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R Sparkes

Publications and source records attributed to R Sparkes.

At least 19 recordsLinked to original sources

Report of the European Network of Forensic Science Institutes (ENSFI): formulation and testing of principles to evaluate STR multiplexes.

This paper describes a collaborative exercise organised under the auspices of the European Network of Forensic Science Institutes (ENFSI). The purpose of this EU (European Union) funded group is to carry out research to enable STR loci to be compared between European laboratories, ultimately leading to the formation of a pan-European database. Accordingly, an exercise was designed to evaluate a prototype STR multiplex system manufactured by Applied Biosystems (ABD). Each laboratory was sent 12 samples to analyse along with a multiplex kit. Of specific interest was the definition of parameters to define the efficiency of the system. Stutter, split allelic peaks (differing by one base), pull-up, heterozygous balance and between locus balance were all objectively measured. Once the important parameters are defined it is possible to directly compare performances of different multiplexes and the different laboratories carrying out the tests. Since the multiplex used was a prototype system, this exercise cannot be regarded as a proficiency test.

Alleles↗

Validation studies of an immunochromatographic 1-step test for the forensic identification of human blood.

An immunochromatographic 1-step test for the detection of fecal occult blood was evaluated for applicability for the forensic identification of human blood in stained material. The following experiments were conducted: 1) determination of the sensitivity and specificity of the assay; 2) evaluation of different extraction media for bloodstains (sterile water, Tris buffer pH 7.5 provided in the test kit, 5% ammonia); 3) analysis of biological samples subjected to a variety of environmental insults; and 4) evaluation of casework samples. This immunochromatographic 1-step occult blood test is specific for human (primate) hemoglobin and is at least an order of magnitude more sensitive than previous methods for detecting human hemoglobin in bloodstains. The antigen is insensitive to a variety of environmental insults, except for exposure to certain detergents and household bleaches and prolonged exposure to certain preparations of luminol. The entire assay can be conducted in field testing conditions within minutes. When in the laboratory the supernatant from a DNA extraction is used for the assay, there is essentially no consumption of DNA for determining the presence of human hemoglobin in a forensic sample. The data demonstrate that this test is robust and suitable for forensic analyses.

Animals↗

Interpreting simple STR mixtures using allele peak areas.

Although existing statistical models can interpret mixtures qualitatively based upon the alleles present, the use of automated sequencers opens the opportunity to take account of quantitative aspects embodied by the peak area. One step in understanding simple mixtures consisting of just two donors is to estimate the mixture ratio. This is relatively easy to do when four-allele mixtures are evident at a given locus. However, if the mixture consists of three or fewer alleles, the process it is not straightforward. We demonstrate that mixture estimates are consistent across all loci in a multiplex system. Once the mixture ratio is known, then the expected peak areas for any given combination of alleles can be estimated using a simple spreadsheet analysis.

Alleles↗

Analysis and interpretation of mixed forensic stains using DNA STR profiling.

The use of multiplex PCR and fluorescent dye technology in the automated detection and analysis of short tandem repeat loci provides not only qualitative information about the profile--i.e. which alleles are present--but can also provide quantitative information on the relative intensities of the bands, and is therefore a measure of the amount of amplified DNA. The availability of this quantitative information allows for the interpretation of mixtures in a detailed way which has not been previously possible with many other human identification systems. In this paper we present a simple approach to the resolution and analysis of mixed STR profiles resulting from the testing of mixed biological stains in forensic casework and highlight factors which can affect it. This approach requires a detailed knowledge--gained through a mixture of experiments and validation studies--of the behaviour of each locus within the multiplex systems described. We summarise the available data from previously published experimental work and validation studies to examine the general principles underlying this approach.

Alleles↗

Prevalence and causes of amblyopia in an adult population.

