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Attenuated familial adenomatous polyposis, which accounts for ~10% of familial adenomatous polyposis, is difficult to diagnose because of its milder course and later onset. In both familial adenomatous polyposis and attenuated familial adenomatous polyposis, duodenal cancer is usually recognized 10-20 years after the diagnosis of colonic polyposis. We present herein a 66-year-old man who received pancreaticoduodenectomy due to ampullary carcinoma 17 years before onset of colonic polyposis. He then received extended right hemicolectoy for ascending colon cancer and â100 polyps located from ceacum to splenic flexure of colon 2 years ago. The patient received Adenomatous polyposis coli (APC) genetic testing and detected a germline pathogenic frameshift variant in the APC gene (NM_000038.6:c.4875delA, ClinVar variant ID (127299)). The variant is classified as likely pathogenic according to the American College of Medical Genetics and Genomics guidelines. APC genetic testing was subsequently performed on his younger children (30 and 26 year old) and they found a same frameshift variant as his father. They were not detected any colonic polyposis by colonoscopy. This is a rare case report of attenuated familial adenomatous polyposis that diagnosed with gastric and colon polyposis >10 years after the diagnosis of ampullary carcinoma and the first report of genetic diagnosis of an attenuated familial adenomatous polyposis variant in young relatives before the onset of the disease.
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Hemophilia A (HA), a rare recessive X-linked bleeding disorder, is caused by either deficiency or dysfunction of coagulation factor VIII (FVIII) resulting from deleterious mutations in the F8 gene encoding FVIII. Over the last 4 decades, the methods aimed at determining the HA carrier status in female relatives of HA patients have evolved from phenotypic studies based on coagulation tests providing merely probabilistic results, via genetic linkage studies based on polymorphic markers providing more accurate results, to next generation sequencing studies enabling highly precise identification of the causative F8 mutation. In parallel, the options for prenatal diagnosis of HA have progressed from examination of FVIII levels in fetal blood samples at weeks 20-22 of pregnancy to genetic analysis of fetal DNA extracted from chorionic villus tissue at weeks 11-14 of pregnancy. In some countries, in vitro fertilization (IVF) combined with preimplantation genetic diagnosis (PGD) has gradually become the procedure of choice for HA carriers who wish to prevent further transmission of HA without the need to undergo termination of pregnancies diagnosed with affected fetuses. In rare cases, genetic analysis of a HA carrier might be complicated by skewed X chromosome inactivation (XCI) of her non-hemophilic X chromosome, thus leading to the phenotypic manifestation of moderate to severe HA. Such skewed XCI may be associated with deleterious mutations in X-linked genes located on the non-hemophilic X chromosome, which should be considered in the process of genetic counseling and PGD planning for the symptomatic HA carrier. Therefore, whole exome sequencing, combined with X-chromosome targeted bioinformatic analysis, is highly recommended for symptomatic HA carriers diagnosed with skewed XCI in order to identify additional deleterious mutations potentially involved in XCI skewing. Identification of such mutations, which may profoundly impact the reproductive choices of HA carriers with skewed XCI, is extremely important.
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Hemofilia A , Humanos , Gravidez , Feminino , Hemofilia A/diagnóstico , Hemofilia A/genética , Fator VIII/genética , Diagnóstico Pré-Natal/métodos , Triagem de Portadores Genéticos , Mutação , HeterozigotoRESUMO
OBJETIVE: To evaluate the effectiveness of vagino-rectal swab autotomy for prenatal screening for GBS infection and to identify the barriers and facilitators encountered by the pregnant woman for this intervention. DESIGN: Cross-sectional study of diagnostic tests. PARTICIPANTS AND SITE: A total of 213 pregnant women who attended the primary care midwife's office in 6 health centers of the Basque Health Service/Osakidetza in Bizkaia, who met the inclusion criteria and agreed to participate in the study, participated in the study. MAIN MEASUREMENTS: The result of the vagino-rectal culture obtained by the pregnant woman was compared with the result of the vagino-rectal culture taken by the midwife in consultation on the same day, and the barriers and facilitators encountered by the women in the self-test were collected. RESULTS: Self-testing as a test for GBS was found to have a sensitivity of 93.3% (95% CI 78.7-98.2), a specificity of 99.4% (95% CI 96.5-99.9), a positive predictive value of 96% (95% CI 82.8-99.4) and a negative predictive value of 98.8% (95% CI 95.6-99.7). 27.3% of respondents encountered some difficulty in the collection, only 4.8% did not feel qualified, 84.2% felt comfortable, 99.5% considered the information provided to be adequate and complete, 94.7% did not find the steps to follow complicated, and 96% were satisfied with the study. CONCLUSIONS: Self-collection of vagino-rectal exudate for GBS detection has proved to be valid and reliable, which would make it possible to offer this option to pregnant women in the systematic screening for GBS infection.
