Biallelic loss-of-function variants of ZFTRAF1 cause neurodevelopmental disorder with microcephaly and hypotonia.

PURPOSE: Neurodevelopmental disorders exhibit clinical and genetic heterogeneity, ergo manifest dysfunction in components of diverse cellular pathways; the precise pathomechanism for the majority remains elusive. METHODS: We studied 5 affected individuals from 3 unrelated families manifesting global developmental delay, postnatal microcephaly, and hypotonia. We used exome sequencing and prioritized variants that were subsequently characterized using immunofluorescence, immunoblotting, pulldown assays, and RNA sequencing. RESULTS: We identified biallelic variants in ZFTRAF1, encoding a protein of yet unknown function. Four affected individuals from 2 unrelated families segregated 2 homozygous frameshift variants in ZFTRAF1, whereas, in the third family, an intronic splice site variant was detected. We investigated ZFTRAF1 at the cellular level and signified it as a nucleocytoplasmic protein in different human cell lines. ZFTRAF1 was completely absent in the fibroblasts of 2 affected individuals. We also identified 110 interacting proteins enriched in mRNA processing and autophagy-related pathways. Based on profiling of autophagy markers, patient-derived fibroblasts show irregularities in the protein degradation process. CONCLUSION: Thus, our findings suggest that biallelic variants of ZFTRAF1 cause a severe neurodevelopmental disorder.

Inactivation of DRG1, encoding a translation factor GTPase, causes a recessive neurodevelopmental disorder.

PURPOSE: Developmentally regulated Guanosine-5'-triphosphate-binding protein 1 (DRG1) is a highly conserved member of a class of GTPases implicated in translation. Although the expression of mammalian DRG1 is elevated in the central nervous system during development, and its function has been implicated in fundamental cellular processes, no pathogenic germline variants have yet been identified. Here, we characterize the clinical and biochemical consequences of DRG1 variants. METHODS: We collate clinical information of 4 individuals with germline DRG1 variants and use in silico, in vitro, and cell-based studies to study the pathogenicity of these alleles. RESULTS: We identified private germline DRG1 variants, including 3 stop-gained p.Gly54∗, p.Arg140∗, p.Lys263∗, and a p.Asn248Phe missense variant. These alleles are recessively inherited in 4 affected individuals from 3 distinct families and cause a neurodevelopmental disorder with global developmental delay, primary microcephaly, short stature, and craniofacial anomalies. We show that these loss-of-function variants (1) severely disrupt DRG1 messenger RNA/protein stability in patient-derived fibroblasts, (2) impair its GTPase activity, and (3) compromise its binding to partner protein ZC3H15. Consistent with the importance of DRG1 in humans, targeted inactivation of mouse Drg1 resulted in preweaning lethality. CONCLUSION: Our work defines a new Mendelian disorder of DRG1 deficiency. This study highlights DRG1's importance for normal mammalian development and underscores the significance of translation factor GTPases in human physiology and homeostasis.

Three siblings with variable degrees of neuromuscular involvement and congenital sideroblastic anemia: A peculiar phenotype and a surprise genotypic explanation.

INTRODUCTION: Congenital sideroblastic anemias (CSAs) are a group of inherited bone-marrow disorders manifesting with erythroid hyperplasia and ineffective erythropoiesis. METHODS: We describe a detailed clinical and genetic characterization of three siblings with CSA. RESULTS: Two of them had limb-girdle myopathy and global developmental delay. The two elder siblings performed allogenic hematopoietic stem-cell transplantation 5 and 3 years prior with stabilization of the hematological features. Exome sequencing in the non-transplanted sibling revealed a novel homozygous nonsense variant in SLC25A38 gene NM_017875.2:c.559C > T; p.(Arg187*) causing autosomal-recessive sideroblastic anemia type-2, and a second homozygous pathogenic previously reported variant in GMPPB gene NM_013334.3:c.458C > T; p.(Thr153Ile) causing autosomal-recessive muscular dystrophy-dystroglycanopathy type B14. With the established diagnosis, hematopoietic stem cell transplantation is now being scheduled for the youngest sibling, and a trial therapy with acetylcholine esterase inhibitors was started for the two neurologically affected patients with partial clinical improvement. CONCLUSION: This family emphasizes the importance of whole-exome sequencing for familial cases with complex phenotypes and vague neurological manifestations.

