Hemophilia A

Introduction to hemophilia A Hemophilia A (HA) is a kind of recessive hereditary hemorrhagic disease caused by X-linked chain clotting factor VIII and abnormal molecular structure. The clinical features are "spontaneous" joint hemorrhage and deep tissue hemorrhage. basic knowledge The proportion of illness: 0.02% - 0.03% Susceptible people: no specific population Mode of infection: non-infectious Complications: cerebral hemorrhage coma muscle atrophy

Hemophilia A cause

(1) Causes of the disease

Women with hemophilia A are extremely rare. Although there are reports of hemophilia A in the sick father and carrier mother and daughter, a significant proportion of the female patients reported are carriers of women due to their normal X chromosome extremes. Random inactivation leads to clinical manifestations of hemophilia; it may also be 2N von Willebrand disease.

There are three possibilities for women with hemophilia to carry hemophilia genes: 1 sure carriers, including daughters of hemophilia patients, mothers with at least 2 hemophilia sons; 1 The mother of the hemophilia son has another hemophilia among the women who have maternal blood relationship with her; 2 very likely carriers, hemophilia patients with no family history of genetic disease become sporadic cases, the mother of sporadic cases is very Possible carriers; 3 possible carriers, women with maternal blood relationship with hemophiliacs and women without hemophilia sons are possible carriers.

The sporadic cases accounted for more than 30% of hemophilia patients, some of which were not found in the family, and partly due to genetic mutations.

(two) pathogenesis

Defects in the FVIII gene leading to defects in FVIII synthesis and abnormalities in the structure of the FVIII cause a decrease or deficiency in FVIII functional activity, which is the underlying pathophysiological basis of hemophilia.

FVIII gene and hemophilia A gene defect, FVIII gene located at the long arm end of X chromosome (Xq28), containing 186 kb, about 0.1% of the full length of X chromosome, 26 exons, total length of about 9kb, intron 25, the mRNA length is about 9029 bp, the FVIII gene defect causing hemophilia A can be a point mutation, partial or total deletion of the gene, insertion of the gene component, generation of point mutations and gene inversion of the stop code, information summarized by Tuddenham et al. Among them, there are more than 80 kinds of point mutations, 6 kinds of insertions, 7 kinds of small deletions, 60 types of large fragments, and there are nearly 400 kinds of FVIII gene defects leading to hemophilia A.

In point mutations, a single base mutation occurs at the restriction enzyme site, and Taq1 recognizes the TCGA sequence. This site mutation can be directly determined by loss of a Taq1 cleavage site.

Mutations also occur frequently in the CpG dinucleotide sequence. The crypto-CGA of arginine is often affected by this sequence mutation. The CT conversion of the CG dinucleotide sequence results in a CGA mutation to TGA, which is the termination code. Since then, the protein is no longer synthesized and often leads to severe hemophilia. The mutation that produces the stop codon is called a nonsense mutation. The GA conversion of the CG dinucleotide sequence results in a CGA mutation to CAA, which is the glutamate code. A non-functional FVIII molecule in which arginine is replaced by glutamate and cannot be normally activated. FVIII: Ag is normal and FVIII: C is less than 1%, and a mutation in which an amino acid is substituted by another amino acid becomes a missense mutation, and there are many errors. The mutations reported that the severity of hemophilia caused by different missense mutations is different. About 5% of hemophilia A is caused by gene deletion and often leads to severe hemophilia. The discovered gene deletions are distributed throughout the FVIII gene. No special segment is more prone to deletion mutation, the length of the deletion can be from 2bp to 210kb. Small deletions can lead to mild hemophilia without changing the reading frame of the gene. Large deletions often lead to the failure of patients to detect FVIII:Ag. , However, patients with other genetic defects anti F antibody also occur, but no F: Ag patient large fragments more susceptible to anti-gene deletion F antibodies.

Insertion is another type of mutation in the FVIII gene defect. Insertion of the human L1 sequence into the FVIII gene can cause hemophilia, and the L1 sequence is a long repeat sequence widely present in the human genome, with retroviral reverse transcription. The DNA of the enzyme is similar. Since it can be moved in the genome and is also called transposon, insertion of a transposon into the FVIII gene leads to hemophilia A, which has been reported in some cases.

