ANOS1 accelerates the progression of esophageal cancer identified by multi-omic approaches.

To assess the role of ANOS1 in esophageal cancer (ESCA) progression, multi-omic analysis and experimental validation were employed. It was revealed that ANOS1 expression is significantly enhanced in ESCA patients and cell lines. The expression level of ANOS1 in ESCA patients can distinguish the malignancy from normal tissue with an area under curve (AUC) >0.75. Moreover, increased expression of ANOS1 is associated with advanced T stage and worse disease-free survival of ESCA patients. Therefore, a clinically applicable nomogram with ANOS1 was established with strong predictive power. Furthermore, high expression of ANOS1 in ESCA is correlated with (i) the enrichment of epithelial-mesenchymal transition by gene set enrichment analysis, (ii) the involvement in hypoxia, angiogenesis, WNT signaling pathway, and TGFß signaling pathway by gene set variation analysis, (iii) the presence of the small insertion and deletion mutational signature ID9, associated with chromothripsis, in the single-nucleotide polymorphism analysis, (iv) the amplification of 11q13.3 in the copy number variants analysis, (v) the enrichment of cancer-associated fibroblasts and mesenchymal stromal cells in the tumor microenvironment. All the results from multi-omic analysis indicate that ANOS1 plays a pivotal role in accelerating the progression of ESCA. Results from in vivo and in vitro experiments show that the knockdown of ANOS1 hampers the proliferation of ESCA cells, further validating the oncogenic role of ANOS1 in ESCA. Additionally, potential chemotherapeutics with sensitivity were identified in the high-ANOS1 group. In conclusion, ANOS1 accelerates the progression of ESCA.

A novel mutation in ANOS1 in a Chinese family with Kallmann syndrome: Case report.

We reported a novel variant in Kallmann syndrome. It not only determines the clinical importance of whole exome sequencing for identification of genetic pathogenic variants, but also enriches the ANOS1 genetic spectrum of CHH patients in Chinese population.

Mutation spectrum and frequency of copy number variations of the ANOS1 gene in patients with Kallmann syndrome or normosmic isolated hypogonadotropic hypogonadism.

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.

Mutation spectrum of Kallmann syndrome: identification of five novel mutations across ANOS1 and FGFR1.

BACKGROUND: Kallmann syndrome (KS) is a common type of idiopathic hypogonadotropic hypogonadism. To date, more than 30 genes including ANOS1 and FGFR1 have been identified in different genetic models of KS without affirmatory genotype-phenotype correlation, and novel mutations have been found. METHODS: A total of 35 unrelated patients with clinical features of disorder of sex development were recruited. Custom-panel sequencing or whole-exome sequencing was performed to detect the pathogenic mutations. Sanger sequencing was performed to verify single-nucleotide variants. Copy number variation-sequencing (CNV-seq) was performed to determine CNVs. The pathogenicity of the identified variant was predicted in silico. mRNA transcript analysis and minigene reporter assay were performed to test the effect of the mutation on splicing. RESULTS: ANOS1 gene c.709 T > A and c.711 G > T were evaluated as pathogenic by several commonly used software, and c.1063-2 A > T was verified by transcriptional splicing assay. The c.1063-2 A > T mutation activated a cryptic splice acceptor site downstream of the original splice acceptor site and resulted in an aberrant splicing of the 24-basepair at the 5' end of exon 8, yielding a new transcript with c.1063-1086 deletion. FRFR1 gene c.1835delA was assessed as pathogenic according to the ACMG guideline. The CNV of del(8)(p12p11.22)chr8:g.36140000_38460000del was judged as pathogenic according to the ACMG & ClinGen technical standards. CONCLUSIONS: Herein, we identified three novel ANOS1 mutations and two novel FGFR1 variations in Chinese KS families. In silico prediction and functional experiment evaluated the pathogenesis of ANOS1 mutations. FRFR1 c.1835delA mutation and del(8)(p12p11.22)chr8:g.36140000_38460000del were assessed as pathogenic variations. Therefore, our study expands the spectrum of mutations associated with KS and provides diagnostic evidence for patients who carry the same mutation in the future.

