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De Barsy syndrome is a recessive progeroid disease classified under the group of cutis laxa syndromes. The disease is attributed to loss-of-function mutations inPYCR1orALDH18A1, leading to premature skin aging. Here we report fivePYCR1pathogenic alleles and a mouse knockout model of the disease. Through these investigations, we have confirmed the key role of PYCR1 in preventing dermal thinning and other connective tissue abnormalities. However, it remains unknown whether endogenous PYCR1 levels undergo changes during normal aging. To address this query, we examined its levels in cultured human cutaneous fibroblasts subjected to induced or replicative senescence. In both instances, PYCR1 levels dropped and correlated with the loss of proliferative capacity. Furthermore, we validated the relevance of these findingsin vivo, by comparing young and chronologically aged human skin, and found that the levels of PYCR1 in the dermis, but not the epidermis, significantly decreased with age. Our results confirm that the loss of PYCR1 is a driver of human skin aging and that its levels in healthy individuals can serve as a biomarker for connective tissues undergoing normal chronological aging.

Cirrhosis is usually a late-onset and life-threatening disease characterized by fibrotic scarring and inflammation that disrupts liver architecture and function. While it is typically the result of alcoholism or hepatitis viral infection in adults, its etiology in infants is much less understood. In this study, we report 14 children from ten unrelated families presenting with a syndromic form of pediatric liver cirrhosis. By genome/exome sequencing, we found recessive variants in FOCAD segregating with the disease. Zebrafish lacking focad phenocopied the human disease, revealing a signature of altered messenger RNA (mRNA) degradation processes in the liver. Using patient’s primary cells and CRISPR-Cas9-mediated inactivation in human hepatic cell lines, we found that FOCAD deficiency compromises the SKI mRNA surveillance pathway by reducing the levels of the RNA helicase SKIC2 and its cofactor SKIC3. FOCAD knockout hepatocytes exhibited lowered albumin expression and signs of persistent injury accompanied by CCL2 overproduction. Our results reveal the importance of FOCAD in maintaining liver homeostasis and disclose a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis.

OBJECTIVE Small fiber neuropathy (SFN) is clinically and etiologically heterogeneous. Although autoimmunity has been postulated to be pathophysiologically important in SFN, few autoantibodies have been described. We aimed to identify autoantibodies associated with idiopathic SFN (iSFN) by a novel high-throughput protein microarray platform that captures autoantibodies expressed in the native conformational state. METHODS Sera from 58 SFN patients and 20 age- and gender-matched healthy controls (HCs) were screened against >1,600 immune-related antigens. Fluorescent unit readout and postassay imaging were performed, followed by composite data normalization and protein fold change (pFC) analysis. Analysis of an independent validation cohort of 33 SFN patients against the same 20 HCs was conducted to identify reproducible proteins in both cohorts. RESULTS Nine autoantibodies were screened with statistical significance and pFC criteria in both cohorts, with at least 50% change in serum levels. Three proteins showed consistently high fold changes in main and validation cohorts: MX1 (FC��=���2.99 and 3.07, respectively, p��=���0.003, q��=���0.076), DBNL (FC��=��2.11 and 2.16, respectively, p��=���0.009, q��

The National Academies of Sciences and Medicine 2020 consensus statement advocates the reinstatement of research in preconception heritable human genome editing (HHGE), despite the ethical concerns that have been voiced about interventions in the germline, and outlines criteria for its eventual clinical application to address monogenic disorders. However, the statement does not give adequate consideration to alternative technologies. Importantly, it omits comparison to fetal gene therapy (FGT), which involves gene modification applied prenatally to the developing fetus and which is better researched and less ethically contentious. While both technologies are applicable to the same monogenic diseases causing significant prenatal or early childhood morbidity, the benefits and risks of HHGE are distinct from FGT though there are important overlaps. FGT has the current advantage of a wealth of robust preclinical data, while HHGE is nascent technology and its feasibility for specific diseases still requires scientific proof. The ethical concerns surrounding each are unique and deserving of further discussion, as there are compelling arguments supporting research and eventual clinical translation of both technologies. In this Opinion, we consider HHGE and FGT through technical and ethical lenses, applying common ethical principles to provide a sense of their feasibility and acceptability. Currently, FGT is in a more advanced position for clinical translation and may be less ethically contentious than HHGE, so it deserves to be considered as an alternative therapy in further discussions on HHGE implementation.

