Genomic Profiling of Congenital Laryngomalacia in Patients Undergoing Supraglottoplasty: A Single-Center Whole-Exome Sequencing Study

Abstract

Background: Laryngomalacia (LM) is a life-threatening condition in infants characterized by stridor. In this study, we aim to identify potential genetic variants associated with LM.

Methods: We conducted a retrospective analysis of whole-exome sequencing (WES) results from 55 LM patients who underwent supraglottoplasty at Shanghai Children’s Hospital between January 2021 and December 2023, and performed a systematic review of published literature on LM, screening for studies involving genetic abnormalities.

Results: We identified 15 likely pathogenic variants from 36 out of the 55 patients (65.45%). Among these 15 likely pathogenic variants, 10 variants were found in 2 or more patients, including 22q11.2 deletion (3 cases), CHD7 (2 cases), COL2A1 (2 cases), COL6A2 (6 cases), COL6A3 (2 cases), FLNB (4 cases), RYR1 (6 cases), KMT2D (2 cases), PLP1 (2 cases), and COLQ (2 cases).

Conclusion: We are the first to report COL6A2, COL6A3, COL2A1, RYR1, PLP1, and COLQ as likely candidate genetic variants for LM. In addition, CHD7-related disorders represent one of the most frequently reported genetic conditions associated with laryngomalacia.

Keywords

laryngomalacia / genetic / prevalence / CHD7 / 22q11.2

1 Introduction

Congenital laryngomalacia (LM) is a common pediatric airway disorder, characterized by airway obstruction due to prolapse of the supraglottic structures during inspiration, affecting up to 10,000 newborns annually. While most cases resolve spontaneously, 5%–20% of cases progress severely, affecting a baby’s breathing and feeding, and even threatening life ^[1].

The etiologic mechanism of LM is complex and remains incompletely understood. LM has been reported to occur in genetically defined disorders, such as 22q11.2 deletion syndrome and CHARGE syndrome (coloboma, heart defects, atresia choanae, retarded growth and development, genital abnormalities, and ear anomalies), which are established to be caused by 22q11.2 deletions and CHD7 mutations, respectively ^[2,^3].

However, these studies predominantly focused on syndromic or atypical features. In these contexts, LM represents part of the clinical phenotype in genetically defined disorders rather than an isolated condition under investigation. A systematic characterization of the genetic landscape of patients primarily diagnosed with LM is required. Whole-exome sequencing (WES) represents a powerful approach to identify genetic alterations associated with diseases^[2]. In this study, we retrospectively analyzed the WES results from 55 pediatric patients who were diagnosed with LM and underwent supraglottoplasty at Shanghai Children’s Hospital from January 2021 to December 2023, to describe the genetic variants identified in this cohort of patients with LM.

2 Materials and methods

2.1 Study population

A total of 55 pediatric patients with LM who underwent supraglottoplasty at Shanghai Children’s Hospital between January 2021 and December 2023 were included in this study. Supraglottoplasty was considered for LM patients who exhibited significant upper airway obstruction accompanied by feeding difficulties, failure to thrive, recurrent cyanotic or apneic episodes, or persistent/worsening respiratory symptoms despite conservative management.

2.2 Data collection

Electronic medical records, including gender, age at which WES was performed, findings on flexible fiberoptic laryngoscopy (FFL), clinical features, comorbidities, and the results of WES, were extracted for analysis. The diagnosis of laryngomalacia relied on both the presence of symptoms and findings on FFL. The type and grade of laryngomalacia were classified as reported previously^[3,^4]. Briefly, type I is characterized by prolapse of the arytenoid mucosa over the airway, type II by shortened aryepiglottic folds causing lateral collapse of the supraglottic structures, and type III by posterior displacement of the epiglottis during inspiration. WES was performed routinely as part of the clinical evaluation process for these patients in our institution.

2.3 Whole-exome sequencing process

WES results were provided by the Department of Laboratory Medicine, Shanghai Children’s Hospital. In short, blood samples were obtained from the patients who underwent supraglottoplasty in our hospital for whole-exome sequencing. After genomic DNA extraction, exon capture, and high-throughput sequencing, WES data were generated. Subsequent analyses were performed, including bioinformatic variant calling and annotation, followed by clinical interpretation according to American College of Medical Genetics and Genomics (ACMG) guidelines.

2.4 Ethics

This study was approved by the Institutional Review Board of Shanghai Children’s Hospital (Approval No. 2021RY024). Written informed consent was obtained from the parents or legal guardians of all patients.

2.5 Systematic review registration

This review was registered in PROSPERO (CRD42024525177) on March 27, 2024.

