Mapping X-linked ophthalmic diseases: II. Linkage relationship of X-linked retinitis pigmentosa to X chromosomal short arm markers

Summary X-linked retinitis pigmentosa (XLRP) is a series of hereditary dystrophic diseases of the retina that occur in three clinically distinguishable variants: the classic form (McK-31360), a type known as choroidoretinal dystrophy (McK-30330), and a variant with golden-metallic or tapetal reflex in the heterozygote (McK30320). Controversy exists as to whether these phenotypic differences are due to clinical variability in disease expression, heterogeneity in disease alleles at a single locus, or a multiplicity of loci for XLRP. We have studied a single large kindred segregating for XLRP with the metallic fundus reflex in the heterozygote with restriction fragment length polymorphisms (RFLPs) from the short arm of the human X chromosome, and found measurable linkage to DXS7 (=12.5 cMorgans at LOD=2.5), the same RFLP previously shown by others to be tightly linked to the other forms of XLRP at =3cM. Although these estimates appeared to be different, each fell just within the 95% probability interval of the other and, therefore, were insufficient to prove or disprove that the metallic sheen form of XLRP is allelic with other forms of XLRP. Additional RFLPs at the DXS43 and the ornithine transcarbamoylase loci provided three-point crosses for determining the relative positions of DXS7 and XLRP, and supported an order that placed this form of XLRP distal to DXS7 on the Xp. Until the question of genetic heterogeneity is resolved, careful phenotypic characterization of the clinical type of XLRP present in families being used for linkage analyses is advisable.Presented in part at the American Society of Human Genetics meeting, Toronto, Canada, November 1, 1984     

A novel locus (RP24) for X-linked retinitis pigmentosa maps to Xq26-27.

Two genetic loci, RP2 and RP3, for X-linked retinitis pigmentosa (XLRP) have been localized to Xp11.3-11.23 and Xp21.1, respectively. RP3 appears to account for 70% of XLRP families; however, mutations in the RPGR gene (isolated from the RP3 region) are identified in only 20% of affected families. Close location of XLRP loci at Xp and a lack of unambiguous clinical criteria do not permit assignment of genetic subtype in a majority of XLRP families; nonetheless, in some pedigrees, both RP2 and RP3 could be excluded as the causative locus. We report the mapping of a novel locus, RP24, by haplotype and linkage analysis of a single XLRP pedigree. The RP24 locus was identified at Xq26-27 by genotyping 52 microsatellite markers spanning the entire X chromosome. A maximum LOD score of 4.21 was obtained with DXS8106. Haplotype analysis assigned RP24 within a 23-cM region between the DXS8094 (proximal) and DXS8043 (distal) markers. Other chromosomal regions and known XLRP loci were excluded by obligate recombination events between markers in those regions and the disease locus. Hemizygotes from the RP24 family have early onset of rod photoreceptor dysfunction; cone receptor function is normal at first, but there is progressive loss. Patients at advanced stages show little or no detectable rod or cone function and have clinical hallmarks of typical RP. Mapping of the RP24 locus expands our understanding of the genetic heterogeneity in XLRP and will assist in development of better tools for diagnosis.     

Normal pharmacological and morphometric parameters in the noradrenergic hyperinnervated mutant mouse, “tottering”

