Single-cell transcription profiles in Bloom syndrome patients link BLM deficiency with altered condensin complex expression signatures.

Bloom syndrome (BS) is an autosomal recessive disease clinically characterized by primary microcephaly, growth deficiency, immunodeficiency and predisposition to cancer. It is mainly caused by biallelic loss-of-function mutations in the BLM gene, which encodes the BLM helicase, acting in DNA replication and repair processes. Here, we describe the gene expression profiles of three BS fibroblast cell lines harboring causative, biallelic truncating mutations obtained by single-cell (sc) transcriptome analysis. We compared the scRNA transcription profiles from three BS patient cell lines to two age-matched wild-type controls and observed specific deregulation of gene sets related to the molecular processes characteristically affected in BS, such as mitosis, chromosome segregation, cell cycle regulation and genomic instability. We also found specific upregulation of genes of the Fanconi anemia pathway, in particular FANCM, FANCD2 and FANCI, which encode known interaction partners of BLM. The significant deregulation of genes associated with inherited forms of primary microcephaly observed in our study might explain in part the molecular pathogenesis of microcephaly in BS, one of the main clinical characteristics in patients. Finally, our data provide first evidence of a novel link between BLM dysfunction and transcriptional changes in condensin complex I and II genes. Overall, our study provides novel insights into gene expression profiles in BS on an sc level, linking specific genes and pathways to BLM dysfunction.

Bi-allelic missense disease-causing variants in RPL3L associate neonatal dilated cardiomyopathy with muscle-specific ribosome biogenesis.

Dilated cardiomyopathy (DCM) belongs to the most frequent forms of cardiomyopathy mainly characterized by cardiac dilatation and reduced systolic function. Although most cases of DCM are classified as sporadic, 20-30% of cases show a heritable pattern. Familial forms of DCM are genetically heterogeneous, and mutations in several genes have been identified that most commonly play a role in cytoskeleton and sarcomere-associated processes. Still, a large number of familial cases remain unsolved. Here, we report five individuals from three independent families who presented with severe dilated cardiomyopathy during the neonatal period. Using whole-exome sequencing (WES), we identified causative, compound heterozygous missense variants in RPL3L (ribosomal protein L3-like) in all the affected individuals. The identified variants co-segregated with the disease in each of the three families and were absent or very rare in the human population, in line with an autosomal recessive inheritance pattern. They are located within the conserved RPL3 domain of the protein and were classified as deleterious by several in silico prediction software applications. RPL3L is one of the four non-canonical riboprotein genes and it encodes the 60S ribosomal protein L3-like protein that is highly expressed only in cardiac and skeletal muscle. Three-dimensional homology modeling and in silico analysis of the affected residues in RPL3L indicate that the identified changes specifically alter the interaction of RPL3L with the RNA components of the 60S ribosomal subunit and thus destabilize its binding to the 60S subunit. In conclusion, we report that bi-allelic pathogenic variants in RPL3L are causative of an early-onset, severe neonatal form of dilated cardiomyopathy, and we show for the first time that cytoplasmic ribosomal proteins are involved in the pathogenesis of non-syndromic cardiomyopathies.

Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are a heterogenous group of epilepsies in which altered brain development leads to developmental delay and seizures, with the epileptic activity further negatively impacting neurodevelopment. Identifying the underlying cause of DEEs is essential for progress toward precision therapies. Here we describe a group of individuals with biallelic variants in DENND5A and determine that variant type is correlated with disease severity. We demonstrate that DENND5A interacts with MUPP1 and PALS1, components of the Crumbs apical polarity complex, which is required for both neural progenitor cell identity and the ability of these stem cells to divide symmetrically. Induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division during neural induction and have an inherent propensity to differentiate into neurons, and transgenic DENND5A mice, with phenotypes like the human syndrome, have an increased number of neurons in the adult subventricular zone. Disruption of symmetric cell division following loss of DENND5A results from misalignment of the mitotic spindle in apical neural progenitors. A subset of DENND5A is localized to centrosomes, which define the spindle poles during mitosis. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state and ultimately shortening the period of neurogenesis. This study provides a mechanism behind DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.

Generation of homozygous Nav1.8 knock-out iPSC lines by CRISPR Cas9 genome editing to investigate a potential new antiarrhythmic strategy.

The sodium channel Nav1.8, encoded by SCN10A, is reported to contribute to arrhythmogenesis by inducing the late INa and thereby enhanced persistent Na+ current. However, its exact electrophysiological role in cardiomyocytes remains unclear. Here, we generated induced pluripotent stem cells (iPSCs) with a homozygous SCN10A knock-out from a healthy iPSC line by CRISPR Cas9 genome editing. The edited iPSCs maintained full pluripotency, genomic integrity, and spontaneous in vitro differentiation capacity. The iPSCs are able to differentiate into iPSC-cardiomyocytes, hence making it possible to investigate the role of Nav1.8 in the heart.

