Myeong-Kyu Kim

Bio: Myeong-Kyu Kim is an academic researcher from Konkuk University. The author has contributed to research in topics: Phenotype & Genotype. The author has co-authored 2 publications.

A Genotype-Phenotype Study of Multiple Hereditary Exostoses in Forty-Three Patients

Abstract: Multiple hereditary exostoses (MHE) is a rare autosomal dominant skeletal disorder with a variety of clinical manifestations. We aimed to evaluate the general clinical phenotypic severity of MHE using our own scoring system and analyzed the risk factors associated with severe clinical phenotypes. In this study, 43 patients from 30 families were analyzed. The mutations were identified by direct sequencing of polymerase chain reaction-amplified genomic DNA or by multiplex ligation-dependent probe amplification. According to a new scoring system devised by the authors, the severity of the phenotype was assessed as mild, moderate, or severe based on the deformity of each segment, number of exostoses, leg length discrepancy, and functional limitations. Of 43 patients from 30 families, 39 patients (90.7%) and 24 families (80%) presented with EXT1 or EXT2 mutations. Patients with EXT1 mutations had a significantly worse phenotype than that of patients with EXT2 mutations or without any detectable mutation. The mean clinical score of patients with an EXT1 mutation (5.76; range, 2.0–8.0; SD = 1.60) was higher than that of patients with an EXT2 mutation (4.06; range, 2.0–7.0; SD = 1.47) or of those without any detectable mutation (4.63; range, 3.0–6.0; SD = 1.44; p = 0.005). According to our classification system, more patients with EXT1 mutations had ‘severe disease’ than those with EXT2 mutations. Deformity scores were also higher in patients with EXT1 mutations (p = 0.018). In the multivariate analysis, the deformity score was found to be associated with the ‘severe’ class (p = 0.031). In conclusion, 90.7% of patients with MHE showed EXT mutations. Our scoring system showed reliable results. We suggest that the extent of deformity is an important factor in determining the phenotype of MHE and close monitoring for the development of severe disease is recommended in patients with high deformity scores.

Modifications using Circular Shift for a Better Bloom Filter

Abstract: The Bloom filter is a hash-based data structure that facilitates membership querying. Computation speed of Bloom filter is affected by hash functions that produce hash outputs. Basically, two operations: 'add' and 'query', consists of the Bloom filter. Previous researches have shown advanced computation speed of Bloom filter since the standard Bloom Filter is published. For example, Double Hash Bloom filter, Single Hash Bloom filter, etc.We propose a system that uses less computation than previous works with similar false positive and which is implemented by using a circular bit shift. This method was implemented with faster calculation speed, compared with previous works. Furthermore, experiments which were compared with previous researches and standard Bloom filter. Therefore, we demonstrate that the proposed system computes faster than previous studies with similar false positive rate.

Clinical and Genetic Analysis of Multiple Osteochondromas in a Cohort of Argentine Patients

Abstract: Multiple Osteochondromatosis (MO, MIM 133700 & 133701), an autosomal dominant O-glycosylation disorder (EXT1/EXT2-CDG), can be associated with a reduction in skeletal growth, bony deformity, restricted joint motion, shortened stature and pathogenic variants in two tumor suppressor genes, EXT1 and EXT2. In this work, we report a cross-sectional study including 35 index patients and 20 affected family members. Clinical phenotyping of all 55 affected cases was obtained, but genetic studies were performed only in 35 indexes. Of these, a total of 40% (n = 14) had a family history of MO. Clinical severity scores were class I in 34% (n:18), class II in 24.5% (n:13) and class III in 41.5% (n:22). Pathogenic variants were identified in 83% (29/35) probands. We detected 18 (62%) in EXT1 and 11 (38%) in EXT2. Patients with EXT1 variants showed a height z-score of 1.03 SD lower than those with EXT2 variants and greater clinical severity (II–III vs. I). Interestingly, three patients showed intellectual impairment, two patients showed a dual diagnosis, one Turner Syndrome and one hypochondroplasia. This study improves knowledge of MO, reporting new pathogenic variants and forwarding the worldwide collaboration necessary to promote the inclusion of patients into future biologically based therapeutics.

Structural Basis of Dual Specificity of Sinorhizobium meliloti Clr, a cAMP and cGMP Receptor Protein

Abstract: Cyclic 3′,5′-AMP (cAMP) and cyclic 3′,5′-GMP (cGMP) are both long known as important nucleotide secondary messengers in eukaryotes. This is also the case for cAMP in prokaryotes, whereas a signaling role for cGMP in this domain of life has been recognized only recently. ABSTRACT In bacteria, the most prevalent receptor proteins of 3′,5′-cyclic AMP (cAMP) and 3′,5′-cyclic GMP (cGMP) are found among transcription factors of the Crp-Fnr superfamily. The prototypic Escherichia coli catabolite activator protein (CAP) represents the main Crp cluster of this superfamily and is known to bind cAMP and cGMP but to mediate transcription activation only in its cAMP-bound state. In contrast, both cyclic nucleotides mediate transcription activation by Sinorhizobium meliloti Clr, mapping to cluster G of Crp-like proteins. We present crystal structures of Clr-cAMP and Clr-cGMP bound to the core motif of the palindromic Clr DNA binding site (CBS). We show that both cyclic nucleotides shift ternary Clr-cNMP–CBS-DNA complexes (where cNMP is cyclic nucleotide monophosphate) to almost identical active conformations, unlike the situation known for the E. coli CAP-cNMP complex. Isothermal titration calorimetry measured similar affinities of cAMP and cGMP binding to Clr in the presence of CBS core motif DNA (equilibrium dissociation constant for cNMP (KDcNMP], ~7 to 11 μM). However, different affinities were determined in the absence of this DNA (KDcGMP, ~24 μM; KDcAMP, ~6 μM). Sequencing of Clr-coimmunoprecipitated DNA as well as electrophoretic mobility shift and promoter-probe assays expanded the list of experimentally proven Clr-regulated promoters and CBS. This comprehensive set of CBS features conserved nucleobases that are consistent with the sequence readout through interactions of Clr amino acid residues with these nucleobases, as revealed by the Clr–cNMP–CBS-DNA crystal structures. IMPORTANCE Cyclic 3′,5′-AMP (cAMP) and cyclic 3′,5′-GMP (cGMP) are both long known as important nucleotide secondary messengers in eukaryotes. This is also the case for cAMP in prokaryotes, whereas a signaling role for cGMP in this domain of life has been recognized only recently. Catabolite repressor proteins (CRPs) are the most ubiquitous bacterial cAMP receptor proteins. Escherichia coli CAP, the prototypic transcription regulator of the main Crp cluster, binds both cyclic mononucleotides, but only the CAP-cAMP complex promotes transcription activation. In contrast, Crp cluster G proteins studied so far are activated by cGMP or by both cAMP and cGMP. Here, we report a structural analysis of the cAMP- and cGMP-activatable cluster G member Clr from Sinorhizobium meliloti, how binding of cAMP and cGMP shifts Clr to its active conformation, and the structural basis of its DNA binding site specificity.

