Genetic and phenotypic variations of inherited retinal diseases in dogs: the power of within- and across-breed studies

Considerable clinical and molecular variations have been known in retinal blinding diseases in man and also in dogs. Different forms of retinal diseases occur in specific breed(s) caused by mutations segregating within each isolated breeding population. While molecular studies to find genes and mutations underlying retinal diseases in dogs have benefited largely from the phenotypic and genetic uniformity within a breed, within- and across-breed variations have often played a key role in elucidating the molecular basis. The increasing knowledge of phenotypic, allelic, and genetic heterogeneities in canine retinal degeneration has shown that the overall picture is rather more complicated than initially thought. Over the past 20?years, various approaches have been developed and tested to search for genes and mutations underlying genetic traits in dogs, depending on the availability of genetic tools and sample resources. Candidate gene, linkage analysis, and genome-wide association studies have so far identified 24 mutations in 18 genes underlying retinal diseases in at least 58 dog breeds. Many of these genes have been associated with retinal diseases in humans, thus providing opportunities to study the role in pathogenesis and in normal vision. Application in therapeutic interventions such as gene therapy has proven successful initially in a naturally occurring dog model followed by trials in human patients. Other genes whose human homologs have not been associated with retinal diseases are potential candidates to explain equivalent human diseases and contribute to the understanding of their function in vision.     

Changes in the human mitochondrial genome after treatment of malignant disease

Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in human cells. The mitochondrial genome encodes essential information for the synthesis of the mitochondrial respiratory chain. Inherited defects of this genome are an important cause of human disease. In addition, the mitochondrial genome seems to be particularly prone to DNA damage and acquired mutations may have a role in ageing, cancer and neurodegeneration. We wished to determine if radiotherapy and chemotherapy used in the treatment of cancer could induce changes in the mitochondrial genome. Such changes would be an important genetic marker of DNA damage and may explain some of the adverse effects of treatment. We studied samples from patients who had received radiotherapy and chemotherapy for point mutations within the mtDNA control region, and for large-scale deletions. In blood samples from patients, we found a significantly increased number of point mutations compared to the control subjects. In muscle biopsies from 7 of 8 patients whom had received whole body irradiation as well as chemotherapy, the level of a specific mtDNA deletion was significantly greater than in control subjects. Our studies have shown that in patients who have been treated for cancer there is an increased level of mtDNA damage.     

A polymorphism in a region with enhancer activity in the second intron of the human apolipoprotein B gene

A 443-base pair fragment (+622 to +1064) from the second intron of the human apolipoprotein B gene was shown to contain a tissue-specific enhancer when placed in front of an apolipoprotein B promoter-chloramphenicol acetyltransferase construct in transfection experiments. To identify potential regulatory mutations in this region of the gene, DNA from various subjects was examined for the presence of point mutations by means of chemical cleavage of mismatched heteroduplexes. An A----G substitution within the second intron of the gene at position +722 was identified in three unrelated subjects and confirmed by DNA sequencing. Although the base substitution was contained within a nuclear protein-binding site, as determined by DNase I footprinting, it did not appear to affect the protein/DNA interaction in its vicinity, as shown by gel retardation experiments. The single base substitution at position +722 abolishes a StyI restriction site, thus creating a StyI polymorphism. Using allele-specific oligonucleotides, we screened the DNA of 172 subjects for the presence of this polymorphism: two other subjects carrying the polymorphism were found. In each of the five unrelated subjects, the polymorphism was associated with the same haplotype.     

Supernumerary chromosomes and spermatogenesis in a human male carrier

Screening for lactate dehydrogenase (LDH) subunit deficiencies was performed on 2880 blood samples from healthy individuals in the Fukuoka Prefecture in Japan by means of electrophoresis. The frequencies of heterozygotes with either LDH-A or LDH-B deficiency were found to be 0.104% at each locus. These estimated frequencies of either LDH-A or LDH-B deficiencies were slightly lower than, but not significantly different from, those found previously in Shizuoka Prefecture. The genetic mutations in individuals heterozygous for LDH-B deficiency were analyzed by the polymerase chain reaction and DNA conformation polymorphism. Abnormal migration patterns were observed in individuals heterozygous for LDH-B deficiency. Subsequent sequence determination of the mutant alleles revealed three novel mutations: an eight-base duplication in exon 3, a four-base duplication in exon 4, and a one-base deletion in exon 7 of the LDH-B gene. These three mutations result in frameshift translation and premature termination. In addition, the mutations resulting in the duplication of eight or four nucleotides appear to cause a decrease in the levels of LDH-B mRNA.     

