- Email: [email protected]
Novel de Novo Large Deletion in Cystic Fibrosis Transmembrane Conductance Regulator Gene Results in a Severe Cystic Fibrosis Phenotype
Novel de Novo Large Deletion in Cystic Fibrosis Transmembrane Conductance Regulator Gene Results in a Severe Cystic Fibrosis Phenotype Aleksandra Norek, PhD, Marta Stremska, BSc, Agnieszka Sobczynska-Tomaszewska, PhD, Katarzyna WertheimTysarowska, MSc, Hanna Dmenska, MD, PhD, and Marta Jurek, MSc We identified c.1521_1523delCTT and c.1679+94_2619+986del8118 in trans in a 6-year-old boy with a severe cystic fibrosis phenotype. The first deletion was inherited maternally, but the latter had arisen de novo. (J Pediatr 2011;159:343-6)
C
ystic fibrosis (CF, MIM: 219700) is one of the most common life-shortening autosomal recessive disorders in the Caucasian population, affecting approximately 1 in 2500 newborns.1 The disease is caused by a dysfunction or a complete deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel (MIM: 602421) in the apical epithelial cell membrane. Since the identification of the CFTR gene in 1989, >1860 different molecular defects were recognized and submitted to Cystic Fibrosis Mutation Database (CFMD, http://www3.genet.sickkids.on.ca/cftr/ app). Most changes described are point mutations and small insertions or deletions. CFMD also lists approximately 48 cases of hereditary large genomic rearrangements, which account for 2.59% of all known CFTR mutations. De novo molecular alterations in the CFTR gene have been previously described, but are rare.2,3,4 Here, we present a novel de novo large deletion, c.1679+94_2619+986del8118 (NM_000492.3), spanning exons 12, 13, and 14a. This rearrangement was identified in a patient with CF who was of Polish origin, carrying the common pathogenic c.1521_1523delCTT mutation (F508del) on the maternal allele.
Methods A patient of Caucasian origin (Polish), aged 6 years, was born at the 40th week of gestation (body weight, 3600 g; length, 56 cm; 10 points on Apgar scale) to healthy, young, unrelated parents (mother 31 years old and father 32 years old). He has 4 siblings with no clinical evidence of CF. The patient was first hospitalized at the age of 2 weeks, when he had a paroxysmal, strenuous cough with no signs of respiratory tract infection. No improvement was observed after symptomatic treatment. At the age of 6 weeks, he was admitted to the hospital because of severe cough persistence and pneumonia. During this hospitalization, his condition gradually worsened with an intensifying paroxysmal dyspnea and a pertusis-like cough. Inflammatory thickening
CF CFMD CFTR PCR STR MLPA
Cystic fibrosis Cystic Fibrosis Mutation Database Cystic fibrosis transmembrane conductance regulator Polymerase chain reaction Short tandem repeat Multiplex Ligation-dependent Probe Assay
was diagnosed in the lung. A pharyngeal swap and trachea aspirate were culture particular Pseudomonas aeruginosa. On the basis of elevated sweat Cl levels (>100 mmol/L) measured with pilocarpine iontophoresis, CF was initially determined. P aeruginosa eradication was obtained after 2 months. As a consequence of severe loss of body weight (at birth, 3600 g; at the age of 3 months, 3000 g), hypoproteinemia, and difficulties with oral feeding, parenteral feeding was introduced through the period of 1.5 months, giving improvement of nutrition status and general condition of the patient. Pancreatic exocrine insufficiency was observed when the patient was 3 years of age. Fecal fat balance was 8 g per 24 hours and 14.19 g per 24 hours at 5 years of age, with the norm being 3.2 g per 24 hours. Liver status was also healthy, without signs of portal hypertension and normal ultrasound scanning liver images. Currently, at the age of 6 years, the patient’s body weight is in the 10th percentile. He remains on a high-calorie diet (Fantomalt, Protifar; Nutricia Nordica AB, Solna, Stockholm, Sweden), pancreatic enzyme (Lipancrea [Polfa Warszawa SA, Warsaw, Poland] 8500U/kg body weight/24 hr), and fatsoluble vitamin supplementation. He receives inhalations (Mucosolvan; Boehringer Ing Pharma GmbH & Co KG, Ingelheim am Rhein, Germany and Pulmozyme; Genentech, San Francisco, California) with physiotherapeutic procedures (bronchial drainage). Bronchial and pulmonary exacerbations are observed 3 to 5 times a year, but they are not lifethreatening and improve after oral antibiotic therapy. Mutational Screening of the CFTR Gene Genomic DNA from the patient and his relatives (parents and 4 siblings) was extracted from peripheral blood lymphocytes, with the method of Miller et al.5 Molecular analysis of the CFTR gene was carried out by using INNO-LiPA CFTR19 and INNO-LiPA CFTR17+Tn kits (Innogenetics NV, Gent, Belgium), sequencing of all 27 exons together with exon/intron junctions (ABI PRISM 3730, Applied Biosystems,
From the Institute of Mother and Child, Warsaw, Poland (A.N., M.S., A.S-T., K.W-T., M.J.); and Children’s Memorial Health Institute, Warsaw, Poland (H.D.) Supported by Polish Ministry of Science and Higher Education (grant NN401 129936). The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright ª 2011 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2011.04.022
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Figure 1. Confirmation of c.1679+94_2619+986del8118 mutation. A, Results of MLPA analysis. Fragment of electropherogram displaying reference runs in lighter color (left) compared with the DNA sample of the patient (right). The deleted fragments are indicated by arrows. B, Results of long-range PCR reaction. The fragment of 964 bp depicts the mutated allele. C, Fragment of electropherogram showing the sequence of mutated allele.
