J Clin Immunol (2013) 33:13251335

DOI 10.1007/s10875-013-9951-9

ORIGINAL RESEARCH

Clinical, Immunological, and Molecular Characterization

of Hyper-IgM Syndrome Due to CD40 Deficiency
in Eleven Patients
Bandar K. Al-Saud & Zobaida Al-Sum & Hanadi Alassiri & Abdulaziz Al-Ghonaium &
Saleh Al-Muhsen & Hasan Al-Dhekri & Rand Arnaout & Osama Alsmadi & Esteban Borrero &
Asma Abu-Staiteh & Faisal Rawas & Hamoud Al-Mousa & Abbas Hawwari

Received: 10 December 2012 / Accepted: 3 October 2013 / Published online: 12 October 2013
# Springer Science+Business Media New York 2013

Abstract
Purpose Hyper-IgM syndrome due to CD40 deficiency
(HIGM3) is a rare form of primary immunodeficiency with
few reported cases. In this study, we further characterize the
clinical, immunological, and molecular profiles of the disease
in a cohort of 11 patients.
Methods Molecular genetic analysis and a comprehensive
clinical review of patients diagnosed with HIGM3 at our
tertiary care center from 1994 to 2011 were undertaken.
Results Eleven patients from seven families were enrolled.
The patients had a median age of 9 years [ranging from 2 to

Electronic supplementary material The online version of this article

(doi:10.1007/s10875-013-9951-9) contains supplementary material,
which is available to authorized users.
B. K. Al-Saud (*) : Z. Al-Sum : A. Al-Ghonaium :
S. Al-Muhsen : H. Al-Dhekri : R. Arnaout : H. Al-Mousa
Section of Allergy and Immunology, Department of Pediatrics,
MBC-58, King Faisal Specialist Hospital and Research Centre,
P.O. Box 3354, Riyadh 11211, Saudi Arabia
e-mail: balsaud@kfshrc.edu.sa
B. K. Al-Saud : R. Arnaout : H. Al-Mousa
College of Medicine, Alfaisal University, Riyadh, Saudi Arabia
H. Alassiri : O. Alsmadi : E. Borrero : A. Abu-Staiteh :
A. Hawwari (*)
Genetics Department, Research Centre, MBC-03, King Faisal
Specialist Hospital and Research Centre, P.O. Box 3354,
Riyadh 11211, Saudi Arabia
e-mail: ahawwari@kfshrc.edu.sa
H. Alassiri : S. Al-Muhsen
King Saud University, Riyadh, Saudi Arabia
F. Rawas
Pathology & Laboratory Medicine Department, King Faisal
Specialist Hospital and Research Centre, Riyadh, Saudi Arabia

22 years old]. All 11 patients had recurrent chest infections at

presentation. Pneumocystis jiroveci pneumonia was confirmed in three patients. Five patients had sclerosing
cholangitis, and five patients had Cryptosporidium isolated
from their stool. Six patients had nasal and sinus infections,
and two of these patients had destructive nasal fungal infections. Eight patients had neutropenia. All of the patients had
low IgG and normal or high IgM levels. IgA was undetectable
in all but three patients. Two novel mutations were found: a
splice site for intron 3 and a missense mutation located in the
coding region of exon 3. Two patients underwent successful
stem cell transplantation from a matched donor. Four patients
are doing well on prophylaxis; two are very sick, one with
protracted diarrhea and persistent Cryptosporidium and the
other with neurological complications. Three patients died
early in life as a result of severe sepsis.
Conclusions To our knowledge, this report provides the largest cohort of patients with this disease with a very long followup period. Our cohort showed variable disease severity.
Keywords Primary immunodeficiency . hyper-IgM
syndrome . novel mutation . neutropenia . Cryptosporidium .
stem cell transplantation

Introduction
Hyper-IgM syndrome (HIGM) is a form of primary immunodeficiency disease that was first described in 1961 [1, 2]. It is
most commonly found in boys and is often characterized by
X-linked inheritance; however, other modes of inheritance
have been observed [3].
Affected patients have a genetic deficit in the immunoglobulin isotype class-switch recombination (CSR) and somatic

