Variants Of Uncertain Significance Research Articles

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

PurposePredicting 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. MethodsFourteen 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 RT-PCR). Results were compared with in silico predictions. ResultsFunctional 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. ConclusionBioinformatic 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.

A pilot study of chlorambucil in pre-treated metastatic pancreatic adenocarcinoma patients bearing germline BRCA or other DNA damage repair system variants

BackgorundPancreatic adenocarcinoma remains a malignancy with a grim prognosis and scarce personalized treatment options. Pathogenic variants of DNA damage repair (DDR) genes are emerging as molecular targets, as they confer a higher sensitivity to DNA-damaging agents. This study aimed at assessing the activity of chlorambucil as salvage therapy in metastatic pancreatic cancer patients bearing a germline pathogenetic variant or variant of uncertain significance on a DDR-related gene. MethodsPlatinum-pretreated metastatic pancreatic cancer patients harbouring a germline variant on a DDR gene received chlorambucil at a daily oral dose of 6 mg/m2 for 42 every 56 days for the first cycle and for 14 every 28 days for the following cycles, until disease progression or unacceptable toxicity. The primary endpoint was 6-month progression-free survival rate (PFS-6). Median progression-free survival (PFS) and overall survival (OS) were secondarily described. ResultsTwenty patients were enrolled between December 2020 and September 2022. PFS-6 was 5%, median PFS and OS were 1.6 months and 3.0 months, respectively. Grade-3 adverse events were observed in 25% of patients, while no Grade-4 toxicity was reported. ConclusionsSingle agent chlorambucil did not show sufficient signal of activity to warrant its further investigation in metastatic pancreatic cancer patients bearing a DDR-related germline alteration.

Evaluation of Bayesian point-based system on the variant classification of hereditary cancer predisposition genes

PurposeTo assess the differences in variant classifications using the American College of Medical Genetics and Genomics and the Association for Molecular Pathology 2015 guidelines and the Bayesian point-based classification system (here referred to as the point system) in 115 hereditary cancer predisposition genes and explore variant subtiering by the point system. MethodsGermline variant classifications for 721 pediatric patients from an in-house panel were retrospectively evaluated using the 2 scoring systems. ResultsA total of 2376 unique variants were identified, with ∼23.5% exhibiting discordant classifications. Unique variants classified by the point system demonstrated a lower rate of variants of uncertain significance (VUS; ∼15%) compared with American College of Medical Genetics and Genomics and the Association for Molecular Pathology 2015 (∼36%). This change is attributed to unique variants with 1 benign supporting evidence (∼12%) or 1 benign strong evidence (∼4%) being classified as likely benign by the point system. Additionally, variants with conflicting/modified evidence (∼5% of 2376) are also resolved by the point system. Subtiering unique variants classified by the point system as VUS (n = 354) indicates ∼77.4% were VUS-Low (0-1 points), whereas the remaining ∼22.6% were VUS-Mid (2-3 points) and VUS-High (4-5 points). ConclusionThe point system reduces the VUS rate and facilitates their subtiering. Future large-scale studies are warranted to explore the impact of the point system on improving VUS reporting and/or VUS clinical management.

Significance of Variants of Uncertain Significance: The Human Cost of Genetic Uncertainty.

This piece narrates the journey of Maria (name of the mother has been altered to protect the family's privacy), a new mother confronting her newborn's unexpected diagnosis of very long chain acyl-CoA dehydrogenase (VLCAD) deficiency, despite undergoing proactive genetic carrier screening within a consanguineous marriage. It highlights the emotional and systemic challenges arising from the lack of diversity in genetic databases, which, in this case, failed to detect pathogenic variants in Maria and her husband. Maria's story sheds light on situations where a masked variant of uncertain significance (VUS) necessitates consultation with a trained genetics specialist and underscores the urgent need for a more equitable healthcare system.

