Population Genetics and New Insight into Range of CAG Repeats of Spinocerebellar Ataxia Type 3 in the Han Chinese Population

Spinocerebellar ataxia type 3 (SCA3), also called Machado-Joseph disease (MJD), is one of the most common SCAs worldwide and caused by a CAG repeat expansion located in ATXN3 gene. Based on the CAG repeat numbers, alleles of ATXN3 can be divided into normal alleles (ANs), intermediate alleles (AIs) and expanded alleles (AEs). It was controversial whether the frequency of large normal alleles (large ANs) is related to the prevalence of SCA3 or not. And there were huge chaos in the comprehension of the specific numbers of the range of CAG repeats which is fundamental for genetic analysis of SCA3. To illustrate these issues, we made a novel CAG repeat ladder to detect CAG repeats of ATXN3 in 1003 unrelated Chinese normal individuals and studied haplotypes defined by three single nucleotide polymorphisms (SNPs) closed to ATXN3. We found that the number of CAG repeats ranged from 13 to 49, among them, 14 was the most common number. Positive skew, the highest frequency of large ANs and 4 AIs which had never been reported before were found. Also, AEs and large ANs shared the same haplotypes defined by the SNPs. Based on these data and other related studies, we presumed that de novo mutations of ATXN3 emerging from large ANs are at least one survival mechanisms of mutational ATXN3 and we can redefine the range of CAG repeats as: ANs≤44, 45 ≤AIs ≤49 and AEs≥50.     

De Novo Mutations in Ataxin-2 Gene and ALS Risk

Pathogenic CAG repeat expansion in the ataxin-2 gene (ATXN2) is the genetic cause of spinocerebellar ataxia type 2 (SCA2). Recently, it has been associated with Parkinsonism and increased genetic risk for amyotrophic lateral sclerosis (ALS). Here we report the association of de novo mutations in ATXN2 with autosomal dominant ALS. These findings support our previous conjectures based on population studies on the role of large normal ATXN2 alleles as the source for new mutations being involved in neurodegenerative pathologies associated with CAG expansions. The de novo mutations expanded from ALS/SCA2 non-risk alleles as proven by meta-analysis method. The ALS risk was associated with SCA2 alleles as well as with intermediate CAG lengths in the ATXN2. Higher risk for ALS was associated with pathogenic CAG repeat as revealed by meta-analysis.     

Long CAG Repeat Sequence and Protein Expression of Androgen Receptor Considered as Prognostic Indicators in Male Breast Carcinoma

Background

The androgen receptor (AR) expression and the CAG repeat length within the AR gene appear to be involved in the carcinogenesis of male breast carcinoma (MBC). Although phenotypic differences have been observed between MBC and normal control group in AR gene, there is lack of correlation analysis between AR expression and CAG repeat length in MBC. The purpose of the study was to investigate the prognostic value of CAG repeat lengths and AR protein expression.

Methods

81 tumor tissues were used for immunostaining for AR expression and CAG repeat length determination and 80 normal controls were analyzed with CAG repeat length in AR gene. The CAG repeat length and AR expression were analyzed in relation to clinicopathological factors and prognostic indicators.

Results

AR gene in many MBCs has long CAG repeat sequence compared with that in control group (P = 0.001) and controls are more likely to exhibit short CAG repeat sequence than MBCs. There was statistically significant difference in long CAG repeat sequence between AR status for MBC patients (P = 0.004). The presence of long CAG repeat sequence and AR-positive expression were associated with shorter survival of MBC patients (CAG repeat: P = 0.050 for 5y-OS; P = 0.035 for 5y-DFS AR status: P = 0.048 for 5y-OS; P = 0.029 for 5y-DFS, respectively).

Conclusion

The CAG repeat length within the AR gene might be one useful molecular biomarker to identify males at increased risk of breast cancer development. The presence of long CAG repeat sequence and AR protein expression were in relation to survival of MBC patients. The CAG repeat length and AR expression were two independent prognostic indicators in MBC patients.     

