Anencephaly in Slovakia and Czech Republic: embryogenesis, risk factors, epidemiology and preventative approaches.

Anencephaly, a fatal anomaly of the central nervous system, belongs to the group of defects of the neural tube (NTDs). It is considered the most common congenital NTD, characterized by concurrent absence of a significant portion of the brain and cranial vault. This deformity occurs between days 23 and 26 after fertilization due to improper closure of the neural tube at its cranial end. Many genetic, epigenetic, and non-genetic factors (nutritional, environmental and geographical factors, parental socioeconomic status) contribute to the etiology of this disease. Despite significant advances in treatment and preventive measures, NTDs continue to pose a significant health and financial burden on patients and society as a whole. This study aimed to examine the incidence of anencephaly in Slovakia compared to the Czech Republic between 2012 and 2020. The authors seek to elucidate the reasons behind the higher incidence of this disease in Slovakia as compared to the Czech Republic, explore the male predominance of anencephaly in Slovakia, and investigate whether the prevention standards used in Slovakia differ from those employed in other countries (Tab. 1, Fig. 2, Ref. 129). Keywords: neural tube defects, anencephaly, risk factors, folic acid, food fortification.

A very early diagnosis of AlstrÓ§m syndrome by next generation sequencing.

BACKGROUND: Alström syndrome is a rare recessively inherited disorder caused by variants in the ALMS1 gene. It is characterized by multiple organ dysfunction, including cone-rod retinal dystrophy, dilated cardiomyopathy, hearing loss, obesity, insulin resistance, hyperinsulinemia, type 2 diabetes mellitus and systemic fibrosis. Heterogeneity and age-dependent development of clinical manifestations make it difficult to obtain a clear diagnosis, especially in pediatric patients. CASE PRESENTATION: Here we report the case of a girl with Alström syndrome. Genetic examination was proposed at age 22 months when suspected macular degeneration was the only major finding. Next generation sequencing of a panel of genes linked to eye-related pathologies revealed two compound heterozygous variants in the ALMS1 gene. Frameshift variants c.1196_1202del, p.(Thr399Lysfs*11), rs761292021 and c.11310_11313del, (p.Glu3771Trpfs*18), rs747272625 were detected in exons 5 and 16, respectively. Both variants cause frameshifts and generation of a premature stop-codon that probably leads to mRNA nonsense-mediated decay. Validation and segregation of ALMS1 variants were confirmed by Sanger sequencing. CONCLUSIONS: Genetic testing makes it possible, even in childhood, to increase the number of correct diagnoses of patients who have ambiguous phenotypes caused by rare genetic variants. The development of high-throughput sequencing technologies offers an exceptionally valuable screening tool for clear genetic diagnoses and ensures early multidisciplinary management and treatment of the emerging symptoms.

Pathogenicity of new BEST1 variants identified in Italian patients with best vitelliform macular dystrophy assessed by computational structural biology.

BACKGROUND: Best vitelliform macular dystrophy (BVMD) is an autosomal dominant macular degeneration. The typical central yellowish yolk-like lesion usually appears in childhood and gradually worsens. Most cases are caused by variants in the BEST1 gene which encodes bestrophin-1, an integral membrane protein found primarily in the retinal pigment epithelium. METHODS: Here we describe the spectrum of BEST1 variants identified in a cohort of 57 Italian patients analyzed by Sanger sequencing. In 13 cases, the study also included segregation analysis in affected and unaffected relatives. We used molecular mechanics to calculate two quantitative parameters related to calcium-activated chloride channel (CaCC composed of 5 BEST1 subunits) stability and calcium-dependent activation and related them to the potential pathogenicity of individual missense variants detected in the probands. RESULTS: Thirty-six out of 57 probands (63% positivity) and 16 out of 18 relatives proved positive to genetic testing. Family study confirmed the variable penetrance and expressivity of the disease. Six of the 27 genetic variants discovered were novel: p.(Val9Gly), p.(Ser108Arg), p.(Asn179Asp), p.(Trp182Arg), p.(Glu292Gln) and p.(Asn296Lys). All BEST1 variants were assessed in silico for potential pathogenicity. Our computational structural biology approach based on 3D model structure of the CaCC showed that individual amino acid replacements may affect channel shape, stability, activation, gating, selectivity and throughput, and possibly also other features, depending on where the individual mutated amino acid residues are located in the tertiary structure of BEST1. Statistically significant correlations between mean logMAR best-corrected visual acuity (BCVA), age and modulus of computed BEST1 dimerization energies, which reflect variations in the in CaCC stability due to amino acid changes, permitted us to assess the pathogenicity of individual BEST1 variants. CONCLUSIONS: Using this computational approach, we designed a method for estimating BCVA progression in patients with BEST1 variants.

