the consanguineous family consists of three men and three women affected with cord ( figure 1a ) . genealogical studies revealed a common ancestor six generations back , born in 1687 , of the parents of patients ii:4 , ii:6 and ii:10 , and the mutation carrier iii:1 . the faroese population has been studied extensively for hereditary ophthalmological disorders , but to our knowledge , no other faroese persons have a diagnosis of cord . pedigree of the family with cord , linkage analysis , genes in the candidate region and mutation analysis of pcdh21 . ( b ) results of the parametric multipoint linkage analysis using genehunter software , which shows a single significant peak on chromosome 10 , with a lod score of 3.1 . ( d ) mutation analysis of pcdh21 shows the homozygous c.524dupa mutation ( left ) , a heterozygous carrier ( middle ) and the wild - type ( right ) . ( e ) analysis of cdna from fibroblasts from a homozygous patient shows that the mutation results in a frameshift ( top ) . it was approved by the faroese ethical committee and carried out in collaboration with the governmental genetics resource centre of the faroe islands . the pedigree analysis provided strong evidence of autosomal recessive mode of inheritance , and all affected persons were assumed homozygous for a mutant allele . rna was extracted from cultured fibroblasts with the rnaeasy mini kit ( qiagen , inc . , valencia , california , usa ) and reverse transcribed to cdna with the superscript ii reverse transcriptase kit ( invitrogen , carlsbad , california , usa ) . dna from the six affected patients ( ii:4 , ii:6 , ii:10 , iv:1 , iv:2 and iv:3 ) was used for a genome - wide search for homozygosity with the affymetrix genechip 50k xba array ( affymetrix inc . , santa clara , california , usa ) . in brief , 250  ng of dna was digested with the restriction enzyme xbai , mixed with xba adapters and ligated with the t4 dna ligase . the purified pcr product was fragmented with dnase i and end - labelled with biotin . the samples were hybridised to an array for 18  h in a hybridisation oven . affymetrix software was used to analyse the data , which were exported to an excel file . multipoint parametric linkage analysis was performed with the genehunter program on the easylinkage v.5.08 plus platform.10 an autosomal recessive mode of inheritance with complete penetrance was assumed , and the disease allele frequency was set at 0.001 . pcr was performed with the promega kit and the following conditions : 0.2  mm dntps , 1 buffer , 1.5  mm mgcl2 , 0.5  mm of each primer , 10  ng template and 1.5  u polymerase in a total volume of 50  l . the pcr program was 94  c for 2  min , 35 cycles of denaturing at 94c for 30  s , annealing at 60c for 30  s and extension at 72c for 30  s and a final extension step of 72c for 7  min . sequencing was performed with abi big dye terminator v. 1.1 cycle sequencing kit ( applied biosystems , foster city , california , usa ) . the sequencing reactions were vacuum - purified with the montage seq96 sequencing reaction cleanup kit ( millipore sa , saintquentin , france ) and analysed on an abi 3130xl gene analyzer ( applied biosystems ) . the data were analysed with sequencing analysis software v.5.2 from applied biosystems , and mutation screening was performed using mutation surveyor v.3.1 software ( softgenetics ) . the consanguineous family consists of three men and three women affected with cord ( figure 1a ) . genealogical studies revealed a common ancestor six generations back , born in 1687 , of the parents of patients ii:4 , ii:6 and ii:10 , and the mutation carrier iii:1 . the faroese population has been studied extensively for hereditary ophthalmological disorders , but to our knowledge , no other faroese persons have a diagnosis of cord . pedigree of the family with cord , linkage analysis , genes in the candidate region and mutation analysis of pcdh21 . ( b ) results of the parametric multipoint linkage analysis using genehunter software , which shows a single significant peak on chromosome 10 , with a lod score of 3.1 . ( d ) mutation analysis of pcdh21 shows the homozygous c.524dupa mutation ( left ) , a heterozygous carrier ( middle ) and the wild - type ( right ) . ( e ) analysis of cdna from fibroblasts from a homozygous patient shows that the mutation results in a frameshift ( top ) . it was approved by the faroese ethical committee and carried out in collaboration with the governmental genetics resource centre of the faroe islands . the pedigree analysis provided strong evidence of autosomal recessive mode of inheritance , and all affected persons were assumed homozygous for a mutant allele . rna was extracted from cultured fibroblasts with the rnaeasy mini kit ( qiagen , inc . , valencia , california , usa ) and reverse transcribed to cdna with the superscript ii reverse transcriptase kit ( invitrogen , carlsbad , california , usa ) . dna from the six affected patients ( ii:4 , ii:6 , ii:10 , iv:1 , iv:2 and iv:3 ) was