AIM:To characterize clinicopathological and familial features of early-onset colorectal cancer(CRC) and compare features of tumors with and without microsatellite instability(MSI).METHODS:Forty-five patients with CRC ...AIM:To characterize clinicopathological and familial features of early-onset colorectal cancer(CRC) and compare features of tumors with and without microsatellite instability(MSI).METHODS:Forty-five patients with CRC aged 45 or younger were included in the study.Clinical information,a three-generation family history,and tumor samples were obtained.MSI status was analyzed and mismatch repair genes were examined in the MSI families.Tumors were included in a tissue microarray and an immunohistochemical study was carried out with a panel of selected antibodies.RESULTS:Early onset CRC is characterized by advanced stage at diagnosis,right colon location,low-grade of differentiation,mucin production,and presence of polyps.Hereditary forms represent at least 21% of cases.Eighty-one percent of patients who died during followup showed a lack of expression of cyclin E,which could be a marker of poor prognosis.β-catenin expression was normal in a high percentage of tumors.CONCLUSION:Early-onset CRC has an important familial component,with a high proportion of tumors showing microsatellite stable.Cyclin E might be a poor prognosis factor.展开更多
The interplay between glucose metabolism and that of the two other primary nutrient classes, amino acids and fatty acids is critical for regulated insulin secretion. Mitochondrial metabolism of glucose, amino acid and...The interplay between glucose metabolism and that of the two other primary nutrient classes, amino acids and fatty acids is critical for regulated insulin secretion. Mitochondrial metabolism of glucose, amino acid and fatty acids generates metabolic coupling factors(such as ATP, NADPH, glutamate, long chain acyl-CoA and diacylglycerol) which trigger insulin secretion. The observation of protein induced hypoglycaemia in patients with mutations in GLUD1 gene, encoding the enzyme glutamate dehydrogenase(GDH) and HADH gene, encoding for the enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase has provided new mechanistic insights into the regulation of insulin secretion by amino acid and fatty acid metabolism. Metabolic signals arising from amino acid and fatty acid metabolism converge on the enzyme GDH which integrates both signals from both pathways and controls insulin secretion. Hence GDH seems to play a pivotal role in regulating both amino acid and fatty acid metabolism.展开更多
Cholate-CoA ligase （,EEL） and bile acid-CoA： amino acid N-acyltransferase （BAAT） sequentially mediate bile-acid amidation. Defects can cause intrahepatic cholestasis. Distinction has required gene sequencing. We ...Cholate-CoA ligase （,EEL） and bile acid-CoA： amino acid N-acyltransferase （BAAT） sequentially mediate bile-acid amidation. Defects can cause intrahepatic cholestasis. Distinction has required gene sequencing. We assessed potential clinical utility of immunostaining of liver for CCL and BAAT. Using commercially available antibodies against BAAT and CCL, we immunostained liver from an infant with jaundice, deficiency of amidated bile acids, and transcription-terminating mutation in BAAT. CCL was normally expressed. BAAT expression was not de- tected. Immunostaining may facilitate diagnosis in bile- acid amidation defects.展开更多
Congenital hyperinsulinism(CHI) is a rare but complex heterogeneous disorder caused by unregulated secre-tion of insulin from the β-cells of the pancreas leading to severe hypoglycaemia and neuroglycopaenia. Swift di...Congenital hyperinsulinism(CHI) is a rare but complex heterogeneous disorder caused by unregulated secre-tion of insulin from the β-cells of the pancreas leading to severe hypoglycaemia and neuroglycopaenia. Swift diagnosis and institution of appropriate management is crucial to prevent or minimise adverse neurodevel-opmental outcome in children with CHI. Histologically there are two major subtypes of CHI, diffuse and focal disease and the management approach will significantly differ depending on the type of the lesion. Patients with medically unresponsive diffuse disease require a near total pancreatectomy, which then leads on to the de-velopment of iatrogenic diabetes mellitus and pancre-atic exocrine insufficiency. However patients with focaldisease only require a limited pancreatectomy to re-move only the focal lesion thus providing complete cure to the patient. Hence the preoperative differentiation of the histological subtypes of CHI becomes paramount in the management of CHI. Fluorine-18L-3, 4-hydroxy-phenylalanine positron emission tomography(18F-DOPA-PET) is now the gold standard for pre-operative differentiation of focal from diffuse disease and locali-sation of the focal lesion. The aim of this review article is to give a clinical overview of CHI, then review the role of dopamine in β-cell physiology and finally discuss the role of 18F-DOPA-PET imaging in the management of CHI.展开更多
