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Objective: Here we try to explore patient genotype with the aim to locate and analyze SPINK5 mutations in 3 chinese patients suffering from Netherton Syndrome. Using immunohistochemistry analysis we tried to observe LEKTI expression in the epidermis and investigate the relationship between phenotypical severity and epidermal LEKTI concentration. We also evaluate and document the effectiveness of IVIG therapy administered in these patients for three consecutive months.
Methods:Our study comprised of three Chinese patients suffering from Netherton Syndrome from non consanguineous families. After a proper written consent was obtained from the parents of the patients, an adequate volume of blood was taken from the patients and rest of the members of their families. This was followed by DNA extraction using a commercially available kit. Direct PCR was performed on the extracted DNA using a PCR kit following the instruction included using all of the 33 exons of SPINK5 gene. PCR was performed under the previously established conditions given in various publications. After obtaining the PCR product it was further analyzed for mutations in SPINK5 gene by using Chromas 240 S systems to find the exact location of the mutation.We also employed Quantitative PCR analysis to reveal any large allelic deletions in proband 1 and 2.
To investigate the possible effect of SPINK5 mutations on the LEKTI expression in our patients we carried out immunohistochemistry on the skin samples from proband 2. After proper consent small skin section from ILC lesion area was taken with a clean incision following locally anaesthetizing the area with subcutaneous lidocaine. For LEKTI detection we used anti-human DB-1 rabbit polyclonal antibody (Santa Cruz bio technology,INC) against LEKTI domains DI-D6(N-terminal)
In our study we also examine the efficacy of IVIG therapy which we used to manage the NS manifestation in our patients.During the course of treatment we administered IVIG infusion at a concentration of 500mg/kg/day each week for at least three months All the patients were able to complete the therapy.
Results: We were successful in analyzing and locating suspected SPINK5 gene mutation in all of the three patients and their parents. Among the three patients, Patient one, a 4 year old female child showed a single nucleotide variation which was a homozygous nonsense c.2260A＞T SPINK5 mutation in exon 24. The amino acid change observed was p.K754X. This mutation has been reported earlier in two siblings of Taiwanese origin by Chao et al. in 2004. Patient 2, a 4 years old female was found to have a compound heterozygous mutation, of which one is IVS11+7A＞T located in intron 11 of the SPINK 5 gene and another is a large fragment deletion 200bp up and down the IVS11+7 on the other allele. This is a novel mutation which has not been reported in earlier publications. In Patient 3, a 2 year old female child, we discovered a homozygous c.80A＞G mutation in exon 2 with amino acid change p.Q27R. This mutation is a novel kind without any mention in earlier literature.
IHC analysis carried out in proband 2 revealed residual LEKTI concentration in the epidermis as opposed to the NULL LEKTI expression in majority of the other IHC evaluations carried out in NS syndrome patients.This, with the support from previous studies we propose might be due the“LEAKY”nature of the mutation in proband 2.Dermoscopy and light microscopic examination of scalp hair demonstrated characteristic bamboo nodes which is a ball and socket deformity of hair shaft.This is a consistent hair shaft deformity found in NS.
The treatment options for managing NS are very limited.Based on previous studies we chose IVIG therapy as the main treatment modality for our patients.We prescribed IVIG infusions at 500 mg/kg/day each week for three months.At the end of therapy we observed a significant improvement of the erythematous skin lesions and edema.There was also a considerably lesser scaling and pruritis.
Conclusion:
Here we report successful unmasking of three distinct mutations in three patients of Chinese origin. Two out of three mutations that we report here are novel in nature. Through IHC we successfully established LEKTI level discrepencies (residual expression) in the skin sample of proband 2. Supported by these IHC results showing truncated LEKTI expression and its impact on cutaneous severity, we conclude that the phenotypical features and severity are directly linked to the epidermal LEKTI levels present in our patients which in turn is directly dependent on the type of SPINK5 mutations inherited.Dermoscopic examination of hair shaft revealed typical bamboo node appearance.This is an integral finding of TI in NS patients We also affirm the effectiveness of IVIG treatment to palliate cutaneous severity in Netherton syndrome following a three month therapy in our patients.Although the degree of remission were different in different patients but significant reduction of erythroderma,scaling,edema and pruritis was seen in all patients.
