The maennelein phenotype was identified among G3 mice of the pedigree R4657, some of which showed reduced body weights compared to wild-type littermates (Figure 1).

Whole exome HiSeq sequencing of the G1 grandsire identified 103 mutations. The body weight phenotype was linked to a mutation in Pappa:  a G to A transition at base pair 65,314,796 (v38) on chromosome 4, or base pair 190,623 in the GenBank genomic region NC_000070 within the donor splice site of intron 16 (5-base pairs from exon 16 [out of 22 total exons]). Linkage was found with a recessive model of inheritance, wherein four variant homozygotes departed phenotypically from six homozygous reference mice and six heterozygous mice with a P value of 3.983 x 10^-8 (Figure 2).  

The effect of the mutation at the cDNA and protein levels has not been examined, but the mutation is predicted to result in the use of a cryptic site in exon 16. The resulting transcript would have a 71-base pair deletion of exon 16, resulting in a frame-shifted protein product beginning after amino acid 1,365 of the protein (which is normally 1,624 amino acids in length) and terminating after the inclusion of 23 aberrant amino acids.

          <--exon 15 exon 16-->                         intron 16-->    exon 17-->

4311 ……GCCCAGCTGAAAG GCAACAAC……AAGCACAAGGTGGGC……CCAAGAA gtaagtgccgcta…… ACGGGCTTT……GGTGACTTGTGA……

1315 ……-A--Q--L--K-- G--N--N-……-K--H--K-------……-------                 -T--G--F-……-G--D--L--*-

                  correct                   deleted                            aberrant

The donor splice site of intron 16, which is destroyed by the maennelein mutation, is indicated in blue lettering and the mutated nucleotide is indicated in red.

Pappa encodes pregnancy-associated plasma protein A (PAPP-A; alternatively, IGFBP4 protease or differentially expressed in placenta 1 [DIPLA1]), a member of the pappalysin subfamily of the metzincin protease family along with PAPP-A2 (see the record for Lilliputian) and ulilysin. PAPP-A is initially translated as a 1,624 proprotein; cleavage of the signal peptide and the propeptide (amino acids 23-80) generates the mature 1,544 amino acid peptide (1). PAPP-A has several domains, including a signaling peptide (amino acids 1-22), a laminin G-like domain, three Lin12/Notch repeats (LNRs), a metalloprotease region (amino acids 272-583), and five complement control protein (CCP) domains (alternatively, short consensus repeat [SCR] or Sushi domains; amino acids 1210-1279, 1280-1341, 1342-1409, 1410-1470, and 1473-1553) (Figure 3) (2).

The maennelein mutation within the donor splice site of intron 16 is predicted to result in a 71-base pair deletion of exon 16, resulting in a frame-shifted protein product beginning after amino acid 1,365 of the protein.

Please see the record caer for more information about Pappa.

PAPP-A is a secreted metalloproteinase that cleaves insulin-like growth factor binding protein 2 (IGFBP2), IGFBP4, and IGFBP5. The IGFBPs regulate the IGF-I signaling pathways by binding IGF-I. IGFBP5 also has IGF-I-independent functions. IGFPB5 is able to bind its putative receptor to enter the cytoplasm and subsequently interact with, and regulate, other proteins. IGFBPs are carrier proteins that regulate the bioavailability of insulin-like growth factors (IGFs) by prolonging their-half-life and circulation turnover. IGFs are essential for the regulation of growth and development by influencing the proliferation, differentiation, and apoptosis of osteoblasts (3;4). IGFs bind to two types of receptors, IGF-IR and IGF-IIR, subsequently activating downstream tyrosine kinase pathways. In IGF-I-associated signaling, both the IRS-1/phosphoinositide 3-kinase/serine–threonine kinase pathway and the Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase pathway are activated, which subsequently promote cell proliferation, tissue differentiation, and protection from apoptosis.

Pappa-deficient (Pappa^-/-) mice showed reduced body sizes and weights (60 to 70% of wild-type mice) as well as delayed bone ossification (5). The phenotype of the maennelein mice mimics that of the Pappa^-/- mice, indicating loss of PAPP-A function. PAPP-A functions as a growth-promoting enzyme through its role in cleaving IGFBPs (and subsequent release of bioactive IGF). IGFBP4 cleavage is required to activate most, if not all, IGF2-mediated growth-promoting activity.

PCR program

1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40x
6) 72°C 10:00
7) 4°C hold

The following sequence of 260 nucleotides is amplified (chromosome 4, + strand):

1   ctgtgtcttc caggcaacaa cagctttctg acctgtatgg aagatggact gtggtccttc
61  ccagaggcct tgtgtgagct catgtgcctc gccccacccc cagttcccaa tgcggaccta
121 cagacagccc ggtgtcgaga gaacaagcac aaggtgggct ccttctgcaa gtacaagtgt
181 aaacctggat accacgtgcc tggctcatct cggaagtcca agaagtaagt gccgctaggg
241 tcatagccat agaccttgag 

Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.