Case Report: Long-Term Survival of a Patient with Cerebral Metastasized Ovarian Carcinoma Treated with a Personalized Peptide Vaccine and Anti-PD-1 Therapy.

Ovarian cancer is one of the most common cancers among women and the most lethal malignancy of all gynecological cancers. Surgery is promising in the early stages; however, most patients are first diagnosed in the advanced stages, where treatment options are limited. Here, we present a 49-year-old patient who was first diagnosed with stage III ovarian cancer. After the tumor progressed several times under guideline therapies with no more treatment options available at that time, the patient received a fully individualized neoantigen-derived peptide vaccine in the setting of an individual healing attempt. The tumor was analyzed for somatic mutations via whole exome sequencing and potential neoepitopes were vaccinated over a period of 50 months. During vaccination, the patient additionally received anti-PD-1 therapy to prevent further disease progression. Vaccine-induced T-cell responses were detected using intracellular cytokine staining. After eleven days of in vitro expansion, four T-cell activation markers (namely IFN-É£, TNF-α, IL-2, and CD154) were measured. The proliferation capacity of neoantigen-specific T-cells was determined using a CFSE proliferation assay. Immune monitoring revealed a very strong CD4+ T-cell response against one of the vaccinated peptides. The vaccine-induced T-cells simultaneously expressed CD154, TNF, IL-2, and IFN-É£ and showed a strong proliferation capacity upon neoantigen stimulation. Next-generation sequencing, as well as immunohistochemical analysis, revealed a loss of Beta-2 microglobulin (B2M), which is essential for MHC class I presentation. The results presented here implicate that the application of neoantigen-derived peptide vaccines might be considered for those cancer stages, where promising therapeutic options are lacking. Furthermore, we provide more data that endorse the intensive investigation of B2M loss as a tumor escape mechanism in clinical trials using anti-cancer vaccines together with immune-checkpoint inhibitors.

McKusick-Kaufman Syndrome: A Case Report With an Emphasis on Perinatal Diagnosis and Genetic Counseling.

McKusick-Kaufman syndrome is a rare genetic disorder that affects limb development, genital formation, and heart function. It is caused by mutations in the MKKS gene on chromosome 20. Individuals with this condition may have extra fingers or toes, fused labia or undescended testes, and, less commonly, severe heart defects. Diagnosis involves a physical examination and genetic testing, while treatment focuses on symptom management, including surgical intervention if necessary. The prognosis varies depending on the severity of associated complications. In a recent case, a 27-year-old woman with fetal hydrometrocolpos gave birth to a female neonate with extra digits on both hands and feet, fused labia, and a small vaginal opening. The neonate also had a large abdominal cystic mass, and echocardiography revealed a patent foramen ovale. Genetic testing confirmed an MKKS gene mutation, and the hydrometrocolpos required surgical management. Early diagnosis and intervention can improve outcomes for individuals with this syndrome.

Biallelic Variants in Seven Different Genes Associated with Clinically Suspected Bardet-Biedl Syndrome.

