LINE-1 elements at the sites of molecular rearrangements in Alport syndrome-diffuse leiomyomatosis.

Deletions encompassing the 5' termini of the paired type IV collagen genes COL4A5 and COL4A6 on chromosome Xq22 give rise to Alport syndrome (AS) and associated diffuse leiomyomatosis (DL), a syndrome of disseminated smooth-muscle tumors involving the esophagus, large airways, and female reproductive tract. In this study, we report isolation and characterization of two deletion junctions. The first, in a patient described elsewhere, arose by a nonhomologous recombination event fusing a LINE-1 (L1) repetitive element in intron 1 of COL4A5 to intron 2 of COL4A6, resulting in a 13.4-kb deletion. The second, in a previously undescribed family, arose by unequal homologous recombination between the same L1 and a colinear L1 element in intron 2 of COL4A6, resulting in a>40-kb deletion. L1 elements have contributed to the emergence of this locus as a site of frequent recombinations by diverse mechanisms. These give rise to AS-DL by disruption of type IV collagen and perhaps other as yet unidentified genes, evidenced by deletions as small as 13.4 kb.     

The role of long-chain fatty-acid-CoA ligase 3 in vitamin D3 and androgen control of prostate cancer LNCaP cell growth

The antiproliferative effect of 1alpha,25(OH)(2)D(3) on human prostate cancer cells is well known, but the mechanism is still not fully understood, especially its androgen-dependent action. Based on cDNA microarray results, we found that long-chain fatty-acid-CoA ligase 3 (FACL3/ACS3) might play an important role in vitamin D(3) and androgen regulation of LNCaP cell growth. The expression of FACL3/ACS3 was found to be significantly upregulated by 1alpha,25(OH)(2)D(3) and the regulation was shown to be time-dependent, with the maximal regulation over 3.5-fold at 96h. FACL3/ACS3 was a dominant isoform of FACL/ACS expressed in LNCaP cells as indicated by measuring the relative expression of each isoform. 1alpha,25(OH)(2)D(3) had no significant effect on the expression of FACL1(FACL2), FACL4 and FACL6 except for its downregulation of FACL5 at 24 and 48h by around twofold. The upregulation of FACL3/ACS3 expression by 1alpha,25(OH)(2)D(3) was accompanied with increased activity of FACL/ACS as demonstrated by enzyme activity assay using a (14)C-labeled substrate preferential for FACL3/ACS3. The growth inhibitory effect of 1alpha,25(OH)(2)D(3) on LNCaP cells was significantly attenuated by FACL3/ACS3 activity inhibitor. Androgen withdrawal (DCC-serum), in the presence of antiandrogen Casodex or in AR-negative prostate cancer cells (PC3 and DU145), vitamin D(3) failed to regulate FACL3/ACS3 expression. The upregulation of FACL3/ACS3 expression by vitamin D(3) was recovered by the addition of DHT in DCC-serum medium. Western blot analysis showed that the expression of androgen receptor (AR) protein was consistent with vitamin D(3) regulation of FACL3/ACS3 expression. Taken together, the data suggest that the upregulation of FACL3/ACS3 expression by vitamin D(3) is through an androgen/AR-mediated pathway and might be one of the contributions of the vitamin D(3) antiproliferative effect in prostate cancer LNCaP cells.     

The LETM1/YOL027 gene family encodes a factor of the mitochondrial K+ homeostasis with a potential role in the Wolf-Hirschhorn syndrome

The yeast open reading frames YOL027 and YPR125 and their orthologs in various eukaryotes encode proteins with a single predicted trans-membrane domain ranging in molecular mass from 45 to 85 kDa. Hemizygous deletion of their human homolog LETM1 is likely to contribute to the Wolf-Hirschhorn syndrome phenotype. We show here that in yeast and human cells, these genes encode integral proteins of the inner mitochondrial membrane. Deletion of the yeast YOL027 gene (yol027Delta mutation) results in mitochondrial dysfunction. This mutant phenotype is complemented by the expression of the human LETM1 gene in yeast, indicating a functional conservation of LetM1/Yol027 proteins from yeast to man. Mutant yol027Delta mitochondria have increased cation contents, particularly K+ and low-membrane-potential Deltapsi. They are massively swollen in situ and refractory to potassium acetate-induced swelling in vitro, which is indicative of a defect in K+/H+ exchange activity. Thus, YOL027/LETM1 are the first genes shown to encode factors involved in both K+ homeostasis and organelle volume control.     

