Actualities in neonatal endocrine and metabolic screening

Abstract

Neonatal metabolic screening has proven to be an important tool for the early detection of innate metabolic errors. Despite the fact that simple and effective methods of testing for metabolic diseases have been identified since the middle of the twentieth century, no consensus has been reached so far on the content of neonatal metabolic screening panels. There are large differences between countries in the number of metabolic diseases identified through national metabolic screening programs, ranging from zero to several tens, the most common testing being for phenylketonuria and congenital hypothyroidism (including in Romania). Given the fact that rare but treatable diseases have been identified in recent decades, reducing the financial burden on the health system, it would be useful to include them in the national neonatal metabolic screening program.

Introduction

Inborn errors of metabolism (IEM) are a category of inherited diseases caused mainly by genetic defects, which conduct to functional defects of some enzymes and other proteins necessary to maintain normal metabolism. IEM results in disruption of biochemical pathways, accumulation of intermediate metabolites, or lack of terminal metabolites. There are several forms of IEM, including disorders of amino acid metabolism (sulfur amino acid metabolism disorders, urea cycle disorders, branched-chain amino acid metabolism abnormalities), organic acidemia, fatty acid oxidation disorders, glucose metabolism disorders, and other conditions. Many genetic metabolic diseases, such as amino acid metabolic disorders, organic acid metabolic disorders, and fatty acid oxidation disorders can be simultaneously detected with high accuracy by determining the amino acid and acylcarnitine content of dried blood on filter paper. A large number of genetic metabolic diseases can be detected at an early stage in newborns due to the widespread popularity of tandem mass spectrometry (MS/MS). The incidence rate of IEM varies between different countries and populations around the world (1).

Hereditary metabolic diseases have high morbidity and mortality, a high risk of recurrence in affected families, but there is the possibility of detecting asymptomatic newborns through neonatal metabolic screening (NBS) programs. NBS programs are internationally recognized secondary prevention interventions that would have a positive impact in the “field of public health”. These screening programs aim at the precocious detection of asymptomatic newborns suffering from certain rare diseases, to find a final diagnosis and apply the appropriate treatment to prevent further complications and sequelae.

History of neonatal metabolic tests

The most significant finding in the history of neonatal metabolic screening was the discovery of phenylketonuria (PKU) by Dr. Asbjørn Folling in 1934 (2). The discovery of phenylketonuria marked the first finding of a biochemical explanation for mental retardation (3).

Guthrie and Susi (4) developed a simple, inexpensive, and effective metabolic test to determine if newborns have phenylketonuria (PKU) in the mid-20^th century. To define the requirements for additional diseases to be included in screening programs, the World Health Organization published the Wilson and Jungner screening criteria in the Disease Screening Principles and Practices in 1968. These criteria were intended to ensure that these programs meet the main objective: maximum benefit with minimum cost. A screening test is cost-effective when it reduces costs or when the cost of the test, lifelong treatment, and follow-up is offset by the health benefits for the patient (5).

Over time, new laboratory tests have been developed to detect additional diseases, including congenital hypothyroidism (CH). Phenylketonuria and congenital hypothyroidism are the most commonly tested diseases in neonatal metabolic screening programs and there are substantial variations in other diseases that are included in screening programs (6).

Metabolic screening of newborns worldwide

Although screening newborns has traditionally been concerned with only a few mental illnesses, screening programs now include disorders that can cause premature death, inherited metabolic diseases, hemoglobin, lysosomal storage disorders, and more.

In 2009, the European Commission started an evaluation of the current NBS services and nominated a network of experts to assess and recommend a central group of disorders to be examined (7). However, at present the variability of the content of screening programs in the European Union is very high, as there are programs that include up to 30 disorders and others that include only 2. In addition, different conditions or diseases vary not only between different European countries but also within the different regions of a given country. There is therefore an urgent need to build a consensus on the diseases that should be included in the 48 European countries (8). For the Middle East and North Africa, comprising 21 countries, there is no consensus on the package of diseases included in the screening program, and NBS by MS/MS (mass spectrometry) is selective or limited, while there is a high coverage for congenital hypothyroidism (9). The 20 countries in Latin America and the 24 countries in the Asia Pacific region do not have a uniform screening package and, except for 1 or 2 countries, all assess congenital hypothyroidism. High-income countries around the world have now adopted NBS programs (10).