Virus-like particles as robust tools for functional assessment: Deciphering the pathogenicity of ABCA4 genetic variants of uncertain significance

The retina-specific ABCA transporter, ABCA4, is essential for vision, and its genetic variants are associated with a wide range of inherited retinal degenerative diseases (IRDs) leading to blindness. Of the 1,630 identified missense variants in ABCA4, ∼50% are of unknown pathogenicity (variants of unknown significance, VUS). This genetic uncertainty presents three main challenges: (i) inability to predict disease-causing variants in relatives of IRD patients with multiple ABCA4 mutations; (ii) limitations in developing variant-specific treatments; and (iii) difficulty in using these variants for future disease prediction, affecting patients' life-planning and clinical trial participation. To unravel the clinical significance of ABCA4 genetic variants at the level of protein function, we have developed a virus-like particle (VLP)-based system that expresses the ABCA4 protein and its variants. We validated the efficacy of this system in the enzymatic characterization (ATPase activity) of VLPs harboring ABCA4 and two variants of established pathogenicity: p.N965S and p.C1488R. Our results were consistent with previous reports and clinical phenotypes. We also applied this platform to characterize the VUS p.Y1779F and observed a functional impairment, suggesting a potential pathogenic impact. This approach offers an efficient, high-throughput method for ABCA4 VUS characterization. Our research points to the significant promise of the VLP-based system in the functional analysis of membrane proteins, offering important perspectives on the disease-causing potential of genetic variants and shedding light on genetic conditions involving such proteins.

Management Targeted Genetic Evaluation of an Idiopathic Neuropathy Cohort Through ATTRv Amyloidosis Screening.

While the reported prevalence of polyneuropathies is 1%-3%, the incidence of hereditary transthyretin amyloidosis in the United States is estimated to be 1 in 100 000 individuals. Polyneuropathies are known to be difficult to treat and lead to significant morbidity. The aim of pain management is symptomatic treatment, with varying approaches to progression prevention being based on the causative pathophysiology.We assessed the prevalence of hereditary amyloid transthyretin variant (ATTRv) amyloidosis, a progressive autosomal dominant multisystem disease caused by the abnormal formation and extracellular deposition of transthyretin protein fibrils in various tissues, in an idiopathic polyneuropathy population by using genetic analysis. Individuals aged 18 and over with an established diagnosis of polyneuropathy, via electromyography testing that was deemed to be idiopathic, at a large, urban neurology clinic consented to an institutional review board-approved protocol for genetic testing. No further exclusions were made regarding age of onset, family history, axonal neuropathy subtype, comorbidities suggestive of ATTRv amyloidosis, etc. Clinical genetic testing was performed on 134 participants via an 81-gene panel associated with inherited neuromuscular disorders or targeted TTR gene sequencing with deletion and duplication analysis. Within our cohort, 38.06% had at least one reportable finding in one of 38 distinct genes, for a total of 76 reported alterations. Four individuals were identified as having a single pathogenic alteration in an autosomal recessive gene, consistent with carrier status for the 4 following disorders: congenital insensitivity to pain with anhidrosis (NTRK1), Charcot-Marie-Tooth disease type IIP (LRSAM1), Brown-Vialetto-Van Laere syndrome type II (SLC52A2), hereditary sensory and autonomic neuropathy type III (IKBKAP). One individual was found to have a variant of uncertain significance (VUS) (p.G103D) in the TTR gene. Precision medicine on the molecular level with genetic testing in the identification of specific neuropathies may provide clinicians with more detailed information for developing a more direct therapeutic and treatment modality for better-targeted management. Further investigation is needed to expand on the knowledge and understanding of the clinical relevance surrounding the alterations found in the genetic evaluation of idiopathic neuropathy.

A Humanized Yeast Model for Studying TRAPP Complex Mutations; Proof-of-Concept Using Variants from an Individual with a TRAPPC1-Associated Neurodevelopmental Syndrome.

