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Michael J Bamshad - One of the best experts on this subject based on the ideXlab platform.
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single nucleotide evolutionary constraint scores highlight disease causing mutations
Nature Methods, 2010Co-Authors: Gregory M Cooper, Michael J Bamshad, Jay Shendure, David L Goode, Arend Sidow, Deborah A NickersonAbstract:Identifying disease-causing genetic variants in individual human genomes is a major challenge, even in protein-coding exons (the `exome'). Analysis of nucleotide-level sequence conservation may help address this challenge, on the assumption that purifying selection `constrains' evolutionary divergence at phenotypically important nucleotides. In contrast to functional classifiers (for example, non-synonymous mutations), constraint scores are quantitative and applicable to any genomic position1. However, it remains unclear if constraint scores can facilitate causal variant discovery, as statistical power is estimated to be marginal at the single-nucleotide level given current genome alignments1,2. We therefore applied and assessed a nucleotide-level evolutionary metric to prioritize causal variants in genomes of 16 individuals. We analyzed exomes from four individuals with Freeman-Sheldon Syndrome (FSS; Online Mendelian Inheritance in Man (OMIM) database identifier 193700), a dominant disease caused by mutations in MYH33; four individuals with Miller Syndrome (OMIM identifier 263750), a recessive disease caused by mutations in DHODH4; and eight HapMap samples3. We generated constraint scores by Genomic Evolutionary Rate Profiling (GERP)1 on the mammalian subset of the 44-way MULTIZ/TBA alignments (http://genome.ucsc.edu; for details see2). For each aligned site, GERP defines a `rejected substitution' (RS) score by estimating the actual number of substitutions at that site and subtracting it from the number expected assuming neutrality (~5.82 substitutions per site). Selectively constrained sites tolerate fewer substitutions than neutral sites and have positive RS scores1,2. We first defined the consensus nucleotide from the chimpanzee, gorilla, orangutan, and macaque genomes as ancestral and determined the derived allele frequency (DAF) for each variant in the eight HapMap exomes. We found a significant inverse correlation between DAF and RS score (Fig. 1; p < 0.0001; R2 = 1.4%, slope estimated as −1% DAF per RS). No correlation existed between DAF and the RS score for the nucleotide adjacent to the variant (Supplementary Fig. 1). While the DAF-RS correlation resulted partly from enrichment for singletons at sites with high RS scores, it was significant even within common variants (p < 0.0001; Supplementary Fig. 2). We also found that segregating sites, regardless of DAF, were enriched at sites with low RS scores and progressively depleted as the RS scores increased (Supplementary Fig. 3). Consistent with previous data2, these results suggest that RS scores enrich site-specifically for deleterious variants and non-variant positions at which new mutations would be deleterious. Figure 1 RS scores inversely correlate with DAF of single-nucleotide variants (n=48,750) in 8 HapMap exomes. The average DAF (Y-axis) is plotted for all variants at a site within a given RS bin (X-axis). Error bars show 1 standard error unit. Next, we tested whether constraint scores could enrich for FSS or Miller Syndrome causal variants. We identified candidate disease genes as those in which the affected individuals had variants not seen in the HapMap exomes that affected a nucleotide with a high constraint score. For a comparison, we used functional definitions of deleteriousness, namely non-synonymous, splice-site, or insertion-deletion (indel)3,4. We first used a threshold of RS > 0 (fewer substitutions than expected), and found that this narrowed candidate gene lists nearly as effectively as functional annotations. For example, there were 21 genes in which all FSS samples had a rare, functionally annotated variant3,4 versus 24 genes in which all FSS samples had a rare variant with RS > 0 (Fig. 2a). Increasing the RS threshold, which cannot readily be done with functional annotations, reduced candidate gene lists. At a threshold of RS > 4, for example, MYH3 was one of only five FSS candidate genes, while DHODH was the only Miller Syndrome candidate (Fig. 2b). Figure 2 Constraint scores enrich for disease-causing genes. (a). Number of genes (Y-axis, log-scale) in which at least the given number of FSS individuals (X-axis) has a rare variant that is functionally defined (white), or with increasing RS scores (light gray ... We note that protein-based approaches could similarly be used to reduce candidate gene lists. For example, there were only seven genes in which all FSS individuals harbored a rare variant annotated by PolyPhen5 as `possibly' or `probably' damaging. However, PolyPhen (and related approaches) is restricted to non-synonymous variants, does not facilitate ranking of candidates (see below), and excluded DHODH as a Miller Syndrome candidate4. Finally, we exploited the quantitative nature of constraint scores and ranked genes by the average score of all rare and deleterious (RS > 0) variants in the affected individuals. MYH3 and DHODH ranked highly for their associated diseases, even under models allowing for the possibility of multiple causal genes. For example, requiring only that at least two individuals shared the same causal gene, MYH3 ranked 9th among 666 genes. If we assumed FSS and Miller Syndrome to be monogenic, MYH3 and DHODH ranked as the top candidates, respectively (Fig. 2). RS scores for known or user-defined variants can be obtained from the Genome Variation Server (http://gvs.gs.washington.edu/GVS/) or SeattleSeq annotation pipeline (http://gvs.gs.washington.edu/SeattleSeqAnnotation/). Constraint scores facilitate threshold flexibility and candidate ranking and do not require functional annotations. Even in exomes, this allows for the possibility that synonymous variants contribute to disease6. More importantly, this independence offers exciting potential for the discovery of causal variation in arbitrary genomic segments (for example, linkage peaks) and ultimately resequenced genomes.
