Neuro-Ophthalmology Panel

Updated
Summary
  • Is a 87 gene panel that includes assessment of non-coding variants.
  • In addition, it also includes the maternally inherited mitochondrial genome. Is ideal for patients with a clinical suspicion / diagnosis of nystagmus, optic atrophy or progressive external ophthalmoplegia.

Analysis methods
  • PLUS
Availability

4 weeks

Number of genes

87

Test code

OP1301

Panel size

Small

CPT code *
81443(1)
* The CPT codes provided are based on AMA guidelines and are for informational purposes only. CPT coding is the sole responsibility of the billing party. Please direct any questions regarding coding to the payer being billed.

Summary

The Blueprint Genetics Neuro-Ophthalmology Panel (test code OP1301):

ICD codes

Commonly used ICD-10 code(s) when ordering the Neuro-Ophthalmology Panel

ICD-10 Disease
F84.2 Rett syndrome
G31.89 Mohr-Tranebjaerg syndrome
H55.00 Nystagmus
H49.40 Progressive external ophthalmoplegia
H55.00 Nystagmus 1, congenital, X-linked
G11.3 Ataxia - oculomotor apraxia
Q87.89 Acro-renal-ocular syndrome
H51.0 Horizontal gaze palsy with progressive scoliosis
E13.8 Wolfram syndrome
Q04.8 Septo-optic dysplasia
H47.20 Optic atrophy
G11.9 Hereditary ataxia
C94.2 Acute Megakaryoblastic Leukemia
K59.8 Chronic Intestinal Pseudoobstruction
T36.5 Adverse effect of aminoglycosides
G93.41 Metabolic Encephalopathy
H49.81 Kearns Sayre Syndrome
E88.42 MERFF Syndrome
H47.013 Nonarteritic Anterior Ischemic Optic Neuropathy
G60.2 Neuropathy in association with hereditary ataxia
G30 Alzheimer's Disease
G25.5 Chorea
G40 Epilepsy and recurrent seizures
I42 Cardiomyopathy
N26.9 Focal Segmental Glomerulosclerosis
G31.82 Leigh's Disease
H47.2 Leber's hereditary optic neuropathy
G71.3 Mitochondrial Myopathy
I42.1 Hypertrophic Cardiomyopathy
E11.9 Non-Insulin Dependent Diabetes Mellitus
Z86.74 Personal history of sudden cardiac arrest
H90.3 Sensorineural Hearing Loss

Sample Requirements

  • Blood (min. 1ml) in an EDTA tube
  • Extracted DNA, min. 2 μg in TE buffer or equivalent
  • Saliva (Please see Sample Requirements for accepted saliva kits)

Label the sample tube with your patient's name, date of birth and the date of sample collection.

We do not accept DNA samples isolated from formalin-fixed paraffin-embedded (FFPE) tissue. In addition, if the patient is affected with a hematological malignancy, DNA extracted from a non-hematological source (e.g. skin fibroblasts) is strongly recommended.

Please note that, in rare cases, mitochondrial genome (mtDNA) variants may not be detectable in blood or saliva in which case DNA extracted from post-mitotic tissue such as skeletal muscle may be a better option.

Read more about our sample requirements here.

Neuro-ophthalmological disorders are a group of diseases that affect vision, control of eye movements, or pupillary reflexes. They include diseases restricted to the visual system and systemic diseases in which the neuro-ophthalmologic sign is accompanied by other neurological symptoms. Optic atrophy affects primarily the retinal ganglion cells and the nerve fiber layer of the retina leading to decreased visual acuity (see the description of the Optic Atrophy Panel for details). Congenital nystagmus is defined as conjugated, spontaneous and involuntary ocular oscillations that appear at birth or during the first three months of life. Binocular vision and color vision are normal and visual acuity is typically better than 6/12. Variants in the X-linked FRMD7 explain approximately 85% of patients with congenital nystagmus. Female mutation carriers can be affected. The prevalence of congenital nystagmus is estimated to be 1:3,000. Examples of syndromes associated with eye movement problems are ataxia with oculomotor apraxia type 1 and 2, caused by variants in APTX and SETX, respectively, and horizontal gaze palsy with progressive scoliosis caused by variants in ROBO3. Variants in TUBB3 are associated with an autosomal dominant strabismus syndrome called congenital fibrosis of the extraocular muscles. Mitochondrial diseases frequently manifest with neuro-ophthalmologic symptoms and signs. Progressive external ophthalmoplegia (PEO) is characterized by weakness of the eye muscles. The symptoms in PEO include drooping eyelids (ptosis) and weakness or paralysis of the muscles that move the eye (ophthalmoplegia), and in some cases skeletal muscle myopathy. Autosomal dominant PEO may be caused by variants in POLGSLC25A4, or C10orf2. Ophthalmoplegia may also be associated with mitochondrial DNA depletion syndromes, which are a genetically and clinically heterogeneous group of autosomal recessive disorders.