1326

hypermutation (SHM) pathways. This genetic deficit can result from a mutated cell surface molecule, such as an autosomal recessive lack of CD40 on the surfaces of B cells and
antigen-presenting cells [4], or as a result of an X-linked
CD40 ligand (CD40L) deficit on the surface of activated T
cells [59]. HIGM can also result from intracellular deficits
intrinsic to only B cells, such as an autosomal recessive
mutation in activation-induced cytidine deaminase (AID)
[10] or uracil-N-glycosylase (UNG) [11], or from intracellular
deficits found in B cells and other immune cells, such as Xlinked mutations in the NF-B essential modulator (NEMO)
[12, 13].
CD40, a 50-kDa member of the tumor necrosis factor
receptor (TNFR) superfamily, is expressed on many immune
cells, including B cells, monocytes, dendritic cells, and T cells,
and on some non-immune cells, such as epithelial cells and
neuronal cells, among others [14]. Patients with HIGM due to
mutations in CD40, CD40L, or NEMO suffer from recurrent
bacterial, viral, and opportunistic infections; in contrast, patients with deficits in AID and UNG have milder forms of the
disease and primarily suffer from bacterial infections [14].
HIGM caused by CD40 deficiency (HIGM3) occurs very
rarely, with only five cases reported in the literature, two (5a,
5b) of which are included in this study [4, 15]. The extreme
rarity of the number of cases reported in the literature may be
due to the misdiagnosis of this syndrome as a common variable immunodeficiency or a true low incidence in the general
population. Nevertheless, the high rate of consanguineous
marriages in the Saudi population [16] provided us with the
opportunity to review a relatively large number of patients
diagnosed with this form of autosomal recessive HIGM.
This study reviews the clinical and immunological features
of this disease in a cohort of 11 Saudi patients with HIGM3
and investigates the molecular basis of the disease.

Methods
Clinical Data
Data were collected by a retrospective chart review. Eleven
patients with HIGM3 who were managed at the King Faisal
Specialist Hospital and Research Centre in Riyadh (KFSHRC),
Kingdom of Saudi Arabia, between 1994 and 2011 were included. Informed consent was obtained from the patients or
their parents, and the study was approved by our hospitals
research advisory committee.
CD40 Expression on B cells Assessed by Flow Cytometry
A 3-ml peripheral blood specimen was collected from each
patient and control. The following antibodies were used
to perform immunofluorescent studies: CD40-PE, CD40L

J Clin Immunol (2013) 33:13251335

(CD154)-PE (Beckman Coulter, Beverly, MA, USA), CD8FITC, CD19-(APC, PerCP, or FITC), CD69-APC, CD3PerCP, and IgG-PE isotype control (Becton Dickinson). For
CD40L expression, we stimulated cells using a calcium ionophore (ionomycin, 300 ng/ml, Sigma, St. Louis, MO, USA)
and phorbol 12-myristate 13-acetate (PMA, 15 ng/ml, Sigma,
St. Louis, MO, USA) as described previously by OGorman
et al. [17]. Specimens were then acquired on a FACS-Calibur
flow cytometer (Becton Dickinson) and analyzed using
CellQuest software; CD40L expression was analyzed on
in vitro-activated CD8-negative cells, whereas CD40 expression was analyzed on CD19+ cells.
DNA Sequencing
Genomic DNA was extracted from whole blood samples
obtained from patients (and their families, when available)
using a Gentra Puregene Blood Extraction kit (Qiagen, Valencia, CA, USA). Promoters, exons, and both the 5 and 3
untranslated regions of CD40 were amplified by polymerase
chain reaction (PCR) using HotStarTaq DNA polymerase
(Qiagen, Valencia, CA, USA). Primers were designed from
within the intron regions to span the splice sites and the exon/
intron boundaries. In addition, M13 sequences were attached
to the 5 end of each primer to allow for forward and reverse
sequencing. See the supplementary material for more details.
Reverse Transcription PCR (RT-PCR)
RNA was extracted as indicated from Epstein-Barr virus
(EBV)-transformed B cell lines using TRIzol reagent (Sigma,
T-9424). Additionally, 5 ml of blood was collected from the
patients (and their family members, when available) into
PAXgene tubes (PreAnalytix, a Qiagen/BD Company, Valencia, CA., USA), and RNA was extracted using a PAXgene
Blood RNA kit (Qiagen for PreAnalytix, Valencia, CA, USA).
The extracted RNA was then synthesized into first-strand
complementary DNA (cDNA) using oligo (dT) primers
(Invitrogen) and the SuperScript III RT enzyme (Invitrogen,
Carlsbad, CA USA). The CD40 gene was amplified by PCR
using HotStarTaq polymerase. Primers were designed to sequence the entire CD40 cDNA and particular exon regions
where a splice site mutation was found to assess the possibility
of exon 3 skipping. See the supplementary material for more
details.
Western Blot Analysis
EBV-transformed B cell lines were lysed using RIPA buffer
(50 mM TrisHCl, 150 mM NaCl, 1 % NP-40, 0.5 % sodium
deoxycholate, and 0.1 % SDS, pH 7.4) containing protease
inhibitors (Complete Ultra tablet, EDTA-free, Roche, Germany). Proteins were quantitated using a Pierce BCA kit (Pierce,

