Disease-causing Missense Mutations Research Articles

Identification of Two Disease-causing Genes TJP2 and GJB2 in a Chinese Family with Unconditional Autosomal Dominant Nonsyndromic Hereditary Hearing Impairment.

Background:There are more than 300 genetic loci that have been found to be related to hereditary hearing impairment (HHI), including 92 causative genes for nonsyndromic hearing loss, among which 34 genes are related to autosomal dominant nonsyndromic HHI (ADNSHHI). Traditional linkage analysis and candidate gene sequencing are not effective at detecting the ADNSHHI, especially for the unconditional families that may have more than one pathogenic cause. This study identified two disease-causing genes TJP2 and GJB2 in a Chinese family with unconditional ADNSHHI.Methods:To decipher the genetic code of a Chinese family (family 686) with ADNSHHI, different gene screening techniques have been performed, including linkage analysis, candidate genes screening, high-throughput sequencing and Sanger sequencing. These techniques were done on samples obtained from this family over a period of 10 years.Results:We identified a pathogenic missense mutation, c. 2081G>A (p.G694E), in TJP2, a gene that plays a crucial role in apoptosis and age-related hearing loss (ARHL). The mutation was co-segregated in this pedigree in all, but not in the two patients who presented with different phenotypes from the other affected family members. In one of the two patients, we confirmed that the compound heterozygosity for p. Y136* and p.G45E in the GJB2 gene may account for the phenotype shown in this patient.Conclusions:We identified the co-occurrence of two genetic causes in family 686. The possible disease-causing missense mutation of TJP2 in family 686 presents an opportunity for further investigation into ARHL. It is necessary to combine various genes screening methods, especially for some unconventional cases.

Tau and neurodegenerative disease: the story so far.

In 1975, tau protein was isolated as a microtubule-associated factor from the porcine brain. In the previous year, a paired helical filament (PHF) protein had been identified in neurofibrillary tangles in the brains of individuals with Alzheimer disease (AD), but it was not until 1986 that the PHF protein and tau were discovered to be one and the same. In the AD brain, tau was found to be abnormally hyperphosphorylated, and it inhibited rather than promoted in vitro microtubule assembly. Almost 80 disease-causing exonic missense and intronic silent mutations in the tau gene have been found in familial cases of frontotemporal dementia but, to date, no such mutation has been found in AD. The first phase I clinical trial of an active tau immunization vaccine in patients with AD was recently completed. Assays for tau levels in cerebrospinal fluid and plasma are now available, and tau radiotracers for PET are under development. In this article, we provide an overview of the pivotal discoveries in the tau research field over the past 40 years. We also review the current status of the field, including disease mechanisms and therapeutic approaches.

Mutational Spectrum Analysis of Neurodegenerative Diseases and Its Pathogenic Implication.

One of the most conspicuous features of neurodegenerative diseases (NDs) is the occurrence of dramatic conformation change of individual proteins. We performed a mutational spectrum analysis of disease-causing missense mutations in seven types of NDs at nucleotide and amino acid levels, and compared the results with those of non-NDs. The main findings included: (i) The higher mutation ratio of G:C→T:A transversion to G:C→A:T transition was observed in NDs than in non-NDs, interpreting the excessive guanine-specific oxidative DNA damage in NDs; (ii) glycine and proline had highest mutability in NDs than in non-NDs, which favor the protein conformation change in NDs; (iii) surprisingly low mutation frequency of arginine was observed in NDs. These findings help to understand how mutations may cause NDs.

SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery.

Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds. In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations. We found that the SDHBR46Q mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4 ± 63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation.Furthermore, HIF1α, but not HIF2α, increased markedly and the cells showed a strong DNA CpG island methylatorphenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines. These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.

Tet oncogene family member 2 gene alterations in childhood acute myeloid leukemia.

