Mayo Medical Laboratories offers Supplemental Newborn Screening for Inherited Metabolic Disorders (Test Code #82594)

Supplemental newborn screening has the benefits of signaling the need for early treatment in the approximate one in 4,000 babies who have a metabolic disorder detectable by tandem mass spectrometry screening. This expanded screening program will test at birth for more than 30 disorders and allow for the initiation of treatment before the infant suffers lasting harm.

Introduction

Laboratory analysis of blood spots dried on filter paper for the purpose of newborn screening plays an important role in preventive medicine. Only a handful of disorders were screened for during its first 30 years of existence. This situation has dramatically changed since the 1990s when tandem mass spectrometry (MS/MS) was introduced into a few newborn screening laboratories worldwide. MS/MS can detect more than 30 additional disorders by simultaneous acylcarnitine and amino acid analyses in a single blood spot (Table 1). These disorders are caused by genetically determined (inherited) defects in protein and fatty acid metabolism. Affected children typically do not appear ill at birth. Symptoms usually manifest acutely during the first year of life, but onset in adulthood has also been described. These acute metabolic decompensations can result in severe long-term complications and often death. The ability to identify affected newborns before the onset of symptoms can dramatically improve the prognosis of most patients.

Brief History of Newborn Screening

Before newborn screening became available, PKU due to a genetic defect of phenylalanine hydroxylase was typically not diagnosed before six months of life when developmental delay or other non-specific neurological symptoms become apparent. Treatment based on a phenylalanine-restricted diet became available in the 1950s. Already incurred neurologic damage, however, could not be reversed but only mitigated. To allow for the presymptomatic identification of PKU patients and timely initiation of dietary intervention, a simple method for the measurement of phenylalanine in blood spots dried on filter paper was developed by Dr. Robert Guthrie. This test was a bacterial inhibition assay. Dried blood spots are placed on agar plates containing a strain of Bacillus subtilis that requires phenylalanine for growth. The agar also contains beta-2-thienylalanine, a phenylalanine analogue that prevents bacterial growth. Only when phenylalanine is present in abnormally high amounts in the serum spot the analogue's action is overcome and the bacterial colonies grow, which is easily detectable. Once this assay was established, newborn screening first began in some regions of New England in 1961 and rapidly spread around the world as the Guthrie Test.

A few additional disorders such as congenital hypothyroidism, galactosemia, and sickle cell disease were also soon added to many newborn screening programs. As of the early 1990s then, the introduction of MS/MS enabled an expansion of newborn screening programs to include more than 30 disorders. However, because the number of disorders screened for in a newborn screening program is at the discretion of each state, a significant discrepancy exists between states, the minimum number of disorders being three and the maximum 34. While individually rare, the incidence for any of the detectable disorders is 1 in 4,000 newborns with Medium-Chain Acyl-Coenzyme A Dehydrogenase (MCAD) deficiency being more frequent than PKU at 1 in 12,000 vs. 1 in 20,000 newborns respectively.

Tandem Mass Spectrometry (MS/MS)

Tandem mass spectrometers allow the rapid analysis of individual compounds in complex mixtures. Two mass spectrometers are coupled, separated only by a collision cell, all within one instrument. In general, the first (MS1) or second analyzer (MS2) can be set either to scan a mass range or to select one or more individual ions of a specific mass-to-charge ratio (m/z). The collision cell is utilized to further breakdown the ions by use of a neutral gas (e.g. nitrogen) and to transmit them to MS2 (Figure). This design enables four different analytical or scan modes, three of which are being used by Mayo's Biochemical Genetics Laboratory for the purpose of newborn screening (Table 2).

For acylcarnitine profiling, MS1 scans a defined mass range and MS2 is set to transmit only fragment ions with a specific m/z value following collision-activated fragmentation. In this mode, the data system then correlates each detected ion to its respective precursor scanned in MS1 (precursor or parent ion scan). In a neutral loss experiment MS1 and MS2 are both scanned at the same rate with a constant m/z difference. The resulting spectrum includes only those compounds, among precursor ions, that fragment with a common neutral loss (a behavior indicating that they belong to a family of structurally related compounds. This scan is used for the generation of amino acid profiles.

The concentrations of a few, selected amino acids and acylcarnitine species are more accurately measured when taking advantage of an MS/MS analysis in selected reaction monitoring (SRM) mode, where the selection of a parent ion in MS1 is followed by a similar process for a specific fragment ion in MS2. The resulting signal corresponds exclusively to the transition from parent to product ion, a process virtually free of any interference irrespective of the specimen analyzed.

