Congenital hereditary endothelial dystrophy with progressive sensorineural deafness: a case report of Harboyan syndrome

ABSTRACT We present the case of a 37-year-old woman who underwent bilateral penetrating keratoplasty for congenital hereditary endothelial dystrophy at the age of 10 years. Over the subsequent 27 years, the patient's vision slowly deteriorated. Our examination revealed decompensation of the right corneal graft. We addressed this with regraft surgery. We then learned that the patient had been suffering from progressive hearing loss since adolescence. Tonal audiometry revealed hearing per ceptive deafness of 25 dB, which was more prominent in the left ear. Because the patterns of progressive sensorineural hearing loss and congenital hereditary endothelial dystrophy have both been linked to the same gene, slc4a11, we tested our patient for mutations in this gene. The test was positive for a heterozygous slc4a11 gene fifth exon mutation on chromosome 20p13-p12, which causes a frameshift. A combined clinical and genetic evaluation confirmed a diagnosis of Harboyan syndrome. After the genetic diagnosis of the disease, she was evaluated for the need for a hearing aid due to her hearing loss. The patient was also informed about genetic counseling.

Rapid and unbiased enrichment of extracellular vesicles via a meticulously engineered peptide.

Extracellular vesicles (EVs) have garnered significant attention in biomedical applications. However, the rapid, efficient, and unbiased separation of EVs from complex biological fluids remains a challenge due to their heterogeneity and low abundance in biofluids. Herein, we report a novel approach to reconfigure and modify an artificial insertion peptide for the unbiased and rapid isolation of EVs in 20 min with ∼80% recovery in neutral conditions. Moreover, the approach demonstrates exceptional anti-interference capability and achieves a high purity of EVs comparable to standard ultracentrifugation and other methods. Importantly, the isolated EVs could be directly applied for downstream protein and nucleic acid analyses, including proteomics analysis, exome sequencing analysis, as well as the detection of both epidermal growth factor receptor (EGFR) and V-Ki-ras2 Kirsten Rat Sarcoma Viral Oncogene Homologue (KRAS) gene mutation in clinical plasma samples. Our approach offers great possibilities for utilizing EVs in liquid biopsy, as well as in various other biomedical applications.

Genomic Pipeline for Analysis of Mutational Events in Bacteria.

Unveiling the strategies of bacterial adaptation to stress constitute a challenging area of research. The understanding of mechanisms governing emergence of resistance to antimicrobials is of particular importance regarding the increasing threat of antibiotic resistance on public health worldwide. In the last decades, the fast democratization of sequencing technologies along with the development of dedicated bioinformatical tools to process data offered new opportunities to characterize genomic variations underlying bacterial adaptation. Thereby, research teams have now the possibility to dive deeper in the deciphering of bacterial adaptive mechanisms through the identification of specific genetic targets mediating survival to stress. In this chapter, we proposed a step-by-step bioinformatical pipeline enabling the identification of mutational events underlying biocidal stress adaptation associated with antimicrobial resistance development using Escherichia marmotae as an illustrative model.

A novel stoploss mutation CYB5R3 c.906A>G(p.*302Trpext*42) involved in the pathogenesis of hereditary methemoglobinemia.

Recessive congenital methemoglobinemia (RCM) is a hereditary autosomal disorder with an extremely low incidence rate. Here, we report a case of methemoglobinemia type I in a patient with congenital persistent cyanosis. The condition was attributed to a novel compound heterozygous mutation in CYB5R3, characterized by elevated methemoglobin levels (13.4 % of total hemoglobin) and undetectable NADH cytochrome b5 reductase (CYB5R3) activity. Whole-exome sequencing (WES) revealed two heterozygous mutations in CYB5R3: a previously reported pathogenic missense mutation c.611G>A(p.Cys204Tyr) inherited from the father, and a novel stop codon mutation c.906A>G(p.*302Trpext*42) from the mother, the latter mutation assessed as likely pathogenic according to ACMG guidelines. In cells overexpressing the CYB5R3 c.906A>G mutant construct, the CYB5R3 mRNA level was significantly lower than in cells overexpressing the wild-type (WT) CYB5R3 construct. However, there was no significant difference in protein expression levels between the mutant and WT constructs. Notably, an additional protein band of approximately 55 kDa was detected in the mutant cells. Immunofluorescence localization showed that, compared to wild-type CYB5R3, the subcellular localization of the CYB5R3 p.*302Trpext*42 mutant protein did not show significant changes and remained distributed in the endoplasmic reticulum and mitochondria. However, the c.906A>G(p.*302Trpext*42) mutation resulted in increased intracellular reactive oxygen species (ROS) levels and decreased NAD+/NADH ratio, suggesting impaired CYB5R3 function and implicating this novel mutation as likely pathogenic.

