|Year : 2020 | Volume
| Issue : 3 | Page : 167-173
Clinical and molecular characterization of lipoid proteinosis in three Indian families
Sahana M Srinivas1, Madhuri Maganthi2, Priya Jeevamani Chandrasekaran3, Aruna Gowdra4, Raghupathy Palany5
1 Department of Pediatric Dermatology, Indira Gandhi Institute of Child Health, Bengaluru, Karnataka, India
2 Department of Pediatrics, Bangalore Baptist Hospital, Bengaluru, Karnataka, India
3 Department of Dermatology, Bangalore Baptist Hospital, Bengaluru, Karnataka, India
4 Department of Biochemistry, Indira Gandhi Institute of Child Health, Bengaluru, Karnataka, India
5 Department of Pediatric Endocrinology, Indira Gandhi Institute of Child Health, Bengaluru, Karnataka, India
|Date of Submission||16-Jan-2020|
|Date of Decision||13-Feb-2020|
|Date of Acceptance||21-Feb-2020|
|Date of Web Publication||30-Jun-2020|
Dr. Sahana M Srinivas
Department of Pediatric Dermatology, Indira Gandhi Institute of Child Health, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Lipoid proteinosis (LP) is a rare autosomal recessive disorder characterized by deposition of hyaline material in the skin, mucous membrane, and multiple organs resulting in varied manifestations such as scarring and thickening of the skin, beaded papules, and hoarseness of voice. It results from mutations in the gene encoding extracellular matrix protein 1 (ECM1). The objectives of this study are to find the mutation spectrum of the ECM1 gene in the Indian children and study the genotype-phenotype correlation. Materials and Methods: This pilot study included three unrelated Indian families with the clinical diagnosis of LP. Next-generation sequencing was done to look for ECM1 gene mutations in the proband and was confirmed by Sanger sequencing in their siblings and parents. Chromosomal microarray was done wherever necessary. Results: All six children from three unrelated consanguineous families had characteristic clinical features of LP. Two children in family 1 exhibited systemic features like temporal lobe epilepsy in one and central precocious puberty in the other. One child from family 1 and one child from family 3 had short stature. Genetic analysis revealed novel homozygous missense variation in exon 7 (c.856T>C) in family 1 and homozygous nonsense variation in exon 8 (c.1327C>T) in family 2 of the ECM1 gene. Family 3 revealed a large homozygous deletion of the ECM1 gene which has not been previously described. Conclusion: All the three families had clinical heterogeneity. This study documented two novel mutations in the ECM1 gene. Skin severity and systemic involvement were associated with missense mutation. Confirmation of ECM1 gene mutation is necessary for proper genetic counseling.
Keywords: Autosomal recessive, ECM1 gene, lipoid proteinosis, novel mutation
|How to cite this article:|
Srinivas SM, Maganthi M, Chandrasekaran PJ, Gowdra A, Palany R. Clinical and molecular characterization of lipoid proteinosis in three Indian families. Indian J Paediatr Dermatol 2020;21:167-73
|How to cite this URL:|
Srinivas SM, Maganthi M, Chandrasekaran PJ, Gowdra A, Palany R. Clinical and molecular characterization of lipoid proteinosis in three Indian families. Indian J Paediatr Dermatol [serial online] 2020 [cited 2020 Aug 7];21:167-73. Available from: http://www.ijpd.in/text.asp?2020/21/3/167/288506
| Introduction|| |
Lipoid proteinosis (LP), also known as Urbach–Wiethe disease or hyalinosis cutis et mucosae (OMIM: 247100), is an autosomal recessive (AR) single gene disorder characterized by progressive deposition of hyaline material in the skin, mucous membrane, and different tissues of the body resulting in varied manifestations. It was first described by Urbach and Wiethe in 1929. It results from mutations in the gene encoding extracellular matrix protein 1 (ECM1) located on chromosome 1q21.2. The most common location of the mutation is at exons 6 and 7. Clinically, it presents as hoarseness of voice within the 1st year of life due to the infiltration of vocal cords, yellowish beaded papules and nodules on the face, eyelid margins, neck and hands, linear or varioliform scarring on the face, trunk and upper extremities, short stature, respiratory distress, neuropsychiatric abnormalities such as epilepsy, mental subnormality, aggressive attitude, and intracranial calcifications., Although the disease is progressive in early adult life, prognosis is good but affects the quality of life. Widespread visceral involvement such as the lungs, kidney, bladder, and gastrointestinal tract (gastrointestinal bleeding, dysphagia) can lead to life-threatening events. The extensive scarring and hoarseness can give rise to severe psychosocial issues leading to absenteeism from school.
