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BACKGROUND: Von Willebrand disease (VWD), the most prevalent hereditary bleeding disorder, results from deficiency of von Willebrand factor (VWF). OBJECTIVES: This large cohort study aims to offer a comprehensive exploration of mutation spectra and laboratory features in quantitative VWF deficiencies, shedding light on genetic underpinnings and genotype-phenotype associations. METHODS: Our cohort consisted of 221 Caucasian index patients with quantitative VWD, along with 47 individuals whose plasma VWF levels fell within the lower normal boundaries (50-70 IU/dL). We conducted comprehensive VWF assays and genetic analyses, encompassing VWF gene sequencing, copy number variation investigations, and bioinformatic assessments. RESULTS: Following International Society on Thrombosis and Haemostasis-Scientific and Standardization Committee VWF guidelines, 77 index patients were characterized as having type 1 VWD (VWF antigen [VWF:Ag] < 30 IU/dL), 111 as having type 1 VWD (VWF:Ag, 30-50 IU/dL), and 33 as having type 3 VWD. Mutation detection rates were 88%, 65%, and 92%, respectively. Notably, blood group O overrepresentation was evident in type 1 with VWF:Ag of 30 to 50 IU/dL, particularly among mutation-negative patients, suggesting a potential causal role of blood group O. A total of 223 VWF variants, comprising 147 distinct variations, were identified in quantitative VWD patients, of which 57 were novel variants (39%). Additionally, approximately 70% of individuals with VWF levels within the lower normal boundaries (50-70 IU/dL) displayed VWF variants. CONCLUSION: Our data advance our understanding of the molecular mechanisms underlying quantitative VWD, offering valuable insights for future research and clinical management. Distinct mutation patterns were observed among subgroups, particularly the contrast between type 1 VWD (VWF:Ag < 30 IU/dL) and type 1 VWD (VWF:Ag, 30-50 IU/dL), an area with limited prior investigation.
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Access to accurate force-field parameters for small molecules is crucial for computational studies of their interactions with proteins. Although a number of general force fields for small molecules exist, e.g., CGenFF, GAFF, and OPLS, they do not cover all common chemical groups and their combinations. The Force Field Toolkit (ffTK) provides a comprehensive graphical interface that streamlines the development of classical parameters for small molecules directly from quantum mechanical (QM) calculations, allowing for force-field generation for almost any chemical group and validation of the fit relative to the target data. ffTK relies on supported external software for the QM calculations, but it can generate the necessary QM input files and parse and analyze the QM output. In previous ffTK versions, support for Gaussian and ORCA QM packages was implemented. Here, we add support for Psi4, an open-source QM package free for all users, thereby broadening user access to ffTK. We also compare the parameter sets obtained with the new ffTK version using Gaussian, ORCA, and Psi4 for three molecules: pyrrolidine, n-propylammonium cation, and chlorobenzene. Despite minor differences between the resulting parameter sets for each compound, most prominently in the dihedral and improper terms, we show that conformational distributions sampled in molecular dynamics simulations using these parameter sets are quite comparable.
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Hepatitis B virus (HBV) is the leading cause of chronic liver pathologies worldwide. HBV nucleocapsid, a key structural component, is formed through the self-assembly of the capsid protein units. Therefore, interfering with the self-assembly process is a promising approach for the development of novel antiviral agents. Applied to HBV, this approach has led to several classes of capsid assembly modulators (CAMs). Here, we report structurally novel CAMs with moderate activity and low toxicity, discovered through a biophysics-guided approach combining docking, molecular dynamics simulations, and a series of assays with a particular emphasis on biophysical experiments. Several of the identified compounds induce the formation of aberrant capsids and inhibit HBV DNA replication in vitro, suggesting that they possess modest capsid assembly modulation effects. The synergistic computational and experimental approaches provided key insights that facilitated the identification of compounds with promising activities. The discovery of preclinical CAMs presents opportunities for subsequent optimization efforts, thereby opening new avenues for HBV inhibition.
