A novel homozygous mutation 371delA in TGM1 leads to a classic lamellar ichthyosis phenotype

Malformation of the cornified cell envelope (CCE) arising from mutations of the transglutaminase (TGase) 1 gene (TGM1) is the cause of some cases of lamellar ichthyosis (LI). However, genotype/phenotype correlation in TGM1 mutations has not yet been fully clarified. We report a typical case of LI caused by a novel mutation in TGM1. The patient, a 33-year-old woman, showed thick, lamellar scales on the entire body surface. Immunofluorescence labelling with anti-TGase 1 antibodies was negative in the patient's epidermis. In situ TGase activity assay detected markedly reduced TGase activity in granular layers of the patient's epidermis. Electron microscopy revealed incomplete thickening of the CCE during keratinization in the epidermis. Sequencing of the entire exons and exon-intron borders of TGM1 revealed that the patient was a homozygote for a novel deletion mutation 371delA in exon 3. This mutation leads to a frameshift resulting in a premature termination codon 43 bp downstream from the mutation site. According to the protein modelling of TGase 1, the truncated protein from this mutated allele loses the entire catalytic core domain of TGase 1. Thus, the present homozygous mutation is expected to cause total loss of TGase 1 activity, resulting in large, dark, lamellar scales on the entire body, the classic phenotype of LI, in this patient.     

Structural, enzymatic and molecular studies in a series of nonbullous congenital ichthyosiform erythroderma patients

Causative gene defects have not been demonstrated in the majority of nonbullous congenital ichthyosiform erythroderma (NBCIE) cases. The purpose of this study was to further elucidate the pathogenesis of NBCIE. Immunohistochemical and ultrastructural observations, transglutaminase activity assays and sequencing of TGM1 were performed in five patients from four NBCIE families. Transglutaminase 1 (TGase 1), involucrin and loricrin expression and in situ transglutaminase activity were present in all of the cases. Ultrastructurally, two cases out of five showed incomplete thickening of the cornified cell envelope (CCE) during keratinization and the other three exhibited abnormal lipid droplets in the cornified cells and malformed lamellar granules. No TGM1 mutation was found in any of the four families by direct sequence analysis. NBCIE cases with normal TGase 1 seemed to have two distinct patterns of abnormality, one with abnormal lipid droplets and malformed lamellar granules and the other with defective CCE formation.     

Novel mutations of the transglutaminase 1 gene in lamellar ichthyosis

Lamellar ichthyosis, one form of congenital autosomal recessive ichthyosis, is caused by mutations in the gene (TGM1) encoding the transglutaminase 1 enzyme. Mutations, deletions, or insertion of TGM1 have been reported so far. Here we report that three novel mutations of TGM1, D101V, N288T, and R306W, cause lamellar ichthyosis in two different families. The patient in family LI-KD has N288T and R306W mutations, and the patient in family LI-LK has D101V and R306W mutations. The activity of the transglutaminase 1 enzyme of the patient in family LI-LK was only about 15% of normal. Also, three-dimensional structural prediction analyses revealed that the N288T and R306W mutations, and possibly the D101V mutation, cause misfolding in the central catalytic core domain of the transglutaminase 1 enzyme that would probably result in reduced enzyme activity. Our data suggest that the greatly reduced transglutaminase 1 activities are due to disruptions of the native folding of transglutaminase 1, and that these mutations may play a critical role in the pathology of lamellar ichthyosis.     

