Neurol Sci DOI 10.1007/s10072-015-2121-5
ORIGINAL ARTICLE
A frameshift mutation in HTRA1 expands CARASIL syndrome and peripheral small arterial disease to the Chinese population Bin Cai • Jiabin Zeng • Yi Lin • Yu Lin • WenPing Lin Wei Lin • Zhiwen Li • Ning Wang
•
Received: 13 December 2014 / Accepted: 20 February 2015 Ó Springer-Verlag Italia 2015
Abstract Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a rare hereditary cerebral artery disease. The HtrA serine protease 1 (HTRA1) gene has been identified as the causative gene of CARASIL. Here, we report a novel mutation in the HTRA1 gene in a CARASIL pedigree and explore its pathogenesis at the protein level. Subcutaneous tissue biopsy and HTRA1 gene analysis were performed in a CARASIL patient, and HTRA1 and TGF-b1 protein expression in subcutaneous tissue and cultured fibroblasts from the proband were detected by immunohistochemistry and western blotting. A 28-year-old male proband and his brother experienced recurrent stroke, hair loss and low back pain. Abnormalities in the proband were found in the elastic plate of subcutaneous small arteries, and a novel homozygous frameshift mutation (c.161_162insAG), leading to the formation of a stop codon 159 amino acids downstream of the insertion (p.Gly56Alafs*160) was detected. Reduced HTRA1 protein and increased TGF-b1
B. Cai, J. Zeng and Y. Lin contributed equally. B. Cai Y. Lin Y. Lin W. Lin Z. Li (&) N. Wang (&) Department of Neurology and Institute of Neurology, First Affiliated Hospital, Fujian Medical University, 20 Chazhong Road, Fuzhou 350005, Fujian, China e-mail: [email protected] N. Wang e-mail: [email protected] J. Zeng Department of Paediatrics, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, China W. Lin Department of Orthopedic Surgery, Second Affiliated Hospital, Fujian Medical University, Quanzhou, Fujian, China
expression were detected in subcutaneous tissue and in cultured fibroblasts. A frameshift mutation in the HTRA1 gene detected in a CARASIL pedigree resulted in reduced HTRA1 protein and increased TGF-b1 expression, which may cause severe CARASIL and peripheral small arterial disease. Keywords Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) Fibroblasts Gene mutation Genetic disease HTRA1 TGF
Introduction Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a rare single-gene disorder directly affecting the cerebral small vessels, is characterized by nonhypertensive cerebral small vessel arteriopathy with subcortical infarcts, alopecia and spondylosis, and has an onset in early adulthood [1, 2]. The first CARASIL patients were most likely described in preliminary reports in 1965, and approximately 50 patients, primarily from Japan, have been reported; more patients are also being reported in other populations [3–5]. In 2009, the HtrA serine protease 1 gene (HTRA1) was identified by Hara et al. [6] as the causative gene of CARASIL. The HTRA1 protein is a serine protease that represses signaling by TGF-b family members. Some new mutations were reported later, though to the best of our knowledge, only one frameshift mutation has been reported. Additionally, few studies on the function of the mutation are available. Here, we report a patient with CARASIL harboring a frameshift mutation in HTRA1 that leads to the production of an inactive HTRA1 protein
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Neurol Sci
A 28-year-old male proband (II-10) was admitted to a local hospital in 2012 because of sudden lower limb weakness and thigh numbness for 2 months. Brain MRI revealed multiple abnormal signals, and the patient was diagnosed with multiple sclerosis (MS) and treated with 500 mg methylprednisone for 5 days. He experienced hair loss, starting 10 years prior, which developed into alopecia (Fig. 1a). The patient experienced low back pain and was diagnosed with a lumbar herniated disc by CT scan. No hypertension, diabetes mellitus or dyslipidemia was recorded. He had no history of cigarette smoking or alcohol
use. His parents had a consanguineous marriage, and their neuroimaging examination was normal. The proband was subsequently admitted in our department with the presence of diffuse pyramidal signs, including bilateral Babinski sign and left ankle clonus. A follow-up MRI showed diffuse leukoencephalopathy, subcortical infarcts and microbleeds (Fig. 1b); VEP was normal, and oligoclonal bands in CSF were absent. The diagnosis of MS was ruled out, and the hypothesis of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) was first considered. Transcranial Doppler (TCD) indicated increased cerebrovascular resistance. Ultrasonic Doppler showed bilateral vertebral arteries supplying a high-resistance vascular bed. Single-photon emission computed tomography (SPECT) revealed reduced perfusion in the left fronto-temporal lobe. Additionally, spine MRI showed degenerative disc disease (Fig. 1c, d). However, no NOTCH3 mutation in the blood or ultrastructural granular osmiophilic material (GOM) on the vascular
