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December 2022; 8 (6) Research ArticleOpen Access

Heterozygous HTRA1 Mutations Cause Cerebral Small Vessel Diseases

Genetic, Clinical, and Pathologic Findings From 3 Chinese Pedigrees

Tingyan Yao, Junge Zhu, Xiao Wu, Xuying Li, Yongjuan Fu, Yuan Wang, Zhanjun Wang, Fanci Xu, Hong Lai, Aini He, Lianghong Teng, Chaodong Wang, Haiqing Song
First published December 6, 2022, DOI: https://doi.org/10.1212/NXG.0000000000200044
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Abstract

Background and Objectives Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a rare hereditary cerebrovascular disease caused by homozygous or compound heterozygous variations in the high-temperature requirement A serine peptidase 1 (HTRA1) gene. However, several studies in recent years have found that some heterozygous HTRA1 mutations also cause cerebral small vessel disease (CSVD). The current study aims to report the novel genotypes, phenotypes, and histopathologic results of 3 pedigrees of CSVD with heterozygous HTRA1 mutation.

Methods Three pedigrees of familiar CSVD, including 11 symptomatic patients and 3 asymptomatic carriers, were enrolled. Whole-exome sequencing was conducted in the probands for identifying rare variants, which were then evaluated for pathogenicity according to the American College of Medical Genetics and Genomics guidelines. Sanger sequencing was performed for validation of mutations in the probands and other family members. The protease activity was assayed for the novel mutations. All the participants received detailed clinical and imaging examinations and the corresponding results were concluded. Hematoma evacuation was performed for an intracerebral hemorrhage patient with the p.Q318H mutation, and the postoperative pathology including hematoma and cerebral small vessels were examined.

Results Three novel heterozygous HTRA1 mutations (p.Q318H, p.V279M, and p.R274W) were detected in the 3 pedigrees. The protease activity was largely lost for all the mutations, confirming that they were loss-of-function mutations. The patients in each pedigree presented with typical clinical and imaging features of CVSD, and some of them displayed several new phenotypes including color blindness, hydrocephalus, and multiple arachnoid cysts. In addition, family 1 is the largest pedigree with heterozygous HTRA1 mutation so far and includes homozygous twins, displaying some variation in clinical phenotypes. More importantly, pathologic study of a patient with p.Q318H mutation showed hyalinization, luminal stenosis, loss of smooth muscle cells, splitting of the internal elastic lamina, and intramural hemorrhage/dissection-like structures.

Discussion These findings broaden the mutational and clinical spectrum of heterozygous HTRA1-related CSVD. Pathologic features were similar with the previous heterozygous and homozygous cases. Moreover, clinical heterogeneity was revealed within the largest single family, and the mechanisms of the phenotypic heterogenetic remain unclear. Overall, heterozygous HTRA1-related CSVD should not be simply taken as a mild type of CARASIL as previously considered.

Glossary

CARASIL=
cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy;
CSVD=
cerebral small vessel disease;
DMEM=
Dulbecco's Modified Eagle Medium;
H&E=
hematoxylin and eosin;
HEK=
human embryonic kidney;
HTRA1=
high-temperature requirement A serine peptidase 1;
ICH=
intracerebral hemorrhage;
LP=
likely pathogenic;
LP=
pathogenic;
MoCA=
Montreal Cognitive Assessment;
SMCs=
smooth muscle cells;
SWI=
susceptibility-weighted sequences;
WES=
whole-exome sequencing;
WT=
wild type

Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a hereditary cerebral small vessel disease (CSVD) caused by biallelic mutations in the gene coding for high-temperature requirement A serine peptidase 1 (HTRA1).1,2 CARASIL is clinically characterized by stroke, dementia, alopecia, and spondylosis deformans and radiologically featured by leukoencephalopathy, multiple lacunar infarctions, microbleeds, and brain atrophy.3,4 CARASIL is initially and most commonly reported in Japan,1,2,5 and subsequently, some sporadic cases were identified worldwide, including China, India, the United States, Italy, and Spain.5,-,7 CARASIL is extremely rare, with only 28 families reported to date.8 However, recently, increasing reports have shown that heterozygous HTRA1 mutations are also associated with CSVD, showing that heterozygous HTRA1 mutations account for 0.8%–6.5% of CSVD cases without a NOTCH3 mutation worldwide.9,-,12

