Targeted Exonic Sequencing Identifies Novel Causes of Disease in a Cerebral Small Vessel Disease Cohort

doi:10.21203/rs.3.rs-1333758/v1

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Targeted Exonic Sequencing Identifies Novel Causes of Disease in a Cerebral Small Vessel Disease Cohort

https://doi.org/10.21203/rs.3.rs-1333758/v1

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Cerebral small vessel diseases (CSVDs) are a set of conditions that affect the small blood vessels in the brain and can cause severe neurological pathologies such as stroke and vascular dementia. The most common monogenic CSVD is cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) which is caused by mutations in NOTCH3. However, only 15-20% of CADASIL cases referred for genetic testing have pathogenic mutations in NOTCH3. Investigation of other monogenic forms of CSVD caused by HTRA1, COL4A1, COL4A2, CTSA, TREX1 and GLA were rarely requested as follow-up to CADASIL diagnostic testing. We performed whole exome sequencing for 50 individuals suspected of having CADASIL, but did not exhibit a disease-causing mutation in NOTCH3, and applied targeted analysis of all monogenic forms of CSVD including NOTCH3, HTRA1, COL4A1, COL4A2, CTSA, TREX1 and GLA to look for rare, protein-altering mutations. We identified three mutations affecting the Collagen type IV genes in three individuals likely to be causative of CSVD. This suggests that screening for all monogenic forms of CSVD when one monogenic form is clinically suspected may improve diagnosis in clinically suspected monogenic CSVD. However, despite these findings, the majority of NOTCH3 negative CSVD cases did not have candidate mutations in known CSVD genes, suggesting that additional genetic factors contributing to the disease are yet to be identified.

Cerebral small vessel disease

CADASIL

Collagen Type IV

Targeted exonic sequencing

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is a cerebral small vessel disease (CSVD) which is the most common inherited monogenic cause of stroke and vascular dementia in the world[1]. It has an estimated prevalence of 2-4 per 100,000, although this is thought to be under-representative of incidence[2, 3]. The clinical manifestations of CADASIL can be heterogeneous and include subcortical ischaemic events (60-80%); cognitive impairment including dementia and apathy (40-60%); migraine with/without aura (20-40%); mood disturbances including severe depression and manic episodes (~20%); motor disturbances such as gait disturbances (90%), urinary incontinence (80-90%) and pseudobulbar palsy (50%); and other neurological manifestations such as epilepsy/seizures (~10%)[4, 5].

Mutations in NOTCH3 were originally identified to be cysteine altering in the epidermal growth factor like repeats (EGFR), primarily within exons 3 and 4, however with the advent of next generation sequencing (NGS) technology, there has been an increase in the number and type of mutations found to cause the disease[6]. Further work has also identified that the severity of CADASIL may also be related to the position of the change in the EGFR of CADASIL, where mutations in EGFR 1 - 6 are thought to be causative of a more severe phenotype and mutations in EGFR 7 - 24 thought to be associated with milder phenotypes[7, 8]. Despite our current understanding of the clinical presentation of CADASIL as well as the strong monogenic causal link, genetic testing of clinically referred individuals by the Genomics Research Centre (GRC) has identified mutations in 15-22% of patients, with up to 85% of patients unable to receive a definitive diagnosis[9, 10].

One potential reason for this relatively low diagnostic yield may be phenotypic overlap with other CSVDs as well as other complex disorders such as familial hemiplegic migraine, motor neuron disease, multiple sclerosis and Alzheimer’s disease (AD)[11]. The overlapping symptoms of these disorders with CADASIL has been well documented. In particular, six genes have been established to cause five CSVDs with overlapping clinical presentations to CADASIL[12]. The first of these genes is High-Temperature Requirement Serine Peptidase 1 (HTRA1) where mutations are known to cause the recessive form of CADASIL, cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL)[13]. The second is Cathepsin-A (CTSA), causing Cathepsin-A related arteriopathy with strokes and leukoencephalopathy (CARASAL) where symptoms include migraine, hypertension, sinusitis, asthma and depression with a family history of stroke and cognitive decline, as well as observed white matter hyper-intensities seen via magnetic resonance imaging (MRI)[14].

