B-cell receptor stereotypy in chronic lymphocytic leukemia

Opsežne imunogenetičke studije sprovedene tokom proteklih nekoliko decenija u oblasti hronične limfocitne leukemije (HLL) su identifikovale grupe pacijenata koji eksprimiraju visoko homologne, skoro identične imunoglobulinske molekule koji ulaze u sastav B-ćelijskog receptora (BĆR IG). Ovaj fenomen, nazvan „BĆR stereotipija“, obuhvata oko 30% HLL i predstavlja najvažniju potvrdu uloge antigenske stimulacije u patogenezi i evoluciji bolesti. Na osnovu sekvence CDR3 regiona teških lanaca IG definisani su stereotipni subsetovi koji ispoljavaju konzistentne subset-specifične biološke i kliničke karakteristike. Neki od najzastupljenijih subsetova imaju i jasan prognostički značaj, tako da se čak mogu smatrati različitim varijantama bolesti. Subklasifikacija HLL bazirana na BĆR stereotipiji je omogućila kompartmentalizovan pristup u istraživanju ove bolesti, i otvorila mogućnost za precizniju stratifikaciju pacijenata, kao i uspešniju prognostiku i lečenje, uprkos izrazitoj kliničkoj heterogenosti HLL.During the past several decades, extensive immunogenetic research of chronic lymphocytic leukemia (CLL) led to the identification of groups of patients expressing highly homologous, almost identical B-cell receptor immunoglobulin molecules (BCR IG). This phenomenon, termed „BCR stereotypy“, accounts for around 30% of all CLL and represents the strongest evidence for the role of antigenic stimulation in CLL pathogenesis and evolution. Based on the sequence of CDR3 region within IG heavy chains multiple stereotyped subsets have been defined, which display consistent subset-specific biological and clinical characteristics. Some of the most frequent subsets also exert strong prognostic significance and can be considered to be distinct disease variants. Stereotypy-based CLL subclassification enabled compartmentalized approach in the research of this disease, and offered the possibility of refining stratification of patients that can improve prognostication and clinical decision-making despite extreme heterogeneity of CLL

Molecular basis of chronic lymphocytic leukemia: correlation between the immunoglobulin heavy chain mutational status and the expression of apoptotic genes

