Clinical features and genotype-phenotype correlations in epilepsy patients with de novo DYNC1H1 variants.

OBJECTIVE: DYNC1H1 variants are involved on a disease spectrum from neuromuscular disorders to neurodevelopmental disorders. DYNC1H1-related epilepsy has been reported in small cohorts. We dissect the electroclinical features of 34 patients harboring de novo DYNC1H1 pathogenic variants, identify subphenotypes on the DYNC1H1-related epilepsy spectrum, and compare the genotype-phenotype correlations observed in our cohort with the literature. METHODS: Patients harboring de novo DYNC1H1 pathogenic variants were recruited through international collaborations. Clinical data were retrospectively collected. Latent class analysis was performed to identify subphenotypes. Multivariable binary logistic regression analysis was applied to investigate the association with DYNC1H1 protein domains. RESULTS: DYNC1H1-related epilepsy presented with infantile epileptic spasms syndrome (IESS) in 17 subjects (50%), and in 25% of these individuals the epileptic phenotype evolved into Lennox-Gastaut syndrome (LGS). In 12 patients (35%), focal onset epilepsy was defined. In two patients, the epileptic phenotype consisted of generalized myoclonic epilepsy, with a progressive phenotype in one individual harboring a frameshift variant. In approximately 60% of our cohort, seizures were drug-resistant. Malformations of cortical development were noticed in 79% of our patients, mostly on the lissencephaly-pachygyria spectrum, particularly with posterior predominance in a half of them. Midline and infratentorial abnormalities were additionally reported in 45% and 27% of subjects. We have identified three main classes of subphenotypes on the DYNC1H1-related epilepsy spectrum. SIGNIFICANCE: We propose a classification in which pathogenic de novo DYNC1H1 variants feature drug-resistant IESS in half of cases with potential evolution to LGS (Class 1), developmental and epileptic encephalopathy other than IESS and LGS (Class 2), or less severe focal or genetic generalized epilepsy including a progressive phenotype (Class 3). We observed an association between stalk domain variants and Class 1 phenotypes. The variants p.Arg309His and p.Arg1962His were common and associated with Class 1 subphenotype in our cohort. These findings may aid genetic counseling of patients with DYNC1H1-related epilepsy.

Homozygous loss-of-function variants in FILIP1 cause autosomal recessive arthrogryposis multiplex congenita with microcephaly.

Arthrogryposis multiplex congenita forms a broad group of clinically and etiologically heterogeneous disorders characterized by congenital joint contractures that involve at least two different parts of the body. Neurological and muscular disorders are commonly underlying arthrogryposis. Here, we report five affected individuals from three independent families sharing an overlapping phenotype with congenital contractures affecting shoulder, elbow, hand, hip, knee and foot as well as scoliosis, reduced palmar and plantar skin folds, microcephaly and facial dysmorphism. Using exome sequencing, we identified homozygous truncating variants in FILIP1 in all patients. FILIP1 is a regulator of filamin homeostasis required for the initiation of cortical cell migration in the developing neocortex and essential for the differentiation process of cross-striated muscle cells during myogenesis. In summary, our data indicate that bi-allelic truncating variants in FILIP1 are causative of a novel autosomal recessive disorder and expand the spectrum of genetic factors causative of arthrogryposis multiplex congenita.

New insights into the clinical and molecular spectrum of the novel CYFIP2-related neurodevelopmental disorder and impairment of the WRC-mediated actin dynamics.

PURPOSE: A few de novo missense variants in the cytoplasmic FMRP-interacting protein 2 (CYFIP2) gene have recently been described as a novel cause of severe intellectual disability, seizures, and hypotonia in 18 individuals, with p.Arg87 substitutions in the majority. METHODS: We assembled data from 19 newly identified and all 18 previously published individuals with CYFIP2 variants. By structural modeling and investigation of WAVE-regulatory complex (WRC)-mediated actin polymerization in six patient fibroblast lines we assessed the impact of CYFIP2 variants on the WRC. RESULTS: Sixteen of 19 individuals harbor two previously described and 11 novel (likely) disease-associated missense variants. We report p.Asp724 as second mutational hotspot (4/19 cases). Genotype-phenotype correlation confirms a consistently severe phenotype in p.Arg87 patients but a more variable phenotype in p.Asp724 and other substitutions. Three individuals with milder phenotypes carry putative loss-of-function variants, which remain of unclear pathogenicity. Structural modeling predicted missense variants to disturb interactions within the WRC or impair CYFIP2 stability. Consistent with its role in WRC-mediated actin polymerization we substantiate aberrant regulation of the actin cytoskeleton in patient fibroblasts. CONCLUSION: Our study expands the clinical and molecular spectrum of CYFIP2-related neurodevelopmental disorder and provides evidence for aberrant WRC-mediated actin dynamics as contributing cellular pathomechanism.