Maternal drinking during pregnancy can result in a wide spectrum of cognitive and behavioral abnormalities termed fetal alcohol spectrum disorders (FASD). The heterogeneity observed in FASD-related phenotypes can be a...Maternal drinking during pregnancy can result in a wide spectrum of cognitive and behavioral abnormalities termed fetal alcohol spectrum disorders (FASD). The heterogeneity observed in FASD-related phenotypes can be attributed to a number of environmental and genetic factors;however, ethanol dose and timing of exposure may have significant influences. Here, we report the behavioral effects of acute, binge-like ethanol exposure at three neurodevelopmental times corresponding to the first, second, and third trimester of human development in C57BL/6J mice. Results show that developmental ethanol exposure consistently delays the development of basic motor skill reflexes and coordination as well as impairs spatial learning and memory. Observed changes in activity and anxiety-related behaviors, however, appear to be dependent on timing of alcohol exposure. The variability in behaviors between different treatment models suggests that these may be useful in evaluating the mechanisms disrupted by ethanol at specific neurodevelopmental times. The results provide further evidence that, regardless of developmental stage, the developing brain is acutely sensitive to alcohol exposure.展开更多
AIM: To investigate the clinical presentations associated with bile acid synthesis defects and to describe identification of individual disorders and diagnostic pitfalls. METHODS: We describe semiquantitative determin...AIM: To investigate the clinical presentations associated with bile acid synthesis defects and to describe identification of individual disorders and diagnostic pitfalls. METHODS: We describe semiquantitative determination of 16 urinary bile acid metabolites by electrospray ionization-tandem mass spectrometry. Sample preparation was performed by solid-phase extraction. The total analysis time was 2 min per sample. We determined bile acid metabolites in 363 patients with suspected defects in bile acid metabolism. RESULTS: Abnormal bile acid metabolites were found in 36 patients. Two patients had bile acid synthesis defects but presented with atypical presentations. In 2 other patients who were later shown to be affected by biliary atresia and cystic fibrosis the profile of bile acid metabolites was initially suggestive of a bile acid synthesis defect. Three adult patients suffered from cerebrotendinous xanthomatosis. Nineteen patients had peroxisomal disorders, and 10 patients had cholestatic hepatopathy of other cause. CONCLUSION: Screening for urinary cholanoids should be done in every infant with cholestatic hepatopathy as well as in children with progressive neurological disease to provide specific therapy.展开更多
The application of microarray-based techniques for the diagnosis of genomic rearrangements has been steadily growing in popularity since its introduction in 2004.Given the many advantages of these techniques over conv...The application of microarray-based techniques for the diagnosis of genomic rearrangements has been steadily growing in popularity since its introduction in 2004.Given the many advantages of these techniques over conventional cytogenetics,there is increasing pressure towards their application in prenatal diagnosis.However,there remain several important issues that must be addressed.For example,microarray-based techniques(comparative genomic hybridization-based arrays and single nucleotide polymorphism-based arrays) allow detection of even very small genomic imbalances that can determine pathological clinical conditions.In addition,there are other copy number variations which represent normal variation,with no detectable effects on phenotype.Given the still incomplete knowledge of the changes in our genome and the associated phenotypes,microarray-based diagnosis is likely to find variants of uncertain and unknown clinical significance.The interpretation of these variants is now a major challenge for the medical geneticist,who often find it difficult to establish precise correlations between genotype and phenotype.There is sufficient available evidence to justify the use of microarray-based diagnostics for a select number of specific conditions,but there is also an inevitable trend towards ever wider application.It is very important that this drift does not progress in an unchecked and uncontrolled manner under the thrust of commercial interests.Therefore,we recommend that scientific societies be vigilant and take an advisory role in the adopting of these technologies as new scientific knowledge becomes available.展开更多