2.6 Search strategy

Two reviewers conducted a systematic review in PubMed, Web of Science, Embase and the Cochrane Library databases. Up until February 15, 2024, articles were searched for with the following terms: (e.g., gene, mutation, microarray, sequencing, syndrome, trisomy, deletion, duplication) AND (laryngomalacia). The reference lists of selected articles, other related studies, and review articles were checked for eligible studies. Authors were contacted if the study was unpublished or had inadequate data.

2.7 Study inclusion and exclusion criteria

Published studies that addressed the following question were included in the review: pediatric patients with genetic abnormalities who were found to have laryngomalacia. Case series, randomized clinical trials, and cohort studies reporting genetic disorders in which laryngomalacia was described as a clinical manifestation were included, whereas animal or laboratory studies, non-English-language articles, conference abstracts, and expert opinions were excluded.

2.8 Data extraction

For the comprehensive analysis, we extracted the first author, publication year, genetic risk factors examined, and the number of cases and participants from each eligible study. Data extraction was independently conducted by two researchers and discrepancies were resolved through discussion and consensus.

3 Results

3.1 Clinical characteristics and the WES results of our patients

A total of 55 patients were included in the study; 35 were males and 20 were females, markedly consistent with the male-to-female ratio reported in other LM cohort studies, near 1.6:1–2.3:1^[5]. Clinical characteristics of these patients were presented in Table 1. Briefly, the age of the study population ranged from 1 month to 12 months, with an average age of 6.1 months. The types of laryngomalacia were observed as follows: 36 cases of type I+II, 10 cases of type II, 6 cases of type II+III, and 3 cases of type I. Following the American College of Medical Genetics and Genomics (ACMG) criteria, among the 55 patients, we identified 35 likely pathogenic variants across 15 disease-related genes (22q11.2 deletion, CHD7, COL6A2, COL6A3, FLNB, RYR1, KMT2D, COLQ, COL2A1, 13q21.32 deletion, KAT6A, FRAS1, MUC5B, SMARCA4, PLP1). Among these 15 disease-related genes, 10 genes were observed in 2 or more patients: COL6A2 (6 cases),FLNB (4 cases), RYR1 (6 cases), 22q11.2 deletion(3 cases), CHD7 (2 cases), COL2A1 (2 cases), COL6A3 (2 cases), KMT2D (2 cases), PLP1 (2 cases), and COLQ (2 cases). Notably, variants in COL6A2, COL6A3, COL2A1, RYR1, PLP1, and COLQ were not previously described in similar patient cohorts. The detailed loci of mutations were presented in Table 1. The potential functional effects of these pathogenic variant genes were scrutinized by literature review and discussed in Table 2. Briefly, these genes have been validated to be related to muscle, cartilage, or neuromuscular junction development, supporting the etiologic theories that LM may be a consequence of altered or underdeveloped muscle, cartilaginous, or neural structures^[5]. These observations provide a descriptive overview of the genetic landscape in patients primarily diagnosed with LM.

3.2 Reported genetic abnormalities associated with congenital laryngomalacia

To further strengthen the evidence on the impact of gene mutations on the association of LM, we conducted a systematic search (Fig. 1). A total of 29 studies were obtained. The characteristics and outcomes of the included studies were presented in Suppl. Table 1. Briefly, the results showed that CHD7 mutations, 22q11.2 deletion syndrome, trisomy 13 or 18, and trisomy 21 were the most common genetic abnormalities that have been reported to be associated with congenital laryngomalacia. Due to insufficient patients for rare mutations, we focused on these four relatively common mutations. 20 studies were finally confirmed: 7 studies evaluated trisomy 21, 3 studies evaluated the CHD7 mutation, 7 studies evaluated the 22q11.2 deletion syndrome, and 3 studies evaluated the trisomy 13 or 18. Trisomy 21 had the highest number of cases (including 7 included studies, 889 cases). 22q11.2 deletion syndrome had the second highest number of LM cases (including 7 included studies, 185 cases). CHD7 mutations were the most frequently described genetic abnormalities associated with laryngomalacia. These findings support the potential contribution of genetic and developmental pathways to laryngomalacia. However, these studies primarily investigated the occurrence of laryngomalacia among patients with established genetic abnormalities, the prevalence of specific genetic abnormalities among patients with laryngomalacia remains to be determined in appropriately designed cohort studies.

The flow diagram illustrates the process of identifying, screening, and including studies in the literature identification. A total of 2,377 records were retrieved from databases and 29 studies met the inclusion criteria for the final analysis.

