Gene mutant for early obesity in Sichuan and Chongqing regions and application of gene mutant

Abstract

The invention discloses a premature obesity gene mutant in Sichuan and Chongqing regions and application thereof, and particularly relates to the technical field of medical molecular biology. The mutant is a CAMKK2 (c.1614dup) mutant and / or an AIM2 (c.1029dup) mutant, and the mutant is a CAMKK2 (c.1614dup) mutant and / or an AIM2 (c.1029dup) mutant. The invention finds that CAMKK2 (c.1614dup) and AIM2 (c.1029dup) mutants carried in premature obesity patients in Sichuan and Chongqing regions are potential pathogenic gene mutations. A patient carrying the gene mutation is poor in weight reduction effect after a weight reduction operation. According to the invention, an individualized treatment scheme can be determined by detecting specific gene mutation, for example, a patient carrying CAMKK2 (c.1614dup) and / or AIM2 (c.1029dup) mutants can enhance the weight loss effect in combination with exercise intervention and diet intervention after a weight loss operation.

Description

Technical Field

[0001] The present invention relates to the technical field of medical molecular biology, and in particular to a Sichuan-Chongqing region early-onset obesity gene mutant and application thereof. Background Art

[0002] With the rapid development of social economy and the great changes in residents' lifestyles, the prevalence of overweight and obesity has increased at an alarming rate worldwide and has become the main cause of chronic diseases worldwide, including type 2 diabetes, cardiovascular disease and certain cancers [Circulation research 2020; 126(11): 1477-1500]. Early-onset obesity refers to the onset of obvious obesity symptoms in childhood or adolescence, which is more serious than ordinary obesity. Previous studies have found that genetic factors play a key role in early-onset obesity. Genome-wide association studies (GWAS) and whole exome sequencing (WES) have discovered many genetic mutation sites associated with obesity [Nat Rev Genet 2022; 23(2): 120-133]. However, a considerable proportion of patients with genetic obesity cannot be explained by the identified genetic mutation sites. This means that there are more genetic mutation sites that have not yet been discovered, and racial, cultural and regional differences have strengthened this genetic diversity. At the same time, bariatric metabolic surgery is becoming the most effective treatment option for obese patients, which can effectively improve complications and quality of life [Annals of surgery 2018; 268(1): 106-113]. It has been reported that patients with known early-onset obesity gene mutations lose less weight after bariatric surgery, indicating that early-onset obesity gene mutations have a long-term negative impact on bariatric surgery [Obes Surg 2019; 29(4): 1169-1173.].

[0003] At present, a large number of patients with early-onset obesity cannot be explained by known gene mutations, and most of the early-onset obesity gene mutations identified by WES are based on the genetic characteristics of Western populations. However, the unique dietary structure in Sichuan and Chongqing, China may lead to different obesity genetic patterns. There are no reports on the use of WES combined with clinical weight loss data to identify early-onset obesity gene mutations in the population of Sichuan and Chongqing, China. Summary of the invention

[0004] To this end, the present invention provides a Sichuan-Chongqing region early-onset obesity gene mutant and application thereof, so as to solve the problem that there is no report on the Sichuan-Chongqing region early-onset obesity gene.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] According to the first aspect of the present invention, a gene mutant for early-onset obesity in Sichuan and Chongqing is provided, wherein the mutant is a CAMKK2 (c.1614dup) mutant.

[0007] According to the second aspect of the present invention, a mutant of early-onset obesity gene in Sichuan and Chongqing is provided, and the mutant is an AIM2 (c.1029dup) mutant.

[0008] According to the third aspect of the present invention, a gene mutant for early-onset obesity in Sichuan and Chongqing is provided, wherein the mutant is a CAMKK2 (c.1614dup) mutant, the sequence of which is shown in SEQ ID NO.1, and the mutation site is 1615; an AIM2 (c.1029dup) mutant, the sequence of which is shown in SEQ ID NO.2, and the mutation site is 1030.

[0009] According to the fourth aspect of the present invention, a mutant of a gene for early-onset obesity in Sichuan and Chongqing is provided for use in preparing a biological sample product for screening early-onset obesity in Sichuan and Chongqing.