We have performed a complete screening of the Parkin gene (PRKN2) and looked for p.Gly2019Ser (G2019S) and p.Arg1441Gly (R1441G) LRRK2/dardarin gene mutations in twenty seven patients with Parkinson’s disease (PD) wi...We have performed a complete screening of the Parkin gene (PRKN2) and looked for p.Gly2019Ser (G2019S) and p.Arg1441Gly (R1441G) LRRK2/dardarin gene mutations in twenty seven patients with Parkinson’s disease (PD) with an age at onset younger than 50 years (EOPD), living in Gipuzkoa (Basque Country, Spain). Thirteen of them (48%) were PRKN2 mutation carriers. The c.255-256DelA mutation was the most frequent, followed by a deletion involving exons 3 and 4. A deletion involving exons 3 and 12 of the PRKN2 gene and R1441G LRRK2 mutation was found together in one PD patient. Four out of fourteen PRKN2 negative patients carried the p.G2019S mutation. Both PRKN2 mutation carriers and non-carriers presented frequently with family history (10 PRKN2 mutation carriers and 8 PRKN2 non-carriers);in fact, five patients without a known gene mutation had a first degree relative affected, suggesting another monogenic disease. PRKN2 carriers presented with a younger age at onset (36.7 vs. 41.7) and more benign disease progression. Indeed, those PD patients younger than forty who initially presented with unilateral tremor became shortly bilateral. Relatively, symmetric parkinsonism and slow disease progression carried more frequently PRKN2 mutations than patients with unilateral akinetic rigid parkinsonism and age at onset later than 40 years. As expected in a recessive disease, PRKN2 patients present more often with affected siblings and unaffected patients. The G2019S LRRK2 mutation, less prevalent than R1441G in our area, may be also a frequent cause of PD in EOPD (4 patients).展开更多
Adams-Oliver Syndrome (AOS) is a rare genetic disease characterized by combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD), often accompanied by defects in scalp and skull ossifica...Adams-Oliver Syndrome (AOS) is a rare genetic disease characterized by combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD), often accompanied by defects in scalp and skull ossification. Different clinical phenotypes may be related to variable severity both of aplasia cutis and TTLD, and of minor clinical features as cutis marmorata telangiectatica congenita (CMTC), congenital cardiac defect and vascular anomalies. The treatment is multidisciplinary: dermatologic, orthopedic and surgical consult should be required. It still remains unclear how to treat patients with a large skin defect that can‘t be closed primarly and may require both surgical and conservative management. We report two cases of AOS with typical limb defects and an area of aplasia cutis over vertex of the scalp managed conservatively with two different dermatologic devices.展开更多
For the analysis of microRNA, a common approach is to first extract microRNA from cellular samples prior to any specific microRNA detection. Thus, it is important to determine the quality and yield of extracted microR...For the analysis of microRNA, a common approach is to first extract microRNA from cellular samples prior to any specific microRNA detection. Thus, it is important to determine the quality and yield of extracted microRNA. In this study, solid-phase extraction was used to isolate small RNA (? Green II staining. Testing for contamination of any small DNA fragments, RNase and cellular peptides or proteins were systematically carried out. By scanning the gel image obtained from PAGE analysis, the average percentage of total microRNA (19 - 25 nt) in the extracted RNA samples was determined to be equal to 2.3 ± 0.5%. The yield of total microRNA was calculated to be ~0.5ng of microRNA per milligram of frozen mouse brain tissue. In comparison to other methods that require the use of expensive specialized instrumentation, the approach of combining the standard UV absorbance and PAGE analysis represents a simple and viable method for evaluating the quality and yield of microRNA extraction from tissue samples.展开更多
Several nuclear genes have been found to be linked to ichthyosis, and Next Generation Sequencing approach on panels of targeted genes has turned out to be particularly useful in analyzing diseases characterized by sig...Several nuclear genes have been found to be linked to ichthyosis, and Next Generation Sequencing approach on panels of targeted genes has turned out to be particularly useful in analyzing diseases characterized by significant genetic and phenotypic heterogeneity. We developed a panel of 26 genes to be screened with the Ion Personal Genome Machine (PGM) for causative mutations relating to ichthyosis. Sequencing runs were obtained from a patient with ichthyosis using the Ion Torrent PGM and then processed with Ion Torrent Suite, Variant Caller, Coverage Analysis and wANNOVER tools. No causative mutations were found using Variant Caller and wANNOVER softwares, whereas the “Coverage Analysis” tool revealed a common large deletion in STS gene in a patient with X-linked ichthyosis. Identification of indels in Next Generation Sequencing (NGS) data is a veritable challenge. This study demonstrates the efficacy and effectiveness of using NGS approach to detect large deletions without resorting to specific algorithms for “indel” detection. Our results indicate that the NGS panel is a useful, rapid and cost-effective screening test for patients whose features are suggestive of a genetic etiology involving one of the genes embedded in the panel. It is an excellent alternative to Sanger sequencing as for costs, ease of analysis, and turnaround time.展开更多
