Tracking COL1A1 RNA in osteogenesis imperfecta. splice-defective transcripts initiate transport from the gene but are retained within the SC35 domain.

This study illuminates the intra-nuclear fate of COL1A1 RNA in osteogenesis imperfecta (OI) Type I. Patient fibroblasts were shown to carry a heterozygous defect in splicing of intron 26, blocking mRNA export. Both the normal and mutant allele associated with a nuclear RNA track, a localized accumulation of posttranscriptional RNA emanating to one side of the gene. Both tracks had slightly elongated or globular morphology, but mutant tracks were cytologically distinct in that they lacked the normal polar distribution of intron 26. Normal COL1A1 RNA tracks distribute throughout an SC-35 domain, from the gene at the periphery. Normally, almost all 50 COL1A1 introns are spliced at or adjacent to the gene, before mRNA transits thru the domain. Normal COL1A1 transcripts may undergo maturation needed for export within the domain such as removal of a slow-splicing intron (shown for intron 24), after which they may disperse. Splice-defective transcripts still distribute thru the SC-35 domain, moving approximately 1-3 micrometer from the gene. However, microfluorimetric analyses demonstrate mutant transcripts accumulate to abnormal levels within the track and domain. Hence, mutant transcripts initiate transport from the gene, but are impeded in exit from the SC-35 domain. This identifies a previously undefined step in mRNA export, involving movement through an SC-35 domain. A model is presented in which maturation and release for export of COL1A1 mRNA is linked to rapid cycling of metabolic complexes within the splicing factor domain, adjacent to the gene. This paradigm may apply to SC-35 domains more generally, which we suggest may be nucleated at sites of high demand and comprise factors being actively used to facilitate expression of associated loci.

The genetic legacy of Paleolithic Homo sapiens sapiens in extant Europeans: a Y chromosome perspective.

A genetic perspective of human history in Europe was derived from 22 binary markers of the nonrecombining Y chromosome (NRY). Ten lineages account for >95% of the 1007 European Y chromosomes studied. Geographic distribution and age estimates of alleles are compatible with two Paleolithic and one Neolithic migratory episode that have contributed to the modern European gene pool. A significant correlation between the NRY haplotype data and principal components based on 95 protein markers was observed, indicating the effectiveness of NRY binary polymorphisms in the characterization of human population composition and history.

Defective splicing of mRNA from one COL1A1 allele of type I collagen in nondeforming (type I) osteogenesis imperfecta.

Osteogenesis imperfecta (OI) type I is the mildest form of heritable bone fragility resulting from mutations within the COL1A1 gene. We studied fibroblasts established from a child with OI type I and demonstrated underproduction of alpha 1 (I) collagen chains and alpha 1 (I) mRNA. Indirect RNase protection suggested two species of alpha 1 (I) mRNA, one of which was not collinear with fully spliced alpha 1 (I) mRNA. The noncollinear population was confined to the nuclear compartment of the cell, and contained the entire sequence of intron 26 and a G-->A transition in the first position of the intron donor site. The G-->A transition was also identified in the genomic DNA. The retained intron contained an in-frame stop codon and introduced an out-of-frame insertion within the collagen mRNA producing stop codons downstream of the insertion. These changes probably account for the failure of the mutant RNA to appear in the cytoplasm. Unlike other splice site mutations within collagen mRNA that resulted in exon skipping and a truncated but inframe RNA transcript, this mutation did not result in production of a defective collagen pro alpha 1 (I) chain. Instead, the mild nature of the disease in this case reflects failure to process the defective mRNA and thus the absence of a protein product from the mutant allele.

Molecular basis of nanomelia, a heritable chondrodystrophy of chicken.

