Angela Michelucci

Torque Teno Virus Viremia Load Size in Patients with Selected Congenital Defects of Innate Immunity

ABSTRACT Plasma loads of torque teno virus (TTV) among individuals differ extensively beginning early in life, suggesting a role for innate immunity. Here, congenital mannose-binding lectin deficiencies, but not deficiencies in respiratory ciliary function, correlated with increased TTV loads. Notably, however, the presence of either disorder was associated with particularly high TTV loads.

Pharmacogenetics of anti-estrogen treatment of breast cancer

A major effort is underway to select genetic polymorphisms potentially relevant to the clinical efficacy and safety of endocrine treatment of breast cancer. Genetic factors of the host that affect the metabolism of tamoxifen, a widely used drug for the adjuvant treatment of breast cancer, have received particular attention. Cytochrome P450 isoform 2D6 (CYP2D6) is a key step in the metabolism of tamoxifen to its active moiety endoxifen. Women with functionally deficient genetic variants of CYP2D6 who are given drugs that inhibit CYP2D6 are exposed to low endoxifen plasma levels and may enjoy reduced benefits from tamoxifen treatment. Therefore, CYP2D6 status may be an important predictor of the benefits of tamoxifen to an individual; unfortunately, the data are not uniformly concordant, and definitive evidence that would suggest the routine analysis of CYP2D6 before commencing tamoxifen treatment is not yet available. Recent research has focused on the role UDP-glucuronosyltransferases, a family of metabolizing enzymes that play an important role in the metabolic clearance of tamoxifen and of the aromatase inhibitors as well, and how interindividual differences in these enzymes may play a role in the clinical outcome upon administration of anti-estrogen treatment. In conclusion, whether a pharmacogenetic profile should be obtained prior to initiating tamoxifen therapy is currently a matter of debate, although summing up all the scientific evidence available on this issue it appears that the genetic screening would be an useful support for clinical decision making in selected patients.

Gene editing of DNAH11 restores normal cilia motility in primary ciliary dyskinesia

Background Primary ciliary dyskinesia (PCD) is a rare autosomal recessive genetic disorder characterised by dysfunction of motile cilia. Ciliary dysmotility causes poor mucociliary clearance and leads to impairment of pulmonary function and severe respiratory infections. PCD has no specific therapy. With the aim to permanently restore gene function and normalise ciliary motility, we used gene editing to replace mutated with wild-type sequence in defective cells. Methods The target gene was dynein heavy chain 11 (DNAH11), an essential component of ciliary structure. Airway ciliated cells were collected from two patients with PCD with DNAH11 nonsense mutations and altered ciliary beating and pattern. Repair of the genetic defect was performed ex vivo by site-specific recombination using transcription activator-like effector nucleases (TALENs). Results In an epithelial cell line engineered to contain the DNAH11 target site, TALENs cleaved over 80% of the mutated DNAH11 sequence and replaced the mutated sequence with wild-type sequence in about 50% of cells. In airway ciliated cells of patients with PCD, site-specific recombination and normalisation of ciliary beating and pattern occurred in 33% and 29% of cells, respectively. Conclusion This study demonstrates that gene editing can rescue ciliary beating ex vivo, opening up new avenues for treating PCD.

Ambiguous external genitalia due to defect of 5-α-reductase in seven Iraqi patients: prevalence of a novel mutation.

We report on seven Iraqi patients with 46,XY karyotype and ambiguous genitalia characterized by perineo-scrotal hypospadias, bifid scrotum, clitoris like phallus, palpable testes in inguinal canal and pseudovagina. Patients were raised five as females and two as males. They are all unrelated with the exception of two couples of brothers. The diagnosis of 5-α-reductase-2 deficiency syndrome was first hypothesized on clinical grounds and then confirmed by molecular analysis. Direct sequencing analysis of the SRD5A2 gene revealed in five patients a novel homozygous frame-shift mutation (c.453delC) and in two related patients a previous reported missense mutation. The presence of the same mutation in unrelated patients of the same population suggests a possible founder effect. This report brings the 5-α-reductase-2 deficiency syndrome to the attention of clinical geneticists and child surgeons and discusses the appropriate clinical and surgical strategies for treating these patients.

Mannose-binding lectin 2 gene polymorphism and lung damage in primary ciliary dyskinesia.

Mannose‐binding lectin (MBL) plays an important role in innate immunity and has been reported to be associated with the age‐related decline in lung function in cystic fibrosis.

Identification of three novel mutations in the CHD7 gene in patients with clinical signs of typical or atypical CHARGE syndrome

CHARGE syndrome is an autosomal dominant disorder characterized by features represented in its acronym: Coloboma, Heart defect, Atresia of the choanae, Retarded growth and development, Genital abnormalities, Ear anomalies/deafness. We report two patients with a diagnosis of typical CHARGE syndrome and one with atypical clinical diagnosis. All the three patients had uni- or bilateral choanal atresia and sensorineural hearing loss. The patients were screened for CHD7 gene mutations. Three novel occurring de novo heterozygous mutations were identified: a mutation in the donor splice site of intron 24, a missense mutation in exon 2 and a deletion in exon 11.

