Detection of 13 Hypervariable Region 1 (Hv1) SNPs using single-base extension (Sbe) primers in parallel with Sanger sequencing.

The aim of this study was to determine whether single-base extension (SBE) chemistry can be applied to forensic practice of testing the target single nucleotide polymorphisms (SNPs) of the mitochondrial DNA (mtDNA) Hypervariable Region 1 (HV1). Despite itsweak discrimination power, high copy number of mtDNA per cell and its stability against degradation guarantee mtDNA testing a place in modern forensic genetics. In this research, buccal swab samples were obtained from 294 individuals from Bosnia and Herzegovina. Following DNA isolation using QIAamp® DNA Mini Kit, full sequencing of HV1 was completed using chain-termination Sanger sequencing method. SBE reactions were then performed by targeting 13 SNPs that were identified to be the most frequent in the study population. Uniplex SBE reactions for each individual SNP, as well as two multiplex reactions were prepared for both pure and mixed samples. The results showed complete agreement of the Sanger sequencing results with SBE reactions for both uniplex and multiplex reactions. The results obtained with SBE were encouraging in regard to multiplexing and processing of the mixed samples, since the allele of the minor contributor to the sample was observed in SBE electropherogram in all prepared mixtures. SBE method is limited by the fact that only target SNPs of interest will be analyzed, meaning that they must be carefully selected and curated for each population. However, typing with SBE protocol is accurate, as compared to the golden standard of Sanger sequencing, but was more time- and labor-efficient and simpler to analyze, therefore offering a suitable alternative when Sanger sequencing is not available, given that polymorphic SNPs are known for the population of interest.

Novel Therapeutic Effects in Rat Spinal Cord Injuries: Recovery of the Definitive and Early Spinal Cord Injury by the Administration of Pentadecapeptide BPC 157 Therapy.

Recently, marked therapeutic effects pertaining to the recovery of injured rat spinal cords (1 min compression injury of the sacrocaudal spinal cord (S2-Co1) resulting in tail paralysis) appeared after a single intraperitoneal administration of the stable gastric pentadecapeptide BPC 157 at 10 min post-injury. Besides the demonstrated rapid and sustained recovery (1 year), we showed the particular points of the immediate effect of the BPC 157 therapy that began rapidly after its administration, (i) soon after injury (10 min), or (ii) later (4 days), in the rats with a definitive spinal cord injury. Specifically, in counteracting spinal cord hematoma and swelling, (i) in rats that had undergone acute spinal cord injury, followed by intraperitoneal BPC 157 application at 10 min, we focused on the first 10-30 min post-injury period (assessment of gross, microscopic, and gene expression changes). Taking day 4 post-injury as the definitive injury, (ii) we focused on the immediate effects after the BPC 157 intragastric application over 20 min of the post-therapy period. Comparable long-time recovery was noted in treated rats which had definitive tail paralysis: (iii) the therapy was continuously given per orally in drinking water, beginning at day 4 after injury and lasting one month after injury. BPC 157 rats presented only discrete edema and minimal hemorrhage and increased Nos1, Nos2, and Nos3 values (30 min post-injury, (i)) or only mild hemorrhage, and only discrete vacuolation of tissue (day 4, (ii)). In the day 4-30 post-injury study (iii), BPC 157 rats rapidly presented tail function recovery, and no demyelination process (Luxol fast blue staining).

Therapy Effect of the Stable Gastric Pentadecapeptide BPC 157 on Acute Pancreatitis as Vascular Failure-Induced Severe Peripheral and Central Syndrome in Rats.

