General Information: The SALSA MS-MLPA Probemix ME-033 TNDM is a research use only (RUO) assay for the detection of aberrant methylation of one or more sequences of the PLAGL1 gene on chromosomal region 6q24. This probemix can also be used to detect deletions/duplications in the PLAGL1 gene; the chromosomal regions 6q22 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:
1. Paternal uniparental disomy of chromosome 6. This accounts for approximately ~40% of the 6q24-related TNDM cases and can be detected by an absence of methylation of the three PLAGL1 promoter region probes that have an HhaI site. These three probes target a maternally imprinted genomic area: the maternal allele is methylated, while the paternal allele is unmethylated in normal control samples. As compared to reference probes that do not contain an HhaI site, the signal of the MS-MLPA probes in imprinted regions is reduced by 50% upon HhaI digestion in DNA samples from normal individuals. While it is reduced by 100% in TNDM cases, due to the inheritance of two unmethylated paternal alleles and no imprinted maternal allele.
2. Duplication of 6q24 paternal allele. This accounts for ~30% of the 6q24-related TNDM cases and can be detected by a copy number change of the PLAGL1 specific probes and one or more other 6q24 probes.
3. Hypomethylation of the maternal PLAGL1 differentially methylated region. This accounts for approximately ~30% of the 6q24-related TNDM cases and can be detected by a methylation change of the three PLAGL1 promoter region probes that have an HhaI site. Approximately half of the hypomethylation cases are due to a defect ZFP57 gene. The four ZFP57-specific MLPA probes in ME033 detect copy number changes of ZFP57. Recessive mutations of ZFP57 have been identified in ~10% of all TNDM patients.

More information is available at https://www.ncbi.nlm.nih.gov/books/NBK1534/

Copy number probes for two other genes are included in this probemix because of their involvement in TNMD: INS (11p15.5) and KCNJ11 (11p15.1). Recessive loss of function mutations in the INS gene have been reported in several patients with TNDM, whereas activating mutations in KCNJ11 have been reported as a possible cause of TNDM. Additionally probes for ZC2HC1B (6q24.2, downstream of PLAGL1) are included to determine copy numbers of the 6q24 region.

Probemix content: The SALSA MLPA Probemix ME033-A1 TNDM contains 39 (MS-)MLPA probes with amplification products between 130 and 436 nt. Three MS-MLPA probes contain a HhaI recognition site and provide information on the methylation status of the PLAGL1 gene, specifically the PLAGL1_TSS_alt-DMR. All probes present will also give information on copy number changes in the analysed sample. In addition, 10 reference probes are included which are not affected by HhaI digestion and detect genes located outside the PLAGL1_TSS_alt-DMR region. Also, two digestion control probe(s) 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 is 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 MLPA General Protocol and online at www.mrcholland.com.
.