Cystic fibrosis-causing variants in Chinese patients with congenital absence of the vas deferens: a cohort and meta-analysis

Yi Lu; Jing Wang; Zhong-Lin Cai; Teng-Yan Li; Hong-Jun Li; Bin-Bin Wang

doi:10.4103/aja2024124

. 2025 Mar 11;27(5):611–620. doi: 10.4103/aja2024124

Cystic fibrosis-causing variants in Chinese patients with congenital absence of the vas deferens: a cohort and meta-analysis

Yi Lu ^1,^*, Jing Wang ^2,^*, Zhong-Lin Cai ³, Teng-Yan Li ⁴, Hong-Jun Li ^1,^✉, Bin-Bin Wang ^4,^✉

PMCID: PMC12422572 PMID: 40065563

Abstract

Individuals with congenital absence of the vas deferens (CAVD) may transmit cystic fibrosis (CF)-causing variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to their offspring through assisted reproductive technology (ART). We aimed to delineate the spectrum and estimate the prevalence of CF-causing variants in Chinese individuals with CAVD through a cohort analysis and meta-analysis. CFTR was sequenced in 145 Chinese individuals with CAVD. CFTR variants were classified as CF-causing or non-CF-causing variants regarding clinical significance. A comprehensive genotype analysis was performed in Chinese individuals with CAVD, incorporating previous studies and our study cohort. The prevalence of CF-causing variants was estimated through meta-analysis. In our cohort, 56 different CFTR variants were identified in 108 (74.5%) patients. Twenty variants were categorized as CF-causing and were detected in 28 (19.3%) patients. A comprehensive genotype analysis of 867 patients identified 174 different CFTR variants. Sixty-four were classified as CF-causing variants, 56.3% of which had not been previously reported in Chinese patients with CF. Meta-analysis showed that 14.8% (95% confidence interval [CI]: 11.0%–18.9%) CAVD cases harbored one CF-causing variant, and 68.6% (95% CI: 65.1%–72.0%) CAVD cases carried at least one CFTR variant. Our study underscores the urgent need for extensive CFTR screening, including sequencing of whole exons and flanking regions and detection of large rearrangements and deep intronic CF-causing variants, in Chinese individuals with CAVD before undergoing ART. The established CF-causing variants spectrum may aid in the development of genetic counseling strategies and preimplantation diagnosis to prevent the birth of a child with CF.

Keywords: congenital absence of the vas deferens, cystic fibrosis, cystic fibrosis transmembrane conductance regulator, genetic counseling

INTRODUCTION

Congenital absence of the vas deferens (CAVD), which leads to obstructive azoospermia, is one of the primary causes of male infertility.1 CAVD is predominantly caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR) gene,2 with the secondary causes including hemizygous variants in the adhesion G protein-coupled receptor G2 (ADGRG2) gene.3

Cystic fibrosis (CF) is among the most common, recessive inherited, life-threatening diseases in the White population.4 It is caused by bi-allelic CFTR variants that severely disrupt the CFTR function. Despite obstructive infertility, individuals with CAVD can conceive through assisted reproductive technology (ART). However, these patients may transmit CFTR variants, particularly severe or CF-causing variants, to their offspring, thereby increasing the risk of CF occurrence compared with the general population.5 Therefore, genetic testing and counseling before ART are particularly important for individuals with CAVD and their partners. In White populations, the spectrum of CF-causing variants has been well established, with c.1521_1523del; p.Phe508del (ΔF508) variant being the most common, accounting for nearly 70% of all variant alleles.6 Most individuals with isolated CAVD of European descent possess a CF-causing variant.7 In several European countries, CFTR mutation screening has become routine for preimplantation genetic diagnosis (PGD) to prevent the birth of children with CF.8

In contrast, owing to the extremely low prevalence of CF in China, the CFTR variant spectrum is not well established, and no such screening strategy has been implemented. However, an increasing number of Chinese CAVD couples undergoing ART are at elevated risk of transmitting CF-causing variants to their offspring or having child with CF.9 It is crucial to delineate the spectrum of CFTR variants, particularly those causing CF, in individuals with CAVD to inform future CFTR screening and genetic counseling strategies in Chinese population. In this study, we used targeted sequencing to identify CF-causing variants in a cohort of Chinese individuals with CAVD. Furthermore, we delineated the spectrum and estimated the overall prevalence of CFTR and specifically CF-causing variants through a systematic review and meta-analysis that includes both the present cohort and previous studies on the Chinese CAVD population.

PARTICIPANTS AND METHODS

Participants

A cohort of individuals with isolated CAVD was recruited from the Department of Urology, Peking Union Medical College Hospital (Beijing, China) between 2015 and 2022. The inclusion criteria were: (1) impalpable vas deferens by physical examination and absence of vas deferens confirmed by ultrasound examination; (2) azoospermia with a low pH value (<7.2) and fructose concentration (<25 μmol per ejaculate); (3) normal serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels; and (4) no absence of typical CF symptoms. A total of 145 patients diagnosed with congenital bilateral absence of the vas deferens (CBAVD) met the eligibility criteria for the mutational analysis of known genes associated with CAVD. This study was approved by the Ethics Committee of the National Research Institute for Family Planning (Beijing, China; Approval No. 2021010). Written informed consent was obtained from all patients.

Mutational analysis by targeted sequencing

Genomic DNA was extracted from peripheral blood samples. All coding exons and flanking introns (±100 base pairs) of known disease-associated genes CFTR and ADGRG2 were amplified using multiplex polymerase chain reaction (PCR) to construct the sequencing library (MultiSeq, iGeneTech Corp., Beijing, China). Subsequently, massively parallel sequencing was conducted on a NovaSeq6000 platform (Illumina, San Diego, CA, USA). Single nucleotide variants (SNVs) and indels in the target regions were detected using the Genome Analysis Toolkit (GATK version 3.8.0; https://github.com/broadinstitute/gatk).

Classification of clinical significance and pathogenicity

The clinical significance of CFTR variants was determined based on classifications in the CFTR2 (https://www.cftr2.org) and CFTR-France (https://cftr.iurc.montp.inserm.fr/cftr) databases. CFTR variants were categorized into five groups: CF-causing, CFTR-related disorder (RD)-causing, varying clinical consequence (VCC), variant of unknown clinical significance (VUS), and nondisease-causing (Supplementary Table 1). A variant classified as CF-causing in at least one database was designated as CF-causing. Nonsense, frameshift, or canonical splice site (i.e., ±1, ±2) variants, if not listed in the databases, were also classified as CF-causing due to their disruption of the encoded protein. These CF-causing variants are referred to as CF-causing variants. Variants classified as VCC, CFTR-RD, or VUS were grouped as non-CF-causing (NCF) variants.

Supplementary Table 1.

Determination criteria of clinical significance for cystic fibrosis transmembrane conductance regulator variants

Clinical significance	Explanation	Final determination criteria^a
CF-causing	When in trans with another CF-causing variant, will result in CF	CF-causing classification in at least one database^b
VCC	When in trans with another CF-causing variant, can either result in CF or in a CFTR-RD	VCC classification in at least one database No CF-causing classification in either database
CFTR-RD causing	When in trans with a CF-causing variant, will result in CFTR-RD such as chronic pancreatitis, bronchiectasis, CRS-NP or CBAVD	CFTR-RD-causing classification in at least one database No CF-causing or VCC classifications in either databases
Nondisease-causing	When in trans with a CF-causing variant, will not cause CF, nor CFTR-RD	Nondisease-causing classification in both databases
VUS	Unclassified because of insufficient data	Conditions other than the above criteria^c

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^aAccording to classifications in CFTR2 database and CFTR-France database; ^bNonsense, frameshift and canonical splicing variants, if not recorded in both databases, will be classified as CF-causing; ^cMissense, in-frame insertion/deletion, and noncanonical splicing variants, if not recorded in both databases, will be classified as VUS. VCC: varying clinical significance; CF: cystic fibrosis; VUS: variant of unknown clinical significance; CFTR: CF transmembrane conductance regulator; CFTR-RD: CFTR-related disorder; CRS-NP: chronic rhinosinusitis with or without nasal polyposis; CBAVD: congenital bilateral absence of vas deferens

Sanger sequencing

Variants identified through targeted sequencing are considered highly reliable because of their substantial coverage of thousands of mapping reads. Sanger sequencing was not used for validation, except for the CFTR c.1210-34_1210-6TG[m]T[n] variant, also known as IVS9-(TG)m(T)n. For this variant, all participants were genotyped using the primers: forward, 5’-GCTTTGAAAGAGGAGGAT-3’; and reverse, 5’-CAAGACACTACACCCATAC-3’.

Literature review and meta-analysis

We performed a comprehensive literature review to delineate the spectrum of CFTR variants and CF-causing variants in Chinese individuals with CAVD and performed a meta-analysis to estimate their prevalence. YL and JW searched for studies that identified CFTR variants in Chinese individuals with CAVD on PubMed, CNKI, and Wanfang databases prior to June 2024 using a predefined search syntax (Supplementary Table 2). Only the articles published in English or Chinese were included. The eligibility of each study identified by literature search was judged according to inclusion and exclusion criteria (Supplementary Table 2). For all eligible studies, the CAVD phenotype and CFTR genotype of each patient were extracted. The clinical significance of each CFTR variant was assessed according to the aforementioned criteria. All conflicts were resolved through discussion.

Supplementary Table 2.

Search strategy and inclusion/exclusion criteria for meta-analysis

Search strategy
Term 1	‘Congenital bilateral aplasia of the vas deferens’[MeSH] OR ‘Congenital bilateral aplasia of the vas deferens’[tiab] OR ‘CBAVD’[tiab] OR ‘congenital unilateral absence of the vas deferens’[tiab] OR ‘CUAVD’[tiab] OR ‘congenital absence of the vas deferens’[tiab] OR ‘congenital aplasia of the vas deferens’[tiab] OR ‘CAVD’[tiab]
Term 2	‘Cystic Fibrosis Transmembrane Conductance Regulator’[MeSH] OR ‘Cystic Fibrosis Transmembrane Conductance Regulator’[tiab] OR ‘CFTR’[tiab]
Search syntax	Term 1#2
Inclusion criteria
1	Conducted in Chinese isolated CAVD patients, including those with CBAVD, CUAVD or CPAVD
2	Involved sequencing of the entire CFTR gene, including whole exons and flanking intronic regions, using direct sequencing or next-generation sequencing
3	Reported the CFTR genotype for each CAVD patient
Exclusion criteria
1	Only genotyped specific polymorphisms (e.g., IVS9-polyT and p.Met470Val) or sequenced only parts of the gene (e.g., selected exons and promoter regions)
2	Case reports and pedigree analyses

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CAVD: congenital absence of the vas deferens; CBAVD: congenital bilateral absence of vas deferens; CFTR: cystic fibrosis transmembrane conductance regulator; CUAVD: congenital unilateral absence of vas deferens; CPAVD: congenital bilateral partial aplasia of vas deferens

We estimated the percentages of patients carrying CFTR variants, two variant alleles, and one variant allele as well as the proportions of patients harboring CF-causing variants, two NCF variants (NCF/NCF), and one NCF variant (NCF/none) through meta-analysis. The allele frequency of the IVS9-5T variant and the proportions of patients with homozygous and heterozygous IVS9-5T variants were also calculated. Additionally, these analyses were conducted separately for individuals with CBAVD and those with congenital unilateral absence of the vas deferens (CUAVD) or congenital bilateral partial aplasia of the vas deferens (CPAVD). The meta-analysis was performed using the metaprop package in STATA 16.0 (StataCorp, College Station, TX, USA).10

RESULTS

CFTR variants in CAVD cohort

The age (mean ± standard deviation [s.d.]) of the 145 participants with CAVD was 29.4 ± 4.5 years. Total testis volume and serum hormone levels were within normal ranges, whereas semen volume and pH were below the lower limits (Table 1).

