A homozygous STIM1 mutation impairs store-operated calcium entry and natural killer cell effector function without clinical immunodeficiency.

To the Editor:

Stromal interaction molecule 1 (STIM1) is a transmembrane protein pivotal to store-operated calcium entry (SOCE) that localizes to either the cell or endoplasmic reticulum (ER) membranes, with the N-terminus in either the extracellular space or the ER, respectively. Plasma membrane ORAI calcium release–activated calcium modulator 1 (ORAI1) Ca2+ channels are activated by STIM1. Families previously described with recessive STIM1 mutations (MIM #612783) had life-threatening viral, bacterial, and fungal infections; developmental myopathy; hypohidrosis; and amelogenesis imperfecta (AI; generalized developmental enamel abnormalities).1, 2, 3 We investigated a consanguineous family, segregating a novel syndrome of recessive AI and hypohidrosis by using autozygosity mapping and clonal sequencing. A homozygous rare missense mutation in STIM1 (p.L74P) in the EF-hand domain was identified (see the Methods and Results sections in this article's Online Repository at www.jacionline.org).

The family was re-evaluated, with particular attention paid to features associated with recessive STIM1 mutations (Table I and see Table E1, Table E2, Table E3 in this article's Online Repository at www.jacionline.org). The 2 affected cousins (18 and 11 years old, respectively) did not have overt clinical immunodeficiency. Further evaluation of their immune systems showed a normal immunoglobulin profile with an adequate specific antibody response to both nonlive (pneumococcus, tetanus and, Hib) and live (mumps, measles, and rubella) vaccinations. In addition, both subjects had detectable IgG against varicella zoster virus after a previous uncomplicated primary infection. The younger cousin was also found to have IgG against EBV viral capsid antigen, suggesting previous exposure, but neither showed any evidence of acute infection or previous exposure to cytomegalovirus.

Table I

Summary of the main clinical and clinical immunologic features in subjects with either homozygous or heterozygous STIM1 c.221T>C mutations

Feature	V:3	IV:4	IV:3	V:2
STIM1 genotype	Homozygous c.221T>C	Heterozygous c.221T>C	Heterozygous c.221T>C	Homozygous c.221T>C
Age at evaluation (y)	18-21	45-48	41	9-12
Persistent infections	None	None	None	None
Other infections	Infancy: repeated chest infections but not thereafter	No reported issues	No reported issues	Infancy: chest, urinary tract, gastrointestinal tract, ear, and eye infections but not thereafter
Autoimmune disorder	Transient ITP aged 2.5 y	None	Sjogren syndrome	None
Lymphocytes
Total (×109/L [1.00-2.80])	1.50	2.30	2.64	2.34
CD4 (absolute; × 109/L [0.300-1.400])	0.841	1.091	1.502	0.908
CD8 (absolute; × 109/L [0.200-0.900])	0.055	0.236	0.415	0.488
CD4/CD8 (1.07-1.87)	15.29	4.62	3.62	1.86
NK (absolute; × 109/L [0.090-0.600])	0.238	0.581	0.252	0.252
Immunization history	Full schedule without adverse events	Not assessed	Not assessed	Full schedule without adverse events
Musculoskeletal	Muscle bulk, tone, power, and reflexes normal; hypermobility in upper and lower limbs; CK normal	No issues evident; not formally examined	No issues evident; not formally examined	Muscle bulk, tone, power, and reflexes normal; hypermobility in upper and lower limbs; CK normal
Pupil reaction	Normal	Normal	Normal	Normal
Sweating	Diminished sweating recognized from infancy onward; insufficient sample for sweat test analysis	No reported issues	No reported issues	Diminished sweating recognized from infancy onward; reduced sweating on starch and iodine testing
Dental enamel	Generalized hypomineralized AI	Enamel within normal limits	Enamel within normal limits	Generalized hypomineralized AI
Development	Global development normalHeight, 156 cm (<0.4th percentile)Weight, 40.3 kg (<0.4th percentile)Head circumference, 51.5 cm (<0.4th percentile)	Not assessed	Not assessed	Global development normalHeight, 122 cm (0.4th-2nd percentile)Weight 24 kg (2nd-9th percentile)Head circumference, 50 cm (0.4th percentile)

Further details are presented in Table E1, Table E2, Table E3. Values in boldface are outside the reference ranges.

CK, Creatine kinase; ITP, idiopathic thrombocytopenic purpura.

