Biomedical subjects

Frans P M Cremers

Publications and source records attributed to Frans P M Cremers.

At least 19 recordsLinked to original sources

PubMed2025

Non-coding single-nucleotide and structural variants affecting the EYS putative promoter cause autosomal recessive retinitis pigmentosa.

PURPOSE: Variants in untranslated genomic regions are difficult to identify as pathogenic but are capable of causing disease by interfering with gene expression. This study aimed to characterize the effect of variants identified in the 5'-untranslated region of EYS in patients with autosomal recessive retinitis pigmentosa (RP). METHODS: Variant screening included gene panels, Sanger, exome, and genome sequencing. Functional validation included an electrophoretic mobility shift assay and various luciferase assays. RESULTS: Patients with RP from 6 EYS biallelic Arab-Muslim families harbored a 5' noncoding EYS variant, c.-453G>T, and 4 harbored a structural variant affecting the 5' noncoding exons. Electrophoretic mobility shift assay analysis revealed an effect on binding of transcription factors for c.-453G>T and a neighboring variant c.-454G>T. Dual luciferase assays using overexpression of various transcription factors showed distinct effects on expression. c.-453G>T was associated with higher luciferase expression with CRX overexpression and c.-454G>C with OTX2 overexpression. In addition, the 2 variants were found to influence translation by affecting upstream initiation codons. Interestingly, visual function of EYS RP patients who harbor c.-453G>T are better than those with biallelic null EYS variants. CONCLUSION: Our analysis revealed both single-nucleotide and structural variants in the EYS promoter as the cause of autosomal recessive RP. These variants may affect EYS expression via a dual mechanism by altering transcription factor binding affinity at the EYS promoter and by affecting upstream open reading frames.

Humans↗

PubMed2006

Towards understanding CRUMBS function in retinal dystrophies.

Mutations in the Crumbs homologue 1 (CRB1) gene cause autosomal recessive retinitis pigmentosa (arRP) and autosomal Leber congenital amaurosis (arLCA). The crumbs (crb) gene was originally identified in Drosophila and encodes a large transmembrane protein required for maintenance of apico-basal cell polarity and adherens junction in embryonic epithelia. Human CRB1 and its two paralogues, CRB2 and CRB3, are highly conserved throughout the animal kingdom. Both in Drosophila and in vertebrates, the short intracellular domain of Crb/CRB organizes an evolutionary conserved protein scaffold. Several lines of evidence, obtained both in Drosophila and in mouse, show that loss-of-function of crb/CRB1 or some of its intracellular interactors lead to morphological defects and light-induced degeneration of photoreceptor cells, features comparable to those observed in patients lacking CRB1 function. In this review, we describe how understanding Crb complex function in fly and vertebrate retina enhances our knowledge of basic cell biological processes and might lead to new therapeutic approaches for patients affected with retinal dystrophies caused by mutations in the CRB1 gene.

Amino Acid Sequence↗

PubMed2006

Development of a genotyping microarray for Usher syndrome.

BACKGROUND: 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, eight genes have been implicated in the syndrome, together comprising 347 protein-coding exons. METHODS: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele-specific oligonucleotides corresponding to all 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. RESULTS: Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III 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. CONCLUSION: The Usher genotyping microarray is 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.

DNA↗

PubMed2006

Choroideremia: variability of clinical and electrophysiological characteristics and first report of a negative electroretinogram.

