Fetal-onset IPEX: Report of two families and review of literature

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ava i l ab l e a t www.sc i enced i r ec t . com

C l i n i ca l Immuno logy

www.e l sev i e r . com/ l o ca t e / y c l im

Clinical Immunology (2014) xx, xxx–xxx

YCLIM-07389; No. of pages: 10; 4C:

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Fetal-onset IPEX: Report of two families andreview of literature

OMariana Moraes Xavier-da-Silva a, Carlos A. Moreira-Filhob, Edson Suzuki c,Francy Patriciod, Antonio Coutinhoe, Magda Carneiro-Sampaiob,⁎
O
PRa Universidade Estadual de Londrina (UEL), Londrina, PR, Brazil

b Department of Pediatrics, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, SP, Brazilc Faculdade de Medicina Estadual de Marilia (FAMEMA), Marília, SP, Brazild Dept of Pathology, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brazile Instituto Gulbenkian de Ciência, Oeiras, Portugal

DReceived 8 August 2014; accepted with revision 18 December 2014

C

⁎ Corresponding author at: Instituto dada FMUSP, Av. Dr. Enéas Carvalho de AguSP, Brazil.

E-mail address: [email protected] (

http://dx.doi.org/10.1016/j.clim.2011521-6616/© 2014 Published by Elsevi

Please cite this article as: M.M. Xavier-http://dx.doi.org/10.1016/j.clim.201

E

ECTKEYWORDS

Fetal IPEX;Primary immunodeficiency;FOXP3;Type I diabetes mellitus;Miscarriages;Fetal hydrops

Abstract Early-life autoimmunity is an IPEX characteristic, however intrauterine forms hadnot yet been described. Here, two unrelated families with clear evidence of fetal-onset IPEX arereported. One had 5 miscarriages of males in two generations, and a newborn presenting type-1diabetes mellitus immediately after birth, diarrhea, thrombocytopenia, eczematous dermatitis,eosinophilia, high IgE levels and autoantibodies to pancreatic islet antigens at 4-days-old.Maternal serology was negative. He presented a FOXP3 mutation, c.1189CNT, p.Arg397Trp,previously described only in another family with IPEX at birth. The second family had severalmiscarriages of males in three consecutive generations and a novel FOXP3 c.319_320delTC

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mutation was observed in two miscarried monochorionic twin male fetuses. These twins died at21 weeks of gestation due to hydrops, and CD3+ infiltrating lymphocytes were found in theirpancreas. We demonstrate that: i) IPEX may develop in fetal life; and ii) c.1189CNT andc.319_320delTC mutations are associated with early-onset phenotype.© 2014 Published by Elsevier Inc.

Criança—Hospital das Clínicasiar, 647, 05403-900 São Paulo,

M. Carneiro-Sampaio).

4.12.007er Inc.

da-Silva, et al., Fetal-onset IPEX4.12.007

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UN1. Introduction

Immune dysregulation, polyendocrinopathy, enteropathy,x-linked (IPEX) syndrome is a rare monogenic primaryimmunodeficiency (PID), characterized by early-onset

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life-threatening multisystemic autoimmunity [1–4]. IPEXsyndrome is caused by mutation of FOXP3, mapped onXp11.23, which codifies for the forkhead box protein 3(FOXP3) [5,6]. The transcription-repressor FOXP3 was shownto play a key role in the differentiation and function ofCD4+CD25+ natural regulatory T cells (Tregs), that hadproven to be essential for the establishment and mainte-nance of natural tolerance [7–10]. In IPEX, the carriermothers are healthy, even those presenting lower Treg cellnumbers in peripheral blood [3,4].

eport of two families and review of literature, Clin. Immunol. (2014),

mailto:[email protected]

http://dx.doi.org/10.1016/j.clim.2014.12.007

http://dx.doi.org/


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2 M.M. Xavier-da-Silva et al.

