Functional characterization vs in silico prediction for TBX5 missense and splice variants in Holt-Oram syndrome.

PURPOSE: Predicting effects of genomic variants has become a real challenge in the diagnosis of rare human diseases. Holt-Oram syndrome is an autosomal condition characterized by the association of radial and heart defects, due to variants in TBX5. Most variants are predicted to be truncating and result in haploinsufficiency. The pathogenicity of missense or splice variants is harder to demonstrate. METHODS: Fourteen TBX5 variants of uncertain significance (5 missense, 9 splice) and 6 likely pathogenic missense variants were selected for functional testing, depending on the variant-type (immunolocalization, western blot, reporter assays, minigene splice assays, and reverse transcription-polymerase chain reaction). Results were compared with in silico predictions. RESULTS: Functional tests allowed to reclassify 9/14 variants of uncertain significance in TBX5 as likely pathogenic, confirming their role in Holt-Oram syndrome. We demonstrated loss of function (n = 8) or gain of function (n = 1) for 9 of the 11 missense variants, whereas no functional impact was shown for the 2 variants: p.(Gly195Ala) and p.(Ser261Cys), as suggested by contradictory predictions of in silico approaches. Of 9 splice variants predicted to affect splicing by SpliceAI, we observed partial or complete exon skipping (n = 6), intron retention (n = 2) or exon shortening (n = 1), inducing frame shifting with premature stop codons. CONCLUSION: Bioinformatic and biological approaches are complementary, together with a good knowledge of clinical conditions, for accurate American College of Medical Genetics and Genomics classification in human rare diseases.

RPL26 variants: A rare cause of Diamond-Blackfan anemia syndrome with multiple congenital anomalies at the forefront.

PURPOSE: Diamond-Blackfan anemia syndrome (DBS) is a rare congenital disorder originally characterized by bone marrow failure with or without various congenital anomalies. At least 24 genes are implicated, the vast majority encoding for ribosomal proteins. RPL26 (ribosomal protein L26) is an emerging candidate (DBA11, MIM#614900). We aim to further delineate this rare condition. METHODS: Patients carrying heterozygous RPL26 variants were recruited. In one of them, erythroid proliferation and differentiation from peripheral blood CD34+ cells were studied by flow cytometry, and RPL26 expression by quantitative reverse transcription polymerase chain reaction and immunoblotting. RESULTS: We report on 8 affected patients from 4 families. Detailed phenotyping reveals that RPL26 is mainly associated with multiple congenital anomalies (particularly radial ray anomalies), albeit with variable expression. Mandibulofacial dysostosis and neural tube defects are potential features in DBA11, expanding the growing list of DBS abnormalities. In 1 individual, we showed that RPL26 haploinsufficiency was responsible for subclinical impairment in erythroid proliferation and enucleation. The absence of hematological involvement in 4 adults from this series contributes to the mounting evidence that bone marrow failure is not universally central to all DBS genes. CONCLUSION: We confirm RPL26 as a DBS gene and expand the phenotypic spectrum of the gene and the disease.

Expanded phenotypic spectrum of UDP-glucose-6-dehydrogenase recessive neurodevelopmental disorder: Two novel descriptions with or without epileptic encephalopathy.

Recent advances in the understanding of infantile developmental epileptic encephalopathies (IDEE) have revealed the association of biallelic pathogenic variants in UGDH. In this study, we report two novel combinations identified by exome sequencing: p.(Arg135Trp) with p.(Arg65*) and p.(Arg102Trp) with p.(Arg65*). Both combinations share a common pathogenic nonsense variant, with the missense variants strategically located in the NAD-binding domain of the UGDH protein, predicted in structural models to create new interactions with the central domain. The first patient exhibited the typical UGDH-related disease phenotype and progressive microcephaly, a rarely reported feature. In contrast, the second patient presented an atypical phenotype, including absence of seizure, severe intellectual disability, ataxic gait, and abnormal eye movements. This comprehensive analysis extends the phenotypic spectrum of UGDH syndrome beyond early infantile intractable encephalopathy to include intellectual disability without epilepsy.

Novel Genetic and Phenotypic Expansion in Ameliorated PUF60-Related Disorders.

Heterozygous variants in the Poly(U) Binding Splicing Factor 60kDa gene (PUF60) have been associated with Verheij syndrome, which has the key features of coloboma, short stature, skeletal abnormalities, developmental delay, palatal abnormalities, and congenital heart and kidney defects. Here, we report five novel patients from unrelated families with PUF60-related disorders exhibiting novel genetic and clinical findings with three truncating variants, one splice-site variant with likely reduced protein expression, and one missense variant. Protein modeling of the patient's missense variant in the PUF60 AlphaFold structure revealed a loss of polar bonds to the surrounding residues. Neurodevelopmental disorders were present in all patients, with variability in speech, motor, cognitive, social-emotional and behavioral features. Novel phenotypic expansions included movement disorders as well as immunological findings with recurrent respiratory, urinary and ear infections, atopic diseases, and skin abnormalities. We discuss the role of PUF60 in immunity with and without infection based on recent organismic and cellular studies. As our five patients showed less-severe phenotypes than classical Verheij syndrome, particularly with the absence of key features such as coloboma or palatal abnormalities, we propose a reclassification as PUF60-related neurodevelopmental disorders with multi-system involvement. These findings will aid in the genetic counseling of patients and families.

