A gene for X-linked idiopathic congenital nystagmus (NYS1) maps to chromosome Xp11.4-p11.3

Congenital nystagmus (CN) is a common oculomotor disorder (frequency of 1/1,500 live births) characterized by bilateral uncontrollable ocular oscillations, with onset typically at birth or within the first few months of life. This condition is regarded as idiopathic, after exclusion of nervous and ocular diseases. X-linked, autosomal dominant, and autosomal recessive modes of inheritance have been reported, but X-linked inheritance is probably the most common. In this article, we report the mapping of a gene for X-linked dominant CN (NYS1) to the short arm of chromosome X, by showing close linkage of NYS1 to polymorphic markers on chromosome Xp11.4-p11.3 (maximum LOD score of 3.20, over locus DXS993). Because no candidate gene, by virtue of its function, has been found in this region of chromosome Xp, further studies are required, to reduce the genetic interval encompassing the NYS1 gene. It is hoped that the complete gene characterization will address the complex pathophysiology of CN.     

Assignment of the gene for complete X-linked congenital stationary night blindness (CSNB1) to Xp11.3

X-linked congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder characterized by a presumptive defect of neurotransmission between the photoreceptor and bipolar cells. Carriers are not clinically detectable. A new classification for CSNB includes a complete type, which lacks rod function by electroretinography and dark adaptometry, and an incomplete type, which shows some rod function on scotopic testing. The refraction in the complete CSNB patients ranges from mild to severe myopia; the incomplete ranges from moderate hyperopia to moderate myopia. To map the gene responsible for this disease, we studied eight multigeneration families, seven with complete CSNB (CSNB1) and one with incomplete CSNB, by linkage analysis using 17 polymorphic X-chromosome markers. We found tight genetic linkage between CSNB1 and an Xp11.3 DNA polymorphic site, DXS7, in seven families with CSNB1 (LOD 7.35 at theta = 0). No recombinations to CSNB1 were found with marker loci DXS7 and DXS14. The result with DXS14 may be due to the small number of scored meioses (10). No linkage could be shown with Xq loci PGK, DXYS1, DXS52, and DXS15. Pairwise linkage analysis maps the gene for CSNB1 at Xp11.3 and suggests that the CSNB1 locus is distal to another Xp11 marker, TIMP, and proximal to the OTC locus. Five-point analysis on the eight families supported the order DXS7-CSNB1-TIMP-DXS225-DXS14. The odds in favor of this order were 9863:1. Removal of the family with incomplete CSNB (F21) revealed two most favored orders, DXS7-CSNB1-TIMP-DXS255-DXS14 and CSNB1-DXS7-TIMP-DXS255-DXS14. Heterogeneity testing using the CSNB1-M27 beta and CSNB1-TIMP linkage data (DXS7 was not informative in F21) was not significant to support evidence of genetic heterogeneity (P = 0.155 and 0.160, respectively).     

Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.Abbreviations cDNA Complementary DNA - ESTs Expressed sequence tagsCommunicated by I.S. Chung     

Genetic heterogeneity of FG syndrome: a fourth locus (FGS4) maps to Xp11.4-p11.3 in an Italian family

FG syndrome (FGS, MIM 305450) is a rare X-linked recessive disorder comprising mental retardation and multiple malformations. Various families have been described to date, increasing our knowledge of the phenotype variability and making the clinical diagnosis complex, especially in sporadic patients. The first locus for FG syndrome (FGS1) was linked to chromosome region Xq12-q21.31, but other families have been excluded from this locus. The genetic heterogeneity of FG syndrome has been confirmed by analysis of an X chromosome inversion [inv(X)(q11q28)] in an affected boy and in his mentally retarded maternal uncle, suggesting that an additional locus for FG syndrome (FGS2, MIM 300321) is located at either Xq11 or Xq28. Recently, a third locus (FGS3) has been mapped to Xp22.3. We have identified and clinically characterized an Italian FG family, including 31 members with three affected males in two generations and two obligate carriers. We have excluded linkage to known FGS loci, whereas an extensive study of the whole X chromosome has yielded a maximum LOD score (Z(max)) of 2.66 (recombination fraction=0) for markers between DXS8113 and sWXD805. This new locus for FG syndrome corresponds to a region of approximately 4.6 Mb on the X chromosome.     

