Professor Giancarlo la Marca of the University of Florence and Meyer Children’s Hospital, Florence, Italy, speaks with Professor Gianluca Giorgi, University of Siena, Italy, and Dr. Vera Köster for ChemViews Magazine about the importance and potential of newborn screening and why it is more than a useful biochemical test.
What does newborn screening mean?
Newborn screening (NBS) is known to be a biochemical test that enables the identification of many inborn errors of metabolism (IEM) a few days after birth. But considering that NBS requires expert lab technicians, chemists, biologists, nutritionists, and medical specialists for metabolic disorders, it cannot only be considered a useful biochemical test, rather it should be considered a complex and integrated program.
The objective of a newborn screening program is to detect some IEM before the clinical manifestation of the associated symptoms. As a consequence, medical doctors can start the best available treatment and have the best prognosis by modifying the natural course of the disease.
Why is it important to test children for inherited metabolic disorders?
If metabolic disorders are not diagnosed and treated early, most of them can cause mental and/or growth retardation, severe permanent sequelae, and in some cases death. If they are treated on time, the majority of children can have a normal life.
Treatment does not mean surgical intervention or other invasive approaches. In the majority of cases, affected newborns require dietetic therapy and/or a drug supplement.
When was the first NBS used?
The history of NBS as a population-based test dates back from the beginning of the 1960s when the biologist Robert Guthrie developed a simple and inexpensive bacterial inhibition assay based on a filter paper spot able to identify the most frequent aminoacidopathy: the PKU or phenylketonuria.
Here phenylalanine cannot be metabolized to tyrosine, phenylalanine accumulates and is converted into phenylpyruvate (phenylketone). This can be detected in the urine. Untreated PKU can, for example, lead to intellectual disability, and seizures.
In the following decade, some other clinical labs both in the United States and in Europe added congenital hypothyroidism (CH) to their panel, again by using a single drop of whole blood on paper.
The subsequent development of electrospray tandem mass spectrometry in the 1990s permitted the introduction of this new technology into clinical chemistry laboratories, in particular for newborn screening purposes. MS/MS is a versatile, specific, and sensitive technology that gives technicians the ability to measure many biomarkers in a single and fast analytical run. People working in the field of newborn screening understood the possibility of passing from one dried blood spot (DBS) for one test and one disorder to one DBS for one multiplex test for many disorders.
For which kinds of disorders and inborn errors is it possible to carry out a diagnosis in the first days of life?
In fact, today MS/MS can easily identify and quantify—in a run of two minutes or less—several metabolites such as acylcarnitines, aminoacids, succinylacetone, and more recently, some purines. Nowadays, including pilot projects and regionally governed and structured national NBS programs, many labs all around the world screen for more than 40 or more IEM with a single test.
The number of potential identifiable disorders is not a technological problem, rather it depends on regional or national public health strategies. Some expanded newborn screening programs are now not only screening for PKU, CH, and, more recently, for cystic fibrosis (CF), and congenital adrenal hyperplasia (CAH), but also for other like aminoacidopathies, β-oxidation fatty acid defects, organic acidurias, urea cycle defects, and, since 2011, for some severe combined immunodeficiencies (SCID).
What is the impact of newborn screening on population in terms of benefits, health, and quality of life?
Since the 1960s, population newborn screening programs have greatly reduced the morbidity and mortality of the diseases screened for at birth; PKU is the classic example. Despite the universal success in addressing this disease, significant challenges arose and have remained open.
In particular, the diagnosis and management of PKU and mild forms of hyperphenylalaninemias (HPAs) deserve a short comment. Patients with mild HPA do not need treatment, but during the weaning period they should undergo close clinical and biochemical follow-up for assessment of the need to control protein intake. In addition, it is now clear that adherence to diet is especially important during pregnancy in adult females with even mild HPA if teratogenic effects are to be avoided. Because of the increased intake of protein during pregnancy and of the transplacental fetal/maternal gradient (1.5 for the fetus), the phenylalanine (Phe) levels in fetal blood could be elevated. Children are not affected by PKU, but are born with microcephaly, congenital malformations, and global developmental delay caused by their in utero exposure to elevated levels of phenylalanine. This has become known as Maternal PKU Syndrome.
