TY  - JOUR
AB  - Purpose: To investigate the molecular basis of Bardet-Biedl syndrome (BBS) in five consanguineous families of Paki­stani origin. Methods: Linkage in two families (A and B) was established to BBS7 on chromosome 4q27, in family C to BBS8 on chromosome 14q32.1, and in family D to BBS10 on chromosome 12q21.2. Family E was investigated directly with exome sequence analysis. Results: Sanger sequencing revealed two novel mutations and three previously reported mutations in the BBS genes. These mutations include two deletions (c.580_582delGCA, c.1592_1597delTTCCAG) in the BBS7 gene, a missense mutation (p.Gln449His) in the BBS8 gene, a frameshift mutation (c.271_272insT) in the BBS10 gene, and a nonsense mutation (p.Ser40*) in the MKKS (BBS6) gene. Conclusions: Two novel mutations and three previously reported variants, identified in the present study, further extend the body of evidence implicating BBS6, BBS7, BBS8, and BBS10 in causing BBS.
AU  - Ullah, A.*
AU  - Umair, M.
AU  - Yousaf, M.*
AU  - Khan, S.A.*
AU  - Nazim-Ud-Din, M.*
AU  - Shah, K.*
AU  - Ahmad, F.*
AU  - Azeem, Z.*
AU  - Ali, G.*
AU  - Alhaddad, B.
AU  - Rafique, A.*
AU  - Jan, A.*
AU  - Haack, T.B.
AU  - Strom, T.M.
AU  - Meitinger, T.
AU  - Ghous, T.*
AU  - Ahmad, W.*
C1  - 51631
C2  - 43379
SP  - 482-494
TI  - Sequence variants in four genes underlying Bardet-Biedl syndrome in consanguineous families.
JO  - Mol. Vis.
VL  - 23
PY  - 2017
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - PURPOSE: Within a mutagenesis screen, we identified the new mouse mutant Aey80 with small eyes; homozygous mutants were not obtained. The aim of the study was its molecular characterization. METHODS: We analyzed the offspring of paternally N-ethyl-N-nitrosourea (ENU)-treated C3HeB/FeJ mice for dysmorphology parameters, which can be observed with the naked eye. The Aey80 mutant (abnormality of the eye) was further characterized with laser interference biometry, Scheimpflug imaging, and optical coherence tomography. Linkage analysis of the Aey80 mutant was performed using a panel of single nucleotide polymorphisms different among C3HeB/FeJ and C57BL/6J mice. The Aey80 mutation was identified with sequence analysis of the positional candidate gene. RESULTS: We identified a new mutant characterized by an obvious small-eye phenotype; homozygotes are not viable after birth. Embryos at embryonic day 15.5 demonstrate a clear gene-dosage effect: Heterozygotes have small eyes, whereas homozygous mutants do not have eyes. In adult mice, the lenses and the entire eyes of the heterozygous mutants were significantly smaller than those of the wild-types (p<0.01). No other ocular phenotypes were observed; the lenses were fully transparent, and no adhesion to the cornea was observed. The mutation was mapped to chromosome 2; markers between 70.8 MB and 129.5 MB showed significant linkage to the mutation resulting in paired box gene 6 (Pax6) as an excellent candidate gene. We amplified cDNAs from the embryonic eyes and observed an additional band while amplifying the region corresponding to exons 7 and 8. The additional band included an alternative exon of 141 bp, which was associated with a G->A exchange four bases downstream of the end of the alternative exon. The alternative exon in the mutants is predicted to encode 30 novel amino acids and three stop codons. This alternative exon kept the paired domain intact but led to a loss of the homeodomain and the C-terminal proline-serine-threonine (PST) domain. The mutation cosegregated in the mutant line, since all five additional small-eyed mice from this line showed the same mutation. A general polymorphism at the mutated site was excluded with sequence analysis of seven other wild-type mouse strains different from C3HeB/FeJ. CONCLUSIONS: These findings demonstrate a novel allele of the paired box gene 6 (Pax6) that affects lens development in a semidominant manner leading to a classical small-eye phenotype. However, the site of the mutation more than 1 kb downstream of exon 7 and resulting in an alternative exon is quite unusual. It indicates the importance of sequence analysis of cDNA for mutation detection; mutations like this are unlikely to be identified by analyzing genomic sequences only. Moreover, this particular mutation demonstrates how a novel exon can be created by only a single base-pair exchange.  