J Clin Immunol (2013) 33:13251335

USA). Following standard protocols, 10 and 30 g of protein

were used for Western blot analysis. See the supplementary
material for more detail.

Results
Clinical Features
Eleven patients from seven families of Arab descent were
studied in this cohort. Six patients (2, 3, 4a, 4b, 4c, and 6)
were from one tribe. All the patients resulted from consanguineous marriages. The median age was 9 years (ranging
from 2 to 22 years old), and 60% were male (Table I). All the
patients initially presented with respiratory infections; viruses
were the most common causal organisms, followed by bacterial organisms. Pneumocystis jiroveci was isolated as the
causative organism of pneumonia (PJP) in three patients before the diagnosis of HIGM3, representing 25% of the patients
(3, 5a, and 6). Six patients had nasal and sinus infections; in
two patients (1b and 7), a destructive nasal infection due to a
Candida species and Pseudomonas aeruginosa required
prolonged intravenous treatment. Failure to thrive was a
prominent feature in most of the patients. Protracted or recurrent diarrhea was common and was found in over half of the
patients. Cryptosporidium was the most frequently isolated
pathogen (Table I). Sclerosing cholangitis occurred in almost
half of the patients, and Cryptosporidium was identified as the
cause in four out of the five patients (Table I).
Unfortunately, with respect to the clinical outcome, patients
4b and 4c died at the ages of one and a half and 4 years,
respectively, both secondary to severe gastroenteritis and chest
infections; patient 4c also suffered from liver cirrhosis secondary to sclerosing cholangitis. Patient 7 had poor compliance with prophylactic intravenous immunoglobulin (IVIG)
and granulocyte colony-stimulating factor (G-CSF) and died
at the age of 2 years secondary to Pseudomonas sepsis. Two
patients, 4a and 5a, are currently chronically ill with diarrhea
and sclerosing cholangitis; patient 5a, at the age of 16, also
developed headaches, right-sided weakness, and expressive
aphasia. Brain CT scan showed a left temporal occipital
stroke, and work-up showed that this patient had protein C
and S deficiencies, most likely secondary to chronic liver
disease. Four patients are currently doing relatively well at
ages of 2, 9, 17, and 22 years on conventional therapy with
prophylactic IVIG, antibiotics, and G-CSF for episodes of
neutropenia. For patients 3, 5b, and 6, no fully matched sibling
donors were identified, and therefore, they did not receive
hematopoietic stem cell transplantation (HSCT). Two patients
(1b and 2) underwent HSCT from a matched sibling donor at
19 months and 3 years of age, respectively.
We used myeloablative conditioning (busulfan 16 mg/kg
and cyclophosphamide 200 mg/kg) and cyclosporine as

