Direct, ensemble FRET approaches to monitor transient state kinetics of human DNA polymerase δ holoenzyme assembly and initiation of DNA synthesis.

In humans, DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, proliferating cell nuclear antigen (PCNA), carry out DNA synthesis during lagging strand replication, the initiation of leading strand DNA replication as well as most of the major DNA damage repair pathways. In each of these contexts, pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process that involves the PCNA clamp loader, replication factor C and, depending on the DNA synthesis pathway, the major single strand DNA-binding protein complex, replication protein A (RPA). In a recent report from our laboratory, we designed and utilized direct, ensemble Förster Resonance Energy Transfer approaches to monitor the transient state kinetics of pol δ holoenzyme assembly and initiation of DNA synthesis on P/T junctions engaged by RPA. In this chapter, we detail the original approaches and discuss adaptations that can be utilized to monitor fast kinetic reactions in the millisecond (ms) timescale. All approaches described in this chapter utilize a commercially-available fluorescence spectrophotometer, can be readily evolved for alternative DNA polymerases and P/T DNA substrates, and permit incorporation of protein posttranslational modifications, accessory factors, DNA covalent modifications, accessory factors, enzymes, etc. Hence, these approaches are widely accessible and broadly applicable for characterizing DNA polymerase holoenzyme assembly and initiation of DNA synthesis during any PCNA-dependent DNA synthesis pathway.

RFC2 may contribute to the pathogenicity of Williams syndrome revealed in a zebrafish model.

Williams syndrome (WS) is a rare multisystemic disorder caused by recurrent microdeletions on 7q11.23, characterized by intellectual disability, distinctive craniofacial and dental features, and cardiovascular problems. Previous studies have explored the roles of individual genes within these microdeletions in contributing to WS phenotypes. Here, we report five patients with WS with 1.4 Mb-1.5 Mb microdeletions that include RFC2, as well as one patient with a 167 kb microdeletion involving RFC2 and six patients with intragenic variants within RFC2. To investigate the potential involvement of RFC2 in WS pathogenicity, we generate a rfc2 knockout (KO) zebrafish using CRISPR-Cas9 technology. Additionally, we generate a KO zebrafish of its paralog gene, rfc5, to better understand the functions of these RFC genes in development and disease. Both rfc2 and rfc5 KO zebrafish exhibit similar phenotypes reminiscent of WS, including small head and brain, jaw and dental defects, and vascular problems. RNA-seq analysis reveals that genes associated with neural cell survival and differentiation are specifically affected in rfc2 KO zebrafish. In addition, heterozygous rfc2 KO adult zebrafish demonstrate an anxiety-like behavior with increased social cohesion. These results suggest that RFC2 may contribute to the pathogenicity of Williams syndrome, as evidenced by the zebrafish model.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS): a family with five affected sibs from Turkey.

BACKGROUND: Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), a relatively common cause of late-onset progressive ataxia, is a genetic disease characterised by biallelic pentanucleotide AAGGG repeat expansion in intron 2 of the replication factor complex subunit 1 gene. Herein, we describe the first molecularly confirmed CANVAS family with five affected siblings from Turkey. CASE PRESENTATION: The family comprised seven siblings born from healthy non-consanguineous parents. CANVAS phenotype was present in five of them; two were healthy and asymptomatic. Chronic cough was the first symptom reported in all five siblings, followed by the development of sensory symptoms, oscillopsia and imbalance. Clinical head impulse test (HIT) was positive in all cases and video HIT performed on three patients revealed very low vestibulo-ocular reflex gains bilaterally. Magnetic resonance imaging and nerve conduction studies revealed cerebellar atrophy and sensory neuronopathy, respectively. RP-PCR confirmed the homozygous presence of the AAGGG repeat expansion in all five cases. CONCLUSION: Genetic screening for CANVAS should be considered in all patients with late-onset ataxia, sensory disturbances and vestibular involvement, especially in the presence of chronic cough.

