Tissue and plasma proteomic profiling indicates AHSG as a potential biomarker for ascending thoracic aortic aneurysms.

BACKGROUND: Thoracic Aortic Aneurysms (TAAs) develop asymptomatically and are characterized by dilatation of the aorta. This is considered a life-threatening vascular disorder due to the risk of aortic dissection and rupture. There is an urgent need to identify blood-borne biomarkers for the early detection of TAA. The goal of the present study was to identify potential protein biomarkers associated with TAAs, using proteomic analysis of aortic tissue and plasma samples. METHODS: Extracted proteins from 14 aneurysmal and 12 non-aneurysmal thoracic aortic tissue specimens as well as plasma samples from six TAA patients collected pre-and postoperatively and six healthy controls (HC), were analyzed by liquid chromatography-tandem mass spectrometry. Proteomic data were further processed and following filtering criteria, one protein was selected for verification and validation in a larger cohort of patients and controls using a targeted quantitative proteomic approach and enzyme-linked immunosorbent assay, respectively. RESULTS: A total of 1593 and 363 differentially expressed proteins were identified in tissue and plasma samples, respectively. Pathway enrichment analysis on the differentially expressed proteins revealed a number of dysregulated molecular pathways that might be implicated in aneurysm pathology including complement and coagulation cascades, focal adhesion, and extracellular matrix receptor interaction pathways. Alpha-2-HS glycoprotein (AHSG) was selected for further verification in 36 TAA and 21 HC plasma samples using targeted quantitative proteomic approach. The results showed a significantly decreased concentration of AHSG (p = 0.0002) in the preoperative plasma samples compared with HC samples. Further analyses using a larger validation dataset revealed that AHSG protein levels were significantly lower (p = 0.03) compared with HC. Logistic regression analysis on the validation dataset revealed males, advanced age, hypertension and hyperlipidaemia as significant risk factors for TAA. CONCLUSION: AHSG concentrations distinguish plasma samples derived from TAA patients and controls. The findings of this study suggest that AHSG may be a potential biomarker for TAA that could lead to better diagnostic capabilities.

Sex-specific age association with primary DNA transfer.

Practicing forensic scientists who are called to provide expert witness testimony are often asked to explain both the presence and the absence of DNA on objects that have been handled by perpetrators with bare hands. Unwashed hands, depending on what they have come in contact with previously, may become the vehicle of both primary and secondary transfer of DNA. In this study, we investigated the propensity of primary and secondary transfer of DNA from unwashed bare hands of 128 individuals onto plastic tubes. Our experiments, carried out in triplicate, have shown that DNA was not detected on all the touched tubes, secondary transfer of DNA, through unwashed hands, was small, and in the majority of cases primary DNA transfer could be distinguished from secondary DNA transfer. A statistically significant association was demonstrated between percent DNA profile deposited on plastic tubes, through unwashed hands, and the age of male individuals.

Deciphering the maternal ancestral lineage of Greek Cypriots, Armenian Cypriots and Maronite Cypriots.

Cyprus was conquered from several populations because of its special geographical location. In this study, 406 unrelated Cypriot samples were tested based on their mitochondrial DNA. In more detail, 185 were Greek Cypriots, 114 Armenian Cypriots and 107 Maronite Cypriots. This is the first time where the mitochondrial DNA of Greek Cypriots, Armenian Cypriots and Maronite Cypriots is compared with the aim of characterizing the maternal ancestry of Cypriots. The control region of the mtDNA is the most informative in terms of studying maternal ancestry and consists of three hypervariable regions (HVS-I, HVS-II, HVS-III). The hypervariable regions can provide important information regarding the maternal ancestor of the tested samples. The entire control region of the mtDNA was used to determine the mitotypes and subsequently the haplogroups of all the Cypriot DNA samples. Based on the aforementioned analyses, Greek Cypriots were found to be genetically closer to Armenian Cypriots, while Greek Cypriots and Armenian Cypriots showed moderate genetic differentiation with Maronite Cypriots. The most prevalent haplogroups among Cypriots were haplogroups H and U, while R0 is common but in different frequencies for Greek Cypriots, Armenian Cypriots and Maronite Cypriots. It is proposed that the maternal ancestor may have originated during the Neolithic period and/or the Bronze age.

Correction: Comparative Y-chromosome analysis among Cypriots in the context of historical events and migrations.

[This corrects the article DOI: 10.1371/journal.pone.0255140.].

