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

Journal Pre-proofs Short communication A novel splice site variant in ANOS1 gene leads to Kallmann syndrome in three siblings Xiaohui Jiang, Dingming Li, Yanzi Gao, Xueguang Zhang, Xiang Wang, Yihong Yang, Ying Shen PII: DOI: Reference:

S0378-1119(19)30836-4 https://doi.org/10.1016/j.gene.2019.144177 GENE 144177

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28 August 2019 16 October 2019 16 October 2019

Please cite this article as: X. Jiang, D. Li, Y. Gao, X. Zhang, X. Wang, Y. Yang, Y. Shen, A novel splice site variant in ANOS1 gene leads to Kallmann syndrome in three siblings, Gene Gene (2019), doi: https://doi.org/10.1016/j.gene. 2019.144177

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A novel splice site variant in ANOS1 gene leads to Kallmann

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syndrome in three siblings

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Xiaohui Jianga,b,1, Dingming Lia,b,1, Yanzi Gaoc, Xueguang Zhangd, Xiang Wangd,

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Yihong Yange, Ying Shend,*

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aHuman

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Chengdu 610041, China

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bKey

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(Sichuan University), Ministry of Education, Chengdu 610041, China

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cWest

Sperm Bank, West China Second University Hospital, Sichuan University,

Laboratory of Birth Defects and Related Disease of Women and Children

China School of stomatology, Sichuan University, Chengdu, 610041, China

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dDepartment

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(SCU-CUHK), Key Laboratory of Obstetric, Gynecologic and Pediatric Diseases

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and Birth Defects of Ministry of Education, West China Second University Hospital,

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Sichuan University, Chengdu 610041, China

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eCenter

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University, Chengdu 610041, China

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*Corresponding

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*Correspondence address: Tel.: +86-15982083665; E-mail: [email protected]

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(Y. Shen)

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1The authors consider that the first two authors should be regarded as joint First Authors.

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of Obstetrics/Gynecology, Joint Laboratory of Reproductive Medicine

of Reproductive Medicine, West China Second University Hospital, Sichuan

author.

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Highlight

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A novel mutation in ANOS1 gene was identified in a Kallmann syndrome family.

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This splice site variant results in a truncated protein due to a frameshift.

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Timely hormone replacement therapy of KS depends largely on precise genetic

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diagnosis.

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Abstract

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Idiopathic hypogonadotropic hypogonadism (IHH) is a rare genetic disease caused

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by low doses of hypothalamic gonadotropin-releasing hormone (GnRH), leading to

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absence or delayed sexual development. Kallmann syndrome (KS) is characterized by

34

IHH with anosmia or hyposmia. Here, we identified a novel splice site variant (c.

35

726+2T>G) of ANOS1 gene in three siblings with KS from a Chinese Han family by

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whole-exome sequencing (WES). In this family, KS is classified as an X-linked

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recessive inheritance pattern. This mutation was inherited from the mother by Sanger

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sequencing. An in vitro functional experiment has identified the deleterious effect of

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this mutation on the transcriptional level of ANOS1 gene. Importantly, the effectiveness

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of timely hormone replacement therapy was evaluated on the three siblings. Hence,

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finding genetic causes could be helpful in the early diagnosis and timely treatment of

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KS.

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Abbreviations:

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46 47 48 49 50 51

IHH, Idiopathic hypogonadotropic hypogonadism; HPG, hypothalamo-pituitarygonadal; KS, Kallmann syndrome; WES, whole-exome sequencing; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; nIHH, normosmic IHH; CDGP, constitutional delay of growth and puberty; WAP, whey acidic protein; FnIII, fibronectin type III; HCG, human chorionic gonadotrophin; HMG, human menopausal gonadotrophin

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Key words: idiopathic hypogonadotropic hypogonadism, Kallmann syndrome, ANOS1

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gene, whole exome sequencing, mutation

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1. Introduction

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Idiopathic hypogonadotropic hypogonadism (IHH) is a heterogeneous disorder

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characterized by delayed or absent puberty and infertility and is often accompanied by

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other developmental abnormities, such as renal agenesis or hypoplasia, cleft lip/palate,

64

dental agenesis, ear anomalies, hearing impairment and bimanual synkinesis or skeletal

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anomalies (Quinton, et al., 2001). The causative factors of IHH are complicated.