Summary Pharmacological and anatomical analyses of central monoaminergic and cholinergic neurons were performed in the tottering mouse, an autosomal recessive neurologic gene mutation that results in an overproduction of axons of the locus coeruleus and an increase in norepinephrine content in specific terminal fields. Except for the previously reported increase in norepinephrine content, all pharmacological parameters measured, including tyrosine hydroxylase activity, norepinephrine turnover, serotonin content, and choline acetyltransferase activity, in targets hyperinnervated by the locus coeruleus were normal. Immunocytochemical staining for tyrosine hydroxylase demonstrated the pronounced hyperinnervation in the tottering brain, whereas both serotonin and choline acetyltransferase immunostaining were similar between tottering and wild type. The volume of 3 target areas that are hyperinnervated by the locus coeruleus in the tottering mouse, the hippocampus, cerebellum, and cochlear nuclei, were normal. In addition, neuronal number and somal size in the locus coeruleus were found to be unchanged in the mutant genotype. These data demonstrate several features of the effects of the tottering gene: 1) compensatory changes in several adrenergic pharmacological parameters do not occur in response to the hyperinnervation of targets by locus coeruleus axons; 2) neither direct effects of the tottering gene on, nor compensatory changes in, the extent of cholinergic or serotonergic innervation of several targets of the locus coeruleus appear to occur; and 3) the lack of changes in size of the targets of the locus coeruleus suggest that the hyperinnervation in the tottering mouse is due to a direct genetic alteration of axonal growth by the locus coeruleus neurons, rather than to selective shrinkage of targets in the presence of normal terminal arbors.     

A recombination outside the BB deletion refines the location of the X linked retinitis pigmentosa locus RP3.

Genetic loci for X-linked retinitis pigmentosa (XLRP) have been mapped between Xp11.22 and Xp22.13 (RP2, RP3, RP6, and RP15). The RP3 gene, which is responsible for the predominant form of XLRP in most Caucasian populations, has been localized to Xp21.1 by linkage analysis and the map positions of chromosomal deletions associated with the disease. Previous linkage studies have suggested that RP3 is flanked by the markers DXS1110 (distal) and OTC (proximal). Patient BB was thought to have RP because of a lesion at the RP3 locus, in addition to chronic granulomatous disease, Duchenne muscular dystrophy (DMD), mild mental retardation, and the McLeod phenotype. This patient carried a deletion extending approximately 3 Mb from DMD in Xp21.3 to Xp21.1, with the proximal breakpoint located approximately 40 kb centromeric to DXS1110. The RP3 gene, therefore, is believed to reside between DXS1110 and the proximal breakpoint of the BB deletion. In order to refine the location of RP3 and to ascertain patients with RP3, we have been analyzing several XLRP families for linkage to Xp markers. Linkage analysis in an American family of 27 individuals demonstrates segregation of XLRP with markers in Xp21.1, consistent with the RP3 subtype. One affected mate shows a recombination event proximal to DXS1110. Additional markers within the DXS1110-OTC interval show that the crossover is between two novel polymorphic markers, DXS8349 and M6, both of which are present in BB DNA and lie centromeric to the proximal breakpoint. This recombination places the XLRP mutation in this family outside the BB deletion and redefines the location of RP3.     

Dihydropteridine reductase variation in man and the characid fish “Cheirodon axelrodi”: Evidence for a dimeric enzyme structure

Summary Genetic evidence for a dimeric structure of dihydropteridine reductase in man and in the fish species Cheirodon axelrodi and Salmo irideus is presented. A single locus in man and two loci in the fishes examined encode this enzyme. Zymograms revealed two alleles for the locus in man and two alleles for each locus in the fish Cheirodon axelrodi. The liver homogenate of a patient with dihydropteridine reductase deficiency showed no detectable activity in the gel, while his parents showed the normal electrophoretic phenotype.     

Icelandic families with autosomal dominant polycystic kidney disease: families unlinked to chromosome 16p13.3 revealed by linkage analysis

We have mainly used 3 highly polymorphic DNA markers, 3HVR (D16S85), 16AC2.5 (D16S291) and SM7 (D16S283), flanking the PKD1 region on chromosome 16p13.3 to establish linkage status in seven Icelandic families with autosomal dominant polycystic kidney disease (ADPKD). In four families, the disease locus is in the PKD1 region, and three families are unlinked to chromosome 16p13.3. In one of the unlinked families, the disease locus is excluded from a part of the long arm of chromosome 2, and we support a theory of more than 2 loci being responsible for ADPKD. Our data confirm the location of the locus YNH24 (D2S44) to chromosome 2q13-q24.     

