Novel CHP1 mutation in autosomal-recessive cerebellar ataxia: autopsy features of two siblings.,Acta Neuropathologica Communications

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Novel CHP1 mutation in autosomal-recessive cerebellar ataxia: autopsy features of two siblings.
Acta Neuropathologica Communications ( IF 7.1 ) Pub Date : 2020-08-12 , DOI: 10.1186/s40478-020-01008-2
Rie Saito ₁ , Norikazu Hara ₂ , Mari Tada ₁ , Yoshiaki Honma ₃ , Akinori Miyashita ₂ , Osamu Onodera ₄ , Takeshi Ikeuchi ₂ , Akiyoshi Kakita ₁

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(To the editor)

Recent genetic studies have led to the discovery of many novel causative genes of autosomal recessive cerebellar ataxias (ARCAs), which represent an extensive group of clinically and genetically heterogeneous neurodegenerative disorders that are manifested mostly in children [1]. For example, biallelic mutation in Calcineurin homologous protein-1 (CHP1) has been identified recently in two siblings of a consanguineous family showing cerebellar atrophy, spastic ataxia, motor neuropathy, intellectual disability, and slow ocular saccades (ARCA-CHP1) [2]. CHP1 serves as an essential cofactor of NHE1 (Na⁺ / H⁺ exchanger 1) which is a major regulator of intracellular ions and pH homeostasis. In vitro and in vivo studies of ARCA-CHP1 have shown that alteration of CHP1 and NHE1 expression could affect crucially ARCA pathomechanisms [2, 3]. However, the neuropathologic features and alteration of CHP1 and NHE1 expression remain unknown in patients with ARCA-CHP1. Here we investigated the clinicopathologic and biochemical features of two autopsied siblings with ARCA harboring a novel homozygous missense mutation in CHP1 identified through whole-exome sequencing (WES).

Two siblings (patients 1 and 2) developed cerebellar ataxic gait and speech at the ages of 30 and 56 years, respectively, followed by cognitive decline, pyramidal signs, loss of deep tendon reflexes and hearing loss. In patients 1 and 2, brain CT images revealed diffuse cerebellar atrophy (Supplementary Fig. 1). Their clinical features were summarized in Table 1, and described in detail in Additional file 1.

Table 1 Clinical features of patients with ARCA-CHP1

Full size table

The histopathologic features of the nervous system in patients 1 and 2 were quite similar, being characterized by marked degeneration of the cerebellum and dorsal column pathway. These changes were more severe in the patient with younger disease onset. Atrophy of the cerebellar hemispheres was more severe than that of the vermis (Fig. 1a). Microscopically, severe loss of Purkinje cells with Bergman gliosis, being more prominent in the cerebellar hemisphere than in the vermis, was evident (Fig. 1b and c). Immunoreactivity of calbindin-D28k in the remaining Purkinje cells was decreased (Fig. 1d and Fig. 2j), whereas that of parvalbumin in stellate cells and basket cell was relatively preserved (Fig. 1e). In the dentate nucleus, although the neurons were shrunken, neuronal loss was not obvious (Fig. 1f). Regarding the cerebellar afferent system, degeneration of the inferior olivary nuclei and pontine nucleus was unremarkable. No neuronal loss or focal gliosis was observed in the other regions of the brainstem and cerebrum, except for moderate neuronal loss (Fig. 1g) and gliosis (Fig. 1h) in layers II and III of the frontal cortex. There were no expanded polyglutamine-positive or ubiquitinated inclusions in the affected areas. The brains showed no pathological features suggestive complications arising from Alzheimer’s disease or Parkinson’s disease. The spinal cord and dorsal roots were atrophic (Fig. 1i). The gracile fasciculus showed loss of myelinated fibers extending from the cervical to the lumbar level (Fig. 1j), and the dorsal root ganglia showed severe loss of ganglion cells (Fig. 1k). Severe loss of myelinated fibers in the sural nerve was also evident (Fig. 1l).

