REGULAR ARTICLE
Accurate, simple, and inexpensive assays to diagnose F8 gene inversion
mutations in hemophilia A patients and carriers
Debargh Dutta,1 Devi Gunasekera,1 Margaret V. Ragni,2,3 and Kathleen P. Pratt1
1
Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD; 2Division Hematology/Oncology, University of Pittsburgh Medical Center,
Pittsburgh, PA; and 3Hemophilia Center of Western Pennsylvania, Pittsburgh, PA
Key Points
• Improved assays to detect intron 22 and intron 1 inversions in the
F8 gene have been
developed.
• These assays can efficiently detect or rule
out the most common
genetic mutations
resulting in hemophilia A.
The most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 gene
inversion, which together cause ;50% of severe hemophilia A cases. We report a simple and
accurate RNA-based assay to detect these mutations in patients and heterozygous carriers.
The assays do not require specialized equipment or expensive reagents; therefore, they may
provide useful and economic protocols that could be standardized for central laboratory
testing. RNA is purified from a blood sample, and reverse transcription nested polymerase
chain reaction (RT-NPCR) reactions amplify DNA fragments with the F8 sequence spanning
the exon 22 to 23 splice site (intron 22 inversion test) or the exon 1 to 2 splice site (intron 1
inversion test). These sequences will be amplified only from F8 RNA without an intron 22 or
intron 1 inversion mutation, respectively. Additional RT-NPCR reactions are then carried out
to amplify the inverted sequences extending from F8 exon 19 to the first in-frame stop codon
within intron 22 or a chimeric transcript containing F8 exon 1 and the VBP1 gene. These
latter 2 products are produced only by individuals with an intron 22 or intron 1 inversion
mutation, respectively. The intron 22 inversion mutations may be further classified (eg, as
type 1 or type 2, reflecting the specific homologous recombination sites) by the standard
DNA-based “inverse-shifting” PCR assay if desired. Efficient Bcl I and T4 DNA ligase enzymes
that cleave and ligate DNA in minutes were used, which is a substantial improvement over
previous protocols that required overnight incubations. These protocols can accurately
detect F8 inversion mutations via same-day testing of patient samples.
Introduction
Hemophilia A (HA) is an X-linked bleeding disorder that occurs in 1 of every 5000 males. The disease is
caused by mutations of the factor VIII gene, F8, which is located in the Xq28 region and consists of 26
exons and 25 introns.1-3 The most frequent HA-causing mutations are either an intron 22 gene inversion
(Inv22),4,5 which is responsible for ;45% of severe HA cases, or an intron 1 gene inversion (Inv1),6
which is responsible for 2% to 3% of severe HA cases. In Inv22, the first 22 F8 exons become separated
from exons 23 to 26 due to a recombination event during meiosis in germ cells.7 A 9.5-kb region in intron
22 (int22h1) undergoes homologous recombination with either of 2 extragenic homologs, int22h2
(proximal) or int22h3 (distal), which are located ;500 kb and 600 kb upstream of the F8 gene,
respectively8 (Figure 1). These 2 recombination events are referred to as type 1 and type 2 Inv22
mutations, respectively.9 In Inv1, an intragenic region in intron 1 (int1h1) of the F8 gene undergoes
homologous recombination with int1h2, which is located ;100 kb in the telomeric direction, between
the genes VBP1 and BRCC3.10 Additional rare mutations involve partial F8 gene duplications combined
with an inversion.11,12 Consistent with recommendations of Jenkins et al in 1994, hemophilia genotyping
Submitted 23 September 2016; accepted 3 November 2016. DOI 10.1182/
bloodadvances.2016001651.
27 DECEMBER 2016 x VOLUME 1, NUMBER 3
231
Figure 1. Schematic of F8 intron 22 and intron 1 inversion
A
h3 h2
mutations. (A-C) Light gray rectangles indicate exons 1 to 22,
h1
22 23 26
1
dark gray rectangles indicate exons 23 to 26, and black
rectangles indicate the homologous sequences int22h1,
Normal F8 gene
int22h2, and int22h3, which are abbreviated h1, h2, and h3,
B
1
22
23 26
respectively. Introns and extragenic DNA are indicated by
Homologous
Recombination
black lines. Exons and introns are not drawn to scale. (A) The
normal F8 gene. (B) Homologous recombination between
int22h1 and either int22h2 or int22h3 separates exons 1 to 22
C
1
22
from exons 23 to 26. (C) The inverted F8 gene. (D-F) Light
23 26
Intron 22 gene inversion
gray rectangles indicate exons 1 to 26, dark gray rectangles
indicate the facultative exons F1 and F2 and VBP1 and BRCC3
genes, and black rectangles indicate the homologous
sequences int1h1 and int1h2. Introns and extragenic DNA are
D
indicated by black lines.
int1h2
int1h2
BRCC3
F1F2VBP1
1
26
1
26
E
Normal F8 gene
Homologous
recombination
F
F1F2VBP1
1
BRCC3
26
Intron 1 gene inversion
centers worldwide determine intron 22 inversion status as a first-line
test for patients with severe HA and family members that may
be carriers of or affected by the disorder.13 Determination of the
HA-causing mutation (ie, “hemophilia genotyping”) is useful for the
clinical management of patients and their families, helping both
patients and providers determine appropriate clinical courses.
For many years, intron 22 inversion status was determined by
Southern blotting,4,5 which, although accurate, is labor and time
intensive and must be carried out in specialized laboratories.
Modern testing utilizes inverse-shifting polymerase chain reaction
(IS-PCR)14 and, less frequently, long-range PCR15,16 on DNA
isolated from blood samples to determine patients’ intron 22 or
intron 1 inversion status. These methods, especially when carried
out in an accredited laboratory, are accurate and they represent
the current standard of care in developed countries. Although
widely accepted as diagnostic tests, they are time consuming and
have proven somewhat difficult to standardize; therefore, there are
still some inter- and intralaboratory variations. Next-generation
sequencing may be used to detect inversion mutations; however,
it is not yet used for routine clinical assays. RNA-based methods to
detect Inv22 and Inv1 mutations10,17 have been used in research
and have also been proposed for clinical genotyping of HA
patients.18 These methods have been based on the presence or
absence of PCR-amplified complementary DNA products spanning
the normal exon 22 to 23 or exon 1 to 2 splice site, respectively.
