Mutation in ITCH Gene Can CauseSyndromic Multisystem AutoimmuneDisease With Acute Liver FailureNicola Kleine-Eggebrecht, MD,a Christian Staufner, MD,b Simone Kathemann, MD,a Magdeldin Elgizouli, MD,c

Robert Kopajtich, PhD,d Holger Prokisch, PhD,d Elke Lainka, MDa

abstractPediatric intractable autoimmune hepatitis is rare and may be responsible foracute liver failure. Mutations in the itchy E3 ubiquitin protein ligase (ITCH)gene (located on chromosome 20q11.22) can lead to a deficiency of theencoded protein, resulting in increased T-cell activity with lack of immunetolerance and manifestation of a complex systemic autoimmune disease. A 1-year-old girl of consanguineous parents received a liver transplant (LT)because of acute liver failure attributed to a drug-induced hypereosinophilicsyndrome with positive liver-kidney-mikrosome-2 antibodies. Notablefindings were syndromic features, dystrophy, short stature, psychomotorretardation, and muscular hypotonia. Later, we saw corticosteroid-sensitiverejections as well as a systemic autoimmune disease with detection of specificantibodies (de novo autoimmune hepatitis, thyroiditis with exophthalmos,diabetes mellitus type 1, and immune neutropenia). Histologically, livercirrhosis with lobular inflammatory infiltrates, giant-cell hepatitis, andductopenia was verified in chronic cholestasis. Shortly after a second LT,a comparable liver histology could be detected, and viral, bacterial, andmycotic infections deteriorated the general health condition. Because ofrefractory pancytopenia related to portal hypertension and hypersplenism,a posttransplant lymphoproliferative disorder was excluded. One year afterthe second LT, epidural and subdural bleeding occurred. Three monthsafterward, the girl died of sepsis. Postmortem, whole-exome sequencingrevealed a homozygous mutation in the ITCH gene. A biallelic mutation inITCH can cause a severe syndromic multisystem autoimmune disease with theabove phenotypic characteristics and acute liver failure because ofautoimmune hepatitis. This case reveals the importance of ubiquitinpathways for regulation of the immune system.

The immune system is regulated andinfluenced by various factors. Animportant mechanism is theubiquitination of proteins and theassociated proteasomal degradation.1,2

Ubiquitin is a small protein present inall eukaryotic cells that is involved inprotein quality control, transduction ofsignals, and cell cycle control.Ubiquitination of T-cell receptorsmediates their downregulation by

altering signaling components; thus,interleukin -2 production and T-cellproliferation are inhibited.3,4 Thesemechanisms promote immunologictolerance and T-cell anergy.5

The regulation of ubiquitination isconducted stringently via substrate-specific E3 ubiquitin ligases, one ofwhich is encoded by the itchy E3ubiquitin protein ligase (ITCH) gene

Departments of aPediatrics, Gastroenterology, andHepatology and cHuman Genetics, University of Duisburg-Essen, Essen, Germany; bDepartment of Pediatrics,University of Heidelberg, Heidelberg, Germany; anddInstitute of Human Genetics, Klinikum rechts der Isar,Technical University Munich, Munich, Germany

Dr Kleine-Eggebrecht designed the study and draftedthe initial manuscript; Dr Staufner was responsiblefor the genetic diagnosis, interpretation of geneticfindings, and critical revision of the manuscript; DrKathemann was responsible for the medicaltreatment, was involved in diagnostics, and revisedthe manuscript; Dr Elgizouli counseled the familyand critically revised the manuscript; Dr Kopajtichconfirmed the splicing effect in fibroblasts andcritically revised the manuscript; Dr Prokisch wasresponsible for the genetic analysis andinterpretation of the results; Dr Lainka wasresponsible for medical care, drafted the initialmanuscript, and critically revised the manuscript;and all authors read and approved the finalmanuscript as submitted and agree to beaccountable for all aspects of the work.

DOI: https://doi.org/10.1542/peds.2018-1554

Accepted for publication Oct 8, 2018

Address correspondence to Elke Lainka, MD,Department of Pediatrics, University of Duisburg-Essen, Hufelandstrasse 55, 45147 Essen, Germany.E-mail: elke.lainka@uk-essen.de

PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online,1098-4275).

Copyright © 2019 by the American Academy ofPediatrics

FINANCIAL DISCLOSURE: The authors have indicatedthey have no financial relationships relevant to thisarticle to disclose.

FUNDING: This work was supported by the DietmarHopp Foundation (23011235 to Dr Staufner).

