Guiding the design of SARS-CoV-2 genomic surveillance by estimating the resolution of outbreak detection.

Genomic surveillance of SARS-CoV-2 has been essential to inform public health response to outbreaks. The high incidence of infection has resulted in a smaller proportion of cases undergoing whole genome sequencing due to finite resources. We present a framework for estimating the impact of reduced depths of genomic surveillance on the resolution of outbreaks, based on a clustering approach using pairwise genetic and temporal distances. We apply the framework to simulated outbreak data to show that outbreaks are detected less frequently when fewer cases are subjected to whole genome sequencing. The impact of sequencing fewer cases depends on the size of the outbreaks, and on the genetic and temporal similarity of the index cases of the outbreaks. We also apply the framework to an outbreak of the SARS-CoV-2 Delta variant in New South Wales, Australia. We find that the detection of clusters in the outbreak would have been delayed if fewer cases had been sequenced. Existing recommendations for genomic surveillance estimate the minimum number of cases to sequence in order to detect and monitor new virus variants, assuming representative sampling of cases. Our method instead measures the resolution of clustering, which is important for genomic epidemiology, and accommodates sampling biases.

Co-infection with SARS-CoV-2 Omicron and Delta variants revealed by genomic surveillance.

Co-infections with different variants of SARS-CoV-2 are a key precursor to recombination events that are likely to drive SARS-CoV-2 evolution. Rapid identification of such co-infections is required to determine their frequency in the community, particularly in populations at-risk of severe COVID-19, which have already been identified as incubators for punctuated evolutionary events. However, limited data and tools are currently available to detect and characterise the SARS-CoV-2 co-infections associated with recognised variants of concern. Here we describe co-infection with the SARS-CoV-2 variants of concern Omicron and Delta in two epidemiologically unrelated adult patients with chronic kidney disease requiring maintenance haemodialysis. Both variants were co-circulating in the community at the time of detection. Genomic surveillance based on amplicon- and probe-based sequencing using short- and long-read technologies identified and quantified subpopulations of Delta and Omicron viruses in respiratory samples. These findings highlight the importance of integrated genomic surveillance in vulnerable populations and provide diagnostic pathways to recognise SARS-CoV-2 co-infection using genomic data.

Biallelic AOPEP Loss-of-Function Variants Cause Progressive Dystonia with Prominent Limb Involvement.

BACKGROUND: Monogenic causes of isolated dystonia are heterogeneous. Assembling cohorts of affected individuals sufficiently large to establish new gene-disease relationships can be challenging. OBJECTIVE: We sought to expand the catalogue of monogenic etiologies for isolated dystonia. METHODS: After the discovery of a candidate variant in a multicenter exome-sequenced cohort of affected individuals with dystonia, we queried online platforms and genomic data repositories worldwide to identify subjects with matching genotypic profiles. RESULTS: Seven different biallelic loss-of-function variants in AOPEP were detected in five probands from four unrelated families with strongly overlapping phenotypes. In one proband, we observed a homozygous nonsense variant (c.1477C>T [p.Arg493*]). A second proband harbored compound heterozygous nonsense variants (c.763C>T [p.Arg255*]; c.777G>A [p.Trp259*]), whereas a third proband possessed a frameshift variant (c.696_697delAG [p.Ala234Serfs*5]) in trans with a splice-disrupting alteration (c.2041-1G>A). Two probands (siblings) from a fourth family shared compound heterozygous frameshift alleles (c.1215delT [p.Val406Cysfs*14]; c.1744delA [p.Met582Cysfs*6]). All variants were rare and expected to result in truncated proteins devoid of functionally important amino acid sequence. AOPEP, widely expressed in developing and adult human brain, encodes a zinc-dependent aminopeptidase, a member of a class of proteolytic enzymes implicated in synaptogenesis and neural maintenance. The probands presented with disabling progressive dystonia predominantly affecting upper and lower extremities, with variable involvement of craniocervical muscles. Dystonia was unaccompanied by any additional symptoms in three families, whereas the fourth family presented co-occurring late-onset parkinsonism. CONCLUSIONS: Our findings suggest a likely causative role of predicted inactivating biallelic AOPEP variants in cases of autosomal recessive dystonia. Additional studies are warranted to understand the pathophysiology associated with loss-of-function variation in AOPEP. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Facioscapulohumeral muscular dystrophy type 2: an update on the clinical, genetic, and molecular findings.

