Northern Virginia Community College Early Western Civilizations Project In class we have analyzed various images, including graffiti and manuscript illumin

Northern Virginia Community College Early Western Civilizations Project In class we have analyzed various images, including graffiti and manuscript illuminations. The “meme” or GIF could be considered to be a more modern version of historical graffiti or illuminations. feel free to create a meme or GIF commenting on the impact of a major event or figure in Early Western Civilization covered and discussed in this class. The Journal of Molecular Diagnostics, Vol. 21, No. 4, July 2019
jmd.amjpathol.org
A Reference System for BRCA Mutation
Detection Based on Next-Generation Sequencing
in the Chinese Population
Shoufang Qu,* Qiong Chen,y Yuting Yi,z Kang Shao,x{ Wenxin Zhang,* Yin Wang,k Jian Bai,k Xuchao Li,** Zhiyuan Liu,**
Xiaowen Wang,yy Ruilin Jing,yy Yanfang Guan,z Xin Yi,z Miaoli Yan,x{ Boyang Cao,x{ Feng Chen,zz Shida Zhu,x{ Xuexi Yang,y
Yingsong Wu,y and Jie Huang*
From the Department of in Vitro Diagnostic Reagent,* National Institutes for Food and Drug Control (NIFDC), Beijing; the Institute of Antibody
Engineering,y School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou; the Department of Research,z Geneplus-Beijing
Ltd., Beijing; the Department of Research,x BGI-Shenzhen, Shenzhen; the China National GeneBank,{ BGI-Shenzhen, Shenzhen; the Department of Research,k
BerryGenomics Co., Ltd., Beijing; the Department of Research,** Amoy Diagnostics Co., Ltd., Xiamen; the Department of Research,yy Annoroad Gene
Technology Co., Ltd., Beijing; and the Research Institute,zz Guangzhou Darui Biotechnology Co., Ltd., Guangzhou, People’s Republic of China
Accepted for publication
March 13, 2019.
Address correspondence to Jie
Huang, Ph.D., National Institutes for Food and Drug
Control (NIFDC), No.2,
Tiantan Xili Dongcheng District, Beijing 10050, People’s
Republic of China; or Yingsong
Wu, Ph.D., Life Science Bldg.
9F, 1023 S. Shatai Rd.,
Guangzhou, Guangdong
510515, People’s Republic of
China. E-mail: jhuang5522@
nifdc.org.cn or wg@smu.edu.
cn.
The absence of interpretation guidelines and limited data on BRCA1/2 mutations in the Chinese population have impeded the detection of BRCA variants based on next-generation sequencing (NGS) in
China. This study was performed to establish a reference system for performance evaluation of BRCA
genetic testing and variant interpretation, which includes interpretation rules, reference materials
(RMs), and a reference database (RD). BRCA1/2 mutations identified in cell lines and clinical cases were
selected to establish RMs. All mutations were detected by NGS and validated by Sanger sequencing.
Variant call format files and standard variant data sets were collected and annotated to build the RD.
Participant laboratories were invited to validate this reference system. Interpretation rules for BRCA
variants in the Chinese population were generated as a standard for BRCA variant interpretation.
