Reply to Fry, L.E.; MacLaren, R.E. Comment on "Di Giosaffatte et al. A Novel Hypothesis on Choroideremia-Manifesting Female Carriers: Could CHM In-Frame Variants Exert a Dominant Negative Effect? A Case Report. Genes 2022, 13, 1268".

We express our gratitude to Dr. Fry and Prof. McLaren [...].

CCND2 mutations in atypical chronic myeloid leukemia: a report of two cases.

Atypical chronic myeloid leukemia (aCML) is a rare MDS/MPN disease characterized by the absence of BCR::ABL1 rearrangement and well known typical mutations associated with myeloproliferative disorders. Mutational landscape associated with this disease was recently described with frequent involvement of SETBP1 and ETNK1 mutations. CCND2 mutations have not been frequently detected in MPN or MDS/MPN patients. We describe two cases of aCML with two CCND2 mutations in 280 and 281 codons which rapidly develop progressive characteristics, and we reviewed the literature about this unfavorable association, suggesting a role as a new possible marker of aggressive disease.

Germline NUP98 Variants in Two Siblings with a Rothmund-Thomson-Like Spectrum: Protein Functional Changes Predicted by Molecular Modeling.

Two adult siblings born to first-cousin parents presented a clinical phenotype reminiscent of Rothmund-Thomson syndrome (RTS), implying fragile hair, absent eyelashes/eyebrows, bilateral cataracts, mottled pigmentation, dental decay, hypogonadism, and osteoporosis. As the clinical suspicion was not supported by the sequencing of RECQL4, the RTS2-causative gene, whole exome sequencing was applied and disclosed the homozygous variants c.83G>A (p.Gly28Asp) and c.2624A>C (p.Glu875Ala) in the nucleoporin 98 (NUP98) gene. Though both variants affect highly conserved amino acids, the c.83G>A looked more intriguing due to its higher pathogenicity score and location of the replaced amino acid between phenylalanine-glycine (FG) repeats within the first NUP98 intrinsically disordered region. Molecular modeling studies of the mutated NUP98 FG domain evidenced a dispersion of the intramolecular cohesion elements and a more elongated conformational state compared to the wild type. This different dynamic behavior may affect the NUP98 functions as the minor plasticity of the mutated FG domain undermines its role as a multi-docking station for RNA and proteins, and the impaired folding can lead to the weakening or the loss of specific interactions. The clinical overlap of NUP98-mutated and RTS2/RTS1 patients, accounted by converging dysregulated gene networks, supports this first-described constitutional NUP98 disorder, expanding the well-known role of NUP98 in cancer.

Two unrelated cases with biallelic CHEK2 variants:a novel condition with constitutional chromosomal instability?

Constitutional heterozygous mutations in CHEK2 gene have been associated with hereditary cancer risk. To date, only a few homozygous CHEK2 mutations have been reported in families with cancer susceptibility. Here, we report two unrelated individuals with a personal and familial cancer history in whom biallelic CHEK2 alterations were identified. The first case resulted homozygous for the CHEK2 c.793-1 G > A (p.Asp265Thrfs*10) variant, and the second one was found to be compound heterozygous for the c.1100delC (p.Thr367Metfs*15) and the c.1312 G > T (p.Asp438Tyr) variants. Multiple cytogenetic anomalies were demonstrated on peripheral lymphocytes of both patients. A literature revision showed that a single other CHEK2 homozygous variant was previously associated to a constitutional randomly occurring multi-translocation karyotype from peripheral blood in humans. We hypothesize that, at least some biallelic CHEK2 mutations might be associated with a novel disorder, further expanding the group of chromosome instability syndromes. Additional studies on larger cohorts are needed to confirm if chromosomal instability could represent a marker for CHEK2 constitutionally mutated recessive genotypes, and to investigate the cancer risk and the occurrence of other anomalies typically observed in chromosome instability syndromes.

Long-term outcomes of COVID-19 vaccination in patients with rare and complex connective tissue diseases: The ERN-ReCONNET VACCINATE study.

Background: Vaccination is one of the most important measures to contain the COVID-19 pandemic, especially for frail patients. VACCINATE is a multicentre prospective observational study promoted by the European Reference Network on Rare and Complex Connective Tissue and Musculoskeletal Diseases (ERN ReCONNET) aimed at assessing the long-term outcomes of COVID-19 vaccination in patients with rare and complex connective tissue diseases (rcCTDs) in terms of efficacy and safety. Methods: Adult rcCTDs patients were eligible for recruitment. Demographic, clinical and vaccination data were collected at enrolment. Follow-up visits were scheduled 4, 12, 24, 36 and 48 weeks after completion of the first vaccination cycle; data on adverse events, disease exacerbations and the occurrence of new SARS-CoV-2 infections were collected at these time-points. Findings: 365 rcCTDs patients (87 % female, mean age 51.8 ± 14.6 years) were recruited. Overall, 200 patients (54.8 %) experienced at least one adverse event, generally mild and in most cases occurring early after the vaccination. During follow-up, 55 disease exacerbations were recorded in 39 patients (10.7 %), distributed over the entire observation period, although most frequently within 4 weeks after completion of the vaccination cycle. The incidence of new SARS-CoV-2 infections was 8.9 per 1000 person-months, with no cases within 12 weeks from vaccine administration and an increasing trend of infections moving away from the primary vaccination cycle. Only one case of severe COVID-19 was reported during the study period. Interpretation: COVID-19 vaccination seems effective and safe in rcCTDs patients. The rate of new infections was rather low and serious infections were uncommon in our cohort. No increased risk of disease flares was observed compared to previous disease history; however, such exacerbations may be potentially severe, emphasising the need for close monitoring of our patients.

A pathogenic variant in the FLCN gene presenting with pure dementia: is autophagy at the intersection between neurodegeneration and cancer?

Introduction: Folliculin, encoded by FLCN gene, plays a role in the mTORC1 autophagy cascade and its alterations are responsible for the Birt-Hogg-Dubé (BHD) syndrome, characterized by follicle hamartomas, kidney tumors and pneumothorax. Patient and results: We report a 74-years-old woman diagnosed with dementia and carrying a FLCN alteration in absence of any sign of BHD. She also carried an alteration of MAT1A gene, which is also implicated in the regulation of mTORC1. Discussion: The MAT1A variant could have prevented the development of a FLCN-related oncological phenotype. Conversely, our patient presented with dementia that, to date, has yet to be documented in BHD. Folliculin belongs to the DENN family proteins, which includes C9orf72 whose alteration has been associated to neurodegeneration. The folliculin perturbation could affect the C9orf72 activity and our patient could represent the first human model of a relationship between FLCN and C9orf72 across the path of autophagy.

Expanding the Spectrum of KDM5C Neurodevelopmental Disorder: A Novel De Novo Stop Variant in a Young Woman and Emerging Genotype-Phenotype Correlations.

As a consequence of the implementation of NGS technologies, the diagnostic yield of neurodevelopmental disorders has dramatically increased during the past two decades. Among neurodevelopmental genes, transcription-related genes and chromatin remodeling genes are the most represented category of disease-causing genes. Indeed, the term "chromatinopathies" is now widely used to describe epigenetic disorders caused by mutations in these genes. We hereby describe a twenty-seven-year-old female patient diagnosed with moderate intellectual disability comorbid with other neuropsychiatric and behavioral issues carrying a de novo heterozygous stop variant in the KDM5C gene (NM_004187.5: c. 3847G>T, p.Glu1283*), encoding a histone demethylase that specifically acts on the H3K4 lysines. The gene is located on the X chromosome and has been associated with Claes-Jensen-type intellectual disability, an X-linked syndromic disorder. We discuss our case in relation to previously reported affected females harboring pathogenic mutations in the KDM5C gene with the objective of delineating genotype-phenotype correlations and further defining a common recognizable phenotype. We also highlight the importance of reverse phenotyping in relation to whole-exome sequencing results.

Congenital Defects in a Patient Carrying a Novel Homozygous AEBP1 Variant: Further Expansion of the Phenotypic Spectrum of Ehlers-Danlos Syndrome Classical-like Type 2?

In 2018, a new clinical subtype, caused by biallelic variants in the AEBP1 gene, encoding the ACLP protein, was added to the current nosological classification of the Ehlers-Danlos Syndromes (EDS). This new phenotype, provisionally termed EDS classical-like type 2 (clEDS2), has not yet been fully characterized, as only nine cases have been reported to date. Here we describe a patient, homozygous for a novel AEBP1 pathogenic variant (NM_001129.5 c.2123_2124delTG (p.Val708AlafsTer5)), whose phenotype is reminiscent of classical EDS but also includes previously unreported multiple congenital malformations. Furthermore, we briefly summarize the current principal clinical manifestations of clEDS2 and the molecular evidence surrounding the role of AEBP1 in the context of extracellular matrix homeostasis and connective tissue development. Although a different coexisting etiology for the multiple congenital malformations of our patient cannot be formally excluded, the emerging role of ACLP in TGF-ß and WNT pathways may explain their occurrence and the phenotypical variability of clEDS2.

Genetic Dysruption of the Histaminergic Pathways: A Novel Deletion at the 15q21.2 locus Associated with Variable Expressivity of Neuropsychiatric Disorders.

The involvement of the Histaminergic System (HS) in neuropsychiatric disease is not well-documented, and few studies have described patients affected by different neuropsychiatric conditions harbouring disruptions in genes involved in the HS. In humans, histamine is synthetised from histidine by the histidine decarboxylase enzyme encoded by the HDC gene (OMIM*142704). This is the sole enzyme in our organism able to synthetise histamine from histidine. Histamine is also contained in many different food types. We hereby describe a twenty-one-year-old female diagnosed with a borderline intellectual disability with autistic traits and other peculiar neuropsychological features carrying a 175-Kb interstitial deletion on chromosome 15q21.2. The deletion was inherited from the mother, who was affected by a severe anxiety disorder. The deleted region contains entirely the HDC and the SLC27A2 genes and partially the ATP8B4 gene. The HDC gene has been previously associated with Tourette Syndrome (TS). Based on the functional role of the HDC, we propose this gene as the best candidate to explain many traits associated with the clinical phenotype of our patient and of her mother.

A Novel Hypothesis on Choroideremia-Manifesting Female Carriers: Could CHM In-Frame Variants Exert a Dominant Negative Effect? A Case Report.

Choroideremia is an X-linked recessive condition presenting in males, with progressive degeneration of retinal and choroidal tissues leading to progressive visual loss. Its pathological mechanism is due to alterations in the CHM gene that encodes for REP1, a protein required for prenylation of Rab by the Rab geranylgeranyl transferase (RGGT). Even though female carriers are predicted to be not affected by the disease, a wide phenotypic spectrum ranging from mild to severe cases has been reported in women. The reason why Choroideremia manifests in female carriers remains elusive. While X chromosome inactivation (XCI) skewing has been proposed as a leading putative mechanism, emerging evidence has shown that CHM could variably escape from XCI. We described a family with an initial clinical suspicion of Retinitis Pigmentosa in which a novel CHM pathogenic splicing variant was found by exome sequencing. The variant, initially found in the 63-year-old female presenting with impaired visual acuity and severe retinal degeneration, segregated in the 31-year-old daughter and the 37-year-old son, both presenting with fundus anomalies. mRNA studies revealed a shorter in-frame CHM isoform lacking exon 10. Molecular modeling of the ternary REP1/Rab/RGGT protein complex predicted significant impairing of REP1/Rab binding without alteration of REP1/RGGT interaction. We suggest that, in our female cases, the biallelic expression of CHM may have led to the production of both the mutant and wild type REP1. The mutant isoform, sequestrating RGGT, could reduce its available amount for Rab prenylation, thus exerting a dominant-negative effect. If confirmed with further studies and in large cohorts of female carriers, the here proposed molecular mechanism could help to explain the complexity of manifestation of Choroideremia in females.

First Italian experience using the automated craniofacial gestalt analysis on a cohort of pediatric patients with multiple anomaly syndromes.

BACKGROUND: In this study, we used the novel DeepGestalt technology powered by Face2Gene (FDNA Inc., MA, USA) in suggesting a correct diagnosis based on the facial gestalt of well-known multiple anomaly syndromes. Only molecularly characterized pediatric patients were considered in the present research. SUBJECTS AND METHODS: A total of 19 two-dimensional (2D) images of patients affected by several molecularly confirmed craniofacial syndromes (14 monogenic disorders and 5 chromosome diseases) and evaluated at the main involved Institution were analyzed using the Face2Gene CLINIC application (vs.19.1.3). Patients were cataloged into two main analysis groups (A, B) according to the number of clinical evaluations. Specifically, group A contained the patients evaluated more than one time, while in group B were comprised the subjects with a single clinical assesment. The algorithm's reliability was measured based on its capacity to identify the correct diagnosis as top-1 match, within the top-10 match and top-30 matches, only based on the uploaded image and not any other clinical finding or HPO terms. Failure was represented by the top-0 match. RESULTS: The correct diagnosis was suggested respectively in 100% (8/8) and 81% (9/11) of cases of group A and B, globally failing in 16% (3/19). CONCLUSION: The tested tool resulted to be useful in identifying the facial gestalt of a heterogeneous group of syndromic disorders. This study illustrates the first Italian experience with the next generation phenotyping technology, following previous works and providing additional observations.

Longitudinal dynamics of SARS-CoV-2 anti-receptor binding domain IgG antibodies in a wide population of health care workers after BNT162b2 vaccination.

OBJECTIVES: With the availability of vaccines, commercial assays detecting anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies evolved toward quantitative assays directed to the spike glycoprotein or its receptor-binding domain (RBD). The objective was to perform a large-scale, longitudinal study involving health care workers (HCWs), with the aim of establishing the kinetics of immune response throughout the 9-month period after receipt of the second dose of the BNT162b2 vaccine. METHODS: Quantitative determination of immunoglobulin (Ig) G antibodies against the RBD of the S1 subunit of the spike protein of SARS-CoV-2 on the Alinity systems. RESULTS: The highest levels of anti-RBD IgG were measured after 1 month from full vaccination (median: 1432 binding antibody units/ml [BAU/ml]); subsequently, a steep decrease (7.4-fold decrease) in IgG levels was observed at 6 months (median: 194.3 BAU/ml), with a further 2.5-fold decrease at 9 months (median: 79.3 BAU/ml). Furthermore, the same data, when analyzed for sex, showed significant differences between male and female participants at both 1 and 9 months from vaccination, but not at 6 months. CONCLUSION: Our results confirm the tendency of anti-RBD antibodies to decrease over time, also when extending the analysis up to 9 months, and highlight a better ability of the female sex to produce antibodies 1 month and 9 months after vaccination. Overall, these data, obtained in a wide population of HCWs, support the importance of having increased the vaccine doses.

Atypical variants in COL1A1 and COL3A1 associated with classical and vascular Ehlers-Danlos syndrome overlap phenotypes: expanding the clinical phenotype based on additional case reports.

The vast majority of reported (likely) pathogenic missense variants in the genes coding for the fibrillar collagens leads to the substitution of one of the obligatory glycine residues in the Gly-Xaa-Yaa repeat sequence of the triple helical domain. Their phenotypic consequences and deleterious effects have been well-documented. However, with increasing access to molecular diagnostic testing based on next-generation sequencing techniques, such as sequencing of multi-gene panels and whole-exome sequencing, non-glycine substitutions are more frequently identified in individuals suspected to have a heritable collagen disorder, but their pathogenic effect is often difficult to predict.Some specific non-glycine substitutions in the proα1(I)- (p.(Arg312Cys)) and proα1(III)- (glutamic acid to lysine at different positions) collagen chain have been identified in a number of individuals presenting a phenotype showing features of both classical and vascular Ehlers-Danlos syndrome. The number of reported individuals with these defects is currently very low, and several of these non-glycine substitutions had initially been categorised as variants of unknown significance (VUS), complicating early diagnosis, accurate counselling, management guidelines, and correct classification. This collaborative study reports on the phenotype of 22 and 7 individuals harbouring these rare variants in COL1A1 and COL3A1, respectively, expanding our knowledge on clinical presentation, phenotypic variability, and natural history, and informing on the risk for potentially life-threatening events, such as vascular, gastro-intestinal, and pregnancy-related complications.

Loss-of-function variants in exon 4 of TAB2 cause a recognizable multisystem disorder with cardiovascular, facial, cutaneous, and musculoskeletal involvement.

PURPOSE: This study aimed to describe a multisystemic disorder featuring cardiovascular, facial, musculoskeletal, and cutaneous anomalies caused by heterozygous loss-of-function variants in TAB2. METHODS: Affected individuals were analyzed by next-generation technologies and genomic array. The presumed loss-of-function effect of identified variants was assessed by luciferase assay in cells transiently expressing TAB2 deleterious alleles. In available patients' fibroblasts, variant pathogenicity was further explored by immunoblot and osteoblast differentiation assays. The transcriptomic profile of fibroblasts was investigated by RNA sequencing. RESULTS: A total of 11 individuals from 8 families were heterozygotes for a novel TAB2 variant. In total, 7 variants were predicted to be null alleles and 1 was a missense change. An additional subject was heterozygous for a 52 kb microdeletion involving TAB2 exons 1 to 3. Luciferase assay indicated a decreased transcriptional activation mediated by NF-κB signaling for all point variants. Immunoblot analysis showed a reduction of TAK1 phosphorylation while osteoblast differentiation was impaired. Transcriptomic analysis identified deregulation of multiple pleiotropic pathways, such as TGFß-, Ras-MAPK-, and Wnt-signaling networks. CONCLUSION: Our data defined a novel disorder associated with loss-of-function or, more rarely, hypomorphic alleles in a restricted linker region of TAB2. The pleiotropic manifestations in this disorder partly recapitulate the 6q25.1 (TAB2) microdeletion syndrome and deserve the definition of cardio-facial-cutaneous-articular syndrome.

Comparison of Two Blood-Based Genotyping Tests to Investigate the KRAS G12C Mutation in Patients with Non-Small-Cell Lung Cancer at Failure of First-Line Treatments.

Although molecular profiling at diagnosis has traditionally relied on direct sampling of neoplastic tissue, cancer clonal evolution represents a critical obstacle to use primary tissue biopsies to guide clinical decision-making at the time of progressive disease. Liquid biopsies might offer enormous advantages over tissue biopsies, tracking in real-time temporal-based tumor dynamics following each line of treatment. Here, we compared two liquid biopsy assays, specifically real-time polymerase chain reaction and next-generation sequencing, to track the KRAS G12C mutation at onset of progression from previous lines of therapy. The KRAS G12C mutation was acquired at the time of progressive disease in 24% of patients. Furthermore, all patients with KRAS G12C mutation-positive tissue became negative in ctDNA at progressive disease. The presence of other somatic mutations in all these samples confirmed the tumor origin of the circulating DNA. This pilot study suggests that in the assessment of the plasma KRAS G12C mutation as a druggable target, real-time PCR assay Idylla might be a suitable approach to better match patients to interventional biomarker-targeted therapies.

Clinical and Molecular Aspects of the Neurodevelopmental Disorder Associated with PAK3 Perturbation.

X-linked intellectual disability can be diagnosed in about 10-12% of intellectually disabled males. In the past, mutations affecting the PAK3 gene (p21 protein-activated kinase 3, MIM#300142) have been associated with a non-syndromic form of X-linked intellectual disability, which has to date been identified in a limited number of families.Since this neurodevelopmental disorder mostly afflicts males, descriptions of symptomatic female carriers are quite rare.We describe a female patient with neurodevelopmental delay and a novel PAK3 variant. Interestingly, she manifests craniofacial anomalies, including microcephaly, representing the second reported microcephalic female but the first for whom a detailed clinical description is available. She also displays other uncommon clinical findings, which we illustrate.Moreover, a comprehensive clinical and molecular review of all to date published patients has been made. This study contributes to further delineate the PAK3-related phenotype, which can be considered a non-syndromic X-linked intellectual disability, with seemingly recurrent craniofacial abnormalities.