The Influence of Tissue Plasminogen Activator I/D Polymorphism on the tPA Response to Exercise.

The purpose was to determine if the Alu-insertion (I)/deletion (D) polymorphism of the tissue plasminogen activator (tPA) gene influences the tPA response to maximal exercise. Fifty male subjects (age = 23.6 ± 4.7 yrs) completed a maximal treadmill exercise test. Blood samples were drawn before and immediately after exercise for determination of plasma tPA antigen and activity. Isolated DNA was amplified via polymerase chain reaction, electrophoresed, and visually amplified to determine tPA genotype. Subjects were classified as possessing the D allele (D) (n = 28) or being homozygous for the I allele (I) (n = 22). Differences in tPA antigen and activity were assessed using a two-factor (genotype and time) repeated measures analysis of variance. There were significant main effects for time for tPA antigen and activity (p < 0.05), but no main effect for genotype. Furthermore, there was no genotype x time interaction due to a similar increase in tPA antigen in the D group (pre-exercise = 5.83 + 2.39 ng/ml, post-exercise = 21.88 + 7.38 ng/ml) and the I group (pre-exercise = 5.61 + 2.82 ng/ml, post-exercise = 19.05 + 7.67 ng/ml) and a similar increase in tPA activity in the D group (pre-exercise = 0.39 ± 0.19 IU/ml, post-exercise = 9.73 ± 4.22 IU/ml) and I group (pre-exercise = 0.45 ± 0.29 IU/ml, post-exercise = 9.76 ± 5.50 IU/ml). The I/D polymorphism of the tPA gene does not influence the tPA antigen nor tPA activity responses to maximal exercise in healthy, young, sedentary males.

Neuropsychological performance measures as intermediate phenotypes for attention-deficit/hyperactivity disorder: A multiple mediation analysis.

Genetic influences on dopaminergic neurotransmission have been implicated in attention-deficit hyperactivity disorder (ADHD) and are theorized to impact cognitive functioning via alterations in frontal-striatal circuitry. Neuropsychological functioning has been proposed to account for the potential associations between dopamine candidate genes and ADHD. However, to date, this mediation hypothesis has not been directly tested. Participants were 498 youth ages 6-17 years (mean M = 10.8 years, SD = 2.4 years, 55.0% male). All youth completed a multistage, multiple-informant assessment procedure to identify ADHD and non-ADHD cases, as well as a comprehensive neuropsychological battery. Youth provided a saliva sample for DNA analyses; the 480 base pair variable number of tandem repeat polymorphism of the dopamine active transporter 1 gene (DAT1) and the 120 base pair promoter polymorphism of the dopamine receptor D4 gene (DRD4) were genotyped. Multiple mediation analysis revealed significant indirect associations between DAT1 genotype and inattention, hyperactivity-impulsivity, and oppositionality, with specific indirect effects through response inhibition. The results highlight the role of neurocognitive task performance, particularly response inhibition, as a potential intermediate phenotype for ADHD, further elucidating the relationship between genetic polymorphisms and externalizing psychopathology.

Variation in an Iron Metabolism Gene Moderates the Association Between Blood Lead Levels and Attention-Deficit/Hyperactivity Disorder in Children.

Although attention-deficit/hyperactivity disorder (ADHD) is a heritable neurodevelopmental condition, there is also considerable scientific and public interest in environmental modulators of its etiology. Exposure to neurotoxins is one potential source of perturbation of neural, and hence psychological, development. Exposure to lead in particular has been widely investigated and is correlated with neurodevelopmental outcomes, including ADHD. To investigate whether this effect is likely to be causal, we used a Mendelian randomization design with a functional gene variant. In a case-control study, we examined the association between ADHD symptoms in children and blood lead level as moderated by variants in the hemochromatosis (HFE) gene. The HFE gene regulates iron uptake and secondarily modulates lead metabolism. Statistical moderation was observed: The magnitude of the association of blood lead with symptoms of ADHD was altered by functional HFE genotype, which is consistent with a causal hypothesis.

