ORIGINAL ARTICLE
The Clinical Respiratory Journal
A new compound heterozygous CFTR mutation in a Chinese family with cystic fibrosis Yingjun Xie1, Xueqiong Huang2, Yujian Liang2, Lingling Xu2, Yuxin Pei2, Yucai Cheng2, Lidan Zhang2 and Wen Tang2 1 Department of Prenatal Diagnosis, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China 2 Department of Pediatrics, Pediatric Intensive Care Unit, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China
Abstract Introduction: Cystic fibrosis (CF) is the most common autosomal recessive disease among Caucasians but is rarer in the Chinese population, because mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Objectives: To elucidate the causative role of a novel compound heterozygous mutation of CF. Materials and Methods: In this study, clinical samples were obtained from two siblings with recurrent airway infections, clubbed fingers, salt-sweat and failure to gain weight in a non-consanguineous Chinese family. Next-generation sequencing was performed on the 27 coding exons of CFTR in both children, with confirmation by Sanger sequencing. Results: Next-generation sequencing showed the same compound heterozygous CFTR mutation (c.865A>T p.Arg289X and c.3651_3652insAAAT p.Tyr1219X) in both children. Conclusions: As this mutation is consistent with the clinical manifestations of CF and no other mutations were detected after scanning the gene sequence, we suggest that the CF phenotype is caused by compound heterozygosity for c.865A>T and c.3651_3652insAAAT. As c865A>T is not currently listed in the “Cystic Fibrosis Mutation Database”, this information about CF in a Chinese population is of interest. Please cite this paper as: Xie Y, Huang X, Liang Y, Xu L, Pei Y, Cheng Y, Zhang L and Tang W. A new compound heterozygous CFTR mutation in a Chinese family with cystic fibrosis. Clin Respir J 2015; 00: 000–000. DOI:10.1111/crj.12401.
Key words CFTR – Chinese family – cystic fibrosis – compound heterozygous mutation – gene mutation – heterozygosity – next-generation sequencing Correspondence Wen Tang, MD, Department of Pediatrics, Pediatric Intensive Care Unit, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China. Tel: 186 020 87755766 ext. 8922 Fax: 101186-02-87333122 email: [email protected] Received: 30 May 2015 Revision requested: 30 September 2015 Accepted: 05 October 2015 DOI:10.1111/crj.12401 Authorship and contributorship Yingjun Xie and Xueqiong Huang contributed equally to the manuscript and should be considered co-first authors. Ethics Ethical approval was obtained for this study from the Ethics Committee of the First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). Conflict of interest All authors state no potential conflicts.
Introduction Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which most commonly occur among Caucasians of Northern European descent; an estimated 1 in 2500 Caucasian births are affected (1). Approximately 30 000 Americans have CF, with an estimated 1000 new cases diagnosed each year (Cystic Fibrosis Foundation. About Cystic Fibrosis: What you Need to Know.
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Available at http://www.cff.org/AboutCF/accessed 11 January 2010). It is characterised by nasal polyposis, recurrent pneumonia and bronchiolitis (2). To date, more than 1900 CFTR gene mutations have been identified (Cystic Fibrosis Mutation Database. http:// www.genet.sickkids.on). Mutations in the CFTR gene are responsible for the clinical presentation of CF (3). CFTR comprises 27 exons and encodes a membrane protein of 1480 amino
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Figure 1. (A) Chest CT of the elder sibling showing bilateral bronchiectasis and a thickened bronchus wall. (B) and (C) Chest CT of the younger sibling showing multiple bronchiectasis and a thickened bronchus wall. The pathological changes worsened in B. (D) Chest X-ray showing diffuse lesions in younger sibling.
acids (4). It is expressed on the apical surface of epithelial cells lining the sweat glands, pancreatic glands, intestines, airway and vas deferens (5). In addition to regulating chloride transport through adenosine triphosphate (ATP)-driven conformational changes, CFTR plays a role in the transport of sodium and water through the inhibition of epithelial sodium channel activity (6). In CF, dysfunctional CFTR protein leads to absent or decreased chloride secretion, increased sodium and water absorption and liquid depletion on the surface of airways (7). Although CF is common among Caucasians, the incidence of CF is extremely low in the Chinese population. To date, only 20 CF cases have been reported in Chinese people, and 15 of those cases were diagnosed through genetic tests (8–16). Seven of the fifteen cases presented with symptoms of sinusitis (8, 10, 12, 13) (46.7%), and pancreatic insufficiency was identified in five cases (9–11, 13, 15) (33.3%), which suggested phenotypic variations associated with geographic or ethnic variations. Here, we present a Chinese family in which two children carried the same compound heterozygous mutation (c.865A>T and c.3651_3652insAAAT) in CFTR, with clinical characteristics such as recurrent airway infections, hypoxemia and obstructive ventilatory impairment, sinusitis, clubbed fingers, white sweat 2
(white powder-like crystals of sweat on the surface of the skin) and steatorrhea. In this report, we elucidate the causative role of a novel compound heterozygous mutation in the pathogenesis of a severe form of CF with a review of previous reported cases.
