Other acceptable sample types: saliva (please call 267-426-1447 for kits), fresh or flash-frozen tissues, cultured fibroblasts, extracted DNA (300 ng of 25ng/µl DNA), urine, and buccal swabs.
Specimen Preparation
Please provide detailed clinical history and features for both the proband and the maternal family member (if submitting a sample). A separate order is required for the proband and the maternal family member.
Regarding Blood Specimens: Heparinized specimens, severely hemolyzed specimens, frozen, clotted or possibly commingled specimens, blood in non-sterile or leaky containers, mislabeled or inappropriately labeled specimens.
Do not heat, freeze or centrifuge blood before shipment. Refrigerate sample until shipment.
Please call 6-1447 with any questions regarding non-blood specimens.
Storage/Transport Temperature
For CHOP Phlebotomy: Samples can be collected throughout the week. Samples collected on weekends or holidays are held in Central Labs and sent to the Genomic Diagnostic Lab the following business day.
For External Clients: Refrigerate sample until shipment. Send the sample at room temperature with overnight delivery for receipt Monday through Friday, optimally within 24 hours of collection.
Please contact the lab (267-426-1447) with questions regarding non-blood specimens.
Volume Required
3 mL whole blood or 8µg DNA
Minimum Required
2 mL whole blood
Phlebotomy Draw
Yes
Clinical Features
Mitochondrial disorders are a clinically and genetically heterogeneous group of conditions that manifest with variable symptoms, often affecting skeletal and cardiac muscle, the endocrine system, and the central nervous system (including vision and hearing) [Chinnery 2014, PMID: 20301403; Koenig 2008, PMID: 18410845; Zeviani 2004, PMID: 15358637]. Phenotypic variability is common. Individuals with an identical mitochondrial variant may present with different clinical manifestations, age of onset, and degree of severity, even within the same family [Liang 2014, PMID: 24239706; Molnar 2017, PMID: 28987165].
The clinical sensitivity for the MitoGenome Sequencing and Deletion Analysis testing is variable, depending on several factors including the age of onset, clinical features, and family history. Mitochondrial disorders are the result of both nuclear and mitochondrial DNA alterations, with an estimated 70-85% being the result of a nuclear etiology [Chinnery 2014, PMID: 20301403; Koenig 2008, PMID: 18410845; Molnar 2017, PMID: 28987165].
Order the Maternal MitoGenome Seq + Del (https://www.testmenu.com/chop/Tests/1061707) to submit the proband's biological mother as a comparator. The order should be placed in the family member’s medical record, not the patient’s medical record.
For more information contact the lab at 6-1447 or by sending an email to DGDGeneticCounselor@email.chop.edu.
Performing Lab
Division of Genomic Diagnostics
Performed
Monday-Friday, 9:00am - 4:00pm
Reported
14 days
Detection Rate
This analysis is based on the current understanding of mitochondrial disease. Clinical implications of some variants may be uncertain or unknown at the time of this report and may change over time. Interpretations are made with the assumption that provided clinical information and family relationships are accurate.
The MitoGenome Sequencing and Deletion Analysis includes sequence analysis of the entire mitochondrial genome and deletion analysis (detecting deletions greater than 1 kb in size). This test is validated to identify single nucleotide variants and large deletions at heteroplasmy levels of >1% and >15%, respectively. Large duplications cannot be detected by this test. The sensitivity and lower detection limit for indels is not known at this time. Deletion heteroplasmy levels will be assessed for large deletions that remove the MT-ND4 gene (specifically chrM:11217-11320 region) but does not affect the MT-RNR2 gene (specifically chrM:1863-1965 region). For samples that have mtDNA carrying reciprocal duplications in addition to mtDNA carrying deletions, deletion heteroplasmy assessment may not be accurate.
This analysis does not include assessment of nuclear genes that are associated with mitochondrial disease.
Utility
The clinical utility of the assay is to support a clinical diagnosis of mitochondrial disease related phenotypes due to defects in the mitochondrial genome, facilitate genetic counseling, and assess the risk to first-degree relatives as well as other at-risk family members.
