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Seckel Syndrome, Primary Microcephaly and Familial Cutaneous Telangiectasia and Cancer Syndrome via ATR Gene Sequencing with CNV Detection

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

Sequencing with CNV

Test Code Test Copy GenesPriceCPT Code Copy CPT Codes
7979 ATR$890 81479,81479 Add to Order

New York State Approved Test

Pricing Comments

Our favored testing approach is exome based NextGen sequencing with CNV analysis. This will allow cost effective reflexing to PGxome or other exome based tests. However, if full gene Sanger sequencing is desired for STAT turnaround time, insurance, or other reasons, please see link below for Test Code, pricing, and turnaround time information. If the Sanger option is selected, CNV detection may be ordered through Test #600.

The Sanger Sequencing method for this test is NY State approved.

For Sanger Sequencing click here.
Targeted Testing

For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 26 days.

Clinical Sensitivity

The clinical sensitivity of ATR germline mutations in patients with Seckel syndrome, primary microcephaly or familial cutaneous telangiectasia and cancer syndrome is currently unknown as only a small number of individual cases have been reported.

The clinical sensitivity of ATR germline deletions and duplications in patients with Seckel syndrome, primary microcephaly or familial cutaneous telangiectasia and cancer syndrome is currently unknown as only a small number of individual cases have been reported. No large deletions and duplications have been reported for Seckel, syndrome and FCTCS, but a single gross deletion has been reported for primary microcephaly (Mokrani-Benhelli et al. 2013).

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Clinical Features

Seckel syndrome is a rare autosomal recessive disorder that is characterized by pre- and post-natal growth delay resulting in dwarfism. Other characteristics include microcephaly, mild to moderate mental retardation, characteristic narrow bird-like face with a beak-like nose, large eyes, micrognathia, delayed bone age, hip dysplasia, and elbow dislocation (Faivre et al. 2002). Germline pathogenic mutations in the ATR gene, which is causative for Seckel syndrome, have also been described as causative for primary microcephaly (Verloes et al. 1993; Mokrani-Benhelli et al. 2013). In addition, a germline mutation has also been shown to segregate in a five generation family with autosomal dominant oropharyngeal cancer syndrome/familial cutaneous telangiectasia and cancer syndrome (FCTCS) (Tanaka et al. 2012). Affected individuals displayed telangiectases, alopecia, and other malignancies such as nonmelanoma skin cancer, and less commonly breast cancer and cervical cancer. Interestingly, individuals with primary microcephaly and Seckel syndrome do not display an increased risk of malignancy.

Genetics

Seckel syndrome, primary microcephaly, and FCTCS are disorders that are caused by mutations in the ATR gene (Kalay et al. 2011; Tanaka et al. 2012; Verloes et al. 1993). The ATR gene encodes a serine/threonine-protein kinase which is involved in DNA repair through phosphorylation of proteins (e.g. p53, Rad17, Nbs1 and H2AX) similar to those phosphorylated by the ATM kinase (O’Driscoll et al. 2003). ATM is activated after DNA double-strand breaks, whereas ATR is activated by DNA single strands, such as those that occur during response to stalled replication forks (Tanaka et al. 2012); ATR cell lines from affected individuals exhibit defective DNA damage response (O’Driscoll et al. 2003). Limited reported pathogenic variants include missense, nonsense, splicing, small and large deletions (Human Gene Mutation Database).

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.

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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

This test provides full coverage of all coding exons of the ATR gene plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define full coverage as >20X NGS reads or Sanger sequencing.

Since this test is performed using exome capture probes, a reflex to any of our exome based tests is available (PGxome, PGxome Custom Panels).

Indications for Test

Individuals suspected of having Seckel syndrome, primary microcephaly or familial cutaneous telangiectasia and cancer syndrome. Also individuals with a family history of these disorders. This test may also be considered for the reproductive partners of individuals who carry pathogenic variants in ATR.