Patients and methods Subjects The patient, a 15-year-old boy, developed a recurrent respiratory infection at approximately 1 month of age and manifested a productive cough of yellow-green sputum at age 2. At 7 years of age, he developed shortness of breath at rest, and he was diagnosed with bronchiectasis using a chest computed tomography (CT) scan at 10 years of age (Fig. 1D). He did not undergo further CT scans for economic reasons. Long-term yellow-green purulent nasal discharge and steatorrhea have been described, but pancreatic enzyme replacement was not performed, as the diagnosis of CF was uncertain at that time. White powder-like crystals were detected on the skin and hair roots after sweating. As there is no sweat chloride test available in many Chinese hospitals, sweat test values are not available. Phlegm sounds, but no rales, were present in the bilateral lungs. The heart and abdomen were normal, and clubbing of the fingers and toes was observed.
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Currently, the patient receives care in the home and relies on an extra oxygen supply of 2 L/min day and night. Physical examination revealed malnutrition, with a body weight of 33 kg (22.2 standard deviation), a height of 155 cm (23.3 standard deviation), and a weight for height value of 21.7 standard deviation. The sibling, a 12-year-old boy, complained of a frequent productive cough lasting for 4 months and shortness of breath for 1 month when he came to the hospital. His symptoms worsened despite treatment with mechanical ventilation, broad-spectrum antibiotics and bronchial lavage at several major local hospitals. The boy died of severe pneumonia at age 12. Pseudomonas aeruginosa was cultured from the sputum and bronchoalveolar lavage fluid. His parent revealed that he developed recurrent lower respiratory infections with yellowish-greenish sputum at 5 years of age. However, he was well developed and relatively healthy, with a body weight of 38 kg (20.8 standard deviation), a height of 155 cm (10.8 standard deviation) and a weight for height value of 22.1 standard deviation. Coarse crackles were audible in the lower chest, and clubbed fingers were observed. A chest CT scan obtained at 11 years of age revealed bronchiectasis, which was more severe than was observed 4 years ago (Fig. 1A–C). However, sweat test values, nasal potential difference values, lung function recordings and pancreatic function were not available for these two boys. Although CF is quite rare in China, based on the observation of classical respiratory symptoms and white sweat and the exclusion of chronic lung disease, the diagnosis of CF was suspected in the 15-year-old boy. The healthy non-consanguineous parents were both Cantonese and had no personal or family history of CF, following a healthy pregnancy and no prenatal complications. Ethical approval was obtained for this study from the Ethics Committee of the First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). All data were collected with the informed consent of the patients.
Next-generation sequencing Next-generation sequencing of the 27 coding exons of CFTR in both children was performed using standard protocols. The reference sequence (NM_000492) was obtained from GenBank (http://www.ncbi.nlm.nih. gov/gene/?term5NM_000492). Genomic DNA samples were extracted from peripheral blood using the Flexi Gene DNA Kit (Qiagen, USA), followed by hybridisation with the NimbleGen 2.1 probe sequence capture array to enrich for exonic DNA. The sequenc-
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ing data were captured and independently verified in each sample using the Illumina Hiseq 2000 platform (San Diego, CA). Raw image files were processed using Illumina Pipeline software (version 1.3.4) for base calling with the default parameters, and sequences were generated for each individual in the form of 90 bp reads.
Sanger sequencing To verify the DNA sequence variants in exons 7 and 22 detected by next generation sequencing (NGS), we amplified the target sites and flanking sequences of the patient’s DNA individually with specific primers designed using Primer 6.0. The forward (50 -TCTGGCACATAGGAGGCATT-30 ) and reverse (50 -AAATAACACCCTGGACCAACTAC-30 ) primers for exon 7 were predicted to generate a 517 bp product, whereas the forward (50 -CCCGACAAATAACCAAGTGACA-30 ) and reverse (50 -GATTCTGCTAACACATTGCTTCAG-30 ) primers for exon 22 were predicted to generate a 459 bp product. Sanger sequencing of both exons in the parents was performed according to the manufacturer’s instructions using the Big Dye Terminator Cycle Sequencing Kit (version 3.1) and analysed using a 3130xl Genetic DNA analyzer, ABI Prism.
Results The capture and sequencing of genomic DNA indicated that both children had the same mutations in the CFTR gene. Sanger sequencing confirmed that both brothers carried the same compound heterozygous mutation (c.865A>T and c.3651_3652insAAAT) in CFTR (Fig. 2A, B). Furthermore, sequencing of the parental DNA showed that the c.3651_3652insAAAT mutation was inherited from the father (Fig. 2D), whereas the c.865A>T mutation was inherited from the mother (Fig. 2C).