Linkage analysis of X-linked cone-rod dystrophy: localization to Xp11.4 and definition of a locus distinct from RP2 and RP3.

Progressive X-linked cone-rod dystrophy (COD1) is a retinal disease affecting primarily the cone photoreceptors. The COD1 locus originally was localized, by the study of three independent families, to a region between Xp11.3 and Xp21.1, encompassing the retinitis pigmentosa (RP) 3 locus. We have refined the COD1 locus to a limited region of Xp11.4, using two families reported elsewhere and a new extended family. Genotype analysis was performed by use of eight microsatellite markers (tel-M6CA, DXS1068, DXS1058, DXS993, DXS228, DXS1201, DXS1003, and DXS1055-cent), spanning a distance of 20 cM. Nine-point linkage analysis, by use of the VITESSE program for X-linked disorders, established a maximum LOD score (17.5) between markers DXS1058 and DXS993, spanning 4.0 cM. Two additional markers, DXS977 and DXS556, which map between DXS1058 and DXS993, were used to further narrow the critical region. The RP3 gene, RPGR, was excluded on the basis of two obligate recombinants, observed in two independent families. In a third family, linkage analysis did not exclude the RPGR locus. The entire coding region of the RPGR gene from two affected males from family 2 was sequenced and was found to be normal. Haplotype analysis of two family branches, containing three obligate recombinants, two affected and one unaffected, defined the COD1 locus as distal to DXS993 and proximal to DXS556, a distance of approximately 1.0 Mb. This study excludes COD1 as an allelic variant of RP3 and establishes a novel locus that is sufficiently defined for positional cloning.     

Large-scale evaluation of candidate genes identifies associations between VEGF polymorphisms and bladder cancer risk

Common genetic variation could alter the risk for developing bladder cancer. We conducted a large-scale evaluation of single nucleotide polymorphisms (SNPs) in candidate genes for cancer to identify common variants that influence bladder cancer risk. An Illumina GoldenGate assay was used to genotype 1,433 SNPs within or near 386 genes in 1,086 cases and 1,033 controls in Spain. The most significant finding was in the 5′ UTR of VEGF (rs25648, p for likelihood ratio test, 2 degrees of freedom = 1 × 10⁻⁵). To further investigate the region, we analyzed 29 additional SNPs in VEGF, selected to saturate the promoter and 5′ UTR and to tag common genetic variation in this gene. Three additional SNPs in the promoter region (rs833052, rs1109324, and rs1547651) were associated with increased risk for bladder cancer: odds ratio (95% confidence interval): 2.52 (1.06–5.97), 2.74 (1.26–5.98), and 3.02 (1.36–6.63), respectively; and a polymorphism in intron 2 (rs3024994) was associated with reduced risk: 0.65 (0.46–0.91). Two of the promoter SNPs and the intron 2 SNP showed linkage disequilibrium with rs25648. Haplotype analyses revealed three blocks of linkage disequilibrium with significant associations for two blocks including the promoter and 5′ UTR (global p = 0.02 and 0.009, respectively). These findings are biologically plausible since VEGF is critical in angiogenesis, which is important for tumor growth, its elevated expression in bladder tumors correlates with tumor progression, and specific 5′ UTR haplotypes have been shown to influence promoter activity. Associations between bladder cancer risk and other genes in this report were not robust based on false discovery rate calculations. In conclusion, this large-scale evaluation of candidate cancer genes has identified common genetic variants in the regulatory regions of VEGF that could be associated with bladder cancer risk.     

