Pediatric intractable autoimmune hepatitis is rare and may be responsible for acute liver failure. Mutations in the itchy E3 ubiquitin protein ligase (ITCH) gene (located on chromosome 20q11.22) can lead to a deficiency of the encoded protein, resulting in increased T-cell activity with lack of immune tolerance 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 hypereosinophilic syndrome with positive liver-kidney-mikrosome-2 antibodies. Notable findings were syndromic features, dystrophy, short stature, psychomotor retardation, and muscular hypotonia. Later, we saw corticosteroid-sensitive rejections as well as a systemic autoimmune disease with detection of specific antibodies (de novo autoimmune hepatitis, thyroiditis with exophthalmos, diabetes mellitus type 1, and immune neutropenia). Histologically, liver cirrhosis with lobular inflammatory infiltrates, giant-cell hepatitis, and ductopenia was verified in chronic cholestasis. Shortly after a second LT, a comparable liver histology could be detected, and viral, bacterial, and mycotic infections deteriorated the general health condition. Because of refractory pancytopenia related to portal hypertension and hypersplenism, a posttransplant lymphoproliferative disorder was excluded. One year after the second LT, epidural and subdural bleeding occurred. Three months afterward, the girl died of sepsis. Postmortem, whole-exome sequencing revealed a homozygous mutation in the ITCH gene. A biallelic mutation in ITCH can cause a severe syndromic multisystem autoimmune disease with the above phenotypic characteristics and acute liver failure because of autoimmune hepatitis. This case reveals the importance of ubiquitin pathways for regulation of the immune system.

The immune system is regulated and influenced by various factors. An important mechanism is the ubiquitination of proteins and the associated proteasomal degradation.1,2 Ubiquitin is a small protein present in all eukaryotic cells that is involved in protein quality control, transduction of signals, and cell cycle control. Ubiquitination of T-cell receptors mediates their downregulation by altering signaling components; thus, interleukin -2 production and T-cell proliferation are inhibited.3,4 These mechanisms promote immunologic tolerance and T-cell anergy.5 

The regulation of ubiquitination is conducted stringently via substrate-specific E3 ubiquitin ligases, one of which is encoded by the itchy E3 ubiquitin protein ligase (ITCH) gene (Online Mendelian Inheritance in Man [OMIM] entry *606409; chromosome 20q11.22). Biallelic mutations in the ITCH gene can cause severe deficiency of the respective E3 ubiquitin ligase. This leads to increased T-cell activity with a lack of immune tolerance in mouse models.6,8 Lohr et al9 demonstrated for the first time that human ITCH E3 deficiency can cause a syndromic multisystem autoimmune disease (SMAD) in humans, which includes major phenotypic features (OMIM entry #613385).

A 1-year-old girl of consanguineous parents received a living-donor liver transplant (LT) from her mother because of acute liver failure, which was suspected to be caused by drug-induced hypereosinophilic syndrome with positive liver-kidney-mikrosome-2 (LKM-2) antibodies. The liver histology of the explanted liver revealed autoimmune hepatitis with giant-cell hepatitis and accompanying cholangitis (Fig 1). Previously, there had been a regular ingestion of amoxicillin because of respiratory infections. Notable was also dystrophy (body weight below the third percentile; BMI 14.2), short stature (body height below the third percentile), choanal atresia, psychomotor retardation, and muscular hypotonia as well as characteristic craniofacial features, such as frontal bossing, prominent eyes, orbital ptosis, and small chin (Fig 2). She carried a 120 bp microduplication in chromosomal region 22q12.3 (detected on a comparative genomic hybridization microarray), which she inherited from a healthy father of no clinical significance. After 8 weeks, the first corticosteroid-sensitive graft rejection reaction was observed. After successful treatment with corticosteroids, the patient developed an alloimmune eosinophilic liver disease again with elevated liver enzymes. We changed the immunosuppressive medication basiliximab and cyclosporine A to tacrolimus and mycophenolate mofetil and administered high doses of steroids. Two years after the LT, complete liver cirrhosis with lobular inflammatory infiltrates, giant-cell hepatitis, and ductopenia was verified in chronic cholestasis. Because of the persistence of abnormal liver function, the patient received 3 cycles of rituximab, which had a positive but temporary effect on liver inflammation. In the meantime, she developed a systemic autoimmune disease with detection of specific antibodies that affected different organ systems (de novo autoimmune hepatitis with donor specific auto-antibodies, Hashimoto thyroiditis, and diabetes mellitus type 1). After a second LT 2 years later, a comparable liver histology could be detected. Infectious complications were adenovirus hepatitis, methicillin-resistant Staphylococcus aureus, and Escherichia coli sepsis. The girl also suffered from enteropathy with chronic diarrhea and recurring pulmonal infections, including pulmonary aspergillosis. At the age of 5 years, refractory pancytopenia with portal hypertension and hypersplenism was diagnosed. A posttransplant lymphoproliferative disease was excluded by bone marrow biopsy. The progressive pancytopenia necessitated regular transfusions of erythrocyte and thrombocyte concentrates and substitution of immunoglobulin. Because of the multisystem autoimmune features, we considered a bone marrow transplant, but liver failure and, later, multiple organ failures limited the treatment options over time, and a bone marrow transplant was not realizable. Five months afterward, epidural and subdural bleeding occurred during thrombocytopenia followed by craniotomy and anticonvulsive therapy for seizures of partial onset. Three months later, the girl died of sepsis at home. Within a multicenter study on genetic causes of indeterminate acute liver failure lead by the Children’s Hospital Heidelberg and the Institute of Human Genetics of the Technical University of Munich, trio whole-exome sequencing was performed on genomic DNA of the patient and her parents. Postmortem, a homozygous splice mutation in the ITCH gene (NM_001257137.1, c.1693-1G>A) was identified, predicted to cause a loss of the splice acceptor site of exon 18, and rated as likely pathogenic. This homozygous mutation was confirmed by Sanger sequencing, and a segregation analysis revealed that both parents were heterozygous. A real-time polymerase chain reaction analysis of a patient fibroblast ITCH complementary DNA fragment covering exons 15 to 19 revealed a shorter amplicon, indicative of a splice defect. Sanger sequencing was used to confirm that the splice acceptor of exon 19 was used instead of the canonical splice acceptor of exon 18, resulting in a skipping of exon 18 (Fig 3).