Role of Haptoglobin in Polycystic Ovary Syndrome (PCOS), Obesity and Disorders of Glucose Tolerance in Premenopausal Women

Background

Hp² alleles of the haptoglobin α–chain polymorphism reduce the anti-oxidant properties and increase the pro-inflammatory actions of this acute-phase protein in a gene-dosage fashion. We hypothesized that the haptoglobin polymorphism might contribute to the increased oxidative stress and low-grade chronic inflammation frequently associated with polycystic ovary syndrome, obesity, and abnormalities of glucose tolerance.

Methodology/Principal Findings

Serum haptoglobin and the haptoglobin α–chain polymorphism were determined in 141 patients with polycystic ovary syndrome and 102 non-hyperandrogenic women. Of the whole group of 243 premenopausal women, 117 were obese and 51 showed abnormal glucose tolerance. Although serum haptoglobin concentrations were similar in PCOS patients and controls, the former presented with an increased frequency of Hp² alleles (62% vs. 52%, P = 0.023). Circulating haptoglobin levels increased with obesity (P<0.001), yet no association was found between obesity and haptoglobin genotypes. No differences were observed in haptoglobin levels or genotype frequencies depending on glucose tolerance. Fifty percent of the variation in serum haptoglobin concentrations was explained by the variability in serum C-reactive protein concentrations, BMI, insulin sensitivity and haptoglobin genotypes.

Conclusions/Significance

Serum haptoglobin concentrations in premenopausal women are largely dependent on the haptoglobin polymorphism and on the presence of obesity, with insulin resistance and chronic inflammation possibly modulating this relationship. The association of polycystic ovary syndrome with Hp² alleles suggests that the anti-oxidant and anti-inflammatory properties of haptoglobin may be reduced in these patients.     

Intermediate CAG Repeat Expansion in the ATXN2 Gene Is a Unique Genetic Risk Factor for ALS?A Systematic Review and Meta-Analysis of Observational Studies

Amyotrophic lateral sclerosis (ALS) is a rare degenerative condition of the motor neurons. Over 10% of ALS cases are linked to monogenic mutations, with the remainder thought to be due to other risk factors, including environmental factors, genetic polymorphisms, and possibly gene-environmental interactions. We examined the association between ALS and an intermediate CAG repeat expansion in the ATXN2 gene using a meta-analytic approach. Observational studies were searched with relevant disease and gene terms from MEDLINE, EMBASE, and PsycINFO from January 2010 through to January 2014. All identified articles were screened using disease terms, gene terms, population information, and CAG repeat information according to PRISMA guidelines. The final list of 17 articles was further evaluated based on the study location, time period, and authors to exclude multiple usage of the same study populations: 13 relevant articles were retained for this study. The range 30–33 CAG repeats in the ATXN2 gene was most strongly associated with ALS. The meta-analysis revealed that the presence of an intermediate CAG repeat (30-33) in the ATXN2 gene was associated with an increased risk of ALS [odds ratio (OR) = 4.44, 95%CI: 2.91–6.76)] in Caucasian ALS patients. There was no significant difference in the association of this CAG intermediate repeat expansion in the ATXN2 gene between familial ALS cases (OR = 3.59, 1.58–8.17) and sporadic ALS cases (OR = 3.16, 1.88–5.32). These results indicate that the presence of intermediate CAG repeat expansion in the ATXN2 gene is a specific genetic risk factor for ALS, unlike monogenic mutations with an autosomal dominant transmission mode, which cause a more severe phenotype of ALS, with a higher prevalence in familial ALS.     

Repeat Associated Non-AUG Translation (RAN Translation) Dependent on Sequence Downstream of the ATXN2 CAG Repeat

Spinocerebellar ataxia type 2 (SCA2) is a progressive autosomal dominant disorder caused by the expansion of a CAG tract in the ATXN2 gene. The SCA2 disease phenotype is characterized by cerebellar atrophy, gait ataxia, and slow saccades. ATXN2 mutation causes gains of toxic and normal functions of the ATXN2 gene product, ataxin-2, and abnormally slow Purkinje cell firing frequency. Previously we investigated features of ATXN2 controlling expression and noted expression differences for ATXN2 constructs with varying CAG lengths, suggestive of repeat associated non-AUG translation (RAN translation). To determine whether RAN translation occurs for ATXN2 we assembled various ATXN2 constructs with ATXN2 tagged by luciferase, HA or FLAG tags, driven by the CMV promoter or the ATXN2 promoter. Luciferase expression from ATXN2-luciferase constructs lacking the ATXN2 start codon was weak vs AUG translation, regardless of promoter type, and did not increase with longer CAG repeat lengths. RAN translation was detected on western blots by the anti-polyglutamine antibody 1C2 for constructs driven by the CMV promoter but not the ATXN2 promoter, and was weaker than AUG translation. Strong RAN translation was also observed when driving the ATXN2 sequence with the CMV promoter with ATXN2 sequence downstream of the CAG repeat truncated to 18 bp in the polyglutamine frame but not in the polyserine or polyalanine frames. Our data demonstrate that ATXN2 RAN translation is weak compared to AUG translation and is dependent on ATXN2 sequences flanking the CAG repeat.     