[0010] According to a fifth aspect of the present invention, a system for screening biological samples susceptible to early-onset obesity in Sichuan and Chongqing is provided, comprising:

[0011] A nucleic acid extraction device, wherein the nucleic acid extraction device is used to extract a nucleic acid sample from the biological sample;

[0012] a nucleic acid sequence determination device, the nucleic acid sequence determination device being connected to the nucleic acid extraction device and used for analyzing the nucleic acid sample so as to determine the nucleic acid sequence of the nucleic acid sample;

[0013] A judgment device is connected to the nucleic acid sequence determination device so as to judge whether the biological sample is susceptible to early-onset obesity based on whether the nucleic acid sequence of the nucleic acid sample is compared with the sequence of the existing early-onset obesity gene mutant gene in Sichuan and Chongqing, and whether it has CAMKK2 (c.1614dup) and / or AIM2 (c.1029dup) mutants.

[0014] According to the sixth aspect of the present invention, a kit for screening biological samples susceptible to early-onset obesity in Sichuan and Chongqing is provided, comprising reagents suitable for detecting early-onset obesity gene mutants in Sichuan and Chongqing, and CAMKK2 (c.1614dup) mutants and / or AIM2 (c.1029dup) mutants.

[0015] According to a seventh aspect of the present invention, a construct is provided, comprising the early-onset obesity gene mutant in Sichuan and Chongqing as described above.

[0016] The present invention has the following advantages:

[0017] This invention is the first to explore the gene mutations of early-onset obesity in Sichuan and Chongqing, China through high-throughput sequencing, and verify it using the patients' clinical weight loss data.

[0018] The present invention discovered a new pathogenic gene for early-onset obesity in Sichuan and Chongqing. Based on this, it can be used for early screening of pathogenic mutation gene carriers of early-onset obesity gene mutants in Sichuan and Chongqing, and then early intervention treatment before the carriers develop the disease; it can also be used for molecular diagnosis of patients with early-onset obesity in Sichuan and Chongqing and differential diagnosis of related diseases. This technology has the advantages of being fast, accurate, efficient, simple, and having a high early diagnosis rate. The test results can provide a scientific basis for the early diagnosis and differential diagnosis of early-onset obesity in Sichuan and Chongqing, and the development of therapeutic drugs for early-onset obesity in Sichuan and Chongqing.

[0019] The present invention found that the CAMKK2 (c.1614dup) and AIM2 (c.1029dup) mutants carried by patients with early-onset obesity in Sichuan and Chongqing are potential pathogenic gene mutations. Patients carrying this gene mutation have poor weight loss effects after bariatric surgery. According to the present invention, individualized treatment plans can be determined by detecting specific gene mutations, such as patients carrying CAMKK2 (c.1614dup) and / or AIM2 (c.1029dup) mutants can be combined with exercise intervention and dietary intervention to enhance the weight loss effect after bariatric surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0021] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0022] Figure 1 The weight loss rate of a patient with 5 candidate gene mutations provided in Example 1 of the present invention;

[0023] Figure 2 A schematic diagram of the structure of a system for screening biological samples for early-onset obesity in Sichuan and Chongqing provided in Example 2 of the present invention;

[0024] Figure 3 It is a schematic diagram of the structure of the nucleic acid extraction device of the present invention;

[0025] Figure 4 It is a schematic diagram of the structure of the library construction unit of the present invention. DETAILED DESCRIPTION

[0026] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] 1. Sample collection: The present invention involves the collection of peripheral blood samples from patients with early-onset obesity and non-early-onset obesity who underwent weight-loss surgery. The patients and their fathers / mothers were all from Sichuan and Chongqing. A total of 62 patients with early-onset obesity and 9 patients with non-early-onset obesity were included in this study.

[0028] 2. Extraction of total DNA: The present invention uses a blood genomic DNA extraction kit for extracting total DNA from blood samples. NanoDrop is used to detect the purity of the extracted genomic total DNA. The specific steps are as follows:

[0029] 2.1 Add 200 μl of buffer GB and 20 μl of proteinase K premix solution to 200 μl of human blood sample, mix thoroughly by inversion, and place at 56°C for 10 min, inverting and mixing several times, until the solution becomes clear;

[0030] 2.2 After standing at room temperature for 2-5 minutes, add 350 μl buffer BD and mix thoroughly by inversion. Flocculent precipitation may appear at this time;

[0031] 2.3 Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CG2, centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CG2 into the collection tube;

