Beatriz Buentello-Volante

Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements

Abstract Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling.

Gene Therapy for Retinitis Pigmentosa Caused by MFRP Mutations: Human Phenotype and Preliminary Proof of Concept

Autosomal recessive retinitis pigmentosa (RP), a heterogeneous group of degenerations of the retina, can be due to mutations in the MFRP (membrane-type frizzled-related protein) gene. A patient with RP with MFRP mutations, one of which is novel and the first splice site mutation reported, was characterized by noninvasive retinal and visual studies. The phenotype, albeit complex, suggested that this retinal degeneration may be a candidate for gene-based therapy. Proof-of-concept studies were performed in the rd6 Mfrp mutant mouse model. The fast-acting tyrosine-capsid mutant AAV8 (Y733F) vector containing the small chicken β-actin promoter driving the wild-type mouse Mfrp gene was used. Subretinal vector delivery on postnatal day 14 prevented retinal degeneration. Treatment rescued rod and cone photoreceptors, as assessed by electroretinography and retinal histology at 2 months of age. This AAV-mediated gene delivery also resulted in robust MFRP expression predominantly in its normal location within the retinal pigment epithelium apical membrane and its microvilli. The clinical features of MFRP-RP and our preliminary data indicating a response to gene therapy in the rd6 mouse suggest that this form of RP is a potential target for gene-based therapy.

Homozygosity Mapping Identifies the Crumbs Homologue 1 (Crb1) Gene as Responsible for a Recessive Syndrome of Retinitis Pigmentosa and Nanophthalmos

The association of retinitis pigmentosa (RP) and microphthalmia has been reported in a number of familial and isolated cases. Here, the results of genetic analysis in a familial case of early RP associated with nanophthalmos are described. Two affected sibs were ascertained from an endogamous population in Mexico. A genome‐wide linkage analysis was performed by means of an Affymetrix 250K microarray. Five large regions of homozygosity were demonstrated. The largest interval comprised 15.08 Mb at chromosome 1q31–32.1 and contained the Crumbs homologue‐1, CRB1, a gene responsible for a number of recessive retinal dystrophies. Nucleotide sequence analysis demonstrated a c.1125C>G transversion in CRB1 exon 5, predicting a novel p.Tyr375X variant. To our knowledge this is the first instance in which a CRB1 mutation has been associated with early RP and nanophthalmos. Our results suggest a role for CRB1 in promoting axial growth of the eye. Clinical analysis of additional subjects with retinal dystrophies due to CRB1 mutations will help to identify if the high hyperopia, a frequently observed trait in these subjects, could be related to decreased eye axial length (nanophthalmos).

Erratum to: Next generation sequencing uncovers a missense mutation in COL4A1 as the cause of familial retinal arteriolar tortuosity

Objectives Our aim was to determine the molecular cause of autosomal dominant familial retinal arteriolar tortuosity (FRAT) in a family with three affected subjects.

Mutational screening of FOXE3, GDF3, ATOH7, and ALDH1A3 in congenital ocular malformations. Possible contribution of the FOXE3 p.VAL201MET variant to the risk of severe eye malformations.