A novel approach for identifying candidate imprinted genes through sequence analysis of imprinted and control genes

Through the sequence analysis of 27 imprinted human genes and a set of 100 control genes we have developed a novel approach for identifying candidate imprinted genes based on the differences in sequence composition observed. The imprinted genes were found to be associated with significantly reduced numbers of short interspersed transposable element (SINE) Alus and mammalian-wide interspersed repeat (MIR) repeat elements, as previously reported. In addition, a significant association between imprinted genes and increased numbers of low-complexity repeats was also evident. Numbers of the Alu classes AluJ and AluS were found to be significantly depleted in some parts of the flanking regions of imprinted genes. A recent study has proposed that there is active selection against SINE elements in imprinted regions. Alternatively, there may be differences in the rates of insertion of Alu elements. Our study indicates that this difference extends both upstream and downstream of the coding region. This and other consistent differences between the sequence characteristics of imprinted and control genes has enabled us to develop discriminant analysis, which can be used to screen the genome for candidate imprinted genes. We have applied this function to a number of genes whose imprinting status is disputed or uncertain.     

A gene for X-linked optic atrophy is closely linked to the Xp11.4-Xp11.2 region of the X chromosome.

The aim of this study was to identify the chromosomal location of the disease-causing gene in a family apparently segregating X-linked optic atrophy. A large family of 45 individuals with a four-generation history of X-linked optic atrophy was reexamined in a full ophthalmic as well as electrophysiological examination. A DNA linkage analysis of the family was undertaken in order to identify the chromosomal location of the disease-causing gene. Linkage analysis was performed with 26 markers that spanned the entire X chromosome. The affected males showed very early onset and slow progression of the disease. Ophthalmic study of the female carriers did not reveal any abnormalities. Close linkage without recombination was found at the MAOB locus (maximum LOD score [Zmax] 4.19). The Zmax - 1 support interval was found at a recombination fraction of .076 distal and .018 proximal to MAOB. Multipoint linkage analysis placed the optic atrophy-causing gene in the Xp11.4-p11.21 interval between markers DXS993 and DXS991, whereas any other localization along the X chromosome could be excluded.     

Cloning and sequence analysis of candidate human natural killer-enhancing factor genes

A cytosol factor from human red blood cells enhances natural killer (NK) activity. This factor, termed NK-enhancing factor (NKEF), is a protein of 44000 M _r consisting of two subunits of equal size linked by disulfide bonds. NKEF is expressed in the NK-sensitive erythroleukemic cell line K562. Using an antibody specific for NKEF as a probe for immunoblot screening, we isolated several clones from a gt11 cDNA library of K562. Additional subcloning and sequencing revealed that the candidate NKEF cDNAs fell into one of two categories of closely related but non-identical genes, referred to as NKEF A and B. They are 88% identical in amino acid sequence and 71% identical in nucleotide sequence. Southern blot analysis suggests that there are two to three NKEF family members in the genome. Analysis of predicted amino acid sequences indicates that both NKEF A and B are cytosol proteins with several phosphorylation sites each, but that they have no glycosylation sites. They are significantly homologous to several other proteins from a wide variety of organisms ranging from prokaryotes to mammals, especially with regard to several well-conserved motifs within the amino acid sequences. The biological functions of these proteins in other species are mostly unknown, but some of them were reported to be induced by oxidative stress. Therefore, as well as for immunoregulation of NK activity. NKEF may be important for cells in coping with oxidative insults.     

Large-scale identification and characterization of genetic variants in asthma candidate genes