As the majority of carriers of heterozygous HTRA1 mutations are clinically asymptomatic, it is still elusive why certain heterozygous mutations, and what kind of the mutations, could cause CSVD with a dominant mode of inheritance. The heterozygous and homozygous HTRA1 mutations do not form segregated clusters, but are interspersed with one another, sharing the same hotspot position (amino acids 250–300).8,12,-,14 Moreover, with the limited numbers of cases, the clinical and imaging features of patients with heterozygous HTRA1 mutations remain largely unclear. Previously, it was roughly considered a mild type of typical CARASIL.8,12,13 However, with limited number of cases, the phenotypic diversities remain unclear. Previously, 2 autopsy reports from Japan with heterozygous HTRA1 mutations have been reported, displaying similar pathologic manifestations with CARASIL.10,15 However, it has remained obscure whether patients with heterozygous mutations show variability of the vascular changes between mutations and ethnicities and whether there is a genotype-phenotype correlation.

In the current study, 3 pedigrees of heterozygous HTRA1 mutations were enrolled. We investigated the genetic, clinical, and imaging manifestations among the patients and asymptomatic mutation carriers. In addition, the pathologic findings of cerebral small vessels were revealed in a patient with the heterozygous p.Q318H mutation.

Methods

Participants

A total of 11 symptomatic patients and 3 asymptomatic carriers in 3 Chinese pedigrees of familial CSVD with a pathogenic heterozygous HTRA1 mutation were enrolled. All the symptomatic patients and asymptomatic carriers received standard neurologic examination and detailed clinical assessments by a team of senior neurologists, using the Mini-Mental State Examination, Montreal Cognitive Assessment (MoCA), Hamilton Anxiety Rating Scale, and Hamilton Depression Rating Scale. All the participants underwent brain and lumbar MRI scans. In addition, 1,381 individuals free of neurologic disorders were enrolled as ethnically matched healthy controls. During the study, a patient with a heterozygous HTRA1 mutation suffered a severe intracerebral hemorrhage (ICH) and received emergency hematoma evacuation. After the operation, the hematoma specimen containing some cerebral small vessels was sent for pathologic studies.

Whole-Exome and Sanger Sequencing

Using the DNA from the patients and their relatives, whole-exome sequencing (WES) was performed (Running Gene Medicine Tech, Beijing, China) according to the manufacturer's protocol. Whole-exome capture was performed using the Agilent SureSelect Human All Exon Kit (Agilent Technologies, Santa Clara, CA), and high-throughput sequencing was conducted using the Illumina HiSeq 2000 sequencer (Illumina, San Diego, CA). The Genome Analysis Toolkit was applied for identifying the variants. Synonymous variants were excluded. The pathogenicity of the single-nucleotide variants was predicted by PolyPhen-2, Sorting Intolerant From Tolerant (SIFT), and MutationTaster. All detected changes were confirmed by Sanger sequencing, which was performed with PCR primers to sequence exons of HTRA1. All the variants were sequentially evaluated for the population frequency data, literature/database query and in-house evidences (including specificity of phenotype or family history, case-control data/rare variant, segregation data, de novo variants, allelic data, and experimental evidence), variant type–specific profiles, pathogenicity prediction algorithms and hotspot, and/or functional domain evidences, according to the American College of Medical Genetics/Association for Molecular Pathology (ACMG/AMP) guideline and recommendations developed by the ClinGen Sequence Variant Interpretation Working Group and various disease-specific variant curation expert panels. Each variant was classified into 1 of the following 5 categories: pathogenic, likely pathogenic (LP), uncertain significance, likely benign, and benign.

Plasmid Constructs

Expression constructs (pcDNA3.1 vector) for the wild-type (HTRA1-WT) and mutated HTRA1 (HTRA1 mutants) were generated using a human full-length HTRA1 clone with the overlapping PCR and homologous recombination (pEASY®-Basic Seamless Cloning and Assembly Kit; Transgen, China). The primer sequences were provided in eTable 1, links.lww.com/NXG/A566.

