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.

A Homozygous NDUFS6 Variant Associated with Neuropathy and Optic Atrophy.

Background: The NADH dehydrogenase [ubiquinone] iron-sulfur protein 6 (NDUFS6) gene encodes for an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I). Bi-allelic NDUFS6 variants have been linked with a severe disorder mostly reported as a lethal infantile mitochondrial disease (LMID) or Leigh syndrome (LS). Objective: Here, we identified a homozygous variant (c.309 + 5 G > A) in NDUFS6 in one male patient with axonal neuropathy accompanied by loss of small fibers in skin biopsy and further complicated by optic atrophy and borderline intellectual disability. Methods: To address the pathogenicity of the variant, biochemical studies (mtDNA copy number quantification, ELISA, Proteomic profiling) of patient-derived leukocytes were performed. Results: The analyses revealed loss of NDUFS6 protein associated with a decrease of three further mitochondrial NADH dehydrogenase subunit/assembly proteins (NDUFA12, NDUFS4 and NDUFV1). Mitochondrial copy number is not altered in leukocytes and the mitochondrial biomarker GDF15 is not significantly changed in serum. Conclusions: Hence, our combined clinical and biochemical data strengthen the concept of NDUFS6 being causative for a very rare form of axonal neuropathy associated with optic atrophy and borderline intellectual disability, and thus expand (i) the molecular genetic landscape of neuropathies and (ii) the clinical spectrum of NDUFS6-associated phenotypes.

Impact of cfDNA Reference Materials on Clinical Performance of Liquid Biopsy NGS Assays.

BACKGROUND: Liquid biopsy enables the non-invasive analysis of genetic tumor variants in circulating free DNA (cfDNA) in plasma. Accurate analytical validation of liquid biopsy NGS assays is required to detect variants with low variant allele frequencies (VAFs). METHODS: Six types of commercial cfDNA reference materials and 42 patient samples were analyzed using a duplex-sequencing-based liquid biopsy NGS assay. RESULTS: We comprehensively evaluated the similarity of commercial cfDNA reference materials to native cfDNA. We observed significant differences between the reference materials in terms of wet-lab and sequencing quality as well as background noise. No reference material resembled native cfDNA in all performance metrics investigated. Based on our results, we established guidelines for the selection of appropriate reference materials for the different steps in performance evaluation. The use of inappropriate materials and cutoffs could eventually lead to a lower sensitivity for variant detection. CONCLUSION: Careful consideration of commercial reference materials is required for performance evaluation of liquid biopsy NGS assays. While the similarity to native cfDNA aids in the development of experimental protocols, reference materials with well-defined variants are preferable for determining sensitivity and precision, which are essential for accurate clinical interpretation.

Diagnostic yield and clinical relevance of expanded germline genetic testing for nearly 7000 suspected HBOC patients.

Here we report the results of a retrospective germline analysis of 6941 individuals fulfilling the criteria necessary for genetic testing of hereditary breast- and ovarian cancer (HBOC) according to the German S3 or AGO Guidelines. Genetic testing was performed by next-generation sequencing using 123 cancer-associated genes based on the Illumina TruSight® Cancer Sequencing Panel. In 1431 of 6941 cases (20.6%) at least one variant was reported (ACMG/AMP classes 3-5). Of those 56.3% (n = 806) were class 4 or 5 and 43.7% (n = 625) were a class 3 (VUS). We defined a 14 gene HBOC core gene panel and compared this to a national and different internationally recommended gene panels (German Hereditary Breast and Ovarian Cancer Consortium HBOC Consortium, ClinGen expert Panel, Genomics England PanelsApp) in regard of diagnostic yield, revealing a diagnostic range of pathogenic variants (class 4/5) from 7.8 to 11.6% depending on the panel evaluated. With the 14 HBOC core gene panel having a diagnostic yield of pathogenic variants (class 4/5) of 10.8%. Additionally, 66 (1%) pathogenic variants (ACMG/AMP class 4 or 5) were found in genes outside the 14 HBOC core gene set (secondary findings) that would have been missed with the restriction to the analysis of HBOC genes. Furthermore, we evaluated a workflow for a periodic re-evaluation of variants of uncertain clinical significance (VUS) for the improvement of clinical validity of germline genetic testing.