used for a genome - wide search for homozygosity with the affymetrix genechip 50k xba array ( affymetrix inc . , santa clara , california , usa ) . in brief , 250  ng of dna was digested with the restriction enzyme xbai , mixed with xba adapters and ligated with the t4 dna ligase . the purified pcr product was fragmented with dnase i and end - labelled with biotin . the samples were hybridised to an array for 18  h in a hybridisation oven . affymetrix software was used to analyse the data , which were exported to an excel file . multipoint parametric linkage analysis was performed with the genehunter program on the easylinkage v.5.08 plus platform.10 an autosomal recessive mode of inheritance with complete penetrance was assumed , and the disease allele frequency was set at 0.001 . pcr was performed with the promega kit and the following conditions : 0.2  mm dntps , 1 buffer , 1.5  mm mgcl2 , 0.5  mm of each primer , 10  ng template and 1.5  u polymerase in a total volume of 50  l . the pcr program was 94  c for 2  min , 35 cycles of denaturing at 94c for 30  s , annealing at 60c for 30  s and extension at 72c for 30  s and a final extension step of 72c for 7  min . sequencing was performed with abi big dye terminator v. 1.1 cycle sequencing kit ( applied biosystems , foster city , california , usa ) . the sequencing reactions were vacuum - purified with the montage seq96 sequencing reaction cleanup kit ( millipore sa , saintquentin , france ) and analysed on an abi 3130xl gene analyzer ( applied biosystems ) . the data were analysed with sequencing analysis software v.5.2 from applied biosystems , and mutation screening was performed using mutation surveyor v.3.1 software ( softgenetics ) . the three oldest affected siblings ( patients ii:4 , ii:6 and ii:10 ) reported decreased vision from around age 17  years . in addition to cord , two of the patients ( patients iv:1 and iv:3 ) had oculocutaneous albinism , which caused nystagmus and reduced visual acuity from early infancy . the genetic background for their albinism is unknown , but no mutations were found by sequencing tyr and oca2 . patient ii:10 complained of impaired vision at night and of delayed adaptation to sudden changes in light intensity . visual acuity varied between 0.67 and 0.02 ( table 1 ) . in patient ii:4 , large inter - ocular differences in visual acuity were observed due to a large scar in the left macula , which was likely caused by a foetal toxoplasmosis infection . the inter - ocular difference in visual acuity in patient ii:10 clinical and electrophysiological characteristics of patients with cord caused by pcdh21 mutations erg normal values . cone scotopic flicker response : amplitude median 94  v , range 53145  v ( 595% ) , implicit time median 29.0  ms , range 27.034.0  ms ( 595% ) . rod low - intensity flash response : b - wave amplitude median 210  v , range 124302  v ( 595% ) , implicit time median 103.0 ms , range 81.0123.0  ms ( 595% ) . cone - rod high - intensity flash response : a  b - wave amplitude median 427  v , range 285674  v ( 595% ) , implicit time median 48.0  ms , range 41.055.0  ms ( 595% ) . the foveal changes consisted of irregular pigmentation and atrophy of the rpe with a horizontal oval - shaped or ill - defined  moth - eaten  appearance . in patient iv:2 , the foveal regions had an extensive sheen . in the two patients with oculocutaneous albinism ( patients iv:1 and iv:3 ) , the changes in the foveal regions were less obvious due to the scarcity of pigment . the peripheral pigment disturbances included diffuse rpe atrophy and black hyperpigmentations of polymorphic shape , some of which were filiform and sheeted the vessels . the amount of pigmentation varied considerably among patients of the same age but showed some correlation to the extent of visual field constriction . in the oldest patients , the optic discs looked moderately atrophic , and the retinal vessels were universally constricted . ( a ) diffuse atrophy of the retinal pigment epithelium ( rpe ) and extensive sheen in the macular region ( patient ii:4 ) . ( b ) numerous , polymorphic , intraretinal hyperpigmentations in the upper temporal midperiphery ( patient ii:4 ) . ( d ) irregular rpe atrophy in the macular region , attenuated arterioles and paleness of the optic nerve head ( patient ii:10 ) . visual field measurements with a large object showed considerable variation ; patient ii:4 had only narrow peripheral remnants left in the lower visual fields and a paracentral preserved island of 10 , whereas patients ii:6 and ii:10 had normal or slightly constricted outer field limits . in patient ii:6 , colour vision was severely impaired in the two patients tested ( patients ii:6 and ii:10 at ages 36 and 38  years , respectively ) . dark adaptation was tested in two patients ( patients ii:10 and iv:2 at ages 38 and 15  years , respectively ) . it showed a monophasic and protracted course , with only a slight elevation of the final threshold of 0.51 log unit in patient