Kit for detecting hemochromia related gene mutation and SNP (Single Nucleotide Polymorphism) sites

Abstract

The invention discloses a kit for detecting hemochromia related gene mutation and SNP (Single Nucleotide Polymorphism) sites. The kit comprises a detection reagent for mutation sites and SNP sites of hemochromia related genes, and the related genes comprise one or more of an HFE gene, an HJV gene, an HAMP gene, a TFR2 gene and an SLC40A1 gene. The kit provided by the invention can detect a plurality of mutation sites and SNP sites of five related genes of hemochromia, is comprehensive in detection, high in accuracy, quicker and more sensitive, is simple and convenient to operate, can comprehensively reflect hemochromia, and is beneficial to guiding clinical medication.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a kit for detecting hemochromatosis-related gene mutations and SNP sites. Background Art

[0002] Hemochromatosis (HC) refers to a liver metabolic disorder caused by gene mutation, which has congenital, lifelong and familial characteristics. Hemochromatosis, also known as hereditary hemochromatosis (HHC), is a rare autosomal recessive genetic disease. Its pathogenesis is that mutations in iron metabolism-related genes lead to excessive iron absorption in the small intestine, and excessive iron accumulation in the body causes degeneration and diffuse fibrosis, metabolism and dysfunction in the liver, kidney, brain, pancreas, etc., leading to clinical symptoms such as cirrhosis, diabetes, cardiomyopathy, pituitary damage, testicular atrophy, joint diseases and skin pigmentation.

[0003] Among the five genes currently known to be associated with hereditary hemochromatosis (HAMP, HJV, TFR2, FPN and HFE), they can be divided into: HFE (type I), HJV (type IIA), HAMP (type IIB), TFR2 (type III) and SLC40A1 (type IV) according to different gene mutation types. Types I to III are all autosomal recessive inheritance; type IV is autosomal dominant inheritance. All proteins encoded by TFR2, HAMP and HFE2 / HJV genes are involved in the regulation of hepcidin-related signaling pathways, thereby affecting the distribution of iron in the body; while the iron transporter (Ferroportin) encoded by the SLC40A1 gene is the only intracellular iron transporter, which exists in intestinal epithelial cells, macrophages, hepatocytes and placental syncytiotrophoblasts, and is inherited in a dominant manner. This gene mutation either causes obvious hereditary hemochromatosis or obvious hemochromatosis.

[0004] The incidence of hereditary hemochromatosis is high in the Caucasian population in Northern Europe. Studies have shown that the mutation sites of HH patients in my country may be mainly non-type I HH, which is quite different from those in European and American countries. In Asian populations, especially Japanese patients with hereditary hemochromatosis, the frequency of TFR2 gene mutations is very high. Therefore, the Japanese believe that the TFR2 gene has a strong pathogenic gene for hemochromatosis, especially in middle-aged people without HFE mutations. The internal sequencing data of our laboratory showed that there is no p.C321X mutation in the general Chinese population, suggesting that the p.C321X homozygous mutation on the HJV gene may be the pathogenic mutation of this HHC patient. Consistent with this, the site was wild-type homozygous or heterozygous in the four family members who did not suffer from HHC, and none of them was p.C321X homozygous. Therefore, it is believed that the HJV p.C321X homozygous mutation can cause HHC. SLC40A1 (type IV) hereditary hemochromatosis is an autosomal dominant genetic disease with a high incidence rate. It is easier to detect positive specimens through gene mutation detection. Foreign studies have confirmed that SLC40A1 has many mutation points, but there is still little research on this gene in China.

[0005] Hemochromatosis is often asymptomatic before middle age. 80% to 90% of male patients have a total iron reserve of >10g before symptoms appear. Symptoms in female patients often occur after menopause. Since iron loss during menstruation and pregnancy reduces iron load, the intrahepatic iron content of female patients who have menopause before the age of 50 is increased. Despite blood loss during pregnancy and menstruation, women can have complete clinical manifestations of hemochromatosis. Since the clinical manifestations of sequelae caused by excessive iron load occur in the later stage, early clinical laboratory tests are the best way to evaluate iron storage. Nonspecific systemic symptoms such as fatigue can be seen in female patients in the early stage; in male patients, cirrhosis or diabetes is common in the early stage. Clinical manifestations of worsening iron deposition may include hepatocellular dysfunction, cirrhosis, copper pigmentation of the skin, diabetes (complication rate is 50% to 60%), and cardiomyopathy with clinical manifestations of cardiac hypertrophy, heart failure, and arrhythmias or conduction block. Pituitary failure is common and can lead to testicular atrophy and loss of libido. Abdominal pain, arthritis, and uncommon chondrocalcinosis. Although the increased incidence of diabetes in families suggests that factors other than pancreatic iron deposition may play a role, these lesions are due to parenchymal cell iron deposition. The incidence of hepatocellular carcinoma in patients with long-standing hemochromatosis (about 14%) is higher than that of any type of cirrhosis.

[0006] Because hemochromatosis is insidious, progresses slowly, and the degree of tissue involvement varies, the disease is often diagnosed in the late stage of the disease after significant tissue damage, and non-hereditary iron overload such as congenital hemolytic anemia (sickle cell anemia, thalassemia) should be excluded. In hereditary hemochromatosis, serum iron is elevated (>300mg / dl). Serum transferrin saturation is a sensitive indicator reflecting increased iron. When >50%, it has diagnostic value. Serum ferritin is elevated and erythrocyte ferritin is elevated by >200 attograms / erythrocyte. The use of the chelating agent desferrioxamine (500-1000mg intramuscular injection, the dose is calculated according to the patient's body size) can increase urinary iron excretion (>2mg / 24h). In some cases, when diagnosis is difficult, this treatment can be used as a diagnostic test. Increased intrahepatic iron content can be reflected by magnetic resonance imaging. Liver biopsy was once an important diagnostic measure. Currently, this examination only provides a basis for diagnosing fibrosis (cirrhosis). The diagnosis can be confirmed when hepatic iron deposition and increased iron content (mean hepatic iron index > 2; mean hepatic iron concentration > 250 μmol / gm) are detected. The use of C282Y (most common mutation) and H63D (less common mutation) tests has simplified the clinical diagnosis of genotype and the appropriate screening method for first-degree relatives.

[0007] HJV mRNA is mainly expressed in the liver, heart, and skeletal muscle. Among them, the expression of HJV in hepatic parenchymal cells has an important regulatory function on the secretion of hepcidin, thereby maintaining the balance of iron metabolism in the body. HJV exists in two forms: the membrane form (m-HJV) and the soluble form (s-HJV). When the glycosylphosphatidylinositol (GPI) anchoring domain at the tail of the membrane form HJV is cut off, a soluble HJV protein can be formed. The regulatory functions of the two on hepcidin are opposite. Among them, the membrane form HJV, as a co-receptor of bone morphogenetic protein (BMP), can upregulate the expression of hepcidin through the BMP / SMAD pathway; while the soluble HJV can compete with the membrane form HJV to bind to BMP, resulting in the downregulation of hepcidin expression. In recent years, a variety of different HJV mutations have been found in patients with hemochromatosis. Among them, various HJV mutations can significantly reduce the expression level of attached HJV. At the same time, the study also found that when carrying HJV mutations, the expression of hepcidin is abnormally reduced, which further suggests the role of HJV mutations in the occurrence of hemochromatosis.

[0008] Huang et al. reported that the HJV mutation p.C321X was present in a young Chinese female patient with hereditary hemochromatosis. Unlike this study, the mutation p.C321X detected by Huang et al. in the patient was heterozygous and originated from her phenotypically normal mother. This mutant allele formed a compound heterozygous with another HJV mutant allele (p.I281T) from her phenotypically normal father, ultimately leading to hereditary hemochromatosis. Among them, the p.I281T homozygous mutation has been reported to cause hemochromatosis. The p.C321X mutation has similar functions to the previously reported p.R326X mutation. Both can cause the HJV protein to lack the glycosylphosphatidylinositol (GPI) anchoring domain at the tail, thereby forming soluble HJV, and ultimately causing abnormal iron metabolism.