PURPOSE: To analyze the variability of clinical and electrophysiological characteristics in X-linked choroideremia and provide the first report of a negative electroretinogram in choroideremia. DESIGN: Retrospective study. PARTICIPANTS: The records of 18 male patients with choroideremia and 8 female carriers were evaluated. METHODS: The data were reviewed regarding visual acuity (VA), color vision, perimetry, fundus autofluorescence, and full-field electroretinography (according to standards of the International Society for Clinical Electrophysiology of Vision). MAIN OUTCOME MEASURES: Morphological and functional phenotype characteristics, fundus autofluorescence, electroretinography, and Rab escort protein 1 (REP-1) mutations. RESULTS: Four unrelated families with choroideremia (9 affected males, 7 carriers) and 10 unrelated individuals (9 affected males, 1 carrier) were included. Mutational analysis, performed in 2 families and 3 individual males, revealed REP-1 mutations in all except 1 male. The age of the males ranged from 5.9 to 63.0 years (mean, 33.9), and VA ranged from hand movements to 1.0 (median, 0.7). Fundus autofluorescence (n = 7) showed defects in the retinal pigment epithelium in all males. Electroretinography (n = 13) was almost undetectable in 6 males and reduced in 6, indicating a rod-cone dystrophy. A further male showed a negative electroretinogram, with a b:a wave ratio of 0.5. Visual acuity of the 8 carriers (age, 4.8-56.8 years [mean, 24.0]) ranged from light perception to 1.2 (median, 1.0). Light perception was present in 1 carrier manifesting choroideremia with distinct chorioretinal atrophy. Pigmentary stippling, seen in the other carriers, was seen in fundus autofluorescence (n = 1) with a distinct speckled pattern. Electroretinograms were normal in 6 of 7 and reduced in the manifesting carrier. Defects in color vision and visual field were found in affected males and in the female carriers. CONCLUSIONS: The phenotype of choroideremia presents with high variability. In addition to the previously reported findings, we observed a negative electroretinogram, indicating a postreceptoral retinal dysfunction, in 1 affected male; severe course of choroideremia with early blindness in 1 manifesting carrier; color vision deficits in the majority of affected males and carriers; and characteristic alterations in fundus autofluorescence.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is one of the main causes of childhood blindness. To date, mutations in eight genes have been described, which together account for approximately 45% of LCA cases. We localized the genetic defect in a consanguineous LCA-affected family from Quebec and identified a splice defect in a gene encoding a centrosomal protein (CEP290). The defect is caused by an intronic mutation (c.2991+1655A-->G) that creates a strong splice-donor site and inserts a cryptic exon in the CEP290 messenger RNA. This mutation was detected in 16 (21%) of 76 unrelated patients with LCA, either homozygously or in combination with a second deleterious mutation on the other allele. CEP290 mutations therefore represent one of the most frequent causes of LCA identified so far.

Alleles↗

PubMed2006

The DFNB31 gene product whirlin connects to the Usher protein network in the cochlea and retina by direct association with USH2A and VLGR1.

Mutations in the DFNB31 gene encoding the PDZ scaffold protein whirlin are causative for hearing loss in man and mouse. Whirlin is known to be essential for the elongation process of the stereocilia of sensory hair cells in the inner ear, though its complete spatial and temporal expression patterns remained elusive. Here, we demonstrate that, in embryonic development, the gene is not only expressed in the inner ear, but also in the developing brain and the retina. Various isoforms of whirlin are widely and differentially expressed, and we provide evidence that whirlin directly associates with USH2A isoform b and VLGR1b, two proteins that we previously reported to be part of the Usher protein interactome. These proteins co-localize with whirlin at the synaptic regions of both photoreceptor cells and outer hair cells in the cochlea. These findings indicate that whirlin is part of a macromolecular PDZ protein scaffold that functions in the organization of the pre- and/or postsynaptic side of photoreceptor and hair cell synapses. Whirlin might be involved in synaptic adhesion through interaction with USH2A and VLGR1b as well as in synaptic development as suggested by its spatial and temporal expression patterns. In addition, we demonstrate that whirlin, USH2A and Vlgr1b co-localize at the connecting cilium and the outer limiting membrane of photoreceptor cells and in spiral ganglion neurons of the inner ear. Our data show that whirlin is connected to the dynamic Usher protein interactome and indicate that whirlin has a pleiotropic function in both the retina and the inner ear.

Animals↗

PubMed2006

Mutations in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene cause autosomal recessive nonsyndromic hearing loss.

