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. 2022 Mar 29;13(4):611.
doi: 10.3390/genes13040611.

WT1, NR0B1, NR5A1, LHX9, ZFP92, ZNF275, INSL3, and NRIP1 Genetic Variants in Patients with Premature Ovarian Insufficiency in a Mexican Cohort

Luis Ramos  1
Affiliations

WT1, NR0B1, NR5A1, LHX9, ZFP92, ZNF275, INSL3, and NRIP1 Genetic Variants in Patients with Premature Ovarian Insufficiency in a Mexican Cohort

Luis Ramos. Genes (Basel). .

Abstract

Premature ovarian insufficiency (POI) is one of the main causes of female premature infertility. POI is a genetically heterogeneous disorder with a complex molecular etiology; as such, the genetic causes remain unknown in the majority of patients. Therefore, this study aimed to identify mutations and characterize the associated molecular contribution of gonadogenesis-determinant genes to POI. Genomic assays, including PCR-SSCP and Sanger sequencing, followed by in silico analyses were used to investigate the underpinnings of ovarian deficiency in 11 women affected by POI. Large deletions and nucleotide insertions and duplications were excluded by PCR. Thirteen genetic variants were identified in the WT1 (c.213G>T, c.609T>C, c.873A>G, c.1122G>A), NR0B1 (c.353C>T, c.425G>A), NR5A1 (c.437G>C, IVS4-20C>T), LHX9 (IVS2-12G>C, IVS3+13C>T, c.741T>C), ZNF275 (c.969C>T), and NRIP1 (c.3403C>T) genes. Seven novel genetic variants and five unpublished substitutions were identified. No genetic aberrations were detected in the ZFP92 and INSL3 genes. Each variant was genotyped using PCR-SSCP in 100 POI-free subjects, and their allelic frequencies were similar to the patients. These analyses indicated that allelic variation in the WT1, NR0B1, NR5A1, LHX9, ZFP92, ZNF275, INSL3, and NRIP1 genes may be a non-disease-causing change or may not contribute significantly to the genetics underlying POI disorders. Findings support the polygenic nature of this clinical disorder, with the SNVs identified representing only a probable contribution to the variability of the human genome.

Keywords: fertility; non-coding SNVs; non-synonymous SNVs; ovary; polygenic; synonymous SNVs.

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Conflict of interest statement

The author declare no conflict of interest.

Figures

Figure 1
Figure 1
Representative banding patterns for the WT1 (NM_024426.6), NR0B1 (NM_000475.5), NR5A1 (NM_004959.5), LHX9 (NM_020204.3), ZFP92 (NM_001136273.2), ZNF275 (NM_001080485.4), INSL3 (NM_001265587.2), and NRIP1 (NM_003489.4) genes. All exons were amplified by (α-32P)-dCTP-PCR, and one representative exon is shown for each gene. The arrows indicate 500 bp, and 100 bp indicates the molecular weight marker (100–1500 bp). C1 and C2 are unrelated healthy subjects. P1–P11 are patients clinically classified with POI. The expected sizes for the amplified exons are shown on the right.
Figure 2
Figure 2
Genetic variants identified using (α-32P)-dCTP-PCR-SSCP analysis of the WT1 (NM_024426.6), NR0B1 (NM_000475.5), NR5A1 (NM_004959.5), LHX9 (NM_020204.3), ZNF275 (NM_001080485.4), and NRIP1 (NM_003489.4) genes from patients with POI (P) and healthy subjects (C). Different SSCP patterns are marked by asterisks. Heterozygous and homozygous subjects are indicated by two and one asterisks, respectively.
Figure 3
Figure 3
Schematic illustration of amplified exons from the human WT1 (NM_024426.6) gene located on chromosome 11. Exons (1–10) are represented by blue boxes. Partial nucleotide sequences of the human WT1 gene were obtained by bidirectional Sanger sequencing from patients with POI. The human genome sequence revealed that the WT1 gene contains four synonymous polymorphisms.
Figure 4
Figure 4
X-chromosome and exon representations of the human NR0B1 (NM_000475.5) gene. Exons (1 and 2) were analyzed by capillary electrophoresis. Human NR0B1 gene sequence variants were identified in exon 1 (blue box) from patients with POI. The presented fragment of the nucleic acid sequence of the NR0B1 gene contains both the non-synonymous and synonymous variants.
Figure 5
Figure 5
Partial nucleotide sequence of the human NR5A1 (NM_004959.5) gene and a structural diagram of its location on chromosome 9. Genotypic variants in the coding/non-coding regions are located in exon 4 and intron 4 (blue boxes), respectively. Exons are indicated with blue boxes.
Figure 6
Figure 6
Allelic variants of the human LHX9 (NM_020204.3) gene located on the long arm of chromosome 1. Five exons (blue boxes) were assessed by PCR-SSCP analysis and the coding/non-coding variants were identified by DNA sequencing. The locations of the substitutions are indicated above the electropherogram.
Figure 7
Figure 7
The ZNF275 (NM_001080485.4) gene variant on the long arm of the X chromosome was verified by Sanger sequencing. A homozygous variant (TGT) and heterozygous variant (TGT/TGC) were found in exon 3 (blue box).
Figure 8
Figure 8
Exonic region of the human NRIP1 (NM_003489.4) gene located on the long arm of chromosome 21. Nucleotide sequence analysis identified a polymorphic variant located in the end region of the intronless NRIP1 gene (blue box).
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