Blue cone monochromatism: a phenotype and genotype assessment with evidence of progressive loss of cone function in older individuals

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Blue cone monochromatism: a phenotype and genotype assessment with evidence of progressive loss of cone function in older individuals
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  Blue conemonochromatism:a phenotype andgenotypeassessment withevidenceof progressive lossof cone function inolder individuals M Michaelides 1 , S Johnson 1 , MP Simunovic 2 ,K Bradshaw 3 , G Holder 4 , JD Mollon 2 , AT Moore 1 and DM Hunt 1 Abstract  Aim  To perform a detailed clinical andpsychophysical assessment of the members ofthree British families affected with blue conemonochromatism (BCM), and to determine themolecular basis of disease in these families.  Methods  Affected and unaffectedmembers of three families with BCM wereexamined clinically and underwentelectrophysiological and detailedpsychophysical testing. Blood samples weretaken for DNA extraction. The strategy for molecular analysis was to amplify the codingregions of the long wavelength-sensitive (L)and middle wavelength-sensitive (M) coneopsin genes and the upstream locus controlregion by polymerase chain reaction, and toexamine these fragments for mutations bydirect sequencing. Results  We have confirmed the reportedfinding of protan-like D-15 arrangements ofpatients with BCM. In addition, we havedemonstrated that the Mollon–Reffin (MR)Minimal test is a useful colour-discriminationtest to aid in the diagnosis of BCM. Affectedmales were shown to fail the protan anddeutan axes, but retained good discriminationon the tritan axis of the MR test, a compellingevidence for residual colour vision in BCM.This residual tritan discrimination was alsoreadily detected with HRR plates. In twofamilies, psychophysical testing demonstratedevidence for progression of disease. In twopedigrees, BCM could be linked to unequalcrossovers within the opsin gene array thatresulted in a single 5 0 -L/M-3 0 hybrid gene, withan inactivating Cys203Arg mutation. Thecausative mutations were not identified in thethird family. Conclusions  The MR test is a useful methodof detecting BCM across a wide range of agegroups; residual tritan colour discrimination isclearly demonstrated and allows BCM to bedistinguished from rod monochromatism.BCM is usually classified as a stationary conedysfunction syndrome; however, two of our families show evidence of progression. This isthe first report of progression associated with agenotype consisting of a single 5 0 -L/M-3 0 hybrid gene carrying an inactivating mutation.We have confirmed that the Cys203Arginactivating mutation is a common sequencechange in blue cone monochromats. Eye  (2005)  19,  2–10. doi:10.1038/sj.eye.6701391Published online 16 April 2004 Keywords:  monochromatism; cone; dysfunction;phenotype; genotype Introduction In order to derive colour vision, the normalhuman visual system compares the rate of quantum catchesinthreeclassesofcone,the short(S or blue) wavelength-sensitive, middle (M orgreen) wavelength-sensitive and long (L or red)wavelength-sensitive cones, which are maximallysensitive to light at 430, 535, and 565nm,respectively. This triad of cone types provides thephysiological substrate for trichromacy. 1–3 Received: 19 August 2003Accepted: 10 November2003Published online: 16 April2004 1 Institute of OphthalmologyUniversity College LondonLondon, UK 2 Department ofExperimental PsychologyUniversity of CambridgeCambridge, UK 3 Department ofOphthalmologyAddenbrooke’s HospitalCambridge, UK 4 Moorfields Eye HospitalCity Road London, UKCorrespondence:Professor DM HuntInstitute of OphthalmologyUniversity College London11-43 Bath StreetLondon EC1V 9EL, UKTel:  þ 44 (0)20 7608 6820Fax:  þ 44 (0)20 7608 6863E-mail: Eye (2005) 19,  2–10 &  2005 Nature Publishing Group All rights reserved 0950-222X/05 $  C L I    NI     C AL  S T  UDY   Each cone class contains its own visual pigmentcomposed of an opsin protein linked by a Schiff base tothe chromophore retinal. In humans, the L- and M-opsinsare encoded by genes on the X chromosome, and the S-opsin by a gene located on chromosome 7. 