Allele Assignment Research Articles

VMD2 Promoter Requires Two Proximal E-box Sites for Its Activity in Vivo and Is Regulated by the MITF-TFE Family

The retinal pigment epithelium (RPE) is crucial for the function and survival of retinal photoreceptors. VMD2 encodes bestrophin, an oligomeric chloride channel that is preferentially expressed in the RPE and, when mutated, causes Best macular dystrophy. Previously, we defined the VMD2 upstream region from -253 to +38 bp as being sufficient to direct RPE-specific expression in the eye, and we suggested microphthalmia-associated transcription factor (MITF) as a possible positive regulator. Here we show that in transgenic mice the -154 to +38 bp region is sufficient for RPE expression, and mutation of two E-boxes, 1 and 2, within this region leads to loss of promoter activity. A yeast one-hybrid screen using bait containing E-box 1 identified clones encoding MITF, TFE3, and TFEB, and chromatin immunoprecipitation with antibodies against these proteins enriched the VMD2 proximal promoter. Analysis using in vivo electroporation with constructs containing mutation of each E-box indicated that expression in native RPE requires both E-boxes, yet in vitro DNA binding studies suggested that MITF binds well to E-box 1 but only minimally to E-box 2. MITF knockdown by small interfering RNA (siRNA) in cell culture revealed a strong correlation between MITF and VMD2 mRNA levels. Sequential transfection of a luciferase construct with expression vectors following MITF siRNA revealed that TFE3 and TFEB can also transactivate the VMD2 promoter. Taken together, we suggest that VMD2 is regulated by the MITF-TFE family through two E-boxes, with E-box 1 required for a direct interaction of MITF-TFE factors and E-box 2 for binding of the as yet unidentified factor(s).

Unbiased Interpretation of Haplotypes at Duplicated Microsatellites

The Y-chromosome is a powerful tool for population geneticists to study human evolutionary history. Haploid and largely non-recombining, it should contain a simple record of past mutational events. However, this apparent simplicity is compromised by Y-linked duplicons, which make up approximately 35% of this chromosome; 25% of these duplicons are large inverted repeats (palindromes). For microsatellites lying in these palindromes, two loci cannot be easily distinguished due to PCR co-amplification, and this order misspecification of alleles generates an additional variance component. Due to this ambiguity, population geneticists have traditionally used an arbitrary method to assign the alleles (shorter allele to locus 1, larger allele to locus 2). Here, we simulate these posterior estimate distributions under three different novel allele assignment priors and compare this with the original method. We use a sample of 33 human populations, typed for duplicated microsatellites lying within palindrome P8, to illustrate our approach. We show that both intra- and inter-population statistics can be dramatically affected by order misspecification. Surprisingly, matrices of pairwise F-statistics or distance estimates appear far less sensitive to order misspecification and remain relatively unchanged under the priors considered, suggesting that these microsatellites can be considered as useful markers for population genetic studies using an appropriate data treatment. Duplicated microsatellites represent an attractive source of information to investigate the extensive structural polymorphism observed among human Y chromosomes, as well as processes of intra-chromosomal gene conversion acting between duplicons.

The Role of the STAS Domain in the Function and Biogenesis of a Sulfate Transporter as Probed by Random Mutagenesis

Sulfate transporters in plants represent a family of proteins containing transmembrane domains that constitute the catalytic part of the protein and a short linking region that joins this catalytic moiety with a C-terminal STAS domain. The STAS domain resembles an anti-sigma factor antagonist of Bacillus subtilis, which is one distinguishing feature of the SLC26 transporter family; this family includes transporters for sulfate and other anions such as iodide and carbonate. Recent work has demonstrated that this domain is critical for the activity of Arabidopsis thaliana sulfate transporters, and specific lesions in this domain, or the exchange of STAS domains between different sulfate transporters, can severely impair transport activity. In this work we generated a Saccharomyces cerevisiae expression library of the A. thaliana Sultr1;2 gene with random mutations in the linking region-STAS domain and identified STAS domain lesions that altered Sultr1;2 biogenesis and/or function. A number of mutations in the beta-sheet that forms the core of the STAS domain prevented intracellular accumulation of Sultr1;2. In contrast, the linking region and one surface of the STAS domain containing N termini of the first and second alpha-helices have a number of amino acids critical for the function of the protein; mutations in these regions still allow protein accumulation in the plasma membrane, but the protein is no longer capable of efficiently transporting sulfate into cells. These results suggest that the STAS domain is critical for both the activity and biosynthesis/stability of the transporter, and that STAS sub-domains correlate with these specific functions.

