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Abstract Background APP duplication is a rare genetic cause of Alzheimer disease and cerebral amyloid angiopathy (CAA). We aimed to evaluate the phenotypes of APP duplications carriers. Methods Clinical, radiological, and neuropathological features of 43 APP duplication carriers from 24 French families were retrospectively analyzed, and MRI features and cerebrospinal fluid (CSF) biomarkers were compared to 40 APP-negative CAA controls. Results Major neurocognitive disorders were found in 90.2% symptomatic APP duplication carriers, with prominent behavioral impairment in 9.7%. Symptomatic intracerebral hemorrhages were reported in 29.2% and seizures in 51.2%. CSF Aβ42 levels were abnormal in 18/19 patients and 14/19 patients fulfilled MRI radiological criteria for CAA, while only 5 displayed no hemorrhagic features. We found no correlation between CAA radiological signs and duplication size. Compared to CAA controls, APP duplication carriers showed less disseminated cortical superficial siderosis (0% vs 37.5%, p = 0.004 adjusted for the delay between symptoms onset and MRI). Deep microbleeds were found in two APP duplication carriers. In addition to neurofibrillary tangles and senile plaques, CAA was diffuse and severe with thickening of leptomeningeal vessels in all 9 autopsies. Lewy bodies were found in substantia nigra, locus coeruleus, and cortical structures of 2/9 patients, and one presented vascular amyloid deposits in basal ganglia. Discussion Phenotypes associated with APP duplications were heterogeneous with different clinical presentations including dementia, hemorrhage, and seizure and different radiological presentations, even within families. No apparent correlation with duplication size was found. Amyloid burden was severe and widely extended to cerebral vessels as suggested by hemorrhagic features on MRI and neuropathological data, making APP duplication an interesting model of CAA.

Abstract Epidermal growth factor receptor (EGFR) amplification and EGFR variant III (EGFRvIII, deletion of exons 2–7) are of clinical interest for glioblastoma. The aim was to develop a digital PCR (dPCR)-based method using locked nucleic acid (LNA)-based hydrolysis probes, allowing the simultaneous detection of the EGFR amplification and EGFRvIII variant. Sixty-two patients were included. An exploratory cohort (n = 19) was used to develop the dPCR assay using three selected amplicons within the EGFR gene, targeting intron 1 (EGFR1), junction of exon 3 and intron 3 (EGFR2) and intron 22 (EGFR3). The copy number of EGFR was estimated by the relative quantification of EGFR1, EGFR2 and EGFR3 amplicon droplets compared to the droplets of a reference gene. EGFRvIII was identified by comparing the copy number of the EGFR2 amplicon to either the EGFR1 or EGFR3 amplicon. dPCR results were compared to fluorescence in situ hybridization (FISH) and next-generation sequencing for amplification; and to RT-PCR-based method for EGFRvIII. The dPCR assay was then tested in a validation cohort (n = 43). A total of 8/19 EGFR-amplified and 5/19 EGFRvIII-positive tumors were identified in the exploratory cohort. Compared to FISH, the EGFR3 dPCR assay detected all EGFR-amplified tumors (8/8, 100%) and had the highest concordance with the copy number estimation by NGS. The concordance between RT-PCR and dPCR was also 100% for detecting EGFRvIII using an absolute difference of 10.8 for the copy number between EGFR2 and EGFR3 probes. In the validation cohort, the sensitivity and specificity of dPCR using EGFR3 probes were 100% for the EGFR amplification detection compared to FISH (19/19). EGFRvIII was detected by dPCR in 8 EGFR-amplified patients and confirmed by RT-PCR. Compared to FISH, the EGFR2/EGFR3 dPCR assay was estimated with a one-half cost value. These results highlight that dPCR allowed the simultaneous detection of EGFR amplification and EGFRvIII for glioblastoma.

Abstract Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a strong genetic component whose knowledge evolves quickly. Next-generation sequencing is the only effective technology to deal with the high genetic heterogeneity of ASD in a clinical setting. However, rigorous criteria to classify rare genetic variants conferring ASD susceptibility are currently lacking. We have performed whole-exome sequencing to identify both nucleotide variants and copy number variants (CNVs) in 253 ASD patients, including 68 patients with intellectual disability (ID) and 90 diagnosed as Asperger syndrome. Using explicit criteria to classify both susceptibility genes and susceptibility variants we prioritized 217 genes belonging to the following categories: syndromic genes, genes with an excess of de novo protein truncating variants and genes targeted by rare CNVs. We obtained a susceptibility variant detection rate of 19.7% (95% CI: [15–25.2%]). The rate for CNVs was 7.1% (95% CI: [4.3–11%]) and 12.6% (95% CI: [8.8–17.4%]) for nucleotide variants. The highest rate (30.1%, 95% CI: [20.2–43.2%]) was obtained in the ASD + ID subgroup. A strong contributor for at risk nucleotide variants was the recently identified set of genes (n = 81) harboring an excess of de novo protein truncating variants. Since there is currently no evidence that the genes targeted here are necessary and sufficient to cause ASD, we recommend to avoid the term “causative of ASD” when delivering the information about a variant to a family and to use instead the term “genetic susceptibility factor contributing to ASD”.

