The annexin A5 protective shield model revisited: inherited carriage of the M2/ANXA5 haplotype in placenta as a predisposing factor for the development of obstetric antiphospholipid antibodies.

This concise review summarizes the role of reduced ANXA5 expression through carriage of the M2/ANXA5 haplotype as a predisposing factor for various thrombophilia related obstetric complications. A revised ANXA5 'protective shield' model is emphasized, where decreased coverage resulting of M2 carriage at placental villi could lead directly to the observed pathology and on the other hand through exposing of antiphospholipid antigenic determinants, to the development of antiphospholipid antibodies (aPL). The aPL then can further disrupt the ANXA5 protective shield. Available and prospective evidence for this revised model is discussed. Conclusions are made about the diagnostic implications of M2 carriage and possible therapeutic strategies with anticoagulants, proven successful in obstetric antiphospholipid syndrome (APS) treatment.

Microsatellite instability and promoter hypermethylation of MLH1 and MSH2 in patients with sporadic colorectal cancer.

PURPOSE: Inactivation of the genes involved in DNA mismatch repair (MMR) is associated with microsatellite instability (MSI) and loss of heterozygosity (LOH). The aim of the current study was to assess the presence of MSI and promoter hypermethylation of MLH1 and MSH2 in Bulgarian PATIENTS WITH SPORADIC COLORECTAL CANCER (CRC) AND TO ANALYZE THEIR POSSIBLE EFFECT ON THE DEVELOPMENT, PROGRESSION AND PROGNOSIS OF THE DISEASE. METHODS: We examined MSI in 126 patients with sporadic CRC and the methylation status of the MLH1 and MSH2 promoter regions in the cases with MSI/LOH by using a panel of 5 microsatellite markers (BAT26, D5S346, D18S35, D2S123 and FGA) and methyl-specific PCR (MSP) of bisulfite converted DNA. RESULTS: MSI/LOH was found in 36 (28.6%) patients. Among them, 30 were analyzed for promoter hypermethylation of MLH1 and we detected hypermethylation in 15 (50%) of them, whereas promoter hypermethylation of MSH2 was observed only in one case. The presence of MSI/LOH was associated with younger age (p=0.002), more advanced stage (III/IV stage) (p=0.029), lower degree of differentiation (p=0.001), and right-sided tumor localization (p=0.0002), but not with overall survival (log rank, p=0.566). CONCLUSION: Our data suggest that sporadic CRCs with MSI/LOH are more aggressive, develop earlier and progress faster to more advanced stage. The most frequent cause of failure of DNA MMR system appeared to be the hypermethylation of CpG islands of the promoter region of MLH1, whereas the methylation of MSH2 was a rare event.

CHEK2 I157T and colorectal cancer in Bulgaria.

PURPOSE: Germline variants of the CHEK2 gene have been shown to act as low-penetrance cancer susceptibility alleles for a wide range of human malignancies. CHEK2 I157T has particularly been linked to colorectal cancer (CRC) risk. We aimed at establishing the population frequency and contribution of this variant to colorectal carcinogenesis in Bulgaria. METHODS: We have genotyped 802 population controls and 343 CRC patients from Bulgaria for the CHEK2 I157T variant. RESULTS: Heterozygous were 9 of 343 patients (2.62%, odds ratio/OR=1.0, 95% confidence interval/CI = 0.42 - 2.33, p=0.99% and 21 of 802 controls (2.62%). Higher frequencies were found among patients with multiple polyposis (2/40, 5%, p=0.28) and the rarer mucinous histology (1/11, 9.09%, p= 0.26). CONCLUSION: We conclude that CHEK2 I157T is not relevant for CRC risk in Bulgaria, but studies on a larger scale might help evaluate its possible significance in respect to disease characteristics.

[Low PAPP-A levels between 11 and 13(+6) weeks of gestation and the risk of preeclampsia].

