Donor splice-site mutation in CUL4B is likely cause of X-linked intellectual disability.

X-linked intellectual disability is the most common form of cognitive disability in males. Syndromic intellectual disability encompasses cognitive deficits with other medical and behavioral manifestations. Recently, a large family with a novel form of syndromic X-linked intellectual disability was characterized. Eight of 24 members of the family are male and had cognitive dysfunction, short stature, aphasia, skeletal abnormalities, and minor anomalies. To identify the causative gene(s), we performed exome sequencing in three affected boys, both parents, and an unaffected sister. We identified a haplotype consisting of eight variants located in cis within the linkage region that segregated with affected members in the family. Of these variants, two were novel. The first was at the splice-donor site of intron 7 (c.974+1G>T) in the cullin-RING ubiquitin ligase (E3) gene, CUL4B. This variant is predicted to result in failure to splice and remove intron 7 from the primary transcript. The second variant mapped to the 3'-UTR region of the KAISO gene (c.1127T>G). Sanger sequencing validated the variants in these relatives as well as in three affected males and five carriers. The KAISO gene variant was predicted to create a binding site for the microRNAs miR-4999 and miR-4774; however, luciferase expression assays failed to validate increased targeting of these miRNAs to the variant 3'-UTR. This SNP may affect 3'-UTR structure leading to decreased mRNA stability. Our results suggest that the intellectual disability phenotype in this family is caused by aberrant splicing and removal of intron 7 from CUL4B gene primary transcript.

Whole-exome sequencing of DNA from peripheral blood mononuclear cells (PBMC) and EBV-transformed lymphocytes from the same donor.

BACKGROUND: The creation of lymphoblastoid cell lines (LCLs) through Epstein-Barr virus (EBV) transformation of B-lymphocytes can result in a valuable biomaterial for cell biology research and a renewable source of DNA. While LCLs have been used extensively in cellular and genetic studies, the process of cell transformation and expansion during culturing may introduce genomic changes that may impact their use and the interpretation of subsequent genetic findings. RESULTS: We performed whole exome sequencing on a tetrad family using DNA derived from peripheral blood mononuclear cells (PBMCs) and LCLs from each individual. We generated over 4.7 GB of mappable sequence to a 125X read coverage per sample. An average of 19,354 genetic variants were identified. Comparison of the two DNA sources from each individual showed an average concordance rate of 95.69%. By lowering the variant calling parameters, the concordance rate between the paired samples increased to 99.82%. Sanger sequencing of a subset of the remaining discordant variants did confirm the presence of de novo mutations arising in LCLs. CONCLUSIONS: By varying software stringency parameters, we identified 99% concordance between DNA sequences derived from the two different sources from the same donors. These results suggest that LCLs are an appropriate representation of the genetic material of the donor and suggest that EBV transformation can result in low-level generation of de novo mutations. Therefore, use of PBMC or early passage EBV-transformed cells is recommended. These findings have broad-reaching implications, as there are thousands of LCLs in public biorepositories and individual laboratories.

Relationship between beta4 hydrogen bond and beta6 hydrophobic interactions during aggregate, fiber or crystal formation in oversaturated solutions of hemoglobin A and S.

Oversaturated deoxy-alpha(2)beta(2)(T4V) aggregated instantly without a delay time, which is in contrast to the delay time before the generation of fibers of deoxy-HbS and deoxy-alpha(2)beta(2)(E6V,D73H). Solubility of deoxy-alpha(2)beta(2)(T4V) was approximately 10-fold lower than that of deoxy-HbS and was similar to oxy- and deoxy-alpha(2)beta(2)(E6V,T4V). These results indicate that beta4Val in HbA in the oxy and deoxy forms with or without beta6Val facilitates hydrophobic interaction of the A-helix with the EF helix of adjacent molecules without forming a beta4/beta73 hydrogen bond. Deoxy-HbA generated crystals following aggregation as does HbC-Harlem(alpha(2)beta(2)(E6V,D73N)), while alpha(2)beta(2)(T4V) and alpha(2)beta(2)(D73H) as well as HbS, alpha(2)beta(2)(E6V,D73H) and alpha(2)beta(2)(E6V,T4V) in the oxy and deoxy forms did not form crystals, indicating in addition to the strength of beta6 amino acid hydrophobicity that the synergism between the beta4Thr hydrogen bond and beta6 hydrophobic interaction free energies on the A-helix play a critical role in formation of fibers versus crystalline nuclei during phase transition.

