Human Mutation

Pompe disease is an autosomal recessive disorder caused by GAA variants leading to acid alpha-glucosidase deficiency. Diagnosis is challenging due to the variable phenotypic presentation and overlap with other conditions. Traditionally, diagnosis relies on measuring enzyme activity, but next-generation sequencing (NGS) advancements have improved accuracy. However, interpreting variants is complex, especially because pseudodeficiency alleles mimic disease-causing variants. We present two patients harboring the pseudodeficiency allele NM_000152.5(GAA):c.271G>A, p.Asp91Asn, which is confusing due to inaccurate reports and results related to enzymatic activity. The first case was a recently published controversial case of a 700-year-old mummy in which the authors classified the variant as pathogenic. The second patient had symptoms compatible with late-onset Pompe disease and was homozygous for the variant. We aimed to determine the correct variant classification using GAA:c.271G>A as a model and to achieve a genetic diagnosis of the second patient. This variant was analyzed following international guidelines (ACMG-AMP) and reviewed with the Lysosomal Diseases Variant Curation Expert Panel. The second patient underwent NGS. We demonstrated that GAA:c.271G>A meets the criterion of being classified as benign for Pompe. Additionally, the second patient carried a heterozygous pathogenic PABPN1 variant associated with oculopharyngeal muscular dystrophy, which better explained the clinical features. This underscores the importance of expanding the genetic analysis in the presence of pseudodeficiency alleles that can mask the true cause of the disease and highlights the fact that an accurate diagnosis should adhere to guidelines on variant curation to reduce the risk of misdiagnosis, which could result in inadequate care and risky medical decisions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/24314698v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@12a720borg.highwire.dtl.DTLVardef@1a53d15org.highwire.dtl.DTLVardef@14d4729org.highwire.dtl.DTLVardef@7b0186_HPS_FORMAT_FIGEXP M_FIG C_FIG

Heterozygous loss-of-function variants in GCK cause persistent, mildly elevated plasma glucose beginning at birth. Recently, clinical phenotype created by deleterious variants in GCK is called GCK-MODY, although currently registered as "MODY, type 2" (maturity-onset diabetes of the young type 2, MODY2, MIM # 125851) in OMIM. The hyperglycemia of GCK-MODY is a benign and non-progressive condition, usually suspected upon detection of hyperglycemia in adulthood. Medication is not essential, nevertheless, pregnant females may require special attention. Here, we report recurrent and novel GCK variants detected in 43 pediatric patients who were investigated for hyperglycemia by the referring pediatric endocrinologist. Electronic medical records (EMR) of the patients were collected and reviewed retrospectively. All patients applied to Ankara City Hospital between April 2019 and June 2022. GCK variants were screened on NovaSeq 6000 next-generation sequencing platform (Illumina). Variants detected in GCK were checked with recent literature for a proper pathogenicity classification. In 43 patients, 28 distinct GCK variants were identified. Of these variants, 25 were classified as likely pathogenic/pathogenic variants (c.1342G>A, c.112C>T, c.1178T>C, c.130G>A, c.565A>G, c.208+3A>T, c.349G>C, c.863+5G>A, c.214G>A, c.1292C>A, c.830_831delTG, c.106C>T, c.572G>A, c.107G>C, c.454T>C, c.793G>A, c.645C>A, c.377T>A, c.667G>A, c.46-1G>A, c.149A>C, c.1079C>G, c.401T>C, c.758T>G, c.467_483+6del; NM_000162.5), while remaining 3 variants (c.950A>C, c.186G>T, c.188G>A; NM_000162.5) were classified as variant of uncertain significance. According to the variant effect, missense variants accounted for the majority (71%; 20/28), followed by splice junction (14%; 4/28), premature termination (7%; 2/28), frameshift (4%; 1/28), and synonymous (4%; 1/28) alterations. There were 3 novel variants: c.830_831del, c.377T>A, c.467_483+6del.