Discussion The CFTR genetic mutation analysis revealed a previously unreported compound heterozygous mutation. Normative population databases (e.g. 1000 Genomes SNP database and HapMap) were used for comparison, and this same mutation has not been previously observed in CF patients. The mutation identified in this study is associated with the clinical manifestations of CF, and no other mutations were detected after scanning the gene sequence. The non-sense mutation c.865A>T, because a thymine to adenine transversion, located at nucleotide 865 in exon 7 and called p.Arg289X, replaces the normal arginine at 289 with a terminal code. This 3
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Figure 2. (A, B) Next generation sequencing showing that both siblings carried the c.865A>T/c.3651_3652insAAAT CFTR mutation. (C) Sanger sequencing showing that the mother carried the c.865A>T CFTR mutation. (D) Sanger sequencing showing that the father carried the c.3651_3652insAAAT CFTR mutation.
mutation falls in the class I category and creates inframe stop signals termed premature termination codons (PTCs) located in the fourth transmembrane domain, which blocks protein production by causing the premature cessation of translation and accelerating mRNA degradation because non-sense-mediated interruption (17). Thus, most CFTR protein cannot reach
the plasma membrane, thus, losing the regulation of channel lining and pore function. Several studies have explored CFTR mutations in exon 7 (Table 1) involving non-sense and missense mutations. Some nonsense mutations disrupt protein production through PTC mutations, such as c.825C>G, c.828C>A and c.868C>T; some cases with non-sense mutation such
Table 1. Clinical and genetic manifestations in people with detected mutations in exon 7 cDNA name
Protein
Legacy name
Allele 2
Age
Clinical manifestations
Reference
c.825C>G c.828C>A c.868C>T c.769G>A c.772A>G c.794T>G c.829T>A c.842T>C
p.Tyr275X p.Cys276X p.Gln290X p.Glu257Lys p.Arg258Gly p.Met265Arg p.Trp277Arg p.Met281Thr
NA C276X Q290X E257K R258G M265R W277R M281T
NA NA F508del NA NA F508del F508del NA
NA NA 18Y NA NA 1D 11Y 38Y
Ron Agatep* Ferec et al.* Ferec et al.* Rohlfs* Mercier et al. (18) Schwarz et al.* Ramirez et al. (19) Casals et al.*
c.829T>A c.859A>T
p.Ile285Phe p.Asn287Tyr
I285F N287Y
1497G>A F508del
1Y 3.5Y
c.861C>G
p.Asn287Lys
NA
NA
NA
NA NA NA NA CBAVD MI Mild lung disease, PI Chronic pancreatitis, PS, sweat Cl was 62 mEq/L Chronic respiratory problems Rectal prolapse, PS, sweat Cl was 75/81 mEq/L NA
Schrijver et al. (20) Shrimpton et al. (21) *
Age, age at diagnosis; MI, meconium ileus; PS, pancreatic sufficiency; PI, pancreatic insufficiency; NA, not available; sweat Cl, sweat chloride level (mE/L). * Cited from CFTR mutations database (http://www.genet.sickkids.on.ca/cftr).
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Table 2. Clinical and genetic manifestations in people with detected mutations in exon 22 cDNA name
Protein
Legacy name
c.3657_3658 insA
p.Thr1220AsnfsX45 3789insA
c.3691delT
p.Ser1231ProfsX4
c.3617C>G c.3617C>A
p.Ser1206X p.Ser1206X
c.3618_3619 delAG c.3623delG
p.Gly1208ProfsX56 p.Gly1208AlafsX3
c.3627A>C
p.Gln1209His
c.3629T>A
p.Met1210Lys
c.3630G>A
p.Met1210Ile
c.3634G>A
p.Val1212Ile
c.3634G>T c.3648A>C c.3659C>T c.3659delC c.3664_3665insTCAA c.3674C>T
p.Val1212Phe NA p.Thr1220Ile p.Thr1220LysfsX8 p.Gly1222ValfsX44 p.Ala1225Val
Allele 2
Age
F508del
6Y
Clinical manifestations
Mild-moderate lung disease, PI, sweat Cl was 95 mEq/L 3821-3823del T S945L 29Y PS, sweat Cl was 60 mEq/L S1206X NA NA Healthy CF carrier S1206X(C>A) TG13-T5 adult Bronchiectasis, P. Aeruginosa infection, sinusitis, sweat Cl was 82/89 mEq/L 3750delAG NA NA NA 3755delG NA 3M Severe lung disease, PI NA # 8M Metabolic alkalosis, sweat Cl was 60 mEq/L M1210K R1066C 1Y PI, sweat Cl was 118 mEq/L M1210I NA NA Diffuse panbronchiolitis V1212I F508del 23Y Mild lung disease, P. Aeruginosa infection, CBAVD, sweat Cl was 90 mEq/L NA NA NA NA 3780 A/C NA adult Healthy CF carrier 3791C/T NA NA NA 3791delC NA NA PI NA 2789 1 5G>A NA PS NA NA NA NA
Reference Schaedel et al.*
Wong* Ferec et al.* Claustres M et al.*
Mercier et al.* Claustres et al.* *
Claustres et al.* Nukiwa et al.* Macek et al.*
* Picci* Ghanem et al.* Macek et al.* Ruslan et al.* Myrto Poulou et al.*