Gene inversion is another genetic alteration of hemophilia A found in recent years. There is one CpG island (two-way transcriptionally active promoter) in the FVIII gene intron 22 structure, and two FVIIIs associated with this island. Gene-related gene, 1 is F8A gene, its transcription direction is opposite to that of FVIII gene, and the other is F8B gene, its transcription direction is the same as that of FVIII gene, and F8A gene has three copies at the end of X chromosome (Xq). One is located in the intron 22 of the FVIII gene (the gene in the gene), the other two are located 500 kb upstream of the FVIII gene, and the F8A in the FVIII gene can undergo homologous recombination with any of the two upstream F8A genes. Recombination reverses the end of Xq, dividing the FVIII gene into two opposite parts, the promoter to exon 22 as part, and exon 23 to exon 26 as another part, which are about 500 kb apart. Leading to severe hemophilia A, it is now believed that the FVIII gene inversion accounts for about 50% of severe hemophilia A.

Hemophilia A prevention

1. Establish genetic counseling, strict premarital examination, and strengthen prenatal diagnosis to reduce the birth of children with hemophilia.

2. Patients with hemophilia should avoid activities that are severe or prone to injury, exercise and work to reduce the risk of bleeding.

3. In severe cases, alternative treatment should be given before traumatic examination and traumatic treatment.

Hemophilia A complications Complications cerebral hemorrhage coma muscle atrophy

Intracranial hemorrhage and peripheral nervous system symptoms are the most common complications of this disease. Intracranial hemorrhage is also the most common cause of death. There is a history of trauma, but sometimes the trauma is light and does not attract attention. The bleeding site can be epidural, subdural. And in the brain, it can be manifested as a gradually increasing headache, gradual coma and symptoms and localization of increased intracranial pressure. Many patients have central nervous system symptoms several days after trauma, so patients with cerebral hemorrhage may have head trauma. Early replacement therapy should be used, and EEG abnormalities suggest that subclinical cerebral hemorrhage may have occurred before.

The peripheral nervous system often causes severe pain due to hemorrhagic invasion or hematoma compression, numbness and dysfunction, and muscle atrophy.

Hemophilia A symptoms Common symptoms Muscle hemorrhage Repeated bleeding Joint malformation Abortion Paralysis Respiratory obstruction Low heat shock

The clinical features of hemophilia A are abnormal excessive bleeding in different parts of the body, especially soft tissue hematoma and joint bleeding are the characteristics of this disease. The severity of bleeding is parallel with the level of FVIII: C in patients, and the bleeding of patients with hemophilia A Injury-related, but because the damage is extremely mild and not noticed, and considered "spontaneous" bleeding, Table 1 is the classification criteria for hemophilia developed in China, although there is some overlap between the types, but this type of classification still Widely used, it has important significance in clinical diagnosis and treatment. International classification standards are not consistent. Hougie is divided into 3 types, no subclinical type, clinical characteristics are similar to Chinese standards, but heavy FVIII: C is less than 1%, light FVIII: C 6% to 30%, Wintrobe and China's standard except for subclinical FVIII: C 25% to 50% are the same, there are several types, the difference is in the range of FVIII: C level, foreign to Hougie classification It is widely used.

Severe hemophilia A often experiences bleeding without obvious trauma caused by daily activities. Children have no joint bleeding before retreat. Soft tissue hemorrhage is common. Joint bleeding begins to occur frequently after walking. If there is no effective alternative treatment, repeated Joint bleeding can often lead to chronic hemophilic arthropathy in patients years ago, which is characteristic of severe patients, but even in patients with severe hemophilia, the onset of bleeding is intermittent, not occurring for weeks, months or even years. Severe bleeding is not uncommon. There are also severe cases diagnosed in adulthood. In addition to cerebral hemorrhage, sudden death from bleeding is rare.

Moderate cases can have hematoma and joint bleeding, and are often caused by definite trauma, a few may have joint deformities, but rarely appear before adulthood.

Mild cases rarely have joint bleeding, no joint deformity, bleeding episodes are not easy to occur, often caused by obvious trauma, many patients have only a slight history of easily overlooked bleeding, often diagnosed by surgery-induced bleeding.

