JRI 
Vol. 22, Issue 4, / October-December 2021
(Case Report, pages 295-301)

Pooja Chauhan
- Centre for Genetic Disorders, Institute of Science, Banaras Hindu University, Varanasi, India
Anjali Rani
- Department of Obstetrics and Gynaecology, Institute of Medical Science, Banaras Hindu University, Varanasi, India
Amit Kumar Rai Corresponding Author
- Centre for Genetic Disorders, Institute of Science, Banaras Hindu University, Varanasi, India

Received: 10/24/2020 Accepted: 2/14/2021 - Publisher : Avicenna Research Institute

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Abstract

Background: Inhibin and activin regulate the follicle stimulating hormone level by their antagonistic actions and thus have been considered as strong candidate genes in the etiology of ovarian dysgenesis. In the present study, two cases of primary amenorrhea with poorly developed secondary sexual characteristics were reported. The purpose of the study was to identify mutations in candidate gene.
Case Presentation: In this paper, clinical, genetic, biochemical, and molecular findings in female patients with primary amenorrhea were reported. Whole blood culture and G-banding for karyotyping, sequencing, and in silico analysis were performed following the standard protocol. Both cases were cytogenetically characterized as normal females with 46,XX, chromosome constitution. Hormonal assay revealed high level of follicle stimulating hormone and luteinizing hormone. DNA sequence analysis of inhibin identified two novel heterozygous missense mutations of c.975T>A and c.1156G>A which were translated into p.I310N and p.D386N, respectively. These identified positions were highly conserved across species during evolution. In silico prediction tools, intramolecular hydrogen bonding pattern and hydrophobicity analysis, revealed deleterious effect of p.I310N and neutral effect of p.D386N mutation.
Conclusion: Our observation suggested that identified novel mutation in the first case might be the reason for ovarian dysgenesis and provides additional support to the previously reported genotype-phenotype correlations.



Keywords: Amenorrhea, Follicle stimulating hormone, Gonadal dysgenesis, Inhibins, Luteinizing hormone, Mutation


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Full Text

Introduction
Inhibins are dimeric glycoproteins and members of transforming growth factor (TGF)-β family which regulate synthesis and secretion of follicle stimulating hormone (FSH) from pituitary. Inhibin suppresses FSH secretion and lowers concentration of serum inhibin which has been associated with follicular depletion (1) and increased FSH level (Ovarian reserve marker) (2-4). Inhibin exists in two forms of inhibin A and inhibin B, and is composed of heterodimer of inhibin α subunit and two related inhibin β subunits (Inhibin βA and inhibin βB, respectively) (Figure 1). Inhibin A suppresses the secretion of FSH from pituitary whereas inhibin B initiates the folliculogenesis (5). Inhibin A is predominantly produced during mid menstrual cycle while inhibin B is produced by granulosa cells during early follicular phase. Interestingly, homodimer of inhibin beta subunit can also form another hormone, activin which exerts opposite effect on FSH synthesis and secretion than inhibin and participates in menstrual cycle regulation, cell proliferation, and differentiation (5). Balance of inhibin and activin is required for regulation of reproductive cycle in females (6).
Several studies on infertility reported that over-expression of inhibin α subunit gene in rats results in decreased rate of folliculogenesis with approximately 50% reduction in oocyte maturation that ultimately leads to reduced fertility (7). The study by shelling et al. in 2000 reported all inhibin subunits encoding genes as candidate genes for premature ovarian failure (POF) (8). Furthermore, a missense mutation of p.A257T in INHA serves as a susceptibility factor for POF because it impairs the bioactivity of inhibin B (9). This missense mutation was studied in different ethnic groups by several studies (8, 10, 11).
In the present study, two novel mutations, p.I310N and p.D386N, in inhibin βB subunit were identified in the cases with primary amenorrhea that might lead to decreased bioactive inhibin level. Decreased inhibin level might result in elevation of FSH by disturbing the negative feedback to pituitary, leading to ovarian dysgenesis.

