阅读说明：本技术 一种用于儿童威尔森氏症快速筛查的参数组合和试剂盒 (Parameter combination and kit for rapidly screening children Wilson's disease ) 是由 周建利 周少明 陈沫先 赵煜桢 于 2021-07-20 设计创作，主要内容包括：本发明提供了一种用于儿童威尔森氏症快速筛查的参数组合和试剂盒。该参数组合包括ALT水平、血浆铜蓝蛋白水平和24小时尿铜水平中一种或者多种。试剂盒,其包括检测ALT水平、血浆铜蓝蛋白水平和/或尿铜水平的试剂；和一说明书。本发明提供的参数组合物和试剂盒通过参考ALT升高、血浆铜蓝蛋白水平降低和24小时尿铜升高三个参数,可以适用于威尔森氏症的早期诊断和筛查,操作简单易行,可信度高,便于早期的诊断和介入治疗,将有利于大大提高生存率并具有良好的预后。(The invention provides a parameter combination and a kit for rapidly screening children Wilson's disease. The combination of parameters includes one or more of ALT levels, ceruloplasmin levels, and 24-hour urine copper levels. A kit comprising reagents for detecting ALT levels, ceruloplasmin levels, and/or urine copper levels; and an instruction sheet. The parameter composition and the kit provided by the invention can be suitable for early diagnosis and screening of Wilson's disease by referring to three parameters of ALT (acute respiratory syndrome), plasma ceruloplasmin level reduction and 24-hour urine copper increase, are simple and easy to operate, have high reliability, are convenient for early diagnosis and interventional therapy, are beneficial to greatly improving survival rate and have good prognosis.)

1. A combination of parameters for rapid screening of wilson's disease in children comprising one or more of ALT levels, ceruloplasmin levels and 24 hour urine copper levels.

2. The combination of parameters of claim 1, wherein an elevation of ALT, a decrease in ceruloplasmin levels, and/or an elevation of 24 hours urine copper is a preliminary diagnosis of wilson's disease.

3. A kit for rapidly screening children Wilson's disease is characterized by comprising reagents for detecting ALT level, ceruloplasmin level and/or urine copper level; and an instruction sheet.

4. The kit of claim 3, wherein the instructions comprise instructions for:

a. detecting ALT level, ceruloplasmin level and 24-hour urine copper level; and

b. judging whether the preliminary diagnosis of the Wilson's disease can be made: if ALT is increased, the level of ceruloplasmin is reduced and/or urine copper is increased in 24 hours, then the initial diagnosis of Wilson's disease can be made; if any index meeting the above standard does not exist, the patient can be judged not to have the Wilson's disease.

5. The kit of claim 4, wherein the instructions further comprise instructions for:

c. if the initial diagnosis is Wilson's disease, the gene mutation test of ATP7B gene is used to determine if Wilson's disease is present.

Technical Field

The invention relates to the technical field of medical detection, in particular to a parameter combination and a kit for rapidly screening children Wilson's disease.

Background

Hepatolenticular degeneration (HLD), also known as wilson's disease/Wilson's Disease (WD), is an autosomal recessive genetic disorder. The disease occurs worldwide, with a prevalence in the population of 1/30,000 in live births^[1]. WD is encoded by intracellular copper transport on chromosome 13Caused by mutations in the ATP7B gene of the protein, resulting in impaired intracellular copper output^[2]. ATP7B is a P-type atpase, which is mainly expressed in the liver. ATP7B binds copper to its N-terminal domain and is responsible for copper transport across membranes, using ATP as its energy source. Studies have shown that mutations at different sites affect ATPase activity. To date, more than 800 different mutations at the ATP7B site have been found in WD patients^[3]. In particular, mutation of WD ATP7B gene affects interaction between copper ions and ceruloplasmin, and further affects excretion of copper in bile, and is the main pathway of liver copper excretion. If copper excretion in bile is reduced, copper will be deposited in the area around the liver, causing damage to hepatocytes, which are first clinically diagnosed as elevated alanine Aminotransferase (ALT). Subsequent copper deposition on the brain, cornea and kidneys causes damage to the respective organs, with various clinical symptoms^[4]. Over time, the liver is gradually damaged by the deposited copper, and some patients eventually develop cirrhosis or liver failure, as well as severe neurological, hematological, and psychiatric symptoms^[4]。

