Next-generation phenotyping integrated in a national framework for patients with ultrarare disorders improves genetic diagnostics and yields new molecular findings.

Individuals with ultrarare disorders pose a structural challenge for healthcare systems since expert clinical knowledge is required to establish diagnoses. In TRANSLATE NAMSE, a 3-year prospective study, we evaluated a novel diagnostic concept based on multidisciplinary expertise in Germany. Here we present the systematic investigation of the phenotypic and molecular genetic data of 1,577 patients who had undergone exome sequencing and were partially analyzed with next-generation phenotyping approaches. Molecular genetic diagnoses were established in 32% of the patients totaling 370 distinct molecular genetic causes, most with prevalence below 1:50,000. During the diagnostic process, 34 novel and 23 candidate genotype-phenotype associations were identified, mainly in individuals with neurodevelopmental disorders. Sequencing data of the subcohort that consented to computer-assisted analysis of their facial images with GestaltMatcher could be prioritized more efficiently compared with approaches based solely on clinical features and molecular scores. Our study demonstrates the synergy of using next-generation sequencing and phenotyping for diagnosing ultrarare diseases in routine healthcare and discovering novel etiologies by multidisciplinary teams.

A syndromic neurodevelopmental disorder caused by rare variants in PPFIA3.

PPFIA3 encodes the protein-tyrosine phosphatase, receptor-type, F-polypeptide-interacting-protein-alpha-3 (PPFIA3), which is a member of the LAR-protein-tyrosine phosphatase-interacting-protein (liprin) family involved in synapse formation and function, synaptic vesicle transport, and presynaptic active zone assembly. The protein structure and function are evolutionarily well conserved, but human diseases related to PPFIA3 dysfunction are not yet reported in OMIM. Here, we report 20 individuals with rare PPFIA3 variants (19 heterozygous and 1 compound heterozygous) presenting with developmental delay, intellectual disability, hypotonia, dysmorphisms, microcephaly or macrocephaly, autistic features, and epilepsy with reduced penetrance. Seventeen unique PPFIA3 variants were detected in 18 families. To determine the pathogenicity of PPFIA3 variants in vivo, we generated transgenic fruit flies producing either human wild-type (WT) PPFIA3 or five missense variants using GAL4-UAS targeted gene expression systems. In the fly overexpression assays, we found that the PPFIA3 variants in the region encoding the N-terminal coiled-coil domain exhibited stronger phenotypes compared to those affecting the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 function is partially conserved in the fly. However, two of the tested variants failed to rescue the lethality at the larval stage and one variant failed to rescue lethality at the adult stage. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant-negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Hemizygous variants in protein phosphatase 1 regulatory subunit 3F (PPP1R3F) are associated with a neurodevelopmental disorder characterized by developmental delay, intellectual disability and autistic features.

Protein phosphatase 1 regulatory subunit 3F (PPP1R3F) is a member of the glycogen targeting subunits (GTSs), which belong to the large group of regulatory subunits of protein phosphatase 1 (PP1), a major eukaryotic serine/threonine protein phosphatase that regulates diverse cellular processes. Here, we describe the identification of hemizygous variants in PPP1R3F associated with a novel X-linked recessive neurodevelopmental disorder in 13 unrelated individuals. This disorder is characterized by developmental delay, mild intellectual disability, neurobehavioral issues such as autism spectrum disorder, seizures and other neurological findings including tone, gait and cerebellar abnormalities. PPP1R3F variants segregated with disease in affected hemizygous males that inherited the variants from their heterozygous carrier mothers. We show that PPP1R3F is predominantly expressed in brain astrocytes and localizes to the endoplasmic reticulum in cells. Glycogen content in PPP1R3F knockout astrocytoma cells appears to be more sensitive to fluxes in extracellular glucose levels than in wild-type cells, suggesting that PPP1R3F functions in maintaining steady brain glycogen levels under changing glucose conditions. We performed functional studies on nine of the identified variants and observed defects in PP1 binding, protein stability, subcellular localization and regulation of glycogen metabolism in most of them. Collectively, the genetic and molecular data indicate that deleterious variants in PPP1R3F are associated with a new X-linked disorder of glycogen metabolism, highlighting the critical role of GTSs in neurological development. This research expands our understanding of neurodevelopmental disorders and the role of PP1 in brain development and proper function.

