Janus kinase 3 deficiency caused by a homozygous synonymous exonic mutation that creates a dominant splice site

Accepted Manuscript JAK3 deficiency caused by a homozygous synonymous exonic mutation that creates a dominant splice site Craig D. Platt, MD, PhD, Michel J. Massaad, PhD, Brittany Cangemi, BA, Birgitta Schmidt, MD, Hasan Aldhekri, MD, Raif S. Geha, MD PII:

S0091-6749(16)31445-2

DOI:

10.1016/j.jaci.2016.09.057

Reference:

YMAI 12495

To appear in:

Journal of Allergy and Clinical Immunology

Received Date: 18 July 2016 Revised Date:

23 August 2016

Accepted Date: 9 September 2016

Please cite this article as: Platt CD, Massaad MJ, Cangemi B, Schmidt B, Aldhekri H, Geha RS, JAK3 deficiency caused by a homozygous synonymous exonic mutation that creates a dominant splice site, Journal of Allergy and Clinical Immunology (2017), doi: 10.1016/j.jaci.2016.09.057. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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JAK3 deficiency caused by a homozygous synonymous exonic mutation that creates a

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dominant splice site

3 Craig D. Platt, MD, PhD1 Michel J. Massaad, PhD1 Brittany Cangemi, BA1 Birgitta Schmidt, MD1

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Hasan Aldhekri, MD2 Raif S. Geha, MD1

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Division of Immunology, Boston Children’s Hospital, Harvard Medical School, Boston, MA. 2

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Section of Pediatric Allergy & Immunology, King Faisal Specialist Hospital & Research Center, Riyadh, Saudi Arabia.

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Address correspondence to: Raif S. Geha, One Blackfan Circle, Boston, Massachusetts 02115,

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(617) 919-2482; [email protected]

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Capsule Summary – Two siblings were found to have JAK3 deficiency due to a synonymous

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exonic mutation. The mutation does not change the predicted JAK3 amino acid sequence, though it

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is pathogenic as it creates a new donor splice site that disrupts protein expression.

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Key words: JAK3 deficiency, mRNA splicing, synonymous mutation, silent mutation, aberrant

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splicing, SCID, skin granuloma.

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19 Abbreviations:

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BLCL: B-lymphoblastoid cell line

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CID: combined immunodeficiency

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JAK3: Janus kinase 3

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SCID: severe combined immunodeficiency

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HSCT: hematopoietic stem cell transplantation

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Supported by: National Institute of Health grants T32 AI007512 (C.P.), K12 HD052896-10 (C.P.),

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and the Perkins Fund (R.S.G.).

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To the editor: Janus kinase 3 (JAK3) is a cytosolic tyrosine kinase that is primarily expressed in

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hematopoietic cells.1 Null mutations in JAK3 typically manifest as T-B+NK- severe combined

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immunodeficiency (SCID), while hypomorphic mutations can present with a broader range of

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immune dysregulation.1,2 Here we report two siblings with a homozygous synonymous exonic

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mutation in JAK3 that creates a dominant splice site and dramatically decreases expression of WT

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protein. This mutation resulted in T-B+NKlow SCID in one patient and in a combined

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immunodeficiency (CID) with granulomatous skin disease in her sibling.

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Four children resulted from the consanguineous union of two parents from Saudi Arabia

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(Fig 1A). Given a family history of SCID in an older sibling, the family’s fourth child (Patient 1)

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was screened shortly after birth by lymphocyte flow cytometry and diagnosed with T-B+NKlow

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SCID. Her 6-year-old brother (Patient 2) had a persistent violaceous, granulomatous dermatitis on

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his arms since 2 years of age (see Fig E1 in this article’s Online Repository at www.jacionline.org)

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but no history of frequent or unusual infections. Skin biopsy revealed a prominent dermal nodular

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infiltrate and poorly formed granulomas extending to the deep dermis (see Fig E2 in this article’s

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Online Repository at www.jacionline.org). Special stains for organisms performed with Periodic

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acid–Schiff, Gram, Grocott's methenamine silver, and Fite were negative (data not shown). As

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granulomatous skin disease has been associated with CID2,3 an immunologic evaluation of both

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siblings was performed.