Characterization of insecticide resistance and their mechanisms in field populations of the German cockroach (Blattodea: Ectobiidae) in Taiwan under different treatment regimes.

This study investigated how management strategies influence resistance profiles in German cockroach (Blattella germanica (L.)) populations and their impact on the performance of commercial gel baits containing fipronil, imidacloprid, and indoxacarb. Field populations from premises managed under 3 different strategies: Baiting, random insecticide (RI) used, and insecticide rotation (IR) were tested. Almost all populations under RI and IR were resistant to deltamethrin, but low to moderate resistance was observed under the Baiting approach. Cytochrome P450 monooxygenases (P450) were involved in deltamethrin resistance in these resistant populations. All individuals under Baiting and RI were homozygous for the L993F mutation, but the populations under IR lacked homozygous-resistant individuals. Eighty-three percent of field populations with complete homozygosity for the Rdl mutation displayed low mortality upon exposure to 3× LD95 fipronil. The effect of P450 and the Rdl mutation conferred high fipronil resistance in populations under the Baiting approach, recording moderate performance indices (PI) of 44-67 in fipronil bait. By contrast, those populations under RI and IR, in which involve glutathione S-transferases in fipronil resistance, had high PIs of 78-93. Almost 80% of populations exhibited over 90% mortality at 3× LD95 indoxacarb treatment, accompanied by high PIs of 90-100 in indoxacarb bait. Partial mortality from 1× LD95 imidacloprid occurred across all field populations due to the involvement of P450. PIs of imidacloprid bait ranged 5-57 and 20-94 in populations under RI and IR, respectively. Field populations demonstrate different resistance profiles depending on the treatment regimes, and the resistance mechanisms involved influenced gel bait's effectiveness.

Oral sialadenoma papilliferum with kras mutation in a patient with linear nevus sebaceous syndrome.

Linear nevus sebaceous syndrome (LNSS) is a rare neurocutaneous syndrome part of the epidermal nevus syndromes group, characterized by the presence of sebaceous nevi and other extracutaneous lesions genetically related to RAS family gene mutations. Sialadenoma papilliferum (SP) is a rare benign intraoral neoplasm which is usually BRAF or HRAS mutated. We report a case of a young female girl diagnosed with a LNSS who developed a SP which had a KRAS mutation. This is the first case of SP with a KRAS mutation in the context of a LNSS.

Association of various RAS codon mutations and prognostic outcomes of patients with colorectal liver metastases after hepatectomy.

PURPOSE: The prognostic and predictive value of RAS mutations in patients with colorectal liver metastases (CRLM) who have undergone hepatectomy holds substantial importance. The present study aimed to investigate the impact of different RAS codon mutations on long-term survival in CRLM patients. METHODS: A retrospective analysis was conducted on clinicopathological data from 399 CRLM patients with RAS mutations who underwent hepatectomy between January 2000 and December 2020. The RAS mutation gene status was assessed in KRAS codons (G12, G13, Q61, and A146) and NRAS codons (G12, G13, and Q61). Survival curves were generated using the Kaplan-Meier plotter and compared using the log-rank test. Univariate and multivariate analyses were performed to analyze the clinicopathological data. RESULTS: In the entire cohort, patients with KRAS G12 mutations exhibited the most favorable prognosis (p = 0.018). Comparatively, patients harboring KRAS Q61 mutations experienced poorer overall survival (OS) with a median of 15 months versus 33 months (p = 0.011) when compared to those with KRAS G12 mutations. Moreover, patients with NRAS Q61 mutations also showed decreased OS with a median of 26 months versus 33 months (p = 0.020) in comparison to KRAS G12 mutation patients. The results of multivariate analysis showed that both KRAS Q61 mutation (HR 2.130; 95% CI 1.088-4.168; p = 0.027) and NRAS Q61 mutation (HR 2.877; 95% CI 1.398-5.922; p = 0.004) were independent influencing factors of OS. Based on all identified risk factors, patients with RAS mutation were divided into high-risk and low-risk groups. Notably, in the high-risk group, the incorporation of postoperative chemotherapy was associated with longer OS, while it did not improve the survival of patients in the low-risk group. CONCLUSIONS: KRAS Q61 and NRAS Q61 mutations are promising predictors for OS in CRLM patients after hepatectomy. Postoperative chemotherapy may significantly benefit CRLM patients with RAS mutations, particularly those identified as high-risk.

