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Alport Syndrome (AS) Sequencing Panel

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

NGS Sequencing

Test Code Test Copy GenesCPT Code Copy CPT Codes
1388 COL4A3 81408 Add to Order
COL4A4 81407
COL4A5 81408
COL4A6 81479
Full Panel Price* $1540.00
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

A large panel study showed that the overall detection rate is 53%, and a likely causative mutation was identified in 82% (23/28) of families with clear X-linked cases (Hertz 2009). Approximately 57% of clinical suspected autosomal recessive Alport syndrome patients have mutations in COL4A3 or COL4A4 (Zhang et al. 2012).

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Deletion/Duplication Testing via aCGH

Test Code Test Copy GenesIndividual Gene PriceCPT Code Copy CPT Codes
600 COL4A3$690.00 81479 Add to Order
COL4A4$690.00 81479
COL4A5$690.00 81407
COL4A6$690.00 81479
Full Panel Price* $840.00
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

Large deletions/duplications and complex large rearrangements account for ~15% of pathogenic mutations found in COL4A5 (King et al. 2006; Human Gene Mutation Database). However, only a few large deletions involving COL4A3 and COL4A4 have been identified (Oka et al. 2014; Human Gene Mutation Database).

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Clinical Features

Alport syndrome (AS) is a hereditary nephritis caused by defects in collagen type IV protein, which is responsible for basement membrane formation in the kidney, ear and eye. The disease affects approximately 1 in 50,000 individuals and is characterized by progressive renal failure, sensorineural hearing loss and eye abnormalities. The most common symptoms include persistent microhematuria in early childhood, progressive proteinuria, bilateral high frequency sensorineural hearing loss and anterior lenticonus in late childhood and adolescence (Kashtan 2013).

Genetics

Approximately 80% of Alport syndrome cases are X-linked caused by mutations in the COL4A5 gene; the remaining cases are inherited in an autosomal recessive (15%) and autosomal dominant manner (5%) caused by mutations in either in COL4A3 or COL4A4. Heterozygous mutations in COL4A3 and COL4A4 can also cause benign familial hematuria (also called Thin-Basement Membrane Nephropathy) (van der Loop et al. 2000; Mochizuki et al. 1994; Nagel et al. 2005; Hertz et al. 2009). Large deletions involving 5’ ends of the COL4A5 gene and a breakpoint within intron 2 of the COL4A6 gene were reported to cause diffuse leiomyomatosis (Uliana et al. 2011). A missense mutation in the COL4A6 gene was reported to cause X-linked non-syndromic hearing loss in one Hungarian family (Rost et al. 2014). Some heterozygous female carriers of the COL4A5 pathogenic mutations may develop some clinical features, and their clinical manifestations are variable largely determined by random X-inactivation. ~95% of female carriers have hematuria (Jais et al. 2003).

To date, more than 600 unique causative COL4A5 mutations have been reported throughout the gene including missense (~42%, more than 85% of these missense mutations affect a glycine residue), nonsense (6%), splicing (17%), small deletion/insertion (22%), and large deletion/insertion (~13%) and complex large rearrangements (1%) (King et al. 2006; Human Gene Mutation Database). Almost 140 pathogenic COL4A3 mutations have been reported. They are: missense (~44%, more than 70% of these missense mutations affect a glycine residue), nonsense (12%), splicing (15%), small deletion/insertion (28%), and only two large deletions, respectively (Oka et al. 2014; Human Gene Mutation Database). More than 90 pathogenic COL4A4 mutations have been reported throughout the gene. They are: missense (~54%, more than 70% of these missense mutations affect a glycine residue), nonsense (8%), splicing (10%), small deletion/insertion (25%), and only one large deletion (Oka et al. 2014; Human Gene Mutation Database).

Testing Strategy

This Next Generation Sequencing (NGS) panel involves the simultaneous sequencing of COL4A3, COL4A4, COL4A5 and COL4A6 that have been implicated in Alport syndrome and related disorders. For this NGS panel, each coding exon plus ~20bp of flanking non-coding DNA are simultaneously sequenced for each of the genes listed below. Sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization method, 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, undocumented and questionable variant calls are confirmed by Sanger sequencing.