Table 1.

Clinical characteristics of congenital absence of the vas deferens patients

Clinical parameter	CAVD patients (total=145), mean±s.d.	Normal range
Age (year)	29.4±4.5	-
Total testis volume (ml)	25.01±4.41	15-25
Semen volume (ml)	1.11±0.80	>1.5
Semen pH	6.56±0.37	>7.2
FSH (mIU ml⁻¹)	5.23±2.85	1.27–11.96
LH (mIU ml⁻¹)	3.89±1.97	1.24–8.62
PRL (mIU ml⁻¹)	193.72±81.76	56–278
E2 (pg ml⁻¹)	33.39±15.94	20–75
Testosterone (ng ml⁻¹)	3.82±1.39	1.75–7.80

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CAVD: congenital absence of the vas deferens; E2: estradiol; FSH: follicle-stimulating hormone; LH: luteinizing hormone; PRL: prolactin; s.d.: standard deviation; -: not applicable

Through targeted sequencing, we identified 56 different CFTR variants (184 variant alleles) in 108 of the 145 patients (Table 2). The CFTR genotypes of each individual with CAVD are listed in Supplementary Table 3. These variants included 37 missense, 10 splicing, 5 nonsense, and 4 indel variants (Table 2 and Figure 1a). The most prevalent variant was c.1210-12T[5], also known as IVS9-5T, which accounted for 94 variant alleles found in 76 individuals, yielding an allele frequency of 32.4%. Specifically, 61 alleles of the TG12T5 variant and 33 alleles of the TG13T5 variant were identified, respectively. Among the sequenced patients, 18 (12.4%) were homozygous for the IVS9-5T variant, and 58 (40.0%) were heterozygous.

Table 2.

Cystic fibrosis transmembrane conductance regulator variants identified in congenital absence of the vas deferens cohort

Variant (legacy name)	Type	Number of alleles (n)	AF in Chinese patients (%)		AF in gnomAD^b		Clinical significance

			CAVD (n=145)	CF (n=202)^a	Total	East Asian	CFTR2^c	CFTR-France^d	Final determination
c.50del; p.Phe17Serfs^*8 (182delT)	Frameshift deletion	1	0.34	0	6.20×10⁻⁷	0	CF-causing	NR	CF-causing
c.91C>T; p.Arg31Cys (R31C)	Missense	3	1	0	1.51×10⁻³	6.76×10⁻³	Non-CF-causing	VUS	VUS
c.92G>A; p.Arg31His (R31H)	Missense	1	0.34	0	2.48×10⁻⁵	8.93×10⁻⁵	NR	NR	VUS
c.215C>A; p.Ala72Asp (A72D)	Missense	1	0.34	0	0	0	NR	VUS	VUS
c.223C>T; p.Arg75^* (R75X)	Nonsense	2	0.69	1.29	1.18×10⁻⁵	4.46×10⁻⁵	CF-causing	CF-causing	CF-causing
c.263T>G; p.Leu88^* (L88X)	Nonsense	1	0.34	2.58	6.45×10⁻⁷	0	CF-causing	CF-causing	CF-causing
c.264_268del; p.Leu88Phefs^*21 (396delATATT)	Frameshift deletion	1	0.34	0.77	0	0	NR	NR	CF-causing^e
c.293A>G; p.Gln98Arg (Q98R)	Missense	2	0.69	3.09	3.72×10⁻⁶	0	CF-causing	CF-causing	CF-causing
c.320C>A; p.Ala107Asp (A107D)	Missense	1	0.34	0.26	0	0	NR	NR	VUS
c.326A>G; p.Tyr109Cys (Y109C)	Missense	1	0.34	0.77	0	0	NR	VUS	VUS
c.400A>G; p.Arg134Gly (R134G)	Missense	1	0.34	0	0	0	NR	NR	VUS
c.558C>G; p.Asn186Lys (N186K)	Missense	2	0.69	0.52	6.32×10⁻⁷	0	NR	VUS	VUS
c.592G>C; p.Ala198Pro (A198P)	Missense	1	0.34	0	6.20×10⁻⁷	0	NR	NR	VUS
c.650A>G; p.Glu217Gly (×10217G)	Missense	4	1.38	1.55	2.06×10⁻³	1.10×10⁻²	NR	VUS	VUS
c.869+5G>A (1001+5G>A)	Splicing	2	0.69	0	1.24×10⁻⁶	2.24×10⁻⁵	NR	CF-causing	CF-causing
c.890G>A; p.Arg297Gln (R297Q)	Missense	1	0.34	0	8.85×10⁻⁴	2.68×10⁻⁴	NR	VUS	VUS
c.926C>G; p.Ala309Gly (A309G)	Missense	1	0.34	0	0	0	NR	VUS	VUS
c.1040G>A; p.Arg347His (R347H)	Missense	1	0.34	0.26	5.21×10⁻⁵	0	CF-causing	VCC	CF-causing
c.1069G>A; p.Ala357Thr (A357T)	Missense	1	0.34	0	0	0	NR	VUS	VUS
c.1209+1G>C (1341+1G>C)	Splicing	2	0.69	0	0	0	CF-causing	NR	CF-causing
c.1210-12T[5] (5T)	Splicing	94	32.4	0	-	-	VCC	VCC	VCC
c.1210-34_1210-6TG[12]T[5] (5T; TG12)	Splicing	61	21	-	-	-	-	-	-
c.1210-34_1210-6TG[13]T[5] (5T; TG12)	Splicing	33	11.4	-	-	-	-	-	-
c.1210-6T>A (1342-6T>A)	Splicing	2	0.69	0	2.85×10⁻⁵	0	NR	VUS	VUS
c.1351G>A; p.Gly451Arg (G451R)	Missense	1	0.34	0	3.10×10⁻⁶	6.69×10⁻⁵	NR	NR	VUS
c.1403T>C; p.Leu468Pro (L468P)	Missense	1	0.34	0	0	0	NR	NR	VUS
c.1405A>G; p.Met469Val (M469V)	Missense	1	0.34	0	7.48×10⁻⁶	6.70×10⁻⁵	NR	Disease-causing	VUS
c.1520T>A; p.Ile507Asn (I507N)	Missense	1	0.34	0	0	0	NR	NR	VUS
c.1521_1523del; p.Phe508del (ΔF508)	In-frame deletion	1	0.34	1.8	1.19×10⁻²	2.23×10⁻⁵	CF-causing	CF-causing	CF-causing
c.1586A>C; p.Asp529Ala (D529A)	Missense	1	0.34	0	0	0	NR	NR	VUS
c.1666A>G; p.Ile556Val (I556V)	Missense	7	2.41	1.55	1.27×10⁻³	4.20×10⁻²	NR	CFTR-RD-causing	CFTR-RD-causing
c.1766+1G>T (1898+1G>T)	Splicing	1	0.34	0.26	0	0	CF-causing	NR	CF-causing
c.1767-1G>A (1899-1G>A)	Splicing	1	0.34	0	0	0	NR	NR	CF-causinge
c.1775G>T; p.Cys592Phe (C592F)	Missense	1	0.34	0	0	0	NR	NR	VUS
c.1837G>A; p.Ala613Thr (A613T)	Missense	1	0.34	0	3.77×10⁻⁶	2.23×10⁻⁵	CF-causing	NR	CF-causing
c.1986_1989del; p.Thr663Argfs^*8 (2118delAACT)	Frameshift deletion	2	0.69	0	5.58×10⁻⁶	0	CF-causing	CF-causing	CF-causing
c.2042A>T; p.Glu681Val (×10681V)	Missense	2	0.69	0.26	5.89×10⁻⁵	2.07×10⁻³	NR	NR	VUS
c.2125C>T; p.Arg709^* (R709X)	Nonsense	1	0.34	0.77	1.12×10⁻⁵	0	CF-causing	CF-causing	CF-causing
c.2374C>T; p.Arg792^* (R792X)	Nonsense	1	0.34	0.77	3.29×10⁻⁶	0	CF-causing	CF-causing	CF-causing
c.2646G>A; p.Trp882^* (W882X)	Nonsense	1	0.34	0	0	0	CF-causing	NR	CF-causing
c.2684G>A; p.Ser895Asn (S895N)	Missense	1	0.34	0.52	7.31×10⁻⁵	2.43×10⁻³	NR	NR	VUS
c.2812G>T; p.Val938Leu (V938L)	Missense	1	0.34	0.26	1.24×10⁻⁶	4.46×10⁻⁵	NR	NR	VUS
c.2834C>T; p.Ser945Leu (S945L)	Missense	1	0.34	1.03	4.09×10⁻⁵	0	CF-causing	VCC	CF-causing
c.2907A>C; p.Ala969Ala (NULL)	Splicing	1	0.34	0.26	1.24×10⁻⁶	4.46×10⁻⁵	NR	NR	VUS
c.2909G>A; p.Gly970Asp (G970D)	Missense	4	1.38	12.11	5.94×10⁻⁶	1.58×10⁻⁴	CF-causing	CF-causing	CF-causing
c.2918T>G; p.Leu973Arg (L973R)	Missense	1	0.34	0	0	0	NR	NR	VUS
c.2936A>C; p.Asp979Ala (D979A)	Missense	2	0.69	0.26	8.79×10⁻⁶	2.91×10⁻⁴	NR	VUS	VUS
c.2950G>A; p.Asp984Asn (D984N)	Missense	1	0.34	0	6.28×10⁻⁷	0	NR	NR	VUS
c.3068T>G; p.Ile1023Arg (I1023R)	Missense	1	0.34	2.84	0	0	NR	NR	VUS
c.3140-26A>G (3272-26A>G)	Splicing	1	0.34	0.52	1.02×10⁻⁴	0	CF-causing	VCC	CF-causing
c.3208C>T; p.Arg1070Trp (R1070W)	Missense	1	0.34	0	8.00×10⁻⁵	2.20×10⁻⁵	VCC	VCC	VCC
c.3367+2T>C (3499+2T>C)	Splicing	1	0.34	0	0	0	CF-causing	NR	CF-causing
c.3406G>A; p.Ala1136Thr (A1136T)	Missense	1	0.34	0.26	1.43×10⁻⁵	1.34×10⁻⁴	NR	VUS	VUS
c.3454G>C; p.Asp1152His (D1152H)	Missense	1	0.34	0	3.98×10⁻⁴	0	VCC	VCC	VCC
c.3469-3C>A (3601-3C>A)	Splicing	2	0.69	0	6.82×10⁻⁶	2.45×10⁻⁴	NR	NR	VUS
c.4056G>C; p.Gln1352His (Q1352H)	Missense	12	4.14	1.03	5.87×10⁻⁴	1.92×10⁻²	NR	CFTR-RD-causing	CFTR-RD-causing
c.4091C>T; p.Ala1364Val (A1364V)	Missense	1	0.34	0	1.80×10⁻⁵	6.68×10⁻⁵	NR	VUS	VUS
c.4262T>A; p.Val1421Glu (V1421Q)	Missense	1	0.34	0	0	0	NR	NR	VUS

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^aChinese CF patients reported and summarized by Shen et al.14 ^bGnomAD v4.1.0 (https://gnomad.broadinstitute.org). ^cLast accessed on April 7, 2023 (https://www.cftr2.org). ^dLast accessed on September 22, 2023 (https://cftr.iurc.montp.inserm.fr/cftr). ^eFrameshift and canonical splicing variants, despite not recorded in both databases, are also deemed as CF-causing. AF: allele frequency; CAVD: congenital absence of vas deferens; CF: cystic fibrosis; CFTR: CF transmembrane conductance regulator; CFTR-RD: CFTR-related disorders; NR: not recorded; VCC: varying clinical consequence; VUS: variant of unknown clinical significance; -: not applicable

Supplementary Table 3.

