Bioinformatics of germline variant discovery for rare disease diagnostics: current approaches and remaining challenges.

Next-generation sequencing (NGS) has revolutionized the field of rare disease diagnostics. Whole exome and whole genome sequencing are now routinely used for diagnostic purposes; however, the overall diagnosis rate remains lower than expected. In this work, we review current approaches used for calling and interpretation of germline genetic variants in the human genome, and discuss the most important challenges that persist in the bioinformatic analysis of NGS data in medical genetics. We describe and attempt to quantitatively assess the remaining problems, such as the quality of the reference genome sequence, reproducible coverage biases, or variant calling accuracy in complex regions of the genome. We also discuss the prospects of switching to the complete human genome assembly or the human pan-genome and important caveats associated with such a switch. We touch on arguably the hardest problem of NGS data analysis for medical genomics, namely, the annotation of genetic variants and their subsequent interpretation. We highlight the most challenging aspects of annotation and prioritization of both coding and non-coding variants. Finally, we demonstrate the persistent prevalence of pathogenic variants in the coding genome, and outline research directions that may enhance the efficiency of NGS-based disease diagnostics.

Are trait-associated genes clustered together in a gene network?

Genome-wide association studies (GWAS) have provided an abundance of information about the genetic variants and their loci that are associated to complex traits and diseases. However, due to linkage disequilibrium (LD) and noncoding regions of loci, it remains a challenge to pinpoint the causal genes. Gene network-based approaches, paired with network diffusion methods, have been proposed to prioritize causal genes and to boost statistical power in GWAS based on the assumption that trait-associated genes are clustered in a gene network. Due to the difficulty in mapping trait-associated variants to genes in GWAS, this assumption has never been directly or rigorously tested empirically. On the other hand, whole exome sequencing (WES) data focuses on the protein-coding regions, directly identifying trait-associated genes. In this study, we tested the assumption by leveraging the recently available exome-based association statistics from the UK Biobank WES data along with two types of networks. We found that almost all trait-associated genes were significantly more proximal to each other than randomly selected genes within both networks. These results support the assumption that trait-associated genes are clustered in gene networks, which can be further leveraged to boost the power of GWAS such as by introducing less stringent p value thresholds.

Whole-exome sequencing identifies rare genetic variants associated with human plasma metabolites.

Metabolite levels measured in the human population are endophenotypes for biological processes. We combined sequencing data for 3,924 (whole-exome sequencing, WES, discovery) and 2,805 (whole-genome sequencing, WGS, replication) donors from a prospective cohort of blood donors in England. We used multiple approaches to select and aggregate rare genetic variants (minor allele frequency [MAF] < 0.1%) in protein-coding regions and tested their associations with 995 metabolites measured in plasma by using ultra-high-performance liquid chromatography-tandem mass spectrometry. We identified 40 novel associations implicating rare coding variants (27 genes and 38 metabolites), of which 28 (15 genes and 28 metabolites) were replicated. We developed algorithms to prioritize putative driver variants at each locus and used mediation and Mendelian randomization analyses to test directionality at associations of metabolite and protein levels at the ACY1 locus. Overall, 66% of reported associations implicate gene targets of approved drugs or bioactive drug-like compounds, contributing to drug targets' validating efforts.

Association of novel DNAH11 variants with asthenoteratozoospermia lead to male infertility.