The present study introduces a simple protocol in which RNA isolated
from a whole blood sample or from peripheral blood mononuclear
cells (PBMCs) is subjected to reverse transcription nested PCR
(RT-NPCR) to detect specific amplicons indicating the subject does
or does not have an Inv22 or Inv1 mutation. We also describe an
improved IS-PCR assay that may be performed to diagnose an
232
DUTTA et al
inversion mutation or as a second-line test if knowledge of the
specific homologous recombination site for an Inv22 mutation is
desired.5 Either or both of these assays, the first RNA based and the
second DNA based, may be used for same-day testing of blood
samples to identify inversion mutations in HA patients and carriers.
The RNA-based assay requires only PCR amplification and the
ability to run and visualize a stained agarose gel or minigel.
Materials and methods
This study was approved by institutional review boards at the University of
Pittsburgh, Bloodworks NW, University of Washington, and the Uniformed
Services University of the Health Sciences.
Human subjects
All subjects provided written, informed consent according to the Principles
of Helsinki. PBMCs were from subjects enrolled in the “INHIBIT” feasibility
study (National Heart, Lung, and Blood Institute [NHLBI] HL114674, M.V.R.,
principal investigator [PI]), the Grifols-funded study “Mechanisms of Immune
Tolerance to factor VIII” (K.P.P., PI), or NHLBI 1RC2 HL101851 (T. Howard
and K.P.P., PIs). Normal control buffy-coat samples were from the National
Institutes of Health blood bank. PBMCs from 8 HA-Inv22, 4 carrier-Inv22,
2 HA-Inv1, 1 carrier-Inv1, and 16 non-HA control subjects were used as
sources of RNA and DNA. RNA was also isolated from whole blood from
2 HA-Inv22, 1 carrier-Inv22, and 1 non-HA control subject.
Primer design
Primers for amplification of RNA (isolated from PBMCs or whole blood) were
designed using DNASTAR software (DNASTAR, Inc., Madison, WI). The F8
RNA transcript sequence (National Center for Biotechnology Information
reference sequence: NM_000132.3) was analyzed using the DNASTAR
SeqBuilder program. All primer sequences were checked for specificity
to their target sequences using BLAST (http://www.ncbi.nlm.nih.gov/tools/
primer-blast). For the Inv22 test, primers exon 22 forward 1 (exon 22 FWD1)
27 DECEMBER 2016 x VOLUME 1, NUMBER 3
Table 1. Oligonucleotide primer sequences and expected RT-NPCR amplicons
Primer name
Sequence (59-39)
Nested PCR reaction
(product size)
Diagnostic for
To test F8 exon 22-23 integrity and detect
inverted F8 exon 22 to intron 22 mRNA
product
Exon 22 FWD1
GTGGATCTGTTGGCACCAATG
First PCR
Exon 24 REV
CTCCCTTGGAGGTGAAGTCG
(428 bp)
Exon 22 FWD2
ACCAATGATTATTCACGGCATCAAGA
Second PCR
Exon 23 REV
TGCAAACGGATGTATCGAGCAATAA
(225 bp)
Exon 19 FWD1
TCCAAAGCTGGAATTTGGCG
First PCR
INT22 REV
CAATTCTTTCCATTTTCCAAGACACCGTG
(428 bp)
Exon 19 FWD2
TGCTGGGATGAGCACACTTTT
Second PCR
INT22 REV
CAATTCTTTCCATTTTCCAAGACACCGTG
(378 bp)
Exon 22-23 junction
Exon 22-23 junction
Intron 22 inverted RNA
Intron 22 inverted RNA
To test F8 Exon1-2 integrity and detect inverted
F8 exon1-VBP1 chimeric mRNA product
SP FWD
GCACATCCAGTGGGTAAAGTTC
First PCR
Exon 3 REV
AGACTGACAGGATGGGAAGC
(509 bp)
SP FWD
GCACATCCAGTGGGTAAAGTTC
Second PCR
Exon 2 REV
GGCCTTGGCTTAGCGATGTT
(413 bp)
Exon 1 FWD1
TGGGAGCTAAAGATATTTTAGAGAA
First PCR
VBP1 REV1
ACTTATACTTCTGGTACTGTTCATCCAGC
(590 bp)
Exon 1 FWD2
GAATTAACCTTTTGCTTCTCCAGTTGAAC
Second PCR
VBP1 REV2
TGTTTCATGAAGGAATCTAC
(500 bp)
Exon 1-2 junction
Exon 1-2 junction
F8-VBP1 chimeric RNA
F8-VBP1 chimeric RNA
The protocol for both the reverse transcription PCR and the nested PCR steps of the Inv22 assay was as follows: 50°C, 30 min; 95°C, 15 min; 30 cycles of 94°C (30 s), 50°C (45 s), and
72°C (60 s) followed by a 10-min incubation at 72°C. The protocol for both the reverse transcription PCR and the nested PCR steps of the Inv1 assay was as follows: 50°C, 30 min; 95°C, 15 min;
30 cycles of 94°C (30 s), 59°C (45 s), and 72°C (60 s) followed by a 10-min incubation at 72°C.
and exon 24 reverse (Exon 24 REV) were designed to amplify a region
spanning F8 exons 22 to 24, generating an expected 428-bp product.