ii:10 ( figure 3 ) . the dark adaptation curves in patients ii:10 and iv:2 show a protracted initial phase and an absent mesopic break , resulting in a monophasic , straightened course compared to the normal diphasic course . the erg of patient ii:10 showed severely reduced amplitudes and prolonged photopic implicit times ( table 1 ) . patient iv:1 had normal amplitudes at the age of 2  years but prolonged cone implicit times . eight years later , the erg showed absent response to low - intensity stimuli and a 50% reduction in the response to high - intensity flash . in patient iv:2 , the erg response was absent at age 17  years . a frequent cause of cord is mutations in abca4 , and we , therefore , analysed the snp data for linkage to abca4 . assuming autosomal recessive inheritance and a homozygous or compound heterozygous mutation , we did not find evidence of linkage to abca4 ( see online supplementary table 1 ) . genome - wide analysis of snp data for homozygosity revealed a single homozygous region on chromosome 10q23.1q23.2 ( see online supplementary table 2 ) ; a significant lod score of 3.1 was found by multipoint linkage analysis ( figure 1b ) . the linkage interval had a size of 4.2  mb and was flanked by the heterozygous markers rs6584455 and rs868042 ( figure 1c ) . the region encompasses 11 genes , of which three were candidate genes : rgr , which had previously been shown to cause autosomal recessive retinitis pigmentosa in humans , lrit1 , which is highly expressed in retina , and pcdh21 , where a knockout mouse model had shown loss of cone and rod photoreceptor cells.1113 no mutations were found in rgr or lrit1 . sequencing of pcdh21 revealed a 1  bp insertion , c.524dupa ( figure 1d ) . the six patients were homozygous for the mutation , and 12 family members were heterozygous carriers ( figure 1a ) . individuals ii:1 and ii:3 did not carry the mutation ; no dna was available from the remaining family members . the 1  bp duplication is expected to cause a frameshift resulting in a premature stop codon ( p.q175qfsx47 ) , which was confirmed by analysis of cdna from fibroblasts ( figure 1e ) . screening of 159 anonymous faroese controls for the mutation revealed three heterozygous carriers , corresponding to a carrier frequency of 1.9% . the three oldest affected siblings ( patients ii:4 , ii:6 and ii:10 ) reported decreased vision from around age 17  years . in addition to cord , two of the patients ( patients iv:1 and iv:3 ) had oculocutaneous albinism , which caused nystagmus and reduced visual acuity from early infancy . the genetic background for their albinism is unknown , but no mutations were found by sequencing tyr and oca2 . patient ii:10 complained of impaired vision at night and of delayed adaptation to sudden changes in light intensity . visual acuity varied between 0.67 and 0.02 ( table 1 ) . in patient ii:4 , large inter - ocular differences in visual acuity were observed due to a large scar in the left macula , which was likely caused by a foetal toxoplasmosis infection . the inter - ocular difference in visual acuity in patient ii:10 clinical and electrophysiological characteristics of patients with cord caused by pcdh21 mutations erg normal values . cone scotopic flicker response : amplitude median 94  v , range 53145  v ( 595% ) , implicit time median 29.0  ms , range 27.034.0  ms ( 595% ) . rod low - intensity flash response : b - wave amplitude median 210  v , range 124302  v ( 595% ) , implicit time median 103.0 ms , range 81.0123.0  ms ( 595% ) . cone - rod high - intensity flash response : a  b - wave amplitude median 427  v , range 285674  v ( 595% ) , implicit time median 48.0  ms , range 41.055.0  ms ( 595% ) . the foveal changes consisted of irregular pigmentation and atrophy of the rpe with a horizontal oval - shaped or ill - defined  moth - eaten  appearance . in patient iv:2 , the foveal regions had an extensive sheen . in the two patients with oculocutaneous albinism ( patients iv:1 and iv:3 ) , the changes in the foveal regions were less obvious due to the scarcity of pigment . the peripheral pigment disturbances included diffuse rpe atrophy and black hyperpigmentations of polymorphic shape , some of which were filiform and sheeted the vessels . the amount of pigmentation varied considerably among patients of the same age but showed some correlation to the extent of visual field constriction . in the oldest patients , the optic discs looked moderately atrophic , and the retinal vessels were universally constricted . ( a ) diffuse atrophy of the retinal pigment epithelium ( rpe ) and extensive sheen in the macular region ( patient ii:4 ) . ( b ) numerous , polymorphic , intraretinal hyperpigmentations in the upper temporal midperiphery ( patient ii:4 ) . ( d ) irregular rpe atrophy in the macular region , attenuated arterioles and paleness of the optic nerve head ( patient ii:10 ) . visual field measurements with a large object showed considerable variation ; patient ii:4 had only