Restriction of the Global IgM Repertoire in Antiphospholipid Syndrome.

The typical anti-phospholipid antibodies (APLA) in the anti-phospholipid syndrome (APS) are reactive with the phospholipid-binding protein ß2GPI as well as a growing list of other protein targets. The relation of APLA to natural antibodies and the fuzzy set of autoantigens involved provoked us to study the changes in the IgM repertoire in APS. To this end, peptides selected by serum IgM from a 7-residue linear peptide phage display library (PDL) were deep sequenced. The analysis was aided by a novel formal representation of the Igome (the mimotope set reflecting the IgM specificities) in the form of a sequence graph. The study involved women with APLA and habitual abortions (n=24) compared to age-matched clinically healthy pregnant women (n=20). Their pooled Igomes (297 028 mimotope sequences) were compared also to the global public repertoire Igome of pooled donor plasma IgM (n=2 796 484) and a set of 7-mer sequences found in the J regions of human immunoglobulins (n=4 433 252). The pooled Igome was represented as a graph connecting the sequences as similar as the mimotopes of the same monoclonal antibody. The criterion was based on previously published data. In the resulting graph, identifiable clusters of vertices were considered related to the footprints of overlapping antibody cross-reactivities. A subgraph based on the clusters with a significant differential expression of APS patients' mimotopes contained predominantly specificities underrepresented in APS. The differentially expressed IgM footprints showed also an increased cross-reactivity with immunoglobulin J regions. The specificities underexpressed in APS had a higher correlation with public specificities than those overexpressed. The APS associated specificities were strongly related also to the human peptidome with 1 072 mimotope sequences found in 7 519 human proteins. These regions were characterized by low complexity. Thus, the IgM repertoire of the APS patients was found to be characterized by a significant reduction of certain public specificities found in the healthy controls with targets representing low complexity linear self-epitopes homologous to human antibody J regions.

Clinical impact of copy number variation changes in bladder cancer samples.

The aim of the present study was to detect copy number variations (CNVs) related to tumour progression and metastasis of urothelial carcinoma through whole-genome scanning. A total of 30 bladder cancer samples staged from pTa to pT4 were included in the study. DNA was extracted from freshly frozen tissue via standard phenol-chloroform extraction and CNV analysis was performed on two alternative platforms (CytoChip Oligo aCGH, 4x44K and Infinium OncoArray-500K BeadChip; Illumina, Inc.). Data were analysed with BlueFuse Multi software and Karyostudio, respectively. The results highlight the role of genomic imbalances in regions containing genes with metastatic and proliferative potential for tumour invasion. A high level of genomic instability in uroepithelial tumours was observed and a total of 524 aberrations, including 175 losses and 349 gains, were identified. The most prevalent genetic imbalances affected the following regions: 1p, 1q, 2q, 4p, 4q, 5p, 5q, 6p, 6q, 7q, 8q, 9p, 9q, 10p, 10q, 11q, 13q and 17q. High-grade tumours more frequently harboured genomic imbalances (n=227) than low-grade tumours (n=103). A total of 36 CNVs in high-grade bladder tumours were detected in chromosomes 1-5, 8-11, 14, 17, 19 and 20. Furthermore, five loss of heterozygosity variants containing 176 genes were observed in high-grade bladder cancer and may be used as potential targets for precision therapy. Revealing specific chromosomal regions related to the metastatic potential of uroepithelial tumours may lay a foundation for implementing molecular CNV profiling of bladder tumours as part of a routine progression risk estimation strategy, thus expanding the personalized therapeutic approach.

Centenarian Exomes as a Tool for Evaluating the Clinical Relevance of Germline Tumor Suppressor Mutations.

OBJECTIVES: The aim of the present study was to evaluate the clinical relevance of mutations in tumor suppressor genes using whole-exome sequencing data from centenarians and young healthy individuals. METHODS: Two pools, one of centenarians and one of young individuals, were constructed and whole-exome sequencing was performed. We examined the whole-exome sequencing data of Bulgarian individuals for carriership of tumor suppressor gene variants. RESULTS: Of all variants annotated in both pools, 5080 (0.06%) are variants in tumor suppressor genes but only 46 show significant difference in allele frequencies between the two studied groups. Four variants (0.004%) are pathogenic/risk factors according to single nucleotide polymorphism database: rs1566734 in PTPRJ, rs861539 in XRCC3, rs203462 in AKAP10, and rs486907 in RNASEL. DISCUSSION: Based on their high minor allele frequencies and presence in the centenarian group, we could reclassify them from pathogenic/risk factors to benign. Our study shows that centenarian exomes can be used for re-evaluating the clinically uncertain variants.