Fig. 1 The pedigree, brain MRI and gene analysis of the proband. a The proband developed alopecia with male pattern baldness. b Brain MRI of the proband: T1-weighted (a; e), T2-weighted (b; f) and fluid attenuated inversion recovery (FLAIR), c MRI showed diffuse leukoencephalopathy involving the periventricular and deep white matter, including the anterior temporal lobes and external capsules, and multiple lacunar infarcts in the brain hemispheres and brainstem. No significant strengthening of enhanced lesions (g); susceptibility-weighted image (SWI), d of MRI showed multiple cerebral microbleeds (CMBs) on the pons, basal ganglia and hemispheric subcortical white matter. A brain magnetic resonance
angiogram (MRA) showed no significant stenosis of large cerebral arteries (h). c, d Cervical and lumbar spine MRI of the proband. T2weighted images show degenerative disc disease, including disc herniations and the degeneration of vertebral bodies. e The pedigree of the Chinese family with CARASIL. The proband is indicated by the arrow; the solid symbols denote the patient with CARASIL, and the symbol with a slash indicates the deceased individual. Double horizontal lines consanguineous marriage; hash unaffected individuals with a heterozygous c.161_162insAG mutation. f Mutation analysis of HTRA1 in the proband identified a novel homozygous mutation in exon 1 (c.161_162insAG)
(p.Gly56Alafs*160). Additionally, we investigated the pathology of small arteries in the subcutaneous tissue and expression of HTRA1 and TGF-b1 in fibroblasts and subcutaneous tissue from the proband by western blotting and immunohistochemistry (IHC).
Case report
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Neurol Sci
wall was found, which excluded CADASIL. Regardless, scalp alopecia in his teenage years and acute lower back pain, which are characteristic extraneurologic signs of CARASIL, suggested the diagnosis of CARASIL; thus, HTRA1 gene analysis was performed to confirm the diagnosis. The older brother of the proband was admitted to our department because of a similar neurological history (Fig. 1e) in 2002, when he was 29 years old. He also developed alopecia and a lumbar herniated disc. The neurological examination and MRI results were similar to the proband, and he was diagnosed with MS and treated with prednisone. However, pseudobulbar syndrome and tetraparesis progressed until he was bedridden at age 30 and died at age 32.
HTRA1 and TGF-b1 protein expression in subcutaneous tissue and cultured fibroblasts Fibroblasts from subcutaneous tissue of the proband were cultured in vitro. HTRA1 and TGF-b1 protein expression in the fibroblasts and subcutaneous tissue were detected by immunohistochemistry (IHC) using rabbit anti-HTRA1 polyclonal (1:50, Proteintech) or rabbit anti-TGF-b1 polyclonal (1:100, Santa Cruz) primary antibody. HTRA1 and TGF-b1 protein expression in the subcutaneous tissue was also detected by western blotting, as previously described [7], using primary antibodies against HTRA1 (1:300) and TGF-b1 (1:200). Horseradish peroxidase-labeled anti-rabbit secondary antibody (1:3000, Sigma, St Louis, MO, USA) was used to detect immunoreactivity.
Materials and methods Results This study was approved by the Ethics Committee of the First Affiliated Hospital, Fujian Medical University. Written informed consent was obtained from the proband, his family members and the control subjects. Genetic analysis Blood samples of the proband and his family members and fibroblasts from the subcutaneous tissue of the proband were obtained, and genomic DNA was extracted using QIAamp DNA Blood Mini Kit (Qiagen, Germany). Mutation analysis in the proband and his parents was performed on the entire coding region and exon boundaries of the HTRA1 gene by direct sequencing. Additionally, 120 unrelated healthy individuals of matching Han Chinese ancestry were enrolled in this study. These control subjects were between 50 and 70 years of age and had no history of stroke or leukoencephalopathy, as identified by clinical evaluation. Pathological analysis of subcutaneous tissue To assess the degree of vessel occlusion, formalin-fixed, paraffin-embedded subcutaneous tissue slides of lower leg samples from the proband and control were examined by H&E staining or elastic van Gieson staining according to a standardized histological protocol. We carried out the general two-step IHC staining on subcutaneous tissue slides according to the manufacturer’s protocol; a rabbit anti-actin polyclonal antibody (1:100, Santa Cruz) was incubated with the samples overnight at 4 °C. Control subcutaneous tissue was acquired from a leg injury patient.
