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.

Episignatures in practice: independent evaluation of published episignatures for the molecular diagnostics of ten neurodevelopmental disorders.

Variants of uncertain significance (VUS) are a significant issue for the molecular diagnosis of rare diseases. The publication of episignatures as effective biomarkers of certain Mendelian neurodevelopmental disorders has raised hopes to help classify VUS. However, prediction abilities of most published episignatures have not been independently investigated yet, which is a prerequisite for an informed and rigorous use in a diagnostic setting. We generated DNA methylation data from 101 carriers of (likely) pathogenic variants in ten different genes, 57 VUS carriers, and 25 healthy controls. Combining published episignature information and new validation data with a k-nearest-neighbour classifier within a leave-one-out scheme, we provide unbiased specificity and sensitivity estimates for each of the signatures. Our procedure reached 100% specificity, but the sensitivities unexpectedly spanned a very large spectrum. While ATRX, DNMT3A, KMT2D, and NSD1 signatures displayed a 100% sensitivity, CREBBP-RSTS and one of the CHD8 signatures reached <40% sensitivity on our dataset. Remaining Cornelia de Lange syndrome, KMT2A, KDM5C and CHD7 signatures reached 70-100% sensitivity at best with unstable performances, suffering from heterogeneous methylation profiles among cases and rare discordant samples. Our results call for cautiousness and demonstrate that episignatures do not perform equally well. Some signatures are ready for confident use in a diagnostic setting. Yet, it is imperative to characterise the actual validity perimeter and interpretation of each episignature with the help of larger validation sample sizes and in a broader set of episignatures.

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.

A postzygotic de novo NCDN mutation identified in a sporadic FTLD patient results in neurochondrin haploinsufficiency and altered FUS granule dynamics.

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.

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".

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.

Characterization of XPR1/SLC53A1 variants located outside of the SPX domain in patients with primary familial brain calcification.

Primary familial brain calcification (PFBC) is a rare neurological disease characterized by deposits of calcium phosphate in the basal ganglia and other regions of the brain. Pathogenic variants in the XPR1/SLC53A1 gene, which encodes the only known inorganic phosphate exporter, cause an autosomal dominant form of PFBC. These variants are typically located in the SPX N-terminal domain of the protein. Here, we characterize three XPR1 variants outside of SPX in three PFBC patients with an apparently sporadic presentation: c.1375C > T p.(R459C), c.1855A > G p.(N619D) and c.1886T > G p.(I629S), with the latter identified as the first XPR1/SLC53A1 de novo mutation to occur in a PFBC proband. When tested in an in vitro physiological complementation assay, the three XPR1 variants were impaired in phosphate export function, although they were normally expressed at the cell surface and could serve as functional receptors for retrovirus entry. Moreover, peripheral blood cells from the p.N619D patient could be assayed ex vivo and displayed significantly impaired phosphate export. Our results establish for the first time the clinical and molecular characteristics of XPR1 variants located outside the SPX domain and assert a direct link between these variants, deficient phosphate export, and PFBC. Moreover, we unveiled new structural features in XPR1 C-terminal domain that play a role in phosphate export and disease.

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.

Identification of potential genetic risk factors for bipolar disorder by whole-exome sequencing.

This study aims at assessing the burden of rare (minor allele frequency < 1%) predicted damaging variants in the whole exome of 92 bipolar I disorder (BD) patients and 1051 controls of French ancestry. Patients exhibiting an extreme phenotype (earlier onset and family history of mood disorder) were preferentially included to increase the power to detect an association. A collapsing strategy was used to test the overall burden of rare variants in cases versus controls at the gene level. Only protein-truncating and predicted damaging missense variants were included in the analysis. Thirteen genes exhibited p values exceeding 10-3 and could be considered as potential risk factors for BD. Furthermore, the validity of the association was supported when the Exome Aggregation Consortium database non-Finnish European population was used as controls for eight of them. Their gene products are involved in various cerebral processes, some of which were previously implicated in BD and belong to pathways implicated in the therapeutic effect of lithium, the main mood stabilizer. However, exome-wide threshold for association study was not reached, emphasizing that larger samples are needed.

Somatic variants in autosomal dominant genes are a rare cause of sporadic Alzheimer's disease.

INTRODUCTION: A minority of patients with sporadic early-onset Alzheimer's disease (AD) exhibit de novo germ line mutations in the autosomal dominant genes such as APP, PSEN1, or PSEN2. We hypothesized that negatively screened patients may harbor somatic variants in these genes. METHODS: We applied an ultrasensitive approach based on single-molecule molecular inversion probes followed by deep next generation sequencing of 11 genes to 100 brain and 355 blood samples from 445 sporadic patients with AD (>80% exhibited an early onset, <66 years). RESULTS: We identified and confirmed nine somatic variants (allele fractions: 0.2%-10.8%): two APP, five SORL1, one NCSTN, and one MARK4 variants by independent amplicon-based deep sequencing. DISCUSSION: Two of the SORL1 variant might have contributed to the disease, the two APP variants were interpreted as likely benign and the other variants remained of unknown significance. Somatic variants in the autosomal dominant AD genes may not be a common cause of sporadic AD, including early onset cases.