Cystic fibrosis transmembrane conductance regulator and adhesion G protein-coupled receptor G2 variants in each congenital absence of the vas deferens case of the present study

Patient ID	CFTR (NM_000492.3)		ADGRG2(NM_001079858.2)^b

	IVS9-(TG)m(T)n	Other variants (legacy name)^a
CN108	TG12T7/TG11T7	c.890G>A; p.Arg297Gln (R297Q)
CN109	TG12T5/TG11T7	c.4056G>C; p.Gln1352His (Q1352H)
CN110	TG12T5/TG11T7	c.2042A>T; p.Glu681Val (E681V)
CN111	TG12T7/TG11T7	c.264_268del; p.Leu88Phefs*21 (396delATATT)
CN113	TG12T7/TG11T7	c.1666A>G; p.Ile556Val (I556V)
CN114	TG13T5/TG11T7	c.91C>T; p.Arg31Cys (R31C)
CN115	TG12T7/TG12T7	c.926C>G; p.Ala309Gly (A309G), c.1986_1989del; p.Tdr663Argfs^*8 (2118delAACT)
CN117	TG13T5/TG12T7	c.263T>G; p.Leu88^* (L88X)
CN118	TG13T5/TG13T5
CN119	TG12T7/TG12T5	c.2909G>A; p.Gly970Asp (G970D)
CN120	TG12T7/TG11T7
CN121	TG13T5/TG12T7	c.223C>T; p.Arg75^* (R75X)
CN122	TG12T7/TG11T7	c.1666A>G; p.Ile556Val (I556V)
CN123	TG12T7/TG12T7	c.1210-6T>A (1342-6T>A)
CN124	TG13T7/TG12T7	c.400A>G; p.Arg134Gly (R134G), c.3469-3C>A (3601-3C>A)	c.2312A>G; p.Asn771Ser
CN126	TG13T5/TG12T7	c.592G>C; p.Ala198Pro (A198P)
CN128	TG12T7/TG11T7
CN129	TG12T5/TG10T9	c.1521_1523del; p.Phe508del (ΔF508)
CN130	TG13T5/TG12T5
CN131	TG12T7/TG11T7
CN132	TG12T5/TG12T5
CN133	TG12T7/TG12T7
CN134	TG12T5/TG12T5
CN135	TG12T5/TG12T7	c.2909G>A; p.Gly970Asp (G970D)
CN136	TG13T5/TG11T7	c.4056G>C; p.Gln1352His (Q1352H)
CN137	TG12T7/TG11T7
CN138	TG12T5/TG12T7	c.2936A>C; p.Asp979Ala (D979A)	c.2312A>G; p.Asn771Ser
CN139	TG12T5/TG12T7
CN140	TG12T7/TG11T7	c.869+5G>A (1001+5G>A), c.1210-6T>A (1342-6T>A)
CN141	TG12T7/TG12T7
CN142	TG12T7/TG12T7	c.1351G>A; p.Gly451Arg (G451R), c.3208C>T; p.Arg1070Trp (R1070W)
CN143	TG13T5/TG12T5
CN144	TG13T5/TG12T5
CN145	TG12T5/TG11T7
CN146	TG12T5/TG12T7	c.2936A>C; p.Asp979Ala (D979A)
CN147	TG12T7/TG12T7	c.4056G>C; p.Gln1352His (Q1352H)
CN148	TG12T7/TG12T7	c.2684G>A; p.Ser895Asn (S895N)
CN149	TG12T5/TG11T7	c.1666A>G; p.Ile556Val (I556V)
CN150	TG12T7/TG12T7
CN151	TG13T5/TG12T7	c.2909G>A; p.Gly970Asp (G970D)
CN152	TG12T7/TG11T7	c.215C>A; p.Ala72Asp (A72D), c.1040G>A; p.Arg347His (R347H)
CN153	TG12T5/TG11T7	c.1775G>T; p.Cys592Phe (C592F), c.4262T>A; p.Val1421Glu (V1421E)
CN154	TG12T5/TG11T7
CN155	TG12T5/TG12T7	c.3140-26A>G (3272-26A>G)
CN157	TG12T7/TG12T7
CN158	TG11T7/TG11T7		c.1829G>A; p.Gly610Asp
CN159	TG13T5/TG12T5
CN160	TG12T7/TG12T7
CN161	TG11T7/TG11T7	c.1666A>G; p.Ile556Val (I556V)	c.2312A>G; p.Asn771Ser
CN162	TG12T7/TG12T7
CN163	TG12T5/TG11T7	c.558C>G; p.Asn186Lys (N186K)
CN164	TG12T7/TG11T7
CN165	TG12T7/TG12T7	c.223C>T; p.Arg75^* (R75X)
CN166	TG12T5/TG12T7	c.1837G>A; p.Ala613Thr (A613T)
CN167	TG12T7/TG11T7	c.3367+2T>C (3499+2T>C)
CN168	TG11T7/TG11T7	c.1767-1G>A (1899-1G>A), c.4056G>C; p.Gln1352His (Q1352H)
CN169	TG12T7/TG12T7	c.650A>G; p.Glu217Gly (E217G)
CN170	TG12T5/TG11T7	c.2374C>T; p.Arg792^* (R792X)
CN171	TG13T5/TG11T7	c.1666A>G; p.Ile556Val (I556V)
CN172	TG12T5/TG12T7	c.293A>G; p.Gln98Arg (Q98R)
CN173	TG12T5/TG12T5
CN174	TG12T7/TG12T6
CN175	TG12T5/TG11T7	c.869+5G>A (1001+5G>A)
CN176	TG12T5/TG12T7
CN177	TG12T5/TG11T7	c.1403T>C; p.Leu468Pro (L468P)
CN178	TG13T5/TG13T5
CN179	TG12T5/TG12T7	c.320C>A; p.Ala107Asp (A107D)
CN180	TG12T5/TG12T7
CN181	TG13T5/TG12T5
CN182	TG12T5/TG11T7	c.2646G>A; p.Trp882^* (W882X)
CN183	TG13T5/TG12T7
CN184	TG12T7/TG12T7
CN185	TG12T5/TG12T7
CN186	TG11T7/TG11T7
CN187	TG11T7/TG11T7
CN188	TG12T7/TG11T7
CN189	TG12T7/TG11T7	c.4056G>C; p.Gln1352His (Q1352H)
CN190	TG12T7/TG12T7
CN191	TG11T7/TG11T7	c.1069G>A; p.Ala357Thr (A357T), c.4056G>C; p.Gln1352His (Q1352H)
CN192	TG12T5/TG11T9
CN193	TG12T7/TG11T7	c.91C>T; p.Arg31Cys (R31C)
CN194	TG12T5/TG12T7	c.650A>G; p.Glu217Gly (E217G), c.3406G>A; p.Ala1136Thr (A1136T), c.4056G>C; p.Gln1352His (Q1352H)	c.2312A>G; p.Asn771Ser
CN195	TG12T7/TG12T6	c.92G>A; p.Arg31His (R31H)
CN196	TG12T7/TG12T7
CN197	TG12T5/TG11T7	c.4056G>C; p.Gln1352His (Q1352H)
CN198	TG11T7/TG11T7
CN199	TG12T7/TG12T7	c.650A>G; p.Glu217Gly (E217G), c.1209+1G>C (1341+1G>C)
CN200	TG12T7/TG12T7
CN201	TG12T5/TG11T7
CN202	TG13T5/TG12T5
CN203	TG12T7/TG11T7
CN204	TG12T5/TG12T7	c.2909G>A; p.Gly970Asp (G970D)
CN205	TG11T7/TG11T7	c.2042A>T; p.Glu681Val (E681V), c.2812G>T; p.Val938Leu (V938L), c.2907A>C; p.Ala969Ala
CN206	TG11T7/TG11T7	c.4056G>C; p.Gln1352His (Q1352H)
CN207	TG13T5/TG12T7	c.3469-3C>A (3601-3C>A)
CN208	TG12T5/TG12T7		c.1969C>T; p.Arg657Trp
CN209	TG11T7/TG11T7
CN211	TG12T7/TG12T7
CN212	TG11T7/TG11T7
CN213	TG12T5/TG11T7
CN214	TG12T7/TG11T7	c.1666A>G; p.Ile556Val (I556V)
C-1	TG12T5/TG12T7
C-2	TG12T7/TG11T7
C-3	TG12T7/TG11T7
C-4	TG11T7/TG10T7
C-5	TG12T5/TG11T7	c.1520T>A; p.Ile507Asn (I507N)
C-6	TG11T7/TG11T7
C-7	TG12T5/TG12T7
C-8	TG12T7/TG11T7	c.91C>T; p.Arg31Cys (R31C), c.1586A>C; p.Asp529Ala (D529A)
C-9	TG12T7/TG11T7
C-10	TG12T7/TG12T7
C-11	TG13T5/TG12T5
C-12	TG12T5/TG12T7	c.2918T>G; p.Leu973Arg (L973R)
C-13	TG13T5/TG11T7	c.558C>G; p.Asn186Lys (N186K)
C-14	TG12T5/TG12T7	c.2950G>A; p.Asp984Asn (D984N)
C-15	TG12T5/TG12T7	c.1986_1989del; p.Thr663Argfs^*8 (2118delAACT)
C-16	TG13T5/TG11T7	c.1766+1G>T (1898+1G>T)
C-17	TG11T7/TG11T7		c.2336G>A; p.Trp779^*
C-18	TG12T5/TG12T7
C-19	TG12T7/TG12T7		c.2312A>G; p.Asn771Ser
C-20	TG12T5/TG12T7	c.50del; p.Phe17Serfs^*8 (182delT)
C-21	TG12T7/TG11T7	c.3454G>C; p.Asp1152His (D1152H)
C-22	TG13T5/TG13T5
C-23	TG12T5/TG12T7		c.2312A>G; p.Asn771Ser
C-24	TG11T7/TG11T7	c.4091C>T; p.Ala1364Val (A1364V)
C-25	TG13T5/TG12T5
C-26	TG12T7/TG11T9	c.1405A>G; p.Met469Val (M469V)
C-27	TG13T5/TG13T5
C-28	TG13T5/TG11T7	c.326A>G; p.Tyr109Cys (Y109C), c.650A>G; p.Glu217Gly (E217G)
C-29	TG12T7/TG11T7	c.293A>G; p.Gln98Arg (Q98R), c.4056G>C; p.Gln1352His (Q1352H)
C-30	TG13T5/TG12T7	c.1209+1G>C (1341+1G>C)
C-31	TG13T5/TG12T5
C-32	TG13T5/TG13T5
C-33	TG11T7/TG11T7	c.1666A>G; p.Ile556Val (I556V)
C-34	TG13T7/TG12T7
C-35	TG12T7/TG11T7
C-36	TG12T7/TG12T7
C-37	TG13T5/TG12T5
C-38	TG13T5/TG12T7	c.3068T>G; p.Ile1023Arg (I1023R)
C-39	TG12T7/TG12T7
C-40	TG12T7/TG11T7	c.4056G>C; p.Gln1352His (Q1352H)
C-41	TG12T5/TG12T7
C-42	TG12T5/TG11T7	c.4056G>C; p.Gln1352His (Q1352H)
C-43	TG12T5/TG12T7	c.2834C>T; p.Ser945Leu (S945L)
C-44	TG12T5/TG11T7	c.2125C>T; p.Arg709^* (R709X)

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^aAll variants are heterozygous unless indicated as homozygous. ^bAll variants are hemizygous; CFTR: cystic fibrosis transmembrane conductance regulator; ADGRG2: adhesion G protein-coupled receptor G2

*CFTR* variant spectrum in CAVD cohort. (a) Distribution and allele frequency of different *CFTR* variant types. (b) Individuals (n, %) carrying *CFTR* and *ADGRG2* variants. (c) Distribution and allele frequency of *CFTR* variants of different clinical significance. (d) Combination of *CFTR* variants (n, %) by clinical significance. (e) *CFTR* variants with 2 or more variant alleles. (f) Distribution of CF-causing, VCC, and CFTR-RD-causing variants across *CFTR* gene. CF-causing variants that were not reported in Chinese CF patients were highlighted in red. VCC and CFTR-RD-causing variants were highlighted in yellow. NCF including VCC, CFTR-RD-causing, and VUS. *CFTR*: cystic fibrosis transmembrane conductance regulator; CAVD: congenital absence of the vas deferens; CF: cystic fibrosis; *ADGRG2*: adhesion G protein-coupled receptor G2; CFTR-RD: CFTR-related disorder; VCC: varying clinical consequence; VUS: variant of unknown clinical significance; ABC: ATP-binding cassette.