WES analysis uncovered 6672 variants that segregated in an autosomal-recessive manner, in which seven candidates were prioritized (Table 2 and Additional file 1). Among them, we focused on a homozygous missense variant, p.Arg91Cys (c.271C > T), in CHP1. The variant has not been found in publicly available databases and exhibited the highest CADD score [4] – 32.0 – of all the candidates, indicating a highly predictively-damaging effect, and considered “likely pathogenic” based on the ACMG guidelines [5]. The variant was confirmed by Sanger sequencing (Fig. 2a and b). CHP1 immunoreactivity was detected in the membrane and cytoplasm of neurons and the neuropil in controls, but was completely lost in the cerebellar and cerebral cortex of the patients (Fig. 2c-i). Indeed, immunoblot analysis revealed that expression of CHP1 protein in the patients was reduced in the cerebellum by 80% and in the frontal cortex by 60% relative to the controls. Moreover, we confirmed a severe reduction of NHE1 protein in those tissues (Fig. 2j and k). The levels of CHP1 and NHE1 expression remained consistently decreased when normalized against those of calbindin-D28k or neurofilament. These results indicated that the reduction of CHP1 and NHE1 was not due to loss of Purkinje cells or other neurons. Details of methods are in Additional file 1.

Table 2 Profiles of candidate variants segregating in the studied patients

Full size table

Assuming a compound heterozygous model, we found four candidate variants located in two genes, ZNF440 and ZNF804A (Supplementary Table 2). However, both variants in ZNF440 appeared benign, because their CADD scores were lower than 10. For ZNF804A, we failed to find any study that had associated it with cerebellar ataxia. Accordingly, the homozygous variant in CHP1 was considered most likely to be linked with the present phenotype based on the genetic and pathological findings.

Comparing the present patients with the reported two siblings harboring a homozygous p.Lys19del CHP1 mutation [2], despite sharing other clinical manifestations, the age at onset differed considerably between the two families and the most significant clinical feature in the present patients was onset of ataxia in middle age and cognitive decline, in contrast to the infantile-onset ataxia and intellectual disability in the reported patients (Table 1). Such differences in the clinical features might have been a consequence of the different pathogenic variants. Indeed, in vitro experiments have revealed that the pathogenic variant p.Lys19del led to almost complete loss of the CHP1 protein [2], whereas immunoblotting in our patients demonstrated incomplete reduction of CHP1 protein in the brain tissue harboring the p.Arg91Cys CHP1 mutation. This remaining protein expression could have resulted in the milder phenotype.

Based on our observations, the CHP1 insufficiency was presumed to have been linked to neuronal loss in the cerebellar and frontal cortex, which would have been associated with cerebellar ataxia and cognitive decline, respectively. Indeed, Chp1 deficiency in zebrafish causes cerebellar hypoplasia, movement disorder and motor axon abnormalities, which can be ameliorated by co-injection with wild-type human CHP1 mRNA [2]. Furthermore, we demonstrated a definite reduction in the levels of CHP1 and NHE1 expression in the affected brain tissue, resembling the findings in mice with a homozygous point mutation of chp1 [3]. Since neither the p.Arg91Cys variant nor the p.Lys19del found in the reported siblings is located in EF-hand motifs that preferentially bind to Ca²⁺ [6], a direct role of these variants in the calcium-dependent interaction between CHP1 and NHE1 would appear to be unlikely. However, the CHP1 p.Lys19del variant failed to form functional protein complexes and showed a tendency for aggregation, resulting in decreased levels of soluble CHP1 and membrane expression of NHE1 in cultured cells [2]. It can be speculated that similar mechanisms might have been operating in the present siblings.

