Abstract
Objective
This study was performed to investigate the molecular characteristics and frequency of copy number variations (CNVs) of ANOS1 in patients with Kallmann syndrome (KS) or normosmic isolated hypogonadotropic hypogonadism (nIHH) using multiplex ligation-dependent probe amplification (MLPA) analysis and sequencing.
Methods
Among 45 patients from 43 independent families, Sanger sequencing, next-generation sequencing (NGS), or microarray was performed in 24 patients from 23 families, and MLPA was performed in 19 patients who did not show rare sequence variants (n = 18) or ANOS1 amplification by PCR (n = 1).
Results
Seven patients (four patients with KS, one patient with nIHH, one prepubertal boy with anosmia, and one newborn patient) from six families (6/43, 14%) harbored molecular defects in ANOS1 including a nonsense mutation (c.1140G>A (p.W380*)), a frameshift mutation (c.1260del (p.Q421Kfs*61)), a splice site mutation (c.1449+1G>A), an exon 7 deletion, a complete deletion, and 7.9 Mb-sized inversion encompassing ANOS1. The complete deletion of ANOS1 was identified in a neonate with a micropenis and cryptorchidism. Unilateral renal agenesis was found in three patients, whereas only one patient displayed both synkinesia and sensorineural hearing loss. There was no reversal of hypogonadotropic hypogonadism in any patient during 9.1 ± 2.9 years of treatment with testosterone enanthate.
Conclusions
Molecular defects in the ANOS1 gene could be identified in 14% of probands including various types of CNVs (3/43, 7.0%). Comprehensive analysis using sequencing and analysis for CNVs is required to detect molecular defects in ANOS1.
Introduction
Isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD) is a rare, complex genetic disorder caused by defects in endogenous hypothalamic GnRH production, secretion, or action (1). Since the development of the GnRH neurons is accompanied by the development and/or migration of the olfactory system in the early fetus, patients with Kallmann syndrome (KS) manifest a combined dysfunction of the GnRH and olfactory systems (2). IGD is also associated with a normal sense of smell in approximately 50% of patients, referred to as normosmic isolated hypogonadotropic hypogonadism (nIHH) (3). With recent advances in next-generation sequencing (NGS) techniques, more than 60 causative genes for IGD with a diverse mode of inheritance have been identified (4). Previous studies using high-throughput NGS have reported genetic defects in about 30–50% of patients, which varied depending on the number of genes sequenced or whether additional testing was done for copy number variations (CNVs) (5, 6, 7).
The ANOS1 gene, previously known as KAL1, comprises 14 exons and encodes a 680-amino acid extracellular adhesion protein, anosmin-1, which controls the growth and migrational guidance of GnRH and olfactory neurons. ANOS1 was the first gene to be described as causative for KS in 1991 (8, 9). Mutations in ANOS1 have been found in approximately 5–10% of KS patients and in some nIHH cases (7, 10, 11, 12, 13). To date, more than 200 pathogenic or likely pathogenic variants in this gene have been reported, including missense, nonsense, splice variants, and deletions of the entire gene to a single exon (https://www.ncbi.nlm.nih.gov/clinvar/). CNVs of ANOS1 have been reported to various degrees comprising approximately 10% of the mutations in this gene, including deletions of a single exon to that of the entire gene via gross deletions at Xp22.31 as a contiguous gene deletion syndrome (11, 14, 15, 16, 17). ANOS2P, a homolog of ANOS1, is a pseudogene located on the long arm of the Y chromosome, and gene conversion or translocation from ANOS2P to ANOS1 can potentially affect the function of ANOS1, leading to KS or nIHH (18). However, there have been a few studies that have systematically evaluated CNVs in ANOS1 (11, 17, 19).
