This study aimed to determine if MALDI-TOF mass spectrometry (MALDI-TOF-MS) of DNA from dried blood spots (DBS) could detect the hemizygous 22q11.2 deletion associated with DiGeorge syndrome in newborns. Samples from 54 patients with 22q11DS and 100 controls were analyzed using PCR and MALDI-TOF-MS. A cutoff value of 0.7 correctly identified all patients with the deletion with 100% sensitivity and specificity. The assay requires only a small amount of DNA and could potentially be added to newborn screening to detect this and other genetic mutations. However, full clinical validation in larger populations is still needed before applying to routine new

1. Introduction
Methods
Discussion
Conclusions
References
MALDI-TOF-MS Assay to Detect the Hemizygous 22q11.2 Deletion
in DNA from Dried Blood Spots
Meredith Herman, Monica Hessler, Jonathan Horton, Abigail Howard, Emily Hubbard, Tyler Jackson, Darek Kalisz, Amanda Kucharzyk, Colar Kuhns, Chelsea Lentz, Caitlyn Libiran, Jacob Malsch, Melissa Manning
Results
• DiGeorge Syndrome is a congenital defect caused
by deletions at site 22q11.2.
• Screening in newborns currently includes FISH and
a genome-wide comparative genomic hybridization
assay.
• DBS and MALDI-TOF-MS method is much faster
• Can DBS and MALDI-TOF-MS replace FISH and
hybridization assays in detecting DiGeorge
Syndrome?
• Samples collected included 54 patients previously
diagnosed with 22q11DS, 100 control samples, all
prepared as DBS.
• PCR Targets: UFD1L (sequence within deleted
region) and chromosome 18 (reference sequence)
were selected.
• Two separate extension primers were created with a
single nucleotide difference (SND*1 and SND*2).
• Target and reference sequences from patient DBS
amplified through PCR and extended.
• Extension products analyzed on MSArray workstation
with MALDI-TOF mass spectrometer.
• MassArray Typer 4.0 was used to analyze the data to
determine the genotyping call for each sample.
• MALDI-TOF-MS analysis generated by a spectrogram in signal peaks indicative of oligonucleotide mass
and signal intensity
• For each sample, the ratio of peak intensities for each SND from the target sequence (22q11DS from
chromosome 22) to that of the reference sequence (chromosome 18) was used.
• Healthy controls were expected to have a ratio of 1.0 (2:2)
• Hemizygous deletions were expected to have a ration of 0.5 (1:2)
• Initial experiment with 2 separate cohorts of 10 CB-DBS and 10 22q11DS DBS yielded good internal
consistency:
• There was no overlap between ratios of the controls or the affected 22q11DS in any run
• A larger sample size encompassing 100 CB-DBS and 54 22q11DS-DBS patients was used to test the
validity of the assay:
• A cutoff value of 0.7 showed 100% diagnostic sensitivity and specificity, correctly identifying all patients
with the deletion
• Only a small amount of DNA and single set of
primers for the target and reference genes is
required.
• Correctly detected all 22q11DS samples from the
control samples.
• Assay multiplexing can occur at the PCR stage
or after PCR has been completed.
• Use of an internal genomic sequence as the
competitor ensures the ratio between target and
competitor is in the optimal range.
• MALDI-TOF-MS could be added to newborn
screening to correctly detect hemizygous deletions
(DiGeorge Syndrome).
• MALDI-TOF-MS has a future in helping detect
other mutations in newborn screening.
• Full clinical validation of the MALDI-TOF-MS
assay will require testing large populations before
it can be applied to routine newborn screening.
• Kobrynski, Lisa J., Golriz K. Yazdanpanah, Deborah Koontz, Francis K. Lee,
and Robert F. Vogt. "MALDI-TOF-MS Assay to Detect the Hemizygous
22q11.2 Deletion in DNA from Dried Blood Spots." Clinical Chemistry 62.1
(2016): 287-92. Web
• Singhal N, Kumar M, Kanaujia PK and Virdi JS (2015) MALDI-TOF mass
spectrometry: an emerging technology for microbial identification and
diagnosis. Front. Microbiol. 6:791. doi: 10.3389/fmicb.2015.00791
CB-DBS Median peak ratio 22q11DS
Cohort # 1
SND*1 1.06 0.55
SND*2 0.98 0.56
Cohort # 2
SND*1 1.15 0.60
SND*2 0.89 0.51
SND*1 SND*2
CB-DBS 0.96 0.99
22q11DS patients 0.36 0.53
Methods Advantages Disadvantages
FISH (Fluorescent
in situ
Hydridization)
-Rapid detection and
identification directly from
slide smears
-Fast and ease-of use of
conventional staining
methods combined with
specificity of molecular
methods
- Test limited by the
availability of specific
antigens for detection
MALDI-TOF MS -Fast
-Accurate
-Less expensive than
molecular and
immunological-based
detection methods
-Trained laboratory
personnel not required
- High initial cost of the
MALDI-TOF equipment
Figure 1: Spectrogram from MALDI-TOF-MS analysis. 4 distinct peaks were identified on all tested samples from Chromosome 22 and
Chromosome 18 (2 each)T=target sequence, R= reference sequence
Table 1: CB-DBS vs. 22q11DS Median peak ratios of target : reference sequence between 2 cohorts
Table 2: Median peak ratios of target : reference sequence
Table 3: Comparative methods of chromosomal deletions