Lamis Yehia

Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

Background: Patients with heterozygous germline mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) experience autoimmunity and lymphoid hyperplasia. Objectives: Because regulation of the phosphoinositide 3‐kinase (PI3K) pathway is critical for maintaining regulatory T (Treg) cell functions, we investigate Treg cells in patients with heterozygous germline PTEN mutations (PTEN hamartoma tumor syndrome [PHTS]). Methods: Patients with PHTS were assessed for immunologic conditions, lymphocyte subsets, forkhead box P3 (FOXP3)+ Treg cell levels, and phenotype. To determine the functional importance of phosphatases that control the PI3K pathway, we assessed Treg cell induction in vitro, mitochondrial depolarization, and recruitment of PTEN to the immunologic synapse. Results: Autoimmunity and peripheral lymphoid hyperplasia were found in 43% of 79 patients with PHTS. Immune dysregulation in patients with PHTS included lymphopenia, CD4+ T‐cell reduction, and changes in T‐ and B‐cell subsets. Although total CD4+FOXP3+ Treg cell numbers are reduced, frequencies are maintained in the blood and intestine. Despite pathogenic PTEN mutations, the FOXP3+ T cells are phenotypically normal. We show that the phosphatase PH domain leucine‐rich repeat protein phosphatase (PHLPP) downstream of PTEN is highly expressed in normal human Treg cells and provides complementary phosphatase activity. PHLPP is indispensable for the differentiation of induced Treg cells in vitro and Treg cell mitochondrial fitness. PTEN and PHLPP form a phosphatase network that is polarized at the immunologic synapse. Conclusion: Heterozygous loss of function of PTEN in human subjects has a significant effect on T‐ and B‐cell immunity. Assembly of the PTEN‐PHLPP phosphatase network allows coordinated phosphatase activities at the site of T‐cell receptor activation, which is important for limiting PI3K hyperactivation in Treg cells despite PTEN haploinsufficiency. GRAPHICAL ABSTRACT Figure. No caption available.

Germline Compound Heterozygous Poly-glutamine Deletion in USF3 May Be Involved in Predisposition to Heritable and Sporadic Epithelial Thyroid Carcinoma

Abstract Cowden syndrome (CS) is an autosomal dominant disorder that predisposes to breast, thyroid, and other epithelial cancers. Differentiated thyroid carcinoma (DTC), as one of the major component cancers of CS, is the fastest rising incident cancer in the USA, and the most familial of all solid tumours. To identify additional candidate genes of CS and potentially DTC, we analysed a multi-generation CS-like family with papillary thyroid cancer (PTC), applying a combined linkage-based and whole-genome sequencing strategy and identified an in-frame germline compound heterozygous deletion, p.[Gln1478del];[Gln1476-Gln1478del] in USF3 (previously known as KIAA2018). Among 90 unrelated CS/CS-like individuals, 29% were found to have p.[Gln1478del];[Gln1476-Gln1478del]. Of 497 TCGA PTC individuals, 138 (27%) were found to carry this germline compound deletion, with somatically decreased tumour USF3 expression. We demonstrate an increased migration phenotype along with enhanced epithelial-to-mesenchymal transition (EMT) signature after USF3 knockdown or USF3 p.[Gln1478del];[Gln1476-Gln1478del] overexpression, which sensitizes cells to the endoplasmic reticulum (ER) stress. Loss of USF3 function induced cell necrosis-like features and impaired respiratory capacity while providing a glutamine-dependent cell survival advantage, strongly suggests a metabolic survival and migration-favouring microenvironment for carcinogenesis. Therefore, USF3 may be involved in the predisposition of thyroid cancer. Importantly, the results that glutamine-dependent survival and sensitivity to ER stress in USF3-deficient cells provide avenues for therapeutic and adjunct preventive interventions for both sporadic cancer as well as cancer predisposition syndromes with similar mechanisms.

Exome sequencing reveals germline gain-of-function EGFR mutation in an adult with Lhermitte-Duclos disease

Lhermitte–Duclos disease (LDD) is a rare cerebellar disorder believed to be pathognomonic for Cowden syndrome. Presently, the only known etiology is germline PTEN mutation. We report a 41-yr-old white female diagnosed with LDD and wild-type for PTEN. Exome sequencing revealed a germline heterozygous EGFR mutation that breaks a disulfide bond in the receptors extracellular domain, resulting in constitutive activation. Functional studies demonstrate activation of ERK/AKT signaling pathways, mimicking PTEN loss-of-function downstream effects. The identification of EGFR as a candidate LDD susceptibility gene contributes to advancement of molecular diagnosis and targeted therapy for this rare condition with limited treatment options.