In addition, COL4A1 and COL4A2 cause brain small vessel disease with or without ocular anomalies where clinical features include neurological problems such as hemiparesis, porencephaly, vascular lesions in the brain and peripheral organs, transient ischaemic attacks, adult onset haemorrhagic stroke, periventricular brain abnormalities, white matter hyperintensities, leukoencephalopathy (including cerebellar hypoplasia, cerebral atrophy and vascular changes) and ophthalmological signs such as small/tilted discs, myopia or amblyopia[15, 16]. TREX1 causes retinal vasculopathy with cerebral leukoencephalopathy where major features of the disease can include retinopathy, cognitive impairment, psychiatric disease, seizures and white matter disease. Finally, GLA which encodes for Galactosidase Alpha, is known to cause Fabry disease, cerebrovascular manifestations of which include early stroke/transient ischaemic attacks, white matter lesions, hemiparesis, vertigo or dizziness and other complications associated with vascular disease (diplopia, hemiataxia, memory loss, hearing loss, etc.)[17, 18].

Previous work utilising diagnostic genetic testing of CADASIL patients found evidence to suggest that some CADASIL referred patients with no pathogenic NOTCH3 mutations had mutations in other monogenic forms of CSVD which caused a CADASIL-like phenotypes[9]. Due to the overlapping phenotypic presentation of these diseases and CADASIL, we sought to investigate the likelihood of other genetic causes of CSVD in a cohort of NOTCH3 negative patients with a clinical referral for CADASIL.

Patient Cohort:

Blood samples were chosen from CADASIL referred patients (n=50) with no pathogenic NOTCH3 mutations. All patients had approved diagnostic testing for CADASIL with their doctors and ethical approval for this study was obtained through the QUT HREC along with appropriate consents for the patient cohort (Approval Number 1800000611). Diagnostic testing was performed using the Genomics Research Centre (GRC) custom 5-gene panel where only the notch receptor 3 (NOTCH3) gene was analysed. Further screening of for calcium voltage-gated channel subunit alpha 1A (CACNA1A), ATPase Na⁺/K⁺ Transporting subunit Alpha 2 (ATP1A2), sodium voltage-gated channel alpha subunit 1 (SCN1A), and Potassium two pore domain channel subfamily K member 18 (KCNK18)[10] which have all previously been associated with other diseases such as familial hemiplegic migraine and migraine with/without aura, episodic ataxia type 2, spinocerebellar ataxia type 6 and epilepsy was also performed to rule out any conditions with overlapping symptoms to CADASIL.

Whole Exome Sequencing:

We used an input of 80ng of DNA used for whole exome sequencing (WES) using the Ion AmpliSeq Exome RDY-kits (Carlsbad, Ca., USA) for library preparation, according to manufactures’ instructions (MAN0010084). Completed libraries from samples were quantified using an Invitrogen Qubit 3 Fluorometer (Venlo, Netherlands) and combined at equimolar concentration of 100pMol prior to sequencing. Template preparation, enrichment and chip loading was performed using the Ion P1 Hi-Q Chef Kit (Cat. Number A30011) and 540 Chips on the Thermo Fisher Scientific Ion Chef (Carlsbad, Ca., USA) targeted at 200bp lengths. Sequencing was performed using the Ion Proton and Ion S5+ platforms with sequencing alignment (Hg19) and variant calling was completed via the Ion Torrent software (Carlsbad, Ca., USA).

Analysis and variant Validation

An in-house developed analysis pipeline (VCF-DART) was utilised for targeted analysis of non-amyloidal monogenic CSVD genes NOTCH3, HTRA1, COL4A1, COL4A2, CTSA, TREX1 and GLA (Table 1) [19]. Variants were filtered based on potential functional impacted including non-synonymous, frameshifts, stop-gain or losses, or in canonical splice sites, and with a minor allele frequency (MAF) <0.001. Furthermore, in silico pathogenicity tools including Sorting Intolerant From Tolerant (SIFT), Polymorphism Phenotyping v2, Mutation Taster and PredictSNP2 were also used to determine likely pathogenicity [20-23]. Variants were then stratified as potentially disease causing based on the American College of Medical Genetics (ACMG) Guidelines for determining pathogenicity [24]. Suspected disease-causing mutations were validated by Sanger sequencing using BigDye Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems) and analysed using the ABI3500 Genetic Analyser (Life Technologies/ThermoFisher Scientific).