TTBK2 T3290C mutation in spinocerebellar ataxia 11 interferes with ciliogenesis.

This study aimed to elucidate the impact of the TTBK2 T3290C mutation (MUT) associated with Spinocerebellar Ataxia 11 (SCA11) on TTBK2 expression, function, and ciliogenesis. Lymphocytes were isolated from peripheral blood samples of SCA11 family members with the MUT and healthy controls (wild-type, WT). HEK-293 cells transfected with either WT or MUT TTBK2 plasmids were used to assess the MUT's impact on TTBK2 protein expression, enzymatic activity, and its binding to Cep164 protein. Mouse embryonic fibroblast cells transfected with WT or MUT TTBK2 plasmids examined the MUT's effect on cilia formation. Clinically, there was no significant difference in the expression of TTBK2 between the SCA11 patients and healthy individuals. The TTBK2 T3290C MUT did not affect protein expression or enzymatic activity but did reduce ciliary formation in embryonic cells and decreased binding affinity to Cep164. Therefore, our data suggested that the TTBK2 T3290C MUT in SCA11 may impair ciliogenesis by weakening the interaction with Cep164.

Identification and analysis of gamma-irradiation-induced Stemphylium blight tolerant lentil (Lens culinaris) mutant.

In the short-season winter environment of India and Bangladesh, lentil growth and seed yield are significantly hindered by foliar blight caused by Stemphylium botryosum. As the international germplasm pool lacks a resistance source, the study aims to develop a mutant population to identify a high-yielding mutant resistance against the pathogen. A gamma-irradiated population was developed based on its GR50 dose of 248.8 Gy. The screening of almost 130,000 M2 plants identified a tolerant lentil mutant, MM216. The multi-location trials revealed that MM216 showed an impressive and robust resistance; the selected mutant line could be recommended as a donor in the lentil breeding program against the pathogen globally. A 100 g seed was exposed to a GR50 dose to develop the M1 population. At maturity, at least 100 M2 seeds of each 1300 M1 plant were harvested individually. So, almost 130,000 M2 plants were screened in the disease hot spot. The selected mutants were advanced to M7 by screening in the field and challenged in controlled conditions with the pure pathogen isolate. A resistance mutant, MM216, with a per cent disease index (PDI) of <10, was identified where the mean of the check varieties, WBL 77, was >55. The resistance ability was confirmed further in controlled conditions. The fungal and plant DNA ratio was almost negligible in the tolerant mutant, whereas it was 0.17 in WBL77 at 196 h post-inoculation. The selected mutant did not display any yield penalty, but there was a delay in flowering by a week compared to WBL77.

Protein engineering of an alkaline protease from Bacillus licheniformis (BLAP) for efficient and specific chiral resolution of the racemic ethyl tetrahydrofuroate.

Enzymatic resolution of ethyl tetrahydrofuroate to produce (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid is a green biomanufacturing strategy. However, enzymatic activity and selectivity are still limiting factors of their industrial applications and development. In previous study, we incidentally found that a Bacillus licheniformis alkaline protease (BLAP), not a lipase, could specifically resolve ethyl tetrahydrofuroate to produce (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid. In this study, the point-saturation-mutation libraries based on the seven amino acid sites (L105, I113, P114, L115, V309, Y310, and M326) were constructed and screened using the molecular docking technology. It was found that activity of the mutant BLAPY310E reached 182.78 U/mL with high stereoselectivity, 3.14 times higher than that of the wild-type BLAP. Further simulated mutation analysis showed that the Y310E mutation increased the distance from the substrate ligand to the binding pocket from 2.3 Što 4.5 Å, reducing steric hindrance to the active center. Under the optimal conditions and after 3.5 h of reaction catalyzed by BLAPY310E, 200 mM ethyl tetrahydrofuroate was converted to (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid with the ee values of 99.9 % and 68.63 %, respectively. The enantiomeric ratio of BLAPY310E was 105.5, which was 30.23 times higher than that of BLAP. This study advances the comprehension of protease activity and selectivity mechanisms in resolving ester substances and lays a robust foundation for the industrial production of the optically pure (S)-2-ethyl tetrahydrofuroate and (R)-2-tetrahydrofuroic acid via biological enzymatic methods.