BackgroundCystic fibrosis (CF) is an autosomal recessive disease resulting from pathogenic CF transmembrane conductance regulator (CFTR) pathogenic gene variants. While CFs frequency varies among ethnicities, its epidemiology, clinical manifestations, and mutational profiles in Africa still must be explored due to the absence of a comprehensive public health strategy there. This study postulates that complete sequencing of CFTR using Oxford Nanopore Technology (ONT)-based long-read sequencing enhances the diagnostic yield. MethodsTo amplify [~]25-kb fragments covering the whole CFTR gene (NM_000492.4), we designed 11 primer pairs, and barcoded libraries were prepared and sequenced on ONT flow cells (R10.4.1) using an Mk1C device. Variant pathogenicity was assessed by expressing the variant channel in HEK293 cells and examining expression through immuno-blotting. ResultsWith sequencing data obtained from 9 Moroccan individuals (6 probands with suspected CF diagnoses and 3 parents), we identified the following variants: c.680T>G p.Leu227Arg, c.1521_1523del p.Phe508del, c.3484C>T p.Arg1162*, c.1090T>C p.Ser364Pro, c.3233T>C p.Phe1078Ser and c.2991G>C p.Leu997Phe. The analytical pipeline we developed allowed the phasing of the variants. Sanger sequencing confirmed all these results. The previously uncharacterised CFTR variants p.Ser364Pro and p.Phe1078Ser exhibit diminished expression in HEK293 cells, substantiating their pathogenic nature, with p.Phe1078Ser responding positively to the in vitro treatment with CFTR-modulator molecules. ConclusionsThis study demonstrates the potential of long-read sequencing using ONT as an efficient means to detect CF-causing variants in African populations. Given the significant genetic heterogeneity in Africa, this technique can serve as an affordable molecular screening tool for CF, especially in areas with constrained access to genetic screening. HighlightsO_LIHere, we demonstrate the efficiency of sequencing the complete CFTR gene using Oxford Nanopore Technology, an information-rich and accurate method. C_LIO_LINot-yet-reported CFTR variants of unknown significance were identified from a small cohort of nine cystic fibrosis patients and parents from Fez, Morocco. C_LIO_LIThe biochemical characterisation of the CFTR variants p.Ser364Pro, p.Phe1078Ser, and p.Leu997Phe highlights the importance of conducting expression studies when genetic variants are identified. C_LIO_LIOur proposed approach will enhance diagnostic outcomes, particularly in populations with significant genetic diversity, such as those in Africa. C_LI

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations to the CF transmembrane conductance regulator (CFTR). Symptoms and severity of the disease vary shown that modifier genes influence disease severity and clinical course. We previously reported epithelial sodium channel (ENaC) genes as modifiers of disease severity in long-term non-progressors sharing deltaF508 homozygous for CFTR genotype. Here we describe the opposite, modifier genes that may be associated with rapidly progressing CF (RPCF) in a cohort of patients with the shared deltaF508 homozygous genotype. We have identified three rare missense SLC26A9 variants in four individuals (out of six) deemed to have RPCF: c.229G>A; p.G77S (present in two patients), c.1885C>T; p.P629S and c.2546G>A; p.R849Q. By analyzing publicly available single cell sequencing dataset from LungMAP, we revealed that both SLC26A9 and CFTR mRNA are highly enriched in Alveolar type 2 (AT2) cells, with a few (greater than 1%) in respiratory airway secretory (RAS) cells and ionocytes. Structural modeling suggests deleterious effects of these mutations as they are in critical protein domains which might affect the ion transportation capability of SLC26A9. The enrichment of rare and potentially deleterious SLC26A9 mutations in patients with RPCF suggests SLC26A9 is a modifier gene associated with RPCF.