# p.Asp110Glu In cis, p.Ser737Phe in trans. Age, age at diagnosis; MI, meconium ileus; PS, pancreatic sufficiency; PI, pancreatic insufficiency; NA, not available; sweat Cl, sweat chloride level (mE/L). * Cited from CFTR mutations database (http://www.genet.sickkids.on.ca/cftr)
as 794T>G, 829T>A, 829T>A and 859A>T, compounded with the second mutation in allele 2, are associated with mild lung disease or chronic pancreatitis. Thus, mutation in exon 7 seems to induce clinically mild pancreatitis. The other allele (c.3651_3652insAAAT) in exon 22, in which four nucleotides (AAAT) are inserted between 3651 and 3652, causes a frame shift mutation. This mutation causes the substitution of a stop codon for a tyrosine (p.Tyr1219X), resulting in truncation of the CFTR protein by 1219 amino acids from the sec-
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ond nuclear binding domain (NBF2). As the NBF2 domain contains a number of highly conserved motifs predicted to bind and hydrolyze ATP, the c.3651_3652insAAAT mutation interrupts that process and alters the conductance of the chloride channel, suggesting that the mutation might cause an imbalance between salt and water (22, 23). However, previous reports of mutation in exon 22 (Table 2) show two healthy CF carriers with c.3617C>G and c.3648A>C. In this study, one of the probands did not manifest chronic steatorrhea, suggesting that a mutation in 5
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Table 3. Cystic fibrosis CFTR mutations and clinical manifestations in Chinese patients Case 1 2 3 4 5 6 7 8 9 10
Gender F F F M F M F F F F
Age 6 months 8 years 23 years 17 years 14 years 18 months 41 months 14 years 13 years 10 years
Family history #
1 PCM NA 1# 1# 1# NA NA NA NA
Sinusitis
Pancreatic insufficiency
CFTR mutation
Reference
NA NA 2 NA NA 1 NA 1 1 1
1 2 2 2 2 NA 1 2 NA 1
1898 1 5G>T/2215insG 1898 1 5G>T/1898 1 5G>T 451-458del8/3041G>A 1898 1 5G>T/2215insG 1898 1 5G>T/2215insG G151T/989-992insA R553X/R553X 699C>A/3821-3823delT 263T>G/2909G>A 3196C>T/R1066C
Wang et al. (15) Zielenskietal (26) Wagner et al. (14) Wu et al. (16) Wu et al. (16) Alper et al. (8) Chen et al. (9) Li et al. (12) Liu et al. (13) Liu et al. (13)
1, positive; 2, negative; 1#, sibling with similar symptoms; PCM, parental consanguineous marriage; F, female; M, male; NA, not available; Case 4 and case 5 is sibling.
exon 22 can present with symptoms on a spectrum from life threatening to nearly asymptomatic. There are different mutations in each exon, with a wide spectrum of clinical manifestations ranging from mild to severe disease. Some CFTR mutations are categorised as being associated with pancreatic insufficiency and other CFTR mutations with pancreatic insufficiency. However, additional measurements of pancreatic function are needed. In addition, geographic distribution, regional origin and consanguinity are the primary influences on the incidence of CF (24, 25). No definitive CF mutation has been identified in China because of the different genetic mutation spectrum and low incidence of CF (Table 3). The CFTR gene database revealed that the proportion of non-sense mutations accounts for 8.24% of all CFTR mutations (http://www.genet.sickkids.on.ca/cftr). G542X is the second most common CFTR mutation after F508del, and this mutation is prevalent in Hispanic but not in Chinese populations (27). However, Chinese patients often have severe lung disease, although most of them have normal pancreatic function; and, CF might be diagnosed late in these patients. Because the spectrum of mutations is different from that of the European and American populations, genetic screening in Chinese people tends to be negative. At the same time, although the Chinese incidence of CF disease is lower than the Caucasian incidence, the absolute value of the mutant alleles may be quite large. As a result, next-generation sequencing of the coding exons of CFTR would be the best choice for these patients (28). In conclusion, we report a compound heterozygous mutation (c.865A>T/c.3651_3652insAAAT) in CF patients. Although the incidence of CF is low, carriers of CF alleles have been identified in China. Additional genetic analyses are needed to understand the different 6
spectrum of mutations and clinical manifestations in Chinese CF patients compared with those of European and American patients.
Acknowledgements The authors would like to thank the family for their contribution to this study as well as all of the members of the exome capture group and the high-throughput sequencing facilities of BGI- Shenzhen.
