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.

Deep Learning for Instance Retrieval: A Survey.

In recent years a vast amount of visual content has been generated and shared from many fields, such as social media platforms, medical imaging, and robotics. This abundance of content creation and sharing has introduced new challenges, particularly that of searching databases for similar content - Content Based Image Retrieval (CBIR) - a long-established research area in which improved efficiency and accuracy are needed for real-time retrieval. Artificial intelligence has made progress in CBIR and has significantly facilitated the process of instance search. In this survey we review recent instance retrieval works that are developed based on deep learning algorithms and techniques, with the survey organized by deep feature extraction, feature embedding and aggregation methods, and network fine-tuning strategies. Our survey considers a wide variety of recent methods, whereby we identify milestone work, reveal connections among various methods and present the commonly used benchmarks, evaluation results, common challenges, and propose promising future directions.

Rhythmic Haptic Cueing Using Wearable Devices as Physiotherapy for Huntington Disease: Case Study.

BACKGROUND: Huntington disease (HD) is an inherited genetic disorder that results in the death of brain cells. HD symptoms generally start with subtle changes in mood and mental abilities; they then degenerate progressively, ensuing a general lack of coordination and an unsteady gait, ultimately resulting in death. There is currently no cure for HD. Walking cued by an external, usually auditory, rhythm has been shown to steady gait and help with movement coordination in other neurological conditions. More recently, work with other neurological conditions has demonstrated that haptic (ie, tactile) rhythmic cues, as opposed to audio cues, offer similar improvements when walking. An added benefit is that less intrusive, more private cues are delivered by a wearable device that leaves the ears free for conversation, situation awareness, and safety. This paper presents a case study where rhythmic haptic cueing (RHC) was applied to one person with HD. The case study has two elements: the gait data we collected from our wearable devices and the comments we received from a group of highly trained expert physiotherapists and specialists in HD. OBJECTIVE: The objective of this case study was to investigate whether RHC can be applied to improve gait coordination and limb control in people living with HD. While not offering a cure, therapeutic outcomes may delay the onset or severity of symptoms, with the potential to improve and prolong quality of life. METHODS: The approach adopted for this study includes two elements, one quantitative and one qualitative. The first is a repeated-measures design with three conditions: before haptic rhythm (ie, baseline), with haptic rhythm, and after exposure to haptic rhythm. The second element is an in-depth interview with physiotherapists observing the session. RESULTS: In comparison to the baseline, the physiotherapists noted a number of improvements to the participant's kinematics during her walk with the haptic cues. These improvements continued in the after-cue condition, indicating some lasting effects. The quantitative data obtained support the physiotherapists' observations. CONCLUSIONS: The findings from this small case study, with a single participant, suggest that a haptic metronomic rhythm may have immediate, potentially therapeutic benefits for the walking kinematics of people living with HD and warrants further investigation.

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.

Chitotriosidase deficiency in the Cypriot population: Identification of a novel deletion in the CHIT1 gene.

OBJECTIVES: The purpose of this study was to determine the normal range of chitotriosidase activity in the Cypriot population and the frequency of the 24bp duplication polymorphism. Furthermore, we compared the allele frequency of this polymorphism in two locations with different malaria endemicity in the past. DESIGN AND METHODS: Plasma chitotriosidase activity was measured using a fluorogenic substrate. The 24bp polymorphism was detected using PCR analysis of exon 10 of the CHIT1 gene. Additional mutations were detected using direct sequencing. RESULTS: The normal range of chitotriosidase activity was found to be 9.5-44.0nmol/ml/hr. Among 114 normal individuals genotyped for the 24bp duplication, 7% were found to be homozygous, 36% heterozygous and 57% wild type (allele frequency 0.25). The allele frequency of this polymorphism in individuals originating from two locations with different malaria endemicity in the past was not significantly different. A novel deletion mutation in the CHIT1 gene was identified associated with loss of chitotriosidase activity. This new deletion eliminates 29 nucleotides from exon 9 resulting in the generation of a premature stop codon, probably leading to the production of an aberrant protein molecule. CONCLUSIONS: The normal range of chitotriosidase activity and the allele frequency of the 24bp duplication polymorphism in the Cypriot population were found to be similar to those of other Mediterranean populations. No evidence for an association between the presence of the 24bp duplication polymorphism and susceptibility to malaria was found. A novel deletion in exon 9 of the CHIT1 gene was identified (allele frequency 0.01).