Identification and characterization of the mutation HTRA1 mutation screening in blood samples and fibroblasts from the proband revealed a novel homozygous frameshift mutation in exon 1 (c.161_162insAG) (Fig. 1e). We believe that the novel homozygous mutation is not a gene polymorphism because it was not detected in 240 control chromosomes and was not described in SNP databases. This mutation leads to a frameshift that results in a stop codon 159 amino acids downstream of the insertion (p.Gly56Alafs*160), producing an inactivate HTRA1 protein (p.Gly56Alafs*160). The identical heterozygous c.161_162insAG mutation was detected in the patient’s unaffected parents (I-1 and I-2), one sister (II-4) and his son (III-5) (Fig. 1f). Unfortunately, the brother (II-2) who developed the typical clinical manifestations of CARASIL passed away 9 years ago, and no DNA sample was available for the mutation analysis. To rule out CADASIL, the exons of the NOTCH3 gene were screened in the proband, and no pathogenic mutation was identified. Pathology of subcutaneous tissue Compared with the control (Fig. 2a–c), the pathological analysis of the subcutaneous tissue from the proband demonstrated intimal thickening of the arterioles (Fig. 2d), and elastic van Gieson staining demonstrated a discontinuous internal elastic membrane (Fig. 2e). IHC with an anti-actin antibody revealed arteries with widespread loss of vascular smooth muscle cells (SMCs) (Fig. 2f).
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Neurol Sci
Reduced HTRA1 and increased TGF-b1 protein in the proband’s subcutaneous tissue and cultured fibroblasts HTRA1 protein was detectable in the subcutaneous tissue from the control but not from the proband by IHC and western blotting; conversely, TGF-b1 protein was detected in the subcutaneous tissue from the proband but not from the control (Fig. 3a, c). The results were similar for the cultured fibroblasts by immunohistochemical analysis: HTRA1 protein was detectable in the cultured fibroblasts from the control but not from the proband, whereas TGF-b1 protein was detected in the cultured fibroblasts from the proband but not from the control (Fig. 3b).
Fig. 2 Pathological analysis of the proband’s subcutaneous tissue. H&E staining demonstrated intimal thickening of the arterioles (d); elastic van Gieson staining demonstrated discontinuous internal elastic membrane (e); IHC with an anti-actin antibody showed arteries with widespread loss of vascular smooth muscle cells (SMCs) (f). The wavy appearance of the internal elastic lamina is a normally occurring artifact caused by the contraction of medial SMCs at the time of tissue fixation (b). Scale bar 100 lm)
Fig. 3 HTRA1 and TGF-b1 protein expression in the proband’s subcutaneous tissue and cultured fibroblasts. a, b IHC of subcutaneous tissue: subcutaneous tissue (a) or cultured fibroblasts (b) obtained from the proband and the control stained with an antiHTRA1 or anti-TGF-b1 antibody. c Western blot of the subcutaneous tissue obtained from the proband and the control using an anti-HTRA1 or anti-TGF-b1 antibody
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Discussion Both of our cases were misdiagnosed and initially treated as MS because of cerebral white matter lesions on MRI. Nonetheless, the diagnosis of CARASIL was confirmed by a novel homozygous mutation in exon 1 (c.161_162insAG) of the HTRA1 gene detected in the proband. The deleterious effect of this mutation is supported by the following arguments. First, this mutation leads to a frameshift that results in the formation of a stop codon 159 amino acids downstream of the insertion (p.Gly56Alafs*160) and produces a truncated protein without the proteolytic (trypsin-like) and PDZ domains. As the serine protease activity of HTRA1 is required to inhibit TGF-b family signaling, the loss of
Neurol Sci