Methods:Our study comprised of three Chinese patients suffering from Netherton Syndrome from non consanguineous families. After a proper written consent was obtained from the parents of the patients, an adequate volume of blood was taken from the patients and rest of the members of their families. This was followed by DNA extraction using a commercially available kit. Direct PCR was performed on the extracted DNA using a PCR kit following the instruction included using all of the 33 exons of SPINK5 gene. PCR was performed under the previously established conditions given in various publications. After obtaining the PCR product it was further analyzed for mutations in SPINK5 gene by using Chromas 240 S systems to find the exact location of the mutation.We also employed Quantitative PCR analysis to reveal any large allelic deletions in proband 1 and 2.
To investigate the possible effect of SPINK5 mutations on the LEKTI expression in our patients we carried out immunohistochemistry on the skin samples from proband 2. After proper consent small skin section from ILC lesion area was taken with a clean incision following locally anaesthetizing the area with subcutaneous lidocaine. For LEKTI detection we used anti-human DB-1 rabbit polyclonal antibody (Santa Cruz bio technology,INC) against LEKTI domains DI-D6(N-terminal)
In our study we also examine the efficacy of IVIG therapy which we used to manage the NS manifestation in our patients.During the course of treatment we administered IVIG infusion at a concentration of 500mg/kg/day each week for at least three months All the patients were able to complete the therapy.
Results: We were successful in analyzing and locating suspected SPINK5 gene mutation in all of the three patients and their parents. Among the three patients, Patient one, a 4 year old female child showed a single nucleotide variation which was a homozygous nonsense c.2260A＞T SPINK5 mutation in exon 24. The amino acid change observed was p.K754X. This mutation has been reported earlier in two siblings of Taiwanese origin by Chao et al. in 2004. Patient 2, a 4 years old female was found to have a compound heterozygous mutation, of which one is IVS11+7A＞T located in intron 11 of the SPINK 5 gene and another is a large fragment deletion 200bp up and down the IVS11+7 on the other allele. This is a novel mutation which has not been reported in earlier publications. In Patient 3, a 2 year old female child, we discovered a homozygous c.80A＞G mutation in exon 2 with amino acid change p.Q27R. This mutation is a novel kind without any mention in earlier literature.
IHC analysis carried out in proband 2 revealed residual LEKTI concentration in the epidermis as opposed to the NULL LEKTI expression in majority of the other IHC evaluations carried out in NS syndrome patients.This, with the support from previous studies we propose might be due the“LEAKY”nature of the mutation in proband 2.Dermoscopy and light microscopic examination of scalp hair demonstrated characteristic bamboo nodes which is a ball and socket deformity of hair shaft.This is a consistent hair shaft deformity found in NS.
The treatment options for managing NS are very limited.Based on previous studies we chose IVIG therapy as the main treatment modality for our patients.We prescribed IVIG infusions at 500 mg/kg/day each week for three months.At the end of therapy we observed a significant improvement of the erythematous skin lesions and edema.There was also a considerably lesser scaling and pruritis.
Conclusion:
Here we report successful unmasking of three distinct mutations in three patients of Chinese origin. Two out of three mutations that we report here are novel in nature. Through IHC we successfully established LEKTI level discrepencies (residual expression) in the skin sample of proband 2. Supported by these IHC results showing truncated LEKTI expression and its impact on cutaneous severity, we conclude that the phenotypical features and severity are directly linked to the epidermal LEKTI levels present in our patients which in turn is directly dependent on the type of SPINK5 mutations inherited.Dermoscopic examination of hair shaft revealed typical bamboo node appearance.This is an integral finding of TI in NS patients We also affirm the effectiveness of IVIG treatment to palliate cutaneous severity in Netherton syndrome following a three month therapy in our patients.Although the degree of remission were different in different patients but significant reduction of erythroderma,scaling,edema and pruritis was seen in all patients.