Variants in FGF10 cause early onset of severe childhood interstitial lung disease: A detailed description of four affected children.

INTRODUCTION: Fibroblast growth factor 10 (FGF10) is a signaling molecule with a well-established role for lung branching morphogenesis. Rare heterozygous, deleterious variants in the FGF10 gene are known causes of the lacrimo-auriculo-dento-digital (LADD) syndrome and aplasia of lacrimal and salivary glands. Previous studies indicate that pathogenic variants in FGF10 can cause childhood Interstitial Lung Disease (chILD) due to severe diffuse developmental disorders of the lung, but detailed reports on clinical presentation and follow-up of affected children are lacking. METHODS: We describe four children with postnatal onset of chILD and heterozygous variants in FGF10, each detected by exome or whole genome sequencing. RESULTS: All children presented with postnatal respiratory failure. Two children died within the first 2 days of life, one patient died at age of 12 years due to right heart failure related to severe pulmonary hypertension (PH) and one patient is alive at age of 6 years, but still symptomatic. Histopathological analysis of lung biopsies from the two children with early postpartum demise revealed diffuse developmental disorder representing acinar dysplasia and interstitial fibrosis. Sequential biopsies of the child with survival until the age of 12 years revealed alveolar simplification and progressive interstitial fibrosis. DISCUSSION: Our report extends the phenotype of FGF10-related disorders to early onset chILD with progressive interstitial lung fibrosis and PH. Therefore, FGF10-related disorder should be considered even without previously described syndromic stigmata in children with postnatal respiratory distress, not only when leading to death in the neonatal period but also in case of persistent respiratory complaints and PH.

Synthesis of prebiotic organics from CO2 by catalysis with meteoritic and volcanic particles.

The emergence of prebiotic organics was a mandatory step toward the origin of life. The significance of the exogenous delivery versus the in-situ synthesis from atmospheric gases is still under debate. We experimentally demonstrate that iron-rich meteoritic and volcanic particles activate and catalyse the fixation of CO2, yielding the key precursors of life-building blocks. This catalysis is robust and produces selectively aldehydes, alcohols, and hydrocarbons, independent of the redox state of the environment. It is facilitated by common minerals and tolerates a broad range of the early planetary conditions (150-300 °C, ≲ 10-50 bar, wet or dry climate). We find that up to 6 × 108 kg/year of prebiotic organics could have been synthesized by this planetary-scale process from the atmospheric CO2 on Hadean Earth.

Rare variants in PPFIA3 cause delayed development, intellectual disability, autism, and epilepsy.

PPFIA3 encodes the Protein-Tyrosine Phosphatase, Receptor-Type, F Polypeptide-Interacting Protein Alpha-3 (PPFIA3), which is a member of the LAR protein-tyrosine phosphatase-interacting protein (liprin) family involved in synaptic vesicle transport and presynaptic active zone assembly. The protein structure and function are well conserved in both invertebrates and vertebrates, but human diseases related to PPFIA3 dysfunction are not yet known. Here, we report 14 individuals with rare mono-allelic PPFIA3 variants presenting with features including developmental delay, intellectual disability, hypotonia, autism, and epilepsy. To determine the pathogenicity of PPFIA3 variants in vivo , we generated transgenic fruit flies expressing either human PPFIA3 wildtype (WT) or variant protein using GAL4-UAS targeted gene expression systems. Ubiquitous expression with Actin-GAL4 showed that the PPFIA3 variants had variable penetrance of pupal lethality, eclosion defects, and anatomical leg defects. Neuronal expression with elav-GAL4 showed that the PPFIA3 variants had seizure-like behaviors, motor defects, and bouton loss at the 3 rd instar larval neuromuscular junction (NMJ). Altogether, in the fly overexpression assays, we found that the PPFIA3 variants in the N-terminal coiled coil domain exhibited stronger phenotypes compared to those in the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin- α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 protein function is partially conserved in the fly. However, the PPFIA3 variants failed to rescue lethality. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Germline mutations in WNK2 could be associated with serrated polyposis syndrome.