Genes in the Neuro-Ophthalmology Panel and their clinical significance

Gene Associated phenotypes Inheritance ClinVar HGMD
ACO2 Optic atrophy, Infantile cerebellar-retinal degeneration AR 16 15
AFG3L2* Spastic ataxia, Spinocerebellar ataxia AD/AR 22 40
ANTXR1 Hemangioma, capillary infantile, susceptibility to, Growth retardation, alopecia, pseudoanodontia, and optic atrophy (GAPO syndrome) AD/AR 6 14
APTX Ataxia, early-onset, with oculomotor apraxia and hypoalbuminemia AR 14 46
ATAD3A* Harel-Yoon syndrome AD/AR 4 17
AUH 3-methylglutaconic aciduria AR 12 11
C10ORF2 Perrault syndrome, Mitochondrial DNA depletion syndrome, Progressive external ophthalmoplegia with mitochondrial DNA deletions, autosomal dominant, 3 AR 37 80
C12ORF65 Spastic paraplegia, Combined oxidative phosphorylation deficiency AR 10 11
C19ORF12 Spastic Paraplegia, Neurodegeneration with brain iron accumulation AR 15 37
CHN1 Duane retraction syndrome 2 AD 11 11
CISD2* Wolfram syndrome 2 AR 2 4
DNAJC19 3-methylglutaconic aciduria AR 3 6
DNM1L Encephalopathy due to defective mitochondrial and peroxisomal fission 1 AD 17 20
FDXR Auditory neuropathy and optic atrophy AR 5 19
FRMD7 Nystagmus, infantile periodic alternating XL 15 95
GPR143 Nystagmus, congenital, Ocular albinism XL 22 181
HESX1 Septooptic dysplasia, Pituitary hormone deficiency, combined AR/AD 15 26
KIF21A Fibrosis of extraocular muscles, congenital 1 AD 9 17
MECR Dystonia, childhood-onset, with optic atrophy and basal ganglia abnormalities (DYTOABG) AR 7 6
MFN2 Hereditary motor and sensory neuropathy, Charcot-Marie-Tooth disease AD/AR 70 223
MT-ATP6 Neuropathy, ataxia, and retinitis pigmentosa, Leber hereditary optic neuropathy, Ataxia and polyneuropathy, adult-onset, Cardiomyopathy, infantile hypertrophic, Leigh syndrome, Striatonigral degeneration, infantile, mitochondrial Mitochondrial 19
MT-ATP8 Cardiomyopathy, apical hypertrophic, and neuropathy, Cardiomyopathy, infantile hypertrophic Mitochondrial 4
MT-CO1 Myoglobinuria, recurrent, Leber hereditary optic neuropathy, Sideroblastic anemia, Cytochrome C oxidase deficiency Mitochondrial 17
MT-CO2 Cytochrome c oxidase deficiency Mitochondrial 8
MT-CO3 Cytochrome c oxidase deficiency, Leber hereditary optic neuropathy Mitochondrial 9
MT-CYB Leber hereditary optic neuropathy Mitochondrial 69
MT-ND1 Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes, Leber hereditary optic neuropathy, Leber optic atrophy and dystonia Mitochondrial 21
MT-ND2 Leber hereditary optic neuropathy, Mitochondrial complex I deficiency Mitochondrial 6
MT-ND3 Leber optic atrophy and dystonia, Mitochondrial complex I deficiency Mitochondrial 7
MT-ND4 Leber hereditary optic neuropathy, Leber optic atrophy and dystonia, Mitochondrial complex I deficiency Mitochondrial 11
MT-ND4L Leber hereditary optic neuropathy Mitochondrial 2
MT-ND5 Myoclonic epilepsy with ragged red fibers, Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes, Leber hereditary optic neuropathy, Mitochondrial complex I deficiency Mitochondrial 19
MT-ND6 Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes, Oncocytoma, Leber hereditary optic neuropathy, Leber optic atrophy and dystonia, Mitochondrial complex I deficiency Mitochondrial 16
MT-RNR1 Deafness, mitochondrial Mitochondrial 3
MT-RNR2 Chloramphenicol toxicity/resistance Mitochondrial 2
MT-TA Leber hereditary optic neuropathy, Mitochondrial multisystemic disorder, Progressive external ophthalmoplegia, Dilated cardiomyopathy (DCM) Mitochondrial 4
MT-TC Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes Mitochondrial 3
MT-TD Mitochondrial multisystemic disorder Mitochondrial 1
MT-TE Diabetes-deafness syndrome, Mitochondrial myopathy, infantile, transient, Mitochondrial myopathy with diabetes Mitochondrial 5
MT-TF Myoclonic epilepsy with ragged red fibers, Nephropathy, tubulointerstitial, Encephalopathy, mitochondrial, Epilepsy, mitochondrial, Myopathy, mitochondrial, Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes Mitochondrial 7
MT-TG Hypertrophic cardiomyopathy, Encephalopathy, Myopathy Mitochondrial 3
