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While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism., POLRMT is key for transcription of the mitochondrial genome, yet has not been implicated in mitochondrial disease to date. Here, the authors identify mutations in POLRMT in individuals with mitochondrial disease-related phenotypes and characterise underlying defects in mitochondrial transcription.

BackgroundClassic Fabry disease (FD) is caused by GLA mutations that result in enzymatic deficiency of alpha-galactosidase A (AGAL), lysosomal storage of globotriaosylceramide, and a resulting multisystemic disease. In non-classic later-onset FD, patients have some preserved AGAL activity and a milder disease course, though female carriers may also be affected. While FD pathogenesis has been mostly attributed to catalytic deficiency of mutated AGAL, lysosomal storage and impairment of lysosomal functions, other pathogenic factors may be important, especially in non-classic later-onset FD.MethodsWe characterized the clinical, biochemical, genetic, molecular, cellular and organ pathology correlates of the p.L394P AGAL variant that was identified in six individuals with end-stage kidney disease by the Czech national screening program for FD and by further screening of 25 family members.ResultsClinical findings revealed a milder clinical course with ~15% residual AGAL activity. Laboratory investigations documented intracellular retention of mutated AGAL with resulting ER stress and the unfolded protein response (UPR). Kidney biopsies did not show lysosomal storage. We observed similar findings of ER stress and UPR with several other classic and non-classic FD missense GLA variants.ConclusionsWe identified defective proteostasis of mutated AGAL resulting in chronic ER stress and UPR of AGAL expressing cells (hereafter referred to as AGALopathy) as an important contributor to FD pathogenesis. These findings provide insight into non-classic later-onset FD and may better explain clinical manifestations with implications for pathogenesis, clinical characterization and treatment of all FD forms.Significance statementCatalytic deficiency of mutated AGAL is responsible for classicFabry disease (FD) pathogenesis but does not fully explain the findings in non-classic later-onset FD, in which affected individuals and female carriers develop clinical manifestations despite some AGAL activity and variably mitigated lysosomal storage. In this investigation of individuals with the p.L394P AGAL variant, we identified defective proteostasis of mutated AGAL resulting in chronic endoplasmic reticulum stress and the unfolded protein response as significant contributors to pathogenesis of non-classic later-onset FD. Similar effects were documented also in other AGAL variants identified in classic and non-classicFD. Endoplasmic reticulum stress and the unfolded protein response therefore play an important role in FD.

The recent description of biallelic DNAJC30 variants in Leber hereditary optic neuropathy (LHON) and Leigh syndrome challenged the longstanding assumption for LHON to be exclusively maternally inherited and broadened the genetic spectrum of Leigh syndrome, the most frequent paediatric mitochondrial disease. Herein, we characterize 28 so far unreported individuals from 26 families carrying a homozygous DNAJC30 p.Tyr51Cys founder variant, 24 manifesting with LHON, two manifesting with Leigh syndrome, and two remaining asymptomatic. This collection of unreported variant carriers confirms sex-dependent incomplete penetrance of the homozygous variant given a significant male predominance of disease and the report of asymptomatic homozygous variant carriers. The autosomal recessive LHON patients demonstrate an earlier age of disease onset and a higher rate of idebenone-treated and spontaneous recovery of vision in comparison to reported figures for maternally inherited disease. Moreover, the report of two additional patients with childhood- or adult-onset Leigh syndrome further evidences the association of DNAJC30 with Leigh syndrome, previously only reported in a single childhood-onset case.

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.1668F 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.

Amyloid A amyloidosis is a serious clinical condition resulting from the systemic deposition of amyloid A originating from serum amyloid A proteins with the kidneys being the most commonly and earliest affected organ. Previously described amyloid A amyloidosis is linked to increased production and deposition of serum amyloid A proteins secondary to inflammatory conditions arising from infectious, metabolic, or genetic causes. Here we describe a family with primary amyloid A amyloidosis due to a chr11:18287683 T>C (human genome version19) mutation in the SAA1 promoter linked to the amyloidogenic SAA1.1 haplotype. This condition leads to a doubling of the basal SAA1 promoter activity and sustained elevation of serum amyloid A levels that segregated in an autosomal dominant pattern in 12 genetically affected and in none of six genetically unaffected relatives, yielding a statistically significant logarithm of odds (LOD) score over 5. Affected individuals developed proteinuria, chronic kidney disease and systemic deposition of amyloid composed specifically of the SAA1.1 isoform. Tocilizumab (a monoclonal antibody against the interleukin-6 receptor) had a beneficial effect when prescribed early in the disease course. Idiopathic forms represent a significant and increasing proportion (15-20%) of all diagnosed cases of amyloid A amyloidosis. Thus, genetic screening of the SAA1 promoter should be pursued in individuals with amyloid A amyloidosis and no systemic inflammation, especially if there is a positive family history.

