Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

The role of TDP-43 and related proteins in neurodegenerative diseases

Introduction: TDP-43 (TAR DNA-binding protein-43), encoded by the TARDBP gene, has recently been identified as the molecular link, which connects the, until recently considered, separate entities that currently form the FTD/ALS spectrum. Specifically, TDP-43 is the main component of ubiquitinated inclusions found in both FTD and ALS. The direct causal relationship of TDP-43 with the neurodegenerative process was suggested by the discovery of pathogenic variants in the TARDBP gene, in families with members presenting with FTD/ALS. Furthermore, TDP‐43 proteinopathy has been observed in carriers of pathogenic variants in additional genes associated with both FTD and ALS, as well as in patients with an Alzheimer’s disease (AD) subtype, thus demonstrating a more general role of TDP‐43 in the neurodegenerative process. The purpose of this dissertation is to investigate TDP‐43 proteinopathy in Greek patients with neurodegenerative diseases and in particular: 1. To measure TDP-43 protein levels in the cerebrospinal fluid (CSF) as a candidate diagnostic biomarker for the FTD/ALS spectrum. 2. To detect genetic changes in the TARDBP gene, as well as in other genes associated with FTD/ALS and to evaluate their contribution to the clinical phenotype. 3. To correlate genetic changes not only with the TDP-43 levels in the CSF, but also with the levels of "classical" biomarkers for Alzheimer's disease (AD), i.e. total tau protein (τΤ), its phosphorylated form (τΡ-181) and the β-amyloid peptide with 42 amino acids (Αβ42). Patients and Methods: The study population consisted of 54 patients with FTD, 61 patients with ALS and 14 patients with FTD-ALS, who were diagnosed with the most recent diagnostic criteria, supported by the “classical” AD biomarkers (τΤ, τΡ-181 and Αβ42), in the CSF, to rule out the inclusion of patients with frontal and logopenic variant of AD in the FTD subgroup. The quantitative determination of TDP-43 levels and “classical” biomarkers in the CSF was performed by double-sandwich enzyme-linked immunosorbent assay (ELISA). Patients were screened for pathogenic hexanucleotide repeat expansion (GGGGCC)n in the C9orf72 gene, using the repeat-primed polymerase chain reaction (PCR) method, while C9orf72 (-) patients underwent whole exome sequencing. The levels of biomarkers were compared with a control group of 28 healthy individuals without mental or motor impairment and the genetic changes with an already very well-characterized sample of 81 healthy aged adults from Crete. Also, 100 patients from Crete diagnosed with dementia (of the AD type) were tested for pathogenic variants in FTD/ALS genes. Results: Statistically significant higher TDP-43 levels in the CSF were found in all patient groups (FTD, ALS, FTD/ALS), compared to healthy controls. Patients with ALS showed a tendency to have higher CSF TDP-43 levels compared to patients with FTD. Significantly higher levels of τΤ were found in all groups, compared to healthy controls, with the FTD group showing increased levels compared to the ALS group. The levels of τΡ-181 and Aβ42 did not differ significantly among patient groups. ROC analysis showed that the combination of TDP-43 with τ proteins, as expressed by the formula TDP-43 × τΤ/τΡ-181 has high sensitivity and specificity (> 80%), for separation of ALS/FTD spectrum patients from healthy controls.Genetic analysis revealed pathogenic or likely pathogenic variants in 18.6% (24/129) of patients in the ALS/FTD spectrum. Genes harboring these variants included, in a decreasing order of