Table E1

Additional clinical features of the 2 subjects with homozygous STIM1 c.221T>C mutations

Feature	V3	V2
Birth and neonatal period	Full-term (2.3 kg) by using forceps for fetal distress	Emergency cesarean section because of fetal decelerations at 36/40 wkBirth weight, 1.94 kg (<3rd percentile)Apgar score, 9 at 1 and 5 minutes, respectivelySpecial care baby unit for 1 mo, establishing feeds with nasogastric tube feeds for the first 2 wkDuring this time, there was 1 episode of unexplained fever.Unexplained neonatal hypercalcemia settled spontaneously.
Nails and hair	Normal	Normal
Dysmorphic features	None	None
Other medical history	Asthma diagnosed in infancyEvaluated in infancy for cystic fibrosis (negative) after repeated chest infectionsAt age 17 y, had a spontaneous pneumothorax of the left lung requiring pleurodesisFour apical bullae were found on imaging.Five months later, he had a right pneumothorax secondary to an apical bulla also requiring pleurodesis.At aged 18 y, V3 was evaluated with regard to macular pigmentation and bilateral drusen on both maculae, and mild congenital lens opacities were identified.	Asthma diagnosed in infancyEczemaGeneralized problems with increased leg fatigability and clumsinessTight Achilles tendons of unknown causeBilateral pes cavusHypermobility in upper limbs
Allergies	Allergic to red food coloring	None

Table E2

Summary of clinical immunologic data in subjects with either homozygous or heterozygous STIM1 c.221T>C mutations

Feature	VI:2		V:1		V:2	VI:3
Homozygous c.221T>C		Heterozygous c.221T>C		Heterozygous c.221T>C	Homozygous c.221T>C
Year of evaluation	2011	2014	2011	2014	2011	2012	2013
Bacterial antibodies
Pneumococcal (μg/mL)	209.0	−	101.0	−	82.2	181.0	−
Tetanus (IU/mL)	0.890	−	5.320	−	0.960	0.620	−
Haemophilus species (μg/mL)	0.230	−	0.240	−	<0.110	0.430	−
Viral antibodies
HSV IgG	ND	ND	−	+ve	−	−	−
VZV IgG	+ve	+ve	−	+ve	−	+ve	−
CMV IgM	ND	−	−	ND	−	−	−
CMV IgG	ND	ND	−	+ve	−	ND	−
EBV VCA IgM	ND	−	−	ND	−	−	−
EBV VCA IgG	ND	−	−	+ve	−	+ve	−
Measles IgG	−	+ve	−	+ve	−	−	−
Mumps IgG	−	+ve	−	+ve	−	+ve	−
Rubella IgG	−	+ve	−	+ve	−	+ve	−
Viral PCR
EBV	−	ND	−	−	−	−	−
CMV	−	ND	−	−	−	−	−
Adenovirus	−	ND	−	−	−	−	−
Lymphocytes
Total (×109/L [1.00-2.80])	1.50	1.32	2.30	2.35	2.64	2.34	2.20
CD4/CD8 (1.07-1.87)	15.29*	10.58	4.62	5.87	3.62	1.86	1.9
CD3 (absolute; × 109/L [0.700-2.100])	0.921	0.949	1.365	1.605	1.968	1.504	1.457
CD8 (absolute; × 109/L [0.200-0.900])	0.055	0.080	0.236	0.231	0.415	0.488	0.489
NK (absolute; × 109/L [0.090-0.600])	0.238	0.191	0.581	0.449	0.252	0.252	0.152
CD4 (absolute; × 109/L [0.300-1.400])	0.841	0.846	1.091	1.355	1.502	0.908	0.929
CD19 (absolute; × 109/L [0.100-0.500])	0.258	0.127	0.245	0.282	0.381	0.574	0.564
CD3+ cells (%)	71	75	64	68	75	64	66
CD4+ cells (%)	66	66	51	57	57	39	41
CD8+ cells (%)	4	6	11	10	16	21	21
CD56+CD16+ cells (%)	14	14	24	19	10	11	7
CD19+ cells (%)	15	9	10	12	14	24	26
CD4+FOXP3+ cells	Normal	Normal	−	−	−	−	−
T-cell proliferation after stimulation
PHA	Normal	Normal	−	Normal	−	Normal	−
Anti-CD3 antibody	Normal	Normal	−	Normal	−	Normal	−
Immunoglobulins
IgG (g/dL [6.0-16.0])	11.8	−	11.8	−	12.4	9.6	−
IgG1 (g/L [3.62-10.27])	7.48	−	−	−	−	−	−
IgG2 (g/L [0.81-4.72])	2.66	−	−	−	−	−	−
IgG3 (g/L [0.138-1.058])	0.420	−	−	−	−	−	−
IgG4 (g/L [0.049-1.085])	0.224	−	−	−	−	−	−
IgA (g/dL [0.80-4.00])	1.85	−	2.53	−	3.51	3.95	−
IgM (g/dL [0.25-2.00])	2.08	−	0.77	−	1.20	1.12	−
IgE (kU/L [0.5-120.0])	<2.0	−	195.0	−	24.4	157.0	−
Other antibodies
ANA†	−ve	−ve	−ve	−ve	+ve†	+ve‡	−
dsDNA (IU/mL [0-50])	−	−ve	−	−	−ve	−ve	−
Rheumatoid factor (IU/mL [<20])	<15	<15	<15	−	122	−	−
Complement
C3 (g/dL [0.75-1.65])	1.14	−	1.02	−	1.34	−	−
C4 (g/dL [0.12-0.40])	0.28	−	0.31	−	0.44	−	−

