Molecular analysis and genotype-phenotype correlation in patients affected by hyperphenylalaninemias in southern italy

L’Iperfenilalaninemia (HPA), una malattia genetica autosomica recessiva, è il risultato di un difetto dell’attività dell’enzima Fenilalanina Idrossilasi (PAH), con conseguente accumulo plasmatico di Fenilalanina. Nella maggior parte dei casi (circa il 98%) l’espressione fenotipica della patologia è il risultato della presenza di mutazioni a carico del gene PAH, codificante per l’enzima fenilalanina idrossilasi, con locus 12q24.1 sul cromosoma 12. La patologia ha una frequenza nella popolazione caucasica di circa 1 ogni 10.000, corrispondente ad una frequenza di portatori di circa 1 a 50; la maggior parte degli individui affetti sono identificati nel periodo neonatale mediante programmi di screening (obbligatori in diversi Paesi, quali l'Italia) sulla popolazione generale. La HPA è espressa con una significativa variabilità di espressione fenotipica e con differenti gradi di gravità. La terapia alimentare, basata su una ristretta assunzione di fenilalanina, previene i danni neurologici. I fenotipi dell’Iperfenilalaninemia sono definiti in base ai livelli di fenilalanina nel sangue alla nascita: Fenilchetonuria classica (PKU), con valori di fenilalanina nel plasma >1200 μmol/L (>20mg/dl); forma lieve di PKU con livelli di fenilalaninemia compresi tra 600 and 1200 μmol/L (10-20 mg/dl); Iperfenilalaninemia non-PKU (HPA- non PKU o MHP) quando i valori di fenilalaninemia sono al di sotto di 600 μmol/L (<10 mg/dl). Ad oggi, tramite l’analisi molecolare del gene PAH, sono state individuate circa 600 mutazioni (la maggior parte di tipo missenso) e oltre il 50% in condizione di eterozigosi composita. Il numero considerevole di mutazioni individuate rappresenta una consistente evidenza di eterogeneità allelica alla base della patologia che ne giustifica l’espressività variabile (anche nell’ambito di una stessa famiglia). Il progetto ha avuto, innanzitutto, l’obiettivo di incrementare le conoscenze sui meccanismi molecolari e biochimici, ancora oggi non del tutto noti, che sono alla base dell’eziopatogenesi ed espressività variabile dell’HPA basandoci su un'attenta analisi delle mutazioni responsabili della patologia in pazienti provenienti dal Sud Italia. La caratterizzazione genotipica dei pazienti con elevati livelli di Phe individuati con lo screening neonatale viene spesso eseguita per completare la diagnosi. Da questi dati, abbiamo anche correlato i genotipi con le attività enzimatiche residue (PRA) ottenute in esperimenti di espressione in vitro ed elencate nel database PAHdb (http://www.pahdb.mcgill.ca/), eseguendo una correlazione genotipo-fenotipo. Nella sezione finale della tesi, si è puntato l’attenzione sulle implicazioni della PKU a livello metabolico, con particolare attenzione alla risposta al BH4: la quantificazione dell’attività dell’enzima PAH, espresso in cellule in coltura, è stata eseguita tramite la tecnica della spettrometria di massa tandem presso la sezione di malattie metaboliche della clinica universitaria di Heidelberg (Germania). La spettrometria di massa consente l'uso di isotopi stabili per la quantificazione della fenilalanina e della tirosina e la misura dell'attività dell’enzima PAH delle mutazioni PKU, consentendo di testare e predire la loro risposta al BH4 in un sistema cellulare di mammifero. In sintesi, i dati ottenuti in questo studio sulla frequenza e la distribuzione delle mutazioni del gene PAH rafforzano l’idea della notevole eterogeneità delle mutazioni nei pazienti HPA, con particolare riferimento al Sud Italia. Questo lavoro ha portato anche alla conclusione che il genotipo è il principale determinante del fenotipo biochimico nella maggior parte dei pazienti con deficit di PAH ed ha un grande valore nel determinare la risposta al cofattore. Inoltre, il calcolo dell’attività residua enzimatica proveniente dalle informazioni ottenute dai nostri esperimenti in vitro e da quelle disponibili nel database potrebbe essere utile per la previsione e/o l'esclusione di potenziali candidati per la terapia con BH4. I risultati qui presentati forniscono quindi una delucidazione sui genotipi PKU, sui fenotipi, e sulla risposta al BH4 come riferimento per i medici, operatori sanitari e ricercatori per la diagnosi e la definizione di un trattamento su misura dei pazienti. Un numero significativo di pazienti affetti da PKU potrebbero trarre beneficio dalla terapia col BH4 che, combinata con una dieta meno rigida, o usata in casi particolari come monoterapia, potrebbe ridurre al minimo le carenze nutrizionali e le disfunzioni neurologiche e psicologiche, contribuendo ad una migliore qualità di vita di questi pazienti.The hyperphenylalaninemia (HPA), an autosomal recessive genetic disorder, is the result of a defect of enzyme phenylalanine hydroxylase (PAH), resulting in the accumulation of phenylalanine (Phe) in the blood. In most cases (about 98%), the phenotypic expression of disease is the result of the presence of mutations in the PAH gene, coding for the enzyme PAH, with 12q24.1 locus on chromosome 12. The disease has a frequency in the Caucasian population of about 1 in 10,000 live births, corresponding to a carrier frequency of about 1 to 50; the majority of affected individuals are identified in the neonatal period by screening programs (mandatory in several countries, such as Italy) on the general population. HPA is expressed with a significant variability of phenotypic expression and with different degrees of severity. Food therapy, based on a limited intake of phenylalanine, prevents neurological damage. Phenotypes of hyperphenylalaninemia are defined based on the levels of phenylalanine in the blood at birth: classical phenylketonuria (PKU), with values of Phe in the plasma >1200 μmol/L (>20 mg/dl); mild form of PKU with phenylalaninemia levels comprised between 600 and 1200 μmol/L (10-20 mg/dl); Non-PKU hyperphenylalaninemia (HPA-non PKU or MHP) when the values of phenylalaninemia are below 600 μmol/L (<10 mg/dl). To date, through the molecular analysis of PAH gene, about 600 mutations have been identified (the majority are missense) and over 50% of patients are composite heterozygous. The considerable number of identified mutations is a substantial evidence of allelic heterogeneity of the underlying pathology that justifies the variable expressivity (even within the same family). The project aims first to increase the knowledge about the molecular and biochemical mechanisms of the disease, still not fully known, which are the basis of the etiology and variable expressivity of HPA, relying on the analysis of gene mutations responsible of the disease in patients from Southern Italy. Genotyping of patients with elevated Phe levels detected in newborn screening is often performed to complete diagnosis. From these data, we also correlated genotypes with predicted residual activities (PRA) from in vitro expression experiments tabulated in PAHdb (http://www.pahdb.mcgill.ca/), performing a genotype–phenotype correlation. The molecular bases of PKU and their implications at the metabolic level with focus on BH4 responsiveness were addressed in the final section of thesis. The quantification of PAH activity expressed in cultured cells was performed by a tandem mass spectrometry assay at the section of Dietmar-Hopp-Metabolic Center of Universitatsklinikum of Heidelberg (Germany). Mass spectrometry allows the use of stable isotopes for Phe and Tyr quantification and PAH activity measurement of PKU mutations. In summary, the data obtained in this study on the frequency and distribution of mutations in the PAH gene reinforce the idea of considerable heterogeneity of mutations in patients HPA, with particular reference to Southern Italy. This work has also led to the conclusion that the genotype is the main determinant of the biochemical phenotype in most patients with PAH deficiency and has greater value in estimation of BH4-responsiveness. In addition, calculating the residual PAH activity from the information obtained from our in vitro experiments and those available in the database may be useful for predicting and/or exclusion of potential candidates for BH4 therapy. The results presented herein provide then a clarification on PKU genotypes, on phenotypes, and response to BH4 as a reference available for clinicians, health care professionals and researchers for diagnosis and establishment of tailored treatment of patients. A significant number of PKU patients is likely to benefit from BH4 treatment which, combined with a less strict diet, or in some cases as monotherapy, may reduce nutritional deficiencies and neurological and psychological dysfunctions, contributing to a better quality of life of these patients

