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.

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.

Osteogenesis imperfecta at the beginning of bone and joint decade.

Osteogenesis imperfecta (OI), or brittle bone disease, is a heritable disorder characterized by increased bone fragility. Four different types of the disease are commonly distinguished, ranging from a mild condition (type I) to a lethal one (type II). Types III and IV are the severe forms surviving the neonatal period. In most cases, there is a reduction in the production of normal type I collagen or the synthesis of abnormal collagen as a result of mutations in the type I collagen genes. These classic forms of OI are described in this review. There are instances, however, where alterations in bone matrix components, other than type I collagen, are the basic abnormalities of the OI. Recently, three such discrete types have been identified by histomorphometric evaluation (types V and VI) and linkage analysis (Rhizomelic OI). They provide evidence for the as yet poorly understood complexity of the phenotype-genotype correlation in OI. We also discuss bisphosphonates treatment as well as fracture management and surgical correction of deformities observed in the patients with OI. However, ultimately, strengthening bone in OI will involve steps to correct the underlying genetic mutations that are responsible for this disorder. Thus, we also describe different genetic therapeutic approaches that have been tested either on OI cells or on available OI murine models.

DNA typing from skeletal remains: evaluation of multiplex and megaplex STR systems on DNA isolated from bone and teeth samples.

AIM: To evaluate the performance of three multiplex short tandem repeat (STR) systems (AmpflSTR Profiler, AmpflSTR Profiler Plus, and AmpflSTR COfiler), and a megaplex STR system (PowerPlex 16) on DNA extracted from the skeletal remains. By performing a microbial DNA challenge study, we also evaluated the influence of microbial DNA on human DNA typing. METHODS: A subset of 86 DNA extracts isolated from 8-50 years old bone and teeth samples, corresponding to 20 identification cases from mass graves in Croatia and Bosnia and Herzegovina, and to 4 paternity cases involving deceased parents in Spain, were analyzed by the above systems. RESULTS: Bone samples with no detectable human DNA (tested with Quantiblot), as well as teeth samples with detectable human DNA, were successfully amplified. Surprisingly, even in highly degraded samples, PowerPlex 16 offered very robust amplification for the both Penta E and Penta D markers. We observed a few non-specific extra peaks of 202 and 308 base pairs, which appeared to match 16S rRNA of the Pseudomonas halodenitrificans. CONCLUSION: AmpflSTR Profiler Kit, AmpflSTR Profiler Plus Kit, the AmpflSTR COfiler Kit, and the PowerPlex 16 system are very sensitive multiplex STR amplification systems, which can be successfully used to obtain a multilocus STR profile from old teeth and bone samples with minimal amounts (pg) of human DNA or even with no detectable human DNA.

Population variation of human mitochondrial DNA hypervariable regions I and II in 105 Croatian individuals demonstrated by immobilized sequence-specific oligonucleotide probe analysis.

AIM: To detect sequence variation in 105 Croatian individuals by the use of duplex polymerase chain reaction amplification of full-length hypervariable region I and II (HVI/HVII) products and subsequent hybridization to a linear array of 27 immobilized sequence-specific oligonucleotide (SSO) probes, which targets six regions within HVI and HVII, and two additional sites, 189 and 16093. METHODS: Chelex-extracted bloodstains were used for amplification of HV regions. In all cases, a single robust amplification was sufficient for immobilized SSO probe typing and subsequent direct sequence analysis for both HVI and HVII. This method, suitable for a range of forensic samples (including shaft portions of single hairs), was also applied to the analysis of 18 skeletal elements recovered from a mass grave. Using a panel of immobilized SSO probes, we have developed a rapid screening approach to mitochondrial DNA (mtDNA) haplotyping before direct sequence analysis. RESULTS: We established a reference sequence database of mtDNA haplotypes for 105 randomly selected Croatian individuals. Fifty different mitotypes were observed (33 unique). The most frequent mitotypes occurred 18 times or approximately 17.1% [111111 189 (A) 16093 (T)] and 11 times or approximately 10.5% [131111 189 (A) 16093 (T)]; all other mitotypes occurred 5% or less. The corresponding genetic diversity value for this database was approximately 0.952. The usefulness of establishing an mtDNA reference database with immobilized SSO probe testing has been demonstrated by determining the strength of a match comparison obtained for one skeletal element and a corresponding maternal reference from 18 specimens recovered from a mass grave. CONCLUSION: The sequence variation detected by the panel of immobilized SSO probes is sufficiently diverse to be used for identification of human skeletal remains from mass graves. The immobilized SSO typing strip targets polymorphic regions within HVI and HVII and is a useful identification tool for mass grave and mass disaster analysis, as well as for criminal casework testing.

Etoposide as the basic and interferon-alpha as the maintenance therapy for Langerhans cell histiocytosis: a RTC.

