An Effective and Universal Long-Read Sequencing-Based Approach for SMN1 2 + 0 Carrier Screening through Family Trio Analysis

Abstract

Background

Population-wide carrier screening for spinal muscular atrophy (SMA) is recommended by professional organizations to facilitate informed reproductive options. However, genetic screening for SMN1 2 + 0 carriers, accounting for 3%–8% of all SMA carriers, has been challenging due to the large gene size and long distance between the 2 SMN genes.

Methods

Here we repurposed a previously developed long-read sequencing-based approach, termed comprehensive analysis of SMA (CASMA), to identify SMN1 2 + 0 carriers through haplotype analysis in family trios (CASMA-trio). Bioinformatics pipelines were developed for accurate haplotype analysis and SMN1 2 + 0 deduction. Seventy-nine subjects from 24 families composed of, at the minimum, 3 were enrolled, and CASMA-trio was employed to determine whether an index subject with 2 SMN1 copies was a 2 + 0 carrier in these families. For the proof-of-principle, SMN2 2 + 0 was also analyzed.

Results

Among the 16 subjects with 2 SMN1 copies, CASMA-trio identified 5 subjects from 4 families as SMN1 2 + 0 carriers, which was consistent with pedigree analysis involving an affected proband. CASMA-trio also identified SMN2 2 + 0 in six out of 43 subjects with 2 SMN2 copies. Additionally, CASMA-trio successfully determined the distribution pattern of SMN1 and SMN2 genes on 2 alleles in all 79 subjects.

Conclusions

CASMA-trio represents an effective and universal approach for SMN1 2 + 0 carriers screening, as it does not reply on the presence of an affected proband, certain single-nucleotide polymorphisms, ethnicity-specific haplotypes, or complicated single-nucleotide polymorphism analysis across 3 generations. Incorporating CASMA-trio into existing SMA carrier screening programs will greatly reduce residual risk ratio.

Introduction

Spinal muscular atrophy (SMA; OMIM#253300) is a severe autosomal recessive neuromuscular disease characterized by progressive symmetrical limb and trunk muscle weakness due to progressive degeneration and loss of motor neurons. The genetic basis of SMA involves biallelic pathogenic variants including copy number loss and intragenic variants in the survival motor neuron 1 (SMN1) gene, with the copy number of the survival motor neuron 2 (SMN2) gene modifying disease severity (1). SMN1 and SMN2 are 28-kb highly homologous genes, respectively, located in 2 duplicated 500-kb DNA segments on chromosome 5 in an area prone to rearrangements and deletions (2). Notably, a single paralogous sequence variant at c.840 in exon 7 results in altered SMN2 splicing and reduced production of functional protein (3).

SMA poses a significant health burden, with an incidence of approximately 1 in 10 000 live births and carrier frequencies of 1/35 to 1/117 in various ethnic groups (4). Due to the high incidence rate and disease severity, population-wide SMA carrier screening is recommended by the American College of Medical Genetics and Genomics (ACMG) and the American College of Obstetricians and Gynecologists to facilitate informed reproductive options (5–7). Among SMA carriers, approximately 90%–95% exhibit a single copy of SMN1 (1 + 0), while 2% carry an allele with an intragenic SMN1 variant (1 + 1^D and 2 + 1^D), and the additional 3%–8% are silent (2 + 0) carriers (4, 8, 9). Various dosage-sensitive methods, including quantitative PCR (10), multiplex ligation-dependent probe amplification (MLPA) (11, 12), digital PCR (13, 14), TaqMan quantitative technology (15), and next-generation sequencing (16), have been employed for the screening of SMN1 1 + 0 carriers. However, these methods use the copy number of c.840C on exon 7 as a proxy for SMN1 and may generate false-positive results in individuals who have intragenic deletions (17–19). In terms of SMN1 intragenic variants, techniques such as allele-specific reverse transcription PCR or long-range (LR) nested PCR plus Sanger sequencing have been utilized. However, these methods are labor-intensive and not feasible for large-scale carrier screening (20–22). Recently, we have developed a feasible long-read sequencing (LRS) approach termed comprehensive analysis of SMA (CASMA), which can simultaneously screen for SMN1 1 + 0, 1 + 1^D, and 2 + 1^D carriers by analyzing full-length SMN1 (23).