−, Not investigated; ANA, antinuclear antibody; CMV, cytomegalovirus; dsDNA, double-strand DNA; ND, not detected; RNP, ribonucleoprotein; VCA, viral capsid antigen; +ve, positive; −ve, negative; VZV, varicella zoster virus. Values in boldface are outside the reference ranges.

On resampling 3 months later: CD4/CD8 ratio, 14.36; CD8, 0.092.

Positive (homogenous: weak RNP antibody positive).

Positive (nucleolar).

Table E3

Summary of key clinical findings associated with individual reported recessive STIM1 mutations and summarized key clinical findings associated with dominant STIM1 mutations

Feature	Recessive homozygous mutations						Dominant mutations
Reference	Picard et al, 2009E8	Byun et al, 2010E9	Fuchs et al, 2012E10	Wang et al 2014E11	Schaballie et al, 2015E12	This study	Bohm et al, 2013E13	Morin et al, 2014E14
Individual (AR) or diagnosis (AD)	Pr1, Pr2, and Pr3∗	Pr4†	Pr5 and Pr6	Pr7	Pr8 and Pr9	V2 and V3	Tubular aggregate myopathy‡	Stormorken syndrome‡
Predicted protein effect of mutation	No protein	No protein	p.429 R>C	p. 146A>V	p.165P>Q	p.74 L>P	All missense in the EF-hand	p.304 R>W
Age at last examination (y)	1, 5, 6, and 9	2	1.7 and 6	6	8 and 21	11 and 21	Various	Various
Immune deficiency	Life-threatening infections	Life-threatening infections	Life-threatening infections	History of frequent throat infections: no immunologic evaluation performed	Life-threatening infections	No persistent severe infection	NR	NR
Other immune dysregulation	AIHAITP	AIHA	AIHAITP	NR	Colitis, psoriasis	V3 transient ITP	NR	NR
Skeletal muscle	Developmental skeletal myopathy with hypotonia, profound	NR	Developmental skeletal myopathy with hypotonia, mild	NR	Developmental skeletal myopathy, profound	No abnormalities	Clinical myopathy except with 1 mutation Increased CK typical	Clinical myopathyIncreased CK
Mydriasis	Yes	NR	Yes	NR	No	No	NC	Yes
Sweat glands	NC	NR	Anhidrosis	NR	Anhidrosis	Hypohidrosis	NC	NC
Dental enamel	Abnormal	NR	Abnormal	Abnormal	Abnormal	Abnormal	NC	NC
Died	Pr1 died 9 y (during HSCT)Pr2 died 1.5 y (encephalitis)	Pr4 died 2 y (Kaposi sarcoma)	Pr6 died 1.7 y (sepsis)	NR	NA	NA	NA	NA
Alive	Pr3 alive at 6 y (HSCT at 1.3 y)	NA	Pr5 alive (HSCT)	Pr7 lost to follow-up at 5 y	Pr8 and Pr9 alive	V2 and V3 alive	All alive	All alive

AIHA, Autoimmune hemolytic anemia; AD, autosomal dominant; AR, autosomal recessive; CK, creatine kinase; HSCT, hematopoietic stem cell transplantation; ITP, idiopathic thrombocytopenic purpura; NA, not applicable; NC, no comment made; NR, comment made but feature not recognized.

Mutation confirmed in Pr1 and Pr3; no DNA sample available for Pr2.

Mutation identified after death.

