Karin W. Littink

Homozygosity mapping in patients with cone-rod dystrophy: novel mutations and clinical characterizations.

PURPOSE To determine the genetic defect and to describe the clinical characteristics in a cohort of mainly nonconsanguineous cone-rod dystrophy (CRD) patients. METHODS One hundred thirty-nine patients with diagnosed CRD were recruited. Ninety of them were screened for known mutations in ABCA4, and those carrying one or two mutations were excluded from further research. Genome-wide homozygosity mapping was performed in the remaining 108. Known genes associated with autosomal recessive retinal dystrophies located within a homozygous region were screened for mutations. Patients in whom a mutation was detected underwent further ophthalmic examination. RESULTS Homozygous sequence variants were identified in eight CRD families, six of which were nonconsanguineous. The variants were detected in the following six genes: ABCA4, CABP4, CERKL, EYS, KCNV2, and PROM1. Patients carrying mutations in ABCA4, CERKL, and PROM1 had typical CRD symptoms, but a variety of retinal appearances on funduscopy, optical coherence tomography, and autofluorescence imaging. CONCLUSIONS Homozygosity mapping led to the identification of new mutations in consanguineous and nonconsanguineous patients with retinal dystrophy. Detailed clinical characterization revealed a variety of retinal appearances, ranging from nearly normal to extensive retinal remodeling, retinal thinning, and debris accumulation. Although CRD was initially diagnosed in all patients, the molecular findings led to a reappraisal of the diagnosis in patients carrying mutations in EYS, CABP4, and KCNV2.

Mutations in C2ORF71 Cause Autosomal-Recessive Retinitis Pigmentosa

With a worldwide prevalence of 1 in 4,000, retinitis pigmentosa (RP) is the most common form of hereditary retinal degeneration. More than 30 genes and loci have been implicated in nonsyndromic autosomal-recessive (ar) RP. Genome-wide homozygosity mapping was conducted in one Dutch and one Israeli family affected by arRP. The families were found to share a 5.9 Mb homozygous region on chromosome 2p23.1-p23.3. A missense variant in one of the genes residing in this interval, C2ORF71, has recently been reported to be associated with RP. C2ORF71, encoding a putative protein of 1,288 amino acids, was found to be specifically expressed in human retina. Furthermore, RT-PCR analysis revealed that in the mouse eye, C2orf71 is expressed as early as embryonic day 14. Mutation analysis detected a 1 bp deletion (c.946 del; p.Asn237MetfsX5) segregating with RP in the Dutch family, whereas a nonsense mutation (c.556C > T; p.Gln186X) was identified in the Israeli family. Microsatellite-marker analysis in additional Israeli families revealed cosegregation of a C2ORF71-linked haplotype in one other family, in which a 13 bp deletion (c.2756_2768 del; p.Lys919ThrfsX) was identified. Clinically, patients with mutations in C2ORF71 show signs of typical RP; these signs include poor night vision and peripheral field loss, typical retinal bone-spicule-type pigment deposits, pale appearance of the optic disk, and markedly reduced or completely extinguished electroretinograms. In conclusion, truncating mutations in C2ORF71 were identified in three unrelated families, thereby confirming the involvement of this gene in the etiology of arRP.

High-resolution homozygosity mapping is a powerful tool to detect novel mutations causative of autosomal recessive RP in the Dutch population

