Analysis of genetic and clinical characteristics of a Chinese Kallmann syndrome cohort with ANOS1 mutations

in European Journal of Endocrinology
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  • 1 Department of Endocrinology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Key laboratory of Endocrine, Ministry of Health, Beijing, China
  • | 2 Department of Physiology, State Key Laboratory of Medical Molecular Biology, School of Basic Medicine, Graduate School of Peking Union Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing, China

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Objective

To analyze ANOS1 gene mutations in a large Chinese Kallmann syndrome (KS) cohort and to characterize the clinical presentation of the disease in patients with ANOS1 mutations.

Patients and methods

Chinese patients with KS, including 187 sporadic and 23 pedigree cases were recruited. Patients’ ANOS1 gene sequences were analyzed by direct sequencing of PCR-amplified products. In silico analysis was used to assess functional relevance of newly identified missense mutations. Patients’ clinical characteristics were analyzed retrospectively.

Result(s)

Fifteen nonsynonymous rare ANOS1 variants were found in 13 out of 187 sporadic and 8 out of 23 familial IHH probands. Seven novel (C86F, C90Y, C151W, Y379X, c.1062 + 1G > A, Y579L fs 591X, R597X) and eight recurrent ANOS1 mutations (S38X, R257X, R262X, R423X, R424X, V560I, c.1843-1G > A, p.R631X) were identified. All the novel mutations were predicted to be pathogenic. The prevalence of cryptorchidism was high (38.1%) and occurred in patients with different kind of ANOS1 mutations, while the patients with the same mutation did not present with cryptorchidism uniformly.

Conclusion(s)

The prevalence of ANOS1 gene mutations is low in sporadic KS patients, but is much higher in familial KS patients. In the present study, we identify seven novel ANOS1 mutations, including two mutations in the CR domain, which are probably pathogenic. These mutations expand the ANOS1 mutation spectrum and provide a foundation for prenatal diagnosis and genetic counseling.

Abstract

Objective

To analyze ANOS1 gene mutations in a large Chinese Kallmann syndrome (KS) cohort and to characterize the clinical presentation of the disease in patients with ANOS1 mutations.

Patients and methods

Chinese patients with KS, including 187 sporadic and 23 pedigree cases were recruited. Patients’ ANOS1 gene sequences were analyzed by direct sequencing of PCR-amplified products. In silico analysis was used to assess functional relevance of newly identified missense mutations. Patients’ clinical characteristics were analyzed retrospectively.

Result(s)

Fifteen nonsynonymous rare ANOS1 variants were found in 13 out of 187 sporadic and 8 out of 23 familial IHH probands. Seven novel (C86F, C90Y, C151W, Y379X, c.1062 + 1G > A, Y579L fs 591X, R597X) and eight recurrent ANOS1 mutations (S38X, R257X, R262X, R423X, R424X, V560I, c.1843-1G > A, p.R631X) were identified. All the novel mutations were predicted to be pathogenic. The prevalence of cryptorchidism was high (38.1%) and occurred in patients with different kind of ANOS1 mutations, while the patients with the same mutation did not present with cryptorchidism uniformly.

Conclusion(s)

The prevalence of ANOS1 gene mutations is low in sporadic KS patients, but is much higher in familial KS patients. In the present study, we identify seven novel ANOS1 mutations, including two mutations in the CR domain, which are probably pathogenic. These mutations expand the ANOS1 mutation spectrum and provide a foundation for prenatal diagnosis and genetic counseling.

Introduction

The hypothalamic–pituitary–gonadal (HPG) axis plays a crucial role in the development, progression and maintenance of normal reproductive function (1). The pulsatile secretion of gonadotropin-releasing hormone (GnRH) regulates the synthesis and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in the pituitary, which subsequently stimulate the gonads to produce sex steroids and gametes. Isolated hypogonadotropic hypogonadism (IHH) is caused by defects in the HPG axis, resulting in low levels of sex steroids, delayed puberty or absence of puberty and sterility. IHH is divided into Kallmann syndrome (KS) and normosmic idiopathic hypogonadotropic hypogonadism (nIHH). Approximately 50% of all IHH cases are due to KS (2).

KS is a congenital disorder characterized by IHH and hyposmia/anosmia. Both GnRH neurons and olfactory neurons originate from the nasal placode. During normal development, GnRH neurons migrate along olfactory neuron axons to the hypothalamus. In KS, defects in GnRH neuron migration lead to IHH, and the absence or hypoplasia of the olfactory bulb and its axonal tracts results in hyposmia/anosmia. Some KS patients may also have other associated abnormalities, such as renal agenesis, synkinesis, cleft lip, cleft palate, dental agenesis, shortening of metacarpals, sensory neural hearing loss and seizures (3). The etiology of KS is not quite clear, but mutations in genes that regulate GnRH neuron development, migration and function are important causative factors (4).

The ANOS1 gene is the pathogenic gene found to cause X-linked KS. It is located on the X chromosome (Xp22.3) adjacent to pseudoautosomal region 1 (PAR1), a highly variable and unstable region of the chromosome. ANOS1 encodes the protein anosmin-1, an extracellular matrix protein. Anosmin-1 comprises an N-terminal cysteine-rich domain (Cys-box), followed by a whey acidic protein (WAP) domain, four fibronectin type III (FnIII) domains and a histidine-rich C terminal region (5). Anosmin-1 promotes neuronal cell adhesion, neurite outgrowth, axonal guidance and CNS projection neuron branching. Additionally, it plays a role in the migration of multiple types of neuronal precursors, including GnRH-producing neurons and oligodendrocyte precursors. ANOS1 mutations are found in approximately 10–20% of familial and sporadic KS patients. Nearly seventy mutations have, hitherto, been identified in the ANOS1 gene, which are spread widely throughout the entire gene with no mutation ‘hot spots’ in the affected regions.

