Molecular Genetic Basis of Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic disease of the myocardium characterized by a hypertrophic left ventricle with a preserved or increased ejection fraction. Cardiac hypertrophy is often asymmetrical, which is associated with left ventricular outflow tract obstruction. Myocyte hypertrophy, disarray, and myocardial fibrosis constitute the histological features of HCM. HCM is a relatively benign disease but an important cause of sudden cardiac death in the young and heart failure in the elderly. Pathogenic variants (PVs) in genes encoding protein constituents of the sarcomeres are the main causes of HCM. PVs exhibit a gradient of effect sizes, as reflected in their penetrance and variable phenotypic expression of HCM. MYH7 and MYBPC3, encoding ß-myosin heavy chain and myosin binding protein C, respectively, are the two most common causal genes and responsible for ≈40% of all HCM cases but a higher percentage of HCM in large families. PVs in genes encoding protein components of the thin filaments are responsible for ≈5% of the HCM cases. Whereas pathogenicity of the genetic variants in large families has been firmly established, ascertainment causality of the PVs in small families and sporadic cases is challenging. In the latter category, PVs are best considered as probabilistic determinants of HCM. Deciphering the genetic basis of HCM has enabled routine genetic testing and has partially elucidated the underpinning mechanism of HCM as increased number of the myosin molecules that are strongly bound to actin. The discoveries have led to the development of mavacamten that targets binding of the myosin molecule to actin filaments and imparts beneficial clinical effects. In the coming years, the yield of the genetic testing is expected to be improved and the so-called missing causal gene be identified. The advances are also expected to enable development of additional specific therapies and editing of the mutations in HCM.

Hypermorphic and hypomorphic AARS alleles in patients with CMT2N expand clinical and molecular heterogeneities.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes implicated in several dominant and recessive disease phenotypes. The canonical function of ARSs is to couple an amino acid to a cognate transfer RNA (tRNA). We identified three novel disease-associated missense mutations in the alanyl-tRNA synthetase (AARS) gene in three families with dominant axonal Charcot-Marie-Tooth (CMT) disease. Two mutations (p.Arg326Trp and p.Glu337Lys) are located near a recurrent pathologic change in AARS, p.Arg329His. The third (p.Ser627Leu) is in the editing domain of the protein in which hitherto only mutations associated with recessive encephalopathies have been described. Yeast complementation assays demonstrated that two mutations (p.Ser627Leu and p.Arg326Trp) represent loss-of-function alleles, while the third (p.Glu337Lys) represents a hypermorphic allele. Further, aminoacylation assays confirmed that the third mutation (p.Glu337Lys) increases tRNA charging velocity. To test the effect of each mutation in the context of a vertebrate nervous system, we developed a zebrafish assay. Remarkably, all three mutations caused a pathological phenotype of neural abnormalities when expressed in zebrafish, while expression of the human wild-type messenger RNA (mRNA) did not. Our data indicate that not only functional null or hypomorphic alleles, but also hypermorphic AARS alleles can cause dominantly inherited axonal CMT disease.

LRSAM1-mediated ubiquitylation is disrupted in axonal Charcot-Marie-Tooth disease 2P.

Charcot-Marie-Tooth (CMT) disease type 2 is a genetically heterogeneous group of inherited neuropathies characterized by motor and sensory deficits as a result of peripheral axonal degeneration. We recently reported a frameshift (FS) mutation in the Really Interesting New Gene finger (RING) domain of LRSAM1 (c.2121_2122dup, p.Leu708Argfs) that encodes an E3 ubiquitin ligase, as the cause of axonal-type CMT (CMT2P). However, the frequency of LRSAM1 mutations in CMT2 and the functional basis for their association with disease remains unknown. In this study, we evaluated LRSAM1 mutations in two large Dutch cohorts. In the first cohort (n = 107), we sequenced the full LRSAM1 coding exons in an unbiased fashion, and, in the second cohort (n = 468), we specifically sequenced the last, RING-encoding exon in individuals where other CMT-associated genes had been ruled out. We identified a novel LRSAM1 missense mutation (c.2120C > T, p.Pro707Leu) mapping to the RING domain. Based on our genetic analysis, the occurrence of pathogenic LRSAM1 mutations is estimated to be rare. Functional characterization of the FS, the identified missense mutation, as well as of another recently reported pathogenic missense mutation (c.2081G > A, p.Cys694Tyr), revealed that in vitro ubiquitylation activity was largely abrogated. We demonstrate that loss of the E2-E3 interaction that is an essential prerequisite for supporting ubiquitylation of target substrates, underlies this reduced ubiquitylation capacity. In contrast, LRSAM1 dimerization and interaction with the bona fide target TSG101 were not disrupted. In conclusion, our study provides further support for the role of LRSAM1 in CMT and identifies LRSAM1-mediated ubiquitylation as a common determinant of disease-associated LRSAM1 mutations.