BACKGROUND: Bi-allelic variants in DNAH11 have been identified as causative factors in Primary Ciliary Dyskinesia, leading to abnormal respiratory cilia. Nonetheless, the specific impact of these variants on human sperm flagellar and their involvement in male infertility remain largely unknown. METHODS: A collaborative effort involving two Chinese reproductive centers conducted a study with 975 unrelated infertile men. Whole-exome sequencing was employed for variant screening, and Sanger sequencing confirmed the identified variants. Morphological and ultrastructural analyses of sperm were conducted using Scanning Electron Microscopy and Transmission Electron Microscopy. Western Blot Analysis and Immunofluorescence Analysis were utilized to assess protein levels and localization. ICSI was performed to evaluate its efficacy in achieving favorable pregnancy outcomes for individuals with DNAH11 variants. RESULTS: In this study, we identified seven novel variants in the DNAH11 gene in four asthenoteratozoospermia subjects. These variants led the absence of DNAH11 proteins and ultrastructure defects in sperm flagella, particularly affecting the outer dynein arms (ODAs) and adjacent structures. The levels of ODA protein DNAI2 and axoneme related proteins were down regulated, instead of inner dynein arms (IDA) proteins DNAH1 and DNAH6. Two out of four individuals with DNAH11 variants achieved clinical pregnancies through ICSI. The findings confirm the association between male infertility and bi-allelic deleterious variants in DNAH11, resulting in the aberrant assembly of sperm flagella and contributing to asthenoteratozoospermia. Importantly, ICSI emerges as an effective intervention for overcoming reproductive challenges caused by DNAH11 gene variants.

Assessing the contribution of genes involved in monogenic bone disorders to the etiology of atypical femoral fractures.

BACKGROUND: Recent studies suggested that genetic variants associated with monogenic bone disorders were involved in the pathogenesis of atypical femoral fractures (AFF). Here, we aim to identify rare genetic variants by whole exome sequencing in genes involved in monogenic rare skeletal diseases in 12 women with AFF and 4 controls without any fracture. RESULTS: Out of 33 genetic variants identified in women with AFF, eleven (33.3%) were found in genes belonging to the Wnt pathway (LRP5, LRP6, DAAM2, WNT1, and WNT3A). One of them was rated as pathogenic (p.Pro582His in DAAM2), while all others were rated as variants of uncertain significance according to ClinVar and ACMG criteria. CONCLUSIONS: Osteoporosis, rare bone diseases, and AFFs may share the same genes, thus making it even more difficult to identify unique risk factors.

Whole genome sequencing increases the diagnostic rate in Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is one of the most common and genetically heterogeneous inherited neurological diseases, with more than 130 disease-causing genes. Whole genome sequencing (WGS) has improved diagnosis across genetic diseases, but the diagnostic impact in CMT is yet to be fully reported. We present the diagnostic results from a single specialist inherited neuropathy centre, including the impact of WGS diagnostic testing. Patients were assessed at our specialist inherited neuropathy centre from 2009 to 2023. Genetic testing was performed using single gene testing, next-generation sequencing targeted panels, research whole exome sequencing and WGS and, latterly, WGS through the UK National Health Service. Variants were assessed using the American College of Medical Genetics and Genomics and Association for Clinical Genomic Science criteria. Excluding patients with hereditary ATTR amyloidosis, 1515 patients with a clinical diagnosis of CMT and related disorders were recruited. In summary, 621 patients had CMT1 (41.0%), 294 CMT2 (19.4%), 205 intermediate CMT (CMTi, 13.5%), 139 hereditary motor neuropathy (HMN, 9.2%), 93 hereditary sensory neuropathy (HSN, 6.1%), 38 sensory ataxic neuropathy (2.5%), 72 hereditary neuropathy with liability to pressure palsies (HNPP, 4.8%) and 53 'complex' neuropathy (3.5%). Overall, a genetic diagnosis was reached in 76.9% (1165/1515). A diagnosis was most likely in CMT1 (96.8%, 601/621), followed by CMTi (81.0%, 166/205) and then HSN (69.9%, 65/93). Diagnostic rates remained less than 50% in CMT2, HMN and complex neuropathies. The most common genetic diagnosis was PMP22 duplication (CMT1A; 505/1165, 43.3%), then GJB1 (CMTX1; 151/1165, 13.0%), PMP22 deletion (HNPP; 72/1165, 6.2%) and MFN2 (CMT2A; 46/1165, 3.9%). We recruited 233 cases to the UK 100 000 Genomes Project (100KGP), of which 74 (31.8%) achieved a diagnosis; 28 had been otherwise diagnosed since recruitment, leaving a true diagnostic rate of WGS through the 100KGP of 19.7% (46/233). However, almost half of the solved cases (35/74) received a negative report from the study, and the diagnosis was made through our research access to the WGS data. The overall diagnostic uplift of WGS for the entire cohort was 3.5%. Our diagnostic rate is the highest reported from a single centre and has benefitted from the use of WGS, particularly access to the raw data. However, almost one-quarter of all cases remain unsolved, and a new reference genome and novel technologies will be important to narrow the 'diagnostic gap'.