Foster, California) and Multiplex Ligation-Dependent Probe Assay (MLPA P091; MRC-Holland, Amsterdam, The Netherlands and ABI PRISM 3730) were performed according to the manufacturer’s recommendations. Long-Range Polymerase Chain Reaction The reaction was carried out to multiply the CFTR gene fragment (sized 13 895 base pairs) containing the region from exon 11 to exon 14a with flanking sequences. The fragment was amplified with these primers: 5’-CCT TTC AAA TTC AGA TTG AGC ATA-3’ (forward) and 5’-CAA GTC TCC ACC AGA TTA ACT AGA-3’ (reverse). The polymerase chain reaction (PCR) protocol comprised 30 cycles of 98 C for 10 seconds and 68 C for 15 minutes. Before the first cycle, there was the initial denaturation step of 94 C for 1 minute, and after the last cycle, the extension step of 10 minutes at 72 C. After PCR, the reaction products were analyzed with agarose gel electrophoresis. Breakpoints Identification To specify the size of deletion and amplify only the fragment flanking the breakpoints, PCR in restricted conditions, with 344
the same primers as in long-range PCR, was performed: 35 cycles of 95 C for 30 seconds, 59 C for 30 seconds, 72 C for 60 seconds, the initial denaturation step of 95 C for 6 minutes, and the extension step for 7 minutes at 72 C. Finally, the reaction product was sequenced with 5’-GAT GTG CCT TTC AAA TTC AGA-3’ forward primer. Capillary electrophoresis was carried out by means of an automatic sequencer ABI PRISM 3730. The resulting sequences were analyzed with Mutation Surveyor Software version 3.1 (SoftGenetics, State College, Pennsylvania). Paternity Test A paternity test was carried out at the Department of Forensic Medicine, Warsaw Medical Academy. Isolated DNA of the patient and his parents was amplified with the use of PowerPlex 16 system (Promega, Madison, Wisconsin) and AmpFlSTR SEfiler Plus kit (Applied Biosystems) These 19 markers were tested: D3S13, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, vWa D8S1179, TPOX, FGA, D2S1338, D19S433, SE33. The products of short tandem repeat (STR) sets amplification reaction were then subjected to Norek et al
August 2011
CLINICAL AND LABORATORY OBSERVATIONS
electrophoresis in denaturing polyacrylamide gel with the use of ABI 377 (Applied Biosystems). Comparative analysis (patient-mother-father) of the particular STRs was performed. STR Loci Analysis Additionally, we designed microsatellites to confirm biparental inheritance of chromosome 7 in the proband and his siblings. These STR loci localized on chromosome 7 were analyzed: D7S480, D7S481, D7S799, D7S2462, and D7S2497. Selection criteria of STR markers were position on chromosome 7 and heterozygosity index >0.8, according to GeneCards (http://genecards.weizmann.ac.il/geneloc/index.shtml). Fragments were amplified with a pair of self-designed primers, from which the forward ones were marked with 6-FAM at 5’ end. Primers sequences are listed in the Table (available at www.jpeds.com). Gene scan electrophoresis was performed on an automatic sequencer ABI PRISM 3730.