Internal primers exon 22 forward 2 (exon 22 FWD2) and exon 23 reverse
(exon 23 REV) were designed to amplify a shorter F8 sequence spanning
exons 22 to 23, generating an expected 225-bp product. To test for the
presence of the 39 end of the truncated F8 mRNA product (containing exons
1-22 plus additional bases within the intron 22 sequence) that is expressed
only in individuals with an Inv22 mutation, primers exon 19 forward 1 (exon
19 FWD1) and intron 22 reverse (INT22 REV) were designed to amplify a
F8 sequence spanning the exon 22 to intron 22 junction, generating
an expected 390-bp product. Internal primers exon 19 forward 2 (exon 19
FWD2) and INT22 REV were designed to amplify a shorter F8 sequence
spanning this same junction to generate an expected 378-bp product.
For the Inv1 RT-NPCR test, primers signal peptide forward (SP FWD) and
exon 3 reverse (exon 3 REV) were designed to amplify a region spanning the
F8 signal peptide sequence and exon 3, generating an expected 509-bp
product. Internal primers SP FWD and exon 2 reverse (exon 2 REV) were
designed to amplify a shorter sequence that also spans the F8 exon 1 to 2
junction, generating an expected 413-bp product. To test for the presence
of the chimeric F8-VBP1 chimeric mRNA product (containing the F8 signal
peptide sequence, 2 facultative exons, F8 Exon 1 and VBP1 gene exon
sequences), which is expressed only in individuals with an Inv1 mutation,
primer sets from Bagnall et al10 were used. Primer names and sequences,
expected PCR products, and their diagnostic indications are listed in
Table 1. Table 2 lists primers for the IS-PCR assays.
PBMCs. PBMCs were harvested from blood samples by Ficoll-Paque
PLUS underlay (GE Healthcare, Pittsburg, PA) and used immediately or
frozen in 0.4% dimethyl sulfoxide Hybrid-Max (Sigma, St. Louis, MO) in 1mL
of heat-inactivated fetal bovine serum (FBS; Gibco BRL, Green Island, NY)
27 DECEMBER 2016 x VOLUME 1, NUMBER 3
in liquid nitrogen. RNA yields from frozen cells could optionally be improved
by culturing thawed cells for 24 hours before RNA isolation, as follows: Vials
containing ;10 million PBMCs were thawed at 37°C and then transferred
into a 15-mL tube. Next, 2 mL of 50% heat-inactivated FBS in RPMI 1640
(Gibco BRL) containing Pen-strep (Gibco BRL) and 2 mM glutamate (Gibco
BRL) was added drop-wise while gently mixing the cells. Then, 7 mL RPMI
1640 was slowly added to this mix and centrifuged at 329g for 8 min with the
brakes off. After aspiration of the supernatant, the cells were resuspended in
10 mL of freshly prepared 5% FBS in RPMI 1640. Cells were centrifuged
and resuspended in 10% FBS in RPMI 1640 and then counted using 0.45%
Trypan blue (Gibco BRL) and the cell concentration was adjusted to
1 million cells per milliliter. Finally, 10 million PBMCs in 5 mL of 10% FBS in
RPMI 1640 were seeded in 2 wells of a 6-well plate and cultured for
24 hours at 37°C in a 5% CO2 incubator for 24 hours.
Table 2. Primers for IS-PCR tests to detect Inv1 and Inv22 mutations
Primer name
Sequence (59-39)
1U
CCTTTCAACTCCATCTCCAT
2U
ACGTGTCTTTTGGAGAAGTC
3U
CTCACATTGTGTTCTTGTAGTC
1D
ACATACGGTTTAGTCACAAGT
ED
TCCAGTCACTTAGGCTCAG
1-IU
GCCGATTGCTTATTTATATC
1-ID
TCTGCAACTGGTACTCATC
1-ED
GCCTTTACAATCCAACACT
F8 GENE INVERSION DETECTION IN HEMOPHILIA A
233
Table 3. Expected molecular weights (bp) of amplicons from IS-PCR
and RT-NPCR assays to detect intron 22 inversions
(Qiagen). Sequencing was performed in the Uniformed Services University of
the Health Sciences Biomedical Instrument Center Genomic Core facility.
IS-PCR
IS-PCR diagnostic complementary
First
Second
Inv22
Inv22
RT-NPCR RT-NPCR
Simplified IS-PCR. IS-PCR was performed as described elsewhere,14 with the following modifications. Digestion of 1 to 2 mg genomic
DNA was carried out using a FastDigest Bcl I kit (Thermo Fisher Scientific)
according to the supplier’s specifications for 5 min at 37°C. This step replaced
the overnight incubation that was required using the earlier version of the Bcl I
enzyme. The enzyme was then inactivated at 80°C for 20 min. DNA fragments
were circularized using a Rapid DNA Ligation kit (Thermo Fisher Scientific)
with T4 DNA ligase at 22°C for 15 min (DNA preconcentration by phenolchloroform/ethanol precipitation was not required). A total of 5 mL of the
circularized DNA product was subjected to PCR using polymerase from a
One-Step RT-PCR Kit (Qiagen) with 1 mM concentration of each primer as
shown in Tables 3 and 4. Then, 1U, 2U, 3U, 1D, and ED primers were used for
Inv22 screening, while 1-IU, 1-ID, and 1-ED primers were used for Inv1
screening, as described previously.14 PCR cycling steps were as follows:
95°C (15 min); 35 cycles of (94°C [30 s], 56°C [1 min], and 72°C [1.5 min])
followed by a 2-min incubation at 94°C and 5-min incubation at 72°C. The
IS-PCR amplified product solutions were loaded on 2% to 2.15% agarose
gels and electrophoresed at 100 mV for 25 min using a Smart minigel
electrophoresis system (Luminous Biosciences), and gel images were
obtained on a FluorChem imager (ProteinSimple, San Jose, CA).