Background: The pleomorphic clinical presentation makes the diagnosis of desminopathy difficult. We aimed to describe the prevalence, phenotypic expression, and mitochondrial function of individuals with putative disease-causing desmin (DES) variants identified in patients with an unexplained etiology of cardiomyopathy. Methods: A total of 327 Czech patients underwent whole exome sequencing and detailed phenotyping in probands harboring DES variants. Results: Rare, conserved, and possibly pathogenic DES variants were identified in six (1.8%) probands. Two DES variants previously classified as variants of uncertain significance (p.(K43E), p.(S57L)), one novel DES variant (p.(A210D)), and two known pathogenic DES variants (p.(R406W), p.(R454W)) were associated with characteristic desmin-immunoreactive aggregates in myocardial and/or skeletal biopsy samples. The individual with the novel DES variant p.(Q364H) had a decreased myocardial expression of desmin with absent desmin aggregates in myocardial/skeletal muscle biopsy and presented with familial left ventricular non-compaction cardiomyopathy (LVNC), a relatively novel phenotype associated with desminopathy. An assessment of the mitochondrial function in four probands heterozygous for a disease-causing DES variant confirmed a decreased metabolic capacity of mitochondrial respiratory chain complexes in myocardial/skeletal muscle specimens, which was in case of myocardial succinate respiration more profound than in other cardiomyopathies. Conclusions: The presence of desminopathy should also be considered in individuals with LVNC, and in the differential diagnosis of mitochondrial diseases.

Objectives Phosphoribosylpyrophosphate synthetase (PRPS1) superactivity is an X-linked disorder characterized by urate overproduction Online Mendelian Inheritance in Man (OMIM) gene reference 300661. This condition is thought to rarely affect women, and when it does, the clinical presentation is mild. We describe a 16-year-old African American female who developed progressive tophi, nephrolithiasis and acute kidney failure due to urate overproduction. Family history included a mother with tophaceous gout who developed end-stage kidney disease due to nephrolithiasis and an affected sister with polyarticular gout. The main aim of this study was to describe the clinical manifestations of PRPS1 superactivity in women. Methods Whole exome sequencing was performed in affected females and their fathers. Results Mutational analysis revealed a new c.520 G > A (p.G174R) mutation in the PRPS1 gene. The mutation resulted in decreased PRPS1 inhibition by ADP. Conclusion Clinical findings in previously reported females with PRPS1 superactivity showed a high clinical penetrance of this disorder with a mean serum urate level of 8.5 (4.1) mg/dl [506 (247) μmol/l] and a high prevalence of gout. These findings indicate that all women in families with PRPS1 superactivity should be genetically screened for a mutation (for clinical management and genetic counselling). In addition, women with tophaceous gout, gout presenting in childhood, or a strong family history of severe gout should be considered for PRPS1 mutational analysis.

The formation of the vertebrate brain requires the generation, migration, differentiation and survival of neurons. Genetic mutations that perturb these critical cellular events can result in malformations of the telencephalon, providing a molecular window into brain development. Here we report the identification of an N-ethyl-N-nitrosourea-induced mouse mutant characterized by a fractured hippocampal pyramidal cell layer, attributable to defects in neuronal migration. We show that this is caused by a hypomorphic mutation in Vps15 that perturbs endosomal-lysosomal trafficking and autophagy, resulting in an upregulation of Nischarin, which inhibits Pak1 signaling. The complete ablation of Vps15 results in the accumulation of autophagic substrates, the induction of apoptosis and severe cortical atrophy. Finally, we report that mutations in VPS15 are associated with cortical atrophy and epilepsy in humans. These data highlight the importance of the Vps15-Vps34 complex and the Nischarin-Pak1 signaling hub in the development of the telencephalon.

