Figure 3.

Mutations Affecting DNA Binding Sites of Human BHLHA9 and Homologous bHLH Proteins

(A) Model of human BHLHA9 with depiction of the basic helix-loop-helix region as indicated in the NCBI Protein database.

(B) Mapping of MSSD-associated missense mutations to the basic nucleic acid binding site of BHLHA9 enclosing the E-box specificity site identified with the CCD program,³⁵ projected onto an amino acid sequence alignment of orthologous BHLHA9 sequences from various vertebrates and of human (Hsap) TCF3, TCF4, TCF12, TWIST1, HAND2, and NEUROD1 sequences generated by ClustalW.³⁶ Numbering of amino acids refers to human (Hsap) BHLHA9. NCBI sequence identifiers (gi) are indicated. The amino acids mutated in the MSSD-affected individuals (red arrows) are conserved in all aligned proteins. Species abbreviations are as follows: Mmus, Mus musculus; Ggal, Gallus gallus; Drer, Danio rerio.

(C) Amino acid substitutions in the DNA-binding region of the basic domain of helix 1 associated with somatic mutations in cancer listed in the COSM database (green arrows) and/or clinical Mendelian phenotypes (red arrows).

TWIST1: p.Arg118Cys, in Saethre-Chotzen syndrome (SCS)²³ and COSM1449859; p.Gln119Pro, SNP rs104894057, in Saethre-Chotzen syndrome;^23,24 p.Arg120Cys in COSM1269548.

NEUROD1: p.Arg109Leu in COSM719047; p.Glu119Gly in COSM402132; p.Arg111Leu, rs104893649, in diabetes mellitus type II (NIDDM); p.Arg113His in COSM1217138.

TCF4: p.Arg576Gly, p.Arg576Gln, p.Arg578Pro, p.Arg578His, p.Arg580Trp, p.Arg580Gln in Pitt-Hopkins syndrome (PTHS), references in Sepp et al.;³⁷ p.Arg578Cys in COSM563719 and COSM1141464.

The association of detrimental missense mutations affecting homologous amino acids in the basic DNA-binding region of these bHLH proteins with clinical phenotypes adds weight to the notion that the comparable mutations in BHLHA9 cause MSSD.