Development of genomic reference materials for
Huntington disease genetic testing
Lisa Kalman, PhD1, Monique A. Johnson, PhD2, Jeanne Beck, PhD3, Elizabeth Berry-Kravis, MD, PhD4,
Arlene Buller, PhD5, Brett Casey, MD6, Gerald L. Feldman, MD, PhD7, James Handsfield, MPH1,
John P. Jakupciak, PhD8, Samantha Maragh, BS8, Karla Matteson, PhD9, Kasinathan Muralidharan, PhD10,
Kristy L. Richie, MA8, Elizabeth M. Rohlfs, PhD11, Frederick Schaefer, PhD12, Tina Sellers, MS, CGC3,
Elaine Spector, PhD13, and C. Sue Richards, PhD2
Purpose: Diagnostic and predictive testing for Huntington disease requires an accurate measurement of CAG
repeats in the HD (IT15) gene. However, precise repeat sizing can be technically challenging, and is complicated
by the lack of quality control and reference materials (RM). The aim of this study was to characterize genomic DNA
from 14 Huntington cell lines available from the National Institute of General Medical Sciences Human Genetic Cell
Repository at the Coriell Cell Repositories for use as reference materials for CAG repeat sizing. Methods: Fourteen
Huntington cell lines were selected for study. The alleles in these materials represent a large range of sizes that
include important diagnostic cutoffs and allele combinations. The allele measurement study was conducted by ten
volunteer laboratories using a variety of polymerase chain reaction-based in-house developed methods and by DNA
sequence analysis. Results: The Huntington alleles in the 14 genomic DNA samples range in size from 15 to 100
CAG repeats. There was good agreement among the ten laboratories, and thus, the 95% confidence interval was
small for each measurement. The allele size determined by DNA sequence analysis agreed with the laboratory
developed tests. Conclusion: These DNA materials, which are available from Coriell Cell Repositories, will facilitate
accurate and reliable Huntington genetic testing. Genet Med 2007:9(10):719–723.
Key Words: quality control, Huntington disease, genetic testing, reference materials, CAG repeats
Huntington disease (HD), which affects approximately 1 in
10,000 individuals, is an adult-onset, autosomal dominant
neurodegenerative disease. People affected by this fatal disor-
various psychotic and behavioral symptoms. A juvenile form
sion has also been observed.1
67 exons spanning 180 kb3and encodes a 348 kDa protein of
unknown function.4HD is caused by an expansion of an un-
stable polymorphic trinucleotide (CAG)nrepeat in the first
Mutable normal alleles have 27–35 repeats and can be meioti-
penetrance, whereas alleles with 40 or more repeats are fully
The discovery of the HD gene and the association of the
(CAG)nrepeat length with the risk for developing HD made
predictive genetic testing possible. The molecular detection of
Prevention, Atlanta, Georgia;2Department of Molecular and Medical Genetics, Oregon Health
and Science University, Portland, Oregon;3Coriell Institute for Medical Research, Coriell Cell
Repositories, Camden, New Jersey;4Departments of Pediatrics, Neurological Sciences, and Bio-
chemistry, Rush University Medical Center, Chicago, Illinois;5Molecular Genetics, Quest Diag-
nostics, San Juan Capistrano, California;6Department of Pathology, Children’s and Women’s
Health Center of British Columbia, Vancouver, British Columbia, Canada;7Clinical Genetics,
troit, Michigan;8National Institute of Standards and Technology, Biochemical Science Division,
Gaithersburg, Maryland;9Department of Medical Genetics, Graduate School of Medicine, Uni-
versity of Tennessee Medical Center, Knoxville, Tennessee;10Department of Human Genetics,
G23, Atlanta, GA 30333. E-mail: LKalman@cdc.gov.
Use of trade names and commercial sources is for identification only and does not imply
endorsement by the Centers for Disease Control and Prevention or the US Department of
Health and Human Services.
and is not subject to copyright. Certain commercial equipment, instruments, materials, or
companies are identified in this paper to specify the experimental procedure. Such identifica-
tion does not imply recommendation or endorsement by NIST, nor does it imply that the
materials or equipment identified are the best available for this purpose.
Disclosure: The authors declare no conflict of interest.
SAS/STAT® is a registered trademark of SAS Institute Inc. QIAquick® is a registered trade-
mark of Qiagen GMBH. BigDye®, GeneAmp®, ABI PRISM® are registered trademarks, and
POP-6™ is a trademark of Applera Corporation. PERFORMA® DTR is a registered trade-
mark of Edge Biosystems.
