Reaction-restriction Fragment Length Polymorphism Approach Research Articles

Fungal Identifier (FId): An Updated Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Approach to Ease Ascomycetous Yeast Isolates' Identification in Ecological Studies.

Culturomics has been temporarily exceeded by the advent of omics approaches such as metabarcoding and metagenomics. However, despite improving our knowledge of microbial population composition, both metabarcoding and metagenomics are not suitable for investigating and experimental testing inferences about microbial ecological roles and evolution. This leads to a recent revival of culturomics approaches, which should be supported by improvements in the available tools for high-throughput microbial identification. This study aimed to update the classical PCR-RFLP approach in light of the currently available knowledge on yeast genomics. We generated and analyzed a database including more than 1400 ascomycetous yeast species, each characterized by PCR-RFLP profiles obtained with 143 different endonucleases. The results allowed for the in silico evaluation of the performance of the tested endonucleases in the yeast species' identification and the generation of FId (Fungal Identifier), an online freely accessible tool for the identification of yeast species according to experimentally obtained PCR-RFLP profiles.

Diversity of dung beetle-associated yeasts from pristine environments of Botswana.

Yeast-insect interactions are increasingly becoming an attractive source of discovery for previously unknown, unique, diverse, and industrially relevant yeast species. Despite a wealth of studies that have recently focused on yeasts in symbiotic association with Hymenopteran insects, yeasts associated with Coleopteran insects, such as lignocellulosic-rich dung-dependent beetles, remain poorly studied. Trends in yeast discovery suggest that species richness and diversity can be attributed to the ecological niche of the insect. Here, we considered the potential of dung beetles inhabiting the extreme environments of Botswana, characterized by desert-like conditions (semi-arid to arid and hot) as well as protected pristine environments, as possible attribute niches that can shape the extremophilic and diverse life history strategies of yeasts. We obtained a total of 97 phylogenetically diverse yeast isolates from six species of dung beetles from Botswana's unexplored environments, representing 19 species belonging to 11 genera. The findings suggest that the guts of dung beetles are a rich niche for non-Saccharomyces yeast species. Meyerozyma and Pichia were the most dominant genera associated with dung beetles, representing 55% (53 out of 97) of the yeast isolates in our study. Trichosporon and Cutaneotrichosporon genera represented 32% (31 out of 97) of the isolates. The remaining isolates belonged to Apiotrichum, Candida, Diutina, Naganishia, Rhodotorula, and Wickerhamiella genera (12 out of 97). We found out that about 62% (60 out of 97) of the isolates were potentially new species because of their low internal transcribed spacer (ITS) sequence similarity when compared to the most recent optimal species delineation threshold. A single isolate was unidentifiable using the ITS sequences. Using an in silico polymerase chain reaction-restriction fragment length polymorphism approach, we revealed that there was genetic diversity within isolates of the same species. Our results contribute to the knowledge and understanding of the diversity of dung beetle-associated yeasts.

Risk Association, Linkage Disequilibrium, and Haplotype Analyses of β-Like Globin Gene Polymorphisms with Malaria Risk in the Sabah Population of Malaysian Borneo.

Single nucleotide polymorphisms (SNPs) in the β-like globin gene of the human hosts to the risk of malaria are unclear. Therefore, this study investigates these associations in the Sabah population, with a high incidence of malaria cases. In brief, DNA was extracted from 188 post-diagnostic blood samples infected with Plasmodium parasites and 170 healthy controls without a history of malaria. Genotyping of the β-like globin C-158T, G79A, C16G, and C-551T SNPs was performed using a polymerase chain reaction-restriction fragment length polymorphism approach. Risk association, linkage disequilibrium (LD), and haplotype analyses of these SNPs were assessed. This study found that the variant allele in the C-158T and C16G SNPs were protective against malaria infections by 0.5-fold, while the variant allele in the G79A SNP had a 6-fold increased risk of malaria infection. No SNP combination was in perfect LD, but several haplotypes (CGCC, CGCT, and CGGC) were identified to link with different correlation levels of malaria risk in the population. In conclusion, the C-158T, G79A, and C16G SNPs in the β-like globin gene are associated with the risk of malaria. The haplotypes (CGCC, CGCT, and CGGC) identified in this study could serve as biomarkers to estimate malaria risk in the population. This study provides essential data for the design of malaria control and management strategies.

Molecular identification of Candida species isolated from candiduria and its risk factors in neonates and children.