SMN1 2 + 0 carriers were first deduced in families that had an affected child and one parent with 2 SMN1 copies (9, 24). However, this approach relies on the presence of an SMA proband, making it impractical for carrier screening. An alternative approach, using short tandem repeat linkage, enables the determination of SMN1 2 + 0 carriers through haplotype analysis across 3 generations of family members or 2 generations of multichild families (25). However, the clinical feasibility of this approach is limited, due to the requirement of multiple family members and the complexity of short tandem repeat analysis. Another approach to screen for a SMN1 2 + 0 carrier is to detect the copy number of SMN1 in semen samples by digital PCR, but it is not applicable for screening female silent carriers (26). In Ashkenazi Jewish and African American ethnicities, the single-nucleotide polymorphism (SNP) g.27134T > G has been identified to be tightly linked to SMN1 duplication alleles (27). A recent study using whole-genome LRS further defined the specific haplotypes containing the SNP g.27134T>G that had high correlation with SMN1 duplication alleles in African ethnicity (28). An updated ACMG practice guideline recommended the analysis of this SNP for enhanced SMA carrier screening (29). However, g.27134T>G had weak linkage to SMN1 duplication alleles in other ethnicities such as Caucasian, Hispanic, and Asian (16, 25, 27, 28). The LRS-based CASMA assay identified signature variants downstream of the SMN1 gene (fusion of SMN1 gene and SMN2 downstream sequences) in duplication alleles, which could represent a universal approach to identify SMN1 2 + 0 carriers that result from conversion of SMN2 to SMN1 (23). In our pilot study, this approach demonstrated approximately 50% sensitivity in identifying SMN1 2 + 0 carriers. However, it also identified the 2 + 0 pattern in 5.0% of deletion alleles and in 7.4% of normal alleles, indicating a low positive predictive value and limited clinical feasibility for SMN1 2 + 0 carrier screening. Consequently, an effective and universal approach for comprehensive SMA carrier screening including SMN1 2 + 0 is still lacking.

Taking advantage of long sequencing reads, CASMA can obtain the haplotypes of full-length SMN1, which could potentially be used for deducing 2 + 0 carriers through family trio analysis. To test the hypothesis, 24 families of least 3 were enrolled and subjected to CASMA assay (CASMA-trio), then an in-house developed bioinformatics pipeline was employed to determine whether an index subject with 2 SMN1 copies was a 2 + 0 carrier. Additionally, for the proof-of-principle, the possibilities of SMN2 2 + 0 were also analyzed in these families. Moreover, the distribution of SMN1 and SMN2 haplotypes on 2 alleles were determined in all the subjects.

Materials and Methods

Study Subjects

A total of 79 subjects from 24 families were retrospectively enrolled in the study, including 16 subjects with 2 copies of SMN1, and 43 subjects with 2 copies of SMN2. Among them, 5 individuals from 4 families were identified as SMN1 2 + 0 carriers through genotypic analysis of family members with an affected proband. The SMN1 and SMN2 copy numbers were determined with the SALSA MLPA Kit P060-B2 and Coffalyser software (MRC-Holland). All the families included in this study met the following criteria: (a) each family consisted of at least a trio (father, mother, and child); and (b) at least one member of the trio had 2 copies of SMN1 or SMN2 for analysis of SMN1 2 + 0 or SMN2 2 + 0, respectively. The samples used in this study were previously collected and analyzed by CASMA assay (23). This study was performed with the approval of the Ethics Review Committee of International Peace Maternity and Child Health Hospital (GKLW2022-13). Written informed consent was obtained from all the subjects or their legal guardians.

Full-Length Sequencing of SMN1/2 by CASMA Assay

Long-range PCR, library preparation, and LRS were performed as previously described (23). Briefly, the full-length (SMN1/2-FL, 28.5-kb) and downstream regions (SMN1/2-D, 26.1-kb) of SMN1 and SMN2 genes were amplified from genomic DNA samples. The amplified products were then ligated to unique PacBio barcoded adaptor by one-step end-repair and ligation, digested with exonucleases to removed failed ligation products, and followed by purification, quantification, and pooling to form single-molecule real-time dumbbell (SMRTbell) library. The SMRTbell library was prepared using the Sequel II Binding Kit v.3.2 (Pacific Biosciences), and then sequenced on the Sequel IIe platform (Pacific Biosciences) for 30 hours using the circular consensus sequencing (CCS) mode.

