28 research outputs found
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
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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