Facioscapulohumeral muscular dystrophy (FSHD) is a common genetic disease of the skeletal muscle with a characteristic pattern of weakness. Facioscapulohumeral muscular dystrophy type 2 (FSHD2) accounts for approximately 5% of all cases of FSHD and describes patients without a D4Z4 repeat contraction on chromosome 4. Phenotypically FSHD2 shows virtually no difference from FSHD1 and both forms of FSHD arise via a common downstream mechanism of epigenetic derepression of the transcription factor DUX4 in skeletal muscle cells. This results in expression of DUX4 and target genes leading to skeletal muscle toxicity. Over the past decade, major progress has been made in our understanding of the genetic and epigenetic architecture that underlies FSHD2 pathogenesis, as well as the clinical manifestations and disease progression. These include the finding that FSHD2 is a digenic disease and that mutations in the genes SMCHD1, DNMT3B, and more recently LRIF1, can cause FSHD2. FSHD2 is complex and it is important that clinicians keep abreast of recent developments; this review aims to serve as an update of the clinical, genetic, and molecular research into this condition.

Diagnostic Yield of Whole Genome Sequencing After Nondiagnostic Exome Sequencing or Gene Panel in Developmental and Epileptic Encephalopathies.

OBJECTIVE: To assess the benefits and limitations of whole genome sequencing (WGS) compared to exome sequencing (ES) or multigene panel (MGP) in the molecular diagnosis of developmental and epileptic encephalopathies (DEE). METHODS: We performed WGS of 30 comprehensively phenotyped DEE patient trios that were undiagnosed after first-tier testing, including chromosomal microarray and either research ES (n = 15) or diagnostic MGP (n = 15). RESULTS: Eight diagnoses were made in the 15 individuals who received prior ES (53%): 3 individuals had complex structural variants; 5 had ES-detectable variants, which now had additional evidence for pathogenicity. Eleven diagnoses were made in the 15 MGP-negative individuals (68%); the majority (n = 10) involved genes not included in the panel, particularly in individuals with postneonatal onset of seizures and those with more complex presentations including movement disorders, dysmorphic features, or multiorgan involvement. A total of 42% of diagnoses were autosomal recessive or X-chromosome linked. CONCLUSION: WGS was able to improve diagnostic yield over ES primarily through the detection of complex structural variants (n = 3). The higher diagnostic yield was otherwise better attributed to the power of re-analysis rather than inherent advantages of the WGS platform. Additional research is required to assist in the assessment of pathogenicity of novel noncoding and complex structural variants and further improve diagnostic yield for patients with DEE and other neurogenetic disorders.

The Lifeact-EGFP mouse is a translationally controlled fluorescent reporter of T cell activation.

It has become increasingly evident that T cell functions are subject to translational control in addition to transcriptional regulation. Here, by using live imaging of CD8+ T cells isolated from the Lifeact-EGFP mouse, we show that T cells exhibit a gain in fluorescence intensity following engagement of cognate tumour target cells. The GFP signal increase is governed by Erk1/2-dependent distal T cell receptor (TCR) signalling and its magnitude correlates with IFN-γ and TNF-α production, which are hallmarks of T cell activation. Enhanced fluorescence was due to increased translation of Lifeact-EGFP protein, without an associated increase in its mRNA. Activation-induced gains in fluorescence were also observed in naïve and CD4+ T cells from the Lifeact-EGFP reporter, and were readily detected by both flow cytometry and live cell microscopy. This unique, translationally controlled reporter of effector T cell activation simultaneously enables tracking of cell morphology, F-actin dynamics and activation state in individual migrating T cells. It is a valuable addition to the limited number of reporters of T cell dynamics and activation, and opens the door to studies of translational activity and heterogeneities in functional T cell responses in situ.

Linkage analysis and whole exome sequencing reveals AHNAK2 as a novel genetic cause for autosomal recessive CMT in a Malaysian family.