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Complicações Infecciosas na Gravidez , Infecções Estreptocócicas , Estudos Transversais , Exsudatos e Transudatos , Feminino , Humanos , Gravidez , Complicações Infecciosas na Gravidez/diagnóstico , Diagnóstico Pré-Natal , Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiaeRESUMO
With the expansion of carrier screening to general preconception and prenatal patient populations, most patients will receive negative results, which we define as indicating <25% risk of having a child with a genetic condition. Because there is limited experience with expanded carrier screening, it is important to understand how receiving negative results affects patients, especially as providers, payers, and policymakers consider whether to offer it. In this mixed-methods study, we asked preconception patients enrolled in the NextGen study about their expectations and experiences receiving negative expanded carrier screening results. Participants completed surveys at study enrollment (n = 110 women, 51 male partners), after receiving carrier results (n = 100 women, 38 male partners), after receiving secondary findings (n = 98 women, 36 male partners), and 6 months after receiving results (n = 95 women, 28 male partners). We also interviewed a subset of participants 12 to 24 months after receiving results (n = 24 women, 12 male partners). We found minimal negative emotional impact and privacy concerns, increased confidence in reproductive plans, and few changes to health behaviors, although some patients made health decisions based on misunderstandings of their results. These findings suggest that expanded carrier screening causes minimal psychosocial harms, but systems are needed to reduce the risk of misinterpreting results.
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Triagem de Portadores Genéticos , Aconselhamento Genético/psicologia , Participação do Paciente/psicologia , Diagnóstico Pré-Natal/psicologia , Adulto , Feminino , Humanos , Masculino , Resultados Negativos , Gravidez , Inquéritos e QuestionáriosRESUMO
To increase accessibility to genetics services for low-urgency patients seeking Ashkenazi Jewish (AJ) carrier screening, we designed an interactive computer (IC) module that provides pre-test genetics education and allows genetics professionals to order the test without meeting the patients beforehand. We compared this module with in-person genetic counseling (GC) using a randomized trial. AJ individuals were randomized to undergo genetics education via the IC module (n = 26) or GC (n = 28). We compared post-interventional genetics knowledge, perceived genetic risk, and anxiety between the two groups, after accounting for pre-interventional scores, using ANCOVA. Wilcoxon Rank-Sum test was used to compare post-interventional satisfaction. Post-interventional genetics knowledge, risk perception, or anxiety were not significantly different between the two groups after accounting for baseline scores (p = 0.50-0.54), although the data are inconclusive regarding the module's non-inferiority at a 5% margin. Post-intervention satisfaction scores were generally higher in the GC group than the IC module group. Our IC module has the potential to improve access to clinical genetics services for patients and staff, but it is not suitable for all AJ patients and cannot completely replace the benefits of in-person consultations.
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Triagem de Portadores Genéticos , Aconselhamento Genético , Judeus/genética , Feminino , Humanos , Internet , Masculino , Fatores de RiscoRESUMO
PURPOSE: An injector equipped with a bead capture and a bead detection system is presented. In the context of magnetic resonance navigation (MRN), in which MRI gradients are used to steer intravascular therapeutic carriers, fast and reliable injection is essential. In this paper, we present a prototype of injector to control and to detect the release of magnetic beads. METHODS: The injector relies on two distinct subsystems: (1) the capture subsystem, which creates local magnetic force to stop the flow of magnetic beads; and (2) the detection subsystem, which detects flowing beads and generates a trigger signal to start MRI gradient pulses. Both systems rely on small microcoils wound on the tubing. RESULTS: Five-turn microcoils show the best compromise between size and performance. Less than 5 mW of power is required to capture 0.8-mm beads moving in a flow above 5 mL min-1 or when a gradient above 200 mT m-1 is applied. The detection system is not sensitive to noise and detects every 0.8-mm bead in flow rates up to 14 mL m-1 . CONCLUSION: The prototype of injector shows performance above the requirements inherent to magnetic resonance navigation. This system is a step toward in vivo multibifurcation MRN. Magn Reson Med 77:444-452, 2017. © 2016 Wiley Periodicals, Inc.
