ANOS1 variants in a large cohort of Chinese patients with congenital hypogonadotropic hypogonadism

Abstract

Objective

Congenital hypogonadotropic hypogonadism (CHH) is a rare congenital gonadal dysplasia caused by defects in the synthesis, secretion or signal transduction of hypothalamic gonadotropin releasing hormone. The main manifestations of CHH are delayed or lack puberty, low levels of sex hormones and gonadotropins, and may be accompanied with other clinical phenotypes. Some patients with CHH are also accompanied with anosmia or hyposmia, which is called Kalman syndrome (KS). ANOS1, located on X chromosome, is the first gene associated with CHH in an X-linked recessive manner. This study aims to provide a basis for the genetic diagnosis of CHH by analyzing the gene variant spectrum of ANOS1 in CHH and the relationship between clinical phenotype and genotype.

Methods

In this study, whole exome sequencing (WES) was used to screen rare sequencing variants (RSVs) of ANOS1 in a Chinese cohort of 165 male CHH patients. Four commonly used in silico tools were used to predict the function of the identified RSVs in coding region, including Polyphen2, Mutation Taster, SIFT, and Combined Annotation Dependent Depletion (CADD). Splice Site Prediction by Neural Network (NNSPLICE) was employed to predict possibilities of intronic RSVs to disrupt splicing. American College of Medical Genetics and Genomics (ACMG) guidelines was used to assess the pathogenicity of the detected RSVs. The ANOS1 genetic variant spectrum of CHH patients in Chinese population was established. The relationship between clinical phenotype and genotype was analyzed by collecting detailed clinical data.

Results

Through WES analysis for 165 CHH patients, ANOS1 RSVs were detected in 17 of them, with the frequency of 10.3%. A total of 13 RSVs were detected in the 17 probands, including 5 nonsense variants (p.T76X, p.R191X, p.W257X, p.R262X, and p.W589X), 2 splicing site variants (c.318+3A>C, c.1063-1G>C), and 6 missense variants (p.N402S, p.N155D, p.P504L, p.C157R, p.Q635P, and p.V560I). In these 17 CHH probands with ANOS1 RSVs, many were accompanied with other clinical phenotypes. The most common associated phenotype was cryptorchidism (10/17), followed by unilateral renal agenesis (3/17), dental agenesis (3/17), and synkinesia (3/17). Eight RSVs, including p.T76X, p.R191X, p.W257X, p.R262X, p.W589X, c.318+3A>C, c.1063-1G>C, and p.C157R, were predicted to be pathogenic or likely pathogenic ANOS1 RSVs by ACMG. Eight CHH patients with pathogenic or likely pathogenic ANOS1 variants had additional features. In contrast, only one out of nine CHH patients with non-pathogenic (likely benign or uncertain of significance) ANOS1 variants according to ACMG exhibited additional features. And function of the non-pathogenic ANOS1 variants accompanied with other CHH-associated RSVs.

Conclusion

The ANOS1 genetic spectrum of CHH patients in Chinese population is established. Some of the correlations between clinical phenotype and genotype are also established. Our study indicates that CHH patients with pathogenic or likely pathogenic ANOS1 RSVs tend to exhibit additional phenotypes. Although non-pathogenic ANOS1 variants only may not be sufficient to cause CHH, they may function together with other CHH-associated RSVs to cause the disease.

The impulse secretion of gonadotropin releasing hormone (GnRH) in the hypothalamus plays a critical role in the initiation and maintenance of normal puberty and reproductive function in human. GnRH stimulates the pituitary to secrete gonadotropins of luteinizing hormone (LH) and follicle stimulating hormone (FSH), which lead to the production of sex hormones ^{[ 1- 2]}. Deficient in the synthesis, secretion, or action of GnRH leads to congenital hypogonadism hypogonadism (CHH, OMIM 140116), a rare disorder characterized by delayed or absent puberty. CHH was first reported in association with olfactory defects and named Kallmann syndrome (KS, OMIM 300836) ^{[ 3]}. Subsequently, a number of CHH patients with normal olfaction were defined as normosmic CHH (nCHH). Additional abnormalities, such as midline defects, ataxia, renal agenesis, synkinesia, and digital anomalies, have also been reported as variable characteristics of this disease ^{[ 4- 5]}.