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IPEX typically manifests very early in life: symptom onsetoccurs in the first year of life in 93% of the cases [4]. Part ofthe patients presents clinical features already at birth ordevelops symptoms shortly after birth, however intrauterineIPEX has not been well characterized. The most commonIPEX findings are enteropathy (around 95% of the patients),type-1 diabetes mellitus (65%), skin disease, commonlyatopic-like dermatitis (65%), sepsis or recurrent infections(37%), cytopenias (31%), and thyroiditis (20%) [4]. Failure tothrive has been described in most patients. Hyper IgE levelsand eosinophilia have been identified in almost all patientsin whom these analyses were performed. Less commonadditional features include pneumonitis, nephritis, hepati-tis, vasculitis, arthritis, myositis, bronchial asthma, alopeciaas well as lymphadenopathy and splenomegaly. Tissuelesions are caused both by autorreactive T lymphocytesand by autoantibodies [11,12]. These disorders often appearsequentially rather than simultaneously, and the affectedorgan spectrum varies substantially from patient to patient[3]. The life expectancy of patients with IPEX syndromewithout hematopoietic stem cell transplant rarely extendsbeyond infancy.

The Human Gene Mutation Database (http://www.hgmd.org/) scores 64 different mutations in FOXP3 until 2014.Database and literature reviews show that most of theaffected individuals have mutations altering the C-terminalforkhead DNA-binding domain of the protein [2,4,13].Genotype–phenotype correlations – considering diseaseseverity, age of onset, target organs – have been difficultto establish in IPEX, with suggestions that environmental andepigenetic factors might participate in determining clinicalpicture and outcome. Clinical manifestations may be highlyheterogeneous in patients with the same mutation, differingeven within the same family [14].

In this article, we describe two unrelated IPEX familieswith clear evidence of intrauterine disease onset, charac-terized by autoimmune diabetes mellitus present at birth,history of multiple miscarriages of males – some of thosecases were due to hydrops, and two miscarried twinspresented CD3+ infiltrating lymphocytes in the pancreas –prematurity and restriction of intrauterine growth. Aliterature review was conducted in search of independentobservations of fetal-onset IPEX.
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2.1. Study subjects

2.1.1. Family 1The proband was a male infant, born after 36 weeks ofgestation, without a known cause for prematurity, in goodvital conditions, but presenting intrauterine growth restric-tion, low weight of 2160 g (Z score b −3) and length of46 cm (Z score b −2) [15], light meconium amniotic fluid,and a very thin umbilical cord. Hyperglycemia was observedat the first hour of life: shortly after birth, after receivingmaternal milk, his capillary glycemia was 130 mg/dL,170 mg/dL after 1 h, and reaching 300 mg/dL within 6 h.Although receiving intravenous insulin infusion in theneonatal intensive care unit, glycemia remained difficultto control varying in a range of 30 to 480 mg/dL. Anti-GAD

Please cite this article as: M.M. Xavier-da-Silva, et al., Fetal-onset IPEX: Rhttp://dx.doi.org/10.1016/j.clim.2014.12.007

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and anti-islet (ICA) antibodies were positive, with lowpeptide C at the 4th day of life, whereas maternal serumwas negative for both analyses. Immediately after birth healso presented tachydyspnea, and needed mechanicalventilation for 50 days due to respiratory distress. He alsohad feeding intolerance since the first hours of life, andevolved to watery diarrhea, sometimes with melena feces.Generalized pruritic desquamating dermatitis was observedsince the first days of life. He presented neonatal sepsis andwas treated with broad spectrum antibiotics and anti-fungaldrugs. He also had recurrent respiratory tract infections.Microbiological studies found Staphylococcus aureus (isolat-ed from a blood sample), Klebsiella pneumoniae (isolatedfrom urine, and probably a nosocomial infection) andCandida sp. (isolated from oral and rectal swabs). Labora-tory findings showed: hemoglobin level of 7.1 g/L (positiveCoombs test), thrombocytopenia (4000 platelets/mm3),leukocytosis of 18,000 cells/mm3 with 40% lymphocytes, CD4+cells: 874/mm3 (normal: 1580–4850) and CD8+: 206/mm3

(normal: 680–2470), eosinophilia (34%); albumin level of2.5 mg/dL; IgE N 2000 IU/mL (normal ≤ 17 IU/mL) and IgA,IgG and IgM were within normal range for age; normal thyroidfunction. Hewas dependent on red cell transfusions tomaintainan adequate hemoglobin level, and had also received transfu-sions of platelets.

