Transposon mutagenesis was used to isolate an Escherichia coli mutant that released into the exte... more Transposon mutagenesis was used to isolate an Escherichia coli mutant that released into the external medium a heterologous protein, the Aeromonas hydrophila aerolysin. Genetic mapping and phenotypic characterization of E. coli CM209 showed that Tn5 is inserted in the tolQ gene. This mutant strain released a significant amount of unprocessed (proaerolysin) protein into the external medium, together with other periplasmic proteins. Similar levels of the toxin were detected in the intracellular compartments of the parental and mutant strains. Whereas inactivation of the tolQ gene itself was responsible for some of the phenotypic properties of strain CM209, such as tolerance to group A colicins or to filamentous bacteriophages, the leaky phenotype was associated with a polar effect exerted by Tn5 on distal genes of the tolQRA cluster.Key words: Tn5, tolQ gene, aerolysin, leaky mutants.
We report the occurrence of Tn5 secondary transpositions in Escherichia coli HB101 hha::Tn5(pANN2... more We report the occurrence of Tn5 secondary transpositions in Escherichia coli HB101 hha::Tn5(pANN202-312). Tn5-induced hha mutants (hemolysin oveiproducers) segregated at high frequency either hemolysin-negative clones or clones showing the parental hemolytic phenotype (reduced hemolysin production). Secondary transpositions of Tn5 appeared to be responsible for the phenotypes of both types of derivatives. The hemolysin-negative clones no longer harboured Tn5 in the hha gene, but rather Tn5 was in the hly genes of the hemolytic plasmid pANN202-312. The derivatives that recovered the parental hemolytic phenotype contained a single copy of Tn5 in a chromosomal location different from that of hha. Both kinds of transpositional events appeared to restore the function of the hha gene.Key words: Tn5, transposition, Escherichia coli, excision.
Background: In vivo confocal microscopy is an imaging technique that has been applied to the stud... more Background: In vivo confocal microscopy is an imaging technique that has been applied to the study of the ocular surface. However, confocal microscopes dedicated to eye examination are routinely adopted only in ophthalmology reference centres and do not allow an examination of periocular tissue, nor a fluorescence examination. Methods: We applied for the first time the two in vivo confocal microscopes commonly used in dermatology (VivaScope ® 1500 and 3000, CALIBER, distributed in Europe by Mavig GmbH, Munich, Germany) to observe the cornea, the bulbar and tarsal conjunctiva, the eyelid margin, the lacrimal punctum and the palpebral skin of healthy volunteers. Tumoral, inflammatory and infectious diseases of the ocular mucosa and periocular skin from more than 200 patients were observed under the same microscopes. Both microscopes have a reflectance mode. VivaScope ® 1500 allows an additional fluorescent examination and its placement on the ocular surface was made possible by the creation of a special interface between the microscope and the ocular apparatus. Results: Thanks to its compact and flexible configuration, the handheld camera VivaScope ® 3000 allowed to access more easily to the ocular and periocular tissues. Diagnosis of benign and malignant tumors (melanoma, squamous cell carcinoma and basal cell carcinoma), as well as infectious and genetic disease (storage diseases), could be evocated. The detection of parasites (Demodex folliculorum) on eyelids was possible. Confocal images correlated well with conventional histopathology. The fluorescence examination of corneal squamous cell carcinoma by VivaScope 1500 was characterized by extravasation of fluorescein after intravenous injection. Conclusions: Confocal microscopes dedicated to the skin offer new perspectives for the diagnosis, optimization of treatments, and follow-up of the ocular diseases. They will allow dermatologists to examine conjunctival and eyelid tumors, as it is for skin or genital mucosa. In addition, thanks to some adaptations of the dermatological device VivaScope ® 1500, it is possible for the first time to perform a fluorsecnte examination of the ocular and peri-ocular tissue, opening a new era in the clinical imaging of the ocular surface. A new semiology remains to be learned.