Background and Purpose: The present study was performed to raise attention on the frequency of Candida spp. and evaluation of risk factors of candiduria in neonates and children.Materials and Methods: In total, 60 urine samples were collected from the suspected neonates and children. Identification of Candida at species level was performed using the polymerase chain reaction-restriction fragment length polymorphism approach. Results: The restriction fragment length polymorphism fingerprint analysis revealed that Candida parapsilosis (n=17; 28.33 %) is the most prevalent isolated species followed by Candida albicans (n=9; 15%), Candida tropicalis (n=4; 9.52%), and C. glabrata (n=2; 4.76%). All of the C. albicans and C. parapsilosis complex strains were identified as C. albicans with HWP1 gene primers and using the NlaIII restriction enzyme activity, respectively. In this study, none of the mentioned factors was the cause of infection, but they could be considered risk factors. The mean hospital stay was 21 days (range: 7-21 days). More than 90% of the patients had a urinary catheter, and about 26% of them received antibiotics. Regarding the risk factors, there was no significant difference between the two groups of candidiasis in terms of C. albicans and non-albicans Candida (P<0.01). Conclusion: Candiduria has always been a challenging issue, especially in children admitted to hospitals. Outcome of candiduria in patients with generally healthy is little.

Toward Decentralized Agrigenomic Surveillance? A Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Approach for Adaptable and Rapid Detection of User-Defined Fungal Pathogens in Potato Crops

Agrigenomics is one of the emerging focus areas for omics sciences. Yet, agrigenomics differs from medical omics applications such as pharmacogenomics and precision medicine, by virtue of vastly distributed geography of applications at the intersection of agriculture, nutrition, and genomics research streams. Crucially, agrigenomics can address diagnostics and safety surveillance needs in remote and rural farming communities or decentralized food, crop, and environmental monitoring programs for prompt, selective, and differential identification of pathogens. A case in point is the potato crop that serves as a fundamental nutritional source worldwide. Decentralized potato crop and plant protection facilities are pivotal to minimize unnecessary, preemptive use of broad-spectrum fungicides, thus helping to curtail the costs, environmental burden, and the development of resistance in opportunistic human pathogenic fungi. We report here a polymerase chain reaction-restriction fragment length polymorphism approach that is sensitive and adaptable in detection and broad identification of fungal pathogens in potato crops, with a view to future decentralized agrigenomic surveillance programs. Notably, the fingerprinting patterns obtained by the method fully differentiated 12 fungal species examined in silico, with 10 of them also tested in vitro. The method can be scaled up through improvements in electrophoresis and enzyme panel for adaption to other crops and/or pathogens. We suggest that decentralized and integrated agrosurveillance programs and translational agrigenomic programs can inform future innovations in multidomain biosecurity, particularly across omics applications from agriculture and nutrition to clinical medicine and environmental biosafety.

Improving Biosurveillance Systems to Enable Situational Awareness During Public Health Emergencies.

Improving Biosurveillance Systems to Enable Situational Awareness During Public Health Emergencies.

Scat detection dogs, DNA and species distribution modelling reveal a diminutive geographical range for the Vulnerable small red brocket deerMazama bororo

AbstractThe small red brocket deerMazama bororois endemic to the Brazilian Atlantic Forest, a biome that has been greatly fragmented and altered by human activities. This elusive species is morphologically similar to the red brocket deerMazama americanaand the Brazilian dwarf brocket deerMazama nana, and genetic typing is necessary for reliable identification. To determine the geographical range ofM. bororomore accurately, we conducted non-invasive genetic sampling using scat detection dogs trained to locate deer faeces. We surveyed 46 protected areas located within the species’ potential distribution and collected a total of 555 scat samples in 30 of the protected areas. Using a polymerase chain reaction–restriction fragment length polymorphism approach, we genotyped 497 scat samples (89%) and detectedM. bororoin seven localities in three Brazilian states. The results support a range extension of the small red brocket deer to latitudes 23 and 28°S and longitudes 47 and 49°W. We show that the species’ distribution is associated with 37,517 km2of the Ombrophilous Dense Forest in the Brazilian Atlantic Forest, and this conclusion is supported by species distribution modelling. The small red brocket deer is the largest endemic species in Brazil and may have the smallest geographical distribution of any Neotropical deer species. This species occupies fragmented landscapes and is threatened by human encroachment, poaching, and predation by dogs, and based on our findings we recommend policy intervention for conservation planning of the Ombrophilous Dense Forest.

