CN116694756A - Diagnostic marker for familial adult myoclonus epilepsy and application - Google Patents

Abstract

The invention discloses a diagnostic marker and application of familial adult myoclonic epilepsy, belonging to the technical field of medical diagnosis. In the present invention, through two FAME families, PKHD1L1 gene exon23:c.2602A>T heterozygous mutation exists in the diseased genomes of the two families, and the PKHD1L1 gene exon23:c.2602A>T heterozygous mutation is confirmed genetically It may be the causative gene of FAME. Based on this, the present invention can be used for screening or diagnosing FAME, especially the genetic diagnosis of familial cortical tremor with epilepsy by detecting whether the subject carries the above mutation. The detection kit provided by the invention can be used to quickly and effectively predict or diagnose familial cortical tremor with epilepsy. The invention lays an important foundation for the research on the pathogenesis of familial cortical tremor with epilepsy, and provides a new theoretical basis for the treatment of patients with familial cortical tremor with epilepsy.

Description

Diagnostic markers and application of familial adult myoclonic epilepsy

technical field

The invention belongs to the technical field of medical diagnosis, and in particular relates to a diagnostic marker and application of familial adult myoclonic epilepsy.

Background technique

Familial adult myoclonic epilepsy (FAME) was first named by Japanese scholar Okuma in 1997. Its clinical features are as follows: (1) autosomal dominant inheritance, onset in adulthood, a few onset in adolescence, no significant gender difference; (2) myoclonic seizures with or without generalized tonic-clonic seizures; (3) ) Fine tremor of distal limbs, antiepileptic drugs are effective, but β-blockers are ineffective; (3) non-progressive course, generally not accompanied by ataxia and dementia; (4) electrophysiological studies suggest tremor and/or Myoclonus is of cortical origin. So far, there are only dozens of family reports in the world, mainly concentrated in Japan and Europe, and there are fewer reports in my country. There are two subtypes of FAME with similar clinical manifestations, such as benign adult familial myoclonic epilepsy (BAFME) and familial cortical tremor and epilepsy (FCTE). So far, there have been many screenings for FAME-causing genes at home and abroad, but no further verification and functional research has been carried out. Therefore, the real FAME-causing genes have not been discovered yet.

The PKHD1L1 gene (NM_001034931) is located on human chromosome 8q23, accounting for about 168kb. The RNA of PKHD1L1 gene has multiple splicing modes and produces multiple transcripts. The longest transcript consists of 78 exons and a total of 13079bp encodes a protein Fibrocystin-L consisting of 4243 amino acids. The results of bioinformatics prediction indicated that Fibrocystin-L is a single transmembrane receptor-like protein. Its carboxyl terminal is located in the cell and consists of 8 amino acids. Contains a potential protein kinase C (proteinkinase C, PKC) phosphorylation site. The extracellular region of Fibrocystin-L consists of 4212 amino acids, and the signal peptide, 14 tandem T IG domains and TEME homology region composed of 9 PbH1 repeat sequences are arranged sequentially from the N-terminus. Preliminary RT-PCR results showed that PKHD1L1 was widely expressed in various tissues and organs of humans and mice. As a newly discovered and defined gene, the spatiotemporal expression and subcellular localization of PKHD1L1 in the growth and development of mammals are still unknown, and the characteristics and biological functions of the PKHD1L1 gene and its products are also unknown. It has been reported in the literature that PKHD1L1 gene is mostly related to polycystic kidney disease, obesity, and thyroid cancer, but the correlation with epilepsy has not been reported.

Contents of the invention

The purpose of the present invention is to provide diagnostic markers and applications of familial adult myoclonic epilepsy, and to verify the possible pathogenic gene of FAME with exon23:c.2602A>T heterozygous mutation of PKHD1L1 gene from the perspective of genetics.

The invention provides an application of a reagent for verifying the genotype of the exon23:c.2602 site of the PKHD1L1 gene in the preparation of a diagnostic tool for familial adult myoclonic epilepsy.

Preferably, when there is an A>T mutation at the exon23:c.2602 site of the PKHD1L1 gene, it is considered to have familial adult myoclonic epilepsy.

Preferably, the method for verifying the genotype at exon23:c.2602 of the PKHD1L1 gene includes sequencing.