PacBio Data Analysis for SMN1/2 Haplotype and Copy Number

The sequencing reads were processed and underwent quality control as previously described except for haplotype analysis (23). The raw subreads were processed to obtain high-quality CCS reads, debarcoded to individual samples, and then aligned to the hg38 reference build using the software suite (smrtlink 10.1.0.119588, Pacific Biosciences). The SMN1 and SMN2 genes were discriminated by the functional paralogous sequence variant at c.840. For haplotype analysis, each CCS read was aligned to the reference gene in order to detect any SNPs that were present. SNPs present in a repeat region were masked. SNPs with a variant allele frequency <20% or >80% were filtered to generate a SNP matrix that had SNPs on each CCS read. The SNPs were recursively used to divide the CCS reads into 2 groups until further division was not possible. Each final group was a specific haplotype and the read numbers of each haplotype were counted. The copy numbers of SMN1/2 were calculated using a Poisson distribution-based caller with haplotype number and read count as input, as previously reported (23).

SMN1 or SMN2 2 + 0 Analysis through the CASMA-trio Pipeline

A family trio with father-mother-child was required for SMN1 or SMN2 2 + 0 analysis. The individuals who had 2 copies of SMN1 or SMN2 were used to perform 2 + 0 analysis and were referred as index subjects. The pipeline to determine whether an index subject was a SMN1 2 + 0 carrier is displayed in Fig. 1. SMN-FL haplotypes were used in the pipeline. However, in rare cases where both parents shared the same SMN-FL haplotype, the SMN-D haplotypes were then employed. If the index subject was the child, there were 2 possible scenarios: (a) the SMN1 haplotypes of the index subject were inherited from only one parent, then the index subject was a SMN1 2 + 0 carrier; and (b) the SMN1 haplotypes of the index subject were inherited from both parents, then the index subject was not a SMN1 2 + 0 carrier. If the index subject was a parent, there were 3 possible scenarios: (a) all or (b) no SMN1 haplotypes of the index subject were inherited by the child, then the index subject was a SMN1 2 + 0 carrier; or (c) only part of the SMN1 haplotypes of the index subject were inherited by the child, then the index subject was not a SMN1 2 + 0 carrier. The analysis of SMN2 2 + 0 followed a similar pipeline as SMN1 2 + 0. Additionally, for index subjects with 3 copies of SMN1 or SMN2, the possibility of 3 + 0 was also determined with the same pipeline.

Results

Applying CASMA-trio to Analyze SMN1 and SMN2 Distribution Pattern

To validate the hypothesis that CASMA-trio could be used for SMN1/2 2 + 0 screening through haplotype analysis, 79 subjects from 24 families were enrolled and subjected to the CASMA assay. The SMN1 and SMN2 copy numbers identified by CASMA was 100% concordant with MLPA (Table 1). For each index subject with 2 copies of SMN1 or SMN2, CASMA-trio bioinformatics pipeline analysis was performed to determine the distribution pattern of the 2 SMN1 or SMN2 copies (Fig. 1). The CASMA analysis was able to construct the haplotype of the full-length SMN genes by analyzing the SNPs (Supplemental Tables 1 and 2). Among the 16 subjects from 13 families that had 2 SMN1 copies, CASMA-trio identified SMN1 2 + 0 in 5 subjects from 4 families, which was concordant with the pedigree analysis involving an affected proband (Table 1). Similarly, among the 43 subjects from 21 families that had 2 SMN2 copies, CASMA-trio identified the distribution pattern of SMN2 2 + 0 in 6 subjects from 5 families (Table 1). Furthermore, the distribution pattern of SMN1 and SMN2 genes on 2 alleles was successfully determined by CASMA-trio for all the 79 subjects. Given that most individuals had 3–4 SMN copies in total and 1–2 SMN copies on each allele (30), allelic analysis of SMN2 could provide reinforcement for the SMN1 distribution analysis. Indeed, for the 9 subjects with 2 SMN1 copies and 2 SMN2 copies, 8 exhibited SMN2 1 + 1 and one displayed SMN2 0 + 2, which correlated with SMN1 1 + 1 and SMN1 2 + 0, respectively. Among the other 7 subjects with 2 SMN1 copies and only one SMN2 copy, 3 had SMN2 and one copy of SMN1 located on the same allele, and 4 had private SMN2 on one allele, providing further support for SMN1 1 +1 and SMN1 2 + 0, respectively.