Gene

Official Gene Symbol OMIM ID
ATR 601215
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Test

Name
LIG4 Syndrome via LIG4 Gene Sequencing with CNV Detection

CONTACTS

Genetic Counselors
Geneticist
Citations
  • Faivre L, Merrer M Le, Lyonnet S, Plauchu H, Dagoneau N, Campos-Xavier AB, Attia-Sobol J, Verloes A, Munnich A, Cormier-Daire V. 2002. Clinical and genetic heterogeneity of Seckel syndrome. American Journal of Medical Genetics 112: 379–383. PubMed ID: 12376940
  • Human Gene Mutation Database (Bio-base).
  • Kalay E, Yigit G, Aslan Y, Brown KE, Pohl E, Bicknell LS, Kayserili H, Li Y, Tüysüz B, Nürnberg G, Kiess W, Koegl M, et al. 2011. CEP152 is a genome maintenance protein disrupted in Seckel syndrome. Nature Genetics 43: 23–26. PubMed ID: 21131973
  • Mokrani-Benhelli H, Gaillard L, Biasutto P, Guen T Le, Touzot F, Vasquez N, Komatsu J, Conseiller E, Pïcard C, Gluckman E, Francannet C, Fischer A, et al. 2013. Primary Microcephaly, Impaired DNA Replication, and Genomic Instability Caused by Compound Heterozygous ATR Mutations. Human Mutation 34: 374–384. PubMed ID: 23111928
  • O’Driscoll M, Ruiz-Perez VL, Woods CG, Jeggo PA, Goodship JA. 2003. A splicing mutation affecting expression of ataxia–telangiectasia and Rad3–related protein (ATR) results in Seckel syndrome. Nature Genetics 33: 497–501. PubMed ID: 12640452
  • Tanaka A, Weinel S, Nagy N, O’Driscoll M, Lai-Cheong JE, Kulp-Shorten CL, Knable A, Carpenter G, Fisher SA, Hiragun M, Yanase Y, Hide M, et al. 2012. Germline Mutation in ATR in Autosomal- Dominant Oropharyngeal Cancer Syndrome. The American Journal of Human Genetics 90: 511–517. PubMed ID: 22341969
  • Verloes A, Drunat S, Gressens P, Passemard S. 1993. Primary Autosomal Recessive Microcephalies and Seckel Syndrome Spectrum 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: 20301772
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TEST METHODS

Exome Sequencing with CNV Detection

Test Procedure

For PGxome® we use Next Generation Sequencing (NGS) technologies to cover the coding regions of targeted genes plus 10 bases of flanking non-coding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. As required, genomic DNA is extracted from patient specimens. Patient DNA corresponding to these regions is captured using Agilent Clinical Research Exome hybridization probes. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads (Illumina, San Diego, CA, USA). The following quality control metrics are generally achieved: >97% of target bases are covered at >20x, and mean coverage of target bases >120x. Data analysis and interpretation is performed by the internally developed software Titanium-Exome. In brief, the output data from the NovaSeq 6000 is converted to fastqs by Illumina Bcl2Fastq, and mapped by BWA. Variant calls are made by the GATK Haplotype caller and annotated using in house software and SnpEff. Variants are filtered and annotated using VarSeq (www.goldenhelix.com).

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.

Copy number variants (CNVs) are also detected from NGS data. We utilize a CNV calling algorithm that compares mean read depth and distribution for each target in the test sample against multiple matched controls. Neighboring target read depth and distribution and zygosity of any variants within each target region are used to reinforce CNV calls. All CNVs are confirmed using another technology such as aCGH, MLPA, or PCR before they are reported.

Analytical Validity

NextGen Sequencing: 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.

Copy Number Variant Analysis: The PGxome test detects most larger deletions and duplications including intragenic CNVs and large cytogenetic events; however aberrations in a small percentage of regions may not be accurately detected due to sequence paralogy (e.g., pseudogenes, segmental duplications), sequence properties, deletion/duplication size (e.g., 1-3 exons vs. 4 or more exons), and inadequate coverage. In general, sensitivity for single, double, or triple exon CNVs is ~70% and for CNVs of four exon size or larger is >95%, but may vary from gene-to-gene based on exon size, depth of coverage, and characteristics of the region.

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 sequencing does not reveal any heterozygous differences from the reference sequence, we cannot be certain that we were able to detect both patient alleles.

For technical reasons, the PGxome test is not 100% sensitive. Some exons cannot be efficiently captured, and some genes cannot be accurately sequenced because of the presence of multiple copies in the genome. Therefore, a small fraction of sequence variants will not be detected.

We sequence coding exons for all available transcripts plus 10 bp of flanking non-coding DNA for each exon. We also sequence other regions within or near genes in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere.  Unless specifically indicated, test reports contain no information about other portions of genes.

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 amplification.

Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes if taken 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.

Balanced translocations or inversions are only rarely detected.

Certain types of sex chromosome aneuploidy may not be detected.  

Our ability to detect CNVs due to somatic mosaicism is limited.

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.

A negative finding does not rule out a genetic diagnosis.

Genetic counseling to help to explain test results to the patients and to discuss reproductive options is recommended.

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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
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