GENE-40115; No. of pages: 3; 4C: Gene xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short communication
Novel ABCA12 mutations in harlequin ichthyosis: A journey from photo diagnosis to prenatal diagnosis☆ Shagun Aggarwal a,b, Anjana Kar b,d, Philip Bland c, David Kelsell c, Ashwin Dalal b,⁎ a
Department of Medical Genetics, Nizam's Institute of Medical Sciences, Hyderabad, India Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad, India Department of Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK d Manipal University, Manipal, India b c
a r t i c l e
i n f o
Article history: Received 13 October 2014 Received in revised form 22 November 2014 Accepted 1 December 2014 Available online xxxx
a b s t r a c t Harequin ichthyosis is a severe autosomal recessive ichthyosis of congenital onset caused by biallelic mutations in the ABCA12 gene. We report two neonates of Indian origin with harlequin ichthyosis. The parents were retrospectively found to have novel mutations in ABCA12 gene after neonatal demise, which helped in providing prenatal diagnosis in subsequent pregnancies. © 2014 Elsevier B.V. All rights reserved.
Keywords: Harlequin ichthyosis ABCA12 mutations Molecular testing
1. Introduction
2. Materials and methods
Harlequin ichthyosis (HI) is the severest form of autosomal recessive ichthyosis of congenital onset (Richard and Bale, 1993–2014). The affected neonates have armor-like thick skin with deep fissures, severe facial abnormalities, microcephaly and joint contractures. The neonatal period is complicated by prematurity, respiratory difficulty, dyselectrolemia, temperature instability and infections. Most neonates don't survive, however with aggressive neonatal care, survival and evolution into a milder ichthyosis have been reported (Rajpopat et al., 2011). More than 93% of patients with harlequin ichthyosis have been reported to have mutations in the ABCA12 gene (Richard and Bale, 1993–2014; Rajpopat et al., 2011). Here we report two distinct cases of parents who were retrospectively found to be carriers of ABCA12 gene mutations after neonatal demise with harlequin ichthyosis.
Two families had attended for genetic counseling in view of previous neonatal deaths. Family 1 was consanguineous, while family 2 was nonconsanguineous. The diagnosis of the previous children had not been established. In both the cases, photographs of the affected children were present and these were suggestive of a diagnosis of harlequin ichthyosis (Supplementary Fig. 1a & b). As this disorder is an autosomal recessive condition, both sets of parents were presumed obligatory carriers for the same. Bidirectional Sanger sequencing of the coding exons and flanking intronic regions of ABCA12 gene was done on parental DNA.
Abbreviations: ABCA12, ATP-binding cassette sub-family A member 12; SNP, single nucleotide polymorphism; HI, harlequin ichthyosis; LI, lamellar ichthyosis; SIFT, mutation prediction software; Glc-NAc, N acetylglucosamine; mRNA, messenger RNA; CIE, congenital ichthyosiform erythroderma; dbSNP, single nucleotide polymorphism database; PTC, premature termination codon ☆ The authors have no conflict of interest to declare. ⁎ Corresponding author at: Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, 4-1-714, Tuljaguda Complex Mozamzahi Road, Nampally, Hyderabad Andhra Pradesh 500001 India. E-mail address: [email protected] (A. Dalal).
3. Results Both the partners of family 1 were found to have heterozygous mutation c.7317_7318insACAAA in the exon 49 of ABCA12 gene (Fig. 1a). This mutation causes frameshift, introducing PTC (premature termination codon) at the 2465 position (p.C2440Tfs*26). Wild type protein is of 2596 aminoacids and mutant is of 2465 aminoacids (Fig. 1c). On the basis of Mutation Taster and SIFT Indel in silico predictions, these amino acid sequence changes suggest that there is loss of ABC transporter 2 domain and potential loss of carbohydrate N-linked GlcNAc. This mutation has not been reported in mutation databases, neither as a SNP in dbSNP. Family 2 was found to be heterozygous carriers for the c.4051delG mutation in exon 28 of ABCA12 (Fig. 1b). This leads to a frameshift causing a PTC at the 1366 amino acid position (p.V1351Sfs*16). Mutation
http://dx.doi.org/10.1016/j.gene.2014.12.002 0378-1119/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Aggarwal, S., et al., Novel ABCA12 mutations in harlequin ichthyosis: A journey from photo diagnosis to prenatal diagnosis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.002
2
S. Aggarwal et al. / Gene xxx (2014) xxx–xxx
Fig. 1. a: Sequence chromatogram of control and parents from families 1 and 2. b: Schematic presentation of cDNA of ABCA12 and normal ABCA12 protein. c: Schematic presentation of consequences of mutation c.7317_7318insACAA causing loss of ABC Transporter I Domain and c.4051_4051delG causing nonsense mediated decay.
