Angelica Leccese

Mutation of the angiopoietin-1 gene (ANGPT1) associates with a new type of hereditary angioedema

Background: Hereditary angioedema (HAE) is a rare genetic disease usually caused by mutation in the C1 inhibitor or the coagulation Factor XII gene. However, in a series of patients with HAE, no causative variants have been described, and the pathophysiology of the disease remains unknown (hereditary angioedema with yet unknown genetic defect [U‐HAE]). Identification of causative genes in patients with U‐HAE is valuable for understanding the cause of the disease. Objective: We conducted genetic studies in Italian patients with U‐HAE to identify novel causative genes. Methods: Among patients belonging to 10 independent families and unrelated index patients with U‐HAE recruited from the Italian Network for C1‐INH‐HAE (ITACA), we selected a large multiplex family with U‐HAE and performed whole‐exome sequencing. The angiopoietin‐1 gene (ANGPT1) was investigated in all patients with familial or sporadic U‐HAE. The effect of ANGPT1 variants was investigated by using in silico prediction and plasma and transfected cells from both patients and control subjects. Results: We identified a missense mutation (ANGPT1, c.807G>T, p.A119S) in a family with U‐HAE. The ANGPT1 p.A119S variant was detected in all members of the index family with U‐HAE but not in asymptomatic family members or an additional 20 patients with familial U‐HAE, 22 patients with sporadic U‐HAE, and 200 control subjects. Protein analysis of the plasma of patients revealed a reduction of multimeric forms and a reduced ability to bind the natural receptor tunica interna endothelial cell kinase 2 of the ANGPT1 p.A119S variant. The recombinant mutated ANGPT1 p.A119S formed a reduced amount of multimers and showed reduced binding capability to its receptor. Conclusion: ANGPT1 impairment is associated with angioedema, and ANGPT1 variants can be the basis of HAE.

In vitro residual activity of phenylalanine hydroxylase variants and correlation with metabolic phenotypes in PKU.

Hyperphenylalaninemias (HPAs) are genetic diseases predominantly caused by a wide range of variants in the phenylalanine hydroxylase (PAH) gene. In vitro expression analysis of PAH variants offers the opportunity to elucidate the molecular mechanisms involved in HPAs and to clarify whether a disease-associated variant is genuinely pathogenic, while investigating the severity of a metabolic phenotype, and determining how a variant exerts its deleterious effects on the PAH enzyme. To study the effects of gene variants on PAH activity, we investigated eight variants: c.611A>G (p.Y204C), c.635T>C (p.L212P), c.746T>C (p.L249P), c.745C>T (p.L249F), c.809G>A (p.R270K), c.782G>C (p.R261P), c.587C>A (p.S196Y) and c.1139C>T (p.T380M), associated with different phenotypic groups. Transient expression of mutant full-length cDNAs in COS-7 cells yielded PAH proteins with PAH activity levels between 7% and 51% compared to the wild-type enzyme. With one exception (p.Y204C, which had no significant impact on PAH function), lower PAH activity was associated with a more severe phenotype (e.g. p.L249P with 7% PAH activity, 100% of classic PKU and no BH4 responsiveness), while higher activity correlated with milder phenotypes (e.g. p.T380M with 28% PAH activity, 97% of mild HPA and 83% of BH4 responsiveness). The results of the in vitro residual PAH activity have major implications, both for our understanding of genotype-phenotype correlations, and thereby existing inconsistencies, but also for the elucidation of the molecular basis of tetrahydrobiopterin (BH4) responsiveness.

Lack of genotypephenotype correlation in congenital adrenal hyperplasia due to a CYP21A2-like gene

CONTEXT Congenital Adrenal Hyperplasia (CAH) due to 21-hydroxylase deficiency, encoded by CYP21A2 gene, is an autosomal recessive disorder. The CYP21A2 gene, localized in a genetic unit defined RCCX module, is considered one of the most polymorphic of human genes. OBJECTIVES We considered new evidences about the presence of a RCCX trimodular haplotype with a CYP21A2-like gene to explain the lack of a genotype-phenotype correlation in individuals of two different families. DESIGN AND METHODS To verify gene duplication we used Multiplex Ligation Probe-Dependent Amplifications (MLPA) and to confirm the presence of a CYP21A2-like gene downstream TNXA gene we used previously described amplification and restriction strategy followed by the sequencing of the CYP21A2 gene downstream TNXB gene. RESULTS The amplification strategy and restriction analysis of CYP21A1P/CYP21A2-TNXA PCR product in association with MLPA assay and sequencing of CYP21A2 gene downstream TNXB were able to identify the presence of the CYP21A2-like gene in healthy subjects of the two families, wherein the direct sequencing of CYP21A2 gene showed genotypes correlated to pathological phenotypes. CONCLUSIONS The strategy suggested is useful to facilitate molecular testing in CAH patients, considering the new evidence about possible different haplotypes.

