Unique noncoding variants upstream of PRDM13 are associated with a spectrum of developmental retinal dystrophies including progressive bifocal chorioretinal atrophy.

The autosomal dominant progressive bifocal chorioretinal atrophy (PBCRA) disease locus has been mapped to chromosome 6q14-16.2 that overlaps the North Carolina macular dystrophy (NCMD) locus MCDR1. NCMD is a nonprogressive developmental macular dystrophy, in which variants upstream of PRDM13 have been implicated. Whole genome sequencing was performed to interrogate structural variants (SVs) and single nucleotide variants (SNVs) in eight individuals, six affected individuals from two families with PBCRA, and two individuals from an additional family with a related developmental macular dystrophy. A SNV (chr6:100,046,804T>C), located 7.8 kb upstream of the PRDM13 gene, was shared by all PBCRA-affected individuals in the disease locus. Haplotype analysis suggested that the variant arose independently in the two families. The two affected individuals from Family 3 were screened for rare variants in the PBCRA and NCMD loci. This revealed a de novo variant in the proband, 21 bp from the first SNV (chr6:100,046,783A>C). This study expands the noncoding variant spectrum upstream of PRDM13 and suggests altered spatio-temporal expression of PRDM13 as a candidate disease mechanism in the phenotypically distinct but related conditions, NCMD and PBCRA.

Cost-effective molecular inversion probe-based ABCA4 sequencing reveals deep-intronic variants in Stargardt disease.

PURPOSE: Stargardt disease (STGD1) is caused by biallelic mutations in ABCA4, but many patients are genetically unsolved due to insensitive mutation-scanning methods. We aimed to develop a cost-effective sequencing method for ABCA4 exons and regions carrying known causal deep-intronic variants. METHODS: Fifty exons and 12 regions containing 14 deep-intronic variants of ABCA4 were sequenced using double-tiled single molecule Molecular Inversion Probe (smMIP)-based next-generation sequencing. DNAs of 16 STGD1 cases carrying 29 ABCA4 alleles and of four healthy persons were sequenced using 483 smMIPs. Thereafter, DNAs of 411 STGD1 cases with one or no ABCA4 variant were sequenced. The effect of novel noncoding variants on splicing was analyzed using in vitro splice assays. RESULTS: Thirty-four ABCA4 variants previously identified in 16 STGD1 cases were reliably identified. In 155/411 probands (38%), two causal variants were identified. We identified 11 deep-intronic variants present in 62 alleles. Two known and two new noncanonical splice site variants showed splice defects, and one novel deep-intronic variant (c.4539+2065C>G) resulted in a 170-nt mRNA pseudoexon insertion (p.[Arg1514Lysfs*35,=]). CONCLUSIONS: smMIPs-based sequence analysis of coding and selected noncoding regions of ABCA4 enabled cost-effective mutation detection in STGD1 cases in previously unsolved cases.

Deep-intronic ABCA4 variants explain missing heritability in Stargardt disease and allow correction of splice defects by antisense oligonucleotides.

PURPOSE: Using exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify the missing heritability. METHODS: Sequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects. RESULTS: In 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects. CONCLUSION: Our data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.

North Carolina Macular Dystrophy Is Caused by Dysregulation of the Retinal Transcription Factor PRDM13.

