Mutation Detection In Gene DKC1Responsible For Dyskeratosis Congeita And Telomere Length In Patients With Dyskeratosis Congenita

Part1Mutation detection in DKCl gene responsible for DCDyskeratosis congenita (DC, MIM305000) is a rare inherited syndrome exhibiting marked clinical feature and genetic heterogeneity, which associated with a triad of mucocutaneous features:nail dystrophy, oral leukoplakia, and abnormal reticulate skin pigmentation. Besides, DC patients can present with various other features including bone marrow failure, aplastic anemia, pulmonary fibrosis, malignancy. Clinical features of DC often appear in childhood. Progressive bone narrow failure develops in more than80%of the patients by the age of30years old, which is the main cause of early mortality. Hoyeraal-Hreidarsson(HH) syndrome is a severe phenotype of DC due to DKC1mutations, which characterized by aplastic anaemia, immunodeficiency, microcephaly, cerebellar-hypoplasia and growth retardation. There are three modes of inheritance, including X-linked, autosomal dominant and autosomal recessive inheritance. Approximately50%of DC patients remain genetically uncharacterized. The most common one is the X-linked, caused by mutations of the DKC1, located at Xq28. DKC1encodes a514-amino-acid protein, dyskerin, a nucleolar protein, highly conserved in evolution, which is an essential structural component of small nucleolar RNA-protein complexes (snoRNPs) and of the mammalian telomerase enzyme complex. DKC1consists of15exons spanning a region of15kb. X-linded DC is predominantly caused by missense mutations in DKC1. Targeted disruption DKC1causes embryonic lethality in mice. The mutation p.A353V, nucleotide substitution C>T at positon1058in exon11, is a hotspot for mutation in DKC1. The p.A353V mutation was detected in30%of the male patitents with X-linked DC. Telomerase contains telomerase RNA component(TERC) and telomerase reverse transcriptase(TERT). The terozygous TERC and TERT mutation have been identified in autosomal dominant DC patients, bone marrow failure, and aplastic anemia. TERC and TERT mutations lead telomerase function defective and this is associated with short telomeres. There are2patients with DC among20members family. The proband was a16-year-old male with reticular hyperpigmentation on the neck, extremities and chest areas when he was4-year-old. Examination of proband revealed typical mucosal leukoplakia on tongue, abnormal skin pigmentation in the trunk and the extremities. His fingernails and toenails were dystrophic. Blood routine, gastroscope, chest X-ray, ECG, and bone marrow cell showed normal respectively. The diagnosis was made by typical clinical. Because of the patients were all males, whose parents presented with normal phenotype, they might be the X-linked recessive inheritance in this family. So we chose DKC1for mutation research. Genomic DNA was extracted from peripheral blood of2affected individuals, other unaffected individuals and50controls using a Qiagen kit. Mutational analysis of DKC1was performed by sequencing the polymerase chain reaction (PCR) products of all exons (exons1-15) and flanking intronic regions with primers designed by Oligo6.0. We identified novel transition mutation of c.1192C>G (p.L398V) in exon12of DKC1. This mutation was detected in two affected individuals who exhibited mild phenotype but was absent in all unaffected family members and the50unrelated controls. Their mother carried a heterozygous c.1192C＞G (p.L398V) mutation.Part2Telomere length in patients with dyskeratosis congenitaIn1999. Mitchell JR found that DC peripheral blood cells have a lower level of telomerase RNA, which produce lower levels of telomerase activity and have shorter telomeres than matched normal cells. The pathology of DC is consistent with compromised telomerase function leading to a defect in telomere maintenance, which may limit the proliferative capacity of human somatic cells in epithelia and blood. Dyskerin required both for ribosomal RNA (rRNA) pseudouridine modification and for cellular accumulation of telomerase RNA. Dyskerin mutations reduced accumulation of telomerase RNA, and the reduction telomerase RNA lead to the inability of telomerase to maintain telomere length. Mutations in varied genes that encode compents of the telomerase complex(TERC、TERT、NHP2、NOP10) and telomere shelterin(TINF2) can led short telomere. Studies over the last10years had demonstrated that DC is principally a disease of defective telomere maintenance. However, there was no correlation between telomere length and clinical. Patients of DKC1and TINF2mutations are present at a younger age and have more disease features than those with TERC or TERT mutations, but there is no difference in telomere length. Especially, temolere length was normal in some uncharacterized DC. So, nowdays the relationship between DC and telomere is not clear. Clinical heterogeneity is not explained by gene mutations and short temomere. To investigate the relationship between telomere length and phenotype of patients with X-linked DC in a Chinese family. Telomere length was measured by fluorescent quantitative Polymerase Chain Reaction (PCR) in patients with dyskeratosis congenita, unaffected relatives in the family and control subjects.36B4is single copy gene(S), which encodes acidic ribosomal phosphoprotein, is located on chromosome12. Telomere(T) PCR and single copy gene(S) PCR were always performed in separate wells. Repeated measures of T/S ratio in the same DNA sample. The average relative ratio of T/S reflect relative length differences in all individuals. Besides, examination, hematology and lung function of all affected subjects were done. There were no significant difference between unaffected relatives and control subjects (t=0.847,P=0.412), no difference between patients, unaffected member and controls. In a word the telomere length had no shorter in the patients with slight phenotype of dykseratosis congenital than that in the control subjects.