In family F01, the father-mother-son trio all had 2 SMN1 and 2 SMN2 copies, which were distinguished by 2 haplotypes in each subject (Fig. 2A). When using the son (GS003) as the index subject, SMN1-FL haplotype analysis revealed that one SMN1 copy was inherited from the father (Hap-F1), while the other SMN1 copy was inherited from the mother (Hap-M1), indicating that the son's 2 SMN1 copies were distributed on 2 alleles (SMN1 1 + 1). Similarly, when using either parent (GS001 or GS002) as the index subject, only one of the 2 SMN1 copies was inherited by the son, indicating that the parent also had an SMN1 1 + 1 distribution. The distribution of the 2 SMN2 copies followed the same pattern for all 3 family members (Fig. 2A). Through haplotype analysis, CASMA-trio was also able to determine the allelic distribution between SMN1 and SMN2 in all 3 subjects. For example, in the father, SMN1-Hap-F1 and SMN2-Hap-F1 were on one allele, while SMN1-Hap-F2 and SMN2-Hap-F2 were located on the other allele.

Family F07 consisted of 2 parents (GS022 and GS023) who were SMN1 1 + 0 carriers, a sister (GS024) with 1 copy of SMN1, and an affected son with no intact SMN1. The father had 2 SMN2 copies, both the mother and sister had 1 SMN2 copy, while the son had 3 SMN2 copies. When using the father as the index subject, it was observed that both the 2 SMN2 copies were inherited by the son, indicating that they were located on the same allele (SMN2 2 + 0) (Fig. 2B). CASMA-trio analysis further confirmed that the 2 SMN2 copies and 1 SMN1 copy were located on separate alleles.

CASMA-trio Analysis in Families with SMN1 2 + 0 Carriers

Family F05 had an affected daughter (III-1) with no SMN1. MLPA analysis showed that the father (II-2) was a SMN1 1 + 0 carrier and the mother (II-1) had 2 copies of SMN1 (Fig. 3A). Pedigree analysis deduced that the mother should be a SMN1 2 + 0 carrier. To validate this, the mother was selected as the index subject in (maternal grandfather)–(maternal grandmother)–mother trio for CASAM-trio analysis. The results showed that both SMN1 haplotypes of the mother were inherited from the maternal grandmother (I-2), confirming that she was indeed a SMN1 2 + 0 carrier (Fig. 3B), consistent with conventional pedigree analysis. Similarly, CASMA-trio analysis indicated that II-1 had 2 SMN2 copies inherited from I-1 on the same allele. This analysis also revealed that II-1 had 2 SMN1 copies on one allele, and 2 SMN2 copies on the other allele.

Family F09 had an affected SMA patient (III-2) who died at infancy. The father (II-2) was a SMN1 1 + 0 carrier and the mother (II-1) had 2 SMN1 copies (Fig. 3C). Pedigree analysis deduced that the mother should be a SMN1 2 + 0 carrier. The distribution pattern of SMN1 in II-1 could be analyzed using either the (maternal grandfather)–(maternal grandmother)–mother trio or father–mother–daughter trio by CASMA-trio. Both analysis pipelines consistently indicated that the mother was indeed a SMN1 2 + 0 carrier, as well as the daughter (III-1) (Fig. 3D). Both I-2 and II-2 had 2 SMN2 copies distributed on separate alleles. Furthermore, the distribution of SMN1 and SMN2 on 2 alleles were also determined in all the subjects (Fig. 3B and D).

CASMA-trio Analysis Using the SMN1/2-D Fragment

CASMA-trio successfully determined the inheritance of SMN1 and SMN2 haplotypes in 23 out of 24 families by analyzing SMN1/2-FL haplotypes (Supplemental Tables 1 and 2). However, in family F03, SMN1/2-D haplotype analysis was needed to supplement for better discrimination. The mother had 2 SMN1 copies, while both the father and daughter had one SMN1 copy. When using SMN1-FL haplotypes in CASMA-trio pipeline, it was clear that one SMN1 haplotype of the mother was not inherited by the daughter (Fig. 4). However, the other SMN1 haplotype of the mother was exactly the same as that of the father and the daughter, making it impossible to deduce the inheritance. Subsequently, SMN1-D haplotypes were applied in CASMA-trio pipeline. The 2 parents had different SMN1-D haplotypes, and CASMA-trio indicated that one of the SMN1-D haplotypes was inherited by the daughter, indicating that the mother had a SMN1 1 + 1 pattern. Both SMN1-FL and SMN2-D haplotype analysis by the CASMA-trio pipeline confirmed that all 3 family members had one SMN2 copy on each allele.

