REVIEWS Pediatric Dermatology Vol. 31 No. 5 539–546, 2014
Recent Advances in the Genetics and Management of Harlequin Ichthyosis Hera Ahmed*,† and Edel A. O’Toole, M.B., Ph.D., F.R.C.P.*,† *Department of Dermatology, Barts Health National Health Service Trust, Whitechapel, London, UK, †Centre for Cutaneous Research, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK
Abstract: Harlequin ichthyosis (HI) is the most severe and devastating form of the autosomal recessive congenital ichthyoses (ARCIs). Mutations in the ABCA12 gene result in disruption of intercellular lipid deposition in the stratum corneum and a major skin barrier defect. Patients present at birth, often premature, with cutaneous thick, yellow, hyperkeratotic plates with deep erythematous fissures, causing a typical facial appearance. Harlequin ichthyosis has often been considered to be fatal, and management tends to be palliative, but follow-up of 45 affected infants has shown that with good neonatal care and early introduction of oral retinoids, survival rates are improving. Because ABCA12 mutations have been identified, known carriers are able to undergo preventative preimplantation and prenatal genetic testing. Experimental studies have shown recovery of lipid secretion in lamellar granules using corrective gene therapy. Further research is needed to develop alternative therapies to retinoids in HI.
Autosomal recessive congenital ichthyoses (ARCIs) are a heterogeneous group of skin disorders seen at birth that are characterized by scaling and hyperkeratosis. The majority of neonates with ARCIs are born with a collodion membrane, a shiny filmlike outer layer of skin that sheds over the first month of life, and are known as collodion babies. Ten percent of these are self-healing. The eventual phenotypes of the collodion baby vary from mild to severe congenital ichthyosiform erythroderma (CIE) and lamellar ichthyosis (LI) or an
overlap phenotype. The distinctive clinical features of harlequin ichthyosis (HI) (see below) are unlikely to be confused with the less severe appearance of the collodion baby, but a small proportion of affected neonates fall between the two presentations. These have been referred to as “chrysalis babies” and may account for a milder phenotype subgroup of HI (1). Across the spectrum of ARCI, the most common mutation seen is in the transglutaminase 1 gene (TGM1), seen in 32% of a cohort of 520 families
Address correspondence to Edel O’Toole, M.B., Ph.D., F.R.C.P., Centre for Cutaneous Research, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London E1 1BB, UK, or e-mail: [email protected]. DOI: 10.1111/pde.12383
© 2014 Wiley Periodicals, Inc.
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with ARCI (2), which includes missense, nonsense, and splice site mutations (3). Seven other causative genes have been identified, including two lipoxygenases (ALOX12B, ALOXE3) typically associated with CIE or intermediate LI/CIE phenotypes, a potential receptor involved in lipid metabolism and cell–cell (ichthyin [NIPAL4]) adhesion, a gene from the cytochrome P450 family (CYP4F22), CERS3 (involved in sphingolipid metabolism), PNPLA1 (involved in epidermal barrier formation), and the adenosine triphosphate (ATP)-binding cassette transporter gene, ABCA12 (2–5). It is now widely accepted that mutations in the ABCA12 gene cause HI because loss of this epidermal lipid transporter results in severe skin barrier dysfunction (6). ABCA12 mutations are also seen in some cases of LI and CIE and account for 5% of ARCIs (2,3). The molecular genetics, use of early retinoids, and clinical outcomes in survivors with HI have been assessed to gain a deeper understanding of management (7–10). This article aims to review the most recent molecular findings that assist in prenatal diagnosis and prevention strategies for affected families and to provide insight into why survival rates are improving. EPIDEMIOLOGY Reverend Oliver Hart first described a case of HI in South Carolina in 1750. An incidence of approximately 1 in 300,000 births has been calculated. Approximately 200 cases have been reported internationally, and fewer than 5 cases are reported annually in the United Kingdom (11). A multicenter retrospective study of 45 cases (12) that provided demographic data for patients with ABCA12 mutations resulting in HI found a female:male ratio of 1.15:1 and a diverse range of ethnic backgrounds, including British Pakistani (22.2%), British white (11.1%), British Somali (6.7%), British Bangladeshi (4.4%), North American (4.4%), Balochistani (4.4%), Swedish white (4.4%), Turkish (4.4%), Iranian (4.4%), Swiss white (4.4%), British Indian (2.2%), Bangladeshi (2.2%), Israeli (2.2%), Jamaican Eritrean (2.2%), Egyptian (2.2%), Hmong Laotian (2.2%), Kosovan (2.2%), Afghani Iranian (2.2%), South American (2.2%), Native American (2.2%), Central American (2.2%), Irish white (2.2%), and Arab other (2.2%). Consanguinity may explain the higher incidence in some ethnic groups. Mortality data showed that there were 25 survivors (56%), ages 10 months to 25 years and 20 deaths (44%), the age of death ranging from 1 to 52 days.