frequency, the C9orf72, TARDBP, GRN, VCP, SOD1 and FUS genes. The average age of disease onset in patients with the C9orf72 repeat expansion was about 10 years earlier compared to other patients. In 2 patients from Crete initially diagnosed as AD we identified the p.Ile383Val variant. Patients with ALS, either alone or concurrently with FTD, harboring the C9orf72 repeat expansion and the MAPT p.Asp285As change had significantly lower levels of τΡ-181 compared to the other patients. Also, carriers of rare APP variants had lower levels of Aβ42, compared to non-carriers. Conclusions: TDP-43 proteinopathy is a disease entity with significant clinical, biochemical and genetic heterogeneity. It has been traditionally thought to be present in younger patients compared to other neurodegenerative disorders; however, recently it is increasingly recognized among the elderly. The combination of TDP-43 with tau proteins in the CSF, as expressed by the formula TDP-43×τT /τP-181, has high sensitivity and specificity and is therefore a potential biomarker of TDP-43 proteinopathy. The most common gene variant associated with TDP-43 proteinopathy in the Greek population is the C9orf72 repeat expansion with a high frequency of 10,8% (14/129) compared to other European populations and is associated with the disease onset 10 years earlier than the sporadic forms. The variant p.Ile383Val in the TARDBP gene is another common pathogenic variant in the Greek population.Εισαγωγή: Η TDP-43 (TAR DNA-binding protein-43) πρωτεΐνη έχει πλέον αναγνωριστεί ως o μοριακός κρίκος, ο οποίος συνδέει πολλές από τις μέχρι πρόσφατα θεωρούμενες ξεχωριστές οντότητες νοσημάτων, που σήμερα συγκροτούν το φάσμα μετωποκροταφικής άνοιας / πλάγιας μυατροφικής σκλήρυνσης FTD/ALS. Η TDP-43 είναι το κύριο συστατικό των ουβικουιτινωμένων εγκλείστων που ανευρίσκονται τόσο στην FTD όσο και στην ALS. Η αιτιώδης συνάφεια της νευροεκφυλιστικής διεργασίας με την TDP‐43 πρωτεΐνη, προήλθε από την αναγνώριση παθογόνων μεταλλάξεων στο γονίδιο TARDBP, που την κωδικοποιεί, σε οικογένειες του φάσματος FTD/ALS. Εντούτοις, TDP‐43 πρωτεϊνοπάθεια έχει παρατηρηθεί και σε φορείς μεταλλάξεων και άλλων γονιδίων που σχετίζονται τόσο με την FTD όσο και με την ALS, αλλά και με άλλες νόσους, όπως μια μορφή της νόσου Alzheimer (AD), καταδεικνύοντας έτσι έναν γενικότερο καθοριστικό ρόλο της TDP‐43 στην νευροεκφυλιστική διεργασία. Σκοπός της παρούσας διατριβής είναι η διερεύνηση της TDP‐43 πρωτεϊνοπάθειας και ειδικότερα: 1. Η μέτρηση των επιπέδων της TDP-43 πρωτεΐνης στο εγκεφαλονωτιαίο υγρό (ΕΝΥ) ως υποψήφιος διαγνωστικός βιοδείκτης για το φάσμα FTD/ALS. 2. Η ανίχνευση γενετικών αλλαγών στο γονίδιο TARDBP, αλλά και σε άλλα γονίδια σχετιζόμενα ή όχι με TDP-43 πρωτεϊνοπάθεια (C9οrf72, GRN, VCP κτλ.) και η αξιολόγηση της συνεισφοράς των γενετικών αλλαγών αυτών στον κλινικό φαινότυπο. 3. Η πιθανή συσχέτιση των γενετικών αλλαγών με τα επίπεδα της TDP-43 στο ΕΝΥ, αλλά και με τα επίπεδα των “κλασσικών” βιοδεικτών για την AD, δηλαδή την ολική πρωτεΐνη (τΤ), την φωσφορυλιωμένη μορφή αυτής (τΡ-181) και το β-αμυλοειδές πεπτίδιο με 42 αμινοξέα (Αβ42). Ασθενείς και Μέθοδοι: Ο πληθυσμός της μελέτης αποτελείται από 54 ασθενείς με FTD, 61 ασθενείς με ALS και 14 ασθενείς με FTD-ALS, οι οποίοι διαγνώστηκαν με τα πλέον πρόσφατα διαγνωστικά κριτήρια, υποστηριζόμενα από τους “κλασσικούς” βιολογικoύς δείκτες για την AD προκειμένου, ειδικά για την ομάδα FTD, να αποκλεισθεί η παρείσφρηση της μετωπιαίας και λογοπενικής παραλλαγής της AD. Ο ποσοτικός προσδιορισμός των επίπεδων της TDP-43 και των κλασικών βιοδεικτών στο ΕΝΥ έγινε με μεθόδους ενζυμικής ανοσοπροσροφητικής δοκιμασίας διπλού σάντουιτς [double-sandwich