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Immunological workup of Patient 1 revealed nearly absent T cells (56 cells/µl), normal B

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cells (1600 cells/µl), low NK cells (54 cells/µl) and negligible T cell proliferation to PHA, ConA

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and anti-CD3 (Table 1). All detectable T cells were of maternal origin (data not shown). Patient 2

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had low numbers of T cells (566 cells/µl), B cells (120 cells/µl) and NK cells (17 cells/µl),

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consistent with a CID. He also had reduced percentages of naïve T cells, and inversion of the 3

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CD4:CD8 ratio (Table 1). Proliferation to mitogens and antigens was below the lower limit of

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normal (Table 1). There was no evidence of maternal engraftment (data not shown). Patient 2 had

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normal TCR Vβ usage that did not suggest a restricted T cell repertoire (see Fig E3 in this article’s

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Online Repository at www.jacionline.org), and normal immunoglobulins (Table 1), but poor

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responses to tetanus and pneumococcal vaccines (data not shown).

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To determine the underlying genetic defect, genomic DNA from Patient 1 was evaluated in

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a commercial laboratory using massive parallel sequencing of 46 genes associated with SCID/CID

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that included JAK3. While no pathogenic variants were reported, the possibility of a non-coding

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mutation in JAK3 that affects protein expression was pursued, as mutations in this gene typically

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present with T-B+NK- SCID. Immunoblotting of lysates from Epstein Barr virus-transformed B-

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lymphoblastoid cell lines (BLCLs) revealed a dramatic decrease in JAK3 expression in both

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patients (Fig 1B). STAT5 phosphorylation in response to IL-2 and IL-15 stimulation of BLCLs was

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severely diminished in the patients, confirming a functional defect in JAK3 signaling (Fig 1C).

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A comprehensive list of all exonic variants found by the commercial laboratory was

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requested. It included a single homozygous, synonymous variant in exon 19 of JAK3 (c.2652C>T;

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pV884V) that had been presumed benign as it did not alter the predicted amino acid sequence.

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However, in silico analysis using the independent splice site prediction tools MaxEntScan4 and

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Human Splicing Finder5 indicated that the variant likely created a donor splice site (see Table E1 in

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this article’s Online Repository at www.jacionline.org). The variant was confirmed by Sanger

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sequencing to be homozygous in both patients, and heterozygous in the parents and healthy sibling

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(Fig 1D).

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To evaluate whether the mutation impacted splicing of JAK3 mRNA, a 218 bp cDNA

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fragment surrounding the mutation site was amplified by RT-PCR (Fig 1E, top panel). mRNA

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purified from control PBMCs produced a single band of the expected molecular weight (Fig 1E, 4

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bottom panel). In contrast, two bands were amplified from PBMC mRNA isolated from Patient 1: a

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barely detectable full-length band, and a much more abundant, lower molecular weight band (Fig

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1E, bottom panel). Sanger sequencing revealed that the full-length band contained WT cDNA,

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while the lower band contained two cDNA species with deletions of either 29 or 30 bp at the 3’ end

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of exon 19 (Fig 1F). The 29 bp deletion resulted in a frameshift with a premature stop codon

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(p.K884Pfs*73). Immunoblotting of patient BLCL lysates using a mAb specific for the N-terminus

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of JAK3 revealed no truncated protein at the predicted molecular weight of approximately 95 kDa,

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indicating that the mutant protein was not expressed or was rapidly degraded (Fig 1B). The 30 bp

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deletion removes 10 terminal amino acids of exon 19 (p.V884_P893del), thereby encoding a mutant

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protein ~1.2 kDa smaller than WT JAK3. Due to the closeness of their predicted molecular weight

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(115 kDa versus 114 kDa), the WT and p.V884_P893del mutant protein would be expected to

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migrate as a single band of ~115 kDa on gel electrophoresis. The dramatic decrease in the intensity

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of this band strongly suggests that the p.V884_P893del mutation decreases protein stability.

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It is notable that Patient 1 and Patient 2 had identical homozygous mutations in JAK3 with

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similar reductions in protein expression but different phenotypes (see Table E2 in this article’s

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Online Repository at www.jacionline.org for additional clinical details on all siblings). The

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relatively normal TCR Vβ repertoire of Patient 2, residual proliferation to mitogens, and lack of

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significant infections indicates that his T cells retain some function and are not merely expanded

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from a limited number of clones. Such phenotypic variability has been documented in a variety of

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other PID genes6. Modifier genes, epigenetic factors, or environmental exposures may all play a

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role in determining the clinical phenotype.6

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Synonymous codon variants are generally thought to have little significance7 and are often

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filtered out during analysis of the vast amount of data generated though next generation