Germline TTN variants are enriched in PTEN-wildtype Bannayan-Riley-Ruvalcaba syndrome

Bannayan–Riley–Ruvalcaba syndrome (BRRS) is a rare congenital disorder classically characterized by macrocephaly in combination with intestinal hamartomatous polyposis, vascular malformations, lipomas, and genital lentiginosis. Germline PTEN mutations have been reported in up to 60% of BRRS patients. The remaining cases are of unknown genetic etiology. We exome-sequenced 35 unrelated PTEN-wildtype patients with classic presentation of BRRS and identified TTN germline missense variants in 12/35 (34%) patients. TTN encodes TITIN, a key structural and functional muscle protein. Exome and TTN-targeted sequencing in an additional unrelated series of 231 BRRS-like patients revealed 37 (16%) additional patients with germline TTN variants. All variants were predicted to be deleterious and equally distributed between the A-band and I-band protein domains. Rare TTN variants (MAF ≤ 0.0001) are enriched in classic BRRS patients compared to BRRS-like (OR = 2.7, 95% CI 1.21-5.94, p = 1.6 × 10-2) and multiple population controls (OR = 2.2, 95% CI 1.01-4.20, p = 4.7 × 10-2). Germline TTN mutations of different genotypes, inheritance patterns, and protein domain enrichment have been identified in multiple cardiac and/or skeletal muscular disorders. Functional interrogation of I-band variant p.Cys5096Arg identified in one of our classic BRRS patients, using CRISPR-Cas9 genome-edited cell lines, reveals an increased growth and lack of contact inhibition phenotype associated with increased levels of or phosphorylation of focal adhesion kinase (FAK) in mutant cells. These findings suggest that TITIN could play a role in overgrowth-relevant pathways and phenotypes. In summary, our observations suggest TTN as a candidate predisposing gene in classic PTEN-wildtype BRRS patients, perhaps suggesting this syndrome join the growing list of Titinopathies.Rare disease: New gene linked to Bannayan-Riley-Ruvalcaba syndromeTTN variants seem to explain many cases of a rare hereditary condition previously linked only to mutations in the unrelated gene PTEN. Charis Eng and team from the Cleveland Clinic, Ohio, USA, sequenced the protein-coding DNA from 35 patients with Bannayan-Riley-Ruvalcaba syndrome (BRRS), a disease characterized by large head size, tumors and other overgrowth problems. All of these individuals showed no mutations in PTEN, a tumor suppressor that explains 60% of BRRS cases. However, one-third had variants in TTN, which encodes a protein called TITIN that’s involved in muscle elasticity. Additional testing revealed more BRRS-like patients with TTN variants, and lab experiments indicated one possibility of how TTN variants lead to increased cellular growth. The authors suggest BRRS could be considered a “titinopathy” along with other cardiac and skeletal diseases caused by TTN mutations.

Unexpected cancer-predisposition gene variants in Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome patients without underlying germline PTEN mutations

Patients with heritable cancer syndromes characterized by germline PTEN mutations (termed PTEN hamartoma tumor syndrome, PHTS) benefit from PTEN-enabled cancer risk assessment and clinical management. PTEN-wildtype patients (~50%) remain at increased risk of developing certain cancers. Existence of germline mutations in other known cancer susceptibility genes has not been explored in these patients, with implications for different medical management. We conducted a 4-year multicenter prospective study of incident patients with features of Cowden/Cowden-like (CS/CS-like) and Bannayan-Riley-Ruvalcaba syndromes (BRRS) without PTEN mutations. Exome sequencing and targeted analysis were performed including 59 clinically actionable genes from the American College of Medical Genetics and Genomics (ACMG) and 24 additional genes associated with inherited cancer syndromes. Pathogenic or likely pathogenic cancer susceptibility gene alterations were found in 7 of the 87 (8%) CS/CS-like and BRRS patients and included MUTYH, RET, TSC2, BRCA1, BRCA2, ERCC2 and HRAS. We found classic phenotypes associated with the identified genes in 5 of the 7 (71.4%) patients. Variant positive patients were enriched for the presence of second malignant neoplasms compared to patients without identified variants (OR = 6.101, 95% CI 1.143–35.98, p = 0.035). Germline variant spectrum and frequencies were compared to The Cancer Genome Atlas (TCGA), including 6 apparently sporadic cancers associated with PHTS. With comparable overall prevalence of germline variants, the spectrum of mutated genes was different in our patients compared to TCGA. Intriguingly, we also found notable enrichment of variants of uncertain significance (VUS) in our patients (OR = 2.3, 95% CI 1.5–3.5, p = 0.0002). Our data suggest that only a small subset of PTEN-wildtype CS/CS-like and BRRS patients could be accounted for by germline variants in some of the known cancer-related genes. Thus, the existence of alterations in other and more likely non-classic cancer-associated genes is plausible, reflecting the complexity of these heterogeneous hereditary cancer syndromes.