WES was performed on 50 diagnostic samples referred for CADASIL gene testing that had been screened for disease causing mutations by the GRC custom 5-gene NGS panel (NOTCH3, CACNA1A, ATP1A2, SCN1A and KCNK18), but reported negative for pathogenic mutations in those genes. Targeted analysis of WES data for NOTCH3 and the other CSVD genes (COL4A1, COL4A2, HTRA1, TREX, CTSA and GLA) identified 10 rare mutations in the exonic regions of COL4A1, COL4A2, NOTCH3, CTSA and GLA (Table 3). There were no rare exonic mutations identified in HTRA1 or TREX1 in our cohort which suggests that there may be other genes involved.

Through stratification via the ACMG guidelines for determining pathogenicity, we have identified two likely pathogenic mutations in DGR320 (COL4A1 – c.3574G>C p.Gly1192Arg) and DGR343 (COL4A2 – c.290G>A p.Gly97Glu) (Table 3). A third mutation was also identified as a candidate causal mutation in DGR028 (COL4A2 c.4802C>T p.Ala1601Val) due to the predominantly damaging/disease causing effect that was predicted, although it could only be classified as a variant of unknown significance (VoUS) by the ACMG guidelines.

Clinical information available for the 3 participants is shown in Table 2. Both DGR028 and DGR320 were identified via imaging suggesting small vessel disease, including white matter hyperintensities in DGR320. DGR028 also showed progressive cognitive decline, as well as mood disturbances and a history of psychiatric illnesses. DGR320 had a strong family history of stroke and white matter hyperintensities which appears to follow a dominant inheritance pattern with the symptomology also observed in the proband’s sibling and child (Figure 1). DGR343 had no clinical notes provided or made available on request.

A novel mutation was identified in COL4A1 c.3574G>C p.Gly1192Arg seen in DGR320. Mutations previously reported in COL4A2, c.290G>A p.Gly97Glu (rs749501904) and c.4802C>T p.Ala160Val (rs754118201) were present in DGR343 and DGR028, respectively. Familial analysis was completed to include the mother of DGR320 where the same COL4A1 mutation was identified (Figure 1). Clinical investigation of the mother of the proband showed no obvious clinical signs and symptoms associated with CSVD. Clinical testing of the sibling of the proband identified identical MRI findings, however genetic testing was declined with clinical information of the sibling suffering a stroke before the age of 55.

Another 5 samples had 8 rare (MAF <0.001) exonic mutations that were identified across COL4A1, NOTCH3, CTSA and GLA, however, these were considered as unlikely to be causative of the CSVD pathology. From these mutations, those identified in DGR332 (GLA c.48T>G p.Leu16Leu), DGR342 (NOTCH3 c.4914A.G p.Glu1638Glu), DGR351 (CTSA c.939T>A p.Thr313Thr) and DGR360 (NOTCH3 c.120C>G p.Ala40Ala) were synonymous and considered as likely benign according to the ACMG guidelines. There was also two missense mutations identified in cis in DGR360 in NOTCH3 c.154G>A p.Gly52Arg and c.133G>C p.Asp45His which were classified as VoUS. These changes were in not considered as candidate mutations for CSVD in this study as there were multiple lines of computation evidence which supported a benign/tolerated effect and the amino acid change was not typical for CADASIL. Despite this, these changes in NOTCH3 should be functionally assessed as it may give evidence of non-Cysteine altering mutations causing CADASIL in some individuals.

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[15]. 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[26].

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[27]. 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[25]. 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[28]. 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[29]. 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.

Overall, three probands were identified to have mutations in COL4A1 and COL4A2 causing related CSVD conditions in a NOTCH3 negative cohort. While we were able to identify clinically relevant mutations in these three individuals, we were not able to identify CSVD mutations in the remaining 47 probands. Further investigation of the exome sequencing data of these individuals may yield other potential novel causes of CSVD. These results show that genetic investigation of patients with a clinical indication for CADASIL should also include the CSVD genes, COL4A1 and COL4A2, and that there may be other novel causes of these diseases that are yet to be discovered.

Funding:

This work was supported through the National Health and Medical Research Council of Australia (GNT1168601) Dora Lush Biomedical Postgraduate Research scholarship.

Conflicts of Interest:

The authors declare that there are no conflicts of interest

Availability of Data:

All data relevant for this study is included within this manuscript or has been submitted to relevant databases. This includes genetic mutation information which has been submitted to ClinVar and dbSNP. Due to the medical nature and ethical agreements for use of samples, any further information may be made available on request.

