Upstream open reading frame-introducing variants in patients with primary familial brain calcification.

More than 50% of patients with primary familial brain calcification (PFBC), a rare neurological disorder, remain genetically unexplained. While some causative genes are yet to be identified, variants in non-coding regions of known genes may represent a source of missed diagnoses. We hypothesized that 5'-Untranslated Region (UTR) variants introducing an AUG codon may initiate mRNA translation and result in a loss of function in some of the PFBC genes. After reannotation of exome sequencing data of 113 unrelated PFBC probands, we identified two upstream AUG-introducing variants in the 5'UTR of PDGFB. One, NM_002608.4:c.-373C>G, segregated with PFBC in the family. It was predicted to create an upstream open reading frame (ORF). The other one, NM_002608.4:c.-318C>T, was found in a simplex case. It was predicted to result in an ORF overlapping the natural ORF with a frameshift. In a GFP reporter assay, both variants were associated with a dramatic decrease in GFP levels, and, after restoring the reading frame with the GFP sequence, the c.-318C>T variant was associated with a strong initiation of translation as measured by western blotting. Overall, we found upstream AUG-introducing variants in the 5'UTR of PDGFB in 2/113 (1.7%) undiagnosed PFBC cases. Such variants thus represent a source of putative pathogenic variants.

Biallelic NAA60 variants with impaired n-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications.

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning.

Assessment of Mendelian and risk-factor genes in Alzheimer disease: A prospective nationwide clinical utility study and recommendations for genetic screening.

PURPOSE: To assess the likely pathogenic/pathogenic (LP/P) variants rates in Mendelian dementia genes and the moderate-to-strong risk factors rates in patients with Alzheimer disease (AD). METHODS: We included 700 patients in a prospective study and performed exome sequencing. A panel of 28 Mendelian and 6 risk-factor genes was interpreted and returned to patients. We built a framework for risk variant interpretation and risk gradation and assessed the detection rates among early-onset AD (EOAD, age of onset (AOO) ≤65 years, n = 608) depending on AOO and pedigree structure and late-onset AD (66 < AOO < 75, n = 92). RESULTS: Twenty-one patients carried a LP/P variant in a Mendelian gene (all with EOAD, 3.4%), 20 of 21 affected APP, PSEN1, or PSEN2. LP/P variant detection rates in EOAD ranged from 1.7% to 11.6% based on AOO and pedigree structure. Risk factors were found in 69.5% of the remaining 679 patients, including 83 (12.2%) being heterozygotes for rare risk variants, in decreasing order of frequency, in TREM2, ABCA7, ATP8B4, SORL1, and ABCA1, including 5 heterozygotes for multiple rare risk variants, suggesting non-monogenic inheritance, even in some autosomal-dominant-like pedigrees. CONCLUSION: We suggest that genetic screening should be proposed to all EOAD patients and should no longer be prioritized based on pedigree structure.

High diagnostic potential of short and long read genome sequencing with transcriptome analysis in exome-negative developmental disorders.

Exome sequencing (ES) has become the method of choice for diagnosing rare diseases, while the availability of short-read genome sequencing (SR-GS) in a medical setting is increasing. In addition, new sequencing technologies, such as long-read genome sequencing (LR-GS) and transcriptome sequencing, are being increasingly used. However, the contribution of these techniques compared to widely used ES is not well established, particularly in regards to the analysis of non-coding regions. In a pilot study of five probands affected by an undiagnosed neurodevelopmental disorder, we performed trio-based short-read GS and long-read GS as well as case-only peripheral blood transcriptome sequencing. We identified three new genetic diagnoses, none of which affected the coding regions. More specifically, LR-GS identified a balanced inversion in NSD1, highlighting a rare mechanism of Sotos syndrome. SR-GS identified a homozygous deep intronic variant of KLHL7 resulting in a neoexon inclusion, and a de novo mosaic intronic 22-bp deletion in KMT2D, leading to the diagnosis of Perching and Kabuki syndromes, respectively. All three variants had a significant effect on the transcriptome, which showed decreased gene expression, mono-allelic expression and splicing defects, respectively, further validating the effect of these variants. Overall, in undiagnosed patients, the combination of short and long read GS allowed the detection of cryptic variations not or barely detectable by ES, making it a highly sensitive method at the cost of more complex bioinformatics approaches. Transcriptome sequencing is a valuable complement for the functional validation of variations, particularly in the non-coding genome.

Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as risk factors for Alzheimer's disease.

Alzheimer's disease (AD), the leading cause of dementia, has an estimated heritability of approximately 70%1. The genetic component of AD has been mainly assessed using genome-wide association studies, which do not capture the risk contributed by rare variants2. Here, we compared the gene-based burden of rare damaging variants in exome sequencing data from 32,558 individuals-16,036 AD cases and 16,522 controls. Next to variants in TREM2, SORL1 and ABCA7, we observed a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk, and a suggestive signal in ADAM10. Additionally, the rare-variant burden in RIN3, CLU, ZCWPW1 and ACE highlighted these genes as potential drivers of respective AD-genome-wide association study loci. Variants associated with the strongest effect on AD risk, in particular loss-of-function variants, are enriched in early-onset AD cases. Our results provide additional evidence for a major role for amyloid-ß precursor protein processing, amyloid-ß aggregation, lipid metabolism and microglial function in AD.

SPiP: Splicing Prediction Pipeline, a machine learning tool for massive detection of exonic and intronic variant effects on mRNA splicing.

Modeling splicing is essential for tackling the challenge of variant interpretation as each nucleotide variation can be pathogenic by affecting pre-mRNA splicing via disruption/creation of splicing motifs such as 5'/3' splice sites, branch sites, or splicing regulatory elements. Unfortunately, most in silico tools focus on a specific type of splicing motif, which is why we developed the Splicing Prediction Pipeline (SPiP) to perform, in one single bioinformatic analysis based on a machine learning approach, a comprehensive assessment of the variant effect on different splicing motifs. We gathered a curated set of 4616 variants scattered all along the sequence of 227 genes, with their corresponding splicing studies. The Bayesian analysis provided us with the number of control variants, that is, variants without impact on splicing, to mimic the deluge of variants from high-throughput sequencing data. Results show that SPiP can deal with the diversity of splicing alterations, with 83.13% sensitivity and 99% specificity to detect spliceogenic variants. Overall performance as measured by area under the receiving operator curve was 0.986, better than SpliceAI and SQUIRLS (0.965 and 0.766) for the same data set. SPiP lends itself to a unique suite for comprehensive prediction of spliceogenicity in the genomic medicine era. SPiP is available at: https://sourceforge.net/projects/splicing-prediction-pipeline/.

Deep intronic NIPBL de novo mutations and differential diagnoses revealed by whole genome and RNA sequencing in Cornelia de Lange syndrome patients.

Cornelia de Lange syndrome (CdLS; MIM# 122470) is a rare developmental disorder. Pathogenic variants in 5 genes explain approximately 50% cases, leaving the other 50% unsolved. We performed whole genome sequencing (WGS) ± RNA sequencing (RNA-seq) in 5 unsolved trios fulfilling the following criteria: (i) clinical diagnosis of classic CdLS, (ii) negative gene panel sequencing from blood and saliva-isolated DNA, (iii) unaffected parents' DNA samples available and (iv) proband's blood-isolated RNA available. A pathogenic de novo mutation (DNM) was observed in a CdLS differential diagnosis gene in 3/5 patients, namely POU3F3, SPEN, and TAF1. In the other two, we identified two distinct deep intronic DNM in NIPBL predicted to create a novel splice site. RT-PCRs and RNA-Seq showed aberrant transcripts leading to the creation of a novel frameshift exon. Our findings suggest the relevance of WGS in unsolved suspected CdLS cases and that deep intronic variants may account for a proportion of them.

uORF-introducing variants in the 5'UTR of the NIPBL gene as a cause of Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS) is a clinically-recognizable rare developmental disorder. About 70% of patients carry a missense or loss-of-function pathogenic variant in the NIPBL gene. We hypothesized that some variants in the 5'-untranslated region (UTR) of NIPBL may create an upstream open reading frame (uORF), putatively leading to a loss of function. We searched for NIPBL 5'-UTR variants potentially introducing uORF by (i) reannotating NGS data of 102 unsolved CdLS patients and (ii) literature and variant databases search. We set up a green fluorescent protein (GFP) reporter assay and studied NIPBL expression in a lymphoblastoid cell line (LCL). We identified two variants introducing a novel ATG codon sequence in the 5'-UTR of NIPBL, both predicted to introduce uORF: a novel c.-457_-456delinsAT de novo mutation in a 15-year-old male with classic CdLS, and a c.-94C>T variant in a published family. Our reporter assay showed a significant decrease of GFP levels in both mutant contexts, with similar levels of messenger RNA (mRNA) as compared to wt constructs. Assessment of LCL of one patient showed consistent results with decreased NIPBL protein and unchanged mRNA levels. 5'-UTR uORF-introducing NIPBL variants may represent a rare source of pathogenic variants in unsolved CdLS patients.

