X-linked severe combined immunodeficiency: localization within the region Xq13.1-q21.1 by linkage and deletion analysis.

X-linked severe combined immunodeficiency (SCID) (McKusick 30040; IMD4) is a disease of unknown pathogenesis characterized by severe and persistent infections from early in life that are due to absence of both cellular and humoral immune function. Although the disease has been provisionally mapped to proximal Xq, high lethality and lack of a carrier test have limited the number of scorable meioses. We performed linkage analysis in six new kindreds with X-linked SCID, using a random pattern of T-cell X inactivation to rule out the carrier state in at-risk women. Our linkage results, combined with analysis of Xq interstitial deletions, confirmed the regional assignment of X-linked SCID, narrowed the boundaries within which this locus lies to Xq13.1-q21.1, and established the locus order DXS159-(PGK1, SCID)-DXS72-DXS3, defining flanking markers for prenatal diagnosis and carrier testing.     

X-linked alpha-thalassemia/mental retardation (ATR-X) syndrome: localization to Xq12-q21.31 by X inactivation and linkage analysis.

We have examined seven pedigrees that include individuals with a recently described X-linked form of severe mental retardation associated with alpha-thalassemia (ATR-X syndrome). Using hematologic and molecular approaches, we have shown that intellectually normal female carriers of this syndrome may be identified by the presence of rare cells containing HbH inclusions in their peripheral blood and by an extremely skewed pattern of X inactivation seen in cells from a variety of tissues. Linkage analysis has localized the ATR-X locus to an interval of approximately 11 cM between the loci DXS106 and DXYS1X (Xq12-q21.31), with a peak LOD score of 5.4 (recombination fraction of 0) at DXS72. These findings provide the basis for genetic counseling, assessment of carrier risk, and prenatal diagnosis of the ATR-X syndrome. Furthermore, they represent an important step in developing strategies to understand how the mutant ATR-X allele causes mental handicap, dysmorphism, and down-regulation of the alpha-globin genes.     

Structural organization and complete sequence of the human alpha-N-acetylgalactosaminidase gene: homology with the alpha-galactosidase A gene provides evidence for evolution from a common ancestral gene.

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc; EC 3.2.1.49), the lysosomal glycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties in glycoconjugates, is encoded by a gene localized to chromosome 22q13----qter. The deficient activity of this enzyme results in Schindler disease, an autosomal recessive disorder characterized by the increased urinary excretion of glycopeptides and oligosaccharides containing alpha-N-acetylgalactosaminyl moieties. Recently, the 3.6-kb full-length alpha-GalNAc cDNA sequence was isolated and found to have remarkable nucleotide and predicted amino acid homology (55.8 and 46.9%, respectively) with the human alpha-galactosidase A (alpha-Gal A) cDNA. To investigate the possible evolutionary relatedness of the two glycosidases, the alpha-GalNAc chromosomal gene was isolated and characterized. Screening of a human genomic DNA cosmid library resulted in the identification of a clone, gAGB-1, with an approximately 35-kb insert that contained the entire alpha-GalNAc gene. A single approximately 15-kb EcoRI fragment of gAGB-1, which contained the complete 3.6-kb cDNA sequence, was digested and the subcloned fragments were sequenced in both orientations. The 13,709-bp alpha-GalNAc gene had nine exons ranging from 95 to 2028 bp and intronic sequences of 304 to 2684 bp. All exon/intron junctions conformed to the GT/AG consensus rule. Analysis of 1.4 kb of 5' flanking sequence revealed three Sp1 and two CAAT-like promoter elements. This region was GC-rich (56%), but no HTF island was identified. The gene contained six Alu-repetitive elements, all in the reverse orientation. Comparison of the structural organization of the alpha-GalNAc and the alpha-Gal A genes revealed that all six alpha-Gal A introns were identically positioned in the homologous alpha-GalNAc exonic sequence. Two additional introns, 1 and 8, were identfied in the alpha-GalNAc gene. The predicted amino acid sequences of alpha-GalNAc exons 2 through 7 and those of corresponding alpha-Gal A exons 1 through 6 were 46.2 to 62.7% identical. In contrast, there was little, if any, similarity between the deduced amino acid sequences of alpha-Gal A exon 7 and alpha-GalNAc exons 8 and 9. The remarkable amino acid identity and the identical exonic interruption by six introns of the alpha-GalNAc and alpha-Gal A genes suggest that this region in both genes is evolutionarily related and arose through duplication and divergence from a common ancestral gene.     

