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10 Mild cystic fibrosis phenotype in adult patients with novel 3272–16T>A mutation
S58
1. Genetics
Posters
9 Analysis of cystic fibrosis gene mutations in children with cystic fibrosis in a regional center in Eastern Romania
11 The risk of incorrect reporting or misinterpretation of genotype results
L.M.S. Trandafir1 , A.A.M. Tudose2 , D.T. Paduraru Anton1 . 1 Sfanta Maria Clinical Emergency Hospital, Pediatric, Iasi, Romania; 2 Mavromati Clinical Emergency Hospital, Neonatology, Botosani, Romania
J. Wilson1,2 , E. Williams1 , T. Kotsimbos1,2 . 1 Alfred Health, Cystic Fibrosis Service, Prahran, Australia; 2 Monash University, Prahran, Australia
Objectives: The purpose of this study was to describe the genetic mutations identified in patients of pediatric age with cystic fibrosis (CF) diagnosed in a cystic fibrosis center in north-eastern Romania. Methods: We performed a retrospective study which included 37 children with CF, followed between December 2012 and December 2014. In the absence of neonatal screening, diagnosis was based on sweat test in patients who presented respiratory or digestive clinical manifestations. The 32 patients (21 boys and 11 girls) involved were performed genetic tests only to identify 38 mutations using Real time PCR method. Results: Following the genetic tests performed, there were identified: homozygous (F508del − 11 patients, G542X − 1 patient, 1898 + 1G>A − 1 patient), heterozygous compounds (F508del/G542X − 4 patients, F508del 7T/9T − 2 patients, F508del 9T/9T − 4 patients, D1152H 7T/7T − 1 patient) and other types of mutations 7T/7T − 8 patients. F508del mutation frequency was 65.62% in the study group. F508del mutation was associated with mixed forms (digestive and pulmonary) of CF in 13 patients and with pulmonary form of CF in 8 patients. Mutation 1898 + 1G>A was associated with a severe form of digestive disease and pulmonary of CF with unfavorable evolution. Conclusion: F508del mutation frequency was similar compared to the frequency of this particular mutation in other European countries (70%). In Romania, it is necessary to introduce a genetic screening test for an early FC diagnosis and also for establishing the genetic profile of CF.
Objectives: To determine the possibility and likelihood of inaccurate genotype results in CF. Methods: Over 500 genotype results were collected at a centre for adult CF care. Genotype results were entered into a database to generate study data. Genotype results were repeated if a. the result was unclear and repeat testing was requested by the genetic testing laboratory, b. the managing clinician had significant concerns regarding genotype/phenotype correlation or c. results held in the database were incomplete, i.e. one single mutation was identified and no second mutation was found, using an older (smaller) panel of probes. Results: From over 500 genotypes collected between 1991 and 2014, there were 5 results that were found to be inaccurate/misread on repeat testing. AB was described as having the same mutation as her brother CD, being F508del/F508del. On subsequent testing, he had R117H/unk. F508del was not proven on re-testing. EF had been treated as CF. On repeat testing he was found to be homogygous for R117H. GH was found to be 3454G>C with a typographical error. IJ was identified as unk/unk, and extended testing was unhelpful, however like siblings AB and CD, was found to have a gene abnormality (unspecified) like his brother. Patient
Initial
Subsequent
Change status
AB CD EF GH IJ
F508del/F508del F508del/F508del R117H/unk F508del/345G>C unk/unk
unk/unk RII7H/unk R117H/R117H F508del/3454G>C unk/unk
Y Y Y N N
Conclusion: Genotype results may be incorrect or misinterpreted in 1% of patients. Retesting and consultation is important if doubt exists.