Charcot-Marie-Tooth (CMT) disease is a form of inherited peripheral neuropathy that affects motor and sensory neurons. To identify the causative gene in a consanguineous family with autosomal recessive CMT (AR-CMT), we employed a combination of linkage analysis and whole exome sequencing. After excluding known AR-CMT genes, genome-wide linkage analysis mapped the disease locus to a 7.48-Mb interval on chromosome 14q32.11-q32.33, flanked by the markers rs2124843 and rs4983409. Whole exome sequencing identified two non-synonymous variants (p.T40P and p.H915Y) in the AHNAK2 gene that segregated with the disease in the family. Pathogenic predictions indicated that p.T40P is the likely causative allele. Analysis of AHNAK2 expression in the AR-CMT patient fibroblasts showed significantly reduced mRNA and protein levels. AHNAK2 binds directly to periaxin which is encoded by the PRX gene, and PRX mutations are associated with another form of AR-CMT (CMT4F). The altered expression of mutant AHNAK2 may disrupt the AHNAK2-PRX interaction in which one of its known functions is to regulate myelination.

De Novo Variants Disrupting the HX Repeat Motif of ATN1 Cause a Recognizable Non-Progressive Neurocognitive Syndrome.

Polyglutamine expansions in the transcriptional co-repressor Atrophin-1, encoded by ATN1, cause the neurodegenerative condition dentatorubral-pallidoluysian atrophy (DRPLA) via a proposed novel toxic gain of function. We present detailed phenotypic information on eight unrelated individuals who have de novo missense and insertion variants within a conserved 16-amino-acid "HX repeat" motif of ATN1. Each of the affected individuals has severe cognitive impairment and hypotonia, a recognizable facial gestalt, and variable congenital anomalies. However, they lack the progressive symptoms typical of DRPLA neurodegeneration. To distinguish this subset of affected individuals from the DRPLA diagnosis, we suggest using the term CHEDDA (congenital hypotonia, epilepsy, developmental delay, digit abnormalities) to classify the condition. CHEDDA-related variants alter the particular structural features of the HX repeat motif, suggesting that CHEDDA results from perturbation of the structural and functional integrity of the HX repeat. We found several non-homologous human genes containing similar motifs of eight to 10 HX repeat sequences, including RERE, where disruptive variants in this motif have also been linked to a separate condition that causes neurocognitive and congenital anomalies. These findings suggest that perturbation of the HX motif might explain other Mendelian human conditions.

Genome sequencing as a first-line genetic test in familial dilated cardiomyopathy.

PURPOSE: We evaluated genome sequencing (GS) as an alternative to multigene panel sequencing (PS) for genetic testing in dilated cardiomyopathy (DCM). METHODS: Forty-two patients with familial DCM underwent PS and GS, and detection rates of rare single-nucleotide variants and small insertions/deletions in panel genes were compared. Loss-of-function variants in 406 cardiac-enriched genes were evaluated, and an assessment of structural variation was performed. RESULTS: GS provided broader and more uniform coverage than PS, with high concordance for rare variant detection in panel genes. GS identified all PS-identified pathogenic or likely pathogenic variants as well as two additional likely pathogenic variants: one was missed by PS due to low coverage, the other was a known disease-causing variant in a gene not included on the panel. No loss-of-function variants in the extended gene set met clinical criteria for pathogenicity. One BAG3 structural variant was classified as pathogenic. CONCLUSION: Our data support the use of GS for genetic testing in DCM, with high variant detection accuracy and a capacity to identify structural variants. GS provides an opportunity to go beyond suites of established disease genes, but the incremental yield of clinically actionable variants is limited by a paucity of genetic and functional evidence for DCM association.

A 1.35 Mb DNA fragment is inserted into the DHMN1 locus on chromosome 7q34-q36.2.

Distal hereditary motor neuropathies predominantly affect the motor neurons of the peripheral nervous system leading to chronic disability. Using whole genome sequencing (WGS) we have identified a novel structural variation (SV) within the distal hereditary motor neuropathy locus on chromosome 7q34-q36.2 (DHMN1). The SV involves the insertion of a 1.35 Mb DNA fragment into the DHMN1 disease locus. The source of the inserted sequence is 2.3 Mb distal to the disease locus at chromosome 7q36.3. The insertion involves the duplication of five genes (LOC389602, RNF32, LMBR1, NOM1, MNX1) and partial duplication of UBE3C. The genomic structure of genes within the DHMN1 locus are not disrupted by the insertion and no disease causing point mutations within the locus were identified. This suggests the novel SV is the most likely DNA mutation disrupting the DHMN1 locus. Due to the size and position of the DNA insertion, the gene(s) directly affected by the genomic re-arrangement remains elusive. Our finding represents a new genetic cause for hereditary motor neuropathies and highlights the growing importance of interrogating the non-coding genome for SV mutations in families which have been excluded for genome wide coding mutations.

