ROZMARIC Tomaz

Detection of Vitamin B12 Deficiency in Newborns

Early diagnostics and treatment are crucial in preventing irreversible neurological damage caused by vitamin B12 deficiency (B12-D), which is most probably strongly underdiagnosed in Austria. In newborns, B12-D is indicated by elevated propionylcarnitine or altered methionine values in the screening for organic acidurias and different forms of homocystinurias, as performed by the regular newborn screening program in Austria with a considerable number of false-positive results. Though B12-D screening may be justified due to fulfilling of the Wilson and Jungner criteria defining conditions that should be screened for, it is rarely listed in regular newborn screening panels and mostly regarded as a secondary condition or incidental finding.

The aim of this study is to provide the basis for a newborn screening strategy directly targeting B12-D that is independent of technically demanding and expensive mass-spectrometric methods. The underlying hypothesis is that epigenetic alterations in peripheral blood of newborns can be utilized to identify vitamin B12-deficient newborns.

The first step of the intended project aims at implementation and evaluation of screening for B12-D from dried blood spots of newborns. The screening will be based on adaption of published strategies of a second-tier measurement (i.e. a second test from same dried blood spots suspicious in primary parameters) of total homocysteine (tHCY) using high pressure liquid chromatography-coupled tandem mass spectrometry (HPLC-MS). Frequency of detected cases as well as the positive predictive value will be compared to data published from other screening labs as well as to the conventional one-tier strategy currently performed in the Austrian newborn screening to optimize decision criteria for recalls of suspicious cases. In addition, the possibility and value of measurement of an additional parameter, methylmalonic acid, from dried blood spots, will be assessed. As the next step, the methylome of the first dried blood spot card of newborns diagnosed with profound B12-D will be compared to that of newborns with normal metabolic values. The genome-wide methylation profile will be performed by reduced representation bisulfite sequencing (RRBS). By using bioinformatical analysis, sites with most significant differences between the two groups will be identified and selected for the feasibility to quantify these differences by a technically simple and commercially feasible method such as methylation-specific quantitative real-time PCR. Finally, this method will be evaluated for its capability to identify children at risk of B12-D.

The study will be performed in the research lab of the Austrian Newborn Screening in collaboration with Goran Mitulović, Clinical Institute of Laboratory Medicine, Gerda Egger, Clinical Institute for Pathology and Christoph Bock, Biomedical Sequencing Facility, CeMM.

Methods and Skills:

Mass spectrometry; liquid chromatography; NGS sequencing

Publications:

Kovač J, Šutuš Temovski T, Rozmarič T, Horvat S, Beltram J, Trebušak Podkrajšek K, Battelino T, Kotnik P. DEPTOR promoter genetic variants and insulin resistance in obese children and adolescents. Pediatr Diabetes 18: 152-158, 2017