PURPOSE: To identify specific mutations causing North Carolina macular dystrophy (NCMD). DESIGN: Whole-genome sequencing coupled with reverse transcription polymerase chain reaction (RT-PCR) analysis of gene expression in human retinal cells. PARTICIPANTS: A total of 141 members of 12 families with NCMD and 261 unrelated control individuals. METHODS: Genome sequencing was performed on 8 affected individuals from 3 families affected with chromosome 6-linked NCMD (MCDR1) and 2 individuals affected with chromosome 5-linked NCMD (MCDR3). Variants observed in the MCDR1 locus with frequencies <1% in published databases were confirmed using Sanger sequencing. Confirmed variants absent from all published databases were sought in 8 additional MCDR1 families and 261 controls. The RT-PCR analysis of selected genes was performed in stem cell-derived human retinal cells. MAIN OUTCOME MEASURES: Co-segregation of rare genetic variants with disease phenotype. RESULTS: Five sequenced individuals with MCDR1-linked NCMD shared a haplotype of 14 rare variants spanning 1 Mb of the disease-causing allele. One of these variants (V1) was absent from all published databases and all 261 controls, but was found in 5 additional NCMD kindreds. This variant lies in a DNase 1 hypersensitivity site (DHS) upstream of both the PRDM13 and CCNC genes. Sanger sequencing of 1 kb centered on V1 was performed in the remaining 4 NCMD probands, and 2 additional novel single nucleotide variants (V2 in 3 families and V3 in 1 family) were identified in the DHS within 134 bp of the location of V1. A complete duplication of the PRDM13 gene was also discovered in a single family (V4). The RT-PCR analysis of PRDM13 expression in developing retinal cells revealed marked developmental regulation. Next-generation sequencing of 2 individuals with MCDR3-linked NCMD revealed a 900-kb duplication that included the entire IRX1 gene (V5). The 5 mutations V1 to V5 segregated perfectly in the 102 affected and 39 unaffected members of the 12 NCMD families. CONCLUSIONS: We identified 5 rare mutations, each capable of arresting human macular development. Four of these strongly implicate the involvement of PRDM13 in macular development, whereas the pathophysiologic mechanism of the fifth remains unknown but may involve the developmental dysregulation of IRX1.

Clinical Characteristics and Risk Factors of Extensive Macular Atrophy with Pseudodrusen: The EMAP Case-Control National Clinical Trial.

PURPOSE: To assess the association of clinical and biological factors with extensive macular atrophy with pseudodrusen (EMAP) characterized by bilateral macular atrophy occurring in patients aged 50 to 60 years and a rapid progression to legal blindness within 5 to 10 years. DESIGN: A national matched case-control study. PARTICIPANTS: Participants were recruited in 10 French Departments of Ophthalmology and their associated clinical investigation centers. All 115 patients with EMAP had symptoms before the age of 55 years due to bilateral extensive macular atrophy with a larger vertical axis and diffuse pseudodrusen. Three controls without age-related macular degeneration (AMD) or retinal disease at fundus examination were matched for each patient with EMAP by gender, age, and geographic area (in total 415). METHODS: Subjects and controls underwent an eye examination including color, red-free autofluorescent fundus photographs and spectral-domain optical coherence tomography with macular analysis. The interviews collected demographic, lifestyle, family and personal medical history, medications, and biological data. Associations of risk factors were estimated using conditional logistic regression. MAIN OUTCOME MEASURES: Extensive macular atrophy with pseudodrusen status (cases vs. controls). RESULTS: Extensive macular atrophy with pseudodrusen most frequently affected women (70 women, 45 men). After multivariate adjustment, family history of glaucoma or AMD was strongly associated with EMAP (odds ratio [OR], 2.3, P = 0.008 and OR, 1.5, P = 0.01, respectively). No association was found with cardiac diseases or their risk factors. Mild and moderate kidney disease and higher neutrophil rate were associated with a reduced risk of EMAP (OR, 0.58, P = 0.04; OR, 0.34, P = 0.01; and OR, 0.59, P = 0.003, respectively). On the contrary, eosinophilia (OR, 1.6; P = 0.0002), lymphocytosis (OR, 1.84; P = 0.0002), increased erythrocyte sedimentation rate (OR, 6.5; P = 0.0005), decreased CH50 (P = 0.001), and high plasma C3 level (P = 0.023) were significantly associated with a higher risk of EMAP. CONCLUSIONS: This study documents an association between EMAP and family history of AMD and glaucoma, a clear female predominance, and a systemic inflammatory profile. The reduced CH50 and increased C3 plasma values could reflect a more severe complement pathway dysfunction than in AMD, leading to early pseudodrusen and rapid development of geographic atrophy. There is no association of EMAP with AMD cardiac diseases or cardiac risks, including cigarette smoking.