ANOS1, the first gene associated with KS, was surfaced during the investigation of KS brothers with a large contiguous gene deletion on the X chromosome ^{[ 6]}. Then ANOS1 part deletion was detected in a male infant with abnormal genitalia, hypogonadotropic hypogonadism, agenesis of the olfactory bulbs and tracts (i.e., KS) associated with chondrodysplasia puncta and ichthyosis ^{[ 7]}. ANOS1, located on chromosome Xp22.31, comprises 14 exons and encodes a 680-amino acid extracellular cell adhesion protein. The anosmin-1 protein encoded by ANOS1 is comprised of an N-terminal cysteine-enriched Cys-box domain, a whey acidic protein (WAP) domain, 4 fibronectin type III (FnIII) domains, and a histidine-rich C terminal region ( Figure 1) ^{[ 8]}.

Figure 1. Distribution of RSVs in ANOS1 .

ANOS1 promotes neuronal cell adhesion, neurite outgrowth, axonal guidance, and central nervous system (CNS) projection neuron branching. The expression of ANOS1 during embryonic development has been detected at various brain sites including the olfactory bulbs and the cerebellum, as well as other sites such as spinal cord, inner ear, and kidney ^{[ 6, 9- 10]}. Accordingly, defects including renal agenesis, cleft palate, mirror movements, and hearing loss were often associated with KS caused by ANOS1 ^{[ 11- 15]} . About 160 variants have been identified in the ANOS1 gene, and most of them are loss-of-function variants (frameshift, nonsense and splice site) presumed to be pathogenic. However, the contribution of missense and likely benign ANOS1 variants to the CHH is largely unknown.

In this study, we aim to investigate the prevalence of ANOS1 in a large cohort of male CHH patients from China, and evaluate the contribution of ANOS1 variants to CHH by using detailed phenotyping and co-segregation analysis.

1. Patients and methods

1.1. Patients and clinical evaluation

A total of 165 male CHH probands from China (115 KS and 50 nCHH) were recruited at Xiangya Hospital (Changsha, China) and the People’s Hospital of Henan Province (Zhengzhou, China). All patients or their guardians gave written informed consents for hormonal, anthropometric, and genetic detection. The studies involving human samples were approved by the Ethics Committee of School of Life Sciences, Central South University (No. 2017030801). This study also consisted of a control group of 450 unrelated, ethically matched male Chinese participants from Xiangya Hospital.

The diagnosis of CHH was defined by: absence of pubertal development by 18 years of age, or previously medical induced puberty under this age, or neonatal micropenis with cryptorchidism; low sex-steroid levels in association with inappropriately low/normal gonadotropin levels and an abnormal/normal response to a GnRH stimulation test; inappropriately low/normal gonadotropin levels; otherwise normal anterior pituitary anatomy and function; excluded secondary causes of hypogonadism. Olfactory function was assessed by self-reported for KS or interpreted by University of Pennsylvania Smell Identification Test (UPSIT score <5th percentile of age) for nCHH/KS. The brain and olfactory bulb structure were examined using magnetic resonance imaging (MRI) if available. Comprehensive auditory functions were assessed by a pure tone test if suspicious. Other clinical manifestations often seen in CHH, such as cleft lip, synkinesis, cleft palate, dental agenesis, renal agenesis, skeletal deformities, and seizures were carefully analyzed. Additionally, bone densitometry, weight, height, skin texture, testis size, sperm count and medication history for phenotype-genotype analysis were also registered. Familial co-segregation was performed whenever available.

1.2. Whole exome sequencing

Whole exome sequencing (WES) in CHH and control cohorts was performed using previously described methods ^{[ 16]}. Non-synonymous rare sequencing variants (RSVs) with minor allele frequency (MAF)<1% in the dbSNP, ExAC, Genome Aggregation Database (gnomAD), Chinese Millionome Database (CMDB), and 1000 Genomes in ANOS1 as well as a panel of genes involved in CHH, including CCDC141, AXL, DUSP6, FEZF1, FGF8, FGF17, FGFR1, FLRT3, GNRH1, GNRHR, HESX1, HS6ST1, IL17RD, KISS1, KISS1R, NSMF, PROK2, PROKR2, SEMA3A, SEMA3E, PLXNA1, SEMA7A, SPRY4, TAC3, TACR3, WDR11, LEP, LEPR, NR0B1, PCSK1, STUB1, CHD7, DMXL2, SOX2, PNPLA6, RNF216, SOX10, and OTUD4.