Whole Genome Sequencing Identifies a 78 kb Insertion from Chromosome 8 as the Cause of Charcot-Marie-Tooth Neuropathy CMTX3.

With the advent of whole exome sequencing, cases where no pathogenic coding mutations can be found are increasingly being observed in many diseases. In two large, distantly-related families that mapped to the Charcot-Marie-Tooth neuropathy CMTX3 locus at chromosome Xq26.3-q27.3, all coding mutations were excluded. Using whole genome sequencing we found a large DNA interchromosomal insertion within the CMTX3 locus. The 78 kb insertion originates from chromosome 8q24.3, segregates fully with the disease in the two families, and is absent from the general population as well as 627 neurologically normal chromosomes from in-house controls. Large insertions into chromosome Xq27.1 are known to cause a range of diseases and this is the first neuropathy phenotype caused by an interchromosomal insertion at this locus. The CMTX3 insertion represents an understudied pathogenic structural variation mechanism for inherited peripheral neuropathies. Our finding highlights the importance of considering all structural variation types when studying unsolved inherited peripheral neuropathy cases with no pathogenic coding mutations.

Pathogenic mechanisms underlying X-linked Charcot-Marie-Tooth neuropathy (CMTX6) in patients with a pyruvate dehydrogenase kinase 3 mutation.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. An X-linked form of CMT (CMTX6) is caused by a missense mutation (R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. PDK3 is one of 4 isoenzymes that negatively regulate the activity of the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation of its first catalytic component pyruvate dehydrogenase (designated as E1). Mitochondrial PDC catalyses the oxidative decarboxylation of pyruvate to acetyl CoA and links glycolysis to the energy-producing Krebs cycle. We have previously shown the R158H mutation confers PDK3 enzyme hyperactivity. In this study we demonstrate that the increased PDK3 activity in patient fibroblasts (PDK3(R158H)) leads to the attenuation of PDC through hyper-phosphorylation of E1 at selected serine residues. This hyper-phosphorylation can be reversed by treating the PDK3(R158H) fibroblasts with the PDK inhibitor dichloroacetate (DCA). In the patient cells, down-regulation of PDC leads to increased lactate, decreased ATP and alteration of the mitochondrial network. Our findings highlight the potential to develop specific drug targeting of the mutant PDK3 as a therapeutic approach to treating CMTX6.

MORC2 mutations cause axonal Charcot-Marie-Tooth disease with pyramidal signs.

OBJECTIVE: To use linkage analysis and whole exome sequencing to identify the genetic mutation in a multigenerational Australian family with Charcot-Marie-Tooth disease type 2 (CMT2) and pyramidal signs. METHODS: Genome-wide linkage analysis was performed to map the locus. Whole exome sequencing was undertaken on selected individuals (3 affected, 1 normal), and segregation analysis and mutation screening were carried out using high-resolution melt analysis. The GEM.app database was queried to identify additional families with mutations. RESULTS: Significant linkage (2-point LOD score ≥ +3) and haplotype analysis mapped a new locus for CMT2 and pyramidal signs to a 6.6Mb interval on chromosome 22q12.1-q12.3. Whole exome sequencing identified a novel mutation (p.R252W) in the microrchidia CW-type zinc finger 2 (MORC2) gene mapping within the linkage region. The mutation fully segregated with the disease phenotype in the family. Screening additional families and querying unsolved CMT2 exomes, we identified the p.R252W mutation in 2 unrelated early onset CMT2 families and a second mutation p.E236G in 2 unrelated CMT2 families. Both the mutations occurred at highly conserved amino acid residues and were absent in the normal population. INTERPRETATION: We have identified a new locus in which MORC2 mutations are the likely pathogenic cause of CMT2 and pyramidal signs in these families. MORC2 encodes the human CW-type zinc finger 2 protein, which is a chromatin modifier involved in the regulation of DNA repair as well as gene transcription.

Improved inherited peripheral neuropathy genetic diagnosis by whole-exome sequencing.

