Streamlined two-step fragment analysis PCR and exome sequencing of RFC1 for diagnostic testing of suspected CANVAS patients.

Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is caused by biallelic pathogenic expansions, or compound heterozygosity with other pathogenic variants in the RFC1 gene. CANVAS is estimated to be underdiagnosed, both because of the lack of formal diagnostic criteria and molecular challenges that translate to lesser access and high cost of routine testing. Our aim was to address the need for making CANVAS genetic testing routine, by designing a streamlined two-step PCR consisting of a short-allele screening PCR and a confirmatory PCR with fragment capillary electrophoresis detection. Exome sequencing of RFC1 was additionally foreseen to resolve potential compound heterozygosity cases. Specificity of our approach was evaluated using ataxia patients with known non-CANVAS diagnoses, and optimized using Southern blot confirmed CANVAS patients. We evaluated our approach by testing patients consecutively referred for clinically suspected CANVAS using first the two-step PCR, followed by exome sequencing. Our approach was able to accurately identify negative and confirm positive cases in prospectively collected suspected CANVAS patients presenting with at least three typical clinical signs. The proposed testing approach provides an alternative method able to clearly distinguish between CANVAS negative and positive cases and can be easily incorporated into the genetic diagnostic laboratory workflow.

The Expanding Phenotypical Spectrum of WARS2-Related Disorder: Four Novel Cases with a Common Recurrent Variant.

Biallelic variants in the mitochondrial form of the tryptophanyl-tRNA synthetases (WARS2) can cause a neurodevelopmental disorder with movement disorders including early-onset tremor-parkinsonism syndrome. Here, we describe four new patients, who all presented at a young age with a tremor-parkinsonism syndrome and responded well to levodopa. All patients carry the same recurrent, hypomorphic missense variant (NM_015836.4: c.37T>G; p.Trp13Gly) either together with a previously described truncating variant (NM_015836.4: c.797Cdel; p.Pro266ArgfsTer10), a novel truncating variant (NM_015836.4: c.346C>T; p.Gln116Ter), a novel canonical splice site variant (NM_015836.4: c.349-1G>A), or a novel missense variant (NM_015836.4: c.475A>C, p.Thr159Pro). We investigated the mitochondrial function in patients and found increased levels of mitochondrially encoded cytochrome C Oxidase II as part of the mitochondrial respiratory chain as well as decreased mitochondrial integrity and branching. Finally, we conducted a literature review and here summarize the broad phenotypical spectrum of reported WARS2-related disorders. In conclusion, WARS2-related disorders are diagnostically challenging diseases due to the broad phenotypic spectrum and the disease relevance of a relatively common missense change that is often filtered out in a diagnostic setting since it occurs in ~0.5% of the general European population.

DNMT3B rs2424913 as a Risk Factor for Congenital Heart Defects in Down Syndrome.

Impairments of the genes that encode enzymes that are involved in one-carbon metabolism because of the presence of gene polymorphisms can affect the methylation pattern. The altered methylation profiles of the genes involved in cardiogenesis may result in congenital heart defects (CHDs). The aim of this study was to investigate the association between the MTHFR rs1801133, MTHFR rs1801131, MTRR rs1801394, DNMT1 rs2228611, DNMT3A rs1550117, DNMT3B rs1569686, and DNMT3B rs2424913 gene polymorphisms and congenital heart defects in Down syndrome (DS) individuals. The study was conducted on 350 participants, including 134 DS individuals with CHDs (DSCHD+), 124 DS individuals without CHDs (DSCHD-), and 92 individuals with non-syndromic CHD. The genotyping was performed using the PCR-RFLP method. A statistically significant higher frequency of the DNMT3B rs2424913 TT in the DSCHD+ individuals was observed. The DNMT3B rs2424913 TT genotype, as well as the T allele, had significantly higher frequencies in the individuals with DS and atrial septal defects (ASDs) in comparison with the individuals with DS and other CHDs. Furthermore, our results indicate a statistically significant effect of the DNMT3B rs1569686 TT genotype in individuals with non-syndromic CHDs. The results of the study suggest that the DNMT3B rs2424913 TT genotypes may be a possible predisposing factor for CHDs in DS individuals, and especially those with ASDs.

Mutations in SCN3A cause early infantile epileptic encephalopathy.