HTRA1 protein caused by this mutation will result in the failure to repress signaling by the TGF-b family, which increases TGF-b1 expression. TGF-b family signaling is closely associated with vascular angiogenesis and remodeling; thus, the increased TGF-b1 expression will result in ischemic cerebral small vessel disease. Second, SPECT showed reduced regional cerebral blood flow, and Ultrasonic Doppler and TCD showed increased cerebrovascular resistance (data not shown); all of these tests suggested the stenosis of cerebral small arteries. Arteriosclerosis observed in cerebral small arteries, including the thickening and splitting of the internal elastic lamina, the loss of vascular SMCs and hyaline degeneration of the media, are characteristic features of CARASIL [8]. Arteriosclerosis was also reported in the peripheral small arteries in some other organs but not in the skin. We performed a skin biopsy and found peripheral small arterial disease in the subcutaneous tissue, including the loss of vascular SMCs and thickening and splitting of the internal elastic lamina, which was consistent with the results reported previously [3, 9]. Third, we found decreased HTRA1 and increased TGFb1 protein expression in the subcutaneous tissue and cultured fibroblasts by IHC and western blotting, results that may be attributable to the HTRA1 mutation. We consider that the small arterial disease in the subcutaneous tissue may be caused by the mutation in HTRA1 because this protein is expressed in the skin as well as in the brain [10]. CARASIL should be considered in the differential diagnosis of patients with previously suspected atypical MS and the presence of extraneurological signs, including scalp alopecia in the teens and acute mid to lower back pain. The diagnosis can be confirmed by HTRA1 molecular genetic testing. In conclusion, a new frameshift mutation in the HTRA1 gene detected in a CARASIL pedigree resulted in decreased HTRA1 and increased TGF-b1 protein expression, which may cause CARASIL and peripheral small arterial disease. Acknowledgments This work was Grant No. 81171114 of Natural Science Foundation of China (BC), Grant No. 2014-ZQN-ZD-18 of Fujian Provincial Medical Project for Middle-aged and Young Talents
(BC), Grant No. 2011-CXB-12 of Fujian Provincial medical innovation Project (BC), Grant No. 81100838 of Natural Science Foundation of China(YL), Grant No. 81201403 of Natural Science Foundation of China (WPL), National key clinical specialty discipline construction program and Fujian key clinical specialty discipline construction program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to acknowledge Ji Ming-kai (Department of Dermatology, First Affiliated Hospital, Fujian Medical University) for performing the skin biopsy. Conflict of interest of interest.
The authors declare that they have no conflict
References 1. Yanagawa S, Ito N, Arima K, Ikeda S (2002) Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy. Neurology 58(5):817–820 2. Nozaki H, Nishizawa M, Onodera O (2014) Features of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy. Stroke. doi:10.1161/STROKEAHA.114. 004236 3. Wang XL, Li CF, Guo HW, Cao BZ (2012) A novel mutation in the HTRA1 gene identified in Chinese CARASIL pedigree. CNS Neurosci Ther 18(10):867–869 4. Mendioroz M, Fernandez-Cadenas I, Del RA et al (2010) A missense HTRA1 mutation expands CARASIL syndrome to the Caucasian population. Neurology 75(22):2033–2035 5. Bianchi S, Di PC, Gallus GN et al (2014) Two novel HTRA1 mutations in a European CARASIL patient. Neurology 82(10): 898–900 6. Hara K, Shiga A, Fukutake T et al (2009) Association of HTRA1 mutations and familial ischemic cerebral small-vessel disease. N Engl J Med 360(17):1729–1739 7. Cai B, Lin Y, Xue XH, Fang L, Wang N, Wu ZY (2011) TATmediated delivery of neuroglobin protects against focal cerebral ischemia in mice. Exp Neurol 227(1):224–231 8. Oide T, Nakayama H, Yanagawa S, Ito N, Ikeda S, Arima K (2008) Extensive loss of arterial medial smooth muscle cells and mural extracellular matrix in cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Neuropathology 28(2):132–142 9. Yanagawa S, Ito N, Arima K, Ikeda S (2002) Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy. Neurology 58(5):817–820 10. De Luca A, De Falco M, Severino A et al (2003) Distribution of the serine protease HtrA1 in normal human tissues. J Histochem Cytochem 51(10):1279–1284
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ORIGINAL ARTICLE
A frameshift mutation in HTRA1 expands CARASIL syndrome and peripheral small arterial disease to the Chinese population Bin Cai • Jiabin Zeng • Yi Lin • Yu Lin • WenPing Lin Wei Lin • Zhiwen Li • Ning Wang
•
Received: 13 December 2014 / Accepted: 20 February 2015 Ó Springer-Verlag Italia 2015
