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Blepharophimosis-Ptosis-Epicanthus Inversus syndrome (BPES) via the FOXL2 Gene

  • 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
1673 FOXL2$610.00 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

A FOXL2 mutation analysis in two different studies identified mutations in 65-72% of the  blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) patients (De Baere et al. 2001; Beysen et al. 2008; De Baere 2009)

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 FOXL2$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

Blepharophimosis-Ptosis-Epicanthus Inversus syndrome (BPES) is a dominantly inherited developmental disorder of the eyelids, which may severely impair visual function (Amati et al. 1995). BPES is clinically characterized by four major ophthalmic manifestations that are present at birth: blepharophimosis (shortening of the horizontal palpebral fissure), ptosis (droopy eyelids), epicanthus inversus (a vertical fold of the skin that stretches from the lower eyelid near the inner corner of the eye and towards either side of the nose), and telecanthus (lateral displacement of the inner canthi that leads to abnormal interpupillary distance) (Zahanova et al. 2012). Clinically, two types of BPES have been recognized. BPES Type I includes female infertility as a result of premature ovarian failure (POF) along with the four major ophthalmic manifestations described above. BPES Type II is limited to the four major eyelid abnormalities (Zahanova et al. 2012; Alao et al. 2012). Patients with BPES have a high incidence of bilateral strabismus, amblyopia and refractive errors (Choi et al. 2006). Ptosis with strabismus doubles the risk of amblyopia. Patients who have severe ptosis are recommended to have surgery before 3 years of age, and all other patients should undergo surgical repair before 5 years of age (Beckingsale et al. 2003). Also, premature ovarian failure can be corrected with hormone replacement therapy. Other symptoms of BPES include lacrimal duct anomalies, a broad nasal bridge, low-set ears, and a short philtrum (abnormal distance between the upper lip and the nose) (De Baere 2009).

Genetics

Blepharophimosis-Ptosis-Epicanthus Inversus syndrome is a dominantly inherited developmental disorder. Mutations in the FOXL2 gene, which is located on chromosome 3q23, has been shown to be causative for both BPES Type I and II. FOXL2 belongs to the winged helix /forkhead (FH) transcription factor gene family, which is known to be involved in a diverse range of developmental processes such as establishment of the body axis and the development of tissues from all three germ layers (Lehmann et al. 2003). FOXL2 is a single-exon gene encoding a highly conserved protein that contains a 110-amino-acid DNA-binding forkhead domain and a poly alanine tract of 14 residues that is conserved in mammals. FOXL2 is been reported to be expressed in the mesenchyme of the developing eyelids and in fetal and adult ovarian follicles (Crisponi et al. 2001; Beysen et al. 2009). Currently, four of the 11 human FH genes that are shown to be causative for human hereditary developmental disorders exhibit an ocular phenotype (Verdin and De Baere 2012).

Of all genetic defects found, approximately 71% are intragenic mutations of FOXL2, 10–12% are deletions encompassing FOXL2, and 5% are deletions located outside its transcription unit (Beysen et al., 2009; D’haene et al. 2009; D’haene et al. 2010). Genotype-phenotype correlations indicated that the patients with mutations that result in proteins with truncation before the poly-Ala tract are at high risk for developing POF (BPES type I). Expansion of poly-Ala tract may lead to BPES type II. However, several exceptions to this correlation were found (De Baere et al. 2003). The largest group in the intragenic mutations is frameshift mutations (44%) followed by in-frame changes (33%), nonsense mutations (12%) and finally missense mutations (11%). The majority of the in-frame mutations (93%) lead to polyalanine expansions, representing the most important mutational hotspot in FOXL2 (Verdin and De Baere 2012; De Baere et al. 2003). BPES Patients with large interstitial deletions encompassing FOXL2 may present other clinical findings, such as microcephaly, mild mental retardation and growth delay (D’haene et al., 2009; de Ru et al. 2005). So far, about 200 FOXL2 pathogenic sequence variations (missense, nonsense, small and gross insertions/duplications and Complex rearrangements) have been reported in BPES (Human Gene Mutation Database). Mutations in the core promoter region as well as the 3'-UTR of FOXL2 have been reported in BPES patients (Li et al. 2009; Chawla et al. 2013)

Testing Strategy

This test involves bidirectional DNA Sanger sequencing of FOXL2 single coding exon plus ~20 bp of flanking non-coding DNA on either side that exon is sequenced. This test also covers the three noncoding FOXL2 variants (c.*86C>T; c.*619C>A; c.-251G>A) that have been reported to be causative for BPES (Human Gene Mutation Database). We will also sequence any single portion (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 blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) types I and II.

