Detection of mutations in the CYP21A2 gene: genotype-phenotype correlation in Slovenian couples with conceiving problems

The objective of this study was to compare the CYP 21A2 genetic profiles of couples with unexplained fertility problems (UFP) with genetic profiles of healthy controls (HCs). Furthermore, we analyzed associations between mutations in the CYP21A2 gene and various clinical and laboratory parameters. Allele-specific polymerase chain reaction (PCR) was used in 638 probands with UFP and 200 HCs. Statistic analysis with χ2 was used to study the association of mutations with infertility. The effect of mutations on particular clinical and laboratory parameters was assessed with the analysis of variance (ANOVA) test. With regard to the CYP21A2 gene, 0.6% of probands with UFP and 0.5% of HCs were positive for the c.290-13A/C>G mutation; 0.6% of probands with UFP and 1.5% of HCs were positive for the p.I172N mutation; there were no probands with UFP positive for the p.P30L mutation, whereas 0.5% of HCs were; and 0.2% of probands with UFP and 0.5% of HCs were found to have the p.V281L mutation. We found a significant association between c.290-13A/C>G mutation and the frequency of significant hormone deviations (χ2 = 6.997, p = 0.008). Similar association was also observed between the c.29013A/C>G mutation and the frequency of polycystic ovary syndrome (PCOS) (χ2 = 16.775, p = 0.000). Our findings indicate that no significant difference in the prevalence of CYP 21A2 mutations can be found in probands with UFP when compared with HCs without infertility history. The results also imply the significant association of the c.290-13A/ C>G mutation in the CYP21A2 gene, not only with the frequency of PCOS, but also with the frequency of significant hormone deviations

De novo KMT2D heterozygous frameshift deletion in a newborn with a congenital heart anomaly

Kabuki syndrome (KS) is characterized by typical facial features and patients are also affected by multiple congenital anomalies, of which congenital heart anomalies (CHAs) are present in 28.0 to 80.0%. In approximately 75.0% of patients, the genetic causes of KS are caused by mutation in the KMT2D gene. Although KS is a well-characterized syndrome, reaching the diagnosis in neonates is still challenging. Namely, newborns usually display mild facial features; therefore the diagnosis is mainly based on congenital malformations. In our case, a newborn was referred for next generation sequencing (NGS) testing due to the prenatally observed CHA. After birth, a ventricular septal defect (VSD), vesicoureteral reflux, muscular hypotonia, cleft palate, mild microcephaly, and some dysmorphic features, were noted. The NGS analysis was performed on the proband’s genomic DNA using the TruSight One Sequencing Panel, which enriches exons of 4813 genes with clinical relevance to the disease. After variant calling, NGS data analysis was predominantly focused on rare variants in genes involved in VSD, microcephaly, and muscular hypotonia; features observed predominantly in our proband. With the aforementioned protocol, we were able to determine the previously unreported de novo frameshift deletion in the KMT2D gene resulting in translation termination. Although our proband is a typical representative of KS, his diagnosis was reached only after NGS analysis. Our proband thus represents the importance of genotypephenotype driven NGS analysis in diagnosis of patients with congenital anomalies

MTHFR C677T and A1298C Genotypes and Haplotypes in Slovenian Couples with Unexplained Infertility Problems and in Embryonic Tissues from Spontaneous Abortions

The objective of this study was to analyze the methylenetetrahydrofolate reductases (MTHFRs) C677T and A1298C genotype distributions in couples with unexplained fertility problems (UFP) and healthy controls, and to analyze the genotype and haplotype distribution in spontaneously aborted embryonic tissues (SAET) using allele specific polymerase chain reaction (PCR) in 200 probands with UFP, 353 samples of SAET and 222 healthy controls. The analysis revealed a significant overall representation of the 677T allele in male probands from couples with UFP (p = 0.036). The combined genotype distribution for both MTHFR polymorphisms was also significantly altered (χ2 21.73, p <0.001) although female probands made no contribution (c2 1.33, p = 0.72). The overall representation of the 677T allele was more pronounced in SAET (0.5 vs. 0.351 in controls, p <0.001) regardless of the karyotype status (aneuploidy vs. normal karyotype). In addition, the frequencies of the CA and CC haplotypes were significantly lower than in the control group (p = 0.021 and p = 0.001, respectively), whereas the frequency of the TC haplotype was significantly higher than in controls (p <0.0001). The presented findings indicate that only male probands contribute to the association of MTHFR mutations with fertility problems in grown adults and demonstrate a high prevalence of mutated MTHFR genotypes in SAET