[0009] Li et al. reported a Chinese middle-aged male hemochromatosis patient carrying a heterozygous HJV mutant allele p.C321X, which was derived from his healthy mother. However, the patient did not carry other HJV gene mutations, nor mutations in other hemochromatosis-related genes (HAMP, TFR2, FPN, and HFE), a conclusion that contradicts the previous understanding that hemochromatosis is a recessive genetic disease. Li et al. further analyzed other members of the patient's family and found that one of the patient's sisters and one of his brothers also inherited the allele carrying the p.C321X mutation from his mother. Interestingly, the patient's brother was suspected of having hemochromatosis, while his sister was in good health. Because healthy men have never been reported to carry the mutant allele p.C321X, Li et al. believe that genes and environmental factors jointly affect the occurrence of the disease. Men carrying an abnormal HJV allele (p.C321X) are also very likely to develop hemochromatosis, but the age of onset may be later than patients carrying homozygous or compound heterozygous mutations of the HJV gene; while women carrying an abnormal allele p.C321X basically will not develop the disease.

[0010] The TFR2 gene is also an iron overload-related gene and helps diagnose hereditary hemochromatosis in patients without HFE gene mutations. In the process of human iron regulation, the TFR2 protein plays an independent function and has no clear correlation with the HFE / TFRC signaling pathway. The TFR2 gene has two transcripts, α and β, which have different expressions and functions. The α transcript is mainly expressed in the liver, and the expressed protein is a transmembrane protein; while the β transcript is a spectrum-low-expression version. The protein produced by this transcript lacks cytoplasmic domains, transmembrane domains, and extracellular domains, so it is an intracellular protein. So far, gene mutation detection has become increasingly clear and important in the diagnosis of hereditary hemochromatosis.

[0011] Excessive iron deposition in the human body and related iron-related diseases are more likely to induce malignant tumors, especially in patients with SLC40A1 gene mutations. There have been clear reports that even mutations in the first intron of the gene (c.44-24G>C) exist in both homozygous and heterozygous patients with abnormal iron metabolism, indicating that they have clear physiological significance. Many recent studies have focused on the 5 ‘ The study of CGGn microsatellite of UTR may have important pathological significance. Ferroportin binds to hepcidin on the cell membrane surface, which leads to tyrosine phosphorylation of the cytoplasmic membrane end of ferroportin, resulting in internal localization, dephosphorylation and finally ubiquitination degradation of ferroportin. Hepcidin can thus participate in the distribution, localization and deposition of iron. The different mutation sites of the SLC40A1 gene affect the binding function with hepcidin, so patients have different manifestations. The deletion of the 241 amino acids at the C-terminus of the protein can only lead to mild anemia and mild hemochromatosis phenotypes in patients. The iron load is mainly concentrated in stem cells and Kupffer cells. The hemoglobin level recovers very slowly after bloodletting.

[0012] Since hemochromatosis is insidious, progresses slowly, and the degree of tissue involvement varies, the disease is often diagnosed in the late stage of the disease after significant tissue damage, and non-hereditary iron overload such as congenital hemolytic anemia (sickle cell anemia, thalassemia) should be excluded. Therefore, diagnosis is difficult and there are multiple diseases that are confused; and the diagnosis is delayed, and the appearance of typical symptoms is accompanied by severe deterioration of the disease. Therefore, rapid diagnosis at the molecular level can help clinicians make accurate judgments, provide timely treatment, and improve survival rates. In addition, because the diagnosis of hereditary hemochromatosis is very difficult, the statistical data on the incidence in China is inaccurate. Genetic diagnosis can improve the statistics on the incidence of the disease in China, which has very strong social benefits. Timely diagnosis of the categories of patients with hereditary hemochromatosis can better serve the clinic, and is also responsible for many suspected hereditary hemochromatosis, which has important medical and social benefits for better treatment and post-treatment maintenance. Summary of the invention

[0013] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a kit for detecting hemochromatosis-related gene mutations and SNP sites, so as to solve the problems in the prior art.

[0014] To achieve the above purpose, the present invention specifically adopts the following technical solutions.

[0015] The first aspect of the present application provides a kit for detecting mutations and SNP sites of hemochromatosis-related genes, the kit comprising detection reagents for mutation sites and SNP sites of hemochromatosis-related genes, and the related genes comprise one or more of the HFE gene, HJV gene, HAMP gene, TFR2 gene and SLC40A1 gene.

[0016] The gene mutations in this application include point mutations, deletion mutations, and insertion mutations.

[0017] In the first aspect of the present invention, the mutation site of the HFE gene is selected from one or more of D129N, S65C, C282Y, H63D, Y230C, C225*, Q238P, E277K, V295, H109Y, I287S, Y46X and A356fs.

[0018] In the first aspect of the present invention, the SNP point of the HFE gene is selected from one or more of rs2071303, rs1800708, rs1572982 and rs3817623.

[0019] Preferably, the primer set comprises specific primers for amplifying the exon and / or UTR segment to which the mutation site of the HFE gene belongs.

[0020] More preferably, the specific primers are used to amplify exon 1 of the HFE gene.

[0021] More preferably, the specific primers for amplifying exon 1 of the HFE gene include an upstream primer and a downstream primer.

[0022] More preferably, in the specific primers used to amplify exon 1 of the HFE gene, the sequence of the upstream primer is shown as SEQ ID NO.1, and the sequence of the downstream primer is shown as SEQ ID NO.2.

[0023] More preferably, the specific primers are used to amplify exon 2 of the HFE gene.

[0024] Further preferably, the specific primers for amplifying exon 2 of the HFE gene include an upstream primer and a downstream primer.

[0025] More preferably, in the specific primers used to amplify exon 2 of the HFE gene, the sequence of the upstream primer is shown as SEQ ID NO.3, and the sequence of the downstream primer is shown as SEQ ID NO.4.

[0026] More preferably, the specific primers are used to amplify exon 3 of the HFE gene.

[0027] Further preferably, the specific primers for amplifying exon 3 of the HFE gene include an upstream primer and a downstream primer.

[0028] More preferably, in the specific primers used to amplify exon 3 of the HFE gene, the sequence of the upstream primer is shown as SEQ ID NO.5, and the sequence of the downstream primer is shown as SEQ ID NO.6.

[0029] More preferably, the specific primers are used to amplify exons 4-5 of the HFE gene.

[0030] Further preferably, the specific primers for amplifying exons 4-5 of the HFE gene include an upstream primer and a downstream primer.

[0031] More preferably, in the specific primers used to amplify exons 4-5 of the HFE gene, the sequence of the upstream primer is shown as SEQ ID NO.7, and the sequence of the downstream primer is shown as SEQ ID NO.8.

[0032] More preferably, the specific primers are used to amplify exon 6 of the HFE gene.

[0033] Further preferably, the specific primers for amplifying exon 6 of the HFE gene include an upstream primer and a downstream primer.

[0034] More preferably, in the specific primers used to amplify exon 6 of the HFE gene, the sequence of the upstream primer is shown as SEQ ID NO.9, and the sequence of the downstream primer is shown as SEQ ID NO.10.

[0035] In the first aspect of the present invention, the mutation site of the HJV gene is selected from one or more of E3D, Q6H, N129*, I281T, G320V, C321X and R326X.

[0036] Preferably, the primer set comprises specific primers for amplifying the exons and / or UTR segments to which the mutation site of the HJV gene belongs.

[0037] Preferably, specific primers are used to amplify exon 1 of the HJV gene.

[0038] More preferably, the specific primers for amplifying Exon 1 of the HJV gene include an upstream primer and a downstream primer.

[0039] Further preferably, in the specific primers used to amplify exon 1 of the HJV gene, the sequence of the upstream primer is shown in SEQ ID NO.11, and the sequence of the downstream primer is shown in SEQ ID NO.12

[0040] Preferably, specific primers are used to amplify exon 2 of the HJV gene.

[0041] More preferably, the specific primers for amplifying Exon 2 of the HJV gene include an upstream primer and a downstream primer.