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congenital contracture Syndrome caused by mutation in embryonic myosin heavy chain characterized by significant changes in adult muscle contractility
Biophysical Journal, 2010Co-Authors: Alice W Ward, Michael J Bamshad, Anita E Beck, Michael RegnierAbstract:Congenital contracture Syndromes affect 1 out of every 1000 live births, and of those Syndromes, distal arthrogryposis (DA), characterized by contractures of the hands and feet, is the most predominant. In one subtype of DA, Freeman Sheldon Syndrome (FSS), 97% of the cases are caused by mutations in the embryonic myosin heavy chain gene, MYH3. To assess the effects of this mutation on adult muscle contractility, skeletal muscle was obtained from a needle biopsy of the gastrocnemius muscle in an FSS individual (MYH3 R672C) and a control subject were performed and skinned single muscle fibers were dissected for measurements of contractile performance as the [Ca2+] of physiological solutions was varied. The magnitude of passive stiffness was 2x greater for patient fibers. There was no difference in maximal Ca2+ activated force found in the affected adult muscle fibers (0.204uN ± 0.044) compared to normal adult muscle fibers (0.259uN ± 0.028). However specific force was 69% less; this was attributable to hypertrophy of the patient fibers (159um ± 8 as compared to normal control myofibers of 87um ± 3). Little to no change was observed in Ca2+ sensitivity (pCa50) or in cooperativity of the force-pCa relationship. Relaxation was dramatically slower in patient fibers, taking 4x longer to reach 50% relaxation and 10x longer to reach 90% relaxation. Control experiments suggested this is not due to the larger patient fiber size. Preliminary analysis, using a 12.5% agarose gel, and Western Blots, indicated that these differences were not fiber type dependent. Interestingly, we have identified that embryonic myosin (MYH3) is present in single adult muscle cells. This work was supported by HL65497 (Regnier) and HD48895 (Bamshad).
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mutations in embryonic myosin heavy chain myh3 cause freeman sheldon Syndrome and sheldon hall Syndrome
Nature Genetics, 2006Co-Authors: Reha M Toydemir, Ann Rutherford, Frank G Whitby, Lynn B Jorde, John C Carey, Michael J BamshadAbstract:The genetic basis of most conditions characterized by congenital contractures is largely unknown. Here we show that mutations in the embryonic myosin heavy chain (MYH3) gene cause Freeman-Sheldon Syndrome (FSS), one of the most severe multiple congenital contracture (that is, arthrogryposis) Syndromes, and nearly one-third of all cases of Sheldon-Hall Syndrome (SHS), the most common distal arthrogryposis. FSS and SHS mutations affect different myosin residues, demonstrating that MYH3 genotype is predictive of phenotype. A structure-function analysis shows that nearly all of the MYH3 mutations are predicted to interfere with myosin's catalytic activity. These results add to the growing body of evidence showing that congenital contractures are a shared outcome of prenatal defects in myofiber force production. Elucidation of the genetic basis of these Syndromes redefines congenital contractures as unique defects of the sarcomere and provides insights about what has heretofore been a poorly understood group of disorders.