Localization of the microsatellite probe DXS426 between DXS7 and DXS255 on Xp and linkage to X-linked retinitis pigmentosa.

The microsatellite marker DXS426 maps to the interval Xp21.1-Xp11.21, the chromosomal region which contains two loci for X-linked retinitis pigmentosa (XLRP; RP2 and RP3). We have refined the localization of DXS426 both physically, by mapping it to a deletion which spans the interval Xp21.3-Xp11.23, and genetically, by studying multiply informative crossovers which indicate that DXS426 lies between DXS7 and DXS255 (i.e., Xp11.4-Xp11.22). As this is the region which contains the RP2 gene, RP2 families could be identified on the basis of linkage of XLRP to DXS426. Multiply informative crossovers in two RP2 families indicate that the most likely location of the RP2 gene is between DXS426 and DXS7. DXS426 is therefore an important highly informative marker for the purposes of carrier detection and early diagnosis of RP2 and for the localization of the disease gene.     

One haplotype is associated with the Swiss type of hereditary persistence of fetal hemoglobin in the Yugoslavian population

Summary Blood samples from normal adults and from members of seven families with the Swiss type of hereditary persistence of fetal hemoglobin (HPFH) from Yugoslavia were analyzed for their fetal hemoglobin (Hb F) and ^G levels, while haplotyping defined the chromosomes at eight or nine polymorphic restriction sites. The data indicate that Swiss-HPFH, characterized by slightly elevated Hb F and ^G levels and no recognizable hematological abnormality, is associated with a chromosome whose restriction enzyme haplotype is identical to the no. 3 (Senegal) haplotype found in black sickle cell (SS) patients. Many adults with this chromosome have high ^G but normal Hb F levels. It is suggested that the Swiss-HPFH phenotype results from the action of more than one factor; one is linked to the -globin gene cluster and causes high ^G values, while others result in an increased Hb F production and are perhaps of different origins.     

Plant development in long-term embryogenic callus lines of Cucurbita pepo

Embryogenic callus derived from pumpkin hypocotyl segments was induced and maintained for 15 years on MS medium supplemented with the auxins IBA (4.9 M), 2, 4-D (4.5 M) or IAA (5.7 M). On induction media continued embryo maturation and development of adult plants typically failed. Therefore, small embryogenic clumps and individually isolated embryos were subcultured two to four times on one of the conversion media: MS supplemented with 1.5% sucrose and (a) no hormone, (b) 2.9 M IAA, (c) 5.7 M IAA, (d) 11.4 M IAA, (e) 12 M IEt, (f) 3.8 M ABA or (g) 2% activated charcoal. The cell line and the kind of auxin used in the induction and maintenance medium, both had a marked influence on the development of plantlets. The best result was achieved with a line that has been induced and maintained for 15 years on MS with IBA. In the IBA line, out of 100 embryos, 77 developed into plantlets on MS medium supplemented with 11.4 M IAA.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid - 2, 4-D 2, 4-di-chlorophenoxyacetic acid - IEt indole-3-etha-nol - MS Murashige and Skoog (1962) medium     

Effect of oxygen on growth and respiration of potato tuber callus

Growth and oxygen uptake of potato callus is faster in an oxygen-enriched atmosphere (70% oxygen, v/v; oxygen-callus) than in air (20% oxygen, v/v; air-callus). Especially the non-mitochondrial, so-called residual respiration is increased in oxygen-callus. The capacities of the mitochondrial respiratory pathways (cytochrome pathway, V_cyt and alternative pathway, V_alt) are also higher in this callus. In both callus types only a small part of the alternative pathway capacity is used in uninhibited respiration. The lower oxygen uptake of air-callus at normal air oxygen pressures is partially due to diffusional impedance. Measurement of the respiratory parameters of air-callus in oxygen-saturated medium leads to higher values than measurement in air-saturated medium, although these values are still lower than those of oxygen-callus.ATP-production was calculated from the oxygen-uptake data and compared with the dry weight production of the callus to give values of 10.0 and 10.8 g dry weight produced.-mol ATP^-1, for air-callus and oxygen-callus respectively. As no harmful side-effects are observed, cultivation of callus under elevated oxygen pressures may be useful, when rapid callus-growth is necessary.Abbreviations AA antimycin A; A; - BHAM benzohydroxamate - DW dry weight - FW fresh weight - 8-OHQ 8-hydroxyquinolin - RC respiratory control - SHAM salicylhydroxamate     