In two large Turkish consanguineous families, a locus for autosomal recessive nonsyndromic hearing loss (ARNSHL) was mapped to chromosome 6p21.3 by genome-wide linkage analysis in an interval overlapping with the loci DFNB53 (COL11A2), DFNB66, and DFNB67. Fine mapping excluded DFNB53 and subsequently homozygous mutations were identified in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene, also named tetraspan membrane protein of hair cell stereocilia (TMHS) gene, which was recently shown to be mutated in the "hurry scurry" mouse and in two DFNB67-linked families from Pakistan. In one family, we found a homozygous one-base pair deletion, c.649delG (p.Glu216ArgfsX26) and in the other family we identified a homozygous transition c.494C>T (p.Thr165Met). Further screening of index patients from 96 Turkish ARNSHL families and 90 Dutch ARNSHL patients identified one additional Turkish family carrying the c.649delG mutation. Haplotype analysis revealed that the c.649delG mutation was located on a common haplotype in both families. Mutation screening of the LHFPL5 homologs LHFPL3 and LHFPL4 did not reveal any disease causing mutation. Our findings indicate that LHFPL5 is essential for normal function of the human cochlea.

Amino Acid Sequence↗

PubMed2006

Microarray-based mutation detection and phenotypic characterization of patients with Leber congenital amaurosis.

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 GUCY2D-associated 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.

Adaptor Proteins, Signal Transducing↗

PubMed2006

CRB1 heterozygotes with regional retinal dysfunction: implications for genetic testing of leber congenital amaurosis.

PURPOSE: To test human CRB1 heterozygotes for possible clinical or functional retinal changes and to evaluate whether a patient with Leber congenital amaurosis (LCA) with CRB1 mutations not consistent with previously described CRB1 phenotypes carried a modifier allele in another LCA gene. METHODS: Seven unrelated heterozygous carriers of CRB1 mutations underwent phenotyping by full eye examinations (indirect ophthalmoscopy and slit lamp biomicroscopy) and functional testing (standard full-field electroretinography [ERG] and multifocal ERG). For genotyping of the LCA patients and their parents, denaturing high-performance liquid chromatography (dHPLC) analyses were performed, followed by sequence analysis of CRB1, followed by sequence analysis of the AIPL1 and CRX genes to identify a putative modifier effect in a patient with an atypical CRB1 phenotype. RESULTS: Reduced full-field ERG b-wave amplitudes were observed with scotopic -2 dB flash (140 microV; P < 0.05), normal full-field cone ERGs, and significant regional retinal dysfunction on mfERG in five of seven carriers of CRB1 mutations. A known AIPL1 mutation (p. R302L) was identified as a potential modifier allele in a patient with LCA carrying two CRB1 mutations and with a prominent maculopathy. CONCLUSIONS: In human heterozygotes of CRB1 mutations (parents of offspring with LCA), distinctive regional retinal dysfunctions were found by multifocal ERG measurements that were consistent with the focal histologic abnormalities reported for the two CRB1 knockout mice models. This phenotypic finding may identify CRB1 carriers and point to the causal gene defect in affected LCA offspring, significantly facilitating the molecular diagnostic process. Evidence suggests a modifier allele in AIPL1 in a patient with LCA with prominent atrophic macular lesions and homozygous defects in CRB1.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Erosive vitreoretinopathy and wagner disease are caused by intronic mutations in CSPG2/Versican that result in an imbalance of splice variants.