Variants in membrane trafficking proteins are known to cause rare disorders with severe symptoms. The highly conserved transport protein particle (TRAPP) complexes are key membrane trafficking regulators that are also involved in autophagy. Pathogenic genetic variants in specific TRAPP subunits are linked to neurological disorders, muscular dystrophies, and skeletal dysplasias. Characterizing these variants and their phenotypes is important for understanding the general and specialized roles of TRAPP subunits as well as for patient diagnosis. Patient-derived cells are not always available, which poses a limitation for the study of these diseases. Therefore, other systems, like the yeast Saccharomyces cerevisiae, can be used to dissect the mechanisms at the intracellular level underlying these disorders. The development of CRISPR/Cas9 technology in yeast has enabled a scar-less editing method that creates an efficient humanized yeast model. In this study, core yeast subunits were humanized by replacing them with their human orthologs, and TRAPPC1, TRAPPC2, TRAPPC2L, TRAPPC6A, and TRAPPC6B were found to successfully replace their yeast counterparts. This system was used for studying the first reported individual with an autosomal recessive disorder caused by biallelic TRAPPC1 variants, a girl with a severe neurodevelopmental disorder and myopathy. We show that the maternal variant (TRAPPC1 p.(Val121Alafs*3)) is non-functional while the paternal variant (TRAPPC1 p.(His22_Lys24del)) is conditional-lethal and affects secretion and non-selective autophagy in yeast. This parallels defects seen in fibroblasts derived from this individual which also showed membrane trafficking defects and altered Golgi morphology, all of which were rescued in the human system by wild-type TRAPPC1. This study suggests that humanized yeast can be an efficient means to study TRAPP subunit variants in the absence of human cells and can assign significance to variants of unknown significance (VUS). This study lays the foundation for characterizing further TRAPP variants through this system, rapidly contributing to disease diagnosis.

Calidad de los fenotipos humanos en pacientes con discapacidad intelectual con estudios de secuenciación de exoma en un hospital pediátrico peruano

Introduction. Massive sequencing and the Human Phenotype Ontology have revolutionized genetic diagnosis, allowing for a clearer understanding of diseases. The phenotypes described in medical records can be specific or general, the more detailed and specific the description, the higher the quality of the information. Objectives. To determine the relationship between the quality of human phenotypes in patients with intellectual disability and the findings from massive sequencing test at the Instituto Nacional de Salud del Niño. Methods. We analyzed 124 patients with intellectual disabilities with exome sequencing. The quality of human phenotypes was evaluated using different quality index. Results. A total of 278 human phenotype terms were identified. Phenotypic precision improved after filtering, highlighting the importance of specific phenotypic descriptions (from 103 to 86 unique terms). The specificity index of the dataset was lower in the group with variants of uncertain significance compared to the group with pathogenic variants (0 vs. 0.05). The most frequent phenotypes were psychomotor delay (12.9% - 15.9%) and microcephaly (4.5% - 5.2%). Conclusions. The importance of evaluating the quality of human phenotypes is highlighted. The presence of uncertain variants suggests a genotype-phenotype complexity, emphasizing the need for greater clarity in genetic interpretation. Consistency in confidence intervals and the specificity index of the dataset underscores the competence of local medical-genetic personnel.

Genetic findings in people with schwannomas who do not meet clinical diagnostic criteria for NF2-related schwannomatosis

BackgroundMost schwannomas are isolated tumours occurring in otherwise healthy people. However, bilateral vestibular schwannomas (BVS) or multiple non-vestibular schwannomas indicate an underlying genetic predisposition. This is most commonly NF2-related schwannomatosis...

Next-generation sequencing in Charcot-Marie-Tooth: a proposal for improvement of ACMG guidelines for variant evaluation

BackgroundThe application of massive parallel sequencing technologies in the molecular analysis of Charcot-Marie-Tooth (CMT) has enabled the rapid and cost-effective identification of numerous potentially significant variants for diagnostic purposes. The...

Phenotypic features, prevalence of KCNJ11-MODY in Chinese patients with early-onset diabetes and a literature review.