There are more than 300 cases published in worldwide literature, but only a few cases have been genetically confirmed, especially from the Indian subcontinent. The purpose of this study was to understand the molecular basis of LP in Indian children. The phenotype-genotype correlation will help to predict the severity and early interventions of at-risk individuals may prevent complications and improve the quality of life. Genetic confirmation will also facilitate genetic counseling, carrier screening, and prenatal diagnosis. We studied six affected children with varied clinical features along with ECM1 gene mutations.
| Materials and Methods|| |
This study was a prospective observational study of six children diagnosed with LP belonging to three unrelated consanguineous families with Indian ethnicity presenting to the department of pediatric dermatology and clinical genetics at two tertiary care hospitals in south India during the period from January 2018 to December 2018. A detailed history, pedigree analysis, and thorough cutaneous and systemic examination were done in affected children as well as their unaffected siblings and parents. All the demographic details were recorded on a predesigned proforma. Ophthalmology, otorhinolaryngology, and neurology opinions were taken. Complete hemogram, biochemistry profile, random blood sugars, thyroid profile, and computed tomography (CT) scan were done in all children. Magnetic resonance imaging (MRI), electrocardiogram, and two-dimensional echocardiography were done when necessary. Indirect laryngoscopy and skin biopsy were done in all children. All children diagnosed clinically with LP, unaffected siblings and parents were subjected to genetic analysis. Next-generation sequencing (NGS) was performed in the proband and the identified mutation was confirmed by Sanger sequencing in the proband, affected, and unaffected siblings and parents. Chromosomal microarray analysis (CMA) was performed to confirm the deletion. The Institutional Review Board and Ethical committee approval were taken for the study.
- DNA extracted from peripheral blood was used to perform targeted gene capture using a custom capture kit.
- Targeted gene sequencing: Selective capture and sequencing of the protein-coding regions of the genes were performed. The libraries were sequenced to mean >80–100X coverage on illumina sequencing platform. The sequences obtained were aligned to human reference genome (GRch37/hg19) using BWA program and analyzed using Picard and GATK version 3.6. It is a Genome Analysis Toolkit from Broad Institute of Harvard and MIT, USA. to identify variants relevant to the clinical indication. Gene annotation of the variants was performed using visual evoked potential program against the Ensembl release 87 human gene model. Clinically, relevant mutations were annotated using published variants in literature and a set of diseases databases – ClinVar, OMIM, GWAS, HGMD, and SwissVar. Common variants were filtered based on allele frequency in 1000Genome Phase 3, ExAC, EVS, dbSNP147, 1000 Japanese Genome, and our Indian population database. Nonsynonymous variant effect was calculated using multiple algorithms such as Polyphen-2, Protein Variation Effect Analyzer (PROVEAN), SIFT, and Mutation Taster2.
- Sanger sequencing: exon 7 and exon 8 of the ECM1 gene was polymerase chain reaction-amplified, and the product was sequenced using Sanger sequencing. The detection limits of Sanger sequencing for the presence of variation were ~12%. The sequencing was aligned to available reference sequence ENST00000369049 to detect variation using the analysis software programs.
- CMA to detect large deletion was performed using the Affymetrix Cytoscan™ XON array, Thermo Fisher Scientific, Waltham, USA. Genomic DNA (100 ng) was isothermally amplified. Amplified DNA was fragmented to product size of 25 bp–125 bp, and re-precipitated. Resuspended DNA was labeled and hybridized on Cytoscan™ XON gene chip and then scanned. Data were analyzed using the chromosome analysis suite. The analysis is based on the human reference genome (GRch37/hg19).
| Results|| |
The clinical and molecular findings of all six children with LP are summarized in [Table 1].
Family 1 (cases 1-3)
Three female siblings aged 8 years, 16 years, and 17 years presented with skin lesions on the face, trunk, and extremities from infancy. They were born at term to third-degree consanguineous marriage [Figure 1]a. Recurrent blisters on the face were seen in cases 2 and 3. Case 1 had a mild developmental delay with poor scholastic performance. All these girls had hoarseness of voice, beaded eyelid papules, and characteristic facial scarring [Figure 1]b, [Figure 1]c, [Figure 1]d, [Figure 1]e. Hyperkeratosis on the elbows and knees was present in case 1. Thick lips, macroglossia with a restricted protrusion, and thick sublingual frenulum were seen in cases 1 and 3 [Figure 1]c. Yellowish nodules on the palate were present in all children. Systemic involvement was seen in case 1 (central precocious puberty, hypothyroidism, and short stature) and case 3 (temporal lobe epilepsy). Routine blood tests were normal. CT scan and MRI showed calcifications in the amygdala and hippocampus in all children. Indirect laryngoscopy revealed vocal cord hypertrophy. Case 1 was treated with thyroxine replacement and three monthly GnRH analog injection.
