New insights into the phenotypes of 6q deletions.

Deletions of chromosome 6q are rare. We report 3 new patients with 6q deletions. Case 1 is a male with an interstitial deletion [del(6)(q13q14.2)], hypotonia, speech delays, and minor anomalies. Case 2 is a male with an interstitial deletion [del(6)(q16.2q22.32)] and malformations, including truncus arteriosus and bilateral oligodactyly. Case 3 is a male with a terminal deletion [del(6)(q25.2)] with retinal pits, hydrocephalus, atrioventricular canal, and hydronephrosis. The findings in our patients and those from 57 previously reported cases demonstrated 3 phenotypic groups associated with 6q deletions. Group A [del(6)(q11-q16)] had a high incidence of hernias, upslanting palpebral fissures, and thin lips with lower frequency of microcephaly, micrognathia, and heart malformations. Group B [del(6)(q15-q25)] was associated with increased intrauterine growth retardation, abnormal respiration, hypertelorism, and upper limb malformations. Group C [del(6)(q25-qter)] was associated with retinal abnormalities, cleft palate, and genital hypoplasia. The only universal finding among all patients with 6q deletions was mental retardation. Other findings common to all 3 groups included ear anomalies (90%), hypotonia (82%), and postnatal growth retardation (68%).

Human Ah receptor (AHR) gene: localization to 7p15 and suggestive correlation of polymorphism with CYP1A1 inducibility.

The mammalian aromatic hydrocarbon receptor (AHR) is a ubiquitous ligand-activated transcription factor. AHR ligands include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin), benzo[a]pyrene, and polychlorinated and polybrominated biphenyls; the endogenous ligand is not yet known. Following ligand binding, the AHR transcriptionally activates genes encoding drug-metabolizing enzymes important in both the metabolic potentiation of substrates to genotoxic reactive intermediates and ultimate carcinogens, and the detoxification of toxic or carcinogenic drugs and other environmental pollutants. AHR-mediated gene expression is also involved in many critical life processes (e.g. cell type-specific differentiation, cell division, apoptosis) by signal transduction mechanisms. Similar to mice, human populations exhibit a > 20-fold range of the CYP1A1 inducibility/AHR affinity phenotype. In the present study, we localized the human AHR gene to chromosome 7p15, using fluorescence in situ hybridization (FISH). Performing linkage analysis in a three-generation family, we show with good probability that the high CYP1A1 inducibility phenotype segregates with the 7p15 region. Sequencing 93 nt (31 amino acids) of the human AHR gene's exon 9, which is the region correlated with the mouse A375V polymorphism responsible for the major portion of high vs low CYP1A1 inducibility/AHR affinity, we found no nucleotide differences; Val-381 was present in all five individuals examined (four related and one unrelated), two of whom show "high' and three of whom show "low' CYP1A1 inducibility. These data indicate that the "high' and "low' CYP1A1 inducibility trait, in the population studied, cannot be explained by a difference among these 31 amino acids in exon 9 of the AHR gene.

Ullrich-Turner syndrome and neurofibromatosis-1.

There is a well-known association between neurofibromatosis-1 (NF1) and Noonan syndrome-like manifestations, including short stature, short broad neck, and hypertelorism. These anomalies are thought to be due to variable expression of the NF1 gene. We report on two girls with NF1 who were found to have the Ullrich-Turner syndrome. Case 1, a 12-year-old white girl, was followed in a Neurofibromatosis Clinic because of multiple café-au-lait spots and a family history of NF1 in her mother and sister. On examination, she had short stature, hypertelorism, and short neck with low posterior hairline. Karyotype was 86% 46,XY/14% 45,X. Case 2, the first child of a woman with NF1, presented at birth with lymphedema of hands and feet and a short broad neck. Karyotype was 45,X. At age 23 months she was short, had epicanthic folds, hypertelorism, narrow palate, right simian crease, 19 café-au-lait spots, and axillary freckling. We conclude that chromosome studies should be performed in girls with NF1 who have short stature and Noonan- or Ullrich-Turner-like findings. Dilemmas raised by the dual diagnoses of NF1 and Ullrich-Turner syndrome include potential risks of growth hormone therapy and estrogen replacement therapy.

Prenatal diagnosis of ring chromosome 6 in a fetus with hydrocephalus.

