Congresso Nazionale del Trentennale – Ferrara, 2016

Genetica e Malattia Venosa Cronica

Roberto Pola, MD PhD Divisione di Clinica Medica e Malattie Vascolari Fondazione Policlinico Universitario A. Gemelli Università Cattolica del Sacro Cuore, Roma

Family Studies

Cornu-Thenard A, Boivin P, Baud JM, De Vincenzi I, Carpentier PH. Importance of the familial factor in varicose disease. Clinical study of 134 families. J Dermatol Surg Oncol 1994.

Risk of developing varicose veins:

- 90% when both parents had varicose veins

- 62% in women with one parent that had varicose veins

- 25% in men with one parent that had varicose veins

prospective case-control study

Family Studies

Ahti TM, Makivaara LA, Luukkaala T, Hakama M, Laurikka JO. Effect of family history on the incidence of varicose veins: a population-based follow-up study in Finland. Angiology 2009.

Risk of developing varicose veins:

- 1,6 fold-increase in those with a family history of varicosities

longitudinal study

Family Studies

Methods used in genetic studies of Chronic Venous Disease (CVD)

1. Family studies: Positive family history in patients with venous disease has been reported as early as 1855.

2. Gene expression studies:

Candidate gene approach: A candidate gene approach is based on the search for mutations in those genes for which there is a reasonable likelihood of involvement in CVD. This would be based on prior assumptions, for example, looking for variation in a gene known to influence a key relevant enzyme or protein structure or critical pathway.

Genome-wide analysis: A technology in which many (500,000–5,000,000) single-nucleotide polymorphism (SNP) markers are identified across the whole genome for an individual in a single analysis. Algorithms are applied that compare the frequencies of SNPs between disease and control cohorts.

3. Next generation sequencing: New technology in which thousands-to-many-millions of sequencing reactions are produced in parallel. It makes possible to examine a large number of genes in a single reaction and to perform large-scale analyses of the patient’s genome in a short time.

Which Genes? The Candidate Gene approach

The C677T methylenetetrahydrofolate reductase (MTHFR) (marker of cardiovascular risk): 98 patients with varicose veins and 297 controls. They found a significantly higher prevalence of subjects with at least one C677T MTHFR allele among those with varicose veins than among a control group (odds ratio 1.74). (Mol Genet Metab 1998;63:35–6). Thrombomodulin (TM) (Marker of endothelial injury): 327 patients with venous thrombosis and 398 controls. 19% of the VT patients investigated had varicose veins. VT patients with the del TT allele were more likely to have varicose veins than patients with the wild-type allele (33% versus 17%, P <0.007). (this post hoc analysis was not originally designed to identify risk factors for varicose veins). (Arterioscler Thromb Vasc Biol 1999;19:1098–104). FOXC2 gene mutation was the cause of the rare inherited condition of lymphoedema distichiasis a condition in which varicose veins are a common feature. (marker of primary venous valve failure) 2060 female twin pairs they concluded that there was a functional variant within the vicinity of the FOXC2 gene which predisposed individuals to varicose veins. A further study by these workers showed that every participant (18) with the FOXC2 mutation had evidence of venous reflux on duplex ultrasound scanning, compared with one in 12 controls (P, 0.0001). The valves in the superficial veins were always affected by the FOXC2 mutation and in 78% of the deep system. (Circulation 2007;115:1912–20)

Which Genes? The Candidate Gene approach

Which Genes? The Candidate Gene approach

• Three studies agree that SNPs in FOXC2, a transcription factor involved in lymphatic and vascular development, provide a strong link toward venous varicosity, valve failure, and hemorrhoids.

• Polymorphism in the HFE gene increases the risk of venous ulceration by almost 7-fold, while also

causing the known dysregulation in iron absorption. • The MMPs (and, to a lesser extent, their inhibitors, TIMPs) are perhaps the best studied in venous

disease. MMPs are involved in ECM degradation and structural vein wall changes, ultimately contributing to venous remodeling, dilation, and valve dysfunction. But the exact mechanisms have yet to be elucidated and further research is required in this area.

“Considering our limited knowledge of the biological pathways involved in the pathogenesis of venous diseases, we are not in a strong position to formulate

truly erudite a priori candidate gene hypothesis-directed studies”.

Genome Wide Analysis

None of the single-nucleotide polymorphisms in these genes have

shown to have significant association in this study.

GWAS of self-reported varicose veins in 20,000 cases and 65,000 controls of European ancestry. 1000 Genomes reference haplotypes were imputed. Several novel associations for varicose veins were found:

- the strongest association was with SNP rs507666 in the ABO gene, which encodes a protein defining the ABO blood group. Variation in SNP rs507666 determines A1 blood subgroup status. This finding is consistent with a previous study that reported the association between the A blood group and varicose veins

- SNP rs11121615 in the CASZ1 gene, associated with blood pressure variation

- SNP rs1433196 in the ANGPT1 gene, that encodes angiopoietin 1

- SNP rs966562, which is near the ANGPT2 gene, that encodes angiopoietin 2

- SNP rs6905288, which is near the VEGF-A gene, which is important for angiogenesis, vasculogenesis and endothelial cell growth.