1.3. Gross or partial deletions sequencing

Copy number variations (CNV) analysis was performed using ExomeDepth algorithm as described previously ^{[ 17]}.

1.4. Bioinformatics analysis

RSVs were interpreted according to American College of Medical Genetics and Genomics (ACMG) guidelines ^{[ 18]}. Missense variants were automated in InterVar ( http://wintervar.wglab.org/) and re-interpretation was made through manual adjustment according to phenotypes, segregation, inheritance, and statistical difference compared with control. RSVs were divided into 5 types: Pathogenic (P), likely pathogenic (LP), of uncertain significance (U), likely benign (LB), and benign (B). Furthermore, 4 commonly used in silico tools were used to predict the function of the identified missense variants, including Polyphen2, MutationTaster, SIFT, and Combined Annotation Dependent Depletion (CADD). Splice Site Prediction by Neural Network (NNSPLICE) ^{[ 19]} was employed to predict possibilities of intronic RSVs to disrupt splicing.

1.5. Statistical analysis

A gene-collapsed RSV association test on CHH, versus 450 controls, was performed to compare rare-variant allele frequencies by means of a two-tailed Fisher’s exact test. A Fisher’s exact test was also used to compare the percentages of patients with different groups as appropriate. The significance level was set at P<0.05.

2. Results

2.1. Enrichment of ANOS1 RSVs in CHH patients

Overall, we identified 13 non-synonymous ANOS1 RSVs in 17 CHH patients ( Table 1 and Figure 1). Among them, 5 nonsense RSVs, p.T76X, p.R191X, p.W257X, p.R262X, and p.W589X, have been previously reported. Two novel splicing variants (c.318+3A>C and c.1063-1G>C) were predicted to disrupt the normal splicing. Additionally, 4 novel missense RSVs (p.N402S, p.N155D, p.P504L, and p.C157R) and 2 previously reported missense variants (p.Q635P and p.V560I) were identified in 10 unrelated patients. No CNV or intragenic deletions of ANOS1 were found in this study. For the patients with nonsense and splicing ANOS1 RSVs, only proband 4 carried p.E2009K CHD7 RSV and p.R191X ANOS1 RSV, whereas other patients only carried the ANOS1 RSVs. In contrast, most patients (proband 11, 13, 14, 15, 16, and 17, Table 1) with missense RSVs also carried additional RSVs in another CHH associated genes.

Table 1.

Molecular genetic characteristics of the CHH probands with identified ANOS1 variants