MAGI-ACMG: Algorithm for the Classification of Variants According to ACMG and ACGS Recommendations.

We have developed MAGI-ACMG, a classification algorithm that allows the classification of sequencing variants (single nucleotide or small indels) according to the recommendations of the American College of Medical Genetics (ACMG) and the Association for Clinical Genomic Science (ACGS). The MAGI-ACMG classification algorithm uses information retrieved through the VarSome Application Programming Interface (API), integrates the AutoPVS1 tool in order to evaluate more precisely the attribution of the PVS1 criterion, and performs the customized assignment of specific criteria. In addition, we propose a sub-classification scheme for variants of uncertain significance (VUS) according to their proximity either towards the "likely pathogenic" or "likely benign" classes. We also conceived a pathogenicity potential criterion (P_POT) as a proxy for segregation criteria that might be added to a VUS after posterior testing, thus allowing it to upgrade its clinical significance in a diagnostic reporting setting. Finally, we have developed a user-friendly web application based on the MAGI-ACMG algorithm, available to geneticists for variant interpretation.

PacMAGI: A pipeline including accurate indel detection for the analysis of PacBio sequencing data applied to RPE65.

Third generation sequencing methods, like PacBio, provide information about structural variants, introns, enhancers and promoters. We developed an automated pipeline, called PacMAGI, including quality control, alignment, SNV, INDELs, structural variant calling, phasing, annotation and variant interpretation, for the analysis of PacBio data for any target region. Bi-allelic mutations in the RPE65 gene are associated with different inherited retinal dystrophies, such as Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Diagnostic panel-based NGS analysis is performed on coding regions and intron/exon junctions of genes. To obtain a more conclusive diagnosis, we applied PacMAGI to obtain a second hit on RPE65 in LCA or RP patients who showed a single heterozygous variant by NGS. We used PacBio to sequence the full gene and identify putative second-hits in intronic, problematic and promoter regions. All variants identified in the diagnostic setting with NGS were correctly detected by the pipeline, and thanks to our custom algorithm for INDELs, a previously undetected 'Pathogenic' frameshift variant was found in a RP patient already identified to carry a 'Likely Pathogenic' variant.

Acetylcholine receptor pathway mutations explain various fetal akinesia deformation sequence disorders.

Impaired fetal movement causes malformations, summarized as fetal akinesia deformation sequence (FADS), and is triggered by environmental and genetic factors. Acetylcholine receptor (AChR) components are suspects because mutations in the fetally expressed gamma subunit (CHRNG) of AChR were found in two FADS disorders, lethal multiple pterygium syndrome (LMPS) and Escobar syndrome. Other AChR subunits alpha1, beta1, and delta (CHRNA1, CHRNB1, CHRND) as well as receptor-associated protein of the synapse (RAPSN) previously revealed missense or compound nonsense-missense mutations in viable congenital myasthenic syndrome; lethality of homozygous null mutations was predicted but never shown. We provide the first report to our knowledge of homozygous nonsense mutations in CHRNA1 and CHRND and show that they were lethal, whereas novel recessive missense mutations in RAPSN caused a severe but not necessarily lethal phenotype. To elucidate disease-associated malformations such as frequent abortions, fetal edema, cystic hygroma, or cardiac defects, we studied Chrna1, Chrnb1, Chrnd, Chrng, and Rapsn in mouse embryos and found expression in skeletal muscles but also in early somite development. This indicates that early developmental defects might be due to somite expression in addition to solely muscle-specific effects. We conclude that complete or severe functional disruption of fetal AChR causes lethal multiple pterygium syndrome whereas milder alterations result in fetal hypokinesia with inborn contractures or a myasthenic syndrome later in life.