Identification of two novel nonsense mutations in the transglutaminase 1 gene in a Hungarian patient with congenital ichthyosiform erythroderma

Congenital ichthyosiform erythroderma (CIE) belongs together with lamellar ichthyosis (LI) to the group of autosomal recessive congenital ichthyoses (ARCI). Mutations in the transglutaminase (TGase) 1 gene (TGM1) have been identified in several families with LI and in some families with CIE. We report a case of CIE with two new nonsense mutations: a C7780G transversion in exon 11 resulting in a premature stop codon at aminoacid residue Y503X and a C8533G transversion in exon 13 leading to a nonsense mutation at S669X. These mutations were also identified in a heterozygous pattern in the unaffected parents. These two termination-codons result in the translation of a truncated protein at the C-terminal end domain of the TGM 1 molecule. B.C1 monoclonal antibody failed to detect TGase 1 in the patient's skin sample, and TGase activity measured by monodansyl cadaverine-incorporation showed the reduced TGase activity at the distribution of TGase 1 in the epidermis.     

Acral self-healing collodion baby: report of a new clinical phenotype caused by a novel TGM1 mutation

A minority of collodion babies, called 'self-healing collodion babies', heal spontaneously. We describe a novel clinical phenotype of acral self-healing collodion baby caused by a new TGM1 mutation. The proband, born to healthy parents, presented at birth as a collodion baby strictly localized to the extremities. The skin condition returned to normal at the age of 3 weeks. The older sister was born as a generalized collodion baby; the condition then developed into lamellar ichthyosis. Molecular analysis of TGM1 revealed three novel mutations in the family. The proband was compound heterozygous for the p.Val359Met and p.Arg396His mutations, whereas the older sister was compound heterozygous for p.Arg396His and a deletion mutation c.1922_1926+2delGGCCTGT. Structural modelling of the p.Val359Met mutation suggested a minor disruption of the protein structure, whereas a modification of protein–protein interaction was predicted for p.Arg396His. These predictions corroborated the analysis of recombinant transglutaminase (TGase)-1 proteins carrying the p.Val359Met and p.Arg396His mutations. Both showed decreased levels of protein expression: p.Val359Met displayed residual activity (12·8%), while p.Arg396His caused a dramatic loss of activity (3·3%). These observations demonstrate for the first time that TGM1 mutations can be associated with acral self-healing collodion baby, and expand the clinical spectrum of TGase-1 deficiency.     

Unique and recurrent mutations in the filaggrin gene in Singaporean Chinese patients with ichthyosis vulgaris

Filaggrin is an abundant protein of the outer epidermis that is essential for terminal differentiation of keratinocytes and formation of an effective barrier against water loss and pathogen/allergen/irritant invasion. Recent investigations in Europe and Japan have revealed null mutations in the filaggrin gene (FLG) as the underlying cause of ichthyosis vulgaris (IV), a common skin disorder characterised by dry skin, palmar hyperlinearity and keratosis pilaris. Following the development of a strategy for the comprehensive analysis of FLG, we have identified five unique mutations and one recurrent mutation in Singaporean Chinese IV patients. Mutation 441delA is located in the profilaggrin S100 domain, whereas two additional frameshift mutations, 1249insG and 7945delA, occur in the first partial filaggrin repeat ("repeat 0") and in filaggrin repeat 7, respectively. Both nonsense mutations Q2147X and E2422X are found in filaggrin repeat 6, whereas R4307X was found on one of the longer size variant alleles of FLG, within duplicated repeat 10.2. Mutation E2422X, previously found in a single Dutch patient, was found in one Singaporean IV patient and at a low frequency in Asian population controls. Our study confirms the presence of population-specific as well as recurrent FLG mutations in Singapore.     

板层状鱼鳞病TGM1基因突变研究

目的 探讨一个板层状鱼鳞病家系转谷氨酰胺酶1基因(TGM1)的突变.方法 提取板层状鱼鳞病患者及家族成员的基因组DNA,采用PCR扩增TGM1基因所有的外显子及其邻近的剪切点并进行双向直接测序,并对TGM1基因的同源性进行分析.结果 板层状鱼鳞病患者TGM1基因存在异常:外显子3的第504位碱基由胞嘧啶突变为胸腺嘧啶,使第142位氨基酸由精氨酸(R)转变为半胱氨酸(C),即R142C错义突变;外显子7的第1122位碱基由胞嘧啶突变为胸腺嘧啶,使348位氨基酸由精氨酸(R)突变为终止密码(R348X),导致其编码的蛋白缺失了C端的470个氨基酸.其父亲为R142C杂合子,母亲为R348X突变杂合子;R142C错义突变位于TGM1基因保守区域.结论 该板层状鱼鳞病患者存在转谷氨酰胺酶1基因的R142C错义突变和R348X无义突变.     