A missense change reported in tubular aggregate myopathy and the missense change reported as the cause of Stormorken syndrome have also been identified as the causes of York platelet syndrome, which is characterized by myopathy and platelet abnormalities (Markello et al, 2015).

Lymphocyte studies showed stable CD8 T-cell depletion in the older affected subject only. Other lymphocyte subsets, including CD4 T, natural killer (NK), and B cells, were within the normal range (Table I). However, despite normal PHA and anti-CD3 simulation responses, T-lymphocyte and NK cell SOCE was grossly abnormal, which is consistent with disruption of the Ca2+-binding EF-hand and in keeping with previous reports for recessive STIM1 mutations (see Fig E1, A, in this article's Online Repository at www.jacionline.org).1, 2, 3 The defect in NK cell SOCE was associated with impaired NK cell effector function, as shown by assays of granule exocytosis and intracellular IFN-γ production in response to K562 tumor cells (see Fig E1, B). After recently published mouse studies, which confirmed the importance of STIM1 to neutrophil SOCE and associated functions, we also evaluated neutrophil function. This was found to be within normal limits.

Fig E1

Defective SOCE and impaired NK cell function in STIM1-Leu74Pro patients' cells. A, Calcium flux in lymphocytes after anti-CD3/anti-CD16, 1 μmol/L thapsigargin, or 500 nmol/L ionomycin administration. B, Granule exocytosis and IFN-γ production of purified NK cells after stimulation with K562 tumor target cells alone or with IL-12 and IL-18. Results are representative of 2 experiments performed in duplicate and corrected for unstimulated control values.

Despite abnormal immune system SOCE, the affected subjects in this case appear to be able to compensate for this deficit and avoid overt immunodeficiency. It is possible that the relative preservation of T-cell function might compensate for NK cell dysfunction. Neither might yet have encountered a pathogen that would expose this particular immune system limitation (see Table E2). An ability to mount a partial response to viral infections was reported in a family with clinical immunodeficiency and a history of viral infections caused by a homozygous missense R429C change affecting the STIM1 cytoplasmic domain. A mouse model characterized by conditional knockout of Stim1 and Stim2 in both CD4+ and CD8+ T cells has recently provided further insight into the importance of Stim1 in immune system development and virus-specific memory and recall responses, which prevent acute viral infections from becoming chronic.

Recessive STIM1 mutations can be associated with other immune dysregulations, including autoimmune disease. The older cousin had a transient episode of idiopathic thrombocytopenic purpura when 2 years old that might have been unrelated to the STIM1 mutations. There were no other clinical or serologic markers consistent with autoimmune disease, and regulatory T-cell numbers were normal.

Both cousins were intolerant of warm environments and aware of their inability to sweat normally. This limited the older cousin's ability to participate in sport. There was no clinical or serologic evidence of myopathy. This is in contrast to other recessive STIM1 mutations and also to dominant STIM1 mutations affecting the EF-hand that cause tubular aggregate myopathy (MIM #160565). Hypomineralized AI affected the primary and secondary dentitions of both affected cousins (see Fig E2 in this article's Online Repository at www.jacionline.org), which is in keeping with reports of other recessive STIM1 mutations. The cousins were physically small (height, weight, and head circumference <0.4th percentile) when assessed at 18 years and 9 years, 10 months of age, respectively. Without comparable data from other subjects with recessive STIM1 mutations, it is unclear whether this is a cosegregating feature.

Fig E2

Hypomineralized AI as the presenting feature in a family with STIM1 L74P change. A, Pedigree of the consanguineous family investigated. The 2 affected cousins with AI and hypohidrosis are shaded black. Genotypes of the c.221T>C variant are indicated underneath each family member available for sequencing. Representative electropherograms are shown alongside the pedigree. B, The hypomineralized AI was characterized by opaque discolored enamel on clinical examination, with radiographs of unerupted teeth consistent with a near-normal volume of enamel and a clear difference in radiodensity between enamel and dentine. *Teeth that have been restored. C, Schematic illustration of STIM1 protein showing the domain structure. Positions of the AI and hypohidrosis-associated L74P mutation (red), dominant TAM or Stormorken syndrome mutations (grey), and recessive syndromic immunodeficiency mutations (black) are indicated above the protein. E-rich, Glutamate-rich region; K, lysine-rich region; MLS, microtubule tip localization signal; P, proline/serine-rich region; SAM, sterile α-motif domain; SOAR, STIM1 Orai1-activating region; TM, transmembrane domain. D, Alignment of STIM1 EF-hand orthologous protein sequences. Although p.L74 is conserved in mammals, it is not as strongly conserved as amino acids mutated in dominant TAM. E, NMR structure of STIM1. L74 is shown in red, TAM mutations are shown in dark gray, and Ca2+ binding residues, mutation of which cause constitutive STIM1 activation, are shown in yellow. Substitution of leucine 74 for proline is anticipated to distort the EF-hand loop, interfering with conformational changes in the presence/absence of Ca2+.

