Beijing Key Laboratory for Genetics of Birth Defects, Beijing Pediatric Research Institute; MOE Key Laboratory of Major Diseases in Children; Genetics and Birth Defects Control Center, Beijing Children’s Hospital, Capital Medical University; National Center for Children’s Health, Beijing 100045, China 2 China National Clinical Research Center of Respiratory Diseases, Respiratory Department of Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing 100045, China 3 Henan Key Laboratory of Pediatric Inherited & Metabolic Diseases, Henan Children’s Hospital, Zhengzhou Hospital of Beijing Children’s Hospital, Zhengzhou 450000, China.
These authors contributed equally to this work. Chanjuan Hao, Ruolan Guo and Jun Liu should be considered joint first author.
The high clinical and genetic heterogeneity makes it difficult to reach a confirmative diagnosis of suspected pediatric respiratory inherited diseases. Many patients with monogenic respiratory disorders could be missed without genetic The high clinical and genetic heterogeneity makes it difficult to reach a confirmative diagnosis of suspected pediatric respiratory inherited diseases. Many patients with monogenic respiratory disorders could be missed without genetic testing. We performed a single-center study in Beijing Childrens Hospital to demonstrate the clinical utility of exome sequencing (ES) as a first-tier test by evaluating the diagnostic yields of ES for inherited diseases with respiratory symptoms. A total of 107 patients were recruited in this study. We identified 51 pathogenic or likely pathogenic variants in 37 patients by ES (with or without copy number variants sequencing). The overall diagnostic yield was 34.6% (37/107). The most frequent disorders in our cohort were primary immunodeficiency disease (PIDs) (18/37, 48.6%) and primary ciliary dyskinesia (PCD) (9/37, 24.3%). We further reviewed the directive outcomes of genetic testing on the 37 positive cases. Our study demonstrated the effectiveness of ES as a first-tier test in China for diagnosing monogenic diseases of the respiratory system. In the era of precision medicine, ES as a first-tier test can rapidly make a molecular diagnosis and direct the intervention of the positive cases in pediatric respiratory medicine. Graphical abstract
Among 107 patients with respiratory symptoms, We identified 51 pathogenic or likely pathogenic variants in 37 patients by ES (with or without copy number variants sequencing). Our study demonstrated the effectiveness of ES as a first-tier test in China for diagnosing monogenic diseases of respiratory system
Next-generation sequencing (NGS) is now being widely incorporated into genetic diagnosis of pediatric monogenic disorders (Hu et al., 2018; Meng et al., 2017; Scocchia et al., 2019; Yang et al., 2013). Compared with other medical disciplines, the application of genetic testing in respiratory medicine remains in its infancy. Nevertheless, advances in sequencing techniques and the knowledge on genetics and genomics have already promoted the applications of NGS in monogenic respiratory disorders such as primary immunodeficiency disease (PIDs) and primary ciliary dyskinesia (PCD) (Arts et al., 2019; Cifaldi et al., 2019; Fassad et al., 2020; Marshall et al., 2015; Rudilla et al., 2019). Mendelian disorders in respiratory medicine mainly consist of the following three categories: (i) pulmonary disorders, including airway disease, pulmonary parenchymal disease, and pulmonary vascular disease; (ii) sleep disorder; (iii) other monogenic diseases with respiratory system involvement, including PIDs, neuromuscular diseases, and inherited metabolic disease (Yao & Shen, 2017). The clinical manifestation of a majority of monogenic respiratory disorders is often nonspecific and overlaps with other common respiratory diseases including cough, asthma, pneumonia, bronchitis, bronchopneumonia and bronchiectasis. These atypical phenotypes pose a challenge to the early diagnosis and disease management. In this sense, to obtain a definite diagnosis in suspected cases can be laborious and, in many occasions, ineffective (Cifaldi et al., 2019; Damseh, Quercia, Rumman, Dell, & Kim, 2017; Fassad et al., 2020; Marshall et al., 2015; Rudilla et al., 2019). Exome sequencing (ES) or genome sequencing (GS) has shown advantages for expeditious detection of pediatric disorders (Sanford et al., 2019; Scocchia et al., 2019; Stavropoulos et al., 2016). However, the feasibility of NGS in pediatric respiratory diseases has not been systematically explored. In our cohort, 107 patients with suspected inherited diseases were recruited and diagnostic genomic testing was performed. Of the 107 patients tested, 34.6% (37/107) received a molecular diagnosis. The two most frequent monogenic respiratory disorders were PIDs and PCD. This is the first single center study on the clinical application of NGS to evaluate its feasibility in pediatric monogenic respiratory diseases in China, revealing the monogenic disease spectrum of hospitalized patients in respiratory medicine.