PurposeThe pathogenicity of intragenic duplications depends on their structural configuration. Tandem duplications often disrupt reading frames and cause gene loss-of-function, whereas interspersed (non-tandem) duplications are largely benign. When the configuration cannot be determined, current guidelines presume a tandem structure, leading to some laboratories automatically classifying such variants as likely pathogenic or pathogenic. This study evaluates the validity of this presumption for DMD, in patients with and without clinical indications of dystrophinopathy. MethodsWe performed high-coverage whole-genome long-read sequencing on 15 patients with intragenic DMD duplications. Four patients had clinically indicated dystrophinopathy testing, while in the remaining 11 patients, the duplications were detected without clear indications of dystrophinopathy (e.g., "incidentally detected" through carrier screening). ResultsAll four patients with clinical indications had tandem duplications. In contrast, 64% (7/11) of the incidentally detected cases had interspersed duplications, with four subsequently re-classified as likely benign, two likely pathogenic, and one uncertain. These duplications were often complex, involving co-duplications or co-deletions with other regions. ConclusionOur findings challenge the presumption that intragenic DMD duplications are predominantly tandem. This highlights the need for a cautious variant interpretation approach, particularly in carrier screening and other settings where variants are identified without indications of dystrophinopathy.

Introduction/AimsLimb-girdle muscular dystrophy R1 (LGMDR1) calpain 3-related is one of the most common forms of LGMD. It is typically recessively inherited and associated with progressive weakness of proximal limb-girdle muscles. Recently, several families with an autosomal dominant inheritance transmission pattern have been reported (LGMDD4). Camptocormia is a common clinical feature in these patients. In these families, different mutations in CAPN3 have been identified, including in-frame deletions and missense mutations. In particular, two patients presenting with camptocormia carried the p.Lys254del missense mutation without a second identified mutation in CAPN3. MethodsAmong our patients, we identified two LGMDR1 families as carriers of the p.Lys254del mutation by DNA sequencing, both in homozygous and compound heterozygous states and clinically analyzed the family members carrying this mutation. ResultsInterestingly, these patients did not present the myopathic characteristics described in the so-called dominant patients. No camptocormia or any other severe clinical symptoms were observed. DiscussionAccordingly, we conclude that the p.Lys254del mutation per se cannot be solely responsible for the camptocormia observed in dominant patients. Other additional undisclosed factors might regulate the phenotype associated to a dominant inheritance pattern in CAPN3 mutation carriers.

BackgroundMissense variants in TTN pose a major challenge in genetic diagnostics due to their high frequency in the general population, the large size of the gene, and the complex multidomain architecture of the titin protein. While the contribution of truncating variants (TTNtv) to titinopathies is well established, the role of rare TTN missense variants remains poorly defined. Advances in computational prediction and functional testing offer new tools to assess their potential pathogenicity, which however are currently not fully utilized for clinical application. MethodsWe analyzed an international cohort of unsolved myopathy cases selected based on the presence of a rare missense variant in trans with a TTNtv. Clinical data were collected from neuromuscular centers worldwide. In silico predictions were generated using AlphaMissense and complemented by MAF and exon usage information. Additional inclusion criteria were based on a Minor Allele Frequency < 0.010 and an AlphaMissense score [&ge;] 0.792 for the missense variant, in accordance with the latest ClinGen guidelines. Selected missense variants were characterized in vitro through protein expression and cell imaging assays to assess their effects on domain solubility and aggregation. ResultsThirty patients with TTNtv/missense combinations were identified, presenting with heterogeneous myopathic phenotypes, ranging from congenital to adult onset. An in-depth analysis on AlphaMissense predictions highlighted those changes most frequently predicted as possibly pathogenic. Functional assays showed that three selected variants with changes to proline, located in {beta}-sheets of Ig domains, led to impaired folding, cytoplasmic aggregation and co-localisation with proteostasis markers. In our cohort, all non-proline mutations occurred at buried sites, while some proline substitutions affected exposed residues. Notably, the variant p.(Gln7023Pro) was identified in 5 unrelated families sharing a conserved haplotype, indicating a common ancestor. This variant and the previously reported p.(Arg25480Pro) variant have been reclassified as likely pathogenic. ConclusionsBy integrating clinical, computational, and functional evidence, we propose a framework for interpreting TTN missense variants. Combining multiple lines of evidence is essential for variants classification and interpretation, especially given TTN complexity. Advancing diagnostic accuracy will require tailored interpretation guidelines and a global effort in data sharing and functional validation.