Introduction: Postoperative pancreatic fistula (POPF) is the major complication after distal panc... more Introduction: Postoperative pancreatic fistula (POPF) is the major complication after distal pancreatectomy (DP). In our recent randomized trial we showed that perioperative hydrocortisone (HC) treatment reduces Clavien-Dindo 3-5 complications after pancreaticoduodenectomy (Laaninen et al, Annals of Surgery 2016). Aims: In this study we aimed to find out whether perioperative hydrocortisone treatment prevents the risk for POPF after DP. Patients & methods: 40 patients planned for DP at Tampere University Hospital were randomized to receive perioperative treatment with intravenous HC 100 mg or plasebo. The first dose was given at the induction of anaestesia. 31 patients underwent DP and continued in the study. All had a high-risk, soft pancreas (>40% acini in the pancreatic transsection line as analysed peroperatively). They continued to receive HC/plasebo every 8 hours for two days postoperatively. All complications were graded, the primary endpoint being POPF. Results: Median age was 68 (39-82) years and 35% were men. The groups were similar for age, sex and ASA class distribution. 90-day mortality was zero. With HC treatment the rate of clinically significant POPF (grades B/C) were significantly reduced compared to plasebo (5.9% vs 28.6%, p¼0.016). The rate of overall Clavien-Dindo III-V complications were 5.9% and 21.4% in the HC and placebo group, respectively (ns; p¼0.058) Conclusion: Perioperative HC treatment decreases the rate of clinically relevant POPF even after distal pancreatectomy.
Background Recently, TERT promoter mutations were identified at high frequencies in cutaneous mel... more Background Recently, TERT promoter mutations were identified at high frequencies in cutaneous melanoma tumor samples and cell lines. The mutations were found to have a UV-signature and to lead to increased TERT gene expression. We analyzed a large cohort of melanoma patients for the presence and distribution of TERT promoter mutations and their association with clinico-pathological characteristics. Methods 410 melanoma tumor samples were analyzed by Sanger sequencing for the presence of TERT promoter mutations. An analysis of associations between mutation status and various clinical and pathologic variables was performed. Results TERT promoter mutations were identified in 154 (43%) of 362 successfully sequenced melanomas. Mutation frequencies varied between melanoma subtype, being most frequent in melanomas arising in nonacral skin (48%) and melanomas with occult primary (50%), and less frequent in mucosal (23%), and acral (19%) melanomas. Mutations carried a UV signature (C>T or CC>TT). The presence of TERT promoter mutations was associated with factors such as BRAF or NRAS mutation (P < .001), histologic type (P = .002), and Breslow thickness (P < .001). TERT promoter mutation was independently associated with poorer overall survival in patients with nonacral cutaneous melanomas (median survival 80 months vs 291 months for wild-type; hazard ratio corrected for other covariates 2.47; 95% confidence interval [CI] = 1.29 to 4.74; P = .006). Conclusions UV-induced TERT promoter mutations are one of the most frequent genetic alterations in melanoma, with frequencies varying depending on melanoma subtype. In nonacral cutaneous melanomas, presence of TERT promoter mutations is independently associated with poor prognosis.
We report the occurrence of Tn5 secondary transpositions in Escherichia coli HB101 hha::Tn5(pANN2... more We report the occurrence of Tn5 secondary transpositions in Escherichia coli HB101 hha::Tn5(pANN202-312). Tn5-induced hha mutants (hemolysin oveiproducers) segregated at high frequency either hemolysin-negative clones or clones showing the parental hemolytic phenotype (reduced hemolysin production). Secondary transpositions of Tn5 appeared to be responsible for the phenotypes of both types of derivatives. The hemolysin-negative clones no longer harboured Tn5 in the hha gene, but rather Tn5 was in the hly genes of the hemolytic plasmid pANN202-312. The derivatives that recovered the parental hemolytic phenotype contained a single copy of Tn5 in a chromosomal location different from that of hha. Both kinds of transpositional events appeared to restore the function of the hha gene.Key words: Tn5, transposition, Escherichia coli, excision.
The structure of the cornea is vital to its transparency, and dystrophies that disrupt corneal or... more The structure of the cornea is vital to its transparency, and dystrophies that disrupt corneal organization are highly heritable. To understand the genetic aetiology of Fuchs endothelial corneal dystrophy (FECD), the most prevalent corneal disorder requiring transplantation, we conducted a genome-wide association study (GWAS) on 1,404 FECD cases and 2,564 controls of European ancestry, followed by replication and meta-analysis, for a total of 2,075 cases and 3,342 controls. We identify three novel loci meeting genome-wide significance (Po5 Â 10 À 8): KANK4 rs79742895, LAMC1 rs3768617 and LINC00970/ATP1B1 rs1200114. We also observe an overwhelming effect of the established TCF4 locus. Interestingly, we detect differential sex-specific association at LAMC1, with greater risk in women, and TCF4, with greater risk in men. Combining GWAS results with biological evidence we expand the knowledge of common FECD loci from one to four, and provide a deeper understanding of the underlying pathogenic basis of FECD.
