Inherited metabolic disorders in Cyprus.

Selective screening for inherited metabolic disorders (IMD) began in Cyprus in 1990. Over the last thirty-three years 7388 patients were investigated for IMD and 200 diagnoses were made (diagnostic yield 2.7%). The existence of a single laboratory of Biochemical Genetics for the whole island facilitated the creation of a national registry for IMD. The minimal prevalence of IMD in Cyprus is 53.3 cases per 100,000 live births. The most common group are disorders of amino acid metabolism (41.0%), followed by disorders of carbohydrate metabolism (16.5%), disorders of complex molecule degradation (16.5%), mitochondrial disorders (10.5%) and disorders of vitamin and co-factor metabolism (5.5%). Hyperphenylalaninaemia is the most common IMD (14.0%) followed by galactosaemia (7.0%), glutaric aciduria type I (5.5%) and MSUD (4.0%). Some disorders were found to have a relatively high incidence in specific communities, for example Sandhoff disease among the Cypriot Maronites and GM1 gangliosidosis in one particular area of the island. Other disorders were found to have a relatively higher overall incidence, compared to other Caucasian populations, for example galactosaemia, glutaric aciduria type I and MSUD, while fatty acid oxidation defects, Gaucher disease and classic PKU were found to have a relatively lower incidence. Molecular characterization of selected disorders revealed many novel genetic variants, specific to the Cypriot population.

A novel large intragenic DPYD deletion causing dihydropyrimidine dehydrogenase deficiency: a case report.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD), is the initial and rate-limiting enzyme in the catabolic pathway of pyrimidines. Deleterious variants in the DPYD gene cause DPD deficiency, a rare autosomal recessive disorder. The clinical spectrum of affected individuals is wide ranging from asymptomatic to severely affected patients presenting with intellectual disability, motor retardation, developmental delay and seizures. DPD is also important as the main enzyme in the catabolism of 5-fluorouracil (5-FU) which is extensively used as a chemotherapeutic agent. Even in the absence of clinical symptoms, individuals with either complete or partial DPD deficiency face a high risk of severe and even fatal fluoropyrimidine-associated toxicity. The identification of causative genetic variants in DPYD is therefore gaining increasing attention due to their potential use as predictive markers of fluoropyrimidine toxicity. METHODS: A male infant patient displaying biochemical features of DPD deficiency was investigated by clinical exome sequencing. Bioinformatics tools were used for data analysis and results were confirmed by MLPA and Sanger sequencing. RESULTS: A novel intragenic deletion of 71.2 kb in the DPYD gene was identified in homozygosity. The deletion, DPYD(NM_000110.4):c.850 + 23455_1128 + 8811del, eliminates exons 9 and 10 and may have resulted from a non-homologous end-joining event, as suggested by in silico analysis. CONCLUSIONS: The study expands the spectrum of DPYD variants associated with DPD deficiency. Furthermore, it raises the concern that patients at risk for fluoropyrimidine toxicity due to DPYD deletions could be missed during pre-treatment genetic testing for the currently recommended single nucleotide polymorphisms.

GAA variants associated with reduced enzymatic activity but lack of Pompe-related symptoms, incidentally identified by exome sequencing.

Pompe disease is a rare metabolic myopathy caused by pathogenic variants affecting the activity of the lysosomal glycogen-degrading enzyme acid alpha-glucosidase (GAA). Impaired GAA function results in the accumulation of undegraded glycogen within lysosomes in multiple tissues but predominantly affects the skeletal, smooth and cardiac muscle. The degree of residual enzymatic activity appears to roughly correlate with the age of onset and the severity of the clinical symptoms. Here, we report four siblings in which the GAA variants NM_000152.5:c.2237G > C p.(Trp746Ser) and NM_000152.5:c.266G > A p.(Arg89His) were identified as an incidental finding of clinical exome sequencing. These variants are listed in the ClinVar and the Pompe disease GAA variant databases but are reported here for the first time in compound heterozygosity. All four siblings displayed normal urine tetrasaccharide levels and no clinical manifestations related to Pompe disease. Nevertheless, GAA enzymatic activity was within the range for late onset Pompe patients. Our report shows an association between a novel genotype and attenuated GAA enzymatic activity. The clinical significance can only be established by the regular monitoring of these individuals. The study highlights the major challenges for clinical care arising from incidental findings of next generation sequencing.