[0042] Further preferably, in the specific primers used to amplify exon 2 of the HJV gene, the sequence of the upstream primer is shown as SEQ ID NO.13, and the sequence of the downstream primer is shown as SEQ ID NO.14.

[0043] Preferably, specific primers are used to amplify exon 3 of the HJV gene.

[0044] More preferably, the specific primers for amplifying Exon 3 of the HJV gene include an upstream primer and a downstream primer.

[0045] Further preferably, in the specific primers used to amplify exon 3 of the HJV gene, the sequence of the upstream primer is shown as SEQ ID NO.15, and the sequence of the downstream primer is shown as SEQ ID NO.16.

[0046] Preferably, specific primers are used to amplify exon 4 of the HJV gene.

[0047] More preferably, the specific primers for amplifying Exon 4 of the HJV gene include an upstream primer and a downstream primer.

[0048] Further preferably, in the specific primers used to amplify exon 4 of the HJV gene, the sequence of the upstream primer is shown as SEQ ID NO.17, and the sequence of the downstream primer is shown as SEQ ID NO.18.

[0049] In the first aspect of the present invention, the mutation site of the HAMP gene is selected from one or more of c.-25GNA, c.-25GNA, c.-92GNA and G71D.

[0050] Preferably, the primer set comprises specific primers for amplifying the exon and / or UTR segment to which the mutation site of the HAMP gene belongs.

[0051] Preferably, the specific primers are used to amplify exon 1 of the HAMP gene.

[0052] More preferably, the specific primers for amplifying Exon 1 of the HAMP gene include an upstream primer and a downstream primer.

[0053] Further preferably, in the specific primers used to amplify exon 1 of the HAMP gene, the sequence of the upstream primer is shown as SEQ ID NO.19, and the sequence of the downstream primer is shown as SEQ ID NO.20.

[0054] Preferably, the specific primers are used to amplify exons 2-3 of the HAMP gene.

[0055] More preferably, the specific primers for amplifying Exon 2-3 of the HAMP gene include an upstream primer and a downstream primer.

[0056] Further preferably, in the specific primers used to amplify exons 2-3 of the HAMP gene, the sequence of the upstream primer is shown as SEQ ID NO.21, and the sequence of the downstream primer is shown as SEQ ID NO.22.

[0057] In the first aspect of the present invention, the mutation site of the SLC40A1 gene is selected from one or more of IVS+10del GTT splicing mutation, V221V, c.44-24G>C, V162del, A77D and Q248H.

[0058] In the first aspect of the present invention, the SNP site of the SLC40A1 gene is selected from one or more of rs2304704, rs4287798, rs1156835, rs13008848 and rs2304704.

[0059] Preferably, the primer set comprises specific primers for amplifying the exon and / or UTR segment to which the mutation site of the SLC40A1 gene belongs.

[0060] Preferably, the specific primers are used to amplify exon 1 of the SLC40A1 gene.

[0061] More preferably, the specific primers for amplifying Exon 1 of the SLC40A1 gene include an upstream primer and a downstream primer.

[0062] Further preferably, in the specific primers used to amplify exon 1 of the SLC40A1 gene, the sequence of the upstream primer is shown as SEQ ID NO.23, and the sequence of the downstream primer is shown as SEQ ID NO.24.

[0063] Preferably, the specific primers are used to amplify exon 2 of the SLC40A1 gene.

[0064] More preferably, the specific primers for amplifying Exon 2 of the SLC40A1 gene include an upstream primer and a downstream primer.

[0065] Further preferably, in the specific primers used to amplify exon 2 of the SLC40A1 gene, the sequence of the upstream primer is shown as SEQ ID NO.25, and the sequence of the downstream primer is shown as SEQ ID NO.26.

[0066] Preferably, the specific primers are used to amplify exon 3 of the SLC40A1 gene.

[0067] More preferably, the specific primers for amplifying exon 3 of the SLC40A1 gene include an upstream primer and a downstream primer.

[0068] Further preferably, in the specific primers used to amplify exon 3 of the SLC40A1 gene, the sequence of the upstream primer is shown as SEQ ID NO.27, and the sequence of the downstream primer is shown as SEQ ID NO.28.

[0069] Preferably, the specific primers are used to amplify exon 4 of the SLC40A1 gene.

[0070] More preferably, the specific primers for amplifying Exon 4 of the SLC40A1 gene include an upstream primer and a downstream primer.

[0071] Further preferably, in the specific primers used to amplify exon 4 of the SLC40A1 gene, the sequence of the upstream primer is shown as SEQ ID NO.29, and the sequence of the downstream primer is shown as SEQ ID NO.30.

[0072] Preferably, the specific primers are used to amplify exon 5 of the SLC40A1 gene.

[0073] More preferably, the specific primers for amplifying Exon 5 of the SLC40A1 gene include an upstream primer and a downstream primer.

[0074] Further preferably, in the specific primers used to amplify exon 5 of the SLC40A1 gene, the sequence of the upstream primer is shown as SEQ ID NO.31, and the sequence of the downstream primer is shown as SEQ ID NO.32.

[0075] Preferably, the specific primer No. 6 is used to amplify the SLC40A1 gene.

[0076] More preferably, the specific primers for amplifying Exon 6 of the SLC40A1 gene include an upstream primer and a downstream primer.

[0077] Further preferably, in the specific primers used to amplify exon 6 of the SLC40A1 gene, the sequence of the upstream primer is shown as SEQ ID NO.33, and the sequence of the downstream primer is shown as SEQ ID NO.34.

[0078] Preferably, the specific primers are used to amplify exon 7 of the SLC40A1 gene.

[0079] More preferably, the specific primers for amplifying Exon 7 of the SLC40A1 gene include an upstream primer and a downstream primer.

[0080] Further preferably, in the specific primers used to amplify exon 7 of the SLC40A1 gene, the sequence of the upstream primer is shown as SEQ ID NO.35, and the sequence of the downstream primer is shown as SEQ ID NO.36.

[0081] Preferably, the specific primers are used to amplify exon 8 of the SLC40A1 gene.

[0082] More preferably, the specific primers for amplifying Exon 8 of the SLC40A1 gene include an upstream primer and a downstream primer.

[0083] Further preferably, in the specific primers used to amplify exon 8 of the SLC40A1 gene, the sequence of the upstream primer is shown as SEQ ID NO.37, and the sequence of the downstream primer is shown as SEQ ID NO.38.

[0084] In the first aspect of the present invention, the mutation site of the TFR2 gene is selected from one or more of Y250X, E60X, T515A, M172K, L490R, V561X, I238M, A617A, L750P, A777V, G792R, Q306*, Q672*, L99V, V277L, N540N, T740M, R455Q, R468H, G792R, T740M and R752H.

[0085] In the first aspect of the present invention, the SNP site of the TFR2 gene is selected from one or more of rs41295912, rs148902192, rs80338885, rs2075674, rs41302360, rs41295915 and rs35142847.

[0086] Preferably, the primer set comprises specific primers for amplifying the exon and / or UTR segment to which the mutation site of the TFR2 gene belongs.

[0087] Preferably, the specific primers are used to amplify exons 1-2 of the TFR2 gene.

[0088] More preferably, the specific primers used to amplify exons 1-2 of the TFR2 gene are an upstream primer and a downstream primer.

[0089] Further preferably, in the specific primers used to amplify exons 1-2 of the TFR2 gene, the sequence of the upstream primer is shown as SEQ ID NO.39, and the sequence of the downstream primer is shown as SEQ ID NO.40.

[0090] Preferably, the specific primers are used to amplify exon 3 of the TFR2 gene.

[0091] More preferably, the specific primers used to amplify exon 3 of the TFR2 gene are an upstream primer and a downstream primer.

[0092] Further preferably, in the specific primers used to amplify exon 3 of the TFR2 gene, the sequence of the upstream primer is shown as SEQ ID NO.41, and the sequence of the downstream primer is shown as SEQ ID NO.42.

[0093] Preferably, the specific primers are used to amplify exons 4 to 6 of the TFR2 gene.

[0094] More preferably, the specific primers used to amplify exons 4 to 6 of the TFR2 gene are upstream primers and downstream primers.