Small-fiber neuropathy (SFN) is a disorder that exclusively affects the small nerve fibers, sparing the large nerve fibers. Thinly myelinated Aδ-fibers and unmyelinated C-fibers are damaged, leading to development of neuropathic pain, thermal dysfunction, sensory symptoms, and autonomic disturbances. Although many SFNs are secondary and due to immunological causes or metabolic disturbances, the etiology is unknown in up to half of the patients. Over the years, this proportion of "idiopathic SFN" has decreased, as familial and genetic causes have been discovered, thus shifting a proportion of once "idiopathic" cases to the genetic category. After the discovery of SCN9A-gene variants in 2012, SCN10A and SCN11A variants have been found to be pathogenic in SFN. With improved accessibility of SFN diagnostic tools and genetic tests, many non-SCN variants and genetically inherited systemic diseases involving the small nerve fibers have also been described, but only scattered throughout the literature. There are 80 SCN variants described as causing SFN, 8 genes causing hereditary sensory autonomic neuropathies (HSAN) described with pure SFN, and at least 7 genes involved in genetically inherited systemic diseases associated with SFN. This systematic review aims to consolidate and provide an updated overview on the genetic variants of SFN to date---SCN genes and beyond. Awareness of these genetic causes of SFN is imperative for providing treatment directions, prognostication, and management of expectations for patients and their health-care providers.

Altering the human epigenome with gene-editing technology in attempt to treat a variety of diseases and conditions seems scientifically feasible. We explore some of the ethical and regulatory issues related to the clinical translation of human epigenetic editing arguing that such approaches should be considered akin to somatic therapies.

The concept of vulnerability has played an important role in theoretical bioethics as well as in numerous authoritative guidelines on research ethics. The concept helps to identify situations in which research participants and other individuals may be at a heightened risk of experiencing harm. However, existing guidance documents on the ethics of human germline gene editing largely fail to make any reference to considerations of vulnerability. In this article, we discuss this oversight and we highlight the role that vulnerability can play in ethical debates about human heritable genome editing. Future guidance documents on germline gene editing should pay attention to considerations of vulnerability and reference these appropriately.

Colorectal cancer (CRC) is a high incidence cancer and major cause of cancer mortality. Though disease-causing tumor suppressors for major syndromes are well characterized, about 10% of CRC is familial but without mutations in known tumor suppressors. We exhaustively screened 100 polyposis families for APC germline mutations and identified 13, which are APC mutation-negative, microsatellite-stable (MSS), and with undetectable mutation in known tumor suppressors. Whole exome sequencing in three probands uncovered two with germline frameshift NR0B2 mutations, c.293_301delTTGGGTTGGinsAC and c.227delT. Sanger Sequencing identified a third proband with NR0B2 c.157_166delCATCGCACCT frameshift mutation. All three mutations deleted the C-terminus activation/repression domain of NR0B2, thus are loss-of-function mutations. Real-time RT-PCR performed on tumor and matched mucosa of one patient revealed that NR0B2 downstream targets, SMAD3 was derepressed while GLI1 was downregulated in the colonic mucosa compared to healthy controls. Truncated NR0B2 molecule was predicted to have weakened binding with interacting partners SMAD3, GLI1, BCL2, and RXRα, implying perturbation of TGF-β, Hedgehog, anti-apoptotic and nuclear hormone receptor signaling pathways. Immunostaining also revealed nuclear retention of the most severely truncated NR0B2 molecule compared to the wildtype. Microsatellite and sequencing analysis did not detect loss of wildtype allele in probands' tumors. The patient who acquired somatic KRAS mutation progressed rapidly whist the other two patients manifested with late-onset obesity and diabetes. We propose that haploinsufficiency of NR0B2 is associated with a novel CRC syndrome with metabolic phenotypes.

