CADASIL2 and CARASIL via the HTRA1 Gene
- Summary and Pricing
- Clinical Features and Genetics
|Test Code||Test Copy Genes||Individual Gene Price||CPT Code Copy CPT Codes|
Our most cost-effective testing approach is NextGen sequencing with Sanger sequencing supplemented as needed to ensure sufficient coverage and to confirm NextGen calls that are pathogenic, likely pathogenic or of uncertain significance. If, however, full gene Sanger sequencing only is desired (for purposes of insurance billing or STAT turnaround time for example), please see link below for Test Code, pricing, and turnaround time information.
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The great majority of tests are completed within 20 days.
HTRA1 pathogenic variants have been reported in about 5% of patients with a clinical diagnosis of cerebral small vessel disease and no pathogenic variants in the NOTCH3 gene, referred to as CADASIL2 (Nozaki et al. 2016. PubMed ID: 27164673).
Homozygous and compound heterozygous HTRA1 pathogenic variants have been reported in several families with CARASIL (Cerebral Autosomal Recessive Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), which appears to be rare. To date, about 50 families with a history of the disease have been reported worldwide.
CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) was previously referred to as hereditary multi-infarct dementia, chronic familial vascular encephalopathy, and familial subcortical dementia with arteriopathic leukoencephalopathy. It is a primary vascular disease due to the degeneration of vascular smooth muscle cells (VSMC) mainly in the small arteries penetrating the white matter of the brain. The progressive loss of VSMC results in the thickening of the walls of the affected vessels and accumulation of granular osmiophilic material (GOM). This leads to reduced cerebral blood flow and progressive neurological deterioration (Stevens et al. 1977. PubMed ID: 69080; Davous and Fallet-Bianco. 1991. PubMed ID: 1853035).
CADASIL is clinically heterogeneous even among related affected individuals. Symptoms usually begin during the third and fourth decades of life (Chabriat et al. 1995. PubMed ID: 7564728). However, childhood and juvenile onset have been documented (Hartley et al. 2010. PubMed ID: 20197270; Cleves et al. 2010. PubMed ID: 21078731). The most common features are migraine, usually with aura; transient and recurrent subcortical ischemic strokes in the absence of hypertension; gait disturbance; urinary incontinence, psychiatric disturbances ranging from mood changes to severe depression; and progressive cognitive decline characterized by deterioration of executive function and working memory, and eventually dementia (Ruchoux et al. 1995. PubMed ID: 7676806; Lesnik Oberstein et al. 2001. PubMed ID: 11571335; Amberla et al. 2004. PubMed ID: 15143298).
Additional features include acute encephalopathy characterized by confusion, headache, numbness, fever, seizures, and coma; epilepsy; subclinical peripheral neuropathy; and asymptomatic retinal vascular abnormalities. Cardiovascular events have been reported in some patients (Lesnik Oberstein et al. 2003. PubMed ID: 12861102). MRI findings consist of lacunar infarcts and hyperintense lesions that appear first in the subcortical white matter and basal ganglia (Chabriat et al. 1995. PubMed ID: 7564728). Microbleeds have been documented (Choi et al. 2006. PubMed ID: 17135568; Rutten and Lesnik Oberstein, 2016. PubMed ID: 20301673).
The prevalence of CADASIL is unknown. However, it has been reported to account for ~11% of cases of lacunar infarcts with coexisting leukoencephalopathy in patients younger than 50 years of age (Dong et al. 2003. PubMed ID: 12511775). CADASIL affects individuals from various ethnic and geographic backgrounds (Ducros et al. 1996. PubMed ID: 8554054).
CADASIL is classified in two subtypes, CADASIL1 and 2, based on the genetic cause.
Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) was previously referred to as Maeda syndrome. It is a disease of the small blood vessels of the brain that ultimately leads to vascular dementia. The progressive loss of vascular smooth muscle cells (VSMC) results in the thickening and splitting of the internal elastic lamina. In CARASIL, there is no accumulation of granular osmiophilic material (GOM) that is characteristic of CADASIL (Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy).