Characterization of genetically modified organisms through determination of zygosity and transgene integration concerning both copy number and genome site is important for breeding a transgenic line and the use ...Characterization of genetically modified organisms through determination of zygosity and transgene integration concerning both copy number and genome site is important for breeding a transgenic line and the use of these organisms in the purpose for which it was obtained.Southern blot,fluorescence in situ hybridization or mating are demanding and time-consuming techniques traditionally used in the characterization of transgenic organisms and,with the exception of mating,give ambiguous results.With the emergence of the real-time quantitative PCR technology,different applications have been described for the analysis of transgenic organisms by determination of several parameters to transgenic analysis.However,the accuracy in quantitation by this method can be influenced in all steps of analysis.This review focuses on the aspects that influence pre-analytical steps(DNA extraction and DNA quantification methods),quantification strategies and data analysis in quantification of copy number and zygosity in transgenic animals.展开更多
Many marine invertebrate phyla are characterized by indirect development.These animals transit from planktonic larvae to benthic spats via settlement and metamorphosis,which contributes to their adaption to the marine...Many marine invertebrate phyla are characterized by indirect development.These animals transit from planktonic larvae to benthic spats via settlement and metamorphosis,which contributes to their adaption to the marine environment.Studying the biological process of metamorphosis is,thus,key to understanding the origin and evolution of indirect development.Although numerous studies have been conducted on the relationship between metamorphosis and the marine environment,microorganisms,and neurohormones,little is known about gene regulation network(GRN)dynamics during metamorphosis.Metamorphosis-competent pediveligers of the Pacific oyster Crassostrea gigas were assayed in this study.By assaying gene expression patterns and open chromatin region changes of different samples of larvae and spats,the dynamics of molecular regulation during metamorphosis were examined.The results indicated significantly different gene regulation networks before,during and post-metamorphosis.Genes encoding membrane-integrated receptors and those related to the remodeling of the nervous system were upregulated before the initiation of metamorphosis.Massive biogenesis,e.g.,of various enzymes and structural proteins,occurred during metamorphosis as inferred from the comprehensive upregulation of the protein synthesis system post epinephrine stimulation.Hierarchical downstream gene networks were then stimulated.Some transcription factors,including homeobox,basic helix–loop–helix and nuclear receptors,showed different temporal response patterns,suggesting a complex GRN during the transition stage.Nuclear receptors,as well as their retinoid X receptor partner,may participate in the GRN controlling oyster metamorphosis,indicating an ancient role of the nuclear receptor regulation system in animal metamorphosis.展开更多
Background:Lynch syndrome(LS)is a hereditary condition characterized by a high risk of colorectal cancer,endometrial cancer,and other neoplasia associated with germline alterations in DNA mismatch repair genes.The cla...Background:Lynch syndrome(LS)is a hereditary condition characterized by a high risk of colorectal cancer,endometrial cancer,and other neoplasia associated with germline alterations in DNA mismatch repair genes.The classical genetic diagnostic strategy for LS consists of the Sanger sequencing of genes associated with the suspected syndrome.Next-generation sequencing(NGS)enables the simultaneous sequencing of a large number of hereditary cancer genes.Here,we aimed to study whether other germline pathogenic variants of hereditary cancer genes are present in patients with LS.Methods:A cohort of 84 probands with a previous genetic diagnosis of LS by Sanger sequencing was reanalyzed using NGS via a commercial panel of 94 hereditary cancer genes by hybrid capture.The American College of Medical Genetics and Genomics criteria were used to classify the clinical significance of the variants.The findings of NGS were confirmed by Sanger sequencing.When possible,genetic analyses of the new findings in the proband’s relativeswere also performed by Sanger sequencing.Results:We identified five families(6%),out of 84,with at least two germline pathogenic variants conferring to high or moderate risk in different dominant cancer-predisposing genes:[MLH1-BRCA2-NBN],[MLH1-BRCA1],[MSH2-ATM],[MSH6-NF1],and[MLH1-FANCA].Interestingly,only one out of these five families exhibited a clinical phenotype associated with the new pathogenic variants.The family with three pathogenic variants of the[MLH1-BRCA2-NBN]genes showed a high aggregation of tumors associated with LS and breast and ovarian cancer syndrome.Conclusions:Our results showed that the co-occurrence of more than one pathogenic variant in cancer-predisposing genes was remarkable among cases of LS.Inmost cases,no clinicial manifestations were associated with the secondary pathogenic variants.Further studies are needed to confirm these findings and elucidate their clinical impact.Reanalysis of LS families should be considered only in families with mixed clinical phenotypes.展开更多