Does 5HTTLPR Genotype Moderate the Association of Family Environment With Child Attention-Deficit Hyperactivity Disorder Symptomatology?

Problematic family dynamics are common among youth with attention-deficit hyperactivity disorder (ADHD). Multiple mechanisms, including diathesis-stress (vulnerability) and differential susceptibility Gene × Environment interaction effects (G × E), have been proposed to account for this association. G × E effects for ADHD were examined via interactions between a genetic marker hypothesized to influence sensitivity to the environment (the promoter polymorphism of the serotonin transporter gene -5HTTLPR) and family conflict and cohesion in predicting ADHD symptoms. There were 498 youth ages 6-17 years (251 ADHD, 213 non-ADHD) and their parents who completed a multistage, multi-informant assessment (including parent and youth reports on the Family Environment Scale), and saliva sample collection for genotyping. Linear regression analyses examined interactions between 5HTTLPR genotype and the Family Environment Scale scales of conflict and cohesion reported by parent and child. Criteria laid out by Roisman et al. ( 2012 ) were applied to evaluate diathesis stress versus differential susceptibility G × E mechanisms. Results demonstrated interactions between 5HTTLPR genotype and both conflict and cohesion in predicting inattention but not hyperactivity-impulsivity. Both interactions were highly consistent with differential susceptibility models of G × E effects. 5HTTLPR genotype appeared to moderate the relationship between family conflict/cohesion and inattentive symptoms. Interactions highlight the role of 5HTTLPR genotype as a potential marker of environmental sensitivity and provide support for differential susceptibility models of G × E effects for ADHD.

Challenges and solutions for gene identification in the presence of familial locus heterogeneity.

Next-generation sequencing (NGS) of exomes and genomes has accelerated the identification of genes involved in Mendelian phenotypes. However, many NGS studies fall short of identifying causal variants, with estimates for success rates as low as 25% for uncovering the pathological variant underlying disease etiology. An important reason for such failures is familial locus heterogeneity, where within a single pedigree causal variants in two or more genes underlie Mendelian trait etiology. As examples of intra- and inter-sibship familial locus heterogeneity, we present 10 consanguineous Pakistani families segregating hearing impairment due to homozygous variants in two different hearing impairment genes and a European-American pedigree in which hearing impairment is caused by four variants in three different genes. We have identified 41 additional pedigrees with syndromic and nonsyndromic hearing impairment for which a single previously reported hearing impairment gene has been identified but only segregates with the phenotype in a subset of affected pedigree members. We estimate that locus heterogeneity occurs in 15.3% (95% confidence interval: 11.9%, 19.9%) of the families in our collection. We demonstrate novel approaches to apply linkage analysis and homozygosity mapping (for autosomal recessive consanguineous pedigrees), which can be used to detect locus heterogeneity using either NGS or SNP array data. Results from linkage analysis and homozygosity mapping can also be used to group sibships or individuals most likely to be segregating the same causal variants and thereby increase the success rate of gene identification.

Pedigree structure and kinship measurements of a mid-Michigan community: a new North American population isolate identified.

Previous studies identified a cluster of individuals with an autosomal recessive form of deafness that reside in a small region of mid-Michigan. We hypothesized that affected members from this community descend from a defined founder population. Using public records and personal interviews, we constructed a genealogical database that includes the affected individuals and their extended families as descendants of 461 settlers who emigrated from the Eifel region of Germany between 1836 and 1875. The genealogical database represents a 13-generation pedigree that includes 27,747 descendants of these settlers. Among these descendants, 13,784 are presumed living. Many of the extant descendants reside in a 90-square-mile area, and 52% were born to parents who share at least one common ancestor. Among those born to related parents, the median kinship coefficient is 3.7 × 10(-3). While the pedigree contains 2,510 founders, 344 of the 461 settlers accounted for 67% of the genome in the extant population. These data suggest that we identified a new population isolate in North America and that, as demonstrated for congenital hearing loss, this rural mid-Michigan community is a new resource to discover heritable factors that contribute to common health-related conditions.