Gain-of-function (GOF) variants of KCNJ11 cause neonate diabetes and maturity-onset diabetes of the young (KCNJ11-MODY), while loss-of-function (LOF) variants lead to hyperinsulinemia hypoglycemia and subsequent diabetes. Given the limited research of KCNJ11-MODY, we aimed to analyse its phenotypic features and prevalence in Chinese patients with early-onset type 2 diabetes (EOD). We performed next-generation sequencing on 679 Chinese EOD patients to screen for KCNJ11 exons variants. Bioinformatics prediction and the American College of Medical Genetics and Genomics guidelines was used to determine the pathogenicity and diagnosed KCNJ11-MODY. A literature review was conducted to investigate the phenotypic features of KCNJ11-MODY. We identified six predicted deleterious rare variants in six EOD patients (0.88%). They were classified as uncertain significance (variant of uncertain significance [VUS]), but more common in this EOD cohort than a general Chinese population database, however, without significant difference (53/10,588, 0.50%) (p = .268). Among 80 previously reported patients with KCNJ11-MODY, 23.8% (19/80) carried 9 (32.1%) LOF variants, who had significantly older age at diagnosis, higher birthweight and higher fasting C-peptide compared to patients with GOF variants. Many patients carrying VUS were not correctly diagnosed. Some rare variants of KCNJ11 might contribute to the development of Chinese EOD, although available evidence has not enough power to support them as cause of KCNJ11-MODY. The clinical features of LOF variants were different from GOF variants in KCNJ11-MODY patients. It is necessary to evaluate the pathogenicity of VUS through function experiments.

Variant of Unknown Significance in the EGLN-1 Gene Associated With Familial Erythrocytosis

Variant of Unknown Significance in the EGLN-1 Gene Associated With Familial Erythrocytosis

Germline variant profiling of CHEK2 sequencing variants in breast cancer patients

The cell cycle checkpoint kinase 2 (CHEK2) is a tumor suppressor gene coding for a protein kinase with a role in the cell cycle and DNA repair pathways. Variants within CHEK2 are associated with an increased risk of developing breast, colorectal, prostate and several other types of cancer. Comprehensive genetic risk assessment leads to early detection of hereditary cancer and provides an opportunity for better survival. Multigene panel screening can identify the presence of pathogenic variants in hereditary cancer predisposition genes (HCPG), including CHEK2. Multigene panels, however, also result in large quantities of genetic data some of which cannot be interpreted and are classified as variants of uncertain significance (VUS). A VUS provides no information for use in medical management and leads to ambiguity in genetic counseling. In the absence of variant segregation data, in vitro functional analyses can be used to clarify variant annotations, aiding in accurate clinical management of patient risk and treatment plans. In this study, we performed whole exome sequencing (WES) to investigate the prevalence of germline variants in 210 breast cancer (BC) patients and conspicuously among the many variants in HCPGs that we found, we identified 16 individuals with non-synonymous or frameshift CHEK2 variants, sometimes along with additional variants within other BC susceptibility genes. Using this data, we investigated the prevalence of these CHEK2 variants in African American (AA) and Caucasian (CA) populations identifying the presence of two novel frameshift variants, c.1350delA (p.Val451Serfs*18) and c.1528delC (p.Gln510Argfs*3) and a novel missense variant, c262C>T (p.Pro88Ser). Along with the current clinical classifications, we assembled available experimental data and computational predictions of function for these CHEK2 variants, as well as explored the role these variants may play in polygenic risk assessment.