Results At the age of 3 months, the patient was referred for CFTR molecular testing. Initial report indicated c.1521_1523delCTT in one CFTR allele. Molecular analysis with INNO-LiPA CFTR19 and INNO-LiPA CFTR17+Tn kits followed by sequencing of all 27 CFTR exons with exon/intron junctions did not establish a CF genotype in the patient. In the next step, according to our diagnostic procedure, MLPA analysis was carried out to look for any possible gene rearrangements. As a result, deletion of a large CFTR gene fragment including 3 exons, 12, 13, and 14a, was recognized (Figure 1, A). To verify MLPA reaction results, long-range PCR was carried out, and the deletion was confirmed (Figure 1, B). Sequencing of the 964 bp reaction product enabled the precise mapping of the breakpoints (Figure 1, C). To verify bi-parental inheritance of the CFTR identified defects and to determine the carrier status, molecular tests were performed for the proband’s relatives (Figure 2). The c.1521_1523delCTT mutation was identified in the mother and older brother in one of the CFTR alleles. The c.1679+94_2619+986del8118 rearrangement was not found in any allele of the biological father, mother, nor any of the siblings, suggesting that the mutation had arisen de novo. With paternity tests and STR loci analysis on chromosome 7, paternal origin of the mutated allele in the patient was unanimously confirmed. De novo large deletions in CFTR gene have not been described so far. To understand the mechanism of the de novo mutation presented here, we examined sequence architecture in the breakpoint’s proximity and scanned it for repetitive elements. We found no microhomology between the breakpoints (5’TATTCTGTTTCTGGAAT3’ and 5’CCACTCTGG CTGTGCG3’; deleted fragment underlined), which excluded the involvement of replication-based mechanisms of DNA repair.6 Subsequently, we used UCSC Genome Browser on Human Mar. 2006 (NCBI36/hg18) Assembly (http:// genome.ucsc.edu) to scan for repetitive elements, which established that the proximal breakpoint lays in the unique
Figure 2. Pedigree analysis of patient’s family showing the results of microsatellite haplotypes (D7S481, D7S2497, D7S799, D7S480, D7S2462) and the two mutations detected: m 1 (c.1521_1523delCTT) and m2 (c.1679+94_2619+986del8118). wt, wild type allele.
DNA sequence, whereas the distal one is positioned in the long-terminal repeat repetitive element (long-terminal repeat element 12C, family endogenous retroviral sequence 1). We also used the non-B DB database (http://nonb.abcc.ncifcrf. gov/) to predict possible alternative DNA structures. We found no architectural clues in the sequence, which might predispose for the emergence of this deletion.
Discussion Autosomal recessive disorders may arise although only one parent is a carrier. Our data demonstrate the appearance of a de novo deletion spanning the region from intron 11 to intron 14a in a Polish patient with CF. Mutations occurring de novo in CFTR are extremely rare. There have been 3 published cases of de novo mutations in CFTR gene to date: c.3197G>A (R1066H),2 c.2551C>T (R851X),3 and c.3194T>G (L1065R).4 All of them are point mutations situated at the mutational hot spot regions. The c.1679+94_2619+986del8118 mutation is a de novo large rearrangement in CFTR gene. We hypothesize that this deletion could appear as a result of non-homologous end joining taking place during the DNA double-strand breaks repair. Non-homologous recombination in the CFTR gene has been mentioned as a direct cause of 5 various CFTR gene rearrangements.7 In all published de novo mutations, similarly to ours, the new mutation had appeared on the paternal chromosome. Casals et al4 proposed that this phenomenon may reflect a higher mutation rate in paternal gametes. This tendency was noticed in several other genes (eg, fibroblast growth factor receptor 2).8
Novel de Novo Large Deletion in Cystic Fibrosis Transmembrane Conductance Regulator Gene Results in a Severe Cystic Fibrosis Phenotype
345
THE JOURNAL OF PEDIATRICS
www.jpeds.com