Mutational analysis demonstrated that BRCA2 mutations (55%) were more common than BRCA1 mutations (45%) in Chinese patients. Eliminating duplicates from 19,886 variants, the RD contained 750
unique BRCA mutations. Most BRCA1/2 mutations in the reference system were pathogenic or likely
pathogenic (RMs, 77.5%; RD, 57%). In total, 91 novel pathogenic/likely pathogenic variants were
identified in the RD. The reference system can contribute to NGS performance and high-quality interpretation to facilitate clinical decision making. It could also accelerate the development and application of BRCA mutation detection technologies in China. (J Mol Diagn 2019, 21: 677e686; https://
doi.org/10.1016/j.jmoldx.2019.03.003)
Despite years of intensive study and substantial progress in
understanding susceptibility to breast and ovarian cancers,
these diseases remain a major public health problem and are
an important cause of death in women. Approximately 5%
to 10% of breast cancer cases and 10% to 15% of ovarian
cancer cases are thought to be hereditary, caused by
abnormal genes passed from parent to child.1,2 The genes
BRCA1 and BRCA2 have been associated with susceptibility
to breast and ovarian cancers.3 The carrier rate of BRCA1/2
mutations accounts for approximately 0.2% to 0.3% in the
general population, approximately 3% in breast cancer, and
10% in ovarian cancer.4 Patients predisposed to breast and
ovarian cancers exhibit hereditary breast and ovarian cancer
syndrome, an autosomal dominantly inherited disease with
variable penetrance, which has also been associated with
deleterious mutations in BRCA1/2.5,6 Mutations in either of
Supported by National Important Research Project on Prevention and
Control of Reproductive Health and Major Birth Defects in People’s Republic of China grant 2016YFC1000301 (S.Q.).
S.Q., Q.C., Y.Y., and K.S. contributed equally to this work.
Disclosures: None declared.
Copyright ª 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
https://doi.org/10.1016/j.jmoldx.2019.03.003
Qu et al
these genes confer a 60% and 85% lifetime risk of breast
cancer and a 15% to 40% lifetime risk of ovarian cancer.7,8
Research has suggested that the peak breast cancer onset
occurs at 30 to 40 and 40 to 50 years of age in BRCA1 and
BRCA2 mutation carriers, respectively. After onset, the
morbidity rate remains constant at 20% to 30% per year
until 80 years of age.9 Therefore, BRCA was developed as a
biomarker for individual therapeutics. Previous studies have
confirmed that patients with BRCA-mutant ovarian cancer
are sensitive to platinum chemotherapy, and these patients
exhibit a good prognosis after treatment with poly (ADPribose) polymerase inhibitors.10,11 The poly (ADP-ribose)
polymerase inhibitors olaparib (Lynparza), rucaparib
(Rubraca), and niraparib (Zejula) have been approved by the
Food and Drug Administration.
BRCA1 is located on 17q21 and contains 1863 amino
acids and 24 exons (NM_00,724.3),5 whereas BRCA2 is
located on 13q12e13 and contains 3418 amino acids and 27
exons (NM_000059.3).12 There are no obvious mutation hot
spots in BRCA except in the Ashkenazi population.13,14
Consequently, the entire coding region of the BRCA
sequence compared with the present large sample database
was adopted to screen mutations in BRCA. Sanger
sequencing, which is considered the gold standard of DNA
sequencing, is commonly performed for BRCA testing.
However, limited throughput and lower cost-effectiveness
are drawbacks of Sanger sequencing, restricting the development of this technology in clinical genetic testing.
Meanwhile, the development of next-generation sequencing
(NGS) technologies has provided unprecedented opportunities for researchers to investigate the role of genetic variations in common diseases. With the obvious advantage of
high-throughput screening, NGS has been applied in the
clinical diagnosis of BRCA mutations. Early clinical studies
on BRCA testing have shown that NGS offers high sensitivity, specificity, and cost-effectiveness compared with
current approaches.15e18 However, NGS introduces complexities arising from the choice of components of the
BRCA testing workflow, such as the NGS platform,
enrichment method, and bioinformatics analysis. Different
NGS sequencing platforms have applied independent technologies in clinical diagnosis, such as sequencing by synthesis (Illumina, San Diego, CA), ion torrent (Thermo
Fisher, Carlsbad, CA), and combinatorial probeeanchor
synthesis (Beijing Genomics Institute, Shenzhen, Guangdong, China), which use different enrichment methods and
produce different forms of sequencing data. The resulting
sequencing data have different setting requirements for use
in analysis software. As a result, it is challenging to assess
the accuracy of interpreting complex BRCA sequencing
data.