Long-term follow-up of two patients with oligocone trichromacy.

INTRODUCTION: Oligocone trichromacy (OT) is an uncommon cone dysfunction disorder, the mechanism of which remains poorly understood. OT has been thought to be non-progressive, but its long-term visual outcome has been seldom reported in the literature. Our aim was to present two OT patients followed at our institution over 18 years. MATERIALS AND METHODS: Complete ocular examination, color vision, visual fields, and full-field electroretinography (ERG) were performed at initial presentation and follow-up. Spectral-domain optical coherence tomography (OCT) was performed during follow-up when available at our institution. RESULTS: Initial ocular examination showed satisfactory visual acuities with normal fundus examination and near-to-normal color vision. However, computerized perimetry demonstrated a ring-shaped scotoma around fixation, and ERG showed a profound cone dysfunction. The discrepancy between preserved color vision and profound cone dysfunction leads to the diagnosis of OT. Subsequent follow-ups over 18 years showed subtle degradation of visual acuities along with progression of the myopia in both patients and slight worsening of color vision in one patient. Initial OCT revealed a focal interruption of the ellipsoid line along with decreased thickness of the perifoveal macula. Subsequent OCT imaging performed 2 years later did not show any macular changes. CONCLUSION: Although OT is known to be a non-progressive cone dysfunction, our results suggest that subtle degradation of the visual function might happen over time.

Enrichment of LOVD-USHbases with 152 USH2A genotypes defines an extensive mutational spectrum and highlights missense hotspots.

Alterations of USH2A, encoding usherin, are responsible for more than 70% of cases of Usher syndrome type II (USH2), a recessive disorder that combines moderate to severe hearing loss and retinal degeneration. The longest USH2A transcript encodes usherin isoform b, a 5,202-amino-acid transmembrane protein with an exceptionally large extracellular domain consisting notably of a Laminin N-terminal domain and numerous Laminin EGF-like (LE) and Fibronectin type III (FN3) repeats. Mutations of USH2A are scattered throughout the gene and mostly private. Annotating these variants is therefore of major importance to correctly assign pathogenicity. We have extensively genotyped a novel cohort of 152 Usher patients and identified 158 different mutations, of which 93 are newly described. Pooling this new data with the existing pathogenic variants already incorporated in USHbases reveals several previously unappreciated features of the mutational spectrum. We show that parts of the protein are more likely to tolerate single amino acid variations, whereas others constitute pathogenic missense hotspots. We have found, in repeated LE and FN3 domains, a nonequal distribution of the missense mutations that highlights some crucial positions in usherin with possible consequences for the assessment of the pathogenicity of the numerous missense variants identified in USH2A.

Frequency and clinical pattern of vitelliform macular dystrophy caused by mutations of interphotoreceptor matrix IMPG1 and IMPG2 genes.

