Polymorphism of DNA sequence adjacent to human beta-globin structural gene: relationship to sickle mutation.

Abstract

Restriction endonuclease mapping of the human globin genes revealed a genetic variation in a Hpa I recognition site about 5000 nucleotides from the 3' end of the P-glo in structural gene. Instead of a normal 7.6-kilobase (kb) fragment which contains the globin structural gene, 7.0-kb and 13.0-kb variants were detecte. Both variants were found in eople of African origin and were not detected in Asians or Caucasians. The 13.0-kb variant is frequently associated with the sickle hemoglobin mutation and may be useful for the prediction of the sickle cell gene in prenatal diagnosis. Polymorphism in a restriction enzyme site could be considered as a new class of genetic marker and may offer a new approach to linkage analysis and anthropological studies. Polymorphism in structural genes in the human is a well-known phenomenon which has been utilized for many types of genetic analyses. Usually, the normal and variant genes or gene products are identified by structural studies, functional assays, or immunological methods. Recently, Southern (1) introduced a new method for analysis of DNA consisting of restriction endonuclease digestion of the genomic DNA, electrophoretic separation of the DNA fragments, and identification of the structural genes in these fragments by hybridization analysis. This method has been used to study the organization of many eukaryotic genomes, including the human globin genes (2, 3).During study of the human globin genes by the Southern method, we detected genetic variations of a restriction endonuclease site close to the human fl-globin structural gene. The variations were found in people of African origin, and one variant was commonly associated with the fl-globin structural mutation, hemoglobin S. This type of polymorphism may be useful for linkage analysis, prenatal diagnosis, or anthropological studies.MATERIALS AND METHODS Preparation of Cellular DNA. DNA was prepared from leukocytes, placentas, and cultured fibroblasts according to described methods (4,5), except that 100,ug of proteinase K per ml was used to digest the cells at 550C, and the phenol-extracted DNA was extensively dialyzed against 1 mM Tris-HCI, pH 7. Electrophoresis and Identification of Globin Genes.Samples (10 ;g) of digested DNAs were applied to 6-mm-thick horizontal 0.8% agarose (SeaKem) gels in a buffer (pH 8.05) containing 0.04 M Tris-acetate, 0.02 M Na acetate, 0.018 M NaCl, and 0.02 M EDTA and were electrophoresed at 50 V for 14 hr. The gels were stained for 30 min in ethidium bromide (10 Mg/ml in H20) and photographed under ultraviolet light.The DNAs were transferred to nitrocellulose filters with 0.90 M NaCl/0.09 M Na citrate for 40 hr (1). The filters were dried in a vacuum oven at 80°C for 2 hr and presoaked in 2-4 ml of 50% (vol/vol) formamide/0.45 M NaCI/0.045 M Na citrate containing 200 Mg of yeast tRNA and 200 ,ug of denatured salmon sperm DNA per ml and 1% (vol/vol) Denhardt's solution (9). The filters were wrapped in adhesive polyethylene (Saran-Wrap) and incubated at 41°C for 16 hr.[32P]cDN...

Direct mutation analysis by high-throughput sequencing: From germline to low-abundant, somatic variants

“…The emergence of assays to directly analyze DNA sequence variation enabled investigators to identify mutations at the molecular level and explore their mechanisms of action[18]. The first DNA-based assays screened for mutations at restriction sites that resulted in a restriction fragment length polymorphism (RFLP)[19]. The development of nucleotide sequencing methods subsequently permitted the analysis of small sections of genomic DNA for allelic variants after cloning[20, 21].…”

IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts

“…They are caused by mutated or reduced expression of the adult-stage beta-globin gene (1,2) and manifested when gene expression in the betaglobin locus sequentially switches from fetal to adult types (3) around the time of birth. It is predicted that maintaining fetal hemoglobin (HbF) at levels above 30% in circulating red cells should prevent the manifestation of most sickle-cell disease complications (4).…”

An appraisal of the application of recombinant DNA techniques to chromosome defects

“…In 1978, Kan and Dozy reported that many Blacks suffering from sickle cell anaemia lacked a HpaI site at the 3' flanking region of their ,B-globin gene (Kan & Dozy, 1978). The linkage disequilibrium between a restriction fragment length polymorphism (RFLP) and the sickle cell mutation in this ethnic group has led to the more extensive studies of DNA polymorphisms in the ,B-globin cluster.…”