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Cápside , Virus de la Hepatitis B , Cápside/metabolismo , Proteínas de la Cápside , Ensamble de Virus , NucleocápsideRESUMEN
Treatment of mixed phosphonium-iodonium ylides featuring a six-membered phenoxaphosphonium fragment with aqueous tetrafluoroboronic acid induces a rearrangement, resulting in expansion of the phosphacycle and oxidation of the phosphorus atom. The target difficult-to-access dibenzo[b,f][1,4]oxaphosphepine oxides (3 examples) were isolated in excellent yields (up to 95%) as mixtures of stereoisomers. Hydrolysis of a five-membered mixed ylide, a dibenzophosphole derivative, predominantly preserves the phosphole system with cycle expansion occurring as a side process.
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Congenital factor VII (FVII) deficiency is a rare genetic bleeding disorder characterized by deficient or reduced activity of coagulation FVII. It is caused by genetic variants in the F7 gene. We aimed to evaluate the rate of detection of pathogenic variants in the F7 gene in a large group of patients with FVII deficiency and investigate the correlations between the F7 genotype and FVII activity (FVII:C). Moreover, the influence of the common genetic variant rs6046: c.1238G>A; p.(Arg413Gln), designated as the M2 allele, on FVII:C was investigated. Genetic analysis of the F7 gene was performed on 704 index patients (IPs) using either direct Sanger- or next-generation sequencing. Genetic variants were detected in 390 IPs, yielding a variant detection rate (VDR) of 55%. Notably, the VDR exhibited a linear decline with increasing FVII:C levels. We identified 124 genetic variants, of which 48 were not previously reported. Overall, the frequency of the M2 allele was considerably higher in patients with mild deficiency (FVII:C > 20 IU/dl). Furthermore, IPs lacking an identified pathogenic variant exhibited a significantly higher prevalence of the M2 allele (69%) compared to IPs with a disease-causing variant (47%). These results strongly support the association of the M2 allele with decreased FVII:C levels. This study shows the utility of FVII:C as a predictive marker for identifying pathogenic variants in patients with FVII deficiency. The M2 allele contributes to the reduction of FVII:C levels, particularly in cases of mild deficiency.
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Deficiencia del Factor VII , Humanos , Deficiencia del Factor VII/genética , Mutación , Fenotipo , Factor VII/genética , GenotipoRESUMEN
BACKGROUND: The characterization of inherited mild factor XIII deficiency is more imprecise than its rare, inherited severe forms. It is known that heterozygosity at FXIII genetic loci results in mild FXIII deficiency, characterized by circulating FXIII activity levels ranging from 20% to 60%. There exists a gap in information on 1) how genetic heterozygosity renders clinical bleeding manifestations among these individuals and 2) the reversal of unexplained bleeding upon FXIII administration in mild FXIII-deficient individuals. OBJECTIVES: To assess the prevalence and burden of mild FXIII deficiency among the apparently healthy German-Caucasian population and correlate it with genetic heterozygosity at FXIII and fibrinogen gene loci. METHODS: Peripheral blood was collected from 752 donors selected from the general population with essentially no bleeding complications to ensure asymptomatic predisposition. These were assessed for FXIII and fibrinogen activity, and FXIII and fibrinogen genes were resequenced using next-generation sequencing. For comparison, a retrospective analysis was performed on a cohort of mild inherited FXIII deficiency patients referred to us. RESULTS: The prevalence of mild FXIII deficiency was high (â¼0.8%) among the screened German-Caucasian population compared with its rare-severe forms. Although no new heterozygous missense variants were found, certain combinations were relatively dominant/prevalent among the mild FXIII-deficient individuals. CONCLUSION: This extensive, population-based quasi-experimental approach revealed that the burden of heterozygosity in FXIII and fibrinogen gene loci causes the clinical manifestation of inherited mild FXIII deficiency, resulting in ''unexplained bleeding'' upon provocation.