ObjectiveUse next-generation sequencing (NGS) technology to improve our diagnostic yield in patients with suspected genetic disorders in the Asian setting.DesignA diagnostic study conducted between 2014 and 2019 (and ongoing) under the Singapore Undiagnosed Disease Program. Date of last analysis was 1 July 2019.SettingInpatient and outpatient genetics service at two large academic centres in Singapore.PatientsInclusion criteria: patients suspected of genetic disorders, based on abnormal antenatal ultrasound, multiple congenital anomalies and developmental delay. Exclusion criteria: patients with known genetic disorders, either after clinical assessment or investigations (such as karyotype or chromosomal microarray).InterventionsUse of NGS technology—whole exome sequencing (WES) or whole genome sequencing (WGS).Main outcome measures(1) Diagnostic yield by sequencing type, (2) diagnostic yield by phenotypical categories, (3) reduction in time to diagnosis and (4) change in clinical outcomes and management.ResultsWe demonstrate a 37.8% diagnostic yield for WES (n=172) and a 33.3% yield for WGS (n=24). The yield was higher when sequencing was conducted on trios (40.2%), as well as for certain phenotypes (neuromuscular, 54%, and skeletal dysplasia, 50%). In addition to aiding genetic counselling in 100% of the families, a positive result led to a change in treatment in 27% of patients.ConclusionGenomic sequencing is an effective method for diagnosing rare disease or previous ‘undiagnosed’ disease. The clinical utility of WES/WGS is seen in the shortened time to diagnosis and the discovery of novel variants. Additionally, reaching a diagnosis significantly impacts families and leads to alteration in management of these patients.

Point mutations are a common form of genetic variation and have been identified as important disease biomarkers. Conventional methods for analyzing point mutations, e.g., polymerase chain reaction (PCR), are based on differences in thermal stability of the DNA duplex, which require extensive optimization of the reaction condition and nontrivial design of sequence-selective primers. This motivated the design of molecular translators to convert molecular inputs into generic output sequences, which allows for the target recognition and signal generation regions to be designed independently. In this work, we propose a translator design based on the concept of split proximity circuit (SPC) to achieve both high sequence selectivity and assay robustness using a universal reaction condition, i.e., room temperature and constant ionic concentration. We discussed the design aspects of the SPC recognition regions and demonstrated its plug-and-play capability to discriminate different point mutations for both DNA (seven G6PD mutations) and RNA (let-7 microRNA family members) targets while retaining the same signal generation region. Despite its simple design and nonstringent assay condition requirements, the SPC retained good analytical performance to detect subnanomolar target concentration within a reasonable time of an hour.

Background and Aim: Spinal muscular atrophy (SMA) is a lower motor neuron disease with autosomal recessive inheritance caused by homozygous SMN1 deletions. Although SMA has been considered as incurable, newly developed drugs improve life prognoses and motor functions of patients. To maximize the efficacy of the drugs, SMA patients should be treated before symptoms become apparent. Thus, newborn screening for SMA is strongly recommended. In this study, we aim to establish a new simple screening system based on DNA melting peak analysis. Materials and Methods: A total of 124 dried blood spot (DBS) on FTA® ELUTE cards (51 SMN1-deleted patients with SMA, 20 carriers, and 53 controls) were punched and subjected to direct amplification of SMN1 and CFTR (reference gene). Melting peak analyses were performed to detect SMN1 deletions from DBS samples. Results: A combination of allele-specific polymerase chain reaction (PCR) and melting peak analyses clearly distinguished the DBS samples with and without SMN1. Compared with the results of fresh blood samples, our new system yielded 100% sensitivity and specificity. The advantages of our system include (1) biosafe collection, transfer, and storage for DBS samples, (2) obviating the need for DNA extraction from DBS preventing contamination, (3) preclusion of fluorescent probes leading to low PCR cost, and (4) fast and high-throughput screening for SMN1 deletions. Conclusion: We demonstrate that our system would be applicable to a real-world newborn screening program for SMA, because our new technology is efficient for use in routine clinical laboratories that do not have highly advanced PCR instruments.

Objective Cluster genes, specifically the class 3 semaphorins ( SEMA3) including SEMA3C, have been associated with the development of Hirschsprung disease (HSCR) in Caucasian populations. We aimed to screen for rare and common variants in SEMA3C in Indonesian patients with HSCR. Methods In this prospective clinical study, we analyzed SEMA3C gene variants in 55 patients with HSCR through DNA sequencing and bioinformatics analyses. Results Two variants in SEMA3C were found: p.Val337Met (rs1527482) and p.Val579 = (rs2272351). The rare variant rs1527482 (A) was significantly overrepresented in our HSCR patients (9.1%) compared with South Asian controls in the 1000 Genomes (4.7%) and Exome Aggregation Consortium (ExAC; 3.5%) databases. Our analysis using bioinformatics tools predicted this variant to be evolutionarily conserved and damaging to SEMA3C protein function. Although the frequency of the other variant, rs2272351 (G), also differed significantly in Indonesian patients with HSCR (27.3%) from that in South Asian controls in 1000 Genomes (6.2%) and ExAC (4.6%), it is a synonymous variant and not likely to affect protein function. Conclusions This is the first comprehensive report of SEMA3C screening in patients of Asian ancestry with HSCR and identifies rs1527482 as a possible disease risk allele in this population.