一例火棉胶样婴儿TGM1基因变异的致病性研究

【摘要】 目的 探讨1个先天性常染色体隐性遗传性鱼鳞病家系的遗传学病因。方法 对1例火棉胶样婴儿进行全外显子组测序,Sanger测序验证基因变异位点。应用PolyPhen?2、PROVEAN 和Mutation Taster软件以及蛋白同源建模方法预测基因变异的性质;实时荧光定量PCR和Western印迹法分析变异对等位基因mRNA与蛋白表达量的影响。结果 全外显子组测序与Sanger验证结果证实患儿TGM1基因第6外显子存在c.919C>T（p.Arg307Trp）变异,第7外显子存在c.1019G>A（p.Gly340Glu）变异,且两变异等位基因分别遗传自其母亲与父亲。生物信息学预测提示两种变异均对蛋白结构有害,蛋白同源建模结果进一步证实上述结论。体外实验显示转染野生型质粒与转染c.919C>T或c.1019G>A突变型质粒的293T细胞TGM1基因mRNA表达量差异无统计学意义（t值分别为1.97、1.28,P值分别为0.12、0.27）,但转染突变型TGM1质粒的细胞TGase1蛋白含量显著下降。结论 TGM1基因c.919C>T与c.1019G>A变异可能是该严重鱼鳞病患儿的分子遗传学病因,其编码的错义氨基酸可能通过破坏TGase1蛋白结构,从而影响蛋白功能。     

Keratin 9 gene mutations in five Korean families with epidermolytic palmoplantar keratoderma

Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant disease characterized clinically by localized palmoplantar thickening and histopathologically by granular degeneration of the epidermis. Recent molecular biological studies have revealed that EPPK is caused by mutations of the keratin 9 gene in sequences mainly encoding the highly conserved 1 A rod domain. Here we demonstrate a novel mutation of N160H (position 8 of the 1 A domain) and two other previously reported mutations, R162W and N160S, in five unrelated Korean families with EPPK. The three-dimensional structure of the 1 A domain of the related vimentin intermediate filament protein chain is now known. Based on its likely similarity to the keratin 9 chain, we predict that inappropriate amino acid substitutions in position 10 of 1 A will likely interfere with coiled-coil dimer stability, and those in position 8 will interfere with tetramer stability. Accordingly, these mutations compromise the structural integrity of the keratin intermediate filaments leading to the pathology of EPPK.     

Lamellar ichthyosis

A 6-year-old African boy with a history of a collodion membrane presented with scale in a generalized distribution and flexural accentuation. Large, brown, polygonal scales were present on the forehead, lateral aspects of the face, and extremities. The nature of the scales and the lack of erythroderma in this patient are consistent with a mild form of lamellar ichthyosis (LI). LI and nonbullous congenital ichthyosiform erythroderma (NBCIE) represent phenotypes at the poles of the autosomal recessive ichthyosis spectrum. Mutations in genes encoding transglutaminase 1 (TGM1), the ABCA12 transporter (ABCA12), ichthyin, lipoxygenase 3 (ALOXE3), and 12(R)-lipoxygenase (ALOX12B) have been shown to underlie both NBCIE and LI.     

