Forms

Erythropoietic Protoporphyria via the FECH Gene

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

Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
1691 FECH$840.00 81479 Add to Order
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 18 days.

Clinical Sensitivity

Causative mutations in the FECH gene were found in 140 of 151 patients with protoporphyia with the remaining 11 demonstrating X-linked forms through mutation in the ALAS2 gene (Balwani et al. 2013). Analytical sensitivity for detection of FECH mutations is ~90% as gross deletions cannot be detected by this method and are responsible for ~10% of EPP cases (Whatley et al. 2007; Gouya et al. 2006).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 FECH$690.00 81479 Add to Order
Pricing Comment

# of Genes Ordered

Total Price

1

$690

2

$730

3

$770

4-10

$840

11-30

$1,290

31-100

$1,670

Over 100

Call for quote

Turnaround Time

The great majority of tests are completed within 28 days.

Clinical Features

Erythropoietic protoprophyria (EPP) is an inborn error of heme biosynthesis resulting in cutaneous photosensitivity. Symptoms include acute painful photosensitivity with stinging and burning sensations upon sunlight exposure but without blistering. Chronic permanent skin lesions, anemia, iron deficiency, cholelithiasis, chronic liver disease are potentially life threatening secondary symptoms of EPP (Lecha et al. 2009). EPP onset is during the first year of life, but mean diagnosis age is 22 underlying the difficulties in accurately diagnosing EPP (Wahlin et al. 2010). Genetic testing can be helpful in the differential diagnosis of EPP from phototoxic drug reactions, hydroa vacciniforme, solar urticarial, contact dermatitis, angioedema and other forms of porphyria. Standard treatments include avoidance of sun exposure and beta carotene therapy.

Genetics

EPP is inherited in an autosomal recessive manner through mutations in the FECH gene. In greater than 90% of cases, a pathogenic variant is co-inherited in trans with the hypomorphic variant c.315-48T>C (IVS3-48C) (Balwani et al. 2012). Patients homozygous for the c.315-48C>T variant have been reported to have EPP in rare cases (Whatley et al. 2004), but the penetrance is low. The c.315-48C>T variant, which reduces FECH transcript levels through generation of a cryptic acceptor splice site, leads to nonsense mediated decay and lower FECH protein levels (Gouya et al. 2006; Gouya et al. 2002). Pathogenic variants reported in trans to the c.315-48C>T variant are missense, splice site alterations, small insertion/deletions, and nonsense mutations making up 32%, 22%, 19%, and 14% of EPP mutations (Gouya et al. 2006). The minor allele frequency for the c.315-48C>T variant is 43%, 31%, 11%, 2.7%, and <1% in Japanese, southeast Asian, Caucasian French, North African, and African American populations with EPP being nearly absent in the later ethic cohorts. Gross deletions in the FECH gene have been identified in ~10% of cases of EPP (Whatley et al. 2007). An X-linked form of protoporphyria representing ~2% of cases has been reported to occur through gain of function mutations in the ALAS2 gene (Balwani et al. 2013). Somatic mutations have been reported in the FECH gene in patients with myelodysplasia (Aplin et al. 2001). The FECH protein catalyzes the insertion of ferrous iron into protoporphyrin IX to form heme. When protoporphyrin accumulates in tissues it becomes photo-activated resulting in excess energy to be transferred to oxygen. This leads to heightened reactive oxygen species causing cellular damage and disease (Lecha et al. 2009).

Testing Strategy

Our DNA sequencing test involves bidirectional Sanger sequencing of the entire FECH gene plus ~20bp of flanking non-coding DNA on either side of each exon, 300bp upstream of the start codon, and coverage of the c.315-48T>C intronic variant. We will also sequence any single exon (Test#100) or pair of exons (Test#200) in family members of patients with known mutation or to confirm research results.

Indications for Test

Patients displaying painful photosensitivity with erythema and burning without blisters are hallmark characteristics of EPP. Plasma fluorescence peaks at 634nm confirms presence of high free protoporphrin levels. Ideal candidates have biochemical evidence demonstrating impaired FECH activity (Lecha et al. 2009).

Gene

Official Gene Symbol OMIM ID
FECH 612386
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Disease

Name Inheritance OMIM ID
Erythropoietic Protoporphyria 177000

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Aplin C, Whatley SD, Thompson P, Hoy T, Fisher P, Singer C, Lovell CR, Elder GH. 2001. Late-onset erythropoietic porphyria caused by a chromosome 18q deletion in erythroid cells. Journal of investigative dermatology 117: 1647–1649. PubMed ID: 11886534
  • Balwani M, Bloomer J, Desnick R. 2012. Erythropoietic Protoporphyria, Autosomal Recessive. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 23016163
  • Balwani M, Bloomer J, Desnick R. 2013. X-Linked Protoporphyria. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews(®), Seattle (WA): University of Washington, Seattle. PubMed ID: 23409301
  • Gouya L, Martin-Schmitt C, Robreau A-M, Austerlitz F, Silva V Da, Brun P, Simonin S, Lyoumi S, Grandchamp B, Beaumont C, others. 2006. Contribution of a common single-nucleotide polymorphism to the genetic predisposition for erythropoietic protoporphyria. The American Journal of Human Genetics 78: 2–14. PubMed ID: 16385445
  • Gouya L, Puy H, Robreau A-M, Bourgeois M, Lamoril J, Silva VD, Grandchamp B, Deybach J-C. 2002. The penetrance of dominant erythropoietic protoporphyria is modulated by expression of wildtype FECH. Nature Genetics 30: 27–28. PubMed ID: 11753383
  • Lecha M, Puy H, Deybach J-C. 2009. Erythropoietic protoporphyria. Orphanet Journal of Rare Diseases 4: 19. PubMed ID: 19744342
  • Wahlin S, Floderus Y, Stål P, Harper P. 2011. Erythropoietic protoporphyria in Sweden: demographic, clinical, biochemical and genetic characteristics: EPP in Sweden. Journal of Internal Medicine 269: 278–288. PubMed ID: 20412370
  • Whatley SD, Mason NG, Holme SA, Anstey AV, Elder GH, Badminton MN. 2007. Gene Dosage Analysis Identifies Large Deletions of the FECH Gene in 10% of Families with Erythropoietic Protoporphyria. Journal of Investigative Dermatology 127: 2790–2794. PubMed ID: 17597821
  • Whatley SD, Mason NG, Khan M, Zamiri M, Badminton MN, Missaoui WN, Dailey TA, Dailey HA, Douglas WS, Wainwright NJ, Elder GH. 2004. Autosomal recessive erythropoietic protoporphyria in the United Kingdom: prevalence and relationship to liver disease. J. Med. Genet. 41: e105. PubMed ID: 15286165
Order Kits
TEST METHODS

Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (http://www.hgvs.org).  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 20 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of March 2016, we compared 17.37 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 12 years of our lab operation we have Sanger sequenced roughly 8,800 PCR amplicons. Only one error has been identified, and this was due to sequence analysis error.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 20 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all 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 for example 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 PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Deletion/Duplication Testing Via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

Order Kits

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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