Hronična limfocitna leukemija (HLL), najčešći tip leukemije u Evropi i Severnoj Americi, se manifestuje kao klonska ekspanzija zrelih CD5+ CD19+ CD23+ sIgM+/- B limfocita i karakteriše se izuzetno heterogenim kliničkim tokom. Leukemični, kao i normalni B limfociti, na svojoj površini eksprimiraju imunoglobulinski antigenski receptor. Struktura varijabilnog regiona njegovog teškog lanca (IGH) je determinisana specifičnim rearanžmanima između IGHV, IGHD i IGHJ gena, koji se odvijaju tokom diferencijacije B ćelija. Nakon susreta sa antigenom, B limfociti ulaze u proces afinitetnog sazrevanja u germinalnim centrima sekundarnih limfnih folikula, tokom koga dolazi do akumulacije somatskih hipermutacija u IGHV-IGHD-IGHJ rearanžmanima. Pokazano je da je mutacioni status rearanžiranih gena za varijabilni region teških lanaca imunoglobulina (IGHV) najpouzdaniji molekularni marker u HLL, koji definiše dva podtipa bolesti: M-HLL i N-HLL. Pacijenti sa malim procentom ili bez IGHV mutacija (N-HLL) obično imaju mnogo agresivniji tok bolesti i lošiju prognozu od pacijenata sa mutiranim IGH rearanžmanima (M-HLL). Razlike u IGHV genskom repertoaru između M-HLL i N-HLL klonova, populacione varijacije u učestalosti određenih IGHV gena u HLL rearanžmanima kao i ekspresija visoko homologih, ˝stereotipnih˝ rearanžmana, ukazuju na ulogu antigenske stimulacije u patogenezi HLL.HLL se smatra tipičnim primerom maligniteta uzrokovanog poremećajima u procesu apoptoze. U HLL su detektovane genetičke promene i aberantna ekspresija brojnih proteina regulatora apoptoze, koji su uključeni kako u spoljašnji tako i u unutrašnji put aktivacije ovog procesa. Smatra se da je smanjeni apoptotski potencijal HLL klonova uzrokovan, između ostalog, i poremećajima u ekspresiji proteina Bcl2 familije. U ovom radu je analiziran IGHV mutacioni status i genski repertoar IGHV-IGHD-IGHJ rearanžmana kod pacijenata obolelih od HLL. Pored toga, analizirana je ekspresija gena Bcl2 familije, kao osnovnih regulatora unutrašnjeg puta aktivacije apoptoze, u cilju utvrđivanja njihove uloge u apoptotskoj rezistenciji HLL B limfocita. qRT-PCR metodom je merena ekspresija Bcl2, Bax i Bcl2L12 gena, i ispitivana asocijacija nivoa njihove ekspresije sa odabranim kliničkim i molekularnim prognostičkim faktorima (IGHV mutacionim statusom, ekspresijom CD38 i lipoprotein lipaze). Bcl2L12 je novi član Bcl2 familije apoptotskih proteina čija pro- ili anti-apoptotska funkcija još uvek nije razjašnjena. Pored analize ekspresije Bcl2L12 gena u HLL, jedan od ciljeva ovog rada je bilo i definisanje promotorskog regiona i mesta starta transkripcije Bcl2L12, što je neophodno za proučavanje mehanizama njegove transkripcione regulacije. Određivanjem IGHV mutacionog statusa je pokazano da 55.3% analiziranih pacijenata pripada M-HLL, a 44.7% N-HLL podtipu, kao i da u N-HLL preovlađuju pacijenti sa progresivnim oblikom bolesti. U analiziranim rearanžmanima su sa najvećom frekvencom bili zastupljeni geni IGHV3 familije (55.7%), a zatim IGHV1 (27.3%), IGHV4 (12.5%), IGHV5 (2.3%), IGHV2 (1.1%) i IGHV6 (1.1%) geni. Pokazano je prisustvo tzv. ˝stereotipnih˝ rearanžmana kod 15.3% pacijenata, predominantno u N-HLL podtipu. Na osnovu određenih frekvenci IGHV, IGHD i IGHJ gena i genskih familija je zaključeno da je IGH genski repertoar leukemičnih klonova kod pacijenata iz Srbije veoma sličan repertoaru detektovanom kod pacijenata iz zemalja mediteranskog područja, sa izuzetkom gena IGHV4 familije koji su kod pacijenata u ovoj studiji zastupljeni sa manjom učestalošću.Analizom ekspresije Bcl2, Bax i Bcl2L12 su detektovani znatno viši nivoi ekspresije sva tri gena kod HLL pacijenata u odnosu na zdrave kontrole, pri čemu je povećanje ekspresije bilo najizraženije u slučaju Bcl2 gena. Povišena ekspresija Bcl2 je pokazala asocijaciju sa nepovoljnim prognostičkim parametrima: progresivnim tipom bolesti, visokim nivoom serumskog β2-mikroglobulina i povišenim nivoom ekspresije gena za lipoprotein lipazu (LPL). Ekspresija Bax je pokazala korelaciju samo sa ekspresijom LPL, dok je ekspresija Bcl2L12 bila relativno homogena među HLL pacijentima i, kao takva, nije ispoljila značajnu asocijaciju sa većinom kliničkih i molekularnih prognostičkih faktora. Ekspresija Bcl2, Bax i Bcl2L12 je bila viša u grupi N-HLL pacijenata u odnosu na M-HLL pacijente, ali ova razlika nije dostigla statističku značajnost.Kloniranjem fragmenata 5' kraja Bcl2L12 gena i funkcionalnom analizom reporterskih konstrukata, detektovan je region koji ispoljava jaku promotorsku aktivnost. Ovaj region se prostire od 338 nukleotida uzvodno, do 148 nizvodno od početka kodirajućeg dela egzona 1, a esejom usporene elektroforetske pokretljivosti (˝EMSA˝) je pokazano da se za njega vezuju Sp1 i GATA-1 transkripcioni faktori. Metodom elongacije reverznog prajmera (˝primer extension˝) je određen položaj starta transkripcije Bcl2L12 gena, 33 nukleotida uzvodno od translacionog start kodonaChronic