[0032] 2.4 Add 500 μl buffer GDB to the adsorption column CG2, centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CG2 in the collection tube;

[0033] 2.5 Add 600 μl of rinsing solution PWB to the adsorption column CG2, centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CG2 in the collection tube;

[0034] 2.6 Repeat the previous step;

[0035] 2. Centrifuge at 712,000 rpm for 2 min and discard the waste liquid. Place the adsorption column CG2 at room temperature for 2 min to completely dry the residual rinse liquid in the adsorption material;

[0036] 2.8 Transfer the adsorption column CG2 into a 1.5 ml centrifuge tube, add 50-200 μl of elution buffer TB to the middle of the adsorption membrane, let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 2 minutes, and collect the solution into the centrifuge tube.

[0037] 2.9 Use Nanodrop to detect DNA concentration and purity.

[0038] 3. High-throughput sequencing: The present invention amplifies, builds a library and performs high-throughput sequencing (Illumina HiSeq 2500, double-end sequencing) on ​​the qualified total DNA in (2). (This step is sent to a sequencing company for further processing)

[0039] 4. Sequencing data analysis: The present invention uses Burrow-Wheeler aligner (BWA) to align all reads with the hg38 reference genome. The GATK process is used to screen gene mutants, and the dbNSFP database is used to annotate the harmfulness of non-synonymous mutations. The pathogenicity is evaluated according to the American College of Medical Genetics and Genomics (ACMG) guidelines. The specific steps are as follows:

[0040] 4.1 Genome alignment: Use BWA software to align DNA sequences to the hg38 reference genome, including index construction (FM-index construction for fasta) and mem alignment.

[0041] 4.2 Screening mutants: Filter out ① variants that appear in the non-early-onset group; ② variants with a minimum gene frequency ≤5% in the 1000Genomes and ExAC databases.

[0042] 4.3 Analysis of harmfulness of mutants: Synonymous mutations are considered benign and removed. Frameshift mutations, premature termination and extended termination are considered harmful and retained. The harmfulness prediction of non-synonymous mutations uses the dbNSFP database annotation, including the annotation results of 13 prediction softwares: SIFT, Polyphen2_HDIV, Polyphen2_HVAR, LRT, MutationTaster, MutationAssessor, FATHMM, PROVEAN, MetaSVM, MetaLR, M-CAP, CADD, fathmm-MKL. If for a certain mutant, ≥ half of the annotated prediction software shows harmfulness, then the mutant is considered harmful, otherwise it is harmless.

[0043] 4.4 Pathogenicity analysis: According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), benign and likely benign variants were removed, and pathogenic, likely pathogenic, and variants of uncertain significance were retained.

[0044] 5. Analysis of patient clinical data: Combined with literature data, candidate genes were preliminarily screened. According to whether they carried the candidate gene, patients with early-onset obesity were divided into a control group and several carrier groups (each candidate gene was a carrier group). The weight of the patients was followed up 1 month, 3 months, 6 months, and 12 months after weight loss surgery, and the weight loss rate at each time point was calculated. Calculate whether the weight loss rate between the control group and the carrier group is different.

[0045] 6. Statistical analysis: Independent sample t-test was used to compare the weight loss rates of the two groups. IBM SPSS statistical software was used for statistical analysis.

[0046] Example 1

[0047] The present invention detected 567 harmful gene mutations in patients with early-onset obesity, including 11 pathogenic and potentially pathogenic gene mutations and 60 gene mutations of uncertain importance. Combined with literature reports, a total of 5 candidate gene mutations were screened out, and combined with the patient's clinical weight loss data (weight 1 month, 3 months, 6 months, and 12 months after weight loss surgery), two gene mutations: the mutant is the CAMKK2 (c.1614dup) mutant, the sequence is shown in SEQ ID NO.1, and the mutation site is 1615; the AIM2 (c.1029dup) mutant, the sequence is shown in SEQ ID NO.2, and the mutation site is 1030; were identified as potential pathogenic gene mutations. The list of 5 candidate gene mutations is shown in Table 1; the weight loss rate of patients carrying the 5 candidate gene mutations is shown in Table 1. Figure 1 shown.

[0048] Table 1

[0049]

[0050]

[0051] The clinical information of patients carrying CAMKK2 (c.1614dup) and AIM2 (c.1029dup) is shown in Table 2 .