Recessive multiple epiphyseal dysplasia and Stargardt disease in two sisters.

BACKGROUND: The rapid spread of genome-wide next-generation sequencing in the molecular diagnosis of rare genetic disorders has produced increasing evidence of multilocus genomic variations in cases with a previously well-characterized molecular diagnosis. Here, we describe two patients with a rare combination of skeletal abnormalities and retinal dystrophy caused by variants in the SLC26A2 and ABCA4 genes, respectively, in a family with parental consanguinity. METHODS: Next-generation sequencing and Sanger sequencing were performed to obtain a molecular diagnosis for the retinal and skeletal phenotypes, respectively. RESULTS: Genetic testing revealed that the sisters were homozygous for the p.(Cys653Ser) variant in SLC26A2 and heterozygous for the missense p.(Pro68Leu) and splice donor c.6386+2C>G variants in ABCA4. Segregation analysis confirmed the carrier status of the parents. CONCLUSION: Despite low frequency of occurrence, the detection of multilocus genomic variations in a single disease gene-oriented approach can provide accurate diagnosis even in cases with high phenotypic complexity. A targeted sequencing approach can detect relationships between observed phenotypes and underlying genotypes, useful for clinical management.

Variant Selection and Interpretation: An Example of Modified VarSome Classifier of ACMG Guidelines in the Diagnostic Setting.

Variant interpretation is challenging as it involves combining different levels of evidence in order to evaluate the role of a specific variant in the context of a patient's disease. Many in-depth refinements followed the original 2015 American College of Medical Genetics (ACMG) guidelines to overcome subjective interpretation of criteria and classification inconsistencies. Here, we developed an ACMG-based classifier that retrieves information for variant interpretation from the VarSome Stable-API environment and allows molecular geneticists involved in clinical reporting to introduce the necessary changes to criterion strength and to add or exclude criteria assigned automatically, ultimately leading to the final variant classification. We also developed a modified ACMG checklist to assist molecular geneticists in adjusting criterion strength and in adding literature-retrieved or patient-specific information, when available. The proposed classifier is an example of integration of automation and human expertise in variant curation, while maintaining the laboratory analytical workflow and the established bioinformatics pipeline.

Modeling the effect of 3 missense AGXT mutations on dimerization of the AGT enzyme in primary hyperoxaluria type 1.

INTRODUCTION: Mutations of the AGXT gene encoding the alanine:glyoxylate aminotransferase liver enzyme (AGT) cause primary hyperoxaluria type 1 (PH1). Here we report a molecular modeling study of selected missense AGXT mutations: the common Gly170Arg and the recently described Gly47Arg and Ser81Leu variants, predicted to be pathogenic using standard criteria. METHODS: Taking advantage of the refined 3D structure of AGT, we computed the dimerization energy of the wild-type and mutated proteins. RESULTS: Molecular modeling predicted that Gly47Arg affects dimerization with a similar effect to that shown previously for Gly170Arg through classical biochemical approaches. In contrast, no effect on dimerization was predicted for Ser81Leu. Therefore, this probably demonstrates pathogenic properties via a different mechanism, similar to that described for the adjacent Gly82Glu mutation that affects pyridoxine binding. CONCLUSION: This study shows that the molecular modeling approach can contribute to evaluating the pathogenicity of some missense variants that affect dimerization. However, in silico studies--aimed to assess the relationship between structural change and biological effects--require the integrated use of more than 1 tool.

