Single-Tube Screen for Rapid Detection of Repeat Expansions in Seven Common Spinocerebellar Ataxias.

BACKGROUND: The autosomal dominantly inherited and genetically heterogeneous spinocerebellar ataxias (SCAs) exhibit highly similar clinical presentations. Many are caused by repeat expansions, of which at least 8 involve CAG repeats. Repeat expansion detection is the only method to confirm disease status in symptomatic individuals. We present a novel strategy to simultaneously screen for the presence of CAG repeat expansion in the genes responsible for SCAs 1, 2, 3, 6, 7, 12, and dentatorubral-pallidoluysian atrophy using a simplified single-tube assay. METHODS: The method employs differentially labeled locus-specific primers and a common triplet-primed primer. Amplified products from each locus are distinguished by a combination of the product size and the fluorophore tag. The upper size limit of the normal allele range was used as the cutoff for distinguishing normal from potentially affected samples, with repeat expansion detected by presence of electrophoretic peaks extending beyond the cutoff. RESULTS: Blinded evaluation of the assay on 60 genotype-known DNA samples correctly detected repeat expansion in the expected SCA repeat locus for all 31 DNA samples. CONCLUSIONS: In principle, this strategy can be applied to the simultaneous screening of any group of disease genes sharing the same repetitive units and/or their reverse complement.

Global DNA hypermethylation in down syndrome placenta.

Down syndrome (DS), commonly caused by an extra copy of chromosome 21 (chr21), occurs in approximately one out of 700 live births. Precisely how an extra chr21 causes over 80 clinically defined phenotypes is not yet clear. Reduced representation bisulfite sequencing (RRBS) analysis at single base resolution revealed DNA hypermethylation in all autosomes in DS samples. We hypothesize that such global hypermethylation may be mediated by down-regulation of TET family genes involved in DNA demethylation, and down-regulation of REST/NRSF involved in transcriptional and epigenetic regulation. Genes located on chr21 were up-regulated by an average of 53% in DS compared to normal villi, while genes with promoter hypermethylation were modestly down-regulated. DNA methylation perturbation was conserved in DS placenta villi and in adult DS peripheral blood leukocytes, and enriched for genes known to be causally associated with DS phenotypes. Our data suggest that global epigenetic changes may occur early in development and contribute to DS phenotypes.

Enhanced Detection and Sizing of the HTT CAG Repeat Expansion in Huntington Disease Using an Improved Triplet-Primed PCR Assay.

BACKGROUND: Accurate determination of the CAG repeat number is crucial for diagnostic and predictive testing for Huntington disease (HD). Current PCR-based assays can accurately size up to ∼110 HTT CAG repeats. OBJECTIVE: To develop an improved assay capable of detecting larger CAG repeat expansions. METHODS: A triplet-primed PCR (TP-PCR) assay was optimized and validated on 14 HD reference DNAs, including a sample carrying a large expansion of ∼180 CAG repeats. RESULTS: All 14 HD reference samples showed 100% concordance with the previously verified allele sizes. For alleles under 45 CAGs, identical repeat sizes were obtained, while alleles larger than 46 CAGs were sized to within ±1 CAG. The improved TP-PCR assay successfully detected the ∼180 CAG repeat allele in an affected sample. CONCLUSION: This method extends the detection limit of large expanded alleles to at least ∼175-180 CAG repeats, thus reducing the likelihood of requiring Southern blot analysis for any HD-affected sample.

Simplified strategy for rapid first-line screening of fragile X syndrome: closed-tube triplet-primed PCR and amplicon melt peak analysis.