Case Presentation
Human subjects: Two cases suspected with primary amenorrhea were recruited for the study and they were referred from the Department of Obstetrics and Gynecology, Sir Sunderlal Hospital, Varanasi, India to our centre (Centre for genetic Disorders, Banaras Hindu University, Varanasi, India) in 2016-2017. Clinical history and photographs were collected from each case after receiving informed written consent. This study was approved by institutional ethics committee (No.: F.Sc./Ethics Committee/2015-16/7). Both cases had the following clinical history; the first case was a 17 year old girl with primary amenorrhea along with poorly developed secondary sexual characteristics, breast Tanner stage I, absent pubic and axillary hair. She was diagnosed with absence of uterus during ultrasound scan. The second case was a 21 year old female who had primary amenorrhea along with poorly developed secondary sexual characteristics. Ultrasound showed absence of uterus and ovaries.
Cytogenetic analysis: Whole blood cultures were set in RPMI-1640 medium at pH=7.2 (Sigma- Aldrich, USA) with 10% fetal bovine serum (Himedia, India) and induced by phytohaemagglutinin-M (Sigma-Aldrich, USA). Next, 30-50 metaphases were analyzed by Saline Trypsin Giemsa (STG) method using Ikaros karyotyping system—Metasystems software (Carl Zeiss Microscopy GmbH, Germany). Karyotyping was performed according to ISCN nomenclature.
Hormonal assay: Serum was isolated from each sample which was stored at -80°C until use for hormonal assay. Follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured by chemiluminescence method (Immulite-1000 Analyser, Siemens Healthcare Diagnostics Products, USA) as per the manufacturer’s instructions.
Polymerase chain reaction (PCR) and sequencing: All exons and exon-intron boundaries of all subunits of inhibin gene (INHA, INHBA, and INHBB) were amplified and directly sequenced. Primers were designed by primer blast software (https:// www.ncbi.nlm.nih.gov/tools/primer-blast/) (Table 1). PCR products were sequenced in an automated 3130 Genetic Analyzer (Applied Biosystems, USA) using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) according to the manufacturer’s protocol. The identified mutations were screened in 150 control individuals from the same population by Sanger sequencing.
In silico analysis: Obtained nucleotide substitutions were further analyzed using MutationTaster online tool (www.mutationtaster.org) and other prediction tools like predictSNP, SIFT, Panther, SNAP, Polyphen-1, Polyphen-2, PhD-SNP, and MAPP. Hydrophobicity profile and helix-forming properties of wild-type and mutant INHBB protein were also examined by ProtScale program available at the ExPASy web server.
Homology modeling of inhibin βB subunit: The inhibin βB subunit was homology modeled by using coordinates from Protein Data Bank. Three protein structures named 3rjr_A, 5hly_A, and 5hlz_A were utilized for modeling using Modeller 9.15 and selection of the best model was done by using four different web servers and tools, i.e. RAMPAGE (http://mordred.bioc.cam.ac.uk/~rapper/ rampage.php) for Ramachandran plot analysis, ERRAT program (http://services.mbi.ucla.edu/ ERRAT/) for verifying protein structures determined by crystallography, PDBsum (http://www. ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html) for structural analyses, and Modeller 9.15 to find out the best model among all. The figures were produced using standalone tool, DeepView–Swiss-PdbViewer (http://spdbv.vital-it.ch/).

Results
Cytogenetic analysis revealed 46,XX karyotype signifying the normal chromosome constitution in both cases (Figures 2A, 3A). Hormonal assay from serum showed FSH and LH levels of 90.3 and 18.5 mIU/ml, respectively in case 1. In case 2, the measured level of FSH and LH was 55.95 and 16.09 mIU/ml, respectively. Higher level of both FSH and LH, biomarker of gonadal dysgenesis, was observed in both cases. Resequencing of INHA, INHBA and INHBB genes identified two novel non-synonymous substitutions of c.975T>A in case 1 and c.1156G>A in case 2. These variations were translated into p.I310N and p.D386N, respectively (Figures 2B, 3B). Both isoleucine at position 310 and aspartic acid at 386 position were also highly conserved across species during evolution except a few of them (Figures 2C, 3C). P.I310N variation was predicted to be pathogenic by several in silico prediction tools (MutationTaster, SIFT, SNAP, Polyphen-1, Polyphen-2, PhD-SNP, MAPP and predictSNP) (Figure 2D) while p.D386N mutation was predicted as neutral (Figure 3D).
The study of intramolecular hydrogen bonding pattern of p.I310N mutation revealed the formation of three new hydrogen bonds in mutant protein with TYR327, ARG313, and LEU314 (Figures 4A, 4B). The analysis of alteration in hydrophobicity of the protein due to this mutation revealed minor changes in protein microenvironment (Figures 5A, 5B). On the other hand, intramolecular hydrogen bonding pattern and hydrophobicity analysis of p.D386N mutation showed the neutral effect (Figures 4C, 4D, 5C, 5D).

