Autosomal Recessive Retinitis Pigmentosa genetic testing

The Autosomal Recessive Retinitis Pigmentosa (AR-RP) test is the most comprehensive genetic test available for screening mutations in a number of genes associated with the disease: CERKL, CNGA1, CNGB1, MERTK, PDE6A, PDE6B, PNR, RDH12, RGR, RLBP1, SAG, TULP1, CRB, RPE65, USH2A, USH3A, LRAT. The number of mutations to be tested by AR-RP test is 501.


Wt, heterozygous and homozygous nucleotide change G>A in position 2843 of CRB1 gene analyzed by APEX. The signals corresponding to A in the sense strand and T in the antisense strand are indicative for mutation. The mutation causes amino acid change C948Y.
 
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Requirements for the DNA samples

• DNA quality needs to be ensured (an agarose-gel image from the high molecular weight DNA should be provided with the DNA samples)
• 6 mg of genomic DNA is required for AR-RP chip analysis 
• Preferred concentration range of the DNA is 100-250 ng/ul 
• DNA samples should be provided in pure sterile water 

DNA sample submitting 

• For speedy and secure delivery, international courier services, for example DHL, UPS and FedEx, are recommended; alternatively, you can send samples by air mail as a small parcel. 

• Since high quality DNA samples are stable, there is no need for shipment on dry or wet ice.
  Care should be taken to avoid drying out; please use either screw cap tubes or wrap the caps of each Eppendorf tube with parafilm. 

• In order to avoid damage to the tubes during shipment, a tube storage box made of plastic or cardboard, and doubling it with a padded envelope, is recommended. Please avoid using round containers, such as 50 ml Corning tubes, for tube protection. 

• Send samples to the following address:
         Asper Biotech
         Oru 3
         Tartu 51014
         Estonia
         Ph: +372 7 441 556 

• Please fill in the DNA sample submission form, which improves and accelerates the handling of DNA samples submitted to Asper and include it in the package as you ship samples. Download the form in Microsoft Word or Adobe Acrobat (pdf) format. 

• Notify us by email (info@asperophthalmics.com, or the respective project manager), including the number of samples, which test is to be performed, and shipment tracking data).

• Enclose in the package the list of samples, which test is to be performed and DNA quality data, if available. 

• Please make sure that the declared value for the package in the shipment documents does not exceed 10 EUR (USD).

Additional services

Additional verification by DNA Sequencing
To confirm the results with secondary method, Asper provides verification of the APEX findings by dideoxy sequencing. Sequencing will be performed under strict quality control regulations by professionally trained personnel on Applied Biosystems 3130 Genetic Analyzer.
Hard copies of the reports on official blank
Asper can provide the formatted results on company’s official letter blank upon request. The hard copy will be signed and sealed by head of the lab and sent out by registered mail. 
Storage of DNA samples at Asper's DNA bank
Asper always performs the screening with as limited amount of DNA as possible. If there will be enough remained DNA; it can be storaged in Asper’s DNA bank. The DNA can be used for further analysis by other tests or just for re-screening. The amount of remained DNA will be measured and report will be sent to partner. The data of DNA samples will be recorded in our laboratory information system and stored under strict quality controlled manner. 
Returning of DNA samples
The remained DNA can be also sent back to partners either by regular mail or by courier. 

Turnaround Time

Express delivery – The results will be delivered in 3 – 5  working days after the arrival of samples. Please note that the cost of the express delivery differs from the standard delivery. 
Standard delivery – The results will be delivered approximately in 3 – 6 weeks after the arrival of samples.

For further information

1. Please contact info@asperophthalmics.com
2. AR-RP genetic test (pdf, 141 kb)
3. Payment details

Publications

1. Microarray-based mutation analysis of the ABCA4 (ABCR) gene in autosomal recessive cone-rod dystrophy and retinitis pigmentosa. 
  
Klevering BJ, Yzer S, Rohrschneider K, Zonneveld M, Allikmets R, van den Born LI, Maugeri A, Hoyng CB, Cremers FP.
European Journal of Human Genetics (2004) 12, 1024–1032.

2. Genotyping microarray (disease chip) for leber congenital amaurosis: detection of modifier alleles.

Zernant J, Kulm M, Dharmaraj S, den Hollander AI, Perrault I, Preising MN, Lorenz B, Kaplan J, Cremers FP, Maumenee I, Koenekoop RK, Allikmets R.
Invest Ophthalmol Vis Sci. 2005 Sep;46(9):3052-9.

PURPOSE: Leber congenital amaurosis (LCA) is an early-onset inherited disorder of childhood blindness characterized by visual impairment noted soon after birth. Variants in at least six genes (AIPL1, CRB1, CRX, GUCY2D, RPE65, and RPGRIP1) have been associated with a diagnosis consistent with LCA or early-onset retinitis pigmentosa (RP). Genetically heterogeneous inheritance complicates the analyses of LCA cases, especially in patients without a family history of the disorder, and conventional methods are of limited value. METHODS: To overcome these limitations, arrayed primer extension (APEX) technology was used to design a genotyping microarray for early-onset, severe retinal degenerations that includes all of the >300 disease-associated variants currently described in eight genes (in addition to the six just listed, the early-onset RP genes LRAT and MERTK were added). The resultant LCA array allows simultaneous detection of all known disease-associated alleles in any patient with early-onset RP. The array was validated by screening 93 confirmed patients with LCA who had known mutations. Subsequently, 205 novel LCA cases were screened on the array, followed by segregation analyses in families, if applicable. RESULTS: The microarray was >99% effective in determining the existing genetic variation and yielded at least one disease-associated allele in approximately one third of the novel patients. More than two (expected) variants were discovered in a substantial fraction (22/300) of the patients, suggesting a modifier effect from more than one gene. In support of the latter hypothesis, the third allele segregated with a more severe disease phenotype in at least five families. CONCLUSIONS: The LCA genotyping microarray is a robust and cost-effective screening tool, representing the prototype of a disease chip for genotyping patients with a genetically heterogeneous condition. Simultaneous screening for all known LCA-associated variants in large LCA cohorts allows systematic detection and analysis of genetic variation, facilitating prospective diagnosis and ultimately predicting disease progression.

3. Development of a Genotyping Microarray for Usher Syndrome
Cremers FP, Kimberling WJ, Kulm M, de Brouwer A, van Wijk E, Te Brinke H, Cremers CW, Hoefsloot LH, Banfi S, Simonelli F, Fleischhauer JC, Berger W, Kelley PM, Haralambous E, Bitner-Glindzicz M, Webster AR, Saihan Z, Debaere E, Leroy BP, Silvestri G, McKay G, Koenekoop RK, Millan JM, Rosenberg T, Joensuu T, Sankila EM, Weil D, Weston MD, Wissinger B, Kremer H.
J Med Genet. 2006 Sep 8

Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, 8 genes have been implicated, which together comprise 347 protein-coding exons. Therefore, sequence analysis and the most routinely used mutation scanning techniques are not cost-effective for molecular diagnostics of Usher syndrome. To improve DNA-diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. METHODS: Allele-specific oligonucleotides corresponding to 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. The accuracy of the microarray was analysed using DNAs from 158 patients with known mutations; the efficiency of the microarray was analysed using DNAs from 370 novel European and American patients with Usher syndrome. RESULTS: Validation of the microarray yielded an accuracy of >98%. Among the novel patients, sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I (USH1), 45/189 (24%) patients with Usher syndrome type II (USH2), 6/21 (29%) patients with Usher syndrome type III (USH3), and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. DISCUSSION: The Usher genotyping microarray represents a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.