Long-term chemoprevention in patients with adenomatous polyposis coli: an observational study.

Prospective short-term studies on effectiveness of non-steroidal anti-inflammatory drugs (NSAIDs) point towards a decrease in the number and size of polyps. Effectiveness and safety in the prevention of progression in familial polyposis with NSAIDs in long-term use, which is the prerequisite for therapeutic evaluation in prospective studies, is unknown. The total absolute observation period of 54 patients under sulindac was 399 patient years with a mean of 7.4 (2-19) years per patient. 36 patients (66.7%) showed a fast decrease of polyp burden, 8 (14.8%) were slow responders, and 9 (16.7%) had stable disease; one patient had a slow progression. Upper gastrointestinal (GI) polyp burden remained stable in 47% patients, increased in 31%, and improved in 22%. Advanced adenomas were found in 8 patients only within the first 5 years of chemoprevention, no patient developed desmoid disease, anamnestically evaluated on every follow-up. There were no life-threatening side-effects. Dosage and delivery pattern were essential for effectiveness. This study provides evidence that chemoprevention with sulindac is effective and safe and can, either alone or in combination with other drugs, become a long-term management option in cases of adenomatous polyposis. These results justify further long-term prospective chemoprevention studies to elaborate treatment protocols and guidelines.

Highly sensitive liquid biopsy Duplex sequencing complements tissue biopsy to enhance detection of clinically relevant genetic variants.

Background: Liquid biopsy (LB) is a promising complement to tissue biopsy for detection of clinically relevant genetic variants in cancer and mosaic diseases. A combined workflow to enable parallel tissue and LB analysis is required to maximize diagnostic yield for patients. Methods: We developed and validated a cost-efficient combined next-generation sequencing (NGS) workflow for both tissue and LB samples, and applied Duplex sequencing technology for highly accurate detection of low frequency variants in plasma. Clinically relevant cutoffs for variant reporting and quantification were established. Results: We investigated assay performance characteristics for very low amounts of clinically relevant variants. In plasma, the assay achieved 100% sensitivity and 92.3% positive predictive value (PPV) for single nucleotide variants (SNVs) and 91.7% sensitivity and 100% PPV for insertions and deletions (InDel) in clinically relevant hotspots with 0.5-5% variant allele frequencies (VAFs). We further established a cutoff for reporting variants (i.e. Limit of Blank, LOB) at 0.25% VAF and a cutoff for quantification (i.e. Limit of Quantification, LOQ) at 5% VAF in plasma for accurate clinical interpretation of analysis results. With our LB approach, we were able to identify the molecular cause of a clinically confirmed asymmetric overgrowth syndrome in a 10-year old child that would have remained undetected with tissue analysis as well as other molecular diagnostic approaches. Conclusion: Our flexible and cost-efficient workflow allows analysis of both tissue and LB samples and provides clinically relevant cutoffs for variant reporting and precise quantification. Complementing tissue analysis by LB is likely to increase diagnostic yield for patients with molecular diseases.

Autosomal dominant frontometaphyseal dysplasia: Delineation of the clinical phenotype.