In conclusion, our findings suggest that CHP1 insufficiency resulting from p.Arg91Cys mutation in the affected tissue might have caused loss of neurons in the cerebellum and frontal cortex mediated by the reduction of NHE1 expression. Further studies are needed to clarify the significance of CHP1 p.Arg91Cys mutation in the context of CHP1-related neurodegeneration. Our findings broaden the clinicopathologic and pathophysiologic heterogeneity of ARCA. When encounting patients with middle-aged-onset ARCA accompanied by cognitive decline, ARCA-CHP1 should be considered.

The datasets used and analysed during the current study available from the corresponding author on reasonable request.

ARCA:: Autosomal recessive cerebellar ataxia
CHP1 :: Calcineurin homologous protein-1 gene
NHE1:: Na⁺ / H⁺ exchanger 1; WES: whole-exome sequencing

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Supported in part by JSPS grants-in-aid for Scientific Research to RS (19 K21314), MT (19 K07972) and AK (19H01061, 19H05559), and AMED grant to TI (JP19dk0207045).

Author notes

Rie Saito and Mari Tada contributed equally to this work.

Affiliations

Department of Pathology, Brain Research Institute, Niigata University, 1-757 Asahimachi, Chuo-ku, Niigata, 951-8585, Japan
Rie Saito, Mari Tada & Akiyoshi Kakita
Department of Molecular Genetics, Brain Research Institute, Niigata University, 1-757 Asahimachi, Chuo-ku, Niigata, 951-8585, Japan
Norikazu Hara, Akinori Miyashita & Takeshi Ikeuchi
Department of Neurology, Sado General Hospital, 161 Chigusa, Sado, Niigata, 952-1209, Japan
Yoshiaki Honma
Department of Neurology, Brain Research Institute, Niigata University, 1-757 Asahimachi, Chuo-ku, Niigata, 951-8585, Japan
Osamu Onodera

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Rie SaitoView author publications
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Norikazu HaraView author publications
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Mari TadaView author publications
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Yoshiaki HonmaView author publications
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Akinori MiyashitaView author publications
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Osamu OnoderaView author publications
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Takeshi IkeuchiView author publications
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Contributions

RS, MT, AK designed research project, and performed pathological analysis and drafted the manuscript for intellectual content. NH, AM and TI designed the molecular experiments and performed those. YH collected clinical data. OO discussed the results and commented on the manuscript text. The authors read and approved the final manuscript.

Corresponding authors

Correspondence to Rie Saito or Mari Tada.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of Niigata University (G2015–0676). Written informed consent for autopsy including the use of tissues for research purposes was obtained from the patients’ family.

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Family members have consented to publication.

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The authors declare that they have no competing interests.

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Saito, R., Hara, N., Tada, M. et al. Novel CHP1 mutation in autosomal-recessive cerebellar ataxia: autopsy features of two siblings. acta neuropathol commun 8, 134 (2020). https://doi.org/10.1186/s40478-020-01008-2

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Received: 13 May 2020
Accepted: 31 July 2020
Published: 12 August 2020
DOI: https://doi.org/10.1186/s40478-020-01008-2

Keywords

CHP1
NHE1
Autosomal recessive cerebellar ataxia
Middle-aged onset
Neuropathology

中文翻译：

常染色体隐性小脑共济失调中的新CHP1突变：两个兄弟姐妹的尸检特征。

（致编辑）

最近的遗传研究导致发现了常染色体隐性小脑共济失调（ARCA）的许多新型致病基因，这些基因代表了广泛的临床和遗传异质性神经退行性疾病，主要表现在儿童中[1]。例如，最近在一个血缘家族的两个兄弟姐妹中发现了钙调神经磷酸同源蛋白1（CHP1）的双等位基因突变，表现出小脑萎缩，痉挛性共济失调，运动神经病，智力残疾和慢眼球扫视（ARCA- CHP1）[2] 。CHP1是NHE1（Na ⁺ / H ⁺交换剂1）是细胞内离子和pH稳态的主要调节剂。ARCA- CHP1的体外和体内研究表明，CHP1和NHE1表达的改变可能会严重影响ARCA的发病机制[2，3]。但是，ARCA- CHP1患者的神经病理学特征以及CHP1和NHE1表达的改变仍然未知。在这里，我们调查了通过全外显子组测序（WES）鉴定的两个带有ARCA的尸体的临床病理学和生化特征，其中ARCA携带着CHP1中的新型纯合错义突变。