Pseudodominance in RFC1-Spectrum Disorder.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and disease spectrum is an autosomal recessive disorder associated with biallelic repeat expansion (RE) in the RFC1 gene. A high carrier frequency in the healthy population determines the possibility of having affected members in two consecutive generations. We describe pseudodominance in two families affected with RFC1 disorder (10 affected, 5 oligo/asymptomatic individuals). In Family A, after the 75-year-old index case was diagnosed with CANVAS, the 73-year-old wife decided to undergo screening for carrier testing. Although she did not report any symptoms, she resulted positive for the biallelic AAGGG RE thus leading to a diagnosis in the asymptomatic offspring as well and revealing a pseudodominant pattern of inheritance. In Family B pseudodominance was suspected after the identification of the RFC1 RE in the proband affected by sensitive neuropathy because of a positive family history for undetermined polyneuropathy in the mother. The post-mortem identification of the RFC1 RE in a sample specimen from the deceased mother, who had been under our care, allowed the solution of a "cold case". Our report suggests that pseudodominance is a confounding phenomenon to consider in RFC1-spectrum disorder and genetic counselling is instrumental in families with affected individuals.

Alteration in folate carrier expression via histone deacetylase inhibition in BeWo human placental choriocarcinoma cells.

Folates are essential nutrients for fetal development during pregnancy. Valproic acid (VPA), an inhibitor of histone deacetylases (HDACs), alters the expression of folate carriers in placental cells; however, the underlying mechanisms remain unclear. Here, we aimed to determine the profiles of folate carriers (folate receptor alpha [FOLR1], solute carrier [SLC]-19A1, and SLC46A1) after inhibition of HDACs, especially class I and IIa HDACs, using different inhibitors and gene knockdown tests. Quantitative polymerase chain reaction revealed that BeWo cells (a trophoblast model) expressed HDACs and folate carriers, similar to human placental villi. FOLR1 expression was upregulated by VPA, apicidin, and trichostatin A, but downregulated by MS-275 after 24 h treatment. VPA and apicidin upregulated the expression of SLC46A1. These inhibitors downregulated SLC19A1 expression. TMP269 (a class IIa inhibitor) did not affect folate carrier levels. HDAC1/2 knockdown upregulated FOLR1 and SLC46A1 levels, whereas HDAC1/3 knockdown downregulated FOLR1 levels. Our findings suggest that the pharmacological inhibition of class I HDACs alters the expression of folate carriers in BeWo cells. By contrast, HDAC inhibitors exert different regulatory effects on folate carriers. Moreover, HDAC1/2 inhibition may be a potential mechanism involved in altering FOLR1 and SLC46A1 levels.

Electrophysiological features of the peripheral neuropathy in patients with pathologic biallelic RFC1 repeat expansions.

INTRODUCTION/AIMS: Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) is caused by RFC1 expansions. Sensory neuronopathy, polyneuropathy, and involvement of motor, autonomic, and cranial nerves have all been described with RFC1 expansions. We aimed to describe the electrodiagnostic features of patients with RFC1 expansions through multimodal electrophysiological investigations. METHODS: Thirty-five patients, with a median age of 70 years, and pathologic biallelic repeat expansions in the RFC1 gene, were tested for motor and sensory nerve conduction, flexor carpi radialis (FCR) and soleus H-reflexes, blink reflex, electrochemical skin conductance, sympathetic skin response (SSR), and heart rate variability with deep breathing (HRV). RESULTS: Only 16 patients (46%) exhibited the full clinical CANVAS spectrum. Distal motor amplitudes were normal in 30 patients and reduced in the legs of five patients. Distal sensory amplitudes were bilaterally reduced in a non-length dependent manner in 30 patients. Conduction velocities were normal. Soleus H-reflexes were abnormal in 19/20 patients of whom seven had preserved Achilles reflexes. FCR H-reflexes were absent or decreased in amplitude in 13/14 patients. Blink reflex was abnormal in 4/19 patients: R1 latencies for two patients and R2 latencies for two others. Fourteen out of 31 patients (45%) had abnormal results in at least one autonomic nervous system test, either for ESC (12/31), SSR (5/14), or HRV (6/19). DISCUSSION: Less than half of the patients with RFC1 expansions exhibited the full clinical CANVAS spectrum, but nearly all exhibited typical sensory neuronopathy and abnormal H-reflexes. Involvement of small nerve fibers and brainstem neurons was less common.

High Expression of SRSF10 Promotes Colorectal Cancer Progression by Aberrant Alternative Splicing of RFC5.