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Recently, it was reported that known genetic defects account for 50% of all IHH

67

patients (Crowley, et al., 2008). To date, approximately 50 genes are associated with

68

IHH (Topaloglu, 2017). IHH can be divided into IHH with anosmia/hyposmia

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(Kallmann syndrome [KS]) and normosmic IHH (nIHH). In addition, KS accounts for

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half of all IHH cases (Boehm, et al., 2015). The reason that KS patients suffer from

4

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anosmia/hyposmia is the abnormal migration of GnRH-specific neurons from their

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origin in the olfactory placode to the forebrain (Schwanzel-Fukuda, et al., 1989;

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Teixeira, et al., 2010). Therefore, loss-of-function mutations in genes related to GnRH

74

neuron migration will lead to KS. The major genes and their relative genetic models

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involved in KS include ANOS1 gene (KAL1) in the X-linked form; FGFR1, FGF8,

76

WDR11, SOX10 and CHD7 in the autosomal-dominant form; and PROK2 and PROKR2

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in the autosomal-recessive form (Boehm, et al., 2015). Among these genes, ANOS1

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gene, is the second most commonly mutated gene, accounting for approximately 10–

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20% of familial and sporadic KS patients. Currently, approximately 70 mutations in

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ANOS1 gene have been identified, all of which are spread throughout the entire gene,

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and no mutation “hot spots” have been found in the affected regions (Nie, et al., 2017).

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The diagnosis of IHH is very important because hormone therapy is effective in

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many IHH patients. Consequently, with the exception of typical manifestations, we

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should also pay attention to genetic factors to facilitate IHH diagnosis because it is often

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difficult to distinguish IHH from other similar diseases, such as the constitutional delay

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of growth and puberty (CDGP) (Boehm, et al., 2015).

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In this study, we report a novel splice site variant in ANOS1 gene leading to KS in

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three siblings of a family. The results of functional experiments in vitro show that this

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mutation results in ANOS1 protein truncation and further confirms the pathogenicity of

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this variant. Thus, genetic testing is a good method to diagnose IHH, and effective

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treatment can be provided in a timely manner.

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2. Materials and methods

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2.1 Study Participants

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We describe three subjects in this study. The three patients are from one family and

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diagnosed with infertility at Human Sperm Bank of West China Second University

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Hospital. This study was approved by the Ethical Review Board of West China Second

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University Hospital, Sichuan University (Project number: 2019019). Informed consent

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was obtained from each subject in our study.

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2.2 Whole-exome sequencing (WES) and Sanger sequencing

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Blood samples were obtained from this family, and genomic DNA was extracted

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using a whole blood DNA purification kit (QIAGEN, Germany). Whole-exome

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sequencing (WES) was performed on the three patients. The candidate causative

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mutation found by WES was confirmed in this family through Sanger sequencing. In

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addition, mutation detection in 200 normal controls was performed by Sanger

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sequencing analysis. The primers are: F 5'

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R 5'

CTATTTTGAGGCATAGCAAGT

TCAGATGTGAAACCCTGTATG

3';

3'.

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2.3 Minigene assay

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A functional splicing reporter minigene assay was used to assess the impact of

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sequence variants on splicing. A genomic segment encompassing exon 5, intron 5 and

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exon 6 of ANOS1 gene was PCR-amplified from patient genomic DNA as well as the

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normal control and was cloned into the minigene vector pSPL3. After transient

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transfection into cultured cells, the splicing patterns of the transcripts generated from

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the wild-type and variant constructs were compared by RT-PCR analysis and

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sequencing.

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ACGGGATCACCAGAATTCTGGAGCTCTTTGCTTTGCAGTGTTTTCC 3'; R 5'

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GGCCCAAACATTATGTACCTCTGTATCATATGGCAAAGCCACCTGTTGACT

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A 3'. The primers for RT-PCR are: SD6(F) 5' TCTGAGTCACCTGGACAACC 3';

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SA(R) 5' ATCTCAGTGGTATTTGTCAGC 3'.

The

primers

for

genomic

amplification

are:

F

5'

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3. Results

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3.1 Case description

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We report a non-consanguineous Chinese Han family that has six children, three of

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whom are infertile (III-3, III-6, III-7) (Fig. 1). To identify the cause of the sterility of

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the three patients, we carried out several related clinical examinations on them. The

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karyotypes of the three siblings were 46, XY. However, the sex hormone examination

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indicated that testosterone, FSH, and LH were all decreased. In addition, they showed

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low peak LH and FSH responses after GnRH stimulation (Brito, et al., 1999). Moreover,

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the three patients presented with smooth skin and a thin voice without facial hair or an

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Adam's apple (Fig. 2). According to Tanner stages, all the testicles of the three siblings

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were PH1G2, presenting as a small penis; bilateral testicles of III-3 and III-7 were 2 ml;

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the left side of III-6 was 2 ml, and the right side had cryptorchidism (Fig. 2). Thus, we

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initially diagnosed the three siblings with IHH depending on their low sex hormone

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levels and poor development of secondary sexual characteristics. We further performed

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a T&T olfactometer test on them, and the results of the test indicated that the three

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siblings had varying degrees of olfactory abnormalities. The brain MRI of III-6 showed

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a pituitary that was small and thin, and the other two brothers were normal (Fig. 2). The

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details of the clinical features of the three siblings are shown in Table 1. There was no

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phenotypic abnormality discovered in the other siblings or their parents.