[0095] Further preferably, in the specific primers used to amplify exons 4 to 6 of the TFR2 gene, the sequence of the upstream primer is shown as SEQ ID NO.43, and the sequence of the downstream primer is shown as SEQ ID NO.44.

[0096] Preferably, the specific primers are used to amplify exons 7-8 of the TFR2 gene.

[0097] More preferably, the specific primers used to amplify exons 7-8 of the TFR2 gene are an upstream primer and a downstream primer.

[0098] Further preferably, in the specific primers used to amplify exons 7-8 of the TFR2 gene, the sequence of the upstream primer is shown as SEQ ID NO.45, and the sequence of the downstream primer is shown as SEQ ID NO.46.

[0099] Preferably, the specific primers are used to amplify exon 9 of the TFR2 gene.

[0100] More preferably, the specific primers used to amplify exon 9 of the TFR2 gene are an upstream primer and a downstream primer.

[0101] Further preferably, in the specific primers used to amplify exon 9 of the TFR2 gene, the sequence of the upstream primer is shown as SEQ ID NO.47, and the sequence of the downstream primer is shown as SEQ ID NO.48.

[0102] Preferably, the specific primers are used to amplify exon 10 of the TFR2 gene.

[0103] More preferably, the specific primers used to amplify Exon 10 of the TFR2 gene are an upstream primer and a downstream primer.

[0104] Further preferably, in the specific primers used to amplify exon 10 of the TFR2 gene, the sequence of the upstream primer is shown as SEQ ID NO.49, and the sequence of the downstream primer is shown as SEQ ID NO.50.

[0105] Preferably, the specific primers are used to amplify exons 11-15 of the TFR2 gene.

[0106] More preferably, the specific primers used to amplify exons 11 to 15 of the TFR2 gene are upstream primers and downstream primers.

[0107] Further preferably, in the specific primers used to amplify exons 11 to 15 of the TFR2 gene, the sequence of the upstream primer is shown as SEQ ID NO.51, and the sequence of the downstream primer is shown as SEQ ID NO.52.

[0108] Preferably, the specific primers are used to amplify exon 16 of the TFR2 gene.

[0109] More preferably, the specific primers used to amplify Exon 16 of the TFR2 gene are an upstream primer and a downstream primer.

[0110] Further preferably, in the specific primers used to amplify exon 16 of the TFR2 gene, the sequence of the upstream primer is shown as SEQ ID NO.53, and the sequence of the downstream primer is shown as SEQ ID NO.54.

[0111] Preferably, the specific primers are used to amplify exon 17 of the TFR2 gene.

[0112] More preferably, the specific primers for amplifying exon 17 of the TFR2 gene are an upstream primer and a downstream primer.

[0113] Further preferably, in the specific primers used to amplify exon 17 of the TFR2 gene, the sequence of the upstream primer is shown as SEQ ID NO.55, and the sequence of the downstream primer is shown as SEQ ID NO.56.

[0114] Preferably, the specific primers are used to amplify exon 18 of the TFR2 gene.

[0115] More preferably, the specific primers used to amplify Exon 18 of the TFR2 gene are an upstream primer and a downstream primer.

[0116] Further preferably, in the specific primers used to amplify exon 18 of the TFR2 gene, the sequence of the upstream primer is shown as SEQ ID NO.57, and the sequence of the downstream primer is shown as SEQ ID NO.58.

[0117] In the first aspect of the present invention, the primers in the primer set may be present in the form of each amplification primer alone, or may be a mixture of forward amplification primers and a mixture of reverse amplification primers. Preferably, the primer set consists of a mixture of forward amplification primers and a mixture of reverse amplification primers.

[0118] Preferably, in the mixture formed by the forward amplification primers, the concentration of each forward amplification primer is 0.5-8 pmol / μL. Preferably, the concentration can be 0.5-1.5 pmol / μL, or 1-6 pmol / μL, or 5-8 pmol / μL. In a preferred embodiment, it is 5 pmol / μL.

[0119] Preferably, in the mixture formed by the reverse amplification primers, the concentration of each reverse amplification primer is 0.5-8 pmol / μL. Preferably, the concentration can be 0.5-1.5 pmol / μL, or 1-6 pmol / μL, or 5-8 pmol / μL. In a preferred embodiment, it is 5 pmol / μL.

[0120] In the first aspect of the present invention, the kit further comprises a reagent for extracting genomic DNA from the sample to be tested. The reagent for extracting genomic DNA from the sample can be an existing kit.

[0121] Preferably, the sample to be tested is from human. More preferably, the sample to be tested is selected from peripheral blood.

[0122] In the first aspect of the present invention, the kit further comprises one or both of a PCR Mix reagent and deionized water. Since such commonly used PCR reagents can be purchased separately from the market or prepared by oneself, which reagents need to be assembled into the kit can be configured according to the actual needs of the customer, and for convenience, all of them can be assembled into the kit.

[0123] In the PCR amplification system, primers, PCR Mix reagents, and deionized water are all common components, and their contents are also normal.

[0124] The PCR amplification system can be configured by itself, or can be obtained by directly adding primers to commercially available primer-free and universal PCR amplification solutions. For example, the kit can also contain PCR Mix reagents and deionized water. The PCR amplification system can be obtained by adding the primers of the present invention and the sample DNA to be tested.

[0125] In the first aspect of the present invention, the kit further comprises one or both of a positive control and a negative control.

[0126] Preferably, the positive control contains some of the following mutation sites:

[0127] HFE: D129N, S65C, C282Y, H63D, Y230C, C225*, Q238P, E277K, V295, H109Y, I287S, Y46X, A356fs, rs2071303, rs1800708, rs1572982, rs3817623;

[0128] HJV: E3D, Q6H, N129*, I281T, G320V, C321X, R326X;

[0129] HAMP: c.-25GNA, c.-25GNA, c.-92GNA, G71D, rs3817623;

[0130] TFR2: Y250X, E60X, T515A, M172K, L490R, V561X, I238M, A617A, L750P, A777V, G792R, Q306*, Q672*, L99V, V277L, N540N, T740M, R 455Q, R468H, G792R, T740M, R752H, rs41295912, rs148902192, rs80338885, rs2075674, rs41302360, rs41295915, rs35142847;

[0131] SLC40A1: IVS+10del GTT splicing mutation, V221V, c.44-24G>C, rs2304704, rs4287798, rs1156835, rs13008848, rs2304704, V162del, A77D, Q248H.

[0132] Preferably, the negative control may be deionized water.

[0133] The second aspect of the present invention protects the use of the primer set as described above or the kit as described above in detecting mutation sites and SNP sites of hemochromatosis-related genes or in preparing products for detecting hemochromatosis.

[0134] In the second aspect of the present invention, the product comprises a chip or a detection system.

[0135] The third aspect of the present invention provides a method for detecting hemochromatosis-related gene mutations and SNP sites, comprising the following steps:

[0136] Extracting genomic DNA from the sample to be tested;

[0137] Perform PCR amplification using the kit described above to obtain a PCR amplification product;

[0138] Sequencing the PCR amplification product to obtain a sequencing result;

[0139] The sequencing results are compared with the gene sequences published in the database to obtain gene mutation detection results.

[0140] In the fifth aspect of the present invention, the conditions of the PCR amplification reaction are: 95°C for 5 minutes; 95°C for 30 seconds, 56°C for 30 seconds, 72°C for 30 seconds, 40 cycles; 72°C for 7 minutes.

[0141] In the third aspect of the present invention, the method is a non-disease diagnosis or treatment method.

[0142] Compared with the prior art, the beneficial effects of this application are:

[0143] 1) The kit of the present invention is based on the characteristics of gene mutations that often occur during the onset of hereditary hemochromatosis, combined with the latest molecular biology and statistical principles, and has clear gene selection targets, strong applicability, and is comprehensive and centralized. It can be used for long-term detection of hereditary hemochromatosis, especially long-term and periodic detection of families with a clear genetic background, and is more suitable for newborns and adolescents;

[0144] 2) The kit of the present invention has strong specificity, clear gene-related mutation sites, simple detection process, high accuracy, and is more rapid and sensitive. The detection results are clear and purposeful.