Symptoms usually begin during the second and third decades of life. Most common symptoms include alopecia, ischemic strokes with no hypertension, premature baldness, severe low back pain, personality changes, cognitive deficit and dementia. Additional features include gait disturbance resulting from spasticity of the lower extremities, facial palsy, seizures, convulsions, keratosis, xeroderma, and pigmentary nevus. MRI findings consist of diffuse white matter changes and multiple lacunar infarctions in the basal ganglia and thalamus (Fukutake 2011. PMID: 21215656; Nozaki et al. 2014., PMID: 25116877)
CARASIL is rare and affects more males than females. To date about 50 cases have been reported in the literature (Hara et al. 2009. PubMed ID: 19387015; Tikka et al. 2014. PubMed ID: 25323668).
See also Onodera et al. 2014. PubMed ID: 20437615.
CADASIL is inherited in an autosomal dominant manner with high penetrance. It is classified in two subtypes, CADASIL1 and 2, based on the genetic cause.
CADASIL1 is caused by pathogenic variants in the NOTCH3 gene (Joutel et al. 1996. PubMed ID: 8878478). CADASIL 2 results from pathogenic variants in the HTRA1 gene (Verdura et al. 2015. PubMed ID: 26063658). CADASIL2 differs from CADASIL1 by a later age of onset, usually during the fifth and sixth decades in life.
To date, 13 missense and one splicing variants in the HTRA1 gene have been reported in several families with a history of autosomal dominant cerebral small vessel disease (CSVD), and features similar to that of NOTCH3-related CADASIL, with no pathogenic variants in NOTCH3 (Verdura et al. 2015. PubMed ID: 26063658; Nozaki et al. 2016. PubMed ID: 27164673). Subsequently, the phenotype was denoted CADASIL2.
CARASIL is inherited in an autosomal recessive manner. It is caused by pathogenic homozygous or compound heterozygous variants in the HTRA1 gene (Hara et al. 2009. PubMed ID: 19387015). Penetrance appears to be incomplete. White matter lesions of various severities have been observed in a number of clinically unaffected parents of CARASIL patients (Bianchi et al. 2014. PubMed ID: 24500651; Chen et al. 2013. PubMed ID: 23963851).
Although alopecia is a hallmark feature of CARASIL, HTRA1 pathogenic variants have been reported in three patients with a clinical diagnosis of CARASIL and no alopecia (Nishimoto et al. 2011. PubMed ID: 21482952; Bianchi et al. 2014. PubMed ID: 24500651).
A total of twelve pathogenic variants have been reported to date, which include 8 missense and 4 truncating variants. No large deletions or duplications have been reported (Human Gene Mutation Database).
Pathogenic variants in the HTRA1 have been reported in CARASIL patients from various ethnic and geographic backgrounds including Japanese, Chinese, Spanish, Portuguese, Turkish and Romanian (Tikka et al. 2014. PubMed ID: 25323668; Bianchi et al. 2014. PubMed ID: 24500651; Khaleeli et al. 2015. PubMed ID: 25957642; Menezes et al. 2015. PubMed ID: 25712943).
The HTRA1 gene encodes a serine protease that regulates transforming growth factor–beta signaling. In CARASIL, HTRA1 pathogenic variants result in decreased protease activity, which leads to dysregulation of TGF-beta signaling (Hara et al. 2009. PubMed PMID: 19387015). In CADASIL2 patients, HTRA1 variants result in decreased protease activity and in inhibited wild-type HTRA1 activity (Nozaki et al. 2016. PubMed ID: 27164673).
For this Next Generation Sequencing (NGS) test, sequencing is accomplished by capturing specific regions with an optimized solution-based hybridization kit, followed by massively parallel sequencing of the captured DNA fragments. Additional Sanger sequencing is performed for regions not captured or with insufficient number of sequence reads. All reported pathogenic, likely pathogenic, and variants of uncertain significance are confirmed by Sanger sequencing.