Submitted for publication: March 9, 2007.
Accepted for publication: June 8, 2007.
October 2007 ? Vol. 9 ? No. 10
a r t i c l e
Genetics IN Medicine
an important, and potentially life-altering effect on patients
and their families. Because the difference between a normal
as one CAG repeat, it is especially important for clinical assays
to be very accurate.
gene is technically challenging and difficult to interpret. There
ing were available. Thus, each clinical laboratory offering this
test develops its own in-house polymerase chain reaction
QC and RMs are urgently needed by the genetic testing com-
munity to facilitate accurate size detection of HD alleles.
RMs are essential for many aspects of genetic testing. Regu-
latory requirements and recommendations of professional so-
cieties stipulate that positive controls should be run at least
once each day in which samples are run and in the same man-
ner as patient specimens to detect errors due to test system
failure or operator error.6,9–18In addition, QC and RMs are
needed for test development and validation, lot-testing of new
reagent batches, and for performance evaluation (proficiency
testing and external quality assurance [PT/EQA]) programs.
The lack of available QC and RMs for genetic testing has
been recognized as a critical need of the genetic testing com-
and the National Institute of Standards and Technology
(NIST) held a series of three meetings to develop a sustainable
process to collect, store, validate, and distribute RMs (mainly
focused on genomic DNA) at a reasonable cost.10
Based on recommendations from the meeting participants,
a new CDC-based program, the Genetic Testing Reference
Material Coordination Program21(GeT-RM, formerly called
the Genetic Testing Quality Control Materials Program
[GTQC]), was established in partnership with the genetics
community. The goal of this program is to coordinate a self-
sustaining community process to improve the availability of
appropriate and characterized RMs for QC, proficiency test-
ing, test development, and research. The GeT-RM Program is
coordinated by the CDC, but all of the actual work, including
decisions about RM priorities, mutation confirmation
schemes, specimen collection, material development, and
characterization, occurs through voluntary cooperation of the
laboratories in the genetics community.
lines by the GeT-RM program and the genetics community.
Cell lines and DNA preparation
Fourteen Epstein-Barr virus-transformed lymphoblast cell
100 were selected from the National Institute of General Med-
ical Sciences Human Genetic Cell Repository at Coriell Cell
Repositories. The cell lines were cultured using previously de-
from each of the selected cell lines by Coriell Cell Reposito-
A total of 10 clinical genetic laboratories that offer HD test-
ing volunteered to participate in this mutation confirmation
Vancouver, British Columbia, Canada.
Each of the 10 clinical laboratories used their own in-house
samples. PCR primers were designed by each laboratory to
hybridize just 5? and 3? of the CAG repeat region in exon 1.
The location of primers for each laboratory is summarized
in Figure 1.
In addition, three of the laboratories utilized a second 3?
repeat region (green text, Fig. 1) that is just 3? to the CAG
tion between alleles when the CAG repeat sizes seemed to be
homozygous. In one laboratory (Laboratory 5, Fig. 1), the size
of the CCG repeat region was only determined in the case of
apparent homozygosity. Laboratory 5 used downstream
primer 5?GGCTGAGGAAGCTGAGGAG for this indication.
Two other laboratories routinely measured the CCG repeat
region in each sample. One laboratory (Laboratory 1, Fig. 1)
ABI3100 capillary electrophoresis instrument. This laboratory
utilized primers pairs: (1) flanking only the CAG repeat
(5?CCTTCGAGTCCCTCAAGTCCTTC and 5?HEX-GGCG-
GCGGTGGCGGCTGTTG), (2) flanking only the CCG repeat
(5?NED- AGCAGCAGCAGCAACAGCC and 5?GGCTGAG-
GAAGCTGAGGAG), and (3) flanking both repeats (5?CCT-
TCGAGTCCCTCAAGTCCTTC and 5?6FAM-GGCTGAG-
1) utilized a second downstream primer 5?GCGGCTGAG-
Four of the laboratories used fluorescently labeled PCR
primers, and the PCR products were analyzed using capillary
electrophoresis on an automated DNA sequencer. A fifth lab-
oratory also used fluorescently labeled PCR primers, but the
PCR products were analyzed using an automated gel based
by automated analysis. The remaining five laboratories either
labeled PCR products with radioisotopes and separated the
products using polyacrylamide gel electrophoresis or gener-
ated unlabeled PCR products and later hybridized with a ra-
dioisotope labeled probe specific to the CAG repeat region.