Role of the beta4Thr-beta73Asp hydrogen bond in HbS polymer and domain formation from multinucleate-containing clusters.

Fiber formation and domain formation from deoxy-HbS as well as from beta4 and beta73 HbS variants were investigated after temperature jump using DIC microscopy to gain a basic understanding of the determinants involved. Oversaturated deoxy-HbS generated numerous 14-stranded fibers and formed ovoid-shaped, multispherulitic domains. Domain number increased linearly as a function of time. Oversaturated deoxy-alpha2beta2(E6V,T4S) also generated time-dependent, ovoid-shaped spherulitic domains like HbS and alpha 2beta2(E6V,D73H) in the deoxy form. In contrast, alpha 2beta2(E6V,T4Y) and HbC-Harlem (alpha2beta2(E6V,D73N)) in the deoxy form generated time-dependent, ball-shaped domains containing many straight, crystalline-like fibers without evidence of branching. Some of these domains formed large needlelike crystals after overnight incubation. The inhibitory effect on polymer formation by beta4Tyr in HbS was stronger than that by beta4Ser but weaker than that by beta73Asn or beta73Leu. In contrast, both deoxy- and oxy-alpha2beta2(E6V,T4V) promoted formation of tiny, disordered amorphous aggregates without a delay time like oxy-HbS, which is in contrast to formation after a delay time of needlelike fibers for alpha 2beta2(E6V,D73L). Solubilities for both deoxy- and oxy-alpha 2beta2(E6V,T4V) were similar to that of deoxy-alpha 2beta2(E6V,D73H) but approximately 10-fold lower than that of deoxy-HbS. These results suggest that the strength of the hydrogen bond between beta4Thr and beta73Asp and the balance between the hydrogen bond and beta6Val hydrophobic interactions in deoxy-HbS polymers control formation of different types of fibers in a single domain or lead to formation of disordered, non-nucleated amorphous aggregates. These results also lead to a model in which multinucleation rather than a single-nucleation event occurs in a single cluster to generate numerous fibers growing from a single domain.

Genotyping beta-globin gene mutations on copolymer-coated glass slides with the ligation detection reaction.

BACKGROUND: Methods are needed to analyze small amounts of samples for variation in disease-causing genes. One means is to couple the sensitivity and multiplexing capability of the ligation detection reaction (LDR) with the use of simple glass slides specifically functionalized with a novel polymer coating to enhance sensitivity. METHODS: We developed an array-based genotyping assay based on glass slides coated with copolymer (N,N-dimethylacrylamide, N,N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate). The assay consists of an LDR with genomic DNA followed by a universal PCR (U-PCR) of genomic DNA-templated LDR product. The LDR occurs in the presence of 3 primers for each sequence variant under investigation: 2 distinguishing primers (allele specific and perfectly complementary to wild-type and mutant alleles) and 1 common locus-specific primer. The 2 allele-specific primers have different capture sequences for binding different complementary probes on a tag array. The LDR product templated from genomic DNA is made fluorescent during the U-PCR via incorporation of a Cy5-labeled universal primer into all LDR products; detection occurs on the coated glass slides. RESULTS: The assay was designed to detect 7 prevalent mutations in the beta-globin gene (HBB, hemoglobin, beta) in a multiplex format, and signals for the different alleles are detected by their fluorescence. The assay was applied to 40 genomic DNA samples from both control individuals and patients with known beta-thalassemia mutations. Results show good correspondence between the patients' genotypes as assessed by DNA sequence analysis and those generated from the LDR assays. CONCLUSIONS: The developed technology allows accurate identification of sequence variants in a simple, cost-effective way and offers good flexibility for scaling to other applications with different numbers of single-nucleotide polymorphisms or mutations to be detected.

Applications of nanoparticles to diagnostics and therapeutics in colorectal cancer.