[0145] 3) The test specimen of the kit of the present invention is peripheral blood and the amount used is small, easy to obtain, and is particularly friendly to the human body, especially children, which is beneficial to the test subject itself, uses less experimental equipment, and saves costs;

[0146] 4) The kit of the present invention uses a low-toxic fluorescent dye, does not require radioactive labeling, and has good safety;

[0147] 5) The kit of the present invention adopts the currently internationally recognized gold standard for gene mutation detection: the first-generation sequencing method. The detection results are accurate and efficient, and can effectively reduce false positives. The detection results can effectively serve clinical services.

[0148] 6) The detection system of the kit of the present invention covers the key sites of all core domains of the disease, which is the most comprehensive detection project so far, and can completely detect all possible mutation hotspots of HFE (type I), HJV (type IIA), HAMP (type IIB), TFR2 (type III) and SLC40A1 (type IV) genes.

[0149] 7) The kit of the present invention adopts the classic first-generation sequencing method, covering all pathogenic genes of the five subtypes of hereditary hemochromatosis, and only relies on the high specificity of primers to creatively put the five genes involved into a PCR system, avoiding cross-detection, primer dimers that highly affect the detection effect, and repeated detection. It has high accuracy and can obtain very clear sequencing peaks, avoiding multiple PCR reactions. It has the advantages of high sensitivity, high precision, low cost, and the ability to quickly detect and obtain results. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] Figure 1 Shown is the domain distribution diagram of the protein encoded by the relevant gene HFE of the present invention.

[0151] Figure 2 Shown is the domain distribution diagram of the protein encoded by the relevant gene HJV of the present invention.

[0152] Figure 3 Shown is the domain distribution diagram of the protein encoded by the related gene HAMP of the present invention.

[0153] Figure 4 Shown is the domain distribution diagram of the protein encoded by the related gene TFR2 of the present invention.

[0154] Figure 5 Shown is the domain distribution diagram of the protein encoded by the gene SLC40A1 related to the present invention.

[0155] Figures 6A-6E Shown is a specific analysis diagram of the primers corresponding to the relevant genes of the present invention.

[0156] Figure 7 Shown is a flow chart of the use of the kit of the present invention.

[0157] Figure 8 Shown is a diagram of the gene mutation analysis results in Example 3 of the present invention.

[0158] Fig. 9 Shown is a graph showing the analysis results of Comparative Example 1 of the present invention.

[0159] Fig.10 Shown is a graph showing the analysis results of Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0160] The genes that cause hemochromatosis in Europe, America and Asia may be different, and family screening is difficult, especially in remote areas in the south where there are many suspected patients.

[0161] In view of the low frequency of gene mutation and low false positive rate, once the mutation occurs, it can provide clinicians with useful information about HHC, accurately targeting the disease, especially differentiating it from other diseases, and providing strong guidance for the use of drugs by clinicians in the later stage, thus avoiding other additional tests. Therefore, the detection of genes for this type of disease is very important, which can effectively reduce the economic burden and improve the survival rate for patients and their families. In particular, it provides a safe environment for families with a history of genetic diseases, reduces the psychological pressure of people with a family history, and improves the quality of life of potential patients.

[0162] Based on the classic hot spot mutations of hemochromatosis-related genes, this application adds some special mutation sites, including splicing sites and SNP sites, extends the corresponding detection area, and even includes the UTR region. A total of 69 mutation sites can be detected, which can comprehensively reflect hemochromatosis, reduce the difficulty and cost of detection, and can effectively guide clinical drug use.

[0163] The source of the hemochromatosis-related gene sequence of the present invention is the NCBI (National Center for Biotechnology Information) gene library, and the specific identification and function are shown in Table 1 below.

[0164] Table 1

[0165]

[0166]

[0167] The domain distribution diagram of the protein encoded by the hemochromatosis-related gene of the present invention is as follows: Figure 1-5 The domain distribution of proteins encoded by related genes HFE, HJV, HAMP, TFR2 and SLC40A1 are shown in Figure 1 , 2 , 3, 4 and 5.

[0168] The hemochromatosis-related genes of the present invention, such as HFE (type I), HJV (type IIA), HAMP (type IIB), TFR2 (type III) and SLC40A1 (type IV) gene mutations and SNP sites are shown in Table 2 below.

[0169] Table 2

[0170]

[0171] The primers in the kit of the present invention are shown in Table 3 below.

[0172] Table 3

[0173]

[0174]

[0175]

[0176] like Fig. 6A This is a primer specific analysis diagram of the EXON1 exon of the HFE gene, such as Figure 6B This is a primer specific analysis diagram of the EXON1 exon of the HAMP gene, such as Figure 6C This is the primer specificity analysis diagram of the EXON1 exon of the HJV gene. Fig.6D This is a primer specificity analysis diagram for EXON1 of the SLC40A1 gene, such as Fig. 6E This is a primer specificity analysis diagram for the EXON1-2 exons of the TFR2 gene.

[0177] The present invention provides a kit for hemochromatosis-related gene mutations and SNP sites. The kit can detect hereditary hemochromatosis-related gene mutations by combining PCR and gene sequencing technology. The specific exon segments and / or UTR segments of HFE (type I), HJV (type IIA), HAMP (type IIB), TFR2 (type III) and SLC40A1 (type IV) genes in genomic DNA are amplified by specific primers, and then the target fragments obtained by amplification are sequenced. Finally, the sequencing results are compared with the gene sequences published by NCBI, so as to analyze and obtain the gene mutation detection results. The detection results can assist in the medication guidance of patients with hemochromatosis-related genes.

[0178] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0179] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0180] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0181] Example 1

[0182] This embodiment provides a kit and a detection method for hemochromatosis-related gene mutation sites and SNP sites, such as Figure 7 shown.

[0183] The following steps are involved:

[0184] 1.1. Extraction of genomic DNA from samples to be tested

[0185] Collect blood and extract genomic DNA from the sample to be tested. The procedure is as follows: Collect blood from the patient as the sample to be tested. The genomic DNA can be extracted using a conventional kit column extraction method. Follow the instructions of the kit to collect 50 μL of a DNA solution with a concentration of 50 to 200 ng / μL, which can be used directly for testing or stored at -20°C.

[0186] 2.1. PCR amplification

[0187] Use the specific primers in Table 3 to perform PCR amplification on the genomic DNA of the sample to be tested in step 1.1.

[0188] First, a primer mixture was prepared using the specific primers in Table 3, wherein the concentration of each specific primer in the upstream primer mixture was 5 pmol / μL; the concentration of each specific primer in the downstream primer mixture was 5 pmol / μL. Then, a PCR amplification system was prepared according to Table 4.

[0189] Table 4

[0190] Components Volume (μL) Genomic DNA 1 PCR Mix 10 Upstream primer 1 Downstream primer 1 Deionized water 8 total 21

[0191] PCR amplification can be performed using a common PCR instrument, and the amplification reaction program is shown in Table 5 below. For example, a BIPCR instrument is used.

[0192] Table 5

[0193]

[0194] To ensure the PCR amplification results, the PCR amplification products can be subjected to electrophoresis detection before sequencing. The blank control is a sample with no amplified bands. If the sample band size is correct and the band is single, the PCR amplification products can be sent for detection sequencing.

[0195] 1.3. Sequencing of amplified products

[0196] Sequence the PCR amplification product obtained in step 1.2.

[0197] The PCR amplification products were sequenced using a 3500XL sequencer according to conventional operating procedures to obtain sequencing results.

[0198] 1.4 Gene mutation comparison

[0199] Compare the sequencing results obtained in step 1.3 with the corresponding gene sequences published in the database to obtain the gene mutation detection results.

[0200] The operation is: use Mutation Surveyor software and VECTOR NTI software to compare and analyze the sequencing results in the NCBI database, find gene mutation sites, and obtain detection results.

[0201] If there is no mutation site, the sequencing result is consistent with the sequence in the NCBI database.

