Human ABL1 deficiency syndrome (HADS) is a recognizable syndrome distinct from ABL1-related congenital heart defects and skeletal malformations syndrome.

Germline gain of function variants in the oncogene ABL1 cause congenital heart defects and skeletal malformations (CHDSKM) syndrome. Whether a corresponding ABL1 deficiency disorder exists in humans remains unknown although developmental defects in mice deficient for Abl1 support this notion. Here, we describe two multiplex consanguineous families, each segregating a different homozygous likely loss of function variant in ABL1. The associated phenotype is multiple congenital malformations and distinctive facial dysmorphism that are opposite in many ways to CHDSKM. We suggest that a tight balance of ABL1 activity is required during embryonic development and that both germline gain of function and loss of function variants result in distinctively different allelic congenital malformation disorders.

Actionable secondary findings in arrhythmogenic right ventricle cardiomyopathy genes: impact and challenge of genetic counseling.

BACKGROUND: Comprehensive genetic analysis yields in a higher diagnostic rate but also in a higher number of secondary findings (SF). American College of Medical Genetics and Genomics (ACMG) published a list of 59 actionable genes for which disease causing sequence variants are recommended to be reported as SF including 27 genes linked to inherited cardiovascular disease (CVD) such as arrhythmia syndromes, cardiomyopathies and vascular and connective tissue disorders. One of the selected conditions represented in the actionable gene list is the arrhythmogenic right ventricle cardiomyopathy (ARVC), an inherited heart muscle disease with a particularly high risk of sudden cardiac death (SCD). Since clinical symptoms are frequently absent before SCD, a genetic finding is a promising option for early diagnosis and possible intervention. However, the variant interpretation and the decision to return a SF is still challenging. METHODS: To determine the frequency of medically actionable SF linked to CVD we analyzed data of 6,605 individuals who underwent high throughput sequencing for noncardiac diagnostic requests. In particular, we critically assessed and classified the variants in the ARVC genes: DSC2, DSG2, DSP, PKP2 and TMEM43 and compared our findings with the population-based genome Aggregation Database (gnomAD) and ARVC-afflicted individuals listed in ClinVar and ARVC database. RESULTS: 1% (69/6,605) of tested individuals carried pathogenic SF in one of the 27 genes linked to CVD, of them 13 individuals (0.2%) carried a pathogenic SF in a ARVC gene. Overall, 582 rare variants were identified in all five ARVC genes, 96% of the variants were missense variants and 4% putative LoF variants (pLoF): frameshift, start/stop-gain/loss, splice-site. Finally, we selected 13 of the 24 pLoF variants as pathogenic SF by careful data interpretation. CONCLUSIONS: Since SF in actionable ARVC genes can allow early detection and prevention of disease and SCD, detected variant must undergo rigorous clinical and laboratory evaluation before it can be described as pathogenic and returned to patients. Returning a SF to a patient should be interdisciplinary, it needs genetic counselling and clinicians experienced in inherited heart disease.

HPO-driven virtual gene panel: a new efficient approach in molecular autopsy of sudden unexplained death.

BACKGROUND: Molecular autopsy represents an efficient tool to save the diagnosis in up to one-third of sudden unexplained death (SUD). A defined gene panel is usually used for the examination. Alternatively, it is possible to carry out a comprehensive genetic assessment (whole exome sequencing, WES), which also identifies rare, previously unknown variants. The disadvantage is that a dramatic number of variants must be assessed to identify the causal variant. To improve the evaluation of WES, the human phenotype ontology (HPO) annotation is used internationally for deep phenotyping in the field of rare disease. However, a HPO-based evaluation of WES in SUD has not been described before. METHODS: We performed WES in tissue samples from 16 people after SUD. Instead of a fixed gene panel, we defined a set of HPO terms and thus created a flexible "virtual gene panel", with the advantage, that recently identified genes are automatically associated by HPO terms in the HPO database. RESULTS: We obtained a mean value of 68,947 variants per sample. Stringent filtering ended up in a mean value of 276 variants per sample. Using the HPO-driven virtual gene panel we developed an algorithm that prioritized 1.4% of the variants. Variant interpretation resulted in eleven potentially causative variants in 16 individuals. CONCLUSION: Our data introduce an effective diagnostic procedure in molecular autopsy of SUD with a non-specific clinical phenotype.

Location matters - Genotype-phenotype correlation in LRSAM1 mutations associated with rare Charcot-Marie-Tooth neuropathy CMT2P.