Distribution and Severity of Neuropathology in ß-Mannosidase-Deficient Mice is Strain Dependent.

Neurological dysfunction is common in humans and animals with lysosomal storage diseases. ß-Mannosidosis, an autosomal recessive inherited disorder of glycoprotein catabolism caused by deficiency of the lysosomal enzyme ß-mannosidase, is characterized by intracellular accumulation of small oligosaccharides in selected cell types. In ruminants, clinical manifestation is severe, and neuropathology includes extensive intracellular vacuolation and dysmyelination. In human cases of ß-mannosidosis, the clinical symptoms, including intellectual disability, are variable and can be relatively mild. A ß-mannosidosis knockout mouse was previously characterized and showed normal growth, appearance, and lifespan. Neuropathology between 1 and 9 months of age included selective, variable neuronal vacuolation with no hypomyelination. This study characterized distribution of brain pathology in older mutant mice, investigating the effects of two strain backgrounds. Morphological analysis indicated a severe consistent pattern of neuronal vacuolation and disintegrative degeneration in all five 129X1/SvJ mice. However, the mice with a mixed genetic background showed substantial variability in the severity of pathology. In the severely affected animals, neuronal vacuolation was prominent in specific layers of piriform area, retrosplenial area, anterior cingulate area, selected regions of isocortex, and in hippocampus CA3. Silver degeneration reaction product was prominent in regions including specific cortical layers and cerebellar molecular layer. The very consistent pattern of neuropathology suggests metabolic differences among neuronal populations that are not yet understood and will serve as a basis for future comparison with human neuropathological analysis. The variation in severity of pathology in different mouse strains implicates genetic modifiers in the variable phenotypic expression in humans.

A novel actin mRNA splice variant regulates ACTG1 expression.

Cytoplasmic actins are abundant, ubiquitous proteins in nucleated cells. However, actin expression is regulated in a tissue- and development-specific manner. We identified a novel cytoplasmic-γ-actin (Actg1) transcript that includes a previously unidentified exon (3a). Inclusion of this exon introduces an in-frame termination codon. We hypothesized this alternatively-spliced transcript down-regulates γ-actin production by targeting these transcripts for nonsense-mediated decay (NMD). To address this, we investigated conservation between mammals, tissue-specificity in mice, and developmental regulation using C2C12 cell culture. Exon 3a is 80% similar among mammals and varies in length from 41 nucleotides in humans to 45 in mice. Though the predicted amino acid sequences are not similar between all species, inclusion of exon 3a consistently results in the in the introduction of a premature termination codon within the alternative Actg1 transcript. Of twelve tissues examined, exon 3a is predominantly expressed in skeletal muscle, cardiac muscle, and diaphragm. Splicing to include exon 3a is concomitant with previously described down-regulation of Actg1 in differentiating C2C12 cells. Treatment of differentiated C2C12 cells with an inhibitor of NMD results in a 7-fold increase in exon 3a-containing transcripts. Therefore, splicing to generate exon 3a-containing transcripts may be one component of Actg1 regulation. We propose that this post-transcriptional regulation occurs via NMD, in a process previously described as "regulated unproductive splicing and translation" (RUST).

