Analysis of salivary DNA evidence from a bite mark on a body submerged in water.

A female body was recovered after approximately 5.5 h in a river with slow-moving current. On the victim's right breast, a patterned injury was discovered and determined to be from human adult teeth. Evidence was collected according to established techniques including recovery of saliva from the bite mark area despite the body being found submerged in water. DNA analysis by PCR using polymorphic STR markers revealed a DNA profile of mixed origin. In addition to the victim's DNA profile, a genotype contribution from the perpetrator was identified as a minor component. The DNA typing results from the bite mark correlated with the DNA typing results obtained from other biological trace evidence identified from the victim's genital samples. The bite mark and the DNA evidence were used to screen suspects and played an important role in obtaining resolution of this case. Consequently, it is advisable that investigators routinely swab for salivary DNA in bite mark cases, even when the amount is thought to be minimal.

Removal of a PCR inhibitor and resolution of DNA STR types in mixed human-canine stains from a five year old case.

The analysis of biological trace evidence from a reopened investigation into a 1991 murder from Vernon, B.C. revealed mixed human and dog bloodstains on blue jean pants that contained a PCR inhibitory substance. The presence of the inhibitory substance was detected by the inhibition caused from adding a small aliquot of the test DNA extract into a PCR reaction designed to produce a known standard product. The removal of the PCR inhibitory substance was accomplished by treating the extracted DNA with Thiopropyl Sepharose 6B beads. DNA profiles from two human contributors and a canine were obtained using species specific polymorphic STR markers. The two human DNA profiles obtained from blue jean pants were resolved, one matched the suspect and the other matched the victim. The DNA profile from the canine component matched that obtained from the known sample of the victim's dog who was also slain during the assault. This evidence along with other DNA typing evidence was critical in obtaining a resolution of the case.

Genomic structure of the human D-site binding protein (DBP) gene.

The human gene for the D-Site Binding Protein (DBP) has been sequenced and characterized. This gene is a member of the b/ZIP family of transcription factors and is one of three genes forming the PAR subfamily. DBP has been implicated in the diurnal regulation of a variety of liver-specific genes. Examination of the genomic structure of DBP reveals that the gene is divided into four exons and is contained within a relatively compact region of approximately 6 kb. These exons appear to correspond to functional divisions of the DBP protein. Exon 1 contains a long 5' UTR, and conservation between the rat and the human genes of the presence of small open reading frames within this region suggests that it may play a role in translational control. Exon 2 contains a limited region of similarity to the other PAR domain genes, which may be part of a potential activation domain. Exon 3 contains the PAR domain and differs by only 1 of 71 amino acids between rat and human. Exon 4, containing both the basic and the leucine zipper domains, is likewise highly conserved. The overall degree of homology between the rat and the human cDNA sequences is 82% for the nucleic acid sequence and 92% for the protein sequence. Comparison of the rat and human proximal promoters reveals extensive sequence conservation, with two previously characterized DNA binding sites being conserved at the functional and sequence levels.

Isolation of a novel G protein-coupled receptor (GPR4) localized to chromosome 19q13.3.

We present the cloning and sequencing of the human gene for a novel G-protein coupled receptor (GPR4), from the critical myotonic dystrophy (DM) region on chromosome 19q13.3. The homologous porcine gene was isolated and sequenced as well. The genes of both species are intronless and contain an open reading frame encoding a protein of 362 amino acids. In human, two isoforms of GPR4 are expressed, differing in their 3' untranslated region due to the use of alternate polyadenylation signals and measuring approximately 2.8 and 1.8 kb, respectively. Northern blot analysis showed that GPR4 is widely expressed, with higher levels in kidney, heart, and especially lung, where it is at least fivefold greater than in other tissues. Sequence analysis suggests that GPR4 is a peptide receptor and shares strongest homologies with purinergic receptors and receptors for angiotensin II, platelet activating factor, thrombin, and bradykinin.

The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy.

The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.

The 1.5-Mb region spanning the myotonic dystrophy locus shows uniform recombination frequency.