The Cyprus Institute of Neurology and Genetics, an emerging paradigm of a gender egalitarian organisation.

Females are underrepresented in the science, technology, engineering, mathematics and medicine (STEMM) disciplines globally and although progress has been made, the gender gap persists. Our aim was to explore gender parity in the context of gender representation and internal collaboration at the Cyprus Institute of Neurology and Genetics (CING), a leading national biomedical organisation accredited as an equal opportunity employer. Towards this aim we (1) explored trends in gender parity within the different departments, positions and qualifications and in student representation in the CING's postgraduate school and, (2) investigated the degree of collaboration between male and female researchers within the Institute and the degree of influence within its co-authorship network. We recorded an over-representation of females both in the CING employees and the postgraduate students. The observed female over-representation in pooled CING employees was consistent with a similar over-representation in less senior positions and was contrasted with an observed male over-representation in only one middle rank and culminated in gender equality in the top rank in employee hierarchy. In terms of collaboration, both males and females tended to collaborate with each other without any significant preference to either inter-group or intra-group collaboration. Further comparison of the two groups with respect to their influence in the network in terms of occupying the positions of highest centrality scores, indicated that both gender and seniority level (head vs non-head) were significant in shaping the authors' influence, with no significant difference in those belonging in the same seniority level with respect to their gender. To conclude, our study has validated the formal recognition of the CING's policies and procedures pertinent to its egalitarian culture through the majority of the metrics of gender equality assessed in this study and has provided an extendable paradigm for evaluating gender parity in academic organizations.

Comparative Y-chromosome analysis among Cypriots in the context of historical events and migrations.

Y-chromosome analysis provides valuable information regarding the migration patterns of male ancestors, ranging from the Paleolithic age to the modern humans. STR and SNP genotyping analysis provides data regarding the genetic and geographical ancestry of the populations studied. This study focused on the analysis of the Y-chromosome in Maronite Cypriots and Armenian Cypriots, who came to the island as a result of different historical events. The aim was to provide information on the paternal genetic ancestry of Maronites and Armenians of Cyprus and investigate any affinity with the Greek Cypriots and Turkish Cypriots of the island. Since there is limited information in the current literature, we proceeded and used 23 Y-chromosome STRs and 28 Y-chromosome SNPs to genotype 57 Maronite Cypriots and 56 Armenian Cypriots, which were then compared to data from 344 Greek Cypriots and 380 Turkish Cypriots. All samples were assigned to eight major Y-haplogroups but the most frequent haplogroup among all Cypriots is haplogroup J in the major subclade J2a-L559. The calculated pairwise genetic distances between the populations show that Armenian Cypriots are genetically closer to Greek and Turkish Cypriots compared to Maronite Cypriots. Median Joining Network analysis in 17 Y-STR haplotypes of all Cypriots assigned to J2a-L559, revealed that Cypriots share a common paternal ancestor, prior to the migration of the Armenians and Maronites to Cyprus, estimated in the Late Bronze Age and Early Iron Age.

Genetic variation in genes interacting with BRCA1/2 and risk of breast cancer in the Cypriot population.

Inability to correctly repair DNA damage is known to play a role in the development of breast cancer. Single nucleotide polymorphisms (SNPs) of DNA repair genes have been identified, which modify the DNA repair capacity, which in turn may affect the risk of developing breast cancer. To assess whether alterations in DNA repair genes contribute to breast cancer, we genotyped 62 SNPs in 29 genes in 1,109 Cypriot women with breast cancer and 1,177 age-matched healthy controls. Five SNPs were associated with breast cancer. SNPs rs13312840 and rs769416 in the NBS1 gene were associated with a decrease in breast cancer risk (OR TT vs. TC/CC = 0.58; 95% CI, 0.37-0.92; P = 0.019 and OR GG vs. GT/TT = 0.23, 95% CI 0.06-0.85, P = 0.017, respectively). The variant allele of MRE11A rs556477 was also associated with a reduced risk of developing the disease (OR AA vs. AG/GG = 0.76; 95% CI, 0.64-0.91; P = 0.0022). MUS81 rs545500 and PBOV1 rs6927706 SNPs were associated with an increased risk of developing breast cancer (OR GG vs. GC/CC = 1.21, 95% CI, 1.02-1.45; P = 0.031; OR AA vs. AG/GG = 1.53, 95% CI, 1.07-2.18; P = 0.019, respectively). Finally, haplotype-based tests identified significant associations between specific haplotypes in MRE11A and NBS1 genes and breast cancer risk. Further large-scale studies are needed to confirm these results.