Code availability:

Not applicable

Author’s contributions:

Work was conceived by PJD, LRG and LMH. Laboratory methods to generated whole exome sequencing was completed by PJD and NM and previous diagnostic testing and analysis completed by NM and RAS. Analytical approaches included input from HS and PJD. Writing and editing of the work was completed by PJD, NM, RAS, HGS, LMH and LRG.

Ethics Approval:

Ethical approval was completed through the Queensland University of Technology (QUT) human research ethics committee (HREC) (Approval Number 1800000611)

Consent to Participate:

Informed consent was obtained in line with the ethics approval through the QUT HREC (Approval Number 1800000611) and is in line with the 1964 Declaration of Helsinki and its later amendments

Consent for Publication:

Consent for publication was obtained in line with the ethics approval through the QUT HREC (Approval Number 1800000611)

Acknowledgements:

We would like to acknowledge support for this study through the NHMRC Dora Lush Biomedical Postgraduate Research scholarship. Furthermore, the team at the Genomics Research Centre for all their work and many discussions. Finally, the patients and clinicians who helped make it possible to complete this work.

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Table 1 - Tier 1 gene list with associated OMIM listed conditions

Genes	Condition	MIM#	References
*NOTCH3*	Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)	125310	[1, 39-41]
*HTRA1*	Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, type 2 (CADASIL, type 2)	600142, 616779	[13, 42-44] [45]
*GLA*	Fabry Disease	301500	[12, 41, 46, 47]
*COL4A1*	[Haemorrhage, intracerebral, susceptibility to] Brain Small vessel disease with or without ocular anomalies Microangiopathy and leukoencephalopathy, pontine, autosomal dominant (PADMAL)	614519, 175780, 618564	[12, 16, 28, 48]
*COL4A2*	[Haemorrhage, intracerebral, susceptibility to] Brain Small vessel disease 2	614519, 614483	[15, 16, 28]
*TREX1*	Retinal Vasculopathy with Cerebral Leukoencephalopathy	192315	[49-51]
*CTSA*	Cathepsin-A Related Arteriopathy with Strokes and Leukoencephalopathy	N/A	[44, 52]

Table 2 - Clinical information for participants with the Tier 1 mutations detected. ^a CADASIL phenotype of stroke/cognitive decline/abnormal MRI were assumed as they were referred by a clinician for NOTCH3 testing originally. PTSD – post traumatic stress disorder, GAD – generalized anxiety disorder, MDD – major depressive disorder, OCD – obsessive compulsive disorder.

Sample	Sex	Age	Clinical Indication
DGR028	M	64	Progressive cognitive decline over 10 years (worst past 6months). Hx Migraines. Background of PTSD, GAD, MDD, OCD. Declining function at home. Query CADASIL. MRI: Small vessel disease
DGR320	F	49	No clinical notes provided with initial testing- Patient provided: white matter lesions + blurry vision on/off. Sister and daughter (10 years old) also have white matter lesions. Sister has had previous strokes. Son has migraines w/aura. Clinical consultation letter included right optic disc swelling. MRI identified multiple areas of white matter lesions, including optic nerve - suspicious of multiple sclerosis. MRI remained unchanged over time and there are no lesions on spinal cord. CADASIL skin Bx was negative. Sister's MRI scan identical indicating a hereditary condition. PDGFB/R mutations screening was also recommended.
DGR332	F	20	Recurrent hemiparesis. Tests requested: CADASIL genotype, haemophilia screen
DGR336	M	51	FHx CVAs (early). Meiparetic migraine. MRI - white matter changes around corpus callosum. Skin Bx EM -ve
DGR342	F	65	Query central nervous system demyelination/stroke. 18/12 memory loss. MRI features present of WMH. Few vascular risks. Patient suffers from headaches
DGR343	F	39	No clinical notes^a
DGR351	F	64	No clinical notes^a
DGR360	M	60	Extensive MRI brain ischemic changes Querying CADASIL gene positive

Table 3 - Mutations detected with in silico pathogenicity scores and population frequencies included for rare exonic mutations (MAF < 0.001) from the monogenic CSVD targeted analysis. Green highlighted cells indicate the a predicted benign/tolerated effect, whilst pink highlighted cells indicate a damaging/deleterious effect. Blue highlighted cells indicate and unknown predicted effect for that in silico tool.

No competing interests reported.

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Targeted Exonic Sequencing Identifies Novel Causes of Disease in a Cerebral Small Vessel Disease Cohort

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