A complete deletion of the factor IX gene and new TaqI variant in a hemophilia B kindred

Summary We report a hemophilia B kindred in which the proband has a complete deletion of the factor IX gene extending a minimum of 80 kilobase pairs (kb) 3 of the gene. This individual has severe factor IX deficiency with no detectable circulating factor IX protein. In common with one previous report, despite a total deletion of the factor IX gene, this patient has not developed antibodies to factor IX. The mother of the proband was found to have a new TaqI variant of the factor IX gene on the nondeletion-bearing X chromosome. The location of the altered TaqI site was found to be 5 of exon IV between residues 9731-9734 and does not affect the function of the factor IX protein. The familial natures of both the variant allele and the deletion were established. In addition a study of this kindred at the DXS99 locus demonstrated the first reported recombination event between this site and the factor IX gene.     

Assignment by in situ hybridization of a fibroblast growth factor receptor gene to human chromosome band 10q26

Summary A 2.3-kb cDNA probe for the human bek fibroblast growth factor receptor was used to determine the chromosomal localization of the corresponding gene by in situ hybridization. The results show that this gene, a form of which is amplified in some poorly differentiated stomach cancers, is localized on chromosome region 10q26. The two previously identified fibroblast growth factor receptor genes are thus not on the same chromosome, as the related fig (fms-like gene) fibrovblast growth factor receptor gene has previously been mapped to human chromosome region 8p12.     

The human PDGF receptor α-subunit gene maps to chromosome 4 in close proximity to c-kit

Summary The gene encoding the -subunit of the human platelet-derived growth factor receptor (PDGFRA) maps to band q11–q12 of chromosome 4 by in situ hybridization, which was confirmed by Southern analysis of a Chinese hamster × human cell hybrid that retains only human chromosome 4.     

Two sisters with a distal deletion at the Xq26/Xq27 interface: DNA studies indicate that the gene locus for factor IX is present

Summary Two sisters with premature menopause and a small deletion of the long arm of one of their X chromosomes [del (X)(pterq26.3:)] were investigated with polymorphic DNA probes near the breakpoint. The deleted chromosome retained the factor IX (F9) locus and the loci DXS51 (52A) and DXS100 (pX45h), which are proximal to F9. However, the factor VIII (F8) locus was not present, nor were two loci tightly linked to this locus, DXS52 (St14) and DXS15 (DX13) This deletion refines the location of the F9 locus to Xq26 or to the interface Xq26/Xq27, thus placing it more proximally than has been previously reported. The DNA obtained from these patients should be valuable in the mapping of future probes derived from this region of the X chromosome.     

Molecular and cytogenetic analysis of an interstitial 20p deletion associated with syndromic intrahepatic ductular hypoplasia (Alagille syndrome)

Summary High-resolution chromosome analysis of a 19-year-old female proband with syndromic intrahepatic ductular hypoplasia (Alagille syndrome, AWS) revealed an interstitial deletion of chromosome 20p with breakpoints provisionally located in or close to p11.22 and p12.2. Southern blots from digests of DNA of the proband and her chromosomally normal parents were hybridized with the human DNA probes pR12.21, HuPrPcDNA2, and pDS6-SgI, which have been mapped to the region 20 (p12-pter), and rehybridized with the F IX probe for calibration. Comparing the hybridization signals of the normally sized DNA fragments of the familiy, we found no evidence for loss of any of the three tested distal chromosome 20p loci in our proband. Furthermore, in situ hybridization with HuPrPcDNA2 revealed a specific accumulation of grains at or around the faint distal G band suspected to represent all or most of band p12.3 of the proband's deleted 20p and at p12 of the normal chromosome 20. Thus the AWS of our proband is associated with an interstitial deletion that preserved the three tested distal loci on 20p. Since nine further reported cases of 20p deletion are clinically similar, we propose AWS as a further contiguous gene syndrome and assign it to an approximately 8-Mb-large chromosome 20p segment (provisionally, p11.23–p12.1).     