The present invention also provides a set of primer pairs for amplifying the PKHD1L1 gene, including an upstream primer whose nucleotide sequence is shown in SEQ ID No.1 and a downstream primer shown in SEQ ID No.2.

The present invention also provides a diagnostic kit for familial adult myoclonic epilepsy, comprising the above primer pair and other reagents for PCR amplification.

Preferably, when the diagnostic kit is used for detection, the genomic DNA of the sample is extracted, amplified using the primer pair, and the amplified product is sequenced;

The amplification procedure includes: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 55-60°C for 35 seconds, extension at 72°C for 40-50 seconds, and 35 cycles; extension at 72°C for 5-8 minutes.

Preferably, when there is an A>T mutation at the exon23:c.2602 site of the PKHD1L1 gene in the sequence of the amplified product, the sample is from a patient with familial adult myoclonic epilepsy.

Beneficial effects: the present invention has confirmed the PKHD1L1 gene exon23:c. 2602A>T heterozygous mutation may be the causative gene of FAME. Based on this, the present invention can be used for screening or diagnosing FAME, especially the genetic diagnosis of familial cortical tremor with epilepsy by detecting whether the subject carries the above-mentioned mutations, guiding treatment, and prenatal and postnatal care. The detection kit provided by the invention can be used to quickly and effectively predict or diagnose familial cortical tremor with epilepsy. The invention lays an important foundation for the research on the pathogenesis of familial cortical tremor with epilepsy, and provides a new theoretical basis for the treatment of patients with familial cortical tremor with epilepsy. The invention can provide a possible drug target for treating familial cortical tremor with epilepsy.

Description of drawings

Fig. 1 is the pedigree chart of family one in the embodiment 1 of the present invention;

Figure 2 is a graph showing the amplification results of the PKHD1L1 gene;

Figure 3 is a diagram of the wild type and mutant sequencing results of the PKHD1L1 gene;

Fig. 4 is the pedigree diagram of the second family of the present invention.

Detailed ways

The invention provides an application of a reagent for verifying the genotype of the exon23:c.2602 site of the PKHD1L1 gene in the preparation of a diagnostic tool for familial adult myoclonic epilepsy.

The present invention finds through genome sequencing technology that there is a PKHD1L1 gene exon23:c.2602A>T mutation in patients of two FAME families, but this mutation does not exist in normal people, so it has been confirmed genetically that the PKHD1L1 gene exon23: c. Pathogenicity of the 2602A>T mutation. All patients in the two FAME families mentioned in the present invention have myoclonus with or without generalized tonic-clonic seizures, with or without fine tremors in the distal limbs, and the seizures are all adult-onset. Excitability increased, antiepileptic drug treatment can effectively control seizures, and the course of the disease was benign.

The method for verifying the genotype of the exon23:c.2602 site of the PKHD1L1 gene described in the present invention includes sequencing.

The present invention also provides a set of primer pairs for amplifying the PKHD1L1 gene, including an upstream primer whose nucleotide sequence is shown in SEQ ID No.1 and a downstream primer shown in SEQ ID No.2.

The information of the primer pair of the present invention is preferably as follows:

Upstream primer: 5'-ACTAAAATGTTGCATATGGGTTG-3'

Downstream primer 5'-ATCATAGAAAAGAGAGAGGTGTGTT-3'.

The primer pair of the present invention is used for PCR amplification, and the amplified product is 257bp.

The present invention also provides a diagnostic kit for familial adult myoclonic epilepsy, comprising the above primer pair and other reagents for PCR amplification.

When the present invention uses the diagnostic kit for detection, it preferably includes: extracting the genomic DNA of the sample, using the primer pair to amplify, and sequencing the amplified product; the amplification procedure includes: pre-denaturation at 95°C for 3 minutes; Denaturation at 94°C for 30s, annealing at 55-60°C for 35s, extension at 72°C for 40-50s, 35 cycles; extension at 72°C for 5-8min.

In the present invention, when using the program for amplification, when there is a PKHD1L1 gene exon23:c.2602 site A>T mutation in the sequence of the amplification product, the sample is derived from a patient with familial adult myoclonic epilepsy . The present invention does not specifically limit the type of sample, as long as DNA can be extracted, such as biological samples, blood, etc., blood is used as an example in the embodiment for illustration, but it cannot only be identified as the entirety of the present invention. protected range.