AU  - Puk, O.
AU  - Yan, X.
AU  - Sabrautzki, S.
AU  - Fuchs, H.
AU  - Gailus-Durner, V.
AU  - Hrabě de Angelis, M.
AU  - Graw, J.
C1  - 24018
C2  - 31306
SP  - 877-884
TI  - Novel small-eye allele in paired box gene 6 (Pax6) is caused by a point mutation in intron 7 and creates a new exon.
JO  - Mol. Vis.
VL  - 19
PB  - Molecular Vision
PY  - 2013
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - Purpose: Porcine retina is an excellent model for studying diverse retinal processes and diseases. The morphologies of porcine retinal ganglion cells (RGCs) have, however, not yet been described comprehensively. The aim of the present study was to create a classification of the RGCs using the 1, 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) tracing method. Methods: About 170 RGCs were retrogradely labeled by injecting DiI into the optic nerve of postmortem eyes and statistically analyzed by two different clustering methods: Ward's algorithm and the K-means clustering. Major axis length of the soma, soma area size, and dendritic field area size were selected as main parameters for cluster classification. Results: RGC distribution in clusters was achieved according to their morphological parameters. It was feasible to combine both statistical methods, thereby obtaining a robust clustering distribution. Morphological analysis resulted in a classification of RGCs in three groups according to the soma size and dendritic field: A (large somas and large dendritic fields), B (medium to large somas and medium to large dendritic fields), C (medium to small somas and medium to small dendritic fields). Within groups, fine clustering defined several subgroups according to dendritic arborization and level of stratification. Additionally, cells stratifying in two different levels of the inner plexiform layer were observed within the clusters. Conclusions: This comprehensive study of RGC morphologies in the porcine retina provides fundamental knowledge about RGC cell types and provides a basis for functional studies toward selective RGC cell degeneration in retinal disorders.
AU  - Veiga-Crespo, P.*
AU  - del Río, P.
AU  - Blindert, M.
AU  - Ueffing, M.*
AU  - Hauck, S.M.
AU  - Vecino, E.*
C1  - 24689
C2  - 31637
SP  - 904-916
TI  - Phenotypic map of porcine retinal ganglion cells.
JO  - Mol. Vis.
VL  - 19
PB  - Molecular Vision
PY  - 2013
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - PURPOSE: Identification of mitochondrial DNA (mtDNA) variations in the inherited cataract patients from south India. METHODS: Three families with inherited cataract of maternal origin were evaluated. Clinical and ophthalmologic examinations were performed on available affected as well as unaffected family members. Samples of mtDNA were amplified using 24 pairs of overlapping primers to analyze the entire mitochondrial genome to screen for variations and analyzed for both coding and non-coding regions. Bioinformatic analysis was performed to evaluate the effect of nucleotide variations. RESULTS: DNA sequence analysis of inherited cataract families showed 72 nucleotide variations, of which 15 were observed in the major non-coding D-loop region, 3 in the tRNA genes, 5 in the rRNA genes, and 49 in the protein coding region. Among these variations 56 were reported previously and 16 were novel of which, 12 synonymous substitutions, 2 non-synonymous substitutions along with a frameshift mutation, and one was in the non-coding region. Nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit (ND) gene of mtDNA was highly altered, in general, and found to contain 4 variations specific for cataract patients of the first family, six for the second, and one for the third family. CONCLUSIONS: Seventy-two variations were observed in three inherited cataract families. Four variations were specific for cataract patients of the first family, six for the second, and one for the third family. This is perhaps the first report on the presence of mitochondrial mutations in inherited cataracts.
AU  - Roshan, M.*
AU  - Kabekkodu, S.P.*
AU  - Vijaya, P.H.*
AU  - Manjunath, K.*
AU  - Graw, J.
AU  - Gopinath, P.M.*
AU  - Satyamoorthy, K.*
C1  - 7304
C2  - 29665
SP  - 181-193
TI  - Analysis of mitochondrial DNA variations in Indian patients with congenital cataract.
JO  - Mol. Vis.
