The complex landscape of DMD mutations: moving towards personalized medicine.

Duchenne muscular dystrophy (DMD) is a severe genetic disorder characterized by progressive muscle degeneration, with respiratory and cardiac complications, caused by mutations in the DMD gene, encoding the protein dystrophin. Various DMD mutations result in different phenotypes and disease severity. Understanding genotype/phenotype correlations is essential to optimize clinical care, as mutation-specific therapies and innovative therapeutic approaches are becoming available. Disease modifier genes, trans-active variants influencing disease severity and phenotypic expressivity, may modulate the response to therapy, and become new therapeutic targets. Uncovering more disease modifier genes via extensive genomic mapping studies offers the potential to fine-tune prognostic assessments for individuals with DMD. This review provides insights into genotype/phenotype correlations and the influence of modifier genes in DMD.

An atypical Aymé-Gripp phenotype detected by exome sequencing.

Aymé-Gripp Syndrome (AGS) is an ultra-rare syndrome characterized by peculiar facial traits combined with early bilateral cataracts, sensorineural hearing loss, and variable neurodevelopmental abnormalities. Only a few cases carrying a pathogenic variant in MAF have been described to date. A significant effort is then required to expand the genotypic and phenotypic spectrum of this condition. In this paper, we report the peculiar case of a 6-year-old girl carrying a de novo missense pathogenic variant in MAF, being the first case reported to show a milder phenotype with no cataracts and deafness displayed. Furthermore, we performed a systematic review of previously published cases, focusing on clinical manifestation and genotype.

A new genetic cause of spastic ataxia: the p.Glu415Lys variant in TUBA4A.

Tubulinopathies encompass neurodevelopmental disorders caused by mutations in genes encoding for different isotypes of α- and ß-tubulins, the structural components of microtubules. Less frequently, mutations in tubulins may underlie neurodegenerative disorders. In the present study, we report two families, one with 11 affected individuals and the other with a single patient, carrying a novel, likely pathogenic, variant (p. Glu415Lys) in the TUBA4A gene (NM_006000). The phenotype, not previously described, is that of spastic ataxia. Our findings widen the phenotypic and genetic manifestations of TUBA4A variants and add a new type of spastic ataxia to be taken into consideration in the differential diagnosis.

Alu-Mediated Insertions in the DMD Gene: A Difficult Puzzle to Interpret Clinically.

Disrupting variants in the DMD gene are associated with Duchenne or Becker muscular dystrophy (DMD/BMD) or with hyperCKemia, all of which present very different degrees of clinical severity. The clinical phenotypes of these disorders could not be distinguished in infancy or early childhood. Accurate phenotype prediction based on DNA variants may therefore be required in addition to invasive tests, such as muscle biopsy. Transposon insertion is one of the rarest mutation types. Depending on their position and characteristics, transposon insertions may affect the quality and/or quantity of dystrophin mRNA, leading to unpredictable alterations in gene products. Here, we report the case of a three-year-old boy showing initial skeletal muscle involvement in whom we characterized a transposon insertion (Alu sequence) in exon 15 of the DMD gene. In similar cases, the generation of a null allele is predicted, resulting in a DMD phenotype. However, mRNA analysis of muscle biopsy tissue revealed skipping of exon 15, which restored the reading frame, thus predicting a milder phenotype. This case is similar to very few others already described in the literature. This case further enriches our knowledge of the mechanisms perturbing splicing and causing exon skipping in DMD, helping to properly guide clinical diagnosis.

Functional analysis of three new alpha-thalassemia deletions involving MCS-R2 reveals the presence of an additional enhancer element in the 5' boundary region.