The myotonic dystrophy (DM) mutation has been identified as a heritable unstable CTG trinucleotide repeat sequence. The intergenerational amplification of this sequence is an example of a new class of dynamic mutations responsible for human genetic diseases. To ascertain whether recombination activity influences, or is affected by, the presence of this unique sequence, a comprehensive study of the physical and genetic mapping data for the 1.5-Mb region of human chromosome 19q13.3, which contains the DM locus, was conducted. The recombination rate for this region was examined by correlating genetic distance to physical distance for six selected marker loci. The following markers span the DM region: 19qCEN-p alpha 1.4 (D19S37)-APOC2-CKM-pE0.8 (D19S115)-pGB2.6 (DM)-p134c (D19S51)-19qTER. Initial linear regression analysis of these two parameters failed to reveal a significant linear correlation (coefficient of determination, r2 = .19), suggesting nonuniform rates of recombination. However, the presence of a recombination hot spot was believed to be unlikely, as the marker-to-marker pairs that showed the greatest deviation in recombination frequency were not restricted to a specific region of the 1.5 Mb studied and had relatively broad confidence intervals, as reflected by low LOD values. A second linear regression analysis using only marker intervals with high LOD scores (Zmax > 22) showed linear correlation (r2 = .68) for the entire 1.5-Mb region. This analysis indicated a relatively uniform recombination frequency in the 1.5-Mb region spanning the DM locus. Furthermore, the recombinations observed were neither under- nor overrepresented on DM chromosomes. Consequently, recombination activity is unlikely to influence, or be affected by, the presence of the DM mutation.

Physical mapping and cloning of the proximal segment of the myotonic dystrophy gene region.

The myotonic dystrophy (DM) region has been recently shown to be bracketed by two key recombinant events. One recombinant occurs in a Dutch DM family, which maps the DM locus distal to the ERCC1 gene and D19S115 (pE0.8). The other recombinant event is in a French Canadian DM family, which maps DM proximal to D19S51 (p134c). To further resolve this region, we initiated a chromosome walk in a telomeric direction from pE0.8, a proximal marker tightly linked to DM, toward the genetic locus. An Alu-PCR approach to chromosome walking in a cosmid library from flow-sorted chromosome 19 was used to isolate DM region cosmids. This effort has resulted in the cloning of a 350-kb genomic contig of human chromosome 19q13.3. New genetic and physical mapping information has been generated using the newly cloned markers from this study. As a result of this new mapping information, the minimal area that is to contain the DM gene has been redefined. Approximately 200 kb of sequence between pE0.8 and the closest proximal marker to DM, pKEX0.8, that would have otherwise been screened for DM candidate genes, has been eliminated as containing the DM gene.

Myotonic dystrophy mutation: an unstable CTG repeat in the 3' untranslated region of the gene.

Myotonic dystrophy (DM) is the most common inherited neuromuscular disease in adults, with a global incidence of 1 in 8000 individuals. DM is an autosomal dominant, multisystemic disorder characterized primarily by myotonia and progressive muscle weakness. Genomic and complementary DNA probes that map to a 10-kilobase Eco RI genomic fragment from human chromosome 19q13.3 have been used to detect a variable length polymorphism in individuals with DM. Increases in the size of the allele in patients with DM are now shown to be due to an increased number of trinucleotide CTG repeats in the 3' untranslated region of a DM candidate gene. An increase in the severity of the disease in successive generations (genetic anticipation) is accompanied by an increase in the number of trinucleotide repeats. Nearly all cases of DM (98 percent or 253 of 258 individuals) displayed expansion of the CTG repeat region. These results suggest that DM is primarily caused by mutations that generate an amplification of a specific CTG repeat.

Cloning of the essential myotonic dystrophy region and mapping of the putative defect.

Myotonic dystrophy is a common dominant disorder (global incidence of 1:8,000) with variable onset and a protean nature of symptoms mainly involving progressive muscle wasting, myotonia and cataracts. To define the molecular defect, we have cloned the essential region of chromosome 19q13.3, including proximal and distal markers in a 700-kilobase contig formed by overlapping cosmids and yeast artificial chromosomes (YACs). The central part of the contig bridges an area of about 350 kilobases between two new flanking crossover borders. This segment has been extensively characterized through the isolation of five YAC clones and the subsequent subcloning in cosmids from which a detailed EcoRI, HindIII, MluI and NotI restriction map has been derived. Two genomic probes and two homologous complementary DNA probes were isolated using the cosmids. These probes are all situated within approximately 10 kilobases of genomic DNA and detect an unstable genomic segment in myotonic dystrophy patients. The length variation in this segment shows similarities to the instability seen at the fragile X locus. The physical map location and the genetic characteristics of the length polymorphism is compatible with a direct role in the pathogenesis of myotonic dystrophy.

