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ELEONORA GAMBINERI, MD UNIVERSITY of FLORENCE DEPARTMENT of PEDIATRICS “A. MEYER” CHILDREN’S HOSPITAL FLORENCE, ITALY. ESID Prague Spring Meeting May 14 and 15, 2007 Prague. A FAMILY AFFECTED BY ATYPICAL IPEX SYNDROME .

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ELEONORA GAMBINERI, MD

UNIVERSITY of FLORENCE

DEPARTMENT of PEDIATRICS

“A. MEYER” CHILDREN’S HOSPITAL

FLORENCE, ITALY

ESID Prague Spring Meeting

May 14 and 15, 2007

Prague

A FAMILY AFFECTED BY

ATYPICAL IPEX SYNDROME

slide2

ImmunedysregulationPolyendocrinopathy EnteropathyX-linked

IPEX

A rare genetic disorder of immune regulation characterized by overwhelming systemic autoimmunity

It is caused by mutations in the FOXP3 gene , located on X-chromosome, key molecular factor driving T cell tolerance and crucially important for CD4+CD25+ regulatory T cell development and function

what does foxp3 do

IPEX

FOXP3

What does FOXP3 do?
  • Plays an essential role in the development
  • and function of CD4+CD25+ Regulatory
  • T cells
  • Hori S, et al., Science 2003
  • Khattri R, et al., Nat. Immunol. 2003
  • Fontenot JD, et al., Nat. Immunol. 2003
  • FOXP3 may function as a transcriptional
  • repressor of cytokine promoters
  • Schubert L, et al., J. Biol. Chem. 2001
  • Rheostat of the immune response
  • Khattri R, et al., J. Immunol. 2001
slide4

2

N

C

LEUCINE

ZIPPER

PROLINE-RICH DOMAIN

ZINC-FINGER

FORKHEAD DOMAIN

IPEX

FOXP3

cd4 cd25 regulatory t cells

REGULATORY T CELLS

IPEX

CD4+CD25+ Regulatory T Cells

Make up 5-10% of the normal CD4+ T cell population.

Characterized by expression of CD4 and CD25bright at baseline and of intracytoplasmatic expression of FOXP3. They are positive for Cytotoxic T Lymphocyte associated Antigen-4 (CTLA-4) e Glucocorticoid-Induced Tumor-necrosis factor receptor-Related protein (GITR); recently, it has been reported that they have low expression of CD127.

Require activation and cell contact to repress proliferation of other T cells but do not appear to proliferate themselves after activation.

slide6

IPEX

CLINICAL & LABORATORY FEATURES

BASIC Clinical & Laboratory Features:

Other Clinical & Laboratory Features:

slide7

F373A

Bacchetta et al 2006

ATG>ATA Bacchetta et al 2006

ATG>AGG Myers AK et al 2006

-76fsX108

Owen CJ et al 2003

del-6247_-4859

Torgerson TR et al 2007

IVS9+4G>A

Chatila TA et al 2000

T108M & 454+4A>G

De Benedetti F et al 2006

Locations of FOXP3 mutations reported

IPEX

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

slide9

CLINICAL CASE

IPEX

Male born to healthy unrelated parents after 37 wks of pregnancy, complicated with diabetes and IUGR.

At birth, weight was 2580g. The neonatal period was unremarkable.

At age of 1 month he was admitted to our hospital for vomiting and severe bloody diarrhea

Lab evaluation resulted negative for common GI infections, total protein were low (3.6 g/dl) and anemia was present (Hb 10g/dl)

Severe and intractable diarrhea persisted and because of his progressive clinical deterioration total parenteral nutrition as well as treatments of support were started.

Intestinal biopsies showed histological findings compatible with autoimmune enteropathy

Clinical improvement was slowly obtained when immunosuppressive therapy was started (high-dose intravenous corticosteroids combined to oral cyclosporin)

Although absence of any endocrine dysfunction and skin lesions, due to life threatening enteropathy, a diagnostic procedure for IPEX syndrome was considered.

Unfortunately the patient died prematurely at age of 5 month while he was on immunosuppression

slide10

Molecular analysis of FOXP3

Ex5

Ex5

IPEX

Wild Type

543 C>T

Patient

Bennet C.et al. Curr Opin Pediatr, 2001

Gambineri E.et al.Curr Opin Rheumatol, 2003

slide11

3%

7.8%

9.9%

CD4+

CD25+

2.7%

CD4+ gated

CD25+

FOXP3+

FOXP3 PROTEIN EXPRESSION by Flow Cytometry

IPEX

Proband

Normal donor

slide12

FAMILY SCREENING

?

?

IPEX

I

1

2

II

1

2

3

4

5

6

7

III

1

2

3

4

5

6

Age 58

Chronic diarrhea since infancy

Age 45

No symptoms reported

IV

1

2

3

4

5

6

7

Age 24

Gilbert Syndrome

Mild eczema

Ovaric Polycistosis

V

Proband

slide13

FAMILY SCREENING

3%

Normal

IV-1

III-3

III-1

IV-2

IV-7

CD4+

2%

2.1%

2.5%

1.6%

1.5%

CD25+

2.3%

CD25+

2,5%

1,8%

2.3%

1.7%

1.7%

FOXP3+

2.7%

IPEX

FOXP3 PROTEIN EXPRESSION

by Flow Cytometry

CD4+ gated

slide14

ESEfinder

Wild Type

543 C>T

SF2/ASF

SC35

SRp

4

0

SRp

5

5

IPEX

Disruption of exonic splicing enhancer (ESE) motifs and modification of SR protein binding sites

in mutated of FOXP3

slide15

III-3

Neg

ND

1

2

3

4

5

6

7

8

9

10

11

F2R1

F2R1

F2R3

1400bp

F3R2

F4R1

F6R1

F2R3

FL

  • Lacks full-length isoform

447bp

D2

F3R2

540bp

  • No amplification of fragments

F3R2 and F4R1

F4R1

520bp

F6R1

747bp

IPEX

FOXP3 cDNA

FOXP3 cDNA fragments @ PCR amplification

cDNA

  • Lower cDNA expression
slide16

Conclusion & Future Perspectives

IPEX

  • In the majority of cases IPEX is a severe disease. Patients develop symptoms early in infancy and most die within the first 2 years of life. Milder and late-onset forms of the disease have been described.
  • We found a family harboring a silent mutation within a splice site that do not alter protein expression but seems to affect mRNA transcription. Although only 2 members, among those with mutated FOXP3, manifest an incomplete form of IPEX with different disease severity.

IS IPEX UNDERESTIMATED OR MISDIAGNOSED?

IS REALLY FOXP3 RESPONSIBLE FOR THE DISEASE?

WHAT IS MODULATING THE PHENOTYPE VARIABILITY?

slide17

Acknowledgments

IPEX

University of Florence, Department of Pediatrics

“A. MEYER” Children’s Hospital - Florence, Italy

Immunology Lab: Gastroenterology Unit:

Lucia Bianchi, PhD Paolo Lionetti,MD

Sara Ciullini Mannurita, BS Francesca Bronzini,MD

Anna Maria Grazia Gelli, PhD

Chiara Azzari, MD, PhD

Perroni Lucia - Genoa

Tommasini Alberto - Trieste

Badolato Raffaele - Brescia

Bacchetta Rosa, Roncarolo Maria Grazia - Milan

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