We report three novel deletions involving the Multispecies Conserved Sequences (MCS) R2, also known as the Major Regulative Element (MRE), in patients showing the α-thalassemia phenotype. The three new rearrangements showed peculiar positions of the breakpoints. 1) The (αα)ES is a telomeric 110 kb deletion ending inside the MCS-R3 element. 2) The (αα)FG, 984 bp-long, ends 51 bp upstream to MCS-R2; both are associated with a severe α-thalassemia phenotype. 3) The (αα)CT, 5058 bp-long starts at position +93 of MCS-R2 and is the only one associated to a mild α-thalassemia phenotype. To understand the specific role of different segments of the MCS-R2 element and of its boundary regions we carried out transcriptional and expression analysis. Transcriptional analysis of patients' reticulocytes showed that (αα)ES was unable to produce α2-globin mRNA, while a high level of expression of the α2-globin genes (56%) was detected in (αα)CT deletion, characterized by the presence of the first 93 bp of MCS-R2. Expression analysis of constructs containing breakpoints and boundary regions of the deletions (αα)CT and (αα)FG, showed comparable activity both for MCS-R2 and the boundary region (-682/-8). Considering that the (αα)CT deletion, almost entirely removing MCS-R2, has a less severe phenotype than the (αα)FG α0thalassemia deletion, removing both MCS-R2 almost entirely and an upstream 679 bp, we infer for the first time that an enhancer element must exist in this region that helps to increase the expression of the α-globin genes. The genotype-phenotype relationship of other previously published MCS-R2 deletions strengthened our hypothesis.

Next-Generation Sequencing (NGS) Analysis Illustrates the Phenotypic Variability of Collagen Type IV Nephropathies.

Mutations in COL4A3-A5 cause a spectrum of glomerular disorders, including thin basement membrane nephropathy (TBMN) and Alport syndrome (AS). The wide application of next-generation sequencing (NGS) in the last few years has revealed that mutations in these genes are not limited to these clinical entities. In this study, 176 individuals with a clinical diagnosis of inherited kidney disorders underwent an NGS-based analysis to address the underlying cause; those who changed or perfected the clinical diagnosis after molecular analysis were selected. In 5 out of 83 individuals reaching a molecular diagnosis, the genetic result was unexpected: three individuals showed mutations in collagen type IV genes. These patients showed the following clinical pictures: (1) familial focal segmental glomerulosclerosis; (2) end-stage renal disease (ESRD) diagnosed incidentally in a 49-year-old man, with diffuse cortical calcifications on renal imaging; and (3) dysmorphic and asymmetric kidneys with multiple cysts and signs of tubule-interstitial defects. Genetic analysis revealed rare heterozygote/compound heterozygote COL4A4-A5 variants. Our study highlights the key role of NGS in the diagnosis of inherited renal disorders and shows the phenotype variability in patients carrying mutations in collagen type IV genes.

Neurofibromatosis Type 1: Pediatric Aspects and Review of Genotype-Phenotype Correlations.

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition, with a birth incidence of approximately 1:2000-3000, caused by germline pathogenic variants in NF1, a tumor suppressor gene encoding neurofibromin, a negative regulator of the RAS/MAPK pathway. This explains why NF1 is included in the group of RASopathies and shares several clinical features with Noonan syndrome. Here, we describe the main clinical characteristics and complications associated with NF1, particularly those occurring in pediatric age. NF1 has complete penetrance and shows wide inter- and intrafamilial phenotypic variability and age-dependent appearance of manifestations. Clinical presentation and history of NF1 are multisystemic and highly unpredictable, especially in the first years of life when penetrance is still incomplete. In this scenario of extreme phenotypic variability, some genotype-phenotype associations need to be taken into consideration, as they strongly impact on genetic counseling and prognostication of the disease. We provide a synthetic review, based on the most recent literature data, of all known genotype-phenotype correlations from a genetic and clinical perspective. Molecular diagnosis is fundamental for the confirmation of doubtful clinical diagnoses, especially in the light of recently revised diagnostic criteria, and for the early identification of genotypes, albeit few, that correlate with specific phenotypes.

Spectrum of Genetic Variants in the Dystrophin Gene: A Single Centre Retrospective Analysis of 750 Duchenne and Becker Patients from Southern Italy.