基金Supported by The Instituto de Salud Carlos Ⅲ, Ministerio de Sanidad y Consumo (FIS01-04, project P047-04)
文摘AIM:To characterize clinicopathological and familial features of early-onset colorectal cancer(CRC) and compare features of tumors with and without microsatellite instability(MSI).METHODS:Forty-five patients with CRC aged 45 or younger were included in the study.Clinical information,a three-generation family history,and tumor samples were obtained.MSI status was analyzed and mismatch repair genes were examined in the MSI families.Tumors were included in a tissue microarray and an immunohistochemical study was carried out with a panel of selected antibodies.RESULTS:Early onset CRC is characterized by advanced stage at diagnosis,right colon location,low-grade of differentiation,mucin production,and presence of polyps.Hereditary forms represent at least 21% of cases.Eighty-one percent of patients who died during followup showed a lack of expression of cyclin E,which could be a marker of poor prognosis.β-catenin expression was normal in a high percentage of tumors.CONCLUSION:Early-onset CRC has an important familial component,with a high proportion of tumors showing microsatellite stable.Cyclin E might be a poor prognosis factor.
文摘The interplay between glucose metabolism and that of the two other primary nutrient classes, amino acids and fatty acids is critical for regulated insulin secretion. Mitochondrial metabolism of glucose, amino acid and fatty acids generates metabolic coupling factors(such as ATP, NADPH, glutamate, long chain acyl-CoA and diacylglycerol) which trigger insulin secretion. The observation of protein induced hypoglycaemia in patients with mutations in GLUD1 gene, encoding the enzyme glutamate dehydrogenase(GDH) and HADH gene, encoding for the enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase has provided new mechanistic insights into the regulation of insulin secretion by amino acid and fatty acid metabolism. Metabolic signals arising from amino acid and fatty acid metabolism converge on the enzyme GDH which integrates both signals from both pathways and controls insulin secretion. Hence GDH seems to play a pivotal role in regulating both amino acid and fatty acid metabolism.
基金Supported by Great Ormond Street Hospital Children’s Charity, to Clayton PTNational Institutes of HealthGrant R01 DK58214, to Bull LN
文摘Cholate-CoA ligase （,EEL） and bile acid-CoA： amino acid N-acyltransferase （BAAT） sequentially mediate bile-acid amidation. Defects can cause intrahepatic cholestasis. Distinction has required gene sequencing. We assessed potential clinical utility of immunostaining of liver for CCL and BAAT. Using commercially available antibodies against BAAT and CCL, we immunostained liver from an infant with jaundice, deficiency of amidated bile acids, and transcription-terminating mutation in BAAT. CCL was normally expressed. BAAT expression was not de- tected. Immunostaining may facilitate diagnosis in bile- acid amidation defects.
文摘Congenital hyperinsulinism(CHI) is a rare but complex heterogeneous disorder caused by unregulated secre-tion of insulin from the β-cells of the pancreas leading to severe hypoglycaemia and neuroglycopaenia. Swift diagnosis and institution of appropriate management is crucial to prevent or minimise adverse neurodevel-opmental outcome in children with CHI. Histologically there are two major subtypes of CHI, diffuse and focal disease and the management approach will significantly differ depending on the type of the lesion. Patients with medically unresponsive diffuse disease require a near total pancreatectomy, which then leads on to the de-velopment of iatrogenic diabetes mellitus and pancre-atic exocrine insufficiency. However patients with focaldisease only require a limited pancreatectomy to re-move only the focal lesion thus providing complete cure to the patient. Hence the preoperative differentiation of the histological subtypes of CHI becomes paramount in the management of CHI. Fluorine-18L-3, 4-hydroxy-phenylalanine positron emission tomography(18F-DOPA-PET) is now the gold standard for pre-operative differentiation of focal from diffuse disease and locali-sation of the focal lesion. The aim of this review article is to give a clinical overview of CHI, then review the role of dopamine in β-cell physiology and finally discuss the role of 18F-DOPA-PET imaging in the management of CHI.