lymphocytic leukemia (CLL), the most common type of leukemia in Western countries, manifests as clonal expansion of mature CD5+ CD19+ CD23+ sIgMlow B lymphocytes and it is characterized by an extremely heterogeneous clinical course. Leukemic, as well as normal B lymphocytes, express immunoglobulin antigenic receptor at their surface. The structure of its heavy chain (IGH) variable region is being formed during B-cell differentiation, through rearrangements between IGHV, IGHD and IGHJ genes. After antigen encouner, B lymphocytes undergo the process of affinity maturation in germinal centers of secondary lymphoid follicles, durring which IGHV-IGHD-IGHJ rearrangements accumulate somatic hypermutations. It has been shown that the mutational status of rearranged immunoglobulin heavy variable genes (IGHV) represents the most reliable molecular marker in CLL, which defines two CLL subsets: M-CLL and U-CLL. The patients without or with a small percentage of IGHV mutations (UCLL) usually have more agressive disease and inferior prognosis in comparison to patients expressing mutated IGH rearrangements (M-CLL). Biased IGHV gene repertoire between MCLL and U-CLL clones, population differences in IGHV gene usage, as well as the expression of highly homologous, ˝stereotyped˝ rearrangements, strongly imply the role of antigenic stimulation in pathogenesis of CLL.CLL typifies the malignancy caused by defective apoptosis. Genetic alterations and aberrant expression of numerous proteins involved in extrinsic and intrinsic pathways of apoptosis regulation have been described in CLL. Reduced apoptotic capacity of CLL clones is, in part, caused by disturbances in the expression of Bcl2 family proteins. In this study, we analized IGHV mutational status and gene repertoire of IGHV-IGHD-IGHJ rearrangements in CLL patients. Furthermore, the expression of Bcl2 family genes, the key regulators of intrinsic apoptotic pathway, has been studied, in order to elucidate their role in resistance of CLL B lymphocytes to apoptosis. The expression levels of Bcl2, Bax and Bcl2L12 genes were measured by qRT-PCR, and association of their expression with clinical and molecular prognostic factors (IGHV mutational status, expression of CD38 and lipoprotein lipase) was analized. Bcl2L12 is a novel member of Bcl2 family of apoptotic proteins, whose pro- or anti-apoptotic function has not been elucidated yet. Besides the expression analysis of Bcl2L12 gene in CLL, one of the aims of this dissertation was defining the promoter region and transcription start site of Bcl2L12, which is essential for the study of mechanisms involved in its transcriptional regulation. The analysis of IGHV mutational status showed that 55.3% of patients enrolled in this study belonged to M-CLL, and 44.7% to U-CLL subset, and that in U-CLL predominated patients with the progressive form of the disease. The most frequently expressed genes were those belonging to IGHV3 family (55.7%), followed by IGHV1 (27.3%), IGHV4 (12.5%), IGHV5 (2.3%), IGHV2 (1.1%) i IGHV6 (1.1%) genes. The presence of ˝stereotyped˝ rearrangements was detected in 15.3% of patients, predominantly in U-CLL subset. Based on the determined frequencies of IGHV, IGHD and IGHJ genes and gene families, we concluded that the IGH gene repertoire of leukemic clones of Serbian CLL patients closely resembles the repertoire observed in patients from Mediterranean countries, with the exception of IGHV4 family, which was underrepresented in our cohort. The expression analysis of Bcl2, Bax and Bcl2L12 showed that those three genes were overexpressed in CLL patients compared to healthy controls, and that elevation of expression level was the most prominent in the case of Bcl2 gene. High expression levels of Bcl2 were associated with unfavorable prognostic parameters: progressive form of the disease, elevated β2- microglobulin and high expression of lipoprotein lipase gene (LPL). The expression of Bax was correlated only with the expression of LPL, while the expression of Bcl2L12 turned out to be relatively homogenous among CLL patients and, as such, failed to show association with the majority of clinical and molecular prognostic factors. Expression levels of Bcl2, Bax and Bcl2L12 were higher in U-CLL in comparison to M-CLL group of patients, but this difference did not reach statistical significance.Cloning of fragments corresponding to 5' end of Bcl2L12 gene and functional analysis of reporter constructs, led to identification of a region which exerts high promoter activity. This region extends from 338 nucleotides upstream, to 148 nucleotides downstream relative to the beginning of exon 1 coding sequence, and electromobility shift assay (˝EMSA˝) showed that it binds Sp1 and GATA-1 transcription factors. Transcription start site of Bcl2L12 gene was determined by primer extension assay, which showed that it is located 33 nucleotides upstream of translation start codon