Nanomelia is a recessively inherited connective tissue disorder of chicken affecting cartilage development. Other investigators have demonstrated that it involves low aggrecan production and diminished aggrecan mRNA levels. Based on genetic linkage studies showing a high likelihood that the mutation responsible for the nanomelic phenotype lay within the aggrecan gene, a series of experiments was performed to define the molecular basis of the trait. Aggrecan mRNA was present in the nucleus of the nanomelic chondrocyte but greatly reduced in the cytoplasmic compartment, a finding suggestive of a premature stop codon within the aggrecan transcript. Since no defect in mRNA splicing could be demonstrated by ribonucleasease protection studies, direct DNA sequencing was initiated by polymerase chain reaction of the mRNA and of genomic DNA. A stop codon was demonstrated at codon 1513, which is located in the eighth repeat of the chondroitin sulfate 2 domain of the large tenth exon. The mutation creates a unique BasBI restriction site which readily distinguishes the mutant and wild-type alleles.

Interferon-induced growth arrest is mediated by membrane structural changes.

Interferon-gamma (IFN-gamma) is an immunomodulator shown to augment the expression of major histocompatibility (MHC) class I/class II antigens on the cell surface. In previous studies, we have demonstrated that the enhanced expression of these antigens on the cell surface is in part due to IFN-gamma-mediated abrogation of antigen shedding. In this study, we demonstrate that IFN-gamma induces structural changes in the cell membrane by altering the cholesterol/phospholipid ratio. Furthermore, such changes not only mediate enhanced expression of antigen on the cell surface but may drive the cells to growth arrest and apoptosis. These results were obtained by employing x-ray diffraction, electron microscopy, and DNA analysis.

Platelet function analysis in children with Schönlein-Henoch syndrome.

BACKGROUND: Schönlein-Henoch syndrome (SHS) or anaphylactic purpura in childhood is the result of pathologic and immunologic responses to different antigens. These antigens could induce the formation of immune complexes responsible for vasculitis and their precipitation on the endothelium of small blood vessels. Purpuric bruises, hematuria, hematemesis, melena, or hematochesis may suggest coagulation disturbances. Increasing bleeding tendency may suggest platelet function disturbance. To examine the qualitative function of platelets in children with SHS, we decided to analyze its aggregation function. METHODS: Using the Born method of testing, we analyzed platelet aggregation in 24 children with SHS. RESULTS: Based on the aggregograms examined, we observed that most patients had abnormal aggregation curves, in which platelets demonstrated a block of release of the endogenous ADP, with or without disaggregation. CONCLUSIONS: One clinical symptom of SHS appearing in most patients is a mild or increased tendency toward bleeding. On measuring induced aggregation of platelets in children with SHS, we observed that the qualitative function of platelets was disturbed.

Northern and southern Croatian population data on seven PCR-based loci.

Northern and southern Croatian sample populations were typed at seven PCR-based loci -LDLR, GYPA, HBGG, D7S8, Gc, HLA-DQA1 and D1S80. The results show that all loci meet Hardy-Weinberg expectations and that there is little evidence for association of alleles between loci. Allelic frequency distributions at all loci, except HLA-DQA1, show no differences between the northern and southern Croatian sample populations. Moreover, the population data for Croatians are similar to U.S. Caucasians; only HLA-DQA1 for southern Croatians was statistically different compared with U.S. Caucasians. A Croatian population database(s) has been created and can be used for forensic analyses to estimate the frequency of a multiple locus DNA profile.

Identification of war victims from mass graves in Croatia, Bosnia, and Herzegovina by use of standard forensic methods and DNA typing.

The postmortem remains of sixty-one war victims were excavated from 6 mass graves in Bosnia and Herzegovina one and a half years after interment Using standard identification methods, including the matching of medical and dental records, the recognition of distinguishing characteristics such as the use of clothing and belongings, and video superimposition, 35 persons were identified. For the remaining 26 persons identification efforts continue. DNA typing was performed at the HLA DQA1 locus and five PM system loci. Results from DNA typing were confirmed by other methods. DNA profiles of family members of 150 missing persons are now being developed using the 6 loci. These DNA profiles will then be compared with those generated from the bone and teeth remains of the unidentified victims.

Spine and spinal cord war injuries during the war in Croatia.