Premutation and full-mutation hyperexpansion of CGG-triplets in the X-linked Fragile X Mental Retardation 1 (FMR1) gene have been implicated in fragile X-associated tremor/ataxia syndrome, fragile X-associated primary ovarian insufficiency, and fragile X syndrome (FXS), respectively. The currently available molecular diagnostic tests are either costly or labour-intensive, which prohibits their application as a first-line FMR1 test in large-scale population-based screening programs. In this study, we demonstrate the utility of a simplified closed-tube strategy for rapid first-line screening of FXS based on melt peak temperature (Tm) analysis of direct triplet-primed polymerase chain reaction amplicons (dTP-PCR MCA). In addition, we also evaluated the correlation between Tm and CGG-repeat size based on capillary electrophoresis (CE) of dTP-PCR amplicons. The assays were initially tested on 29 FMR1 reference DNA samples, followed by a blinded validation on 107 previously characterised patient DNA samples. The dTP-PCR MCA produced distinct melt profiles of higher Tm for samples carrying an expanded allele. Among the samples tested, we also observed a good correlation between Tm and CGG-repeat size. In the blinded validation study, dTP-PCR MCA accurately classified all normal and expansion carriers, and the FMR1 genotypic classification of all samples was completely concordant with the previously determined genotypes as well as the dTP-PCR CE results. This simple and cost-effective MCA-based assay may be useful as a first-line FXS screening tool that could rapidly screen out the large majority of unaffected individuals, thus minimising the number of samples that need to be analysed by Southern blot analysis.

Single-tube nonaplex microsatellite PCR panel for preimplantation genetic diagnosis of Hb Bart's hydrops fetalis syndrome.

OBJECTIVE: To develop a single-tube multi-marker assay for improved preimplantation genetic diagnosis (PGD) of deletional and/or non-deletional Hb Bart's hydrops fetalis syndrome, providing haplotype confirmation of deletional status, and maximization of linkage informativity. METHODS: We performed in silico mining to identify novel microsatellites within 1 Mb flanking the alpha-globin gene cluster, and optimized a single-tube assay combining detection of α(0) -thalassemia deletions with multi-marker linkage analysis. We performed validation on 100 single cells prior to clinical PGD application. RESULTS: Of 42 markers encompassing the α-globin gene cluster that were identified in silico, 9 were highly polymorphic (0.68 ≤ polymorphism information content ≤ 0.92; 0.66 ≤ Ho ≤ 0.90; 10 ≤ alleles ≤ 35) and optimized to co-amplify directly from a single cell. A validation analysis of 100 single lymphoblasts yielded 100% amplification success for all markers, and individual marker allele drop-out (ADO) rates of 0-5%. Clinical application of the assay in PGD for Hb Bart's (2 cases/cycles) resulted in a twin pregnancy and healthy live birth of two baby girls. CONCLUSIONS: This single-tube nonaplex microsatellite PCR panel can be applied directly to PGD of most deletional Hb Bart's without the need for deletion-specific customization, and to linkage-based PGD of non-deletional Hb Bart's.

Left Ventricular Non-compaction: Is It Genetic?

Left ventricular non-compaction (LVNC) is reported to affect 0.14 % of the pediatric population. The etiology is heterogeneous and includes a wide number of genetic causes. As an illustration, we report two patients with LVNC who were diagnosed with a genetic syndrome. We then review the literature and suggest a diagnostic algorithm to evaluate individuals with LVNC. Case 1 is a 15-month-old girl who presented with hypotonia, global developmental delay, congenital heart defect (including LVNC) and facial dysmorphism. Case 2 is a 7-month-old girl with hypotonia, seizures, laryngomalacia and LVNC. We performed chromosomal microarray for both our patients and detected chromosome 1p36 microdeletion. We reviewed the literature for other genetic causes of LVNC and formulated a diagnostic algorithm, which includes assessment for syndromic disorders, inborn error of metabolism, copy number variants and non-syndromic monogenic disorder associated with LVNC. LVNC is a relatively newly recognized entity, with heterogeneity in underlying etiology. For a systematic approach of evaluating the underlying cause to improve clinical care of these patients, a diagnostic algorithm for genetic evaluation of patients with LVNC is proposed.

Clinical application of targeted long read sequencing in prenatal beta-thalassemia testing and genetic counseling.