Mutations in the tet oncogene family member 2 gene (TET2) are frequently found in adult patients with acute myeloid leukemia (AML). Reports of TET2 mutations in children are limited. We assessed the prevalence of TET2 mutations in Taiwanese children with AML and analyzed their prognosis. Between 1997 and 2010, a total of 69 consecutive children with AML were enrolled at the National Taiwan University Hospital. The analysis for TET2 mutations was performed using direct sequencing. Clinical characteristics and overall survival (OS) were compared between patients with and without TET2 alterations. Intronic and missense mutations were identified. No nonsense or frameshift mutations were observed. Two putative disease-causing missense mutations (S609C and A1865G) were identified in one patient. We estimated the prevalence of TET2 mutations in the current patient population to be 1.4%. The most common polymorphism was I1762V (45%), followed by V218M (12%), P29R (6%), and F868L (6%). Patients with polymorphism I1762V had an increased 10-year survival rate compared with patients without I1762V (48.4% vs. 25.7%, p=0.049) by Chi-square test; OS was not different when examined using the Kaplan-Meier method (p=0.104). The prevalence of TET2 mutations in children with AML compared with adults with AML was lower and less complex. Patient prognosis associated with TET2 mutations in children requires further investigation.

Targeted Resequencing of 29 Candidate Genes and Mouse Expression Studies Implicate ZIC3 and FOXF1 in Human VATER/VACTERL Association.

The VATER/VACTERL association describes the combination of congenital anomalies including vertebral defects, anorectal malformations, cardiac defects, tracheoesophageal fistula with or without esophageal atresia, renal malformations, and limb defects. As mutations in ciliary genes were observed in diseases related to VATER/VACTERL, we performed targeted resequencing of 25 ciliary candidate genes as well as disease-associated genes (FOXF1, HOXD13, PTEN, ZIC3) in 123 patients with VATER/VACTERL or VATER/VACTERL-like phenotype. We detected no biallelic mutation in any of the 25 ciliary candidate genes; however, identified an identical, probably disease-causing ZIC3 missense mutation (p.Gly17Cys) in four patients and a FOXF1 de novo mutation (p.Gly220Cys) in a further patient. In situ hybridization analyses in mouse embryos between E9.5 and E14.5 revealed Zic3 expression in limb and prevertebral structures, and Foxf1 expression in esophageal, tracheal, vertebral, anal, and genital tubercle tissues, hence VATER/VACTERL organ systems. These data provide strong evidence that mutations in ZIC3 or FOXF1 contribute to VATER/VACTERL.

Misfolding caused by the pathogenic mutation G47R on the minor allele of alanine:glyoxylate aminotransferase and chaperoning activity of pyridoxine

Liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5′-phosphate (PLP) enzyme, exists as two polymorphic forms, the major (AGT-Ma) and the minor (AGT-Mi) haplotype. Deficit of AGT causes Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive rare disease. Although ~one-third of the 79 disease-causing missense mutations segregates on AGT-Mi, only few of them are well characterized. Here for the first time the molecular and cellular defects of G47R-Mi are reported. When expressed in Escherichia coli, the recombinant purified G47R-Mi variant exhibits only a 2.5-fold reduction of its kcat, and its apo form displays a remarkably decreased PLP binding affinity, increased dimer–monomer equilibrium dissociation constant value, susceptibility to thermal denaturation and to N-terminal region proteolytic cleavage, and aggregation propensity. When stably expressed in a mammalian cell line, we found ~95% of the intact form of the variant in the insoluble fraction, and proteolyzed (within the N-terminal region) and aggregated forms both in the soluble and insoluble fractions. Moreover, the intact and nicked forms have a peroxisomal and a mitochondrial localization, respectively. Unlike what already seen for G41R-Mi, exposure of G47R-Mi expressing cells to pyridoxine (PN) remarkably increases the expression level and the specific activity in a dose-dependent manner, reroutes all the protein to peroxisomes, and rescues its functionality. Although the mechanism of the different effect of PN on the variants G47R-Mi and G41R-Mi remains elusive, the chaperoning activity of PN may be of value in the therapy of patients bearing the G47R mutation.

Parkinson disease: α-synuclein mutational screening and new clinical insight into the p.E46K mutation.