Nanotechnology has considerable promise for the detection, staging and treatment of cancer. Here, we outline one such promising application: the use of nanostructures with surface-bound ligands for the targeted delivery and ablation of colorectal cancer (CRC), the third most common malignancy and the second most common cause of cancer-related mortality in the US. Normal colonic epithelial cells as well as primary CRC and metastatic tumors all express a unique surface-bound guanylyl cyclase C (GCC), which binds the diarrheagenic bacterial heat-stable peptide enterotoxin ST. This makes GCC a potential target for metastatic tumor ablation using ST-bound nanoparticles in combination with thermal ablation with near-infrared or radiofrequency energy absorption. Furthermore, the incorporation of iron or iron oxide into such structures would provide advantages for magnetic resonance imaging (MRI). Although the scenarios outlined in this article are hypothetical, they might stimulate ideas about how other cancers could be attacked using nanotechnology.

The Hb A variant (beta73 Asp-->Leu) disrupts Hb S polymerization by a novel mechanism.

Polymerization of a 1:1 mixture of hemoglobin S (Hb S) and the artificial mutant HbAbeta73Leu produces a dramatic morphological change in the polymer domains in 1.0 M phosphate buffer that are a characteristic feature of polymer formation. Instead of feathery domains with quasi 2-fold symmetry that characterize polymerization of Hb S and all previously known mixtures such as Hb A/S and Hb F/S mixtures, these domains are compact structures of quasi-spherical symmetry. Solubility of Hb S/Abeta73Leu mixtures was similar to that of Hb S/F mixtures. Kinetics of polymerization indicated that homogeneous nucleation rates of Hb S/Abeta73Leu mixtures were the same as those of Hb S/F mixtures, while exponential polymer growth (B) of Hb S/Abeta73Leu mixtures were about three times slower than those of Hb S/F mixtures. Differential interference contrast (DIC) image analysis also showed that fibers in the mixture appear to elongate between three and five times more slowly than in equivalent Hb S/F mixtures by direct measurements of exponential growth of mass of polymer in a domain. We propose that these results of Hb S/Abeta73Leu mixtures arise from a non-productive binding of the hybrid species of this mixture to the end of the growing polymer. This "cap" prohibits growth of polymers, but by nature is temporary, so that the net effect is a lowered growth rate of polymers. Such a cap is consistent with known features of the structure of the Hb S polymer. Domains would be more spherulitic because slower growth provides more opportunity for fiber bending to spread domains from their initial 2-fold symmetry. Moreover, since monomer depletion proceeds more slowly in this mixture, more homogeneous nucleation events occur, and the resulting gel has a far more granular character than normally seen in mixtures of non-polymerizing hemoglobins with Hb S. This mixture is likely to be less stiff than polymerized mixtures of other hybrids such as Hb S with HbF, potentially providing a novel approach to therapy.

Combinatorial sequencing-by-hybridization: analysis of the NF1 gene.

Neurofibromatosis type 1 (NF1), one of the most common autosomal dominant disorders, is caused by mutations in the NF1 gene. A variety of methods are currently used in clinical settings to define disease-causing mutations. We describe microarray-based combinatorial sequencing-by-hybridization (cSBH), which overcomes some disadvantages associated with other techniques. Sequence readout of 2 kb was achieved on a single slide, with detection of base substitutions, insertions and small deletions. In addition, cSBH analysis of the entire NF1 gene demonstrates reproducibility, efficiency and reduced time; therefore, representing an alternative to extensive DNA sequence characterization.

Nanobiotechnology: the promise and reality of new approaches to molecular recognition.

Nanobiotechnology is the convergence of engineering and molecular biology that is leading to a new class of multifunctional devices and systems for biological and chemical analysis with better sensitivity and specificity and a higher rate of recognition. Nano-objects with important analytical applications include nanotubes, nanochannels, nanoparticles, nanopores and nanocapacitors. Here, we take a critical look at the subset of recent developments in this area relevant to molecular recognition. Potential benefits of using nano-objects (nanotubes, quantum dots, nanorods and nanoprisms) and nanodevices (nanocapacitors, nanopores and nanocantilevers) leading to an expanded range of label multiplexing are described along with potential applications in future diagnostics. We also speculate on further pathways in nanotechnology development and the emergence of order in this somewhat chaotic, yet promising, new field.

Molecular diagnostics: hurdles for clinical implementation.