A patient with ring chromosome 6/monosomy 6 mosaicism is presented. At 25 weeks' gestation, ultrasound examination demonstrated fetal hydrocephalus. Amniocentesis was performed. The fetal karyotype was 45,XY,-6/ 45,XY,-6,+f/46,XY,r(6)(p25q27). Delivery of this male infant was by Caesarean section at 37 weeks' gestation. The karyotype in peripheral blood lymphocytes was 46,XY,r(6)(p25q27) with no indications of mosaicism. The infant had hydrocephalus which required treatment with a ventriculoperitoneal shunt at 22 days of age. He had no other obvious serious congenital anomalies. By 17 months he had developed microcephaly, seizures, severe bilateral hearing loss, and global development delay. This patient provides information regarding phenotypic variability of ring chromosome 6 and also reinforces the importance of offering amniocentesis if fetal hydrocephalus is detected as an isolated anomaly.

Cloning and regional assignment of the human myosin heavy chain 12 (MYH12) gene to chromosome band 15q21.

Sequences encoding 1,235 bp of the human myosin heavy chain 12 (MYH12) gene have been cloned from a human brain cDNA library by PCR amplification. The human sequence is 95.8% identical to the mouse sequence at the amino acid level, indicating that the MYH12 gene has been evolutionarily well conserved. Somatic cell hybrid analysis and in situ hybridization place the MYH12 gene on human chromosome 15, at band q21, and extend distally the known region of chromosome 15 linkage homology on mouse chromosome 9.

Hemizygosity at the insulin-like growth factor I receptor (IGF1R) locus and growth failure in the ring chromosome 15 syndrome.

The ring chromosome 15 syndrome is characterized by mild-to-severe growth failure. We evaluated the status of the insulin-like growth factor I receptor (IGF1R) gene, which had previously been assigned to band 15q26 in several patients with de novo ring 15 chromosomes, to investigate a possible correlation between disruption or loss of the IGF1R gene with the severe growth failure phenotype. The presence or absence of the IGF1R gene on the ring 15 chromosomes of five patients was ascertained by in situ hybridization and gene-dosage (Southern) blotting. The location of the breakpoints was determined by typing polymorphic markers from the distal end of the long arm of chromosome 15 in both the probands and their parents. Deletion mapping determined that all breakpoints were distal to D15S100 and that the IGF1R gene is located between D15S107 and D15S87. Three patients who had suffered severe growth failure in early childhood were hemizygous at the IGF1R locus, while one patient with borderline short stature had two copies of the IGF1R gene. The correlation between IGF1R gene dosage and growth retardation demonstrated here in our ring chromosome 15 patients suggests a possible role for heterozygous IGF1R gene mutations or deletions in other cases of unexplained growth failure.

Gene for a tissue-specific transcriptional activator (EBF or Olf-1), expressed in early B lymphocytes, adipocytes, and olfactory neurons, is located on human chromosome 5, band q34, and proximal mouse chromosome 11.

Murine B lymphocytes, adipocytes, and olfactory neurons contain a DNA-binding protein that participates in the regulation of genes encoding tissue-specific components of signal transduction. Purification and cloning of this protein, termed early B-cell factor (EBF), from murine B lymphocytes and independent cloning of a protein, termed Olf-1, from olfactory neuronal cells revealed virtual complete amino acid sequence identity between these proteins. As a first step towards identifying a human genetic disorder or mouse mutation for which EBF could be a candidate gene, we have chromosomally mapped the corresponding locus in both species. By Southern hybridization analyses of somatic cell hybrid panels with murine cDNA probe, fluorescence chromosomal in situ hybridization (FISH) of human genomic clones, and analysis of recombinant inbred mouse strains, we have found single sites for EBF homologous sequences on human Chromosome (Chr) 5, band q34, and on proximal mouse Chr 11, in an evolutionarily conserved region.

Chromosome localizations of genes for five cAMP-specific phosphodiesterases in man and mouse.

Cyclic nucleotides are important second messengers that mediate a number of cellular responses to external signals. Cyclic nucleotide phosphodiesterases play a role in signal transduction by regulating the cellular concentrations of these messengers. Here, we have applied Southern analyses of somatic cell hybrid lines and of recombinant inbred (RI) mouse strains as well as fluorescence chromosomal in situ hybridization (FISH) to chromosomally localize five cAMP-specific nucleotide phosphodiesterase genes in human and mouse. Genes DPDE1, DPDE2, DPDE3, and DPDE4 that share sequence homology with the Drosophila dunce gene were assigned to human chromosome 19 (DPDE1 and DPDE2), 5q12 (DPDE3), and 1p31 (DPDE4) and to mouse chromosomes 8, 9, 13, and 4, respectively. The high-affinity cAMP-specific phosphodiesterase gene (HCP1) was mapped to human chromosome 8q13-q22. Since these genes are potential candidates for involvement in psychiatric or behavioral disorders, knowledge of their chromosomal localizations will facilitate the discovery of their association with disease genes as they are being mapped by linkage studies.

