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.

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.

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.

The added value of a European Reference Network on rare and complex connective tissue and musculoskeletal diseases: insights after the first 5 years of the ERN ReCONNET.

In order to address the main challenges related to the rare diseases (RDs) the European Commission launched the European Reference Networks (ERNs), virtual networks involving healthcare providers (HCPs) across Europe. The mission of the ERNs is to tackle low prevalence and RDs that require highly specialised treatment and a concentration of knowledge and resources. In fact, ERNs offer the potential to give patients and healthcare professionals across the EU access to the best expertise and timely exchange of lifesaving knowledge, trying to make the knowledge travelling more than patients. For this reason, ERNs were established as concrete European infrastructures, and this is particularly crucial in the framework of rare and complex diseases in which no country alone has the whole knowledge and capacity to treat all types of patients.It has been five years since their kick-off launch in Vilnius in 2017. The 24 ERNs have been intensively working on different transversal areas, including patient management, education, clinical practice guidelines, patients' care pathways and many other fundamental topics. The present work is therefore aimed not only at reporting a summary of the main activities and milestones reached so far, but also at celebrating the first 5 years of the ERN on Rare and Complex Connective Tissue and Musculo-skeletal Diseases (ReCONNET), in which the members of the network built together one of the 24 infrastructures that are hopefully going to change the scenario of rare diseases across the EU.

Koolen-de Vries syndrome in the first adulthood patient of Southern India ancestry.

Koolen-de Vries syndrome (KdVS, MIM#610443) is a rare malformation condition mainly characterized by cognitive impairment in association with craniofacial and visceral anomalies. The core phenotype is caused by mutations in the chromatin remodeler KANSL1 (MSL1V1, KIAA1267, KAT8 Regulatory NSL Complex Subunit 1, MIM#612452), which maps to 17q21.31 critical genomic region (Koolen et al., Nature Genetics 2012;44:639-641). Considering its molecular basis, KdVS is included in the group of Developmental Disorders of Chromatin Remodeling (DDCRs), also termed chromatinopathies. We describe the first KdVS patient of Southern India ethnicity, harboring the typical de novo 17q21.31 microdeletion, including KANSL1. Observed facial features and congenital anomalies are in line with the already reported KdVS phenotype, suggesting that phenotypic features are consistent across different ethnicities.

COL1-related overlap disorder: A novel connective tissue disorder incorporating the osteogenesis imperfecta/Ehlers-Danlos syndrome overlap.

The 2017 classification of Ehlers-Danlos syndromes (EDS) identifies three types associated with causative variants in COL1A1/COL1A2 and distinct from osteogenesis imperfecta (OI). Previously, patients have been described with variable features of both disorders, and causative variants in COL1A1/COL1A2; but this phenotype has not been included in the current classification. Here, we expand and re-define this OI/EDS overlap as a missing EDS type. Twenty-one individuals from 13 families were reported, in whom COL1A1/COL1A2 variants were found after a suspicion of EDS. None of them could be classified as affected by OI or by any of the three recognized EDS variants associated with COL1A1/COL1A2. This phenotype is dominated by EDS-related features. OI-related features were limited to mildly reduced bone mass, occasional fractures and short stature. Eight COL1A1/COL1A2 variants were novel and five recurrent with a predominance of glycine substitutions affecting residues within the procollagen N-proteinase cleavage site of α1(I) and α2(I) procollagens. Selected variants were investigated by biochemical, ultrastructural and immunofluorescence studies. The pattern of observed changes in the dermis and in vitro for selected variants was more typical of EDS rather than OI. Our findings indicate the existence of a wider recognizable spectrum associated with COL1A1/COL1A2.

The facial dysmorphology analysis technology in intellectual disability syndromes related to defects in the histones modifiers.