Long non-coding RNA MSTRG.5970.28 regulates proliferation and apoptosis of goose follicle granulosa cells via the miR-133a-3p/ANOS1 pathway.

The development of follicles in the ovaries is a critical determinant of poultry egg production. There are existing studies on the follicular development patterns in poultry, but the specific regulatory mechanisms still need further study. In a previous study, we identified long non-coding RNA (lncRNA) MSTRG.5970.28, anosmin 1 (ANOS1), and its predicted target miR-133a-3p that may be associated with goose ovary development. However, the function of MSTRG.5970.28 in goose granulosa cells and its regulatory mechanisms affecting granulosa cell proliferation and apoptosis have not been reported. In the present study, MSTRG.5970.28 and miR-133a-3p overexpression and interference vectors were constructed. Combined with reverse-transcription real-time quantitative PCR (RT-qPCR), a dual luciferase activity assay, Cell Counting Kit-8 (CCK-8), and flow cytometric analysis, we investigated the role of the MSTRG.5970.28-miR-133a-3p-ANOS1 axis in goose follicular granulosa cells and the associated regulatory mechanisms. MSTRG.5970.28 was found to be localized in the cytoplasm and its expression was influenced by reproductive hormones. The targeting relationship among MSTRG.5970.28, ANOS1, and miR-133a-3p were verified by a dual luciferase activity assay. CCK-8 and apoptosis assays showed that MSTRG.5970.28 inhibited the proliferation and promoted apoptosis of goose granulosa cells. The regulatory role of miR-133a-3p on granulosa cell proliferation and apoptosis was opposite to MSTRG.5970.28. We found that the proliferative and apoptotic effects of granulosa cells caused by MSTRG.5970.28 overexpression were attenuated by miR-133a-3p. MSTRG.5970.28 functions as a competitive endogenous RNA that regulates ANOS1 expression by sponging miR-133a-3p and thus exerts regulatory functions in granulosa cells. In sum, the present study identified lncRNA MSTRG.5970.28 as associated with goose ovary development, which affects the expression of ANOS1 by targeting miR-133a-3p, thereby influencing the proliferation and apoptosis of goose granulosa cells.

ANOS1 variants in a large cohort of Chinese patients with congenital hypogonadotropic hypogonadism. / ä¸­å›½å ˆå¤©æ€§ä½Žä¿ƒæ€§è ºæ¿€ç´ æ€§æ€§è ºåŠŸèƒ½å‡é€€ç—‡æ‚£è€ ANOS1的突变.

OBJECTIVES: 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. CONCLUSIONS: 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.

Integrative RNA profiling of TBEV-infected neurons and astrocytes reveals potential pathogenic effectors.

Tick-borne encephalitis virus (TBEV), the most medically relevant tick-transmitted flavivirus in Eurasia, targets the host central nervous system and frequently causes severe encephalitis. The severity of TBEV-induced neuropathogenesis is highly cell-type specific and the exact mechanism responsible for such differences has not been fully described yet. Thus, we performed a comprehensive analysis of alterations in host poly-(A)/miRNA/lncRNA expression upon TBEV infection in vitro in human primary neurons (high cytopathic effect) and astrocytes (low cytopathic effect). Infection with severe but not mild TBEV strain resulted in a high neuronal death rate. In comparison, infection with either of TBEV strains in human astrocytes did not. Differential expression and splicing analyses with an in silico prediction of miRNA/mRNA/lncRNA/vd-sRNA networks found significant changes in inflammatory and immune response pathways, nervous system development and regulation of mitosis in TBEV Hypr-infected neurons. Candidate mechanisms responsible for the aforementioned phenomena include specific regulation of host mRNA levels via differentially expressed miRNAs/lncRNAs or vd-sRNAs mimicking endogenous miRNAs and virus-driven modulation of host pre-mRNA splicing. We suggest that these factors are responsible for the observed differences in the virulence manifestation of both TBEV strains in different cell lines. This work brings the first complex overview of alterations in the transcriptome of human astrocytes and neurons during the infection by two TBEV strains of different virulence. The resulting data could serve as a starting point for further studies dealing with the mechanism of TBEV-host interactions and the related processes of TBEV pathogenesis.