ID	Dx	c.HGVS	p.HGVS	Domain	MAF		NNSPLICE	ACMG criteria	In silico prediction
					matched	all			CADD	SIFT	Polyphen2	MutationTaster
1	KS	ANOS1 c.C1511T	p.P504L	FnIII	0.002718	0.0002901		U	11.3	T	B	N
		CHD7 c.2214A>C	p.E738D		0	0		U	14.7	T	B	D
2	KS	ANOS1 463A>G	p.N155D	WAP	0.008177	0.000676		LB	8.8	T	B	N
		FGF8 c.581C>T	p.T194M		0	0		U	28.8	D	D	D
3	KS	ANOS1 1904A>C	p.Q635P	FnIII	0	0		U	24.8	D	D	D
4	KS	ANOS1 1766G>A	p.W589X	FnIII	0	0		P	39	T	.	A
5	KS	ANOS1 1678 G>A	p.V560I	FnIII	0.009956	0.007762		LB	2.6	T	B	N
		PCSK1 704T>C	p.V235A		0.002199	0.0001566		LP	24.4	D	D	D
6	KS	ANOS1 1678 G>A	p.V560I	FnIII	0.009956	0.007762		LB	2.6	T	B	N
		PNPLA6 1720G>A	p.V574I		0	0.00000827		U	13.2	T	B	D
7	KS	ANOS1 463A>G	p.N155D	WAP	0.008177	0.000676		LB	8.8	T	B	N
8	KS	ANOS1 1205A>G	p.N402S	FnIII	0	0		U	4.3	T	B	N
9	KS	ANOS1: c.318+3A>C		Cysteine	0	0	0.89					A
10	KS	ANOS1 469T>C	p.C157R	WAP	0	0		P	26.3	D	D	D
11	KS	ANOS1 1678 G>A	p.V560I	FnIII	0.009956	0.007762		LB	2.6	T	B	N
		CCDC141 c.2611C>T	p.Q871X		0	0		P	35	T		A
12	KS	ANOS1 769C>T	p.R257X	FnIII	0	0		P	35	T		A
13	KS	ANOS1 C228C>A	p.T76X	Cysteine	0	0		P	35	T		A
14	nCHH?	ANOS1 C571C>T	p.R191X	FnIII	0	0		P	35	T		A
		CHD7 6025G>A	p.E2009K		0	0		U	28	D	D	D
15	KS	ANOS1 784C>T	p.R262X	FnIII	0	0		P	35	T		A
16	KS	ANOS1 c.C1511T	p.P504L	FnIII	0.002718	0.0002901		U	11.3	T	B	N
		PNPLA6 c.3534G>C	p.W1178C		0	0		U	31	D	D	D
17	KS	ANOS1 c.1063-1G>C		FnIII	0	0	0.98	LP				A

Dx: Diagnosis; KS: Kallmann syndrome; nCHH: Normosmic congenital hypogonadotropic hypogonadism; HGVS: Human Genome Variation Society; Homo: Homozygous; Het: Heterozygous; N/A: Not available; WAP: Whey acidic protein domain; FnIII: Four fibronectin type III domains; Cysteine: N-terminalCys-box domain rich of cysteine; MAF: Minor allele frequency in ExAC database; matched: Ethnically-matched population in ExAC; all: All populations in ExAC; NNSPLICE: Splice Site Prediction by Neural Network; P: Pathogenic; B: Benign; D: Damaging; U: Uncertain significance; LP: Likely pathogenic; LB: Likely benign; CADD: Combined Annotation Dependent Depletion; SIFT: T, Tolerated; D, Deleterious; Polyphen2: B, Benign; D, Damaging; Mutation Taster: N, polymorphism; D, Disease_causing; A, Disease_causing_automatic.

Most RSVs were detected only in a single pedigree or case, except 3 missenses (p.N155D, p.P504L, and p.V560I). It should be noted that p.N155D and p.V560I were also found in one out of 450 ethnically matched control. The prevalence of ANOS1 RSVs was significantly higher in CHH cohort compared to ethnically matched controls (10.3%, 17/165 vs 0.4%, 2/450; P<0.001).

Co-segregation was analyzed in 14 available pedigrees. As shown in Figure 2 and Table 1, probands 2, 6, and 17 carried de novo RSVs, whereas other probands inherited ANOS1 variants from their mothers ( Figure 2). According to the ACMG guidelines and NNSPLICE, 8 variants were pathogenic or likely pathogenic, 3 variants were of uncertain significance, and 2 were likely benign ( Table 1).

Figure 2. Informative pedigrees of CHH probands carrying ANOS1 RSVs identified in this study.

Proband of each family is indicated by an arrow. The available genotypes are indicated below each individual. Squares depict males and circles depict females. F: Family; CHH: Congenital hypogonadism hypogonadism; RSVs: Rare sequencing variants; WT: Wild type.

2.2. Genotype-phenotype correlation

Clinical data from the 17 probands with ANOS1 RSVs are summarized in Table 2. The mean age at diagnosis was 18.2 years. Sixteen of them were affected with KS while proband 4 showed normal olfaction on formal test and alleged normal sense of smell in daily life. However, he was not available for MRI examination. All of the 8 probands who harbored pathogenic or likely pathogenic ANOS1 variants exhibited additional features, including unilateral or bilateral cryptorchidism, dental agenesis, unilateral renal agenesis, synkinesia, and hearing loss ( Table 2).

