Anna Maria Pinto

Phenylbutyrate increases SMN gene expression in spinal muscular atrophy patients

Spinal muscular atrophy (SMA) is caused by insufficient levels of survival motor neuron (SMN) protein. Recently, we found that sodium 4-phenylbutyrate (PB), a well-tolerated FDA approved drug, enhances SMN gene expression in vitro. We provide here the first evidence that oral administration of PB (triButyrate®) significantly increases SMN expression in leukocytes of SMA patients. This finding provides a strong rationale to further investigate the effects of PB as also supported by preliminary clinical data.

Alport syndrome: impact of digenic inheritance in patients management

Alport syndrome (ATS) is a genetically heterogeneous nephropathy with considerable phenotypic variability and different transmission patterns, including monogenic (X‐linked/autosomal) and digenic inheritance (DI). Here we present a new series of families with DI and we discuss the consequences for genetic counseling and risk assessment. Out of five families harboring variants in more than one COL4 gene detected by next generation sequencing (NGS), minigene‐splicing assay allowed us to identify four as true digenic. Two families showed COL4A3/A4 mutations in cis, mimicking an autosomal dominant inheritance with a more severe phenotype and one showed COL4A3/A4 mutations in trans, mimicking an autosomal recessive inheritance with a less severe phenotype. In a fourth family, a de novo mutation (COL4A5) combined with an inherited mutation (COL4A3) triggered a more severe phenotype. A fifth family, predicted digenic on the basis of silico tools, rather showed monogenic X‐linked inheritance due to a hypomorphic mutation, in accordance with a milder phenotype. In conclusion, this study highlights the impact of DI in ATS and explains the associated atypical presentations. More complex inheritance should be therefore considered when reviewing prognosis and recurrence risks. On the other side, these findings emphasize the importance to accompany NGS with splicing assays in order to avoid erroneous identification of at risk members.

Exploiting the potential of next-generation sequencing in genomic medicine.

ABSTRACT Introduction: The review highlights the impact of next-generation sequencing (NGS) on genomic medicine and the consequences of the progression from a single-gene panel technology to a whole exome sequencing approach. Areas covered: We brought together literature-based evidences, personal unpublished data and clinical experience to provide a critical overview of the impact of NGS on our daily clinical practice. Expert commentary: NGS has changed the role of clinical geneticist and has broadened the view accomplishing a transition from a monogenic Mendelian perspective to an oligogenic approach to disorders. Thus, it is a compelling new expertise which combines clinical evaluation with big omics data interpretation and moves forward to phenotype re-evaluation in light of data analysis. We introduced the term, ‘exotyping’, to highlight this holistic approach. Further, the review discusses the impact that the combination of genetic reprogramming and transcriptome analysis will have on the discovery of evidence-based therapies.

Sporadic hereditary motor and sensory neuropathies: Advances in the diagnosis using next generation sequencing technology

Hereditary motor and sensory neuropathies (HMSN) are genetically heterogeneous disorders affecting peripheral motor and sensory functions. Many different pathogenic variants in several genes involved in the demyelinating, the axonal and the intermediate HMSN forms have been identified, for which all inheritance patterns have been described. The mutation screening currently available is based on Sanger sequencing and is time-consuming and relatively expensive due to the high number of genes involved and to the absence of mutational hot spots. To overcome these limitations, we have designed a custom panel for simultaneous sequencing of 28 HMSN-related genes. We have applied this panel to three representative patients with variable HMSN phenotype and uncertain diagnostic classifications. Using our NGS platform we rapidly identified three already described pathogenic heterozygous variants in MFN2, MPZ and DNM2 genes. Here we show that our pre-custom platform allows a fast, specific and low-cost diagnosis in sporadic HMSN cases. This prompt diagnosis is useful for providing a well-timed treatment, establishing a recurrence risk and preventing further investigations poorly tolerated by patients and expensive for the health system. Importantly, our study illustrates the utility and successful application of NGS to mutation screening of a Mendelian disorder with extreme locus heterogeneity.

Compound heterozygous missense and deep intronic variants in NDUFAF6 unraveled by exome sequencing and mRNA analysis

Biallelic mutations in NDUFAF6 have been identified as responsible for cases of autosomal recessive Leigh syndrome associated with mitochondrial complex I deficiency. Here we report two siblings and two unrelated subjects with Leigh syndrome, in which we found the same compound heterozygous missense (c.532G>C:p.A178P) and deep intronic (c.420+784C>T) variants in NDUFAF6. We demonstrated that the identified intronic variant creates an alternative splice site, leading to the production of an aberrant transcript. A detailed analysis of whole-exome sequencing data together with the functional validation based on mRNA analysis may reveal pathogenic variants even in non-exonic regions.

Combined ultrasound and exome sequencing approach recognizes Opitz G/BBB syndrome in two malformed fetuses.

Orofacial clefts are the most common congenital craniofacial anomalies and can occur as an isolated defect or be associated with other anomalies such as posterior fossa anomalies as a part of several genetic syndromes. We report two consecutive voluntary pregnancy interruptions in a nonconsanguineous couple following the fetal ultrasound finding of cleft lip and palate and posterior fossa anomalies confirmed by means of post-termination examination on the second fetus. The quantitative fluorescent PCR, the karyotype, and the comparative genomic hybridization-array analysis after amniocentesis were normal. Exome sequencing on abortive material from both fetuses detected a missense mutation in MID1, resulting in a clinical diagnosis of Opitz G/BBB syndrome. The same mutation was found in the mother and in her brother, who both revealed cerebellar anomalies at an MRI examination. Our study supports the efficacy of exome sequencing in the presence of both a family history suggestive of an inherited disorder and well-documented ultrasound findings. It reveals the importance of a synergistic effort between gynecologists and geneticists aimed at the integration of the most sophisticated ultrasound techniques with the next-generation sequencing tools to provide a definite diagnosis essential to orient the final decision and to estimate a proper recurrence risk.

Urine-derived podocytes-lineage cells: A promising tool for precision medicine in Alport Syndrome

Alport Syndrome (ATS) is a rare genetic disorder caused by collagen IV genes mutations, leading to glomerular basement membrane damage up to end‐stage renal disease. Podocytes, the main component of the glomerular structure, are the only cells able to produce all the three collagens IV alpha chains associated with ATS and thus, they are key players in ATS pathogenesis. However, podocytes‐targeted therapeutic strategies have been hampered by the difficulty of non‐invasively isolating them and transcripts‐based diagnostic approaches are complicated by the inaccessibility of other COL4 chains‐expressing cells. We firstly isolated podocyte‐lineage cells from ATS patients’ urine samples, in a non‐invasive way. RT‐PCR analysis revealed COL4A3, COL4A4, and COL4A5 expression. Transcripts analysis on RNA extracted from patients urine derived podocyte‐lineage cells allowed defining the pathogenic role of intronic variants, namely one mutation in COL4A3 (c.3882+5G>A), three mutations in COL4A4 (c.1623+2T>A, c.3699_3706+1del, c.2545+143T>A), and one mutation in COL4A5 (c.3454+2T>C). Therefore, our cellular model represents a novel tool, essential to unequivocally prove the effect of spliceogenic intronic variants on transcripts expressed exclusively at a glomerular level. This process is a key step for providing the patient with a definite molecular diagnosis and with a proper recurrence risk. The established system also opens up the possibility of testing personalized therapeutic approaches on disease‐relevant cells.

iPSC-derived neurons profiling reveals GABAergic circuit disruption and acetylated α-tubulin defect which improves after iHDAC6 treatment in Rett syndrome

ABSTRACT Mutations in MECP2 gene have been identified in more than 95% of patients with classic Rett syndrome, one of the most common neurodevelopmental disorders in females. Taking advantage of the breakthrough technology of genetic reprogramming, we investigated transcriptome changes in neurons differentiated from induced Pluripotent Stem Cells (iPSCs) derived from patients with different mutations. Profiling by RNA‐seq in terminally differentiated neurons revealed a prominent GABAergic circuit disruption along with a perturbation of cytoskeleton dynamics. In particular, in mutated neurons we identified a significant decrease of acetylated &agr;‐tubulin which can be reverted by treatment with selective inhibitors of HDAC6, the main &agr;‐tubulin deacetylase. These findings contribute to shed light on Rett pathogenic mechanisms and provide hints for the treatment of Rett‐associated epileptic behavior as well as for the definition of new therapeutic strategies for Rett syndrome. HIGHLIGHTSDevelopment of a patient‐specific neuronal model for Rett syndrome using iPSCs.GABAergic circuits up‐regulation in Rett iPSC‐derived neurons.Reduction of acetylated &agr;‐tubulin in Rett neurons, reverted by selective HDAC6 inhibitors.

Parent-of-origin effect of hypomorphic pathogenic variants and somatic mosaicism impact on phenotypic expression of retinoblastoma

Retinoblastoma is the most common eye cancer in children. Numerous families have been described displaying reduced penetrance and expressivity. An extensive molecular characterization of seven families led us to characterize the two main mechanisms impacting on phenotypic expression, as follows: (i) mosaicism of amorphic pathogenic variants; and (ii) parent-of-origin-effect of hypomorphic pathogenic variants. Somatic mosaicism for RB1 splicing variants (c.1960+5G>C and c.2106+2T>C), leading to a complete loss of function was demonstrated by high-depth NGS in two families. In both cases, the healthy carrier parent (one with retinoma) showed a variant frequency lower than that expected for a heterozygous individual, indicating a 56–60% mosaicism level. Previous evidences of a ~3-fold excess of RB1 maternal canonical transcript led us to hypothesize that this differential allelic expression could influence phenotypic outcome in families at risk for RB onset. Accordingly, in five families, we identified a higher tumor risk associated with paternally inherited hypomorphic pathogenic variants, namely a deletion resulting in the loss of 37 amino acids at the N-terminus (c.608-16_608del), an exonic substitution with a “leaky” splicing effect (c.1331A>G), a partially deleterious substitution (c.1981C>T) and a truncating C-terminal variant (c.2663+2T>C). The identification of these mechanisms changes the genetic/prenatal counseling and the clinical management of families, indicating a higher recurrence risk when the hypomorphic pathogenic variant is inherited from the father, and suggesting the need for second tumor surveillance in unaffected carriers at risk of developing adult-onset cancer such as osteosarcoma or leiomyosarcoma.

Low-level TP53 mutational load antecedes clonal expansion in chronic lymphocytic leukaemia

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