Malattia Leventinese and Doyne Honeycomb Retinal Dystrophy via the EFEMP1 Gene
- Summary and Pricing
- Clinical Features and Genetics
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The great majority of tests are completed within 18 days.
A mutation screening showed that 99% of the ML and DHRD affected members (161 out of 162 affected patients in 37 families) had the EFEMP1 p.Arg345Trp variant, which was absent in 477 control individuals and in 494 unrelated patients with age-related macular degeneration. Out of 161,160 were heterozygotes and one patient was homozygous for the p.Arg345Trp variant. No difference was observed between the homozygous patient and heterozygotes of similar age. (Stone et al. 1999).
Malattia Leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD) are autosomal dominant disorders and have phenotypic similarity with age-related macular degeneration (AMD). The hallmark feature of both ML and DHRD is early-onset drusen (yellow-white deposit) between the retinal pigment epithelium (RPE) and Bruch’s membrane, which is a typical AMD feature (Stone et al. 1999).
Autosomal dominant ML and DHRD are due to a single pathogenic variant in the EFEMP1 gene, which encodes the epidermal growth factor containing fibrillin-like extracellular matrix protein 1 (also known as fibulin-3). This protein is expressed in the tissues that are closest to the site of drusen formation. Mutation screening showed that all affected families had the p.Arg345Trp variant, which was not present in 477 control individuals or in 494 patients with age-related macular degeneration (Stone et al. 1999). In another study, 14 unrelated individuals diagnosed with early onset of multiple drusen and no apparent family history of the disease, did not have p.Arg345Trp or any other EFEMP1 variant. These results suggest that EFEMP1 is not associated with other types of early onset drusen phenotypes or typical AMD (Sauer et al. 2001; Guymer et al. 2002; Narendran et al. 2005).
This test involves bidirectional DNA Sanger sequencing of all coding exons and ~ 20 bp of flanking noncoding sequence of EFEMP1 gene. We will also sequence any single exon (Test #100) in family members of patients with a known mutation or to confirm research results.
Indications for Test
All patients with symptoms suggestive of early onset drusen are candidates.
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- Genetic Counselor Team - email@example.com
- Madhulatha Pantrangi, PhD - firstname.lastname@example.org
- Guymer RH, McNeil R, Cain M, Tomlin B, Allen PJ, Dip CL, Baird PN. 2002. Analysis of the Arg345Trp disease-associated allele of the EFEMP1 gene in individuals with early onset drusen or familial age-related macular degeneration. Clin. Experiment. Ophthalmol. 30: 419–423. PubMed ID: 12427233
- Narendran N, Guymer RH, Cain M, Baird PN. 2005. Analysis of the EFEMP1 gene in individuals and families with early onset drusen. Eye 19: 11–15. PubMed ID: 15218514
- Sauer CG, White K, Kellner U, Rudolph G, Jurklies B, Pauleikhoff D, Weber BH. 2001. EFEMP1 is not associated with sporadic early onset drusen. Ophthalmic Genet. 22: 27–34. PubMed ID: 11262647
- Stone EM, Lotery AJ, Munier FL, Héon E, Piguet B, Guymer RH, Vandenburgh K, Cousin P, Nishimura D, Swiderski RE, Silvestri G, Mackey DA, Hageman GS, Bird AC, Sheffield VC, Schorderet DF. 1999. A single EFEMP1 mutation associated with both Malattia Leventinese and Doyne honeycomb retinal dystrophy. Nature genetics 22: 199–202. PubMed ID: 10369267
Bi-Directional Sanger Sequencing
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.
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).
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.
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.