Because of the presence of a pseudogene, multiplex ligation-dependent probe amplification (MLPA) analysis, as well as sequencing of ANOS1, is recommended for identifying genetic defects in this gene, including rare sequence variants and CNVs (11). Although advanced molecular genetic techniques have expanded the mutation spectrum of ANOS1 in patients with IGD, CNVs such as deletions and duplications are not detected by Sanger sequencing, targeted gene sequencing, or whole-exome sequencing (WES). We hypothesized that high-degree sequence homology between the ANOS1 and ANOS2P can lead to more frequent CNVs than other genes causing IGD. Thus, this study aimed to demonstrate the frequency of rare sequence variants and CNVs in ANOS1 in an IGD cohort using MLPA analysis as well as NGS or Sanger sequencing.
Patients and methods
Patients
This study included 45 patients (38 males and 7 females) from 43 families, 39 of whom were diagnosed with IGD, 5 of whom were prepubertal children who attended our center due to anosmia, and 1 neonate treated at our center due to cryptorchidism and a micropenis. The diagnosis of IGD was based on previously described criteria (6) as follows: (i) absent or incomplete pubertal development by the age of 17 years in females and 18 years in males; (ii) serum testosterone levels of <1.0 ng/mL in males or serum estradiol levels of <20 pg/mL in females with low or normal levels of gonadotropin; (iii) normalcy in other pituitary hormones; (iv) no structural lesions in the hypothalamus or pituitary gland; and (v) no sex chromosomal abnormalities.
Among our study cohort, 21 patients from 20 families harbored pathogenic or likely pathogenic mutations in known IGD genes other than ANOS1 (Supplementary Fig. 1). The patients were evaluated by targeted gene panel sequencing (n = 15) or WES (n = 6). Comprehensive analysis of ANOS1 was performed in 24 patients (20 males and 4 females) from 23 families including Sanger sequencing or NGS, and MLPA analysis. Our study cohort contained 24 patients, including 13 KS cases, 8 nIHH cases, 2 prepubertal children with anosmia, and 1 newborn patient with micropenis and cryptorchidism. Three previously reported patients with ANOS1 mutations (6, 20) were included to delineate the clinical and mutation spectrum in our cohort. The study was approved by the institutional review board at Asan Medical Center (IRB No. 2021-0855). Informed consent was obtained from patients or their parents.
Clinical and endocrinological evaluations
The clinical information on the study patients that was retrospectively reviewed included family history, the presence of micropenis and cryptorchidism, accompanying anomalies, and sense of smell. The pubertal stage was rated according to the Marshall and Tanner criteria (21), and the testicular volume was measured using a Prader orchidometer. Olfactory function was evaluated using 12-item smell identification testing in one patient or the 16-item Korean version of the Sniffin’ sticks test II in two cases (22, 23). The remaining patients were assessed by self-reporting. The luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured via an immunoradiometric assay (Diasource, Louvain-la-Neuve, Belgium), while the testosterone (TESTO-CT2; CisBioassays, Codolet, France) and estradiol (Coat-A-Count; Siemens Healthineers, Erlangen, Germany) levels were measured by radioimmunoassay.
Molecular genetic testing of the ANOS1 gene
To detect single nucleotide variants (SNVs), Sanger sequencing or NGS was performed. MLPA or chromosomal microarray was conducted for the detection of CNVs. Some CNVs were identified by bioinformatic algorithms for CNV detection using NGS data. The detailed genetic testing flow is as follows (Fig. 1). Among the 24 patients from 23 families in whom comprehensive analysis of the ANOS1 gene was conducted, Sanger sequencing was initially performed in 12 patients from 11 families. Nine patients without sequence variants by Sanger sequencing underwent targeted gene panel sequencing (n = 5) or WES (n = 4). NGS was initially performed in 11 patients, including targeted gene panel sequencing (n = 8) or WES (n = 3). Whole-genome sequencing (WGS) was carried out in four patients without any sequence variant by targeted gene panel sequencing or WES. MLPA analysis was performed in 19 patients who showed no sequence variants in ANOS1 by Sanger sequencing or NGS. In a newborn patient, the complete deletion of ANOS1 was detected by G-scanning, a commercial genetic screening test that uses a single nucleotide polymorphism (SNP) array (Boryung Biopharma Co., Seoul, Korea). Re-validation of the CNVs was performed using a CytoScan Dx Assay (Affymetrix), which contains 2,696,550 probes, including 743,304 SNPs and 1,953,246 nonpolymorphic probes. The average probe spacing for the RefSeq gene is 880 bp, which can detect 25–50 kb of copy number changes.