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Injeções/instrumentação , Imageamento por Ressonância Magnética/métodos , Imãs , Modelos Teóricos , Cirurgia Assistida por Computador/métodos , Desenho de Equipamento , MicroesferasRESUMO
Reproductive techniques such as prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD), although debated, are legally forbidden in France in case of Lynch syndrome. The preference of mutation carriers about their reproductive options is not systematically considered in France. We aimed to prospectively assess the reproductive preferences of mismatch repair mutation carriers consulting in our institution (2003-2010, n = 100). We also considered the short- and long-term post-disclosure psychological impact using the Impact of Events Scale-Revised questionnaire to measure the prevalence of posttraumatic stress disorder (PTSD) in those patients. Complete data were obtained for 34 respondents (17 males, 17 females, median age of 33.5 years [22-59]). Seventeen respondents (57 %) preferred spontaneous natural conception versus 28 % and 35 % choosing PND and PGD, respectively. At results disclosure, respondents mainly explained their distress by fear of premature death (43 %) and transmitting mutated genes (42 %). One year later, this last fear remained predominant in 55 % of subjects. None of the main socio-demographical, psychological or medical variables (including fear of transmitting mutations) was significantly associated with the reproductive preferences. Results disclosure had a real and time-decreasing psychological impact on mutation carriers. Reproductive techniques, expected to decrease the hereditary risk, were not significantly preferred to natural conception.
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Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/psicologia , Tomada de Decisões , Mutação , Reprodução , Adulto , Reparo de Erro de Pareamento de DNA , Revelação , Feminino , França , Humanos , Masculino , Pessoa de Meia-Idade , Diagnóstico Pré-Implantação , Diagnóstico Pré-Natal/psicologia , Transtornos de Estresse Pós-Traumáticos/psicologia , Inquéritos e Questionários , Adulto JovemRESUMO
Duchenne Muscular Dystrophy (DMD) is an X-linked neuromuscular disorder in which the detection of female carriers is of the utmost importance for genetic counseling. Haplotyping with polymorphic markers and quantitation of creatine kinase levels (CK) allow tracking of the at-risk haplotype and evidence muscle damage, respectively. Such approaches are useful for carrier detection in cases of unknown mutations. The lack of informative markers and the inaccuracy of CK affect carrier detection. Therefore, herein we designed novel mini-STR (Short Tandem Repeats) assays to amplify 10 loci within the DMD gene and estimated allele frequencies and the polymorphism information content among other parameters in 337 unrelated individuals from three Mexican populations. In addition, we tested the utility of the assays for carrier detection in three families. Moreover, given that serum levels of miR-206 discern between DMD patients and controls with a high area under the curve (AUC), the potential applicability for carrier detection was assessed. The serum levels of miR-206 of non-carriers (n = 24) and carriers (n = 23) were compared by relative quantitation using real-time PCR (p < 0.05), which resulted in an AUC = 0.80 in the Receiver Operating Characteristic curve analysis. In conclusion, miR-206 has potential as a "liquid biopsy" for carrier detection and genetic counseling in DMD.