Next generation sequencing: The mitochondrial genome is specifically enriched by amplifying the entire mtDNA as a single linear amplicon using a high-fidelity long-range PCR system (LR-PCR). LR-PCR products are purified using the AMPure XP beads and subjected to library preparation using SureSelect QXT followed by NGS on the Illumina NovaSeq 6000 system (150bp paired-end reads) with an average sequencing depth of 20,000X. Two different mtDNA LR-PCR products are sequenced for each proband to increase sensitivity and specificity. Only one amplicon is sequenced for an accompanying maternal relative specimen, if provided. Sequences are aligned to the revised Cambridge Reference Sequence (rCRS, NC_012920.1) [Andrews 1999, PMID: 10508508]. Variant calls, single nucleotide and small insertion/deletion heteroplasmy quantification and haplogroup determination are achieved using an in-house custom-built bioinformatics pipeline.
Deletion detection: An in-house screening algorithm based on read depth is used to call deletions in the mitochondrial genes. The heteroplasmy level of mtDNA deletions identified by NGS is assessed using droplet digital PCR (ddPCR). The ddPCR assay quantifies the fraction of non-deleted to full-length mtDNA molecules, which is used to infer the heteroplasmy level of mtDNA molecules carrying the deletion.
Molecular Testing Notes
This analysis includes sequencing and deletion analysis of the genes of the mitochondrial genome as well as a heteroplasmy calcuation and haplogroup assessment. The MitoGenome Sequencing and Deletion Analysis includes the following genes: MT-ATP6, MT-ATP8, MT-CO1, MT-CO2, MT-CO3, MT-CYB, MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-RNR1, MT-RNR2, MT-TA, MT-TC, MT-TD, MT-TE, MT-TF, MT-TG, MT-TH, MT-TI, MT-TK, MT-TL1, MT-TL2, MT-TM, MT-TN, MT-TP, MT-TQ, MT-TR, MT-TS1, MT-TS2, MT-TT, MT-TV, MT-TW, and MT-TY.
CPT Codes
81460 and 81465
Collection
Collect
Whole blood (2-3 mL in Lavender top, EDTA tube)
Other acceptable sample types: saliva (please call 267-426-1447 for kits), fresh or flash-frozen tissues, cultured fibroblasts, extracted DNA (300 ng of 25ng/µl DNA), urine, and buccal swabs.
Specimen Preparation
Please provide detailed clinical history and features for both the proband and the maternal family member (if submitting a sample). A separate order is required for the proband and the maternal family member.
Regarding Blood Specimens: Heparinized specimens, severely hemolyzed specimens, frozen, clotted or possibly commingled specimens, blood in non-sterile or leaky containers, mislabeled or inappropriately labeled specimens.
Do not heat, freeze or centrifuge blood before shipment. Refrigerate sample until shipment.
Please call 6-1447 with any questions regarding non-blood specimens.
Storage/Transport Temperature
For CHOP Phlebotomy: Samples can be collected throughout the week. Samples collected on weekends or holidays are held in Central Labs and sent to the Genomic Diagnostic Lab the following business day.
For External Clients: Refrigerate sample until shipment. Send the sample at room temperature with overnight delivery for receipt Monday through Friday, optimally within 24 hours of collection.
Please contact the lab (267-426-1447) with questions regarding non-blood specimens.
Volume Required
3 mL whole blood or 8µg DNA
Minimum Required
2 mL whole blood
Phlebotomy Draw
Yes
Ordering
Clinical Features
Mitochondrial disorders are a clinically and genetically heterogeneous group of conditions that manifest with variable symptoms, often affecting skeletal and cardiac muscle, the endocrine system, and the central nervous system (including vision and hearing) [Chinnery 2014, PMID: 20301403; Koenig 2008, PMID: 18410845; Zeviani 2004, PMID: 15358637]. Phenotypic variability is common. Individuals with an identical mitochondrial variant may present with different clinical manifestations, age of onset, and degree of severity, even within the same family [Liang 2014, PMID: 24239706; Molnar 2017, PMID: 28987165].
