Yonago Acta medica 2014;57:37–43

Original Article

Expansion of Genetic Testing in the Division of Functional Genomics, ResearchCenter for Bioscience and Technology, Tottori University from 2000 to 2013
Kaori Adachi*†*Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, Yonago 683-8503, Japan and†Division of Child Neurology, Department of Brain and Neuroscience, School of Medicine, Tottori University Faculty of Medicine,
Yonago 683-8503, Japan

ABSTRACTIn Japan, however, genetic testing has not yet become a common practice in the medical field, since
public insurance covers only screenings for mutationsin 36 genes. Mutation hunting on several genes is conducted on a research basis, which as expected depends
on the research project and is not ensured to be provided
in the future.
In the Division of Functional Genomics, ResearchCenter for Bioscience and Technology, Tottori University, we have established a genetic testing facility thatcan provide the same screening procedures conductedworldwide. This system started as a service for screening
mutations responsible for several types of inherited disorders of children, and now has expanded to cover morethan 60 inherited disorders. Human DNA samples havebeen provided not only from Tottori University Hospital
but also from other medical facilities all over Japan andforeign countries. Here the development of the systemfrom January 2000 to September 2013 is reported.

Background At the Division of Functional Genomics,
Research Center for Bioscience and Technology, Tottori
University, we have been making an effort to establish agenetic testing facility that can provide the same screening procedures conducted worldwide.
Methods Direct sequencing of PCR products is themain method to detect point mutations, small deletions
and insertions. Multiplex ligation-dependent probe amplification (MLPA) was used to detect large deletions
or insertions. Expansion of the repeat was analyzed
for triplet repeat diseases. Original primers were constructed for 41 diseases when the reported primers failed
to amplify the gene. Prediction of functional effect ofhuman nsSNPs (PolyPhen) was used for evaluation ofnovel mutations.
Results From January 2000 to September 2013, a totalof 1,006 DNA samples were subjected to genetic testingin the Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University.
The hospitals that requested genetic testing were locatedin 43 prefectures in Japan and in 11 foreign countries.
The genetic testing covered 62 diseases, and mutations
were detected in 287 out of 1,006 with an average mutation detection rate of 28.5%. There were 77 samples forprenatal diagnosis. The number of samples has rapidly
increased since 2010.
Conclusion In 2013, the next-generation sequencers
were introduced in our facility and are expected to provide more comprehensive genetic testing in the near future. Nowadays, genetic testing is a popular and powerfultool for diagnosis of many genetic diseases. Our genetic
testing should be further expanded in the future.

SUBJECTS AND METHODS
Subjects

line mutation

From January 2000 to September 2013, a total of 1,006
DNA samples were subjected to genetic testing in ourfacility. They were from 13 departments at Tottori University Hospital, 123 hospitals in Japan and 11 in foreigncountries (Table 1). Most of the DNA was extracted
from peripheral blood. Exceptions include 24 from cultured fibroblasts and 3 from tissue. For prenatal diagnosis, 40 DNA samples were from chorionic villi, 36 from
cultured amnionic cells and 1 from umbilical blood. The
DNA was subjected to genetic testing for 62 inherited
diseases (Table 2).
The samples were collected according to “Guideline for Genetic Testing” (August 2005) approved by
the Association of Genetic Medicine (10 societies and

The development of new technologies such as microarray analysis and next-generation sequencing has enabledmore comprehensive screening for mutations that cause
inherited diseases.1, 2 According to GeneTests (http://
www.genetests.org/), screening methods covering morethan 2,000 genes are currently available.