Prevailing Plasmodium falciparum dihydrofolate reductase 108-asparagine in Hodeidah, Yemen: A questionable sulfadoxine–pyrimethamine partner within the artemisinin-based combination therapy

Given that the evolution and spread of resistance to sulfadoxine–pyrimethamine (SP) have been documented at a quick pace worldwide, the present study investigated the mutant Plasmodium falciparum dihydrofolate reductase 108-asparagine (dhfr 108N) as a key marker of resistance to the combination among parasite isolates from Hodeidah. The association of parasitologic indices with the dhfr 108N mutant allele was also studied. Ninety patients with microscopically confirmed P. falciparum infection from Hodeidah were included in the present study. Polymerase chain reaction-restriction fragment length polymorphism approach was adopted for the molecular detection of this marker. The dhfr 108N was detected among about 61% of P. falciparum isolates, in its pure and mixed-type forms, from Hodeidah. Age, gender and residence of patients were not significant predictors for the presence of the mutant allele among parasite isolates. In contrast, a history of malaria and antimalarial drug intake in the year preceding the study as well as frequent antimalarial drug intake were significantly associated with this mutant allele. The high frequency of dhfr 108N among parasites isolates makes the role of SP questionable as a partner with outstanding effectiveness within the ACT, at least, in the near future. SP plus artesunate should be monitored for its antimalarial efficacy at regular intervals, preferably through the molecular detection of resistance-associated mutations.

Polymorphism of the CD14 and TLR4 Genes and Post-treatment Apical Periodontitis

IntroductionThis study investigated the association of CD14 -260C>T and TLR4 +896A>G gene polymorphisms with post-treatment apical periodontitis in Brazilian individuals. MethodsThe study population consisted of 41 patients with post-treatment apical periodontitis and 42 individuals with root canal–treated teeth exhibiting healed/healing periradicular tissues (controls). All teeth had apical periodontitis lesions at the time of treatment, which was completed at least 1 year previously. Saliva was collected from the participants; DNA was extracted and used for CD14 and TLR4 genotyping using the polymerase chain reaction–restriction fragment length polymorphism approach and a real-time polymerase chain reaction TaqMan assay (Applied Biosystems, Foster City, CA), respectively. ResultsNo specific genotype or allele of the CD14 and TLR4 genes or any combination thereof was positively associated with post-treatment apical periodontitis (P > .05). ConclusionsData from the present study suggest that polymorphisms in the CD14 and TLR4 genes do not influence the response to endodontic treatment of teeth with apical periodontitis.

Effects of EPHX1, SCN1A and CYP3A4 genetic polymorphisms on plasma carbamazepine concentrations and pharmacoresistance in Chinese patients with epilepsy

The pharmacokinetics and pharmacodynamics of carbamazepine (CBZ) vary widely among patients with epilepsy. In this study, we sought to investigate the effects of genetic polymorphisms of the microsomal epoxide hydrolase (EPHX1), the sodium channel α subunit type I (SCN1A) and the cytochrome P450 3A4 (CYP3A4) genes on plasma CBZ concentrations and pharmacoresistance in Chinese patients with epilepsy. The EPHX1 c.337T>C, c.416A>G, SCN1A IVS5-91G>A or CYP3A4*1G polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism approach or direct DNA sequencing in 83 Chinese patients treated with CBZ monotherapy. Patients with the variant EPHX1 c.416A>G genotypes had higher adjusted plasma CBZ concentrations compared to those with the wild type genotype (P<0.05). In contrast, the SCN1A IVS5-91G>A and CYP3A4*1G variant alleles had no significant effects on CBZ maintenance doses or adjusted CBZ concentrations. There were no associations between all the studied genotypes involving EPHX1 c.337T>C, c.416A>G, SCN1A IVS5-91G>A or CYP3A4*1G polymorphisms and pharmacoresistance in this patient cohort. These results suggest that the EPHX1 c.416A>G polymorphism affects CBZ metabolism in Chinese patients with epilepsy. However, whether it contributes to CBZ resistance needs to be further investigated in a larger cohort of patients.