BACKGROUND: Beta thalassemia, related to HBB mutation and associated with elevated hemoglobin A2 (HbA2), is an important genetic hemoglobinopathy with high incidences of disease and carrier rates in Singapore. Carrier screening is essential to facilitate prenatal counseling and testing. However, when individuals with elevated HbA2 do not have an identifiable HBB disease-associated variant, there is ambiguity on risk to their offspring. METHODS: We describe a case report of a proband with elevated HbA2, no identifiable HBB disease-associated variant, whose partner was a beta thalassemia carrier. Through clinical HBB gene sequencing, multiplex ligation-dependent probe amplification (MLPA) analysis, as well as targeted Nanopore long read sequencing of selected genes, we performed a complete analysis of HBB including the promoter region, 5'UTR and coding gene sequence, as well as evaluation for potential modifier variants and other rare structural variants. RESULTS: This process identified that the proband was heterozygous for KLF1:c.544T>C (p.Phe182Leu), a potential functional polymorphism previously known to be associated with benign elevated HbA2 levels. The presence of disease variants in the HBB locus was excluded. CONCLUSION: This finding provided clarity and enabled family planning for the proband and her family.

Machine learning unveils an immune-related DNA methylation profile in germline DNA from breast cancer patients.

BACKGROUND: There is an unmet need for precise biomarkers for early non-invasive breast cancer detection. Here, we aimed to identify blood-based DNA methylation biomarkers that are associated with breast cancer. METHODS: DNA methylation profiling was performed for 524 Asian Chinese individuals, comprising 256 breast cancer patients and 268 age-matched healthy controls, using the Infinium MethylationEPIC array. Feature selection was applied to 649,688 CpG sites in the training set. Predictive models were built by training three machine learning models, with performance evaluated on an independent test set. Enrichment analysis to identify transcription factors binding to regions associated with the selected CpG sites and pathway analysis for genes located nearby were conducted. RESULTS: A methylation profile comprising 51 CpGs was identified that effectively distinguishes breast cancer patients from healthy controls achieving an AUC of 0.823 on an independent test set. Notably, it outperformed all four previously reported breast cancer-associated methylation profiles. Enrichment analysis revealed enrichment of genomic loci associated with the binding of immune modulating AP-1 transcription factors, while pathway analysis of nearby genes showed an overrepresentation of immune-related pathways. CONCLUSION: This study has identified a breast cancer-associated methylation profile that is immune-related to potential for early cancer detection.

Rapid prenatal diagnosis of common beta-thalassemia mutations in Southeast Asia using pyrosequencing.

OBJECTIVE: Current methods of prenatal diagnosis to detect beta-thalassemia are Sanger sequencing and reverse dot blot. These methods are time-consuming and can prolong assay turnaround time. We aim to develop a sensitive and rapid method to detect 27 beta-thalassemia mutations using pyrosequencing. METHOD: Pyrosequencing primer pairs and sequencing primers were designed to detect 27 most common beta-thalassemia mutations found in Singapore. Pyrosequencing was performed on 191 DNA samples with known beta-thalassemia mutations isolated from 143 peripheral blood and 48 prenatal samples (seven chorionic villus biopsies, 26 cultured amniocytes, 15 uncultured amniocytes). All mutations were validated with Sanger sequencing. RESULTS: Pyrosequencing identified 210 alleles with beta-thalassemia mutations and 82 alleles without mutations with 100% sensitivity (lower 95% confidence interval [CI], 97.8%) and 100% specificity (lower 95% CI, 94.4%). All pyrosequences were concordant with Sanger-based sequences. Pyrosequencing was able to detect DNA concentrations as low as 2 ng, obviating the need for cell culture in volume-restricted samples. Sample receipt-to-report assay turnaround times were 16 to 18 h (Sanger sequencing) and 4 to 6 h (pyrosequencing). CONCLUSION: Pyrosequencing is a rapid and sensitive method to detect common beta-thalassemia mutations without the need for cell culture, thus reducing the assay turnaround time.

Degradation of methylation signals in cryopreserved DNA.