文摘Maternal drinking during pregnancy can result in a wide spectrum of cognitive and behavioral abnormalities termed fetal alcohol spectrum disorders (FASD). The heterogeneity observed in FASD-related phenotypes can be attributed to a number of environmental and genetic factors;however, ethanol dose and timing of exposure may have significant influences. Here, we report the behavioral effects of acute, binge-like ethanol exposure at three neurodevelopmental times corresponding to the first, second, and third trimester of human development in C57BL/6J mice. Results show that developmental ethanol exposure consistently delays the development of basic motor skill reflexes and coordination as well as impairs spatial learning and memory. Observed changes in activity and anxiety-related behaviors, however, appear to be dependent on timing of alcohol exposure. The variability in behaviors between different treatment models suggests that these may be useful in evaluating the mechanisms disrupted by ethanol at specific neurodevelopmental times. The results provide further evidence that, regardless of developmental stage, the developing brain is acutely sensitive to alcohol exposure.
基金Supported by Grants from the United States National Institutes of Health (GM069338 and HL20948) awarded to Russell DW
文摘AIM: To investigate the clinical presentations associated with bile acid synthesis defects and to describe identification of individual disorders and diagnostic pitfalls. METHODS: We describe semiquantitative determination of 16 urinary bile acid metabolites by electrospray ionization-tandem mass spectrometry. Sample preparation was performed by solid-phase extraction. The total analysis time was 2 min per sample. We determined bile acid metabolites in 363 patients with suspected defects in bile acid metabolism. RESULTS: Abnormal bile acid metabolites were found in 36 patients. Two patients had bile acid synthesis defects but presented with atypical presentations. In 2 other patients who were later shown to be affected by biliary atresia and cystic fibrosis the profile of bile acid metabolites was initially suggestive of a bile acid synthesis defect. Three adult patients suffered from cerebrotendinous xanthomatosis. Nineteen patients had peroxisomal disorders, and 10 patients had cholestatic hepatopathy of other cause. CONCLUSION: Screening for urinary cholanoids should be done in every infant with cholestatic hepatopathy as well as in children with progressive neurological disease to provide specific therapy.
文摘The application of microarray-based techniques for the diagnosis of genomic rearrangements has been steadily growing in popularity since its introduction in 2004.Given the many advantages of these techniques over conventional cytogenetics,there is increasing pressure towards their application in prenatal diagnosis.However,there remain several important issues that must be addressed.For example,microarray-based techniques(comparative genomic hybridization-based arrays and single nucleotide polymorphism-based arrays) allow detection of even very small genomic imbalances that can determine pathological clinical conditions.In addition,there are other copy number variations which represent normal variation,with no detectable effects on phenotype.Given the still incomplete knowledge of the changes in our genome and the associated phenotypes,microarray-based diagnosis is likely to find variants of uncertain and unknown clinical significance.The interpretation of these variants is now a major challenge for the medical geneticist,who often find it difficult to establish precise correlations between genotype and phenotype.There is sufficient available evidence to justify the use of microarray-based diagnostics for a select number of specific conditions,but there is also an inevitable trend towards ever wider application.It is very important that this drift does not progress in an unchecked and uncontrolled manner under the thrust of commercial interests.Therefore,we recommend that scientific societies be vigilant and take an advisory role in the adopting of these technologies as new scientific knowledge becomes available.
