Tuberous sclerosis complex (TSC) is a multisystem disorder associated with hamartomas formation in the brain, skin, heart, and other viscera [1] [2] , with Epilepsy as the most common presenting symptom [3] . The incidence of TSC is estimated to be 1 in 6000 to 1 in 10,000 live birth [4] . The autosomal dominant pattern of TSC inheritance comes from a mutation in either the TSC1 or TSC2 gene [5] [6] . Both serve as tumor suppressors and inactivating mutations in either gene is associated with the formation of hamartomas [7] . Although familial TSC shows an equivalent representation of mutations in TSC1 and TSC2, sporadic cases are more associated with mutations in the TSC2 gene and have severe symptoms [1] [5] [8] .

Missense mutations in TSC2 which disrupt its ability to bind to TSC1 have been identified in TSC patients [1] [9] . TSC2 located on chromosome 16p13.3.38, contains 41 exons and encodes a 200-kDa protein called tuberin [1] [4] . Mutations in TSC2 are four times more common and have more severe clinical symptoms than those of TSC1 [5] [10] . According to Human Genome Mutation Data Base (www.HGMD.org) roughly more than 45% of TSC1 & TSC2 mutations are deletions/duplications.

Renal problems have been observed in some sporadic patients as a relatively sever phenotype caused by a large deletion involving both TSC2 and PKD1 locus which is located 60bp downstream of TSC2 in a tail-to-tail orientation [6] [11] .

Scientists are looking for a genius method to screen TSC2 and TSC1 gene mutations through populations as a fast cost benefit screening test before entire gene direct sequencing. Multiplex ligation dependent probe assay (MLPA) has been recently developed by MRC Co. as a method to identify deletion/duplication mutations as copy number variations (CNVs). This method enables accurate copy number determination of multiple (up to 40) genomic regions, by utilizing an oligonucleotide ligation assay with embedded universal primer sequences [10] [11] .

In this study we applied commercially available TSC2 MLPA probe sets to enable comprehensive analysis of all exons within TSC2 for deletions and duplications in 81 Iranian TSC patients.

Clinical and Para clinical Manifestations of 28 girls and 53 boys affected with TSC showed a range of 7 - 180 months for age of the patients. The most common manifestation were Hypo pigmented malecules (82.7%). About 29.6% of the patients have positive familial history of TSC but only sixteen families (19.8%) had a consanguineous marriage. In the other word about 70.4% showed a sporadic form without positive history of TSC in pedigree.

We used MLPA technique to screen 81 TSC patient DNA samples for genomic deletions and duplications of TSC2 gene and contiguous deletions of TSC2 and the adjacent PKD1. 29 cases (35.8%) were identified with Deletions/duplications in TSC2 gene (Figure 1). Only three of the identified mutations were genomic duplications, while all others were deletions. Considering 26 deletions, 18 (69.2%) were defined as large deletions, of which two of them extended to 5’UTR, 4 (15.4%) were deletions of two adjacent exons, 3 (11.5%) were deletions of a just a single exon, there were also one sample (3.8%) showing deletion only in 5’UTR. Exons 21 - 41 were defined as mutational hotspots. Five samples showed deletion in PKD1 gene, these samples had no retinal cysts. Four of these were sporadic and one of them had a familial history of TSC. Genotype-phenotype analysis showed that none of five patients with retinal problems had deletions in PKD1. 57 (70.37%) patients had no familial history of Tuberous Sclerosis, including those cases with only one of symptoms of TSC, and 24 (29.63%) with at least one affected family member (Table 1). All clinical aspects of studied patients have been previously announced [3] .

Tuberous sclerosis complex is an autosomal dominant disorder caused by mutations in either TSC1 or TSC2 gene. Screening these mutations in different populations could help to unravel complexities of the disease and find a new and faster way of diagnosis for TSC. Studies on TSC and many other human diseases have shown that MLPA is a highly sensitive technique to identify many aspects of widespread deletions/duplications [12] [13] . We used MLPA to detect deletion/duplication mutation in TSC2 gene of 81 patients with TSC. We also checked for probable contiguous deletions of TSC2 and the adjacent PKD1. We found that 64.5% of all patients had mutations in TSC2; these data are similar to those previously done in different populations and showed strong relation between TSC2 deletions/duplications and Tuberous Sclerosis [7] [10] [14] . The samples with no mutations in TSC2 are going to be analyzed for mutations in TSC1 gene. Those with deletions duplications in TSC2 are going to undergo target analysis for detection of the main area of mutation, in our future studies.

In the present study we found that duplications are much less common than deletions (only 10.3% with duplications). Our finding is consistent with the results that Kozlowski et al. revealed; they had a comprehensive study on large deletions/duplications of TSC1 and TSC2 in patients with TSC, which could find only two duplications in TSC2 [10] .

From all patients of our study 8 were diagnosed with renal problems, but none of them had deletions in PKD1, and all PKD1 deletions were found in patients with no signs of kidney disorders, not considering one sample whose father had renal function impairment. According to previous studies, which have shown a strong relation between PKD1 deletions and kidney problems [10] [11] , we should also consider the patients with deletion in PKD1 for kidney disorders in following studies (Figure 2).

Sporadic cases covering 70.37% of our study, which is in consistent with previous studies that estimated two thirds of tuberous sclerosis patients are sporadic [7] .

This project was supported by grants from skin research center, Shahid Beheshti Medical Science University, Tehran, Iran. We thank to Dr. Toosi, head of skin research center with our great pleasure for scientific help.