Corresponding author: Kaori Adachi
adachika@med.tottori-u.ac.jp
Received 2013 December 5
Accepted 2013 December 19
Abbreviations: DHPLC, denaturing high performance liquid
chromatography; MLPA, multiplex ligation-dependent probe
amplification; PolyPhen, prediction of functional effect of humannsSNPs; SSCP, single strand conformation polymorphism

Key words

genetic testing; prenatal diagnosis; germ-37

K. Adachi
Table 1. The number of medical facilities that requested for genetic testing in our laboratory*
Area

NumberHokkaido
Tohoku
Kanto
Chubu
Kinki
Chugoku
Shikoku
Kyusyu

5
10
27
20
20
19
3
19

Total

123

Foreign countries

11

Prefecture or Country
Aomori 1, Iwate 1, Miyagi 3, Akita 1, Yamagata 2, Fukushima 2
Ibaraki 1, Tochigi 3, Gunma 2, Saitama 4, Chiba 2, Tokyo 11, Kanagawa 4
Niigata 2, Ishikawa 2, Fukui 1, Yamanashi 1, Nagano 1, Gifu 1, Shizuoka 4, Aichi 8
Mie 3, Shiga 3, Kyoto 2, Osaka 8, Hyogo 3, Nara 1
Tottori 4, Shimane 1, Okayama 5, Hiroshima 6, Yamaguchi 3
Tokushima 1, Kagawa 2
Fukuoka 5, Saga 1, Nagasaki 2, Kumamoto 1, Oita 4, Miyazaki 1, Kagoshima 3, Okinawa 2
United Kingdom 1, Macedonia 1, United Arab Emirates 1, United States 1, India 1, Singapore 1, Turkey 1,
Thailand 1, China 1, Belgium 1, Russia 1*Tottori University Hospital excluded.
PCR

1 committee), and also according to the “Guidelinesfor Genetic Tests and Diagnoses in Medical Practice”(February 2011) approved by the Japanese Associationof Medical Sciences. Prenatal diagnosis was approvedby the Ethical Committee of Tottori University.

Before 2009, the primers were designed according to published data with variable rates of successful amplification.
From 2010, almost all primers were designed originally in
our laboratory using Primer3 software utilizing the gene
information from Entrez Gene and RefSeq, resulting insuccessful amplification in almost all cases. The coding
sequence with 70 bp of the flanking area of each exon was
amplified by PCR. The annealing temperature was set
relatively high (around 63 ˚C) (Table 2). The sequences ofthe primers are available on request.
AmpliTaq Gold (Life Technologies, Carlsbad, CA)
was routinely used except for the amplification of GC
rich sequences (more than 70%). For GC rich sequences,
TaKaRa LA Taq (Takara Bio, Otsu, Japan) was used.
The reaction mixture contained in a 10 µL reaction volume 10 to 100 ng of template DNA, 2.5 U AmpliTaq
Gold, 250 µM or 400 µM dNTP, 1 µM of each primer(forward and reverse) and was run at the following conditions: initial denaturation at 95 ˚C for 5 min, followed
by 30 cycles with denaturaion at 95 ˚C for 1 min, annealing at 60 ˚C for 1 min, extension at 72 ˚C for 1 min andfinal extension at 72 ˚C for 5 min. For GC-rich or relatively long sequences, 0.5 U TaKaRa LA Taq was usedinstead of AmpliTaq Gold. The reaction contained 400µM dNTP and was run as follows: initial denaturation at
94 ˚C for 1 min, followed by 30 cycles with denaturaion
at 94 ˚C for 30 s, annealing at 60 ˚C for 30 s, extension
at 72 ˚C for 2 min and final extension at 72 ˚C for 5 min.
For diagnosis of incontinentia pigmenti andFukuyama-type congenital muscular dystrophy, long
insertion or deletion was detected according to the methods described by Bardaro et al.3 and Kato et al.,4 respectively.

