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INTRACELLULAR DEFECTS AND PRIMARY IMMUNODEFICIENCIES

Rayna Doll, DO, Robert W Hostoffer, DO

Allergy/Immunology Fellowship

University Hospitals, Cleveland Medical Center, Cleveland, Ohio

AGENDA

Will discuss mutations of NEMO

Will discuss mutations of IPEX

Will discuss new primary immunodeficiency involving SEC61A1

NEMO

NF-kappa-B essential modulator

Aka inhibitor of nuclear factor kappa-B kinase subunit gamma (IKK-γ)

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MUTATIONS:

A very rare form of immunodeficiency caused by a mutation of the NEMO (NF-kappa-B essential modulator) gene on chromosome Xq28.

Hypomorphic mutations in the NFκB essential modulator (NEMO) impair NFκB function and are linked to both immunodeficiency and ectodermal dysplasia (ED) as well as susceptibility to atypical mycobacterial infections.

Estimated incidence of NEMO-ID is 1:250,000 live male births.

All patients had serious pyogenic bacterial illnesses early in life and the median age of first infection was 8.1mos.

Most boys developed mycobacterial disease (median age=84mos) and a minority had herpes viral infections.

Initial immunological assessments showed hypogammaglobulinemia (median IgG=170mg/dl) with variable IgM (median=41mg/dl) and IgA (median=143mg/dl).

Two patients developed hyper-IgM and five developed hyper-IgA.

All patients evaluated had normal lymphocyte subsets with impaired proliferative responses, specific-antibody production, and NK cell function.

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NEMO

NEMO-ID joins a growing list of human genetic defects that impair NK cell function.

Infectious susceptibilities common to these disorders suggest an important role for NK cells in host defense.

Boys with a NEMO mutation and evidence of impaired specific antibody production should be treated with intravenous immunoglobulin.

MAC prophylaxis should be considered because of a high incidence of this infection.

Viral disease caused by herpesviruses should be treated aggressively, and a chemoprophylaxis regimen should also be considered.

At this time it is premature to comment on stem cell transplantation due to limited experience. Only one patient with Nemo-ID who has undergone successful stem cell transplantation.

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NEMO

NEMO-ID is characterized by specific infectious susceptibilities and immunologic impairments and has opened doors to clinical considerations of a new facet of innate immune deficiency, highlighting the importance of innate immunity.

These observations also suggest that defects in innate immunity probably are responsible for a portion of the infant mortality rate and that targeted diagnosis of these disorders in families having concerning histories will be beneficial.

IMMUNE DYSREGULATION POLYENDOCRINOPATHY ENTEROPATHY X- LINKED

Clinical syndrome described by Powell et al (1982).

First described in 1982 by Powell et al. as a syndrome of diarrhea, polyendocrinopathy, and fatal infection in infancy.

Neonatal onset diabetes mellitus

Hypothyroidism

Enteritis (diarrhea/villous atrophy)

Hemolytic anemia & thrombocytopenia.

Dermatitis: erythema -> eczema psoriasiform

Death by 1-2 years of age

IPEX – OUTSIDE (CLINICAL FINDINGS)

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IPEX CLINICAL AND LABATORY FEATURES

ENTEROPATHY Watery diarrhea (rarely bloody) Villious atrophy on biopsy Inflammatory bowel disease

DERMATITIS Eczema Erithroderma Psoriasiform dermatitis Alopecia

ENDOCRINOPATHIES

Type 1 diabetes

Thyroid

LABATORY FINDINDS

Elevated IgE

OTHER CLINICAL AND LABORATORY FINDINGS

HEMATOLOGIC

Coombs (+) hemolytic anemia

Autoimmune thrombocytopenia

Autoimmune neutropenia

RENAL

Nephrosis or nephritis

HEPATIC

Autoimmune hepatitis

RARE

Lymphadenopathy

Arthritis and vasculitis

AUTOANTIBODIES MAY BE PRESENT

AIE-75 (Antibody against gut and kidney specific antigen)

ANA

Antibody against different organs (thyroid, pancreatic islets, erythrocytes, platelets, smooth muscle)

SKIN DISEASE IN IPEX

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IPEX – INSIDE (AUTOPSY FINDINGS)

PANCREAS: Islets of Langerhans absent with

lymphocytic infiltrates

INTESTINE: Villous atrophy with

lmyphocytic infiltrates

LIVER: Cholangitis

SPLEEN: Enlarged

LYMPH NODES: Follicular hyperplasia

THYROID: Lymphocytic infiltrates

LUNGS: Consolidation/inflammation

THYMUS: Atrophy

IPEX

(n=43)

IPEX-Like

(n=27)

Clinical Features No.

Affected

%

Affected

No.