HLA and MICA associations with head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a very aggressive tumour arising from the epithelial lining of the upper aerodigestive tract. The precise mechanisms involved in the pathogenesis of HNSCC have not been elucidated. Previous studies observed aberrant HLA expression patterns on HNSCC tumour cells and this study focused on the allelic polymorphism of HLA genes and the MHC class I chain related gene A (MICA) and HNSCC. We investigated whether associations with HLA and/or MIC alleles or haplotypes are involved in the pathogenesis of HNSCC and could explain the observed HLA expression patterns. Patients and controls were typed for HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 with sequence specific priming (SSP), supplemented with sequencing based typing (SBT). MICA allelic polymorphism was included and MICA allele assignment was based upon the combination of high resolution SBT of exons 2-4 in combination with repeat analysis and nucleotide polymorphism of exon 5. HLA-B *35 (p=0.014, OR=0.31) and HLA-B *40 (p=0.013, OR=2.9) were significantly associated in respectively the metastasized patients and the oral cavity patients. In addition, the HLA-B *40-DRB1 *13 haplotype (p=0.016, OR=4.1) was more often observed in the oral cavity patient group. The biological significance of the prevalence of specific HLA haplotypes in patients with oral cavity HNSCC and metastasizing HNSCC requires further investigation.

Sequence-Based Typing of the Complete Coding Sequence of DQA1 and Phenotype Frequencies in the Dutch Caucasian Population

Typing of DQA1 by sequencing has been a challenge because of a 3-nucleotide deletion in exon 2 in half of the alleles. Furthermore, 19 of the 28 alleles cannot be identified on basis of exon 2 alone, but need additional exon information. With the sequencing strategy presented here the complete exons 1–4 are sequenced heterozygously, enabling identification of all DQA1 alleles by sequence-based typing (SBT). Exons 1–4 were amplified and sequenced separately, the combined sequences were used for automated allele assignment. The method was validated by typing 21 individuals with all possible different allele group combinations. In addition 26 quality control samples were correctly typed by this method. To determine the phenotype frequencies 155 unrelated Dutch Caucasian individuals were DQA1 typed. In total 15 known and two new DQA1 alleles were identified. DQA1*0103 and *0505 were the most frequent alleles with phenotype frequencies of 30% and 29%, respectively. The SBT method presented here is an improvement compared to already existing protocols in that the complete exon sequence is obtained for all coding exons, using identical polymerase chain reaction conditions. Furthermore, all exons are sequenced heterozygously, facilitating allele assignment and reducing the number of amplification reactions.

Low‐cost, simultaneous, single‐sequence genotyping of the HLA‐A, HLA‐B and HLA‐C loci

New automated DNA sequencing technology has enabled the development of an assay for genotyping the three major HLA class 1 loci from a single sequence of each gene containing exon 3, intron 2 and exon 2. The assay allows 31 subjects (with 3 negative controls) to be genotyped at all three loci simultaneously, using a 96-well plate format. Genotypes were assigned by comparing each sequence to a database of 307 HLA-A, 563 HLA-B and 166 HLA-C alleles. Unequivocal, 4-digit allele assignments were made for 40 of 130 HLA-A genes, 82 of 130 HLA-B genes and 97 of 130 HLA-C genes from 21 European, 20 Tongan and 24 Niuean subjects. Ambiguity in interpretation of the sequence contributed to 66 of the 170 equivocal allele assignments, and 105 equivocal assignments were due to polymorphisms outside exons 2 and 3. All known alternative interpretations of ambiguous genotypes were identified, and seven HLA-B and two HLA-C ambiguities were resolved by reading the out-of-phase exon 2 sequence that followed an indel in intron 2. The genotypes of a subgroup of 27 heterozygous subjects, whose genotypes contained all of the alleles identified in this study, were confirmed with commercial, generic PCR-SSP typing. In European subjects, the repertoire of HLA-B/HLA-C haplotypes was almost identical to previously published data. We identified five new HLA-B/HLA-C haplotypes in the Polynesian subjects, and the remaining haplotypes were of Asian origin. In summary, we are describing a low-cost, sequencing assay for the three major HLA class I loci that provides a level of resolution that is comparable with a commercial PCR-SSP assay.

