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RLBP1-RELATED DISORDERS VIA THE RLBP1 GENE

Clinical Features

Retinitis pigmentosa (RP; OMIM # 268000) or rod cone dystrophies (RCDs) represent a group of hereditary retinal dystrophies with a worldwide prevalence of ~1 in 4000 (Booij et al. J Med Genet 42 (11): e67, 2005). RP is clinically characterized by retinal pigment deposits visible on fundus examination, nyctalopia (night blindness), followed by progressive loss of peripheral vision in daylight, which eventually leads to blindness (van Soest et al. Surv Ophthalmol 43(4):321-334,1999).
     A unique atypical variant of RP denoted Bothnia dystrophy (BD; OMIM # 607475) has been identified in the northern Swedish population with an estimated prevalence of ~1 in 2500 due to founder effect (Köhn et al. Invest Ophthalmol Vis Sci 49(7):3172-3177, 2008). BD is characterized by early, severe nyctalopia in early childhood, fundus changes similar to retinitis punctata albescens (RPA), and progressive macular and peripheral retinal degeneration with progressive reduction of visual acuity (VA) and visual fields (VF) that leads to legal blindness in early adulthood (Burstedt et al. Arch Ophthalmol 119(2):260-267, 2001; Burstedt et al. Golovleva Arch Ophthalmol 128(8):989-995, 2010).
     RPA (OMIM #136880) is an autosomal recessive (ar) form of RP characterized by nyctalopia, discrete uniform white flecks in the fundus, reduced VA and VF, progressive attenuation of retinal arterioles, abnormal fundus pigmentation and abnormal electroretinogram (ERG) amplitudes (Fishman et al. Arch Ophthalmol 122(1):70-75, 2004).
     Fundus albipunctatus (FA; OMIM #136880) is an infrequent form of apparently stationary nyctalopia. FA is characterized by the presence of numerous white flecks in the fundus with a greater concentration in the mid and peripheral regions of the retina, which is comparable to RPA (Naz et al. Br J Ophthalmol 95(7):1019-1024, 2011).
    Newfoundland rod–cone dystrophy (NFRCD; OMIM # 607476) ophthalmoscopic appearance is similar to that of RPA with a different age of onset. NFRCD is typically evident in the first decade of life with rapid progression, leading to legal blindness by the second to fourth decades (Eichers et al. Am J Hum Genet 70(4):955-964. 2002).

Citations
  • Booij JC. 2005. Identification of mutations in the AIPL1, CRB1, GUCY2D, RPE65, and RPGRIP1 genes in patients with juvenile retinitis pigmentosa. Journal of Medical Genetics 42: e67–e67. PubMed ID: 16272259
  • Burstedt, M.S. and Golovleva, I. (2010). "Central retinal findings in Bothnia dystrophy caused by RLBP1 sequence variation." Arch Ophthalmol 128(8):989-995. PubMed ID: 20696998
  • Burstedt, M.S. et al. (2001). "Ocular phenotype of bothnia dystrophy, an autosomal recessive retinitis pigmentosa associated with an R234W mutation in the RLBP1 gene." Arch Ophthalmol 119(2):260-267. PubMed ID: 11176989
  • Eichers, E. R. et al. (2002). "Newfoundland rod-cone dystrophy, an early-onset retinal dystrophy, is caused by splice-junction mutations in RLBP1." Am J Hum Genet 70(4):955-964. PubMed ID: 11868161
  • Fishman, G.A. et al. (2004). "Novel mutations in the cellular retinaldehyde-binding protein gene (RLBP1) associated with retinitis punctata albescens: evidence of interfamilial genetic heterogeneity and fundus changes in heterozygotes." Arch Ophthalmol 122(1):70-75. PubMed ID: 14718298
  • Köhn, L. et al. (2008). "Carrier of R14W in carbonic anhydrase IV presents Bothnia dystrophy phenotype caused by two allelic mutations in RLBP1." Invest Ophthalmol Vis Sci 49(7):3172-3177. PubMed ID: 18344446
  • Naz, S. et al. (2011). "Mutations in RLBP1 associated with fundus albipunctatus in consanguineous Pakistani families." Br J Ophthalmol 95(7):1019-1024. PubMed ID: 21447491
  • van Soest S, Westerveld A, Jong PT de, Bleeker-Wagemakers EM, Bergen AA. 1999. Retinitis pigmentosa: defined from a molecular point of view. Surv Ophthalmol 43: 321–334. PubMed ID: 10025514