CLINICAL PRESENTATION HI presents clinically at birth. The mean gestational age at birth is known to be preterm (35 wks, range 30– 39 wks) (12). Neonates born with HI have the typical appearance of generalized, thick, yellowish, hyperkeratotic plates with deep erythematous fissures, particularly on the trunk (Fig. 1). The constricting skin causes severe ectropion (eversion of the lower eyelids) and eclabium (eversion of the lips), rudimentary ears with loss of the retroauricular fold, and nasal hypoplasia. Digits may be hypoplastic or autoamputated and limbs have limited mobility with flexion contractures due to the encasement of constricting bands. The taut skin may also result in a microcephaly-like appearance (3,11,12). Babies who survive into infancy tend to shed their outer layer of hyperkeratotic plaques, leaving severe erythroderma with generalized scaling, resembling severe CIE (3,7,12). Other clinical features in infancy include palmoplantar keratoderma, alopecia, and persistent ectropion and eclabium. Harlequin ichthyosis is now recognized as a skin disease with a wide range of complications, and a multidisciplinary approach is required. In the neonatal period, patients are prone to electrolyte imbalances such as hypernatremia, hypocalcemia, and hypoglycemia. Infants and children can develop hearing difficulties caused by blockage of the external auditory canal from skin debris, which requires regular microsuction. Ophthalmologic complications are also common and include recurrent conjunctivitis, exposure keratitis, squint, and nystagmus. Developmental delay is seen in approximately one-third of patients in speech and language and fine and gross motor skills. In early childhood, recurrent skin infections are common; implicated organisms include methicillin-resistant
Figure 1. Typical appearance of neonate with harlequin ichthyosis.
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Staphylococcus aureus, S. aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, and Klebsiella. Severe anemia affecting a minority of patients with HI requires regular blood transfusions. Children may also need urologic intervention for urinary incontinence caused by an unstable bladder. Patients who survive into their teens may develop inflammatory arthritis and joint contractures requiring systemic disease-modifying therapy. Vitamin D deficiency can cause rickets and osteomalacia in these patients, so monitoring of vitamin D levels and providing supplements is essential (12). PATHOPHYSIOLOGY The mutated protein of interest in HI and a subgroup of ARCIs is the ATP-binding cassette transporter A12 (ABCA12), which functions to transport lipid glucosylceramides to the extracellular space through lamellar granules (13). Lamellar granules secrete lipid glucosylceramides and hydrolytic enzymes (e.g., proteases, lipases) and proteins (e.g., corneodesmosin), which are required for desquamation (skin shedding) (8). The lipid glucosylceramides are hydrolyzed to form hydroxyceramides, which are covalently linked to cornified envelope proteins to form the extracellular lamellar membrane, which is needed to keep the skin barrier permeable (13). In HI, lamellar granules are distorted, reduced, or absent, resulting in disruption of intercellular lipid deposition in the stratum corneum (14). Extreme hyperkeratosis results as a compensatory mechanism for the reduced lipid layer. The skin barrier is impaired, leading to defective desquamation and permeability, which may be responsible for complications seen in HI such as dehydration and sepsis. The metabolism of triacylglycerol generates fatty acids for the synthesis of acylceramides, and its role during the formation of a functional skin barrier has recently been discovered (15). Two recent studies have also identified a role for the Nrf2 transcription factor in skin barrier homeostasis and antioxidant defense (16,17). GENETICS The HI locus was mapped to chromosome 2q35 using single-nucleotide polymorphism (SNP) array technology (18). More than 70 mutations in ABCA12 associated with ARCI have been described, with 51 of these resulting in HI phenotypes (Fig. 2) (19–29). Previous studies of individuals with HI using sequence analysis of the 53 coding exons of the ABCA12 gene have shown that the insertion of a premature stop codon results in nonsense substitutions in the majority of patients tested. Frameshift mutations have also
been identified arising from four single base pair deletions and one single base pair insertion (9). The ABCA12 mutations in HI are compound heterozygous or homozygous and result in truncated or deleted regions of the C terminal end of the ABCA12 protein in the second nucleotide-binding fold, located between amino acids 2282 and 2467 (9,10). If the mutation in ABCA12 is found in the first nucleotidebinding fold between amino acids 1370 and 1554 and results in only one amino acid alteration, patients develop the less severe phenotype, LI type 2 (9,19). In individuals with CIE, at least one mutation on each allele is a missense mutation and also results in only one amino acid alteration (19). In patients in whom ABCA12 mutations are found on only one allele, more complex methods must be used to find the mutation on the other allele because the disease is recessive. Deletion mutations in compound heterozygotes are undetectable using standard polymerase chain reaction (PCR) and sequencing, so multiplex PCR and oligonucleotide array analysis have been used and have identified a previously undetected heterozygous deletion of exon 8 (9). Because exome sequencing is now affordable, many laboratories are using it for the diagnosis of HI. This can be a targeted capture or whole-exome approach (30). Continued reductions in costs should allow molecular diagnosis to be part of routine investigations. Some mutations seem to be more prevalent in certain ethnic groups. Two individuals of Caucasian origin with HI were found to have the same mutation (2025delG) in exon 16 (9), whereas the mutation 7322delC, the most commonly reported mutation in HI, has been identified only in patients of Pakistani origin (18,19). This founder mutation effect is also seen in patients with LI because the same missense mutation (p.Asn1380Ser) has been identified in five African families (19). A comprehensive study analyzing molecular findings in 45 cases of HI (12) found that all deaths were associated with homozygous mutations (the same mutation was found on both alleles). In 21 survivors, 52% were compound heterozygotes (the mutation was different on each allele) compared with 48% homozygotes. These data suggest a survival advantage for compound heterozygotes over homozygotes. Compound heterozygotes may have less penetrance of the disease because the two recessive alleles have mutations at different locations. In combination, this may be less deleterious, as this effect has been seen in other autosomal recessive conditions (31). The increasing number of patients with HI found to have the ABCA12 mutation provides strong
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Figure 2. Schematic of known ABCA12 mutations.