VL  - 18
IS  - 21
PB  - Molecular Vision
PY  - 2012
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - PURPOSE: To study susceptibility to glaucoma injury as it may be affected by mutations in ocular connective tissue components. METHODS: Mice homozygous for an N-ethyl-N-nitrosourea induced G257D exchange (Gly to Asp) missense mutation (Aca23) in their collagen 8A2 gene were studied to measure intraocular pressure (IOP), axial length and width, number of retinal ganglion cells (RGC), and inflation responses. Three month old homozygous Aca23 mutant and wild type (WT) mice had 6 weeks exposure to elevated IOP induced by polystyrene microbead injection. Additional Aca23 and matched controls were studied at ages of 10 and 18 months. RESULTS: Aca23 mice had no significant difference from WT in IOP level, and in both strains IOP rose with age. In multivariable models, axial length and width were significantly larger in Aca23 than WT, became larger with age, and were larger after exposure to glaucoma (n=227 mice). From inflation test data, the estimates of scleral stress resultants in Aca23 mice were similar to age-matched and younger WT C57BL/6 (B6) mice, while the strain estimates for Aca23 were significantly less than those for either WT group in the mid-sclera and in some of the more anterior scleral measures (p<0.001; n=29, 22, 20 eyes in Aca23, older WT, younger WT, respectively). With chronic IOP elevation, Aca23 eyes increased 9% in length and 7% in width, compared to untreated fellow eyes (p<0.05, <0.01). With similar elevated IOP exposure, WT eyes enlarged proportionately twice as much as Aca23, increasing in length by 18% and in nasal-temporal width by 13% (both p<0.001, Mann-Whitney test). In 4 month old control optic nerves, mean RGC axon number was not different in Aca23 and WT (46,905±7,592, 43,628±11,162, respectively; p=0.43, Mann-Whitney test, n=37 and 29). With chronic glaucoma, Aca23 mice had a mean axon loss of only 0.57±17%, while WT mice lost 21±31% (median loss: 1% versus 10%, n=37, 29, respectively; p=0.001; multivariable model adjusting for positive integral IOP exposure). CONCLUSIONS: The Aca23 mutation in collagen 8α2 is the first gene defect found to alter susceptibility to experimental glaucoma, reducing RGC loss possibly due to differences in mechanical behavior of the sclera. Detailed study of the specific changes in scleral connective tissue composition and responses to chronic IOP elevation in this strain could produce new therapeutic targets for RGC neuroprotection.
AU  - Steinhart, M.R.*
AU  - Cone, F.E.*
AU  - Nguyen, C.*
AU  - Nguyen, T.D.*
AU  - Pease, M.E.*
AU  - Puk, O.
AU  - Graw, J.
AU  - Oglesby, E.N.*
AU  - Quigley, H.A.*
C1  - 8398
C2  - 30090
SP  - 1093-1106
TI  - Mice with an induced mutation in collagen 8A2 develop larger eyes and are resistant to retinal ganglion cell damage in an experimental glaucoma model.
JO  - Mol. Vis.
VL  - 18
PB  - Molecular Vision
PY  - 2012
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - PURPOSE: Within a mutagenesis screen, we identified the new mouse mutant Aca47 with small lenses and reduced axial eye lengths. The aim of the actual study was the molecular and morphological characterization of the mouse mutant Aca47. METHODS: We analyzed the offspring of paternally N-ethyl-N-nitrosourea (ENU) treated C57BL/6J mice for eye-size parameters by non-invasive in vivo laser interference biometry. Linkage analysis of the eye size mutant Aca47 was performed using single nucleotide polymorphisms and microsatellite markers. The Aca47 mutation was identified by sequence analysis of positional candidate genes. A general polymorphism at the mutated site was excluded by restriction analysis. Eyes of the Aca47 mouse mutant were characterized by histology. Visual properties were examined in the virtual drum. RESULTS: We identified a new mutant characterized by a significantly smaller lens and reduced axial eye length without any changes for cornea thickness, anterior chamber depth or aqueous humor size. The smaller size of lens was more pronounced in the homozygous mutants, which further developed congenital cataracts in the lens nucleus. The mutation was mapped to chromosome 7 between the markers D7Mit247 and D7Mit81. Using a positional candidate approach, the lens intrinsic integral membrane protein MP19 encoding gene Lim2 was sequenced; a T→C exchange at cDNA position 151 leads to a cysteine-to-arginine substitution at position 51 of the Lim2 protein. Eye histology of adult heterozygous mutants did not show alterations on the cellular level. However, homozygous lenses revealed irregularly arranged lens fiber layers in the cortex. Virtual vision tests indicated that visual properties are not affected by reduced eye size of heterozygous individuals. CONCLUSIONS: These findings demonstrate a novel missense mutation in the Lim2 gene that affects lens development in a semidominant manner. Since homozygous mutants develop congenital lens opacities, this line can be used as a model for inherited cataract formation in humans.