The deletion we describe includes amino acids from 561 to 873 (in frame), which correspond to partial loss of nucleotide-binding domain 1, transmembrane domain 2, and complete loss of regulatory (R-domain) domain.9 All these components have been shown to play a crucial role in CFTR channel function,1 which may explain the severity of CF symptoms in the patient. CFMD currently lists >1860 molecular defects. This case emphasizes the importance of comprehensive mutation screening of CFTR gene, especially in the cases in which a patient exhibits clinical manifestation of CF and only one mutated CFTR allele is identified. However, analyses of large CFTR gene rearrangements have only recently come in clinical practice and are not included in conventional CFTR mutation panels. Thus such mutations can easily escape detection and lead to confusion in genetic counseling.10 Definitive identification of both mutated CFTR alleles confirms the clinical diagnosis and facilitates patient treatment. Testing of proband’s parents and relatives is of importance for genetic counseling purposes and planning of consecutive pregnancies.11 Systematic segregation studies in CF would assist in detection of more de novo mutations than are currently described. n We thank Pawel Stankiewicz and Katarzyna Derwi nska for their bioinformatic support, Michal Milewski for his suggestions concerning methods in breakpoints identification, and Katarzyna Niepokoj and Aleksandra Susek for their technical assistance in microsatellite analysis. Submitted for publication Sep 8, 2010; last revision received Mar 22, 2011; accepted Apr 18, 2011. Reprint requests: Aleksandra Norek, PhD, Institute of Mother and Child, Department of Medical Genetics, Kasprzaka 17A, 01-211 Warsaw, Poland. E-mail: [email protected]
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References 1. Rowntree RK, Harris A. The phenotypic consequences of CFTR mutations. Ann Hum Genet 2003;67(5):471-85. 2. Cremonesi L, Cainarca S, Rossi A, Padoan R, Ferrari M. Detection of a de novo R1066H mutation in an Italian patient affected by cystic fibrosis. Hum Genet 1996;98:119-21. 3. White MB, Leppert M, Nielsen D, Zielenski J, Gerrard B, Stewart C, et al. A de novo cystic fibrosis mutation: CGA (Arg) to TGA (stop) at codon 851 of the CFTR gene. Genomics 1991;11:778-9. 4. Casals T, Ramos MD, Gimenez J, Larriba S, Nunez V, Estivill X. High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes. Hum Genet 1997;101:365-70. 5. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16(3):1215. 6. Stankiewicz P, Lupski JR. Structural variation in the human genome and its role in disease. Annu Rev Med 2010;61:437-55. 7. Audrezet MP, Chen J-M, Raguenes O, Chuzhanova N, Giteau K, Le Marechal C, et al. Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms. Hum Mut 2004;23:343-57. 8. Zlotogora J. Parents of children with autosomal recessive diseases are not always carriers of the respective mutant alleles. Hum Genet 2004;114: 521-6. 9. Zielenski J. Genotype and phenotype in cystic fibrosis. Respiration 2000; 67:117-33. 10. Svensson AM, Chou LS, Miller CE, Robles JA, Swensen JJ, Voelkerding KV, et al. Detection of large rearrangements in the cystic fibrosis transmembrane conductance regulator gene by multiplex ligation-dependant probe amplification assay when sequencing fails to detect two disease-causing mutations. Genet Test Mol Biomark 2010; 14(2):171-4. 11. Dequeker E, Stuhrmann M, Morris MA, Casals T, Castellani C, Claustres M, et al. Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFR-related disorders—updated European recommendations. Eur J Hum Genet 2009;17:51-65.
Norek et al
CLINICAL AND LABORATORY OBSERVATIONS
August 2011
Table. Short tandem repeant markers used in this study Primer name D7S481i5 D7S481i3 D7S2497i5 D7S2497i3 D7S799i5 D7S799i3 D7S480i5 D7S480i3 D7S2462i5 D7S2462i3
Heterozygosity*
Primer sequence
Amplification temperature ( C)
Product size (bp)†
0.83
5’-6-FAM/GATTCTCATTCTCACCCCCA-3’ 5’-ATCCCCCACTGTCTCCAAAA-3’ 5’-6-FAM/CCTGGTCTGTTTCTACTGCG-3’ 5’-ATGGCTCTTTCTCTGTGGA-3’ 5’-6-FAM/ATACTACATTACAGACAC-3’ 5’-GGTCCAATCCACTCACTTCAT-3’ 5’-6-FAM/GCAGTTTCCCCCTCCCCA-3’ 5’-ATAGGGCTGGAGGGAGGAG-3’ 5’-6-FAM/GCACCACTACACTCCAGCC-3’ 5’-CTGAAGCCTACATAACACATAC-3’
62
196
53
163
51
163
62
113
53
155
0.85 0.87 0.86 0.84
Amplification temperature and product size are listed. *http://genecards.weizmann.ac.il/geneloc/index.shtml. †According to the reference sequence (NM_000492.3).