两名兄弟姐妹（患者1和2）分别在30岁和56岁时出现了小脑共济失调的步态和言语，随后出现认知能力下降，锥体束征，深肌腱反射丧失和听力下降。在患者1和2中，脑部CT图像显示弥漫性小脑萎缩（补充图1）。表1总结了它们的临床特征，并在附加文件1中进行了详细描述。

表1 ARCA- CHP1患者的临床特征

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患者1和2的神经系统组织病理学特征非常相似，其特征是小脑和背柱通路明显变性。这些疾病的发作在年轻患者中更为严重。小脑半球的萎缩比the骨的严重（图1a）。镜下可见，Bergman神经胶质细胞大量丢失浦肯野细胞，在小脑半球比在mis骨更明显（图1b和c）。其余的浦肯野细胞中钙结合蛋白-D28k的免疫反应性降低（图1d和图2j），而星状细胞和篮状细胞中小白蛋白的免疫反应性相对保留（图1e）。在齿状核中，尽管神经元收缩，但神经元损失并不明显（图1f）。关于小脑传入系统下橄榄核和桥脑核变性不明显。在脑干和大脑的其他区域没有观察到神经元丢失或局灶性胶质增生，除了额叶皮层第二层和第三层中度神经元丢失（图1g）和神经胶质增生（图1h）。受影响地区没有扩大的聚谷氨酰胺阳性或泛素化包涵体。大脑未显示任何病理特征，提示阿尔茨海默氏病或帕金森氏病引起的并发症。脊髓和背根萎缩（图1i）。细纹束显示从颈椎延伸至腰椎的髓鞘纤维丢失（图1j），而背根神经节显示神经节细胞严重丢失（图1k）。腓肠神经中髓鞘纤维的严重丧失也很明显（图1l）。在脑干和大脑的其他区域没有观察到神经元丢失或局灶性胶质增生，除了额叶皮层第二层和第三层中度神经元丢失（图1g）和神经胶质增生（图1h）。受影响地区没有扩大的聚谷氨酰胺阳性或泛素化包涵体。大脑未显示任何病理特征，提示阿尔茨海默氏病或帕金森氏病引起的并发症。脊髓和背根萎缩（图1i）。细纹束显示从颈椎延伸至腰椎的髓鞘纤维丢失（图1j），而背根神经节显示神经节细胞严重丢失（图1k）。腓肠神经中髓鞘纤维的严重丧失也很明显（图1l）。在脑干和大脑的其他区域没有观察到神经元丢失或局灶性胶质增生，除了额叶皮层第二层和第三层中度神经元丢失（图1g）和神经胶质增生（图1h）。受影响地区没有扩大的聚谷氨酰胺阳性或泛素化包涵体。大脑未显示任何病理特征，提示阿尔茨海默氏病或帕金森氏病引起的并发症。脊髓和背根萎缩（图1i）。细纹束显示从颈椎延伸至腰椎的髓鞘纤维丢失（图1j），而背根神经节显示神经节细胞严重丢失（图1k）。腓肠神经中髓鞘纤维的严重丧失也很明显（图1l）。除了额叶皮层II和III的中度神经元丢失（图1g）和神经胶质增生（图1h）。受影响地区没有扩大的聚谷氨酰胺阳性或泛素化包涵体。大脑没有显示出任何病理特征，提示阿尔茨海默氏病或帕金森氏病引起的并发症。脊髓和背根萎缩（图1i）。细纹束显示从颈椎延伸至腰椎的髓鞘纤维丢失（图1j），而背根神经节显示神经节细胞严重丢失（图1k）。腓肠神经中髓鞘纤维的严重丧失也很明显（图1l）。除了额叶皮层II和III中度神经元丢失（图1g）和神经胶质增生（图1h）。受影响地区没有扩大的聚谷氨酰胺阳性或泛素化包涵体。大脑未显示任何病理特征，提示阿尔茨海默氏病或帕金森氏病引起的并发症。脊髓和背根萎缩（图1i）。细纹束显示从颈椎延伸至腰椎的髓鞘纤维丢失（图1j），而背根神经节显示神经节细胞严重丢失（图1k）。腓肠神经中髓鞘纤维的严重丧失也很明显（图1l）。受影响地区没有扩大的聚谷氨酰胺阳性或泛素化包涵体。大脑未显示任何病理特征，提示阿尔茨海默氏病或帕金森氏病引起的并发症。脊髓和背根萎缩（图1i）。细纹束显示从颈椎延伸至腰椎的髓鞘纤维丢失（图1j），而背根神经节显示神经节细胞严重丢失（图1k）。腓肠神经中髓鞘纤维的严重丧失也很明显（图1l）。受影响地区没有扩大的聚谷氨酰胺阳性或泛素化包涵体。大脑未显示任何病理特征，提示阿尔茨海默氏病或帕金森氏病引起的并发症。脊髓和背根萎缩（图1i）。细纹束显示从颈椎延伸至腰椎的髓鞘纤维丢失（图1j），而背根神经节显示神经节细胞严重丢失（图1k）。腓肠神经中髓鞘纤维的严重丧失也很明显（图1l）。背根神经节显示神经节细胞严重丢失（图1k）。腓肠神经中髓鞘纤维的严重丧失也很明显（图1l）。背根神经节显示神经节细胞严重丢失（图1k）。腓肠神经中髓鞘纤维的严重丧失也很明显（图1l）。

