Search

We aimed to evaluate the clinical and laboratory features and MEFV allele distribution in Crimean Tatar familial Mediterranean fever patients and to compare them with Turkish familial Mediterranean fever patients and healthy controls.All newly diagnosed familial Mediterranean fever patients with Crimean Tatar nationality (n = 18) in Children's Regional Hospital in Simferopol were enrolled in the study and were compared to 40 familial Mediterranean fever cases followed up at Hacettepe University, Ankara, Turkey. The distribution of MEFV alleles was assessed in the 127 unrelated healthy Crimean Tatar adults aged 20 years or more from different parts of the Crimea peninsula.Age and gender distribution, the frequency of colchicine resistance, and colchicine intolerance were similar between Turkish and Crimean Tatar children with familial Mediterranean fever. The duration of familial Mediterranean fever attack was shorter in Turkish patients than in Crimean Tatar (2.0 vs. 3.0 days, P.001). Chest pain was more frequent in Turkish familial Mediterranean fever patients, whereas arthralgia, arthritis, and erysipeloid rash were more common in Crimean TatarT. MEFV allele distribution in Crimean Tatar was M694V-81%, M680I and V726A 9.5% both, and 68.6%, 14.3%, and 12.9% in Turkish, consequently. Homozygous carriers were 11%, compound-heterozygous was 6%, and heterozygous was 83%, compared to Turkish being 45%, 30%, and 25%, respectively. The allele distribution in healthy Crimean Tatar and Turkish was 10.2% and M694V was 7.1%, M680I was 1.6%, and V726A was 1.6%.The similar MEFV allele prevalence in both populations suggests the high prevalence of familial Mediterranean fever and the high number of undiagnosed patients in the Crimea peninsula. Younger age at onset, shorter duration of attacks, the prevalence of articular involvement, and erysipeloid rash were distinctive features of familial Mediterranean fever in Crimean Tatar.

Background Many cancer patients undergo sophisticated laboratory testing, which requires proper interpretation and interaction between different specialists. Case presentation We describe a patient with an extensive family history of cancer, who was diagnosed with bilateral breast cancer and two lung cancer lumps by the age of 40 years. She submitted a lung cancer specimen to a genetic profiling service, which reported the presence of the EGFR mutation (a combination of G719S and L833V substitutions) and the TP53 с.322_327del (p.G108_F109del) mutation in the tumor tissue. Possible therapeutic options were discussed at a medical conference, where one of the discussants raised a concern that the identified TP53 mutation may not necessarily be somatic, but reflect the germ-line status of the gene. Review of clinical records and follow-up dialog with the patient revealed, that she previously provided her blood for DNA analysis in two laboratories. The first laboratory utilized a custom NGS assay and did not detect the TP53 mutation, instead pointed to a potential pathogenic significance of the MSH6 c.2633 T > C (p.V878A) allele. The second laboratory revealed the TP53 с.322_327del (p.G108_F109del) allele but stated in the written report that it has an unknown pathogenic significance. To resolve the possible uncertainty regarding the role of the TP53 с.322_327del (p.G108_F109del) variant, we suggested that the patient invite her second cousin for genetic testing, as she was affected by neuroblastoma at the age of 3 years. This analysis revealed the presence of the same TP53 variant. Conclusion We provide point-by-point discussion, reviewing multiple laboratory mistakes and clinical misinterpretations occurred with this patient. This case report exemplifies the need to involve rigorous clinical expertise in the daily practice of medical laboratory facilities.

Germline variants in known breast cancer (BC) predisposing genes explain less than half of hereditary BC cases. This study aimed to identify missing genetic determinants of BC. Whole exome sequencing (WES) of lymphocyte DNA was performed for 49 Russian patients with clinical signs of genetic BC predisposition, who lacked Slavic founder mutations in BRCA1, BRCA2, CHEK2, and NBS1 genes. Bioinformatic analysis of WES data was allowed to compile a list of 229 candidate mutations. 79 of these mutations were subjected to a three-stage case–control analysis. The initial two stages, which involved up to 797 high-risk BC patients, 1504 consecutive BC cases, and 1081 healthy women, indicated a potentially BC-predisposing role for 6 candidates, i.e., USP39 c.*208G > C, PZP p.Arg680Ter, LEPREL1 p.Pro636Ser, SLIT3 p.Arg154Cys, CREB3 p.Lys157Glu, and ING1 p.Pro319Leu. USP39 c.*208G > C was strongly associated with triple-negative breast tumors (p = 0.0001). In the third replication stage, we genotyped the truncating variant of PZP (rs145240281) and the potential splice variant of USP39 (rs112653307) in three independent cohorts of Russian, Byelorussian, and German ancestry, comprising a total of 3216 cases and 2525 controls. The data obtained for USP39 rs112653307 supported the association identified in the initial stages (the combined OR 1.72, p = 0.035). This study suggests the role of a rare splicing variant in BC susceptibility. USP39 encodes an ubiquitin-specific peptidase that regulates cancer-relevant tumor suppressors including CHEK2. Further epidemiological and functional studies involving these gene variants are warranted.

Primary immune deficiencies are usually attributed to genetic defects and, therefore, frequently referred to as inborn errors of immunity (IEI). We subjected the genomic DNA of 333 patients with clinical signs of IEI to next generation sequencing (NGS) analysis of 344 immunity-related genes and, in some instances, additional genetic techniques. Genetic causes of the disease were identified in 69/333 (21%) of subjects, including 11/18 (61%) of children with syndrome-associated IEIs, 45/202 (22%) of nonsyndromic patients with Jeffrey Modell Foundation (JMF) warning signs, 9/56 (16%) of subjects with periodic fever, 3/30 (10%) of cases of autoimmune cytopenia, 1/21 (5%) of patients with unusually severe infections and 0/6 (0%) of individuals with isolated elevation of IgE level. There were unusual clinical observations: twins with severe immunodeficiency carried a de novo CHARGE syndrome-associated SEMA3E c.2108C>T (p.S703L) allele; however, they lacked clinical features of CHARGE syndrome. Additionally, there were genetically proven instances of Netherton syndrome, Х-linked agammaglobulinemia, severe combined immune deficiency (SCID), IPEX and APECED syndromes, among others. Some patients carried recurrent pathogenic alleles, such as AIRE c.769C>T (p.R257*), NBN c.657del5, DCLRE1C c.103C>G (p.H35D), NLRP12 c.1054C>T (p.R352C) and c.910C>T (p.H304Y). NGS is a powerful tool for high-throughput examination of patients with malfunction of immunity.

Whole exome sequencing (WES) is a powerful tool for the cataloguing of population-specific genetic diseases. Within this proof-of-concept study we evaluated whether analysis of a small number of individual exomes is capable of identifying recurrent pathogenic alleles. We considered 106 exomes of subjects of Russian origin and revealed 13 genetic variants, which occurred more than twice and fulfilled the criteria for pathogenicity. All these alleles turned out to be indeed recurrent, as revealed by the analysis of 1045 healthy Russian donors. Eight of these variants (NAGA c.973GA, ACADM c.985AC, MPO c.2031-2AC, SLC3A1 c.1400TC, LRP2 c.6160GA, BCHE c.293AG, MPO c.752TC, FCN3 c.349delC) are non-Russian-specific, as their high prevalence was previously demonstrated in other European populations. The remaining five disease-associated alleles appear to be characteristic for subjects of Russian origin and include CLCN1 c.2680CT (myotonia congenita), DHCR7 c.453GA (Smith-Lemli-Opitz syndrome), NUP93 c.1162CT (steroid-resistant nephrotic syndrome, type 12), SLC26A2 c.1957TA (multiple epiphyseal dysplasia) and EIF3F c.694TG (mental retardation). These recessive disease conditions may be of particular relevance for the Russian Federation and other countries with a significant Slavic population.