OBJECTIVE: The study aimed to determine the prevalence, causes, and associations with amblyopia in a defined older population. DESIGN: In a population-based study, 3654 persons 49 years of age or older from an area west of Sydney, Australia, underwent a detailed eye examination and history, including objective and subjective refraction, cover testing, and retinal and lens photography. Amblyopia was diagnosed in eyes with reduced best-corrected visual acuity in the absence of any other cause. RESULTS: Amblyopia was diagnosed in 118 participants, or 3.2% of the population using a visual acuity criterion of 20/30 or less and 2.9% using a visual acuity criterion of 20/40 or less. Using a two-line visual acuity difference between the eyes, the amblyopia prevalence was 2.6% and 2.5%, respectively, for the above criteria. The underlying amblyogenic causes assessed were anisometropia (50%), strabismus (19%), mixed strabismus and anisometropia (27%), and visual deprivation (4%). The visual acuity of the amblyopic eye was 20/200 or worse (19%), 20/80 to 20/160 (19%), 20/40 to 20/63 (52%), and 20/30 (11%). No statistically significant associations were found between amblyopia and gender or eye affected. The most frequent pattern of strabismus was esotropia, whereas hypermetropia was the most frequent refractive error in amblyopic eyes. The mean age at diagnosis was earlier for strabismic and mixed amblyopia (7.4 years) than for anisometropic amblyopia (12.7 years). CONCLUSION: This study has provided prevalence and cases of amblyopia in an older population. Amblyopia is a frequent cause of lifelong unilateral visual impairment.

Aged↗

Development of guidelines to designate alleles using an STR multiplex system.

We have carried out extensive validation of a multiplex system of 6 STR loci and the Amelogenin sex test to investigate the following characteristics: relative peak areas of heterozygote peaks; non-specific artefacts; stutters caused by slippage of the Taq enzyme; pull-up (an electronic artefact often associated with software). The purpose of this paper is to demonstrate how interpretation guidelines can be formulated by experimentation and analysis using casework samples. Automation allows data relating to band position (in base pairs), peak area and peak height to be easily collected and manipulated in spreadsheets or computer programs. This paves the way to designing expert systems to carry out the interpretative process.

Alleles↗

The validation of a 7-locus multiplex STR test for use in forensic casework. (I). Mixtures, ageing, degradation and species studies.

We have evaluated a multiplex STR system for routine forensic use, which co-amplifies six short tandem repeat (STR) loci; HUMTH01, D21S11, D18S51, D8S1179, HUMVWF31/A and HUMFIBRA (FGA), in conjunction with the X-Y homologous gene Amelogenin. Analysis of PCR products employs denaturing polyacrylamide gels coupled with fluorescent labelled primers and detection is undertaken on ABD 373A automated sequencers. The technique was shown to be robust and reproducible when samples were analysed under conditions consistent with those encountered in a forensic environment. The system was demonstrated to be human specific and is suitable for use with both aged and degraded material. Somatic stability was proven with a wide range of tissue types and we were able to detect mixtures at ratios between 1:10 and 10:1. During this study no incidence of sample mis-typing due to allelic or locus drop-out was observed. Furthermore, although additional artefact bands were occasionally encountered these did not interfere with the interpretation of results. The performance of the system with poor quality samples demonstrated its suitability as a powerful tool in forensic investigation.

Adult↗

The validation of a 7-locus multiplex STR test for use in forensic casework. (II), Artefacts, casework studies and success rates.

PCR-based DNA typing of biological evidence is now widely used in forensic analyses due to the obvious advantages of enhanced sensitivity, the ability to distinguish discrete alleles and efficacy with degraded samples. A multiplex short tandem repeat (STR) system has been previously developed which successfully co-amplifies six STR loci HUMTH01, D21S11, D18S51, D8S1179, HUMVWF31/A and HUMFIBRA (FGA) in conjunction with the X-Y homologous gene Amelogenin. This is known as the second generation multiplex system (SGM). Detection of the PCR products is undertaken on ABD 373A or 377 automated sequencers using denaturing polyacrylamide gels coupled with fluorescent-based technology. We have evaluated this system for routine forensic use and demonstrated that the technique is robust and reproducible under conditions consistent with those encountered in a forensic environment. A total of 132 stains from simulated and actual casework were analysed, together with relevant control areas and reference samples. The success rate was high with 76% of stains giving full profiles; we were also able to successfully detect and interpret mixtures. No mistyping was observed. A detailed examination of each of these profiles has assisted in the development of guidelines for casework interpretation. Although artefacts, stutter peaks and undenatured DNA were occasionally observed, these did not interfere with the accuracy of interpretation. In addition 38 samples, previously examined using the quadruplex system, were analysed with the SGM to enable a direct comparison to be made between the systems. The performance of the system with poor quality samples demonstrated its use as a rapid and powerful technique for individual identification.