Non-canonical role of cancer-associated mutant SEC23B in the ribosome biogenesis pathway

Abstract SEC23B is a component of coat protein complex II (COPII) vesicles that transport secretory proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Loss-of-function SEC23B mutations cause a rare form of anemia, resulting from decreased SEC23B levels. We recently identified germline heterozygous SEC23B variants as potentially cancer-predisposing. Mutant SEC23B associated with ER stress-mediated tumorigenesis, without decreased SEC23B expression. However, our understanding of the processes behind these observations remain limited. Here, we show mutant SEC23B exists within nucleoli, in addition to classical distribution at the ER/Golgi. This occurs independent of other COPII proteins and does not compromise secretory function. Mutant cells have increased ribosomal protein and translation-related gene expression, and enhanced translational capacity, in the presence of ER stress. We show that mutant SEC23B binds to UBF transcription factor, with increased UBF transcription factor binding at the ribosomal DNA promoter. Our data indicate SEC23B has potential non-canonical COPII-independent function, particularly within the ribosome biogenesis pathway, and that may contribute to the pathogenesis of cancer-predisposition.

Preliminary report: Late seizure recurrence years after epilepsy surgery may be associated with alterations in brain tissue transcriptome

We recently proposed that the maturation of a new epileptic focus (epileptogenesis) may explain late seizure recurrences, starting months to years after resective epilepsy surgery. We explore here the hypothesis that inherent transcriptomic changes may distinguish such “late relapsers.” An in‐depth clinical review of 2 patients with recurrent seizures starting years after surgery is contrasted to 4 controls who remained seizure‐free postoperatively. This clinical analysis is combined with RNA sequencing from the resected brain tissue, followed by unsupervised hierarchical clustering, independent pathway analysis, and multidimensional scaling analysis. Late‐recurrence patients clustered apart from seizure‐free patients, with late recurrence patients clustering together in the central space, whereas the seizure‐free patients clustered together in the periphery. We utilized RNA‐seq to identify differentially expressed genes between late‐recurrence and seizure‐free samples. We found 29 annotated genes with statistically significant differential expression (q < 0.05). The top canonical pathways identified as distinctly separating the late‐recurrence patients from the seizure‐free patients included the intrinsic prothrombin activation pathway (p = 1.55E‐06), the complement system (p = 4.57E‐05), and the atherosclerosis signaling pathway (p = 4.57E‐05). Our observations suggest that late recurrences after epilepsy surgery may be influenced partly by differences in gene expression in neuroinflammatory and brain healing/remodeling pathways. Such a hypothesis needs to be validated in the future.

Lucking into science

Before I embarked on my Ph.D. journey in cancer genomics, I had never thought of a career as a scientist. I had dreamed of becoming a physician for as long as I could remember. But the Fulbright scholarship I won to pursue Ph.D. training in the United States was the chance of a lifetime to continue

Thyroglobulin in Metastatic Thyroid Cancer: Culprit or Red Herring?

To the Editor: Siraj et al. have presented an interesting paper on a highly relevant topic—understanding the biological relevance of diagnostic or prognostic markers for papillary thyroid cancer (PTC). The study included a large PTC series (>800) from Saudi Arabia, a geographic region known to be highly enriched with thyroid cancer. In addition to identifying genes in the RAS/MAPK signaling pathway (BRAF [MIM: 164757], KRAS [MIM: 190070], NRAS [MIM: 164790], and HRAS [MIM 190020]), which are well established as oncogenic drivers in PTC, the authors found other genes that were somatically mutated in their cohort. Of these genes, the authors focused on thyroglobulin (TG [MIM: 188450]), shown to be somatically mutated in ~3% of thyroid tumors. Statistical analysis of clinicopathological parameters revealed that TG somatic mutations were associated with poorer clinical outcome. Finally, the authors noted a higher enrichment of TG somatic mutations in metastatic PTC tissues and suggested clonal expansion from the primary tumors. Although these findings are compelling and translationally relevant, some fundamental aspects remain to be addressed. First, the authors’ finding that TG is recurrently mutated in PTC is not novel in and of itself. As they noted, the frequency of TG mutations in The Cancer Genome Atlas (TCGA) PTC study was 2.24%, whereas their study series showed 3.05%. Importantly, the TCGA series excluded individuals with aggressive disease, and yet TG somatic mutations would seem overrepresented in these tumors, as in the reported study (odds ratio 1⁄4 1.372; 95% confidence interval 1⁄4 0.653–3.105; p 1⁄4 0.4269). Second, TG secretion is often associated with advanced and differentiated thyroid carcinomas, including PTC. Interestingly, the authors reported that the majority (20/27 or 74.1%) of affected individuals harbored frameshift or splicing mutations, which would be expected to result in loss-of-function (LOF) effects. This is of paramount importance for clinical management post-operatively, and it would be valuable to evaluate thyroglobulin levels in these individuals with LOF TG mutations. Third, two thyroid tumors seemed to harbor two TGmutations, which is either a confirmation of its role in thyroid carcinogenesis or a consequence of random mutational events due to the large size of TG (genomic sequence 1⁄4 267,939 bp; coding sequence 1⁄4 8,453 bp). In order to exclude the latter, it is highly recommended to inspect the mutation frequency and spectrum of TG in unrelated