Maximization of STR DNA typing success for touched objects

In order to produce database-eligible DNA profiles from touched objects, each testing procedure including sample recovery, extraction, amplification and separation was evaluated and optimized. The developed methodologies were tested on control samples as well on fingerprints deposited on a variety of substrates such as credit cards, keys, and pens. All samples were amplified in triplicate to confirm the presence of each allele and to detect drop-ins. Overall the modifications implemented produced reproducible results for DNA titrated to 20 pg. For DNA dilutions, 25 pg routinely resulted in full profiles, and 12.5 pg determined 76.9% of the database loci tested. Similarly, for the touched objects, 75.8% of the 20-pg to 100-pg samples yielded database-eligible profiles; the remaining samples either were mixtures or contained an insufficient number of allelic calls. Here, the three-amplification approach was crucial and produced more complete profiles with confidence in the allelic assignments. DNA amounts below 20 pg did show partial profiles with correct allelic determinations that could have been compared in a specific case but were often too incomplete for database entry.

A Typing System for the Major Histocompatibility Complex Class I Chain Related Genes A and B Using Polymerase Chain Reaction with Sequence-Specific Primers

The Major Histocompatibility Complex (MHC) class I chain related (MIC) A and B genes are important additional loci within the MHC. We have developed a MICA and MICB typing system using the polymerase chain reaction with sequence-specific primers (PCR-SSP), which operates under the same conditions as our routine HLA-A, -B, and -C typing method. We designed 95 primers in 84 SSP mixtures for MICA and 39 primers in 29 mixtures for MICB. This detected and differentiated all 55 MICA and 19 MICB alleles (except MICA*00701 from MICA*026, MICA*00201 from MICA*020, and three MICB alleles, which are intronic variations). A computer program confirmed the MICA amplification reactivity of each SSP mixture and evaluated the typing set for MICA allele combination ambiguities. Seventy-six "reference" DNA samples were used for validation: 50 from International Histocompatibility Workshop B lymphoblastoid cell lines (IHW BCLs) and 26 MICA-typed samples from two laboratories. The reference material identified 28 out of the 55 MICA alleles and 13 of the 19 MICB alleles, and directly validated 62 of the 84 MICA and 20 of the 29 MICB SSP mixtures. Our genotyping agreed with 283 out of the 286 (98.95%) MICA and MICB reference laboratories' allele assignments or the consensus assignments. Two of the discrepancies remain unresolved, whereas one was probably due to a reference laboratory's failure to differentiate alleles differing in exon 5 of the MICA gene. A comparison of the MICA and MICB allele assignments between laboratories identified a "disagreement rate" of 19.4% for MICA alleles and 13.1% for MICB alleles. Accordingly, we have compiled "consensus" MICA and MICB genotypes for the 50 IHW BCLs tested, which have been confirmed by our typing. We also typed 166 random blood donors. Their MICA and MICB carriage and allele frequencies and HLA-B, MICA, MICB linkage disequilibrium parameters and haplotype frequencies largely concurred with other published data on United Kingdom subjects, further supporting the validity of our typing system. This PCR-SSP system is a simple, reliable and rapid technique for typing MICA and MICB alleles. It is easily updated as new alleles are identified but clearly requires a continuing validation review until all known MICA and MICB alleles have been identified.