Heterogeneous clinical and functional features of GRIN2D-related developmental and epileptic encephalopathy.

N-methyl d-aspartate receptors are ligand-gated ionotropic receptors mediating a slow, calcium-permeable component of excitatory synaptic transmission in the CNS. Variants in genes encoding NMDAR subunits have been associated with a spectrum of neurodevelopmental disorders. Here we report six novel GRIN2D variants and one previously-described disease-associated GRIN2D variant in two patients with developmental and epileptic encephalopathy. GRIN2D encodes for the GluN2D subunit protein; the GluN2D amino acids affected by the variants in this report are located in the pre-M1 helix, transmembrane domain M3, and the intracellular carboxyl terminal domain. Functional analysis in vitro reveals that all six variants decreased receptor surface expression, which may underline some shared clinical symptoms. In addition the GluN2D(Leu670Phe), (Ala675Thr) and (Ala678Asp) substitutions confer significantly enhanced agonist potency, and/or increased channel open probability, while the GluN2D(Ser573Phe), (Ser1271Phe) and (Arg1313Trp) substitutions result in a mild increase of agonist potency, reduced sensitivity to endogenous protons, and decreased channel open probability. The GluN2D(Ser573Phe), (Ala675Thr), and (Ala678Asp) substitutions significantly decrease current amplitude, consistent with reduced surface expression. The GluN2D(Leu670Phe) variant slows current response deactivation time course and increased charge transfer. GluN2D(Ala678Asp) transfection significantly decreased cell viability of rat cultured cortical neurons. In addition, we evaluated a set of FDA-approved NMDAR channel blockers to rescue functional changes of mutant receptors. This work suggests the complexity of the pathological mechanisms of GRIN2D-mediated developmental and epileptic encephalopathy, as well as the potential benefit of precision medicine.

De novo variants in GABRA2 and GABRA5 alter receptor function and contribute to early-onset epilepsy.

GABAA receptors are ligand-gated anion channels that are important regulators of neuronal inhibition. Mutations in several genes encoding receptor subunits have been identified in patients with various types of epilepsy, ranging from mild febrile seizures to severe epileptic encephalopathy. Using whole-genome sequencing, we identified a novel de novo missense variant in GABRA5 (c.880G > C, p.V294L) in a patient with severe early-onset epilepsy and developmental delay. Targeted resequencing of 279 additional epilepsy patients identified 19 rare variants from nine GABAA receptor genes, including a novel de novo missense variant in GABRA2 (c.875C > A, p.T292K) and a recurrent missense variant in GABRB3 (c.902C > T, p.P301L). Patients with the GABRA2 and GABRB3 variants also presented with severe epilepsy and developmental delay. We evaluated the effects of the GABRA5, GABRA2 and GABRB3 missense variants on receptor function using whole-cell patch-clamp recordings from human embryonic kidney 293T cells expressing appropriate α, ß and γ subunits. The GABRA5 p.V294L variant produced receptors that were 10-times more sensitive to GABA but had reduced maximal GABA-evoked current due to increased receptor desensitization. The GABRA2 p.T292K variant reduced channel expression and produced mutant channels that were tonically open, even in the absence of GABA. Receptors containing the GABRB3 p.P301L variant were less sensitive to GABA and produced less GABA-evoked current. These results provide the first functional evidence that de novo variants in the GABRA5 and GABRA2 genes contribute to early-onset epilepsy and developmental delay, and demonstrate that epilepsy can result from reduced neuronal inhibition via a wide range of alterations in GABAA receptor function.

Introducing a Specific Term to Present Caries Experience in Populations with Low Caries Prevalence: Specific Affected Caries Index (SaC).