Structural basis of bifunctionality of Sinorhizobium meliloti Clr, a cAMP and cGMP receptor protein

Abstract: In bacteria, Crp-Fnr superfamily transcription factors are the most ubiquitous receptor proteins of 3’,5’-cyclic adenosine monophosphate (cAMP) and 3’,5’-cyclic guanosine monophosphate (cGMP). The prototypic Escherichia coli CAP protein represents the main CRP subclass and is known to bind cAMP and cGMP, but to mediate transcription activation only in its cAMP-bound state. In contrast, both cyclic nucleotides mediate transcription activation by CRP subclass G protein Clr of Sinorhizobium meliloti. We present crystal structures of apo-Clr, and Clr•cAMP and Clr•cGMP bound to the core motif of the palindromic Clr DNA binding site (CBS). We show that both cyclic nucleotides shift ternary Clr•cNMP•CBS-DNA complexes to almost identical active conformations. Unlike the situation known for the E. coli CAP•cNMP complex, in the Clr•cNMP complex, the nucleobases of cGMP and cAMP are in the syn- and anti-conformation, respectively, allowing a shift to the active conformations in both cases. Isothermal titration calorimetry measured similar affinities of cAMP and cGMP binding to Clr in presence of CBS core motif DNA (KDcNMP 16 μM). However, different affinities were determined in absence of this DNA (KDcGMP 24 μM; KDcGMP 6 μM). Sequencing of Clr co-immunoprecipitated DNA as well as Electrophoretic Mobility Shift and promoter-probe assays expanded the list of experimentally proven Clr-regulated promoters and CBS. This comprehensive set of CBS features conserved nucleobases, which are in agreement with the sequence readout through interactions of Clr amino acid residues with these nucleobases, as revealed by the Clr•cNMP•CBS-DNA crystal structures. IMPORTANCE Cyclic 3’,5’-adenosine monophosphate (cAMP) and cyclic 3’,5’-guanosine monophosphate (cGMP) are both long known as important nucleotide second messenger in eukaryotes. This is also the case for cAMP in prokaryotes, whereas a signaling role for cGMP in this domain of life has been recognized only recently. Catabolite repressor proteins (CRPs) are the most ubiquitous bacterial cAMP receptor proteins. Escherichia coli CAP, the prototypic transcription regulator of the main CRP subclass, binds both cyclic mononucleotides, but only the CAP•cAMP complex promotes transcription activation. In contrast, CRP subclass G proteins studied so far, are activated by cGMP, or both by cAMP and cGMP. Here, we report a structural analysis of the bifunctional cAMP- and cGMP-activatable Clr from Sinorhizobium meliloti, how binding of cAMP and cGMP shifts Clr to its active conformation, and the structural basis of its DNA binding site specificity.

Hereditary multiple exostoses caused by a chromosomal inversion removing part of EXT1 gene

Abstract: Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder characterized by the development of multiple, circumscript and usually symmetric bony protuberances called osteochondromas. Most HME are caused by EXT1 and EXT2 loss of function mutations. Most pathogenic mutations are nonsense followed by missense mutations and deletions.Here we report on a patient with a rare and complex genotype resulting in a typical HME phenotype. Initial point mutation screening in EXT1 and EXT2 genes by Sanger sequencing did not reveal any pathogenic variants. The patient along with the healthy parents was subsequently referred for karyotype and array-Comparative Genomic Hybridization (CGH) analyses. Chromosomal analysis revealed two independent de novo apparently balanced rearrangements: a balanced translocation between the long arms of chromosomes 2 and 3 at breakpoints 2q22 and 3q13.2 and a pericentric inversion with breakpoints at 8p23.1q24.1. Both breakpoints were confirmed by Fluorescence In Situ Hybridization (FISH). Subsequently, array-CGH revealed a novel heterozygous deletion within the EXT1 gene at one of the inversion breakpoints, rendering the inversion unbalanced. The mode of inheritance, as well as the size of the deletion were further investigated by Quantitative Real-time PCR (qPCR), defining the deletion as de novo and of 3.1 kb in size, removing exon 10 of EXT1. The inversion in combination with the 8p23.1 deletion most likely abolishes the transcription of EXT1 downstream of exon 10 hence resulting in a truncated protein.The identification of a rare and novel genetic cause of HME, highlights the importance of additional comprehensive investigation of patients with typical clinical manifestations, even when EXT1 and EXT2 mutation analysis is negative.