Abstract While de novo variants (DNV) are overall at low risk of recurrence in subsequent pregnancies, a subset is at high risk due to parental mosaicism. Accurately identifying cases of parental mosaicism is therefore important for genetic counseling in clinical care. Some studies have investigated the rate of parental mosaics, but most were either limited by the sensitivity of the techniques (i.e. exome or genome sequencing), or focused on specific types of disease such as epileptic syndromes. This study aimed to determine the proportion of parental mosaicism among the DNV causing neurodevelopmental disorders (NDDs) in a series not enriched in epilepsy syndromes. We collected 189 patients with NDD-associated DNV. We applied a smMIP enrichment method and sequenced parental blood DNA samples to an average depth of 7000x. Power simulation indicated that mosaicism with an allelic fraction of 0.5% would have been detected for 87% of positions with 90% power. We observed seven parental mosaic variants (3.7% of families), of which four (2.1% of families) had an allelic fraction of less than 1%. In total, our study identifies a relatively low proportion of parental mosaicism in NDD-associated DNVs and raises the question of a biological mechanism behind the higher rates of parental mosaicism detected in other studies, particularly those focusing on epileptic syndromes.

Abstract Frontotemporal dementia (FTD) is a heterogeneous clinical disorder characterized by progressive abnormalities in behavior, executive functions, personality, language and/or motricity. A neuropathological subtype of FTD, frontotemporal lobar degeneration (FTLD)-FET, is characterized by protein aggregates consisting of the RNA-binding protein fused in sarcoma (FUS). The cause of FTLD-FET is not well understood and there is a lack of genetic evidence to aid in the investigation of mechanisms of the disease. The goal of this study was to identify genetic variants contributing to FTLD-FET and to investigate their effects on FUS pathology. We performed whole-exome sequencing on a 50-year-old FTLD patient with ubiquitin and FUS-positive neuronal inclusions and unaffected parents, and identified a de novo postzygotic nonsense variant in the NCDN gene encoding Neurochondrin (NCDN), NM_014284.3:c.1206G > A, p.(Trp402*). The variant was associated with a ~ 31% reduction in full-length protein levels in the patient’s brain, suggesting that this mutation leads to NCDN haploinsufficiency. We examined the effects of NCDN haploinsufficiency on FUS and found that depleting primary cortical neurons of NCDN causes a reduction in the total number of FUS-positive cytoplasmic granules. Moreover, we found that these granules were significantly larger and more highly enriched with FUS. We then examined the effects of a loss of FUS function on NCDN in neurons and found that depleting cells of FUS leads to a decrease in NCDN protein and mRNA levels. Our study identifies the NCDN protein as a likely contributor of FTLD-FET pathophysiology. Moreover, we provide evidence for a negative feedback loop of toxicity between NCDN and FUS, where loss of NCDN alters FUS cytoplasmic dynamics, which in turn has an impact on NCDN expression.

Abstract The prevalence of congenital hydrocephalus has been estimated at 1.1 per 1000 infants when including cases diagnosed before 1 year of age after exclusion of neural tube defects. Classification criteria are based either on CSF dynamics, pathophysiological mechanisms or associated lesions. Whereas inherited syndromic hydrocephalus has been associated with more than 100 disease-causing genes, only four genes are currently known to be linked to congenital hydrocephalus either isolated or as a major clinical feature: L1CAM, AP1S2, MPDZ and CCDC88C. In the past 10 years, pathogenic variants in CCDC88C have been documented but the neuropathology remains virtually unknown. We report the neuropathology of two foetuses from one family harbouring two novel compound heterozygous pathogenic variants in the CCDC88C gene: a maternally inherited indel in exon 22, c.3807_3809delinsACCT;p.(Gly1270Profs*53) and a paternally inherited deletion of exon 23, c.3967-?_c.4112-?;p.(Leu1323Argfs*10). Medical termination of pregnancy was performed at 18 and 23 weeks of gestation for severe bilateral ventriculomegaly. In both fetuses, brain lesions consisted of multifocal atresia-forking along the aqueduct of Sylvius and the central canal of the medulla, periventricular neuronal heterotopias and choroid plexus hydrops. The second fetus also presented lumbar myelomeningocele, left diaphragmatic hernia and bilateral renal agenesis. CCDC88C encodes the protein DAPLE which contributes to ependymal cell planar polarity by inhibiting the non-canonical Wnt signaling pathway and interacts with MPDZ and PARD3. Interestingly, heterozygous variants in PARD3 result in neural tube defects by defective tight junction formation and polarization process of the neuroepithelium. Besides, during organ formation Wnt signalling is a prerequisite for planar cell polarity pathway activation, and mutations in planar cell polarity genes lead to heart, lung and kidney malformations. Hence, candidate variants in CCDC88C should be carefully considered whether brain lesions are isolated or associated with malformations suspected to result from disorders of planar cell polarity.

Abstract Background Infantile hypercalcemia is an autosomal recessive disorder caused either by mutations in the CYP24A1 gene (20q13.2) or in the SLC34A1 gene (5q35.3). This disease is characterized by hypercalcemia, hypercalciuria and nephrocalcinosis in paediatric patients. Maternal uniparental disomy of chromosome 20 [UPD(20)mat], resulting in aberrant expression of imprinted transcripts at the GNAS locus, is a poorly characterized condition. UPD(20)mat patients manifest a phenotype similar to that of Silver-Russell syndrome and small for gestational age-short stature. Case presentation We report here the genetic and clinical characterization of a male child with a phenotype of infantile hypercalcemia, postnatal growth retardation, and minor dysmorphic features. Genetic analysis using a next generation sequencing panel revealed a homozygous pathogenic variant of CYP24A1. The absence of the variant in the father led to microsatellite segregation analysis, suggestive of UPD. SNP-array revealed a large terminal copy neutral loss of heterozygosity leading to CYP24A1 homozygosity. SNP-array data of parent–child trio confirmed a UPD(20)mat responsible for both infantile hypercalcemia and Silver-Russell syndrome-like traits. Conclusion This is the first report of uniparental disomy of chromosome 20 revealed by infantile hypercalcemia related to CYP24A1 biallelic homozygous variants, underlying the importance of controlling allelic segregation in cases of homozygosity.