Association of Chemokine and Chemokine Receptor Gene Polymorphis (MCP1 A-2518G and CCR2-V64I) with Urinary Bladder Cancer: A Study in Kashmiri Population

Epidemiological evidence points to a connection between inflammation and a predisposition for the development of cancer. Several pro-inflammatory gene products have been identified that lead to variations in the production and concentration of inflammatory proteins. A case-control study was conducted in chemokine and chemokine receptor genes (MCP-1 A-2518G and CCR2-V64I) to elucidate the possible role of these SNPs as risk factors in urinary bladder cancer (UBC) development. Using the polymerase chain reaction-restriction fragment length polymorphism approach, we investigated the genotype distribution of 120 bladder cancer patients in comparison with 155 cancer-free controls from the same geographical region. Significant differences were observed in the distribution of genotypes in MCP-1 A-2518G between the control and bladder cancer patients (0.36 vs 0.47; p=0.000). Homozygous variant GG frequency of MCP-1 A-2518G in cases was found to be 20% as against 5.1% in controls (p<0.05). No significant association was found in CCR2-V64I genotypes (p = 0.09) between the controls and patients with the frequency of homozygous variant (64I) being 15% and 20% (0.15 vs. 0.20) respectively. Interestingly, CCR2-V64I heterozygote (wt/64I+64I) frequencies were significantly increased in the higher grades/stages of bladder cancer patients (p< 0.05). Based on above mentioned results, we conclude that the MCP-1 -2518A/G polymorphism is associated with genetic susceptibility to the risk for bladder cancer.

Chronic renal impairment and DDAH2-1151 A/C polymorphism determine ADMA levels in type 2 diabetic subjects

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). Increased levels of ADMA cause impaired vasodilation, leading to endothelial dysfunction and a higher risk for cardiovascular events. In patients with a chronic kidney disease, increased ADMA levels are reported to play a role in the pathogenesis of accelerated atherosclerosis and are an independent risk marker leading to end-stage renal disease and mortality. Circulating ADMA is metabolized by the action of dimethylarginine dimethylamino hydrolase (DDAH) and DDAH2 isoform is the most prevalent in tissues expressing endothelial NOS. DDAH and NOS are co-expressed in the same kidney regional sites supporting the hypothesis that a strict and specific regulation of intracellular ADMA levels is crucial for NO generation in the kidney. Starting from these findings, the study aims to investigate the role of DDAH2 gene promoter polymorphism at position -1151 A/C in determining the levels of ADMA in type 2 diabetic patients (T2DM) with chronic renal impairment. Three groups of carefully selected subjects of both sexes were enrolled and compared. The first group (control subjects) comprised 286 non-diabetic subjects (mean age 55.8 ± 11.4 years), the second group (T2DM uncomplicated subjects) was made up of 322 T2DM subjects without complications (mean age 64.9 ± 9.6 years) whereas the third group (T2DM CRF subjects) included 110 T2DM patients with chronic renal impairment. The rs805304 DDAH2-1151 A/C promoter polymorphism was determined by a polymerase chain reaction-restriction fragment length polymorphism approach. Results T2DM CRF subjects showed significant increased plasma levels of ADMA with respect to those of T2DM uncomplicated subjects and control subjects (0.51 versus 0.39 versus 0.37 μmol/L, P = 0.002, respectively). Analysis of variance showed an interaction between DDAH2-1151 C carrier and groups on ADMA plasma levels (F = 4.36; P < 0.05). ADMA plasma levels were also dependent on groups (F = 4.96; P < 0.01). Our work demonstrates that rs805304 DDAH2-1151 polymorphism plays a central role in determining ADMA in diabetic renal impairment, where patients with DDAH2-1151 C carriers showed the highest ADMA levels. This unfavourable genetic profile is highlighted by pathological kidney conditions such as diabetic CRF. These findings could open new insights on the pathways involving ADMA/DDAH/NOS in the development and progression of chronic renal impairment and therefore of the other micro- macrovascular diabetic complications.

Impact of codon 72 Arg &gt; Pro single nucleotide polymorphism in TP53 gene in the risk of kangri cancer: a case control study in Kashmir

Kangri cancer found only in Kashmir (north India) is a unique thermally induced squamous cell carcinoma of the skin that develops because of chronic and persistent irritation due to the use of a kangri (a brazier) by the Kashmiri people to combat the chilling cold temperature during winter. Being unique to this region, the molecular etiology of the invasive kangri cancer is not known fully. The TP53 gene, codon 72 polymorphism (Arg72Pro), has been found to be associated with cancer susceptibility but has not been investigated in kangri cancer risk. A case control study was conducted to find the genotype distribution of TP53 Arg72Pro SNP and to elucidate the possible role of this SNP as risk factor in kangri cancer development. Using the polymerase chain reaction-restriction fragment length polymorphism approach, we tested the genotype distribution of 106 kangri cancer patients in comparison with 200 cancer-free controls from the same geographical region. A significant difference was observed between the control and kangri cancer patients with odds ratio = 2.02 and 95% confidence interval = 1.2-3.3 (p = 0.01). Interestingly, the proline form was abundantly observed in advanced-grade tumors (p < 0.05). We also found a significant association of the variant allele (GC + CC) with male subjects and patients >45 years of age (p < 0.05). Thus, it is evident from our study that Arg72Pro SNP is implicated in kangri cancer and that the rare, proline-related allele is connected with higher susceptibility to kangri cancer.