10 Mild cystic fibrosis phenotype in adult patients with novel 3272−16T>A mutation
12 Reverse hybridization enables fast and reliable detection of 25 common CF mutations
S. Krasovskiy1 , E. Amelina1 , M. Usacheva1 , A. Stepanova2 , A. Poliakov2 , M.-P. Audr´ezet3 , C. F´erec3 , N. Kashirskaya2 . 1 Research Institute of Pulmonology, Moscow, Russian Federation; 2 Federal State Budgetary Institution “Research Centre for Medical Genetics”, Moscow, Russian Federation; 3 CHU de Brest, Laboratory of Molecular Genetics, Brest, France
S. Weidler1 , J. Hammermann1 , P. Binkenstein1 , C. M¨uller2 , G. Rottwinkel2 , M. Stopsack3 , M.A. Lee-Kirsch1 . 1 Medizinische Fakult¨at Carl Gustav Carus, Technische Universit¨at Dresden, Department of Pediatrics, Dresden, Germany; 2 Astra Biotech GmbH, Berlin, Germany; 3 Technische Universit¨ at Dresden, Institute for Clinical Chemistry and Laboratory Medicine, Dresden, Germany
Objectives: Patients who have milder symptoms or atypical cystic fibrosis (CF) may have at least one so called class V mutation. This class includes mutations that leave residual levels of normal CFTR transcripts and protein. Recently by sequencing of the coding region of the CFTR gene and exon-intron junctions we’ve revealed 3272−16T>A mutation in Russian adult CF patients. As far as we know this mutation hasn’t been described (before December 2014) by any other geneticists and it is not in the CFTR1 and CFTR2 lists. 3272−16T>A mutation is a splicing mutation in intron 19 of CFTR gene (thymine to adenine substitution). Aim: To reveal the clinical characteristics of 3272−16T>A mutation in CF adult patients. Methods: Adult CF patients carrying 3272−16T>A mutation were included in the study. The following data were evaluated: the age at CF diagnosis, pancreatic status, the presence of the following complications: meconium ileus or DIOS, diabetes and liver cirrhosis. Results: We’ve found 10 patients (6 men) carried 3272−16T>A mutation and the following mutations in the other CFTR allele: F508del − 6 patients, 394delTT − 2 patients, 2184insA − 1 patient, not identified − 1 patient. The average age of patients was 27.4±5.5 years (from 18.4 to 35.0 years). Age at diagnosis ranged from 3.5 to 26.9 years (12.3±7.0 yrs). All 10 were pancreatic sufficient. None of them had meconium ileus in anamnesis or DIOS, diabetes and liver cirrhosis. Conclusion: 3272−16T>A mutation is a mild mutation associated with pancreatic sufficiency and lack of development of some complications specific to the classical CF genotypes.
Objectives: Nationwide CF newborn screening (CFNBS) will be established in Germany in 2015. IRT is a reliable marker for the 1st tier of CFNBS. Although sweat testing represents the gold standard for CF diagnostics, it is of limited reliability in (preterm) neonates. A fast and simple assay to screen for common CF mutations using newborn screening cards would be beneficial. Methods: Analysis of 25 common CFTR mutations was performed using the CFcheck EU-25 kit (Astra Biotech) on 66 samples with known CFTR genotype. Fluorescent probes were generated in two consecutive multiplex PCR reactions. Pooled PCR products were hybridized to a slide containing targets corresponding to wild type and mutant CFTR sequences. Signal intensities were measured with a laser scanner. Results: The mutation detection rate was 100% (66/66). False positive signals occurred in 5 patients and led to 7.6% repeat analyses. The specificity was 99.8% according to the high number of screened mutations on each sample (25 on 2 alleles/sample). Conclusion: The reliable and fast detection of 25 CF mutations using newborn screening cards following positive CFNBS results may complement present CFNBS programs. Early knowledge of genotype, reduced number of recalls and sweat tests may outweigh unwanted heterozygous carrier detection.
1. Genetics
Posters
9 Analysis of cystic fibrosis gene mutations in children with cystic fibrosis in a regional center in Eastern Romania
11 The risk of incorrect reporting or misinterpretation of genotype results
L.M.S. Trandafir1 , A.A.M. Tudose2 , D.T. Paduraru Anton1 . 1 Sfanta Maria Clinical Emergency Hospital, Pediatric, Iasi, Romania; 2 Mavromati Clinical Emergency Hospital, Neonatology, Botosani, Romania
J. Wilson1,2 , E. Williams1 , T. Kotsimbos1,2 . 1 Alfred Health, Cystic Fibrosis Service, Prahran, Australia; 2 Monash University, Prahran, Australia
Objectives: The purpose of this study was to describe the genetic mutations identified in patients of pediatric age with cystic fibrosis (CF) diagnosed in a cystic fibrosis center in north-eastern Romania. Methods: We performed a retrospective study which included 37 children with CF, followed between December 2012 and December 2014. In the absence of neonatal screening, diagnosis was based on sweat test in patients who presented respiratory or digestive clinical manifestations. The 32 patients (21 boys and 11 girls) involved were performed genetic tests only to identify 38 mutations using Real time PCR method. Results: Following the genetic tests performed, there were identified: homozygous (F508del − 11 patients, G542X − 1 patient, 1898 + 1G>A − 1 patient), heterozygous compounds (F508del/G542X − 4 patients, F508del 7T/9T − 2 patients, F508del 9T/9T − 4 patients, D1152H 7T/7T − 1 patient) and other types of mutations 7T/7T − 8 patients. F508del mutation frequency was 65.62% in the study group. F508del mutation was associated with mixed forms (digestive and pulmonary) of CF in 13 patients and with pulmonary form of CF in 8 patients. Mutation 1898 + 1G>A was associated with a severe form of digestive disease and pulmonary of CF with unfavorable evolution. Conclusion: F508del mutation frequency was similar compared to the frequency of this particular mutation in other European countries (70%). In Romania, it is necessary to introduce a genetic screening test for an early FC diagnosis and also for establishing the genetic profile of CF.