Amongst Parkinson's disease-causing genetic factors, missense mutations and genomic multiplications in the gene encoding α-synuclein are well established causes of the disease, although genetic data in populations with a high degree of admixture, such as the Brazilian one, are still scarce. In this study, we conducted a molecular screening of α-synuclein point mutations and copy number variation in the largest cohort of Brazilian patients with Parkinson's disease (n=549) and also in twelve Portuguese and one Bolivian immigrants. Genomic DNA was isolated from peripheral blood leukocytes or saliva, and the mutational screening was performed by quantitative and qualitative real-time PCR. The only alteration identified was the p.E46K mutation in a 60-year-old man, born in Bolivia, with a familial history of autosomal dominant Parkinson's disease. This is the second family ever reported, in which this rare pathogenic mutation is segregating. The same mutation was firstly described ten years ago in a Spanish family with a neurodegenerative syndrome combining parkinsonism, dementia and visual hallucinations. The clinical condition of our proband reveals a less aggressive phenotype than previously described and reinforces that marked phenotypic heterogeneity is common among patients with Parkinson's disease, even among those carriers sharing the same mutation. Our findings add new insight into the preexisting information about α-synuclein p.E46K, improving our understanding about the endophenotypes associated to this mutation and corroborate that missense alterations and multiplications in α-synuclein are uncommon among Brazilian patients with Parkinson's disease.

Structural snapshots of GALC: The Krabbe Disease enzyme

Glycosphingolipids are ubiquitous components of mammalian cell membranes, and defects in their catabolism by lysosomal enzymes cause a diverse array of diseases. Deficiencies in the enzyme β-galactocerebrosidase (GALC) cause Krabbe disease, a devastating genetic disorder characterized by widespread demyelination and rapid, fatal neurodegeneration[1]. Many disease-causing mutations throughout the GALC gene have been identified; rather than rendering the enzyme catalytically incompetent, many of these are predicted to destabilise the protein, compromising lysosomal delivery by causing retention in the endoplasmic reticulum. In these cases, pharmacological chaperone therapy (PCT) may be an appropriate treatment strategy, whereby the specific binding of a small molecule may stabilise folding of the mutant protein sufficiently to correct trafficking defects. Here we present a series of high resolution crystal structures that illustrate key steps in the catalytic cycle of GALC, including enzyme-substrate, enzyme-intermediate and enzyme-product complexes[2]. These structures reveal active site conformational changes accompanying the catalytic steps and provide key mechanistic insights. Building on this, we identify two candidate molecules that stabilise GALC by specific binding to the active site. The nature of several disease-causing missense mutations is also explored using secretion and activity assays, and we show that mutations can be classified into two groups based on defects in catalysis or trafficking. Overall, this study provides an atomic framework for the rational design of GALC pharmacological chaperones, and helps to identify which mutations may be suitable for PCT, bringing the prospect of a drug-based therapy one step closer.

Kinase and channel activity of TRPM6 are co-ordinated by a dimerization motif and pocket interaction

Mutations in the gene that encodes the atypical channel-kinase TRPM6 (transient receptor potential melastatin 6) cause HSH (hypomagnesaemia with secondary hypocalcaemia), a disorder characterized by defective intestinal Mg2+ transport and impaired renal Mg2+ reabsorption. TRPM6, together with its homologue TRPM7, are unique proteins as they combine an ion channel domain with a C-terminally fused protein kinase domain. How TRPM6 channel and kinase activity are linked is unknown. Previous structural analysis revealed that TRPM7 possesses a non-catalytic dimerization motif preceding the kinase domain. This interacts with a dimerization pocket lying within the kinase domain. In the present study, we provide evidence that the dimerization motif in TRPM6 plays a critical role in regulating kinase activity as well as ion channel activity. We identify mutations within the TRPM6 dimerization motif (Leu1718 and Leu1721) or dimerization pocket (L1743A, Q1832K, A1836N, L1840A and L1919Q) that abolish dimerization and establish that these mutations inhibit protein kinase activity. We also demonstrate that kinase activity of a dimerization motif mutant can be restored by addition of a peptide encompassing the dimerization motif. Moreover, we observe that mutations that disrupt the dimerization motif and dimerization pocket interaction greatly diminish TRPM6 ion channel activity, in a manner that is independent of kinase activity. Finally, we analyse the impact on kinase activity of ten disease-causing missense mutations that lie outwith the protein kinase domain of TRPM6. This revealed that one mutation lying nearby the dimerization motif (S1754N), found previously to inhibit channel activity, abolished kinase activity. These results provide the first evidence that there is structural co-ordination between channel and kinase activity, which is mediated by the dimerization motif and pocket interaction. We discuss that modulation of this interaction could comprise a major regulatory mechanism by which TRPM6 function is controlled.