BACKGROUND: Blood-based DNA methylation has shown great promise as a biomarker in a wide variety of diseases. Studies of DNA methylation in blood often utilize samples which have been cryopreserved for years or even decades. Therefore, changes in DNA methylation associated with long-term cryopreservation can introduce biases or otherwise mislead methylation analyses of cryopreserved DNA. However, previous studies have presented conflicting results with studies reporting hypomethylation, no effect, or even hypermethylation of DNA following long-term cryopreservation. These studies may have been limited by insufficient sample sizes, or by their profiling of methylation only on an aggregate global scale, or profiling of only a few CpGs. RESULTS: We analyzed two large prospective cohorts: a discovery (n = 126) and a validation (n = 136) cohort, where DNA was cryopreserved for up to four years. In both cohorts there was no detectable change in mean global methylation across increasing storage durations as DNA. However, when analysis was performed on the level of individual CpG methylation both cohorts exhibited a greater number of hypomethylated than hypermethylated CpGs at q-value < 0.05 (4049 hypomethylated but only 50 hypermethylated CpGs in discovery, and 63 hypomethylated but only 6 hypermethylated CpGs in validation). The results were the same even after controlling for age, storage duration as buffy coat prior to DNA extraction, and estimated cell type composition. Furthermore, we find that in both cohorts, CpGs have a greater likelihood to be hypomethylated the closer they are to a CpG island; except for CpGs at the CpG islands themselves which are less likely to be hypomethylated. CONCLUSION: Cryopreservation of DNA after a few years results in a detectable bias toward hypomethylation at the level of individual CpG methylation, though when analyzed in aggregate there is no detectable change in mean global methylation. Studies profiling methylation in cryopreserved DNA should be mindful of this hypomethylation bias, and more attention should be directed at developing more stable methods of DNA cryopreservation for biomedical research or clinical use.

Screening for CGG repeat expansion in the FMR1 gene by melting curve analysis of combined 5' and 3' direct triplet-primed PCRs.

BACKGROUND: CGG repeat expansions in the FMR1 (fragile X mental retardation 1) gene are associated with fragile X syndrome, fragile X-associated tremor/ataxia syndrome, and fragile X-associated primary ovarian insufficiency. We evaluated the use of melting curve analysis (MCA) of triplet-primed PCR (TP-PCR) assays as a rapid screening tool for the positive identification of expanded FMR1 alleles in men and women. METHODS: Both 5'- and 3'-weighted direct TP-PCRs (dTP-PCRs) were evaluated on 29 cell line-derived DNA samples and 44 blinded clinical samples. The presence of expansions was identified by the melting curve profiles generated automatically through MCA on the LightCycler 480 Real-Time PCR System. All samples were also analyzed by capillary electrophoresis to confirm the identities of the PCR fragments that gave rise to the observed melt peak profiles. RESULTS: The presence of expanded alleles in samples from both males and females produced melt peak profiles that were distinct from those of individuals with the normal allelic form. In the blinded test, positive and negative calls for the presence of an expanded allele corroborated with previously determined genotype classifications for all samples. CONCLUSIONS: The approach of dTP-PCR plus MCA offers a single-step strategy with high diagnostic sensitivity and specificity for rapid screening detection of FMR1 CGG repeat expansions, regardless of sex. The combined use of 5'- and 3'-weighted dTP-PCR assays minimizes the incidence of false-negative results arising from repeat-flanking deletions.

High-Throughput Methylation-Specific Triplet-Primed PCR and Melting Curve Analysis for Selective and Reliable Identification of Actionable FMR1 Genotypes.