To cite: Kleine-Eggebrecht N, Staufner C,Kathemann S, et al. Mutation in ITCH Gene CanCause Syndromic Multisystem AutoimmuneDisease With Acute Liver Failure. Pediatrics.2019;143(2):e20181554

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(Online Mendelian Inheritance in Man[OMIM] entry *606409; chromosome20q11.22). Biallelic mutations in theITCH gene can cause severedeficiency of the respective E3ubiquitin ligase. This leads toincreased T-cell activity with a lack ofimmune tolerance in mousemodels.6–8 Lohr et al9 demonstratedfor the first time that human ITCH E3deficiency can cause a syndromicmultisystem autoimmune disease(SMAD) in humans, which includesmajor phenotypic features (OMIMentry #613385).

CASE REPORT

A 1-year-old girl of consanguineousparents received a living-donor livertransplant (LT) from her motherbecause of acute liver failure, whichwas suspected to be caused by drug-induced hypereosinophilic syndromewith positive liver-kidney-mikrosome-2 (LKM-2) antibodies.The liver histology of the explantedliver revealed autoimmune hepatitiswith giant-cell hepatitis andaccompanying cholangitis (Fig 1).Previously, there had been a regularingestion of amoxicillin because ofrespiratory infections. Notable wasalso dystrophy (body weight belowthe third percentile; BMI 14.2), shortstature (body height below the thirdpercentile), choanal atresia,

psychomotor retardation, andmuscular hypotonia as well ascharacteristic craniofacial features,such as frontal bossing, prominenteyes, orbital ptosis, and small chin(Fig 2). She carried a 120 bpmicroduplication in chromosomalregion 22q12.3 (detected ona comparative genomic hybridizationmicroarray), which she inheritedfrom a healthy father of no clinicalsignificance. After 8 weeks, the firstcorticosteroid-sensitive graftrejection reaction was observed. After

successful treatment withcorticosteroids, the patient developedan alloimmune eosinophilic liverdisease again with elevated liverenzymes. We changed theimmunosuppressive medicationbasiliximab and cyclosporine A totacrolimus and mycophenolatemofetil and administered high dosesof steroids. Two years after the LT,complete liver cirrhosis with lobularinflammatory infiltrates, giant-cellhepatitis, and ductopenia was verifiedin chronic cholestasis. Because of the

FIGURE 1Liver histology of the explanted liver (scale bar,100 µm): giant-cell hepatitis with infiltration oflymphocytes and plasma cells, which is com-patible with autoimmune hepatitis and ac-companying cholangitis with reactiveproliferation of bile ducts (by kind permissionof Professor H.A. Baba, Institute of Pathology,University of Duisburg-Essen, Essen, Germany).

FIGURE 2Picture of our index patient showing ptosis, small chin, and frontal bossing (used with family’sconsent).

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persistence of abnormal liverfunction, the patient received 3 cyclesof rituximab, which had a positive buttemporary effect on liverinflammation. In the meantime, shedeveloped a systemic autoimmunedisease with detection of specificantibodies that affected differentorgan systems (de novo autoimmunehepatitis with donor specific auto-antibodies, Hashimoto thyroiditis, anddiabetes mellitus type 1). Aftera second LT 2 years later,a comparable liver histology could bedetected. Infectious complicationswere adenovirus hepatitis,methicillin-resistant Staphylococcus

aureus, and Escherichia coli sepsis.The girl also suffered fromenteropathy with chronic diarrheaand recurring pulmonal infections,including pulmonary aspergillosis. Atthe age of 5 years, refractorypancytopenia with portalhypertension and hypersplenism wasdiagnosed. A posttransplantlymphoproliferative disease wasexcluded by bone marrow biopsy. Theprogressive pancytopenianecessitated regular transfusions oferythrocyte and thrombocyteconcentrates and substitution ofimmunoglobulin. Because of themultisystem autoimmune features,

we considered a bone marrowtransplant, but liver failure and, later,multiple organ failures limited thetreatment options over time, anda bone marrow transplant was notrealizable. Five months afterward,epidural and subdural bleedingoccurred during thrombocytopeniafollowed by craniotomy andanticonvulsive therapy for seizures ofpartial onset. Three months later, thegirl died of sepsis at home. Withina multicenter study on genetic causesof indeterminate acute liver failurelead by the Children’s HospitalHeidelberg and the Institute ofHuman Genetics of the Technical

FIGURE 3A, Genetic results: visualization of the sequence data through the Integrative Genomics Viewer. The ITCH variant was detected in the index patient inhomozygous form; the parents are heterozygous carriers. This variant is predicted to interfere with the splice acceptor site of exon 18. B, Functionalvalidation: polymerase chain reaction amplification of an ITCH complementary DNA fragment (ranging from exon 15 to exon 19) yielded a shorter productin the patient compared with a control sample. C, Sanger sequencing is used to confirm the loss of the splice acceptor, which leads to skipping ofexon 18.