Methods

Direct Sequencing of the PCR product is the main
method to detect point mutations, small deletions andinsertions. In the past, single strand conformation polymorphism (SSCP) and denaturing high performanceliquid chromatography (DHPLC) were used in mutation
screenings for porphyria, holoprosencephaly and Joubert
Syndrome. Mutations in the aberrant PCR product detected by SSCP or DHPLC were confirmed by direct
sequencing. SSCP and DHPLC were used from January 2000 to October 2001, and from July 2004 to April
2010, respectively.
The length of the triplet repeat was determined in
the diagnosis of myotonic dystrophy, fragile X syndrome
and dentatorubral-pallidoluysian atrophy. Multiplex
ligation-dependent probe amplification (MLPA) wasused for diagnosis of Fabry disease, Rett syndrome,
Pelizaeus-Merzbacher disease, Duchenne muscular dystrophy, von Hippel-Lindau disease and spinal muscularatrophy, because these diseases are often caused by large
deletions and insertions. Real-time PCR was speciallyused to detect deletions or insertions in the genes thatcause Pelizaeus-Merzbacher disease, Gaucher disease,
Menkes disease and metachromatic leukodystorophy.
Disease and gene information

Information on diseases and causative genes was obtained from Online Mendelian Inheritance in Man andGene database provided by the National Center for Biotechnology Information.

Direct sequencing

PCR products were run on 1.5% of agarose gel electro38

Expansion of genetic testing
Table 2. List of inherited diseases surveyed for genetic mutations in our facility
DiseaseGene symbol

Number of genetic cases

Myotonic dystrophy
Fragile X syndrome
Niemann-Pick disease type C
GM1 gangliosidosis
Joubert syndrome
Gaucher disease
Tay-Sachs disease
Porphyria
Pompe disease
Cowden syndrome
Fabry disease
Menkes disease
Metachromatic leukodystrophy
Rett syndrome/MECP2-related disorders
X-linked hydrocephalus
Pelizaeus-Merzbacher disease
Fukuyama-type congenital muscular dystrophy
Nephrogenic diabetes insipidus (NDI)
Myotubular myopathy
Galactosialidosis
Duchenne muscular dystrophy
Von Hippel-Lindau disease
Spinal muscular atrophy
Dentatorubral-pallidoluysian atrophy (DRPLA)
Tuberous sclerosis
Limb-girdle muscular dystrophy type 2B (LGMD2B)
Pendred syndrome
Enlarged parietal foramina
Sialidosis
Congenital muscular dystrophy-dystroglycanopathy
Methylmalonic acidemia
Niemann-Pick disease type C2
Benign hereditary chorea
Li-Fraumeni syndrome
Mucopolysaccharidosis type IVB (Morquio)
Dyschromatosis symmetrica hereditaria
Incontinentia pigmenti
Melanoma
Calcium-sensing receptor
Dopamine transporter
Familial dysautonomia
Holoprosencephaly
Lesch-Nyhan syndrome
Mitochondrial diseases
Papillon-Lefevre disease
Werner syndrome
Ceroid lipofuscinosis, neuronal, 1
Feingold syndrome
Gardner syndrome
GM2-gangliosidosis, AB variant
Greig cephalopolydactyly syndrome
Growth hormone receptor
Klippel-Feil syndrome
Leber hereditary optic neuropathy
Limb-girdle muscular dystrophy type 1C (LGMD1C)
Nevoid basal cell carcinoma syndrome
Niemann-Pick disease type B
Ornithine carbamoyltransferase deficiency disease
Pseudohypoparathyroidism
Sialic acid storage disease
Townes-Brocks syndrome
Zellweger syndrome

DMPK
FMR1
NPC1†
GLB1
NPHP1†, CEP290†, AHI1†, TMEM67, RPGRIP1L
GBA
HEXA
HMBS†, PPOX†, CPOX†
GAA
PTEN
GLA
ATP7A†
ARSA†
MECP2
L1CAM
PLP1
FKTN
AVPR2
MTM1†
CTSA
DMD
VHL†
SMN1
ATN1
TSC1†, TSC2†
DYSF†
SLC26A4†
ALX4†, MSX2†
NEU1
FKRP†
MMAA†, MMAB†, MMADHC†, MUT†
NPC2†
NKX2-1
TP53
GLB1
ADAR†
IKBKG
CDKN2A†CASR
SLC6A3IKBKAP
SHH, ZIC2†
HPRT1
–
CTSC†
WRN†
PPT1†
MYCN†
AXIN1†
GM2A†
GLI3†
GHR
GDF6†
–
CAV3
PTCH1†
SMPD1†OTC†
GNAS†
SLC17A5†
SALL1
PEX26†