Inherited peripheral neuropathies (IPNs) are a group of related diseases primarily affecting the peripheral motor and sensory neurons. They include the hereditary sensory neuropathies (HSN), hereditary motor neuropathies (HMN), and Charcot-Marie-Tooth disease (CMT). Using whole-exome sequencing (WES) to achieve a genetic diagnosis is particularly suited to IPNs, where over 80 genes are involved with weak genotype-phenotype correlations beyond the most common genes. We performed WES for 110 index patients with IPN where the genetic cause was undetermined after previous screening for mutations in common genes selected by phenotype and mode of inheritance. We identified 41 missense sequence variants in the known IPN genes in our cohort of 110 index patients. Nine variants (8%), identified in the genes MFN2, GJB1, BSCL2, and SETX, are previously reported mutations and considered to be pathogenic in these families. Twelve novel variants (11%) in the genes NEFL, TRPV4, KIF1B, BICD2, and SETX are implicated in the disease but require further evidence of pathogenicity. The remaining 20 variants were confirmed as polymorphisms (not causing the disease) and are detailed here to help interpret sequence variants identified in other family studies. Validation using segregation, normal controls, and bioinformatics tools was valuable as supporting evidence for sequence variants implicated in disease. In addition, we identified one SETX sequence variant (c.7640T>C), previously reported as a putative mutation, which we have confirmed as a nonpathogenic rare polymorphism. This study highlights the advantage of using WES for genetic diagnosis in highly heterogeneous diseases such as IPNs and has been particularly powerful in this cohort where genetic diagnosis could not be achieved due to phenotype and mode of inheritance not being previously obvious. However, first tier testing for common genes in clinically well-defined cases remains important and will account for most positive results.

Analysis of dynein intermediate chains, light intermediate chains and light chains in a cohort of hereditary peripheral neuropathies.

The cytoplasmic dynein heavy chain (DYNC1H1) gene has been increasingly associated with neurodegenerative disorders including axonal Charcot-Marie-Tooth disease (CMT2), intellectual disability and malformations of cortical development. In addition, evidence from mouse models (Loa, catabolite repressor-activator (Cra) and Sprawling (Swl)) has shown that mutations in Dync1h1 cause a range of neurodegenerative phenotypes with motor and sensory neuron involvement. In this current study, we examined the possible contribution of other cytoplasmic dynein subunits that bind to DYNC1H1 as a cause of inherited peripheral neuropathy. We focused on screening the cytoplasmic dynein intermediate, light intermediate and light chain genes in a cohort of families with inherited peripheral neuropathies. Nine genes were screened and ten variants were detected, but none was identified as pathogenic, indicating that cytoplasmic dynein intermediate, light intermediate and light chains are not a cause of neuropathy in our cohort.

A new locus for X-linked dominant Charcot-Marie-Tooth disease (CMTX6) is caused by mutations in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene.

Hereditary motor and sensory disorders of the peripheral nerve form one of the most common groups of human genetic diseases collectively called Charcot-Marie-Tooth (CMT) neuropathy. Using linkage analysis in a three generation kindred, we have mapped a new locus for X-linked dominant CMT to chromosome Xp22.11. A microsatellite scan of the X chromosome established significant linkage to several markers including DXS993 (Zmax = 3.16; θ = 0.05). Extended haplotype analysis refined the linkage region to a 1.43-Mb interval flanked by markers DXS7110 and DXS8027. Whole exome sequencing identified a missense mutation c.G473A (p.R158H) in the pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) gene. The change localized within the 1.43-Mb linkage interval, segregated with the affected phenotype and was excluded in ethnically matched control chromosomes. PDK3 is one of the four isoenzymes regulating the pyruvate dehydrogenase complex (PDC), by reversible phosphorylation, and is a nuclear-coded protein located in the mitochondrial matrix. PDC catalyzes the oxidative decarboxylation of pyruvate to acetyl CoA and is a key enzyme linking glycolysis to the energy-producing Krebs cycle and lipogenic pathways. We found that the R158H mutation confers enzyme hyperactivity and binds with stronger affinity than the wild-type to the inner-lipoyl (L2) domain of the E2p chain of PDC. Our findings suggest a reduced pyruvate flux due to R158H mutant PDK3-mediated hyper-phosphorylation of the PDC as the underlying pathogenic cause of peripheral neuropathy. The results highlight an important causative link between peripheral nerve degeneration and an essential bioenergetic or biosynthetic pathway required for the maintenance of peripheral nerves.