PURPOSE To determine the genetic defects underlying autosomal recessive retinitis pigmentosa (arRP) in the Dutch population and in a subset of patients originating from other countries. The hypothesis was that, because there has been little migration over the past centuries in certain areas of The Netherlands, a significant fraction of Dutch arRP patients carry their genetic defect in the homozygous state. METHODS High-resolution genome-wide SNP genotyping on SNP arrays and subsequent homozygosity mapping were performed in a large cohort of 186 mainly nonconsanguineous arRP families living in The Netherlands. Candidate genes residing in homozygous regions were sequenced. RESULTS In ~94% of the affected individuals, large homozygous sequences were identified in their genome. In 42 probands, at least one of these homozygous regions contained one of the 26 known arRP genes. Sequence analysis of the corresponding genes in each of these patients revealed 21 mutations and two possible pathogenic changes, 14 of which were novel. All mutations were identified in only a single family, illustrating the genetic diversity within the Dutch population. CONCLUSIONS This report demonstrates that homozygosity mapping is a powerful tool for identifying the genetic defect underlying genetically heterogeneous recessive disorders like RP, even in populations with little consanguinity.

BBS1 mutations in a wide spectrum of phenotypes ranging from nonsyndromic retinitis pigmentosa to Bardet-Biedl syndrome

OBJECTIVE To investigate the involvement of the Bardet-Biedl syndrome (BBS) gene BBS1 p.M390R variant in nonsyndromic autosomal recessive retinitis pigmentosa (RP). METHODS Homozygosity mapping of a patient with isolated RP was followed by BBS1 sequence analysis. We performed restriction fragment length polymorphism analysis of the p.M390R allele in 2007 patients with isolated RP or autosomal recessive RP and in 1824 ethnically matched controls. Patients with 2 BBS1 variants underwent extensive clinical and ophthalmologic assessment. RESULTS In an RP proband who did not fulfill the clinical criteria for BBS, we identified a large homozygous region encompassing the BBS1 gene, which carried the p.M390R variant. In addition, this variant was detected homozygously in 10 RP patients and 1 control, compound heterozygously in 3 patients, and heterozygously in 5 patients and 6 controls. The 14 patients with 2 BBS1 variants showed the entire clinical spectrum, from nonsyndromic RP to full-blown BBS. In 8 of 14 patients, visual acuity was significantly reduced. In patients with electroretinographic responses, a rod-cone pattern of photoreceptor degeneration was observed. CONCLUSIONS Variants in BBS1 are significantly associated with nonsyndromic autosomal recessive RP and relatively mild forms of BBS. As exemplified in this study by the identification of a homozygous p.M390R variant in a control individual and in unaffected parents of BBS patients in other studies, cis - or trans -acting modifiers may influence the disease phenotype. CLINICAL RELEVANCE It is important to monitor patients with an early diagnosis of mild BBS phenotypes for possible life-threatening conditions.

Mutation spectrum of EYS in Spanish patients with autosomal recessive retinitis pigmentosa

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies characterised ultimately by the loss of photoreceptor cells. We have recently identified a new gene (EYS) encoding an ortholog of Drosophila spacemaker (spam) as a commonly mutated gene in autosomal recessive RP. In the present study, we report the identification of 73 sequence variations in EYS, of which 28 are novel. Of these, 42.9% (12/28) are very likely pathogenic, 17.9% (5/28) are possibly pathogenic, whereas 39.3% (11/28) are SNPs. In addition, we have detected 3 pathogenic changes previously reported in other populations. We are also presenting the characterisation of EYS homologues in different species, and a detailed analysis of the EYS domains, with the identification of an interesting novel feature: a putative coiled‐coil domain. Majority of the mutations in the arRP patients have been found within the domain structures of EYS. The minimum observed prevalence of distinct EYS mutations in our group of patients is of 15.9% (15/94), confirming a major involvement of EYS in the pathogenesis of arRP in the Spanish population. Along with the detection of three recurrent mutations in Caucasian population, our hypothesis of EYS being the first prevalent gene in arRP has been reinforced in the present study.

Mutations in the EYS gene account for approximately 5% of autosomal recessive retinitis pigmentosa and cause a fairly homogeneous phenotype.