Newborn Screening and Mayo's Biochemical Genetics Laboratory

Historically, testing for inborn errors of metabolism (IEM) has been provided by research laboratories, each offering analyses only for disorders in line with their scientific interest. With increasing awareness of genetics in medicine and hundreds of IEMs identified to date, Clinical Biochemical Genetics is now recognized as a laboratory discipline concerned with the evaluation and diagnosis of patients and families with inherited metabolic disease, monitoring of treatment, and distinguishing heterozygous carriers from non-carriers by metabolite and enzymatic analysis of physiological fluids and tissues.

The Biochemical Genetics Laboratory (BGL) at Mayo is an interdisciplinary group of laboratorians, geneticists, and pediatricians. The mission of BGL is to provide biochemical testing and result interpretation of the highest quality for the diagnosis, study, and clinical care of patients with inborn errors of metabolism, high risk of cardiac disease, malabsorption, and malnutrition disorders. The BGL routinely performs qualitative detection and quantitative determination of diagnostic markers based on a variety of manual, automated, and chromatographic methods, including MS/MS.

In 2001, more than 400,000 tests were performed using over 159 different procedures, the majority of tests aimed to the biochemical diagnosis of IEM. Several of these procedures are screening tests, such as the analyses of amino acids, organic acids and acylcarnitines, and are performed on specimens from patients presenting with symptoms reminiscent of an IEM (high-risk screening), postmortem samples obtained primarily from children that have died suddenly without apparent reason (postmortem screening), as well as for the purpose of prenatal diagnosis.

While there have always been discrepancies between the various state-mandated newborn screening programs, the inconsistent integration of MS/MS has widened the gap. With proven expertise and following the formation of a collaborative agreement with Neo Gen Screening, Inc., an independent laboratory with experience in high-throughput newborn screening using MS/MS, Mayo's Biochemical Genetics Laboratory bridges the gaps in state screening programs by offering the Mayo Supplemental Newborn Screening Program (Unit Code: 82954) to interested hospitals. In addition, Mayo Medical Laboratories offers the benefit of a full spectrum of follow-up testing. Although the sensitivity and specificity of MS/MS in the identification of a detectable disorder is excellent and its positive predictive value higher than that of other technologies, it remains a screening test that requires further testing to confirm a disorder (usually on a sample other than a blood spot).

Newborn Screening by MS/MS and Reporting of Results

The quantitative measurements of the various amino acids and acylcarnitines support the interpretation of the complete profile but are not diagnostic by themselves. The interpretation is by pattern recognition. This concept sets apart a biochemical genetics labarotory from conventional clinical chemistry and most newborn screening laboratories that are used to provide quantitative results without interpretation.

Abnormal results of a screening test are typically not sufficient to conclusively establish a diagnosis of a particular disease. To verify a preliminary diagnosis, independent biochemical (i.e., in vitro enzyme assay) or molecular genetic analyses are required, many of which are offered by the Mayo Clinic. As opposed to other screening laboratories, we do not recommend sending another blood spot for follow up of abnormal results because normal blood spot values for newborns older than one week are generally not available and important time in the diagnostic process may be lost to the detriment of affected children.

The reports from Mayo's Biochemical Genetics Laboratory will be in text form only and values for the more than 60 analytes and analyte ratios will not be provided when the screening result is normal.

A report for an abnormal screening result will include:

• A quantitative result of the abnormal metabolites,
• A detailed interpretation of the results, including an overview of the results' significance, possible differential diagnoses, recommendations for additional biochemical testing and confirmatory studies (enzyme assay, molecular analysis), name and phone number of contacts at the Mayo Clinic, and
• A phone number for one of the laboratory directors if the referring physician has additional questions.
  

Future Enhancement of Newborn Screening Programs

The BGL is continuously developing new methods, which allow for either more accurate diagnoses or further expansion of the spectrum of identifiable disorders. This includes the testing of newborn blood spots. For example, screening for congenital adrenal hyperplasia (CAH) is in place in 37 states at this time. The remaining states do not screen for this inherited endocrinopathy because the available immunoassays (ie, fluorescent immunoassay) for the determination of 17-alpha-hydroxy progesterone (17OHP) have a poor positive predictive value causing approximately 200 false positives for each truly abnormal result. This translates into a significant financial burden on the health care system and, more importantly, unnecessary blood draws for the predominantly premature babies and emotional stress for the involved families.

To overcome this unfavorable situation, the BGL in collaboration with Mayo's Endocrine Laboratory recently developed a new MS/MS based method for the determination of 17OHP and other relevant steroids. Retrospective testing of more than 700 blood spots indicates an improvement of the positive predictive value from 0.5% to higher than 50%. This steroid analysis will soon be included in Mayo's Supplemental Newborn Screening Program (SNS).