Generation and cardiac differentiation of an induced pluripotent stem cell line from a patient with arrhythmia-induced cardiomyopathy.

Arrhythmia-induced cardiomyopathy (AIC) is characterized by left-ventricular systolic dysfunction caused by persistent arrhythmia. To date, genetic or pathological drivers causing AIC remain unknown. Here, we generated induced pluripotent stem cells (iPSCs) from an AIC patient. The AIC-iPSCs exhibited full pluripotency and differentiation characteristics and maintained a normal karyotype after reprogramming. The AIC-iPSCs differentiated into functional beating AIC-iPSC-cardiomyocytes (CMs), which represents the cell-type of interest to study molecular, genetic and functional aspects of AIC.

Generation of homozygous CRISPRa human induced pluripotent stem cell (hiPSC) lines for sustained endogenous gene activation.

CRISPR/Cas9 technology is a powerful tool, owing to its robust on-target activity and high fidelity. Mutated Cas9 without nuclease activity (dCas9) fused to transcriptional modulators, can function as transcriptional inhibitors or activators (CRISPRa). We generated homozygous human induced pluripotent stem cell (hiPSC) lines with an inserted CRISPRa cassette into the AAVS1 locus whilst maintaining pluripotency and genomic integrity, the ability to differentiate into all three germ layers, generate functional cardiomyocytes, and validated Cas9-mediated induction of endogenous gene expression. Our generated hiPSC-CRISPRa offers a valuable tool for studying endogenous transcriptional modulation with single and multiplexed possibilities in all human cell types.

A newborn with hereditary haemorrhagic telangiectasia and an unusually severe phenotype.

UNLABELLED: Hereditary haemorrhagic telangiectasia (HHT), associated with arteriovenous malformations, is a genetic disease of the vascular system with a frequency of approx. 1:10,000. Genetic diagnosis serves to identify individuals at risk of developing the disease and is a useful tool for genetic counselling purposes. QUESTIONS UNDER STUDY: Here we report on a child presenting severe arteriovenous malformations leading to heart failure. Her mother and grandmother present fewer symptoms of hereditary haemorrhagic telangiectasia. In this study we identify the cause of HHT in the family. METHODS: Clinical examination, PCR, DNA sequencing, quantitative PCR, Southern blot, xray, ultrasound, cardiac catheterisation and angiocardiography. RESULTS: Initially the sequence variant in c.392C>T in the endoglin gene was detected in the grandmother, but not in other affected family members. Further analyses revealed a deletion of exon 1 of endoglin, segregating with the phenotype. CONCLUSIONS: This report points out the need for careful evaluation of molecular genetic findings, particularly in diseases with highly variable phenotype.

An exceptional complex chromosomal rearrangement (CCR) with eight breakpoints involving four chromosomes (1;3;9;14) in an azoospermic male with normal phenotype.

A 27-year-old man was referred for chromosome analysis due to infertility caused by azoospermia. Chromosome analysis by conventional karyotyping, multicolour FISH (M-FISH) and multicolour banding (MCB) analysis revealed an apparently balanced translocation between chromosomes 1, 3, 9 and 14 as well as an additional inverted insertion of 3q material with a total of eight breakpoints. Due to the diversity of theoretically unbalanced products of meiotic recombination in this exceptional complex chromosomal rearrangement a successful result of assisted reproduction seems unlikely.

Novel mutations in the ENG and ACVRL1 genes causing hereditary hemorrhagic teleangiectasia.

Hereditary haemorrhagic teleangiectasia (HHT) is an autosomal dominantly inherited disorder characterised by cutaneous and mucosal telangiectasias, epistaxis and arteriovenous malformations in lung, liver, central nervous system and gastrointestinal tract. Mutations in the genes for endoglin (ENG) and for activin A receptor type II-like kinase 1 (ACVRL1) have been identified to cause HHT. We performed molecular diagnosis in clinically affected probands of 52 HHT families and detected mutations in 34 cases. We report on a total of 19 novel disease-causing mutations, 7 in ENG and 12 in ACVRL1. Three of the novel mutations affected acceptor splice-sites in the ENG gene. RNA analyses in these three patients and in two further patients described before resulted in reduction of the transcript or in a shortened transcript. Furthermore, we identified a family with the mutation c.199C>T in the ACVRL1 gene with liver AVMs. This is the fifth family with this mutation and liver AVMs, clearly indicating a genotype-phenotype correlation for this mutation.

