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Heterozygous missense HTRA1 mutations have been associated with an autosomal dominant cerebral small vessel disease (CSVD) whereas the pathogenicity of heterozygous HTRA1 stop codon variants is unclear. We performed a targeted high throughput sequencing of all known CSVD genes, including HTRA1, in 3853 unrelated consecutive CSVD patients referred for molecular diagnosis. The frequency of heterozygous HTRA1 mutations leading to a premature stop codon in this patient cohort was compared with their frequency in large control databases. An analysis of HTRA1 mRNA was performed in several stop codon carrier patients. Clinical and neuroimaging features were characterized in all probands. Twenty unrelated patients carrying a heterozygous HTRA1 variant leading to a premature stop codon were identified. A highly significant difference was observed when comparing our patient cohort with control databases: gnomAD v3.1.1 [P = 3.12 × 10-17, odds ratio (OR) = 21.9], TOPMed freeze 5 (P = 7.6 × 10-18, OR = 27.1) and 1000 Genomes (P = 1.5 × 10-5). Messenger RNA analysis performed in eight patients showed a degradation of the mutated allele strongly suggesting a haploinsufficiency. Clinical and neuroimaging features are similar to those previously reported in heterozygous missense mutation carriers, except for penetrance, which seems lower. Altogether, our findings strongly suggest that heterozygous HTRA1 stop codons are pathogenic through a haploinsufficiency mechanism. Future work will help to estimate their penetrance, an important information for genetic counselling. [ABSTRACT FROM AUTHOR]

Background Cerebral cavernous malformations (CCMs) are vascular malformations mostly located within the central nervous system. Most deleterious variants are loss of function mutations in one of the three CCM genes. These genes code for proteins that form a ternary cytosolic complex with CCM2 as a hub. Very few CCM2 missense variants have been shown to be deleterious by modifying the ternary CCM complex stability. Objectives To investigate the causality of novel missense CCM2 variants detected in patients with CCM. Methods The three CCM genes were screened in 984 patients referred for CCM molecular screening. Interaction between CCM1 and CCM2 proteins was tested using co-immunoprecipitation experiments for the CCM2 missense variants located in the phosphotyrosine binding (PTB) domain. Results 11 distinct CCM2 rare missense variants were found. Six variants predicted to be damaging were located in the PTB domain, four of them were novel. When co-transfected with CCM1 in HEK293T cells, a loss of interaction between CCM1 and CCM2 was observed for all six variants. Conclusion We showed, using co-immunoprecipitation experiments, that CCM2 missense variants located in the PTB domain were actually damaging by preventing the normal interaction between CCM1 and CCM2. These data are important for diagnosis and genetic counselling, which are challenging in patients harbouring such variants. [ABSTRACT FROM AUTHOR]

Background Moyamoya angiopathy (MMA) is characterised by a progressive stenosis of the terminal part of the internal carotid arteries and the development of abnormal collateral deep vessels. Its pathophysiology is unknown. MMA can be the sole manifestation of the disease (moyamoya disease) or be associated with various conditions (moyamoya syndrome) including some Mendelian diseases. We aimed to investigate the genetic basis of moyamoya using a whole exome sequencing (WES) approach conducted in sporadic cases without any overt symptom suggestive of a known Mendelian moyamoya syndrome. Methods A WES was performed in four unrelated earlyonset moyamoya sporadic cases and their parents (trios). Exome data were analysed under dominant de novo, autosomal recessive and X-linked hypotheses. A panel of 17 additional sporadic cases with early-onset moyamoya was available for mutation recurrence analysis. Results We identified two germline de novo mutations in CBL in two out of the four trio probands, two girls presenting with an infancy-onset severe MMA. Both mutations were predicted to alter the ubiquitin ligase activity of the CBL protein that acts as a negative regulator of the RAS pathway. These two germline CBL mutations have previously been described in association with a developmental Noonan-like syndrome and susceptibility to juvenile myelomonocytic leukaemia (JMML). Notably, the two mutated girls never developed JMML and presented only subtle signs of RASopathy that did not lead to evoke this diagnosis during follow-up. Conclusions These data suggest that CBL gene screening should be considered in early-onset moyamoya, even in the absence of obvious signs of RASopathy. [ABSTRACT FROM AUTHOR]