Comprehensive analysis of Novel mutations in CCM1/KRIT1 and CCM2/MGC4607 and their clinical implications in Cerebral Cavernous malformations

BackgroundCerebral Cavernous Malformations (CCM) is a genetic disease characterized by vascular abnormalities in the brain and spinal cord, affecting 0.4-0.5 % of the population. We identified two novel pathogenic mutations, CCM1/KRIT1 c.811delT (p.Trp271GlyfsTer5) and CCM2/MGC4607 c.613_614insGG p.Glu205GlyfsTer31), which disrupt crucial protein domains and potentially alter disease progression. ObjectiveThe study aims to comprehensively analyze a Brazilian cohort of CCM patients, integrating genetic, clinical, and structural aspects. Specifically, we sought to identify novel mutations within the CCM complex, and explore their potential impact on disease progression. MethodsWe conducted a detailed examination of neuroradiological and clinical features in both symptomatic and asymptomatic CCM patients, performing genetic analyses through sequencing of the CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10 genes In silico structural predictions were carried out using PolyPhen-2, SIFT, and Human Genomics Community tools. Protein-protein interactions and docking analyses were explored using the STRING database. ResultsGenetic analysis identifies 6 pathogenic mutations, 4 likely pathogenic, 1 variants of uncertain significance, and 7 unclassified mutations, including the novel mutations in CCM1 c.811delT and CCM2 c.613_614insGG. In silico structural analysis revealed significant alterations in protein structure, supporting their pathogenicity. Protein-protein interaction analysis indicated nuanced impacts on cellular processes. Clinically, we observed a broad spectrum of symptoms, including seizures and focal neurological deficits. However, no statistically significant differences were found in lesion burden, age of first symptom onset, or sex between the identified CCM1/KRIT1 and CCM2/MGC4607 mutations among all patients studied. ConclusionThis study enhances the understanding of CCM by linking clinical variability, genetic mutations, and structural effects. The identification of these novel mutations opens new avenues for research and potential therapeutic strategies.

Mutation Spectrum of GAA Gene in Pompe Disease: Current Knowledge and Results of an Italian Study.

Studying a patient with Pompe disease (PD) is like opening Pandora's box. The specialist is faced with numerous clinical features similar to those of several diseases, and very often the symptoms are well hidden and none is associated with this rare disease. In recent years, scientific interest in this disease has been growing more and more, but still no symptom is recognized as key to a correct diagnosis of it, nor is there any specific disease marker to date. New diagnostic/therapeutic proposals on disease allow for the diffusion of knowledge of this pathology for timely diagnosis of the patient. Due to unawareness and difficulty in diagnosis, many adults with PD are diagnosed with great delay. In this article, we report and discuss current knowledge of PD and provide new data from work conducted on a cohort of 2934 Italian subjects recruited in recent years. A genetic analysis of the GAA gene was performed on patients with significant clinical signs and pathological enzyme activity to define the genetic profile of subjects. This identified 39 symptomatic PD subjects with low acid alpha-glucosidase enzyme activity and the presence of two causative mutations in GAA gene regions. Furthermore, 22 subjects with genetic variants of uncertain significance (GVUS) were identified.

Association of Genetic Alterations with Prognosis in Extramammary Paget's Disease: Insights into the Involvement of somatic CDKN2A variants in Poor Prognosis.

Previous studies reported the mutational landscape in extramammary Paget's disease (EMPD); however, the prognostic implications of genetic alterations remain unexplored. While CDKN2A loss is known to be associated with tumor progression or poor prognosis in some types of cancer, its significance in EMPD has not been investigated. To examine the association between common genetic alterations and prognosis in EMPD. This is a retrospective cohort study analyzing EMPD cases registered until January 2024 in the Center for Cancer Genomics and Advanced Therapeutics database, which is a nationwide database recording clinical data and comprehensive genomic profiling (CGP) test results in Japan. A total of 167 cases were recorded in the database, with CDKN2A loss being the most frequent genetic variant. Survival analysis was conducted on 127 cases. Survival from chemotherapy initiation was analyzed with adjusting for length bias inherent in the database using the Kaplan-Meier estimator, an established adjustment method. Cases with BRCA2-mutant tumors (n=18) had a worse prognosis than those with BRCA2-wild-type tumors (n=109; HR=2.97, 95% CI 1.46-6.01, p=0.003). Additionally, CDKN2A-mutant group (n=72) had a significantly worse prognosis than those with CDKN2A-wild-type group (n=55; HR=1.81, 95% CI 1.06-3.07, p=0.029). Most CDKN2A variants were pathogenic, primarily characterized by loss, while most BRCA2 variants were variants of uncertain significance. In the analysis of survival from CGP enrollment based on Eastern Cooperative Oncology Group performance status (ECOG-PS), cases with ECOG-PS 1 at the time of CGP enrollment had significantly poorer prognosis than those with ECOG-PS 0 (p=0.034; median survival time, 531 vs. 259 days). Somatic CDKN2A variant, mainly exhibiting loss, may be associated with poor prognosis in EMPD. Cases with BRCA2-mutant cancer might also have a worse prognosis in EMPD. In addition, CGP testing before PS deteriorates is preferable, considering the observed median survival of individuals undergoing CGP tests in an ECOG-PS-1 condition was less than 9 months.