In the coming years, molecular diagnostics will continue to be of critical importance to public health worldwide. It will facilitate the detection and characterization of disease, as well as monitoring of the drug response, and will assist in the identification of genetic modifiers and disease susceptibility. A wide range of molecular-based tests is available to assess DNA variation and changes in gene expression. However, there are major hurdles to overcome before the implementation of these tests in clinical laboratories, such as which test to employ, the choice of technology and equipment, and issues such as cost-effectiveness, accuracy, reproducibility, personnel training, reimbursement by third-party payers and intellectual property. At present, PCR-based testing predominates; however, alternative technologies aimed at reducing genome complexity without PCR are anticipated to gain momentum in the coming years. Furthermore, development of integrated chip devices ("lab-on-a-chip") should allow point-of-care testing and facilitate genetic readouts from single cells and molecules. Together with proteomic-based testing, these advances will improve molecular diagnostic testing and will present additional challenges for implementing such testing in health care settings.

Identification of APC gene mutations in colorectal cancer using universal microarray-based combinatorial sequencing-by-hybridization.

Familial adenomatous polyposis (FAP) is an autosomal dominant inherited form of colorectal cancer, caused mostly by mutations in the APC gene. Due to the wide variety of mutations found and the large size of the APC gene, several methods of mutation detection are used, which can be time consuming and costly. Here we demonstrate a new method of mutation detection in the APC gene using an array-based approach termed combinatorial sequencing-by-hybridization (cSBH). In cSBH, a universal probe set is attached to a support and a second one is in solution. Two-probe ligation occurs when a DNA strand from the target PCR product consecutively anneals to both unlabeled array-bound and solution-phase dye-labeled probe, creating all target-complementary long-labeled probes attached to the surface. A standard array reader scores fluorescent signals at each array position. Cell lines and patient DNA with known APC gene mutations were analyzed using a cSBH-based HyChip trade mark product. Results show that this universal hexamer (6-mer) chip can successfully detect a range of mutations. Results are very robust for a continuous readout of 3.6 kb from a PCR target, with 99.97% accuracy on a single HyChip trade mark slide. cSBH is a fast, cost-efficient method for first stage mutation screening in the APC or any other gene.

Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss.

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .

Erythroid-induced commitment of K562 cells results in clusters of differentially expressed genes enriched for specific transcription regulatory elements.

Understanding regulation of fetal and embryonic hemoglobin expression is critical, since their expression decreases clinical severity in sickle cell disease and beta-thalassemia. K562 cells, a human erythroleukemia cell line, can differentiate along erythroid or megakaryocytic lineages and serve as a model for regulation of fetal/embryonic globin expression. We used microarray expression profiling to characterize transcriptomes from K562 cells treated for various times with hemin, an inducer of erythroid commitment. Approximately 5,000 genes were expressed irrespective of treatment. Comparative expression analysis (CEA) identified 899 genes as differentially expressed; analysis by the self-organizing map (SOM) algorithm clustered 425 genes into 8 distinct expression patterns, 322 of which were shared by both analyses. Differential expression of a subset of genes was validated by real-time RT-PCR. Analysis of 5'-flanking regions from differentially expressed genes by PAINT v3.0 software showed enrichment in specific transcription regulatory elements (TREs), some localizing to different expression clusters. This finding suggests coordinate regulation of cluster members by specific TREs. Finally, our findings provide new insights into rate-limiting steps in the appearance of heme-containing hemoglobin tetramers in these cells.

Mechanisms of endothelial cell attachment, proliferation, and differentiation on 4 types of platinum-based endovascular coils.

OBJECTIVE: A subarachnoid hemorrhage is neurologically devastating, with 50% of patients becoming disabled or deceased. Advent of Guglielmi detachable coils in 1995 permitted endovascular treatment of cerebral aneurysms. Coiling is efficacious and safe, but durability needs improvement, as nearly 20% of patients require further invasive intervention secondary to aneurysm recurrence. The aim of this study is to develop an in vitro model of endothelial cell (EC) proliferation and differentiation on four types of platinum-based coils, using gene expression profiling to understand EC biology as they colonize and differentiate on coils. METHODS: Human umbilical vein ECs were grown in vitro on platinum coil segments. Growth patterns were assessed as a function of coil type. Gene expression profiles for coil attached versus coil unattached ECs were determined using immunohistochemistry and gene array analysis. RESULTS: ECs showed rapid, robust attachment to all coil types. Some detachment occurred within 24-48 hours. Significant growth of remaining attached cells occurred during the next week, creating a confluence on coils and within coil grooves. Similar growth curve results were obtained with human brain ECs on platinum-based coil surfaces. Differentiation markers in attached cells (α(1), α(2), ß(1) integrins) were expressed on immunostaining, whereas microarray gene expression revealed 48 up-regulated and 68 down-regulated genes after 24-hour growth on coils. Major pathways affected as a function of time of colonization on coils and coil type included those involved in regulation of cell cycle and cell signaling. CONCLUSIONS: We developed an in vitro model for evaluating endothelialization of platinum coils to optimize coil design to support robust EC colonization and differentiation.