A presumptive diagnosis of IPEX was given, and the patientwas treated with immunosuppressors tacrolimus and cyclo-sporine. After immunosuppression, he had hepatomegaly andhepatitis due to drug toxicity with high levels of AST (480 U/L)and ALT (390 U/L), as well as gamma-GT (1250 U/L). When8 months old, the FOXP3 mutation having been confirmed, hereceived hematopoietic stem cell transplantation (bonemarrow from a matched unrelated donor), but died due tosepsis complications (lung bleeding) 7 days after transplanta-tion. Before transplantation, his weight was 3650 g.

The proband was the third child of a non-consanguineousItalian-descent family. His mother had a first baby who is ahealthy 4 year-old girl, but her second gestation ended up asa miscarriage of a male fetus with hydrops, at the 27th weekof gestation. Maternal family history revealed multiple maleinfant deaths: mother's sister had 2 miscarriages (bothmales) and the grandmother had 3 miscarriages of males(Fig. 1).

DNA sequencing analysis of FOXP3 (see the descriptionbelow) revealed that patient III-3 (Fig. 1) had mutation inexon 12. The mutation was a substitution of cytosine forthymine at nucleotide position 1189 (c.1189CNT), resultingin the substitution of arginine for tryptophan (p.Arg397Trp).The patient's mother and grandmother are heterozygouscarriers of the c.1189CNT mutation. Probably the patient'saunt, who had two miscarriages of male fetuses, may also bea heterozygous carrier. This mutation was firstly describedby Wildin et al. [6] and Levy-Lahad and Wildin [17] in two fullbrothers and their maternal half-brother, all born withdiabetes mellitus type-1, enteropathy, hypothyroidism andthrombocytopenia. The two eldest patients died at a fewweeks of age and the third, treated more promptly, diedafter 5 months.

2.1.2. Family 2The proband (IV-2 in Fig. 2) was a male infant, born at31 weeks of gestational age, without a known cause for


http://www.hgmd.org/

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Figure 1 Heredogram of the family 1. Triangle indicates miscarriage; bold P indicates the proband; P inside the circle indicatescurrent pregnancy.

3Fetal-onset IPEX: Report of two families and review of literature

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prematurity, weighting 1500 g. He had meconium in amni-otic fluid and presented untreatable diarrhea since the firsthours of life, failure to thrive and precocious sepsis. He diedafter 51 days of life due to complications of sepsis. Apresumptive diagnosis of IPEX was given only after death.

The proband's mother had a previous male baby, also bornwith 31 weeks of gestational age, and with approximately1500 g, and also with meconium in amniotic fluid. He diedafter 2 h of life without a known cause. After these twopreterm babies, she got pregnant of two monochorionicmale twins and had a miscarriage at 21 weeks of gestation.Each of the fetuses weighted around 290 g and bothpresented hydrops with congestive heart failure (no congen-ital cardiac anomaly was detected). Necropsy revealedgeneralized edema, ascites, bilateral pleural effusions, andmyocardial lesions (edema in the myocardium with areas ofischemic necrosis of myocytes), besides lesions of anoxia.The pathological findings were quite similar in the twofetuses. The skin was extremely pale. No abnormalities werefound in the gastrointestinal tract. The liver had markeddegree of chronic passive congestion with zone 3 necrosis,hemorrhagic areas, and diffuse foci of hematopoiesis. Adiffuse CD3+ lymphocytic interstitial infiltration, containingCD4+ and CD8+ cells, was observed in the pancreaticparenchyma (Figs. 3A, B and C). The spleen presentedintense hematopoiesis with white pulp depletion. Thymicarchitecture and cellularity in HE staining preparations were

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Figure 2 Heredogram of the family 2. Triangle indicates miscarricurrent pregnancy.


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The proband's family was non-consanguineous and alsohad an Italian-descent. Maternal familial history revealedseveral miscarriages of males in three consecutive genera-tions (Fig. 2).