The clinical manifestations of children with WD may vary due to the time of initiation of treatment^[5,6]. In general, the copper excretion mechanism of newborn infants is not fully developed and becomes more effective within one year after birth. However, the failure of the critical pathway for copper excretion in WD patients to develop or to malfunction, leads to copper accumulation in the patient's lifetime, and gradually leads to various clinical symptoms^[7]. Generally, WD patients are diagnosed at ages 5-35 years (13 years on average)^[8]But also young and older patients (>70) Need to diagnose^[4,8-10]. In addition, a study of 143 WD children showed that 21 children (15%) developed symptoms or findings of liver dysfunction before the age of 5 years^[10]. The most common initial manifestation of WD children at an average age of 9-13 years is liver disease^[11,12]While WD accounts for 8% -10% of children with chronic active hepatitis^[13]. In other patients, liver disease may progress asymptomatic, and multiple complications of cirrhosis or acute liver failure often do not appear until puberty. In addition, in some casesIn cases, neurological or psychiatric manifestations may also occur prior to overt liver disease. In summary, children and elderly patients are more likely to be diagnosed with liver and nervous system manifestations, respectively. The average age of elderly patients presenting with nervous system symptoms is 15-21 years^[6,11,14,15]. The age difference in WD onset may reflect changes in gene mutation and penetrance, extragenic factors and other environmental factors (e.g., diet)^[16]. In the prior art, gene mutation detection and analysis are mainly used, and patients are mostly detected after the disease condition is characterized, so that the method is not suitable for early physical examination screening. Therefore, high precision early diagnosis is crucial for WD patients and their prognosis.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a parameter combination and a kit for rapidly screening children Wilson's disease.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, a parameter set for rapid screening of Wilson's disease in children is provided, comprising one or more of ALT levels, ceruloplasmin levels and 24-hour urine copper levels.

Furthermore, a preliminary diagnosis of Wilson's disease can be made by elevation of ALT, reduction of ceruloplasmin levels and/or elevation of urine copper at 24 hours.

The second aspect of the invention provides a kit for rapidly screening children Wilson's disease, which comprises reagents for detecting ALT level, ceruloplasmin level and/or urine copper level; and

an instruction book.

Further, the above description includes the following indications:

a. detecting ALT level, ceruloplasmin level and 24-hour urine copper level; and

b. judging whether the preliminary diagnosis of the Wilson's disease can be made: if ALT is increased, the level of ceruloplasmin is reduced and/or urine copper is increased in 24 hours, then the initial diagnosis of Wilson's disease can be made; if any index meeting the above standard does not exist, the patient can be judged not to have the Wilson's disease.

Further, the above specification also includes the following indications:

c. if the initial diagnosis is Wilson's disease, the gene mutation test of ATP7B gene is used to determine if Wilson's disease is present.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the parameter composition and the kit provided by the invention can be suitable for early diagnosis and screening of Wilson's disease by referring to three parameters of ALT (acute respiratory syndrome), plasma ceruloplasmin level reduction and 24-hour urine copper increase, are simple and easy to operate, have high reliability, are convenient for early diagnosis and interventional therapy, are beneficial to greatly improving survival rate and have good prognosis.

Detailed Description

The invention provides a parameter combination and a kit for rapidly screening children Wilson's disease. The combination of parameters includes one or more of ALT levels, ceruloplasmin levels, and 24-hour urine copper levels. An elevation in ALT, a decrease in ceruloplasmin levels and/or an elevation in urine copper over 24 hours can be a preliminary diagnosis of Wilson's disease.

The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.

In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.