enzyme-linked immunosorbent assay (ELISA). Στους ασθενείς πραγματοποιήθηκε επίσης έλεγχος για τον εντοπισμό παθολογικών εξανουκλεοτιδικών επαναλήψεων (GGGGCC)n του γονιδίου C9orf72, με την μέθοδο αλυσιδωτής αντίδρασης πολυμεράσης (PCR) και PCR με τη χρήση εκκινητών επαναλαμβανόμενης αλληλουχίας (repeat-primed PCR), σε συνδυασμό με αυτοματοποιημένη ανάλυση τμημάτων DNA. Στους ασθενείς με απουσία παθολογικού αριθμού εξανουκλεοτιδικών επαναλήψεων πραγματοποιήθηκε αλληλούχιση ολόκληρου του εξώματος (whole exome sequencing). Τα επίπεδα των βιοδεικτών συγκρίθηκαν με ομάδα ελέγχου 28 υγιών ατόμων, χωρίς νοητική ή κινητική διαταραχή και οι γενετικές αλλαγές με ένα ήδη πολύ καλά χαρακτηρισμένο δείγμα 81 νοητικά υγιών μαρτύρων από την Κρήτη. Επίσης, αναζητήθηκαν αλλαγές σε FTD/ALS γονίδια σε ένα πληθυσμό 100 με AD ασθενών με άνοια (AD τύπου) από την Κρήτη (Μελέτη Γήρανσης της Κρήτης). Αποτελέσματα: Στατιστικά σημαντικά υψηλότερες τιμές TDP-43 στο ΕΝΥ ανευρέθηκαν σε όλες τις ομάδες ασθενών (FTD, ALS, FTD-ALS), σε σχέση με τους υγιείς μάρτυρες. Τα επίπεδα TDP-43 των ασθενών με ALS ήταν αυξημένα σε σχέση με αυτά των ασθενών με FTD, ωστόσο αυτή η διαφορά δεν ήταν στατιστικά σημαντική. Στατιστικά σημαντικά υψηλότερα επίπεδα τT ανευρέθηκαν σε όλες τις ομάδες ασθενών, σε σχέση με τους υγιείς μάρτυρες, με τα επίπεδα των ασθενών με FTD να είναι αυξημένα σε σχέση με αυτά των ασθενών με ALS. Τα επίπεδα της τP-181 και του Αβ42 δεν διέφεραν σημαντικά μεταξύ των ομάδων ασθενών. Η ανάλυση ROC έδειξε ότι ο συνδυασμός των επίπεδων της TDP-43 στο ΕΝΥ με τις πρωτεΐνες T(tau), εκφραζόμενος με τον τύπο TDP-43×τT/τP-181 έχει υψηλή ευαισθησία και ειδικότητα (>80%), για τον διαχωρισμό ασθενών του φάσματος ALS/FTD από υγιείς μάρτυρες. Με την γενετική ανάλυση εντοπίστηκαν παθογόνες και πιθανά παθογόνες αλλαγές στο 18,6% (24/129) των ασθενών του φάσματος ALS/FTD. Τα γονίδια που εμφανίζονταν αυτές οι αλλαγές ήταν με φθίνουσα σειρά συχνότητας, τα γονίδια C9orf72, TARDBP, GRN, VCP, SOD1 και FUS. Η μέση ηλικία έναρξης της νόσου των ασθενών με παθολογική επέκταση εξανουκλεοτιδίου στο C9orf72 ήταν κατά 10 περίπου έτη μικρότερη σε σχέση με τους υπόλοιπους ασθενείς. Η p.Ile383Val TARDBP αλλαγή βρέθηκε και σε 2 ασθενείς διαγνωσμένους με άνοια στην Κρήτη. Εξετάζοντας τα επίπεδα της TDP-43, Aβ42, τΤ και τp-181 διαπιστώσαμε ότι οι φέροντες μετάλλαξη (επέκταση εξανουκλεοτιδίου) του C9orf72 γονιδίου ασθενείς με ALS (με ή χωρίς FTD), όπως και οι φορείς της συνώνυμης αλλαγής p.Asp285Asp στο γονίδιο MAPT, παρουσίαζαν στατιστικώς σημαντικά χαμηλότερα επίπεδα τΡ-181 σε σχέση με τους υπολοίπους ασθενείς. Επίσης, οι φορείς σπάνιων αλλαγών του APP είχαν χαμηλότερα επίπεδα Αβ42, σε σχέση με τους ασθενείς που δεν έφερες αντίστοιχες αλλαγές. Συμπεράσματα: Η TDP-43 πρωτεϊνοπάθεια είναι νόσος με σημαντική κλινική και γενετική ετερογένεια. Εκφράζεται κυρίως στο προγεροντικό φάσμα ηλικιών και στον Ελληνικό πληθυσμό, εντούτοις γεροντική εμφάνιση αναγνωρίζεται ολοένα και περισσότερο. Τα υψηλά επίπεδα της TDP-43 στο ΕΝΥ σε συνδυασμό με τις πρωτεΐνες T (tau) με τον τύπο TDP-43×τT/τP-181 έχουν υψηλή ευαισθησία και ειδικότητα και ως εκ τούτου αποτελούν έναν εν δυνάμει βιοδείκτη της TDP-43 πρωτεϊνοπάθειας. Το πιο συχνό γονίδιο που συνδέεται με TDP-43 πρωτεϊνοπάθεια στον Ελληνικό πληθυσμό είναι το C9orf72, με υψηλή συχνότητα 10,8% (14/129) σε σχέση με άλλους Ευρωπαϊκούς πληθυσμούς και το οποίο φέρει την ηλικία έναρξης της νόσου 10 χρόνια νωρίτερα σε σχέση με τις σποραδικές μορφές. Η αλλαγή p.Ile383Val στο γονίδιο της TARDBP ίσως είναι πιο συχνή στον Ελληνικό πληθυσμό σε σχέση με άλλους πληθυσμούς και σε ασθενείς του γεροντικού φάσματος ηλικιών και χρήζει περαιτέρω διερεύνησης

