NF1/SPRED1 Next Generation Sequencing and Deletion/Duplication - Clinical Genetic Test - GTR

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NF1/SPRED1 Next Generation Sequencing and Deletion/Duplication

Clinical Genetic Test

offered by

UAB Medical Genomics Laboratory

View lab's test page

GTR Test Accession: GTR000501087.8

INHERITED DISEASEDYSMORPHOLOGYINHERITED DISEASE SUSCEPTIBILITY ... View more

Last updated in GTR: 2024-07-23

View version history

GTR000501087.8, last updated: 2024-07-23

GTR000501087.7, last updated: 2023-06-07

GTR000501087.6, last updated: 2022-06-08

GTR000501087.5, last updated: 2021-06-14

GTR000501087.4, last updated: 2019-06-20

GTR000501087.3, last updated: 2018-07-10

GTR000501087.2, last updated: 2015-02-09

GTR000501087.1, registered in GTR: 2013-04-11

Last annual review date for the lab: 2023-06-07 Past due

At a Glance

Test purpose:

Diagnosis; Screening

Conditions (4):

Neurofibromatosis, type 1; Cafe au lait spots, multiple; Legius syndrome more...

Genes (2):

NF1 (17q11.2); SPRED1 (15q14)

Methods (2):

Molecular Genetics - Deletion/duplication analysis: Multiplex Ligation-dependent Probe Amplification (MLPA); ...

Target population:

Patients with multiple CALMs w/wo skinfold freckling and no other …

Clinical validity:

>95% detection rate for non-founder patients who meet NIH diagnostic …

Clinical utility:

Establish or confirm diagnosis

Ordering Information

Offered by:

UAB Medical Genomics Laboratory
View lab's website
View lab's test page

Test short name:

NFSP-NG

Specimen Source:

Cord blood
Fibroblasts
Isolated DNA
Peripheral (whole) blood
Saliva
View specimen requirements

Who can order:

Genetic Counselor
Health Care Provider
Licensed Physician
Nurse Practitioner
Physician Assistant

Test Order Code:

NFSP-NG
View other test codes

CPT codes:

**AMA CPT codes notice

Lab contact:

Brandon Shaw, MS, CGC, Certified Genetic counselor, CGC, Genetic Counselor
brandonshaw@uabmc.edu
205-934-1520
Victoria Moy, MS, Genetic Counselor
vmoy@uabmc.edu
205-934-5528

Contact Policy:

Pre-test email/phone consultation regarding genetic test results and interpretation is provided to patients/families.

How to Order:

Additional information regarding the specific details needed for test submission can be found on our website
Order URL

Test service:

Clinical Testing/Confirmation of Mutations Identified Previously
Custom Deletion/Duplication Testing
Custom Sequence Analysis
Result interpretation

Test additional service:

Custom mutation-specific/Carrier testing

Test development:

Test developed by laboratory but exempt from FDA oversight (eg. NYS CLEP approved, offered within a hospital or clinic)

Informed consent required:

Based on applicable state law

Test strategy:

The DNA-based NF1/SPRED1-only by NGS involves sequencing as well as deletion/duplication analysis of the entire coding NF1 region plus the alternatively spliced exons 9br, 23a and 48a (67 exons total), as well as sequencing and deletion/duplication analysis for SPRED1. The test uses an extensively customized and optimized set of Agilent … The DNA-based NF1/SPRED1-only by NGS involves sequencing as well as deletion/duplication analysis of the entire coding NF1 region plus the alternatively spliced exons 9br, 23a and 48a (67 exons total), as well as sequencing and deletion/duplication analysis for SPRED1. The test uses an extensively customized and optimized set of Agilent HaloPlex capture probes, followed by sequencing of overlapping amplicons within the regions of interest using 300bp paired-end Illumina sequencing chemistry. Each coding exon plus ~50bp of flanking intronic sequence are simultaneously sequenced. 5’ and 3’ untranslated sequences are not included. The average coverage is >1800x with >98.7% of the NF1 coding region ≥350x and 99.9% ≥200x, allowing detection of very low level mosaicism, down to 3-5% variant allele fraction respectively (regions covered by ≥350x respectively ≥200x) with 95% confidence. Variant and copy number calls are made using a unique bioinformatics pipeline detecting all types of variants including single nucleotide substitutions, indels, and frameshifts caused by deletion/ duplication up to 112bp.
Based on >15 years of experience with comprehensive RNA-based NF1 testing, we designed the customized and optimized NGS NF1-component of the assay to comprise all regions encountered through analysis of >15,000 unrelated individuals including >8,100 NF1-variant-positive individuals carrying 1 out of >3,100 different unique NF1 variants identified in the UAB MGL cohort. Included in the NGS assay are the regions covering >65 different deep intronic splice variants (which reside beyond the +/-50 intronic base pairs that flank all exons). Validation of the full panel included, besides substitutions (missense, nonsense, splice variants), the most challenging variants such as insertions/deletions/duplications of 1-112bp (~25% of the UAB NF1 cohort) and one-to-multiple exon deletions/duplications (~2.8% of the UAB NF1 cohort). The analytical sensitivity of our NGS testing approach was 100% for substitutions as well as insertion/deletions up to 112bp. This panel has not yet been validated to identify deletions/duplications >112bp and <1 exon, but such variants have not yet been found in the UAB cohort, and therefore are likely very rare. The panel has been validated for the detection of germline (heterozygous) single-exon deletions/duplications as well as multi-exon deletions/duplications, however mosaic single-exon deletion/duplications validation is still pending. Single exon deletions/duplications are present in ~0.45% of NF1-positive patients from the UAB cohort with 9% of these individuals being mosaic (~0.045% of all in the UAB NF1-positive cohort). Detection of Alu/LINE insertions, identified in 0.25% of patients from the UAB NF1-positive cohort, has not yet been validated using the current NGS approach.
With the largest dataset of NF1 genotypes matched with phenotypes, any genotype-phenotype correlations identified will be reported in real time.
Confirmatory testing of reportable variants is performed by Sanger sequencing or other orthogonal methods.
For novel NF1 variants of unknown significance, we offer free of charge targeted RNA-based testing to assess the effect of the variant on splicing and enhance the correct classification/ interpretation.
Relevant family members of a proband with any (novel or previously identified) variant of unknown significance are offered free of charge targeted analysis as long as accurate phenotypic data are provided by a health care professional to enhance the interpretation. There is no limitation to the number of relatives that can be tested free of charge.
Mosaicism is often present in sporadic patients with an NF1 microdeletion and has important repercussions for counseling. Free of charge evaluation by FISH analysis on 200 interphase chromosomes is offered in such cases. View more

View citations (10)

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Messiaen LM, et al. Hum Mutat. 2000;15(6):541-55. doi:10.1002/1098-1004(200006)15:6<541::AID-HUMU6>3.0.CO;2-N. PMID: 10862084.
Pitfalls of automated comparative sequence analysis as a single platform for routine clinical testing for NF1. Messiaen LM, et al. J Med Genet. 2005;42(5):e25. PMID: 15863657.
Spectrum of single- and multiexon NF1 copy number changes in a cohort of 1,100 unselected NF1 patients. Wimmer K, et al. Genes Chromosomes Cancer. 2006;45(3):265-76. doi:10.1002/gcc.20289. PMID: 16283621.
Upadhyaya M, Huson SM, Davies M, Thomas N, Chuzhanova N, Giovannini S, Evans DG, Howard E, Kerr B, Griffiths S, Consoli C, Side L, Adams D, Pierpont M, Hachen R, Barnicoat A, Li H, Wallace P, Van Biervliet JP, Stevenson D, Viskochil D, Baralle D, Haan E, Riccardi V, Turnpenny P, Lazaro C, Messiaen L. An absence of cutaneous neurofibromas associated with a 3-bp inframe deletion in exon 17 of the NF1 gene (c.2970-2972 delAAT): evidence of a clinically significant NF1 genotype-phenotype correlation. Am J Hum Genet. 2007;80(1):140-51. doi:10.1086/510781. Epub 2006 Dec 08. PMID: 17160901.
Extensive in silico analysis of NF1 splicing defects uncovers determinants for splicing outcome upon 5' splice-site disruption. Wimmer K, et al. Hum Mutat. 2007;28(6):599-612. doi:10.1002/humu.20493. PMID: 17311297.
Maertens O, De Schepper S, Vandesompele J, Brems H, Heyns I, Janssens S, Speleman F, Legius E, Messiaen L. Molecular dissection of isolated disease features in mosaic neurofibromatosis type 1. Am J Hum Genet. 2007;81(2):243-51. doi:10.1086/519562. Epub 2007 Jun 20. PMID: 17668375.
Mosaic type-1 NF1 microdeletions as a cause of both generalized and segmental neurofibromatosis type-1 (NF1). Messiaen L, et al. Hum Mutat. 2011;32(2):213-9. doi:10.1002/humu.21418. PMID: 21280148.
The diagnostic evaluation and multidisciplinary management of neurofibromatosis 1 and neurofibromatosis 2. Gutmann DH, et al. JAMA. 1997;278(1):51-7. PMID: 9207339.
Huson, SM; Hughes, RAC: The Neurofibromatoses: a Pathogenetic and Clinical Overview. London: Chapman & Hall Medical, 1994
Messiaen LM and Wimmer K (2008) NF1 Mutational Spectrum, in Kaufmann D (ed): Neurofibromatoses. Monogr Hum Genet. Basel, Karger, Vol 16:63-77

