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IntroductionCompared with breast cancer (BC) in women, BC in men is a rare disease with genetic and molecular peculiarities. Therapeutic approaches for male BC (MBC) are currently extrapolated from the clinical management of female BC, although the disease does not exactly overlap in males and females. Data on specific molecular biomarkers in MBC are lacking, cutting out male patients from more appropriate therapeutic strategies. Growing evidence indicates that Next Generation Sequencing (NGS) multigene panel testing can be used for the detection of predictive molecular biomarkers, including Tumor Mutational Burden (TMB) and Microsatellite Instability (MSI).MethodsIn this study, NGS multigene gene panel sequencing, targeting 1.94 Mb of the genome at 523 cancer-relevant genes (TruSight Oncology 500, Illumina), was used to identify and characterize somatic variants, Copy Number Variations (CNVs), TMB and MSI, in 15 Formalin-Fixed Paraffin-Embedded (FFPE) male breast cancer samples.Results and discussionA total of 40 pathogenic variants were detected in 24 genes. All MBC cases harbored at least one pathogenic variant. PIK3CA was the most frequently mutated gene, with six (40.0%) MBCs harboring targetable PIK3CA alterations. CNVs analysis showed copy number gains in 22 genes. No copy number losses were found. Specifically, 13 (86.7%) MBCs showed gene copy number gains. MYC was the most frequently amplified gene with eight (53.3%) MBCs showing a median fold-changes value of 1.9 (range 1.8-3.8). A median TMB value of 4.3 (range 0.8-12.3) mut/Mb was observed, with two (13%) MBCs showing high-TMB. The median percentage of MSI was 2.4% (range 0-17.6%), with two (13%) MBCs showing high-MSI. Overall, these results indicate that NGS multigene panel sequencing can provide a comprehensive molecular tumor profiling in MBC. The identification of targetable molecular alterations in more than 70% of MBCs suggests that the NGS approach may allow for the selection of MBC patients eligible for precision/targeted therapy.

Background Variants of GATOR1-genes represent a recognised cause of focal cortical dysplasia (FCD), the most common structural aetiology in paediatric drug-resistant focal epilepsy. Reports on familial cases of GATOR1-associated FCD are limited, especially with respect to epilepsy surgery outcomes. Methods We present phenotypical manifestations of four unrelated patients with drug-resistant focal epilepsy, FCD and a first-degree relative with epilepsy. All patients underwent targeted gene panel sequencing as a part of the presurgical work up. Literature search was performed to compare our findings to previously published cases. Results The children (probands) had a more severe phenotype than their parents, including drug-resistant epilepsy and developmental delay, and they failed to achieve seizure freedom post-surgically. All patients had histopathologically confirmed FCD (types IIa, IIb, Ia). In Patient 1 and her affected father, we detected a known pathogenic NPRL2 variant. In patients 2 and 3 and their affected parents, we found novel likely pathogenic germline DEPDC5 variants. In family 4, we detected a novel variant in NPRL3. We identified 15 additional cases who underwent epilepsy surgery for GATOR1-associated FCD, with a positive family history of epilepsy in the literature; in 8/13 tested, the variant was inherited from an asymptomatic parent. Conclusion The presented cases displayed a severity gradient in phenotype with children more severely affected than the parents. Although patients with GATOR1-associated FCD are considered good surgical candidates, post-surgical seizure outcome was poor in our familial cases, suggesting that accurate identification of the epileptogenic zone may be more challenging in this subgroup of patients.

Familial hypophosphatemic rickets (FHR) is a disorder characterized by phosphate wasting and hypophosphatemia due to defects in renal phosphate transport regulation. There are 4 known inherited forms of FHR that differ in their molecular causes. Very few studies have been conducted that focused on the molecular analysis of FHR in Koreans. Eighteen mutations of the PHEX gene have been identified to this date in Korea. Herein, we report the clinical case of a 24-month-old boy presenting with bowed legs and short stature. The biochemical profile showed hypophosphatemia with decreased tubular reabsorption of phosphate. Several family members were identified with short stature and genu varum. Therefore, he was diagnosed with FHR. To identify the molecular causes of FHR, we performed targeted gene panel sequencing and found a novel hemizygous missense variant, c.1949T>C (p.Leu650Pro), in the PHEX gene. This variant was also detected in the boy’s mother who exhibited genu varum and short stature.

