POLR3B-associated leukodystrophy: clinical, neuroimaging and molecular-genetic analyses in four patients: clinical heterogeneity and novel mutations in POLR3B gene

Introduction and aim of the study. White matter disorders represent a spectrum of neurological diseases frequently associated with an unfavourable prognosis and a delay in diagnostics. We report the broad phenotypic spectrum of a rare hypomyelinating leukodystrophy and three novel mutations. Further, we aim to explore the role of the combined clinical and neuroimaging diagnostic approach in the era of whole exome sequencing. Materials and methods. We present a clinical, neuroimaging and molecular-genetic characterisation of four patients from three families suffering from a rare genetic leukoencephalopathy. Two severely affected siblings (P1, P2) manifested a profound developmental delay, cerebellar symptomatology, microcephaly, failure to thrive, short stature and delayed teeth eruption with oligodontia. The other two patients (P3, P4), on the contrary, suffer from substantially less serious impairment with mild to moderate developmental delay and cerebellar symptomatology, delayed teeth eruption, or well-manageable epilepsy. In all four patients, magnetic resonance revealed cerebellar atrophy and supratentorial hypomyelination with T2-weight hypointensities in the areas of the ventrolateral thalamic nuclei, corticospinal tract and the dentate nuclei. Results. Using whole-exome sequencing in P1, P2 and P3, and targeted sequencing in P4, pathogenic variants were disclosed in POLR3B, a gene encoding one of 17 subunits of DNA-dependent RNA polymerase III — all patients were compound heterozygotes for point mutations. Three novel mutations c.727A>G (p.Met243Val) and c.2669G>A (p.Arg890His) (P1, P2), and c.1495G>A (p.Met499Val) (P3) were found. Magnetic resonance revealed the characteristic radiological pattern of POLR3-leukodystrophies in our patients. Conclusion and clinical implications. The diagnosis of POLR3-associated leukodystrophies can be significantly accelerated using the combined clinical and neuroradiological recognition pattern. Therefore, it is of crucial importance to raise the awareness of this rare disorder among clinicians. Molecular-genetic analyses are indispensable for a swift diagnosis confirmation in cases of clear clinical suspicion, and for diagnostic search in patients with less pronounced symptomatology. They represent an invaluable tool for unravelling the complex genetic background of heritable white matter disorders

Mutation of Nogo-B Receptor, a Subunit of cis-Prenyltransferase, Causes a Congenital Disorder of Glycosylation

SummaryDolichol is an obligate carrier of glycans for N-linked protein glycosylation, O-mannosylation, and GPI anchor biosynthesis. cis-prenyltransferase (cis-PTase) is the first enzyme committed to the synthesis of dolichol. However, the proteins responsible for mammalian cis-PTase activity have not been delineated. Here we show that Nogo-B receptor (NgBR) is a subunit required for dolichol synthesis in yeast, mice, and man. Moreover, we describe a family with a congenital disorder of glycosylation caused by a loss of function mutation in the conserved C terminus of NgBR-R290H and show that fibroblasts isolated from patients exhibit reduced dolichol profiles and enhanced accumulation of free cholesterol identically to fibroblasts from mice lacking NgBR. Mutation of NgBR-R290H in man and orthologs in yeast proves the importance of this evolutionarily conserved residue for mammalian cis-PTase activity and function. Thus, these data provide a genetic basis for the essential role of NgBR in dolichol synthesis and protein glycosylation

Development of a human mitochondrial oligonucleotide microarray (h-MitoArray) and gene expression analysis of fibroblast cell lines from 13 patients with isolated F1Fo ATP synthase deficiency