Western Blotting

After transfecting with HTRA1-WT and mutants for 12 hours, HEK293T cells were cultured with serum-free culture media for 48 hours. The serum-free culture media were collected and centrifuged for 10 minutes at 1,000 rpm, and the secreted HTRA1 proteins were analyzed by Western blotting. Conditioned supernatant proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore). Next, the PVDF membranes were blocked in 5% skim milk for 1 hour at room temperature and cultured with anti-HTRA1 primary antibody (anti-HTRA1; Abcam, Britain) overnight at 4°C. On the second day, the membranes were cultured with HRP-coupled secondary antibody (goat anti-rabbit IgG; ZSGB Bio, China) for 1 hour at room temperature, and the immunoreactive bands were detected by the Fusion FX7 system (Vilber Lourmat).

Cell Culture and Transfection

Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco) with 10% fetal bovine serum (Gibco) and 1% PS (100 U/mL penicillin and 100 μg/mL streptomycin; Thermo Fisher) and were incubated in a 37°C constant temperature incubator containing 5% CO2. For transfection, HEK 293T cells were seeded in 6-well plates. After 24 hours of culturing in the incubator, plasmids of pcDNA3.1 vector, pcDNA3.1-HTRA1-WT, and pcDNA3.1-HTRA1 mutants were transfected into HEK 293T cells with 3 μg per well in 6-well plates by Lipofectamine 8000 reagent (Lipo 8000; Beyotime, Shanghai, China), according to the manufacturer's protocol.

HTRA1 Protease Assay

After 12 hours of transfection of HTRA1-WT and HTRA1 mutants in the HEK 293T cells, the total DMEM medium was replaced by serum-free medium with 0.4 mg/mL denatured bovine serum albumin (BSA; Sigma) (0.4 mg/mL BSA was denatured by the addition of 1.5 mM dithiothreitol) and 1% penicillin-streptomycin (1% PS) and incubated for 24 hours at 37°C. The conditioned supernatants were collected and centrifuged for 15 minutes at 12,000 rpm to remove the cell debris, and the BSA degradation was evaluated by SDS-PAGE and Coomassie brilliant blue staining.

MRI Scan

Brain MRI data were acquired at a 3T scanner (Magnetom Skyra; Siemens, Germany) using a 20-channel receiver head and neck joint coil (open 16 channels). The white matter hyperintensity was quantified on T2-weighted images or fluid-attenuated inversion recovery sequences, whereas cerebral microbleeds were analyzed on susceptibility-weighted sequences (SWI).

Pathologic Examination

Hematoma specimens were fixed in 10% formalin, processed with embedding paraffin wax. Four-micrometer-thick sections were cut and stained with hematoxylin and eosin (H&E), Elastica-Goldner, and Masson. Immunohistochemical staining was performed using primary antibodies for CD34 (Zymed; dilution 1:50) and α-smooth muscle actin (GBI; dilution 1:50).

Standard Protocol Approvals, Registrations, and Patient Consents

All the recruited participants had given their informed consent for participation in this study. For cognitively impaired patients, detailed written consents were obtained from their first-degree relatives. The study was approved by the Institutional Ethics Committees of Xuanwu Hospital of Capital Medical University.

Data Availability

Data not published within the article are available in a public repository, and anonymized data will be available by reasonable request from any qualified investigator.

Results

Demographic Data of Patients Carrying the Heterozygous HTRA1 Mutations

WES was performed for the 3 probands and all their family members at risk, totally 25 participants including 11 symptomatic patients and 14 asymptomatic individuals (Figure 1). Individuals simultaneously having a pathogenic HTRA1 mutation, neurologic symptoms, and CSVD radiologic features were defined as symptomatic patients. In contrast, pathogenic mutation carriers without clinical symptoms of CSVD were defined as asymptomatic carriers. Totally, 11 symptomatic patients and 3 asymptomatic carriers from 3 Chinese pedigrees were identified. The symptomatic patients included 8 males and 3 females. All the patients were from Northern China. Among these families, family 1 was the largest pedigree of heterozygous HTRA1-related CSVD reported thus far, which spanned 5 generations and included 9 extant patients and 2 asymptomatic carriers (Figure 1A). All the 3 pedigrees showed an autosomal dominant inheritance pattern.