Angiotensinogen promoter polymorphisms predict low diffusing capacity in U.S. and Spanish IPF cohorts.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in angiotensinogen (AGT) at positions -20 and -6 are associated with increased severity and progression of various fibrotic diseases. Our earlier work demonstrated that the progression of idiopathic pulmonary fibrosis (IPF) was associated with the A-6 allele. This study examined the hypothesis that the homozygous CC genotype at -20 and the AA genotype at -6 would confer worse measures of pulmonary function (measured by pulmonary function tests) in IPF. METHODS: Multiple logistic regression analysis was applied to a NIH Lung Tissue Research Consortium cohort and a Spanish cohort, while also adjusting for covariates to determine the effects of these SNPs on measures of pulmonary function. RESULTS: Analysis demonstrated that the CC genotype at -20 was strongly associated with reduced diffusing capacity in males in both cohorts (p = 0.0028 for LTRC and p = 0.017 for the Spanish cohort). In females, the AA genotype was significantly associated with lower FVC (p = 0.0082) and V alv (p = 0.022). In males, the haplotype CA at -20 and -6 in AGT was also strongly associated with reduced diffusing capacity in both cohorts. CONCLUSIONS: This study is the first to demonstrate an association of AGT polymorphisms (-20A > C and -6G > A) with lower measures of pulmonary function in IPF. It is also the first to relate the effect of gender in lung fibrosis with polymorphisms in AGT.

Actin in hair cells and hearing loss.

Hereditary deafness is genetically heterogeneous such that mutations of many different genes can cause hearing loss. This review focuses on the evidence and implications that several of these deafness genes encode actin-interacting proteins or actin itself. There is a growing appreciation of the contribution of the actin interactome in stereocilia development, maintenance, mechanotransduction and malfunction of the auditory system.

Life stressors and 5-HTTLPR interaction in relation to midpregnancy depressive symptoms among African-American women.

OBJECTIVE: In earlier analyses of nonHispanic White women we found a stronger relation between abuse history and midpregnancy elevated depressive symptoms in women with the serotonin transporter (5-HTTLPR) S/S genotype. Here, we focus on African-American women (N=698). Our inquiry is motivated by racial differences in depression diagnosis/treatment, stressors, and frequency of major 5-HTTLPR alleles (S, LA, LG). MATERIALS AND METHODS: Stressful life events (lifetime) and depressive symptoms (current) were ascertained at 15-27 weeks gestation. A Center for Epidemiological Studies Depression Score of more than or equal to 18 was considered 'elevated'. Life events were scored together and separated into six subconstructs. 5-HTTLPR genotypes were grouped as follows: (i) L and S alleles, (ii) S-LG equivalence ('triallelic to biallelic'), and (iii) LA/LA, all others, S/S ('high/intermediate/low'). Odds ratios (OR) for 'elevated' depressive symptoms-life events (total and subconstructs) relations were calculated for each genotype grouping. RESULTS: The prevalence of 'elevated' depressive symptoms did not vary by genotype. The relation between stressful life events and 'elevated' depressive symptoms was stronger in S/S compared with LA/LA genotype (interaction P=0.11). Of the six subconstructs, only abuse showed a statistically significant gene-environment interaction. The OR for the abuse-'elevated' depressive symptoms association was greater for S/S vs. LA/LA genotype (interaction P=0.03) and in the 'triallelic to biallelic' grouping (interaction P=0.04). In the 'high/intermediate/low' grouping, 'low' (S/S) had a higher OR (5.5) than both 'intermediate' and 'high' (ORs≤2.3) (interaction P=0.10). CONCLUSIONS: These results show the importance of examining racial groups, specific stressful events, and different 5-HTTLPR genotype groupings when exploring gene-environment interactions in depression.

Identification of ATPAF1 as a novel candidate gene for asthma in children.