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Deficiencia del Factor XIII , Factor XIII , Hemostáticos , Humanos , Factor XIII/genética , Deficiencia del Factor XIII/diagnóstico , Deficiencia del Factor XIII/genética , Fibrinógeno/genética , Hemorragia/diagnóstico , Hemorragia/genética , Estudios RetrospectivosRESUMEN
ABSTRACT: Allogeneic hematopoietic cell transplantation (HCT) is a curative therapy for hematological malignancies for which graft-versus-host disease (GVHD) remains a major complication. The use of donor T-regulatory cells (Tregs) to prevent GVHD appears promising, including in our previous evaluation of an engineered graft product (T-reg graft) consisting of the timed, sequential infusion of CD34+ hematopoietic stem cells and high-purity Tregs followed by conventional T cells. However, whether immunosuppressive prophylaxis can be removed from this protocol remains unclear. We report the results of the first stage of an open-label single-center phase 2 study (NCT01660607) investigating T-reg graft in myeloablative HCT of HLA-matched and 9/10-matched recipients. Twenty-four patients were randomized to receive T-reg graft alone (n = 12) or T-reg graft plus single-agent GVHD prophylaxis (n = 12) to determine whether T-reg graft alone was noninferior in preventing acute GVHD. All patients developed full-donor myeloid chimerism. Patients with T-reg graft alone vs with prophylaxis had incidences of grade 3 to 4 acute GVHD of 58% vs 8% (P = .005) and grade 3 to 4 of 17% vs 0% (P = .149), respectively. The incidence of moderate-to-severe chronic GVHD was 28% in the T-reg graft alone arm vs 0% with prophylaxis (P = .056). Among patients with T-reg graft and prophylaxis, CD4+ T-cell-to-Treg ratios were reduced after transplantation, gene expression profiles showed reduced CD4+ proliferation, and the achievement of full-donor T-cell chimerism was delayed. This study indicates that T-reg graft with single-agent tacrolimus is preferred over T-reg graft alone for the prevention of acute GVHD. This trial was registered at www.clinicaltrials.gov as #NCT01660607.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Humanos , Tacrolimus/uso terapéutico , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/prevención & control , Enfermedad Injerto contra Huésped/patología , Inmunosupresores/uso terapéutico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Donantes de TejidosRESUMEN
IgA nephropathy (IgAN) is an autoimmune disorder which is believed to be non-monogenic. We performed an exome-wide association study of 70 children with IgAN and 637 healthy donors. The HLA allele frequencies were compared between the patients and healthy donors from the bone marrow registry of the Pirogov University. We tested 78,020 gene markers for association and performed functional enrichment analysis and transcription factor binding preference detection. We identified 333 genetic variants, employing three inheritance models. The most significant association with the disorder was observed for rs143409664 (PRAG1) in the case of the additive and dominant models (PBONF = 1.808 × 10-15 and PBONF = 1.654 × 10-15, respectively), and for rs13028230 (UBR3) in the case of the recessive model (PBONF = 1.545 × 10-9). Enrichment analysis indicated the strongly overrepresented "immune system" and "kidney development" terms. The HLA-DQA1*01:01:01G allele (p = 0.0076; OR, 2.021 [95% CI, 1.322-3.048]) was significantly the most frequent among IgAN patients. Here, we characterized, for the first time, the genetic background of Russian IgAN patients, identifying the risk alleles typical of the population. The most important signals were detected in previously undescribed loci.
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Glomerulonefritis por IGA , Ubiquitina-Proteína Ligasas , Niño , Humanos , Estudios de Casos y Controles , Exoma/genética , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Glomerulonefritis por IGA/genética , Glomerulonefritis por IGA/diagnóstico , Polimorfismo de Nucleótido Simple , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Serum albumin is currently in the focus of biomedical research as a promising platform for the creation of multicomponent self-assembling systems due to the presence of several sites with high binding affinity of various compounds in its molecule, including lipophilic oligonucleotide conjugates. In this work, we investigated the stoichiometry of the dodecyl-containing oligonucleotides binding to bovine and human serum albumins using an electrophoretic mobility shift assay. The results indicate the formation of the albumin-oligonucleotide complexes with a stoichiometry of about 1 : (1.25 ± 0.25) under physiological-like conditions. Using atomic force microscopy, it was found that the interaction of human serum albumin with the duplex of complementary dodecyl-containing oligonucleotides resulted in the formation of circular associates with a diameter of 165.5 ± 94.3 nm and 28.9 ± 16.9 nm in height, and interaction with polydeoxyadenylic acid and dodecyl-containing oligothymidylate resulted in formation of supramolecular associates with the size of about 315.4 ± 70.9 and 188.3 ± 43.7 nm, respectively. The obtained data allow considering the dodecyl-containing oligonucleotides and albumin as potential components of the designed self-assembling systems for solving problems of molecular biology, biomedicine, and development of unique theranostics with targeted action.
