Study on DEHAL1 Mutations in Patients with Congenital Hypothyroidism and Thyroid Goiter

The objective of this research is to study the types and characteristics of DEHAL1 gene mutation in patients with congenital hypothyroidism (CH) and thyroid goiter from Shandong Province, which can provide some evidence for gene diagnosis of CH. 47 cases of patients who were diagnosed as CH combined with thyroid goiter by neonatal screening and 100 normal controls were selected as subjects and their genome DNA were extracted. All the exons were amplified by polymerase chain reaction (PCR) and PCR products were sequenced by direct sequencing (Sanger sequencing). DNA sequencing results were compared to the DEHAL1 gene reference sequence to see whether there was mutation, and χ2 test was used on the gene frequency of discovered Single Nucleotide Polymorphisms (SNP). The results showed that no DEHAL1 gene mutation was found in 60 cases of CH with thyroid goiter patients and 100 normal controls, however, two SNPs were found(rs672766, IVS3+129C>T; rs2076292, IVS3+142C>T) in intron region. There was no significant difference between the SNP rate in CH patients and normal controls (P > 0.05). It can be concluded that DEHAL1 gene mutation rate is very low which may not be the main factor leading to the congenital hypothyroidism (CH) with thyroid goiter in Shandong Province, China.

Patients with iodotyrosine dehalogenase deficiency (ITDD) had been described in the 1950s on the basis of chromatographic studies with radioactive-labeled compounds and measurement of the enzymatic activity in goitrous thyroid gland [18] . In 2002, the use of Serial Analysis of Gene Expression (SAGE) [19] applied to human thyroid tissue, allows the cloning of DEHAL1 81 gene [20] . DEHAL1 is organized in 6 exons (NM_001164694), encoding 293 amino acid, spanning over 35 kb on human chromosome 6p 24. DEHAL1 is mainly expressed in thyroid, in addition, it is also present in the liver, kidney, and colon at low levels.
DEHAL1 gene encodes iodotyrosine deiodinase which is responsible for the deiodination of MIT and DIT ensuring iodine recycling for thyroid hormone biosynthesis [21] . In 2008, the first DEHAL1 mutations were reported in three different consanguineous families, 3 homozygous mutations, two missense (R101W,I116T) and one inframe-deletion of three base pairs(F105-I106L) [22] .  (Table.1). PCR products were separated by 15g/L agarose gel and scanned by UVP gel imaging instrument. PCR products whose band are single and bright can be sequenced. .

Agarose gel electrophoresis of PCR products
15g/L agarose gel was used to test the PCR products quality, whose band was single and bright can be sequenced ( Figure 1).
Order from left to right lane is MarkerⅠ, the amplified products of DEHAL1 gene exon 1 to 6

Bioinformatics and Statistical analysis
Two SNPs (rs672766, IVS3+129C>T; rs2076292, IVS3+142C>T) were found in intron region, this change has no influence on the protein function. The former was found in 7 cases (variation frequency: 11.7%) and 21 normal controls (variation frequency:21%). the latter was found in 4 cases (variation frequency:6.7%) and 17 normal controls (variation frequency:17%). SNP variation frequencies of the CH patients and normal control group were analyzed by chi square test (υ=1), and the result showed that there was no significant difference between the CH patients and normal control group (the former: χ 2 =2.26, P > 0.05; the latter: χ 2 =3.51, P > 0.05).

Discussion
DEHAL1 gene is located in human chromosome 6q25.1, which contains 6 exons (NM_001164694) and encodes 293 amino acids. In addition to thyroid gland, DEHAL1 gene has a low level expression in liver, kidney and trachea. The mRNA of DEHAL1 has two subtypes, DEHAL1 and DEHAL1B. Studies have shown that the length difference between the two subtypes is 102 bp and the function of DEHAL1B is inactive [18] . Thyroid hormone plays a vital role in promoting individual growth, as a result, the lack of thyroid hormone at birth or at an early age will result in cretinism. As we all know, iodine is the essential material involving in thyroid hormone biosynthesis.
Two highly specialized systems have been built to ensure the adequate supply of iodine in human body.
One is the iodine accumulation system, the iodine derived from digestion and absorption of food will be transported to thyroid by the NIS (sodium/iodine symporter). The other one is the deiodination system, the deiodination of MIT and DIT by iodotyrosine deiodinase can make sure the recovery of iodine, so that the efficiency of the thyroid hormone synthesis can be improved. Once the iodine is absent, the thyroid hormone synthesis will be decreased, the level of serum T4 will decline and TSH will rise, which will lead to CH combined with thyroid goiter.
Patients with ITDD had been found in 1950s [17] , Rosenberg [22] had isolated and purified the DEHAL1 from bovine thyroid in 1979. People begin to study the DEHAL1 gene in molecular level with the development of molecular biology. DEHAL1 (also known as IYD) gene was cloned from human thyroid tissue by the method of SAGE [20] in 2002 [18] . In 2008, Moreno JC [21] found three homozygous mutations of DEHAL1 gene in patients from three unrelated families, including two missense mutations and a frame deletion mutation.
Functional studies in vitro showed that the activity of iodotyrosine deiodinase with these three mutations decreased. Afink [17] found a mutation (c.658G>A, p.Ala220Thr) in a consanguineous Moroccan family in 2008. This mutation existed not only in the homozygous but also in a 14 year old boy, which indicated that mutation may have dominant effect.
Ainhoa Iglesias [23]  therefore, the sample size should be expanded to verify in future research. On the other hand, there may be regional bias, so the study objects should be selected in different regions to eliminate the regional bias. At present, with the development and application of WES (whole genome sequencing technology) [24] , which could improve the efficiency of gene sequencing, the diagnosis of some diseases is easier than before. So the WES could be used to detect mutation of some genes related to CH which will provide some convenience for studying the pathogenesis of CH.