Comprehensive Analysis of Juvenile Nasopharyngeal Angiofibromas via Whole-Exome Sequencing.

INTRODUCTION: The molecular basis and mechanisms of juvenile nasopharyngeal angiofibromas (JNA) pathogenesis are still unknown. Despite being a rare and benign neoplasm, JNA is a locally aggressive and potentially destructive head and neck neoplasm, typically found in young males. The advancement of genome technologies and analytical tools has provided an unparalleled opportunity to explore the intricacy of JNA. The present study provides the first evidence of the involvement of Y-chromosome genes in JNA. METHODS: A total of 13 JNA patients at an advanced disease stage and five age-matched male controls were registered for this study. Whole-exome sequencing (WES) analysis was conducted followed by functional analysis to understand the molecular mechanism of the JNA. RESULTS: WES analysis revealed a high prevalence of mutations in 14 genes within the protein-coding, male-specific region of the Y-chromosome of young males (mean age: 13.8 ± 2.4) with JNA. These mutations, occurring at 28 distinct positions, were characterized as moderate to high impact and were prevalent in nine JNA patients but not in the control group. The most frequently mutated genes were USP9Y and UTY, followed by KDM5D, DDX3Y, and TSPY4. The expression of USP9Y, UTY, and DDX3Y was found to be co-modulated, implying their coordinated regulation as a complex. Furthermore, somatic mutations were detected in genes previously linked to JNA. CONCLUSION: The wide array of genetic mutations in the Y-chromosome male-specific region, along with the somatic alterations identified in JNA, provides novel insights into JNA pathophysiology.

Novel, pathogenic insertion variant of GSDME associates with autosomal dominant hearing loss in a large Chinese pedigree.

Nonsyndromic hearing loss (NSHL) is a genetically diverse, highly heterogeneous condition characterised by deafness, and Gasdermin E (GSDME) variants have been identified as directly inducing autosomal dominant NSHL. While many NSHL cases associated with GSDME involve the skipping of exon 8, there is another, less understood pathogenic insertion variant specifically found in Chinese pedigrees that causes deafness, known as autosomal dominant 5 (DFNA5) hearing loss. In this study, we recruited a large Chinese pedigree, conducted whole-exome and Sanger sequencing to serve as a comprehensive clinical examination, and extracted genomic DNA samples for co-segregation analysis of the members. Conservation and expression analyses for GSDME were also conducted. Our clinical examinations revealed an autosomal dominant phenotype of hearing loss in the family. Genetic analysis identified a novel insertion variant in GSDME exon 8 (GSDME: NM_004403.3: c.1113_1114insGGGGTGCAGCTTACAGGGTGGGTGT: p. P372fs*36). This variant is segregated with the deafness phenotype of this pedigree. The GSDME gene was highly conserved in the different species we analysed, and its mRNA expression was ubiquitously low in different human tissues. In conclusion, we have successfully identified a novel pathogenic insertion variant of GSDME in a Chinese pedigree that causes deafness, shedding light on the genetic basis of hearing loss within this specific family. Our findings expand the spectrum of known variants associated with GSDME-related deafness and may further support both the underlying gain-of-function mechanism and functional associations of GSDME hearing loss variants.

Novel mutation in EFEMP1 identified from two Chinese POAG families differentially activated endoplasmic reticulum stress markers and induced glaucoma in mouse.