BACKGROUND: Patients with serrated polyposis syndrome (SPS) have multiple and/or large serrated colonic polyps and higher risk for colorectal cancer. SPS inherited genetic basis is mostly unknown. We aimed to identify new germline predisposition factors for SPS by functionally evaluating a candidate gene and replicating it in additional SPS cohorts. METHODS: After a previous whole-exome sequencing in 39 SPS patients from 16 families (discovery cohort), we sequenced specific genes in an independent validation cohort of 211 unrelated SPS cases. Additional external replication was also available in 297 SPS cases. The WNK2 gene was disrupted in HT-29 cells by gene editing, and WNK2 variants were transfected using a lentiviral delivery system. Cells were analysed by immunoblots, real-time PCR and functional assays monitoring the mitogen-activated protein kinase (MAPK) pathway, cell cycle progression, survival and adhesion. RESULTS: We identified 2 rare germline variants in the WNK2 gene in the discovery cohort, 3 additional variants in the validation cohort and 10 other variants in the external cohorts. Variants c.2105C>T (p.Pro702Leu), c.4820C>T (p.Ala1607Val) and c.6157G>A (p.Val2053Ile) were functionally characterised, displaying higher levels of phospho-PAK1/2, phospho-ERK1/2, CCND1, clonogenic capacity and MMP2. CONCLUSION: After whole-exome sequencing in SPS cases with familial aggregation and replication of results in additional cohorts, we identified rare germline variants in the WNK2 gene. Functional studies suggested germline WNK2 variants affect protein function in the context of the MAPK pathway, a molecular hallmark in this disease.

Next-generation phenotyping contributing to the identification of a 4.7 kb deletion in KANSL1 causing Koolen-de Vries syndrome.

Next-generation phenotyping (NGP) is an application of advanced methods of computer vision on medical imaging data such as portrait photos of individuals with rare disorders. NGP on portraits results in gestalt scores that can be used for the selection of appropriate genetic tests, and for the interpretation of the molecular data. Here, we report on an exceptional case of a young girl that was presented at the age of 8 and 15 and enrolled in NGP diagnostics on the latter occasion. The girl had clinical features associated with Koolen-de Vries syndrome (KdVS) and a suggestive facial gestalt. However, chromosomal microarray (CMA), Sanger sequencing, multiplex ligation-dependent probe analysis (MLPA), and trio exome sequencing remained inconclusive. Based on the highly indicative gestalt score for KdVS, the decision was made to perform genome sequencing to also evaluate noncoding variants. This analysis revealed a 4.7 kb de novo deletion partially affecting intron 6 and exon 7 of the KANSL1 gene. This is the smallest reported structural variant to date for this phenotype. The case illustrates how NGP can be integrated into the iterative diagnostic process of test selection and interpretation of sequencing results.

Solving the genetic aetiology of hereditary gastrointestinal tumour syndromes- a collaborative multicentre endeavour within the project Solve-RD.