Adult↗

A new method of STR interpretation using inferential logic--development of a criminal intelligence database.

A short tandem repeat (STR) system consisting of seven multiplexed loci has recently been introduced in the UK to support a National strategy to create large DNA databases for criminal intelligence purposes. The process uses automated sequencers, employing dye-labelled primers. Identification of tetrameric loci such as HUMTH01 are straightforward. Sizing windows are estimated by running a series of control allelic ladders on several gels and 'unknown' samples are designated if they fall within a defined window. However, utilisation of complex STRs (eg. D21S11) characteristically have common variants which differ by just 2 bp. In addition, rare alleles are encountered which may differ by just 1 bp from a common variant. To assist with the identification of alleles, we have introduced a series of allelic ladders, so that direct comparisons with 'unknown' samples can be made on the same gel. To designate an allele, it should be within 0.5 bp of an allelic ladder marker. Not all alleles (in particular rare alleles) can be included within an allelic ladder, however their expected positions can be easily calculated by reference to existing alleles in the ladder. Measurement of band shift is also a useful diagnostic tool. A series of guidelines are described to enable reliable allelic identification. These guidelines can be converted into computer programmes which form the basis of an expert system.

Alleles↗

U-type exchange in a paracentric inversion as a possible mechanism of origin of an inverted tandem duplication of chromosome 8.

A mentally retarded male with dysmorphic features was found to have a de novo 46,XY,inv dup(8) (p.23.1-->12). Confirmation of the segments duplicated in the rearrangement was achieved by biochemical analysis of glutathione reductase, which maps to 8p21.1, and DNA studies using the chromosome specific probe y-19-1D (D85131), which maps to 8p21. Assay of cathepsin B, which has been localised to 8p22, did not differ from controls with normal chromosomal constitution. DNA studies using the Defensin 1 gene probe, which maps to 8p23, showed a previously undetected deletion of that segment. We propose that the inverted tandem duplication/deletion arose as a single U-type exchange within an inversion loop.

Abnormalities, Multiple↗

A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostosis.

Craniosynostosis, the premature fusion of calvarial sutures, is a common developmental anomaly that causes abnormal skull shape. The locus for one autosomal dominant form of craniosynostosis has been mapped to chromosome 5qter. The human MSX2 gene localizes to chromosome 5, and a polymorphic marker in the MSX2 intron segregates in a kindred with the disorder with no recombination. Moreover, a histidine substitutes for a highly conserved proline at position 7 of the MSX2 homeodomain exclusively in affected members. In the mouse, transcripts of the Msx2 gene are localized to calvarial sutures. These results provide compelling evidence that the mutation causes this craniosynostosis syndrome.

Acrocephalosyndactylia↗

Characterization and chromosomal mapping of the gene encoding the cellular DNA binding protein HTLF.

A region of the human T-cell leukemia virus long terminal repeat (HTLV-I LTR) located between -155 and -117 is important in the regulation of gene expression by the ets family of transcription factors. In an attempt to identify additional cellular transcription factors that bind to this portion of the HTLV-I LTR, we used lambda gt11 expression cloning with oligonucleotides corresponding to this element. A 1239-bp cDNA was isolated from a Jurkat cDNA library, which encoded a protein capable of binding to this purine-rich region. This protein, which we designated human T-cell leukemia virus enhancer factor (HTLF), contains a domain with homology to the recently described fork head DNA binding domain. Chromosome mapping of the HTLF gene demonstrated that it was localized to human chromosome 2p16-p22. HTLF is a unique cellular gene that may function in the transcriptional regulation of HTLV-I LTR.

Amino Acid Sequence↗

Characterization and chromosomal mapping of the gene encoding the cellular DNA binding protein ILF.

