Background The effects of Toll-like receptor 2 (TLR2) in grafting-related immune diseases have attracted more and more attention.Blocking TLR2 signal pathway can extend the survival time of heart and kidney grafts.However, the effects of anti-TLR2 monoclonal antibody on corneal graft have not been confirmed.Objective This study was to investigate the influence of anti-TLR2 monoclonal antibody on corneal graft survival in the rats received penetrating keratoplasty (PKP).Methods Allograft corneal transplantation was performed on the right eyes of 24 SPF female Wistar rats to establish PKP models,with 12 SD rats as donors.The model eyes were randomized into the TLR2 monoclonal antibody group and the model group.Anti-TLR2 monoclonal antibody of 15 μg/30 μl was subconjunctivally injected on day 0,2,4,6 and 8 following the modeling in the TLR2 monoclonal antibody group,and equal amount of normal saline was injected in the same way in the model group.The edema,transparency and neovascularization were observed under the slit lamp microscope after surgery, and rejection index (RI) was scored based on the criteria of Holland.Corneal tissue sections of the rats were prepared for the histopathological examination on day 9 and 15 after operation.The research protocol was approved by the Southern Medical University Ethics Committee.Results Mild corneal edema was found in the two groups 1-4 days after operation.A lot of new blood vessels, edema and opacification of corneas were seen in the model group 9-14 days after operation,but in the TLR2 monoclonal antibody group,corneal opacification was found 15 days after operation.The RI scores were significantly higher in the model group than those in the TLR2 monoclonal antibody group 5,9,15 days after operation (t=4.183,4.954,13.506;all at P＜0.05).The survival time in the TLR2 monoclonal antibody group was 15.5 days,with the 95％ confidence interval (CI) 14.9-16.1;while that in the model group was 9.5 days,with the 95％ CI 8.7-10.3, showing a significant difference between the two groups (Z =12.728,P =0.001).The corneal histopathological examination revealed that corneal stromal edema,infiltration of inflammatory cells and vascular lumen were more prominent 9 and 15 days after operation in the model group than those in the TLR2 monoclonal antibody group.Conclusions Anti-TLR2 monoclonal antibody can inhibit inflammatory response after allograft corneal transplantation and therefore extend the survival time of graft in rats.

Two patients with coexistence of thyroid disease and suspected Gitelman's syndrome underwent SLC12A3 gene analysis. The results confirmed that both patients were compound heterozygotes of SLC12A3 gene mutation. Three novel variants of SLC12A3 were found in this study. This report suggests that Gitelman's syndrome may coexist with other disorders associated with hypokalemia, such as Graves' disease.

Objective To analyze and identify the mutations of ATP6V0A4 and ATP6V1B1 gene in autosomal recessive distal renal tubular acidosis (rdRTA) children,and study the association of genotype and phenotype. Methods Genome DNA was amplified by PCR.Mutations of ATP6V0A4 and ATP6V1B1 gene in 3 children from 3 families were examined by direct sequencing.One hundred unrelated healthy subjects were selected to evaluate all mutations found in this study. Results A novel homozygous nonsense mutation was identified in ATP6VOA4 gene in one child, and a novel heterozygous nonsense variant and a frame-shift alteration were found in another child.No mutation of both genes was found in the third child.Conclusions Study of mutant genes of rdRTA in Chinese patients is helpful to understand the association in genotype and phenotype and increase the level of cognition and treatment to this disease.