Schematic diagram of the genetic testing workflow used in this study. Among the 45 from 43 families included patients, Sanger sequencing or next-generation sequencing (NGS) was performed on 23 patients from 22 families. MLPA analysis was performed in 19 patients who did not show rare sequence variants (n = 18) or amplified product of ANOS1 by polymerase chain reaction (n = 1). One newborn patient was analyzed using a single nucleotide polymorphism (SNP) array.
Citation: Endocrine Connections 12, 5; 10.1530/EC-22-0413
For Sanger sequencing, genomic DNA was extracted from peripheral blood leukocytes using the Puregene DNA isolation kit (Qiagen). All coding exons and exon–intron boundaries in the ANOS1 gene were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers (Supplementary Table 1, see section on supplementary materials given at the end of this article). The obtained PCR products were sequenced with an ABI3130xl Genetic Analyzer (Applied Biosystems).
Targeted gene panel sequencing was performed using a customized panel, which includes 69 genes related to GnRH development and migration and rare syndromes or neurologic disorders causing hypogonadotropic hypogonadism as previously described (6). For WES, SureSelect Human All Exon V5 (Agilent Technologies) or MGI Easy Exome (MGI Tech Co., San Jose, CA, USA) was used for library construction. Sequencing was performed using the MGI DNBSEQ-G400 or MGI DNBSEQ-T7 platform (MGI Tech Co.), and sequenced reads were aligned to the human reference genome (hg19) using the Burrow-Wheeler Alignment program (version 0.7.17). To detect SNVs and insertion-deletion variants, Genome Analysis Toolkit (GATK version 4.1.8) was used. Annotation was performed with a Variant Effect Predictor (24). For WGS, MGIEasy FS DNA Library Prep Set (MGI Tech Co.) was used for library preparation, and sequencing was performed on the MGI DNBSEQ-T7 platform (MGI Tech Co.). The sequenced reads were aligned to the human reference genome (hg19) using the Burrow-Wheeler Alignment program (version 0.7.17). Variant calling was performed with the Genome Analysis Tool kit (GATK version 4.1.8), and annotation was done with Variant Effect Predictor (24), dbNSFP version 4.1. Copy-number variant (CNV) calling was performed using the Parliament2 (25) pipeline and annotated using AnnotSV version 3.0.2 (26).
Sequence variants were classified according to the standards and guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (27). Sequence variants found via targeted gene panel sequencing or WES were validated by Sanger sequencing.
MLPA analysis was conducted to detect deletions or duplications of ANOS1 using the SALSA MLPA kit P132 Kallmann-1 (MRC Holland, Amsterdam, Netherlands) in accordance with the manufacturer’s instructions. Briefly, genomic DNA was denatured and hybridized with the MLPA probe mix, and subsequently, a ligation reaction was carried out to ligate hybridized probes, after which PCR amplification of ligated products was performed. PCR amplicons were then run on an ABI3130xl Genetic Analyzer (Applied Biosystems), and the gene dosage was determined using GeneMarker software version 1.7 (SoftGenetics, State College, PA, USA).
