Elena Aller

A novel gene for Usher syndrome type 2: mutations in the long isoform of whirlin are associated with retinitis pigmentosa and sensorineural hearing loss

Usher syndrome is an autosomal recessive condition characterized by sensorineural hearing loss, variable vestibular dysfunction, and visual impairment due to retinitis pigmentosa (RP). The seven proteins that have been identified for Usher syndrome type 1 (USH1) and type 2 (USH2) may interact in a large protein complex. In order to identify novel USH genes, we followed a candidate strategy, assuming that mutations in proteins interacting with this “USH network” may cause Usher syndrome as well. The DFNB31 gene encodes whirlin, a PDZ scaffold protein with expression in both hair cell stereocilia and retinal photoreceptor cells. Whirlin represents an excellent candidate for USH2 because it binds to Usherin (USH2A) and VLGR1b (USH2C). Genotyping of microsatellite markers specific for the DFNB31 gene locus on chromosome 9q32 was performed in a German USH2 family that had been excluded for all known USH loci. Patients showed common haplotypes. Sequence analysis of DFNB31 revealed compound heterozygosity for a nonsense mutation, p.Q103X, in exon 1, and a mutation in the splice donor site of exon 2, c.837+1G>A. DFNB31 mutations appear to be a rare cause of Usher syndrome, since no mutations were identified in an additional 96 USH2 patients. While mutations in the C-terminal half of whirlin have previously been reported in non-syndromic deafness (DFNB31), both alterations identified in our USH2 family affect the long protein isoform. We propose that mutations causing Usher syndrome are probably restricted to exons 1–6 that are specific for the long isoform and probably crucial for retinal function. We describe a novel genetic subtype for Usher syndrome, which we named USH2D and which is caused by mutations in whirlin. Moreover, this is the first case of USH2 that is allelic to non-syndromic deafness.

An Update on the Genetics of Usher Syndrome

Usher syndrome (USH) is an autosomal recessive disease characterized by hearing loss, retinitis pigmentosa (RP), and, in some cases, vestibular dysfunction. It is clinically and genetically heterogeneous and is the most common cause underlying deafness and blindness of genetic origin. Clinically, USH is divided into three types. Usher type I (USH1) is the most severe form and is characterized by severe to profound congenital deafness, vestibular areflexia, and prepubertal onset of progressive RP. Type II (USH2) displays moderate to severe hearing loss, absence of vestibular dysfunction, and later onset of retinal degeneration. Type III (USH3) shows progressive postlingual hearing loss, variable onset of RP, and variable vestibular response. To date, five USH1 genes have been identified: MYO7A (USH1B), CDH23 (USH1D), PCDH15 (USH1F), USH1C(USH1C), and USH1G(USH1G). Three genes are involved in USH2, namely, USH2A (USH2A), GPR98 (USH2C), and DFNB31 (USH2D). USH3 is rare except in certain populations, and the gene responsible for this type is USH3A.

Identification of 14 novel mutations in the long isoform of USH2A in Spanish patients with Usher syndrome type II

Mutations in USH2A gene have been shown to be responsible for Usher syndrome type II, an autosomal recessive disorder characterised by hearing loss and retinitis pigmentosa. USH2A was firstly described as consisting of 21 exons, but 52 novel exons at the 3’ end of the gene were recently identified. In this report, a mutation analysis of the new 52 exons of USH2A gene was carried out in 32 unrelated patients in which both disease-causing mutations could not be found after the screening of the first 21 exons of the USH2A gene. On analysing the new 52 exons, fourteen novel mutations were identified in 14 out of the 32 cases studied, including 7 missense, 5 frameshift, 1 duplication and a putative splice-site mutation.

Genetic analysis of 2299delG and C759F mutations (USH2A) in patients with visual and/or auditory impairments

The most common mutation in the USH2A gene (Usherin), 2299delG, causes both typical Usher (USH) syndrome type II and atypical USH syndrome, two autosomal recessive disorders, characterised by moderate to severe sensorineural hearing loss and retinitis pigmentosa (RP). Furthermore, the C759F mutation in the USH2A gene has been described in 4.5% of patients with nonsyndromic recessive RP. We have investigated the presence of the 2299delG and/or the C759F mutations in 191 unrelated Spanish patients with different syndromic and nonsyndromic retinal diseases, or with nonsyndromic hearing impairment. The 2299delG mutation was observed in patients with clinical signs of USHII or of atypical USH syndrome, whereas the C759F mutation, regardless of being associated with the 2299delG mutation or not, was identified in cases with nonsyndromic RP, as well as in patients with RP associated with a variability of hearing impairment. The comparative analysis of both phenotypic and genotypic data supports the hypothesis that sensorineural hearing loss in patients with RP may depend on the nature and on the association of the USH2A allele variants present.