OBJECTIVE: The present report summarizes the experience of an evacuation hospital in southern Croatia in treating 96 patients with spine and spinal cord war injuries. METHODS: A retrospective review was done for 96 wounded persons (86 soldiers, 10 civilians) with spinal cord injuries from August 1991 through December 1995. The ages ranged from 15 to 59 years (mean, 28.3 years for soldiers, 38.5 years for civilians). Diagnostic procedures were plain radiography, computed tomography, and computed tomographic myelography. However, in most cases a more conservative surgical approach was used. RESULTS: The highest rates of admittance were recorded in 1992 (N = 38) and 1993 (N = 47). The lumbar spine was injured in 55% of the patients, the cervical and thoracic spines in 17.7%. All injuries were caused by projectiles from automatic rifles and sniper fire (51%) and from fragments of explosive devices (49%). Blast injury of the spinal cord was found in 10 patients. The most frequent complications caused by the fragments were wound infection, urinary tract infection, decubitus, and pneumonia. Four patients (4.2%) died in the hospital, and 43.0% of patients survived but were severely handicapped. CONCLUSION: Careful clinical examination combined with modern diagnostic imaging and use of broad-spectrum antibiotics reduced the need for surgical intervention in patients with spinal cord injuries.

Orbitocranial war injuries: report of 14 cases.

OBJECTIVE: In this study, we review the initial clinical and radiological management and early outcomes of 14 patients with orbitocranial war injuries treated at the University Hospital Split between 1991 and 1995. METHODS: This investigation involves 14 patients (13 soldiers and 1 civilian) with orbitocranial war injuries. The mean patient age was 31 years (range, 23-54 years). The penetrating object was a metal shrapnel fragment in 8 patients and a bullet in 6 patients. The results of clinical and radiological management were retrospectively analyzed. RESULTS: The mean time from the moment of wounding to hospital admission was 6 hours (range, 1-30 hours). The mean Glasgow Coma Scale score was 8 (range, 3-14). Craniotomy was the basic neurosurgical procedure, and three patients were treated with simple scalp wound debridement and closure. Osteoplastic operations of the orbital bones were performed in 13 patients. Enucleation/evisceration was performed in 6 patients (42.8%). At discharge, the mean Glasgow Outcome Scale score was 13, and 1 patient died in the hospital. Blindness (including amaurosis and anophthalmus) was present in nine eyes (8 patients), light-perception positivity and projection positivity were present in four eyes, and visual acuity was at 0.1 in 1 patient. CONCLUSION: An early multidisciplinary therapeutic approach and computed tomography as a diagnostic procedure are necessary for a good result in the treatment of orbitocranial war injuries.

Psychological characteristics of wounded and disabled Croatian war veterans.

OBJECTIVE: Evaluation of the psychological state of a group of 119 Croatian disabled war veterans who suffered grave traumatic war experiences during the war in Croatia (1991-1992). METHODS: Semistructured Clinical Interview, Profile Index Emotions test, and Zung's Self-Rating Depression Scale were used to assess disabled war veterans accommodated in special institutions for rehabilitation. RESULTS: Changes in psychological functioning were established in 63.8% of disabled war veterans, the most frequent among them being indisposition, irritability, anxiety, and fear. In nearly half of the veterans there were changes in their relationships with close persons, difficulties in accepting the reality of their situations, and increased aggressiveness. Higher average values on the depression index (0.52) were also established. CONCLUSION: The established change in the psychological profile of disabled Croatian war veterans was lower than expected. However, because of their great vulnerability, it is necessary to increase efforts to secure their complete psychosocial recovery.

Analysis of medical aid to Croatian Army soldiers wounded at the front line.

OBJECTIVE: Analysis of medical aid to Croatian Army soldiers at the front lines. METHODS: This investigation concerned 115 severely wounded Croatian soldiers (males, median age 29 years, range 17-57 years) at three rehabilitation institutions. All had been wounded at the front line by various explosive devices or bullets. In the investigation, semi-structured clinical interviews were used. RESULTS: First aid was provided to 47 soldiers (40.9%) within 15 minutes and to 44 soldiers (38.3%) within 1 hour. First aid was either self-administered or given by fellow combatants in 55 cases (47.8%), by physicians in 37 cases (23.2%), and by medical technicians and orderlies in 21 cases (18.2%). The basic procedures of medical aid were hemostasis, hemodynamic substitution, immobilization, and analgesia. First surgical operations were performed at health stations on 4 soldiers (3.5%), at war hospitals on 43 soldiers (37.4%), and at evacuation hospitals on 68 soldiers (59.1%). CONCLUSION: First aid either self-administered or provided by fellow combatants is the most important procedure at the front lines before medical aid is administered.