Autosomal recessive neuromuscular disorder in a transgenic line of mice.

We have generated a line of transgenic mice that when homozygous for the transgene develop a severe, adult-onset neuromuscular disorder. This mutation is likely the result of the insertional inactivation of an endogenous gene by the transgene integration. The mutant mice have a gait abnormality with stiffened and/or splayed hind legs, and adopt a hunched posture with some exhibiting kyphosis of the thoracic spine. These symptoms progress gradually to severe motor dysfunction. Pathologic changes were found in skeletal muscle and peripheral nerve of the mutant animals. In young mice the muscles from both upper and lower extremities show necrosis and phagocytosis. In older mice, regeneration with muscle fiber splitting, internally located nuclei, and variable fiber size are conspicuous features. Interactions between Schwann cells and axons also appear disrupted in these animals. Although many peripheral axons are well myelinated, the nerve and nerve roots contain very large bundles of juxtaposed, bare axons, reminiscent of Schwann cell-axon interactions in early development. Within these bundles there are axons large enough to be myelinated. The relationship between the pathologic changes in the muscles and nerves is not clear. The phenotypic abnormalities of these animals resemble those that occur in the spontaneous mouse mutants dystrophia muscularis and myodystrophy. Nevertheless, the chromosomal position of the transgene integration site, which was mapped by fluorescent in situ hybridization to chromosome 11, indicates that this disorder represents a new neuromuscular mutation.

Structure and localization on the X chromosome of the gene coding for the human filopodial protein moesin (MSN).

Moesin is a member of a recently discovered family of closely related proteins that includes ezrin, radixin, and merlin. It is widely expressed in different tissues and cells and has been localized to filopodia and other membranous protrusions that are important for cell-cell recognition and signaling and cell movement. Here, we have localized the coding gene (MSN) to Xq11.2-q12 by Southern and Western blot analyses of Chinese hamster x human somatic cell hybrids and by fluorescence chromosomal in situ hybridization. Moesin-like sequences were identified on chromosomes 5 and 6. The murine Msn locus was mapped to the X chromosome as well by studying a rodent x mouse hybrid panel. The structure of the human moesin gene has been determined. The 12 exons are distributed over > 30 kb, and the exon/intron junctions demarcate individual highly conserved domains. Primer extension analysis revealed two major start transcription sites, 184 and 133 bp upstream of the initiation codon. The 5'-flanking region is GC-rich, lacks a TATA box, and contains four SP1 and one AP1 binding sites.

Gene for the alpha-subunit of the human interleukin-3 receptor (IL3RA) localized to the X-Y pseudoautosomal region.

Interleukin-3 (IL3) and granulocyte/macrophage colony-stimulating factor (CSF2) stimulate proliferation and differentiation of various hemopoietic cell types. As is characteristic of the cytokine receptor family, the receptors for these proteins comprise alpha- and beta-subunits. While IL3 and CSF2 receptors each have unique alpha- subunits, they share a common beta-subunit. By Southern analysis of somatic cell hybrid panels, pulsed-field gel electrophoresis (PFGE), and fluorescence chromosomal in situ hybridization, we have mapped the cloned sequence for the IL3 receptor alpha (IL3RA) to the X-Y pseudoautosomal region at bands Xp22.3 and Yp11.3, near the gene for the alpha-subunit of the CSF2 receptor (CSF2RA). The CSF2RA and IL3RA genes are so close that their order could not be determined by two-color interphase in situ hybridization. They share PFGE fragments generated by different restriction enzymes down to the 50-100-kb size range. Pseudoautosomal inheritance was demonstrated by an EcoRI RFLP detected with the IL3RA cDNA probe.

Human dystroglycan: skeletal muscle cDNA, genomic structure, origin of tissue specific isoforms and chromosomal localization.

