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Anophthalmia / Microphthalmia Sequencing Panel

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
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TEST METHODS

NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
2637 ALDH1A3 81479 Add to Order
BCOR 81479
BMP4 81479
BMP7 81479
CRYBA4 81479
FOXE3 81479
GDF6 81479
HCCS 81479
MITF 81479
OTX2 81479
RAX 81479
SIX6 81479
SMOC1 81479
SOX2 81479
STRA6 81479
TENM3 81479
VSX2 81479
Full Panel Price* $1490.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
2637 Genes x (17) $1490.00 81479(x17) Add to Order
Pricing Comment

If you would like to order a subset of these genes contact us to discuss pricing.

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 28 days.

Clinical Sensitivity

Heterozygous loss-of-function variants in SOX2 and OTX2 (Wyatt A. et al. 2008) are the most common genetic pathologies associated with severe eye malformations. For information regarding the clinical sensitivity of other genes please see the table below. For a few genes, due to the limited number of cases, estimation of clinical sensitivity is difficult.

Gene Clinical sensitivity
ALDH1A3 Unknown
BCOR ~1%
BMP4 2%
BMP7 Unknown
HCCS Unknown
MITF Unknown
RAX 3%
SIX6 Unknown
SOX2 15-20%
STRA6 >1%
VSX2 ~2%
TENM3 (ODZ3) Unknown
FOXE3 3%
GDF6 (KFS1) ~1%
CRYBA4 Unknown
OTX2 2-5%
SMOC1 Unknown

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 BCOR$690.00 81479 Add to Order
FOXE3$690.00 81479
GDF6$690.00 81479
MITF$690.00 81479
OTX2$690.00 81479
SOX2$690.00 81479
Full Panel Price* $840.00
Test Code Test Copy Genes Total Price CPT Codes Copy CPT Codes
600 Genes x (6) $840.00 81479(x6) 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 Sensitivity

No large deletions or insertions have been reported in GDF6 that are causative for A/M (Human Gene Mutation Database). Gross deletions have been reported frequently in BCOR (HGMD; Hilton et al. 2009).

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Clinical Features

Anophthalmia (A; absence of a globe in the orbit) and microphthalmia (M; reduced size of the globe) are severe and rare developmental defects of the globe with an estimated incidence of 0.2–0.4/10 000 and ~1.5/10 000 live births, respectively (Källén and Tornqvist 2005). Both A/M may be unilateral or bilateral, and over 50% of A/M affected individuals have systemic abnormalities such as hypothalamic–pituitary disorder, mild dysmorphic facial features and short stature, urogenital anomalies, cryptorchidism and/or micropenis in males, developmental delay, seizures, oesophageal atresia or tracheoesophageal fistula and hearing loss (Ragge et al. 2005), but only 25% of these are part of distinct and well-defined syndromes (Bakrania et al. 2007). Unilateral A/M cases often have developmental anomalies of the other eye; including coloboma, lens, and optic nerve (Ragge et al. 2007).