Up to now, indices like the mean dmft/DMFT and the SiC (Significant Caries Index) have been used to depict caries experience in populations with high prevalence. With the caries decline, particularly for populations with low caries levels, these indices reach their statistical limits. This paper aims to introduce a specific term, the Specific affected Caries Index (SaC) for the risk groups in populations with low caries prevalence and to illustrate its use based on the consecutive German National Oral Health Survey (GNOHS) in children. In groups with a caries prevalence less than one-third of the population, many caries-free children (DMFT = 0) are included in the SiC (risk group), which calls for a new way of illustration. Mean caries experience (DMFT), caries prevalence, the SiC and SaC were portrayed for 12-year-olds in the GNOHS from 1994/95 to 2016. The SaC describes the mean caries experience (DMFT) in the group presenting caries experience (DMFT > 0). In 12-year-old 6th graders in Germany, the mean caries experience decreased from 2.4 (1994/95) to 0.4 DMFT (2016), with a recent prevalence of 21.2% (DMFT > 0, 2016). In 2016, the mean number of affected teeth in children with DMFT > 0 (SaC) was 2.1, while the SiC including 12% DMFT-free children in the risk group was 1.3. The SiC fails to reflect the caries severity in children in a population with low caries prevalence. Therefore, the newly introduced term Specific affected Caries Index (SaC) may be used to describe accurately caries experience in caries risk children in populations presenting low caries prevalence.

Caries Trends in the Primary Dentition of 6- to 7-Year-old Schoolchildren in Germany from 1994 to 2016: Results from the German National Oral Health Surveys in Children.

Long-term nationally representative caries data in the primary dentition are rare, but nonetheless central to assess needs in caries prevention and treatment. This study evaluated the prevalence and trends of caries levels in the primary dentition of 6- to 7-year-olds in Germany as a whole and its federal states individually. In 2016, employing a randomized cluster selection, 6- to 7-year-old first graders were included in the National German Oral Health Survey performed regularly since 1994/95. Children were examined by 482 calibrated dentists in all 17 German regions using the WHO criteria for the decayed, missing, and filled teeth (dmft) including the assessment of initial carious lesions (it). In total, 151,555 6- to 7-year-olds were examined. Caries prevalence in the primary dentition dropped from 65% in 1994 to 44% in 2016, while the mean caries experience dropped from 2.89 to 1.73 dmft (dt = 0.74, mt = 0.19, ft = 0.80). When initial lesions were included, the mean caries experience increased to idmft = 2.12 (it = 0.38). In 2016, 49.7% of the examined 6- to 7-year-olds were caries-free including initial lesions. The Care Index at the tooth level was 57.5%, and the Significant Caries Index was 4.84 dmft. Depending on the German region, the mean dmft varied considerably, ranging from 1.37 to 2.31. In conclusion, despite the overall caries decline in 6- to 7-year-olds in Germany, only minor caries reductions were observed over the last decade, with a still existing high proportion of untreated dental decay. This calls for more effective preventive and restorative efforts with focus on the primary dentition in Germany.

40-Year Longitudinal Caries Development in German Adolescents in the Light of New Caries Measures.

This study assessed the 40-year longitudinal caries development in German adolescents in the light of the sixth National Oral Health Survey in Children (NOHSC, 2016) employing initial DMFT (IDMFT), Significant Caries Index (SiC) and Specific Affected Caries Index (SaC). On the basis of the current NOHSC (randomized cluster selection using school list or regional community school surveys, 55,956 12-year-old sixth-graders examined by 482 calibrated community/study dentists) DMFT, SiC, a novel IDMFT including initial lesions (IT) and the recently introduced SaC were calculated and also recalculated for national and international surveys from the last 4 decades. In 2016, 78.8% of children were caries-free (DMFT = 0), 65.5% including IT lesions. The mean DMFT was 0.44 (single components: DT = 0.14, MT = 0.02, FT = 0.29, IT = 0.52) showing a clear association with the school type as marker for the socio-economic status. The mean number of affected teeth in children with DMFT >0 was 2.07 (SaC) in comparison to almost 9 teeth in the 1970s. The current care index on the tooth level was 66.3%, leaving only 7.7% of children with restorative treatment needs. Longitudinally, a continuous caries decline of more than 80%, including the risk groups (SiC/SaC), to an internationally extremely low level was observed. In conclusion, the National Oral Health Surveys reveal a continuous caries decline to a very low caries level in 12-year-old 6th-graders in Germany even if IT lesions are included (IDMFT). In spite of proportional reductions in the risk groups (SiC/SaC), the polarized caries distribution according to socio-economic parameters reveals the need for targeted preventive programmes.

