Mutations in bone morphogenetic protein receptor 1B cause brachydactyly type A2

Abstract

Brachydactyly (BD) type A2 is an autosomal dominant hand malformation characterized by shortening and lateral deviation of the index fingers and, to a variable degree, shortening and deviation of the first and second toes. We performed linkage analysis in two unrelated German families and mapped a locus for BD type A2 to 4q21-q25. This interval includes the gene bone morphogenetic protein receptor 1B (BMPR1B), a type I transmembrane serinethreonine kinase. In one family, we identified a T599 --> A mutation changing an isoleucine into a lysine residue (I200K) within the glycine/serine (GS) domain of BMPR1B, a region involved in phosphorylation of the receptor. In the other family we identified a C1456 --> T mutation leading to an arginine-to-tryptophan amino acid change (R486W) in a highly conserved region C-terminal of the BMPR1B kinase domain. An in vitro kinase assay showed that the I200K mutation is kinase-deficient, whereas the R486W mutation has normal kinase activity, indicating a different pathogenic mechanism. Functional analyses with a micromass culture system revealed a strong inhibition of chondrogenesis by both mutant receptors. Overexpression of mutant chBmpR1b in vivo in chick embryos by using a retroviral system resulted either in a BD phenotype with shortening and/or missing phalanges similar to the human phenotype or in severe hypoplasia of the entire limb. These findings imply that both mutations identified in human BMPR1B affect cartilage formation in a dominant-negative manner.

Fig. 1.

Phenotype of BDA2. (A and C) Short and medially deviated second fingers and clinodactyly of the fifth fingers. (B) Radiograph of the triangular-shaped middle phalanx of the second finger (Right) and missing middle phalanx of the second finger (Left). (D) Short, broad, and laterally deviated first toe and medial deviation of the second toe. The pictures were taken from persons A–D as indicated in Fig. 2.

Fig. 2.

BDA2 pedigrees. Affected persons are indicated by filled symbols. Overlined symbols indicate individuals who underwent clinical examination and mutation analysis.

Fig. 3.

Schematic BMPR1B structure and mutations. Shown is the structure of BMPR1B showing the functional domains and the position of mutations (indicated by arrows). The amino acid sequences of BMPR1B are from different species, demonstrating the high degree of conservation within both domains. The two different mutations within the domains are underlined.

Fig. 4.

Retroviral overexpression of BmpR1B mutants in micromass cultures and in chicken embryos. (A) Alcian blue-stained micromass cultures after 5 days of cultivation transfected with control [enhanced GFP (EGFP)] virus, WT Bmpr1b, or the indicated mutant constructs. In comparison with cultures expressing enhanced GFP^– or the WT gene, those expressing either the I200K or R486W mutation genes exhibited a strong inhibition of chondrogenesis. (B) The quantification of Alcian blue incorporation into the extracellular matrix of micromass cultures reflecting the production of proteoglycan-rich cartilaginous matrix measured at days 5 and 6 is shown. Both mutations strongly inhibit cartilage formation. Note the stronger effect of the I200K mutation. The Q249R mutation described in the Booroola strain of Mérino sheep was also tested but shows no significant difference to WT BmpR1b. (C) Transfection of mutant chBmpR1b in chicken limbs induces a variable phenotypic severity, presumably due to infection efficacy. Infected limbs (Right) are shown along with the uninfected contralateral control limbs (Left). The milder phenotypes observed for both mutations (Top) display a marked BD with absent middle phalanges (R486W) or missing distal phalanges (1200K). In limbs with a high infection rate, a much stronger phenotype was observed, resulting in extreme shortening of all skeletal elements (I200K, Bottom). pg, pelvic girdle; fe, femur; ti, tibia; mt, metatarsal; d, digit.

Fig. 5.

Immunodetection of BmpR1b constructs expressed in COS7 cells. COS7 cells were transfected with HA-tagged mouse BmpR1b constructs containing WT BmpR1b (A), I200K BmpR1b (B), and R486W BmpR1b (C). Immunostaining against HA shows that WT and both mutant BmpR1b constructs are located at the cell surface.

Fig. 6.

In vitro kinase assay. COS7 cells were transiently transfected with mouse BmpR1B constructs containing WT BmpR1b, a kinase-inactivated receptor (KR), and the mutations I200K and R486W. Cells were solubilized, and BMPRs were immunoprecipitated by anti-HA antibodies and then subjected to in vitro kinase assays in the presence of [γ-³²P]ATP. The kinase-inactivated receptor and I200K mutant showed no kinase activity; mutant R486W revealed a unchanged kinase activity.