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sequencing.6 Nevertheless, synonymous mutations in genes such as IL10RA and MRE11A have 5

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previously been reported to cause disease through disruption of known splice sites.8,9 Our case

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illustrates the importance of evaluating synonymous mutations even when they are distant from

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known splice sites, as they may create new donor or acceptor splice sites. The significance of a synonymous variant can be determined through a series of steps. First,

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splicing prediction tools can determine the probability that a variant is pathogenic. Next, cDNA

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amplification and sequencing can definitively demonstrate that splicing is affected. Finally,

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functional analysis is necessary to prove that that a change in splicing is responsible for the patient

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phenotype. As whole exome sequencing and targeted gene panels become an increasingly important

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part of the clinical immunologist’s toolbox, the need to evaluate the biologic relevance of

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synonymous variants will continue to increase.

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Craig D. Platt, MD, PhD1

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Michel J. Massaad, PhD1

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Birgitta Schmidt, MD1 Hasan Aldhekri, MD2 Raif S. Geha, MD1

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Brittany Cangemi, BA1

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Division of Immunology, Boston Children’s Hospital, Harvard Medical School, Boston, MA.

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Section of Pediatric Allergy & Immunology,
King Faisal Specialist Hospital & Research Center,


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Riyadh, Saudi Arabia.

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Table 1: Immune profile of patients Patient 1

Patient 2

Age 3 months

Normal Values a for Age

Age 6 years

Normal Values for a Age

56

2500-5600

566

1000-2600

154

530-1500

3.90%

0.4%-2.6%

5.60%

57.1%-84.9%

38.50%

3.3%-15.2%

52%

11.3%-26.7%

+ +

CD3 CD4

+

7

1600-4000

+

+

-

16.90%

0.1-1.9%

+

+

+

11.90%

76.7-91.4%

+

-

-

54.20%

1.1-5.3%

+

-

+

16.90%

6.7-15.6%

+

34.30%

59.7- 82.4%

37

500-1700

344

330-1100

0.90%

1.5-22.7%

15.40%

9.1-49.1%

CD4 CD45RA CCR7 (TEMRA) CD4 CD45RA CCR7 (naïve) CD4 CD45RA CCR7 (effector memory) CD4 CD45RA CCR7 (central memory) +

+

+

CD3 CD8

+

+

+

-

+

+

+

+

-

-

+

-

+

CD8 CD45RA CCR7 (TEMRA) CD8 CD45RA CCR7 (naïve) CD8 CD45RA CCR7 (effector memory) CD8 CD45RA CCR7 (central memory) CD19+ +

-

+

+

IgD CD27 (naïve) IgD CD27 (unswitched memory ) IgD-CD27+ (switched memory) hi

hi

low

hi

CD24 CD38 (plasmablasts) low

hi

low

CD19 CD21 CD38 CD24 CD38

(marginal zone-like)

CD16+/CD56+ Immunoglobulins IgG (mg/dL) IgA (mg/dL) IgE (kU/L)

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IgM (mg/dL)

low

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+

T cell proliferation (counts per minute) Phytohemagglutinin Concanavalin A Anti-CD3 Tetanus Candida Background

6.60%

45.3-63.6%

0.30%

62.1-94.0%

2.30%

28.4-80.6%

96.30%

1.3- 19.5%

71.30%

6.2-29.3%

2.50%

0.9-5.6%

11.00%

1.0-4.5%

1600

300-2000

120

270-860

n.d.

94.10%

47.30-77.00%

n.d.

3.00%

5.20-20.40%

n.d.

1.00%

10.90-30.40%

n.d.

19.90%

7.2-23.8%

n.d.

0.00%

0.4-5.2%

n.d.

5.60%

2.5-9.4%

n.d.

9.80%

12.6-36.0%

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CD24 CD38 (transitional)

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CD4 CD31 CD45RA (recent thymic emigrants)

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CD3

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Lymphocyte subsets (cells per μl)

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160-1100

17

70-480

809* <7

280-750 6-50

775 104

639-1344 70-312

39

15-70

171

34-210

<1

0-30

5

0-200

750 657 1966 n.d. n.d. 1273

96,090-358,179 65,699-239,344 62,927-217,761

26,802 24,681 34,629 1239 17348 322

96,090-358,179 65,699-239,344 62,927-217,761 8544 – 102,895 6231-197,940 204-2104

204-2104

a

normal values from age-matched controls in the Boston Children’s Hospital clinical immunology laboratory *on IVIG n.d. = not done

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Figure Legends Fig 1. Characterization of the JAK3 mutation. (A) Pedigree. (B) Immunoblot of lysates

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from BLCLs derived from patients and controls (left). JAK3 expression relative to controls (right).

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***p<0.001, ns=not significant (one-way ANOVA). (C) Percentage of pSTAT5+ BLCLs after

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stimulation for 10 minutes with IL-2 or IL-15. ****p<0.0001 (two-way ANOVA). (D) Sanger

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sequencing of c.2652C>T variant. (E) RT-PCR amplification of JAK3 mRNA encompassing the

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patient mutation site. (F) Diagram of mRNA splice variants induced by mutation. For B and C n=2

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for controls, n=2 for patients; data pooled from 2 independent experiments. Columns/lines and bars

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represent means and SEM.

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Extended Figure Legends

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Fig E1. Rash on forearm and hand of Patient 2.

Fig E2. H&E staining of biopsy of a skin lesion from Patient 2 at 10x (A) and 20x (B) magnification, showing granulomatous infiltrate. Scale bars = 200 µm.

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Fig E3: Quantitative analysis of the TCR Vβ repertoire of T lymphocytes from patient 2.

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PBMCs were labeled using the IOTest Beta Mark TCR V beta repertoire kit. Analysis of CD4+ and

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CD8+ T cells was performed by flow cytometry.

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Table E1. In silico splicing prediction scores for JAK3 c.2652C>T:pV884V WT allele

Mutant allele

C

T

1. MaxEntScan score

-5.24

2.51

2. Human Splicing Finder score

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66.67

1. MaxEntScan: http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html

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Score range -20 to +20. A higher score indicates a greater potential for splice site. +3 is the

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threshold for a likely splice site.

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2. Human Splicing Finder: http://www.umd.be/HSF3/index.html

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Score range 0–100. A higher score indicates a greater potential for splice site. 65 is the threshold for

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a likely splice site. If the WT score is under the threshold and the score variation is above +10%, the

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mutation is likely to create a new splice site. In this case, the difference is +67%, supporting the

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presence of new donor splice site.

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Table E2. Summary of clinical history and treatment outcomes for Patient 1, Patient 2 and

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their siblings. Sex

Genotype

Presenting History

Treatment Outcome

Female

c.2652C>T homozygous

T-B+NKlow SCID detected by flow cytometry immediately after birth given clinical history of Affected Sister (see below).

Patient 2

Male

c.2652C>T homozygous

Granulomatous skin disease starting at 2 years of age.

Healthy Brother Affected Sister

Male

c.2652C>T heterozygous Presumed c.2652C>T homozygous

No significant past medical history. Hospitalized with pneumonia and disseminated BCG at 6 months of age.

Underwent hematopoietic stem cell transplantation (HSCT) with a 9/10 HLA-A mismatched unrelated donor after myeloablative conditioning. Post transplant course was complicated by severe gut GVHD, which was well controlled by pulse dose steroids. She is currently alive and well. Currently undergoing evaluation for HSCT. Continues to have no history of recurrent or opportunistic infections. He has been started on trimethoprim/sulfamethoxazole and IVIG. n/a

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Died at 19 months of age from complications of HSCT.

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Female

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Family Member Patient 1

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Supplemental Methods: Immunoblotting. Cell lysates were immunoblotted using a mAb raised against the N-

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terminus of JAK3 (clone D7B12, Cell Signaling) or anti-actin (clone ACTN05 (C4), Abcam)

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followed by HRP-conjugated goat anti–rabbit antibody or goat anti–mouse antibody conjugated to

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horseradish peroxidase. Densitometry was performed using ImageJ (NIH). JAK3:actin ratios were

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calculated and normalized to the JAK3:actin ratios of control EBV lines from healthy donors.

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STAT5 Phosphorylation. PBMCs from Patient 1, Patient 2 and two healthy controls were

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infected with EBV in the presence of cyclosporine A to generate BLCLs. BLCLs were stimulated

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with the indicated concentrations of human IL-2 (Peprotech) or IL-15 (Peprotech) for 10 min at

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37oC, before fixation using Lyse/Fix Phosflow buffer (BD Biosciences) and permeabilization using

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BD PhosflowTM Perm Buffer III according to the manufacturer’s instructions. Cells were stained

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with anti-phospho-STAT5 (Y694-PE) (clone SRBCZX, eBiosciences) and analyzed using a

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LSRFortessa (BD Biosciences).

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Semi-quantitative RT-PCR. Total RNA was prepared from PBMCs cells from Patient 1 or

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a healthy control using the RNeasy kit (Qiagen). cDNA synthesis and amplification were performed

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from total RNA using the Superscript III One Step RT-PCR System with Platinum Taq DNA

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polymerase. Primers used for JAK3 were: forward primer 5’- GACTTTCAGCGGGAGATTCA-3’

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(exon 19), and reverse primer 5’- TACTCCATGC CCTTGCAGAT-3’ (spans exon 19 and 20).

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References:

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1.

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Immunity 2012;36:515–28. 2.

Scarselli A, Di Cesare S, Di Matteo G, De Matteis A, Ariganello P, Romiti ML, et al.

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Casanova JL, Holland SM, Notarangelo LD. Inborn Errors of Human JAKs and STATs.

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Combined immunodeficiency due to JAK3 mutation in a child presenting with skin

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granuloma. J Allergy Clin Immunol 2016;137:648–51. 3.

Harp J, Coggshall K, Ruben BS, Ramirez-Valle F, He SY, Berger TG. Cutaneous

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granulomas in the setting of primary immunodeficiency: A report of four cases and review of

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the literature. Int J Dermatol 2015;54:617–25.

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Yeo G, Burge CB. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol 2004;11:377–94.

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Desmet FO, Hamroun D, Lalande M, Collod-Bëroud G, Claustres M, Béroud C. Human Splicing Finder: An online bioinformatics tool to predict splicing signals. Nucleic Acids Res

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2009;37:1–14.

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Platt C, Geha RS, Chou J. Gene hunting in the genomic era: Approaches to diagnostic dilemmas in patients with primary immunodeficiencies. J Allergy Clin Immunol

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2014;134:262–8. 7.

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Hunt RC, Simhadri VL, Iandoli M, Sauna ZE, Kimchi-Sarfaty C. Exposing synonymous

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mutations. Trends Genet 2014;30:308–21. 8.

Matsumoto Y, Miyamoto T, Sakamoto H, Izumi H, Nakazawa Y, Ogi T, et al. Two unrelated

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patients with MRE11A mutations and Nijmegen breakage syndrome-like severe

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microcephaly. DNA Repair 2011;10:314–21.

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9.

Oh SH, Baek J, Liany H, Foo JN, Kim KM, Yang SC-O, et al. A Synonymous Variant in IL10RA Affects RNA Splicing in Pediatric Patients with Refractory Inflammatory Bowel 12

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Disease. J Crohns Colitis 2016 [Epub ahead of print].

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Figure 1 B

*** ***

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JAK3 % of Control

A

50

ns

C

on tr

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ol s Pt 1 Pt 2

0

50

10

IL-15 (ng/mL)

IL-2 (ng/mL)

D

0

0

00

0

15

30

60

0

0

5

2

5

Controls Patients

****

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10

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0. 4

Controls Patients

% pSTAT5+ cells

20

****

% pSTAT5+ cells

C

F

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Exon 19

218 bp 189/190 bp

Exon 20

mRNA 1

WT

mRNA 2

p. K884Pfs*73

mRNA3

p. V884_P893del

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Figure E1

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Figure E2

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A

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Vβ 5. 3 Vβ Vβ 3 7. 1 Vβ 9 Vβ 1 Vβ 6 1 Vβ 7 18 Vβ 2 Vβ 0 5 Vβ . 1 13 .1 V Vβ β8 13 Vβ . 6 5. Vβ 2 12 Vβ Vβ 2 Vβ 2 3 21 .3 Vβ Vβ 1 1 Vβ 1 1 Vβ 4 Vβ 2 2 13 Vβ . 2 7. 2 Vβ 4

%Vβ usage in CD8+ T cells 0.1

CD8 +

100

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Vβ 5. 3 Vβ Vβ 3 7. 1 Vβ 9 Vβ 1 Vβ 6 1 Vβ 7 18 Vβ 2 Vβ 0 5 Vβ . 1 13 .1 V Vβ β8 13 Vβ . 6 5. Vβ 2 12 Vβ Vβ 2 Vβ 2 3 21 .3 Vβ Vβ 1 1 Vβ 1 1 Vβ 4 Vβ 2 2 13 Vβ . 2 7. 2 Vβ 4

%Vβ usage in CD4+ T cells

CD4 +

100

1

normal range

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Figure E3

percent of T cells with indicated Vβ usage