Soft tissue angiofibroma: Clinicopathologic, immunohistochemical and molecular analysis of 14 cases.

Soft tissue angiofibroma is rare and has characteristic histomorphological and genetic features. For diagnostic purposes, there are no specific antibodies available. Fourteen lesions (6 females, 8 males; age range 7-67 years) of the lower extremities (12) and trunk (2) were investigated by immunohistochemistry, including for the first time NCOA2. NCOA2 was also tested in a control group of other spindle cell lesions. The known fusion-genes (AHRR-NCOA2 and GTF2I-NCOA2) were examined using RT-PCR in order to evaluate their diagnostic value. Cases in which no fusion gene was detected were additionally analysed by RNA sequencing. All cases tested showed nuclear expression of NCOA2. However, this was not specific since other spindle cell neoplasms also expressed this marker in a high percentage of cases. Other variably positive markers were EMA, SMA, desmin and CD34. STAT6 was negative in the cases tested. By RT-PCR for the most frequently observed fusions, an AHRR-NCOA2 fusion transcript was found in 9/14 cases. GTF2I-NCOA2 was not detected in the remaining cases (n = 3). RNA sequencing revealed three additional positive cases; two harbored a AHRR-NCOA2 fusion and one case a novel GAB1-ABL1 fusion. Two cases failed molecular analysis due to poor RNA quality. In conclusion, the AHRR-NCOA2 fusion is a frequent finding in soft tissue angiofibroma, while GTF2I-NCOA2 seems to be a rare genetic event. For the first time, we report a GAB1-ABL1 fusion in a soft tissue angiofibroma of a child. Nuclear expression of NCOA2 is not discriminating when compared with other spindle cell neoplasms.

tRNA splicing endonuclease mutations cause pontocerebellar hypoplasia.

Pontocerebellar hypoplasias (PCH) represent a group of neurodegenerative autosomal recessive disorders with prenatal onset, atrophy or hypoplasia of the cerebellum, hypoplasia of the ventral pons, microcephaly, variable neocortical atrophy and severe mental and motor impairments. In two subtypes, PCH2 and PCH4, we identified mutations in three of the four different subunits of the tRNA-splicing endonuclease complex. Our findings point to RNA processing as a new basic cellular impairment in neurological disorders.

The hippo pathway is activated and is a causal mechanism for adipogenesis in arrhythmogenic cardiomyopathy.