Cognition in (pre)symptomatic Dutch-type hereditary and sporadic cerebral amyloid angiopathy.

INTRODUCTION: Cerebral amyloid angiopathy (CAA) is a main cause of cognitive dysfunction in the elderly. We investigated specific cognitive profiles, cognitive function in the stage before intracerebral hemorrhage (ICH), and the association between magnetic resonance imaging (MRI) based cerebral small vessel disease (cSVD) burden in CAA because data on these topics are limited. METHODS: We included Dutch-type hereditary CAA (D-CAA) mutation carriers with and without ICH, patients with sporadic CAA (sCAA), and age-matched controls. Cognition was measured with a standardized test battery. Linear regression was performed to assess the association between MRI-cSVD burden and cognition. RESULTS: D-CAA ICH- mutation carriers exhibited poorer global cognition and executive function compared to age-matched controls. Patients with sCAA performed worse across all cognitive domains compared to D-CAA ICH+ mutation carriers and age-matched controls. MRI-cSVD burden is associated with decreased processing speed. DISCUSSION: CAA is associated with dysfunction in multiple cognitive domains, even before ICH, with increased MRI-cSVD burden being associated with slower processing speed. HIGHLIGHTS: Cognitive dysfunction is present in early disease stages of cerebral amyloid angiopathy (CAA) before the occurrence of symptomatic intracerebral hemorrhage (sICH). Presymptomatic Dutch-type CAA (D-CAA) mutation carriers show worse cognition than age-matched controls. More early awareness of cognitive dysfunction in CAA before first sICH is needed. Increased cerebral small vessel disease CAA-burden on magnetic resonance imaging is linked to a decrease in processing speed.

AVM: A Manually Curated Database of Aerosol-transmitted Virus Mutations, Human Diseases, and Drugs.

Aerosol-transmitted viruses possess strong infectivity and can spread over long distances, earning the difficult-to-control title. They cause various human diseases and pose serious threats to human health. Mutations can increase the transmissibility and virulence of the strains, reducing the protection provided by vaccines and weakening the efficacy of antiviral drugs. In this study, we established a manually curated database (termed AVM) to store information on aerosol-transmitted viral mutations (VMs). The current version of the AVM contains 42,041 VMs (including 2613 immune escape mutations), 45 clinical information datasets, and 407 drugs/antibodies/vaccines. Additionally, we recorded 88 human diseases associated with viruses and found that the same virus can target multiple organs in the body, leading to diverse diseases. Furthermore, the AVM database offers a straightforward user interface for browsing, retrieving, and downloading information. This database is a comprehensive resource that can provide timely and valuable information on the transmission, treatment, and diseases caused by aerosol-transmitted viruses (http://www.bio-bigdata.center/AVM).

Experimental evidence of inbreeding depression for competitive ability and its population-level consequences in a mixed-mating plant.

Inbreeding depression is a key factor regulating the evolution of self-fertilization in plants. Despite predictions that inbreeding depression should evolve with selfing rates as deleterious alleles are increasingly exposed and removed by selection, evidence of purging the genetic load in wild populations is equivocal at best. This discordance could be explained, in part, if the load underlying inbreeding depression is subject to soft selection, i.e., the fitness of selfed individuals depends on the frequency and density of selfed vs. outcrossed individuals in the population. Somewhat counterintuitively, this means that populations with contrasting mutation load can have similar fitness. Soft selection against selfed individuals may be expected when there is inbreeding depression for competitive ability in density-regulated populations. We tested population-level predictions of inbreeding depression in competitive ability by creating a density series of potted plants consisting of either purely outcrossed, purely selfed, or mixed (50% outcrossed, 50% selfed) seed of the mixed-mating biennial Sabatia angularis (Gentianaceae) representing ecological neighborhoods. Focusing on the growth and survival of juveniles, we show that mean plant size is independent of neighborhood composition when resources are limiting, but greatest in outcrossed neighborhoods at low densities. Across a range of densities, this manifests as stronger density-dependence in outcrossed populations compared to selfed or mixed ones. We also found significantly greater size inequalities among individuals in mixed neighborhoods, even at high densities where mean juvenile size converged, a key signature of asymmetric competition between outcrossed and selfed individuals. Our work illustrates how soft selection could shelter the genetic load underlying inbreeding depression and its demographic consequences.