“Diet for life” is now the mantra for treatment of all babies with PKU identified during newborn screening — not only because of the sequelae of maternal PKU but also because it is well known that adults with PKU, both males and females, continue to face significant medical, cognitive, and psychiatric challenges including osteopenia, B12 deficiency, cognitive decline, particularly in areas of higher executive functions, and psychiatric problems including depression and anxiety disorders. In spite of these challenges, the principles of universal newborn screening for PKU remain the gold standard against which the addition of new tests for newborn screening has been measured.
Which criteria exist?
In fact, it is well known that one fundamental worldwide-approved criterion is that an IEM can be screened only if a related treatment is available. For all disorders included in the newborn screening programs a therapy should be possible even if, in some cases, it is not completely curative.
If a therapy is not completely curative, the early detection of the disorder and a subsequent immediate correct treatment should give babies the possibility to have a better quality of life, to extend life expectancy, and when required, to allow suitable genetic counseling especially for prenatal diagnoses. Moreover, an early diagnosis could relieve families with a severely ill child from difficult diagnostic procedures.
The qualifier “not completely” is one relevant reason why newborn screening panels are not all the same worldwide. The question of what to screen for appears to be evolving as new treatment options become available.
Can you say something on the costs?
The prototype inborn error of metabolism that has led to the dramatic expansion of universal newborn screening has been medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, the frequency of which is higher in most countries than PKU (1:8000). Here the body’s ability to break down medium-chain fatty acids into acetyl-CoA is damaged. This leads to hypoglycemia and sudden death without timely intervention, most often caused by periods of fasting or vomiting.
At the time of a child’s first febrile episode, this fatty acid oxidation defect is fatal in up to 25 % of children not previously screened at birth and treated presymptomatically. In more than 80 % of cases there is at least one episode of coma and about 50 % entail neurological damage. In the first 24 to 36 months of life, treatment protocols are based on avoidance of prolonged fasting and supportive therapy to prevent hypoglycemia.
There is no question that presymptomatic detection by newborn screening can prevent deaths or permanent neurological sequelae in MCAD. Some studies evaluated the cost of surviving babies with neurological illness as being up to 200,000 to 400,000 euros per patient per year. This money can cover a regional NBS program of screening with mass spectrometry (MS) for 25,000 to 30,000 newborns. To screen or not to screen, that is the crucial question.
What do you personally think?
My opinion is that even if some published data have suggested that some newborn children with metabolic disorders can remain asymptomatic their whole lives without treatment – including MCAD patients – or can die from fatal clinical manifestations in the first few hours of life before the results of newborn screening are available, benefits in terms of life expectancy and improved quality of life for babies and families are more relevant. When we are discussing the cost of neonatal screening, perhaps we should ask ourselves how these procedures save public money and contribute to our overall health.
What are the main challenges and what are future trends in newborn screening?
Avoiding false-negative results by using specific biomarkers and reducing the false-positive rate by using second-tier tests is a big challenge for a successful NBS program. Advances in hardware, software, sensitivity, and automation of analytical instruments have resulted in the proliferation of potential new NBS tests in recent years. Furthermore, improvements in treatments such as bone-marrow transplantation, gene therapy, and enzyme replacement, as well as newer pharmacological approaches such as chaperone therapy and read-through of premature stop codons have raised the status of some disorders as candidates for NBS. NBS tests have been proposed for lysosomal storage disorders, Duchenne muscular dystrophy, Wilson’s disease, and severe combined immunodeficiencies, to name a few. While pilot projects have demonstrated the potential of these tests, it remains to be evaluated how effectively they can be applied in most NBS centers and how effective the newer treatments are. Each new test adds an additional complexity to the NBS program, and some of the treatments are both expensive and long-term.
What is your main field of research?
I work at Meyer Children’s University Hospital of Florence and my main field of research is newborn screening. My group consists of about 15 researchers, among them chemists, pharmacologists, biologists, and technicians. All of them work both in routine diagnostics and research. The main part of our research activity is directed toward finding new biomarkers and/or new tests for NBS.