F8 allele
Male
N
487
457; 405
225
—
Inv22-1
333
559; 457
—
378
Inv22-2
385
559; 405
—
378
Inv22-3*
NR
NR
—
378
Dup 1-22*
487
559; 457; 405
225
378
Del 22-1*
333
457
—
—
Del 22-2*
385
405
—
—
Female (or male
with >1 F8 gene or
Xq28 segment)
487
457; 405
225
—
N/Inv22-1
487; 333
559; 457; 405
225
378
N/Inv22-2
487; 385
559; 457; 405
225
378
N/Inv22-3*
NR
NR
225
378
N/Dup 1-22*
487
559; 457; 405
225
378
N/Del 22-1*
487; 333
457; 405
225
—
N/Del 22-2*
487; 385
457; 405
225
—
N/N
Del22-1 and Del 22-2, F8 exon 1-22 deletion involving 0.5 Mb from int22h-1 and the most
centromeric of the homologs int22h-2, int22h-3. Dup 1-22, tandem duplication(s) of an F8
gene segment spanning int22h-1 to the most centromeric copy of int22h (int22h-2 or int22h-3).
Inv1, intron 1 inversion; Inv22, intron 22 inversion; Inv22-3, type 3 inversions11,12 involve
homologous recombination of int22h-1 with a duplicated int22h sequence (a rare genetic
event, ,1% of F8 inversion mutations); N, normal F8 gene; NR, not reported here, as this
family of rare mutations would produce amplicons of various lengths.
*Additional F8 gene mutations/rearrangements resulting from alternative pairings among
the int22h homologs int22h-1, int22h-2, and int22h-3.14,24
RNA and DNA isolation from whole blood or PBMCs. Total cellular
RNA from 2.5 mL whole blood cryopreserved in PAXgene RNA tubes (Applied
Biosystems, Foster City, CA) or from PBMCs was purified using the RNeasy
Plus Mini Kit (Qiagen, Gaithersburg, MD), with removal of contaminating
genomic DNA using an RNase-free DNase Set (Qiagen) following the
manufacturer’s protocols. Genomic DNA from whole blood or PBMCs was
purified using the PureLink Genomic DNA mini kit (Invitrogen, Carlsbad, CA)
as per the instruction manual. The purified nucleic acid concentrations were
determined using a NanoPhotometer P-Class (Implen, Westlake Village, CA).
RT-NPCR. PCR runs were carried out on a GeneAmp PCR System
9700 (Applied Biosystems). DNase- and RNase-free distilled water was used
for all reactions. The PCR mix, which was freshly made before each PCR run,
consisted of 2 mL reverse transcriptase, 10 mL reaction buffer, and 2 mL
nucleotide mix. For the first (outer) PCR reaction of each nested PCR
experiment, 0.4 to 0.5 mg template RNA and the volumes of each outer primer
needed for a final concentration of 1 mM were added to the PCR mix, and the
total volume was brought to 50 mL with distilled water. For the second (inner)
PCR reaction, 5 mL of the first PCR reaction product was added to a new PCR
mix plus the volumes of each outer primer needed for a final concentration of
1 mM, and the total volume was brought to 50 mL with distilled water. Primers
used for each RT-NPCR reaction are summarized in Table 1.
Agarose gel electrophoresis and complementary DNA sequencing.
A total of 20 mL of each RT-NPCR product was run on a 2% agarose/tris
acetate-EDTA gel (LE Agarose, BioExpress (Kaysville, UT) using a Smart mini
gel electrophoresis system 1 (Luminous Biosciences, Rockville, MD). DNA
bands were visualized by ethidium bromide (Fisher Scientific, Fair Lawn, NJ)
staining, cut from the gel and purified using a QIAquick PCR purification kit
234
DUTTA et al
Results
RT-NPCR
RNA was analyzed immediately or stored as 0.5-mg aliquots
at 280°C. For all of the RNA samples, the absorbance (l) 5 260/
280 nm ratio was between 2 and 2.2 and the l 5 260/230 nm ratio
was between 2 and 2.2. Thus, highly pure total RNA was obtained.
RT-NPCR assays were carried out for all subjects. Figure 2 shows
representative results of Inv22 diagnostic assays. The 225-bp
product indicating an intact exon 22 to 23 junction was amplified
from all non-HA samples and from the 4 carrier-Inv22 samples, but
not from 9 of the 10 HA-Inv22 samples. Interestingly, RNA from
1 HA-Inv22 subject amplified both the 225-bp and the 378-bp
bands (not shown); DNA sequencing and IS-PCR indicated a
partial F8 gene duplication in addition to an Inv22 mutation. The
378-bp amplicon produced only from F8 mRNA in which intron 22
was not spliced and removed was amplified from all HA-Inv22 and
carrier-Inv22 samples, but not from any of the non-HA samples. DNA
sequencing of the bands confirmed that they were indeed the expected
amplicons. Figure 3 shows representative results of Inv1 diagnostic
Table 4. Expected molecular weights (bp) of amplicons from IS-PCR
and RT-NPCR assays to detect intron 1 inversions
IS-PCR
diagnostic Inv1
First
RT-NPCR
Second
RT-NPCR
N
304
413
—
Inv1
224
—
500
—
—
—
F8 allele
Male
Del 1-2
Female (or male with >1
F8 gene or Xq28 segment)
N/N
N/Inv1
N/Del 1-2
304
413
—
304; 224
413
500
304
413
—
Del 1-2, any F8 gene deletion that significantly alters or removes the exon 1 to 2 splice site
sequences and that does not produce a chimeric F8 exon 1/VBP1 sequence. Other
abbreviations are explained in Table 3.