Objective: Cerebral small vessel disease (cSVD) is a heterogeneous group of disorders. Screening of known cSVD genes identifies the causative mutation in <15% of familial cSVD cases. We sought to identify novel causes of cSVD.Methods: We used linkage analysis and exome sequencing to identify the causal mutation in a French cSVD family. The identified candidate gene was then screened in 202 cSVD unrelated probands, including 1 proband from the first reported pontine autosomal dominant microangiopathy with leukoencephalopathy (PADMAL) family. Sanger sequencing was used to confirm variants in all mutated probands and analyze their segregation in probands' relatives. Mutation consequences were assessed with luciferase reporter assays and real-time quantitative polymerase chain reaction (RT-qPCR).Results: A candidate heterozygous variant located in a predicted miR-29 microRNA binding site, within the 3' untranslated region of COL4A1, was identified in the large French cSVD family. Five additional unrelated probands, including the PADMAL proband, harbored heterozygous variants in this microRNA binding site. Variants cosegregated with the affected phenotype, and cumulative logarithm of odds score reached 6.03, establishing linkage to this locus. A highly significant difference was observed when comparing the number of variants within this binding site in cases and controls (p = 1.77 × 10E-12). RT-qPCR analyses of patients' primary fibroblasts and luciferase reporter assays strongly favor an upregulation of COL4A1 mediated by disruption of miR-29 binding to its target site. Magnetic resonance imaging features were characterized by the presence of multiple pontine infarcts in all symptomatic mutation carriers.Interpretation: Mutations upregulating COL4A1 expression lead to PADMAL, a severe early onset ischemic cSVD, distinct from the various phenotypes associated with COL4A1 missense glycine mutations. Ann Neurol 2016;80:741-753. [ABSTRACT FROM AUTHOR]

Heterozygous missense HTRA1 mutations have been associated with an autosomal dominant cerebral small vessel disease (CSVD) whereas the pathogenicity of heterozygous HTRA1 stop codon variants is unclear. We performed a targeted high throughput sequencing of all known CSVD genes, including HTRA1, in 3853 unrelated consecutive CSVD patients referred for molecular diagnosis. The frequency of heterozygous HTRA1 mutations leading to a premature stop codon in this patient cohort was compared with their frequency in large control databases. An analysis of HTRA1 mRNA was performed in several stop codon carrier patients. Clinical and neuroimaging features were characterized in all probands. Twenty unrelated patients carrying a heterozygous HTRA1 variant leading to a premature stop codon were identified. A highly significant difference was observed when comparing our patient cohort with control databases: gnomAD v3.1.1 [P = 3.12 × 10-17, odds ratio (OR) = 21.9], TOPMed freeze 5 (P = 7.6 × 10-18, OR = 27.1) and 1000 Genomes (P = 1.5 × 10-5). Messenger RNA analysis performed in eight patients showed a degradation of the mutated allele strongly suggesting a haploinsufficiency. Clinical and neuroimaging features are similar to those previously reported in heterozygous missense mutation carriers, except for penetrance, which seems lower. Altogether, our findings strongly suggest that heterozygous HTRA1 stop codons are pathogenic through a haploinsufficiency mechanism. Future work will help to estimate their penetrance, an important information for genetic counselling., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Moyamoya angiopathy (MMA) is a cerebral angiopathy affecting the terminal part of internal carotid arteries. Its prevalence is 10 times higher in Japan and Korea than in Europe. In East Asian countries, moyamoya is strongly associated to the R4810K variant in the RNF213 gene that encodes for a protein containing a RING-finger and two AAA+ domains. This variant has never been detected in Caucasian MMA patients, but several rare RNF213 variants have been reported in Caucasian cases. Using a collapsing test based on exome data from 68 European MMA probands and 573 ethnically matched controls, we showed a significant association between rare missense RNF213 variants and MMA in European patients (odds ratio (OR)=2.24, 95% confidence interval (CI)=(1.19-4.11), P=0.01). Variants specific to cases had higher pathogenicity predictive scores (median of 24.2 in cases versus 9.4 in controls, P=0.029) and preferentially clustered in a C-terminal hotspot encompassing the RING-finger domain of RNF213 (P<10 -3 ). This association was even stronger when restricting the analysis to childhood-onset and familial cases (OR=4.54, 95% CI=(1.80-11.34), P=1.1 × 10 -3 ). All clinically affected relatives who were genotyped were carriers. However, the need for additional factors to develop MMA is strongly suggested by the fact that only 25% of mutation carrier relatives were clinically affected.

