Physical mapping of 49 microsatellite markers on chromosome 19 and correlation with the genetic linkage map.

We have regionally localized 49 microsatellite markers developed by Généthon using a panel of previously characterized somatic cell hybrids that retain fragments from chromosome 19. The tight correlation observed between the physical and the genetic orders of the microsatellites provide cytogenetic anchorages to the genetic map data. We propose a position for the centromere just above D19S415, from the study of two hybrids, each of which retains one of the two derivatives of a balanced translocation t(1;19)(q11;q11). Microsatellites, which can be identified by a standard PCR protocol, are useful tools for the localization of disease genes and for the establishment of YAC or cosmid contigs. These markers can also judiciously be used for the characterization of new hybrid cell line panels. We report such a characterization of 11 clones, 8 of which were obtained by irradiation-fusion. Using the whole hybrid panel, we were able to define the order of 12 pairs of genetically colocalized microsatellites. As examples of gene mapping by the combined use of microsatellites and hybrid cell lines, we regionally assigned the PVS locus between the 19q13.2 markers D19S417 and D19S423 and confirmed the locations of fucosyltransferase loci FUT1, FUT2, and FUT5.

Regional assignment of 68 new human gene transcripts on chromosome 11.

We have tested 80 expressed sequence-tagged site (eSTS) markers assigned to human chromosome 11 by the Genexpress program on a panel of somatic cell hybrids containing parts of this chromosome, characterized by cytogenetic data, reference markers, and with respect to the Généthon microsatellite genetic map. Sixty-eight new gene transcripts have been assigned to 25 subregions, one of which was newly defined by five of the eSTS markers. The markers are distributed on the short and long arms in agreement with their physical length. The genic map thus obtained has been integrated with the cytogenetic, genetic, and disease maps. Two eSTS markers have been further mapped with respect to a yeast artificial chromosome (YAC) contig close to the brain-derived neurotrophic factor (BDNF) gene and thus provide potential candidate genes for the mental retardation phenotype of WAGR (Wilms' tumor, aniridia, genitourinary abnormalities and mental retardation) syndrome. Altogether, the 68 new gene transcripts localized here represent more than a threefold increase in the number of unknown regionalized genes that could reveal potential candidate genes for the numerous orphan pathologies associated with chromosome 11.

Relative positions of two clusters of human alpha-L-fucosyltransferases in 19q (FUT1-FUT2) and 19p (FUT6-FUT3-FUT5) within the microsatellite genetic map of chromosome 19.

Five on the seven cloned human fucosyltransferase genes have been mapped to two clusters, one on 19q and the other on 19p. Comparative DNA sequence analysis showed the Généthon microsatellite D19S596 lies 2.2 kb downstream of the coding region of FUT1, indicating that the cluster comprising the closely linked FUT1 and FUT2 genes is located 4 cM distal to D19S412 (lod score 13.7) and 9 cM proximal to D19S571 (lod score 11.7). Polymorphic markers of FUT3, FUT5, and FUT6 were used for linkage analysis with 14 Généthon microsatellites in Indonesian families. These three loci constitute a cluster on 19p, located between the Généthon microsatellites D19S216 and D19S567, which are known to be only 1 cM distant from each other. Two cross-overs, one between FUT6 and FUT3 and the other between FUT3 and FUT5, suggest the gene order 19pter-D19S216-FUT6-FUT3-FUT5-D19S567++ +-cen. Comparison of genetic and physical maps suggests that the FUT6-FUT3-FUT5 cluster is located on 19p13.3 and the FUT1-FUT2 cluster on 19q13.3. FUT6, FUT3 and FUT5 genes share more than 85% homology and encode three similar, but distinct alpha(1,3) fucosyltransferases. FUT1 and FUT2 share about 70% homology and encode two distinct alpha(1,2)fucosyltransferases. No sequence homology was found between the genes of the two clusters. The members of each of these two clusters have probably emerged by duplication and divergent evolution of two unrelated ancestor genes.

A 1.7-Mb YAC contig around the human BDNF gene (11p13): integration of the physical, genetic, and cytogenetic maps in relation to WAGR syndrome.