HI is diagnosed clinically from appearance at birth (32). For parents who are carriers, prenatal diagnosis is possible using fetal DNA analysis owing to genetic advances (33). Ultrasound performed in the second trimester can also identify features of a harlequin fetus, but before prenatal diagnosis is performed, parents need to discuss whether they would opt for termination if HI is diagnosed. Fetal skin biopsy is no longer used in prenatal diagnosis of HI.
routine ultrasound screening has identified the clinical features of the disease in the prenatal period. The first report of prenatal sonographic features of HI was in 1989 (34). Features that have been observed include ectropion and eclabium, dysplastic ears, fixed position of the hands, absent nose, edematous thighs and feet, intrauterine growth retardation, polyhydramnios, and the “snowflake sign” (skin particles floating in the amniotic cavity) (4,35), but the phenotypic appearance is not usually apparent until the third trimester. A study reporting three cases of HI found that an ultrasonographic diagnosis could be made in the second trimester before HI features such as ectropion and eclabium manifested by using a short foot to femur length as a diagnostic pointer (36).
Ultrasonographic Diagnosis
Fetal DNA Analysis
Fetal DNA analysis for diagnosis of HI is limited to families whose older children have been affected or are known carriers, but for those with de novo mutations,
Since the identification of the ABCA12 gene mutation, fetal skin biopsy has been replaced with direct DNA sequence analysis using fetal DNA from chorionic
evidence of its role in the disease process and allows high-risk families to undergo prenatal or preimplantation diagnosis if using in vitro fertilization. DIAGNOSIS
Ahmed and O’Toole: Harlequin Ichthyosis 543
villi or amniotic fluid samples (37). DNA analysis has advantages over fetal skin biopsy in that it is the most definitive diagnostic tool, can be performed earlier, and is more widely available. Fetal DNA can also be compared with parental DNA to delineate pathogenic mutations and determine uniparental disomy, nonpaternity, de novo mutations, and germline mosaicism (33). Amniocentesis is safest performed after 15 weeks, but carries a 1% risk of miscarriage (38). Chorionic villus sampling can be performed earlier, at 10 weeks, but is associated with a slightly higher miscarriage rate (39). Preimplantation Genetic Diagnosis The discovery of the causative link between ABCA12 mutations and HI has allowed for preimplantation diagnosis of possible carriers who wish to start a family using in vitro fertilization. The technique uses DNA analysis of a blastomere biopsy of the 6- to 10cell embryo, as well as single blastomeres. A selection of disease-free embryos is then transferred to the uterus for implantation (40). This innovative procedure can be used as a preventative measure in highrisk families and avoids the possibility of termination. MANAGEMENT It was once thought that a baby born with HI would die in the first few days or weeks of life. Although it is a rare disease, improved neonatal care has increased survival (22,41). Treatment and supportive care is required throughout the patient’s life to address complex needs and involves a multidisciplinary team of pediatricians, dermatologists, ophthalmologists, plastic surgeons, psychologists, dieticians, geneticists, and ear, nose, and throat specialists (42). The management of patients with HI can be divided into the critical care received early in life to optimize survival and the on-going supportive care required in infancy and beyond. Neonatal Period All patients with HI need to be sent immediately to a neonatal intensive care unit to prevent complications such as respiratory distress, dehydration, electrolyte imbalance, impaired thermoregulation, systemic bacterial infections, and feeding difficulties (2). Severe eclabium makes it difficult for the baby to suck, in which case enteral nutrition must be used. The patient is placed in a humidified incubator to maintain heat and given supportive intravenous fluids to avoid
dehydration. Peripheral access may be difficult, in which case umbilical cannulation may be necessary. Bathing twice daily is important; emulsifying ointments and antiseptic solutions can be added to the bath water. Topical emollients are applied regularly to the skin. Antibiotic and hypromellose eye drops are given to prevent bacterial conjunctivitis and keratitis (43). Neonates are usually obligate nasal breathers; in the case of complete nasal occlusion, intubation may be required (41). Early referral to a dietician is required because they may need nasogastric feeding because of jaw immobility. Breastfeeding is encouraged if possible. Current treatment usually includes acitretin, an oral retinoid that has widely replaced its parent compound etretinate in the treatment of disorders of keratinization because of its shorter half-life (44). The first case of its use in HI was reported in 2001 (45). Reports have shown significant improvement after 1 month of use when started by day 7 of life. Positive changes seen in the skin include softening and separation of hyperkeratotic yellow plaques, revealing an erythematous layer resembling CIE (3,7,12). As the skin contractures loosen, mobility of the limbs is greatly improved, ectropion and eclabium gradually improve, and the child is able to breastfeed (45). It has been suggested that patients treated early with acitretin may have a survival advantage. In a