PolyQ repeat expansions in ATXN2 associated with ALS are CAA interrupted repeats

Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive disease leading to paralysis and death. Recently, intermediate length polyglutamine (polyQ) repeats of 27-33 in ATAXIN-2 (ATXN2), encoding the ATXN2 protein, were found to increase risk for ALS. In ATXN2, polyQ expansions of ≥ 34, which are pure CAG repeat expansions, cause spinocerebellar ataxia type 2. However, similar length expansions that are interrupted with other codons, can present atypically with parkinsonism, suggesting that configuration of the repeat sequence plays an important role in disease manifestation in ATXN2 polyQ expansion diseases. Here we determined whether the expansions in ATXN2 associated with ALS were pure or interrupted CAG repeats, and defined single nucleotide polymorphisms (SNPs) rs695871 and rs695872 in exon 1 of the gene, to assess haplotype association. We found that the expanded repeat alleles of 40 ALS patients and 9 long-repeat length controls were all interrupted, bearing 1-3 CAA codons within the CAG repeat. 21/21 expanded ALS chromosomes with 3CAA interruptions arose from one haplotype (GT), while 18/19 expanded ALS chromosomes with <3CAA interruptions arose from a different haplotype (CC). Moreover, age of disease onset was significantly earlier in patients bearing 3 interruptions vs fewer, and was distinct between haplotypes. These results indicate that CAG repeat expansions in ATXN2 associated with ALS are uniformly interrupted repeats and that the nature of the repeat sequence and haplotype, as well as length of polyQ repeat, may play a role in the neurological effect conferred by expansions in ATXN2.     

Thrombotic Antiphospholipid Syndrome Shows Strong Haplotypic Association with SH2B3-ATXN2 Locus

Background

Thrombotic antiphospholipid syndrome is defined as a complex form of thrombophilia that is developed by a fraction of antiphospholipid antibody (aPLA) carriers. Little is known about the genetic risk factors involved in thrombosis development among aPLA carriers.

Methods

To identify new loci conferring susceptibility to thrombotic antiphospholipid syndrome, a two-stage genotyping strategy was performed. In stage one, 19,000 CNV loci were genotyped in 14 thrombotic aPLA+ patients and 14 healthy controls by array-CGH. In stage two, significant CNV loci were fine-mapped in a larger cohort (85 thrombotic aPLA+, 100 non-thrombotic aPLA+ and 569 healthy controls).

Results

Array-CGH and fine-mapping analysis led to the identification of 12q24.12 locus as a new susceptibility locus for thrombotic APS. Within this region, a TAC risk haplotype comprising one SNP in SH2B3 gene (rs3184504) and two SNPs in ATXN2 gene (rs10774625 and rs653178) exhibited the strongest association with thrombotic antiphospholipid syndrome (p-value = 5,9 × 10⁻⁴ OR 95% CI 1.84 (1.32–2.55)).

Conclusion

The presence of a TAC risk haplotype in ATXN2-SH2B3 locus may contribute to increased thrombotic risk in aPLA carriers.     

Mutational Profile of GNAQ Q209 in Human Tumors

Background

Frequent somatic mutations have recently been identified in the ras-like domain of the heterotrimeric G protein α-subunit (GNAQ) in blue naevi 83%, malignant blue naevi (50%) and ocular melanoma of the uvea (46%). The mutations exclusively affect codon 209 and result in GNAQ constitutive activation which, in turn, acts as a dominant oncogene.