Bardet-Biedl syndrome (BBS) is a rare clinically and genetically heterogeneous autosomal recessive multi-systemic disorder with 22 known genes. The primary clinical and diagnostic features include six different hallmarks, such as rod-cone dystrophy, learning difficulties, renal abnormalities, male hypogonadism, post-axial polydactyly, and obesity. Here, we report nine consanguineous families and a non-consanguineous family with several affected individuals presenting typical clinical features of BBS. In the present study, 10 BBS Pakistani families were subjected to whole exome sequencing (WES), which revealed novel/recurrent gene variants, including a homozygous nonsense mutation (c.94C>T; p.Gln32Ter) in the IFT27 (NM_006860.5) gene in family A, a homozygous nonsense mutation (c.160A>T; p.Lys54Ter) in the BBIP1 (NM_001195306.1) gene in family B, a homozygous nonsense variant (c.720C>A; p.Cys240Ter) in the WDPCP (NM_015910.7) in family C, a homozygous nonsense variant (c.505A>T; p.Lys169Ter) in the LZTFL1 (NM_020347.4) in family D, pathogenic homozygous 1 bp deletion (c.775delA; p.Thr259Leufs*21) in the MKKS/BBS5 (NM_170784.3) gene in family E, a pathogenic homozygous missense variant (c.1339G>A; p.Ala447Thr) in BBS1 (NM_024649.4) in families F and G, a pathogenic homozygous donor splice site variant (c.951+1G>A; p?) in BBS1 (NM_024649.4) in family H, a pathogenic bi-allelic nonsense variant in MKKS (NM_170784.3) (c.119C>G; p.Ser40*) in family I, and homozygous pathogenic frameshift variants (c.196delA; p.Arg66Glufs*12) in BBS5 (NM_152384.3) in family J. Our findings extend the mutation and phenotypic spectrum of four different types of ciliopathies causing BBS and also support the importance of these genes in the development of multi-systemic human genetic disorders.

Crystal structure of the phosphorylated Arabidopsis MKK5 reveals activation mechanism of MAPK kinases.

The mitogen-activated protein kinase (MAPK) signaling pathways are highly conserved in eukaryotes, regulating various cellular processes. The MAPK kinases (MKKs) are dual specificity kinases, serving as convergence and divergence points of the tripartite MAPK cascades. Here, we investigate the biochemical characteristics and three-dimensional structure of MKK5 in Arabidopsis (AtMKK5). The recombinant full-length AtMKK5 is phosphorylated and can activate its physiological substrate AtMPK6. There is a conserved kinase interacting motif (KIM) at the N-terminus of AtMKK5, indispensable for specific recognition of AtMPK6. The kinase domain of AtMKK5 adopts active conformation, of which the extended activation segment is stabilized by the phosphorylated Ser221 and Thr215 residues. In line with sequence divergence from other MKKs, the αD and αK helices are missing in AtMKK5, suggesting that the AtMKK5 may adopt distinct modes of upstream kinase/substrate binding. Our data shed lights on the molecular mechanisms of MKK activation and substrate recognition, which may help design specific inhibitors targeting human and plant MKKs.

Bardet-Biedl syndrome: The pleiotropic role of the chaperonin-like BBS6, 10, and 12 proteins.

Bardet-Biedl syndrome (BBS) is a rare pleiotropic disorder known as a ciliopathy. Despite significant genetic heterogeneity, BBS1 and BBS10 are responsible for major diagnosis in western countries. It is well established that eight BBS proteins, namely BBS1, 2, 4, 5, 7, 8, 9, and 18, form the BBSome, a multiprotein complex serving as a regulator of ciliary membrane protein composition. Less information is available for BBS6, BBS10, and BBS12, three proteins showing sequence homology with the CCT/TRiC family of group II chaperonins. Even though their chaperonin function is debated, scientific evidence demonstrated that they are required for initial BBSome assembly in vitro. Recent studies suggest that genotype may partially predict clinical outcomes. Indeed, patients carrying truncating mutations in any gene show the most severe phenotype; moreover, mutations in chaperonin-like BBS proteins correlated with severe kidney impairment. This study is a critical review of the literature on genetics, expression level, cellular localization and function of BBS proteins, focusing primarily on the chaperonin-like BBS proteins, and aiming to provide some clues to understand the pathomechanisms of disease in this setting.

Apparent but unconfirmed digenism in an Iranian consanguineous family with syndromic Retinal Disease.