|Figure 1: (a) Pedigree of family 1, (b) case 1 showing beaded papules, facial scarring with thick and coarse facies, (c) macroglossia with the restricted protrusion in case 1, (d and e) case 2 and 3 showing diffuse facial scarring and beaded papules with coarse facies|
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Molecular analysis in all siblings showed homozygous missense variation in exon 7 of the ECM1 gene, resulting in the amino acid substitution of arginine for cysteine at codon 286 (p. Cys286Arg; ENST00000369049) [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d. This ECM1 variant has not been reported in the Human Gene Mutation Database (HGMD) Professional 2018.4, dbSNP, 1000 genomes, ExAC, and Indian databases. We also excluded it from 300 normal healthy Indians. The in silico predictions of the variant is probably damaging by Polyphen-2 (with a score of 1.00), PROVEAN (predicted it to be deleterious with a score of −9.18), SIFT and Mutation Taster2. The reference codon is conserved across mammals. The identical mutation was present in the heterozygous condition in the unaffected parents.
|Figure 2: The Integrative Genomics Viewer and chromatogram views of the variation of case 1. (a) Excerpt of next-generation sequencing data visualized with integrative genomic viewer showing homozygous missense variation in exon 7 of the extracellular matrix protein 1. (b) Sequence chromatogram and alignment to the reference sequence showing the variation in exon 7 of the extracellular matrix protein 1 gene (chr1:150483999T>C; c. 856T>C) of the index child, and heterozygous carrier mother (c), and father (d) indicated by red arrow|
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Family 2 (cases 4 and 5)
Two male siblings aged 17 years and 18 years, born at term to third-degree consanguineous parents presented with skin lesions on the face and trunk from infancy [Figure 3]a. Both children had normal developmental milestones. Clinically, both children had hoarseness of voice from birth, beaded eyelid papules, facial scarring, acneiform scarring, hyperkeratosis of the elbows and knees, partial alopecia, and yellowish nodules on the palate [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]e, [Figure 3]f, [Figure 4]a and [Figure 4]b. Case 4 had skin-colored papules on the neck along with thick and fissured lips, macroglossia, and thick sublingual frenulum [Figure 3]c and [Figure 3]f. There was no systemic involvement in both the children. CT scan showed intracranial calcifications in the amygdala in both the children. Molecular testing in both boys showed homozygous nonsense variation in exon 8 of the ECM1 gene, resulting in a stop codon and premature truncation of the protein at codon 443(p. Arg443Ter; ENST00000369049) [Figure 5]a, [Figure 5]b, [Figure 5]c, [Figure 5]d. The p. Arg443Ter variant has been reported in HGMD Professional 2018.4. The in silico predictions of the variant are damaging by Polyphen-2, SIFT, MutationTaster2 and PROVEAN (predicted it to be deleterious with a score of − 8.54). The reference codon is conserved across species. An identical mutation was detected in the heterozygous condition in unaffected three siblings and parents.
|Figure 3: (a) Pedigree of family 2, case 4 showing (b) facial scarring, beaded papules (c) thick lips, macroglossia with indentations, (d and e) hyperkeratosis on the elbow and knee joints. (f) Skin colored to yellowish papules on the neck|
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|Figure 4: Case 5 showing (a) beaded papules and facial scarring, (b) yellowish nodules on the hard palate|
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|Figure 5: Integrative Genomics Viewer and chromatogram views of the variation of case 4. (a) Excerpt of next-generation sequencing data visualized with the Integrative Genomics Viewer showing homozygous nonsense variation in exon 8 of the extracellular matrix protein 1 gene. (b) Sequence chromatogram and alignment to the reference sequence showing the variation in exon 8 of the extracellular matrix protein 1 gene (chr1:150484990C>T; c. 1327C>T) of the index child, and heterozygous carrier mother (c), and father (d) indicated by red arrow|
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Family 3 (case 6)
This 14-year-old boy, born at term presented with generalized blisters from 4 months of age and hoarseness of voice from 7 months of age. He is the 5th child born to third-degree consanguineous parents with uneventful birth and neonatal period [Figure 6]a. His developmental milestones were normal. There was a history of two sibling deaths at 2 years of age who had similar skin lesions, hoarseness of voice, and seizures. However, there were no medical records available to document the cause of death. There was no history of similar illness in the extended family. He was malnourished with short stature with no systemic involvement. Cutaneous examination showed crusting, erosions on the scalp, face, trunk and extremities, beaded eyelid papules, facial scarring, hyperkeratosis on the elbows and knees, sparse hair, macroglossia with restricted protrusion and thick, and short sublingual frenulum [Figure 6]b and [Figure 6]c. Indirect laryngoscopy revealed granular deposits over the laryngeal inlet. CT scan showed coarse, clumpy, and symmetrical amygdala calcification. Molecular analysis by NGS showed homozygous large deletion of the ECM1 gene. Confirmatory test with CMA detected a homozygous loss of 11 kbp comprising the ECM1 gene at chromosome 1 at cytoregion q21.3, suggestive of large deletion (arr (hg19 1q 21.3 (150, 480, 075–150, 490, 826)) ×0) [Figure 6]d. We were not able to confirm this deletion in unaffected siblings and parents as they did not give the consent for further testing.