Background Spinal muscular atrophy (SMA) is an inherited neuromuscular disease affecting 1 in 8,000 newborns. The majority of patients carry bi‐allelic variants in the survival of motor neuron 1 gene (SMN1). SMN1 is located in a duplicated region on chromosome 5q13 that contains Alu elements and is predisposed to genomic rearrangements. Due to the genomic complexity of the SMN region and genetic heterogeneity, approximately 50% of SMA patients remain without genetic diagnosis that is a prerequisite for genetic treatments. In this work we describe the diagnostic odyssey of one SMA patient in whom routine diagnostics identified only a maternal heterozygous SMN1Δ(7–8) deletion. Methods We characterized SMN transcripts, assessed SMN protein content in peripheral blood mononuclear cells (PBMC), estimated SMN genes dosage, and mapped genomic rearrangement in the SMN region. Results We identified an Alu‐mediated deletion encompassing exons 2a‐5 of SMN1 on the paternal allele and a complete deletion of SMN1 on the maternal allele as the cause of SMA in this patient. Conclusion Alu‐mediated rearrangements in SMN1 can escape routine diagnostic testing. Parallel analysis of SMN gene dosage, SMN transcripts, and total SMN protein levels in PBMC can identify genomic rearrangements and should be considered in genetically undefined SMA cases., Spinal muscular atrophy (SMA) is devastating inherited neuromuscular disease resulting from variants of SMN1 and deficiency of the survival motor neuron protein (SMN). In this work, we describe the diagnostic odyssey for one SMA patient in whom routine diagnostic procedures identified only a heterozygous, maternally inherited deletion of exons 7 and 8 in SMN1. Using an individual approach we found in this case that SMA was caused by a novel Alu‐mediated deletion.

Background Hereditary mutations in the CHEK2 gene (which encodes CHK2 kinase) contribute to a moderately increased risk of breast cancer (BC) and other cancers. Large variations in the frequency of CHEK2 mutations and the occurrence of variants of unknown clinical significance (VUS) complicate estimation of cancer risk in carriers of germline CHEK2 mutations. Patients and methods We performed mutation analysis of 1,526 high-risk Czech BC patients and 3,360 Czech controls. Functional analysis was performed for identified VUS using a model system based on a human RPE1-CHEK2-KO cell line harboring biallelic inactivation of endogenous CHEK2. Results The frequency of ten truncating CHEK2 variants differed markedly between BC patients (2.26%) and controls (0.11%; p = 4.1 × 1012). We also found 23 different missense variants in 4.5% patients and in 4.0% of controls. The most common was p.I157T, which was found in patients and controls with the same frequency. Functional analysis identified nine functionally deleterious VUS, another nine functionally neutral VUS, and four intermediate VUS (including p.I157T). We found that carriers of truncating CHEK2 mutations had a high BC risk (OR 8.19; 95% CI 4.11-17.75), and that carriers of functionally deleterious missense variants had a moderate risk (OR 4.06; 95% CI, 1.37-13.39). Carriers of these mutations developed BC at 44.4 and 50.7 years, respectively. Functionally neutral and functionally intermediate missense variants did not increase the BC risk. BC in CHEK2 mutation carriers was frequently ER-positive and of higher grade. Notably, carriers of CHEK2 mutations developed second cancers more frequently than BRCA1/BRCA2/PALB2/p53 or mutation non-carriers. Conclusion Hereditary CHEK2 mutations contribute to the development of hereditary BC. The associated cancer risk in mutation carriers increases with the number of affected individuals in a family. Annual follow-up with breast ultrasound, mammography, or magnetic resonance imaging is recommended for asymptomatic mutation carriers from the age of 40. Surgical prevention and specific follow-up of other tumors should be considered based on family cancer history. The work was supported by grants from the Czech Health Research Council of the Ministry of Health of the Czech Republic NR 15-28830A, 16-29959A, NV19-03-00279, projects of the PROGRES Q28/LF1, GAUK 762216, SVV2019 / 260367, PRIMUS/17/MED/9, UNCE/MED/016, Progress Q26, LQ1604 NPU II and project AVCR Qualitas. The analysis of a set of unselected controls was made possible by the existence and support of the scientific infrastructure of the National Center for Medical Genomics (LM2015091) and its project aimed at creating a reference database of genetic variants of the Czech Republic (CZ.02.1.01/0.0/0.0/16_013/0001634). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 2. 4. 2019 Accepted: 14. 5. 2019.