250*
173
73*
56*
42*
40*
38*
38
32
30
28
23*
19*
13
13*
11*
10*
10
9
8
7*
5
5*
4
4
4
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2*
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1

115‡
19‡
24‡
20‡
9‡
9‡
12‡
3‡
8‡
6‡
5‡
3‡
4‡
4‡
3‡
3‡
4‡
3‡
1‡
4‡
3‡
3‡
0‡
2‡
2‡
1‡
3‡
2‡
2‡
0‡
0‡
0‡
2‡
2‡
2‡
1‡
1‡
1‡
0‡
0‡
0‡
0‡
0‡
0‡
0‡
1‡
0‡
0‡
0‡
0‡
0‡
0‡
0‡
0‡
0‡
0‡
0‡
0‡
0‡
0‡
0‡
0‡

1006

287‡

Total		
*The number includes the cases of prenatal diagnosis†The PCR primers were originally designed in our laboratory‡The number indicates the cases of mutation.

39

K. Adachi
Table 3. The number of cases subjected to genetic
testing from January 2000 to September 2013

phoresis and were purified by Ultrafree-DA centrifugal
filter unit (Millipore, Billerica, MA). The sequence wasdetermined by using BigDye Terminator v3.1 Cycle Sequencing Kit and capillary sequencer 3130xL Genetic
Analyzer (Life Technologies).

		
Year
Total

Number of cases
Tottori University Other
Hospital
hospitals

2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013*

6
6
2
7
18
21
44
43
87
74
128
219
198
153

6
6
2
7
18
21
25
9
18
18
55
78
70
54

0
0
0
0
0
0
19
34
69
56
73
141
128
99

Fragment analysis for triplet repeat diseases

Total

1006

387

619

PCR in a total volume of 10 µL contained 100 ng ofDNA, 2.5 U AmpliTaq Gold DNA Polymerase, 250 µM
dNTP, 10% DMSO, 1 µM each primer (forward andreverse), 0.1 µM of the fluorescent labeled probe (FAM,
HEX or NED) and was run as follows: initial denaturation at 94 ˚C for 5 min, followed by 30 cycles withdenaturation at 95 ˚C for 1 min, annealing at 55 ˚C for
1 min, extension at 72 ˚C for 1 min, and final extension
at 72 ˚C for 5 min. The PCR products were mixed with
GeneScan 500 ROX Size Standard (Life Technologies).
After incubation at 95 ˚C for 5 min, the samples wereimmediately cooled and electrophoresed by 3130xL Genetic Analyzer. Before 2005, ALFred DNA Sequencer(Amersham Pharmacia Biotech, Piscataway, NJ) wasused.

* January to September 2013.

SSCP

As described previously, PCR products were separated
on 12% polyacrylamide gel at room temperature or 4 ˚C,
and were visualized by silver staining.5
DHPLC

A WAVE-MD Mutation Detection System (Transgenomic,
Omaha, NE) based on the DHPLC method was used
to detect heteroduplex states according to the manufacturer’s instructions.