ABSTRACT To elucidate the etiology of hypertrophic cardiomyopathy (HC) in humans, we analyzed the... more ABSTRACT To elucidate the etiology of hypertrophic cardiomyopathy (HC) in humans, we analyzed the δ-sarcoglycan gene (SG), which is reported to be the causal gene for HC in the Syrian hamster BIO14.6. We performed polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses on the δ-SG in 102 patients with HC. SSCP was detected in exon 2 of the gene, but not in the other exons. The direct sequencing analysis of exon 2 revealed a C→T substitution at nucleotide residue 84 (TAC→TAT) with no amino acid alteration (Tyr→Tyr). There were no significant differences in allele frequencies of C/T between the patients with HC and the control group. Patients with HC were classified into 4 subgroups: obstructive HC, nonobstructive HC, apical HC, and familial HC. The allele frequency of C/T polymorphism in each of these groups was compared with that of the control group. The obstructive HC group showed a significantly greater frequency of the allele T than in the control group (31.6% vs 15.1%, RR = 2.6, p = 0.023). No other significant differences were observed. Thus, amino acid alteration in δ-SG may not be a common cause of HC in Japanese patients.
The clinical course of 66 boys and 49 girls with autosomal recessive polycystic kidney disease re... more The clinical course of 66 boys and 49 girls with autosomal recessive polycystic kidney disease recruited from departments of paediatric nephrology was investigated over a mean observation period of 4.92 years. This is a selected study group of children from departments of paediatric nephrology who in most cases survived the neonatal period, since birth clinics did not participate. The median age at diagnosis was 29 days (prenatal to 14.5 years). We observed decreased glomerular filtration rates (GFRs) in 72% (median age at onset of decrease of GFR < 2SD, 0.6 years; range. 0‐18.7 years), and 11 patients developed end‐stage renal disease. Hypertension requiring drug treatment was found in 70% (median age at start of medication, 0.5 years; range, 0‐16.7 years). Kidney length was above the 97th centile in 68% of patients, and kidney length did not increase with age or deterioration of renal function. Urinary tract infections occurred in 30%, growth retardation in 25%. and clinical si...
BACKGROUND: Porphyria cutanea tarda (PCT) occurs in sporadic (sPCT) and familial (fPCT) forms, wh... more BACKGROUND: Porphyria cutanea tarda (PCT) occurs in sporadic (sPCT) and familial (fPCT) forms, which are generally clinically indistinguishable and have traditionally been differentiated by erythrocyte uroporphyrinogen decarboxylase (UROD, EC 4.1.1.37) activity. We used UROD gene sequencing as the reference standard in assessing the diagnostic accuracy of UROD activity, evaluating the mutation spectrum of the UROD gene, determining the frequency and disease attributes of PCT and its subtypes in Norway, and developing diagnostic models that use clinical and laboratory characteristics for differentiating fPCT and sPCT. METHODS: All consecutive patients with PCT diagnosed within a 6-year period were used for incidence calculations. UROD activity analysis, UROD gene sequencing, analysis of hemochromatosis mutations, and registration of clinical and laboratory data were carried out for 253 patients. RESULTS: Fifty-three percent of the patients had diseaserelevant mutations, 74% of which were c.578GϾC or c.636ϩ1GϾC. The UROD activity at the optimal cutoff had a likelihood ratio (LR) of 9.2 for fPCT, whereas a positive family history had an LR of 19. A logistic regression model indicated that low UROD activity, a high uroporphyrin-heptaporphyrin ratio, a young age at diagnosis, male sex, and low alcohol consumption were predictors of fPCT. The incidence of PCT was 1 in 100 000. CONCLUSIONS: Two commonly occurring mutations are responsible for the high frequency of fPCT in Norway. UROD activity has a high diagnostic accuracy for differentiating the 2 PCT types, and a model that takes into account both clinical information and laboratory test results can be used to predict fPCT.