Impaired SorLA maturation and trafficking as a new mechanism for SORL1 missense variants in Alzheimer disease.

The SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer's disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aß peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of the SorLA protein. An initial screening, based on the overexpression of 70 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variants (R332W, S577P, and R654W) and two maturation-competent variants (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs was associated with a clear increase of Aß secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Furthermore, structural analysis of the impact of missense variants on SorLA protein suggested that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein 3D structure resulting from changes in the interatomic interactions.

Clinical and neuropathological diversity of tauopathy in MAPT duplication carriers.

Microduplications of the 17q21.31 chromosomal region encompassing the MAPT gene, which encodes the Tau protein, were identified in patients with a progressive disorder initially characterized by severe memory impairment with or without behavioral changes that can clinically mimic Alzheimer disease. The unique neuropathological report showed a primary tauopathy, which could not be unanimously classified in a given known subtype, showing both 4R- and 3R-tau inclusions, mainly within temporal cortical subregions and basal ganglia, without amyloid deposits. Recently, two subjects harboring the same duplication were reported with an atypical extrapyramidal syndrome and gait disorder. To decipher the phenotypic spectrum associated with MAPT duplications, we studied ten carriers from nine families, including two novel unrelated probands, gathering clinical (n = 10), cerebrospinal fluid (n = 6), MRI (n = 8), dopamine transporter scan (n = 4), functional (n = 5), amyloid (n = 3) and Tau-tracer (n = 2) PET imaging data as well as neuropathological examination (n = 4). Ages at onset ranged from 37 to 57 years, with prominent episodic memory impairment in 8/10 patients, associated with behavioral changes in four, while two patients showed atypical extrapyramidal syndrome with gait disorder at presentation, including one with associated cognitive deficits. Amyloid imaging was negative but Tau imaging showed significant deposits mainly in both mesiotemporal cortex. Dopaminergic denervation was found in 4/4 patients, including three without extrapyramidal symptoms. Neuropathological examination exclusively showed Tau-immunoreactive lesions. Distribution, aspect and 4R/3R tau aggregates composition suggested a spectrum from predominantly 3R, mainly cortical deposits well correlating with cognitive and behavioral changes, to predominantly 4R deposits, mainly in the basal ganglia and midbrain, in patients with prominent extrapyramidal syndrome. Finally, we performed in vitro seeding experiments in HEK-biosensor cells. Morphological features of aggregates induced by homogenates of three MAPT duplication carriers showed dense/granular ratios graduating between those induced by homogenates of a Pick disease and a progressive supranuclear palsy cases. These results suggest that MAPT duplication causes a primary tauopathy associated with diverse clinical and neuropathological features.

Detection of copy-number variations from NGS data using read depth information: a diagnostic performance evaluation.

The detection of copy-number variations (CNVs) from NGS data is underexploited as chip-based or targeted techniques are still commonly used. We assessed the performances of a workflow centered on CANOES, a bioinformatics tool based on read depth information. We applied our workflow to gene panel (GP) and whole-exome sequencing (WES) data, and compared CNV calls to quantitative multiplex PCR of short fluorescent fragments (QMSPF) or array comparative genomic hybridization (aCGH) results. From GP data of 3776 samples, we reached an overall positive predictive value (PPV) of 87.8%. This dataset included a complete comprehensive QMPSF comparison of four genes (60 exons) on which we obtained 100% sensitivity and specificity. From WES data, we first compared 137 samples with aCGH and filtered comparable events (exonic CNVs encompassing enough aCGH probes) and obtained an 87.25% sensitivity. The overall PPV was 86.4% following the targeted confirmation of candidate CNVs from 1056 additional WES. In addition, our CANOES-centered workflow on WES data allowed the detection of CNVs with a resolution of single exons, allowing the detection of CNVs that were missed by aCGH. Overall, switching to an NGS-only approach should be cost-effective as it allows a reduction in overall costs together with likely stable diagnostic yields. Our bioinformatics pipeline is available at: https://gitlab.bioinfo-diag.fr/nc4gpm/canoes-centered-workflow .