Mutation analysis of ATP2C1 gene in Taiwanese patients with Hailey-Hailey disease

BACKGROUND: Hailey-Hailey disease (HHD) is an autosomal dominant disorder with recurrent eruption of vesicles and bullae involving predominantly the neck, groin and axillary regions. Histopathology shows suprabasal cleavage in epidermal cells. Recent studies have revealed that HHD is caused by mutations in the ATP2C1 gene encoding a novel Ca2+ pump. OBJECTIVES: To analyse the mutations of the ATP2C1 gene in Taiwanese patients with HHD. METHODS: In total, five familial and two sporadic cases of HHD were retrieved from the medical records. The diagnosis of HHD was made based on the characteristic clinical features and histopathological evidence. All 27 exons and flanking intron boundaries were amplified by polymerase chain reaction and products analysed by direct sequencing. RESULTS: We identified six novel mutations and one reported mutation: three deletion mutations (nt884-904del, 1459delCTCA, 1975delA), two non-sense mutations (R39X, R783X), one mis-sense mutation (A730T) and one splicing mutation (483 + 2T-->A). The non-sense mutation R39X had been reported previously; the other six mutations are novel mutations. CONCLUSIONS: These results demonstrate that a spectrum of ATP2C1 gene mutations is present in Taiwanese HHD patients.     

Paternal germline mosaicism in Herlitz junctional epidermolysis bullosa

We studied a single patient with the lethal (Herlitz) type of junctional epidermolysis bullosa (H-JEB). Screening for mutations in the LAMB3 gene in the patient revealed the previously described hotspot mutation R635X and a novel one basepair deletion in exon 10. The single basepair deletion 1094delA could be detected in the clinically unaffected mother, while the nonsense mutation R635X could not be found in the peripheral blood DNA of either parent. After excluding non-paternity by microsatellite analysis using random markers on chromosomes 3, 8 and 18, we determined that the mutation R635X in the proband was most likely the result of a de novo event or alternatively, germline mosaicism. The parents requested prenatal diagnosis for a second pregnancy, and while the maternal mutation 1094delA could not be detected in DNA from the fetus, unexpectedly, the mutation R635X was present in the chorionic villus DNA. These findings were most consistent with paternal germline mosaicism for the recessive mutation R635X. The results have had a significant impact on the genetic counseling in this family. To our knowledge, this study represents the first documented case of germline mosaicism in junctional epidermolysis bullosa, and serves as a reminder that germline mosaicism should be considered in cases in which a 'new' mutation is found in the offspring of a clinically and/or genetically unaffected parent.     

Fibroblast apoptosis in a patient affected by lamellar ichthyosis

Background:  Lamellar ichthyosis (LI) is a congenital recessive skin disorder characterized by generalized scaling and hyperkeratosis. The pathology may be caused by mutations in transglutaminase 1 (TGM1) gene that encodes an enzyme critical for terminally differentiating keratinocytes. Because of evidences that transglutaminase enzymes are involved in programmed cell death, we investigated morphological and biochemical apoptotic parameters in cultured skin fibroblasts from a patient with a severe LI and homozygous for the TGM1 R142H mutation.
Method:  The principle apoptotic signals (mitochondrial membrane potential, analysis of oxygen consumption, DNA fragmentation and Bax/Bcl-2 gene expression) were analyzed in cultured fibroblasts from a LI patient, his mother (TGM1 mutation carrier) and a control subject.
Results:  LI fibroblasts showing a reduction of fibronectin expression evidenced a strong inhibition of oxygen consumption, a dramatic drop in the mitochondrial membrane potential (Δψ_m), and a higher apoptotic index.
Conclusion:  The present results suggest a possible connection between the alterations in the keratinization process leading to LI and the observed increased fibroblast apoptosis.     

Epidermolysis bullosa simplex Dowling-Meara due to an arginine to cysteine substitution in exon 1 of keratin 14

Epidermolysis bullosa simplex (EBS) is a blistering disorder affecting the basal layer of the epidermis usually inherited in an autosomal dominant fashion. Most cases are caused by mutations in the genes encoding keratin 5 (K5) and keratin 14 (K14) and are characterized by cytolysis within the basal layer of the epidermis. We report a patient manifesting the Dowling-Meara variant of EBS in whom we characterized a cytosine to thymine transition at codon 125 (R125C) in K14. This missense mutation is located at the amino terminus of the helical rod domain of the keratin 14 molecule, resulting in defective pairing with K5, thereby disrupting keratin tonofibril integrity.     