WAGR (Wilms tumor, aniridia, genito-urinary abnormalities, mental retardation) syndrome in humans is associated with deletions of the 11p13 region. The brain-derived neurotrophic factor (BDNF) gene maps to this region, and its deletion seems to contribute to the severity of the patients' mental retardation. Yeast artificial chromosomes (YACs) carrying the BDNF gene have been isolated and characterized. Localization of two known exons of this gene leads to a minimal estimation of its size of about 40 kb. Chimerism of the BDNF YACs has been investigated by fluorescence in situ hybridization and chromosome assignment on somatic cell hybrids. Using the BDNF gene, YAC end sequence tagged sites (STS), and Généthon microsatellite markers, we constructed a 1.7-Mb contig and refined the cytogenetic map at 11p13. The resulting integrated physical, genetic, and cytogenetic map constitutes a resource for the characterization of genes that may be involved in the WAGR syndrome.

Assignment of 112 microsatellite markers to 23 chromosome 11 subregions delineated by somatic hybrids: comparison with the genetic map.

Using a panel of 25 somatic cell hybrids, we have regionally localized 112 microsatellite markers generated by Généthon and assigned to chromosome 11. A genetic map of 74 of them was produced using linkage analysis of the eight largest CEPH (Centre d'Etude du Polymorphisme Humain) families. They could be ordered on chromosome 11 with an average distance of 2.1 cM. The tight correlation observed between the genetic order and the physical assignment of these microsatellites reinforces the genetic map data. These newly localized markers identified by the PCR method using a standardized protocol represent useful tools for mapping YAC clones and establishing YAC contigs and for studying genetic diseases or cancers associated with specific genes and/or germinal/somatic rearrangements of chromosome 11.

Molecular genetics of alpha-L-fucosyltransferase genes (H, Se, Le, FUT4, FUT5 and FUT6).

Six human alpha-L-fucosyltransferase genes have been registered in the GDB as FUT1 to FUT6 according to the chronology of their description. FUT1 and FUT2 encode the alpha(1,2)fucosyltransferases H and Se respectively. The FUT2 gene has not been cloned, but it is expected to be closely linked to FUT1 on the long arm of chromosome 19. FUT3, FUT4, FUT5 and FUT6 encode different alpha(1,3)fucosyltransferases which share between 60 and 90% homology with each other, but none with FUT1. Missense and nonsense point mutations have been found to inactivate the cognate enzymes of FUT1, FUT3 and FUT6. FUT3 and FUT6 are closely linked on the short arm of chromosome 19 and encode the Lewis and plasma enzymes respectively. The FUT5 gene has been cloned and sequenced, but its tissue expression has not been defined as yet. FUT4 has been mapped to 11q21 and encodes a monomorphic myeloid enzyme. All but FUT4 are genetically polymorphic. The deficient alleles of FUT1 and FUT6 have a very low incidence and they have been found mainly around the Indian Ocean. A myeloid enzyme is present in 5 to 10 week old human embryos and is later progressively replaced by different patterns of adult fucosyltransferase enzymes in all tissues, except in leukocytes and brain which continue to express a FUT4 like enzyme in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

The gene encoding myeloid alpha-3-fucosyl-transferase (FUT4) is located between D1 1S388 and D11S919 on 11q21.

The last step in the biosynthesis of Le(x) antigen, the addition of a fucose to precursor polysaccharides, can be catalyzed by different alpha-3-fucosyltransferases. We localized the gene (FUT4) encoding myeloid alpha-3-fucosyltransferase by PCR assay using panels of somatic cell and radiation hybrids which retain different rearrangements of chromosome 11. FUT4 was assigned to chromosome band 11q21 between D11S388 and D11S919.

Chromosomal localization of 9 KOX zinc finger genes: physical linkages suggest clustering of KOX genes on chromosomes 12, 16, and 19.

Nine KOX zinc finger genes were localized on four human chromosomes by in situ hybridization of cDNA probes to metaphase chromosomes. KOX1 (ZNF10), KOX11 (ZNF18), and KOX12 (ZNF19) were mapped to chromosome bands 12q24.33, 17p13-p12, and 16q22-q23, respectively. Six other KOX genes were localized on chromosome 19: KOX6 (ZNF14) and KOX13 (ZNF20) to 19p13.3-p13.2, KOX5 (ZNF13) and KOX22 (ZNF27) to 19q13.2-qter, and KOX24 (ZNF28) and KOX28 (ZNF30) to 19q13.4. Pulsed field gel electrophoresis experiments showed that the pairs of KOX genes found on the chromosome bands 12q24.33, 16q22-q23, 19p13.3-p13.2, or 19q13.3-qter lie within 200-300 kb DNA fragments. This suggests the existence of KOX gene clusters on these chromosomal bands.