Mammalian TBX1 preferentially binds and regulates downstream targets via a tandem T-site repeat.

Haploinsufficiency or mutation of TBX1 is largely responsible for the etiology of physical malformations in individuals with velo-cardio-facial/DiGeorge syndrome (VCFS/DGS/22q11.2 deletion syndrome). TBX1 encodes a transcription factor protein that contains an evolutionarily conserved DNA binding domain termed the T-box that is shared with other family members. All T-box proteins, examined so far, bind to similar but not identical consensus DNA sequences, indicating that they have specific binding preferences. To identify the TBX1 specific consensus sequence, Systematic Evolution of Ligands by Exponential Enrichment (SELEX) was performed. In contrast to other TBX family members recognizing palindrome sequences, we found that TBX1 preferentially binds to a tandem repeat of 5′-AGGTGTGAAGGTGTGA-3′. We also identified a second consensus sequence comprised of a tandem repeat with a degenerated downstream site. We show that three known human disease-causing TBX1 missense mutations (F148Y, H194Q and G310S) do not alter nuclear localization, or disrupt binding to the tandem repeat consensus sequences, but they reduce transcriptional activity in cell culture reporter assays. To identify Tbx1-downstream genes, we performed an in silico genome wide analysis of potential cis-acting elements in DNA and found strong enrichment of genes required for developmental processes and transcriptional regulation. We found that TBX1 binds to 19 different loci in vitro, which may correspond to putative cis-acting binding sites. In situ hybridization coupled with luciferase gene reporter assays on three gene loci, Fgf8, Bmper, Otog-MyoD, show that these motifs are directly regulated by TBX1 in vitro. Collectively, the present studies establish new insights into molecular aspects of TBX1 binding to DNA. This work lays the groundwork for future in vivo studies, including chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) to further elucidate the molecular pathogenesis of VCFS/DGS.

BOR-Syndrome-Associated Eya1 Mutations Lead to Enhanced Proteasomal Degradation of Eya1 Protein

Mutations in the human EYA1 gene have been associated with several human diseases including branchio-oto (BO) and branchio-oto-renal (BOR) syndrome, as well as congenital cataracts and ocular anterior segment anomalies. BOR patients suffer from severe malformations of the ears, branchial arches and kidneys. The phenotype of Eya1-heterozygous mice resembles the symptoms of human patients suffering from BOR syndrome. The Eya1 gene encodes a multifunctional protein that acts as a protein tyrosine phosphatase and a transcriptional coactivator. It has been shown that Eya1 interacts with Six transcription factors, which are also required for nuclear translocation of the Eya1 protein. We investigated the effects of seven disease-causing Eya1 missense mutations on Eya1 protein function, in particular cellular localization, ability to interact with Six proteins, and protein stability. We show here that the BOR-associated Eya1 missense mutations S454P, L472R, and L550P lead to enhanced proteasomal degradation of the Eya1 protein in mammalian cells. Moreover, Six proteins lead to a significant stabilization of Eya1, which is caused by Six-mediated protection from proteasomal degradation. In case of the mutant L550P, loss of interaction with Six proteins leads to rapid protein degradation. Our observations suggest that protein destabilization constitutes a novel disease causing mechanism for Eya1.

Hsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteins

We examined the role of Hsp90 in expression and maturation of wild-type (WT) and mutant ether-a-go-go related gene (HERG) proteins by using Hsp90 inhibitors, geldanamycin (GA) and radicicol, and Hsp90 overexpression. The proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analysed by western blotting, immunoprecipitation, immunofluorescence, and whole-cell patch-clamp techniques. GA and radicicol suppressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harbouring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the WT; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquitinated form, increased its immunoreactivity in the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins. Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for long QT syndrome 2 caused by trafficking abnormalities of HERG proteins.