AU  - Puk, O.
AU  - Ahmad, N.
AU  - Wagner, S.
AU  - Hrabě de Angelis, M.
AU  - Graw, J.
C1  - 6333
C2  - 27891
SP  - 1164-1171
TI  - Microphakia and congenital cataract formation in a novel Lim2C51R mutant mouse.
JO  - Mol. Vis.
VL  - 17
IS  - 131
PB  - Molecular Vision
PY  - 2011
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous retinal disorder. Despite tremendous knowledge about the genes involved in RP, little is known about the genetic causes of RP in Indonesia. Here, we aim to identify the molecular genetic causes underlying RP in a small cohort of Indonesian patients, using genome-wide homozygosity mapping. METHODS: DNA samples from affected and healthy individuals from 14 Indonesian families segregating autosomal recessive, X-linked, or isolated RP were collected. Homozygosity mapping was conducted using Illumina 6k or Affymetrix 5.0 single nucleotide polymorphism (SNP) arrays. Known autosomal recessive RP (arRP) genes residing in homozygous regions and X-linked RP genes were sequenced for mutations. RESULTS: In ten out of the 14 families, homozygous regions were identified that contained genes known to be involved in the pathogenesis of RP. Sequence analysis of these genes revealed seven novel homozygous mutations in ATP-binding cassette, sub-family A, member 4 (ABCA4), crumbs homolog 1 (CRB1), eyes shut homolog (Drosophila) (EYS), c-mer proto-oncogene tyrosine kinase (MERTK), nuclear receptor subfamily 2, group E, member 3 (NR2E3) and phosphodiesterase 6A, cGMP-specific, rod, alpha (PDE6A), all segregating in the respective families. No mutations were identified in the X-linked genes retinitis pigmentosa GTPase regulator (RPGR) and retinitis pigmentosa 2 (X-linked recessive; RP2). CONCLUSIONS: Homozygosity mapping is a powerful tool to identify the genetic defects underlying RP in the Indonesian population. Compared to studies involving patients from other populations, the same genes appear to be implicated in the etiology of recessive RP in Indonesia, although all mutations that were discovered are novel and as such may be unique for this population.
AU  - Siemiatkowska, A.M.*
AU  - Arimadyo, K.*
AU  - Moruz, L.M.*
AU  - Astuti, G.D.*
AU  - de Castro-Miró, M.*
AU  - Zonneveld, M.N.*
AU  - Strom, T.M.
AU  - de Wijs, I.J.*
AU  - Hoefsloot, L.H.*
AU  - Faradz, S.M.*
AU  - Cremers, F.P.*
AU  - den Hollander, A.I.*
AU  - Collin, R.W.*
C1  - 6817
C2  - 29312
SP  - 3013-3024
TI  - Molecular genetic analysis of retinitis pigmentosa in Indonesia using genome-wide homozygosity mapping.
JO  - Mol. Vis.
VL  - 17
PB  - Molecular Vision
PY  - 2011
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - PURPOSE: To identify the genetic defect in two Pakistani families with autosomal recessive achromatopsia. METHODS: Two families (RP26 and RP44) were originally diagnosed with retinal dystrophy based upon their medical history. To localize the causative genes in these families, homozygosity mapping was performed using Affymetrix 10K single nucleotide polymorphism (SNP) arrays. Sequence analysis was used to find the mutations in candidate genes cyclic nucleotide-gated channel alpha-3 (CNGA3; family RP26) and cyclic nucleotide-gated channel beta-3 (CNGB3; family RP44). Control individuals were analyzed by allele-specific PCR for the CNGA3 mutation and BstXI restriction analysis for the CNGB3 mutation. After genetic analysis, clinical diagnosis was re-evaluated by electroretinography and color vision testing. During the course of this study, selected affected members of family RP26 were given pink glasses as supportive therapy. RESULTS: Sequence analysis of the positional candidate genes identified a novel missense mutation in CNGA3 (c.822G>T; p.R274S) in family RP26, and a novel CNGB3 frameshift mutation (c.1825delG; p.V609WfsX9) in family RP44. Clinical re-evaluation after genetic analysis revealed that both families have segregating autosomal recessive achromatopsia. CONCLUSIONS: Genetic analysis of two Pakistani families with retinal disease enabled the establishment of the correct diagnosis of achromatopsia. Two novel mutations were identified in CNGA3 and CNGB3 that are both specifically expressed in cone photoreceptors. Re-evaluation of the clinical status revealed that both families had achromatopsia. The use of pink glasses in patients was helpful in reducing photophobia and enabled rod-mediated vision.