Genetics

Anophthalmia/Microphthalmia (A/M) may be inherited as an autosomal dominant, autosomal recessive, or X-linked trait. A/M has a complex aetiology with a wide range of causes, including chromosomal abnormalities, as well as environmental factors (Pedace et al. 2009). Chromosomal duplications, deletions and translocations account for 23–30% of A/M cases. Bakrania et al. reported whole SOX2 gene deletions in ~10% of their A/M patients cohort (Bakrania et al. 2007), which emphasizes the necessity of careful chromosomal analysis (particularly the 3q region that comprises the SOX2 gene) (Guichet et al. 2004). SOX2 has been identified as a major causative gene in which heterozygous, loss of function variants account for 15–20% of the A/M cases (Reis et al. 2010; Faivre et al. 2006; Ragge et al. 2005; Williamson 2014). The majority of the causative SOX2 sequence variations are de novo (FitzPatrick 2009). Occasional cases result from parental gonosomal mosaicism (Faivre et al. 2006; Schneider et al. 2008).Heterozygous loss-of-function variants in SOX2 and OTX2 (Wyatt A. et al. 2008) are the most common genetic pathologies associated with severe eye malformations. Bi-allelic loss-of-function variants in STRA6 (Gerth-Kahlert et al. 2013) are confirmed as an emerging cause of nonsyndromal eye malformations. Other genes involved in A/M include ALDH1A3 (Yahyavi et al. 2013), BCOR (Ng et al. 2004), BMP4 (Reis et al. 2011), BMP7 (Wawersik et al. 1999), HCCS (Wimplinger et al. 2006), MITF (Tassabehji et al. 1994), RAX (Abouzeid et al. 2012), SIX6 (Aldahmesh MA. et al. 2013), VSX2 (Reis et al. 2011), TENM3 (Aldahmesh et al. 2012), FOXE3 (Reis et al. 2010), GDF6 (Asai-Coakwell M. et al. 2009), CRYBA4 (Billingsley G. et al. 2006), and SMOC1 (Okada I. et al. 2011). Gene-associated M/A inheritance pattern, total number of reported pathogenic variants, mutation spectrum, gene alternative names and their percentage of pathonic variants accounting for the MAC cases are listed in the Table below (Bardakjian et al. 2013; Human Gene Mutation Database). FOXE3 has been reported to have autosomal dominant or autosomal recessive inheritance. Dominant variants are those (e.g., c.958T>C (p.*320Argext*72) and c.942dupG (p.Leu315Alafs*117)), which result in extension of the open reading frame beyond the normal stop codon and are reported to have dominant negative effect (Semina et al. 2001; Iseri et al. 2009). Recessive variants (e.g., missense variants) result in altered protein interactions (Iseri et al. 2009). See individual gene test descriptions for more information on molecular biology of gene products.

Gene Pattern of Inheritance Reported Mutation Spectrum Variants
ALDH1A3 Autosomal Recessive(AR) ~15 Missense, nonsense and splicing
BCOR X-linked (XL) ~40 Missense, splicing, nonsense, small and gross deletions/insertions
BMP4 Autosomal Dominant (AD) ~10 Missense, small deletions and insertions and gross deletions
BMP7 AD ~5 Missense, regulatory and small deletions
HCCS XL Over 5 Missense, nonsense, small and gross deletions
MITF AD ~20 Missense, nonsense, splicing, small delettions
RAX AR Over 10 Missense, nonsense, splicing, small and gross deletions
SIX6 AR Over 5 Missense, small and gross deletions
SOX2 AD Over 70 Missense, nonsense, small del/ins and complex rearrangements
STRA6 AR 25 Missense, nonsense, splicing, small and large deletions, small insertions
VSX2 AR Over 10 Missense, nonsense, splicing, small and large deletions, small insertions
TENM3 (ODZ3) AR One Small insertion so far
FOXE3 AD/AR Over 10 Missense, nonsense, small deletions and insertions
GDF6 (KFS1) AD ~10 Missense, complex rearrangements
CRYBA4 AD ~5 Only missense
OTX2 AD Over 40 Missense, nonsense, splicing, small deletion/insertion and large deletion
SMOC1 AR Over 10 Missense, nonsense, splicing, small deletions/ insertions
Testing Strategy

For this Next Generation (NextGen) panel, the full coding regions plus ~20 bp of non-coding DNA flanking each exon are sequenced for each of the genes listed below. 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 any regions not captured or with insufficient number of sequence reads. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.

Indications for Test

Candidates for this test are patients with symptoms consistent Microphthalmia/anophthalmia/coloboma.

Genes

Official Gene Symbol OMIM ID
ALDH1A3 600463
BCOR 300485
BMP4 112262
BMP7 112267
CRYBA4 123631
FOXE3 601094
GDF6 601147
HCCS 300056
MITF 156845
OTX2 600037
RAX 601881
SIX6 606326
SMOC1 608488
SOX2 184429
STRA6 610745
TENM3 610083
VSX2 142993
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