We revealed the therapy effect of the stable gastric pentadecapeptide BPC 157 (10 µg/kg, 10 ng/kg ig or po) with specific activation of the collateral rescuing pathways, the azygos vein, on bile duct ligation in particular, and acute pancreatitis as local disturbances (i.e., improved gross and microscopy presentation, decreased amylase level). Additionally, we revealed the therapy's effect on the acute pancreatitis as vascular failure and multiorgan failure, both peripherally and centrally following "occlusion-like" syndrome, major intoxication (alcohol, lithium), maintained severe intra-abdominal hypertension, and myocardial infarction, or occlusion syndrome, and major vessel occlusion. The application-sacrifice periods were ligation times of 0-30 min, 0-5 h, 0-24 h (cured periods, early regimen) and 4.30 h-5 h, 5 h-24 h (cured periods, delayed regimen). Otherwise, bile duct-ligated rats commonly presented intracranial (superior sagittal sinus), portal and caval hypertension and aortal hypotension, gross brain swelling, hemorrhage and lesions, heart dysfunction, lung lesions, liver and kidney failure, gastrointestinal lesions, and severe arterial and venous thrombosis, peripherally and centrally. Unless antagonized with the key effect of BPC 157 regimens, reversal of the inferior caval and superior mesenteric vein congestion and reversal of the failed azygos vein activated azygos vein-recruited direct delivery to rescue the inferior-superior caval vein pathway; these were all antecedent to acute pancreatitis major lesions (i.e., acinar, fat necrosis, hemorrhage). These lesions appeared in the later period, but were markedly attenuated/eliminated (i.e., hemorrhage) in BPC 157-treated rats. To summarize, while the innate vicious cycle may be peripheral (bile duct ligation), or central (rapidly developed brain disturbances), or peripheral and central, BPC 157 resolved acute pancreatitis and its adjacent syndrome.

DNA extraction method from bones using Maxwell® 16.

This paper describes the automated purification of DNA extracted from human bones using Maxwell® 16 bench top instrument. Analysis of nuclear short tandem repeats (STR) is invaluable in identification of human remains exhumed from mass graves in Croatia. Up to today 4683 skeletal remains have been recovered and for 897 human remains identity has not been determined. DNA has been extracted from 70% of all unidentified samples. For more than 90% of the samples nuclear STR profiles have been obtained using either organic phenol/chloroform method or silica-column purification for the extraction of DNA from bones or teeth. In order to evaluate a Maxwell® 16 DNA extraction performance 40 bone samples with different stage of decomposition were analyzed. The efficacy of manual silica based extraction and an automated purification was compared. The DNA yield per gram of starting material, removal of inhibitors and the quality of resulting STR profiles of the Maxwell extracts from duplicate amplifications were evaluated. The results show that Maxwell 16 platform can be used instead of manual DNA extraction procedures.

Functional promoter polymorphism of the neuronal isoform of tryptophan hydroxylase (Tph2) in suicide.

The association between suicide and G-703T polymorphism of the tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, was studied in a sample of 291 suicide victims and 280 healthy subjects of Croatian origin. No significant differences were found between the groups. Obtained results do not support involvement of the investigated polymorphism in the susceptibility to suicide completion.

Sequence polymorphism of the mitochondrial DNA control region in the population of Vojvodina Province, Serbia.

In order to generate and establish the database for forensic identification purposes in Vojvodina Province (Serbia), the sequence of the hypervariable regions 1 (HV1) and 2 (HV2) of the mtDNA control region were determined in a population of 104 unrelated individuals from Vojvodina Province, using a fluorescent-based capillary electrophoresis sequencing method. A total of 93 different haplotypes were found, of these 83 mtDNA types were unique, nine haplotypes were shared by two individuals and one haplotype by three individuals. The variation of mtDNA HV1 and HV2 regions was confined to 116 nucleotide positions, of which 72 were observed in the HV1 and 44 in the HV2. A statistical estimate of the results for this population showed the genetic diversity of 0.9977 and the random match probability of 1.18%. Haplogroup H was the most common haplogroup (43.3%). Haplogroups observed at intermediate levels included clusters U (13.5%), T (10.6%), J (8.6%) and W (5.8%).

Evaluation of population variation at 17 autosomal STR and 16 Y-STR haplotype loci in Croatians.