PURPOSE: To assess the frequency of and to characterize the clinical spectrum and optical coherence tomography findings of vitelliform macular dystrophy linked to IMPG1 and IMPG2, 2 new causal genes expressed in the interphotoreceptor matrix. DESIGN: Retrospective epidemiologic, clinical, electrophysiologic, and molecular genetic study. PARTICIPANTS: The database of a national referral center specialized in genetic sensory diseases was screened for patients with a macular vitelliform dystrophy without identified mutation or small deletion or large rearrangement in BEST1 and PRPH2 genes. Forty-nine families were included. METHODS: Clinical, imaging, and electro-oculogram findings were reviewed. Mutation screening of IMPG1 and IMPG2 genes were performed systematically. MAIN OUTCOME MEASURES: Frequency, inheritance, and clinical pattern of vitelliform dystrophy associated with IMPG1 and IMPG2 mutations were characterized. RESULTS: IMPG1 was the causal gene in 3 families (IMPG1 1-3, 11 patients) and IMPG2 in a fourth family (2 patients). With an autosomal dominant transmission, families 1 and 2 had the c.713T→G (p.Leu238Arg) mutation in IMPG1 and family 4 had the c.3230G→T (p.Cys1077Phe) mutation in IMPG2. Patients with IMPG1 or IMPG2 mutations had a late onset and moderate visual impairment (mean visual acuity, 20/40; mean age of onset, 42 years), even in the sporadic case of family 3 with a presumed recessive transmission (age at onset, 38 years; mean visual acuity, 20/50). Drusen-like lesions adjacent to the vitelliform deposits were observed in 9 of 13 patients. The vitelliform material was above the retinal pigment epithelium (RPE) at any stage of the macular dystrophy, and this epithelium was well preserved and maintained its classical reflectivity on spectral-domain optical coherence tomography (SD-OCT). Electro-oculogram results were normal or borderline in 9 cases. CONCLUSIONS: IMPG1 and IMPG2 are new causal genes in 8% of families negative for BEST1 and PRPH2 mutations. These genes should be screened in adult-onset vitelliform dystrophy with (1) moderate visual impairment, (2) drusen-like lesions, (3) normal reflectivity of the RPE line on SD-OCT, and (4) vitelliform deposits located between ellipsoid and interdigitation lines on SD-OCT. These clinical characteristics are not observed in the classical forms of BEST1 or PRPH2 vitelliform dystrophies.

Phenotypic variability in a French family with a novel mutation in the BEST1 gene causing multifocal best vitelliform macular dystrophy.

AIMS: To describe genetic and clinical findings in a French family affected by best vitelliform macular dystrophy (BVMD). METHODS: We screened eight at-risk members of a family, including a BVMD-affected proband, by direct sequencing of 11 bestrophin-1 (BEST1) exons. Individuals underwent ophthalmic examination and autofluorescent fundus imaging, indocyanine green angiography, electro-oculogram (EOG), electroretinogram (ERG), multifocal ERG, optical coherence tomography (OCT), and where possible, spectral domain OCT. RESULTS: The sequence analysis of the BEST1 gene revealed one previously unknown mutation, c.15C>A (p.Y5X), in two family members and one recently described mutation, c.430A>G (p.S144G), in five family members. Fundus examination and electrophysiological responses provided no evidence of the disease in the patient carrying only the p.Y5X mutation. Three patients with the p.S144G mutation did not show any preclinical sign of BVMD except altered EOGs. Two individuals of the family exhibited a particularly severe phenotype of multifocal BVMD-one individual carrying the p.S144G mutation heterozygously and one individual harboring both BEST1 mutations (p.S144G inherited from his mother and p.Y5X from his father). Both of these family members had multifocal vitelliform autofluorescent lesions combined with abnormal EOG, and the spectral domain OCT displayed a serous retinal detachment. In addition, ERGs demonstrated widespread retinal degeneration and multifocal ERGs showed a reduction in the central retina function, which could be correlated with the decreased visual acuity and visual field scotomas. CONCLUSIONS: A thorough clinical evaluation found no pathological phenotype in the patient carrying the isolated p.Y5X mutation. The patients carrying the p.S144G variation in the protein exhibited considerable intrafamilial phenotypic variability. Two young affected patients in this family exhibited an early onset, severe, multifocal BVMD with a diffuse distribution of autofluorescent deposits throughout the retina and rapid evolution toward the loss of central vision. The other genetically affected relatives had only abnormal EOGs and displayed no or extremely slow electrophysiological evolution.

Kjellin syndrome: long-term neuro-ophthalmologic follow-up and novel mutations in the SPG11 gene.