The L74P STIM1 change within the EF-hand domain precedes the first Ca2+-binding aspartate residue by 2 amino acids (see Fig E2) and therefore might be expected to distort the Ca2+-binding region of the protein. Therefore we compared the response of mutant YFP-STIM1 (L74P) with the depletion of Ca2+ stores after thapsigargin or cyclopiazonic acid (CPA) treatment with that of wild-type YFP-STIM1 and the previously published EF-hand mutant YFP-STIM1 (D76A, see Fig E3 in this article's Online Repository at www.jacionline.org).

Fig E3

STIM1 localization and Ca2+ flux in cells transfected with STIM1 constructs. A, TIRFM of HEK293 cells transfected with either wild-type (WT), D76 A mutant, or L74P mutant YFP-STIM1 after treatment with 2 μmol/L thapsigargin to deplete ER Ca2+ stores. The graph on the bottom left shows changes in TIRF fluorescence within single puncta areas indicated by white circles on the images (ROI1-3). The graph on the bottom right shows average footprint fluorescence for cells transfected with WT STIM1 (n = 39), D76 A (n = 30), or L74P (n = 31) constructs. B, Representative recordings of cytosolic calcium ([Ca2+]i) made in HEK293 cells doubly transfected with ORAI1-CFP and either WT (n = 12), D76 A (n = 12), or L74P (n = 13) STIM1-YFP constructs. C, Bar graphs indicating mean ± SEM [Ca2+]i relating to the presence of extracellular Ca2+ or SERCA inhibition by CPA. Top row, Mean baseline Ca2+ levels in the presence and absence of extracellular Ca2+. Bottom row, Peak responses to CPA, integral of the CPA-evoked response, and peak value of capacitative Ca2+ entry (CCE). *P < .01 compared with control with control values (ANOVA).

Using total internal reflection fluorescence microscopy (TIRFM), we replicated previous observations that wild-type YFP-STIM1 relocalizes to puncta proximal to the plasma membrane after treatment of transfected HEK293 cells with 2 μmol/L thapsigargin to deplete ER Ca2+ stores through sarcoendoplasmic reticulum calcium transport ATPase (SERCA) inhibition (see Fig E3, A). The EF-hand mutant YFP-STIM1 (D76 A) was present in these puncta before thapsigargin treatment, with no observable response to thapsigargin (see Fig E3, A). Similarly, mutant YFP-STIM1 (L74P) showed no response to thapsigargin but also appeared to form constitutive puncta, which was less distinct in appearance than that for the D76A mutant (see Fig E3).

We compared Ca2+ fluctuations in HEK293 cells transfected with ORAI-CFP and either wild-type YFP-STIM1, mutant YFP-STIM1 (D76A), or mutant YFP-STIM1 (L74P; see Fig E3, B and C). Both YFP-STIM1 (D76A) and YFP-STIM1 (L74P) transfected cells had increased basal Ca2+ concentrations compared with wild-type YFP-STIM1 and reduced peak and integral responses to CPA-induced SERCA inhibition (see Fig E3, B and C). However, in contrast to the EF-hand mutant YFP-STIM1 (D76A), YFP-STIM1 (L74P) did not demonstrate reduced SOCE after CPA washout and Ca2+ restoration, suggesting that the previously reported desensitization of SOCE observed with the YFP-STIM1 (D76A) mutant does not occur with the YFP-STIM1 (L74P) mutant form. Therefore the L74P mutation appears to result in a distinct molecular phenotype compared with the loss of function observed in immunodeficient patients and the constitutive activation observed in patients with myopathy.

This study is the first to report recessive STIM1 mutations in patients presenting with AI and hypohidrosis without overt clinical immunodeficiency or myopathy. Clinical immunologic investigations were consistent with abnormal NK cell and T-lymphocyte function that might be expected to be associated with ongoing clinical immunodeficiency. However, despite severely abnormal SOCE, this was not the case in these patients. Missense mutations affecting the EF-hand can have very different clinical phenotypes with respect to the immune system, muscle, sweating, and enamel formation. This has important implications for clinical evaluation, as well as understanding the biological functions of STIM1.