Kathleen Delgrosso

Simple two-color array-based approach for mutation detection

The ability to analyze multiple polymorphic/mutation sites rapidly and accurately is pivotal in all areas of genetic analysis. We have applied single nucleotide primer extension (SNE) for detection of multiple point mutations in a micro-array format using two-color, fluorescent dye-tagged dideoxynucleoside triphosphate terminators (ddNTPs). The oligonucleotide primer ending one nucleotide short of the mutation site being probed is bound to the slide and single-base extended in place with two different Cy5/Cy3 dye-tagged terminators using solution-phase, locus-specific, single-stranded complementary templates generated by PCR from genomic DNA. The composite fluorescence produced contains peaks of distinct wave lengths corresponding to each Cy dye-tagged terminator incorporated, resulting in a fluorescent ‘fingerprint’ for each DNA target. DNA polymerase-catalyzed incorporation of Cy dye-tagged dideoxynucleoside triphosphates was dependent on the particular dyes, the specific ddNTP, the DNA target concentration, sequence of the template, on-slide temperature cycling and washing conditions. Results from analysis of mutations in the human hemochromatosis and connexinxa026 genes show that this approach has several advantages over existing methods and is simple, rapid, robust, cost effective and accurate with potential applications in many areas of genetic analysis.

Genotyping β-Globin Gene Mutations on Copolymer-Coated Glass Slides with the Ligation Detection Reaction

BACKGROUNDnMethods are needed to analyze small amounts of samples for variation in disease-causing genes. One means is to couple the sensitivity and multiplexing capability of the ligation detection reaction (LDR) with the use of simple glass slides specifically functionalized with a novel polymer coating to enhance sensitivity.nnnMETHODSnWe developed an array-based genotyping assay based on glass slides coated with copolymer (N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate). The assay consists of an LDR with genomic DNA followed by a universal PCR (U-PCR) of genomic DNA-templated LDR product. The LDR occurs in the presence of 3 primers for each sequence variant under investigation: 2 distinguishing primers (allele specific and perfectly complementary to wild-type and mutant alleles) and 1 common locus-specific primer. The 2 allele-specific primers have different capture sequences for binding different complementary probes on a tag array. The LDR product templated from genomic DNA is made fluorescent during the U-PCR via incorporation of a Cy5-labeled universal primer into all LDR products; detection occurs on the coated glass slides.nnnRESULTSnThe assay was designed to detect 7 prevalent mutations in the beta-globin gene (HBB, hemoglobin, beta) in a multiplex format, and signals for the different alleles are detected by their fluorescence. The assay was applied to 40 genomic DNA samples from both control individuals and patients with known beta-thalassemia mutations. Results show good correspondence between the patients genotypes as assessed by DNA sequence analysis and those generated from the LDR assays.nnnCONCLUSIONSnThe developed technology allows accurate identification of sequence variants in a simple, cost-effective way and offers good flexibility for scaling to other applications with different numbers of single-nucleotide polymorphisms or mutations to be detected.

DETECTION OF A NOVEL DNA POLYMORPHISM IN THE β-GLOBIN CLUSTER AND EVIDENCE FOR SITE-SPECIFIC RECOMBINATION

Within the εγδβ-globin gene region a non-random association of polymorphic restriction sites 5′ and 3′ to the δ globin gene has been observed. Between these two clusters lies a 9 kb region including the δ-globin gene which may contain sites of increased recombination. Analysis of DNA from an Albanian family revealed a novel Rsa I site 550 bp 5′ to the β-globin gene within the 9 kb region. One maternal and one paternal chromosome were identical at 9 polymorphic sites but differed at the Rsa site. This finding suggests that this polymorphism is randomly associated with previously defined haplotypes. Population screening showed the presence of this site in people of northern European, Mediterranean, Middle Eastern, African, Southeast Asian and Asian Indian descent, with an overall frequency of 0.39. DNA regions with βA, βS,βE and β-thalassemia alleles carrying and lacking the Rsa site were also identified. In one individual sequence analysis showed 28 alternating purine-pyrimidine dinucleotides in the region containing the Rsa site, rather than the 26 previously found in other individuals, suggesting that unequal crossing-over in this immediate region may lead to randomization of 5′ and 3′ polymorphic clusters.

Interpretation of fetal hemoglobin only on newborn screening for hemoglobinopathy.

Newborn screening for hemoglobinopathies rarely produces a fetal hemoglobin only result; it is most consistent with beta-thalassemia major, although other diagnoses are possible. The authors describe two unrelated African-American babies born in North Carolina whose newborn screening revealed fetal hemoglobin only. Both had a relatively benign clinical and hematologic picture. Molecular analyses indicated that both children are compound heterozygotes for beta-thalassemia and pancellular (deletional) hereditary persistence of fetal hemoglobin, a rare and apparently benign condition. Accurate interpretation of the fetal hemoglobin only result on newborn screening requires thorough evaluation, including family studies and molecular analysis.

Identification of the Nucleotide Change (CAC→CGC) Responsible for Hb P-Galveston [β117(G19)His→Arg]

Hb P-Galveston is a P-globin variant first identified in 1957 by Schneider and Haggard (1). The properties of this hemoglobin (Hb) variant were examined by Schneider et al in 1969 (2) and Di Iorio et a1 in 1975 (3). Hb P Galveston is associated with minimal hematologic abnormalities: target cells, hypochromia and anisopoikilocytosis. The molecule shows only a minimally increased oxygen affinity and normal stability (3). Until now, the nucleotide change responsible for this substitution was only presumed. We now demonstrate that the single nucleotide change of A+G at the second position of codon 1 17 is responsible for this variant. A child was referred after newborn screening identified an abnormal variant by high performance liquid chromatography (HPLC). Hematologic parameters at 8 months of age were as follows: Hb 9.9 g/dL, MCV 79 fL, MCHC 26.0 g/dL, reticulocytes 2.1%, RDW 12.9%. Genomic DNA was isolated from the child’s peripheral blood, and the P-globin gene was amplified by polymerase chain reaction (PCR); the third exon was examined by automated fluorescence-based DNA sequence analysis. Our results show heterozygosity for an

THE SILENT CARRIER ALLELE: β-THALASSEMIA WITHOUT A MUTATION IN THE β-GLOBIN GENE REGION

The silent carrier of β thalassemia has a decreased β/α globin synthesis ratio, but normal Hb A2 and Hb F levels and red cell indices. We have restudied the first described family (N. Engl. J. Med. 281:1327, 1969), in which the father is a silent carrier,the mother has high Hb A2 β-thalassemia trait, and both children have β thalassemia of intermediate clinical severity. The relative excess α globin in this family is not due to an increase in α-globin gene numbers. The maternal and paternal β-globin genes were cloned from the daughters genomic DNA. The maternal gene contains a previously reported IVS-1 splice junction βo thalassemia mutation. Sequence analysis of the paternal gene failed to reveal any base changes of functional significance. In HeLa cells the gene was expressed at normal levels with proper processing of RNA. Haplotype analysis revealed that the affected son and daughter inherited different εγδβ-globin gene clusters from the father. However, the father was homozygous for all polymorphic restriction sites downstream from a Taq I site approximately 3 kb 5 to the δ-globin gene. Two explanations for these results are: (1) recombination within primordial germ cells of the father occurred downstream from the Taq I site (2) the paternal silent carrier allele is not linked to the β-globin cluster. Studies which distinguish between these possibilities are in progress. In either case, the mutation responsible for β thalassemia is outside the region analyzed by structural and functional studies.