AU  - Azam, M.*
AU  - Collin, R.W.J.*
AU  - Shah, S.T.A.*
AU  - Shah, A.A.*
AU  - Khan, M.I.*
AU  - Hussain, A.*
AU  - Sadeque, A.*
AU  - Strom, T.M.
AU  - Thiadens, A.A.H.J.*
AU  - Roosing, S.*
AU  - den Hollander, A.I.*
AU  - Cremers, F.P.M.*
AU  - Qamar, R.*
C1  - 528
C2  - 27507
SP  - 774-781
TI  - Novel CNGA3 and CNGB3 mutations in two Pakistani families with achromatopsia.
JO  - Mol. Vis.
VL  - 16
IS  - 87
PB  - Molecular Vision
PY  - 2010
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - PURPOSE: Identification of causal mutation in the crystallin, connexin, and paired box gene 6 (PAX6) genes associated with childhood cataract in patients from India. METHODS: In this study, forty eight members from seventeen families and 148 sporadic cases of childhood cataract were evaluated. Clinical and ophthalmologic examinations were performed on available affected and unaffected family members. Samples of genomic DNA were PCR amplified to screen for mutations in the candidate genes viz., alpha-A crystallin (CRYAA), beta- B2 crystallin (CRYBB2), gamma-A crystallin (CRYGA), gamma-B crystallin (CRYGB), gamma-C crystallin (CRYGC), gamma-D crystallin (CRYGD), gap junction alpha-3 (GJA3), gap junction alpha-8 (GJA8), and PAX6 based on polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) analysis. Samples showing any band mobility shift were subjected to bidirectional sequencing to confirm the variation. Co-segregation of the observed change with the disease phenotype was further tested by restriction fragment length polymorphism (RFLP) for the appropriate restriction site. RESULTS: DNA sequencing analysis of CRYAA, CRYBB2, CRYGA-D, GJA3, GJA8, and PAX6 of the affected members of a family (C-35) showed a novel heterozygous missense mutation C>A at position 229 in CRYGD in three affected members of family C-35 with anterior polar coronary cataract. This variation C229A substitution created a novel restriction site for AluI and resulted in a substitution of highly conserved arginine at position 77 by serine (R77S). AluI restriction site analysis confirmed the transversion mutation. Analysis of the available unaffected members of the family (C-35) and 100 unrelated control subjects (200 chromosomes) of the same ethnic background did not show R77S variation. Data generated using ProtScale and PyMOL programs revealed that the mutation altered the stability and solvent-accessibility of the CRYGD protein. CONCLUSIONS: We describe here a family having anterior polar coronary cataract that co-segregates with the novel allele R77S of CRYGD in all the affected members. The same was found to be absent in the ethnically matched controls (n=100) studied. Interestingly the residue Arg has been frequently implicated in four missense (R15C, R15S, R37S, and R59H) and in one truncation mutation (R140X) of CRYGD. In two of the reported mutations Arg residues have been replaced with Serine. This finding further expands the mutation spectrum of CRYGD in association with childhood cataract and demonstrates a possible mechanism of cataractogenesis. Screening of other familial (n=48) and sporadic (n=148) cases of childhood cataract, did not reveal any previously reported or novel mutation in the candidate genes screened.
AU  - Roshan, M.*
AU  - Vijaya, P.H.*
AU  - Lavanya, G.R.*
AU  - Shama, P.K.*
AU  - Santhiya, S.T.*
AU  - Graw, J.
AU  - Gopinath, P.M.*
AU  - Satyamoorthy, K.*
C1  - 5365
C2  - 27620
SP  - 887-896
TI  - A novel human CRYGD mutation in a juvenile autosomal dominant cataract.
JO  - Mol. Vis.