Kalman et al.
Genetics IN Medicine
Each laboratory received an aliquot of DNA from a previ-
ously characterized Huntington cell line, CD0002224with
were also permitted to use their own in-house validated con-
trols. None of the laboratories reported making adjustments
for abnormal migration of HD amplicons during electro-
guish the band of interest from other “stutter bands” depend-
ing on the method of detection. Laboratories using separation
by gel electrophoresis based their selection on both the inten-
sity (brightest) and the position of the bands. Often, but not
always, the band of interest is the highest band. For laborato-
ries using capillary electrophoresis, the prominent peak that
satisfies the threshold was selected.
DNA from each of the 14 Huntington cell lines being tested as
in each of the 14 samples was not revealed to the laboratories.
The laboratories measured the CAG repeat lengths in each
DNA sample in three separate assays using their in-house
crepancies and compiled the data for statistical analysis.
Mean repeat values and 95% confidence intervals were
calculated using SAS/STAT® PROC GLM. Modes were de-
termined by counting the most common CAG repeat length
reported from each allele.
DNA sequence analysis
DNA sequence analysis was performed to determine the
CAG repeat length in each of the 14 DNA samples. PCR prod-
ucts were gel-purified using a filter column (QIAquick® gel
extraction kit, Qiagen, Valencia, CA). Purified DNA samples
were cycle sequenced (unidirectional) using BigDye® Termi-
nator sequencing kit, version 1.1 (Applied Biosystems, Foster
City, CA), using the forward primer. Cycle sequencing reac-
tions were performed on a PE Applied Biosystems GeneAmp®
PCR System 9700 thermal cycler. DNA sequencing was per-
formed in triplicate using an ABI PRISM® Model 310 Genetic
Analyzer with POP-6™ polymer system and 47 cm ? 50 ?m
capillary. Sequencing data were analyzed with Sequencing
Analysis Software, version 3.3.
The ABI sequencing data were analyzed by DNASTAR Inc
(Madison, WI) Lasergene6.1 SeqManII software to determine
the quality scores.25
RM needs for HD genetic testing were identified through
discussions with clinical laboratory directors and other ex-
Fig. 1. Location of PCR primers flanking the CAG repeat region of the HD gene. The CAG trinucleotide repeats are indicated by shaded boxes. Calculation of the number
of CAG repeats is specific to the primers used. The forward and reverse primers used by each laboratory are indicated. The numbers next to each primer correspond to
individual laboratories. Primer set 11 (NIST) was used for amplification and DNA sequence analysis. The arrows indicate the direction of DNA synthesis from the primers.
Primers used to measure the CCG repeats are not shown but are described in “Methods.”
Huntington disease reference materials
October 2007 ? Vol. 9 ? No. 10
perts. Fourteen cell lines were selected for study. These cell
lines contained a large range of allele sizes and combinations
including normal alleles and alleles at important diagnostic
cutoffs, e.g., 35–36 and 39–40 repeats. Additionally, some of
the cell lines were included because they contained HD alleles
useful for technical reasons, such as homozygous alleles, two
large CAG repeat sizes.
All 10 testing laboratories were able to report data from
three independent size determinations for 27 of the 28 Hun-
the mean and mode CAG repeat number. The size of the re-
maining allele, approximately 100 CAG repeats, proved more
problematic to measure. Eight laboratories were able to report
CAG repeat length from three independent assays for this al-
lele, one laboratory reported results from only one of three
available data (25 measurements) were used to determine the
mean and mode repeat length of this allele.
Using the composite data from the 10 clinical laboratories,
the mean CAG repeat length, 95% confidence intervals, and
the modal values for each cell line were determined (Table 1).
indicates that the values had a normal distribution and were not
skewed. We did not see differences between different detection
The CAG repeat length for each DNA sample was also de-
repeat lengths determined by DNA sequencing and laboratory
analysis were identical for 25 of the 28 alleles. For the remain-
ing three alleles, the value determined by DNA sequence anal-
ysis fell within the 95% confidence limits of the mean values
determined by the 10 laboratories. Quality scores were gener-
were in the acceptable range (?40) for all alleles, except for an
allele in sample NA20207 with 21 repeats. It is likely that the
score for this allele (37.8) was low because of difficulty physi-
cally separating the PCR products for the two alleles (19/21)
before sequence analysis. It is also possible that there is some
minor contamination or mosaicism in sample NA20207,
ity score for this allele.