Whole-exome sequencing (WES) has become the strategy of choice to identify causal variants in monogenic disorders. However, the list of candidate variants can be quite large, including false positives generated by sequencing errors. To reduce this list of candidate variants to the most relevant ones, a cost-effective strategy would be to focus on regions of linkage identified through linkage analysis conducted with common polymorphisms present in WES data. However, the non-uniform exon coverage of the genome and the lack of knowledge on the power of this strategy have largely precluded its use so far. To compare the performance of linkage analysis conducted with WES and SNP chip data in different situations, we performed simulations on two pedigree structures with, respectively, a dominant and a recessive trait segregating. We found that the performance of the two sets of markers at excluding regions of the genome were very similar, and there was no real gain at using SNP chip data compared with using the common SNPs extracted from WES data. When analyzing the real WES data available for these two pedigrees, we found that the linkage information derived from the WES common polymorphisms was able to reduce by half the list of candidate variants identified by a simple filtering approach. Conducting linkage analysis with WES data available on pedigrees and excluding among the candidate variants those that fall in excluded linkage regions is thus a powerful and cost-effective strategy to reduce the number of false-positive candidate variants.

Objective: To compare clinical features of CCM1, CCM2, and CCM3 mutation carriers., Methods: A detailed clinical and molecular analysis of 163 consecutive cerebral cavernous malformation (CCM) families was performed., Results: A deleterious mutation was detected in 128 probands. Three hundred thirty-three mutation carriers were identified (238 CCM1, 67 CCM2, and 28 CCM3). Ninety-four percent of the probands with an affected relative had a mutation compared with 57% of the probands with multiple lesions but no affected relative (p < 0.001). The number of affected individuals per family was lower in CCM3 families (p < 0.05). The proportion of patients with onset of symptoms before 15 years of age was higher in the CCM3 group (p < 0.0025). Cerebral hemorrhage was the most common initial presentation in CCM3 patients. The average number of T2-weighted imaging lesions was similar in the three groups, in contrast with a significantly lower number of gradient-echo sequence lesions in CCM2 patients (p < 0.05). The number of gradient-echo sequence lesions increased more rapidly with age in CCM1 than in CCM2 patients (p < 0.05)., Interpretation: Despite similarities among the three groups, there is a significantly lower number of affected individuals in CCM3 pedigrees, CCM3 mutations may confer a higher risk for cerebral hemorrhage, particularly during childhood, and the increment of gradient-echo sequence lesions with age differs between CCM1 and CCM2 patients.

Mammalian hepatitis B viruses encode an essential regulatory protein, termed X, which may also be implicated in liver cancer development associated with chronic infection. X protein, also referred to as HBx in human virus and WHx in woodchuck virus, has been reported to bind to a number of cellular proteins, including the DDB1 subunit of the damaged DNA-binding (DDB) complex. Our previous work provided genetic evidence for the importance of WHx-DDB1 interaction in both the activity of the X protein and establishment of viral infection in woodchucks. In the present study, a direct action of DDB1 on the X protein is documented. Physical interaction between the two proteins leads to an increase in X protein stability. This effect results from protection of the viral protein from proteasome-mediated degradation. Protection of WHx is overcome in the presence DDB2, the second subunit of the DDB heterodimer. In keeping with observations reported for HBx, DDB2 was found to directly bind to WHx. Nonetheless, the counteracting effect of DDB2 on X stabilization requires DDB2-DDB1 interaction. Taken together, these findings substantiate the physical and functional connection between the X protein and the DDB1-DDB2 heterodimer, leading to the regulation of the pool of the viral protein.