Dystrophinopathies are X-linked recessive muscle disorders caused by mutations in the dystrophin (DMD) gene that include deletions, duplications, and point mutations. Correct diagnosis is important for providing adequate patient care and family planning, especially at this time when mutation-specific therapies are available. We report a large single-centre study on the spectrum of DMD gene variants observed in 750 patients analyzed for suspected Duchenne (DMD) or Becker (BMD) muscular dystrophy, over the past 30 years, at the Cardiomyology and Medical Genetics of the University of Campania. We found 534 (71.21%) large deletions, 73 (9.73%) large duplications, and 112 (14.93%) point mutations, of which 44 (5.9%) were small ins/del causing frame-shifts, 57 (7.6%) nonsense mutations, 8 (1.1%) splice site and 3 (0.4%) intronic mutations, and 31 (4.13%) non mutations. Moreover, we report the prevalence of the different types of mutations in patients with DMD and BMD according to their decade of birth, from 1930 to 2020, and correlate the data to the different techniques used over the years. In the most recent decades, we observed an apparent increase in the prevalence of point mutations, probably due to the use of Next-Generation Sequencing (NGS). In conclusion, in southern Italy, deletions are the most frequent variation observed in DMD and BMD patients followed by point mutations and duplications, as elsewhere in the world. NGS was useful to identify point mutations in cases of strong suspicion of DMD/BMD negative on deletions/duplications analyses. In the era of personalized medicine and availability of new causative therapies, a collective effort is necessary to enable DMD and BMD patients to have timely genetic diagnoses and avoid late implementation of standard of care and late initiation of appropriate treatment.

A Novel Homozygous Loss-of-Function Variant in SPRED2 Causes Autosomal Recessive Noonan-like Syndrome.

Noonan syndrome is an autosomal dominant developmental disorder characterized by peculiar facial dysmorphisms, short stature, congenital heart defects, and hypertrophic cardiomyopathy. In 2001, PTPN11 was identified as the first Noonan syndrome gene and is responsible for the majority of Noonan syndrome cases. Over the years, several other genes involved in Noonan syndrome (KRAS, SOS1, RAF1, MAP2K1, BRAF, NRAS, RIT1, and LZTR1) have been identified, acting at different levels of the RAS-mitogen-activated protein kinase pathway. Recently, SPRED2 was recognized as a novel Noonan syndrome gene with autosomal recessive inheritance, and only four families have been described to date. Here, we report the first Italian case, a one-year-old child with left ventricular hypertrophy, moderate pulmonary valve stenosis, and atrial septal defect, with a clinical suspicion of RASopathy supported by the presence of typical Noonan-like facial features and short stature. Exome sequencing identified a novel homozygous loss-of-function variant in the exon 3 of SPRED2 (NM_181784.3:c.325del; p.Arg109Glufs*7), likely causing nonsense-mediated decay. Our results and the presented clinical data may help us to further understand and dissect the genetic heterogeneity of Noonan syndrome.

Mystery(n) Phenotypic Presentation in Europeans: Report of Three Further Novel Missense RNF213 Variants Leading to Severe Syndromic Forms of Moyamoya Angiopathy and Literature Review.

Moyamoya angiopathy (MMA) is a rare cerebral vasculopathy in some cases occurring in children. Incidence is higher in East Asia, where the heterozygous p.Arg4810Lys variant in RNF213 (Mysterin) represents the major susceptibility factor. Rare variants in RNF213 have also been found in European MMA patients with incomplete penetrance and are today a recognized susceptibility factor for other cardiovascular disorders, from extracerebral artery stenosis to hypertension. By whole exome sequencing, we identified three rare and previously unreported missense variants of RNF213 in three children with early onset of bilateral MMA, and subsequently extended clinical and radiological investigations to their carrier relatives. Substitutions all involved highly conserved residues clustered in the C-terminal region of RNF213, mainly in the E3 ligase domain. Probands showed a de novo occurring variant, p.Phe4120Leu (family A), a maternally inherited heterozygous variant, p.Ser4118Cys (family B), and a novel heterozygous variant, p.Glu4867Lys, inherited from the mother, in whom it occurred de novo (family C). Patients from families A and C experienced transient hypertransaminasemia and stenosis of extracerebral arteries. Bilateral MMA was present in the proband's carrier grandfather from family B. The proband from family C and her carrier mother both exhibited annular figurate erythema. Our data confirm that rare heterozygous variants in RNF213 cause MMA in Europeans as well as in East Asian populations, suggesting that substitutions close to positions 4118-4122 and 4867 of RNF213 could lead to a syndromic form of MMA showing elevated aminotransferases and extracerebral vascular involvement, with the possible association of peculiar skin manifestations.

Genotype-Phenotype Correlations in Neurofibromatosis Type 1: Identification of Novel and Recurrent NF1 Gene Variants and Correlations with Neurocognitive Phenotype.

Neurofibromatosis type 1 (NF1) is one of the most common genetic tumor predisposition syndrome, caused by mutations in the NF1. To date, few genotype-phenotype correlations have been discerned in NF1, due to a highly variable clinical presentation. We aimed to study the molecular spectrum of NF1 and genotype-phenotype correlations in a monocentric study cohort of 85 NF1 patients (20 relatives, 65 sporadic cases). Clinical data were collected at the time of the mutation analysis and reviewed for accuracy in this investigation. An internal phenotypic categorization was applied. The 94% of the patients enrolled showed a severe phenotype with at least one systemic complication and a wide range of associated malignancies. Spine deformities were the most common complications in this cohort. We also reported 66 different NF1 mutations, of which 7 are novel mutations. Correlation analysis identified a slight significant inverse correlation between age at diagnosis and delayed acquisition of psychomotor skills with residual multi-domain cognitive impairment. Odds ratio with 95% confidence interval showed a higher prevalence of learning disabilities in patients carrying frameshift mutations. Overall, our results aim to offer an interesting contribution to studies on the genotype-phenotype of NF1 and in genetic management and counselling.

Therapeutic homology-independent targeted integration in retina and liver.

Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs.

A Translational Approach to Spinal Neurofibromatosis: Clinical and Molecular Insights from a Wide Italian Cohort.

Spinal neurofibromatosis (SNF), a phenotypic subclass of neurofibromatosis 1 (NF1), is characterized by bilateral neurofibromas involving all spinal roots. In order to deepen the understanding of SNF's clinical and genetic features, we identified 81 patients with SNF, 55 from unrelated families, and 26 belonging to 19 families with at least 1 member affected by SNF, and 106 NF1 patients aged >30 years without spinal tumors. A comprehensive NF1 mutation screening was performed using NGS panels, including NF1 and several RAS pathway genes. The main features of the SNF subjects were a higher number of internal neurofibromas (p < 0.001), nerve root swelling (p < 0.001), and subcutaneous neurofibromas (p = 0.03), while hyperpigmentation signs were significantly less frequent compared with the classical NF1-affected cohorts (p = 0.012). Fifteen patients underwent neurosurgical intervention. The histological findings revealed neurofibromas in 13 patients and ganglioneuromas in 2 patients. Phenotypic variability within SNF families was observed. The proportion of missense mutations was higher in the SNF cases than in the classical NF1 group (21.40% vs. 7.5%, p = 0.007), conferring an odds ratio (OR) of 3.34 (CI = 1.33−10.78). Two unrelated familial SNF cases harbored in trans double NF1 mutations that seemed to have a subclinical worsening effect on the clinical phenotype. Our study, with the largest series of SNF patients reported to date, better defines the clinical and genetic features of SNF, which could improve the management and genetic counseling of NF1.

Intermittent macrothrombocytopenia in a novel patient with Takenouchi-Kosaki syndrome and review of literature.

Takenouchi-Kosaki syndrome (TKS) is a recently delineated syndromic form of thrombocytopenia strictly related to an hot-spot missense variant, p.Tyr64Cys, in CDC42 (Cell Division Control protein 42). Herein we report an additional patient with the p.Tyr64Cys aminoacidic substitution who showed the well-defined phenotypical TKS features and an intermittent, very mild, macrothrombocytopenia at 10.7 years of age (93,000/mL), that was only retrospectively valorized. Outside of this value the PLT count had always been higher than 100,000/mL. We also review literature data from patients carrying this recurrent variant. Our female patient presented with prenatal onset of short stature and microcephaly, camptodactyly, heart defects, typical facial gestalt, developmental delay, and not specific brain abnormalities. After several genetic investigations (karyotype, CGH-Array, targeted NGS analysis for short stature genes), by whole exome sequencing we identified the p.Tyr64Cys in CDC42, occurring de novo. The case presented here provides further evidence that macrothrombocytopenia can be intermittent and thus it might escape attention of clinicians. Without this key feature, TKS clinical presentation can overlap other syndromic forms of short stature. Immunodeficiency, autoimmunity, and malignancies were recently reported in patients with the p.Tyr64Cys substitution, making imperative an early diagnosis of Takenouchi-Kosaki syndrome to organize the most proper follow-up of these pediatric patients. The whole exome sequencing can be a solving tool in the challenge to the rare diseases.

Clinical variability of neurofibromatosis 1: A modifying role of cooccurring PTPN11 variants and atypical brain MRI findings.

Neurofibromatosis 1 (NF1) is a disorder characterized by variable expressivity caused by loss-of-function variants in NF1, encoding neurofibromin, a protein negatively controlling RAS signaling. We evaluated whether concurrent variation in proteins functionally linked to neurofibromin contribute to the variable expressivity of NF1. Parallel sequencing of a RASopathy gene panel in 138 individuals with molecularly confirmed clinical diagnosis of NF1 identified missense variants in PTPN11, encoding SHP2, a positive regulator of RAS signaling, in four subjects from three unrelated families. Three subjects were heterozygous for a gain-of-function variant and showed a severe expression of NF1 (developmental delay, multiple cerebral neoplasms and peculiar cortical MRI findings), and features resembling Noonan syndrome (a RASopathy caused by activating variants in PTPN11). Conversely, the fourth subject, who showed an attenuated presentation, carried a previously unreported PTPN11 variant that had a hypomorphic behavior in vitro. Our findings document that functionally relevant PTPN11 variants occur in a small but significant proportion of subjects with NF1 modulating disease presentation, suggesting a model in which the clinical expression of pathogenic NF1 variants is modified by concomitant dysregulation of protein(s) functionally linked to neurofibromin. We also suggest targeting of SHP2 function as an approach to treat evolutive complications of NF1.

Poikiloderma With Neutropenia and Mastocytosis: A Case Report and a Review of Dermatological Signs.

Poikiloderma with neutropenia (PN) is a very rare genetic disorder mainly characterized by poikiloderma and congenital neutropenia, which explains the recurrence of respiratory infections and risk of developing bronchiectasis. Patients are also prone to develop hematological and skin cancers. Here, we present the case of a patient, the only child of apparently unrelated Serbian parents, affected by PN resulting from the homozygous mutation NM_024598.3:c.243G>A (p.Trp81Ter) of USB1; early onset of poikiloderma (1 year of age) was associated with cutaneous mastocytosis. We also provide a review of the literature on this uncommon condition with a focus on dermatological findings.

Nephroplex: a kidney-focused NGS panel highlights the challenges of PKD1 sequencing and identifies a founder BBS4 mutation.

BACKGROUND: Genetic testing of patients with inherited kidney diseases has emerged as a tool of clinical utility by improving the patients' diagnosis, prognosis, surveillance and therapy. METHODS: The present study applied a Next Generation Sequencing (NGS)-based panel, named NephroPlex, testing 115 genes causing renal diseases, to 119 individuals, including 107 probands and 12 relatives. Thirty-five (poly)cystic and 72 non (poly)cystic individuals were enrolled. The latter subgroup of patients included Bardet-Biedl syndrome (BBS) patients, as major components. RESULTS: Disease-causing mutations were identified in 51.5 and 40% of polycystic and non-polycystic individuals, respectively. Autosomal dominant polycystic kidney disease (ADPKD) patients with truncating PKD1 variants showed a trend towards a greater slope of the age-estimated glomerular filtration rate (eGFR) regression line than patients with (i) missense variants, (ii) any PKD2 mutations and (iii) no detected mutations, according to previous findings. The analysis of BBS individuals showed a similar frequency of BBS4,9,10 and 12 mutations. Of note, all BBS4-mutated patients harbored the novel c.332+1G>GTT variant, which was absent in public databases, however, in our internal database, an additional heterozygote carrier was found. All BBS4-mutated individuals originated from the same geographical area encompassing the coastal provinces of Naples. DISCUSSION: In conclusion, these findings indicate the potential for a genetic panel to provide useful information at both clinical and epidemiological levels.