Figure 1
Figure 1 Pedigree Charts and Electropherograms of 3 Heterozygous HTRA1 Mutations

Pedigree charts of 3 families and corresponding electropherogram of each HTRA1 mutation: Family 1 (A), Family 2 (B), Family 3 (C). Squares indicate men; circles, women; filled symbols, clinically and MRI-proven affected individual; empty symbol, clinically healthy individual; diagonal black line, deceased individual; syringe symbol, members undergoing genetic testing. Asterisks symbol, deceased members with a history of a stroke under 60 years of age; black dot, asymptomatic mutation carrier. Ages at examination are displayed in the lower-left corner of each pedigree icons. Ages at onset are displayed in the lower-right corner of each pedigree icons. HTRA1 = high-temperature requirement A serine peptidase 1.

Identification of 3 Heterozygous HTRA1 Mutations

In the analysis of the WES data, rare coding and damaging variants were called for all the genes including those pathogenic for small vessel diseases (NOTCH3, HTRA1, COL4A1/2, TREX1, etc.). After standard interpretations according to the ACMG/AMP guidelines, 3 novel heterozygous mutations in HTRA1 were detected in the 3 families. Among them, the c.954G>C (p.Q318H) variant and the c.835G>A (p.V279M) variant were not reported before. The c.820C>T (p.R274W) variant has different nucleic acid but with the same amino acid position from the c.821G>A (p.R274Q) variant reported in 2 CARASIL cases whose heterozygous family members were reported clinically asymptomatic.16,17 Moreover, the p.Q318H variant was absent from the Genome Aggregation Database (gnomAD), whereas the p.V279M and p.R274W variants were present in gnomAD with an extremely low allele frequency of 1/251004 and 3/250870, respectively. Furthermore, all the 3 variants were absent in our 1,381 Chinese healthy controls. All the variants were predicted to be pathogenic by 3 bioinformatics prediction tools (SIFT, MutationTaster, and PolyPhen-2). Importantly, the variant c.954G>C was cosegregated with the CSVD phenotypes in family 1, and the other 2 families were too small to analyze the cosegregation. Overall, the 3 HTRA1 variants can be classified as LP or pathogenic according to the guideline of the ACMG/AMP. In addition, all these mutations are missense mutations in exon 4, which encodes the trypsin-like serine protease domain (Table 1).

View this table:
Table 1

Heterozygous HTRA1 Mutations Identified in This Study

Protease Activity of the HTRA1 Mutants

To investigate the alterations of the HTRA1 protease activity of the 3 novel mutations, we first detected the secretion of HTRA1-WT and mutants in the serum-free culture media. Our results indicated that HTRA1-WT and all mutants were secreted efficiently in the cell culture media, and they presented varying degrees of degradation (Figure 2A). Next, to investigate the alterations of the HTRA1 protease activity of the 3 novel mutations, the conditioned supernatants with the protease substrate (BSA) of transiently transfected HEK 293T cells (HTRA1-WT and HTRA1 mutants) were collected and analyzed by SDS-PAGE and Coomassie staining. Reduced BSA degradation can reflect the interference of the protease activity by the mutations. It showed that the levels of the protease activities of the p.R274W, p.V279M, and p.Q318H mutant proteins were markedly decreased compared with the HTRA1-WT protein (Figure 2, B and C).

Figure 2
Figure 2 Significant Decreases of the Protease Activity of the 3 Novel Heterozygous Mutated HTRA1

(A) Expression of secreted HTRA1-WT and mutants in conditioned supernatants of HEK293T cells. The molecular weight of full-length HTRA1 was about 51 KDa, and the degradation products presented as the lower-molecular-weight bands. Anti-HTRA1 antibody was used for detecting HTRA1-WT and mutants in Western blot assay. (B) The proteolytic activity of HTRA1s was evaluated by analyzing the BSA degradation via SDS-PAGE and Coomassie Blue staining. (C) Quantitative analysis of the HTRA1 protease activities using 1-way ANOVA. n = 3, **p < 0.01 and ***p < 0.001 compared with WT. Control: without any treatment; vector: pcDNA 3.1 empty vector transfection; WT: pcDNA 3.1-HTRA1 wild-type transfection; p.Arg274Trp: pcDNA 3.1-HTRA1-p.Arg274Trp transfection; p.Val279Met: pcDNA 3.1-HTRA1-p.Val279Met transfection; p.Gln318His: pcDNA 3.1-HTRA1-p.Gln318His transfection. HTRA1 = high-temperature requirement A serine peptidase 1.