Most BRCA1/2 variations do not increase the risk of
cancer. Thus, the clinical significance of BRCA variants is
judged by pathogenicity. The American College of Medical
Genetics and Genomics (ACMG) classifies variants into five
tiers: pathogenic, likely pathogenic, variant of uncertain
significance (VUS), likely benign, and benign. Only variants considered pathogenic or likely pathogenic have clinical significance. Although the ACMG has provided
directive opinions and advice for this classification,19
variant data interpretation should be evaluated synthetically with the use of information from population and disease databases, literature reports, and patient medical
histories. Consequently, with the lack of a sophisticated
evaluation system for BRCA variant detection and interpretation, analysis of the large-scale data obtained from
NGS and defining the clinical significance of these variants
remains challenging at present.
To date, no large-scale study based on the mutation frequency and characteristics of BRCA in the Chinese population has been performed. It is estimated that the breast
cancer information core database contains >1700 and >2000
distinct variants of BRCA1 and BRCA2, respectively, and
these variants include only 79 BRCA1 (4.44%) and 67
BRCA2 (3.35%) mutations identified in the Chinese population (https://research.nhgri.nih.gov/projects/bic, last
accessed May 8, 2017). The large sample database of
BRCA variants consists primarily of data from European
and American populations. This has increased the
difficulty of annotating BRCA variants in China.
Meanwhile, clinical applications of BRCA mutation
detection are also under investigation in a large number of
laboratories in China. Therefore, it will be important to
adopt a standard to evaluate the performance of NGSbased BRCA variant screening that is clinically actionable.
The primary aim in this study was to establish a reference
system to evaluate the performance of BRCA genetic testing
and variant interpretation. The reference system contained
BRCA mutation reference materials (RMs), interpretation
rules, and a reference database (RD). BRCA mutation RMs
were DNA samples that harbored BRCA mutations, which
are used to evaluate the performance of BRCA mutation
testing, data analysis, and variant interpretation. The BRCA
mutation RD, which supplements the RMs, includes NGS
data from clinical cases, which are applied to evaluate the
performances of data analysis and variant interpretation.
Interpretation rules are developed based on the progress of
RD preparation, which is suitable for interpreting BRCA1/2
variants in the Chinese population.
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Materials and Methods
Interpretation Standard Setting
To provide a standard of BRCA variant interpretation to
build the RMs and RD, experts in the interpretation of
sequence variants in China reached a consensus on BRCA
variant interpretation in the Chinese population. The judgment criteria for the clinical significance of BRCA mutations
were defined according to the guidelines of ACMG,19 and
the characteristics of BRCA variants in the Chinese population. From the information on BRCA single nucleotide
–
The Journal of Molecular Diagnostics
Reference System for BRCA Detection
polymorphisms from noninvasive prenatal testing of
100,000 samples, the characteristics of BRCA variants specific to the Chinese population were defined. BRCA variants
were filtered with single nucleotide polymorphism >0.003,
which were identified as normal in the Chinese population.
Items in the ACMG that were not applicable for BRCA
variant interpretation were also canceled.
pathogenic genetic mutations were taken as the requirement
of specificity. The RM assays were performed in triplicate to
meet accuracy and specificity requirements.
The DNA samples were kept at 20 C and transported
on dry ice. To test DNA stability, two sets of RMs were kept
for 3 or 7 days at 37 C, separately, and one set was frozen
and thawed three times. An additional three sets were
selected to verify the uniformity.
RM Preparation
RD Preparation
To establish the BRCA mutation RMs, eight cell lines that
harbor multiple BRCA mutations, 16 clinical cases with
BRCA1 or BRCA2 mutations, and one case without BRCA
mutations were selected. Of the 16 clinical subjects, six
were women from the pedigrees of BRCA families. Clinical
sample collection was approved by the participating centers
(Annoroad Gene Technology, Beijing, China; Darui Technology, Guangzhou, China; Top Gene Technology,
Shanghai, China). Written informed consent was obtained
from all subjects, and 40 mL of whole blood was collected
from each participant and kept at 20 C.