OBJECTIVE: Voltage-gated sodium (Na+ ) channels underlie action potential generation and propagation and hence are central to the regulation of excitability in the nervous system. Mutations in the genes SCN1A, SCN2A, and SCN8A, encoding the Na+ channel pore-forming (α) subunits Nav1.1, 1.2, and 1.6, respectively, and SCN1B, encoding the accessory subunit ß1 , are established causes of genetic epilepsies. SCN3A, encoding Nav1.3, is known to be highly expressed in brain, but has not previously been linked to early infantile epileptic encephalopathy. Here, we describe a cohort of 4 patients with epileptic encephalopathy and heterozygous de novo missense variants in SCN3A (p.Ile875Thr in 2 cases, p.Pro1333Leu, and p.Val1769Ala). METHODS: All patients presented with treatment-resistant epilepsy in the first year of life, severe to profound intellectual disability, and in 2 cases (both with the variant p.Ile875Thr), diffuse polymicrogyria. RESULTS: Electrophysiological recordings of mutant channels revealed prominent gain of channel function, with a markedly increased amplitude of the slowly inactivating current component, and for 2 of 3 mutants (p.Ile875Thr and p.Pro1333Leu), a leftward shift in the voltage dependence of activation to more hyperpolarized potentials. Gain of function was not observed for Nav1.3 variants known or presumed to be inherited (p.Arg1642Cys and p.Lys1799Gln). The antiseizure medications phenytoin and lacosamide selectively blocked slowly inactivating over transient current in wild-type and mutant Nav1.3 channels. INTERPRETATION: These findings establish SCN3A as a new gene for infantile epileptic encephalopathy and suggest a potential pharmacologic intervention. These findings also reinforce the role of Nav1.3 as an important regulator of neuronal excitability in the developing brain, while providing additional insight into mechanisms of slow inactivation of Nav1.3. Ann Neurol 2018;83:703-717.

Altered LINE-1 Methylation in Mothers of Children with Down Syndrome.

Down syndrome (DS, also known as trisomy 21) most often results from chromosomal nondisjunction during oogenesis. Numerous studies sustain a causal link between global DNA hypomethylation and genetic instability. It has been suggested that DNA hypomethylation might affect the structure and dynamics of chromatin regions that are critical for chromosome stability and segregation, thus favouring chromosomal nondisjunction during meiosis. Maternal global DNA hypomethylation has not yet been analyzed as a potential risk factor for chromosome 21 nondisjunction. This study aimed to asses the risk for DS in association with maternal global DNA methylation and the impact of endogenous and exogenous factors that reportedly influence DNA methylation status. Global DNA methylation was analyzed in peripheral blood lymphocytes by quantifying LINE-1 methylation using the MethyLight method. Levels of global DNA methylation were significantly lower among mothers of children with maternally derived trisomy 21 than among control mothers (P = 0.000). The combination of MTHFR C677T genotype and diet significantly influenced global DNA methylation (R2 = 4.5%, P = 0.046). The lowest values of global DNA methylation were observed in mothers with MTHFR 677 CT+TT genotype and low dietary folate. Although our findings revealed an association between maternal global DNA hypomethylation and trisomy 21 of maternal origin, further progress and final conclusions regarding the role of global DNA methylation and the occurrence of trisomy 21 are facing major challenges.

Down syndrome: parental origin, recombination, and maternal age.

The aims of the present study were to assess (1) the parental origin of trisomy 21 and the stage in which nondisjunction occurs and (2) the relationship between altered genetic recombination and maternal age as risk factors for trisomy 21. The study included 102 cases with Down syndrome from the Croatian population. Genotyping analyses were performed by polymerase chain reaction using 11 short tandem repeat markers along chromosome 21q. The vast majority of trisomy 21 was of maternal origin (93%), followed by paternal (5%) and mitotic origin (2%). The frequencies of maternal meiotic I (MI) and meiotic II errors were 86% and 14%, respectively. The highest proportion of cases with zero recombination was observed among those with maternal MI derived trisomy 21. A higher proportion of telomeric exchanges were presented in cases with maternal MI errors and cases with young mothers, although these findings were not statistically significant. The present study is the first report examining parental origin and altered genetic recombination as a risk factor for trisomy 21 in a Croatian population. The results support that trisomy 21 has a universal genetic etiology across different human populations.

MTHFR C677T and A1298C polymorphisms as a risk factor for congenital heart defects in Down syndrome.

BACKGROUND: Congenital heart defects (CHD) are present in most, but not all, cases of Down syndrome (DS). The presence of methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms has been reported as a risk factor for CHD in DS. The aims of the present study were to assess (i) the frequency of MTHFR C677T and A1298C polymorphisms in DS individuals in the Croatian population; (ii) the relationship between the two maternal MTHFR polymorphisms and CHD-affected DS children; and (iii) the transmission frequencies of the variant alleles of the two MTHFR polymorphisms in CHD-affected DS. METHODS: The study population included 112 DS subjects and 221 controls. CHD were present in 48% of the DS subjects (54/112). The mothers of 107 DS individuals were available for the study; none was a periconceptional folic acid user. Allele transmission was analyzed in 34 complete parent-offspring triads. RESULTS: The frequencies of the allele, individual, and combined genotypes of MTHFR C677T and A1298C in DS subjects were not statistically different compared to the normal healthy Croatian controls. The maternal MTHFR polymorphisms were not found to be a risk factor for DS-related CHD. The allele transmission of the two MTHFR polymorphisms showed no deviations from random segregation. CONCLUSIONS: Because the fetus is lost in a great proportion of trisomy 21 pregnancies, both maternal and fetal, not only live-born MTHFR C677T and A1298C, as well as maternal nutrition and lifestyle during pregnancy, should be analyzed to asses the impact on CHD in DS.