4 Discussion

In this study, we reviewed WES results from 55 patients with LM to comprehensively identify gene mutations that might be associated with LM. The male-to-female ratio in our cohort (including 35 males and 20 females) was markedly consistent with that reported in other LM cohort studies, near 1.6:1–2.3:1^[5]. It indicated a potential association between sex disparity and prevalence rates. However, among the 15 likely candidate genetic variants identified in 36 of 55 patients, there was no statistically significant difference in the distribution between sexes. Therefore, the underlying mechanisms driving this sex discrepancy require further investigation.

COL6A2/COL6A3 (8 cases) and RYR1 (6 cases) were the most frequently identified genes in our study. Previous studies have demonstrated that mutations in COL6 genes could cause early-onset progressive muscle weakness, with Ullrich congenital muscular dystrophy (UCMD) being the most severe form^[5]. Pathogenic variants in RYR1 could cause a spectrum of rare congenital myopathies, which are also characterized by muscle weakness^[16]. COLQ (2 cases) is also linked to muscle tone. Mutations in COLQ result in the extended residence of acetylcholine in the synaptic space, which leads to prolonged synaptic currents and action potentials and presents as muscle weakness^[17]. Our findings may provide a clue for the hypothesis that hypotonia contributes to LM by causing dynamic prolapse of tissue into the larynx during inspiration^[18]. However, standardized evaluations of systemic muscle tone were not performed for our cohort. Therefore, we cannot determine whether the affected patients exhibited generalized systemic hypotonia or if LM could be an isolated or initial manifestation of these neuromuscular disorders. Further studies incorporating systematic neuromuscular assessments are needed to clarify the genotype-phenotype correlations, which may provide the potential for early diagnosis.

The second most frequently identified gene group includes COL2A1 (2 cases) and FLNB (4 cases), both of which are involved in cartilage and skeletal development. COL2A1 mutations were reported to be responsible for chondrodysplasia^[19], and FLNB mutations cause skeletal disorders affecting chondrocyte development^[20]. These observations support the theory that LM may result from anatomical abnormalities of the laryngeal cartilage, weakening airway support and resulting in airway obstruction and stridor^[21].

We also found 7 variants in genes associated with syndromes, including CHD7 (2 cases), KMT2D (2 cases), and 22q11.2 deletion (3 cases). CHD7, an ATP-dependent chromatin remodeler, is mutated in CHARGE syndrome. Many previous studies have reported that LM was prevalent in CHARGE syndrome^[6,^7]. In our cohort of 55 patients, 2 patients were found to have CHARGE syndrome. Further investigation is needed to better define the link between CHARGE syndrome and LM. Mutations in KMT2D cause Kabuki syndrome, a congenital multisystem developmental disorder characterized by distinctive facial gestalt and skeletal abnormalities^[22]. To our knowledge, this is the first to report the presence of LM in Kabuki syndrome, expanding its phenotypic spectrum. 22q11.2 deletion causes 22q11.2 deletion syndrome, characterized by craniofacial defects, cardiovascular anomalies, velopharyngeal insufficiency, and skeletal muscle hypotonia^[23]. Previous reports have suggested that LM is an occasional finding in 22q11.2 deletion syndrome^[9]. However, three LM cases were found to have 22q11.2 deletion syndrome in our cohort, indicating that many LM cases present in a syndromic context, which may lead to underestimation of the actual prevalence of LM. The prevalence of LM varied across studies of 22q11.2 deletion syndrome, possibly because most affected children did not undergo routine FFL, resulting in a low detection rate. We encourage them to clarify whether the affected patients exhibited generalized systemic hypotonia or if LM could be an isolated or initial manifestation of these neuromuscular disorders. Addressing this would help clinicians better understand the genotype-phenotype correlations and the potential for early diagnosis.

In conclusion, we conducted a systematic analysis to explore genetic variants that might be associated with LM and identified variants in COL6A2, COL6A3, COL2A1, RYR1, PLP1, and COLQ in a subset of patients. Previous studies have suggested that abnormalities in these genes might cause muscle, bone and cartilage, nervous system, or neuromuscular junction abnormalities. However, standardized evaluations of systemic muscle tone, as well as other specific muscular, neuromuscular, skeletal, auditory, ocular, and craniofacial phenotypes were not performed for our cohort. Therefore, studies with functional validation and systematic whole-body evaluation are required to elucidate these genotype–phenotype correlations and facilitate early diagnosis in future work. These findings may provide deeper insights into the genomic landscape of LM and may help improve future diagnostic evaluation of LM patients.

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