Frontometaphyseal dysplasia (FMD) is caused by gain-of-function mutations in the X-linked gene FLNA in approximately 50% of patients. Recently we characterized an autosomal dominant form of FMD (AD-FMD) caused by mutations in MAP3K7, which accounts for the condition in the majority of patients who lack a FLNA mutation. We previously also described a patient with a de novo variant in TAB2, which we hypothesized was causative of another form of AD-FMD. In this study, a cohort of 20 individuals with AD-FMD is clinically evaluated. This cohort consists of 15 individuals with the recently described, recurrent mutation (c.1454C>T) in MAP3K7, as well as three individuals with missense mutations that result in substitutions in the N-terminal kinase domain of TGFß-activated kinase 1 (TAK1), encoded by MAP3K7. Additionally, two individuals have missense variants in the gene TAB2, which encodes a protein with a close functional relationship to TAK1, TAK1-associated binding protein 2 (TAB2). Although the X-linked and autosomal dominant forms of FMD are very similar, there are distinctions to be made between the two conditions. Individuals with AD-FMD have characteristic facial features, and are more likely to be deaf, have scoliosis and cervical fusions, and have a cleft palate. Furthermore, there are features only found in AD-FMD in our review of the literature including valgus deformity of the feet and predisposition to keloid scarring. Finally, intellectual disability is present in a small number of subjects with AD-FMD but has not been described in association with X-linked FMD.

Recurrent de novo BICD2 mutation associated with arthrogryposis multiplex congenita and bilateral perisylvian polymicrogyria.

Autosomal dominantly inherited mutations of BICD2 are associated with congenital-onset spinal muscular atrophy characterised by lower limb predominance. A few cases have also showed upper motor neuron pathology, including presenting with features resembling hereditary spastic paraplegia. The age-of-onset for the published families is usually at birth but also included cases with childhood- and adult-onset disease. In this report we described two isolated probands that presented in utero with features associated with reduced fetal movements. Both cases were diagnosed at birth with arthrogryposis multiplex congenita (AMC) and hypotonia. Other variable features included congenital fractures, hip dislocation, micrognathia, respiratory insufficiency, microcephaly and bilateral perisylvian polymicrogyria. Patient 1 is 4 years of age and stable, but shows significant motor developmental delay and delayed speech. Patient 2 passed away at 7 weeks of age. Through next generation sequencing we identified the same missense substitution in BICD2 (p.Arg694Cys) in both probands. Sanger sequencing showed that in both cases the mutation arose de novo. The in utero onset in both cases suggests that the p.Arg694Cys substitution may have a more deleterious effect on BICD2 function than previously described mutations. Our results broaden the phenotypes associated with BICD2 mutations to include AMC and cortical malformations and therefore to a similar phenotypic spectrum to that associated with its binding partner DYNC1H1.

Mutations in MAP3K7 that Alter the Activity of the TAK1 Signaling Complex Cause Frontometaphyseal Dysplasia.

Frontometaphyseal dysplasia (FMD) is a progressive sclerosing skeletal dysplasia affecting the long bones and skull. The cause of FMD in some individuals is gain-of-function mutations in FLNA, although how these mutations result in a hyperostotic phenotype remains unknown. Approximately one half of individuals with FMD have no identified mutation in FLNA and are phenotypically very similar to individuals with FLNA mutations, except for an increased tendency to form keloid scars. Using whole-exome sequencing and targeted Sanger sequencing in 19 FMD-affected individuals with no identifiable FLNA mutation, we identified mutations in two genes-MAP3K7, encoding transforming growth factor ß (TGF-ß)-activated kinase (TAK1), and TAB2, encoding TAK1-associated binding protein 2 (TAB2). Four mutations were found in MAP3K7, including one highly recurrent (n = 15) de novo mutation (c.1454C>T [ p.Pro485Leu]) proximal to the coiled-coil domain of TAK1 and three missense mutations affecting the kinase domain (c.208G>C [p.Glu70Gln], c.299T>A [p.Val100Glu], and c.502G>C [p.Gly168Arg]). Notably, the subjects with the latter three mutations had a milder FMD phenotype. An additional de novo mutation was found in TAB2 (c.1705G>A, p.Glu569Lys). The recurrent mutation does not destabilize TAK1, or impair its ability to homodimerize or bind TAB2, but it does increase TAK1 autophosphorylation and alter the activity of more than one signaling pathway regulated by the TAK1 kinase complex. These findings show that dysregulation of the TAK1 complex produces a close phenocopy of FMD caused by FLNA mutations. Furthermore, they suggest that the pathogenesis of some of the filaminopathies caused by FLNA mutations might be mediated by misregulation of signaling coordinated through the TAK1 signaling complex.