27 DECEMBER 2016 x VOLUME 1, NUMBER 3
A
Exon 22
Exon 23
Exon 23
Exon 22
CAAGGTGGATCTGTTGGCACCAATGATTATTCACGGCATCAAGA
CTTATCGAGGAAATTCCACTGGAACCTTAATGGTCTTCTTTGGCAAT
Exon 22 FWD1 primer
Exon 22 FWD2 primer
Exon 23
Exon 24
Exon 24
CGACTTCACCTCCAAGGGAG
TTTAAATAGTTGCAGCATGCCAT
TTATTGCTCGATACATCCGTTTGCA
Exon 24 REV primer
Exon 23 REV primer
B
Normal F8 mRNA transcript
Exons 1-18
C
GACCTCAG
Exon 19
Exon 20
Exon 21
Exon 22
Exon 23
Exon 24
Exon 25
Exon 26
225 bp
MW N
HA
428 bp
C
RT-NPCR products
225 bp
F8 intron-22 inverted mRNA transcript
D
Exon 19
Exon 20 Exon 21
Exon 19
TCCAAAGCTGGAATTTGGCG
TGCTGGGATGAGCACACTTTT
Exon 19 FWD1 primer
Exon 19 FWD2 primer
Exon 22
Exon 22
CAAGGTGG
GTGTTTT
Exon 22
Intron 22
Intron 22
TCCACTGGAACCTTAATGCAACGTGTCTTGGAAAATGGAAAGAATTGGAAGCCTTCCTACCGCTGGTGA
E
INT22 REV primer
STOP
Exons 1-18
Exon 19
Exon 20
F
MW
N
HA
Exon 21
Exon 22
RT-NPCR products
C
Intron 22
378 bp
390 bp
378 bp
Figure 2. RT-NPCR to detect F8 intron 22 inversion mutations. (A) Schematic representation of exons 22 to 24 in WT F8 mRNA. Primers (gray arrows and font) were
designed to hybridize within F8 exons 22 (pink), 23 (turquoise), and 24 (green). (B) WT-F8 mRNA transcript. RT-NPCR produces 428-bp (black line, often not visible on the gel)
and 225-bp (gray bar) bands that are both diagnostic of an intact F8 exon 22 to 23 junction sequence. (C) RT-NPCR Inv22 test 1, representative result. MW, 1 kb plus DNA
ladder (Invitrogen); N, HA, C, normal control, HA-Inv22, and carrier-Inv22 subjects, respectively. The 225-bp band is amplified from the N and C samples. (The weak ;350-bp band
seen in lane N is due to nonspecific binding of the primers.) (D) Schematic representation of F8 exons 19 to 22 plus part of the transcribed intron 22 sequence in F8 mRNA from
an individual with an Inv22 mutation. Primers were designed to hybridize within F8 exon 19 (blue) and the F8 intron 22 sequence (orange). Note that 51 bases 39 to the end of
the exon 22 sequence, terminating in a TGA stop codon within intron 22, are transcribed as a consequence of the inversion mutation. (E) RT-NPCR produces 390-bp (black
line, often not visible on the gel) and 378-bp (gray bar) bands that are both diagnostic of an unspliced F8 exon 22 to intron 22 sequence. (F) RT-NPCR Inv22 test 2, representative
result. MW, molecular weight ladder; N, HA, C, normal control, HA-Inv22, and carrier-Inv22 subjects, respectively. The 378-bp band is amplified from the HA and C samples.
(The weaker ;750-bp band is due to nonspecific binding of the primers.)
27 DECEMBER 2016 x VOLUME 1, NUMBER 3
F8 GENE INVERSION DETECTION IN HEMOPHILIA A
235
A
Signal Peptide (SP)
Exon 1
Exon 1
SP
GCTTA
ATAAGTCATGCAA
GCACATCCAGTGGGTAAGTTC
SP FWD primer
Exon 1
Exon 3
Exon 2
Exon 2
Exon 3
AACATCGCTAAGCCAAGGCC
CGCAAGATTTCC
CATGGGTCTG
GCTTCCCATCCTGTCAGTCT
Exon 2 REV primer
B
CTGAGG
Exon 3 REV primer
Normal F8 mRNA transcript
SP
Exon 1
Exon 2
Exon 3
Exons 4-26
413 bp
509 bp
RT-NPCR products
C
MW
N
HA
C
509 bp
413 bp
F8 intron-1 inverted, chimeric mRNA transcript
D
Exon 1
SP
SP
Exon 1
TGGGAGCTAAAGATATTTTAGAGAAGAATTAACCTTTTGCTTCTCCAGTTGAAC
ATAAGTCATGCAA
GCTTA
Exon 1 FWD1 primer
Exon 1
Exon 1 FWD2 primer
Facultative Facultative VBP1 Exon 1
VBP1 Exon 1 VBP1 Exon 2
VBP1 Exon 2
GTAGATTCCTTCATCAAACA
GCTGGATGAACAGTACCAGAAGTATAAGT
VBP1 REV2 primer
E
Exon 1
SP
Facultative
VBP1 REV1 primer
Facultative VBP1 Exon 1 VBP1 Exon 2
VBP1 Exons 3-6
500 bp
590 bp
F
RT-NPCR products
MW N
HA
C
500 bp
Figure 3. RT-NPCR to detect F8 intron-1 inversion mutations. (A) Schematic representation of wild-type F8 (WT-F8) mRNA from the signal peptide (SP) sequence
through exon 3. Primers (gray arrows and font) were designed to hybridize within F8 signal peptide (SP) sequence (violet) and F8 exons 1 (yellow), 2 (brown), and 3 (light blue). (B)
WT-F8 mRNA transcript. RT-NPCR produces 509-bp (black line, often not visible on the gel) and 413-bp (gray bar) bands that are both diagnostic of an intact F8 exon 1 to 2
junction sequence. (C) RT-NPCR Inv1 test 1, representative result. MW, molecular weight ladder; N, HA, C, normal control, HA-Inv1, and carrier-Inv1 subjects, respectively.