NLRP12-related autoinflammatory disease (NLRP12-AID) is an exceptionally rare autosomal dominant disorder caused by germline mutations in NLRP12 gene. Very few patients with NLRP12-AD have been identified worldwide; therefore, there is a scarcity of data on phenotypic presentation of this syndrome. Here we provide evidence that NLRP12-AID may have clinical manifestations characteristic for primary immune deficiencies (PID). 246 children with periodic fever (PF) of unknown origin were subjects to the next generation sequencing (NGS) analysis; 213 of these patients had signs of primary immunodeficiency (PID) manifested by recurrent infections, while 33 kids had isolated PF. The NGS panel was composed of 302 genes implicated in PID and/or AID. 15 patients (9 girls and 6 boys) with NLRP12-AID were identified. Median age of first AID-related fever episode was 12 months, ranging from 2 months to 13 years. Main clinical features of NLRP12-related AID were periodic fever (100%), abdominal pain and diarrhea (47%), arthralgia (20%), headache (20%) and failure to thrive (33%). Nine patients demonstrated increased susceptibility to infection and two children suffered from Crohn's disease. Administration of short courses of NSAID or corticosteroids resulted in resolution of the disease flare. In one severe case, canakinumab (anti-interleukin-1β antibody) was successfully used. Significant number of patients with genetically assigned diagnosis of NLPR12-AID has clinical features which close resemble primary immune deficiency. This phenotypic overlap may result in underdiagnosis of NLPR12-AID among patients with PID.

Bloom syndrome (BS) is an exceptionally rare hereditary disease. Typical manifestations of BS usually include growth deficiency, a characteristic facial appearance, skin hypersensitivity to ultraviolet irradiation, and a strong predisposition to early-onset cancers. We have previously described a recurrent BLM c.1642C>T (p.Q548X) mutation, which is present in heterozygous state in 0.2-0.6% of individuals of Slavic origin. Despite the high occurrence of this founder allele, BS has not yet been described in patients of Slavic ethnicity. Here, we present 2 cases of BS, which were missed by standard genetic counseling and were eventually identified entirely due to chance. Our patients show the need for further investigations to confirm whether the atypical appearance of BS is indeed characteristic for biallelic carriers of the c.1642C>T (p.Q548X) allele and whether the absence of skin manifestations contributes to the underdiagnosis of the disease in Russia. Therefore, we suggest that all Slavic patients with only one single clinical feature of BS are to be screened for this allele and subjected to further analysis wherever appropriate. In addition to identifying new BS patients, this effort will help to clarify the frequency of “atypical BS” with incomplete phenotypic manifestations.

Background Inflammatory myofibroblastic tumors (IMTs) are exceptionally rare neoplasms, which are often driven by rearranged tyrosine kinases. Methods This study considered 33 consecutive patients with IMT (median age, 6.6; age range, 0.6-15.8 years). RNA and cDNA were successfully obtained in 29 cases. The molecular analysis included sequential tests for 5'/3'-end unbalanced gene expression, variant-specific PCR, and next-generation sequencing (NGS). Results 5'/3'-end unbalanced ALK expression was revealed in 15/29 (52%) IMTs. Strikingly, all these tumors demonstrated high amount of ALK protein detected by immunohistochemistry. Variant-specific PCR was capable of identifying the type of ALK rearrangement in 11/15 IMTs with 5'/3'-end unbalanced ALK expression. The remaining four tumors were analyzed by NGS; two known and two novel (CLTC-ins6del84-ALK and EEF1G-ALK) ALK rearrangements were detected. Five IMTs demonstrated 5'/3'-end unbalanced ROS1 expression, and all these tumors carried TFG-ROS1 fusion. Nine tumors, which were negative for 5'/3'-end unbalanced ALK/ROS1 expression, were subjected to further analysis. Variant-specific PCR revealed two additional tumors with gene rearrangements (TFG-ROS1 and ETV6-NTRK3). The remaining seven IMTs were tested by NGS; single instances of TFG-ROS1 and novel SRF-PDGFRb translocations were detected. Conclusions Twenty-four of 29 IMTs (83%) were shown to have druggable rearrangements involving tyrosine kinases, 20 of these 24 gene fusions were detectable by simple and inexpensive PCR assay, which is based on the detection 5'/3'-end unbalanced gene expression.

Background: Chronic non-bacterial osteomyelitis (CNO) is Data about incidence, prevalence and clinical and genetic features of chronic non-bacterial osteomyelitis (CNO) in Russia is scarce. Objectives: The aim of our study was to evaluate clinical and genetic peculiarities of CNO in Russia. Methods: The diagnosis of CNO was made with criteria, proposed by Jansson [1, 2], after the exclusion of other causes of bone disease. Our cohort consists of three main subtypes: i) early-onset ( Results: We selected a subgroup of the CNO patients having the following features: 1) early disease onset ( In the subgroup patients with early onset we performed genetic tests. Rare variants of PID genes were detected in 7/22 (32%) patients. Mutations affecting the genes previously associated with CNO were found only in two patients: one of them carried heterozygous variant IL1RN c.170G>T (p.C57F) and another had IL1RN c.512T>C (p.V171A). No mutation of LPIN2 was revealed. Other detected variants included one pathogenic MEFV p.M694V mutation in heterozygous state and a number of VUS in CD40LG, NLRP12, CR2, NLRP3, IL12B, PLCG2, SH3BP2, CARD14, IRF8, CASP10 and NFKB1A genes. Conclusion: We have found the unique regional subtype of CNO with early-onset in North Caucasus region with at least 10 times higher prevalence. We have not yet seen similar patients in other nationalities of Russia. Mutations in known genes were detected only in a minor fraction of CNO patients from Dagestan and Chechnya. This work supported by the Russian Foundation for Basic Research (grant No 18-515-57001) and by Japan Medical Research Foundation (grant No 18jmrf001). References [1] Jansson AF, Muller TH, Gliera L, Ankerst DP, Wintergerst U, Belohradsky BH, Jansson V. Clinical score for nonbacterial osteitis in children and adults. Arthritis Rheum. 2009;60(4):1152-9. [2] Jansson AF, Grote V; ESPED Study Group. Nonbacterial osteitis in children: data of a German Incidence Surveillance Study. Acta Paediatr. 2011;100(8):1150-7. Disclosure of Interests: None declared

Hereditary non-polyposis colorectal cancer (HNPCC), also known as Lynch syndrome (LS), is a common cancer-predisposing syndrome. This study aimed to investigate the spectrum of germ-line mutations in Russian LS patients. LS-related mismatch repair (MMR) genes were analyzed in 16 patients, who were forwarded to genetic testing due to strong clinical features of LS and had high-level microsatellite instability (MSI-H) in the tumor (n = 14) or unknown MSI status (n = 2). In addition, 672 consecutive colorectal cancer (CRC) cases were screened for family history; 15 patients were younger than 50 years and reported 2 or more instances of LS-related cancers in 1st- or 2nd-degree relatives. Seven of these cases demonstrated MSI-H and therefore were subjected to DNA germ-line testing. Overall, 17/23 (74%) subjects carried LS-associated gene variants (MLH1: 10; MSH2: 4; MSH6: 2; PMS2: 1), with 2 alleles (MLH1 c.677G > T and MSH2 с.1906G > C) detected twice. Testing for recurrent mutations of 30 consecutive MSI-H CRCs led to the identification of 2 additional subjects with LS. The analysis of all relevant publications identified 28 unrelated LS patients presented in Russian medical literature and 3 unrelated Russian LS subjects described in international journals. Overall, 15/49 (31%) genetic defects revealed in Russian LS patients were represented by six recurrent alleles (MLH1: c.350C > T, c.677G > T, c.1852_1854del; MSH2: c.942+3A > T, c.1861C > T, с.1906G > C). We conclude that the founder effect for LS in Russia is seemingly less pronounced than the one for hereditary breast-ovarian cancer syndrome, however testing for recurrent LS mutations may be considered feasible in some circumstances.