Genetic analysis of intellectual disability and autism.

BACKGROUND AND AIM: Intellectual disability (ID) and autism spectrum disorders (ASD) are neurodevelopmental conditions that often co-exist and affect children from birth, impacting on their cognition and adaptive behaviour. Social interaction and communication ability are also severely impaired in ASD. Almost 1-3% of the population is affected and it has been estimated that approximately 30% of intellectual disability and autism is caused by genetic factors. The aim of this review is to summarize monogenic conditions characterized by intellectual disability and/or autism for which the causative genes have been identified. METHODS AND RESULTS: We identified monogenic ID/ASD conditions through PubMed and other NCBI databases. Many such genes are located on the X chromosome (>150 out of 900 X-linked protein-coding genes), but at least 2000 human genes are estimated to be involved in ID/ASD. We selected 174 genes (64 X-linked and 110 autosomal) for an NGS panel in order to screen patients with ID and/or ASD, after fragile X syndrome and significant Copy Number Variants have been excluded. CONCLUSIONS: Accurate clinical and genetic diagnosis is required for precise treatment of these disorders, but due to their genetic heterogeneity, most cases remain undiagnosed. Next generation sequencing technologies have greatly enhanced the identification of new genes associated with intellectual disability and autism, ultimately leading to the development of better treatment options.

Expanding the phenotype of thrombocytopenia absent radius syndrome with hypospadias.

Rare genetic diseases and syndromes may appear with unique features in some patients. In genetically-solved cases, this situation indicates a phenotypic expansion of the syndrome with additional features (i.e. the disease-associated gene gives rise to unusual clinical presentation). However, this situation can also hide a multilocus pathogenic variation that cannot be solved genetically except by a massive sequencing approach, such as exome sequencing. Here we describe the case of a child with bilateral radial aplasia, transient thrombocytopenia and anemia, cow's milk intolerance, hypospadias, facial dysmorphism, mild hypothyroidism and umbilical and inguinal hernia. Bilaterally absent radius, presence of thumbs and low platelet count are pathognomonic of thrombocytopenia absent radius (TAR) syndrome, but the child also showed other features beyond those reported in the literature. Since various diseases resembling the proband's phenotype required differential diagnosis, clinical exome sequencing was performed. The results showed compound heterozygous mutations in the RBM8A gene, confirming the suspicion of TAR syndrome. A truncating heterozygous variant in the DUOX2 gene, known to be associated with transient thyroid dyshormonogenesis type 6 (TDH6), was also detected and may explain the proband's mild hypothyroidism.

Hydroxytyrosol: A natural compound with promising pharmacological activities.

Hydroxytyrosol is a phenolic phytochemical with antioxidant properties in vitro. It is a natural compound that can be found in olive leaves and oil. The main dietary source of hydroxytyrosol is extra virgin olive oil. Due to its bioavailability, chemical properties and easy formulation along with its lack of toxicity, hydroxytyrosol is considered an excellent food supplement by the nutraceutical and food industries. The purpose of this review is to discuss the potential therapeutic effects of hydroxytyrosol in vivo. To do so, we conducted an electronic search in PubMed and other literature databases using "hydroxytyrosol", "beneficial effect/s", "pharmacology" as key-words. From this search, we found that hydroxytyrosol has anti-inflammatory, anti-tumor, antiviral, antibacterial and antifungal properties. Hydroxytyrosol also improves endothelial dysfunction, decreases oxidative stress, and is neuro- and cardio-protective. Due to all these biological properties, hydroxytyrosol is currently the most actively investigated natural phenol. The evidence presented in this review suggests that hydroxytyrosol has great pharmacological potential.

Oguchi type I caused by a homozygous missense variation in the SAG gene.