[0202] If there is a point mutation, a mutation peak will appear at the corresponding position (a mutation peak refers to a peak that changes color or two peaks exist at the same position).

[0203] Example 2

[0204] In this example, the kit of Example 1 is verified in terms of accuracy, sensitivity, precision, etc., including the following:

[0205] 2.1. Source of inspection procedure (method)

[0206] Int J Hematol, 2014, 99(4): 487-492; Int J Hematol. 2016 Nov 28; Int J Hematol. 2016 Nov 28; Blood Cells Mol Dis. 2013 Jan; 50(1): 31-2; Blood Cells Mol Dis. 2014 Feb-Mar; 52(2-3); Blood Cells Mol Dis. 2001 Jan-Feb; 27(1): 279-84; Acta Haematol 2007; 118: 237–241, etc.

[0207] 2.2. Verification Purpose

[0208] By analyzing and evaluating the accuracy, sensitivity, and precision of the methodology, the test procedure (methodology) is validated to meet the needs of clinical trials.

[0209] 2.3 Experimental Materials

[0210] 2.3.1 Specimen Type

[0211] DNA extracted from peripheral blood.

[0212] 2.3.2 Reagents

[0213] The main reagent information is shown in Table 6.

[0214] Table 6. Reagent information for the detection and validation of hereditary hemochromatosis-related gene mutations

[0215]

[0216] 2.3.3 Instruments

[0217] 1) BIPCR instrument instrument number: HT-LE-133

[0218] 2) 3500xl sequencer instrument number: HT-LE-386

[0219] 2.3.4 Consumables

[0220] 1.5mL centrifuge tubes, 500μL centrifuge tubes, 200μL centrifuge tubes, 96-well plates, 1000μL Tip tips, 200μL Tip tips, 10μL Tip tips.

[0221] 2.3.5 Specimens

[0222] Eight specimens were used, of which four were positive (confirmed with the test results of the clinically confirmed specimens provided) and four were negative. The specimen type was DNA extracted from peripheral blood using a whole blood genomic DNA rapid extraction kit (spin column type). See Table 7 for specimen information.

[0223] Table 7

[0224]

[0225]

[0226] 2.4 Verification Experimental Results

[0227] 2.4.1 Accuracy

[0228] Take samples that have been tested before in this laboratory. Such specimens fully meet the clinical diagnosis and re-test them. The consistency of the test results must reach more than 95%.

[0229] As shown in Tables 8 and 9 below, 2 gene fragments were selected from each of the 8 samples, and the consistency of the test results reached 100%, which met the requirements. Negative samples were not detected.

[0230] Table 8. HHC (SLC40A1) detection accuracy verification results NM_014585

[0231]

[0232] Table 9. HHC (TFR2) test accuracy verification results NM_003227

[0233]

[0234] 2.4.2 Sensitivity

[0235] The positive sample numbered P17040841 in Table 4 was diluted with the negative DNA sample (Cx) in Table 4 to make the mutant DNA content 100%, 80%, 50%, 20%, 10%, 5%, 2%, etc. The diluted DNA was subjected to PCR and sequencing to detect the synonymous mutation of exon 16 of TFR2. The test results were compared. The results are shown in Table 10.

[0236] Table 10 HHC (TFR2) detection sensitivity verification results

[0237] Negative DNA content (ng / μL) Mutant DNA content (ng / μL) Mutation status (yes or no) 0 100 have 20 80 have 50 50 have 80 20 have 90 10 Yes, the mutation peak is low and can be interpreted 95 5 Yes, the mutation peak is low and difficult to interpret 98 2 none

[0238] As shown in Table 10, when the mutation sample is diluted to 2%, no mutation can be detected. When the mutation rate of the sample is lower than 5%, the mutation cannot be detected by the first generation sequencing method; and accurate detection results can be obtained when the dilution concentration is ≥ 10%.

[0239] 2.4.3 Precision

[0240] 1) Intra-batch precision

[0241] The three target exons of one sample were tested three times every day for three consecutive days. The three samples were tested with mutant DNA contents of 50%, 20% and 10% in the sensitivity test, respectively. The results are shown in Table 11 below.

[0242] Table 11 Results of intra-batch precision verification of HHC (TFR2) test

[0243]

[0244]

[0245] As can be seen from Table 11, the three tests for each specimen were conducted independently, and the test results were consistent, but the peak graph of DNA with a mutant DNA content of 10% was generally low, which would affect the interpretation results. The precision met the requirements.

[0246] 2)Batch-to-batch precision

[0247] The operation is: one person per day for three consecutive days, the same three specimens are tested. The requirement is: the consistency of the results must reach more than 95%; the test results are shown in Table 12.

[0248] Table 12 HHC (TFR2) test batch precision comparison results

[0249]

[0250] As can be seen from Table 12, different personnel tested the same specimens and the results were completely consistent. The batch-to-batch precision met the experimental requirements and satisfied the clinical platform testing requirements.

[0251] 2.4.4. Verification Results Summary

[0252] The results of the above verification experiments are summarized in Table 13.

[0253] Table 13 Summary of verification results

[0254]

[0255] 2.5 Conclusion

[0256] The sample used in this method is DNA extracted from peripheral blood.

[0257] The qualitative accuracy of this method is 100%.

[0258] The sensitivity of this method is: when the mutation rate is ≥10%, sequencing can detect it, but the peak graph is low and difficult to interpret; when the mutation rate is <10%, the sequencing result is inaccurate and needs to be repeated or the template is added for re-testing.

[0259] The precision of this method was 100%.

[0260] Through the verification of the accuracy, sensitivity, and precision of the method, the test procedure (method) was verified to meet the requirements of clinical experiments, and this laboratory has the ability to carry out HHC-related gene mutation detection.

[0261] Example 3

[0262] The test kit and detection method of Implementation 1 were used to test the cases for verification.

[0263] The patient was 45 years old and had severe skin pigmentation. The exposed skin color was bronze, and the oral mucosa was severely pigmented. He also had symptoms such as fatigue, drowsiness, abdominal pain, joint pain, loss of libido, arrhythmia, joint swelling, tenderness, and loss of body hair. Laboratory tests showed serum iron of 50 μmol / L and transferrin saturation of 90%. He was clinically diagnosed with hereditary hemochromatosis.

[0264] The peripheral blood was collected and tested using the kit of the present application, and the sequencing results were compared with the sequences in the NCBI database. Figure 8 If there is no mutation site, the sequence to be tested is consistent with the sequence in the NCBI database; if there is a point mutation, two peaks will appear at the corresponding position.

[0265] from Figure 8 It can be seen that there are multiple mutations in the HFE (type I), HJV (type IIA), HAMP (type IIB), TFR2 (type III) and SLC40A1 (type IV) genes, which is considered to be hemochromatosis, which is consistent with the clinical diagnosis.

[0266] Comparative Example 1

[0267] The difference between this comparative example 1 and example 1 is that the primers for the mutation site of the SLC40A1 gene are different, and the primer sequences are shown below. They are mixed with other primers, and then amplified using the same method as example 1 with healthy human genomic DNA as a template. The results are shown in Fig. 9 Among them, A is the specific analysis diagram of SEQ ID NO.59 and SEQ ID NO.60 primers; B is the sequencing result obtained by using SEQ ID NO.59 and SEQ ID NO.60 primers to replace SEQ ID NO.23 and SEQ ID NO.24 primers in Table 3 for amplification; C is the sequencing result of Example 1.

[0268] SLC40A1E1F:5'-GCAAGGTTGACGGGAGC-3'(SEQ ID NO.59)

[0269] SLC40A1E1R:5'-ACAGCAGAGCCACATTCC-3'(SEQ ID NO.60)

[0270] from Fig. 9 It can be seen that although the primers of SEQ ID NO.59 and SEQ ID NO.60 have stronger specificity than the primers of SEQ ID NO.23 and SEQ ID NO.24, the peak graph of amplified sequencing is not ideal; while the primer set of Example 1 can amplify a peak graph of a single band.