FXY2/MID2, a gene related to the X-linked Opitz syndrome gene FXY/MID1, maps to Xq22 and encodes a FNIII domain-containing protein that associates with microtubules

Opitz G/BBB syndrome (OS) is a genetically heterogeneous disorder with an X-linked locus and an autosomal locus linked to 22q11.2. OS affects multiple organ systems with often variable severity even between siblings. The clinical features, which include hypertelorism, cleft lip and palate, defects of cardiac septation, hypospadias, and anorectal anomalies, indicate an underlying disturbance of the developing ventral midline of the embryo. The gene responsible for X-linked OS, FXY/MID1, is located on the short arm of the human X chromosome within Xp22.3 and encodes a protein with both an RBCC (RING finger, B-box, coiled coil) and a B30.2 domain. The Fxy gene in mice is also located on the X chromosome but spans the pseudoautosomal boundary in this species. Here we describe a gene closely related to FXY/MID1, called FXY2, which also maps to the X chromosome within Xq22. The mouse Fxy2 gene is located on the distal part of the mouse X chromosome within a region syntenic to Xq22. Analysis of genes flanking both FXY/MID1 and FXY2 (as well as their counterparts in mouse) suggests that these regions may have arisen as a result of an intrachromosomal duplication on an ancestral X chromosome. We have also identified in both FXY2 and FXY/MID1 proteins a conserved fibronectin type III domain located between the RBCC and B30.2 domains that has implications for understanding protein function. The FXY/MID1 protein has previously been shown to colocalize with microtubules, and here we show that the FXY2 protein similarly associates with microtubules in a manner that is dependent on the carboxy-terminal B30.2 domain.     

Genetic characterization and molecular mapping of a Hessian fly-resistance gene transferred from T. turgidum ssp. dicoccum to common wheat

A gene (temporarily designated Hdic) conferring resistance to the Hessian fly (Hf) [Mayetiola destructor (Say)] was previously identified from an accession of German cultivated emmer wheat [Triticum turgidum ssp. dicoccum (Schrank ex Schübler) Thell] PI 94641, and was transferred to the Hf-resistant wheat germplasm KS99WGRC42. The inheritance of Hdic resistance exhibited incomplete penetrance because phenotypes of some heterozygous progenies are fully resistant and the others are fully susceptible. Five simple sequence repeat (SSR) markers (Xgwm136,Xcfa2153, Xpsp2999,Xgwm33, and Xbarc263) were linked to the Hdic gene on the short arm of wheat chromosome 1A in the same region as the H9, H10, and H11 loci. Flanking markers Xgwm33 and Xcfa2153 were mapped at distances 0.6 cM proximal and 1.4 cM distal, respectively. Marker analysis revealed that a very small intercalary chromosomal segment containing Hdic was transferred from emmer wheat to KS99WGRC42. This is the first emmer-derived Hf-resistance gene that has been mapped and characterized. The Hdic gene confers a high level of antibiosis to biotypes GP and L, as well as to strains vH9 and vH13 of the Hf, which is different from the biotype reaction patterns of the known Hf-resistance genes on chromosome 1A (H5 and H11 susceptible to biotype L, H9 and H10 susceptible to strain vH9). These results suggested that Hdic is either a new gene or a novel allele of a known H gene on chromosome 1A. The broad spectrum of resistance conferred by the Hdic gene makes it valuable for developing Hf resistant wheat cultivars. Mention of commercial or proprietary product does not constitute an endorsement by USDA.     

Cloning and characterization of RNF6, a novel RING finger gene mapping to 13q12.

Myeloproliferative disorders frequently show deletions or rearrangements of the long arm of chromosome 13. We report here the cloning of RNF6, a new gene that maps close to the chromosome 13 breakpoint in a case of myelofibrosis with a t(4;13)(q26;q12). RNF6 is predicted to encode a 685-amino-acid protein with a coiled-coil domain and a RING-H2 finger at the amino and carboxy terminis, respectively. In addition, we have identified a novel motif, Lys-X-X-Leu/Ile-X-X-Leu/Ile (KIL motif), that is located shortly upstream of a subset of RING-H2 proteins, including RNF6. Drosophila g1, rat Neurodap1, and mouse Praja1. FISH and physical mapping indicated that RNF6 is located at 13q12.2 close to marker D13S1121, and it is oriented from telomere to centromere. RNF6 is not disrupted by the t(4;13).     

PPM-X: a new X-linked mental retardation syndrome with psychosis, pyramidal signs, and macroorchidism maps to Xq28.

We report a three-generation family manifesting a previously undescribed X-linked mental retardation syndrome. Four of the six moderately retarded males have had episodes of manic-depressive psychosis. The phenotype also includes pyramidal signs, Parkinsonian features, and macroorchidism, but there are no characteristic dysmorphic facial features. Affected males do not show fragile sites at distal Xq on cytogenetic analysis, nor do they have expansions of the CGG repeats at the FRAXA, FRAXE, or FRAXF loci. Linkage analyses were undertaken, and a maximal LOD score of 3.311 at theta = .0 was observed with the microsatellite marker DXS1123 in Xq28. A recombination was detected in one of the affected males with DXS1691 (Xq28), which gives the proximal boundary of the localization. No distal recombination has been detected at any of the loci tested.     