Discussion
The present study reports two mutations (p.I310N and p.D386N) in inhibin βB subunit, identified in females with primary amenorrhea. The first identified mutation results in alteration in intramolecular hydrogen bonding pattern as well as protein microenvironment due to hydrophobicity change while the second mutation does not show any pathogenic effect. The alteration in protein microenvironment might interfere with normal function of inhibin B protein that leads to disturbance in inhibin and activin balance. Activin and inhibin balance plays an important role in regulation of ovarian cycle through FSH level. As inhibin βB subunit involves in multiple complexes with antagonistic functions, the consequences of mutations related to loss-of-function or gain-of-function of protein might be difficult to predict. However, there is no report till date that mutation in inhibin βB subunit is involved in ovarian dysgenesis in females. An independent study reported that p.M370T substitution is involved in spermatogenic impairment in males (12). Furthermore, very low level of inhibin B in prepubertal girls is a biomarker for premature ovarian failure (13, 14). INHA has been more studied in ovarian failure and several mutations have been reported by different studies (10, 11, 15-17).
A nucleotide substitution (INHA 769G>A) in inhibin α subunit is well studied in different populations with reference to premature ovarian failure (8, 11, 15-17). Dixit et al. also reported three different mutations (p.S92N, p.H175Q, p.A182D) in INHA gene in patients with premature ovarian failure (10). A p.H175Q mutation in INHA was observed by Dixit et al. in a patient who experienced menopause at the age of 30 years with high level of FSH and LH (78 IU/l and 47 IU/l, respectively). Interestingly, another patient of this study with p.A182D mutation in INHA never menstruated and had underdeveloped secondary sexual characteristics.

Conclusion
Our study has demonstrated for the first time that mutation in INHBB subunit might lead to ovarian dysgenesis. Mutations in inhibin gene might affect the FSH level either through inhibition by inhibin or stimulation by activin through disturbing the ratio between inhibin and activin. Inhibin is suggested as a marker for ovarian reserve in premature ovarian failure patients and early screening of inhibin gene is necessary for identification of ovarian reserve to manage ovarian failure.

Acknowledgement
We thank the guardians for giving consent to publish the data. We are also thankful to Centre for Genetic Disorders, BHU, Varanasi, India for chromosomal analysis, Taq inhouse as a gift by CGD and Genetic analyser ABI3130, innovative programme of UGC for hormonal assay experiment by Immulite 1000.

Conflict of Interest
The authors have no conflict of interest.




Figures, Charts, Tables

High Resolution
Figure 1. INHA, INHBA, and INHBB produce precursor form of proteins containing N-terminal signal peptide (Black), a propeptide (Gray), and a subunit chain (Red, orange, and blue). These subunits dimerize by disulfide bond to form functional protein

Figure 1. INHA, INHBA, and INHBB produce precursor form of proteins containing N-terminal signal peptide (Black), a propeptide (Gray), and a subunit chain (Red, orange, and blue). These subunits dimerize by disulfide bond to form functional protein



High Resolution
Figure 2. A) Representative karyotype (46,XX) of case 1, B) Electropherogram of the c.975T>A (p.I310N), C) The multiple sequence alignment of the p.I310N mutant protein reveals the conservation of isoleucine at this position throughout evolution, D) In silico prediction of both variants using prediction tools of SIFT, SNAP, Polyphen-1, Polyphen-2, PhD-SNP, MAPP, and PredictSNP