VL  - 16
IS  - 99
PB  - Molecular Vision
PY  - 2010
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - PURPOSE: The aim of the study was to resolve the genetic etiology in families having inherited cataracts. METHODS: Families afflicted with congenital/childhood cataracts were registered in Chennai and Orissa (India). Blood samples were collected from the probands and available family members. Selected functional candidate genes were amplified by polymerase chain reaction (PCR) and characterized by direct sequencing. Putative mutations were confirmed in healthy controls. RESULTS: We observed interesting new polymorphisms of ethnic specificity, some of frequent nature, such as a 3-bp deletion in intron 3 of CRYBB2 (encoding βB2-crystallin) and IVS1+9 c>t variation in HSF4 (encoding heat-shock factor 4). Some rare single nucleotide polymorphisms (SNPs) co-segregate with the respective phenotype such as IVS3+120c>a of CRYBB2, while M44V of CRYGD (encoding γD-crystallin), although found in association with blue dot opacity was seen in a few healthy controls too. We identified two new mutations co-segregating along with the respective cataract phenotype within the families that were not seen in healthy controls from India or Germany. These include two missense mutations; one in GJA3 (encoding gap junction protein α3, which is also referred to as connexin 46); the mutation affects codon 19 (T19M), and the corresponding phenotype is a posterior-polar cataract. The other missense mutation affects CRYBB2 (W59C; total cataract). Additionally, a cDNA variation (G54A) identified in a zonular cataract affects a highly conserved splice site of CRYBB2. This mutation, however, showed reduced penetrance in the family, which might be explained by different molecular consequences in the affected family members: nonsense-mediated decay of the mutated mRNA might have no clinical phenotype in heterozygotes, whereas the translation of the mutated mRNA is predicted to lead to a small hybrid protein (consisting of 16 amino acids of the βB2-crystallin and 18 new amino-acids), which might have a dominant-negative function in the lens. CONCLUSIONS: This report identifies in families with childhood cataract some new alleles, which may be considered as causative for cataracts. Furthermore, we report some geographically restricted rare polymorphic sites, whose significance might be considered in some context as modifiers or alleles in sensitizing ocular lens toward cataractogenesis.
AU  - Santhiya, S.T.*
AU  - Kumar, G.S.*
AU  - Sudhakar, P.*
AU  - Gupta, N.*
AU  - Klopp, N.
AU  - Illig, T.
AU  - Söker, T.
AU  - Groth, M.*
AU  - Platzer, M.*
AU  - Gopinath, P.M.*
AU  - Graw, J.
C1  - 5993
C2  - 27761
SP  - 1837-1847
TI  - Molecular analysis of cataract families in India: New mutations in the CRYBB2 and GJA3 genes and rare polymorphisms.
JO  - Mol. Vis.
VL  - 16
IS  - 199
PB  - Molecular Vision
PY  - 2010
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - The aim of this study was the genetic, cellular, and physiological characterization of a connexin50 (CX50) variant identified in a child with congenital cataracts. METHODS: Lens material from surgery was collected and used for cDNA production. Genomic DNA was prepared from blood obtained from the proband and her parents. PCR amplified DNA fragments were sequenced and characterized by restriction digestion. Connexin protein distribution was studied by immunofluorescence in transiently transfected HeLa cells. Formation of functional channels was assessed by two-microelectrode voltage-clamp in cRNA-injected Xenopus oocytes. RESULTS: Ophthalmologic examination showed that the proband suffered from bilateral white, diffuse cataracts, but the parents were free of lens opacities. Direct sequencing of the PCR product produced from lens cDNA showed that the proband was heterozygous for a G>T transition at position 741 of the GJA8 gene, encoding the exchange of methionine for isoleucine at position 247 of CX50 (CX50I247M). The mutation was confirmed in the genomic DNA, but it was also present in the unaffected mother. When expressed in HeLa cells, both wild type CX50 and CX50I247M formed gap junction plaques. Both CX50 and CX50I247M induced gap junctional currents in pairs of Xenopus oocytes. CONCLUSIONS: Although the CX50I247M substitution has previously been suggested to cause cataracts, our genetic, cellular, and electrophysiological data suggest that this allele more likely represents a rare silent, polymorphic variant.
AU  - Graw, J.
AU  - Schmidt, W.*
AU  - Minogue, P.J.*
AU  - Rodriguez, J.*
AU  - Tong, J.J.*
AU  - Klopp, N.
AU  - Illig, T.
AU  - Ebihara, L.*
AU  - Berthoud, V.M.*
AU  - Beyer, E.C.*
C1  - 271
C2  - 26459
SP  - 1881-1885
TI  - The GJA8 allele encoding CX50I247M is a rare polymorphism, not a cataract-causing mutation.