In this study, we sequenced the ANOS1 gene in 187 sporadic and 23 familial cases of KS from the Chinese patient population to determine the prevalence of ANOS1 mutations in a large Chinese KS cohort and to analyze the clinical characteristics of KS patients with ANOS1 mutations.

Subjects and methods

Subjects

Twenty-three familial and 187 unrelated sporadic Chinese patients with KS were recruited from Peking Union Medical hospital between January 2009 and December 2016. All patients were male and diagnosed preliminarily with KS based on clinical manifestation and sex hormone data. The study was approved by the PUMCH’s Ethics Committee for Human Research and complies with the Declaration of Helsinki. Inclusion criteria were (1) absence of pubertal development by 18 years of age, (2) low concentration of sex steroids, (3) low or inappropriately normal gonadotropin levels at hypogonadal levels, (4) documented absence of sense of smell (anosmia) or deficient sense of smell (hyposmia), (5) normal function of growth hormone-IGF1 (insulin-like growth factor 1) axis, pituitary–adrenal axis and pituitary–thyroid axis and (6) other conditions leading to secondary hypogonadism were excluded from the study.

After obtaining informed consent, blood samples from all patients and some parents were collected for genetic testing. The clinical manifestations, including synkinesis, cleft lip, cleft palate, dental agenesis, shortening of metacarpals, renal agenesis, sensory neural hearing loss and seizures, testis size, the sperm counting and medication history, were collected and analyzed retrospectively.

Hormonal assays

Basal serum FSH, LH and testosterone were measured using chemiluminescent immunoassays (Bayor Diagnostics Corporation, USA). The intra- and inter-assay variation coefficients were 3.9% and 4.5% for FSH, 2.3% and 2.8% for LH and 5.6% and 6.6% for total testosterone respectively. The lowest measurable limits were 0.23 IU/L, 0.07 IU/L and 5.2 ng/dL for FSH, LH and total testosterone respectively.

Mutation screening

Genomic DNA was extracted from leukocytes of peripheral blood using a QIAGEN Mini Blood kit according to the manufacturer’s instructions. Fourteen exons and the exon–intron boundaries of the ANOS1 gene were amplified by PCR. The PCR products were purified with QIAquick PCR Purification Kit, and subsequently sequenced using a Taq big dye terminator sequencing kit and an ABI3730 automated sequencer (Applied Biosystems). The entire coding region, including the exon–intron boundaries of the ANOS1 gene, was sequenced in both forward and reverse directions in all patients. All sequencing primers are available on request.

The resulting sequences were analyzed using Chromas pro V1.7 (Applied Biosystems) and a BLAST search was performed with the reference sequences GenBank NG_007088.1 (ANOS1, g.DNA), GenBank NM_000216.2 (ANOS1, c.DNA) and GenBank NP_000207.2 (ANOS1, p.protein). For genomic DNA and cDNA numbering, the A nucleotide of the ATG translation initiation codon was designated +1. The variants were identified as mutations if they were not found in 100 control subjects, dbSNP database in NCBI (http://www.ncbi.nlm.nih.gov/snp/) or exome variant sever (http://evs.gs.washington.edu/EVS/).

Patients with identified ANOS1 mutations were screened for digenic/oligogenic mutations by sequencing additional genes related to the hypothalamic–pituitary–gonadal axis (FGF8, FGFR1, GNRH1, GNRHR, KISS1, KISS1R, NELF, PROK2, PROKR2, TAC3, TACR3, LEP, LEPR, WDR11, HS6ST1, CHD7 and SEMA3A) (all primer sequences and PCR conditions are available upon request) to try to explain the reason why the patients with the same mutation presented with not exact the same clinical manifestations.

Bioinformatics analysis of novel missense variants

Evolutionary conservation at the affected amino acids was assessed by a multiple sequence alignment of anosmin-1 orthologs (MSA ORTHO). The human anosmin-1 sequence, as annotated in the UniProt database (http://www.uniprot.org/), was aligned with 26 orthologous sequences within the same database (May 2013) from closely and distantly related species (6). MSA ORTHO was built using the Bioedit program (https://www.bioedit.com/).