Objective To describe the clinical characteristics of one child with primary hyperoxaluria types Ⅲ, and to analyze the potential mutant genes in his family.Methods AGXT, GRHPR and HOGA1 genes were analyzed by direct sequencing analysis in this family.One hundred unrelated healthy subjects were also analyzed as controls.Results The child had early onset of symptoms (0.8 year).His principal clinical manifestation included nephrolithiasis and obstructive nephropathy, however his nephrocalcinosis was mild.And he presented high urine oxalate, high urine calcium, and lower citrate levels.Two novel heterozygous mutations in HOGA1 were identified (compound heterozygous), one mutation was a 2-bp substitution at the last position in exon 6 and the first position of intron 6 respectively (c.834_834 + 1GG ＞ TT);another was a guanine to adenine substitution of the last nucleotide of exon 6 (c.834G ＞ A).Both of these variants found in this study probably acted as splicing mutations.Direct sequencing analysis failed to find these mutations in 100 unrelated healthy subjects.In addition, a SNP (c.715G ＞ A, p.V239I) was found in this family.There were no mutations detected in AGXT and GRHPR.Conclusions Two novel mutations are identified probably in association with PH Ⅲ.This is the first description and investigation on mutant gene analysis of PHⅢ in Asia.

Objective To describe the clinical characteristics,and to analyze the AGXT gene mutation in three siblings with primary hyperoxaluria type I (PHI).Methods AGXT gene mutation was analyzed by direct sequencing analysis in this family,and the minor allele status was also tested.One hundred unrelated healthy subjects were also analyzed as controls.Results Three mutations in AGXT were identified in each of three patients including two novel heterozygous missense mutations and one previously reported variant.One mutation was a methionine to leucine substitution at position 49 (p.M49L,c.145A ＞ C) in exon 1,one was an asparagine to isoleucine transition at codon 72 (p.N72I,c.215A ＞ T) in exon 2,and another was a heterozygous nonsense mutation at codon 333 (p.R333*).Both p.M49L and p.R333* occured in cis configuration with the minor allele IVS1 +74 bp.Conclusions Two novel mutations are identified probably in association with PHI,however their pathogenicity and potential molecular mechanisms should be explored by further investigations.This is the first investigation on mutant gene analysis of PHI in China.

Objective To analyze the characteristics of the genotype, phenotype, and follow-up of Gitelman's syndrome (GS) in the largest group of Chinese patients. Methods Sixty seven patients with GS underwent SLC12A3 gene analysis. Clinical characteristics and biochemical findings at the first presentation as well as follow-up were reviewed. Additionally, the associations of genotypes and phenotypes were explored. Results Forty-one different SLC12A3 mutations were identified in 67 patients with GS, including 11 novel ones, and 5 recurrent ones. 3 families (5. 7% ) had triple SLC12A3 mutations. Typical hypocalciuria and hypomagnesemia were not found in 6(9% ) and 8 (11. 9% )patients, respectively. In addition, male patients had an earlier age of onset and a higher urinary fraction excretion of electrolytes. 2 patients presented with chronic kidney disease, 13 (19. 4% ) with type 2 diabetes, 14 (20. 9% )with impaired glucose tolerance, and 5(7. 5% ) with impaired fasting glucose. Conclusion This study revealed 41 mutations in 67 Chinese patients with GS, including 11 novel variants and 5 high-frequency ones. Fraction excretion of electrolyte in urine may be more sensitive in the evaluation of phenotype compared with those of blood. It is difficult to correct hypokalemia and hypomagnesemia in GS. Patients with GS are at higher risk of the development of diabetes than ordinary people.

Objective To analyze the mutations of causal genes in 5 children with primary distal renal tubular acidosis (dRTA),and explore their association of genotype and phenotype,so as to raise the awareness of the disease.Methods The whole exome sequencing was used to identify mutations in these 5 children from 5 families.Results A total of 4 different mutations of ATP6V0A4 gene were found in 2 dRTA children,including a novel heterozygous intron mutation (c.639 + 1G＞ A),a reported heterozygous nonsense variant (c.580C ＞T,p.Arg194*) and 2 novel heterozygous duplications (c.1504dupT,p.Tyr502Leufs*22;c.2351dupT,p.Phe785Ilefs*28).Two novel heterozygous missense mutations of ATP6V 1B 1 gene (c.409C ＞ T,p.Pro 137Ser;c.904C ＞ T,p.Arg302Trp) were identified in the third child,and a heterozygous missense mutation of SLC4A1 gene (c.1765C ＞ A,p.Arg589Ser) previously reported was found in the fourth child.No mutation of the dRTA-related causal genes was found in the fifth child.Furthermore,the mutations of causal genes in each of the first three children were compound heterozygous,which were consistent with the autosomal recessive inheritance pattern,and the variant from the fourth child was de novo.Conclusions The present study has found 7 mutations,including 5 novel variants,which enriches the human gene mutation database (HGMD) and contributes to a better understanding of the disease mechanisms.