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University of Munich, trio whole-exome sequencing was performed ongenomic DNA of the patient and herparents. Postmortem, a homozygoussplice mutation in the ITCH gene(NM_001257137.1, c.1693-1G.A)was identified, predicted to causea loss of the splice acceptor site ofexon 18, and rated as likelypathogenic. This homozygousmutation was confirmed by Sangersequencing, and a segregationanalysis revealed that both parentswere heterozygous. A real-timepolymerase chain reaction analysis ofa patient fibroblast ITCHcomplementary DNA fragmentcovering exons 15 to 19 revealeda shorter amplicon, indicative ofa splice defect. Sanger sequencingwas used to confirm that the spliceacceptor of exon 19 was used insteadof the canonical splice acceptor ofexon 18, resulting in a skipping ofexon 18 (Fig 3).

DISCUSSION

Through different signaling pathways,ubiquitination plays an importantrole in immune regulation andcontrols T-cell responsiveness.1,6 InItch2/2 mice, elevated levels ofcirculating immunoglobulins, anti-nuclear antibodies, and lymphocytescould be verified, and several studiesrevealed that mouse mutations in Itchcan lead to fatal autoimmune diseaseswith histiocyte and lymphocyteinfiltration of different organsystems.10,11

The human phenotype of ITCHdeficiency is SMAD with morphologicand developmental abnormalities. Itis based on 10 Old Order Amishchildren of consanguineous parentswho were all homozygous fora truncating mutation in ITCH (OMIMentry 606409). A sequence analysis ofITCH revealed a homozygous singlebase pair insertion in exon 6(c.394_395insA). This frameshiftmutation was predicted to truncateITCH at amino acid position 139. All

10 patients had a similar phenotype,comparable to our case. Interestingly,participation of the immune systemwas found in only 60% of patients(Table 1). Diabetes mellitus,autoimmune hepatitis, andenteropathy with detection ofperinuclear anti-neutrophilcytoplasmic antibodies, smooth-muscle antibodies, anti-enterocyteantibodies, and a lymphocyticinflammation of the gastrointestinaltract were identified in just 10% to30% of the children.9 Our indexpatient was suffering fromautoimmune diseases in severalorgans with partial detection ofantibodies: chronic lung disease,enteropathy, diabetes mellitus(elevated levels of islet-cellantibodies, glutamic aciddecarboxylase antibodies, and insulinauto-antibodies), autoimmunehepatitis (LKM-2 antibodies), andhypothyroidism (thyroid peroxidaseantibodies). In contrast to thepatients described by Lohr et al,9 whohad repeatedly normal blood cellcounts, our patient developedprogressive pancytopenia associatedwith hypersplenism and portalhypertension caused by livercirrhosis. Additionally, we describethe first case of ITCH deficiency inhumans with autoimmune hepatitisthat progressed to acute liver failurerequiring a LT. It may be speculatedwhether an inappropriate activation

of T cells due to ITCH deficiency wasthe only reason for the severe livercomplications seen in our patient.Results of oncological, hepatological,and immunologic research areneeded to understand thepathophysiology of ITCH deficiencyand may inspire novel therapeuticoptions targeting the ubiquitinationsystem.12

The fact that our patient was muchmore affected suggests that there mayhave been additional etiologic factorsor genetic predispositionscompromising, for instance, thefunction of other ubiquitin ligases.9

There are no functional tests availablethat can help to elucidate thecausality between this newhomozygous mutation in ITCH andthe described (liver) phenotype.Special assays will be needed fordetermining the fate of ubiquitinatedsubstrates (such as Jun proto-oncogene, AP-1 transcription factorsubunit in the cell) and for studyingdownstream signaling pathways, suchas those involving NOTCH.9 Besides,the reasons for pediatric acute liverfailure are diverse. Otherimmunologic and genetic diseasesassociated with autoimmune hepatitisare known (Table 2). In children, 30%to 50% of acute liver failures remainindeterminate despite detaileddiagnostic workup. Genetic diseasesare increasingly considered to be

TABLE 1 Clinical Characteristics Seen in Our Girl and 10 Index Patients From Lohr et al