Dystroglycan is a novel laminin binding component of the dystrophin-glycoprotein complex which provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. Here we report the cDNA and genomic structure of human dystroglycan. The human dystroglycan is encoded by a single gene (DAG1) mapped to chromosome 3 band p21. The coding sequence is organized into two exons, separated by a large intron. The predicted amino acid sequence of human and rabbit dystroglycan are 93% identical with predicted glycosylation sites being conserved. Human dystroglycan is expressed in a variety of fetal and adult tissues. Our data suggest that muscle and non-muscle isoforms of dystroglycan differ by carbohydrate moieties but not protein sequence. Therefore, we hypothesize that variable glycosylation of the conserved protein core might modulate laminin binding. The relationship of dystroglycan to human diseases is discussed.

cDNA cloning of the two subunits of human CAAX farnesyltransferase and chromosomal mapping of FNTA and FNTB loci and related sequences.

The CAAX farnesyltransferase is a heterodimeric enzyme that attaches a farnesyl group to a single cysteine in several cellular proteins. Substrates include the p21ras proteins, nuclear lamins, and several retinal proteins, all of which end with a "CAAXbox," where C is cysteine, A is an aliphatic amino acid, and X is methionine or serine. Full-length cDNAs for the alpha and beta subunits of the rat farnesyltransferase have been cloned, and both have been shown to be essential for catalytic activity. Here we have used the rat cDNAs to clone cDNAs for the human alpha and beta subunits. Comparison of the human and rat amino acid sequences revealed a remarkable degree of conservation (93% identity for the alpha subunit and 96% identity for the beta subunit). The functional genes for the alpha and beta subunits of human farnesyltransferase (gene symbols, FNTA and FNTB) were localized to human chromosome bands 8p22-q11 and 14q23-q24, respectively, by Southern blot hybridization and PCR analyses of panels of human x Chinese hamster somatic cell hybrid lines and by fluorescence chromosomal in situ hybridization. We also found several related farnesyltransferase genes. FNTAL1 was assigned to 11q13.4-q14.1, FNTAL2 to chromosome 13, and FNTBL1 to chromosome 9.

Structural comparison and chromosomal localization of the human and mouse IL-13 genes.

The genomic structure of the recently described cytokine IL-13 has been determined for both human and mouse genes. The nucleotide sequence of a 4.6-kb DNA segment of the human gene is described. The human IL-13 gene (IL13) occurs as a single copy in the haploid genome and maps to human chromosome 5. A 4.3-kb DNA fragment of the mouse IL-13 gene (IL13) has been sequenced and found to occur as a single copy, mapping to mouse chromosome 11. Intrachromosomal mapping studies revealed that both genes contain four exons and three introns and show a high degree of sequence identity throughout their length. Potential recognition sequences for transcription factors that are present in the 5'-flanking region and are conserved between both genes include IFN-responsive elements, binding sites for AP-1, AP-2 and AP-3, and NF-IL 6 site, and a TATA-like sequence. Both genes map to chromosomal locations adjacent to genes encoding other cytokines, including IL-3, GM-CSF, IL-5, and IL-4, suggesting that IL-13 is another member of this cytokine gene family that may have arisen by gene duplication.

The Vin-1 gene, identified by provirus insertional mutagenesis, is the cyclin D2.

The Vin-1 gene was initially identified as a gene whose expression is altered by the integration of proviruses in the Vin-1 common site of integration in retrovirus-induced rodent T-cell leukemias. We have now isolated the Vin-1 cDNA. Sequencing of the Vin-1 cDNA and Vin-1 exons revealed that the proviruses are integrated at the 5' end of the Vin-1 gene in an inverse transcriptional orientation. The sequence of the Vin-1 gene is identical to that of the recently identified G1-phase cyclin D2 gene. The human homolog of the Vin-1/cyclin D2 gene (CCND2) was mapped to chromosome 12, band p13.3, by in situ hybridization, confirming previous mapping data. Our results strongly support a role of the cyclin D2 gene in oncogenesis and thereby implicate altered cell cycle regulation in transformation.

Molecular cloning, cDNA sequence, and chromosomal assignment of the human radixin gene and two dispersed pseudogenes.

