Congenital Erythropoietic Porphyria via the UROS Gene
- Summary and Pricing
- Clinical Features and Genetics
|Test Code||Test Copy Genes||Price||CPT Code Copy CPT Codes|
Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.
For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.
The great majority of tests are completed within 20 days.
In a series of unrelated patients with CEP, pathogenic variants in the UROS gene were identified in 24 of 27 cases (Katugampola et al. 2012). Analytical sensitivity should be high as nearly all reported variants are detectable by sequencing. Only one case of a gross deletion encompassing exons 2-3 has been reported and is not detectable by sequencing (Katugampola et al. 2012).
Congenital Erythropoietic Porphyria (CEP), also known as Günther’s disease, is a metabolic disorder due to impairment of the fourth enzyme, uroporphyrinogen III synthase (UROS), in the heme biosynthetic pathway. Symptom onset is variable and corollary to the degree of UROS activity and amount of sun exposure. In severe cases, infants present at birth with skin blistering, hydrops fetalis, and are prone to secondary dermal infections which can lead to scarring. Other symptoms include corneal ulcers, reddish-brown coloring of teeth, mild bone loss, hemolytic anemia and bone marrow expansion. Milder cases of CEP present in adulthood with cutaneous photosensitivity (Katugampola et al. 2012). Vitamin D deficiency can be a secondary effect of CEP due to avoidance of sun exposure. Treatments for CEP include avoidance of sun exposure, vitamin D supplementation, and transfusions if hemolytic anemia is severe. Bone marrow transplantation is currently the only curative therapy. Genetic testing is helpful in the differential diagnosis of CEP from other types of porphyria, epidermolysis bullosa, and myelodysplastic syndrome (Erwin et al. 2013; Karim et al. 2015).
CEP is inherited in an autosomal recessive manner through pathogenic variants in the UROS gene. Clinical phenotype is related to the degree of UROS activity with severe individuals having undetectable enzymatic levels. Biallelic pathogenic variants in the UROS gene are fully penetrant with the majority of cases presenting during infancy. Missense pathogenic variants occur in the majority of cases and have been reported throughout the coding region. The c.217T>C (p.Cys73Arg) variant is present in about a third of cases and is the most commonly found pathogenic variant in the UROS gene (Fortian et al. 2009; Erwin et al. 2013; Warner et al. 2002). Splice site alterations, small insertions/deletions, and promoter variants have also been reported in a minority of cases of CEP (Solis et al. 2001; Xu et al. 1995). Gross deletions encompassing one or more exons have only been reported in one case (Katugampola et al. 2012). The UROS gene encodes the uroporphyrinogen III synthase with catalyzes hydroxymethylbilane to uroporphyrinogen III in the heme biosynthetic pathway. Deposition of porphyrin isomers in tissues become photo-activated leading to oxygen radical formation and damage (Karim et al. 2015).
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.
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.
This test provides full coverage of all coding exons of the UROS gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.
Indications for Test
Candidates for testing include individuals with non-immune hydrops fetalis and cutaneous photosensitivity. Biochemical findings indicative of CEP include decreased uroporphyrinogen III synthase activity in erythrocytes (typically less than 10% of normal), and increased urinary uroporphyrin I and coproporphyrin I isomers (Erwin et al. 1993; Katugampola et al. 2012).
|Official Gene Symbol||OMIM ID|
- Genetic Counselor Team - firstname.lastname@example.org
- Luke Drury, PhD - email@example.com
- Erwin A. et al. 2013. Congenital Erythropoietic Porphyria. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, Smith RJ, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 24027798
- Fortian A. et al. 2009. Biochemistry. 48: 454-61. PubMed ID: 19099412
- Karim Z. et al. 2015. Clinics and Research in Hepatology and Gastroenterology. 39: 412-25. PubMed ID: 26142871
- Katugampola R.P. et al. 2012. The British Journal of Dermatology. 167: 901-13. PubMed ID: 22816431
- Solis C. et al. 2001. The Journal of Clinical Investigation. 107: 753-62. PubMed ID: 11254675
- Warner C.A. et al. 1992. The Journal of Clinical Investigation. 89: 693-700. PubMed ID: 1737856
- Xu W. et al. 1995. The Journal of Clinical Investigation. 95: 905-12. PubMed ID: 7860775
NextGen Sequencing using PG-Select Capture Probes
We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~10 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from the patient specimen. For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes. Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA). Regions with insufficient coverage by NGS are often covered 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.
Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).
(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign Variants
Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org). Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.
As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.
In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.
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 Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele. Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).
We sequence all coding exons for each given transcript, plus ~10 bp of flanking non-coding DNA for each exon. Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.
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 PCR.
Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes 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.
Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.
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.
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.
- 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.
(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.
(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.
(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.