The German 19-item version of the Child Oral Health Impact Profile: translation and psychometric properties.

OBJECTIVES: This study seeks to develop and validate the 19-item German version of the Child Oral Health Impact Profile (COHIP-G19), an instrument to assess the oral health-related quality of life (OHRQoL) in children and adolescents. MATERIALS AND METHODS: The 19 items of the original English-language COHIP were translated into German using an established forward-backward approach. For the assessment of the psychometric properties of the COHIP-G19, children and adolescents aged 7-17 years came from two samples: 112 patients were consecutively recruited at a university-based orthodontic clinic and 313 came from a convenience sample of students in public schools. RESULTS: Internal consistency of the COHIP-G19 was satisfactory in both populations (Cronbach's alpha, 0.78/0.80; average inter-item correlation, 0.16/0.17). The COHIP-G19 summary scores were correlated in the expected direction with a global oral health rating (r = 0.46/0.40) and two measures for perceived general health (EQ-5D-Y: r = 0.26/0.29; KIDSCREEN-27: r = 0.40/0.33). While COHIP-G19 summary scores did not significantly differ with respect to the presence of caries or gingivitis (p > 0.05), malocclusion and insufficient oral hygiene behavior were related to more impaired OHRQoL, represented in significantly lower COHIP-G19 summary scores in students in public schools (p < 0.05), but not in orthodontic patients. CONCLUSIONS: While this study revealed some potential to improve reliability and validity in scores of the German version of the COHIP-19, overall, the study proved the instrument has sufficient psychometric properties and is well comparable to the original English-language version. CLINICAL RELEVANCE: The COHIP-G19 is a valid and reliable instrument to assess OHRQoL in German children and adolescents in clinical and community settings.

Inequalities in dental caries experience among 6-year-old German children after the caries decline.

OBJECTIVE: As in many other countries, caries decline in Germany has left pockets of persisting caries prevalence. This study aims to assess the benefit of a 10-year community-based prophylaxis program, focused on regular toothbrushing with fluoridated toothpastes or gels and involving institutions noted as having the highest caries levels. MATERIALS AND METHODS: The caries data (d3mft/D3MFT) was extracted from the results of the compulsory school entry examinations in Greifswald/Germany (2003/2004-2012/2013) involving ∼280 6-7-year-olds each year. Data from schools that include children with the highest caries levels and coming from low-SES families were analyzed independently and used for comparisons. Additionally, caries trends from Greifswald were compared to data from representative national surveys (2004-2009). RESULTS: Data from 2871 children were available for analysis. The baseline d3mft value (2003/2004) was 3.2±3.8; the d3-component corresponded to 70% of the index. The latest caries data (2012/2013) showed a strong reduction (43.8%) in caries prevalence (d3mft=1.8±2.5). Similarly, the SiC-Index declined significantly from 2003/2004 (7.9±2.3) to 2012/2013 (4.8±2.3; p<0.001). Nevertheless, in all analyzed years the d3mft values and the SiC-Index were significantly higher in the institutions that included children coming from lower-SES families (p<0.05). The amount of caries reduction between 2004 and 2009 corresponded to 38% in Greifswald as compared to 13% in Germany. CONCLUSIONS: This strategy involving a combination of regular toothbrushing and fluoride application has achieved an overall substantial caries reduction, thereby indicating that caries-control strategies for heterogeneous risk groups can be highly successful as setting approach. However, activities targeting high risk groups still need to be strengthened.

Clinical spectrum and treatment outcome of 95 children with continuous spikes and waves during sleep (CSWS).