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MicroRNAs/sangue , MicroRNAs/genética , Repetições de Microssatélites/genética , Distrofia Muscular de Duchenne/sangue , Distrofia Muscular de Duchenne/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Segregação de Cromossomos/genética , Feminino , Variação Genética , Heterozigoto , Humanos , Desequilíbrio de Ligação/genética , Masculino , México , Pessoa de Meia-Idade , Linhagem , Adulto JovemRESUMO
This study aims to determine the way to predict the haemophilia A (HA) carrier status and the potential severity in six females with low FVIII: C levels (<0.50 IU mL(-1) ), F8 gene variations and without family history of HA. Except p.Ser577Tyr, F8 gene variations that we reported have never been described (p.Leu107His, p.Pro521Leu, p.Val682Leu, p.Leu2032Pro, p.Ala315dup). Prediction of their potential causal impact was studied by two strategies: bioinformatics approaches and site-directed mutagenesis followed by FVIII cellular expression into COS-1 cell. FVIII clotting assay ( FVIII: C) and antigen ( FVIII: Ag) were assayed in vitro. In silico analysis showed the probably damaging effect of all substitutions and the full conservation of the residues across mammalian species, except for p.Leu2032Pro. The in vitro variant expression model showed abnormal intra and/or extracellular FVIII: C and FVIII: Ag levels for five mutations, which suggest their causality in HA and provide informations about the involved mechanism. We suspect a defect in synthesis and secretion for p.Leu107His, p.Ala315dup and p.Pro521Leu. The mutation p.Val682Leu only affects the FVIII function while p.Ser577Tyr alters function and synthesis. The variant p.Leu2032Pro is probably a polymorphism because no alteration of the FVIII protein expression was observed in vitro. In vitro results suggest that mutations p.Ser577Tyr and p.Ala315dup could led to a severe HA in men. This study demonstrates the ability of this in vitro cellular expression model to contribute to the diagnosis strategy for female suspected of being HA carrier, without HA family history and with a novel F8 gene variation and to provide new criteria for the genetic counselling.
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Fator VIII/genética , Expressão Gênica , Hemofilia A/diagnóstico , Hemofilia A/genética , Heterozigoto , Mutação , Animais , Testes de Coagulação Sanguínea , Células COS , Linhagem Celular , Chlorocebus aethiops , Fator VIII/imunologia , Fator VIII/metabolismo , Feminino , Hemofilia A/sangue , Humanos , Técnicas In Vitro , Masculino , Mutação de Sentido Incorreto , FenótipoRESUMO
Linkage analysis in autosomal inherited von Willebrand disease (VWD) is important to diagnose the carriers and reduce the burden of severe type VWD. The study was designed to identify the carriers and estimate the frequency of variable number of tandem repeats (VNTR) instability in VWD families. Carrier detection was performed in eight recessive type 3 VWD (VWD3) families using VNTRs VWF1 and VWF2, RsaI (789Thr/Ala) linkage markers, multimer analysis and DNA sequencing. Moreover, five dominant VWD families were studied through DNA sequencing and multimer analysis. Frequency of VWF VNTR instability was investigated in 20 VWD families. In VWD3 families, a total of 22 (81.5%) carriers were identified using VWF1 and VWF2 markers. However, only 13(48.1%) carriers were identified through RsaI markers. Mutation screening revealed 22(81.5%) carriers in VWD3 and 4 (33.3%) carriers in VWD2 families. In comparison to DNA sequencing, the accuracy of VWF1 and VWF2 markers in VWD3 was 85.7% while RsaI could identify 68.2% carriers accurately. Mutations p.R1205H and p.C1272R were identified as de novo in families. Multimer analysis confirmed the identified carriers in VWD2 families. Three VWD families were found to be carrying VNTR instability for VWF1 and VWF2 locus. VNTRs could be an effective linkage markers for carrier detection in VWD3 families. However, in the event of germline de novo mutations and VNTR instability, it may confound risk of misdiagnosis of carriers. Multimer analysis could be an alternative way of carrier detection in dominant type 2A and type 2B VWD families.