To validate inversion, PCR amplicons were designed to amplify the identified breakpoints. The primers were designed as region-specific primers, and primers for breakpoint 1 (Fig. 2C and D) were 5’-CCGAAGTCAGATGAAGCACA-3’ (forward) and 5’-GCCTGGTTACGGTCCACTTA-3’ (reverse). Primers for breakpoint 2 were 5’-ATATGTGGAGATGCCCTGA-3’ (forward) and 5’-CCTAGGGGTTTTGTGTGGTG-3’ (reverse). Target sequences were amplified by PCR using PrimeSTAR® GXL DNA polymerase (Takara Bio Inc.). Agarose gel electrophoresis was used to separate PCR products. Then, PCR products were extracted from the gel using a gel extraction protocol (MEGAquick-spinTM plus fragment DNA purification kit, iNtRON Biotechnology, Korea). Amplicons of inversion alleles were sequenced by direct sequencing using ABI3130xl Genetic Analyzer (Applied Biosystems). Primers for genotyping at breakpoint 1 were 5’-CTTGCAGATGGTCACCAGA-3’ (forward) and 5’-CCTTGGGAATCTTTGGGAAT-3’ (reverse). Primers for genotyping breakpoint 2 were 5’-TTTGGCTTTGGAGGTACCAG-3’ (forward) and 5’-AGGAGCAAATGCCCTTTCTT-3’ (reverse).
Distribution of mutations in the ANOS1 gene in the study series. (A) Structure of the ANOS1 gene. Coding exons are indicated by boxes, introns are indicated by black lines. (B) Structure of ANOS1 protein and mutations in the ANOS1 gene found in the study cohort. Cys, cysteine-rich region domain; FNIII, fibronectin type III domain; H, histidine-rich region domain; S, signal peptide; WAP, whey acidic protein-like domain. (C) Schematic view of the inversion variant of ANOS1 in the Integrative Genomic Viewer (https://software.broadinstitute.org/software/igv/). The green and blue read pairs indicate both ends were mapped on the forward and reverse strands, respectively. (D) Mapping of the breakpoints by Sanger sequencing. PCR primers were designed to amplify the normal (A–B and C–D) pairs and mutant (A–C and B–D) pairs. The mutant amplicon was amplified in patient 7. Sanger sequencing of mutant products revealed breakpoints of inversion.
Citation: Endocrine Connections 12, 5; 10.1530/EC-22-0413
Statistics
Continuous variables were presented as mean ± s.d. The chi-squared tests or Fisher exact tests were used to compare categorical variables of clinical characteristics between patients with and without mutations in ANOS1. Mann–Whitney U test was used to compare the mean values of demographic and biochemical characteristics of the two groups. Statistical analyses were performed using SPSS version 21.0 for Windows (IBM).
Results
Molecular analysis of the ANOS1 gene
Among the study cohort of 45 patients from 43 families, 6 different genetic variants in ANOS1 were identified in 7 male patients from 6 independent families (6/43 families, 14%). These included a nonsense mutation (c.1140G>A (p.W380*)), frameshift mutation (c.1260del (p.Q421Kfs*61)), splice site mutation (c.1449+1G>A), single exon deletion, a complete deletion of the gene, and inv(X)(p22.33p22.2) encompassing the ANOS1 gene (Fig. 2).
In patient 1, exon 7 of ANOS1 was found to be absent by PCR amplification before Sanger sequencing, and this was confirmed by MLPA analysis (Supplementary Fig. 2). This deletion has not been previously reported and is not found in the Genome Aggregation Database (https://gnomad.broadinstitute.org). Two sibling patients (patients 2 and 3) were found to be hemizygous for c.1140G>A (p.W380*) in ANOS1, which has been classified previously as pathogenic (20). Patient 4 was found to harbor a frameshift mutation, c.1260del (p.Q421Kfs*61), which was also previously reported to be pathogenic (6). In patient 5, a splice site mutation, c.1449+1G>A, was identified by WES and was classified as pathogenic (9). Patient 6 was referred during the neonatal period due to a suspected ANOS1 deletion revealed by G-scanning, which was undertaken in a primary clinic because of micropenis and cryptorchidism. By SNP array, we identified a complete deletion of the ANOS1 gene through the loss of an approximately 948 kb region in the chromosome Xp22.31 (chrX: 8,135,644–9,083,909) that incorporates the VCX2, ANOS1, FAM9A, and FAM9B genes. In patient 7, WGS identified a novel structural variation of 7.9 Mb-sized inversion encompassing ANOS1. The inversion was validated by PCR using two sets of specific primers, including both breakpoints of inversion (Fig. 2C and D).