[0271] Comparative Example 2

[0272] The difference between this comparative example 2 and example 1 is that the amplification primers for the mutation site of the TFR2 gene are different. The primer sequences are shown below. They are mixed with other primers, and then amplified using the same method as example 1 with healthy human genomic DNA as a template. The results are shown in Fig.10 Among them, A is the specific analysis diagram of SEQ ID NO.61 and SEQ ID NO.62 primers; B is the sequencing result obtained by amplifying using SEQ ID NO.61 and SEQ ID NO.62 primers instead of SEQ ID NO.39 and SEQ ID NO.40 primers in Table 3; C is the sequencing result of Example 1.

[0273] TFR2

[0274] TFR2E1-2F:5'-GGAGCAGCCTTGGTTCA-3'(SEQ ID NO.61)

[0275] TFR2E1-2R:5'-GCGGGTGGCAAGATG-3'(SEQ ID NO.62)

[0276] from Fig.10 It can be seen that the primer specificity of SEQ ID NO.61 and SEQ ID NO.62 is similar to that of SEQ ID NO.39 and SEQ ID NO.40, but the peak diagram of amplification sequencing is not ideal, and false positives occur.

[0277] The detection kit of the present invention can be used for patients with complications of hereditary hemochromatosis, especially for patients with hidden or possible familial hereditary hemochromatosis.

[0278] Hereditary Hemochromatosis (HHC) is a rare autosomal recessive genetic disease. Due to the insidious nature of hereditary hemochromatosis, slow progression, and variable degree of tissue involvement, the disease is often diagnosed in the late stage of the disease after significant tissue damage, and non-hereditary iron overload such as congenital hemolytic anemia (sickle cell anemia, thalassemia) should be excluded. Therefore, diagnosis is difficult and there are multiple diseases that are confused; and the diagnosis is delayed, and the appearance of typical symptoms is accompanied by severe deterioration of the disease. Therefore, rapid diagnosis at the molecular level can help clinicians make accurate judgments, provide timely treatment, and improve survival rates. In addition, because the diagnosis of hereditary hemochromatosis is very difficult, the statistical data on the incidence in China is inaccurate, and the family disease is not taken seriously, resulting in incomplete data, and many mutation sites are difficult to obtain. Genetic diagnosis can improve the statistics of the incidence of the disease in China, which has very strong social benefits.

[0279] The panel designed by the present invention for five genes related to hereditary hemochromatosis (HAMP, HJV, TFR2, TFR2 and HFE) has low cost and provides a blessing for the society and patients to improve the quality of life of patients.

[0280] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present application.

Claims

1. A kit for detecting hemochromatosis-related gene mutations and SNP sites, It is characterized in that The kit comprises detection reagents for mutation sites and SNP sites of hemochromatosis-related genes, and the hemochromatosis-related genes comprise one or more of HFE gene, HJV gene, HAMP gene, TFR2 gene and SLC40A1 gene.

2. The kit according to claim 1, It is characterized in that Include at least one of the following technical features: A1) the mutation site of the HFE gene is selected from one or more of D129N, S65C, C282Y, H63D, Y230C, C225*, Q238P, E277K, V295, H109Y, I287S, Y46X and A356fs; A2) the SNP site of the HFE gene is selected from one or more of rs2071303, rs1800708, rs1572982 and rs3817623; A3) the mutation site of the HJV gene is selected from one or more of E3D, Q6H, N129*, I281T, G320V, C321X and R326X; A4) the mutation site of the HAMP gene is selected from one or more of c.-25GNA, c.-25GNA, c.-92GNA and G71D; A5) the SNP site of the HAMP gene is selected from rs3817623; A6) the mutation site of the TFR2 gene is selected from one or more of Y250X, E60X, T515A, M172K, L490R, V561X, I238M, A617A, L750P, A777V, G792R, Q306*, Q672*, L99V, V277L, N540N, T740M, R455Q, R468H, G792R, T740M and R752H; A7) the SNP site of the TFR2 gene is selected from one or more of rs41295912, rs148902192, rs80338885, rs2075674, rs41302360, rs41295915 and rs35142847; A8) the mutation site of the SLC40A1 gene is selected from one or more of IVS+10del GTT shear mutation, V221V, c.44-24G>C, V162del, A77D and Q248H; A9) The SNP site of the SLC40A1 gene is selected from one or more of rs2304704, rs4287798, rs1156835, rs13008848 and rs2304704.

3. The kit according to claim 1 or 2, It is characterized in that The detection reagent comprises specific primers for amplifying exons and / or UTR segments belonging to the HFE gene, HJV gene, HAMP gene, TFR2 gene or SLC40A1 gene.

4. The kit according to claim 3, It is characterized in that Specific primers comprising at least one of the following: B1) specific primers for amplifying exon 1 of the HFE gene, specific primers for amplifying exon 2 of the HFE gene, specific primers for amplifying exon 3 of the HFE gene, specific primers for amplifying exons 4-5 of the HFE gene, and specific primers for amplifying exon 6 of the HFE gene; B2) a specific primer for amplifying exon 1 of the HJV gene, a specific primer for amplifying exon 2 of the HJV gene, a specific primer for amplifying exon 3 of the HJV gene, and a specific primer for amplifying exon 4 of the HJV gene; B3) specific primers for amplifying exon 1 of the HAMP gene, and specific primers for amplifying exons 2-3 of the HAMP gene; B4) specific primers for amplifying exon 1 of the SLC40A1 gene, specific primers for amplifying exon 2 of the SLC40A1 gene, specific primers for amplifying exon 3 of the SLC40A1 gene, specific primers for amplifying exon 4 of the SLC40A1 gene, specific primers for amplifying exon 5 of the SLC40A1 gene, specific primers for amplifying exon 6 of the SLC40A1 gene, specific primers for amplifying exon 7 of the SLC40A1 gene, and specific primers for amplifying exon 8 of the SLC40A1 gene; B5) specific primers for amplifying exons 1-2 of the TFR2 gene, specific primers for amplifying exons 3 of the TFR2 gene, specific primers for amplifying exons 4-6 of the TFR2 gene, and specific primers for amplifying exons 7-8 of the TFR2 gene; Specific primers for amplifying exon 9 of the TFR2 gene, specific primers for amplifying exon 10 of the TFR2 gene, specific primers for amplifying exons 11-15 of the TFR2 gene, specific primers for amplifying exon 16 of the TFR2 gene, specific primers for amplifying exon 17 of the TFR2 gene, and specific primers for amplifying exon 18 of the TFR2 gene.

5. The kit according to claim 4, It is characterized in that Also includes any one or more of the following features: C1) The specific primers for amplifying the first exon of the HFE gene include an upstream primer and a downstream primer; C2) The specific primers for amplifying the second exon of the HFE gene include an upstream primer and a downstream primer; C3) The specific primers for amplifying the third exon of the HFE gene include an upstream primer and a downstream primer; C4) The specific primers for amplifying the fourth and fifth exons of the HFE gene include an upstream primer and a downstream primer; C5) The specific primers for amplifying the sixth exon of the HFE gene include an upstream primer and a downstream primer; C6) The specific primers for amplifying the first exon of the HJV gene include an upstream primer and a downstream primer; C7) The specific primers for amplifying the second exon of the HJV gene include an upstream primer and a downstream primer; C8) The specific primers for amplifying the third exon of the HJV gene include an upstream primer and a downstream primer; C9) The specific primers for amplifying the fourth exon of the HJV gene include an upstream primer and a downstream primer; C10) The specific primers for amplifying the first exon of the HAMP gene include an upstream primer and a downstream primer; C11) specific primers for amplifying exons 2-3 of the HAMP gene include an upstream primer and a downstream primer; C12) specific primers for amplifying exon 1 of the SLC40A1 gene include an upstream primer and a downstream primer; C13) specific primers for amplifying exon 2 of the SLC40A1 gene include an upstream primer and a downstream primer; C14) specific primers for amplifying exon 3 of the SLC40A1 gene include an upstream primer and a downstream primer; C15) specific primers for amplifying exon 4 of the SLC40A1 gene include an upstream primer and a downstream primer; C16) specific primers for amplifying exon 5 of the SLC40A1 gene include an upstream primer and a downstream primer; C17) specific primers for amplifying exon 6 of the SLC40A1 gene include an upstream primer and a downstream primer; C18) specific primers for amplifying exon 7 of the SLC40A1 gene include an upstream primer and a downstream primer; C19) specific primers for amplifying exon 8 of the SLC40A1 gene include an upstream primer and a downstream primer; C20) specific primers upstream primer and downstream primer for amplifying exons 1-2 of TFR2 gene; C21) specific primers upstream primer and downstream primer for amplifying exon 3 of TFR2 gene; C22) specific primers upstream primer and downstream primer for amplifying exons 4-6 of TFR2 gene; C23) specific primers upstream primer and downstream primer for amplifying exon 7-8 of TFR2 gene; C24) specific primers upstream primer and downstream primer for amplifying exon 9 of TFR2 gene; C25) specific primers upstream primer and downstream primer for amplifying exon 10 of TFR2 gene; C26) an upstream primer and a downstream primer for amplifying exons 11 to 15 of the TFR2 gene; C27) an upstream primer and a downstream primer for amplifying exon 16 of the TFR2 gene; C28) specific primers upstream primer and downstream primer for amplifying exon 17 of TFR2 gene; C29) Specific primers for amplifying exon 18 of the TFR2 gene: upstream primer and downstream primer.