Long-term follow-up of a female patient with non-classical 11ß-hydroxylase deficiency and two novel mutations in CYP11B1.

11ß-Hydroxylase deficiency is the second most common enzyme disorder after 21-hydroxylase deficiency causing congenital adrenal hyperplasia (CAH11ß). In females, the clinical phenotype of CAH11ß classic forms is associated with ambiguous genitalia, virilization and hypertension, while most common complaints in milder non-classic forms include hirsutism, acne, menstrual disturbances, and infertility. Herein, we present clinical and genetic characteristics of an adult woman with 11ß-hydroxylase deficiency, hypertension and infertility; she has been followed up from her first pregnancy to her early menopause. Genetic analyses of the patient revealed a compound-heterozygosity due to two variants in the CYP11B1 gene p.Val316Met and p.Asp480ThrfsTer2. Both mutations have not been previously reported as pathogenic in the literature. Emerging questions concerning the clinical management, fertility potential, mineral corticoid abnormalities and perimenopausal transition in patients with non-classic CAH11ß have also been briefly discussed. The presented case of an adult woman with CAH11ß shows that the proper diagnosis and close monitoring of patients with different CAH forms might ensure good therapy adherence and successful fertility.

Novel Hypomorphic Mutation in FANCD2 Gene Observed in a Fetus with Multiple Congenital Anomalies.

Congenital anomalies affect 1% to 2% of the newborns. The urinary tract and the kidneys are involved in 4-5% of the cases while upper-extremities abnormalities are present in 10%. Certain anomalies occur in isolation, whereas others are associated with systemic conditions. The prenatal detection of fetal anomalies compatible with life is a challenge for both the parents and the physician. The prognosis for the fetus/newborn and the reproductive decisions of the family largely depend on the causes underlying the disease. The reported case is of a G2P1 pregnant woman referred for routine ultrasound scan at 24 weeks of gestation (w.g.). The fetus had growth retardation, right kidney agenesis, bilateral absence of radial bones and thumbs, radial deviation of the wrists, and short humeri. Nuchal fold thickness was 5 mm and there was a single umbilical artery. After termination of pregnancy, SNP array genotyping and next-generation sequencing of targeted candidate-genes were performed trying to clarify the etiology of the fetal polymalformative syndrome. A new hypomorphic mutation in FANCD2 gene was found to underlie this fetal anomaly. The case illustrates that patients/families affected by rare monogenic disorders may benefit from application of modern technologies like microarrays and NGS.

Next-generation sequencing of BRCA1 and BRCA2 in breast cancer patients and control subjects.

Breast cancer is currently the most common type of cancer in females. The majority of the hereditary forms of breast cancer are caused by mutations in the BRCA1 and BRCA2 genes, whose main function is the DNA repair of double-strand breaks. Genetic testing of females with a family history of breast cancer is recommended to determine their hereditary predisposition for this type of cancer. The variants with no clear clinical significance may represent a diagnostic challenge when performing targeted resequencing. In this study, DNA samples were obtained from 24 breast cancer patients (mean age, 35±10 years) with a positive family history and from 71 age-matched healthy controls. Informed consent was obtained from all the subjects. Sequence-targeted BRCA1 and BRCA2 libraries were prepared using the TruSeq Custom Amplicon method and sequenced on the Illumina MiSeq system. A wide range of variants were identified in the BRCA1 and BRCA2 genes. Two pathological/presumably pathological variants were detected in the breast cancer patient group: a mutation in BRCA2 at the chromosomal (chr) position chr13:32890665, which affected the first position of the 5' splice region following exon 2; and a mutation in BRCA1 at chr17:41219635, causing an in-frame triple nucleotide deletion of valine 1688 (8.3%). In the patient and control groups, 7 likely polymorphic variants and 13 common variants were detected in the BRCA1 and BRCA2 genes. To the best of our knowledge, this study was the first to identify 3 common polymorphisms in BRCA2, characteristic solely of the Bulgarian population, including chr13:32973737, T/-, a single-nucleotide polymorphism (SNP) within the 3'-UTR of exon 27; chr13:32973280, A/-, a mononucleotide deletion within the 5'-UTR of exon 27; and chr13:32973924, T/-, a mononucleotide deletion downstream of the gene sequence. To the best of our knowledge, this study was the first to apply next-generation sequencing of the BRCA1 and BRCA2 genes in a Bulgarian population, prompting further investigation for local founder mutations and variants characteristic for this particular region.