Methodology

To assess if the mutations are present in other tumor types we performed a systematic mutational profile of the GNAQ exon 5 in a panel of 922 neoplasms, including glioblastoma, gastrointestinal stromal tumors (GIST), acute myeloid leukemia (AML), blue naevi, skin melanoma, bladder, breast, colorectal, lung, ovarian, pancreas, and thyroid carcinomas.

Principal Findings

We detected the previously reported mutations in 6/13 (46%) blue naevi. Changes affecting Q209 were not found in any of the other tumors. Our data indicate that the occurrence of GNAQ mutations display a unique pattern being present in a subset of melanocytic tumors but not in malignancies of glial, epithelial and stromal origin analyzed in this study.     

Frequent MAGE Mutations in Human Melanoma

Background

Cancer/testis (CT) genes are expressed only in the germ line and certain tumors and are most frequently located on the X-chromosome (the CT-X genes). Amongst the best studied CT-X genes are those encoding several MAGE protein families. The function of MAGE proteins is not well understood, but several have been shown to potentially influence the tumorigenic phenotype.

Methodology/Principal Findings

We undertook a mutational analysis of coding regions of four CT-X MAGE genes, MAGEA1, MAGEA4, MAGEC1, MAGEC2 and the ubiquitously expressed MAGEE1 in human melanoma samples. We first examined cell lines established from tumors and matching blood samples from 27 melanoma patients. We found that melanoma cell lines from 37% of patients contained at least one mutated MAGE gene. The frequency of mutations in the coding regions of individual MAGE genes varied from 3.7% for MAGEA1 and MAGEA4 to 14.8% for MAGEC2. We also examined 111 fresh melanoma samples collected from 86 patients. In this case, samples from 32% of the patients exhibited mutations in one or more MAGE genes with the frequency of mutations in individual MAGE genes ranging from 6% in MAGEA1 to 16% in MAGEC1.

Significance

These results demonstrate for the first time that the MAGE gene family is frequently mutated in melanoma.     

Frequency of KCNC3 DNA variants as causes of spinocerebellar ataxia 13 (SCA13)

Background

Gain-of function or dominant-negative mutations in the voltage-gated potassium channel KCNC3 (Kv3.3) were recently identified as a cause of autosomal dominant spinocerebellar ataxia. Our objective was to describe the frequency of mutations associated with KCNC3 in a large cohort of index patients with sporadic or familial ataxia presenting to three US ataxia clinics at academic medical centers.

Methodology

DNA sequence analysis of the coding region of the KCNC3 gene was performed in 327 index cases with ataxia. Analysis of channel function was performed by expression of DNA variants in Xenopus oocytes.

Principal Findings

Sequence analysis revealed two non-synonymous substitutions in exon 2 and five intronic changes, which were not predicted to alter splicing. We identified another pedigree with the p.Arg423His mutation in the highly conserved S4 domain of this channel. This family had an early-onset of disease and associated seizures in one individual. The second coding change, p.Gly263Asp, subtly altered biophysical properties of the channel, but was unlikely to be disease-associated as it occurred in an individual with an expansion of the CAG repeat in the CACNA1A calcium channel.

Conclusions

Mutations in KCNC3 are a rare cause of spinocerebellar ataxia with a frequency of less than 1%. The p.Arg423His mutation is recurrent in different populations and associated with early onset. In contrast to previous p.Arg423His mutation carriers, we now observed seizures and mild mental retardation in one individual. This study confirms the wide phenotypic spectrum in SCA13.     

Serum Testosterone Levels and Androgen Receptor CAG Polymorphism Correlate with Hepatitis B Virus (HBV)-Related Acute Liver Failure in Male HBV Carriers

Background

Augmentation of androgen/androgen receptor (AR) pathway may influence chronic hepatitis B (CHB) more likely in males. AR activity is modulated by a polymorphic CAG repeat sequence in AR exon 1. This study aimed to investigate the relationship between serum testosterone levels, CAG repeat numbers and hepatitis B virus (HBV)-related acute liver failure (ALF).

Methods

Three hundred and seventy eight male CHB patients with ALF and 441 asymptomatic HBV carriers (AsCs) were recruited. AR CAG repeats numbers were analyzed. The serum testosterone levels of AsCs, ALFs and patients with hepatitis B flare groups, and sequential serum samples, were assessed quantitatively.