Discussion

Pan-ethnic SMA carrier screening is recommended for all couples by the ACMG and the American College of Obstetricians and Gynecologists to facilitate informed reproductive options due to high carrier rate and disease severity. However, genetic screening for SMN1 2 + 0 carrier, accounting for 3%–8% of all SMA carriers, has been technically challenging. In this study, we employed CASMA-trio, a modified version of the previously published CASMA approach, to enable effective and universal screening of SMN1 2 + 0 carriers through family trio analysis. In this pilot study, CASMA-trio successfully identified whether subjects with 2 SMN1 copies (n = 16) and SMN2 copies (n = 43) exhibited the 2 + 0 pattern. Reconciliation of the data from both SMN1 and SMN2 further strengthened the analysis. Importantly, CASMA-trio did not rely on the presence of an affected proband, specific SNPs or haplotypes that might be exclusive to some particular ethnicities, or complicated SNP analysis involving 3 generations of family members. In fact, none of the enrolled samples had the g.27134T>G variant, which is known for its high predictive value for SMN1 duplication alleles in the Ashkenazi Jewish population. SMN1 3 + 0 carriers have also been inferred or reported (30, 31). While this study did not identify any subjects that were SMN1/2 3 + 0 carriers, the CASMA-trio pipeline theoretically has the capability to analyze the 3 + 0 pattern. Additionally, the same principle could potentially be applied to screen for 2 + 0 carriers of other disease-causing genes, such as CYP21A2 (32).

The CASMA assay utilized 2 fragments, namely SMN-FL and SMN-D, to maximally capture the sequence information of 2 SMN genes by long-range PCR and LRS. The SMN-FL enabled analysis of copy number variations and intragenic variants in the SMN genes, while SMN-D provided supplementary haplotype information and could identify SMN1 2 + 0 carrier with approximately 50% sensitivity (23). However, the clinical utility of the fragment SMN-D was limited due to low positive prediction value for 2 + 0 and doubled labor and cost. The SMN-FL fragment, which was a large amplicon spanning 28.5-kb, facilitated the distinction of haplotypes between 2 parents in most families. Here, only one couple out of the 24 families had indistinguishable SMN1-FL haplotypes (Fig. 4). In such cases, SMN1-D haplotypes were employed to determine whether the index subject was a SMN1 2 + 0 carrier or not. Alternatively, to overcome the limitation of haplotype variation, CASMA-trio could be applied to analyze different family trios, including the index subject and both parents, or index subject, the spouse, and child.

Accurate sequencing, variant calling, and haplotype analysis are key attributes for determining success of CASMA-trio analysis. The sequencing polymerase employed by PacBio exhibits an impressive capacity to read sequences exceeding 200 kb, with a mean sequencing length of approximately 70–100 kb, which allows for the generation of multiple sequencing passes of the 28.5-kb SMN insert within the dumbbell-shaped SMRTbell library, resulting in high-fidelity CCS reads. The integration of a high sequencing depth can further act as a proofing mechanism against random sequencing errors. The software pbampliconclustering (Pacific Biosciences) facilitates the clustering of PacBio CCS reads by collapsing the same nucleotides, k-mer counts, and clustering algorithms to distinguish different SMN haplotypes, which has been employed in the previously described CASMA assay (23). However, while this haplotype clustering approach is generally effective, it might result in a loss of certain SNP information during the collapsing step. This issue was addressed here with a novel recursive SNP method. In the case of sample SA0139, the 2 SMN2 haplotypes were accurately distinguished by the c.834+485G>T (AGGTGC > AGTTGC) variant (Supplemental Fig. 1). Notably, both the reference sequence AGGTGC and the variant sequence AGTTGC would be collapsed to AGTGC, rendering them indistinguishable by pbampliconclustering (23).

