Correspondence:
Dr. Clementien L.Vermont
Erasmus MC University Medical Center-Sophia Children’s Hospital
PO Box 2060, 3000 CB
Rotterdam the Netherlands
Email:
c.vermont@erasmusmc.nl
Main text: 1248 words
Number of tables and figures: 2
Supplemental data: 1 Figure and methods
Disclosure of conflicts of interest
The authors have no conflict of interest.
Financial Support
The authors received no financial support for the research, authorship
or publication of this article.
Main text:
To the editor,
Mendelian susceptibility to mycobacterial disease (MSMD) is a rare
inherited disorder characterized by infections with weakly virulent
mycobacteria in otherwise healthy individuals.1 MSMD
is caused by genetic defects in genes coding for proteins in the
IL-12/IFN-γ signaling pathway.2 IFN-γR1 deficiency is
a common cause of MSMD.1 The pathogenic variants inIFNGR1 can be either autosomal recessive (OMIM #209950) or
dominant (OMIM #615978),3 resulting in complete or
partial functional defects.4 Lack of surface
expression leads to a complete loss of function, while expression of
hypomorphic IFN-γR1 can lead to either partial loss or complete loss of
function. Complete loss of function in IFN-γR signaling results in
severe mycobacterial infections in early childhood and poor survival,
while variants with partial loss of function may result in a milder
clinical phenotype that manifests later in childhood. A general approach
in diagnosing patients with MSMD has been proposed in literature using
flow cytometry, lymphocyte immunophenotyping and sequencing of genes
known to be involved in mycobacterial host
immunity.4,5 However, since recent advances in genomic
analysis technologies, a genotype-first approach for patients with a
suspicion of an inborn error of immunity has currently become a more
typical approach.6 While having the advantage of
analyzing multiple genes simultaneously, some variants can be missed and
the interpretation of the significance of novel variants or new
combination of hypomorphic variants can be a diagnostic challenge. Here,
we describe three patients with MSMD with novel or new combinations of
hypomorphic variants in which functional assays were crucial to
determine the extent of the interferon gamma receptor signaling defects.
Patient 1 is a 6-year old girl who was admitted to our pediatric ward
with progressive cough, fever and tachypnea. Her medical history was
remarkable with more than 20 hospital admissions for bronchial
hyperreactivity, lower respiratory tract infections and a coarctectomy
at the age of 2. Imaging showed mediastinal and hilar lymphadenopathy, a
large right upper lobe mass and lymph nodes compressing the trachea
(Supplementary Fig S1A). An ultrasound showed three focal lesions in the
spleen and hepatomegaly. Because of her severely compromised airway,
steroids were given, improving intraluminal tracheal diameter.
Histological examination on the lymph node ruled out malignant lymphoma.
Sputum, stomach fluid, lymph node biopsy and bone marrow culture
revealed Mycobacterium avium . Plasma IFN-γ level on admission was
elevated (311 pg/ml). She was treated with azithromycin, rifampicin and
ethambutol. Amikacine was added for the first 2 months. She recovered
after a year of treatment. Patient 2 is a 7-year old boy was admitted
with unexplained and recurrent low-grade fever, neck and leg pain,
vasculitis-like skin lesions and polydipsia for several months. He had a
medical history with recurrent bronchial hyperreactivity, recurrent
parotitis, parechovirus-associated radiculitis and cluster headache. A
brain MRI, CT scans of thorax and vertebrae, total body X-ray studies
and abdominal ultrasound showed a hypophysitis leading to
panhypopituarism, several bone lesions in skull, vertebrae and hip,
hilar lymphadenopathy and a single lesion in the spleen. Tuberculin skin
test showed induration of 18mm. Cultures of a bone lesion and sputum
grew Mycobacterium persicum . Serum IFN-γ levels were
undetectable. He started treatment with rifampicin, isoniazid and
ethambutol as well as hydrocortisone, levothyroxine and desmopressin for
his panhypopituarism, and has improved very well. Patient 3 is a 1-year
old girl with a swollen right upper arm and axillary lymphadenopathy.
She had a BCG scar that looked inflamed. Her medical history showed
bronchial hyperactivity and eczema. A CT scan showed multiple active
hilar and axillary lymph nodes, a splenic lesion and osteolytic lesions
of her right humerus, two ribs and a vertebral lesion in L2. Pathology
of a bone biopsy from the right humerus showed lesions compatible with
Langerhans cell histiocytosis (LCH). Treatment for multifocal LCH was
started using prednisone and vinblastine. The bone lesions showed
improvement but new skin lesions appeared at the site of the BCG scar,
head and back. Repeated CT scans showed multiple bone lesions in
scapulae, ribs and progression of hilar and axillary lymphadenopathy.
Skin biopsies of the lesions showed granulomatous infiltration and grewMycobacterium bovis BCG strain. The previous diagnosis of LCH was
re-evaluated and a new diagnosis of disseminated BCG-itis was made.
Treatment with rifampicin and isoniazid resulted in rapid improvement on
skin and bone lesions as well as on lymphadenopathy.
A diagnostic whole exome sequencing (WES)-based PID gene panel analysis
was used as first line diagnostic approach. In Patient 1 one
heterozygous pathogenic variant in the IFNGR1 gene
NM_000416.2(IFNGR1):c.188T>G, p.(Val63Gly) was identified.
