Figure 1. Umbilical cord blood (UCB) (n = 33) and peripheral blood (n = 112, 0 to 51 years) samples were analyzed. TRECs in different age groups are shown. TRECs were significantly higher in umbilical cord blood (6.2 3.2 10 3 copies/mg DNA) and infants (5.8 2.3 10 3 copies/mg DNA) as com- pared with other age groups of children (3.5 2.8 10 3 copies/mg DNA in 2 to 6 years old, 2.0 1.4 10 3 copies/mg DNA in 7 to 12 years old, 8.2 6.3 10 2 copies/mg DNA in 13 to 18 years old) and adults (3.4 3.6 10 2 copies/mg DNA). Morinishi et al, J.Pediatrics 2009, 155: 829-833 TREC正常値 sjKREC正常値 FIG E2. KREC levels were analyzed in genomic DNA samples extracted from peripheral blood of control subjects at different age groups (n 5 158; age range, 1 month to 55 years). KREC levels were significantly higher in infants (17.9 6 3.9 3 10 3 copies/mg DNA) compared with other children’s age groups (8.9 6 1.3 3 10 3 copies/mg DNA in the 1- to 6-year-old group and 3.6 6 3.8 3 10 3 copies/mg DNA in the 7- to 18-year-old group) and adults (2.0 6 3.3 3 10 3 copies/mg DNA; P < .0001). 1mo-11mo(n=15) 1y-6y(n=35) 7y-18y(n=56) 19y-(n=52) 10 0 1 10 2 10 3 10 4 10 5 10 6 sjKREC copies/ gDNA KREC copies/ g DNA Undetectable Kamae et al, J. Allergy Clin Immunol 2013, 131: 1437-1440.e1-5 mean: 17.9 8.9 3.6 2.0 max: 442 min: 2.8 349 0.99 12.1 0.29 12.0 0.22
Transcript
40.6! 2.3 ng/mL (mean! SEM) (range, 13 to 167 ng/mL),7.8! 2.8 ng/mL (2.9 to 13.0 ng/mL), and 5.3! 0.2 ng/mL(0.6 to 20.2 ng/mL), respectively.
Quantitative real-time PCR for dRec-jJa sjTRECs wasperformed using the same primers and dRec probes asreported by Hazenberg et al.14
As an internal control, RNaseP gene was amplified in eachsample tested using TaqMan RNaseP Primer-Probe (VICdye) Mix (Applied Biosystems, Foster City, California).
Statistical AnalysisAn exponential regression model was used to quantify the re-lationship between age and TRECs levels (per mg DNA andper RNaseP). Goodness-of-fit of the model was evaluatedby R2. The Dunnett multiple comparison test was conductedto test the differences of each age group (0 to 1, 2 to 6, 7 to 12,13 to 18 years and adults) versus umbilical cord blood com-parisons serving as a control group (Figure 1). RNaseP andTRECs levels of patients with SCID and control patientswere compared by an unpaired Student t test (if the variancewas equal) or Welch t test (if the variance was different).
All statistical analyses were performed using GraphPadPrism Version 4.00 (GraphPad Software, San Diego, Califor-nia). P < .05 denotes a statistically significant difference.
Results
TRECs were detectable in all DNA samples from whole bloodof normal control patients, including umbilical cord blood(n = 33), healthy infants (0 to 1 year old, n = 12), children(2 to 18 years old, n = 63), and adults (n = 37). TRECs inwhole blood were found to decline with increasing age(r = 0.851). TRECs of umbilical cord blood were significantlyhigher than those of children and adults but were not signif-icantly different from those of infants (Figure 1). We founda strong correlation between TRECs copies/mg DNA andTRECs/RNaseP ratio (r = 0.979).
TRECs of peripheral blood samples from all 14 patientswith SCID before hematopoietic stem cell transplantationwere below detectable levels (<10 copies/mg DNA) with theexception of 1 (P18, see below), in contrast to high levelsof control infants (5.8! 0.7" 103/mg DNA, n = 12)(P < .0001) (Figure 2, A).
Next, we analyzed TRECs of dried blood spots fromnormal control neonates using simulated Guthrie cardsfrom cord blood (n = 31), neonatal Guthrie cards obtainedduring this study period (January 2005 to December 2007)(n = 26), and those stored in a neonatal mass screening centerfor less than 5 years (n = 300). TRECs were detectable in alldried blood spots: in cord blood (1.3! 0.1" 104 copies/mgDNA, mean! SEM), in neonatal Guthrie cards(2.3! 0.2" 104 copies/mg DNA), and in stored neonatalGuthrie cards (3.6! 0.2" 105 copies/mg DNA).