Perinatal outcomes after a prenatal diagnosis of a fetal copy number variant: a retrospective population-based cohort study

BackgroundThere are no established guidelines for the follow up of infants born after a prenatal diagnosis of a genomic copy number variant (CNV), despite their increased risk of developmental issues. The aims of this study were (i) to determine the perinatal outcomes of fetuses diagnosed with and without a CNV, and (ii) to establish a population-based paediatric cohort for long term developmental follow up.MethodsAn Australian state-wide research database was screened for pregnant individuals who had a prenatal chromosomal microarray (CMA) between 2013–2019 inclusive. Following linkage to laboratory records and clinical referrer details, hospital records were manually reviewed for study eligibility. Eligible participants were mother–child pairs where the pregnancy resulted in a livebirth, the mother was able to provide informed consent in English (did not require a translator) and the mother was the primary caregiver for the child at hospital discharge after birth. Research invitations were sent by registered post at an average of six years after the prenatal diagnostic test. Statistical analysis was performed in Stata17.ResultsOf 1832 prenatal records examined, 1364 (74.5%) mother–child pairs were eligible for recruitment into the follow up cohort. Of the 468 ineligible, 282 (60.3%) had ‘no live pregnancy outcome’ (209 terminations of pregnancy (TOP) and 73 miscarriages, stillbirths, and infant deaths), 157 (33.5%) required a translator, and 29 (6.2%) were excluded for other reasons. TOP rates varied by the type of fetal CNV detected: 49.3% (109/221) for pathogenic CNVs, 18.2% (58/319) for variants of uncertain significance and 3.3% (42/1292) where no clinically significant CNV was reported on CMA. Almost 77% of invitation letters were successfully delivered (1047/1364), and the subsequent participation rate in the follow up cohort was 19.2% (201/1047).ConclusionsThis study provides Australia’s first population-based data on perinatal outcomes following prenatal diagnostic testing with CMA. The relatively high rates of pregnancy loss for those with a prenatal diagnosis of a CNV presented a challenge for establishing a paediatric cohort to examine long term outcomes. Recruiting a mother–child cohort via prenatal ascertainment is a complex and resource-intensive process, but an important step in understanding the impact of a CNV diagnosis in pregnancy and beyond.Trial registrationACTRN12620000446965p; Registered on April 6, 2020.

Relationship between Capillaroscopic Architectural Patterns and Different Variant Subgroups in Fabry Disease: Analysis of Cases from a Multidisciplinary Center.

