General information
The SALSA MLPA
Probemix ME033 TNDM is a
research use only (RUO) assay for the detection of aberrant methylation of one or more sequences of the
PLAGL1 gene on the 6q24 chromosomal region. This probemix can also be used to detect deletions/duplications in the
PLAGL1 gene; the chromosomal regions 6p22 and 6q24, as well as the chromosomal region 11p15.
Genomic imprinting is the monoallelic expression of genes, dependent on the parental origin of the chromosome. It plays a role in growth and development. Imprinting disorders like Transient Neonatal Diabetes Mellitus (TNDM) originate from a disturbance in this monoallelic expression by disruption or epimutation of imprinted genes (Ishida et al. 2013).
TNDM is a form of diabetes that occurs in infants and is characterised by severe intra-uterine growth retardation, hyperglycemia, dehydration and absence of ketoacidosis.
Three different genetic mechanisms have been described as major causes of TNDM (Temple et al. 2005) (also see Figure 1): paternal uniparental disomy of chromosome 6 (pUPD6) (~40% of TNDM cases), duplication of the 6q24 paternal allele (~30% of TNDM cases), and hypomethylation of the maternal
PLAGL1 differentially methylated region (DMR) (
PLAGL1:alt-TSS-DMR) (~30% of TNDM cases). In this last group, hypomethylation can result from either an isolated imprinting variant (only affecting the
PLAGL1:alt-TSS-DMR), or as part of multi-locus imprinting disturbances (MLIDs). Approximately half of the TNDM-MLID cases are due to a defect in the
ZFP57 gene.
In order to detect the majority of TNDM cases, several
PLAGL1-specific probes, of which four methylation-specific probes targeting the
PLAGL1:alt-TSS-DMR, as well as other probes targeting the 6q24 region, have been included in ME033-B1. Additionally, five
ZFP57-specific MLPA probes detect copy number changes of
ZFP57.
Copy number probes for two other genes are included in this probemix because of their involvement in TNDM:
INS and
KCNJ11 (11p15). Recessive loss of function mutations in the
INS gene have been reported in several patients with TNDM (Støy et al. 2021), whereas activating mutations in
KCNJ11 have been reported as a possible cause of TNDM (Gloyn et al. 2006). Additionally, probes for
ZC2HC1B (6q24, downstream of
PLAGL1),
SF3B5 (6q24, upstream of
PLAGL1) and several other genes in the flanking region are included to determine copy numbers of the 6q24 chromosomal region.
More information is available at
https://www.ncbi.nlm.nih.gov/books/NBK1534/.
This SALSA MLPA probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes.
Probemix content
The SALSA MLPA Probemix ME033-B1 TNDM contains 43 (methylation-specific) probes with amplification products between 124 and 445 nucleotides (nt). 21 probes target the 6q24 chromosomal region, of which ten target the
PLAGL1 gene. Four of the probes targeting
PLAGL1 are methylation-specific probes which contain an HhaI recognition site and provide information on the methylation status of the
PLAGL1:alt-TSS-DMR. In addition, there are five probes targeting the
ZFP57 gene on 6p22 and five probes targeting
INS and
KCNJ11 on 11p15. All probes present will also give information on copy number changes in the analysed sample. In addition, ten reference probes are included that are not affected by HhaI digestion and detect genes located outside the
PLAGL1 gene and the 6p22, 6q24 and 11p15 chromosomal regions. Also, two digestion control probes are included in this probemix indicating whether or not restriction endonuclease digestion in the MS-MLPA reaction was complete. Complete probe sequences and the identity of the genes detected by the reference probes are available online (
www.mrcholland.com).
This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MS-MLPA General Protocol and online at
www.mrcholland.com.