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Oligonucleótidos , Albúmina Sérica , Animales , Bovinos , Humanos , Oligonucleótidos/química , Albúmina Sérica/metabolismo , Microscopía de Fuerza Atómica , Ensayo de Cambio de Movilidad ElectroforéticaRESUMEN
BamA, the core component of the ß-barrel assembly machinery (BAM) complex, is an outer-membrane protein (OMP) in Gram-negative bacteria. Its function is to insert and fold substrate OMPs into the outer membrane (OM). Evidence suggests that BamA follows the asymmetric hybrid-barrel model where the first and last strands of BamA separate, a process known as lateral gate opening, to allow nascent substrate OMP ß-strands to sequentially insert and fold through ß-augmentation. Recently, multiple lead compounds that interfere with BamA's function have been identified. We modeled and then docked one of these compounds into either the extracellular loops of BamA or the open lateral gate. With the compound docked in the loops, we found that the lateral gate remains closed during 5 µs molecular dynamics simulations. The same compound when docked in the open lateral gate stays bound to the ß16 strand of BamA during the simulation, which would prevent substrate OMP folding. In addition, we simulated mutants of BamA that are resistant to one or more of the identified lead compounds. In these simulations, we observed a differing degree and/or frequency of opening of BamA's lateral gate compared to BamA-apo, suggesting that the mutations grant resistance by altering the dynamics at the gate. We conclude that the compounds act by inhibiting BamA lateral gate opening and/or binding of substrate, thus preventing subsequent OMP folding and insertion.
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Proteínas de la Membrana , Pliegue de Proteína , Simulación de Dinámica Molecular , MutaciónRESUMEN
BACKGROUND: Elicitation of allergic contact dermatitis (ACD), an inflammatory type 4 hypersensitivity disease, induces skin infiltration by polyclonal effector CD8 αß T cells and precursors of tissue-resident memory T (TRM) cells. Because TRM have long-term potential to contribute to body-surface immunoprotection and immunopathology, their local regulation needs a fuller understanding. OBJECTIVE: We sought to investigate how TRM-cell maturation might be influenced by innate-like T cells pre-existing within many epithelia. METHODS: This study examined CD8+ TRM-cell maturation following hapten-induced ACD in wild-type mice and in strains harboring altered compartments of dendritic intraepidermal γδ T cells (DETCs), a prototypic tissue-intrinsic, innate-like T-cell compartment that reportedly regulates ACD, but by no elucidated mechanism. RESULTS: In addition to eliciting CD8 TRM, ACD induced DETC activation and an intimate coregulatory association of the 2 cell types. This depended on DETC sensing IFN-γ produced by CD8 cells and involved programmed death-ligand 1 (PD-L1). Thus, in mice lacking DETC or lacking IFN-γ receptor solely on γδ cells, ACD-elicited CD8 T cells showed enhanced proliferative and effector potentials and reduced motility, collectively associated with exaggerated ACD pathology. Comparable dysregulation was elicited by PD-L1 blockade in vitro, and IFN-γ-regulated PD-L1 expression was a trait of human skin-homing and intraepithelial γδ T cells. CONCLUSIONS: The size and quality of the tissue-infiltrating CD8 T-cell response during ACD can be profoundly regulated by local innate-like T cells responding to IFN-γ and involving PD-L1. Thus, interindividual and tissue-specific variations in tissue-intrinsic lymphocytes may influence responses to allergens and other challenges and may underpin inflammatory pathologies such as those repeatedly observed in γδ T-cell-deficient settings.