Oguchi disease, is a very rare form of night blindness caused by biallelic variations in the SAG or GRK1 genes, both involved in rod restoration after light stimuli. Here we report the clinical and genetic findings of an 8-year old boy with a history of reduced visual acuity, nyctalpia and hemeralopia. Clinical findings, in particular the Mizuo-Nakamura phenomenon, were compatible with a diagnosis of Oguchi disease. Genetic testing revealed a novel missense homozygous variation in the SAG gene. This is the first evidence that the disease can be caused by missense variations in this gene.

Diagnostic and therapeutic implements based on advanced Biotechnology should be available in low-income countries.

N.A.

Syndromic infertility.

Infertility due to genetic mutations that cause other defects, besides infertility, is defined as syndromic. Here we describe three of these disorders for which we perform genetic tests. 1) Hypopituitarism is an endocrine syndrome characterized by reduced or absent secretion of one or more anterior pituitary hormones with consequent dysfunction of the corresponding peripheral glands. Deficiencies in all the hormones is defined as pan-hypopituitarism, lack of two or more hormones is called partial hypopituitarism, whereas absence of a single hormone is defined as selective hypopituitarism. Pan-hypopituitarism is the rarest condition, whereas the other two are more frequent. Several forms exist: congenital, acquired, organic and functional. 2) The correct functioning of the hypothalamic-pituitary-gonadal axis is fundamental for sexual differentiation and development during fetal life and puberty and for normal gonad function. Alteration of the hypothalamic-pituitary system can determine a condition called hypogonadotropic hypogonadism, characterized by normal/low serum levels of the hormones FSH and LH. 3) Primary ciliary dyskinesia is frequently associated with infertility in males because it impairs sperm motility (asthenozoospermia). Primary ciliary dyskinesia is a group of genetically and phenotypically heterogeneous disorders that show morpho-structural alterations of the cilia. Adult women with primary ciliary dyskinesia can be subfertile and have an increased probability of extra-uterine pregnancies. This is due to delayed transport of the oocyte through the uterine tubes.

A SALL4 zinc finger missense mutation predicted to result in increased DNA binding affinity is associated with cranial midline defects and mild features of Okihiro syndrome.

Truncating mutations of the gene SALL4 on chromosome 20q13.13-13.2 cause Okihiro and acro-renal-ocular syndromes. Pathogenic missense mutations within the SALL4 or SALL1 genes have not yet been reported, raising the question which phenotypic features would be associated with them. Here we describe the first missense mutation within the SALL4 gene. The mutation results in an exchange of a highly conserved zinc-coordinating Histidine crucial for zinc finger (ZF) structure within a C2H2 double ZF domain to an Arginine. Molecular modeling predicts that this exchange does not result in a loss of zinc ion binding but leads to an increased DNA-binding affinity of the domain. The index patient shows mild features of Okihiro syndrome, but in addition cranial midline defects (pituitary hypoplasia and single central incisor). This finding illustrates that the phenotypic and functional effects of SALL4 missense mutations are difficult to predict, and that other SALL4 missense mutations might lead to phenotypes not overlapping with Okihiro syndrome.

Supernumerary ring chromosome 8: clinical and molecular cytogenetic characterization in a case report.

We report on a 3-year-old male with developmental delay, autistic behavior, and minor abnormalities consistent with trisomy 8 syndrome whose cytogenetic analysis revealed mosaicism for a supernumerary ring chromosome (SRC). Fluorescence in situ hybridization (FISH) studies, using centromeric and yeast artificial chromosome (YAC) probes, were performed to characterize further the supernumerary chromosome. The ring origin has been detected from the short arm of chromosome 8, resulting in r(8)(p10p23.1). Moreover, uniparental disomy (UPD) using microsatellite analysis was excluded. To our knowledge a total of 25 cases, confirmed by FISH, have been reported with either supernumerary marker or ring chromosome 8. We present a detailed clinical and molecular cytogenetic characterization of this additional case in order to better define the genotype-phenotype correlation.