In our CAVD cohort, 37 patients had only one CFTR variant allele, and 71 had more than one variant alleles. Additionally, we found that three patients carried a pathogenic or likely pathogenic hemizygous variant of ADGRG2, with two patients having no concurrent CFTR variants and one patient also having a heterozygous CFTR IVS9-5T variant (Supplementary Table 3 and 4). In total, two CFTR variant alleles, one CFTR variant allele, and hemizygous ADGRG2 variants accounted for the pathogenic cause in 49.0%, 24.8%, and 2.1% of individuals with CAVD, respectively (Figure 1b).

Supplementary Table 4.

Frequency and in silico prediction of variants in adhesion G protein-coupled receptor G2

Variant	Number of alleles	gnomAD frequency		In silico prediction			ACMG classification

		Total	East Asian	SIFT	PolyPhen2	MutationTastor
c.1829G>A; p.Gly610Asp	1	0	0	Deleterious	Probably damaging	Disease-causing	Likely pathogenic
c.1969C>T; p.Arg657Trp	1	1.09×10⁻⁵	0	Deleterious	Probably damaging	Disease-causing	Likely pathogenic
c.2312A>G; p.Asn771Ser	6	2.11×10⁻³	6.16×10⁻²	Tolerated	Benign	Polymorphism	Benign
c.2336G>A; p.Trp779*	1	0	0	-	-	-	Pathogenic

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VUS: variant of unknown significance; ACMG: American College of Medical Genetics and Genomics; SIFT: Sorting Intolerant From Tolerant; -: not applicable

Clinical significance of CFTR variants in CAVD cohort

We further assessed the clinical significance of CFTR variants found in our CAVD cohort according to annotations from the CFTR2 and CFTR-France databases. Of the 56 CFTR variants identified, 20 were categorized as CF-causing variants, three as VCC variants, two as CFTR-RD-causing variants, and 31 as VUS (Table 2 and Figure 1c).

Eighteen different variants, including three indels, five missense, five nonsense, and five splicing variants, were annotated as CF-causing in either or both databases. Notably, two noncanonical splicing variants (c.869+5G>A and c.3140-26A>G) were shown to dramatically reduce the levels of normal CFTR transcripts through functional experiments (Supplementary Table 5)11,12 and have been annotated as CF-causing variants in databases. Three additional noncanonical splicing variants (c.1210-6T>A, c.2907A>C, and c.3469-3C>A) were in silico predicted or experimentally validated to cause aberrant splicing (Supplementary Table 5).13 However, they were still categorized as VUS because of the lack of quantitative data on their missplicing rates. One frameshift deletion (c.264_268del; p.Leu88Phefs^*21) and one canonical splicing variant (c.1767-1G>A), although not recorded, were also considered as CF-causing variants. Thus, 20 different CF-causing variants were identified in our CAVD cohort, eight (40.0%) of which had not been previously reported in Chinese CF patients.14

Supplementary Table 5.

Effect of noncanonical splicing variants of cystic fibrosis transmembrane conductance regulator

Variant	Effect	Missplicing rate	Evidence
c.869+5G>A	Exon 7 skipped	81.45%	In vitro study11
c.1210-6T>A	21 base pairs deletion from the 5’ end of exon 10 (in-frame)	Not available	NetGene2 prediction
c.2907A>C	Exon 17 skipped	Not determined	In vitro study13
c.3140-26A>G	25 base pairs extension of the 5’ end of exon 20 (out-of-frame, leading to a premature termination)	100%	In vivo study12
c.3469-3C>A	151 base pairs deletion from the 5’ end of exon 22 (out-of-frame, leading to a premature termination)	Not available	Human splicing finder prediction

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Two missense variants, c.1666A>G; p.Ile556Val and c.4056G>C; p.Gln1352His, were categorized as CFTR-RD-causing variants. In addition to the IVS9-5T variant, two other variants (c.3208C>T; p.Arg1070Trp, and c.3454G>C; p.Asp1152His) were also classified as VCC, which means that they can either result in CF or in a CFTR-RD when in trans with another CF-causing variant. The remaining variants were mostly in silico predicted to be deleterious, whereas none were predicted to affect splicing patterns (Supplementary Table 6). They were classified as VUS since they lacked sufficient genetic and functional evidence for clinical significance in causing CF or CFTR-RD.

Supplementary Table 6.

In silico prediction of variant of unknown significance variants of cystic fibrosis transmembrane conductance regulator in congenital absence of the vas deferens cohort

Variant	SIFT	PolyPhen2	MutationTastor	CADD score	SpliceAI Δ score	Pangolin Δ score
c.91C>T; p.Arg31Cys	Deleterious	Probably damaging	Disease-causing	27.3	0.01	0.03
c.92G>A; p.Arg31His	Tolerated	Benign	Disease-causing	22.5	0	0.01
c.215C>A; p.Ala72Asp	Deleterious	Probably damaging	Disease-causing	34	0.05	0.03
c.320C>A; p.Ala107Asp	Deleterious	Probably damaging	Disease-causing	26.5	0.01	0
c.326A>G; p.Tyr109Cys	Deleterious	Probably damaging	Disease-causing	26	0.03	0
c.400A>G; p.Arg134Gly	Deleterious	Probably damaging	Disease-causing	23.5	0.23	0.05
c.558C>G; p.Asn186Lys	Tolerated	Probably damaging	Disease-causing	25.3	0	0.01
c.592G>C; p.Ala198Pro	Deleterious	Probably damaging	Disease-causing	24.3	0	0.01
c.650A>G; p.Glu217Gly	Deleterious	Benign	Polymorphism	24.5	0.21	0.18
c.890G>A; p.Arg297Gln	Deleterious	Benign	Disease-causing	24.4	0.06	0.05
c.926C>G; p.Ala309Gly	Tolerated	Benign	Disease-causing	22.8	0.07	0.03
c.1069G>A; p.Ala357Thr	Deleterious	Probably damaging	Disease-causing	25.3	0.03	0.04
c.1351G>A; p.Gly451Arg	Deleterious	Probably damaging	Disease-causing	25	0.01	0.03
c.1403T>C; p.Leu468Pro	Deleterious	Probably damaging	Disease-causing	29.6	0.04	0.02
c.1405A>G; p.Met469Val	Tolerated	Probably damaging	Disease-causing	23	0.01	0.01
c.1520T>A; p.Ile507Asn	Deleterious	Probably damaging	Disease-causing	28.1	0	0
c.1586A>C; p.Asp529Ala	Deleterious	Probably damaging	Disease-causing	23.8	0.01	0
c.1775G>T; p.Cys592Phe	Deleterious	Probably damaging	Disease-causing	25	0.04	0.03
c.2042A>T; p.Glu681Val	Deleterious	Probably damaging	Disease-causing	22.8	0.36	0.25
c.2684G>A; p.Ser895Asn	Tolerated	Benign	Polymorphism	0.682	0.04	0.01
c.2812G>T; p.Val938Leu	Tolerated	Probably damaging	Disease-causing	24.9	0.04	0.03
c.2918T>G; p.Leu973Arg	Deleterious	Probably damaging	Disease-causing	28.4	0.02	0.04
c.2936A>C; p.Asp979Ala	Deleterious	Probably damaging	Disease-causing	28.3	0.01	0.02
c.2950G>A; p.Asp984Asn	Deleterious	Probably damaging	Disease-causing	31	0.19	0.13
c.3068T>G; p.Ile1023Arg	Tolerated	Probably damaging	Disease-causing	25.1	0.08	0.03
c.3406G>A; p.Ala1136Thr	Deleterious	Probably damaging	Disease-causing	25.3	0.01	0.02
c.4091C>T; p.Ala1364Val	Deleterious	Probably damaging	Disease-causing	35	0.13	0.06
c.4262T>A; p.Val1421Glu	Deleterious	Probably damaging	Disease-causing	27.6	0.02	0.01

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CADD (https://cadd.gs.washington.edu). SpliceAI and Pangolin scores are used for evaluation for potential impact on splicing (https://spliceailookup.broadinstitute.org). Δ scores range from 0 to 1 and can be interpreted as the probability that the variant affects splicing at any position within a window around it. For SpliceAI, a detailed characterization of the Δ scores is provided for 0.2 (high recall), 0.5 (recommended), and 0.8 (high precision) cutoffs. CADD: combined annotation dependent depletion; SIFT: Sorting Intolerant From Tolerant

Overall, 28 patients carried one CF-causing variant, either alone or in combination with NCF variants, resulting in a 19.3% prevalence of CF-causing variants in our cohort (Figure 1d). Additionally, 31.7% of the patients had two NCF variants and 23.4% harbored only one NCF variant. In addition to the IVS9-5T VCC variant, no significant CFTR hotspot variants were found in the CAVD cohort (Figure 1e). The most common CF-causing variant, c.2909G>A; p.Gly970Asp (G970D), was found in four patients, followed by p.Arg75^*, p.Gln98Arg, c.869+5G>A, c.1209+1G>C, and p.Thr663Argfs^*8, each identified in two patients (Figure 1e). The other 14 CF-causing variants were detected in only one patient each. The spectra of CF-causing, VCC, and CFTR-RD-causing variants in our CAVD cohort are shown in Figure 1f.

Literature review of CFTR variants in Chinese individuals with CAVD

Our literature search identified 11 studies that investigated CFTR variants in Chinese individuals with CAVD and met the inclusion criteria of our meta-analysis (Supplementary Figure 1^{(146.4KB, tif)}).15,16,17,18,19,20,21,22,23,24,25 Including our cohort, a total of 12 studies encompassing 867 Chinese individuals with CAVD were analyzed (Supplementary Table 7). The detailed CFTR genotypes of the 722 individuals with CAVD from 11 previous studies are provided in Supplementary Table 8.

Supplementary Table 7.