Introduction-Melanoma origin has always been a debated subject, as well as the role of adjacent m... more Introduction-Melanoma origin has always been a debated subject, as well as the role of adjacent melanocytic nevi. Epidemiological and histopathological studies point to melanomas arising either de novo or from a nevus. Methods-Sixty-one melanomas found in association with a preexisting nevus were microdissected, after careful selection of cell subpopulations and submitted to Sanger sequencing of the BRAF, NRAS, C-KIT, PPP6C, STK19 and RAC1 genes. Each gene was evaluated twice in all samples by sequencing or by sequencing and another confirmation method, allele-specific fluorescent polymerase chain reaction (PCR) and capillary electrophoresis detection, or by SNaPshot Analysis. Only mutations confirmed via two different molecular methods or twice by sequencing were considered positive. Results-The majority of cases presented concordance of mutational status between melanoma and the associated nevus for all 6 genes (40/60; 66.7%). Nine cases presented concomitant BRAF
Journal of Inherited Metabolic Disease, Jun 8, 2023
Wilson disease (WD) is a complex disease in which diagnosis and long‐term metabolic copper contro... more Wilson disease (WD) is a complex disease in which diagnosis and long‐term metabolic copper control remains challenging. The absence of accurate biomarkers requires the combination of different parameters to ensure copper homeostasis. Exchangeable copper and its ratio (REC) have been suggested to be useful biomarkers in this setting. We aimed at introducing these measurements and evaluate their performance and accuracy in our real‐world cohort of WD patients. Exchangeable copper and REC were measured in 48 WD patients and 56 control individuals by inductively coupled plasma‐mass‐spectrometry. Demographic and clinical characteristics were collected. REC was shown to be significantly higher among WD patients compared to controls and useful for WD identification by using the previously established cutoffs: 71.4% of WD patients with a recent diagnosis had REC ≥18.5% and 95.1% of long‐term treated WD had REC ≥14%; only four patients of the cohort presented discordant levels. Moreover, REC values were below 15% in all the control individuals. Exchangeable copper was significantly higher in WD patients compared to controls and tended to be reduced among WD patients who were compliant to medication. This real‐life study confirmed that exchangeable copper and REC are useful serum biomarkers that can be used as complementary tests to ensure WD diagnosis (REC) and copper homeostasis whithin time (exchangeable copper). The desirable target levels for this last objective still needs to be validated in prospective cohorts.
Fragile X syndrome is the commonest familial form of inherited mental retardation. The molecular ... more Fragile X syndrome is the commonest familial form of inherited mental retardation. The molecular defect is an expansion of the CGG trinucleotide repeats in the 5 0 untranslated region of the FMR1 gene that is inherited in an unstable fashion in fragile X families. In an attempt to provide more information about the CGG tract intergenerational variation, we have evaluated 642 transmissions in 175 Fragile X families. PCR and Southern blot (StB12.3) was used to analyse the CGG number. Among premutated alleles, 90.2% showed expansion, two-thirds to a full mutation while the rest remained in the premutation range, 5.5% of alleles did not vary and finally 4.3% of them reduced in size. Premutated females showed an increased risk of expansion to the full mutation depending on the CGG tract. The estimated risk for 80 triplets is more than seven times that of a woman carrying 59 CGG, the risk being 100% for alleles of >100 repeats. Fifty-nine repeats was the smallest allele that expanded to full mutation. Contractions were detected more frequently in males than in females, being statistically significant. This study contributes to the literature by increasing the data available regarding transmissions in Fragile X families and it allows us to perform more precise genetic counselling for women with the CGG repeat in the premutation range.
Fragile-X-associated tremor/ataxia syndrome (FXTAS) is a late-onset multisystem neurological diso... more Fragile-X-associated tremor/ataxia syndrome (FXTAS) is a late-onset multisystem neurological disorder characterized by intention tremor and cerebellar ataxia. We hypothesized that in FMR1 premutation females with FXTAS, a normal X chromosome might more frequently be inactivated; therefore, the aim of this study was to determine the relationship between skewed X chromosome inactivation (XCI) and FXTAS. We studied the XCI patterns of cases of FMR1 premutation in 10 women with FXTAS and 21 without FXTAS. The distribution of XCI patterns in the FXTAS and no-FXTAS groups showed differences regarding the allele presenting severe skewed XCI. In the FXTAS group, all cases preferentially inactivated the non-expanded X chromosome, whereas in the no-FXTAS group, all inactivated the expanded X chromosome. Nevertheless, no significant differences were found on comparing XCI frequencies among FMR1 premutation carriers with and without FXTAS. As expected, we found statistically significant differences in the skewed XCI on comparing FMR1 premutation women and controls. Although the reduced sample size and blood XCI patterns are two limitations of this study, our results suggest that the skewed XCI of the normal FMR1 allele may be a risk factor for the development of FXTAS. Furthermore, our findings also support the protective effect of the expression of a normal FMR1 allele.
INTRODUCTION Autosomal dominant polycystic kidney disease (ADPKD) is one of the commonest genetic... more INTRODUCTION Autosomal dominant polycystic kidney disease (ADPKD) is one of the commonest genetic diseases in man, affecting 1/1,000 individuals. It is char-acterised by the progressive development and enlarge-ment of multiple fluid filled cysts in the kidney that may ...
