FAM111A Mutations Result in Hypoparathyroidism and Impaired Skeletal Development

Kenny-Caffey syndrome (KCS) and the similar but more severe osteocraniostenosis (OCS) are genetic conditions characterized by impaired skeletal development with small and dense bones, short stature, and primary hypoparathyroidism with hypocalcemia. We studied five individuals with KCS and five with OCS and found that all of them had heterozygous mutations in FAM111A. One mutation was identified in four unrelated individuals with KCS, and another one was identified in two unrelated individuals with OCS; all occurred de novo. Thus, OCS and KCS are allelic disorders of different severity. FAM111A codes for a 611 amino acid protein with homology to trypsin-like peptidases. Although FAM111A has been found to bind to the large T-antigen of SV40 and restrict viral replication, its native function is unknown. Molecular modeling of FAM111A shows that residues affected by KCS and OCS mutations do not map close to the active site but are clustered on a segment of the protein and are at, or close to, its outer surface, suggesting that the pathogenesis involves the interaction with as yet unidentified partner proteins rather than impaired catalysis. FAM111A appears to be crucial to a pathway that governs parathyroid hormone production, calcium homeostasis, and skeletal development and growth.     

Analysis of short stature homeobox-containing gene (<Emphasis Type="Italic">SHOX</Emphasis>) and auxological phenotype in dyschondrosteosis and isolated Madelung deformity

Dyschondrosteosis (DCO; also called Léri-Weill syndrome) is a skeletal dysplasia characterised by disproportionate short stature because of mesomelic shortening of the limbs. Madelung deformity is a feature of DCO that is distinctive, variable in expressivity and frequently observed. Mutations of the SHOX (short stature homeobox-containing) gene have been previously described as causative in DCO. Isolated Madelung deformity (IMD) without the clinical characteristics of DCO has also been described in sporadic and a few familial cases but the genetic defect underlying IMD is unknown. In this study, we have examined 28 probands with DCO and seven probands with IMD for mutations in the SHOX gene by using polymorphic CA-repeat analysis, fluorescence in situ hybridisation (FISH), Southern blotting, direct sequencing and fibre-FISH analyses. This was combined with auxological examination of the probands and their family members. Evaluation of the auxological data showed a wide intra- and interfamilial phenotype variability in DCO. Out of 28 DCO probands, 22 (79%) were shown to have mutations in the SHOX gene. Sixteen unrelated DCO families had SHOX gene deletions. Four novel DCO-associated mutations were found in different families. In two additional DCO families, the previously described nonsense mutation (Arg195Stop) was detected. We conclude that mutations in the SHOX gene are the major factor in the pathogenesis of DCO. In a female proband with severe IMD and her unaffected sister, we detected an intrachromosomal duplication of the SHOX gene.     

Structural mechanisms of the degenerate sequence recognition by Bse634I restriction endonuclease

Restriction endonuclease Bse634I recognizes and cleaves the degenerate DNA sequence 5'-R/CCGGY-3' (R stands for A or G; Y for T or C, '/' indicates a cleavage position). Here, we report the crystal structures of the Bse634I R226A mutant complexed with cognate oligoduplexes containing ACCGGT and GCCGGC sites, respectively. In the crystal, all potential H-bond donor and acceptor atoms on the base edges of the conserved CCGG core are engaged in the interactions with Bse634I amino acid residues located on the α6 helix. In contrast, direct contacts between the protein and outer base pairs are limited to van der Waals contact between the purine nucleobase and Pro203 residue in the major groove and a single H-bond between the O2 atom of the outer pyrimidine and the side chain of the Asn73 residue in the minor groove. Structural data coupled with biochemical experiments suggest that both van der Waals interactions and indirect readout contribute to the discrimination of the degenerate base pair by Bse634I. Structure comparison between related enzymes Bse634I (R/CCGGY), NgoMIV (G/CCGGC) and SgrAI (CR/CCGGYG) reveals how different specificities are achieved within a conserved structural core.     

Experimental approaches to understanding variation in grain size in Panicum miliaceum (broomcorn millet) and its relevance for interpreting archaeobotanical assemblages

The dimensions of archaeobotanical grains identified as Panicum miliaceum (broomcorn millet) vary greatly in size. This is illustrated by the remains from the archaeological site of Zanovskoe in eastern Ukraine (5th–1st centuries cal. b.c.). We carried out experimental work on broomcorn millet plants and grains, aiming at a comprehensive understanding of factors that may have contributed to variation in the grain size of broomcorn millet in archaeobotanical assemblages. We analyzed the dependence of grain size variation on selected environmental and taphonomic factors. Our results indicate that immaturity is more likely than environmental stress to account for small grain size in broomcorn millet plants. Depending on charring temperature and time, immature broomcorn millet grains can withstand charring and are potentially preserved in archaeological assemblages. Depending on maturity level, such grains vary in size and shape. These results are potentially important for accurate identification of archaeobotanical specimens.     

Mutations in short stature homeobox containing gene (<Emphasis Type="Italic">SHOX</Emphasis>) in dyschondrosteosis but not in hypochondroplasia

Dyschondrosteosis (DCO) and hypochondroplasia (HCH) are common skeletal dysplasias characterized by disproportionate short stature. The diagnosis of these conditions might be difficult to establish especially in early childhood. Point mutations and deletions of the short stature homeobox containing gene (SHOX) are detected in DCO and idiopathic short stature with some rhizomelic body disproportion, whereas mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are found in 40-70% of HCH cases. In this study, we performed mutational analysis of the coding region of the SHOX gene in five DCO and 18 HCH patients, all of whom tested negative for the known HCH-associated FGFR3 mutations. The polymorphic CA-repeat analysis, direct sequencing and Southern blotting were used for detection of deletions and point mutations. The auxological and radiological phenotype of these patients was carefully determined. Three novel mutations in DCO patients were found: (1) a deletion of one base (de1272G) (according to GenBank accession nos. Y11536, Y11535), resulting in a premature stop codon at position 75 of the amino acid sequence; (2) the transversion C485G resulting in the substitution Leu132Val; and (3) the transversion G549T causing an Arg153Leu substitution. These substitutions segregate with the DCO phenotype and affect evolutionarily conserved homeodomain residues, based on a comparison of homeobox containing proteins in 13 species. Moreover, these changes were not found in 80 unrelated, unaffected individuals. This strongly suggests that these mutations are pathogenic. The phenotype of our patients with DCO and HCH varied from mild to severe shortness and body disproportion. These results further support clinical and genetic heterogeneity of dyschondrosteosis and hypochondroplasia.     

The crystal structure of the rare-cutting restriction enzyme SdaI reveals unexpected domain architecture

Rare-cutting restriction enzymes are important tools in genome analysis. We report here the crystal structure of SdaI restriction endonuclease, which is specific for the 8 bp sequence CCTGCA/GG ("/" designates the cleavage site). Unlike orthodox Type IIP enzymes, which are single domain proteins, the SdaI monomer is composed of two structural domains. The N domain contains a classical winged helix-turn-helix (wHTH) DNA binding motif, while the C domain shows a typical restriction endonuclease fold. The active site of SdaI is located within the C domain and represents a variant of the canonical PD-(D/E)XK motif. SdaI determinants of sequence specificity are clustered on the recognition helix of the wHTH motif at the N domain. The modular architecture of SdaI, wherein one domain mediates DNA binding while the other domain is predicted to catalyze hydrolysis, distinguishes SdaI from previously characterized restriction enzymes interacting with symmetric recognition sequences.