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 22q11DSCohort # 1    

SND*1 1.06 0.55SND*2 0.98 0.56

Cohort # 2    SND*1 1.15 0.60SND*2 0.89 0.51

  SND*1 SND*2CB-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