Clinical and endocrine characteristics of the patients harboring mutations in ANOS1
The clinical and endocrine characteristics of our study patients are summarized in Table 1. Among the seven cases in our series showing ANOS1 mutations, four were diagnosed with KS and one was categorized as an nIHH patient. As mentioned, the neonate with a micropenis and cryptorchidism (patient 6) had a complete deletion of the ANOS1 gene, and a prepubertal boy with anosmia (patient 7) had an inversion including in ANOS1.
Clinical and molecular characteristics of the included patients with mutations in the ANOS1 gene.
| Family no. | Patient no. | Age at diagnosis (years) | Phenotype | Olfaction | Basal hormone levels | Brain MRI | Non-reproductive phenotype | ANOS1 mutation |
|---|---|---|---|---|---|---|---|---|
| 1 | 1 | 19.5 | KS | Anosmia (self-reported) | LH 0.7 mIU/mL, FSH 0.92 mIU/mL, testosterone 0.05 ng/mL | Absence of olfactory bulbs Hypoplastic left olfactory sulcus | None | Exon 7 deletion |
| 2 | 2 | 16.4 | KS | Anosmia (2/16, KVSS II) | LH <0.1 mIU/mL, FSH 0.4 mIU/mL, testosterone 0.67 ng/mL | Hypoplasia of bilateral olfactory bulbs and absence of olfactory sulci | None | c.1140G>A (W380*) |
| 3 | 14.9 | KS | Hyposmia (4/16, KVSS II) | LH <0.1 mIU/mL, FSH 0.7 mIU/mL, testosterone 0.69 ng/mL | Absence of bilateral olfactory sulci and bulbs | Right renal agenesis | c.1140G>A (W380*) | |
| 3 | 4 | 16.9 | nIHH | Normosmia (self-reported) | LH 1.1 mIU/mL, FSH 2.0 mIU/mL, testosterone 0.11 ng/mL | Normal olfactory bulbs and sulci | None | c.1260del (p.Q421Kfs*61) |
| 4 | 5 | 17.9 | KS | Anosmia (self-reported) | LH <0.1 mIU/mL, FSH 0.4 mIU/mL, testosterone 0.06 ng/mL | ND | Right renal agenesis | c.1449+1G>A |
| 5 | 6 | 0.5 | Micropenis, cryptorchidism | NA | LH 0.82 mIU/mL, FSH 0.75 mIU/mL, testosterone 0.04 ng/mL | ND | None | Complete deletion |
| 6 | 7 | 13.5 | Anosmia | Anosmia (2/12, KVSS) | LH 1.3 mIU/mL, FSH 0.62 mIU/mL, testosterone 0.26 ng/mL | Hypoplastic olfactory bulbs | Synkinesia, bilateral SNHL, developmental delay, ptosis, myopia, left renal agenesis | NC_000023.10:g.(8665932T>C;8665932_16596011inv) |
FSH, follicle-stimulating hormone; KS, Kallmann syndrome; KVSS, Korean version of Sniffin’ Sticks test; LH, luteinizing hormone; MRI, magnetic resonance imaging; NA, not assessed; ND, not done; nIHH, normosmic isolated hypogonadotropic hypogonadism; No, number; SNHL, sensorineural hearing loss.