MYO7A mutation screening in Usher syndrome type I patients from diverse origins

Usher syndrome (USH) (OMIM 276901) is an autosomal recessive disorder characterised by hearing impairment associated with retinitis pigmentosa and in some cases vestibular dysfunction. This disease accounts for approximately 50% of individuals with combined deafness and blindness in developed countries. The estimated prevalence of USH ranges from 3.8 to 6.2/100 000.1–3 Phenotypically, three clinical types of Usher syndrome have been defined according to the severity of hearing impairment, age of retinitis pigmentosa onset and the presence or absence of vestibular response. Usher syndrome type I (USH1) is the most serious type, characterised by severe to profound congenital sensorineural hearing loss, balance deficiency and prepubertal onset of retinitis pigmentosa leading to blindness. USH2 is characterised by moderate to severe hearing impairment, normal vestibular function and later onset of retinal degeneration than USH1. USH3 displays progressive hearing loss, retinitis pigmentosa and variable vestibular phenotype. Six loci for USH1 (USH1B–USH1G) have been mapped and, to date, five genes have been identified.4,5 The MYO7A gene was found to be responsible for USH1B6 and is the most common subtype of USH1, accounting for approximately 50% of cases.7–9 Defects in MYO7A also cause autosomal dominant non-syndromic sensorineural hearing impairment (DFNA11) (MIM 601317),10 autosomal recessive deafness (DFNB2) (MIM 600060)11,12 as well as atypical types of Usher syndrome which are clinically similar to USH3.13 The MYO7A gene has 49 exons, of which 48 are coding, and spans approximately 87 kb of genomic sequence on chromosome 11q13.5. The encoded protein is an unconventional myosin, the myosin VIIA,14 predicted to consist of 2215 amino acids and has a molecular mass of 254 kDa. This protein contains three typical domains: the N terminal head or motor; the neck or regulatory domain consisting of five IQ motifs; and the tail …

Altered Antioxidant-Oxidant Status in the Aqueous Humor and Peripheral Blood of Patients with Retinitis Pigmentosa

Retinitis Pigmentosa is a common form of hereditary retinal degeneration constituting the largest Mendelian genetic cause of blindness in the developed world. It has been widely suggested that oxidative stress possibly contributes to its pathogenesis. We measured the levels of total antioxidant capacity, free nitrotyrosine, thiobarbituric acid reactive substances (TBARS) formation, extracellular superoxide dismutase (SOD3) activity, protein, metabolites of the nitric oxide/cyclic GMP pathway, heme oxygenase-I and inducible nitric oxide synthase expression in aqueous humor or/and peripheral blood from fifty-six patients with retinitis pigmentosa and sixty subjects without systemic or ocular oxidative stress-related disease. Multivariate analysis of covariance revealed that retinitis pigmentosa alters ocular antioxidant defence machinery and the redox status in blood. Patients with retinitis pigmentosa present low total antioxidant capacity including reduced SOD3 activity and protein concentration in aqueous humor. Patients also show reduced SOD3 activity, increased TBARS formation and upregulation of the nitric oxide/cyclic GMP pathway in peripheral blood. Together these findings confirmed the hypothesis that patients with retinitis pigmentosa present reduced ocular antioxidant status. Moreover, these patients show changes in some oxidative-nitrosative markers in the peripheral blood. Further studies are needed to clarify the relationship between these peripheral markers and retinitis pigmentosa.

Mutation screening of USH3 gene (clarin‐1) in Spanish patients with Usher syndrome: low prevalence and phenotypic variability

Usher syndrome type III is an autosomal recessive disorder clinically characterized by the association of retinitis pigmentosa (RP), variable presence of vestibular dysfunction and progressive hearing loss, being the progression of the hearing impairment the critical parameter classically used to distinguish this form from Usher syndrome type I and Usher syndrome type II. Usher syndrome type III clinical subtype is the rarest form of Usher syndrome in Spain, accounting only for 6% of all Usher syndrome Spanish cases. The gene responsible for Usher syndrome type III is named clarin‐1 and it is thought to be involved in hair cell and photoreceptor cell synapses. Here, we report a screening for mutations in clarin‐1 gene among our series of Usher syndrome Spanish patients. Clarin‐1 has been found to be responsible for the disease in only two families: the first one is a previously reported family homozygous for Y63X mutation and the second one, described here, is homozygous for C40G. This accounts for 1.7% of Usher syndrome Spanish families. It is noticeable that, whereas C40G family is clinically compatible with Usher syndrome type III due to the progression of the hearing loss, Y63X family could be diagnosed as Usher syndrome type I because the hearing impairment is profound and stable. Thus, we consider that the progression of hearing loss is not the definitive key parameter to distinguish Usher syndrome type III from Usher syndrome type I and Usher syndrome type II.

