BREACHing new grounds in fragile X syndrome: Trinucleotide expansion linked to genome-wide heterochromatin domains and genome misfolding.

In a recent study in Cell, Malachowski et al.1 show that the trinucleotide expansion in the FMR1 gene underlying fragile X syndrome triggers formation of large heterochromatin domains across the genome, resulting in the repression of synaptic genes housed within these domains.

High-Resolution NMR Structures of Intrastrand Hairpins Formed by CTG Trinucleotide Repeats.

The CAG and CTG trinucleotide repeat expansions cause more than 10 human neurodegenerative diseases. Intrastrand hairpins formed by trinucleotide repeats contribute to repeat expansions, establishing them as potential drug targets. High-resolution structural determination of CAG and CTG hairpins poses as a long-standing goal to aid drug development, yet it has not been realized due to the intrinsic conformational flexibility of repetitive sequences. We herein investigate the solution structures of CTG hairpins using nuclear magnetic resonance (NMR) spectroscopy and found that four CTG repeats with a clamping G-C base pair was able to form a stable hairpin structure. We determine the first solution NMR structure of dG(CTG)4C hairpin and decipher a type I folding geometry of the TGCT tetraloop, wherein the two thymine residues form a T·T loop-closing base pair and the first three loop residues continuously stack. We further reveal that the CTG hairpin can be bound and stabilized by a small-molecule ligand, and the binding interferes with replication of a DNA template containing CTG repeats. Our determined high-resolution structures lay an important foundation for studying molecular interactions between native CTG hairpins and ligands, and benefit drug development for trinucleotide repeat expansion diseases.

Structural Dynamics Role of AGG Interruptions in Inhibition CGG Repeat Expansion Associated with Fragile X Syndrome.

Abnormal expansion of trinucleotide CGG repeats is responsible for Fragile X syndrome. AGG interruptions in CGG repeat tracts were found in most healthy individuals, suggesting a crucial role in preventing disease-prone repeat expansion. Previous biophysics studies emphasize a difference in the secondary structure affected by AGG interruptions. However, the mechanism of how AGG interruptions impede repeat expansion remains elusive. We utilized single-molecule fluorescence resonance energy transfer spectroscopy to investigate the structural dynamics of CGG repeats and their AGG-interrupted variants. Tandem CGG repeats fold into a stem-loop hairpin structure with the capability to undergo a conformational rearrangement to modulate the length of the overhang. However, this conformational rearrangement is much more retarded when two AGG interruptions are present. Considering the significance of hairpin slippage in repeat expansion, we present a molecular basis suggesting that the internal loop created by two AGG interruptions acts as a barrier, obstructing the hairpin slippage reconfiguration. This impediment potentially plays a crucial role in curbing abnormal expansion, thereby contributing to the genomic stability.

Number of CAG repeats and mortality in middle aged and older men.

DESIGN: The androgen receptor (AR) mediates peripheral effects of testosterone. Previous data suggests an association between the number of CAG repeats in exon-1 of the AR gene and AR transcriptional activity. The aim of this analysis was to determine the association between the number of AR CAG repeats and all-cause mortality in men and the influence of testosterone level on the association. PATIENTS AND MEASUREMENTS: Follow-up data to 27 January 2018 were available for men aged 40-79 years recruited across six countries of the European Male Aging Study between 2003 and 2005. Cox proportional hazards modelling was used to determine the association between CAG repeat number/mortality. Results were expressed as hazard ratios (HR)/95% confidence intervals (CI). RESULTS: One thousand nine hundred and seventy-seven men were followed up. Mean baseline age was 60 ± 11.1 years. Mean duration of follow-up was 12.2 years. At follow up 25.1% of men had died. CAG repeat length ranged from 6 to 39, with the highest proportion of CAG repeat number at 21 repeats (16.4%). In a multivariable model, compared to men with 22-23 AR CAG repeats: for men with <22 and >23 AR CAG HR, 95% CI for mortality were, <22 CAG repeats 1.17 (0.93-1.49) and >23 CAG repeats 1.14 (0.88-1.47). In a post-hoc analysis, the association was significant for men in the lowest tertile of baseline testosterone (<14.2 nmol/L) with >23 CAG repeats: in the adjusted model for <22 and >23 CAG repeats, respectively, 1.49 (0.97-2.27) and 1.68 (1.06-2.67) versus 22-23 repeats. CONCLUSIONS: Our European-wide cohort data overall found no association of androgen receptor CAG repeat number and mortality in men. However, post hoc analysis suggested that an association might be present in men with lower baseline testosterone concentrations, which merits further investigation.