6. The kit according to claim 5, It is characterized in that Also includes any one or more of the following features: D1) Among the specific primers used to amplify exon 1 of the HFE gene, the sequence of the upstream primer is shown as SEQ ID NO.1, and the sequence of the downstream primer is shown as SEQ ID NO.2; D2) Among the specific primers used to amplify exon 2 of the HFE gene, the sequence of the upstream primer is shown in SEQ ID NO.3, and the sequence of the downstream primer is shown in SEQ ID NO.4; D3) Among the specific primers used to amplify exon 3 of the HFE gene, the sequence of the upstream primer is shown in SEQ ID NO.5, and the sequence of the downstream primer is shown in SEQ ID NO.6; D4) Among the specific primers used to amplify exons 4-5 of the HFE gene, the sequence of the upstream primer is shown in SEQ ID NO.7, and the sequence of the downstream primer is shown in SEQ ID NO.8; D5) Among the specific primers used to amplify exon 6 of the HFE gene, the sequence of the upstream primer is shown in SEQ ID NO.9, and the sequence of the downstream primer is shown in SEQ ID NO.10; D6) Among the specific primers used to amplify exon 1 of the HJV gene, the sequence of the upstream primer is shown in SEQ ID NO.11, and the sequence of the downstream primer is shown in SEQ ID NO.12; D7) in the specific primers for amplifying exon 2 of the HJV gene, the sequence of the upstream primer is shown in SEQ ID NO.13, and the sequence of the downstream primer is shown in SEQ ID NO.14; D8) in the specific primers for amplifying exon 3 of the HJV gene, the sequence of the upstream primer is shown in SEQ ID NO.15, and the sequence of the downstream primer is shown in SEQ ID NO.16; D9) in the specific primers for amplifying exon 4 of the HJV gene, the sequence of the upstream primer is shown in SEQ ID NO.17, and the sequence of the downstream primer is shown in SEQ ID NO.18; D10) in the specific primers for amplifying exon 1 of the HAMP gene, the sequence of the upstream primer is shown in SEQ ID NO.19, and the sequence of the downstream primer is shown in SEQ ID NO.20; D11) in the specific primers for amplifying exons 2-3 of the HAMP gene, the sequence of the upstream primer is shown in SEQ ID NO.21, and the sequence of the downstream primer is shown in SEQ ID NO.22; D12) in the specific primers used to amplify exon 1 of the SLC40A1 gene, the sequence of the upstream primer is shown in SEQ ID NO.23, and the sequence of the downstream primer is shown in SEQ ID NO.24; D13) in the specific primers used to amplify exon 2 of the SLC40A1 gene, the sequence of the upstream primer is shown in SEQ ID NO.25, and the sequence of the downstream primer is shown in SEQ ID NO.26; D14) in the specific primers used to amplify exon 3 of the SLC40A1 gene, the sequence of the upstream primer is shown in SEQ ID NO.27, and the sequence of the downstream primer is shown in SEQ ID NO.28; D15) in the specific primers for amplifying exon 4 of the SLC40A1 gene, the sequence of the upstream primer is shown in SEQ ID NO.29, and the sequence of the downstream primer is shown in SEQ ID NO.30; D16) in the specific primers used to amplify exon 5 of the SLC40A1 gene, the sequence of the upstream primer is shown in SEQ ID NO.31, and the sequence of the downstream primer is shown in SEQ ID NO.32; D17) in the specific primers used to amplify exon 6 of the SLC40A1 gene, the sequence of the upstream primer is shown in SEQ ID NO.33, and the sequence of the downstream primer is shown in SEQ ID NO.34; D18) in the specific primers used to amplify exon 7 of the SLC40A1 gene, the sequence of the upstream primer is shown in SEQ ID NO.35, and the sequence of the downstream primer is shown in SEQ ID NO.36; D19) in the specific primers used to amplify exon 8 of the SLC40A1 gene, the sequence of the upstream primer is shown in SEQ ID NO.37, and the sequence of the downstream primer is shown in SEQ ID NO.38; D20) in the specific primers for amplifying exons 1-2 of the TFR2 gene, the sequence of the upstream primer is shown in SEQ ID NO.39, and the sequence of the downstream primer is shown in SEQ ID NO.40; D21) in the specific primers for amplifying exon 3 of the TFR2 gene, the sequence of the upstream primer is shown in SEQ ID NO.41, and the sequence of the downstream primer is shown in SEQ ID NO.42; D22) in the specific primers for amplifying exons 4 to 6 of the TFR2 gene, the sequence of the upstream primer is shown in SEQ ID NO.43, and the sequence of the downstream primer is shown in SEQ ID NO.44; D23) in the specific primers for amplifying exons 7-8 of the TFR2 gene, the sequence of the upstream primer is shown in SEQ ID NO.45, and the sequence of the downstream primer is shown in SEQ ID NO.46; D24) in the specific primers used to amplify exon 9 of the TFR2 gene, the sequence of the upstream primer is shown in SEQ ID NO.47, and the sequence of the downstream primer is shown in SEQ ID NO.48; D25) in the specific primers for amplifying exon 10 of the TFR2 gene, the sequence of the upstream primer is shown in SEQ ID NO.49, and the sequence of the downstream primer is shown in SEQ ID NO.50; D26) in the specific primers for amplifying exons 11 to 15 of the TFR2 gene, the sequence of the upstream primer is shown in SEQ ID NO.51, and the sequence of the downstream primer is shown in SEQ ID NO.52; D27) in the specific primers for amplifying exon 16 of the TFR2 gene, the sequence of the upstream primer is shown in SEQ ID NO.53, and the sequence of the downstream primer is shown in SEQ ID NO.54; D28) in the specific primers for amplifying exon 17 of the TFR2 gene, the sequence of the upstream primer is shown in SEQ ID NO.55, and the sequence of the downstream primer is shown in SEQ ID NO.56; D29) In the specific primers used to amplify exon 18 of the TFR2 gene, the sequence of the upstream primer is shown in SEQ ID NO.57, and the sequence of the downstream primer is shown in SEQ ID NO.

58.

7. The kit according to claim 1, It is characterized in that The kit further comprises one or both of a PCR Mix reagent and deionized water.

8. The kit according to claim 1, It is characterized in that The kit also contains one or both of a positive control and a negative control.

9. Use of the kit according to any one of claims 1 to 8 in detecting mutation sites and SNP sites of hemochromatosis-related genes or in preparing products for detecting hemochromatosis.

10. A method for detecting hemochromatosis-related gene mutations and SNP sites, It is characterized in that The steps include: Extracting genomic DNA from the sample to be tested; Perform PCR amplification using the kit according to any one of claims 1 to 8 to obtain a PCR amplification product; Sequencing the PCR amplification product to obtain a sequencing result; The sequencing results are compared with the gene sequences published in the database to obtain gene mutation detection results.

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