Lethal genes on the sex chromosomes concealed in a population of the mosquito Aedes aegypti L.

A population has been examined in which an overall parity between the sexes hides considerable between-family variation in sex ratio. A proportion of families show highly distorted sex ratios, with either an excess of females or an excess of males. Distorted sex ratios are invariably associated with mortality in the immature stages at a level appropriate to the action of recessive lethal genes. It has been shown that 26% of M-bearing (Y) chromosomes and at least 24% of m-bearing (X) chromosomes carry a recessive lethal gene.Two such genes have been investigated. l kills males and, in a cross between two heterozygotes, gives rise to a sex ratio close to 2:1 (excess families). k kills females and, in a cross between two heterozygotes, gives rise to a sex ratio close to 1:2 (excess families). Selection for excess or excess did not increase the level of sex ratio distortion.No crossing over occurs between k and the M/m locus whereas l shows 5–10% recombination with M/m. A test for allelism confirmed that l and k are not allelic. The penetrance of k is complete whereas l shows somewhat less than full penetrance. The penetrance of l has been improved by selection.The high frequency of lethals remained in the population during the two year period of study. There was evidence for heterosis preserving this frequency, the heterozygotes living longer and producing more progeny. However lethals were no longer to be found after four further years of laboratory culture.     

Microdeletions in interval 6 of the Y chromosome of males with idiopathic sterility point to disruption of AZF,a human spermatogenesis gene

Summary For males with idiopathic sterility, a molecular screen specific for small lesions (microdeletions) in interval 6 of the Y chromosome was set up using 29 Y-DNA probes. A de novo microdeletion in Y interval 6 was detected in 2 out of 19 chromosomally normal sterile males. The first microdeletion includes the Y-DNA probes pY6HP35 and 12f3; the second microdeletion includes the Y-DNA probes pY6HP52, 49f, FR15-II and the subinterval C of probe 50f2. A probe of the pY6H sequence family is present in both deletions. Sequences of this family cross-hybridize to dhMiF1, a DNA sequence of a fertility gene structure on the Y chromosome of Drosophila hydei. It was possible to map the position of the Y-deletion of one patient to the distal part of Yq11.22 or the proximal part of Yq11.23, and the deletion of the second patient to the distal part of Yq11.23. These microdeletions probably do not overlap. Since AZF, a human spermatogenesis gene, has been mapped to Y interval 6, we postulate that the microdeletions detected in this chromosome region affect the functional DNA structure of the AZF gene. If this holds true, it is possible that the AZF locus, cytogenetically mapped to distal Yq11, contains two spermatogenesis genes (AZFa and AZFb) or a large gene structure comparable to the Y fertility genes of Drosophila.     

Heterogeneity Analysis in 40 X-linked Retinitis Pigmentosa Families

Analysis of genetic heterogeneity in 40 kindreds with X-linked retinitis pigmentosa (XLRP), with 20 polymorphic markers, showed that significant heterogeneity is present (P=.001) and that 56% of kindreds are of RP3 type and that 26% are of RP2 type. The location of the RP3 locus was found to be 0.4 cM distal to OTC in the Xp21.1 region, and that of the RP2 locus was 6.5 cM proximal to DXS7 in Xp11.2-p11.3. Bayesian probabilities of linkage to RP2, RP3, or to neither locus were calculated. This showed that 20 of 40 kindreds could be assigned to one or the other locus, with a probability >.70 (14 kindreds with RP3 and 6 kindreds with RP2 disease). A further three kindreds were found to be unlinked to either locus, with a probability >.8. The remaining 17 kindreds could not be classified unambiguously. This highlights the difficulty of classifying families in the presence of genetic heterogeneity, where the two loci are separated by an estimated 16 cM.     