Toward localization of the Werner syndrome gene by linkage disequilibrium and ancestral haplotyping: lessons learned from analysis of 35 chromosome 8p11.1-21.1 markers.

Werner syndrome (WS) is an autosomal recessive disorder characterized by premature onset of a number of age-related diseases. The gene for WS, WRN, has been mapped to the 8p 11.1-21.1 region with further localization through linkage disequilibrium mapping. Here we present the results of linkage disequilibrium and ancestral haplotype analyses of 35 markers to further refine the location of WRN. We identified an interval in this region in which 14 of 18 markers tested show significant evidence of linkage disequilibrium in at least one of the two populations tested. Analysis of extended and partial haplotypes covering 21 of the markers studied supports the existence of both obligate and probable ancestral recombinant events which localize WRN almost certainly to the interval between D8S2196 and D8S2186, and most likely to the narrower interval between D8S2168 and D8S2186. These haplotype analyses also suggest that there are multiple WRN mutations in each of the two populations under study. We also present a comparison of approaches to performing disequilibrium tests with multiallelic markers, and show that some commonly used approximations for such tests perform poorly in comparison to exact probability tests. Finally, we discuss some of the difficulties introduced by the high mutation rate at microsatellite markers which influence our ability to use ancestral haplotype analysis to localize disease genes.     

Lowe oculocerebrorenal syndrome: DNA-based linkage of the gene to Xq24-q26, using tightly linked flanking markers and the correlation to lens examination in carrier diagnosis.

The Lowe syndrome (LS), or oculocerebrorenal syndrome, has been studied using DNA-based linkage analysis, and the findings have been correlated with the result of a thorough ophthalmologic examination. It was found that the LS gene was linked to markers in the Xq24-q26 region and that the locus DXS42 was the most closely linked marker, giving a LOD score of 3.12 at zero recombination distance. Combined with earlier data, this forms the basis for carrier detection and prenatal diagnosis by using tightly linked flanking markers. A summary of our and other data suggests that the loci DXS17, DXS11, DXS87, and DXS42 are located on the proximal side, and DXS86 and DXS10 on the distal side of the Lowe locus. In isolated cases of LS the question of whether the mother is a carrier of the mutation arises. It was found that a lens examination with slit-lamp illumination and a count of the total number of lenticular opacities is a reliable method of ascertaining the carrier state.     

Functional identification of a Leishmania gene related to the peroxin 2 gene reveals common ancestry of glycosomes and peroxisomes.

Glycosomes are membrane-bounded microbody organelles that compartmentalize glycolysis as well as other important metabolic processes in trypanosomatids. The compartmentalization of these enzymatic reactions is hypothesized to play a crucial role in parasite physiology. Although the metabolic role of glycosomes differs substantially from that of the peroxisomes that are found in other eukaryotes, similarities in signals targeting proteins to these organelles suggest that glycosomes and peroxisomes may have evolved from a common ancestor. To examine this hypothesis, as well as gain insights into the function of the glycosome, we used a positive genetic selection procedure to isolate the first Leishmania mutant (gim1-1 [glycosome import] mutant) with a defect in the import of glycosomal proteins. The mutant retains glycosomes but mislocalizes a subset glycosomal proteins to the cytoplasm. Unexpectedly, the gim1-1 mutant lacks lipid bodies, suggesting a heretofore unknown role of the glycosome. We used genetic approaches to identify a gene, GIM1, that is able to restore import and lipid bodies. A nonsense mutation was found in one allele of this gene in the mutant line. The predicted Gim1 protein is related the peroxin 2 family of integral membrane proteins, which are required for peroxisome biogenesis. The similarities in sequence and function provide strong support for the common origin model of glycosomes and peroxisomes. The novel phenotype of gim1-1 and distinctive role of Leishmania glycosomes suggest that future studies of this system will provide a new perspective on microbody biogenesis and function.     

Isolation and initial characterization of a novel zinc finger gene, DNMT3L, on 21q22.3, related to the cytosine-5-methyltransferase 3 gene family

We have isolated the DNMT3L gene that is related to the cytosine-5-methyltransferase 3 (DNMT3) family. The gene is located on chromosome 21q22.3 between the AIRE and the KIAA0653 genes and spans approximately 16 kb of genomic sequence. The encoded protein of 387 amino acids has a cysteine-rich region containing a novel-type zinc finger domain that is conserved in DNMT3A and DNMT3B but also in ATRX, a member of the SNF2 protein family. The novel domain, called an ADD (ATRX, DNMT3, DNMT3L)-type zinc finger, contains two subparts: a C2C2 and an imperfect PHD zinc finger. Expression of the DNMT3L mRNA was not detectable by Northern blotting; however, RT-PCR amplification revealed that it is expressed at low levels in several tissues including testis, ovary, and thymus.