retinoidtreated group of 20 patients, 83% survived, compared with 76% mortality in an untreated group of 21 patients, most deaths occurring in the first 3 days of life (12). An initial dose of 1 mg/kg/day encourages skin shedding, but it has been recommended to reduce the dose thereafter to 0.5 mg/kg/day to avoid any long-term side effects of musculoskeletal, neurologic, or gastrointestinal toxicity (46). A complete blood count, serum electrolytes, serum lipids, and liver function tests should be conducted at baseline, 1 month after initiating therapy, and then every 3 months (12,45). Oral liarozole, a retinoic acid metabolism-blocking agent, may be an alternative to oral acitretin, although small randomized clinical trials in ARCI have not shown a difference in efficacy, and larger clinical trials are needed to assess any benefit (47). Infancy and Beyond Patients who survive the neonatal period will live with a chronic skin disease that needs to be managed on a daily basis with the frequent use of topical emollients and sometimes intermittent use of oral acitretin. Surgical correction may be required for persistent
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ectropion, but recurrence is frequent. Infants having difficulty maintaining caloric intake will need nasogastric tube feeding until they can eat a high-calorie diet. Monitoring of vitamin D is important to avoid rickets because sun exposure may be limited. Children with HI tend to have frequent hospital admissions, which can interrupt development of their speech, language, and social skills (12), and speech and language therapy input may be beneficial. More than half of children with HI in the United Kingdom need a statement of special needs so that additional help is provided in school (12). It is important not to forget that, as with any dermatologic disease, support from a psychologist may help those who feel self-conscious about their appearance. Awareness of their condition, regular use of emollients, and a nutritious diet are all necessary to living a healthy, independent life with HI, as survivors have shown. Corrective Gene Therapy The discovery of the HI gene allows the possibility of corrective gene therapy. Autologous keratinocyte stem cells are harvested, genetically modified, and generated into sheets of disease-free epithelium with an integrating vector. This method has been shown to restore lipid secretion in lamellar granules in HI keratinocytes (10,13). An experimental study found that electron microscopic observation of HI keratinocytes cultured for 1 week showed poor lipid secretion of lamellar granules, with 7.0% of cells showing a normal distribution of glucosylceramide staining (10). After corrective ABCA12 gene transfer, this number increased significantly to 16.7%. Although these findings are promising, there is a risk of insertional mutagenesis with corrective gene therapy. Experimental studies in epidermolysis bullosa have explored the use of engineered transcription activator-like effector nucleases for precise gene correction and have shown normal protein expression in vivo in patient-specific gene mutations (48). An alternative method used for precise genome editing is homologous recombination using recombinant adenoassociated virus vectors. This has been shown to produce low rates of random integration (49). Readthrough Compounds Nonsense mutations in HI result in generation of a premature termination codon that leads to degradation of the messenger RNA template and to the production of a nonfunctional, truncated protein. Recoding of a premature termination codon allows
readthrough and production of normal protein. This mechanism was first established using aminoglycosides in the 1960s. Newer readthrough compounds are in clinical trials for Duchenne muscular dystrophy (50). Discovery of Alternate Pathways A greater understanding of the pathways that lead to hyperkeratosis in inherited ichthyoses will create more opportunities for treatment. As discussed earlier, if the mutation in ABCA12 is found in a different location, patients develop a less severe hyperkeratotic disease, LI type 2. Studies investigating individuals with LI with TGM1 mutations have found that interleukin-1-a expression is high in these patients, and the use of interleukin-1 receptor antagonists led to a dose-dependent decrease in hyperkeratosis in in vitro models (51). Because HI shares the same genetic pathogenesis, there may be other pathways involved that also cause high cytokine expression, creating an opportunity for targeted therapeutics. Although such biologic therapies can act on several pathways, causing adverse effects, the discovery of new therapies may reduce the need for constant application of emollients in scaling disorders. REFERENCES 1. O’Toole EA, Kelsell DP. Harlequin ichthyosis. In: Irvine AD, Hoeger PH, Albert CY, eds. Harper’s textbook of pediatric dermatology. Oxford: WileyBlackwell, doi: 10.1002/9781444345384.ch13. 2. Fischer J. Autosomal recessive congenital ichthyosis. J Invest Dermatol 2009;129:1319–1321. 3. Richard G, Bale SJ. Autosomal recessive congenital ichthyosis. 2001 Jan 10 [updated 2012 Sep 13]. In: Pagon RA, Adam MP, Bird TD et al, eds. GeneReviews [online]. Seattle: University of Washington, 1993–2014. Available at http://www.ncbi.nlm.nih.gov/books/ NBK1420/. 4. Radner FP, Marrakchi S, Kirchmeier P et al. Mutations in CERS3 cause autosomal recessive congenital ichthyosis in humans. PLoS Genet 2013;9:e1003536. 5. Grall A, Guaguere E, Planchais S et al. PNPLA1 mutations cause autosomal recessive congenital ichthyosis in golden retriever dogs and humans. Nat Genet 2012;44:140–147. 6. Habib A, Pasha W, Raza N et al. Harlequin ichthyosis in two siblings. J Coll Physicians Surg Pak 2011;21:503– 505. 7. Haftek M, Cambazard F, Dhouailly D et al. A longitudinal study of a harlequin infant presenting clinically as non-bullous congenital ichthyosiform erythroderma. Br J Dermatol 1996;135:448–453. 8. Ishida-Yamamoto A, Simon M, Kishibe M et al. Epidermal lamellar granules transport different cargoes