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mutations in tnnt3 cause multiple congenital contractures a second locus for distal arthrogryposis type 2b
American Journal of Human Genetics, 2003Co-Authors: Sandy S Sung, Michael J Bamshad, Lynn B Jorde, John C Carey, Anna Marie E Brassington, P A KrakowiakAbstract:To the Editor: We recently reported that distal arthrogryposis type 1 (DA1 [MIM 108120]) and distal arthrogryposis type 2B (DA2B [MIM 601680]), both of which are characterized by congenital contractures of the hands/wrists and feet/ankles (Bamshad et al. 1996), are caused by mutations in TNNI2 and TPM2, respectively (Sung et al. 2003). TNNI2 encodes an isoform of troponin I; this isoform and the isoforms of troponin T (TnT) and troponin C constitute the troponin complex of fast-twitch myofibers. This complex is the primary sensor of intracellular Ca+2 ion concentration in skeletal muscle, and, consequently, it is an important regulator of muscle contraction. The troponin complex of fast-twitch myofibers exerts its effect on muscle contraction by binding to actin and β-tropomyosin, the product encoded by TPM2 (Clark et al. 2002). These findings led us to hypothesize that mutations in genes encoding other contractile-apparatus proteins specific to fast-twitch myofibers might also cause multiple congenital contractures. We now report the discovery of a mutation, in TNNT3 (the gene encoding TnT specific to fast-twitch myofibers), that causes DA2B. We sequenced TNNT3 in 47 families with either DA2A (classical Freeman-Sheldon Syndrome [MIM 193700]) or DA2B. We found a G→A missense mutation, at nucleotide position 188 in exon 9 of the TNNT3 cDNA (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_006757","term_id":"184172387","term_text":"NM_006757"}}NM_006757), that causes an arginine-to-histidine substitution at amino acid residue 63 (R63H) of TnT in a mother with DA2B and her two affected children (fig. 1). For several reasons, this mutation is probably disease causing. First, the mutation identified in the proband was also present in all affected family members (fig. 1). There is, however, a probability of 1/4 that this pattern occurred by chance. The inference that R63H causes DA2B would be strengthened by demonstrating that this mutation did not occur in the unaffected parents of I-2 (i.e., that it is a de novo mutation). However, the only living parent of I-2 is unavailable for study. Second, this change was not found in 488 chromosomes from an ethnically matched control group that we screened. Third, R63H results in the substitution of an amino acid residue that is conserved in all known isoforms of TnT (fig. 2), implying that this difference is likely to have structural and/or functional consequences. Fourth, substitution of the homologous amino acid residue in the cardiac-specific form of TnT causes cardiomyopathy (Varnava et al. 1999). Figure 1 Electropherogram demonstrating heterozygosity for a G→A missense mutation at nucleotide position 188 in exon 9 of TNNT3 in a family with DA2B. To confirm the presence of this mutation, we incorporated a MluI restriction site into the amplicon ... Figure 2 Amino acid sequences of fast-twitch TnT in human, mouse, and bird, aligned with amino acid sequences of human slow-twitch TnT and human cardiac TnT. Because mutations in TNNI2 have been found in only ∼10% of cases of DA2B, we suspected that DA2B is a genetically heterogeneous condition (Sung et al. 2003). To date, however, linkage studies have not identified any candidate regions other than chromosome 11p15.5 (Krakowiak et al. 1997). The observation that DA2B can be caused by mutations in either TNNI2 or TNNT3 confirms that DA2B is genetically heterogeneous. Because TNNI2 and TNNT3 are located within several hundred kilobases of one another on chromosome 11p15.5, this conclusion is also consistent with the results of our prior linkage studies (Sung et al. 2003). Nevertheless, the absence of mutations in TNNI2 or TNNT3 in most cases of DA2B suggests either that regulatory regions of these genes harbor mutations or that mutations in genes yet to be identified also cause DA2B. Although the cause of DA2B can be distinguished by direct testing of TNNT3 and TNNI2, there appear to be few, if any, ways to distinguish, on the basis of only clinical characteristics, which gene is responsible. There may, however, be sufficient phenotypic differences between DA2B and DA1 to distinguish between them. In addition to the facial features (e.g., small mouth and prominent nasolabial folds) common to DA2B but lacking in individuals with DA1, several characteristics (e.g., vertical talus and scoliosis) are more frequent in DA2B than in DA1. Additionally, the hand and foot contractures in patients with DA2B appear to be more resilient to medical intervention (e.g., occupational therapy and casting). It should be cautioned, however, that mutations have been found in too few families with both DA1 and DA2B to lend much credibility to broad generalizations about genotype-phenotype relationships. The mechanism by which the R63H substitution in TnT in fast-twitch myofibers causes congenital contractures is unknown. Missense mutations in TNNT2—a TNNT3 paralogue, encoding a cardiac-specific form of TnT—cause ∼15% of cases of familial hypertrophic cardiomyopathy (Watkins et al. 1995). One of these mutations is an arginine-to-leucine substitution of amino acid residue 94 (R94L), which is homologous to amino acid residue 63 in fast-twitch myofiber TnT (Varnava et al. 1999). The R94L substitution perturbs tropomyosin-dependent functions of TnT, including the binding of tropomyosin to actin (Palm et al. 2001), an effect that might be due, in part, to impaired flexibility of the N-terminal tail of TnT (Hinkle and Tobacman 2003). The R63H substitution may have a similar effect on TnT in fast-twitch myofibers. The theme that is emerging from this and our previous studies is that perturbation of the function of the contractile apparatus of skeletal muscle during fetal development can cause multiple congenital contractures in individuals with an otherwise normal neuromuscular examination. On the basis of this result, it seems plausible that polymorphisms in one or more of the genes encoding the proteins of the troponin-tropomyosin complex of fast-twitch myofibers may influence an individual’s susceptibility to isolated contractures (e.g., idiopathic clubfoot) or modify the phenotype of common myopathic disorders (e.g., Duchenne muscular dystrophy). At minimum, this report underscores the existence of a new class of genetic muscle diseases that lack many of the findings typical of a heritable myopathy.