Haploinsufficiency of the Primary Familial Brain Calcification Gene SLC20A2 Mediated by Disruption of a Regulatory Element.

OBJECTIVE: Primary familial brain calcification (PFBC) is a rare cerebral microvascular calcifying disorder with diverse neuropsychiatric expression. Five genes were reported as PFBC causative when carrying pathogenic variants. Haploinsufficiency of SLC20A2, which encodes an inorganic phosphate importer, is a major cause of autosomal-dominant PFBC. However, PFBC remains genetically unexplained in a proportion of patients, suggesting the existence of additional genes or cryptic mutations. We analyzed exome sequencing data of 71 unrelated, genetically unexplained PFBC patients with the aim to detect copy number variations that may disrupt the expression of core PFBC-causing genes. METHODS: After the identification of a deletion upstream of SLC20A2, we assessed its consequences on gene function by reverse transcriptase droplet digital polymerase chain reaction (RT-ddPCR), an ex vivo inorganic phosphate uptake assay, and introduced the deletion of a putative SLC20A2 enhancer mapping to this region in human embryonic kidney 293 (HEK293) cells by clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 9 (Cas9). RESULTS: The 8p11.21 deletion, segregating with PFBC in a family, mapped 35 kb upstream of SLC20A2. The deletion carriers/normal controls ratio of relative SLC20A2 mRNA levels was 60.2% (P < 0.001). This was comparable with that of patients carrying an SLC20A2 premature stop codon (63.4%; P < 0.001). The proband exhibited a 39.3% decrease of inorganic phosphate uptake in blood (P = 0.015). In HEK293 cells, we observed a 39.8% decrease in relative SLC20A2 mRNA levels after normalization on DNA copy number (P < 0.001). DISCUSSION: We identified a deletion of an enhancer of SLC20A2 expression, with carriers showing haploinsufficiency in similar ranges to loss-of-function alleles, and we observed reduced mRNA levels after deleting this element in a cellular model. We propose a 3-step strategy to identify and easily assess the effect of such events. © 2020 International Parkinson and Movement Disorder Society.

Rare genetic susceptibility variants assessment in autism spectrum disorder: detection rate and practical use.

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".

Xq28 copy number gain causing moyamoya disease and a novel moyamoya syndrome.

BACKGROUND: The molecular anomalies causing moyamoya disease (MMD) and moyamoya syndromes (MMS) are unknown in most patients. OBJECTIVE: This study aimed to identify de novo candidate copy number variants (CNVs) in patients with moyamoya. METHODS: Rare de novo CNVs screening was performed in 13 moyamoya angiopathy trios using whole exome sequencing (WES) reads depth data and whole genome high density SNP array data. WES and SNP array data from an additional cohort of 115 unrelated moyamoya probands were used to search for recurrence of these rare de novo CNVs. RESULTS: Two de novo CNVs were identified in two unrelated probands by both methods and confirmed by qPCR. One of these CNVs, located on Xq28, was detected in two additional families. This interstitial Xq28 CNV gain is absent from curated gold standard database of control genomic variants and gnomAD databases. The critical region contains five genes, including MAMLD1, a major NOTCH coactivator. Typical MMD was observed in the two families with a duplication, whereas in the triplicated patients of the third family, a novel MMS associating moyamoya and various systemic venous anomalies was evidenced. CONCLUSION: The recurrence of this novel Xq28 CNV, its de novo occurrence in one patient and its familial segregation with the affected phenotype in two additional families strongly suggest that it is pathogenic. In addition to genetic counselling application, its association with pulmonary hypertension is of major importance for clinical care. These data also provide new insights into the genomic architecture of this emblematic, non-atherosclerotic, large vessel disease.

Causative Mutations and Genetic Risk Factors in Sporadic Early Onset Alzheimer's Disease Before 51 Years.