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A new compound heterozygous CFTR mutation in a Chinese family with cystic fibrosis Yingjun Xie1, Xueqiong Huang2, Yujian Liang2, Lingling Xu2, Yuxin Pei2, Yucai Cheng2, Lidan Zhang2 and Wen Tang2 1 Department of Prenatal Diagnosis, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China 2 Department of Pediatrics, Pediatric Intensive Care Unit, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China
Abstract Introduction: Cystic fibrosis (CF) is the most common autosomal recessive disease among Caucasians but is rarer in the Chinese population, because mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Objectives: To elucidate the causative role of a novel compound heterozygous mutation of CF. Materials and Methods: In this study, clinical samples were obtained from two siblings with recurrent airway infections, clubbed fingers, salt-sweat and failure to gain weight in a non-consanguineous Chinese family. Next-generation sequencing was performed on the 27 coding exons of CFTR in both children, with confirmation by Sanger sequencing. Results: Next-generation sequencing showed the same compound heterozygous CFTR mutation (c.865A>T p.Arg289X and c.3651_3652insAAAT p.Tyr1219X) in both children. Conclusions: As this mutation is consistent with the clinical manifestations of CF and no other mutations were detected after scanning the gene sequence, we suggest that the CF phenotype is caused by compound heterozygosity for c.865A>T and c.3651_3652insAAAT. As c865A>T is not currently listed in the “Cystic Fibrosis Mutation Database”, this information about CF in a Chinese population is of interest. Please cite this paper as: Xie Y, Huang X, Liang Y, Xu L, Pei Y, Cheng Y, Zhang L and Tang W. A new compound heterozygous CFTR mutation in a Chinese family with cystic fibrosis. Clin Respir J 2015; 00: 000–000. DOI:10.1111/crj.12401.
Key words CFTR – Chinese family – cystic fibrosis – compound heterozygous mutation – gene mutation – heterozygosity – next-generation sequencing Correspondence Wen Tang, MD, Department of Pediatrics, Pediatric Intensive Care Unit, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China. Tel: 186 020 87755766 ext. 8922 Fax: 101186-02-87333122 email: [email protected] Received: 30 May 2015 Revision requested: 30 September 2015 Accepted: 05 October 2015 DOI:10.1111/crj.12401 Authorship and contributorship Yingjun Xie and Xueqiong Huang contributed equally to the manuscript and should be considered co-first authors. Ethics Ethical approval was obtained for this study from the Ethics Committee of the First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). Conflict of interest All authors state no potential conflicts.
Introduction Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which most commonly occur among Caucasians of Northern European descent; an estimated 1 in 2500 Caucasian births are affected (1). Approximately 30 000 Americans have CF, with an estimated 1000 new cases diagnosed each year (Cystic Fibrosis Foundation. About Cystic Fibrosis: What you Need to Know.
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Available at http://www.cff.org/AboutCF/accessed 11 January 2010). It is characterised by nasal polyposis, recurrent pneumonia and bronchiolitis (2). To date, more than 1900 CFTR gene mutations have been identified (Cystic Fibrosis Mutation Database. http:// www.genet.sickkids.on). Mutations in the CFTR gene are responsible for the clinical presentation of CF (3). CFTR comprises 27 exons and encodes a membrane protein of 1480 amino
1
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Figure 1. (A) Chest CT of the elder sibling showing bilateral bronchiectasis and a thickened bronchus wall. (B) and (C) Chest CT of the younger sibling showing multiple bronchiectasis and a thickened bronchus wall. The pathological changes worsened in B. (D) Chest X-ray showing diffuse lesions in younger sibling.
acids (4). It is expressed on the apical surface of epithelial cells lining the sweat glands, pancreatic glands, intestines, airway and vas deferens (5). In addition to regulating chloride transport through adenosine triphosphate (ATP)-driven conformational changes, CFTR plays a role in the transport of sodium and water through the inhibition of epithelial sodium channel activity (6). In CF, dysfunctional CFTR protein leads to absent or decreased chloride secretion, increased sodium and water absorption and liquid depletion on the surface of airways (7). Although CF is common among Caucasians, the incidence of CF is extremely low in the Chinese population. To date, only 20 CF cases have been reported in Chinese people, and 15 of those cases were diagnosed through genetic tests (8–16). Seven of the fifteen cases presented with symptoms of sinusitis (8, 10, 12, 13) (46.7%), and pancreatic insufficiency was identified in five cases (9–11, 13, 15) (33.3%), which suggested phenotypic variations associated with geographic or ethnic variations. Here, we present a Chinese family in which two children carried the same compound heterozygous mutation (c.865A>T and c.3651_3652insAAAT) in CFTR, with clinical characteristics such as recurrent airway infections, hypoxemia and obstructive ventilatory impairment, sinusitis, clubbed fingers, white sweat 2
(white powder-like crystals of sweat on the surface of the skin) and steatorrhea. In this report, we elucidate the causative role of a novel compound heterozygous mutation in the pathogenesis of a severe form of CF with a review of previous reported cases.
