Supplementary Materials
Generation of cell lines: PBMCs were prepared from available patients (1a and 6),
parents and unrelated normal controls. Cells, after washing, were cultured with
Epstein-Barr virus (EBV), PHA, and cyclosporine in RPMI complete media for 3- 4
weeks and frozen until ready to use.
Genomic DNA amplifications were performed by touchdown PCR. Annealing
temperatures ranged from 55 to 68°C. PCR products were purified by using the
AgencourtR AMPureR XP PCR Purification Kit (Beckman Coulter, Beverly,
Massachusetts), and sequenced with the BigDyeR Terminator v3.1 Cycle Sequencing
Kit (Applied Biosystems, Foster City, California) using the M13 Forward primer. After
the sequencing reaction, the DNA was purified using the AgencourtR CleanSeqR Kit
(Beckman Coulter, Beverly, Massachusetts) and run on a 3730xl DNA Analyzer
(Applied Biosystems, Foster City, California). All kits were used in accordance with
the manufacturer’s instructions. Sequencing data were analyzed for mutation detection
using SeqMan II software (DNA Star Inc., Madison, WI). Samples with mutations
were confirmed by sequencing the reverse strand using M13 Reverse primer. Once a
mutation was found, one parent’s or both parent’s DNA, if available, were sequenced to
determine their heterozygote (carrier) status for the same mutations as their children
siblings. To rule out the possibility that the mutations detected were unreported normal
variations within the Saudi population, 250 DNA samples from normal individuals
obtained from the blood bank of the King Faisal Specialist Hospital and Research
Centre were sequenced.
Reverse transcription (RT)-PCR was performed with the following primers: Primers
1: 5’-CAGGGGAGTCAGCAGAGG-3’ (forw); 5’-ACAGGCCTCACTCGTACAGT-3’
(rev).
Primers
2:
5’-ACCTAGGGCTTCGGGTC-3’
(forw);
5’ACATCAGTCTTGTTTGTGCC-3’
(rev).
Primers
3:
5’AAATGTCACCCTTGGACAAG-3’ (forw); 5’-CTCTGGCCAACTGCCTG-3’ (rev).
Primer
4:
5’-GCATCTCAGTGCAGGAGAGA-3’
(forw);
5’AACAATGCTGCAATGGGC-3’ (rev). Primer 5: 5’-CATCATGGTGGCTTCCCT-3’
(forw); 5’-AGACCCTTCTTCCCCACC 3’ (rev). In addition, M13 sequences were
attached to the 5’ end of each primer to allow forward and reverse sequencing. The PCR
conditions were 1 cycle at 94°C for 3 min, 35 cycles at 94°C for 30 s, at 55°C for 30 s,
and at 72°C for 30 s, and 1 cycle at 72°C for 3 min. To assess exon 3 skipping, RT-PCR
was performed in a patient and one family member of that patient from exon 1 to exon 4
using
primers
5’-CAGGGGAGTCAGCAGAGG-3’
(forw)
and
5’AACAATGCTGCAATGGGC-3’ (rev), from exon 2 to exon 4 using primers 5’ACCTAGGGCTTCGGGTC-3’ (forw) and 5’-AACAATGCTGCAATGGGC-3’ (rev),
and from exon 3 to exon 4 using primers 5’-AAATGTCACCCTTGGACAAG-3’
(forw) and 5’-AACAATGCTGCAATGGGC-3’ (rev).
Western blot analysis was performed as follows. Briefly, Laemmli sample buffer
(BioRad, USA) supplemented with 1/20 volume of Beta-mercaptoethanol (βME;
Sigma, USA) was added to the protein samples at a 1:1 ratio and boiled for 5 minutes.
Proteins were then separated on a 12% ready-made Criterion XT Bis-Tris gel (BioRad,
USA) and transferred to a Trans-Blot Transfer medium 0.2 m nitrocellulose membrane
(BioRad, USA). Taking advantage of the difference in size between CD40 (50 kD) and
STAT5 (90 kD), filters were cut horizontally in two halves and the bottom half was
probed with an affinity-purified rabbit polyclonal antibody to the carboxyl- (SC-975,
Santa Cruz,CA, USA) which recognizes only the 40 kD variant and/or amino- (5853,
Abcam) terminus of the CD40 protein. . The top half was probed with an affinitypurified rabbit polyclonal antibody against the C-terminus of the signal transducer and
activator of transcription 5 (STAT5) protein (SC-835; Santa Cruz, USA) as a loading
control. HRP-goat anti-rabbit antibody (65-6420, Invitrogen, USA) was used as a
secondary antibody. The blots were developed using Super Signal West-Pico
chemiluminescence (Pierce, USA).
Supplementary Fig. 1: Flow cytometry analysis of CD40 expression on B cells for
control compare to all 11 patients. Panels show double staining for PE anti-CD40 and
APC, PerCP, or FITC anti-CD19.
Supplementary Fig. 2: c. 256+2T>C mutation. Electrograms of the four family
members, sequenced as indicated. The line along the electrograms is placed next to the
mutation site. The arrow points to the mutation. P = patient.
Supplementary Fig. 3: (a) The sequence shows the sequence of the cryptic splice site
within exon 3. Below the sequence, the output of the splice prediction program is shown.
(b) A sequence showing the result of the misspliced transcript and the expected protein
sequence. (c) RT-PCR of two additional NCs showing the correctly spliced cDNA.
Supplementary Fig. 4: T57M mutation: (a) Electrograms of the two family members,
sequenced as indicated. P = patient. (b) Alignment of protein sequence of the indicated
species around the mutated amino acid. The affected amino acid (T) is bolded, showing
its conservation among the many species indicated. (c) Output of PolyPhen2 prediction.
The output predicts a very high probability of the mutation being damaging, as indicated
by a score of 1.
Supplementary Table: in vitro T cell proliferation in response to PHA
(cpm)
Patient
1a
1b
2
3
4a
4b
4c
5a
5b
6
7
Patients
PHA(cpm)
NA
84,424
69498
115516
NA
70253
NA
92020
48649
NA
40391
Control –1
PHA(cpm)
Control – 2
PHA(cpm)
% of Control
142,755
104,435
138,510
140,082
123,703
138,510
60%
61%
84%
101,709
103,327
68%
97583
81310
101595
76070
92%
62%
126,609
169,539
27%
** 2.5×105 mononuclear cells were cultured in 0.5 mL of RPMI 1640 plus 10% AB serum for 3
days in the presence of PHA (10 μg/mL). The results of triplicate cultures are expressed as the
mean counts per minute of [3H] thymidine incorporation (±1 SD) cpm (counts per minute).