Effects of UGT1A6, UGT2B7, and CYP2C9 Genotypes on Plasma Concentrations of Valproic Acid in Chinese Children with Epilepsy

Valproic acid (VPA) is one of the most commonly prescribed drugs for the treatment of epilepsy. Interindividual variability in VPA dose and plasma concentration may reflect functional consequences of genetic polymorphisms in genes encoding drug-metabolizing enzymes. The aim of this study was to determine the relationship between plasma concentrations of VPA and single nucleotide polymorphisms (SNPs) involving uridine diphosphate glucuronosyltransferase (UGT) 1A6 (UGT1A6), UGT2B7, and cytochrome P450 2C9 (CYP2C9) genes in Chinese children with epilepsy. UGT1A6, UGT2B7, and CYP2C9 polymorphisms were identified by the polymerase chain reaction-restriction fragment length polymorphism approach or direct automated DNA sequencing in 98 epileptic patients treated with VPA monotherapy. Patients with double heterozygosities at nucleotide positions T19G, A541G and A552C in the UGT1A6 gene, were associated with higher VPA doses compared to those with wild type or single heterozygosity (p = 0.010). Lower adjusted plasma VPA concentrations were also observed in patients with UGT1A6 double heterozygosities than those with single heterozygosity (p = 0.027). There were no differences in VPA dose or adjusted plasma VPA concentrations among the UGT2B7*2 or CYP2C9*3 genotypic groups. These results suggest that UGT1A6 mutations affect VPA metabolism in epileptic children. It needs to be further investigated in a larger cohort of patients.

Association analysis of −429T/C and −374T/A polymorphisms of receptor of advanced glycation end products (RAGE) gene in Malaysian with type 2 diabetic retinopathy

Conflicting results have been reported in different populations on the association between two particular RAGE gene polymorphisms (−429T/C and −374T/A) and retinopathy in diabetic patients. Therefore this study was designed to assess the association between both gene polymorphisms with retinopathy in Malaysian diabetic patients. A total of 342 type 2 diabetic patients [171 without retinopathy (DNR) and 171 with retinopathy (DR)] and 235 healthy controls were included in this study. Genomic DNA was obtained from blood samples and the screening for the gene polymorphisms was done using polymerase chain reaction-restriction fragment length polymorphism approach. Overall, the genotype distribution for both polymorphisms was not statistically different (p>0.05) among the control, DNR and DR groups. The −429C minor allele frequency of DR group (12.0%) was not significantly different (p>0.05) when compared to DNR group (16.1%) and healthy controls (11.3%). The −374A allele frequency also did not differ significantly between the control and DNR (p>0.05), control and DR (p>0.05) as well as DNR and DR groups (p>0.05). This is the first study report on RAGE gene polymorphism in Malaysian DR patients. In conclusion, −429T/C and −374T/A polymorphisms in the promoter region of RAGE gene were not associated with Malaysian type 2 DR patients.

Role of TP53 Arg72Pro polymorphism in urinary bladder cancer predisposition and predictive impact of proline related genotype in advanced tumors in an ethnic Kashmiri population

Among various polymorphic variants of TP53 gene, codon 72 polymorphism (Arg72Pro) has been found to be associated with cancer susceptibility, but only few studies have investigated their effect on bladder cancer risk. A case–control study was conducted and we observed the genotype distribution of TP53 Arg72Pro SNP, to elucidate the possible role of this SNP as risk factor in urinary bladder cancer (UBC) development and to examine its correlation with the clinicopathologic variables of UBC cases. Using the polymerase chain reaction-restriction fragment length polymorphism approach, we tested the genotype distribution of 108 bladder cancer patients in comparison with 138 cancer-free controls from the same geographical region. We observed significant differences between the control and bladder cancer patients with odds ratio = 2.9 and 95% confidence interval = 1.5–4.5 ( P = 0.00001). Interestingly, the proline form was abundantly observed in advanced tumors ( P < 0.05). We also found a significant association of the variant allele (GC+CC) with male subjects and ever smokers ( P = 0.001). Thus, it is evident from our study that Arg72Pro SNP is implicated in bladder cancer, and that the rare, proline-related allele is connected with higher susceptibility to bladder cancer.