MT-TH Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes Mitochondrial 4
MT-TI Progressive external ophthalmoplegia Mitochondrial 7
MT-TK Myoclonic epilepsy with ragged red fibers Mitochondrial 5
MT-TL1 Cytochrome c oxidase deficiency, Myoclonic epilepsy with ragged red fibers, Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes, Diabetes-deafness syndrome, Cyclic vomiting syndrome, SIDS, susceptibility to Mitochondrial 14
MT-TL2 Progressive external ophthalmoplegia, Mitochondrial multisystemic disorder Mitochondrial 5
MT-TM Mitochondrial Myopathy, Leigh syndrome, Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes Mitochondrial 1
MT-TN Progressive external ophthalmoplegia Mitochondrial 3
MT-TP Mitochondrial multisystemic disorder Mitochondrial 2
MT-TQ Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes, Encephalopathy Mitochondrial 2
MT-TR Dilated cardiomyopathy (DCM) Mitochondrial 2
MT-TS1 Myoclonic epilepsy with ragged red fibers, Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes Mitochondrial 10
MT-TS2 Mitochondrial multisystemic disorder Mitochondrial 2
MT-TT Mitochondrial 5
MT-TV Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes Mitochondrial 3
MT-TW Leigh syndrome, Mitochondrial Myopathy Mitochondrial 8
MT-TY Mitochondrial 4
MTPAP Spastic ataxia AR 1 2
NDUFS1 Mitochondrial complex I deficiency AR 22 28
NR2F1 Bosch-Boonstra optic atrophy syndrome AD 23 34
OPA1 Optic atrophy, Optic atrophy 1, Optic atrophy with or without deafness, Ophthalmoplegia, myopathy, ataxia, and neuropathy, Behr synrome, Mitochondrial DNA depletion syndrome 14 AD/AR 96 390
OPA3 Optic atrophy, 3-methylglutaconic aciduria AD/AR 13 15
OTX2 Microphthalmia, syndromic, Pituitary hormone deficiency, combined, Retinal dystrophy, early-onset, and pituitary dysfunction AD 23 73
PAX6 Aniridia, cerebellar ataxia, and mental retardation (Gillespie syndrome), Keratitis, Coloboma, ocular, Cataract with late-onset corneal dystrophy, Morning glory disc anomaly, Foveal hypoplasia, Aniridia, Optic nerve hypoplasia, Peters anomaly AD 144 550
PHOX2A Fibrosis of extraocular muscles, congenital, 2 AR 3 5
POLG POLG-related ataxia neuropathy spectrum disorders, Sensory ataxia, dysarthria, and ophthalmoparesis, Alpers syndrome, Progressive external ophthalmoplegia with mitochondrial DNA deletions, Mitochondrial DNA depletion syndrome AD/AR 89 290
PRPS1* Phosphoribosylpyrophosphate synthetase I superactivity, Arts syndrome, Charcot-Marie-Tooth disease, X-linked recessive, 5, Deafness, X-linked 1 XL 27 32
ROBO3 Gaze palsy, horizontal, with progressive scoliosis AR 16 40
RRM2B Progressive external ophthalmoplegia with mitochondrial DNA deletions, Mitochondrial DNA depletion syndrome AD/AR 41 41
RTN4IP1 Optic atrophy 10 with or without ataxia, mental retardation, and seizures AR 2 12
SALL4 Acro-renal-ocular syndrome, Duane-radial ray/Okohiro syndrome AD 21 56
SETX Ataxia with oculomotor apraxia, Amyotrophic lateral sclerosis, juvenile, Spinocerebellar ataxia AD/AR 36 210
SLC25A4 Progressive external ophthalmoplegia with mitochondrial DNA deletions, Mitochondrial DNA depletion syndrome AD/AR 12 14
SLC25A46 Neuropathy, hereditary motor and sensory, type VIB AR 14 17
SLC38A8 Foveal hypoplasia 2 AR 11 18
SOX2* Microphthalmia, syndromic AD 34 104
SPG7 Spastic paraplegia AR 69 111
TIMM8A* Mohr-Tranebjaerg syndrome, Jensen syndrome, Opticoacoustic nerve atrophy with dementia XL 11 21
TK2# Mitochondrial DNA depletion syndrome AR 38 52
TMEM126A Optic atrophy AR 3 1
TSFM# Combined oxidative phosphorylation deficiency AR 6 6
TUBB3* Fibrosis of extraocular muscles, congenital, Cortical dysplasia, complex, with other brain malformations AD/AR 28 25
TYMP Mitochondrial DNA depletion syndrome AR 84 94
UCHL1 Parkinson disease 5, autosomal dominant, Spastic paraplegia 79, autosomal recessive AD/AR 5 5
WFS1 Wolfram syndrome, Deafness, Wolfram-like syndrome, autosomal dominant, Deafness, autosomal dominant 6/14/38, Cataract 41 AD/AR 69 362
YME1L1* Optic atrophy 11 1 1
ZNHIT3# PEHO syndrome 5 1