Anderson-Fabry disease (AFD) is a genetic lysosomal storage disorder caused by mutations in the α-galactosidase A gene, leading to impaired lysosomal function and resulting in both macrovascular and microvascular alterations. AFD patients often exhibit increased intima-media thickness (IMT) and reduced flow-mediated dilation (FMD), indicating non-atherosclerotic arterial thickening and the potential for cardiovascular events. Nailfold capillaroscopy, a non-invasive diagnostic tool, has shown potential in diagnosing and monitoring microcirculatory disorders in AFD, despite limited research. This study evaluates nailfold capillaroscopy findings in AFD patients, exploring correlations with GLA gene variant subgroups (associated with classical or late-onset phenotypes and variants of uncertain significance (VUSs)), and assessing morpho-functional differences between sexes. It aims to determine whether capillaroscopy can assist in the early identification of individuals with multiorgan vascular involvement. A retrospective observational study was conducted with 25 AFD patients from AOUP "G. Rodolico-San Marco" in Catania (2020-2023). Patients underwent genetic testing, enzyme activity evaluation, and nailfold capillaroscopy using Horus basic HS 200 videodermatoscopy. Parameters like angiotectonic disorder, vascular areas, capillary density, and intimal thickening were assessed. The study identified significant differences in capillaroscopy findings among patients with different GLA gene variant subgroups. Classic AFD variant patients showed reduced capillary length and signs of erythrocyte aggregation and dilated subpapillary plexus. No correlation was found between enzymatic activity and capillaroscopy parameters. However, Lyso-Gb3 levels were positively correlated with average capillary length (ῤ = 0.453; p = 0.059). Sex-specific differences in capillaroscopy findings were observed in neoangiogenesis and average capillary length, with distinct implications for men and women. This study highlights the potential of nailfold capillaroscopy in the diagnostic process and clinical management of AFD, particularly in relation to specific GLA gene mutations, as a valuable tool for the early diagnosis and monitoring of AFD.

Calibration of variant effect predictors on genome-wide data masks heterogeneous performance across genes

In silico variant effect predictions are available for nearly all missense variants but played a minimal role in clinical variant classification because they were deemed to provide only supporting evidence. Recently, the ClinGen Sequence Variant Interpretation (SVI) Working Group updated recommendations for variant effect prediction use. By analyzing control pathogenic and benign variants across all genes, they were able to compute evidence strength for predictor score intervals with some intervals generating moderate, strong, or even very strong evidence. However, this genome-wide approach could obscure heterogeneous predictor performance in different genes. We quantified the gene-by-gene performance of two top predictors, REVEL and BayesDel, by analyzing control variants in each predictor score interval in 3,668 disease-relevant genes. Approximately 10% of intervals had sufficient control variants for analysis, and ∼70% of these intervals exceeded the maximum number of incorrect predictions implied by the SVI recommendations. These trending discordant intervals arose owing to the divergence of the gene-specific distribution of predictions from the genome-wide distribution, suggesting that gene-specific calibration is needed in many cases. Approximately 22% of ClinVar missense variants of uncertain significance in genes we analyzed (REVEL= 100,629, BayesDel= 71,928) had predictions in trending discordant intervals. Thus, genome-wide calibrations could result in many variants receiving inappropriate evidence strength. To facilitate a review of the SVI's calibrations, we developed a web application enabling visualization of gene-specific predictions and trending concordant and discordant intervals.

Delayed Wound Healing in a Young Patient with TNXB Variants and the Role of WES

After decades of quality improvement efforts, surgical site infections and abnormal wound healing remain intermittent complications in surgery. In both cases, the complication and abnormality may be superficial, restricted to the skin, or deeper within the tissue. The synthesis of connective tissue proteins, critical for structural support as part of the extracellular matrix, is under the control of multiple genes, forming a complex adaptive network. We report an unusual case of delayed and abnormal wound healing with chronic infection in a 16-year-old patient following surgery for scoliosis. Skin patch test showed that he is allergic to cobalt, a component of fixation hardware used in his surgery. He underwent evaluation by clinical genetics with whole exome sequencing (WES) which showed two (maternally and paternally inherited) variants in the TNXB ( Tenasin XB) gene. These two variants are variants of uncertain significance (VUS). TNXB encodes tenascin-XB which is an extracellular matrix glycoprotein located primarily in the basement membrane. Autosomal recessive pathogenic and likely pathogenic variants in TNXB cause TNXB-related classical-like Ehlers-Danlos Syndrome. At present, there are no causal linkages of these VUSs in TNXB to abnormal wound healing. However, as WES becomes more affordable, its use will increase and therefore it is important that we start to document such associations.