Acid Ceramidase Depletion Impairs Neuronal Survival and Induces Morphological Defects in Neurites Associated with Altered Gene Transcription and Sphingolipid Content.

The ASAH1 gene encodes acid ceramidase (AC), an enzyme that is implicated in the metabolism of ceramide (Cer). Mutations in the ASAH1 gene cause two different disorders, Farber disease (FD), a rare lysosomal storage disorder, and a rare form of spinal muscular atrophy combined with progressive myoclonic epilepsy (SMA-PME). In the absence of human in vitro neuronal disease models and to gain mechanistic insights into pathological effects of ASAH1 deficiency, we established and characterized a stable ASAH1 knockdown (ASAH1KD) SH-SY5Y cell line. ASAH1KD cells displayed reduced proliferation due to elevated apoptosis and G1/S cell cycle arrest. Distribution of LAMP1-positive lysosomes towards the cell periphery and significantly shortened and less branched neurites upon differentiation, implicate AC for lysosome positioning and neuronal development, respectively. Lipidomic analysis revealed changes in the intracellular levels of distinct sphingolipid species, importantly without Cer accumulation, in line with altered gene transcription within the sphingolipid pathway. Additionally, the transcript levels for Rho GTPases (RhoA, Rac1, and Cdc42), which are key regulators of axonal orientation, neurite branching and lysosome positioning were found to be dysregulated. This study shows the critical role of AC in neurons and suggests how AC depletion leads to defects seen in neuropathology of SMA-PME and FD.

A novel mutation deep within intron 7 of the GBA gene causes Gaucher disease.

BACKGROUND: Mutations in the GBA gene that encodes the lysosomal enzyme acid ß-glucocerebrosidase cause Gaucher disease (GD), the most common lysosomal storage disorder. Most of the mutations are missense/nonsense, however, a few splicing mutations within or close to conserved consensus donor or acceptor splice sites have also been described. The aim of the study was to identify the mutation(s) in a Cypriot patient with type I GD. METHODS: The genomic DNA of the proband was screened for nine common mutations using Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. All exons and exon-intron boundaries, and the 5'UTR and 3'UTR regions of the GBA gene, were investigated by Sanger sequencing. RNA analysis was performed using standard procedures, and the abnormal transcript was further cloned into pGEM-T-Easy plasmid vector and sequenced. The relevant intronic region was further sequenced by the Sanger method to identify the genetic variant. RESULTS: A novel point mutation, g.12599C > A (c.999 + 242C > A), was detected deep in intron 7 of the GBA gene. This type of mutation has been previously described for other diseases but this is the first time, as far as we know, that it is described for GD. This mutation creates a new donor splice site leading to aberrant splicing and resulting in the insertion of the first 239nt of intron 7 as a pseudoexon in the mRNA, creating a premature stop codon. CONCLUSION: This study expands the mutation spectrum of GD and highlights the importance of RNA sequencing for the molecular diagnosis of patients bearing mutations in nonexonic regions.

Biochemical Characterization of the GBA2 c.1780G>C Missense Mutation in Lymphoblastoid Cells from Patients with Spastic Ataxia.

The GBA2 gene encodes the non-lysosomal glucosylceramidase (NLGase), an enzyme that catalyzes the conversion of glucosylceramide (GlcCer) to ceramide and glucose. Mutations in GBA2 have been associated with the development of neurological disorders such as autosomal recessive cerebellar ataxia, hereditary spastic paraplegia, and Marinesco-Sjogren-Like Syndrome. Our group has previously identified the GBA2 c.1780G>C [p.Asp594His] missense mutation, in a Cypriot consanguineous family with spastic ataxia. In this study, we carried out a biochemical characterization of lymphoblastoid cell lines (LCLs) derived from three patients of this family. We found that the mutation strongly reduce NLGase activity both intracellularly and at the plasma membrane level. Additionally, we observed a two-fold increase of GlcCer content in LCLs derived from patients compared to controls, with the C16 lipid being the most abundant GlcCer species. Moreover, we showed that there is an apparent compensatory effect between NLGase and the lysosomal glucosylceramidase (GCase), since we found that the activity of GCase was three-fold higher in LCLs derived from patients compared to controls. We conclude that the c.1780G>C mutation results in NLGase loss of function with abolishment of the enzymatic activity and accumulation of GlcCer accompanied by a compensatory increase in GCase.

hCINAP is an atypical mammalian nuclear adenylate kinase with an ATPase motif: structural and functional studies.

