First report of whole CFTR gene duplication in a healthy newborn carrying R74W and V855I variants on the same allele.

Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder among Caucasians. The improvement of genetic techniques has allowed the identification of an increasing number of genetic variants, including large rearrangements such as duplications. We report the first case of a whole CFTR gene duplication in a healthy newborn, who had normal sweat test, also carrying R74W and V855I variants on the same allele. Familial segregation analysis and the observed frequencies of all the CFTR gene variants, revealed that R74W and V855I were probably both present in a cis arrangement on the allele also containing the duplication (i.e., in a double complex allele). Since R74W is a "variant of varying clinical consequence" its arrangement in trans with one pathogenic variant may not be sufficient to cause a classic CF disease phenotype. Moreover, its duplication could even be an advantage that could compensate for the effect of the alteration.

Impact of High-to-Moderate Penetrance Genes on Genetic Testing: Looking over Breast Cancer.

Breast cancer (BC) is the most common cancer and the leading cause of cancer death in women worldwide. Since the discovery of the highly penetrant susceptibility genes BRCA1 and BRCA2, many other predisposition genes that confer a moderate risk of BC have been identified. Advances in multigene panel testing have allowed the simultaneous sequencing of BRCA1/2 with these genes in a cost-effective way. Germline DNA from 521 cases with BC fulfilling diagnostic criteria for hereditary BC were screened with multigene NGS testing. Pathogenic (PVs) and likely pathogenic (LPVs) variants in moderate penetrance genes were identified in 15 out of 521 patients (2.9%), including 2 missense, 7 non-sense, 1 indel, and 3 splice variants, as well as two different exon deletions, as follows: ATM (n = 4), CHEK2 (n = 5), PALB2 (n = 2), RAD51C (n = 1), and RAD51D (n = 3). Moreover, the segregation analysis of PVs and LPVs into first-degree relatives allowed the detection of CHEK2 variant carriers diagnosed with in situ melanoma and clear cell renal cell carcinoma (ccRCC), respectively. Extended testing beyond BRCA1/2 identified PVs and LPVs in a further 2.9% of BC patients. In conclusion, panel testing yields more accurate genetic information for appropriate counselling, risk management, and preventive options than assessing BRCA1/2 alone.

Genotypes and phenotypes heterogeneity in PIK3CA-related overgrowth spectrum and overlapping conditions: 150 novel patients and systematic review of 1007 patients with PIK3CA pathogenetic variants.

BACKGROUND: Postzygotic activating PIK3CA variants cause several phenotypes within the PIK3CA-related overgrowth spectrum (PROS). Variant strength, mosaicism level, specific tissue involvement and overlapping disorders are responsible for disease heterogeneity. We explored these factors in 150 novel patients and in an expanded cohort of 1007 PIK3CA-mutated patients, analysing our new data with previous literature to give a comprehensive picture. METHODS: We performed ultradeep targeted next-generation sequencing (NGS) on DNA from skin biopsy, buccal swab or blood using a panel including phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin pathway genes and GNAQ, GNA11, RASA1 and TEK. Additionally, 914 patients previously reported were systematically reviewed. RESULTS: 93 of our 150 patients had PIK3CA pathogenetic variants. The merged PROS cohort showed that PIK3CA variants span thorough all gene domains, some were exclusively associated with specific PROS phenotypes: weakly activating variants were associated with central nervous system (CNS) involvement, and strongly activating variants with extra-CNS phenotypes. Among the 57 with a wild-type PIK3CA allele, 11 patients with overgrowth and vascular malformations overlapping PROS had variants in GNAQ, GNA11, RASA1 or TEK. CONCLUSION: We confirm that (1) molecular diagnostic yield increases when multiple tissues are tested and by enriching NGS panels with genes of overlapping 'vascular' phenotypes; (2) strongly activating PIK3CA variants are found in affected tissue, rarely in blood: conversely, weakly activating mutations more common in blood; (3) weakly activating variants correlate with CNS involvement, strong variants are more common in cases without; (4) patients with vascular malformations overlapping those of PROS can harbour variants in genes other than PIK3CA.

The recurrent SETBP1 c.2608G > A, p.(Gly870Ser) variant in a patient with Schinzel-Giedion syndrome: an illustrative case of the utility of whole exome sequencing in a critically ill neonate.