The ClinGen Hereditary Breast, Ovarian and Pancreatic Cancer (HBOP) Variant Curation Expert Panel (VCEP) is composed of internationally recognized experts in clinical genetics, molecular biology and variant interpretation. This VCEP made specifications for ACMG/AMP guidelines for the ataxia telangiectasia mutated (ATM) gene according to the Food and Drug Administration (FDA)-approved ClinGen protocol. These gene-specific rules for ATM were modified from the American College of Medical Genetics and Association for Molecular Pathology (ACMG/AMP) guidelines and were tested against 33 ATM variants of various types and classifications in a pilot curation phase. The pilot revealed a majority agreement between the HBOP VCEP classifications and the ClinVar-deposited classifications. Six pilot variants had conflicting interpretations in ClinVar and reevaluation with the VCEPs ATM-specific rules resulted in four that were classified as benign, one as likely pathogenic and one as a variant of uncertain significance (VUS) by the VCEP, improving the certainty of interpretations in the public domain. Overall, 28 the 33 pilot variants were not VUS leading to an 85% classification rate. The ClinGen-approved, modified rules demonstrated value for improved interpretation of variants in ATM.

Mutations in ATP-binding cassette transporter type A4 (ABCA4) have been linked to several forms of inherited retinal diseases (IRDs) besides the classically defined Stargardt disease (STGD), known as ABCA4 retinopathies. ABCA4 is a sizeable locus harboring 50 exons; thus, its analysis has revealed a rich area of genetic information comprising at least 1,200 disease-causing mutations of varied severity and types. Due to the clinical and genetic heterogeneity, diagnosing ABCA4 retinopathies is challenging. To date, no ABCA4-retinopathy has been detected in Lebanon. Using next-generation sequencing, we sought to pinpoint the mutation spectrum in seven families with different forms of IRDs: STGD, rod-cone and cone-rod dystrophies (RCD and CRD, respectively). Eight ABCA4 mutations were found, including one novel; c.4330G>C; p.(Trp1408Cys). Three families were diagnosed with CRD, two with STGD, and two others with RCD. In conclusion, our study revealed a novel ABCA4 mutation and showed significant genotypic and phenotypic heterogeneity in Lebanon.

BackgroundLoss-of-function variants in MME (membrane metalloendopeptidase) are a known cause of recessive Charcot-Marie-Tooth Neuropathy (CMT). A deep intronic variant, MME c.1188+428A>G (NM_000902.5), was identified through whole genome sequencing (WGS) of two Australian families with recessive inheritance of axonal CMT using the seqr platform. MME c.1188+428A>G was detected in a homozygous state in Family 1, and in a compound heterozygous state with a known pathogenic MME variant (c.467del; p.Pro156Leufs*14) in Family 2. AimsWe aimed to determine the pathogenicity of the MME c.1188+428A>G variant through segregation and splicing analysis. MethodsThe splicing impact of the deep intronic MME variant c.1188+428A>G was assessed using an in vitro exon-trapping assay. ResultsThe exon-trapping assay demonstrated that the MME c.1188+428A>G variant created a novel splice donor site resulting in the inclusion of an 83 bp pseudoexon between MME exons 12 and 13. The incorporation of the pseudoexon into MME transcript is predicted to lead to a coding frameshift and premature termination codon (PTC) in MME exon 14 (p.Ala397ProfsTer47). This PTC is likely to result in nonsense mediated decay (NMD) of MME transcript leading to a pathogenic loss-of-function. InterpretationTo our knowledge, this is the first report of a pathogenic deep intronic MME variant causing CMT. This is of significance as deep intronic variants are missed using whole exome sequencing screening methods. Individuals with CMT should be reassessed for deep intronic variants, with splicing impacts being considered in relation to the potential pathogenicity of variants.