Identification of a developmental gene expression signature, including HOX genes, for the normal human colonic crypt stem cell niche: overexpression of the signature parallels stem cell overpopulation during colon tumorigenesis.

Our goal was to identify a unique gene expression signature for human colonic stem cells (SCs). Accordingly, we determined the gene expression pattern for a known SC-enriched region--the crypt bottom. Colonic crypts and isolated crypt subsections (top, middle, and bottom) were purified from fresh, normal, human, surgical specimens. We then used an innovative strategy that used two-color microarrays (∼18,500 genes) to compare gene expression in the crypt bottom with expression in the other crypt subsections (middle or top). Array results were validated by PCR and immunostaining. About 25% of genes analyzed were expressed in crypts: 88 preferentially in the bottom, 68 in the middle, and 131 in the top. Among genes upregulated in the bottom, ∼30% were classified as growth and/or developmental genes including several in the PI3 kinase pathway, a six-transmembrane protein STAMP1, and two homeobox (HOXA4, HOXD10) genes. qPCR and immunostaining validated that HOXA4 and HOXD10 are selectively expressed in the normal crypt bottom and are overexpressed in colon carcinomas (CRCs). Immunostaining showed that HOXA4 and HOXD10 are co-expressed with the SC markers CD166 and ALDH1 in cells at the normal crypt bottom, and the number of these co-expressing cells is increased in CRCs. Thus, our findings show that these two HOX genes are selectively expressed in colonic SCs and that HOX overexpression in CRCs parallels the SC overpopulation that occurs during CRC development. Our study suggests that developmental genes play key roles in the maintenance of normal SCs and crypt renewal, and contribute to the SC overpopulation that drives colon tumorigenesis.

Use of linkage analysis, genome-wide association studies, and next-generation sequencing in the identification of disease-causing mutations.

For the past two decades, linkage analysis and genome-wide analysis have greatly advanced our knowledge of the human genome. But despite these successes the genetic architecture of diseases remains unknown. More recently, the availability of next-generation sequencing has dramatically increased our capability for determining DNA sequences that range from large portions of one individual's genome to targeted regions of many genomes in a cohort of interest. In this review, we highlight the successes and shortcomings that have been achieved using genome-wide association studies (GWAS) to identify the variants contributing to disease. We further review the methods and use of new technologies, based on next-generation sequencing, that are becoming increasingly used to expand our knowledge of the causes of genetic disease.

High-resolution SNP arrays in mental retardation diagnostics: how much do we gain?

We used Affymetrix 6.0 GeneChip SNP arrays to characterize copy number variations (CNVs) in a cohort of 70 patients previously characterized on lower-density oligonucleotide arrays affected by idiopathic mental retardation and dysmorphic features. The SNP array platform includes approximately 900,000 SNP probes and 900,000 non-SNP oligonucleotide probes at an average distance of 0.7 Kb, which facilitates coverage of the whole genome, including coding and noncoding regions. The high density of probes is critical for detecting small CNVs, but it can lead to data interpretation problems. To reduce the number of false positives, parameters were set to consider only imbalances >75 Kb encompassing at least 80 probe sets. The higher resolution of the SNP array platform confirmed the increased ability to detect small CNVs, although more than 80% of these CNVs overlapped to copy number 'neutral' polymorphism regions and 4.4% of them did not contain known genes. In our cohort of 70 patients, of the 51 previously evaluated as 'normal' on the Agilent 44K array, the SNP array platform disclosed six additional CNV changes, including three in three patients, which may be pathogenic. This suggests that about 6% of individuals classified as 'normal' using the lower-density oligonucleotide array could be found to be affected by a genomic disorder when evaluated with the higher-density microarray platforms.