Gene

Official Gene Symbol OMIM ID
FOXL2 605597
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Alao MJ, Lalèyè A, Lalya F, Hans C, Abramovicz M, Morice-Picard F, Arveiler B, Lacombe D, Rooryck C. 2012. Blepharophimosis, ptosis, epicanthus inversus syndrome with translocation and deletion at chromosome 3q23 in a black African female. Eur J Med Genet 55: 630–634. PubMed ID: 22906557
  • Amati P, Chomel JC, Nivelon-Chevalier A, Gilgenkrantz S, Kitzis A, Kaplan J, Bonneau D. 1995. A gene for blepharophimosis-ptosis-epicanthus inversus syndrome maps to chromosome 3q23. Hum. Genet. 96: 213–215. PubMed ID: 7635472
  • Beckingsale PS, Sullivan TJ, Wong VA, Oley C. 2003. Blepharophimosis: a recommendation for early surgery in patients with severe ptosis. Clin. Experiment. Ophthalmol. 31: 138–142. PubMed ID: 12648048
  • Beysen D, Jaegere S De, Amor D, Bouchard P, Christin-Maitre S, Fellous M, Touraine P, Grix AW, Hennekam R, Meire F, Oyen N, Wilson LC, et al. 2008. Identification of 34 novel and 56 known FOXL2 mutations in patients with Blepharophimosis syndrome. Hum. Mutat. 29: E205–219. PubMed ID: 18642388
  • Beysen D, Paepe A De, Baere E De. 2009. FOXL2 mutations and genomic rearrangements in BPES. Hum. Mutat. 30: 158–169. PubMed ID: 18726931
  • Chawla B, Bhadange Y, Dada R, Kumar M, Sharma S, Bajaj MS, Pushker N, Chandra M, Ghose S. 2013. Clinical, Radiologic, and Genetic Features in Blepharophimosis, Ptosis, and Epicanthus Inversus Syndrome in the Indian Population. Investigative ophthalmology & visual science 54: 2985–2991. PubMed ID: 23513057
  • Choi K-H, Kyung S, Oh SY. 2006. The factors influencing visual development in blepharophimosis-ptosis-epicanthus inversus syndrome. J Pediatr Ophthalmol Strabismus 43: 285–288. PubMed ID: 17022162
  • Crisponi L, Deiana M, Loi A, Chiappe F, Uda M, Amati P, Bisceglia L, Zelante L, Nagaraja R, Porcu S, Ristaldi MS, Marzella R, et al. 2001. The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome. Nat. Genet. 27: 159–166. PubMed ID: 11175783
  • D’haene B, Attanasio C, Beysen D, Dostie J, Lemire E, Bouchard P, Field M, Jones K, Lorenz B, Menten B, Buysse K, Pattyn F, et al. 2009. Disease-causing 7.4 kb cis-regulatory deletion disrupting conserved non-coding sequences and their interaction with the FOXL2 promotor: implications for mutation screening. PLoS Genet. 5: e1000522. PubMed ID: 19543368
  • D’haene B, Nevado J, Pugeat M, Pierquin G, Lowry RB, Reardon W, Delicado A, García-Miñaur S, Palomares M, Courtens W, Stefanova M, Wallace S, et al. 2010. FOXL2 copy number changes in the molecular pathogenesis of BPES: unique cohort of 17 deletions. Hum. Mutat. 31: E1332–1347. PubMed ID: 20232352
  • de Baere E, Beysen D, Oley C, Lorenz B, Cocquet J, Sutter P De, Devriendt K, Dixon M, Fellous M, Fryns J-P, Garza A, Jonsrud C, et al. 2003. FOXL2 and BPES: mutational hotspots, phenotypic variability, and revision of the genotype-phenotype correlation. Am. J. Hum. Genet. 72: 478–487. PubMed ID: 12529855
  • de Baere E, Dixon MJ, Small KW, Jabs EW, Leroy BP, Devriendt K, Gillerot Y, Mortier G, Meire F, Maldergem L Van, Courtens W, Hjalgrim H, et al. 2001. Spectrum of FOXL2 gene mutations in blepharophimosis-ptosis-epicanthus inversus (BPES) families demonstrates a genotype--phenotype correlation. Hum. Mol. Genet. 10: 1591–1600. PubMed ID: 11468277
  • de Baere E. 2009. Blepharophimosis, Ptosis, and Epicanthus Inversus. 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: 20301614
  • de Ru MH, Gille JJP, Nieuwint AWM, Bijlsma JB, Blij JF van der, Hagen JM van. 2005. Interstitial deletion in 3q in a patient with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and microcephaly, mild mental retardation and growth delay: clinical report and review of the literature. Am. J. Med. Genet. A 137: 81–87. PubMed ID: 16015581
  • Human Gene Mutation Database (Bio-base).
  • Lehmann OJ, Sowden JC, Carlsson P, Jordan T, Bhattacharya SS. 2003. Fox’s in development and disease. Trends Genet. 19: 339–344. PubMed ID: 12801727
  • Li D, Zeng W, Tao J, Li S, Liang C, Chen X, Mu W, Wang X, Qin Y, Jie Y, Wei W. 2009. Mutations of the transcription factor FOXL2 gene in Chinese patients with blepharophimosis-ptosis-epicanthus inversus syndrome. Genet Test Mol Biomarkers 13: 257–268. PubMed ID: 19371227
  • Verdin H, Baere E De. 2012. FOXL2 impairment in human disease. Horm Res Paediatr 77: 2–11. PubMed ID: 22248822
  • Zahanova S, Meaney B, Łabieniec B, Verdin H, Baere E De, Nowaczyk MJM. 2012. Blepharophimosis-ptosis-epicanthus inversus syndrome plus: deletion 3q22.3q23 in a patient with characteristic facial features and with genital anomalies, spastic diplegia, and speech delay. Clin. Dysmorphol. 21: 48–52. PubMed ID: 21934608
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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