Studies detecting cystic fibrosis transmembrane conductance regulator variants in Chinese individual with congenital absence of the vas deferens that were included in meta-analysis

Study	Patients	Sequencing method	Target
Li et al. 201215	73 CBAVD	Sanger sequencing	All exons and flanking intronic regions of CFTR
Du et al. 201216	9 CBAVD	Sanger sequencing	All exons and flanking intronic regions of CFTR
Yang et al. 201517	11 CBAVD, 6 CUAVD, 2 CPAVD	Sanger sequencing	All exons and flanking intronic regions of CFTR
Yuan et al. 201918	50 CBAVD, 21 CUAVD, 1 CPAVD	Sanger sequencing	All exons and flanking intronic regions of CFTR and ADGRG2
Wang et al. 202019	38 CBAVD	Targeted NGS	5’ and 3’ UTR, coding regions, flanking introns, and pathogenic deep introns of CFTR and ADGRG2
Luo et al. 202120	266 CBAVD, 10 CUAVD	Multiplex PCR + NGS	5’ UTR, coding regions and flanking introns of CFTR
Tan et al. 202221	13 CBAVD, 9 CUAVD	WGS	Whole genome
Cheng et al. 202222	46 CBAVD	WES	Whole exome
Fang et al. 202223	46 CBAVD, 4 CUAVD	WES	Whole exome
Qu et al. 202324	97 CBAVD, 7 CUAVD	WES	Whole exome
Tang et al. 202425	13 CBAVD	WES	Whole exome
Present study	145 CBAVD	Multiplex PCR + NGS	All exons and flanking introns of CFTR and ADGRG2

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CBAVD: congenital bilateral absence of vas deferens; CUAVD: congenital unilateral absence of vas deferens; CPAVD: congenital bilateral partial aplasia of vas deferens; NGS: next-generation sequencing; PCR: polymerase chain reaction; WES: whole-exome sequencing; WGS: whole-genome sequencing; CFTR: cystic fibrosis transmembrane conductance regulator; ADGRG2: adhesion G protein-coupled receptor G2; UTR: untranslated region

Supplementary Table 8.

Cystic fibrosis transmembrane conductance regulator variants in each Chinese congenital absence of the vas deferens case from previous studies

graphic file with name AJA-27-611-g002.jpg

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Overall, 174 different CFTR variants were identified among Chinese individuals with CAVD, comprising 64 CF-causing variants, nine VCC variants, three CFTR-RD-causing variants, 97 VUS, and one non-disease-causing variant (Supplementary Table 9). Of the 64 CF-causing variants, 41 were recorded and annotated in either the CFTR2 or CFTR-France databases or both. The remaining 23 unrecorded variants, including nonsense, canonical splicing, and frameshift indels, were also classified as CF-causing variants. Notably, 36 (56.3%) of the CF-causing variants identified in individuals with CAVD have not previously been reported in Chinese CF patients.14

Supplementary Table 9.

Cystic fibrosis transmembrane conductance regulator variants identified in Chinese individuals with congenital absence of vas deferens from previous studies and the present study

Variant	Legacy name	Number of alleles	AF in Chinese patients (%)		AF in gnomAD^b		Clinical significance