Introduction. Variability in the motivation to approach or withdraw from others displayed in infancy is thought to have long-term effects on human social development. Frontal brain asymmetry has been linked to motivational processes in infants and adults, with greater left frontal asymmetry reflecting motivation to approach and greater right frontal asymmetry reflecting motivation to withdraw. We examined the hypothesis that variability in infants’ social motivation is linked to genetic variation in the endogenous oxytocin system. Specifically, we measured infants’ frontal brain asymmetry and later looking preferences to smiling and frowning individuals and assayed a single-nucleotide polymorphism in the CD38 gene (rs3796863) linked to autism spectrum disorder and reduced peripheral oxytocin levels. Methods. 77 11-month-old infants’ (36 female) brain responses were measured via functional near-infrared spectroscopy (fNIRS) while viewing four individuals display either smiles or frowns directed toward or away from them. This was followed by a person preference test using eyetracking. Results. Frontal brain asymmetry patterns evoked by direct-gaze faces significantly differed as a function of CD38 genotype. Specifically, while non-risk A-allele carriers displayed greater left lateralization to smiling faces (approach) and greater right lateralization to frowning faces (withdrawal), infants with the CC (ASD risk) genotype displayed withdrawal from smiling faces. During eyetracking, A-allele carriers showed a heightened preference for the individual who smiled, while CC infants preferred the individual who frowned.Conclusions. Our findings demonstrate that, from early in human ontogeny, genetic variation in the oxytocin system is linked to variability in brain and behavioral markers of social motivation.

Converging evidence suggests that oxytocin (OT) is associated with creative thinking (CT) and that release of OT depends on ADP ribosyl-cyclases (CD38 and CD157). Neural mechanisms of CT and OT show a strong association with dopaminergic (DA) pathways, yet the link between CT and CD38, CD157, dopamine receptor D2 (DRD2) and catechol-O-methyltransferase (COMT) peripheral gene expression remain inconclusive, thus limiting our understanding of the neurobiology of CT. To address this issue, two principal domains of CT, divergent thinking (AUT), were assessed. In men, both AUT is associated with gene expression of CD38, CD157, and their interaction CD38 × CD157. There were no significant associations for DA expression (DRD2, COMT, DRD2 × COMT) on both CT measures. However, analysis of the interactions of OT and DA systems reveal significant interactions for AUT in men. The full model explained a sizable 39% of the variance in females for the total CT score. The current findings suggest that OT and DA gene expression contributed significantly to cognition and CT phenotype. This provides the first empirical foundation of a more refined understanding of the molecular landscape of CT.

Colorectal cancer (CRC) is a high incidence cancer and major cause of cancer mortality. Though disease-causing tumor suppressors for major syndromes are well characterized, about 10% of CRC is familial but without mutations in known tumor suppressors. We exhaustively screened 100 polyposis families for APC germline mutations and identified 13, which are APC mutation-negative, microsatellite-stable (MSS), and with undetectable mutation in known tumor suppressors. Whole exome sequencing in three probands uncovered two with germline frameshift NR0B2 mutations, c.293_301delTTGGGTTGGinsAC and c.227delT. Sanger Sequencing identified a third proband with NR0B2 c.157_166delCATCGCACCT frameshift mutation. All three mutations deleted the C-terminus activation/repression domain of NR0B2, thus are loss-of-function mutations. Real-time RT-PCR performed on tumor and matched mucosa of one patient revealed that NR0B2 downstream targets, SMAD3 was derepressed while GLI1 was downregulated in the colonic mucosa compared to healthy controls. Truncated NR0B2 molecule was predicted to have weakened binding with interacting partners SMAD3, GLI1, BCL2, and RXRα, implying perturbation of TGF-β, Hedgehog, anti-apoptotic and nuclear hormone receptor signaling pathways. Immunostaining also revealed nuclear retention of the most severely truncated NR0B2 molecule compared to the wildtype. Microsatellite and sequencing analysis did not detect loss of wildtype allele in probands' tumors. The patient who acquired somatic KRAS mutation progressed rapidly whist the other two patients manifested with late-onset obesity and diabetes. We propose that haploinsufficiency of NR0B2 is associated with a novel CRC syndrome with metabolic phenotypes.