Assembly of recently translated full-length and C-terminal truncated human gamma-globin chains with a pool of alpha-globin chains to form Hb F in a cell-free system.

Assembly of alpha-globin with translated, full-length and C-terminal truncated human gamma-globin to form Hb F was assessed in a cell-free transcription/translation system. Polysome profiles showed two amino acid C-terminal-truncated gamma-chains retained on polysomes can assemble with unlabeled holo alpha-chains only after puromycin-induced chain release. Two amino acid C-terminal truncated gamma-chains encoded from vectors containing a stop codon at the translation termination site were released from polysomes and assembled with alpha-chains in the absence of puromycin addition, while removal of 11 or more amino acids from the gamma-chain carboxy-terminus inhibited assembly with alpha-chains. These results suggest that amino acids in the HC- and H-helix gamma-chain regions including amino acids 135-144 at the C-terminus in the translated gamma-chains play a key role in assembly with alpha-chains, and that assembly occurs soon after exit of translated gamma-chains from the ribosome tunnel and release from polysomes thereby preventing stable gamma(2) homo-dimer formation.

Opportunities for near-infrared thermal ablation of colorectal metastases by guanylyl cyclase C-targeted gold nanoshells.

Colorectal cancer is the third most common malignancy and the second most common cause of cancer-related mortality worldwide. While surgery remains the mainstay of therapy, approximately 50% of patients who undergo resection develop parenchymal metastatic disease. Unfortunately, current therapeutic regimens offer little improvement to the survival of patients with parenchymal metastases in the liver and lung. In that context, there is a significant unrealized opportunity at the intersection of engineering and biology for the development of novel targeted therapeutic approaches to colorectal cancer metastases. This opportunity exploits the discovery that an intestinal receptor, guanylyl cyclase C, which mediates diarrhea induced by bacterial heat-stable enterotoxins (STs), is over-expressed by metastatic colorectal tumors only. Moreover, it leverages recent advances in the fabrication of metal nanoshells with defined thicknesses absorb near-infrared (NIR) light, resulting in resonance and transfer of thermal energies of more than 40 degrees C. Thus, the conjugation of ST to gold nanoshells, which can undergo resonance excitation by NIR light and emit heat, represents a previously unrecognized approach for the targeted therapy of parenchymal colorectal cancer metastases, specifically to the liver and lung. This article discusses the potential of ST-targeted nanoshells for NIR thermal ablation of metastatic colorectal tumors and highlights the significant challenges and solutions linked to the translation of this emerging technology to patient care.

Inhibition of hemoglobin S polymerization in vitro by a novel 15-mer EF-helix beta73 histidine-containing peptide.

Our mutational studies on Hb S showed that the Hb S beta73His variant (beta6Val and beta73His) promoted polymerization, while Hb S beta73Leu (beta6Val and beta73Leu) inhibited polymerization. On the basis of these results, we speculated that EF-helix peptides containing beta73His interact with beta4Thr in Hb S and compete with Hb S, resulting in inhibition of Hb S polymerization. We, therefore, studied inhibitory effects of 15-, 11-, 7-, and 3-mer EF-helix peptides containing beta73His on Hb S polymerization. The delay time prior to Hb S polymerization increased only in the presence of the 15-mer His peptide; the higher the amount, the longer the delay time. DIC image analysis also showed that the fiber elongation rate for Hb S polymers decreased with increasing concentration of the 15-mer His peptide. In contrast, the same 15-mer peptide containing beta73Leu instead of His and peptides shorter than 11 amino acids containing beta73His including His alone showed little effect on the kinetics of polymerization and elongation of polymers. Analysis by protein-chip arrays showed that only the 15-mer beta73His peptide interacted with Hb S. CD spectra of the 15-mer beta73His peptide did not show a specific helical structure; however, computer docking analysis suggested a lower energy for interaction of Hb S with the 15-mer beta73His peptide compared to peptides containing other amino acids at this position. These results suggest that the 15-mer beta73His peptide interacts with Hb S via the beta4Thr in the betaS-globin chain in Hb S. This interaction may influence hydrogen bond interaction between beta73Asp and beta4Thr in Hb S polymers and interfere in hydrophobic interactions of beta6Val, leading to inhibition of Hb S polymerization.