Affected

%

Affected

Enteropathy 42 98 27 100

Skin Disease 38 88 18 67

Endocrinopathy 30 70 16 59

Diabetes 23 77 7 43

Thyroid Disease 13 43 6 38

Hematologic Disease 24 56 5 18

Nephropathy 15 35 3 11

Hepatitis 9 22 3 11

Neurologic Disease 19 44 9 33

Serious Infections 24 56 14 53

Other

Lymphadenopathy 2 5 3 11

Arthritis/Vasculitis 2 5 3 11

IPEX VS. IPEX-LIKE PATIENTS – CLINICAL 1

IPEX

(n=43)

IPEX-Like

(n=27)

No. % No. %

Immunologic

Elevated IgE 19/26 74 6/10 60

Elevated IgA 20/30 67 1/9 11

Outcomes

Alive 28 53 25 93

BMT 11 26 2 7

Dead 15 35 2 7

IPEX VS. IPEX-LIKE PATIENTS – IMMUNOLOGIC FEATURES AND OUTCOMES

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IPEX

From Ochs HD et al. Immunol Rev. 2005 Feb;203:156-64. Review.

Locations of FOXP3 mutations reported

IVS9+4G>A Chatila TA et al 2000

-76fsX108 Owen CJ et al 2003

ATG>ATA Bacchetta et al 2006 ATG>AGG Myers AK et al 2006

F373A Bacchetta et al 2006

T108M & 454+4A>G De Benedetti F et al 2006

del-6247_-4859 Torgerson TR et al 2007

PROLINE-RICH DOMAIN ZINC-FINGER

LEUCINE

ZIPPER FORKHEAD DOMAIN

N C

Clustering of Missense Mutations in FOXP3

XMEN

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XMEN

Primary immunodeficiency

Group of genetic abnormalities that impairs both humoral and cell-mediated immunity

Loss of function mutation of the gene encoding the magnesium transporter

MAGT1

2 major consequences: suboptimal T-cell activation and loss of NKG2D on NK and cytotoxic T cells

MAGT1

MAGT1 is a cell surface protein that regulates the balance of Mg2+ between the extracellular fluid and the intracellular free basal pool

In T and B cells, MAGT1 participates in intracellular Mg2+ homeostasis

Defect leads to decreased intracellular free Mg2+

T CELL ACTIVATION

Figure 1 - Li F-Y, Chaigne-Delalande B, Su H, Uzel G, Matthews H, Lenardo MJ. XMEN disease: a new primary immunodeficiency affecting Mg2+ regulation of immunity against Epstein-Barr virus. Blood. 2014;123(14):2148-2152. doi:10.1182/blood-2013-11-538686.

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LOSS OF NKG2D

(C) Model of role of MAGT1 in Mg2+ homeostasis and NKG2D expression

Chronic decrease in the basal level of free Mg2+ leads to loss of expression of NKG2D

NKG2D- a receptor on NK and cytotoxic T cells involved in antiviral and antitumor cytotoxicity. Control of EBV-infected cells and tumor surveillance.

XMEN is the first PID associated with decreased expression of NKG2D.

Figure 1 - Li F-Y, Chaigne-Delalande B, Su H, Uzel G, Matthews H, Lenardo MJ. XMEN disease: a new primary immunodeficiency affecting Mg2+ regulation of immunity against Epstein-Barr virus. Blood. 2014;123(14):2148-2152. doi:10.1182/blood-2013-11-538686.

PATIENT PRESENTATION

Figure 1 - Li F-Y, Chaigne-Delalande B, Su H, Uzel G, Matthews H, Lenardo MJ. XMEN disease: a new primary immunodeficiency affecting Mg2+ regulation of immunity against Epstein-Barr virus. Blood. 2014;123(14):2148-2152. doi:10.1182/blood-2013-11-538686.

REPORTED CASES

7 XMEN patients characterized thus far Because XMEN is an X-linked disease, all patients with clinical disease to date have

been males.

Most consistent clinical feature among patients is persistent high level of EBV along with increased susceptibility for developing EBV-positive lymphomas

Age at diagnosis varies from 3 to 45 years

EBV-associated lymphomas have been the only reason some patients with XMEN disease come to medical attention

All 4 postpubertal patients have various EBV-associated B-cell lymphoproliferative disorders

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DIAGNOSIS

Suspect in cases with: unexplained chronic, especially high level, elevations in EBV viremia (PCR), splenomegaly, and a personal or family history of EBV-positive B cell lymphoproliferative diseases

A final diagnosis confirmed by examining MAGT1 mRNA expression or MAGT1 exon sequencing Cincinnati Children’s Molecular Genetics Laboratory or through the National Institutes

of Health Clinical Center

TREATMENT

Oral supplementation with high dose magnesium L-threonate

Restored basal intracellular free Mg2+along with NKG2D expression and decreased the number of EBV-infected cells in two XMEN patients

Rituximab (anti-CD20 antibody) will likely reduce the level of EBV-infected cells in the blood