Novel GLA Deletion in a Cypriot Female Presenting with Cornea Verticillata.

Fabry disease is an X-linked lysosomal storage disorder resulting from a deficiency of the hydrolytic enzyme α-galactosidase A (α-Gal-A). It is characterized by progressive lysosomal accumulation of globotriaosylceramide (Gb3) and multisystem pathology, affecting the skin, nervous and cerebrovascular systems, kidneys, and heart. Heterozygous females typically exhibit milder symptoms and a later age of onset than males. Rarely, they may be relatively asymptomatic throughout a normal life span or may have symptoms as severe as those observed in males with the classic phenotype. We report on a 17-year-old female in whom cornea verticillata was found during a routine ophthalmological examination but with no other clinical symptoms. Leucocyte α-galactosidase activity was within the overlap range between Fabry heterozygotes and normal controls. Sanger sequencing of the GLA gene failed to reveal any pathogenic variants. Multiplex Ligation-dependent Probe Amplification (MLPA) analysis revealed a deletion of exon 7. Using a long-range PCR walking approach, we managed to identify the deletion breakpoints. The deletion spans 1182 bp, with its 5' end located within exon 6 of the GLA gene and its 3' end located 612 bp downstream of exon 7. This finding represents a novel deletion identified in the first reported Cypriot female carrier of Fabry disease.

Molecular analysis of Cypriot patients with Glutaric aciduria type I: identification of two novel mutations.

OBJECTIVES: The purpose of this study was to identify the mutations in the glutaryl-CoA dehydrogenase gene (GCDH) in ten Cypriot patients with Glutaric aciduria type I (GAI). DESIGN AND METHODS: Molecular analysis of the GCDH gene was performed by direct sequencing of the patients' genomic DNA. In silico tools were applied to predict the effect of the novel variants on the structure and function of the protein. RESULTS: All disease alleles were characterized (mutation detection rate 100%). Five missense mutations were identified: c.192G>T (p.Glu64Asp) and c.803G>T (p.Gly268Val), which are novel, and three previously described mutations, c.1123T>C (p.Cys375Arg), c.1204C>T (p.Arg402Trp) and c.1286C>T (p.Thr429Met). CONCLUSIONS: Two novel mutations, p.Glu64Asp and p.Gly268Val, account for the majority of disease alleles (76.5%) in Cypriot patients with Glutaric aciduria type I. A founder effect for the p.Glu64Asp and the p.Gly268Val can be suggested based on the place of origin of the carriers of these mutations. Identification of the causative mutations of GAI in Cypriot patients will facilitate carrier detection as well as post- and pre-natal diagnosis.

The first family with Tay-Sachs disease in Cyprus: Genetic analysis reveals a nonsense (c.78G>A) and a silent (c.1305C>T) mutation and allows preimplantation genetic diagnosis.

Tay-Sachs disease (TSD) is a recessively inherited neurodegenerative disorder caused by mutations in the HEXA gene resulting in ß-hexosaminidase A (HEX A) deficiency and neuronal accumulation of GM2 ganglioside. We describe the first patient with Tay-Sachs disease in the Cypriot population, a juvenile case which presented with developmental regression at the age of five. The diagnosis was confirmed by measurement of HEXA activity in plasma, peripheral leucocytes and fibroblasts. Sequencing the HEXA gene resulted in the identification of two previously described mutations: the nonsense mutation c.78G>A (p.Trp26X) and the silent mutation c.1305C>T (p.=). The silent mutation was reported once before in a juvenile TSD patient of West Indian origin with an unusually mild phenotype. The presence of this mutation in another juvenile TSD patient provides further evidence that it is a disease-causing mutation. Successful preimplantation genetic diagnosis (PGD) and prenatal follow-up were provided to the couple.

The spectrum of mutations identified in Cypriot patients with phenylalanine hydroxylase deficiency detected through neonatal screening.