Primary brain calcification: an international study reporting novel variants and associated phenotypes.

Primary familial brain calcification (PFBC) is a rare cerebral microvascular calcifying disorder with a wide spectrum of motor, cognitive, and neuropsychiatric symptoms. It is typically inherited as an autosomal-dominant trait with four causative genes identified so far: SLC20A2, PDGFRB, PDGFB, and XPR1. Our study aimed at screening the coding regions of these genes in a series of 177 unrelated probands that fulfilled the diagnostic criteria for primary brain calcification regardless of their family history. Sequence variants were classified as pathogenic, likely pathogenic, or of uncertain significance (VUS), based on the ACMG-AMP recommendations. We identified 45 probands (25.4%) carrying either pathogenic or likely pathogenic variants (n = 34, 19.2%) or VUS (n = 11, 6.2%). SLC20A2 provided the highest contribution (16.9%), followed by XPR1 and PDGFB (3.4% each), and PDGFRB (1.7%). A total of 81.5% of carriers were symptomatic and the most recurrent symptoms were parkinsonism, cognitive impairment, and psychiatric disturbances (52.3%, 40.9%, and 38.6% of symptomatic individuals, respectively), with a wide range of age at onset (from childhood to 81 years). While the pathogenic and likely pathogenic variants identified in this study can be used for genetic counseling, the VUS will require additional evidence, such as recurrence in unrelated patients, in order to be classified as pathogenic.

Biallelic Loss of Function of SORL1 in an Early Onset Alzheimer's Disease Patient.

Heterozygous SORL1 protein truncating variants (PTV) are a strong risk factor for early-onset Alzheimer's disease (EOAD). In case control studies performed at the genome-wide level, PTV definition is usually straightforward. Regarding splice site variants, only those affecting canonical sites are typically included. Some other variants, not annotated as PTV, could, however, affect splicing and hence result in a loss of SORL1 function. We took advantage of the whole exome sequencing data from the 9/484 patients with a previously reported SORL1 PTV in the French EOAD series and searched for a second variant which may affect splicing and eventually result in more than 50% loss of function overall. We found that one patient, known to carry a variant predicted to disrupt the canonical 5' splice site of exon 8, also carried a second novel intronic variant predicted to affect SORL1 splicing of exon 29. Segregation analysis showed that the second variant was located in trans from the known PTV. We performed ex vivo minigene splicing assays and showed that both variants led to the generation of transcripts containing a premature stop codon. This is therefore the first evidence of a human carrying biallelic SORL1 PTV. This patient had a family history of dementia in both maternal and paternal lineages with later ages of onset than the proband himself. However, his 55 years age at onset was in the same ranges as previously published SORL1 heterozygous PTV carriers. This suggests that biallelic loss of SORL1 function is an extremely rare event that was not associated with a dramatically earlier age at onset than heterozygous SORL1 loss-of-function variant carriers, in this single patient.

Brain calcifications and PCDH12 variants.

OBJECTIVE: To assess the potential connection between PCDH12 and brain calcifications in a patient carrying a homozygous nonsense variant in PCDH12 and in adult patients with brain calcifications. METHODS: We performed a CT scan in 1 child with a homozygous PCDH12 nonsense variant. We screened DNA samples from 53 patients with primary familial brain calcification (PFBC) and 26 patients with brain calcification of unknown cause (BCUC). RESULTS: We identified brain calcifications in subcortical and perithalamic regions in the patient with a homozygous PCDH12 nonsense variant. The calcification pattern was different from what has been observed in PFBC and more similar to what is described in in utero infections. In patients with PFBC or BCUC, we found no protein-truncating variant and 3 rare (minor allele frequency <0.001) PCDH12 predicted damaging missense heterozygous variants in 3 unrelated patients, albeit with no segregation data available. CONCLUSIONS: Brain calcifications should be added to the phenotypic spectrum associated with PCDH12 biallelic loss of function, in the context of severe cerebral developmental abnormalities. A putative role for PCDH12 variants remains to be determined in PFBC.

Contribution to Alzheimer's disease risk of rare variants in TREM2, SORL1, and ABCA7 in 1779 cases and 1273 controls.