236
DUTTA et al
27 DECEMBER 2016 x VOLUME 1, NUMBER 3
A
B
RT-NPCR test for intron 22 inversion
N
1
2
HA
type 1
HA
type 2
6
8
C
3
4
5
7
9
IS-PCR test for intron 22 inversion
N
10
1
2
C
3
4 5
HA
type 1
HA
type 2
6
8
7
9 10
559
487
405
333
378
457
385
225
C
RT-NPCR test for intron 1 inversion
C
N
1
2
3
4
D
HA
5
6
IS-PCR test for intron 1 inversion
1
7
N
C
HA
2
8
500
413
304
224
Figure 4. Side-by-side comparison of RT-NPCR (RNA test) and IS-PCR (DNA test) results. See Tables 3 and 4 for an explanation of the bands visualized on these agarose
gels. (A) Representative RT-NPCR to detect Inv22 mutations. Lane 1, molecular weight (MW) ladder; lanes 2 and 3, normal control (N) subject; lanes 4 and 5, carrier-Inv22 (C)
subject with a type 2 Inv22 mutation; lanes 6 and 7, HA subject with a type 1 Inv22 mutation; lanes 8 and 9, HA subject with a type 2 Inv22 mutation. The 225-bp band is
amplified from the N and C samples. The 378-bp band is amplified from the C and HA-Inv22 type 1 or type 2 mutation samples. (B) Representative IS-PCR to detect Inv22 mutations
using DNA samples from the same subjects shown in panel A. Lanes 2, 4, 6, and 8 show diagnostic IS-PCR results for the N, C, HA-Inv22-type 1, and HA-Inv22-type 2 samples,
respectively. Lanes 3, 5, 7, and 9 show complementary IS-PCR tests for the same samples. Lanes 1 and 10, MW ladder. (C) Representative RT-NPCR to detect Inv1
mutations. Lane 1, MW ladder; lanes 2 and 3, normal control (N) subject; lanes 4 and 5, carrier-Inv1 (C) subject; lanes 6 and 7, HA-Inv1 subject; lane 8, MW ladder. The 413-bp
band is amplified from the N and C samples. The 500-bp band is amplified from the C and HA-Inv1 samples. (D) Representative IS-PCR to detect Inv1 mutations using DNA
samples from the same subjects shown in panel C. Lanes 1 and 2, MW ladder; lanes N, C, and HA show IS-PCR results for the N, C, and HA-Inv1 subjects.
assays. The 413-bp product indicating an intact exon 1 to 2 junction
was amplified from all carrier-Inv1 and non-HA control samples, but not
from the 2 HA-Inv1 samples. The 500-bp amplicon produced only from
F8 mRNA in which intron 1 was not spliced and removed was amplified
from the 2 HA-Inv1 samples and the carrier-Inv1 sample, but not
from the non-HA control samples. DNA sequencing of these
bands confirmed that they were indeed the expected amplicons.
IS-PCR
IS-PCR assays were carried out using DNA isolated from 11 of the
subjects whose RNA was used for the RT-NPCR assays described
above: 4 HA-Inv22 and 3 carrier-Inv22 subjects, 1 HA-Inv1 subject,
1 HA-Inv carrier, and 2 non-HA controls. Results of IS-PCR using
the revised protocol described above were comparable to published
results of assays using the classic IS-PCR method.14 In each case, the
HA genotype determined by IS-PCR was consistent with that
obtained using the RT-NPCR assay, indicating the RT-NPCR method
is an acceptable alternative or corollary to IS-PCR genotyping to
detect F8 gene inversion mutations. Figure 4 shows side-by-side
comparisons of RT-NPCR and IS-PCR test results using RNA and
DNA, respectively, from normal controls and from HA and carrier
subjects with an Inv22 or Inv1 mutation.
Discussion
Many hemophilia patients, especially in developing countries, have
limited options for diagnosis and treatment, although the World
Federation for Hemophilia and other organizations are working to
improve access to appropriate medical care. Genotyping of hemophilia
patients and family members (ie, determining the hemophilia-causing
mutation) is important to patients and their families, as well as to
physicians who provide their clinical care.19,20 Genotyping can aid in
clinical management by predicting the potential severity of the bleeding
disorder and by indicating (albeit with less accuracy) the relative risk that
patients may develop neutralizing anti–factor VIII antibodies.21 Because
most HA carriers possess both a normal and a mutated X chromosome,
these carriers have a 50% chance of transmitting the mutated copy to
their children. Sons who inherit the variant X chromosome will be
affected by HA, while daughters of a non-HA father and a carrier mother
who inherit the variant gene from their mother will be carriers. Many
family members wish to have their carrier status determined through
genetic testing. We report here a set of simple and relatively inexpensive
assays to detect Inv22 and Inv1 mutations, which are the causative
mutation in approximately half of severe HA cases. These assays can
provide same-day or next-day results, thereby improving the efficiency of
diagnosing inversion mutations.
Figure 3. (continued) The 413-bp band is amplified from the N and C samples. (The 509-bp outer RT-NPCR product is also seen on this gel.) (D) Schematic representation
of the chimeric F8-VBP1 mRNA from an individual with an Inv1 mutation, showing the transcript from the F8 SP sequence to the 59 region of the VBP1 gene. Primers were designed
to hybridize within F8 exon 1 (yellow), and VBP1 exon 2 (turquoise). Note that 2 facultative exons between F8 exon 1 and VBP1 exon 1 are also contained in this chimeric transcript
formed as a consequence of the inversion mutation. Outer primers exon 1 FWD and VBP1 REV1 were designed to amplify a 590-bp product. Inner primers exon 1 FWD2
and VBP1 REV2 were designed to amplify a 500-bp product. (E) RT-NPCR produces 590-bp (black line, often not visible on the gel) and 500-bp (gray bar) bands that are both
diagnostic of the chimeric RNA resulting from an Inv1 mutation. (F) RT-NPCR Inv1 test 2, representative result. MW, molecular weight ladder; N, HA, C, normal control, HA-Inv1, and
carrier-Inv1 subjects, respectively. The 500-bp band is amplified from the HA and C samples.