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3.2 Molecular genetic analysis

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Approximately 50% of all IHH cases are caused by genetic defects (Crowley, et al.,

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2008). To elucidate the underlying genetic cause of the three siblings, we performed

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WES on them. Additionally, the pedigree analysis revealed an X-linked recessive

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inheritance in this family (Fig. 1). Thus, we focused on homozygous mutations in these

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patients. Briefly, variants were considered if (1) the minor allele frequency was <1% in

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any database, including the gnomAD, ExAC Browser and 1000 Genomes Project; (2)

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the variant affected coding regions or canonical splice sites; and (3) the variant was

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predicted as damaging by PolyPhen-2, SIFT and MutationTaster tools. Consequently,

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a novel deleterious hemizygous splice site variant (c. 726+2T>G) was identified in the

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ANOS1 gene on the X chromosome, which is a definitely causative gene associated

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with KS.

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To confirm the putative contribution of this mutation, we investigated this variant

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site by Sanger sequencing in the whole family (Fig. 3). The unaffected mother (II-2)

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and one sister (III-2) carried a heterozygous mutation. The unaffected father (II-1) and

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the other two sisters (III-5, III-8) did not have this mutation. Furthermore, we did not

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detect this mutation in a sample of 200 Chinese Han controls, supporting the

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pathogenicity of this variant. Thus, we conclude that this is a pathogenic variant

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according to the American College of Medical Genetics guidelines leading to KS in this

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family.

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3.3 Mutation effect analysis

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By bioinformatics analysis, we predicted that this splice site variant (c. 726+2T>G)

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was likely to damage splicing and lead to abnormal protein expression. Unfortunately,

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the ANOS1 gene is hardly expressed in blood. Therefore, we could not check the

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damage using the patients’ blood samples. To this end, we carried out a functional

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splicing reporter minigene assay to uncover the harmful effect of this mutation on

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ANOS1 gene splicing. As shown in Fig. 4A, the RT-PCR product of the variant was

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longer than that of the wild-type. Sequence analysis revealed that the variant caused 38

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nucleotides of intron 5 to be retained between exon 5 and exon 6, which was expected

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to result in a truncated protein due to a frameshift and premature termination codon (p.

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Thr243Glyfs*8) (Fig. 4B). Thus, the splice site mutation in intron 5 was the genetic

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cause of KS in this family.

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4. Discussion

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IHH is a rare genetic disorder with a prevalence of 1:30,000 in males and 1:125,000

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in females, which mainly impairs sexual development in puberty and subsequently

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results in infertility in adults (Laitinen, et al., 2011). Therefore, it is necessary to provide

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an effective treatment for IHH patients during puberty or preadolescence, which

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depends on the accurate and efficient diagnosis of IHH. Hitherto, the diagnosis of IHH

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has been based on clinical, biochemical, and imaging examinations. However, it is

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particularly challenging to differentiate IHH and CDGP in early adolescence. Therefore,

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genetic testing is a good way to diagnose IHH. By next-generation sequencing, nearly

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50 genes have been identified to be related to IHH (Boehm, et al., 2015). Here, by WES,

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we detected a novel intronic ANOS1 gene mutation in three siblings with KS, which

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changes the splice site and forms a truncated protein.

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KS is a form of IHH with anosmia or hyposmia. Mutations in genes regulating

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GnRH neuron development, migration, and function are the key causative factors

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(Topaloglu and Kotan, 2016). Additionally, ANOS1 gene is the first described

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pathogenic gene associated with KS, and mutations are low in sporadic KS patients but

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much higher in familial KS patients (Hamada, et al., 2013). ANOS1 gene is located on

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the X chromosome and encodes an extracellular glycoprotein called Anosmin-1, which

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contains four domains: an N-terminal cysteine-rich (Cys-box) domain, a whey acidic

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protein (WAP) domain, four fibronectin type III (FnIII) domains, and a histidine-rich

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C terminal region (del Castillo, et al., 1992). Anosmin-1 plays several roles in the