Transposon mutagenesis was used to isolate an Escherichia coli mutant that released into the exte... more Transposon mutagenesis was used to isolate an Escherichia coli mutant that released into the external medium a heterologous protein, the Aeromonas hydrophila aerolysin. Genetic mapping and phenotypic characterization of E. coli CM209 showed that Tn5 is inserted in the tolQ gene. This mutant strain released a significant amount of unprocessed (proaerolysin) protein into the external medium, together with other periplasmic proteins. Similar levels of the toxin were detected in the intracellular compartments of the parental and mutant strains. Whereas inactivation of the tolQ gene itself was responsible for some of the phenotypic properties of strain CM209, such as tolerance to group A colicins or to filamentous bacteriophages, the leaky phenotype was associated with a polar effect exerted by Tn5 on distal genes of the tolQRA cluster.Key words: Tn5, tolQ gene, aerolysin, leaky mutants.
We report the occurrence of Tn5 secondary transpositions in Escherichia coli HB101 hha::Tn5(pANN2... more We report the occurrence of Tn5 secondary transpositions in Escherichia coli HB101 hha::Tn5(pANN202-312). Tn5-induced hha mutants (hemolysin oveiproducers) segregated at high frequency either hemolysin-negative clones or clones showing the parental hemolytic phenotype (reduced hemolysin production). Secondary transpositions of Tn5 appeared to be responsible for the phenotypes of both types of derivatives. The hemolysin-negative clones no longer harboured Tn5 in the hha gene, but rather Tn5 was in the hly genes of the hemolytic plasmid pANN202-312. The derivatives that recovered the parental hemolytic phenotype contained a single copy of Tn5 in a chromosomal location different from that of hha. Both kinds of transpositional events appeared to restore the function of the hha gene.Key words: Tn5, transposition, Escherichia coli, excision.
Background: In vivo confocal microscopy is an imaging technique that has been applied to the stud... more Background: In vivo confocal microscopy is an imaging technique that has been applied to the study of the ocular surface. However, confocal microscopes dedicated to eye examination are routinely adopted only in ophthalmology reference centres and do not allow an examination of periocular tissue, nor a fluorescence examination. Methods: We applied for the first time the two in vivo confocal microscopes commonly used in dermatology (VivaScope ® 1500 and 3000, CALIBER, distributed in Europe by Mavig GmbH, Munich, Germany) to observe the cornea, the bulbar and tarsal conjunctiva, the eyelid margin, the lacrimal punctum and the palpebral skin of healthy volunteers. Tumoral, inflammatory and infectious diseases of the ocular mucosa and periocular skin from more than 200 patients were observed under the same microscopes. Both microscopes have a reflectance mode. VivaScope ® 1500 allows an additional fluorescent examination and its placement on the ocular surface was made possible by the creation of a special interface between the microscope and the ocular apparatus. Results: Thanks to its compact and flexible configuration, the handheld camera VivaScope ® 3000 allowed to access more easily to the ocular and periocular tissues. Diagnosis of benign and malignant tumors (melanoma, squamous cell carcinoma and basal cell carcinoma), as well as infectious and genetic disease (storage diseases), could be evocated. The detection of parasites (Demodex folliculorum) on eyelids was possible. Confocal images correlated well with conventional histopathology. The fluorescence examination of corneal squamous cell carcinoma by VivaScope 1500 was characterized by extravasation of fluorescein after intravenous injection. Conclusions: Confocal microscopes dedicated to the skin offer new perspectives for the diagnosis, optimization of treatments, and follow-up of the ocular diseases. They will allow dermatologists to examine conjunctival and eyelid tumors, as it is for skin or genital mucosa. In addition, thanks to some adaptations of the dermatological device VivaScope ® 1500, it is possible for the first time to perform a fluorsecnte examination of the ocular and peri-ocular tissue, opening a new era in the clinical imaging of the ocular surface. A new semiology remains to be learned.