RATIONALE: Mutations in the intercalated disc proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (AC). AC is characterized by the replacement of cardiac myocytes by fibro-adipocytes, cardiac dysfunction, arrhythmias, and sudden death. OBJECTIVE: To delineate the molecular pathogenesis of AC. METHODS AND RESULTS: Localization and levels of selected intercalated disc proteins, including signaling molecules, were markedly reduced in human hearts with AC. Altered protein constituents of intercalated discs were associated with activation of the upstream Hippo molecules in the human hearts, in Nkx2.5-Cre:Dsp(W/F) and Myh6:Jup mouse models of AC, and in the PKP2 knockdown HL-1 myocytes (HL-1(PKP2:shRNA)). Level of active protein kinase C-α isoform, which requires PKP2 for activity, was reduced. In contrast, neurofibromin 2 (or Merlin), a molecule upstream of the Hippo pathway and that is inactivated by protein kinase C-α isoform, was activated. Consequently, the downstream Hippo molecules mammalian STE20-like protein kinases 1/2 (MST1/2), large tumor suppressor kinases 1/2 (LATS1/2), and Yes-associated protein (YAP) (the latter is the effector of the pathway) were phosphorylated. Coimmunoprecipitation detected binding of phosphorylated YAP, phosphorylated ß-catenin, and junction protein plakoglobin (the latter translocated from the junction). RNA sequencing, transcript quantitative polymerase chain reaction, and reporter assays showed suppressed activity of SV40 transcriptional enhancer factor domain (TEAD) and transcription factor 7-like 2 (TCF7L2), which are transcription factors of the Hippo and the canonical Wnt signaling, respectively. In contrast, adipogenesis was enhanced. Simultaneous knockdown of Lats1/2, molecules upstream to YAP, rescued inactivation of YAP and ß-catenin and adipogenesis in the HL-1(PKP2:shRNA) myocytes. CONCLUSIONS: Molecular remodeling of the intercalated discs leads to pathogenic activation of the Hippo pathway, suppression of the canonical Wnt signaling, and enhanced adipogenesis in AC. The findings offer novel mechanisms for the pathogenesis of AC.

A frameshift mutation in LRSAM1 is responsible for a dominant hereditary polyneuropathy.

Despite the high number of genes identified in hereditary polyneuropathies/Charcot-Marie-Tooth (CMT) disease, the genetic defect in many families is still unknown. Here we report the identification of a new gene for autosomal dominant axonal neuropathy in a large three-generation family. Linkage analysis identified a 5 Mb region on 9q33-34 with a LOD score of 5.12. Sequence capture and next-generation sequencing of the region of interest identified five previously unreported non-synonymous heterozygous single nucleotide changes or indels, four of which were confirmed by Sanger sequencing. Two sequence variants co-segregated with the disease, and one, a 2 bp insertion in the last exon of LRSAM1, was also absent in 676 ethnicity-matched control chromosomes. This frameshift mutation (p.Leu708Argfx28) is located in the C-terminal RING finger motif of the encoded protein. Ubiquitin ligase activity in transfected cells with constructs carrying the patient mutation was affected as measured by a higher level of abundance of TSG101, the only reported target of LRSAM1. Injections of morpholino oligonucleotides in zebrafish embryos directed against the ATG or last splice site of zebrafish Lrsam1 disturbed neurodevelopment, showing a less organized neural structure and, in addition, affected tail formation and movement. LRSAM1 is highly expressed in adult spinal cord motoneurons as well as in fetal spinal cord and muscle tissue. Recently, a homozygous mutation in LRSAM1 was proposed as a strong candidate for the disease in a family with recessive axonal polyneuropathy. Our data strongly support the hypothesis that LRSAM1 mutations can cause both dominant and recessive forms of CMT.

A rare loss-of-function SCN5A variant is associated with lidocaine-induced ventricular fibrillation.

The human genome contains over 4 million variant sites, as compared with the reference genome, including rare sequence variants, which have the potential to exert large phenotypic effects, such as susceptibility to drug toxicity. We report identification and functional characterization of a rare non-synonymous (p.A1427S) variant in the SCN5A gene that was associated with incessant and lethal ventricular tachycardia and fibrillation after administration of lidocaine to a patient with acute myocardial infarction. The variant, located in a highly conserved domain distinct from the predicted lidocaine-binding site, decreased peak current density of the sodium channel. With the increasing availability of the whole exome and whole genome sequencing data, it would be possible to identify and characterize rare variants in SCN5A that might predispose to lethal ventricular arrhythmias.

Recessive MYL2 mutations cause infantile type I muscle fibre disease and cardiomyopathy.