Primary open-angle glaucoma (POAG) is the most common type of glaucoma. Using whole-exome sequencing, we identified two independent families diagnosed as POAG from the China with a novel EFEMP1 variant (Exon3, c.175A>C p.Met59Leu); Three previously reported variants c.1160G>A p.R387Q, c.1189T>C p.Y397H, and c.1429C>T p.R477C in EFEPM1 from 55 sporadic POAG individuals were also identified. The variant c.175A>C p.Met59Leu co-segregated with the disease phenotype within the families. Immunoprecipitation and western blot assays showed that all three EFEMP1 mutants (p.Met59Leu, pArg140Trp, pArg345Trp) increased intracellular protein aggregations, and pMet59Leu and pArg140Arg also enhanced their extracellular proteins secretion, compared to WT in HEK293T. The differential regulations to endoplasmic reticulum (ER) stress markers ATF4, GPR78/94, and CHOP, and differential phosphorylation activations to CREB at Ser133, AKT at Ser473, p44/42 at Thr202/Tyr204, and STAT3 at Tyr705, were also detected among the mutants and WT. Finally, we revealed a significant increment of intraocular pressure and obvious reduction of RGC cells at the sixth week following intravitreal injection of adenovirus 5 (Ad5) expressing in pMet59Leu compared to WT and GFP controls. Together, variant c.175A>C p.Met59Leu in EFEMP1 is pathogenic and different mutants in EFEMP1 triggered distinct signaling pathways, explaining the reason of mutation-dependent disease phenotypes of EFEMP1.

Identification of a Novel Homozygous GLS Gene Variant Associated with Developmental and Epileptic Encephalopathy (DEE) Type 71.

Developmental and epileptic encephalopathy (DEEs) (OMIM#618,328) is characterized by seizures, hypotonia, and brain abnormalities, often arising from mutations in genes crucial for brain function. Among these genes, GLS stands out due to its vital role in the central nervous system (CNS), with homozygous variants potentially causing DEE type 71. Using Whole Exome Sequencing (WES) on a patient exhibiting symptoms of epileptic encephalopathy, we identified a novel homozygous variant, NM_014905.5:c.1849G > T; p.(Asp617Tyr), in the GLS gene. The 5-year-old patient, born to consanguineous parents, presented with developmental delay, encephalopathy, frequent seizures, and hypotonia. Sanger sequencing further validated the GLS gene variant in both the patient and his family. Furthermore, our bioinformatics analysis indicated that this missense variant could lead to alteration of splicing, resulting in the activation of a cryptic donor site and potentially causing loss of protein function. Our finding highlights the pathogenic significance of the GLS gene, particularly in the context of brain disorders, specifically DEE71.

Clinical genomics expands the link between erroneous cell division, primary microcephaly and intellectual disability.

Primary microcephaly is a rare neurogenic and genetically heterogeneous disorder characterized by significant brain size reduction that results in numerous neurodevelopmental disorders (NDD) problems, including mild to severe intellectual disability (ID), global developmental delay (GDD), seizures and other congenital malformations. This disorder can arise from a mutation in genes involved in various biological pathways, including those within the brain. We characterized a recessive neurological disorder observed in nine young adults from five independent consanguineous Pakistani families. The disorder is characterized by microcephaly, ID, developmental delay (DD), early-onset epilepsy, recurrent infection, hearing loss, growth retardation, skeletal and limb defects. Through exome sequencing, we identified novel homozygous variants in five genes that were previously associated with brain diseases, namely CENPJ (NM_018451.5: c.1856A > G; p.Lys619Arg), STIL (NM_001048166.1: c.1235C > A; p.(Pro412Gln), CDK5RAP2 (NM_018249.6 c.3935 T > G; p.Leu1312Trp), RBBP8 (NM_203291.2 c.1843C > T; p.Gln615*) and CEP135 (NM_025009.5 c.1469A > G; p.Glu490Gly). These variants were validated by Sanger sequencing across all family members, and in silico structural analysis. Protein 3D homology modeling of wild-type and mutated proteins revealed substantial changes in the structure, suggesting a potential impact on function. Importantly, all identified genes play crucial roles in maintaining genomic integrity during cell division, with CENPJ, STIL, CDK5RAP2, and CEP135 being involved in centrosomal function. Collectively, our findings underscore the link between erroneous cell division, particularly centrosomal function, primary microcephaly and ID.

