The genetic framework of primary ciliary dyskinesia assessed by soft computing analysis.

BACKGROUND: International guidelines disagree on how best to diagnose primary ciliary dyskinesia (PCD), not least because many tests rely on pattern recognition. We hypothesized that quantitative distribution of ciliary ultrastructural and motion abnormalities would detect most frequent PCD-causing groups of genes by soft computing analysis. METHODS: Archived data on transmission electron microscopy and high-speed video analysis from 212 PCD patients were re-examined to quantitate distribution of ultrastructural (10 parameters) and functional ciliary features (4 beat pattern and 2 frequency parameters). The correlation between ultrastructural and motion features was evaluated by blinded clustering analysis of the first two principal components, obtained from ultrastructural variables for each patient. Soft computing was applied to ultrastructure to predict ciliary beat frequency (CBF) and motion patterns by a regression model. Another model classified the patients into the five most frequent PCD-causing gene groups, from their ultrastructure, CBF and beat patterns. RESULTS: The patients were subdivided into six clusters with similar values to homologous ultrastructural phenotype, motion patterns, and CBF, except for clusters 1 and 4, attributable to normal ultrastructure. The regression model confirmed the ability to predict functional ciliary features from ultrastructural parameters. The genetic classification model identified most of the different groups of genes, starting from all quantitative parameters. CONCLUSIONS: Applying soft computing methodologies to PCD diagnostic tests optimizes their value by moving from pattern recognition to quantification. The approach may also be useful to evaluate atypical PCD, and novel genetic abnormalities of unclear disease-producing potential in the future.

The BRCA1 c.4096+1G>A Is a Founder Variant Which Originated in Ancient Times.

Approximately 30-50% of hereditary breast and ovarian cancer (HBOC) is due to the presence of germline pathogenic variants in the BRCA1 (OMIM 113705) and BRCA2 (OMIM 600185) onco-suppressor genes, which are involved in DNA damage response. Women who carry pathogenic BRCA1 variants are particularly likely to develop breast cancer (BC) and ovarian cancer (OC), with a 45-79 percent and 39-48 percent chance, respectively. The BRCA1 c.4096+1G>A variant has been frequently ascertained in Tuscany, Italy, and it has also been detected in other Italian regions and other countries. Its pathogenetic status has been repeatedly changed from a variant of uncertain significance, to pathogenic, to likely pathogenic. In our study, 48 subjects (38 of whom are carriers) from 27 families were genotyped with the Illumina OncoArray Infinium platform (533,531 SNPs); a 20 Mb region (24.6 cM) around BRCA1, including 4130 SNPs (21 inside BRCA1) was selected for haplotype analysis. We used a phylogenetic method to estimate the time to the most recent common ancestor (MRCA) of BRCA1 c.4096+1G>A founder pathogenic variant. This analysis suggests that the MRCA lived about 155 generations ago-around 3000 years ago.

In Tandem Intragenic Duplication of Doublesex and Mab-3-Related Transcription Factor 1 (DMRT1) in an SRY-Negative Boy with a 46,XX Disorder of Sex Development.

Disorders of sexual development (DSDs) encompass a group of congenital conditions associated with atypical development of internal and external genital structures. Among those with DSDs are 46,XX males, whose condition mainly arises due to the translocation of SRY onto an X chromosome or an autosome. In the few SRY-negative 46,XX males, overexpression of other pro-testis genes or failure of pro-ovarian/anti-testis genes may be involved, even if a non-negligible number of cases remain unexplained. A three-year-old boy with an SRY-negative 46,XX karyotype showed a normal male phenotype and normal prepubertal values for testicular hormones. A heterozygous de novo in tandem duplication of 50,221 bp, which encompassed exons 2 and 3 of the Doublesex and Mab-3-related transcription factor 1 (DMRT1) gene, was detected using MPLA, CGH-array analysis, and Sanger sequencing. Both breakpoints were in the intronic regions, and this duplication did not stop or shift the coding frame. Additional pathogenic or uncertain variants were not found in a known pro-testis/anti-ovary gene cascade using a custom NGS panel and whole genome sequencing. The duplication may have allowed DMRT1 to escape the transcriptional repression that normally occurs in 46,XX fetal gonads and thus permitted the testicular determination cascade to switch on. So far, no case of SRY-negative 46,XX DSD with alterations in DMRT1 has been described.

Cutaneous lesions and other non-endocrine manifestations of Multiple Endocrine Neoplasia type 1 syndrome.