Pathogenic effects of missense mutations identified in this study were also analyzed using an extensive set of prediction tools since different principles and algorithms were used in different in silico analysis tools, and the prediction result is not exactly same for a specific missense mutation. The following in silico analysis tools were included in this study. (1) MutationTaster. It assesses the functional impact based on evolutionary conservation of the affected amino acid in protein homologs (7) (http://www.mutationtaster.org/). (2) SIFT. Its prediction is based on the degree of conservation of amino acid residues in sequence alignments derived from closely related sequences (8) (http://sift.jcvi.org/). (3) PolyPhen. It predicts possible impact of an amino acid substitution on the structure and function using straightforward physical and comparative considerations (9) (http://genetics.bwh.harvard.edu/pph2/). (4) Mutation assessor. It uses the evolutionary information deriving from protein family alignments of large numbers of homologous sequences and 3D structures of sequence homologs to assess the function of missense variant (10) (http://mutationassessor.org/). (5) Panther. It evaluates a single substitution at a specific amino acid position in a protein based on the information about evolutionarily related proteins (11) (http://www.pantherdb.org/tools/csnpScoreForm.jsp). (6) SNAP. The prediction is based on the evolutionary information taken from an automatically generated multiple sequence alignment, structural features such as predicted secondary structure and solvent accessibility (12) (https://rostlab.org/services/snap). (7) SNPs & Go. It utilizes sequence information, evolutionary information derived in different ways and the defined functional GO score to estimate possible impact of an amino acid replacement (13) (http://snps-and-go.biocomp.unibo.it/). (8) MutPred. It evaluates the influence of an amino acid displacement upon protein sequence and molecular models change of structural features and functional sites between wild-type and mutant sequences (14) (http://mutpred.mutdb.org/). (9) Provean. Its prediction is based on an alignment-based score, which measures the change in sequence similarity of a query sequence to a protein sequence homolog before and after the introduction of an amino acid variation to the query sequence (15) (http://provean.jcvi.org/index.php) and (10) Sapred. It assesses the effect of missense variant through the structural neighbor profiles, nearby functional sites, aggregation properties and disordered regions (16) (http://sapred.cbi.pku.edu.cn/).

Results

Mutation screening and oligogenetic analysis

Overall, our mutation screening of ANOS1 revealed fifteen mutations in 8 probands out of 23 KS pedigree and 13 out of 187 sporadic IHH cases. Among 21 KS patients, four missense, eight nonsense, two splicing site and one insertion mutations were identified (Fig. 1), of which eight mutations (S38X, R257X, R262X, R423X, R424X, V560I, c.1843-1G > A and R631X) were reported previously. Three missense substitutions (c.257 G > T/p.C86F, c.269 G > A/p.C90Y, c.453 C > G/p.C151W), one splice-site mutation (c.1062 + 1G > A), two nonsense mutations (Y379X, R597X) and one insertion (c.1736 ins T, Y579Lfs591X) were novel (i.e. absent from the queried databases and the 100 controls). Each mutation was detected only in a single pedigree or case, with the exception of c.1678 G > A/p.V560I (three sporadic patients), c.1270 C > T/p.R424X (one sporadic patient and one pedigree) and c.784 C > T/p.R262X (two sporadic patients). These mutations are widely distributed in nine different exons or at intron–exon boundaries; that is, we found no mutation ‘hot spot’ region in ANOS1 (Fig. 2A). All the mutations we identified locate in different functional domains, except FnIII-3 (Fig. 2B).

Figure 1
Figure 1

The sequencing chromatogram of the mutation in ANOS1 gene. Dash indicates mutated nucleotide. Novel mutations were shown in red.

Citation: European Journal of Endocrinology 177, 4; 10.1530/EJE-17-0335

Figure 2
Figure 2

Distribution of mutations in ANOS1 gene and anosmin1 protein. (A) Blue box stands for the exons. (B) Recurrent mutations are in the upper of the box, novel mutations found in this study are in the below of the box and shown in red. (C) Distribution of missense or nonsense mutations in relation to the anosmin1 protein. Novel mutations were shown in red.

Citation: European Journal of Endocrinology 177, 4; 10.1530/EJE-17-0335

ANOS1 mutations including C90Y, R257X, R262X, R424X, R597X, Y579Lfs591X and R631X were found in familial patients, and their mother was found to be a carrier. The detailed pedigree chart of the familial patients is shown in Fig. 3.

Figure 3
Figure 3

KS pedigree (1–6) with ANOS1 mutation are shown. Affected individuals with KS are shown as completely shaded. The arrow indicates the proband of each pedigree. All individuals marked numbers were screened for the mutation of ANOS1 gene.

Citation: European Journal of Endocrinology 177, 4; 10.1530/EJE-17-0335

One patient was found to harbor not only two ANOS1 mutations (c.1062 + 1G > A and E552K) but also heterozygotic KISS1R L200V mutation. Two patients possessed both ANOS1 C86F and KISS1R Tyr323His (Het) mutations and both ANOS1 V560I and FGF8 E176K mutations respectively.

Bioinformatics analysis of novel missense variants

The cysteine residue at the position 86th, 90th and 151st of anosmin-1 protein is conserved across twenty-six different species (Fig. 4) Ten in silico analysis tools for missense variants classified C86F, C90Y and C151W as pathogenic variants (Table 1).

Figure 4
Figure 4

The sequence alignment of anosmin-1 proteins from different species. The cysteine residue at the position 86th, 90th and 151st of anosmin-1 protein is conserved across twenty-six different species.

Citation: European Journal of Endocrinology 177, 4; 10.1530/EJE-17-0335

Table 1

Functional prediction of missense mutation found in KS patients.

SiftPolyphenPantherSNAPMutation assessorMutation tastingSNPs and GoMutPredProveanSapred
C86F++++++++++
C90Y++++++++++
C151W++++++++++

+: pathogenic.

Patient characteristics

Data collected from the 21 patients who harbored ANOS1 gene mutations are summarized in Table 2. All these patients have low LH, FSH and testosterone. Gynecomastia and agenesis of right kidney occurred in one patient. Thirteen out of 21 patients’ medical histories included MRI test results and all of them (100.0%) had absence/hypoplasia of olfactory bulbs and olfactory tracts. The prevalence of cryptorchidism was high among ANOS1 mutation-harboring patients (8 out of 21, 38.1%) and was found in patients with nonsense mutations (S38X, R257X, R262X, R424X, R597X, R631X), frame-shift mutations (Y579L fs 591X) and missense mutations (C90Y, V560I). However, we also found that patients with the same mutation (V560I, R424X, R597X) do not uniformly present with cryptorchidism. Only one patient displayed agenesis of right kidney, and no patients showed synkinesis.