Two novel compound heterozygous loss-of-function mutations cause fetal IRAK-4 deficiency presenting with Pseudomonas Aeruginosa sepsis.

PURPOSE: To report a case of a five-month-old Chinese infant who died of interleukin-1 receptor-associated kinase-4 (IRAK-4) deficiency presenting with rapid and progressive Pseudomonas aeruginosa sepsis. METHODS: The genetic etiology of IRAK-4 deficiency was confirmed through trio-whole exome sequencing and Sanger sequencing. Functional consequences were invested using an in vitro minigene splicing assay. RESULTS: Trio-whole exome sequencing of genomic DNA identified two novel compound heterozygous mutations, IRAK-4 (NM_016123.3): c.942-1G > A and c.644_651+ 6delTTGCAGCAGTAAGT in the proband, which originated from his symptom-free parents. These mutations were predicted to cause frameshifts and generate three truncated proteins without enzyme activity. CONCLUSIONS: Our findings expand the range of IRAK-4 mutations and provide functional support for the pathogenic effects of splice-site mutations. Additionally, this case highlights the importance of considering the underlying genetic defects of immunity when dealing with unusually overwhelming infections in previously healthy children and emphasizes the necessity for timely treatment with wide-spectrum antimicrobials.

Germline genetic variants in Turkish familial multiple myeloma/monoclonal gammopathy of undetermined significance cases.

Multiple myeloma (MM) is a haematological malignancy primarily affecting the elderly, with a striking male predilection and ethnic disparities in incidence. Familial predisposition to MM has long been recognized, but the genetic underpinnings remain elusive. This study aimed to investigate germline variants in Turkish families with recurrent MM cases. A total of 37 MM-affected families, comprising 77 individuals, were included. Targeted next-generation sequencing analysis yielded no previously reported rare variants. Whole exome sequencing analysis in 11 families identified rare disease-causing variants in various genes, some previously linked to familial MM and others not previously associated. Notably, genes involved in ubiquitination, V(D)J recombination and the PI3K/AKT/mTOR pathway were among those identified. Furthermore, a specific variant in BNIP1 (rs28199) was found in 13 patients across nine families, indicating its potential significance in MM pathogenesis. While this study sheds light on genetic variations in familial MM in Turkey, its limitations include sample size and the absence of in vivo investigations. In conclusion, familial MM likely involves a polygenic inheritance pattern with rare, disease-causing variants in various genes, emphasizing the need for international collaborative efforts to unravel the intricate genetic basis of MM and develop targeted therapies.

Genomic and transcriptomic profiling of inflammatory breast cancer reveals distinct molecular characteristics to non-inflammatory breast cancers.

PURPOSE: Inflammatory breast cancer (IBC), a rare and highly aggressive form of breast cancer, accounts for 10% of breast cancer-related deaths. Previous omics studies of IBC have focused solely on one of genomics or transcriptomics and did not discover common differences that could distinguish IBC from non-IBC. METHODS: Seventeen IBC patients and five non-IBC patients as well as additional thirty-three Asian breast cancer samples from TCGA-BRCA were included for the study. We performed whole-exon sequencing (WES) to investigate different somatic genomic alterations, copy number variants, and large structural variants between IBC and non-IBC. Bulk RNA sequencing (RNA-seq) was performed to examine the differentially expressed genes, pathway enrichment, and gene fusions. WES and RNA-seq data were further investigated in combination to discover genes that were dysregulated in both genomics and transcriptomics. RESULTS: Copy number variation analysis identified 10 cytobands that showed higher frequency in IBC. Structural variation analysis showed more frequent deletions in IBC. Pathway enrichment and immune infiltration analysis indicated increased immune activation in IBC samples. Gene fusions including CTSC-RAB38 were found to be more common in IBC. We demonstrated more commonly dysregulated RAS pathway in IBC according to both WES and RNA-seq. Inhibitors targeting RAS signaling and its downstream pathways were predicted to possess promising effects in IBC treatment. CONCLUSION: We discovered differences unique in Asian women that could potentially explain IBC etiology and presented RAS signaling pathway as a potential therapeutic target in IBC treatment.

