DFold: PCR design that minimizes secondary structure and optimizes downstream genotyping applicationsFredman, David; Jobs, M.; Strömqvist, L.; Brookes, A.J.
doi: 10.1002/humu.20066pmid: 15221783
Secondary structures in polymerase chain reaction (PCR) target sequences have a negative impact on amplification success rates and on downstream uses of PCR products. For example, signal strength and allele discrimination in single nucleotide polymorphism (SNP) genotyping methods can be compromised by allele‐biased amplification and/or by PCR product folding that limits access of interrogating probes. To increase the fidelity and robustness of PCR, and to aid follow‐on applications, we have developed DFold (http://dfold.cgb.ki.se)—a generalized software solution that creates PCR oligonucleotide primer designs devoid of stable secondary structures. We demonstrate the effectiveness of the tool by applying it to a range of dynamic allele‐specific hybridization (DASH) assay designs, many of which we evaluate in the laboratory. We further consider how the system throughput may be made sufficiently high for use upon millions of target sequences in order to support whole‐genome analyses. Hum Mutat 24:1–8, 2004. © 2004 Wiley‐Liss, Inc.
Characterization of a mutagenic B1 retrotransposon insertion in the jittery mouseGilbert, Nicolas; Bomar, Jamee M.; Burmeister, Margit; Moran, John V.
doi: 10.1002/humu.20060pmid: 15221784
B1 elements are an abundant class of short interspersed elements (SINEs) in the mouse genome and mobilize by a process known as retrotransposition. Here, we report the characterization of a mutagenic B1 insertion into exon 4 of the Atcay gene, which was previously shown to be responsible for the jittery mouse. Mutations in the human ortholog of this gene, ATCAY, are responsible for Cayman ataxia. The B1 insertion is ∼150‐bp long, ends in a 45–50‐bp polyadenylic acid (poly A) tail, is flanked by a perfect 13‐bp target‐site duplication, and is inserted into a sequence that resembles a LINE‐1 endonuclease consensus cleavage site. Computational analysis indicates that the mutagenic insertion is most closely related to elements of the B1‐C subfamily, and we have identified two possible progenitor B1 sequences on mouse chromosome 19. Together, these data demonstrate that B1 retrotransposition is ongoing in the mouse genome and is consistent with the hypothesis that the reverse transcriptase and endonuclease encoded by LINE‐1 elements mediate B1 mobility. Hum Mutat 24:9–13, 2004. © 2004 Wiley‐Liss, Inc.
Haplotypes of the angiotensin II receptor genes AGTR1 and AGTR2 in women with normotensive pregnancy and women with preeclampsiaPlummer, Sally; Tower, Clare; Alonso, Pedro; Morgan, Linda; Baker, Phil; Broughton‐Pipkin, Fiona; Kalsheker, Noor
doi: 10.1002/humu.20050pmid: 15221785
Angiotensin II (AII) acts as a growth factor in local systems, mediating diverse effects such as cellular proliferation and apoptosis. These effects are controlled through two main receptor subtypes: AGTR1 and AGTR2. We studied the haplotype frequencies of both receptor genes in women with preeclamptic pregnancies and normotensive pregnant women. We also looked for any association between AGTR1 genotype at sites in the 5′ flanking region and binding of AII to platelets, which express AGTR1, in 58 normotensive pregnant women. There were nine common haplotypes of AGTR1, with no significant difference in haplotype frequency between the two groups of women. Platelet AII binding in normotensive pregnant women was associated with the genotype at g.5245C>T in the 5′ flanking region of AGTR1 (GenBank AF245699.1), with CC homozygotes at g.5245 having the lowest levels, and g.5245 TT homozygotes having the highest levels (P=0.05). Two novel polymorphisms were identified in AGTR2 (GenBank AY324607.1) at nucleotides g.1701T>C and g.2184A>T. Variation of AGTR2 could be explained by the existence of four common haplotypes. There was evidence for a significant increase in the frequency of the haplotype TAATGC at nucleotides g.1701, g.2041, g.2184, g.4673, g.4679, and g.4975, respectively (P=0.004), in women with preeclampsia. Hum Mutat 24:14–20, 2004. © 2004 Wiley‐Liss, Inc.
