GCK-MODY diabetes associated with protein misfolding, cellular self-association and degradation

GCK-MODY, dominantly inherited mild fasting hyperglycemia, has been associated with >600 different mutations in the glucokinase (GK)-encoding gene (GCK). When expressed as recombinant pancreatic proteins, some mutations result in enzymes with normal/near-normal catalytic properties. The molecular mechanism(s) of GCK-MODY due to these mutations has remained elusive. Here, we aimed to explore the molecular mechanisms for two such catalytically 'normal' GCK mutations (S263P and G264S) in the F260-L270 loop of GK. When stably overexpressed in HEK293 cells and MIN6 β-cells, the S263P- and G264S-encoded mutations generated misfolded proteins with an increased rate of degradation (S263P>G264S) by the protein quality control machinery, and a propensity to self-associate (G264S>S263P) and form dimers (SDS resistant) and aggregates (partly Triton X-100 insoluble), as determined by pulse-chase experiments and subcellular fractionation. Thus, the GCK-MODY mutations S263P and G264S lead to protein misfolding causing destabilization, cellular dimerization/aggregation and enhanced rate of degradation. In silico predicted conformational changes of the F260-L270 loop structure are considered to mediate the dimerization of both mutant proteins by a domain swapping mechanism. Thus, similar properties may represent the molecular mechanisms for additional unexplained GCK-MODY mutations, and may also contribute to the disease mechanism in other previously characterized GCK-MODY inactivating mutations.     

Discovery of a novel site regulating glucokinase activity following characterization of a new mutation causing hyperinsulinemic hypoglycemia in humans

Type 2 diabetes is a global problem, and current ineffective therapeutic strategies pave the way for novel treatments like small molecular activators targeting glucokinase (GCK). GCK activity is fundamental to beta cell and hepatocyte glucose metabolism, and heterozygous activating and inactivating GCK mutations cause hyperinsulinemic hypoglycemia (HH) and maturity onset diabetes of the young (MODY) respectively. Over 600 naturally occurring inactivating mutations have been reported, whereas only 13 activating mutations are documented to date. We report two novel GCK HH mutations (V389L and T103S) at residues where MODY mutations also occur (V389D and T103I). Using recombinant proteins with in vitro assays, we demonstrated that both HH mutants had a greater relative activity index than wild type (6.0 for V389L, 8.4 for T103S, and 1.0 for wild type). This was driven by an increased affinity for glucose (S(0.5), 3.3 ± 0.1 and 3.5 ± 0.1 mm, respectively) versus wild type (7.5 ± 0.1 mm). Correspondingly, the V389D and T103I MODY mutants had markedly reduced relative activity indexes (<0.1). T103I had an altered affinity for glucose (S(0.5), 24.9 ± 0.6 mm), whereas V389D also exhibited a reduced affinity for ATP and decreased catalysis rate (S(0.5), 78.6 ± 4.5 mm; ATP(K(m)), 1.5 ± 0.1 mm; K(cat), 10.3 ± 1.1s(-1)) compared with wild type (ATP(K(m)), 0.4 ± <0.1; K(cat), 62.9 ± 1.2). Both Thr-103 mutants showed reduced inhibition by the endogenous hepatic inhibitor glucokinase regulatory protein. Molecular modeling demonstrated that Thr-103 maps to the allosteric activator site, whereas Val-389 is located remotely to this position and all other previously reported activating mutations, highlighting α-helix 11 as a novel region regulating GCK activity. Our data suggest that pharmacological manipulation of GCK activity at locations distal from the allosteric activator site is possible.     