Odd-even disparity in the population of slipped hairpins in RNA repeat sequences with implications for phase separation.

Low-complexity nucleotide repeat sequences, which are implicated in several neurological disorders, undergo liquid-liquid phase separation (LLPS) provided the number of repeat units, n, exceeds a critical value. Here, we establish a link between the folding landscapes of the monomers of trinucleotide repeats and their propensity to self-associate. Simulations using a coarse-grained Self-Organized Polymer (SOP) model for (CAG)n repeats in monovalent salt solutions reproduce experimentally measured melting temperatures, which are available only for small n. By extending the simulations to large n, we show that the free-energy gap, ΔGS, between the ground state (GS) and slipped hairpin (SH) states is a predictor of aggregation propensity. The GS for even n is a perfect hairpin (PH), whereas it is a SH when n is odd. The value of ΔGS (zero for odd n) is larger for even n than for odd n. As a result, the rate of dimer formation is slower in (CAG)30 relative to (CAG)31, thus linking ΔGS to RNA-RNA association. The yield of the dimer decreases dramatically, compared to the wild type, in mutant sequences in which the population of the SH decreases substantially. Association between RNA chains is preceded by a transition to the SH even if the GS is a PH. The finding that the excitation spectrum-which depends on the exact sequence, n, and ionic conditions-is a predictor of self-association should also hold for other RNAs (mRNA for example) that undergo LLPS.

The relationship between the number of CAG repeats and clinical manifestations: a survey of Chinese DRPLA family.

OBJECTIVE: To describe the clinical, radiological, and genetic characteristics of a Chinese family with dentatorubropallidoluysian atrophy (DRPLA). Explore the distribution of CAG repeat size to the clinical features of patients. METHODS: We collected the clinical symptoms and DNA analysis for the DRPLA gene was performed on the family members. DRPLA patients reported in the literature were reviewed to analyze the association between CAG repeat size and clinical features. RESULTS: Six family members were confirmed by genetic analysis. The number of CAG repeat in the proband, her sister, her grandmother, her father, her uncle, and her cousin, was determined respectively as 63, 75, 50, 50, 50, 54. In our family, the sister of the proband had the earliest onset age and the most severe clinical symptoms, followed by the proband, and other family members showed no obvious clinical symptoms. Consistent with the conclusion of previous studies, the more repeats CAG, the earlier the age of onset and the severer phenotypes are. CONCLUSION: We found six family members have CAG repeat expansion in the DRPLA gene on chromosome 12p13. Even in the same family, patients have different clinical presentations. The size of CAG repeats is negatively correlated with the age of onset and positively correlated with symptom severity. When the number of repeats is ≥ 63, the age at onset is < 21 years old, and obvious clinical symptoms generally appear. It seems to say the more repeats CAG, the earlier the age of onset and the severer phenotypes are. LIMITATIONS: With a small number of cases in our family, the conclusion that the more CAG repeats, the earlier the onset and the more severe the clinical symptoms cannot be fully proved.

Differential expression of the androgen receptor gene is correlated with CAG polymorphic repeats in patients with prostate cancer.