OBJECTIVE To determine the prevalence of mutations in the EYS gene in a cohort of patients affected by autosomal recessive retinitis pigmentosa (RP) and to describe the associated phenotype. DESIGN Case series. PARTICIPANTS Two hundred forty-five patients affected by autosomal recessive RP. METHODS All coding exons of EYS were screened for mutations by polymerase chain reaction amplification and sequence analysis. All 12 patients carrying mutations in EYS were re-examined, which included Goldmann kinetic perimetry, electroretinography, and high-resolution spectral-domain optical coherence tomography (OCT). MAIN OUTCOME MEASURES DNA sequence variants, best-corrected visual acuity, fundus appearance, visual field assessments using Goldmann kinetic perimetry, electroretinogram responses, and OCT images. RESULTS Nine novel truncating mutations and one previously described mutation in EYS were identified in 11 families. In addition, 18 missense changes of uncertain pathogenicity were found. Patients carrying mutations in EYS demonstrated classic RP with night blindness as the initial symptom, followed by gradual constriction of the visual field and a decline of visual acuity later in life. The onset of symptoms typically occurred between the second and fourth decade of life. The fundus displayed bone spicules increasing in density with age and generalized atrophy of the retinal pigment epithelium and choriocapillaris with relative sparing of the posterior pole until later in the disease process, when atrophic macular changes occurred. CONCLUSIONS Mutations in EYS account for approximately 5% of autosomal recessive RP patients in a cohort of patients consisting predominantly of patients of western European ancestry. The EYS-associated RP phenotype is typical and fairly homogeneous in most patients.

A novel nonsense mutation in CEP290 induces exon skipping and leads to a relatively mild retinal phenotype.

PURPOSE. To identify the genetic defect in a family with variable retinal phenotypes. The proband had a diagnosis of Leber congenital amaurosis (LCA), whereas her two cousins had an early-onset severe retinal dystrophy (EOSRD) with useful vision. A distant family member had retinitis pigmentosa (RP). METHODS. DNA samples of the affected family members were genotyped with 250 K genome-wide SNP microarrays. Genetic defects were localized by linkage analysis and homozygosity mapping, and candidate genes were analyzed by sequencing. Patients underwent a full ophthalmic examination. RESULTS. Compound heterozygous mutations in CEP290 were identified in the proband and her two cousins: the frequent c.2991+1655A>G founder mutation and a novel nonsense mutation in exon 7 (c.451C>T, p.Arg151X). The proband had nystagmus, hyperopia, a flat electroretinogram (ERG), and decreased visual acuity (20/250) from birth. The two cousins had minimal scotopic ERG responses at the age of 2. In one of these patients, visual acuity had reached a level of 20/32 at age 5, which is high for patients with CEP290 mutations. Analysis of the CEP290 mRNA in affected individuals revealed altered splice forms in which either exon 7 or exons 7 and 8 were skipped. In both mutant cDNA products, the open reading frame was not disrupted. Furthermore, homozygosity mapping and mutation analysis in the distant family member affected by RP revealed a homozygous mutation in MERTK, but no CEP290 mutations. This MERTK mutation was heterozygously present in the most severely affected (LCA) patient, but was absent in the two more mildly affected cousins. CONCLUSIONS. A novel nonsense mutation in CEP290 results in nonsense-associated altered splicing. That the remaining open reading frame is intact may explain the less severe phenotype observed in the two affected cousins. The additional heterozygous mutation in MERTK may clarify the more severe phenotype in the proband. This study extends the phenotypic spectrum of CEP290-associated diseases at the mild end.

Novel null mutations in the EYS gene are a frequent cause of autosomal recessive retinitis pigmentosa in the Israeli population.