To determine whether this method can identify patientswith SCID, we quantified TRECs using 15 stored neonatal Gu-thrie cards from patients with SCID (patients 1 through 11, 13through 15, and patient 17). RNaseP levels were high in all neo-natal Guthrie cards from patients with SCID (1.8! 0.3" 106
copies/mg DNA, n = 15), which were similar to control levels(2.3! 0.1" 106 copies/mg DNA, n = 26) (P = .184), indicat-ing an appropriate amount of genomic DNA was extractedfrom the neonatal Guthrie cards (Figure 2, B). In contrast,TRECs were below detection levels in all patients (P < .0001)except 1 (patient 11). This patient with SCID had compoundheterozygous mutations of ADA (Gln119Stop/Arg34Ser). Hehad detectable but significantly lower levels of TRECs(6.2" 102 copies/mg DNA) than those of control neonates(2.3! 0.2" 104 copies/mg DNA, n = 26) (Figure 2, B). At 1month of age, the TRECs from the peripheral blood of patient11 were below detectable levels (Table and Figure 2, A).
These results indicate that neonatal mass screening ofSCID by quantitative real-time PCR for TRECs using neona-tal Guthrie cards is feasible.
We analyzed TRECs in 4 patients with SCID with maternalT-cell engraftment (patients 12 through 15, CD3+ cells: 40 to1326/mL). We found that all patients had undetectable levelsof TRECs in neonatal Guthrie cards (patients 13 through 15)and peripheral blood (patients 12 and 14). Patient 12 hada normal lymphocyte count (3250/mL) on admission aswell as a significant number of T, B, and NK cells (Table).His peripheral blood TRECs level was below the detection
Figure 1. Umbilical cord blood (UCB) (n = 33) and peripheralblood (n = 112, 0 to 51 years) samples were analyzed. TRECsin different age groups are shown. TRECs were significantlyhigher in umbilical cord blood (6.2! 3.2" 103 copies/mgDNA) and infants (5.8! 2.3" 103 copies/mg DNA) as com-pared with other age groups of children (3.5! 2.8" 103
copies/mg DNA in 2 to 6 years old, 2.0! 1.4" 103 copies/mgDNA in 7 to 12 years old, 8.2! 6.3" 102 copies/mg DNA in 13to 18 years old) and adults (3.4! 3.6" 102 copies/mg DNA).
December 2009 ORIGINAL ARTICLES
Identification of Severe Combined Immunodeficiency by T-Cell Receptor Excision Circles QuantificationUsing Neonatal Guthrie Cards
831
Morinishi et al, J.Pediatrics 2009, 155: 829-833 TREC正常値 sjKREC正常値
1m
o-1
1m
o(n
=15)
1y-6
y(n
=35)
7y-1
8y(n
=56)
19y-(n=52)
100
101
102
103
104
105
106
sjK
RE
C
co
pie
s/
gD
NA
mean: 17.9 8.9 3.6 2.0 max: 442 min: 2.8
349 0.99
x103 copies/ g DNA 12.1 0.29
12.0 0.22
KR
EC
co
pie
s/
g D
NA
FIG E2. KREC levels were analyzed in genomic DNA samples extractedfrom peripheral blood of control subjects at different age groups (n 5 158;age range, 1 month to 55 years). KREC levels were significantly higher ininfants (17.9 6 3.9 3 103 copies/mg DNA) compared with other children’sage groups (8.9 6 1.3 3 103 copies/mg DNA in the 1- to 6-year-old groupand 3.66 3.83 103 copies/mg DNA in the 7- to 18-year-old group) and adults(2.0 6 3.3 3 103 copies/mg DNA; P < .0001).
J ALLERGY CLIN IMMUNOL
MAY 2013
1440.e2 LETTERS TO THE EDITOR
1m
o-1
1m
o(n
=15)
1y-6
y(n
=35)
7y-1
8y(n
=56)
19y-(n=52)
100
101
102
103
104
105
106
sjK
RE
C
co
pie
s/
gD
NA
mean: 17.9 8.9 3.6 2.0 max: 442 min: 2.8
349 0.99
x103 copies/ g DNA 12.1 0.29
12.0 0.22
KR
EC
co
pie
s/
g D
NA
FIG E2. KREC levels were analyzed in genomic DNA samples extractedfrom peripheral blood of control subjects at different age groups (n 5 158;age range, 1 month to 55 years). KREC levels were significantly higher ininfants (17.9 6 3.9 3 103 copies/mg DNA) compared with other children’sage groups (8.9 6 1.3 3 103 copies/mg DNA in the 1- to 6-year-old groupand 3.66 3.83 103 copies/mg DNA in the 7- to 18-year-old group) and adults(2.0 6 3.3 3 103 copies/mg DNA; P < .0001).