Mapping around the Xq13.1 breakpoints of two X/A translocations in hypohidrotic ectodermal dysplasia (EDA) female patients.

Cellular hybrids were obtained from a t(X;12) identified in a female patient with hypohidrotic ectodermal dysplasia (EDA). This rearrangement had the same Xq13.1 cytogenetic breakpoint as a t(X;9) found in a previously observed EDA patient. A comparative analysis of these two rearrangements with nine probes was performed at the molecular level. These probes could define three subregions: three are proximal, two are distal, and four are between the two breakpoints. These last probes should prove useful for cloning the gene.

A multidrug-resistant ovarian carcinoma cell line with a malignant suppressed phenotype is a CD44 gene expression defective mutant.

A multidrug-resistant cell subline (OV1/VCR) derived from an ovarian adenocarcinoma cell line (OV1/P) was characterized by a typical suppressed malignant phenotype and by a unique karyotypic change: del(11)(p13). In an attempt to discern some genetic alteration of 11p genes that may be relevant to the phenotypic shift, cells were analyzed with DNA probes mapped in the deleted region and with monoclonal antibodies (MoAbs) against 11p-encoded membrane molecules. Southern blot did not detect abnormal restriction patterns of the probed sequences. OV1/VCR cells did not express the CD44 epitope (11p13 MIC4 locus) recognized by the F10-44 MoAb and did not accumulate RNAs of the CD44 (Hermes) core peptide. This defect was not detected in another OV1/P-derived drug-resistant subline that retained the malignant behavior and did not have the del(11p) marker. It may have contributed to phenotypic reversion because evidence shows that CD44 membrane molecule is involved in cell-cell interaction and growth regulation of cancer cells.

Five specificity patterns of (1----3)-alpha-L-fucosyltransferase activity defined by use of synthetic oligosaccharide acceptors. Differential expression of the enzymes during human embryonic development and in adult tissues.

The use of synthetic trisaccharides as acceptors led to the definition of five main (1----3)-alpha-L-fucosyltransferase activity patterns in human adult tissues: (I). Myeliod cells, granulocytes, monocytes, and lymphoblasts, transfer an alpha-L-fucopyranosyl group to O-3 of a 2-acetamido-2-deoxy-D-glucosyl residue of H blood-group Type 2 oligosaccharide [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----4)-beta-D-GlcpNAc----R] with Mn2+ as activator. (II) Brain has the same acceptor specificity pattern as myeloid cells, but can also use Co2+ as activator. (III) Plasma and liver transfer an alpha-L-furopyranosyl group to H blood-group Type 2 and to sialyl-N-acetyllactosamine [alpha-NeuAc-(2----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc----R]. (IV) Intestine, gall bladder, kidney, and milk have the same activity as (III), but also transfer an alpha-L-fucopyranosyl group to O-4 of a 2-acetamido-2-deoxy-D-glucose residue of H blood-group Type 1 [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc----R] and sialyl Type 1 [alpha-NeuAc-(1----3)-beta-D-Galp-(1----3)-beta-D-GlcpNAc----R]. (V) Stomach mucosa is not able to use sialyl-N-acetyllactosamine, but can transfer an alpha-L-fucopyranosyl group to the other Type 1 and Type 2 acceptors. Unlike in adult tissue, a single myeloid-like pattern of (1----3)-alpha-L-fucosyltransferase activity was found at early stages of development in all tissues tested. This embryonic enzyme is later progressively replaced by enzymes or mixtures of enzymes having the corresponding adult patterns of enzyme expression. All lymphoblastoid cell lines and half of the tumor epithelial cell lines tested expressed the myeloid-like pattern of enzyme found in normal embryonic tissues. The remaining tumor epithelial cell lines expressed different forms of (1----3/4)-alpha-L-fucosyltransferase acceptor specificity patterns.

Molecular studies of a translocated (X;22) DiGeorge patient using somatic cell hybridization.