The gene encoding L1, a neural adhesion molecule of the immunoglobulin family, is located on the X chromosome in mouse and man

The murine and human genes for the L1 neural adhesion molecule were shown to lie on conserved regions of the X chromosome to which genes responsible for several neuromuscular diseases have been mapped and which are adjacent to the fragile site (FRAXA) associated with mental retardation. By pulsed-field gel mapping we have demonstrated physical linkage between the L1 gene and other genes located in Xq28: L1 lies between the eye pigment RCP, GCP locus and the glucose-6-phosphate dehydrogenase (G6PD) gene. This location is compatible with the implication of the L1 molecule in one of the X-linked neuromuscular diseases mapped to this region.     

Generation of 'humanized' hCYP1A1_1A2_Cyp1a1/1a2(-/-) mouse line

Human/rodent CYP1A1 and CYP1A2 orthologs are well known to exhibit species-specific differences in substrate preferences and rates of metabolism. This lab previously characterized a BAC-transgenic mouse carrying the human CYP1A1_CYP1A2 locus; in this line, human dioxin-inducible CYP1A1 and basal vs dioxin-inducible CYP1A2 have been shown to be expressed normally (with regard to mRNAs, proteins and three enzyme activities) in every one of nine mouse tissues studied. The mouse Cyp1a1 and Cyp1a2 genes are oriented head-to-head and share a bidirectional promoter region of 13,954 bp. Using Cre recombinase and loxP sites inserted 3' of the stop codons of both genes, we show here a successful interchromosomal excision of 26,173 bp that ablated both genes on the same allele. The Cyp1a1/1a2(-) double-knockout allele was bred with the "humanized" line; the final product is the hCYP1A1_1A2_Cyp1a1/1a2(-/-) line on a theoretically >99.8% C57BL/6J genetic background-having both human genes replacing the mouse orthologs. This line will be valuable for human risk assessment studies involving any environmental toxicant or drug that is a substrate for CYP1A1 or CYP1A2.     

A further study of a possible locus for schizophrenia on the X chromosome

Several studies suggest that the X chromosome may contain a gene for schizophrenia. In the present study, we recruited 142 male schizophrenic patients and their biological mothers from all parts of the United Kingdom to detect a genetic association for the SYP/CACNA1F locus in the Xp11 region and the FACL4 locus in the Xq22.3-Xq23 region. The haplotype-based haplotype relative risk (HHRR) analysis showed allelic association for rs2071316 (chi2=6.85, P=0.009) and rs5905724 (chi2=5.3, P=0.021) at the CACNA1F locus, but not for rs5943414 and rs1324805 at the FACL4 locus and rs3817678 at the SYP locus. The haplotype analysis showed a weak association for the rs3817678-rs2071316-rs5905724 haplotypes (chi2=12.19, df=4, P=0.016) but did not show such an association for the rs5943414-rs1324805 haplotypes (chi2=3.96, df=2, P=0.138). Because the linkage disequilibrium signal was detected only at the CACNA1F locus, this gene should perhaps be considered as being a candidate for schizophrenia although further work is needed to draw firm conclusions.     

Deletions in Xq26.3–q27.3 including FMR1 result in a severe phenotype in a male and variable phenotypes in females depending upon the X inactivation pattern

High resolution cytogenetics, microsatellite marker analyses, and fluorescence in situ hybridization were used to define Xq deletions encompassing the fragile X gene, FMR1, detected in individuals from two unrelated families. In Family 1, a 19-year-old male had facial features consistent with fragile X syndrome; however, his profound mental and growth retardation, small testes, and lover limb skeletal defects and contractures demonstrated a more severe phenotype, suggestive of a contiguous gene syndrome. A cytogenetic deletion including Xq26.3–q27.3 was observed in the proband, his phenotypically normal mother, and his learning-disabled non-dysmorphic sister. Methylation analyses at the FMR1 and androgen receptor loci indicated that the deleted X was inactive in > 95% of his mother’s white blood cells and 80–85% of the sister’s leukocytes. The proximal breakpoint for the deletion was approximately 10 Mb centromeric to FMR1, and the distal breakpoint mapped 1 Mb distal to FMR1. This deletion, encompassing ∼13 Mb of DNA, is the largest deletion including FMR1 reported to date. In the second family, a slightly smaller deletion was detected. A female with moderate to severe mental retardation, seizures, and hypothyroidism, had a de novo cytogenetic deletion extending from Xq26.3 to q27.3, which removed ∼12 Mb of DNA around the FMR1 gene. Cytogenetic and molecular data revealed that ∼50% of her white blood cells contained an active deleted X. These findings indicate that males with deletions including Xq26.3–q27.3 may exhibit a more severe phenotype than typical fragile X males, and females with similar deletions may have an abnormal phenotype if the deleted X remains active in a significant proportion of the cells. Thus, important genes for intellectual and neurological development, in addition to FMR1, may reside in Xq26.3–q27.3. One candidate gene in this region, SOX3, is thought to be involved in neuronal development and its loss may partly explain the more severe phenotypes of our patients. Received: 19 December 1996 / Accepted: 13 March 1997     