OBJECTIVE: Continuous spikes and waves during sleep (CSWS) is an epileptic encephalopathy characterized by generalised epileptiform activity and neurocognitive dysfunction. Causes and outcome are diverse and treatment is mainly empirical. METHODS: Retrospective descriptive analysis of clinical and EEG data of children with CSWS diagnosed between 1998 and 2018 at the University Hospital Heidelberg. RESULTS: Ninety-five children were included with a median age at diagnosis of 5.4 years. A structural/metabolic aetiology was found in 43.2%, genetic alterations in 17.9%, while it remained unknown in 38.9%. The proportion of patients with genetic aetiology increased from 10.3% (1998-2007) to 22.8% (2008-2018). On average, each patient received 5 different treatments. CSWS was refractory in >70% of cases, steroids and neurosurgery were most effective. No difference was observed between children with CSWS or Near-CSWS (Spike-Wave-Index 40-85%). CONCLUSIONS: Our cohort confirms CSWS as an age-dependent epileptic encephalopathy. Structural brain abnormalities were most frequent, but genetic causes are increasingly identified. More specific criteria for the diagnosis and treatment goals should be elaborated and implemented based on evidence. SIGNIFICANCE: This study is the largest monocentric observational study on treatment effects in children with CSWS, providing data for diagnostic and therapeutic decisions.

Long-term seizure outcome in pediatric patients with focal cortical dysplasia undergoing tailored and standard surgical resections.

PURPOSE: Focal cortical dysplasia (FCD) is the major cause of focal intractable epilepsy in childhood. Here we analyze the factors influencing the success of surgical treatment in a large cohort of children with histologically ascertained FCD. METHOD: A retrospective study of the effects of FCD type, surgical intervention, and age at surgery in a pediatric cohort. RESULTS: A total of 113 patients (71 male; mean age at surgery 10.3 years; range 0-18) were analyzed; 45 had undergone lesionectomy, 42 lobectomy, 18 multi-lobectomy, and eight hemispherotomy. Complete seizure control (Engel Ia) was achieved in 56% after two years, 52% at five years, and 50% at last follow-up (18-204 months). Resections were more extensive in younger patients (40% of the surgeries affecting more than one lobe in patients aged nine years or younger vs. 22% in patients older than nine years). While resections were more limited in older children, their long-term outcome tended to be superior (42% seizure freedom in patients aged nine years or younger vs. 56% in patients older than nine years). The outcome in FCD I was not significantly inferior to that in FCD II. CONCLUSIONS: Our data confirm the long-term efficacy of surgery in children with FCD and epilepsy. An earlier age at surgery within this cohort did not predict a better long-term outcome, but it involved less-tailored surgical approaches. The data suggest that in patients with an unclear extent of the dysplastic area, later resections may offer advantages in terms of the precision of surgical-resection planning.

Diagnosis of peritonitis using near-infrared optical imaging of in vivo labeled monocytes-macrophages.

Peritonitis is an inflammatory process characterized by massive monocytes-macrophages infiltration. Since early diagnosis is important for a successful therapeutic outcome, the feasibility for a selective labeling and imaging of macrophages for highly sensitive optical imaging was assessed. After in vitro incubation of mouse macrophages J774A.1 with the far-red/near-infrared fluorochrome DY-676, distinct fluorescence intensities (1026+/-142 a.u.) were detected as compared to controls (552+/-54 a.u.) using a whole-body small animal near-infrared fluorescence (NIRF) imaging system. Macrophage labeling was confirmed by confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting, (FACS). The fluorochrome was also found to be predominantly distributed within compartments in the cytoplasm. Additionally, peritonitis was induced in mice by intraperitoneal injection of zymosanA. After intravenous injection of fluorochrome (55 nmol/kg) and using whole-body fluorescence imaging, higher fluorescence intensities (869+/-151 a.u.) were detected in the peritoneal area of diseased mice as compared to controls (188+/-41 a.u.). Furthermore, cells isolated from peritoneal lavage revealed the presence of labeled monocytes-macrophages. The results indicate that in vivo diagnosis of peritonitis by near-infrared optical imaging of labeled monocytes-macrophages is feasible. Possibly, early stages of other inflammatory diseases could also be detected by the proposed diagnostic method in the long term.

Design and synthesis of two pyrazole libraries based on o-hydroxyacetophenones.

Two new solid-phase syntheses of substituted pyrazoles are described. The first includes supporting an o-hydroxyacetophenone on Merrifield resin, Vilsmeier-Haack formylation on the methyl group and cyclization with a substituted hydrazine to afford a pyrazole ring with two diversity centers. The second starts from o-hydroxyacetophenone supported on Wang resin, which undergoes a Claisen condensation with a carboxylic acid ester to yield a 1,3-dicarbonyl compound that cyclizes to a pyrazole using a hydrazine. Both methods have been used to synthesize two small pyrazole libraries.