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25. Nawaz S, Tariq M, Ahmad I et al. Non-bullous congenital ichthyosiform erythroderma associated with homozygosity for a novel missense mutation in an ATP binding domain of ABCA12. Eur J Dermatol 2012;22:178–181. 26. Shimizu Y, Sugiura K, Aoyama Y et al. Novel ABCA12 missense mutation p.Phe2144Ser underlies congenital ichthyosiform erythroderma. J Dermatol 2013;40:581–582. 27. Goldsmith T, Fuchs-Telem D, Israeli S et al. The sound of silence: autosomal recessive congenital ichthyosis caused by a synonymous mutation in ABCA12. Exp Dermatol 2013;22:251–254. 28. Fukuda A, Hamada T, Ishii N et al. Novel adenosine triphosphate (ATP)-binding cassette, subfamily A, member 12 (ABCA12) mutations associated with congenital ichthyosiform erythroderma. Br J Dermatol 2012;166:218–221. 29. Peterson H, Lofgren S, Bremmer S et al. Novel ABCA12 mutations leading to recessive congenital ichthyosis. Pediatr Dermatol 2013;30:e236–e237. 30. Scott CA, Plagnol V, Nitoiu D et al. Targeted sequence capture and high-throughput sequencing in the molecular diagnosis of ichthyosis and other skin diseases. J Invest Dermatol 2013;133:573–576. 31. Negishi I, Kato G, Moriwaki S et al. Compound heterozygosity for the xeroderma pigmentosum complementation group A gene associated with a mild phenotype. Eur J Dermatol 2002;12:536–539. 32. Zapalowicz K, Wygledowska G, Roszkowski T et al. Harlequin ichthyosis—difficulties in prenatal diagnosis. J Appl Genet 2006;47:195–197. 33. Ashton GH, Eady RA, McGrath JA. Prenatal diagnosis for inherited skin diseases. Clin Dermatol 2000;18:643– 648. 34. Berg C, Geipel A, Kohl M et al. Prenatal sonographic features of harlequin ichthyosis. Arch Gynecol Obstet 2003;268:48–51. 35. Meizner I. Prenatal ultrasonic features in a rare case of congenital ichthyosis (harlequin fetus). J Clin Ultrasound 1992;20:132–134. 36. Suresh S, Vijayalakshmi R, Indrani S et al. Short foot length: a diagnostic pointer for harlequin ichthyosis. J Ultrasound Med 2004;23:1653–1657. 37. Yanagi T, Akiyama M, Sakai K et al. DNA-based prenatal exclusion of harlequin ichthyosis. J Am Acad Dermatol 2008;58:653–656. 38. Eddleman KA, Malone FD, Sullivan L et al. Pregnancy loss rates after midtrimester amniocentesis. Obstet Gynecol 2006;108:1067–1072. 39. Tabor A, Alfiveric Z. Update on procedure-related risks for prenatal diagnosis techniques. Fetal Diagn Ther 2010;27:1–7. 40. Shimizu H, Suzumori K. Prenatal diagnosis as test for genodermatoses: its past, present and future. J Dermatol Sci 1999;19:1–8. 41. Milstone LM, Choate KA. Improving outcomes for harlequin ichthyosis. J Am Acad Dermatol 2013;69: 808–809. 42. Kitts C, Eckles D, Romano L. Emerging science: new hope in harlequin ichthyosis. J Obstet Gynecol Neonatal Nurs 2010;39(Suppl 1):S132–S133.
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43. Ward PS, Jones RD. Successful treatment of a harlequin fetus. Arch Dis Child 1989;64:1309–1311. 44. Grønhøj Larsen F, Steinkjer B, Jakobsen P et al. Acitretin is converted to etretinate only during concomitant alcohol intake. Br J Dermatol 2000;143:1164–1169. 45. Singh S, Bhura M, Maheshwari A et al. Successful treatment of harlequin ichthyosis with acitretin. Int J Dermatol 2001;40:472–483. 46. Brecher AR, Orlow SJ. Oral retinoid therapy for dermatological conditions in children and adolescents. J Am Acad Dermatol 2003;49:171–182. 47. Hernandez-Martin A, Aranegui B, Martin-Santiago A et al. A systematic review of clinical trials of treatments for the congenital ichthyoses, excluding ichthyosis vulgaris. J Am Acad Dermatol 2013;69: 544–549.
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Recent Advances in the Genetics and Management of Harlequin Ichthyosis Hera Ahmed*,† and Edel A. O’Toole, M.B., Ph.D., F.R.C.P.*,† *Department of Dermatology, Barts Health National Health Service Trust, Whitechapel, London, UK, †Centre for Cutaneous Research, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK
Abstract: Harlequin ichthyosis (HI) is the most severe and devastating form of the autosomal recessive congenital ichthyoses (ARCIs). Mutations in the ABCA12 gene result in disruption of intercellular lipid deposition in the stratum corneum and a major skin barrier defect. Patients present at birth, often premature, with cutaneous thick, yellow, hyperkeratotic plates with deep erythematous fissures, causing a typical facial appearance. Harlequin ichthyosis has often been considered to be fatal, and management tends to be palliative, but follow-up of 45 affected infants has shown that with good neonatal care and early introduction of oral retinoids, survival rates are improving. Because ABCA12 mutations have been identified, known carriers are able to undergo preventative preimplantation and prenatal genetic testing. Experimental studies have shown recovery of lipid secretion in lamellar granules using corrective gene therapy. Further research is needed to develop alternative therapies to retinoids in HI.
