Inorganic phosphorus (Pi) in CSF is a biomarker for SLC20A2-associated idiopathic basal ganglia calcification (IBGC1)

Accepted Manuscript Inorganic phosphorus (Pi) in CSF is a biomarker for SLC20A2-associated idiopathic basal ganglia calcification (IBGC1)

Isao Hozumi, Hisaka Kurita, Kazuhiro Ozawa, Nobuyuki Furuta, Masatoshi Inden, Shin-ichiro Sekine, Megumi Yamada, Yuichi Hayashi, Akio Kimura, Takashi Inuzuka, Mitsuru Seishima PII: DOI: Reference:

S0022-510X(18)30130-8 doi:10.1016/j.jns.2018.03.014 JNS 15829

To appear in:

Journal of the Neurological Sciences

Received date: Revised date: Accepted date:

12 July 2017 7 February 2018 6 March 2018

Please cite this article as: Isao Hozumi, Hisaka Kurita, Kazuhiro Ozawa, Nobuyuki Furuta, Masatoshi Inden, Shin-ichiro Sekine, Megumi Yamada, Yuichi Hayashi, Akio Kimura, Takashi Inuzuka, Mitsuru Seishima , Inorganic phosphorus (Pi) in CSF is a biomarker for SLC20A2-associated idiopathic basal ganglia calcification (IBGC1). The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jns(2018), doi:10.1016/j.jns.2018.03.014

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Inorganic phosphorus (Pi) in CSF is a biomarker for SLC20A2-associated idiopathic basal ganglia calcification (IBGC1)

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Isao Hozumia, * , Hisaka Kuritaa, Kazuhiro Ozawab, Nobuyuki Furutac, Masatoshi Indena, Shin- ichiro

Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu, 501-1196, Japan

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Sekinea, Megumi Yamadad, Yuichi Hayashid, Akio Kimurad, Takashi Inuzukad, Mitsuru Seishimac

Nursing Collaboration Center, Gifu College Nursing, 3047-1, Hashima, Gifu, 501-6295, Japan

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Department of Informative Clinical Medicine, Gifu University, Graduate School of Medicine, 1-1 Yanagido Gifu 501-1194, Japan

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Department of Neurology and Geriatrics, Gifu University, Graduate School of Medicine, 1-1 Yanagido Gifu 501-1194, Japan

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* Correspondening author at: Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu

Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu, 501-1196, Japan

Emal address: hozumi＠gifu-pu.ac.jp (I. Hozumi) Tel & Fax: 81-58-230-8121

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Keywords: Idiopathic basal ganglia calcification (IBGC), Fahr’s disease, Primary familial brain calcification (PFBC), Inorganic phosphorus (Pi), Cerebrospinal fluid (CSF)

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ABSTRACT

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Introduction: Idiopathic basal ganglia calcification (IBGC), also called Fahr’s disease or recently

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primary familial brain calcification (PFBC), is characterized by abnormal deposits of minerals including calcium mainly and phosphate in the brain. Mutations in SLC20A2 (IBGC1 (merged with

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former IBGC2 and IBGC3)), which encodes PiT-2, a phosphate transporter, is the major cause of

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IBGC. Recently, Slc20a2-KO mice have been showed to have elevated levels of inorganic

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phosphorus (Pi) in cerebrospinal fluid (CSF); however, CSF Pi levels in patients with IBGC have not been fully examined.

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Methods: We investigated the cases of 29 patients with IBGC including six patients with SLC20A2

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mutation and three patients with PDGFB mutation, and 13 controls. The levels of sodium (Na), potassium (K), chloride (Cl), calcium (Ca), and Pi in sera and CSF were determined by potentiometry and colorimetry. Moreover, clinical manifestations were investigated in the IBGC patients with high Pi levels in CSF. Results: The study revealed that the average level of Pi in the CSF of the total group of patients with IBGC is significantly higher than that of the control group, and the levels of Pi in CSF of the IBGC patients with SLC20A2 mutations are significantly higher than those of the IBGC patients with

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PDGFB mutations, the other IBGC patients and controls. Conclusion: Results of this study suggest that the levels of CSF Pi will be a good biomarker for

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IBGC1.