Prostate cancer (PCa) is one of the most common types of cancer in men. The aetiology of the disease is not well established, but it has been related to one of the main pathways of regulation of prostate proliferation, mediated by androgens. The androgen receptor (AR) gene encodes the androgen-receptor protein, which functions as a transactivation factor for steroid hormones. It has been proposed that the AR gene transcription levels are mediated by short tandem repeats corresponding to the CAG sequence. However, there are conflicting results in this relationship. We evaluated the expression levels of the AR gene and identified the number of CAG repeats (CAGn) in the Mexican population, establishing the relationship between expression levels and increase in the number of CAG repeats. We evaluated the expression levels of AR in tissue samples of PCa and benign prostate disease, such as benign prostatic hyperplasia, or prostatitis, to determine the difference in their expression levels. Our results showed a statistically insignificant underexpression of 0.64-fold decrease in AR levels of PCa patients compared to benign prostate disease patients (P = 0.623) and suggest that the number of CAGn was correlated with the relative expression of the AR gene (P = 0.009) and this correlation was positive, moderate, and proportional (ρ = 0.467) and no correlation was found between CAGn with other clinical features.

Reduced-penetrance Huntington's disease-causing alleles with 39 CAG trinucleotide repeats could be a genetic factor of amyotrophic lateral sclerosis.

BACKGROUND: Expanded HTT alleles with 40 or more CAG repeats were recently found to be a rare cause of frontotemporal dementia and amyotrophic lateral sclerosis (ALS) spectrum diseases. The aim of this study was to investigate the role of HTT repeat expansions in a Taiwanese cohort with ALS. METHODS: We analyzed the numbers of CAG repeats in exon 1 of HTT in a cohort of 410 Taiwanese patients with ALS and 1514 control individuals by utilizing polymerase chain reaction and amplicon fragment length analysis. RESULTS: Only one of the 410 ALS patients carried a reduced-penetrance HD-causing allele with 39 CAG repeats, and none had an expanded HTT CAG repeats ≥40. The patient presented with rapidly progressive bulbar-onset ALS with disease onset at the age of 64 years. He had neither chorea nor cognitive impairment. He had a family history of chorea, but no other family member manifested with ALS. None of the 1514 control individuals carried an HTT expanded allele with CAG repeats larger than 37 repeats. CONCLUSION: The HTT allele with 39 CAG repeats could be a genetic factor linked to ALS susceptibility.

Unraveling the role of relative telomere length and CAG expansion on initial symptoms of juvenile Huntington disease.

BACKGROUND AND PURPOSE: Juvenile-onset Huntington disease (JHD) is defined when symptoms initiate before 20 years of age. Mechanisms explaining differences between juvenile and adult onset are not fully understood. Our aim was to analyze the distribution of initial symptoms in a cohort of JHD patients and to explore its relationship with CAG expansion and relative telomere length (RTL). METHODS: A total of 84 JHD patients and 54 neurologically healthy age and sex matched individuals were recruited. CAG length was measured by southern blot or triplet repeat primed polymerase chain reaction. RTL was measured using the Cawthon method. RESULTS: Psychiatric symptoms were most frequent when considering the entire cohort. When divided into onset before or after 10 years, cognitive symptoms were more frequent in the youngest, whilst in the older group psychiatric symptoms prevailed. Motor symptoms were rare in the youngest and epilepsy was observed only in this group as well as a larger CAG expansion. RTL analysis revealed shorter telomeres in JHD patients compared to controls. This difference is not influenced by age, initial symptoms, time of disease or CAG expansion. CONCLUSIONS: To the best of our knowledge this is the largest cohort of JHD patients reported. Psychiatric manifestations deserve special attention when JHD is suspected and epilepsy is especially important in the youngest patients. Initial symptoms seem to be influenced by CAG expansion and therefore age of onset. RTL is significantly reduced in JHD patients which can influence the characteristic neurodegeneration of JHD and contribute to the clinical discrepancy between adult and juvenile forms of Huntington disease.