Identification of ten novel mutations in factor VIII gene: A study of a cohort of 52 haemophilia A patients

Introduction: Haemophilia A (HA) is the most common X-linked recessive genetic disease caused by mutations in the gene coding for coagulation factor VIII (FVIII) resulting in spontaneous bleeding. Aim: The aim of our study is to provide additional information about the genetic causes of HA describing the correlation between the observed mutations and the clinical phenotype in a cohort of 52 patients suffering from HA to different degrees. Methods: First we performed a search of inversion 22 (IVS22) and of the intron 1 (IVS1) for severe HA patients; inversions negative and moderate/mild HA patients were screened by direct sequencing of the coding regions and exon-intron junctions of FVIII gene. Where no PCR amplification was observed we used the Multiplex Ligation-dependent Probe Amplification (MLPA) and, to confirm a large deletion, we performed an array-comparative Genomic Hybridization (array CGH). Results: 42% of severe HA patients had IVS22 while the remainder had missense mutations, large deletions and small insertions. All but one moderate/mild HA patients had missense mutations. Only two patients did not show good correlation between the genotype and the clinical phenotype reported to us by the respective centers of haemophilia. We also identified 10 novel mutations not previously described in the most common mutation database. Conclusion: We studied a group of 52 HA patients and we found 26 mutations in the FVIII gene of which 10 are new mutations. These results confirm the great heterogeneity of the molecular defects gene responsible for the deficiency of FVIII.

The first case of a small supernumerary marker chromosome derived from chromosome 10 in an adult woman with an apparently normal phenotype

Abstract Small supernumerary marker chromosomes (sSMCs) originating from chromosome 10 are rare. A limited number of cases are documented. We report a new diagnosis of a mosaic sSMC (10) in a normal female who asked for genetic evaluation before undergoing controlled ovarian hyperstimulation, in vitro fertilization, and embryo transfer. Chromosome preparations from peripheral lymphocyte cultures were performed according to standard procedures. QFQ-banded chromosomes confirmed the presence of an sSMC: 47,XX,+mar[49]/46,XX[51]. FISH and array CGH analysis showed that the sSMC consisted of chromosome 10 with a gain of the 10p11.1p11.21 (2.5 Mb) chromosomal region. The presence of sSMC (10) was also confirmed in the patient’s mother and sister. It did not appear to affect the phenotype of the women who were phenotypically normal and healthy, and at the time of writing the woman became pregnant naturally. Phenotypes associated with an sSMC vary from normal to severely abnormal. It has been shown that variations in the chromosomal region of sSMCs result in observable differences in clinical outcome. The phenotypical consequences of sSMCs are difficult to predict because of differences in euchromatic DNA content, chromosomal origin, and varying degrees of mosaicism. Therefore, the continued investigation of a larger number of sSMC cases, in particular those originating from chromosome 10 that are the infrequently encountered and characterized, and a better understanding of the genetic content is important in order to improve the delineation of karyotype-phenotype correlation, contributing to a more informed prenatal counseling or prognosis.

Intra-familiar discordant PKU phenotype explained by mutation analysis in three pedigrees.

Classical phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are two phenotypes of phenylalanine hydroxylase (PAH) deficiency with different degrees of severity. We have analyzed three families in which classical PKU, MHP and a normal phenotype occurred within each family due to the different combinations of three mutations segregating within the family. Indeed, sequence PAH analysis revealed three different alleles segregating in each family. This report suggests that when discordant phenotypes occur in a family, complete analysis of the PAH gene may be performed in order to support the diagnosis and assist in accurate genetic counseling and patient management. We further support the marked heterogeneity of hyperphenylalaninemia primarily due to allelic heterogeneity at the PAH locus.