A new locus for congenital cataract, microcornea, microphthalmia, and atypical iris coloboma maps to chromosome 2.

OBJECTIVE: To report a novel phenotype of autosomal dominant atypical congenital cataract associated with variable expression of microcornea, microphthalmia, and iris coloboma linked to chromosome 2. Molecular analysis of this phenotype may improve our understanding of anterior segment development. DESIGN: Observational case study, genome linkage analysis, and gene mutation screening. PARTICIPANTS: Three families, 1 Egyptian and 2 Belgians, with a total of 31 affected were studied. METHODS: Twenty-one affected subjects and 9 first-degree relatives underwent complete ophthalmic examination. In the Egyptian family, exclusion of PAX6, CRYAA, and MAF genes was demonstrated by haplotype analysis using microsatellite markers on chromosomes 11, 16, and 21. Genome-wide linkage analysis was then performed using 385 microsatellite markers on this family. In the 2 Belgian families, the PAX6 gene was screened for mutations by direct sequencing of all exons. MAIN OUTCOME MEASURES: Phenotype description, genome-wide linkage of the phenotype, linkage to the PAX6, CRYAA, and MAF genes, and mutation detection in the PAX6 gene. RESULTS: Affected members of the 3 families had bilateral congenital cataracts inherited in an autosomal dominant pattern. A novel form of hexagonal nuclear cataract with cortical riders was expressed. Among affected subjects with available data, 95% had microcornea, 39% had microphthalmia, and 38% had iris coloboma. Seventy-five percent of the colobomata were atypical, showing a nasal superior location in 56%. A positive lod score of 4.86 was obtained at theta = 0 for D2S2309 on chromosome 2, a 4.9-Mb common haplotype flanked by D2S2309 and D2S2358 was obtained in the Egyptian family, and linkage to the PAX6, CRYAA, or MAF gene was excluded. In the 2 Belgian families, sequencing of the junctions and all coding exons of PAX6 did not reveal any molecular change. CONCLUSIONS: We describe a novel phenotype that includes the combination of a novel form of congenital hexagonal cataract, with variably expressed microcornea, microphthalmia, and atypical iris coloboma, not caused by PAX6 and mapping to chromosome 2. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

Selection processes in a citrus hybrid population using RAPD markers

The objective of this work was to evaluate the processes of selection in a citrus hybrid population using segregation analysis of RAPD markers. The segregation of 123 RAPD markers between 'Cravo' mandarin (Citrus reticulata Blanco) and 'Pêra' sweet orange (C. sinensis (L.) Osbeck) was analysed in a F1 progeny of 94 hybrids. Genetic composition, diversity, heterozygosity, differences in chromosomal structure and the presence of deleterious recessive genes are discussed based on the segregation ratios obtained. A high percentage of markers had a skeweness of the 1:1 expected segregation ratio in the F1 population. Many markers showed a 3:1 segregation ratio in both varieties and 1:3 in 'Pêra' sweet orange, probably due to directional selection processes. The distribution analysis of the frequencies of the segregant markers in a hybrid population is a simple method which allows a better understanding of the genetics of citrus group.

A pathogenic mechanism in Huntington"s disease involves small CAG-repeated RNAs with neurotoxic activity

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches

Identifying the parent-of-origin of de novo SNVs in schizophrenia

Several studies over the last few years have shown that newly arising (de novo) mutations contribute to the genetics of schizophrenia (SZ), autism (ASD) and other developmental disorders. The strongest evidence comes from studies of de novo Copy Number Variation (CNV), where the rate of new mutations is shown to be increased in cases when compared to controls [23, 24]. Research on de novo point mutations and small insertion-deletions (indels) has been more limited, but with the development of next-generation sequencing (NGS) technology, such studies are beginning to provide preliminary evidence that de novo single-nucleotide mutations (SNVs) might also increase risk of SZ and ASD [25, 26] Advanced paternal age is a major source of new mutations in human beings [27] and could thus be associated with increased risk for developing SZ, ASD or other developmental disorders. Indeed, advanced paternal age is found to be a risk factor for developing SZ and ASD in the offspring [28, 29] and new mutations related to advanced paternal age have been implicated as a cause of sporadic cases in several autosomal dominant diseases, some neurodevelopmental diseases, including SZ and ASD, and social functioning. New single-base substitutions occur at higher rates at males compared to females and this difference increases with paternal age. This is due to the fact that sperm cells go through a much higher number of cell divisions (~840 by the age of 50), which increases the risk for DNA copy errors in the male germ line [30] . By contrast, the female eggs (oocytes) undergo only 24 cell divisions and all but the last occur during foetal life. The aim of my project is to determine the parent-of-origin of de novo SNVs, using large samples of parent-offspring trios affected with schizophrenia (SZ). From whole exome sequencing of 618 Bulgarian proband-offspring trios affected, nearly 1000 de novo (SNVs or small indels) have been identified and from these, the parent-of-origin of at least 60% of the mutations (N=600) can be established. This project is contained in a main one that consists on the determination of the parental origin of different types of de novo mutations (SNVs, small indels and large CNVs).