Genetic syndromes are frequently associated with Intellectual Disability (ID), as well as craniofacial dysmorphisms. A group of ID syndromes with typical abnormal face related to chromatin remodeling defects, have been recognized, coining the term chromatinopathies. This is a molecular heterogeneous subset of congenital disorders caused by mutations of the various components of the Chromatin-Marking System (CMS), including modifiers of DNA and chromatin remodelers. We performed a phenotypic study on a sample of 120 individuals harboring variants in genes codifying for the histones enzymes, using the DeepGestalt technology. Three experiments (two multiclass comparison experiments and a frontal face-crop analysis) were conducted, analyzing respectively a total of 181 pediatric images in the first comparison experiment and 180 in the second, all individuals belonging predominantly to Caucasian population. The classification results were expressed in terms of the area under the curve (AUC) of the receiver-operating-characteristic curve (ROC). Significant values of AUC and low p-values were registered for all syndromes in the three experiments, in comparison with each other, with other ID syndromes characterized by recognizable craniofacial dysmorphisms and with unaffected controls. Final findings indicated that this group of diseases is characterized by distinctive dysmorphisms, which result pathognomonic. A correct interrogation and use of adequate informatics aids, could become a valid support for clinicians.

Tubulin-related cerebellar dysplasia: definition of a distinct pattern of cerebellar malformation.

OBJECTIVE: To determine the neuroimaging pattern of cerebellar dysplasia (CD) and other posterior fossa morphological anomalies associated with mutations in tubulin genes and to perform clinical and genetic correlations. METHODS: Twenty-eight patients harbouring 23 heterozygous pathogenic variants (ten novel) in tubulin genes TUBA1A (n = 10), TUBB2B (n = 8) or TUBB3 (n = 5) were studied by a brain MRI scan performed either on a 1.5 T (n = 10) or 3 T (n = 18) MR scanner with focus on the posterior fossa. RESULTS: Cerebellar anomalies were detected in 24/28 patients (86%). CD was recognised in 19/28 (68%) including cortical cerebellar dysplasia (CCD) in 18/28, either involving only the cerebellar hemispheres (12/28) or associated with vermis dysplasia (6/28). CCD was located only in the right hemisphere in 13/18 (72%), including four TUBB2B-, four TUBB3- and five TUBA1A-mutated patients, while in the other five TUBA1A cases it was located only in the left hemisphere or in both hemispheres. The postero-superior region of the cerebellar hemispheres was most frequently affected. CONCLUSIONS: The cerebellar involvement in tubulinopathies shows specific features that may be labelled as 'tubulin-related CD'. This pattern is unique and differs from other genetic causes of cerebellar dysplasia. KEY POINTS: • Cortical cerebellar dysplasia without cysts is suggestive of tubulin-related disorder. • Cerebellar dysplasia in tubulinopathies shows specific features labelled as 'tubulin-related CD'. • Focal and unilateral involvement of cerebellar hemispheres has important implications for counselling.

Phenotypic variability of PINK1 expression: 12 Years' clinical follow-up of two Italian families.

BACKGROUND: Mutations in the PINK1 gene are the second most frequent cause of autosomal recessive early-onset parkinsonism. METHODS: We evaluated five affected PINK1 homozygous and 14 heterozygous mutation carriers from two large Italian families over a 12-year follow-up period. Motor, nonmotor, cognitive, psychiatric, and behavioral profiles were systematically assessed. Four homozygotes and eight heterozygotes underwent magnetic resonance imaging. RESULTS: All homozygotes showed a mild progression of motor signs and a persistent excellent response to levodopa. All but one patient complained of nonmotor symptoms and sleep impairment. Three presented impulse control disorders and two anxiety and apathy. All obtained abnormal scores at Montreal Cognitive Assessment (MoCA) and in tests sensitive to frontal functions; one presented a global cognitive impairment. Three heterozygotes showed motor signs and were diagnosed as possibly affected. They had nonmotor symptoms and cognitive impairment, and two of them showed mild bilateral temporal atrophy. Five unaffected heterozygotes reported abnormal scores at MoCA and low performances at tests sensitive to frontal functions. CONCLUSION: We expanded the phenotypic profile of PINK1-related parkinsonism, including psychiatric and cognitive features as part of clinical presentation.