* Some, or all, of the gene is duplicated in the genome. Read more.

# The gene has suboptimal coverage (means <90% of the gene’s target nucleotides are covered at >20x with mapping quality score (MQ>20) reads), and/or the gene has exons listed under Test limitations section that are not included in the panel as they are not sufficiently covered with high quality sequence reads.

The sensitivity to detect variants may be limited in genes marked with an asterisk (*) or number sign (#)

Gene refers to the HGNC approved gene symbol; Inheritance refers to inheritance patterns such as autosomal dominant (AD), autosomal recessive (AR), mitochondrial (mi), X-linked (XL), X-linked dominant (XLD) and X-linked recessive (XLR); ClinVar refers to the number of variants in the gene classified as pathogenic or likely pathogenic in this database (ClinVar); HGMD refers to the number of variants with possible disease association in the gene listed in Human Gene Mutation Database (HGMD). The list of associated, gene specific phenotypes are generated from CGD or Mitomap databases.

Non-coding variants covered by Neuro-Ophthalmology Panel

Gene Genomic location HG19 HGVS RefSeq RS-number
ANTXR1 Chr2:69472345 c.1435-12A>G NM_032208.2
FRMD7 ChrX:131228285 c.285-118C>T NM_194277.2
GPR143 ChrX:9708630 c.885+748G>A NM_000273.2
GPR143 ChrX:9711844 c.659-131T>G NM_000273.2
OPA1 Chr3:193334932 c.449-34dupA NM_130837.2
OPA1 Chr3:193374829 c.2179-40G>C NM_130837.2
PAX6 Chr11:31685945 c.*125537G>T NM_000280.4 rs606231388
PAX6 Chr11:31812434 c.1033-42_1033-26delATGTGTTCCTCAGTAACinsG NM_000280.4
PAX6 Chr11:31816377 c.524-41T>G NM_000280.4
PAX6 Chr11:31823338 c.142-14C>G NM_000280.4 rs1131692291
PAX6 Chr11:31828391 c.-52+5delG NM_000280.4
PAX6 Chr11:31828391 c.-52+3_-52+6delAAGTinsTG NM_000280.4
PAX6 Chr11:31828392 c.-52+3_-52+4delAA NM_000280.4
PAX6 Chr11:31828395 c.-52+1delG NM_000280.4
PAX6 Chr11:31828396 c.-52+1G>A NM_000280.4
PAX6 Chr11:31828456 c.-115_-112delACTA NM_000280.4 rs1011844558
PAX6 Chr11:31828461 c.-118_-117delTT NM_000280.4
PAX6 Chr11:31828469 c.-125dupG NM_000280.4
PAX6 Chr11:31828474 c.-128-1G>T NM_000280.4
PAX6 Chr11:31828474 c.-128-2delA NM_000280.4 rs1131692282
PAX6 Chr11:31832372 c.-138_-129+3delCCTCATAAAGGTG NM_000280.4
PAX6 Chr11:31832374 c.-129+2T>A NM_000280.4
PAX6 Chr11:31832375 c.-129+1G>A NM_000280.4
TIMM8A ChrX:100601671 c.133-23A>C NM_004085.3 rs869320666
WFS1 Chr4:6271704 c.-43G>T NM_006005.3

Added and removed genes from the panel

Genes added Genes removed
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
MT-TY

Test Strengths

The strengths of this test include:
  • CAP accredited laboratory
  • CLIA-certified personnel performing clinical testing in a CLIA-certified laboratory
  • Powerful sequencing technologies, advanced target enrichment methods and precision bioinformatics pipelines ensure superior analytical performance
  • Careful construction of clinically effective and scientifically justified gene panels
  • Some of the panels include the whole mitochondrial genome (please see the Panel Content section)