寻常型鱼鳞病一家系Filaggrin基因突变检测

目的探讨一家系寻常型鱼鳞病(ichthyosis vulgaris,IV)丝聚合蛋白(filaggrin,FLG)基因的突变。方法提取IV患者及其家庭成员和100例健康对照者基因组DNA,采用PCR及直接测序法,对FLG基因已报道的13个突变位点(3321delA,441delA,1249insG,E1795X,S3296X,R501X,2282del4,R2447X,S2889X,7945delA,3702delG,Q2417X,R4307X)进行测序。结果三代7位成员中4例IV患者同时检测到FLG(441delA)基因突变。结论患者FLG(441delA)基因突变可能导致其发病。     

Lamellar ichthyosis with a novel homozygous C-terminal mutation in the transglutaminase-1 gene

Background  The majority of cases of Lamellar ichthyosis (LI) are caused by mutations in the transglutaminase-1 ( TGM1 ) gene. The mutations in the β-barrel domains of the TGM1 gene are found very infrequently and several authors have suggested that these domains are not essential for the function of the enzyme. Other authors have postulated that mutations in these loci are pathogenic but they imply a less severe clinical picture of LI.
Case report  We report a patient with a severe phenotype of LI who had a homozygous mutation affecting the β-barrel 2 domain of the TGM1.
Conclusions  This finding indicates that the integrity of β-barrel domains is important for the correct function of the enzyme and that we are still far away from a consistent genotype-phenotype correlation.     

The clinical spectrum of nonbullous congenital ichthyosiform erythroderma and lamellar ichthyosis

Until about 20 years ago, the term lamellar ichthyosis (LI) represented all nonbullous autosomal recessive ichthyoses except for harlequin ichthyosis and ichthyosis syndromes. Since the 1980s, nonbullous autosomal recessive ichthyoses have been divided into two major clinical entities, nonbullous congenital ichthyosiform erythroderma (NBCIE) and LI. The nature of scaling and intensity of erythroderma are important clinical features that distinguish between NBCIE and LI. However, a considerable number of cases show an intermediate phenotype between the two classic clinical features. Histologically, parakeratosis and inflammatory cell infiltration are seen more frequently in NBCIE than in LI and the stratum corneum is usually thicker in LI than in NBCIE. However, neither histopathological findings nor ultrastructural features seem to help clearly distinguish between NBCIE and LI. Mutations in any of the three known causative genes, TGM1, ALOXE3 or ALOX12B, can lead either to NBCIE or LI. Candidate genes specific to either NBCIE or LI alone have not been identified. Based on these facts, it might be better to consider NBCIE and LI as variations of a single keratinization disorder, although the classification of these autosomal recessive congenital ichthyosis patients into NBCIE or LI depending on their clinical features is still useful for practical patient management.     

Analyses of the transglutaminase 1 gene mutation and ultrastructural characteristics in a Japanese patient with lamellar ichthyosis

We described a Japanese female with lamellar ichthyosis whose transglutaminase 1 gene (TGM1 gene) was mutated. DNA sequence analysis revealed that the patient had a homozygous mutation, i.e. a point mutation from G to A at nucleotide 1494 resulting in the substitution of glycine for arginine at codon 143. Her mother was heterozygous for this mutation. In situ transglutaminase assay in the patient's skin showed loss of enzyme activity. Ultrastructural examination revealed incomplete formation of cornified cell envelopes and electron-dense materials adjacent to plasma membranes. These results suggest that defective transglutaminase activity caused by homozygous TGM1 gene mutation (G143R) results in disruption of cornified envelope assembly and the clinical phenotype of lamellar ichthyosis.