Molecular characteristics, phenotypic diversity and genotype-estimated therapeutic responsiveness of Serbian patients with phenylketonuria

Fenilketonurija (PKU) jeste retko metaboličko oboljenje koje se nasleđuje autozomalno recesivno. Uzrok PKU su mutacije u genu koji kodira za enzim fenilalanin-hidroksilazu (PAH). U ovom revijskom radu su opisane najznačajnije metode za detekciju mutacija u PAH genu. Budući da slika o PKU prevazilazi "jednostavno" monogensko oboljenje, prikazani su i podaci o varijantama u nekodirajućim regionima PAH gena, kao i genima modifikatorima za PKU. Iako postoji značajna korelacija između PAH genotipa i PKU fenotipa, uočene su i neusaglašenosti. U ovom revijskom radu su izneti mogući uzroci za neusaglašenost između genotipa i fenotipa, kao što su: propust u detekciji više od dve mutacije prisutne u PAH genotipu pacijenta, neusaglašenost u fenotipskoj klasifikaciji pacijenata (koncentracija fenilalanina u serumu pre terapije i tolerancija fenilalanina), fenomen interalelske komplementacije (pozitivne i negativne). Novi terapijski pristup, terapija tetrahidrobiopterinom (BH4), predstavlja važnu inovaciju u lečenju pacijenata sa PKU. Međutim, u zemljama u kojima test opterećenja sa BH4 i terapija sa BH4 nisu dostupni, procena odgovora na terapiju koja je zasnovana na genotipu predstavlja koristan pristup. Ovakav pristup omogućava procenu potencijalne koristi od terapije sa BH4 što je bitna informacija, kako za pacijenta, tako i na nivou populacije. Predložen je i optimalni algoritam za molekularnu dijagnostiku, uspostavljen prema objavljenim učestalostima mutacija kod pacijenata sa PKU u Srbiji. U budućnosti, molekularno-genetički algoritam za PKU bi mogao da bude proširen tako da uključi i različite transkripcione regulatorne elemente u nekodirajućim regionima PAH gena, kao i gene modifikatore koji tek treba da budu otkriveni.Phenylketonuria (PKU) is a rare, inherited metabolic disease which is transmitted in an autosomal recessive pattern. PKU is caused by mutations in the gene encoding the phenylalanine hydroxylase (PAH) enzyme. This review cites the most prominent methods for the detection of mutations in the PAH gene. Since the image of PKU transcends "simple" monogenic disease, the known data on non-coding PAH gene variants and their role and PKU modifier genes have been further reviewed. It has been shown that there is a significant correlation between mutant PAH genotypes and PKU phenotypes. However, genotype-phenotype correlation inconsistencies have also been found. This review discusses the possible causes of phenotypic inconsistencies, such as oversight of more than two mutations present in the patient's PAH genotype, pitfalls of patient phenotypic classification (plasma phenylalanine concentration and phenylalanine tolerance), the inter-allelic complementation (positive and negative) phenomenon. A new therapeutic approach, tetrahydrobiopterin (BH4) supplementation therapy, is an important innovation in the course of PKU patients' treatment. However, in countries where the BH4-loading test and BH4-supplementation therapy are not available, a genotype-based estimation of responsiveness to the therapy is a valuable approach. It enables BH4-potential benefit estimation, which provides vital information both for the patient and for the population. An optimal molecular diagnostics algorithm, established according to the published mutation frequencies in Serbian PKU patients, has been suggested. In the future, the molecular-genetic algorithm for PKU could be expanded to include a variety of transcriptional regulatory elements located in noncoding PAH gene regions and yet to be discovered modifier genes

Characterization of transcription factors binding to-120 GATA motif of rat βbminy-globin promoter