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Dermatitis Alérgica por Contacto , Interferón gamma , Animales , Humanos , Ratones , Antígeno B7-H1 , Linfocitos T CD8-positivos/patología , Dermatitis Alérgica por Contacto/patología , Piel/patologíaRESUMEN
OBJECTIVE: To compare incoming general surgery interns' performance on a basic skills assessment before and after the COVID pandemic. DESIGN: A retrospective cohort study compared surgical skill performances of incoming general surgery interns. Each underwent an evidence-based standardized assessment (pretest) with 12-basic surgical knot tying and suturing tasks. A post-test was administered after a 3-month self-directed skills curriculum. Student's t-tests compared proficiency scores from pre-COVID vs. COVID-era general surgery interns before and after curriculum completion. p < 0.05 was significant. SETTING: Data was collected from surgical residents in an academic general surgery program in the United States. PARTICIPANTS: General surgery interns from 2017 to 2019 (pre-COVID) and 2021 to 2022 (COVID-era) were included. Interns with missing data or extreme outliers were excluded. A total of 100 interns in general surgery were included in the pretest cohort (59 pre-COVID, 41 COVID-era) and 101 interns were in the post-test cohort (66 pre-COVID, 35 COVID-era). RESULTS: COVID-era interns scored significantly lower on the pretest compared to pre-COVID interns (COVID-era 721.9+/-268.8 vs. pre-COVID 935.9+/- 228.0, p < 0.001). After the skills curriculum both cohorts improved their proficiency scores. However COVID-era interns still scored significantly lower (COVID-era 1255.0+/-166.3 vs. pre-COVID 1369.8+/-165.6, pâ¯=â¯0.001). CONCLUSIONS: This analysis objectively described deficits in fundamental surgical skills for incoming interns whose medical school education was disrupted by the COVID-19 pandemic. A targeted surgical skills curriculum partially remediated these deficiencies. However, many surgical interns may need additional intervention and potentially more time in order to fully develop their surgical skills and meet the competency requirements required for advancement.
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COVID-19 , Cirugía General , Internado y Residencia , Humanos , Estados Unidos , Estudios Retrospectivos , Pandemias , COVID-19/epidemiología , Curriculum , Competencia Clínica , Cirugía General/educación , Educación de Postgrado en MedicinaRESUMEN
Viral outbreaks remain a serious threat to human and animal populations and motivate the continued development of antiviral drugs and vaccines, which in turn benefits from a detailed understanding of both viral structure and dynamics. While great strides have been made in characterizing these systems experimentally, molecular simulations have proven to be an essential, complementary approach. In this work, we review the contributions of molecular simulations to the understanding of viral structure, functional dynamics, and processes related to the viral life cycle. Approaches ranging from coarse-grained to all-atom representations are discussed, including current efforts at modeling complete viral systems. Overall, this review demonstrates that computational virology plays an essential role in understanding these systems.
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Simulación de Dinámica Molecular , Estructuras Virales , Animales , HumanosRESUMEN
Large-scale cortical beta (ß) oscillations were implicated in the learning processes, but their exact role is debated. We used MEG to explore the dynamics of movement-related ß-oscillations while 22 adults learned, through trial and error, novel associations between four auditory pseudowords and movements of four limbs. As learning proceeded, spatial-temporal characteristics of ß-oscillations accompanying cue-triggered movements underwent a major transition. Early in learning, widespread suppression of ß-power occurred long before movement initiation and sustained throughout the whole behavioral trial. When learning advanced and performance reached asymptote, ß-suppression after the initiation of correct motor response was replaced by a rise in ß-power mainly in the prefrontal and medial temporal regions of the left hemisphere. This post-decision ß-power predicted trial-by-trial response times (RT) at both stages of learning (before and after the rules become familiar), but with different signs of interaction. When a subject just started to acquire associative rules and gradually improved task performance, a decrease in RT correlated with the increase in the post-decision ß-band power. When the participants implemented the already acquired rules, faster (more confident) responses were associated with the weaker post-decision ß-band synchronization. Our findings suggest that maximal beta activity is pertinent to a distinct stage of learning and may serve to strengthen the newly learned association in a distributed memory network.
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Aprendizaje , Movimiento , Humanos , Adulto , Aprendizaje/fisiología , Tiempo de Reacción/fisiología , Movimiento/fisiología , Lóbulo Temporal , Cognición , Ritmo beta/fisiología , MagnetoencefalografíaRESUMEN
The clonal selection theory describes key features of adaptive immune responses of B and T cells. For αß T cells and B cells, antigen recognition and selection principles are known at a detailed molecular level. The precise role of the antigen receptor in γδ T cells remains less well understood. To better understand the role of the γδ T cell receptor (TCR), we generate an orthotopic TCRδ transgenic mouse model. We demonstrate a multi-layered functionality of γδ TCRs and diverse roles of CDR3δ-mediated selection during γδ T cell development. Whereas epithelial populations using Vγ5 or Vγ7 chains are almost unaffected in their biology in the presence of the transgenic TCRδ chain, pairing with Vγ1 positively selects γδ T cell subpopulations with distinct programs in several organs, thereby distorting the repertoire. In conclusion, our data support dictation of developmental tropism together with adaptive-like recognition principles in a single antigen receptor.