Turner's syndrome--correlation between karyotype and phenotype

Turner's syndrome is defined as a congenital disease determining by quantitative and/or structural aberrations of one from two X chromosomes with frequent presence of mosaicism. Clinically it is characterized by growth and body proportion abnormalities, gonadal dysgenesis resulting in sexual infantilism, primary amenorrhoea, infertility, characteristic stigmata, anomalies of heart, renal and bones and the presence of some diseases like Hashimoto thyroiditis with hypothyroidism, diabetes mellitus type 2, osteoporosis, hypertension. Turner's syndrome occurs in 1:2000 to 1:2500 female livebirth. The most frequent X chromosome aberrations in patients with phenotype of Turner syndrome are as follows: X monosomy - 45,X; mosaicism (50-75%), including 45,X/46,XX (10-15%), 45,X/46,XY (2-6%), 45,X/46,X,i(Xq), 45,X/46,X,del(Xp), 45,X/46,XX/47,XXX; aberration of X structure: total or partial deletion of short arm of X chromosome (46,X,del(Xp)) isochromosom of long arm of X chromosome (46,X,(i(Xq)), ring chromosome (46, X,r(X)), marker chromosome (46,X+m). Searching of X chromosome and mapping and sequencing of genes located at this chromosome (such as SHOX, ODG2, VSPA, SOX 3) have made possible to look for linkage between phenotypes and adequate genes or regions of X chromosome. In this paper current data concerning correlation between phenotype and karyotype in patients with TS have been presented.     

Identification of four highly conserved genes between breakpoint hotspots BP1 and BP2 of the Prader-Willi/Angelman syndromes deletion region that have undergone evolutionary transposition mediated by flanking duplicons

Prader-Willi and Angelman syndromes (PWS and AS) typically result from an approximately 4-Mb deletion of human chromosome 15q11-q13, with clustered breakpoints (BP) at either of two proximal sites (BP1 and BP2) and one distal site (BP3). HERC2 and other duplicons map to these BP regions, with the 2-Mb PWS/AS imprinted domain just distal of BP2. Previously, the presence of genes and their imprinted status have not been examined between BP1 and BP2. Here, we identify two known (CYFIP1 and GCP5) and two novel (NIPA1 and NIPA2) genes in this region in human and their orthologs in mouse chromosome 7C. These genes are expressed from a broad range of tissues and are nonimprinted, as they are expressed in cells derived from normal individuals, patients with PWS or AS, and the corresponding mouse models. However, replication-timing studies in the mouse reveal that they are located in a genomic domain showing asynchronous replication, a feature typically ascribed to monoallelically expressed loci. The novel genes NIPA1 and NIPA2 each encode putative polypeptides with nine transmembrane domains, suggesting function as receptors or as transporters. Phylogenetic analyses show that NIPA1 and NIPA2 are highly conserved in vertebrate species, with ancestral members in invertebrates and plants. Intriguingly, evolutionary studies show conservation of the four-gene cassette between BP1 and BP2 in human, including NIPA1/2, CYFIP1, and GCP5, and proximity to the Herc2 gene in both mouse and Fugu. These observations support a model in which duplications of the HERC2 gene at BP3 in primates first flanked the four-gene cassette, with subsequent transposition of these four unique genes by a HERC2 duplicon-mediated process to form the BP1-BP2 region. Duplicons therefore appear to mediate genomic fluidity in both disease and evolutionary processes.     