Radixin is a cytoskeletal protein that may be important in linking actin to the plasma membrane. Recent cloning of the murine and porcine radixin cDNAs revealed a protein highly homologous to ezrin and moesin. We have cloned and sequenced the human radixin cDNA and found the predicted amino acid sequence for the human protein to be nearly identical to those predicted for radixin in the two other species. By Southern analyses of Chinese hamster x human somatic cell hybrid DNA and of PCR products derived from hybrids, the coding gene (RDX) was mapped to 11q. Fluorescence chromosomal in situ hybridization with a cDNA plasmid further localized this gene to band 11q23. However, PCR amplification with "radixin-specific" primers on the hybrid DNA panel yielded an additional, very similar DNA sequence that was further characterized by direct sequencing of PCR products. This sequence represents a truncated version and the respective locus (RDXP2) was assigned to Xp21.3. Furthermore, by employing a different set of primers, a third sequence was found that was 90% identical to the radixin sequence but contained termination codons and seemed to lack introns. This pseudogene (RDXP1) was mapped to 11p by Southern and PCR analyses.

Genes for the dimerization cofactor of hepatocyte nuclear factor-1 alpha (DCOH) are on human and murine chromosomes 10.

Hepatocyte nuclear factor-1 alpha (HNF-1 alpha; gene symbol, TCF1) forms dimers with itself as well as with HNF-1 beta and regulates the expression of several liver-specific genes. Recently, a dimerization cofactor of hepatocyte nuclear factor-1 alpha, called DCOH, has been identified. Here, we report the chromosomal localization of the genes for this cofactor to chromosomes 10 in both humans and mice by Southern blot analyses of somatic cell hybrids.

A serine/proline-rich protein is fused to HRX in t(4;11) acute leukemias.

Translocations involving chromosome band 11q23 in acute leukemias have recently been shown to involve the HRX gene that codes for a protein with significant similarity to Drosophila trithorax. HRX gene alterations are consistently observed in t(4;11) (q21;q23)-carrying leukemias and cell lines by Southern blot analyses and are accompanied by HRX transcripts of anomalous size on Northern blots. HRX-homologous cDNAs were isolated from a library prepared from t(4;11)-carrying acute leukemia cells. cDNAs representative of transcription products from the derivative 11 chromosome were shown to contain HRX sequences fused to sequences derived from chromosome band 4q21. Fragments of the latter were used to clone and analyze cDNAs for wild-type 4q21 transcripts that predicted a 140-Kd basic protein (named FEL) that is rich in prolines, serines, and charged amino acids. FEL contains guanosine triphosphate-binding and nuclear localization consensus sequences and uses one of two possible 5' exons encoding the first 12 or 5 amino acids. After t(4;11) translocations, 913 C-terminal amino acids of FEL are fused in frame to the N-terminal portion of HRX containing its minor groove DNA binding motifs. These features are similar to predicted t(11;19) fusion proteins, suggesting that HRX consistently contributes a novel DNA-binding motif to at least two different chimeric proteins in acute leukemias.

The CD40 ligand, gp39, is defective in activated T cells from patients with X-linked hyper-IgM syndrome.

The prominent role of the CD40 receptor in B cell responses led us to investigate the role of the gp39-CD40 interaction in a group of primary immunodeficient patients with defective antibody production. Here we report that patients with hyper-IgM syndrome (HIM) have a defective gp39-CD40 interaction. B cells from HIM patients express functional CD40, but their T cells do not bind CD40-Ig. These patients expressed normal levels of gp39 mRNA, but these mRNAs encode defective gp39 proteins owing to mutations in the extracellular domain of gp39. Soluble recombinant forms of gp39 containing these mutations were unable to bind CD40 and drive normal B cell proliferation. The gene encoding gp39 was mapped to Xq26, the X chromosome region where the gene responsible for HIM had previously been mapped. These data suggest that a defect in gp39 is the basis of X-linked HIM.

Serotonin receptor 1c gene assigned to X chromosome in human (band q24) and mouse (bands D-F4).

In the mammalian nervous system, serotonin (5-hydroxytryptamine) binds to distinct cell surface receptor subtypes that are defined by their ligand binding and effector-coupling properties. The 5HT1c receptor is a G-protein coupled receptor that stimulates phospholipase C-catalyzed hydrolysis of phosphatidylinositol bisphosphate, leading to the mobilization of intracellular calcium and to the activation of protein kinase C. By using somatic cell hybrid analysis and FISH, we have mapped the HTR1C locus to the human X chromosome, band q24 and to the mouse X chromosome region D-F4. Comparison of these map positions offers new insights into the evolution of human and murine X chromosomes. Since HTR1C is expressed in certain parts of the central nervous system and abnormal function of the serotoninergic system has been implicated in affective disorders, obsessive-compulsive disorder and epilepsy, establishing the precise map position of HTR1C is an important first step toward evaluating this locus as a candidate for mutations in these syndromes and in X-linked mental disorders.