Abstract Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a rare hereditary cerebral artery disease. The HtrA serine protease 1 (HTRA1) gene has been identified as the causative gene of CARASIL. Here, we report a novel mutation in the HTRA1 gene in a CARASIL pedigree and explore its pathogenesis at the protein level. Subcutaneous tissue biopsy and HTRA1 gene analysis were performed in a CARASIL patient, and HTRA1 and TGF-b1 protein expression in subcutaneous tissue and cultured fibroblasts from the proband were detected by immunohistochemistry and western blotting. A 28-year-old male proband and his brother experienced recurrent stroke, hair loss and low back pain. Abnormalities in the proband were found in the elastic plate of subcutaneous small arteries, and a novel homozygous frameshift mutation (c.161_162insAG), leading to the formation of a stop codon 159 amino acids downstream of the insertion (p.Gly56Alafs*160) was detected. Reduced HTRA1 protein and increased TGF-b1
B. Cai, J. Zeng and Y. Lin contributed equally. B. Cai Y. Lin Y. Lin W. Lin Z. Li (&) N. Wang (&) Department of Neurology and Institute of Neurology, First Affiliated Hospital, Fujian Medical University, 20 Chazhong Road, Fuzhou 350005, Fujian, China e-mail: [email protected] N. Wang e-mail: [email protected] J. Zeng Department of Paediatrics, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, China W. Lin Department of Orthopedic Surgery, Second Affiliated Hospital, Fujian Medical University, Quanzhou, Fujian, China
expression were detected in subcutaneous tissue and in cultured fibroblasts. A frameshift mutation in the HTRA1 gene detected in a CARASIL pedigree resulted in reduced HTRA1 protein and increased TGF-b1 expression, which may cause severe CARASIL and peripheral small arterial disease. Keywords Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) Fibroblasts Gene mutation Genetic disease HTRA1 TGF
Introduction Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a rare single-gene disorder directly affecting the cerebral small vessels, is characterized by nonhypertensive cerebral small vessel arteriopathy with subcortical infarcts, alopecia and spondylosis, and has an onset in early adulthood [1, 2]. The first CARASIL patients were most likely described in preliminary reports in 1965, and approximately 50 patients, primarily from Japan, have been reported; more patients are also being reported in other populations [3–5]. In 2009, the HtrA serine protease 1 gene (HTRA1) was identified by Hara et al. [6] as the causative gene of CARASIL. The HTRA1 protein is a serine protease that represses signaling by TGF-b family members. Some new mutations were reported later, though to the best of our knowledge, only one frameshift mutation has been reported. Additionally, few studies on the function of the mutation are available. Here, we report a patient with CARASIL harboring a frameshift mutation in HTRA1 that leads to the production of an inactive HTRA1 protein
123
Neurol Sci
A 28-year-old male proband (II-10) was admitted to a local hospital in 2012 because of sudden lower limb weakness and thigh numbness for 2 months. Brain MRI revealed multiple abnormal signals, and the patient was diagnosed with multiple sclerosis (MS) and treated with 500 mg methylprednisone for 5 days. He experienced hair loss, starting 10 years prior, which developed into alopecia (Fig. 1a). The patient experienced low back pain and was diagnosed with a lumbar herniated disc by CT scan. No hypertension, diabetes mellitus or dyslipidemia was recorded. He had no history of cigarette smoking or alcohol
use. His parents had a consanguineous marriage, and their neuroimaging examination was normal. The proband was subsequently admitted in our department with the presence of diffuse pyramidal signs, including bilateral Babinski sign and left ankle clonus. A follow-up MRI showed diffuse leukoencephalopathy, subcortical infarcts and microbleeds (Fig. 1b); VEP was normal, and oligoclonal bands in CSF were absent. The diagnosis of MS was ruled out, and the hypothesis of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) was first considered. Transcranial Doppler (TCD) indicated increased cerebrovascular resistance. Ultrasonic Doppler showed bilateral vertebral arteries supplying a high-resistance vascular bed. Single-photon emission computed tomography (SPECT) revealed reduced perfusion in the left fronto-temporal lobe. Additionally, spine MRI showed degenerative disc disease (Fig. 1c, d). However, no NOTCH3 mutation in the blood or ultrastructural granular osmiophilic material (GOM) on the vascular