Nucleotide substitutions at the -6 position in the promoter region of the factor IX gene result in different severity of hemophilia B Leyden: consequences for genetic counseling

Mutations in the promoter region of the factor IX gene result in hemophilia B Leyden, which is characterized by considerable improvement in the disease after puberty. We have found that distinct nucleotide substitutions at the -6 position in the Leyden-specific (LS) region are associated with a different severity of hemophilia B. The proband (aged 2) from one family is a severe hemophiliac with factor IX activity (F.IXC) and antigen (F.IXAg) levels less than 1.0U/dl. F.IXC and F.IXAg levels in two affected uncles are approximately 30% of normal levels. The LS region was targeted for analysis because the phenotypes suggested the inheritance of a factor IX Leyden gene. An abnormal TaqI digestion pattern was found in amplified DNA from the proband, and sequencing showed a G (-6) to C transversion that was linked to the disease in the family. In another family, two brothers (aged 8 and 9) suffer from mild hemophilia with F.IXC ranging from 7 to 10 U/dl and F.IXAg from 3 to 4 U/dl. They are the only documented members of the family with a bleeding tendency. Denaturing gradient gel electrophoresis on amplified fragments from one of the patient's genomic DNA corresponding to the 8 exons and flanking sequences of the factor IX gene suggested a defect only in a segment from the 5 region. This segment showed an altered TaqI digestion pattern, and sequencing demonstrated a G(-6) to A transition that was traced to the patients's mother and a grandmother. The different phenotypes associated with the G (-6) to A purine nucleotide transition compared with a G(-6) to C transversion provide evidence that this area is directly involved in the regulation of the human factor IX gene expression in vivo by binding of regulatory factors. The ability to predict that the conditions of a hemophilia B patient will improve with age has important implications for genetic counseling of the family. Therefore, the LS region should always be included when scanning the factor IX gene for mutations.     

Mapping of the human gene encoding the mutual signal-transducing subunit (β-chain) of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and interleukin-5 (IL-5) receptor complexes to chromosome 22q13.1

The human gene encoding the mutual signal-transducing subunit (-chain) of granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5 receptor complexes has been mapped to chromosome 22q13.1 by the fluorescence in situ hybridization method.     

Mutational analysis of hemophilia B in Russia: Molecular-genetic study

Hemophilia B is a hereditary X-linked coagulation disorder. This pathology is caused by various defects in the factor IX gene, which is, being about 34 kb long and consisting of eight exons, localized in the Xq27 locus of the X-chromosome long arm. Mutations were revealed in 56 unrelated patients with hemophilia B in this study by using direct sequencing of factor IX gene functionally important fragments. Forty-six mutations were found with prevailing missense mutations (n = 30). The rest of the mutations were nonsense (n = 4) and splicing (n = 4) mutations, large deletions (n = 3), microdeletions (n = 2), microinsertions (n = 2), and promoter mutations (n = 1). Eleven of 46 mutations were previously unknown for human populations.     

T296→M,a common mutation causing mild hemophilia B in the Amish and others: founder effect,variability in factor IX activity assays,and rapid carrier detection

Summary By direct genomic sequencing, we have delineated the causative mutation in 64 families of European descent with hemophilia B. Six (9%) had a CT transition at base 31008, which substitutes methionine for threonine 296 (T²⁹⁶M) in the catalytic domain of factor IX. Five of the patients had the same haplotype (frequency of 16% in the northern European population). These individuals are of Amish/German descent and they are likely to share a common ancestor. The sixth patient had a different haplotype, which indicates that his mutation had an independent origin. The data highlight the importance of clinical criteria for the classification of hemophilia B. All six patients had clinically mild disease and their factor IX coagulant activities were in the range of 3%–6% when tested simultaneously in one laboratory, yet the factor IX activities provided with patient records varied 40-fold. Due to the high frequency of this mutation, we have utilized the technique of polymerase chain reaction amplification of specific alleles (PASA) to perform rapid and inexpensive carrier diagnoses in the families with this mutation. This is of particular importance for the Amish since the mutation should account for much of, if not all, the mild hemophilia B that is commonly found in this population.     

Monoclonal antibodies to coagulation factor IX define a high-frequency polymorphism by immunoassays.

Monoclonal antibodies have been used to demonstrate a polymorphism of human plasma coagulation factor IX antigen in double antibody solid-phase immunoradiometric assays. This polymorphism is detected in an assay where a monoclonal antibody (A-1) adsorbed to microtiter wells is used to bind factor IX from diluted plasma samples. Plasma samples with the factor IX polymorphism have less than 0.2 U/ml of apparent antigen when tested with the A-1 antibody, while assays with other monoclonal antibodies and assays with goat antisera to factor IX show normal amounts of factor IX antigen. Factor IX coagulant activity was normal in samples from donors with the polymorphism. The thin-layer polyacrylamide gel isoelectric focusing pattern of factor IX purified from a donor with the factor IX polymorphism (IXp) was identical to that obtained with factor IX prepared from a donor who did not have the polymorphism (IXn). Purified radiolabeled factor IX prepared from a donor with the polymorphism showed a Ka for the A-1 antibody that was threefold less than that measured for IXn. The gene frequency of IXp in male blood donors is 0.25. This polymorphism may be useful as a marker for the X chromosome in genetic studies on plasma samples. Further studies are necessary to determine the explanation for decreased reaction of IXp with the A-1 monoclonal antibody.     