Mutations in genes encoding the cadherin receptor-ligand pair DCHS1 and FAT4 disrupt cerebral cortical development.

The regulated proliferation and differentiation of neural stem cells before the generation and migration of neurons in the cerebral cortex are central aspects of mammalian development. Periventricular neuronal heterotopia, a specific form of mislocalization of cortical neurons, can arise from neuronal progenitors that fail to negotiate aspects of these developmental processes. Here we show that mutations in genes encoding the receptor-ligand cadherin pair DCHS1 and FAT4 lead to a recessive syndrome in humans that includes periventricular neuronal heterotopia. Reducing the expression of Dchs1 or Fat4 within mouse embryonic neuroepithelium increased progenitor cell numbers and reduced their differentiation into neurons, resulting in the heterotopic accumulation of cells below the neuronal layers in the neocortex, reminiscent of the human phenotype. These effects were countered by concurrent knockdown of Yap, a transcriptional effector of the Hippo signaling pathway. These findings implicate Dchs1 and Fat4 upstream of Yap as key regulators of mammalian neurogenesis.

A novel germline KIT mutation (p.L576P) in a family presenting with juvenile onset of multiple gastrointestinal stromal tumors, skin hyperpigmentations, and esophageal stenosis.

Familial gastrointestinal stromal tumor (GIST) syndrome is a rare autosomal dominant genetic disorder. We report on a kindred in which 3 family members carry a germline mutation (c.1727T>C, p.L576P) in exon 11 of the KIT gene. This mutation was not reported so far in familial GISTs. Apart from multiple GISTs in 2 of the mutation carriers, all of them had multiple hyperpigmented skin macules and a history of achalasia-like stenosis of the esophagus in early childhood. In the index patient >100 tumors and a diffuse Cajal cell hyperplasia of the small bowel occurred. Sequencing of DNA extracted from tumor tissue of one of his GISTs revealed the KIT mutation in exon 11 (c.1727T>C). By array comparative genomic hybridization whole chromosomal gains 3, 5, 7, 9, 12, 15, and 18 were detected. In addition, we could identify a gain on chromosome 4, spanning the KIT gene. Together with the family described here, 24 unrelated cases with proven germline mutations in KIT have been reported. In these families the diagnosis was established from the age of 30 years onwards. Because in 1 patient reported here the GIST was a coincidental finding at the age of 15 years, the tumors might occur at a very young age and remain unnoticed until they-either due to increasing size, ulceration, or malignant progression-become symptomatic. Therefore, we propose to start screening patients with known KIT mutations from a younger age.

Germline truncating-mutations in BRCA1 and MSH6 in a patient with early onset endometrial cancer.

BACKGROUND: Hereditary Breast and Ovarian Cancer Syndrome (HBOCS) and Hereditary Non-Polyposis Colorectal Cancer Syndrome (HNPCC, Lynch Syndrome) are two tumor predisposition syndromes responsible for the majority of hereditary breast and colorectal cancers. Carriers of both germline mutations in breast cancer genes BRCA1 or BRCA2 and in mismatch repair (MMR) genes MLH1, MSH2, MSH6 or PMS2 are very rare. CASE PRESENTATION: We identified germline mutations in BRCA1 and in MSH6 in a patient with increased risk for HBOC diagnosed with endometrial cancer at the age of 46 years. CONCLUSIONS: Although carriers of mutations in both MMR and BRCA genes are rare in Caucasian populations and anamnestical and histopathological findings may guide clinicians to identify these families, both syndromes can only be diagnosed through a complete gene analysis of the respective genes.