Figure 2. A) Representative karyotype (46,XX) of case 1, B) Electropherogram of the c.975T>A (p.I310N), C) The multiple sequence alignment of the p.I310N mutant protein reveals the conservation of isoleucine at this position throughout evolution, D) In silico prediction of both variants using prediction tools of SIFT, SNAP, Polyphen-1, Polyphen-2, PhD-SNP, MAPP, and PredictSNP



High Resolution
Figure 3. A) Representative karyotype (46,XX) of the second case, B) Electropherogram of the c.1156G>A (p.D386N), C) The multiple sequence alignment of the p.D386N mutant protein reveals the conservation of isoleucine at this position throughout evolution, D) In silico prediction of both variants using prediction tools of SIFT, SNAP, Polyphen-1, Polyphen-2, PhD-SNP, MAPP, and PredictSNP

Figure 3. A) Representative karyotype (46,XX) of the second case, B) Electropherogram of the c.1156G>A (p.D386N), C) The multiple sequence alignment of the p.D386N mutant protein reveals the conservation of isoleucine at this position throughout evolution, D) In silico prediction of both variants using prediction tools of SIFT, SNAP, Polyphen-1, Polyphen-2, PhD-SNP, MAPP, and PredictSNP



High Resolution
Figure 4. The tertiary structure of the INHBB protein predicted by Modeller and visualized on the standalone tool Deep-View–Swiss-PdbViewer, A) the wild-type INHBB protein with I310, B) the mutant N310 INHBB protein formation of three new hydrogen bonds occurs with TYR327, ARG313, and LEU314, C) the wild-type INHBB protein with D386, B) the mutant N386 INHBB protein shows no change in hydro-gen bonding pattern

Figure 4. The tertiary structure of the INHBB protein predicted by Modeller and visualized on the standalone tool Deep-View–Swiss-PdbViewer, A) the wild-type INHBB protein with I310, B) the mutant N310 INHBB protein formation of three new hydrogen bonds occurs with TYR327, ARG313, and LEU314, C) the wild-type INHBB protein with D386, B) the mutant N386 INHBB protein shows no change in hydro-gen bonding pattern



High Resolution
Figure 5. Hydrophobicity profile of wild-type and mutant p.I310N and p.D386N in inhibin βB subunit predicted by the ProtScale program at the ExPASy server, A) the black circle represents the hydrophobicity around I310 in the wild-type protein, B) the black circle represents the hydrophobicity around N310 in the mutant protein showing a mild shift in hydrophobicity compared to the wild-type, C) black circle represents the hydrophobicity around D386 in the wild-type protein, B) the black circle represents the hydrophobicity around N386 in the mutant protein showing no shift in hydrophobicity compared to the wild-type

Figure 5. Hydrophobicity profile of wild-type and mutant p.I310N and p.D386N in inhibin βB subunit predicted by the ProtScale program at the ExPASy server, A) the black circle represents the hydrophobicity around I310 in the wild-type protein, B) the black circle represents the hydrophobicity around N310 in the mutant protein showing a mild shift in hydrophobicity compared to the wild-type, C) black circle represents the hydrophobicity around D386 in the wild-type protein, B) the black circle represents the hydrophobicity around N386 in the mutant protein showing no shift in hydrophobicity compared to the wild-type