Autosomal recessive congenital ichthyoses (ARCIs) are a heterogeneous group of skin disorders seen at birth that are characterized by scaling and hyperkeratosis. The majority of neonates with ARCIs are born with a collodion membrane, a shiny filmlike outer layer of skin that sheds over the first month of life, and are known as collodion babies. Ten percent of these are self-healing. The eventual phenotypes of the collodion baby vary from mild to severe congenital ichthyosiform erythroderma (CIE) and lamellar ichthyosis (LI) or an
overlap phenotype. The distinctive clinical features of harlequin ichthyosis (HI) (see below) are unlikely to be confused with the less severe appearance of the collodion baby, but a small proportion of affected neonates fall between the two presentations. These have been referred to as “chrysalis babies” and may account for a milder phenotype subgroup of HI (1). Across the spectrum of ARCI, the most common mutation seen is in the transglutaminase 1 gene (TGM1), seen in 32% of a cohort of 520 families
Address correspondence to Edel O’Toole, M.B., Ph.D., F.R.C.P., Centre for Cutaneous Research, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London E1 1BB, UK, or e-mail: [email protected]. DOI: 10.1111/pde.12383
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with ARCI (2), which includes missense, nonsense, and splice site mutations (3). Seven other causative genes have been identified, including two lipoxygenases (ALOX12B, ALOXE3) typically associated with CIE or intermediate LI/CIE phenotypes, a potential receptor involved in lipid metabolism and cell–cell (ichthyin [NIPAL4]) adhesion, a gene from the cytochrome P450 family (CYP4F22), CERS3 (involved in sphingolipid metabolism), PNPLA1 (involved in epidermal barrier formation), and the adenosine triphosphate (ATP)-binding cassette transporter gene, ABCA12 (2–5). It is now widely accepted that mutations in the ABCA12 gene cause HI because loss of this epidermal lipid transporter results in severe skin barrier dysfunction (6). ABCA12 mutations are also seen in some cases of LI and CIE and account for 5% of ARCIs (2,3). The molecular genetics, use of early retinoids, and clinical outcomes in survivors with HI have been assessed to gain a deeper understanding of management (7–10). This article aims to review the most recent molecular findings that assist in prenatal diagnosis and prevention strategies for affected families and to provide insight into why survival rates are improving. EPIDEMIOLOGY Reverend Oliver Hart first described a case of HI in South Carolina in 1750. An incidence of approximately 1 in 300,000 births has been calculated. Approximately 200 cases have been reported internationally, and fewer than 5 cases are reported annually in the United Kingdom (11). A multicenter retrospective study of 45 cases (12) that provided demographic data for patients with ABCA12 mutations resulting in HI found a female:male ratio of 1.15:1 and a diverse range of ethnic backgrounds, including British Pakistani (22.2%), British white (11.1%), British Somali (6.7%), British Bangladeshi (4.4%), North American (4.4%), Balochistani (4.4%), Swedish white (4.4%), Turkish (4.4%), Iranian (4.4%), Swiss white (4.4%), British Indian (2.2%), Bangladeshi (2.2%), Israeli (2.2%), Jamaican Eritrean (2.2%), Egyptian (2.2%), Hmong Laotian (2.2%), Kosovan (2.2%), Afghani Iranian (2.2%), South American (2.2%), Native American (2.2%), Central American (2.2%), Irish white (2.2%), and Arab other (2.2%). Consanguinity may explain the higher incidence in some ethnic groups. Mortality data showed that there were 25 survivors (56%), ages 10 months to 25 years and 20 deaths (44%), the age of death ranging from 1 to 52 days.
CLINICAL PRESENTATION HI presents clinically at birth. The mean gestational age at birth is known to be preterm (35 wks, range 30– 39 wks) (12). Neonates born with HI have the typical appearance of generalized, thick, yellowish, hyperkeratotic plates with deep erythematous fissures, particularly on the trunk (Fig. 1). The constricting skin causes severe ectropion (eversion of the lower eyelids) and eclabium (eversion of the lips), rudimentary ears with loss of the retroauricular fold, and nasal hypoplasia. Digits may be hypoplastic or autoamputated and limbs have limited mobility with flexion contractures due to the encasement of constricting bands. The taut skin may also result in a microcephaly-like appearance (3,11,12). Babies who survive into infancy tend to shed their outer layer of hyperkeratotic plaques, leaving severe erythroderma with generalized scaling, resembling severe CIE (3,7,12). Other clinical features in infancy include palmoplantar keratoderma, alopecia, and persistent ectropion and eclabium. Harlequin ichthyosis is now recognized as a skin disease with a wide range of complications, and a multidisciplinary approach is required. In the neonatal period, patients are prone to electrolyte imbalances such as hypernatremia, hypocalcemia, and hypoglycemia. Infants and children can develop hearing difficulties caused by blockage of the external auditory canal from skin debris, which requires regular microsuction. Ophthalmologic complications are also common and include recurrent conjunctivitis, exposure keratitis, squint, and nystagmus. Developmental delay is seen in approximately one-third of patients in speech and language and fine and gross motor skills. In early childhood, recurrent skin infections are common; implicated organisms include methicillin-resistant
Figure 1. Typical appearance of neonate with harlequin ichthyosis.