Characterization of OsMIK in a rice mutant with reduced phytate content reveals an insertion of a rearranged retrotransposon.

The rice low phytic acid (lpa) mutant Os-lpa-XS110-1(XS-lpa) has ~45 % reduction in seed phytic acid (PA) compared with the wild-type cultivar Xiushui 110. Previously, a single recessive gene mutation was shown to be responsible for the lpa phenotype and was mapped to a region of chromosome 3 near OsMIK (LOC_Os03g52760) and OsIPK1 (LOC_Os03g51610), two genes involved in PA biosynthesis. Here, we report the identification of a large insert in the intron of OsMIK in the XS-lpa mutant. Sequencing of fragments amplified through TAIL-PCRs revealed that the insert was a derivative of the LINE retrotransposon gene LOC_Os03g56910. Further analyses revealed the following characteristics of the insert and its impacts: (1) the inserted sequence of LOC_Os03g56910 was split at its third exon and rejoined inversely, with its 5' and 3' flanking sequences inward and the split third exon segments outward; (2) the LOC_Os03g56910 remained in its original locus in XS-lpa, and the insertion probably resulted from homologous recombination repair of a DNA double strand break; (3) while the OsMIK transcripts of XS-lpa and Xiushui 110 were identical, substantial reductions of the transcript abundance (~87 %) and the protein level (~60 %) were observed in XS-lpa, probably due to increased methylation in its promoter region. The above findings are discussed in the context of plant mutagenesis, epigenetics and lpa breeding.

Are myotonic dystrophy and peripheral neuropathy associated? : morphological, morphometric and electrophysiological analysis in DM1 transgenic mice