The clinical sensitivity for the MitoGenome Sequencing and Deletion Analysis testing is variable, depending on several factors including the age of onset, clinical features, and family history. Mitochondrial disorders are the result of both nuclear and mitochondrial DNA alterations, with an estimated 70-85% being the result of a nuclear etiology [Chinnery 2014, PMID: 20301403; Koenig 2008, PMID: 18410845; Molnar 2017, PMID: 28987165].
Order the Maternal MitoGenome Seq + Del (https://www.testmenu.com/chop/Tests/1061707) to submit the proband's biological mother as a comparator. The order should be placed in the family member’s medical record, not the patient’s medical record.
For more information contact the lab at 6-1447 or by sending an email to DGDGeneticCounselor@email.chop.edu.
Performing Lab
Division of Genomic Diagnostics
Performed
Monday-Friday, 9:00am - 4:00pm
Reported
14 days
Detection Rate
This analysis is based on the current understanding of mitochondrial disease. Clinical implications of some variants may be uncertain or unknown at the time of this report and may change over time. Interpretations are made with the assumption that provided clinical information and family relationships are accurate.
The MitoGenome Sequencing and Deletion Analysis includes sequence analysis of the entire mitochondrial genome and deletion analysis (detecting deletions greater than 1 kb in size). This test is validated to identify single nucleotide variants and large deletions at heteroplasmy levels of >1% and >15%, respectively. Large duplications cannot be detected by this test. The sensitivity and lower detection limit for indels is not known at this time. Deletion heteroplasmy levels will be assessed for large deletions that remove the MT-ND4 gene (specifically chrM:11217-11320 region) but does not affect the MT-RNR2 gene (specifically chrM:1863-1965 region). For samples that have mtDNA carrying reciprocal duplications in addition to mtDNA carrying deletions, deletion heteroplasmy assessment may not be accurate.
This analysis does not include assessment of nuclear genes that are associated with mitochondrial disease.
Utility
The clinical utility of the assay is to support a clinical diagnosis of mitochondrial disease related phenotypes due to defects in the mitochondrial genome, facilitate genetic counseling, and assess the risk to first-degree relatives as well as other at-risk family members.
Next generation sequencing: The mitochondrial genome is specifically enriched by amplifying the entire mtDNA as a single linear amplicon using a high-fidelity long-range PCR system (LR-PCR). LR-PCR products are purified using the AMPure XP beads and subjected to library preparation using SureSelect QXT followed by NGS on the Illumina NovaSeq 6000 system (150bp paired-end reads) with an average sequencing depth of 20,000X. Two different mtDNA LR-PCR products are sequenced for each proband to increase sensitivity and specificity. Only one amplicon is sequenced for an accompanying maternal relative specimen, if provided. Sequences are aligned to the revised Cambridge Reference Sequence (rCRS, NC_012920.1) [Andrews 1999, PMID: 10508508]. Variant calls, single nucleotide and small insertion/deletion heteroplasmy quantification and haplogroup determination are achieved using an in-house custom-built bioinformatics pipeline.
Deletion detection: An in-house screening algorithm based on read depth is used to call deletions in the mitochondrial genes. The heteroplasmy level of mtDNA deletions identified by NGS is assessed using droplet digital PCR (ddPCR). The ddPCR assay quantifies the fraction of non-deleted to full-length mtDNA molecules, which is used to infer the heteroplasmy level of mtDNA molecules carrying the deletion.
Molecular Testing Notes
This analysis includes sequencing and deletion analysis of the genes of the mitochondrial genome as well as a heteroplasmy calcuation and haplogroup assessment. The MitoGenome Sequencing and Deletion Analysis includes the following genes: MT-ATP6, MT-ATP8, MT-CO1, MT-CO2, MT-CO3, MT-CYB, MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, MT-RNR1, MT-RNR2, MT-TA, MT-TC, MT-TD, MT-TE, MT-TF, MT-TG, MT-TH, MT-TI, MT-TK, MT-TL1, MT-TL2, MT-TM, MT-TN, MT-TP, MT-TQ, MT-TR, MT-TS1, MT-TS2, MT-TT, MT-TV, MT-TW, and MT-TY.