Identification of four novel mutations in VSP13A in Iranian patients with Chorea-acanthocytosis (ChAc).

Chorea-acanthocytosis (ChAc) is a rare autosomal recessive neurodegenerative disorder characterized by a variety of involuntary movements, predominantly chorea, and the presence of acanthocytosis in peripheral blood smears. ChAc is caused by mutations in the vacuolar protein sorting-associated protein 13A (VPS13A) gene. The aim of the present study was to conduct a clinical and genetic analysis of five patients with suspected ChAc in Iran. This study included five patients who were referred to the genetic department of the Endocrinology and Metabolism Research Institute between 2020 and 2022, with a suspicion of ChAc. Clinical features and the presence of characteristic MRI findings were evaluated in the patients. Whole-exome sequencing (WES) followed by Sanger sequencing was employed to identify the disease-causing variants. The functional effects of novel mutations were analyzed by specific bioinformatics prediction tools. WES and data analysis revealed the presence of five distinct VPS13A mutations in the patients, four of which were novel. These included one nonsense mutation (p.L984X), and three splice site mutations (c.755-1G>A, c.144+1 G>C, c.2512+1G>A). All mutations were validated by Sanger sequencing, and in silico analysis predicted that all mutations were pathogenic. This study provides the first molecular genetic characteristics of Iranian patients with ChAc, identifying four novel mutations in the VPS13A gene. These findings expand the VPS13A variants spectrum and confirm the clinical variability in ChAc patients.

Functional characterisation of human recessive DIS3 variants in premature ovarian insufficiency.

Premature ovarian insufficiency (POI) is characterised by the loss or complete absence of ovarian activity in women under the age of 40. Clinical presentation of POI varies with phenotypic severity ranging from premature loss of menses to complete gonadal dysgenesis. POI is genetically heterogeneous with >100 causative gene variants identified thus far. The aetiology of POI varies from syndromic, idiopathic, monogenic to autoimmune causes the condition. Genetic diagnoses are beneficial to those impacted by POI as it allows for improved clinical management and fertility preservation. Identifying novel variants in candidate POI genes, however, is insufficient to make clinical diagnoses. The impact of missense variants can be predicted using bioinformatic algorithms but computational approaches have limitations and can generate false positive and false negative predictions. Functional characterisation of missense variants, is therefore imperative, particularly for genes lacking a well-established genotype:phenotype correlation. Here we used whole-exome sequencing (WES) to identify the first case of a homozygous missense variant in DIS3 (c.2320C > T; p.His774Tyr) a critical component of the RNA exosome in a POI patient. This adds to the previously described compound heterozygous patient. We perform the first functional characterisation of a human POI-associated DIS3 variant. A slight defect in mitotic growth was caused by the variant in a Saccharomyces cerevisiae model. Transgenic rescue of Dis3 knockdown in Drosophila melanogaster with human DIS3 carrying the patient variant led to aberrant ovarian development and egg chamber degeneration. This supports a potential deleterious impact of the human c.2320C > T; p.His774Tyr variant.

Noninvasive electrical stimulation as a neuroprotective strategy in retinal diseases: a systematic review of preclinical studies.