Genomic DNA was extracted from the enrolled subjects.
All mutations were detected by NGS and validated by
Sanger sequencing. DNA was added to 180 tubes (20-mL
samples of 10 ng/mL) as the RMs of BRCA variants. Four
participating centers (BGI-Shenzhen, Shenzhen, China;
Darui Technology; Berry Genomics, Beijing, China;
Annoroad) were invited to validate the performance of the
RMs. Three sets of RMs were sent to each laboratory to
assess the accuracy and specificity of detecting and interpreting BRCA variants and the consistency of repeat testing.
Detection methods were performed according to the standard operating procedure and instructions of the BRCA
variant detection kit of each laboratory. Detected variants
were marked and interpreted according to the interpretation
rules of BRCA genetic variants. Because pathogenic (class
5) and likely pathogenic (class 4) tiers had the same clinical
significance in terms of the clinical decision, the detection
results were judged by whether the variants were interpreted
correctly, which will directly affect the clinical decisions.
Table 1 shows the judgment standard for the interpretation
errors. Undetected noncharacterized pathogenic and likely
Table 1
To establish the BRCA mutation RD, variant call format
(VCF) files and standard variant data sets (Excel format;
Microsoft, Redman, WA) obtained by NGS sequencing of
clinical cases were collected by five participating laboratories (BGI; Geneplus Technology, Beijing, China; Darui
Technology; Berry Genomics; AmoyDx, Xiamen, China).
Subjects eligible for the RD included at least one BRCA
variant screened with the use of the entire coding region of
BRCA obtained by NGS, without the limitations of clinical
significance and testing samples (germline or somatic).
Eligible VCF files were reviewed by five participating
centers to assess the accuracy of detection of BRCA1/2
mutations and variant interpretation. VCF files were named
according to the sample names, with VCF ver. 4.0 and
coverage region of BRCA sequencing in CDS 15. Each
sample should match one VCF file, and the variant callings
in the VCF files should be kept consistent in the variant data
set. The following fields of the variant data sets were
required to be submitted: sample ID, condition, origin,
sample type, standard genome [hg19/(GRCh37)], Chr, Pos
(1 base), Ref base, alter base, zygosity, minor allele frequency, depth, gene, ExIn_ID, function, transcript
(BRCA1:NM_007294.3, BRCA2:NM_000059.3), changes
in nucleotides (cHGVS), changes in amino acids (pHGVS),
clinical significance, evidence level, interpretation, submitter, detection method, platform, validation, method of
validation, and date of submission. Standard gene variant
nomenclature published by the Human Genome Variation
Society
(HGVS;
http://varnomen.hgvs.org/
recommendations/checklist, last accessed July 2, 2018)
was required in cHGVS and pHGVS. Annotation of the
Judgment Rules of Interpretation Error
Classification
Excluded interpretation error
Interpretation error
Deduction, points
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
Class
5
5
5
5
5
2
5
5
2
5
5
4
4
3
3
2
1
1
4
4
5
5
2
2
3, class 1
2
2
3
2,
3
2,
5,
1
5,
5,
3
class 1
class 1
class 4
class 4
class 4
Class 5, pathogenic; class 4, likely pathogenic; class 3, variant of uncertain significance; class 2, likely benign; class 1, benign.
The Journal of Molecular Diagnostics
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Qu et al
Table 2
The Specific Differences between the Chinese Developed Rules and ACMG Rules
BRCA variant interpretation
Chinese-developed rules
ACMG rules
Deleted items
Not applicable
Detailed items
PVS1 Caveats: As for the variants at the
extreme 30 end of a gene, if there is no report
at this variant and none of null
pathogenic variants were reported after this
variant, it is not formed as PVS1 evidence.
PP3: Only all predictive results could be used as an
evidence. Predictive results could not be used
as the unique evidence for clinical classification
and clinical decision.