Functional analysis of polymorphisms in the promoter regions of genes on 22q11Hoogendoorn, Bastiaan; Coleman, Sharon L.; Guy, Carol A.; Smith, S. Kaye; O'Donovan, Michael C.; Buckland, Paul R.
doi: 10.1002/humu.20061pmid: 15221787
Segmental aneusomy, which includes chromosome 22 deletion syndrome (del(22)(q11.2q11.2)), has been associated with DiGeorge syndrome (DGS), velocardiofacial syndrome (VCFS), conotruncal anomaly face (CAF) syndrome, cat‐eye syndrome (CES), der(22) syndrome, and duplication of the del(22)(q11.2q11.2) syndrome's typically deleted region. Adults with del(22)(q11.2q11.2) may develop psychiatric illnesses, including schizophrenia, schizoaffective disorder, and bipolar disorder, suggesting that lower gene dosage leads to a predisposition to these illnesses. In a bid to identify important regulatory polymorphisms (SNPs) that may emulate changes in gene dosage of the genes within the common deletion, we have analyzed the promoter region of 47 genes (44 of which encode a protein with known function) encoding proteins in and around 22q11 for sequence variants. A total of 33 of the promoters contained polymorphisms. Of those, 25 were cloned into a reporter gene vector, pGL3. The relative ability of each promoter haplotype to promote transcription of the luciferase gene was tested in each of two human cell lines (HEK293t and TE671), using a cotransfected CMV‐SPAP plasmid as an internal control. Five genes (PRODH, DGCR14, GSTT2, SERPIND1, and a gene tentatively called DKFZP434P211) showed activity differences between haplotypes of greater than 1.5‐fold. Of those, PRODH, which encodes proline dehydrogenase, has previously been highlighted in relation to schizophrenia, and the functional promoter polymorphism reported here may be involved in pathogenic mechanisms. Hum Mutat 24:35–42, 2004. © 2004 Wiley‐Liss, Inc.
Harmonized microarray/mutation scanning analysis of TP53 mutations in undissected colorectal tumorsFavis, Reyna; Huang, Jianmin; Gerry, Norman P.; Culliford, Alfred; Paty, Philip; Soussi, Thierry; Barany, Francis
doi: 10.1002/humu.20069pmid: 15221790
Both the mutational status and the specific mutation of TP53 (p53) have been shown to impact both tumor prognosis and response to therapies. Molecular profiling of solid tumors is confounded by infiltrating wild‐type cells, since normal DNA can interfere with detection of mutant sequences. Our objective was to identify TP53 mutations in 138 stage I–IV colorectal adenocarcinomas and liver metastases without first enriching for tumor cells by microdissection. To achieve this, we developed a harmonized protocol involving multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) with Universal DNA microarray analysis and endonuclease V/ligase mutation scanning. Sequences were verified using dideoxy sequencing. The harmonized protocol detected all 66 mutations. Dideoxy sequencing detected 41 out of 66 mutations (62%) using automated reading, and 59 out of 66 mutations (89%) with manual reading. Data analysis comparing colon cancer entries in the TP53 database (http://p53.curie.fr) with the results reported in this study showed that distribution of mutations and the mutational events were comparable. Hum Mutat 24:63–75, 2004. © 2004 Wiley‐Liss, Inc.
Two‐color multiplex ligation‐dependent probe amplification: Detecting genomic rearrangements in hereditary multiple exostosesWhite, Stefan J.; Vink, Geraldine R.; Kriek, Marjolein; Wuyts, Wim; Schouten, Jan; Bakker, Bert; Breuning, Martijn H.; Dunnen, Johan T. den
doi: 10.1002/humu.20054pmid: 15221792
Genomic deletions and duplications play an important role in the etiology of human disease. Versatile tests are required to detect these rearrangements, both in research and diagnostic settings. Multiplex ligation‐dependent probe amplification (MLPA) is such a technique, allowing the rapid and precise quantification of up to 40 sequences within a nucleic acid sample using a one‐tube assay. Current MLPA probe design, however, involves time‐consuming and costly steps for probe generation. To bypass these limitations we set out to use chemically synthesized oligonucleotide probes only. The inherent limitations of this approach are related to oligonucleotide length, and thus the number of probes that can be combined in one assay is also limited. This problem was tackled by designing a two‐color assay, combining two sets of probes, each amplified by primers labeled with a different fluorophore. In this way we successfully combined 28 probes in a single reaction. The assay designed was used to screen for the presence of deletions and duplications in patients with hereditary multiple exostoses (HME). Screening 18 patients without detectable point mutations in the EXT1 and EXT2 genes revealed five cases with deletions of one or more exons: four in EXT1 and one in EXT2. Our results show that a two‐color MLPA assay using only synthetic oligonucleotides provides an attractive alternative for probe design. The approach is especially suited for cases in which the number of patients to be tested is limited, making it financially unattractive to invest in cloning. Hum Mutat 24:86–92, 2004. © 2004 Wiley‐Liss, Inc.