Selective Screening of Children for Hereditary Metabolic Diseases by Tandem Mass Spectrometry in Kazakhstan

Inborn errors of metabolism (IEM) are not have specific clinical signs, they masquerade as other diseases, and are difficult to diagnose using only clinical manifestations or routine laboratory tests. IEM most commonly manifest in early infancy and childhood. Despite the fact that most IEM are rare in the population, they occupy one of the first places in the structure of childhood pathology, early infant mortality and disability. IEM often remains undiagnosed, while timely diagnosis and timely treatment started can prevent severe systemic damage leading to death and disability. The appointment of a special treatment (diet therapy, cofactors, enzyme replacement therapy) prevents or significantly inhibits the development of the pathological process, especially if the diagnosis is made in the early stages of the disease. To start pathogenetic treatment as early as possible, it is necessary to diagnose IEM as accurately and as early as possible.

Among the diseases included in mass screening programs IEM are especially important due to the development of disability and early mortality in the absence of timely diagnosis and treatment, as well as a high risk of recurrence in burdened families. In this connection, the main goals of mass screening - the prevention of disability in children and the reduction of early infant mortality - dictate the need to introduce modern technologies for preclinical diagnosis of IEM.

Based on the results of the study, it is planned to scientifically substantiate the need for the introduction of selective screening of children for hereditary metabolic diseases using the technology of tandem mass spectrometry in the Republic of Kazakhstan for timely diagnosis, therapy of IEM and prevention of disability. The introduction of a selective newborn screening program for IEM should always be preceded by a study aimed at studying the prevalence of the disease in a certain region, determining regional reference values of the studied metabolites. Local incidence and outcome data can be used to persuade health officials to prioritize screening in health care spending.

The main scientific question and hypothesis of the project is whether it is necessary to introduce tandem mass spectrometry technology in the neonatal screening program for IEM.

In Kazakhstan, the use of MS/MS in metabolic screening programs is in development. This is due to the high cost of equipment and consumables, as well as the lack of special screening centers and specialists.

Selective screening is an important tool for diagnosing various types of inborn errors of metabolism (IEM). Currently, MS/MS-based technologies are widely used, which allow the simultaneous determination of the amount of several metabolites (amino acids, acylcarnitines) from a very small amount of a biological sample. However, age reference cutoff ranges for each analyte should be established first for each population prior to screening as cutoffs depend on various factors such as genetic background, population geographic location, diet, sex, age.

Data on the prevalence of most IEMs, with the exception of phenylketonuria, are not available in Kazakhstan. The data obtained in this study will make it possible to conduct a comparative analysis of the prevalence of IEM in Asia and establish reference values for the concentrations of amino acids and acylcarnitines in the blood of newborns for the Kazakhstan population. Newborn sreening is currently being conducted in Kazakhstan for two hereditary diseases - phenylketonuria and congenital hypothyroidism, which are the most represented screening diseases in most countries. A nationwide newborn screening program for two mandatory diseases, congenital hypothyroidism and phenylketonuria (PKU), began in 2007. ENBS on aminoacid disorders (AAD), organic acidemias (OA) and fatty acid oxidation defects (FAOD) is not conducted in Kazakhstan.

For Kazakhstan, as a country that does not have a mandatory ENBS program, selective screening can be an important tool for diagnosing IEM. The present study aims to develop and validate an MS/MS method for the simultaneous determination of 51 metabolites in DBS for IEM screening, as well as the determination of age-related amino acid ranges, acylcarnitines and succinylacetone in children's population of Kazakhstan. Our selective screening was initiated to obtain data on the incidence of IEM in children at risk in Western Kazakhstan. From October 2022 to December 2024, the frequency of 37 AAD, OA and FAOD will be assessed using LC-MS/MS technology in a group of children at high risk. A summary report will be presented on the screening of high-risk children for IEM in Western Kazakhstan using the LC-MS/MS method.

The results of selective screening tests in different age groups of subjects should be interpreted by comparison with the reference values and/or cut-off levels established for these groups. It is generally accepted that children need to use reference populations that reflect changes associated with growth and development. Therefore, one of the objectives of this study is to establish reference intervals for the concentration of amino acids and acylcarnitines in dry blood spots for different age groups in the range from 1 day to 18 years.

The goal of this observational study is to сonduct selective screening for IEM among children of Kazakhstan aged from 1 day to 18 years using tandem mass spectrometry to determine the prevalence of IEM and reference values of metabolites followed by subsequent substantiation of the need to introduce selective screening of newborns for IEM by MS/MS.

The main questions it aims to answer are:

question 1: what are the reference values of metabolites (amino acids and acylcarnitines) in different age groups of children in Kazakhstan; question 2: what is the contribution of IEM to the structure of the pathology of the nervous system in young children and is it necessary to introduce tandem mass spectrometry technology in the neonatal screening program for IEM?

Eligibility Criteria:
Inclusion Criteria:

Referense group: Healthy male and female children aged 1 day to 18 years. Newborns born during the study period (all newborns will be included in this study will meet all selected inclusion criteria to ensure that they do not suffer from any disorder or disease. Healthy male and female newborns must weigh within 2500 -4000 g, gestational age 37-42 weeks, Apgar score above 7 for 10 min.) Sample group: children aged 1 day to 18 years will be selected if one of the main criteria or two or more additional criteria (symptoms) is identified.

Main criteria (symptoms): 1) Sudden deterioration in the clinical condition of the child after a period of normal development (days, weeks, months): acute metabolic encephalopathy, lethargy (coma), seizures resistant to antiepileptic therapy. 2) Hepatomegaly (hepatosplenomegaly). 3) Metabolic acidosis with an increase in the anion gap. 4) Multiple fractures. 5) Child mortality in the family from diseases with similar symptoms.

Additional criteria (symptoms): Treatment-resistant seizures; Abnormal muscle tone: dystonia, hyperkinesis, hypotension; Speech delay; Mental retardation of unknown cause; Cardiomyopathy; Tachypnoea; Frequent spitting up (vomiting); Osteo-articular abnormalities (joint stiffness, chest deformity, rickets-like changes); Hernias (umbilical, inguinal-scrotal); Persistent or recurrent hypoglycemia; Metabolic alkalosis; Increase in ketone bodies in the blood and (or) urine; Hyperammonemia; Increase in the level of liver enzymes (AlAT, AST) more than 1.5 times the norm; Increase in the level of creatine phosphokinase (CPK) more than 2 times the norm; Decrease in the level of alkaline phosphatase (AP) below the age norm; Imaging or electrophysiological studies suggesting metabolic disorders; Leukopenia; Thrombocytopenia; Abnormal urine, body, ear wax, any unusual smell; Hair growth disorders, alopecia; Ophthalmological anomalies; Unusual appearance, dysmorphic features; History of previous sibling death of unknown cause; Parents' consanguinity; A positive family history of metabolic disorders.

Exclusion Criteria:

The study will exclude patients who has:

1. perinatal brain injury,
2. brain injuries,
3. infections of the central nervous system,
4. toxicological diseases,
5. tumors,
6. chromosomal abnormalities,
7. with the symptoms specified in the inclusion criteria, but with a confirmed diagnosis of any disease other than amino acids disorders (AAD), fatty acid oxidation defects (FAOD) or organic academias (OA).