More than 80 genes are known to be associated with Charcot-Marie-Tooth disease (CMT). Mutations of LRSAM1 were identified as a rare cause and define the subgroup of axonal neuropathy CMT2P. We identified additional 14 patients out of 12 families. Clinical and electrophysiological data confirm a late-onset axonal neuropathy with a predominance of sensorimotor impairment. The patients harbored ten different variants in LRSAM1, seven of which were novel. Due to variable inheritance patterns and clustering of pathogenic variants in 3´-prime exons, interpretation of genetic variants in LRSAM1 is challenging. The majority follows dominant inheritance, whereas recessive inheritance has been described for one variant. Variants at the 3`end may or may not escape from nonsense-mediated decay, thereby defining the pattern of inheritance. Our data emphasize the importance of the C-terminal RING domain, which exerts a dominant-negative effect on protein function, whenever affected by an altered or truncated protein. In conclusion, CMT2P is a rare, but nevertheless relevant cause of adult-onset axonal and painful neuropathy. ACMG (American College of Medical Genetics and genomics) criteria should be carefully applied in variant interpretation, with special attention to premature termination codon-introducing variants and their location within the gene.

Critical assessment of secondary findings in genes linked to primary arrhythmia syndromes.

As comprehensive sequencing technologies gain widespread use, questions about so-called secondary findings (SF) require urgent consideration. The American College of Medical Genetics and Genomics has recommended to report SF in 59 genes (ACMG SF v2.0) including four actionable genes associated with inherited primary arrhythmia syndromes (IPAS) such as catecholaminergic polymorphic ventricular tachycardia, long QT syndrome, and Brugada syndrome. Databases provide conflicting results for the purpose of identifying pathogenic variants in SF associated with IPAS at a level of sufficient evidence for clinical return. As IPAS account for a significant proportion of sudden cardiac deaths (SCD) in young and apparently healthy individuals, variant interpretation has a great impact on diagnosis and prevention of disease. Of 6381 individuals, 0.4% carry pathogenic variants in one of the four actionable genes related to IPAS: RYR2, KCNQ1, KCNH2, and SCN5A. Comparison of the databases ClinVar, Leiden Open-source Variant Database, and Human Gene Mutation Database showed impactful differences (0.2% to 1.3%) in variant interpretation improvable by expert-curation depending on database and classification system used. These data further highlight the need for international consensus regarding the variant interpretation, and subsequently management of SF in particular with regard to treatable arrhythmic disorders with increased risk of SCD.

HADHA and HADHB gene associated phenotypes - Identification of rare variants in a patient cohort by Next Generation Sequencing.

The heterooctameric mitochondrial trifunctional protein (MTP), composed of four α- and ß-subunits harbours three enzymes that each perform a different function in mitochondrial fatty acid ß-oxidation. Pathogenic variants in the MTP genes (HADHA and HADHB) cause MTP deficiency, a rare autosomal recessive metabolic disorder characterized by phenotypic heterogeneity ranging from severe, early-onset, cardiac disease to milder, later-onset, myopathy and neuropathy. Since metabolic myopathies and neuropathies are a group of rare genetic disorders and their associated muscle symptoms may be subtle, the diagnosis is often delayed. Here we evaluated data of 161 patients with myopathy and 242 patients with neuropathy via next generation sequencing (NGS) and report the diagnostic yield in three patients of this cohort by the detection of disease-causing variants in the HADHA or HADHB gene. The mitigated phenotypes of this treatable disease were missed by the newborn screening, highlighting the importance of phenotype-based NGS analysis in patients with rare and clinically very variable disorders such as MTP deficiency.

Mitochondrial and nuclear disease panel (Mito-aND-Panel): Combined sequencing of mitochondrial and nuclear DNA by a cost-effective and sensitive NGS-based method.

BACKGROUND: The diagnosis of mitochondrial disorders is challenging because of the clinical variability and genetic heterogeneity of these conditions. Next-Generation Sequencing (NGS) technology offers a robust high-throughput platform for nuclear and mitochondrial DNA (mtDNA) analyses. METHOD: We developed a custom Agilent SureSelect Mitochondrial and Nuclear Disease Panel (Mito-aND-Panel) capture kit that allows parallel enrichment for subsequent NGS-based sequence analysis of nuclear mitochondrial disease-related genes and the complete mtDNA genome. Sequencing of enriched mtDNA simultaneously with nuclear genes was compared with the separated sequencing of the mitochondrial genome and whole exome sequencing (WES). RESULTS: The Mito-aND-Panel permits accurate detection of low-level mtDNA heteroplasmy due to a very high sequencing depth compared to standard diagnostic procedures using Sanger sequencing/SNaPshot and WES which is crucial to identify maternally inherited mitochondrial disorders. CONCLUSION: We established a NGS-based method with combined sequencing of the complete mtDNA and nuclear genes which enables a more sensitive heteroplasmy detection of mtDNA mutations compared to traditional methods. Because the method promotes the analysis of mtDNA variants in large cohorts, it is cost-effective and simple to setup, we anticipate this is a highly relevant method for sequence-based genetic diagnosis in clinical diagnostic applications.

The Variant p.(Arg183Trp) in SPTLC2 Causes Late-Onset Hereditary Sensory Neuropathy.