OBJECTIVE: Kjellin's syndrome is a hereditary neuro-ophthalmologic syndrome. We describe the clinical phenotypes of 7 patients, identifying the responsible mutations for 4 of them. A 10-year ophthalmologic and neurologic follow-up of 5 patients allowed us to describe the disease's characteristics, early symptoms and progression, associated ocular signs, and retinal changes in carriers. DESIGN: Retrospective clinical study and molecular genetics investigation. PARTICIPANTS: The records of 7 patients with Kjellin's syndrome were analyzed retrospectively. METHODS: All patients underwent full neurologic and ophthalmologic examinations. The neurologic examinations included assessments of initial symptoms, intelligence quotient tests, psychologic tests, and either magnetic resonance imaging or computed tomography. The ophthalmologic examinations included visual acuity on an Early Treatment Diabetic Retinopathy Study chart, intraocular pressure color vision assessment, slit-lamp and fundus examination, Goldmann perimetry, fundus autofluorescence, optical coherence tomography and fluorescein angiography, electro-oculography, electroretinography, and flash visual evoked potentials. Direct sequencing of the SPG11 and SPG15 genes and gene-dosage analysis for the former were performed for 4 of these index patients. MAIN OUTCOME MEASURES: Identification of new mutations in the SPG11 gene, validating its implication in Kjellin's syndrome. RESULTS: The first signs appear before the age of 10 years, with late verbal development and difficulty running and walking. Life expectancy is between 30 and 40 years. The secondary ophthalmologic symptoms only moderately affect visual acuity. In addition to the classic symptoms, 3 of the 7 patients displayed small whitish lens opacities, and 3 neurologically unaffected parents (father or mother), all heterozygous carriers, exhibited whitish retinal dots. All the patients who were tested carried SPG11, not SPG15, mutations. CONCLUSIONS: Neurologic signs of SPG11 mutations emerge in early infancy, with walking and language difficulties. Onset of paraplegia occurs at the end of the first decade or during the second decade. Retinal changes, an integral part of SPG11 mutations in this series of patients, are only observed once the paraplegia has become apparent.

Systematic screening of BEST1 and PRPH2 in juvenile and adult vitelliform macular dystrophies: a rationale for molecular analysis.

PURPOSE: To evaluate a genetic approach of BEST1 and PRPH2 screening according to age of onset, family history, and Arden ratio in patients with juvenile vitelliform macular dystrophy (VMD2) or adult-onset vitelliform macular dystrophy (AVMD), which are characterized by autofluorescent deposits. DESIGN: Clinical, electrophysiologic, and molecular retrospective study. PARTICIPANTS: The database of a clinic specialized in genetic sensory diseases was screened for patients with macular vitelliform dystrophy. Patients with an age of onset less than 40 years were included in the VMD2 group (25 unrelated patients), and patients with an age of onset more than 40 years were included in the AVMD group (19 unrelated patients). METHODS: Clinical, fundus photography, and electro-oculogram (EOG) findings were reviewed. Mutation screening of BEST1 and PRPH2 genes was systematically performed. MAIN OUTCOME MEASURES: Relevance of age of onset, family history, and Arden ratio were reviewed. RESULTS: Patients with VMD2 carried a BEST1 mutation in 60% of the cases. Seven novel mutations in BEST1 (p.V9L, p.F80V, p.I73V, p.R130S, pF298C, pD302A, and p.179delN) were found. Patients with VMD2 with a positive family history or a reduced Arden ratio carried a BEST1 mutation in 70.5% of cases and in 83% if both criteria were fulfilled. Patients with AVMD carried a PRPH2 mutation in 10.5% of cases and did not carry a BEST1 mutation. The probability of finding a PRPH2 mutation increased in the case of a family history (2/5 patients). Electro-oculogram was normal in 3 of 15 patients with BEST1 mutations and reduced in the 3 patients with PRPH2 mutations. CONCLUSIONS: Age of onset is a major criterion to distinguish VMD2 from AVMD. Electro-oculogram is not as relevant because decreased or normal Arden ratios have been associated with mutations in both genes and diseases. A positive family history increased the probability of finding a mutation. BEST1 screening should be recommended to patients with an age of onset less than 40 years, and PRPH2 screening should be recommended to patients with an age of onset more than 40 years. For an onset between 30 and 40 years, PRPH2 can be screened if no mutation has been detected in BEST1. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Spectrum of SPATA7 mutations in Leber congenital amaurosis and delineation of the associated phenotype.