BACKGROUND: Asthma is a common disease of children with a complex genetic origin. Understanding the genetic basis of asthma susceptibility will allow disease prediction and risk stratification. OBJECTIVE: We sought to identify asthma susceptibility genes in children. METHODS: A nested case-control genetic association study of children of Caucasian European ancestry from a birth cohort was conducted. Single nucleotide polymorphisms (SNPs, n = 116,024) were genotyped in pools of DNA samples from cohort children with physician-diagnosed asthma (n = 112) and normal controls (n = 165). A genomic region containing the ATPAF1 gene was found to be significantly associated with asthma. Additional SNPs within this region were genotyped in individual samples from the same children and in 8 independent study populations of Caucasian, African American, Hispanic, or other ancestries. SNPs were also genotyped or imputed in 2 consortia control populations. ATPAF1 expression was measured in bronchial biopsies from asthmatic patients and controls. RESULTS: Asthma was found to be associated with a cluster of SNPs and SNP haplotypes containing the ATPAF1 gene, with 2 SNPs achieving significance at a genome-wide level (P = 2.26 × 10(-5) to 2.2 × 10(-8)). Asthma severity was also found to be associated with SNPs and SNP haplotypes in the primary population. SNP and/or gene-level associations were confirmed in the 4 non-Hispanic populations. Haplotype associations were also confirmed in the non-Hispanic populations (P = .045-.0009). ATPAF1 total RNA expression was significantly (P < .01) higher in bronchial biopsies from asthmatic patients than from controls. CONCLUSION: Genetic variation in the ATPAF1 gene predisposes children of different ancestries to asthma.

Interplay of cytokine polymorphisms and bacterial vaginosis in the etiology of preterm delivery.

Recent findings suggest that the association between inflammation-related genes and preterm delivery may be stronger in the presence of bacterial vaginosis (BV). Tumor necrosis factor-alpha (TNFα) and interleukin 1-beta (IL-1ß) are pro-inflammatory cytokines capable of inducing preterm labor in non-human primates. In this study the authors tested associations among two TNFα promoter polymorphisms (-G308A and -G238A), a single IL-1ß polymorphism (+C3954T), vaginal microbial findings, and risk of preterm delivery. Data were from the Pregnancy Outcomes and Community Health (POUCH) Study (n=777 term and n=230 preterm deliveries). Vaginal smears collected at mid-pregnancy (15-27 weeks gestation) were scored according to Nugent's criteria. A Nugent score of ≥ 4 was modeled as the cut-point for intermediate and positive BV. Logistic regression was used to estimate odds ratios for associations among independent covariates (vaginal flora, genotype) and preterm delivery. Results showed that women with a Nugent score of≥ 4 and the TNFα -238 A/G or A/A were at increased risk of delivering preterm (race/ethnicity adjusted OR 2.6, 95% CI 1.2, 5.8). The p-value for the genotype and Nugent score interaction=0.02. This study points to one more example of a potential gene-environment interaction in a preterm delivery pathway. Future tests of this finding will determine the robustness of these results.

Ion-dependent polymerization differences between mammalian beta- and gamma-nonmuscle actin isoforms.

beta- and gamma-nonmuscle actins differ by 4 amino acids at or near the N terminus and distant from polymerization interfaces. beta-Actin contains an Asp(1)-Asp(2)-Asp(3) and Val(10) whereas gamma-actin has a Glu(1)-Glu(2)-Glu(3) and Ile(10). Despite these small changes, conserved across mammals, fish, and birds, their differential localization in the same cell suggests they may play different roles reflecting differences in their biochemical properties. To test this hypothesis, we established a baculovirus-driven expression system for producing these actins in isoform-pure populations although contaminated with 20-25% insect actin. Surprisingly, Ca-gamma-actin exhibits a slower monomeric nucleotide exchange rate, a much longer nucleation phase, and a somewhat slower elongation rate than beta-actin. In the Mg-form, this difference between the two is much smaller. Ca-gamma-actin depolymerizes half as fast as does beta-actin. Mixing experiments with Ca-actins reveal the two will readily co-polymerize. In the Ca-form, phosphate release from polymerizing beta-actin occurs much more rapidly and extensively than polymerization, whereas phosphate release lags behind polymerization with gamma-actin. Phosphate release during treadmilling is twice as fast with beta- as with gamma-actin. With Mg-actin in the initial stages, phosphate release for both actins correlates much more closely with polymerization. Calcium bound in the high affinity binding site of gamma-actin may cause a selective energy barrier relative to beta-actin that retards the equilibration between G- and F-monomer conformations resulting in a slower polymerizing actin with greater filament stability. This difference may be particularly important in sites such as the gamma-actin-rich cochlear hair cell stereocilium where local mm calcium concentrations may exist.