Numberofdiseases
2
4
1
3
7
8
13
5
17
20
24
29
26
27

tations were confirmed using the human genome mutation database (BIOBASE, Halchtersche, Germany) and
SNP database. Prediction of functional effect of humannsSNPs (PolyPhen) was used for evaluation of novel mutations.6
Fragment analysis data was analyzed by Peak Scanner Software v1.0 (Life Technologies) for the repeat
size. MLPA data was analyzed by Coffalyzer Software(MRC-Holland).
The personale and running processes of the facility

After October 2010, the working force to run the facility
consists of 1 senior medical genetist, 1 assistant profes-

MLPA
250

Real-time PCRWhen there was any inconsistency in the results of mutations in family members, real-time PCR was used toconfirm the presence of deletions or duplications aroundthe mutations.

35
30

200

25
150

20
15

100

10
50
5
0

*
00 01 02 03 04 05 06 07 08 09 10 11 12 13
20 20 20 20 20 20 20 20 20 20 20 20 20 20

Number of diseases

Number of cases

An MLPA kit (MRC-Holland, Amsterdam, the Netherlands) was used according to the manufacturer’s instructions. In brief, 80 mg of DNA in 6% of glycerol was incubated at 98 ˚C for 15 min for initial denaturation. The
final reaction mixture was electrophoresed by a 3130xL
Genetic Analyzer using a 50 cm-long capillary POP-7.

0

Year

Data analysis

Fig. 1. The number of DNA samples subjected to genetic testingfrom January 2000 to September 2013. Vertical bars show thenumber of cases. Light gray shows the number from other hospitals, dark gray shows the number from Tottori University Hospital.
Dots show the number of diseases.

Sequence data was analyzed by Sequence Scanner (LifeTechnologies) and Genetyx Software (Genetyx, Shibuya,
Japan). Variant Reporter Software (Life Technologies)
was used for detection of mutations from 2011. The mu40

Expansion of genetic testingFig. 2. A flow chart of the genetic diagnosis using direct sequencing of PCR products. When the system has not been established, it isnecessary to obtain the genetic information and design new PCR primers. In this case, it takes at least 3 weeks to detect mutations. Whenthe system has already been established, it takes 2 weeks.

Flow chart of genetic testing

sor and 6 part-time staff. The diagnostic procedures are
separated into steps such as DNA preparation, PCR,
purification, etc., each of which takes approximately 3
working hours, and the samples were processed sequentially by the part-time staff. The senior medical genetist
is responsible for genetic counseling to the patients andfamily members, as well as conformation of mutations
and preparation of the final report.

After 2010, it started to take at least 2 weeks for each genetic test to be completed. An additional one and a halfweeks is required if it is necessary to settle new screening procedures (Fig. 2). PolyPhen software was used
to predict whether any mutation detected was diseasecausing or not.
DISCUSSION
We have received a number of requests for genetic testing from all over Japan. Institutes that provide genetic
testing are rare, and our institute provides a unique function for the Japanese scientific and medical fields. Actually, genetic testing has contributed to several scientificreports.7–18 The number of cases of myotonic dystrophy
was particularly high partly because the genetic testing
was approved by Ministry of Health, Labour and Welfare of Japan as “highly advanced medical treatment”.
It may also be because our facility is the only one approved for conducting prenatal diagnosis of this diseasein Japan.19 The numbers of cases of fragile X syndrome,
Niemann-Pick disease type C, GM1-gangliosidosis andother lysosomal storage diseases are also high because
these diseases were the subjects of active research at
Tottori University.20–22
Direct sequencing by a capillary sequencer is themain method of genetic testing and the size of the repeat
was analyzed for triplet repeat diseases. MLPA wasused for deletions or insertions of exons. For Joubert
syndrome, 19 or more disease-causing genes were reported. Direct sequencing of every exon of all genes isimpractical. In such cases, SSCP or DHPLC was used

RESULTS
Distribution of the hospitals that requested for genetic testing

The hospitals that requested for genetic testing were located 43 in prefectures of Japan and in 11 foreign countries (Table 1).
The number of DNA samples subjected to genetic
testing