Recently we isolated a cellular DNA binding protein, designated interleukin enhancer binding factor (ILF), that binds to purine-rich regulatory motifs in both the HIV-1 LTR and the IL2 promoter. Further analysis of the ILF gene reveals the existence of two mRNA species, both of which encode proteins containing the recently described fork head DNA binding domain. Gel retardation analysis demonstrates that the portion of the ILF protein with homology to the fork head domain is sufficient to mediate DNA binding to a number of related purine-rich sequences. ILF mRNA is expressed constitutively in both lymphoid and nonlymphoid tissues. Chromosomal mapping localizes the ILF gene to human chromosome 17q25, which is a site of chromosomal translocations in some cases of human acute myelogous leukemias. These studies further characterize the structure of the cellular DNA binding protein ILF and may prove valuable in the molecular analysis of possible translocations affecting this gene.

Amino Acid Sequence↗

Segregation and linkage analyses of Tourette's syndrome and related disorders.

Segregation and linkage analyses were performed with data from a large Tourette's syndrome (TS) multigenerational kindred. Results of segregation analyses were remarkably similar to some reported earlier and suggest that the mode of transmission is consistent with autosomal dominant inheritance. The analyses were done using three diagnostic schemes to specify affected family members (TS only; TS or chronic tics [CT]; and TS, CT or obsessive compulsive disorder [OCD]). The estimates of penetrance for the genotypes AA, Aa and aa (A denotes the susceptibility allele) in the analyses including relatives with TS, CT or OCD were 0.99, 0.99 and 0.00, respectively, for males and 0.70, 0.70 and 0.00 for females. Pairwise linkage analyses with 140 marker loci failed to identify a linked marker. However, approximately 30 percent of the genome was excluded as the site of the hypothesized locus for TS.

Child↗

Gene mapping in the idiopathic generalized epilepsies: juvenile myoclonic epilepsy, childhood absence epilepsy, epilepsy with grand mal seizures, and early childhood myoclonic epilepsy.

Idiopathic generalized epilepsies, i.e., juvenile myoclonic epilepsy (JME), childhood absence epilepsy, and epilepsy with grand mal [generalized tonic-clonic seizures (GTCS)], are the most common genetic epilepsies. Linkage studies using Bf, HLA serologic, and DNA markers by three independent investigators, one from Los Angeles and two from Berlin, have localized the JME locus to the short arm of chromosome 6 (6p). Because members of the same JME family have the same JME phenotype of childhood absence epilepsy, epilepsy with grand mal (GTCS) seizures, or early childhood myoclonic epilepsy (ECME), our observations give evidence for a single-locus etiology in 6p for JME and for at least some of the childhood absence seizures, epilepsy with grand mal (GTCS) seizures, and ECME. Studies should now address whether locus heterogeneity exists within childhood absence epilepsy, epilepsy with grand mal (GTCS) seizures, or ECME. Markers linked to JME (Bf, HLA serologic, and DNA markers in the DQ region) can be used to resolve etiologic heterogeneity. Using such markers, both linked and unlinked forms of phenotypes that are clinically indistinguishable may be detected and provide evidence for etiologic heterogeneity. Studies should also concentrate on narrowing the JME locus to 2 to 3 cm by screening families with recombinant events using RFLPs, candidate genes, and new expressed sequences on chromosome 6.

Adolescent↗

Male-enhanced antigen gene is phylogenetically conserved and expressed at late stages of spermatogenesis.

The male-enhanced antigen gene (Mea) was previously isolated from a mouse testicular cDNA library by using a pool of specific antisera against the serological H-Y antigen. The present studies characterize the human and mouse cDNAs and indicate that the MEA gene is conserved at both nucleic acid and protein levels. The corresponding mRNA encodes proteins of 18-20 kDa. The phylogenetic conservation could be extended to other mammalian species by Southern blot analysis. Although the Mea gene was transcribed as a 1-kilobase mRNA in most tissues, it was expressed at the highest level in adult testis. The testis-enhanced expression of the Mea gene was associated with germ cell development at late stages of spermatogenesis. Chromosome walking experiments identified two linked genes, A and B, located within 38 kilobases of human genomic sequence. Like the MEA gene, genes A and B were coordinately transcribed in the testis, which suggests that MEA and genes A and B are members of a gene family. In situ hybridization studies localized the MEA gene to the short arm of human chromosome 6 at band p21.1-21.3, close to the major histocompatibility complex locus. The genetic conservation and testis-specific expression of the MEA gene support the hypothesis that it plays an important role in mammalian spermatogenesis and/or testis development.

Amino Acid Sequence↗