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Birt-Hogg-Dube Syndrome via FLCN Gene Sequencing with CNV Detection

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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TEST METHODS

Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
8409 FLCN$890.00 81479,81479 Add to Order
Pricing Comment

Our favored testing approach is exome based NextGen sequencing with CNV analysis. This will allow cost effective reflexing to PGxome or other exome based tests. However, if full gene Sanger sequencing is desired for STAT turnaround time, insurance, or other reasons, please see link below for Test Code, pricing, and turnaround time information. If the Sanger option is selected, CNV detection may be ordered through Test #600.

For Sanger Sequencing click here.
Targeted Testing

For ordering targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 26 days.

Clinical Sensitivity

FLCN causative mutations will be detected in approximately 88% of individuals with BHDS by sequencing. Almost half of the individuals with BHDS will have a deletion (c.1285delC) or duplication (c.1285dupC) of a C nucleotide in the polycytosine tract in exon 11 (Toro. GeneReviews. 2008). Gross deletions or duplications that are not normally detected via sequencing will not be detected using this method.

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Clinical Features

Birt-Hogg-Dubé syndrome (BHDS) includes cutaneous manifestations, such as fibrofolliculomas (yellowish dome-shaped papules), trichodiscomas (multiple small papules), and acrochordons (skin tags). Skin features typically appear in the third to fourth decade of life and continually grow in size and number with age. BHDS is also characterized by pulmonary cysts/history of pneumothorax, and various types of renal tumors. The renal tumors that develop are usually bilateral and multifocal, and slow growing (Toro. GeneReviews. 2008). Tumor types include renal oncocytoma, chromophobe renal cell carcinoma, oncocytic hybrid tumor, and a minority of clear cell renal cell carcinoma. The median diagnosis of renal tumors is 48 years of age (range 31-71 years) (Schmidt et al. Am J Hum Genet 76:1023–33, 2005). Some families have renal tumor and/or autosomal dominant spontaneous pneumothorax without cutaneous manifestations. Early diagnosis is important for intervention and improving patient morbidity, since mutations in the FLCN gene may be associated with other cancers (Palmirotta et al. Anticancer Research 30: 751-758 2010; Toro et al. J Med Genet 45:321–331, 2008).

Genetics

BHDS is inherited in an autosomal dominant pattern with high penetrance and is caused by mutations in the FLCN gene. The proportion of de novo versus inherited mutations in FLCN is currently unknown. FLCN encodes the folliculin protein, which is highly expressed in skin, type 1 pneumocytes, and distal nephrons of the kidney (Toro. GeneReviews. 2008). Its exact function is unknown, although it may act downstream of rapamycin (mTOR), adenosine monophosphate-activated protein kinase (AMPK), and have a role in the modulation of energy/nutrient sensing and signaling pathways (Hartnan Oncogene 28(13): 1594-1604, 2009). It has also been suggested to have a role in tumor suppression from the observation that germline mutations in FLCN cause renal tumors, and the presence of somatic mutations and loss of heterozygosity in tumors tissues. The most frequent causative mutation occurs within a polycytosine C8 tract of exon 11 . This mutation probably occurs due to DNA polymerase slippage resulting in gains or losses of repeat units leading to truncated folliculin. Other causative mutations are located throughout the FLCN gene (Palmirotta et al. Anticancer Research 30: 751-758 2010). These include missense, nonsense, splicing, small and large insertions and deletions (Human Gene Mutation Database).

Testing Strategy

For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads. 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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This test provides full coverage of all coding exons of the FLCN gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Since this test is performed using exome capture probes, a reflex to any of our exome based tests is available (PGxome, PGxome Custom Panels).

Indications for Test

Molecular genetic testing is suggested for individuals known to have or suspected of having BHDS, including individuals with either one major or two minors findings as listed below (Menko et al. Lancet 10:1199, 2009):

Major criteria

• At least five fibrofolliculomas or trichodiscomas (at least one histologically confirmed)

Minor criteria

• Multiple lung cysts: bilateral basally located lung cysts with no other apparent cause, with or without spontaneous primary pneumothorax

• Renal cancer: early onset (<50 years) or multifocal or bilateral renal cancer, or renal cancer of mixed chromophobe and oncocytic histology

• A first-degree relative with BHDS

Gene

Official Gene Symbol OMIM ID
FLCN 607273
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
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Interstitial Lung Disease Sequencing Panel with CNV Detection
Neonatal Crisis Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Hartnan et al. 2008. The role of the Birt-Hogg-Dubé protein in mTOR activation and renal tumorigenesis. Oncogene 28(13): 1594-1604. PubMed ID: 19234517
  • Human Gene Mutation Database (Bio-base).
  • Menko et al. (2009) "Birt-Hogg-Dubé syndrome: diagnosis and management."  Lancet 10:1199. PubMed ID: 19959076
  • Palmirotta et al. (2010) "Association between Birt Hogg Dube syndrome and cancer predisposition." Anticancer Research 30: 751-758. PubMed ID: 20392993
  • Schmidt et al. (2005) "Germline BHD-mutation spectrum and phenotype analysis of a large cohort of families with Birt-Hogg-Dubé syndrome." Am J Hum Genet 76:1023–33,. PubMed ID: 15852235
  • Toro et al. (2008) "BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dubé syndrome: a new series of 50 families and a review of published reports." J Med Genet 45:321–331. PubMed ID: 18234728
  • Toro JR. 1993. Birt-Hogg-Dubé Syndrome. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle,. PubMed ID: 20301695
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TEST METHODS

Exome Sequencing with CNV Detection

Test Procedure

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 on the NovaSeq 6000 using 2x150 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 NovaSeq 6000 is converted to fastqs by Illumina Bcl2Fastq, 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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

Analytical Validity

Copy Number Variant Analysis: The PGxome test detects most larger deletions and duplications including intragenic CNVs and large cytogenetic events; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g., 1-3 exons vs. 4 or more exons), and inadequate coverage. In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is >95%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.

Analytical Limitations

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.

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 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.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.  

In nearly all cases, our ability to determine the exact copy number change within a targeted region is limited.

Our ability to detect CNVs due to somatic mosaicism is limited.

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 sample 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.

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Ordering Options


myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
REQUISITION FORM
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.

SPECIMEN TYPES
WHOLE BLOOD

(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.

DNA

(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.

CELL CULTURE

(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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