The mean age at presentation of the five IGD patients (four KS and one nIHH) was 17.1 ± 1.7 years (range, 14.9–19.5 years). In these cases, the mean basal LH and FSH levels were 0.4 ± 0.5 and 0.8 ± 0.7 mIU/mL, respectively, and the testosterone levels were 0.3 ± 0.3 ng/mL. Among the four IGD patients who underwent a brain MRI, aplasia or hypoplasia of the olfactory bulbs and sulci was documented in three cases, and all showed hyposmia or anosmia. One patient with nIHH (patient 4) revealed normal olfactory bulbs and sulci (Table 1). Unilateral renal agenesis was detected in two IGD patients. Gynecomastia was observed in patient 1, and he underwent a mastectomy. Synkinesia and sensorineural hearing loss were found only in a prepubertal patient with anosmia (patient 7). Unilateral renal agenesis was more common in patients with ANOS1 mutations compared to those without ANOS1 mutations (P = 0.036; Table 2). The five IGD patients have been treated with monthly testosterone enanthate injections since the age of 15.9 ± 1.4 years. During a 9.1 ± 2.9 year follow-up period (current age, 26.8 ± 1.3 years), there was no observed reversal of the hypogonadotropic hypogonadism, and the testicular volume changed from 2.2 ± 0.5 to 2.5 ± 1.0 mL (P = 0.190).
Comparison of clinical characteristics of patients with or without mutations in ANOS1.
| Characteristics | Patients without ANOS1 mutation (n = 17) | Patients with ANOS1 mutation (n = 7) | P |
|---|---|---|---|
| Male, n (%) | 12 (70.6%) | 7 (100%) | 0.541 |
| Mean age ± s.d., years | 15.0 ± 4.1 | 16.5 ± 2.1 | 0.821 |
| LHa, mIU/mL | 1.49 ± 1.19 | 0.42 ± 0.464 | 0.27 |
| FSHa, mIU/mL | 0.51 ± 0.24 | 0.83 ± 0.72 | 0.903 |
| Testosteronea, ng/mL | 0.15 ± 0.13 | 0.31 ± 0.33 | 0.624 |
| Hyposmia/anosmia, n (%) | 9/17 (52.9%) | 5/6 (83.3%) | 0.351 |
| Olfactory bulb hypoplasia/aplasia | 3/14 (21.4%) | 4/5 (80%) | 0.26 |
| Unilateral renal agenesis, n (%) | 0/12 | 3/7 (42.9%) | 0.036 |
| Hearing defect, n (%) | 1 (5.9%) | 1 (14.3%) | 0.481 |
| Synkinesia, n (%) | 0 | 1 (14.3%) | 0.273 |
aThese levels were determined in five patients with IGD. FSH, follicle-stimulating hormone; LH, luteinizing hormone.
Patient 6, a neonate who presented with micropenis and cryptorchidism, was treated with a 3-month course of intramuscular testosterone enanthate at a monthly dose of 25 mg during the age of 4–6 months, and the stretched penile length, in this case, increased from 1.3 to 2.5 cm. Both testes were palpable in the inguinal canal at 1 month of age, and then spontaneously descended into the scrotum at 1 year of age.
Discussion
This study identified that molecular defects in ANOS1 were identified in 14% (6 of 43 families) of the patients in our current study series, and CNVs were found in approximately 7.0% (3/43 families) of patients in our cohort. Abnormalities in the olfactory bulbs/sulci usually accompany mutations in the ANOS1 gene. Renal anomaly was more common in patients with ANOS1 mutations; however, only one patient manifested synkinesia and hearing defect. Traditional automatic sequencing cannot detect the CNVs of ANOS1; therefore, comprehensive analysis of ANOS1 using both sequencing and MLPA analysis can increase diagnostic yields.