4 There existthree types of inherited colour vision deficiency in whichvision is dichromatic; each type corresponds to aselective defect in one of the three receptormechanisms. 1,2 Protanopia and deuteranopia arecharacterised by defects of the red- and green-sensitivemechanisms, respectively. They are among the commonX-linked disorders of colour vision whose associationwith alterations in the visual pigment gene cluster atXq28 identified those genes as encoding the red- andgreen-sensitive visual pigments. 4,5 The wild-typearrangement of the L- and M-opsin genes consists of ahead-to-tail tandem array of two or more repeat units of 39kb on chromosome Xq28 that are 98% identical at theDNA level. 5 This high level of identity would appear topre-dispose the L and M-opsin genes to unequal inter-and intragenic recombination. Transcriptional regulationof the L and M genes is controlled by an upstream locuscontrol region (LCR). 6 In contrast, tritanopia is anautosomal dominant disorder that is characterised by a selective defect of the blue-sensitive mechanismand is due to dominant mutations in the S-coneopsin gene.Blue cone monochromatism (BCM), or X-linkedincomplete achromatopsia, is a rare congenital stationarycone dysfunction syndrome, affecting less than 1 in100000 individuals, characterised by the absence of L-and M-cone function. 2 Thus, blue-cone monochromatspossess rod vision and a normal short-wavelength-sensitive cone mechanism. Photopic vision is mediated by the blue cones, and without a comparison betweendifferent classes of the cone photoreceptor, affectedindividuals are reported to have poor colourdiscrimination. Mutations in the L- and M-opsin genearray that result in the lack of functional L- and M-pigments, and thus inactivate the corresponding cones,have been identified in the majority of BCM casesstudied. 6,7 As in rod monochromacy (RM), BCM typicallypresents with reduced visual acuity, pendularnystagmus, and photophobia. Visual acuity is of theorder of 6/24–6/60. Eccentric fixation may be presentand myopia is a common finding. 8 BCM is distinguishedfrom RM via psychophysical or electrophysiologicaltesting. The photopic electroretinogram (ERG) isprofoundly reduced in both, but the S-cone ERG is wellpreserved in BCM. 9 BCM patients have highFarnsworth–Munsell 100-Hue scores, but have fewererrors in the vertical (tritan) axis when compared to RM.At the molecular level, mutation analyses have provedhighly efficient at establishing the molecular basis forBCM. 6,7 The mutations in the L- and M-opsin gene arraythat cause BCM fall into two classes. In the first class, anormal L- and M-opsin gene array is inactivated by adeletion in the LCR, located upstream of the L-opsingene. A deletion in this region would appear to abolishtranscription of all genes in the opsin gene array andtherefore inactivates both L- and M-cones. 10 In thesecond class of mutations, the LCR is preserved, butchanges within the L- and M-pigment gene array lead toloss of functional pigment production. The most commongenotype in this class consists of a single inactivated L/M hybrid gene. The first step in this second mechanism isthought to be unequal crossing over that reduces thenumber of genes in the array to one, followed by amutation that inactivates the remaining gene. A frequentinactivating mutation that has been described is thethymine-to-cytosine transition at nucleotide 648, whichresults in a cysteine-to-arginine substitution at codon 203,a mutation known to disrupt the folding of cone opsinmolecules. 