A total of 1,006 genetic tests covering 62 diseases were
performed from January 2000 to September 2013 (Table2). The numbers of samples exceeded 50 in myotonic
dystrophy, fragile X syndrome, Niemann-Pick diseasetype C and GM1-gangliosidois. Original primers wereconstructed for 41 diseases. The number of genetic tests
gradually increased year by year and did so rapidly after
we introduced a new service system in 2010 (Table 3,
Fig. 1). All samples were analyzed individually, andmutations were detected in 287 out of 1,006 with anaverage mutation detection rate of 28.5%. Prenatal diagnosis covered 13 diseases and 77 DNA samples wereanalyzed.
41

K. Adachi

for initial screening. These methods are prone to givefalse-negative results and it will be better to use nextgeneration sequencers,23–25 which were introduced to
Tottori University in 2013.
The efficiency of genetic testing has improved andnew lines of genetic testing have been enabled since2010 by the improvement of running processes. Genetic
testing is often still conducted based on research, and theexpense is usually paid for by research funds. Because
the expense is limited and genetic testing is necessary
for clinical diagnosis of many inherited diseases, thecosts should be included in medical expenses. At Tottori
University Hospital, we have settled on basic original
rules that enable us to refer the cost to the patient or client.
Genetic testing performed for research lacks thehigh quality required in clinics. In the United Statesand Europe, the quality of genetic testing is secured by
Clinical Laboratory Improvement Amendments.26 Unfortunately there is no guideline for quality control inJapan. At our facility, testing is performed according to
the Standard Operating Procedures and the informationis shared by each member. Genetic testing is now a popular and powerful tool for diagnosis of many inherited
diseases. We should promote it further in the future.

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7 Fujimoto S, Manabe Y, Fujii D, Kozai Y, Matsuzono K,
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8 Chiba Y, Komori H, Takei S, Hasegawa-Ishii S, Kawamura
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2013 May 27. [Epub ahead of print]. PMID: 23711246.
9 Sekijima Y, Nakamura K, Kishida D, Narita A, Adachi K,
Ohno K, et al. Clinical and serial MRI findings of a sialidosis
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Genotype/phenotype of 6 Chinese cases with Niemann-Pickdisease type C. Gene. 2012;498:332-5. PMID: 22326530.
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12 Higaki K, Li L, Bahrudin U, Okuzawa S, Takamuram A,
Yamamoto K, et al. Chemical chaperone therapy: chaperone effect on mutant enzyme and cellular pathophysiology
in -galactosidase deficiency. Hum Mutat. 2011;32:843-52.
PMID: 21520340.
13 Watanabe T, Yoshida Y, Adachi K, Nanba E, Yamamoto O.
Multiple subcutaneous hard nodules. Clin Exp Dermatol.
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17 Maeda N, Horie Y, Adachi K, Nanba E, Kawasaki H, Daimon
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Acknowledgments: I would like to thank: Professor Eiji Nanba
and all technical staff of the Division of Functional Genomics,
Research Center for Bioscience and Technology for building oursystem of genetic testing and helpful discussion; Dr. Kousaku
Ohno, Emeritus Professor of Tottori University and the staff of theDivision of Child Neurology, Department of Brain and Neuroscience, Faculty of Medicine for collection of the samples and helpful discussion; and Professors Tatsuo Watanabe, Ichiro Hisatomeand Haruaki Ninomiya for helpful discussion. I would also liketo acknowledge all the doctors at our hospital and other hospitals
who provided samples. Finally, thanks to the Center for Promoting
Next-Generation Highly Advanced Medicine at Tottori University
Hospital, which supported the acceptance of samples and sending
reports.
This work was supported by a JSPS Grant-in-Aid for Scientific Research (B) 21659257, 22390207 and 25293230, Health
Labour Sciences Research Grant (Research on Measures for Intractable Diseases) H21-Intractable-public-022, H22-Intractablepublic-002, H22-Intractable-public-014 and H22-Intractablepublic-126.
The author declares no conflict of interest.
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