The GnRH neurons have a unique developmental process as they arise outside of the brain, in the primitive olfactory code, and subsequently, migrate into the intracranial hypothalamus. Anosmin-1, which is encoded by ANOS1, is a glycoprotein and an axon guidance molecule during fetal development (28) and is transiently expressed in the developing human fetus, including the olfactory bulbs and along the migratory pathway for GnRH neurons (29). In addition, anosmin-1 regulates neuronal cell function, such as neurite branching, outgrowth, and migration through the activation of FGFR1 (30, 31), and promotes angiogenesis through vascular endothelial growth factor (VEGFR)-related signaling as well as migration (32). Hence, in the absence of anosmin-1, olfactory bulb development and axon navigation are impaired, resulting in hypogonadotropic hypogonadism. Based on the detection of anosmin-1 in the mesonephric duct and ureteric bud in the 6- to 7-week-old human embryo, unilateral renal agenesis and/or ipsilateral ductus deferens agenesis are presumed to be related to developmental failure (33).
Anosmin-1 also promotes the outgrowth and branching of the Purkinje axons in the developing rat cerebellum, suggesting that it may be involved in synkinesia and cerebellar ataxia (34). The transient expression of anosmin-1 in numerous tissues of the developing human fetus thereby contributes to various non-reproductive phenotypes, although anosmin-1 is mainly involved in GnRH and olfactory cell development. Hyposmia or anosmia are the typical characteristics of patients with mutations in ANOS1. However, in our current study cohort, the olfactory function was preserved in one patient who developed normal olfactory bulbs. This is not a common finding among patients harboring an ANOS1 mutation (12, 35). In a previous report, normosmic patients with an ANOS1 mutation showed a decrease in olfactory function with age, although hypoplastic olfactory bulbs were observed on MRI in those cases (35). Thus, the development of olfactory bulbs and tracts can be variably affected by the mutation in ANOS1, leading to variable degrees of olfactory dysfunction (36). It has been suggested that environmental as well as genetic factors play a role in olfactory function (35). However, the olfactory function was assessed by self-reported history in two previously reported patients (12) and one patient in our cohort (Supplementary Table 1). Therefore, a possible misjudgment by the patients could not be excluded.
Previous large-scale IGD cohort studies using NGS technologies have demonstrated that numerous genes contribute to the development of IGD (7, 13, 37, 38, 39). In a prior study with 130 patients in a Brazilian cohort using targeted gene panel sequencing, about 10% of the patients harbored ANOS1 mutations (7). A single-center study of 210 Chinese patients using direct sequencing of ANOS1 reported a 9% of prevalence of mutations in this gene (13). Missense mutations were the most frequently reported; followed by frameshift, nonsense, or deletions, but no mutational hot spots have been found. The anosmin-1 protein comprises an N-terminus, a cysteine-rich (cys-box) domain, a whey acidic protein-like domain, and four fibronectin III (FNIII) domains (40). Although only a small number of patients were included in this present study, most of the identified mutations were located in the FNIII domain, which is essential for the binding of anosmin-1 to FGFR1 and necessary for the activation of VEGFR2 (32). In terms of clinical characteristics, ANOS1 mutations are associated with not only a severe reproductive phenotype (7) but also non-reproductive phenotypes such as bimanual synkinesia or renal abnormality (1, 4). In the present study, unilateral renal agenesis and synkinesia were more commonly observed in patients with ANOS1 variants.
The sequence homologies between active genes and their corresponding pseudogenes can lead to frequent homologous recombination events, including gene conversions, deletions, and duplications, as found in CYP21A2, GBA, and PKD1 (18). In previous studies, exon 14 in ANOS1 was reported to be sometimes replaced by a homologous segment in ANOS2P (18, 41). In our current cohort, we identified two patients with CNVs, including a single exon deletion and a whole gene deletion. There are some prior studies that have systematically evaluated molecular defects in ANOS1 via sequencing and MLPA (11, 17). In a previous report on 115 Brazilian patients with KS, 17 cases were found to harbor ANOS1 mutations (11). Of these, 29.4% (5 of 17) had an intragenic deletion, and one patient (5.8%) had a whole gene deletion (11). In several other studies, the frequency of ANOS1 mutations was either too small or MLPA analysis was not conducted, making it difficult to assess the mutation spectrum of this gene (37, 39, 42).