The differences between the recommendations for the screening of metabolic disorders are dependent on sociocultural, epidemiological, and especially economic factors. The different conditions specific to each country or region lead, not only to differences in the recommendations for the diseases that should be tested, but also to differences in the organization and funding of NBS programs (Table 1) (11,12).

Table 1.

Comparison of national NBS programs for South Eastern European countries (adapted from Koracin V. et al. Current status of newborn screening in southeastern Europe 2021)

Country	Number of screening centers	Diseases included in the national NBS and the year of introduction	Method used	Screening age (hours)
Federation of Bosnia and Herzegovina (excluding Sarajevo)	2	CH (2000, 2005) PKU (2001, 2005)	D, F	48–96
Federation of Bosnia and Herzegovina - Republika Srpska	1	CH (2007), PKU (2007)	D, F	48–72
Bulgaria	2	CAH, CH, PKU (1978–1979)	F	24
Croatia	1	CH (1985), CUD (2017), GAI (2017), IVA (2017), VLCADD (2017), LCHADD (2017), MCADD (2017), PKU (1978)	D, TMS (MS/ MS), GT for confirmation	48-72
Greece	1	CH (1979), GALT (2006), PKU (1974), G6PD def. (1977)	Genetic Screening Processor (Perkin Elmer), Home (G6PD)	72-120
Hungary	2	CH (1980), CUD (2007), GALT (1975), PA / MMA(2007), GAI (2007), GAII (2007), IVA (2007), VLCADD (2007), LCHADD (2007), MCADD(2007), MSUD (2007), FAH (2007), 3MCC (2007), PKU (1975), BTD (1980), CTNI (2007)	D, F, TMS (MS/ MS)	48-72
Kosovo	0	/	/	/
Northern Macedonia	1	CH (2007), CF (2018)	D, TMS (MS/ MS)	48
Malta	1	CH (1989), HBP (1989)	D, HPLC	72-120
Montenegro	1	CH (2007)	D	48-72
Romania	5	CH (2010), PKU (2010)	F, TMS (MS/ MS)	24-72
Serbia	1	CH (1983); PKU (1983)	D, F	48-72
Slovenia	1	CH (1981), PKU (1979), CUD (2018), GAI (2018), GAII (2018), PA / MMA (2018), IVA (2018), VLCADD (2018), MCADD (2018), LCHADD(2018), MSUD (2018), FAH (2018), 3MCC (2018), CPDI (2018), CPDII (2018), 3HMGA (2018), HSD (2018), BKT (2018)	D, F, TMS (MS / MS), NGS	48-72

ARG, arginase deficiency; BKT, b -ketothiolase deficiency; BTD, biotinidase deficiency; CAH, congenital adrenal hyperplasia; cap., capita; CF, cystic fibrosis; CH, congenital hypothyroidism; CITI, citrullinemia type 1; CITII, citrullinemia type 2; CPDI, carnitine palmitoyltransferase deficiency type 1; CPDII, carnitine palmitoyltransferase deficiency type 2; CTNI, cardiac troponin I; CUD, carnitine uptake defective; CW, country wide; D, Delfia method; EL, entity level; F, fluorimetric method; FAH, tyrosinemia type 1; GAI, glutaric acidemia type I; GAII, glutaric acidemia type II; GALT, classic galactosemia; GDP, gross domestic product; G6PD def., Glucose-6-phosphate dehydrogenase deficiency; GT, genetic testing; BPH, haemoglobinopathy; HCY, homocystinuria; 3HMGA, 3-hydroxy-3-methylglutaric aciduria; H-PHE, hyperphenylalaninemia; HPLC, high-performance liquid chromatography; HSD, holocarboxylase synthethase deficiency; IEM, inborn errors of metabolism; IVA, isovaleric acidaemia/ 2-methylbutyrylglycinuria; Lab., Laboratory; LCHADD, long-chain L-3-hydroxyacyl-CoA dehydrogenase deficiency/ trifunctional protein deficiency; MAL, malonic acidemia; MCADD, medium-chain acyl-CoA dehydrogenase deficiency; 3MCC, 3-hydroxy-methylglutaric aciduria; MET, hypermethioninemia; MSUD, maple syrup urine disease; NGS, next generation sequencing; NKH, non-ketotic hyperglycinemia; PA / MMA, propionic / methylmalonic aciduria; PKU, phenylketonuria; TYR, tyrosinemia; VLCADD, very long-chain acyl-CoA dehydrogenase deficiency.