In the last few years, for example, we studied how we could solve the problem of false-positive and false-negative results in the detection of tyrosinemia type I in NBS programs. It is an autosomal recessive disorder that is due to a deficiency of fumarylacetoacetic hydrolase, the last enzyme in the tyrosine metabolism. This condition, if not treated, is characterized by severe liver failure and renal and neurological impairment. The availability of an effective treatment has increased the need to improve early detection and has made this disease an eligible candidate for newborn screening by tandem mass spectrometry.
Up to a few years ago, tyrosine had been the common marker for tyrosinemia type I, causing many false-positive and false-negative results. Indeed a great number of affected infants have normal tyrosine levels at the time of the routine heel stick for newborn screening. For this reason, some authors have concluded that tyrosine is not a good marker for detecting tyrosinemia type 1. Others, in spite of the availability of an effective treatment, eliminated tyrosinemia type I from newborn screening panels. In other programs second-tier testing that utilized succinylacetone (SA), a specific pathognomonic marker for this disease, has been introduced to avoid excessive recalls. These methods converted and extracted SA by adding hydroxylamine hydrochloride or dansylhydrazine.
However, false-negative results cannot be avoided by this approach because most cases of tyrosinemia type I, with tyrosine levels within the normal ranges, would not qualify for a second-tier test. For this reason, we studied a modified protocol devised to incorporate SA testing into a classic amino acid (AA) and acylcarnitine (AC) screening program by adding hydrazine to the DBS extractive solution. A screening method that incorporated SA into the metabolite panel would save time and also eliminate false-negatives. This novel approach is now used in many countries all around the world.
In 2011 and 2014 two new inexpensive methods capable of quantifying some purines along with other metabolites were developed by our group. The measurements of these new metabolites allow the detection of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies at birth. ADA and PNP are fatal autosomal recessive forms of SCID caused by an inherited disorder of purine metabolism. Both ADA and PNP-SCID comply with all the criteria for inclusion in a newborn screening program. The Secretary’s Advisory Committee on Heritable Disorders in Newborns and Children in 2010 and the EU tender in 2011 recommend the inclusion of all SCIDs in the newborn screening panels. Since 2008 a polymerase chain reaction (PCR)-based test is available for the detection of all T-SCIDs. This method presents the indubitable advantage of screening for the entire panel of T-SCIDs but, with respect to MS, it is quite expensive as RT-PCR machines, kits, and molecular biologists are needed. Moreover, it has been recently demonstrated that it is not sensitive to late/delayed ADA-SCID forms and that it can detect some disorders not covered by the NBS worldwide approved criteria.
What is most fascinating for you about your job or research?
I believe that applied research and discoveries are fascinating. Working in a pediatric hospital gives us different challenges every day. We have new clinical problems to solve, new biochemical pathways to study, new biomarkers to discover, and new tests to develop very often. It is not so easy to explain, but my collaborators and I have the chance to utilize our knowledge in chemistry and technology every day, even if only to offer our contribution to stating whether or not a newborn is affected or not affected. This is both a big responsibility and a big honor for us.
What do you do in your spare time?
My job is really time consuming, therefore, I love to spend my leisure time with family, and when possible I like to swim and read books.
Thank you very much for the interview.
Giancarlo la Marca, born 1974 in Manfredonia (FG), Italy, graduated summa cum laude in Pharmaceutical Chemistry and Technology from the University of Florence, Italy, in 2000 and received a specialization in Hospital Pharmacy, also summa cum laude, from the University of Florence, Italy, in 2005. In 2001, la Marca moved to the Mass Spectrometry Lab in the Newborn Screening Centre of Meyer Children’s Hospital, Florence, where he coordinated the pilot project on expanded newborn screening.