BACKGROUND: Bardet-Biedl syndrome is an autosomal recessive disease characterized by rod-cone dystrophy, postaxial polydactyly, kidney defects, obesity, mental retardation and hypogonadism. Here, we report different genotypes in two Bardet-Biedl syndrome affected sisters with a different clinical phenotype regarding severity. MATERIALS AND METHODS: The proband of the family was examined by Next Generation Sequencing (NGS) using clinical exome and filtering by syndromic and non-syndromic genes associated with retinal dystrophies. RESULTS: Targeted NGS revealed two novel variants in the MKKS and CEP290 genes in homozygosis state in the proband. Segregation analysis revealed the presence of the same MKKS homozygous variant in her younger affected sister but not the CEP290 variant. Both sisters presented different clinical manifestation, at different ages, with a more severe renal and retinal defect in the case of the sister carrying mutations in both genes. Another unaffected sister showed only homozygosity for the CEP290 variant, thus supporting the non-pathogenic role of this mutation in BBS phenotype. CONCLUSIONS: In this study, NGS proved to be a powerful and efficient sequencing method to identify causal variants in different genes. However, it remarks the importance of the segregation analysis and clinical information to establish the pathogenicity of new variants. The two affected sisters present different genotypes and clinical manifestation, suggesting that the novel CEP290 variant could be acting as a modifier, making the phenotype more severe in the sister homozygote for MKKS and CEP290 genes. On the other hand, the difference in the age of both sisters highlight the important role of monitoring disease progression also to confirm the modifier role of genetic variants.

Genome-Wide Identification and Analysis of MKK and MAPK Gene Families in Brassica Species and Response to Stress in Brassica napus.

Mitogen-activated protein kinase (MAPK) cascades are common and conserved signal transduction pathways and play important roles in various biotic and abiotic stress responses and growth and developmental processes in plants. With the advancement of sequencing technology, more systematic genetic information is being explored. The work presented here focuses on two protein families in Brassica species: MAPK kinases (MKKs) and their phosphorylation substrates MAPKs. Forty-seven MKKs and ninety-two MAPKs were identified and extensively analyzed from two tetraploid (B. juncea and B. napus) and three diploid (B. nigra, B. oleracea, and B. rapa) Brassica species. Phylogenetic relationships clearly distinguished both MKK and MAPK families into four groups, labeled A-D, which were also supported by gene structure and conserved protein motif analysis. Furthermore, their spatial and temporal expression patterns and response to stresses (cold, drought, heat, and shading) were analyzed, indicating that BnaMKK and BnaMAPK transcript levels were generally modulated by growth, development, and stress signals. In addition, several protein interaction pairs between BnaMKKs and C group BnaMAPKs were detected by yeast two-hybrid assays, in which BnaMKK3 and BnaMKK9 showed strong interactions with BnaMAPK1/2/7, suggesting that interaction between BnaMKKs and C group BnaMAPKs play key roles in the crosstalk between growth and development processes and abiotic stresses. Taken together, our data provide a deeper foundation for the evolutionary and functional characterization of MKK and MAPK gene families in Brassica species, paving the way for unraveling the biological roles of these important signaling molecules in plants.

Legg-Calvé-Perthes disease in a patient with Bardet-Biedl syndrome: A case report of a novel MKKS/BBS6 mutation.

This article reports a girl with Bardet-Biedl syndrome (BBS) having a novel causative mutation who developed Legg-Calvé-Perthes disease (LCPD). There exists a possibility that the prognosis of LCPD had been adversely affected by the concomitant BBS.

Novel mutation in MKKS/BBS6 linked with arRP and polydactyly in a family of North Indian origin.