Background:While efficacy of tofacitinib (TOF) has been proven in many adult immune-mediated conditions, the information on its’ safety and efficacy in the pediatric population is limited.Objectives:to evaluate the safety and efficacy of TOF in children with immune-mediated diseases.Methods:from 23 children whom TOF has been initiated, 17 children with treatment duration of > 6 months were extracted including 16 girls and 1 boy with the following diagnosis: JIA (n=10), autoinflammatory diseases (AID) (n=5) and juvenile dermatomyositis (JDM) (n=2) due to impossibility to taper corticosteroids (CS) or previous biologic treatment failed. The treatment outcome was classified according to the opinion of the attending physicians as complete response (CR) i.e., the absence of disease activity, partial response (PR) – a significant improvement of symptoms and disease activity or no response (NR) - no changes in disease activity.Results:Mean duration of TOF treatment was 25.4±18.9 months. TOF was used as monotherapy in 3 cases, in combination with methotrexate (MTX) in 6, and in combination with other biologics in 3 children: tocilizumab (n=2) and canakinumab (n=1). Nine patients received CS. (Table 1). In two JIA patients with alopecia TOF induced intensive hair growth and controlled joint inflammation. 9 patients had CR: AID (n=3), JIA (n=4) and JDM(n=1): 7 patients had PR and 1 was NR. 13 patients had a previous history of several subsequent failed biologic: 4 biologics (n=1), 3 biologics (n=6), 2 biologics (n=1), 1 biologic (n=5). TOF treatment allowed discontinuation of CS in patient#6 and reducing the CS in 8/10 patients from 0.4 ±0.27 mg/kg to 0.15±0.1 mg/kg in 3.7±3.4 times: in 2 cases the tapering of steroids failed (Figure 1). 4 patients had side effects not requiring treatment discontinuation: liver enzymes elevation (n=2), hypercholesterolemia (n=1), lymphadenitis (n=1). In pt#6 after achievement of the remission the TOF dosage was decreased up to 2 times and tocilizumab intervals were increased up to 6 weeks.Table 1.#DiagnosisIndicationPrevious biologicsCurrent treat-mentTOF, dose, mg/kgDurationof TOF treatment, monthsGeneticvariantsdetectedEfficacy1AIDSevere inflamma-tion, aortitis, colitisINX, TCZ, ADACS, TOF0.57NOD2 c.2578G>A (p.A860T); NOD2 c.2722G>C (p.G908R)ADA2 c.927G>A (p.M309I)PR2JDMrecurrent skin rashCS, TOF, MTX0.55PR3JDMskin involvement, ulcerationCS, MTX, TOF0.77NLRP12c.154G>A (p.G52S)CR4AID,IFPskin rash, recurrent inflammation, failure to thriveCAN, TCZCS, TOF, CAN0.532RNASEH2Bc.916dupA (p.I309Nfs*7)CR5JIA,polysevere arthritisETA,TCZ, ADATOF,TCZ0.2722PR6soJIAresistant to CS and biologic systemic inflammation, arthritisTCZ, ABC, CANTOF, TCZ0.423PR7JIA,poly (RF+) alopeciaSevere arthritis, lung involvement,alopeciaETA, ABC, TCZ, ADATOF, MTX, CS0.337IL1RN c.10G>C (p.A4P); NLRP3 c.2113C>A (p.Q705L); MEFV c.1105C>T (p.P369S)CR8IFP, CANDLE-likerecurrent inflammation, digital ischemia, ulcers, CS-dependencyETA, RTX, CANTOF, CS0.2543MDA5NLRP3CR9AID, IFPsystemic inflammation, ulcersETATOF, CS0.513СR10JIA, ERAarthritisETATOF, MTX0.244CR11JIA, polyarthritisABC, ETATOF, MTX0.2538CR12JIA, polyArthritis, alopeciaETATOF0.1531PR13soJIA + MASsystemic inflammation, arthritisTCZ, CAN, ETATOF, CS0.538NR14JIA, polyarthritisINX, ETA, ADA, TCZTOF, MTX0.224PR15AIDSystemic inflammation, CS-dependencyTCZTOF,CS0.521STAT3, c1343A>CPR16JIA, ERAarthritisETATOF0.2521PR17JIA, polyarthritisADA, TCZ, ETATOF0.1539СRFigure 1.Conclusion:Tofacitinib is a promising agent in treating pediatric rheumatic diseases. In our study the best results were in AID patients with rare alleles in interferon pathway genes, patients with arthritis and alopecia and in children with JDM. Future studies are needed to identify clear indications for treatment with JAK-inhibitors.Acknowledgements:This work was supported by the RSF grant №20-45-01005.Disclosure of Interests:None declared.

Background:Chronic non-bacterial osteomyelitis (CNO) is an immune-mediated chronic inflammatory bone disease which predominantly affects children and adolescents. The pathogenesis of CNO related to imbalance between pro-inflammatory and anti-inflammatory cytokines. Interferon-I mediated pathway is associated with pathogenesis of different pediatric rheumatic diseases, such as juvenile systemic lupus erythematosus (jSLE), juvenile dermatomyositis (JDM), systemic onset of juvenile idiopathic arthritis (soJIA), and, most of all, with macrophage activation syndrome. The data on interferon-I- regulated pathway in CNO is absent. NSAIDs, non-biologic and biologic anti-inflammatory drugs and bisphosphonates (BF) are treatment options for patients with CNO. The main adverse event of BF is a flu-like syndrome probably caused by the excessive cytokine release stimulated by BF.Objectives:The aim of our study was to evaluate activity of Interferon-I mediated pathway in CNO patients and it’s dynamics after BF treatment.Methods:This prospective study included children with CNO requiring BF treatment (n=9), patients with soJIA (n=8), JDM (n=11) and jSLE (n=40) and healthy controls (HC, n=21). The activity of Interferon-I mediated pathway was assessed using interferon I score (IFN1 score). The score represented the median expression of 5 IFN1-regulated genes (IFI44L, IFI44, IFIT3, LY6E, MX1) measured by quantitative real-time PCR. Patients with CNO were treated with standard 3-day regimen (1 mg/kg/day). We measured interferon score before pamidronate (Day 0, n=9) and after (Day 3, n=7).Results:Median interferon score was 1.09 (0.96; 1.67) in CNO patients, 1.95 (1.3; 5.75) in soJIA, 7.6 (1.78; 29.0) in JDM and 16.9 (2.55; 40.3) in jSLE and 0.95 (0.82; 1.17) in HC (p=0.00001). Where were no difference in the IFN1 score between CNO and HC (p=0.222). In 6/7 CNO patients interferon score increased after pamidronate (p=0.015). The median interferon score after pamidronate increased and became 3.06 (0.87; 4,9, p=0.043); this may possibly explain the development of BF-related flu-like symptoms (cytokine release syndrome).Conclusion:While interferon I-regulated pathway is not directly associated with CNO pathogenesis, BF likely activates interferon-I-regulated pathway and thus could be a possible cause of flu-like syndrome.This work supported by the Russian Foundation for Basic Research (grant № 18-515-57001).Disclosure of Interests:None declared