Since 2004 he has coordinated the Expanded Newborn Screening in the Tuscany Region, since 2006 the Expanded Newborn Screening for the Umbria Region, and in 2012 the Expanded Newborn Screening for the Sardinia Region. La Marca is a member of the Italian Society for Newborn Screening, the Italian Society for the Study of the Inborn Errors of Metabolism, the Italian Chemical Society, and the Italian Society of Pharmacology. He is a member of the Directive Council of the Mass Spectrometry Centre, University of Florence, Italy. He has authored more than 80 peer-reviewed publications dealing with areas of pharmacology, clinical chemistry, mass spectrometry, and pediatrics.
- Porcellino Prize 2011 by City of Florence for research activity in pediatric preventative medicine
- Young Researchers Prize 2012 Meyer Hospital/University of Florence
- Paul Harris Prize 2012, for research in pediatric preventive medicine
- “Excellence of the Year” prize 2013
- Young Researchers Prize 2013: For his contribution in the discovery of new methods for the early diagnosis of ADA-SCID
- Rapid 2nd-Tier Test for Measurement of 3-OH-Propionic and Methylmalonic Acids on Dried Blood Spots: Reducing the False-Positive Rate for Propionylcarnitine during Expanded Newborn Screening by Liquid Chromatography–Tandem Mass Spectrometry,
G. la Marca, S. Malvagia, E. Pasquini, M. Innocenti, M. A. Donati, E. Zammarchi,
Clin. Chem. 2007, 53, 1364–1369.
- Progress in Expanded Newborn Screening for Metabolic Conditions by LC–MS/MS in Tuscany: Update on Methods to Reduce False Tests,
G. la Marca, S. Malvagia, B. Casetta, E. Pasquini, M. A. Donati, E. Zammarchi,
J. Inherited Metab. Dis. 2008, 31, 395–404.
- New Strategy for the Screening of Lysosomal Storage Disorders: The Use of the Online Trapping-and-Cleanup Liquid Chromatography/Mass Spectrometry,
G. la Marca, B. Casetta, S. Malvagia, R. Guerrini, E. Zammarchi,
Anal. Chem. 2009, 81, 6113–6121.
- Neonatal Screening for Severe Combined Immunodeficiency Due to Adenosine Deaminase Defect: A Reliable and Inexpensive Method Using Tandem Mass Spectrometry,
C. Azzari, G. la Marca, M. Resti,
J. Allergy Clin. Immunol. 2011, 127, 1394–1399.
- LC–MS/MS Method for Simultaneous Determination on a Dried Blood Spot of Multiple Analytes Relevant for Treatment Monitoring in Patients with Tyrosinemia Type I,
G. la Marca, S. Malvagia, S. Materazzi, M. L. Della Bona, S. Boenzi, D. Martinelli, C. Dionisi-Vici,
Anal. Chem. 2012, 84, 1184–1188.
- Tandem Mass Spectrometry, but Not T-Cell Receptor Excision Circle Analysis, Identifies Newborns with Late-Onset Adenosine Deaminase Deficiency,
G. la Marca, C. Canessa, E. Giocaliere, F. Romano, M. Duse, S. Malvagia, F. Lippi, S. Funghini, L Bianchi, M. L. Della Bona, C. Valleriani, D. Ombrone, M. Moriondo, F. Villanelli, C. Speckmann, S. Adams, B. H. Gaspar, M. Hershfield, I. Santisteban, L. Fairbanks, G. Ragusa, M. Resti, M. de Martino, R. Guerrini, C. Azzari,
J. Allergy Clin. Immunol. 2013, 131, 1604–1610.
- Diagnosis of Immunodeficiency Caused by a Purine Nucleoside Phosphorylase Defect by Using Tandem Mass Spectrometry on Dried Blood Spots,
G. la Marca, C. Canessa, E. Giocaliere, F. Romano, S. Malvagia, S. Funghini, M. Moriondo, C. Valleriani, F. Lippi, D. Ombrone, M. L. Della Bona, C. Speckmann, S. Borte, N. Brodszki, A. R. Gennery, K. Weinacht, F. Celmeli, J. Pagel, M. de Martino, R. Guerrini, H. Wittkowski, I. Santisteban, P. Bali, A. Ikinciogullari, M. Hershfield, L. D. Notarangelo, M. Resti, C. Azzari,
J. Allergy Clin. Immunol. 2014, 134, 155–159.