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development of the central nervous system: promoting neuronal cell adhesion, neurite

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outgrowth, axonal guidance, CNS projection neuron branching, and the migration of

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multiple types of neuronal precursors, including GnRH-producing neurons and

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oligodendrocyte precursors (Nie, et al., 2017). Mutations in ANOS1 gene disrupt the

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protein structure and impair its functions, thus leading to KS (Hu, et al., 2003). In our

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study, the splice site variant (c. 726+2T>G) causes a truncated ANOS1 protein and

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destroys the FnIII domain (Fig. 5), which is involved in cell adhesion, tyrosine kinases

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and phosphatases, which are implicated in neuronal migration and in axon guidance

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(Legouis, et al., 1993). Therefore, dysfunctional Anosmin-1 is unable to mediate the

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normal function of GnRH neurons and causes a defect in GnRH production, secretion,

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or action, consequently resulting in KS.

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On the Y chromosome, there is a pseudogene of ANOS1 gene named ANOS2P.

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Although ANOS2P could not produce a functional protein, the sequences of ANOS1

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gene and ANOS2P have a high degree of similarity, ranging from 86.2%-98.3% for

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exons and from 86.3%-99.1% for introns (del Castillo, et al., 1992). Accordingly, when

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screening the ANOS1 gene variants in male IHH patients through PCR sequencing, the

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pseudogene ANOS2P should not be amplified. Herein, we designed primers located in

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the region where the two genes are different to validate the splice site variant in this

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family by PCR sequencing. The results of PCR sequencing are consistent with WES

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results.

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In approximately 10% of KS patients, hormone levels, testis or penile volumes

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recover or grow spontaneously (Topaloglu and Kotan, 2016). Most cases of IHH are

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caused by a spectrum of abnormal GnRH secretory patterns, including deficient

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synthesis or secretion of endogenous GnRH (Spratt, et al., 1987), and so it is very

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responsive to hormonal therapy. Consequently, for the remaining patients, hormone

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replacement therapy is extremely essential. Approximately 92% of IHH patients can

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reach normal adult testicular volume and have sperm production after treatment with a

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GnRH pituitary pump (Pitteloud, et al., 2002). In the present study, the GnRH

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stimulation test showed that the pituitary glands of the three siblings responded to

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exogenous GnRH, which provided a laboratory basis for subsequent treatment and

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prognosis. The three siblings were treated with 2000 U of human chorionic

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gonadotrophin (HCG) and 75 U of human menopausal gonadotrophin (HMG) twice

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weekly. Eight months later, the Tanner stage of the three patients changed from PH2G2

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to PH2G3 or PH3G3. Specifically, the testicular size increased, and nocturnal penile

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tumescence became frequent, and spermatorrhea was occasional. Taken together, our

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results show that it is necessary to diagnose IHH and treat it in a timely manner, and

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most IHH patients will have a good prognosis.

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5. Conclusion

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In conclusion, in this study, we identified a novel pathogenic mutation of ANOS1

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gene in three siblings from a Chinese Han family, thus expanding the known spectrum

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of ANOS1 gene mutations. The in vitro functional experiment elucidated the

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pathogenesis of this mutation in KS. In this family, hormone replacement therapy

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effectively induced sexual maturation. Therefore, genetic analysis plays a crucial role

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in IHH diagnosis and prognosis and further provides beneficial knowledge related to

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genetic counselling.

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Acknowledgements

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We thank all the patients who provided samples to this research. This work was

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supported by General Program of China Post-doctoral Science Foundation

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(2018M640920), Postdoctoral fund of Sichuan University (20826041B4090) and

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Sichuan Science & Technology Program (2018SZ0144).

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Authors’ roles

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X.J. and D.L. collected blood samples and conducted the clinical evaluations. X.W.,

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X.Z. and Y.Y. performed PCR, minigene assay and sanger sequencing analysis. Y.S.

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designed this study and wrote manuscript.

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Declaration of interest

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The authors declare that they have no conflicts of interest with the contents of this

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article.

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Reference

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Giacobini P, Hardelin JP, Juul A et al. Expert consensus document: European

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Consensus Statement on congenital hypogonadotropic hypogonadism--pathogenesis,

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del Castillo I, Cohen-Salmon M, Blanchard S, Lutfalla G, Petit C. Structure of the X-

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linked Kallmann syndrome gene and its homologous pseudogene on the Y

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chromosome. Nat Genet 1992;2: 305-310.