Syntheses of alpha-dystroglycan derived glycosyl amino acids carrying a novel mannosyl serine/threonine linkage

-Dystroglycan (-DG) is a membrane-associated, extracellular glycoprotein. It is anchored to the cell-membrane by binding to the transmembrane glycoprotein -dystroglycan (-DG) to form an /-DG-complex. It was discovered that the bovine peripheral nerve -DG possesses the Ser/Thr linked tetrasaccharide as the major constituent of the O-linked carbohydrates, which was proposed to contribute laminin binding activity of this glycoprotein.This structure has a striking feature in terms of the mode of linkage between oligosaccharide and the core protein. It has a mannose residue linked to the core protein through Ser/Thr residue. A similar structure was proposed to exist in brain derived HNK-1 immunoreactive O-glycans. Being interested in the structural novelty and potential biological significance of this type of glycan chains, the chemical synthesis of Ser/Thr linked mannose containing tetrasaccharide was investigated. Tetrasaccharide donor was constructed from monosaccharide blocks and coupled with Ser/Thr derivatives. Subsequent deprotection afforded target tetraosyl serine. Furthermore, synthetic routes to lower homologues, namely Gal--(1,4)-GlcNAc--(1,2)-Man--Ser and GlcNAc--(1,2)-Man--Ser were also provided.     

Three novel mutations causing a truncated protein within the RP2 gene in Italian families with X-linked retinitis pigmentosa

X-linked retinitis pigmentosa (XLRP) results from mutations in a number of loci, including RP2 at Xp11.3, and RP3 at Xp21.1. RP2 and RP3 genes have been identified by positional cloning. RP2 mutations are found in about 10% of XLRP patients. We performed a mutational screening of RP2 gene inpatients belonging to seven unrelated families in linkage with the RP2 locus. SSCP analysis detected three conformation variants, within exon 2 and 3. Direct sequencing of exon 2, disclosed a G-->A transition at nucleotide 449 (W150X), and a G-->T transversion in position 547 (E183X). Sequence analysis of exon 3 variant revealed an insertion (853/854insG), leading to a frameshift. In this patient, we detected an additional sequence alteration (A-->G at nucleotide 848, E283G). Each mutation was co-segregating with the disease in the affected family members available for the study. These mutations are expected to introduce a stop codon within the RP2 coding sequence probably resulting in a truncated or unstable protein.     

Remapping of the RP15 locus for X-linked cone-rod degeneration to Xp11.4-p21.1, and identification of a de novo insertion in the RPGR exon ORF15

X-linked forms of retinitis pigmentosa (XLRP) are among the most severe, because of their early onset, often leading to significant vision loss before the 4th decade. Previously, the RP15 locus was assigned to Xp22, by linkage analysis of a single pedigree with "X-linked dominant cone-rod degeneration." After clinical reevaluation of a female in this pedigree identified her as affected, we remapped the disease to a 19.5-cM interval (DXS1219-DXS993) at Xp11.4-p21.1. This new interval overlapped both RP3 (RPGR) and COD1. Sequencing of the previously published exons of RPGR revealed no mutations, but a de novo insertion was detected in the new RPGR exon, ORF15. The identification of an RPGR mutation in a family with a severe form of cone and rod degeneration suggests that RPGR mutations may encompass a broader phenotypic spectrum than has previously been recognized in "typical" retinitis pigmentosa.     

Mutants in yeast affecting ethidium bromide induced rho- formation and their effects on transmission and recombination of mitochondrial genes.