BACKGROUND: Pathogenic variants in the autosomal dominant genes PSEN1, PSEN2, or APP, APOE4 alleles, and rare variants within TREM2, SORL1, and ABCA7 contribute to early-onset Alzheimer's disease (EOAD). However, sporadic EOAD patients have been insufficiently studied to define the probability of being a carrier of one of these variants. OBJECTIVE: To describe the proportion of each genetic variation among patients with very young-onset sporadic AD. METHODS: We first screened PSEN1, PSEN2, and APP in 154 EOAD patients with an onset before 51 years and a negative family history. Among 99 patients with no mutation (NMC), whole exome sequencing (WES) was performed. We analyzed the APOE genotype and rare protein-truncating or missense predicted damaging variants of TREM2, SORL1, and ABCA7. Neurological examination and cerebrospinal fluid (CSF) biomarkers were systematically retrieved. RESULTS: Nineteen (12.3%) mutation carriers (MC) harbored an APP or PSEN1 pathogenic or likely pathogenic variant. Among the NMC, 54/99 carried at least one genetic risk factor, including 9 APOE4/E4 homozygous, 37 APOE4 heterozygous, and 14 with a rare variant in another risk factor gene: 3 SORL1, 4 TREM2, and 9 ABCA7. MC presented an earlier disease onset (p < 0.0001) and associated neurologic symptoms more frequently (p < 0.002). All but one patient had at least 2 CSF biomarkers in abnormal ranges. CONCLUSION: The genetic component of very early sporadic EOAD gathers a substantial proportion of pathogenic variants in autosomal dominant genes and an even higher proportion of patients carrying genetic risk factors, suggesting an oligogenic determinism, even at this range of ages.

Confirmation and further delineation of the SMG9-deficiency syndrome, a rare and severe developmental disorder.

INTRODUCTION: SMG9 deficiency is an extremely rare autosomal recessive condition originally described in three patients from two families harboring homozygous truncating SMG9 variants in a context of severe syndromic developmental disorder. To our knowledge, no additional patient has been described since this first report. METHODS: We performed exome sequencing in a patient exhibiting a syndromic developmental delay and in her unaffected parents and report the phenotypic features. RESULTS: Our patient presented with a syndromic association of severe global developmental delay and diverse malformations, including cleft lip and palate, facial dysmorphic features, brain abnormalities, heart defect, growth retardation, and severe infections. She carried a novel SMG9 homozygous variant NM_019108.3:c.1177C>T, p.(Gln393*), while her unaffected parents were both heterozygous. CONCLUSIONS: We confirm that bi-allelic truncating SMG9 variants cause a severe developmental syndrome including brain and heart malformations associated with facial dysmorphic features, severe growth and developmental delay with or without ophthalmological abnormalities, severe feeding difficulties, and life-threatening infections.

A Simple, Universal, and Cost-Efficient Digital PCR Method for the Targeted Analysis of Copy Number Variations.

BACKGROUND: Rare copy number variations (CNVs) are a major cause of genetic diseases. Simple targeted methods are required for their confirmation and segregation analysis. We developed a simple and universal CNV assay based on digital PCR (dPCR) and universal locked nucleic acid (LNA) hydrolysis probes. METHODS: We analyzed the mapping of the 90 LNA hydrolysis probes from the Roche Universal ProbeLibrary (UPL). For each CNV, selection of the optimal primers and LNA probe was almost automated; probes were reused across assays and each dPCR assay included the CNV amplicon and a reference amplicon. We assessed the assay performance on 93 small and large CNVs and performed a comparative cost-efficiency analysis. RESULTS: UPL-LNA probes presented nearly 20000000 occurrences on the human genome and were homogeneously distributed with a mean interval of 156 bp. The assay accurately detected all the 93 CNVs, except one (<200 bp), with coefficient of variation <10%. The assay was more cost-efficient than all the other methods. CONCLUSIONS: The universal dPCR CNV assay is simple, robust, and cost-efficient because it combines a straightforward design allowed by universal probes and end point PCR, the advantages of a relative quantification of the target to the reference within the same reaction, and the high flexibility of the LNA hydrolysis probes. This method should be a useful tool for genomic medicine, which requires simple methods for the interpretation and segregation analysis of genomic variations.

Identification of mobile retrocopies during genetic testing: Consequences for routine diagnosis.