Predominantly antibody deficiencies (PAD) are a heterogeneous group of disorders characterized by dysfunctional antibody production, low immunoglobulin levels in serum and impaired vaccine responses. The clinical picture is variable, ranging from mild symptoms to severe complications, which may include autoimmunity, gastrointestinal disease, allergy, and malignancies. If left untreated, PAD patients are at risk of enduring disease progression, irreversible organ damage, and reduced life expectancy. A timely diagnosis has been shown to significantly improve disease prognosis. Here, we report on our experience using targeted gene panel sequencing by employing Agilent’s HaloPlex or SureSelect and Illumina’s MiSeq technologies in a cohort of 291 individuals who presented with low or absent immunoglobulin levels in combination with or without other clinical features. In total, we have detected over 57 novel or previously reported relevant mutations in ADA, ADA2, BTK, CTLA4, LRBA, NFKB1, NFKB2, PIK3CD, STAT3, and TNFRSF13B. Overall, a genetic diagnosis could be made in 24.7% of the investigated patients. The percentage of coverage for the targeted regions ranged from 90% to 98% in this study. Moreover, functional assays were performed on a defined group of the patients carrying candidate variants in CTLA4, LRBA, NFKB1 and BTK, which confirmed their deleterious effect on protein expression and/or function. This study reiterates that the immunological heterogeneity of predominantly antibody deficiencies may have a diverse genetic origin, although certain clinical features may hint towards a specific group of defects. Employing targeted sequencing panels proves to be a very time- and cost-efficient, yet reliable, method for the establishment of a genetic diagnosis in individuals with PAD. However, in case of negative panel results, or if functional testing reveals inconspicuous observations in patients with a clear indication for genetic testing, further work-up including whole exome or whole genome sequencing should be considered.

Predominantly antibody deficiencies (PAD) are a heterogeneous group of disorders characterized by dysfunctional antibody production, low immunoglobulin levels in serum and impaired vaccine responses. The clinical picture is variable, ranging from mild symptoms to severe complications, which may include autoimmunity, gastrointestinal disease, allergy, and malignancies. If left untreated, PAD patients are at risk of enduring disease progression, irreversible organ damage, and reduced life expectancy. A timely diagnosis has been shown to significantly improve disease prognosis. Here, we report on our experience using targeted gene panel sequencing by employing Agilent’s HaloPlex or SureSelect and Illumina’s MiSeq technologies in a cohort of 291 individuals who presented with low or absent immunoglobulin levels in combination with or without other clinical features. In total, we have detected over 57 novel or previously reported relevant mutations in ADA, ADA2, BTK, CTLA4, LRBA, NFKB1, NFKB2, PIK3CD, STAT3, and TNFRSF13B. Overall, a genetic diagnosis could be made in 24.7% of the investigated patients. The percentage of coverage for the targeted regions ranged from 90% to 98% in this study. Moreover, functional assays were performed on a defined group of the patients carrying candidate variants in CTLA4, LRBA, NFKB1 and BTK, which confirmed their deleterious effect on protein expression and/or function. This study reiterates that the immunological heterogeneity of predominantly antibody deficiencies may have a diverse genetic origin, although certain clinical features may hint towards a specific group of defects. Employing targeted sequencing panels proves to be a very time- and cost-efficient, yet reliable, method for the establishment of a genetic diagnosis in individuals with PAD. However, in case of negative panel results, or if functional testing reveals inconspicuous observations in patients with a clear indication for genetic testing, further work-up including whole exome or whole genome sequencing should be considered.

Purpose We developed a new workflow design which included results from both biochemical and targeted gene sequencing analysis interpreted comprehensively. We then conducted a pilot study to evaluate the benefit of this new approach in newborn screening (NBS) and demonstrated the efficiency of this workflow in detecting causative genetic variants. Materials and methods Ten patients in Group 1 were diagnosed clinically using biochemical assays only, and 10 newborns in Group 2 were diagnosed with suspected inherited metabolic disease (IMD) in NBS. We applied NewbornDiscovery (SD Genomics), an integrated workflow design that encompasses analyte-phenotype-gene, single nucleotide variant/small insertion and deletion/copy number variation analyses along with clinical interpretation of genetic variants related to each participant's condition. Results A molecular genetic diagnosis was established in 95% (19/20) of individuals. In Group 1, 13 and 7 of 20 alleles were classified as pathogenic and likely pathogenic, respectively. In Group 2, 11 and 6 of 17 alleles with identified causative variants were pathogenic and likely pathogenic, respectively. There were no variants of uncertain significance. For each individual, the NewbornDiscovery and biochemical analysis results reached 100% concordance, since the single newborn testing negative for causative genetic variant in Group 2 showed a benign clinical course. Conclusion This integrated diagnostic workflow resulted in a high yield. This approach not only enabled early confirmation of specific IMD, but also detected conditions not included in the current NBS.