BACKGROUND: Colorectal cancer (CRC) remains a global health concern with persistently high incidence and mortality rates. However, the specific pathogenesis of CRC remains poorly understood. This study aims to investigate the role and pathogenesis of serine and arginine rich splicing factor 10 (SRSF10) in colorectal cancer. METHODS: Bioinformatics analysis was employed to predict SRSF10 gene expression in CRC patients. Functional experiments involving SRSF10 knockdown and overexpression were conducted using CCK8, transwell, scratch assay, and flow cytometry. Additionally, the PRIdictor website was utilized to predict the SRSF10 interaction site with RFC5. The identification of different transcripts of SRSF10-acting RFC5 pre-mRNA was achieved through agarose gel electrophoresis. RESULT: The knockdown of SRSF10 inhibited the proliferation and migration ability of CRC cells, while promoting apoptosis and altering the DNA replication of CRC cells. Conversely, when SRSF10 was highly expressed, it enhanced the proliferation and migration ability of CRC cells and caused changes in the cell cycle of colorectal cancer cells. This study revealed a change in the replicating factor C subunit 5 (RFC5) gene in colorectal cancer cells following SRSF10 knockdown. Furthermore, it was confirmed that SRSF10 increased RFC5 exon2-AS1(S) transcription variants, thereby promoting the development of colorectal cancer through AS1 exclusion to exon 2 of RFC5. CONCLUSION: In summary, this study demonstrates that SRSF10 promotes the progression of colorectal cancer by generating an aberrantly spliced exclusion isoform of AS1 within RFC5 exon 2. These findings suggest that SRSF10 could serve as a crucial target for the clinical diagnosis and treatment of CRC.

Genetic profiles of multiple system atrophy revealed by exome sequencing, long-read sequencing and spinocerebellar ataxia repeat expansion analysis.

BACKGROUND AND PURPOSE: Multiple system atrophy (MSA) is a progressive, adult-onset neurodegenerative disorder clinically characterized by combinations of autonomic failure, parkinsonism, cerebellar ataxia and pyramidal signs. Although a few genetic factors have been reported to contribute to the disease, its mutational profiles have not been systemically studied. METHODS: To address the genetic profiles of clinically diagnosed MSA patients, exome sequencing and triplet repeat detection was conducted in 205 MSA patients, including one familial case. The pathogenicity of variants was determined according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines. RESULTS: In the familial patient, a novel heterozygous COQ2 pathogenic variant (p.Ala351Thr) was identified in the MSA pedigree. In the sporadic patients, 29 pathogenic variants were revealed in 21 genes, and the PARK7 p.Ala104Thr variant was significantly associated with MSA (p = 0.0018). Moreover, burden tests demonstrated that the pathogenic variants were enriched in cerebellar ataxia-related genes in patients. Furthermore, repeat expansion analyses revealed that two patients carried the pathogenic CAG repeat expansion in the CACNA1A gene (SCA6), one patient carried the (ACAGG)exp/(ACAGG)exp expansion in RFC1 and one carried the GAA-pure expansion in FGF14 gene. CONCLUSION: In conclusion, a novel COQ2 pathogenic variant was identified in a familial MSA patient, and repeat expansions in CACNA1A, RFC1 and FGF14 gene were detected in four sporadic patients. Moreover, a PARK7 variant and the burden of pathogenic variants in cerebellar ataxia-related genes were associated with MSA.

Identification of RFC4 as a potential biomarker for pan-cancer involving prognosis, tumour immune microenvironment and drugs.

RFC4 is required for DNA polymerase δ and DNA polymerase ε to initiate DNA template expansion. Downregulated RFC4 inhibits tumour proliferation by causing S-phase arrest and inhibiting mitosis, resulting in the reduction of tumour cells. RFC4 has been implicated that it plays an important role in the initiation and progression of cancers, but a comprehensive analysis of the role of RFC4 in cancer has not been performed. We comprehensively analysed the expression, prognosis, methylation level, splicing level, relationship of RFC4 and immune infiltration, and pan-cancer immunotherapy response used various databases (including TCGA, GTEx, UALCAN, Oncosplicing, TIDE, TISCH, HPA and CAMOIP), and experimented its biological function in HCC. Through pan-cancer analysis, we found that RFC4 is significantly upregulated in most tumours. The tumour patients with high expression of RFC4 have poor prognosis. The methylation level and variable splicing level of RFC4 were abnormal in most tumours compared with the adjacent tissues. Furthermore, RFC4 was closely associated with immune cell infiltration in various cancers. RFC4 was significantly co-expressed with immune checkpoints and other immune-related genes. The expression of RFC4 could indicate the immunotherapy efficacy of some tumours. The RFC4 expression was associated with sensitivity to specific small molecule drugs. Cell experiments have shown that downregulated RFC4 can inhibit cell cycle and tumour cell proliferation. We conducted a systematic pan-cancer analysis of RFC4, and the results showed that RFC4 can serve as a biomarker for cancer diagnosis and prognosis. These findings open new perspectives for precision medicine.