Here, we propose a comprehensive and universal workflow for SMA carrier screening (Fig. 5). The CASMA assay could be performed for couples seeking SMA carrier screening. If one partner is a carrier of SMN1 1 + 0 or 1 + 1^D, and the spouse has 2 or 3 normal copies of SMN1, the CASMA-trio would be recommended to determine whether the spouse is a SMN1 2/3 + 0 carrier. If the spouse is not a carrier, the couple is at low risk for SMA. If the spouse is a SMN1 2/3 + 0 carrier, the couple is considered high risk for SMA. For low-risk couples, routine preconception or prenatal care can be followed. However, for high-risk couples, genetic counseling should be offered, and in vitro fertilization and preimplantation genetic testing or prenatal diagnosis can be suggested with informed consent. This workflow enables the screening of nearly 100% of SMA carriers, including SMN1 1 + 0, 1 + 1^D, and 2/3 + 0.

There are certain limitations of CASMA-trio analysis. First, the efficacy of this method requires a trio analysis, which may not always be feasible as trios are not readily available. In cases where only a single parent is available, a definitive conclusion can only be made when the index subject does not have any haplotypes that are the same as the parent, thus indicating a 2 + 0 carrier status (Supplemental Fig. 2). In situations where shared haplotypes exist, our method can provide a likelihood prediction but not a definitive answer. To enhance the accuracy of prediction, it may be beneficial to calculate the frequency of each specific haplotype by accumulating data from a large-scale population. Additionally, for couples seeking carrier testing at assisted reproduction centers, another possibility is to use the couple and embryo(s) as trios for analysis. While amplifying a 28.5-kb SMN fragment from embryo samples can be very challenging, it may be worth exploring the possibility of amplifying smaller yet sufficient fragments to distinguish various haplotypes, especially considering that the haplotype information of the couple is already available. Second, in rare circumstances where the parents have the same SMN1-FL and SMN-D haplotypes, the results may be inconclusive. Last, de novo rearrangements are found in 2% of index patients with SMA (33), indicating that a parent with 2 SMN1 copies may not necessarily always be a 2 + 0 carrier when 2 or no SMN1 haplotypes are inherited by the child.

Conclusion

LRS and haplotype-informed CASMA-trio analysis represents an effective and universal approach for screening SMN1 2 + 0 carriers. Implementation of CASMA-trio analysis into the CASMA assay or existing SMA carrier screening programs will facilitate comprehensive screening for all types of carriers and greatly reduce residual risk ratio.

Supplemental Material

Supplemental material is available at Clinical Chemistry online.

Nonstandard Abbreviations

SMA, spinal muscular atrophy; CASMA, comprehensive analysis of spinal muscular atrophy; SMN1, survival of motor neuron 1; SMN2, survival of motor neuron 2; ACMG, American College of Medical Genetics and Genomics; MLPA, multiplex ligation-dependent probe amplification; LRS, long-read sequencing; SNP, single-nucleotide polymorphisms; SMRTbell single-molecule real-time dumbbell; CCS, circular consensus sequencing; FL, full-length; D, downstream.

Human Genes

SMN1, survival of motor neuron 1, telomeric; SMN2, survival of motor neuron 2, centromeric; CYP21A2, cytochrome P450 family 21 subfamily A member 2.

Author Contributions

The corresponding author takes full responsibility that all authors on this publication have met the following required criteria of eligibility for authorship: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Nobody who qualifies for authorship has been omitted from the list.

Authors’ Disclosures or Potential Conflicts of Interest

Upon manuscript submission, all authors completed the author disclosure form.

Research Funding

This work was supported by the Shanghai Municipal Science and Technology Commission (22Y11902300, 22Y11906700), Clinical Research Plan of IPMCH (IPMCH2022CR1-02), the National Natural Science Foundation of China (No. 81971401, 81871136), the Shanghai Municipal Key Clinical Specialty and Shanghai Municipal Health and Family Planning Committee (20204Y0233), the Shanghai Municipal Key Clinical Specialty, and the National Key R&D Program of China (No. 2022YFC2703400).

Disclosures

A. Mao and J. Zhan are employees of Berry Genomics Corporation.

Role of Sponsor

The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, preparation of manuscript, or final approval of manuscript.

Acknowledgments

The authors acknowledge all the subjects for participating in this study.

References

Author notes