A cardioskeletal myopathy with onset and death in infancy, morphological features of muscle type I hypotrophy with myofibrillar disorganization and dilated cardiomyopathy was previously reported in three Dutch families. Here we report the genetic cause of this disorder. Multipoint parametric linkage analysis of six Dutch patients identified a homozygous region of 2.1 Mb on chromosome 12, which was shared between all Dutch patients, with a log of odds score of 10.82. Sequence analysis of the entire linkage region resulted in the identification of a homozygous mutation in the last acceptor splice site of the myosin regulatory light chain 2 gene (MYL2) as the genetic cause. MYL2 encodes a myosin regulatory light chain (MLC-2V). The myosin regulatory light chains bind, together with the essential light chains, to the flexible neck region of the myosin heavy chain in the hexameric myosin complex and have a structural and regulatory role in muscle contraction. The MYL2 mutation results in use of a cryptic splice site upstream of the last exon causing a frameshift and replacement of the last 32 codons by 20 different codons. Whole exome sequencing of an Italian patient with similar clinical features showed compound heterozygosity for two other mutations affecting the same exon of MYL2, also resulting in mutant proteins with altered C-terminal tails. As a consequence of these mutations, the second EF-hand domain is disrupted. EF-hands, assumed to function as calcium sensors, can undergo a conformational change upon binding of calcium that is critical for interactions with downstream targets. Immunohistochemical staining of skeletal muscle tissue of the Dutch patients showed a diffuse and weak expression of the mutant protein without clear fibre specificity, while normal protein was absent. Heterozygous missense mutations in MYL2 are known to cause dominant hypertrophic cardiomyopathy; however, none of the parents showed signs of cardiomyopathy. In conclusion, the mutations in the last exon of MYL2 are responsible for a novel autosomal recessive lethal myosinopathy due to defects changing the C-terminal tail of the ventricular form of the myosin regulatory light chain. We propose 'light chain myopathy' as a name for this MYL2-associated myopathy.

Mechanisms for nonrecurrent genomic rearrangements associated with CMT1A or HNPP: rare CNVs as a cause for missing heritability.

Genomic rearrangements involving the peripheral myelin protein gene (PMP22) in human chromosome 17p12 are associated with neuropathy: duplications cause Charcot-Marie-Tooth disease type 1A (CMT1A), whereas deletions lead to hereditary neuropathy with liability to pressure palsies (HNPP). Our previous studies showed that >99% of these rearrangements are recurrent and mediated by nonallelic homologous recombination (NAHR). Rare copy number variations (CNVs) generated by nonrecurrent rearrangements also exist in 17p12, but their underlying mechanisms are not well understood. We investigated 21 subjects with rare CNVs associated with CMT1A or HNPP by oligonucleotide-based comparative genomic hybridization microarrays and breakpoint sequence analyses, and we identified 17 unique CNVs, including two genomic deletions, ten genomic duplications, two complex rearrangements, and three small exonic deletions. Each of these CNVs includes either the entire PMP22 gene, or exon(s) only, or ultraconserved potential regulatory sequences upstream of PMP22, further supporting the contention that PMP22 is the critical gene mediating the neuropathy phenotypes associated with 17p12 rearrangements. Breakpoint sequence analysis reveals that, different from the predominant NAHR mechanism in recurrent rearrangement, various molecular mechanisms, including nonhomologous end joining, Alu-Alu-mediated recombination, and replication-based mechanisms (e.g., FoSTeS and/or MMBIR), can generate nonrecurrent 17p12 rearrangements associated with neuropathy. We document a multitude of ways in which gene function can be altered by CNVs. Given the characteristics, including small size, structural complexity, and location outside of coding regions, of selected rare CNVs, their identification remains a challenge for genome analysis. Rare CNVs may potentially represent an important portion of "missing heritability" for human diseases.

Presence of C11orf95-MKL2 fusion is a consistent finding in chondroid lipomas: a study of eight cases.