Materials and methods Editorial policies and ethical considerations
The study was approved by the institutional review board of the Hospital (BCH, Approval No. 2015-26). All the patients or legal guardians provided written informed consent for this study. For the patients undergoing ES tests, they or their legal guardians have the right to decide whether or not to be informed of the results of secondary findings.
Patients in this research were initially referred to the Department of Respiratory inclusion criteria were hospitalized patients, respiratory involvement, and suspected genetic disorders. The assessment for the option of genomic testing assays was carried out by a panel of trained physicians and geneticists at Beijing was performed for suspected monogenic disorders with respiratory system involvement. Parallel testing by ES and copy number variants sequencing (CNV-seq) were performed for suspected syndromic disorders, as the likelihood of a chromosomal condition was not excluded by the specialist team.
Exome sequencing and variant interpretation pipeline
Genomic DNA was isolated from peripheral blood obtained from the probands and their parents using a Gentra Puregene Blood Kit (QIAGEN, Hilden, Germany). Libraries of genomic DNA were captured using the Agilent Sureselect Human All Exon v6 kit (Agilent Technologies Inc. Mississauga, ON, Canada) and were sequenced on an Illumina NovaSeq. 6000 Analyzer (Illumina, San Diego, CA, USA) -bp paired-end runs. The exome sequencing resulted in over 12 Gb of clean data. The average sequencing depth was more than 100X. Quality score fulfilled Q20>95% and Q30>90%. Sequencing reads (fastq files) were aligned to the GRCh37/hg19 human reference genome sequence using Burrows-Wheeler Aligner (BWA) and bam files were created by Picard. Genome Analysis Toolkit (GATK) software was used to perform the variant calling. Variants were annotated and filtered by TGex (https://geneyx.com/geneyxanalysis/). The analysis regions included all exonic nucleotides and the 50-bp flanking intronic nucleotides. The mainly reference databases of TGex included population databases (dbSNP https://www.ncbi.nlm.nih.gov/snp/, 1000G https://www.internationalgenome.org/, gnomAD http://gnomad.broadinstitute.org/) and disease databases (HGMD https://portal.biobase-international.com/hgmd/, ClinVar https://www.ncbi.nlm.nih.gov/clinvar/, OMIM https://omim.org/; and MalaCards https://www.malacards.org/). All variants were classified according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) interpretation standards and guidelines (Richards et al., 2015). Copy number variants (CNVs) based on exome sequencing data were generated using the CNV detection program: CNVkit. The clinical significance of CNVs was analyzed and interpreted on the basis of Database of Genomic Variants, DECIPHER database, ClinVar, OMIM, and ClinGen (Clinical Genome Resource), and subsequently classified according to the ACMG and ClinGen recommendation (Kearney et al., 2011; Riggs et al., 2020).
Multiplex Ligation-dependent Probe Amplification (MLPA)
This article is protected by copyright. All rights reserved. Accepted Article Multiplex Ligation-dependent Probe Amplification (MLPA) For one or more exons deletion or duplication of gene level, we use MLPA to make a further verification of the copy number variations (CNVs). The MLPA probes, reaction and data analysis were performed according to the recommendation of the MRC-Holland protocol (www.mrcholland.com).
Parallel testing by exome sequencing and copy number variation sequencing
For the patients with suspected syndromic disorders, we performed parallel ES and CNV-seq. In this strategy, CNVs detection and calling were performed using our in-house pipeline. In brief, CNVs detection was based on two sequencing datasets. One dataset was derived from the sequencing of targeted exome library captured by the uniquely designed probes. The probes (iGeneTech, Beijing, China) were added per 50 bp in intergenic regions and intronic regions to better call CNVs from ES data. CNVkit was used to call the CNVs (>250kb) and the parameters were as default. The other part of data was derived from the directly sequencing of the library without capture. Each sample yielded one giga base (Gb) raw data. CNV analysis based on low-coverage genome sequencing (0.3X) was used to call the large CNVs and the healthy parents were used as the reference control samples. The procedure was described in details (Hu et al., 2020). The analysis and interpretation of CNVs have been described before and were carried out the same as described above.