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Cataract 23 (CTRCT23) via the CRYBA4 Gene
Cataract Type 11 via the PITX3 Gene
Congenital Abnormalities of the Kidney and Urinary Tract (CAKUT) Sequencing Panel with CNV Detection
Congenital Cataracts Sequencing Panel
Disorders of Sex Development and Infertility Sequencing Panel with CNV Detection
Disorders of Sex Development Sequencing Panel with CNV Detection
Female Infertility Sequencing Panel with CNV Detection
Klippel-Feil Syndrome via the GDF6 Gene
Leber Congenital Amaurosis Sequencing Panel with CNV Detection
Male Infertility Sequencing Panel with CNV Detection
Microphthalmia and Anophthalmia via the RAX Gene
Oculocutaneous Albinism Sequencing Panel
Oculofaciocardiodental Syndrome and Lenz Microphthalmia Syndrome via the BCOR Gene
Syndromic Microphthalmia via the OTX2 Gene
Waardenburg Syndrome Sequencing Panel
Waardenburg Syndrome Type IIA via the MITF Gene
X-Linked Intellectual Disability Sequencing Panel with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Abouzeid H. et al. 2012. Molecular Vision. 18: 1449-56. PubMed ID: 22736936
  • Aldahmesh M.A. et al. 2012. Genetics in Medicine : Official Journal of the American College of Medical Genetics. 14: 900-4. PubMed ID: 22766609
  • Aldahmesh M.A. et al. 2013. Clinical Genetics. 84: 198-9. PubMed ID: 23167593
  • Asai-Coakwell M. et al. 2009. Human Molecular Genetics. 18: 1110-21. PubMed ID: 19129173
  • Bakrania P. et al. 2007. The British Journal of Ophthalmology. 91: 1471-6.  PubMed ID: 17522144
  • Bardakjian et al. 2013. Microphthalmia/Anophthalmia/Coloboma Spectrum. 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: 20301552
  • Billingsley G. et al. 2006. American Journal of Human Genetics. 79: 702-9. PubMed ID: 16960806
  • Faivre L. et al. 2006. American Journal of Medical Genetics. Part A. 140: 636-9. PubMed ID: 16470798
  • FitzPatrick. 2009. SOX2-Related Eye Disorders. 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: 20301477
  • Gerth-Kahlert C. et al. 2013. Molecular Genetics & Genomic Medicine. 1: 15-31. PubMed ID: 24498598
  • Guichet A. et al. 2004. Prenatal Diagnosis. 24: 828-32. PubMed ID: 15503273
  • Hilton E. et al. 2009. European Journal of Human Genetics : Ejhg. 17: 1325-35. PubMed ID: 19367324
  • Human Gene Mutation Database (Bio-base).
  • Iseri S.U. et al. 2009. Human Mutation 30: 1378-86. PubMed ID: 19708017
  • Källén B, Tornqvist K. 2005. European Journal of Epidemiology. 20: 345–350. PubMed ID: 15971507
  • Ng D. et al. 2004. Nature Genetics. 36: 411-6. PubMed ID: 15004558
  • Okada I. et al. 2011. American Journal of Human Genetics. 88: 30-41. PubMed ID: 21194678
  • Pedace et al. 2009. European Journal of Medical Genetics. 52: 273–276. PubMed ID: 19254784
  • Ragge N.K. et al. 2005. American Journal of Medical Genetics. Part A. 135: 1-7; discussion 8.  PubMed ID: 15812812
  • Ragge N.K. et al. 2007. Eye (london, England). 21: 1290-300.  PubMed ID: 17914432
  • Reis L.M. et al. 2010. Molecular Vision 16: 768-73. PubMed ID: 20454695
  • Reis LM. et al. 2011. Human Genetics. 130: 495-504. PubMed ID: 21340693
  • Schneider A. et al. 2008. American Journal of Medical Genetics Part A 146A: 2794-8. PubMed ID: 18831064
  • Semina E.V. et al. 2001. Human Molecular Genetics. 10: 231-6. PubMed ID: 11159941
  • Tassabehji M. et al. 1994. Nature Genetics. 8: 251-5. PubMed ID: 7874167
  • Wawersik S. et al. 1999. Developmental Biology. 207: 176-88. PubMed ID: 10049573
  • Williamson K.A., FitzPatrick DR. 2014. European Journal of Medical Genetics. 57: 369-80.  PubMed ID: 24859618
  • Wimplinger I. et al. 2006. American Journal of Human Genetics. 79: 878-89. PubMed ID: 17033964
  • Wyatt A. et al. 2008. Human Mutation 29: E278–E283. PubMed ID: 18781617
  • Yahyavi M. et al. 2013. Human Molecular Genetics. 22: 3250-8. PubMed ID: 23591992
Order Kits
TEST METHODS

NextGen Sequencing using PG-Select Capture Probes

Test Procedure

We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 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 covered by Sanger sequencing.  All pathogenic, likely pathogenic, or 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.

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, Common 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.

Analytical Validity

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.   

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 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 ~20 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.

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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