Table 2.

Clinical and laboratory findings of the CHH patients carrying ANOS1 mutations

No.	Age/years	Gender	Dx	Other features	At diagnosis				OB	Sperm
					E2/ (pg·mL -1)	T/ (ng·mL -1)	FSH/ (mIU·mL -1)	LH/ (mIU·mL -1)
1	12	M	KS	Cryptorchidism, dental agenesis, kidney agenesis,hypospadias	9.9	0.4	0.2	0.1	NA	—
2	25	M	KS	—	28	0.2	0.3	0.3	NA	5.9×10 6/mL
3	17	M	KS	Cryptorchidism,	13	0.9	0.5	0.4	Anosmia	2-6
				synkinesia
4	18	M	KS	Right cryptorchidism	5	1.1	0.3	0.2	Anosmia	4×10 6/mL
5	20	M	KS	Blue color blindness	17	0.4	1.3	0.9	NA	5.2×10 6/mL
6	17	M	KS	Right cryptorchidism	15	0.3	0.4	0.4	Hyposmia	NA
7	16	M	KS	—	17	0.3	1.8	0.8	Hyposmia	6.9×10 6/mL
8	19	M	KS	—	1.7	0.3	0.6	0.3	Hyposmia	3.0×10 6/mL
9	18	M	KS	Cryptorchidism	5.6	0.5	1.8	0.2	Anosmia	0
				Right kidney deficiency
10	22	M	KS	Left renal agenesis, cryptorchidism, left hearing loss	16	1	0.3	0.4	Aosmia	0.9×10 6/mL
11	22	M	KS	—	20	1	0.3	0.4	Hyposmia	5.6×10 6/mL
12	23	M	KS	Scoliosis, synkinesia cryptorchidism	18	0.1	0.3	0.2	Aosmia	0
13	4	M	KS	Strabismus, synkinesia optic nerve dysplasia cryptorchidism	14	0.04	0.4	<0.1	NA	—
14	28	M	nCHH?	Dental agenesis, ptosis, microphthalmos	14	0.57	2.26	0.52	NA	4.9×10 6/mL
15	8	M	KS	Dental agenesis, cryptorchidism	20	0.02	0.3	0.1	NA	—
16	36	M	KS	—	11	0.1	2.2	0.7	Aosmia	NA
17	2	M	KS	Umbilical fistula, cryptorchidism, development delay	16	0.82	0	0.07	Aosmia	—

Age means the age of diagnosis. M: Male; Dx: Diagnosis; KS: Kallmann syndrome; nCHH: Normosmic congenital hypogonadotropic hypogonadism; OB: Olfactory bulb; NA: Not applicable. Reference range for testosterone (T) in normal adult men is 1.75-7.81 ng/mL, for estradiol (E₂) <53 pg/mL, for luteinizing hormone (LH) 1.2-8.6 mIU/mL, and for follicle-stimulating hormone (FSH) 1.3-19.3 mIU/mL.

Proband 1, a four-years-old boy, inherited a p.T76X ANOS1 variant from his unaffected mother. He had strabismus, synkinesia, optic nerve dysplasia, and bilateral cryptorchidism as well as KS. Proband 2 carried a de novo c.318+3A>C ANOS1 RSV and showed bilateral cryptorchidism and right renal deficiency other than KS.

Proband 3 inherited a c.1063-1G>C ANOS1 RSV from his unaffected mother and manifested bilateral cryptorchidism and developmental delay. Proband 4 carrying p.E2009K CHD7 RSV and p.R191X ANOS1 RSV showed dental agenesis, potosis, and microphthalmia besides CHH. He admitted normal sense of smell, but unfortunately refused to perform olfactory bulb MRI. His mother (II꞉2 in Family 4, Figure 2) with p.E2009K CHD7 and p.R191X ANOS1 RSVs manifested scarce pubic hair and bicornate uterus but showed normal puberty and fertility. His maternal uncle (II:5 in Family 4, Figure 2) who carried p.E2009K CHD7 RSV is unaffected. Proband 5, an eight-years-old boy with p.R262X ANOS1 RSV, showed cryptorchidism, dental agenesis, and KS. The individual II:3 in family 5 also carried p.R262X ANOS1 RSV and was diagnosed with KS without associated phenotypes, whereas 2 women (I:2 and II:2, Figure 2) with this RSV were unaffected.