Cilj ovog rada je bio rasvetljavanje regulacije transkripcije adultnog βbminy-globinskog gena pacova. Da bi se okarakterisali transkripcioni faktori uključeni u ovu regulaciju, upotrebljena je metodologija koja je obuhvatila "footprint" (otisak proteina na DNK), "gel shift" (metoda usporene pokretljivosti u gelu) i "supershift" (metoda kompeticije antitelom u "gel shift"-u) eseje. U ovom radu je analiziran GATA motiv lociran na-120 bp distalnog promotora βbminy-globinskog gena. "Footprint" analiza je otkrila vezivanje nuklearnih faktora iz MEL ćelija za GATA motiv. Uz pomoć "gel shift" eseja detektovana su dva proteinska kompleksa. Kompleks koji je brže migrirao u gelu je eritroidno-specifičan i zastupljeniji je u diferencirajućim MEL ćelijama. Eksperimenti kompeticije sa GATA-1 oligonukleotidom i GATA-1 antitelima potvrdili su vezivanje GATA-1 transkripcionog faktora za GATA motiv na poziciji-120 adultnog βbminy-globinskog gena pacova.The aim of this study was to elucidate the regulation of rat adult βbminy-globin gene transcription. We used DNasel foot printing, gel mobility shift and super shift assays to characterize transcription factors involved in this regulation. In this study we analyzed GATA motif at-120 bp in the distal promoter of βbminy-globin gene. Footprint analysis revealed the binding of nuclear factors from MEL cells to the GATA motif. By using gel mobility shift assay two protein complexes were detected. The faster migrating complex was erythroid-specific and more abundant in differentiating MEL cells. Competition experiments with GATA-1 oligonucleotides and GATA-1 protein antibodies confirmed binding of GATA-1 transcription factor to GATA motif at - 120 bp regulation of rat adult βbminy-globin gene

Application of Next-Generation Sequencing Technology and Establishment of Biobanks

Institute of Molecular Genetics and Genetic Engineering has become widely recognized as an expert centre for rare diseases (RD). It is the first institution is Serbia that applied NGS methodology in research and diagnostics of RD. We are also the institution with RD biobank collections containing DNA, RNA, mononuclear cells and tissue samples from over 2000 of patients affected with 50 different diseases. An accurate diagnosis was provided to over than 100 RD patients who were undiagnosed for years. We used Clinical-Exome Sequencing TruSightOne Gene Panel (4813 clinicaly-relevant genes), Illumina MiSeq instrument and Illumina VariantStudio. For monogenic diseases, filtration and prioritization of variants were performed according to “in-house” pipeline, using virtual gene panels. Variants were analyzed by various in silico softwares and classified according to ACMG guidelines. Variants selected by these criteria were confirmed by conventional Sanger sequencing and parents’ samples were analyzed whenever available. Furthermore, novel variants in DNAI1, MUT, PAH, PCCB, SLC37A4, SPAG16 and SPAG17 genes were functionally characterized in adequate in vitro systems such as immortalized patients’ fibroblasts or CRISPR /Cas9 edited commercial cell lines. Clinical-exome sequencing enabled diagnosis of more than 50 different diagnosis (hematological, metabolic, endocrinological, pulmonary, immunological, orthopedic, dermatological, ophthalmological, cardiological, epileptic encephalopathies etc.). It was particularly important for genetically heterogeneous diseases, such as glycogen storage diseases, branched-chain organic acidurias, primary ciliary dyskinesia, MODY or mitochondriopathies. Moreover, different diseases with overlapping clinical manifestations were accurately diagnosed. Also, we used TruSeq-Amplicon Cancer Panel to analyse different childhood and adult rare hematological malignancies. Besides studying diagnostic and prognostic malignancy markers, we designed “in-house” virtual pharamocogenomic panel, and performed association studies of pharmacogenomic markers and the course and outcome of rare hematological malignancies, resulting in recommendations for therapeutic modalities in accordance with genomic profile of the patien

Association of mitochondrial DNA variants and cognitive impairment of phenylketonuria patients