Vertebrate eye development is a highly organized process requiring the participation of a number of genes that act coordinately during embryogenesis to produce functional visual structures. Disturbances in this genetic program can produce different forms of ocular malformations with a range of severity that depends on the stage at which development is altered. Congenital eye malformations are heterogeneous and include the microphthalmia-anophthalmia-coloboma (MAC) spectrum and other defects such as congenital cataract and anterior segment dysgenesis, which frequently results in serious visual disability. In recent years it has been recognized that molecular defects in genes such as SOX2, RAX, OTX2, CHX10, and GDF6, among others, are responsible for a highly variable spectrum of congenital eye anomalies, including MAC. Considerable intraand interfamilial phenotypic heterogeneity is frequently associated with mutations in these genes. Four relative newcomers to this group of MAC-related genes are FOXE3, GDF3, ATOH7, and ALDH1A3. These genes play crucial roles during human eye development and their mutations are associated with a wide phenotypic spectrum. In this work, we performed a comprehensive screening of the MAC-causing genes FOXE3, GDF3, ATOH7, and ALDH1A3 in a group of unrelated Mexican subjects suffering from bilateral congenital MAC spectrum and related eye malformations. The study was approved by the Institutional Ethics Committee and patients or their parents gave written consent for molecular analyses. Seventy-six Mexican-Mestizo subjects (68 sporadic and 8 familial cases) with different eye abnormalities (Table 1) were studied. All of them were previously screened and tested negative for mutations in the MAC-associated genes CHX10, GDF6, OTX2, RAX, and SOX2. Due to DNA availability, FOXE3 and GDF3 molecular screening was performed only in a subset of 52 subjects from the initial group of 76 participants. Molecular studies included PCR amplification and direct nucleotide sequencing. Analysis of the complete coding sequence of ATOH7, ALDH1A3, and GDF3 genes in DNA from affected subjects did not identify any deleterious mutation. The novel heterozygous c.213C4A and c.258G4A substitutions in ATOH7, resulting in the p.Gly71Gly and p.Leu86Leu synonymous variants, respectively, were identified in DNA from several patients. A homozygous c.292T4C (p.Y98H) FOXE3 deleterious mutation was identified in a single patient suffering from bilateral microphthalmia and sclerocornea. The previously described c.601G4A (p.Val201Met) substitution in FOXE3 (Figure 1) was

Genome-wide mRNA analysis reveals a TUBD1 isoform profile as a potential biomarker for diabetic retinopathy development

Abstract Diabetic retinopathy (DR) affects approximately one third of all diabetic subjects and is the leading cause of blindness in young to middle‐aged adults in the developed world. While early diagnosis is crucial for preventing DR‐associated visual loss, the identification of accessible biomarkers that could lead to presymptomatic recognition of the disease is of great clinical importance. The aim of this work was to investigate the possible involvement of alternative splicing events in DR development by performing a genome‐wide transcriptional profiling comparing blood‐derived RNA from DR subjects and from diabetic‐non DR controls. A total of 95 RNA samples, 67 from patients with bilateral DR and 28 from diabetic patients without DR after a period of at least 10 years with type 2 DM, were compared in a genome‐wide transcriptome analysis using the GeneChip® Human Gene 2.0 ST Array which contains probe sets covering all exons of ˜33,500 coding transcripts of annotated genes. Microarray data analysis followed by RT‐PCR and cDNA sequencing identified important differential splicing events in TUBD1 (Tubulin, Delta‐1) isoforms between DR and DM samples. Specifically, the co‐expression of particular TUBD1 isoforms was significantly associated with NPDR risk (p = 0.039 by Pearsons chi‐squared test; OR (CI 95%): 8.1 (1.0–72.7)). Analysis of TUBD1 signal pathways and regulating networks using a MetaCore platform showed that HIF‐1, a molecule playing an important role in the pathogenesis of DR, is a direct regulator of TUBD1 expression. In conjunction, our data suggest that TUBD1 mRNA isoform expression profile in peripheral blood could be an accessible biomarker for predicting the risk for diabetic retinopathy development. HighlightsInvolvement of alternative splicing in diabetic retinopathy (DR) was investigated.Blood RNA samples from DR (n = 67) and from non‐DR patients (n = 28) were compared.GeneChip Human Gene 2.0 ST Arrays were used for genome‐wide transcriptome analysis.Co‐expression of particular TUBD1 isoforms were associated with DR occurrence risk.TUBD1 isoforms profile could be an accessible biomarker for predicting DR development.