Pathologic RFC1 repeat expansions do not contribute to the development of inflammatory neuropathies.

Biallelic expansions of the AAGGG repeat in the replication factor C subunit 1 (RFC1) have recently been described to be responsible for cerebellar ataxia, peripheral neuropathy and vestibular areflexia syndrome. This genetic alteration has also allowed genetic classification in up to one-third of cases with idiopathic sensory neuropathy. Here, we screened a well-characterized cohort of inflammatory neuropathy patients for RFC1 repeat expansions to explore whether RFC1 was increased from background rates and possibly involved in the pathogenesis of inflammatory neuropathy. A total of 259 individuals with inflammatory neuropathy and 243 healthy controls were screened for the AAGGG repeat expansion using short-range flanking PCR and repeat-primed PCR. Cases without amplifiable PCR product on flanking PCR and positive repeat-primed PCR were also tested for the mostly non-pathogenic expansions of the AAAGG and AAAAG repeat units. None of the patients showed biallelic AAGGG expansion of RFC1, and their carrier frequency for AAGGG was comparable with controls [n = 27 (5.2%) and n = 23 (4.7%), respectively; P > 0.5]. Data suggest that the pathologic expansions of AAGGG repeats do not contribute to the development of inflammatory neuropathies nor lead to misdiagnosed cases. Accordingly, routine genetic screening for RFC1 repeat expansion is not indicated in this patient population.

Analysis and occurrence of biallelic pathogenic repeat expansions in RFC1 in a German cohort of patients with a main clinical phenotype of motor neuron disease.

Biallelic pathogenic repeat expansions in RFC1 were recently identified as molecular origin of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) as well as of one of the most common causes of adult-onset ataxia. In the meantime, the phenotypic spectrum has expanded massively and now includes mimics of multiple system atrophy or parkinsonism. After identifying a patient with a clinical diagnosis of amyotrophic lateral sclerosis (ALS) as a carrier of biallelic pathogenic repeat expansions in RFC1, we studied a cohort of 106 additional patients with a clinical main phenotype of motor neuron disease (MND) to analyze whether such repeat expansions are more common in MND patients. Indeed, two additional MND patients (one also with ALS and one with primary lateral sclerosis/PLS) have been identified as carrier of biallelic pathogenic repeat expansions in RFC1 in the absence of another genetic alteration explaining the phenotype, suggesting motor neuron disease as another extreme phenotype of RFC1 spectrum disorder. Therefore, MND might belong to the expanding phenotypic spectrum of pathogenic RFC1 repeat expansions, particularly in those MND patients with additional features such as sensory and/or autonomic neuropathy, vestibular deficits, or cerebellar signs. By systematically analyzing the RFC1 repeat array using Oxford nanopore technology long-read sequencing, our study highlights the high intra- and interallelic heterogeneity of this locus and allows the identification of the novel repeat motif 'ACAAG'.

An Updated Canvas of the RFC1-mediated CANVAS (Cerebellar Ataxia, Neuropathy and Vestibular Areflexia Syndrome).

Proliferation of specific nucleotide sequences within the coding and non-coding regions of numerous genes has been implicated in approximately 40 neurodegenerative disorders. Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), a neurodegenerative disorder, is distinguished by a pathological triad of sensory neuropathy, bilateral vestibular areflexia and cerebellar impairments. It manifests in adults gradually and is autosomal recessive and multi-system ataxia. Predominantly, CANVAS is associated with biallelic AAGGG repeat expansions in intron 2 of the RFC1 gene. Although various motifs have been identified, only a subset induces pathological consequences, by forming stable secondary structures that disrupt gene functions both in vitro and in vivo. The pathogenesis of CANVAS remains a subject of intensive research, yet its precise mechanisms remain elusive. Herein, we aim to comprehensively review the epidemiology, clinical ramifications, molecular mechanisms, genetics, and potential therapeutics in light of the current findings, extending an overview of the most significant research on CANVAS.

An Update on the Adult-Onset Hereditary Cerebellar Ataxias: Novel Genetic Causes and New Diagnostic Approaches.