AIMS: Chondroid lipomas are benign adipose tissue tumours. Their rarity and peculiar morphology can lead to misinterpretation, especially in small biopsies. Based on a recurrent translocation t(11;16)(q13;p13), the C11orf95-MKL2 fusion gene has been found in a few cases. Therefore, it seemed appropriate to look for this fusion gene in a larger cohort. METHODS AND RESULTS: We describe eight further cases from four females and four males with an age range of 21-81 years (median 49 years). The tumours were situated in the lower arm (three), lower leg (two), thigh (one), back (one) and head (one); seven lesions were deep-seated and one was located subcutaneously. Sizes ranged from 3 to 12 cm (median 6.3 cm). All patients were treated by simple excision, and follow-up, available for six patients (range 2 months-12 years; median 15 months), demonstrated recurrence in one case. Histologically, the circumscribed and lobulated tumours showed a variable composition of adipocytes, lipoblasts, hibernoma-like cells and chondroblast-like cells embedded in a chondroid matrix. Immunohistochemistry, performed in four cases, revealed positivity for S-100 and pancytokeratin in two of three neoplasms stained for each marker. A C11orf95-MKL2 fusion gene was shown by RT-PCR analysis in seven of the eight cases. CONCLUSIONS: Molecular analysis can be used to support the diagnosis of chondroid lipoma, especially in small samples. This may be helpful in planning treatment when the differential diagnosis includes malignant lesions.

Strategic approaches to unraveling genetic causes of cardiovascular diseases.

DNA sequence variants are major components of the "causal field" for virtually all medical phenotypes, whether single gene familial disorders or complex traits without a clear familial aggregation. The causal variants in single gene disorders are necessary and sufficient to impart large effects. In contrast, complex traits are attributable to a much more complicated network of contributory components that in aggregate increase the probability of disease. The conventional approach to identification of the causal variants for single gene disorders is genetic linkage. However, it does not offer sufficient resolution to map the causal genes in small families or sporadic cases. The approach to genetic studies of complex traits entails candidate gene or genome-wide association studies. Genome-wide association studies provide an unbiased survey of the effects of common genetic variants (common disease-common variant hypothesis). Genome-wide association studies have led to identification of a large number of alleles for various cardiovascular diseases. However, common alleles account for a relatively small fraction of the total heritability of the traits. Accordingly, the focus has shifted toward identification of rare variants that might impart larger effect sizes (rare variant-common disease hypothesis). This shift is made feasible by recent advances in massively parallel DNA sequencing platforms, which afford the opportunity to identify virtually all common as well as rare alleles in individuals. In this review, we discuss various strategies that are used to delineate the genetic contribution to medically important cardiovascular phenotypes, emphasizing the utility of the new deep sequencing approaches.

Nuclear plakoglobin is essential for differentiation of cardiac progenitor cells to adipocytes in arrhythmogenic right ventricular cardiomyopathy.

RATIONALE: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disease of desmosome proteins characterized by fibroadipogenesis in the myocardium. We have implicated signaling properties of junction protein plakoglobin (PG) in the pathogenesis of ARVC. OBJECTIVE: To delineate the pathogenic role of PG in adipogenesis in ARVC. METHODS AND RESULTS: We generated mice overexpressing PG, either a wildtype (PG(WT)) or a truncated (PG(TR)), known to cause ARVC, in the heart; and PG null (PG⁻/⁻) embryos. PG(WT) and PG(TR) mice exhibited fibro-adiposis, cardiac dysfunction, and premature death. Subcellular protein fractionation and immunofluorescence showed nuclear localization of PG(WT) and PG(TR) and reduced membrane localization of PG(TR). Coimmunoprecipitation showed reduced binding of PG(TR) but not PG(WT) to desmosome proteins DSP and DSG2. Transgene PG(WT) and PG(TR) were expressed in c-Kit+:Sca1+ cardiac progenitor cells (CPCs) isolated from the hearts of PG(WT) and PG(TR) by fluorescence activated cell sorting. CPCs isolated from the transgenic hearts showed enhanced adipogenesis, increased levels of adipogenic factors KLF15, C/EBP-α and noncanonical Wnt5b, and reduced level of CTGF, an inhibitor of adipogenesis. Treatment with BIO activated the canonical Wnt signaling, reversed the proadipogenic transcriptional switch and prevented adipogenesis in a dose-dependent manner. Moreover, c-Kit+ CPCs, isolated from PG⁻/⁻ embryos, were resistant to adipogenesis, expressed high mRNA levels of CTGF and other canonical Wnt signaling targets. CONCLUSIONS: Nuclear PG provokes adipogenesis in c-Kit+ CPCs by repressing the canonical Wnt signaling and inducing a proadipogenic gene expression. The findings suggest that adipocytes in ARVC, at least in part, originate from c-Kit+ CPCs.