Pre-test genetic counseling required:

Decline to answer

Post-test genetic counseling required:

Decline to answer

Conditions

Total conditions: 4

Condition/Phenotype	Identifier

Test Targets

Genes

Total genes: 2

Gene	Associated Condition	Germline or Somatic	Allele (Lab-provided)	Variant in NCBI

Methodology

Total methods: 2

Method Category

Test method

Instrument

Deletion/duplication analysis

Multiplex Ligation-dependent Probe Amplification (MLPA)

Applied Biosystems 3730 capillary sequencing instrument

Sequence analysis of the entire coding region

Next-Generation (NGS)/Massively parallel sequencing (MPS)

Other

Clinical Information

Test purpose:

Diagnosis; Screening

Clinical validity:

>95% detection rate for non-founder patients who meet NIH diagnostic criteria for NF1.

View citations (1)

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Messiaen LM, et al. Hum Mutat. 2000;15(6):541-55. doi:10.1002/1098-1004(200006)15:6<541::AID-HUMU6>3.0.CO;2-N. PMID: 10862084.

Clinical utility:

Establish or confirm diagnosis

View citations (1)

https://www.uab.edu/medicine/genetics/medical-genomics-laboratory/testing-services/nf1-legius-syndrome-and-rasopathies/nfsp-ng

Target population:

Patients with multiple CALMs w/wo skinfold freckling and no other typical NF1 associated features (Lisch nodules, bone abnormalities, neurofibromas, optic pathway gliomas).

Variant Interpretation:

What is the protocol for interpreting a variation as a VUS?
Variants are interpreted based on ACMG criteria and internal protocols. To further investigate a VUS, the laboratory will: 1. Review software predictions (REVEL, SIFT, PolyPhen, SpliceAI, etc). 2. Review internal patient database to determine if the variant has been identified previously in our patient cohort and review all available data. … View more

Are family members with defined clinical status recruited to assess significance of VUS without charge?
Yes.

Will the lab re-contact the ordering physician if variant interpretation changes?
Not provided.

Research:

Is research allowed on the sample after clinical testing is complete?
The laboratory may preform further studies on samples submitted for clinical testing only for disorders that may also help to explain the patient's phenotype.

Recommended fields not provided:

Will the lab re-contact the ordering physician if variant interpretation changes?, Sample negative report, Sample positive report

Technical Information

Test Procedure:

The DNA-based NF1/SPRED1-only by NGS involves sequencing as well as deletion/duplication analysis of the entire coding NF1 region plus the alternatively spliced exons 9br, 23a and 48a (67 exons total), as well as sequencing and deletion/duplication analysis for SPRED1. The test uses an extensively customized and optimized set of Agilent … The DNA-based NF1/SPRED1-only by NGS involves sequencing as well as deletion/duplication analysis of the entire coding NF1 region plus the alternatively spliced exons 9br, 23a and 48a (67 exons total), as well as sequencing and deletion/duplication analysis for SPRED1. The test uses an extensively customized and optimized set of Agilent HaloPlex capture probes, followed by sequencing of overlapping amplicons within the regions of interest using 300bp paired-end Illumina sequencing chemistry. Each coding exon plus ~50bp of flanking intronic sequence are simultaneously sequenced. 5’ and 3’ untranslated sequences are not included. The average coverage is >1800x with >98.7% of the NF1 coding region ≥350x and 99.9% ≥200x, allowing detection of very low level mosaicism, down to 3-5% variant allele fraction respectively (regions covered by ≥350x respectively ≥200x) with 95% confidence. Variant and copy number calls are made using a unique bioinformatics pipeline detecting all types of variants including single nucleotide substitutions, indels, and frameshifts caused by deletion/ duplication up to 112bp. Based on >15 years of experience with comprehensive RNA-based NF1 testing, we designed the customized and optimized NGS NF1-component of the assay to comprise all regions encountered through analysis of >15,000 unrelated individuals including >8,100 NF1-variant-positive individuals carrying 1 out of >3,100 different unique NF1 variants identified in the UAB MGL cohort. Included in the NGS assay are the regions covering >65 different deep intronic splice variants (which reside beyond the +/-50 intronic base pairs that flank all exons). Validation of the full panel included, besides substitutions (missense, nonsense, splice variants), the most challenging variants such as insertions/deletions/duplications of 1-112bp (~25% of the UAB NF1 cohort) and one-to-multiple exon deletions/duplications (~2.8% of the UAB NF1 cohort). The analytical sensitivity of our NGS testing approach was 100% for substitutions as well as insertion/deletions up to 112bp. This panel has not yet been validated to identify deletions/duplications >112bp and <1 exon, but such variants have not yet been found in the UAB cohort, and therefore are likely very rare. The panel has been validated for the detection of germline (heterozygous) single-exon deletions/duplications as well as multi-exon deletions/duplications, however mosaic single-exon deletion/duplications validation is still pending. Single exon deletions/duplications are present in ~0.45% of NF1-positive patients from the UAB cohort with 9% of these individuals being mosaic (~0.045% of all in the UAB NF1-positive cohort). Detection of Alu/LINE insertions, identified in 0.25% of patients from the UAB NF1-positive cohort, has not yet been validated using the current NGS approach. With the largest dataset of NF1 genotypes matched with phenotypes, any genotype-phenotype correlations identified will be reported in real time. Confirmatory testing of reportable variants is performed by Sanger sequencing or other orthogonal methods. For novel NF1 variants of unknown significance, we offer free of charge targeted RNA-based testing to assess the effect of the variant on splicing and enhance the correct classification/ interpretation. Relevant family members of a proband with any (novel or previously identified) variant of unknown significance are offered free of charge targeted analysis as long as accurate phenotypic data are provided by a health care professional to enhance the interpretation. There is no limitation to the number of relatives that can be tested free of charge. Mosaicism is often present in sporadic patients with an NF1 microdeletion and has important repercussions for counseling. Free of charge evaluation by FISH analysis on 200 interphase chromosomes is offered in such cases. View more

View citations (1)

Messiaen et al 2000, Messiaen and Wimmer 2005, Wimmer et al 2007, Messiaen and Wimmer, 2008

Availability:

Tests performed
Entire test performed in-house

Analytical Validity:

Analytical sensitivity of next generation sequencing is typically validated using a minimum of 20 DNA samples. No false positives were detected during analysis. The test uses an extensively customized and optimized set of Agilent HaloPlex capture probes, followed by sequencing of overlapping amplicons within the regions of interest using 300bp … View more

Proficiency testing (PT):

Is proficiency testing performed for this test?
Yes

Method used for proficiency testing:
Alternative Assessment

PT Provider:
reanalysis of internal, blinded specimens

Description of PT method:
Reanalysis of internally blinded specimens

Description of internal test validation method: Help
For details, please review article cited below.

VUS:

Software used to interpret novel variations
Alamut, Google Scholar search, Mastermind literature search, LOVD, evolutionary consevation, Grantham score, splicing prediction software (SpliceAI), disorder specific databases as necessary.

Laboratory's policy on reporting novel variations
The laboratory will issue a report summarizing what is currently known about the variant along with its current classification based on ACMG criteria. If applicable, further studies such as RNA-based testing and/or familial studies will be offered to potentially further clarify the clinical significance of the variant. If further studies … View more

Recommended fields not provided:

Test Confirmation, Assay limitations, Citations for Analytical validity, Major CAP category, CAP category, CAP test list

Regulatory Approval

FDA Review:

Category: FDA exercises enforcement discretion

Additional Information

Reviews:

Genereviews

PubMed Clinical Queries

Clinical resources:

Molecular resources:

View NF1 variations in ClinVar

RefSeqGene

Coriell Institute for Medical Research

Consumer resources:

Genetic Alliance

MalaCards

MedlinePlusGenetics (GHR)

NCATS Office of Rare Diseases Research (GARD)

MedlinePlus

IMPORTANT NOTE: NIH does not independently verify information submitted to GTR; it relies on submitters to provide information that is accurate and not misleading. NIH makes no endorsements of tests or laboratories listed in GTR. GTR is not a substitute for medical advice. Patients and consumers with specific questions about a genetic test should contact a health care provider or a genetics professional.