1327

prophylaxes for graft-versus-host disease (GVHD). Patient

1b is currently 16 months post-HSCT. At the most recent
follow-up, he demonstrated engraftment with mixed chimerism, as indicated by short tandem repeat (STR) loci, and
immune constitution, with CD40 expression on B cells
(74 %) and monocytes (4 %) (Fig. 1a). He is currently off
IVIG and PJP prophylaxes. At the 1-year follow-up for patient
2, STR loci indicated sustained full engraftment, and immune
constitution was observed with CD40 expression on B cells
(100 %) and monocytes (98 %) (Fig. 1b). He is currently off
IVIG and PJP prophylaxes.
Immunological Features (Table II)
Severe neutropenia was a common finding in our cohort; eight
patients had neutrophil counts of less than 500 per L. All
patients had low IgG and normal or high IgM levels; IgA was
undetectable in all but three patients (4a, 4c, and 5b). No
patients expressed CD40 on their B cells (Supplementary
Fig. 1), as assessed by flow cytometry, but all patients demonstrated normal CD40L expression on activated T cells. We
typically found normal distributions of lymphocyte markers,
although some patients had low numbers of NK cells. The
lymphocyte proliferation response to phytohemagglutinin
(PHA) in patients who had the test performed was normal
(more than 50 % of the control), with the exception of patient
7 (Supplementary Table 1).
Mutation Analysis
Two novel mutations (Table III) were identified in seven
patients (1a, 1b, 2, 3, 4a, 4c, and 6). These mutations have
not been described previously and were not found as normal
variants in our 250 healthy controls (data not shown).
The first mutation (c. 256+2 T>C) was homozygous and
was found in five patients (2, 3, 4a, 4c, and 6). It was located at
position +2 of the invariant donor splice site of intron 3
(Supplementary Fig. 2).
To assess the effect of the splice site mutation on the correct
splicing and expression of the CD40 protein, we performed RTPCR and Western blot analysis on patient 6 (Fig. 2a). The RTPCR of the unrelated normal control unexpectedly showed two
bands, with the top band representing the expected normal
splicing (exon 2-exon 3-exon 4) and the bottom band representing exon 3 skipping (exon 2-exon 4). The RT-PCR of the
patient showed only one band that had the same size as the
bottom band of the normal control. No amplification of the
expected normal splicing of the patient cDNA was detected. To
ensure the specificity of these bands, we cloned and sequenced
the three bands using a TA cloning kit. The sequencing result
for the top band of the normal control was as expected (data not
shown) for normal splicing. However, the sequences of the
bottom bands of the normal and patient PCR products were

M
F

M
M
F
M

M
M
M

1a
1b

2
3
4a
4b

4c

5a

5b
6
7

8
12
9

18

12
6
6
4

3
1

Age at presentation
(months)

Yes
Yes
Yes

Yes

Yes

Yes
Yes
Yes
Yes

Yes
Yes

Recurrent
URTI/LRTI

No
No
No

Yes

No

No
No
No
No

No
No

Interstitial
pneumonia

No
Yes
No

Yes

No

No
Yes
No
No

No
No

PJP

Yes
No
Yes

Yes

Yes

No
Yes
Yes
Yes

Yes
Yes

Failure
to thrive

No
No
Yes

Yes

Yes

No
No
Yes
Yes

Yes
Yes

Protracted
diarrhea

No
No
Yes

No

Yes

No
No
Yes
No

Yes
Yes

Cryptosporidium

No
No
No

Yes

Yes

No
No
Yes
No

Yes
Yes

Sclerosing
cholangitis

No
Sinusitis
Destructive
fungal

Sinusitis

No

No
Destructive
fungal
No
No
Mastoiditis
No

ENT/Sinuses

Yes
Yes
Yes

Yes

Yes

Off
Yes
Yes
Yes

Yes
Off

IVIG

Yes
Yes
Yes

Yes

Yes

Off
No
Yes
Yes

No
Off

GSCF

No
No
No

No

No

Yes
No
No
No

For
Yes

HSCT

Alive and well (17)

Alive and well (22)
Deceased at 2 years
of age

Cure (6)
Alive and well (9)
Chronic diarrhea (9)
Deceased at 1 1/
2 years of age
Deceased at 4 years
of age
Stroke (18)

Alive
Cure (5)

Outcome (current
age in years)

URTI upper respiratory tract infection, LRTI lower respiratory tract infection, PJP Pneumocystis jiroveci pneumonia, ENT Ear nose and throat, IVIG Intravenous immunoglobulin, GCSF granulocyte
colony stimulating factor, HSCT hematopoietic stem cell transplantation

Gender

Patient

Table I Demographic and clinical features

1328
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J Clin Immunol (2013) 33:13251335

1329

(a)

Patient 1b postSCT

Control

Patient 1b postSCT

Control

100%

100%

74%

CD 40 expression on B cells

95 %

4%

CD 40 expression on monocytes

(b)

Control

100%

Patient 2 postSCT

100%

CD 40 expression on B cells

Patient 2 postSCT

Control

94%

98%

CD 40 expression on monocytes

Fig. 1 Flow cytometry analysis of CD40 expression on B cells and monocytes from the control compared with patient 1b (a) and patient 2 (b) post-stem
cell transplantation