[0052] Table 2

[0053]

[0054] As shown in Table 2, patients carrying the AIM2 (c.1029dup) mutant all have familial hereditary characteristics, indicating that the AIM2 (c.1029dup) mutant is very likely inherited from their parents, while CAMKK2 (c.1614dup) is a de novo mutation, that is, it has not been passed down through the family to cause disease.

[0055] The present invention explored the early-onset obesity gene mutations in Sichuan and Chongqing, China for the first time by means of high-throughput sequencing, and verified it using the clinical weight loss data of patients. The present invention found that the CAMKK2 (c.1614dup) and AIM2 (c.1029dup) mutants carried by patients with early-onset obesity in Sichuan and Chongqing are potential pathogenic gene mutations. Patients carrying this gene mutation have poor weight loss effects after weight loss surgery. According to the present invention, individualized treatment plans can be determined by detecting specific gene mutations, such as patients carrying CAMKK2 (c.1614dup) and / or AIM2 (c.1029dup) mutants can enhance the weight loss effect by combining exercise intervention and dietary intervention after weight loss surgery.

[0056] Example 2

[0057] A system for screening biological samples for susceptibility to early-onset obesity in Sichuan and Chongqing.

[0058] like Figure 2-4 It can be known that the system for screening biological samples susceptible to early-onset obesity in Sichuan and Chongqing includes a nucleic acid extraction device 100 , a nucleic acid sequence determination device 200 and a judgment device 300 .

[0059] According to an embodiment of the present invention, the nucleic acid extraction device 100 is used to extract a nucleic acid sample from a biological sample. As mentioned above, according to an embodiment of the present invention, the type of nucleic acid sample is not particularly limited. For RNA as a nucleic acid sample, the nucleic acid extraction device further includes an RNA extraction unit 101 and a reverse transcription unit 102, wherein the extraction unit 101 is used to extract an RNA sample from a biological sample, and the reverse transcription unit 102 is connected to the RNA extraction unit 101 and is used to perform a reverse transcription reaction on the RNA sample to obtain a cDNA sample, and the obtained cDNA sample constitutes a nucleic acid sample.

[0060] According to an embodiment of the present invention, the nucleic acid sequence determination device 200 is connected to the nucleic acid extraction device 100, and is used to analyze the nucleic acid sample to determine the nucleic acid sequence of the nucleic acid sample. As shown above, the nucleic acid sequence of the nucleic acid sample can be determined by a sequencing method. Therefore, according to an embodiment of the present invention, the nucleic acid sequence determination device 200 may further include: a library construction unit 201 and a sequencing unit 202. The library construction unit 201 is used to construct a nucleic acid sequencing library for the nucleic acid sample; the sequencing unit 202 is connected to the library construction unit 201, and is used to sequence the nucleic acid sequencing library to obtain a sequencing result consisting of a plurality of sequencing data. As mentioned above, the early-onset obesity exons in Sichuan and Chongqing can be enriched by PCR amplification, and the efficiency of screening biological samples susceptible to early-onset obesity in Sichuan and Chongqing can be further improved. Therefore, the library construction unit 201 may further include a PCR amplification module (not shown in the figure), in which exon-specific primers susceptible to early-onset obesity in Sichuan and Chongqing are provided, so as to use the exon-specific primers susceptible to early-onset obesity in Sichuan and Chongqing to perform PCR amplification on the nucleic acid sample. Therefore, combined with the latest sequencing technology, a higher sequencing depth can be achieved for a single site, and the detection sensitivity and accuracy are greatly improved. Therefore, the high-throughput and deep sequencing characteristics of these sequencing devices can be used to further improve the efficiency of nucleic acid sample detection and analysis, thereby improving the precision and accuracy of subsequent analysis of sequencing data.