Androgen receptor polyQ alleles and COVID-19 severity in men: A replication study.

BACKGROUND: Ample evidence indicates a sex-related difference in severity of COVID-19, with less favorable outcomes observed in men. Genetic factors have been proposed as candidates to explain this difference. The polyglutamine (polyQ) polymorphism in the androgen receptor gene has been recently described as a genetic biomarker of COVID-19 severity. OBJECTIVE: To test the association between the androgen receptor polyQ polymorphism and COVID-19 severity in a large cohort of COVID-19 male patients. MATERIALS AND METHODS: This study included 1136 male patients infected with SARS-CoV-2 as confirmed by positive PCR. Patients were retrospectively and prospectively enrolled from March to November 2020. Patients were classified according to their severity into three categories: oligosymptomatic, hospitalized and severe patients requiring ventilatory support. The number of CAG repeats (polyQ polymorphism) at the androgen receptor was obtained by PCR and patients were classified as either short (<23 repeats) or long (≥23 repeats) allele carriers. The association between polyQ alleles (short or long) and COVID-19 severity was assessed by Chi-squared (Chi2 ) and logistic regression analysis. RESULTS: The mean number of polyQ CAG repeats was 22 (±3). Patients were classified as oligosymptomatic (15.5%), hospitalized (63.2%), and severe patients (21.3%) requiring substantial respiratory support. PolyQ alleles distribution did not show significant differences between severity classes in our cohort (Chi2 test p > 0.05). Similar results were observed after adjusting by known risk factors such as age, comorbidities, and ethnicity (multivariate logistic regression analysis). DISCUSSION: Androgen sensitivity may be a critical factor in COVID-19 disease severity. However, we did not find an association between the polyQ polymorphism and the COVID-19 severity. Additional studies are needed to clarify the mechanism underlying the association between androgens and COVID-19 outcome. CONCLUSIONS: The results obtained in our study do not support the role of this polymorphism as biomarker of COVID-19 severity.

The length of uninterrupted CAG repeats in stem regions of repeat disease associated hairpins determines the amount of short CAG oligonucleotides that are toxic to cells through RNA interference.

Extended CAG trinucleotide repeats (TNR) in the genes huntingtin (HTT) and androgen receptor (AR) are the cause of two progressive neurodegenerative disorders: Huntington's disease (HD) and Spinal and Bulbar Muscular Atrophy (SBMA), respectively. Anyone who inherits the mutant gene in the complete penetrance range (>39 repeats for HD and 44 for SBMA) will develop the disease. An inverse correlation exists between the length of the CAG repeat and the severity and age of onset of the diseases. Growing evidence suggests that it is the length of uninterrupted CAG repeats in the mRNA rather than the length of poly glutamine (polyQ) in mutant (m)HTT protein that determines disease progression. One variant of mHTT (loss of inhibition; LOI) causes a 25 year earlier onset of HD when compared to a reference sequence, despite both coding for a protein that contains an identical number of glutamines. Short 21-22 nt CAG repeat (sCAGs)-containing RNAs can cause disease through RNA interference (RNAi). RNA hairpins (HPs) forming at the CAG TNRs are stabilized by adjacent CCG (in HD) or CUG repeats (in SBMA) making them better substrates for Dicer, the enzyme that processes CAG HPs into sCAGs. We now show that cells deficient in Dicer or unable to mediate RNAi are resistant to the toxicity of the HTT and AR derived HPs. Expression of a small HP that mimics the HD LOI variant is more stable and more toxic than a reference HP. We report that the LOI HP is processed by Dicer, loaded into the RISC more efficiently, and gives rise to a higher quantity of RISC-bound 22 nt sCAGs. Our data support the notion that RNAi contributes to the cell death seen in HD and SBMA and provide an explanation for the dramatically reduced onset of disease in HD patients that carry the LOI variant.

Suppression of trinucleotide repeat expansion in spermatogenic cells in Huntington's disease.