For Sanger sequencing, polymerase chain reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.
This test provides full coverage of all coding exons of the HTRA1 gene, plus ~10 bases of flanking noncoding DNA. We define full coverage as >20X NGS reads or Sanger sequencing.
Indications for Test
It has been suggested that screening for HTRA1 pathogenic variants should be considered in all patients with a hereditary small vessel disease of unknown etiology (Verdura et al. 2015. PubMed ID: 26063658).
|Official Gene Symbol||OMIM ID|
|Cerebral Arteriopathy, Autosomal Dominant, with Subcortical Infarcts and Leukoencephalopathy, Type 2||AD||616779|
- Genetic Counselor Team - email@example.com
- Khemissa Bejaoui, PhD - firstname.lastname@example.org
- Amberla et al. 2004. PubMed ID: 15143298
- Bianchi et al. 2014. PubMed ID: 24500651
- Chabriat et al. 1995. PubMed ID: 7564728
- Chen et al. 2013. PubMed ID: 23963851
- Choi et al. 2006. PubMed ID: 17135568
- Cleves et al. 2010. PubMed ID: 21078731
- Davous and Fallet-Bianco. 1991. PubMed ID: 1853035
- Dong et al. 2003. PubMed ID: 12511775
- Ducros et al. 1996. PubMed ID: 8554054
- Fukutake. 2011. PubMed ID: 21215656
- Hara et al. 2009. PubMed ID: 19387015
- Hartley et al. 2010. PubMed ID: 20197270
- Human Gene Mutation Database (Bio-base).
- Joutel et al. 1996. PubMed ID: 8878478
- Khaleeli et al. 2015. PubMed ID: 25957642
- Lesnik Oberstein et al. 2001. PubMed ID: 11571335
- Lesnik Oberstein et al. 2003. PubMed ID: 12861102
- Menezes et al. 2015. PubMed ID: 25712943
- Nishimoto et al. 2011. PubMed ID: 21482952
- Nozaki et al. 2014. PubMed ID: 25116877
- Nozaki et al. 2016. PubMed ID: 27164673
- Onodera et al. 2014. PubMed ID: 20437615
- Ruchoux et al. 1995. PubMed ID: 7676806
- Rutten and Lesnik Oberstein 2016. PubMed ID: 20301673
- Rutten et al. 2013. PubMed ID: 24000151
- Stevens et al. 1977. PubMed ID: 69080
- Tikka et al. 2014. PubMed ID: 25323668
- Verdura et al. 2015. PubMed ID: 26063658
NextGen Sequencing using PG-Select Capture Probes
We use a combination of Next Generation Sequencing (NGS) and Sanger sequencing technologies to cover the full coding regions of the listed genes plus ~20 bases of non-coding DNA flanking each exon. As required, genomic DNA is extracted from the patient specimen. For NGS, patient DNA corresponding to these regions is captured using an optimized set of DNA hybridization probes. Captured DNA is sequenced using Illumina’s Reversible Dye Terminator (RDT) platform (Illumina, San Diego, CA, USA). Regions with insufficient coverage by NGS are covered by Sanger sequencing. All pathogenic, likely pathogenic, or variants of uncertain significance are confirmed by Sanger sequencing.
For Sanger sequencing, Polymerase Chain Reaction (PCR) is used to amplify targeted regions. After purification of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in both the forward and reverse directions.
Patient DNA sequence is aligned to the genomic reference sequence for the indicated gene region(s). All differences from the reference sequences (sequence variants) are assigned to one of five interpretation categories, listed below, per ACMG Guidelines (Richards et al. 2015).
(1) Pathogenic Variants
(2) Likely Pathogenic Variants
(3) Variants of Uncertain Significance
(4) Likely Benign Variants
(5) Benign, Common Variants
Human Genome Variation Society (HGVS) recommendations are used to describe sequence variants (http://www.hgvs.org). Rare variants and undocumented variants are nearly always classified as likely benign if there is no indication that they alter protein sequence or disrupt splicing.