WES分析发现了以常染色体隐性方式分离的6672个变异体，其中优先选择了7个变异体（表2和附加文件1）。其中，我们集中研究了CHP1中的纯合错义变体p.Arg91Cys（c.271C> T）。。该变体尚未在公开数据库中找到，在所有候选物中均显示出最高的CADD评分[4]-32.0 –表明具有高度预测性的破坏作用，并且根据ACMG准则被认为“可能具有致病性” [5]。通过Sanger测序证实了该变体（图2a和b）。在对照的神经元和神经纤维的膜和细胞质中检测到CHP1免疫反应性，但在患者的小脑和大脑皮层中完全消失（图2c-i）。确实，免疫印迹分析显示，与对照组相比，患者中小脑中CHP1蛋白的表达降低了80％，额叶皮质中的表达降低了60％。此外，我们证实了这些组织中NHE1蛋白的严重减少（图2j和k）。当相对于钙结合蛋白-D28k或神经丝标准化时，CHP1和NHE1表达水平持续降低。这些结果表明CHP1和NHE1的减少不是由于浦肯野细胞或其他神经元的丢失。方法的详细信息在附加文件1中。

表2在研究患者中分离的候选变异的概况

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假设有一个复合杂合模型，我们发现了位于两个基因ZNF440和ZNF804A中的四个候选变体（补充表2）。但是，ZNF440的两个变体均表现为良性，因为其CADD得分低于10。对于ZNF804A，我们找不到任何与小脑性共济失调相关的研究。因此，基于遗传和病理学发现，CHP1中的纯合变异体被认为最有可能与当前表型相关。