Objective To analyze the variants of 42 Chinese patients with Bartter syndrome type 3 (BS3) and explore the characteristics of genotype and phenotype. Methods Forty-two genetically diagnosed patients from 40 Han and one Hui families were collected in the Affiliated Hospital of Qingdao University and the Affiliated Qingdao Municipal Hospital of Qingdao University during the period of June 2012 to October 2018. The second-generation sequencing and multiplex ligase probe-dependent amplification (MLPA) technique were used to analyze the CLCNKB gene variation and its characteristics in children with BS3. The clinical data were collected, and the therapeutic effect and growth improvement were observed and followed up. Thirty eight patients were divided into severe (n=26) and light (n=12) groups according to the severity of genetic variation. The clinical phenotypic characteristics of the two groups were compared. Results Thirty-six variants including 16 novel ones of CLCNKB gene were found. The whole gene deletion of CLCNKB gene was the most frequent mutation (40％), and the rate of large deletions was up to 55％. The most common symptoms included development retardation (38/42), polydipsia and polyuria (35/42), constipation (31/42) and vomiting (27/42). All patients presented with hypokalemia, hypochloremia and metabolic alkalosis. After the medicine treatment that based on indomethacin and potassium chloride, most patients could achieve obvious recovery of growth rate and restoration of hypokalemia. The severe group showed more severe metabolic alkalosis than the light group. Conclusions Thirty-six variants of CLCNKB gene have been found in this study, including 16 novel ones, which enrich the human gene mutation database (HGMD) and provide valuable references to diagnosis, treatment and the genetic counseling of Chinese population.

The gene mutations of a patient with fructose-1,6-bisphosphatase (FBPase) deficiency and her parents were analyzed and her clinical manifestations, laboratory tests, and genetic characteristics were reviewed. The molecular analysis of FBP1 gene showed a G residue duplication at base 960 in exon 7(c. 960dupG) in this patient while her parents carried the heterozygous c. 960dupG mutation. The prominent clinical feature of this patient was the benign course of the disease with age. However, acute attack could be triggered by stress, long-time fasting, a large amounts of fructose intake, etc. The typical clinical manifestations were severe lactic acidosis, hypoglycemia, and elevated liver enzymes.

Objective To describe the clinical characteristics of a family with tuberous sclerosis (TSC) complicated with giant bilateral renal angiomyolipoma,and to analyze the causal genetic mutations.Methods All coding regions of TSC1 gene and TSC2 gene were analyzed by next generation sequencing.The potential effect on abnormal splicing was confirmed by minigene assay based on the pSPL3 exon capture vector and was validated in vivo with the patient's RNA.Results The clinical manifestations of the proband were typical.Bilateral large renal angiomyolipomas were her principal clinical features.Besides,facial angiofibroma,periungual fibroma,lung lymphangioleiomyomatosis and subependymal nodule were also noted.A novel heterozygous variant of TSC2 gene (c.3610G＞A) was identified.On the protein level,this variant was presumed to be missense mutation (p.Glyl204Arg);however,the splicing assay revealed that this mutation also led to aberrant splicing with the whole exon 29 skipping on the transcripts level.Conclusion A novel mutation c.3610G＞A,contributing to aberrant splicing and missense alteration (p.Glyl204Arg),is identified in association with TSC.