Christina A Gurnett - One of the best experts on this subject based on the ideXlab platform.
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exome sequencing identifies an myh3 mutation in a family with distal arthrogryposis type 1
Journal of Bone and Joint Surgery American Volume, 2011Co-Authors: David M Alvarado, Jillian G Buchan, Christina A GurnettAbstract:Distal arthrogryposis is characterized by contractures of the distal regions of the hands and feet1. The severe types of distal arthrogryposis, types 2A (also called Freeman-Sheldon Syndrome) and 2B (also called Sheldon-Hall Syndrome), include facial involvement and scoliosis, whereas distal arthrogryposis type 1 (DA1) does not2. Distal arthrogryposis type 1 affects approximately one in 10,000 children and represents the most common type3. Multiple genes encoding proteins in the sarcomere, including myosin heavy chain 3 (MYH3), myosin heavy chain 8 (MYH8), tropomyosin 2 (TPM2), troponin I2 (TNNI2), troponin T3 (TNNT3), and myosin binding protein C1 (MYBPC1)4-9, have been implicated in distal arthrogryposis Syndromes. Although MYH3 mutations account for nearly all cases of Freeman-Sheldon Syndrome (type 2A) and nearly one-third of all cases of Sheldon-Hall Syndrome (type 2B), to our knowledge MYH3 mutations have not been described in patients with distal arthrogryposis type 14. Previous studies have shown that mutations in known genes are rare causes of distal arthrogryposis type 19,10; therefore, genetic heterogeneity is expected and additional causative genes remain to be identified. Because of the large number of genes as well as the relatively large size of the genes already implicated in distal arthrogryposis Syndromes, new methods to sequence all genes are necessary to effectively define the genetic basis of distal arthrogryposis in individual families. Exome sequencing is a new genetic tool that uses next-generation sequencing methods to identify mutations within the coding exons of the entire human genome (the “exome”)11. Because the exome comprises just 1% of the genome, the cost of sequencing only the exons is currently only a fraction of the cost of whole-genome sequencing. The next-generation exome capture and sequencing methods that were used in this study represent a substantial advance in research methods and, despite their limitations, these methods are likely to soon have a major impact on the diagnosis of patients with inherited musculoskeletal disorders. For instance, our identification of a mutation in MYH3 in the family described in this study broadens the phenotype associated with MYH3 mutations to include distal arthrogryposis type 1 and suggests that there may be substantial overlap between types 1, 2A, and 2B. Distal arthrogryposis type 1 should be considered in the differential diagnosis of isolated clubfoot, particularly in the presence of even minor hand contractures in the patient or family members.
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skeletal muscle contractile gene tnnt3 myh3 tpm2 mutations not found in vertical talus or clubfoot
Clinical Orthopaedics and Related Research, 2009Co-Authors: Christina A Gurnett, Farhang Alaee, David M Desruisseau, Stephanie BoehmAbstract:Arthrogryposis presents with lower limb contractures that resemble clubfoot and/or vertical talus. Recently, mutations in skeletal muscle contractile genes MYH3 (myosin heavy chain 3), TNNT3 (troponin T3), and TPM2 (tropomyosin 2) were identified in patients with distal arthrogryposis DA2A (Freeman-Sheldon Syndrome) or DA2B (Sheldon-Hall Syndrome). We asked whether the contractile genes responsible for distal arthrogryposis are also responsible for cases of familial clubfoot or vertical talus. We determined the frequency of MYH3, TNNT3, and TPM2 mutations in patients with idiopathic clubfoot, vertical talus, and distal arthrogryposis type 1 (DA1). We resequenced the coding exons of the MYH3, TNNT3, and TPM2 genes in 31 patients (five with familial vertical talus, 20 with familial clubfoot, and six with DA1). Variants were evaluated for segregation with disease in additional family members, and the frequency of identified variants was determined in a control population. In one individual with DA1, we identified a de novo TNNT3 mutation (R63H) previously identified in an individual with DA2B. No other causative mutations were identified, though we found several previously undescribed single-nucleotide polymorphisms of unknown importance. Although mutations in MYH3, TNNT3, and TPM2 are frequently associated with distal arthrogryposis Syndromes, they were not present in patients with familial vertical talus or clubfoot. The TNNT3 R63H recurrent mutation identified in two unrelated individuals may be associated with either DA1 or DA2B.
John C Carey - One of the best experts on this subject based on the ideXlab platform.