Identification of novel splice mutation in SMAD3 in two Cypriot families with nonsyndromic thoracic aortic aneurysm. Two case reports.

BACKGROUND: Thoracic aortic aneurysm and dissection (TAA/D) represents a potentially lethal disease group characterized by an increased risk of dissection or rupture. Only a small percentage (approximately 30%) of individuals with nonsyndromic familial TAA/D have a pathogenic variant in one of the genes that have been found to be associated with the disease. METHODS: A targeted sequencing panel and direct sequencing approach were used to identify causative mutations in the index patients and other family members. RESULTS: In this study we report two apparently unrelated Cypriot families with nonsyndromic familial TAA/D. The proband A is a female patient diagnosed with TAA/D and intracranial aneurysm and opted for an elective intervention. The proband B is a male patient who was diagnosed with TAA/D and underwent cardiac surgery. Sequencing analysis identified a novel splice site variant (c.871+1G>A) in SMAD3 which is shown to be associated with the disease. Analysis of mRNA from the patient's tissue confirmed aberrant splicing and exon 6 skipping. CONCLUSION: Our findings expand the mutation spectrum of variants that have been shown to be associated with nonsyndromic familial TAA/D. This study demonstrates the importance of a comprehensive clinical and genetic evaluation aiming at early diagnosis and intervention.

MicroRNAs in ascending thoracic aortic aneurysms.

Thoracic Aortic Aneurysm (TAA) is characterized by the dilation of the aorta and is fatal if not diagnosed and treated appropriately. The underlying genetic mechanisms have not been completely delineated, so better knowledge of the physiopathology of TAAs is needed to improve detection and therapy. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and are known to be involved in cardiovascular diseases (CVDs). The current study aimed to identify miRNAs that can be used as possible biomarkers for the early diagnosis of patients with ascending TAAs (ATAAs). MiRNA expression was profiled by NanoString nCounter technology using 12 samples including tissue and pre- and post-surgical plasma from ATAA patients. Four miRNAs were selected and further validated by real time polymerase chain reaction (RT-PCR) in 22 plasma samples from which three miRNAs (hsa-miR140-5p, hsa-miR-191-5p and hsa-miR-214-3p) showed significant expression level differences between the two types of plasma samples. Further analyses of the corresponding predicted target genes by these miRNAs, revealed two genes (Myotubularin-related protein 4 (MTMR4) and Phosphatase 1 catalytic subunit ß (PPP1CB)) whose expression was inversely correlated with the expression of their respective miRNAs. Overall, in this pilot study, we identified three miRNAs that might serve as potential biomarkers and therapeutic targets in ATAA.

Effect of paraoxonase 1 polymorphisms on the response of lipids and lipoprotein-associated enzymes to treatment with fluvastatin.

BACKGROUND: Decreased paraoxonase 1 (PON1) and increased total serum lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activities are suggested to be risk factors for vascular disease. Common PON1 genetic polymorphisms (Q192R and L55M) significantly affect PON1 activity and may also influence high-density lipoprotein (HDL)-associated Lp-PLA(2) activity. However, little is known about the possible effect of PON1 common genetic polymorphisms on the response of lipids as well as PON1 and Lp-PLA(2) activities to treatment with statins. METHODS: Two hundred two hypercholesterolemic patients were treated with fluvastatin 40 mg/day. Fasting serum lipids, Q192R and L55M PON1 polymorphisms as well as PON1 and Lp-PLA(2) (total serum and HDL-associated) activities were determined before and after 6 months of treatment. RESULTS: Fluvastatin treatment did not affect HDL-cholesterol or apolipoprotein (apo) AI but resulted in significant decreases in total cholesterol, triglycerides, low-density lipoprotein-cholesterol, apo B and apo E, as well as total serum Lp-PLA(2) activity. In contrast, PON1 activity significantly increased. None of these changes was influenced by Q192R or L55M PON1 polymorphisms. Overall, HDL-Lp-PLA(2) did not change but L55M polymorphism significantly influenced its response to fluvastatin. Specifically, LL homozygotes experienced a significant increase, while M carriers (LM or MM) experienced a non-significant decrease in HDL-Lp-PLA(2) activity (p = 0.030 between groups). CONCLUSIONS: Q192R and L55M PON1 polymorphisms did not affect the response of lipids, PON1 and total serum Lp-PLA(2) to treatment with a statin. However, L55M PON1 polymorphism significantly modulated the response of HDL-Lp-PLA(2). It should be noted that this is an association study and therefore provides no proof but only indication that PON1 may also exert Lp-PLA(2) activity in HDL.