A Genetic Population Study of Six Y STRS (Y-Plextm6) in an Uruguayan Sample and Its Comparison with Other Populations

Genomic DNA was extracted from blood samples of 91 unrelated Uruguayan males that were initially spotted on FTAR paper (Whatman, Clifton, NJ). The six loci (DYS393, DYS19, DYS389II, DYS390, DYS391, and DYS385) were amplified using the YPLEXTM6 kit (Reliagene Technologies, Inc., New Orleans, LA) according to the manufacturer’s recommendations and to published experiences (1). The amplified products were analysed by capillary electrophoresis using the ABI Prism 310 DNA Sequencer (PE Biosystems, Foster City, CA). Allele assignment was performed by comparison with self-established allelic ladder, positive and negative controls included in the kit. Several external populations were used for comparison from previously published papers; these are samples from Southern Spain and the Spanish Canary Islands (2), Northern Argentinian Amerindians (3), Italians (4), Mozambique (5), Caucasian Argentinians (6), Brazilians (5), and from Portugal (7). Since duplicated loci such as DYS385 cannot be dealt with in AMOVA, the study had to be confined to the systems DYS393, DYS19, DYS389II, DYS390, DYS391. STR allele frequencies, diverse Y-chromosome diversity indices and population differentiation were tested using software ARLEQUIN version 2.000 (8). The allele frequencies and gene diversity of the STR’s DYS393, DYS19, DYS389II, DYS390, DYS391 y DYS385 from Uruguay are shown in Table 1. We counted 76 different haplotypes in a population sample of 91 Uruguayan males giving a haplotype diversity

Nomenclature for factors of the HLA system, 2004

Nomenclature for factors of the HLA system, 2004

MALDI-TOF MS and TaqMan® assisted SNP genotyping of DNA isolated from formalin-fixed and paraffin-embedded tissues (FFPET)

Formalin-fixed paraffin-embedded tissues (FFPET) from archived clinical samples provide an invaluable source for large-scale molecular genetic studies. Pharmacogenetic investigations that require long-term clinical follow-up data of patients may particularly benefit from FFPET analysis. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and TaqMan-based (Thermus aquaticus polymerase) methodologies have become standard genotyping procedures. However, no data are available on the applicability of MALDI-TOF MS to the genotyping of low quality DNA, as it is usually obtained from FFPET, and data from TaqMan genotyping are limited. We isolated constitutional DNA from 274 FFPET samples (229 patients with breast cancer and 45 patients with benign breast diseases) and genotyped 15 polymorphic loci in 10 genes. Nine SNPs were genotyped by MALDI-TOF MS, and six were genotyped by the TaqMan methodology. We established rates for successful allele assignment for all FFPET, for FFPET prepared prior to 1990, and for FFPET prepared post-1990. Both methodologies showed high success rates ranging between 70.9 and 99.6% (mean: 91.8%) for MALDI-TOF MS and between 82.3 and 97.7% (mean: 91.0%) for TaqMan genotyping. No significant differences in genotyping performances for FFPET prepared prior to 1990 or post-1990 were observed. With the exception of one, all other genotype frequencies were in Hardy-Weinberg equilibrium. Furthermore, genotype frequencies matched those observed in a German breast cancer population and other Caucasian populations. Our study shows for the first time that MALDI-TOF MS and TaqMan genotyping procedures provide reliable data, and are therefore applicable in studies that require large scale FFPET genotyping.

Unusual variant alleles in commonly used short tandem repeat loci

Unusually large variant alleles were observed in the short tandem repeat (STR) systems D3S1358 and D21S11, both of which are included in the international standard set of loci (ISSOL) and routinely typed in National DNA intelligence databases worldwide. The observed alleles fell within the size range of the adjacent STR marker, which could easily cause problems with respect to correct allele assignments for both loci concerned. We compared the amplification and potential interpretation with three different commercially available kits, which are frequently used in forensic work. PCR products were cloned and sequenced in order to determine the structure of these unusual allele variants and confirm their size and designation (D3S1358 allele 26, D21S11 allele 46). In the locus D21S11 we observed an as yet undescribed partial duplication of the constant region.