The treatment of patients who suffer from a disseminated form of Langerhans cell histiocytosis (LCH) is still controversial. So far, few larger randomized studies have been performed. The authors present 3 patients with a disseminated form of LCH--4 months, 9 months, and 2 years old, respectively. The lesional Langerhans cells in each patient showed positive immunohistochemical reaction to S-100 protein and the presence of Birbeck granules was confirmed by electron microscopy. All the patients were treated with etoposide (VP-16), 200 mg/m2 for 3 consecutive days, with 15 cycles at intervals of 3 weeks between each cycle, followed by maintenance therapy with IFN-alpha. All 3 patients reached complete stabile remission. The patients were young, at high risk, with multiple-organ involvement of LCH, and two of them had obvious signs of organ dysfunction at presentation, suggesting a poor prognosis. All remain disease-free several years after therapy. The results suggest that INF-alpha may prevent recurrences in high-risk patients.

Frequency of portal and systemic bacteremia in acute appendicitis.

BACKGROUND: Acute appendicitis is the most common condition requiring an emergency abdominal operation in childhood. In the present study, we analyzed the frequency of portal and systemic bacteremia in 42 patients with acute appendicitis and determined the microbial agents responsible for an acute appendicitis and for portal and systemic bacteremia. METHODS: Appendectomies were performed on 50 young patients (5-18 years of age), as well as clinical and bacteriological tests. Six independent samples from each patient isolated from the peripheral vein, superior mesenteric vein, appendix and peritoneum were obtained prior to surgery, during surgery and after surgery for biochemical, immunologic and bacteriologic examination. RESULTS: Pathohistology confirmed the diagnosis of appendicitis in 42 patients, while in the other eight patients there were no obvious pathologic findings, so they served as a control group. Of 50 patients with a clinical appearance of acute appendicitis, in 19 patients (38%) we detected portal bacteremia in the mesenteric vein, while in only three cases (6%) did we find systemic bacteremia detected from the peripheral vein. Furthermore, bacteriologic analysis revealed that Bacteroides spp. and Escherichia coli were the predominant species isolated. CONCLUSIONS: The results presented in this paper suggests that portal bacteremia did not influence peripheral blood reactions. Furthermore, in the present study we have found a positive correlation between the smear and bacteremia of the superior mesenteric vein, but not with the bacteremia of systemic blood.

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.

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.

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.

Craniocerebral war missile injuries: clinical and radiological study.

In this study we reviewed the initial clinical and radiological management and early outcomes of 176 consecutive patients from the war in Croatia.

Premature termination codon in the aggrecan gene of nanomelia and its influence on mRNA transport and stability.

AIM: To analyze the influence of the premature termination codon on mRNA transport and stability METHODS: Chondrocyte mRNA was isolated from homozygous and heterozygous nanomelic 17-days old embryos and examined by RT-PCR analysis. To analyze aggrecan mRNA stability, mRNA synthesis was inhibited with DRB [5,6 dichloro-1-(-D-ribofuranosyl benzimidazole)], a specific inhibitor of RNA polymerase II. Visualization of the aggrecan alleles was performed by in situ hybridization. RESULTS: The level of mutant aggrecan mRNA within the nucleus was equal to that of the control, but no mutant mRNA was observed in the cytoplasm. RT-PCR revealed that the mutant transcript was only detectable in the nucleus, compared with house-keeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene or collagen type II. A restriction site induced by premature termination codon TAA allowed the distinction of normal and mutant transcripts in chondrocytes derived from embryos heterozygous for the nanomelic mutation. After the treatment with DRB, identical decay rates were demonstrated for both transcripts within the heterozygous nucleus. In situ hybridization showed no abnormal mRNA accumulation. CONCLUSION: This is the first evidence suggesting that the transcript of the mRNA with the premature termination codon within an exon does exit the nucleus.

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.

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.

Allele frequencies of six highly polymorphic DNA loci in the Croatian population.

The allele frequency distributions in a series of Croats were analyzed for six unlinked polymorphic DNA loci: THO1, FESFPS, VWA01, APOB, D1S80, and D17S5. The allele frequencies were determined for 100 unrelated genomic DNA samples. The observed heterozygote frequencies of the loci ranged from 0.63 to 0.76; however, the the expected heterozygosity ranged from 0.68 to 0.82, with only D17S5 having a significant excess of homozygous phenotypes (p < 0.001). The excess homozygosity seen in the D17S5 system may be due to allelic drop-out and warrants further technical analysis of that system, given the uniform lack of significant deviation in the other five systems. The forensic usefulness of these systems can be measured using two different statistics: the power of discrimination and the likelihood of a coincidental match. The power of discrimination ranged from 0.85 to 0.94 for the 6 systems with the combined likelihood of a coincidental match based on these 6 systems of 1 in 3.6 million, or slightly less than the population of Croatia. A second, more conservative estimator of the likelihood of a match is based on the most common phenotype for each system. If someone had the most common phenotype for each of the 6 systems, the chance of a coincidental match would be approximately 1 in 64,000. For paternity testing the usefulness of a system is measured by the average power of exclusion or (1-power of exclusion), the random man not excluded. The average power of exclusion, based on observed heterozygosity, ranged from 0.33 to 0.53, and the average power of exclusion based on the expected heterozygosity ranged from 0.39 to 0.64. The combined average power of exclusion was 99.2% for these 6 systems, using the expected heterozygosity. Based on the results of testing these six systems, there is no significant substructuring within the southern Croatian populations, and these systems provide a useful tool for forensic, paternity, and anthropological applications.

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.