BACKGROUND: Patients and families with suspected, but genetically unexplained (unsolved) genetic tumour risk syndromes lack appropriate treatment and prevention, leading to preventable morbidity and mortality. To tackle this problem, patients from the European Reference Network on Genetic Tumour Risk Syndromes (ERN GENTURIS) are analysed in the European Commission's research project "Solving the unsolved rare diseases" (Solve-RD). The aim is to uncover known and novel cancer predisposing genes by reanalysing available whole-exome sequencing (WES) data of large cohorts in a combined manner, and applying a multidimensional omics approach. APPROACH: Around 500 genetically unsolved cases with suspected hereditary gastrointestinal tumour syndromes (polyposis, early-onset/familial colorectal cancer and gastric cancer) from multiple European centres are aimed to be included. Currently, clinical and germline WES data from 294 cases have been analysed. In addition, an extensive molecular profiling of gastrointestinal tumours from these patients is planned and deep learning techniques will be applied. The ambitious, multidisciplinary project is accompanied by a number of methodical, technical, and logistic challenges, which require the development and implementation of new analysis tools, the standardisation of bioinformatics pipelines, and strategies to exchange data and knowledge. RESULTS: and Outlook. The first re-analysis of 229 known and proposed cancer predisposition genes allowed solving 2-3% of previously unsolved GENTURIS cases. The integration of expert knowledge and new technologies will help to identify the genetic basis of additional unsolved cases within the ongoing project. The ERN GENTURIS approach might serve as a model for other genomic initiatives.

GestaltMatcher facilitates rare disease matching using facial phenotype descriptors.

Many monogenic disorders cause a characteristic facial morphology. Artificial intelligence can support physicians in recognizing these patterns by associating facial phenotypes with the underlying syndrome through training on thousands of patient photographs. However, this 'supervised' approach means that diagnoses are only possible if the disorder was part of the training set. To improve recognition of ultra-rare disorders, we developed GestaltMatcher, an encoder for portraits that is based on a deep convolutional neural network. Photographs of 17,560 patients with 1,115 rare disorders were used to define a Clinical Face Phenotype Space, in which distances between cases define syndromic similarity. Here we show that patients can be matched to others with the same molecular diagnosis even when the disorder was not included in the training set. Together with mutation data, GestaltMatcher could not only accelerate the clinical diagnosis of patients with ultra-rare disorders and facial dysmorphism but also enable the delineation of new phenotypes.

Variant profiling of colorectal adenomas from three patients of two families with MSH3-related adenomatous polyposis.

The spectrum of somatic genetic variation in colorectal adenomas caused by biallelic pathogenic germline variants in the MSH3 gene, was comprehensively analysed to characterise mutational signatures and identify potential driver genes and pathways of MSH3-related tumourigenesis. Three patients from two families with MSH3-associated polyposis were included. Whole exome sequencing of nine adenomas and matched normal tissue was performed. The amount of somatic variants in the MSH3-deficient adenomas and the pattern of single nucleotide variants (SNVs) was similar to sporadic adenomas, whereas the fraction of small insertions/deletions (indels) (21-42% of all small variants) was significantly higher. Interestingly, pathogenic somatic APC variants were found in all but one adenoma. The vast majority (12/13) of these were di-, tetra-, or penta-base pair (bp) deletions. The fraction of APC indels was significantly higher than that reported in patients with familial adenomatous polyposis (FAP) (p < 0.01) or in sporadic adenomas (p < 0.0001). In MSH3-deficient adenomas, the occurrence of APC indels in a repetitive sequence context was significantly higher than in FAP patients (p < 0.01). In addition, the MSH3-deficient adenomas harboured one to five (recurrent) somatic variants in 13 established or candidate driver genes for early colorectal carcinogenesis, including ACVR2A and ARID genes. Our data suggest that MSH3-related colorectal carcinogenesis seems to follow the classical APC-driven pathway. In line with the specific function of MSH3 in the mismatch repair (MMR) system, we identified a characteristic APC mutational pattern in MSH3-deficient adenomas, and confirmed further driver genes for colorectal tumourigenesis.

TBK1 and TNFRSF13B mutations and an autoinflammatory disease in a child with lethal COVID-19.