OBJECTIVES: The purpose of this study was to identify the mutations responsible for phenylalanine hydroxylase deficiency in Cypriot patients detected through neonatal screening. DESIGN AND METHODS: Analysis of the PAH gene was performed by direct sequencing of the patients' genomic DNA, MLPA analysis and real-time PCR. RESULTS: Among 22 independent alleles thirteen previously described mutations were detected (detection rate 100%), all in compound heterozygosity: p.Arg395Gly (18.2%), c.168+5G>C (13.6%), p.EX3del (9%), c.1066-11G>A (9%), p.Ala403Val (9%), p.Glu178Gly (9%), p.Ser70Pro (4.5%), p.Arg241His (4.5%), p.Phe55fs (4.5%), p.Arg158Gln (4.5%), p.Asp222Gly (4.5%), p.Ala300Ser (4.5%), p.Pro225Thr (4.5%). Of the ten different genotypes, three have been previously reported to be associated with a mild clinical phenotype and to respond to tetrahydrobiopterin (BH4) administration. CONCLUSIONS: Marked genetic heterogeneity was found in Cypriot patients with hyperphenylalaninemia with two mutations accounting for 32% of the alleles. Most of the mutations detected have been found in other European and Mediterranean populations.

Maple syrup urine disease in Cypriot families: identification of three novel mutations and biochemical characterization of the p.Thr211Met mutation in the E1alpha subunit.

We report five mutations, three of them novel, responsible for maple syrup urine disease in four unrelated Cypriot families. The five children studied are the first cases of classic maple syrup urine disease to be reported among Cypriots. The first novel mutation identified is a single-base deletion in exon 6 of the Elalpha gene (c.718delG), which leads to a frameshift after Ala240 and to a stop codon 89 residues further downstream. The other two novel mutations identified are in the Elbeta subunit: a two-base deletion in exon 6, c.662_663delCC, which leads to a frameshift after Ala221 and creates a stop codon 17 residues further downstream, as well as a splice mutation, IVS3[+3]delA, which results in the skipping of exon 3. The two known mutations identified are in the Elalpha gene: the G > C transversion at the 3'-splice acceptor site, (IVS5-1G > C), which results in the deletion of the entire exon 6, and the missense mutation in exon 5 (c.632C > T), which corresponds to a p.Thr211Met substitution. The p.Thr211Met substitution is located in a potassium-ion pocket in the E1 component required for stability of the bound cofactor thiamine diphosphate. The mutant E1 protein harboring the p.Thr211Met substitution was shown unable to bind thiamine diphosphate, leading to undetectable E1 activity.

The Arg482His mutation in the beta-galactosidase gene is responsible for a high frequency of GM1 gangliosidosis carriers in a Cypriot village.

GM1 gangliosidosis is a lysosomal storage disorder caused by deficiency of beta-galactosidase. It is mainly characterized by progressive neurodegeneration, and in its most severe infantile form, it leads to death before the age of 4. The GLB1 gene gives rise to two alternatively spliced mRNAs that encode the beta-galactosidase and the elastin binding protein (EBP). The diagnosis of two patients with the infantile form of GM1 gangliosidosis and 11 carriers in a small mountainous village in Cyprus prompted us to carry out a study in order to establish the frequency of carriers in the village and identify the mutations involved. Carrier detection was initially based on the measurement of beta-galactosidase activity in leucocytes. Among 85 random samples from the village, 10 were classified as carriers. Sequencing of the GLB1 gene in a Cypriot patient identified the missense mutation c.1445G>A (p.Arg482His) in the homozygous state. Seven of the 10 carriers identified using the enzyme assay were found to carry the same mutation by NspI restriction enzyme analysis. The three individuals who were negative for the c.1445G>A had borderline enzyme results and were probably wrongly classified as carriers. The frequency of GM1 gangliosidosis carriers in this village is approximately 8% (1:12). Western blot analysis showed a marked decrease of the 64-kDa mature form of the enzyme protein and a similar reduction of the 67-kDa EBP. Our results indicate that the c.1445G>A mutation, which appears to be responsible for all GM1 gangliosidosis alleles in this Cypriot village, affects protein conformation.

Magnetic resonance imaging findings and novel mutations in GM1 gangliosidosis.

Two unrelated children and their siblings of Arab origin were diagnosed as having GM1 gangliosidosis on the basis of clinical features and markedly low levels of beta-galactosidase. The T2-weighted magnetic resonance images of the brain revealed certain characteristic features, including delayed myelination and abnormal appearance of the subcortical white matter, internal capsule, and basal ganglia. Their mutation analysis showed two novel mutations, which have not been described in an Arabic population.