			CAVD (n=867)	CF (n=202)^a	Total	Ease Asian	CFTR2^c	CFTR-France^d	Final determination
c.-34C>T	99C>T	6	0.35	0	1.25×10⁻⁶	4.46×10⁻⁵	NR	VUS	VUS
c.50del; p.Phe17Serfs^*8	182delT	1	0.06	0	6.20×10⁻⁷	0	CF-causing	NR	CF-causing
c.91C>T; p.Arg31Cys	R31C	4	0.23	0	1.51×10⁻³	6.76×10⁻³	Non CF-causing	VUS	VUS
c.92G>A; p.Arg31His	R31H	1	0.06	0	2.48×10⁻⁵	8.93×10⁻⁵	NR	NR	VUS
c.95T>G; p.Leu32Arg	L32R	1	0.06	0	0	0	NR	NR	VUS
c.148T>C; p.Ser50Pro	S50P	1	0.06	0	0	0	NR	CF-causing	CF-causing
c.215C>A; p.Ala72Asp	A72D	1	0.06	0	0	0	NR	VUS4	VUS
c.223C>T; p.Arg75^*	R75X	2	0.12	1.29	1.18×10⁻⁵	4.46×10⁻⁵	CF-causing	CF-causing	CF-causing
c.224G>A; p.Arg75Gln	R75Q	1	0.06	0	3.01×10⁻²	1.12×10⁻⁴	Non-CF-causing	Nondisease-causing	Nondisease-causinge
c.228del; p.Phe78Serfs^*13	360delT	1	0.06	0	6.20×10⁻⁷	0	NR	NR	CF-causing
c.263T>G; p.Leu88^*	L88X	7	0.4	2.58	6.45×10⁻⁷	0	CF-causing	CF-causing	CF-causing
c.264_268del; p.Leu88Phefs^*21	396delATATT	1	0.06	0.77	0	0	NR	NR	CF-causing
c.293A>G; p.Gln98Arg	Q98R	4	0.23	3.09	3.72×10⁻⁶	0	CF-causing	CF-causing	CF-causing
c.320C>A; p.Ala107Asp	A107D	2	0.12	0.26	0	0	NR	NR	VUS
c.326A>G; p.Tyr109Cys	Y109C	1	0.06	0.77	0	0	NR	VUS	VUS
c.328G>T; p.Asp110Tyr	D110Y	1	0.06	0	6.20×10⁻⁷	0	NR	NR	VUS
c.330del; p.Pro111Argfs^*13	462delC	1	0.06	0	6.20×10⁻⁷	0	NR	NR	CF-causing
c.374T>C; p.Ile125Thr	I125T	8	0.46	1.29	1.96×10⁻⁴	6.04×10⁻³	NR	NR	VUS
c.400A>G; p.Arg134Gly	R134G	2	0.12	0	0	0	NR	NR	VUS
c.407T>G; p.Leu136Arg	L136R	1	0.06	0	0	0	NR	NR	VUS
c.418C>T; p.Pro140Ser	P140S	1	0.06	0	2.82×10⁻⁴	2.23×10⁻⁴	NR	NR	VUS
c.461T>G; p.Ile154Arg	I154R	1	0.06	0	0	0	NR	NR	VUS
c.482A>G; p.Tyr161Cys	Y161C	1	0.06	0	1.86×10⁻⁶	2.23×10⁻⁵	NR	NR	VUS
c.520-2A>G	652-2A>G	1	0.06	0	0	0	NR	NR	CF-causing
c.532G>A; p.Gly178Arg	G178R	1	0.06	0.52	7.47×10⁻⁶	0	CF-causing	CF-causing	CF-causing
c.558C>G; p.Asn186Lys	N186K	9	0.52	0.52	6.32×10⁻⁷	0	NR	VUS4	VUS
c.579+4T>C	711+4T>C	1	0.06	0	1.37×10⁻⁶	0	NR	NR	VUS
c.579+5G>A	711+5G>A	1	0.06	0	6.86×10⁻⁷	0	CF-causing	CF-causing	CF-causing
c.579+7A>G	711+7A>G	1	0.06	0	0	0	NR	NR	VUS
c.592G>C; p.Ala198Pro	A198P	5	0.29	0	6.20×10⁻⁷	0	NR	NR	VUS
c.595C>T; p.His199Tyr	H199Y	1	0.06	2.32	1.24×10⁻⁶	0	CF-causing	CF-causing	CF-causing
c.601G>A; p.Val201Met	V201M	1	0.06	0	1.69×10⁻⁴	1.11×10⁻⁴	VUS	CFTR-RD-causing	CFTR-RD-causing
c.650A>G; p.Glu217Gly	E217G	11	0.63	1.55	2.06×10⁻³	1.10×10⁻²	NR	VUS2	VUS
c.764T>A; p.Ile255Asn	I255N	1	0.06	0	2.50×10⁻⁶	4.48×10⁻⁵	NR	NR	VUS
c.799G>A; p.Glu267Lys	E267K	1	0.06	0	0	0	NR	NR	VUS
c.861C>G; p.Asn287Lys	N287K	1	0.06	0.26	3.23×10⁻⁵	9.61×10⁻⁴	NR	VUS3	VUS
c.865A>T; p.Arg289^*	R289X	2	0.12	0.26	0	0	CF-causing	NR	CF-causing
c.868C>T; p.Gln290^*	Q290X	1	0.06	0.26	6.21×10⁻⁷	0	CF-causing	CF-causing	CF-causing
c.869+3A>T	1001+3A>T	2	0.12	0	6.21×10⁻⁷	0	NR	NR	VUS
c.869+5G>A	1001+5G>A	5	0.29	0	1.24×10⁻⁶	2.24×10⁻⁵	NR	CF-causing	CF-causing
c.870-1G>C	1002-1G>C	1	0.06	0.26	0	0	NR	NR	CF-causing
c.890G>A; p.Arg297Gln	R297Q	1	0.06	0	8.85×10⁻⁴	2.68×10⁻⁴	NR	VUS1	VUS
c.926C>G; p.Ala309Gly	A309G	4	0.23	0	0	0	NR	VUS4	VUS
c.935_937del; p.Phe312del	ΔF312	1	0.06	0	0	0	VCC	Disease-causing	VCC
c.988G>T; p.Gly330^*	G330X	1	0.06	0	2.48×10⁻⁶	0	CF-causing	NR	CF-causing
c.1000C>T; p.Arg334Trp	R334W	1	0.06	2.58	2.91×10⁻⁵	0	CF-causing	CF-causing	CF-causing
c.1040G>A; p.Arg347His	R347H	4	0.23	0.26	5.21×10⁻⁵	0	CF-causing	VCC	CF-causing
c.1055G>A; p.Arg352Gln	R352Q	1	0.06	0	1.61×10⁻⁵	0	CF-causing	CF-causing	CF-causing
c.1069G>A; p.Ala357Thr	A357T	5	0.29	0	0	0	NR	VUS2	VUS
c.1070C>T; p.Ala357Val	A357V	1	0.06	0	0	0	NR	VUS	VUS
c.1115A>G; p.Gln372Arg	Q372R	1	0.06	0	0	0	NR	NR	VUS
c.1124T>C; p.Leu375Ser	L375S	1	0.06	0	0	0	NR	NR	VUS
c.1159_1160del; p.Leu387Asnfs^*23	1291delTT	1	0.06	0	0	0	CF-causing	NR	CF-causing
c.1163C>A; p.Thr388Lys	T388K	1	0.06	0	0	0	NR	NR	VUS
c.1209+1G>C	1341+1G>C	3	0.17	0	0	0	CF-causing	NR	CF-causing
c.1209+2T>G	1341+2T>G	1	0.06	0	0	0	CF-causing	NR	CF-causing
c.1210-12T[5]	5T	515	29.7	0	−	−	VCC	VCC	VCC
c.1210-6T>A	1342-6T>A	3	0.17	0	2.85×10⁻⁵	0	NR	VUS4	VUS
c.1231A>G; p.Lys411Glu	K411E	2	0.12	0.26	3.72×10⁻⁵	1.34×10⁻³	NR	NR	VUS
c.1256G>T; p.Arg419Ile	R419I	1	0.06	0	0	0	NR	NR	VUS
c.1330A>T; p.Ile444Phe	I419F	2	0.12	0	0	0	NR	NR	VUS
c.1351G>A; p.Gly451Arg	G451R	5	0.29	0	3.10×10⁻⁶	6.69×10⁻⁵	NR	NR	VUS
c.1357del; p.Leu453Cysfs^*16	1489delT	1	0.06	0	0	0	NR	NR	CF-causing
c.1390A>C; p.Lys464Gln	K464Q	3	0.17	0	0	0	NR	VUS	VUS
c.1394C>A; p.Thr465Asn	T465N	1	0.06	0	1.88×10⁻⁶	0	NR	NR	VUS
c.1394C>T; p.Thr465Ile	T465I	1	0.06	0	6.26×10⁻⁷	0	NR	NR	VUS
c.1403T>C; p.Leu468Pro	L468P	1	0.06	0	0	0	NR	NR	VUS
c.1405A>G; p.Met469Val	M469V	2	0.12	0	7.48×10⁻⁶	6.70×10⁻⁵	NR	Disease-causing	VUS
c.1407del; p.Val470^*	1539delG	1	0.06	0	0	0	NR	NR	CF-causing
c.1407G>T; p.Met469Ile	M469I	5	0.29	0	4.36×10⁻⁶	1.34×10⁻⁴	NR	NR	VUS
c.1409T>A; p.Val470Glu	V470E	2	0.12	1.03	0	0	NR	NR	VUS
c.1423del; p.Leu475Trpfs^*52	1555delC	1	0.06	0.26	0	0	NR	NR	CF-causing
c.1426G>A; p.Glu476Lys	E476K	1	0.06	0	0	0	NR	NR	VUS
c.1453A>T; p.Ser485Cys	S485C	3	0.17	0	1.18×10⁻⁶	4.24×10⁻⁴	NR	VUS	VUS
c.1472G>T; p.Cys491Phe	C491F	1	0.06	0	2.73×10⁻⁵	0	NR	NR	VUS
c.1486T>C; p.Trp496Arg	W496R	1	0.06	0	0	0	NR	NR	VUS
c.1489A>G; p.Ile497Val	I497V	1	0.06	0	6.20×10⁻⁷	0	NR	NR	VUS
c.1520T>A; p.Ile507Asn	I507N	1	0.06	0	0	0	NR	NR	VUS
c.1521_1523del; p.Phe508del	ΔF508	5	0.29	1.8	1.19×10⁻²	2.23×10⁻⁵	CF-causing	CF-causing	CF-causing
c.1558G>A; p.Val520Ile	V520I	2	0.12	0	1.14×10⁻⁴	9.82×10⁻⁴	NR	NR	VUS
c.1586A>C; p.Asp529Ala	D529A	2	0.12	0	0	0	NR	NR	VUS
c.1591T>C; p.Ser531Pro	S531P	1	0.06	0	0	0	NR	NR	VUS
c.1657C>T; p.Arg553^*	R553X	6	0.35	3.61	9.71×10⁻⁵	2.23×10⁻⁵	CF-causing	CF-causing	CF-causing
c.1666A>G; p.Ile556Val	I556V	44	2.54	1.55	1.27×10⁻³	4.20×10⁻²	NR	CFTR-RD-causing	CFTR-RD-causing
c.1669T>A; p.Ser557Thr	S557T	1	0.06	0	3.76×10⁻⁶	1.12×10⁻⁴	NR	NR	VUS
c.1673T>C; p.Leu558Ser	L558S	1	0.06	0	1.26×10⁻⁶	0	CF-causing	CF-causing	CF-causing
c.1680-1G>A	1812-1G>A	1	0.06	0	1.87×10⁻⁶	2.25×10⁻⁵	CF-causing	CF-causing	CF-causing
c.1697C>T; p.Ala566Val	A566V	1	0.06	0	0	0	NR	NR	VUS
c.1731C>A; p.Tyr577^*	Y577X	1	0.06	0	0	0	CF-causing	NR	CF-causing
c.1766+1G>T	1898+1G>T	1	0.06	0.26	0	0	CF-causing	NR	CF-causing
c.1766+5G>T	1898+5G>T	9	0.52	5.41	3.75×10⁻⁶	1.12×10⁻⁴	CF-causing	CF-causing	CF-causing
c.1767-2A>C	1899-2A>C	1	0.06	0	6.42×10⁻⁷	1.19×10⁻⁵	NR	NR	CF-causing
c.1767-1G>A	1899-1G>A	1	0.06	0	0	0	NR	NR	CF-causing
c.1775G>T; p.Cys592Phe	C592F	2	0.12	0	0	0	NR	NR	VUS
c.1798A>G; p.Arg600Gly	R600G	1	0.06	0	0	0	NR	VUS	VUS
c.1810A>C; p.Thr604Pro	T604P	1	0.06	0.52	0	0	NR	NR	VUS
c.1826A>G; p.His609Arg	H609R	1	0.06	0	1.25×10⁻⁶	0	CF-causing	Disease-causing	CF-causing
c.1837G>A; p.Ala613Thr	A613T	1	0.06	0	3.77×10⁻⁶	2.23×10⁻⁵	CF-causing	NR	CF-causing
c.1865G>A; p.Gly622Asp	G622D	1	0.06	0	1.20×10⁻⁴	5.58×10⁻⁴	VCC	VCC	VCC
c.1900C>T; p.Gln634^*	Q634X	1	0.06	0	0	0	CF-causing	NR	CF-causing
c.1925C>G; p.Ser642^*	S642X	1	0.06	0	0	0	NR	NR	CF-causing
c.1979C>A; p.Ser660^*	S600X	1	0.06	0	0	0	NR	NR	CF-causing
c.1986_1989del; p.Thr663Argfs^*8	2118delAACT	2	0.12	0	5.58×10⁻⁶	0	CF-causing	CF-causing	CF-causing
c.2036G>A; p.Trp679^*	W679X	2	0.12	1.03	1.86×10⁻⁶	0	CF-causing	NR	CF-causing
c.2042A>T; p.Glu681Val	E681V	7	0.4	0.26	5.89×10⁻⁵	2.07×10⁻³	NR	NR	VUS
c.2046dup; p.Gln685Thrfs^*4	2178insA	1	0.06	0	3.41×10⁻⁵	0	NR	NR	CF-causing
c.2080_2081insG; p.Glu695Glyfs^*35	2212insG	1	0.06	0	6.20×10⁻⁷	2.23×10⁻⁵	NR	NR	CF-causing
c.2125C>T; p.Arg709^*	R709X	1	0.06	0.77	1.12×10⁻⁵	0	CF-causing	CF-causing	CF-causing
c.2147del; p.Lys716Argfs^*6	2279delA	2	0.12	0	0	0	NR	NR	CF-causing
c.2213T>G; p.Leu738^*	L738X	1	0.06	0	0	0	NR	NR	CF-causing
c.2249C>T; p.Pro750Leu	P750L	1	0.06	0	6.04×10⁻⁴	6.69×10⁻⁵	VCC	CFTR-RD-causing	VCC
c.2354G>A; p.Arg785Gln	R785Q	1	0.06	0	3.47×10⁻⁵	2.26×10⁻⁵	NR	NR	VUS
c.2374C>T; p.Arg792^*	R792X	1	0.06	0.77	3.29×10⁻⁶	0	CF-causing	CF-causing	CF-causing
c.2540A>G; p.Asn847Ser	N847S	1	0.06	0	1.18×10⁻⁵	3.80×10⁻⁴	NR	VUS	VUS
c.2551C>T; p.Arg851^*	R851X	1	0.06	0.52	2.48×10⁻⁶	0	CF-causing	CF-causing	CF-causing
c.2552G>A; p.Arg851Gln	R851Q	1	0.06	0	2.42×10⁻⁵	3.80×10⁻⁴	NR	NR	VUS
c.2646G>A; p.Trp882^*	W882X	1	0.06	0	0	0	CF-causing	NR	CF-causing
c.2672A>G; p.Asp891Gly	D891G	1	0.06	0	1.49×10⁻⁵	0	NR	VUS	VUS
c.2684G>A; p.Ser895Asn	S895N	7	0.4	0.52	7.31×10⁻⁵	2.43×10⁻³	NR	NR	VUS
c.2797A>G; p.Arg933Gly	R933G	1	0.06	0	1.24×10⁻⁶	0	VCC	CFTR-RD-causing	VCC
c.2812G>T; p.Val938Leu	V938L	6	0.35	0.26	1.24×10⁻⁶	4.46×10⁻⁵	NR	NR	VUS
c.2812G>A; p.Val938Met	V938M	1	0.06	0	1.24×10⁻⁶	4.46×10⁻⁵	NR	NR	VUS
c.2834C>T; p.Ser945Leu	S945L	1	0.06	1.03	4.09×10⁻⁵	0	CF-causing	VCC	CF-causing
c.2846A>C; p.His949Pro	H949P	1	0.06	0	0	0	NR	VUS	VUS
c.2907A>C; p.Ala969Ala	NULL	4	0.23	0.26	1.24×10⁻⁶	4.46×10⁻⁵	NR	NR	VUS
c.2909G>A; p.Gly970Asp	G970D	25	1.44	12.11	5.94×10⁻⁶	1.58×10⁻⁴	CF-causing	CF-causing	CF-causing
c.2918T>G; p.Leu973Arg	L973R	1	0.06	0	0	0	NR	NR	VUS
c.2927T>C; p.Phe976Ser	F976S	1	0.06	0	1.27×10⁻⁶	0	NR	NR	VUS
c.2929T>C; p.Ser977Pro	S977P	3	0.17	0	0	0	NR	NR	VUS
c.2936A>C; p.Asp979Ala	D979A	11	0.63	0.26	8.79×10⁻⁶	2.91×10⁻⁴	NR	VUS4	VUS
c.2950G>A; p.Asp984Asn	D984N	1	0.06	0	6.28×10⁻⁷	0	NR	NR	VUS
c.2977G>T; p.Asp993Tyr	D993Y	1	0.06	1	6.38×10⁻⁷	0	NR	CF-causing	CF-causing
c.2988+2T>C	3120+2T>C	2	0.12	1	0	0	NR	NR	CF-causing
c.3021dup; p.Val1008Cysfs^*39	3153insT	1	0.06	0	0	0	NR	NR	CF-causing
c.3062C>T; p.Pro1021Leu	P1021L	3	0.17	0.52	0	0	NR	NR	VUS
c.3068T>G; p.Ile1023Arg	I1023R	4	0.23	2.84	0	0	NR	VUS4	VUS
c.3084_3085del; p.Met1028Ilefs^*18	3216delGT	1	0.06	0	0	0	NR	NR	CF-causing
c.3140-26A>G	3272-26A>G	2	0.12	0.52	1.02×10⁻⁴	0	CF-causing	VCC	CF-causing
c.3193C>T; p.Leu1065Phe	L1065F	1	0.06	0	6.20×10⁻⁷	0	NR	NR	VUS
c.3200C>T; p.Ala1067Val	A1067V	1	0.06	0	7.50×10⁻⁵	0	NR	VUS	VUS
c.3205G>A; p.Gly1069Arg	G1069R	5	0.29	0	1.36×10⁻⁵	1.14×10⁻⁴	VCC	CFTR-RD-causing	VCC
c.3208C>T; p.Arg1070Trp	R1070W	2	0.12	0	8.00×10⁻⁵	2.20×10⁻⁵	VCC	VCC	VCC
c.3209G>A; p.Arg1070Gln	R1070Q	1	0.06	0	3.07×10⁻⁴	1.56×10⁻⁴	VCC	VCC	VCC
c.3289C>T; p.Arg1097Cys	R1097C	1	0.06	0	6.14×10⁻⁵	1.76×10⁻³	NR	NR	VUS
c.3315G>T; p.Met1105Ile	M1105I	1	0.06	0	0	0	NR	NR	VUS
c.3350T>C; p.Ile1117Thr	I1117T	1	0.06	0	0	0	NR	NR	VUS
c.3367+2T>C	3499+2T>C	1	0.06	0	0	0	CF-causing	NR	CF-causing
c.3406G>A; p.Ala1136Thr	A1136T	3	0.17	0.26	1.43×10⁻⁵	1.34×10⁻⁴	NR	VUS2	VUS
c.3407C>T; p.Ala1136Val	A1136V	1	0.06	0	2.48×10⁻⁶	2.23×10⁻⁵	NR	NR	VUS
c.3454G>C; p.Asp1152His	D1152H	1	0.06	0	3.98×10⁻⁴	0	VCC	VCC	VCC
c.3468+1G>A	3600+1G>A	1	0.06	0	0	0	CF-causing	NR	CF-causing
c.3469-3C>A	3601-3C>A	9	0.52	0	6.82×10⁻⁶	2.45×10⁻⁴	NR	NR	VUS
c.3484C>T; p.Arg1162^*	R1162X	1	0.06	0.52	4.59×10⁻⁵	0	CF-causing	CF-causing	CF-causing
c.3623G>A; p.Gly1208Asp	G1208D	1	0.06	0	1.24×10⁻⁶	0	NR	NR	VUS
c.3635del; p.Val1212Alafs^*16	3767delT	1	0.06	0.77	0	0	NR	NR	CF-causing
c.3659C>T; p.Thr1220Ile	T1220I	1	0.06	0.26	5.58×10⁻⁵	1.03×10⁻³	NR	NR	VUS
c.3700A>G; p.Ile1234Val	I1234V	2	0.12	0.52	6.82×10⁻⁶	0	CF-causing	CF-causing	CF-causing
c.3710G>A; p.Gly1237Asp	G1237D	1	0.06	0	1.12×10⁻⁵	2.90×10⁻⁴	NR	NR	VUS
c.3717+45G>A	3849+45G>A	1	0.06	0	0	0	NR	NR	VUS
c.3829A>G; p.Ile1277Val	I1277V	1	0.06	0	7.44×10⁻⁶	4.46×10⁻⁵	NR	NR	VUS
c.3878T>C; p.Val1293Ala	V1293A	1	0.06	0	0	0	NR	NR	VUS
c.3874-4A>G	4006-4A>G	1	0.06	0	0	0	NR	VUS	VUS
c.3907A>C; p.Asn1303His	N1303H	1	0.06	0	0	0	NR	NR	VUS
c.3946T>C; p.Trp1316Arg	W1316R	1	0.06	0	2.49×10⁻⁶	0	NR	NR	VUS
c.3961G>A; p.Glu1321Lys	E1321K	1	0.06	0	1.89×10⁻⁶	4.48×10⁻⁵	NR	NR	VUS
c.3987_3988del; p.Gln1330Valfs^*6	4119delAC	1	0.06	0	0	0	NR	NR	CF-causing
c.4028G>T; p.Gly1343Val	G1343V	1	0.06	0	2.48×10⁻⁶	0	NR	NR	VUS
c.4056G>C; p.Gln1352His	Q1352H	65	3.75	1.03	5.87×10⁻⁴	1.92×10⁻²	NR	CFTR-RD-causing	CFTR-RD-causing
c.4091C>T; p.Ala1364Val	A1364V	2	0.12	0	1.80×10⁻⁵	6.68×10⁻⁵	NR	VUS4	VUS
c.4137-4C>A	4269-4C>A	1	0.06	0	1.93×10⁻⁶	6.74×10⁻⁵	NR	NR	VUS
c.4262T>A; p.Val1421Glu	V1421Q	2	0.12	0	0	0	NR	NR	VUS
c.4297G>A; p.Glu1433Lys	E1433K	2	0.12	0	1.49×10⁻⁵	0	NR	VUS	VUS
c.4297G>T; p.Glu1433^*	E1433X	1	0.06	0	1.24×10⁻⁶	0	NR	NR	CF-causing
c.4433C>G; p.Thr1478Arg	T1478R	3	0.17	0	1.86×10⁻⁶	4.46×10⁻⁵	NR	VUS	VUS