ANOS1 variants in a large cohort of Chinese patients with congenital hypogonadotropic hypogonadism / 中南大学学报(医学版)

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.

A Novel Noncanonical Splicing Mutation of ANOS1 Gene in Siblings with Kallmann Syndrome Identified by Whole-Exome Sequencing.

Kallmann syndrome (KS) is a rare genetic disorder that is characterized by idiopathic hypogonadotropic hypogonadism associated with anosmia. Genetic variants in ANOS1 gene are the most common mutations associated with X-linked recessive form of KS. Canonical ± 1 or 2 splice site variants in ANOS1 have been described to be responsible for KS. Here, we identified a novel noncanonical splice site variant (c.1062+4T>C) in ANOS1 gene in two siblings with KS by whole-exome sequencing (WES). Sanger sequencing showed this mutation was inherited from their mother, whose brother was a KS patient as well. Through the functional assay in vitro, we found that this mutation resulted in a 50-bp deletion of exon 7, which caused frameshift mutation leading to a premature termination of translation and a truncated anosmin-1 protein. Our results revealed that this noncanonical splice site variant is involved in KS. Thus, it is suggested that we should pay attention to the noncanonical splice site variants when using molecular genetic diagnostics of KS.

A Family With Novel X-Linked Recessive Homozygous Mutation in ANOS1 (c.628_629 del, p.1210fs∗) in Kallmann Syndrome Associated Unilateral Ptosis: Case Report and Literature Review.

OBJECTIVE: Kallmann syndrome (KS) may be accompanied by anosmia or hyposmia and midline defects. We present an overweight 16-year-old boy with a lack of puberty, anosmia, congenital right eye ptosis, and normal intellectual function. METHODS: Testicular ultrasonography was performed. Whole-exome sequencing was performed on peripheral blood specimens. Genetic results were confirmed by Sanger sequencing. Anosmia was evaluated quantitatively using the Korean version of the Sniffin' stick test II. RESULTS: Our patient presented with a complaint of lack of body hair growth and small penile size with no remarkable medical history. He was the second son of third-degree consanguineous healthy parents. Physical examination revealed pubertal Tanner stage I. Congenital right eye ptosis and obesity were noted. Anosmia was confirmed. The laboratory evaluation revealed a low serum level of testosterone, follicle-stimulating hormone, and luteinizing hormone. An X-linked recessive homozygous mutation, c.628_629 del (p.1210fs∗) in exon 5 of the ANOS1 gene was revealed and was also found in the patient's uncle and great uncle on the mother's side. CONCLUSION: To date, approximately 28 ANOS1 mutations producing KS phenotypes have been described. However, to the best of our knowledge, this particular X-linked recessive mutation has not been previously reported in KS. Furthermore, ptosis is a rare finding in KS literature. Identification of these cases increases awareness of the phenotypic heterogeneity in novel forms of KS, thereby expediting early definitive treatment, which may prevent the development of further complications.

Molecular diagnosis of Kallmann syndrome with diabetes by whole exome sequencing and bioinformatic approaches.

BACKGROUND: Kallmann syndrome (KS) is a hypogonadotropic hypogonadism accompanied by anosmia or hyposmia. It is associated with the low secretion of gonadotropins which can lead to other abnormal endocrine metabolism disorders such as diabetes. Through genetic and molecular biological methods, more than 10 KS pathogenic genes have been found. AIM: To identify the existing mutation sites of KS with diabetes and reveal the relationship between genotype and phenotype. METHODS: We studied KS pathogenesis through high-throughput exome sequencing on four diabetes' patients with KS for screening the potential pathogenic sites and exploring the genotype-phenotype correlation. Clinical data and peripheral blood samples were collected from the patients. White blood cells were separated and genomic DNA was extracted. High-throughput sequencing of all exons in the candidate pathogenic genes of probands was performed, and the results obtained were analyzed. RESULTS: Sequencing revealed mutations in the KLB p.T313M, ANOS1 p.C172F, and IGSF10 gene (p.Lys1819Arg and p.Arg1035Thr) at different sites, which may have been associated with disease onset. CONCLUSION: The diagnosis of KS is challenging, especially in early puberty, and the clinical manifestations reflect physical delays in development and puberty. Timely diagnosis and treatment can induce puberty, thereby improving sexual, bone, metabolic and mental health.

