FMRP Levels in Human Peripheral Blood Leukocytes Correlates with Intellectual Disability

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. FXS is an X-linked, neurodevelopmental disorder caused by a CGG trinucleotide repeat expansion in the 5′ untranslated region (UTR) of the Fragile X Mental Retardation gene, FMR1. Greater than 200 CGG repeats results in epigenetic silencing of the gene leading to the deficiency or absence of Fragile X mental retardation protein (FMRP). The loss of FMRP is considered the root cause of FXS. The relationship between neurological function and FMRP expression in peripheral blood mononuclear cells (PBMCs) has not been well established. Assays to detect and measure FMR1 and FMRP have been described; however, none are sufficiently sensitive, precise, or quantitative to properly characterize the relationships between cognitive ability and CGG repeat number, FMR1 mRNA expression, or FMRP expression measured in PBMCs. To address these limitations, two novel immunoassays were developed and optimized, an electro-chemiluminescence immunoassay and a multiparameter flow cytometry assay. Both assays were performed on PMBCs isolated from 27 study participants with FMR1 CGG repeats ranging from normal to full mutation. After correcting for methylation, a significant positive correlation between CGG repeat number and FMR1 mRNA expression levels and a significant negative correlation between FMRP levels and CGG repeat expansion was observed. Importantly, a high positive correlation was observed between intellectual quotient (IQ) and FMRP expression measured in PBMCs.

Is FMR1 CGG Repeat Number Polymorphism Associated With Phenotypic Variation in the General Population? Report From a Cohort of 5,499 Adults

FMR1 CGG repeat length was assayed in 5499 research participants (2637 men and 2862 women) in the Wisconsin Longitudinal Study (WLS), a population-based cohort. Most past research has focused on clinically-ascertained individuals with expansions in CGG repeats, either those with fragile X syndrome (> 200 CGG repeats), the FMR1 premutation (55–200 repeats), or in the gray zone (variously defined as 45–54 or 41–54 repeats). In contrast, the WLS is a unique source of data that was obtained from an unselected cohort of individuals from the general population for whom FMR1 CGG repeat length was assayed. The WLS is a random sample of one-third of all high school seniors in the state of Wisconsin in 1957. The most recent round of data collection was in 2011; thus, the study spanned over 50 years. Saliva samples were obtained from 69% of surviving members of the cohort in 2008 and 2011, from which CGG repeats were assayed. With one exception, the CGG repeat length of all members of this cohort was below 100 (ranging from 7 to 84). The present study evaluated the genotype-phenotype associations of CGG repeat number and IQ, college graduation, age at menopause, number of biological children, having a child with intellectual or developmental disabilities, and the likelihood of experiencing an episode of depression during adulthood. Linear and curvilinear effects were probed. Although effect sizes were small, significant associations were found between CGG repeat length and high school IQ score, college graduation, number of biological children, age at menopause, and the likelihood of having an episode of depression. However, there was no significant association between repeat length and having a child diagnosed with an IDD condition. This study demonstrates a continuum of phenotype effects with FMR1 repeat lengths and illustrates how research inspired by a rare genetic condition (such as fragile X syndrome) can be used to probe genotype-phenotype associations in the general population.

Serum neurofilament light chain as a severity marker for spinocerebellar ataxia

Since the serum neurofilament light (NfL) chain is known as a promising biomarker in neurodegenerative diseases, we aimed to evaluate serum NfL as a biomarker indicating neuronal damage in autosomal-dominant (AD) spinocerebellar ataxia (SCA). We reviewed patients diagnosed with AD SCA in the outpatient clinic of Seoul National University Hospital’s (SNUH) Department of Neurology between May and August of 2019. We reviewed the demographic data, clinical characteristics, Scale for the Assessment and Rating of Ataxia (SARA) score, and brain magnetic resonance imaging (MRI) scans. The serum NfL was measured by electrochemiluminescence (ECL) immunoassay. Forty-nine patients with AD SCA were reviewed and their serum NfL level was determined. The median serum NfL level (109.5 pg/mL) was higher than control (41.1 pg/mL) (p-value < 0.001). Among the AD SCA patients, there was a positive correlation between the serum NfL level and the trinucleotide repeat number (r = 0.47, p-value = 0.001), disease duration (r = 0.35, p-value = 0.019), disease duration/age × trinucleotide repeat number (r = 0.330, p-value = 0.021), and SARA score (n = 33; r = 0.37, p-value = 0.033). This study shows that serum NfL is elevated in AD SCA patients and correlates with clinical severity.

Congenital Phenotypes and DMPK CTG Repeat Number in Mothers and Children With Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is an autosomal dominant genetic disease. In DM1, the mutant allele expands during gametogenesis, and an extended CTG repeat sequence is inherited by the offspring. This often results in increased severity of DM1 symptoms in the affected offspring and may cause congenital myotonic dystrophy (CDM). This study aimed to clarify whether CTG repeat number predicts CDM in offspring. This retrospective study examined 14 women with DM1, their pregnancy and labor histories, and their 14 children diagnosed with DM1. There were 12 CDM patients and 2 non-CDM patients. Correlations between CDM onset and CTG repeat numbers of the mother and child were analyzed. Women who bore a child with CDM (infants with detected polyhydramnios during pregnancy, hypotonia, respiratory insufficiency, or suckling failure at birth) had a mean repeat number of 643 (standard deviation [SD] 436). For women who bore a child without CDM, the mean repeat number was 950 (SD 71), and no significant between-group difference was detected. The mean (SD) CTG repeat numbers observed in children with and without CDM were 1,646 (324) and 1,700 (565), respectively. CDM cannot be predicted based on the CTG repeat numbers of mothers or children.