BACKGROUND: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by degeneration or loss of lower motor neurons. The survival of motor neuron (SMN) 1 gene, which produces the SMN protein, has been identified as a responsible gene for the disease. SMN is ubiquitously expressed in any tissue and may play an important role on the metabolism in the human body. However, no appropriate biomarkers reflecting the alteration in the metabolism in SMA have been identified. METHODS: Low-molecular-weight metabolites were extracted from plasma of 20 human infants (9 SMA type 1 patients and 11 controls) and 9 infant mice (5 SMA-model mice, 4 control mice), and derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide. Finally, the derivatized products were applied to Gas Chromatography/Mass Spectrometry apparatus. To confirm the metabolite abnormality in SMA type 1 patients, we performed SMN-silencing experiment using a hepatocyte-derived cell line (HepG2). RESULTS: We performed a comprehensive metabolomics analysis of plasma from the patients with SMA type 1 and controls, and found that phosphoethanolamine (PEA) was significantly higher in the patients than in the controls. HepG2 experiment also showed that SMN-silencing increased PEA levels. However, comprehensive metabolomics analysis of plasma from SMA-model mice and control mice showed different profile compared to human plasma; there was no increase of PEA even in the SMA-model mice plasma. CONCLUSION: Our data suggested that PEA was one of the possible biomarkers of human SMA reflecting metabolic abnormalities due to the SMN protein deficiency.

Background Spinal muscular atrophy (SMA) is a neuromuscular disease caused by homozygous deletion of SMN1 exons 7 and 8. However, exon 8 is retained in some cases, where SMN2 exon 7 recombines with SMN1 exon 8, forming a hybrid SMN gene. It remains unknown how the hybrid SMN gene contribute to the SMA phenotype. Method We analyzed 515 patients with clinical suspicion for SMA. SMN1 exons 7 and 8 deletion was detected by PCR followed by enzyme digestion. Hybrid SMN genes were further analyzed by nucleotide sequencing. SMN2 copy number was determined by real-time PCR. Results SMN1 exon 7 was deleted in 228 out of 515 patients, and SMN1 exon 8 was also deleted in 204 out of the 228 patients. The remaining 24 patients were judged to carry a hybrid SMN gene. In the patients with SMN1 exon 7 deletion, the frequency of the severe phenotype was significantly lower in the patients with hybrid SMN gene than in the patients without hybrid SMN gene. However, as for the distribution of SMN2 exon 7 copy number among the clinical phenotypes, there was no significant difference between both groups of SMA patients with or without hybrid SMN gene. Conclusion Hybrid SMN genes are not rare in Japanese SMA patients, and it appears to be associated with a less severe phenotype. The phenotype of patients with hybrid SMN gene was determined by the copy number of SMN2 exon 7, as similarly for the patients without hybrid SMN gene.

BACKGROUND: Polymerase chain reaction (PCR) analysis using DNA from dried blood spot (DBS) samples on filter paper is a critical technique for spinal muscular atrophy (SMA) newborn screening. However, DNA extraction from DBS is time-consuming, and elimination of PCR inhibitors from DBS is almost impossible. METHODS: Exon 7 of the two homologous SMA-related genes, survival motor neuron (SMN) 1 and SMN2, of five SMA patients and five controls were amplified by PCR with a punched-out circle of the DBS paper. Two types of DNA preparation methods were tested; DNA-extraction (extracted DNA was added in a PCR tube) and non-DNA-extraction (a punched-out DBS circle was placed in a PCR tube). As for the DNA polymerases, two different enzymes were compared; TaKaRa Ex Taq(™) and KOD FX Neo(™). To test the diagnostic quality of PCR products, RFLP (Restriction fragment length polymorphism) analysis with DraI digestion was performed, differentiating SMN1 and SMN2. RESULTS: In PCR using extracted DNA, sufficient amplification was achieved with TaKaRa Ex Taq(™) and KOD FX Neo(™), and there was no significant difference in amplification efficiency between them. In direct PCR with a punched-out DBS circle, sufficient amplification was achieved when KOD FX Neo(™) polymerase was used, while there was no amplification with TaKaRa Ex Taq(™). RFLP analysis of the direct PCR products with KOD FX Neo(™) clearly separated SMN1 and SMN2 sequences and proved the presence of both of SMN1 and SMN2 in controls, and only SMN2 in SMA patients, suggesting that the direct PCR products with KOD FX Neo(™) were of sufficient diagnostic quality for SMA testing. CONCLUSION: Direct PCR with DNA polymerases like KOD FX Neo(™) has potential to be widely used in SMA newborn screening in the near future as it obviates the DNA extraction process from DBS and can precisely amplify the target sequences in spite of the presence of PCR inhibitors.