BACKGROUND: Electrical activity has a crucial impact on the development and survival of neurons. Numerous recent studies have shown that noninvasive electrical stimulation (NES) has neuroprotective action in various retinal disorders. OBJECTIVE: To systematically review the literature on in vivo studies and provide a comprehensive summary of the neuroprotective action and the mechanisms of NES on retinal disorders. METHODS: Based on the PRISMA guideline, a systematic review was conducted in PubMed, Web of Science, Embase, Scopus and Cochrane Library to collect all relevant in vivo studies on "the role of NES on retinal diseases" published up until September 2023. Possible biases were identified with the adopted SYRCLE's tool. RESULTS: Of the 791 initially gathered studies, 21 articles met inclusion/exclusion criteria for full-text review. The results revealed the neuroprotective effect of NES (involved whole-eye, transcorneal, transscleral, transpalpebral, transorbital electrical stimulation) on different retinal diseases, including retinitis pigmentosa, retinal degeneration, high-intraocular pressure injury, traumatic optic neuropathy, nonarteritic ischemic optic neuropathy. NES could effectively delay degeneration and apoptosis of retinal neurons, preserve retinal structure and visual function with high security, and its mechanism of action might be related to promoting the secretion of neurotrophins and growth factors, decreasing inflammation, inhibiting apoptosis. The quality scores of included studies ranged from 5 to 8 points (a total of 10 points), according to SYRCLE's risk of bias tool. CONCLUSION: This systematic review indicated that NES exerts neuroprotective effects on retinal disease models mainly through its neurotrophic, anti-inflammatory, and anti-apoptotic capabilities. To assess the efficacy of NES in a therapeutic setting, however, well-designed clinical trials are required in the future.

The recommendation of re-classification of variants of uncertain significance (VUS) in adult genetic disorders patients.

Since variants of uncertain significance (VUS) reported in genetic testing cannot be acted upon clinically, this classification may delay or prohibit precise diagnosis and genetic counseling in adult genetic disorders patients. Large-scale analyses about qualitatively distinct lines of evidence used for VUS can make them re-classification more accurately. We analyzed 458 Chinese adult patients WES data, within 15 pathogenic evidence PS1, PS2, PM1, PM6 and PP4 were not used for VUS pathogenic classification, meanwhile the PP3, BP4, PP2 were used much more frequently. The PM2_Supporting was used most widely for all reported variants. There were also 31 null variants (nonsense, frameshift, canonical ±1 or 2 splice sites) which were probably the disease-causing variants of the patients were classified as VUS. By analyzed the evidence used for all VUS we recommend that appropriate genetic counseling, reliable releasing of in-house data, allele frequency comparison between case and control, expanded verification in patient family, co-segregation analysis and functional assays were urgent need to gather more evidence to reclassify VUS. We also found adult patients with nervous system disease were reported the most phenotype-associated VUS and the lower the phenotypic specificity, the more reported VUS. This result emphasized the importance of pretest genetic counseling which would make less reporting of VUS. Our result revealed the characteristics of the pathogenic classification evidence used for VUS in adult genetic disorders patients for the first time, recommend a rules-based process to evaluate the pathogenicity of VUS which could provide a strong basis for accurately evaluating the pathogenicity and clinical grade information of VUS. Meanwhile, we further expanded the genetic spectrum and improve the diagnostic rate of adult genetic disorders.

Hidden Aberrant Transcripts in TTC37 Cause Trichohepatoenteric Syndrome.

The patient had clinical suspicion of THES. Complex genetic analyzes using WES, WGS were performed without success in the diagnosis. Further molecular analyzes using RNA and protein was necessary to reach the final correct THES diagnosis.

A novel homozygous FAM92A gene (CIBAR1) variant further confirms its association with non-syndromic postaxial polydactyly type A9 (PAPA9).

Polydactyly is a very common digit anomaly, having extra digits in hands and/or toes. Non-syndromic polydactyly in both autosomal dominant and autosomal recessive forms are caused by disease-causing variants in several genes, including GLI1, GLI3, ZNF141, FAM92A, IQCE, KIAA0825, MIPOL1, STKLD1, PITX1, and DACH1. Whole exome sequencing (WES) followed by bi-directional Sanger sequencing was performed for the single affected individual (II-1) of the family to reveal the disease causative variant/gene. 3D protein modeling and structural molecular docking was performed to determine the effect of the identified mutation on the overall protein structure. WES revealed a novel biallelic missense variant (c.472G>C; p.Ala158Pro) in exon 6 of the FAM92A gene. The identified variant segregated perfectly with the disease phenotype using Sanger sequencing. Furthermore, Insilco analysis revealed that the variant significantly changes the protein secondary structure, and substantially impact the stability of FAM92A. We report the second FAM92A disease-causing mutation associated with recessive non-syndromic postaxial polydactyly. The data further confirms the contribution of FAM92A in limb development and patterning.