Mutation in LIM2 Is Responsible for Autosomal Recessive Congenital Cataracts

PurposeTo identify the molecular basis of non-syndromic autosomal recessive congenital cataracts (arCC) in a consanguineous family.MethodsAll family members participating in the study received a comprehensive ophthalmic examination to determine their ocular phenotype and contributed a blood sample, from which genomic DNA was extracted. Available medical records and interviews with the family were used to compile the medical history of the family. The symptomatic history of the individuals exhibiting cataracts was confirmed by slit-lamp biomicroscopy. A genome-wide linkage analysis was performed to localize the disease interval. The candidate gene, LIM2 (lens intrinsic membrane protein 2), was sequenced bi-directionally to identify the disease-causing mutation. The physical changes caused by the mutation were analyzed in silico through homology modeling, mutation and bioinformatic algorithms, and evolutionary conservation databases. The physiological importance of LIM2 to ocular development was assessed in vivo by real-time expression analysis of Lim2 in a mouse model.ResultsOphthalmic examination confirmed the diagnosis of nuclear cataracts in the affected members of the family; the inheritance pattern and cataract development in early infancy indicated arCC. Genome-wide linkage analysis localized the critical interval to chromosome 19q with a two-point logarithm of odds (LOD) score of 3.25. Bidirectional sequencing identified a novel missense mutation, c.233G>A (p.G78D) in LIM2. This mutation segregated with the disease phenotype and was absent in 192 ethnically matched control chromosomes. In silico analysis predicted lower hydropathicity and hydrophobicity but higher polarity of the mutant LIM2-encoded protein (MP19) compared to the wild-type. Moreover, these analyses predicted that the mutation would disrupt the secondary structure of a transmembrane domain of MP19. The expression of Lim2, which was detected in the mouse lens as early as embryonic day 15 (E15) increased after birth to a level that was sustained through the postnatal time points.ConclusionA novel missense mutation in LIM2 is responsible for autosomal recessive congenital cataracts.     

A Mutation in the Mitochondrial Fission Gene Dnm1l Leads to Cardiomyopathy

Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease.     

Linkage of the Steroid Sulfatase Gene to the Sex-Reversed Mutation in the Mouse

Dosage studies and the inheritance pattern of the gene for steroid sulfatase (Sts) in the mouse have previously provided indirect evidence for a functional Y-linked allele which recombines obligatorily with its X-linked allele in male meiosis. In this study, we have investigated the linkage relationship of Sts and the sex-reversed mutation (Sxr), a gene which is known to reside in the pairing region of the Y chromosome. The results clearly demonstrate that Sxr and Sts are linked in a region of obligatory recombination and Sts maps proximal to Sxr with most recombinants occurring proximal to the two genes.     

A Novel Nonsense Mutation in the MIP Gene Linked to Congenital Posterior Polar Cataracts in a Chinese Family

Purpose

To detect the causative mutation for congenital posterior polar cataracts in a five-generation Chinese family and further explore the potential pathogenesis of this disease.

Methods

Coding exons, with flanking sequences of five candidate genes, were screened using direct DNA sequencing. The identified mutations were confirmed by restriction fragment length polymorphism (RFLP) analysis. A full-length wild-type or an Y219* mutant aquaporin0 (AQP0) fused with an N-terminal FLAG tag, was transfected into HEK293T cells. For co-localization studies, FLAG-WT-AQP0 and Myc-Y219*-AQP0 constructs were co-transfected. Quantitative real-time RT-PCR, western blotting and immunofluorescence studies were performed to determine protein expression levels and sub-cellular localization, respectively.

Results

We identified a novel nonsense mutation in MIP (c.657 C>G; p.Y219*) (major intrinsic protein gene) that segregates with congenital posterior polar cataract in a Chinese family. This mutation altered a highly conserved tyrosine to a stop codon (Y219*) within AQP0.When FLAG-WT-AQP0 and FLAG-Y219*-AQP0 expression constructs were singly transfected into HEK 293T cells, mRNA expression showed no significant difference between the wild-type and the mutant, while Y219*-AQP0 protein expression was significantly lower than that of wild-type AQP0. Wild-type AQP0 predominantly localized to the plasma membrane, while the mutated protein was abundant within the cytoplasm of HEK293T cells. However, when FLAG-WT-AQP0 andMyc-MU-AQP0were co-expressed, both proteins showed high fluorescence in the cytoplasm.