IMGT/GENE-DB: a comprehensive database for human and mouse immunoglobulin and T cell receptor genes

IMGT/GENE-DB is the comprehensive IMGT genome database for immunoglobulin (IG) and T cell receptor (TR) genes from human and mouse, and, in development, from other vertebrates. IMGT/GENE-DB is the international reference for the IG and TR gene nomenclature and works in close collaboration with the HUGO Nomenclature Committee, Mouse Genome Database and genome committees for other species. IMGT/GENE-DB allows a search of IG and TR genes by locus, group and subgroup, which are CLASSIFICATION concepts of IMGT-ONTOLOGY. Short cuts allow the retrieval gene information by gene name or clone name. Direct links with configurable URL give access to information usable by humans or programs. An IMGT/GENE-DB entry displays accurate gene data related to genome (gene localization), allelic polymorphisms (number of alleles, IMGT reference sequences, functionality, etc.) gene expression (known cDNAs), proteins and structures (Protein displays, IMGT Colliers de Perles). It provides internal links to the IMGT sequence databases and to the IMGT Repertoire Web resources, and external links to genome and generalist sequence databases. IMGT/GENE-DB manages the IMGT reference directory used by the IMGT tools for IG and TR gene and allele comparison and assignment, and by the IMGT databases for gene data annotation. IMGT/GENE-DB is freely available at http://imgt.cines.fr.

High-throughput molecular haplotype analysis (allelic assignment) of single-nucleotide polymorphisms by fluorescent polymerase chain reaction

High-throughput molecular haplotype analysis (allelic assignment) of single-nucleotide polymorphisms by fluorescent polymerase chain reaction

Testing for parentage and kinship

Parentage analyses are of interest to workers in health care, law enforcement, immigration and other fields. This review describes recent applications, technical advances, and quality improvements. Mutations at short tandem repeat sequence loci confound interpretations of genetic data used to assess all blood relationships. Rates of the usual mutation type (change in repeat number) are probably related to specific alleles at each locus as well as to allele length, locus, and gender. Short tandem repeat sequences have relatively limited information content per locus. Intermediate tandem repeat sequence loci may be better. In immigration proceedings, probabilities can be calculated for excluding parentage in blood relatives who might impersonate the biologic parent. Unrelated immigrants from a subpopulation may appear to be related, but it is now possible to statistically determine the effect of population substructure on kinship determinations. In forensic analyses, sex chromosomal (X and Y) short tandem repeat sequences and mitochondrial DNA sequence variations have helped identify the parental lineages of human remains. Recent laboratory quality improvements include a way to estimate the frequency of common mother-child specimen mislabeling in routine paternity cases. In prenatal testing there are now methods for avoiding erroneous assignment of contaminant maternal alleles to the fetus. False paternity exclusions can be avoided by adhering to a standard of the American Association of Blood Banks requiring duplicate DNA isolation and retesting of excluded men. Laboratory technology and quality have advanced, but genetic tests with greater information content are needed. Better communication is highly desirable between persons requesting tests and parentage laboratories.

Five-Locus HLA Typing of Hematopoietic Stem Cell Donor Volunteers Using PCR Sequence Specific Primers

We have developed a strategy for five-locus human leukocyte antigen (HLA) typing of hematopoeitic stem cell (HSC) donors using the polymerase chain reaction with sequence-specific primers (PCR-SSP). The PCR-SSP method is robust, reproducible, and accurate. New PCR-SSP mixtures can be added as required and all reactions are carried out under the same conditions, which can easily be applied to the typing of other loci, e.g., ABO blood groups. Initially, 127 PCR-SSP reactions were used to detect simultaneously HLA-A, -B, -C, -DRB1/3/4/5, and DQB1 alleles, differentiated generally to the level of the first two digits of the allele name, essentially equivalent to the serological split specificity. Approximately 40% of subjects were tested against a further 29 HLA-A, -B SSP mixtures to exclude rare alleles and unambiguously assign a two-digit HLA allele family. This gave an overall typing resolution equivalent to or greater than the split specificity level and covered all HLA-A, -B, -C, -DRBland DQB1 alleles listed in the WHO's Nomenclature for Factors of the HLA System, 2000. The Welsh Bone Marrow Donor Registry has used this strategy to HLA type over 35,000 HSC donors over 9 years. Comprehensive and accurate five-locus HLA typing allows confident and rapid identification of potential matched HSC donors for patients requiring stem cell transplantation generally without the need for typing additional loci. This allows resources to be focused directly on allele level typing of DRB1 and other loci. This strategy decreases overall donor work-up time, which is a major benefit to patients.