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Hamada AJ, Esteves SC, Agarwal A. A comprehensive review of genetics and genetic testing in azoospermia. Clinics (Sao Paulo) 2013;68 Suppl 1: 39-60. Hu Y, Tanriverdi F, MacColl GS, Bouloux PM. Kallmann's syndrome: molecular pathogenesis. Int J Biochem Cell Biol 2003;35: 1157-1162.

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Laitinen EM, Vaaralahti K, Tommiska J, Eklund E, Tervaniemi M, Valanne L, Raivio

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T. Incidence, phenotypic features and molecular genetics of Kallmann syndrome in

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Finland. Orphanet J Rare Dis 2011;6: 41.

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Legouis R, Lievre CA, Leibovici M, Lapointe F, Petit C. Expression of the KAL gene

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in multiple neuronal sites during chicken development. Proc Natl Acad Sci U S A

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Nie M, Xu H, Chen R, Mao J, Wang X, Xiong S, Zheng J, Yu B, Cui M, Ma W et al.

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Analysis of genetic and clinical characteristics of a Chinese Kallmann syndrome

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cohort with ANOS1 mutations. Eur J Endocrinol 2017;177: 389-398.

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Pitteloud N, Hayes FJ, Dwyer A, Boepple PA, Lee H, Crowley WF, Jr. Predictors of

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Quinton R, Duke VM, Robertson A, Kirk JM, Matfin G, de Zoysa PA, Azcona C,

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MacColl GS, Jacobs HS, Conway GS et al. Idiopathic gonadotrophin deficiency:

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Schwanzel-Fukuda M, Bick D, Pfaff DW. Luteinizing hormone-releasing hormone

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(LHRH)-expressing cells do not migrate normally in an inherited hypogonadal

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(Kallmann) syndrome. Brain Res Mol Brain Res 1989;6: 311-326.

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conditions. J Clin Invest 2010;120: 3668-3672.

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Topaloglu AK. Update on the Genetics of Idiopathic Hypogonadotropic Hypogonadism. J Clin Res Pediatr Endocrinol 2017;9: 113-122. Topaloglu AK, Kotan LD. Genetics of Hypogonadotropic Hypogonadism. Endocr Dev

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2016;29: 36-49.

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Figure 1. Family pedigree. Dark box: Infertile man (III-3, III-6 and III-7).

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Figure 2. Phenotypes of three siblings with KS. All of the three patients with smooth

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skin and barrenly facial hair or without an Adam's apple; the brain MRI showed III-6

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presenting a small and thin pituitary (red arrow) and the pituitaries of III-3 and III-7

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were normal (white arrows).

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Figure 3. Sanger sequencing results of this family. The black arrows point to the

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mutation site (c. 726+2T>G) in ANOS1 gene.

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Figure 4. Functional effect of the splice site mutation on ANOS1 gene transcript. (A)

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Agarose gel electrophoresis of RT-PCR fragments obtained from wild-type plasmid

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and mutant plasmid. A measurable increase in molecular weight was observed in

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mutant plasmid (4) compared with wild-type plasmid (3). A 100-bp ladder molecular-

341

weight marker (1) is shown. 2, blank control; 5, empty vector. (B) Sequence analysis

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of the RT-PCR product obtained from mutant plasmid demonstrated the gain of 38

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nucleotides from intron 5 between exon 5 and exon 6.

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Figure 5. The domains of ANOS1 protein and the position of truncated protein. ANOS1

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protein contains a WAP super family and three FN3 super family.

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Table 1. Clinical and hormonal characteristics of patients in the family

Subject

III-7

III-6

Age(years) Gender Height/weight (cm/Kg)

21 Male 176/64

26 Male 179/70.5

Tanner stage

PH1G2

PH1G2

Testicular size (ml)

bilateral 2ml

Left 2ml, right cryptordism

17

Penis length

micropenis

micropenis

T&T olfactometer test

1.60

2.40

Karyotype

46, XY

46, XY

E2 (pg/ml)

11.8

34.0

T (ng/ml)

0.42

0.30

FSH (IU/L)

0.6

0.3

LH (IU/L)

0.1

0.4

LH (IU/L; basal/stimulated for 15 min/30min/60min)

0.2/0.7/1.1/1.0

0.1/0.65/1.3/1.7

FSH (IU/L; basal/stimulated for 15 min/30min/60min)

0.4/1.0/1.9/2.7

0.15/0.9/1.6/1.9

Basal hormone concentrations

GnRH excitatory experiment

352 353 354 355

E2, Estradiol; T, testosterone; FSH, follicle-stimulating hormone; LH, luteinizing hormone; GnRH, gonadotropin-releasing hormone.