General information
The SALSA MLPA Probemix P357 MODY Mix 2 is a research use only (RUO) assay for the detection of deletions or duplications in the KLF11, NEUROD1, PAX4, CEL, INS, PDX1 and HNF1B genes, which are associated with Maturity-Onset Diabetes of the Young.

Maturity-Onset Diabetes of the Young (MODY) is a distinct form of non-insulin-dependent diabetes mellitus (NDDM), also known as type II diabetes. MODY has an autosomal dominant inheritance and it generally develops in individuals under 25 years of age. Approximately 5% of all diabetes patients suffer from MODY. As described in more detail below, 14 forms of MODY have now been identified with the highest prevalence for MODY 2 and 3. Each form of MODY has been associated with one gene.

This P357-A3 MODY Mix 2 probemix contains probes for the PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4 and INS genes. This probemix is therefore specific for MODY 4-10. The widely used probemix P241 MODY Mix 1 contains probes for the HNF4A, GCK, HNF1A and HNF1B genes involved in MODY types 1-3 and 5, the more frequent causes of MODY. For these genes a clear relation has been described between copy number changes of the genes and the MODY phenotype or the renal cysts and diabetes (RCAD) syndrome.

The MODY genes
• MODY 1 is a result of defects in the Hepatocyte nuclear factor-4-alpha gene (HNF4A) on chromosome 20q13. This gene comprises 12 exons and spans about 30 kb of genomic DNA. The HNF4A protein regulates the expression of HNF1A. MLPA probes for the HNF4A gene are present in the P241 probemix.

• MODY 2 is caused by mutations in Glucokinase gene (GCK) on chromosome 7p13. This gene comprises 12 exons and spans about 45 kb of genomic DNA. MLPA probes for GCK are present in the P241 probemix.

• MODY 3 is caused by defects in the HNF1 Homeobox A gene (HNF1A) which comprises 10 exons and spans 24 kb of genomic DNA on chromosome 12q24. MLPA probes for HNF1A are present in the P241 probemix.

• MODY 4 has been linked to defects in the Pancreas/Duodenum homeobox protein 1 gene (PDX1). The PDX1 gene (two exons) spans ~6 kb of genomic DNA and is located on chromosome 13q12.2, ~27 Mb from the p-telomere.

• MODY 5 have been associated with the HNF1 Homeobox B gene (HNF1B). The HNF1B gene (nine exons) spans ~59 kb of genomic DNA and is located on chromosome 17q12, ~33 Mb from the p-telomere. The HNF1B probes in this P357 probemix detect the same sequences as the HNF1B probes in P241.

• MODY 6 has been linked to defects in the neuronal differentiation 1 gene (NEUROD1). The NEUROD1 gene (two exons) spans ~4 kb of genomic DNA and is located on chromosome 2q31.3, ~182 Mb from the p-telomere.

• MODY 7 is caused by mutations in the Krüppel-Like Factor 11 gene (KLF11) on chromosome 2p25.1. The KLF11 gene (four exons) spans ~11 kb of genomic DNA and is located on chromosome 2p25.1, ~10 Mb from the p-telomere.

• MODY 8 has been associated with defects in Carboxyl-ester lipase gene (CEL). The CEL gene (11 exons) spans ~10 kb of genomic DNA and is located on chromosome 9q34.2, ~135 Mb from the p-telomere. Due to sequence similarity with the CEL pseudogene CELP, only probes for exons 2 to 7 of CEL are included. Compared to CEL, CELP lacks a 4.8-kb fragment containing exons 2 to 7. The CEL gene is located in a CNV region (http://dgv.tcag.ca/dgv/app/home). We observed duplications of this gene in DNA samples of several healthy individuals.

• MODY 9 is caused by defects in the Paired box 4 gene (PAX4). The PAX4 gene (12 exons) spans ~8 kb of genomic DNA and is located on chromosome 7q32.1, ~127 Mb from the p-telomere.

• MODY 10 has been linked to mutations in the insulin gene (INS). The INS gene (three exons) spans ~1.4 kb of genomic DNA and is located on chromosome 11p15.5, ~138 Mb from the p-telomere. Please note that INS gene mutations that cause MODY are all heterozygous missense mutations with a dominant-negative mode of action; a heterozygous deletion of the INS gene might not result in diabetes. The ME033 probemix contains INS probes that target the same sequences as the INS probes in this P357 probemix.

• MODY 11 has been linked to defects in the B lymphoid tyrosine kinase gene (BLK). BLK has 13 exons and spans about 71 kb of genomic DNA on chromosome 8p23.1. No probes for this gene are included in P241 or P357.

• MODY 12 has been linked to defects in the ATP binding cassette subfamily C member 8 gene (ABCC8). The ABCC8 gene consists of 39 exons and spans 84 kb of genomic DNA on chromosome 11p15.1. Probes for this gene are included in the P117 probemix.

• MODY 13 has been linked to defects in the potassium inwardly rectifying channel subfamily J member 11 gene (KCNJ11). The KCNJ11 gene consists of one exon and spans 3.4 kb of genomic DNA on chromosome 11p15.1. Probes for this gene are included in the ME033 probemix.

• MODY 14 has been linked to defects in the gene encoding adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1). The APPL1 gene consists of 22 exons and spans 46 kb of genomic DNA on chromosome 3p14.3. No probes for this gene are included in P241 or P357.

More information is available at https://www.ncbi.nlm.nih.gov/books/NBK500456/.

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 P357-A3 MODY Mix 2 contains 45 MLPA probes with amplification products between 131 and 490 nucleotides (nt). This includes four probes for the KLF11 gene (one for each exon), two probes for the NEUROD1 gene (one for each exon), nine probes for the PAX4 gene (for exons 4 to 12), five probes for the CEL gene, three probes for the INS gene (one for each exon), two probes for the PDX1 gene (one for each exon) and ten probes for the HNF1B gene (one for each exon and two for exon 4). In addition, ten reference probes are included that detect autosomal chromosomal locations. 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 MLPA General Protocol and online at www.mrcholland.com.