BACKGROUND Ovarian cancer is a disease with high mortality. Approximately 1,000 women are diagnosed with ovarian cancer in the Czech Republic annually. Women harboring a mutation in cancer-predisposing genes face an increased risk of tumor development. Mutations in BRCA1, BRCA2, BRIP1, and Lynch syndrome genes (RAD51C, RAD51D, and STK11) are associated with a high risk of ovarian cancer, and mutations in ATM, CHEK2, NBN, PALB2, and BARD1 appear to increase the risk. Our aim was to examine the frequency of mutations in cancer-predisposing genes in the Czech Republic. MATERIALS AND METHODS We analyzed 1,057 individuals including ovarian cancer patients and 617 non-cancer controls using CZECANCA panel next-generation sequencing on the Illumina platform. Pathogenic mutations in high-risk genes, including CNVs, were detected in 30.6% of patients. The mutation frequency reached 25.0% and 18.2% in subgroups of unselected ovarian cancer patients and patients with a negative family cancer history, respectively. The most frequently mutated genes were BRCA1 and BRCA2. The overall frequency of mutations in non-BRCA genes was comparable to that in BRCA2. The mutation frequency in ovarian cancer patients aged >70 years was three times higher than that in patients diagnosed before the age of 30. CONCLUSION Ovarian cancer is a heterogeneous disease with a high proportion of hereditary cases. The lack of efficient screening for early diagnosis emphasizes the importance of identifying carriers of mutations in ovarian cancer-predisposing genes; this is because proper follow-up and prevention strategies can reduce overall ovarian cancer-related mortality. This work was supported by grants AZV 15-27695A, SVV2019/260367, PROGRES Q28/LF1. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 7. 3. 2019 Accepted: 24. 4. 2019.

Adult-onset neuronal ceroid lipofuscinoses (ANCL, Kufs disease) are rare hereditary neuropsychiatric disorders characterized by intralysosomal accumulation of ceroid in tissues. The ceroid accumulation primarily affects the brain, leading to neuronal loss and progressive neurodegeneration. Although several causative genes have been identified (DNAJC5, CLN6, CTSF, GRN, CLN1, CLN5, ATP13A2), the genetic underpinnings of ANCL in some families remain unknown. Here we report one family with autosomal dominant (AD) Kufs disease caused by a 30 bp in-frame duplication in DNAJC5, encoding the cysteine-string protein alpha (CSPα). This variant leads to a duplication of the central core motif of the cysteine-string domain of CSPα and affects palmitoylation-dependent CSPα sorting in cultured neuronal cells similarly to two previously described CSPα variants, p.(Leu115Arg) and p.(Leu116del). Interestingly, the duplication was not detected initially by standard Sanger sequencing due to a preferential PCR amplification of the shorter wild-type allele and allelic dropout of the mutated DNAJC5 allele. It was also missed by subsequent whole-exome sequencing (WES). Its identification was facilitated by reanalysis of original WES data and modification of the PCR and Sanger sequencing protocols. Independently occurring variants in the genomic sequence of DNAJC5 encoding the cysteine-string domain of CSPα suggest that this region may be more prone to DNA replication errors and that insertions or duplications within this domain should be considered in unsolved ANCL cases.

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.

Aim: Genetic variants affecting statin uptake, metabolism or predisposing to muscular diseases may confer susceptibility to statin-induced myopathy. Besides the SLCO1B1 rs4149056 genotype, common genetic variants do not seem to determine statin-associated myopathy. Here we aimed to address the potential role of rare variants. Methods: We performed whole exome sequencing in 88 individuals suffering from statin-associated myopathy and assessed the burden of rare variants using candidate-gene and exome-wide association analysis. Results: In the novel candidate gene CLCN1, we identified a heterozygote truncating mutation p.R894* in four patients. In addition, we detected predictably pathogenic case-specific variants in MYOT, CYP3A5, SH3TC2, FBXO32 and RBM20. Conclusion: These findings support the role of rare variants and nominate loci for follow-up studies.

Autosomal dominant tubulointerstitial kidney disease caused by mucin-1 gene (We performed MUC1fs immunostaining on urinary cell smears and various tissues from ADTKD-After technique refinement, the sensitivity and specificity for MUC1fs immunostaining of urinary cell smears were 94.2% and 88.6%, respectively. Further genetic testing on 17 families with positive MUC1fs immunostaining revealed six families with five novelWe developed a noninvasive immunohistochemical method to detect MUC1fs that, after further validation, may be useful in the future for diagnostic testing. Production of the MUC1fs protein may be central to the pathogenesis of ADTKD