11 Our aim was to perform a detailed clinical andpsychophysical assessment of members of three Britishfamilies affected with BCM, and subsequently todetermine the molecular basis of BCM in these families. Patients and methods Affected and unaffected members of three families withBCM were examined clinically and underwentpsychophysical and electrophysiological testing. Afterinformed consent was obtained, blood samples weretaken, genomic DNA was isolated from whole bloodusing an extraction kit (Nucleon s Biosciences), andmolecular genetic analysis was performed. Clinical assessment  The pedigrees of the families studied can be seen inFigures 1a–c. A full medical and ophthalmic history wastaken in all examined members. A full ophthalmologicalexamination was performed. All affected individuals hadan ERG performed. Adults had an ERG that conformedto the ISCEV standard, but the children had a modifiedprotocol using skin electrodes. Colour vision testingincluded the use of Hardy, Rand and Rittler (HRR)plates, SPP2 plates for acquired colour deficiency,Farnsworth–Munsell (FM) 100-hue test, Farnsworth D-15, the Mollon-Reffin (MR) minimal test, 12 acomputerised colour vision test, 13,14 and anomaloscopy.The FM 100-hue, Farnsworth D-15 and the MR testwere all performed under CIE Standard Illuminant Cfrom a MacBeth Easel lamp. The MR minimal test is asaturation discrimination-type test. 12 The caps used in Blue cone monochromatism M Michaelides  et al  3 Eye  this test are of a similar design to those in the D-15 andFM 100-hue tests. The test features a series of caps that liealong protan, deutan, and tritan lines, respectively. Inaddition, there is one demonstration cap, which does notlie along a dichromatic confusion axis. The remainder of the caps are all neutral, but have varying lightness (thereare a total of nine grey caps). The examiner places onecoloured cap among a group of neutral caps and asks thesubject to tap the side of the cap that is coloured. Thecoloured caps vary in their saturation, so the severity of the defect can be assessed. It can be performed bychildren as young as 5 years.  Molecular genetic analysis The underlying strategy for molecular analysis was toamplify the coding regions of the L- and M-cone opsingenes and the upstream LCR by PCR, and to examinethese fragments for mutations by direct sequencing.Exonic sequences of the L- and M-genes are 98%identical at the nucleotide level and, although there may be less evolutionary pressure for intronic sequences toremain stable, the introns of the two genes have alsoremained almost identical ( 4 99.9% identical at thenucleotide level). It was not possible, therefore, to designPCR primers that amplify only L- or only M-opsin exonicfragments from genomic DNA. Consequently, the primerpairs used in this study amplified L and M sequencessimultaneously, and individual nucleotide differences between the genes were subsequently utilised fordifferentiation between the genes in sequence analysis.(i) PCR amplification of the L- and M- opsin genes:Intronic forward and reverse PCR primers (Table 1) weredesigned to amplify the LCR, and all six exons of the L-and M-genes from genomic DNA. LCR primers weredesigned based on the published sequence, 10 andencompassed the LCR core sequence. Primer pairs thatwould co-amplify both L- and M-opsin exonic sequenceswere designed within each intron approximately 50bpfrom the intron–exon junction, in order that the whole of each exon, some flanking DNA, and the splice sites wereamplified. The design of these primers was based on thepublished sequences of the L- and M-opsin genessequence. 5 PCR reactions (50 m l) were performed asfollows: 1   NH 4  buffer, 1mM MgCl 2 , 200 m M each Figure 1  Family pedigrees showing X-linked recessive inheri-tance pattern. Affected males are represented by filled-insymbols, carrier females by a central black dot. (a) Family A:Subject III:2 is deuteranomalous and her father is reported to bered-green colour blind. This is represented by a shadedquadrant. (b) Family B (c) Family C. Table 1  Primers for amplification of the LCR and L/R exons 1 to 6 Primer name Sequence  T a 1 C Product size (bp) LCR1 þ  5 0 -ggcaaatggccaaatggt-3 0 49 884LCR1- 5 0 -ccatgctatttggaagcc-3 0 L/M.Ex1F 5 0 -ggtgggaggaggaggtctaa-3 0 64 334L/M.Ex1R 5 0 -ggtggcccccagtgcagcc-3 0 L/M.Ex2F 5 0 -ggtatagacaggcggtgctg-3 0 60 400L/M.Ex2R 5 0 -gtgaatgagtggtttccgcc-3 0 L/M.Ex3F 5 0 -gtctaagcaggacagtgggaagctttgctt-3 0 60 302L/M.Ex3R 5 0 -taaggtcacagagtctgacc-3 0 L/M.Ex4F 5 0 -acaaaccccacccgagttgg-3 0 58 340L/M.Ex4R 5 0 -aggagtctcagtggactcat-3 0 L/M.Ex5F 5 0 -cctctcctcctccccacaac-3 0 62 402L/M.Ex5R 5 0 -caggtggggccatcactgca-3 0 L/M.Ex6F 5 0 -agggaaggctcgggcacgta-3 0 60 283L/M.Ex6R 5 0 -gataaattacatttattttacaggg-3 0 Blue cone monochromatism M Michaelides  et al  4 Eye  dNTP, 10pmols each of sense and antisense primers,200ng-1 m g DNA, 1U Bio Taq ,  X  m l dH 2 O F with theexception that the concentrations of MgCl 2  used in LCR,exon 3 and 6 PCR reactions were optimised at 0.5mM,0.5mM and 2mM respectively F and with annealing atthe exon-specific temperatures (Table 1). PCR productswere visualised by electrophoresis using low meltingtemperature agarose gel. Target products were thenexcised and eluted.(ii) Mutation analysis of the PCR-amplified exons andLCR was carried out by direct sequencing using a cycle-sequencing kit. After ethanol precipitation, the DNAproducts were analysed on an ABI 373A automated DNAsequencer and examined for alterations, utilising Sequencing Analysis  (ABI Prism TM ) and  GeneWorks TM software. Results PhenotypeFamily A  (Figure 1a)  Patient III:3 F This 7-year-old boy(proband) was srcinally seen at 1 year of age. He wasfound to have horizontal pendular nystagmus, normalfundi, and clear media. A family history of nystagmus,photophobia, and ‘colour blindness’ affecting males of the family, including his grandfather (I:1) and malecousin (III:1), was established. ERG testing revealedabsent cone responses, but normal rod responses.He had a visual acuity of 3/36 in each eye with hismyopic correction. Psychophysical testing on the MR testand HRR plates revealed reasonable discrimination onlyalong the tritan axis. On computerised testing, his colour-discrimination ellipses were oriented along the angle thatone would expect of someone making colourdiscriminations based on a comparison of quantumcatches in the rods and S-cones.Patient I:1 F This 60-year-old man was found to have avisual acuity of 6/36 in the right eye and 6/60 in the left.He was found to have clear lenses and mild macularretinal pigment epithelial (RPE) changes. As a child, hehad obvious nystagmus, but this had improvedthroughout life, to the point that it was not at allnoticeable. He felt that his vision had continued to slowlydeteriorate throughout life. Cone ERG responses wereabsent, but rod responses were normal. On the basis of his results on the HRR plates, the D-15, the MR minimaltest, and Nagel anomaloscope, it was concluded that hehad no residual colour vision.His 12-year-old grandson (III:1) had a visual acuity of 6/60 in the right eye and 6/36 in the left and displayedevidence of residual colour discrimination. He had clearmedia and normal fundi. He showed reasonablediscrimination along both the tritan line of MR test andalso on the SPP2 tritan plates. He displayed a protanordering of the D-15. On computerised testing hisellipses were oriented along the angle that is expected forcolour discriminations based solely upon a comparisonofquantumcatches intherodsandtheS-cones(Figures2,4 and 5).Patient III:2 F This 14-year-old daughter of II:2 wasasymptomatic. On the Nagel anomaloscope, she wasfound to be deuteranomalous. On the MR minimal test,she showed good discrimination on the tritan axis, butwas badly impaired on both protan and deutan axes.Patients II:2 and II:3 were both asymptomatic, and ondetailed psychophysical testing were found to havenormal colour vision. The father of patients III:1 and III:2was not available for psychophysical testing, but wasreported to be ‘colour blind’.A consistent psychophysical hypothesis from theseobservations would be that the proband III:3 and III:1have inherited from their maternal grandfather I:1, viatheir mothers, an X-chromosome with