Y-chromosomal analysis of Greek Cypriots reveals a primarily common pre-Ottoman paternal ancestry with Turkish Cypriots.

Genetics can provide invaluable information on the ancestry of the current inhabitants of Cyprus. A Y-chromosome analysis was performed to (i) determine paternal ancestry among the Greek Cypriot (GCy) community in the context of the Central and Eastern Mediterranean and the Near East; and (ii) identify genetic similarities and differences between Greek Cypriots (GCy) and Turkish Cypriots (TCy). Our haplotype-based analysis has revealed that GCy and TCy patrilineages derive primarily from a single gene pool and show very close genetic affinity (low genetic differentiation) to Calabrian Italian and Lebanese patrilineages. In terms of more recent (past millennium) ancestry, as indicated by Y-haplotype sharing, GCy and TCy share much more haplotypes between them than with any surrounding population (7-8% of total haplotypes shared), while TCy also share around 3% of haplotypes with mainland Turks, and to a lesser extent with North Africans. In terms of Y-haplogroup frequencies, again GCy and TCy show very similar distributions, with the predominant haplogroups in both being J2a-M410, E-M78, and G2-P287. Overall, GCy also have a similar Y-haplogroup distribution to non-Turkic Anatolian and Southwest Caucasian populations, as well as Cretan Greeks. TCy show a slight shift towards Turkish populations, due to the presence of Eastern Eurasian (some of which of possible Ottoman origin) Y-haplogroups. Overall, the Y-chromosome analysis performed, using both Y-STR haplotype and binary Y-haplogroup data puts Cypriot in the middle of a genetic continuum stretching from the Levant to Southeast Europe and reveals that despite some differences in haplotype sharing and haplogroup structure, Greek Cypriots and Turkish Cypriots share primarily a common pre-Ottoman paternal ancestry.

Allele distribution of 15 STR loci used for human identity purposes in the Greek Cypriot population of the island of Cyprus.

Allele frequencies for 15 STRs (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, THO1, Penta E, D16S539, CSF1PO, Penta D and TPOX) in the PowerPlex-16 System (Promega Corporation) were derived from a sample of 1475 unrelated Greek Cypriot individuals from the island of Cyprus.

Apolipoprotein E genotype in matched men and women with coronary heart disease.

Apolipoprotein E (apo E) plays an important role in lipid metabolism and its polymorphism may be a risk determinant of coronary heart disease (CHD). Since evidence suggested a gender-specific effect of apo E polymorphism, we studied the influence of gender-specific interaction of the polymorphism on CHD. From a total of 463 Greek Caucasians (314 men and 149 postmenopausal women) with angiographically documented CHD, we selected 79 women (68+/- 9 yr old) and 79 men (66+/- 9 yr old) who were matched for clinical characteristics. Apo E genotyping was performed by PCR and RFLP analysis. Biochemical parameters were also measured. The results were as follows: the E3/3 genotype occurred in 78.5% of the patients, followed by E3/4, E2/3, E2/4, and E4/4 genotypes, which occurred in 9.5%, 9.5%, 1.9%, and 0.6% of the patients, respectively. No significant differences were observed in the apo E allele or apo E genotype distributions between the matched Greek men and women with CHD. The E3/3 men patients were more frequently part of a family with a history of CHD, compared to women (p=0.035).

Association of apolipoprotein E genotype with early onset of coronary heart disease in Greek men.

Apolipoprotein (apo) E polymorphism has been associated with coronary heart disease (CHD) although its relation to the age of CHD onset is still not defined. The age of onset of established CHD was obtained from 502 Greek men and compared to 103 healthy men. The age grouping was based on the age of CHD onset (earlier < or =44 years, n = 73, intermediate 45-64 years, n = 321, and later > or =65 years, n = 108). Apo E genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the lipid profile was assessed. No differences in genotype and allele frequencies were found within the CHD groups. The apo epsilon3/4 genotype and the apo epsilon4 allele were less frequent in the earlier-onset group than in healthy men (11.0 % vs 22.3%, Pearson Chi-Square p = 0.028 and 6.8% vs 13.6%, Pearson Chi-Square p = 0.023, respectively). The lipid profile was similar in all genotypes of all groups except for high-density lipoprotein cholesterol levels, which were higher in epsilon2 carriers compared to non-epsilon2 carriers (in mg/dL [+/-SD]; 44 [9] vs 39 [10], in mmol/L [+/-SD]; 1.1 [0.2] vs 1.0 [0.3] p = 0.005). There is an association between apo E genotype and early onset of CHD in Greek men. In the earlier CHD onset group, the apo epsilon3/4 genotype was less frequent compared to healthy men. This supports that the apo epsilon3/4 genotype is associated with decreased risk of premature CHD. Because the results of similar studies are not consistent, it may be that the relationship between apo E genotype and CHD is related to ethnicity rather than a universal phenomenon.