Trinucleotide repeats (TNRs) are dispersed throughout the human genome. About 20 loci are related to human diseases, such as Huntington's disease (HD). A larger TNR instability is predominantly observed in the paternal germ cells in some TNR disorders. Suppressing the expansion during spermatogenesis can provide a unique opportunity to end the vicious cycle of genetic anticipation. Here, using an in vitro differentiation method to derive advanced spermatogenic cells, we investigated the efficacy of two therapeutic agents, araC (cytarabine) and aspirin, on stabilizing TNRs in spermatogenic cells. Two WT patient-derived induced pluripotent stem cell (iPSC) lines and two HD hiPSC lines, with 44 Q and 180 Q, were differentiated into spermatogonial stem cell-like cells (SSCLCs). Both HD cell lines showed CAG tract expansion in SSCLC. When treated with araC and aspirin, HD1 showed moderate but not statistically significant stabilization of TNR. In HD2, 10 nM of aspirin and araC showed significant stabilization of TNR. All cell lines showed increased DNA damage response (DDR) gene expression in SSCLCs while more genes were significantly induced in HD SSCLC. In HD1, araC and aspirin treatment showed general suppression of DNA damage response genes. In HD2, only FAN1, OGG1, and PCNA showed significant suppression. When the methylation profile of HD cells was analyzed, FAN1 and OGG1 showed significant hypermethylation after the aspirin and araC treatment in SSCLC compared to the control. This study underscores the utility of our in vitro spermatogenesis model to study and develop therapies for TNR disorders such as HD.

The mechanism of replication stalling and recovery within repetitive DNA.

Accurate chromosomal DNA replication is essential to maintain genomic stability. Genetic evidence suggests that certain repetitive sequences impair replication, yet the underlying mechanism is poorly defined. Replication could be directly inhibited by the DNA template or indirectly, for example by DNA-bound proteins. Here, we reconstitute replication of mono-, di- and trinucleotide repeats in vitro using eukaryotic replisomes assembled from purified proteins. We find that structure-prone repeats are sufficient to impair replication. Whilst template unwinding is unaffected, leading strand synthesis is inhibited, leading to fork uncoupling. Synthesis through hairpin-forming repeats is rescued by replisome-intrinsic mechanisms, whereas synthesis of quadruplex-forming repeats requires an extrinsic accessory helicase. DNA-induced fork stalling is mechanistically similar to that induced by leading strand DNA lesions, highlighting structure-prone repeats as an important potential source of replication stress. Thus, we propose that our understanding of the cellular response to replication stress may also be applied to DNA-induced replication stalling.

[The role of alleles with an intermediate number of trinucleotide repeats in Parkinson's disease and other neurodegenerative disorders]. / Rol' allelei s promezhutochnym kolichestvom trinukleotidnykh povtorov pri bolezni Parkinsona i drugikh neirodegenerativnykh zabolevaniyakh.

Genetic factors underlie the pathological processes that cause the manifestation of a wide range of neurodegenerative diseases. The pathological expansion of unstable trinucleotide repeats is known to lead monogenic neurological diseases such as Huntington's disease, Kennedy's disease, spinocerebellar ataxia, and others. However, the latest data suggests individuals with intermediate allele (IA) repeat length have a risk of developing common neurological phenotype, for example, Parkinson's disease, Alzheimer's disease. In this study, we review the current knowledge on intermediate alleles of HTT gene for pathogenesis and clinical features of neurodegenerative diseases, with the focus on Parkinson's disease. Early diagnosis of neurodegenerative disease and genetic counselling of the family can be improved via the implementation of specific management strategies of IA carriers by team of highly experienced professionals in the fields of neurology and genetics.

The number of androgen receptor CAG repeats and mortality in men.

