Marga Belle White

Detecting single base substitutions as heteroduplex polymorphisms

We have developed a sensitive technique for detecting single base substitutions in polymerase chain reaction (PCR) products from individuals heterozygous for polymorphisms or new mutations. This technique takes advantage of the formation of heteroduplexes in the PCR between different alleles from heterozygous individuals. These heteroduplexes can be detected on polyacrylamide gels because they migrate slower than their corresponding homoduplexes. Using PCR, we have generated a series of point mutations in a defined region of DNA in the equine infectious anemia virus (EIAV). Each mutation is the result of a single base substitution. By mixing the PCR products amplified from these mutations with one another, as well as with wildtype PCR products, we can generate heteroduplexes in which the identity of the mismatched bases is known. We detected eight of nine point mutations using this technique. We have also modified the electrophoretic conditions to optimize the detection of these heteroduplexes. In addition, the usefulness of this technique is demonstrated by its ability to detect a mutation in the cystic fibrosis gene that is the result of a single base substitution. This technique should prove useful for rapidly screening large numbers of individuals for new mutations or polymorphisms.

Multiple mutations in highly conserved residues are found in mildly affected cystic fibrosis patients

We have identified three different point mutations in the coding region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Each mutation segregates with the disease in two- or three-generation pedigrees and is not found on the normal chromosome of any documented cystic fibrosis carrier. One of the mutations is found in two independent families that contain at least one individual with a mild course of disease. All of these alterations replace charged amino acids with less polar residues and are found in the putative transmembrane sections of the molecule. The mutated amino acids are found to be conserved in both rodents and amphibians and lie in a region of CFTR that is believed to form a channel in the membrane. Although these alterations are rare, they provide important clues to functionally important regions of the molecule.

Typing of HLA-DQA1 and DQB1 using DNA single-strand conformation polymorphism

The technique of single-strand conformation polymorphism (SSCP), which is capable of distinguishing DNA sequence variability, was adapted to the identification of the HLA-DQA1 and DQB1 alleles. Eight DQA1 alleles and 12 DQB1 alleles were distinguished by amplifying the second exon of the genes in the presence of radioactive deoxynucleotide, denaturing the products with heat, and separating the single strands by electrophoresis in nondenaturing gels. For DQA1, it was possible to distinguish the eight alleles with standard bis-acrylamide or with a Hydrolink gel matrix. Twelve DQB1 alleles were identified by a protocol employing a combination of oligohybridization and SSCP using products amplified by specific DQB1 primers.

Detection of three rare frameshift mutations in the cystic fibrosis gene in an African-American (CF444delA), an Italian (CF2522insC), and a Soviet (CF3821delT)1 ☆

We have identified three new frameshift mutations in the CFTR gene in patients with cystic fibrosis (CF). The first one involves the deletion of an adenine nucleotide in exon 4 in an African-American patient (CF444delA), the second involves the insertion of a cytosine nucleotide in exon 13 in an Italian patient (CF2522insC), and the third results from the deletion of a thymidine nucleotide in exon 19 in a Soviet patient (CF3821delT). Each mutation is predicted to result in premature termination of the CFTR protein.

The use of DNA heteroduplex patterns to map recombination within the HLA class II region

Differential DNA heteroduplex patterns were used to investigate inheritance of HLA class II region genes in a family where a living related kidney transplant was under consideration. Serologic typing of the family members for HLA class I (HLA-A, B, and C) and class II (HLA-DR and DQ) alleles indicated that the patient (109) and one sibling (126) had inherited the same maternal and paternal HLA alleles. However, a strong reciprocal mixed lymphocyte response implied that these two individuals were not completely HLA identical. Serologic typing for HLA-DQ was confirmed by allele-specific oligonucleotide typing family members for HLA-DQ alpha and beta genes. To assess a possible nonidentical gene(s), DNA was amplified from all family members at the second exon of the DR beta, DQ alpha, DP alpha, and DP beta genes and the products were analyzed by DNA heteroduplex formation. This method showed that individuals 109 and 126 were identical at DR and DQ but differed at DP. This difference was confirmed by allele-specific oligonucleotide hybridization and indicated that 126 had inherited a recombinant maternal chromosome with a cross-over occurring in the region between the DQ beta and DP alpha genes. These data demonstrate the applicability of DNA heteroduplex patterns in establishing identity-nonidentity of alleles in the major histocompatibility complex.

Identification of Cystic Fibrosis Mutations

Using oligonucleotide primers from the sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene we have employed the polymerase chain reaction to amplify several coding exons. These regions have been examined from 110 patients that contain 127 chromosomes without the common CF mutation (DeltaF508). Eight additional mutations have been identified in this group, in a total of four different exons. Most of the mutations were initially identified using an assay for single-stranded conformation polymorphisms. All mutations were subsequently characterized by direct sequencing of the amplified DNA, and can be assayed by restriction enzyme digestion or allele-specific oligonucleotide hybridization.