PURPOSE: Linkage intervals for erosive vitreoretinopathy (ERVR) and Wagner disease previously were found to overlap at 5q14.3. In a Japanese family with Wagner disease, a CSPG2/Versican splice site mutation (c.4004-2A-->G) was recently reported that resulted in a 39-nucleotide exon 8 in-frame deletion. We investigated whether CSPG2/Versican was mutated in six Dutch families and one Chinese family with Wagner disease and in a family with ERVR. METHODS: In all families, extensive ophthalmic examinations, haplotype analysis of the 5q14.3 region, and sequence analysis of CSPG2/Versican were performed. The effects of splice site mutations were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (QPCR). RESULTS: Three novel intron 7 sequence variants (c.4004-5T-->C, c.4004-5T-->A, c.4004-1G-->A) were identified in seven families. The c.4004-5T-->C variant was identified in four families with Wagner disease and a family with ERVR. The families were shown to carry the same 5q14.3 haplotype, strongly suggesting that this is a common Dutch founder variant. All three changes segregated with the disease in the respective families and were absent in 250 healthy individuals. In patients with the c.4004-5T-->A and c.4004-1G-->A variants, RT-PCR analysis of CSPG2/Versican showed activation of a cryptic splice site resulting in a 39-nt exon 8 in-frame deletion in splice variant V0. QPCR revealed a highly significant (P < 0.0001) and consistent increase of the V2 (>38-fold) and V3 (>12-fold) splice variants in all patients with intron 7 nucleotide changes and in a Chinese Wagner disease family, in which the genetic defect remains to be found. CONCLUSIONS: Wagner disease and ERVR are allelic disorders. Seven of the eight families exhibit a variant in intron 7 of CSPG2/Versican. The conspicuous clustering of sequence variants in the splice acceptor site of intron 7 and the consistent upregulation of the V2 and V3 isoforms strongly suggest that Wagner disease and ERVR may belong to a largely overlooked group of diseases that are caused by mRNA isoform balance shifts, representing a novel disease mechanism.

Adolescent↗

PubMed2005

Interaction of nephrocystin-4 and RPGRIP1 is disrupted by nephronophthisis or Leber congenital amaurosis-associated mutations.

RPGR-interacting protein 1 (RPGRIP1) is a key component of cone and rod photoreceptor cells, where it interacts with RPGR (retinitis pigmentosa GTPase regulator). Mutations in RPGRIP1 lead to autosomal recessive congenital blindness [Leber congenital amaurosis (LCA)]. Most LCA-associated missense mutations in RPGRIP1 are located in a segment that encodes two C2 domains. Based on the C2 domain of novel protein kinase C epsilon (PKC epsilon), we built a 3D-homology model for the C-terminal C2 domain of RPGRIP1. This model revealed a potential Ca2+-binding site that was predicted to be disrupted by a missense mutation in RPGRIP1, which was previously identified in an LCA patient. Through yeast two-hybrid screening of a retinal cDNA library, we found this C2 domain to specifically bind to nephrocystin-4, encoded by NPHP4. Mutations in NPHP4 are associated with nephronophthisis and a combination of nephronophthisis and retinitis pigmentosa called Senior-Løken syndrome (SLSN). We show that RPGRIP1 and nephrocystin-4 interact strongly in vitro and in vivo, and that they colocalize in the retina, matching the panretinal localization pattern of specific RPGRIP1 isoforms. Their interaction is disrupted by either mutations in RPGRIP1, found in patients with LCA, or by mutations in NPHP4, found in patients with nephronophthisis or SLSN. Thus, we provide evidence for the involvement of this disrupted interaction in the retinal dystrophy of both SLSN and LCA patients.

Animals↗

PubMed2005

Fine mapping of autosomal dominant nonsyndromic hearing impairment DFNA21 to chromosome 6p24.1-22.3.

Previously, the DFNA21 locus was positioned telomeric to the DFNA13 locus based on testing of candidate loci. One family member in this region did not carry the at risk haplotype, although he had the same nonspecific midfrequency hearing impairment as other affected family members. Hence, we performed a whole genome linkage scan excluding other regions of the genome and confirming the localization of DFNA21 to 6p22.3-24.1. The DFNA21 interval was determined to span 12.4 Mb (approximately 22 cM) and is delimited on the telomeric side by BV097155 and on the centromeric side by D6S1691. A maximum lod score of 3.51 (theta = 0.066), was calculated for marker D6S1721. The DFNA21 region does not overlap the adjacent DFNA31 and DFNA13 loci and contains 31 known genes. The coding regions and exon-intron boundaries of four candidate genes, SOX4, MYLIP, CAP2, and RPEL1, were sequenced, but no mutations were identified.