Introduction: Postoperative pancreatic fistula (POPF) is the major complication after distal panc... more Introduction: Postoperative pancreatic fistula (POPF) is the major complication after distal pancreatectomy (DP). In our recent randomized trial we showed that perioperative hydrocortisone (HC) treatment reduces Clavien-Dindo 3-5 complications after pancreaticoduodenectomy (Laaninen et al, Annals of Surgery 2016). Aims: In this study we aimed to find out whether perioperative hydrocortisone treatment prevents the risk for POPF after DP. Patients & methods: 40 patients planned for DP at Tampere University Hospital were randomized to receive perioperative treatment with intravenous HC 100 mg or plasebo. The first dose was given at the induction of anaestesia. 31 patients underwent DP and continued in the study. All had a high-risk, soft pancreas (>40% acini in the pancreatic transsection line as analysed peroperatively). They continued to receive HC/plasebo every 8 hours for two days postoperatively. All complications were graded, the primary endpoint being POPF. Results: Median age was 68 (39-82) years and 35% were men. The groups were similar for age, sex and ASA class distribution. 90-day mortality was zero. With HC treatment the rate of clinically significant POPF (grades B/C) were significantly reduced compared to plasebo (5.9% vs 28.6%, p¼0.016). The rate of overall Clavien-Dindo III-V complications were 5.9% and 21.4% in the HC and placebo group, respectively (ns; p¼0.058) Conclusion: Perioperative HC treatment decreases the rate of clinically relevant POPF even after distal pancreatectomy.
Background Recently, TERT promoter mutations were identified at high frequencies in cutaneous mel... more Background Recently, TERT promoter mutations were identified at high frequencies in cutaneous melanoma tumor samples and cell lines. The mutations were found to have a UV-signature and to lead to increased TERT gene expression. We analyzed a large cohort of melanoma patients for the presence and distribution of TERT promoter mutations and their association with clinico-pathological characteristics. Methods 410 melanoma tumor samples were analyzed by Sanger sequencing for the presence of TERT promoter mutations. An analysis of associations between mutation status and various clinical and pathologic variables was performed. Results TERT promoter mutations were identified in 154 (43%) of 362 successfully sequenced melanomas. Mutation frequencies varied between melanoma subtype, being most frequent in melanomas arising in nonacral skin (48%) and melanomas with occult primary (50%), and less frequent in mucosal (23%), and acral (19%) melanomas. Mutations carried a UV signature (C>T or CC>TT). The presence of TERT promoter mutations was associated with factors such as BRAF or NRAS mutation (P < .001), histologic type (P = .002), and Breslow thickness (P < .001). TERT promoter mutation was independently associated with poorer overall survival in patients with nonacral cutaneous melanomas (median survival 80 months vs 291 months for wild-type; hazard ratio corrected for other covariates 2.47; 95% confidence interval [CI] = 1.29 to 4.74; P = .006). Conclusions UV-induced TERT promoter mutations are one of the most frequent genetic alterations in melanoma, with frequencies varying depending on melanoma subtype. In nonacral cutaneous melanomas, presence of TERT promoter mutations is independently associated with poor prognosis.
We report the occurrence of Tn5 secondary transpositions in Escherichia coli HB101 hha::Tn5(pANN2... more We report the occurrence of Tn5 secondary transpositions in Escherichia coli HB101 hha::Tn5(pANN202-312). Tn5-induced hha mutants (hemolysin oveiproducers) segregated at high frequency either hemolysin-negative clones or clones showing the parental hemolytic phenotype (reduced hemolysin production). Secondary transpositions of Tn5 appeared to be responsible for the phenotypes of both types of derivatives. The hemolysin-negative clones no longer harboured Tn5 in the hha gene, but rather Tn5 was in the hly genes of the hemolytic plasmid pANN202-312. The derivatives that recovered the parental hemolytic phenotype contained a single copy of Tn5 in a chromosomal location different from that of hha. Both kinds of transpositional events appeared to restore the function of the hha gene.Key words: Tn5, transposition, Escherichia coli, excision.
The structure of the cornea is vital to its transparency, and dystrophies that disrupt corneal or... more The structure of the cornea is vital to its transparency, and dystrophies that disrupt corneal organization are highly heritable. To understand the genetic aetiology of Fuchs endothelial corneal dystrophy (FECD), the most prevalent corneal disorder requiring transplantation, we conducted a genome-wide association study (GWAS) on 1,404 FECD cases and 2,564 controls of European ancestry, followed by replication and meta-analysis, for a total of 2,075 cases and 3,342 controls. We identify three novel loci meeting genome-wide significance (Po5 Â 10 À 8): KANK4 rs79742895, LAMC1 rs3768617 and LINC00970/ATP1B1 rs1200114. We also observe an overwhelming effect of the established TCF4 locus. Interestingly, we detect differential sex-specific association at LAMC1, with greater risk in women, and TCF4, with greater risk in men. Combining GWAS results with biological evidence we expand the knowledge of common FECD loci from one to four, and provide a deeper understanding of the underlying pathogenic basis of FECD.