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1. Introduction Idiopathic basal ganglia calcification (IBGC), also called Fahr ’s disease [1] or recently primary

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familial brain calcification (PFBC) [2], is a rare and intractable disease. It is characterized by

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abnormal deposits of minerals including calcium mainly and phosphate in the basal ganglia and other

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brain regions such as the thalamus and cerebellum. For the diagnosis of IBGC, other secondary causes of calcification should be excluded [1, 2]. The causative genes for familial IBGC (FIBGC)

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have been successively identified: SLC20A2 (IBGC3) [3-5]. PDGFRB (IBGC4) [6], PDGFB

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(IBGC5) [7], and XPR1 (IBGC6) [8]. However, IBGC3 is now changed to IBGC1, which thus

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comprises SLC20A2-associated IBGCs. Mutations in SLC20A2, which encodes type III sodium-dependent phosphate transporter 2 (PiT-2), are the major cause of IBGC [2]. We have

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already reported that type III sodium-dependent transporters (PiT-1 and PiT-2) are the major

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sodium-dependent phosphate transporters in the mice and human brain [9]. XPR-1 also encodes a transporter which exports inorganic phosphorus (Pi) out of cells [8]. The causative molecules suggest that IBGC could be mainly caused by an impaired phosphate homeostasis in the brain. Recently, Slc20a2-KO mice have been shown to have elevated levels of inorganic phosphate (Pi) in cerebrospinal fluid (CSF) [10, 11]. Jensen et al. have as the first reported that Slc20a2-KO mice showed calcification in the basal ganglia and the cortex in the brain, and downregulation of PiT-2 alone can lead to brain calcification [12]. Therefore, in this study we examined the levels of Pi in

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CSF of patients with IBGC compared with those of controls.

2. Methods

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2.1. Subjects and samples

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We obtained CSF sample from 29 patients with IBGC. Genetic mutations of SLC20A2 and

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PDGFB

in the patients were examined previously. Six patients presented with mutations in SLC20A2 [4, 5] as

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shown in Table 2 and three patients in PDGFB (data not shown). The other 20 patients with IBGC

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showed neither mutations in SLC20A2, PDGFB, PDGFRB nor XPR1. Controls were selected from

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people without any organic neurological disease and calcification in the brain CT. The patients with IBGC included 16 males and 13 females, and the controls included 7 males and 6 females. The mean

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respectively.

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ages of the patients with IBGC and controls were 42.9 ± 18.6 (SD) and 42.9 ± 19.6 (SD) years,

2.2. Biochemical analyses

The levels of sodium (Na), potassium (K), chloride (Cl), calcium (Ca), and Pi in sera and CSF were determined by potentiometry and colorimetry. 2.3. Ethics Clinical examinations were performed in accordance with the latest version of the Helsinki Declaration. All participants involved in this study provided their written informed consent. All

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investigations carried out in this present study were approved by the Ethics Committee of the Gifu University Graduate School of Medicine and Gifu Pharmaceutical University. 2.4. Statistical analyses

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The relationship among age and the levels of biometals in CSF and sera were statistically

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analyzed using Pearson product moment correlation. The relationship between sex, and IBGC and

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controls groups was analyzed by Fisher’s exact test. The comparisons among disease groups and controls were analyzed using Student’s t-test and Tukey’s honestly significant difference (HSD) test.

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A significance level of 0.05 was used for all statistical tests (two-tailed). Statistical analyses were

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performed using IBM SPSS Statistics 24.0 (IBM Corp., Armonk, NY).

3.1. Bioelements in CSF

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3. Results

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In Figure 1, Student’s t-test showed that the levels of Pi in CSF in all the patients with IBGC were significantly higher than those of controls (p = 0.001). Comparison among IBGC patients with SLC20A2 mutations, IBGC patients with PDGFB mutations, other IBGC patients without mutations in SLC20A2, PDGBF, PDGFRB nor XPR1, and controls using Tukey’s HSD test showed that the levels of Pi in CSF in the IBGC patients with SLC20A2 mutations were significantly higher than those in controls, the IBGC patients with PDGFB mutations and the other IBGC patients, respectively (p < 0.001, p = 0.018, p < 0.001). The levels of Pi in the IBGC patients with PDGFB

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mutations seem to lie between the levels of the IBGC patients with SLC20A2 mutations and those of the other IBGC patients, however, the number of the patients with PDGFB mutations are too small to draw a conclusion. (Figure 1 and Table 1)

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The level of Pi in CSF showed no correlation with that in sera in the IBGC patients and controls

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(ρ = -0.005, p = 0.980, n = 29; ρ = -0.335, p = 0.314, n = 11, respectively). The level of Pi in CSF

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showed no correlation with the ages of the IBGC patients and controls (ρ = 0.148, p = 0.444, n = 29; ρ = 0.297, p = 0.324, n = 13, respectively).