Peripheral nerve war injuries.

OBJECTIVE: The purpose of this study is to evaluate peripheral nerve war injuries sustained during the war in southern Croatia and Bosnia and Herzegovina. PATIENTS AND METHODS: During the war in Croatia, 713 patients (99% male and 1% female) with wounds inflicted by firearms were examined at the Laboratory of Neurophysiology, University Hospital, Split. The patients, soldiers and civilians alike, ranged in age from 6 to 73 years (average, 28 years). All patients with firearm nerve war injuries underwent detection by electromyography and plurisegmental examination of the damaged peripheral nerve (neurography). The patients were examined and controlled on three occasions: within 2 months after wounding; up to 6 months after wounding; and more than 6 months after wounding. RESULTS: Single peripheral nerve lesions were present in 80% of the patients, and multiple peripheral nerve or plexus lesions were present in 20% of the patients. Peroneal and ulnar nerves were most often involved (20.9% and 19.8%, respectively). Associated massive injuries to the muscles, large blood vessels, or vital organs were present in 45% of the patients. Wounds were inflicted by shell fragments in 80% of the patients and by projectiles in 20% of the patients. CONCLUSION: According to our results, better recovery was achieved with conservative treatment and when physical therapy was initiated early with maximal patient cooperation. Electromyoneurographic findings were the most valid in the prognostic classification of war-inflicted peripheral nerve injuries.

Genetic analysis of the glucose-6-phosphate dehydrogenase deficiency in a southern Croatia.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. G6PD Mediterranean is caused by a C-->T transition at nucleotide 563, is characterized with less than 10% of normal enzyme activity and is classified as severe G6PD deficiency. Nineteen unrelated males from Southern Croatia with severe G6PD deficiency were tested, by enzyme digestion, for the presence of the Mediterranean mutation. Individuals with G6PD Mediterranean were further screened for the silent C-->T transition at nucleotide 1311. Four of the nineteen individuals were positive for the Mediterranean mutation (21%) and all four had the silent mutation.

Early intracranial infections after brain missile injuries--the role of computer tomography in diagnosis and treatment.

During the war in Croatia, from August 1991 until December 1994, 138 soldiers were treated at the Split University Hospital for different brain injuries inflicted by missiles. Nine of these 138 patients developed intracranial infection. This retrospective study reports on the outcome of these 9 intracranial infections caused by penetrating missile head injuries. In case of clinically suspected infection, computerized tomography scan was obtained at two time points during the course of infection in the same patient. Scans were obtained with and without contrast media, 7 to 14 days after the injury and the 4 weeks later. The role of computerized tomography in the detection and follow-up of various intracranial infections and long-term consequences were evaluated.

Association of NOS3 gene variants and clinical contributors of hypoxic-ischemic encephalopathy.

The aim of this study was to analyze the association of different clinical contributors of hypoxic-ischemic encephalopathy with NOS3 gene polymorphisms. A total of 110 children with hypoxic-ischemic encephalopathy and 128 control children were selected for this study. Association of gender, gestational age, birth weight, Apgar score, cranial ultrasonography, and magnetic resonance imaging findings with genotypic data of six haplotype-tagging single nucleotide polymorphisms and the most commonly investigated rs1800779 and rs2070744 polymorphisms was analyzed. The TGT haplotype of rs1800783, rs1800779, and rs2070744 polymorphisms was associated with hypoxic-ischemic encephalopathy. Children with the TGT haplotype were infants below 32 weeks of gestation and they had the most severe brain damage. Increased incidence of the TT genotype of the NOS3 rs1808593 SNP was found in the group of hypoxic-ischemic encephalopathy patients with medium and severe brain damage. The probability of brain damage was twice as high in children with the TT genotype than in children with the TG genotype of the same polymorphism. Furthermore, the T allele of the same polymorphism was twice as frequent in children with lower Apgar scores. This study strongly suggests associations of NOS3 gene polymorphism with intensity of brain damage and severity of the clinical picture in affected children.