Most carriers have no bleeding symptoms. FVIII: C less than 45% can cause hemorrhagic abnormalities after surgery and major trauma. Very few FVIII: C less than 5% are clinically as moderate hemophilia A, sometimes diagnosed as female hemophilia. .

1. Joint bleeding is one of the typical bleeding symptoms of this disease. It is found in more than 2/3 cases. It often occurs in trauma. After walking and exercising, the most common sites of joint bleeding are the knee joint and the ankle joint. , elbow joint, hip joint, shoulder joint and wrist joint, joint bleeding is related to the weight bearing and activity intensity of the joint. There is very little joint bleeding in children before the toddler. Joint bleeding often comes from the synovial blood vessels of the joint, and blood enters the joint. In the cavity and osteophytes, there is slight joint discomfort at the beginning of joint bleeding, which lasts for several minutes to several hours, then gradually the pain is aggravated, the joints are locally swollen, and the fever is hot. When the blood penetrates into the skin or under the skin, there may be redness and ecchymosis. Limit, sometimes patients may have low fever, but obvious long-lasting fever often suggests co-infection. Due to the relationship between joint structure, joint bleeding is often self-limiting. When bleeding stops, some patients' blood in the joint gradually absorbs within days to weeks. The joint function is gradually restored. Repeated joint bleeding causes chronic damage to the joint, synovitis, articular cartilage destruction, bone hyperplasia and atrophy. Facial lip hyperplasia and osteophyte formation, joint space stenosis and osteonecrosis and cystic changes, leading to joint deformity and dysfunction, severe claudication, if early replacement therapy for joint bleeding, often allow patients to stop bleeding early And pain and accelerate blood absorption, to avoid severe hemophilic joint disease and patient disability, hemophilia A patients with small joint bleeding, and spinal joints rarely bleeding.

2. Muscle hemorrhage and hematoma are characteristics of hemophilia and other clotting factor deficiencies. Other hemorrhagic diseases are rare, often occur after trauma or activity, or can occur in cases where the trauma is not obvious, can occur in any part, but Forced muscle groups are more likely to occur, about 75% of patients have muscle bleeding and hematoma, subcutaneous and muscle bleeding have a tendency to spread around, hematoma can gradually increase, severe cases, especially retroperitoneal hemorrhage can cause anemia and shock, hematoma compression is important Serious organ consequences, retroperitoneal hemorrhage can cause paralytic intestinal obstruction, hematoma into the chest or neck can cause airway obstruction, lower abdominal hematoma leading to urinary tract obstruction can affect renal function, hematoma compression nerve can cause nerve damage, axillary site bleeding Often disabling, the frequency of muscle bleeding is calf, thigh, buttocks, forearms and abdomen.

3. Skin and mucosal bleeding The bleeding of the skin and mucous membranes is not a characteristic of this disease. Other hemorrhagic diseases often have bleeding in the skin and mucous membranes. Hemorrhagic disease is characterized by no bleeding, but flaky siltation. Spot, and often accompanied by subcutaneous induration, a small hematoma formed by bleeding below the dermis, often caused by minor trauma, often bleeding when the skin has a large wound, mucosal bleeding is common, small wounds in the mucosa often cause continuous bleeding, no It is not easy to stop the replacement treatment. The small wounds in the gums, tongue and other oral mucosa often continue to bleed. If the replacement treatment is not used, it can lead to severe blood loss. The gastrointestinal bleeding is not common. The bleeding is often serious, and the upper digestive tract can be damaged due to food. Mucosal or peptic ulcer caused by 5 times the incidence of peptic ulcer in adult hemophilia patients.

4. The incidence of false tumors is about 2%. It is more common in patients with severe hemophilia A deficiency replacement therapy. The common parts are thighs, pelvis and iliopsoas, which can also occur in the buttocks, calves, feet, forearms and hands. After traumatic hemorrhage, a cystic hematoma is formed under the periosteum and under the tendon of the tendon. If the blood in the hematoma is not absorbed, the blood is destroyed and the local osmotic pressure is increased. The repeated hemorrhage in the capsule often increases in volume within a few years, thus compressing and destroying. Corrosion of surrounding tissues to form pseudotumor, pseudotumor is a complication of hemophilia risk, it can be divided into three types, the first is a simple cyst, the pedicle is connected to the muscle fascia, the second is cystic at the beginning, However, due to the vascular supply affecting the nearby bone and periosteum, bone absorption and cyst formation are caused, and the third is the separation of periosteum and bone due to subperiosteal hemorrhage.