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No significant differences in sex or age were observed among IBGC patients and controls in the

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study (Table 1). The levels of Na, K, Cl, Ca, and P i in sera were within the normal range in all

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3.2. Clinical manifestation

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patients with IBGC and controls (data not shown).

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The clinical manifestation of the patients with SLC20A2 mutations are shown in Table 2. The CT images of patients #2 and #3 are shown in Figure 2. The CT images of patients #1 and #4, and #6 are shown in reference [4] and [5], respectively. No significant relationship among Pi levels in CSF, ages and total calcification score (TCS) [13] was found. The CT images and clinical manifestation of IBGC patients #7, #8, #9, and #10 whose mutations have not been found are shown in Figure 2 and Table 3.

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Discussion In this study, the findings in the CSF of Slc20a2-KO mice was confirmed in human samples. The data analyses showed no increase in Pi levels with age. The number of IBGC patients with PDGFB

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mutations are too small to draw a conclusion. A larger number of patients as well as controls should

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be examined before a definitive conclusion can be drawn. Recently Paucar et al. presented a case

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carrying the R467X mutation in SLC20A2 and type I diabetes mellitus with poor glycemic control. She showed increased level of Pi in CSF [14].

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We have previously demonstrated that the levels of Cu, Zn, and Mg but not those of Ca were

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elevated in the CSF of three IBGC patients [15]. To measure these heavy metals for the aid of

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diagnosis of IBGC, pretreatment and inductively coupled plasma mass spectrometry (ICP-MS) are required to obtain precise data [15, 16]. On the other hand, the concentration of Pi in CSF can be

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examined by common methods in general hospitals. Therefore, Pi levels in CSF provide a more

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efficient and more easily employed clinical biomarker for IBGC. Pi level in CSF can be a marker for the screenings of patients for IBGC, especially those with SLC20A2-associated mutations (IBGC1). In addition, this may also be used as an indicator of the effect of a therapeutic agent for the patients with high Pi in CSF, especially IBGC1 patients. Not all IBGC patients showed high levels of Pi. Although the high level of Pi in CSF is not necessarily specific to IBGC, higher levels are associated with SLC20A2-associated IBGC. Although IBGC is a rare disease and we don’t have many CSF samples from patients, higher tendency of Pi in

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CSF can be seen in the patients with IBGC. This suggests that the high level of Pi in CSF promotes the progression of the disease at a certain level. On the other hand, PiT-2 has been reported to be highly expressed in the choroid plexus, and results suggest it removes P i from the CSF [10, 11]. It

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was recently shown that at least some PiT-2 mutations, could act in a dominant negative manner on

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PiT-2 Pi-transport function [17], thus one possible explanation for differences in the CSF Pi

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concentration is that it can depend on the type of PiT-2 mutation. The levels of Pi might also be influenced by other factors including diurnal variation. Large cohort and international cooperation

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researches will be needed to solve the problems in future.

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The pathway in which CSF and interstitial fluid (ISF) are communicated is proposed as a

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glymphatic pathway by Nedergaard [18]. Jensen et al. and Wallingford et al. proposed that the glymphatic pathway is associated with vascular calcification [10, 11]. The detailed routes of CSF

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reaching cervical lymph nodes have not been clarified yet. Morris et al. using tracer demonstrated a

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perivascular lymphatic drainage pathway in which ISF flow into the perivascular space of blood capillaries between the basement membrane and the foot processes of astrocytes. And then the flow enters into the space between the basement membranes of smooth muscle fibers in the tunica media of small arteries [19]. These concepts may play useful roles in understanding brain calcification, and is compatible with the microscopic findings in the autopsies of patients with IBGC [20-23]. By the observation in the microscopic examination, calcification (to put it more precisely, mineral deposits) in IBGC was seen in the perivascular space of blood capillaries [20, 21], the walls of small blood

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vessels coalescent together, the cytoplasm of pericytes, and glial process [21]. Mineralization was also observed in the tunica media of small-sized artery [20, 23]. Mineral deposits were not seen in the bodies of neurons and astrocytes, or veins [20-23].