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Portador Sadio/diagnóstico , Análise Mutacional de DNA , Ligação Genética/genética , Marcadores Genéticos/genética , Mutação em Linhagem Germinativa/genética , Doenças de von Willebrand/diagnóstico , Doenças de von Willebrand/genética , Feminino , Loci Gênicos/genética , Instabilidade Genômica/genética , Humanos , Masculino , Linhagem , Sequências de Repetição em Tandem/genéticaRESUMO
Timely detection of carbapenem-resistant Acinetobacter baumannii (CRAB) carriers is essential to direct infection control measures. In this work, we aimed to develop a practical protocol to detect CRAB from screening samples. To choose a selective medium that detects CRAB with high sensitivity and specificity, 111 A. baumannii clinical isolates were inoculated on three types of agar: mSuperCARBA (SC), CHROMagar Acinetobacter (CaA), and modified CHROMagar Acinetobacter (mCaA) containing 4.5 mg/mL meropenem. SC was non-selective, CaA was the most sensitive (100%), but only moderately specific (72%), and mCaA was highly specific (97%) and sensitive (98%). Confirmation of the carbapenem-resistant phenotype using PCR-based detection of blaOXA-23, blaOXA-24, and blaOXA-58 genes was specific but not sensitive, detecting only 58% of CRAB isolates. Identification of A. baumannii using either gyrB or blaOXA-51 PCR was excellent. Next, we used the same methodology in routine screening for CRAB carriage. mCaA had the best yield, with high sensitivity but moderate specificity to differentiate between CRAB and other carbapenem-resistant organisms. Skin sampling using sponges and 6 hour enrichment was highly sensitive (98%), while other body sites had poor sensitivity (27%- 41%). Shorter incubation had slightly lower yield, and longer incubation did not improve the detection. Performing PCR for blaOXA-51 and gyrB on colonies growing on modified mCaA differentiated between CRAB and other species with high accuracy (98% and 99%, respectively). Based on our results, we present a procedure for easy and reliable detection of CRAB carriage using skin sampling, short enrichment, selection on mCaA, and PCR-based identification. IMPORTANCE: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a substantial cause of nosocomial infections, classified among the most significant multidrug-resistant pathogens by the World Health Organization and by the US Centers for Disease Control. Limiting the spread of CRAB is an important goal of infection control, but laboratory methods for identification of CRAB carriers are not standardized. In this work, we compared different selective agar plates, tested the efficiency of A. baumannii identification by PCR for species-specific genes, and used PCR-based detection of common resistance genes to confirm the carbapenem-resistant phenotype. During a prospective study, we also determined the optimal sample enrichment time. Based on our results, we propose a simple and efficient protocol for the detection of CRAB carriage using skin sampling, short enrichment, selection on appropriate agar plates, and PCR-based identification, resulting in a turn-around time of 24 hours.
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Acinetobacter baumannii , beta-Lactamases , beta-Lactamases/genética , Estudos Prospectivos , Ágar , Testes de Sensibilidade Microbiana , Carbapenêmicos/farmacologiaRESUMO
CONTEXT: Multiplex ligation probe amplification (MLPA) is a new technique to identify deletions and duplications and can evaluate all 79 exons in dystrophin gene in patients with Duchenne muscular dystrophy (DMD). Being semi-quantitative, MLPA is also effective in detecting duplications and carrier testing of females; both of which cannot be done using multiplex PCR. It has found applications in diagnostics of many genetic disorders. AIM: To study the utility of MLPA in diagnosis and carrier detection for DMD. MATERIALS AND METHODS: Mutation analysis and carrier detection was done by multiplex PCR and MLPA and the results were compared. RESULTS AND CONCLUSIONS: We present data showing utility of MLPA in identifying mutations in cases with DMD/BMD. In the present study using MLPA, we identified mutations in additional 5.6% cases of DMD in whom multiplex PCR was not able to detect intragenic deletions. In addition, MLPA also correctly confirmed carrier status of two obligate carriers and revealed carrier status in 6 of 8 mothers of sporadic cases.
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Carrier screening for autosomal recessive variants has become a cornerstone of community and public health genetics. While the first carrier screening programs were confined to conditions with relatively high prevalence, and hence well-known carrier frequency, the number of candidate genes has increased greatly since the advent of high-throughput DNA sequencing technologies. The epidemiological database of the ensuing gene panels is mostly sparse, and judgement of their performance is, therefore, anything but straightforward. We therefore derived estimates of the carrier detection probabilities among non-consanguineous and consanguineous couples as expected using the 'Tier 3' carrier screening gene panel recently recommended by the American College of Medical Genetics (ACMG). For non-Finnish Europeans, the respective estimate for unrelated couples equals 0.63%, implying that the ACMG Tier 3 panel accounts for over 90% of the genetic load for autosomal recessive diseases in this population. Among the offspring of first cousins, the corresponding incidence is expected to be tenfold higher, an increase still consistent with previous estimates of the overall risk of birth defects for this type of mating. Our considerations are intended to aid the implementation of carrier screening programs and to provide additional support to reproductive counselling and to obtaining informed consent.
