Homozygous in-frame deletion in CATSPERE in a man producing spermatozoa with loss of CatSper function and compromised fertilizing capacity.

STUDY QUESTION: Does a man (patient 1) with a previously described deficiency in principle cation channel of sperm (CatSper) function have a mutation in the CatSper-epsilon (CATSPERE) and/or CatSper-zeta (CATSPERZ) gene? SUMMARY ANSWER: Patient 1 has a homozygous in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE. WHAT IS KNOWN ALREADY: CatSper is the principal calcium channel of mammalian spermatozoa. Spermatozoa from patient 1 had a specific loss of CatSper function and were unable to fertilize at IVF. Loss of CatSper function could not be attributed to genetic abnormalities in coding regions of seven CatSper subunits. Two additional subunits (CatSper-epsilon (CATPSERE) and CatSper-zeta (CATSPERZ)) were recently identified, and are now proposed to contribute to the formation of the mature channel complex. STUDY DESIGN, SIZE, DURATION: This was a basic medical research study analysing genomic data from a single patient (patient 1) for defects in CATSPERE and CATSPERZ. PARTICIPANTS/MATERIALS, SETTING, METHODS: The original exome sequencing data for patient 1 were analysed for mutations in CATSPERE and CATSPERZ. Sanger sequencing was conducted to confirm the presence of a rare variant. MAIN RESULTS AND THE ROLE OF CHANCE: Patient 1 is homozygous for an in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE that is predicted to be highly deleterious. LIMITATIONS, REASONS FOR CAUTION: The nature of the molecular deficit caused by the rs761237686 variant and whether it is exclusively responsible for the loss of CatSper function remain to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS: Population genetics are available for a significant number of predicted deleterious variants of CatSper subunits. The consequence of homozygous and compound heterozygous forms on sperm fertilization potential could be significant. Selective targeting of CatSper subunit expression maybe a feasible strategy for the development of novel contraceptives. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by project grants from the MRC (MR/K013343/1 and MR/012492/1), Chief Scientist Office/NHS research Scotland. This work was also supported by NIH R01GM111802, Pew Biomedical Scholars Award 00028642 and Packer Wentz Endowment Will to P.V.L. C.L.R.B is the editor-in-chief of Molecular Human Reproduction, has received lecturing fees from Merck and Ferring, and is on the Scientific Advisory Panel for Ohana BioSciences. C.L.R.B was chair of the World Health Organization Expert Synthesis Group on Diagnosis of Male infertility (2012-2016).

Mutation in the guanine nucleotide-binding protein beta-3 causes retinal degeneration and embryonic mortality in chickens.

PURPOSE: To identify the gene defect that causes blindness and the predisposition to embryonic death in the retinopathy globe enlarged (rge) chicken. METHODS: Linkage analysis, with previously uncharacterized microsatellite markers from chicken chromosome 1, was performed on 138 progeny of an rge/+ and an rge/rge cross, and candidate genes were sequenced. RESULTS: The rge locus was refined and the gene for guanine nucleotide-binding protein beta-3 (GNB3), which encodes a cone transducin beta subunit, was found to have a 3-bp deletion (D153del) that segregated with the rge phenotype. This mutation deleted a highly conserved aspartic acid residue in the third of seven WD domains in GNB3. In silico modeling suggested that this mutation destabilized the protein. Furthermore, a 70% reduction was found in immunoreactivity to anti-GNB3 in the rge-affected retina. CONCLUSIONS: These findings implicate the beta-subunit of cone transducin as the defective protein underlying the rge phenotype. Furthermore, GNB3 is ubiquitously expressed, and the c.825C-->T GNB3 splicing variant (MIM 139130) has been associated with hypertension, obesity, diabetes, low birth weight, coronary heart disease, and stroke in the human population. It therefore seems likely that the defect underlying these human diseases also causes reduced embryonic viability in the rge chicken, making it a powerful model for studying the pathology involved in these associations.

Correction of the Pathogenic Alternative Splicing, Caused by the Common GNB3 c.825C>T Allele, Using a Novel, Antisense Morpholino.