PURPOSE To characterize the role of EYS, a recently identified retinal disease gene, in families with inherited retinal degenerations in the Israeli and Palestinian populations. METHODS Clinical and molecular analyses included family history, ocular examination, full-field electroretinography (ERG), perimetry, autozygosity mapping, mutation detection, and estimation of mutation age. RESULTS Autozygosity mapping was performed in 171 consanguineous Israeli and Palestinian families with inherited retinal degenerations. Large homozygous regions, harboring the EYS gene, were identified in 15 of the families. EYS mutation analysis in the 15 index cases, followed by genotyping of specific mutations in an additional 121 cases of inherited retinal degenerations, revealed five novel null mutations, two of which are founder mutations, in 10 Israeli and Palestinian families with autosomal recessive retinitis pigmentosa (arRP). The most common mutation identified was a founder mutation in the Moroccan Jewish subpopulation. The ESTIAGE program produced an estimate that the age of the most recent common ancestor was 26 generations. The retinal phenotype in most patients was typical yet relatively severe RP, with an early age of onset and nonrecordable ERGs on presentation. CONCLUSIONS The results demonstrate that EYS is currently the most commonly mutated arRP gene in the Israeli population, mainly due to founder mutations. EYS mutations were associated with an RP phenotype in all patients. The authors concluded that the gene plays only a minor role in causing other retinal phenotypes.

A Homozygous Frameshift Mutation in LRAT Causes Retinitis Punctata Albescens

PURPOSE To determine the genetic defect and to describe the clinical characteristics in patients with retinitis punctata albescens (RPA) and fundus albipunctatus (FAP). DESIGN Case series/observational study. PARTICIPANTS We included 13 patients affected by RPA or FAP. METHODS Thirteen patients were collected from 8 families with a retinal dystrophy characterized by tiny, yellow-white dots on funduscopy, typical for FAP or RPA. All patients underwent full ophthalmologic examinations, including visual field assessment. Fundus photography, and electroretinography were performed in 12 patients, and optical coherence tomography and fundus autofluorescence were performed in 4 patients. DNA samples of all patients were screened for mutations in RLBP1 and for mutations in RDH5 in patients who did not carry mutations in RLBP1. DNA samples of 2 sibling pairs of nonconsanguineous families who carried mutations neither in RLBP1 nor in RDH5 were analyzed by genome-wide homozygosity mapping. Sequence analysis was performed of LRAT, a candidate gene in a shared homozygous region. MAIN OUTCOME MEASURES We assessed DNA sequence variants, best-corrected visual acuity, fundus appearance, visual field measurements, electroretinogram responses, optical coherence tomography, and fundus autofluorescence. RESULTS A homozygous frameshift mutation was identified in LRAT in 4 patients with RPA. Mutations in RLBP1 were identified in 7 patients with RPA and in 1 patient with FAP and cone dystrophy. One patient had compound heterozygous mutations in RDH5 and suffered from FAP with mild maculopathy. CONCLUSIONS A genetic defect was identified in LRAT as a novel cause of RPA. LRAT is therefore the fourth gene involved in the visual cycle that may cause a white-dot retinopathy. We also revealed that mutations in RLBP1 may lead to FAP with cone dystrophy.

The power of homozygosity mapping: discovery of new genetic defects in patients with retinal dystrophy

Retinal dystrophies (RD) represent a group of inherited ophthalmic diseases, which are characterized by dysfunction or progressive loss of photoreceptor cells, often accompanied by fundus abnormalities. To date, approximately 115 genes are known to be mutated in these diseases that together are estimated to account for ∼50% of the inherited RD. Knowledge of the genetic defect used to be beneficial for the patient only in terms of genetic counseling, and receiving a more accurate disease diagnosis and prognosis. The first successful clinical trials using gene augmentation therapy in RD patients with RPE65 mutations however have given an enormous boost to the development of several types of genetic therapies for RD. As a consequence, the identification of the genetic causes of RD has become more important than ever, also for the individual patient. One of the methods to discover novel mutations is genome-wide homozygosity mapping. This method has been a regularly used method to identify the genetic defect in patients from consanguineous families. In this mini-review, we will provide an overview of our own research that leads to the conclusion that homozygosity mapping can also be a powerful method to identify the genetic defect in patients from nonconsanguineous families.