Abstract: Myotonic dystrophy (DM1), also known as Steinert disease, is an inherited autosomal dominant disease. It is characterized by myotonia, muscular weakness and atrophy, but DM1 may have manifestations in other organs such as eyes, heart, gonads, gastrointestinal and respiratory tracts, as well as brain. In 1992, it was demonstrated that this complex disease results from the expansion of CTG repeats in the 3' untranslated region of the DM protein kinase (DMPK) gene on chromosome 19. The size of the inherited expansion is critically linked to the severity of the disease and the age of onset. Although several electrophysiological and histological studies have been carried out to verify the possible involvement of peripheral nerve abnormality with DM1, the results have not been univocal. Therefore, at present the possible association between peripheral neuropatliy and DM1 remains debated. Recently, transgenic mice have been generated, that carry the human genomic DM1 region with 300 CTG repeats, and display the human DMl phenotype. The generation of these DM1 transgenic mice provides a useful tool to investigate the type and incidence of structural abnormalities in the peripheral nervous system associated with DM1 disease. By using the DM1 transgenic mice, we investigated the presence/absence of the three major peripheral neuropathies: axonal degeneration, axonal demyelination and neuronopathy. The morphological and morphometric analysis of sciatic, sural and phrenic nerves demonstrated the absence of axonal degeneration or demyelination. The morphometric analysis also ruled out any loss in the numbers of sensory or motor neurons in lumbar dorsal root ganglia and lumbar spinal cord enlargement respectively. Moreover, the éxamination of serial hind limb muscle sections from DMl mice showed a normal intramuscular axonal arborization as well as the absence of changes in the number and structure of endplates. Finally, the electrophysiological tests performed in DM1 transgenic mice showed that the compound muscle axon potentials (CMAPs) elicited in the hind limb digits in response to a stimulation of the sciatic nerve with anear-nerve electrode were similar to thosé obtained in wild type mice. On the basis of all our results, we hypothesized that 300 CTG repeats are not sufficient to induce disorder in the peripheral nervous system of this DM1 transgenic mouse model. Résumé La dystrophie myotonique (DM1), connue aussi sous le nom de maladie de Steinert, est une maladie héréditaire autosornale dominante. Elle est caractérisée par une myotonie, une faiblesse et une atrophie musculaires, mais peut aussi se manifester dans d'autres organes tels que les yeux, les voies digestive et respiratoire, ou le cerveau. En 1992, il a été montré que cette maladie complexe résultait de l'expansion d'une répétition de CTG dans une partie non traduite en 3' du gène codant pour la protéine kinase DM (DMPK), sur le chromosome 19. La taille de l'expansion héritée est étroitement liée à la sévérité et l'âge d'apparition de DM1. Bien que plusieurs études électrophysiologiques et histologiques aient été menées, pour juger d'une implication possible d'anomalies au niveau du système nerveux périphérique dans la DM1, les résultats n'ont jusqu'ici pas été univoques. Aujourd'hui, la question d'une neuropathie associée avec la DM1 reste donc controversée. Des souris transgéniques ont été élaborées, qui portent la séquence DM1 du génome humain avec 300 répétitions CTG et expriment le phénotype des patients DM1: Ces souris transgéniques DMl procurent un outil précieux pour l'étude du type et de l'incidence d'éventuelles anomalies du système nerveux périphérique dans la DM1. En utilisant ces souris transgéniques DM1, nous avons étudié la présence ou l'absence des trois principaux types de neuropathies périphériques: la dégénération axonale, la démyélinisation axonale et la neuronopathie. Les études morphologiques et morphométrique des nerfs sciatiques, suraux et phréniques ont montré l'absence de dégénération axonale ou de démyélinisation. L'analyse du nombre de cellules neuronales n'a pas dévoilé de diminution des nombres de neurones sensitifs dans les ganglions des racines dorsales lombaires ou de neurones moteurs dans la moëlle épinière lombaire des souris transgéniques DMl. De plus, l'examen de coupes sériées de muscle des membres postérieurs de souris DM1 a montré une arborisation axonale intramusculaire normale, de même que l'absence d'irrégularité dans le nombre ou la structure des plaques motrices. Enfin, les tests électrophysiologiques effectués sur les souris DMl ont montré que les potentiels d'action de la composante musculaire (CMAPs) évoqués dans les doigts des membres postérieurs, en réponse à une stimulation du nerf sciatique à l'aide d'une électrode paranerveuse, étaient identiques à ceux observées chez les souris sauvages. Sur la base de l'ensemble de ces résultats, nous avons émis l'hypothèse que 300 répétitions CTG ne sont pas suffisantes pour induire d'altérations dans le système nerveux périphérique du modèle de souris transgéniques DM 1.

Syndrome of birt-hogg-dubé, a histopathological pitfall with similarities to tuberous sclerosis: a report of three cases.

: Birt-Hogg-Dubé Syndrome (BHD) is a rare condition, transmitted as an autosomal-dominant trait. The etiology is due to a mutation in the BHD gene, which encodes folliculin (FLCN), located on chromosome 17p. The skin changes observed are benign skin tumors consisting of hamartomas of the hair follicle with dermal changes. Patients with BHD have an increased risk of spontaneous pneumothorax due to rupture of lung cysts and an increased risk of kidney tumors. We report 3 new cases of BHD and discuss their clinical features, histopathological findings, and molecular diagnostics. We highlight the importance of genetic analysis to confirm the diagnosis because of the clinical pitfalls involved in establishing a diagnosis. Finally, we discuss the histopathological features in BHD and tuberous sclerosis complex and focus on their overlapping criterias. A correct diagnosis is essential as it can be life saving for patients.