Characterization of common BRCA1 and BRCA2 variants

Many missense variants identified in BRCA1 and BRCA2, two genes responsible for the majority of hereditary breast and ovarian cancer, are of unclear clinical significance. Characterizing the significance of such variants is important for medical management of patients in whom they are identified. The aim of this study was to characterize eight of the most common reported missense mutations in BRCA1 and BRCA2 occurring in patients tested for hereditary risk of breast and ovarian cancers. The prevalence of each variant in a control population, co-segregation of the variant with cancer within families, location of the variant within the gene, the nature of the amino acid substitution and conservation of the wild-type amino acid among species were considered. In a control population, the BRCA1 variants M1652I, R1347G, and S1512I, were each observed at a frequency of 4.08%, 2.04%, and 2.04%, respectively, and the BRCA2 variants A2951T, V2728I, and D1420Y, were seen at 1.02%, 0.68%, and 0.34%, respectively. Although the BRCA2 variants T598A and R2034C were not seen in this group of controls, other clinical and published observations indicate that these variants are not deleterious. Based on epidemiological and biological criteria, we therefore conclude that the BRCA1 missense mutations R1347G, S1512I and M1652I, and the BRCA2 missense mutations T598A, D1420Y, R2034C, V2728I, and A2951T, are not deleterious mutations.     

HbA1c-based diabetes diagnosis among patients with glucokinase mutation (GCK-MODY) is affected by a genetic variant of glucose-6-phosphatase (G6PC2)

Aims Genetic variation at the rs560887 locus of the glucose-6-phosphatase, catalytic?2 gene (G6PC2) is known to affect regulation of fasting glycaemia. We determined the rs560887 genotype of patients with monogenic diabetes and glucokinase gene mutations (GCK-MODY) and correlated the genotypes with HbA(1c) levels. Methods Patients from families with GCK-MODY were recruited from two large cohorts from Poland (n?=?128) and the Czech Republic (n?=?154). Genotypes at the rs560887 polymorphic site in G6PC2 were examined using real-time quantitative polymerase chain reaction. The effect of rs560887 genotype on age at diagnosis of GCK-MODY and initial HbA(1c) levels were evaluated separately within both cohorts. Following that, a meta-analysis of rs560887 genotype-HbA(1c) associations of both Polish and Czech cohorts was performed to confirm homogeneity of findings and validate cohort-specific results. Results GG homozygosity at rs560887 was associated with marginally elevated HbA(1c) levels (P?=?0.07 in both cohorts). The effects observed in both groups were very homogeneous (Q?=?0.18; P?=?0.68). Meta-analysis showed that GG homozygosity at rs560887 was associated with mean HbA(1c) levels higher by 2.4?mmol/mol (0.24%), 95%?CI 0.5-4.4?mmol/mol (0.05-0.44%) than in individuals with other genotypes. Additionally, meta-analysis of both cohorts showed that GG homozygous individuals had higher odds of reaching the 48?mmol/mol (6.5%) diagnostic threshold of diabetes; (odds ratio 1.90; 95%?CI 1.07-3.36; P?=?0.03). No such effects were observed for age at diagnosis of diabetes. Conclusions Variation at the rs560887 locus of G6PC2 is associated with worse glycated haemoglobin levels in individuals with GCK mutations; GG homozygotes are more likely to meet diagnostic criteria for diabetes based on HbA(1c) level.     

Functional Analysis Helps to Define KCNC3 Mutational Spectrum in Dutch Ataxia Cases

Spinocerebellar ataxia type 13 (SCA13) is an autosomal dominantly inherited neurodegenerative disorder of the cerebellum caused by mutations in the voltage gated potassium channel KCNC3. To identify novel pathogenic SCA13 mutations in KCNC3 and to gain insights into the disease prevalence in the Netherlands, we sequenced the entire coding region of KCNC3 in 848 Dutch cerebellar ataxia patients with familial or sporadic origin. We evaluated the pathogenicity of the identified variants by co-segregation analysis and in silico prediction followed by biochemical and electrophysiological studies. We identified 19 variants in KCNC3 including 2 non-coding, 11 missense and 6 synonymous variants. Two missense variants did not co-segregate with the disease and were excluded as potentially disease-causing mutations. We also identified the previously reported p.R420H and p.R423H mutations in our cohort. Of the remaining 7 missense variants, functional analysis revealed that 2 missense variants shifted Kv3.3 channel activation to more negative voltages. These variations were associated with early disease onset and mild intellectual disability. Additionally, one other missense variant shifted channel activation to more positive voltages and was associated with spastic ataxic gait. Whereas, the remaining missense variants did not change any of the channel characteristics. Of these three functional variants, only one variant was in silico predicted to be damaging and segregated with disease. The other two variants were in silico predicted to be benign and co-segregation analysis was not optimal or could only be partially confirmed. Therefore, we conclude that we have identified at least one novel pathogenic mutation in KCNC3 that cause SCA13 and two additionally potential SCA13 mutations. This leads to an estimate of SCA13 prevalence in the Netherlands to be between 0.6% and 1.3%.     