narrow peripheral remnants left in the lower visual fields and a paracentral preserved island of 10 , whereas patients ii:6 and ii:10 had normal or slightly constricted outer field limits . in patient ii:6 , colour vision was severely impaired in the two patients tested ( patients ii:6 and ii:10 at ages 36 and 38  years , respectively ) . dark adaptation was tested in two patients ( patients ii:10 and iv:2 at ages 38 and 15  years , respectively ) . it showed a monophasic and protracted course , with only a slight elevation of the final threshold of 0.51 log unit in patient ii:10 ( figure 3 ) . the dark adaptation curves in patients ii:10 and iv:2 show a protracted initial phase and an absent mesopic break , resulting in a monophasic , straightened course compared to the normal diphasic course . the erg of patient ii:10 showed severely reduced amplitudes and prolonged photopic implicit times ( table 1 ) . patient iv:1 had normal amplitudes at the age of 2  years but prolonged cone implicit times . eight years later , the erg showed absent response to low - intensity stimuli and a 50% reduction in the response to high - intensity flash . in patient iv:2 , the erg response was absent at age 17  years . a frequent cause of cord is mutations in abca4 , and we , therefore , analysed the snp data for linkage to abca4 . assuming autosomal recessive inheritance and a homozygous or compound heterozygous mutation , we did not find evidence of linkage to abca4 ( see online supplementary table 1 ) . genome - wide analysis of snp data for homozygosity revealed a single homozygous region on chromosome 10q23.1q23.2 ( see online supplementary table 2 ) ; a significant lod score of 3.1 was found by multipoint linkage analysis ( figure 1b ) . the linkage interval had a size of 4.2  mb and was flanked by the heterozygous markers rs6584455 and rs868042 ( figure 1c ) . the region encompasses 11 genes , of which three were candidate genes : rgr , which had previously been shown to cause autosomal recessive retinitis pigmentosa in humans , lrit1 , which is highly expressed in retina , and pcdh21 , where a knockout mouse model had shown loss of cone and rod photoreceptor cells.1113 sequencing of pcdh21 revealed a 1  bp insertion , c.524dupa ( figure 1d ) . the six patients were homozygous for the mutation , and 12 family members were heterozygous carriers ( figure 1a ) . individuals ii:1 and ii:3 did not carry the mutation ; no dna was available from the remaining family members . the 1  bp duplication is expected to cause a frameshift resulting in a premature stop codon ( p.q175qfsx47 ) , which was confirmed by analysis of cdna from fibroblasts ( figure 1e ) . screening of 159 anonymous faroese controls for the mutation revealed three heterozygous carriers , corresponding to a carrier frequency of 1.9% . to our knowledge , this is the first report of mutations in pcdh21 as a cause of human disease . in a previous study , two heterozygous missense mutations were found in three patients with autosomal recessive rp , but a second mutation was not found ; hence , the pathogenicity of the mutations is unclear.14 the pcdh21 mutation was identified in a small consanguineous family from the faroe islands , and a carrier frequency of 1:53 ( 1.9% ) was found . a high carrier frequency is found in other disorders in the faroe islands that are caused by founder mutations , for example cystic fibrosis ( 1:24 ) and mitochondrial encephalomyopathy with elevated methylmalonic acid ( 1:33).8 9 pcdh21 was identified by rattner et al by subtractive hybridisation and high - throughput screening in a search for genes specifically expressed in the retina.13 at the same time , nakajima et al identified pcdh21 in a search for genes that encode proteins with cadherin domains.15 pcdh21 is conserved among different vertebrate species but has not been found in invertebrates . it is a member of the protocadherin family , the largest subgroup within the cadherin superfamily of large cell surface proteins characterised by a variable number of extracellular cadherin domains.16 the cadherins are involved in calcium - dependent cell cadherin / protocadherin genes have previously been shown to be involved in disorders of the retina : mutations in the protocadherin gene pcdh15 are responsible for usher syndrome type 1f , and mutations in the cadherin genes cdh23 and cdh3 were shown to cause usher syndrome type 1d and hypotrichosis with juvenile macular dystrophy , respectively.1719 pcdh21 has six predicted cadherin repeats and an intracellular domain , which is not homologous to that of other cadherins . pcdh21 is highly expressed the retina , more specifically in the junction between the inner and outer segments ( os ) of rod and cone photoreceptors ; the expression is also high in olfactory bulbs , with a lower expression in the septum and olfactory cortex.13 15 pcdh21 expression is very low or absent in heart , kidney , liver , lung , spleen , testis and skeletal muscle . the photoreceptor os