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^aChinese CF patients reported and summarized by Shen et al.;14 ^bgnomAD v4.1.0 (https://gnomad.broadinstitute.org); ^cUpdated date: April 7, 2023 (https://www.cftr2.org); ^dUpdated date: September 22, 2023 (https://cftr.iurc.montp.inserm.fr/cftr); ^eClassified as nondisease-causing considering the classifications in CFTR2 (non-CF-causing) and CFTR-France (nondisease-causing) and its high population frequency (3%). AF: allele frequency; CAVD: congenital absence of vas deferens; CF: cystic fibrosis; CFTR: CF transmembrane conductance regulator; CFTR-RD: CFTR-related disorders; NR: not recorded; VCC: varying clinical consequence; VUS: variant of unknown clinical significance

As shown in Figure 2a, the most prevalent CFTR variant in Chinese individuals with CAVD was IVS9-5T VCC, which present in 515 alleles. However, there was no obvious hotspot of CF-causing variants, as they were distributed throughout CFTR gene (Figure 2b). The most common CF-causing variant was c.2909G>A; p.Gly970Asp (G970D), which had an allele frequency of 1.4% and was the only CF-causing variant with a frequency >1%. Seventeen CF-causing variants were identified in two or more patients, while the remaining variants were detected in only one patient each. None of the patients harbored more than one CF-causing variant. The distributions of CF-causing, VCC, and CFTR-RD-causing variants among all Chinese individuals with CAVD are illustrated in Figure 2c.

*CFTR* variant spectrum in 867 Chinese individuals with CAVD from previous studies and present cohort. (a) NCF variants with ≥5 alleles. (b) CF-causing variants with ≥2 alleles. (c) Distribution of CF-causing, VCC, and CFTR-RD-causing variants across *CFTR* gene. CF-causing variants that were not reported in Chinese CF patients were highlighted in red. VCC and CFTR-RD-causing variants were highlighted in yellow. NCF including VCC, CFTR-RD-causing, and VUS. *CFTR*: cystic fibrosis transmembrane conductance regulator; CAVD: congenital absence of the vas deferens; CF: cystic fibrosis; CFTR-RD: CFTR-related disorder; VCC: varying clinical consequence; VUS: variant of unknown clinical significance; ABC: ATP-binding cassette.

Meta-analysis

We conducted a meta-analysis to estimate the prevalence of CFTR variants in Chinese individuals with CAVD, focusing on the clinical significance, allele number, and IVS9-T polymorphism (Supplementary Table 10). Pooled analysis demonstrated that 14.8% (95% confidence interval [CI]: 11.0%–18.9%) CAVD cases harbored one CF-causing variant, 28.7% (95% CI: 24.4%–33.2%) CAVD cases had the NCF/NCF combination, and 22.8% (95% CI: 19.5%–26.2%) CAVD cases carried only one NCF variant (Figure 3). Regarding allele number, 68.6% (95% CI: 65.1%–72.0%) of individuals with CAVD carried at least one CFTR variant allele, 43.3% (95% CI: 38.5%–48.2%) had two CFTR variant alleles, and 24.2% (95% CI: 20.7%–27.9%) harbored only one CFTR variant allele (Figure 4). The allele frequency of the IVS9-5T variant was estimated to be 29.5% (95% CI: 27.3%–31.7%), with 11.1% (95% CI: 8.9%–13.4%) of individuals with CAVD being homozygous and 35.4% (95% CI: 29.9%–41.1%) being heterozygous for the IVS9-5T variant (Supplementary Figure 2^{(166.6KB, tif)}).

Supplementary Table 10.

Meta-analysis of prevalence of cystic fibrosis transmembrane conductance regulator variants in Chinese individuals with congenital absence of vas deferens

CFTR variant carrier	CAVD (n=867)	CBAVD (n=760)^a	CUAVD/CPAVD (n=57)^a	P^b
Clinical significance
[CF]	14.8% (11.0–18.9)	15.5% (11.2–20.3)	5.8% (0–18.8)	0.229
[NCF]/[NCF]^c	28.7% (24.4–33.2)	27.9% (23.0–33.1)	38.0% (25.0–51.9)	0.120
[NCF]/none^c	22.8% (19.5–26.2)	23.5% (20.4–26.7)	23.7% (12.6–36.7)	0.790
Allele number
Variant carrier	68.6% (65.1–72.0)	69.2% (65.4–72.9)	77.1% (58.4–92.1)	0.465
Two variant alleles	43.3% (38.5–48.2)	43.4% (37.5–49.4)	47.3% (33.6–61.2)	0.584
One variant allele	24.2% (20.7–27.9)	25.0% (21.6–28.5)	25.3% (13.9–38.4)	0.790
IVS9-5T variant
AF	29.5% (27.3–31.7)	29.9% (27.2–32.6)	28.3% (19.6–37.8)	0.837
Homozygous 5T	11.1% (8.9–13.4)	10.4% (8.1–12.9)	16.8% (7.1–28.8)	0.107
Heterozygous 5T	35.4% (29.9–41.1)	37.8% (31.3–44.6)	23.0% (8.2–41.3)	0.150

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^aThe study by Fang et al.23 was excluded from subgroup analysis since the detailed vas deferens phenotype of each patient was not reported. ^bComparison between CBAVD and CUAVD/CPAVD. ^cExcluding carriers of [CF] variants. [CF]: CF-causing variants; [NCF]: including VCC, CFTR-RD-causing variants, and VUS. CAVD: congenital absence of vas deferens; CBAVD: congenital bilateral absence of vas deferens; CPAVD: congenital bilaterial partial aplasia of vas deferens; CUAVD: congenital unilateral absence of vas deferens; CF: cystic fibrosis; CFTR: CF transmembrane conductance regulator; CFTR-RD: CFTR-related disorders; VCC: varying clinical consequence; VUS: variant of unknown clinical significance; NCF: non-CF-causing; AF: allele frequency

Forest plot of proportions of patients carrying *CFTR* variants regarding clinical significance in Chinese CAVD population. NCF: including VCC, CFTR-RD-causing, and VUS. *CFTR*: cystic fibrosis transmembrane conductance regulator; CAVD: congenital absence of the vas deferens; CF: cystic fibrosis; CI: confidence interval; VCC: varying clinical consequence; VUS: variant of unknown clinical significance; ES: effect size.

Forest plot of proportions of patients carrying *CFTR* variants regarding allele number in Chinese CAVD population. *CFTR*: cystic fibrosis transmembrane conductance regulator; CAVD: congenital absence of the vas deferens; CI: confidence interval; ES: effect size.

Further meta-analysis was performed in subgroups of individuals with CBAVD and CUAVD/CPAVD (Supplementary Table 10). CF-causing variants were detected in 15.5% (95% CI: 11.2%–20.3%) of CBAVD (Supplementary Figure 3^{(166.1KB, tif)}) and in 5.8% (95% CI: 0–18.8%) of CUAVD/CPAVD (Supplementary Figure 4^{(108.6KB, tif)}). The proportion of patients harboring at least one CFTR variant was 69.2% (95% CI: 65.4%–72.9%; Supplementary Figure 5^{(166.8KB, tif)}) in the CBAVD subgroup and 77.1% (95% CI: 58.4%–92.1%; Supplementary Figure 6^{(114.3KB, tif)}) in the CUAVD/CPAVD subgroup. The allele frequency of the IVS9-5T variant was 29.9% (95% CI: 27.2%–32.6%; Supplementary Figure 7^{(167.1KB, tif)}) in the CBAVD subgroup and 28.3% (95% CI: 19.6%–37.8%; Supplementary Figure 8^{(112.1KB, tif)}) in the CUAVD/CPAVD subgroup. No significant differences were observed in these proportions between the CBAVD and CUAVD/CPAVD subgroups (Supplementary Table 10).

Seven previous studies, along with the current study, sequenced ADGRG2 in a total of 448 individuals with CBAVD (Supplementary Table 11). The hemizygous ADGRG2 variant was found in seven patients, accounting for 1.6% of all individuals with CBAVD.

Supplementary Table 11.