Asthma is a chronic inflammatory disorder of the airways, and a number of genetic loci are associated with the disease. Candidate gene association studies have been regarded as effective tools to study complex traits. Knowledge of the sequence variation and structure of the candidate genes is required for association studies. Thus, we investigated the genetic variants of 32 asthma candidate genes selected by colocalization of positional and functional candidate genes. We screened all exons and promoter regions of those genes using 12 healthy individuals and 12 asthma patients and identified a total of 418 single nucleotide polymorphisms (SNPs), including 270 known SNPs and 148 novel SNPs. Levels of nucleotide diversity varied from gene to gene (0.72×10⁻⁴–14.53×10⁻⁴), but the average nucleotide diversity between coding SNPs (cSNPs) and noncoding SNPs was roughly equivalent (4.63×10⁻⁴ vs 4.69×10⁻⁴). However, nucleotide diversity of cSNPs was strongly correlated to codon degeneracy. Nucleotide diversity was much higher at fourfold degenerate sites than at nondegenerate sites (9.42×10⁻⁴ vs 3.14×10⁻⁴). Gene-based haplotype analysis of asthma-associated genes in this study revealed that common haplotypes (frequency >5%) represented 90.5% of chromosomes, and they could be uniquely identified with five or fewer haplotype-tagging SNPs per gene. Therefore, our results may have important implications for the selection of asthma candidate genes and SNP markers for comprehensive association studies using large sample populations.     

Identification of drought-inducible genes and differentially expressed sequence tags in barley

Drought limits cereal yields in several regions of the world and plant water status plays an important role in tolerance to drought. To investigate and understand the genetic and physiological basis of drought tolerance in barley, differentially expressed sequence tags (dESTs) and candidate genes for the drought response were mapped in a population of 167 F₈ recombinant inbred lines derived from a cross between Tadmor (drought tolerant) and Er/Apm (adapted only to specific dry environments). One hundred sequenced probes from two cDNA libraries previously constructed from drought-stressed barley (Hordeum vulgare L., var. Tokak) plants and 12 candidate genes were surveyed for polymorphism, and 33 loci were added to a previously published map. Composite interval mapping was used to identify quantitative trait loci (QTL) associated with drought tolerance including leaf relative water content, leaf osmotic potential, osmotic potential at full turgor, water-soluble carbohydrate concentration, osmotic adjustment, and carbon isotope discrimination. A total of 68 QTLs with a limit of detection score 2.5 were detected for the traits evaluated under two water treatments and the two traits calculated from both treatments. The number of QTLs identified for each trait varied from one to 12, indicating that the genome contains multiple genes affecting different traits. Two candidate genes and ten differentially expressed sequences were associated with QTLs for drought tolerance traits.     

Large-scale identification of expressed sequence tags involved in rice and rice blast fungus interaction

To better understand the molecular basis of the defense response against the rice blast fungus (Magnaporthe grisea), a large-scale expressed sequence tag (EST) sequencing approach was used to identify genes involved in the early infection stages in rice (Oryza sativa). Six cDNA libraries were constructed using infected leaf tissues harvested from 6 conditions: resistant, partially resistant, and susceptible reactions at both 6 and 24 h after inoculation. Two additional libraries were constructed using uninoculated leaves and leaves from the lesion mimic mutant spl11. A total of 68,920 ESTs were generated from 8 libraries. Clustering and assembly analyses resulted in 13,570 unique sequences from 10,934 contigs and 2,636 singletons. Gene function classification showed that 42% of the ESTs were predicted to have putative gene function. Comparison of the pathogen-challenged libraries with the uninoculated control library revealed an increase in the percentage of genes in the functional categories of defense and signal transduction mechanisms and cell cycle control, cell division, and chromosome partitioning. In addition, hierarchical clustering analysis grouped the eight libraries based on their disease reactions. A total of 7,748 new and unique ESTs were identified from our collection compared with the KOME full-length cDNA collection. Interestingly, we found that rice ESTs are more closely related to sorghum (Sorghum bicolor) ESTs than to barley (Hordeum vulgare), wheat (Triticum aestivum), and maize (Zea mays) ESTs. The large cataloged collection of rice ESTs in this study provides a solid foundation for further characterization of the rice defense response and is a useful public genomic resource for rice functional genomics studies.     