Fig. 1 The pedigree, brain MRI and gene analysis of the proband. a The proband developed alopecia with male pattern baldness. b Brain MRI of the proband: T1-weighted (a; e), T2-weighted (b; f) and fluid attenuated inversion recovery (FLAIR), c MRI showed diffuse leukoencephalopathy involving the periventricular and deep white matter, including the anterior temporal lobes and external capsules, and multiple lacunar infarcts in the brain hemispheres and brainstem. No significant strengthening of enhanced lesions (g); susceptibility-weighted image (SWI), d of MRI showed multiple cerebral microbleeds (CMBs) on the pons, basal ganglia and hemispheric subcortical white matter. A brain magnetic resonance
angiogram (MRA) showed no significant stenosis of large cerebral arteries (h). c, d Cervical and lumbar spine MRI of the proband. T2weighted images show degenerative disc disease, including disc herniations and the degeneration of vertebral bodies. e The pedigree of the Chinese family with CARASIL. The proband is indicated by the arrow; the solid symbols denote the patient with CARASIL, and the symbol with a slash indicates the deceased individual. Double horizontal lines consanguineous marriage; hash unaffected individuals with a heterozygous c.161_162insAG mutation. f Mutation analysis of HTRA1 in the proband identified a novel homozygous mutation in exon 1 (c.161_162insAG)
(p.Gly56Alafs*160). Additionally, we investigated the pathology of small arteries in the subcutaneous tissue and expression of HTRA1 and TGF-b1 in fibroblasts and subcutaneous tissue from the proband by western blotting and immunohistochemistry (IHC).
Case report
123
Neurol Sci
wall was found, which excluded CADASIL. Regardless, scalp alopecia in his teenage years and acute lower back pain, which are characteristic extraneurologic signs of CARASIL, suggested the diagnosis of CARASIL; thus, HTRA1 gene analysis was performed to confirm the diagnosis. The older brother of the proband was admitted to our department because of a similar neurological history (Fig. 1e) in 2002, when he was 29 years old. He also developed alopecia and a lumbar herniated disc. The neurological examination and MRI results were similar to the proband, and he was diagnosed with MS and treated with prednisone. However, pseudobulbar syndrome and tetraparesis progressed until he was bedridden at age 30 and died at age 32.
HTRA1 and TGF-b1 protein expression in subcutaneous tissue and cultured fibroblasts Fibroblasts from subcutaneous tissue of the proband were cultured in vitro. HTRA1 and TGF-b1 protein expression in the fibroblasts and subcutaneous tissue were detected by immunohistochemistry (IHC) using rabbit anti-HTRA1 polyclonal (1:50, Proteintech) or rabbit anti-TGF-b1 polyclonal (1:100, Santa Cruz) primary antibody. HTRA1 and TGF-b1 protein expression in the subcutaneous tissue was also detected by western blotting, as previously described [7], using primary antibodies against HTRA1 (1:300) and TGF-b1 (1:200). Horseradish peroxidase-labeled anti-rabbit secondary antibody (1:3000, Sigma, St Louis, MO, USA) was used to detect immunoreactivity.
Materials and methods Results This study was approved by the Ethics Committee of the First Affiliated Hospital, Fujian Medical University. Written informed consent was obtained from the proband, his family members and the control subjects. Genetic analysis Blood samples of the proband and his family members and fibroblasts from the subcutaneous tissue of the proband were obtained, and genomic DNA was extracted using QIAamp DNA Blood Mini Kit (Qiagen, Germany). Mutation analysis in the proband and his parents was performed on the entire coding region and exon boundaries of the HTRA1 gene by direct sequencing. Additionally, 120 unrelated healthy individuals of matching Han Chinese ancestry were enrolled in this study. These control subjects were between 50 and 70 years of age and had no history of stroke or leukoencephalopathy, as identified by clinical evaluation. Pathological analysis of subcutaneous tissue To assess the degree of vessel occlusion, formalin-fixed, paraffin-embedded subcutaneous tissue slides of lower leg samples from the proband and control were examined by H&E staining or elastic van Gieson staining according to a standardized histological protocol. We carried out the general two-step IHC staining on subcutaneous tissue slides according to the manufacturer’s protocol; a rabbit anti-actin polyclonal antibody (1:100, Santa Cruz) was incubated with the samples overnight at 4 °C. Control subcutaneous tissue was acquired from a leg injury patient.