Introduction: Sporadic clinical episodes of malignant hyperthermia (MH) that develop during general anaesthesia (GA) have been reported in Singapore. However, there is no published local report of a confirmed case of MH susceptibility (MHS) by skeletal muscle contracture tests and/or molecular tests. Materials and Methods: We report 2 patients from an extended family who developed signs of clinical MH while under GA. The MH episodes were successfully treated with intravenous dantrolene sodium. Sequence analysis of the entire Ryanodine Receptor Type 1 (RyR1) coding gene was carried out in an index patient. Results: The index patient was found to carry a c.7373G>A (p.Arg2458His) mutation in exon 46. This particular mutation satisfies the criteria for a MHS causative mutation. Hence, the index patient was considered to be MHS and did not need to undergo further muscle contracture testing. The same mutation was also found in 3 other members of his extended family. Conclusion: This is the first report of a Singaporean family with at least 4 members carrying a MH-causative mutation in RyR1 gene. This report serves to highlight the existence of the putative gene for MH in Singapore, and the need for clinical vigilance during anaesthesia involving the use of triggering agents. Key words: Dantrolene, Inhalational agent, Suxamethonium

Background and purpose Most spinal muscular atrophy (SMA) patients are homozygous for survival of motor neuron 1 gene (SMN1) deletion. However, some SMA patients carry an intragenic SMN1 mutation. Such patients provide a clue to understanding the function of the SMN protein and the role of each domain of the protein. We previously identified mutations in the Tudor domain and C-terminal region of the SMN protein in three Japanese SMA patients. To clarify the effect of these mutations on protein stability, we conducted expression assays of SMN with mutated domains. Patients and methods Patients A and B carried a mutation in SMN1 exon 3, which encodes a Tudor domain, c.275G>C (p.Trp92Ser). Patient C carried a mutation in SMN1 exon 6, which encodes a YG-box; c.819_820insT (p.Thr274Tyrfs). We constructed plasmid expression vectors containing wild-type and mutant SMN1 cDNAs. After transfection of HeLa cells with the expression plasmids, RNA and protein were isolated and analyzed by reverse-transcription PCR and western blot analysis. Results The abundance of wild-type and mutant SMN1 transcripts in HeLa cells was almost the same. However, western blot analysis showed lower levels of mutant SMN proteins compared with wild-type SMN. In mutant SMN proteins, it is noteworthy that the level of the p.Thr274Tyrfs mutant was much reduced compared with that of the p.Trp92Ser mutant. Conclusions SMN mutations may affect the stability and levels of the protein.

BACKGROUND: Spinal Muscular Atrophy (SMA) is a common autosomal recessive disorder caused by SMN1 gene deletion. SMA has been considered an incurable disease. However, a newly-developed antisense oligonucleotide drug, nusinersen, brings about a good outcome to SMA patients in the clinical trials. Now, a screening for SMA is required for early diagnosis and early treatment so as to give a better clinical outcome to the patients. We have invented a new technology, mCOP-PCR, for SMA screening using dried blood spot (DBS) on the filter paper. One of the problems encountered in SMA screening is poor quality and quantity of DNA extracted from DBS. METHODS: DNA was extracted from DBS of six individuals. Fresh blood DNA of each individual had already been genotyped using PCR/RFLP. The fragments including the sequence of SMN1/SMN2 exon 7 were pre-amplified with conventional PCR. To determine which pre-amplified product is a better template for the real-time mCOP-PCR, we did pre-amplification with a single PCR or pre-amplification with a nested PCR. RESULTS: The real-time mCOP-PCR using pre-amplified products with a single PCR brought about ambiguous results in some SMN1-carrying individuals. However, the results of real-time mCOP-PCR following pre-amplification with a nested PCR were completely matched with those of PCR-RFLP. CONCLUSION: In our study on the real-time mCOP-PCR screening system for SMA, a nested PCR secured the DNA template quality and quantity, leading to unambiguous results of SMA screening.