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mutations in embryonic myosin heavy chain myh3 cause freeman sheldon Syndrome and sheldon hall Syndrome
Nature Genetics, 2006Co-Authors: Reha M Toydemir, Ann Rutherford, Frank G Whitby, Lynn B Jorde, John C Carey, Michael J BamshadAbstract:The genetic basis of most conditions characterized by congenital contractures is largely unknown. Here we show that mutations in the embryonic myosin heavy chain (MYH3) gene cause Freeman-Sheldon Syndrome (FSS), one of the most severe multiple congenital contracture (that is, arthrogryposis) Syndromes, and nearly one-third of all cases of Sheldon-Hall Syndrome (SHS), the most common distal arthrogryposis. FSS and SHS mutations affect different myosin residues, demonstrating that MYH3 genotype is predictive of phenotype. A structure-function analysis shows that nearly all of the MYH3 mutations are predicted to interfere with myosin's catalytic activity. These results add to the growing body of evidence showing that congenital contractures are a shared outcome of prenatal defects in myofiber force production. Elucidation of the genetic basis of these Syndromes redefines congenital contractures as unique defects of the sarcomere and provides insights about what has heretofore been a poorly understood group of disorders.
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mutations in tnnt3 cause multiple congenital contractures a second locus for distal arthrogryposis type 2b
American Journal of Human Genetics, 2003Co-Authors: Sandy S Sung, Michael J Bamshad, Lynn B Jorde, John C Carey, Anna Marie E Brassington, P A KrakowiakAbstract:To the Editor: We recently reported that distal arthrogryposis type 1 (DA1 [MIM 108120]) and distal arthrogryposis type 2B (DA2B [MIM 601680]), both of which are characterized by congenital contractures of the hands/wrists and feet/ankles (Bamshad et al. 1996), are caused by mutations in TNNI2 and TPM2, respectively (Sung et al. 2003). TNNI2 encodes an isoform of troponin I; this isoform and the isoforms of troponin T (TnT) and troponin C constitute the troponin complex of fast-twitch myofibers. This complex is the primary sensor of intracellular Ca+2 ion concentration in skeletal muscle, and, consequently, it is an important regulator of muscle contraction. The troponin complex of fast-twitch myofibers exerts its effect on muscle contraction by binding to actin and β-tropomyosin, the product encoded by TPM2 (Clark et al. 2002). These findings led us to hypothesize that mutations in genes encoding other contractile-apparatus proteins specific to fast-twitch myofibers might also cause multiple congenital contractures. We now report the discovery of a mutation, in TNNT3 (the gene encoding TnT specific to fast-twitch myofibers), that causes DA2B. We sequenced TNNT3 in 47 families with either DA2A (classical Freeman-Sheldon Syndrome [MIM 193700]) or DA2B. We found a G→A missense mutation, at nucleotide position 188 in exon 9 of the TNNT3 cDNA (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_006757","term_id":"184172387","term_text":"NM_006757"}}NM_006757), that causes an arginine-to-histidine substitution at amino acid residue 63 (R63H) of TnT in a mother with DA2B and her two affected children (fig. 1). For several reasons, this mutation is probably disease causing. First, the mutation identified in the proband was also present in all affected family members (fig. 1). There is, however, a probability of 1/4 that this pattern occurred by chance. The inference that R63H causes DA2B would be strengthened by demonstrating that this mutation did not occur in the unaffected parents of I-2 (i.e., that it is a de novo mutation). However, the only living parent of I-2 is unavailable for study. Second, this change was not found in 488 chromosomes from an ethnically matched control group that we screened. Third, R63H results in the substitution of an amino acid residue that is conserved in all known isoforms of TnT (fig. 2), implying that this difference is likely to have structural and/or functional consequences. Fourth, substitution of the homologous amino acid residue in the cardiac-specific form of TnT causes cardiomyopathy (Varnava et al. 1999). Figure 1 Electropherogram demonstrating heterozygosity for a G→A missense mutation at nucleotide position 188 in exon 9 of TNNT3 in a family with DA2B. To confirm the presence of this mutation, we incorporated a MluI restriction site into the amplicon ... Figure 2 Amino acid sequences of fast-twitch TnT in human, mouse, and bird, aligned with amino acid sequences of human slow-twitch TnT and human cardiac TnT. Because mutations in TNNI2 have been found in only ∼10% of cases of DA2B, we suspected that DA2B is a genetically heterogeneous condition (Sung et al. 2003). To date, however, linkage studies have not identified any candidate regions other than chromosome 11p15.5 (Krakowiak et al. 1997). The observation that DA2B can be caused by mutations in either TNNI2 or TNNT3 confirms that DA2B is genetically heterogeneous. Because TNNI2 and TNNT3 are located within several hundred kilobases of one another on chromosome 11p15.5, this conclusion is also consistent with the results of our prior linkage studies (Sung et al. 2003). Nevertheless, the absence of mutations in TNNI2 or TNNT3 in most cases of DA2B suggests either that regulatory