D19S51 is closely linked with and maps distal to the myotonic dystrophy locus on 19q.

Recent genetic linkage studies have mapped the myotonic dystrophy (DM) locus to 19q13.3. All closely linked DM markers identified to date have been located on the centromeric side of the disease locus, with a relatively large genetic interval (9 cM) observed between the nearest distal marker and DM. We show here that the recently described marker p134C is tightly linked to DM (peak lod score 35.8 at peak recombination fraction .006) and confirm the previous suggestion that the p134C locus, D19S51 maps distal to the disease locus. D19S51 and the closest proximal flanking loci, ERCC1 and D19S115 (pE0.8), define a small genetic interval of less than 2 cM that contains the DM locus.

Physical and genetic mapping of a novel chromosome 19 ERCC1 marker showing close linkage with myotonic dystrophy.

Recent genetic linkage analyses have mapped the myotonic dystrophy locus to the region of 19q13.2-13.3 lying distal to the gene for creatine kinase subunit M (CKM). The human excision repair gene ERCC1 has also been mapped to this region of chromosome 19. A novel polymorphic DNA marker, pEO.8, has been isolated from a chromosome 19 ERCC1-containing cosmid that maps to a 300-kb NotI fragment encompassing both CKM and ERCC1. Genetic linkage analysis reveals close linkage between pEO.8 and myotonic dystrophy (DM) (zmax = 19.3, theta max = 0.01). Analysis of two key recombinant events suggests a mapping of DM distal to pEO.8 and CKM.

Hae III--a suitable restriction endonuclease for restriction fragment length polymorphism analysis of biological evidence samples.

Hae III has been selected by our laboratories as the restriction endonuclease of choice for restriction fragment length polymorphism analysis of forensic science samples. The enzyme is compatible with the D2S44 probe system and generates relatively small DNA fragments for that marker system. Similarly, Hae III is compatible with several other independent polymorphic loci, including D1S7, D4S139, D16S85, D17S74, D17S79, D14S13, and D20S15. Hae III is functional under a variety of adverse conditions for DNA digestion and is not affected by the methylation pattern in mammals. Finally, Hae III is a relatively inexpensive restriction endonuclease.

A simple and sensitive method for quantifying human genomic DNA in forensic specimen extracts.

The analysis of DNA restriction fragment length polymorphisms by Southern blot hybridization requires that sufficient quantities of high molecular weight genomic DNA be extracted from biological specimens. Prior to analysis, it is necessary to determine the quantity and quality of the extracted DNA. For many applications, it is also desirable to determine the amount of DNA which is of human origin. In this report, we describe a simple and highly sensitive procedure for the specific quantification of human genomic DNA in forensic extracts or any biological sample. A small fraction of the extract is immobilized onto a nylon membrane and subsequently hybridized to p17H8 (D17Z1), a cloned probe which detects highly repetitive, primate-specific alpha satellite DNA. The procedure requires less than four hours to complete and can be used to quantify subnanogram amounts of hybridizable human genomic DNA.

Bloodstain characterization in the EAP, Hp, Hb, AK and Glo I typing systems using minigels and the PhastSystem.

Application of minigels and the PhastSystem to obtain phenotyping results from bloodstains in the EAP, Hp, AK, and Glo I typing systems was investigated. Nonequilibrium isoelectric focusing with 4-6.5 PhastGel produced readily interpretable phenotypes in the EAP typing system. Both 4-6.5 and 5-8 PhastGel produced AK typing system phenotypes using nonequilibrium isoelectric focusing conditions. The 8-25% PAG PhastGel developed by two staining techniques allowed discrimination of phenotypes for the Hp typing system. Phenotypes from the Glo I typing system were also obtained with this gel type. Variant haemoglobins could be detected on pH 5-8 PhastGel using isoelectric focusing conditions. Much potential for standardized, rapid phenotyping of bloodstains was found to exist utilizing the PhastSystem.