Rare single base pair changes in genes are an important cause of disease, as they can reside in key regions of the gene influencing biological function by impacting the protein conformation and protein interactions. Generation of the necessary experimental evidence to define the outcome of the presence of these gene variants is time consuming and costly. These challenges have led to the development of a plethora of in silico prediction tools. These tools frequently use similar sources of information and are trained on overlapping multi-gene truth datasets. However, frequently there has been no quantitative validation of the performance of these in silico tools for individual genes. Here we have applied the ClinGen Sequence Variant Interpretation Working Groups recommended in silico score thresholds to a set of predisposition gene variants with established pathogenicity/benignity. Of the genes assessed (BRCA1, BRCA2, TP53, TERT and ATM), in silico tool predictions showed inferior sensitivity (<65%) for pathogenic TERT variants and inferior sensitivity ([&le;]81%) for benign TP53 variants. This validation study highlights in silico tool performance can be gene-specific and is dependent on the training set on which the algorithm is built. Where there are sufficient numbers of established benign and pathogenic missense variants based on clinical and functional evidence, the use of in silico tool scores should be validated for individual genes. For genes where this is not possible and gene-agnostic in silico score cut offs are used, consideration of missense variant-protein structural impact relationships is suggested.

Oculocutaneous albinism (OCA) is genetically and clinically heterogeneous recessive disorders with at least 23 associated genes. Isolated OCA is characterized by hypopigmentation in the skin, hair, and eyes combined with ocular abnormalities. Hermansky Pudlak syndrome (HPS) and Chediak-Higaski syndrome are syndromic forms of OCA, distinguished by immunological and hematological symptoms in addition to hypopigmentation and ocular anomalies. Targeted clinical care is crucial for the patients and molecular genetic diagnosis is important for classification of patients. Current diagnostic yield is approximately 70%, and a high prevalence of patients, heterozygous for pathogenic variants in OCA genes, might suggest presence of disease-causing non-coding variants. We describe here NGS analysis, including CNV analysis, of 28 consanguineous families, comprising a total of 136 individuals presenting with OCA. We provide a molecular genetic diagnosis in all 28 families. Noteworthy, five families (18 %) had pathogenic variants in a gene associated with HPS. Furthermore, we report the first deep intron variant in TYR causing OCA and show by minigene analysis that the variant causes inclusion of a pseudoexon.

BackgroundRare genetic diseases (RGDs) affect individuals, families, and healthcare systems worldwide. Population-scale genomic data remain largely restricted to Western cohorts with estimated 10,000 RGDs. South Asian populations remain underrepresented in molecular, clinical, and genomic databases. This study presents the first molecular epidemiological analysis of RGDs in the Punjabi population of Pakistan. MethodsData were collected from the provincial RGD registry at the Punjab Thalassemia and Genetic Disorders Prevention and Research Institute (PTGDPRI), Lahore. Families diagnosed using next-generation sequencing (NGS) between 2021 and 2023 were enrolled. Structured questionnaires captured clinical, demographic, and socioeconomic information, and statistical and genetic analyses were performed to assess the inheritance patterns, allele frequencies, and disease distribution. ResultsThe registry included 167 families with 72 distinct RGDs, with a mean burden of 0.81{+/-}0.24 affected children per family. Niemann-Pick disease (NP), progressive familial intrahepatic cholestasis (PFIC), and mucopolysaccharidosis (MPS) were the most common diseases. Consanguinity was observed in 89% of families, 77% of which involved first-cousin marriages, and was significantly associated with RGD incidence. Most families belonged to low-income groups despite high literacy rates, underscoring inequity in healthcare. The primary and secondary variants included 131 variants including CNV and SNVs annotated pathogenic, likely pathogenic or variants of unknown significance across 110 genes, including 24 founder pathogenic variants. ConclusionsThis study provides the first genomic and epidemiological overview of RGDs in the Punjabi population. The findings reveal how genetic, socioeconomic, and cultural factors converge to amplify the RGD burden and highlight the need for affordable molecular diagnostics, inclusive genomic databases, and regional genomic surveillance initiatives in South-Asia.