Taster and SIFT Indel in silico analysis indicated that this mutation produces a stop codon 66 bp upstream of the exon–intron junction, which may lead to nonsense-mediated mRNA decay (NMD) (Fig. 1c). This mutation is also novel and has not been reported previously. In both families, prenatal diagnosis could be provided in subsequent pregnancy resulting in birth of normal offsprings. 4. Discussion Autosomal recessive congenital ichthyosis are a group of disorders which present as ichthyosis at birth and subsequently evolve into phenotypes of varying severity like lamellar ichthyosis (LI) and congenital ichthyosiform erythroderma (CIE) (Richard and Bale, 1993–2014; Oji et al., 2010). The phenotype at birth is a colloidion baby, where the neonate is covered with a taut, shiny, membrane-like skin or a severe phenotype called harlequin ichthyosis (HI). HI presents with a thick armor-like skin with large cracks covering the whole body, facial abnormalities in the form of severe eclabium and ectropion, joint deformities/ contractures and microcephaly. These children suffer from complications like infections, temperature instability, dyselectrolemia, dehydration, respiratory difficulty and majority succumb in the neonatal life (Richard and Bale, 1993–2014; Rajpopat et al., 2011). Adequate
supportive care with attention to barrier protection, infection control and maintenance of fluid–electrolyte balance along with oral retinoid therapy can lead to survival beyond this period and subsequently these children evolve into a mild phenotype of CIE (Rajpopat et al., 2011, Rossi and Mesia, 2011). Majority (N 93%) of individuals with HI have been found to have mutations in the ABCA12 gene (Richard and Bale, 1993–2014; Rajpopat et al., 2011; Akiyama, 2010; Kelsell et al., 2005; Thomas et al., 2006). This gene encodes for a 2595 amino acid protein which functions as a membrane transporter in the epidermis. It transports lipid molecules and lamellar bodies in the organelles of the stratum corneum cells and hence helps in maintaining the epidermal lipid barrier that is essential to the skin integrity (Akiyama, 2014). Mutations in this gene leading to HI phenotype have been loss of function mutations, either nonsense type or deletions ranging from 1–35 exons. Approximately 56 mutations have been reported till date. A small proportion of individuals with LI have also been reported to have ABCA12 mutations, but these are primarily missense type in a particular domain of the protein (Richard and Bale, 1993–2014; Rajpopat et al., 2011; Akiyama, 2010; Kelsell et al., 2005; Thomas et al., 2006). Here, we report two novel mutations of ABCA12 gene in individuals of Indian origin. Both mutations lead to frameshift, downstream
Please cite this article as: Aggarwal, S., et al., Novel ABCA12 mutations in harlequin ichthyosis: A journey from photo diagnosis to prenatal diagnosis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.002
S. Aggarwal et al. / Gene xxx (2014) xxx–xxx
premature termination codon with presumed formation of a truncated non-functional protein or non-sense mediated decay. This is in corroboration with previous genotype–phenotype reports where severe ABCA12 mutations leading to complete destruction of function or loss of production of ABCA12 protein cause HI phenotype, whereas milder mutations lead to LI phenotype (Richard and Bale, 1993–2014; Rajpopat et al., 2011). In both the cases discussed here, the affected neonate was demised and no biological tissue was available. The causative mutations were identified in the parents on the basis of a diagnosis made from clinical photographs. The identification of mutations confirmed the photodiagnosis and helped in providing prenatal diagnosis by molecular testing in both the families resulting in birth of normal offsprings. To conclude, we report two cases of HI with identification of novel ABCA12 mutations in individuals of Indian origin. The report reiterates the importance of preserving clinical photographs in cases of neonatal demise, which may provide appropriate diagnostic information of utility for subsequent prenatal diagnosis. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.12.002. Acknowledgments We acknowledge the laboratory team of the University Medical Centre Nijmegen St Radboud, The Netherlands who helped to perform the
3