An intellectually disabled patient with the 5q14.3q15 microdeletion syndrome associated with an apparently de novo t(2;5)(q13;q14)

An Intellectually Disabled PatientWith the 5q14.3q15 Microdeletion Syndrome Associated With an Apparently De Novo t(2;5)(q13;q14) Jaime Toral-L opez, Beatriz Buentello-Volante, Marta M. Balderas-Minor, Carmen Amezcua-Herrera, Juan M. Valdes-Miranda, Luz Ma. Gonz alez-Huerta, Marco Gudi~no, Sergio A. Cuevas-Covarrubias, and Juan Carlos Zenteno* Department of Genetics, Centro M edico Ecatepec, ISSEMYM, Mexico, Mexico Department of Genetics-Research Unit, Institute of Ophthalmology ‘‘Conde de Valenciana’’ Mexico City, Mexico Department of Neuropediatrics, Centro M edico Ecatepec, ISSEMYM, Mexico, Mexico Nuclear Magnetic Resonance service, Hospital General de M exico, Mexico City, Mexico Department of Genetics, Hospital General de M exico, Faculty of Medicine, UNAM, Mexico City, Mexico Department of Experimental Medicine, UNAM, Mexico City, Mexico Department of Biochemistry, Faculty of Medicine, National Autonomous University of Mexico (UNAM), Mexico City, Mexico

Acro-spondylo-pubic dysostosis associated with cataracts, microcephaly, and normal intelligence

We report on an adult male with normal intelligence who exhibited an unusual combination of microcephaly, dysostoses of limbs, vertebrae, patellae, and pubic bone, camptodactyly of all fingers, and syndactyly of toes, absent nails on thumbs and some fingers, bilateral cataract, cryptorchidism, polythelia, and nipple‐like skin pigmentations of shoulders and upper back. We have been unable to find a description of a similar combination of manifestations in literature. The cause of the anomalies remains unknown.

A rapidly progressive defective spermatogenesis in a Mexican family affected by spino-bulbar muscular atrophy

ABSTRACT Spino-bulbar muscular atrophy (SBMA) is an X-linked recessive adult progressive disorder affecting motor neurons. It is caused by a poly-glutamine tract expansion in the androgen receptor (AR) which generates protein aggregates that cannot be processed by proteasomes. A secondary mild androgen resistance is developed by AR dysfunction and patients present endocrine abnormalities including gynecomastia and poor function of testosterone in tissues; however, normally they are fertile. In this report we describe a Mexican family with three affected brothers with primary infertility caused by a progressive impairment of spermatogenesis leading to azoospermia before 40 years of age. They presented common features associated to patients affected by SMBA, such as gynecomastia, high level of CPK, muscle cramps, fasciculations, muscle wastage, and impaired swallowing. Two intracytoplasmic sperm injection (ICSI) cycles were performed in one of the patients resulting in fertilization failure. Molecular analysis of AR gene exon 1 revealed 54 CAG repeats in DNA extracted from leukocytes in affected patients and 22 repeats in the fertile non-affected brother. Severe impaired spermatogenesis of rapid progression has not been associated before to SBMA. This is the first report of assisted reproduction techniques indicated by male infertility in patients with this rare disorder. Further studies are required to confirm the unusual result of intracytoplasmic sperm injection cycles. We discuss the implications and possible pathogenesis of these unique features of SBMA in this family.

Familial Gelatinous Drop-Like Corneal Dystrophy Caused by a Novel Nonsense TACSTD2 Mutation.

Purpose: To describe the clinical findings and results of molecular analysis in a Mexican family diagnosed with gelatinous drop-like corneal dystrophy (GDLD). Methods: Ophthalmological examination was performed in 1 unaffected and 4 affected relatives. Molecular analysis included polymerase chain reaction amplification and direct nucleotide sequencing of the entire TACSTD2 gene-coding region in genomic DNA. Results: The corneal phenotype in adult patients was characterized by white-yellow nodular lesions on the corneal surface and stromal neovascularization. Lesions were incipient in an affected relative aged 14 years. Nucleotide sequencing of the TACSTD2 gene demonstrated a homozygous c.331G>T transversion, which predicts a novel p.(E111*) nonsense mutation, in DNA of all 4 GDLD relatives. Conclusions: Our results expand the mutational spectrum of TACSTD2 in patients with GDLD. To the best of our knowledge, this is the first clinical and molecular description of patients with GDLD from Latin America.