SARS-CoV-2 infection has been playing havoc with emerging omicron variants of concern (VoC). Here, we report sequencing of the omicron variant in 13 patients from India using Oxford Nanopore Technology (ONT) Minion, wherein a rapid amplicon based sequence analysis was performed to assess and compare with existing 34 mutations in spike glycoprotein. We highlight and discuss the nature of these mutations that are unique and common to other populations. This is perhaps the first report on omicron variants from India using a long read sequencing chemistry.

The molecular diagnosis of albinism is hampered by a significant number of genetic variants of unknown significance (VUS) including a majority of missense and in-frame insertion deletion variants. This contributes to the high rate of unresolved genetic diagnosis for this disease. We designed a straightforward test of missense VUS in albinism genes based on functional rescue. As a proof of concept, the assay was set up for testing variants in the TYR gene associated with oculocutaneous albinism type 1. The TYR gene was knocked-out in the human melanogenic MNT1 cell line and the resulting unpigmented clones used as host cells for rescue experiments. Selected VUS and control sequences were run through the assay. Expression of tyrosinase was quantified by Western-blot, melanin synthesis was evaluated by direct observation as well as absorbance monitoring. One VUS, p.Ser270Phe (S270F) can be classified as pathogenic as it fails to restore pigmentation, whereas rescue was achieved with D305E and A391T. The two most frequent missense VUS of TYR, S192Y and R402Q, were also tested independently or in combination confirming the pathogenic effect of their association in cis. All in all, this new assay is straightforward enough to be transposed in diagnosis laboratories and can be considered for testing variants in other albinism genes such as TYRP1 and SLC45A2.

BackgroundIncontinentia pigmenti (IP) is a rare, hereditary multisystemic disorder affecting 1.2 in 100,000 live births, predominantly females. Conventional genetic analyses through short-read sequencing are complicated in case of IP due to the presence of a highly homologous pseudogene. Traditionally, long-range PCR is employed in order to overcome this challenge, however, detection of skewed X-Inactivation can also aid in correctly assign a variant to IKBKG. MethodsWe employed a comprehensive multi-method approach, incorporating whole-exome sequencing (WES), long-range PCR, RT-PCR, X-inactivation analysis, and nanopore sequencing of genomic DNA, to identify and accurately phase a small heterozygous deletion NM_001099857.5:c.363_367del, p.(Leu122Glyfs*14) to the IKBKG gene in a family affected with IP. ResultsThe deletion was initially detected through WES and skewed X-inactivation was observed in both the proband and her mother. Long-range PCR specific to IKBKG was utilized to verify that the variant is located in IKBKG and not in its highly homologous pseudogene. On RNA level, the variant was undetectable, suggesting nonsense-mediated decay (NMD) of the transcript containing the variant. We further utilized nanopore sequencing not only to pinpoint and accurately map the variant to the IKBKG gene but also to analyze methylation status of both alleles. This allowed us to confirm the skewed X-inactivation, with the variant-carrying allele found to be predominantly inactivated. ConclusionNanopore sequencing serves as a valuable tool in genetic diagnosis, enabling the precise localization of the variant in either the gene or the pseudogene. Furthermore, in females with skewed X-inactivation, this method facilitates the determination of whether the variant is predominantly located on the activated or inactivated X-chromosome.