molecular testing in family 2. The Council of Scientific and Industrial Research (1061130842) funded the fellowship for AK. SHHiRT, the Harlequin Ichthyosis Charity funded the PhD studentship for PB. References Akiyama, M., 2010. ABCA12 mutations and autosomal recessive congenital ichthyosis: a review of genotype/phenotype correlations and of pathogenetic concepts. Hum. Mutat. 31, 1090–1096. Akiyama, M., 2014. The roles of ABCA12 in epidermal lipid barrier formation and keratinocyte differentiation. Biochim. Biophys. Acta 1841 (3), 435–440 (Mar). Kelsell, D.P., Norgett, E.E., Unsworth, H., Teh, M.-T., Cullup, T., Mein, C.A., DoppingHepenstal, P.J., Dale, B.A., Tadini, G., Fleckman, P., Stephens, K.G., Sybert, V.P., et al., 2005. Mutations in ABCA12 underlie the severe congenital skin disease harlequin ichthyosis. Am. J. Hum. Genet. 76, 794–803. Oji, V., Tadini, G., Akiyama, M., Bardon, C.B., Bodemer, C., Bourrat, E., Coudiere, P., DiGiovanna, J.J., Elias, P., Fischer, J., Fleckman, P., Gina, M., et al., 2010. Revised nomenclature and classification of inherited ichthyoses: results of the first ichthyosis consensus conference in Soreze 2009. J. Am. Acad. Dermatol. 63, 607–641. Rajpopat, S., Moss, C., Mellerio, J., Vahlquist, A., Ganemo, A., Hellstrom-Pigg, M., Ilchyshyn, A., Burrows, N., Lestringant, G., Taylor, A., Kennedy, C., Paige, D., et al., 2011. Harlequin ichthyosis: a review of clinical and molecular findings in 45 cases. Arch. Dermatol. 147, 681–686. Richard, G., Bale, S.J., 1993–2014. Autosomal recessive congenital ichthyosis. In: Pagon, R.A., Adam, M.P., Bird, T.D., Dolan, C.R., Fong, C.T., Smith, R.J.H., Stephens, K. (Eds.), GeneReviews® [Internet]. University of Washington, Seattle, Seattle (WA) (2001). Rossi, G., Mesia, D., 2011. Management of harlequin ichthyosis in low-income countries. Ann. Trop. Paediatr. 31 (3), 247–249. Thomas, A.C., Cullup, T., Norgett, E.E., Hill, T., Barton, S., Dale, B.A., Sprecher, E., Sheridan, E., Taylor, A.E., Wilroy, R.S., DeLozier, C., Burrows, N., et al., 2006. ABCA12 is the major harlequin ichthyosis gene. J. Invest. Dermatol. 126, 2408–2413.
Please cite this article as: Aggarwal, S., et al., Novel ABCA12 mutations in harlequin ichthyosis: A journey from photo diagnosis to prenatal diagnosis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.002
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short communication
Novel ABCA12 mutations in harlequin ichthyosis: A journey from photo diagnosis to prenatal diagnosis☆ Shagun Aggarwal a,b, Anjana Kar b,d, Philip Bland c, David Kelsell c, Ashwin Dalal b,⁎ a
Department of Medical Genetics, Nizam's Institute of Medical Sciences, Hyderabad, India Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad, India Department of Centre for Cutaneous Research, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK d Manipal University, Manipal, India b c
a r t i c l e
i n f o
Article history: Received 13 October 2014 Received in revised form 22 November 2014 Accepted 1 December 2014 Available online xxxx
a b s t r a c t Harequin ichthyosis is a severe autosomal recessive ichthyosis of congenital onset caused by biallelic mutations in the ABCA12 gene. We report two neonates of Indian origin with harlequin ichthyosis. The parents were retrospectively found to have novel mutations in ABCA12 gene after neonatal demise, which helped in providing prenatal diagnosis in subsequent pregnancies. © 2014 Elsevier B.V. All rights reserved.
Keywords: Harlequin ichthyosis ABCA12 mutations Molecular testing
1. Introduction
2. Materials and methods
Harlequin ichthyosis (HI) is the severest form of autosomal recessive ichthyosis of congenital onset (Richard and Bale, 1993–2014). The affected neonates have armor-like thick skin with deep fissures, severe facial abnormalities, microcephaly and joint contractures. The neonatal period is complicated by prematurity, respiratory difficulty, dyselectrolemia, temperature instability and infections. Most neonates don't survive, however with aggressive neonatal care, survival and evolution into a milder ichthyosis have been reported (Rajpopat et al., 2011). More than 93% of patients with harlequin ichthyosis have been reported to have mutations in the ABCA12 gene (Richard and Bale, 1993–2014; Rajpopat et al., 2011). Here we report two distinct cases of parents who were retrospectively found to be carriers of ABCA12 gene mutations after neonatal demise with harlequin ichthyosis.
Two families had attended for genetic counseling in view of previous neonatal deaths. Family 1 was consanguineous, while family 2 was nonconsanguineous. The diagnosis of the previous children had not been established. In both the cases, photographs of the affected children were present and these were suggestive of a diagnosis of harlequin ichthyosis (Supplementary Fig. 1a & b). As this disorder is an autosomal recessive condition, both sets of parents were presumed obligatory carriers for the same. Bidirectional Sanger sequencing of the coding exons and flanking intronic regions of ABCA12 gene was done on parental DNA.
