Identification Of The Causative Mutations For Two Families With Inherited Red Cell Defects

Congenital red blood cell disease is the most common hereditary disease worldwide and mostly including enzyme disorders,hemoglobinopathy,membrane disorders.In this study,we collected two pedigrees with hereditary red blood cell disease,conducted molecular studies on these two families,identified pathogenic mutations,and performed genotypic and phenotypic learning.Part 1 A novel large deletion was identified in a ??0-thalassemia patientBackgroundBeta-thalassemia,mostly inherited in autosomal recessive manner,is usually caused by mutations in HBB gene.Reduced HBB expression in individuals with the mutation would result in imbalance ratio of ?-and ?-globin chains.The excess a-globin chains would attached to the erythrocyte membrane and destroyed red blood cells.The Chinese G??A????0-deletion and S.E.A-HPHF are the most common large deletion reported in southern China.In this study,we found a thalassemia major patient who inheried CD41-42?HBB:c.126₁29del CTTT?from his father,and a new large fragment of the ?-globin gene cluster from the mother.In this paper,molecular identification of large fragment deletions of this ?-globin gene cluster was carried out by family studies,and its effect on ?-globin gene expression and its phenotype was analyzed.Materials and methods1.Subject:The proband was a 3-year-old girl from Jiangxi province,China,clinical diagnosed as thalassemia major?TM?,who has suffered severe anemia since 4-month old and received blood transfusion since 6-month-old.Sanger sequencing showed that she was homozygous for the CD41-42?HBB:c.126 129del CTTT?mutation.However,a heterozygous CD41-42?HBB:c.126₁29del CTTT?mutation was found in her father but not in her mother.A large deletion in beta globin gene cluster was found in both the proband and her mother by MLPA.2.Experimental methods:NGS was performed in the proband and was compared with two unrelated normal controls.Gap-PCR and Sanger sequencing were used to identify breakpoints of the deletion in the family.ResultsThe proband was diagnosed as thalassemia major?TM?,inherited a CD41-42?HBB::c.126₁29del CTTT?munation and a large deletion from father and mother respectively.NGS predicted that the deletion was about 222 kb in size.Unique elements of recombinant DNA were amplified and sequenced.The deletion was identified to be chr11:5031238-5254163?hg19?,222,920 bp in size,with a micro homology of four nucleotides?GTCT?in both breakpoints.Conclusions1.Our study found a novel deletion in beta globin gene cluster named ?223 kbdel,which enriched the mutational spectrum of thalassemia,and can deepen the understanding of the types of thalassemia in Chinese population.2.MLPA and NGS technologies can help improve detection rate of thalassemia carriers with rare or novel copy number variations.Part ? Identification of the causative mutation of a hereditary spherocytosis patientBackgroundHereditary spherocytosis?HS?has been reported worldwide and is the most common inherited red cell membrane disorder characterized by anemia,jaundice,splenomegaly and spherical-shaped erythrocytes on blood smear.Genetic analysis can provide strong evidence for clinical diagnosis.In this study,we conducted molecular genetic studies on a family with HS and found a frameshift mutation?ANK1:c.2394 2397del CAGT?.Materials and methods1.Subjects:The proband was a 6-year-old male from Guangdong provinces with anemia.Preliminary examination revealed that the spleen of the proband was enlarged,and the father of the proband had a total splenectomy at the age of 6 due to unexplained hemolysis.Spherical red blood cells in the peripheral blood were found in both proband and his father.2.Methods:Peripheral blood of the family members were collected.Then,SDS-PAGE and sanger sequencing was performed to investigate the pathogenic mutation.ResultsSDS-PAGE confirmed the diagnosis of HS and suggested that the ankyrin deficiency caused the disease.Sanger sequencing found a frameshift mutation?ANK1:c.2394₂397del CAGT?in both proband and the father which.Sequencing of cDNA showed a slight chaotic peak,which was consistent with the mutation allele DNA sequence.Conclusion1.We diagnosed a patient with HS and found a frameshift mutation ANK1:c.2394₂397del CAGT.On the other hand,cDNA was studied and the result suggested that mRNA of mutant allele was expressed in vivo but the level was low.2.ANK1:c.2394 2397del CAGT resulted in reducded expression of ankyrin,which may be the main cause of small spherical red blood cells.3.Our study demonstrates that SDS-PAGE and Sanger sequencing can be performed for genetic analysis.