Adhesion G protein-coupled receptor G2 variants in Chinese individuals with congenital bilateral absence of vas deferens from studies sequencing adhesion G protein-coupled receptor G2

Study	Patient	ADGRG2 variant	Concurrent CFTR variant
Yuan et al.18 2019	16	c.2452A>T; p.Lys818^*	No
	17	c.3023G>A; p.Arg1008Gln	No
Wang et al.19 2020	Lib2100	c.2038C>T; p.His680Tyr	No
Tan et al.21 2022	None	-	-
Cheng et al.22 2022	None	-	-
Fang et al.23 2022	None	-	-
Qu et al.24 2023	P73	c.2041A>G; p.Met681Val	No
Tang et al.25 2024	None	-	-
Present study	CN158	c.1829G>A; p.Gly610Asp	No
	CN208	c.1969C>T; p.Arg657Trp	IVS9-5T
	C-17	c.2336G>A; p.Trp779^*	No

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ADGRG2: Adhesion G protein-coupled receptor G2; CFTR: cystic fibrosis transmembrane conductance regulator

DISCUSSION

Through targeted sequencing of a cohort of individuals with CAVD, we identified 56 different CFTR variants, of which 20 were categorized as CF-causing variants detected in 28 individuals. A comprehensive analysis covers 867 Chinese individuals from 11 previous studies and the current cohort was performed. This analysis identified 64 CF-causing variants, 56.3% of which had not been previously reported in Chinese patients with CF. Meta-analysis revealed that 14.8% of Chinese individuals with CAVD harbored a CF-causing variant, and 68.6% carried at least one CFTR variant. These findings underscore the urgent need for CFTR variant screening and genetic counseling in Chinese individuals with CAVD who wish to have children through ART.

CF is rare in the Asian populations. To date, approximately 200 genetically confirmed cases of CF of Chinese origin have been documented in the literature, with most cases diagnosed in recent years, suggesting an underestimation of its incidence.14 Despite the low prevalence of CF in China, CFTR screening and genetic counseling for CAVD couples desiring children should not be overlooked. Our study indicates that 14.8% of Chinese individuals with CAVD carry a CF-causing variant. Considering the large number of individuals with CAVD, this proportion could lead to a significant increase in CF-causing carriers within the general population and a potential rise in CF patients as more Chinese couples with CAVD opt for ART.

The spectrum of CF-causing variants in the Chinese CAVD population shows overlap and divergence from that observed in Chinese CF patients. The most predominant CF-causing, p.Gly970Asp (G970D), was found in 25 (2.9%) of the 867 individuals with CAVD, accounting for 19.1% of the 131 CF-causing variant alleles. It is the most common variant among Chinese CF patients, with an allele frequency of 12.11%.14 In contrast, more than half of the CF-causing variants identified in individuals with CAVD, including c.869+5G>A (five alleles), c.1209+1G>C (three alleles), c.1986_1989del; p.Thr663Argfs^*8 (two alleles), and c.2147del; p.Lys716Argfs^*6 (two alleles), have not been detected in CF patients. These findings largely expand the known spectrum of CF-causing variants in the Chinese population.

Our findings indicated significant differences in the spectra of CFTR and CF-causing variants between Chinese and White CAVD populations. Approximately 87% of White patients harbor one CF-causing variant, with p.Phe508del (ΔF508) being the most prevalent.7 In contrast, only 14.8% of the Chinese patients carried a CF-causing variant, with numerous low-frequency variants distributed throughout the gene. These distinctions may necessitate the use of different strategies for CFTR screening. The American College of Medical Genetics and Genomics (ACMG) has established a core panel of the 23 most frequent (>0.1%) CF-causing variants for CFTR mutation carrier screening in the American White population.26 This panel covers 84% of all the mutated alleles and was recently updated to 100 variants.27 Owing to the variation in CF-causing variant distribution across racial and ethnic groups, many expanded variant panels have been developed for different screening programs.28,29 However, these panels are not well-suited to the Chinese population. Among the 23 genes in the ACMG panel, only five (p.Phe508del, p.Arg553^*, p.Arg1162^*, c.1766+1G>A, and p.Arg334Trp) were identified in 14 Chinese individuals with CAVD. Conversely, the predominant CF-causing variants in Chinese individuals with CAVD, such as p.Gly970Asp, c.1766+5G>T, p.Leu88^*, and c.869+5G>A, are extremely rare in White populations, each with an allele frequency of less than 0.01% according to CFTR2 database. Given that the majority of CF-causing variants identified in the Chinese CAVD and CF populations were detected only once, developing a CFTR variant panel that encompasses most mutated alleles appears unfeasible. Therefore, extensive CFTR analysis through the sequencing of whole exons and flanking regions may be more appropriate, although the high costs and challenges of interpreting the clinical significance of novel variants pose potential obstacles.

The prevalence of CF-causing variants may have been underestimated for several reasons. Deep intronic variants, such as c.1680-886A>G (also known as 1811+1.6kbA>G), c.2989-313A>T, c.3469-1304C>G (3600+11.5kbC>G), c.3718-2477C>T (3849+10kbC>T), and c.3874-4522A>G (4005+5727A>G), can result in various phenotypes, including typical CF.30,31,32 Moreover, large deletions and duplications in CFTR have been identified in individuals with CAVD.33,34 However, most of the individuals with CAVD included in our study were not tested for deep introns regions or large rearrangements. Detection of large rearrangements and deep intronic CF-causing variants should be considered when establishing CFTR screening strategy in the Chinese population. Furthermore, many missense and noncanonical splicing variants are currently classified as VUS. Although in silico predicted to disrupt splicing patterns or have deleterious impact on protein function, their clinical significance cannot be fully determined because of insufficient functional and genetic evidence. As the evidence accumulates, some of these variants may be reclassified as CF-causing variants.

This meta-analysis estimates the prevalence of CFTR variants, CF-causing variants, and IVS9-5T alleles in Chinese individuals with CAVD. This highlights the urgent need for extensive CFTR analysis, encompassing whole exons and flanking regions, large rearrangements, and deep intronic CF-causing variants, in Chinese individuals with CAVD before ART. The established spectrum of CF-causing variants may contribute to the development of genetic counseling strategies and preimplantation diagnosis to prevent the birth of a child with CF.

AUTHOR CONTRIBUTIONS

HJL and BBW were involved in the conception and design of the study. YL and JW performed literature search and data extraction and wrote original draft. ZLC performed meta-analysis. TYL performed Sanger sequencing. All authors read and approved the final manuscript.

COMPETING INTERESTS

All authors declare no competing interests.

Supplementary Figure 1

Flowchart diagram of literature search.

AJA-27-611_Suppl1.tif^{(146.4KB, tif)}

Supplementary Figure 2

Forest plot of allele frequency of IVS9-5T variant Chinese CAVD population; CAVD: congenital absence of the vas deferens; CI: confidence interval; ES: effect size; het: heterozygous; homo: homozygous.

AJA-27-611_Suppl2.tif^{(166.6KB, tif)}

Supplementary Figure 3

Forest plot of proportions of patients carrying CFTR variants regarding clinical significance in Chinese CBAVD population (n = 760). CBAVD: congenital bilateral absence of the vas deferens; CFTR: cystic fibrosis transmenbrane conductance regulator; CI: confidence interval; ES: effect size; [CF]: CF-causing variant; [NCF]: non-CF-causing variant.

AJA-27-611_Suppl3.tif^{(166.1KB, tif)}

Supplementary Figure 4

Forest plot of proportions of patients carrying CFTR variants regarding clinical significance in Chinese CUAVD/CPAVD population (n = 57). CFTR: cystic fibrosis transmenbrane conductance regulator; CUAVD: congenital unilateral absence of vas deferens; CPAVD: congenital bilateral partial aplasia of vas deferens; CI: confidence interval; ES: effect size; [CF]: CF-causing variant; [NCF]: non-CF-causing variant.

AJA-27-611_Suppl4.tif^{(108.6KB, tif)}

Supplementary Figure 5

Forest plot of proportions of patients carrying CFTR variants regarding allele number in Chinese CBAVD population (n = 760). CBAVD: congenital bilateral absence of the vas deferens; CFTR: cystic fibrosis transmenbrane conductance regulator; CI: confidence interval; ES: effect size.

AJA-27-611_Suppl5.tif^{(166.8KB, tif)}

Supplementary Figure 6

Forest plot of proportions of patients carrying CFTR variants regarding allele number in Chinese CUAVD/CPAVD population (n = 57). CFTR: cystic fibrosis transmenbrane conductance regulator; CUAVD: congenital unilateral absence of vas deferens; CPAVD: congenital bilateral partial aplasia of vas deferens; CI: confidence interval; ES: effect size.

AJA-27-611_Suppl6.tif^{(114.3KB, tif)}

Supplementary Figure 7

Forest plot of allele frequency of IVS9-5T variant in Chinese CBAVD population (n = 760). CBAVD: congenital bilateral absence of the vas deferens; CFTR: cystic fibrosis transmenbrane conductance regulator; CI: confidence interval; ES: effect size; het: heterozygous; homo: homozygous.

AJA-27-611_Suppl7.tif^{(167.1KB, tif)}

Supplementary Figure 8

Forest plot of allele frequency of IVS9-5T variant in Chinese CUAVD/CPAVD population (n = 57). CFTR: cystic fibrosis transmenbrane conductance regulator; CUAVD: congenital unilateral absence of vas deferens; CPAVD: congenital bilateral partial aplasia of vas deferens; CI: confidence interval; ES: effect size, het: heterozygous; homo: homozygous.

AJA-27-611_Suppl8.tif^{(112.1KB, tif)}

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (grant No. 82171588) and the Fundamental Research Funds for the Central Institutes (grant No. 2023GJZD01). We would like to acknowledge the patients and his family members for participating in our research.

Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure 1

Flowchart diagram of literature search.

AJA-27-611_Suppl1.tif^{(146.4KB, tif)}

Supplementary Figure 2

AJA-27-611_Suppl2.tif^{(166.6KB, tif)}

Supplementary Figure 3

AJA-27-611_Suppl3.tif^{(166.1KB, tif)}

Supplementary Figure 4

AJA-27-611_Suppl4.tif^{(108.6KB, tif)}

Supplementary Figure 5

AJA-27-611_Suppl5.tif^{(166.8KB, tif)}

Supplementary Figure 6

AJA-27-611_Suppl6.tif^{(114.3KB, tif)}

Supplementary Figure 7

AJA-27-611_Suppl7.tif^{(167.1KB, tif)}

Supplementary Figure 8

AJA-27-611_Suppl8.tif^{(112.1KB, tif)}

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PERMALINK

Cystic fibrosis-causing variants in Chinese patients with congenital absence of the vas deferens: a cohort and meta-analysis

Yi Lu

Jing Wang

Zhong-Lin Cai

Teng-Yan Li

Hong-Jun Li

Bin-Bin Wang

Abstract

INTRODUCTION

PARTICIPANTS AND METHODS

Participants

Mutational analysis by targeted sequencing

Classification of clinical significance and pathogenicity

Supplementary Table 1.

Sanger sequencing

Literature review and meta-analysis

Supplementary Table 2.

RESULTS

CFTR variants in CAVD cohort

Table 1.

Table 2.

Supplementary Table 3.

Figure 1.

Supplementary Table 4.

Clinical significance of CFTR variants in CAVD cohort

Supplementary Table 5.

Supplementary Table 6.

Literature review of CFTR variants in Chinese individuals with CAVD

Supplementary Table 7.

Supplementary Table 8.

Supplementary Table 9.

Figure 2.

Meta-analysis

Supplementary Table 10.

Figure 3.

Figure 4.

Supplementary Table 11.

DISCUSSION

AUTHOR CONTRIBUTIONS

COMPETING INTERESTS

ACKNOWLEDGMENTS

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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