Variants of uncertain significance in the era of next-generation sequencing.

ABSTRACT: Next-generation sequencing (NGS) is now widely used in diagnosing rare diseases. However, it has some limitations, such as variants of uncertain significance (VUS). This can present difficulties even for nurse practitioners involved in clinical genetics. We present three cases from our clinical practice: two targeted panel testing and one exome sequencing. Whole blood samples were collected and sent for NGS analysis. In case 1, a VUS was found in the LITAF gene, which is associated with autosomal dominant Charcot-Marie-Tooth disease type 1C. In case 2, a VUS was reported in the MEFV gene, which is associated with autosomal recessive and autosomal dominant familial Mediterranean fever. In these cases, the reported VUS corresponded to the clinical diagnosis. In case 3, two variants in the heterozygous state were found in the ATP7B gene, which is associated with Wilson disease, and the disorder was later clinically recognized. According to the published guidelines, VUSs should not be discussed as a cause for an observed genetic condition. Nevertheless, if the reported variant is in a gene associated with the clinically diagnosed disorder, and there is a strong genotype-phenotype correlation, it could be suggestive of the etiological role of this variant.

Molecular screening for fragile X syndrome in children with unexplained intellectual disability and/or autistic behaviour.

INTRODUCTION: Fragile X syndrome (FXS, OMIM #300624) is the most common inherited form of intellectual disability and the leading monogenic cause of autism.

16p11.2 Duplication Syndrome - a Case Report.

16p11.2 duplication syndrome is a rare disorder, often associated with intellectual disability, attention deficit, hyperactivity disorder, and a predisposition to epilepsy and schizophrenia. There are no specific dysmorphic features for this genetic condition, but micro-cephaly, micrognathia and hypertelorism could be present. We report a case of 16p11.2 duplication syndrome which has the typical clinical presentation - slight facial dysmorphism, impaired intellectual development, and autistic behavior. Whole-exome sequencing was performed, but no pathogenic or likely pathogenic mutations were identified. Array comparative genomic hybridization analysis established the diagnosis of 16p11.2 duplication syndrome, which illustrates the importance of this method when diagnosing children with unexplained intellectual disability.

The clinical significance of A2ML1 variants in Noonan syndrome has to be reconsidered.

The RASopathies are a group of clinically and genetically heterogeneous developmental disorders caused by dysregulation of the RAS/MAPK signalling pathway. Variants in several components and regulators of this pathway have been identified as the pathogenetic cause. In 2015, missense variants in A2ML1 were reported in three unrelated families with clinical diagnosis of Noonan syndrome (NS) and a zebrafish model was presented showing heart and craniofacial defects similar to those caused by a NS-associated Shp2 variant. However, a causal role of A2ML1 variants in NS has not been confirmed since. Herein, we report on 15 individuals who underwent screening of RASopathy-associated genes and were found to carry rare variants in A2ML1, including variants previously proposed to be causative for NS. In cases where parental DNA was available, the respective A2ML1 variant was found to be inherited from an unaffected parent. Seven index patients carrying an A2ML1 variant presented with an alternate disease-causing genetic aberration. These findings underscore that current evidence is insufficient to support a causal relation between variants in A2ML1 and NS, questioning the inclusion of A2ML1 screening in diagnostic RASopathy testing.

Comparison between thrombophilic gene polymorphisms among high risk patients.

INTRODUCTION: The purpose of this study was to compare the role of the thrombophilic variants among two groups of high risk patients with vascular disorders and recurrent pregnancy loss. METHODS: 200 patients, including 76 with thrombotic accidents and 124 with two or more idiopathic recurrent miscarriage during the first trimester, were tested for the presence of Factor V (F V) Leiden G1691A, Factor II (F II) G20210A, plasminogen activator inhibitor (PAI) 4G/5G, and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms using Real time polymerase chain reaction (RT - PCR) in the Laboratory of Medical Genetics, Varna, Bulgaria between June 2016 and May 2019. Frequencies of thrombophilic gene polymorphisms were compared among the two populations and to the expected genotype frequencies. RESULTS: Individuals with a history of vascular disorders had a significantly higher frequency of F V Leiden variant compared to women with recurrent miscariage. There was no statistical difference between the analyzed patients for the other three thrombophilic polymorphisms. The allelic frequencies and the expected genotype frequencies of the F V, F II and MTHFR polymorphisms were calculated according to Hardy-Weinberg equilibrium. The percentages of the homozygotes for F V and F II were higher than expected in the two groups of patients. For the MTHFR there was no difference. CONCLUSION: F V Leiden remains the strongest risk factor for vascular disorders and recurrent pregnancy loss. Screening for this variant should be recommended to patients with thrombotic accidents and women with repeated miscarriage. The role of F II, PAI and MTHFR remains controversial.