Hereditary sensory and autonomic neuropathy 1 (HSAN1) is an autosomal dominant disorder that can be caused by variants in SPTLC1 or SPTLC2, encoding subunits of serine palmitoyl-CoA transferase. Disease variants alter the enzyme's substrate specificity and lead to accumulation of neurotoxic 1-deoxysphingolipids. We describe two families with autosomal dominant HSAN1C caused by a new variant in SPTLC2, c.547C>T, p.(Arg183Trp). The variant changed a conserved amino acid and was not found in public variant databases. All patients had a relatively mild progressive distal sensory impairment, with onset after age 50. Small fibers were affected early, leading to abnormalities on quantitative sensory testing. Sural biopsy revealed a severe chronic axonal neuropathy with subtotal loss of myelinated axons, relatively preserved number of non-myelinated fibers and no signs for regeneration. Skin biopsy with PGP9.5 labeling showed lack of intraepidermal nerve endings early in the disease. Motor manifestations developed later in the disease course, but there was no evidence of autonomic involvement. Patients had elevated serum 1-deoxysphingolipids, and the variant protein produced elevated amounts of 1-deoxysphingolipids in vitro, which proved the pathogenicity of the variant. Our results expand the genetic spectrum of HSAN1C and provide further detail about the clinical characteristics. Sequencing of SPTLC2 should be considered in all patients presenting with mild late-onset sensory-predominant small or large fiber neuropathy.

Human endogenous retroviral long terminal repeat sequences as cell type-specific promoters in retroviral vectors.

The human genome contains more than half a million human endogenous retrovirus (HERV) long terminal repeats (LTRs) that can be regarded as mobile regulatory modules. Many of these HERV LTRs have been recruited during evolution as transcriptional control elements for cellular gene expression. We have cloned LTR sequences from two HERV families, HERV-H and HERV-L, differing widely in their activity and tissue specificity into a murine leukemia virus (MLV)-based promoter conversion vector (ProCon). Various human cell lines were infected with the HERV-MLV hybrid vectors, and cell type-specific expression of the reporter gene was compared with the promoter specificity of the corresponding HERV LTRs in transient-transfection assays. Transcription start site analysis of HERV-MLV hybrid vectors revealed preferential use of the HERV promoter initiation site. Our data show that HERV LTRs function in the context of retroviral vectors in certain cell types and have the potential to be useful as cell type-specific promoters in vector construction.

Low volume amplification and sequencing of mitochondrial DNA on a chemically structured chip.

Low volume (LV) amplification (1 microL) of nuclear DNA (nucDNA) on a chemically structured chip is an appropriate and highly sensitive method to simultaneously amplify amelogenin and 15 forensically relevant short tandem repeats (STR). In this study, a combined method using on-chip LV amplification of mitochondrial DNA (mtDNA) and subsequent on-chip LV cycle sequencing was established to obtain a method, which is sensitive and robust enough to allow reliable analysis of DNA amounts representing the single cell level. All the necessary steps of the procedure--except for the purification of the sequencing products--were accomplished within the same final 2-microL reaction volume.

The potential of retroviral vectors to cotransfer human endogenous retroviruses (HERVs) from human packaging cell lines.

Using a versatile and highly sensitive retroviral microarray, we have investigated particle preparations from three different human packaging cell lines harboring retroviral vector systems based on human immunodeficiency virus (HIV) and murine leukemia virus (MLV). 293Rev/Gag/Pol(i) cells inducibly express high titers of HIV-derived particles for packaging of HIV vectors. The Phoenix-GP and the Anjou 65 cell lines constitutively express MLV vector particles. We compared the transcription profiles of human endogenous retroviruses (HERVs) in all cell lines with the HERV sequences present in the particles. In addition, the influence of the transfected vector plasmid on the copackaging of HERVs was investigated. All particle preparations showed a defined pattern of endogenous retroviral sequences that differed from the cellular HERV expression pattern. HERV transcripts were observed in the particle preparations independent of whether a vector construct was coexpressed or not. Furthermore, our results suggest that particle preparations are frequently contaminated by cellular vesicles (exosomes) containing cellular RNAs including HERV transcripts.

Low-volume amplification on chemically structured chips using the PowerPlex16 DNA amplification kit.

In forensic DNA analysis, improvement of DNA typing technologies has always been an issue. It has been shown that DNA amplification in low volumes is a suitable way to enhance the sensitivity and efficiency of amplification. In this study, DNA amplification was performed on a flat, chemically structured glass slide in 1-microl reaction volumes from cell line DNA contents between 1,000 and 4 pg. On-chip DNA amplification reproducibly yielded full allelic profiles from as little as 32 pg of template DNA. Applicability on the simultaneous amplification of 15 short tandem repeats and of a segment of the Amelogenin gene, which are routinely used in forensic DNA analysis, is shown. The results are compared to conventional in-tube amplification carried out in 25-microl reaction volumes.