SALSA MLPA probemix P241-D2 MODY mix 1 Lot D As compared to version D1 (lot D1-0911), one reference probe has been replaced.

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1 mix P241-D2 MODY mix 1 Lot D As compared to version D1 (lot D1-0911), one reference has been replaced. 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 a clear autosomal inheritance and it generally develops in individuals under age of 25 years. Approximately 5% of all diabetes patients suffer from MODY. As described in more detail on page 2, eleven forms of MODY are identified with the highest prevalence for MODY 2 and 3. Each form of MODY is associated with one gene. This P241-D2 MODY mix contains s for the GCK, HNF1A, HNF1B and HNF4A genes and is therefore specific for MODY 1, 2, 3 and 5. A total of 42 s are present for these four genes. In addition, 8 reference s are included in this mix, detecting 8 different autosomal chromosomal locations. This SALSA mix is designed to detect deletions/duplications of one or more sequences in the aforementioned genes in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that. Note that a mutation or polymorphism in the sequence detected by a can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some signals are more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in these genes are expected to be small (point) mutations which will not be detected by this SALSA test. SALSA mixes and reagents are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. They are not CE/FDA certified for use in diagnostic procedures. Purchase of the test mixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA mix and reagents requires a thermocycler with heated lid and sequence type electrophoresis equipment. Different fluorescent PCR primers are available. The MLPA technique has been first described in Nucleic Acid Research 30, e57 (2002). Related SALSA mixes P117 ABCC8: Contains s for the ABCC8 gene, involved in familial hyperinsulinemic hypoglycemia 1. P357 MODY mix 2: Contains s for PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4 and INS genes, involved in MODY 4 to 10. References for SALSA mix P241 MODY mix 1 Valentínová L. et al., Identification and functional characterisation of novel glucokinase mutations causing maturity-onset diabetes of the young in Slovakia. PLOS One 7: e Carette C. et al., Familial young-onset forms of diabetes related to HNF4A and rare HNF1A molecular aetiologies. Diabet Med. 27: Gasperíková D. et al., Identification of a novel beta-cell glucokinase (GCK) promoter mutation (- 71G>C) that modulates GCK gene expression through loss of allele-specific Sp1 binding causing mild fasting hyperglycemia in humans. Diabetes 58: Garin I. et al., Haploinsufficiency at GCK gene is not a frequent event in MODY2 patients. Clin Endocrinol. 68: More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands SALSA P241 MODY mix 1 mix Page 1 of 7

2 The MODY genes MODY 1 is a result of defects in the Hepatocyte nuclear factor-4-alpha (HNF4A) gene on chromosome 20q13. The HNF4A gene comprises 11 exons and spans about 30 kb of genomic DNA. The protein encoded by this gene regulates the expression of HNF1A. MODY 2 is caused by mutations in the Glucokinase gene (GCK) on chromosome 7p13. This gene comprises 12 exons, spans about 45 kb of genomic DNA. MODY 3 is caused by defects in the HNF1 Homeobox A gene (HNF1A) on chromosome 12q24, which comprises 10 exons and spans about 24 kb of genomic DNA. MODY 4 has been linked to defects in the Pancreas/Duodenum homeobox protein 1 gene (PDX1) on chromosome 13q12.1. This gene comprises 2 exons and spans about 6 kb of genomic DNA. (Probemix P357) MODY 5 (RCAD) have been associated with the HNF1 Homeobox B gene (HNF1B) on chromosome 17q12. The HNF1B gene comprises 9 exons and spans about 59 kb of genomic DNA. MODY 6 has been linked to defects in the NEUROD1 gene on 2q31. This gene, with a size of 4 kb, has 2 exons. (Probemix P357) MODY 7 is caused by mutations in the Kruppel-Like Factor 11 gene (KLF11) on chromosome 2p25.1. It consists of 4 exons and spans approximately 11 kb of genomic DNA. (Probemix P357) MODY 8 has been associated with defects in Carboxyl-ester lipase gene (CEL) on chromosome 9q34, which has a size of 10 kb and has 11 exons. (Probemix P357) MODY 9 is caused by defects in the Paired box 4 gene (PAX4) on chromosome 7q32. This gene spans 5.4 kb of genomic DNA, contains 9 exons. (Probemix P357) MODY 10 has been linked to mutations in the insulin gene (INS) on chromosome 11p15. This gene has 3 exons and has a size of 1.4 kb. (Probemix P357) MODY 11 has been linked to defects in the B lymphoid tyrosine kinase gene (BLK). BLK is comprised of 13 exons spanning about 70 kb. No s for this gene have been included in P241 or P357. Data analysis The P241-D2 MODY mix contains 50 MLPA s with amplification products between 130 and 495 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at nt, three DNA Denaturation control fragments (Dfragments) at nt, one X-fragment at 100 nt and one Y-fragment at 105 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. Data generated by this mix can first be normalised intra-sample by dividing the peak height of each s amplification product by the total height of only the reference s in this mix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised ratio in a sample by the average intra-normalised ratio of all reference samples. Please note that this type of normalisation assumes no changes occurred in the genomic regions recognised by the reference s. Data normalisation should be performed within one experiment. Only samples purified by the same method should be compared. Confirmation of most exons deletions and amplifications can be done by e.g. Southern blotting, long range PCR, qpcr, FISH. Note that Coffalyser, the MLPA analysis tool developed at, can be downloaded free of charge from our website Many copy number alterations in healthy individuals are described in the database of genomic variants: For example, a duplication of a complete gene might not be pathogenic, while a partial duplication or a deletion may result in disease. For some genes, certain in-frame deletions may result in a very mild, or no disease. Copy number changes of reference s are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This mix was developed by. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P241 MODY mix 1 mix Page 2 of 7