Proband 6 with a de novo p.W589X ANOS1 RSV showed KS and right cryptorchidism. Proband 7 carrying a p.R257X ANOS1 RSV was diagnosed as KS with synkinesia, bilateral cryptorchidism, and scoliosis. His maternal cousin (III:2 in Family 7, Figure 2) with the same RSV had KS and synkinesia. Proband 8 with a p.C157R ANOS1 RSV showed mild hearing loss, cryptorchidism, and left renal agenesis along with KS. And his nephew (III:1 in Family 8, Figure 2) with the same RSV was diagnosed as KS along with cryptorchidism. Two women (I:2 and II:2, Figure 2) with this RSV were unaffected. Proband 9 with a p.Q635P ANOS1 RSV displayed right cryptorchidism and synkinesia in addition to KS.

We also identified 5 non-pathogenic ANOS1 RSVs in 8 CHH patients. Six patients also carried RSVs in other CHH-related genes. And only one showed additional symptoms along with KS. A significantly higher proportion of CHH patients with pathogenic or likely pathogenic ANOS1 RSVs showed additional phenotypes, compared with those with non-pathogenic ANOS1 variants (8/8 vs 1/9, P<0.01). Proband 13, with p.P504L ANOS1 and p.E738D CHD7 RSVs, showed KS along with dental agenesis, unilateral renal agenesis, strabismus, and hypospadias. His mother (II:4) and aunt (II:2) who also carried these RSVs manifested no armpit and pubic hair but showed normal puberty and fertility. And his grandfather (I:2) with a p.E738D CHD7 variant was unaffected ( Figure 2).

2.3. Treatment evaluation

The male patients except proband 1, 3, 5, and 13 accepted the treatment with testosterone (T) or human chorionic gonadotropin/human menopausal gonadotropin treatment (HCG/HMG). All of the treatments obviously increased the testicular sizes in males. Spermatogenesis was evaluated in 11 male patients ( Table 2), and sperm appeared in 9 patients (at least 24 months of continuous HCG/HMG therapy). As regards to the 2 patients (proband 2 and 7) with failure of spermatogenesis, they were both found with bilateral cryptorchidism and several neurologic defects.

3. Discussion

In this study, we systematically analyzed the ANOS1 RSVs in a large Chinese cohort of male CHH patients. As a result, we identified 13 ANOS1 RSVs in 17 probands, with the frequency of 10.3%. Besides the common concomitant symptoms of cryptorchidism, synkinesia, and unilateral renal agenesis, dental agenesis was also frequently found in CHH patients with ANOS1 RSVs. The prevalence (10/17) of cryptorchidism in this cohort is higher than that in previous reports ^{[ 15, 20- 22]}.

CHH patients with RSVs in FGFR1 signaling pathways are prone to have additional skeletal phenotypes, such as cleft lip/palate, dental agenesis, syndactyly, and clinodactyly ^{[ 12, 23- 27]}. A previous study ^{[ 4]} found that dental agenesis is significantly enriched in FGFR1 signaling group and is useful for prioritizing genetic screening. Intriguingly, Another study ^{[ 28]} found that anosmin-1, encoded by ANOS1, interacted with FGFR1- FGF-HS6ST1 complex and enhances FGFR1 signaling. In this study, 3 probands (4, 5 and 13) had dental agenesis, reinforcing the potential interaction between anosmin-1 and FGFR1 signaling ^{[ 29- 30]}.

ANOS1 RSVs are linked to KS in almost all cases except one study. In this circumstance, 2 patients who carry truncating or frameshift ANOS1 RSVs show normosmia or borderline olfactory function ^{[ 12]}. In the current study, 16 out of 17 patients with ANOS1 RSVs showed complete absent or decreased olfaction. However, proband 4 with a p.R191X ANOS1 RSV and a p.E2009K CHD7 RSV showed borderline olfaction test and alleged normal olfactory function in daily life. Nevertheless, we cannot rule out the possible slightly hypoplasia of olfactory bulb in this patient without MRI analysis.