Abbreviations: ABCA12, ATP-binding cassette sub-family A member 12; SNP, single nucleotide polymorphism; HI, harlequin ichthyosis; LI, lamellar ichthyosis; SIFT, mutation prediction software; Glc-NAc, N acetylglucosamine; mRNA, messenger RNA; CIE, congenital ichthyosiform erythroderma; dbSNP, single nucleotide polymorphism database; PTC, premature termination codon ☆ The authors have no conflict of interest to declare. ⁎ Corresponding author at: Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, 4-1-714, Tuljaguda Complex Mozamzahi Road, Nampally, Hyderabad Andhra Pradesh 500001 India. E-mail address: [email protected] (A. Dalal).
3. Results Both the partners of family 1 were found to have heterozygous mutation c.7317_7318insACAAA in the exon 49 of ABCA12 gene (Fig. 1a). This mutation causes frameshift, introducing PTC (premature termination codon) at the 2465 position (p.C2440Tfs*26). Wild type protein is of 2596 aminoacids and mutant is of 2465 aminoacids (Fig. 1c). On the basis of Mutation Taster and SIFT Indel in silico predictions, these amino acid sequence changes suggest that there is loss of ABC transporter 2 domain and potential loss of carbohydrate N-linked GlcNAc. This mutation has not been reported in mutation databases, neither as a SNP in dbSNP. Family 2 was found to be heterozygous carriers for the c.4051delG mutation in exon 28 of ABCA12 (Fig. 1b). This leads to a frameshift causing a PTC at the 1366 amino acid position (p.V1351Sfs*16). Mutation
http://dx.doi.org/10.1016/j.gene.2014.12.002 0378-1119/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Aggarwal, S., et al., Novel ABCA12 mutations in harlequin ichthyosis: A journey from photo diagnosis to prenatal diagnosis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.002
2
S. Aggarwal et al. / Gene xxx (2014) xxx–xxx
Fig. 1. a: Sequence chromatogram of control and parents from families 1 and 2. b: Schematic presentation of cDNA of ABCA12 and normal ABCA12 protein. c: Schematic presentation of consequences of mutation c.7317_7318insACAA causing loss of ABC Transporter I Domain and c.4051_4051delG causing nonsense mediated decay.
Taster and SIFT Indel in silico analysis indicated that this mutation produces a stop codon 66 bp upstream of the exon–intron junction, which may lead to nonsense-mediated mRNA decay (NMD) (Fig. 1c). This mutation is also novel and has not been reported previously. In both families, prenatal diagnosis could be provided in subsequent pregnancy resulting in birth of normal offsprings. 4. Discussion Autosomal recessive congenital ichthyosis are a group of disorders which present as ichthyosis at birth and subsequently evolve into phenotypes of varying severity like lamellar ichthyosis (LI) and congenital ichthyosiform erythroderma (CIE) (Richard and Bale, 1993–2014; Oji et al., 2010). The phenotype at birth is a colloidion baby, where the neonate is covered with a taut, shiny, membrane-like skin or a severe phenotype called harlequin ichthyosis (HI). HI presents with a thick armor-like skin with large cracks covering the whole body, facial abnormalities in the form of severe eclabium and ectropion, joint deformities/ contractures and microcephaly. These children suffer from complications like infections, temperature instability, dyselectrolemia, dehydration, respiratory difficulty and majority succumb in the neonatal life (Richard and Bale, 1993–2014; Rajpopat et al., 2011). Adequate
supportive care with attention to barrier protection, infection control and maintenance of fluid–electrolyte balance along with oral retinoid therapy can lead to survival beyond this period and subsequently these children evolve into a mild phenotype of CIE (Rajpopat et al., 2011, Rossi and Mesia, 2011). Majority (N 93%) of individuals with HI have been found to have mutations in the ABCA12 gene (Richard and Bale, 1993–2014; Rajpopat et al., 2011; Akiyama, 2010; Kelsell et al., 2005; Thomas et al., 2006). This gene encodes for a 2595 amino acid protein which functions as a membrane transporter in the epidermis. It transports lipid molecules and lamellar bodies in the organelles of the stratum corneum cells and hence helps in maintaining the epidermal lipid barrier that is essential to the skin integrity (Akiyama, 2014). Mutations in this gene leading to HI phenotype have been loss of function mutations, either nonsense type or deletions ranging from 1–35 exons. Approximately 56 mutations have been reported till date. A small proportion of individuals with LI have also been reported to have ABCA12 mutations, but these are primarily missense type in a particular domain of the protein (Richard and Bale, 1993–2014; Rajpopat et al., 2011; Akiyama, 2010; Kelsell et al., 2005; Thomas et al., 2006). Here, we report two novel mutations of ABCA12 gene in individuals of Indian origin. Both mutations lead to frameshift, downstream