BACKGROUND: Spinal Muscular Atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration or loss of lower motor neurons. More than 95% of SMA patients show homozygous deletion for the survival motor neuron 1 (SMN1) gene. For the screening of SMN1 deletion, it is necessary to differentiate SMN1 from its highly homologous gene, SMN2. We developed a modified competitive oligonucleotide priming-PCR (mCOP-PCR) method using dried blood spot (DBS)-DNA, in which SMN1 and SMN2-specific PCR products are detected with gel-electrophoresis. Next, we added a targeted pre-amplification step prior to the mCOP-PCR step, to avoid unexpected, non-specific amplification. The pre-amplification step enabled us to combine mCOP-PCR and real-time PCR. In this study, we combined real-time mCOP-PCR and PCR-restriction fragment length polymorphism (PCR-RFLP) to develop a new screening system for detection of SMN1 deletion. METHODS: DBS samples of the subjects were stored at room temperature for a period of less than one year. Each subject had already been genotyped by the first PCR-RFLP using fresh blood DNA. SMN1/SMN2 exon 7 was collectively amplified using conventional PCR (targeted pre-amplification), the products of which were then used as a template in the real-time PCR with mCOP-primer sets. To confirm the results, the pre-amplified products were subject to the second PCR-RFLP. RESULTS: The real-time mCOP-PCR separately amplified SMN1 and SMN2 exon7, and clearly demonstrated SMN1 deletion in an SMA patient. The results of the real-time mCOP-PCR using DBS-DNA were completely consistent with those of the first and second PCR-RFLP analysis. CONCLUSION: In our new system for detection of SMN1 deletion, real-time mCOP-PCR rapidly proved the presence or absence of SMN1 and SMN2, and the results were easily tested by PCR-RFLP. This solid genotyping system will be useful for SMA screening.

BACKGROUND: Spinal Muscular Atrophy (SMA) is a common autosomal recessive neuromuscular disease characterized by defects of lower motor neurons. More than 95% of SMA patients show homozygous deletion for the survival motor neuron 1 (SMN1) gene. For the screening of SMN1 deletion using dried blood spot (DBS), we developed a new combined system with real-time “modified competitive oligonucleotide priming”-polymerase chain reaction (mCOP-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). Although our real-time mCOP-PCR method is secured enough to be gene-specific, its amplification efficiency is not as good because the reverse primers carry a nucleotide mismatched with the sequence of the pre-amplified product. The mismatch has consequently been generated in the process of introducing a restriction enzyme site in the pre-amplified products for PCR-RFLP. METHOD: DBS samples of the subjects were stored at room temperature for a period of less than one year. Each subject had already been genotyped by the first PCR-RFLP using fresh blood DNA. SMN1/SMN2 exon 7 was collectively amplified using conventional PCR (targeted pre-amplification). Pre-amplified products were used as template in the real-time mCOP-PCR, and, on the other hand, were digested with DraI enzyme (PCR-RFLP). To improve the amplification efficiency of mCOP-PCR, one nucleotide change was introduced in the original reverse primers (SMN1-COP and SMN2-COP) to eliminate the mismatched nucleotide. RESULTS: The real-time mCOP-PCR with a new primer (SMN1-COP-DRA or SMN2-COP-DRA) more rapidly and specifically amplified SMN1 and SMN2, and clearly demonstrated SMN1 deletion in an SMA patient. With the new primers, the amplification efficiencies of real-time mCOP-PCR were improved and the Cq values of SMN1 (+) and SMN2 (+) samples were significantly lowered. CONCLUSION: In the advanced version of our screening system for homozygous SMN1 deletion using DBS, the real-time mCOP-PCR with newly-designed reverse primers demonstrated the presence or absence of SMN1 and SMN2 within a shorter time, and the results were easily tested by PCR-RFLP. This rapid and accurate screening system will be useful for detection of newborn infants with SMA.