Study on genetic etiology of three cases of Kallmann syndrome using whole-exome sequencing / 中华内分泌代谢杂志

Objective:Kallmann syndrome(KS) is a complex genetic disease characterized by congenital hypogonadotropic hypogonadism and anosmia. More than 20 genes have been reported to be associated with KS. Herein, we explore potential genetic aberration in 3 KS patients using the whole-exome sequencing. The potentially pathogenic variants filtered were validated by Sanger sequencing.Methods:Genomic DNA was extracted from the peripheral blood of 3 patients with KS and their family members. Sanger sequencing and pedigree verification were performed on the pathogenic variants identified using whole-exome sequencing. The function of the mutation sites were analyzed with bioinformatics software.Results:The proband 1 was a 25 years old male, characterized by lower gonadotropin gonad hypofunction, early grey hair and bilateral sensorineural hearing loss. A heterozygous mutation c. 475C>T(p.R159W) of SOX10 gene was detected in the proband 1. His mother, sister and cousin who had KS phenotype were also found carrying this mutation, showing an autosomal dominant inheritance. The proband 2 was a 15-year-old male with hypogonadotropic hypogonadism and unilateral renal agenesis. The proband was hemizygous for c. 844delC(p.R282Vfs*28) of ANOS1 gene, his mother was heterozygous for the mutation, which was consistent with the X-linked recessive inheritance. The proband 3 was a 21 years old female, characterized by hypogonadotropic hypogonadism and anosmia. A heterozygous missense mutation c. 149G>A(p.R50Q) was detected in FGF17 gene. The mutation p. R50Q was predicted to be pathogenic by the SIFT and PolyPhen2 programs, and has not been reported in HGDM database yet, which considered to be a novel mutation.Conclusion:KS is a clinically and genetically heterogeneous disease. In this study, ANOS1 c. 844delC, SOX10 c. 475C>T and FGF17 c. 149G>A mutations were found in 3 patients with KS by whole exome sequencing, which would expand the genotypic and phenotype spectrum of KS.

Identification of two novel mutations in three Chinese families with Kallmann syndrome using whole exome sequencing.

Kallmann syndrome (KS) is a rare developmental disorder that manifests as congenital hypogonadotropic hypogonadism with anosmia. More than 19 genes have been found to be associated with KS. However, approximately 70% of the causes of KS remain unclear. Here, we studied seven KS patients, from three families, who had delayed puberty and olfactory bulb dysplasia. However, the families of these patients showed a range of other unique clinical features, including hearing loss, anosmia (to varying degrees) and unilateral renal agenesis. We performed whole exome sequencing and copy number variation (CNV) sequencing on samples acquired from these patients. We identified two novel mutations (c.844delC in ANOS1, c.475C>T in SOX10) and a novel trigenic pattern, PROKR2/CHD7/FEZF1 (c.337T>C in PROKR2, c.748C>G in FEZF1, c.8773G>A in CHD7). The c.844delC mutation in the ANOS1 gene was predicted to generate a truncated form of the anosmin-1 protein. SIFT and PolyPhen-2 predicted that the c.475C>T mutation in SOX10 had a damaging effect. The PROKR2 mutation (c.337T>C) was previously reported as harmful. No pathogenic copy number alterations were detected. Our study expands the genotypic and phenotypic spectrum of KS, a disease that shows considerable clinical and genetic heterogeneity. The application of whole exome sequencing could facilitate our understanding of the pathogenesis of KS.

Expanding the mutational spectrum of monogenic hypogonadotropic hypogonadism: novel mutations in ANOS1 and FGFR1 genes.