J ALLERGY CLIN IMMUNOL
MAY 2013
1440.e2 LETTERS TO THE EDITOR
Undetectable
Kamae et al, J. Allergy Clin Immunol 2013, 131: 1437-1440.e1-5
1m
o-1
1m
o(n
=15)
1y-6
y(n
=35)
7y-1
8y(n
=56)
19y-(n=52)
100
101
102
103
104
105
106
sjK
RE
C
co
pie
s/
gD
NA
mean: 17.9 8.9 3.6 2.0 max: 442 min: 2.8
349 0.99
x103 copies/ g DNA 12.1 0.29
12.0 0.22
KR
EC
co
pie
s/
g D
NA
FIG E2. KREC levels were analyzed in genomic DNA samples extractedfrom peripheral blood of control subjects at different age groups (n 5 158;age range, 1 month to 55 years). KREC levels were significantly higher ininfants (17.9 6 3.9 3 103 copies/mg DNA) compared with other children’sage groups (8.9 6 1.3 3 103 copies/mg DNA in the 1- to 6-year-old groupand 3.66 3.83 103 copies/mg DNA in the 7- to 18-year-old group) and adults(2.0 6 3.3 3 103 copies/mg DNA; P < .0001).
J ALLERGY CLIN IMMUNOL
MAY 2013
1440.e2 LETTERS TO THE EDITOR
sjKREC正常値
1m
o-1
1m
o(n
=15)
1y-6
y(n
=35)
7y-1
8y(n
=56)
19y-(n=52)
100
101
102
103
104
105
106
sjK
RE
C
co
pie
s/
gD
NA
mean: 17.9 8.9 3.6 2.0 max: 442 min: 2.8
349 0.99
x103 copies/ g DNA 12.1 0.29
12.0 0.22
KR
EC
co
pie
s/
g D
NA
FIG E2. KREC levels were analyzed in genomic DNA samples extractedfrom peripheral blood of control subjects at different age groups (n 5 158;age range, 1 month to 55 years). KREC levels were significantly higher ininfants (17.9 6 3.9 3 103 copies/mg DNA) compared with other children’sage groups (8.9 6 1.3 3 103 copies/mg DNA in the 1- to 6-year-old groupand 3.66 3.83 103 copies/mg DNA in the 7- to 18-year-old group) and adults(2.0 6 3.3 3 103 copies/mg DNA; P < .0001).
J ALLERGY CLIN IMMUNOL
MAY 2013
1440.e2 LETTERS TO THE EDITOR
1m
o-1
1m
o(n
=15)
1y-6
y(n
=35)
7y-1
8y(n
=56)
19y-(n=52)
100
101
102
103
104
105
106
sjK
RE
C
co
pie
s/
gD
NA
mean: 17.9 8.9 3.6 2.0 max: 442 min: 2.8
349 0.99
x103 copies/ g DNA 12.1 0.29
12.0 0.22
KR
EC
co
pie
s/
g D
NA
FIG E2. KREC levels were analyzed in genomic DNA samples extractedfrom peripheral blood of control subjects at different age groups (n 5 158;age range, 1 month to 55 years). KREC levels were significantly higher ininfants (17.9 6 3.9 3 103 copies/mg DNA) compared with other children’sage groups (8.9 6 1.3 3 103 copies/mg DNA in the 1- to 6-year-old groupand 3.66 3.83 103 copies/mg DNA in the 7- to 18-year-old group) and adults(2.0 6 3.3 3 103 copies/mg DNA; P < .0001).
J ALLERGY CLIN IMMUNOL
MAY 2013
1440.e2 LETTERS TO THE EDITOR
Undetectable
Kamae et al, J. Allergy Clin Immunol 2013, 131: 1437-1440.e1-5
1m
o-1
1m
o(n
=15)
1y-6
y(n
=35)
7y-1
8y(n
=56)
19y-(n=52)
100
101
102
103
104
105
106
sjK
RE
C
co
pie
s/
gD
NA
mean: 17.9 8.9 3.6 2.0 max: 442 min: 2.8
349 0.99
x103 copies/ g DNA 12.1 0.29
12.0 0.22
KR
EC
co
pie
s/
g D
NA
FIG E2. KREC levels were analyzed in genomic DNA samples extractedfrom peripheral blood of control subjects at different age groups (n 5 158;age range, 1 month to 55 years). KREC levels were significantly higher ininfants (17.9 6 3.9 3 103 copies/mg DNA) compared with other children’sage groups (8.9 6 1.3 3 103 copies/mg DNA in the 1- to 6-year-old groupand 3.66 3.83 103 copies/mg DNA in the 7- to 18-year-old group) and adults(2.0 6 3.3 3 103 copies/mg DNA; P < .0001).
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