The very common GNB3 c.825C>T polymorphism (rs5443) is present in approximately half of all human chromosomes. Significantly, the presence of the GNB3 825T allele has been strongly associated with predisposition to essential hypertension. Paradoxically the presence of the GNB3 825T allele, in exon 10, introduces a pathogenic alternative RNA splice site into the middle of exon 9. To attempt to correct this pathogenic aberrant splicing, we, therefore, bioinformatically designed, using a Gene Tools(®) algorithm, a GNB3-specific, antisense morpholino. It was hoped that this morpholino would behave in vitro as either a potential splice blocker and/or exon skipper, to both bind and inhibit/reduce the aberrant splicing of the GNB3 825T allele. On transfecting a human lymphoblast cell line homozygous for the 825T allele, with this antisense morpholino, we encouragingly observed both a significant reduction (from ∼58% to ∼5%) in the production of the aberrant smaller GNB3 transcript, and a subsequent increase in the normal GNB3 transcript (from ∼42% to ∼95%). Our results demonstrate the potential use of a GNB3-specific antisense morpholino, as a pharmacogenetic therapy for essential hypertension.

Analysis of the rdd locus in chicken: a model for human retinitis pigmentosa.

PURPOSE: To identify the locus responsible for the blind mutation rdd (retinal dysplasia and degeneration) in chickens and to further characterise the rdd phenotype. METHODS: The eyes of blind and sighted birds were subjected to ophthalmic, morphometric and histopathological examination to confirm and extend published observations. Electroretinography was used to determine age of onset. Birds were crossed to create pedigrees suitable for genetic mapping. DNA samples were obtained and subjected to a linkage search. RESULTS: Measurement of IOP, axial length, corneal diameter, and eye weight revealed no gross morphological changes in the rdd eye. However, on ophthalmic examination, rdd homozygotes have a sluggish pupillary response, atrophic pecten, and widespread pigmentary disturbance that becomes more pronounced with age. Older birds also have posterior subcapsular cataracts. At three weeks of age, homozygotes have a flat ERG indicating severe loss of visual function. Pathological examination shows thinning of the RPE, ONL, photoreceptors and INL, and attenuation of the ganglion cell layer. From 77 classified backcross progeny, 39 birds were blind and 38 sighted. The rdd mutation was shown to be sex-linked and not autosomal as previously described. Linkage analysis mapped the rdd locus to a small region of the chicken Z chromosome with homologies to human chromosomes 5q and 9p. CONCLUSIONS: Ophthalmic, histopathologic, and electrophysiological observations suggest rdd is similar to human recessive retinitis pigmentosa. Linkage mapping places rdd in a region homologous to human chromosomes 9p and 5q. Candidate disease genes or loci include PDE6A, WGN1, and USH2C. This is the first use of genetic mapping in a chicken model of human disease.

Genetic, ophthalmic, morphometric and histopathological analysis of the Retinopathy Globe Enlarged (rge) chicken.

PURPOSE: To identify the locus responsible for rge (retinopathy globe enlarged) in chickens and further characterise the rge phenotype. METHODS: A colony of chickens carrying the rge mutation was rederived from a single heterozygous animal of the original line. The eyes of blind, heterozygous and normal birds were subjected to ophthalmic, morphometric and histopathological examination to confirm and extend published observations. DNA samples were obtained and subjected to a whole genome linkage search. RESULTS: From 138 classified backcross progeny, 56 birds were blind and 82 sighted. Heterozygous birds were indistinguishable from wild type, but homozygotes had sluggish or unresponsive pupils, posterior sub-capsular lens opacities and an atrophic pecten. The fundus appeared normal with no significant pigmentary disturbance, but axial length and eye weight were increased. Pathology revealed focal retinal lesions. Linkage analysis placed the rge locus in a small region of chicken chromosome 1. CONCLUSIONS: rge is a severe recessive retinal dystrophy in chickens, with associated globe enlargement. Linkage mapping has highlighted chicken chromosome 1 in a region most probably homologous to human chromosomes 7q31-35, 21q21 or 22q12-21. Candidate disease loci include RP10 (IMPDH1) and uncharacterised Ushers (USH1E) and glaucoma (GLC1F) loci.

Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display.