INTRODUCTION: The androgen receptor (AR) mediates peripheral effects of testosterone. Evidence suggests that the number of CAG repeats in exon-1 of the AR gene negatively correlates with AR transcriptional activity. The aim of this analysis was to determine the association between CAG repeat number and mortality in men. METHODS: Men aged 40-79 years were recruited from primary care for participation in the UK arm of the European Male Aging Study between 2003 and 2005. Cox proportional hazards modelling was used to determine the association between CAG repeat number/mortality. Results were expressed as hazard ratios(HR)/95% confidence intervals (CI). RESULTS: 312 men were followed up. The mean baseline age was 59.5 years. At follow up, 85/312(27%) men had died. CAG repeat length ranged from 14 to 39, with the highest proportion of CAG repeat number at 21 repeats(16.4%). In a multivariable model, using men with CAG repeat numbers of 22-23 as the reference, men with a lower number of CAG repeats(<22) showed a trend for a higher mortality in the follow-up period (HR 1.46 (0.75, 2.81)) as did men with higher number of repeats (>23) (1.37 (0.65, 2.91)). CONCLUSION: Our data suggest that CAG repeat number may partially influence the risk of mortality in men. Further larger studies are required to quantify the effect.

Novel eGZ-motif formed by regularly extruded guanine bases in a left-handed Z-DNA helix as a major motif behind CGG trinucleotide repeats.

The expansion of d(CGG) trinucleotide repeats (TRs) lies behind several important neurodegenerative diseases. Atypical DNA secondary structures have been shown to trigger TR expansion: their characterization is important for a molecular understanding of TR disease. CD spectroscopy experiments in the last decade have unequivocally demonstrated that CGG runs adopt a left-handed Z-DNA conformation, whose features remain uncertain because it entails accommodating GG mismatches. In order to find this missing motif, we have carried out molecular dynamics (MD) simulations to explore all the possible Z-DNA helices that potentially form after the transition from B- to Z-DNA. Such helices combine either CpG or GpC Watson-Crick steps in Z-DNA form with GG-mismatch conformations set as either intrahelical or extrahelical; and participating in BZ or ZZ junctions or in alternately extruded conformations. Characterization of the stability and structural features (especially overall left-handedness, higher-temperature and steered MD simulations) identified two novel Z-DNA helices: the most stable one displays alternately extruded Gs, and is followed by a helix with symmetrically extruded ZZ junctions. The G-extrusion favors a seamless stacking of the Watson-Crick base pairs; extruded Gs favor syn conformations and display hydrogen-bonding and stacking interactions. Such conformations could have the potential to hijack the MMR complex, thus triggering further expansion.

Association of trinucleotide repeat polymorphisms CAG and GGC in exon 1 of the androgen receptor gene with male infertility: a cross-sectional study.

BACKGROUND: : Infertility is a global problem that brings about serious sexual and social consequences that strain the health sector and society. The expansion of CAG and GGC repeats in androgen receptor (AR) gene (Ensembl number ENSG00000169083) may lead to reduced fertility. Our objective was to determine the association of CAG and GGC repeats with altered sperm parameters in male infertile subjects. METHODS: This was a cross-sectional study conducted at Aga Khan University, Karachi, Pakistan. A total of 376 males were recruited, out of which group A (N = 208) and group B (N = 168) were comprised of subjects with normal and altered sperm parameters, respectively, from 18 to 60 years. The numbers of CAG and GGC repeats were determined by using PCR amplification and sequence analysis using the Molecular Evolutionary Genetic Analysis (MEGA) software version 6.0. Statistical analysis was performed using the SPSS version 20 and the P-value of <0.05 was considered significant. RESULTS: The mean androgen receptor gene CAG repeats were significantly longer in males with altered sperm parameters as compared to male subjects with normal sperm parameters (P < 0.001). There was no significant difference found for GGC repeats for subjects with altered sperm parameters. DISCUSSION: Longer CAG length corresponded to greater severity of spermatogenic defect and may lead to subfertility recommendations.