A human gene that restores the DNA-repair defect in SCID mice is located on 8p11.1→q11.1

In order to map the gene that is responsible for the DNA-repair defect in severe combined immune deficient (SCID) mice, a mixture of microcells independently isolated from mouse A9 cells containing pSV2neo-tagged human chromosomes 5, 7, 8, 9, 11, 15, 18 or 20 were fused with SCID fibroblast cell lines SCVA2 and SCVA4, which were originally established from lung tissue of the C.B.17-scid/scid mouse by SV40 virus transfection. After irradiation with ⁶⁰Co -rays and selection with antibiotic G418, 12 independent clones were obtained, of which 4 contained an intact chromosome 8, 3 clones contained a deleted chromosome 8 [del(8)q22qter or del(8)q23 qter] and remaining 5 had no detectable or specific human chromosome. We further independently transferred a single human chromosome 8 or 11 into the SCVA cells via microcell fusion, and examined the radiation sensitivity of the microcell hybrids. Complementation of the radiation sensitivity was correlated with the presence of human chromosome 8 in microcell hybirds, whereas no correlation was observed in clones following the transfer of human chromosome 11. Thus, the results indicate that human chromosome 8 restored high sensitivity to ionizing radiation. A number of subclones that were radiation resistant or sensitive were isolated from the microcell hybrids. The concordance of the radiation sensitivity with the presence or absence of specific DNA fragments on chromosome 8 indicates that the human gene is located on the centromeric region of chromosome 8, i.e., 8p11.1 q11.1.     

The gene encoding vasoactive intestinal peptide is located on human chromosome 6p21→6qter

Summary Vasoactive intestinal peptide (VIP) is a regulatory neuropeptide involved in a wide variety of functions, among them vasodilation, smooth muscle relaxation, sweat secretion, gastrointestinal peristalsis, and pancreatic function. A deficient VIP-innervation of sweat glands was recently described as a possible pathogenic factor in sweating of cystic fibrosis (CF) patients. To investigate a possible role for a defective VIP-gene in cystic fibrosis, we have used a panel of rodent-human hybrid cells, retaining defined complements of human chromosomes to localize the VIP-gene to the human chromosome region 6p216qter. As the CF gene was recently mapped to chromosome 7, we conclude that the VIP-gene is not the primary gene defect in this disease.     

Organization and genomic distribution of “82H” alpha satellite DNA

Summary We have investigated the organization and genomic distribution of sequences homologous to p82H, a cloned human alpha satellite sequence purported, based on previous in situ hybridization experiments, to exist at the centromere of each human chromosome. We report here that, using Southern blotting analysis under conditions of high stringency, p82H hybridizes solely to a low-copy or single-copy alphoid domain located at or near the centromeric region of human chromosome 14. In contrast, conditions of reduced hybridization stringency permit extensive cross-hybridization with nonidentical, chromosome-specific alpha satellite subsets found elsewhere in the human genome. Thus, the previously described ubiquity of 82H human centromeric sequences reflects the existence of diverse alpha satellite subsets located at the centromeric region of each human chromosome.     

Human high-affinity FcγRI (CD64) gene mapped to chromosome 1q21.2-q21.3 by fluorescence in situ hybridization

The human FcRI gene encodes for a highaffinity Fc receptor that plays pivotal roles in the immune response. We have used fluorescence in situ hybridization analysis to localize the FcRI gene to human chromosome 1. The human FcRI (CD64) gene has been assigned to human chromosome 1q21.2-q21.3 using R-banded human (pro)metaphase chromosomes.     

Mapping parathyroid hormone, β-globin,insulin, and LDH-A genes within the human chromosome 11 short arm by spot blotting sorted chromosomes

Summary Rearranged human chromosomes carrying segments of chromosome 11 were separated from the normal chromosome 11 by high-resolution chromosome sorting. Sorted chromosomes were tested with parathyroid hormone, -globin, insulin, and LDH-A gene-specific probes to determine the genes carried by each chromosome segment. Based on the gene content and karyotypes of these abnormal chromosomes, the parathyroid hormone, -globin, insulin, and LDH-A genes and the unique restriction fragment ADJ-762 are all located on the terminal band of the short arm of human chromosome 11 (band 11p15), with LDH-A proximal to the other loci.     