The USH2A c.2299delG mutation: dating its common origin in a Southern European population

Usher syndrome type II is the most common form of Usher syndrome. USH2A is the main responsible gene of the three known to be disease causing. It encodes two isoforms of the protein usherin. This protein is part of an interactome that has an essential role in the development and function of inner ear hair cells and photoreceptors. The gene contains 72 exons spanning over a region of 800 kb. Although numerous mutations have been described, the c.2299delG mutation is the most prevalent in several populations. Its ancestral origin was previously suggested after the identification of a common core haplotype restricted to 250 kb in the 5′ region that encodes the short usherin isoform. By extending the haplotype analysis over the 800 kb region of the USH2A gene with a total of 14 intragenic single nucleotide polymorphisms, we have been able to define 10 different c.2299delG haplotypes, showing high variability but preserving the previously described core haplotype. An exhaustive c.2299delG/control haplotype study suggests that the major source of variability in the USH2A gene is recombination. Furthermore, we have evidenced twice the amount of recombination hotspots located in the 500 kb region that covers the 3′ end of the gene, explaining the higher variability observed in this region when compared with the 250 kb of the 5′ region. Our data confirm the common ancestral origin of the c.2299delG mutation.

Phosphodiesterase inhibition induces retinal degeneration, oxidative stress and inflammation in cone-enriched cultures of porcine retina

Inherited retinal degenerations affecting both rod and cone photoreceptors constitute one of the causes of incurable blindness in the developed world. Cyclic guanosine monophosphate (cGMP) is crucial in the phototransduction and, mutations in genes related to its metabolism are responsible for different retinal dystrophies. cGMP-degrading phosphodiesterase 6 (PDE6) mutations cause around 4-5% of the retinitis pigmentosa, a rare form of retinal degeneration. The aim of this study was to evaluate whether pharmacological PDE6 inhibition induced retinal degeneration in cone-enriched cultures of porcine retina similar to that found in murine models. PDE6 inhibition was induced in cone-enriched retinal explants from pigs by Zaprinast. PDE6 inhibition induced cGMP accumulation and triggered retinal degeneration, as determined by TUNEL assay. Western blot analysis and immunostaining indicated that degeneration was accompanied by caspase-3, calpain-2 activation and poly (ADP-ribose) accumulation. Oxidative stress markers, total antioxidant capacity, thiobarbituric acid reactive substances (TBARS) and nitric oxide measurements revealed the presence of oxidative damage. Elevated TNF-alpha and IL-6, as determined by enzyme immunoassay, were also found in cone-enriched retinal explants treated with Zaprinast. Our study suggests that this ex vivo model of retinal degeneration in porcine retina could be an alternative model for therapeutic research into the mechanisms of photoreceptor death in cone-related diseases, thus replacing or reducing animal experiments.

The contribution of GPR98 and DFNB31 genes to a Spanish Usher syndrome type 2 cohort

The Hartree-Fock method and the 6-31G** basis set were employed to calculate the molecular properties of artemisinin and 20 derivatives with antimalarial activity. Maps of molecular electrostatic potential (MEPs) and molecular docking were used to investigate the interaction between ligands and the receptor (heme). Principal component analysis and hierarchical cluster analysis were employed to select the most important descriptors related to activity. The correlation between biological activity and molecular properties was obtained using the partial least squares and principal component regression methods. The regression PLS and PCR models built in this study were also used to predict the antimalarial activity of 30 new artemisinin compounds with unknown activity. The models obtained showed not only statistical significance but also predictive ability. The significant molecular descriptors related to the compounds with antimalarial activity were the hydration energy (HE), the charge on the O11 oxygen atom (QO11), the torsion angle O1-O2-Fe-N2 (D2) and the maximum rate of R/Sanderson Electronegativity (RTe+). These variables led to a physical and structural explanation of the molecular properties that should be selected for when designing new ligands to be used as antimalarial agents.