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Phosphate toxicity accelerates the aging process [24]. High Pi levels in sera in patients with

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chronic renal failure are an important factor for prognosis and contribute to the medial calcification

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of small arteries (Mönckeberg-type arteriosclerosis). Patients in dialysis with hyperphosphatemia are also known to have significantly poorer prognosis as compared to patients who have Pi co ntrolled in

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the normal range [25]. Elevated extracellular Pi concentration have been demonstrated to promote

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the production of reactive oxygen (ROS) in mitochondria and cause cytotoxicity [26, 27]. The

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condition is supposed to occur around the capillaries and the small arteries. The role of Pi in the mitochondrial respiratory control and mitochondrial oxidation ha ve been examined in several studies Decreased intracellular Pi concentration in neurons due to the damage of intracellular

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[28-30].

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transport of Pi is also thought to promote the production of ROS in mitochondria and cause cytotoxicity, but without mineral deposits. And, we have recently demonstrated that the cell damage assumed to be occurring due to low Pi in neurons can be rescued by the administration of 5-aminolevulinic acid [31]. The type III Pi transporters are the major Na-dependent Pi transporters in the brain, and the mRNAs of the other Pi transporters, which are encoded by SLC17 (type I) and SLC34 (type II) mRNAs, were hardly detected in the brain [9]. Therefore, the type III Pi transporters are very

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important in the regulation of Pi in the brain. This suggests that the Pi level in ISF and CSF may play an important role in mineralization in the brain. The level of Pi in CSF will be a good biomarker for

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SLC20A2-associated IBGC and an indicator for the effect of therapeutic agents.

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Funding

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This work was supported by grants from the Ministry of Health, Labour and Welfare of Japan: (H26-Nanbyotou (Nan)-Ippan-001, H26-Nanchitoh (Nan)-Ippan-085, and Grants-in Aid from the

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Research Committee of CNS Degenerative Diseases, Research on Policy Planning and Evaluation

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for Rare and Intractable Diseases, Health, Labour and Welfare Sciences Research Grants), and the

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Ministry of Education, Culture, Sports, Science and Technology of Japan (Basic Research (B)

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Conflict of interest

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(17H04198)).

The authors have no conflict of interest in relation to this work.

Acknowledgements The authors thank the patients who supported this research. We thank the involved doctors (Dr. Seiju Kobayashi, Sapporo Medical University Hospital, Dr. Tetsuo Toge, Kagawa University Hospital), and Dr. Akihiro Ueda (Fujita Health Hospital). The authors also thank Ms. Madoka Sato

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for technical assistance.

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[31] N.Takase, M. Inden, S. Sekine, Y. Ishii1, H. Yonemitsu, W. Iwashita, H. Kurita, Y. Taketani, I. Hozumi, Neuroprotective effect of 5-aminolevulinic acid against low inorganic phosphate in

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neuroblastoma SH-SY5Y cells, Sci. Rep. 7(2017)5768.

Levels of Pi in CSF in all patients with IBGC, and IBGC patients with SLC20A2

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Figure 1

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Legends

mutations, with PDGFB mutations, and others (without SLC20A2 or PDGFB mutations), and

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controls. The levels of Pi in CSF of all patients with IBGC were higher than those of controls (p

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= 0.001 in Student’s t–test is shown above the dotted line). The values showing significant

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differences according to Tukey’s HSD test are shown above the solid line. The clinical

Figure 2

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supplementary data.

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manifestation and CT images of the patients indicated by numbers (#) are shown in Table 2 and

CT images of patients (#2, #3, #7, #8, #9, and #10) with high Pi levels in CSF

CT image of patient #1 is shown in Figure 3B in reference [4]. CT images of patients #4 are shown in Figure e-2B (A) in reference [4]. CT images of patients #5 are shown in Figure 2D in reference [4]. CT images of patients #6 are shown in Figure 1b in reference [5]. No significant differences were recognized among total calcification score (TCS), Pi levels in CSF and ages.

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Levels of Na, K, Cl, Ca, and Pi in CSF of three groups of IBGC patients and controls.

Table 2

Clinical features of six IBGC patients with SLC20A2 mutations.

Table 3

Clinical features of patients (#7 #8, #9 and #10) with high Pi levels in CSF.

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controls

Levels of Pi in CSF in patients with IBGC and

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Fig 1

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Levels of Na, K, Cl, Ca and Pi in CSF of patients with IBGC and controls

Multiple

IBGC

comparis Total

1. Control

on

2.

3.