Bi-allelic loss-of-function variants in TMEM147 cause moderate to profound intellectual disability with facial dysmorphism and pseudo-Pelger-Huët anomaly.

The transmembrane protein TMEM147 has a dual function: first at the nuclear envelope, where it anchors lamin B receptor (LBR) to the inner membrane, and second at the endoplasmic reticulum (ER), where it facilitates the translation of nascent polypeptides within the ribosome-bound TMCO1 translocon complex. Through international data sharing, we identified 23 individuals from 15 unrelated families with bi-allelic TMEM147 loss-of-function variants, including splice-site, nonsense, frameshift, and missense variants. These affected children displayed congruent clinical features including coarse facies, developmental delay, intellectual disability, and behavioral problems. In silico structural analyses predicted disruptive consequences of the identified amino acid substitutions on translocon complex assembly and/or function, and in vitro analyses documented accelerated protein degradation via the autophagy-lysosomal-mediated pathway. Furthermore, TMEM147-deficient cells showed CKAP4 (CLIMP-63) and RTN4 (NOGO) upregulation with a concomitant reorientation of the ER, which was also witnessed in primary fibroblast cell culture. LBR mislocalization and nuclear segmentation was observed in primary fibroblast cells. Abnormal nuclear segmentation and chromatin compaction were also observed in approximately 20% of neutrophils, indicating the presence of a pseudo-Pelger-Huët anomaly. Finally, co-expression analysis revealed significant correlation with neurodevelopmental genes in the brain, further supporting a role of TMEM147 in neurodevelopment. Our findings provide clinical, genetic, and functional evidence that bi-allelic loss-of-function variants in TMEM147 cause syndromic intellectual disability due to ER-translocon and nuclear organization dysfunction.

Monoallelic and biallelic variants in LEF1 are associated with a new syndrome combining ectodermal dysplasia and limb malformations caused by altered WNT signaling.

PURPOSE: LEF1 encodes a transcription factor acting downstream of the WNT-ß-catenin signaling pathway. It was recently suspected as a candidate for ectodermal dysplasia in 2 individuals carrying 4q35 microdeletions. We report on 12 individuals harboring LEF1 variants. METHODS: High-throughput sequencing was employed to delineate the genetic underpinnings of the disease. Cellular consequences were characterized by immunofluorescence, immunoblotting, pulldown assays, and/or RNA sequencing. RESULTS: Monoallelic variants in LEF1 were detected in 11 affected individuals from 4 unrelated families, and a biallelic variant was detected in an affected individual from a consanguineous family. The phenotypic spectrum includes various limb malformations, such as radial ray defects, polydactyly or split hand/foot, and ectodermal dysplasia. Depending on the type and location of LEF1 variants, the inheritance of this novel Mendelian condition can be either autosomal dominant or recessive. Our functional data indicate that 2 molecular mechanisms are at play: haploinsufficiency or loss of DNA binding are responsible for a mild to moderate phenotype, whereas loss of ß-catenin binding caused by biallelic variants is associated with a severe phenotype. Transcriptomic studies reveal an alteration of WNT signaling. CONCLUSION: Our findings establish mono- and biallelic variants in LEF1 as a cause for a novel syndrome comprising limb malformations and ectodermal dysplasia.

A homozygous frame-shift variant in PROSER1 is associated with developmental delay, hypotonia, genitourinary malformations, and distinctive facial features.

We report four children from three related families who presented with a similar phenotype characterized by developmental delay, hypotonia, seizures, failure-to-thrive, strabismus, drooling, recurrent otitis media, hearing impairment, and genitourinary malformations. They also shared common facial features including arched eyebrows, prominent eyes, broad nasal bridge, low-hanging columella, open mouth, thick lower lip, protruding tongue, large low-set ears, and parietal bossing. Exome sequencing for affected individuals revealed a homozygous frame-shift variant, c.1833del; p.(Thr612Glnfs*22), in PROSER1 which encodes the proline and serine rich protein 1 (PROSER1). PROSER1 has recently been found to be part of the histone methyltransferases KMT2C/KMT2D complexes. PROSER1 stabilizes TET2, a member of the TET family of DNA demethylases which is involved in recruiting the enhancer-associated KMT2C/KMT2D complexes and mediating DNA demethylation, activating gene expression. Therefore, PROSER1 may play vital and potentially general roles in gene regulation, consistent with the wide phenotypic spectrum observed in the individuals presented here. The consistent phenotype, the loss-of-function predicted from the frame-shift, the co-segregation of the phenotype in our large pedigree, the vital role of PROSER1 in gene regulation, and the association of related genes with neurodevelopmental disorders argue for the loss of PROSER1 to be the cause for a novel recognizable syndrome.