Multiplex targeted high-throughput sequencing in a series of 352 patients with congenital limb malformations.

Congenital limb malformations (CLM) comprise many conditions affecting limbs and more than 150 associated genes have been reported. Due to this large heterogeneity, a high proportion of patients remains without a molecular diagnosis. In the last two decades, advances in high throughput sequencing have allowed new methodological strategies in clinical practice. Herein, we report the screening of 52 genes/regulatory sequences by multiplex high-throughput targeted sequencing, in a series of 352 patients affected with various CLM, over a 3-year period of time. Patients underwent a clinical triage by expert geneticists in CLM. A definitive diagnosis was achieved in 35.2% of patients, the yield varying considerably, depending on the phenotype. We identified 112 single nucleotide variants and 26 copy-number variations, of which 52 are novel pathogenic or likely pathogenic variants. In 6% of patients, variants of uncertain significance have been found in good candidate genes. We showed that multiplex targeted high-throughput sequencing works as an efficient and cost-effective tool in clinical practice for molecular diagnosis of congenital limb malformations. Careful clinical evaluation of patients may maximize the yield of CLM panel testing.

TAR syndrome: Clinical and molecular characterization of a cohort of 26 patients and description of novel noncoding variants of RBM8A.

Thrombocytopenia-absent radius (TAR) syndrome is characterized by radial defect and neonatal thrombocytopenia. It is caused by biallelic variants of RBM8A gene (1q21.1) with the association of a null allele and a hypomorphic noncoding variant. RBM8A encodes Y14, a core protein of the exon junction complex involved in messenger RNA maturation. To date, only two hypomorphic variants have been identified. We report on a cohort of 26 patients affected with TAR syndrome and carrying biallelic variants in RBM8A. Half patients carried a 1q21.1 deletion and one of the two known hypomorphic variants. Four novel noncoding variants of RBM8A were identified in the remaining patients. We developed experimental models enabling their functional characterization in vitro. Two variants, located respectively in the 5'-untranslated region (5'-UTR) and 3'-UTR regions, are responsible for a diminished expression whereas two intronic variants alter splicing. Our results bring new insights into the molecular knowledge of TAR syndrome and enabled us to propose genetic counseling for patients' families.

WNT10B variants in split hand/foot malformation: Report of three novel families and review of the literature.

Split-hand/foot malformation (SHFM) is a genetically heterogeneous congenital limb malformation typically limited to a defect of the central rays of the autopod, presenting as a median cleft of hands and feet. It can be associated with long bone deficiency or included in more complex syndromes. Among the numerous genetic causes, WNT10B homozygous variants have been recently identified in consanguineous families, but remain still rarely described (SHFM6; MIM225300). We report on three novel SHFM families harboring WNT10B variants and review the literature, allowing us to highlight some clinical findings. The feet are more severely affected than the hands and there is a frequent asymmetry without obvious side-bias. Syndactyly of third-fourth fingers was a frequent finding (62%). Polydactyly, which was classically described in SHFM6, was only present in 27% of patients. No genotype-phenotype correlation is delineated but heterozygous individuals might have mild features of SHFM, suggesting a dose-effect of the WNT10B loss-of-function.

FAM46A mutations are responsible for autosomal recessive osteogenesis imperfecta.

BACKGROUND: Stüve-Wiedemann syndrome (SWS) is characterised by bowing of the lower limbs, respiratory distress and hyperthermia that are often responsible for early death. Survivors develop progressive scoliosis and spontaneous fractures. We previously identified LIFR mutations in most SWS cases, but absence of LIFR pathogenic changes in five patients led us to perform exome sequencing and to identify homozygosity for a FAM46A mutation in one case [p.Ser205Tyrfs*13]. The follow-up of this case supported a final diagnosis of osteogenesis imperfecta (OI), based on vertebral collapses and blue sclerae. METHODS AND RESULTS: This prompted us to screen FAM46A in 25 OI patients with no known mutations.We identified a homozygous deleterious variant in FAM46A in two affected sibs with typical OI [p.His127Arg]. Another homozygous variant, [p.Asp231Gly], also classed as deleterious, was detected in a patient with type III OI of consanguineous parents using homozygosity mapping and exome sequencing.FAM46A is a member of the superfamily of nucleotidyltransferase fold proteins but its exact function is presently unknown. Nevertheless, there are lines of evidence pointing to a relevant role of FAM46A in bone development. By RT-PCR analysis, we detected specific expression of FAM46A in human osteoblasts andinterestingly, a nonsense mutation in Fam46a has been recently identified in an ENU-derived (N-ethyl-N-nitrosourea) mouse model characterised by decreased body length, limb, rib, pelvis, and skull deformities and reduced cortical thickness in long bones. CONCLUSION: We conclude that FAM46A mutations are responsible for a severe form of OI with congenital bowing of the lower limbs and suggest screening this gene in unexplained OI forms.