Seventeen autosomal STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818, FGA, Penta E and Penta D) and 16 Y-STR haplotype loci (DYS19, DYS385, DYS389I, DYS398II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and GATA H4.1) were analyzed in the sample of 200 unrelated Croatians. The agreement with HWE was confirmed for all autosomal STR loci. The combined power of discrimination (PD) and the combined power of exclusion (PE) for the 17 autosomal STR loci were 0.999999999999999999682299331476 and 0.99999995, respectively. Penta E proved to be the most informative autosomal STR locus. Among 200 Croatian males, 197 Y-STR haplotypes were identified and haplotype diversity was estimated at 0.9998+/-0.0005.

TPH gene polymorphism and aging: indication of combined effect on the predisposition to violent suicide.

Findings relating serotonin to suicidal behavior suggest the role of genes for tryptophan hydroxylase (TPH) in the genetic substrate of this disorder. Association of Tph1 gene and suicidality, despite considerable research efforts, remains controversial. Polymorphism A218C in intron 7 of Tph1 gene was studied in violent suicide victims (N = 247) and controls (N = 320) of Slavic (Croatian) origin, with specific consideration of the influence of subjects' age. The frequency of, allegedly less active, CC genotype was increased in older (above 65 years) victims as compared to controls (P = 0.0126 and 0.0008, for comparison with age-specific and integral control samples, respectively), while there were no differences between victims under 65 years and controls. Excess of the CC genotype in elderly victims of violent suicide points to the possible combined effect of the respective genetic factor and physiological changes during aging on the predisposition to this disorder.

Variability of the tryptophan hydroxylase gene: study in victims of violent suicide.

Tryptophan hydroxylase (TPH), the enzyme controlling serotonin synthesis, is considered to be a potential contributor to the biological substrate of suicide. The association of the promoter (-7065CT) and intron 7 (218AC) polymorphisms, and the related haplotype, of the Tph1 gene with suicidal behavior was investigated in a sample of 160 victims of violent suicide and 284 healthy controls. All individuals were males of Croatian (Slavic) origin. Allele frequencies of both polymorphisms in Croatian controls were similar to control values reported for other European populations. Alleles at the two loci demonstrated highly significant linkage disequilibrium. No differences between controls and victims for the Tph1 genetic variation, either at single loci, or at a haplotypic level, were demonstrated, albeit there was a tendency, not reaching statistical significance, towards an increase of the intron 7CC genotype in the suicide group. Negative association results on the individual Tph1 loci, in accordance with the majority of previous reports, confirmed the lack of their major effect also in the Slavic ethnicity. Haplotypic results, on the other hand, opposing the previous positive finding, point to the possible influence of ethnicity (or gender) on the association between the Tph1 gene polymorphism and suicide.

Genetic variation at nine short tandem repeat loci among islanders of the eastern Adriatic coast of Croatia.

We have analyzed the extent of genetic variation at nine autosomal short tandem repeat loci (D3S1358, VWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317, D7S820) among six populations from Croatia: five distributed in the islands of the eastern Adriatic coast and one from the mainland. The purpose is to investigate the usefulness of these loci in detecting regional genetic differentiation in the studied populations. Significant heterogeneity among the island and mainland populations is revealed in the distributions of allele frequencies; however, the absolute magnitude of the coefficient of gene differentiation is small but significant. The summary measures of genetic variation, namely, heterozygosity, number of alleles, and allele size variance, do not indicate reduced genetic variation in the island populations compared to the mainland population. In contrast to the two measures of genetic variation, allele size variance and within-locus heterozygosity, the imbalance index (beta) indicates evidence of recent expansion of population sizes in all islands and in the mainland. High mutation rates of the studied loci together with local drift effects are likely explanations for interisland genetic variation and the observed lack of reduced genetic diversity among the island populations.

5HT-2C receptor polymorphism in suicide victims. Association studies in German and Slavic populations.