The genetic correlates of extreme impulsive violence are poorly understood, and there have been no studies that have systematically characterized a large group of affected individuals both clinically and genetically. We performed a genome-wide rare copy number variant (CNV) analysis in 281 males from four Czech prisons who met strict clinical criteria for extreme impulsive violence. Inclusion criteria included age ≥ 18 years, an ICD-10 diagnosis of Dissocial Personality Disorder, and the absence of an organic brain disorder. Participants underwent a structured psychiatric assessment to diagnose extreme impulsive violence and then provided a blood sample for genetic analysis. DNA was genotyped and CNVs were identified using Illumina HumanOmni2.5 single-nucleotide polymorphism array platform. Comparing with 10851 external population controls, we identified 828 rare CNVs (frequency ≤ 0.1% among control samples) in 264 participants. The CNVs impacted 754 genes, with 124 genes impacted more than once (2-25 times). Many of these genes are associated with autosomal dominant or X-linked disorders affecting adult behavior, cognition, learning, intelligence, specifically expressed in the brain and relevant to synapses, neurodevelopment, neurodegeneration, obesity and neuropsychiatric phenotypes. Specifically, we identified 31 CNVs of clinical relevance in 31 individuals, 59 likely clinically relevant CNVs in 49 individuals, and 17 recurrent CNVs in 65 individuals. Thus, 123 of 281 (44%) individuals had one to several rare CNVs that were indirectly or directly relevant to impulsive violence. Extreme impulsive violence is genetically heterogeneous and genomic analysis is likely required to identify, further research and specifically treat the causes in affected individuals.

Contains fulltext : 167296.pdf (Publisher’s version ) (Open Access) 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.

Secondary hyperparathyroidism is a well-known complication of end-stage renal disease (ESRD). Both nodular and diffuse parathyroid hyperplasia occur in ESRD patients. However, their distinct molecular mechanisms remain poorly understood. Parathyroid tissue obtained from ESRD patients who had undergone parathyroidectomy was used for Illumina transcriptome screening and subsequently for discriminatory gene analysis, pathway mapping, and gene annotation enrichment analysis. Results were further validated using quantitative RT-PCR on the independent larger cohort. Microarray screening proved homogeneity of gene transcripts in hemodialysis patients compared with the transplant cohort and primary hyperparathyroidism; therefore, further experiments were performed in hemodialysis patients only. Enrichment analysis conducted on 485 differentially expressed genes between nodular and diffuse parathyroid hyperplasia revealed highly significant differences in Gene Ontology terms and the Kyoto Encyclopedia of Genes and Genomes database in ribosome structure ( P = 3.70 × 10−18). Next, quantitative RT-PCR validation of the top differently expressed genes from microarray analysis proved higher expression of RAN guanine nucleotide release factor (RANGRF; P < 0.001), calcyclin-binding protein ( CACYBP; P < 0.05), and exocyst complex component 8 ( EXOC8; P < 0.05) and lower expression of peptidylprolyl cis/ trans-isomerase and NIMA-interacting 1 (PIN1; P < 0.01) mRNA in nodular hyperplasia. Multivariate analysis revealed higher RANGRF and lower PIN1 expression along with parathyroid weight to be associated with nodular hyperplasia. In conclusion, our study suggests the RANGRF transcript, which controls RNA metabolism, to be likely involved in pathways associated with the switch to nodular parathyroid growth. This transcript, along with PIN1 transcript, which influences parathyroid hormone secretion, may represent new therapeutical targets to cure secondary hyperparathyroidism.

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 (CSPα). These mutationscausing a deletion, p.Leu116del, and an amino acid exchange, p.Leu115Arg, respectivelyare located within the cysteine-string domain of the protein and affect both palmitoylation-dependent sorting and the amount of CSPα in neuronal cells. The resulting depletion of functional CSPα 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 CSPα in humans and demonstrates the need for detailed investigation of CSPα in the neuronal ceroid lipofuscinoses and other neurodegenerative diseases presenting with neuronal protein aggregation.

In the supplementary information PDF originally posted, there were discrepancies from the integrated supplementary information that appeared in the HTML; the former has been corrected as follows. In the legend to Supplementary Fig. 2c, "major organs of the mouse" has been changed to "major organs of the adult mouse." In the legend to Supplementary Fig. 6d,h, "At E14.5 Mbe/Mbe mutants have a smaller percentage of Brdu positive cells in bin 3" has been changed to "At E14.5 Mbe/Mbe mutants have a higher percentage of Brdu positive cells in bin 3."