Indications for Test

Candidates for this test are patients with symptoms consistent with clinical diagnosed Alport syndrome, benign familial hematuria and the family members of patients who have known mutations in COL4A5, COL4A4, COL4A3 and COL4A6.

Genes

Official Gene Symbol OMIM ID
COL4A3 120070
COL4A4 120131
COL4A5 303630
COL4A6 303631
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Tests

Name
Alport Syndrome (AS) via the COL4A3 Gene
Alport Syndrome (AS) via the COL4A4 Gene
Alport Syndrome (AS) via the COL4A5 Gene
Deafness, X-linked 6 (DFNX6) via the COL4A6 Gene

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Hertz JM. 2009. Alport syndrome. Molecular genetic aspects. Dan Med Bull 56: 105–152. PubMed ID: 19728970
  • Human Gene Mutation Database (Bio-base).
  • Jais JP. 2003. X-Linked Alport Syndrome: Natural History and Genotype-Phenotype Correlations in Girls and Women Belonging to 195 Families: A “European Community Alport Syndrome Concerted Action” Study. Journal of the American Society of Nephrology 14: 2603-2610. PubMed ID: 14514738
  • Kashtan CE. 2013. Alport Syndrome and Thin Basement Membrane Nephropathy. 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: 20301386
  • King K, Flinter FA, Green PM. 2006. A two-tier approach to mutation detection in the COL4A5 gene for Alport syndrome. Human Mutation 27: 1061–1061. PubMed ID: 16941480
  • Mochizuki T, Lemmink HH, Mariyama M, Antignac C, Gubler MC, Pirson Y, Verellen-Dumoulin C, Chan B, Schröder CH, Smeets HJ. 1994. Identification of mutations in the alpha 3(IV) and alpha 4(IV) collagen genes in autosomal recessive Alport syndrome. Nat. Genet. 8: 77-81. PubMed ID: 7987396
  • Nagel et al. Novel COL4A5, COL4A4, and COL4A3 mutations in Alport syndrome. Hum Mutat  26(1): 60. 2005. PubMed ID: 15954103
  • Oka M, Nozu K, Kaito H, Fu XJ, Nakanishi K, Hashimura Y, Morisada N, Yan K, Matsuo M, Yoshikawa N, Vorechovsky I, Iijima K. 2014. Natural history of genetically proven autosomal recessive Alport syndrome. Pediatric Nephrology. PubMed ID: 24633401
  • Rost S, Bach E, Neuner C, Nanda I, Dysek S, Bittner RE, Keller A, Bartsch O, Mlynski R, Haaf T, Müller CR, Kunstmann E. 2014. Novel form of X-linked nonsyndromic hearing loss with cochlear malformation caused by a mutation in the type IV collagen gene COL4A6. Eur. J. Hum. Genet. 22: 208-215. PubMed ID: 23714752
  • Uliana V, Marcocci E, Mucciolo M, Meloni I, Izzi C, Manno C, Bruttini M, Mari F, Scolari F, Renieri A, Salviati L. 2011. Alport syndrome and leiomyomatosis: the first deletion extending beyond COL4A6 intron 2. Pediatric Nephrology 26: 717-724. PubMed ID: 21380622
  • Van Der Loop FT, Heidet L, Timmer ED, Den Bosch BJ Van, Leinonen A, Antignac C, Jefferson JA, Maxwell AP, Monnens LA, Schröder CH, others. 2000. Autosomal dominant Alport syndrome caused by a COL4A3 splice site mutation. Kidney international 58: 1870–1875. PubMed ID: 11044206
  • Zhang Y, Wang F, Ding J, Zhang H, Zhao D, Yu L, Xiao H, Yao Y, Zhong X, Wang S. 2012. Genotype-phenotype correlations in 17 Chinese patients with autosomal recessive Alport syndrome. American Journal of Medical Genetics Part A 158A: 2188-2193. PubMed ID: 22887978
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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