Conclusions

The novel nonsense mutation in the MIP gene (c.657 C>G) identified in a Chinese family may cause posterior polar cataracts. The dominant negative effect of the mutated protein on the wild-type protein interfered with the trafficking of wild-type protein to the cell membrane and both the mutant and wild-type protein were trapped in the cytoplasm. Consequently, both wild-type and mutant protein lost their function as a water channel on the cell membrane, and may result in a cataract phenotype. Our data also expands the spectrum of known MIP mutations.     

iRhom2 Mutation Leads to Aberrant Hair Follicle Differentiation in Mice

iRhom1 and iRhom2 are inactive homologues of rhomboid intramembrane serine proteases lacking essential catalytic residues, which are necessary for the maturation of TNFα-converting enzyme (TACE). In addition, iRhoms regulate epidermal growth factor family secretion. The functional significance of iRhom2 during mammalian development is largely unclear. We have identified a spontaneous single gene deletion mutation of iRhom2 in Uncv mice. The iRhom2^Uncv/Uncv mice exhibit hairless phenotype in a BALB/c genetic background. In this study, we observed dysplasia hair follicles in iRhom2^Uncv/Uncv mice from postnatal day 3. Further examination found decreased hair matrix proliferation and aberrant hair shaft and inner root sheath differentiation in iRhom2^Uncv/Uncv mutant hair follicles. iRhom2 is required for the maturation of TACE. Our data demonstrate that iRhom2^Uncv cannot induce the maturation of TACE in vitro and the level of mature TACE is also significantly reduced in the skin of iRhom2^Uncv/Uncv mice. The activation of Notch1, a substrate of TACE, is disturbed, associated with dramatically down-regulation of Lef1 in iRhom2^Uncv/Uncv hair follicle matrix. This study identifies iRhom2 as a novel regulator of hair shaft and inner root sheath differentiation.     

Identification of the Gene Whose Mutation Leads to the Appearance of Recessive Lethal Germlings in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The data are presented on genetic and molecular-genetic analysis of a mutant from the collection of morphological insertion mutants of Arabidopsis thaliana we obtained earlier, which belongs to the phenotypic class of recessive lethal germlings. A nucleotide DNA sequence, 147 bp in size, was identified, which adheres to the left border area of T-DNA insertion. The site of localization of the insertion was determined using computer analysis.__________Translated from Ontogenez, Vol. 36, No. 3, 2005, pp. 222–224.Original Russian Text Copyright © 2005 by Ogarkova, Tomilov, Tomilova, Tarasov.     

Mutations in the EPHA2 Gene Are a Major Contributor to Inherited Cataracts in South-Eastern Australia

Congenital cataract is the most common cause of treatable visual impairment in children worldwide. Mutations in many different genes lead to congenital cataract. Recently, mutations in the receptor tyrosine kinase gene, EPHA2, have been found to cause congenital cataract in six different families. Although these findings have established EPHA2 as a causative gene, the total contribution of mutations in this gene to congenital cataract is unknown. In this study, for the first time, a population-based approach was used to investigate the frequency of disease causing mutations in the EPHA2 gene in inherited cataract cases in South-Eastern Australia. A cohort of 84 familial congenital or juvenile cataract index cases was screened for mutations in the EPHA2 gene by direct sequencing. Novel changes were assessed for segregation with the disease within the family and in unrelated controls. Microsatellite marker analysis was performed to establish any relationship between families carrying the same mutation. We report a novel congenital cataract causing mutation c.1751C>T in the EPHA2 gene and the previously reported splice mutation c.2826-9G>A in two new families. Additionally, we report a rare variant rs139787163 potentially associated with increased susceptibility to cataract. Thus mutations in EPHA2 account for 4.7% of inherited cataract cases in South-Eastern Australia. Interestingly, the identified rare variant provides a link between congenital and age-related cataract.     

