible Dye Terminator (RDT) platform NextSeq 500 using 150 by 100 bp paired end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. Data analysis and interpretation is performed by the internally developed software Titanium-Exome. In brief, the output data from the NextSeq 500 is converted to fastqs by Illumina Bcl2Fastq 1.8.4, and mapped by BWA. Variant calls are made by the GATK Haplotype caller and annotated using in house software and SnpEff. Variants are filtered and annotated using VarSeq (www.goldenhelix.com). Common benign, likely benign, and low quality variants are filtered from analysis. All reported Pathogenic and Likely Pathogenic variants are confirmed by Sanger sequencing.

For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.

Reporting

Reports will consist of four different sections:

  1. Variants that are causative for recessive disease
  2. Medically actionable variants from the ACMG recommended list of genes (if requested) (Kalia et al. 2016)
  3. Variants in other genes that result in a dominant Mendelian disorder (if requested)
  4. Pharmacogenetic variants that may influence drug response (if requested) (currently limited to 15 genes)

All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories (Pathogenic, Likely Pathogenic, Variant of Uncertain Significance, Likely Benign and Benign) per ACMG Guidelines (Richards et al. 2015). All sequence variants in appropriate gene regions will be detected and interpreted, but only Pathogenic and Likely Pathogenic variants will be included in the test report.

Additionally, only actionable pharmacogenetic variants will be reported. The interpretation of and terminology used for identified pharmacogenetic findings are based on CPIC guidelines, PharmGKB, workgroup recommendations, and peer-reviewed literature (Caudle et al. 2016; Kalman et al. 2015). Variants with unknown or uncertain significance in drug response will not be reported.

Likely benign and benign variants are not included in the reports. However, a full list of all sequence variants will be provided to the ordering physician upon request.

Nomenclature for sequence variants comes from Human Genome Variation Society (HGVS) (http://www.hgvs.org).

Limitations and Other Test Notes

Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.

When sequencing does not reveal any heterozygous differences from the reference sequence, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not capture or amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele.

Our Sanger and NGS tests (including PGxome) are generally not capable of detecting Copy Number Variants (CNVs).

Due to technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of causative sequence variants will not be detected. For those planning reproduction, we cannot guarantee a child free of genetic disorders.

We sequence coding exons for most given transcripts, plus ~10 bp of flanking non- coding DNA for each exon. Unless specifically indicated, test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions, uncharacterized alternative exons, chromosomal rearrangements, repeat expansions, epigenetic effects, and mitochondrial genome variants.

In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient's nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during amplification.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes if taken from whole blood). Test reports contain no information about the DNA sequence in other cell-types.

We cannot be certain that the reference sequences are correct.

We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the specimen arrives at PreventionGenetics.

Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended. Results of PGxome testing can be used for both diagnostic and scientific research purposes.

Contacts

Genetic Counselors: GC Team - clinicaldnatesting@preventiongenetics.com

Geneticist: Jerry Machado, PhD, DABMG, FCCMG - jerry.machado@preventiongenetics.com

References

Caudle et al. 2016. Genetics in Medicine. PubMed ID: 27441996

Kalia S.S. et al. 2016. Genetics in Medicine: Official Journal of the American College of Medical Genetics. Advance online publication. doi:10.1038/gim.2016.190. PubMed ID: 27854360

Kalman et al. 2015. Clin Pharmacol Ther. 99(2): 172-85. PubMed ID: 26479518

MacArthur D.G. et al. 2014. Nature. 508: 469-76. PubMed ID: 24759409

Richards S. et al. 2015. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 17: 405-24. PubMed ID: 25741868