Cytogenetic screening of chromosomal abnormalities and genetic analysis of FSH receptor Ala307Thr and Ser680Asn genes in amenorrheic patients

Abstract

Objective: Amenorrhea is a rare reproductive medical condition defined by the absence of menstruation during puberty or later life. This study aims to establish the frequency and pattern of chromosomal abnormalities (CA) in both primary amenorrhea (PA) and secondary amenorrhea (SA), and further to detect the genetic changes in exon 10 at nucleotide positions 919 and 2039 of the genotypes Thr307Ala, and Asn680Ser, respectively.

Design settings and patients: This cross-sectional study was conducted on a sample of seventy amenorrhoeic women according to the Helsinki declaration rules of medical ethics, as divided into 40 (57.14%) with PA and 30 (42.86%) with SA, and 30 healthy women with normal menstruation as the control. The chromosomal karyotyping was performed according to the ISCN, 2020. PCR products were submitted to RFLP and Sanger sequencing for women with normal karyotype and high FSH serum levels.

Results: The classical Turner Syndrome was the most common CA in PA, followed by isochromosome X [46, Xi(X)(q10)], mosaicism of Turner and isochromosome X [45, X /46, Xi(X)(q10)], sex reversal (46, XY) and (46, XX,-3,+der3,-19,del 19 p). Abnormal SA cases were characterized by mosaicism Turner syndrome (45,X/46,XX) and (46,XX,-3,+der3,X,+derX). The homozygous genotypes AA and GG of Ala307Thr (rs6165) in the FSHR gene are most common in PA, while the homozygous genotype AA is more common in SA. GG and AG genotypes of Ser680Asn (rs6166) are more frequent in Iraqi patients with PA and SA compared to the healthy control women. Both PCR-RFLP and Sanger sequencing indicated a marked matching between genotypes.

Conclusions: The study emphasizes the need for cytogenetic analysis to determine the genetic basis of PA and SA. Further, genotyping for women with normal karyotype and high FSH serum concentrations via PCR-RFLP should be considered for the precise diagnosis and development of appropriate management of and counselling for these patients.

Keywords: Amenorrhea; FSHR; Karyotyping; PCR-RFLP.

Figure 1. Ideogram of study design.

Figure 2. Karyotype of pure turner monosomy X: 45,X.

Figure 3. Karyotype of isochromosome of long arm of X: 46,Xi (Xq).

Figure 4. Sex reversal karyotype: 46,XY.

Figure 5. Karyotype of monosomy X (45,X) and (46,XX).

Figure 6. PCR product of FSHR gene (Ala307Thr, 577 bp).

The product was subject to electrophoresis on 1.5% agarose. (A) PCR product of specimen with patients. (B) PCR product of control specimen.

Figure 7. Electrophoretogram of DNA fragments for Ala307Thr polymorphisms after digestion with AhdI restriction enzymes.

(A) PCR-RFLP results of patients; (B) PCR-RFLP results with normal menstruation cycle. Homozygote A/A was shown by the bands of 403 bp, 143 bp and 31 bp. Homozygote G/G was shown by the 403 bp and 174 bp bands. Heterozygote A/G was shown by the bands of 403 bp, 174 bp, 143 bp and 31 bp. Note that the 31 bp bands were run off the gel.

Figure 8. DNA sequencing of the forward strand of Ala307Thr polymorphisms (AA, AG and GG).

Figure 9. The PCR product of Ser680Asn polymorphism gene on exon 10 of FSHR was 520 bp.

(A) PCR product of patients. (B) PCR product of control specimen.

Figure 10. (A–B) Electrophoretogram of DNA fragments for Ser680Asn polymorphisms after digestion with BsrI restriction enzymes.

Homozygote A/A was indicated by the bands at 520 bp. Homozygote G/G was indicated by the bands at 413 bp and 107 bp. Heterozygote A/G was indicated by the bands at 520 bp, 413 bp, and 107 bp.

Figure 11. DNA sequencing of the forward strand of Ser680Asn polymorphisms (AA, AG, and GG).