PAPP-A is one of the 2 biochemical markers of the first trimester Down syndrome serum screening. In Bulgaria this screening was first performed in 2006. The threshold values for evaluation of the risk of development of preeclampsia used by the researchers vary in a wide range. Nevertheless in the last several years the most used value is 0.4 MoM. The aim of represented study is evaluate the low PAPP-A levels as a marker for development of preeclampsia alone, or in combination with some risk factors. The data of 194 singleton pregnancies that underwent a first trimester Down syndrome screening between January 2008 and June 2009 had been analyzed. Twenty three patients (11.6%) developed preeclampsia, of which 8 required delivery before 34th gw. The control group includes 134 women who had term deliveries of healthy babies. Nine out of 23 (39%) patients with preeclampsia, 5 out of 8 (62.5%) patients with early onset preeclampsia and 42 out of 134 (30.4%) controls had PAPP-A levels below 0.4 MoM. These levels are associated with relative risk for development of preeclampsia of 1.5 times. Low PAPP-A levels in the first trimester were associated with a low prognostic value for development of preeclampsia and early preeclampsia before 34th gw. When low PAPP-A levels are added to the maternal age and especially results of Doppler evaluation of the uterine arteries significant prognostic value improvement was observed.

Microsatellite instability in Bulgarian patients with endometrial cancer.

PURPOSE: Microsatellite instability (MSI) is a frequent event in different types of cancer. In several studies MSI was shown to have both clinical and prognostic value. The aim of our study was to determine the frequency of MSI in Bulgarian patients with endometrial cancer (EC) and the possible relation of this phenomenon to their clinicopathological characteristics. PATIENTS AND METHODS: A total of 33 histologically confirmed EC patients were analyzed for tumor MSI using a panel of 6 microsatellite markers. RESULTS: We identified MSI in 30% of endometrial cancer cases. Six of them had high degree of MSI (MSI-H), and 4 displayed low degree of MSI (MSI-L). CONCLUSION: The frequency of MSI in Bulgarian EC patients does not differ significantly from that reported in other European studies.

Evidence for protein splicing in the endoplasmic reticulum-Golgi intermediate compartment.

We present evidence that the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) is a site for protein splicing of cathepsin C: (i) maturation of the enzyme in COS 7 cells is a two-step process starting within the ERGIC, and (ii) the intermediately processed polypeptide retains both termini of the proenzyme and lacks an internal fragment.

Mutation analysis in Duchenne and Becker muscular dystrophy patients from Bulgaria shows a peculiar distribution of breakpoints by intron.

For the first time in Bulgaria, a deletion/duplication screening was performed on a group of 84 unrelated Duchenne/Becker muscular dystrophy patients, and the breakpoint distribution in the dystrophin gene was analyzed. Intragenic deletions were detected in 67.8% of patients, and intragenic duplications in 2.4%. A peculiar distribution of deletion breakpoints was found. Only 13.2% of the deletion breakpoints fell in the "classical" hot spot in intron 44, whereas the majority (> 54%) were located within the segment encompassing introns 45-51, which includes intron 50, the richest in breakpoints (16%) in the Bulgarian sample. Comparison with data from Greece and Turkey points at the probable existence of a deletion hot spot within intron 50, which might be a characteristic of populations of the Balkan region.

Y-chromosomal STR haplotypes in three major population groups in Bulgaria.

A total of 280 unrelated males from the three largest population groups in Bulgaria: Bulgarians, Bulgarian Turks and Gypsies, were analyzed for seven Y-chromosome STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392 and DYS393). Comparison of the allele frequency distributions revealed significant differences between the three ethnic groups which were confirmed with haplotype analysis. This permits us to suggest that population differentiation should be taken into account in forensic case analysis and paternity testing in Bulgaria.

Enzymes and cytomorphological sperm alterations in some diseases of the male reproductive system.