A Novel Triplet-Primed PCR Assay to Detect the Full Range of Trinucleotide CAG Repeats in the Huntingtin Gene (HTT)

The expanded CAG repeat number in HTT gene causes Huntington disease (HD), which is a severe, dominant neurodegenerative illness. The accurate determination of the expanded allele size is crucial to confirm the genetic status in symptomatic and presymptomatic at-risk subjects and avoid genetic polymorphism-related false-negative diagnoses. Precise CAG repeat number determination is critical to discriminate the cutoff between unexpanded and intermediate mutable alleles (IAs, 27–35 CAG) as well as between IAs and pathological, low-penetrance alleles (i.e., 36–39 CAG repeats), and it is also critical to detect large repeat expansions causing pediatric HD variants. We analyzed the HTT-CAG repeat number of 14 DNA reference materials and of a DNA collection of 43 additional samples carrying unexpanded, IAs, low and complete penetrance alleles, including large (>60 repeats) and very large (>100 repeats) expansions using a novel triplet-primed PCR-based assay, the AmplideX PCR/CE HTT Kit. The results demonstrate that the method accurately genotypes both normal and expanded HTT-CAG repeat numbers and reveals previously undisclosed and very large CAG expansions >200 repeats. We also show that this technique can improve genetic test reliability and accuracy by detecting CAG expansions in samples with sequence variations within or adjacent to the repeat tract that cause allele drop-outs or inaccuracies using other PCR methods.

Color Intensity of the Red-Fleshed Berry Phenotype of Vitis vinifera Teinturier Grapes Varies Due to a 408 bp Duplication in the Promoter of VvmybA1

Grapevine (Vitis vinifera) teinturier cultivars are characterized by their typical reddish leaves and red-fleshed berries due to ectopic anthocyanin formation. Wines of these varieties have economic importance as they can be used for blending to enhance the color of red wines. The unique and heritable mutation has been known for a long time but the underlying genetic mechanism still is not yet understood. Here we describe the association of the red-fleshed berry phenotype with a 408 bp repetitive DNA element in the promoter of the VvmybA1 gene (grapevine color enhancer, GCE). Three different clones of ‘Teinturier’ were discovered with two, three and five allelic GCE repeats (MybA1t2, MybA1t3 and MybA1t5). All three clones are periclinal chimeras; these clones share the same L1 layer, but have distinct L2 layers with different quantities of GCE repeats. Quantitative real time PCR and HPLC analysis of leaf and berry samples showed that the GCE repeat number strongly correlates with an increase of the expression of VvmybA1 itself and the VvUFGT gene regulated by it and the anthocyanin content. A model is proposed based on autoregulation of VvmybA1t to explain the red phenotype which is similar to that of red-fleshed apples. This study presents results about the generation and modes of action of three MybA1t alleles responsible for the red-fleshed berry phenotype of teinturier grapevines.

TGM6 L517W is not a pathogenic variant for spinocerebellar ataxia type 35

ObjectiveTo investigate the pathogenicity of the TGM6 variant for spinocerebellar ataxia 35 (SCA35), which was previously reported to be caused by pathogenic mutations in the gene TGM6.MethodsNeurologic assessment and brain MRI were performed to provide detailed description of the phenotype. Whole-exome sequencing and dynamic mutation analysis were performed to identify the genotype.ResultsThe proband, presenting with myoclonic epilepsy, cognitive decline, and ataxia, harbored both the TGM6 p.L517W variant and expanded CAG repeats in gene ATN1. Further analysis of the other living family members in this pedigree revealed that the CAG repeat number was expanded in all the patients and within normal range in all the unaffected family members. However, the TGM6 p.L517W variant was absent in 2 affected family members, but present in 3 healthy individuals.ConclusionsThe nonsegregation of the TGM6 variant with phenotype does not support this variant as the disease-causing gene in this pedigree, questioning the pathogenicity of TGM6 in SCA35.

Genetic and Environmental Influences on Self-Reported Cognitive Functioning: Associations of Diverse Measures of Stress across the FMR1 CGG Repeat Range

Background : The FMR1 gene is essential for neural development and healthy synaptic function. The modal number of CGG repeats in FMR1 is 30, but the range is large with the reported copy number extending down to as few as 6 CGGs and up to over 200 CGGs, conferring fragile X syndrome. Prior work suggests that behavioral phenotypes, including cognitive function, may vary along the continuum of the FMR1 CGG repeat range. Stress may negatively influence cognitive function; however, it is not known whether FMR1 -related variability (i.e., CGG repeat length), in addition to stress, independently influences cognitive function across the CGG range. Methods : Participants included 1275 mothers who had CGGs ranging from 18 to 123 repeats. Participants completed self-report measures of executive function (BRIEF-A), memory, subjective stress (i.e., perceived stress), and objective stress (i.e., number of life events, parenting a child with a disability). Stress and FMR1 -related variability (i.e., CGG repeat length) were examined as predictors of self-reported executive function and memory difficulty. Results : Each measure of stress (i.e., perceived stress, life events, and parenting a child with a disability) significantly predicted greater self-reported difficulties in executive function and the likelihood of memory problems, net of age and level of education. Additionally, above and beyond stress effects, CGG repeat number significantly predicted executive functioning and memory difficulties. There was a linear association of CGG repeat number with executive functioning limitations. The association of CGGs with memory difficulties was curvilinear, with participants in the premutation range having the greatest likelihood of reporting such difficulties. Conclusions : These findings suggest that CGG repeat length confers independent contributions to self-reported executive function difficulty and memory problems over and above indices of stress, suggesting additive effects of genetic variation and environmental exposure. Keywords : Stress, Cognitive Function, Executive Function, Memory, FMR1 , CGG Repeats