Association of apolipoprotein E polymorphism with myocardial infarction in Greek patients with coronary artery disease.

Studies in several populations have indicated that genetic variation at the apolipoprotein E (apoE) structural locus influences the risk of coronary artery disease (CAD) and myocardial infarction (MI). This study aimed at investigating whether apoE polymorphism has an allelic and/or genotypic impact on the risk of MI in Greek patients with CAD. We compared apoE gene polymorphism in a group of patients with angiographically confirmed CAD but not MI [CAD/MI (-)-group, n = 143] and a group of age and sex-matched CAD patients who had experienced a non-fatal Ml [CAD/MI (+)-group, n = 124]. The patients were also compared with a group of healthy younger individuals (n = 240) with no family history of CAD. The apoE genotype distribution differed significantly between the two groups of CAD patients (p = 0.02). The epsilon2 allele was 5.3-fold less frequent in the CAD/ MI (+)-group compared with the CAD/MI (-)-group (1.2% vs. 6.3%, p = 0.01). The frequency of the epsilon2 allele in healthy subjects was 8.1%, which is 6.8-fold higher than in CAD/MI (+)-patients (p = 0.001) and twice as high compared with all CAD patients (p = 0.02). No differences in epsilon4 allele frequencies were observed between CAD/MI (+)- and CAD/MI (-)-patients (10.9% vs. 9.8%), or between patients with CAD and healthy subjects (10.3% vs. 10.2%). In summary, the epsilon4 allele was not found to be associated with an increased risk for CAD or MI. In contrast, a negative association of the epsilon2 allele with Ml was observed among Greek patients with CAD.

The ACE gene and endurance performance during the South African Ironman Triathlons.

PURPOSE: Several studies have suggested that the insertion (I) variant rather than the deletion (D) variant of the human angiotensin-converting enzyme (ACE) gene is associated with elite endurance performance. The aim of this study was to determine whether the ID polymorphism is associated with the performance of the fastest finishers of the South African Ironman Triathlons. METHODS: A total of 447 Caucasian male triathletes of a variety of nationalities and athletic ability who completed either the 2000 or 2001 South African Ironman Triathlons and 199 Caucasian male control subjects were genotyped for the ACE ID polymorphism. RESULTS: There was a significantly higher frequency of the I allele in the fastest 100 South African-born finishers (103 I, 51.5% and 97 D, 48.5%) compared with the 166 South African-born control subjects (140 I, 42.2% and 192 D, 57.8%) (P = 0.036). There was also a significant linear trend for the allele distribution among the fastest 100 finishers (I allele = 51.5%), slowest 100 finishers (I allele = 47.5%), and control (I allele = 42.2%) South African-born subjects (P = 0.033). There was, however, no significant difference in the ACE genotype or allele frequencies when athletes born outside South Africa were analyzed. CONCLUSION: To our knowledge this is the first study that has examined the effect of an athlete's ACE genotype on their actual performance during an ultra-endurance race. The I allele of the ACE gene was associated with the endurance performance of the fastest 100 South African-born finishers in these triathlons.

HDL cholesterol levels in patients with molecularly defined familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is the most common genetic disorder leading to premature atherosclerosis. Typically, it is due to mutations in the LDL receptor gene resulting in elevated total and LDL cholesterol levels. The type of the LDL receptor gene mutations may affect the severity of hypercholesterolemia and consequently the incidence of coronary atherosclerosis. Furthermore, high-density lipoprotein (HDL) cholesterol levels have been recently shown to be an independent risk factor for coronary heart disease in this population. We examined the effect of the type of the LDL receptor gene mutations and of common gene polymorphisms possibly affecting HDL metabolism [cholesterol ester transfer protein (CETP), apolipoprotein A-IV (ApoA-IV), angiotensin converting enzyme (ACE), and apolipoprotein E (ApoE)] on HDL cholesterol levels in patients with molecularly defined heterozygous FH who were attending our lipid clinic (n=84). The nature of the LDL receptor gene mutation (81T>G, n=12; 858C>A, n=13; 1285G>A, n=12; 1646G>A, n=22; and 1775G>A, n=25) did not significantly influence HDL cholesterol levels. Unlike other gene polymorphisms, the apolipoprotein (apo) E gene polymorphism did significantly affect these levels. In fact, the presence of the E4 allele was associated with lower HDL cholesterol levels compared to patients not carrying this allele. We conclude that HDL cholesterol levels in heterozygous FH patients may be affected by the apoE gene polymorphism.