A 6.3 Mb maternally derived microduplication of 20p13p12.2 in a fetus with Brachydactyly type D and related literature review.

BACKGROUND: With the introduction of genetic tests such as chromosomal microarray analysis (CMA) and exome sequencing (ES) into fetal medical practices, genotype-phenotype correlations in intrauterine-onset disorders have substantially improved. The BMP2 gene, located on the long arm of chromosome 20 plays a role in bone and cartilage development and is associated with Brachydactyly type A2, an autosomal dominant disease characterized by malformations of the middle phalanx of the index finger and abnormalities of the second toe. However, the BMP2 gene has so far never been reported as a candidate gene for Brachydactyly type D (BDD) affecting only the thumbs. METHODS AND RESULTS: Here, we report one family possessing a maternally inherited 6.3 Mb microduplication of 20p13p12.2 including the BMP2 gene with discordant phenotypes between the mother and the fetus. The mother was affected with BDD alongside mild facial dysmorphism and learning difficulties, while the female fetus showed BDD, severe symmetric intrauterine growth restriction combined with oligohydramnios. The CMA and Trio ES tests were implemented. Trio ES ruled out other possible monogenic causes for the family. After reviewing cases and literature with duplications within this genomic region, we found that they are extremely rare and most of the cited cases were too small for comparison. The disturbance of the BMP2 gene could explain BDD, but the other clinical presentations in the mother and fetus are not yet fully understood. CONCLUSION: This study provides important evidence for the current understanding of genotype-phenotype association of this 6.3 Mb size duplication in the 20p13p12.2 region. This duplication is a unique CNV occurring so far only in this family. Further cases and research are needed to understand the discordance in the phenotypes between the mother and fetus and establish the relationship between BMP2 gene and BDD.

Biallelic ZNFX1 variants are associated with a spectrum of immuno-hematological abnormalities.

Biallelic changes in the ZNFX1 gene have been recently reported to cause severe familial immunodeficiency. Through a search of our bio/databank with information from genetic testing of >55 000 individuals, we identified nine additional patients from seven families with six novel homozygous ZNFX1 variants. Consistent with the previously described phenotype, our patients suffered from monocytosis, thrombocytopenia, hepatosplenomegaly, recurrent infections, and lymphadenopathy. The two most severely affected probands also had renal involvement and clinical presentations compatible with hemophagocytic lymphohistiocytosis. The disease was less lethal among our patients than previously reported. We identified two missense changes, two variants predicted to result in complete protein loss through nonsense-mediated decay, and two frameshift changes that likely introduce a truncation. Our findings (i) independently confirm the role of ZNFX1 in primary genetic immunodeficiency, (ii) expand the genetic and clinical spectrum of ZNFX1-related disease, and (iii) illustrate the utility of large, well-curated, and continually updated genotype-phenotype databases in resolving molecular diagnoses of patients with initially negative genetic testing findings.

A teaching tool about the fickle p value and other statistical principles based on real-life data.

A poor understanding of statistical analysis has been proposed as a key reason for lack of replicability of many studies in experimental biomedicine. While several authors have demonstrated the fickleness of calculated p values based on simulations, we have experienced that such simulations are difficult to understand for many biomedical scientists and often do not lead to a sound understanding of the role of variability between random samples in statistical analysis. Therefore, we as trainees and trainers in a course of statistics for biomedical scientists have used real data from a large published study to develop a tool that allows scientists to directly experience the fickleness of p values. A tool based on a commonly used software package was developed that allows using random samples from real data. The tool is described and together with the underlying database is made available. The tool has been tested successfully in multiple other groups of biomedical scientists. It can also let trainees experience the impact of randomness, sample sizes and choice of specific statistical test on measured p values. We propose that live exercises based on real data will be more impactful in the training of biomedical scientists on statistical concepts.