Clinical spectrum of rare bone fragility disorders and response to bisphosphonate treatment: a retrospective study.

Osteogenesis Imperfecta (OI) is a clinically and genetically heterogeneous group of diseases characterized by brittle bones. Though genetic mutations in COL1A1 and COL1A2 account for approximately 85-90% of OI cases, there are now more than twenty genes described, responsible for rare forms of OI. Treatment is based on the use of bisphosphonates and though it is well established that they increase lumbar spine (LS) bone mineral density (BMD), the clinical impact on fracture reduction is still debated.In this study, we investigated the clinical characteristics of 38 patients with a bone fragility disorder that had variants in non-COL1A1/COL1A2 genes in order to study genotype-phenotype correlations, as the natural history of these rare forms is still not well known. We then studied the usefulness of bisphosphonate treatment by evaluating the effects on LS BMD, annual non-vertebral fracture rate, bone turnover markers and height. This study enabled us to better define the natural history of patients with non-COL1 pathogenic variants. Patients with CRTAP and TMEM38B variants consistently had a prenatal presentation with a short (<3rd p) and bowed femur. Importantly, this prenatal involvement does not predict the postnatal severity of the disease. Regarding treatment by bisphosphonates, all patients showed a significant increase in LS BMD while treated and this increase was dependent on the dose received. The increase in LS BMD also translated in a reduction of fracture rate during treatment. Finally, our study showed that the earlier bisphosphonates are initiated, the greater the fracture rate is reduced.

Episignatures in practice: independent evaluation of published episignatures for the molecular diagnostics of ten neurodevelopmental disorders.

Variants of uncertain significance (VUS) are a significant issue for the molecular diagnosis of rare diseases. The publication of episignatures as effective biomarkers of certain Mendelian neurodevelopmental disorders has raised hopes to help classify VUS. However, prediction abilities of most published episignatures have not been independently investigated yet, which is a prerequisite for an informed and rigorous use in a diagnostic setting. We generated DNA methylation data from 101 carriers of (likely) pathogenic variants in ten different genes, 57 VUS carriers, and 25 healthy controls. Combining published episignature information and new validation data with a k-nearest-neighbour classifier within a leave-one-out scheme, we provide unbiased specificity and sensitivity estimates for each of the signatures. Our procedure reached 100% specificity, but the sensitivities unexpectedly spanned a very large spectrum. While ATRX, DNMT3A, KMT2D, and NSD1 signatures displayed a 100% sensitivity, CREBBP-RSTS and one of the CHD8 signatures reached <40% sensitivity on our dataset. Remaining Cornelia de Lange syndrome, KMT2A, KDM5C and CHD7 signatures reached 70-100% sensitivity at best with unstable performances, suffering from heterogeneous methylation profiles among cases and rare discordant samples. Our results call for cautiousness and demonstrate that episignatures do not perform equally well. Some signatures are ready for confident use in a diagnostic setting. Yet, it is imperative to characterise the actual validity perimeter and interpretation of each episignature with the help of larger validation sample sizes and in a broader set of episignatures.

TRIT1 deficiency: Two novel patients with four novel variants.

TRIT1 encodes a tRNA isopentenyl transferase that allows a strong interaction between the mini helix and the codon. Recent reports support the TRIT1 bi-allelic alterations as the cause of an autosomal recessive disorder, named combined oxydative phophorylation deficiency 35, with microcephaly, developmental disability, and epilepsy. The phenotype is due to decreased mitochondrial function, with deficit of i6A37 in cytosolic and mitochondrial tRNA. Only 10 patients have been reported. We report on two new patients with four novel variants, and confirm the published clinical TRIT1 deficient phenotype stressing the possibility of both very severe, with generalized pharmaco-resistant seizures, and mild phenotypes.

De novo variants in genes regulating stress granule assembly associate with neurodevelopmental disorders.