Uvod: Fenilketonurija (PKU) je metaboličko oboljenje uzrokovano mutacijama u genu za fenilalanin hidroksilazu (PAH). Ukoliko se ne leče, pacijenti sa fenilketonurijom razvijaju tešku mentalnu retardaciju, koja može biti i posledica neurodegeneracije. Ovo je prva studija koja istražuje prisustvo mitohondrijalnih DNK varijanti kod pacijenata sa fenilketonurijom, m. 10398A, za koju je pokazana povezanost sa neurodegenerativnim oboljenjima, kao i m. 10410T. Metode: U ovoj studiji je analizirano 64 pacijenta sa fenilketonurijom i 50 zdravih kontrola iz srpske populacije. Pacijenti su podeljeni u grupe prema uzrastu u kome je postavljena dijagnoza i odnosu prema dijeti sa niskim sadržajem fenilalanina. IQ je određen pomoću odgovarajućih uzrasnih skala. Rezultati: Mitohondrijalne DNK varijante, m. 10398A i m. 10410T, su detektovane sekvenciranjem. Učestalost m. 10398A je bila jednaka kod pacijenata i zdravih kontrola (82,81% i 82,00%), što ukazuje na isto etničko poreklo ovih ispitanika. U slučaju m. 10410, nisu detektovane različite varijante. U grupi pacijenata kojima je kasno postavljena dijagnoza i koji su neadekvatno lečeni, nije pronađena statistička značajnost u srednjoj vrednosti IQ između pacijenata sa m. 10398A i m. 10398G. Isto je pokazano i za pacijente sa višim IQ koji su otkriveni prilikom neonatalnog skrininga i koji su pravilno lečeni. Međutim, kada su iz ove grupe isključeni pacijenti koji nose p. L48S mutaciju u PAH genu, koja ima nekonzistentan uticaj na fenotip, prisustvo m. 10398A alela je povezano sa nižim IQ. Zaključak: Ova studija je potvrdila važnost neonatalnog skrininga i pravilnog sprovođenja dijete kod pacijenata sa fenilketonurijom. Statističke analize nisu jasno utvrdile uticaj mitohondrijalne DNK varijante, m. 10398A, na IQ ovih pacijenata, osim kad je i PAH genotip uključen u analizu. Studije u većim grupama ce rasvetliti povezanost između mutacija u PAH genu, mitohondrijalnih DNK varijanti i složenog kognitivnog fenotipa kod pacijenata sa PKU.Background: Phenylketonuria (PKU) is a metabolic disorder caused by phenylalanine hydroxylase gene (PAH) mutations. If left untreated, PKU patients develop severe mental retardation potentially due to neurodegeneration. This is the first study that investigates presence of mitochondrial DNA variants in PKU patients, m.10398A, reportedly associated with neurodegenerative diseases and m.10410T. Methods: We analyzed 64 PKU patients and 50 healthy controls from Serbian population. PKU patients were categorized into groups according to time of diagnosis and compliance to low-phenylalanine diet. The IQ was determined according to age-appropriate scales. Results: We detected m.10398A and m.10410T variants by direct sequencing. Frequency of m.10398A was similar in patients and healthy controls (82.81% and 82.00% respectively) suggesting their identical ethnic background. No variation was detected for m.10410. In group with late diagnosis and poorly controlled diet, no statistically significant difference in average IQ was found between patients with m.10398A and m.10398G. The same was shown for PKU patients with higher IQ, diagnosed at neonatal screening and treated with low-phenylalanine diet. However, when patients carrying p.L48S, a PAH mutation with inconsistent effect, were excluded from the study, presence of m.10398A variant was associated with lower IQ. Conclusions: This study emphasizes the importance of neonatal screening and good control of low-phenylalanine diet in PKU patients. Statistical analysis did not indicate clear impact of mitochondrial DNA variant m.10398A on IQ of PKU patients, except when PAH genotype was also considered. Studies in larger cohorts will elucidate the association between PAH gene mutations, mitochondrial DNA variants and complex PKU cognitive phenotype

Мијелодиспластични синдром са приближном тетраплоидијом удруженом са мутацијом гена TP53 – редак случај