<p>Abstract</p> <p>Background</p> <p>To strengthen research and differential diagnostics of mitochondrial disorders, we constructed and validated an oligonucleotide microarray (h-MitoArray) allowing expression analysis of 1632 human genes involved in mitochondrial biology, cell cycle regulation, signal transduction and apoptosis. Using h-MitoArray we analyzed gene expression profiles in 9 control and 13 fibroblast cell lines from patients with F₁F_oATP synthase deficiency consisting of 2 patients with mt9205ΔTA microdeletion and a genetically heterogeneous group of 11 patients with not yet characterized nuclear defects. Analysing gene expression profiles, we attempted to classify patients into expected defect specific subgroups, and subsequently reveal group specific compensatory changes, identify potential phenotype causing pathways and define candidate disease causing genes.</p> <p>Results</p> <p>Molecular studies, in combination with unsupervised clustering methods, defined three subgroups of patient cell lines – M group with mtDNA mutation and N1 and N2 groups with nuclear defect. Comparison of expression profiles and functional annotation, gene enrichment and pathway analyses of differentially expressed genes revealed in the M group a transcription profile suggestive of synchronized suppression of mitochondrial biogenesis and G1/S arrest. The N1 group showed elevated expression of complex I and reduced expression of complexes III, V, and V-type ATP synthase subunit genes, reduced expression of genes involved in phosphorylation dependent signaling along MAPK, Jak-STAT, JNK, and p38 MAP kinase pathways, signs of activated apoptosis and oxidative stress resembling phenotype of premature senescent fibroblasts. No specific functionally meaningful changes, except of signs of activated apoptosis, were detected in the N2 group. Evaluation of individual gene expression profiles confirmed already known <it>ATP6/ATP8 </it>defect in patients from the M group and indicated several candidate disease causing genes for nuclear defects.</p> <p>Conclusion</p> <p>Our analysis showed that deficiency in the ATP synthase protein complex amount is generally accompanied by only minor changes in expression of ATP synthase related genes. It also suggested that the site (mtDNA vs nuclear DNA) and the severity (ATP synthase content) of the underlying defect have diverse effects on cellular gene expression phenotypes, which warrants further investigation of cell cycle regulatory and signal transduction pathways in other OXPHOS disorders and related pharmacological models.</p

Biallelic loss-of-function variants in PLD1 cause congenital right-sided cardiac valve defects and neonatal cardiomyopathy

Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function

Heterozygous Loss-of-Function SEC61A1 Mutations Cause Autosomal-Dominant Tubulo-Interstitial and Glomerulocystic Kidney Disease with Anemia

Autosomal-dominant tubulo-interstitial kidney disease (ADTKD) encompasses a group of disorders characterized by renal tubular and interstitial abnormalities, leading to slow progressive loss of kidney function requiring dialysis and kidney transplantation. Mutations in UMOD, MUC1, and REN are responsible for many, but not all, cases of ADTKD. We report on two families with ADTKD and congenital anemia accompanied by either intrauterine growth retardation or neutropenia. Ultrasound and kidney biopsy revealed small dysplastic kidneys with cysts and tubular atrophy with secondary glomerular sclerosis, respectively. Exclusion of known ADTKD genes coupled with linkage analysis, whole-exome sequencing, and targeted re-sequencing identified heterozygous missense variants in SEC61A1—c.553A>G (p.Thr185Ala) and c.200T>G (p.Val67Gly)—both affecting functionally important and conserved residues in SEC61. Both transiently expressed SEC6A1A variants are delocalized to the Golgi, a finding confirmed in a renal biopsy from an affected individual. Suppression or CRISPR-mediated deletions of sec61al2 in zebrafish embryos induced convolution defects of the pronephric tubules but not the pronephric ducts, consistent with the tubular atrophy observed in the affected individuals. Human mRNA encoding either of the two pathogenic alleles failed to rescue this phenotype as opposed to a complete rescue by human wild-type mRNA. Taken together, these findings provide a mechanism by which mutations in SEC61A1 lead to an autosomal-dominant syndromic form of progressive chronic kidney disease. We highlight protein translocation defects across the endoplasmic reticulum membrane, the principal role of the SEC61 complex, as a contributory pathogenic mechanism for ADTKD

Current methods of genome analysis and their use in identification of genetic determinants of human diseases

The study of rare genetic diseases presents unique opportunity to uncover the genetic and molecular basis of human traits and greatly helped to the identification of genes, to the elucidation of their function and to the characterization of metabolic pathways and cellular processes. Over the past decades, linkage analysis has been appropriate approach to search for the genes causing Mendelian diseases and contributed to the identification of many genes, but the genetic cause of many diseases remains unknown. New methods of studying the human genome, microarray technology and massively parallel sequencing (next generation sequencing), represent a way to efficiently identify the cause of genetically determined diseases, based on direct observation of mutations in the genome of affected individuals. These techniques replaced the traditional method of disease gene identification represented by linkage analysis and sequencing of candidate genes and have become the standard approach to elucidate the molecular basis of diseases. In this work, i describe the the results achieved by using these methods - identification of the genes underlying mucopolysacharidosis type IIIC, isolated defect of ATP synthase, Rotor syndrome, autosomal dominat ANCL and GAPO syndrome