Hypoxanthine guanine phosphoribosyltransferase (HPRT) mutations in the Asian population

Mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome, which is characterized by hyperuricemia, severe motor disability, and self-injurious behavior, or HPRT-related gout (Kelley-Seegmiller syndrome). The marked heterogeneity of HPRT deficiency is well known, with more than 300 mutations at the HPRT gene locus having been reported (deletions, insertions, duplications, abnormal splicing, and point mutations at different sites of the coding region from exons 1 to 9). We have identified mutations in Asian families with patients manifesting different clinical phenotypes, including rare cases of female subjects, by analyzing all nine exons of the HPRT gene (HPRT1) from genomic DNA and reverse-transcribed mRNA using the polymerase chain reaction technique coupled with direct sequencing. We developed suitable methods to detect the mutations identified from respective families with HPRT deficiency. Then, prenatal genetic diagnoses in HPRT-deficient families were carried out using both mRNA and genomic DNA from chorionic villi or amniotic fluid cells. As shown here in the heterogeneity of HPRT mutations, the spectrum of 70 mutations identified in the Asian population fits the four main conclusions that emerged previously from worldwide analysis.     

Progressive Retinal Atrophy in the Border Collie: A new XLPRA

Background

Several forms of progressive retinal atrophy (PRA) segregate in more than 100 breeds of dog with each PRA segregating in one or a few breeds. This breed specificity may be accounted for by founder effects and genetic drift, which have reduced the genetic heterogeneity of each breed, thereby facilitating the identification of causal mutations. We report here a new form of PRA segregating in the Border Collie breed. The clinical signs, including the loss of night vision and a progressive loss of day vision, resulting in complete blindness, occur at the age of three to four years and may be detected earlier through systematic ocular fundus examination and electroretinography (ERG).

Results

Ophthalmic examinations performed on 487 dogs showed that affected dogs present a classical form of PRA. Of those, 274 have been sampled for DNA extraction and 87 could be connected through a large pedigree. Segregation analysis suggested an X-linked mode of transmission; therefore both XLPRA1 and XLPRA2 mutations were excluded through the genetic tests.

Conclusion

Having excluded these mutations, we suggest that this PRA segregating in Border Collie is a new XLPRA (XLPRA3) and propose it as a potential model for the homologous human disease, X-Linked Retinitis Pigmentosa.

     

Rapid generation of plant traits via regulation of DNA mismatch repair

The reversible inhibition of DNA repair is a novel approach to maximize genetic diversity within a plant's genome in order to generate offspring exhibiting important de novo output traits. This process is based on the inhibition of the evolutionarily conserved mismatch repair (MMR) system. In this process, a human dominant negative MMR gene allele is introduced into the germline of a target plant, yielding progeny that can be screened to identify variants with commercially important agronomic output traits. Using this novel strategy, we generated MMR-deficient Arabidopsis thaliana plants that showed genome-wide instability of nucleotide repeats associated with chromosomal microsatellites, in addition to base substitution mutations. Functional screenings of the MMR-deficient Arabidopsis offspring identified variants expressing selectable traits (ethylene insensitivity and salt tolerance), as well as plants exhibiting altered morphologic traits (albinos and dwarfs). We determined by segregation analyses of variant plants that the de novo phenotypes were due to both recessive and dominant genetic mutations. Mutations caused by MMR deficiency showed a different spectrum compared with those derived using ethylmethane sulphonate (EMS) mutagenesis. Our finding demonstrates the feasibility of using reversible MMR deficiency via transient expression of a single human gene product to enhance genetic diversity in plants.     

A visual pigment expressed in both rod and cone photoreceptors.

Rods and cones contain closely related but distinct G protein-coupled receptors, opsins, which have diverged to meet the differing requirements of night and day vision. Here, we provide evidence for an exception to that rule. Results from immunohistochemistry, spectrophotometry, and single-cell RT-PCR demonstrate that, in the tiger salamander, the green rods and blue-sensitive cones contain the same opsin. In contrast, the two cells express distinct G protein transducin alpha subunits: rod alpha transducin in green rods and cone alpha transducin in blue-sensitive cones. The different transducins do not appear to markedly affect photon sensitivity or response kinetics in the green rod and blue-sensitive cone. This suggests that neither the cell topology or the transducin is sufficient to differentiate the rod and the cone response.     