BACKGROUND: Schinzel-Giedion syndrome (SGS) is a multiple malformation syndrome mainly characterized by severe intellectual disability, distinctive facial features, and multiple congenital anomalies, including skeletal abnormalities, genitourinary and renal malformations, cardiac defects, as well as an increased pediatric cancer risk. Recently, SGS has been associated with de novo heterozygous deleterious variants in the SETBP1 gene; to date, nine different variants, clustering in exon 4 of SETBP1, have been identified in 25 patients. CASE PRESENTATION: In this study, by using Whole Exome Sequencing (WES), we identified a patient with a recurrent missense mutation in SETBP1, the c.2608G > A, p.(Gly870Ser) variant, previously reported as likely pathogenic. This finding allowed us to confirm the suspected clinical diagnosis of SGS. Clinical features of patients carrying the same variant, including our patient, were evaluated by a review of medical records. CONCLUSIONS: Our study confirms SGS as a severe disorder potentially presenting at birth as a critically ill neonate and demonstrates the causal role of the c.2608G > A, p.(Gly870Ser) variant in the etiology of the syndrome. Moreover, although the cohort of SETBP1-patients reported in the literature is still small, our study reports for the first time the prevalence of the variant (about 27%, 7/26). Finally, given the heterogeneity of clinical presentations of affected patients hospitalized in Neonatal Intensive Care Units (NICU) and/or Pediatric Intensive Care Units (PICU), in agreement with emerging data from the literature, we suggest that WES should be used in the diagnosis of unexplained syndromic conditions, and even as part of a standard first-line diagnostic approach, as it would allow a better diagnosis, counseling and management of affected patients and their families.

Accurate Classification of NF1 Gene Variants in 84 Italian Patients with Neurofibromatosis Type 1.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant genetic diseases. It is caused by mutations in the NF1 gene encoding for the large protein, neurofibromin. Genetic testing of NF1 is cumbersome because 50% of cases are sporadic, and there are no mutation hot spots. In addition, the most recognizable NF1 clinical features—café-au-lait (CALs) spots and axillary and/or inguinal freckling—appear early in childhood but are rather non-specific. Thus, the identification of causative variants is extremely important for early diagnosis, especially in paediatric patients. Here, we aimed to identify the underlying genetic defects in 72 index patients referred to our centre for NF1. Causative mutations were identified in 58 subjects, with 29 being novel changes. We evaluated missense and non-canonical splicing mutations with both protein and splicing prediction algorithms. The ratio of splicing mutations detected was higher than that reported in recent patients’ series and in the Human Gene Mutation Database (HGMD). After applying in silico predictive tools to 41 previously reported missense variants, we demonstrated that 46.3% of these putatively missense mutations were forecasted to alter splicing instead. Our data suggest that mutations affecting splicing can be frequently underscored if not analysed in depth. We confirm that hamartomas can be useful for diagnosing NF1 in children. Lisch nodules and cutaneous neurofibromas were more frequent in patients with frameshifting mutations. In conclusion, we demonstrated that comprehensive in silico analysis can be a highly specific method for predicting the nature of NF1 mutations and may help in assuring proper patient care.

Genetic basis of single-suture synostoses: genes, chromosomes and clinical implications.

BACKGROUND: Non syndromic craniosynostoses are the most frequent craniofacial malformations worldwide. They represent a wide and heterogeneous group of entities, in which the dysmorphism may occur in a single (simple forms) or in multiple sutures (complex forms). Simple forms present a higher birth prevalence and are classified according to the involved suture and to the corresponding abnormal cranial shape: scaphocephaly (SC; sagittal suture), trigonocephaly (TC; metopic suture), anterior plagiocephaly (unilateral coronal suture), posterior plagiocephaly (unilateral lambdoid suture). They occur commonly as sporadic forms, although a familiar recurrence is sometimes observed, suggesting a mendelian inheritance. The genetic causes of simple craniosynostosis are still largely unknown, as mutations in common craniosynostosis-associated genes and structural chromosomal aberrations have been rarely found in these cases. AIMS: This review is intended to dissect comprehensively the state-of-the art on the genetic etiology of single suture craniosynostoses, in the attempt to categorize all known disease-associated genes and chromosomal aberrations. Possible genotype/phenotype correlations are discussed as useful clues towards the definition of optimized clinical management flowcharts.

Detection of premature segregation of centromeres in persons exposed to ionizing radiation.

We have analyzed the frequency of premature centromeric division (PCD) in medical personnel professionally exposed to low doses of radiation. They had chromosome aberrations (CAs) involving dicentric chromosomes, ring chromosomes, acentric fragments, chromosome breaks, and chromatid breaks. The study included 30 exposed subjects and 23 controls who were each analyzed by a conventional cytogenetics procedure and subsequently by fluorescent in situ hybridization (FISH). The latter was applied particularly in order to verify PCD in a specific chromosome (chromosome 18) in both metaphases and interphase nuclei. The results revealed a significant difference (p < 0.001) in frequencies between the two groups (exposed and controls) for all the observed variables (CAs), metaphases with PCD (MPCD), total number of chromosomes with PCD (TPCD), number of PCD metaphases in acrocentric chromosomes (MAPCD), and the total number of acrocentric chromosomes with PCD (TAPCD). The doses of ionizing radiation absorbed by the subjects' bodies were measured with thermoluminescent dosimeters once a month during the duration of occupational exposure. They were expressed in mSv, as mean annual effective doses for the period of exposure. The Spearman rank test showed a high positive correlation between total life effective dose and frequency of CAs and PCD. Based on the results obtained in this study, we suggest that PCD, as a phenomenon manifesting chromosomal instability (CIN), should be considered as a suitable cytogenetic biomarker for individuals occupationally exposed to ionizing radiation.

Influence of some detoxification enzyme polymorphisms on cytogenetic biomarkers between individuals exposed to very low doses of 1,3-butadiene.