Human coilin interacting nuclear ATPase protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins UsnRNPs and snoRNPs. hCINAP has previously been designated as an adenylate kinase (AK6), but is very atypical as it exhibits unusually broad substrate specificity, structural features characteristic of ATPase/GTPase proteins (Walker motifs A and B) and also intrinsic ATPase activity. Despite its intriguing structure, unique properties and cellular localization, the enzymatic mechanism and biological function of hCINAP have remained poorly characterized. Here, we offer the first high-resolution structure of hCINAP in complex with the substrate ADP (and dADP), the structure of hCINAP with a sulfate ion bound at the AMP binding site, and the structure of the ternary complex hCINAP-Mg(2+) ADP-Pi. Induced fit docking calculations are used to predict the structure of the hCINAP-Mg(2+) ATP-AMP ternary complex. Structural analysis suggested a functional role for His79 in the Walker B motif. Kinetic analysis of mutant hCINAP-H79G indicates that His79 affects both AK and ATPase catalytic efficiency and induces homodimer formation. Finally, we show that in vivo expression of hCINAP-H79G in human cells is toxic and drastically deregulates the number and appearance of CBs in the cell nucleus. Our findings suggest that hCINAP may not simply regulate nucleotide homeostasis, but may have broader functionality, including control of CB assembly and disassembly in the nucleus of human cells.

The nuclear ATPase/adenylate kinase hCINAP is recruited to perinucleolar caps generated upon RNA pol.II inhibition.

hCINAP is an atypical nucleoplasmic enzyme, combining structural features of adenylate kinases and ATPases, which exhibits dual enzymatic activity. It interacts with the Cajal Body marker coilin and its level of expression and enzymatic activity influence Cajal Body numbers. Here we show that upon specific transcriptional inhibition of RNA pol.II, hCINAP segregates in perinuclear caps identified as Dark Nucleolar Caps (DNCs). These are distinct from perinucleolar caps where coilin and fibrillarin (both Cajal Body components) accumulate. In DNCs, hCINAP co-localizes with Paraspeckle Protein (PSP1) and also co-segregates with PSP1, and not coilin, in nuclear and nucleolar foci upon UV irradiation.

Characterization of hCINAP, a novel coilin-interacting protein encoded by a transcript from the transcription factor TAFIID32 locus.

Coilin is a marker protein for the Cajal body, a subnuclear domain acting as a site for assembly and maturation of nuclear RNA-protein complexes. Using a yeast two-hybrid screen to identify coilin-interacting proteins, we have identified hCINAP (human coilin interacting nuclear ATPase protein), a nuclear factor of 172 amino acids with a P-loop nucleotide binding motif and ATPase activity. The hCINAP protein sequence is highly conserved across its full-length from human to plants and yeast and is ubiquitously expressed in all human tissues and cell lines tested. The yeast orthologue of CINAP is a single copy, essential gene. Tagged hCINAP is present in complexes containing coilin in mammalian cells and recombinant, Escherichia coli expressed hCINAP binds directly to coilin in vitro. The 214 carboxyl-terminal residues of coilin appear essential for the interaction with hCINAP. Both immunofluorescence and fluorescent protein tagging show that hCINAP is specifically nuclear and distributed in a widespread, diffuse nucleoplasmic pattern, excluding nucleoli, with some concentration also in Cajal bodies. Overexpression of hCINAP in HeLa cells results in a decrease in the average number of Cajal bodies per nucleus, consistent with it affecting either the stability of Cajal bodies and/or their rate of assembly. The hCINAP mRNA is an alternatively spliced transcript from the TAF9 locus, which encodes the basal transcription factor subunit TAFIID32. However, hCINAP and TAFIID32 mRNAs are translated from different ATG codons and use distinct reading frames, resulting in them having no identity in their respective protein sequences.