Background: Multiple Endocrine Neoplasia type 1 is a rare genetic syndrome mainly caused by mutations of MEN1 gene and characterized by a combination of several endocrine and non-endocrine manifestations. The objective of this study was to describe cutaneous lesions and other non-endocrine manifestations of MEN1 in a cohort of patients with familial (F) and sporadic (S) MEN1, compare the prevalence of these manifestations between the two cohorts, and investigate the correlation with MEN1 mutation status. Methods: We collected phenotypic and genotypic data of 185 patients with F-MEN1 and S-MEN1 followed from 1997 to 2022. The associations between F-MEN1 and S-MEN1 or MEN1 mutation-positive and mutation-negative patients and non-endocrine manifestations were determined using chi-square or Fisher's exact tests or multivariate exact logistic regression analyses. Results: The prevalence of angiofibromas was significantly higher in F-MEN1 than in S-MEN1 in both the whole (p < 0.001) and index case (p = 0.003) cohorts. The prevalence of lipomas was also significantly higher in F-MEN1 than in S-MEN1 (p = 0.009) and in MEN1 mutation-positive than in MEN1 mutation-negative (p = 0.01) index cases. In the whole cohort, the prevalence of lipomas was significantly higher in MEN1 mutation-positive compared to MEN1 mutation-negative patients (OR = 2.7, p = 0.02) and in F-MEN1 than in S-MEN1 (p = 0.03), only after adjustment for age. No significant differences were observed for the other non-endocrine manifestations between the two cohorts. Hibernoma and collagenoma were each present in one patient (0.5%) and meningioma and neuroblastoma in 2.7% and 0.5%, respectively. Gastric leiomyoma was present in 1.1% of the patients and uterine leiomyoma in 14% of women. Thyroid cancer, breast cancer, lung cancer, basal cell carcinoma, melanoma, and colorectal cancer were present in 4.9%, 2.7%, 1.6%, 1.6%, 2.2%, and 0.5% of the whole series, respectively. Conclusions: We found a significantly higher prevalence of angiofibromas and lipomas in F-MEN1 compared with S-MEN1 and in MEN1 mutation-positive compared to MEN1 mutation-negative patients. In patients with one major endocrine manifestation of MEN1 , the presence of cutaneous lesions might suggest the diagnosis of MEN1 and a possible indication for genetic screening.

A Multisystem Mitochondrial Disease Caused by a Novel MT-TL1 mtDNA Variant: A Case Report.

BACKGROUND: Mitochondrial tRNA (MTT) genes are hotspot for mitochondrial DNA mutation and are responsible of half mitochondrial disease. MTT mutations are associated with a broad spectrum of phenotype often with complex multisystem involvement and complex genotype-phenotype correlations. MT-TL1 mutations, among which the m.3243A>G mutation is the most frequent, are associated with myopathy, maternal inherited diabetes and deafness, MELAS, cardiomyopathy, and focal segmental glomerulosclerosis. CASE STUDY: Here we report the case of an Italian 49-years old female presenting with encephalomyopathy, chronic proteinuric kidney disease and a new heteroplasmic m.3274_3275delAC MT-TL1 gene mutation. CONCLUSIONS: Our case demonstrates a systemic mitochondrial disease caused by the heteroplasmic m.3274_3275delAC MT-TL1 gene mutation, not yet described in the literature. A mitochondrial disease should be suspected in case of complex multisystem phenotypes, including steroid-resistant nephrotic syndrome with multisystemic involvement.

Longitudinal Nitric Oxide Levels and Infections by Ultrastructure and Genotype in Primary Ciliary Dyskinesia.

BACKGROUND: We hypothesized that differences in nasal nitric oxide (nNO) and fractional exhaled nitric oxide (Feno) relate to prognosis in primary ciliary dyskinesia (PCD). RESEARCH QUESTION: What is the relationship between baseline values and longitudinal evolution of nNO and Feno and ultrastructure, genotype, and respiratory infections in PCD? STUDY DESIGN AND METHODS: Prospective, longitudinal, single-center study in adults and children evaluated biannually for up to 10 years. We compared cross-sectional and longitudinal values of nNO and Feno in ultrastructural (inner dynein arm [IDA] and microtubular disorganization [MTD]) and genetic (CCDC39 and CCDC40) groups known to have worse pulmonary function with patients within the ultrastructural and genetic groups with a better prognosis. Linear mixed-effects models were used to evaluate longitudinal associations. RESULTS: One hundred forty-one patients with PCD underwent 1,014 visits. At enrollment, no differences were found in children in nNO or Feno between the IDA and MTD group and the other ultrastructural groups. In adults, nNO (P = .038) and Feno (P = .032) were significantly lower in the IDA and MTD group than in all other combined ultrastructural groups. Feno values were significantly lower in the CCDC39 and CCDC40 group than in the DNAH5 and DNAH11 combined genotype group (P = .033) and in all other genotypes (P = .032). The IDA and MTD group showed a significant decline in nNO with age (P < .01) compared with other ultrastructural groups who showed stable levels. The CCDC39 and CCDC40 group showed the steepest decline in nNO over time (P < .01) compared with all other genotypes. A higher nNO was associated with lower likelihood of any positive bacterial isolate from the lower respiratory tract (P = .008). Changes in Feno over time did not differ between structural groups or genotypes. INTERPRETATION: Lower nNO in patients with PCD with genetic and ultrastructural changes associated with greater lung function decline may be related to worse prognosis, but whether a low nNO is causal needs further study. If lower nNO directly results in a poorer prognosis, strategies augmenting upper airway nitric oxide production may be worth evaluating.