BACKGROUND: To identify the underlying genetic defect in a fourth-generation autosomal recessive retinitis pigmentosa (arRP) family. Detailed family history and clinical data were collected from nine members, including three affected, from an arRP family. METHODS: Whole-exome sequencing (WES) was performed on DNA sample of an affected individual IV: 2. Variants obtained by WES were annotated using Ion Reporter Software (ver. 5.2). Potential pathogenic variants detected in an affected member were validated in other affected and unaffected family members by Sanger sequencing. Further 150 ethnically-matched controls were tested for the variant that co-segregated completely with disease in the family, so as to exclude it as a polymorphism. Various web-based bioinformatics tools were also applied to access pathogenic potential of the observed variant. RESULTS: All the three patients had RP with polydactyly of both hands and feet, however, they did not show other symptoms of Bardet-Biedl syndrome (BBS) or McKusick-Kaufmann Syndrome (MKKS). A novel missense mutation, that is, c.518A>C (p.His173Pro) was identified in MKKS/BBS6 that co-segregated completely with the disease phenotype in all the three affected members and was not observed in six unaffected members of the family. Also the c.518A>C change was not observed in 150 ethnically matched controls (300 chromosomes), hence excluding it as a polymorphism. CONCLUSIONS: Present study is the second report of identifying a novel mutation in MKKS/BBS6 that is linked with arRP in association with polydactyly, however, with no other signs of BBS or MKKS. These findings further expand the mutation spectrum of MKKS/BBS6 for arRP with polydactyly.

Bardet-Biedl Syndrome as a Chaperonopathy: Dissecting the Major Role of Chaperonin-Like BBS Proteins (BBS6-BBS10-BBS12).

Bardet-Biedl syndrome (BBS) is a rare genetic disorder that belongs to the group of ciliopathies, defined as diseases caused by defects in cilia structure and/or function. The six diagnostic features considered for this syndrome include retinal dystrophy, obesity, polydactyly, cognitive impairment and renal and urogenital anomalies. Furthermore, three of the 21 genes currently known to be involved in BBS encode chaperonin-like proteins (MKKS/BBS6, BBS10, and BBS12), so BBS can be also considered a member of the growing group of chaperonopathies. Remarkably, up to 50% of clinically-diagnosed BBS families can harbor disease-causing variants in these three genes, which highlights the importance of chaperone defects as pathogenic factors even for genetically heterogeneous syndromes such as BBS. In addition, it is interesting to note that BBS families with deleterious variants in MKKS/BBS6, BBS10 or BBS12 genes generally display more severe phenotypes than families with changes in other BBS genes. The chaperonin-like BBS proteins have structural homology to the CCT family of group II chaperonins, although they are believed to conserve neither the ATP-dependent folding activity of canonical CCT chaperonins nor the ability to form CCT-like oligomeric complexes. Thus, they play an important role in the initial steps of assembly of the BBSome, which is a multiprotein complex essential for mediating the ciliary trafficking activity. In this review, we present a comprehensive review of those genetic, functional and evolutionary aspects concerning chaperonin-like BBS proteins, trying to provide a new perspective that expands the classical conception of BBS only from a ciliary point of view.

Whole-exome sequencing identified compound heterozygous variants in MMKS in a Chinese pedigree with Bardet-Biedl syndrome.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized by retinal dystrophy, polydactyly, obesity, developmental delay, and renal defects. At least 21 candidate BBS-associated genes (BBS1-19, NPHP1, and IFT172) have previously been identified, and all of them play important roles in ciliary function. Here, we collected a BBS pedigree with four members and performed whole-exome sequencing on the proband. The variants were analyzed and evaluated to confirm their pathogenicity. We found compound heterozygous variants (c.1192C>T, p.Q398* and c.1175C>T, p.T392M) in MKKS in both the siblings, and these were likely to be pathogenic variants. We also found a missense variant (c.2029G>C, p.E677Q) in NPHP1 and a missense variant (c.2470C>T, p.R824C) in BBS9 in the proband only, which are variants of uncertain significance. The compound heterozygous variants were probably responsible for the BBS phenotype in this Chinese pedigree and the missense mutations in NPHP1 and BBS9 might contribute to the mutation load.

Rare renal ciliopathies in non-consanguineous families that were identified by targeted resequencing.