Through linkage analysis and candidate gene sequencing, we identified three unrelated families with the autosomal-dominant inheritance of early onset anemia, hypouricosuric hyperuricemia, progressive kidney failure, and mutations resulting either in the deletion (p.Leu16del) or the amino acid exchange (p.Leu16Arg) of a single leucine residue in the signal sequence of renin. Both mutations decrease signal sequence hydrophobicity and are predicted by bioinformatic analyses to damage targeting and cotranslational translocation of preprorenin into the endoplasmic reticulum (ER). Transfection and in vitro studies confirmed that both mutations affect ER translocation and processing of nascent preprorenin, resulting either in reduced (p.Leu16del) or abolished (p.Leu16Arg) prorenin and renin biosynthesis and secretion. Expression of renin and other components of the renin-angiotensin system was decreased accordingly in kidney biopsy specimens from affected individuals. Cells stably expressing the p.Leu16del protein showed activated ER stress, unfolded protein response, and reduced growth rate. It is likely that expression of the mutant proteins has a dominant toxic effect gradually reducing the viability of renin-expressing cells. This alters the intrarenal renin-angiotensin system and the juxtaglomerular apparatus functionality and leads to nephron dropout and progressive kidney failure. Our findings provide insight into the functionality of renin-angiotensin system and stress the importance of renin analysis in families and individuals with early onset hyperuricemia, anemia, and progressive kidney failure.

Blindness due to retinal degeneration affects millions of people worldwide, but many disease-causing mutations remain unknown. ​PNPLA6 encodes the ​patatin-like phospholipase domain containing protein 6, also known as ​neuropathy target esterase (​NTE), which is the target of toxic organophosphates that induce human paralysis due to severe axonopathy of large neurons. Mutations in ​PNPLA6 also cause human spastic paraplegia characterized by motor neuron degeneration. Here we identify ​PNPLA6 mutations in childhood blindness in seven families with retinal degeneration, including Leber congenital amaurosis and Oliver McFarlane syndrome. ​PNPLA6 localizes mostly at the inner segment plasma membrane in photoreceptors and mutations in Drosophila ​PNPLA6 lead to photoreceptor cell death. We also report that lysophosphatidylcholine and lysophosphatidic acid levels are elevated in mutant Drosophila. These findings show a role for ​PNPLA6 in photoreceptor survival and identify phospholipid metabolism as a potential therapeutic target for some forms of blindness.

The significance of borderline changes in kidney allograft biopsies is widely debated. To help resolve this, we studied differences in intrarenal gene expression patterns between early clinical and 3-month protocol biopsies, all of which had borderline histologic changes. The gene expression profiles in training set of patients by microarray analysis and data were validated in a larger cohort using RT-qPCR. There was greater expression of immunity- and inflammation-related genes in the early clinical biopsies compared to the 3-month protocol biopsies with borderline changes. In early clinically manifested borderline changes, graft deterioration within 24 months due to chronic rejection was associated with increased activation of immune, defense, and inflammatory processes. Regression modeling identified higher donor age and expression of macrophage receptor CLEC5A as risk factors for progression. In the 3-month protocol biopsies with borderline changes, graft dysfunction was associated with increased expression of fibrinogen complex transcripts. The discrimination power of fibrinogen was confirmed by cross-validation on two independent cohorts. Thus, our study highlights variations in gene expression between clinical and subclinical borderline changes despite similar histological findings. The data also support a recommendation for frequent patient monitoring, especially in those with borderline changes who received grafts from older donors.

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.

We carried out whole-genome homozygosity mapping, gene expression analysis and DNA sequencing in individuals with isolated mitochondrial ATP synthase deficiency and identified disease-causing mutations in TMEM70. Complementation of the cell lines of these individuals with wild-type TMEM70 restored biogenesis and metabolic function of the enzyme complex. Our results show that TMEM70 is involved in mitochondrial ATP synthase biogenesis in higher eukaryotes.