The Ser434Phe Androgen Receptor Gene Mutation Does Not Affect Fertility but is Associated with Increased Prolactin.

Introduction: Prolactin is a hormone secreted by the anterior pituitary gland essential for lactation. Non-physiological hyperprolactinemia characterized by serum prolactin levels exceeding 20 ng/mL in men and 25 ng/mL in women, often results from medication use or pituitary gland tumors. In a minority of cases, the cause of hyperprolactinemia remains unknown despite clinical investigations. Familial idiopathic hyperprolactinemia may stem from mutations in genes encoding prolactin (PRL) and its receptor (PRLR). Methods: This study investigated genetic polymorphisms in PRL and PRLR genes using polymerase chain reaction (PCR) and Sanger sequencing in three sisters affected by familial idiopathic hyperprolactinemia. No mutations were found in these genes, prompting whole exome sequencing (WES) of the proband to identify other potentially involved genes. Results: WES revealed a heterozygous missense substitution c.1301C>T (p.Ser434Phe) in the androgen receptor (AR) gene. Next-generation sequencing (NGS) for the AR gene confirmed that the proband and her two affected sisters, along with three asymptomatic sisters, were all heterozygous carriers of the mutation. Their father was hemizygous, while their mother had a normal genotype. Conclusion: The heterozygous missense mutation in the AR gene found in this family with familial idiopathic hyperprolactinemia is not yet explained. Hence, further research is warranted to elucidate the functional implications of this mutation on AR and its role in the pathogenesis of hyperprolactinemia.

Trousseau's Syndrome in Lung Cancer Patients: A Retrospective Study in a Japanese Community Hospital.

Trousseau's syndrome is a cancer-associated thromboembolism that significantly impacts patients' prognosis and quality of life (QOL). This study aimed to investigate the frequency, characteristics, and prognosis of Trousseau's syndrome in lung cancer patients at a Japanese community hospital and examine the effects of therapeutic agents on this condition. We retrospectively reviewed the electronic medical records of lung cancer patients diagnosed with thrombotic complications at the time of diagnosis in our department between August 2013 and April 2019. Patients' characteristics, thromboembolism sites, treatments, and prognosis were analyzed. Among 956 lung cancer patients, 19 (2%) had Trousseau's syndrome. The median age was 65 years, and adenocarcinoma was the most common histologic type (78.9%). The most common site of thromboembolism was the brain (84.2%). The median survival time was 84 days, and 52.6% of patients died within 90 days of diagnosis. Patients who survived longer than 90 days tended to have a higher frequency of non-adenocarcinoma histology, EGFR gene mutations, and therapeutic induction with immune checkpoint inhibitors (ICI). Trousseau's syndrome in lung cancer patients is associated with poor prognosis. Histologic type, EGFR mutation status, and treatment with ICI may influence the prognosis. Future larger-scale studies are needed to validate these potential prognostic factors and to develop personalized treatment strategies.

Exceptional sustained long-term complete response to Tepotinib in a MET-amplified advanced intrahepatic biliary tract cancer failing Durvalumab plus Cisplatin and Gemcitabine.