Cataracts and Microphthalmia Caused by a Gja8 Mutation in Extracellular Loop 2

The mouse semi-dominant Nm2249 mutation displays variable cataracts in heterozygous mice and smaller lenses with severe cataracts in homozygous mice. This mutation is caused by a Gja8^R205G point mutation in the second extracellular loop of the Cx50 (or α8 connexin) protein. Immunohistological data reveal that Cx50-R205G mutant proteins and endogenous wild-type Cx46 (or α3 connexin) proteins form diffuse tiny spots rather than typical punctate signals of normal gap junctions in the lens. The level of phosphorylated Cx46 proteins is decreased in Gja8^R205G/R205G mutant lenses. Genetic analysis reveals that the Cx50-R205G mutation needs the presence of wild-type Cx46 to disrupt lens peripheral fibers and epithelial cells. Electrophysiological data in Xenopus oocytes reveal that Cx50-R205G mutant proteins block channel function of gap junctions composed of wild-type Cx50, but only affect the gating of wild-type Cx46 channels. Both genetic and electrophysiological results suggest that Cx50-R205G mutant proteins alone are unable to form functional channels. These findings imply that the Gja8^R205G mutation differentially impairs the functions of Cx50 and Cx46 to cause cataracts, small lenses and microphthalmia. The Gja8^R205G mutation occurs at the same conserved residue as the human GJA8^R198W mutation. This work provides molecular insights to understand the cataract and microphthalmia/microcornea phenotype caused by Gja8 mutations in mice and humans.     

Gene Targeting in Penicillium chrysogenum: Disruption of the lys2 Gene Leads to Penicillin Overproduction

Two strategies have been used for targeted integration at the lys2 locus of Penicillium chrysogenum. In the first strategy the disruption of lys2 was obtained by a single crossing over between the endogenous lys2 and a fragment of the same gene located in an integrative plasmid. lys2-disrupted mutants were obtained with 1.6% efficiency when the lys2 homologous region was 4.9 kb, but no homologous integration was observed with constructions containing a shorter homologous region. Similarly, lys2-disrupted mutants were obtained by a double crossing over (gene replacement) with an efficiency of 0.14% by using two lys2 homologous regions of 4.3 and 3.0 kb flanking the pyrG marker. No homologous recombination was observed when the selectable marker was flanked by short lys2 homologous DNA fragments. The disruption of lys2 was confirmed by Southern blot analysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. The lys2-disrupted mutants lacked α-aminoadipate reductase activity (encoded by lys2) and showed specific penicillin yields double those of the parental nondisrupted strain, Wis 54-1255. The α-aminoadipic acid precursor is channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at the α-aminoadipate reductase level.     

snthr-1Bao稀毛小鼠突变基因的精确定位及克隆鉴定

snthr^-1Bao稀毛小鼠是本实验室培育的呈单基因隐性遗传的突变系小鼠,突变基因已被初步定位于第9号染色体末端;为了精确定位并鉴定snthr^-1Bao稀毛小鼠的突变基因,将(C57BL/6J×snthr^-1Bao)F₁代互交繁殖F₂代小鼠4 400余只,其中稀毛小鼠1 100只,并在2个微卫星、35个可能的简单序列重复标记（simple sequence repeat,SSR）及3个酶切扩增多态性序列（c1eaved amplified polymorphic sequences,CAPS）标记中找到4个合适的基因组标记。利用这些标记及F₂代稀毛小鼠将突变基因精确定位到第9号染色体距着丝粒117.763 kb及119.129 kb之间1.367 Mb的范围内,在其间的21个基因中确定Plcd1为稀毛突变的强力候选基因。通过对基因组的直接测序,发现snthr^-1Bao稀毛小鼠基因组上有一个14 883 bp的缺失,这一缺失包含了Plcd1基因的4—15号外显子及Vill基因的10—19号外显子。推测极可能是Plcd1基因缺失导致snthr^-1Bao小鼠出现稀毛表型。     

