key: cord-0314718-k2o6fzjx authors: Whittaker, E.; Thrippleton, S.; Chong, L. Y.; Collins, V. C.; Ferguson, A. C.; Henshall, D. E.; Lancastle, E.; Wilkinson, T.; Wilson, B.; Wilson, K.; Sudlow, C.; Wardlaw, J.; Rannikmäe, K. title: Systematic Review of Cerebral Phenotypes Associated with Monogenic Cerebral Small Vessel Disease date: 2021-11-14 journal: nan DOI: 10.1101/2021.11.12.21266276 sha: 56cc0bf95c4a804cf13820649a18bff622483078 doc_id: 314718 cord_uid: k2o6fzjx Background: Cerebral small vessel disease (cSVD) is an important cause of stroke and vascular dementia. Most cases are multifactorial, but an emerging minority have a monogenic cause. While NOTCH3 is the best-known gene, several others have been reported. We aimed to summarise the cerebral phenotypes associated with these more recent cSVD genes. Methods: We performed a systematic review (PROSPERO: CRD42020196720), searching Medline/Embase (conception to July 2020) for any language publications describing COL4A1/2, TREX1, HTRA1, ADA2, or CTSA pathogenic variant carriers. We extracted data about individuals characteristics, clinical and vascular radiological cerebral phenotypes. We summarised phenotype frequencies per gene, comparing patterns across genes. Results: We screened 6,485 publications including 402, and extracted data on 390 COL4A1, 123 TREX1, 44 HTRA1 homozygous, 41 COL4A2, 346 ADA2, 82 HTRA1 heterozygous, and 14 CTSA individuals. Mean age ranged from 15 (ADA2) to 59 years (HTRA1 heterozygotes). Clinical phenotype frequencies varied widely: stroke 9% (TREX1) to 52% (HTRA1 heterozygotes), cognitive features 0% (ADA2) to 64% (HTRA1 homozygotes), psychiatric features 0% (COL4A2; ADA2) to 57% (CTSA). Among individuals with neuroimaging, vascular radiological phenotypes appeared common, ranging from 62% (ADA2) to 100% (HTRA1 homozygotes; CTSA). White matter lesions were the most common pathology, except in ADA2 and COL4A2 cases, where ischaemic and haemorrhagic lesions dominated, respectively. Conclusions: There appear to be differences in cerebral manifestations across cSVD genes. Vascular radiological changes were more common than clinical neurological phenotypes, and present in the majority of individuals with reported neuroimaging. However, these results may be affected by age and biases inherent to case reports. In the future, better characterisation of associated phenotypes, as well as insights from population-based studies, should improve our understanding of monogenic cSVD to inform genetic testing, guide clinical management, and help unravel underlying disease mechanisms. 2 should improve our understanding of monogenic cSVD to inform genetic testing, guide clinical management, and help unravel underlying disease mechanisms. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 14, 2021. Cerebral small vessel disease (cSVD) is recognised as an important cause of stroke and vascular cognitive impairment worldwide. The term cSVD describes a group of pathological processes which affect the small arteries, arterioles, venules, and capillaries within the brain (1) . Features of cSVD on neuroimaging include subcortical infarcts, white matter lesions (WML), deep intracerebral haemorrhage (ICH), enlarged perivascular spaces (PVS), cerebral microbleeds and brain atrophy (2) . Despite the increase in cSVD burden amongst an ageing population, the underlying disease mechanisms are incompletely understood, and therapeutic options limited, with vascular risk factor management remaining the mainstay of cSVD prevention and treatment (3) . While the majority of cSVD cases are thought to result from the interaction of multiple genetic variants and environmental factors, an important minority of cases are monogenic, i.e., caused by a pathogenic rare variant in a single gene. NOTCH3 is the best known of these genes and is implicated in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) (4) . However, since NOTCH3 was first described in 1996, several additional cSVD genes have been identified, including COL4A1, TREX1, HTRA1, COL4A2, ADA2 and, most recently, CTSA. Pathogenic rare variants in these genes have been associated with various clinical phenotypes alongside cSVD, including extracerebral manifestations, as well as certain radiological features seen on neuroimaging (5) . Better characterisation of these rare disorders, including which radiological and clinical phenotypes are associated with specific genes, can inform genetic testing and counselling, including the appropriate selection of patients and screening of family members. This knowledge can also aid in the management of affected individuals, for example by guiding appropriate screening for certain associated phenotypes. Furthermore, an improved understanding of monogenic cSVD may offer insights into the disease mechanisms . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266276 doi: medRxiv preprint 4 underlying sporadic cSVD, as there is increasing evidence to suggest an overlap of disease pathways involved in both sporadic and monogenic disease (6) (7) (8) . Observations from largescale genetic association studies have also shown common variation in monogenic cSVD genes to be associated with sporadic cSVD. Examples include COL4A2 single nucleotide polymorphisms' (SNPs) association with lacunar ischemic stroke and deep ICH, HTRA1 SNPs association with ischaemic stroke, and possibly association of NOTCH3 SNPs with WMLs (9-12). We undertook a systematic literature review with the aim of identifying all reported individuals with putative pathogenic rare variants in any of the following monogenic cSVD genes: COL4A1, TREX1, HTRA1, COL4A2, ADA2 and CTSA. We aimed to summarise and compare both clinical and vascular radiological cerebral phenotypes associated with each monogenic cSVD gene. We have registered a PROSPERO protocol (ID: CRD42020196720) at https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020196720 (13) . We followed the PRISMA guidelines (14) . We searched the MEDLINE and EMBASE databases using OvidSP (from conception to July 2020) for publications about individuals with pathogenic rare variants in any of our genes of interest: COL4A1, TREX1, HTRA1, COL4A2, ADA2 or CTSA. We did not restrict the search by language or publication date; we limited it to human studies; we included conference abstracts. We used a previously-published search strategy (see Data Supplement) (5) . In summary, the search included: . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint • Study authors considered the rare variant to be probably or definitely pathogenic . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint We selected the list of clinical cerebral phenotypes to extract to represent known manifestations of cSVD, including stroke, and the broad categories of cognitive and psychiatric features. We additionally included headache as phenotype-of-interest because of its association with several monogenic cSVD genes in OMIM (ADA2, COL4A1, TREX1, HTRA1). Finally, we also noted any other cerebral clinical phenotypes on our data extraction form. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266276 doi: medRxiv preprint 7 We selected the list of vascular radiological cerebral phenotypes to extract to represent known manifestations of cSVD and again noted any other features on our data extraction form. Finally, we noted any specific radiological patterns to lesion location or severity that might help identify cases in everyday clinical practice. To assess agreement in data extraction, at least 2 members of the team extracted data from 10% of publications, working independently, and blinded to each other's decisions. Where radiological imaging findings were described, the terminology used across publications varied widely, as has been noted previously in the literature (2) . We made an effort to sort the imaging descriptions into our pre-specified categories to deal with the variable terminology (see Supplemental Methods for a list of decisions and assumptions), discussing uncertainties with an expert neuroradiologist (JW). For each gene, we summarised the total number of relevant publications, pedigrees, individuals and rare variants, and the individuals' characteristics. We summarised data on the presence or absence of each cerebral phenotype (clinical and vascular radiological) as well as cumulative evidence of any vascular radiological feature, to assess their apparent frequency. We compared findings between genes, highlighting shared patterns and differences in the frequencies of associated phenotypes. We stratified the presence of clinical stroke and any vascular feature(s) on neuroimaging by presence of one or more vascular risk factors. We used the Chi-squared test (significance threshold of 0.05) to assess differences in phenotype frequency in patients with and without vascular risk factors. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266276 doi: medRxiv preprint We used the Ensembl Variant Effect Predictor (VEP) (15) to assess the consequences of the genetic variants included in our systematic review. We extracted information on the variants based on the following VEP sub-components: (i) SnpEff variant annotation and effect prediction tool to assess variant impact (16) ; (ii) ClinVar to assess variant's clinical significance (17) ; (iii) SIFT to predict whether an amino acid substitution is likely to affect protein function (18) ; and, (iv) PolyPhen-2 to predict the effect of an amino acid substitution on the structure and function of a protein (19) . Where conflicting evidence was provided for the same variant (usually because an allele may have a different effect in different transcripts), we selected the category with a more significant / negative effect. We calculated the results (expressed as percentages) among variants per each individual VEP subcomponent. We included 402 publications from 6485 identified for screening ( Figure 1 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint ≥ 86% for all genes except ADA2 (62%). Figure 4 shows the proportion of individuals with specific features suggestive of vascular brain disease, and Supplementary Table II shows the breakdown of these features by location and severity. Ischaemia: Presence ranged from 0% (COL4A2) to 66% (HTRA1 HetZ ). Ischaemia was the commonest radiological manifestation for ADA2 individuals (45%). Location was reported for most individuals (80%), and as expected, where reported was mainly in deep/lacunar areas. Most individuals (70%) had multiple lesions. ICH: Presence ranged from 0% (TREX1) to 68% (COL4A2). It was predominantly present in COL4A1/2 individuals. However, ICH was also present in a small minority (7% to 10%) of HTRA1, ADA2 and CTSA individuals. Porencephaly was present in COL4A1/2 individuals only (61% and 76%, respectively) and intraventricular haemorrhage was present in COL4A1 individuals only (7%). Location, where reported, was mostly deep. The burden is less clear: single lesions were common, though a minority of individuals did have multiple lesions. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266276 doi: medRxiv preprint WML: Presence ranged from 3% (ADA2) to 100% (CTSA). WMLs were the commonest radiological manifestation for 5/7 genes (not COL4A2 and ADA2). Location was poorly reported, though where reported was common in the temporal regions in several genes. CTSA individuals appear to have lesions mainly in the frontal and parietal regions (though numbers are low). The burden of WML, where reported, was mostly severe, though the burden was not reported well (data missing for 51% individuals). The exception to this was HTRA1 HetZ individuals, who appear to have less severe WMLs. All CTSA individuals with WML with known location had temporal lobe sparing. Microbleeds: Presence ranged from 1% (TREX1; ADA2) to 30% (HTRA1 HomZ ). Microbleeds were also common in HTRA1 HetZ individuals (27%). Location, where reported, was mostly deep. All individuals had multiple lesions where burden was reported. Atrophy: Presence ranged from 0% (COL4A2) to 71% (CTSA). Location and burden were poorly described overall, and the low numbers make it difficult to make any conclusions. Variant pathogenicity assessment VEP produced results from ≥ 1 of its sub-components for 15% to 66% of variants overall (SnpEff 66%, ClinVar 15%, SIFT 60%, and PolyPhen-2 62%), although there was substantial variability for these estimates across different genes. While the percentage of variants with supporting evidence of pathogenicity was high (81% to 99%) when studying only the group of variants with data available, this appeared much lower when including all variants regardless of whether or not VEP was able to process them (12% to 65%). Again, there was substantial variability across individual genes (Supplementary Table 3 ). . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266276 doi: medRxiv preprint Vascular changes are commonly seen on neuroimaging in individuals with rare variant(s) in cSVD genes. Where data are available, the most frequent radiological manifestations are WMLs and ischaemic changes and, as expected, most lesions are deep. Common clinical phenotypes include clinical stroke, psychiatric symptoms and, most frequently reported, cognitive decline. Overall, radiological vascular phenotypes were more common than clinical neurological phenotypes. However, when interpreting these results, it is important to bear in mind that variation in the mean age of affected individuals may explain some of the differences in phenotypes between genes (e.g., increased age is a risk factor for both clinical stroke and vascular cerebral phenotypes on neuroimaging). Both ICH and ischaemic stroke were described for all cSVD genes, although the most common stroke subtype was haemorrhagic for COL4A1/2, and ischaemic for the remaining genes. Enlarged perivascular spaces were infrequently reported, which may reflect this feature being less apparent with older imaging modalities, difficult to differentiate from other lesions such a lacunes (2) , and/or less commonly reported on neuroimaging. The frequency of both clinical stroke and vascular radiological features on neuroimaging was higher for those with at least one vascular risk factor, compared to those with no reported risk factors. However, vascular risk factors were generally poorly reported (therefore their presence cannot be excluded in most cases), age is highly likely to be a confounding factor, and individuals presenting with stroke/vascular radiological features are more likely to be investigated for vascular risk factors. More research is needed to understand the role for a focussed effort on addressing modifiable vascular risk factors in the management of monogenic cSVDs. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint We identified only 14 individuals with a putative pathogenic variant in CTSA. This is likely (at least partly) explained by the relatively recent description of its association with cSVD, but the small overall number of affected individuals limit the conclusions that can be drawn about its phenotype associations. The strengths of our study are: (1) a comprehensive search strategy, including foreignlanguage papers and abstracts; (2) systematic data extraction following a pre-set spreadsheet with a comprehensive list of variables to be collected, while also allowing for novel phenotypes to be recorded; (3) inclusion of several cSVD genes allowing comparisons to be made across these. This research also has some limitations. Firstly, reporting for some variables was poor. For example, region of origin as a marker of ethnicity was frequently poorly reported and therefore often had to be assumed based on information such as the location of the authors' institute. It is possible that some true differences between ethnicities may not have been revealed due to incorrect categorisation. The frequency of neuroimaging reporting was also low for some genes, and it is unknown if neuroimaging was not reported due to lack of positive findings, or whether it was not done at all. Secondly, case reports and case series have many inherent biases which are difficult to control for (e.g. testing bias, publication bias and reporting bias). In addition, the case reports included in this research appeared to lack use of a reporting structure. Current guidelines such as CARE (22, 23) don't work so well in the field of rare genetic diseases and so new, tailored guidelines could help improve the consistency of reporting. The frequency profile of clinical cerebral phenotypes associated with monogenic cSVDs suggests it is important to consider a broader spectrum of manifestations when identifying potential patients for genetic testing. Specifically, cognitive involvement appeared even more frequently than clinical stroke for several genes. Our results also show that in monogenic . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266276 doi: medRxiv preprint cSVD a radiological vascular phenotype is described more frequently than clinical cerebral phenotypes suggesting a potential benefit of radiological screening, both for patients and for at-risk family members. It is also notable that across several monogenic cSVDs, WMLs were commonly identified in the temporal region, a feature which has previously been associated with CADASIL (caused by NOTCH3 mutations) (24). It is therefore important to also consider other cSVD genes in the presence of this feature. Finally, according to OMIM (https://www.omim.org), headache is a known phenotype associated with TREX1 rare variants, thus its high frequency in TREX1 individuals was expected. However other genes associated in OMIM with headache (COL4A1, ADA2 and HTRA1) were not found to have a clear association with this phenotype in our review. 43% of CTSA individuals (albeit among a total of only 14 individuals) also reported headache, which is more than the expected population prevalence of 15% (25), suggesting a potentially novel associated phenotype. Epilepsy was another common phenotype in COL4A1/2, as suggested by OMIM and previous literature (26). VEP predicted 81% to 99% of the processed variants to have a high likelihood of being pathogenic. However, since these percentages are calculated only among variants with data available, this introduces a bias, as some variants without data (e.g., synonymous SNPs) have a lower prior likelihood of being pathogenic. Adjusting these calculations to include all variants resulted in only 12% to 65% of variants having supporting evidence of pathogenicity, with substantial variability for results across individual genes. Also, it is possible that some variants have been submitted to ClinVar based on the same casereport/case-series included in our review. This makes it difficult to draw robust conclusions about included variants' pathogenicity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266276 doi: medRxiv preprint The findings summarised here have potential clinical implications for the diagnosis and follow up of monogenic cSVDs, especially in conjunction with previous data of associated extracerebral phenotypes (5) . Having said this, to get a more comprehensive and less biased overview of the clinical and radiological consequences of monogenic cSVDs, further work should address these same questions using a genotype-first approach (i.e., studying this in a population-based setting and among individuals selected based on carrying the variant of interest, regardless of their phenotype). The emergence of prospective population-based studies with bio-samples yielding genetic data at scale, such as the UK Biobank (https://www.ukbiobank.ac.uk), will make this possible and complement our study findings. In summary, we found that individuals with rare variant(s) in our genes of interest appear to develop vascular features on neuroimaging. Clinical stroke, cognitive and psychiatric features are also common. The phenotype profiles appear to differ across monogenic cSVD genes, however, these results may be affected by age and other biases inherent to case reports. In the future, better characterisation of associated phenotypes, as well as insights from populationbased studies, should improve our understanding of monogenic cSVD to inform genetic testing, guide clinical management, and help unravel underlying disease mechanisms. Ethics: As a systematic review based on data from published studies this work does not require approval from an ethical standards committee. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266276 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266276 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266276 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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Individual reported to have a different region of origin/ancestry to that of the country they lived in was considered to be from their region of origin (e.g., Chinese origin person living in USA was considered Asian). †If mean age was available for a group of individuals, the overall summary estimate was weighted by group size. For 78/123 TREX1 individuals, only mean age was reported, therefore they were included in the calculations for mean but not for median age/age range. Turkey was reported on specifically because of high proportion of ADA2 individuals from there.. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprintThe copyright holder for this this version posted November 14, 2021. ; https://doi.org/10.1101/2021.11.12.21266276 doi: medRxiv preprint