In order to better characterize the chromosomic rearrangement of an unbalanced 45XX t(X;22) (q28;q11) DiGeorge patient, a somatic hybrid clone segregating the translocated chromosome was constructed and investigated using X and 22 linked markers. Our study demonstrated that this de novo translocation was from paternal origin. The breakpoint was assigned between DXS296 and IDS loci at Xq28 and between D22S9 and BCRL2 at 22q11. This observation and published data allow to locate a "critical region" for DiGeorge syndrome between these two last loci on 22q11. Our hybrid clone may be a useful tool for mapping new probes arising in this region.

Human chromosome 16 encodes a factor involved in induction of class II major histocompatibility antigens by interferon gamma.

Interferon gamma (IFN-gamma) induces expression of class II major histocompatibility complex (MHC)-encoded antigens in immunocompetent cells. To gain further insight into the mechanism of this induction, we prepared somatic cell hybrids between different human cell lines and a murine cell line, RAG, that does not express murine class II MHC antigens before or after treatment with murine IFN-gamma. Some of the resulting cell hybrids express murine class II MHC antigens when treated with murine IFN-gamma. This inducible phenotype is correlated with the presence of human chromosome 16. It has been shown previously that the induction of class I MHC antigens by human IFN-gamma in human-rodent hybrids requires the presence of species-specific factors encoded by chromosome 6, which bears the gene for the human IFN-gamma receptor, and chromosome 21, whose product(s) is necessary for the transduction of human IFN-gamma signals. In this report, we show that the induction of murine class II MHC antigens by human IFN-gamma in the human-RAG cell hybrids requires, likewise, the presence of human chromosomes 6 and 21, in addition to chromosome 16. In some of these hybrids, when all three of these human chromosomes were present, induction of cell-surface HLA-DR antigens was also observed. Our results demonstrate that human chromosome 16 encodes a non-species-specific factor involved in the induction of class II MHC antigens by IFN-gamma.

Chromosome 11q localization of one of the three expected genes for the human alpha-3-fucosyltransferases, by somatic hybridization.

Seventy-one human x mouse hybrid cell lines were used to map the locus of a human alpha-3-fucosyltransferase to 11q. The enzyme transfers fucose onto H type 2 more efficiently than onto sialyl-N-acetyllactosamine, suggesting that it is the myeloid type of alpha-3-fucosyltransferase (Mollicone et al., 1990), which makes the 3-fucosyllactosamine epitope on polymorphonuclear cells and monocytes. This epitope is also known as CD15 (Tetteroo et al., 1987).

More precise localization of the gene for Hunter syndrome.

A linkage analysis between the Hunter syndrome locus (IDS) and four polymorphic loci of the Xq27-Xq28 region, DXS105, DXS98, DXS304, and DXS52, was performed in large families. A significant lod score was obtained between DXS304 and the Hunter gene (Zmax = 6.57 at theta max = 0.0). The Hunter gene can be localized within 7 cM of this marker. In addition, the translocation breakpoint of the Hunter female case described by J. Mossman et al. (1986, Arch. Dis. Child. 58: 911-915) was localized between DXS98 and DXS304 using somatic cell hybrids. These two results are in agreement and give the following order: DXS105-DXS98-IDS-DXS304-DXS52. Probes for these marker loci can thus be used for carrier detection.

Strategy for constructing somatic hybrids isolating the two derivative chromosomes in X;autosome translocations. Application to a female patient t(X;5) with Hunter syndrome.

X; autosomal translocations are excellent tools for genetic analysis because of the easy selection of clones isolating the derivative bearing the HPRT gene in somatic cell hybrids. We have developed a strategy to select clones isolating the other derivative avoiding fastidious and time consuming technics, mainly based on immunofluorescent screening using MIC 2 and MIC 5 antigenic markers and we have succeeded in isolating in a rodent context the two X;5 translocated derivative chromosomes of a female patient with Hunter syndrome. The location of MIC 5 gene was specified between the IDS and G6PD DXS369 (RN1), DXS296 (VK21c), and DXS304 (U62), DXS52 and F8c (F814) are proximal and distal from the breakpoint disrupting the IDS gene respectively.

[21-deoxycortisol. A new marker of virilizing adrenal hyperplasia caused by 21-hydroxylase deficiency]. / Le 21-désoxycortisol. Un nouveau marqueur de l'hyperandrogénie surrénalienne par déficit en 21-hydroxylase.