Table 1. Primer list used for sequence analysis of INHA, INHBA, and INHBB gene

Table 1. Primer list used for sequence analysis of INHA, INHBA, and INHBB gene



References

  1. MacNaughton J, Banah M, McCloud P, Hee J, Burger H. Age related changes in follicle stimulating hormone, luteinizing hormone, oestradiol and immunoreactive inhibin in women of reproductive age. Clin Endocrinol (Oxf). 1998;36(4):339-45.   [PubMed]
  2. Burger HG, Cahir N, Robertson DM, Groome NP, Dudley E, Green A, et al. Serum inhibins A and B fall differentially as FSH rises in perimenopausal women. Clin Endocrinol (Oxf). 1998;48(6):809-13.   [PubMed]
  3. Reame NE, Wyman TL, Phillips DJ, de Kretser DM, Padmanabhan V. Net increase in stimulatory input resulting from a decrease in inhibin B and an increase in activin A may contribute in part to the rise in follicular phase follicle-stimulating hormone of aging cycling women. J Clin Endocrinol Metab. 1998;83(9):3302-7.   [PubMed]
  4. Farnworth PG, Robertson DM, de Kretser DM, Burger HG. Effects of 31 kDa bovine inhibin on FSH and LH in rat pituitary cells in vitro: antagonism of gonadotrophin-releasing hormone agonists. J Endocrinol. 1988;119(2):233-41.   [PubMed]
  5. Harris SE, Chand AL, Winship IM, Gersak K, Nishi Y, Yanase T, et al. INHA promoter polymorphisms are associated with premature ovarian failure. Mol Hum Reprod. 2005;11(11):779-84.   [PubMed]
  6. Stenvers KL, Findlay JK. Inhibins: from reproductive hormones to tumor suppressors. Trends Endocrinol Metab. 2009;21(3):174-80.   [PubMed]
  7. Cho BN, McMullen ML, Pei L, Yates CJ, Mayo KE. Reproductive deficiencies in transgenic mice expressing the rat inhibin alpha-subunit gene. Endocrinology. 2001;142(11):4994-5004.   [PubMed]
  8. Shelling AN, Burton KA, Chand AL, Van Ee CC, France JT, Farquhar CM, et al. Inhibin: a candidate gene for premature ovarian failure. Hum Reprod. 2000;15(12):2644-9.   [PubMed]
  9. Chand AL, Harrison CA, Shelling AN. Inhibin and premature ovarian failure. Hum Reprod Update. 2010;16(1):39-50.   [PubMed]
  10. Dixit H, Rao KL, Padmalatha V, Kanakavalli M, Deenadayal M, Gupta N, et al. Expansion of the germline analysis for the INHA gene in Indian women with ovarian failure. Hum Reprod. 2006;21(6):1643-4.   [PubMed]
  11. Prakash GJ, Ravi Kanth VV, Shelling AN, Rozati R, Sujatha M. Mutational analysis of inhibin alpha gene revealed three novel variations in Indian women with premature ovarian failure. Fertil Steril. 2010;94(1):90-8.   [PubMed]
  12. Lopes AM, Aston KI, Thompson E, Carvalho F, Goncalves J, Huang N, et al. Human spermatogenic failure purges deleterious mutation load from the autosomes and both sex chromosomes, including the gene DMRT1. PLoS Genet. 2013;9(3):e1003349.   [PubMed]
  13. Hagen CP, Main KM, Kjaergaard S, Juul A. FSH, LH, inhibin B and estradiol levels in Turner syndrome depend on age and karyotype: longitudinal study of 70 Turner girls with or without spontaneous puberty. Hum Reprod, 2010;25(12):3134-41.   [PubMed]
  14. Gravholt CH, Naeraa RW, Andersson AM, Christiansen JS, Skakkebaek NE. Inhibin A and B in adolescents and young adults with Turner’s syndrome and no sign of spontaneous puberty. Hum Reprod. 2002;17(8):2049-53.   [PubMed]
  15. Chand AL, Ooi GT, Harrison CA, Shelling AN, Robertson DM. Functional analysis of the human inhibin a subunit variant A257T and its potential role in premature ovarian failure. Hum Reprod. 2007;22(12):3241-8.   [PubMed]
  16. Dixit H, Deendayal M, Singh L. Mutational analysis of the mature peptide region of inhibin genes in Indian women with ovarian failure. Hum Reprod. 2004;19(8):1760-4.   [PubMed]
  17. Marozzi A, Porta C, Vegetti W, Crosignani PG, Tibiletti MG, Dalprà L, et al. Mutation analysis of the inhibin alpha gene in a cohort of Italian women affected by ovarian failure. Hum Reprod. 2002;17(7):1741-5.   [PubMed]

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