The hereditary cerebellar ataxias (HCAs) are rare, progressive neurologic disorders caused by variants in many different genes. Inheritance may follow autosomal dominant, autosomal recessive, X-linked or mitochondrial patterns. The list of genes associated with adult-onset cerebellar ataxia is continuously growing, with several new genes discovered in the last few years. This includes short-tandem repeat (STR) expansions in RFC1, causing cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS), FGF14-GAA causing spinocerebellar ataxia type 27B (SCA27B), and THAP11. In addition, the genetic basis for SCA4, has recently been identified as a STR expansion in ZFHX3. Given the large and growing number of genes, and different gene variant types, the approach to diagnostic testing for adult-onset HCA can be complex. Testing methods include targeted evaluation of STR expansions (e.g. SCAs, Friedreich ataxia, fragile X-associated tremor/ataxia syndrome, dentatorubral-pallidoluysian atrophy), next generation sequencing for conventional variants, which may include targeted gene panels, whole exome, or whole genome sequencing, followed by various potential additional tests. This review proposes a diagnostic approach for clinical testing, highlights the challenges with current testing technologies, and discusses future advances which may overcome these limitations. Implementing long-read sequencing has the potential to transform the diagnostic approach in HCA, with the overall aim to improve the diagnostic yield.

Screening of lung cancer serum biomarkers based on Boruta-shap and RFC-RFECV algorithms.

OBJECTIVE: This study aimed to identify a set of serum miRNAs as potential biomarkers for lung cancer diagnosis using algorithmic approaches. METHODS: Serum miRNA expression data from lung cancer patients and non-tumor controls were obtained. The top six miRNAs were selected using Boruta-shap and RFC-RFECV algorithms. A Gaussian Naive Bayes (NB) classifier was trained and evaluated using cross-validation, ROC curve analysis, and evaluation metrics. RESULTS: Six miRNAs (hsa-miRNA-144, hsa-miRNA-107, hsa-miRNA-484, hsa-miRNA-103, hsa-miRNA-26b, and hsa-miRNA-641) were identified as feature genes. The NB classifier achieved an area under curve (AUC) of 0.8966 and a mean AUC of 0.88 in cross-validation. Accuracy, recall, and F1 scores exhibited promising results, with an accuracy of 82%. In the validation set, the AUC values for the NB and SVC classifiers were 0.9345 and 0.9423, respectively, with a mean AUC of 0.95 in cross-validation. The classifiers demonstrated an accuracy of 89% in diagnosing lung cancer. CONCLUSION: This study identified a panel of six serum miRNAs with potential as non-invasive biomarkers for lung cancer diagnosis. These miRNAs show promise in providing sensitive and specific tools for detecting lung cancer. SIGNIFICANCE: Lung cancer is one of the top cancers worldwide, threatening the health and lives of tens of thousands of people. miRNA is a biomarker, which can be used as a potential clinical tool for diagnosis and prognosis of cancer patients. Therefore, the use of multiple miRNAs to construct diagnostic models may be one of the future methods of accurate diagnosis of lung cancer. In this study, we used the Boruta-shap and RFC-RFECV algorithms to automatically identify and extract characteristic miRNAs highly associated with lung cancer, thereby establishing an accurate classifier for the diagnosis of lung cancer with characteristic miRNAs.

Bilateral vestibulopathy as the initial presentation of CANVAS.

Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a slowly progressing autosomal recessive ataxic disorder linked to an abnormal biallelic intronic (most commonly) AAGGG repeat expansion in the replication factor complex subunit 1 (RFC1). While the clinical diagnosis is relatively straightforward when the three components of the disorder are present, it becomes challenging when only one of the triad (cerebellar ataxia, neuropathy or vestibular areflexia) manifests. Isolated cases of Bilateral Vestibulopathy (BVP) or vestibular areflexia that later developed the other components of CANVAS have not been documented. We report four cases of patients with chronic imbalance and BVP that, after several years, developed cerebellar and neuropathic deficits with positive genetic testing for RFC1. Our report supports the concept that CANVAS should be considered in every patient with BVP of unknown etiology, even without the presence of the other triad components. This is especially important given that about 50% of cases in many BVP series are diagnosed as idiopathic, some of which may be undiagnosed CANVAS.

Raman Flow Cytometry and Its Biomedical Applications.

Raman flow cytometry (RFC) uniquely integrates the "label-free" capability of Raman spectroscopy with the "high-throughput" attribute of traditional flow cytometry (FCM), offering exceptional performance in cell characterization and sorting. Unlike conventional FCM, RFC stands out for its elimination of the dependency on fluorescent labels, thereby reducing interference with the natural state of cells. Furthermore, it significantly enhances the detection information, providing a more comprehensive chemical fingerprint of cells. This review thoroughly discusses the fundamental principles and technological advantages of RFC and elaborates on its various applications in the biomedical field, from identifying and characterizing cancer cells for in vivo cancer detection and surveillance to sorting stem cells, paving the way for cell therapy, and identifying metabolic products of microbial cells, enabling the differentiation of microbial subgroups. Moreover, we delve into the current challenges and future directions regarding the improvement in sensitivity and throughput. This holds significant implications for the field of cell analysis, especially for the advancement of metabolomics.