Novel de Novo Large Deletion in Cystic Fibrosis Transmembrane Conductance Regulator Gene Results in a Severe Cystic Fibrosis Phenotype
346.e1
C
ystic fibrosis (CF, MIM: 219700) is one of the most common life-shortening autosomal recessive disorders in the Caucasian population, affecting approximately 1 in 2500 newborns.1 The disease is caused by a dysfunction or a complete deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel (MIM: 602421) in the apical epithelial cell membrane. Since the identification of the CFTR gene in 1989, >1860 different molecular defects were recognized and submitted to Cystic Fibrosis Mutation Database (CFMD, http://www3.genet.sickkids.on.ca/cftr/ app). Most changes described are point mutations and small insertions or deletions. CFMD also lists approximately 48 cases of hereditary large genomic rearrangements, which account for 2.59% of all known CFTR mutations. De novo molecular alterations in the CFTR gene have been previously described, but are rare.2,3,4 Here, we present a novel de novo large deletion, c.1679+94_2619+986del8118 (NM_000492.3), spanning exons 12, 13, and 14a. This rearrangement was identified in a patient with CF who was of Polish origin, carrying the common pathogenic c.1521_1523delCTT mutation (F508del) on the maternal allele.
Methods A patient of Caucasian origin (Polish), aged 6 years, was born at the 40th week of gestation (body weight, 3600 g; length, 56 cm; 10 points on Apgar scale) to healthy, young, unrelated parents (mother 31 years old and father 32 years old). He has 4 siblings with no clinical evidence of CF. The patient was first hospitalized at the age of 2 weeks, when he had a paroxysmal, strenuous cough with no signs of respiratory tract infection. No improvement was observed after symptomatic treatment. At the age of 6 weeks, he was admitted to the hospital because of severe cough persistence and pneumonia. During this hospitalization, his condition gradually worsened with an intensifying paroxysmal dyspnea and a pertusis-like cough. Inflammatory thickening
CF CFMD CFTR PCR STR MLPA
Cystic fibrosis Cystic Fibrosis Mutation Database Cystic fibrosis transmembrane conductance regulator Polymerase chain reaction Short tandem repeat Multiplex Ligation-dependent Probe Assay
was diagnosed in the lung. A pharyngeal swap and trachea aspirate were culture particular Pseudomonas aeruginosa. On the basis of elevated sweat Cl levels (>100 mmol/L) measured with pilocarpine iontophoresis, CF was initially determined. P aeruginosa eradication was obtained after 2 months. As a consequence of severe loss of body weight (at birth, 3600 g; at the age of 3 months, 3000 g), hypoproteinemia, and difficulties with oral feeding, parenteral feeding was introduced through the period of 1.5 months, giving improvement of nutrition status and general condition of the patient. Pancreatic exocrine insufficiency was observed when the patient was 3 years of age. Fecal fat balance was 8 g per 24 hours and 14.19 g per 24 hours at 5 years of age, with the norm being 3.2 g per 24 hours. Liver status was also healthy, without signs of portal hypertension and normal ultrasound scanning liver images. Currently, at the age of 6 years, the patient’s body weight is in the 10th percentile. He remains on a high-calorie diet (Fantomalt, Protifar; Nutricia Nordica AB, Solna, Stockholm, Sweden), pancreatic enzyme (Lipancrea [Polfa Warszawa SA, Warsaw, Poland] 8500U/kg body weight/24 hr), and fatsoluble vitamin supplementation. He receives inhalations (Mucosolvan; Boehringer Ing Pharma GmbH & Co KG, Ingelheim am Rhein, Germany and Pulmozyme; Genentech, San Francisco, California) with physiotherapeutic procedures (bronchial drainage). Bronchial and pulmonary exacerbations are observed 3 to 5 times a year, but they are not lifethreatening and improve after oral antibiotic therapy. Mutational Screening of the CFTR Gene Genomic DNA from the patient and his relatives (parents and 4 siblings) was extracted from peripheral blood lymphocytes, with the method of Miller et al.5 Molecular analysis of the CFTR gene was carried out by using INNO-LiPA CFTR19 and INNO-LiPA CFTR17+Tn kits (Innogenetics NV, Gent, Belgium), sequencing of all 27 exons together with exon/intron junctions (ABI PRISM 3730, Applied Biosystems,
From the Institute of Mother and Child, Warsaw, Poland (A.N., M.S., A.S-T., K.W-T., M.J.); and Children’s Memorial Health Institute, Warsaw, Poland (H.D.) Supported by Polish Ministry of Science and Higher Education (grant NN401 129936). The authors declare no conflicts of interest. 0022-3476/$ - see front matter. Copyright ª 2011 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2011.04.022
343
THE JOURNAL OF PEDIATRICS
www.jpeds.com
Vol. 159, No. 2
Figure 1. Confirmation of c.1679+94_2619+986del8118 mutation. A, Results of MLPA analysis. Fragment of electropherogram displaying reference runs in lighter color (left) compared with the DNA sample of the patient (right). The deleted fragments are indicated by arrows. B, Results of long-range PCR reaction. The fragment of 964 bp depicts the mutated allele. C, Fragment of electropherogram showing the sequence of mutated allele.