Human airway epithelial cell targeting peptides were identified by biopanning on 1HAEo-cells, a well characterised epithelial cell line. Bound phage were recovered after three rounds of binding, high stringency washing and elution, leading to the production of an enriched phage peptide population. DNA sequencing of 56 clones revealed 14 unique sequences. Subsequent binding analysis revealed that 13 of these peptides bound 1HAEo-cells with high affinity. Three peptides, SERSMNF, YGLPHKF and PSGAARA were represented at high frequency. Three clearly defined families of peptide were identified on the basis of sequence motifs including (R/K)SM, L(P/Q)HK and PSG(A/T)ARA. Two peptides, LPHKSMP and LQHKSMP contained two motifs. Further detailed sequence analysis by comparison of peptide sequences with the SWISSPROT protein database revealed that some of the peptides closely resembled the cell binding proteins of viral and bacterial pathogens including Herpes Simplex Virus, rotavirus, Mycoplasma pneumoniae and rhinovirus, the latter two being respiratory pathogens, as well as peptide YGLPHKF having similarity to a protein of unknown function from the respiratory pathogen Legionella pneumophila. Peptides were incorporated into gene delivery formulations with the cationic lipid Lipofectin and plasmid DNA and shown to confer a high degree of transfection efficiency and specificity in 1HAEo-cells. Improved transfection efficiency and specificity was also observed in human endothelial cells, fibroblasts and keratinocytes. Therefore, on the basis of clone frequency after biopanning, cell binding affinity, peptide sequence conservation and pathogenic similarity, we have identified 3 novel peptide families and 5 specific peptides that have the potential for gene transfer to respiratory epithelium in vivo as well as providing useful in vitro transfection reagents for primary human cell types of scientific and commercial interest.

The D153del mutation in GNB3 gene causes tissue specific signalling patterns and an abnormal renal morphology in Rge chickens.

BACKGROUND: The GNB3 gene is expressed in cone but not rod photoreceptors of vertebrates, where it acts as the ß transducin subunit in the colour visual transduction process. A naturally occurring mutation 'D153del' in the GNB3 gene causes the recessively inherited blinding phenotype retinopathy globe enlarged (rge) disease in chickens. GNB3 is however also expressed in most other vertebrate tissues suggesting that the D153del mutation may exert pathological effects that outlie from eye. PRINCIPAL FINDINGS: Recombinant studies in COS-7 cells that were transfected with normal and mutant recombinant GNB3 constructs and subjected to cycloheximide chase showed that the mutant GNB3d protein had a much shorter half life compared to normal GNB3. GNB3 codes for the Gß3 protein subunit that, together with different Gγ and Gα subunits, activates and regulates phosphorylation cascades in different tissues. As expected, the relative levels of cGMP and cAMP secondary messengers and their activated kinases such as MAPK, AKT and GRK2 were also found to be altered significantly in a tissue specific manner in rge chickens. Histochemical analysis on kidney tissue sections, from rge homozygous affected chickens, showed the chickens had enlargement of the glomerular capsule, causing glomerulomegaly and tubulointerstitial inflammation whereas other tissues (brain, heart, liver, pancreas) were unaffected. SIGNIFICANCE: These findings confirm that the D153del mutation in GNB3 gene targets GNB3 protein to early degradation. Lack of GNB3 signalling causes reduced phosphorylation activity of ERK2 and AKT leading to severe pathological phenotypes such as blindness and renal abnormalities in rge chickens.

Mpdz null allele in an avian model of retinal degeneration and mutations in human leber congenital amaurosis and retinitis pigmentosa.

PURPOSE: To identify the defective gene in the sex-linked, recessively inherited retinal dysplasia and degeneration (rdd) chicken and to search for the human equivalent disease. METHODS: Microsatellites from chicken chromosome Z were genotyped in 77 progeny of a carrier male (rdd/+) and an affected female (rdd/W), and candidate genes were sequenced. Retinal cross-sections from rdd and wild-type birds were analyzed by immunohistology. The human orthologous gene was screened in a panel of archival DNAs from 276 patients with retinitis pigmentosa (RP) or Leber congenital amaurosis (LCA) using melting curve analysis and DNA sequencing. RESULTS: The rdd locus was refined to an approximately 3-Mb region on chromosome Z. Sequence analysis identified a C→T change in the mpdz gene that created a premature stop codon (c.1372C→T, p.R458X), which segregated with the disease phenotype. As expected, the full-length mpdz protein was absent in rdd retinas, but in wild-type birds, it localized to the retinal outer limiting membrane, where it may have a role in the interactions between photoreceptors and Müller glia cells. The screen to identify the human equivalent disease found 10 heterozygous variants in the orthologous gene in patients with RP (three missense and two null alleles) and LCA (four missense and one null allele). CONCLUSIONS: These findings reveal that MPDZ is essential for normal development of the retina and may have a role in maintaining photoreceptor integrity. The identification of human mutations suggests that MPDZ plays a role in human retinal disease, but the precise nature of this role remains to be determined.