27 DECEMBER 2016 x VOLUME 1, NUMBER 3
F8 GENE INVERSION DETECTION IN HEMOPHILIA A
237
Most current inversion assays rely on either IS-PCR or long-range
PCR of DNA samples. Both methods have been validated in
accredited laboratories. However, the substantial time required to
conduct these assays, and difficulties with amplifying products for
the long-range PCR,14 indicated that diagnostic assays could be
improved further. Several recent publications have reported
alternative methods of identifying Inv22 mutations. A capillary gel
electrophoresis system to detect PCR products has been described,
which has sufficient sensitivity to distinguish between a 584-bp product
(Inv22 positive) and a 512-bp product (Inv22 negative).22 Kumar et al
have reported a quantitative reverse transcription PCR method that is
diagnostic for Inv22 in HA patients. However, quantitative reverse
transcription PCR without gel electrophoresis cannot be used to
diagnose Inv22 carriers.18 Gau et al reported a method that used a
different set of primers to amplify an intact exon 22 to 23 splice site
PCR product23; a potential drawback to using this method is that
lack of an exon 22 to 23 diagnostic band could also be caused by
improper PCR conditions or other experimental artifacts.
rearrangements include an int22h1/int22h2-mediated duplication
that is associated with cognitive impairment (X-linked intellectual
disability).25,26 This mutation has not been associated with HA,
presumably because the normal F8 gene is not directly affected.
Similarly, Del22-1 and Del 22-2 mutations have been described in
females with skewed X-chromosome inactivation but are not associated
with a clinical phenotype, whereas they are embryonic lethal in males.
Our RT-NPCR method offers several advantages to improve sensitivity
and accuracy, specifically (1) nested PCR using the primers reported
here allows reliable amplification of PCR products without requiring
specialized equipment or more expensive reagents required for
quantitative PCR, and (2) in addition to testing the integrity of exon
22 to 23 and exon 1 to 2 junction regions, the method includes a
second set of RT-NPCR reactions to amplify specific products that
are seen only in samples from HA patients and carriers who have an
intron 22 or intron 1 inversion mutation, respectively. In other words, the
diagnosis of an Inv22 or Inv1 mutation requires both the lack of a PCR
band corresponding to the normal splice site (RT-NPCR assay 1) and
the presence of a specific PCR product indicating the chimeric RNA
region resulting from an inversion mutation (RT-NPCR assay 2). RNA
from individuals without an inversion mutation will produce specific PCR
products in the first, but not the second, of each pair of reactions. RNA
from carriers who are heterozygous for the normal and mutated F8 allele
will produce both the normal and mutated RT-NPCR products. As can
be seen in Figure 4, these optimized RT-NPCR assays produce minimal
off-target, nonspecific PCR products, making the analysis of agarose
gels straightforward. Taking advantage of the availability of improved Bcl
I and T4 DNA ligase enzymes, we also report that the “gold standard”
IS-PCR assay may now be accomplished in a significantly shorter time
than the previous iteration of this useful method.
We propose that the RT-NPCR method reported here may be
used as an accurate and efficient means of screening for F8 gene
inversion mutations. IS-PCR may be used as an alternative method
or as a confirmatory step (eg, to distinguish between type 1 and
type 2 Inv22 mutations). Both assays may be carried out in 1 or
2 days, allowing same-day or next-day diagnosis of patients. The
relatively low cost of reagents and the rapidity of these assays
should encourage their widespread use in HA genotyping, including in
resource-poor environments where current costs may be prohibitive.
Tables 3 and 4 list the expected amplicons produced following
IS-PCR and the new RT-NPCR method to analyze DNA or RNA
samples, respectively, from HA patients with a hemizygous F8
mutation and from carriers. Table 3 also includes the expected results
for F8 gene duplications and deletions resulting from alternative
pairings of int22-h1, int22-h2, and int22-h3, some of which can result
in severe HA. The Xq28 region that includes F8 is highly polymorphic,
with multiple possible duplications and deletions.24 Proper analysis of
these rare variants requires DNA-based assays, including IS-PCR
and copy-number variation analysis. The RNA-based assays
described here can accurately detect the most common HA-causing F8
inversion mutations and some but not all of the alternative, rare
int22h-mediated DNA rearrangements. As noted in “Results,” 1 of
the 10 HA-Inv22 subjects analyzed here had an atypical Inv22 plus
a partial F8 duplication mutation that was detected by RT-NPCR
and confirmed by sequencing and IS-PCR. Other previously noted Xq28
238
DUTTA et al
The RT-NPCR assays to diagnose an Inv22 mutation will produce
neither the 225-bp nor the 378-bp amplicon in assays of male HA
patients with a major F8 gene deletion that significantly alters or
removes the exon 22 to 23 splice site sequences. Such a negative
PCR result would suggest, but not prove, the presence of a F8
deletion mutation spanning the exon 22 to 23 junction region (because
lack of the diagnostic amplicons could also result from incorrect PCR
conditions). Similarly, the RT-NPCR assays to diagnose an Inv1
mutation will produce neither the 413-bp nor the 500-bp amplicon in
assays of male HA patients with a major F8 gene deletion that
significantly alters or removes the exon 1 to 2 splice site sequences.
Acknowledgments
The authors thank Zhaozhang Li for primers and sequencing data.
This work was funded by National Institutes of Health, National
Heart, Lung, and Blood Institute research grants 1R01-HL130448,
1RC2-HL101851, and U34-HL114674, and a hemophilia research
grant from Grifols, Inc. The funders had no role in study design, data
collection and analysis, or manuscript preparation.
The opinions or assertions contained herein are the private ones
of the authors and are not to be construed as official or reflecting
the views of the Department of Defense or the Uniformed Services
University of the Health Sciences.
Authorship
Contribution: D.D. designed and performed experiments, analyzed
data, and wrote the paper; K.P.P. designed experiments, analyzed
data, wrote the paper; D.G. purified PBMCs and reviewed the paper;
and M.V.R. enrolled subjects and reviewed the paper.
Conflict-of-interest disclosure: K.P.P. is an inventor on a patent
application related to this study. The remaining authors declare no
competing financial interests.
ORCID profiles: D.D., 0000-0003-1517-0670; D.G., 0000-00025147-7744; K.P.P., 0000-0001-6837-6133.