比较本病患者与报告的两个携带纯合p.Lys19del CHP1的兄弟姐妹突变[2]，尽管有其他临床表现，但两个家庭的发病年龄差异很大，目前的患者中最显着的临床特征是中年共济失调发作和认知能力下降，而婴儿期共济失调和已报告患者的智力障碍（表1）。临床特征的这种差异可能是不同病原体变异的结果。确实，体外实验表明，致病性变体p.Lys19del导致CHP1蛋白几乎完全丧失[2]，而我们患者的免疫印迹法表明，携带p.Arg91Cys CHP1的脑组织中CHP1蛋白的还原不完全。突变。这种剩余的蛋白表达可能导致了较温和的表型。

根据我们的观察，假设CHP1功能不全与小脑和额叶皮层的神经元丢失有关，这分别与小脑性共济失调和认知能力下降有关。确实，斑马鱼的Chp1缺乏会导致小脑发育不全，运动障碍和运动轴突异常，可通过与野生型人类CHP1 mRNA共同注射来缓解[2]。此外，我们证明了受影响的脑组织中CHP1和NHE1表达的水平明显降低，类似于在chp1纯合点突变的小鼠中的发现[3]。由于在所报道的同胞中发现的p.Arg91Cys变体和p.Lys19del都不位于优先与Ca ²⁺结合的EF手基序中[6]，这些变体在CHP1和NHE1之间的钙依赖性相互作用中的直接作用似乎不太可能。然而，CHP1 p.Lys19del变体未能形成功能性蛋白复合物，并显示出聚集的趋势，从而导致培养细胞中可溶性CHP1的水平降低和NHE1的膜表达降低[2]。可以推测，目前的兄弟姐妹中可能已经在运行类似的机制。

总之，我们的发现表明，受感染组织中p.Arg91Cys突变引起的CHP1功能不全可能导致NHE1表达减少介导的小脑和额叶皮质神经元丢失。需要进一步的研究来阐明在CHP1相关的神经变性的背景下CHP1 p.Arg91Cys突变的重要性。我们的发现扩大了ARCA的临床病理和病理生理异质性。当考虑中年发病的ARCA伴有认知功能减退的患者时，应考虑ARCA- CHP1。

在当前研究期间使用和分析的数据集可应合理要求从相应的作者处获得。

ARCA：: 常染色体隐性小脑共济失调
CHP1：: 钙调磷酸酶同源蛋白1基因
NHE1：: Na ⁺ / H ⁺交换剂1; WES：全外显子组测序

1。
Anheim M，Trenchant C，Koenig M（2012）常染色体隐性性小脑共济失调。英格兰医学杂志366：636–646
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下载参考

不适用。

JSPS为科学研究（RS（19 K21314），MT（19 K07972）和AK（19H01061、19H05559）提供科研资助，并为TI（JP19dk0207045）提供了AMED资助。

作者须知

齐藤理惠和Mari Tada对这项工作做出了同样的贡献。

隶属关系

新泻大学脑科学研究所病理学部，新泻市中央区旭町1-757，日本951-8585
齐藤理惠，田田麻里＆秋田明吉
新泻大学脑科学研究所分子遗传学系，新泻市中央区旭町1-757，日本951-8585
原纪敏，宫下昭典＆池内健
佐渡综合医院神经内科，新泻佐渡市千户市161，日本952-1209
本间佳明
新泻大学脑科学研究所神经内科，新泻市中央区旭町1-757，日本951-8585
小寒修

作者

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会费

RS，MT，AK设计了研究项目，并进行了病理分析并起草了具有知识性内容的手稿。NH，AM和TI设计并进行了分子实验。YH收集了临床数据。OO讨论了结果并评论了手稿文本。作者阅读并批准了最终手稿。

通讯作者

与斋藤理惠或玛丽·多田对应。

道德规范的批准和同意参加

本研究得到新泻大学伦理学委员会的批准（G2015-0676）。从患者家属那里获得了尸检的书面知情同意书，包括将组织用于研究目的。

同意发表

家庭成员已同意出版。

利益争夺

作者宣称他们没有竞争利益。

发行人须知

对于已发布地图和机构隶属关系中的管辖权主张，Springer Nature保持中立。

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