  • Our Nucleus online portal providing transparent and easy access to quality and performance data at the patient level
  • Our publicly available analytic validation demonstrating complete details of test performance
  • ~2,000 non-coding disease causing variants in our clinical grade NGS assay for panels (please see ‘Non-coding disease causing variants covered by this panel’ in the Panel Content section)
  • Our rigorous variant classification scheme
  • Our systematic clinical interpretation workflow using proprietary software enabling accurate and traceable processing of NGS data
  • Our comprehensive clinical statements

Test Limitations

The following exons are not included in the panel as they are not sufficiently covered with high quality sequence reads: TK2 (NM_001271934:3), TSFM (NM_001172696:5), ZNHIT3 (NM_001281432:5). Genes with suboptimal coverage in our assay are marked with number sign (#) and genes with partial, or whole gene, segmental duplications in the human genome are marked with an asterisk (*) if they overlap with the UCSC pseudogene regions. Gene is considered to have suboptimal coverage when >90% of the gene’s target nucleotides are not covered at >20x with mapping quality score (MQ>20) reads. The technology may have limited sensitivity to detect variants in genes marked with these symbols (please see the Panel content table above).

This test does not detect the following:
  • Complex inversions
  • Gene conversions
  • Balanced translocations
  • Some of the panels include the whole mitochondrial genome but not all (please see the Panel Content section)
  • Repeat expansion disorders unless specifically mentioned
  • Non-coding variants deeper than ±20 base pairs from exon-intron boundary unless otherwise indicated (please see above Panel Content / non-coding variants covered by the panel).
This test may not reliably detect the following:
  • Low level mosaicism in nuclear genes (variant with a minor allele fraction of 14.6% is detected with 90% probability)
  • Stretches of mononucleotide repeats
  • Low level heteroplasmy in mtDNA (>90% are detected at 5% level)
  • Indels larger than 50bp
  • Single exon deletions or duplications
  • Variants within pseudogene regions/duplicated segments
  • Some disease causing variants present in mtDNA are not detectable from blood, thus post-mitotic tissue such as skeletal muscle may be required for establishing molecular diagnosis.

The sensitivity of this test may be reduced if DNA is extracted by a laboratory other than Blueprint Genetics.

For additional information, please refer to the Test performance section and see our Analytic Validation.

The genes on the panel have been carefully selected based on scientific literature, mutation databases and our experience.

Our panels are sectioned from our high-quality, clinical grade NGS assay. Please see our sequencing and detection performance table for details regarding our ability to detect different types of alterations (Table).