Demographics and test settings
In the 107 patients, there were 73 males (68.2%) and 34 females (31.8%) (sex 5), with a median age of 15 months (ranging from 1 month to 18 years) at the time of sample collection. Detailed phenotypic data were collected and anonymously according to the standardized Human Phenotype Ontology terms. Of the 107 patients, 96 (89.7%) (mainly with respiratory system involvement) were performed with ES, with 94 receiving trio-ES and 2 (P54 and P106) proband-ES due to unavailability of their parental samples; 11 (10.3%) with multiple congenital malformations or syndromic phenotypes were performed with parallel tests of ES and CNV-seq (Figure 1). The overall average turnaround time (TAT) to issue an initial report was 4 weeks (ranging from 3~6 weeks).
Mutation types and inheritance
We identified 51 pathogenic or likely pathogenic variants in 37 patients (Table 1). The criteria for establishing a positive test included: (i) variants were classified by following the ACMG/AMP guidelines into pathogenic/likely pathogenic (P/LP) ones; (ii) variants were in line with the segregation law (a heterozygous P/LP variant was detected in the gene with dominant inheritance pattern; or homozygous/compound heterozygous P/LP variants were detected in the gene with recessive inheritance pattern) The overall diagnostic yield of 107 patients was 34.6% (37/107). Among these 37 cases, two of them (Patient 44 and Patient 73) have been previously described and published (Hu et al., 2019; Liu et al., 2019). Autosomal dominant (AD), autosomal recessive (AR), X-linked dominant (XLD) and X-linked recessive (XLR) disorders were observed in 12 (32.4%), 18 (48.6%), 1 (2.7%) and 6 (16.2%) patients, respectively (Figure 2A). 37.2% (19/51) SNVs and CNVs were not reported in previous literature or public databases. Of the 12 AD cases, 10 (83.3%) had de novo variants and 2 (Patient 98 and Patient 100, 16.7%) had inherited variants. Reviewing the family history of Patient 98, we found the father had recurrent epistaxis during childhood, which was a characteristic feature of ENG mutation. The father of Patient 100, with a mutation in SFTPC, has thus far experienced no symptoms, which is reasonable and acceptable as pulmonary surfactant metabolism dysfunction-2 (SMDP2) caused by SFTPC mutation has a highly variable phenotype and reduced penetrance. Among the 18 AR disorders, there were 4 (22.2%) cases of homozygosity, and 14 (77.8%) cases of compound heterozygosity. Of the 6 XLR cases, 1 (16.7%) variant was de novo and 5 (83.3%) were inherited from maternal carriers. The XLD de novo variant was identified in FLNA gene in Patient 12 (female). Two CNVs were observed in 2 individuals. The CNV detected in Patient 73 by parallel tests was de novo(Hu et al., 2019). Patient 6 had a homozygous ~200 kb deletion (ranging chr9: 214929-418085, GRCh37/hg19) encompassing exons 1~30 of DOCK8 gene that were inherited from the consanguineous parents. This deletion analysis was performed based on the ES reads depth data and further confirmed by MLPA (using the SALSA MLPA P385-A2 DOCK8 probe mix)
Precision diagnosis and its clinical management
Of the diagnosed disorders, PIDs was diagnosed in 48.6% (18/37) patients, which was the most frequent monogenic disorders in respiratory medicine. The second most frequent disorder was PCD accounting for 24.3% (9/37) of the patients, followed by 16.2% (6/37) syndromic disorders (Figure 2B). The molecularly definitive diagnosis was classified into four categories (Figure 2C). A, 21.6% (8/37) patients who had no clinical diagnosis due to nonspecific phenotypes obtained definitive diagnosis. Of these, five cases presented with multiple congenital malformations, one case was diagnosed as interstitial lung disease (ILD), one with achondroplasia, and one with platelet glycoprotein IV deficiency. B, 8.1% patients (3/37) changed the initial diagnosis after obtaining molecular diagnosis. C, 15 patients (40.5%) who were clinically diagnosed as PIDs were molecularly confirmed and classified into different subtypes, for which specific treatment options were considered. D, 11 patients whose previous diagnoses were confirmed according to genetic testing results, mostly as diagnoses of PCD (6/11). Molecular findings guided the clinicians to reach a definitive diagnosis, which is directive to the patient care and effective therapeutic measures. A subsequent change in clinical management based on the molecular diagnosis was shown in the following aspects: (i) treatment planning; (ii) further examination of respiratory system; (iii) referral for systemic evaluation; (iv) provision of appropriate genetic counseling (Table 1). Here, we list some examples. For 12 patients who were molecularly confirmed to have PIDs, bone marrow transplantation was a recommended treatment. Notably, Patient 53 with