Advances in newborn metabolic screening technology

There was a revolution in these programs in the early 1990s with the introduction of tandem mass spectrometry (MS/MS) in screening laboratories. This technique is mainly aimed at detecting oxidative disorders of amino acids, organic acids, and mitochondrial fatty acids. It is a multi-analytical method that allows the simultaneous detection and measurement of more than 50 metabolites and thus the screening of over 40 innate metabolic errors using a single dry blood sample. The emergence of this technology was a paradigm shift, moving from the conventional “test> a metabolite> a disease” to MS/MS “a test> more metabolites> more diseases”. Tandem mass spectrometry became a key method for detecting inherited metabolic disorders. This novel approach requires a reconsideration of the classical criteria set by Wilson and Jungner that are still in use, as they have been defined in a different context and therefore need to be adapted. Potentially treatable diseases with high morbidity and mortality could be candidates for inclusion in an extensive newborn screening panel, even if their prevalence is low (13).

When it comes to costs, there is debate about neonatal screening for innate metabolic diseases using MS/MS in newborns without risk factors, as it may not be cost-effective. This needs to be confirmed using data on observed mortality and reducing the effects of disability. The results determine the need for a meticulous analysis before promoting neonatal screening using MS / MS (14).

With the constant growth of metabolic disorders added to national NBS programs, the number of different laboratory testing methods used is also increasing. It soon became apparent that the use of different methods would result in a much more difficult task for laboratories. The progress of genetics was accelerated by the development of next-generation sequencing methods (NGS). Therefore, many authors see it as the method that could allow the next extension of the spectrum of diseases included in metabolic screening and the methodological standardization of screening programs. As the cost of sequencing steadily decreases, the feasibility of implementing neonatal metabolic screening programs with the use of NGS is becoming increasingly possible (15).

The role of genetic testing in neonatal screening

NGS allows the simultaneous processing of a large number of samples and is easy to extend from a few genes to the entire genome. As the number of diseases added to the national NBS programs increases, the number of different laboratory methods used is also increasing. It is obvious that the use of different methods results in a much higher workload for laboratories. The progress of genetics was accelerated by the introduction of next-generation sequencing (NGS) methods. As the cost of NGS decreases, the feasibility of introducing NBS with the use of NGS is becoming increasingly possible. The first technical concern is the ability to prepare, analyze, interpret, and report results quickly enough to meet NBS requirements. However, newer optimized protocols allow the results to be reported on the 4^th day after receiving the samples in the laboratory. There is evidence to suggest that NGS could not provide a unified platform for NBS, as some of the diseases currently being examined have a very weak genetic background or highly variable penetration. For example, in CH only 10-15% of patients have a genetic background. Other examples are some fatty acid metabolism disorders and Pompe disease, which does not have a clear genotype-phenotype correlation (15).

What should we add to the metabolic screening of newborns?

Currently, in Romania, metabolic screening of newborns consists of testing for phenylketonuria and congenital hypothyroidism. In addition to metabolic screening, newborns are subjected to another type of screening, that of congenital hearing impairment. These screening programs should be updated by adding others for other metabolic disorders with a major impact on long-term development and survival.

Spinal Muscular Atrophy (SMA) Screening

SMA is a rare and devastating disease

SMA affects about 1 in 10,000 people. Most cases of SMA are generated by the absence of a segment of a gene called SMN1, leading to the inability of the gene to produce proteins. SMN1 is responsible for the production of motor neuron survival protein - SMN, which is needed to maintain the normal function of motor neurons. Without enough SMN protein, progressive loss of controlled muscle movement will appear. Usually, the earlier the onset of the disease, the more severe the symptoms. SMA can affect the baby’s ability to swallow, breathe and walk (16).

New treatments that modify the disease have recently been approved, and early treatment has been linked to a better outcome. In this context, several newborn screening programs have been implemented in some countries. Dangouloff Tamara et al. conducted a survey and contacted experts from 152 countries, receiving 87 responses. The results were published in 2021. They identified 9 screening programs that also included SMA testing. They detected 288 newborns with SMA out of approximately 3,670,000 newborns tested. Many respondents pointed to the lack of cost-benefit data as a major obstacle to the implementation of SMA screening tests in neonatal screening programs. For the next four years, the data obtained are expected to cover 24% of newborns in countries where a drug that changes the course of the disease is available and 8.5% coverage in countries without drugs that change the course of the disease. The annual proportion of newborns to be tested is expected to increase steadily. Experts expressed the strong need to implement tests for early detection of AMS in neonatal screening programs as a means of improving the outcome of patients suffering from AMS (17).