Foster, California) and Multiplex Ligation-Dependent Probe Assay (MLPA P091; MRC-Holland, Amsterdam, The Netherlands and ABI PRISM 3730) were performed according to the manufacturer’s recommendations. Long-Range Polymerase Chain Reaction The reaction was carried out to multiply the CFTR gene fragment (sized 13 895 base pairs) containing the region from exon 11 to exon 14a with flanking sequences. The fragment was amplified with these primers: 5’-CCT TTC AAA TTC AGA TTG AGC ATA-3’ (forward) and 5’-CAA GTC TCC ACC AGA TTA ACT AGA-3’ (reverse). The polymerase chain reaction (PCR) protocol comprised 30 cycles of 98 C for 10 seconds and 68 C for 15 minutes. Before the first cycle, there was the initial denaturation step of 94 C for 1 minute, and after the last cycle, the extension step of 10 minutes at 72 C. After PCR, the reaction products were analyzed with agarose gel electrophoresis. Breakpoints Identification To specify the size of deletion and amplify only the fragment flanking the breakpoints, PCR in restricted conditions, with 344
the same primers as in long-range PCR, was performed: 35 cycles of 95 C for 30 seconds, 59 C for 30 seconds, 72 C for 60 seconds, the initial denaturation step of 95 C for 6 minutes, and the extension step for 7 minutes at 72 C. Finally, the reaction product was sequenced with 5’-GAT GTG CCT TTC AAA TTC AGA-3’ forward primer. Capillary electrophoresis was carried out by means of an automatic sequencer ABI PRISM 3730. The resulting sequences were analyzed with Mutation Surveyor Software version 3.1 (SoftGenetics, State College, Pennsylvania). Paternity Test A paternity test was carried out at the Department of Forensic Medicine, Warsaw Medical Academy. Isolated DNA of the patient and his parents was amplified with the use of PowerPlex 16 system (Promega, Madison, Wisconsin) and AmpFlSTR SEfiler Plus kit (Applied Biosystems) These 19 markers were tested: D3S13, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, vWa D8S1179, TPOX, FGA, D2S1338, D19S433, SE33. The products of short tandem repeat (STR) sets amplification reaction were then subjected to Norek et al
August 2011
CLINICAL AND LABORATORY OBSERVATIONS
electrophoresis in denaturing polyacrylamide gel with the use of ABI 377 (Applied Biosystems). Comparative analysis (patient-mother-father) of the particular STRs was performed. STR Loci Analysis Additionally, we designed microsatellites to confirm biparental inheritance of chromosome 7 in the proband and his siblings. These STR loci localized on chromosome 7 were analyzed: D7S480, D7S481, D7S799, D7S2462, and D7S2497. Selection criteria of STR markers were position on chromosome 7 and heterozygosity index >0.8, according to GeneCards (http://genecards.weizmann.ac.il/geneloc/index.shtml). Fragments were amplified with a pair of self-designed primers, from which the forward ones were marked with 6-FAM at 5’ end. Primers sequences are listed in the Table (available at www.jpeds.com). Gene scan electrophoresis was performed on an automatic sequencer ABI PRISM 3730.