Sustainable observations suggest that suicidal behaviour by itself may have biological correlates, among which those related to the serotonergic synapse hold the key position. Based on the association of suicide and serotonergic dysfunction, it was proposed that genetic mechanisms affecting suicidal behaviour could be related to the alterations of the genes encoding the elements of 5HT synapse. The present study tested the association of the polymorphism in the serotonin 2C (5HT-2C) receptor coding region (Cys23Ser) with suicide commitment. Study was based on two independent samples, one of German (284 suicide victims versus 297 controls) and other of Slavic/Croatian (118 suicide victims versus 275 controls) ethnicity. No significant differences in allele or genotype frequencies between victims and controls were demonstrated. Results did not provide supporting evidence for the potential involvement of the investigated variants of 5HT-2C receptor in the susceptibility to suicide.

Serotonin transporter gene promoter (5-HTTLPR) and intron 2 (VNTR) polymorphisms in Croatian suicide victims.

BACKGROUND: Disturbances in serotonin (5HT) transmission are the most frequently reported neurobiological substrates of suicidal behavior. Because 5HT transporter plays a central role in the regulation of 5HT synaptic function and its gene contains two functional polymorphisms (5-HTTLPR in the promoter region and VNTR in the second intron), it represents an interesting candidate for association studies in suicidal behavior. METHODS: In this study, a possible association of 5-HTTLPR and intron 2-VNTR polymorphisms of the 5HT transporter gene with suicidal behavior was investigated in a sample of 135 suicide victims and 299 healthy control subjects of Croatian/southern Slavic origin. RESULTS: There were no significant differences in 5-HTTLPR and intron 2-VNTR genotype- and allele- frequency distributions between suicide victims and healthy control subjects; however, a tendency toward an increase of 5-HTTLPR allele L and VNTR-allele 10 were observed in suicide group. Analysis of distribution of estimated haplotype frequencies revealed differences between suicide victims and control subjects, with an excess of haplotype L10 among suicide victims (p =.0112). CONCLUSIONS: Our results provide modest evidence for a possible association of the 5HT transporter gene with a completed suicide. Further studies are needed to determine whether alterations in 5HTt gene expression are involved in suicidal behavior.

Alterations in CDKN2A locus as potential indicator of melanoma predisposition in relatives of non-familial melanoma cases.

AIM: To examine constitutional alterations of CDKN2A/p16INK4A locus as a potential indicator of melanoma predisposition among the first-degree relatives of patients with malignant melanoma. METHOD: The study included eight families with a single member affected with melanoma. Members of the families were screened for allelic cosegregation with 9p21 region polymorphic markers IFNA, D9S126, and D9S104. The patient's tumors were screened for loss of heterozygosity (LOH) with the same markers, as well as for single strand conformational polymorphism (SSCP) variability of CDKN2A. In suspect cases, constitutional DNA was examined by SSCP and direct sequencing. RESULTS: LOH was detected in four cases, and SSCP indicated variability in at least one CDKN2A exon in these tumor samples. In three of four LOH cases, the remaining allele cosegregated within the family, which was interpreted as a preliminary indicator of potential genetic predisposition. In one of these three families, we found constitutional CDKN2A mutations in the patient and one of the relatives. In the second family, only the patient had the constitutionally altered gene, whereas no constitutional CDKN2A alterations were detected in the third family. All significant mutations were different and had not been reported before. CONCLUSION: We detected one case of melanoma predisposition among unaffected family members, which corresponded to statistical expectations for such a small number of screened families. Since constitutional mutations of CDKN2A exons have limited incidence, our stepwise approach seemed to be more informative and more affordable than straightforward CDKN2A sequencing of all subjects.

Clinical case of acral hemorrhagic Darier's disease is not caused by mutations in exon 15 of the ATP2A2 gene.

