Multiplex Ligation-dependent Probe Amplification analysis of Copy Number Variants in mentally retarded patients

The aetiology of mental retardation and dysmorphism is still poorly understood in over half of the affected individuals. Recent studies have shown that genomic mutations such as Copy Number Variants (CNV) can be important factors predisposing to the highly heterogeneous clinical conditions observed in affected individuals. Genome−wide array−Comparative Genomic Hybridization (aCGH) is the best technology available so far for a first screening of CNVs in genomic DNA in patients and control individuals. However, CNVs detected by aCGH need validation by an independent method such as Multiplex Ligation-dependent Probe Amplification (MLPA). In this report we present the results of a MLPA analysis performed on 120 mentally retarded patients and their parents and 400 samples from a reference population. Patients who underwent MLPA were previously scored positive to aCGH. Our results show that, overall, from the 150 CNVs identified by aCGH, 123 were confirmed by our MLPA analysis and overall account for 65 gains (size range: from 3,6 Kb to 3,0 Mb) and 58 losses (size range: from 2,2 Kb to 1,9 Mb). At least 16 CNVs are de novo (11 loss, 5 gain). Inheritance from a healthy parent could be established only for 83 CNVs. For 24 CNVs it was not possible to establish if they were de novo or inherited because one or both parents were unavailable for the analysis. In the majority of cases, a given CNV appear to be unique to a particular patient as far their chromosomal location and size is concerned. In contrast five 3 CNVs are shared each by two patients. One CNV_loss in chromosome 15q11.2 was shared by six unrelated patients. To investigate on the possible pathogenic role played by CNVs in this phenotypically and genetically heterogeneous population of mentally retarded patients and thus select the strongest candidate CNVs we have applied a multi-step strategy. Briefly, we first excluded CNVs also occurring in the general population. To this end, we screened by MLPA 400 neurotypical geographically- and ethnically-matched individuals. Second only CNVs occurring as de novo events were considered. Third, we queried a large body of data stored in the literature and in several databases to exclude CNVs previously associated to mental retardation (MR) or described as phenotypically unexpressed polymorphisms in the general population. The best candidate CNVs selected as just described were then analyzed for their gene content and to assess by in silico functional annotation analysis the role of these genes in nervous system. This approach led in some cases to the identification of several CNV, genes and statistically significant ontologies related to nervous system thus supporting their pathogenic role. A result of special interest was the identification in one MR patient with a complex phenotype of a duplicated microRNA (hsa-miR-150) overlapping a CNV on chromosome 19. The functions and diseases associated to the individual target genes/mRNA regulated by this microRNA appear to be highly correlated to the clinical phenotypes displayed 4 by this patient and suggest the occurrence of a new MR syndrome caused by a duplicated microRNA. In summary, the results of our study: (i) emphasize the importance of MLPA to exclude false positives generated by aCGH, (ii) describe the identification of novel CNVs and genes potentially implicated in MR. We believe that the results of this study will significantly contribute to the discovery of new microdeletion syndromes and a more precise correlation of the mutant genotype with the highly variable phenotype displayed by mentally retarded patients

LDOC1 expression in fibroblasts of patients with Down syndrome

Abstract Down syndrome (DS) is characterised by intellectual disability and is caused by trisomy 21. Apoptosis is a programmed cell death process and is involved in neurodegenerative diseases such as Alzheimer. People with DS can develop some traits of Alzheimer disease at an earlier age than subjects without trisomy 21. The leucine zipper, down regulated in cancer 1 (LDOC1) appears to be involved in the apoptotic pathways. The aim of the present work was to detect the presence of intracellular synthesis of LDOC1 protein and LDOC1 mRNA in fibroblast cultures from DS subjects. The western blot shows the presence of LDOC1 protein in fibroblasts of DS subjects but no evidence of LDOC1 protein in fibroblasts of normal subjects. LDOC1 gene mRNA expression is increased in fibroblasts from DS subjects compared to fibroblasts from normal subjects. The data obtained from this study strengthen the hypothesis that the over-expression of LDOC1 gene could play a role in determining the phenotype of individuals with DS but does not exclude that this results from apoptotic mechanisms

A de novo mutation of KRT1 in a baby girl causing epidermolytic ichthyosis with impressive epidermolytic palmoplantar keratoderma

We report a 6-year-old girl showing epidermolytic ichthyosis/epidermolytic hyperkeratosis (EI/EH). Targeted Next Generation Sequencing revealed a de novo, previously unidentified KRT1 mutation. The findings of this study expands the clinical and  spectrum and genotype-phenotype correlation associated with EI/EH

Implementation of Sample Pooling Procedure Using a Rapid SARS-CoV-2 Diagnostic Real-Time PCR Test Performed Prior to Hospital Admission of People with Intellectual Disabilities

Reliability, accuracy, and timeliness of diagnostic testing for SARS-CoV-2 infection have allowed adequate public health management of the disease, thus notably helping the timely mapping of viral spread within the community. Furthermore, the most vulnerable populations, such as people with intellectual disability and dementia, represent a high-risk group across multiple dimensions, including a higher prevalence of pre-existing conditions, lower health maintenance, and a propensity for rapid community spread. This led to an urgent need for reliable in-house rapid testing to be performed prior to hospital admission. In the present study, we describe a pooling procedure in which oropharyngeal and nasopharyngeal swabs for SARS-CoV-2 detection (performed prior to hospital admission using rapid RT-PCR assay) are pooled together at the time of sample collection. Sample pooling (groups of 2–4 samples per tube) allowed us to significantly reduce response times, consumables, and personnel costs while maintaining the same test sensitivity

Allelic Variations in the Human Genes TMPRSS2 and CCR5, and the Resistance to Viral Infection by SARS-CoV-2

During the first wave of COVID-19 infection in Italy, the number of cases and the mortality rates were among the highest compared to the rest of Europe and the world. Several studies demonstrated a severe clinical course of COVID-19 associated with old age, comorbidities, and male gender. However, there are cases of virus infection resistance in subjects living in close contact with infected subjects. Thus, to explain the predisposition to virus infection and to COVID-19 disease progression, we must consider, in addition to the genetic variability of the virus and other environmental or comorbidity conditions, the allelic variants of specific human genes, directly or indirectly related to the life cycle of the virus. Here, we analyzed three human genetic polymorphisms belonging to the TMPRSS2 and CCR5 genes in a sample population from Sicily (Italy) to investigate possible correlations with the resistance to viral infection and/or to COVID-19 disease progression as recently described in other human populations. Our results did not show any correlations of the rs35074065, rs12329760, and rs333 polymorphisms with SARS-CoV-2 infection or with COVID-19 disease severity. Further studies on other human genetic polymorphisms should be performed to identify the major human determinants of SARS-CoV-2 viral resistance

Next Generation Sequencing and Electromyography Reveal the Involvement of the <i>P2RX6</i> Gene in Myopathy

Ion channelopathies result from impaired ion channel protein function, due to mutations affecting ion transport across cell membranes. Over 40 diseases, including neuropathy, pain, migraine, epilepsy, and ataxia, are associated with ion channelopathies, impacting electrically excitable tissues and significantly affecting skeletal muscle. Gene mutations affecting transmembrane ionic flow are strongly linked to skeletal muscle disorders, particularly myopathies, disrupting muscle excitability and contraction. Electromyography (EMG) analysis performed on a patient who complained of weakness and fatigue revealed the presence of primary muscular damage, suggesting an early-stage myopathy. Whole exome sequencing (WES) did not detect potentially causative variants in known myopathy-associated genes but revealed a novel homozygous deletion of the P2RX6 gene likely disrupting protein function. The P2RX6 gene, predominantly expressed in skeletal muscle, is an ATP-gated ion channel receptor belonging to the purinergic receptors (P2RX) family. In addition, STRING pathways suggested a correlation with more proteins having a plausible role in myopathy. No previous studies have reported the implication of this gene in myopathy. Further studies are needed on patients with a defective ion channel pathway, and the use of in vitro functional assays in suppressing P2RX6 gene expression will be required to validate its functional role

Prader–Willi Syndrome with Angelman Syndrome in the Offspring

We report the second case, to the best of our knowledge, of a mother with Prader–Willi syndrome (PWS) who gave birth to a daughter with Angelman syndrome (AS). The menarche occurred when she was 16, and the following menstrual cycles were irregular, but she never took sexual hormone replacement therapy. At the age of 26, our patient with PWS became pregnant. The diagnosis was confirmed by molecular genetic testing that revealed a ~5.7 Mb deletion in the 15q11.1–15q13 region on the paternal allele in the mother with PWS and the maternal one in the daughter with AS, respectively. Both the mother with PWS and the daughter with AS showed peculiar clinical and genetic features of the two syndromes. Our case report reaffirms the possible fertility in PWS; therefore, it is very important to develop appropriate socio-sexual education programs and fertility assessments in order to guarantee the expression of a healthy sexuality

Novel deletion of the E3A ubiquitin protein ligase gene detected by multiplex ligation-dependent probe amplification in a patient with Angelman syndrome

Angelman syndrome (AS) is a severe neurobehavioural disorder caused by failure of expression of the maternal copy of the imprinted domain located on 15q11-q13. There are different mechanisms leading to AS: maternal microdeletion, uniparental disomy, defects in a putative imprinting centre, mutations of the E3 ubiquitin protein ligase (UBE3A) gene. However, some of suspected cases of AS are still scored negative to all the latter mutations. Recently, it has been shown that a proportion of negative cases bear large deletions overlapping one or more exons of the UBE3A gene. These deletions are difficult to detect by conventional gene-scanning methods due to the masking effect by the non-deleted allele. In this study, we have used for the first time multiplex ligation-dependent probe amplification (MLPA) and comparative multiplex dosage analysis (CMDA) to search for large deletions affecting the UBE3A gene. Using this approach, we identified a novel causative deletion involving exon 8 in an affected sibling. Based on our results, we propose the use of MLPA as a fast, accurate and inexpensive test to detect large deletions in the UBE3A gene in a small but significant percentage of AS patients

Exome sequencing in a child with neurodevelopmental disorder and epilepsy: Variant analysis of the AHNAK2 gene

Background: The AHNAK2 gene encodes a large nucleoprotein expressed in several tissues, including brain, squamous epithelia, smooth muscle, and neuropil. Its role in calcium signaling has been suggested and to date, clear evidence about its involvement in the pathogenesis of clinical disorders is still lacking.Methods: Here, we report a female 24-year-old patient diagnosed with a cardio-facio-cutaneous-like phenotype (CFC-like), characterized by epilepsy, psychomotor development delay, atopic dermatitis, congenital heart disease, hypotonia, and facial dysmorphism, who is compound heterozygote for two missense mutations in the AHNAK2 gene detected by exome sequencing.Results: This patient had no detectable variant in any of the genes known to be associated with the cardio-facia-cutaneous syndrome. Moreover, the mode of inheritance does not appear to be autosomal dominant, as it is in typical CFC syndrome. We have performed in silico assessment of mutation severity separately for each missense mutation, but this analysis excludes a severe effect on protein function. Protein structure predictions indicate the mutations are located in flexible regions possibly involved in molecular interactions.Conclusion: We discuss an alternative interpretation on the potential involvement of the two missense mutations in the AHNAK2 gene on the expression of CFC-like phenotype in this patient based on inter-allelic complementation

LDOC1 expression in fibroblasts of patients with Down syndrome

Down syndrome (DS) is characterised by intellectual disability and is caused by trisomy 21. Apoptosis is a programmed cell death process and is involved in neurodegenerative diseases such as Alzheimer. People with DS can develop some traits of Alzheimer disease at an earlier age than subjects without trisomy 21. The leucine zipper, down regulated in cancer 1 (LDOC1) appears to be involved in the apoptotic pathways. The aim of the present work was to detect the presence of intracellular synthesis of LDOC1 protein and LDOC1 mRNA in fibroblast cultures from DS subjects. The western blot shows the presence of LDOC1 protein in fibroblasts of DS subjects but no evidence of LDOC1 protein in fibroblasts of normal subjects. LDOC1 gene mRNA expression is increased in fibroblasts from DS subjects compared to fibroblasts from normal subjects. The data obtained from this study strengthen the hypothesis that the over-expression of LDOC1 gene could play a role in determining the phenotype of individuals with DS but does not exclude that this results from apoptotic mechanisms