Among children, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are typically mild. Here, we describe the case of a 3.5-year-old girl with an unusually severe presentation of coronavirus disease (COVID-19). The child had an autoinflammatory disorder of unknown etiology, which had been treated using prednisolone and methotrexate, and her parents were half cousins of Turkish descent. After 5 days of nonspecific viral infection symptoms, tonic-clonic seizures occurred followed by acute cardiac insufficiency, multi-organ insufficiency, and ultimate death. Trio exome sequencing identified a homozygous splice-variant in the gene TBK1, and a homozygous missense variant in the gene TNFRSF13B. Heterozygous deleterious variants in the TBK1 gene have been associated with severe COVID-19, and the variant in the TNFRSF13B gene has been associated with common variable immunodeficiency (CVID). We suggest that the identified variants, the autoinflammatory disorder and its treatment, or a combination of these factors probably predisposed to lethal COVID-19 in the present case.

Total Femoral Replacement- A Case Report.

Total femoral replacement(TFR) is a well-recognized salvage procedure performed after multiple failed endoprosthetic replacements, which result in severely compromised bone stock and damaged structural integrity. TFR is performed as an alternative to lower limb amputation, restoring femoral integrity and enabling patients to resume ambulation. TFR is expected to be performed more frequently as the worldwide rate of revision arthroplasty increases due to improved patient survival rates and their underlying diseases, exceeding the functional life of endoprosthetic arthroplasty. We present a 74-year-old, overweight woman with an extensive surgical history with respect to her right knee. Her right lower extremity x-rays showed a long-cemented stem knee tumour prosthesis and a Garden 4 subcapital fracture of the ipsilateral hip. Due to multiple surgeries of the knee and femur in the past, a total femoral replacement was required to be performed. The procedure was successful, and the expected outcome was met. A successful procedure performed by a skilled and experienced surgical team, a thorough rehabilitation program, and prompt post-operative management of complications, can overcome the high incidence of infection and dislocation to preserve the limb with improved functionality and reduce pain. TFR is a drastic operative intervention that can preserve the quality of life for most patients.

Two Faces of Vitamin C in Hemodialysis Patients: Relation to Oxidative Stress and Inflammation.

Hemodialysis (HD) is the most common method of renal replacement therapy. Besides toxins, it eliminates nutrients from the circulation, such as ascorbic acid (AA). HD-patients present AA deficiency more often than representatives of the general population, also due to dietary restrictions. This condition aggravates oxidative stress and inflammation related to uremia and extracorporeal circulation and increases cardiovascular risk followed by mortality. Supplementation of AA seems to be a promising approach in the treatment of hemodialysis patients. Many successful interventions restored plasma AA concentration in HD patients by enteral or intravenous supplementation, concomitantly inhibiting oxidative stress and inflammation. A significant number of studies reported opposite, serious pro-oxidant effects of AA. In this narrative review, we present studies, commenting on their limitations; on AA plasma or serum concentration and the influence of its supplementation on protein and lipid peroxidation, DNA damage, reactive oxygen species generation, paraoxonase activity, advanced glycation endproducts, and C-reactive protein (CRP) concentration. Moreover, in terms of safety, the possible development of oxalosis in HD patients regarding the intravenous or enteral route of AA administration is discussed. Unequivocal clinical results of recent studies on hemodialysis patients are displayed.

Nine newly identified individuals refine the phenotype associated with MYT1L mutations.

Both point mutations and deletions of the MYT1L gene as well as microdeletions of chromosome band 2p25.3 including MYT1L are associated with intellectual disability, obesity, and behavioral problems. Thus, MYT1L is assumed to be the-at least mainly-causative gene in the 2p25.3 deletion syndrome. Here, we present comprehensive descriptions of nine novel individuals bearing MYT1L mutations; most of them single nucleotide variants (SNVs). This increases the number of known individuals with causative deletions or SNVs of MYT1L to 51. Since eight of the nine novel patients bear mutations affecting MYT1L only, the total number of such individuals now nearly equals the number of individuals with larger microdeletions affecting additional genes, allowing for a comprehensive phenotypic comparison of these two patient groups. For example, 55% of the individuals with mutations affecting MYT1L only were overweight or obese as compared to 86% of the individuals with larger microdeletions. A similar trend was observed regarding short stature with 5 versus 35%, respectively. However, these differences were nominally significant only after correction for multiple testing, further supporting the hypothesis that MYT1L haploinsufficiency is central to the 2p25.3 deletion phenotype. Most importantly, the large number of individuals with MYT1L mutations presented and reviewed here allowed for the delineation of a more comprehensive clinical picture. Seizures, postnatal short stature, macrocephaly, and microcephaly could be shown to be over-represented among individuals with MYT1L mutations.

