A Genetic Study Of Surviving Patient With Hb Bart’s Hydrops Fetalis Syndrome Due To High Expression Of ζ-globin

Part 1 A genetic study of surviving patient with Hb Bart’s hydrops fetalis Syndrome due to high expression of ζ-globinBackgroundα-thalassemia is an inherited autosomal recessive genetic disease,the most common single-gene disease in the world’s population,characterized by small-cell hypochromic anemia.The reduction or deletion of α-globin chains can lead to excessive unpaired β(β)-like globin chains forming insoluble homotetramers,resulting in intracellular precipitation,ineffective hematopoiesis and severe acute hemolytic anemia.Thalassemia is characterized by insufficient production of α-globin chains of hemoglobin.Individuals with mutations affecting the α-globin gene on a single chromosome accompanied by mild anemia are said to have "silent" α-thalassemia(if one gene is involved)or α-thalassemia traits(when two genes are involved),While compound heterozygotes or homozygotes expressing moderate to severe hemolytic anemia are said to have HbH disease.Non-functional β-chain tetramers(β4 tetramers)formed by excessive β-like globulin chains in adults are called HbH,and γ-chain tetramers(γ4 tetramers)formed during the fetal period are called Hb Bart’s.The most serious form of α-thalassemia is Hb Bart’s Hydrops Foetalis syndrome,a disease without α-globin gene expression.The vast majority fetus with Hb Bart’s fetal edema syndrome dies in utero during 30 to 40 weeks of pregnancy(average 34 weeks),or shortly after delivery.The body had severe edema,ascites,and frog abdomen.In a few cases,there was no edema and ascites.In this study,we found a patient with severe a thalassemia whose parents were both--SEA/carriers.The patient also inherited the parent’s a large deletion gen and became homozygous.In this paper,the expression of zeta globin in patients was quantitatively detected through pedigree research,and the regulatory mechanism of its up-regulational expression as well as the effect on the patient’s phenotype were analyzed.Materials and methods1.Research subject:The proband was a 9-year-old female from Hunan Province.She was born prematurely due to hypoxia without edema.She received blood transfusion for the first time from the 15th day after birth and was diagnosed with severe thalassemia.At the age of 4,HBA gene was sequenced and the result showed she was--SEA/homozygote.The parents and the younger brother of the proband were all carriers of the large fragment deletion gene.2.Experimental method:MLPA was used to detect the deletion of large fragments,and specific products were amplified by PCR based on Gap-PCR principles and methods.Sanger sequencing was used to identify the positions of large fragment deletions and to determine the genotypes of family members.The mRNA levels were detected by quantitative PCR;the expression level of ζ-globin in the experimental group and the control group were quantified using Western Blot,reversed-phase liquid chromatography.Second-generation sequencing of the proband’s entire genome was conducted to speculate possible regulatory sites in genes that could affect its survival.ResultsThe proband was clinically diagnosed as α-thalassemia major(TM)with a genotype of--SEA/--SEA and was unable to produce α-globin normally.The mRNA level and protein expression of ζ-globin are higher than normal people and other--SEA/carriers.In the double-labled immunofluorescence experiment,it can be seen that the fluorescence intensity of ζ-globin is significantly stronger than that of the control samples.The up-regulation of ζ-globin led to the patient’s survival.The ultrasound indicators and growth and development data of the probands in each period in the uterus are within three times the standard deviation of the normal population standard value.Conclusions1.This study reported on a surviving patient with Hb Bart’s syndrome.The analysis results showed that the survival of the patient without any intrauterine intervention was due to the up-regulation of compensatory expression of ζ-globin.2.The cause of ζ-globin in the proband can be expressed after birth,and the specific regulatory mechanism need to be further explored and analyzed.Part Ⅱ Identification of int22 inversion type in a group of hemophiliacsBackgroundHemophilia A(hemophilia A,HA)is a hereditary hemorrhagic disease,the most common X-linked recessive genetic disease,and the incidence of males is about 1/5000.Patients with hemophilia could have bleeding in any part of the body,but most bleeding is concentrated in the joint muscles;other parts have mucosal bleeding,such as nose bleeding,oral bleeding,gastrointestinal bleeding,urinary tract bleeding,etc.;severe visceral bleeding,intracranial bleeding can be life threatening.Severe cases have obvious bleeding,often without incentives;mild cases only show bleeding tendency.Hemophilia A can be divided into three categories according to the genetic mutation:the first type involves a small number of base mutations,mainly base substitution,including missense mutations,nonsense mutations,splice mutations,gene polymorphisms,etc.;The second type of gene mutation involves changes in chromosomal structure,often involving gene fragments ranging in size from 1 kb to 3 Mb;the third type is the deletion and repetition of large fragments.The most common mutation found in approximately 40%of patients with severe hemophilia A is the intron 22 inversion mutation.When Int22h-1 in intron 22 is homologously recombined with one of the two homologous regions of F8 gene(Int22h-2 or Int22h-3),and between Int22h-2 and Int22h-3 After homologous recombination with Int22h-1,four different types of inversion are generated.In this study,we collected 103 patients with hemophilia A from nanfang hospital,and the gene variation type was int22 inversion.The inversion type was classified and correlation analysis of genotype and phenotype was conducted.Materials and methods1.Subjects:DNA samples of 103 inverted patients with Inv22 from nanfang hospital were collected.2.Methods:All samples were genotyped by LD-PCR.Meanwhile,phenotypic information of all patients was collected for genotype-phenotypic analysis.Chi-square test was used to analyze whether there is statistical difference.ResultsThe genotypes of 103 patients with inversion mutations were identified by LD-PCR,and 98 patients were found as Inv22-Ⅰ,accounting for 95.15%,5 inv22-Ⅱ,accounting for 4.85%.Chi-square test results showed that P value of difference in distribution of the two genotypes at different ages and FⅧ levels are P=0.670(age);P=0.414(FⅧ factor activity),respectively.There is no statistical difference in the distribution of the frequency of the two genotypes at different ages and FⅧ.Conclusion1.According to the current typing results,most of the types of int22 inversion in F8 gene in Chinese population are type Ⅰ,type Ⅱ is relatively rare,and type Ⅲ andⅣ have not been found.2.In subsequent studies,it is necessary to expand the sample amount and enrich clinical information,and conduct statistical analysis to determine whether the classification of rearrangement of int22h homologous sequence has clinical significance.