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Mucopolysaccharidosis type II is an X-linked lysosomal storage disorder caused by mutations in the IDS gene that encodes the iduronate-2-sulfatase enzyme. The IDS gene is located on the long arm of the X-chromosome, comprising 9 exons, spanning approximately 24 kb. The analysis of carriers, in addition to detecting mutations in patients, is essential for genetic counseling, since the risk of recurrence for male children is 50%. Mosaicism is a well-known phenomenon described in many genetic disorders caused by a variety of mechanisms that occur when a mutation arises in the early development of an embryo. Sanger sequencing is limited in detecting somatic mosaicism and sequence change levels of less than 20% may be missed. The Next Generation Sequencing (NGS) has been increasingly used in diagnosis. It is a sensitive and fast method for the detection of somatic mosaicism. Compared to Sanger sequencing, which represents a cumulative signal, NGS technology analyzes the sequence of each DNA read in a sample. NGS might therefore facilitate the detection of mosaicism in mothers of MPS II patients. The aim of this study was to reanalyze, by NGS, all MPS II mothers that showed to be non-carriers by Sanger analysis. Twelve non-carriers were selected for the reanalysis on the Ion PGM and Ion Torrent S5 platform, using a custom panel that includes the IDS gene. Results were visualized in the Integrative Genomics Viewer (IGV). We were able to detected the presence of the variant previously found in the index case in three of the mothers, with frequencies ranging between 13 and 49% of the reads. These results suggest the possibility of mosaicism in the mothers. The use of a more sensitive technology for detecting low-level mosaic mutations is essential for accurate recurrence-risk estimates. In our study, the NGS analysis showed to be an effective methodology to detect the mosaic event.
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Hemophilia A (HA) is the most common congenital X-linked coagulopathy caused by mutations in the factor VIII gene. One in 5000 to 10 000 male persons worldwide suffer from HA. It is the archetype of high-cost, low-volume disease. Therefore, identification of carriers is crucial to avoid the birth of affected males. Tracking of the defective X chromosome through indirect linkage analysis represents the most practical method for screening for carriers in developing countries. In this study, 227 individuals from 41 families with HA and 100 normal participants were recruited from the Kurdistan region of Iraq and evaluated for intron 18 BclI, intron 19 HindIII, and IVS7 nt 27 markers by polymerase chain reaction restriction fragment length polymorphism and direct sequencing. Among the studied women, 49%, 42%, and 14% were discovered to be heterozygous for BclI, HindIII, and IVS7 markers, respectively. Using BclI, HindIII, and IVS7 markers, 56%, 46%, and 17% of the families were informative, respectively. The combined informativity of these polymorphic sites reaches 66%. The current study illustrates the effectiveness of the BclI and HindIII markers for the diagnosis of HA carriers among the Iraqi Kurdish population.
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Triagem de Portadores Genéticos , Ligação Genética , Hemofilia A/diagnóstico , Íntrons/genética , Fator VIII/genética , Feminino , Marcadores Genéticos , Hemofilia A/genética , Humanos , Iraque , Masculino , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Spinal muscular atrophy (SMA) is a degenerative neuromuscular disease associated with progressive symmetric weakness and atrophy of the limb muscles. In view of the involvement of numerous point mutations and deletions associated with the disease, the application of polymorphic markers flanking the SMA critical region could be valuable in molecular diagnosis of the disease. In the present study, D5S351 and D5S1414 polymorphic markers located at the SMA critical region in the Iranian populations were characterized. Genotyping of the markers indicated the presence of six and nine different alleles for D5S351 and D5S1414, respectively. Haplotype frequency estimation in 25 trios families and 75 unrelated individuals indicated the presence of six informative haplotypes with frequency higher than 0.05 in the studied population. Furthermore, the D' coefficient and the χ(2) value for D5S351 and D5S1414 markers revealed the presence of linkage disequilibrium between the two markers in the Iranians. These data suggested that D5S351 and D5S1414 could be suggested as informative markers for linkage analysis and molecular diagnosis of SMA in the Iranian population.