Table 2

Clinical and laboratory finding of the KS patients bearing ANOS1 mutations.

No.Family members with KSOther clinical featuresMRIaLH (IU/L) (basal)FSH (IU/L) (basal)TS (nmol/L)Nucleotide changeProtein changeAge at the treatment initiation (years)TreatmentDurationTestis size (mL) (L,R)→after treatedSperm
S0001Cryptorchid (right), maldescent (left)nt0.11.11.01c.113 C > Ap.S38X21TS2 years1,0→ntnt
S000221.32.81.18c.1678 G > Ap.V560I31HCG + HMG6.5 years5,5→12,122.63*106/mL
FGF8, E176K(Het)
S0003Cryptorchid (left)nt0.10.30.69c.1678 G > Ap.V560I20HCG + HMG3 years0,1→8,86.79*106/mL
S0004nt00.10.50c.1678 G > Ap.V560I30HCG + HMG19 months3,3→10,106–8/HP
S0005nt01.00.40c.1267 C > Tp.R423X25HCG + HMG2 months1,1→ntnt
S000610.10.790.80c.453 C > Gp.C151W20HCG + TS2 years1,1→4,4nt
HCG + HMG3 years
S00071000.64c.1062 + 1G > Ap.E552K20HCG + HMG2 years1,1→2,2nt
KISS1R, L200V(Het)
S000830.31.30.90c.1137C > Gp.Y379X20TS5 years1,1→8,80–2/HP
GnRH4 months
S000912.22.53.31c.1843-1G > A21TS8 months1,1→16,1694.4*106/mL
Letrozole4 months
TS6 months
S001010.00.90.86c.784 C > Tp.R262X20HCG + HMG4 years2,2→8,813.05*106/mL
S0011Cryptorchid (both) + gynecomastiant0.20.7ntc.784 G > Tp.R262X34TS11 years1,1→1,1nt
S0012100.21.10c.1270 C > Tp.R424X19TS2 years1,1→2,20
HCG10 months
HCG + HMG1 year
S0013nt0.1s0.81.00c.257 G > Tp.C86F20TS11 years2,2→4,4nt
KISS1R, Y323H(Het)
F0001Cousin, maternal uncleCryptorchid (both)nt00.20.56c.269 G > Ap.C90Y19TSHCGHCG + HMG2 years1.5 years2.5 years1,1→2,8nt
F0002-10Brother100.50.45c.1789A > Tp.R597X19TS4 months2,2→4,40
HCG9 months
HCG + HMG1.5 years
GnRH2 months
F0002-11BrotherCryptorchid (both)100.30.0c.1789A > Tp.R597X18TS4 years2,2→4,40.7*106/mL
HCG + HMG1 year
F0003-3BrotherCryptorchid (both)10.51.60.97c.1270 C > Tp.R424X25TS6 months2,0→2,0nt
HCG1 year
F0003-1Brothernt0.291.70.66c.1270 C > Tp.R424X19TS4 months1.5,1.5→2,2nt
F0004Maternal uncle, cousinCryptorchid (right), agenesis of right kidney1000.74c.769C > Tp.R257X20TSHCGHCG + HMG2 years9 months3 years2,2→8,80
F0005Maternal uncle100.90.34c.1736 ins TY579Lfs591X20TSHCGHCG + HMG5 years5 months46 months1,1→2.5,2.5nt
F0006Maternal uncle, cousinCryptorchid (both)1001.35c.1891 C > Tp.R631X18TS4 months1,1→ntnt

a1: absence of olfactory bulbs and olfactory tracts; 2: absence of olfactory bulbs and hypoplasia of olfactory tracts; 3: hypoplasia of olfactory bulbs and absence of olfactory tracts.−, negative; GnRH, gonadotropin-releasing hormone; HCG, human chorionic gonadotropin; HMG, human menopausal gonadotropin; nt, not test; TS, testosterone.

All patients accepted the treatment of testosterone or HCG/HMG. HCG/HMG therapy markedly increased the testicular sizes, except the patients with history of cryptorchid. Spermatogenesis was evaluated in ten patients and sperm appeared in seven patients (70%).

Discussion

In this study, we analyzed the ANOS1 gene variants in a large Chinese cohort of KS patients from a single medical center and found eight recurrent and seven novel mutations. The clinical characteristics of the KS patients with ANOS1 mutation (the prevalence of cryptorchidism and olfactory bulb/sulci) were also investigated.

The prevalence of ANOS1 mutations is very low in sporadic patients (13 out of 187, 6.95%); however, it is much higher in familial patients (6 out of 23, 26.1%). This result is similar with previous reports. In those studies, the incidence of the ANOS1 mutations was reported to be 3–8% in sporadic IHH (17, 18) and about 30% in familial IHH (19, 20). Of the fifteen mutations found in this study, four are missense mutations. Two naturally occurring mutations (C86F, C90Y) located in the Cys-box domain of anosmin-1 were identified for the first time. These novel missense mutants will provide us a valuable molecular model to investigate the key functional sites of the anosmin-1 protein.