Methylated FMR1 full-mutation expansions cause fragile X syndrome. FMR1 premutation carriers are susceptible to other late-onset conditions, and women with premutation are at risk of transmitting a fully expanded FMR1 allele to offspring. Identification of individuals with actionable FMR1 genotypes (full-mutation males and females, and premutation females at risk for primary ovarian insufficiency and/or having fragile X-affected offspring) can enable timely access to intervention services and genetic counseling. This study presents a rapid, first-tier test based on melting curve analysis of methylation-specific triplet-primed PCR amplicons (msTP-PCR MCA) for concurrent detection of FMR1 CGG-repeat expansions and their methylation status. The msTP-PCR MCA assay was optimized on 20 fragile X reference samples, and its performance was evaluated on 111 peripheral blood-derived DNA samples from patients who have undergone prior molecular testing with PCR and/or Southern blot analysis. The msTP-PCR MCA assay detected all samples with a methylated FMR1 CGG-repeat expansion, and had sensitivity, specificity, positive predictive value, and negative predictive values of 100%, 92.06%, 91.1%, and 100%, respectively. The msTP-PCR MCA assay identified premutation/full-mutation mosaicism down to 1%, detected skewed inactivation in females with FMR1 expansions, and enabled selective identification of all individuals with an actionable FMR1 genotype. The msTP-PCR MCA assay may aid in fragile X screening of at-risk populations and newborns and voluntary carrier screening of women of reproductive age.

Rapid Molecular Screen of Spinocerebellar Ataxia Types 1, 2, and 3 by Triplet-Primed PCR and Melting Curve Analysis.

The autosomal dominantly inherited spinocerebellar ataxias (SCAs) can be caused by dynamic mutations of short tandem repeats within various genes. Because of the significant clinical overlap among the various SCA types, molecular screening of multiple genetic loci by fluorescent PCR and capillary electrophoresis is necessary to identify the causative repeat expansion. We describe a simple, rapid, and inexpensive strategy to screen for CAG repeat expansion mutations at the ATXN1, ATXN2, and ATXN3 loci using melting curve analysis of triplet-primed PCR products. Plasmid DNAs of known repeat sizes were used to generate threshold melt peak temperatures, which rapidly and effectively distinguish samples carrying an expanded allele from those carrying nonexpanded alleles. Melting curve analysis-positive samples were confirmed by capillary electrophoresis sizing of the triplet-primed PCR products. All three assays achieved 100% sensitivity, with 95% CIs of 67.86% to 100% (SCA1), 74.65% to 100% (SCA2), and 91.58% to 100% (SCA3). The SCA1 assay also achieved 100% specificity (95% CI, 97.52%-100%), whereas the SCA2 and SCA3 assays achieved specificity of 99.46% (95% CI, 96.56%-99.97%) and 99.32% (95% CI, 95.70%-99.96%), respectively. These screening assays provide robust and highly accurate detection of expanded alleles and are amenable to large-scale screening while minimizing the need for capillary electrophoresis sizing for every sample.

Simultaneous Screening of the FRAXA and FRAXE Loci for Rapid Detection of FMR1 CGG and/or AFF2 CCG Repeat Expansions by Triplet-Primed PCR.

Moderate to hyper-expansion of trinucleotide repeats at the FRAXA and FRAXE fragile sites, with or without concurrent hypermethylation, has been associated with intellectual disability and other conditions. Unlike molecular diagnosis of FMR1 CGG repeat expansions in FRAXA, current detection of AFF2 CCG repeat expansions in FRAXE relies on low-throughput and otherwise inefficient techniques combining Southern blot analysis and PCR. A novel triplet-primed PCR assay was developed for simultaneous screening for trinucleotide repeat expansions at the FRAXA and FRAXE fragile sites, and was validated using archived clinical samples of known FMR1 and AFF2 genotypes. Population samples and FRAXE-affected samples were sequenced for the evaluation of variations in the AFF2 CCG repeat structure. The duplex assay accurately identified expansions at the FMR1 and AFF2 trinucleotide repeat loci. On Sanger sequencing of the AFF2 CCG repeat, the single-nucleotide polymorphism variant rs868914124(C) that effectively adds two CCG repeats at the 5'-end, was enriched in the Malay population and with short repeats (<11 CCGs), and was present in all six expanded AFF2 alleles of this study. All expanded AFF2 alleles contained multiple non-CCG interruptions toward the 5'-end of the repeat. A sensitive, robust, and rapid assay has been developed for the simultaneous detection of expansion mutations at the FMR1 and AFF2 trinucleotide repeat loci, simplifying screening for FRAXA- and FRAXE-associated disorders.