3 Table 1. P241-D2 MODY mix 1 mix Chromosomal position reference GCK HNF1A HNF1B HNF4A Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA D-fragments: Low signal of 88 or 96 nt fragment indicates incomplete denaturation 100 X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 130 Reference L q HNF1B L GCK L (4) 147 HNF1A L HNF4A L (7) 160 HNF1B L GCK L (7) 172 HNF1A L ± GCK L (12) 184 HNF1A L «Reference L p HNF1A L HNF1B L HNF4A L (8) 214 HNF4A L (5) 220 GCK L HNF1A L GCK L (11) 238 HNF1B L * Reference L q HNF1A L HNF4A L (11) 265 GCK L (6) 274 Reference L p HNF1A L HNF4A L (10) 294 GCK L (9) 301 HNF1B L Reference L q HNF4A L (3) 326 HNF1A L GCK L (5) 346 HNF1A L HNF4A L (9) 364 HNF1B L Reference L q GCK L (10) 386 HNF1B L HNF4A L (4) 400 ๑ HNF4A L (2) 409 HNF4A L (6) 418 HNF1A L GCK L HNF1B L Reference L q GCK L (8) 463 HNF1B L HNF1A L HNF1B L Reference L q12 * New in version D2 (from lot D onwards). «This is located within, or close to, a very strong CpG island. A low signal of this can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. SALSA P241 MODY mix 1 mix Page 3 of 7

4 ๑ The significance of exon 3 deletions is not clear as this exon is not present in the MODY1 associated transcript variant 5 NM_ ± SNP rs is reported to influence the signal in two unrelated cases. In case of apparent deletions, it is recommended to sequence the region targeted by this. Notes The exon numbering for the GCK and HNF4A genes has changed. From description 19 onwards, we have adopted the NCBI exon numbering that is present in the Genbank NM_ sequence for the GCK gene and the Genbank NG_ sequence for the HNF4A gene. This exon numbering used here may differ from literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 1 and Table 2. The identity of the genes detected by the reference s is available on request: info@mlpa.com. Table 2. P241 s arranged according to chromosomal location Table 2a. GCK gene GCK NM_ next start codon (ex 1) L GCTAGGATGTGA-GAGACACAGTCA 0.4 kb L ACAGAGCCAGGA-TGGAGGCCGCCA 35.5 kb L (4) AGACGGATGCAG-AAGGAGATGGAC 1.0 kb L (5) TGGGTGGCACTA-ACTTCAGGGTGA 1.4 kb L (6) GCATCAGATGAA-ACACAAGAAGCT 0.2 kb L (7) CAGAAGGGAACA-ATGTCGTGGGGC 1.0 kb L (8) GCTACTACGAAG-ACCATCAGTGCG 2.1 kb L (9) reverse CCAGGCGGTCAT-ACTCCAGCAGGA 1.1 kb L (10) TAGGTGGCAAGT-ACATGGGCGAGC 0.9 kb L (11) CTACAACATCCT-GAGCACGCTGGG 0.9 kb 178 ± L (12) TCACTCAGGACT-TTGATGCATTTC stop codon (ex 10) ± SNP rs is reported two influence the signal in two unrelated cases. In case of apparent deletions, it is recommended to sequence the region targeted by this. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Note: The GCK exon numbering has changed. From description version 19 onwards, we have adopted the NCBI exon numbering that is present in the Genbank NM_ sequence. The exon numbering used in previous versions of this product description can be found between brackets in Table 1 and Table 2. Table 2b. HNF1A gene HNF1A NM_ next start codon (ex 1) L GCCATGGTTTCT-AAACTGAGCCAG 0.5 kb L nt after ex 1 CTTGGAGGTTTG-AGCCTCCAGCCC 9.7 kb L CACCTGTCCCAA-CACCTCAACAAG 4.6 kb L AGGTGATGAGCT-ACCAACCAAGAA 0.7 kb L CGGCGCAAAGAA-GAAGCCTTCCGG 2.0 kb L GAGACTGCAGAA-GTACCCTCAAGC 0.3 kb L AGCTTGGAGCAG-ACATCCCCAGGC 1.0 kb L reverse GCACAGGTGGCA-TGAGCGGCTGCT 1.7 kb L reverse CACCTCGGGCTT-GTGGCTGTAGAG 0.2 kb L reverse GCCTCAGTGTCT-GAGGTGAAGACC 1.6 kb L CTCCAGCAGCCT-GGTGCTGTACCA stop codon (ex 10) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. SALSA P241 MODY mix 1 mix Page 4 of 7