Clinical and Imaging Features of Patients Carrying the Heterozygous HTRA1 Mutations

There were 9 symptomatic patients and 2 asymptomatic carriers in family 1. The onset age of neurologic symptoms in the 7 male and 2 female patients was 44.1 ± 5.9 years, ranging from 35 to 52 years. More specifically, the onset of cerebrovascular events was at 44.4 ± 5.6 years, whereas the onset of cognitive impairment was at 48.8 ± 7.4 years. Cerebrovascular ischemic events, including stroke and TIA, were the most common onset symptom in 8 of 9 patients while only 1 patient onset with cognitive decline. For the non-neurologic symptoms, spinal spondylosis or herniated discs were detected in 7 patients, and hair sparse at early age was complained by 2 patients and 1 asymptomatic carrier. Except for patient F3-II-3 who has hypertension and uses nifedipine to control the blood pressure, other patients have no hypertension or hyperlipidemia (Table 2). In the imaging findings, all the 9 patients in family 1 displayed moderate or severe leukoencephalopathy. The lesions were most commonly seen in the periventricular regions (9/9), followed by the centrum semiovale (7/9), the external capsule (4/9), and the anterior temporal pole (1/9). Moreover, multiple lacunar infarcts were also seen in all the 9 patients, broadly involving various brain regions including subcortical regions, basal ganglia, and brainstem. In addition, microbleeds in susceptibility-weighted sequences were also detected in all the patients, mostly seen in the basal ganglia region. However, no abnormalities were found in large vessels for all these patients through brain MR angiography.

View this table:
Table 2

Clinical Characteristics of the 11 Patients With Heterozygous HTRA1 Mutations

Besides common clinical and imaging features of CSVDs, we reported several novel phenotypes in family 1. First, monozygotic twins (IV-4 and IV-5) of heterozygous HTRA1-relative CSVD were recruited. They both developed recurrent stroke, cognitive decline, spondylosis lesions, and color blindness, with the same age at onset and very close scores of clinical scales (Table 2 and Figure 3, A and B). Furthermore, multiple arachnoid cysts were detected in patient IV-3, who presented specifically impaired visuospatial/executive function, with normal functions in other cognitive domains and a 27/30 scores for MoCA (Figure 3F). Brain MRI showed mild ventriculomegaly and multiple arachnoid cysts, among which a large one compressed his right prefrontal cortex (Figure 3E). Moreover, severe hydrocephalus was observed in patient IV-8, who developed severe dementia after recurrent stroke and now is almost in a decorticate rigidity state (Figure 3H). Brain MRI showed obviously dilated lateral and 3rd ventricles (Figure 3G). His intracranial pressure was 180 mm H2O with normal level of protein in CSF. After a 50 mL/d draining of CSF by lumbar puncture for 3 days, no clear clinical remission was observed. However, it can not be excluded that the dilated ventricles may be caused by the diffuse brain atrophy. Notably, severe ICH was observed in IV-6, who had only mild gait disturbance before the hemorrhage. He got sudden hemiplegia and aphasia at age 42 years, and the NIH Stroke Scale score in the emergence apartment was 19. Brain CT showed massive cerebral hemorrhage in the left basal ganglia, and the hemorrhage volume was about 70 mL (Figure 3I). Hematoma evacuation was performed to prevent brain herniation.