Development of a 1.4-Mb BAC/PAC contig and physical map within the critical region for complete X-linked congenital stationary night blindness in Xp11.4

A physical map internal to the markers DXS1368 and DXS228 was developed for the p11.4 region of the human X chromosome. Twenty-four BACs and 10 PACs with an average insert size of 149 kb were aligned to form a contig across an estimated 1.4 Mb of DNA. This contig, which has on average fourfold clone coverage, was assembled by STS and EST content analysis using 46 markers, including 8 ESTs, two retinally expressed genes, and 22 new STSs developed from BAC- and PAC-derived DNA sequence. The average intermarker distance was 30 kb. This physical map provides resources for high-resolution mapping as well as suitable clones for large-scale sequencing efforts in Xp11.4, a region known to contain the gene for complete X-linked congenital stationary night blindness.     

Refined mapping of X-linked reticulate pigmentary disorder and sequencing of candidate genes

X-linked reticulate pigmentary disorder with systemic manifestations in males (PDR) is very rare. Affected males are characterized by cutaneous and visceral symptoms suggestive of abnormally regulated inflammation. A genetic linkage study of a large Canadian kindred previously mapped the PDR gene to a greater than 40 Mb interval of Xp22–p21. The aim of this study was to identify the causative gene for PDR. The Canadian pedigree was expanded and additional PDR families recruited. Genetic linkage was performed using newer microsatellite markers. Positional and functional candidate genes were screened by PCR and sequencing of coding exons in affected males. The location of the PDR gene was narrowed to a ∼4.9 Mb interval of Xp22.11–p21.3 between markers DXS1052 and DXS1061. All annotated coding exons within this interval were sequenced in one affected male from each of the three multiplex families as well as one singleton, but no causative mutation was identified. Sequencing of other X-linked genes outside of the linked interval also failed to identify the cause of PDR but revealed a novel nonsynonymous cSNP in the GRPR gene in the Maltese population. PDR is most likely due to a mutation within the linked interval not affecting currently annotated coding exons.     

Analysis of expressed genes in ethylene-treated potato leaves using expressed sequence tags

To isolate useful and interesting plant genes in large quantities, random sequencing of cDNA clones from potato leaf library treated with ethylene was performed. Partial sequences of randomly selected 210 clones with the insert of longer than 500 base pair (bp) as well as poly (A) tail have been compared with sequences in GeneBank, EMBL and DDBJ nucleic acid databases and fostered 193 expressed sequence tags (ESTs). The 210 cDNA clones identified are related to various aspect of metabolic pathways such as glycolysis, amino acid synthesis, translation mechanism, ribosome synthesis, hormone response, stress response, regulation of gene expression, and signal transduction. Among the 193 ESTs, 12 ESTs (29 cDNA clones) appeared more than once and 181 ESTs appeared once regarded as a solitary group. Out of 210 clones, 29 clones (13.8%) have no similarity to the known nucleotide sequences and could serve as a potentially useful resource for plant molecular biology referring to particular genes. Nucleotide sequencing to generate more ESTs from ethylene-induced as well as non-induced potato leaf is in progress as well.     

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.     

Multipoint linkage analysis of steroid sulfatase (X-linked ichthyosis) and distal Xp markers

Six families with steroid sulfatase deficiency (STS; X-linked ichthyosis) have been studied with the Xg blood group (XG) and the DNA markers dic56 (DXS143), 782 (DXS85), pD2 (DXS43), and GMGX9. Carrier status of females was determined by assay of STS in hair roots. GMGX9 detects a frequent restriction fragment length polymorphism and also identifies a deletion in the majority of families with STS deficiency, including five of the six reported here. The linkage relationship of this marker to the others was studied in normal three-generation families yielding 32 phase-known meioses informative for two or more markers. No recombinants were observed between STS and GMGX9, giving a maximum lod score of 8.73 at zero recombination. Multipoint linkage analysis taking STS and GMGX9 as a single locus and incorporating two-point marker data and STS-XG data from published studies gave the map (Sequence: see text). This order was 2.4 times more likely than with (STS,GMGX9) and dic56 reversed and is supported by our findings in a male with steroid sulfatase deficiency due to a deletion of Xp22.3 which encompasses the XG locus. He is deleted for GMGX9 but shows normal hybridization to dic56 and 782.