Generation of N-ethyl-N-nitrosourea (ENU) diabetes models in mice demonstrates genotype-specific action of glucokinase activators

We performed genome-wide mutagenesis in C57BL/6J mice using N-ethyl-N-nitrosourea to identify mutations causing high blood glucose early in life and to produce new animal models of diabetes. Of a total of 13 new lines confirmed by heritability testing, we identified two semi-dominant pedigrees with novel missense mutations (Gck(K140E) and Gck(P417R)) in the gene encoding glucokinase (Gck), the mammalian glucose sensor that is mutated in human maturity onset diabetes of the young type 2 and the target of emerging anti-hyperglycemic agents that function as glucokinase activators (GKAs). Diabetes phenotype corresponded with genotype (mild-to-severe: Gck(+/+) < Gck(P417R/+), Gck(K140E)(/+) < Gck(P417R/P417R), Gck(P417R/K140E), and Gck(K140E/K140E)) and with the level of expression of GCK in liver. Each mutant was produced as the recombinant enzyme in Escherichia coli, and analysis of k(cat) and tryptophan fluorescence (I(320/360)) during thermal shift unfolding revealed a correlation between thermostability and the severity of hyperglycemia in the whole animal. Disruption of the glucokinase regulatory protein-binding site (GCK(K140E)), but not the ATP binding cassette (GCK(P417R)), prevented inhibition of enzyme activity by glucokinase regulatory protein and corresponded with reduced responsiveness to the GKA drug. Surprisingly, extracts from liver of diabetic GCK mutants inhibited activity of the recombinant enzyme, a property that was also observed in liver extracts from mice with streptozotocin-induced diabetes. These results indicate a relationship between genotype, phenotype, and GKA efficacy. The integration of forward genetic screening and biochemical profiling opens a pathway for preclinical development of mechanism-based diabetes therapies.     

Insights into the structure and regulation of glucokinase from a novel mutation (V62M), which causes maturity-onset diabetes of the young

Glucokinase (GCK) serves as the pancreatic glucose sensor. Heterozygous inactivating GCK mutations cause hyperglycemia, whereas activating mutations cause hypoglycemia. We studied the GCK V62M mutation identified in two families and co-segregating with hyperglycemia to understand how this mutation resulted in reduced function. Structural modeling locates the mutation close to five naturally occurring activating mutations in the allosteric activator site of the enzyme. Recombinant glutathionyl S-transferase-V62M GCK is paradoxically activated rather than inactivated due to a decreased S0.5 for glucose compared with wild type (4.88 versus 7.55 mM). The recently described pharmacological activator (RO0281675) interacts with GCK at this site. V62M GCK does not respond to RO0281675, nor does it respond to the hepatic glucokinase regulatory protein (GKRP). The enzyme is also thermally unstable, but this lability is apparently less pronounced than in the proven instability mutant E300K. Functional and structural analysis of seven amino acid substitutions at residue Val62 has identified a non-linear relationship between activation by the pharmacological activator and the van der Waals interactions energies. Smaller energies allow a hydrophobic interaction between the activator and glucokinase, whereas larger energies prohibit the ligand from fitting into the binding pocket. We conclude that V62M may cause hyperglycemia by a complex defect of GCK regulation involving instability in combination with loss of control by a putative endogenous activator and/or GKRP. This study illustrates that mutations that cause hyperglycemia are not necessarily kinetically inactivating but may exert their effects by other complex mechanisms. Elucidating such mechanisms leads to a deeper understanding of the GCK glucose sensor and the biochemistry of beta-cells and hepatocytes.     