Introduction. Chromosomal numerical aberrations are very common in hematological malignancies, but near-tetraploidy (80-104 chromosomes) is rare in myeloid lineage malignancies, with only a few cases reported in myelodysplastic syndrome (MDS). Due to a small number of cases with this rare cytogenetic abnormality, clinicopathological significance of near-tetraploidy in MDS is still unknown. In this case report we present a case of de novo MDS patient with near-tetraploidy in association with TP53 mutation, and we aimed to elucidate the prognostic significance of this rare genetic feature. Case Outline. In August of 2018 a 71-year-old male presented with severe anemia, thrombocytopenia, and leucopenia and enlarged spleen. Laboratory data were as follows: hemoglobin (Hb) 93 g/L, white blood cells (WBC) 2.8×109/L and platelets 23x109/L. The bone marrow aspirate was hypercellular, megakaryocytes were not found, granulocytic cells were 15% with signs of dysplasia, with 16% of blast cells without Auer rods. The finding was in correlation with diagnosis of MDS, type RAEB2 which was also confirmed by immunophenotyping. Cytogenetic finding was near-tetraploidy (48,XY+mar[10]/92,XXYY[10]), and TP53 mutational analysis showed the presence of mutation in exon 8 (p.D281A; c.842 A>C). The patient received from time-to-time packed red blood cells and platelets, and died four months after initial diagnosis. Conclusion. Near-tetraploidy associated with TP53 mutation has been described only in few MDS cases. Results of these reports including ours suggest that the association of TP53 mutation and near-tetra polyploidy is a poor prognostic factor.Нумеричке аберације хромозома су веома честе код хематолошких малигнитета, али су приближне тетраплои- дије (80–104 хромозома) ретке у малигнитетима мијелоидне лозе, са само неколико случајева пријављених у мијелоди- спластичком синдрому (МДС). Због малог броја случајева са овом ретком цитогенетском абнормалношћу, клиничко- -патолошки значај приближне тетраплоидије у МДС-у је још увек непознат. Овим приказом de novo болесника са МДС-ом, са приближном тетраплоидијом и мутацијом у гену TP53, циљ нам је био да расветлимо прогностички значај ове рет- ке генетске карактеристикe. Приказ болесника Приказан је 71-годишњи болесник који је у августу 2018. године развио симптоме тешке анемије, тромбоцитопеније, леукопеније и увећане слезине. Лабо- раторијске анализе су показале следеће: хемоглобин 93 g/L, леукоцити 2,8 × 109 /L и тромбоцити 23 × 109 /L. Аспират коштане сржи је био хиперћелијски, мегакариоцити нису на- ђени, 15% гранулоцитa је било са знацима дисплазије, 16% бластa без Ауерових штапића. Налаз је одговарао дијагнози МДС-а, типа рефракторне анемије са вишком бласта 2, што је потврђено и имунолошком фенотипизацијом. Цитогенет- ском анализом утврђено је присуство приближне тетрапло- идије (48,XY+mar10/92,XXYY[10]), а анализа мутација у гену TP53 показала је присуство мутације у егзону 8 (p.D281A; c.842 A > C). Болесник је по потреби примао трансфузију еритроцита и тромбоците, а умро је четири месеца након почетне дијагнозе. Закључак Присуство приближне тетраплоидије удружене са мутацијама у гену TP53 описано је само у неколико слу- чајева МДС-а. Резултати ових случајева, као и наши резул- тати, указују на то да приближна тетраплоидија повезана са присуством мутација у гену TP53 представља фактор лоше прогнозе

Clinical significance of TP53 aberrations and IGHV mutational status in chronic lymphocytic leukemia

Hronična limfocitna leukemija (HLL) izuzetno je heterogena bolest varijabilnog kliničkog toka. S jedne strane imamo, pacijente sa agresivnom i rezistentnom bolešću od koje umiru svega par meseci nakon dijagnoze, dok s druge strane spektra postoje pacijenti sa indolentnom, sporo progredirajućom bolešću koja ne zahteva lečenje decenijama. Razlozi su samo delimično poznati i već su decenijama unazad tema mnogobrojnih naučnih istraživanja. Tako je razvijen koncept prognostičkih i prediktivnih faktora u HLL-u, koji imaju za cilj da predvide klinički tok, odnosno terapijski ishod HLL-a. Liste prognostičkih i prediktivnih faktora su, sa boljim poznavanjem patofiziologije ove bolesti, svakom godinom sve duže, ali se i međusobno preklapaju. U ovom revijalnom radu izabrali smo aberacije TP53 gena i mutacioni status rearanžiranih IGHV (engl. immunoglobulin heavy variable) gena kao dva najznačajnija i najproučavanija faktora koji imaju i prognostički i prediktivni značaj.Chronic lymphocytic leukemia (CLL) is a very heterogeneous disease with a variable clinical course. On the one side of the spectrum, there are patients with aggressive and resistant disease, of which they die only a few months after diagnosis while, on the other side, there are patients with an indolent, slowly progressive disease that does not require treatment for decades. The reasons for this are only partially known, but they have been the subject of numerous scientific studies during the last several decades. Consequently, the concept of prognostic and predictive factors in CLL have emerged, which aims to predict the clinical course and its therapeutic outcome. With the improvement of understanding the pathophysiology of this disease, the lists of prognostic and predictive factors are getting longer every year, but they also overlap. In this review, we singled out the aberrations of the TP53 gene and the IGHV (immunoglobulin heavy variable) gene mutational status as the two most important and most studied factors that have both prognostic and predictive significance