PP4 use condition: High clinical sensitivity; there
is less overlap between phenotype and other
disease; benign variant could be distinguished
by the large population frequency library;
family history keep the same with inheritance
patterns.
BA1: Allele frequency is >5% in Exome
Sequencing Project, 1000 Genomes Project, or
Exome Aggregation Consortium, Allele
frequency is 1% for BRCA in 1000 Genomes
Project, or Exome Aggregation Consortium
PVS1 Caveats: Beware of genes in which LOF is
not a known disease mechanism (eg, GFAP,
MYH7); use caution with splice variants that are
predicted to lead to exon skipping but leave the
remainder of the protein intact
PM1: Located in a mutational hot spot and/or
critical and well-established functional domain
(eg, active site of an enzyme) without benign
variation
PM3: For recessive disorders, detected in trans
with a pathogenic variant
Note: This requires testing of parents (or
offspring) to determine phase.
PM6: Assumed de novo but without confirmation
of paternity and maternity
PP2: Missense variant in a gene that has a low
rate of benign missense variation and in which
missense variants are a common mechanism of
disease
BS2: Observed in a healthy adult individual for a
recessive (homozygous), dominant
(heterozygous), or X-linked (hemizygous)
disorder, with full penetrance expected at an
early age
BP5: Variant found in a case with an alternate
molecular basis for disease
PVS1 Caveats: Use caution interpreting LOF
variants at the extreme 30 end of a gene.
PP3: Multiple lines of computational evidence
support a deleterious effect on the gene or gene
product (conservation, evolutionary, splicing
impact, etc.)
Caveat: Because many in silico algorithms use the
same or similar input for their predictions, each
algorithm should not be counted as an
independent criterion. PP3 can be used only
once in any evaluation of a variant.
PP4: Patient’s phenotype or family history is
highly specific for a disease with a single
genetic cause.
BA1: Allele frequency is >5% in Exome
Sequencing Project, 1000 Genomes Project, or
Exome Aggregation Consortium
ACMG, American College of Medical Genetics and Genomics; LOF, loss of function.
clinical significance based on the interpretation rule
consensus was required to classify variants as pathogenic,
likely pathogenic, VUS, likely benign, or benign.
Evidence level of interpretation to the five-tier classification should be listed according to the ACMG guidelines.
At present, 19,886 variants in 2843 cases have been
enrolled to establish the RD. Of the 2843 cases, 1323
were tested with the Illumina sequencing platform,
whereas 1032 and 488 were detected with the BGISEQ500 sequencing platform (BGISEQ, Shenzhen, China)
and Ion Torrent sequencing platform, respectively. In
addition, 987 and 1856 cases were obtained by the NGS
enrichment method of multiple PCR amplification and
hybrid capture targeted technologies, respectively. To
form the RD, duplicate variants were removed from the
variant records.
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The Journal of Molecular Diagnostics
Reference System for BRCA Detection
Table 3
The Variants Information of BRCA1/2 in the Reference Materials
Samples
cHGVS
pHGVS
Function
Distribution
Variant interpretation
BRCA1_1
c.3548A>G
c.1303 G>T
c.5351del
c.7397T>C
c.8021dup
c.4327C>T
c.5351del
c.5073del
c.9097del
c.2429A>C
c.5277þ1 G>A
c.1744A>C
c.6952C>T
c.10070C>G
c.981_982delAT_9
c.5722_5723del
c.3109C>T
c.3333_3333del A
c.5171delT
c.9275_9278del
c.6445_6446delAT
c.52A>G
c.3883C>T
c.5470_5477delATTGGGCA
c.3800T>G
BRCA1/2 wild type
c.994del
c.5156delT
c.276dupA
c.4013delA
c.68_69delCT
EX21_27 Del
p.K1183R
p.D435Y
p.N1784fs
p.V2466A
p.I2675fs
p….
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