JO  - Mol. Vis.
VL  - 15
IS  - 200
PB  - Molecular Vision
PY  - 2009
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - The aim of the study was to characterize the underlying mutation in a consanguineous family having cataracts. METHODS: Family D having congenital cataracts was treated at the University Eye Clinics at Giessen (Germany). Lens material from surgeries was collected, immediately frozen at -80 degrees C, and used for cDNA production. Blood was taken from the proband and available family members. Polymerase chain reaction (PCR)-amplified DNA fragments were characterized by sequencing and restriction digestion. RESULTS: The proband, AD, has a dense, triangular nuclear cataract. The parents are consanguineous, and the mother and grandmother suffer from a discrete, symmetric opacity of the fetal lens nucleus. The proband's lens cDNA showed a homozygous insertion of one G after position 776 of the GJA8 gene, leading to a frame shift and 123 novel amino acids. The homozygous mutation was confirmed in the genomic DNA and is also present in the cataract-operated brother of the proband; all other family members investigated were heterozygous. The mutation could not be detected in 96 healthy controls from Germany. CONCLUSIONS: The ins776G mutation most likely causes a recessive triangular cataract with variable expressivity of a weak phenotype in heterozygotes.
AU  - Schmidt, W.*
AU  - Klopp, N.
AU  - Illig, T.
AU  - Graw, J.
C1  - 5896
C2  - 25221
SP  - 851-856
TI  - A novel GJA8 mutation causing a recessive triangular cataract.
JO  - Mol. Vis.
VL  - 14
IS  - 101-102
PB  - Molecular Vision Inc.
PY  - 2008
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - The study demonstrates the functional candidate gene analysis in a cataract family of German descent. METHODS: We screened a German family, clinically documented to have congenital cataracts, for mutation in the candidate genes CRYG (A to D) and CRYBB2 through polymerase chain reaction analyses and sequencing. RESULTS: Congenital cataract was first observed in a daughter of healthy parents. Her two children (a boy and a girl) also suffer from congenital cataracts and have been operated within the first weeks of birth. Morphologically, the cataract is characterized as nuclear with an additional ring-shaped cortical opacity. Molecular analysis revealed no causative mutation in any of the CRYG genes. However, sequencing of the exons of the CRYBB2 gene identified a sequence variation in exon 5 (383 A>T) with a substitution of Asp to Val at position 128. All three affected family members revealed this change but it was not observed in any of the unaffected persons of the family. The putative mutation creates a restriction site for the enzyme TaiI. This mutation was checked for in controls of randomly selected DNA samples from ophthalmologically normal individuals from the population-based KORA S4 study (n=96) and no mutation was observed. Moreover, the Asp at position 128 is within a stretch of 12 amino acids, which are highly conserved throughout the animal kingdom. For the mutant protein, the isoelectric point is raised from pH 6.50 to 6.75. Additionally, the random coil structure of the protein between the amino acids 126-139 is interrupted by a short extended strand structure. In addition, this region becomes hydrophobic (from neutral to +1) and the electrostatic potential in the region surrounding the exchanged amino acid alters from a mainly negative potential to an enlarged positive potential. CONCLUSIONS: The D128V mutation segregates only in affected family members and is not seen in representative controls. It represents the first mutation outside exon 6 of the human CRYBB2 gene.
AU  - Pauli, S.*
AU  - Söker, T.
AU  - Klopp, N.
AU  - Illig, T.
AU  - Engel, W.*
AU  - Graw, J.
C1  - 5887
C2  - 24583
SP  - 962-967
TI  - Mutation analysis in a German family identified a new cataract-causing allele in the CRYBB2 gene.
JO  - Mol. Vis.