Objectives: To determine the possibility and likelihood of inaccurate genotype results in CF. Methods: Over 500 genotype results were collected at a centre for adult CF care. Genotype results were entered into a database to generate study data. Genotype results were repeated if a. the result was unclear and repeat testing was requested by the genetic testing laboratory, b. the managing clinician had significant concerns regarding genotype/phenotype correlation or c. results held in the database were incomplete, i.e. one single mutation was identified and no second mutation was found, using an older (smaller) panel of probes. Results: From over 500 genotypes collected between 1991 and 2014, there were 5 results that were found to be inaccurate/misread on repeat testing. AB was described as having the same mutation as her brother CD, being F508del/F508del. On subsequent testing, he had R117H/unk. F508del was not proven on re-testing. EF had been treated as CF. On repeat testing he was found to be homogygous for R117H. GH was found to be 3454G>C with a typographical error. IJ was identified as unk/unk, and extended testing was unhelpful, however like siblings AB and CD, was found to have a gene abnormality (unspecified) like his brother. Patient
Initial
Subsequent
Change status
AB CD EF GH IJ
F508del/F508del F508del/F508del R117H/unk F508del/345G>C unk/unk
unk/unk RII7H/unk R117H/R117H F508del/3454G>C unk/unk
Y Y Y N N
Conclusion: Genotype results may be incorrect or misinterpreted in 1% of patients. Retesting and consultation is important if doubt exists.
10 Mild cystic fibrosis phenotype in adult patients with novel 3272−16T>A mutation
12 Reverse hybridization enables fast and reliable detection of 25 common CF mutations
S. Krasovskiy1 , E. Amelina1 , M. Usacheva1 , A. Stepanova2 , A. Poliakov2 , M.-P. Audr´ezet3 , C. F´erec3 , N. Kashirskaya2 . 1 Research Institute of Pulmonology, Moscow, Russian Federation; 2 Federal State Budgetary Institution “Research Centre for Medical Genetics”, Moscow, Russian Federation; 3 CHU de Brest, Laboratory of Molecular Genetics, Brest, France
S. Weidler1 , J. Hammermann1 , P. Binkenstein1 , C. M¨uller2 , G. Rottwinkel2 , M. Stopsack3 , M.A. Lee-Kirsch1 . 1 Medizinische Fakult¨at Carl Gustav Carus, Technische Universit¨at Dresden, Department of Pediatrics, Dresden, Germany; 2 Astra Biotech GmbH, Berlin, Germany; 3 Technische Universit¨ at Dresden, Institute for Clinical Chemistry and Laboratory Medicine, Dresden, Germany
Objectives: Patients who have milder symptoms or atypical cystic fibrosis (CF) may have at least one so called class V mutation. This class includes mutations that leave residual levels of normal CFTR transcripts and protein. Recently by sequencing of the coding region of the CFTR gene and exon-intron junctions we’ve revealed 3272−16T>A mutation in Russian adult CF patients. As far as we know this mutation hasn’t been described (before December 2014) by any other geneticists and it is not in the CFTR1 and CFTR2 lists. 3272−16T>A mutation is a splicing mutation in intron 19 of CFTR gene (thymine to adenine substitution). Aim: To reveal the clinical characteristics of 3272−16T>A mutation in CF adult patients. Methods: Adult CF patients carrying 3272−16T>A mutation were included in the study. The following data were evaluated: the age at CF diagnosis, pancreatic status, the presence of the following complications: meconium ileus or DIOS, diabetes and liver cirrhosis. Results: We’ve found 10 patients (6 men) carried 3272−16T>A mutation and the following mutations in the other CFTR allele: F508del − 6 patients, 394delTT − 2 patients, 2184insA − 1 patient, not identified − 1 patient. The average age of patients was 27.4±5.5 years (from 18.4 to 35.0 years). Age at diagnosis ranged from 3.5 to 26.9 years (12.3±7.0 yrs). All 10 were pancreatic sufficient. None of them had meconium ileus in anamnesis or DIOS, diabetes and liver cirrhosis. Conclusion: 3272−16T>A mutation is a mild mutation associated with pancreatic sufficiency and lack of development of some complications specific to the classical CF genotypes.
Objectives: Nationwide CF newborn screening (CFNBS) will be established in Germany in 2015. IRT is a reliable marker for the 1st tier of CFNBS. Although sweat testing represents the gold standard for CF diagnostics, it is of limited reliability in (preterm) neonates. A fast and simple assay to screen for common CF mutations using newborn screening cards would be beneficial. Methods: Analysis of 25 common CFTR mutations was performed using the CFcheck EU-25 kit (Astra Biotech) on 66 samples with known CFTR genotype. Fluorescent probes were generated in two consecutive multiplex PCR reactions. Pooled PCR products were hybridized to a slide containing targets corresponding to wild type and mutant CFTR sequences. Signal intensities were measured with a laser scanner. Results: The mutation detection rate was 100% (66/66). False positive signals occurred in 5 patients and led to 7.6% repeat analyses. The specificity was 99.8% according to the high number of screened mutations on each sample (25 on 2 alleles/sample). Conclusion: The reliable and fast detection of 25 CF mutations using newborn screening cards following positive CFNBS results may complement present CFNBS programs. Early knowledge of genotype, reduced number of recalls and sweat tests may outweigh unwanted heterozygous carrier detection.