FOXP3 sequencing analysis revealed a mutation at thebeginning of exon 4 in the twin fetuses (individuals IV-3 andIV-4 in Fig. 2): a two base pair deletion (c.319_320delTC)altering the translation frameshift (p.(Ser107/Asnfs*204))predicted by computational analysis using GeneDoc(v. 2.7.000, www.psc.edu/biomed/genedoc). The mother,maternal grandmother, the sister and a first cousin of themother (Fig. 2) are carriers of the same mutation. Thismutation has not been previously described.

The present study was approved by the institutionalethics committee (process 1059/2009).

2.2. FOXP3 gene analysis methodology

Genomic DNA was extracted from peripheral leukocytesusing standard procedures, using QIAamp DNA blood Midi Kit(Qiagen, Hilden, Germany) following manufacturer's instruc-tions. Sequences including all intron–exon boundaries of 11coding exons and the coding part of exon 12 were amplifiedby PCR using the primers and cycling temperatures described

age; bold P indicates the proband; P inside the circle indicates


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Figure 3 Necropsy/histopathological findings in the miscarried male twins (individuals IV. 3 and IV.4 in Fig. 2) carrying the FOXP3mutation c.319_320delTC. In A: pancreas with a diffuse interstitial infiltration of CD3+ lymphocytes that appear in brown(immunoperoxidase, CD3, 400X). B: CD4+ cells (400X); C: CD8+ cells (400X). In D: thymus had normal aspect considering theirgestational age (hematoxilin-eosin, 100X).


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RRECby Kobayashi et al. [16]. The sequencing reactions were

performed using the Big Dye Terminator v3.1 Cycle Sequenc-ing Kit (catalog no. 4337455, Applied Biosystems, Austin,Texas) and precipitated with the Big Dye XTerminatorPurification Kit (catalog no. 4376486, Applied Biosystems,Austin, TX) following the manufacturer's instructions. Singlestrand sequencing was carried out using standard methodson an ABI 3130xl sequencer (Applied Biosystems, Forest City,CA). Sequences were aligned using the GeneiousPro 5.1.7®software. Detection of altered sequences was performedemploying the Mutation Surveyor (version 3.10) software andthe reference FOXP3 gene sequence deposited under NCBIReference Sequence NG_007392.1.

3. Review of literature

We reviewed all articles on IPEX cases published in PubMedlisted journals (2000-14) searching evidence for diseasedevelopment in utero: miscarriages, presence of symptomsat birth, prematurity and low birth weight. We found a totalof 130 reported cases, all with identified FOXP3 mutations[1,2,5,6,11,12,14,16–67].

3.1. Miscarriages

In a recent and extensive review, Barzaghi et al. [4]mentioned that multiple spontaneous abortions in maternal


family may be found in IPEX familial histories. Yet, we couldfind only a few descriptions of miscarriages in the literature,and none of multiple miscarriages. In the large familydescribed by Powell et al. in 1982 [68], where a FOXP3mutation was later identified, there were one miscarriageand one stillbirth. Unfortunately, there is no mention to thegender of these individuals, who were not included amongthe 17 males deceased in the first years of life in that family.In the IPEX family from Morocco described by Baud et al.[21], the mother, who was a carrier of F371C mutation, had7 pregnancies, with an unexplained abortion during thefourth month of gestation, without description of gender.Wildin et al. [1] reported a child with IPEX diagnosed at13 years of age (1040GNA), whose mother experienced onemiscarriage (gender unknown). In one of the familiesreported by Harbuz et al. [54], there is one miscarriage,without mention to the gender. In other IPEX families withdetailed described pedigrees, miscarriages are not men-tioned, or even explicitly denied, in the description offamilial history [5,17,19,20,46,51].