Novel mutations including C86F, C90Y, C151W, c.1062 + 1G > A, Y379X, R597X, Y579Lfs591X were found in this study. Although we did not perform functional in vitro studies of the effects of these mutations, evidence from bioinformatics analysis supports the hypothesis that these mutations are probably pathogenic. Four of the identified mutations (c.1062 + 1G > A, Y379X, R597X, Y579Lfs591X) could disrupt the protein structure, result in loss of anosmin-1 function by changing splice sites, form a truncated protein or cause a frame-shift respectively. For the other three missense mutations we identified (C86F, C90Y, C151W): first, the cysteine residue is conserved across twenty-six different species. Second, all the prediction programs we employed classified these mutations as pathogenic variants. Third, two of these cysteine residues (C86 and C90) are located at the Cys-box domain and C151 is located at the WAP domain. The Cys-box contains a core of five disulphide bridges, which form between residues 49–83, 53–77, 86–105, 90–101 and 116–120. All ten cysteine residues that create the five core disulfide bonds are conserved (21). The WAP domain contains eight conserved cysteine residues that form four intramolecular disulphide bonds including 134–164, 147–168, 151–163 and 157–172. Similarly, the positions of these eight cysteine residues are conserved (22). Previous studies identified five mutations in patients with KS that affect cysteine residues that form disulphide bonds in the WAP domain of anosmin-1 (C134G, C163R, C163Y and C172R) (23, 24, 25), but no mutations that affect cysteine residues in the cysteine-rich N-terminal region of the protein had been reported. In this study, two mutations in the cysteine-rich region and one mutation in the WAP domain are described for the first time. If the cysteines at position 86, 90 or 151 were converted to other amino acids, it would disrupt the highly conserved Cys86–Cys105, Cys90–Cys101 and Cys151–Cys163 intramolecular disulphide bonds, and probably affect the folding or stability of this region.

The prevalence of cryptorchidism in this cohort with ANOS1 mutations is 38.1%. Many studies have explored the incidence of cryptorchidism in KS, with conflicting results. One study of a large cohort of 124 patients with the complete form of IHH reported that 15.3% had cryptorchidism (18), but in KS patients with ANOS1 mutations, 40% had cryptorchidism (26). Renal agenesis presented in 35–40% of X-linked KS cases and an even greater incidence of right renal agenesis occurred in KS patients with a ANOS1 mutation (27, 28). Only one patient in our cohort showed right renal agenesis; therefore, the frequency was lower than previously described (28), possibly due to the different ethnic background of the patients. In two other studies from China (20, 26) and one study from Korea (29), no patients were reported to have renal agenesis. Absence or hypoplasia of the olfactory bulb/tract was found in 100% of patients in this cohort, which is similar to previous studies (84.62%) (29). This result showed that there is a strong correlation between sense of smell and MRI images of olfactory bulb/sulci.

There are not many patients (70%) who have been detected with sperm in this study, which is similar with the results of a meta-analysis about 48 studies of HCG/HMG therapy and 16 studies of pulsatile GnRH therapy for HH patients (30). In that study, the rate of successful spermatogenesis in HH patients treated with HCG/HMG was 68% (95% CI: 58–77%). The reason why spermatogenesis failed may be related with following factors, such as cryptorchidism (31, 32) and the short duration of HCG + HMG treatment (33).

In conclusion, the prevalence of ANOS1 gene mutations is low in sporadic KS patients, but is much higher in familial KS patients. Seven novel ANOS1 mutations that are probably pathogenic were identified in the present study. Two mutations located in the CR domain of anosmin-1 protein were found for the first time. These mutations expand the ANOS1 mutation spectrum and provide information that could be useful for prenatal diagnosis of KS and genetic counseling. Cryptorchidism and the absence or hypoplasia of the olfactory bulb/tract is very high in KS patients with ANOS1 mutations.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this study.

Funding

This work was supported by National Key Research and Development Program of China (2016YFbib905100), CAMS Innovation Fund for Medical Sciences (2016-I2M-1-002) and the National Key Program of Clinical Science (WBYZ2011-873).

Author contribution statement

X W and H Z: conceived and designed the experiments; X W, H X, J M, X W, S X, J Z, W M and Q H: collected blood samples and clinical data; M N, R C, B Y and M C: performed the experiments; M N: wrote the paper.

Acknowledgement

The authors thank the subjects and their family members for their participation in the research.

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    Calabrese R, Capriotti E, Fariselli P, Martelli PL & Casadio R. Functional annotations improve the predictive score of human disease-related mutations in proteins. Human Mutation 2009 30 12371244. (doi:10.1002/humu.21047)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

    Li B, Krishnan VG, Mort ME, Xin F, Kamati KK, Cooper DN, Mooney SD & Radivojac P. Automated inference of molecular mechanisms of disease from amino acid substitutions. Bioinformatics 2009 25 27442750. (doi:10.1093/bioinformatics/btp528)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15

    Choi Y, Sims GE, Murphy S, Miller JR & Chan AP. Predicting the functional effect of amino acid substitutions and indels. PLoS ONE 2012 7 e46688. (doi:10.1371/journal.pone.0046688)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16

    Ye ZQ, Zhao SQ, Gao G, Liu XQ, Langlois RE, Lu H & Wei L. Finding new structural and sequence attributes to predict possible disease association of single amino acid polymorphism (SAP). Bioinformatics 2007 23 14441450. (doi:10.1093/bioinformatics/btm119)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17