BACKGROUND: Congenital hypogonadotropic hypogonadism (CHH) is a rare disease, triggered by defective GnRH secretion, that is usually diagnosed in late adolescence or early adulthood due to the lack of spontaneous pubertal development. To date more than 30 genes have been associated with CHH pathogenesis with X-linked recessive, autosomal dominant, autosomal recessive and oligogenic modes of inheritance. Defective sense of smell is present in about 50-60% of CHH patients and called Kallmann syndrome (KS), in contrast to patients with normal sense of smell referred to as normosmic CHH. ANOS1 and FGFR1 genes are all well established in the pathogenesis of CHH and have been extensively studied in many reported cohorts. Due to rarity and heterogenicity of the condition the mutational spectrum, even in classical CHH genes, have yet to be fully characterized. METHODS: To address this issue we screened for ANOS1 and FGFR1 variants in a cohort of 47 unrelated CHH subjects using targeted panel sequencing. All potentially pathogenic variants have been validated with Sanger sequencing. RESULTS: Sequencing revealed two ANOS1 and four FGFR1 mutations in six subjects, of which five are novel and one had been previously reported in CHH. Novel variants include a single base pair deletion c.313delT in exon 3 of ANOS1, three missense variants of FGFR1 predicted to result in the single amino acid substitutions c.331C > T (p.R111C), c.1964 T > C (p.L655P) and c.2167G > A (p.E723K) and a 15 bp deletion c.374_388delTGCCCGCAGACTCCG in exon 4 of FGFR1. Based on ACMG-AMP criteria reported variants were assigned to class 5, pathogenic or class 4, likely pathogenic. Protein structural predictions, the rarity of novel variants and amino acid conservation in case of missense substitutions all provide strong evidence that these mutations are highly likely to be deleterious. CONCLUSIONS: Despite the fact that ANOS1 and FGFR1 are classical CHH genes and were thoroughly explored in several CHH cohorts we identified new, yet undescribed variants within their sequence. Our results support the genetic complexity of the disorder. The knowledge of the full genetic spectrum of CHH is increasingly important in order to be able to deliver the best personalised medical care to our patients.

A novel splice site variant in ANOS1 gene leads to Kallmann syndrome in three siblings.

Idiopathic hypogonadotropic hypogonadism (IHH) is a rare genetic disease caused by low doses of hypothalamic gonadotropin-releasing hormone (GnRH), leading to absence or delayed sexual development. Kallmann syndrome (KS) is characterized by IHH with anosmia or hyposmia. Here, we identified a novel splice site variant (c. 726+2T>G) of ANOS1 gene in three siblings with KS from a Chinese Han family by whole-exome sequencing (WES). In this family, KS is classified as an X-linked recessive inheritance pattern. This mutation was inherited from the mother by Sanger sequencing. An in vitro functional experiment has identified the deleterious effect of this mutation on the transcriptional level of ANOS1 gene. Importantly, the effectiveness of timely hormone replacement therapy was evaluated on the three siblings. Hence, finding genetic causes could be helpful in the early diagnosis and timely treatment of KS.

A rare ANOS1 variant in siblings with Kallmann syndrome identified by whole exome sequencing.

Kallmann syndrome is a rare genetic condition causing congenital hypogonadotropic hypogonadism. It presents with delayed puberty, anosmia, and infertility. Here, we set out to identify a causative DNA variant for Kallmann syndrome in two affected brothers of Hispanic ancestry. The male siblings presented with a clinical diagnosis of Kallmann syndrome (anosmia, delayed puberty, azoospermia, and undetectable luteinizing hormone and follicle stimulating hormone levels). Genetic variations were investigated by whole exome sequencing. Potentially pathogenic variants were filtered and prioritized followed by validation by Sanger sequencing in the two brothers and their mother. A pathogenic variant was identified in the ANOS1 gene on the X chromosome: c.1267C>T; both brothers were hemizygous, and their mother was heterozygous for the variant. The variant is a single nucleotide change that introduces a stop codon in exon 9 (p.R423*), likely producing a truncated variant of the protein. This variant has only been reported twice in the literature, in the setting of finding genetic causes for other conditions. This result supports the clinical value of whole exome sequencing for identification of genetic pathogenic variants. Genetic diagnosis is the essential first step for genetic counseling, preimplantation diagnosis, and research for a potential treatment.