3.2. IPEX cases with clinical manifestations at birth

Most IPEX patients are born at term after an uneventfulpregnancy, with normal weight and length, and withoutpathological features. However, considering the 96 casesfrom literature with information on “age of onset”, in 11 of



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Table 1t1:1 IPEX patients with symptoms at birth or in utero‡.t1:2t1:3 Pt Mutation Manifestations

at birth orin utero‡

Gestationalage (weeks)

Birthweight

Enteropathy T1DM Skin features Cytopenias Other manifestations Outcome Refs

t1:4 1 a c.1189CNT Eczema 32 2000 g(Zb−2)

+ + Eczema ITP Atonic gut, peritonitis,hypothyroidism

Death at19 days

[1,6,17]

t1:5 2 a c.1189CNT Cachexia, T1DM 37 1350 g(Z b−3),SIGR

+ + no ITP Polyhydramnios,peritonitis

Death at5 weeks

[1,6,17]

t1:6 3 a c.1189CNT T1DM 36 2090 g(Z b−2)

+ + na no Polyhydramnios andmeconium in amnioticfluid, infections

Death at5 months

[1,6,17]

t1:7 4 b c.1-7GNT Hyperglycemia 35 1370 g(Z b−3)

+ + no no Caesarean section dueto SIGR, hypothyroidism

Death at54 days

[29]

t1:8 5 b c.817-1GNA Diarrhea 30 na + + Rash no Copious intestinaldischargeimmediately after birth

Death at79 days

[34]

t1:9 6 b c.1045-3CNG Generalizedeczema

At term Normal + + Eczema AIHA Hypothyroidism,infections

Death at11 month

[11]

t1:10 7 c.1150GNA Watery diarrhea 34 na + + Exfoliativedermatitis

Pancytopenia Hypothyroidism, renalfailure

Death at7 weeks

[46]

t1:11 8 b c.1222GNA T1DM 36 2400 g(Z b−2)

no + no no Nephrotic syndrome Alive at15 years

[51]

t1:12 9 c.227delT T1DM 33 1590 g(Z b−2)

+ + no AIHA, ITP, AIN Hypothyroidism,infections

Death at8 months

[51]

t1:13 10 b g.560CNT Diarrhea na na + no “Skin disease” ITP Hyperthyroidism,gastritis

Death at14 months

[14]

t1:14 11 b c.1121TNG Eczema na na + no Eczema AIHA, ITP Allergy Death at8 years

[14]

t1:15 III.3 Fig 1 c.1189 CNT T1DM 36 2160 g(Z b−3)

+ + Psoriasiformdermatitis

AIHA, ITP Meconium in amnioticfluid, sepsis

Death at8 months

Thisarticle

t1:16 IV.3 Fig 2 c.319_320delTC

Hydrops‡ Miscarriageat 21 weeks

290 g na PancreaticCD3+ cellinfiltration

Pale skin Anemia Edema, ascites Miscarriage Thisarticle

t1:17 IV.4 Fig 2 c.319_320delTC

Hydrops‡ Miscarriageat 21 weeks

290 g na PancreaticCD3+ cellinfiltration

Pale skin Anemia Edema, ascites Miscarriage Thisarticle

a Patients from the same family; Z: Z score.t1:18b The only description of the mutation in literature; T1DM: type-1 diabetes mellitus; SIGR: severe intrauterine growth restriction; na: not available; AIHA: autoimmune hemolytic anemia;

ITP: immune thrombocytopenia; AIN: autoimmune neutropenia.t1:19

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these cases (11.5%), disease manifestations were alreadypresent at birth, or began in the first day of life[1,6,11,14,17,29,34,46,51]. These cases are summarized inTable 1, where the proband of the family 1 (individual III.3,Fig. 1) and the twins of family 2 (individuals IV.3 and IV.4)were also included. We deliberately did not consider casesinformed as “neonatal onset”, since we would like to makesure that IPEX manifestations started in utero.

Considering the FOXP3 mutations listed in Table 1, onlythe family reported by Levy-Lahad & Wildin (cases 1–3,Table 1) [6,17] and the family 1 here described (Fig. 1) bearthe c.1189CNT mutation. Mutations detected in patients 4,5, 6, 8, 10 and 11 (Table 1) were described only once in theliterature. It is worth to note that mutations in the forkheadDNA-binding domain of FOXP3 were present in seven out offourteen cases listed in Table 1, i.e. cases 1–3 and cases 7,8, 11 and III.3.