Singapore Undiagnosed Disease Program: Genomic Analysis aids Diagnosis and Clinical Management.

OBJECTIVE: Use next-generation sequencing (NGS) technology to improve our diagnostic yield in patients with suspected genetic disorders in the Asian setting. DESIGN: A diagnostic study conducted between 2014 and 2019 (and ongoing) under the Singapore Undiagnosed Disease Program. Date of last analysis was 1 July 2019. SETTING: Inpatient and outpatient genetics service at two large academic centres in Singapore. PATIENTS: Inclusion 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). INTERVENTIONS: Use 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. RESULTS: We 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. CONCLUSION: Genomic 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.

High-throughput beta-thalassemia carrier screening by allele-specific Q-primer real-time polymerase chain reaction.

Based on a novel Q-primer real-time polymerase chain reaction (PCR) system, we designed allele-specific Q-primers for the detection of three beta-thalassemia mutations [Cd41/42(-TCTT), IVSI nt5 (G>C), and IVSII nt654 (C>T)] that have a high carrier frequency in Southeast Asia. With clear distinction between heterozygote and wild-type, DeltaC(t) (threshold cycle) values were defined. The results of evaluating 139 blinded samples by our system match perfectly with those obtained by the conventional reverse dot blot (RDB) method. With a 384-well plate that included replicates in the same analysis, our throughput reached 190 reactions per run with a turnaround time as short as 130 min, and the cost of consumables was as low as $1 (US) for each test.

Ethnicity and OPRM variant independently predict pain perception and patient-controlled analgesia usage for post-operative pain.

BACKGROUND: Morphine consumption can vary widely between individuals even for identical surgical procedures. As mu-opioid receptor (OPRM1) is known to modulate pain perception and mediate the analgesic effects of opioid compounds in the central nervous system, we examined the influence of two OPRM polymorphisms on acute post-operative pain and morphine usage in women undergoing elective caesarean delivery. RESULTS: Data on self-reported pain scores and amount of total morphine use according to patient-controlled analgesia were collected from 994 women from the three main ethnic groups in Singapore. We found statistically significant association of the OPRM 118A>G with self-administered morphine during the first 24-hour postoperative period both in terms of total morphine (p = 1.7 x 10(-5)) and weight-adjusted morphine (p = 6.6 x 10(-5)). There was also significant association of this OPRM variant and time-averaged self-rated pain scores (p = 0.024). OPRM 118G homozygotes used more morphine and reported higher pain scores than 118A carriers. Other factors which influenced pain score and morphine usage include ethnicity, age and paying class. CONCLUSION: Our results suggest that ethnicity and OPRM 118A>G genotype are independent and significant contributors to variation in pain perception and postoperative morphine use in patients undergoing cesarean delivery.

alpha-Thalassemia mutation analyses in Mazandaran province, North Iran.

Two hundred and fifty-five patients from Mazandaran Province, Iran, all presenting with hypochromic and microcytic anemia, were selected for alpha-thalassemia (alpha-thal) mutation screening. We detected a total of 274 alpha-globin mutations in 227 (89%) of these patients. Among the 21 different alpha-globin alleles found, the -alpha(3.7) (44.9%), polyadenylation signal 2 (poly A2) (AATAAA>AATGAA) (18.2%), -alpha(4.2) (9.1%), alpha(IVS-I(-5 nt)) (6.5%), - -(MED) (4.3%), and alpha(codon 19 (-G)) (4%) were the most frequent. The other 15 mutations included variants that had not yet been observed in Iran, such as Hb Bleuland [alpha108(G15)ThrAsn, ACC>AAC (alpha2)], as well as a novel mutation on the alpha2 gene, also not described to date [3 ' untranslated region (3 'UTR) nucleotide (nt) 46 (C>A)]. These comprehensive new data are useful for establishing a screening strategy for the effective control of alpha-thal in Mazandaran Province.