is a derivative of a non - motile cilium that contains phototransduction proteins organised in a stack of over 1000 discs . the discs are continuously renewed from the base , and the older discs are shedded from the tip . the proteins are synthesised in the inner segment and transported to the os via the connecting cilium . pcdh21 undergoes a specific proteolytic cleavage , which releases ectodomain as a soluble fragment . in rds knockout mice , where the os assembly is disturbed , the proteolytic cleavage is inhibited and full - length pcdh21 accumulates.20 mutations in prom1 have been identified in patients with autosomal recessive rp , and pcdh21 protein has been shown to interact with prom1 by co - immunoprecipitation studies.21 prom1 is a transmembrane protein associated with plasma membrane protrusions , and it co - localises with pcdh21 to the base of the photoreceptor os . in transgenic mice expressing human prom1 with a heterozygous missense mutation , prom1 and pcdh21 protein was found throughout the photoreceptor layers instead of the normal localisation at the base of the photoreceptor os . in pcdh21 mice , these findings suggest that the interaction between pcdh21 and prom1 is necessary for both proteins to function normally . taken together , these findings suggest that pcdh21 is involved in os structure and/or assembly but not in phototransduction in rods and cones . this hypothesis is supported by the localisation of pcdh21 to the base of the os at the earliest stages of differentiation of rods , before other os proteins , for example , rhodopsin , rp1 and rom1 , are found.20 a pcdh21 knockout mouse model showed normal viability and fertility without anatomical or behavioural abnormalities.13 at age 1  month , the retinas appeared normal , but this was followed by a progressive loss of photoreceptors , and a 50% reduction in the number of photoreceptor cells was observed by age 6  months , which likely is the result of apoptosis . analysis of photoreceptor electrical signalling showed 2- to 3-fold diminished saturating a - wave amplitude , which reflects rod photoreceptor function , in pcdh21 mice compared to pcdh21 and pcdh21 mice . the saturating amplitude of the cone - driven b - wave response was diminished about twofold in pcdh21 mice . electron microscopy showed that in pcdh21 mice , the os were shorter with swirls of membranes with imperfectly stacked discs . the inner segments and the knockout mouse thus had abnormal cone and rod function , but the dysfunction of rods and cones was not as severe as expected from the degree of disorganisation of the os . the erg findings and the loss of photoreceptor cells in patients with pcdh21 mutations were similar to those found in the knockout mouse model . despite the high pcdh21 expression in olfactory bulbs , the patients showed no signs of decreased olfactory function . the frameshift mutation found in pchd21 , one of three sequenced genes of 11 candidate genes in the linkage interval , the segregation of the mutation with the disease in the family , the high and almost exclusive expression of the gene in the retina and the similarity of the phenotype of the patients with that of a knockout mouse heavily imply that autosomal recessive cone - rod dystrophy in this family is caused by pcdh21 mutations . since several of the previously reported genes involved in cord have been shown to be involved in , for example , stargardt disease , leber congenital amaurosis and rp , pcdh21 should be considered a candidate gene for other retinal dystrophies . note around the time of the acceptance of this paper , pcdh21 mutations were reported in two families with retinal dystrophy in henderson rh , li z , abd el aziz mm , mackay ds , eljinini ma , zeidan m , moore at , bhattacharya ss , webster ar .
backgroundcone - rod dystrophy is a retinal dystrophy with early loss of cone photoreceptors and a parallel or subsequent loss of rod photoreceptors . 
 it may be syndromic , but most forms are non - syndromic with autosomal dominant , autosomal recessive or x - linked recessive inheritance.methods and resultswe identified a small consanguineous family with six patients with cone - rod dystrophy from the faroe islands . 
 homozygosity mapping revealed a single homozygous locus of 4.2  mb on chromosome 10q23.1q23.2 , encompassing 11 genes . 
 all patients were homozygous for a 1-bp duplication in pcdh21 , c.524dupa , which results in a frameshift and a premature stop codon ( p.q175qfsx47).conclusionto our knowledge , this is the first report of mutations in pcdh21 as a cause of human disease . 
 pcdh21 is highly expressed in the retinal photoreceptor cells . 
 it encodes protocadherin 21 , which belongs to the cadherin superfamily of large cell surface proteins characterised by a variable number of extracellular cadherin domains . 
 a pcdh21 knockout mouse model has previously shown loss of photoreceptor cells and abnormal cone and rod function , similar to the findings in the patients .