Evaluation of Optical Genome Mapping in Clinical Genetic Testing of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common hereditary muscular dystrophy, caused by the contraction of the D4Z4 repeats on the permissive 4qA haplotype on chromosome 4, resulting in the faulty expression of the DUX4 gene. Traditional diagnostics are based on Southern blotting, a time- and effort-intensive method that can be affected by single nucleotide variants (SNV) and copy number variants (CNV), as well as by the similarity of the D4Z4 repeats located on chromosome 10. We aimed to evaluate optical genome mapping (OGM) as an alternative molecular diagnostic method for the detection of FSHD. We first performed optical genome mapping with EnFocus™ FSHD analysis using DLE-1 labeling and the Saphyr instrument in patients with inconclusive diagnostic Southern blot results, negative FSHD2 results, and clinically evident FSHD. Second, we performed OGM in parallel with the classical Southern blot analysis for our prospectively collected new FSHD cases. Finally, panel exome sequencing was performed to confirm the presence of FSHD2. In two patients with diagnostically inconclusive Southern blot results, OGM was able to identify shortened D4Z4 repeats on the permissive 4qA alleles, consistent with the clinical presentation. The results of the prospectively collected patients tested in parallel using Southern blotting and OGM showed full concordance, indicating that OGM is a useful alternative to the classical Southern blotting method for detecting FSHD1. In a patient showing clinical FSHD but no shortened D4Z4 repeats in the 4qA allele using OGM or Southern blotting, a likely pathogenic variant in SMCHD1 was detected using exome sequencing, confirming FSHD2. OGM and panel exome sequencing can be used consecutively to detect FSHD2.

Diagnostic yield of exome sequencing in myopathies: Experience of a Slovenian tertiary centre.

BACKGROUND: Our aim was to present the experience of systematic, routine use of next generation sequencing (NGS) in clinical diagnostics of myopathies. METHODS: Exome sequencing was performed on patients with high risk for inherited myopathy, which were selected based on the history of the disease, family history, clinical presentation, and diagnostic workup. Exome target capture was performed, followed by sequencing on HiSeq 2500 or MiSeq platforms. Data analysis was performed using internally developed bioinformatic pipeline. RESULTS: The study comprised 86 patients, including 22 paediatric cases (26%). The largest group were patients referred with an unspecified myopathy (47%), due to non-specific or incomplete clinical and laboratory findings, followed by congenital myopathies (22%) and muscular dystrophies (22%), congenital myotonias (6%), and mitochondrial myopathies (3%). Altogether, a diagnostic yield was 52%; a high diagnostic rate was present in paediatric patients (64%), while in patients with unspecified myopathies the rate was 35%. We found 51 pathogenic/likely pathogenic variants in 23 genes and two pathogenic copy number variations. CONCLUSION: Our results provide evidence that phenotype driven exome analysis diagnostic approach facilitates the diagnostic rate of complex, heterogeneous disorders, such as myopathies, particularly in paediatric patients and patients with unspecified myopathies.

Methylenetetrahydrofolate Reductase Dimer Configuration as a Risk Factor for Maternal Meiosis I-Derived Trisomy 21.

BACKGROUND: Evidence suggests that the dimer configuration of methylenetetrahydrofolate reductase (MTHFR) enzyme might be destabilized by polymorphisms in monomers at the positions C677T and A1298C. It has been observed that these polymorphisms may lead to stable (CCAA, CCAC, CCCC) and unstable (CTAA, CTAC, TTAA) enzyme dimer configurations. OBJECTIVE: The aim of this study was to evaluate the association of the MTHFR enzyme dimer configuration and folate dietary intake with the stage of meiotic nondisjunction in mothers of children with maternally derived trisomy 21. METHODS: A total of 119 mothers of children with maternally derived free trisomy 21 were included in the study. The mean maternal age at the time of the birth of the child with trisomy 21 was 32.3 ± 6.4 (range 16-43) years. All mothers were Caucasian. Parental origin of trisomy 21 and meiotic stage of nondisjunction was determined using short tandem repeat markers spanning from the centromere to the telomere of chromosome 21q. The MTHFR C677T and A1298C polymorphism was evaluated by PCR-RFLP. RESULTS: Increased frequency of the MTHFR genotype combinations CTAA, CTAC, and TTAA was found in the group of mothers with meiosis I (MI) nondisjunction (p = 0.007). No differences were found between study participants regarding dietary and lifestyles habits. CONCLUSION: The risk for MI nondisjunction of chromosome 21 was 4.6-fold higher in cases who had CTAA, CTAC, and TTAA MTHFR genotype combinations and who did not used folic acid supplements in the preconception period.