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

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

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

Phenylalanine hydroxylase deficiency in south Italy: Genotype-phenotype correlations, identification of a novel mutant PAH allele and prediction of BH4 responsiveness

We investigated the mutation spectrum of the phenylalanine hydroxylase gene (PAH) in a cohort of patients from 33 Italian PKU families. Mutational screening of the known coding region, including conventional intron splice sites, was performed by direct sequencing of the patients' genomic DNA. Thirty-three different disease causing mutations were identified in our patient group, including 19 missense, 6 splicing, 3 nonsense, 5 deletions, with a detection rate of 100%. The most prevalent mutation was the IVS10-11G>A, accounting for 12.1% of PKU alleles studied. Other frequent mutations were: p.R261Q (9.1%), p.P281L (7.6%), and p.R408W (6.1%). We also identified one novel missense mutation, p.H290Q. A spectrum of 31 different genotypes was observed and a genotype based predictions of BH4-responsiveness were assessed. Among all genotypes, 13 were predicted to be BH4-responsive represented by thirteen PKU families. In addition, genotype-phenotype correlations were performed. This study reveals the importance of a full genotyping of PKU patients and the prediction of BH4-responsiveness, not only because of the definitive diagnosis and prediction of the optimal diet, but also to point out those patients that could benefit from new therapeutic approach. They may potentially benefit from BH4 therapy which, combined with a less strict diet, or eventually in special cases as monotherapy, may contribute to reduce nutritional deficiencies and minimize neurological and psychological dysfunctions