Results

The median CAG repeat (M-CAG) frequency was significantly higher in ALF patients than AsCs (P<0.001). Patients with M-CAG alleles (P<0.001, OR 3.0, 95% CI 2.1–4.2) had the highest risk for ALF. Serum testosterone levels were significantly higher (P<0.001) at hepatitis flare point (8.2±3.0 ng/mL) than inactive phase (6.4±2.0 ng/mL). CHB (8.30±2.71 ng/mL, P = 7.6×10⁻⁶) and ALF group (2.61±1.83 ng/mL, P = 1.7×10⁻¹⁷) had significantly different levels of testosterone in comparison with AsCs group (6.56±2.36 ng/mL). The serum testosterone levels sharply decreased from hepatitis flare phase to liver failure phase, and tended to be normal at the recovery phase. Male AsCs with M-CAG alleles had significantly lower serum testosterone levels (P<0.05).

Conclusions

There was a serum testosterone fluctuation during hepatitis B flare and HBV-related ALF, and the median CAG repeats in AR gene exon 1 were associated with lower serum testosterone levels in asymptomatic HBV carriers and an increased susceptibility to HBV-related ALF.     

The Role of Apolipoprotein E as a Risk Factor for an Earlier Age at Onset for Machado-Joseph Disease Is Doubtful

Machado-Joseph disease (MJD) is an inherited neurodegenerative disease caused by an expanded CAG repeat in the ATXN3 gene. Although the principal genetic determinant of the age at onset (AAO) is the length of the expanded CAG repeat, the additional genetic contribution of MJD toward the AAO has mostly not yet been clarified. It was recently suggested in two independent studies that apolipoprotein E (APOE) might be associated with AAO variability in MJD patients. To identify the potential modifier effect of APOE polymorphisms on the AAO of MJD patients, 403 patients with MJD (confirmed by molecular tests) from eastern and southeastern China were enrolled in the present study. CAG repeats in the ATXN3 and APOE polymorphisms were genotyped. Data were analyzed using a statistical package. No contribution of APOE polymorphisms to the variance in disease onset was observed using ANCOVA (F = 0.183, P = 0.947). However, significant effects on the AAO of MJD were found for the normal ATXN3 allele and for the interaction of mutant and normal ATXN3 alleles in a multiple linear regression model (P = 0.043 and P = 0.035, respectively). Our study does not support a role for APOE as a genetic modifier of the AAO of MJD. Additionally, our study presents evidence that the normal ATXN3 allele and its interaction with mutant alleles contribute toward AAO variance in MJD patients.     

Effect of β2-adrenergic receptor gene (ADRB2) 3′ untranslated region polymorphisms on inhaled corticosteroid/long-acting β2-adrenergic agonist response

Background

Evidence suggests that variation in the length of the poly-C repeat in the 3′ untranslated region (3′UTR) of the β₂-adrenergic receptor gene (ADRB2) may contribute to interindividual variation in β-agonist response. However, methodology in previous studies limited the assessment of the effect of sequence variation in the context of poly-C repeat length. The objectives of this study were to design a novel genotyping method to fully characterize sequence variation in the ADRB2 3′UTR poly-C repeat in asthma patients treated with inhaled corticosteroid and long-acting β₂-adrenergic agonist (ICS/LABA) combination therapy, and to analyze the effect of the poly-C repeat polymorphism on clinical response.

Methods

In 2,250 asthma patients randomized to treatment with budesonide/formoterol or fluticasone/salmeterol in a six-month study (AstraZeneca study code: SD-039-0735), sequence diversity in the ADRB2 poly-C repeat region was determined using a novel sequencing-based genotyping method. The relationship between the poly-C repeat polymorphism and the incidence of severe asthma exacerbations, and changes in pulmonary function and asthma symptoms from baseline to the average during the treatment period, were analyzed.

Results

Poly-C repeat genotypes were assigned in 97% (2,192/2,250) of patients. Of the 13 different poly-C repeat alleles identified, six alleles occurred at a frequency of >5% in one or more population in this study. The repeat length of these six common alleles ranged from 10 to 14 nucleotides. Twelve poly-C repeat genotypes were observed at a frequency of >1%. No evidence of an association between poly-C repeat genotype and the incidence of severe asthma exacerbations was observed. Patients’ pulmonary function measurements improved and asthma symptoms declined when treated with ICS/LABA combination therapy regardless of poly-C repeat genotype.