Assignment of human ferritin genes to chromosomes 11 and 19q13.3→19qter

Summary Extracts of hamster-human and mouse-human hybrids, some with translocations involving chromosome 19, have been assayed for both human spleen ferritin (rich in L subunits) and human heart ferritin (rich in H subunits). Hybrid lines retaining part of the long arm of chromosome 19 including the region 19q13.319qter produced human L type ferritin. This confirms the previous assignment of the ferritin gene to chromosome 19 (Caskey et al. 1983). However, lines retaining chromosome 11 were found to contain human H type ferritin suggesting that the gene for the H subunit is on this chromosome. The presence of chromosome 6 was not necessary for the expression of either H or L type human ferritin. It thus seems unlikely that the gene for idiopathic haemochromatosis is a ferritin gene.     

The pattern of spontaneous germ-line mutation: relative rates of mutation at or near CpG dinucleotides in the factor IX gene

Mutations at CpG dinucleotides were delineated in the factor IX gene of 38 hemophilia B patients. When transitions at CpG were considered with those previously reported by us and those compiled in the factor IX mutation database, the following patterns emerged. Many CpG sites were mutated with high frequency, while two CpG sites were infrequently mutated (R²⁹Q and R¹¹⁶ TGA). Of the 6 possible nonsense mutations and the 14 missense mutations that would produce a nonconservative change at conserved amino acids, all have been observed to cause hemophilia B except A^–10T and R³³⁸Q. By contrast, none of the 6 missense changes at nonconserved amino acids have been observed to cause hemophilia B. At those CpG sites that are frequently mutated, the rate of transitions is estimated to be 20-fold higher than transitions at non-CpG sites. Point mutations in close proximity to CpG dinucleotides did not seem elevated.     

Organization and evolution of alpha satellite DNA from human chromosome 11

The human alpha satellite repetitive DNA family is organized as distinct chromosomal subsets located at the centromeric regions of each human chromosome. Here, we describe a subset of the alpha satellite which is localized to human chromosome 11. The principal unit of repetition of this alpha satellite subset is an 850 bp XbaI fragment composed of five tandem diverged alphoid monomers, each 171 bp in length. The pentamer repeat units are themselves tandemly reiterated, present in 500 copies per chromosome 11. In filter hybridization experiments, the Alpha 11 probes are specific for the centromeric alpha satellite sequences of human chromosome 11. The complete nucleotide sequences of two independent copies of the XbaI pentamer reveal a pentameric configuration shared with the alphoid repeats of chromosomes 17 and X, consistent with the existence of an ancestral pentameric repeat common to the centromeric arrays of at least these three human chromosomes.     

Characterisation of a human Y chromosome repeated sequence and related sequences in higher primates

The human Y chromosome carries 2000 copies of a tandemly repeated sequence, 2.47 kb long, which constitutes about 20% of the DNA of this chromosome. These sequences are localised on the tip of the long arm of the Y chromosome. Related sequences are present in DNA of females with a related but distinguishable restriction pattern. These autosomal sequences are distributed in tandem arrays on a number of autosomes. Related sequences are also present in gorilla and chimpanzee. In gorilla they resemble the human sequences in their restriction map but are not found on the Y chromosome whereas in chimpanzee the related sequences behave as a dispersed repeat. Changes in the level of methylation of this sequence in different tissues of human males can be detected with the lowest levels found in sperm and placental DNA.     

Assignment of the gene encoding the catalytic subunit Cβ of CAMP-dependent protein kinase to the p36 band on chromosome

Summary A cDNA for the human catalytic subunit (C) of cAMP-dependent protein kinase (PKA) has been cloned from a testis cDNA library. In the present study, we have determined the chromosomal localization of this gene using a cDNA for C as a probe. Southern blot analysis of genomic DNA from human/mouse cell hybrids revealed that the presence or absence of a 20-kbXbaI fragment, which hybridized with the C probe, was concordant with the presence of human chromosome 1.In situ hybridization to metaphase chromosome confirmed the somatic cell hybrid data and regionally mapped the C gene of PKA to the p36 band on chromosome 1.