Histone demethylase KDM5 regulates cardiomyocyte maturation by promoting fatty acid oxidation, oxidative phosphorylation, and myofibrillar organization.

Rationale: Human pluripotent stem cell-derived CMs (iPSC-CMs) are a valuable tool for disease modeling, cell therapy and to reconstruct the CM maturation process and identify, characterize factors that regulate maturation. The transition from immature fetal to adult CM entails coordinated regulation of the mature gene programming, which is characterized by the induction of myofilament and OXPHOS gene expression among others. Recent studies in Drosophila , C. elegans, and C2C12 myoblast cell lines have implicated the histone H3K4me3 demethylase KDM5 and its homologs, as a potential regulator of developmental gene program and mitochondrial function. We speculated that KDM5 may potentiate the maturation of iPSC-CMs by targeting a conserved epigenetic program that encompass mitochondrial OXPHOS and other CM specific maturation genes. Objectives: The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation. Methods and Results: Immunoblot analysis revealed that KDM5A, B, and C expression was progressively downregulated in postnatal cardiomyocytes and absent in adult hearts and CMs. Additionally, KDM5 proteins were found to be persistently expressed in iPSC-CMs up to 60 days after the onset of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor-resulted in differential regulation of 2,372 genes including upregulation of Fatty acid oxidation (FAO), OXPHOS, and myogenic gene programs in iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the CUT&RUN assay revealed enriched H3K4me3 peaks at the promoter regions of FAO, OXPHOS, and sarcomere genes. Consistent with the chromatin and gene expression data, KDM5 inhibition led to increased expression of multiple sarcomere proteins, enhanced myofibrillar organization and improved calcium handling. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene, which is known to regulate OXPHOS and cardiomyocyte maturation, and resulted in its increased RNA and protein levels. Finally, KDM5 inhibition increased baseline, peak, and spare oxygen consumption rates in iPSC-CMs. Conclusions: KDM5 regulates the maturation of iPSC-CMs by epigenetically regulating the expression of ESRRA, OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.

Pathogenic variants in three families with distal muscle involvement.

Three families suspected of distal hereditary motor neuropathy underwent genetic screening with the aim to identify the molecular defect underlying the disease. The description of the identification reflects the shift in molecular diagnostics that was made during the last decades. Our candidate gene approach yielded a known pathogenic variant in BSCL2 (p.Asn88Ser) in one family, and via a CMT-capture, in HSPB1 (p.Arg127Trp), in addition to five other variations in Charcot-Marie-Tooth-related genes in the proband of the second family. In the third family, using whole exome sequencing, followed by linkage-by-location, a three base pair deletion in exon 33 of MYH7 (p.Glu1508del) was found, a reported pathogenic allele albeit for a myopathy. After identification of the causative molecular defect, cardiac examination was performed for patients of the third family and this demonstrated abnormalities in three out of five affected family members. Heterogeneity and expansion of clinical phenotypes beyond known characteristics requires a wider set of genes to be screened. Whole exome/genome analysis with limited prior clinical information may therefore be used to precede a detailed clinical evaluation in cases of large families, preventing screening of a too narrow set of genes, and enabling the identification of novel disease-associated genes. In our cases, the variants had been reported, and co-segregation analysis confirmed the molecular diagnosis.

The enigma of genetics etiology of atherosclerosis in the post-GWAS era.

Coronary atherosclerosis is a complex heritable trait with an enigmatic genetic etiology. Genome-wide association studies (GWAS) have successfully led to identification of over 100 different loci for susceptibility to coronary atherosclerosis. Most identified single nucleotide polymorphisms (SNP)s and genes have not been previously implicated in the pathogenesis of atherosclerosis and hence, have modest biological plausibility. The novel discoveries, however, might provide the opportunity for identification of new pathways and consequently novel preventive and therapeutic targets. A notable outcome of GWAS is relatively modest effect sizes of the associated SNPs. Collectively, the identified SNPs account for a relatively small fraction of heritability of coronary atherosclerosis, which raises the question of "missing heritability". Because GWAS test the common disease-comment variant hypothesis, a plausible explanation might be the presence of uncommon and rare variants in the genome that are untested in GWAS but that might exert large effect sizes on the risk of atherosclerosis. The latter, however, remains an empiric question pending validation through experimentation. Alternative mechanisms, such as transgenerational epigenetics including microRNAs, might in part account for the heritability of coronary atherosclerosis. Collectively, the recent findings are indicative of the etiological complexity of coronary atherosclerosis. Hence, it is expected that genetic etiology of coronary atherosclerosis will remain enigmatic in the foreseeable future.