Untreated PKU patients without intellectual disability: SHANK gene family as a candidate modifier

Phenylketonuria (PKU) is an inborn error of metabolism caused by variants in the phenylalanine hydroxylase (PAH) gene and it is characterized by excessively high levels of phenylalanine in body fluids. PKU is a paradigm for a genetic disease that can be treated and majority of developed countries have a population-based newborn screening. Thus, the combination of early diagnosis and immediate initiation of treatment has resulted in normal intelligence for treated PKU patients. Although PKU is a monogenic disease, decades of research and clinical practice have shown that the correlation between the genotype and corresponding phenotype is not simple at all. Attempts have been made to discover modifier genes for PKU cognitive phenotype but without any success so far. We conducted whole genome sequencing of 4 subjects from unrelated non-consanguineous families who presented with pathogenic mutations in the PAH gene, high blood phenylalanine concentrations and near-normal cognitive development despite no treatment. We used cross sample analysis to select genes common for more than one patient. Thus, the SHANK gene family emerged as the only relevant gene family with variants detected in 3 of 4 analyzed patients. We detected two novel variants, p.Pro1591Ala in SHANK1 and p.Asp18Asn in SHANK2, as well as SHANK2:p.Gly46Ser, SHANK2:p.Pro1388_Phe1389insLeuPro and SHANK3:p.Pro1716Thr variants that were previously described. Computational analysis indicated that the identified variants do not abolish the function of SHANK proteins. However, changes in posttranslational modifications of SHANK proteins could influence functioning of the glutamatergic synapses, cytoskeleton regulation and contribute to maintaining optimal synaptic density and number of dendritic spines. Our findings are linking SHANK gene family and brain plasticity in PKU for the first time. We hypothesize that variant SHANK proteins maintain optimal synaptic density and number of dendritic spines under high concentrations of phenylalanine and could have protective modifying effect on cognitive development of PKU patients

Molecular genetic strategy for diagnosis of congenital adrenal hyperplasia in Serbia

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is one of the most common endocrine diseases, yet genetic diagnosis is among the most complicated of all monogenic disorders. It has an overall incidence of 1: 10000-1: 20000, it is inherited in autosomal recessive pattern and caused by mutations affecting CYP21A2 gene. Based on the phenotypic expression, this disease is categorized into severe, classical form revealed at birth and mild, non-classical form. Although diagnosis could be established based on biochemical tests and distinctive clinical features, molecular genetic testing is crucial for diagnosis confirmation, detection of carriers and asymptomatic patients, disease prognosis, as well as for providing proper genetic counselling and prenatal diagnosis. Based on CYP21A2 mutational spectrum and frequencies in Serbia, in this paper we propose an optimal molecular genetic diagnostic algorithm for CAH and discuss genetic mechanisms underlying the disease. The complete diagnostic procedure combines multiplex minisequencing technique (SNaPshot PCR) as a method for rapid detection of common point mutations, direct sequencing of whole CYP21A2 gene and PCR with sequence specific primers (PCR-SSP) for large gene rearrangements detection (CYP21A1P/CYP21A2 chimeras). While SNaPshot PCR assay analyses ten common mutations (c. 290-13A/C gt G, p.P30L, p.R356W, p.G110fs, p.V281L, p.Q318X, p.L307fs, p.I172N, Cluster p.[I236N;V237E;M239K] and p.P453S) which account for over 80% of all CYP21A2 mutations in Serbian population, direct sequencing of CYP21A2 gene is needed to identify potential rare or novel mutations present in Serbian population with frequency of 1.8%. Additionally, large gene rearrangements which are present with frequency of 16.7% make PCR-SSP analysis an unavoidable part of molecular characterization of CAH in Serbia. Described molecular genetic strategy is intended to facilitate correct diagnosis assessment in CAH affected individuals and their families in Serbia but it will also contribute to molecular genetic testing of CAH patients across Europe