Mitochondrial disorders are caused by defects in mitochondrial or nuclear DNA. Although the existence of large deletions in mitochondrial DNA (mtDNA) is well known, deletions affecting whole genes are not commonly described in patients with mitochondrial disorders. Based on the results of whole-genome analyses, copy number variations (CNVs) occur frequently in the human genome and may overlap with many genes associated with clinical phenotypes. We report the discovery of two large heterozygous CNVs on 22q13.33 in two patients with mitochondrial disorders. The first patient harboured a novel point mutation c.667G>A (p.D223N) in the SCO2 gene in combination with a paternally inherited 87-kb deletion. As hypertrophic cardiomyopathy (HCMP) was not documented in the patient, this observation prompted us to compare his clinical features with all 44 reported SCO2 patients in the literature. Surprisingly, the review shows that HCMP was present in only about 50% of the SCO2 patients with non-neonatal onset. In the second patient, who had mitochondrial neurogastrointestinal encephalopathy (MNGIE), a maternally inherited 175-kb deletion and the paternally inherited point mutation c.261G>T (p.E87D) in the TYMP gene were identified.

Contains fulltext : 116575.pdf (Publisher’s version ) (Open Access) The genetic cause of GAPO syndrome, a condition characterized by growth retardation, alopecia, pseudoanodontia, and progressive visual impairment, has not previously been identified. We studied four ethnically unrelated affected individuals and identified homozygous nonsense mutations (c.262C>T [p.Arg88(*)] and c.505C>T [p.Arg169(*)]) or splicing mutations (c.1435-12A>G [p.Gly479Phefs(*)119]) in ANTXR1, which encodes anthrax toxin receptor 1. The nonsense mutations predictably trigger nonsense-mediated mRNA decay, resulting in the loss of ANTXR1. The transcript with the splicing mutation theoretically encodes a truncated ANTXR1 containing a neopeptide composed of 118 unique amino acids in its C terminus. GAPO syndrome's major phenotypic features, which include dental abnormalities and the accumulation of extracellular matrix, recapitulate those found in Antxr1-mutant mice and point toward an underlying defect in extracellular-matrix regulation. Thus, we propose that mutations affecting ANTXR1 function are responsible for this disease's characteristic generalized defect in extracellular-matrix homeostasis. 8 p.

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.

The neuronal ceroid lipofuscinoses (NCLs) are a group of rare genetic diseases characterised clinically by the progressive deterioration of mental, motor and visual functions and histopathologically by the intracellular accumulation of autofluorescent lipopigment – ceroid – in affected tissues. The NCLs are clinically and genetically heterogeneous and more than 14 genetically distinct NCL subtypes have been described to date (CLN1–CLN14) (Haltia and Goebel, 2012 [1]). In this review we will chronologically summarise work which has led over the years to identification of NCL genes, and outline the potential of novel genomic techniques and related bioinformatic approaches for further genetic dissection and diagnosis of NCLs. This article is part of a Special Issue entitled: The Neuronal Ceroid Lipofuscinoses or Batten Disease.

Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted by the liver into bile. Failure of this system can lead to a buildup of conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis of bilirubin excretion and hyperbilirubinemia syndromes is largely understood, but that of Rotor syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8 Rotor-syndrome families and found that Rotor syndrome was linked to mutations predicted to cause complete and simultaneous deficiencies of the organic anion transporting polypeptides OATP1B1 and OATP1B3. These important detoxification-limiting proteins mediate uptake and clearance of countless drugs and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1 polymorphisms have previously been linked to drug hypersensitivities. Using mice deficient in Oatp 1a/1b and in the multispecific sinusoidal export pump Abcc3, we found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp 1a/1b transporters mediate their hepatic reuptake. Transgenic expression of human OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this shuttle may allow flexible transfer of bilirubin conjugates (and probably also drug conjugates) formed in upstream hepatocytes to downstream hepatocytes, thereby preventing local saturation of further detoxification processes and hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may confer substantial drug toxicity risks

Background 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 F1Fo ATP 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. Results 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 ATP6/ATP8 defect in patients from the M group and indicated several candidate disease causing genes for nuclear defects. Conclusion 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.

Background: The cause of Rotor syndrome (RS), a rare-familial conjugated hyperbilirubinaemia with normal liver histology, is unclear. We hypothesized that RS can be an allelic variant of Dubin–Johnson syndrome, caused by mutation in ABCC2, and investigated ABCC2 (gene) and ABCC2 (protein) in two patients with RS. Methods: A 57-year-old male presented with a 5-year history of predominantly conjugated hyperbilirubinaemia (170 μmol/l). Urinary porphyrin excretion was increased; cholescintigraphy revealed no chromoexcretion. A 68-year-old male presented with lifelong conjugated hyperbilirubinaemia (85 μmol/l). Bromosulfophthalein elimination was typical for RS. Both patients had histologically normal liver, without pigment. ABCC2 expression was investigated by confocal fluorescence microscopy. ABCC2 was sequenced from genomic DNA and cDNA, and exon deletions/duplications were sought by comparative genomic hybridization on a custom micro-array. Results: In both patients, ABCC2 was expressed unremarkably at the apical membrane of hepatocytes and no sequence alterations were found in 32 exons, adjacent intronic regions and the promoter region of ABCC2. Conclusions: Rotor-type hyperbilirubinaemia is not an allelic variant of ABCC2 deficiency.