ANOS1 is one of the main causative genes for CHH and loss-of-function ANOS1 RSVs are likely sufficient to cause CHH in an X-linked recessive manner. Indeed, 5 nonsense and 2 frameshift RSVs ( p.T76X, p.R191X, p.W257X, p.R262X, p.W589X, c. 318+3A>C, and c.1063-1G>C) predicted to be loss of function led to CHH only in males. The missense C157R in Family 3 predicted to be pathogenic by all the in silico tools was located in the N-terminal WAP domain of ANOS1. The WAP domain contains 8 conserved cysteine residues forming 4 intramolecular disulphide bonds, which is composed of C134-C164, C15-C163, C147-C168, and C157-C172 forming the structural core of the protein and locating at the putative protease binding site of FGFR1-HS6ST1 complex ^{[ 31]}. Substitutions are predicted to disrupt WAP domain core structure formation, leading to protein instability and loss of interaction with serine proteases. Accordingly, 2 male individuals in Family 8 (II:3 and III:1) carrying the C157R RSV which is predicted to destroy the C157-C172 binding site exhibited bilateral cryptorchidism, sensorineural hearing loss, and KS. Additionally, only II:3 displayed left renal agenesis.

Furthermore, our data suggest that those non-pathogenic ANOS1 RSVs are not the major contributor for CHH per se. Indeed, 6 out of 8 probands (proband 11, 13, 14, 15, 16, 17) with non-pathogenic ANOS1 RSVs also carried RSVs in another CHH gene. However, some non-pathogenic ANOS1 RSVs may play a synergetic role with RSVs in other CHH-related genes in the pathogenesis of CHH. For instance, proband 13 with p.P504L ANOS1 and p.E738D CHD7 RSVs was affected with KS. However, his mother and aunt (II:4, II:2) who also carried these 2 RSVs and his grandfather (I:2) who only had p.E738D CHD7 did not show CHH. Besides, proband 17 carried a second heterozygous p.Q871X CCDC141 RSV, which alone seems not sufficient to cause the disease ^{[ 32- 33]}.

There is another thing worth discussing for the RSV p.N155D ANOS1. In Family 10, all men with p.N155D ANOS1 RSV (II:3, III:1, and III:2) had KS whereas the woman (II:2) with this RSV was unaffected. In Family 11, the proband (IV:1) with p.N155D ANOS1 and T194M FGF8 RSVs was diagnosed as KS, whereas his sister (IV:2) with these 2 RSVs was unaffected. Furthermore, 2 male individuals (III:3 and III:4) in Family 11 were also diagnosed as KS, implying an X-linked recessive inheritance although we have no genetic information for these 2 individuals. However, the p.N155D ANOS1 RSV was predicted to be likely benign by all in silico tools and had a relatively high frequency (0.008177) in the ethnically-matched population in public database. Whether this RSV co-segregating with the CHH phenotype in an X-linked recessive manner or just a coincidence required further analysis in other population or functional experiments. Indeed, although in silico predictions of some RSVs are shown to be benign, the lack of specificity of the in silico programs and the rarity of these RSVs in extensive normative databases indicate that these RSVs must undergo further biological evaluation prior to discounting their pathogenicity.

Taken together, our study indicates that CHH patients with pathogenic or likely pathogenic ANOS1 RSVs tend to exhibit additional phenotypes. Although non-pathogenic ANOS1 RSVs only may not be sufficient to cause CHH, they may function together with other CHH-associated RSVs to cause the disease.

Funding Statement

This work was supported by the National Natural Science Foundation (81770780) and the Natural Science Foundation of Hunan Province (2020JJ4901), China.

Conflict of Interest

The authors declare that they have no competing interests.

AUTHORS’CONTRIBUTIONS

Contributions: ZENG Wang and JIANG Fang Investigation and data curation; WANG Xinying Conceptualization, methodology; LI Jiada Supervision, review; MEN Meichao Data curation, writing-original draft preparation, supervision.

Note

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