Darier's disease (Dyskeratosis follicularis, DD) is a genetic disorder characterized by pathogenetic changes of keratinization with variant forms of cutaneous phenotype. Recently, it has been showed that Darier's disease cause mutations in the ATP2A2 gene, at 12q24.1. The gene encodes sarco-endoplasmic reticulum calcium ATPase type 2 (SERCA2). Mutations in exon 15 are reported to be the most consistent mutations associated with the acral hemorrhagic type of Darier's disease. By direct sequencing we investigated exon 15 of the ATP2A2 gene in a Croation family in which one member had a hemorrhagic Darier's disease, but did not record any mutation in the family we investigated. Our results show that mutations in exon 15 of the ATP2A2 gene are not a necessary prerequisite for acral hemorrhagic type of Darier's disease. Our finding support the variability of clinical manifestations of Darier's disease and lack of genotype/phenotype consistency.

Croatian population data for arylsulfatase a pseudodeficiency-associated mutations in healthy subjects, and in patients with Alzheimer-type dementia and Down syndrome.

BACKGROUND: Arylsulfatase A (ASA) is a lysosomal enzyme involved in catabolism of cerebroside sulfate, whose deficiency causes metachromatic leukodystrophy, a rare autosomal recessive disorder characterized by storage of cerebroside sulfate, mainly in the nervous system. Low ASA activities have also been reported in healthy individuals and several neuropsychiatric disorders due to the condition termed ASA pseudodeficiency. The aim of this study was to establish frequency of two mutations associated with ASA pseudodeficiency in healthy individuals in the Croatian population as well as in persons with Alzheimer-type dementia and Down syndrome. METHODS: Presence of N350S and 1524+95 A-->G pseudodeficiency mutations was detected in genomic DNA extracted from leukocytes of healthy subjects (n = 125) and of patients with Alzheimer-type dementia (n = 18) and Down syndrome (n = 21). Arylsulfatase A activity was measured in leukocyte homogenates by spectrophotometry (lambda = 515 nm) using p-nitrocatechol sulfate as chromogenic substrate. RESULTS: Frequency of N350S mutation and mutation 1524+95 A-->G was estimated at 6.8 and 2.8% for healthy controls, 11 and 5.5% for Alzheimer-type dementia, and 12 and 9.5% for Down syndrome, respectively. Arylsulfatase A activity was slightly but not significantly decreased in leukocytes derived from subjects with dementia and Down syndrome in comparison with age-matched control samples. CONCLUSIONS: Frequency of two mutations associated with ASA pseudodeficiency in the Croatian population is slightly below the range reported for other populations. Additionally, despite the proposed role of arylsulfatase A pseudodeficiency as one of the predisposing factors for neuropsychiatric disorders, our preliminary results did not show significantly higher frequencies of either mutation in Alzheimer-type dementia or Down syndrome.

The identification of war victims by reverse paternity is associated with significant risks of false inclusion.

Since February 2001 the process of DNA identification of war victims in Croatia relies on the database of over 3,000 9-locus (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820) STR genotypes of relatives of missing persons. Instead of a targeted approach to DNA typing, the genotype of each skeletal remains analysed is compared to all genotypes in the database to identify potential parents and children. Although this approach has significantly increased the pace of identification by DNA typing, non-targeted matching in a database containing several thousand genotypes considerably decreases the significance of inclusion, especially when identification is based on reverse paternity analysis. To support this statistical prediction we present 3 cases of 10 STR loci matches and 1 case of 11 STR loci matches between a child, child's mother and skeletal remains that did not originate from a father of that child.

Mutation analysis of the MPZ and PMP22 genes in Croatian patients.

We used single-strand conformation polymorphism analysis for mutational screening in two candidate genes, MPZ and PMP22, which have an important role in the pathogenesis of Charcot-Marie-Tooth disease (CMT) and related peripheral neuropathies. A novel Ser8Ser polymorphism was found in exon 1 of the MPZ gene in two heterozygous subjects, in a father with mild CMT2 phenotype and his daughter with normal clinical data. Thr118Met polymorphism was found in exon 5 of the PMP22 gene. The patient heterozygous for 118Met allele had CMT1 disease. We can conclude that the occurrence of the 118Met allele does not usually cause CMT1 and that it is not a clinically relevant disease marker.