Novel PANK2 mutation in the first Greek compound heterozygote patient with pantothenate-kinase-associated neurodegeneration

Pantothenate-kinase-associated neurodegeneration is the most common autosomal recessive form of neurodegeneration with brain iron accumulation. Less than 100 mutations in PANK2 gene (20p13) are responsible for classic and atypical cases. We report here the first Greek case of atypical pantothenate-kinase-associated neurodegeneration, confirmed by molecular analysis that revealed two trans-acting mutations. Our findings highlight the possible role of rare variants contributing to disease risk and possibly to variable clinical phenotype

Novel PANK2 mutation in the first Greek compound heterozygote patient with pantothenate-kinase-associated neurodegeneration

Pantothenate-kinase-associated neurodegeneration is the most common autosomal recessive form of neurodegeneration with brain iron accumulation. Less than 100 mutations in PANK2 gene (20p13) are responsible for classic and atypical cases. We report here the first Greek case of atypical pantothenate-kinase-associated neurodegeneration, confirmed by molecular analysis that revealed two trans-acting mutations. Our findings highlight the possible role of rare variants contributing to disease risk and possibly to variable clinical phenotype

Cerebrospinal fluid amyloid beta and tau proteins in atypical Parkinsonism: a review

Progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy and dementia with Lewy bodies are the most common causes of atypical Parkinsonism and enter the differential diagnosis of Parkinson’s disease. multiple system atrophy, dementia with Lewy bodies and Parkinson’s disease are synucleinopathies, whereas progressive supranuclear palsy and corticobasal degeneration are tauopathies. Multiple cerebrospinal fluid markers have been applied on cohorts of patients with Parkinsonism, with the aim to develop biomarkers for these disorders. Total tau (τΤ), phosphorylated tau at threonine 181 (τP-181) and amyloid-beta with 42 amino acids (Aβ42) are considered classical biomarkers for Alzheimer’s disease. The aim of the present study is to review the literature regarding these classical cerebrospinal fluid biomarkers in cohorts with Parkinsonism, as well as present data on novel approaches regarding analysis of these proteins

Cerebrospinal Fluid Biomarker Profile in TDP-43-Related Genetic Frontotemporal Dementia

Cerebrospinal fluid (CSF) biomarkers, namely total tau, phospho-tau and amyloid beta peptides, have received much attention specifically regarding Alzheimer’s disease (AD), since they can detect the biochemical fingerprint of AD and serve as a diagnostic tool for accurate and early diagnosis during life. In the same way, biomarkers for other neurodegenerative disease pathologies are also needed. We present a case series of six patients with genetic frontotemporal dementia (FTD), with TDP-43 underlying proteinopathy, in an attempt to assess TDP-43 as a novel biomarker alone and in combination with established AD biomarkers for this specific patient group, based on the principles of personalized and precision medicine. Our results indicate that genetic TDP-43-FTD is characterized by increased CSF TPD-43 and increased TDP-43 × τΤ/τP-181 combination. Hence, TDP-43 combined with tau proteins could be a useful tool for the diagnosis of genetic FTD with TDP-43 underling histopathology, supplementing clinical, neuropsychological and imaging data