LAD-1/variant syndrome is caused by mutations in FERMT3.

Leukocyte adhesion deficiency-1/variant (LAD1v) syndrome presents early in life and manifests by infections without pus formation in the presence of a leukocytosis combined with a Glanzmann-type bleeding disorder, resulting from a hematopoietic defect in integrin activation. In 7 consanguineous families, we previously established that this defect was not the result of defective Rap1 activation, as proposed by other investigators. In search of the genetic defect, we carried out homozygosity mapping in 3 of these patients, and a 13-Mb region on chromosome 11 was identified. All 7 LAD1v families share the same haplotype, in which 3 disease-associated sequence variants were identified: a putative splice site mutation in CALDAGGEF1 (encoding an exchange factor for Rap1), an intronic 1.8-kb deletion in NRXN2, and a premature stop codon (p.Arg509X) in FERMT3. Two other LAD1v patients were found to carry different stop codons in FERMT3 (p.Arg573X and p.Trp229X) and lacked the CALDAGGEF1 and NRXN2 mutations, providing convincing evidence that FERMT3 is the gene responsible for LAD1v. FERMT3 encodes kindlin-3 in hematopoietic cells, a protein present together with integrins in focal adhesions. Kindlin-3 protein expression was undetectable in the leukocytes and platelets of all patients tested. These results indicate that the LAD1v syndrome is caused by truncating mutations in FERMT3.

Heparin-associated anti-Xa activity and platelet-derived prothrombotic and proinflammatory biomarkers in moderate to high-risk patients with acute coronary syndrome.

Heparin compounds, to include fractionated and unfractionated preparations, exert both antithrombotic and antiinflammatory effects through combined inhibition of factor Xa and thrombin. The contribution of modulated platelet activity in vivo is less clearly defined. The SYNERGY library was a prospectively designed repository for candidate clinical, hemostatic, platelet, and molecular biomarkers from patients participating in SYNERGY--a large-scale, randomized clinical trial evaluating the comparative benefits of unfractionated heparin (UFH) and enoxaparin in high-risk patients with acute coronary syndrome (ACS). Samples were collected from 201 patients enrolled at 26 experienced, participating sites and shipped to established core laboratories for analysis of platelet, endothelium-derived, inflammatory and coagulation activity biomarkers. Tissue factor pathway inhibitor (TFPI)--a vascular endothelial cell-derived factor Xa regulatory protein-correlated directly with plasma anti-Xa activity (unadjusted: r = 0.23, P < 0.0001; adjusted: ß = 0.10; P = 0.001), as did TFPI-fXa complexes (unadjusted: r = 0.34, P < 0.0001; adjusted: ß = 0.38; P = < 0.0001). In contrast, there was a direct and inverse relationship between anti-Xa activity and two platelet-derived biomarkers-plasminogen activator inhibitor-1 (unadjusted: r = -0.18, P = 0.0012; adjusted: ß = -0.10; P = 0.021) and soluble CD40 ligand (unadjusted: r = -0.11, P = 0.05; adjusted: ß = -0.13; P = 0.049). All measured analyte relationships persisted after adjustment for age, creatinine clearance, weight, sex, and duration of treatment. Differences in biomarkers between patients receiving UFH and those randomized to enoxaparin were not observed. The ability of heparin compounds to affect the prothrombotic and proinflammatory states which characterize ACS may involve factor Xa-related modulation of platelet activation and expression. Whether this potentially beneficial effect is direct or indirect and achieved, at least in part, through the release of endothelial cell-derived coagulation regulatory proteins will require further investigation.