Phenotypes for monogenic forms of CSVD are known to be similar and often at a clinical examination it is difficult to identify the genetic basis for disease. Whilst more common forms of CSVD can be brought upon by lifestyle and environmental factors, when a monogenic form of disease is suspected, using NGS approaches (WES, WGS or targeted gene panels) should be employed. An example of this strategy working was through targeted WES analysis of CSVD patients screened for NOTCH3 mutations. Three candidate causal mutations in 2 genes (COL4A1 and COL4A2) were identified in three patients related to monogenic CSVD. Mutations in these genes have previously been identified as causing syndromes with overlapping phenotypic traits to CADASIL including brain small vessel disease with or without ocular anomalies (BSVD1) (MIM # 175780), microangiopathy and leukoencephalopathy, pontine, autosomal dominant (PADMAL) (MIM # 618564) and brain small vessel disease 2 (BSVD2) (MIM # 618564). Shared features of these monogenic conditions with CADASIL include incidences of ischaemic stroke and intracerebral haemorrhages, cognitive impairment/dementia, lacunar infarcts, white matter hyperintensities and/or cerebral microbleeds[12, 25].
A heterozygous p.Ala1601Val (rs754118201) in COL4A2 identified in DGR028 was identified as the most likely genetic cause for symptoms. However, based on ACMG guidelines it was only determined to be a VoUS. This was in part due to the COL4A2 mutation affecting the C-terminal tandem repeat region of the COL4A2 protein rather than the characteristic glycine-altering mutations that disrupt the triple-helix structure of the protein previously associated with pathogenesis. Despite this finding, the C-terminal p.Ala1601Val change may still be implicated in the disease as other mutations in the NC1 domain of the protein, such as p. Ala1690Thr, have been identified to impair COL4A2 secretion and therefore may cause decreased COL4A1/COL4A2 heterotrimeric formation in the extracellular matrix.
DGR343 had a clinical suspicion of CADASIL with no additional clinical notes available, but based on criteria for suspected CADASIL diagnosis we assumed that the DGR343 had at least one of the following clinical symptoms including stroke-like episodes with neurological deficits and/or dementia/cognitive decline and/or a mood disorder and/or migraine as well as being <55 years old at presentation. A likely-pathogenic mutation was identified in COL4A2 (rs749501904) due to it being quite rare in gnomAD, with MAF of 7.1x10-5 and an allele count of 20/280896, it being the classical Glycine-altering mutation affecting the Alpha-chain of the protein and multiple levels of in silico prediction tools identifying it as likely damaging/deleterious[24, 25]. However, despite our interpretation it has previously been classified as having uncertain significance in ClinVar (VCV000521905.1).
DGR320 was identified to have a novel characteristic Glycine-altering mutation in COL4A1 affecting the triple-helix domain of the Collagen type IV chain. This type of mutation has previously been linked to characteristic COL4A1 and COL4A2 related small vessel diseases and a recent recommendation from the European Academy of Neurology indicates that all Gly-altering mutations in this region should be denoted as pathogenic. DGR320 also presented with a strong family history of white matter hyperintensities as well as other neurological and neurovascular symptoms such as migraine, vision issues and stroke identified in a sibling. Based on this being a novel mutation, that follows the same classical Glycine-change seen in COL4A1 related SVD, a family history that indicated that the symptoms match a monogenic form of CSVD and multiple in silico tools identifying it as damaging/deleterious with no tools predicting a tolerated effect, it was classified as a likely pathogenic mutation.
However, further investigation of this mutation in the mother of the proband identified the same mutation in COL4A1. This was despite the fact the proband’s mother had no detectable clinical symptoms associated with SVD, including previous known ischaemic/haemorrhagic events, or signs of cognitive decline. Unfortunately, imaging via MRI was not conducted, and it is remains unclear whether she exhibited the same white matter hyperintensities seen in the extended family of the proband. This mutation may also cause variable phenotype, as this is a phenomena that has been examined related to COL4A1 and COL4A2 mutations. Studies have shown that phenotypic heterogeneity has been noted in both broad intra- and interfamilial variation with some evidence suggesting that there are reduced penetrance mechanisms. One example found heterozygous COL4A1/COL4A2 mutations that cause severe phenotypes in affected infants, while parents with the same mutation show no clinical signs of disease. As such, it has been hypothesised that COL4A1/2 mutations may serve as risk factors for CSVD phenotypes and additional risk factors may be required for disease to be seen [29-31]. In line with this observation, other functional mutations identified in DGR320 may hold clues into the some of the clinical features identified.
The Genomic Research Centre has previously found that targeted gene investigations of NGS data is an effective method for identifying causal mutations of monogenic conditions[10, 32, 33]. Furthermore, other labs have successfully used these approaches when investigating individuals that have some of the major symptoms of CSVD including lacunar stroke, cognitive impairment and leukodystrophy[34, 35]. Interestingly, these studies identified HTRA1 mutations as the next most common cause of these conditions, where comparatively our findings failed to identify any causative mutations in this gene[34, 35]. This in turn may be due to the clinical suspicion of CADASIL where only individuals with a perceived autosomal dominant inherited cause of CSVD was suspected.
Whilst we were able to identify candidate mutations within three patients, we were not able to functionally assess these mutations to further elucidate their pathogenesis. Furthermore, only one family member from one of the probands (DGR320) consented for further investigation via segregation analysis. In addition, this study was not able to identify mutations in the remaining 47 probands. It is possible that there are mutations in other genes not investigated in detail, including possible mutations in the mitochondrial genome causative of mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), in some of these individuals that was not detected[36-38]. This is a limitation in using Ion AmpliSeq exome RDY sequencing which does not amplify the mitochondrial genome which limited our scope of investigation. This study was also limited by the number of samples and the lack of more detailed clinical information available for some patients, requiring the assumption of sufficient symptomology being used as a premise for clinical CADASIL testing. This decreased our ability to identify if other genes were indirectly affecting the clinical phenotypes present within the study cohort.
Despite these limitations, we were able to identify monogenic causes of CSVD in a small cohort. Further work should focus on the identification of novel genetic causes of CSVD in conjunction with functional and segregation studies which can then be implemented with diagnostic testing to aid in better treatments for individuals. This work highlights a need for better diagnostic testing, moving away from single gene targeted testing and to further implement multi-gene targeted whole exome or whole genome sequencing for diagnostic testing of CSVD in the future.