5. Urinary tract hemorrhage severe hemophilia A common, urine color can be brown red or bright red, depending on the amount of bleeding, bleeding site is generally in the renal parenchyma, mostly unilateral, but also bilateral renal bleeding Bleeding can also occur in the lower urinary tract, and the amount of bleeding is generally small.

6. Hemorrhagic patients who have not undergone alternative treatment after surgery often cause severe bleeding. The bleeding abnormality is not only in the operation, although it has been fully hemostasis, it is very common to have severe bleeding after hours or even days after surgery. Non-healing or poor healing, whether it is major surgery or minor surgery, must be replaced before surgery until the wound is healed.

Hemophilia A check

Screening for all endogenous coagulation systems can be extended, including partial thromboplastin time (PTT) or activated PTT (APTT), Biggs thromboplastin production test (TGT), simple thromboplastin production test (sTGT), plasma Recalcification time and clotting time (CT), due to differences in sensitivity of different tests, can be normal in mild and subclinical cases, and APTT tests also have sensitivity differences in FVIII:C levels due to different combinations of reagents, prothrombin time ( PT) Thrombin time (TT) is normal, bleeding time (BT) is generally normal, and a few can be slightly prolonged, due to barium sulfate adsorption or aluminum hydroxide gel adsorption of normal plasma containing FVIII: C and FXI: C without FIX :C, thus using normal adsorption plasma and serum for APTT correction test or: Biggs TGT can confirm the lack of hemophilia A, B and FXI, FVIII: C can be determined by one-stage or two-stage method, one-phase method requires no FVIII matrix plasma, while the second-stage method is now less used due to complicated operation. The FVIII:C assay can have a large error due to the reagent or operator proficiency, etc., and the FVIII antigen (FVIII:Ag) assay can determine the FVIII protein. Some suffering Normal or mild reduction and FVIII: C is almost undetectable, indicating the presence of dysfunctional FVIII molecules, also known as CRM, and some patients with FVIII: Ag and FVIII: C are almost undetectable, called CRM-, FVIII: C It is expressed as a percentage of the normal average or a unit (U) per milliliter of plasma, U is equal to 100%, normal human plasma contains FVIII: C1 unit per milliliter, and hemophilia patient vWF: Ag is normal.

Due to the rapid development of molecular biology techniques, although most laboratories are unconditional, it has become possible to diagnose hemophilia at the molecular level and has been used for prenatal diagnosis and carrier testing. Currently, indirect genetic diagnosis and direct genetic diagnosis can be used. The method of indirect diagnosis uses gene linkage analysis, and requires a proband, and the proband mother is heterozygous for the analysis site, and there are three polymorphic genetic markers linked to the gene related to hemophilia A: 1 restriction Endonuclease fragment length polymorphism (RFLP): Polymorphic sites used at home and abroad include BcL I, HindIII, XbaI, BgLI, etc. Using a single polypeptide site, the heterozygote detection rate is less than 50%. Therefore, multiple sites must be used in combination to increase the detection rate, which has reached more than 90% in foreign countries; 2 variable number tandem repeat sequence (VNTR), because the human genome contains some different nucleotide repeat sequences, these tandem repeats have different copy numbers. It can also cause DNA restriction fragment length polymorphism and is used for indirect genetic diagnosis. The VNTR site S52 located outside the FVIII gene and tightly linked within the gene is commonly used for analysis. apI digestion and Southern blotting; 3 short repeat order (STR) has high application value, it has been found that there are 2 STRs in FVIII gene, 1 in intron 13 and 1 in intron 22 in.

RFLP has great practicality and limitations. Sometimes it requires direct genetic diagnosis. Direct measurement of gene sequence can directly detect pathogenic genes, make the most accurate diagnosis of hemophilia, and provide a basis for research on molecular pathogenesis. Direct genetic diagnosis was performed primarily by denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism analysis (SSCP) combined with sequential analysis.