Background:the role of interferon pathways in the pathogenesis of systemic lupus erythematosus (SLE) has been proven over the past years. Existing data suggest that interferon score (IFN I score) may serve as a useful marker of disease activity and patient clinical characteristics.Objectives:to compare characteristics of pediatric SLE patients with high and normal IFN I score.Methods:40 SLE patients (33 girls, 7 boys) under 18 years old were included in the cross-sectional study. In all cases the diagnosis was made using Systemic Lupus International Collaborating Clinics (SLICC) classification criteria. The data on clinical manifestations, disease activity by SLEDAI and ECLAM, laboratory findings in the onset of the disease and at the moment of interferon signature assessment were evaluated. Interferon signature was assessed by real-time PCR quantitation of 5 IFN I-regulated transcripts; median expression of ≥2 was considered as a threshold. The patients were divided into 2 groups depending on the level of interferon score: high (group1, n=31) and normal (group2, n=9).Results:The mean age of the disease onset was 12 (9.5; 14.0) years. The most common symptoms were skin lesions (85%), arthritis (67.5%), fever (55%), mucosa (45%), CNS (37.5%) and kidney (30%) involvement. Anemia, leukopenia and thrombocytopenia were observed in 62.5%, 27.5% and 50% of cases, while 87.5% and 70% of patients had ANA positivity and dsDNA antibodies at the onset. The comparison between the groups with increased and normal IFN I-signature is presented in Table 1.Table 1.ParametersGroup 1(High IFN-s)Group 2(Normal IFN-s)p-valueGirls, n (%)25 (80.7)8 (88.9)0.567The onset age, years12.0(10.0; 14.0)11.0 (9.0; 13.0)0.353Time to IFN-signature study, months from onset18.3 (7.0; 26.5)0.97 (0.87;1.73)0.987Skin involvement, n (%)12 (38.7)4 (44.4)0.837CNS involvement, n(%)8 (25.8)1 (11.1)0.353Arthritis, n(%)11 (35.5)2 (22.2)0.455Anemia, n(%)9 (29.0)2 (22.200.687Leucopenia, n(%)9 (29.0)1 (11.1)0.274ANA-positivity, n (%)27 (87.1)5 (55.6)0.037anti dsDNA antibodies, n(%)12 (38.7)2 (22.2)0.361Rheumatoid factor, n (%)11 (35.5)0 (0.0)0.036Hypocomplementemia, n (%)18/28 (64.3)2/6 (33.3)0.162Ferritin level, mkg/l112.0 (39.0; 271.0)21.0 (5.3; 23.7)0.0008Hematuria, n (%)10 (32.3)0 (0.0)0.049Proteinuria, n (%)11 (35.5)0 (0.0)0.036SELENA-SLEDAI, points9 (2;15)1 (0; 4)0.073ECLAM, points3.0 (1.0; 6.0)1.0 (0.0; 1.5)0.048Treatment with Rituximab or Cyclophosphamide, n (%)22 (71.0)3 (33.3)0.040GCS dose 0,2 mg/kg achievement for 6 months, n (%)9/21 (42.9)5/6 (83.3)0.080Conclusion:high IFN I-signature correlated with kidney involvement, ANA and RF-positivity, ferritinemia, proteinuria and hematuria. Patients with high IFN I-signature received more aggressive treatment and needed longer glucocorticosteroid (GCS) treatment. More meticulous dynamic evaluation of IFN-signature is needed to clarify its role as a predictive and prognostic marker.Acknowledgements:This work was supported by the RSF grant № 20-45-01005.Disclosure of Interests:None declared.

Background:Crimean Tatars is an ethnic group in Russia. The presence of familial Mediterranean fever (FMF) has been recognized since 2016.Objectives:The study aimed to evaluate the prevalence and clinical features as well as genetic aspects of FMF in children of Crimean Tatar (CT) origin and compare them with a cohort from Turkey.Methods:This retrospective study included all FMF cases in patients of CT origin (n=18) diagnosed in Children’s Regional Hospital in Simferopol since 2016. We included 40 consecutive FMF cases between February-March 2020, diagnosed and followed at Hacettepe University, Ankara, Turkey. All children were less than 18 years old at the time of inclusion. The diagnosis of FMF was based on the EULAR criteria (2019). We excluded other autoinflammatory diseases and any doubtful cases. For assessment of MEFV alleles prevalence 127 healthy unrelated CT adults from different parts of Crimea peninsula were included. Sanger sequencing of MEFV exons 2 and 10 was performed in all the patients and controls.Results:FMF in CT was diagnosed with nearly 5 year-delay, despite the earlier age at onset. CT children had more frequent and prolonged fever, joint involvement (arthritis and arthralgia) and erysipeloid rash compared to Turkish, who had more attacks with chest pain and abdominal pain which last longer. (Table 1) CT had higher white blood cell count, C-reactive protein, erythrocyte sedimentation rate and lower hemoglobin. It might be explained by the fact that the majority of Crimean Tatars were admitted to the clinic during an attack, which was not always the case with Turkish children. Distribution of MEFV pathogenic alleles p.M694V, p.M680I, p.V726A in CT children was 81%, 9.5% and 9.5%, respectively, while in Turks it was 68.6%, 14.3% and 12.9%. Among the CT patients, proportion of homozygotes, compound-heterozygotes and heterozygotes were 11%, 6% and 83%, and among Turkish patients were 45%; 30%; and 25%, respectively. MEFV pathogenic variants were detected in 10.2% of healthy CT donors: 7.1% individuals had p.M694V, 1.6% - p.M680I, 1.6% - p.V726A. Comorbid diseases including IgA vasculitis, sacroiliitis, JIA, autoimmune hepatitis and inflammatory bowel disease were reported in 5.6% of CT and 10% of Turks. The colchicine treatment rate and regimen were similar, but CT received biologics more frequently (44%) than Turks (22.5%).Conclusion:CT is an ethnic group with a significant number of MEFV mutation carriers assuming the expected prevalence of FMF to be as high as 1:385. Thus, any periodic fever in CT patients should be considered as a sign of possible FMF. The clinical course of FMF has some peculiarities in CT patients.This work supported by the Russian Foundation for Basic Research (grant № 18-515-57001).Table 1.Comparative data of FMF patients with Turkish and Crimean Tatar originFMF featuresTurkish (n=40)Crimean Tatars (n=18)рFamily history of FMF, n (%)16 (40)9 (50)0.477Consanguinity, n (%)8 (20)6 (33)0.272Onset age, years3.3 (2; 5)1.3 (0; 4)0.040Age at FMF diagnosis, years4.7 (3; 8)9.6 (4; 14)0.005Diagnosis delay, years0.9 (0; 2)5.5 (2; 10)0.00001Fever, n (%)33 (83)18 (100)0.058Episode duration, days2.0 (2; 3)3.0 (3; 6)0.000003Fever duration, hours48.0 (48; 72)72.0 (72; 120)0.000002Chest pain, n (%)12 (30)1 (6)0.039Chest pain duration, hours48.0 (24; 72)0.0 (0; 0)0.000001Abdominal pain, n (%)30 (75)9 (50)0.061Abdominal pain duration, hours48.0 (24; 48)24.0 (24; 24)0.043Arthritis, n (%)10 (25)16 (89)0.000006Arthralgia, n (%)19 (48)17 (94)0.0007Erysipeloid rash, n (%)0 (0)9 (50)0.000001Disclosure of Interests:None declared

Diagnostics of genetic diseases in clinical routine often presents a challenge. In particular, most of hereditary diseases are exceptionally rare and therefore unfamiliar to practicing physicians. Furthermore, even if the diagnosis of a particular genetic condition appears convincing on the level of clinical evidence, the causative mutation often remains unknown due to limitations in DNA testing procedures. Recently developed high-throughput sequencing technologies (Next Generation Sequencing, NGS; synonym: massive parallel sequencing) provide a breakthrough in medical genetics. While in the past genetic testing was limited to a single gene or, at best, to a small number of genes, NGS is compatible with a large-scale DNA analysis. One of the most popular applications of NGS is whole exome sequencing (WES), which allows simultaneous reading of coding sequences (exons) of all known genes. Although this technology exists only for a few years, its use has already led to discovery of the causes of more than 150 genetic syndromes. Furthermore, WES may be recommended for the use in clinical routine for selected patients with orphan disease, especially for the families with multiple affected relative. It is likely that WES will become a powerful screening tool in the near future. This review discusses general principles of WES as well as the applications of this technology in medicine.