In the future, additional inherited diseases (i.e., congenital disorders of glycosylation) that can be diagnosed by mass spectrometry methods will become part of Mayo's SNS.

Table 1. Disorders Detectable by Newborn Screening Using MS/MS

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Effectiveness of early treatment	Risk for acute crisis	Effectiveness of MS/MS
			Disorder
Disorders of amino acid metabolism
PKU	+++	-	+
Other hyperphenylalaninemias	+	-	+
MSUD	+++	+	+
Homocystinuria	+++	-	(+)
Tyrosinemia type I	+++	-	(+)
Tyrosinemia type II	+++	-	(+)
Citrullinemia	+++	+	+
ASA	+++	+	+
Argininemia	+++	-	(+)
Disorders of organic acid metabolism
GA-1	+++	+	+
Propionic acidemia	+++	+	+
Methylmalonic acidemias	+++	+	+
Isovaleric acidemia	+++	+	+
3-MCC deficiency	+++	+	+
3-MGH deficiency	?	+/-	+/-
Multiple carboxylase deficiency	+++	+/-	+/-
2-MBDH deficiency	?	?	+
Disorders of fatty acid metabolism
Carnitine transport defect	+++	+	+/-
CPT-1 deficiency (liver)	+	+	(+)
CACT deficiency	+	+	+
CPT-2 deficiency
neonatal onset	-	+	+
late onset	+	-	+
VLCAD deficiency	+	+	(+)
LCHAD deficiency	+	+	(+)
TFP deficiency	+	+	(+)
MCAD deficiency	+++	+	+
MCKAT deficiency	+	+	(+)
M/SCHAD deficiency	+	+	(+)
SCAD deficiency	+	+	+
Functional SCAD deficiency	?	+	+
SCHAD deficiency (muscle)	+	+	-
SCHAD deficiency (fibroblasts)	+	+	+/-
SCHAD deficiency (liver)	+++	+	(+)
SKAT deficiency	+++	+	(+)
ETF & ETF-QO deficiency (GA-2)
neonatal onset	-	+	+
late onset	+	+	(+)
Riboflavin responsive form(s) (GA-2)	+++	+	(+)
2,4-Dienoyl-CoA reductase deficiency	?	?	(+)
HMG-CoA synthase deficiency	+	+	-
HMG-CoA lyase deficiency	+	+	+
from: Matern D. Endocrinologist 2002;12:50-57

Legend

Abbreviations are as follows, in alphabetical order: 2MBDH, 2-methylbutyryl-CoA dehydrogenase; 3-MCC, 3-Methylcrotonyl-CoA carboxylase; 3MGH, 3-methylglutaconyl-CoA hydratase; ASA, Argininosuccinic aciduria; CACT, carnitine acylcarnitine translocase; CPT, carnitine palmitoyltransferase; ETF, electron transfer flavoprotein; ETF-QO, electron transfer flavoprotein ubiquinone-oxidoreductase; GA-1, glutaric acidemia type I; GA2, glutaric acidemia type II; HMG, 3-hydroxy 3-methylglutaryl; LCHAD, long-chain L-3-hydroxy acyl-CoA dehydrogenase; MCAD, medium-chain acyl-CoA dehydrogenase; MCKAT, medium-chain 3-ketoacyl-CoA thiolase; M/SCHAD, medium/short chain L-3-hydroxy acyl-CoA dehydrogenase; MSUD, maple syrup urine disease; PKU: phenylketonuria; SCAD, short-chain acyl-CoA dehydrogenase; SCHAD, short-chain L-3-hydroxy acyl-CoA dehydrogenase; SKAT, short-chain 3-ketoacyl-CoA thiolase (3-ketothiolase); TFP, trifunctional protein; VLCAD, very long-chain acyl-CoA dehydrogenase.

Effectiveness of treatment: +++, demonstrated; +, dietary and preventive measures, -, no effective treatment; §, liver transplantation; ?, insufficient information.

Risk for acute crisis: +, life-threatening metabolic crisis can occur at any age in untreated patients; -, symptoms usually do not present acutely; +/-, not all patients reported presented with acute symptoms; ?, number of patients reported is too small to allow reliable assessment.

Effectiveness of MS/MS: +, demonstrated in blood spots; (+), expected to be effective, but not yet conclusively demonstrated; +/-, questionable effectiveness; -, not effective

Basic scheme of an MS/MS analysis

Table 2. Scan Modes for MS/MS Analysis

Scan		Purpose
Product ion		Detection of all fragment ions originating from a single precursor
Selective reaction monitoring (SRM)		Detection of a specific fragment ion originating from a single precursor
Precursor ion		Detection of all precursors sharing a common fragment ion
Neutral loss		Detection of all precursors sharing a common neutral fragment