Polymorphisms in thrombophilia and renin-angiotensin system pathways, preterm delivery, and evidence of placental hemorrhage.

OBJECTIVE: The purpose of this study was to analyze functional polymorphisms in candidate genes (methylenetetrahydrofolate reductase [MTHFR]677C>T, MTHFR1298A>C, factor 5 1691G>A [FVL], and angiotensinogen (AGT)-6G>A) in relation to a hypothesized placental hemorrhage pathway to preterm delivery (PTD). STUDY DESIGN: We assessed maternal genotypes, pregnancy outcomes, and placental pathologic evidence among 560 white and 399 black women who were recruited at mid trimester into a prospective cohort study (1998-2004). Odds of dominant genotypes were calculated for PTDs with (n = 56) or without (n = 177) evidence of placental hemorrhage (referent = term) with the use of race-stratified polytomous logistic regression models. RESULTS: Among white women, FVL GA/AA and AGT(-6) GA/AA were both associated with hemorrhage-related PTDs (odds ratio [OR], 4.8; 95% confidence interval [CI], 1.6-14.2 and OR, 3.8; 95% CI, 1.3-10.5, respectively), but not other PTDs (ORs, 1.2 and 0.9, respectively). FVL GA/AA was associated with placental abruption (OR, 5.8; 95% CI, 1.1-30) among white women. All results were null for MTHFR genotypes. CONCLUSION: FVL and AGT variant genotypes were associated specifically with hemorrhage-related PTDs.

Gamma-actin is required for cytoskeletal maintenance but not development.

Beta(cyto)-actin and gamma(cyto)-actin are ubiquitous proteins thought to be essential building blocks of the cytoskeleton in all non-muscle cells. Despite this widely held supposition, we show that gamma(cyto)-actin null mice (Actg1(-/-)) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of beta(cyto)-actin. The surprising viability and normal hearing of young Actg1(-/-) mice means that beta(cyto)-actin can likely build all essential non-muscle actin-based cytoskeletal structures including mechanosensory stereocilia of hair cells that are necessary for hearing. Although gamma(cyto)-actin-deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, gamma(cyto)-actin localizes along the length of stereocilia but re-distributes to sites of F-actin core disruptions resulting from animal exposure to damaging noise. In Actg1(-/-) stereocilia similar disruptions are observed even without noise exposure. We conclude that gamma(cyto)-actin is required for reinforcement and long-term stability of F-actin-based structures but is not an essential building block of the developing cytoskeleton.

SNP discovery and haplotype analysis in the segmentally duplicated DRD5 coding region.

The dopamine receptor 5 gene (DRD5) holds much promise as a candidate locus for contributing to neuropsychiatric disorders and other diseases influenced by the dopaminergic system, as well as having potential to affect normal behavioral variation. However, detailed analyses of this gene have been complicated by its location within a segmentally duplicated chromosomal region. Microsatellites and SNPs upstream from the coding region have been used for association studies, but we find, using bioinformatics resources, that these markers all lie within a previously unrecognized second segmental duplication (SD). In order to accurately analyze the DRD5 locus for polymorphisms in the absence of contaminating pseudogene sequences, we developed a fast and reliable method for sequence analysis and genotyping within the DRD5 coding region. We employed restriction enzyme digestion of genomic DNA to eliminate the pseudogenes prior to PCR amplification of the functional gene. This approach allowed us to determine the DRD5 haplotype structure using 31 trios and to reveal additional rare variants in 171 unrelated individuals. We clarify the inconsistencies and errors of the recorded SNPs in dbSNP and HapMap and illustrate the importance of using caution when choosing SNPs in regions of suspected duplications. The simple and relatively inexpensive method presented herein allows for convenient analysis of sequence variation in DRD5 and can be easily adapted to other duplicated genomic regions in order to obtain good quality sequence data.