Similarities, n = 11 Differences, n = 1

Mean age of first symptoms (4.2 y) PancytopeniaMacrocephaly (90%) Liver cirrhosis with acute liver failureTypical craniofacial features (100%) —

Hepatomegaly and/or splenomegaly (90%) —

Developmental delay (100%) —

Muscular hypotonia (60%) —

Short stature (100%) —

Psychomotor delay (100%) —

Chronic lung disease (90%) —

Autoimmune hepatitis (LKM-2 antibodies) (30%) —

Enteropathy (20%) —

Hypothyroidism (40%) —

Diabetes mellitus type 1 (10%) —

From Lohr NJ, Molleston JP, Strauss KA, et al. Human ITCH E3 ubiquitin ligase deficiency causes syndromic multisystemautoimmune disease. Am J Hum Genet. 2010;86(3):447–453. —, not applicable.

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causative for some of these cases ofindeterminate acute liver failure.13–15

On the basis of the current report, wepropose ITCH deficiency to bea notable differential diagnosis inchildren with SMAD, which caninclude autoimmune hepatitis andpossibly acute liver failure.

Burt et al16 first considered thepossibility of a bone marrowtransplant in patients with severe

autoimmune diseases, whichcollectively form the most rapidlyexpanding indication for stem celltransplantation. We decided againsta bone marrow transplant andsplenectomy because our patient wastoo sick to undergo theseinterventions. Especially, liverfunction was deteriorated to anextent that a bone marrowtransplant was not a realistictherapeutic option.

CONCLUSIONS

It should be pointed out that this isonly the second mutation of ITCHdocumented in humans, apart fromthe 1 variant described by Lohr et al9

in 2010. This case underscores therelevance of mutations in the ITCHgene for the clinical courses andtreatment of children suffering fromcomplex autoimmune diseases. Itsupports the assumption that geneticdiseases can be causative not only forautoimmune hepatitis but also forsome of the indeterminate cases of

acute liver failure. Further researchand functional assays will be neededto detect these children at an earlyage before impairment of liverfunction necessitates an LT orprogressive damage to multiple organsystems makes curative therapeuticoptions, such as a bone marrowtransplant, impossible.

ACKNOWLEDGMENT

Dr Lainka gratefully acknowledgesthe comments made by OliverWeiergräber (ForschungszentrumJülich).

ABBREVIATIONS

ITCH: itchy E3 ubiquitin proteinligase

LKM-2: liver-kidney-mikrosome-2LT: liver transplantOMIM: Online Mendelian

Inheritance in ManSMAD: syndromic multisystem

autoimmune disease

POTENTIAL CONFLICT OF INTEREST: The authors have indicated they have no potential conflicts of interest to disclose.

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TABLE 2 Autoimmune Hepatitis–AssociatedDiseases in Pediatric Patients

ImmunologicDiseases

Hereditary Diseases

IBD APECED, AIRE geneThyreoditis IPEX, FOXP3 geneVitiligo Monosomy 22q13

syndromeDiabetes mellitustype 1

FHL, FHL 1–5 types

Nephrotic syndrome SMAD, ITCH deficiency

Positive family anamnesis for autoimmune diseasesknown in 40% of patients; differential diagnosis as drug-induced liver injury. APECED, autoimmune poly-endocrinopathy-candidiasis-ectodermal dystrophy; FHL,familial hemophagocytic lymphohistiocytosis; IBD, in-flammatory bowel disease; IPEX, immune dysregulation,polyendocrinopathy, enteropathy, X-linked syndrome.

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16. Burt RK, Slavin S, Burns WH, MarmontAM. Induction of tolerance inautoimmune diseases by hematopoieticstem cell transplantation: getting closerto a cure? Blood. 2002;99(3):768–784

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DOI: 10.1542/peds.2018-1554 originally published online January 31, 2019; 2019;143;Pediatrics 

Elgizouli, Robert Kopajtich, Holger Prokisch and Elke LainkaNicola Kleine-Eggebrecht, Christian Staufner, Simone Kathemann, Magdeldin

Disease With Acute Liver Failure Gene Can Cause Syndromic Multisystem AutoimmuneITCHMutation in

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DOI: 10.1542/peds.2018-1554 originally published online January 31, 2019; 2019;143;Pediatrics 

Elgizouli, Robert Kopajtich, Holger Prokisch and Elke LainkaNicola Kleine-Eggebrecht, Christian Staufner, Simone Kathemann, Magdeldin

Disease With Acute Liver Failure Gene Can Cause Syndromic Multisystem AutoimmuneITCHMutation in

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