Cryo-EM reveals a nearly complete PCNA loading process and unique features of the human alternative clamp loader CTF18-RFC.

The DNA sliding clamp PCNA is a multipurpose platform for DNA polymerases and many other proteins involved in DNA metabolism. The topologically closed PCNA ring needs to be cracked open and loaded onto DNA by a clamp loader, e.g., the well-studied pentameric ATPase complex RFC (RFC1-5). The CTF18-RFC complex is an alternative clamp loader found recently to bind the leading strand DNA polymerase ε and load PCNA onto leading strand DNA, but its structure and the loading mechanism have been unknown. By cryo-EM analysis of in vitro assembled human CTF18-RFC-DNA-PCNA complex, we have captured seven loading intermediates, revealing a detailed PCNA loading mechanism onto a 3'-ss/dsDNA junction by CTF18-RFC. Interestingly, the alternative loader has evolved a highly mobile CTF18 AAA+ module likely to lower the loading activity, perhaps to avoid competition with the RFC and to limit its role to leading strand clamp loading. To compensate for the lost stability due to the mobile AAA+ module, CTF18 has evolved a unique ß-hairpin motif that reaches across RFC2 to interact with RFC5, thereby stabilizing the pentameric complex. Further, we found that CTF18 also contains a separation pin to locally melt DNA from the 3'-end of the primer; this ensures its ability to load PCNA to any 3'-ss/dsDNA junction, facilitated by the binding energy of the E-plug to the major groove. Our study reveals unique structural features of the human CTF18-RFC and contributes to a broader understanding of PCNA loading by the alternative clamp loaders.

Folate gene polymorphisms CBS 844ins68 and RFC1 A80G and risk of Down syndrome offspring in young Iranian women: A cross-sectional study.

Background: Cytogenetics and association studies showed that folate gene polymorphisms can increase the risk of chromosomal nondisjunction and aneuploidies. The folate-metabolizing gene polymorphisms in Down syndrome mothers (DSM) have been assessed in a variety of populations. Reduced folate carrier 1 (RFC1) and cystathionine beta-synthase (CBS) are key enzymes in folate metabolism. Objective: 2 common polymorphisms, CBS 844ins68 and RFC1 A80G, were analyzed to determine their probable risk for having Down syndrome (DS) babies in young mothers of Khuzestan province, Iran. Materials and Methods: This study was conducted on 100 mothers who had trisomy 21 DS children. 100 age- and ethnic-matched mothers with at least 2 healthy children and no history of abnormal pregnancies were considered as control. The samples were collected from all the mothers from June 2019 to April 2021. Genomic DNA was extracted from peripheral blood. The CBS-844ins68 and RFC1-A80G were genotyped using polymerase chain reaction-electrophoresis and restriction fragment length polymorphism, respectively. Results: The frequency of RFC1 AG and GG genotypes in DSM was significantly higher than the control mothers (odds ratio [OR] of 2.38 and 3.07, respectively). The heterozygote genotype of CBS 844ins68 was significantly more prevalent among DSM than the control (OR: 2.419). The OR was significantly increased to 6.667 when the homozygote of both variants was found together. Conclusion: Studying polymorphisms possibly increases the susceptibility of having a DS child. However, ethnicity, nutrition, and epistatic interactions are considerable factors to be evaluated in future studies.

RFC1: Motifs and phenotypes.

Biallelic intronic expansions (AAGGG)exp in intron 2 of the RFC1 gene have been shown to be a common cause of late-onset ataxia. Since their first description, the phenotypes, neurological damage, and pathogenic variants associated with the RFC1 gene have been frequently updated. Here, we review the various motifs, genetic variants, and phenotypes associated with the RFC1 gene. We searched PubMed for scientific articles published between March 1st, 2019, and January 15th, 2024. The motifs and phenotypes associated with the RFC1 gene are highly heterogeneous, making molecular diagnosis and clinical screening and investigation challenging. In this review we will provide clues to give a better understanding of RFC1 disease. We briefly discuss new methods for molecular diagnosis, the origin of cough in RFC1 disease, and research perspectives.