According to the condition, clinical manifestations, symptoms, signs, choose ECG, B-ultrasound, X-ray, CT, MRI, biochemistry and other tests.

Hemophilia A diagnosis and identification

1. Diagnostic criteria

(1) Clinical manifestations: 1 male patients, with or without family history, family history with coincident hereditary inheritance, female homozygous type can occur, rarely seen; 2 joints, muscles, deep tissue hemorrhage, spontaneous Generally, there are long-term walking, excessive exercise, history of surgery (including small surgery such as tooth extraction), joint deformity caused by repeated joint bleeding, and repeated tumors caused by repeated bleeding in deep tissues (blood cysts).

(2) Laboratory examination: 1 clotting time (test tube method) heavy extension, medium-sized normal, light, subclinical normal; 2 activated partial thromboplastin time (APTT), severely prolonged, can be normal fresh and adsorbed plasma Correction, mild extension or normal, subclinical normal; 3 platelet count, bleeding time, normal blood clot retraction; 4 normal prothrombin time (PT); 5VIII procoagulant activity (FVIII: C) reduced or minimal; The von Willebrand Factor antigen (vWF: Ag) was normal, and FVIII: C/vWF: Ag was significantly reduced.

(3) Severity classification.

(4) Acquired hemophilia A (acquired FVIII deficiency) caused by FVIII antibody.

2. Prenatal and carrier examinations Genetic testing of family members who may be carriers is important in genetic counseling, but it must be noted that approximately 30% of patients have new mutations rather than genetic gain, recombinant DNA. The technique provides a very advantageous means for prenatal diagnosis and carrying status check. The family that can perform restriction fragment length polymorphism (RFLP) analysis must include 1 male patient and the mother is heterozygous on 1 RFLP marker. of.

(1) Prenatal diagnosis: 1FVIII: C and vWF determination: In the past, depending on the pregnancy 18 to 21 weeks, fetal blood was measured by fetal microscopy to detect the levels of FVIII: C and vWF, but in 1978 to 1983, this was applied in the United States. Methods There were only 92 cases of prenatal diagnosis, and an estimated 300 fetuses had the risk of hemophilia A. The reason why this prenatal diagnosis method was not widely used was that the abortion caused by fetal microscopy was up to 6%. The failure rate of blood collection can reach 13% (12/92). RFLP of 2FVIII provides a reliable method for prenatal diagnosis. Currently, there are two methods for extracting embryonic DNA: sampling of 3 month old embryos; 8 to 11 weeks of chorionic villus sampling, to meet the RFLP conditions, that is, must have a proband, the mother of the proband must be heterozygous for the restriction site, using PCR technology to greatly simplify RFLP analysis, but there are Some limitations, some families have no DNA markers available, can only rely on the detection of FVIII: C and vWF.

(2) Carrier examination: the male and normal female sons are normal, and the daughters born are carriers; the female carrier and the normal male son have a 50% probability of being a hemophilia patient. The daughter has a 50% chance of becoming a carrier of the disease-causing gene; the female carrier and the son born to the male patient have a 50% probability of being a hemophilia patient, and the daughter born is either a carrier of the disease-causing gene, or a patient with hemophilia Both male and female patients are sons and daughters. Fortunately, this probability is extremely rare. Although there are reports of hemophilia in hemophiliacs and hemophilia carriers, female blood It is very rare for patients with AIDS, some female patients with abnormal X chromosome, such as Turner syndrome, X heterozygous and other X chromosome deficiency, may also be associated with hemophilia, if the carrier's normal X chromosome is disproportionately inhibited (Non-balance X inactivation), it can also show symptoms of bleeding friends.

There are three methods for judging female pathogenic gene carriers: 1 surely carriers: daughters of hemophilia A patients; mothers who have more than 2 hemophiliacs; mothers who have 1 hemophilia patient, their families There are 1 or more patients with hemophilia A, 2 may be carriers: a female member of a woman has a hemophilia A patient, and her own son has no hemophilia, or The sons of hemophilia A and the daughters of their babies (the virgins of the patients); the aunts of hemophilia A patients and their daughters (ie the cousins of the patients), 3 are likely to be carriers: Nearly one-third of patients with hemophilia A have sporadic cases, and no one in their mother's family has hemophilia A. However, using modern molecular biology techniques to investigate their families, there are carriers. This may be Because the disease-causing gene is in a state of concealment, it may also be because the next generation of males of the disease-causing gene carriers are less, and they are not shown. The number of hemophiliacs caused by new genetic mutations is very small.