SNF2家族新成员Ercc61的cDNA克隆与表达分析

SNF2家族蛋白在基因组复制、修复与表达中具有重要作用.报道了SNF2家族新成员Ercc61(excision repair crosscomplementing rodent repair deficiency,complementation group 6-like)的cDNA克隆、特性与表达分析.通过表达序列标签(EST)搜索和组装,获得了cDNA全长4002 bp的新基因Ercc6l(GenBank Acc.No AY172688),然后通过RT-PCR在小鼠胚胎心脏成功克隆了该基因.Ercc6l在小鼠基因组中由两个外显子和一个内含子组成,定位于X染色体,最大开放阅读框(ORF)编码一个含l 240个氨基酸的假定蛋白质.该假定蛋白质含有SNF2蛋白的8个保守基序(SNF2结构域).通过与SNF2家族各亚家族的成员进行多重比对,初步确认Ercc6l属于ERCC6亚家族成员.将Ercc6l编码区克隆到pEGFP-C3然后转染HeLa,3T3和B16细胞,融合蛋白主要定位于胞浆.BLAST搜索检索出69条小鼠EST与Erccol同源,这些EST主要来自胚胎和肿瘤组织.对小鼠不同发育时期的多种组织进行RT-PCR,发现Ercc6l在胚胎期强表达,出生产后表达显著下调.这些结果提示Ercc6l在胚胎发育和肿瘤发生中可能具有重要作用.     

Myocardial Overexpression of Mecr,a Gene of Mitochondrial FAS II Leads to Cardiac Dysfunction in Mouse

It has been recently recognized that mammalian mitochondria contain most, if not all, of the components of fatty acid synthesis type II (FAS II). Among the components identified is 2-enoyl thioester reductase/mitochondrial enoyl-CoA reductase (Etr1/Mecr), which catalyzes the NADPH-dependent reduction of trans-2-enoyl thioesters, generating saturated acyl-groups. Although the FAS type II pathway is highly conserved, its physiological role in fatty acid synthesis, which apparently occurs simultaneously with breakdown of fatty acids in the same subcellular compartment in mammals, has remained an enigma. To study the in vivo function of the mitochondrial FAS in mammals, with special reference to Mecr, we generated mice overexpressing Mecr under control of the mouse metallothionein-1 promoter. These Mecr transgenic mice developed cardiac abnormalities as demonstrated by echocardiography in vivo, heart perfusion ex vivo, and electron microscopy in situ. Moreover, the Mecr transgenic mice showed decreased performance in endurance exercise testing. Our results showed a ventricular dilatation behind impaired heart function upon Mecr overexpression, concurrent with appearance of dysmorphic mitochondria. Furthermore, the data suggested that inappropriate expression of genes of FAS II can result in the development of hereditary cardiomyopathy.     

Adenomatous Polyposis Coli Mutation Leads to Myopia Development in Mice

Myopia incidence in China is rapidly becoming a very serious sight compromising problem in a large segment of the general population. Therefore, delineating the underlying mechanisms leading to myopia will markedly lessen the likelihood of other sight compromising complications. In this regard, there is some evidence that patients afflicted with familial adenomatous polyposis (FAP), havean adenomatous polyposis coli (APC) mutation and a higher incidence of myopia. To clarify this possible association, we determined whether the changes in pertinent biometric and biochemical parameters underlying postnatal refractive error development in APC^Min mice are relevant for gaining insight into the pathogenesis of this disease in humans. The refraction and biometrics in APC^Min mice and age-matched wild-type (WT) littermates between postnatal days P28 and P84 were examined with eccentric infrared photorefraction (EIR) and customized optical coherence tomography (OCT). Compared with WT littermates, the APC^Min mutated mice developed myopia (average -4.64 D) on P84 which was associated with increased vitreous chamber depth (VCD). Furthermore, retinal and scleral changes appear in these mice along with: 1) axial length shortening; 2) increased retinal cell proliferation; 3) and decreased tyrosine hydroxylase (TH) expression, the rate-limiting enzyme of DA synthesis. Scleral collagen fibril diameters became heterogeneous and irregularly organized in the APC^Min mice. Western blot analysis showed that scleral alpha-1 type I collagen (col1α1) expression also decreased whereas MMP2 and MMP9 mRNA expression was invariant. These results indicate that defective APC gene function promotes refractive error development. By characterizing in APC^Min mice ocular developmental changes, this approach provides novel insight into underlying pathophysiological mechanisms contributing to human myopia development.     