ABSTRACT To elucidate the etiology of hypertrophic cardiomyopathy (HC) in humans, we analyzed the... more ABSTRACT To elucidate the etiology of hypertrophic cardiomyopathy (HC) in humans, we analyzed the δ-sarcoglycan gene (SG), which is reported to be the causal gene for HC in the Syrian hamster BIO14.6. We performed polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses on the δ-SG in 102 patients with HC. SSCP was detected in exon 2 of the gene, but not in the other exons. The direct sequencing analysis of exon 2 revealed a C→T substitution at nucleotide residue 84 (TAC→TAT) with no amino acid alteration (Tyr→Tyr). There were no significant differences in allele frequencies of C/T between the patients with HC and the control group. Patients with HC were classified into 4 subgroups: obstructive HC, nonobstructive HC, apical HC, and familial HC. The allele frequency of C/T polymorphism in each of these groups was compared with that of the control group. The obstructive HC group showed a significantly greater frequency of the allele T than in the control group (31.6% vs 15.1%, RR = 2.6, p = 0.023). No other significant differences were observed. Thus, amino acid alteration in δ-SG may not be a common cause of HC in Japanese patients.
The clinical course of 66 boys and 49 girls with autosomal recessive polycystic kidney disease re... more The clinical course of 66 boys and 49 girls with autosomal recessive polycystic kidney disease recruited from departments of paediatric nephrology was investigated over a mean observation period of 4.92 years. This is a selected study group of children from departments of paediatric nephrology who in most cases survived the neonatal period, since birth clinics did not participate. The median age at diagnosis was 29 days (prenatal to 14.5 years). We observed decreased glomerular filtration rates (GFRs) in 72% (median age at onset of decrease of GFR < 2SD, 0.6 years; range. 0‐18.7 years), and 11 patients developed end‐stage renal disease. Hypertension requiring drug treatment was found in 70% (median age at start of medication, 0.5 years; range, 0‐16.7 years). Kidney length was above the 97th centile in 68% of patients, and kidney length did not increase with age or deterioration of renal function. Urinary tract infections occurred in 30%, growth retardation in 25%. and clinical si...
BACKGROUND: Porphyria cutanea tarda (PCT) occurs in sporadic (sPCT) and familial (fPCT) forms, wh... more BACKGROUND: Porphyria cutanea tarda (PCT) occurs in sporadic (sPCT) and familial (fPCT) forms, which are generally clinically indistinguishable and have traditionally been differentiated by erythrocyte uroporphyrinogen decarboxylase (UROD, EC 4.1.1.37) activity. We used UROD gene sequencing as the reference standard in assessing the diagnostic accuracy of UROD activity, evaluating the mutation spectrum of the UROD gene, determining the frequency and disease attributes of PCT and its subtypes in Norway, and developing diagnostic models that use clinical and laboratory characteristics for differentiating fPCT and sPCT. METHODS: All consecutive patients with PCT diagnosed within a 6-year period were used for incidence calculations. UROD activity analysis, UROD gene sequencing, analysis of hemochromatosis mutations, and registration of clinical and laboratory data were carried out for 253 patients. RESULTS: Fifty-three percent of the patients had diseaserelevant mutations, 74% of which were c.578GϾC or c.636ϩ1GϾC. The UROD activity at the optimal cutoff had a likelihood ratio (LR) of 9.2 for fPCT, whereas a positive family history had an LR of 19. A logistic regression model indicated that low UROD activity, a high uroporphyrin-heptaporphyrin ratio, a young age at diagnosis, male sex, and low alcohol consumption were predictors of fPCT. The incidence of PCT was 1 in 100 000. CONCLUSIONS: Two commonly occurring mutations are responsible for the high frequency of fPCT in Norway. UROD activity has a high diagnostic accuracy for differentiating the 2 PCT types, and a model that takes into account both clinical information and laboratory test results can be used to predict fPCT.