an altered opsinarray that has led to BCM. III:2 has also inherited thisaltered X-chromosome from her mother, and anX-chromosome that leads to a deuteranomalousphenotype from her father. Since III:3 and III:1 both havesome residual colour discrimination and theirgrandfather has none, it would appear that theircondition is not stationary. III:3 and III:1 can be labelled blue-cone monochromats, whereas their grandfather behaves as a rod monochromat, presumably as a result of continued S-cone loss. The lack of colour vision seen inthe grandfather is highly unlikely to be due to lenticularchanges, since his lenses were found to be clear. Figure 2  Patient III:1 (Family A): Colour-discrimination ellipse,oriented along the angle that would be expected with someonemaking colour discriminations based upon a comparison of quantum catches in the rods and the S-cones. Blue cone monochromatism M Michaelides  et al  5 Eye  Family B  (Figure 1b)  Patient III:2 F This 12-year-old boy(proband) srcinally presented with pendularnystagmus, poor visual acuity (6/24 in both eyes), andmyopia. Ocular media were clear with normal fundi.ERG revealed absent cone but normal rod responses. Onpsychophysical testing, the anomaloscope and Sloan’stest for achromatopsia suggested a rod-dominatedspectral sensitivity function. On the D-15, he showed theprotan-like pattern reported for BCM by Weis &Biersdorf. 15 On the computer test, he failed completelyon the protan and deutan lines, but scored nearlynormally along the tritan line, which modulates the bluecones. In addition, his ellipses were well aligned to thetheoretical S-cone/rod confusion axis, consistent with themechanism whereby colour discriminations are basedupon a comparison between rod and S-cone quantumcatches (Figures 3–5). Exactly the same pattern wasexhibited on the MR minimalist test: he could not findsaturated protan and deutan probes among the greydistractors, but could find the least saturated tritan cap.On the SPP2 plates for acquired colour deficiencies, hepassed the plates that are failed by those with purelyscotopic vision (RM).Patient III:1 F This 14-year-old brother of the probandalso presented with pendular nystagmus, poor visualacuity (6/24 in the right eye and 6/36 in the left), myopia,and photophobia. Ocular media were clear with normalfundi. ERG revealed absent cone responses but normalrod function. He showed reasonable discrimination onlyalong the tritan axis on HRR testing. He declined furtherpsychophysical testing.Patient II:1 F The 50-year-old mother of III:1 and III:2was asymptomatic. ERG and colour vision testing wasnormal.Patient I:1 F The maternal grandfather of thepropositus was said to have had poor eyesight since birthand to have always had great problems with colourvision. The grandfather had an elder brother who hadalso worn glasses and had suffered with poor visionsince infancy. Both were deceased at the time of investigation. Family C   (Figure 1c)  Patient V:4 This 7-year-old boy(proband) srcinally presented as an infant withnystagmus and photophobia. His current visual acuitywas recorded at 6/24 in both eyes. He had clear ocularmedia with normal fundi. He had a family history of nystagmus, poor visual acuity, and colour visionaffecting males of the family including his grandfather Figure 3  Patient III:2 (Family B): Colour-discrimination ellipse,well aligned to the theoretical S-cone/rod confusion axis, andconsistent with the mechanism whereby colour discriminationsare based on a comparison between rod and S-cone quantumcatches. Figure 4  Normal subject: A typical colour-discriminationellipse for a normal trichromat. The subject’s thresholds areplotted as filled circles in the CIE 1974 chromaticity diagram. Figure 5  Rod monochromacy: The results of a rod mono-chromat. Note that the subject fails to discriminate almost all of the most saturated stimuli along each axis tested. Blue cone monochromatism M Michaelides  et al  6 Eye
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