Identification and characterization of the mutation HTRA1 mutation screening in blood samples and fibroblasts from the proband revealed a novel homozygous frameshift mutation in exon 1 (c.161_162insAG) (Fig. 1e). We believe that the novel homozygous mutation is not a gene polymorphism because it was not detected in 240 control chromosomes and was not described in SNP databases. This mutation leads to a frameshift that results in a stop codon 159 amino acids downstream of the insertion (p.Gly56Alafs*160), producing an inactivate HTRA1 protein (p.Gly56Alafs*160). The identical heterozygous c.161_162insAG mutation was detected in the patient’s unaffected parents (I-1 and I-2), one sister (II-4) and his son (III-5) (Fig. 1f). Unfortunately, the brother (II-2) who developed the typical clinical manifestations of CARASIL passed away 9 years ago, and no DNA sample was available for the mutation analysis. To rule out CADASIL, the exons of the NOTCH3 gene were screened in the proband, and no pathogenic mutation was identified. Pathology of subcutaneous tissue Compared with the control (Fig. 2a–c), the pathological analysis of the subcutaneous tissue from the proband demonstrated intimal thickening of the arterioles (Fig. 2d), and elastic van Gieson staining demonstrated a discontinuous internal elastic membrane (Fig. 2e). IHC with an anti-actin antibody revealed arteries with widespread loss of vascular smooth muscle cells (SMCs) (Fig. 2f).
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Reduced HTRA1 and increased TGF-b1 protein in the proband’s subcutaneous tissue and cultured fibroblasts HTRA1 protein was detectable in the subcutaneous tissue from the control but not from the proband by IHC and western blotting; conversely, TGF-b1 protein was detected in the subcutaneous tissue from the proband but not from the control (Fig. 3a, c). The results were similar for the cultured fibroblasts by immunohistochemical analysis: HTRA1 protein was detectable in the cultured fibroblasts from the control but not from the proband, whereas TGF-b1 protein was detected in the cultured fibroblasts from the proband but not from the control (Fig. 3b).
Fig. 2 Pathological analysis of the proband’s subcutaneous tissue. H&E staining demonstrated intimal thickening of the arterioles (d); elastic van Gieson staining demonstrated discontinuous internal elastic membrane (e); IHC with an anti-actin antibody showed arteries with widespread loss of vascular smooth muscle cells (SMCs) (f). The wavy appearance of the internal elastic lamina is a normally occurring artifact caused by the contraction of medial SMCs at the time of tissue fixation (b). Scale bar 100 lm)
Fig. 3 HTRA1 and TGF-b1 protein expression in the proband’s subcutaneous tissue and cultured fibroblasts. a, b IHC of subcutaneous tissue: subcutaneous tissue (a) or cultured fibroblasts (b) obtained from the proband and the control stained with an antiHTRA1 or anti-TGF-b1 antibody. c Western blot of the subcutaneous tissue obtained from the proband and the control using an anti-HTRA1 or anti-TGF-b1 antibody
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Discussion Both of our cases were misdiagnosed and initially treated as MS because of cerebral white matter lesions on MRI. Nonetheless, the diagnosis of CARASIL was confirmed by a novel homozygous mutation in exon 1 (c.161_162insAG) of the HTRA1 gene detected in the proband. The deleterious effect of this mutation is supported by the following arguments. First, this mutation leads to a frameshift that results in the formation of a stop codon 159 amino acids downstream of the insertion (p.Gly56Alafs*160) and produces a truncated protein without the proteolytic (trypsin-like) and PDZ domains. As the serine protease activity of HTRA1 is required to inhibit TGF-b family signaling, the loss of
Neurol Sci
