A Novel COL4A5 Pathogenic Variant Joins the Dots in a Family with a Synchronous Diagnosis of Alport Syndrome and Polycystic Kidney Disease.

Alport Syndrome (AS) is the most common genetic glomerular disease, and it is caused by COL4A3, COL4A4, and COL4A5 pathogenic variants. The classic phenotypic spectrum associated with AS ranges from isolated hematuria to chronic kidney disease (CKD) with extrarenal abnormalities. Atypical presentation of the disorder is possible, and it can mislead the diagnosis. Polycystic kidney disease (PKD), which is most frequently associated with Autosomal Dominant PKD (ADPKD) due to PKD1 and PKD2 heterozygous variants, is emerging as a possible clinical manifestation in COL4A3-A5 patients. We describe a COL4A5 novel familial frameshift variant (NM_000495.5: c.1095dup p.(Leu366ValfsTer45)), which was associated with AS and PKD in the hemizygous proband, as well as with PKD, IgA glomerulonephritis and focal segmental glomerulosclerosis (FSGS) in the heterozygous mother. Establishing the diagnosis of AS can sometimes be difficult, especially in the context of misleading family history and atypical phenotypic features. This case study supports the emerging genotypic and phenotypic heterogeneity in COL4A3-A5-associated disorders, as well as the recently described association between PKD and collagen type IV (Col4) defects. We highlight the importance of the accurate phenotyping of all family members and the relevance of next-generation sequencing in the differential diagnosis of hereditary kidney disease.

Genetic Variability of SOX10-Related Disorders within an Italian Family: Straddling the Line between Kallmann and Waardenburg Syndrome.

Introduction: Kallmann syndrome (KS) is a genetically heterogeneous developmental disorder that most often manifests hypogonadotropic hypogonadism (HH) and hypo-/anosmia due to early embryonic impairment in the migration of gonadotropin-releasing hormone neurons. SOX10 (SRY-Box 10; MIM*602229), a key transcriptional activator involved in the development of neural crest cells, has been associated with KS and is identified as one of the causative genes of Waardenburg syndrome (WS). Case Presentation: A 28-year-old female patient, who was clinically diagnosed with KS in her childhood, presented with HH and anosmia, mild bilateral sensorineural hearing loss (SNHL), and pigmentation abnormalities. Next-generation sequencing analysis detected a missense heterozygous SOX10 pathogenic variant (NM_006941.4:c.506C>T) in the proposita and in her mother, whose phenotype included exclusively anosmia and hypopigmented skin patches. The same variant has been described by Pingault et al. [Clin Genet. 2015;88(4):352-9] in a patient with apparently isolated bilateral severe SNHL. Conclusion: Our finding substantiates the extreme phenotypic variability of SOX10-related disorders, which range from classical KS and/or WS to contracted endophenotypes that could share a common pathway in the development of neural crest cells and highlights the need for careful evaluation and long-term follow-up of SOX10 patients, with special focus on atypical/additional and/or late-onset phenotypic traits.

Prenatal identification of a pathogenic maternal FGFR1 variant in two consecutive pregnancies with fetal forebrain malformations.

OBJECTIVE: Holoprosencephaly (HPE) is the most common aberration of forebrain development, and it leads to a wide spectrum of developmental and craniofacial anomalies. HPE etiology is highly heterogeneous and includes both chromosomal abnormalities and single-gene defects. METHODS: Here, we report an FGFR1 heterozygous variant detected by prenatal exome sequencing and inherited from the asymptomatic mother, in association with recurrent neurological abnormalities in the HPE spectrum in two consecutive pregnancies. RESULTS: Individuals with germline pathogenic variants in FGFR1 (MIM: 136350) show extensive phenotypic variability, which ranges from asymptomatic carriers to hypogonadotropic hypogonadism, arhinencephaly, Kallmann's syndrome with associated features such as cleft lip and palate, skeletal anomalies, isolated HPE, and Hartsfield syndrome. CONCLUSION: The presented case supports the role of exome sequencing in prenatal diagnosis when fetal midline structural anomalies are suggestive of a genetic etiology, as early as the first trimester of gestation. The profound heterogeneity of FGFR1 allelic disorders needs to be considered when planning prenatal screening even in asymptomatic carriers.

Implementing the Risk Stratification and Clinical Management of Breast Cancer Families Using Polygenic Risk Score Evaluation: A Pilot Study.

BACKGROUND: The identification of women at high risk of breast cancer (BC) is crucial for personalized screening strategies. Pathogenic and likely pathogenic variants (PVs/LPVs) in susceptibility risk genes explain part of the individual risk. Moreover, a polygenic background, summarized as a polygenic risk score (PRS), contributes to the risk of BC and may modify the individual risk in carrier and non-carrier members of BC families. METHODS: We performed a retrospective pilot study evaluating PRS in women from a subset of high- (BRCA1 and BRCA2) and moderate-risk (PALB2 and ATM) BC families. We included PVs/LPVs carriers and non-carriers and evaluated a PRS based on 577,113 BC-associated variants. Using BOADICEA, we calculated the adjusted lifetime BC risk. RESULTS: Our data showed that in BRCA1/BRCA2 carriers, PVs have a major role in stratifying the lifetime risk, while PRS improves risk estimation in non-carriers of these families. A different scenario may be observed in PALB2 and ATM families where PRS combined with PV/LPV carrier status gives a more informative lifetime risk. CONCLUSIONS: This study showed that in BC families, the PRS might help to quantify the weight of the genetic familial background, improving the individual risk stratification and contributing to personalized clinical management for carrier and non-carrier women.

Co-Inheritance of Pathogenic Variants in PKD1 and PKD2 Genes Determined by Parental Segregation and De Novo Origin: A Case Report.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease, and it is typically caused by PKD1 and PKD2 heterozygous variants. Nonetheless, the extensive phenotypic variability observed among affected individuals, even within the same family, suggests a more complex pattern of inheritance. We describe an ADPKD family in which the proband presented with an earlier and more severe renal phenotype (clinical diagnosis at the age of 14 and end-stage renal disease aged 24), compared to the other affected family members. Next-generation sequencing (NGS)-based analysis of polycystic kidney disease (PKD)-associated genes in the proband revealed the presence of a pathogenic PKD2 variant and a likely pathogenic variant in PKD1, according to the American College of Medical Genetics and Genomics (ACMG) criteria. The PKD2 nonsense p.Arg872Ter variant was segregated from the proband's father, with a mild phenotype. A similar mild disease presentation was found in the proband's aunts and uncle (the father's siblings). The frameshift p.Asp3832ProfsTer128 novel variant within PKD1 carried by the proband in addition to the pathogenic PKD2 variant was not found in either parent. This report highlights that the co-inheritance of two or more PKD genes or alleles may explain the extensive phenotypic variability among affected family members, thus emphasizing the importance of NGS-based techniques in the definition of the prognostic course.

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males.

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways.Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor.

Severe COVID-19 in Hospitalized Carriers of Single CFTR Pathogenic Variants.

The clinical presentation of COVID-19 is extremely heterogeneous, ranging from asymptomatic to severely ill patients. Thus, host genetic factors may be involved in determining disease presentation and progression. Given that carriers of single cystic fibrosis (CF)-causing variants of the CFTR gene-CF-carriers-are more susceptible to respiratory tract infections, our aim was to determine their likelihood of undergoing severe COVID-19. We implemented a cohort study of 874 individuals diagnosed with COVID-19, during the first pandemic wave in Italy. Whole exome sequencing was performed and validated CF-causing variants were identified. Forty subjects (16 females and 24 males) were found to be CF-carriers. Among mechanically ventilated patients, CF-carriers were more represented (8.7%) and they were significantly (p < 0.05) younger (mean age 51 years) compared to noncarriers (mean age 61.42 years). Furthermore, in the whole cohort, the age of male CF-carriers was lower, compared to noncarriers (p < 0.05). CF-carriers had a relative risk of presenting an abnormal inflammatory response (CRP ≥ 20 mg/dL) of 1.69 (p < 0.05) and their hazard ratio of death at day 14 was 3.10 (p < 0.05) in a multivariate regression model, adjusted for age, sex and comorbidities. In conclusion, CF-carriers are more susceptible to the severe form of COVID-19, showing also higher risk of 14-day death.

High rate of HDR in gene editing of p.(Thr158Met) MECP2 mutational hotspot.

Rett syndrome is a progressive neurodevelopmental disorder which affects almost exclusively girls, caused by variants in MECP2 gene. Effective therapies for this devastating disorder are not yet available and the need for tight regulation of MECP2 expression for brain to properly function makes gene replacement therapy risky. For this reason, gene editing with CRISPR/Cas9 technology appears as a preferable option for the development of new therapies. To study the disease, we developed and characterized a human neuronal model obtained by genetic reprogramming of patient-derived primary fibroblasts into induced Pluripotent Stem Cells. This cellular model represents an important source for our studies, aiming to correct MECP2 variants in neurons which represent the primarily affected cell type. We engineered a gene editing toolkit composed by a two-plasmid system to correct a hotspot missense variant in MECP2, c.473 C > T (p.(Thr158Met)). The first construct expresses the variant-specific sgRNA and the Donor DNA along with a fluorescent reporter system. The second construct brings Cas9 and targets for auto-cleaving, to avoid long-term Cas9 expression. NGS analysis on sorted cells from four independent patients demonstrated an exceptionally high editing efficiency, with up to 80% of HDR and less than 1% of indels in all patients, outlining the relevant potentiality of the approach for Rett syndrome therapy.

AAV-mediated FOXG1 gene editing in human Rett primary cells.

Variations in the Forkhead Box G1 (FOXG1) gene cause FOXG1 syndrome spectrum, including the congenital variant of Rett syndrome, characterized by early onset of regression, Rett-like and jerky movements, and cortical visual impairment. Due to the largely unknown pathophysiological mechanisms downstream the impairment of this transcriptional regulator, a specific treatment is not yet available. Since both haploinsufficiency and hyper-expression of FOXG1 cause diseases in humans, we reasoned that adding a gene under nonnative regulatory sequences would be a risky strategy as opposed to a genome editing approach where the mutated gene is reversed into wild-type. Here, we demonstrate that an adeno-associated viruses (AAVs)-coupled CRISPR/Cas9 system is able to target and correct FOXG1 variants in patient-derived fibroblasts, induced Pluripotent Stem Cells (iPSCs) and iPSC-derived neurons. Variant-specific single-guide RNAs (sgRNAs) and donor DNAs have been selected and cloned together with a mCherry/EGFP reporter system. Specific sgRNA recognition sequences were inserted upstream and downstream Cas9 CDS to allow self-cleavage and inactivation. We demonstrated that AAV serotypes vary in transduction efficiency depending on the target cell type, the best being AAV9 in fibroblasts and iPSC-derived neurons, and AAV2 in iPSCs. Next-generation sequencing (NGS) of mCherry+/EGFP+ transfected cells demonstrated that the mutated alleles were repaired with high efficiency (20-35% reversion) and precision both in terms of allelic discrimination and off-target activity. The genome editing strategy tested in this study has proven to precisely repair FOXG1 and delivery through an AAV9-based system represents a step forward toward the development of a therapy for Rett syndrome.