    Georgopoulos NA, Pralong FP, Seidman CE, Seidman JG, Crowley WJ & Vallejo M. Genetic heterogeneity evidenced by low incidence of KAL-1 gene mutations in sporadic cases of gonadotropin-releasing hormone deficiency. Journal of Clinical Endocrinology and Metabolism 1997 82 213217. (doi:10.1210/jc.82.1.213)

    • Search Google Scholar
    • Export Citation
  • 18

    Bhagavath B, Podolsky RH, Ozata M, Bolu E, Bick DP, Kulharya A, Sherins RJ & Layman LC. Clinical and molecular characterization of a large sample of patients with hypogonadotropic hypogonadism. Fertility and Sterility 2006 85 706713. (doi:10.1016/j.fertnstert.2005.08.044)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19

    Zhang S, Wang T, Yang J, Liu Z, Wang S & Liu J. A fertile male patient with Kallmann syndrome and two missense mutations in the KAL1 gene. Fertility and Sterility 2011 95 17831789. (doi:10.1016/j.fertnstert.2011.05.052)

    • Search Google Scholar
    • Export Citation
  • 20

    Gu WJ, Zhang Q, Wang YQ, Yang GQ, Hong TP, Zhu DL, Yang JK, Ning G, Jin N & Chen K et al. Mutation analyses in pedigrees and sporadic cases of ethnic Han Chinese Kallmann syndrome patients. Experimental Biology and Medicine 2015 240 14801489. (doi:10.1177/1535370215587531)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21

    Hu Y, Sun Z, Eaton JT, Bouloux PM & Perkins SJ. Extended and flexible domain solution structure of the extracellular matrix protein anosmin-1 by X-ray scattering, analytical ultracentrifugation and constrained modelling. Journal of Molecular Biology 2005 350 553570. (doi:10.1016/j.jmb.2005.04.031)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 22

    Robertson A, MacColl GS, Nash JA, Boehm MK, Perkins SJ & Bouloux PM. Molecular modelling and experimental studies of mutation and cell-adhesion sites in the fibronectin type III and whey acidic protein domains of human anosmin-1. Biochemical Journal 2001 357 647659. (doi:10.1042/bj3570647)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23

    Jap TS, Chiu CY, Lirng JF & Won GS. Identification of two novel missense mutations in the KAL1 gene in Han Chinese subjects with Kallmann syndrome. Journal of Endocrinological Investigation 2011 34 5359. (doi:10.1007/BF03346695)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24

    Sato N, Katsumata N, Kagami M, Hasegawa T, Hori N, Kawakita S, Minowada S, Shimotsuka A, Shishiba Y & Yokozawa M et al. Clinical assessment and mutation analysis of Kallmann syndrome 1 (KAL1) and fibroblast growth factor receptor 1 (FGFR1, or KAL2) in five families and 18 sporadic patients. Journal of Clinical Endocrinology and Metabolism 2004 89 10791088. (doi:10.1210/jc.2003-030476)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25

    Oliveira LM, Seminara SB, Beranova M, Hayes FJ, Valkenburgh SB, Schipani E, Costa EM, Latronico AC, Crowley WJ & Vallejo M. The importance of autosomal genes in Kallmann syndrome: genotype-phenotype correlations and neuroendocrine characteristics. Journal of Clinical Endocrinology and Metabolism 2001 86 15321538. (doi:10.1210/jc.86.4.1532)

    • Search Google Scholar
    • Export Citation
  • 26

    Li J, Li N, Ding Y, Huang X, Shen Y, Wang J & Wang X. Clinical characteristics and follow-up of 5 young Chinese males with gonadotropin-releasing hormone deficiency caused by mutations in the KAL1 gene. Meta Gene 2016 7 6469. (doi:10.1016/j.mgene.2015.12.001)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 27

    Kim SH, Hu Y, Cadman S & Bouloux P. Diversity in fibroblast growth factor receptor 1 regulation: learning from the investigation of Kallmann syndrome. Journal of Neuroendocrinology 2008 20 141163. (doi:10.1111/j.1365-2826.2007.01627.x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 28

    Costa-Barbosa FA, Balasubramanian R, Keefe KW, Shaw ND, Al-Tassan N, Plummer L, Dwyer AA, Buck CL, Choi JH & Seminara SB et al. Prioritizing genetic testing in patients with Kallmann syndrome using clinical phenotypes. Journal of Clinical Endocrinology and Metabolism 2013 98 E943E953. (doi:10.1210/jc.2012-4116)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29

    Shin SJ, Sul Y, Kim JH, Cho JH, Kim GH, Kim JH, Choi JH & Yoo HW. Clinical, endocrinological, and molecular characterization of Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism: a single center experience. Annals of Pediatric Endocrinology and Metabolism 2015 20 2733. (doi:10.6065/apem.2015.20.1.27)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30

    Rastrelli G, Corona G, Mannucci E & Maggi M. Factors affecting spermatogenesis upon gonadotropin-replacement therapy: a meta-analytic study. Andrology 2014 2 794808. (doi:10.1111/andr.262)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 31

    Liu PY, Baker HW, Jayadev V, Zacharin M, Conway AJ & Handelsman DJ. Induction of spermatogenesis and fertility during gonadotropin treatment of gonadotropin-deficient infertile men: predictors of fertility outcome. Journal of Clinical Endocrinology and Metabolism 2009 94 801808. (doi:10.1210/jc.2008-1648)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32

    Trsinar B & Muravec UR. Fertility potential after unilateral and bilateral orchidopexy for cryptorchidism. World Journal of Urology 2009 27 513519. (doi:10.1007/s00345-009-0406-0)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 33

    Liu Z, Mao J, Wu X, Xu H, Wang X, Huang B, Zheng J, Nie M & Zhang H. Efficacy and outcome predictors of gonadotropin treatment for male congenital hypogonadotropic hypogonadism: a retrospective study of 223 patients. Medicine 2016 95 e2867. (doi:10.1097/MD.0000000000002867)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation

 

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  • View in gallery

    The sequencing chromatogram of the mutation in ANOS1 gene. Dash indicates mutated nucleotide. Novel mutations were shown in red.