Correspondence: Kathleen P. Pratt, Department of Medicine
A3075, Uniformed Services University of the Health Sciences,
4301 Jones Bridge Rd, Bethesda, MD 20814; e-mail: kathleen.
[email protected].
27 DECEMBER 2016 x VOLUME 1, NUMBER 3
References
1.
Vehar GA, Keyt B, Eaton D, et al. Structure of human factor VIII. Nature. 1984;312(5992):337-342.
2.
Gitschier J, Wood WI, Goralka TM, et al. Characterization of the human factor VIII gene. Nature. 1984;312(5992):326-330.
3.
Toole JJ, Knopf JL, Wozney JM, et al. Molecular cloning of a cDNA encoding human antihaemophilic factor. Nature. 1984;312(5992):342-347.
4.
Lakich D, Kazazian HH Jr, Antonarakis SE, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nat Genet.
1993;5(3):236-241.
5.
Naylor JA, Buck D, Green P, Williamson H, Bentley D, Giannelli F. Investigation of the factor VIII intron 22 repeated region (int22h) and the associated
inversion junctions. Hum Mol Genet. 1995;4(7):1217-1224.
6.
Pruthi RK. Hemophilia: a practical approach to genetic testing. Mayo Clin Proc. 2005;80(11):1485-1499.
7.
Rossiter JP, Young M, Kimberland ML, et al. Factor VIII gene inversions causing severe hemophilia A originate almost exclusively in male germ cells. Hum
Mol Genet. 1994;3(7):1035-1039.
8.
Freije D, Schlessinger D. A 1.6-Mb contig of yeast artificial chromosomes around the human factor VIII gene reveals three regions homologous to probes
for the DXS115 locus and two for the DXYS64 locus. Am J Hum Genet. 1992;51(1):66-80.
9.
Goodeve AC, Preston FE, Peake IR. Factor VIII gene rearrangements in patients with severe haemophilia A. Lancet. 1994;343(8893):329-330.
10. Bagnall RD, Waseem N, Green PM, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A.
Blood. 2002;99(1):168-174.
11. Antonarakis SE, Rossiter JP, Young M, et al. Factor VIII gene inversions in severe hemophilia A: results of an international consortium study. Blood. 1995;
86(6):2206-2212.
12. Lannoy N, Ravoet M, Grisart B, Fretigny M, Vikkula M, Hermans C. Five int22h homologous copies at the Xq28 locus identified in intron22 inversion type 3
of the Factor VIII gene. Thromb Res. 2016;137:224-227.
13. Jenkins PV, Collins PW, Goldman E, et al. Analysis of intron 22 inversions of the factor VIII gene in severe hemophilia A: implications for genetic
counseling. Blood. 1994;84(7):2197-2201.
14. Rossetti LC, Radic CP, Larripa IB, De Brasi CD. Developing a new generation of tests for genotyping hemophilia-causative rearrangements involving
int22h and int1h hotspots in the factor VIII gene. J Thromb Haemost. 2008;6(5):830-836.
15. Liu Q, Sommer SS. Subcycling-PCR for multiplex long-distance amplification of regions with high and low GC content: application to the inversion
hotspot in the factor VIII gene. Biotechniques. 1998;25(6):1022-1028.
16. Bagnall RD, Giannelli F, Green PM. Int22h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test
for their detection. J Thromb Haemost. 2006;4(3):591-598.
17. Gunasekera D, Ettinger RA, Nakaya Fletcher S, et al; Personalized Approaches to Therapies for Hemophilia (PATH) Study Investigators. Factor VIII gene
variants and inhibitor risk in African American hemophilia A patients. Blood. 2015;126(7):895-904.
18. Kumar P, Husain N, Soni P, Faridi NJ, Goel SK. New protocol for detection of intron 22 inversion mutation from cases with hemophilia A. Clin Appl
Thromb Hemost. 2015;21(3):255-259.
19. Peake IR, Lillicrap DP, Boulyjenkov V, et al. Report of a joint WHO/WFH meeting on the control of haemophilia: carrier detection and prenatal diagnosis.
Blood Coagul Fibrinolysis. 1993;4(2):313-344.
20. de Brasi C, El-Maarri O, Perry DJ, Oldenburg J, Pezeshkpoor B, Goodeve A. Genetic testing in bleeding disorders. Haemophilia. 2014;20(Suppl 4):54-58.
21. Gouw SC, van den Berg HM, Oldenburg J, et al. F8 gene mutation type and inhibitor development in patients with severe hemophilia A: systematic review
and meta-analysis. Blood. 2012;119(12):2922-2934.
22. Pan TY, Wang CC, Shih CJ, Wu HF, Chiou SS, Wu SM. Genotyping of intron 22 inversion of factor VIII gene for diagnosis of hemophilia A by inverseshifting polymerase chain reaction and capillary electrophoresis. Anal Bioanal Chem. 2014;406(22):5447-5454.
23. Gau JP, Hsu HC, Ho CH, Chau WK, Chen CC, You JY. Rapid detection of intron 22 inversions of the factor VIII gene in Chinese patients with severe
hemophilia A. J Chin Med Assoc. 2003;66(9):518-522.
24. Pezeshkpoor B, Oldenburg J. F8 gene: embedded in a region of genomic instability representing a hotspot of complex rearrangements. Haemophilia.
2015;21(4):513-515.
25. Vanmarsenille L, Giannandrea M, Fieremans N, et al. Increased dosage of RAB39B affects neuronal development and could explain the cognitive
impairment in male patients with distal Xq28 copy number gains. Hum Mutat. 2014;35(3):377-383.
26. El-Hattab AW, Schaaf CP, Fang P, et al. Clinical characterization of int22h1/int22h2-mediated Xq28 duplication/deletion: new cases and literature
review. BMC Med Genet. 2015;16:12.
27 DECEMBER 2016 x VOLUME 1, NUMBER 3
F8 GENE INVERSION DETECTION IN HEMOPHILIA A
239