Prognostic value of TP53 Pro47Ser and Arg72Pro single nucleotide polymorphisms and the susceptibility to gliomas in individuals from Southeast Brazil

The TP53 tumor suppressor gene codifies a protein responsible for preventing cells with genetic damage from growing and dividing by blocking cell growth or apoptosis pathways. A common single nucleotide polymorphism (SNP) in TP53 codon 72 (Arg72Pro) induces a 15-fold decrease of apoptosis-inducing ability and has been associated with susceptibility to human cancers. Recently, another TP53 SNP at codon 47 (Pro47Ser) was reported to have a low apoptosis-inducing ability; however, there are no association studies between this SNP and cancer. Aiming to study the role of TP53 Pro47Ser and Arg72Pro on glioma susceptibility and oncologic prognosis of patients, we investigated the genotype distribution of these SNPs in 94 gliomas (81 astrocytomas, 8 ependymomas and 5 oligodendrogliomas) and in 100 healthy subjects by the polymerase chain reaction-restriction fragment length polymorphism approach. Chi-square and Fisher exact test comparisons for genotype distributions and allele frequencies did not reveal any significant difference between patients and control groups. Overall and disease-free survivals were calculated by the Kaplan-Meier method, and the log-rank test was used for comparisons, but no significant statistical difference was observed between the two groups. Our data suggest that TP53 Pro47Ser and Arg72Pro SNPs are not involved either in susceptibility to developing gliomas or in patient survival, at least in the Brazilian population.

Spinal muscular atrophy genotyping by gene dosage using multiple ligation-dependent probe amplification

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, causing symmetric proximal muscle weakness. SMA is classified in three clinical types, SMA I, SMA II, and SMA III, based on the severity of the symptoms and the age of onset. About 95% of SMA cases are caused by homozygous deletion of the survival motor neuron 1 (SMN1) gene (5q13), or its conversion to SMN2. The molecular diagnosis of this disease is usually carried out by a polymerase chain reaction-restriction fragment length polymorphism approach able to evidence the absence of both SMN1 copies. However, this approach is not able to identify heterozygous healthy carriers, which show a very high frequency in general population (1:50). We used the multiple ligation-dependent probe amplification (MLPA) approach for the molecular diagnosis of SMA in 19 affected patient and in 57 individuals at risk to become healthy carriers. This analysis detected the absence of the homozygous SMN1 in all the investigated cases, and allowed to discriminate between SMN1 deletion and conversion to SMN2 on the basis of the size showed by the peaks specific for the different genes mapped within the SMA critical region. Moreover, MLPA analysis evidenced a condition of the absence of the heterozygous SMN1 in 33 out of the 57 relatives of the affected patients, demonstrating the usefulness of this approach in the identification of healthy carriers. Thus, the MLPA technique represents an easy, low cost, and high throughput system in the molecular diagnosis of SMA, both in affected patients and in healthy carriers.

Patterns of variation at a mitochondrial sequence-tagged-site locus provides new insights into the postglacial history of European Pinus sylvestris populations.

Due to their maternal mode of inheritance, mitochondrial markers can be regarded as almost 'ideal' tools in evolutionary studies of conifer populations. In the present study, polymorphism was analysed at one mitochondrial intron (nad 1, exon B/C) in 23 native European Pinus sylvestris populations. In a preliminary screening for variation using a polymerase chain reaction-restriction fragment length polymorphism approach, two length variants were identified. By fully sequencing the 2.5 kb region, the observed length polymorphism was found to result from the insertion of a 31 bp sequence, with no other mutations observed within the intron. A set of primers was designed flanking the observed mutation, which identified a novel sequence-tagged-site mitochondrial marker for P. sylvestris. Analysis of 747 trees from the 23 populations using these primers revealed the occurrence of two distinct haplotypes in Europe. Within the Iberian Peninsula, the two haplotypes exhibited extensive population differentiation (PhiST = 0.59; P < or = 0.001) and a marked geographical structuring. In the populations of central and northern Europe, one haplotype largely predominated, with the second being found in only one individual of one population.