Pros and cons of inhibiting cholesteryl ester transfer protein

Whether or not it is desirable to inhibit cholesteryl ester transfer protein (CETP) has been an important question for over fifteen years since genetic CETP deficiency was found. Recently, some epidemiological studies which have been reported in Japan as well as Western countries help to clarify the atherogenicity of human subjects with mutations or polymorphisms in the CETP gene. In addition, some experimental atherosclerosis studies, in which CETP was inhibited in rabbits with different approaches, have been reported. There was a considerable difference in the atherogenicity of human CETP deficiency and CETP-inhibited rabbits. In this review, we summarize the recent advances in this field as well as discussing the significance of CETP in reverse cholesterol transport, a major protective system against atherosclerosis.     

The contribution of blue-sensitive cones to spatial responses of post-receptoral visual channels in man

Psychophysical methods developed for the investigation of spatial and temporal pathways in human vision have been applied in combination with the two-colour increment threshold technique of W. S. Stiles to study the way in which signals from blue-sensitive cones are transmitted along the visual pathways. A flicker sensitive spatio-temporal filter, designated 'ST2', has been examined by background modulation methods, and spatial filters sensitive to bars of a specific width by grating adaptation methods employing dichoptic presentation of stimuli. It is shown that the blue-sensitive (pi 3) spectral mechanism contributes to both classes of filter response, in a manner similar to that observed for the red-sensitive spectral mechanism. The binocularly driven, bar-sensitive filters have broad-band spectral response characteristics, thus the data demonstrate that signals arising in blue-sensitive cones converge onto a luminance channel. The results of this investigation, together with those previously published for a second (ST1) spatio-temporal filter, describe a variety of post-receptoral responses involving the pi 3 spectral mechanism.     

Frequent deletions of the human X chromosome distal short arm result from recombination between low copy repetitive elements

Substantial DNA deletions appear to be the molecular basis of several human genetic disorders but rarely account for the majority of observed mutations at any given locus. Exceptions in which deletions do account for the majority of observed abnormalities include the alpha-thalassemias, Duchenne muscular dystrophy, and steroid sulfatase deficiency. Variable deletion breakpoints have been recognized at the alpha-globin and dystrophin loci, but no information is available regarding STS deletions. We have found that these STS alterations usually involve breakpoints within highly similar sequence elements situated approximately 1.9 megabases apart on the X chromosome. It is surprising that these very large deletions produce such mild clinical abnormalities. These results may provide insight into the molecular mechanism of a number of human genetic defects.     

A germ line mutation within the coding sequence for the putative 5-phosphoribosyl-1-pyrophosphate binding site of hypoxanthine-guanine phosphoribosyltransferase (HPRT) in a Lesch-Nyhan patient: missense mutations within a functionally important region probably cause disease

Lesch-Nyhan syndrome caused by a complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT) is the result of a heterogeneous group of germ line mutations. Identification of each mutant gene provides valuable information as to the type of mutation that occurs spontaneously. We report here a newly identified HPRT mutation in a Japanese patient with Lesch-Nyhan syndrome. This gene, designated HPRT Tokyo, had a single nucleotide change from G to A, as identified by sequencing cDNA amplified by the polymerase chain reaction. Allele specific oligonucleotide hybridization analysis using amplified genomic DNA showed that the mutant gene was transmitted from the maternal germ line. This mutation would lead to an amino acid substitution of Asp for Gly at the amino acid position 140 located within the putative 5-phosphoribosyl-1-pyrophosphate (PRPP) binding region. Missense mutations in human HPRT deficient patients thus far reported tend to accumulate in this functionally active region. However, a comparison of the data suggested that both missense and synonymous mutations can occur at any coding sequence of the human germ line HPRT gene, but that a limited percentage of all the missense mutations cause disease. The probability that a mutation will cause disease tends to be higher when the missense mutation is within a functionally important sequence.     

Quantification of mitochondrial DNA mutation load

Mitochondrial DNA (mtDNA) mutations are an important cause of genetic disease and have been proposed to play a role in the ageing process. Quantification of total mtDNA mutation load in ageing tissues is difficult as mutational events are rare in a background of wild-type molecules, and detection of individual mutated molecules is beyond the sensitivity of most sequencing based techniques. The methods currently most commonly used to document the incidence of mtDNA point mutations in ageing include post-PCR cloning, single-molecule PCR and the random mutation capture assay. The mtDNA mutation load obtained by these different techniques varies by orders of magnitude, but direct comparison of the three techniques on the same ageing human tissue has not been performed. We assess the procedures and practicalities involved in each of these three assays and discuss the results obtained by investigation of mutation loads in colonic mucosal biopsies from ten human subjects.     