Patients and methods Subjects The patient, a 15-year-old boy, developed a recurrent respiratory infection at approximately 1 month of age and manifested a productive cough of yellow-green sputum at age 2. At 7 years of age, he developed shortness of breath at rest, and he was diagnosed with bronchiectasis using a chest computed tomography (CT) scan at 10 years of age (Fig. 1D). He did not undergo further CT scans for economic reasons. Long-term yellow-green purulent nasal discharge and steatorrhea have been described, but pancreatic enzyme replacement was not performed, as the diagnosis of CF was uncertain at that time. White powder-like crystals were detected on the skin and hair roots after sweating. As there is no sweat chloride test available in many Chinese hospitals, sweat test values are not available. Phlegm sounds, but no rales, were present in the bilateral lungs. The heart and abdomen were normal, and clubbing of the fingers and toes was observed.
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Currently, the patient receives care in the home and relies on an extra oxygen supply of 2 L/min day and night. Physical examination revealed malnutrition, with a body weight of 33 kg (22.2 standard deviation), a height of 155 cm (23.3 standard deviation), and a weight for height value of 21.7 standard deviation. The sibling, a 12-year-old boy, complained of a frequent productive cough lasting for 4 months and shortness of breath for 1 month when he came to the hospital. His symptoms worsened despite treatment with mechanical ventilation, broad-spectrum antibiotics and bronchial lavage at several major local hospitals. The boy died of severe pneumonia at age 12. Pseudomonas aeruginosa was cultured from the sputum and bronchoalveolar lavage fluid. His parent revealed that he developed recurrent lower respiratory infections with yellowish-greenish sputum at 5 years of age. However, he was well developed and relatively healthy, with a body weight of 38 kg (20.8 standard deviation), a height of 155 cm (10.8 standard deviation) and a weight for height value of 22.1 standard deviation. Coarse crackles were audible in the lower chest, and clubbed fingers were observed. A chest CT scan obtained at 11 years of age revealed bronchiectasis, which was more severe than was observed 4 years ago (Fig. 1A–C). However, sweat test values, nasal potential difference values, lung function recordings and pancreatic function were not available for these two boys. Although CF is quite rare in China, based on the observation of classical respiratory symptoms and white sweat and the exclusion of chronic lung disease, the diagnosis of CF was suspected in the 15-year-old boy. The healthy non-consanguineous parents were both Cantonese and had no personal or family history of CF, following a healthy pregnancy and no prenatal complications. Ethical approval was obtained for this study from the Ethics Committee of the First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). All data were collected with the informed consent of the patients.
Next-generation sequencing Next-generation sequencing of the 27 coding exons of CFTR in both children was performed using standard protocols. The reference sequence (NM_000492) was obtained from GenBank (http://www.ncbi.nlm.nih. gov/gene/?term5NM_000492). Genomic DNA samples were extracted from peripheral blood using the Flexi Gene DNA Kit (Qiagen, USA), followed by hybridisation with the NimbleGen 2.1 probe sequence capture array to enrich for exonic DNA. The sequenc-
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ing data were captured and independently verified in each sample using the Illumina Hiseq 2000 platform (San Diego, CA). Raw image files were processed using Illumina Pipeline software (version 1.3.4) for base calling with the default parameters, and sequences were generated for each individual in the form of 90 bp reads.
Sanger sequencing To verify the DNA sequence variants in exons 7 and 22 detected by next generation sequencing (NGS), we amplified the target sites and flanking sequences of the patient’s DNA individually with specific primers designed using Primer 6.0. The forward (50 -TCTGGCACATAGGAGGCATT-30 ) and reverse (50 -AAATAACACCCTGGACCAACTAC-30 ) primers for exon 7 were predicted to generate a 517 bp product, whereas the forward (50 -CCCGACAAATAACCAAGTGACA-30 ) and reverse (50 -GATTCTGCTAACACATTGCTTCAG-30 ) primers for exon 22 were predicted to generate a 459 bp product. Sanger sequencing of both exons in the parents was performed according to the manufacturer’s instructions using the Big Dye Terminator Cycle Sequencing Kit (version 3.1) and analysed using a 3130xl Genetic DNA analyzer, ABI Prism.
Results The capture and sequencing of genomic DNA indicated that both children had the same mutations in the CFTR gene. Sanger sequencing confirmed that both brothers carried the same compound heterozygous mutation (c.865A>T and c.3651_3652insAAAT) in CFTR (Fig. 2A, B). Furthermore, sequencing of the parental DNA showed that the c.3651_3652insAAAT mutation was inherited from the father (Fig. 2D), whereas the c.865A>T mutation was inherited from the mother (Fig. 2C).