Leber congenital amaurosis (LCA) is the earliest and most severe retinal degeneration. It may present as a congenital stationary cone-rod dystrophy (LCA type I) or a progressive yet severe rod-cone dystrophy (LCA type II). Twelve LCA genes have been identified, three of which account for Type I and nine for LCA type II. All proteins encoded by these genes but two are preferentially expressed in the retina and are responsible for non-syndromic LCA only. By contrast LCA5 and CEP290 are widely expressed and mutations in this latter result in a variety of phenotypes from non-syndromic retinal degeneration to pleiotropic disorders including senior-Loken (SNLS) and Joubert syndromes (JBTS). Recently, mutations in the widely expressed gene SPATA7 were reported to cause LCA or juvenile retinitis pigmentosa. The purpose of this study was i) to determine the level of expression of two major alternative SPATA7 transcripts in a large range of tissues and ii) to assess the involvement of this novel gene in a large cohort of unrelated patients affected with LCA (n = 134). Here, we report high SPATA7expression levels in retina, brain and testis with differential expression of the two transcripts. SPATA7 mutations were identified in few families segregating non-syndromic LCA (n = 4/134). Six different mutations were identified, four of which are novel; All affected both SPATA7 transcripts. The clinical evaluation of patients suggested that SPATA7 mutations account for the rod-cone dystrophy type of the disease.

Dietary, environmental, and genetic risk factors of Extensive Macular Atrophy with Pseudodrusen, a severe bilateral macular atrophy of middle-aged patients.

EMAP (Extensive Macular Atrophy with Pseudodrusen) is a maculopathy we recently described that shares pseudodrusen and geographic atrophy with Age-related Macular Disease (AMD). EMAP differs from AMD by an earlier age of onset (50-55 years) and a characteristic natural history comprising a night blindness followed by a severe visual loss. In a prospective case-control study, ten referral centers included 115 EMAP (70 women, 45 men) patients and 345 matched controls to appraise dietary, environmental, and genetic risk factors. The incidence of EMAP (mean 2.95/1.106) was lower in Provence-Côte d'Azur with a Mediterranean diet (1.9/1.106), and higher in regions with intensive farming or industrialized activities (5 to 20/1.106). EMAP patients reported toxic exposure during professional activities (OR 2.29). The frequencies of common AMD complement factor risk alleles were comparable in EMAP. By contrast, only one EMAP patient had a rare AMD variant. This study suggests that EMAP could be a neurodegenerative disorder caused by lifelong toxic exposure and that it is associated with a chronic inflammation and abnormal complement pathway regulation. This leads to diffuse subretinal deposits with rod dysfunction and cone apoptosis around the age of 50 with characteristic extensive macular atrophy and paving stones in the far peripheral retina.

Duplication events downstream of IRX1 cause North Carolina macular dystrophy at the MCDR3 locus.

Autosomal dominant North Carolina macular dystrophy (NCMD) is believed to represent a failure of macular development. The disorder has been linked to two loci, MCDR1 (chromosome 6q16) and MCDR3 (chromosome 5p15-p13). Recently, non-coding variants upstream of PRDM13 (MCDR1) and a duplication including IRX1 (MCDR3) have been identified. However, the underlying disease-causing mechanism remains uncertain. Through a combination of sequencing studies on eighteen NCMD families, we report two novel overlapping duplications at the MCDR3 locus, in a gene desert downstream of IRX1 and upstream of ADAMTS16. One duplication of 43 kb was identified in nine families (with evidence for a shared ancestral haplotype), and another one of 45 kb was found in a single family. Three families carry the previously reported V2 variant (MCDR1), while five remain unsolved. The MCDR3 locus is thus refined to a shared region of 39 kb that contains DNAse hypersensitive sites active at a restricted time window during retinal development. Publicly available data confirmed expression of IRX1 and ADAMTS16 in human fetal retina, with IRX1 preferentially expressed in fetal macula. These findings represent a major advance in our understanding of the molecular genetics of NCMD and provide insights into the genetic pathways involved in human macular development.