Maternal LINE-1 DNA Methylation and Congenital Heart Defects in Down Syndrome.

Background: Down syndrome (DS) is one of the most common chromosomal abnormalities associated with congenital heart defects (CHD), with approximately 40 to 60% of cases showing cardiac defects. This study assessed (i) the association between maternal LINE-1 methylation and the occurrence of CHDs in children with DS and (ii) the impact of endogenous maternal factors (MTHFR C677T polymorphism and maternal age) and exogenous maternal factors (cigarette smoking, alcohol intake, medication use, body mass index and dietary habits such as folate intake) on maternal LINE-1 methylation and on the occurrence of CHD in children with DS. Patients and Methods: The study included 90 mothers of children with DS of maternal origin (49% DS-CHD+ mothers/51% DS-CHD- mothers). LINE-1 DNA methylation was analyzed in peripheral blood lymphocytes by quantification of LINE-1 methylation using the MethyLight method. MTHFR C677T polymorphism genotyping was performed using PCR-RFLP. Results: LINE-1 methylation was not significantly different between DS-CHD+ and DS-CHD- mothers (P = 0.997). Combination of MTHFR C677T genotype/diet and BMI were significant independent predictors of LINE-1 DNA methylation in DS-CHD+ mothers (ß -0.40, P = 0.01 and ß -0.32, P = 0.03, respectively). In the analyzed multivariate model (model P = 0.028), these two factors explained around 72% of the variance in LINE-1 DNA methylation in mothers of children with DS and CHD. The group with the highest BMI (≥30 kg/m2) had significantly lower LINE-1 methylation than the group with normal BMI (Bonferroni post hoc P = 0.03) and the overweight group (Bonferroni post hoc P = 0.04). The lowest LINE-1 DNA methylation values were found in DS-CHD+ mothers with the CT+TT genotype and a low-folate diet; the values were significantly lower than the values in mothers with the CC genotype and a folate-rich diet (Bonferroni post hoc P = 0.04). Conclusion: Association between maternal LINE-1 methylation and CHD in children with DS was not found. Study showed that the MTHFR genotype/diet combination and BMI were significantly associated with LINE-1 methylation in mothers of children with DS-CHD+. These results highlight the need for a multifactorial approach to assess the roles of endogenous and exogenous maternal factors in maternal LINE-1 DNA methylation and the consequent pathologies in children. More extensive studies in a larger sample may help elucidate these relationships.

Functional inference of methylenetetrahydrofolate reductase gene polymorphisms on enzyme stability as a potential risk factor for Down syndrome in Croatia.

Understanding the biochemical structure and function of the methylenetetrahydrofolate reductase gene (MTHFR) provides new evidence in elucidating the risk of having a child with Down syndrome (DS) in association with two common MTHFR polymorphisms, C677T and A1298C. The aim of this study was to evaluate the risk for DS according to the presence of MTHFR C677T and A1298C polymorphisms as well as the stability of the enzyme configuration. This study included mothers from Croatia with a liveborn DS child (n=102) or DS pregnancy (n=9) and mothers with a healthy child (n=141). MTHFR C677T and A1298C polymorphisms were assessed by PCR-RFLP. Allele/genotype frequencies differences were determined using chi square test. Odds ratio and the 95% confidence intervals were calculated to evaluate the effects of different alleles/genotypes. No statistically significant differences were found between the frequencies of allele/genotype or genotype combinations of the MTHFR C677T and A1298C polymorphisms in the case and the control groups. Additionally, the observed frequencies of the stable (677CC/1298AA, 677CC/1298AC, 677CC/1298CC) and unstable (677CT/1298AA, 677CT/1298AC, 677TT/1298AA) enzyme configurations were not significantly different. We found no evidence to support the possibility that MTHFR polymorphisms and the stability of the enzyme configurations were associated with risk of having a child with DS in Croatian population.