A rational free energy-based approach to understanding and targeting disease-causing missense mutations

Intellectual disability is a condition characterized by significant limitations in cognitive abilities and social/behavioral adaptive skills and is an important reason for pediatric, neurologic, and genetic referrals. Approximately 10% of protein-encoding genes on the X chromosome are implicated in intellectual disability, and the corresponding intellectual disability is termed X-linked ID (XLID). Although few mutations and a small number of families have been identified and XLID is rare, collectively the impact of XLID is significant because patients usually are unable to fully participate in society. To reveal the molecular mechanisms of various intellectual disabilities and to suggest small molecules which by binding to the malfunctioning protein can reduce unwanted effects. Using various in silico methods we reveal the molecular mechanism of XLID in cases involving proteins with known 3D structure. The 3D structures were used to predict the effect of disease-causing missense mutations on the folding free energy, conformational dynamics, hydrogen bond network and, if appropriate, protein-protein binding free energy. It is shown that the vast majority of XLID mutation sites are outside the active pocket and are accessible from the water phase, thus providing the opportunity to alter their effect by binding appropriate small molecules in the vicinity of the mutation site. This observation is used to demonstrate, computationally and experimentally, that a particular condition, Snyder-Robinson syndrome caused by the G56S spermine synthase mutation, might be ameliorated by small molecule binding.

Identification of amphiphysin 1 as an endogenous substrate for CDKL5, a protein kinase associated with X-linked neurodevelopmental disorder

Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in brain and mutations of its gene are known to be associated with neurodevelopmental disorders such as X-linked West syndrome and Rett syndrome. However, the physiological substrates of CDKL5 that are directly linked to these neurodevelopmental disorders are currently unknown. In this study, we explored endogenous substrates for CDKL5 in mouse brain extracts fractionated by a liquid-phase isoelectric focusing. In conjunction with CDKL5 phosphorylation assay, this approach detected a protein band with an apparent molecular mass of 120kDa that is remarkably phosphorylated by CDKL5. This 120-kDa protein was identified as amphiphysin 1 (Amph1) by LC-MS/MS analysis, and the site of phosphorylation by CDKL5 was determined to be Ser-293. The phosphorylation mimic mutants, Amph1(S293E) and Amph1(S293D), showed significantly reduced affinity for endophilin, a protein involved in synaptic vesicle endocytosis. Introduction of point mutations in the catalytic domain of CDKL5, which are disease-causing missense mutations found in Rett patients, resulted in the impairment of kinase activity toward Amph1. These results suggest that Amph1 is the cytoplasmic substrate for CDKL5 and that its phosphorylation may play crucial roles in the neuronal development.

Enhancing Human Spermine Synthase Activity by Engineered Mutations

Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine → spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing.

Structure–phenotype correlations of human CYP21A2 mutations in congenital adrenal hyperplasia

Mutations in the cytochrome p450 (CYP)21A2 gene, which encodes the enzyme steroid 21-hydroxylase, cause the majority of cases in congenital adrenal hyperplasia, an autosomal recessive disorder. To date, more than 100 CYP21A2 mutations have been reported. These mutations can be associated either with severe salt-wasting or simple virilizing phenotypes or with milder nonclassical phenotypes. Not all CYP21A2 mutations have, however, been characterized biochemically, and the clinical consequences of these mutations remain unknown. Using the crystal structure of its bovine homolog as a template, we have constructed a humanized model of CYP21A2 to provide comprehensive structural explanations for the clinical manifestations caused by each of the known disease-causing missense mutations in CYP21A2. Mutations that affect membrane anchoring, disrupt heme and/or substrate binding, or impair stability of CYP21A2 cause complete loss of function and salt-wasting disease. In contrast, mutations altering the transmembrane region or conserved hydrophobic patches cause up to a 98% reduction in enzyme activity and simple virilizing disease. Mild nonclassical disease can result from interference in oxidoreductase interactions, salt-bridge and hydrogen-bonding networks, and nonconserved hydrophobic clusters. A simple in silico evaluation of previously uncharacterized gene mutations could, thus, potentially help predict the often diverse phenotypes of a monogenic disorder.