Vitamin D3 inhibits fatty acid synthase expression by stimulating the expression of long-chain fatty-acid-CoA ligase 3 in prostate cancer cells

FAS and FACL3 are enzymes of fatty acid metabolism. In our previous studies, we found that FAS and FACL3 genes were vitamin D3-regulated and involved in the antiproliferative effect of 1alpha,25(OH)2D3 in the human prostate cancer LNCaP cells. Here, we elucidated the mechanism behind the downregulation of FAS expression by vitamin D3. Triacsin C, an inhibitor of FACL3 activity, completely abolished the downregulation of FAS expression by vitamin D3, whereas an inhibitor of FAS activity, cerulenin, had no significant effect on the upregulation of FACL3 expression by vitamin D3 in LNCaP cells. In human prostate cancer PC3 cells, in which FACL3 expression is not regulated by vitamin D3, no regulation of FAS expression was seen. This suggests that the downregulation of FAS expression by vitamin D3 is mediated by vitamin D3 upregulation of FACL3 expression. Myristic acid, one of the substrates preferential for FACL3, enhanced the repression of FAS expression by vitamin D3. The action of myristic acid was abrogated by inhibition of FACL3 activity, suggesting that the enhancement in the downregulation of FAS expression by vitamin D3 is due to the formation of myristoyl-CoA. The data suggest that vitamin D3-repression of FAS mRNA expression is the consequence of feedback inhibition of FAS expression by long chain fatty acyl-CoAs, which are formed by FACL3 during its upregulation by vitamin D3 in human prostate cancer LNCaP cells.     

A genetic,deletion, physical,and human homology map of the long fin region on zebrafish linkage group 2

Mutation of the gene long fin causes overgrowth of zebrafish fins. Thus, molecular identification of the gene long fin may reveal the mechanisms involved in normal growth control. We have therefore developed genetic and physical maps of the corresponding region on linkage group 2 (LG2). A single deletion allele (lof(jg)(61)) of the long fin gene was also generated. Examination of this deletion for the presence or absence of ESTs independently mapped to LG2 revealed a contiguous deletion of SSLP, STS, and gene-based markers spanning a physical distance of approximately 500 kb, including at least 10 zebrafish genes. Human orthologs of the zebrafish genes in the long fin region were identified and revealed two synteny segments from human chromosome 1 (Hsa1) and Hsa19. Homology searching for additional genes linked to the human orthologs revealed one additional gene in the long fin deletion region. Thus, our development of the genetic, physical, deletion, and human homology maps of the long fin region provides one of the first high-resolution comparisons of a zebrafish region with a homologous human region, and facilitates the molecular identification of the long fin gene.     

A physical map of the mouse shaker-2 region contains many of the genes commonly deleted in Smith-Magenis syndrome (del17p11.2p11.2)

We report the construction of a physical map of the region of mouse chromosome 11 that encompasses shaker-2 (sh2), a model for the human nonsyndromic deafness DFNB3. DFNB3 maps within the common deletion region of Smith-Magenis syndrome (SMS), del(17)(p11.2p11.2). Eleven of the genes mapping within the SMS common deletion region have murine homologs on the sh2 physical map. The gene order in this region is not perfectly conserved between mouse and human, a finding to be considered as we engineer a mouse model of Smith-Magenis syndrome.     

Expression of macrophage-selective markers in human and rodent adipocytes

CD14, CD68 and/or mouse F4/80 or human epidermal growth factor module-containing mucin-like receptor 1 (EMR1) are widely used as macrophage-specific markers. Since macrophages infiltrate several tissues during inflammatory processes, CD14, CD68 and EMR1-F4/80 have been employed to discriminate between tissue-containing macrophages, like adipose tissue (AT), and other cells. Using real-time PCR experiments, we show that isolated adipocytes from humans and mice AT express high levels of CD14 and CD68 mRNA, whereas EMR1-F4/80 is mainly present in the macrophage-containing stroma-vascular fraction. Furthermore, fibroblasts-like cells (adipoblasts), preadipocytes and adipocytes from the murine cell lines, 3T3-F442A and BFC-1, express CD14 and CD68 mRNA and protein as determined by fluorescence-activated cell sorter, but not F4/80 which, as expected, is strongly expressed in the macrophage cell line RAW264.7. These results reinforce the view that EMR1-F4/80 is the best macrophage marker to date and show that CD14 and CD68 are not macrophage-specific proteins.