  • View in gallery

    Distribution of mutations in ANOS1 gene and anosmin1 protein. (A) Blue box stands for the exons. (B) Recurrent mutations are in the upper of the box, novel mutations found in this study are in the below of the box and shown in red. (C) Distribution of missense or nonsense mutations in relation to the anosmin1 protein. Novel mutations were shown in red.

  • View in gallery

    KS pedigree (1–6) with ANOS1 mutation are shown. Affected individuals with KS are shown as completely shaded. The arrow indicates the proband of each pedigree. All individuals marked numbers were screened for the mutation of ANOS1 gene.

  • View in gallery

    The sequence alignment of anosmin-1 proteins from different species. The cysteine residue at the position 86th, 90th and 151st of anosmin-1 protein is conserved across twenty-six different species.

  • 1

    Herbison AE. Control of puberty onset and fertility by gonadotropin-releasing hormone neurons. Nature Reviews Endocrinology 2016 12 452466. (doi:10.1038/nrendo.2016.70)

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    Boehm U, Bouloux PM, Dattani MT, de Roux N, Dode C, Dunkel L, Dwyer AA, Giacobini P, Hardelin JP & Juul A et al. Expert consensus document: European Consensus Statement on congenital hypogonadotropic hypogonadism – pathogenesis, diagnosis and treatment. Nature Reviews Endocrinology 2015 11 547564. (doi:10.1038/nrendo.2015.112)

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    Kim SH. Congenital hypogonadotropic hypogonadism and Kallmann syndrome: past, present, and future. Endocrinology Metabolism 2015 30 456466. (doi:10.3803/EnM.2015.30.4.456)

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    Topaloglu AK & Kotan LD. Genetics of hypogonadotropic hypogonadism. Endocrine Development 2016 29 3649. (doi:10.1159/000438841)

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    MacColl GS, Quinton R & Bulow HE. Biology of KAL1 and its orthologs: implications for X-linked Kallmann syndrome and the search for novel candidate genes. Frontiers of Hormone Research 2010 39 6277. (doi:10.1159/000312694)

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    Sim NL, Kumar P, Hu J, Henikoff S, Schneider G & Ng PC. SIFT web server: predicting effects of amino acid substitutions on proteins. Nucleic Acids Research 2012 40 W452W457. (doi:10.1093/nar/gks539)

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    Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS & Sunyaev SR. A method and server for predicting damaging missense mutations. Nature Methods 2010 7 248249. (doi:10.1038/nmeth0410-248)

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    Reva B, Antipin Y & Sander C. Predicting the functional impact of protein mutations: application to cancer genomics. Nucleic Acids Research 2011 39 e118. (doi:10.1093/nar/gkr407)

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    Thomas PD & Kejariwal A. Coding single-nucleotide polymorphisms associated with complex vs. Mendelian disease: evolutionary evidence for differences in molecular effects. PNAS 2004 101 1539815403. (doi:10.1073/pnas.0404380101)

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    • Export Citation
  • 12

    Bromberg Y & Rost B. SNAP: predict effect of non-synonymous polymorphisms on function. Nucleic Acids Research 2007 35 38233835. (doi:10.1093/nar/gkm238)

  • 13

    Calabrese R, Capriotti E, Fariselli P, Martelli PL & Casadio R. Functional annotations improve the predictive score of human disease-related mutations in proteins. Human Mutation 2009 30 12371244. (doi:10.1002/humu.21047)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14

    Li B, Krishnan VG, Mort ME, Xin F, Kamati KK, Cooper DN, Mooney SD & Radivojac P. Automated inference of molecular mechanisms of disease from amino acid substitutions. Bioinformatics 2009 25 27442750. (doi:10.1093/bioinformatics/btp528)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15

    Choi Y, Sims GE, Murphy S, Miller JR & Chan AP. Predicting the functional effect of amino acid substitutions and indels. PLoS ONE 2012 7 e46688. (doi:10.1371/journal.pone.0046688)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16

    Ye ZQ, Zhao SQ, Gao G, Liu XQ, Langlois RE, Lu H & Wei L. Finding new structural and sequence attributes to predict possible disease association of single amino acid polymorphism (SAP). Bioinformatics 2007 23 14441450. (doi:10.1093/bioinformatics/btm119)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17

    Georgopoulos NA, Pralong FP, Seidman CE, Seidman JG, Crowley WJ & Vallejo M. Genetic heterogeneity evidenced by low incidence of KAL-1 gene mutations in sporadic cases of gonadotropin-releasing hormone deficiency. Journal of Clinical Endocrinology and Metabolism 1997 82 213217. (doi:10.1210/jc.82.1.213)