1901 (15008500)
1801 (15008500)
268 (15008500)
7980 (15008500)
274 (15008500)
401 (15008500)
215 (15008500)
402 (15008500)
336 (15008500)
621 (15008500)
300 (15008500)

1a (6 month)
1b (11 month)
2 (18 month)
3 (5 years)
4a (33 month)
4b (6 month)
4c (19 month)
5a (24 month)
5b (16 month)
6 (18 month)
7 (15 month)

8153 (50007800)
2953 (50007800)
7314 (33006200)
7315 (23003900)
5617 (23003900)
1443 (50007800)
888 (33006200)
11658 (33006200)
5880 (33006200)
3890 (33006200)
2578 (33006200)

ALC
Cells/uL (normal
reference)a
1.5 (2.46.1)
2.7 (4.48.8)
8.6c (5.59.7)
4.6 (711)
1.8 (710)
1.4 (2.46.1)
1.2 (5.59.7)
1.9 (5.59.7)
0.9 (5.59.7)
<0.5 (5.59.7)
2.5 (5.59.7)

IgG (g/L) (normal

reference)b

On IVIG

Pediatrics 1966; 37:715

Pediatric allergy and immunology 1998; 9:4448

ND not done, ANC Absolute Neutrophil Count, ALC Absolute Lymphocyte Count

ANC
Cells/uL (normal
reference)a

Patient(age at work up)

Table II Immunological features

<0.25 (0.250.46)
<0.25 (0.280.64)
<0.25 (0.260.75)
<0.25 (0.661.2)
0.55 (0.341)
<0.25 (0.250.46)
0.4 (0.260.75)
1.34 (0.260.75)
<0.3 (0.260.75)
<0.2 (0.260.75)
<0.25 (0.260.75)

IgA (g/L) (normal

reference)b

0.81 (0.26.6)
1.23 (0.330.77)
3.12 (0.350.81)
0.85 (0.38-0.74)
2.07 (0.42-0.8)
0.53 (0.26.6)
3.99 (0.350.81)
1.44 (0.350.81)
4 (0.350.81)
1.06 (0.350.81)
1.26 (0.350.81)

IgM(g/L) (normal
reference)b

5250 (31004800)
2274 (31004800)
5047 (22004100)
4974 (22004100)
2920 (16002700)
707 (31004800)
294 (22004100)
8624 (22004100)
4880 (22004100)
2956 (22004100)
2215 (22004100)

CD3/mm3 (normal
reference)a

2039 (11001900)
442 (11001900)
1785 (7001600)
1683 (400800)
2190 (400800)
592 (11001900)
417 (7001600)
2331 (7001600)
588 (7001600)
778 (7001600)
310 (7001600)

CD19/mm3 (normal
reference)a

274 (300700)
90 (300700)
190 (200600)
191 (200400)
393 (200400)
115 (300700)
160 (200600)
233 (200600)
294 (200600)
78 (200600)
26 (200600)

CD16/56/mm3 (normal
reference)a

1330
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1331

Table III Mutation analysis

Patient

Nucleotide

Amino acid

Exon

Mutation type

Reference

1a
1b
2
3
4a

170 C>T
170 C>T
c. 256+2 T>C
c. 256+2 T>C
c. 256+2 T>C

Thr57Met
Thr57Met

3
3
3(+2)
3(+2)
3(+2)

Missense
Missense
Splice site
Splice site
Splice site

Novel
Novel
Novel
Novel
Novel

4b
4c
5a
5b
6
7

N/A
c. 256+2 T>C
294 T>C
294 T>C
c. 256+2 T>C
N/A

N/A

N/A
3(+2)
3
3
3(+2)
N/A

N/A
splice
Missense
Missense
Splice site
N/A

N/A
Novel
Ferrari (2001)
Ferrari (2001)
Novel
N/A

identical but contained the first 11 bases of exon 3 followed

immediately by exon 4 sequences (Fig. 2b). This suggested that

(a) RT-PCR

NC1
UD

1/3

Cys83Arg
Cys83Arg
N/A

exon 3 might contain a cryptic splice site immediately after the

first 11 bases. To investigate this possibility, we submitted the

Patient
1/9

UD

1/3

Father
1/9

UD

1/3 1/9

Mother
UD

1/3

1/9

Exon 2 to 4
GAPDH
(b) Sequence of the bottom band of exon 2 to exon 4 of the RT-PCR
Beginning of exon 4
end of exon 2
Normal
Patient 6