Association of NOS3 gene variants and clinical contributors of hypoxic-ischemic encephalopathy

The aim of this study was to analyze the association of different clinical contributors of hypoxic-ischemic encephalopathy with NOS3 gene polymorphisms. A total of 110 children with hypoxic-ischemic encephalopathy and 128 control children were selected for this study. Association of gender, gestational age, birth weight, Apgar score, cranial ultrasonography, and magnetic resonance imaging findings with genotypic data of six haplotype-tagging single nucleotide polymorphisms and the most commonly investigated rs1800779 and rs2070744 polymorphisms was analyzed. The TGT haplotype of rs1800783, rs1800779, and rs2070744 polymorphisms was associated with hypoxic-ischemic encephalopathy. Children with the TGT haplotype were infants below 32 weeks of gestation and they had the most severe brain damage. Increased incidence of the TT genotype of the NOS3 rs1808593 SNP was found in the group of hypoxic-ischemic encephalopathy patients with medium and severe brain damage. The probability of brain damage was twice as high in children with the TT genotype than in children with the TG genotype of the same polymorphism. Furthermore, the T allele of the same polymorphism was twice as frequent in children with lower Apgar scores. This study strongly suggests associations of NOS3 gene polymorphism with intensity of brain damage and severity of the clinical picture in affected children.

The peopling of modern Bosnia-Herzegovina: Y-chromosome haplogroups in the three main ethnic groups.

The variation at 28 Y-chromosome biallelic markers was analysed in 256 males (90 Croats, 81 Serbs and 85 Bosniacs) from Bosnia-Herzegovina. An important shared feature between the three ethnic groups is the high frequency of the "Palaeolithic" European-specific haplogroup (Hg) I, a likely signature of a Balkan population re-expansion after the Last Glacial Maximum. This haplogroup is almost completely represented by the sub-haplogroup I-P37 whose frequency is, however, higher in the Croats (approximately 71%) than in Bosniacs (approximately 44%) and Serbs (approximately 31%). Other rather frequent haplogroups are E (approximately 15%) and J (approximately 7%), which are considered to have arrived from the Middle East in Neolithic and post-Neolithic times, and R-M17 (approximately 14%), which probably marked several arrivals, at different times, from eastern Eurasia. Hg E, almost exclusively represented by its subclade E-M78, is more common in the Serbs (approximately 20%) than in Bosniacs (approximately 13%) and Croats (approximately 9%), and Hg J, observed in only one Croat, encompasses approximately 9% of the Serbs and approximately 12% of the Bosniacs, where it shows its highest diversification. By contrast, Hg R-M17 displays similar frequencies in all three groups. On the whole, the three main groups of Bosnia-Herzegovina, in spite of some quantitative differences, share a large fraction of the same ancient gene pool distinctive for the Balkan area.

Application of forensic DNA testing in the legal system.

DNA technology has taken an irreplaceable position in the field of the forensic sciences. Since 1985, when Peter Gill and Alex Jeffreys first applied DNA technology to forensic problems, to the present, more than 50,000 cases worldwide have been solved through the use of DNA based technology. Although the development of DNA typing in forensic science has been extremely rapid, today we are witnessing a new era of DNA technology including automation and miniaturization. In forensic science, DNA analysis has become "the new form of scientific evidence" and has come under public scrutiny and the demand to show competence. More and more courts admit the DNA based evidence. We believe that in the near future this technology will be generally accepted in the legal system. There are two main applications of DNA analysis in forensic medicine: criminal investigation and paternity testing. In this article we present background information on DNA, human genetics, and the application of DNA analysis to legal problems, as well as the commonly applied respective mathematics.