5 Table 2c. HNF1B gene HNF1B NM_ next start codon (ex 1) L TTGGAAAATGGT-GTCCAAGCTCAC 5.3 kb L TGCAGCAACACA-ACATCCCCCAGA 5.9 kb L CTGAGCCCACCA-ACAAGAAGATGC 0.2 kb L reverse ACACGGACCTCA-GTGACCAAGTTG 1.9 kb L reverse TACCTGACAGCT-TGTTTGGAGGAG 21.1 kb L nt after ex 5 reverse CTCCAGAGCGAC-AATGGCCCAGGT 5.5 kb L GTCTCAGGAGGA-GGTTTGCCCCCA 4.0 kb L reverse GCTCTGCTGCAT-GAGGGGCTGCTG 2.0 kb L AGCAGCATCAGT-ACACTCACCAAC 11.9 kb L reverse GAGGTGCCAGCA-GGACGTCCGTCA stop codon (ex 9) The NM_ sequence represents variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2d. HNF4A gene HNF4A NM_ start codon 5-7 (ex 1) No 1 No 2 78 nt after ex 3 (NM_ ) 400 ๑ L (2) CTTTGGCACTCA-ACTTTGGGGTGG next SALSA P241 MODY mix 1 mix Page 5 of kb L (3) CAGAAGGCACCA-ACCTCAACGCGC 1.4 kb L (4) TGCAGGCTCAAG-AAATGCTTCCGG 6.1 kb L (5) 162 nt before ex 6 reverse AACTCAGCTGGT-GGAAGAACTTTC 1.1 kb L (6) CATGAAGGAGCA-GCTGCTGGTTCT 3.8 kb L (7) 5 nt before ex 8 TTCCTTCTCTCT-TTCAGGTGGCCC 1.5 kb L (8) 66 nt after ex 9 reverse TACAGAACCCAA-TAGGAGAAGTCT 4.3 kb L (9) AGCAGATCCAGT-TCATCAAGCTCT 4.3 kb L (10) 19 nt after ex 11 AACTCTGGGATT-TTACCTTGCAAA 2.2 kb L (11) CTGAGGTCCTGA-TCAGCTTCAAGG stop codon (ex 10) ๑ The significance of exon 3 deletions is not clear as this exon is not present in the MODY1 associated transcript variant 5 NM_ The NM_ and NM_ sequences represent transcript variants 5 and 2, respectively. Both sequences are reference standards in the NCBI RefSeqGene project. Notes The HNF4A exon numbering has changed. From description version 19 onwards, we have adopted the NCBI exon numbering from the Genbank NG_ sequence. The exon numbering used in previous versions of this product description can be found between brackets in Table 1 and Table 2. The HNF4A transcript variant has changed. From description version 20 onwards, we have indicated the ligation sites for the majority of the HNF4A s according to the NM_ sequence. NM_ encodes the MODY1 associated isoform HNF4alpha8 (Carette et al. 2010). Compared to transcript variant 2, it contains an alternative first exon and translation initiation site. 2 and exon 3 are not present in the NM_ sequence. 1 and exon 2 are not present in the NM_ sequence. Mutations/deletions in exon 1 of the HNF4A gene are known to cause MODY (Colclough et al. 2013, Hum Mutat. 34: ). Please note that copy number changes of HNF4A exon 1 cannot be detected by this mix, as there are no MLPA s included for HNF4A exon 1.

6 Note: numbering used here may differ from literature! Complete sequences are available on request: Please notify us of any mistakes: mix P241-D2 MODY sample picture Figure. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with mix P241-D2 MODY (lot D2-0413). Implemented Changes compared to the previous product description versions. Version 20 (55) 08 April Information added about transcript NM_ of the HNF4A gene. - s in Table 2d adjusted according to the NM_ sequence. - Probe remarks added in Table 1 and Table 2; 178 nt L07439, 189 nt L21306 and 400 nt L Various minor textual and layout changes. Version 19 (54) 15 July numbering for the GCK and HNF4A genes has changed in Table 1 and 2. - s for HNF1B has been changed in Table 2c. Version 18 (54) 04 March New sample picture included in product description. Version 17 (53) - RCAD is added as synonym of MODY5 - Corrected text on data analysis. - Updated link for Database of Genomic Variants. Version 16 (50) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). Version 15 (48) - Partial sequences of the GCK exon 1 s have been corrected. These had been switched in previous versions. Version 14 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. SALSA P241 MODY mix 1 mix Page 6 of 7

7 Version 13 (47) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new picture included). - Various minor textual changes on page 1 Version 12 (46) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new picture included). Version 11 (45) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new picture included). - numbering of the HNF4A and GCK genes has been changed. - s updated according to new version of the NM_reference sequence. - Tables have been numbered. Small changes of lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Various minor textual changes on page 1, various minor layout changes. - Data analysis method has been changed. SALSA P241 MODY mix 1 mix Page 7 of 7