Stress granules (SGs) are cytoplasmic assemblies in response to a variety of stressors. We report a new neurodevelopmental disorder (NDD) with common features of language problems, intellectual disability, and behavioral issues caused by de novo likely gene-disruptive variants in UBAP2L, which encodes an essential regulator of SG assembly. Ubap2l haploinsufficiency in mouse led to social and cognitive impairments accompanied by disrupted neurogenesis and reduced SG formation during early brain development. On the basis of data from 40,853 individuals with NDDs, we report a nominally significant excess of de novo variants within 29 genes that are not implicated in NDDs, including 3 essential genes (G3BP1, G3BP2, and UBAP2L) in the core SG interaction network. We validated that NDD-related de novo variants in newly implicated and known NDD genes, such as CAPRIN1, disrupt the interaction of the core SG network and interfere with SG formation. Together, our findings suggest the common SG pathology in NDDs.

Homozygous Loss-of-Function Mutations in CCDC134 Are Responsible for a Severe Form of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a primary bone fragility disorder with an estimated prevalence of 1 in 15,000 births. The majority of OI cases are inherited in an autosomal-dominant manner, while 5% to 10% have recessive or X-linked inheritance. Up to now, approximately 5% of OI cases remain without mutation demonstrated, supporting the involvement of other genes in the disease spectrum. By whole-exome sequencing, we identified a homozygous variant (c.2T>C) in CCDC134 gene in three patients from two unrelated families with severe bone fragility that did not respond to bisphosphonate treatment, short stature, and gracile long bones with pseudarthroses but no dentinogenesis imperfecta. CCDC134 encodes a secreted protein widely expressed and implicated in the regulation of some mitogen-activated protein kinases (MAPK) signaling pathway. Western blot and immunofluorescence analyses confirmed the absence of CCDC134 protein in patient cells compared with controls. Furthermore, we demonstrated that CCDC134 mutations are associated with increased Erk1/2 phosphorylation, decreased OPN mRNA and COL1A1 expression and reduced mineralization in patient osteoblasts compared with controls. These data support that CCDC134 is a new gene involved in severe progressive deforming recessive osteogenesis imperfecta (type III). © 2020 American Society for Bone and Mineral Research.

A second cohort of CHD3 patients expands the molecular mechanisms known to cause Snijders Blok-Campeau syndrome.

There has been one previous report of a cohort of patients with variants in Chromodomain Helicase DNA-binding 3 (CHD3), now recognized as Snijders Blok-Campeau syndrome. However, with only three previously-reported patients with variants outside the ATPase/helicase domain, it was unclear if variants outside of this domain caused a clinically similar phenotype. We have analyzed 24 new patients with CHD3 variants, including nine outside the ATPase/helicase domain. All patients were detected with unbiased molecular genetic methods. There is not a significant difference in the clinical or facial features of patients with variants in or outside this domain. These additional patients further expand the clinical and molecular data associated with CHD3 variants. Importantly we conclude that there is not a significant difference in the phenotypic features of patients with various molecular disruptions, including whole gene deletions and duplications, and missense variants outside the ATPase/helicase domain. This data will aid both clinical geneticists and molecular geneticists in the diagnosis of this emerging syndrome.

Expanding the clinical spectrum of hereditary fibrosing poikiloderma with tendon contractures, myopathy and pulmonary fibrosis due to FAM111B mutations.

BACKGROUND: Hereditary Fibrosing Poikiloderma (HFP) with tendon contractures, myopathy and pulmonary fibrosis (POIKTMP [MIM 615704]) is a very recently described entity of syndromic inherited poikiloderma. Previously by using whole exome sequencing in five families, we identified the causative gene, FAM111B (NM_198947.3), the function of which is still unknown. Our objective in this study was to better define the specific features of POIKTMP through a larger series of patients. METHODS: Clinical and molecular data of two families and eight independent sporadic cases, including six new cases, were collected. RESULTS: Key features consist of: (i) early-onset poikiloderma, hypotrichosis and hypohidrosis; (ii) multiple contractures, in particular triceps surae muscle contractures; (iii) diffuse progressive muscular weakness; (iv) pulmonary fibrosis in adulthood and (v) other features including exocrine pancreatic insufficiency, liver impairment and growth retardation. Muscle magnetic resonance imaging was informative and showed muscle atrophy and fatty infiltration. Histological examination of skeletal muscle revealed extensive fibroadipose tissue infiltration. Microscopy of the skin showed a scleroderma-like aspect with fibrosis and alterations of the elastic network. FAM111B gene analysis identified five different missense variants (two recurrent mutations were found respectively in three and four independent families). All the mutations were predicted to localize in the trypsin-like cysteine/serine peptidase domain of the protein. We suggest gain-of-function or dominant-negative mutations resulting in FAM111B enzymatic activity changes. CONCLUSIONS: HFP with tendon contractures, myopathy and pulmonary fibrosis, is a multisystemic disorder due to autosomal dominant FAM111B mutations. Future functional studies will help in understanding the specific pathological process of this fibrosing disorder.