Screening of mutations and polymorphisms in the glucokinase gene in Czech diabetic and healthy control populations

Glucokinase (GCK) plays a key role in glucose metabolism. GCK mutations are known as a pathogenic cause of maturity-onset diabetes of the young type 2 (MODY2). These mutations are also found in gestational diabetics. The aim of our study was to assess the variability of the GCK gene in the Czech diabetic and control populations. We screened all 10 exons specific for the pancreatic isoform of glucokinase (1a and 2-10) including the intron flanking regions in 722 subjects (in 12 patients with an unrecognised type of MODY and their 10 family members, 313 patients with diabetes mellitus type 2 (DM2), 141 gestational diabetics (GDM), 130 healthy offspring of diabetic parents, and 116 healthy controls without family history of DM2). In two MODY families we identified two mutations in exon 2 of the GCK gene: a novel mutation Val33Ala and the previously described mutation Glu40Lys. In other subgroups (excluding MODY families) we detected only intronic variants and previously described polymorphisms in exons 6 (Tyr215Tyr) and 7 (Ser263Ser), we did not find any known GCK pathogenic mutation. We observed no difference in the frequencies of GCK polymorphisms between Czech diabetic (DM2, GDM) and non-diabetic populations.     

Low prevalence of glucokinase gene mutations in gestational diabetic patients with good glycemic control

Glucokinase (GCK) plays a key role in glucose homeostasis. Gestational diabetes mellitus increases the risk of gestational complications in pregnant women and fetuses. We screened for mutations in coding and flanking regions of the GCK gene in pregnant women with or without gestational diabetes in a Brazilian population. A sample of 200 pregnant women classified as healthy (control, N = 100) or with gestational diabetes (N = 100) was analyzed for mutations in the GCK gene. All gestational diabetes mellitus patients had good glycemic control maintained by diet alone and no complications during pregnancy. Mutations were detected by single-strand conformation polymorphism and DNA sequencing. Thirteen of the 200 subjects had GCK gene mutations. The mutations detected were in intron 3 (c.43331A>G, new), intron 6 (c.47702T>C, rs2268574), intron 9 (c.48935C>T, rs2908274), and exon 10 (c.49620G>A, rs13306388). None of these GCK mutations were found to be significantly associated with gestational diabetes mellitus. In summary, we report a low frequency of GCK mutations in a pregnant Brazilian population and describe a new intronic variation (c.43331A>G, intron 3). We conclude that mutations in GCK introns and in non-translatable regions of the GCK gene do not affect glycemic control and are not correlated with gestational diabetes mellitus.     

Whole Exome Sequencing Reveals Homozygous Mutations in RAI1, OTOF,and SLC26A4 Genes Associated with Nonsyndromic Hearing Loss in Altaian Families (South Siberia)

Hearing loss (HL) is one of the most common sensorineural disorders and several dozen genes contribute to its pathogenesis. Establishing a genetic diagnosis of HL is of great importance for clinical evaluation of deaf patients and for estimating recurrence risks for their families. Efforts to identify genes responsible for HL have been challenged by high genetic heterogeneity and different ethnic-specific prevalence of inherited deafness. Here we present the utility of whole exome sequencing (WES) for identifying candidate causal variants for previously unexplained nonsyndromic HL of seven patients from four unrelated Altaian families (the Altai Republic, South Siberia). The WES analysis revealed homozygous missense mutations in three genes associated with HL. Mutation c.2168A>G (SLC26A4) was found in one family, a novel mutation c.1111G>C (OTOF) was revealed in another family, and mutation c.5254G>A (RAI1) was found in two families. Sanger sequencing was applied for screening of identified variants in an ethnically diverse cohort of other patients with HL (n = 116) and in Altaian controls (n = 120). Identified variants were found only in patients of Altaian ethnicity (n = 93). Several lines of evidences support the association of homozygosity for discovered variants c.5254G>A (RAI1), c.1111C>G (OTOF), and c.2168A>G (SLC26A4) with HL in Altaian patients. Local prevalence of identified variants implies possible founder effect in significant number of HL cases in indigenous population of the Altai region. Notably, this is the first reported instance of patients with RAI1 missense mutation whose HL is not accompanied by specific traits typical for Smith-Magenis syndrome. Presumed association of RAI1 gene variant c.5254G>A with isolated HL needs to be proved by further experimental studies.     