Alpha-synuclein gene duplication: marked intrafamilial variability in two novel pedigrees.

BACKGROUND: Multiplications of the SNCA gene that encodes alpha-synuclein are a rare cause of autosomal dominant Parkinson's disease (PD). METHODS: Here, we describe 2 novel families in which there is autosomal dominant PD associated with SNCA duplication, and we compare the clinical features of all known patients carrying 3 or 4 SNCA copies. RESULTS: Affected members in family A presented with early onset PD that was variably associated with nonmotor features, such as dysautonomia, cognitive deficits, and psychiatric disturbances. In family B, the clinical presentation ranged from early onset PD-dementia with psychiatric disturbances to late onset PD with mild cognitive impairment. CONCLUSIONS: The presence of 4 SNCA copies is associated with a rich phenotype, characterized by earlier onset of motor and nonmotor features compared with patients who bear 3 SNCA copies. The clinical spectrum associated with SNCA duplications is wide, even within a single family, suggesting a role for as yet unidentified genetic or environmental modifiers.

Defining the epsilon-sarcoglycan (SGCE) gene phenotypic signature in myoclonus-dystonia: a reappraisal of genetic testing criteria.

Mutations or exon deletions of the epsilon-sarcoglycan (SGCE) gene cause myoclonus-dystonia (M-D), but a subset of M-D patients are mutation-negative and the sensitivity and specificity of current genetic testing criteria are unknown. We screened 46 newly enrolled M-D patients for SGCE mutations and deletions; moreover, 24 subjects previously testing negative for SGCE mutations underwent gene dosage analysis. In our combined cohorts, we calculated sensitivity, specificity, positive and negative predictive values, and area under the curve of 2 published sets of M-D diagnostic criteria. A stepwise logistic regression was used to assess which patients' characteristics best discriminated mutation carriers and to calculate a new mutation predictive score ("new score"), which we validated in previously published cohorts. Nine of 46 (19.5%) patients of the new cohort carried SCGE mutations, including 5 novel point mutations and 1 whole-gene deletion; in the old cohort, 1 patient with a complex phenotype carried a 5.9-Mb deletion encompassing SGCE. Current diagnostic criteria had a poor ability to discriminate SGCE-positive from SGCE-negative patients in our cohort; conversely, age of onset, especially if associated with psychiatric features (as included in the new score), showed the best discriminatory power to individuate SGCE mutation carriers, both in our cohort and in the validation cohort. Our results suggest that young age at onset of motor symptoms, especially in association with psychiatric disturbance, are strongly predictive for SGCE positivity. We suggest performing gene dosage analysis by multiple ligation-dependent probe amplification (MLPA) to individuate large SGCE deletions that can be responsible for complex phenotypes.

The Language of Pain in the Hypermobile Ehlers-Danlos Syndrome: Metaphors as a Key to Understanding the Experience of Pain and as a Rehabilitation Tool.

Ehlers-Danlos syndromes are a heterogeneous group of Heritable Connective Tissue Disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. Among the different types, the hypermobile Ehlers-Danlos syndrome is the most frequent and includes generalized joint hypermobility as the major diagnostic criterion. Joint hypermobility in hypermobile Ehlers-Danlos syndrome is often associated with pain that does not always allow the use of effective pain-reducing treatments. Patients with hEDS constantly describe their pain in detail. Eighty-nine patients with hEDS diagnoses were recruited and evaluated. They were asked to describe their pain in writing. The texts were examined through Linguistic Inquiry and Word Count. Correlational analyses were conducted between pain perception and language. A comparison of high/low pain perception and the quality of metaphors was carried out. The results showed that language quality varies depending on how much pain is perceived. The greater the pain is perceived, the lesser the positive effects and the greater the negative effects and dehumanizing metaphors are being used. Moreover, a greater pain seems to be related to a verbal experience of greater isolation and less self-care. In conclusion, the use of metaphors is a useful tool for examining illness experience and may help clinicians in the rehabilitation program.