Assays have been validated for various sample types including EDTA-blood, isolated DNA (excluding from formalin fixed paraffin embedded tissue), saliva and dry blood spots (filter cards). These sample types were selected in order to maximize the likelihood for high-quality DNA yield. The diagnostic yield varies depending on the assay used, referring healthcare professional, hospital and country. Plus analysis increases the likelihood of finding a genetic diagnosis for your patient, as large deletions and duplications cannot be detected using sequence analysis alone. Blueprint Genetics’ Plus Analysis is a combination of both sequencing and deletion/duplication (copy number variant (CNV)) analysis.

Performance of Blueprint Genetics high-quality, clinical grade NGS sequencing assay for panels.

Sensitivity % (TP/(TP+FN) Specificity %
Single nucleotide variants 99.89% (99,153/99,266) >99.9999%
Insertions, deletions and indels by sequence analysis
1-10 bps 96.9% (7,563/7,806) >99.9999%
11-50 bps 99.13% (2,524/2,546) >99.9999%
Copy number variants (exon level dels/dups)
1 exon level deletion (heterozygous) 100% (20/20) NA
1 exon level deletion (homozygous) 100% (5/5) NA
1 exon level deletion (het or homo) 100% (25/25) NA
2-7 exon level deletion (het or homo) 100% (44/44) NA
1-9 exon level duplication (het or homo) 75% (6/8) NA
Simulated CNV detection
5 exons level deletion/duplication 98.7% 100.00%
Microdeletion/-duplication sdrs (large CNVs, n=37))
Size range (0.1-47 Mb) 100% (37/37)
     
The performance presented above reached by Blueprint Genetics high-quality, clinical grade NGS sequencing assay with the following coverage metrics
     
Mean sequencing depth 143X
Nucleotides with >20x sequencing coverage (%) 99.86%


Performance of Blueprint Genetics Mitochondrial Sequencing Assay.

Sensitivity % Specificity %
ANALYTIC VALIDATION (NA samples; n=4)
Single nucleotide variants
Heteroplasmic (45-100%) 100.0% (50/50) 100.0%
Heteroplasmic (35-45%) 100.0% (87/87) 100.0%
Heteroplasmic (25-35%) 100.0% (73/73) 100.0%
Heteroplasmic (15-25%) 100.0% (77/77) 100.0%
Heteroplasmic (10-15%) 100.0% (74/74) 100.0%
Heteroplasmic (5-10%) 100.0% (3/3) 100.0%
Heteroplasmic (<5%) 50.0% (2/4) 100.0%
CLINICAL VALIDATION (n=76 samples)
All types
Single nucleotide variants n=2026 SNVs
Heteroplasmic (45-100%) 100.0% (1940/1940) 100.0%
Heteroplasmic (35-45%) 100.0% (4/4) 100.0%
Heteroplasmic (25-35%) 100.0% (3/3) 100.0%
Heteroplasmic (15-25%) 100.0% (3/3) 100.0%
Heteroplasmic (10-15%) 100.0% (9/9) 100.0%
Heteroplasmic (5-10%) 92.3% (12/13) 99.98%
Heteroplasmic (<5%) 88.9% (48/54) 99.93%
Insertions and deletions by sequence analysis n=40 indels
Heteroplasmic (45-100%) 1-10bp 100.0% (32/32) 100.0%
Heteroplasmic (5-45%) 1-10bp 100.0% (3/3) 100.0%
Heteroplasmic (<5%) 1-10bp 100.0% (5/5) 99,997%
SIMULATION DATA /(mitomap mutations)
Insertions, and deletions 1-24 bps by sequence analysis; n=17
Homoplasmic (100%) 1-24bp 100.0% (17/17) 99.98%
Heteroplasmic (50%) 100.0% (17/17) 99.99%
Heteroplasmic (25%) 100.0% (17/17) 100.0%
Heteroplasmic (20%) 100.0% (17/17) 100.0%
Heteroplasmic (15%) 100.0% (17/17) 100.0%
Heteroplasmic (10%) 94.1% (16/17) 100.0%
Heteroplasmic (5%) 94.1% (16/17) 100.0%
Copy number variants (separate artifical mutations; n=1500)
Homoplasmic (100%) 500 bp, 1kb, 5 kb 100.0% 100.0%
Heteroplasmic (50%) 500 bp, 1kb, 5 kb 100.0% 100.0%
Heteroplasmic (30%) 500 bp, 1kb, 5 kb 100.0% 100.0%
Heteroplasmic (20%) 500 bp, 1kb, 5 kb 99.7% 100.0%
Heteroplasmic (10%) 500 bp, 1kb, 5 kb 99.0% 100.0%
The performance presented above reached by following coverage metrics at assay level (n=66)
Mean of medians Median of medians
Mean sequencing depth MQ0 (clinical) 18224X 17366X
Nucleotides with >1000x MQ0 sequencing coverage (%) (clinical) 100%
rho zero cell line (=no mtDNA), mean sequencing depth 12X