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Staphylococcus aureus, S. aureus, Streptococcus pyogenes, Pseudomonas aeruginosa, and Klebsiella. Severe anemia affecting a minority of patients with HI requires regular blood transfusions. Children may also need urologic intervention for urinary incontinence caused by an unstable bladder. Patients who survive into their teens may develop inflammatory arthritis and joint contractures requiring systemic disease-modifying therapy. Vitamin D deficiency can cause rickets and osteomalacia in these patients, so monitoring of vitamin D levels and providing supplements is essential (12). PATHOPHYSIOLOGY The mutated protein of interest in HI and a subgroup of ARCIs is the ATP-binding cassette transporter A12 (ABCA12), which functions to transport lipid glucosylceramides to the extracellular space through lamellar granules (13). Lamellar granules secrete lipid glucosylceramides and hydrolytic enzymes (e.g., proteases, lipases) and proteins (e.g., corneodesmosin), which are required for desquamation (skin shedding) (8). The lipid glucosylceramides are hydrolyzed to form hydroxyceramides, which are covalently linked to cornified envelope proteins to form the extracellular lamellar membrane, which is needed to keep the skin barrier permeable (13). In HI, lamellar granules are distorted, reduced, or absent, resulting in disruption of intercellular lipid deposition in the stratum corneum (14). Extreme hyperkeratosis results as a compensatory mechanism for the reduced lipid layer. The skin barrier is impaired, leading to defective desquamation and permeability, which may be responsible for complications seen in HI such as dehydration and sepsis. The metabolism of triacylglycerol generates fatty acids for the synthesis of acylceramides, and its role during the formation of a functional skin barrier has recently been discovered (15). Two recent studies have also identified a role for the Nrf2 transcription factor in skin barrier homeostasis and antioxidant defense (16,17). GENETICS The HI locus was mapped to chromosome 2q35 using single-nucleotide polymorphism (SNP) array technology (18). More than 70 mutations in ABCA12 associated with ARCI have been described, with 51 of these resulting in HI phenotypes (Fig. 2) (19–29). Previous studies of individuals with HI using sequence analysis of the 53 coding exons of the ABCA12 gene have shown that the insertion of a premature stop codon results in nonsense substitutions in the majority of patients tested. Frameshift mutations have also
been identified arising from four single base pair deletions and one single base pair insertion (9). The ABCA12 mutations in HI are compound heterozygous or homozygous and result in truncated or deleted regions of the C terminal end of the ABCA12 protein in the second nucleotide-binding fold, located between amino acids 2282 and 2467 (9,10). If the mutation in ABCA12 is found in the first nucleotidebinding fold between amino acids 1370 and 1554 and results in only one amino acid alteration, patients develop the less severe phenotype, LI type 2 (9,19). In individuals with CIE, at least one mutation on each allele is a missense mutation and also results in only one amino acid alteration (19). In patients in whom ABCA12 mutations are found on only one allele, more complex methods must be used to find the mutation on the other allele because the disease is recessive. Deletion mutations in compound heterozygotes are undetectable using standard polymerase chain reaction (PCR) and sequencing, so multiplex PCR and oligonucleotide array analysis have been used and have identified a previously undetected heterozygous deletion of exon 8 (9). Because exome sequencing is now affordable, many laboratories are using it for the diagnosis of HI. This can be a targeted capture or whole-exome approach (30). Continued reductions in costs should allow molecular diagnosis to be part of routine investigations. Some mutations seem to be more prevalent in certain ethnic groups. Two individuals of Caucasian origin with HI were found to have the same mutation (2025delG) in exon 16 (9), whereas the mutation 7322delC, the most commonly reported mutation in HI, has been identified only in patients of Pakistani origin (18,19). This founder mutation effect is also seen in patients with LI because the same missense mutation (p.Asn1380Ser) has been identified in five African families (19). A comprehensive study analyzing molecular findings in 45 cases of HI (12) found that all deaths were associated with homozygous mutations (the same mutation was found on both alleles). In 21 survivors, 52% were compound heterozygotes (the mutation was different on each allele) compared with 48% homozygotes. These data suggest a survival advantage for compound heterozygotes over homozygotes. Compound heterozygotes may have less penetrance of the disease because the two recessive alleles have mutations at different locations. In combination, this may be less deleterious, as this effect has been seen in other autosomal recessive conditions (31). The increasing number of patients with HI found to have the ABCA12 mutation provides strong
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Figure 2. Schematic of known ABCA12 mutations.
HI is diagnosed clinically from appearance at birth (32). For parents who are carriers, prenatal diagnosis is possible using fetal DNA analysis owing to genetic advances (33). Ultrasound performed in the second trimester can also identify features of a harlequin fetus, but before prenatal diagnosis is performed, parents need to discuss whether they would opt for termination if HI is diagnosed. Fetal skin biopsy is no longer used in prenatal diagnosis of HI.
routine ultrasound screening has identified the clinical features of the disease in the prenatal period. The first report of prenatal sonographic features of HI was in 1989 (34). Features that have been observed include ectropion and eclabium, dysplastic ears, fixed position of the hands, absent nose, edematous thighs and feet, intrauterine growth retardation, polyhydramnios, and the “snowflake sign” (skin particles floating in the amniotic cavity) (4,35), but the phenotypic appearance is not usually apparent until the third trimester. A study reporting three cases of HI found that an ultrasonographic diagnosis could be made in the second trimester before HI features such as ectropion and eclabium manifested by using a short foot to femur length as a diagnostic pointer (36).