Regarding the type of clinical manifestation at birth –here only live births were considered, i.e. the twins ofFamily 2 were excluded – 6/12 infants presented type-1diabetes mellitus, three presented diarrhea, and three hadgeneralized eczema as the most precocious feature. In thecase reported by Heltzer et al. [34] (patient 5, Table 1),the authors highlighted that “immediately after birth, hedeveloped copious green discharge from his rectum andnasogastric tube, which evolved to secretory diarrhea”. Thepresence of meconium in amniotic fluid of two preterminfants (cases 3 and III.3, Table 1), and of polyhydramnios inpatients 2 and 3, suggests that enteropathy could havebegun in utero. Among the 10 infants with information ongestational age, 9 were preterm ones. In eight patientswhere information on birth weight was available, four casesmay be classified as having intrauterine growth restriction(patients 2, 4, 9 and 12), and two (cases 2 and 4) presented asevere restriction (Z score b −3). Thus, premature labor andrestriction of fetal growth were frequent findings in thisgroup of infants, and reinforce our proposal that IPEX mayhave its onset in intrauterine life. Eleven of 12 patientslisted in Table 1 evolved to death, 9 of them in the first yearof life.

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4. Discussion

As we have previously pointed out, the findings with IPEXpatients, together with the equivalent mouse mutant scurfy,demonstrate that FOXP3 mutations affecting Treg celldevelopment and function critically impair immune peripheraltolerance and homeostasis [9,10,69–72]. Regulatory T cells,therefore, seem to ensure the central role in physiologicaltolerance to the body tissues. Thus, to the best of ourknowledge, all other mechanisms that participate in theprocess of natural tolerance (clonal deletion/anergy, “innate”sensing of “danger”, tissue-protective mechanisms and otherregulators of inflammation, etc.) are conserved, and yet, alltogether they fail to ensure tolerance in the absence of Tregs.The severity and the presence of IPEX autoimmune manifes-tations from the very beginning of life, on the other hand,demonstrate, if necessary it was, the marked “destructive”properties of the adaptive immune system.

The observations on the IPEX families here described addto those notions, showing that the powerful autoimmune


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process leading to disease and death may have its onset inintrauterine life, such that by birth a large damage to organsand tissues may have been established. Autoimmunediseases in fetal life are, indeed, extremely rare eventsand limited, thus far, to patients with IPEX and Omennsyndrome (OS), where a few patients with disease at birthhave been described [73–75]. Both conditions arelife-threatening diseases, and should be considered aspediatric emergencies [76,77]. Hence, all patients withautoimmune manifestations at birth (or suspected cases),should be investigated for these two PIDs that are stronglyassociated with autoimmune disease, and we have thusdesignated as monogenic autoimmune disorders. It mayseem surprising that “break of tolerance” can occur whenthe immune system is still immature. Rather than being“broken”, however, tolerance was never established, as wesuggest. The elegant experiments of Ohki and Le Douarin[78] demonstrated beyond doubt that the presence of tissuesand organs along with the development of an immune systemdoes not suffice to establish tolerance. In other words,establishment of natural tolerance requires appropriatethymic selection, as we know it now, for the commitmentand differentiation of auto-reactive Tregs [79,80].

In this context, and given the current knowledge on theage-dependent development of the various lymphocyte classesand effector functions, as well as on the ability of fetuses toproduce immune responses [80,81], it is not surprising thatpathological fetal autoimmunity exists, and that there is enoughtime before birth to produce auto-antibodies and high IgElevels, or for the destruction of pancreatic beta-cells. It isnoteworthy that T lymphocytes were observed infiltrating thepancreas of twin fetuses of 21 weeks of gestational age carryinga FOXP3 mutation (Fig. 3A). Although Langerhans islets werestill preserved, this lesion could be interpreted as an infiltrationby autorreactive T lymphocytes and an early stage of type 1diabetes mellitus [11].