Expression of GJB2 and GJB6 is reduced in a novel DFNB1 allele.

In a large kindred of German descent, we found a novel allele that segregates with deafness when present in trans with the 35delG allele of GJB2. Qualitative polymerase chain reaction-based allele-specific expression assays showed that expression of both GJB2 and GJB6 from the novel allele is dramatically reduced. This is the first evidence of a deafness-associated regulatory mutation of GJB2 and of potential coregulation of GJB2 and GJB6.

A novel missense mutation in ACTG1 causes dominant deafness in a Norwegian DFNA20/26 family, but ACTG1 mutations are not frequent among families with hereditary hearing impairment.

The gamma-actin gene (ACTG1) encodes a major cytoskeletal protein of the sensory hair cells of the cochlea. Recently, mutations in ACTG1 were found to cause autosomal dominant, progressive, sensorineural hearing impairment linked to the DFNA20/26 locus on chromosome 17q25.3 in four American families and in one Dutch family. We report here the linkage of autosomal dominant, progressive, sensorineural hearing impairment in a large Norwegian family to the DFNA20/26 locus. Sequencing of ACTG1 identified a novel missense mutation (c.1109T>C; p.V370A) segregating with the hearing loss. Functional analysis in yeast showed that the p.V370A mutation restricts cell growth at elevated temperature or under hyperosmolar stress. Molecular modelling suggested that the p.V370A mutation modestly alters a site for protein-protein interaction in gamma-actin and thereby modestly alters gamma-actin-based cytoskeletal structures. Nineteen Norwegian and Danish families with autosomal, dominant hearing impairment were analyzed for mutations in ACTG1 by sequencing, but no disease-associated mutations were identified. Finally, a long-term follow-up of the hearing loss progression associated with the p.V370A mutation in ACTG1 is provided. The present study expands our understanding of the genotype-phenotype relationship of this deafness gene and provides a sensitive and simple functional assay for missense mutations in this gene, which may assist future molecular diagnosis of autosomal-dominant hearing impairment. Finally, the present results do not indicate that mutations in ACTG1 are a frequent cause of autosomal-dominant postlingual sensorineural hearing impairment in Norway nor Denmark.

Effects of human deafness gamma-actin mutations (DFNA20/26) on actin function.

Six point mutations in non-muscle gamma-actin at the DFNA20/26 locus cause autosomal dominant nonsyndromic hearing loss. The molecular basis for the hearing loss is unknown. We have engineered each gamma-actin mutation into yeast actin to investigate the effects of these mutations on actin function in vivo and in vitro. Cells expressing each of the mutant actins as the sole actin in the cell were viable. Four of the six mutant strains exhibited significant growth deficiencies in complete medium and an inability to grow on glycerol as the sole carbon source, implying a mitochondrial defect(s). These four strains exhibited abnormal mitochondrial morphology, although the mtDNA was retained. All of the mutant cells exhibited an abnormally high percentage of fragmented/non-polarized actin cables or randomly distributed actin patches. Five of the six mutants displayed strain-specific vacuole morphological abnormalities. Two of the purified mutant actins exhibited decreased thermal stability and increased rates of nucleotide exchange, indicative of increased protein flexibility. V370A actin alone polymerized abnormally. It aggregated in low ionic strength buffer and polymerized faster than wild-type actin, probably in part because of enhanced nucleation. Mixtures of wild-type and V370A actins displayed kinetic properties in proportion to the mole fraction of each actin in the mixture. No dominant effect of the mutant actin was observed. Our results suggest that a major factor in the deafness caused by these mutations is an altered ability of the actin filaments to be properly regulated by actin-binding proteins rather than an inability to polymerize.