Results At the age of 3 months, the patient was referred for CFTR molecular testing. Initial report indicated c.1521_1523delCTT in one CFTR allele. Molecular analysis with INNO-LiPA CFTR19 and INNO-LiPA CFTR17+Tn kits followed by sequencing of all 27 CFTR exons with exon/intron junctions did not establish a CF genotype in the patient. In the next step, according to our diagnostic procedure, MLPA analysis was carried out to look for any possible gene rearrangements. As a result, deletion of a large CFTR gene fragment including 3 exons, 12, 13, and 14a, was recognized (Figure 1, A). To verify MLPA reaction results, long-range PCR was carried out, and the deletion was confirmed (Figure 1, B). Sequencing of the 964 bp reaction product enabled the precise mapping of the breakpoints (Figure 1, C). To verify bi-parental inheritance of the CFTR identified defects and to determine the carrier status, molecular tests were performed for the proband’s relatives (Figure 2). The c.1521_1523delCTT mutation was identified in the mother and older brother in one of the CFTR alleles. The c.1679+94_2619+986del8118 rearrangement was not found in any allele of the biological father, mother, nor any of the siblings, suggesting that the mutation had arisen de novo. With paternity tests and STR loci analysis on chromosome 7, paternal origin of the mutated allele in the patient was unanimously confirmed. De novo large deletions in CFTR gene have not been described so far. To understand the mechanism of the de novo mutation presented here, we examined sequence architecture in the breakpoint’s proximity and scanned it for repetitive elements. We found no microhomology between the breakpoints (5’TATTCTGTTTCTGGAAT3’ and 5’CCACTCTGG CTGTGCG3’; deleted fragment underlined), which excluded the involvement of replication-based mechanisms of DNA repair.6 Subsequently, we used UCSC Genome Browser on Human Mar. 2006 (NCBI36/hg18) Assembly (http:// genome.ucsc.edu) to scan for repetitive elements, which established that the proximal breakpoint lays in the unique
Figure 2. Pedigree analysis of patient’s family showing the results of microsatellite haplotypes (D7S481, D7S2497, D7S799, D7S480, D7S2462) and the two mutations detected: m 1 (c.1521_1523delCTT) and m2 (c.1679+94_2619+986del8118). wt, wild type allele.
DNA sequence, whereas the distal one is positioned in the long-terminal repeat repetitive element (long-terminal repeat element 12C, family endogenous retroviral sequence 1). We also used the non-B DB database (http://nonb.abcc.ncifcrf. gov/) to predict possible alternative DNA structures. We found no architectural clues in the sequence, which might predispose for the emergence of this deletion.
Discussion Autosomal recessive disorders may arise although only one parent is a carrier. Our data demonstrate the appearance of a de novo deletion spanning the region from intron 11 to intron 14a in a Polish patient with CF. Mutations occurring de novo in CFTR are extremely rare. There have been 3 published cases of de novo mutations in CFTR gene to date: c.3197G>A (R1066H),2 c.2551C>T (R851X),3 and c.3194T>G (L1065R).4 All of them are point mutations situated at the mutational hot spot regions. The c.1679+94_2619+986del8118 mutation is a de novo large rearrangement in CFTR gene. We hypothesize that this deletion could appear as a result of non-homologous end joining taking place during the DNA double-strand breaks repair. Non-homologous recombination in the CFTR gene has been mentioned as a direct cause of 5 various CFTR gene rearrangements.7 In all published de novo mutations, similarly to ours, the new mutation had appeared on the paternal chromosome. Casals et al4 proposed that this phenomenon may reflect a higher mutation rate in paternal gametes. This tendency was noticed in several other genes (eg, fibroblast growth factor receptor 2).8
Novel de Novo Large Deletion in Cystic Fibrosis Transmembrane Conductance Regulator Gene Results in a Severe Cystic Fibrosis Phenotype
345
THE JOURNAL OF PEDIATRICS
www.jpeds.com