Introduction-Melanoma origin has always been a debated subject, as well as the role of adjacent m... more Introduction-Melanoma origin has always been a debated subject, as well as the role of adjacent melanocytic nevi. Epidemiological and histopathological studies point to melanomas arising either de novo or from a nevus. Methods-Sixty-one melanomas found in association with a preexisting nevus were microdissected, after careful selection of cell subpopulations and submitted to Sanger sequencing of the BRAF, NRAS, C-KIT, PPP6C, STK19 and RAC1 genes. Each gene was evaluated twice in all samples by sequencing or by sequencing and another confirmation method, allele-specific fluorescent polymerase chain reaction (PCR) and capillary electrophoresis detection, or by SNaPshot Analysis. Only mutations confirmed via two different molecular methods or twice by sequencing were considered positive. Results-The majority of cases presented concordance of mutational status between melanoma and the associated nevus for all 6 genes (40/60; 66.7%). Nine cases presented concomitant BRAF
Journal of Inherited Metabolic Disease, Jun 8, 2023
Wilson disease (WD) is a complex disease in which diagnosis and long‐term metabolic copper contro... more Wilson disease (WD) is a complex disease in which diagnosis and long‐term metabolic copper control remains challenging. The absence of accurate biomarkers requires the combination of different parameters to ensure copper homeostasis. Exchangeable copper and its ratio (REC) have been suggested to be useful biomarkers in this setting. We aimed at introducing these measurements and evaluate their performance and accuracy in our real‐world cohort of WD patients. Exchangeable copper and REC were measured in 48 WD patients and 56 control individuals by inductively coupled plasma‐mass‐spectrometry. Demographic and clinical characteristics were collected. REC was shown to be significantly higher among WD patients compared to controls and useful for WD identification by using the previously established cutoffs: 71.4% of WD patients with a recent diagnosis had REC ≥18.5% and 95.1% of long‐term treated WD had REC ≥14%; only four patients of the cohort presented discordant levels. Moreover, REC values were below 15% in all the control individuals. Exchangeable copper was significantly higher in WD patients compared to controls and tended to be reduced among WD patients who were compliant to medication. This real‐life study confirmed that exchangeable copper and REC are useful serum biomarkers that can be used as complementary tests to ensure WD diagnosis (REC) and copper homeostasis whithin time (exchangeable copper). The desirable target levels for this last objective still needs to be validated in prospective cohorts.
Fragile X syndrome is the commonest familial form of inherited mental retardation. The molecular ... more Fragile X syndrome is the commonest familial form of inherited mental retardation. The molecular defect is an expansion of the CGG trinucleotide repeats in the 5 0 untranslated region of the FMR1 gene that is inherited in an unstable fashion in fragile X families. In an attempt to provide more information about the CGG tract intergenerational variation, we have evaluated 642 transmissions in 175 Fragile X families. PCR and Southern blot (StB12.3) was used to analyse the CGG number. Among premutated alleles, 90.2% showed expansion, two-thirds to a full mutation while the rest remained in the premutation range, 5.5% of alleles did not vary and finally 4.3% of them reduced in size. Premutated females showed an increased risk of expansion to the full mutation depending on the CGG tract. The estimated risk for 80 triplets is more than seven times that of a woman carrying 59 CGG, the risk being 100% for alleles of >100 repeats. Fifty-nine repeats was the smallest allele that expanded to full mutation. Contractions were detected more frequently in males than in females, being statistically significant. This study contributes to the literature by increasing the data available regarding transmissions in Fragile X families and it allows us to perform more precise genetic counselling for women with the CGG repeat in the premutation range.
Fragile-X-associated tremor/ataxia syndrome (FXTAS) is a late-onset multisystem neurological diso... more Fragile-X-associated tremor/ataxia syndrome (FXTAS) is a late-onset multisystem neurological disorder characterized by intention tremor and cerebellar ataxia. We hypothesized that in FMR1 premutation females with FXTAS, a normal X chromosome might more frequently be inactivated; therefore, the aim of this study was to determine the relationship between skewed X chromosome inactivation (XCI) and FXTAS. We studied the XCI patterns of cases of FMR1 premutation in 10 women with FXTAS and 21 without FXTAS. The distribution of XCI patterns in the FXTAS and no-FXTAS groups showed differences regarding the allele presenting severe skewed XCI. In the FXTAS group, all cases preferentially inactivated the non-expanded X chromosome, whereas in the no-FXTAS group, all inactivated the expanded X chromosome. Nevertheless, no significant differences were found on comparing XCI frequencies among FMR1 premutation carriers with and without FXTAS. As expected, we found statistically significant differences in the skewed XCI on comparing FMR1 premutation women and controls. Although the reduced sample size and blood XCI patterns are two limitations of this study, our results suggest that the skewed XCI of the normal FMR1 allele may be a risk factor for the development of FXTAS. Furthermore, our findings also support the protective effect of the expression of a normal FMR1 allele.
INTRODUCTION Autosomal dominant polycystic kidney disease (ADPKD) is one of the commonest genetic... more INTRODUCTION Autosomal dominant polycystic kidney disease (ADPKD) is one of the commonest genetic diseases in man, affecting 1/1,000 individuals. It is char-acterised by the progressive development and enlarge-ment of multiple fluid filled cysts in the kidney that may ...
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Papers by Celia Badenas