As of March 2016, 6.36 Mb of sequence (83 genes, 1557 exons) generated in our lab was compared between Sanger and NextGen methodologies. We detected no differences between the two methods. The comparison involved 6400 total sequence variants (differences from the reference sequences). Of these, 6144 were nucleotide substitutions and 256 were insertions or deletions. About 65% of the variants were heterozygous and 35% homozygous. The insertions and deletions ranged in length from 1 to over 100 nucleotides.
In silico validation of insertions and deletions in 20 replicates of 5 genes was also performed. The validation included insertions and deletions of lengths between 1 and 100 nucleotides. Insertions tested in silico: 2200 between 1 and 5 nucleotides, 625 between 6 and 10 nucleotides, 29 between 11 and 20 nucleotides, 25 between 21 and 49 nucleotides, and 23 at or greater than 50 nucleotides, with the largest at 98 nucleotides. All insertions were detected. Deletions tested in silico: 1813 between 1 and 5 nucleotides, 97 between 6 and 10 nucleotides, 32 between 11 and 20 nucleotides, 20 between 21 and 49 nucleotides, and 39 at or greater than 50 nucleotides, with the largest at 96 nucleotides. All deletions less than 50 nucleotides in length were detected, 13 greater than 50 nucleotides in length were missed. Our standard NextGen sequence variant calling algorithms are generally not capable of detecting insertions (duplications) or heterozygous deletions greater than 100 nucleotides. Large homozygous deletions appear to be detectable.
Interpretation of the test results is limited by the information that is currently available. Better interpretation should be possible in the future as more data and knowledge about human genetics and this specific disorder are accumulated.
When Sanger sequencing does not reveal any difference from the reference sequence, or when a sequence variant is homozygous, we cannot be certain that we were able to detect both patient alleles. Occasionally, a patient may carry an allele which does not amplify, due to a large deletion or insertion. In these cases, the report will contain no information about the second allele. Our Sanger and NGS Sequencing tests are generally not capable of detecting Copy Number Variants (CNVs).
We sequence all coding exons for each given transcript, plus ~20 bp of flanking non-coding DNA for each exon. Test reports contain no information about other portions of the gene, such as regulatory domains, deep intronic regions or any currently uncharacterized alternative exons.
In most cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.
Our ability to detect minor sequence variants due to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.
Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR.
Unless otherwise indicated, DNA sequence data is obtained from a specific cell-type (usually leukocytes from whole blood). Test reports contain no information about the DNA sequence in other cell-types.
We cannot be certain that the reference sequences are correct.
Rare, low probability interpretations of sequencing results, such as for example the occurrence of de novo mutations in recessive disorders, are generally not included in the reports.
We have confidence in our ability to track a specimen once it has been received by PreventionGenetics. However, we take no responsibility for any specimen labeling errors that occur before the sample arrives at PreventionGenetics.
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- A completed requisition form must accompany all specimens.
- Billing information along with specimen and shipping instructions are within the requisition form.
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(Delivery accepted Monday - Saturday)
- Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
- For small babies, we require a minimum of 1 ml of blood.
- Only one blood tube is required for multiple tests.
- Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
- During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
- In cold weather, include an unfrozen ice pack in the shipping container as insulation.
- At room temperature, blood specimen is stable for up to 48 hours.
- If refrigerated, blood specimen is stable for up to one week.
- Label the tube with the patient name, date of birth and/or ID number.
(Delivery accepted Monday - Saturday)
- Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
- For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
- DNA may be shipped at room temperature.
- Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
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(Delivery preferred Monday - Thursday)
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- Culture and send at least two T25 flasks of confluent cells.
- Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
- Send specimens in insulated, shatterproof container overnight.
- Cell cultures may be shipped at room temperature or refrigerated.
- Label the flasks with the patient name, date of birth, and/or ID number.
- We strongly recommend maintaining a local back-up culture. We do not culture cells.