Please cite this article as: Aggarwal, S., et al., Novel ABCA12 mutations in harlequin ichthyosis: A journey from photo diagnosis to prenatal diagnosis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.002
S. Aggarwal et al. / Gene xxx (2014) xxx–xxx
premature termination codon with presumed formation of a truncated non-functional protein or non-sense mediated decay. This is in corroboration with previous genotype–phenotype reports where severe ABCA12 mutations leading to complete destruction of function or loss of production of ABCA12 protein cause HI phenotype, whereas milder mutations lead to LI phenotype (Richard and Bale, 1993–2014; Rajpopat et al., 2011). In both the cases discussed here, the affected neonate was demised and no biological tissue was available. The causative mutations were identified in the parents on the basis of a diagnosis made from clinical photographs. The identification of mutations confirmed the photodiagnosis and helped in providing prenatal diagnosis by molecular testing in both the families resulting in birth of normal offsprings. To conclude, we report two cases of HI with identification of novel ABCA12 mutations in individuals of Indian origin. The report reiterates the importance of preserving clinical photographs in cases of neonatal demise, which may provide appropriate diagnostic information of utility for subsequent prenatal diagnosis. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.12.002. Acknowledgments We acknowledge the laboratory team of the University Medical Centre Nijmegen St Radboud, The Netherlands who helped to perform the
3
molecular testing in family 2. The Council of Scientific and Industrial Research (1061130842) funded the fellowship for AK. SHHiRT, the Harlequin Ichthyosis Charity funded the PhD studentship for PB. References Akiyama, M., 2010. ABCA12 mutations and autosomal recessive congenital ichthyosis: a review of genotype/phenotype correlations and of pathogenetic concepts. Hum. Mutat. 31, 1090–1096. Akiyama, M., 2014. The roles of ABCA12 in epidermal lipid barrier formation and keratinocyte differentiation. Biochim. Biophys. Acta 1841 (3), 435–440 (Mar). Kelsell, D.P., Norgett, E.E., Unsworth, H., Teh, M.-T., Cullup, T., Mein, C.A., DoppingHepenstal, P.J., Dale, B.A., Tadini, G., Fleckman, P., Stephens, K.G., Sybert, V.P., et al., 2005. Mutations in ABCA12 underlie the severe congenital skin disease harlequin ichthyosis. Am. J. Hum. Genet. 76, 794–803. Oji, V., Tadini, G., Akiyama, M., Bardon, C.B., Bodemer, C., Bourrat, E., Coudiere, P., DiGiovanna, J.J., Elias, P., Fischer, J., Fleckman, P., Gina, M., et al., 2010. Revised nomenclature and classification of inherited ichthyoses: results of the first ichthyosis consensus conference in Soreze 2009. J. Am. Acad. Dermatol. 63, 607–641. Rajpopat, S., Moss, C., Mellerio, J., Vahlquist, A., Ganemo, A., Hellstrom-Pigg, M., Ilchyshyn, A., Burrows, N., Lestringant, G., Taylor, A., Kennedy, C., Paige, D., et al., 2011. Harlequin ichthyosis: a review of clinical and molecular findings in 45 cases. Arch. Dermatol. 147, 681–686. Richard, G., Bale, S.J., 1993–2014. Autosomal recessive congenital ichthyosis. In: Pagon, R.A., Adam, M.P., Bird, T.D., Dolan, C.R., Fong, C.T., Smith, R.J.H., Stephens, K. (Eds.), GeneReviews® [Internet]. University of Washington, Seattle, Seattle (WA) (2001). Rossi, G., Mesia, D., 2011. Management of harlequin ichthyosis in low-income countries. Ann. Trop. Paediatr. 31 (3), 247–249. Thomas, A.C., Cullup, T., Norgett, E.E., Hill, T., Barton, S., Dale, B.A., Sprecher, E., Sheridan, E., Taylor, A.E., Wilroy, R.S., DeLozier, C., Burrows, N., et al., 2006. ABCA12 is the major harlequin ichthyosis gene. J. Invest. Dermatol. 126, 2408–2413.
Please cite this article as: Aggarwal, S., et al., Novel ABCA12 mutations in harlequin ichthyosis: A journey from photo diagnosis to prenatal diagnosis, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.002