Genetics

Mutations in the RLBP1 gene (OMIM # 607475), which is located on 15q26, are associated with various types of autosomal recessive retinal dystrophies such as RPA (Morimura et al. Invest Ophthalmol Vis Sci 40(5):1000-1004, 1999; Demirci et al. Am J Ophthalmol 138(1):171-173.2004; Fishman et al., 2004; Nakamura et al. Am J Ophthalmol 139(6):1133-1135.2005), arRP (Maw et al. Nat Genet 17(2):198-200, 1997), BD (Burstedt et al. Invest Ophthalmol Vis Sci 40(5):995-1000, 1999 and Burstedt et al., 2001), NFRCD (Eichers et al., 2002) and FA (Naz et al., 2011). RLBP1 encodes the cellular retinaldehyde-binding protein (CRALBP), which is mainly expressed in the retinal pigment epithelium (RPE) and Müller cells of the retina. CRALBP plays a crucial role in retinoid metabolism and visual pigment regeneration in the retina, where it functions as a carrier of endogenous retinoids such as 11-cis-retinol and 11-cis-retinal. Photoisomerization of 11-cis-retinal to all-trans-retinal in the retinal photoreceptor cells triggers phototransduction and ultimately results in visual sensation (Köhn et al., 2008; Stecher et al. Biochemistry 38(41):13542-13550, 1999). There are ~ 20 mutations have been reported in RLBP1, which includes missense, splicing, small deletions and gross deletions  (Human Gene Mutation Database).

Citations
  • Burstedt, M.S. et al. (1999). "Bothnia dystrophy caused by mutations in the cellular retinaldehyde-binding protein gene (RLBP1) on chromosome 15q26." Invest Ophthalmol Vis Sci 40(5):995-1000. PubMed ID: 10102298
  • Burstedt, M.S. et al. (2001). "Ocular phenotype of bothnia dystrophy, an autosomal recessive retinitis pigmentosa associated with an R234W mutation in the RLBP1 gene." Arch Ophthalmol 119(2):260-267. PubMed ID: 11176989
  • Demirci, F.Y. et al. (2004). "A novel compound heterozygous mutation in the cellular retinaldehyde-binding protein gene (RLBP1) in a patient with retinitis punctata albescens." Am J Ophthalmol 138(1):171-173. PubMed ID: 15234312
  • Eichers, E. R. et al. (2002). "Newfoundland rod-cone dystrophy, an early-onset retinal dystrophy, is caused by splice-junction mutations in RLBP1." Am J Hum Genet 70(4):955-964. PubMed ID: 11868161
  • Fishman, G.A. et al. (2004). "Novel mutations in the cellular retinaldehyde-binding protein gene (RLBP1) associated with retinitis punctata albescens: evidence of interfamilial genetic heterogeneity and fundus changes in heterozygotes." Arch Ophthalmol 122(1):70-75. PubMed ID: 14718298
  • Human Gene Mutation Database (Bio-base).
  • Köhn, L. et al. (2008). "Carrier of R14W in carbonic anhydrase IV presents Bothnia dystrophy phenotype caused by two allelic mutations in RLBP1." Invest Ophthalmol Vis Sci 49(7):3172-3177. PubMed ID: 18344446
  • Maw, M.A. et al. (1997). "Mutation of the gene encoding cellular retinaldehyde-binding protein in autosomal recessive retinitis pigmentosa." Nat Genet 17(2):198-200. PubMed ID: 9326942
  • Morimura, H. et al. (1999). "Recessive mutations in the RLBP1 gene encoding cellular retinaldehyde-binding protein in a form of retinitis punctata albescens." Invest Ophthalmol Vis Sci 40(5):1000-1004. PubMed ID: 10102299
  • Nakamura, M. et al. (2005). "Novel mutation in RLBP1 gene in a Japanese patient with retinitis punctata albescens." Am J Ophthalmol 139(6):1133-1135. PubMed ID: 15953459
  • Naz, S. et al. (2011). "Mutations in RLBP1 associated with fundus albipunctatus in consanguineous Pakistani families." Br J Ophthalmol 95(7):1019-1024. PubMed ID: 21447491
  • Stecher, H. et al. (1999). "Isomerization of all-trans-9- and 13-desmethylretinol by retinal pigment epithelial cells." Biochemistry 38(41):13542-13550. PubMed ID: 10521261