|Figure 6: (a) Pedigree of family 3, case 6 showing (b) beaded papules and facial scarring on the face, (c) diffuse erosions and crusting present on the back, (d) cytogenomic microarray analysis showing a homozygous loss of 11 kbp comprising extracellular matrix protein 1 gene at ch1q21.3|
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| Discussion|| |
LP is a rare AR disorder seen worldwide, more commonly reported from South Africa and European regions. The exact prevalence is not known. The largest series of LP has been reported from South Africa. It has variable phenotype ranging from mild clinical features to severe life-threatening complications. In our study, all children showed characteristic cutaneous features of LP. Case 6 had severe cutaneous involvement with generalized erosions, crusting, and scarring along with sparse hair on the scalp. Extracutaneous features were documented in family 1 and family 3. Case 1 had hypothyroidism and central precocious puberty which is not described in literature. Epilepsy has been documented in 25% of cases in various studies., Case 3 had temporal lobe epilepsy which has been associated with LP in literature. In a study by Oguz Akarsu et al., intracranial calcifications were seen in 50% of cases, but in our study, all cases had intracranial calcifications. Short stature was present in case 1 and case 6.
The exact pathogenesis of LP is not known. ECM1 is a glycoprotein that binds to basement membrane proteoglycans, growth factors, and fibrillar protein and has a role in wound healing.ECM1 gene has also a role in tumorigenesis, tumor metastasis and is a risk factor for chronic inflammatory bowel diseases.ECM1 gene is located on chromosome 1q21.2, comprises 10 exons. Till date, there are 66 different pathogenic ECM1 gene mutations described in all 10., Both homozygous and compound heterozygous genotypes have been described. Mutations described are frameshift, missense, nonsense, splice site, small and gross insertions and deletions. Fifty percent of the mutations were in exon 6 and exon 7 and found to have missense or nonsense mutation. Most of the mutations described are specific to individual families and only a few are found to be recurrent. There are only 3 studies from the Indian scenario which has shown frameshift and nonsense mutations in exon 6 and exon 7.,,,, Both inter- and intra-familial clinical heterogeneity has been reported by various studies and most of them have not been able to do genotype–phenotype correlation.,, Genetic modifying factors and environmental factors may play a role in clinical heterogeneity., Similar to other studies, we were unable to do phenotype-genotype correlation due to clinical heterogeneity in families and among study patients and also due to the small sample size.
The study has shown missense, nonsense, and large deletion in the ECM1 gene. Nonsense mutation in exon 8 is predicted to result in loss of function of the ECM1 gene due to nonsense-mediated mRNA decay mechanism., Large deletion affecting part of the ECM1 gene and adjacent long noncoding RNA (5141 bp) has been reported by Lee et al. Ours is the only case in literature having homozygous 11 kbp deletion which deletes the whole ECM1 gene. Cutaneous lesions were more severe in case 6 showing large deletion as compared to other cases with missense and nonsense mutation in our study. Missense mutation was associated with systemic involvement in our study. There is no effective treatment for LP. Oral retinoids have shown to improve hoarseness of voice than cutaneous lesions. Other treatments that have shown good clinical response are oral dimethylsulfoxide and D-pencillamine.,
| Conclusion|| |
Our study showed similar existing features of LP in all cases, but severity and expressivity differed between patients and between patients from the same family. All 3 families had unique mutations. We describe two novel mutations: missense and large deletion increasing the mutation spectrum of the ECM1 gene. Ours is the only study using NGS and CMA to identify ECM1 gene mutations. Analysis by NGS will further increase the knowledge and understanding of the molecular basis of LP which will facilitate proper genetic counseling. The limitation of this study was that we could not perform functional assays. Large population studies are required to predict the severity and to do genotype-phenotype correlation.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that name and initials will not be published and due efforts will be made to conceal identity, but anonymity cannot be guaranteed.
We the authors would like to thank the Bangalore Dermatological Society, Bengaluru, Karnataka, India, for funding the project for genetic analysis and Medgenome Labs LTD for performing genetic analysis for our patients.
Financial support and sponsorship
The study was financially supported by Bangalore Dermatological Society, Bengaluru, Karnataka, India.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]