BRCA1 L1705P (c.5114T>C) has been classified in the NCBI SNP database as the variant with uncertain significance and is absent in major BRCA1 databases. BRCA1 W1837X (c.5511G>A) results in a loss of only last 27 residues of BRCA1 protein, thus its pathogenic role still requires a confirmation. This report describes two breast cancer (BC) patients carrying BRCA1 L1705P and W1837X germ-line mutations, respectively. Significant evidence for BC-predisposing impact of the mentioned mutations have been obtained: (1) both index cases presented with the triple-negative receptor status of BC disease; (2) complete segregation with BRCA1-related cancers was observed in the families of these patients; (3) somatic loss of the remaining (wild-type) BRCA1 allele was detected in tumor tissues of the affected women. The results of this study have to be taken into account while providing genetic counseling to cancer patients and while considering the use of BRCA1-specific therapeutic compounds for BC treatment.

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive genetic disorder. It is characterized by heterogeneous clinical manifestations including primary features of the disease (rod-cone dystrophy, polydactyly, obesity, genital abnormalities, renal defects, and learning difficulties) and secondary BBS characteristics (developmental delay, speech deficit, brachydactyly or syndactyly, dental defects, ataxia or poor coordination, olfactory deficit, diabetes mellitus, congenital heart disease, etc.); most of these symptoms may not be present at birth but appear and progressively worsen during the first and second decades of life. At least 20 BBS genes have already been identified, and all of them are involved in primary cilia functioning. Genetic diagnosis of BBS is complicated due to lack of gene-specific disease symptoms; however, it is gradually becoming more accessible with the invention of multigene sequencing technologies. Clinical management of BBS is largely limited to a symptomatic treatment. Mouse experiments demonstrate that the most debilitating complication of BBS, blindness, can be rescued by topical gene therapy. There is a published case report describing the delay of BBS symptoms by nutritional compensation of the disease-related biochemical deficiencies. Progress in DNA testing technologies is likely to rapidly resolve all limitations in BBS diagnosis; however, much slower improvement is expected with regard to BBS treatment.

Ataxia-telangiectasia (AT) is a severe autosomal recessive orphan disease characterized by a number of peculiar clinical manifestations. Genetic diagnosis of AT is complicated due to a large size of the causative gene, ATM. We used next-generation sequencing (NGS) technology for the ATM analysis in 17 children with the clinical diagnosis of AT. Biallelic mutations in the ATM gene were identified in all studied subjects; these lesions included one large gene rearrangement, which was reliably detected by NGS and validated by multiplex ligation-dependent probe amplification (MLPA). There was a pronounced founder effect, as 17 of 30 (57%) pathogenic ATM alleles in the patients of Slavic origin were represented by three recurrent mutations (c.5932G > T, c.450_453delTTCT, and c.1564_1565delGA). These data have to be taken into account while considering the genetic diagnosis and screening for ataxia-telangiectasia syndrome.

Exomes of 27 Russian subjects were analyzed for the presence of medically relevant alleles, such as protein-truncating variants (PTVs) in known recessive disease-associated genes and pathogenic missense mutations included in the ClinVar database. 36 variants (24 PTVs and 12 amino acid substitutions) were identified and then subjected to the analysis in 897 population controls. 9/36 mutations were novel, however only two of them (POLH c.490delG associated with xeroderma pigmentosum variant (XPV) and CATSPER1 c.859_860delCA responsible for spermatogenic failure) were shown to be recurrent. 27 out of 36 pathogenic alleles were already described in prior genetic studies; seven of them occurred only in the index cases, while 20 demonstrated evidence for persistence in Russian population. In particular, non-random occurrence was revealed for SERPINA1 c.1096G > A (alpha-1 antitrypsin deficiency), C8B c.1282C > T and c.1653G > A (complement component 8B deficiency), ATP7B c.3207C > A (Wilson disease), PROP1 c.301_302delAG (combined pituitary hormone deficiency), CYP21A2 c.844G > T (non-classical form of adrenogenital syndrome), EYS c.1155T > A (retinitis pigmentosa), HADHA c.1528G > C (LCHAD deficiency), SCO2 c.418G > A (cytochrome c oxidase deficiency), OTOA c.2359G > T (sensorineural deafness), C2 c.839_866del (complement component 2 deficiency), ACADVL c.848T > C (VLCAD deficiency), TGM5 c.337G > T (acral peeling skin syndrome) and VWF c.2561 G > A (von Willebrand disease, type 2N). These data deserve to be considered in future medical genetic activities.

Background NLRP12-related autoinflammatory disease (NLRP12-AID) is an exceptionally rare autosomal-dominant disorder caused by germ-line mutations in NLRP12 gene. Very few patients with NLRP12-AD have been identified worldwide, therefore there is a scarcity of data on phenotypic presentation of this syndrome. Objectives Here we provide evidence that NLRP12-AID may have clinical manifestations characteristic for primary immune deficiencies (PID). Methods 246 children with periodic fever (PF) of unknown origin were subjects to the next generation sequencing (NGS) analysis; 213 of these patients had signs of primary immunodeficiency (PID) manifested by recurrent infections, while 33 kids had isolated PF. The NGS panel was composed of 302 genes implicated in PID and/or AID. Results NLRP12 variants were detected in 20 cases (see the table 1). Median age of first AID-related fever episode was 12 months, ranging from 2 months to 13 years. Increased association with immune-mediated diseases (n=15, 75%) was observed. Nine patients demonstrated increased susceptibility to infection and two children suffered from Crohn’s disease. Administration of short courses of NSAID or corticosteroids resulted in resolution of the disease flare. In one severe case canakinumab (anti-interleukine-1β antibody) was successfully used. Conclusions Significant number of patients with genetically assigned diagnosis of NLPR12-AID have clinical features which close resemble PID. This phenotypic overlap may result in underdiagnosis of NLPR12-AID among patients with PID. Acknowledgements This work has been supported by Russian Foundation for Basic Research (grant number RFBR 16–04–00 159a). Disclosure of Interest None declared

Distribution of cancer-predisposing mutations demonstrates significant interethnic variations. This study aimed to evaluate patterns of APC and MUTYH germ-line mutations in Russian patients with colorectal malignancies. APC gene defects were identified in 26/38 (68%) subjects with colon polyposis; 8/26 (31%) APC mutations were associated with 2 known mutational hotspots (p.E1309Dfs*4 [n = 5] and p.Q1062fs* [n = 3]), while 6/26 (23%) mutations were novel (p.K73Nfs*6, p.S254Hfs*12, p.S1072Kfs*9, p.E1547Kfs*11, p.L1564X and p.C1263Wfs*22). Biallelic mutations in MUTYH gene were detected in 3/12 (25%) remaining subjects with polyposis and in 6/90 (6.7%) patients with colorectal cancer (CRC) carrying KRAS p.G12C substitution, but not in 231 early-onset CRC cases negative for KRAS p.G12C allele. In addition to known European founder alleles p.Y179C and p.G396D, this study revealed a recurrent character of MUTYH p.R245H germ-line mutation. Besides that, 3 novel pathogenic MUTYH alleles (p.L111P, p.R245S and p.Q293X) were found. Targeted next-generation sequencing of 7 APC/MUTYH mutation-negative DNA samples identified novel potentially pathogenic POLD1 variant (p.L460R) in 1 patient and known low-penetrant cancer-associated allele CHEK2 p.I157T in 3 patients. The analysis of 1120 healthy subjects revealed 15 heterozygous carriers of recurrent MUTYH mutations, thus the expected incidence of MUTYH-associated polyposis in Russia is likely to be 1:23 000.