Conclusions

The extensive sequence diversity present in the poly-C repeat region of the ADRB2 3′UTR did not predict therapeutic response to ICS/LABA therapy.     

Bone Mass and the CAG and GGN Androgen Receptor Polymorphisms in Young Men

Background

To determine whether androgen receptor (AR) CAG (polyglutamine) and GGN (polyglycine) polymorphisms influence bone mineral density (BMD), osteocalcin and free serum testosterone concentration in young men.

Methodology/Principal Findings

Whole body, lumbar spine and femoral bone mineral content (BMC) and BMD, Dual X-ray Absorptiometry (DXA), AR repeat polymorphisms (PCR), osteocalcin and free testosterone (ELISA) were determined in 282 healthy men (28.6±7.6 years). Individuals were grouped as CAG short (CAG_S) if harboring repeat lengths of ≤21 or CAG long (CAG_L) if CAG >21, and GGN was considered short (GGN_S) or long (GGN_L) if GGN ≤23 or >23. There was an inverse association between logarithm of CAG and GGN length and Ward''s Triangle BMC (r = −0.15 and −0.15, P<0.05, age and height adjusted). No associations between CAG or GGN repeat length and regional BMC or BMD were observed after adjusting for age. Whole body and regional BMC and BMD values were similar in men harboring CAG_S, CAG_L, GGN_S or GGN_L AR repeat polymorphisms. Men harboring the combination CAG_L+GGN_L had 6.3 and 4.4% higher lumbar spine BMC and BMD than men with the haplotype CAG_S+GGN_S (both P<0.05). Femoral neck BMD was 4.8% higher in the CAG_S+GGN_S compared with the CAG_L+GGN_S men (P<0.05). CAG_S, CAG_L, GGN_S, GGN_L men had similar osteocalcin concentration as well as the four CAG-GGN haplotypes studied.

Conclusion

AR polymorphisms have an influence on BMC and BMD in healthy adult humans, which cannot be explained through effects in osteoblastic activity.     

Biomarkers of Endocannabinoid System Activation in Severe Obesity

Background

Obesity is a worldwide epidemic, and severe obesity is a risk factor for many diseases, including diabetes, heart disease, stroke, and some cancers. Endocannabinoid system (ECS) signaling in the brain and peripheral tissues is activated in obesity and plays a role in the regulation of body weight. The main research question here was whether quantitative measurement of plasma endocannabinoids, anandamide, and related N-acylethanolamines (NAEs), combined with genotyping for mutations in fatty acid amide hydrolase (FAAH) would identify circulating biomarkers of ECS activation in severe obesity.

Methodology/Principal Findings

Plasma samples were obtained from 96 severely obese subjects with body mass index (BMI) of ≥40 kg/m², and 48 normal weight subjects with BMI of ≤26 kg/m². Triple-quadrupole mass spectroscopy methods were used to measure plasma ECS analogs. Subjects were genotyped for human FAAH gene mutations. The principal analysis focused on the FAAH 385 C→A (P129T) mutation by comparing plasma ECS metabolite levels in the FAAH 385 minor A allele carriers versus wild-type C/C carriers in both groups. The main finding was significantly elevated mean plasma levels of anandamide (15.1±1.4 pmol/ml) and related NAEs in study subjects that carried the FAAH 385 A mutant alleles versus normal subjects (13.3±1.0 pmol/ml) with wild-type FAAH genotype (p = 0.04), and significance was maintained after controlling for BMI.

Conclusions/Significance

Significantly increased levels of the endocannabinoid anandamide and related NAEs were found in carriers of the FAAH 385 A mutant alleles compared with wild-type FAAH controls. This evidence supports endocannabinoid system activation due to the effect of FAAH 385 mutant A genotype on plasma AEA and related NAE analogs. This is the first study to document that FAAH 385 A mutant alleles have a direct effect on elevated plasma levels of anandamide and related NAEs in humans. These biomarkers may indicate risk for severe obesity and may suggest novel ECS obesity treatment strategies.