In this work the alterations of some acrosomal enzymes and gamma-GT were studied in diseases of the male genital tract. Thirty-two patients with vascular diseases, seven with inflammatory diseases and twenty-four controls were investigated. In all patients the sperm morphology was studied and the following enzymes were assayed: beta-glucuronidase, beta-galactosidase, beta-glucosidase and gamma-GT. In all patients with vascular and inflammatory diseases we found severe hypokinesis and decreased number of spermatozoa. The activity of all four enzymes in both pathological groups was decreased in comparison with the controls, and this decrease was significant for all enzymes in males with vascular diseases and for beta-galactosidase and beta-glucosidase in cases with inflammatory diseases. Our data show that the decrease of spermatozoal count was accompanied by a decreased enzyme activity. The role of decreased sperm plasma enzyme activity, the decreased production of spermatozoa and quantitative changes in structure and acrosome enzyme content are discussed.

Hunter syndrome (Muccopolysaccharridosis Type II) in Macedonia and Bulgaria.

BACKGROUND: Mucopolysaccharidosis II (MPS II) is caused by a deficiency of iduronate-2-sulfatase (IDS; EC 3.1.6.13). METHODS AND RESULTS: We describe 11 boys from Bulgaria and Macedonia detected in the period from 1998 to 2008. The mean age at diagnosis was 4.77+/-1.29 years. All children were severely retarded: IQ ranged from 34-80, and they all had coarse faces and hepatomegaly. In addition, splenomegaly was found in 81.81% patients, dysostosis in 45.45%, kyphosis in 27.27%, deafness in 18.08%, growth below the third percentile in 45.45%, growth below the parental target height in all patients, stiff joints in 56.56% and hypertrophic myocardiopathy in 18.18% children. Two patients died at the age of 11 and 35 years. Plasma iduronate-2-sulfatase was low in all probands and normal in parents and relatives. Two new mutations were discovered: p.K236N (c.708G>C) in a child with a moderately severe phenotype, and p.Q80K (c.238C>A) which resulted in a severe phenotype and early death at the age of 11 years. Heterozygote carriers of the pathogenic allele were 29 female relatives. The calculated incidence rate for MPS II in Macedonia (censuses 1994 and 2002, children under 14 years: 483,923 and 426,280) and Bulgaria (censuses 1992 and 2006, children under 14 years: 1 126, 598 and 1,077,020) are 0.36 and 0.46 respectively, while the calculated prevalence rate are 3.6 and 4.6 per 1,000,000 boys (aged 0-14 years). Correlating phenotype and genotype remains a complex endeavour. CONCLUSIONS: We report calculated incidence and prevalence rates in two South Eastern European countries, and 2 novel genetic alterations correlated with their phenotypes.

Four generations of epilepsy caused by an inherited microdeletion of the SCN1A gene.

OBJECTIVES: The aim of this study was to determine the genetic defect in a 4-generational family with an epileptic disorder characterized by febrile and afebrile polymorphic seizures and mild to severe mental retardation by means of analyzing the neuronal voltage-gated sodium channel alpha-subunit gene SCN1A for mutations. METHODS: A Bulgarian family was ascertained and clinically assessed, followed by mutation analysis of the SCN1A gene using direct sequencing to detect point mutations and multiplex amplicon quantification to identify copy number variations. RESULTS: A microdeletion encompassing the entire SCN1A gene segregating with all affected members was identified in this family. Additional analysis showed that the unaffected father of the proband is mosaic for the deletion. So far, SCN1A deletions, predicted to lead to haploinsufficiency, are exclusively identified in isolated patients with Dravet or contiguous gene syndromes. Because of the severe phenotype, SCN1A deletion carriers are usually not able to live independently and start a family, and hence do not transmit the disease. CONCLUSIONS: We report an inherited SCN1A gene deletion not exclusively associated with Dravet syndrome. Moreover, our results demonstrate that SCN1A haploinsufficiency can cause a significant intrafamilial clinical variability including moderately affected to syndromal patients. The involvement of multiple genetic and environmental factors could be the basis of this difference in phenotype severity.

Type I Gaucher disease (GDI) in three siblings: enzyme replacement treatment (ERT) required.