WES is useful for the clinical diagnosis of rare Mendelian diseases with locus heterogeneity. In the recent study in a large IGD cohort, the overall prevalence of CNVs was ~2% (29/1394), affecting previously known IGD genes using WES. Among them, deletions and duplications in ANOS1 were the most common (0.9%, 12/1394) (4). In the present study, two patients with deletions in ANOS1 were identified; however, there was no duplication in ANOS1. CNVs can be usually detected by MLPA analysis or chromosomal microarray. Recently, bioinformatic algorithms detecting CNVs using NGS data have been introduced, such as read depth analysis, paired-end mapping, or split read approach (43). Therefore, both SNVs and CNVs can be identified by NGS. However, the detection of large or complex rearrangements from WES is complicated and limited in the case of variants in non-coding regions or inversion variants without dosage effects. In the present study, various types of CNVs were discovered by different molecular techniques, including MLPA analysis and WGS. Although WGS is the most comprehensive test to detect both SNVs and CNVs, it is not widely used because of challenging data analysis and high cost.
We here identified a patient with a deletion in the chromosome Xp22.31 region which includes the VCX2, ANOS1, FAM9A, and FAM9B genes. The accepted theory of the evolution of sex chromosomes is that they evolved from a pair of homologous autosomes, and using the ratio of nucleotide divergence per synonymous site for X-Y pairs of genes, ANOS1 and ANOS2P began to differentiate from 45 to 50 million years ago (18, 44). Four members of the VCX and two of the VCY families show a high degree of sequence similarity within each gene family, as well as between the genes on the X and Y chromosomes (45). Low-copy number repeat (LCR) regions around the VCX family genes and STS within the chromosome Xp22.3 region are involved in genomic recombination (46, 47). These sequence homologies within the X chromosome, and between the X and Y chromosomes and LCR regions, eventually cause genomic structural aberrations of the X chromosome. Deletions of STS cause X-linked ichthyosis (OMIM 208100), and several patients with Xp22.3 interstitial deletions have demonstrated a link to intellectual disability (46, 47). In patients with a hypogonadism-ichthyosis phenotype, contiguous gene syndromes should be suspected based on the current understanding of the molecular mechanisms of this highly vulnerable genomic location.
The present study identified a patient with a large inversion variant. The inverted region involved the second intron of ANOS1. The patient displayed developmental delay, sensorineural hearing loss, myopia, and ptosis. This region also contains multiple genes, some of which are responsible for X-linked Opitz G/BBB syndrome (MID1), Joubert syndrome (OFD1), and development delay (AP1S2). Although we did not validate whether the inversion leads to these phenotypes by disruption of coding regions or by affecting gene expression, it is presumed that this variant may cause the patient's neurological and ophthalmic abnormalities.
This study was limited by the small number of patients used to delineate the molecular spectrum of ANOS1 variants and genotype–-phenotype correlations in patients with IGD. Genetic testing was performed consecutively using different modalities such as targeted gene panel sequencing, WES, or WGS. In addition, DNA samples were not available for segregation analysis in the present study. However, this study is the first research to assess the frequency of ANOS1 mutations and CNVs in a relatively large IGD cohort in Korea.
In conclusion, genetic defects in ANOS1 accounted for 14% of the IGD patients analyzed in this study, and various CNVs in ANOS1 were found, including an intragenic deletion, a complete deletion, and an inversion spanning ANOS1. Comprehensive analyses using sequencing and MLPA analysis or WGS are helpful in detecting genetic defects in ANOS1.
Declaration of interest
No potential conflict of interest relevant to this article was reported.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/EC-22-0413.
Funding
This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT) (No. NRF2021R1F1A104593011).
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