Leber Congenital Amaurosis (LCA) genetic testing

Together with our partners a Leber congenital amaurosis (LCA) mutation detection tools has been developed and validated. The LCA microarray chip contains 495 disease-associated sequence variants previously identified in 12 LCA or early-onset RP genes: AIPL1, CRB1, CRX, GUCY2D, LRAT, TULP1, MERTK, CEP290, RDH12, RPGRIP1, LCA5 and RPE65.


The LCA chip has been extensively validated by leading scientific institutions worldwide. Hundreds of DNA samples from patients with LCA of diverse ethnicity were analyzed during the evaluation period. All variants detected by chip, were confirmed by direct sequencing, and no conflicting calls or false positives were identified all variants on chip are detected with 100% accuracy. 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.


Nucleotide change G154T in gene GUCY2D exon 2 analyzed by APEX. The signals corresponding to T in the sense strand and A in the antisense strand are indicative for mutation. The mutation causes amino acid change A52S.


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Requirements for the DNA samples

• the DNA quality needs to be ensured 
• 4 mg of genomic DNA is required for LCA 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 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 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. Leber congenital amaurosis (LCA) genetic testing  (pdf, 96 kb)
3. Payment details

Publications

1. 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.

2. Microarray-Based Mutation Detection and Phenotypic Characterization of Patients with Leber Congenital Amaurosis

Suzanne Yzer, Bart P. Leroy, Elfride De Baere,Thomy J. de Ravel, Marijke N. Zonneveld, Krysta Voesenek, Ulrich Kellner, Jose P. Martinez Ciriano, Jan-Tjeerd H. N. de Faber, Klaus Rohrschneider, Ronald Roepman, Anneke I. den Hollander, Johannes R. Cruysberg, Franc¸oise Meire, Ingele Casteels, Norka G. van Moll-Ramirez, Rando Allikmets, L. Ingeborgh van den Born, and Frans P. M. Cremers

Vis Sci. 2006;47:1167–1176) DOI:10.1167/iovs.05-0848

PURPOSE. To test the efficiency of a microarray chip as a diagnostic tool in a cohort of northwestern European patients with Leber congenital amaurosis (LCA) and to perform a genotype– phenotype analysis in patients in whom pathologic mutations were identified.

METHODS. DNAs from 58 patients with LCA were analyzed using a microarray chip containing previously identified disease- associated sequence variants in six LCA genes. Mutations identified by chip analysis were confirmed by sequence analysis. On identification of one mutation, all protein coding exons of the relevant genes were sequenced. In addition, sequence analysis of the RDH12 gene was performed in 22 patients. Patients with mutations were phenotyped.

RESULTS. Pathogenic mutations were identified in 19 of the 58 patients with LCA (32.8%). Four novel sequence variants were identified. Mutations were most frequently found in CRB1 (15.5%), followed by GUCY2D (10.3%). The p.R768W mutation was found in 8 of 10 GUCY2D alleles, suggesting that it is a founder mutation in the northwest of Europe. In early childhood, patients with AIPL1 or GUCY2D mutations show normal fundi. Those with AIPL1-associated LCA progress to an RP-like fundus before the age of 8, whereas patients with GUCY2Dassociated LCA still have relatively normal fundi in their mid- 20s. Patients with CRB1 mutations present with distinct fundus abnormalities at birth and consistently show characteristics of RP12. Pathogenic GUCY2D mutations result in the most severe form of LCA.

CONCLUSIONS. Microarray-based mutation detection allowed the identification of 32% of LCA sequence variants and represents an efficient first-pass screening tool. Mutations in CRB1, and to a lesser extent, in GUCY2D, underlie most LCA cases in this cohort. The present study establishes a genotype-phenotype correlation for AIPL1, CRB1, and GUCY2D.