Uncommon dihydropyrimidine dehydrogenase mutations and toxicity by fluoropyrimidines: a lethal case with a new variant

DPD is the rate-limiting enzyme involved in the metabolism of 5-fluorouracil and its prodrugs, capecitabine and tegafur. Many cases of severe toxicities by fluoropyrimidines are reported in the literature, sometimes with lethal outcome, due to a poor or null metabolizer phenotype. The exon 14-skipping mutation IVS14+1G>A and the c.2846A>T are the most common deficient variants. However, many additional variants of the DPYD gene with unclear functional significance have been reported. We describe a patient with metastatic breast cancer who received capecitabine and trastuzumab at standard doses. Six days after beginning capecitabine, the patient developed fever, leucopenia and neutropenia, mucositis, hand-foot syndrome, multiple organ dysfunction and eventually died. Since the toxicity profile was compatible with capecitabine administration, complete exon sequencing of DPYD was carried out and the patient was found to be compound heterozygous for the rare mutation c.257C>T in exon 4, c.496A>G in exon 6, the new variant c.1850C>T in exon 14 and c.2194G>A in exon 18. Given the marginal role of c.496A>G and c.2194G>A in DPD deficiency, the cause of death was suggested to be dependent on the novel c.1850C>T in combination with c.257C>T. The complexity of DPD pharmacogenetics suggests the need to develop cost-effective screening approaches to identify patients at risk of severe toxicities.

Short Stature in Isodicentric Y Chromosome and Three Copies of the SHOX Gene: Clinical Report and Review of Literature.

Short stature homeobox gene (SHOX) mutations and pseudoautosomal region 1 (PAR1) deletions encompassing SHOX are known causes of Léri-Weill dyschondrosteosis and isolated short stature, while 3 copies of SHOX in cases with triple sex chromosome constitution are responsible for tall stature. Duplications involving SHOX have been rarely reported, and they were found in individuals with short, normal and tall stature. An adopted boy with short stature, isodicentric Y chromosome and 3 copies of SHOX is described. Normal growth hormone (GH) secretion and insulin-like growth factor 1 (IGF1) increase during an IGF1 generation test were found, ruling out impaired GH-IGF1 axis. No other organic or psychiatric causes of impaired growth were found. GH treatment improved linear growth, as reported in children with SHOX haploinsufficiency. This new report and the review of literature support that SHOX duplication may cause short stature, especially in those children with duplications of the 5′SHOX regulatory elements. Chromosome analysis and detailed molecular characterization of the duplicated region should be warranted in individuals with SHOX duplications in order to investigate the presence of occult chromosome imbalance. Additional reports and follow-up till adult height are needed to give conclusions on long-term efficacy and safety of GH treatment in short children with SHOX duplication.

NR5A1 Gene Mutations: Clinical, Endocrine and Genetic Features in Two Girls with 46,XY Disorder of Sex Development

Background: Steroidogenic factor 1, encoded by the NR5A1 gene, is a key regulator of endocrine function within the hypothalamic-pituitary-steroidogenic axis. Both homozygous, compound heterozygous and heterozygous mutations in the NR5A1 gene may determine 46,XY disorders of sex development (DSD). Patients and Methods:NR5A1 gene sequencing was performed in a cohort of 6 patients with 46,XY DSD without specific diagnosis. Results: Heterozygous NR5A1 gene mutations were found in 2 girls, aged 0.5 years and 14 years. The older girl harbored the c.250C>T transition in exon 4 (p.Arg84Cys), previously reported in a Japanese girl. The younger girl presented a de novo novel exon 6 heterozygous frameshift mutation (c.1074dupG) in codon 359 associated with the p.Gly146Ala polymorphism the latter inherited from her father. This baby showed severe impairment of androgen secretion from the first months of life. Overt adrenal insufficiency did not occur, but the older girl showed subnormal cortisol peak after ACTH stimulation. Conclusions:NR5A1 gene mutations are a relatively frequent cause of 46,XY DSD in humans. Clear indications for management of these individuals remain elusive, mainly when diagnosis is made in infancy. Long-term monitoring of adrenal function should be recommended.

Discovery of novel mutations in the dihydropyrimidine dehydrogenase gene associated with toxicity of fluoropyrimidines and viewpoint on preemptive pharmacogenetic screening in patients

BackgroundDihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme of the metabolic pathway of 5-fluorouracil (5-FU) and other fluoropyrimidines to inactive compounds. For this reason, severe, life-threatening toxicities may occur in patients with deficient DPD activity when administered standard doses of 5-FU and its prodrugs.Materials and methodsWe selected three patients with colorectal adenocarcinoma who displayed unexpected severe adverse reactions after treatment with 5-FU and capecitabine. To investigate the possible involvement of deficient variants of the DPD gene (DPYD), a denaturing HPLC (dHPLC) approach followed by target exon sequencing of DPYD was performed on DNA extracted from peripheral blood.ResultsThree novel non-synonymous mutations of DPYD, c.2509-2510insC, c.1801G>C, and c.680G>A, were detected in these subjects. Due to the absence of other deficient variants of DPYD and the compatibility of adverse reactions with fluoropyrimidine treatment, the novel variants were associated with a poor-metabolizer phenotype.ConclusionsStratification of patients on the basis of their genotype may help prevent toxicity, and the large body of evidence about the pathogenesis of fluoropyrimidine-induced adverse reactions strongly encourages the adoption of best practice recommendations to appropriately address this important clinical issue. This approach is of utmost importance within a preventive, prognostic, and personalized approach to patient care in the oncology setting.