Current methods of genome analysis and their use in identification of genetic determinants of human diseases

The study of rare genetic diseases presents unique opportunity to uncover the genetic and molecular basis of human traits and greatly helped to the identification of genes, to the elucidation of their function and to the characterization of metabolic pathways and cellular processes. Over the past decades, linkage analysis has been appropriate approach to search for the genes causing Mendelian diseases and contributed to the identification of many genes, but the genetic cause of many diseases remains unknown. New methods of studying the human genome, microarray technology and massively parallel sequencing (next generation sequencing), represent a way to efficiently identify the cause of genetically determined diseases, based on direct observation of mutations in the genome of affected individuals. These techniques replaced the traditional method of disease gene identification represented by linkage analysis and sequencing of candidate genes and have become the standard approach to elucidate the molecular basis of diseases. In this work, i describe the the results achieved by using these methods - identification of the genes underlying mucopolysacharidosis type IIIC, isolated defect of ATP synthase, Rotor syndrome, autosomal dominat ANCL and GAPO syndrome

Cerebellar dysfunction in a family harboring the PSEN1 mutation co-segregating with a Cathepsin D variant p.A58V

Presenile dementia may be caused by a variety of different genetic conditions such as familial Alzheimer's disease, prion disease as well as several hereditary metabolic disorders including adult onset neuronal ceroid lipofuscinosis. We report a multigenerational family with autosomal dominant presenile dementia harboring a cerebellar phenotype. Longitudinal clinical work-up in affected family members revealed ataxia accompanied by progressive cognitive decline, rapid loss of global cognition, memory, visuospatial and frontal-executive functions accompanied by progressive motor deterioration and early death. Linkage analysis and exome sequencing identified the p.S170F mutation of Presenilin 1 in all affected individuals, which is known to be associated with very early onset Alzheimer's disease. Additional search for potentially modifying variants revealed in all affected individuals of the third generation a paternally inherited variant p.A58V (rs17571) of Cathepsin D which is considered an independent risk factor for Alzheimer's disease. Involvement of cerebellar and brainstem structures leading to functional decortication in addition to rapid progressive presenile dementia in this PSEN1 family may therefore indicate an epistatic effect of the p.A58V Cathepsin D variant on the deleterious course of this disease

Mutations in DNAJC5, Encoding Cysteine-String Protein Alpha, Cause Autosomal-Dominant Adult-Onset Neuronal Ceroid Lipofuscinosis

Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is characterized by accumulation of autofluorescent storage material in neural tissues and neurodegeneration and has an age of onset in the third decade of life or later. The genetic and molecular basis of the disease has remained unknown for many years. We carried out linkage mapping, gene-expression analysis, exome sequencing, and candidate-gene sequencing in affected individuals from 20 families and/or individuals with simplex cases; we identified in five individuals one of two disease-causing mutations, c.346_348delCTC and c.344T>G, in DNAJC5 encoding cysteine-string protein alpha (CSP alpha). These mutations-causing a deletion, p.Leu116del, and an amino acid exchange, p.Leu115Arg, respectively-are located within the cysteine-string domain of the protein and affect both palmitoylation-dependent sorting and the amount of CSP alpha in neuronal cells. The resulting depletion of functional CSP alpha might cause in parallel the presynaptic dysfunction and the progressive neurodegeneration observed in affected individuals and lysosomal accumulation of misfolded and proteolysis-resistant proteins in the form of characteristic ceroid deposits in neurons. Our work represents an important step in the genetic dissection of a genetically heterogeneous group of ANCLs. It also confirms a neuroprotective role for CSP alpha in humans and demonstrates the need for detailed investigation of CSP alpha in the neuronal ceroid lipofuscinoses and other neurodegenerative diseases presenting with neuronal protein aggregation