Diagnostic yield and clinical utility of a comprehensive gene panel for hereditary tumor syndromes.

BACKGROUND: In a considerable number of patients with a suspected hereditary tumor syndrome (HTS), no underlying germline mutation is detected in the most likely affected genes. The present study aimed to establish and validate a large gene panel for HTS, and determine its diagnostic yield and clinical utility. METHODS: The study cohort comprised 173 patients with suspected, but unexplained, HTS (group U) and 64 HTS patients with a broad spectrum of known germline mutations (group K). All patients in group U presented with early age at onset, multiple tumors, and/or a familial clustering of various tumor types; no germline mutation had been identified during routine diagnostics. Sequencing of leukocyte DNA was performed for the 94 HTS genes of the Illumina TruSight™Cancer Panel and 54 additional HTS genes. RESULTS: The sensitivity of the panel to identify known germline variants was 99.6%. In addition to known mutations, a total of 192 rare, potentially pathogenic germline variants in 86 genes were identified. Neither the proportion of rare variants per patient (group K: 0.9 variants; group U: 0.8 variants) nor the proportion of variants in the most frequently mutated, moderately penetrant genes CHEK2 and ATM showed significant inter-group difference. Four of the five patients from group U with a truncating CHEK2 mutation had thyroid cancer, pointing to a broader tumor spectrum in patients with pathogenic CHEK2 variants. In 22% of patients from group K, a further potential causative variant was identified. Here, the most interesting finding was an NF1 nonsense mutation in a child with a known TP53 frameshift mutation. In 17% of patients from group U, potential causative variants were identified. In three of these patients (2%), mutations in PMS2, PTEN, or POLD1 were considered to be causative. In both groups, incidental findings with presumptive predictive value were generated. CONCLUSIONS: The gene panel identified the genetic cause in some prescreened, unexplained HTS patients and generated incidental findings. Some patients harbored predicted pathogenic mutations in more than one established HTS gene, rendering interpretation of the respective alterations challenging. Established moderate risk genes showed an almost equal distribution among patients with known and unexplained disease.

Basal cell carcinomas developing independently from BAP1-tumor predisposition syndrome in a patient with bilateral uveal melanoma: Diagnostic challenges to identify patients with BAP1-TPDS.

Basal cell carcinomas (BCC) have been recently included into the spectrum of BAP1-tumor predisposition syndrome (TPDS). Uveal melanoma (UM) is also a tumor often observed in patients with this hereditary tumor syndrome, in particular bilateral UM is highly suspicious for BAP1-TPDS although no patient has been reported yet. Based on our index patient with BAP1-TPDS with bilateral UM (choroid OD, oculus dexter; iris OS, oculus sinister), several BCCs and thyroid cancer as well as a family history for cancer, this paper analyzes hints and pitfalls to diagnose this syndrome clinically and histologically. A previously undescribed germline variant, namely a heterozygous deletion of a single nucleotide on position 2001 (c.2001delG;p.[Thr668Profs*24] in exon 16 of the BAP1 gene), was identified. Structural changes in the C-terminal of the BAP1 protein were observed by in silico analysis. While the excised iris melanoma showed loss of BAP1 nuclear staining by immunohistochemical staining, the BCCs of our patient (and in the control group, n = 13) were BAP1 positive. Genetic analysis of the BCC of the ocular adnexae confirmed a remaining intact BAP1 copy. The constellation of (bilateral) UM in combination with BCC should raise suspicion for a BAP1-TPDS. As our BCCs probably developed independently from the BAP1-TPDS and UMs frequently show loss of nuclear BAP1 staining, genetic analysis is mandatory to diagnose this syndrome.