[0061] According to an embodiment of the present invention, the judging device 300 is connected to the nucleic acid sequence determining device 200, and is suitable for comparing the nucleic acid sequence of the nucleic acid sample, so as to judge whether the biological sample is susceptible to early-onset obesity in Sichuan and Chongqing based on the difference between the nucleic acid sequence of the nucleic acid sample and the existing obesity gene. Specifically, based on whether the nucleic acid sequence of the nucleic acid sample is compared with the sequence of the existing gene, whether there is a CAMKK2 (c.1614dup) and / or AIM2 (c.1029dup) mutant, it is judged whether the biological sample is susceptible to early-onset obesity in Sichuan and Chongqing. According to a specific example of the present invention, SOAP software can be used for comparison. Thus, using this system, the aforementioned method for screening biological samples susceptible to early-onset obesity in Sichuan and Chongqing can be effectively implemented, so that biological samples susceptible to early-onset obesity in Sichuan and Chongqing can be effectively screened.

[0062] Example 3

[0063] A kit for screening biological samples susceptible to early-onset obesity.

[0064] The present invention proposes a kit for screening biological samples of early-onset obesity in Sichuan and Chongqing. According to an embodiment of the present invention, the kit for screening biological samples of early-onset obesity in Sichuan and Chongqing includes: a reagent suitable for detecting mutants of early-onset obesity in Sichuan and Chongqing, wherein compared with the sequence of existing genes, whether there is a CAMKK2 (c.1614dup) mutant and / or an AIM2 (c.1029dup) mutant. Utilizing the kit according to an embodiment of the present invention, biological samples susceptible to early-onset obesity in Sichuan and Chongqing can be effectively screened. In this article, the term "reagent suitable for detecting mutants of early-onset obesity genes in Sichuan and Chongqing" used should be understood in a broad sense, that is, it can be a reagent for detecting genes encoding early-onset obesity in Sichuan and Chongqing, or it can be a reagent for detecting mutant polypeptides of early-onset obesity in Sichuan and Chongqing, for example, antibodies that recognize specific sites can be used. Thus, biological samples susceptible to early-onset obesity in Sichuan and Chongqing can be efficiently screened.

[0065] Example 4

[0066] The present invention also proposes a construct. According to an embodiment of the present invention, the construct comprises the aforementioned isolated nucleic acid encoding the early-onset obesity gene mutant in Sichuan and Chongqing, i.e., the early-onset obesity gene mutant in Sichuan and Chongqing of the present invention. Thus, the recombinant cells obtained by transforming the receptor cells with the construct of the present invention can be effectively used to screen drugs for treating early-onset obesity in Sichuan and Chongqing. Wherein, the type of the receptor cells is not particularly limited, for example, it can be an Escherichia coli cell, a mammalian cell, and preferably the receptor cells are derived from mammals.

[0067] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A mutant of early-onset obesity gene in Sichuan and Chongqing, characterized in that: The mutant is a CAMKK2 (c.1614dup) mutant.

2. A gene mutant for early-onset obesity in Sichuan and Chongqing, characterized in that: The mutant is an AIM2 (c.1029dup) mutant.

3. A gene mutant for early-onset obesity in Sichuan and Chongqing, characterized in that: The mutants are CAMKK2 (c.1614dup) mutant and AIM2 (c.1029dup) mutant.

4. Application of a gene mutant of early-onset obesity in Sichuan and Chongqing in the preparation and screening of biological sample products for early-onset obesity in Sichuan and Chongqing.

5. A system for screening biological samples for those susceptible to early-onset obesity in Sichuan and Chongqing, characterized in that: include: A nucleic acid extraction device, wherein the nucleic acid extraction device is used to extract a nucleic acid sample from the biological sample; a nucleic acid sequence determination device, the nucleic acid sequence determination device being connected to the nucleic acid extraction device and used for analyzing the nucleic acid sample so as to determine the nucleic acid sequence of the nucleic acid sample; A judgment device is connected to the nucleic acid sequence determination device so as to judge whether the biological sample is susceptible to early-onset obesity based on whether the nucleic acid sequence of the nucleic acid sample is compared with the sequence of the existing early-onset obesity gene mutant gene in Sichuan and Chongqing, and whether it has CAMKK2 (c.1614dup) and / or AIM2 (c.1029dup) mutants.

6. A kit for screening biological samples susceptible to early-onset obesity in Sichuan and Chongqing, characterized in that: The invention comprises reagents suitable for detecting early-onset obesity gene mutants in Sichuan and Chongqing, and CAMKK2 (c.1614dup) mutants and / or AIM2 (c.1029dup) mutants.

7. A construct, characterized in that Comprising the early-onset obesity gene mutant in Sichuan and Chongqing as described in any one of claims 1-3.

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