Genome-wide linkage and haplotype sharing analysis implicates the MCDR3 locus as a candidate region for a developmental macular disorder in association with digit abnormalities.

BACKGROUND: Developmental macular disorders are a heterogeneous group of rare retinal conditions that can cause significant visual impairment from childhood. Among these disorders, autosomal dominant North Carolina macular dystrophy (NCMD) has been mapped to 6q16 (MCDR1) with recent support for a non-coding disease mechanism of PRDM13. A second locus on 5p15-5p13 (MCDR3) has been implicated in a similar phenotype, but the disease-causing mechanism still remains unknown. METHODS: Two families affected by a dominant developmental macular disorder that closely resembles NCMD in association with digit abnormalities were included in the study. Family members with available DNA were genotyped using the Affymetrix GeneChip Human Mapping 250K Sty array. A parametric multipoint linkage analysis assuming a fully penetrant dominant model was performed using MERLIN. Haplotype sharing analysis was carried out using the non-parametric Homozygosity Haplotype method. Whole-exome sequencing was conducted on selected affected individuals. RESULTS: Linkage analysis excluded MCDR1 from the candidate regions (LOD < -2). There was suggestive linkage (LOD = 2.7) at two loci, including 9p24.1 and 5p15.32 that overlapped with MCDR3. The haplotype sharing analysis in one of the families revealed a 5 cM shared IBD segment at 5p15.32 (p value = 0.004). Whole-exome sequencing did not provide conclusive evidence for disease-causing alleles. CONCLUSIONS: These findings do not exclude that this phenotype may be allelic with NCMD MCDR3 at 5p15 and leave the possibility of a non-coding disease mechanism, in keeping with recent findings on 6q16. Further studies, including whole-genome sequencing, may help elucidate the underlying genetic cause of this phenotype and shed light on macular development and function.

A new autosomal dominant eye and lung syndrome linked to mutations in TIMP3 gene.

To revisit the autosomal dominant Sorsby fundus dystrophy (SFD) as a syndromic condition including late-onset pulmonary disease. We report clinical and imaging data of ten affected individuals from 2 unrelated families with SFD and carrying heterozygous TIMP3 mutations (c.572A > G, p.Y191C, exon 5, in family 1 and c.113C > G, p.S38C, exon 1, in family 2). In family 1, all SFD patients older than 50 (two generations) had also a severe emphysema, despite no history of smoking or asthma. In the preceding generation, the mother died of pulmonary emphysema and she was blind after the age of 50. Her two great-grandsons (<20 years), had abnormal Bruch Membrane thickness, a sign of eye disease. In family 2, eye and lung diseases were also associated in two generations, both occurred later, and lung disease was moderate (bronchiectasis). This is the first report of a syndromic SFD in line with the mouse model uncovering the role of TIMP3 in human lung morphogenesis and functions. The TIMP3 gene should be screened in familial pulmonary diseases with bronchiectasis, associated with a medical history of visual loss. In addition, SFD patients should be advised to avoid tobacco consumption, to practice sports, and to undergo regular pulmonary examinations.

Predominantly Cone-System Dysfunction as Rare Form of Retinal Degeneration in Patients With Molecularly Confirmed Bardet-Biedl Syndrome.

PURPOSE: To describe a series of patients with Bardet-Biedl syndrome (BBS) and predominantly retinal cone dysfunction, a previously only rarely reported association. DESIGN: Retrospective observational case series. METHODS: Seven patients with clinically proven Bardet-Biedl syndrome had undergone detailed ocular phenotyping, which included fundus examination, Goldmann visual fields, fundus autofluorescence imaging (FAF), optical coherence tomography (OCT), and electroretinography (ERG). Mutational screening in the BBS genes was performed either by direct Sanger sequencing or targeted next-generation sequencing. RESULTS: All 7 patients had proven BBS mutations; 1 had a cone dystrophy phenotype on ERG and 6 had a cone-rod pattern of dysfunction. Macular atrophy was present in all patients, usually with central hypofluorescence surrounded by a continuous hyperfluorescent ring on fundus autofluorescence imaging. OCT confirmed loss of outer retinal structure within the atrophic areas. No clear genotype-phenotype relationship was evident. CONCLUSIONS: Patients with Bardet-Biedl syndrome usually develop early-onset retinitis pigmentosa. In contrast, the patients described herein, with molecularly confirmed Bardet-Biedl syndrome, developed early cone dysfunction, including the first reported case of a cone dystrophy phenotype associated with the disorder. The findings significantly expand the phenotype associated with Bardet-Biedl syndrome.