Structure-function analysis of Friedreich's ataxia mutants reveals determinants of frataxin binding and activation of the Fe-S assembly complex

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-S cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k(cat)/K(M) higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k(cat)/K(M) of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.     

Functional characterization of MODY2 mutations in the nuclear export signal of glucokinase

Glucokinase (GCK) plays a key role in glucose homeostasis. Heterozygous inactivating mutations in the GCK gene cause the familial, mild fasting hyperglycaemia named MODY2. Besides its particular kinetic characteristics, glucokinase is regulated by subcellular compartmentation in hepatocytes. Glucokinase regulatory protein (GKRP) binds to GCK, leading to enzyme inhibition and import into the nucleus at fasting. When glucose concentration increases, GCK-GKRP dissociates and GCK is exported to the cytosol due to a nuclear export signal (NES). With the aim to characterize the GCK-NES, we have functionally analysed nine MODY2 mutations located within the NES sequence.Recombinant GCK mutants showed reduced catalytic activity and, in most cases, protein instability. Most of the mutants interact normally with GKRP, although mutations L306R and L309P impair GCK nuclear import in cotransfected cells. We demonstrated that GCK-NES function depends on exportin 1. We further showed that none of the mutations fully inactivate the NES, with the exception of mutation L304P, which likely destabilizes its α-helicoidal structure. Finally, we found that residue Glu300 negatively modulates the NES activity, whereas other residues have the opposite effect, thus suggesting that some of the NES spacer residues contribute to the low affinity of the NES for exportin 1, which is required for its proper functioning.In conclusion, our results have provided functional and structural insights regarding the GCK-NES and contributed to a better knowledge of the molecular mechanisms involved in the nucleo-cytoplasmic shuttling of glucokinase. Impairment of this regulatory mechanism by some MODY2 mutations might contribute to the hyperglycaemia in the patients.     

Molecular analysis of the Retinoic Acid Induced 1 gene (RAI1) in patients with suspected Smith-Magenis syndrome without the 17p11.2 deletion

Smith-Magenis syndrome (SMS) is a complex neurobehavioral disorder characterized by multiple congenital anomalies. The syndrome is primarily ascribed to a ～3.7 Mb de novo deletion on chromosome 17p11.2. Haploinsufficiency of multiple genes likely underlies the complex clinical phenotype. RAI1 (Retinoic Acid Induced 1) is recognized as a major gene involved in the SMS phenotype. Extensive genetic and clinical analyses of 36 patients with SMS-like features, but without the 17p11.2 microdeletion, yielded 10 patients with RAI1 variants, including 4 with de novo deleterious mutations, and 6 with novel missense variants, 5 of which were familial. Haplotype analysis showed two major RAI1 haplotypes in our primarily Caucasian cohort; the novel RAI1 variants did not occur in a preferred haplotype. RNA analysis revealed that RAI1 mRNA expression was significantly decreased in cells of patients with the common 17p11.2 deletion, as well as in those with de novo RAI1 variants. Expression levels varied in patients with familial RAI1 variants and in non-17p11.2 deleted patients without identified RAI1 defects. No correlation between SNP haplotype and RAI1 expression was found. Two clinical features, ocular abnormalities and polyembolokoilomania (object insertion), were significantly correlated with decreased RAI1 expression. While not significantly correlated, the presence of hearing loss, seizures, hoarse voice, childhood onset of obesity and specific behavioral aspects and the absence of immunologic abnormalities and cardiovascular or renal structural anomalies, appeared to be specific for the de novo RAI1 subgroup. Recognition of the combination of these features will assist in referral for RAI1 analysis of patients with SMS-like features without detectable microdeletion of 17p11.2. Moreover, RAI1 expression emerged as a genetic target for development of therapeutic interventions for SMS.     