FIGURE 1

Liver histology of the explanted liver (scale bar, 100 µm): giant-cell hepatitis with infiltration of lymphocytes and plasma cells, which is compatible with autoimmune hepatitis and accompanying cholangitis with reactive proliferation of bile ducts (by kind permission of Professor H.A. Baba, Institute of Pathology, University of Duisburg-Essen, Essen, Germany).

FIGURE 1

Liver histology of the explanted liver (scale bar, 100 µm): giant-cell hepatitis with infiltration of lymphocytes and plasma cells, which is compatible with autoimmune hepatitis and accompanying cholangitis with reactive proliferation of bile ducts (by kind permission of Professor H.A. Baba, Institute of Pathology, University of Duisburg-Essen, Essen, Germany).

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FIGURE 2

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

FIGURE 2

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

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FIGURE 3

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

FIGURE 3

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

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Through different signaling pathways, ubiquitination plays an important role in immune regulation and controls T-cell responsiveness.1,6 In Itch−/− mice, elevated levels of circulating immunoglobulins, anti-nuclear antibodies, and lymphocytes could be verified, and several studies revealed that mouse mutations in Itch can lead to fatal autoimmune diseases with histiocyte and lymphocyte infiltration of different organ systems.10,11 

The human phenotype of ITCH deficiency is SMAD with morphologic and developmental abnormalities. It is based on 10 Old Order Amish children of consanguineous parents who were all homozygous for a truncating mutation in ITCH (OMIM entry 606409). A sequence analysis of ITCH revealed a homozygous single base pair insertion in exon 6 (c.394_395insA). This frameshift mutation was predicted to truncate ITCH at amino acid position 139. All 10 patients had a similar phenotype, comparable to our case. Interestingly, participation of the immune system was found in only 60% of patients (Table 1). Diabetes mellitus, autoimmune hepatitis, and enteropathy with detection of perinuclear anti-neutrophil cytoplasmic antibodies, smooth-muscle antibodies, anti-enterocyte antibodies, and a lymphocytic inflammation of the gastrointestinal tract were identified in just 10% to 30% of the children.9 Our index patient was suffering from autoimmune diseases in several organs with partial detection of antibodies: chronic lung disease, enteropathy, diabetes mellitus (elevated levels of islet-cell antibodies, glutamic acid decarboxylase antibodies, and insulin auto-antibodies), autoimmune hepatitis (LKM-2 antibodies), and hypothyroidism (thyroid peroxidase antibodies). In contrast to the patients described by Lohr et al,9 who had repeatedly normal blood cell counts, our patient developed progressive pancytopenia associated with hypersplenism and portal hypertension caused by liver cirrhosis. Additionally, we describe the first case of ITCH deficiency in humans with autoimmune hepatitis that progressed to acute liver failure requiring a LT. It may be speculated whether an inappropriate activation of T cells due to ITCH deficiency was the only reason for the severe liver complications seen in our patient. Results of oncological, hepatological, and immunologic research are needed to understand the pathophysiology of ITCH deficiency and may inspire novel therapeutic options targeting the ubiquitination system.12 