21-deoxycortisol is a steroid produced mainly by the adrenal gland. Its normal plasma baseline concentrations (0.03 to 0.30 n/ml) and its concentrations after tetracosactide injection (0.15 to 0.76 ng/ml) do not significantly vary with age, sex and phases of the menstrual cycle. 21-deoxycortisol was assayed in plasma by a specific radioimmunological method and its values were compared with those of 17-OH progesterone in heterozygous subjects with the classical and non-classical forms of 21-hydroxylase deficiency, and in the amniotic fluid of foetuses with this deficiency. Baseline concentrations of 21-deoxycortisol in the classical forms of 21-hydroxylase deficiency (n = 12; 55.36 to 186.6 ng/ml) and post-tetracosactide concentrations in non-classical late onset forms (n = 31; 4.04 to 47 ng/ml) were much higher than in normal subjects, thus making this steroid as sensitive as, or even more sensitive than 17-OH progesterone in diagnosing 21-hydroxylase deficiency. Post-tetracosactide assays of 21-deoxycortisol in 84 heterozygous subjects with 21-hydroxylase deficiency (0.70 to 5.40 ng/ml) enabled these subjects to be detected with a more than 90 percent sensitivity, which cannot be obtained with 17-OH progesterone assays. 21-deoxycortisol concentrations in the amniotic fluid of foetuses with 21-hydroxylase deficiency (n = 11; 0.391 to 0.930 ng/ml) were constantly superior to those observed in normal foetuses (n = 38; 0.034 to 0.221 ng/ml), so that the deficiency can be diagnosed with the steroid as easily as with 17-OH progesterone.

CpG islands surround a DNA segment located between translocation breakpoints associated with genitourinary dysplasia and aniridia.

We have isolated a DNA segment absent from all the constitutionally deleted chromosomes 11 of our patients with Wilms tumor. This marker separates two balanced translocations that break in band 11p13: the distal one associated with aniridia (AN2), and the proximal one with genitourinary dysplasia (GUD). The GUD breakpoint maps within the smallest region of overlap (SRO) for the Wilms tumor (WT) gene locus, thus strengthening the previous suggestion of an association between Wilms tumor and other abnormalities of the genitourinary system. The 11p13 translocation breakpoint associated with T-cell acute lymphatic leukemia (T-ALL) is centromeric to the SRO and separated from the WT locus by at least one known gene. This region of the human genome (11p13) is rich in CpG islands that potentially identify genes, some of which may be involved in the various phenotypes associated with the WAGR syndrome. This is consistent with the proposition that the majority of human genes are in G-negative bands.

Characterization of human IFN-gamma response using somatic cell hybrids of hematopoietic and nonhematopoietic origin.

A panel of 27 rodent-human somatic cell hybrids composed of cells of hematopoietic (nonadherent cells) and nonhematopoietic origin (adherent cells) was used to identify the chromosomes involved in the biological response to human IFN-gamma (Hu-IFN-gamma). We found that the stimulation of class-I histocompatibility antigen expression correlates with the presence of human chromosomes 6 and 21 in adherent cell hybrids, while human chromosome 6 alone is sufficient in nonadherent hybrids. Scatchard analysis of the binding of radiolabeled Hu-IFN-gamma to nonadherent cell hybrids gave a Kd value similar to that found on human cell lines. Induction of a reporter gene placed under the transcriptional control of the interferon responsive sequence (IRS) in adherent cell hybrids requires both chromosomes 6 and 21. The antiviral protection by Hu-IFN-gamma in adherent cell hybrids was reached at physiological doses (2 units/ml) when human chromosomes 6 and 21 were present, while higher doses of Hu-IFN-gamma (5000 units/ml) were required for hybrids lacking chromosome 21. Thus, we demonstrate that differences exit in the response to Hu-IFN-gamma depending on the origin of the cell type.

Early prenatal diagnosis of 21-hydroxylase deficiency using amniotic fluid 17-hydroxyprogesterone determination and DNA probes.

The results of early prenatal diagnoses of congenital adrenal hyperplasia are reported. The determination of 17-hydroxyprogesterone values in amniotic fluid taken transabdominally at 11 weeks of gestation enabled prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency. There is a clear-cut difference between normal and pathological values at that time of pregnancy. This method of diagnosis can be combined with genotyping of the fetus by HLA-DNA probes on chorionic villus sampling or can be used alone. Prenatal diagnosis with a 21-OH probe is possible when a preliminary study has demonstrated that the index case is homozygous for the deletion.