OBJECTIVE: To evaluate the variation of some biomarkers related to the level of enzymatic activity dependent on the different polymorphisms. METHODS: We studied 27 butadiene-exposed workers and 37 controls using different biomarkers of the genotoxic effect. The genotypes were determined using polymerase chain reaction and restriction fragment length polymorphism-polymerase chain reaction techniques; the subjects were assigned to a specific group based on the microsomal epoxide hydrolase (mEH) activity predicted by their genotype (low, intermediate, high). RESULTS: The studied biomarkers were not able to discriminate between exposed and control individuals, but sister chromatid exchange (SCE) and high frequency cells were influenced by smoking habits. Smokers having fast microsomal epoxide hydrolase activity showed higher SCE frequency (7.61) respect to those presenting intermediate (5.86) or slow (6.65) enzymatic activity. CONCLUSIONS: On the basis of these results, can we suppose the existence of an "intermediate genotype" advantage (at least for induction of SCE)?

Chromosomal aberrations in subjects exposed to ionizing radiation.

Occupational exposure to low doses of ionizing radiation is a particularly delicate subject for investigation, due to the cumulative effects of chronic exposure. It is extremely important to consider and to measure the biological response to given conditions of exposure. The aim of this study was to establish possible recovery from DNA damage in subjects professionally exposed to radiation in their working area by examinations for chromosomal aberrations (CA) at two different times. The first group (I) was composed of 30 professionally exposed subjects in whom unstable CA (dicentrics, ring, acentric fragments, chromatid, chromosomal breaks, and chromatid interchanges) were identified at time zero. After removal from the radiation area, they were re-examined 9 months later. The second group (II) contained 64 healthy individuals, not professionally exposed to ionizing radiation or other known mutagenic agents. In the group of exposed individuals, five (16.67%) subjects exhibited permanent unstable CAs, even after 9 months absence from the radiation. When the nonexposed and exposed groups were compared, an increase of unstable aberrations (p < 0.05) was observed in the exposed group. Nevertheless, a statistically significant decrease of dicentrics, acentric fragments, and ring frequencies was observed in exposed individuals after 9 months away from the radiation area. However, chromatid and isochromatid break frequencies increased slightly but not significantly after 9 months. The detected CAs corresponded to the total effective doses of radiation measured in our subjects. The existence of CAs in some individuals even after absence from the radiation area suggests that the time necessary for the damaged DNA to recover is extremely variable and indicates interindividual differences in radiosensitivity as well as differences in the cellular-reparation response.

SCE frequency measurement could be useful in the prenatal diagnosis of Roberts syndrome.

In a previously published article (Resta et al., 2006) on Robert's syndrome in prenatal diagnosis, a case of a 36-year-old woman and her 36-year-old, nonconsanguineous husband were presented. Our findings suggest the existence of nonsense mediated decay (NMD) variability which could account for the varying severity reported in carriers of identical mutations. Furthermore, fetal cells were used to evaluate the influence of premature centromere separation (PCS) on the sister chromatid exchange (SCE) and micronucleus (MN) frequency. Given the similar variation observed in the SCE frequencies, dependent on tissue/cell type (amniotic fluid sample, chorionic villus sampling) and duration of in vitro cultures (48 hours or 72 hours), the idea was that this new piece of information could be interesting. It seems that the SCE frequency increased proportionally to the cell cycle increasing (1 degrees < 2 degrees < 3 degrees ... n). Obviously, our observations are too scarce to draw conclusions, but further investigation could be useful to corroborate or dispute these results, considering that the two techniques, (MN and SCE), are simple to perform and do not require expensive laboratory equipment.

Lack of genotoxic effect in workers exposed to very low doses of 1,3-butadiene.

1,3-Butadiene (BD), a probable carcinogen to humans, has been shown to have an ill-defined genotoxicity in occupationally exposed workers. In the present study, the influence of exposure to very low doses of BD and to cigarette smoking was investigated on some cytogenetic endpoints, namely, sister chromatid exchanges (SCE), chromosomal aberrations (CA) and cells with a high frequency of SCE (HFC), in peripheral blood lymphocytes. Twenty-seven male workers employed in a petrochemical plant and 26 matched controls were included in the study. As regards the airborne BD values, there was a significant difference between exposed (median BD value 1.5, min-max 0.2-69.0 microg/m3) and non-exposed workers (median BD value 0.4, min-max <0.1-3.8 microg/m3). Genotoxic biomarkers were not able to distinguish between the two groups. The frequency of SCE was higher in smokers than in non-smokers (p=0.001), with a positive correlation between the number of cigarettes smoked per day and both SCE (r=0.4; p=0.004) and HFC frequency (r=0.3; p=0.04). Multiple regression analysis confirmed the influence of cigarette smoking on the level of SCE and HFC, while these parameters were not affected by personal exposure to BD. Overall, the biomarkers of genotoxic effect investigated in our study were not able to discriminate between workers with a very low exposure to BD and controls, while it was possible to distinguish between smokers and non-smokers on the basis of SCE.