Ultrasonographic Diagnosis
Fetal DNA Analysis
Fetal DNA analysis for diagnosis of HI is limited to families whose older children have been affected or are known carriers, but for those with de novo mutations,
Since the identification of the ABCA12 gene mutation, fetal skin biopsy has been replaced with direct DNA sequence analysis using fetal DNA from chorionic
evidence of its role in the disease process and allows high-risk families to undergo prenatal or preimplantation diagnosis if using in vitro fertilization. DIAGNOSIS
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villi or amniotic fluid samples (37). DNA analysis has advantages over fetal skin biopsy in that it is the most definitive diagnostic tool, can be performed earlier, and is more widely available. Fetal DNA can also be compared with parental DNA to delineate pathogenic mutations and determine uniparental disomy, nonpaternity, de novo mutations, and germline mosaicism (33). Amniocentesis is safest performed after 15 weeks, but carries a 1% risk of miscarriage (38). Chorionic villus sampling can be performed earlier, at 10 weeks, but is associated with a slightly higher miscarriage rate (39). Preimplantation Genetic Diagnosis The discovery of the causative link between ABCA12 mutations and HI has allowed for preimplantation diagnosis of possible carriers who wish to start a family using in vitro fertilization. The technique uses DNA analysis of a blastomere biopsy of the 6- to 10cell embryo, as well as single blastomeres. A selection of disease-free embryos is then transferred to the uterus for implantation (40). This innovative procedure can be used as a preventative measure in highrisk families and avoids the possibility of termination. MANAGEMENT It was once thought that a baby born with HI would die in the first few days or weeks of life. Although it is a rare disease, improved neonatal care has increased survival (22,41). Treatment and supportive care is required throughout the patient’s life to address complex needs and involves a multidisciplinary team of pediatricians, dermatologists, ophthalmologists, plastic surgeons, psychologists, dieticians, geneticists, and ear, nose, and throat specialists (42). The management of patients with HI can be divided into the critical care received early in life to optimize survival and the on-going supportive care required in infancy and beyond. Neonatal Period All patients with HI need to be sent immediately to a neonatal intensive care unit to prevent complications such as respiratory distress, dehydration, electrolyte imbalance, impaired thermoregulation, systemic bacterial infections, and feeding difficulties (2). Severe eclabium makes it difficult for the baby to suck, in which case enteral nutrition must be used. The patient is placed in a humidified incubator to maintain heat and given supportive intravenous fluids to avoid
dehydration. Peripheral access may be difficult, in which case umbilical cannulation may be necessary. Bathing twice daily is important; emulsifying ointments and antiseptic solutions can be added to the bath water. Topical emollients are applied regularly to the skin. Antibiotic and hypromellose eye drops are given to prevent bacterial conjunctivitis and keratitis (43). Neonates are usually obligate nasal breathers; in the case of complete nasal occlusion, intubation may be required (41). Early referral to a dietician is required because they may need nasogastric feeding because of jaw immobility. Breastfeeding is encouraged if possible. Current treatment usually includes acitretin, an oral retinoid that has widely replaced its parent compound etretinate in the treatment of disorders of keratinization because of its shorter half-life (44). The first case of its use in HI was reported in 2001 (45). Reports have shown significant improvement after 1 month of use when started by day 7 of life. Positive changes seen in the skin include softening and separation of hyperkeratotic yellow plaques, revealing an erythematous layer resembling CIE (3,7,12). As the skin contractures loosen, mobility of the limbs is greatly improved, ectropion and eclabium gradually improve, and the child is able to breastfeed (45). It has been suggested that patients treated early with acitretin may have a survival advantage. In a retinoidtreated group of 20 patients, 83% survived, compared with 76% mortality in an untreated group of 21 patients, most deaths occurring in the first 3 days of life (12). An initial dose of 1 mg/kg/day encourages skin shedding, but it has been recommended to reduce the dose thereafter to 0.5 mg/kg/day to avoid any long-term side effects of musculoskeletal, neurologic, or gastrointestinal toxicity (46). A complete blood count, serum electrolytes, serum lipids, and liver function tests should be conducted at baseline, 1 month after initiating therapy, and then every 3 months (12,45). Oral liarozole, a retinoic acid metabolism-blocking agent, may be an alternative to oral acitretin, although small randomized clinical trials in ARCI have not shown a difference in efficacy, and larger clinical trials are needed to assess any benefit (47). Infancy and Beyond Patients who survive the neonatal period will live with a chronic skin disease that needs to be managed on a daily basis with the frequent use of topical emollients and sometimes intermittent use of oral acitretin. Surgical correction may be required for persistent
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ectropion, but recurrence is frequent. Infants having difficulty maintaining caloric intake will need nasogastric tube feeding until they can eat a high-calorie diet. Monitoring of vitamin D is important to avoid rickets because sun exposure may be limited. Children with HI tend to have frequent hospital admissions, which can interrupt development of their speech, language, and social skills (12), and speech and language therapy input may be beneficial. More than half of children with HI in the United Kingdom need a statement of special needs so that additional help is provided in school (12). It is important not to forget that, as with any dermatologic disease, support from a psychologist may help those who feel self-conscious about their appearance. Awareness of their condition, regular use of emollients, and a nutritious diet are all necessary to living a healthy, independent life with HI, as survivors have shown. Corrective Gene Therapy The discovery of the HI gene allows the possibility of corrective gene therapy. Autologous keratinocyte stem cells are harvested, genetically modified, and generated into sheets of disease-free epithelium with an integrating vector. This method has been shown to restore lipid secretion in lamellar granules in HI keratinocytes (10,13). An experimental study found that electron microscopic observation of HI keratinocytes cultured for 1 week showed poor lipid secretion of lamellar granules, with 7.0% of cells showing a normal distribution of glucosylceramide staining (10). After corrective ABCA12 gene transfer, this number increased significantly to 16.7%. Although these findings are promising, there is a risk of insertional mutagenesis with corrective gene therapy. Experimental studies in epidermolysis bullosa have explored the use of engineered transcription activator-like effector nucleases for precise gene correction and have shown normal protein expression in vivo in patient-specific gene mutations (48). An alternative method used for precise genome editing is homologous recombination using recombinant adenoassociated virus vectors. This has been shown to produce low rates of random integration (49). Readthrough Compounds Nonsense mutations in HI result in generation of a premature termination codon that leads to degradation of the messenger RNA template and to the production of a nonfunctional, truncated protein. Recoding of a premature termination codon allows
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