ELANE mutation that caused severe congenital neutropenia (OMIM: 202700) received hematopoietic stem cell transplantation. Patient 92 was a 5-month-old preterm-birth boy with bronchopneumonia, abnormal facial features, hydrocephalus and macrocephaly. Molecular testing identified a de novo PIK3CA mutation, and Cowden syndrome 5 (OMIM: 615108) was diagnosed (Orloff et al., 2013). Molecular findings guided the clinicians to reach a definite diagnosis, which is helpful to the proband’s care and the accumulation of knowledge of rare genetic diseases for clinicians. Patient 44, an 8-month-old boy, was referred to the clinic for recurrent fever. The patient presented mild to moderate anemia, severe neutropenia, elevated erythrocyte sedimentation rate and C-reactive protein. ES-based testing identified pathogenic compound heterozygous variants in LPIN2, which led to the diagnosis of Majeed syndrome (OMIM: 609628) (Liu et al., 2019). New subspecialist care and clinical management combined with hematology, even including additional diagnostic studies was initiated. Patient 100, a 4-month-old boy, was referred to the clinic for tachypnea after birth. Chest HRCT (high-resolution computed tomography) showed bilateral diffuse parenchyma infiltration. Molecular testing found a pathogenic variant in SFTPC, which was a mutational hotspot and resulted in pulmonary surfactant metabolism dysfunction 2 (OMIM: 610913). The molecular findings helped clinicians to identify and confirm specific genetic causes of interstitial lung disease, and provided the timely diagnosis by avoiding invasive lung biopsy. The timely diagnosis has allowed specific therapies and even referral for lung transplantation if indicated.
In the current study, we used exome sequencing (with or without CNV-seq) for molecular diagnosis of inherited disease with respiratory system involvement and identified monogenic disorders of 37 patients (with an overall diagnostic rate of 34.6%). Our study showed that the clinical heterogeneity and complex etiology of the respiratory disorders made ES a valuable first-tier diagnostic tool. Of the positive cases, the two most frequent monogenic disorders were PIDs and PCD respectively, accounting for 73.0% (27/37) of molecular diagnoses, which indicates the monogenic disease spectrum in the Department of Respiratory Medicine of Beijing Children’s Hospital. Although phenotyping is a key step before performing genetic testing, a complete description of the patient’s phenotypes is not easily accessible due to the clinical heterogeneity of the respiratory diseases. Among these monogenic diseases, PIDs are genetically and phenotypically heterogeneous disorders with over 300 candidate genes(Bousfiha et al., 2018). Furthermore, the correlation between the phenotype and genotype in PIDs is complicated. PIDs are grouped into 10 specific subtypes according to the International Union of Immunological Societies (IUIS) PID classification (Bousfiha et al., 2018). Patients with PIDs often require immediate clinical care to prevent recurrent severe infection which may lead to fatality (Picard et al., 2018). The clinical variable phenotype of PIDs makes confirmative diagnosis challenging. Misdiagnosis may delay immediate and precise clinical care. PCD is another rare monogenic disease associated with cilia dysmotility and caused by over 40 causative genes. Children affected by PCD have progressive respiratory disease characterized by bronchiectasis and impaired lung function (Horani, Ferkol, Dutcher, & Brody, 2016; Mitchison & Valente, 2017). The clinical and genetic heterogeneity of PCD makes the diagnosis difficult, despite the availability of sophisticated diagnostic technologies, including nNO (nasal nitric oxide), HSVA (high-speed video-microscopy analysis), TEM (transmission electron microscopy), IP (immunoprecipitation) of ciliary proteins, and genetic testing. The “gold standard” to diagnose PCD was considered to be TEM detection. However, ultrastructural defects were not shown for some patients with genetic diagnosis of PCD which may lead to misdiagnosis for these patients (Horani et al., 2013; Schwabe et al., 2008). The American Thoracic Society has recently released Practice Guidelines on the diagnosis of primary ciliary dyskinesia (Shapiro et al., 2018). The Practice Guidelines propose a diagnostic algorithm for suspected PCD patients. It is recommended that a panel of diagnostic tests is applied to diagnose PCD; however, considering the limitations of testing devices and local clinical expertise for TEM or nNO testing, genomic testing is a cost-effective and time-efficient test. It improves the diagnostic workflow, helping to diagnose PCD when TEM is not available and confirming PCD in patients with inconclusive nNO or TEM results. The guidelines conditionally recommend using panel, but our practice results showed that exome sequencing tests were more time-efficient as sequencing cost is decreasing dramatically. Our results manifested that genomic testing could be applied as a diagnostic alternative, especially in medical institutions with limited medical resources in which the other diagnostic strategies such as TEM equipment or expertise are not available (Rumman et al., 2017; Shoemark et al., 2017). Genetic testing not only provides a definitive diagnosis, but also extends beyond its diagnostic value, to the appropriate genetic counseling of the affected individuals and their family members. Therefore, we prefer to perform exome sequencing tests for suspected PCD, overcoming the pitfalls of other diagnostic measures. In our cohort, the patients with the following clinical presentation are usually considered to be appropriate for genetic testing: (i) family members with similar symptoms or suspected inheritance diseases; (ii) specific or severe clinical phenotype; (iii) multiple deformities; and (iv) exclusion of other factors (Boycott et al., 2015; Yao & Shen, 2017). Except for certain syndromes with respiratory system involvement, the majority of rare monogenic diseases are often difficult to be recognized by respiratory clinicians due to the short-term practice of medical genetics in China. According to our study, 16.2% (6/37) of our diagnosed patients did not receive a clinical diagnosis before the genetic testing. The ES-based genotype-testing approach retrospectively directed the clinical diagnosis without extensive clinical evaluation, enabling physicians to reach a definite diagnosis. One limitation of ES as a first-tier test is that it increases the complexity of data analysis and interpretation compared with panel sequencing methods. These complexities are shown in the following aspects: (i) the interpretation of secondary findings; (ii) the evaluation of de novo or null variants in the non-established disease genes; (iii) the increased number and interpretation of variants of uncertain significance (VUS) in genes related to the primary phenotype. Some measures have been taken to address the limitations. Genetic counseling was needed to inform probands and their parents and to address secondary findings and VUS variants. For the evaluation of genes of unknown significance, especially for suspected candidate genes, functional studies are required, although these are time-consuming and are unable to provide a timely molecular diagnosis. To date, we have found several suspected candidate genes whose pathological mechanisms were not yet clear. Functional studies are ongoing in our laboratory. The limitations of ES also include technical issues affecting sequencing depth and coverage, and bioinformatics challenges to identify CNVs based on ES data. In future work, we will define genes with inherited pulmonary conditions that are not well captured or sequenced at a low depth and further optimize the genomic sequencing pipeline, to reach a higher diagnostic rate. For undiagnosed samples, further investigation is required to be taken into account: (i) to use RNA-seq and genome sequencing to identify the putative pathogenic variants located in non-coding regions (Cummings et al., 2017). (ii) to use a new algorithm to re-analyze CNV variants (Gross et al., 2019). (iii) to re-analyze ES data due to follow-up phenotypes and updated genetic disease databases (Ewans et al., 2018). (iv) to identify genes with poor capture and low coverage during ES due to special gene structure, and fill the gap with Sanger sequencing. According to our survey, the diagnostic yield of patients aged 0-18 m and over 18 m was 29.1% (16/55) and 40.4% (21/52), respectively. We speculate that the gradual evolution of the phenotypes over time increased the diagnostic yield of genetic testing. The yield will be higher and genetic diagnostic results more successful if a more detailed clinical phenotype is available. To the best of our knowledge, this is the first single-center study to investigate the genomic testing of inherited pediatric respiratory diseases in China. While a comprehensive and empirical clinical evaluation is critical, the power of ES-based genomic testing is indispensable in expediting the accurate diagnosis for genetic diseases, particularly for those with heterogeneous and atypical clinical manifestations. What’s more, the increment of approximately 250 new mendelian genes each year (Chong et al., 2015), the emergence of novel phenotypes, and the lack of accessibility to other testing techniques all add further difficulties to the disease diagnosis. Our study suggests that ES as a first-tier test can rapidly identify molecular defects and provide a helpful diagnostic strategy for further individualized patient care and personalized genetic counseling in pediatric respiratory medicine.
CJ Hao, BP Xu and W Li conceived and designed the study. J Liu, Y Yao, ZP Zhao, J Yin, LQ Chen and H Wang recruited their respective patients to this study and provided clinical data regarding their patients. RL Guo, XY Hu, J Guo and Z Qi contributed to the analysis of sequencing data. CJ Hao, RL Guo and J Liu were involved in manuscript editing. All authors reviewed and approved the final version.
This work was partially supported by grants from the Ministry of Science and Technology of China (2016YFC1000306), the Beijing Municipal Science and Technology Commission Foundation (Z181100001918003), the Beijing Municipal Commission of Health and Family Planning Foundation (2018-2-1141, 2020-4-1144) and the Special Fund of the Pediatric Medical Coordinated Development Center of Beijing Hospitals Authority (XTCX201807), Beihang University & Capital Medical University Advanced Innovation Center for Big Data-Based Precision Medicine Plan (BHME-201905).
Web resources Bioinformatics databases and tools utilized for exome sequencing interpretation.
Online Mendelian Inheritance in Man, OMIM. URL: https://omim.org/ ClinVar. URL: https://www.ncbi.nlm.nih.gov/clinvar/ Human Gene Mutation Database, HGMD. URL: https://portal.biobase-international.com/hgmd/ MalaCards: The human disease database. URL: https://www.malacards.org/ GnomAD Browser. URL: http://gnomad.broadinstitute.org/ 1000 Genomes. URL: https://www.internationalgenome.org/ dbSNP. URL: https://www.ncbi.nlm.nih.gov/snp/ PolyPhen-2. URL: http://genetics.bwh.harvard.edu/pph2 SIFT. URL: http://sift.bii.a-star.edu.sg/ CADD. URL: https://cadd.gs.washington.edu/ MutationTaster. URL: http://www.mutationtaster.org/ TGex. URL: https://geneyx.com/geneyxanalysis/
Figure 1. Flow diagram of the number of patients who were enrolled and received genetic testing by ES or parallel tests of ES and CNV-seq. * represented multiple congenital malformations or syndromic phenotypes. ** CNVs detection and calling were performed using in-house pipeline, see Methods. Abbreviations: CNV-seq, copy number variants sequencing; ES, exome sequencing; MLPA, multiplex ligation-dependent probe amplification; RT-PCR, reverse transcription polymerase chain reaction; SNV, single nucleotide variation; Indel, small insertion and deletion.
Figure 2. Characteristics of inheritance patterns, distribution of the disease types and clinical effects in 37 cases who received the molecular diagnoses. (A) Proportion of inheritance patterns. (B) Distribution of the disease types. (C) Distribution of the effects of molecular diagnoses by NGS-based first-tier testing. Category A, to produce new clinical diagnoses and guide the etiology. Category B, to change initial diagnoses and identify specific mendelian disease. Category C, to confirm and further reclassify into specific subtypes. Category D, to confirm the clinical diagnoses. Abbreviations: AD, autosomal dominant; AR, autosomal recessive; ILD, interstitial lung disease; NGS, next-generation sequencing; PCD, primary ciliary dyskinesia; PIDs, primary immunodeficiency disorders; XLD, X linked dominant; XLR, X linked recessive
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