HTRA1 protein caused by this mutation will result in the failure to repress signaling by the TGF-b family, which increases TGF-b1 expression. TGF-b family signaling is closely associated with vascular angiogenesis and remodeling; thus, the increased TGF-b1 expression will result in ischemic cerebral small vessel disease. Second, SPECT showed reduced regional cerebral blood flow, and Ultrasonic Doppler and TCD showed increased cerebrovascular resistance (data not shown); all of these tests suggested the stenosis of cerebral small arteries. Arteriosclerosis observed in cerebral small arteries, including the thickening and splitting of the internal elastic lamina, the loss of vascular SMCs and hyaline degeneration of the media, are characteristic features of CARASIL [8]. Arteriosclerosis was also reported in the peripheral small arteries in some other organs but not in the skin. We performed a skin biopsy and found peripheral small arterial disease in the subcutaneous tissue, including the loss of vascular SMCs and thickening and splitting of the internal elastic lamina, which was consistent with the results reported previously [3, 9]. Third, we found decreased HTRA1 and increased TGFb1 protein expression in the subcutaneous tissue and cultured fibroblasts by IHC and western blotting, results that may be attributable to the HTRA1 mutation. We consider that the small arterial disease in the subcutaneous tissue may be caused by the mutation in HTRA1 because this protein is expressed in the skin as well as in the brain [10]. CARASIL should be considered in the differential diagnosis of patients with previously suspected atypical MS and the presence of extraneurological signs, including scalp alopecia in the teens and acute mid to lower back pain. The diagnosis can be confirmed by HTRA1 molecular genetic testing. In conclusion, a new frameshift mutation in the HTRA1 gene detected in a CARASIL pedigree resulted in decreased HTRA1 and increased TGF-b1 protein expression, which may cause CARASIL and peripheral small arterial disease. Acknowledgments This work was Grant No. 81171114 of Natural Science Foundation of China (BC), Grant No. 2014-ZQN-ZD-18 of Fujian Provincial Medical Project for Middle-aged and Young Talents
(BC), Grant No. 2011-CXB-12 of Fujian Provincial medical innovation Project (BC), Grant No. 81100838 of Natural Science Foundation of China(YL), Grant No. 81201403 of Natural Science Foundation of China (WPL), National key clinical specialty discipline construction program and Fujian key clinical specialty discipline construction program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to acknowledge Ji Ming-kai (Department of Dermatology, First Affiliated Hospital, Fujian Medical University) for performing the skin biopsy. Conflict of interest of interest.
The authors declare that they have no conflict
References 1. Yanagawa S, Ito N, Arima K, Ikeda S (2002) Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy. Neurology 58(5):817–820 2. Nozaki H, Nishizawa M, Onodera O (2014) Features of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy. Stroke. doi:10.1161/STROKEAHA.114. 004236 3. Wang XL, Li CF, Guo HW, Cao BZ (2012) A novel mutation in the HTRA1 gene identified in Chinese CARASIL pedigree. CNS Neurosci Ther 18(10):867–869 4. Mendioroz M, Fernandez-Cadenas I, Del RA et al (2010) A missense HTRA1 mutation expands CARASIL syndrome to the Caucasian population. Neurology 75(22):2033–2035 5. Bianchi S, Di PC, Gallus GN et al (2014) Two novel HTRA1 mutations in a European CARASIL patient. Neurology 82(10): 898–900 6. Hara K, Shiga A, Fukutake T et al (2009) Association of HTRA1 mutations and familial ischemic cerebral small-vessel disease. N Engl J Med 360(17):1729–1739 7. Cai B, Lin Y, Xue XH, Fang L, Wang N, Wu ZY (2011) TATmediated delivery of neuroglobin protects against focal cerebral ischemia in mice. Exp Neurol 227(1):224–231 8. Oide T, Nakayama H, Yanagawa S, Ito N, Ikeda S, Arima K (2008) Extensive loss of arterial medial smooth muscle cells and mural extracellular matrix in cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Neuropathology 28(2):132–142 9. Yanagawa S, Ito N, Arima K, Ikeda S (2002) Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy. Neurology 58(5):817–820 10. De Luca A, De Falco M, Severino A et al (2003) Distribution of the serine protease HtrA1 in normal human tissues. J Histochem Cytochem 51(10):1279–1284
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