Chromosome Mapping↗

PubMed2005

Characterization of the Crumbs homolog 2 (CRB2) gene and analysis of its role in retinitis pigmentosa and Leber congenital amaurosis.

PURPOSE: Mutations in the Crumbs homolog 1 (CRB1) gene cause autosomal recessive retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Database searches reveal two other Crumbs homologs on chromosomes 9q33.3 and 19p13.3. The purpose of this study was to characterize the Crumbs homolog 2 (CRB2) gene on 9q33.3, to analyze its expression pattern, and to determine whether mutations in CRB2 are associated with RP and LCA. METHODS: The CRB2 mRNA and its expression pattern in human tissues were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The cellular expression of Crb2 in the mouse eye was determined by mRNA in situ hybridizations. The open reading frame and splice junctions of CRB2 were analyzed for mutations by single-strand conformation analysis and direct nucleotide sequencing in 85 RP patients and 79 LCA patients. RESULTS: The CRB2 gene consists of 13 exons and encodes a 1285 amino acid transmembrane protein. CRB2 is mainly expressed in retina, brain, and kidney. In mouse retina Crb2 expression was detected in all cell layers. Mutation analysis of the CRB2 gene revealed 11 sequence variants leading to an amino acid substitution. Three of them were not identified in control individuals and affect conserved amino acid residues. However, the patients that carry these sequence variants do not have a second sequence variant on the other allele, excluding autosomal recessive inheritance of CRB2 sequence variants as a cause of their disease. CONCLUSIONS: This study shows that CRB2 sequence variants are not a common cause of autosomal recessive RP and LCA. It is possible that a more complex clinical phenotype is associated with the loss or altered function of CRB2 in humans due to its expression in tissues other than the retina.

Amino Acid Sequence↗

PubMed2005

Novel mutations in CACNA1F and NYX in Dutch families with X-linked congenital stationary night blindness.

PURPOSE: To describe the clinical features and genetic analysis of eight X-linked congenital stationary night blindness (XLCSNB) Dutch patients. METHODS: Electroretinogram (ERG) measurements were assessed in Dutch patients. Molecular genetic testing by denaturing high performance liquid chromatography (DHPLC), single stranded conformation polymorphism (SSCP) analysis, and direct sequencing of the CACNA1F and NYX genes were performed in the patients possessing a negative Schubert Bornschein ERG. RESULTS: Molecular genetic testing of CACNA1F and NYX revealed three novel and two known CACNA1F sequence variants as well as two novel sequence alterations in the NYX gene. While one of the CACNA1F sequence variants (5756G>A, R1919H) has been previously described as a common polymorphism in Japanese families, we did not found this transition in 100 European control alleles. CONCLUSIONS: In a pool of eight diagnosed XLCSNB patients, five showed a sequence variation in the CACNA1F and two in the NYX gene. In only one of the eight patients no sequence alteration could be detected. This might be explained by a mutation in other, as yet unidentified coding or regulatory sequences of NYX or CACNA1F or additional genes.

Adult↗

PubMed2005

CNGB3 mutations account for 50% of all cases with autosomal recessive achromatopsia.

Achromatopsia is a congenital, autosomal recessively inherited disorder characterized by a lack of color discrimination, low visual acuity (<0.2), photophobia, and nystagmus. Mutations in the genes for CNGA3, CNGB3, and GNAT2 have been associated with this disorder. Here, we analyzed the spectrum and prevalence of CNGB3 gene mutations in a cohort of 341 independent patients with achromatopsia. In 163 patients, CNGB3 mutations could be identified. A total of 105 achromats carried apparent homozygous mutations, 44 were compound (double) heterozygotes, and 14 patients had only a single mutant allele. The derived CNGB3 mutation spectrum comprises 28 different mutations including 12 nonsense mutations, eight insertions and/or deletions, five putative splice site mutations, and three missense mutations. Thus, the majority of mutations in the CNGB3 gene result in significantly altered and/or truncated polypeptides. Several mutations were found recurrently, in particular a 1 bp deletion, c.1148delC, which accounts for over 70% of all CNGB3 mutant alleles. In conclusion, mutations in the CNGB3 gene are responsible for approximately 50% of all patients with achromatopsia. This indicates that the CNGB3/ACHM3 locus on chromosome 8q21 is the major locus for achromatopsia in patients of European origin or descent.