The epsilon 2 and 4 alleles of apolipoprotein E and ischemic vascular events in the Greek population--implications for the interpretation of similar studies.

The authors investigated whether apolipoprotein (apo) E polymorphism has an allelic and/or genotypic impact on the risk of an ischemic vascular event (IVE) in Greek patients with cardiovascular diseases (CVD). They compared apo E polymorphisms in 1) a group of 165 patients with IVE [IVE(+)], of whom 107 had survived a myocardial infarction and 58 an ischemic stroke; 2) a group of 165 patients, matched with the first group for age and gender, with angiographically confirmed coronary artery disease but without IVE [IVE(-)]; 3) a group of 240 healthy younger individuals with no family history of CVD. The apo epsilon2 allele was 5.2-fold less frequent in the IVE(+) group compared to the IVE(-) group (1.2% vs 6.2%, p = 0.001). The frequency of the epsilon2 allele in healthy subjects was 8.1%, which is 6.7-fold higher than in the IVE(+) group (p < 0.001), and more than twice as high compared to all CVD patients (p = 0.001). No significant differences in epsilon4 allele frequencies were observed between IVE(+) and IVE(-) patients (9.8% vs 8.4%) or between patients with CVD and healthy subjects (9.1% vs 10.2%). The epsilon4 allele was not associated with an increased risk for CVD or IVE. In contrast, an inverse and beneficial association of the epsilon2 allele with IVE was observed among Greek patients with CVD. These results suggest that the epsilon4 and epsilon2 alleles have a variable significance in terms of predicting the risk of vascular events in different populations. Therefore, it is important to carry out "local" studies.

Spectrum of LDLR gene mutations, including a novel mutation causing familial hypercholesterolaemia, in North-western Greece.

BACKGROUND: Familial Hypercholesterolaemia (FH) is a clinical syndrome characterised by elevated serum low-density lipoprotein (LDL) cholesterol, by tendon xanthomata and clinical manifestations of ischaemic heart disease in early life. Typically, it results from mutations in the low-density lipoprotein receptor (LDLR) gene. Furthermore, there are 3 additional genetic disorders that cause clinical syndromes that mimic FH. These are: 1) familial ligand-defective apolipoprotein (apo)-B (FLDH), 2) familial hypercholesterolaemia type 3 (FH3) and 3) autosomal recessive hypercholesterolaemia (ARH). The aim of this study was to elaborate the impact of the above genetic disorders in Greek patients with a clinical diagnosis of FH. METHODS: In this study, we assessed the contribution of the LDLR, Apo B, ARH and PCSK9 genes in the expression of FH in North-western Greece. Two hundred and fifty-four (254) probands with a clinical diagnosis of FH were included in the study. RESULTS: One hundred and sixty-nine (169) patients had one of the following LDLR gene mutations: 81T>G, 1775G>A, 517T>C, 858C>A, 1352T>C, 1285G>A, 761A>C, 1195G>A, 1646G>A and a deletion mutation g.387-410del24 in exon 4. We sequenced the Apo B, ARH and PCSK9 genes in 40, randomly selected patients, from the 85 patients with no identified LDLR gene defects. In these 40, randomly selected patients, with the exception of benign single nucleotide polymorphisms, no functional mutations were identified for all the above mentioned sequenced genes. CONCLUSION: Our results reveal substantial genetic heterogeneity for FH in North-western Greece with at least ten LDLR gene mutations present in the study population. One of these mutations although quite rare is reported here for the first time in the scientific literature. The detection of these mutations is important as they may be used to design multiplex detection assays for large scale population screening programmes to facilitate primary and secondary prevention of cardiovascular disease in the region. Finally, ARH, Apo B and PCSK9 gene defects were excluded from causing FH in a subgroup of the study population indicating that other yet unrecognized genes may be involved in causing the clinical feature of FH, and/or that large scale deletions/duplications evaded the applied mutation detection techniques of this study.