Unravelling the genetic landscape of cervical insufficiency: Insights into connective tissue dysfunction and hormonal pathways

Abstract

Background: The intricate molecular pathways and genetic factors that underlie the pathophysiology of cervical insufficiency (CI) remain largely unknown and understudied.

Methods: We sequenced exomes from 114 patients in Latvia and Lithuania, diagnosed with a short cervix, CI, or a history of CI in previous pregnancies. To probe the well-known link between CI and connective tissue dysfunction, we introduced a connective tissue dysfunction assessment questionnaire, incorporating Beighton and Brighton scores. The phenotypic data obtained from the questionnaire was correlated with the number of rare damaging variants identified in genes associated with connective tissue disorders (in silico NGS panel). SKAT, SKAT-O, and burden tests were performed to identify genes associated with CI without a priori hypotheses. Pathway enrichment analysis was conducted using both targeted and genome-wide approaches.

Results: No patient could be assigned monogenic connective tissue disorder neither genetically, neither clinically upon clinical geneticist evaluation. Expanding our exploration to a genome-wide perspective, pathway enrichment analysis replicated the significance of extracellular matrix-related pathways as important contributors to CI's development. A genome-wide burden analysis unveiled a statistically significant prevalence of rare damaging variants in genes and pathways associated with steroids (p-adj = 5.37E-06). Rare damaging variants, absent in controls (internal database, n = 588), in the progesterone receptor (PGR) (six patients) and glucocorticoid receptor (NR3C1) (two patients) genes were identified within key functional domains, potentially disrupting the receptors' affinity for DNA or ligands.

Conclusion: Cervical insufficiency in non-syndromic patients is not attributed to a single connective tissue gene variant in a Mendelian fashion but rather to the cumulative effect of multiple inherited gene variants highlighting the significance of the connective tissue pathway in the multifactorial nature of CI. PGR or NR3C1 variants may contribute to the pathophysiology of CI and/or preterm birth through the impaired progesterone action pathways, opening new perspectives for targeted interventions and enhanced clinical management strategies of this condition.

Fig 1. Overview of the study design.

CI–cervical insufficiency. WES–whole exome sequencing. WGS–whole genome sequencing. ACGS–The association for clinical genomic science. P/LP–pathogenic/likely pathogenic variant.

Fig 2. Exploring genetics of cervical insufficiency: Connective tissue perspective.

A) Expression (Y axis) of connective tissue disorder genes (blue) in relation to all genes showing at least some expression within cervix (TPM>0) (light blue) (X axis). B) Beighton (pink) / Brighton (blue) / Total score (green) (Y axis) obtained by each study patient (X axis) in the connective tissue disfunction assessment questionnaire. C) Patients age (X-axis) correlation with Beighton hypermobility score (Y-axis). A larger circle size indicates a greater number of patients with the same age and Beighton score. D) Total score obtained in connective tissue dysfunction assessment questionnaire (X-axis) correlation with number of rare damaging gene variants in connective tissue gene panel (Y-axis). A larger circle size indicates a greater number of patients with the same score and number of rare variants.

Fig 3. PGR (progesterone receptor) variants identified in patients with cervical insufficiency and PGR amino acid positions tolerance map to missense variation.

PGR functional domains: 1) an N-terminal (modulatory) domain. 2) a DNA binding domain. 3) a hormone/ligand binding domain. GnomAD–variant frequency in a GnomAD V4 population database. CADD–combined annotation dependent depletion score, variant effect in silico prediction tool. Revel score–variant effect in silico prediction tool.

Fig 4. Genome wide pathway enrichment analysis results.

A. Genome-wide pathway enrichment conducted by selecting genes with single nucleotide variants present exclusively in the cervical insufficiency group (genes shared between cases and controls). B. Genome-wide pathway enrichment conducted by selecting genes devoid of rare damaging single nucleotide variants in controls (with no gene overlap between cases and controls).

Fig 5. Summary of study design and major outcomes.

We were the first to apply the Beighton/Brighton criteria to test the hypothesis linking the connective tissue dysfunction-driven nature of CI to genetics. While our analysis strengthened the association of CI with connective tissue pathways, further research is needed to explore the relationships between subclinical phenotypic expressions of connective tissue disorders and CI. Our next hypothesis is that the use of Beighton/Brighton criteria, along with the connective tissue dysfunction assessment questionnaire developed by our group, can serve as a predictive tool for CI/PTB, at least for a subset of patients. This investigation is currently underway in our group.