Example 1

This example demonstrates that a combination of parameters including one or more of ALT levels, ceruloplasmin levels, and 24-hour urine copper levels can be used for early rapid screening of wilson's disease, with the following specific procedures and results:

30 children in south China who are shown as WD first-onset and have alanine aminotransferase ALT elevation are recruited for treatment in Shenzhen child hospital, 14 women and 16 men, and retrospective analysis and short-term follow-up study are carried out on the cross section of clinical data and gene detection results.

During the visit, 30 of the above described children patients were subjected to physical examination, and liver enzymes were found to be elevated by 30 cases (100%), and serum Ceruloplasmin (CP) levels were decreased in all 30 cases (100%), while 24-hour urine copper excretion was increased (table 1 below).

TABLE 130 clinical data of pediatric patients

Note: hb: hemoglobin, ALT: glutamic-pyruvic transaminase, AST: glutamic-oxaloacetic transaminase, TB: total bilirubin, ALP: serum alkaline phosphatase, GGT: γ -glutamyl transpeptidase, CP: serum ceruloplasmin.

Finally, using a classical confirmation method, a gene mutation test of ATP7B gene was used for confirmation, in which the mutation frequencies of the two mutation sites, c.2333g > T and c.3443t > C, were the highest, accounting for 23.0% and 10.7% of the total number of mutation sites, respectively (see tables 2 and 3 below).

TABLE 230 mutant types of ATP7B for pediatric patients

TABLE 330 summary of ATP7B mutation types in pediatric patients

Example 2

The embodiment provides a kit for rapidly screening children Wilson's disease, which comprises reagents for detecting ALT level, ceruloplasmin level and/or urine copper level; and an instruction sheet.

The specification includes the following instructions:

a. detecting ALT level, ceruloplasmin level and 24-hour urine copper level; and

b. judging whether the preliminary diagnosis of the Wilson's disease can be made: if ALT is increased, the level of ceruloplasmin is reduced and urine copper is increased in 24 hours, the initial diagnosis of Wilson's disease can be made; if the index does not meet the standard, judging that the patient does not suffer from the Wilson's disease;

c. if the initial diagnosis is Wilson's disease, the gene mutation test of ATP7B gene is used to determine if Wilson's disease is present.

From the above, the kit provided by the invention is convenient for early diagnosis of Wilson's disease, is suitable for routine physical examination, and is suitable for screening patients with negative cranial MRI examination and no nervous system expression.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. It will be appreciated by those skilled in the art that any equivalent modifications and substitutions are within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Reference to the literature

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[3]Chen YC,Yu H,Wang RM,et al.Contribution of intragenic deletions to mutation spectrum in Chinese patients with Wilson's disease and possible mechanism underlying ATP7B gross deletions.Parkinsonism RelatDisord.2019；62:128-133.

[4]European Association for Study of Liver.EASL Clinical Practice Guidelines:Wilson's disease.J Hepatol 2012；56:671.

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[10]Wiernicka A,M,Jańczyk W,et al.Early Onset of Wilson Disease:Diagnostic Challenges.J Pediatr Gastroenterol Nutr 2017；65:555.

[11]Saito T.Presenting symptoms and natural history of Wilson disease.Eur J Pediatr 1987；146:261.

[12]Walshe JM.Wilson's disease presenting with features of hepatic dysfunction:a clinical analysis of eighty-seven patients.Q J Med 1989；70:253.

[13]Gitlin JD.Wilson disease.Gastroenterology 2003；125:1868.

[14]Oder W,Grimm G,Kollegger H,et al.Neurological and neuropsychiatric spectrum of Wilson's disease:a prospective study of 45 cases.J Neurol 1991；238:281.

[15]Ferenci P,A,Merle U,et al.Late-onset Wilson's disease.Gastroenterology 2007；132:1294.

[16]Wiggelinkhuizen M,Tilanus ME,Bollen CW,Houwen RH.Systematic review:clinical efficacy of chelator agents and zinc in the initial treatment of Wilson disease.Aliment PharmacolTher 2009；29:947.