Tuberous sclerosis (TS) is a rare autosomal-dominant genetic disease. TS is manifested by the development of multiple hamartomas, which affect brain, kidneys, retina, skin and other organs. This study aimed to reveal specific features of molecular epidemiology of TS in Russia. Blood DNA samples from 61 patients with definite (n = 53) or probable (n = 8) clinical diagnosis of TS were tested for mutations in TSC1 and TSC2 genes using Sanger sequencing and MLPA analysis. Five TSC1/2 mutation-negative patients were further analyzed by exome sequencing. TSC1/2 mutations were detected in 53/61 patients (87%): 39 (74%) carried mutations in the TSC2 and 14 (26%) in the TSC1. Large rearrangements (exon deletions/duplications) affected exclusively TSC2, accounting for 15% of lesions of this gene. 6/8 (75%) patients with incomplete clinical manifestation of TS carried TSC1/2 gene lesion. Overall, 96% of detected germline TSC1/2 mutations occurred de novo. Patients with no mutation identified (NMI) differed from TSC1/2 mutation carriers, being lacking cortical tubers and subependymal nodules but having higher frequencies of renal angiomyolipomas, rhabdomyomas, and lymphangioleiomyomatosis. Exome sequencing failed to identify overt disease-causing mutation candidates among NMI patients. Russian patients with TS have increased frequency of TSC2 large gene rearrangements and TSC1/2 mutations occurring de novo as compared to other studies. Patients with suspected TS diagnosis but NMI status may represent a distinct disease entity.

Large genomic rearrangements (LGRs) constitute a significant share of pathogenic BRCA1 mutations. Multiplex ligation-dependent probe amplification (MLPA) is a leading method for LGR detection; however, it is entirely based on the use of commercial kits, includes relatively time-consuming hybridization step, and is not convenient for large-scale screening of recurrent LGRs. We developed and validated the droplet digital PCR (ddPCR) assay, which covers the entire coding region of BRCA1 gene and is capable to precisely quantitate the copy number for each exon. 141 breast cancer (BC) patients, who demonstrated evident clinical features of hereditary BC but turned out to be negative for founder BRCA1/2 mutations, were subjected to the LGR analysis. Four patients with LGR were identified, with three cases of exon 8 deletion and one women carrying the deletion of exons 5–7. Excellent concordance with MLPA test was observed. Exon 8 copy number was tested in additional 720 BC and 184 ovarian cancer (OC) high-risk patients, and another four cases with the deletion were revealed; MLPA re-analysis demonstrated that exon 8 loss was a part of a larger genetic alteration in two cases, while the remaining two patients had isolated defect of exon 8. Long-range PCR and next generation sequencing of DNA samples carrying exon 8 deletion revealed two types of recurrent LGRs. Droplet digital PCR is a reliable tool for the detection of large genomic rearrangements.

In a search for new breast cancer (BC) predisposing genes, we performed a whole exome sequencing analysis using six patient samples of familial BC and identified a germline inactivating mutation c.183delG [p. Arg61fs] in an orphan G protein-coupled receptor GPRC5A. An extended case-control study revealed a tenfold enrichment for this mutation in BC patients carrying the 5382insC allele of BRCA1, the major founder mutation in the Russian population, compared to wild-type BRCA1 BC cases [6/117 (5.1%) vs. 8/1578 (0.5%), p = 0.0002]. In mammary tumors (n = 60), the mRNA expression of GPRC5A significantly correlated with that of BRCA1 (p = 0.00018). In addition, the amount of GPRC5A transcript was significantly lower in BC obtained from BRCA1 mutation carriers (n = 17) compared to noncarriers (n = 93) (p = 0.026). Accordingly, a siRNA-mediated knockdown of either BRCA1 or GPRC5A in the MDA-MB-231 human BC cell line reduced expression of GPRC5A or BRCA1, respectively. Knockdown of GPRC5A also attenuated radiation-induced BRCA1- and RAD51-containing nuclear DNA repair foci. Taken together, these data suggest that GPRC5A is a modifier of BC risk in BRCA1 mutation carriers and reveals a functional interaction of these genes.

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive ciliopathy characterized by obesity, postaxial polydactyly, retinitis pigmentosa, mental retardation, and kidney abnormalities. At least 19 genes have been shown to be associated with BBS, and therefore, genetic testing is highly complicated. We used an Illumina MiSeq platform for whole exome sequencing analysis of a family with strong clinical features of BBS. A homozygous c.1967_1968delTAinsC (p.Leu656fsX673; RefSeq NM_176824.2) mutation in BBS7 was identified in both affected children, while their healthy sibling and the non-consanguineous parents were heterozygous for this allele. Genotyping of 2,832 DNA samples obtained from Russian blood donors revealed 2 additional heterozygous subjects (0.07%) with the c.1967_1968delTAinsC mutation. These findings may facilitate the genetic diagnosis for Slavic BBS patients.

Bloom syndrome (BS) is an exceptionally rare hereditary disease. Typical manifestations of BS usually include growth deficiency, a characteristic facial appearance, skin hypersensitivity to ultraviolet irradiation, and a strong predisposition to early-onset cancers. We have previously described a recurrent BLM c.1642C>T (p.Q548X) mutation, which is present in heterozygous state in 0.2–0.6% of individuals of Slavic origin. Despite the high occurrence of this founder allele, BS has not yet been described in patients of Slavic ethnicity. Here, we present 2 cases of BS, which were missed by standard genetic counseling and were eventually identified entirely due to chance. Our patients show the need for further investigations to confirm whether the atypical appearance of BS is indeed characteristic for biallelic carriers of the c.1642C>T (p.Q548X) allele and whether the absence of skin manifestations contributes to the underdiagnosis of the disease in Russia. Therefore, we suggest that all Slavic patients with only one single clinical feature of BS are to be screened for this allele and subjected to further analysis wherever appropriate. In addition to identifying new BS patients, this effort will help to clarify the frequency of “atypical BS” with incomplete phenotypic manifestations.

The BLM gene belongs to the RecQ helicase family and has been implicated in the maintenance of genomic stability. Its homozygous germline inactivation causes Bloom syndrome, a severe genetic disorder characterized by growth retardation, impaired fertility and highly elevated cancer risk. We hypothesized that BLM is a candidate gene for breast cancer (BC) predisposition. Sequencing of its entire coding region in 95 genetically enriched Russian BC patients identified two heterozygous carriers of the c.1642 C>T (Q548X) mutation. The extended study revealed this allele in 17/1,498 (1.1%) BC cases vs. 2/1,093 (0.2%) healthy women (p = 0.004). There was a suggestion that BLM mutations were more common in patients reporting first-degree family history of BC (6/251 (2.4%) vs. 11/1,247 (0.9%), p = 0.05), early-onset cases (12/762 (1.6%) vs. 5/736 (0.7%), p = 0.14) and women with bilateral appearance of the disease (2/122 (1.6%) vs. 15/1376 (1.1%), p = 0.64). None of the BLM-associated BC exhibited somatic loss of heterozygosity at the BLM gene locus. This study demonstrates that BLM Q548X allele is recurrent in Slavic subjects and may be associated with BC risk.

