Implementation of second-tier tests in newborn screening for the detection of vitamin B12 related acquired and genetic disorders: results on 258,637 newborns

Abstract

Background Alteration of vitamin B12 metabolism can be genetic or acquired, and can result in anemia, failure to thrive, developmental regression and even irreversible neurologic damage. Therefore, early diagnosis and intervention is critical. Most of the neonatal cases with acquired vitamin B12 deficiency have been detected by clinical symptoms and only few of them trough NBS programs. We aim to assess the usefulness of the second-tier test: methylmalonic acid (MMA), methylcitric acid (MCA) and homocysteine (Hcys) in our newborn screening program and explore the implications on the detection of cobalamin (vitamin B12) related disorders, both genetic and acquired conditions. Methods A screening strategy using the usual primary markers followed by the analysis of MMA, MCA and Hcys as second tier-test in the first dried blood spot (DBS) was developed and evaluated. Results During the period 2015–2018 a total of 258,637 newborns were screened resulting in 130 newborns with acquired vitamin B12 deficiency (incidence 1:1989), 19 with genetic disorders (incidence 1:13,613) and 13 were false positive. No false negatives were notified. Concerning the second-tier test, the percentage of cases with MMA above the cut-off levels, both for genetic and acquired conditions was very similar (58% and 60%, respectively). Interestingly, the percentage of cases with increased levels of Hcys was higher in acquired conditions than in genetic disorders (87% and 47%, respectively). In contrast, MCA was high only in 5% of the acquired conditions versus in 53% of the genetic disorders, and it was always very high in all patients with propionic acidemia. Conclusions When screening for methylmalonic acidemia and homocystinuria, differential diagnosis with acquired vitamin B12 deficiency should be done. The results of our strategy support the inclusion of this acquired condition in the NBS programs, as it is easily detectable and allows the adoption of corrective measures to avoid the consequences of its deficiency.

Screening for Methylmalonic and Propionic Acidemia: Clinical Outcomes and Follow-Up Recommendations

“…In our data, there was a wide, overlapping range of C3 concentrations in both genetic and acquired vitamin B12 deficiency cases, with some of the highest values found in newborns determined to be unaffected. Two recent papers substantiate our finding that C3 is not a specific marker for disorders of propionate metabolism [ 13 , 33 ]. Within our data, there was also significant overlap in the C3/C2 values between IEM and false positive cases, which differed from the findings of other groups, who have suggested that C3/C2 provides better sensitivity and is more frequently high in genetic rather than acquired conditions [ 33 , 34 ].…”

“…Two recent papers substantiate our finding that C3 is not a specific marker for disorders of propionate metabolism [ 13 , 33 ]. Within our data, there was also significant overlap in the C3/C2 values between IEM and false positive cases, which differed from the findings of other groups, who have suggested that C3/C2 provides better sensitivity and is more frequently high in genetic rather than acquired conditions [ 33 , 34 ]. Our data also show that the methylmalonic acid (MMA) concentration obtained on the second-tier test does not help to distinguish an IEM from acquired disease.…”

“…To our knowledge, no additional false negative cases were reported to the metabolic specialists at either of the two centers in Wisconsin during the study’s time frame. Even lower thresholds for C3 were proposed in the recent paper by Pajares et al to ensure identification of all IEM, making second tier testing all the more crucial to avoid an increase in false positive screens [ 33 ].…”

Spectrum Analysis of Inherited Metabolic Disorders for Expanded Newborn Screening in a Central Chinese Population

“…Methylmalonic acid, methylcitric acid and homocysteine are widely known biomarkers of genetic conditions leading isolated or combined MMA and HCY, or PA. However, elevations of methylmalonic acid, methylcitric acid and homocysteine can also be the result of secondary alterations, such as secondary vitamin B12 deficiency ( Pajares et al, 2021 ). Therefore the second-tier test of methylmalonic acid, methylcitric acid and homocysteine is necessary to differentially diagnose the secondary vitamin B12 deficiency when we screening for MMA and HCY.…”

Expanded Newborn Screening in Italy Using Tandem Mass Spectrometry: Two Years of National Experience

“…Remarkly, CblC showed an incidence of 1: 32,271, making this disease one of the most common inherited metabolic diseases in the Italian population, with one of the highest prevalences in the world. The reported incidence in Portugal was 1:85,000 [ 22 ], in Spain and, in the US, 1:100,000 [ 25 , 26 ], while a pilot study in Beijing, China showed the worldwide highest incidence of 1:11,730 [ 27 ]. Defining the mutation spectrum in CblC cases was beyond the scope of this paper.…”