The deletion we describe includes amino acids from 561 to 873 (in frame), which correspond to partial loss of nucleotide-binding domain 1, transmembrane domain 2, and complete loss of regulatory (R-domain) domain.9 All these components have been shown to play a crucial role in CFTR channel function,1 which may explain the severity of CF symptoms in the patient. CFMD currently lists >1860 molecular defects. This case emphasizes the importance of comprehensive mutation screening of CFTR gene, especially in the cases in which a patient exhibits clinical manifestation of CF and only one mutated CFTR allele is identified. However, analyses of large CFTR gene rearrangements have only recently come in clinical practice and are not included in conventional CFTR mutation panels. Thus such mutations can easily escape detection and lead to confusion in genetic counseling.10 Definitive identification of both mutated CFTR alleles confirms the clinical diagnosis and facilitates patient treatment. Testing of proband’s parents and relatives is of importance for genetic counseling purposes and planning of consecutive pregnancies.11 Systematic segregation studies in CF would assist in detection of more de novo mutations than are currently described. n We thank Pawel Stankiewicz and Katarzyna Derwi nska for their bioinformatic support, Michal Milewski for his suggestions concerning methods in breakpoints identification, and Katarzyna Niepokoj and Aleksandra Susek for their technical assistance in microsatellite analysis. Submitted for publication Sep 8, 2010; last revision received Mar 22, 2011; accepted Apr 18, 2011. Reprint requests: Aleksandra Norek, PhD, Institute of Mother and Child, Department of Medical Genetics, Kasprzaka 17A, 01-211 Warsaw, Poland. E-mail: [email protected]
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References 1. Rowntree RK, Harris A. The phenotypic consequences of CFTR mutations. Ann Hum Genet 2003;67(5):471-85. 2. Cremonesi L, Cainarca S, Rossi A, Padoan R, Ferrari M. Detection of a de novo R1066H mutation in an Italian patient affected by cystic fibrosis. Hum Genet 1996;98:119-21. 3. White MB, Leppert M, Nielsen D, Zielenski J, Gerrard B, Stewart C, et al. A de novo cystic fibrosis mutation: CGA (Arg) to TGA (stop) at codon 851 of the CFTR gene. Genomics 1991;11:778-9. 4. Casals T, Ramos MD, Gimenez J, Larriba S, Nunez V, Estivill X. High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes. Hum Genet 1997;101:365-70. 5. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16(3):1215. 6. Stankiewicz P, Lupski JR. Structural variation in the human genome and its role in disease. Annu Rev Med 2010;61:437-55. 7. Audrezet MP, Chen J-M, Raguenes O, Chuzhanova N, Giteau K, Le Marechal C, et al. Genomic rearrangements in the CFTR gene: extensive allelic heterogeneity and diverse mutational mechanisms. Hum Mut 2004;23:343-57. 8. Zlotogora J. Parents of children with autosomal recessive diseases are not always carriers of the respective mutant alleles. Hum Genet 2004;114: 521-6. 9. Zielenski J. Genotype and phenotype in cystic fibrosis. Respiration 2000; 67:117-33. 10. Svensson AM, Chou LS, Miller CE, Robles JA, Swensen JJ, Voelkerding KV, et al. Detection of large rearrangements in the cystic fibrosis transmembrane conductance regulator gene by multiplex ligation-dependant probe amplification assay when sequencing fails to detect two disease-causing mutations. Genet Test Mol Biomark 2010; 14(2):171-4. 11. Dequeker E, Stuhrmann M, Morris MA, Casals T, Castellani C, Claustres M, et al. Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFR-related disorders—updated European recommendations. Eur J Hum Genet 2009;17:51-65.
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CLINICAL AND LABORATORY OBSERVATIONS
August 2011
Table. Short tandem repeant markers used in this study Primer name D7S481i5 D7S481i3 D7S2497i5 D7S2497i3 D7S799i5 D7S799i3 D7S480i5 D7S480i3 D7S2462i5 D7S2462i3
Heterozygosity*
Primer sequence
Amplification temperature ( C)
Product size (bp)†
0.83
5’-6-FAM/GATTCTCATTCTCACCCCCA-3’ 5’-ATCCCCCACTGTCTCCAAAA-3’ 5’-6-FAM/CCTGGTCTGTTTCTACTGCG-3’ 5’-ATGGCTCTTTCTCTGTGGA-3’ 5’-6-FAM/ATACTACATTACAGACAC-3’ 5’-GGTCCAATCCACTCACTTCAT-3’ 5’-6-FAM/GCAGTTTCCCCCTCCCCA-3’ 5’-ATAGGGCTGGAGGGAGGAG-3’ 5’-6-FAM/GCACCACTACACTCCAGCC-3’ 5’-CTGAAGCCTACATAACACATAC-3’
62
196
53
163
51
163
62
113
53
155
0.85 0.87 0.86 0.84
Amplification temperature and product size are listed. *http://genecards.weizmann.ac.il/geneloc/index.shtml. †According to the reference sequence (NM_000492.3).
Novel de Novo Large Deletion in Cystic Fibrosis Transmembrane Conductance Regulator Gene Results in a Severe Cystic Fibrosis Phenotype
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