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Linfocitos Intraepiteliales , Receptores de Antígenos de Linfocitos T gamma-delta , Ratones , Animales , Linaje de la Célula , Ratones TransgénicosRESUMEN
The paper discusses a novel three-channel method to distinguish the formation oils, water, and uranine-based drilling mud and to monitor the clean-up process in the downhole fluid analysis systems. The proposed method is based on the study of the fluorescence spectra of the formation fluid or drilling mud samples. The experiment for different excitation sources (365 nm, 395 nm LEDs, and 405 nm, 460 nm laser sources) was carried out; the spectra were recorded with a VISION2GO VIS spectrometer. The characteristic features in fluorescence spectra of the crude oil sample and various concentrations of the uranine solution in pH-neutral water (5 mg/l, 10 mg/l, 25 mg/l, 50 mg/l, 100 mg/l) were observed. The sufficient unambiguous criterion for differentiating the type of fluid and clean-up process analysis is presented.
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This study is the first to assess the internal consistency and factor validity of the Abbreviated Math Anxiety Scale (AMAS) in a sample of Russian adolescents as well as gender differences and gender invariance. The study included 4,218 adolescents in grades 7-9 (M = 14.23, SD = 0.92). Internal consistency, measured with Cronbach's alpha, was high. Analysis of the factor structure revealed the best correspondence of the second-order factor model, which included two scales (learning math anxiety and math evaluation anxiety) and the general scale of math anxiety. There were greater gender differences in the all three scales. Analysis of gender invariance demonstrated that the mathematics anxiety construct was uniform in boys and girls. These findings confirm the reliable psychometric properties and validity of the AMAS, enabling its use in adolescents.
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AIM: To compare the indicators of the spatial organization of collagen and its regulating factors between benign and malignant prostate neoplasms. METHODS: The study involved tumor tissue samples from 40 patients with stage II-III prostate cancer (PCa) and 20 patients with benign prostatic hyperplasia (BPH). The localization of collagen was determined with a Masson trichrome stain. To establish quantitative indicators of the spatial organization of collagen, morphometric studies were carried out with the CurveAlign and ImageJ programs. RESULTS: PCa tissue had two times lower collagen density (P<0.0001) and 1.3 times lower levels of collagen alignment (P=0.018) compared with BPH tissue. In PCa tissue, collagen fibers were shorter (by 24.2%; P<0.001) and thicker (by 15.5%; P<0.001). PCa tissue samples showed significantly higher levels of metalloproteinase (MMP)-2 (by 2.4 times; P=0.001), MMP-8 (by 2.3 times; P=0.007), and MMP-13 (by 1.9 times; P=0.004). CONCLUSIONS: Collagen matrix spatial organization features, as well as its regulatory factors, could be potential biomarkers of malignant prostate neoplasms.
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Hiperplasia Prostática , Neoplasias de la Próstata , Masculino , Humanos , Hiperplasia Prostática/patología , Neoplasias de la Próstata/patología , Biomarcadores , ColágenoRESUMEN
Hemophilia A and hemophilia B are rare congenital, recessive X-linked disorders caused by lack or deficiency of clotting factor VIII (FVIII) or IX (FIX), respectively. The severity of the disease depends on the reduction of coagulation FVIII or FIX activity levels, which is determined by the type of the pathogenic variants in the genes encoding the two factors (F8 and F9, respectively). Molecular genetic analysis is widely applied in inherited bleeding disorders. The outcome of genetic analysis allows genetic counseling of affected families and helps find a link between the genotype and the phenotype. Genetic analysis in hemophilia has tremendously improved in the last decades. Many new techniques and modifications as well as analysis softwares became available, which made the genetic analysis and interpretation of the data faster and more accurate. Advances in genetic variant detection strategies facilitate identification of the causal variants in up to 97% of patients. In this review, we discuss the milestones in genetic analysis of hemophilia and highlight the importance of identification of the causative genetic variants for genetic counseling and particularly for the interpretation of the clinical presentation of hemophilia patients.