Analysis Of Gene Mutations With Complicated Hereditary Hemolytic Anemia Of Two Patients And Their Families

Background and ObjectiveHereditary hemolytic anemia?HHA?is a group of hereditary heterogeneous anemia caused by mutations in genes encoding erythrocyte membrane proteins,erythrocyte enzymes,and hemoglobin.Congenital dyserythropoietic anemia?CDA?is a rare erythropoietic abnormal disease,usually characterized by a hemolytic phenotype,so it should be considered in the identification of HHA.Patients with typical HHA caused by common single-gene mutation can be diagnosed by combining their clinical manifestations,routine laboratory screening indicators and positive family history,but more and more studies have shown that some HHA patients have mutations of polygenic and multiple sites,combining with non-hereditary hemolysis factors,these complex hemolysis factors may lead to missed diagnosis or misdiagnosis.So these complicated and rare HHA patients should perform genetic testing to identify pathogenic genes in order to take early treatment or preventive measures.This study is divided into two parts,determine the gene mutation site of two patients and their families with hereditary hemolytic anemia by genetic testing,and analyze the relationship between the gene mutation site and the clinical phenotype,improve the gene mutation spectrum of related diseases.At the same time,the clinical characteristics and diagnosis process of HHA combined with non-hereditary diseases were summarized to reduce the occurrence of missed diagnosis and misdiagnosis of HHA.Section one:Study on hereditary spherocytosis combined with autoimmune hepatitisMethodsCollect the clinical data of a proband with hereditary spherocytosis and her parents and grandmother.Blood cell analysis,liver function and other routine items were performed by blood samples.Then determine the proband's gene mutation site using high-throughput sequencing,the sequence is on the whole-exon of coding region and clipping region of the blood system disease-related.Then the gene mutation site was verified by Sanger sequencing,and the family members perform mutation site detection.ResultsThe results of blood cell analysis showed that RBC and Hb of the proband were normal,MCHC and RET increased,MRV,MCV and MSCV decreased;her father's RBC,Hb and MCHC were normal,RET increased,and her MRV,MCV,MSCV decreased;and her mother's parameters were normal except MRV were slightly decreased;her grandmother had mild anemia,her RBC and MCHC were normal,and her MRV,MCV,MSCV were also reduced;all members except her mother had MSCV<MCV,MRV<95.77fl.Liver function results showed that the proband's AST,ALT,TBIL,IBIL,and DBIL increased simultaneously;her father and grandmother had increased TBIL and IBIL;the mother's bilirubin was normal.In addition,the proband's IgG is increased,and autoimmune liver disease autoantibody profiling shows:anti-nuclear antibody?IgG type?was weakly positive,anti-smooth muscle antibody?ASMA?was positive,titer was 1:320,combined with liver biopsy results,the proband was diagnosed as autoimmune hepatitis?AIH?.High-throughput sequencing results showed that the proband found two mutation sites:c.134G>A?p.R45K?heterozygous mutation and c.6544G>C?p.D2182H?heterozygous mutation were respectively detected in exon 2 and exon 46 for SPTA1 gene,Sanger sequencing confirmed the mutations found by the proband,her mother detected SPTA1c.134G>A heterozygous mutation,her father and grandmother detected SPTA1c.6544G>C heterozygous mutation.Conclution1.The heterozygous mutation of c.6544G>C in SPTA1 gene,may be related to the pathogenesis of HS,the clinical phenotype is light HS,the heterozygous mutationof SPTA1 gene c.134G>A may be a benign variation,and the clinical phenotype of SPTA1 genge c.6544G>C and c.134G>A complex heterozygous mutations is also light HS.These two mutation sites were not reported at home and abroad and they are new mutations.2.The abnormal liver function of the proband was caused by HS combined with AIH.Section two:Study on dehydrated hereditary stomatocytosis combined with congenital dyserythropoietic anemia type II and autoimmune hemolytic anemiaMethodsCollect the clinical data of a proband with dehydrated hereditary stomatocytosis combined with congenital dyserythropoietic anemia type II and her parents,elder brother,elder sister and younger brother.Blood samples were collected,liver function,Coombs test,G6PD and other routine items were performed by blood samples.At the same time,?and?thalassemia gene analysis was performed,and then determine the proband's gene mutation site by high-throughput sequencing,the sequence is on the whole-exon of coding region and clipping region of the blood system disease-related.Then the gene mutation site of proband was verified by Sanger sequencing,and the family members perform mutation site detection.ResultsThe results of blood cell analysis showed that the proband had moderate anemia,RET,MCV and MCHC were normal;his father had no anemia,the RET was slightly increased,MCV and MCHC were normal;his mother,elder brother and elder sister had normal Hb,the RET was normal,RBC was increased,and MCV was decreased;the younger brother's Hb was normal and the MCV was decreased.The results of liver function showed that both the TBIL and IBIL of the proband and her younger brothers were increased,and the other members had no abnormalities.Coombs test results showed that the proband was positive for DAT and diagnosed as autoimmune hemolysis?AIHA?.G6PD activity was normal.The results of?and?thalassemia gene test showed that the?and?globin gene mutation were not detected in the proband and her father,her mother and elder brother detected?-globin gene 3.7 heterozygous mutation and?-globin gene 17 site mutation,the genotype were-?^3.7/??and?¹⁷/?N,and her elder sister detected?-globin gene 17 site mutation,and her younger brother detected?-globin gene 3.7 heterozygous mutation.High-throughput sequencing showed that the proband found three mutation sites:PIEZO1 gene c.4608₄615del?p.Gly1537Glufs*82?frameshift mutation,SEC23B gene c.1571C>T?p.Ala524Val?and c.2149G>T?p.Ala717Ser?complex heterozygous mutation.Sanger sequencing confirmed the above three mutations in the proband.Her father detected the PIEZO1 gene c.4608₄615del and the SEC23B gene c.2149G>T complex heterozygous mutations,and her mother and elder sister detected the SEC23B gene c.1571C>T heterozygous mutation,her elder brother detected the SEC23B gene c.2149G>T heterozygous mutation,her younger brother detected the complex heterozygous mutations c.1571C>T and c.2149G>T for SEC23B gene.Conclution1.The clinical phenotype of PIEZO1 gene c.4608₄615del frameshift mutation is asymptomatic DHSt,this mutation site has not been reported at home and abroad,so it is a new mutation.2.The clinical phenotype of SEC23B gene c.1571C>T heterozygous mutation is asymptomatic CDAII type carrier,this mutation site has been reported in the literature.The clinical phenotype of SEC23B gene c.2149G>T heterozygous mutation is asymptomatic CDAII type carrier,this mutation site has not been reported at home and abroad and it is a new mutation.The SEC23B gene c.1571C>T and c.2149G>T complex heterozygous mutation may be related to the pathogenesis of CDA type II,and the clinical phenotype is mild CDA type II.3.The hemolytic anemia of the proband may be caused by DHSt combined with CDA type II and AIHA,and there are hereditary hemolysis factors and acquired hemolysis factor.