A few founder BRCA1 mutations (5382insC 4154delA 185delAG) account for up to 15% of high-risk (young-onset or familial or bilateral) breast cancer (BC) cases in Russia The impact of non founder BRCA1 mutations in this country is less studied in particular there are no reports analyzing gross rearrangements of this gene in the Russian patient series We selected for the study 95 founder mutation negative high-risk BC cases Combination of high-resolution melting (HRM) and sequencing revealed six presumably BC-associated alleles (2080delA, 4808C > G 5214C > T 5236G > A 5460G > T 5622C > T) and one variant of an unknown significance (4885G > A) The pathogenic role of the 5236G > A mutation leading to G1 706E substitution was further confirmed by the loss of heterozygosity analysis of the corresponding tumor tissue Multiplex ligation-dependent probe amplification (MLPA) revealed two additional BRCA1 heterozygotes which carried BRCA1 deletions involving exons 1-2 and 3-7 respectively Based on the results of this investigation and the review of prior Russian studies three BRCA1 mutations (2080delA 3819de15 3875del4) were considered with respect to their possible founder effect and tested in the additional series of 210 high-risk BC patients two BACA heterozygotes (2080delA and 3819de15) were revealed We conclude that the non-founder mutations constitute the minority of BRCA1 defects in Russia (C) 2010 Elsevier Ireland Ltd All rights reserved

Whole exome sequencing (WES) provides a powerful tool for medical genetic research. Several dozens of WES studies involving patients with hereditary cancer syndromes have already been reported. WES led to breakthrough in understanding of the genetic basis of some exceptionally rare syndromes; for example, identification of germ-line SMARCA4 mutations in patients with ovarian hypercalcemic small cell carcinomas indeed explains a noticeable share of familial aggregation of this disease. However, studies on common cancer types turned out to be more difficult. In particular, there is almost a dozen of reports describing WES analysis of breast cancer patients, but none of them yet succeeded to reveal a gene responsible for the significant share of missing heritability. Virtually all components of WES studies require substantial improvement, e.g. technical performance of WES, interpretation of WES results, mode of patient selection, etc. Most of contemporary investigations focus on genes with autosomal dominant mechanism of inheritance; however, recessive and oligogenic models of transmission of cancer susceptibility also need to be considered. It is expected that the list of medically relevant tumor-predisposing genes will be rapidly expanding in the next few years.

Preoperative therapy provides an advantage for clinical drug assessment, as it involves yet untreated patients and facilitates access to the post-treatment biological material. Testing for Slavic founder BRCA mutations was performed for 225 ovarian cancer (OC) patients, who were treated by platinum-based neoadjuvant therapy. 34 BRCA1 and 1 BRCA2 mutation carriers were identified. Complete clinical response was documented in 12/35 (34%) mutation carriers and 8/190 (4%) non-carriers (P = 0.000002). Histopathologic response was observed in 16/35 (46%) women with the germ-line mutation versus 42/169 (25%) patients with the wild-type genotype (P = 0.02). Somatic loss of heterozygosity (LOH) for the remaining wild-type BRCA1 allele was detected only in 7/24 (29%) post-neoadjuvant therapy residual tumor tissues as compared to 9/11 (82%) BRCA1-associated OC, which were not exposed to systemic treatment before the surgery (P = 0.009). Furthermore, comparison of pre- and post-treatment tumor material obtained from the same patients revealed restoration of BRCA1 heterozygosity in 2 out of 3 sample pairs presenting with LOH at diagnosis. The obtained data confirm high sensitivity of BRCA-driven OC to platinating agents and provide evidence for a rapid selection of tumor cell clones without LOH during the course of therapy.

BRCA1 5382insC variant was repeatedly detected in Jewish breast cancer (BC) families residing in USA and Israel as well as in non-Jewish familial BC patients from Poland, Latvia, Hungary, Russia and some other European countries. However, the distribution of BRCA1 5382insC mutation in unselected BC cases vs. controls has been systematically investigated mainly in Ashkenazi Jews. Here we applied a case-control study design in order to evaluate the impact of BRCA1 5382insC allele on BC incidence in St Petersburg, Russia. High frequency of the BRCA1 5382insC allele was detected in a group of bilateral breast cancer patients (10.4%; 15/144). Randomly selected unilateral BC cases demonstrated noticeable occurrence of BRCA1 5382insC mutation as well (3.7%; 32/857), with evident excess of the carriers in the early-onset (40 years) category (6.1%; 6/99) and in patients reporting breast and/or ovarian tumours in first-degree relatives (11.3%; 11/97). Strikingly, none of 478 middle-aged controls and 344 elderly tumour-free women carried the 5382insC variant. The presented data confirm a noticeable contribution of BRCA1 5382insC mutation in BC development in Russia, that may justify an extended BRCA1 5382insC testing within this population.

The gene for Nijmegen chromosomal breakage syndrome (NBS1) plays a role in a variety of processes protecting chromosomal stability. Recently, it was suggested in a Polish case-control study that the founder hypomorphic mutation in NBS1, 657del5, which occurs in approximately 0.5% of Slavic subjects, may be associated with an increased risk of breast cancer (BC). We attempted to validate these findings in Russian subjects, who are also of Slavic descent. Heterozygous carriers for the 657del5 mutation were detected in 2 of 173 (1.16%) bilateral breast cancer cases, 5 of 700 (0.71%) unilateral breast cancer patients, 2 of 348 (0.57%) healthy middle-aged females and in 0 of 344 elderly tumor-free women. The difference between the “extreme” cohorts, i.e., biBC patients vs. elderly controls, approached the formal limit of statistic significance (p = 0.046). LOH at NBS1 was detected in only 3 of 5 available breast tumors from NBS1 657del5-carriers. In 2 of these tumors, the loss involved the mutant NBS1-allele. Overall, our data suggest that the NBS1 657del5 allele may contribute only to a limited fraction of breast cancer cases in Russia. © 2004 Wiley-Liss, Inc.

The CYP19 gene encodes the enzyme aromatase, which plays a key role in the conversion of androgens to oestrogens. A polymorphism in CYP19 in intron 4 (TTTA)n has been reported to be associated with breast cancer (BC) risk, although conflicting evidence has also been published. Here, we employ a non-traditional, highly demonstrative design of a molecular epidemiological study, where the comparison of BC cases and healthy middle-aged female donors was supplemented by an analysis of groups with extreme characteristics of either BC risk (bilateral breast cancer (biBC) patients) or cancer tolerance (tumour-free elderly women agedor=75 years). None of the (TTTA)n polymorphic variants was significantly overrepresented among the affected women compared with any of the control groups. However, a 3-bp deletion/insertion CYP19 polymorphism, which is located in the same intron approximately 50 bp upstream to the (TTTA)n repeat, was evidently associated with the menopausal status in both the BC and biBC cohorts. In particular, the Delta3(TTTA)(7) allele occurred significantly more frequently in premenopausal than in postmenopausal BC patients (65/172 (38%) versus 67/310 (22%); P=0.0001; Odds Ratio (OR)=2.20 (95% Confidence Interval (CI) 1.46-3.32)), while the perimenopausal cases demonstrated an intermediate value (9/34 (26%)). In the biBC cohort, women who developed both tumours during their premenopausal period had a significantly higher prevalence of the Delta3(TTTA)(7) allele than patients with a postmenopausal onset of bilateral disease (16/46 (35%) versus 8/50 (16%); P=0.035; OR=2.80 (1.08-7.23)); those biBC patients, whose tumours were diagnosed before and after the cessation of menses, displayed an intermediate occurrence of the Delta3(TTTA)(7) allele (7/32 (22%)). Similar tendencies in the Delta3(TTTA)(7) allele distribution in BC and biBC patients suggest that its association with the menopausal status of the patients is truly non-random and thus this observation deserves further detailed investigation.