High prevalence of PRPH2 in autosomal dominant retinitis pigmentosa in france and characterization of biochemical and clinical features.

PURPOSE: To assess the prevalence of PRPH2 in autosomal dominant retinitis pigmentosa (adRP), to report 6 novel mutations, to characterize the biochemical features of a recurrent novel mutation, and to study the clinical features of adRP patients. DESIGN: Retrospective clinical and molecular genetic study. METHODS: Clinical investigations included visual field testing, fundus examination, high-resolution spectral-domain optical coherence tomography (OCT), fundus autofluorescence imaging, and electroretinogram (ERG) recording. PRPH2 was screened by Sanger sequencing in a cohort of 310 French families with adRP. Peripherin-2 protein was produced in yeast and analyzed by Western blot. RESULTS: We identified 15 mutations, including 6 novel and 9 previously reported changes in 32 families, accounting for a prevalence of 10.3% in this adRP population. We showed that a new recurrent p.Leu254Gln mutation leads to protein aggregation, suggesting abnormal folding. The clinical severity of the disease in examined patients was moderate with 78% of the eyes having 1-0.5 of visual acuity and 52% of the eyes retaining more than 50% of the visual field. Some patients characteristically showed vitelliform deposits or macular involvement. In some families, pericentral RP or macular dystrophy were found in family members while widespread RP was present in other members of the same families. CONCLUSIONS: The mutations in PRPH2 account for 10.3% of adRP in the French population, which is higher than previously reported (0%-8%) This makes PRPH2 the second most frequent adRP gene after RHO in our series. PRPH2 mutations cause highly variable phenotypes and moderate forms of adRP, including mild cases, which could be underdiagnosed.

Mutations of VMD2 splicing regulators cause nanophthalmos and autosomal dominant vitreoretinochoroidopathy (ADVIRC).

PURPOSE: To investigate the genetic basis of autosomal dominant vitreoretinochoroidopathy (ADVIRC), a rare, inherited retinal dystrophy that may be associated with defects of ocular development, including nanophthalmos. METHODS: A combination of linkage analysis and DNA sequencing in five families was used to identify disease-causing mutations in VMD2. The effect of these mutations on splicing was assessed using a minigene system. RESULTS: Three pathogenic sequence alterations in VMD2 were identified in five families with nanophthalmos associated with ADVIRC. All sequences showed simultaneous missense substitutions and exon skipping. CONCLUSIONS: VMD2 encodes bestrophin, a transmembrane protein located at the basolateral membrane of the RPE, that is also mutated in Best macular dystrophy. We support that each heterozygous affected individual produces three bestrophin isoforms consisting of the wild type and two abnormal forms: one containing a missense substitution and the other an in-frame deletion. The data showed that VMD2 mutations caused defects of ocular patterning, supporting the hypothesized role for the RPE, and specifically VMD2, in the normal growth and development of the eye.

Can we still hear the Cro-magnon man?

This paper is an essay to connect with the stone Age coastal hunters who sheltered 28,000 years ago in the caves of today's italian Mediterranean sea shore cliffs. We have focused on the archaeological Gravettian layer in the Cavillon cave from Grimaldi occupied by Cro Magnons (informal name for Anatomically Modern Humans of the European Upper Paleolithic), which demonstrates the technical skills of the phase. Cro Magnons, like Neanderthals, were seasonally nomadic; however, the diversity of the resources exploited within the territory along the Grimaldi sea coast suggests a longer term site within which a complex symbolic culture developed.