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XERODERMA PIGMENTOSUM SANGER SEQUENCING PANEL

Clinical Features

Xeroderma pigmentosum (XP) results in skin changes (e.g. blistering due to sunburn (60% of cases), persistent erythema, freckling, and hyper/hypopigmentation), early onset skin cancers and internal cancers, ocular problems (e.g. severe keratitis, eyelid atrophy, and conjunctival inflammatory masses), and neurologic abnormalities (e.g. microcephaly, diminished/absent deep tendon stretch reflexes, progressive sensorineural hearing loss, and cognitive impairment) (Kraemer and DiGiovanna. GeneReviews. 2012). The types of cancers involved are usually non-melanoma skin cancers (basal and squamous cell) and cutaneous melanoma.  The incidence of skin cancer is 1000 times the rate of the general population (Webb. BMJ 23;336(7641):444-6, 2008). Sun exposure must be limited because skin cancer can appear within the first decade of life due to ultraviolet radiation; removal of early pre-cancerous lesions is beneficial. XP occurs in approximately 1 in 250,000 live births in the United States, 2.3 in 1,000,000 live births in Western Europe (Kleijer et al. DNA Repair (Amst) 3;7(5):744-50, 2008), and a higher prevalence of 1 in 22,000 live births in Japan (Hirai et al. Mutat Res 10;601(1-2):171-8, 2006).  Higher prevalence is also seen in North Africa, and the Middle East, possibly due to consanguinity. XP presents complete penetrance, but shows a wide variety of clinical heterogeneity within and between XP groups. Heterogeneity may be due to length of sunlight exposure, complementation group, nature of mutation and unknown factors (Lehmann et al. Orphanet Journal of Rare Diseases 6:70, 2011).

Citations
  • Hirai Y, Kodama Y, Moriwaki S-I, Noda A, Cullings HM, MacPhee DG, Kodama K, Mabuchi K, Kraemer KH, Land CE, Nakamura N. 2006. Heterozygous individuals bearing a founder mutation in the XPA DNA repair gene comprise nearly 1% of the Japanese population. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 601: 171–178. PubMed ID: 16905156
  • Kleijer WJ, Laugel V, Berneburg M, Nardo T, Fawcett H, Gratchev A, Jaspers NGJ, Sarasin A, Stefanini M, Lehmann AR. 2008. Incidence of DNA repair deficiency disorders in western Europe: Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. DNA Repair 7: 744–750. PubMed ID: 18329345
  • Kraemer KH, DiGiovanna JJ. 2013. Xeroderma Pigmentosum. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301571
  • Lehmann AR, McGibbon D, Stefanini M. 2011. Xeroderma pigmentosum. Orphanet J Rare Dis 6: 5. PubMed ID: 22044607
  • Webb S. 2008. Xeroderma pigmentosum. BMJ 336: 444–446. PubMed ID: 18292171

Genetics

Xeroderma pigmentosum is an autosomal recessive disorder caused by mutations in the XPA, ERCC3, XPC, ERCC2, DDB2, ERCC4, and ERCC5 genes, which belong to the XPA, XPB, XPC, XPD, XPE, XPF, and XPG complementation groups respectively. The products of these genes are involved in DNA repair, specifically nucleotide excision repair (NER). This mechanism of repair is involved in removing UV-induced dipyrimidine photoproducts and chemical crosslinks. If the damage is left unchecked cells have the potential for cancer development. Specific genotype-phenotype correlations exist for the XP forms (see for review Kraemer and DiGiovanna. GeneReviews. 2012). The XP variant phenotype, which is caused by POLH mutations lead to affected individuals who have an increased skin cancer incidence and eye abnormalities like most XP patients. Mutations in the POLH gene do not cause aberrant nucleotide excision repair, but have difficulty replicating DNA containing ultraviolet-induced damage (Lehmann et al. Proceedings of the National Academy of Sciences of the United States of America 1975, 72:219-223).
 
The XPC and DDB2 (XPE) protein products are required for initial damage detection. Afterwards the products XPB and XPD open up DNA around the photoproduct. XPA verifies correct protein assembly and then the XPG and XPF nucleases cleave the DNA on either side of the damage for correct repair via the DNA polymerase η (encoded by POLH) (Naegeli and Sugasawa. DNA Repair 10:673-683, 2011; Kraemer and DiGiovanna. GeneReviews. 2012). Two types of NER are performed within the cell, namely, global genome repair and transcription coupled repair.  The former is involved in global genome maintenance, whereas the latter is involved in repair of DNA from transcriptionally active genes. All aforementioned protein products are involved in transcription-coupled repair and most of these gene products are also with global genome repair, with the exception of XPC and XPE. Interestingly, patients with XPC or XPE mutations do not have severe sunlight lesions and neurological abnormalities, and this may have to do with their type of NER pathway involvement.

Citations
  • Kraemer KH, DiGiovanna JJ. 2013. Xeroderma Pigmentosum. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301571
  • Lehmann et al. (1975). "Xeroderma pigmentosum cells with normal levels of excision repair have a defect in DNA synthesis after UV-irradiation." Proceedings of the National Academy of Sciences of the United States of America 72:219-223. PubMed ID: 1054497
  • Naegeli H, Sugasawa K. 2011. The xeroderma pigmentosum pathway: Decision tree analysis of DNA quality. DNA Repair 10: 673–683. PubMed ID: 21684221

Testing Strategy

The DNA repair protein complementing XP-A cells protein is encoded by 7 exons from the XPA gene on chromosome 9q22.3; the TFIIH basal transcription factor complex helicase XPB subunit is encoded by 15 exons from the ERCC3 gene on chromosome 2q21; the DNA repair protein complementing XP-C cells is encoded by 16 exons from the XPC gene on chromosome 3p25.2; the TFIIH basal transcription factor complex helicase XPD subunit is encoded by 23 exons from the ERCC2 gene on chromosome 19q13.3; the DNA damage-binding protein 2 is encoded by 10 exons from the DDB2 gene on chromosome 11p12-p11; the DNA repair endonuclease XPF is encoded by 11 exons from the ERCC4 gene on chromosome 16p13.3; the DNA repair protein complementing XP-G cells is encoded by 15 exons from the ERCC5 gene on chromosome 13q22-q34; and the DNA polymerase eta is encoded by 10 exons (exons 2-11) from the POLH gene on chromosome 6p21.  Testing is accomplished by amplifying each coding exon and ~20 bp of adjacent noncoding sequence, then determining the nucleotide sequence using standard dideoxy sequencing methods and a capillary electrophoresis instrument. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.

Testing first for genes with founder mutations, present in some parts of the world such as Japan (i.e. XPA gene) (Hirai et al. Mutat Res 10;601(1-2):171-8, 2006) and northern Africa (i.e. XPC gene) (Tamura et al, J Invest Dermatol 130(6):1491-3, 2010), may be more cost-effective and time efficient for probands from an area where founder mutations are known. Please let us know if you would like particular genes tested ordered in a specific sequence, otherwise we will sequence based on order of mutation prevalence (see clinical sensitivity section).

Citations
  • Hirai Y, Kodama Y, Moriwaki S-I, Noda A, Cullings HM, MacPhee DG, Kodama K, Mabuchi K, Kraemer KH, Land CE, Nakamura N. 2006. Heterozygous individuals bearing a founder mutation in the XPA DNA repair gene comprise nearly 1% of the Japanese population. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 601: 171–178. PubMed ID: 16905156
  • Tamura et al. (2010). "Founder mutations in xeroderma pigmentosum."  J Invest Dermatol 130(6):1491-3. PubMed ID: 20463673

Indications for Test

Individuals with a clinical presentation of XP. People with a family history of XP wanting to know their XP mutation status can also be tested. Carriers are asymptomatic, but the possibility of increased cancer risk is currently being assessed. Earlier diagnosis may improve patient prognosis through regular screening and treatment for early-onset malignancies. This test is specifically designed for heritable germline mutations and is not appropriate for the detection of somatic mutations in tumor tissue.

Gene(s)

Offical Gene Symbol OMIM Id
DDB2 600811
ERCC2 126340
ERCC3 133510
ERCC4 133520
ERCC5 133530
POLH 603968
XPA 611153
XPC 613208

Disease(s)

Name OMIM Id
Xeroderma Pigmentosum, Complementation Group A 278700
Xeroderma Pigmentosum, Complementation Group B 610651
Xeroderma Pigmentosum, Complementation Group C 278720
Xeroderma Pigmentosum, Complementation Group D 278730
Trichothiodystrophy Photosensitive 601675
Xeroderma Pigmentosum, Complementation Group E 278740
Xeroderma Pigmentosum, Complementation Group F 278760
XFE Progeroid Syndrome 610965
Xeroderma Pigmentosum, Complementation Group G 278780
Xeroderma Pigmentosum, Variant Type 278750

Related Tests

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Geneticist(s)

Test Methods

Bi-directional Sanger Sequencing

Test Number Test Price CPT Code
1140 XPA Sanger Sequencing $680 81479
XPC Sanger Sequencing $970 81479
ERCC3 Sanger Sequencing $970 81479
ERCC4 Sanger Sequencing $840 81479
DDB2 Sanger Sequencing $710 81479
ERCC2 Sanger Sequencing $990 81479
POLH Sanger Sequencing $840 81479
ERCC5 Sanger Sequencing $1,060 81479
Full Panel* $6,000

*In a panel of 3 or more genes, if more than 50% of the genes are tested, then a 15% discount will be applied to all genes.

In addition, Targeted Familial Mutation testing via Sanger sequencing is available for any gene in the panel:

Test Number Test Price CPT Code
100 Targeted Familial Mutations - Single Exon Sequencing $250 81479
200 Targeted Familial Mutations - Double Exon Sequencing $370 81479
300 Targeted Familial Mutations - Triple Exon Sequencing $440 81479

Test Procedure

As required, DNA is extracted from the patient specimen using a 5 Prime ArchivePure DNA Blood Kit. PCR is used to amplify the indicated exons plus additional flanking intronic or other 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. Sequencing is performed separately in both the forward and reverse directions.