However, no second variant was detected. Interestingly, only theIFNGR1 transcript with the p.(Val63Gly) variant was identified
with RNA expression analysis, and a SNP array showed decreased binding
of one probe in exon 3 of IFNGR1 . Long range PCR using primers
surrounding exon 3 showed a heterozygous insertion. Sequence analysis
revealed that this insertion concerned a complex insertion of 136
nucleotides of the right arm and the poly-A tail of an AluY transposable
element combined with a 4-nucleotide duplication in exon 3:
NM_000416.2(IFNGR1):c.210_211ins[136;A[?];207_210] (Fig.
1AB). Patient 2 was compound heterozygous for a known pathogenic variant
NM_000416.2(IFNGR1):c.373+1G>T, p.(?) and variant
NM_000416.2(IFNGR1):c.110T>C, p.(Ile37Thr), classified as
probable pathogenic (Fig. 1AB). In Patient 3 a novel variant in theSTAT1 gene: NM_007315.3(STAT1):c.1379A>T,
p.(Asn460Ile) was identified (Fig. 1C). Parental investigation showed
this was a de novo variant (Fig. 1A).
Functional analysis was performed to determine the effect on the
IFN-γ/STAT1 signaling pathway. The surface expression of IFN-γR1 on
monocytes was severely reduced in Patient 1 and Patient 2, while the
expression was normal or even elevated in Patient 3 (Fig. 2A). In
Patient 1 and 2, the expression of STAT1 was normal (Supplementary Fig.
S1B), while the phosphorylation of STAT1 was severely reduced in
response to stimulation IFN-γ, but normal upon IFN-α stimulation (Fig
2BC). Furthermore, the upregulation of the IFN-gamma regulated geneSOCS1 , was absent after stimulation with IFN-γ (Fig. 2D), and
normal upon IFN-α stimulation (Fig. 2E). In line with that, membrane
expression of the IFN-gamma regulated gene CD64 was not increased after
IFN-γ stimulation in Patient 2. These functional data suggest that
Patient 1 and 2 have a defect in IFN-gamma signaling. For Patient 1 the
life-threatening presentation of disease in combination with the results
of the functional analyses led to consideration of allogeneic
hematopoietic stem cell transplantation as a treatment option, for which
patient and her parents are counselled.
The STAT1 gene variant p.(Asn460Ile) in Patient 3, is located in
the DNA binding domain of the STAT1 protein. Studies on the crystal
structure of STAT1 showed that Asn460 is important for DNA binding since
it is the only amino acid that directly interacts via hydrogen bonds to
DNA, implying that this amino acid is crucial for STAT1
function.7,8 In Patient 3, STAT1 protein expression
was not affected (Supplementary Fig. S1B), but the phosphorylation of
STAT1 was slightly decreased (Fig. 2B). Moreover, it seemed to be
delayed upon IFN-γ and IFN-α stimulation in monocytes compared to HCs
(Fig. 2C). Expression of SOCS1 seemed normal after 0.5h of
stimulation with IFN-γ, but was reduced after 1 and 2 hours of
stimulation (Fig. 2D). Together, these functional data and the fact that
the variant is de novo strongly suggest that Patient 3 has an AD
STAT1 deficiency.
In conclusion, patients presenting with disseminated mycobacterial
infections in early or late childhood with bi-allelic or non-conclusive
genetic variants in genes involved in mycobacterial immunity need to be
phenotyped by functional assays of the IFN-γ /STAT1 signaling pathway in
monocytes. These assays are crucial in assessing the extent of IFN-γ
pathway signaling defects and in clinical decision making.
Key Words : mendelian susceptibility to mycobacterial disease,
IFN-γ receptor, STAT1 , mycobacterial infection, SOCS1
Zijun Zhou,1,2,6 Iris H.I.M.
Hollink,3,6 Arjan Bouman,3,6 Mirthe
S. Lourens,1,6 Rik A. Brooimans,1,6Tjakko J. van Ham,3 Pieter
L.A.Fraaij,4,6 Annemarie M.C. van
Rossum,4,6 Eline A.M. Zijtregtop,5Willem A. Dik,1,6 Virgil A.S.H.
Dalm,1,2,6 P. Martin van Hagen,1,2,6Hanna IJspeert,1,6# Clementien. L.
Vermont4,6#*
1Erasmus MC, University Medical Center Rotterdam,
Laboratory Medical Immunology, department of Immunology, the Netherlands
2 Erasmus MC, University Medical Center Rotterdam,
department of Internal Medicine, Division of Clinical Immunology, the
Netherlands
3Erasmus MC, University Medical Center Rotterdam,
department of Clinical Genetics, the Netherlands
4Erasmus MC, University Medical Center
Rotterdam-Sophia Children’s Hospital, department of Pediatrics, Division
of Pediatric Infectious Disease and Immunology, the Netherlands
5Erasmus MC, University Medical Center
Rotterdam-Sophia Children’s Hospital, department of Pediatrics, Division
of Pediatric Hemato-oncology, the Netherlands
6Erasmus MC, University Medical Center Rotterdam,
Academic Center for Rare Immunological Diseases (RIDC), the Netherlands
# these authors contributed equally
Acknowledgements
The research for this manuscript was performed within the framework of
the Erasmus Postgraduate School Molecular Medicine.