Summary A series of mutants called ebi, less inducible by ethidium bromide than the parental strain for the ^+– mutation have been isolated after E.M.S. mutagenesis. Some of the ebi mutants also show an important accumulation of ^– cells, in the absence of ethidium bromide. Ebi mutations are nuclearly inherited as shown by meiotic segregation. The effects of these mutants on the transmission and recombination of mitochondrial genes among the diploid progeny of crosses have been studied. Some of the ebi mutants show a non coordinated transmission of the oli1 mitochondrial marker with respect to other mitochondrial markers unexpected for homosexual crosses. This bias which is independent from will be discussed in relation to the segregation and recombination. No significant decrease of the frequency of recombinants has been detected.Abbreviations E.B. Ethidium bromide - E.M.S. Ethyl méthane sulfonate - C^S/C^R Allelic forms of the rib 1 locus conferring chloramphenicol sensitivity/resistance - E^S/E^R Allelic forms of the rib 3 locus conferring erythromycine sensitivity/resistance - O^R/O^R Allelic forms of the oli 1 locus conferring oligomycin sensitivity/resistance - P^S/P^R Allelic forms of the par 1 locus conferring paromomycine sensitivity/resistance - D^S/D^R Allelic forms of the diu 1 or diu 2 loci conferring diuron sensitivity/resistance - C^S/C^R Allelic forms of the mitochondrial locus - ⁺ grande or respiratory competent cells - ^– petite or cytoplasmic respiratory deficient cells     

Role of mitochondria in ethanol tolerance of Saccharomyces cerevisiae

The presence of active mitochondria and oxidative metabolism is shown to be essential to maintain low inhibition levels by ethanol of the growth rate (), fermentation rate (v) or respiration rate () of Saccharomyces cerevisiae wild type strain S288C. Cells which have respiratory metabolism show K _i (ethanol inhibition constant) values for , v and , higher (K _i>1 M) than those of petite mutants or grande strains grown in anaerobiosis (K _i=0.7 M). In addition, the relationship between or v and ethanol concentration is linear in cells with respiratory metabolism and exponential in cells lacking respiration. When functional mitochondria are transferred to petite mutants, the resulting strain shows K _i values similar to those of the grande strain and the inhibition of and v by increasing ethanol concentrations becomes linear.     

Structures of asparagine linked oligosaccharides of immunoglobulins (IgY) isolated from egg-yolk of Japanese quail

Structures of the Asn linked oligosaccharides of quail egg-yolk immunoglobulin (IgY) were determined in this study. Asn linked oligosaccharides were cleaved from IgY by hydrazinolysis and labelled withp-aminobenzoic acid ethyl ester (ABEE) afterN-acetylation. The ABEE labelled oligosaccharides were then fractionated by a combination of Concanavalin A-agarose column chromatography and anion exchange, normal phase and reversed phase HPLC before their structures were determined by sequential exoglycosidase digestion, methylation analysis, HPLC, and 500 MHz¹H-NMR spectroscopy. Quail IgY contained only neutral oligosaccharides of the following categories: the glucosylated oligomannose type (0.6%, Glc1-3Glc1-3Man₉GlcNAc₂; 35.6%, Glc1-3Man_7–9GlcNAc₂). oligomannose type (15.0%, with the structure Man_5–9GlcNAc₂) and biantennary complex type with core structures of-Man1-3(-Man1-6)Man1-4GlcNAc1-4GlcNAc (9.9%),-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4GlcNAc (25.1%) and-Man1-3(GlcNAc1-4)(-Man1-6)Man1-4GlcNAc1-4(Fuc1-6)GlcNAc (11.4%). Although never found in mammalian proteins, glucosylated oligosaccharides (Glc₁Man_7–9GlcNAc₂) have been located previously in hen IgY.Abbreviations IgG, IgM, IgA, IgY immunoglobulin G, M, A and Y, respectively - ABEE p-aminobenzoic acid ethyl ester