José M. Millán

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.

USH3A transcripts encode clarin-1, a four-transmembrane-domain protein with a possible role in sensory synapses

Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterised by the association of post-lingual progressive hearing loss, progressive visual loss due to retinitis pigmentosa and variable presence of vestibular dysfunction. Because the previously defined transcripts do not account for all USH3 cases, we performed further analysis and revealed the presence of additional exons embedded in longer human and mouse USH3A transcripts and three novel USH3A mutations. Expression of Ush3a transcripts was localised by whole mount in situ hybridisation to cochlear hair cells and spiral ganglion cells. The full length USH3A transcript encodes clarin-1, a four-transmembrane-domain protein, which defines a novel vertebrate-specific family of three paralogues. Limited sequence homology to stargazin, a cerebellar synapse four-transmembrane-domain protein, suggests a role for clarin-1 in hair cell and photoreceptor cell synapses, as well as a common pathophysiological pathway for different Usher syndromes.

Mutational analysis of the MPZ, PMP22 and Cx32 genes in patients of Spanish ancestry with Charcot-Marie-Tooth disease and hereditary neuropathy with liability to pressure palsies

Abstract Charcot-Marie-Tooth disease (CMT) and hereditary neuropathy with liability to pressure palsies (HNPP) are two inherited peripheral neuropathies. The most prevalent mutations are a reciprocal 1.5-Mb duplication and 1.5-Mb deletion, respectively, at the CMT1A/HNPP locus on chromosome 17p11.2. Point mutations in the coding region of the myelin genes, peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ) or connexin 32 (Cx32) have been reported in CMT patients, including CMT type 1 (CMT1), CMT type 2 (CMT2) and Déjérine-Sottas neuropathy (DS) patients, and only in the coding region of PMP22 in HNPP families lacking a deletion. We have investigated point and small mutations in the MPZ, PMP22 and Cx32 genes in a series of patients of Spanish ancestry: 47 CMT patients without duplications, and 5 HNPP patients without deletions. We found 15 different mutations in 16 CMT patients (34%). Nine different mutations in ten patients were detected in the Cx32 gene, this being the most frequently involved gene in this series, whereas five mutations involved the MPZ gene and only one the PMP22 gene. Six out of nine nucleotide substitutions in the Cx32 gene involved two codons encoding arginine at positions 164 and 183, suggesting that these two codons may constitute two Cx32 regions prone to mutate in the Spanish population. Analysis of HNPP patients revealed a 5′ splicing mutation in intron 1 of the PMP22 gene in a family with autosomal dominance, which confirms allelic heterogeneity in HNPP. Ectopic mRNA analysis on leukocytes suggests that this mutation might behave as a null allele.

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.

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.

Informed consent for whole-genome sequencing studies in the clinical setting. Proposed recommendations on essential content and process

The development of new massive sequencing techniques has now made it possible to significantly reduce the time and costs of whole-genome sequencing (WGS). Although WGS will soon become a routine testing tool, new ethical issues have surfaced. In light of these concerns, a systematic review of papers published by expert authors on IC or specific ethical issues related to IC for WGS analysis in the clinical setting has been conducted using the Pubmed, Embase and Cochrane Library databases. Additionally, a search was conducted for international ethical guidelines for genetic studies published by scientific societies and ethical boards. Based on these documents, a minimum set of information to be provided to patients in the IC form was determined. Fourteen and seven documents from the database search and from scientific societies, respectively, were selected. A very high level of consistency between them was found regarding the recommended IC form content. Pre-test counselling and general information common to all genetic tests should be included in the IC form for WGS for diagnostic purposes, but additional information addressing specific issues on WGS are proposed, such as a plan for the ethical, clinically oriented return of incidental findings. Moreover, storage of additional information for future use should also be agreed upon with the patient in advance. Recommendations for WGS studies in the clinical setting concerning both the elements of information and the process of obtaining the IC as well as how to handle the results obtained are proposed.

Charcot-Marie-Tooth disease Genetic and clinical spectrum in a Spanish clinical series

Objectives: To determine the genetic distribution and the phenotypic correlation of an extensive series of patients with Charcot-Marie-Tooth disease in a geographically well-defined Mediterranean area. Methods: A thorough genetic screening, including most of the known genes involved in this disease, was performed and analyzed in this longitudinal descriptive study. Clinical data were analyzed and compared among the genetic subgroups. Results: Molecular diagnosis was accomplished in 365 of 438 patients (83.3%), with a higher success rate in demyelinating forms of the disease. The CMT1A duplication (PMP22 gene) was the most frequent genetic diagnosis (50.4%), followed by mutations in the GJB1 gene (15.3%), and in the GDAP1 gene (11.5%). Mutations in 13 other genes were identified, but were much less frequent. Sixteen novel mutations were detected and characterized phenotypically. Conclusions: The relatively high frequency of GDAP1 mutations, coupled with the scarceness of MFN2 mutations (1.1%) and the high proportion of recessive inheritance (11.6%) in this series exemplify the particularity of the genetic distribution of Charcot-Marie-Tooth disease in this region.

Retinitis pigmentosa and allied conditions today: a paradigm of translational research

Monogenic human retinal dystrophies are a group of disorders characterized by progressive loss of photoreceptor cells leading to visual handicap. Retinitis pigmentosa is a type of retinal dystrophy where degeneration of rod photoreceptors occurs at the early stages. At present, there are no available effective therapies to maintain or improve vision in patients affected with retinitis pigmentosa, but post-genomic studies are allowing the development of potential therapeutic approaches. This review summarizes current knowledge on genes that have been identified to be responsible for retinitis pigmentosa, the involvement of these genes in the different forms of the disorder, the role of the proteins encoded by these genes in retinal function, the utility of genotyping, and current efforts to develop novel therapies.

Mutation of the XNP/ATR-X gene in a family with severe mental retardation, spastic paraplegia and skewed pattern of X inactivation: demonstration that the mutation is involved in the inactivation bias.

We would like to thank the family members for their cooperation and Dr. Charles Schwartz for helpful discussion. This work was supported by the INSERM program PARMIFR and by the PROGRES network, as well as by Spanish Ministry of Health project FIS98/0170 (Fondo de Investigaciones Sanitarias).

BBS genotype–phenotype assessment of a multiethnic patient cohort calls for a revision of the disease definition†

Bardet‐Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, polydactyly, renal abnormalities, and cognitive impairment for which 15 causative genes have been identified. Here we present the results of a mutational analysis of our multiethnic cohort of 83 families (105 cases); 75.9% of them have their mutations identified including 26 novel changes. Comprehensive phenotyping of these patients demonstrate that the spectrum of clinical features is greater than expected and overlapped with the features of other ciliopathies; specifically Alström and McKusick‐Kauffman syndromes. Hum Mutat 32:1–10, 2011.