Given the current conviction that fetuses are sterile (anotion that has been challenged, but remains widely accepted)[82], fetal disease, on the other hand, proves that “external” or“non-self” antigens, or the immune responses to them, are notnecessary for the establishment of autoimmune disease: in theabsence of Treg cells, the developing adaptive immune systemreacts to, and eliminates self-antigens. It is perhaps moresurprising, however, that severe intestinal inflammation anddestruction occur before birth, for there are several reportsclaiming that colonization by intestinal bacterial flora isnecessary in some experimental models of inflammatorybowel disease, involving cell transfers to immunodeficienthosts [83–85]. Keeping to the classical notion that the fetalintestine is not colonized, the repeated observations of severe,untreatable diarrhea frombirth, oftenwithmelenawithin a fewhours after delivery, of meconium in the amniotic fluid ofpreterm infants, all concur to the fact that intestinalautoimmune inflammation in IPEX may start before bacterialcolonization.

Although only one tenth of all described IPEX casespresented manifestations at birth (here included in Table 1),around 45% of the cases with information about “age ofonset” presented clinical manifestations in neonatal period,suggesting that fetal-onset autoimmunity in IPEX is not anuncommon phenomenon [4]. Regarding autoimmune cyto-penias in neonatal period, for example, the production of



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autoantibodies probably had begun a couple of weeks before.On the other hand, the strong immune stimulation immedi-ately after birth – caused by the exposure to environmentalantigens – may trigger or amplify the underlying defects inimmune regulation/homeostasis, what could explain whyalmost all infants develop clinical features in the first weeks/months of life. Maternal Treg cells could play a role inregulation of the fetal immune system and thus delayingautoimmunemanifestations, either by crossing the placenta orby any other putative mechanism [80,86].

Regarding the possible relationships between the severity ofmutations and the occurrence of fetal-onset IPEX, our findingson the families here reported did not add significant under-standing to this topic, since data about the impact of these raremutations on protein expression or function are not yetavailable. However it is clear that both mutations severelyaffected gene transcription. The mutation c.1189CNT in exon12 alters the FOXP3 forkhead domain, a critical moleculardomain for suppressive Treg cell activity [72], and waspreviously described in another family also with fetal onsetand highly severe phenotype [6,17]. The novel mutationc.319_320delTC in exon 4 alters the translation frameshift, aspredicted by computational analysis.

We found no previous descriptions of multiple miscar-riages of males in IPEX families, although this kind of eventwas a relevant complaint in the two families reported in thispaper. Another distinctive feature of these families was thepresence of fetal hydrops in: i) the twins IV.3 and IV.4 infamily 2, in whom a FOXP3 mutation was identified, andii) individual III.2 in family I, presumptively affected by theFOXP3 mutation found in his mother and brother. IPEX hasnot yet been mentioned as a cause of fetal hydrops [87],however, in the cases here described, it is reasonable tocorrelate the anatomopathological findings with the pres-ence of FOXP3 mutations. Considering that hemolyticanemia is a well-known cause of hydrops and also a frequentIPEX manifestation [4], even in precocious cases, one canhypothesize that fetuses that develop autoimmune hemo-lytic anemia would evolve to hydrops and do not survive.Therefore, IPEX should be considered a cause of recurrentmiscarriages of male fetuses and of fetal hydrops.

In conclusion, the findings in the two families heredescribed, together with the 11 cases of IPEX at birthalready reported in literature, clearly show that autoim-mune aggression may occur already in utero, in a conditionwhere fetal functioning of natural Treg cells is severelyimpaired. Fetal-onset IPEX could represent a particularsubgroup of this disease, and certainly constitutes its mostsevere phenotype.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors are thankful to Silvia Yumi Bando, PhD, andFernanda B. Bertonha, PhD for skillful technical assistance inidentifying the FOXP3 mutations and help in revising themanuscript, to M. Claudia Zerbini, MD and Priscila PizzoCrêm dos Santos, for performing immunohistochemistry


techniques, to Maria Teresa Seixas, MD, for the micropho-tographs showed in Fig. 3, and to all medical doctorsinvolved with the assistance and study of these families,particularly Dr. Diogo Soares, for his valuable help withfamily history, and to FAPESP (Fundação de Amparo àPesquisa do Estado de São Paulo) for financial support (grant2008/58.238-4).

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