Utility of fast mycobacterial interspersed repetitive unit-variable number tandem repeat genotyping in clinical mycobacteriological analysis.

Analysis of variable numbers of tandem repeats (VNTR) of genetic elements called mycobacterial interspersed repetitive units (MIRUs) is a recently described, polymerase chain reaction (PCR)-based method used to genotype Mycobacterium tuberculosis. It is much faster, requires a smaller amount of DNA, and has approximately the same discriminatory power as the standard IS6110 restriction fragment-length polymorphism (RFLP) method. We report the adaptation and optimization of MIRU-VNTR genotyping on a capillary electrophoresis system. We describe its application to 3 typical clinical situations encountered in our laboratory (Institut Pasteur de Bruxelles, Laboratoire Tuberculose et Mycobacteries; Brussels, Belgium). MIRU-VNTR genotyping was performed on heat-inactivated M. tuberculosis cultures obtained from clinical specimens on Lowenstein solid medium or in mycobacteria growth indicator liquid tubes (Becton Dickinson). After amplification of 12 genomic loci using 4 different multiplex PCRs, DNA fragments were separated by capillary electrophoresis using the ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems). Sizing of the PCR fragments and assignment of the various MIRU-VNTR alleles were done using the GeneScan and customized Genotyper software packages (PE Applied Biosystem). Clustering on the basis of IS6110 fingerprinting of isolates from 3 different patients attending the same hospital was confirmed by MIRU-VNTR typing. This concordance between 2 independent, highly discriminatory techniques was decisive in triggering an epidemiological inquiry that led to identification of a bronchoscopy-related tuberculosis nosocomial infection. A mixed tuberculosis infection in a patient whose infection was initially suspected as a result of the IS6110 RFLP method was clearly identified by MIRU-VNTR typing. Finally, automated MIRU-VNTR analysis permitted the identification of laboratory contamination in 6 liquid cultures of M. tuberculosis within several hours. These examples illustrate the utility of this genotyping technique for quick and accurate resolution of problems commonly encountered in clinical mycobacteriology.

Allele Frequency Distribution for 9 STR Loci in the Tamil Population of Malaysia

DNA database was obtained from unrelated random Tamil individuals in Malaysia. The DNA was extracted by the salting out procedure (1). Ten ng target DNA was co-amplified using the commercial typing kits, Promega GeneprintTM STR multiplex (CTT, FFv and STR III) systems, according to the manufacturer’s instructions. Assignment of alleles was made by visual comparison between the commercially supplied reference allelic ladders and the amplified samples at the corresponding locus. Data were analyzed as per the methods already reported (2–4). No deviations from equilibrium were observed. The power of discrimination ranges from 0.8458 to 0.9485. The complete dataset can be accessed at www.ppsk.ugm.mg

A proposed nomenclature for 15 canine-specific polymorphic STR loci for forensic purposes.

We performed a population study on 15 polymorphic STR loci (FH2010, FH2079, PEZ2, VWF.X, FH2054, FH2087Ub, FH2611, WILMS-TF, PEZ12, PEZ15, PEZ6, FH2087Ua, ZUBECA4, ZUBECA6, FH2132) on 131 randomly selected dogs. Alleles were identified and grouped according to their estimated fragment length using fixed allelic bins encompassing one base-pair. The allele assignment was confirmed by sequence analysis of homozygote and cloned heterozygote alleles. In order to develop a uniform repeat-based nomenclature, extensive sequence analysis was performed on a selection of alleles from each STR locus. The proposed nomenclature refers to the internationally recognised recommendations for human-specific STR loci in forensic applications. The 15 canine-specific STR loci were grouped into 3 classes (simple STRs, compound STRs and complex/hypervariable STRs) according to their complexity and variability within the repeat structure. Finally, we evaluated the precision of fragment size estimation on a capillary electrophoresis platform and demonstrated reproducibility of fragment length estimation for single base-pair intermediate alleles.

Allele Frequencies for Markers CSF1PO, TPOX, TH01, F13A01, FESFPS, vWA, D16S539, D7S820, D13S317 in the General Population of Nicaragua

General Hispanic-admixed individuals from Nicaragua.