Photon Factory Activity Report 2016 #34 (2017)

BL-4A/2015G081, 2016G131, 2017G079

Current status of Wilson disease in Japan 2017: challenges toward precise diagnosois bysynchrotron-generated X-ray
Akihiro Matsuura, Miyuki Kinebuchi
Fujita Health University Graduate School of Medicine, Department of Molecular Pathology
1-98 Kutsukake, Toyoake 470-1192, Japan

1. Introduction

2. Current status of diagnosis of Wilson disease,

Copper and iron are essential nutrients for all

challenges and solutionsorganisms. Both metals in excess are toxic with strong

2.1 Brief introduction of Clinical Practice Guideline ofredox-activity. Humans as well as higher vertebrates

Wilson disease in Japan 2015

developed the ingenious strategy to manage these

There has been two guidelines for diagnosis anddouble–edged swords; the effective absorption andtreatment of Wilson disease proposed by societies forexcretion system for copper, and the strictly regulated

liver disease in Europe and North America, EASL andabsorption and no specific excretion system for iron.

AASLD, respectively [1, 2]. Because patients withHowever, medical interests of copper in the beginning

Wilson disease in Japan as well as east asia are

had been focused on the toxic effect, acute poisoning or

relatively similar compared to those in Europe andchronic exposure by mine pollution. Since theNorth America, and because the disease forms,

discovery of mammalian copper transporters, ATP7A

genotypes, and national health care policy are different

and ATP7B in 1993, molecular mechanisms of copper

between nations, the Wilson disease guideline working

homeostasis and trafficking pathways in physiologicalgroup (WG) lead by professor Hiroko Kodama has justand pathological conditions have been widely explored.

published the guideline in Japanese [3]. The approach

In this symposium, we would like to introduce about

to diagnosis of WD is similar in these three guidelines,

what is copper toxicosis, namely Wilson disease (WD),but we include an unique disease form, presymptomaticand several problems encountered in clinics andform as different entity because it is difficult tolaboratories. We then show a trial to solve the copper

diagnose and the number of patients with this form isdetection by synchrotron-based technique. We hopeincreasing in Japan as the health care of children isthat scientists in the field of X-ray imaging optics arerelatively generous [4].

continuing

2.2 Problems in diagnostic tests

persistent

hardworks

whichbrings

biomedical researchers like us up to the top level,

When patients were presented with unexplained liver

beyond “precision medicine”.

disease or with slightly elevated liver enzymes, theattending doctors conducted copper-related laboratory
tests; such as serum ceruloplasmin, serum copper,
urinary copper according to the health care plan inJapan. If these tests are positive, physicians ask
surgeons for invasive liver biopsy to examine liver
copper content and pathological examination. First,

Figure 1 Classic Wilson disease with liver cirrhosis.

biochemical liver copper content measurement is not

Macroscopic (left) and Hematoxylin and eosin stained

easy for most laboratories in hospitals. The samples

pictures (right).

must be transferred to the special center that canPhoton Factory Activity Report 2016 #34 (2017)
perform atomic absorption spectrometry (AAS) or
inductively

coupledplasmaatomicemission

spectroscopy (ICP-AES). In this step, there wereseveralfactors

affectedits

accuracy.

Secondly,although molecular genotyping of ATP7B could be
decisive and precise, certain fraction of patients withclinically apparent WD showed negative results [4].
Thirdly, histochemical copper staining methods wereinsufficient which frequently failed to detect copper

Figure2XEDS

histogramsofFFPE

tissuedeposits [5, 6]. Taken together, we often have criticalspecimens of patient with WD. X axis:energy range.

difficulty in the diagnosis.

Y axis: fluorescence yields

2.3 A possible solution
To overcome these challenges and difficulties, we tried

4. Conclusion

to improve the mutation analysis, standardization ofCombination of several techniques, precise diagnosis ofbiochemical tests, and applied synchrotron-radiation

Wilson disease become possible in the early stages

X-ray fluorescence to pathological examinations [7-9].

even before development of clinical manifestations.

Whereas other tests were negative or unable to perform,

Synchrotron X-ray fluorescence analysis may become acopper could be detected and visualized in some

new tool to estimate copper accumulation in the liver

hard-to-diagnose cases.

evenwithfineneedle

biopsyspecimens.

The

pathogenesis of Wilson disease should be clarified in avariety of aspect [10, 11].

3. Experiments and Results
The double crystal spectrograph placed upstream of thehatch was used to monochromatize the X-ray and to

5 Referenceschange the energy range appropriately for the elements[1] European Association for the Study of the Liver,of interest. The X-ray beam was focused to spot size

EASL Clinical Practice Guidelines: Wilson’s disease. Jwith about 30 square micrometers focused by theHepatol 56, 671 (2008).

poly-capillary

Formalin-fixed[2] Robert EA and Schilsky ML, Diagnosis andparaffin-embedded (FFPE) tissue sections from weretreatment of Wilson disease: an update. Hapatol 47,irradiated by the 11 keV X-ray beam. The silicon drift

2089 (2012).

X-ray detector (SDD, Vortex-EX, Hitachi, Japan), a[3] Kodama H, Clinical Practice Gidelines of Wilson

type of energy dispersive X-ray detector (XEDS),

Disease in Japan 2015, Diag Treat 122-180 (2015)

connected to the current amplifier (Model 428, Keithley,[4] Yamaguchi A et al., Mutations of ATP7B gene inU.S.A.) was placed on the mobile rack that could bewilson disease in Japan: Identification of ninereadily moved to adjust the distance between the tip ofmutations and lack of clear founder effect in a Japaneseprobe and samples. The thermoelectrically cooled SDD

population. Hum Mut 11, S320 (1998).

does not require liquid nitrogen. The resultant[5] Ohya K et al., Presymptomatic diagnosis of Wilson

fluorescence X-ray emission was analyzed with energy

disease associated with a novel mutation of the ATP7B

dispersive spectrometry (XEDS) by spot analysis andgene. Eur J Pediatr 161, 124 (2002).

presented as a histogram. Copper Ka peak was clearly[6] Kitazawa J et al., Hemolytic crisis with fulminantdistinguished by other elemental peaks. This tissue

hepatic failure in Wilson disease without consanguinity.

section was completely negative for copper stained

Pediatr Inter 46, 726 (2004).

deposits. Therefore, the SXRF analysis is much higher[7] Kinebuchi M, Matsuura A, Calcium-overloaded

sensitivity

sympathetic preganglionic neurons in a case of severe

comparedsystem.

totheroutinely

histochemical copper staining methods.

usedsepsis with anorexia nervosa. Acute Med Surg 2, 169

Photon Factory Activity Report 2016 #34 (2017)(2015)[8] Kinebuchi M et al. Diagnostic copper imaging ofMenkes disease by synchrotron radiation-generated
X-ray fluorescence analysis. Sci Rep 6, 33247 (2016).
[9] Matsuura A and Kinebuchi M. Refinement of X-ray
fluorescence analysis of biological specimens. PFACR
2013 #31, 315 (2014)[10] Lang PA et al., Liver cell death and anemia in
Wilson disease involve acid sphingomyelinase andceramide. Nat Med 13, 164 (2007).
[11] Kinebuchi M et al., Contribution of Vα24Vβ11
natural killer T cells in Wilsonian hepatits. Clin ExpImmunol 139, 144 (2005).
* amatsuu@fujita-hu.ac.jp