We performed whole-exome and whole-genome sequencing in 927 late-onset Alzheimer disease (LOAD) cases, 852 early-onset AD (EOAD) cases, and 1273 controls from France. We assessed the evidence for gene-based association of rare variants with AD in 6 genes for which an association with such variants was previously claimed. When aggregating protein-truncating and missense-predicted damaging variants, we found exome-wide significant association between EOAD risk and rare variants in SORL1, TREM2, and ABCA7. No exome-wide significant signal was obtained in the LOAD sample, and significance of the order of 10-6 was observed in the whole AD group for TREM2. Our study confirms previous gene-level results for TREM2, SORL1, and ABCA7 and provides a clearer insight into the classes of rare variants involved. Despite different effect sizes and varying cumulative minor allele frequencies, the rare protein-truncating and missense-predicted damaging variants in TREM2, SORL1, and ABCA7 contribute similarly to the heritability of EOAD and explain between 1.1% and 1.5% of EOAD heritability each, compared with 9.12% for APOE ε4.

Cone dystrophy or macular dystrophy associated with novel autosomal dominant GUCA1A mutations.

PURPOSE: Sixteen different mutations in the guanylate cyclase activator 1A gene (GUCA1A), have been previously identified to cause autosomal dominant cone dystrophy (adCOD), cone-rod dystrophy (adCORD), macular dystrophy (adMD), and in an isolated patient, retinitis pigmentosa (RP). The purpose of this study is to report on two novel mutations and the patients' clinical features. METHODS: Clinical investigations included visual acuity and visual field testing, fundus examination, high-resolution spectral-domain optical coherence tomography (OCT), fundus autofluorescence imaging, and full-field and multifocal electroretinogram (ERG) recordings. GUCA1A was screened by Sanger sequencing in a cohort of 12 French families with adCOD, adCORD, and adMD. RESULTS: We found two novel GUCA1A mutations-one amino acid deletion, c.302_304delTAG (p.Val101del), and one missense mutation, c.444T>A (p.Asp148Glu)-each of which was found in one family. The p.Asp148Glu mutation affected one of the Ca2+-binding amino acids of the EF4 hand, while the p.Val101del mutation resulted in the in-frame deletion of Valine-101, localized between two Ca2+-binding aspartic acid residues at positions 100 and 102 of the EF3 hand. Both families complained of visual acuity loss worsening with age. However, the p.Asp148Glu mutation was present in one family with adCOD involving abnormal cone function and an absence of macular atrophy, whereas p.Val101del mutation was encountered in another family with adMD without a generalized cone defect. CONCLUSIONS: The two novel mutations described in this study are associated with distinct phenotypes, MD for p.Val101del and COD for p.Asp148Glu, with no intrafamilial phenotypic heterogeneity.

APP, PSEN1, and PSEN2 mutations in early-onset Alzheimer disease: A genetic screening study of familial and sporadic cases.

BACKGROUND: Amyloid protein precursor (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) mutations cause autosomal dominant forms of early-onset Alzheimer disease (AD-EOAD). Although these genes were identified in the 1990s, variant classification remains a challenge, highlighting the need to colligate mutations from large series. METHODS AND FINDINGS: We report here a novel update (2012-2016) of the genetic screening of the large AD-EOAD series ascertained across 28 French hospitals from 1993 onwards, bringing the total number of families with identified mutations to n = 170. Families were included when at least two first-degree relatives suffered from early-onset Alzheimer disease (EOAD) with an age of onset (AOO) ≤65 y in two generations. Furthermore, we also screened 129 sporadic cases of Alzheimer disease with an AOO below age 51 (44% males, mean AOO = 45 ± 2 y). APP, PSEN1, or PSEN2 mutations were identified in 53 novel AD-EOAD families. Of the 129 sporadic cases screened, 17 carried a PSEN1 mutation and 1 carried an APP duplication (13%). Parental DNA was available for 10 sporadic mutation carriers, allowing us to show that the mutation had occurred de novo in each case. Thirteen mutations (12 in PSEN1 and 1 in PSEN2) identified either in familial or in sporadic cases were previously unreported. Of the 53 mutation carriers with available cerebrospinal fluid (CSF) biomarkers, 46 (87%) had all three CSF biomarkers-total tau protein (Tau), phospho-tau protein (P-Tau), and amyloid ß (Aß)42-in abnormal ranges. No mutation carrier had the three biomarkers in normal ranges. One limitation of this study is the absence of functional assessment of the possibly and probably pathogenic variants, which should help their classification. CONCLUSIONS: Our findings suggest that a nonnegligible fraction of PSEN1 mutations occurs de novo, which is of high importance for genetic counseling, as PSEN1 mutational screening is currently performed in familial cases only. Among the 90 distinct mutations found in the whole sample of families and isolated cases, definite pathogenicity is currently established for only 77%, emphasizing the need to pursue the effort to classify variants.