Discussion The CFTR genetic mutation analysis revealed a previously unreported compound heterozygous mutation. Normative population databases (e.g. 1000 Genomes SNP database and HapMap) were used for comparison, and this same mutation has not been previously observed in CF patients. The mutation identified in this study is associated with the clinical manifestations of CF, and no other mutations were detected after scanning the gene sequence. The non-sense mutation c.865A>T, because a thymine to adenine transversion, located at nucleotide 865 in exon 7 and called p.Arg289X, replaces the normal arginine at 289 with a terminal code. This 3
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Figure 2. (A, B) Next generation sequencing showing that both siblings carried the c.865A>T/c.3651_3652insAAAT CFTR mutation. (C) Sanger sequencing showing that the mother carried the c.865A>T CFTR mutation. (D) Sanger sequencing showing that the father carried the c.3651_3652insAAAT CFTR mutation.
mutation falls in the class I category and creates inframe stop signals termed premature termination codons (PTCs) located in the fourth transmembrane domain, which blocks protein production by causing the premature cessation of translation and accelerating mRNA degradation because non-sense-mediated interruption (17). Thus, most CFTR protein cannot reach
the plasma membrane, thus, losing the regulation of channel lining and pore function. Several studies have explored CFTR mutations in exon 7 (Table 1) involving non-sense and missense mutations. Some nonsense mutations disrupt protein production through PTC mutations, such as c.825C>G, c.828C>A and c.868C>T; some cases with non-sense mutation such
Table 1. Clinical and genetic manifestations in people with detected mutations in exon 7 cDNA name
Protein
Legacy name
Allele 2
Age
Clinical manifestations
Reference
c.825C>G c.828C>A c.868C>T c.769G>A c.772A>G c.794T>G c.829T>A c.842T>C
p.Tyr275X p.Cys276X p.Gln290X p.Glu257Lys p.Arg258Gly p.Met265Arg p.Trp277Arg p.Met281Thr
NA C276X Q290X E257K R258G M265R W277R M281T
NA NA F508del NA NA F508del F508del NA
NA NA 18Y NA NA 1D 11Y 38Y
Ron Agatep* Ferec et al.* Ferec et al.* Rohlfs* Mercier et al. (18) Schwarz et al.* Ramirez et al. (19) Casals et al.*
c.829T>A c.859A>T
p.Ile285Phe p.Asn287Tyr
I285F N287Y
1497G>A F508del
1Y 3.5Y
c.861C>G
p.Asn287Lys
NA
NA
NA
NA NA NA NA CBAVD MI Mild lung disease, PI Chronic pancreatitis, PS, sweat Cl was 62 mEq/L Chronic respiratory problems Rectal prolapse, PS, sweat Cl was 75/81 mEq/L NA
Schrijver et al. (20) Shrimpton et al. (21) *
Age, age at diagnosis; MI, meconium ileus; PS, pancreatic sufficiency; PI, pancreatic insufficiency; NA, not available; sweat Cl, sweat chloride level (mE/L). * Cited from CFTR mutations database (http://www.genet.sickkids.on.ca/cftr).
4
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Table 2. Clinical and genetic manifestations in people with detected mutations in exon 22 cDNA name
Protein
Legacy name
c.3657_3658 insA
p.Thr1220AsnfsX45 3789insA
c.3691delT
p.Ser1231ProfsX4
c.3617C>G c.3617C>A
p.Ser1206X p.Ser1206X
c.3618_3619 delAG c.3623delG
p.Gly1208ProfsX56 p.Gly1208AlafsX3
c.3627A>C
p.Gln1209His
c.3629T>A
p.Met1210Lys
c.3630G>A
p.Met1210Ile
c.3634G>A
p.Val1212Ile
c.3634G>T c.3648A>C c.3659C>T c.3659delC c.3664_3665insTCAA c.3674C>T
p.Val1212Phe NA p.Thr1220Ile p.Thr1220LysfsX8 p.Gly1222ValfsX44 p.Ala1225Val
Allele 2
Age
F508del
6Y
Clinical manifestations
Mild-moderate lung disease, PI, sweat Cl was 95 mEq/L 3821-3823del T S945L 29Y PS, sweat Cl was 60 mEq/L S1206X NA NA Healthy CF carrier S1206X(C>A) TG13-T5 adult Bronchiectasis, P. Aeruginosa infection, sinusitis, sweat Cl was 82/89 mEq/L 3750delAG NA NA NA 3755delG NA 3M Severe lung disease, PI NA # 8M Metabolic alkalosis, sweat Cl was 60 mEq/L M1210K R1066C 1Y PI, sweat Cl was 118 mEq/L M1210I NA NA Diffuse panbronchiolitis V1212I F508del 23Y Mild lung disease, P. Aeruginosa infection, CBAVD, sweat Cl was 90 mEq/L NA NA NA NA 3780 A/C NA adult Healthy CF carrier 3791C/T NA NA NA 3791delC NA NA PI NA 2789 1 5G>A NA PS NA NA NA NA
Reference Schaedel et al.*
Wong* Ferec et al.* Claustres M et al.*
Mercier et al.* Claustres et al.* *
Claustres et al.* Nukiwa et al.* Macek et al.*
* Picci* Ghanem et al.* Macek et al.* Ruslan et al.* Myrto Poulou et al.*
