Deletion and point mutations of PTHLH cause brachydactyly type E

Abstract

Autosomal-dominant brachydactyly type E (BDE) is a congenital limb malformation characterized by small hands and feet predominantly as a result of shortened metacarpals and metatarsals. In a large pedigree with BDE, short stature, and learning disabilities, we detected a microdeletion of approximately 900 kb encompassing PTHLH, the gene coding for parathyroid hormone related protein (PTHRP). PTHRP is known to regulate the balance between chondrocyte proliferation and the onset of hypertrophic differentiation during endochondral bone development. Inactivation of Pthrp in mice results in short-limbed dwarfism because of premature differentiation of chondrocyte. On the basis of our initial finding, we tested further individuals with BDE and short stature for mutations in PTHLH. We identified two missense (L44P and L60P), a nonstop (X178WextX( *)54), and a nonsense (K120X) mutation. The missense mutation L60P was tested in chicken micromass culture with the replication-competent avian sarcoma leukosis virus retroviral expression system and was shown to result in a loss of function. Thus, loss-of-function mutations in PTHLH cause BDE with short stature.

Figure 1

Pedigrees of Families 1–5 Affected individuals are indicated by black symbols. Symbols with crossed lines indicate individuals of whom material was available for testing, and stars indicate individuals of whom clinical or X-ray images are shown in Figure 2.

Figure 2

Clinical Phenotypes (A–D) Family 1, IV-3 (large deletion). Note severe BDE phenotype with small hands (A and C) and feet (B and D) due to shortening of all metacarpals and second to fifth metatarsals (arrows on X-rays). Note additional shortened middle phalanx of digits II and V (A, stars). (E and F) Family 2, II-1 (missense mutation L60P). Note small hands with shortened third to fifth metacarpals. Middle and distal phalanges of index and fifth finger are short (E, stars). (G) Family 3, II-1 (missense mutation L44P). Besides shortened metacarpals III–V, note cone-shaped epiphyses at the phalanges (arrows) and premature closure of the metacarpal epiphyses at the age of 9 years. Epiphyses of ulnar and radius appear irregular and flattened. (H) Family 4, III-1 (nonstop mutation). Note shortened third and fifth metacarpals and abnormal epiphyses prematurely fused to their metaphyses (arrow). (I) Growth chart of female individuals from family 1 (IV-15) and family 2 (II-1). Note that growth velocity of affected individuals stops prematurely, resulting in small stature.

Figure 3

Mutations Associated with BDE An ∼907 kb genomic microdeletion on chromosome 12p was detected in family 1 affecting six genes (gray bar, top; ISCN2009: array 12p11.23p11.22(27,341,677-28,249,358)x1). Point mutations within the PTHLH gene observed in families 2–5 are indicated (bottom). Genes are shown as dark gray boxes, with transcriptional orientation indicated by arrow heads. Small arrows point to genomic position of qPCR amplicons. Exon structure of PTHLH gene is according to NM_198966. Location of the start codon (ATG) in exon 2 is indicated. Genomic positions are according to hg18. Top: 1 mm represents 5 kb. Bottom: exons, 1 mm represents 10 bp; introns, 1 mm represents 100 bp.

Figure 4

Functional Testing of Missense Mutation L60P in Chicken Micromass System WT Pthlh (B and F) and L60P mutant (C and H) were retrovirally overexpressed in micromass cultures from chicken limb buds and were grown for 7 days (A–C) and 10 days (D–F) in culture medium. In comparison to the WT, the L60P mutant does not show a strong suppression of ALP activity (D and H). However, ALP activity is reduced compared to the uninfected control (D and H).