Cognitive behavioral therapy for panic disorder and comorbidity: more of the same or less of more?

This study compared the effects of a higher dose of cognitive behavioral therapy (CBT) for panic disorder versus CBT for panic disorder combined with "straying" to CBT for comorbid disorders in individuals with a principal diagnosis of panic disorder with or without agoraphobia. Sixty-five participants were randomly assigned to one of two treatment conditions, either CBT focused solely upon panic disorder and agoraphobia or CBT that simultaneously addressed panic disorder and agoraphobia and, to a lesser degree, the most severe comorbid condition. Results indicated a significant reduction in panic disorder severity and a decline in severity of comorbid diagnoses across both treatment conditions. However, individuals receiving CBT focused only on panic disorder were more likely to meet high end-state functioning at post-treatment, even in intent-to-treat analyses, and report zero panic attacks at the 1-year follow-up, although this effect was not retained in intent-to-treat analyses. At follow-up, CBT focused only on panic disorder yielded more substantial improvement in the most severe baseline comorbid condition, although not in intent-to-treat analyses, and a greater proportion of individuals in this treatment condition were rated as having no comorbid diagnoses, even in intent-to-treat analyses. These findings raise the possibility that remaining focused on CBT for panic disorder may be more beneficial for both principal and comorbid diagnoses than combining CBT for panic disorder with 'straying' to CBT for comorbid disorders.

Gender, gender roles, and anxiety: perceived confirmability of self report, behavioral avoidance, and physiological reactivity.

Despite the well-documented gender effect in anxiety, less is known about contributing factors to women's greater risk for anxiety and fears. The present study examined the relationship between gender, gender role orientation (i.e., expressivity/instrumentality) and fear of harmless insects (tarantula), using a multimodal approach of self-report measures, a Behavioral Approach Test (BAT), and physiological reactivity. Participants (144 college students; 67 women, 77 men) completed a questionnaire packet and then were instructed to approach a tarantula. We were unable to replicate Pierce and Kirkpatrick's (1992) findings that men underreport anxiety. Consistent with the literature, women in the study experienced greater anxiety and avoidance compared to men. However, men and women did not differ on physiological reactivity during the first 2 min of the BAT. The concordance across avoidance, anxiety and heart rate reactivity differed by gender, suggesting that men and women have different experiences when faced with a fearful object. Furthermore, instrumentality (masculinity) was negatively related to anticipatory anxiety for women but not for men.

Emotional variability and sustained arousal during exposure.

BACKGROUND AND OBJECTIVES: In traditional exposure therapy for phobias and anxiety disorders, reduction of fear responding is used as an index of learning. However, recent evidence in animal models suggests that sustained arousal and enhanced fear responding throughout exposure may actually predict better long-term outcomes (Rescorla, 2000). METHODS: The effects of sustained arousal during exposure were investigated in a clinical analog sample of 59 participants fearful of public speaking. Participants completed exposure with or without the presence of additional excitatory stimuli which were intended to enhance arousal and fear responding throughout exposure. RESULTS: Group assignment (exposure versus exposure with additional excitatory stimuli) did not significantly predict outcome at 1-week follow-up testing, as measured physiologically, subjectively, and behaviorally. A set of regression analyses investigating whether any exposure process measures predicted outcome indicated that sustained arousal throughout exposure as well as variability in subjective fear responding throughout exposure (e.g., Kircanski et al., 2011) predicted lower levels of fear at follow-up testing (p < 0.05; p < 0.001) after controlling for demographic variables as well as pre-exposure fear levels. LIMITATIONS: The excitatory stimuli used failed to produce the intended effects. However, some participants did maintain elevated arousal throughout exposure and this predicted better outcomes at 1-week follow-up testing. CONCLUSIONS: Sustained arousal throughout exposure as well as variability in subjective fear responding during exposure may be better predictors of long-term outcomes than habituation of fear across exposure.

Clinical relevance of retrieval cues for attenuating context renewal of fear.

The present studies investigated if retrieval cues (reminder objects) can attenuate context renewal of fear. In Study 1, 32 participants completed exposure in one of two contexts; 1-week follow-up testing occurred in a novel or the same context. Results indicated significant renewal of fear for those tested in a novel context. In Study 2, 40 participants completed exposure in one of these contexts; half were presented with cues. One week later, all were tested in a novel context with or without cues. Results indicated weak attenuation of context renewal for participants re-presented with cues. In Study 3, 18 participants completed exposure in one of two maximally distinct contexts; all with cues. One week later, participants were tested in a novel context with or without cues. Results indicated no group differences. These findings suggest that clinical relevance of this set of cues for attenuating context renewal may be limited.