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.

Independent and joint effects of the MAPT and SNCA genes in Parkinson disease.

OBJECTIVE: We studied the independent and joint effects of the genes encoding alpha-synuclein (SNCA) and microtubule-associated protein tau (MAPT) in Parkinson disease (PD) as part of a large meta-analysis of individual data from case-control studies participating in the Genetic Epidemiology of Parkinson's Disease (GEO-PD) consortium. METHODS: Participants of Caucasian ancestry were genotyped for a total of 4 SNCA (rs2583988, rs181489, rs356219, rs11931074) and 2 MAPT (rs1052553, rs242557) single nucleotide polymorphism (SNPs). Individual and joint effects of SNCA and MAPT SNPs were investigated using fixed- and random-effects logistic regression models. Interactions were studied on both a multiplicative and an additive scale, and using a case-control and case-only approach. RESULTS: Fifteen GEO-PD sites contributed a total of 5,302 cases and 4,161 controls. All 4 SNCA SNPs and the MAPT H1-haplotype-defining SNP (rs1052553) displayed a highly significant marginal association with PD at the significance level adjusted for multiple comparisons. For SNCA, the strongest associations were observed for SNPs located at the 3' end of the gene. There was no evidence of statistical interaction between any of the 4 SNCA SNPs and rs1052553 or rs242557, neither on the multiplicative nor on the additive scale. INTERPRETATION: This study confirms the association between PD and both SNCA SNPs and the H1 MAPT haplotype. It shows, based on a variety of approaches, that the joint action of variants in these 2 loci is consistent with independent effects of the genes without additional interacting effects.

Audiological and radiological characteristics of a family with T961G mitochondrial mutation.

OBJECTIVE: The aim of this study was to describe audiological and radiological characteristics, and other secondary aspects, in a family carrying a T961G mutation in the 12S rRNA mitochondrial gene. DESIGN: Case report. STUDY SAMPLE: Six members of a family participated in an audiological evaluation that included pure-tone audiometry, immittance tests, auditory brainstem responses (ABR), and otoacoustic emissions (OAE). The radiological evaluation was conducted through temporal bone CT scans using a Toshiba 16 channels Aquilon Spirale. Neuropsychiatric evaluation was also administered. RESULTS: Three participants were diagnosed with severe sensorineural hearing loss of cochlear origin and cochlear malformations visible in CT scans. One participant had a mild mixed-hearing loss and no cochlear malformations. Two participants had normal audiological and radiological findings. CONCLUSIONS: We believe our study can provide helpful insight on the clinical findings of a rare mutation, of which few data have been presented in literature.

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.

An overlook on the current registries for rare and complex connective tissue diseases and the future scenario of TogethERN ReCONNET.

Background: Patient registries play a crucial role in supporting clinical practice, healthcare planning and medical research, offering a real-world picture on rare and complex connective tissue diseases (rCTDs). ERN ReCONNET launched the first European Registry Infrastructure with the aim to plan, upgrade and link registries for rCTDs, with the final goal to promote a harmonized data collection approach all over Europe for rCTDs. Methods: An online survey addressed to healthcare professionals and patients' representatives active in the field of rCTDs was integrated by an extensive database search in order to build a mapping of existing registries for rCTDs. Findings: A total of 140 registries were found, 38 of which include multiple diseases. No disease-specific registry was identified for relapsing polychondritis, mixed connective tissue disease and undifferentiated connective tissue disease. Discussion: This overview on the existing registries for rCTDs provides a useful starting point to identify the gaps and the strengths of registries on the coverage of rCTDs, and to develop a common data set and data collection approach for the establishment of the TogethERN ReCONNET Infrastructure.