Figure 3
Figure 3 Representative Clinical and Radiologic Findings From Individuals Carrying Heterozygous HTRA1 Mutations

(A–J) Findings from family 1. (K and L) Findings from family 2. (M and N) Findings from family 3. (A and B) Fluid-attenuated inversion recovery (FLAIR) images of the monozygotic twins showed multiple ischemic lesions and white matter hyperintensities (WHMs). (C) Microbleeds were seen in the subcortical region, basal ganglia, brainstem, and cerebellum in susceptibility-weighted sequence (SWI) images of patient IV-7. (D) WHMs in the anterior temporal lobe, spondylotic lesions, and normal large vessels in the magnetic resonance angiography (MRA) were observed in patient IV-9. (E and F) Multiple arachnoid cysts were presented in the FLAIR images of patient IV-3, among which a large one compressed the right prefrontal lobe. His Montreal Cognitive Assessment (MoCA) score was 26/30. (G and H) Severe hydrocephalus (obvious dilatation of the lateral, third, and fourth ventricles) was shown in the T2-weighted images of patient IV-8, who was at a decorticate rigidity-like state. (I) Massive hemorrhage in the basal ganglia was seen in the CT images of patient IV-6, along with WHMs and microbleeds shown in MRIs, while no detectable hemangiomas or vascular malformation in MRA. (J) The 25-year-old asymptomatic carrier (V-6) developed mild white matter lesion and early alopecia. (K and L) Confluent WMHs were seen in the FLAIR images; some microbleeds were seen in the SWI images of the proband of family 2. (M and N) Extensive confluent WMHs and massive microbleeds in both cortical and subcortical regions were observed in the proband of family 3. HTRA1 = high-temperature requirement A serine peptidase 1.

In general, patients of family 2 (II-2) and family 3 (II-3) shared similar major clinical and imaging manifestations with those in family 1, having stroke/TIA and cognitive decline as well as leukoencephalopathy, lacunar infarcts and microbleeds. Moreover, patient II-3 in family 3 also experienced an episode of lobar hemorrhage. Her brain MRI showed an old foci ICH, confluent white matter abnormalities, and large number of microbleeds involving the cerebral cortex, basal ganglia, brainstem, and cerebellum (Figure 3, M and N). In addition, asymptomatic carrier F1-V-6 developed alopecia at age 24 years, with very slight/minimal leukoencephalopathy in the region near the frontal horn of the lateral ventricle (Figure 3J). The other 2 asymptomatic carriers have no clinical symptoms and radiologic abnormalities. The information of asymptomatic carriers was summarized in eTable 2, links.lww.com/NXG/A566.

Pathologic Findings

Emergent hematoma evacuation was performed for patient IV-6 in family 1 with ICH, and some hematoma specimens were sent for pathologic studies. Among the hematoma, large patches of fresh hemorrhage, tissues of choroid plexus, and small arteries were observed (Figure 4A). The vascular endothelial cells were CD34 positive (eFigure 1, links.lww.com/NXG/A566). The small arteries showed hyalinization, marked intimal thickening, narrowing of the lumen, and loss of smooth muscle cells (SMCs) in the tunica media with HE stain (Figure 4B). Hyalinization and loss of SMCs were more clearly shown with Masson stain (Figure 4C) and immunohistochemical stain of α-smooth muscle actin antibody (Figure 4D). With the Elastica-Goldner stain, several arteries showed a splitting and wavy multilayered elastic lamina (Figure 4E). Furthermore, bleeding beneath the internal elastic lamina, forming an intramural hemorrhage-like structure, was observed in a few small arteries (Figure 4F). However, no fibrinoid necrosis and deposit of amyloid were found.

Figure 4
Figure 4 Pathologic Findings of a Patient With Heterozygous p.Q318H Mutation in HTRA1

(A) Fresh hemorrhage seen in the hematoma (sword symbol). (B) Small arteries with marked intimal thickening, hyalinosis, and narrowing of the lumen. (C) Hyalinization of the vascular wall. (D) Loss of medial smooth muscle cells in small arteries (blue arrow symbol). (E) Small arteries with a wavy multilayered elastic lamina (black arrow symbol). (F) Small artery with intramural hematoma-like structure (star symbol). Hematoxylin-eosin stain (A and B). Masson stain (C). Immunohistochemical staining of α-smooth muscle actin antibody (D). Elastica-Goldner stain (E and F). The scale bar is in the bottom right corner of each figure. HTRA1 = high-temperature requirement A serine peptidase 1.