文摘We have performed a complete screening of the Parkin gene (PRKN2) and looked for p.Gly2019Ser (G2019S) and p.Arg1441Gly (R1441G) LRRK2/dardarin gene mutations in twenty seven patients with Parkinson’s disease (PD) with an age at onset younger than 50 years (EOPD), living in Gipuzkoa (Basque Country, Spain). Thirteen of them (48%) were PRKN2 mutation carriers. The c.255-256DelA mutation was the most frequent, followed by a deletion involving exons 3 and 4. A deletion involving exons 3 and 12 of the PRKN2 gene and R1441G LRRK2 mutation was found together in one PD patient. Four out of fourteen PRKN2 negative patients carried the p.G2019S mutation. Both PRKN2 mutation carriers and non-carriers presented frequently with family history (10 PRKN2 mutation carriers and 8 PRKN2 non-carriers);in fact, five patients without a known gene mutation had a first degree relative affected, suggesting another monogenic disease. PRKN2 carriers presented with a younger age at onset (36.7 vs. 41.7) and more benign disease progression. Indeed, those PD patients younger than forty who initially presented with unilateral tremor became shortly bilateral. Relatively, symmetric parkinsonism and slow disease progression carried more frequently PRKN2 mutations than patients with unilateral akinetic rigid parkinsonism and age at onset later than 40 years. As expected in a recessive disease, PRKN2 patients present more often with affected siblings and unaffected patients. The G2019S LRRK2 mutation, less prevalent than R1441G in our area, may be also a frequent cause of PD in EOPD (4 patients).
文摘Adams-Oliver Syndrome (AOS) is a rare genetic disease characterized by combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD), often accompanied by defects in scalp and skull ossification. Different clinical phenotypes may be related to variable severity both of aplasia cutis and TTLD, and of minor clinical features as cutis marmorata telangiectatica congenita (CMTC), congenital cardiac defect and vascular anomalies. The treatment is multidisciplinary: dermatologic, orthopedic and surgical consult should be required. It still remains unclear how to treat patients with a large skin defect that can‘t be closed primarly and may require both surgical and conservative management. We report two cases of AOS with typical limb defects and an area of aplasia cutis over vertex of the scalp managed conservatively with two different dermatologic devices.
文摘For the analysis of microRNA, a common approach is to first extract microRNA from cellular samples prior to any specific microRNA detection. Thus, it is important to determine the quality and yield of extracted microRNA. In this study, solid-phase extraction was used to isolate small RNA (? Green II staining. Testing for contamination of any small DNA fragments, RNase and cellular peptides or proteins were systematically carried out. By scanning the gel image obtained from PAGE analysis, the average percentage of total microRNA (19 - 25 nt) in the extracted RNA samples was determined to be equal to 2.3 ± 0.5%. The yield of total microRNA was calculated to be ~0.5ng of microRNA per milligram of frozen mouse brain tissue. In comparison to other methods that require the use of expensive specialized instrumentation, the approach of combining the standard UV absorbance and PAGE analysis represents a simple and viable method for evaluating the quality and yield of microRNA extraction from tissue samples.
文摘Several nuclear genes have been found to be linked to ichthyosis, and Next Generation Sequencing approach on panels of targeted genes has turned out to be particularly useful in analyzing diseases characterized by significant genetic and phenotypic heterogeneity. We developed a panel of 26 genes to be screened with the Ion Personal Genome Machine (PGM) for causative mutations relating to ichthyosis. Sequencing runs were obtained from a patient with ichthyosis using the Ion Torrent PGM and then processed with Ion Torrent Suite, Variant Caller, Coverage Analysis and wANNOVER tools. No causative mutations were found using Variant Caller and wANNOVER softwares, whereas the “Coverage Analysis” tool revealed a common large deletion in STS gene in a patient with X-linked ichthyosis. Identification of indels in Next Generation Sequencing (NGS) data is a veritable challenge. This study demonstrates the efficacy and effectiveness of using NGS approach to detect large deletions without resorting to specific algorithms for “indel” detection. Our results indicate that the NGS panel is a useful, rapid and cost-effective screening test for patients whose features are suggestive of a genetic etiology involving one of the genes embedded in the panel. It is an excellent alternative to Sanger sequencing as for costs, ease of analysis, and turnaround time.