(Full text is available at http://www.manu.edu.mk/prilozi). This is a family of three children, born to healthy Macedonian parents after uneventful pregnancies and delivery. The index child was an eight-year-old girl admitted for abdominal discomfort and distension: the spleen was 14cm below the costal margin (BCM), the liver 8cm BCM. No bone pain or pathology was reported. There was mild pancytopaenia (hemoglobin 11.2 gm/L; WBC counts 4.6 x 10;3; platelets 70 x 10;3). Liver function tests, renal ultrasound, bone scan, and a chest radiograph were within normal limits. Bone marrow analysis in this child and her two brothers (11 and 6.5 years old) revealed Gaucher cells. Both brothers had only mild anaemia, but the older brother had been splenectomized prior to diagnosis of GD1. Enzyme analysis revealed low activity (2.59, 1.62, and 2.55 nmol/h/mg protein, respectively); plasma chitotriosidase levels were also elevated. Genetic testing revealed homozygosity for the N370S/N370S mutation in all three siblings. In the absence of available enzyme replacement treatment (ERT), the girl was splenectomized. Removing an important immune organ (the spleen) introduces further risk for the patients. In addition, this does not solve the bone involvement characteristic for GD. ERT should be introduced for all GD1 patients in Macedonia. Key words: Gaucher disease, N370S mutation, siblings, enzyme replacement therapy.

Spastin gene mutations in Bulgarian patients with hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is an extremely heterogeneous group of neurodegenerative disorders affecting the longest axons in the central nervous system. The most common genetic form accounting for about 40% of the autosomal-dominant HSP (ADHSP) cases is spastin gene, SPG4. We performed mutation screening of the spastin gene on 36 unrelated HSP patients from three different ethnic groups (Bulgarian, Turks and Gypsies) and found four new mutations and one already reported. The phenotype-genotype correlations in Bulgarian SPG4 patients showed a great difference in the age at disease onset between patients with missense mutations and those harboring deletions and splice-site mutations. Our study is the first to present corroborative clinical data in favor of the general hypothesis that the clinical course of the disease is related to the type of the spastin mutation. The clinical and genealogical findings in Bulgarian SPG4 patients suggest that a positive family history for inheritance as an autosomal-dominant trait is a strong indication for spastin mutation screening.

Screening for phenylketonuria in a totalitarian state.

Living under a totalitarian regime has many effects on the structure, way of thinking, and relations in a society. However, it is the impact on neonatal genetic screening that we discuss in this paper. Genetic screening functions at the interface between health services and society at large. Being involved for over a decade in setting up the Bulgarian PKU screening programme, we have had to deal with ways and attitudes which may be difficult for the western mind to grasp. Yet comprehension is very much needed in the new world we are trying to create.

C283Y mutation and other C-terminal nucleotide changes in the gamma-sarcoglycan gene in the Bulgarian Gypsy population.

Sarcoglycanopathies, affecting the dystrophin-associated sarcoglycan (SG) complex, are a heterogeneous group of neuromuscular disorders. A subgroup of these disorders, limb-girdle muscular dystrophy type 2C (LGMD2C) is an autosomal recessive disorder, clinically manifested as an early onset, severe Duchenne-like muscular dystrophy. LGMD2C is caused by mutations in the gamma-SG gene, localized on 13q12. Recently, a number of mutations have been described in that gene, among which C283Y, a "private" Gypsy mutation (eight codons before the 3' end of the gene) is detected. In this article, we report on a single-strand conformation polymorphism (SSCP) method for fast C283Y mutation detection, using direct dry blood spot amplification. The method permits a large number of samples to be easily screened. To check heterozygote carriers of C283Y mutation among Gypsy population in Bulgaria, the SSCP analysis was applied on 400 Gypsy newborns from northeast Bulgaria. Our results show 2.25% of heterozygosity, which means that 1 in 50 Gypsies carries the mutation. Moreover, new SSCP migration patterns were detected that revealed two polymorphisms still unavailable in the literature. One of these changes was 984G-->A, leading to substitution of conserved serine at position 287 with asparagine and the second one is 1049C-->G at the 3' UTR (untranslated region). The present data could help the understanding the role of these sequences for the protein function.