Contains fulltext : 50018.pdf (Publisher’s version ) (Closed access) Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.

The loss of Surf1 protein leads to a severe COX deficiency manifested as a fatal neurodegenerative disorder, the Leigh syndrome (LSCOX). Surf1 appears to be involved in the early step of COX assembly but its function remains unknown. The aim of the study was to find out how SURF1 gene mutations influence expression of OXPHOS and other pro-mitochondrial genes and to further characterize the altered COX assembly. Analysis of fibroblast cell lines from 9 patients with SURF1 mutations revealed a 70% decrease of the COX complex content to be associated with 32–54% upregulation of respiratory chain complexes I, III and V and accumulation of Cox5a subunit. Whole genome expression profiling showed a general decrease of transcriptional activity in LSCOX cells and indicated that the adaptive changes in OXPHOS complexes are due to a posttranscriptional compensatory mechanism. Electrophoretic and WB analysis showed that in mitochondria of LSCOX cells compared to controls, the assembled COX is present entirely in a supercomplex form, as I–III2–IV supercomplex but not as larger supercomplexes. The lack of COX also caused an accumulation of I–III2 supercomplex. The accumulated Cox5a was mainly present as a free subunit. We have found out that the major COX assembly subcomplexes accumulated due to SURF1 mutations range in size between approximately 85–140kDa. In addition to the originally proposed S2 intermediate they might also represent Cox1-containing complexes lacking other COX subunits. Unlike the assembled COX, subcomplexes are unable to associate with complexes I and III.

Early onset mitochondrial encephalo-cardiomyopathy due to isolated deficiency of ATP synthase is frequently caused by mutations in TMEM70 gene encoding enzyme-specific ancillary factor. Diminished ATP synthase results in low ATP production, elevated mitochondrial membrane potential and increased ROS production. To test whether the patient cells may react to metabolic disbalance by changes in oxidative phosphorylation system, we performed a quantitative analysis of respiratory chain complexes and intramitochondrial proteases involved in their turnover. SDS- and BN-PAGE Western blot analysis of fibroblasts from 10 patients with TMEM70 317-2A > G homozygous mutation showed a significant 82–89% decrease of ATP synthase and 50–162% increase of respiratory chain complex IV and 22–53% increase of complex III. The content of Lon protease, paraplegin and prohibitins 1 and 2 was not significantly changed. Whole genome expression profiling revealed a generalized upregulation of transcriptional activity, but did not show any consistent changes in mRNA levels of structural subunits, specific assembly factors of respiratory chain complexes, or in regulatory genes of mitochondrial biogenesis which would parallel the protein data. The mtDNA content in patient cells was also not changed. The results indicate involvement of posttranscriptional events in the adaptive regulation of mitochondrial biogenesis that allows for the compensatory increase of respiratory chain complexes III and IV in response to deficiency of ATP synthase.

Some patients are susceptible to statin-associated myopathy (SAM) either because of genetic variations affecting statin uptake and metabolism, or because they predispose their carriers to muscular diseases. Among the frequent variants examined using the genome-wide association study approach, SLCO1B1 c.521T>C represents the only validated predictor of SAM in patients treated with high-dose simvastatin. Our aim was to ascertain the overall contribution of large copy-number variations (CNVs) to SAM diagnosed in 86 patients. CNVs were detected by whole genome genotyping using Illumina HumanOmni2.5 Exome BeadChips. Exome sequence data were used for validation of CNVs in SAM-related loci. In addition, we performed a specific search for CNVs in the SLCO1B region detected recently in Rotor syndrome subjects. Rare deletions possibly contributing to genetic predisposition to SAM were found in two patients: one removed EYS associated previously with SAM, the other was present in LARGE associated with congenital muscular dystrophy. Another two patients carried deletions in CYP2C19, which may predispose to clopidogrel-statin interactions. We found no common large CNVs potentially associated with SAM and no CNVs in the SLCO1B locus. Our findings suggest that large CNVs do not play a substantial role in the etiology of SAM.