A de novo unbalanced translocation leading to partial monosomy 9p23-pter and partial trisomy 15q25.3-qter associated with 46,XY complete gonadal dysgenesis, tall stature and mental retardation.

Syndromic forms of disorders of sex development constitute a challenge for clinical and molecular investigations. We report on a 12-year-old girl presenting with lack of pubertal development, tall stature and moderate mental retardation. Conventional karyotyping at the age of 3 years revealed a male karyotype (46,XY). At the age of 12 years, the girl had no signs of puberty, and laboratory values were consistent with hypergonadotropic hypogonadism because of complete gonadal dysgenesis. Histology at the time of gonadectomy revealed fibrous tissue without testicular morphology. Cytogenetic reevaluation at that time showed additional material of unknown origin on the short arm of chromosome 9. Subsequent fluorescence in-situ hybridization and Array-CGH analyses revealed an unbalanced translocation between 9p and 15q resulting in a partial monosomy of 9p and a partial trisomy of 15q. The karyotype was described as 46,XY,der(9)t(9;15)(p23;q25.3). We discuss the clinical and molecular cytogenetic findings with respect to the literature.

Expansion of mutation spectrum, determination of mutation cluster regions and predictive structural classification of SPAST mutations in hereditary spastic paraplegia.

The SPAST gene encoding for spastin plays a central role in the genetically heterogeneous group of diseases termed hereditary spastic paraplegia (HSP). In this study, we attempted to expand and refine the genetic and phenotypic characteristics of SPAST associated HSP by examining a large cohort of HSP patients/families. Screening of 200 unrelated HSP cases for mutations in the SPAST gene led to detection of 57 mutations (28.5%), of which 47 were distinct and 29 were novel mutations. The distribution analysis of known SPAST mutations over the structural domains of spastin led to the identification of several regions where the mutations were clustered. Mainly, the clustering was observed in the AAA (ATPases associated with diverse cellular activities) domain; however, significant clustering was also observed in the MIT (microtubule interacting and trafficking), MTBD (microtubule-binding domain) and an N-terminal region (228-269 residues). Furthermore, we used a previously generated structural model of spastin as a framework to classify the missense mutations in the AAA domain from the HSP patients into different structural/functional groups. Our data also suggest a tentative genotype-phenotype correlation and indicate that the missense mutations could cause an earlier onset of the disease.

Identification of subtelomeric genomic imbalances and breakpoint mapping with quantitative PCR in 296 individuals with congenital defects and/or mental retardation.

BACKGROUND: Submicroscopic imbalances in the subtelomeric regions of the chromosomes are considered to play an important role in the aetiology of mental retardation (MR). The aim of the study was to evaluate a quantitative PCR (qPCR) protocol established by Boehm et al. (2004) in the clinical routine of subtelomeric testing. RESULTS: 296 patients with MR and a normal karyotype (500-550 bands) were screened for subtelomeric imbalances by using qPCR combined with SYBR green detection. In total, 17 patients (5.8%) with 20 subtelomeric imbalances were identified. Six of the aberrations (2%) were classified as causative for the symptoms, because they occurred either de novo in the patients (5 cases) or the aberration were be detected in the patient and an equally affected parent (1 case). The extent of the deletions ranged from 1.8 to approximately 10 Mb, duplications were 1.8 to approximately 5 Mb in size. In 6 patients, the copy number variations (CNVs) were rated as benign polymorphisms, and the clinical relevance of these CNVs remains unclear in 5 patients (1.7%). Therefore, the overall frequency of clinically relevant imbalances ranges between 2% and 3.7% in our cohort. CONCLUSION: This study illustrates that the qPCR/SYBR green technique represents a rapid and versatile method for the detection of subtelomeric imbalances and the option to map the breakpoint. Thus, this technique is highly suitable for genotype/phenotype studies in patients with MR/developmental delay and/or congenital defects.

ALK-1 mutations in liver transplanted patients with hereditary hemorrhagic telangiectasia.

Hereditary haemorrhagic telangiectasia (HHT) is an autosomal dominantly inherited disorder characterized by cutaneous and mucosal telangiectasias, epistaxis and arteriovenous malformations in lung, liver, central nervous system, and gastrointestinal tract. Mutations in the genes for endoglin (ENG) and for activin A receptor type II-like kinase 1 (ALK-1) have been identified to be associated with HHT. Intrahepatic manifestation in HHT might lead to the requirement of liver transplantation. We report here on 6 liver transplanted patients and 2 who were scheduled for liver transplantation due to intrahepatic HHT, in whom both genes were sequenced. Mutation analysis revealed in all patients the presence of mutations in ALK-1. In conclusion, these results are of possible prognostic value concerning the need of liver transplantation in HHT patients.