Human retrocopies, that is messenger RNA transcripts benefitting from the long interspersed element 1 machinery for retrotransposition, may have specific consequences for genomic testing. Next genetration sequencing (NGS) techniques allow the detection of such mobile elements but they may be misinterpreted as genomic duplications or be totally overlooked. We report eight observations of retrocopies detected during diagnostic NGS analyses of targeted gene panels, exome, or genome sequencing. For seven cases, while an exons-only copy number gain was called, read alignment inspection revealed a depth of coverage shift at every exon-intron junction where indels were also systematically called. Moreover, aberrant chimeric read pairs spanned entire introns or were paired with another locus for terminal exons. The 8th retrocopy was present in the reference genome and thus showed a normal NGS profile. We emphasize the existence of retrocopies and strategies to accurately detect them at a glance during genetic testing and discuss pitfalls for genetic testing.

Biallelic MYORG mutation carriers exhibit primary brain calcification with a distinct phenotype.

Primary familial brain calcification (PFBC) is a rare neurogenetic disorder with diverse neuropsychiatric expression. Mutations in four genes cause autosomal dominant PFBC: SLC20A2, XPR1, PDGFB and PDGFRB. Recently, biallelic mutations in the MYORG gene have been reported to cause PFBC with an autosomal recessive pattern of inheritance. We screened MYORG in 29 unrelated probands negatively screened for the autosomal dominant PFBC genes and identified 11 families with a biallelic rare or novel predicted damaging variant. We studied the clinical and radiological features of 16 patients of these 11 families and compared them to that of 102 autosomal dominant PFBC patients carrying a mutation in one of the four known autosomal dominant PFBC genes. We found that MYORG patients exhibited a high clinical penetrance with a median age of onset of 52 years (range: 21-62) with motor impairment at the forefront. In particular, dysarthria was the presenting sign in 11/16 patients. In contrast to patients with autosomal dominant PFBC, 12/15 (80%) symptomatic patients eventually presented at least four of the following five symptoms: dysarthria, cerebellar syndrome, gait disorder of any origin, akinetic-hypertonic syndrome and pyramidal signs. In addition to the most severe clinical pattern, MYORG patients exhibited the most severe pattern of calcifications as compared to the patients from the four autosomal dominant PFBC gene categories. Strikingly, 12/15 presented with brainstem calcifications in addition to extensive calcifications in other brain areas (lenticular nuclei, thalamus, cerebellar hemispheres, vermis, ±cortex). Among them, eight patients exhibited pontine calcifications, which were observed in none of the autosomal dominant PFBC patients and hence appeared to be highly specific. Finally, all patients exhibited cerebellar atrophy with diverse degrees of severity on CT scans. We confirmed the existence of cerebellar atrophy by performing MRI voxel-based morphometry analyses of MYORG patients with autosomal dominant PFBC mutation carriers as a comparison group. Of note, in three families, the father carried small pallido-dentate calcifications while carrying the mutation at the heterozygous state, suggesting a putative phenotypic expression in some heterozygous carriers. In conclusion, we confirm that MYORG is a novel major PFBC causative gene and that the phenotype associated with such mutations may be recognized based on pedigree, clinical and radiological features.

Copy Number Variants in miR-138 as a Potential Risk Factor for Early-Onset Alzheimer's Disease.

Early-onset Alzheimer's disease (EOAD) accounts for 5-10% of all AD cases, with a heritability ranging between 92% to 100%. With the exception of rare mutations in APP, PSEN1, and PSEN2 genes causing autosomal dominant EOAD, little is known about the genetic factors underlying most of the EOAD cases. In this study, we hypothesized that copy number variations (CNVs) in microRNA (miR) genes could contribute to risk for EOAD. miRs are short non-coding RNAs previously implicated in the regulation of AD-related genes and phenotypes. Using whole exome sequencing, we screened a series of 546 EOAD patients negative for autosomal dominant EOAD mutations and 597 controls. We identified 86 CNVs in miR genes of which 31 were exclusive to EOAD cases, including a duplication of the MIR138-2 locus. In functional studies in human cultured cells, we could demonstrate that miR-138 overexpression leads to higher Aß production as well as tau phosphorylation, both implicated in AD pathophysiology. These changes were mediated in part by GSK-3ß and FERMT2, a potential risk factor for AD. Additional disease-related genes were also prone to miR-138 regulation including APP and BACE1. This study suggests that increased gene dosage of MIR138-2 could contribute to risk for EOAD by regulating different biological pathways implicated in amyloid and tau metabolism. Additional studies are now required to better understand the role of miR-CNVs in EOAD.