The FVIII intron 22 inversion was directly detected by PCR or the BclI site in the FVIII gene, and the polymorphisms of the DXS52 (ST14) sites outside the STR and FVIII genes in introns 13 and 22 were genetically linked. Analysis, Wang Xuefeng, Shanghai Institute of Hematology, Shanghai Ruijin Hospital, etc., tested 21 families, of which the diagnostic rate was 94.7%. If the combined gene sequencing test, the diagnostic rate can reach 100%, and the inversion of the intron 22 is directly The diagnostic rate was 47.6%; the diagnostic rate of BclI site was 27.8%; the diagnostic rate of STR in introns 13 and 22 was 28.6% and 29.4%, respectively; the diagnostic rate of DXS52 was 81.3%, therefore, hemophilia The carrier test and prenatal diagnosis of the disease A can first detect the inversion of the intron 22, if the result is positive, the diagnosis can be made; if the inversion of the intron 22 is negative, the FVIII gene can be used. , multiple polymorphism results outside the site for genetic linkage analysis to make the final diagnosis.

Differential diagnosis

1. Classical hemophilia A needs to be differentiated from von Willebrand's disease (vWD). The occurrence of vWD is related to the lack of Fvon in vivo von Willebrand factor (vWF). Therefore, the level of FVIII in vWD It also declines, although the magnitude of the decline may be quite different in different patients. In patients with vWD, although the synthesis of FVIII is normal, the half-life in the body is shortened due to the decrease in the level of its vWF. Other manifestations of vWD and hemophilia A have prolonged bleeding time, decreased vWF antigen levels, and decreased ristocetin-induced platelet aggregation.

2. Differentiation from other hereditary coagulation factor-deficient diseases that cause APTT prolongation, such as coagulation factor IX, XI, XII, kallikrein, lack of high molecular weight kininogen, etc., only factor VIII deficiency and factor IX deficiency Case manifestations have the characteristics of X-linked inheritance, and only the lack of these two factors will affect the joints, resulting in disability. Hemophilia A can only be distinguished from hemophilia B through specific examinations. Men or women can develop factors. XI is deficient and has less bleeding than classic hemophilia.

3. Due to FXII, kallikrein, the lack of high molecular weight kininogen does not cause clinical bleeding, therefore, it is easy to distinguish from classic hemophilia, mild hemophilia (FVIII level is about normal 15%) must take into account the lack of combination of factor V and factor VIII, although both APTT and PT may have a slight prolongation, unless the lack of such a combination is considered, often the correct diagnosis is not obtained.

4. Acquired factor VIII deficiency (acquired hemophilia A) is caused by the presence of anti-FVIII antibodies in the blood. The clinical manifestations of hemorrhage are the same as those of hemophilia methyl, but the degree of bleeding is often heavier. Can occur in healthy people, women (especially during pregnancy), the elderly, and some patients with immune diseases, laboratory tests, APTT and S-CT prolonged, and the same amount of normal plasma can not correct the lack of STGT or BTGT, The titer of the inhibitor (anti-FVIII antibody) is increased, and the identification is more accurate, and the Bethesda method is commonly used in clinical examination.

Hemophilia A, hemophilia B and factor XI deficiency and laboratory characteristics of acquired hemophilia A are seen.

5. Hemorrhagic disease caused by hemophilia A and hematoma may be confused with other diseases when the diagnosis is not clear. For example, deep hematoma is mistaken for suppurative lesions and incision and drainage are performed; iliopsoas hemorrhage is mistaken for appendicitis; Misdiagnosed retroperitoneal hematoma as appendix abscess; hemophilia A joint bleeding was mistaken for tuberculosis, arthritis and sarcoma, etc., hemorrhage or hematoma caused by hemophilia was mistaken for kidney tumor, lung disease, digestive tract ulcer, intra-abdominal hemorrhage As ulcer perforation, intestinal obstruction, etc. have been reported.

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