Clinical Sensitivity

DNA testing confirms diagnosis in ~77% of XP patients. The proportion of XP patients that have mutations in XPA, XPC, ERCC2, ERCC4, and ERCC5 are 25%, 25%, 15%, 6%, and 6%, respectively. Individuals with XP variant will have mutations in POLH in 21% of cases. Mutations in the XPA gene are more common in Japan and rare in the United States and Europe. Mutations in the ERCC3 and DDB2 genes are rare (Kraemer and DiGiovanna. GeneReviews. 2012).

Some individuals have a phenotype with both features of XP and Cockayne syndrome (i.e. increased skin cancer in XP and dysmyelination in CS). These XP/CS individuals have mutations in either the ERCC2, ERCC3 or ERCC5 genes (Rapin et al Neurology 28;55(10):1442-9, 2000).

Citations
  • Kraemer KH, DiGiovanna JJ. 2013. Xeroderma Pigmentosum. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong C-T, and Stephens K, editors. GeneReviews™, Seattle (WA): University of Washington, Seattle. PubMed ID: 20301571
  • Rapin I, Lindenbaum Y, Dickson DW, Kraemer KH, Robbins JH. 2000. Cockayne syndrome and xeroderma pigmentosum. Neurology 55: 1442–1449. PubMed ID: 11185579

Analytical Validity

As of July 2013, we compared 7.3 megabases 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 9.5 years of our lab operation we have Sanger sequenced roughly 2,000 PCR amplicons (~ 1 megabase). No errors have been identified.

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.

Turnaround Time

Maximum of 40 days for the first gene and 10 days for each subsequent gene.

Deletion/Duplication Testing Via Array Comparative Genomic Hybridization

Test Number Test Price CPT Code
600 XPA Deletion/Duplication Testing via aCGH $990 81479
XPC Deletion/Duplication Testing via aCGH $990 81479
ERCC3 Deletion/Duplication Testing via aCGH $990 81479
ERCC4 Deletion/Duplication Testing via aCGH $990 81479
DDB2 Deletion/Duplication Testing via aCGH $990 81479
ERCC2 Deletion/Duplication Testing via aCGH $990 81479
POLH Deletion/Duplication Testing via aCGH $990 81479
ERCC5 Deletion/Duplication Testing via aCGH $990 81479
Full Panel* $1,790

*Prices:  Allele copy number analysis of one gene                                              $ 990
               Allele copy number analysis of two genes                                            $1190
               Allele copy number analysis of three or more genes                          $1290 + $100 for every additional gene
               Reanalysis of existing aCGH for additional genes (up to 5 genes)     $ 690

Test Procedure

As required, DNA is extracted from the patient specimen using a Gentra PUREGENE kit. 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 hours at 65ºC.  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.

PreventionGenetics’ high density gene-centric (HDGC) aCGH is designed to have comprehensive coverage for both coding and non-coding regions for each targeted gene with very high density probe coverage.  The average probe spacing within each exon is 47 bp or a minimum of three probes per exon covering all targeted exons and UTRs.  The average probe spacing is 289 bp covering all intronic, 2kb upstream and downstream regions of each targeted gene.  In addition, the flanking 300-bp intronic sequence on either side of targeted exons has enriched probe coverage.  Therefore, PreventionGenetics’ aCGH enables the detection of relatively small deletion and amplification mutations within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene.

Clinical Sensitivity


Analytical Validity

PreventionGenetics’ high density gene-centric custom designed aCGH enables the detection of relatively small deletion and amplification mutations (down to ~300 bp) within a single exon of a given gene or deletion and amplification mutations encompassing the entire gene. PreventionGenetics has established and verified this test’s accuracy and precision.

Analytical Limitations

Any copy number changes smaller than 300bps (within the targeted region) may not be detected by our array.

This array may not detect deletion and amplification mutations 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 happened 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.

Turnaround Time

Maximum of 40 days, although many tests are completed in 3-4 weeks.

Requisition Form

  • The first three pages of the requisition form must accompany all specimens.
  • Billing information is on the third page.
  • Specimen and shipping instructions are listed on the fourth page.
  • All testing must be ordered by a qualified healthcare provider.

Download the Requisition Form (pdf)

Specimen Types

Whole Blood

(Delivery accepted Monday - Saturday)

  • Collect 2-5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube).  For Test #500-DNA Banking only, collect 10-20 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 good for up to 48 hours.
  • If refrigerated, blood specimen is good 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 a screw cap tube at least 15 µg of purified DNA at a concentration of at least 20 µg/ml.  For tests involving the sequencing of  more than three genes, send an additional 5 µg DNA per gene.  DNA may be shipped at room temperature.
  • Label the tube with the DNA concentration as well as the patient name, date of birth, and/or ID number.
  • Specify the composition of the solute.
  • We only accept genomic DNA for testing.  We do not accept products of whole genome amplification reactions or other amplification reactions.

Cell Culture

(Delivery accepted Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Ship at least two T25 flasks of confluent cells.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We do not culture cells.

Additional Information

Version: 1.2
Last Updated 01/03/2014