    • Search Google Scholar
    • Export Citation
  • 18

    Bhagavath B, Podolsky RH, Ozata M, Bolu E, Bick DP, Kulharya A, Sherins RJ & Layman LC. Clinical and molecular characterization of a large sample of patients with hypogonadotropic hypogonadism. Fertility and Sterility 2006 85 706713. (doi:10.1016/j.fertnstert.2005.08.044)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19

    Zhang S, Wang T, Yang J, Liu Z, Wang S & Liu J. A fertile male patient with Kallmann syndrome and two missense mutations in the KAL1 gene. Fertility and Sterility 2011 95 17831789. (doi:10.1016/j.fertnstert.2011.05.052)

    • Search Google Scholar
    • Export Citation
  • 20

    Gu WJ, Zhang Q, Wang YQ, Yang GQ, Hong TP, Zhu DL, Yang JK, Ning G, Jin N & Chen K et al. Mutation analyses in pedigrees and sporadic cases of ethnic Han Chinese Kallmann syndrome patients. Experimental Biology and Medicine 2015 240 14801489. (doi:10.1177/1535370215587531)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21

    Hu Y, Sun Z, Eaton JT, Bouloux PM & Perkins SJ. Extended and flexible domain solution structure of the extracellular matrix protein anosmin-1 by X-ray scattering, analytical ultracentrifugation and constrained modelling. Journal of Molecular Biology 2005 350 553570. (doi:10.1016/j.jmb.2005.04.031)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 22

    Robertson A, MacColl GS, Nash JA, Boehm MK, Perkins SJ & Bouloux PM. Molecular modelling and experimental studies of mutation and cell-adhesion sites in the fibronectin type III and whey acidic protein domains of human anosmin-1. Biochemical Journal 2001 357 647659. (doi:10.1042/bj3570647)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23

    Jap TS, Chiu CY, Lirng JF & Won GS. Identification of two novel missense mutations in the KAL1 gene in Han Chinese subjects with Kallmann syndrome. Journal of Endocrinological Investigation 2011 34 5359. (doi:10.1007/BF03346695)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24

    Sato N, Katsumata N, Kagami M, Hasegawa T, Hori N, Kawakita S, Minowada S, Shimotsuka A, Shishiba Y & Yokozawa M et al. Clinical assessment and mutation analysis of Kallmann syndrome 1 (KAL1) and fibroblast growth factor receptor 1 (FGFR1, or KAL2) in five families and 18 sporadic patients. Journal of Clinical Endocrinology and Metabolism 2004 89 10791088. (doi:10.1210/jc.2003-030476)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25

    Oliveira LM, Seminara SB, Beranova M, Hayes FJ, Valkenburgh SB, Schipani E, Costa EM, Latronico AC, Crowley WJ & Vallejo M. The importance of autosomal genes in Kallmann syndrome: genotype-phenotype correlations and neuroendocrine characteristics. Journal of Clinical Endocrinology and Metabolism 2001 86 15321538. (doi:10.1210/jc.86.4.1532)

    • Search Google Scholar
    • Export Citation
  • 26

    Li J, Li N, Ding Y, Huang X, Shen Y, Wang J & Wang X. Clinical characteristics and follow-up of 5 young Chinese males with gonadotropin-releasing hormone deficiency caused by mutations in the KAL1 gene. Meta Gene 2016 7 6469. (doi:10.1016/j.mgene.2015.12.001)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 27

    Kim SH, Hu Y, Cadman S & Bouloux P. Diversity in fibroblast growth factor receptor 1 regulation: learning from the investigation of Kallmann syndrome. Journal of Neuroendocrinology 2008 20 141163. (doi:10.1111/j.1365-2826.2007.01627.x)

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 28

    Costa-Barbosa FA, Balasubramanian R, Keefe KW, Shaw ND, Al-Tassan N, Plummer L, Dwyer AA, Buck CL, Choi JH & Seminara SB et al. Prioritizing genetic testing in patients with Kallmann syndrome using clinical phenotypes. Journal of Clinical Endocrinology and Metabolism 2013 98 E943E953. (doi:10.1210/jc.2012-4116)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29

    Shin SJ, Sul Y, Kim JH, Cho JH, Kim GH, Kim JH, Choi JH & Yoo HW. Clinical, endocrinological, and molecular characterization of Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism: a single center experience. Annals of Pediatric Endocrinology and Metabolism 2015 20 2733. (doi:10.6065/apem.2015.20.1.27)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30

    Rastrelli G, Corona G, Mannucci E & Maggi M. Factors affecting spermatogenesis upon gonadotropin-replacement therapy: a meta-analytic study. Andrology 2014 2 794808. (doi:10.1111/andr.262)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 31

    Liu PY, Baker HW, Jayadev V, Zacharin M, Conway AJ & Handelsman DJ. Induction of spermatogenesis and fertility during gonadotropin treatment of gonadotropin-deficient infertile men: predictors of fertility outcome. Journal of Clinical Endocrinology and Metabolism 2009 94 801808. (doi:10.1210/jc.2008-1648)

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 32

    Trsinar B & Muravec UR. Fertility potential after unilateral and bilateral orchidopexy for cryptorchidism. World Journal of Urology 2009 27 513519. (doi:10.1007/s00345-009-0406-0)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 33

    Liu Z, Mao J, Wu X, Xu H, Wang X, Huang B, Zheng J, Nie M & Zhang H. Efficacy and outcome predictors of gonadotropin treatment for male congenital hypogonadotropic hypogonadism: a retrospective study of 223 patients. Medicine 2016 95 e2867. (doi:10.1097/MD.0000000000002867)

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation