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 [...].

Specifications and validation of the ACMG/AMP criteria for clinical interpretation of sequence variants in collagen genes associated with joint hypermobility.

Deleterious variants in collagen genes are the most common cause of hereditary connective tissue disorders (HCTD). Adaptations of the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) criteria are still lacking. A multidisciplinary team was set up for developing specifications of the ACMG/AMP criteria for COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL11A1, COL11A2 and COL12A1, associated with various forms of HCTD featuring joint hypermobility, which is becoming one of the most common reasons of referral for molecular testing in this field. Such specifications were validated against 209 variants, and resulted effective for classifying as pathogenic and likely pathogenic null alleles without downgrading of the PVS1 level of strength and recurrent Glycine substitutions. Adaptations of selected criteria reduced uncertainties on private Glycine substitutions, intronic variants predicted to affect the splicing, and null alleles with a downgraded PVS1 level of strength. Segregation and multigene panel sequencing data mitigated uncertainties on non-Glycine substitutions by the attribution of one or more benignity criteria. These specifications may improve the clinical utility of molecular testing in HCTD by reducing the number of variants with neutral/conflicting interpretations. Close interactions between laboratory and clinicians are crucial to estimate the a priori utility of molecular test and to improve medical reports.

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.

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.

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.

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.

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.

When to test fetuses for RASopathies? Proposition from a systematic analysis of 352 multicenter cases and a postnatal cohort.

PURPOSE: Recent studies have identified suggestive prenatal features of RASopathies (e.g., increased nuchal translucency [NT], cystic hygroma [CH], hydrops, effusions, congenital heart diseases [CHD], polyhydramnios, renal anomalies). Our objective is to clarify indications for RASopathy prenatal testing. We compare genotype distributions between pre- and postnatal populations and propose genotype-phenotype correlations. METHODS: Three hundred fifty-two chromosomal microarray-negative cases sent for prenatal RASopathy testing between 2012 and 2019 were collected. For most, 11 RASopathy genes were tested. Postnatal cohorts (25 patients with available prenatal information and 108 institutional database genotypes) and the NSeuroNet database were used for genotypic comparisons. RESULTS: The overall diagnostic yield was 14% (50/352), with rates >20% for effusions, hydrops, and CHD. Diagnostic yield was significantly improved in presence of hypertrophic cardiomyopathy (HCM), persistent or associated CH, any suggestive finding combined with renal anomaly or polyhydramnios, or ≥2 ultrasound findings. Largest prenatal contributors of pathogenic variants were PTPN11 (30%), RIT1 (16%), RAF1 (14%), and HRAS (12%), which considerably differ from their prevalence in postnatal populations. HRAS, LZTR1, and RAF1 variants correlated with hydrops/effusions, and RIT1 with prenatal onset HCM. CONCLUSION: After normal chromosomal microarray, RASopathies should be considered when any ultrasound finding of lymphatic dysplasia or suggestive CHD is found alone or in association.

Prevalence, Type, and Molecular Spectrum of NF1 Mutations in Patients with Neurofibromatosis Type 1 and Congenital Heart Disease.

The aim of this study was to assess the prevalence and type of congenital heart disease (CHD) and the associated mutation spectrum in a large series of patients with neurofibromatosis type 1 (NF1), and correlate the mutation type with the presence and subgroups of cardiac defects. The study cohort included 493 individuals with molecularly confirmed diagnosis of NF1 for whom cardiac evaluation data were available. CHD was reported in 62/493 (12.6%) patients. Among these patients, 23/62 (37.1%) had pulmonary valve stenosis/dysplasia, 20/62 (32.3%) had mitral valve anomalies, and 10/62 (16.1%) had septal defects. Other defects occurred as rare events. In this NF1 subcohort, three subjects carried a whole-gene deletion, while 59 were heterozygous for an intragenic mutation. A significantly increased prevalence of non-truncating intragenic mutations was either observed in individuals with CHD (22/59, 37.3%) or with pulmonary valve stenosis (13/20, 65.0%), when compared to individuals without CHD (89/420, 21.2%) (p = 0.038) or pulmonary valve stenosis (98/459, 21.4%) (p = 0.002). Similarly, patients with non-truncating NF1 mutations displayed two- and six-fold higher risk of developing CHD (odds ratio = 1.9713, 95% confidence interval (CI): 1.1162-3.4814, p = 0.0193) and pulmonary valve stenosis (odds ratio = 6.8411, 95% CI: 2.6574-17.6114, p = 0.0001), respectively. Noteworthy, all but one patient (19/20, 95.0%) with pulmonary valve stenosis, and 18/35 (51.4%) patients with other CHDs displayed Noonan syndrome (NS)-like features. Present data confirm the significant frequency of CHD in patients with NF1, and provide further evidence for a higher than expected prevalence of NF1 in-frame variants and NS-like characteristics in NF1 patients with CHD, particularly with pulmonary valve stenosis.

Clinical and functional characterization of a novel RASopathy-causing SHOC2 mutation associated with prenatal-onset hypertrophic cardiomyopathy.

SHOC2 is a scaffold protein mediating RAS-promoted activation of mitogen-activated protein kinase (MAPK) signaling in response to extracellular stimuli. A recurrent activating mutation in SHOC2 (p.Ser2Gly) causes Mazzanti syndrome, a RASopathy characterized by features resembling Noonan syndrome and distinctive ectodermal abnormalities. A second mutation (p.Met173Ile) supposed to cause loss-of-function was more recently identified in two individuals with milder phenotypes. Here, we report on the third RASopathy-causing SHOC2 mutation (c.807_808delinsTT, p.Gln269_His270delinsHisTyr), which was found associated with prenatal-onset hypertrophic cardiomyopathy. Structural analyses indicated a possible impact of the mutation on the relative orientation of the two SHOC2's leucine-rich repeat domains. Functional studies provided evidence of its activating role, revealing enhanced binding of the mutant protein to MRAS and PPP1CB, and increased signaling through the MAPK cascade. Differing from SHOC2 S2G , SHOC2 Q269_H270delinsHY is not constitutively targeted to the plasma membrane. These data document that diverse mechanisms in SHOC2 functional dysregulation converge toward MAPK signaling upregulation.