The Contribution of Mosaic Variants to Autism Spectrum Disorder

De novo mutation is highly implicated in autism spectrum disorder (ASD). However, the contribution of post-zygotic mutation to ASD is poorly characterized. We performed both exome sequencing of paired samples and analysis of de novo variants from whole-exome sequencing of 2,388 families. While we find little evidence for tissue-specific mosaic mutation, multi-tissue post-zygotic mutation (i.e. mosaicism) is frequent, with detectable mosaic variation comprising 5.4% of all de novo mutations. We identify three mosaic missense and likely-gene disrupting mutations in genes previously implicated in ASD (KMT2C, NCKAP1, and MYH10) in probands but none in siblings. We find a strong ascertainment bias for mosaic mutations in probands relative to their unaffected siblings (p = 0.003). We build a model of de novo variation incorporating mosaic variants and errors in classification of mosaic status and from this model we estimate that 33% of mosaic mutations in probands contribute to 5.1% of simplex ASD diagnoses (95% credible interval 1.3% to 8.9%). Our results indicate a contributory role for multi-tissue mosaic mutation in some individuals with an ASD diagnosis.     

AICAR potentiates ROS production induced by chronic high glucose: roles of AMPK in pancreatic beta-cell apoptosis

We previously demonstrated that chronic high glucose (33.3 mM) induced beta-cell dysfunction and apoptosis through glucokinase (GCK) downregulation, but the exact mechanisms involved remain unclear. Here, we show that prolonged exposure of 5-aminoimidazole-4-carboxamide (AICA)-riboside potentiated apoptosis induced by high glucose in MIN6N8 pancreatic beta-cells, correlating with enhanced GCK downregulation and decreased production of ATP and insulin. These events are potentiated in AMPK-overexpressing cells, but are prevented in cells transfected with mutant dominant-negative AMPK (AMPK-K45R). Furthermore, AMPK activation increases production of reactive oxygen species (ROS) and loss of mitochondria membrane potential induced by high glucose, which is significantly inhibited by treatment with compound C or by AMPK-K45R overexpression. Overexpression of GCK prevents apoptosis; decreased cellular ATP and insulin secretion, and ROS production enhanced by AICAR, but does not affect AMPK activation. Similar results are obtained using isolated primary islet cells. Collectively, these data demonstrate that AMPK activation potentiates beta-cell apoptosis induced by chronic high glucose through augmented GCK downregulation mediated by enhanced ROS production.     

ATP1A3 Mutations and Genotype-Phenotype Correlation of Alternating Hemiplegia of Childhood in Chinese Patients

Alternating hemiplegia of childhood (AHC) is a rare and severe neurological disorder. ATP1A3 was recently identified as the causative gene. Here we report the first genetic study in Chinese AHC cohort. We performed whole-exome sequencing on three trios and three unrelated patients, and screened additional 41 typical cases and 100 controls by PCR-Sanger sequencing. ATP1A3 mutations were detected in 95.7% of typical AHC patients. At least 93.3% were de novo. Four late onset, atypical AHC patients were also mutation positive, suggesting the need for testing ATP1A3 mutations in atypical cases. Totally, 13 novel missense mutations (T370N, G706R, L770R, T771N, T771I, S772R, L802P, D805H, M806K, P808L, I810N, L839P and G893R) were identified in our study. By homology modeling of the mutant protein structures and calculation of an extensive list of molecular features, we identified two statistically significant molecular features, solvent accessibility and distance to metal ion, that distinguished disease-associated mutations from neutral variants. A logistic regression classifier achieved 92.9% accuracy by the average of 100 times of five-fold cross validations. Genotype-phenotype correlation analysis showed that patients with epilepsy were more likely to carry E815K mutation. In summary, ATP1A3 is the major pathogenic gene of AHC in Chinese patients; mutations have distinctive molecular features that discriminate them from neutral variants and are correlated with phenotypes.     