VL  - 13
IS  - 101-102
PB  - MOLECULAR VISION
PY  - 2007
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - Purpose: The aim of the present study was to investigate the molecular basis underlying a nonsyndromic presenile autosomal dominant cataract in a three-generation pedigree. The phenotype was progressive from a peripheral ring-like opacity to a total cataract with advancing age from teenage to adulthood. The visual impairment started as problem in distant vision at the age of 16 years, to diminishing vision by the age of 24. Methods: Clinical interventions included complete ophthalmological examination, a collection of case history, and pedigree details. Blood samples were collected from available family members irrespective of their clinical status. A functional candidate gene approach was employed for PCR screening and sequencing of the exons and their flanking regions of CRYGC, CRYGD, and CRYAA genes. For structural consequences of the mutated alpha A-crystallin we used the bioinformatics tool of the ExPASy server. Results: Sequence analysis of CRYGC and CRYGD genes excluded possible causative mutations but identified known polymorphisms. Sequencing of the exons of the CRYAA gene identified a sequence variation in exon 2 (292 G-> A) with a substitution of Gly to Arg at position 98. All three affected members revealed this change but it was not observed in the unaffected father or sister. The putative mutation obliterated a restriction site for the enzyme BstDSI. The same was checked in controls representing the general population of the same ethnicity (n=30) and of randomly selected DNA samples from ophthalmologically normal individuals from the population-based KORA S4 study (n=96). Moreover, the Gly at position 98 is highly conserved throughout the animal kingdom. For the mutant protein, the isoelectric point was raised from pH 5.77 to 5.96. Moreover, an extended alpha-helical structure is predicted in this region. Conclusions: The G98R mutation segregates only in affected family members and is not seen in representative controls. It represents very likely the fourth dominant cataract-causing allele in CRYAA. In all reported alleles the basic amino acid Arg is involved, suggesting the major importance of the net charge of the alpha A-crystallin for functional integrity in the lens.
AU  - Santhiya, S.T.*
AU  - Söker, T.
AU  - Klopp, N.
AU  - Illig, T.
AU  - Prakash, M.V.S.*
AU  - Selvaraj, B.*
AU  - Gopinath, P.M.*
AU  - Graw, J.
C1  - 4536
C2  - 23648
SP  - 768-773
TI  - Identification of a novel, putative cataract-causing allele in CRYAA (G98R) in an Indian family.
JO  - Mol. Vis.
VL  - 12
IS  - 86-87
PB  - MOLECULAR VISION
PY  - 2006
SN  - 1090-0535
ER  - 

TY  - JOUR
AB  - Purpose: The 44TNJ mutant mouse was generated by the Tennessee Mouse Genome Consortium (TMGC) using an ENU-based mutagenesis screen to produce recessive mutations that affect the eye and brain. Herein we present its retinal phenotype and genetic basis. Methods: Fourth generation offspring (G(4)) and confirmed mutants were examined using slit lamp biomicroscopy, funduscopy, histology, immunohistochemistry, and electroretinography (ERG). 44TNJ mutant mice were crossed to C3BLiA or DBA/2 mice for chromosomal mapping purposes. Linkage analysis by PCR-based microsatellite marker genotyping was used to identify the disease locus. The Rs1h cDNA and its genomic DNA were sequenced directly. Results: The 44TNJ pedigree was the first mutant pedigree identified by the ocular phenotyping domain of the TMGC. Examination of the fundus revealed numerous small and homogeneous intraretinal microflecks in the peripapillary region, which became courser and more irregular in the periphery. Males were typically more affected than females. Histology and immunohistochemistry revealed a disruption of the lamination of the retina, particularly at both margins of the outer nuclear layer, along with reduced calbindin immunostaining. ERG analyses revealed reduced amplitudes of both a-waves and b-waves. Linkage analysis mapped the 44TNJ mutation to the X chromosome close to the marker DXMit117. Sequence analysis of the positional candidate gene Rs1h revealed a T->C exchange at the second base of intron 2 of the Rs1h gene. Conclusions: We have generated and characterized a mutant mouse line that was produced using ENU-based mutagenesis. The 44TNJ pedigree manifests with photoreceptor dysfunction and concurrent structural and functional aberrations at the post-receptoral level. Genetic analysis revealed a mutation in Rs1h, making this the first murine model of X-linked retinoschisis in which the gene is expressed.
AU  - Jablonski, M.M.*
AU  - Dalke, C.
AU  - Wang, X.F.*
AU  - Lu, L.*
AU  - Manly, K.F.*
AU  - Pretsch, W.
AU  - Favor, J.
AU  - Pardue, M.T.*
AU  - Rinchik, E.M.*
AU  - Williams, R.W.*
AU  - Goldowitz, D.*
C1  - 4295
C2  - 22766
SP  - 569-581
TI  - An ENU-induced mutation in Rs1h causes disruption of retinal structure and function.
JO  - Mol. Vis.
VL  - 11
IS  - 67
PB  - MOLECULAR VISION
PY  - 2005
SN  - 1090-0535
ER  -