BACKGROUND: Nephronophthisis-related ciliopathies (NPHP-RC) are a frequent cause of renal failure for children and adolescents. Although diagnosing these diseases clinically is difficult, a comprehensive genetic screening approach of targeted resequencing can uncover the genetic background in this complicated family of diseases. METHODS: We studied three Japanese female patients with renal insufficiency from non-consanguineous parents. A renal biopsy for clinical reasons was not performed. Therefore, we did not know the diagnosis of these patients from a clinical aspect. We performed comprehensive genetic analysis using the TruSight One Sequencing Panel next generation sequencing technique. RESULTS: We identified three different rare NPHP-RC variants in the following genes: SDCCAG8, MKKS, and WDR35. Patient 1 with SDCCAG8 homozygous deletions showed no ciliopathy-specific extrarenal manifestations, such as retinitis pigmentosa or polydactyly prior to genetic analysis. Patient 2 with a MKKS splice site homozygous mutation and a subsequent 39-amino acid deletion in the substrate-binding apical domain, had clinical symptoms of Bardet-Biedl syndrome. She and her deceased elder brother had severe renal insufficiency soon after birth. Patient 3 with a compound heterozygous WDR35 mutation had ocular coloboma and intellectual disability. CONCLUSIONS: Our results suggest that a comprehensive genetic screening system using target resequencing is useful and non-invasive for the diagnosis of patients with an unknown cause of pediatric end-stage renal disease.

Deciphering intrafamilial phenotypic variability by exome sequencing in a Bardet-Biedl family.

Bardet-Biedl syndrome (BBS) is a model ciliopathy characterized by a wide range of clinical variability. The heterogeneity of this condition is reflected in the number of underlying gene defects and the epistatic interactions between the proteins encoded. BBS is generally inherited in an autosomal recessive trait. However, in some families, mutations across different loci interact to modulate the expressivity of the phenotype. In order to investigate the magnitude of epistasis in one BBS family with remarkable intrafamilial phenotypic variability, we designed an exome sequencing-based approach using SOLID 5500xl platform. This strategy allowed the reliable detection of the primary causal mutations in our family consisting of two novel compound heterozygous mutations in McKusick-Kaufman syndrome (MKKS) gene (p.D90G and p.V396F). Additionally, exome sequencing enabled the detection of one novel heterozygous NPHP4 variant which is predicted to activate a cryptic acceptor splice site and is only present in the most severely affected patient. Here, we provide an exome sequencing analysis of a BBS family and show the potential utility of this tool, in combination with network analysis, to detect disease-causing mutations and second-site modifiers. Our data demonstrate how next-generation sequencing (NGS) can facilitate the dissection of epistatic phenomena, and shed light on the genetic basis of phenotypic variability.

Gene expression in the vascular wall of the aortic arch in spontaneously hypertensive hyperlipidemic model rats using DNA microarray analysis.

AIMS: In recent years, there has been an increase in patients with arteriosclerosis and the risk of lifestyle-related diseases. However, the pathogenesis and medication of atherosclerosis have not been elucidated. We developed a rat model of lifestyle-related diseases by feeding a high-fat diet and 30% sucrose solution (HFDS) to spontaneously hypertensive hyperlipidemic rats (SHHR) and reported that this model is a useful model of early atherosclerosis. In order to elucidate the pathogenesis of early atherosclerosis, we searched for atherosclerosis-related genes by microarray analysis using the aortic arch rat model of lifestyle-related diseases. MAIN METHODS: Four-month-old male Sprague-Dawley rats and SHHR were each divided into two normal diet (ND) groups and two HFDS groups. After a four-month treatment, the expression of mRNA in the aortic arch was detected using the oligo DNA microarray one-color method and quantified using real-time PCR. KEY FINDINGS: In this study, we detected 39 genes in microarray analysis. Esm1, Retnlb Mkks, and Grem2 showed particularly marked changes in gene expression in the SHHR-HFDS group. Compared with the SD-ND group, the SHHR-HFDS group had an increase in Mkks gene expression of about 26-fold and an approximately 22-fold increase in the expression of Grem2. Similarly, the expression of Esm1 increased by about 12-fold and that of Retnlg by about 10-fold as shown by quantitative real-time PCR. SIGNIFICANCE: This study suggested that these four genes might be important in early atherosclerosis development.