regions of these genes harbor mutations or that mutations in genes yet to be identified also cause DA2B. Although the cause of DA2B can be distinguished by direct testing of TNNT3 and TNNI2, there appear to be few, if any, ways to distinguish, on the basis of only clinical characteristics, which gene is responsible. There may, however, be sufficient phenotypic differences between DA2B and DA1 to distinguish between them. In addition to the facial features (e.g., small mouth and prominent nasolabial folds) common to DA2B but lacking in individuals with DA1, several characteristics (e.g., vertical talus and scoliosis) are more frequent in DA2B than in DA1. Additionally, the hand and foot contractures in patients with DA2B appear to be more resilient to medical intervention (e.g., occupational therapy and casting). It should be cautioned, however, that mutations have been found in too few families with both DA1 and DA2B to lend much credibility to broad generalizations about genotype-phenotype relationships. The mechanism by which the R63H substitution in TnT in fast-twitch myofibers causes congenital contractures is unknown. Missense mutations in TNNT2—a TNNT3 paralogue, encoding a cardiac-specific form of TnT—cause ∼15% of cases of familial hypertrophic cardiomyopathy (Watkins et al. 1995). One of these mutations is an arginine-to-leucine substitution of amino acid residue 94 (R94L), which is homologous to amino acid residue 63 in fast-twitch myofiber TnT (Varnava et al. 1999). The R94L substitution perturbs tropomyosin-dependent functions of TnT, including the binding of tropomyosin to actin (Palm et al. 2001), an effect that might be due, in part, to impaired flexibility of the N-terminal tail of TnT (Hinkle and Tobacman 2003). The R63H substitution may have a similar effect on TnT in fast-twitch myofibers. The theme that is emerging from this and our previous studies is that perturbation of the function of the contractile apparatus of skeletal muscle during fetal development can cause multiple congenital contractures in individuals with an otherwise normal neuromuscular examination. On the basis of this result, it seems plausible that polymorphisms in one or more of the genes encoding the proteins of the troponin-tropomyosin complex of fast-twitch myofibers may influence an individual’s susceptibility to isolated contractures (e.g., idiopathic clubfoot) or modify the phenotype of common myopathic disorders (e.g., Duchenne muscular dystrophy). At minimum, this report underscores the existence of a new class of genetic muscle diseases that lack many of the findings typical of a heritable myopathy.
Lynn B Jorde - One of the best experts on this subject based on the ideXlab platform.
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mutations in embryonic myosin heavy chain myh3 cause freeman sheldon Syndrome and sheldon hall Syndrome
Nature Genetics, 2006Co-Authors: Reha M Toydemir, Ann Rutherford, Frank G Whitby, Lynn B Jorde, John C Carey, Michael J BamshadAbstract:The genetic basis of most conditions characterized by congenital contractures is largely unknown. Here we show that mutations in the embryonic myosin heavy chain (MYH3) gene cause Freeman-Sheldon Syndrome (FSS), one of the most severe multiple congenital contracture (that is, arthrogryposis) Syndromes, and nearly one-third of all cases of Sheldon-Hall Syndrome (SHS), the most common distal arthrogryposis. FSS and SHS mutations affect different myosin residues, demonstrating that MYH3 genotype is predictive of phenotype. A structure-function analysis shows that nearly all of the MYH3 mutations are predicted to interfere with myosin's catalytic activity. These results add to the growing body of evidence showing that congenital contractures are a shared outcome of prenatal defects in myofiber force production. Elucidation of the genetic basis of these Syndromes redefines congenital contractures as unique defects of the sarcomere and provides insights about what has heretofore been a poorly understood group of disorders.
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mutations in tnnt3 cause multiple congenital contractures a second locus for distal arthrogryposis type 2b
American Journal of Human Genetics, 2003Co-Authors: Sandy S Sung, Michael J Bamshad, Lynn B Jorde, John C Carey, Anna Marie E Brassington, P A KrakowiakAbstract:To the Editor: We recently reported that distal arthrogryposis type 1 (DA1 [MIM 108120]) and distal arthrogryposis type 2B (DA2B [MIM 601680]), both of which are characterized by congenital contractures of the hands/wrists and feet/ankles (Bamshad et al. 1996), are caused by mutations in TNNI2 and TPM2, respectively (Sung et al. 2003). TNNI2 encodes an isoform of troponin I; this isoform and the isoforms of troponin T (TnT) and troponin C constitute the troponin complex of fast-twitch myofibers. This complex is the primary sensor of intracellular Ca+2 ion concentration in skeletal muscle, and, consequently, it is an important regulator of muscle contraction. The troponin complex of fast-twitch myofibers exerts its effect on muscle contraction by binding to actin and β-tropomyosin, the product encoded by TPM2 (Clark et al. 2002). These findings led us to hypothesize that mutations in genes encoding other contractile-apparatus proteins specific to fast-twitch myofibers might also cause