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Renal Coloboma Syndrome and Isolated Renal Hypoplasia via the PAX2 Gene

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

NGS Sequencing

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
7267 PAX2$690.00 81406 Add to Order
Pricing Comment

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 Sanger Sequencing click here.
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

PAX2 mutations have been reported in approximately 50% of patients with classic findings of renal hypoplasia/dysplasia and abnormalities of the optic nerve (Bower et al. 2011). PAX2 mutations were found in 4-6% of patients/fetuses with renal hypodysplasia in three large cohort studies (Weber et al. 2006; Thomas et al. 2011; Madariaga et al. 2013). In a small cohort of 20 unrelated Japanese patients with bilateral renal hypoplasia, PAX2 mutations were detected in two patients (10%) (Nishimoto et al. 2001).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 PAX2$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

Renal coloboma syndrome (also known as papillorenal syndrome; OMIM# 120330) is an autosomal dominant syndrome characterized by optic nerve abnormalities and renal hypoplasia/dysplasia (Sanyanusin et al. 1995; Bower et al. 2011). Other clinical features include hearing loss and central nervous system anomalies. Extremely variable clinical presentations, even within the same family, have been found in renal coloboma syndrome. Classic ophthalmologic findings include optic nerve coloboma, morning glory anomaly, and excavation of the optic disc. The most common renal findings are renal hypodysplasia, vesicoureteral reflux, renal cysts, and multicystic dysplastic kidneys, all of which are within the typical spectrum of congenital anomalies of the kidney and urinary tract (CAKUT). PAX2 and HNF1B are the two major known CAKUT-causing genes to date (Vivante et al. 2014). PAX2 defects can also cause isolated renal hypoplasia (OMIM# 191830) without optic nerve or hearing abnormalities or with subtle features (Nishimoto et al. 2001).

Genetics

Both renal coloboma syndrome and isolated renal hypoplasia are autosomal dominant disorders caused by defects in the PAX2 gene (Sanyanusin et al. 1995; Nishimoto et al. 2001). PAX2 has 11 coding exons that encode a member of the “paired box” (PAX) family of transcriptional regulator genes. PAX2 contains a paired domain (exons 2 to 4), octapeptide domain (exon 5), a partial homeodomain (exon 7), and the transactivation domain (exons 8 to 10). Genetic defects of PAX2 throughout the whole coding region include missense, nonsense, splicing mutations, small deletion/insertions and large deletions encompassing the whole PAX2 gene (Human Gene Mutation Database). PAX2 defects have been reported in approximately 50% of patients with classic findings of renal hypoplasia/dysplasia and abnormalities of the optic nerve (Bower et al. 2011). Of note, de novo PAX2 mutations occur in about 50% of patients.

Testing Strategy

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. All reported pathogenic, likely pathogenic, and 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.

This test provides full coverage of all coding exons of the PAX2 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.

Indications for Test

Candidates for this test are patients with renal coloboma syndrome or isolated renal hypoplasia or the CAKUT spectrum disorder. Testing is also indicated for family members of patients who have known PAX2 mutations.

Gene

Official Gene Symbol OMIM ID
PAX2 167409
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Diseases

Name Inheritance OMIM ID
Papillorenal Syndrome 120330
Renal Adysplasia 191830

Related Tests

Name
Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Panel
Comprehensive Inherited Retinal Dystrophies (includes RPGR ORF15) Sequencing Panel with CNV Detection
Congenital Abnormalities of the Kidney and Urinary Tract (CAKUT) Sequencing Panel with CNV Detection
Nephrotic Syndrome (NS)/Focal Segmental Glomerulosclerosis (FSGS) Sequencing Panel

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Bower M, Salomon R, Allanson J, Antignac C, Benedicenti F, Benetti E, Binenbaum G, Jensen UB, Cochat P, DeCramer S, Dixon J, Drouin R, et al. 2012. Update of PAX2 mutations in renal coloboma syndrome and establishment of a locus-specific database. Hum. Mutat. 33: 457-466. PubMed ID: 22213154
  • Human Gene Mutation Database (Bio-base).
  • Madariaga L, Morinière V, Jeanpierre C, Bouvier R, Loget P, Martinovic J, Dechelotte P, Leporrier N, Thauvin-Robinet C, Jensen UB, Gaillard D, Mathieu M, et al. 2013. Severe prenatal renal anomalies associated with mutations in HNF1B or PAX2 genes. Clin J Am Soc Nephrol 8: 1179-1187. PubMed ID: 23539225
  • Nishimoto K, Iijima K, Shirakawa T, Kitagawa K, Satomura K, Nakamura H, Yoshikawa N. 2001. PAX2 gene mutation in a family with isolated renal hypoplasia. J. Am. Soc. Nephrol. 12: 1769-1772. PubMed ID: 11461952
  • Sanyanusin P, McNoe LA, Sullivan MJ, Weaver RG, Eccles MR. 1995. Mutation of PAX2 in two siblings with renal-coloboma syndrome. Hum. Mol. Genet. 4: 2183-2184. PubMed ID: 8589702
  • Sanyanusin P, Schimmenti LA, McNoe LA, Ward TA, Pierpont ME, Sullivan MJ, Dobyns WB, Eccles MR. 1995. Mutation of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux. Nat. Genet. 9: 358-364. PMID:  PubMed ID: 7795640
  • Thomas R, Sanna-Cherchi S, Warady BA, Furth SL, Kaskel FJ, Gharavi AG. 2011. HNF1B and PAX2 mutations are a common cause of renal hypodysplasia in the CKiD cohort. Pediatr. Nephrol. 26: 897-903. PMID:  PubMed ID: 21380624
  • Vivante A, Kohl S, Hwang D-Y, Dworschak GC, Hildebrandt F. 2014. Single-gene causes of congenital anomalies of the kidney and urinary tract (CAKUT) in humans. Pediatr. Nephrol. PubMed ID: 24398540
  • Weber S, Moriniere V, Knüppel T, Charbit M, Dusek J, Ghiggeri GM, Jankauskiené A, Mir S, Montini G, Peco-Antic A, Wühl E, Zurowska AM, et al. 2006. Prevalence of mutations in renal developmental genes in children with renal hypodysplasia: results of the ESCAPE study. J. Am. Soc. Nephrol. 17: 2864-2870.  PubMed ID: 16971658
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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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