Discussion

Before this study, there have been only 46 families of heterozygous HTRA1-related CSVD with 38 different mutations reported worldwide, among which most cases were from China (30%), followed by Japan (24%), France (24%), and Italy (15%).14 The present study expands the mutation spectrum with 3 novel heterozygous mutations and expands the clinical and radiologic features of heterozygous HTRA1-related CSVD. Moreover, we delineated the pathologic findings of a case with a brain biopsy showing arteriopathy in cerebral small arteries. These results are important to further elucidate the genotype and phenotype of heterozygous HTRA1-related CSVD and better understand why certain heterozygous mutations are associated with the disease.

The 3 novel HTRA1 heterozygous mutations in this study are all located in the trypsin-like serine protease (trypsin) domain, which is the most frequently affected domain in both CARASIL and CVSD caused by heterozygous HTRA1 mutations.8,13,14 However, as most of the heterozygous HTRA1 mutations are insufficient to cause clinical symptoms of CSVD (except MRI changes), it remains unclear what kinds of heterozygous mutations are pathogenic to cause monogenic CSVD. Previous studies proposed that pathogenic heterozygous HTRA1 mutations may result in an impaired activation cascade of HTRA1 or be unable to form stable trimers through a haploinsufficiency or dominant-negative mechanism.8,10,12 However, these hypotheses cannot explain all the heterozygous HTRA1 mutations causing CSVD.18 Moreover, 6 mutations (R166C, P285L, G295R, R302X, R370X, and V297M) were reported in both heterozygous and homozygous (CARASIL) cases,2,8,19 indicating the mutation sites alone can not fully explain the mechanism. Similarly, the mutation c.820C>T (R274W) in family 3 shared the same mutated amino acid but differed in the changed amino acid with the c.821G>A (R274Q) variant in 2 CARASIL families,16,17 suggesting that not only the mutation positions but also the types of amino acid substitution may contribute to the disease.

We expanded the clinical spectrums with several phenotypes that have never been reported. On one hand, monozygotic twins were not reported in this disease. The high similarity of clinical and radiologic manifestations in the twins confirmed the importance of genetic factor in this hereditary disease. On the other hand, some variation was also observed in cases carrying the same mutation in 1 family, suggesting that mutations alone cannot explain the full picture of disease. Also, color blindness was first reported in HTRA1-related CSVD. The causality was unclear, but HTRA1 mutations do cause a known ophthalmic disease, the age-related macular degeneration.20 Moreover, hydrocephalus was not reported in patients with heterozygous or homozygous HTRA1 mutation. With the concern of unclear mechanism underlying the hydrocephalus, regular surgeries like lumbo- or ventriculoperitoneal shunts were not performed. In addition, multiple arachnoid cysts were also reported for the first time in this disease. The patient with arachnoid cysts showed pronounced visuospatial/executive dysfunction, which may be a symptom of focal compression of the giant arachnoid cyst or just a predilection of vascular dementia.21 We leaned to the former, as his mild severity of CSVD did not appear to match his significant decline in visuospatial/executive function. We are not sure that the hydrocephalus and arachnoid cyst are rare symptoms related to HTRA1 mutations or comorbidities of the disease. These symptoms are also rarely observed in sporadic age-related CSVD. However, hydrocephalus and arachnoid cysts were also reported in another hereditary CSVD, the COL4A1/2-related disorders.22 Notably, ICH was reported in a small number of patients with heterozygous HTRA1 mutations.8,12 Because ICH was reported in up to 21.3% of patients with CADASIL,23 it may be underestimated in the HTRA1-related SCVD. Two patients in our study experienced severe ICH, which reminds the clinicians of the risk of hemorrhage for these patients, especially for those who had marked microbleed and/or were on antiplatelet medications.