(c) Western blot analysis of CD40 protein containing the splice site mutation
Mother

Father

Patient 6

NC

Mother

Father

Patient 6

CD40
Stat 5
Antibody to the N-terminus
Fig. 2 The effect of the c. 256+2 T>C mutation: a Reverse transcription
PCR of GAPDH and CD40, as indicated. We used three dilutions of
cDNA from an unrelated normal control and patient 8 (P8), as shown;
UD = undiluted. b Electropherograms of the sequencing of the bottom

Antibody to the C-terminus

bands of the RT-PCR of the patient and the normal control. c Western blot
analysis of patient 6, the patients father and mother, and an unrelated
control (NC) for CD40 protein expression. STAT5 was used as a loading
control

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J Clin Immunol (2013) 33:13251335

sequence [CTG/GTGAGT] from +9 to +17 of exon 3 to a

splice site prediction protocol at http://ibis.tau.ac.il/ssat/
SpliceSiteFrame.htm. As shown in Supplementary Figure 3a,
the output confirmed that the sequence GTGAGT is a splice
site with a score of 86.49 and a free energy of 7.2, which was
very close to the best splice site consensus with a free energy of
9.6. Comparing our sequence with the best splice site
sequence revealed a difference of one base: GTA (G) AGT.
This aberrant splicing is predicted to result in a stop codon (the
X in Supplementary Fig. 3b).
This result clearly showed that the vast majority of patient
RNA spliced together exon 2, part of exon 3, and exon 4,
skipping the remainder of exon 3 (Fig. 2b and Supplementary
Fig. 3b). The appearance of the lower band in our normal
control was unexpected. To clarify this result, cDNA samples
were prepared from two additional healthy donors (NC2 and
NC3) and amplified by PCR. The same pattern of bands as
that in NC1 was observed (Supplementary Fig. 3c). Again, the
bands were cloned, sequenced, and analyzed. Similar results
were obtained, and sequencing of the genomic DNA of all
three healthy donors (NC1, NC2, and NC3) did not detect any
mutation at the invariant donor splice site of intron 3 (data not
shown).
CD40 protein expression in patient 6 was examined by
Western blots of samples from the patient, the patients father
and mother, and an unrelated normal control (Fig. 2c). The
parents and the normal control expressed the correct size of
the CD40 protein. In contrast, no CD40 protein was detected
in the patient sample, indicating its complete absence. To
assess whether the parents expressed the CD40 protein at the
same level as the normal control, densitometric analysis was
performed using (http://rsbweb.nih.gov/ij/download.html).
The patients mother and father and the healthy control
expressed essentially the same amount of CD40 protein
when normalized to the STAT5 protein. Similar results were
obtained using an antibody to the N-terminus of the CD40
protein (Fig. 2c).
The second mutation identified was a missense mutation
(c. 170C>T) that resulted in an amino acid change from a
polar threonine (T) to a non-polar methionine (M) at position 57 (T57M) and was found in two first-degree cousins

Normal
control

Father

Mother

Patient 1a

CD40
Stat 5
Fig. 3 Western blot analysis of CD40 protein containing the T57M
mutation: Western blot analysis of patient 1a, the patients father and
mother, and an unrelated control (NC) for CD40 protein expression; the
results show reduced CD40 protein expression in the patient. STAT5 was
used as a loading control

(Supplementary Fig. 4a). This amino acid is highly conserved (Supplementary Fig. 4b). PolyPhen-2 (http://
genetics.bwh.harvard.edu/pph2/index.shtml) predicted this
mutation to be highly damaging (Supplementary Fig. 4c).
Furthermore, Western blot analysis of one of the two
patients (patient 1a) showed a substantial reduction in CD40
protein expression, which confirmed the damaging effect of
this substitution (Fig. 3).