The DNA-binding domain (DBD) is essential for NR2E3 dimerization and interaction with Crx

Purpose:NR2E3 (PNR) is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S-) cone syndrome (ESCS) and, more recently, with autosomal dominant retinitis pigmentosa (adRP). NR2E3 acts in concert with the transcription factors Crx and Nrl to repress cone-specific genes and activate rod-specific genes. NR2E3 and Crx have been shown to physically interact by their DNA-binding domain (DBD), which may also be implicated in the dimerization process of the nuclear receptor. However, neither NR2E3 homodimerization nor NR2E3/Crx complex formation has been investigated in detail. Methods:In this present work, we analyzed the dimerization of the NR2E3 protein and its interaction with Crx by bioluminescence resonance energy transfer (BRET2) which utilizes Renilla luciferase (hRluc) protein and its substrate DeepBlueC as an energy donor and a mutant green fluorescent protein (GFP2) as the acceptor. We investigated, on whole intact cells, the role of NR2E3 DBD-mutations in dimerization and association with Crx. Results:We clearly showed that NR2E3 formed homodimers in HEK-293T cells. Moreover, all causative NR2E3 mutations present in the DBD of the protein showed an alteration in dimerization, except for the R76Q and the R104W mutants. Interestingly, the adRP-linked G56R mutant was the only DBD-NR2E3 mutant that showed a correct interaction with Crx. Finally, we observed a decrease in rhodospin gene transactivation for all DBD-NR2E3 mutants tested and no potentiation for the adRP-linked G56R mutant. In addition, the p.G56R mutant enhanced the transrepression of M-opsin promoter, while all other DBD-NR2E3 mutants did not repress M-opsin transactivation. Conclusions:A defect, either in the dimer formation or in the interaction of NR2E3 with Crx, leads to abnormal transcriptional activity on rhodopsin and M-opsin promoter and to an atypical retinal development; while the titration of Crx by p.G56R-NR2E3 leads to low levels of rhodopsin and M-opsin expression and may be responsible for the strong adRP phenotype.

Allele-specific silencing of mutant huntingtin in rodent brain and human stem cells.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder resulting from polyglutamine expansion in the huntingtin (HTT) protein and for which there is no cure. Although suppression of both wild type and mutant HTT expression by RNA interference is a promising therapeutic strategy, a selective silencing of mutant HTT represents the safest approach preserving WT HTT expression and functions. We developed small hairpin RNAs (shRNAs) targeting single nucleotide polymorphisms (SNP) present in the HTT gene to selectively target the disease HTT isoform. Most of these shRNAs silenced, efficiently and selectively, mutant HTT in vitro. Lentiviral-mediated infection with the shRNAs led to selective degradation of mutant HTT mRNA and prevented the apparition of neuropathology in HD rat's striatum expressing mutant HTT containing the various SNPs. In transgenic BACHD mice, the mutant HTT allele was also silenced by this approach, further demonstrating the potential for allele-specific silencing. Finally, the allele-specific silencing of mutant HTT in human embryonic stem cells was accompanied by functional recovery of the vesicular transport of BDNF along microtubules. These findings provide evidence of the therapeutic potential of allele-specific RNA interference for HD.

Breeding strategies for recurrent selection of maize

The objectives of this work were to analyze theoretical genetic gains of maize due to recurrent selection among full-sib and half-sib families, obtained by Design I, Full-Sib Design and Half-Sib Design, and genotypic variability and gene loss with long term selection. The designs were evaluated by simulation, based on average estimated gains after ten selection cycles. The simulation process was based on seven gene systems with ten genes (with distinct degrees of dominance), three population classes (with different gene frequencies), under three environmental conditions (heritability values), and four selection strategies. Each combination was repeated ten times, amounting to 25, 200 simulations. Full-sib selection is generally more efficient than half-sib selection, mainly with favorable dominant genes. The use of full-sib families derived by Design I is generally more efficient than using progenies obtained by Full-Sib Design. Using Design I with 50 males and 200 females (effective size of 160) did not result in improved populations with minimum genotypic variability. In the populations with lower effective size (160 and 400) the loss of favorable genes was restricted to recessive genes with reduced frequencies.

Inheritance of resistance to soybean cyst nematode races 3 and 14 in soybean RIL and F2 populations

The objective of this work was to evaluate the soybean inheritance of resistance to cyst nematode races 3 and 14. The following populations where evaluated: one population of recombinant inbred lines (RILs) [Hartwig (resistant) x Y23 (susceptible line)] for races 3, 14 and 9; one population of families F2:3 [M-SOY 8001 (resistant) x MB/BR 46 - Conquista (susceptible)] for race 3; and one population of families F2:3 [(S5995 (resistant) x BRSMG Renascença (susceptible)] for race 14. In RIL populations, four epistatic genes were identified which conditioned resistance to race 14, and three epistatic ones for resistance to races 3 and 9. The lack of one gene provided moderate resistance under all situations. The highest number of genes for resistance to race 14 points out that genes responsible for lower effects might be involved. In population F2:3 from M-SOY 8001 x MB/BR 46 - Conquista, one recessive gene for moderate resistance and two recessive genes complete resistance to race 3 were identified. Two recessive genes conditioning moderate resistance to race 14 were identified in population F2:3 from the crossing S5995 x BRSMG Renascença. These results will be useful in designing crossings, involving these parentals, with higher possibility to accumulating genes that provide resistance to several SCN races.