Investigation of androgen receptor gene CAG repeat length polymorphism in pubertal gynecomastia.

OBJECTIVES: Androgen receptor gene CAG repeat, AR (CAG)n, polymorphism is thought to have an effect on male reproductive functions and a relationship between long AR (CAG)n and decreased androgenic activity has been shown. Therefore, we hypothesized that in adolescents with long AR CAG repeat the prevalence of pubertal gynecomastia (PG) will be higher and we aimed to investigate the association between AR (CAG)n polymorphism and PG in Turkish adolescents. METHODS: Adolescents with PG between 11 and 19 years of age were enrolled as the study group and healthy individuals without a history of PG, who were at least 14 years of age and Tanner 4 or 5 were enrolled as the control group. The AR (CAG)n length was detected by direct DNA sequencing analysis and reproductive hormones were measured by standardized analyses. RESULTS: The mean AR (CAG)n was 22.3 ± 2.6 (mean ± SD) in the PG group (n=101) and 21.9 ± 3.1 (mean ± SD) in the control group (n=88) (p=0.276). The adolescents with short AR (CAG)n had lower body mass index standard deviation scores (BMI SDS) compared to the adolescents with intermediate and long repeat numbers (p=0.029). CONCLUSIONS: The results of this study showed a lack of direct association between AR (CAG)n and PG. However, the significant relationship between the AR (CAG)n quartiles and BMI SDS suggests that long AR (CAG)n might cause PG indirectly. Further studies are needed to better clarify this relationship.

Beyond Trinucleotide Repeat Expansion in Fragile X Syndrome: Rare Coding and Noncoding Variants in FMR1 and Associated Phenotypes.

FMR1 (FMRP translational regulator 1) variants other than repeat expansion are known to cause disease phenotypes but can be overlooked if they are not accounted for in genetic testing strategies. We collected and reanalyzed the evidence for pathogenicity of FMR1 coding, noncoding, and copy number variants published to date. There is a spectrum of disease-causing FMR1 variation, with clinical and functional evidence supporting pathogenicity of five splicing, five missense, one in-frame deletion, one nonsense, and four frameshift variants. In addition, FMR1 deletions occur in both mosaic full mutation patients and as constitutional pathogenic alleles. De novo deletions arise not only from full mutation alleles but also alleles with normal-sized CGG repeats in several patients, suggesting that the CGG repeat region may be prone to genomic instability even in the absence of repeat expansion. We conclude that clinical tests for potentially FMR1-related indications such as intellectual disability should include methods capable of detecting small coding, noncoding, and copy number variants.

Novel implications of a strictly monomorphic (GCC) repeat in the human PRKACB gene.

PRKACB (Protein Kinase CAMP-Activated Catalytic Subunit Beta) is predominantly expressed in the brain, and regulation of this gene links to neuroprotective effects against tau and Aß-induced toxicity. Here we studied a (GCC)-repeat spanning the core promoter and 5' UTR of this gene in 300 human subjects, consisting of late-onset neurocognitive disorder (NCD) (N = 150) and controls (N = 150). We also implemented several models to study the impact of this repeat on the three-dimensional (3D) structure of DNA. While the PRKACB (GCC)-repeat was strictly monomorphic at 7-repeats, we detected two 7/8 genotypes only in the NCD group. In all examined models, the (GCC)7 and its periodicals had the least range of divergence variation on the 3D structure of DNA in comparison to the 8-repeat periodicals and several hypothetical repeat lengths. A similar inert effect on the 3D structure was not detected in other classes of short tandem repeats (STRs) such as GA and CA repeats. In conclusion, we report monomorphism of a long (GCC)-repeat in the PRKACB gene in human, its inert effect on DNA structure, and enriched divergence in late-onset NCD. This is the first indication of natural selection for a monomorphic (GCC)-repeat, which probably evolved to function as an "epigenetic knob", without changing the regional DNA structure.