Bioinformatics

The target region for each gene includes coding exons and ±20 base pairs from the exon-intron boundary. In addition, the panel includes non-coding and regulatory variants if listed above (Non-coding variants covered by the panel). Some regions of the gene(s) may be removed from the panel if specifically mentioned in the ‘Test limitations” section above. If the test includes the mitochondrial genome the target region gene list contains the mitochondrial genes. The sequencing data generated in our laboratory is analyzed with our proprietary data analysis and annotation pipeline, integrating state-of-the art algorithms and industry-standard software solutions. Incorporation of rigorous quality control steps throughout the workflow of the pipeline ensures the consistency, validity and accuracy of results. Our pipeline is streamlined to maximize sensitivity without sacrificing specificity. We have incorporated a number of reference population databases and mutation databases including, but not limited, to 1000 Genomes Project, gnomAD, ClinVar and HGMD into our clinical interpretation software to make the process effective and efficient. For missense variants, in silico variant prediction tools such as  SIFT, PolyPhen, MutationTaster are used to assist with variant classification. Through our online ordering and statement reporting system, Nucleus, ordering providers have access to the details of the analysis, including patient specific sequencing metrics, a gene level coverage plot and a list of regions with suboptimal coverage (<20X for nuclear genes and <1000X for mtDNA) if applicable. This reflects our mission to build fully transparent diagnostics where ordering providers can easily visualize the crucial details of the analysis process.

Clinical interpretation

We provide customers with the most comprehensive clinical report available on the market. Clinical interpretation requires a fundamental understanding of clinical genetics and genetic principles. At Blueprint Genetics, our PhD molecular geneticists, medical geneticists and clinical consultants prepare the clinical statement together by evaluating the identified variants in the context of the phenotypic information provided in the requisition form. Our goal is to provide clinically meaningful statements that are understandable for all medical professionals regardless of whether they have formal training in genetics.

Variant classification is the corner stone of clinical interpretation and resulting patient management decisions. Our classifications follow the ACMG guideline 2015.

The final step in the analysis is orthogonal confirmation. Sequence and copy number variants classified as pathogenic, likely pathogenic and variants of uncertain significance (VUS) are confirmed using bi-directional Sanger sequencing by orthogonal methods such as qPCR/ddPCR when they do not meet our stringent NGS quality metrics for a true positive call.

Our clinical statement includes tables for sequencing and copy number variants that include basic variant information (genomic coordinates, HGVS nomenclature, zygosity, allele frequencies, in silico predictions, OMIM phenotypes and classification of the variant). In addition, the statement includes detailed descriptions of the variant, gene and phenotype(s) including the role of the specific gene in human disease, the mutation profile, information about the gene’s variation in population cohorts and detailed information about related phenotypes. We also provide links to the references, abstracts and variant databases used to help ordering providers further evaluate the reported findings if desired. The conclusion summarizes all of the existing information and provides our rationale for the classification of the variant.

Identification of pathogenic or likely pathogenic variants in dominant disorders or their combinations in different alleles in recessive disorders are considered molecular confirmation of the clinical diagnosis. In these cases, family member testing can be used for risk stratification. We do not recommend using variants of uncertain significance (VUS) for family member risk stratification or patient management. Genetic counseling is recommended.

Our interpretation team analyzes millions of variants from thousands of individuals with rare diseases. Our internal database and our understanding of variants and related phenotypes increases with every case analyzed. Our laboratory is therefore well-positioned to re-classify previously reported variants as new information becomes available. If a variant previously reported by Blueprint Genetics is re-classified, our laboratory will issue a follow-up statement to the original ordering health care provider at no additional cost.

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