In Summary

There is strong circumstantial evidence that varicose veins and chronic venous disease have a genetic background, as found in several family studies

Investigations have so far produced only partially coherent results.

This is due to the fact that almost all studies have been conducted using the candidate gene approach, with small sample sizes and no replication cohorts

There must be further collaborations in order to successfully advance the field of venous disease genetics.

Future studies are needed to establish exact venous disease classification and control subjects will have to be thoroughly screened for venous insufficiency to avoid major bias.

Next generation sequencing: Il termine “Next Generation Sequencing” (NGS) definisce differenti tecnologie di sequenziamento parallelo del DNA che consentono di analizzare contemporaneamente e velocemente DNA o RNA misti.

Il Progetto Genoma Umano, basato su metodo Sanger e condotto dal 1990 al 2003 con il coinvolgimento di molti laboratori nel mondo ha permesso di sequenziare l’intero genoma. Il NGS rende il sequenziamento del genoma fattibile in circa 30 ore con un sequenziatore di medie dimensioni. Il sistema di più recente introduzione legge 18.000 miliardi di basi di DNA per test. Approcci: - “Single-gene panel approach” and “Target multigene sequencing” (mgTGS): differiscono dalle metodiche

classiche per la riduzione dei tempi necessari per ottenere l’analisi e dal numero di geni che possono essere contemporaneamente sequenziati. Necessitano di un’ipotesi a priori.

- “Whole genome sequencing” e “Whole esome sequencing”: permettono di sequenziare l’intero DNA (WGS) o “l’esoma” (WES) identificando mutazioni associate a condizioni patologiche in assenza di una ipotesi patofisiologica a priori e di geni candidati. Il WES ha permesso di identificare varianti geniche associate a patologie note la cui presenza può influire sulla penetranza e sull’espressività fenotipica della patologia. L’identificazione di nuovi loci associati a patologie note può inoltre svelare nuovi meccanismi patogenetici e nuovi target terapeutici. Utilizzati anche per lo studio del trascrittoma ovvero dell’RNA tradotto dalle cellule.

Guilty by association? or personally guilty?

GWAS are "designed to identify common variants...contributing to common disease."

These common variants -- ie, those that account for more than 5% of the allele

frequency -- are usually located in intronic regions of the chromosome, which don't

code for specific functional proteins. To claim an association between the gene and a

disease, these single-nucleotide polymorphisms (SNPs) have to lie hundreds of

kilobases (kb) up- or downstream from a known causative gene, or the SNPs have to

be in linkage disequilibrium with other SNPs of potential interest. Thus, in GWAS, a

SNP's involvement is inferred due to its being in a place or associated with a gene

known to have an effect on the disease or pathology of interest, much as a person

found in a given place and hanging out with the right (or "wrong") crowd may

become a "person of interest" in a criminal investigation. Whether the SNP is

involved at all, or is a victim of "guilt by association," then becomes an issue that

requires considerable probing.

By contrast, whole-exome sequencing looks at exonic regions of the

chromosome, which code for functional proteins. This is no longer a matter of

keeping bad company, but evidence of how the "crime" was committed, ie, exactly

how the protein was rendered nonfunctional and why a patient might be susceptible

to or actually have the disease in question.

La nostra proposta: il Progetto VENOMA

Illumina MiSeq - GenoSeq

Disponibile presso il nostro centro al Policlinico Gemelli

La nostra proposta: il Progetto VENOMA

Studio multicentrico italiano finalizzato all’identificazione di nuovi pathway fisiopatologici della Malattia Venosa Cronica ed eventuali nuovi target terapeutici: Discovery cohort: 30 soggetti di sesso maschile affetti da Insufficienza venosa cronica

primitiva con BMI <30; 30 soggetti di controllo maschi selezionati tra i familiari di I grado. - Registrazione dei dati anamnestici (età d’insorgenza della patologie,

familiari affetti, attività lavorativa, abitudini etc..). - Stratificazione dei pazienti secondo classificazione CEAP. - Studio ecocolorDoppler per valutazione del reflusso superficiale e

profondo. - Prelievo di sangue periferico

Da effettuare nel centro promotore

"Whole esome sequencing" per identificazione di un pannello focalizzato di alterazioni genetiche associate alla patologia.

Da effettuare nei centri partecipanti

La nostra proposta: il Progetto VENOMA

Replication cohort: 100 soggetti maschi affetti da Insufficienza venosa cronica primitiva con

BMI <30. 100 soggetti maschi di controllo selezionati tra i familiari di I grado con

BMI <30.

Identificazione di un pannello focalizzato di alterazioni genetiche associate alla patologia.

Da effettuare nel centro promotore

”Target multigene sequencing" per confermare l’associazione delle varianti identificate con la Malattia Venosa Cronica.

Arruolamento da effettuare nei centri

partecipanti