Discussion
In this report, we characterized the clinical, immunological,
and molecular features of a large cohort diagnosed with
HIGM3 syndrome, which is, to our knowledge, the largest
cohort ever reported in the literature with the longest followup (Table I). The diagnosis was considered based on a positive
family history of HIGM3 in 4 of the 11 patients (36 %), which
highlights the importance of early screening for all newborns
within the affected kindred.
Chronic diarrhea is a common clinical presentation and
was encountered in 63 % of our cohort (Table I). The usual
cause of diarrhea was Cryptosporidium infection, which was
identified in 45 % of our cohort, in contrast to the 6 % and
19 % rates of Cryptosporidium infection reported in two
cohorts of HIGM1 syndrome from the USA and Europe,
respectively [18, 19]. This difference in the prevalence of
Cryptosporidium could be related to an environmental or
genetic cause. Sclerosing cholangitis is an ominous complication of Cryptosporidium infection. Therefore, in symptomatic patients, it is crucial to long-term outcomes to regularly
monitor stool for Cryptosporidium , peripheral blood for eosinophil levels, and liver status using biochemical analysis and
ultrasound [20].
The spectrum of microbial infection in our cohort was not
limited to Cryptosporidium. In fact, three patients had PJP,
seven had fungal infections, and one had CMV (Table I). Such
infection profiles and susceptibility to opportunistic infections
demonstrate that CD40-CD40L signaling is not only important
for B-cell activation and Ig isotype class switching but is also
central in T-cell-mediated activation [14]. Moreover, Fontana
et al. [21] have presented evidence of functional defects in the
dendritic cells of patients with CD40 deficiency by demonstrating that the lack of CD40-CD40L interaction between dendritic
cells and activated T cells leads to a defect in T-cell priming and
interferon- secretion, which could contribute to susceptibility
to opportunistic infections in HIGM3 patients.
We observed two patients with more severe, destructive
nasal fungal infections, which could most likely be attributed
to the accompanying neutropenia. Thus, aggressive therapy
with IV antibiotics and correcting the neutropenia was
warranted.

J Clin Immunol (2013) 33:13251335

The immunological features (Table II) of our cases demonstrate that although all the patients had low IgG and normal or
high IgM levels, normal levels of IgA were detected in patients
4a and 4c and high levels were found in patient 5b (Table II and
Supplementary Fig. 5) on more than one occasion. This finding
has not been reported previously in patients with HIGM3.
However, in more than one cohort [18, 19, 22] of X-linked
HIGM1 patients, normal and high levels of IgA were reported.
The two patients with normal IgA levels (Patients 4a and 4c)
had a splice-site mutation, whereas the third patient had a
missense mutation [4]; both mutations led to the absence of
CD40 expression. Thus, IgA production does not occur because of residual expression or function of CD40. Moreover,
IgA was undetectable in the other affected member of the same
family, which may indicate that other factors play a role. It is
possible that IgA production could result from T-cellindependent activation in the gut-associated lymphoid tissues
through Toll-like receptor (TLR) stimulation of B cells [23].
The NK cell counts were very low (<130/mm3) in some
patients on more than one occasion (Table II and Supplementary Fig. 5). Ostenstad et al. [24] described a patient with Xlinked HIGM who also had NK cell deficiency. Furthermore,
other investigators have shown that NK cells can express
CD40L, which upon the recognition of CD40 on target cells,
activates NK cells [25]. Thus, the lack of CD40-CD40L interaction may affect NK cell growth. In most patients, the PHA
response was normal (>50 % of controls). This finding is
consistent with a few previously reported cases. The PHA
response was low (less than 50 % of controls) in only one
patient; this was explainable by the high response to PHA in the
controls. Our report shows that this syndrome has a variable
clinical course, which was observed even in the six patients (2,
3, 4a, b, c, and 6) from the same tribe who had the same genetic
mutation, demonstrating the heterogeneity of the disease. Overall, although three patients died secondary to severe sepsis, four
patients are currently doing fairly well with no significant
complications on regular IVIG, prophylactic antibiotics, and
G-CSF to treat episodes of neutropenia. All three patients who
died had chronic diarrhea and hepatitis, and Cryptosporidium
was found in two of the three patients. If present, these features
could be considered poor prognostic factors.
Stem cell transplantation has been reported in two Turkish
patients. In 2003, Kutukculer et al. [26] reported on a female
patient with prior PJP and Cryptosporidium infections. She
received hematopoietic stem cells (HSCs) from her matched
brother and received a non-myeloablative conditioning regimen. The patient showed no evidence of engraftment and died
of respiratory failure. The second patient, reported by
Mazzolari et al. [15] in 2007, was a 3-year-old female with
no previous PJP or Cryptosporidium infection; she was treated successfully. She received HSCs from her matched sibling
and underwent a myeloablative conditioning regimen. We
herein reported two more HSCTs in patients 1b and 2; despite