CSF Aβ42 and Aβ42/Aβ40 Ratio in Alzheimer’s Disease and Frontotemporal Dementias

Background: Alzheimer’s disease dementia (ADD) may manifest with atypical phenotypes, resembling behavioral variant frontotemporal dementia (bvFTD) and corticobasal syndrome (CBS), phenotypes which typically have an underlying frontotemporal lobar degeneration with tau proteinopathy (FTLD-tau), such as Pick’s disease, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), or FTLD with TDP-43 proteinopathy (FTLD-TDP). CSF biomarkers total and phosphorylated tau (τT and τP-181), and amyloid beta with 42 and 40 amino acids (Aβ42 and Aβ40) are biomarkers of AD pathology. The primary aim of this study was to compare the diagnostic accuracy of Aβ42 to Aβ42/Aβ40 ratio in: (a) differentiating ADD vs. frontotemporal dementias; (b) patients with AD pathology vs. non-AD pathologies; (c) compare biomarker ratios and composite markers to single CSF biomarkers in the differentiation of AD from FTD; Methods: In total, 263 subjects were included (ADD: n = 98; bvFTD: n = 49; PSP: n = 50; CBD: n = 45; controls: n = 21). CSF biomarkers were measured by commercially available ELISAs (EUROIMMUN). Multiple biomarker ratios (Aβ42/Aβ40; τT/τP-181; τT/Aβ42; τP-181/Aβ42) and composite markers (t-tau: τT/(Aβ42/Aβ40); p-tau: τP-181/(Aβ42/Aβ40) were calculated. ROC curve analysis was performed to compare AUCs of Aβ42 and Aβ42/Aβ40 ratio and relevant composite markers between ADD and FTD, as defined clinically. BIOMARKAPD/ABSI criteria (abnormal τT, τP-181 Aβ42, and Aβ42/Aβ40 ratio) were used to re-classify all patients into AD pathology vs. non-AD pathologies, and ROC curve analysis was repeated to compare Aβ42 and Aβ42/Aβ40; Results: Aβ42 did not differ from Aβ42/Aβ40 ratio in the differentiation of ADD from FTD (AUCs 0.752 and 0.788 respectively; p = 0.212). The τT/Aβ42 ratio provided maximal discrimination between ADD and FTD (AUC:0.893; sensitivity 88.8%, specificity 80%). BIOMARKAPD/ABSI criteria classified 60 patients as having AD pathology and 211 as non-AD. A total of 22 had discrepant results and were excluded. Aβ42/Aβ40 ratio was superior to Aβ42 in the differentiation of AD pathology from non-AD pathology (AUCs: 0.939 and 0.831, respectively; p 42/Aβ40 ratio is superior to Aβ42 in identifying AD pathology, irrespective of the clinical phenotype. CSF biomarker ratios and composite markers provide higher diagnostic accuracy compared to single CSF biomarkers

Thyroid Autoantibodies in the Cerebrospinal Fluid of Subjects with and without Thyroid Disease: Implications for Hashimoto’s Encephalopathy

Introduction. Plasma antithyroid peroxidase (anti-TPO) and anti-thyroglobulin antibodies (anti-Tg) are widely used in the diagnosis of autoimmune thyroiditis. No research has compared anti-TPO and anti-Tg both in plasma and cerebrospinal fluid (CSF) of healthy individuals vis-à-vis patients with thyroid disease. Methods. We measured anti-TPO and anti-Tg antibodies in plasma and CSF in nine subjects (mean age ± SD: 73 ± 6 years) with hypothyroidism and nine subjects (mean age ± SD: 73 ± 8 years) without thyroid disease. Results. The concentration of anti-TPO autoantibodies in CSF was very low compared to plasma in both subjects with thyroid and without thyroid disease (P=0.007). CSF anti-Tg autoantibodies titers were very low compared to the plasma in subjects with thyroid disease (P=0.004), whereas, in subjects without thyroid disease, this difference did not reach statistical significance (P=0.063). Conclusions. Thyroid autoantibodies levels were low in plasma and CSF; we did not observe any transfer of thyroid autoantibodies from the peripheral blood to the CSF. Therefore, regarding Hashimoto’s encephalopathy, where elevated antithyroid autoantibodies are often measured in blood, it is more likely that thyroiditis and encephalopathy represent nonspecific, but distinct, events of an aggressive immune system