Testing Strategy

This test involves bidirectional DNA Sanger sequencing of all coding exons of the RLBP1 gene. The full coding region of each exon plus ~20 bp of flanking non-coding DNA on either side are sequenced. 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.

Indications for Test

All patients with symptoms suggestive of Retinitis pigmentosa (RP), Bothnia dystrophy (BD), Retinitis Punctata Albescens (RPA), Fundus albipunctatus (FA) and Newfoundland rod–cone dystrophy (NFRCD) are candidates for this test. Family members of patients who have known mutations and carrier testing for at-risk family members are also candidates.

Gene(s)

Offical Gene Symbol OMIM Id
RLBP1 180090

Disease(s)

Name OMIM Id
Retinitis Pigmentosa 268000
Bothnia Retinal Dystrophy 607475
Pigmentary Retinal Dystrophy 136880
Newfoundland Rod-Cone Dystrophy 607476

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Test Methods

Bi-directional Sanger Sequencing

Test Number Test Price CPT Code
673 RLBP1 Sanger Sequencing $650 81479
100 RLBP1 Targeted Familial Mutations - Single Exon Sequencing $250 81479
200 RLBP1 Targeted Familial Mutations - Double Exon Sequencing $370 81479
300 RLBP1 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

All BD (20/20) affected patients from seven Vasterbotten families in northern Sweden were homozygous for the c.700C>T (p.Arg234Trp) mutation in exon 7 of the RLBP1 gene, which explains the founder effect (Burstedt et al. Invest Ophthalmol Vis Sci 40(5):995-1000, 1999). Köhn et al. (2008) also tested 121 individuals affected with arRP in the same population for the c.700C>T mutation and found that 67 were homozygous and 10 were heterozygous for that mutation (Köhn et al. Invest Ophthalmol Vis Sci 49(7):3172-3177, 2008).

Citations
  • Burstedt, M.S. et al. (1999). "Bothnia dystrophy caused by mutations in the cellular retinaldehyde-binding protein gene (RLBP1) on chromosome 15q26." Invest Ophthalmol Vis Sci 40(5):995-1000. PubMed ID: 10102298
  • Köhn, L. et al. (2008). "Carrier of R14W in carbonic anhydrase IV presents Bothnia dystrophy phenotype caused by two allelic mutations in RLBP1." Invest Ophthalmol Vis Sci 49(7):3172-3177. PubMed ID: 18344446

Analytical Validity

As of November 2014, we compared 11.3 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 11 years of our lab operation we have Sanger sequenced roughly 4,000 PCR amplicons (~ 2 megabases).  Only one error was 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, although many tests are completed in 2-3 weeks.

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Deletion/Duplication Testing Via Array Comparative Genomic Hybridization

Test Number Test Price CPT Code
600 RLBP1 Deletion/Duplication Testing via aCGH $990 81479

*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


Targeted testing for relatives of probands tested at PreventionGenetics is available (Test #1400, $340) if we are able to test the del/dup by PCR or MLPA.

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

Gross deletions were found in RLBP1 due to high density of Alu elements. Therefore, a systematic search for deletions is recommended when one or both alleles do not show a point mutation in case of RPA or flecked retinal dystrophy (Humbert et al. Invest Ophthalmol Vis Sci 47(11):4719-4724, 2006).

Citations
  • Humbert, G. et al. (2006). "Homozygous deletion related to Alu repeats in RLBP1 causes retinitis punctata albescens." Invest Ophthalmol Vis Sci 47(11):4719-4724. PubMed ID: 17065479

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.

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Requisition Form

  • The first four 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.

Version: 1.1
Last Updated 09/17/2013