Molecular analysis of mutations in a patient with purine nucleoside phosphorylase deficiency.

Purine nucleoside phosphorylase (PNP) deficiency is an inherited autosomal recessive disorder resulting in severe combined immunodeficiency. The purpose of this study was to determine the molecular defects responsible for PNP deficiency in one such patient. The patient's PNP cDNA was amplified by PCR and sequenced. Point mutations leading to amino acid substitutions were found in both alleles. One point mutation led to a Ser-to-Gly substitution at amino acid 51 and was common to both alleles. In addition, an Asp-to-Gly substitution at amino acid 128 and an Arg-to-Pro substitution at amino acid 234 were found in the maternal and paternal alleles, respectively. In order to prove that these mutations were responsible for the disease state, each of the three mutations was constructed separately by site-directed mutagenesis of the normal PNP cDNA, and each was transiently expressed in COS cells. Lysates from cells transfected with the allele carrying the substitution at amino acid 51 retained both function and immunoreactivity. Lysates from cells transfected with PNP alleles carrying a substitution at either amino acid 128 or amino acid 234 contained immunoreactive material but had no detectable human PNP activity. In summary, molecular analysis of this patient identified point mutations within the PNP gene which are responsible for the enzyme deficiency.     

Heterogeneity of mutations in argininosuccinate synthetase causing human citrullinemia

Citrullinemia is an autosomal recessive disease caused by deficiency of argininosuccinate synthetase. In order to characterize mutations, RNA was isolated from cultured fibroblasts from 13 unrelated patients with neonatal citrullinemia. Ten mutations were identified by sequencing of amplified cDNA. Seven single base missense mutations were identified: Gly14----Ser, Ser180----Asn, Arg157----His, Arg304----Trp, Gly324----Ser, Arg363----Trp, and Gly390----Arg. Six of these missense mutations involved conversion of a CpG dinucleotide in the sense strand to TpG or CpA, and six of the seven mutations alter a restriction enzyme site in the cDNA. Two mutations were observed in which the sequences encoded by a single exon (exon 7 or 13) were absent from the cDNA. One mutation is a G----C substitution in the last position of intron 15 resulting in splicing to a cryptic splice site within exon 16. There is extreme heterogeneity of mutations causing citrulinemia. This heterogeneity may prove typical for less common autosomal recessive human genetic diseases.     

Maroteaux-lamy syndrome: five novel mutations and their structural localization

Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB). Mutation analysis in Maroteaux-Lamy syndrome resulted in the identification of approximately 40 molecular defects underlying a great genetic heterogeneity. Here we report five novel mutations in Italian subjects: S65F, P116H, R315Q, Q503X, P531R; each defect was confirmed by restriction enzyme or amplification refractory mutation system (ARMS) analysis. We also performed a three-dimensional (3-D) structure analysis of the alterations identified by us, and of an additional 22 point mutations reported by other groups, in an attempt to draw helpful information about their possible effects on protein conformation.     

Using the Hard, Randy, and Rittler Test to Evaluate Color Vision in Capuchins (Cebus libidinosus)

The identification of color vision types in primates is fundamental to understanding the evolution and biological function of color perception. The Hard, Randy, and Rittler (HRR) pseudoisochromatic test categorizes human color vision types successfully. Here we provide an experimental setup to employ HRR in a nonhuman primate, the capuchin (Cebus libidinosus), a platyrrhine with polymorphic color vision. The HRR test consists of plates with a matrix composed of gray circles that vary in size and brightness. Differently colored circles form a geometric shape (X, O, or Δ) that is discriminated visually from the gray background pattern. The ability to identify these shapes determines the type of dyschromatopsy (deficiency in color vision). We tested six capuchins in their own cages under natural sunlight. The subjects chose between two HRR plates in each trial: one with the gray pattern only and the other with a colored shape, presented on the left or right side at random. We presented the test 40 times and calculated the 95?% confidence limits for chance performance based on the binomial test. We also genotyped all subjects for exons 3 and 5 of the X-linked opsin genes. The HRR test diagnosed two subjects as protan dichromats (missing or defective L-cone), three as deutan dichromats (missing or defective M-cone), and one female as trichromat. Genetic analysis supported the behavioral data for all subjects. These findings show that the HRR test can be applied to diagnose color vision in nonhuman primates.