TABLE 1

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

Similarities, n = 11Differences, n = 1
Mean age of first symptoms (4.2 y) Pancytopenia 
Macrocephaly (90%) Liver cirrhosis with acute liver failure 
Typical 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%) — 
Similarities, n = 11Differences, n = 1
Mean age of first symptoms (4.2 y) Pancytopenia 
Macrocephaly (90%) Liver cirrhosis with acute liver failure 
Typical 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 multisystem autoimmune disease. Am J Hum Genet. 2010;86(3):447–453. —, not applicable.

The fact that our patient was much more affected suggests that there may have been additional etiologic factors or genetic predispositions compromising, for instance, the function of other ubiquitin ligases.9 There are no functional tests available that can help to elucidate the causality between this new homozygous mutation in ITCH and the described (liver) phenotype. Special assays will be needed for determining the fate of ubiquitinated substrates (such as Jun proto-oncogene, AP-1 transcription factor subunit in the cell) and for studying downstream signaling pathways, such as those involving NOTCH.9 Besides, the reasons for pediatric acute liver failure are diverse. Other immunologic and genetic diseases associated with autoimmune hepatitis are known (Table 2). In children, 30% to 50% of acute liver failures remain indeterminate despite detailed diagnostic workup. Genetic diseases are increasingly considered to be causative for some of these cases of indeterminate acute liver failure.13,15 On the basis of the current report, we propose ITCH deficiency to be a notable differential diagnosis in children with SMAD, which can include autoimmune hepatitis and possibly acute liver failure.

TABLE 2

Autoimmune Hepatitis–Associated Diseases in Pediatric Patients

Immunologic DiseasesHereditary Diseases
IBD APECED, AIRE gene 
Thyreoditis IPEX, FOXP3 gene 
Vitiligo Monosomy 22q13 syndrome 
Diabetes mellitus type 1 FHL, FHL 1–5 types 
Nephrotic syndrome SMAD, ITCH deficiency 
Immunologic DiseasesHereditary Diseases
IBD APECED, AIRE gene 
Thyreoditis IPEX, FOXP3 gene 
Vitiligo Monosomy 22q13 syndrome 
Diabetes mellitus type 1 FHL, FHL 1–5 types 
Nephrotic syndrome SMAD, ITCH deficiency 

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

Burt et al16 first considered the possibility of a bone marrow transplant in patients with severe autoimmune diseases, which collectively form the most rapidly expanding indication for stem cell transplantation. We decided against a bone marrow transplant and splenectomy because our patient was too sick to undergo these interventions. Especially, liver function was deteriorated to an extent that a bone marrow transplant was not a realistic therapeutic option.

It should be pointed out that this is only the second mutation of ITCH documented in humans, apart from the 1 variant described by Lohr et al9 in 2010. This case underscores the relevance of mutations in the ITCH gene for the clinical courses and treatment of children suffering from complex autoimmune diseases. It supports the assumption that genetic diseases can be causative not only for autoimmune hepatitis but also for some of the indeterminate cases of acute liver failure. Further research and functional assays will be needed to detect these children at an early age before impairment of liver function necessitates an LT or progressive damage to multiple organ systems makes curative therapeutic options, such as a bone marrow transplant, impossible.

Dr Kleine-Eggebrecht designed the study and drafted the initial manuscript; Dr Staufner was responsible for the genetic diagnosis, interpretation of genetic findings, and critical revision of the manuscript; Dr Kathemann was responsible for the medical treatment, was involved in diagnostics, and revised the manuscript; Dr Elgizouli counseled the family and critically revised the manuscript; Dr Kopajtich confirmed the splicing effect in fibroblasts and critically revised the manuscript; Dr Prokisch was responsible for the genetic analysis and interpretation of the results; Dr Lainka was responsible for medical care, drafted the initial manuscript, and critically revised the manuscript; and all authors read and approved the final manuscript as submitted and agree to be accountable for all aspects of the work.

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

Dr Lainka gratefully acknowledges the comments made by Oliver Weiergräber (Forschungszentrum Jülich).

     
  • ITCH

    itchy E3 ubiquitin protein ligase

  •  
  • LKM-2

    liver-kidney-mikrosome-2

  •  
  • LT

    liver transplant

  •  
  • OMIM

    Online Mendelian Inheritance in Man

  •  
  • SMAD

    syndromic multisystem autoimmune disease

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Competing Interests

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

FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose.