Analysis of the CYP21A2 gene pathogenic variants in CAH patients from Surgut using next-generation sequencing (NGS)

Abstract Background 21-hydroxylase deficiency is present in 90–95% of cases of congenital adrenal hyperplasia (CAH). Eleven major pathogenic variants account for 93% of all identified variants in the CYP21A2 gene in various clinical forms of the disease. Each population has its own range of significant pathogenic variants. We aimed to study the frequency of pathogenic variants in the CYP21A2 gene using NGS technology and real-time PCR in Surgut patients with different clinical forms of CAH. NGS was performed on 70 patients with salt-wasting and non-classical clinical forms of 21-hydroxylase deficiency, verified by direct Sanger sequencing and PCR–RFLP analysis. Results Eleven different pathogenic variants were found in 68.57% (48/70) of patients. Among 92.86% (13/14) of patients with salt-wasting CAH, variants were found to be homozygous, with CYP21A2 gene deletion as the most frequent mutation (46.4% or 13/28 alleles). In the group with non-classical CAH, pathogenic variants were identified only in 60.71% (34/56) of patients. V282L was discovered to be the most common variant in heterozygous carriers (45.45%, 15/33). NGS method identified 2 variants that were not determined by the standard method for major mutations detection: p.C170* and p.W22X, accounting for 3% of all known pathogenic variants. Conclusion Our data make it possible to clarify the specific spectrum of CYP21A2 gene pathogenic variants in CAH patients from Surgut. The NGS method allows for the identification of rare pathogenic variants (3%) in the CYP21A2 gene that are not included in the conventional PCR–RFLP analysis

A Data-Driven Review of the Genetic Factors of Pregnancy Complications

Over the recent years, many advances have been made in the research of the genetic factors of pregnancy complications. In this work, we use publicly available data repositories, such as the National Human Genome Research Institute GWAS Catalog, HuGE Navigator, and the UK Biobank genetic and phenotypic dataset to gain insights into molecular pathways and individual genes behind a set of pregnancy-related traits, including the most studied ones—preeclampsia, gestational diabetes, preterm birth, and placental abruption. Using both HuGE and GWAS Catalog data, we confirm that immune system and, in particular, T-cell related pathways are one of the most important drivers of pregnancy-related traits. Pathway analysis of the data reveals that cell adhesion and matrisome-related genes are also commonly involved in pregnancy pathologies. We also find a large role of metabolic factors that affect not only gestational diabetes, but also the other traits. These shared metabolic genes include IGF2, PPARG, and NOS3. We further discover that the published genetic associations are poorly replicated in the independent UK Biobank cohort. Nevertheless, we find novel genome-wide associations with pregnancy-related traits for the FBLN7, STK32B, and ACTR3B genes, and replicate the effects of the KAZN and TLE1 genes, with the latter being the only gene identified across all data resources. Overall, our analysis highlights central molecular pathways for pregnancy-related traits, and suggests a need to use more accurate and sophisticated association analysis strategies to robustly identify genetic risk factors for pregnancy complications

Protein Misfolding during Pregnancy: New Approaches to Preeclampsia Diagnostics

Preeclampsia (PE) is a multisystem heterogeneous complication of pregnancy remaining a leading cause of maternal and perinatal morbidity and mortality over the world. PE has a large spectrum of clinical features and symptoms, which make diagnosis challenging. Despite a long period of studying, PE etiology is still unclear and there are no reliable rapid tests for early diagnosis of this disease. During the last decade, it was shown that proteins misfolding and aggregation are associated with PE. Several proteins, including amyloid beta peptide, transthyretin, alpha-1 antitrypsin, albumin, IgG k-free light chains, and ceruloplasmin are dysregulated in PE, resulting in toxic deposition of amyloid-like aggregates in the placenta and body fluids. It is also possible that aggregated proteins induce defective trophoblast invasion, placental ischemia, ER stress, and promote PE manifestation. The fact that protein aggregation is an emerging biomarker of PE provides an opportunity to develop new diagnostic approaches based on amyloids special features, such as Congo red (CR) staining and thioflavin T (ThT) enhanced fluorescence

Diagnostics of preeclampsia based on Congo red binding to urinary components: Rationales and limitations.

Preeclampsia is a disorder that can occur during pregnancy and is one of the leading causes of death among pregnant women. This disorder occurs after the 20th week of pregnancy and is characterized by arterial hypertension, proteinuria, fetoplacental, and multiple organ dysfunctions. Despite the long history of studying preeclampsia, its etiology and pathogenesis remain poorly understood, and therapy is symptomatic. One of the factors of the disorder is believed to be misfolded proteins that are prone to form amyloid aggregates. The CRD tests, utilizing the binding of the amyloid-specific dye Congo red to urine components, demonstrate high efficiency in diagnosing preeclampsia. However, these tests have also been found to be positive in other disorders with proteinuria, presumably associated with concomitant amyloidosis. To assess the limitations of the CRD tests, we examined urine congophilia and protein components mediating Congo red positivity in patients with proteinuria, including preeclampsia, amyloid and non-amyloid nephropathies. We stained the urine samples and calculated congophilia levels. We also assessed the contribution of large protein aggregates to congophilia values using ultracentrifugation and determined the molecular weights of congophilic urinary proteins using centrifugal concentrators. All proteinuric groups demonstrate positive results in the CRD tests and congophilia levels were more than two times higher compared with the control non-proteinuric groups (p <0.01). There was a strong correlation between urine protein excretion and congophilia in amyloid nephropathy (rs = 0.76), non-amyloid nephropathies (rs = 0.90), and preeclampsia (rs = 0.90). Removal of large aggregates from urine did not affect the congophilia levels. Separation of urine protein fractions revealed congophilic components in the range of 30-100 kDa, including monomeric serum albumin. Our results indicate limitations of CRD tests in preeclampsia diagnostics in women with renal disorders and underscore the need for further research on the mechanisms of Congo red binding with urine components

Pilot Screening of Cell-Free mtDNA in NIPT: Quality Control, Variant Calling, and Haplogroup Determination

Clinical tests based on whole-genome sequencing are generally focused on a single task approach, testing one or several parameters, although whole-genome sequencing (WGS) provides us with large data sets that can be used for many supportive analyses. In spite of low genome coverage, data of WGS-based non-invasive prenatal testing (NIPT) contain fully sequenced mitochondrial DNA (mtDNA). This mtDNA can be used for variant calling, ancestry analysis, population studies and other approaches that extend NIPT functionality. In this study, we analyse mtDNA pool from 645 cell-free DNA (cfDNA) samples of pregnant women from different regions of Russia, explore the effects of transportation and storing conditions on mtDNA content, analyse effects, frequency and location of mitochondrial variants called from samples and perform haplogroup analysis, revealing the most common mitochondrial superclades. We have shown that, despite the relatively low sequencing depth of unamplified mtDNA from cfDNA samples, the mtDNA analysis in these samples is still an informative instrument suitable for research and screening purposes

The representation of the CRD tests’ results.

The CRD membrane (A) and CRD paper (B) tests are shown. The representative images displayed are from five pregnant women with preeclampsia (1–5) and five control pregnant women (6–10).</p

Experimental data.

All data used to generate the results for this study. (XLSX)</p