Two patients with XMEN and lymphoma underwent allogeneic HSCT but did not survive because of transplant-related complications

HSCT might still be curative for patients with a good match

These patients have a high incidence of primary and secondary lymphomas

PLASMA CELL DEFICIENCY IN HUMANS WITH HETEROZYGOUS MUTATIONS IN SEC61A1

Desirée Schubert1, 2, Marie-Christine Klein3, Sarah Hassdenteufel3, *Andrés Caballero-

Oteyza1, Linlin Yang1,2, Michele Proietti1, Janine Kemming1, Johannes Kühn1, Sandra Winzer1, Stephan Rusch1, Manfred Fliegauf1, Alejandro A. Schäffer4, Stefan Pfeffer5, Adolfo

Cavalié6, Hongzhi Cao7, Fang Yang7, Yong Li8, Anna Köttgen8, Marta Rizzi9, Hermann Eibel1,Robin Kobbe10, Amy Marks11, Brian P. Peppers11

Robert W. Hostoffer11, Jennifer M. Puck12, Richard Zimmermann3 and Bodo Grimbacher1,13

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HUMORAL DISORDERS

Most common primary immunodeficiency

Typically a defect of antibody production

Defects most typically found in maturation and signaling

We describe the first defect involving plasma cells directly

PLASMA CELLS

Terminal differentiation of B cells

Secret Immunoglobulin

Require endoplasmic reticulum for unfolding and processing of immunoglobulin prior to secretion.

In the ER of plasma cells, pores are used to transfer these immunoglobulin , other proteins and calcium in and out.

One of these pore types is called SEC61 aka translocon

SEC61 has both an alpha and gamma subunit

Each of these subnits have isoforms labeled 1 and etc.

SEC61A1 is located on chromosome 3

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Patient 1

Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10

Patient 0

?

Hypogam/CVID - Hypogam/CVID +

Figure 1: Family Tree

? ?

Patient 11 Patient 12

Figure 2: Individual Illnesses and Ages

Infant: 0-2 years, Childhood: 3-12 years, Adolescent: 13-18 years, Adulthood: 18+ years, RSV: Respiratory Syncytial Virus,

URI: Upper Respiratory Infection, TM: Tympanic membrane

Table 1: Presenting Immunoglobulin Levels, Pneumococcal Response and Tetanus antitoxoid IgG

Patient # IgG mg/dL IgA mg/dL IgM mg/dL Pneumococcal response

Tetanus antitoxoid IgG Ab

1 504 L* 39 L* 33 L* No response 1.34 Protective 3** 771 61 34 L* No response 1.16 Protective 4 693L* 58 L* 14 L* No response N/A 5 468 L* 27 L* 26 L* No response 0.29 Intermediate 6 634 L* 22 L* 22 L* No response N/A 7 417 L* 37 L* 27 L* No response N/A

8 421 L* 31 L* 12 L* No response 0.16 Intermediate

9 204 L* 8 L* 13 L* No response 0.34 Intermediate 10 159 L* <7 L* <10 L* No response*** <0.10 Undetectable

L* Lab values are reported low per individual laboratory standard ranges. **Patient 3 original levels in 2000 were all low: 430/23/9 (IgG/A/M mg/dL) *** Patient 10 was not tested in 2004, later tested in 2011

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Table 2: Individual Immunodeficiency Profiles

Patient 1 3 5 7 8 9 10

CD3% 68 77 68 77 73 65 83 CD3abs 0.605 L* 1.609 1.462 1.694 1.57 1.294 1.992

CD3 + CD4% 27 L* 42 38 49 41 36 49 CD3 + CD4abs 0.240 L* 0.878 0.817 1.078 0.882 0.716 1.176 CD3 + CD8% 39 H* 31 25 27 30 24 31

CD3 + CD8abs 0.347 0.648 0.538 0.594 0.645 0.478 0.744 CD4:CD8 0.69 L* 1.35 1.5 1.8 1.4 1.5 1.6 CD19% 15 17 14 12 13 21 11

CD19abs 0.134 0.355 0.301 0.264 0.28 0.418 0.264 CD16/56 17 6 17 11 13 11 5

CD16/56 abs 0.151 0.125 0.366 0.242 0.28 0.219 0.12 L* or Lab values are reported either low or high per individual laboratory standard ranges.

SEC61 MUTATIONS ASSOCIATED WITH DISEASE

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SEC61A1 MUTATION

Humoral deficiencies are the most common primary immunodeficiencies

Humoral defects are those primarily involved with maturation and signaling

SEC61A1 mutation is the first defects that involves plasma cell function and sustainability.

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CONCLUSIONS

Primary Immunodeficiencies were initially thought to be rare

In reality all of us are associated with a small defect in our immune system

It is just a matter of the right organism at the right time that will make the deficiency obvious.