Multicentric carpotarsal osteolysis is caused by mutations clustering in the amino-terminal transcriptional activation domain of MAFB.

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia characterized by aggressive osteolysis, particularly affecting the carpal and tarsal bones, and is frequently associated with progressive renal failure. Using exome capture and next-generation sequencing in five unrelated simplex cases of MCTO, we identified previously unreported missense mutations clustering within a 51 base pair region of the single exon of MAFB, validated by Sanger sequencing. A further six unrelated simplex cases with MCTO were also heterozygous for previously unreported mutations within this same region, as were affected members of two families with autosomal-dominant MCTO. MAFB encodes a transcription factor that negatively regulates RANKL-induced osteoclastogenesis and is essential for normal renal development. Identification of this gene paves the way for development of novel therapeutic approaches for this crippling disease and provides insight into normal bone and kidney development.

Depistage mutationnel des genes de la peripherine/RDS, rhodopsine et ROM-1 dans 69 cas index de retinite pigmentaire et autres dystrophies retiniennes [Mutational screening of peripherin/RDS genes, rhodopsin and ROM-1 in 69 index cases with retinitis pigmentosa and other retinal dystrophies]

PURPOSE: Phenotypic, genetic and molecular characterization of 69 index patients with retinitis pigmentosa (RP) and various inherited retinal diseases. PATIENTS AND METHOD: patients went through complete ocular examination and blood samples were drawn for mutational screening of three candidate genes: rhodopsin (RHO), peripherin/RDS, and ROM-1. RESULTS: the most frequent type of RP among our population was the autosomal dominant (43.6%). Three RHO mutations were found among the RP patients. A RDS mutation was detected in three unrelated families segregating dominant macular dystrophy. DISCUSSION AND CONCLUSIONS: 18% of the autosomal dominant RP patients presented a RHO mutation; RDS R172W mutation was present in 25% of the dominant macular dystrophies.

Regenerative Medicine at the Single Cell Level

Adult stem cells are instrumental for renewal, regeneration, and repair. Self-renewal and the capacity to generate a tissue for an extended period of time (theoretically a life time) are fundamental properties of adult stem cells that allow longterm tissue reconstruction from a single stem cell as experimentally demonstrated with the bone marrow and the skin. Moreover, human epidermal stem cells (holoclones) can be extensively expanded and manipulated in culture before they are transplanted. We have taken advantage of these unique capacities to demonstrate the feasibility of a single epidermal stem cell approach for ex vivo gene therapy using recessive dystrophic epidermolysis bullosa (RDEB) as a model system. We have demonstrated that is possible to reconstruct a functional epidermis and anchoring fibers from the progeny of a single RDEB epidermal stem cell transduced with a Col7a1 cDNA by means of a SIN retrovirus. Demonstrations of safe proviral insertion, absence of tumorogenicity and of dissemination of the transduced engrafted cells meet regulatory affairs safety requirements.

Mutations in B3GALT6, which encodes a glycosaminoglycan linker region enzyme, cause a spectrum of skeletal and connective tissue disorders.

Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a severe skeletal dysplasia, spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMD-JL1). B3GALT6 loss-of-function mutations were found in individuals with SEMD-JL1 from seven families. In a subsequent candidate gene study based on the phenotypic similarity, we found that B3GALT6 is also responsible for a connective tissue disease, Ehlers-Danlos syndrome (progeroid form). Recessive loss-of-function mutations in B3GALT6 result in a spectrum of disorders affecting a broad range of skeletal and connective tissues characterized by lax skin, muscle hypotonia, joint dislocation, and spinal deformity. The pleiotropic phenotypes of the disorders indicate that B3GALT6 plays a critical role in a wide range of biological processes in various tissues, including skin, bone, cartilage, tendon, and ligament.