SNF2家族新成员Ercc6l的cDNA克隆与表达分析(英)

SNF2家族蛋白在基因组复制、修复与表达中具有重要作用. 报道了SNF2家族新成员Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like)的cDNA克隆、特性与表达分析.通过表达序列标签（EST）搜索和组装,获得了cDNA全长4 002 bp的新基因Ercc6l(GenBank Acc.No AY172688),然后通过RT-PCR在小鼠胚胎心脏成功克隆了该基因.Ercc6l在小鼠基因组中由两个外显子和一个内含子组成,定位于X染色体,最大开放阅读框（ORF）编码一个含1 240个氨基酸的假定蛋白质.该假定蛋白质含有SNF2蛋白的8个保守基序（SNF2结构域）.通过与SNF2家族各亚家族的成员进行多重比对,初步确认Ercc6l属于ERCC6亚家族成员.将Ercc6l编码区克隆到pEGFP-C3然后转染HeLa,3T3 和B16细胞,融合蛋白主要定位于胞浆.BLAST搜索检索出69条小鼠EST与Ercc6l同源,这些EST主要来自胚胎和肿瘤组织.对小鼠不同发育时期的多种组织进行RT-PCR,发现Ercc6l在胚胎期强表达,出生产后表达显著下调.这些结果提示Ercc6l在胚胎发育和肿瘤发生中可能具有重要作用.     

Mutation of Npr2 Leads to Blurred Tonotopic Organization of Central Auditory Circuits in Mice

Tonotopy is a fundamental organizational feature of the auditory system. Sounds are encoded by the spatial and temporal patterns of electrical activity in spiral ganglion neurons (SGNs) and are transmitted via tonotopically ordered processes from the cochlea through the eighth nerve to the cochlear nuclei. Upon reaching the brainstem, SGN axons bifurcate in a stereotyped pattern, innervating target neurons in the anteroventral cochlear nucleus (aVCN) with one branch and in the posteroventral and dorsal cochlear nuclei (pVCN and DCN) with the other. Each branch is tonotopically organized, thereby distributing acoustic information systematically along multiple parallel pathways for processing in the brainstem. In mice with a mutation in the receptor guanylyl cyclase Npr2, this spatial organization is disrupted. Peripheral SGN processes appear normal, but central SGN processes fail to bifurcate and are disorganized as they exit the auditory nerve. Within the cochlear nuclei, the tonotopic organization of the SGN terminal arbors is blurred and the aVCN is underinnervated with a reduced convergence of SGN inputs onto target neurons. The tonotopy of circuitry within the cochlear nuclei is also degraded, as revealed by changes in the topographic mapping of tuberculoventral cell projections from DCN to VCN. Nonetheless, Npr2 mutant SGN axons are able to transmit acoustic information with normal sensitivity and timing, as revealed by auditory brainstem responses and electrophysiological recordings from VCN neurons. Although most features of signal transmission are normal, intermittent failures were observed in responses to trains of shocks, likely due to a failure in action potential conduction at branch points in Npr2 mutant afferent fibers. Our results show that Npr2 is necessary for the precise spatial organization typical of central auditory circuits, but that signals are still transmitted with normal timing, and that mutant mice can hear even with these deficits.     

Mutation of the Diamond-Blackfan Anemia Gene Rps7 in Mouse Results in Morphological and Neuroanatomical Phenotypes

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7^Mtu and Rps7^Zma) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.