Molecular modelling and dynamics of CA2 missense mutations causative to carbonic anhydrase 2 deficiency syndrome

Abstract

Carbonic anhydrase 2 (CA2) enzyme deficiency caused by CA2 gene mutations is an inherited disorder characterized by symptoms like osteopetrosis, renal tubular acidosis, and cerebral calcification. This study has collected the CA2 deficiency causal missense mutations and assessed their pathogenicity using diverse computational programs. The 3D protein models for all missense mutations were built, and analyzed for structural divergence, protein stability, and molecular dynamics properties. We found M-CAP as the most sensitive prediction method to measure the deleterious potential of CA2 missense mutations. Free energy dynamics of tertiary structure models of CA2 mutants with DUET, mCSM, and SDM based consensus methods predicted only 50% of the variants as destabilizing. Superimposition of native and mutant CA2 models revealed the minor structural fluctuations at the amino acid residue level but not at the whole protein structure level. Near native molecular dynamic simulation analysis indicated that CA2 causative missense variants result in residue level fluctuation pattern in the protein structure. This study expands the understanding of genotype-protein phenotype correlations underlying CA2 variant pathogenicity and presents a potential avenue for modifying the CA2 deficiency by targeting biophysical structural features of CA2 protein.

Communicated by Ramaswamy H. Sarma     

High prevalence of SLC6A8 deficiency in X-linked mental retardation

A novel X-linked mental retardation (XLMR) syndrome was recently identified, resulting from creatine deficiency in the brain caused by mutations in the creatine transporter gene, SLC6A8. We have studied the prevalence of SLC6A8 mutations in a panel of 290 patients with nonsyndromic XLMR archived by the European XLMR Consortium. The full-length open reading frame and splice sites of the SLC6A8 gene were investigated by DNA sequence analysis. Six pathogenic mutations, of which five were novel, were identified in a total of 288 patients with XLMR, showing a prevalence of at least 2.1% (6/288). The novel pathogenic mutations are a nonsense mutation (p.Y317X) and four missense mutations. Three missense mutations (p.G87R, p.P390L, and p.P554L) were concluded to be pathogenic on the basis of conservation, segregation, chemical properties of the residues involved, as well as the absence of these and any other missense mutation in 276 controls. For the p.C337W mutation, additional material was available to biochemically prove (i.e., by increased urinary creatine : creatinine ratio) pathogenicity. In addition, we found nine novel polymorphisms (IVS1+26G-->A, IVS7+37G-->A, IVS7+87A-->G, IVS7-35G-->A, IVS12-3C-->T, IVS2+88G-->C, IVS9-36G-->A, IVS12-82G-->C, and p.Y498) that were present in the XLMR panel and/or in the control panel. Two missense variants (p.V629I and p.M560V) that were not highly conserved and were not associated with increased creatine : creatinine ratio, one translational silent variant (p.L472), and 10 intervening sequence variants or untranslated region variants (IVS6+9C-->T, IVS7-151_152delGA, IVS7-99C-->A, IVS8-35G-->A, IVS8+28C-->T, IVS10-18C-->T, IVS11+21G-->A, IVS12+15C-->T, *207G-->C, IVS12+32C-->A) were found only in the XLMR panel but should be considered as unclassified variants or as a polymorphism (p.M560V). Our data indicate that the frequency of SLC6A8 mutations in the XLMR population is close to that of CGG expansions in FMR1, the gene responsible for fragile-X syndrome.     

Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease

Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecond-timescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond-timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence, and ¹⁹F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme’s intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, noncooperative variants.