A standardized flow cytometry network study for the assessment of circulating endothelial cell physiological ranges

Circulating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CVL), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CVL of CD34bright on CEC ~ 30%; CVL of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (medianfemale = 9.31 CEC/mL; medianmale = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions

MOESM1 of Ex-vivo characterization of circulating colon cancer cells distinguished in stem and differentiated subset provides useful biomarker for personalized metastatic risk assessment

Additional file 1: Figure S1. Sensibility, specificity and purity of CTCs detection methodology. The sensitivity of the methodology was calculated through the formula employing mean values (expressed in percentage) for each CTCs subsets identified by the combined expression of CK20 and CD45, found in the total cellular suspension collected from the working density phase. The sensitivity or the capability to detect the real subset of CTCs CK20pos corresponded to 91 %. The specificity, corresponding to the probability of a negative test, was calculated at about 87 %. Finally, the purity was at 75 %. Figure S2. Resolution of CTCs detection methodology. To verify that the collected fraction was enriched in cancer cells, HCT 116 cells were infected with pAdenoVator-CMV-IRES-GFP reporter. Human cancer colon cell lines HCT 116 were cultured in RPMI1640 medium containing 10 % fetal bovine serum (FBS), 2 mmol/l L-glutamine, and 30 mg penicillin G/0.05 g streptomycin. Cells were plated at 8 x 106 per well onto a six-well plate 24 hours before infection, and were infected with adenoviral vector. In order to perform infections, HCT 116 cells were incubated with pAdenoVator-CMV5(CuO)-IRES-GFP (Qbiogene, Carlsbad, CA) in serum free medium for 1 hour at 37 °C. Both vectors were used at multiplicity of infection (m.o.i.) of 3000 physical particles/cell, experimentally determined as the lowest m.o.i. at which the majority of the cell population is infected (as assessed by EGFP expression). Twenty-four hours later, both adherent and floating cells were harvested, washed in PBS and counted. Different concentration of HCT 116-GFP (HCT 116*) were put in entire blood sample (5 ml) and were evaluated through cytometric analysis. The resolution for the minimal concentration of HCT 116* (8 x 103 cell/5 ml) put in a volume of peripheral blood sample of 5ml, useful to detect them in the working density phase, was calculated at 5,8 cells/5 ml (B). Figure S3. DTCs in livers of mice treated with localized and advanced cancer eCTCs. Dot Plots report the expression of CK20 antigen on human colon cancer cells disseminated within liver tissue of mouse submitted to xenograft procedure. In particular, dot plot in (A) shows human colon cancer cell CK20 positive founded in liver tissue of mouse injected with eCTCs-CXCR4negCKneg referred as control. Dot plots in (B) and (C) show human cancer colon cells expressing CK20 marker in liver tissues of mouse injected with eCTCs-CXCR4posCKpos derived from localized (B) and advanced (C) colon cancer cases respectively. Figure S4. xenograft developed with circulating stem cells. Xenograft procedure developed injecting eCTCs-CD45negCD133pos organized in spheres (A). In (B) immunofluorescence positive for CD133 (green staining). In (C) Tumour formations produced within 2 weeks and after 80 days. Immunohistochemical analysis shows the distribution of the cancer colon cells expressing CD133 (brown staining) in the tumour sections of 8 μm (D)

Biallelic ZNFX1 variants are associated with a spectrum of immuno-hematological abnormalities

Biallelic changes in the ZNFX1 gene have been recently reported to cause severe familial immunodeficiency. Through a search of our bio/databank with information from genetic testing of >55 000 individuals, we identified nine additional patients from seven families with six novel homozygous ZNFX1 variants. Consistent with the previously described phenotype, our patients suffered from monocytosis, thrombocytopenia, hepatosplenomegaly, recurrent infections, and lymphadenopathy. The two most severely affected probands also had renal involvement and clinical presentations compatible with hemophagocytic lymphohistiocytosis. The disease was less lethal among our patients than previously reported. We identified two missense changes, two variants predicted to result in complete protein loss through nonsense-mediated decay, and two frameshift changes that likely introduce a truncation. Our findings (i) independently confirm the role of ZNFX1 in primary genetic immunodeficiency, (ii) expand the genetic and clinical spectrum of ZNFX1-related disease, and (iii) illustrate the utility of large, well-curated, and continually updated genotype–phenotype databases in resolving molecular diagnoses of patients with initially negative genetic testing findings