Transcriptional regulation of the ABCC6 gene and the background of impaired function of missense disease-causing mutations

The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein expressed primarily in the liver and to a lesser extent in the kidneys and the intestines. We review here the mechanisms of this restricted tissue-specific expression and the role of hepatocyte nuclear factor 4α which is responsible for the expression pattern. Detailed analyses uncovered further regulators of the expression of the gene pointing to an intronic primate-specific regulator region, an activator of the expression of the gene by binding CCAAT/enhancer-binding protein beta, which interacts with other proteins acting in the proximal promoter. This regulatory network is affected by various environmental stimuli including oxidative stress and the extracellular signal-regulated protein kinases 1 and 2 pathway. We also review here the structural and functional consequences of disease-causing missense mutations of ABCC6. A significant clustering of the missense disease-causing mutations was found at the domain–domain interfaces. This clustering means that the domain contacts are much less permissive to amino acid replacements than the rest of the protein. We summarize the experimental methods resulting in the identification of mutants with preserved transport activity but failure in intracellular targeting. These mutants are candidates for functional rescue by chemical chaperons. The results of such research can provide the basis of future allele-specific therapy of ABCC6-mediated disorders like pseudoxanthoma elasticum or the generalized arterial calcification in infancy.

Disease-causing missense mutations in human DNA helicase disorders

Helicases have important roles in nucleic acid metabolism, and their prominence is marked by the discovery of genetic disorders arising from disease-causing mutations. Missense mutations can yield unique insight to molecular functions and basis for disease pathology. XPB or XPD missense mutations lead to Xeroderma pigmentosum, Cockayne's syndrome, Trichothiodystrophy, or COFS syndrome, suggesting that DNA repair and transcription defects are responsible for clinical heterogeneity. Complex phenotypes are also observed for RECQL4 helicase mutations responsible for Rothmund-Thomson syndrome, Baller-Gerold syndrome, or RAPADILINO. Bloom's syndrome causing missense mutations are found in the conserved helicase and RecQ C-terminal domain of BLM that interfere with helicase function. Although rare, patient-derived missense mutations in the exonuclease or helicase domain of Werner syndrome protein exist. Characterization of WRN separation-of-function mutants may provide insight to catalytic requirements for suppression of phenotypes associated with the premature aging disorder. Characterized FANCJ missense mutations associated with breast cancer or Fanconi anemia interfere with FANCJ helicase activity required for DNA repair and the replication stress response. For example, a FA patient-derived mutation in the FANCJ Iron-Sulfur domain was shown to uncouple its ATPase and translocase activity from DNA unwinding. Mutations in DDX11 (ChlR1) are responsible for Warsaw Breakage syndrome, a recently discovered autosomal recessive cohesinopathy. Ongoing and future studies will address clinically relevant helicase mutations and polymorphisms, including those that interfere with key protein interactions or exert dominant negative phenotypes (e.g., certain mutant alleles of Twinkle mitochondrial DNA helicase). Chemical rescue may be an approach to restore helicase activity in loss-of-function helicase disorders. Genetic and biochemical analyses of disease-causing missense mutations in human helicase disorders have led to new insights to the molecular defects underlying aberrant cellular and clinical phenotypes.

Disease‐causing mutations C277R and C277Y modify gating of human ClC‐1 chloride channels in myotonia congenita

Myotonia congenita is a genetic condition that is caused by mutations in the muscle chloride channel gene CLCN1 and characterized by delayed muscle relaxation and muscle stiffness. We here investigate the functional consequences of two novel disease-causing missense mutations, C277R and C277Y, using heterologous expression in HEK293T cells and patch clamp recording. Both mutations reduce macroscopic anion currents in transfected cells. Since hClC-1 is a double-barrelled anion channel, this reduction in current amplitude might be caused by altered gating of individual protopores or of joint openings and closing of both protopores. We used non-stationary noise analysis and single channel recordings to separate the mutants' effects on individual and common gating processes. We found that C277Y inverts the voltage dependence and reduces the open probabilities of protopore and common gates resulting in decreases of absolute open probabilities of homodimeric channels to values below 3%. In heterodimeric channels, C277R and C277Y also reduce open probabilities and shift the common gate activation curve towards positive potentials. Moreover, C277Y modifies pore properties of hClC-1. It reduces single protopore current amplitudes to about two-thirds of wild-type values, and inverts the anion permeability sequence to I(-) = NO(3)(-) >Br(-)>Cl(-). Our findings predict a dramatic reduction of the muscle fibre resting chloride conductance and thus fully explain the disease-causing effects of mutations C277R and C277Y. Moreover, they provide additional insights into the function of C277, a residue recently implicated in common gating of ClC channels.