1333

patient 1bs previous Cryptosporidium infection and sclerosing cholangitis, both HSCTs were successful. Therefore, we
believe that HSCTs should be considered for all patients who
have an HLA-identical donor. Only one of our patients developed a neurological abnormality secondary to a CNS stroke
and was found to have protein C and S deficiencies (Patient
5a; at the age of 16 years), which could be secondary to her
chronic liver disease. Neurological deterioration was reported
in a 10-year-old female Italian patient, although the neuroimaging of her brain was normal [14]. By contrast, her first
cousin (patient 5b), now 17 years of age, is doing fairly well
with no significant complications.
Prior to this report, four mutations were described and
characterized; two of the described mutations affect splice sites
[4, 27]: a missense mutation [4] and a deletion [15]. Here, we
reported two novel mutations (Table II). Five patients from the
same tribe carried an invariant donor splice site in intron 3. Two
patients (first cousins) were affected by a missense mutation
located in the coding region of exon 3. This is not surprising,
given the high number of consanguineous marriages in our
population. We concluded that each mutation most likely originated in a separate tribe and remained confined to that tribe.
The first of our mutations was found at the second base pair
of the donor splice site of intron 3. We have shown that this
mutation affects the proper splicing of exon 3 to exon 4, which
results in an alternative splice site within exon 3 and the
skipping of most of exon 3 (Fig. 2b). The mechanism for exon
skipping has been reported previously for other genes [27].
No protein expression was detected from the aberrantly
spliced mRNA of exon 3. In our patients, the skipping of exon
3 resulted in aberrant splicing that caused a frame shift and led
to an early termination of the CD40 protein (Fig. 2c). Our
inability to detect the truncated protein using antibodies to the
N-terminus of the protein may be the result of conformational
changes introduced by the skipped codons that prevented the
antibodies from binding to the protein. Alternatively, nonsensemediated decay may prevent the translation of the mutated
RNA in both the patients and normal individuals, which is
supported by the presence of aberrantly spliced RNA in three
normal donors.
Our second mutation, a missense mutation (170C>T;
T57M), was located in the extracellular domain of the CD40
protein (exon 3). This change resulted in a substantial reduction
of the CD40 protein as measured by Western blot using antibodies to both the N- and C-termini. However, no protein was
detected on the surface of the B cells from two of our patients
by flow cytometry. This may be explained by a disruption of
the proper folding of the CD40 protein that affected the localization of the protein to the cellular membrane.
The T57M mutation is located in close proximity to another mutation (294 T>C; C83R) reported by Ferrari et al. [4] in
two other Saudi patients who were first cousins (Table III).
Both of these mutations are located in exon 3, which encodes

1334

part of the extracellular domain of the protein and is highly

conserved among the tumor necrosis factor receptor (TNFR)
family members [28]. Neither we nor the Ferrari group detected any CD40 expression on the surface of B cells, indicating that both of these mutations affect the localization of the
CD40 protein to the B cell surface. However, in our case,
residual CD40 protein expression was detected by Western
blot analysis (Fig. 3), indicating that the T57M mutation
causes protein instability. Nevertheless, no residual CD40
protein localization to the B cell surface was detected. We
propose here that the T57M mutation may cause both protein
instability and affect the localization of the protein to the B cell
surface.

Conclusions
To our knowledge, this case is the largest reported cohort of
HIGM3. Our cohort showed variable disease severity, which
may complicate the decision to attempt stem cell transplantation in asymptomatic patients. A multicenter study with a
larger cohort and long-term follow-up is needed.
Acknowledgments We would like to thank the following individuals:
Dr. Abdelmoneim Eldali for help with the statistical analysis; Dr.
Sriharsha Kantamnini for his advice on technical issues related to Western
blot analysis; Dr. Entissar Al-Suhaibani for her support of the MSc
student, Hanadi Al- Asseri, one of the authors of this manuscript; and
the Flow Cytometry section staff (Ameena Siefeldien, Margarida Gillbee
and Shar Al-Janadi).
Funding King Faisal Specialist Hospital and Research Centre

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