Alleles↗

PubMed2005

Audiometric, vestibular, and genetic aspects of a DFNA9 family with a G88E COCH mutation.

OBJECTIVES: To perform genetic analysis and to analyze cochleovestibular impairment features in a newly identified Dutch family with nonsyndromic autosomal dominant hearing impairment (DFNA9). STUDY DESIGN: Genetic analysis was performed using microsatellite markers and single nucleotide polymorphisms. Audiometric data were collected and analyzed longitudinally. Results were compared with those obtained in previously identified P51S COCH mutation carriers (n = 74). Special attention was also given to a comparison of age-related features such as progressive hearing loss and vestibular impairment. SETTING: Tertiary referral center. PATIENTS: G88E COCH mutation carriers from a Dutch family. MAIN OUTCOME MEASURES: The study of clinical features of a DFNA9 family carrying a G88E COCH mutation and to compare this to the symptoms of those carrying a P51S/COCH mutation. RESULTS: Pure-tone thresholds, phoneme recognition scores, and vestibular responses of the G88E mutation carriers were essentially similar to those previously established in the P51S mutation carriers. Hearing started to deteriorate in G88E mutation carriers from age 46 to 49 years and onward, whereas deterioration of vestibular function started from approximately age 46 years. In the P51S mutation carriers, vestibular impairment started earlier, at approximately age 34 years. However, the difference in age of onset with the G88E mutation carriers was not significant. Remarkably, the proportion of patients who developed complete vestibular areflexia within the age range of 40 to 56 years was significantly lower for the G88E mutation carriers than for the P51S mutation carriers. CONCLUSION: Apart from a significantly lower frequency of vestibular areflexia between the ages of 40 and 56 years, there are no phenotypic differences between carriers of the G88E and P51S mutations in the COCH gene.

Adult↗

PubMed2005

MPP5 recruits MPP4 to the CRB1 complex in photoreceptors.

PURPOSE: Mutations in the human Crumbs homologue 1 (CRB1) gene are a frequent cause of Leber congenital amaurosis (LCA) and various forms of retinitis pigmentosa. CRB1 is thought to organize an intracellular protein scaffold in the retina that is involved in photoreceptor polarity. This study was focused on the identification, subcellular localization, and binding characteristics of a novel member of the protein scaffold connected to CRB1. METHODS: To dissect the protein scaffold connected to CRB1, the yeast two-hybrid approach was used to screen for interacting proteins. Glutathione S-transferase (GST) pull-down analysis and immunoprecipitation were used to verify protein-protein interactions. The subcellular localization of the proteins was visualized by immunohistochemistry and confocal microscopy on human retinas and immunoelectron microscopy on mouse retinas. RESULTS: A novel member of the scaffold connected to CRB1, called membrane palmitoylated protein (MPP) subfamily member 4 (MPP4), a membrane-associated guanylate kinase (MAGUK) protein, was identified. MPP4 was found to exist in a complex with CRB1 through direct interaction with the MPP subfamily member MPP5 (PALS1). 3D homology modeling provided evidence for a mechanism that regulates the recruitment of both homo- and heterodimers of MPP4 and -5 proteins to the complex. Localization studies in the retina showed that CRB1, MPP5, and MPP4 colocalize at the outer limiting membrane (OLM). CONCLUSIONS: These data imply that MPP4 and -5 have a role in photoreceptor polarity and, by association with CRB1, pinpoint the cognate genes as functional candidate genes for inherited retinopathies.

Animals↗

PubMed2005

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

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.

Alleles↗