SLC20A2

PDGFB

Total

4. Others

(Tukey's HSD test)

K, mg/dL

Cl, mg/dL

Ca, mg/dL

Pi, mg/dL

7:6

16 : 13

4:2

144.2 (8.3)

2.9 (0.2)

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***p<0.001.

(19.6)

(18.6)

140.1

146.1

(12.3)

(5.0)

2.8

3.0

118.4

3.7 (1.0)

Mean (SD). *p<0.05, **p<0.01,

42.9

(0.2)

122.3 (7.2)

1.6 (0.4)

42.9

—

1:2

11 : 9

—

44.8

37.0

43.2

(20.6)

(20.5)

(18.6)

144.2

148.3

146.3

(7.1)

(1.2)

(4.6)

2.9

3.2

3.0

(0.2)

(0.1)

(0.4)

(0.1)

124.0

124.5

125.0

123.7

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42.9 (18.7)

20

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23 : 19

3

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6

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Na, mg/dL

29

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Age, y

13

(11.0)

(3.8)

(2.9)

(2.0)

(4.3)

3.2

4.0

4.4

4.0

3.9

(1.5)

(0.6)

(0.6)

(0.3)

(0.7)

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Male : Female

42

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No.

n.s.

n.s.

1 < 3*

n.s.

n.s. 1 < 2***, 1

1.3

1.7

2.2

1.6

1.6

(0.2)

(0.4)

(0.4)

(0.2)

(0.3)

< 4*, 2 > 3*, 2 > 4***

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Clinical features of six patients with SLC20A2 mutations

#1

#3

#2

c.344C>T c.344C>T

#5

#6

T115M

T115M

G120R

R467X

S637R

T616X

Hokkaido

Hokkaido

The main

Shikoku

The main

Shanghai

Island

Island

Island

Island

Island

in China

72 y.o., F

53 y.o., M

63 y.o., F

31 y.o., M

22 y.o., M

28 y.o., M

Age at onset

58

46

62

unknown

13

Age at detection of calcification

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c.358G>C c.1399C>T c.1909A>C c.1848G>A

60

50

63

23

22

25

Age at diagnosis of IBGC1

69

50

63

26

22

26

Onset symptom

Dysarthria

Easy to anger

PKC

-

PKC

Complication

Dementia (MMSE 20)

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Mutation

#4

-

Neuropathy

-

-

-

28

31

20

30

24

38

3.7

3.3

3.9

2.4

2.3

1.9

TCS on Brain CT Pi level

CSF

2.8

Family information

References

2

.4

Her son and 5 relatives

His mother and 5 relatives

His son

Fig. 3B in ref. 4

III-1 in Fig. 3A in ref. 4

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No. of other symptomatic individuals

Gait disturbance

2.8

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3.6

Serum

15

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Age, Sex

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of

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Place residence

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Proband information

His mother His mother Fig. e-2B (A) in ref. 4

Fig. 2D in ref. 4

3.1 1.6

3 relatives M4

in

ref. 5

Abbreviations: MMSE = mini- mental state examination, PKC = paroxysmal kinesigenic choreoathetosis, TCS = total calcification score

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Clinical features of patients with high Pi levels in CSF

Disease Mutation

#7

#8

#9

#10

IBGC

IBGC

IBGC

IBGC

N.D.

N.D.

N.D.

N.D.

Proband’s information The main

The main

The main

Island

Island

Island

Island

29 y.o., M

50 y.o., F

13 y.o., F

38y.o., F

Age at onset

22

40

13

38

Age at detection of calcification

22

17

13

38

Age at diagnosis of the disease

22

13

38

syncope

TCS on brain CT

34

Serum (normal: 2.6 －

4.5

CSF

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No. of other symptomatic individuals

PTSD = Post Traumatic Stress Disorder

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32

-

-

-

3.9

3.7

3.7

2.1

1.9

1.9

0

0

0

0

-

-

-

-

3.3

2.4

Family’s information

Weakness of right side

6

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mg/dL)

Epilepsy

28

-

Pi levels

PTSD

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Complication

References

50

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Onset symptom

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Age, Sex

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The main

Place of residence

ACCEPTED MANUSCRIPT Highlights CSF Pi levels in patients with IBGC are significantly higher than those of controls.



Those in patients with IBGC1 are significantly higher than those of other IBGCs.



The concentration of Pi in CSF can be measured in general hospitals.



The level of CSF Pi will be a good biomarker and indicator for IBGC, especially IBGC1.

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