In order to further illustrate the present invention, the diagnostic markers and applications of familial adult myoclonic epilepsy provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

1. Family one

Family 1 (Fig. 1) has 30 persons in five generations, 6 of them have the disease, all of which conform to the characteristics of autosomal dominant inheritance. All patients had myoclonus with or without generalized tonic-clonic seizures, with or without fine tremors in the distal limbs. The epileptic seizures were all onset in adults. Electrophysiological examination revealed increased cortical excitability, and antiepileptic drug treatment was effective. The attack was effectively controlled, and the course of the disease was benign. All patients in family 1 were diagnosed clearly, and the clinical phenotypes were consistent. Through genome-wide exome sequencing combined with linkage analysis, it was found that 5 patients (1 of 6 patients died) had PKHD1L1 gene exon23:c.2602A>T heterozygous mutation, and 11 controls were all homozygous. separation phenomenon.

Screening of the PKHD1L1 gene in 246 normal populations matched in age, sex, region, and ethnicity found that the mutation site does not exist in the normal population, thus preliminarily confirming that the site in PKHD1L1 is the genetic mutation. Pathogenic mutations in the family.

Table 1 Clinical data of a patient in the family

2. Family II (Figure 4)

Three generations of 34 people, including 13 patients. Among them, only two patients (father-son relationship) cooperated with the examination, and the diagnosis was clear, in line with the diagnosis of familial cortical tremor with epilepsy. Sanger sequencing also found that these two patients had PKHD1L1 gene exon23:c.2602A>T heterozygous mutation.

The present invention verifies the pathogenicity of the mutation site from the perspective of genetics through the existence of PKHD1L1 gene exon23:c.2602A>T heterozygous mutation in two families with definite diagnosis.

Example 2

Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the primer pair shown in SEQ ID No.1 and SEQ ID No.2.

And prepare 50 μL reaction system: blood sample DNA template 1 μL, upstream primer 1 μL, downstream primer 1 μL, dNTP 10mM 1 μL, Taq Buffer 5 μL, 25mM MgCl ₂ 5 μL, Taq enzyme (μl) 5U/μl 0.5 μL, water 35.5 μL.

Set the program for PCR amplification: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 30 s, annealing at 55-60°C for 35 s, extension at 72°C for 40-50 s, 35 cycles; extension at 72°C for 5-8 min.

1% agarose electrophoresis, 150V, 100mA 20min electrophoresis results are shown in Figure 2, PCR product purification and recovery kit (Shenggong SK1141), sequencing, the results are shown in Table 2 and Figure 3.

Table 2 Sample genotype statistics

Sample serial number Test No. ch8-110432824A 1B 1 A/T 2B 2 A/T 11B 3 A/A

Although the foregoing embodiment has described the present invention in detail, it is only a part of the embodiments of the present invention, rather than all embodiments, and people can also obtain other embodiments according to the present embodiment without inventive step, these embodiments All belong to the protection scope of the present invention.

Claims (7)

1. The application of a reagent for verifying the genotype of exon23:c.2602 site of PKHD1L1 gene in the preparation of diagnostic tools for familial adult myoclonic epilepsy. 2. The application according to claim 1, characterized in that, when there is an A>T mutation at the exon23:c.2602 site of the PKHD1L1 gene, it is considered to have familial adult myoclonic epilepsy. 3. The application according to claim 1, wherein the method for verifying the genotype of the exon23:c.2602 site of the PKHD1L1 gene comprises sequencing. 4. A pair of primers for amplifying the PKHD1L1 gene, characterized in that it includes an upstream primer whose nucleotide sequence is shown in SEQ ID No.1 and a downstream primer shown in SEQ ID No.2. 5. A diagnostic kit for familial adult myoclonic epilepsy, characterized in that it comprises the primer pair according to claim 4 and other reagents for PCR amplification. 6. according to the described diagnostic kit of claim 5, it is characterized in that, when utilizing described diagnostic kit to detect, extract sample genome DNA, utilize described primer pair to amplify, amplified product is sequenced; The amplification procedure includes: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 55-60°C for 35 seconds, extension at 72°C for 40-50 seconds, and 35 cycles; extension at 72°C for 5-8 minutes. 7. The diagnostic kit according to claim 5 or 6, wherein, when there is a PKHD1L1 gene exon23:c.2602 site A>T mutation in the sequence of the amplified product, the sample is derived from familial adult muscularis Patients with clonic epilepsy.