Somatic inactivation of the remaining allele is a characteristic feature of cancers arising in BRCA1 and BRCA2 mutation carriers, which determines their unprecedented sensitivity to some DNA-damaging agents. Data on tumor-specific status of the involved gene in novel varieties of hereditary breast cancer (BC) remain incomplete. We analyzed 32 tumors obtained from 30 patients with non-BRCA1/2 BC-associated germ-line mutations: 25 women were single mutation carriers (7 BLM, 15 CHEK2 and 3 NBN/NBS1) and 5 were double mutation carriers (2 BLM/BRCA1, 1 CHEK2/BLM, 1 CHEK2/BRCA1 and 1 NBN/BLM). Losses of heterozygosity affecting the wild-type allele were detected in none of the tumors from BLM mutation carriers, 3/18 (17 %) CHEK2-associated BC and 1/4 (25 %) NBN/NBS1-driven tumors. The remaining 28 BC were subjected to the sequence analysis of entire coding region of the involved gene; no somatic mutations were identified. We conclude that the tumor-specific loss of the wild-type allele is not characteristic for BC arising in CHEK2, NBN/NBS1 and BLM mutation carriers. Rarity of "second-hit" inactivation of the involved gene in CHEK2-, NBN/NBS1- and BLM-associated BC demonstrates their substantial biological difference from BRCA1/2-driven cancers and makes them poorly suitable for the clinical trials with cisplatin and PARP inhibitors.

Purpose: Initiation/promotion of endometrial cancer is known to be associated with estrogenic influence. Therefore, it is possible that some allelic polymorphisms of the genes involved in steroidogenesis or steroid metabolism contribute to endometrial cancer susceptibility. Methods: Here, we compared CYP19 (aromatase) gene polymorphism in 85 endometrial cancer patients and in 110 non-affected women. Results: The genotypes containing the longest alleles (A6 and A7) of CYP19 were found to be over-represented in patients as compared to controls. In addition, these genotypes demonstrated a tendency to be associated with increased concentrations of estradiol and testosterone in postmenopausal patients. Conclusions: Thus, CYP19 polymorphism might be one of the genetic risk factors for endometrial cancer development.

The CYP17 gene encodes an enzyme involved in several critical steps of steroidogenesis. The promoter region of the CYP17 displays a single-nucleotide polymorphism, which is suspected to modulate the expression of the gene and thus may contribute in the interindividual variations of hormonal background. In agreement with this functional hypothesis, the MspA1+ allele (designated as A2) of the CYP17 was shown to render an increased risk of breast cancer (BC). However, the latter observation was disputed by a series of negative reports. Here, we re-evaluated the role of CYP17 MspA1 polymorphism in the BC susceptibility, using a non-traditional design of a case-control study. In addition to randomly selected 183 BC patients and 107 female middle-aged donors, we examined the groups with apparently extreme characteristics of either BC risk or BC resistance, namely the 57 bilateral breast cancer (biBC) patients and 75 elderly (≥75 years old) tumor-free women. Neither BC nor biBC patients showed increased prevalence of ‘unfavorable’ A2 allele as compared with the non-affected cohorts. Moreover, the A2 variant was not significantly associated with the tumor size, nodal involvement and menopausal status in the patients either with the monolateral or bilateral disease. Thus, our data argue against the earlier reported role of the CYP17 in BC predisposition and progression. In addition, usual distribution of the CYP17 alleles in the elderly group indicates a neutral effect of this polymorphism on the longevity in females.

The molecular pathogenesis of various categories of breast cancer (BC) has been well described, but surprisingly few reports have appeared on analysis of somatic mutations in bilateral BC. We have performed a polymerase chain reaction (PCR)-driven investigation of chromosomal regions showing common loss of heterozygosity (LOH) in 23 cases (46 tumors) from patients diagnosed with bilateral BC. LOH was observed in 15/46 (33%) informative tumors for chromosome 1p, 5/32 (16%) for 5q, 12/44 (27%) for 11q, 15/40 (38%) for 13q and 4/24 (17%) for 17p. These values are within the range of interlaboratory variations reported for unilateral BC. There was no strong evidence for concordance of LOH within the same patient for any of the chromosomal loci tested. Atypical for breast carcinomas, 7/46 (15%) tumors accumulated a high frequency (ranging from 11 to 29%) of shortened dinucleotide CA repeats, implying microsatellite instability (MI). Further analysis with the highly informative BAT-26 marker allowed for the classification of two of these tumors as having a replication error positive (RER(+)/MSI-H) phenotype, whereas the remaining five carcinomas harbored so-called borderline MI. Thus an involvement of both RER(+) and borderline MI appears to be a distinct feature of bilateral breast carcinomas compared to unilateral lesions.

L-myc polymorphism was investigated in 95 breast cancer (BC), 63 colorectal cancer (CC) and 58 lung cancer (LC) patients, as well as in 122 healthy, middle-aged blood donors (HBDs) and 184 elderly, tumor-free individuals. The occurrence of the S allele in the BC cohort (57%) was significantly higher than that in middle-aged, healthy females (41%) and elderly, non-affected women (47%), implying involvement of the L-myc genotype in BC susceptibility (age-adjusted OR = 1.74, 95% CI 1.11–2.73, p = 0.016). L-myc allele distribution in CC and LC was similar to that in controls. Contrary to earlier reports, L:S allele frequencies ratio in elderly blood donors (EBDs) did not significantly differ from that in HBDs (0.49:0.51 and 0.54:0.46, respectively). However, the S allele had a tendency to be over-represented among elderly compared with middle-aged smokers (55% vs. 44%; OR = 1.57, 95% CI 0.98–2.50, p = 0.059), which implies that it may be linked with tolerance to smoking effects. Int. J. Cancer 85:747–750, 2000. © 2000 Wiley-Liss, Inc.

One hundred and ninety-five consecutive surgically treated Russian colorectal cancer (CRC) patients were retrospectively analyzed for the presence of mutations in KRAS, NRAS, BRAF and PIK3CA genes as well as for the microsatellite instability status. Comparison between high-resolution melting analysis, co-amplification at lower denaturation temperature PCR, DNA sequencing and allele-specific PCR for the detection of KRAS codon 12/13 mutations revealed that none of these methods alone provided satisfactory results in 100 % of the analyzed cases; this experience supports the use of more than one mutation-detecting technique at least in some circumstances. KRAS codon 12/13 substitutions were detected in 70 (35.9 %) CRC cases. Other mutations in the RAS/RAF genes occurred in 22 (11.3 %) cases and included rare KRAS (n = 6), NRAS (n = 8) and BRAF (n = 8) alterations. 5 BRAF mutations affected codon 600, while the remaining 3 potentially functional substitutions were located in the position 594. Twenty-four (12.3 %) CRC cases carried mutations in the PIK3CA, and 18 of these tumors also contained activating alteration in the RAS/RAF genes (p = 0.007). Only 3 (1.5 %) CRC cases showed high-level microsatellite instability (MSI-H) as determined by a panel of mononucleotide markers. Overall, the distribution of potentially predictive mutations in Russian CRC cases is similar to the one observed in other patient series of European descent. Noticeable occurrence of D594G mutation in BRAF oncogene and low frequency of MSI-H may deserve specific attention.