文摘Characterization of genetically modified organisms through determination of zygosity and transgene integration concerning both copy number and genome site is important for breeding a transgenic line and the use of these organisms in the purpose for which it was obtained.Southern blot,fluorescence in situ hybridization or mating are demanding and time-consuming techniques traditionally used in the characterization of transgenic organisms and,with the exception of mating,give ambiguous results.With the emergence of the real-time quantitative PCR technology,different applications have been described for the analysis of transgenic organisms by determination of several parameters to transgenic analysis.However,the accuracy in quantitation by this method can be influenced in all steps of analysis.This review focuses on the aspects that influence pre-analytical steps(DNA extraction and DNA quantification methods),quantification strategies and data analysis in quantification of copy number and zygosity in transgenic animals.
基金supported by the Oceanographic Data Center,IOCAS.We acknowledge financial support from the Science&Technology Innovation Project of Laoshan Laboratory(LSKJ202203001)the Key Research and Development Program of Shandong(2022LZGC015),and the Taishan Scholars Program.
文摘Many marine invertebrate phyla are characterized by indirect development.These animals transit from planktonic larvae to benthic spats via settlement and metamorphosis,which contributes to their adaption to the marine environment.Studying the biological process of metamorphosis is,thus,key to understanding the origin and evolution of indirect development.Although numerous studies have been conducted on the relationship between metamorphosis and the marine environment,microorganisms,and neurohormones,little is known about gene regulation network(GRN)dynamics during metamorphosis.Metamorphosis-competent pediveligers of the Pacific oyster Crassostrea gigas were assayed in this study.By assaying gene expression patterns and open chromatin region changes of different samples of larvae and spats,the dynamics of molecular regulation during metamorphosis were examined.The results indicated significantly different gene regulation networks before,during and post-metamorphosis.Genes encoding membrane-integrated receptors and those related to the remodeling of the nervous system were upregulated before the initiation of metamorphosis.Massive biogenesis,e.g.,of various enzymes and structural proteins,occurred during metamorphosis as inferred from the comprehensive upregulation of the protein synthesis system post epinephrine stimulation.Hierarchical downstream gene networks were then stimulated.Some transcription factors,including homeobox,basic helix–loop–helix and nuclear receptors,showed different temporal response patterns,suggesting a complex GRN during the transition stage.Nuclear receptors,as well as their retinoid X receptor partner,may participate in the GRN controlling oyster metamorphosis,indicating an ancient role of the nuclear receptor regulation system in animal metamorphosis.
基金supported by grants from the Foundation for the Promotion of Health and Biomedical Research in the Valencian Region,FISABIO(Grants:UGP-14-192 and UGP-16-146)the Carlos Ⅲ Health Institute(Grant AES:PI17/01082).
文摘Background:Lynch syndrome(LS)is a hereditary condition characterized by a high risk of colorectal cancer,endometrial cancer,and other neoplasia associated with germline alterations in DNA mismatch repair genes.The classical genetic diagnostic strategy for LS consists of the Sanger sequencing of genes associated with the suspected syndrome.Next-generation sequencing(NGS)enables the simultaneous sequencing of a large number of hereditary cancer genes.Here,we aimed to study whether other germline pathogenic variants of hereditary cancer genes are present in patients with LS.Methods:A cohort of 84 probands with a previous genetic diagnosis of LS by Sanger sequencing was reanalyzed using NGS via a commercial panel of 94 hereditary cancer genes by hybrid capture.The American College of Medical Genetics and Genomics criteria were used to classify the clinical significance of the variants.The findings of NGS were confirmed by Sanger sequencing.When possible,genetic analyses of the new findings in the proband’s relativeswere also performed by Sanger sequencing.Results:We identified five families(6%),out of 84,with at least two germline pathogenic variants conferring to high or moderate risk in different dominant cancer-predisposing genes:[MLH1-BRCA2-NBN],[MLH1-BRCA1],[MSH2-ATM],[MSH6-NF1],and[MLH1-FANCA].Interestingly,only one out of these five families exhibited a clinical phenotype associated with the new pathogenic variants.The family with three pathogenic variants of the[MLH1-BRCA2-NBN]genes showed a high aggregation of tumors associated with LS and breast and ovarian cancer syndrome.Conclusions:Our results showed that the co-occurrence of more than one pathogenic variant in cancer-predisposing genes was remarkable among cases of LS.Inmost cases,no clinicial manifestations were associated with the secondary pathogenic variants.Further studies are needed to confirm these findings and elucidate their clinical impact.Reanalysis of LS families should be considered only in families with mixed clinical phenotypes.