1.Alkaptonuria or black urine disease is a rare autosomal recessive disorder caused by dysfunctional homogentisate 1,2-dioxygenase (HGD) gene (3q13.33) leading to accumulation of homogentisic acid in the body. This inborn error in metabolism of phenylalanine and tyrosine causes accumulation of homogentisic acid leading to ochronosis, pigmentation in the sclera, ear cartilage, mitral valve calcification and osteoarthropathy. Advances in sequencing technologies have helped us to map genetic variants associated with alkaptonuria in diverse populations and regions. Currently, no centralized resource of all the reported actionable variants with uniformity in annotation exists for the HGD gene. We have compiled HGD exonic variants from various data sources and systematically annotated their pathogenicity according to American College of Medical Genetics and the Association of Molecular Pathologists (ACMG/AMP) variant classification framework. A total of 1686 exonic variants were catalogued and manually curated, creating one of the most comprehensive Alkaptonuria variant databases (AKUHub) which is publicly available.

Menieres Disease (MD) is a chronic inner ear disorder defined by recurring episodes of vertigo, fluctuating sensorineural hearing loss, tinnitus, and/or fullness in the ear. Its prevalence varies by region and ethnicity, with scarce epidemiological data in Brazilian population. Although most MD cases are sporadic, Familial MD (FMD) is observed in 5% to 20% of European cases. By exome sequencing, we have found a rare missense variant in the OTOG gene in a Brazilian MD individual with probable European ancestry (chr11:17599671C>T), which was previously reported in a Spanish cohort. Two additional rare missense heterozygous OTOG variants were found in the same proband. Splice Site analysis showed that chr11:17599671C>T may lead to substantial changes generating exonic cis regulatory elements, and protein modelling revealed structural changes in the presence of chr11:17599671C>T, chr11:17576581G>C and chr11:17594108C>T, predicted to highly destabilize protein structure. These findings indicate that missense variants may have an additive effect leading to an unstable Otogelin and support OTOG gene as a key player in the MD pathophysiology.

Large-scale next-generation sequencing (NGS) germline testing is technically feasible today, but variant interpretation represents a major bottleneck in analysis workflows including the extensive variant prioritization, annotation, and time-consuming evidence curation. The scale of the interpretation problem is massive, and variants of uncertain significance (VUS) are a challenge to personalized medicine. This challenge is further compounded by the complexity and heterogeneity of standards used to describe genetic variants and associated phenotypes when searching for relevant information to inform clinical decision-making. For this purpose, all five Swiss academic Medical Genetics Institutions joined forces with the Swiss Institute of Bioinformatics (SIB) to create SwissGenVar as a user-friendly nationwide repository and sharing platform for genetic variant data generated during routine diagnostic procedures and research sequencing projects. Its objective is to provide a protected environment for expert evidence sharing about individual variants to harmonize and up-scale their significance interpretation at clinical grade following international standards. To corroborate the clinical assessment, the variant-related data are combined with consented high-quality clinical information. Broader visibility will be gained by interfacing with international databases, thus supporting global initiatives in personalized health care.

Germline pathogenic variants in TP53 are associated with Li-Fraumeni syndrome (LFS), an autosomal dominant cancer predisposition disorder associated with high risk of malignancy, including early onset breast cancers, sarcomas, adrenocortical carcinomas and brain tumors. Intense cancer surveillance for individuals with TP53 germline pathogenic variants has been shown to decrease mortality; therefore, accurate and consistent classification of variants across clinical and research laboratories is crucial to patient care. Here, we describe the work performed by the Clinical Genome Resource TP53 Variant Curation Expert Panel (ClinGen TP53 VCEP) focused on specifying the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines for germline variant classification to the TP53 gene. Specifications were applied to twenty ACMG/AMP criteria while nine were deemed not applicable. The original strength level for ten criteria was also adjusted due to current evidence. Use of the TP53-specific guidelines and sharing of clinical data amongst experts and clinical laboratories led to a decrease in variants of uncertain significance from 28% to 12% in comparison with the original guidelines. The ClinGen TP53 VCEP recommends the use of these TP53-specific ACMG/AMP guidelines as the standard strategy for TP53 germline variant classification.