# p.Asp110Glu In cis, p.Ser737Phe in trans. Age, age at diagnosis; MI, meconium ileus; PS, pancreatic sufficiency; PI, pancreatic insufficiency; NA, not available; sweat Cl, sweat chloride level (mE/L). * Cited from CFTR mutations database (http://www.genet.sickkids.on.ca/cftr)
as 794T>G, 829T>A, 829T>A and 859A>T, compounded with the second mutation in allele 2, are associated with mild lung disease or chronic pancreatitis. Thus, mutation in exon 7 seems to induce clinically mild pancreatitis. The other allele (c.3651_3652insAAAT) in exon 22, in which four nucleotides (AAAT) are inserted between 3651 and 3652, causes a frame shift mutation. This mutation causes the substitution of a stop codon for a tyrosine (p.Tyr1219X), resulting in truncation of the CFTR protein by 1219 amino acids from the sec-
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ond nuclear binding domain (NBF2). As the NBF2 domain contains a number of highly conserved motifs predicted to bind and hydrolyze ATP, the c.3651_3652insAAAT mutation interrupts that process and alters the conductance of the chloride channel, suggesting that the mutation might cause an imbalance between salt and water (22, 23). However, previous reports of mutation in exon 22 (Table 2) show two healthy CF carriers with c.3617C>G and c.3648A>C. In this study, one of the probands did not manifest chronic steatorrhea, suggesting that a mutation in 5
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Table 3. Cystic fibrosis CFTR mutations and clinical manifestations in Chinese patients Case 1 2 3 4 5 6 7 8 9 10
Gender F F F M F M F F F F
Age 6 months 8 years 23 years 17 years 14 years 18 months 41 months 14 years 13 years 10 years
Family history #
1 PCM NA 1# 1# 1# NA NA NA NA
Sinusitis
Pancreatic insufficiency
CFTR mutation
Reference
NA NA 2 NA NA 1 NA 1 1 1
1 2 2 2 2 NA 1 2 NA 1
1898 1 5G>T/2215insG 1898 1 5G>T/1898 1 5G>T 451-458del8/3041G>A 1898 1 5G>T/2215insG 1898 1 5G>T/2215insG G151T/989-992insA R553X/R553X 699C>A/3821-3823delT 263T>G/2909G>A 3196C>T/R1066C
Wang et al. (15) Zielenskietal (26) Wagner et al. (14) Wu et al. (16) Wu et al. (16) Alper et al. (8) Chen et al. (9) Li et al. (12) Liu et al. (13) Liu et al. (13)
1, positive; 2, negative; 1#, sibling with similar symptoms; PCM, parental consanguineous marriage; F, female; M, male; NA, not available; Case 4 and case 5 is sibling.
exon 22 can present with symptoms on a spectrum from life threatening to nearly asymptomatic. There are different mutations in each exon, with a wide spectrum of clinical manifestations ranging from mild to severe disease. Some CFTR mutations are categorised as being associated with pancreatic insufficiency and other CFTR mutations with pancreatic insufficiency. However, additional measurements of pancreatic function are needed. In addition, geographic distribution, regional origin and consanguinity are the primary influences on the incidence of CF (24, 25). No definitive CF mutation has been identified in China because of the different genetic mutation spectrum and low incidence of CF (Table 3). The CFTR gene database revealed that the proportion of non-sense mutations accounts for 8.24% of all CFTR mutations (http://www.genet.sickkids.on.ca/cftr). G542X is the second most common CFTR mutation after F508del, and this mutation is prevalent in Hispanic but not in Chinese populations (27). However, Chinese patients often have severe lung disease, although most of them have normal pancreatic function; and, CF might be diagnosed late in these patients. Because the spectrum of mutations is different from that of the European and American populations, genetic screening in Chinese people tends to be negative. At the same time, although the Chinese incidence of CF disease is lower than the Caucasian incidence, the absolute value of the mutant alleles may be quite large. As a result, next-generation sequencing of the coding exons of CFTR would be the best choice for these patients (28). In conclusion, we report a compound heterozygous mutation (c.865A>T/c.3651_3652insAAAT) in CF patients. Although the incidence of CF is low, carriers of CF alleles have been identified in China. Additional genetic analyses are needed to understand the different 6
spectrum of mutations and clinical manifestations in Chinese CF patients compared with those of European and American patients.
Acknowledgements The authors would like to thank the family for their contribution to this study as well as all of the members of the exome capture group and the high-throughput sequencing facilities of BGI- Shenzhen.
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