multiple congenital contractures. We now report the discovery of a mutation, in TNNT3 (the gene encoding TnT specific to fast-twitch myofibers), that causes DA2B. We sequenced TNNT3 in 47 families with either DA2A (classical Freeman-Sheldon Syndrome [MIM 193700]) or DA2B. We found a G→A missense mutation, at nucleotide position 188 in exon 9 of the TNNT3 cDNA (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"NM_006757","term_id":"184172387","term_text":"NM_006757"}}NM_006757), that causes an arginine-to-histidine substitution at amino acid residue 63 (R63H) of TnT in a mother with DA2B and her two affected children (fig. 1). For several reasons, this mutation is probably disease causing. First, the mutation identified in the proband was also present in all affected family members (fig. 1). There is, however, a probability of 1/4 that this pattern occurred by chance. The inference that R63H causes DA2B would be strengthened by demonstrating that this mutation did not occur in the unaffected parents of I-2 (i.e., that it is a de novo mutation). However, the only living parent of I-2 is unavailable for study. Second, this change was not found in 488 chromosomes from an ethnically matched control group that we screened. Third, R63H results in the substitution of an amino acid residue that is conserved in all known isoforms of TnT (fig. 2), implying that this difference is likely to have structural and/or functional consequences. Fourth, substitution of the homologous amino acid residue in the cardiac-specific form of TnT causes cardiomyopathy (Varnava et al. 1999). Figure 1 Electropherogram demonstrating heterozygosity for a G→A missense mutation at nucleotide position 188 in exon 9 of TNNT3 in a family with DA2B. To confirm the presence of this mutation, we incorporated a MluI restriction site into the amplicon ... Figure 2 Amino acid sequences of fast-twitch TnT in human, mouse, and bird, aligned with amino acid sequences of human slow-twitch TnT and human cardiac TnT. Because mutations in TNNI2 have been found in only ∼10% of cases of DA2B, we suspected that DA2B is a genetically heterogeneous condition (Sung et al. 2003). To date, however, linkage studies have not identified any candidate regions other than chromosome 11p15.5 (Krakowiak et al. 1997). The observation that DA2B can be caused by mutations in either TNNI2 or TNNT3 confirms that DA2B is genetically heterogeneous. Because TNNI2 and TNNT3 are located within several hundred kilobases of one another on chromosome 11p15.5, this conclusion is also consistent with the results of our prior linkage studies (Sung et al. 2003). Nevertheless, the absence of mutations in TNNI2 or TNNT3 in most cases of DA2B suggests either that regulatory regions of these genes harbor mutations or that mutations in genes yet to be identified also cause DA2B. Although the cause of DA2B can be distinguished by direct testing of TNNT3 and TNNI2, there appear to be few, if any, ways to distinguish, on the basis of only clinical characteristics, which gene is responsible. There may, however, be sufficient phenotypic differences between DA2B and DA1 to distinguish between them. In addition to the facial features (e.g., small mouth and prominent nasolabial folds) common to DA2B but lacking in individuals with DA1, several characteristics (e.g., vertical talus and scoliosis) are more frequent in DA2B than in DA1. Additionally, the hand and foot contractures in patients with DA2B appear to be more resilient to medical intervention (e.g., occupational therapy and casting). It should be cautioned, however, that mutations have been found in too few families with both DA1 and DA2B to lend much credibility to broad generalizations about genotype-phenotype relationships. The mechanism by which the R63H substitution in TnT in fast-twitch myofibers causes congenital contractures is unknown. Missense mutations in TNNT2—a TNNT3 paralogue, encoding a cardiac-specific form of TnT—cause ∼15% of cases of familial hypertrophic cardiomyopathy (Watkins et al. 1995). One of these mutations is an arginine-to-leucine substitution of amino acid residue 94 (R94L), which is homologous to amino acid residue 63 in fast-twitch myofiber TnT (Varnava et al. 1999). The R94L substitution perturbs tropomyosin-dependent functions of TnT, including the binding of tropomyosin to actin (Palm et al. 2001), an effect that might be due, in part, to impaired flexibility of the N-terminal tail of TnT (Hinkle and Tobacman 2003). The R63H substitution may have a similar effect on TnT in fast-twitch myofibers. The theme that is emerging from this and our previous studies is that perturbation of the function of the contractile apparatus of skeletal muscle during fetal development can cause multiple congenital contractures in individuals with an otherwise normal neuromuscular examination. On the basis of this result, it seems plausible that polymorphisms in one or more of the genes encoding the proteins of the troponin-tropomyosin complex of fast-twitch myofibers may influence an individual’s susceptibility to isolated contractures (e.g., idiopathic clubfoot) or modify the phenotype of common myopathic disorders (e.g., Duchenne muscular dystrophy). At minimum, this report underscores the existence of a new class of genetic muscle diseases that lack many of the findings typical of a heritable myopathy.