BACKGROUND: Biliary tract cancers (BTCs) are a diverse group of malignancies with varied genetic backgrounds. The prevalence of intrahepatic cholangiocarcinoma (iCC) is increasing, particularly in Western countries. Despite advancements in treatments, the prognosis for BTC remains poor. Recent molecular profiling has revealed that up to 40% of iCC cases have targetable genetic alterations. MET amplification, although rare, presents a significant target for therapy. CASE PRESENTATION: A 25-year-old female with a history of ulcerative colitis presented with shoulder pain and a positron emission tomography-computed tomography (PET-CT) scan revealed an enlarged liver and multiple metastases. Histopathological analysis diagnosed poorly differentiated adenocarcinoma. First-line therapy with Cisplatin, Gemcitabine, and Durvalumab resulted in disease progression. Molecular profiling identified a TP53 mutation and MET amplification. Based on these findings, Tepotinib was initiated. Tepotinib treatment led to a significant reduction in tumor size and normalization of CA 19-9 levels within 2 months, achieving a complete metabolic remission lasting up to 17 months. The treatment was well tolerated with minimal side effects. DISCUSSION: MET-amplified BTCs are exceedingly rare, and evidence for targeted treatment is limited. This case demonstrates the efficacy of Tepotinib in a young patient with MET-amplified iCC, showing a long-term response and suggesting a potential new standard treatment option for this molecularly defined entity. This case also highlights the aggressive nature of MET-amplified tumors and the need for targeted second-line therapies. CONCLUSION: Tepotinib showed remarkable efficacy in treating MET-amplified intrahepatic cholangiocarcinoma, underscoring the importance of molecular profiling in BTCs and suggesting a potential new therapeutic approach for this rare cancer subtype.

Wide circulation of type 1 vaccine-derived poliovirus strains in clinical specimens from suspected cases of poliomyelitis, their contacts and in wastewater in Madagascar since late 2020.

Madagascar has faced three major outbreaks of vaccine-derived polioviruses (VDPVs) in recent decades, with VDPV type 1 reemerging in late 2020. Here, we report the molecular characterization of these cVDPV1 strains. WHO protocols were used for poliovirus detection in stool and wastewater samples. Molecular genotyping was based on the 5' non-coding (5'NC), VP1, and 3Dpol regions. From 2020 to 2022, 92 of 5690 stool samples and 129 of 1046 wastewater samples tested positive for cVDPV1. Genetic analysis of the VP1 gene revealed 1.3%-6.1% variability compared to the Sabin strain. Most sequences showed mutations at neurovirulence attenuation sites. Phylogenetic analysis distributed strains into four genogroups originating from Southern Madagascar. All analyzed cVDPV1 strains were recombinant, containing mutated oral polio vaccine sequences in VP1 and type C enterovirus sequences in other regions. This study demonstrated that all strains were closely related during this epidemic.

Alpha-1 Antitrypsin Deficiency in a Young Never Smoker With Novel Pi*Null Homozygous Mutation: a Case Report.

Alpha-1 antitrypsin deficiency is an autosomal codominant disorder caused by SERPINA1 gene mutations. PI*Z and PI*S mutations commonly underlie this deficiency, but rarer homozygous PI*null (Q0) mutations may result in a complete loss of alpha-1 antitrypsin (AAT). Such rare mutations lead to severe AAT deficiency and early onset of lung disease. We present a case of 35-year-old female never-smoker born to consanguineous parents who developed severe panlobular emphysema and end-stage respiratory insufficiency requiring lung transplantation. Subsequent genetic testing identified her as homozygous for a novel c.82del mutation - here named Q0Bani-Yas based on the region of the primary carrier's origin - which resulted in undetectable levels of alpha-1 antitrypsin protein.

Classification of PTEN germline non-truncating variants: a new approach to interpretation.

BACKGROUND: PTEN hamartoma tumour syndrome (PHTS) encompasses distinct syndromes, including Cowden syndrome resulting from PTEN pathogenic variants. Missense variants account for 30% of PHTS cases, but their classification remains challenging. To address these difficulties, guidelines were published by the Clinical Genome Resource PTEN Variant Curation Expert Panel. METHODS: Between 2010 and 2020, the Bergonie Institute reference laboratory identified 76 different non-truncating PTEN variants in 166 patients, 17 of which have not previously been reported. Variants were initially classified following the current guidelines. Subsequently, a new classification method was developed based on four main criteria: functional exploration, phenotypic features and familial segregation, in silico modelling, and allelic frequency. RESULTS: This new method of classification is more discriminative and reclassifies 25 variants, including 8 variants of unknown significance. CONCLUSION: This report proposes a revision of the current PTEN variant classification criteria which at present rely on functional tests evaluating only the phosphatase activity of PTEN and apply a particularly stringent clinical PHTS score.The classification of non-truncating variants of PTEN is facilitated by taking into consideration protein stability for variants with intact phosphatase activity, clinical and segregation criteria adapted to the phenotypic variability of PHTS and by specifying the allelic frequency of variants in the general population. This novel method of classification remains to be validated in a prospective cohort.