ANALYSIS OF GENETIC VARIANTS IN PORPHYRIA: SPLICING REGULATORY SITES, COSEGREGATION OF POLYMORPHIC VARIANTS AND HAPLOTYPE CONSTRUCTIONS. RISK ASSESSMENT AND DIAGNOSIS.

MARCUCCI VALERIA; CERBINO GABRIELA; COLOMBO, FEDERICO; GEREZ, ESTHER; BATLLE, ALCIRA; PARERA, VICTORIA ESTELA; ROSSETTI MARÍA VICTORIA

CORDOBA

Congreso; 2° Congreso Argentino de Bioinformática y Biología Computacional; 2011

Background Human porphyrias are metabolic disorders, hereditary or acquired, resulting from partial enzymes deficiencies of haem biosynthetic pathway.  The low prevalence of porphyrias makes necessary the use of bioinformatics tools: database for determining the allelic frequencies of genetic variants in different populations, its effect and possible associations in cis. Here we present the usefulness of some of these tools for association between single nucleotide polymorphisms (SNPs), its effects on splicing regulatory sites and haplotype construction. Materials and methods We have investigated mutations and polymorphisms in 3 different porphyrias, and their associations on its expression (Table 1).  Polymorphisms were studied in 119 non-consanguineous families and 110 control volunteers. All flanking sequences of genetic variants were amplified by PCR and automatically sequenced, digested with specific endonucleases or HemiNested Aso PCR. To study the effect of variants involved in splicing regulatory elements ESEFinder 3.0 and AidSplicing were used.  For the haplotype construction and determination of cis associations between polymorphic variants, Phase and Haploview software were used.    Table 1 Porphyria Gene Variant (SNPs) Family number Acute Intermittent Porphyria (AIP) Porphobilinogen  Deaminase (PBGD) g.3119 G>T 22 g.3581 A>G g.3982 T>C g.6479 G>T g.7064 C>A g.7539 C>T Porphyria Cutanea Tarda (PCT) Uroporphyrinogen Decarboxylase (URO-D) c.1-263 T>A 79 c.603 A>G c.636+30 G>T Erythropoietic Protoporphyria (EPP) Ferrochelatase (FECH) c.1-251A>G 18 c.68-23C>T c.315-48T>C c.798G>C c.921G>A Results Analysis of splicing regulatory elements suggested that the three variants studied in the URO-D gene modify the activity of these sites, as well as two variants in FECH gene. Only c.636 +30 G> T (Intronic Splicing Silencer) presented significative differences between PCT patients and controls volunteers (allelic frequency 0.802 and 0.698 respectively). A common haplotype that cosegregated with p.G111R   mutation in AIP (the most common in Argentina), was not detected in control. In our population, c.1-251A>G, c.68-23C>T and c.315-48T>C variants of the FECH gene showed high levels of cosegregation, observing prevalence of the haplotype formed by the ancestral variants ACT (69.71%) while polymorphic variant GTC was found in only 16.90% of the alleles. Conclusion Clinically some specific gene sequence variants would seemed neutral leading to problems for genetic counseling. In these cases bioinformatic tools provide approximation that leads better conclusions. Therefore, the knowledge of the role of an observed sequence variant and how it influences the protein functionality could be useful to establish the degree of penetrance of the disease and its possible future clinical complication.