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Dystrophinopathies are X-linked recessive diseases caused by mutations in the DMD gene. Our objective was to identify mutations in this gene by Multiplex Ligation Probe Amplification (MLPA), to confirm the clinical diagnosis and determine the carrier status of at-risk relatives. Also, we aimed to characterize the Dystrophinopathies argentine population and the DMD gene. We analyzed a cohort of 121 individuals (70 affected boys, 11 symptomatic women, 37 at-risk women and 3 male villus samples). The MLPA technique identified 56 mutations (45 deletions, 9 duplications and 2 point mutations). These results allowed confirming the clinical diagnosis in 63% (51/81) of patients and symptomatic females. We established the carrier status of 54% (20/37) of females at-risk and 3 male villus samples. We could establish an association between the most frequent deletion intron breakpoints and the abundance of dinucleotide microsatellites loci, despite the underlying mutational molecular mechanism remains to be elucidated. The MLPA demonstrate, again, to be the appropriate first mutation screening methodology for molecular diagnosis of Dystrophinopathies. The reported results permitted to characterize the Dystrophinopathies argentine population and lead to better understanding of the genetic and molecular basis of rearrangements in the DMD gene, useful information for the gene therapies being developed.
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Distrofina/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Íntrons , Repetições de Microssatélites , Distrofias Musculares/genética , Mutação , Argentina , Estudos de Coortes , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase MultiplexRESUMO
Hemophilia A (HA) is an inherited X-linked coagulation disorder caused by the deficiency of factor VIII (FVIII). Linkage analysis is a common indirect method for the detection of female carriers in families with HA. In the current study, 173 patients from 30 unrelated families with HA were recruited from the Azeri Turkish population of northwest Iran and analyzed for BclI and HindIII markers by polymerase chain reaction-restriction fragment length polymorphism. We investigated the potential of using these markers for the detection of mutation in carriers through linkage analysis, which would be of tremendous use in prenatal diagnosis. Among the tested women, 47% and 35% were found to be heterozygous for BclI and HindIII polymorphic markers, respectively. The BclI and HindIII markers were informative for the detection of 63% and 17% potential carriers, respectively, demonstrating the effectiveness of the BclI marker for the detection of HA carriers among the Azeri Turkish population.
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Triagem de Portadores Genéticos/métodos , Hemofilia A/genética , Heterozigoto , Polimorfismo de Fragmento de Restrição , Desoxirribonucleases de Sítio Específico do Tipo II/química , Feminino , Hemofilia A/etnologia , Humanos , Irã (Geográfico)/etnologia , MasculinoRESUMO
Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/µl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains.
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Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/diagnóstico , Doenças dos Peixes/virologia , Iridoviridae/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Medicina Veterinária/métodos , Animais , Primers do DNA/genética , Infecções por Vírus de DNA/diagnóstico , Infecções por Vírus de DNA/virologia , DNA Viral/química , DNA Viral/genética , Peixes , Genótipo , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
Newborn blood-spot screening to detect potentially treatable disorders is widely practiced across the globe. However, there are great variations in practice, both in terms of disorders covered, screening technologies, disease definition, information provision, parental informed consent, and storage and disposal of residual specimens, partly reflecting the degree to which screening is the subject of explicit legislation (and thus public and media pressure) or is embedded in a general health care system and managed at an executive level. It is generally accepted that disorders to be screened for should comply with the ten Wilson and Jungner criteria, but the way that compliance is assessed ranges from broadly-based opinion surveys to detailed analysis of quantitative data. Consequently, even countries with comparable levels of economic development and health care show large differences in the number of disorders screened for. There are several areas on which there are no generally accepted guidelines: how should parents be informed about screening and to what extent should they be encouraged to regard screening as an option to choose to refuse? Is DNA mutation analysis acceptable as part of a screening protocol? How soon should the blood samples be destroyed once screening has been completed? As technology advances and the potential scope of screening expands at both the metabolite and genome level, challenging policy issues will have to be faced.