Two autopsy cases of Japanese patients with a heterozygous HTRA1 mutation have been reported worldwide,10,15 in which the cerebral small vessels showed similar but less severe histologic changes compared with CARASIL.1,24,25 In the current study, we presented the third pathologic report of patients with heterozygous HTRA1 mutation. We revealed the pathologic features in the Chinese patient with the Q318H mutation, which are generally consistent with the 2 autopsy results of Japanese patients, respectively, carrying p.G283E and p.R302Q mutations.10,15 Of note, hyalinization and loss of SMCs are often seen in nonhereditary CSVD, but splitting of the internal elastic lamina is less common. Without atherosclerosis and lipid hyalinization, the pathogenetic pattern of heterozygous HTRA1-related CSVD seems to have some difference with the sporadic aged-related CSVD. Of interest, arterial dissection–like structure was observed in the patient with ICH in this study, and aneurysm-like structure was observed in a patient with pontine hemorrhage.15 However, similar structures were absent in the other autopsy case who was free of hemorrhage events.10 This phenomenon indicated that the fragility of the vascular wall may lead to not only thrombosis but also hemorrhage in this disease. In addition, recent studies found that HTRA1 colocalize with NOTCH3 deposits in the brain vessels of patients with CADASIL26 and Aβ deposits in the capillaries of patients with cerebral amyloid angiopathy,27 but no deposits of amyloid or granular osmiophilic material were observed in this case and the 2 previously reported cases. It still remains unclear how the HTRA1 mutations eventually cause cerebral small vessel dysfunction and how these hereditary CSVD-related genes interact with each other.

Several cohort studies showed that for patients with CSVD without a NOTCH3 mutation, heterozygous HTRA1 mutations account for 3.5%–6.5% of familial cases and 0.8%–4.4% of sporadic cases worldwide.12 Preliminary screening of HTRA1 had been performed in various populations, showing that the heterozygous HTRA1 may be the second most prevalent genetic factor after NOTCH3.10,12 Here, we highlight the role of heterozygous HTRA1 in CSVD, and it is important to screen the mutations in the young patients with CSVD, atypical cases, and familial cases with unknown etiology. However, at the same time, cautious analyses of the pathogenicity of heterozygous HTRA1 variants are also required, as HTRA1 mutations were commonly seen in the Asian population, and many of them may be insufficient to cause CSVD. Previous studies have shown that both NOTCH3 and HTRA1 mutations will increase the lifetime risks of stroke and white matter lesion, and for NOTCH3, only the cysteine-altering variants are considered to cause CADASIL.28,29 However, for HTRA1, similar rules remain undetected. Thus, it is hard to predict the pathogenicity before classic clinical symptoms emerge, which cause the great difficulty in genetic counseling for individuals with heterozygous HTRA1 mutations.

In conclusion, the current study identified 3 novel mutations in HTRA1 and confirmed that heterozygous HTRA1 mutations are an important cause of CSVD. We also presented several novel phenotypes and pathologic findings by biopsy. The main limitation of this study is the relatively small numbers of cases, which may inflate the clinical heterogeneity. However, as this disease is rare and difficult to diagnose, it is hard to collect adequate cases. With the rapidly increased cases of heterozygous HTRA1-related CSVD, large-scale genetic screenings of HTRA1 in cases with CSVD from multicenters and in-depth experiments for mutations are urged to perform.

Study Funding

This work was funded by the National Natural Science Foundation (No. 82171412), Ministry of Science and Technology (2016YFC1306000), and Special Fund from Key Laboratory of Neurodegenerative Diseases, Ministry of Education of China (PXM2019_026283_000002) to C. Wang and Science and Technology Innovation 2030-Major Project (2021ZD0201806) and the Capital Health Research and Development of Special (2020-2-2014) to H. Song.

Disclosure

The authors declare no disclosures relevant to the manuscript and no competing interests. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NG.

Acknowledgment

The authors are grateful to all the patients and their families for volunteering for this study.

Appendix Authors

Table

Footnotes

  • Funding information and disclosures are provided at the end of the article. Full disclosure form information provided by the authors is available with the full text of this article at Neurology.org/NG.

  • The Article Processing Charge was funded by the authors.

  • ↵* These authors contributed equally to this work as co–first authors.

  • Submitted and externally peer reviewed. The handling editor was Suman Jayadev, MD.

  • Received April 18, 2022.
  • Accepted in final form September 28, 2022.

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

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