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de novo mutations in nalcn cause a Syndrome characterized by congenital contractures of the limbs and face hypotonia and developmental delay
American Journal of Human Genetics, 2015Co-Authors: Jessica X Chong, Margaret J Mcmillin, Kathryn M Shively, Anita E Beck, Colby T Marvin, Jose R Armenteros, Kati J Buckingham, Naomi T Nkinsi, Evan A Boyle, Margaret N BerryAbstract:Freeman-Sheldon Syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal-dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five individuals who had been putatively diagnosed with “DA2A with severe neurological abnormalities” and for whom congenital contractures of the limbs and face, hypotonia, and global developmental delay had resulted in early death in three cases; this is a unique condition that we now refer to as CLIFAHDD Syndrome. Exome sequencing identified missense mutations in the sodium leak channel, non-selective (NALCN) in four families affected by CLIFAHDD Syndrome. We used molecular-inversion probes to screen for NALCN in a cohort of 202 distal arthrogryposis (DA)-affected individuals as well as concurrent exome sequencing of six other DA-affected individuals, thus revealing NALCN mutations in ten additional families with “atypical” forms of DA. All 14 mutations were missense variants predicted to alter amino acid residues in or near the S5 and S6 pore-forming segments of NALCN, highlighting the functional importance of these segments. In vitro functional studies demonstrated that NALCN alterations nearly abolished the expression of wild-type NALCN, suggesting that alterations that cause CLIFAHDD Syndrome have a dominant-negative effect. In contrast, homozygosity for mutations in other regions of NALCN has been reported in three families affected by an autosomal-recessive condition characterized mainly by hypotonia and severe intellectual disability. Accordingly, mutations in NALCN can cause either a recessive or dominant condition characterized by varied though overlapping phenotypic features, perhaps based on the type of mutation and affected protein domain(s).
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de novo mutations in nalcn cause a Syndrome of congenital contractures of the limbs and face with hypotonia and developmental delay
bioRxiv, 2015Co-Authors: Jessica X Chong, Margaret J Mcmillin, Kathryn M Shively, Anita E Beck, Colby T Marvin, Jose R Armenteros, Kati J Buckingham, Naomi T Nkinsi, Evan A Boyle, Margaret N BerryAbstract:Freeman-Sheldon Syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five simplex cases putatively diagnosed with “DA2A with severe neurological abnormalities” in which the proband had Congenital Contractures of the LImbs and FAce, Hypotonia, and global Developmental Delay often resulting in early death, a unique condition that we now refer to as CLIFAHDD Syndrome. Exome sequencing identified missense mutations in sodium leak channel, nonselective (NALCN) in four families with CLIFAHDD Syndrome. Using molecular inversion probes to screen NALCN in a cohort of 202 DA cases as well as concurrent exome sequencing of six other DA cases revealed NALCN mutations in ten additional families with “atypical” forms of DA. All fourteen mutations were missense variants predicted to alter amino acid residues in or near the S5 and S6 pore-forming segments of NALCN, highlighting the functional importance of these segments. In vitro functional studies demonstrated that mutant NALCN nearly abolished the expression of wildtype NALCN, suggesting that mutations that cause CLIFAHDD Syndrome have a dominant negative effect. In contrast, homozygosity for mutations in other regions of NALCN has been reported in three families with an autosomal recessive condition characterized mainly by hypotonia and severe intellectual disability. Accordingly, mutations in NALCN can cause either a recessive or dominant condition with varied though overlapping phenotypic features perhaps depending on the type of mutation and affected protein domain(s).
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congenital contracture Syndrome caused by mutation in embryonic myosin heavy chain characterized by significant changes in adult muscle contractility
Biophysical Journal, 2010Co-Authors: Alice W Ward, Michael J Bamshad, Anita E Beck, Michael RegnierAbstract:Congenital contracture Syndromes affect 1 out of every 1000 live births, and of those Syndromes, distal arthrogryposis (DA), characterized by contractures of the hands and feet, is the most predominant. In one subtype of DA, Freeman Sheldon Syndrome (FSS), 97% of the cases are caused by mutations in the embryonic myosin heavy chain gene, MYH3. To assess the effects of this mutation on adult muscle contractility, skeletal muscle was obtained from a needle biopsy of the gastrocnemius muscle in an FSS individual (MYH3 R672C) and a control subject were performed and skinned single muscle fibers were dissected for measurements of contractile performance as the [Ca2+] of physiological solutions was varied. The magnitude of passive stiffness was 2x greater for patient fibers. There was no difference in maximal Ca2+ activated force found in the affected adult muscle fibers (0.204uN ± 0.044) compared to normal adult muscle fibers (0.259uN ± 0.028). However specific force was 69% less; this was attributable to hypertrophy of the patient fibers (159um ± 8 as compared to normal control myofibers of 87um ± 3). Little to no change was observed in Ca2+ sensitivity (pCa50) or in cooperativity of the force-pCa relationship. Relaxation was dramatically slower in patient fibers, taking 4x longer to reach 50% relaxation and 10x longer to reach 90% relaxation. Control experiments suggested this is not due to the larger patient fiber size. Preliminary analysis, using a 12.5% agarose gel, and Western Blots, indicated that these differences were not fiber type dependent. Interestingly, we have identified that embryonic myosin (MYH3) is present in single adult muscle cells. This work was supported by HL65497 (Regnier) and HD48895 (Bamshad).