Molecular genetic and clinical delineation of 22 patients with congenital hypogonadotropic hypogonadism.

BACKGROUND: Congenital hypogonadotropic hypogonadism (CHH) is classified as Kallmann syndrome (KS) with anosmia/hyposmia or normosmic (n)CHH. Here, we investigated the genetic causes and phenotype-genotype correlations in Japanese patients with CHH. METHODS: We enrolled 22 Japanese patients with CHH from 21 families (18 patients with KS and 4 with nCHH) and analyzed 27 genes implicated in CHH by next-generation and Sanger sequencing. RESULTS: We detected 12 potentially pathogenic mutations in 11 families, with three having a mutation in ANOS1 (X-linked recessive); three and four having a mutation in FGFR1 and CHD7, respectively (autosomal dominant); and one having two TACR3 mutations (autosomal recessive). Among four patients with KS carrying a CHD7 mutation, one had perceptive deafness and two had a cleft lip/palate. CONCLUSIONS: The frequency of CHH genes in the Japanese was compatible with previous reports, except that CHD7 mutations might be more common. Furthermore, partial phenotype-genotype correlations were demonstrated in our cohort.

Expanding the genetic spectrum of ANOS1 mutations in patients with congenital hypogonadotropic hypogonadism.

STUDY QUESTION: What is the prevalence and functional consequence of ANOS1 (KAL1) mutations in a group of men with congenital hypogonadotropic hypogonadism (CHH)? SUMMARY ANSWER: Three of forty-two (7.1%) patients presented ANOS1 mutations, including a novel splice site mutation leading to exon skipping and a novel contiguous gene deletion associated with ichthyosis. WHAT IS KNOWN ALREADY: CHH is characterized by lack of pubertal development and infertility, due to deficient production, secretion or action of GnRH, and can be associated with anosmia/hyposmia (Kallmann syndrome, KS) or with a normal sense of smell (normosmic CHH). Mutations in the anosmin-1 (ANOS1) gene are responsible for the X-linked recessive form of KS. STUDY DESIGN, SIZE, DURATION: This cross-sectional study included 42 unrelated men with CHH (20 with KS and 22 with normosmic CHH). PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were screened for mutations in the ANOS1 gene by DNA sequencing. Identified mutations were further investigated by RT-PCR analysis and multiplex ligation-dependent probe amplification (MLPA) analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Hemizygous mutations were identified in three (7.1%) KS cases: a novel splice acceptor site mutation (c.542-1G>C), leading to skipping of exon 5 in the ANOS1 transcript in a patient with self-reported normosmia (but hyposmic upon testing); a recurrent nonsense mutation (c.571C>T, p.Arg191*); and a novel 4.8 Mb deletion involving ANOS1 and eight other genes (VCX3B, VCX2, PNPLA4, VCX, STS, HDHD1, VCX3A and NLGN4X) in KS associated with ichthyosis. LIMITATIONS, REASONS FOR CAUTION: Objective olfactory testing was not performed in all cases of self-reported normosmia and this may have underestimated the olfactory deficits. WIDER IMPLICATIONS OF THE FINDINGS: This study further expands the spectrum of known genetic defects associated with CHH and suggests that patients with self-reported normal olfactory function should not be excluded from ANOS1 genetic testing. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Portuguese Foundation for Science and Technology. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

ANOS1: a unified nomenclature for Kallmann syndrome 1 gene (KAL1) and anosmin-1.

It is accepted that confusion regarding the description of genetic variants occurs when researchers do not use standard nomenclature. The Human Genome Organization Gene Nomenclature Committee contacted a panel of consultants, all working on the KAL1 gene, to propose an update of the nomenclature of the gene, as there was a convention in the literature of using the 'KAL1' symbol, when referring to the gene, but using the name 'anosmin-1' when referring to the protein. The new name, ANOS1, reflects protein name and is more transferrable across species.