Cystic fibrosis screening

Cystic fibrosis (CF) is the most common autosomal recessive genetic disorder with a high fatality rate in Caucasians, affecting 1 in 3,000 live births. The incidence is related to geographical regions and ethnic groups. The diagnosis of CF is classically based on the clinical symptoms specific to the diagnosis of CF and is also confirmed by genetic analysis and the presence of two mutations in the transmembrane conductance cystic fibrosis ( CFTR ) gene in trans. Screening protocols for CF in newborns are based on the immunoreactive trypsinogen (IRT) as the initial test and the sweat test to confirm or rule out the diagnosis of CF. To improve the results of the screening program, a second-level test is required. The second test is either a second blood sample taken at 21-28 days of age for IRT-2 or a CFTR gene mutation analysis using the first blood spot or a biological measurement of pancreatitis-associated protein (PAP). In the last three decades, a growing number of countries in Europe, North, and South America have introduced NBS for CF, so the number of newborns screened for CF is increasing (18). Today, neonatal screening is an essential component of CF care standards. The majority of newborns in North America, Europe, Australia, and New Zealand and an increasing number in South America are being tested for CF. Newborn screening and appropriate early treatment (replacement of pancreatic enzymes, fat-soluble vitamins, supplementation with salt) have a positive effect on growth and nutritional status and for prevention of the deficiency of fat-soluble vitamins and protein malnutrition. Patients diagnosed with cystic fibrosis using neonatal screening have lower treatment costs and lower rates of hospitalizations for intravenous antibiotic treatment due to exacerbation of lung disease (19). Patients diagnosed with CF through neonatal screening are thought to have better lung function and fewer episodes of Pseudomonas aeruginosa infection. Screening in newborns for CF leads to improved health outcomes and increased population survival for patients with CF. Screening is a cost-effective public health strategy. Early diagnosis with NBS will allow the timely introduction of this therapy in the future (20).

Discussion

The current status of the NBS in the SE region of Europe is highly variable and is underdeveloped or even lacking in some countries. In addition, the situation has not changed much in the last thirty years. Several countries have introduced an extended NBS, while a large part of them continue to test only for CH and PKU, and one of the countries surveyed does not yet have an NBS at all. Very recent researches have confirmed a lack of harmonization of NBS programs between European countries, highlighting the need for more comprehensive guidance at the European level (21). The call for further efforts and support for the adjustment of the status of the NBS in SE Europe through international collaboration and the sharing of practical and theoretical knowledge persists. It is suggested that an international working group be set up to assist in the implementation and harmonization of NBS services wherever needed. First of all, a careful assessment of the current situation is needed and must be included in the relevant state-of-the-art documents and international initiatives. Consequently, more active support should be provided in the implementation of core standards, perhaps starting and/or continuing with initiatives to introduce newborn screening programs where necessary. Improving the cost/benefit ratio can be done by developing 1-2 centers of excellence to provide technology and expertise. In Romania, this center exists within the National Institute for Maternal and Child Health “Alessandrescu-Rusescu”, and the national screening program is coordinated by pediatricians. In addition, a minimum set of disorders could be defined to be examined in any region, regardless of country or continent. These efforts could be further supported in particular by a relevant professional forum and international organizations (22).

In conclusion, metabolic diseases have a large spectrum of nonspecific symptoms in newborns. A large variety of metabolic disorders and the lab tests available for them make it difficult to determine an appropriate test scheme. Early involvement of a geneticist and a specialized laboratory will bring up the most effective test results. Failure to diagnose can lead to developmental issues and could even be fatal. The best way to give an early diagnosis for metabolic diseases is to consider these conditions in a timely perspective and to send the biological samples collected in suitable conditions for the most appropriate tests in the proper laboratories. Using a new technology as NGS there are better chances for adding rare metabolic diseases to NBS. The cost evaluation needs to be done considering the potential benefits of including rare but treatable diseases in NBS programs, which we could now detect without additional direct costs.

Conflict of interest

The author declare that he has no conflict of interest.