in the analytic values obtained for the 14 samples tested using
different HD assays or methodologies among the 10 laborato-
ries. There was also very good agreement between the CAG
repeat sizes obtained by the laboratories and the NIST DNA
Mean CAG repeat length in DNA from 14 HD cell lines
Coriell sample number
Clinical laboratory assay
Meancallele 1/allele 2
(95% confidence interval)
(95% confidence interval)
(% total response)
(% total response)
NA20245 15 (13.7–15.8) 15 (13.7–15.8)15 (77%) 15 (80%)15/15
NA2020617 (15.9–18.3) 18 (17.2–19.1)17 (77%)18 (87%)17/18
NA20207 19 (17.7–20.7)21 (20–22.1) 19 (67%)21 (77%)19/21
NA20246 15 (14.1–15.9)24 (22.3–25.9)15 (80%) 24 (77%) 15/24
NA2024715 (14.1–15.9)29 (28.1–29.9) 15 (80%)29 (80%)15/29
NA2024817 (15.0–19.3) 36 (35.1–37.3)17 (77%)36 (80%)17/36
NA2024922 (21.1–22.8)39 (38.3–39.9)22 (87%) 39 (83%) 22/39
NA20250 15 (14.1–15.8) 40 (39.2–41.0)15 (83%)40 (80%)15/40
NA2020835 (33.4–36.3)45 (43.5–46.5) 35 (80%) 45 (77%)35/45
NA2020945 (43.8–46.2) 47 (46.3–47.8)45 (73%)47 (83%)45/46
NA20251 39 (38.1–40.0)50 (49.1–50.8)39 (80%)50 (83%)39/50
NA2025222 (21.2–22.9) 66 (63.7–67.5)22 (83%)65 (37%) 66 (37%) 22/65
NA2021017 (15.4–18.2)74 (72.0–76.6)17 (77%)74 (50%)17/75
NA2025322 (20.3–23.3)99 (95.8–102.7)22 (73%) 100 (52%)22/101
Values were rounded to nearest whole number.
aMean repeat length calculated from 30 responses per allele (except for allele 2 of cell line NA20253 which had only 25 responses).
bCAG repeat length reported most often out of 30 responses per allele (except for allele 2 of cell line NA20253 which had only 25 responses).
Kalman et al.
Genetics IN Medicine
wasfoundbetweentheresultsofDNAsequencingandclinical Download full-text
assays were larger than 45 repeats, which are more difficult to
size or sequence accurately. Because these three alleles were in
the fully penetrant allele range, the interpretation of alleles in
this size range would be unaffected.
The CAG repeat length of 22 of the 28 HD alleles measured
in this study using the laboratory developed PCR methods
agreed with the allele size designated by the original submitter
of the cell lines to Coriell. However, the CAG repeat size of six
confirm the mutations or alleles in genomic DNA materials
from any source using sequencing or other less equivocal
methods, and also underscores the need for characterization
studies of potential RMs.
For most genetic tests and mutations, there is no publicly
available source of DNA or cell lines that can be used as RMs
for QC, PT/EQA, genetic test development/validation or re-
ries and test developers use residual patient specimens when
produce sufficient and varied challenges and also limits the
development and validation of new tests.
There is evidence that the availability of RMs improves the
accuracy of HD repeat sizing. Data collected by the National
European External Quality Assessment Service indicated a
marked decrease in the variance of HD repeat sizing after a 35
repeat HD RM was provided to the participating laborato-
indicate that laboratories generally are able to accurately mea-
sure the CAG repeat length. In each challenge, however, there
are a variety of repeat lengths reported for a given allele. At
times, the variation is 10–20% or more around the consensus
value, suggesting that the availability of HD RMs may help to
improve analytical performance.
The genomic DNA materials characterized in this project
testing. This is especially important for alleles that are close to
repeat could impact the clinical interpretation. In addition,
these studies validate our voluntary community-based ap-
proach to RM development, and may serve as a model for
similar projects in the future.
DNA samples purified from these cell lines, as well as other
materials developed by GeT-RM, are publicly available from
the Coriell Cell Repositories.27More information about the
program and available QC and RMs can be found on the
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