Long-term Efficacy and Safety of Rifampin in the Treatment of a Patient Carrying a CYP24A1 Loss-of-Function Variant.

BACKGROUND: Pharmacological therapy may be useful in the treatment of moderate to severe hypercalcemia in patients with infantile hypercalcemia-1 (HCINF1) due to pathogenic variants in the cytochrome P450 24 subfamily A member 1 (CYP24A1). Rifampin is an antituberculosis drug that is a potent inducer of cytochrome P450 3 subfamily A member 4, which is involved in an alternative catabolic pathway of vitamin D. The efficacy of rifampin in improving hypercalcemia was previously reported, but many questions remain on the long-term efficacy and safety. The aim of the study is to test the long-term efficacy and safety of rifampin in a patient with HCINF1. METHODS: We report clinical, biochemical, and imaging features of a 23-year-old man affected by HCINF1 with moderate hypercalcemia (12.9 mg/dL), symptomatic nephrolithiasis, nephrocalcinosis, and impaired kidney function [estimated glomerular filtration rate (eGFR) 60 mL/min/1.73 m2] treated with rifampin for an overall period of 24 months. Kidney, liver, and adrenal function were evaluated at every follow-up visit. RESULTS: In 2 months, rifampin induced a normalization of serum calcium (9.6 mg/dL) associated with an improvement of kidney function (eGFR 92 mL/min/1.73 m2) stable during the treatment. After 15 months, rifampin was temporally withdrawn because of asthenia, unrelated to impairment of adrenal function. After 3 months, the timing of drug administration was shifted from the morning to the evening, obtaining the remission of asthenia. At the end of follow-up, the nephrolithiasis disappeared and the nephrocalcinosis was stable. CONCLUSIONS: Rifampin could represent an effective choice to induce a stable reduction of calcium levels in patients with HCINF1, with a good safety profile.

A Single mtDNA Deletion in Association with a LMNA Gene New Frameshift Variant: A Case Report.

BACKGROUND: Proximal muscle weakness may be the presenting clinical feature of different types of myopathies, including limb girdle muscular dystrophy and primary mitochondrial myopathy. LGMD1B is caused by LMNA mutation. It is characterized by progressive weakness and wasting leading to proximal weakness, cardiomyopathy, and hearth conduction block. OBJECTIVE: In this article, we describe the case of a patient who presented with limb-girdle weakness and a double trouble scenario -mitochondrial DNA single deletion and a new LMNA mutation. METHODS: Pathophysiological aspects were investigated with muscle biopsy, Western Blot analysis, NGS nuclear and mtDNA analysis and neuromuscular imaging (muscle and cardiac MRI). RESULTS: Although secondary mitochondrial involvement is possible, a "double trouble" syndrome can not be excluded. CONCLUSION: Implication deriving from hypothetical coexistence of two different pathological conditions or the possible secondary mitochondrial involvement are discussed.

Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants.

Germline mutations in the BRCA1 gene have been reported to increase the lifetime risk of developing breast and/or ovarian cancer (BOC). By new sequencing technologies, numerous variants of uncertain significance (VUS) are identified. It is mandatory to develop new tools to evaluate their functional impact and pathogenicity. As the expression of pathogenic BRCA1 variants in Saccharomyces cerevisiae increases the frequency of intra- and inter-chromosomal homologous recombination (HR), and gene reversion (GR), we validated the two HR and the GR assays by testing 23 benign and 23 pathogenic variants and compared the results with those that were obtained in the small colony phenotype (SCP) assay, an additional yeast-based assay, that was validated previously. We demonstrated that they scored high accuracy, sensitivity, and sensibility. By using a classifier that was based on majority of voting, we have integrated data from HR, GR, and SCP assays and developed a reliable method, named yBRCA1, with high sensitivity to obtain an accurate VUS functional classification (benign or pathogenic). The classification of BRCA1 variants, important for assessing the risk of developing BOC, is often difficult to establish with genetic methods because they occur rarely in the population. This study provides a new tool to get insights on the functional impact of the BRCA1 variants.

RAD52 influences the effect of BRCA1/2 missense variants on homologous recombination and gene reversion in Saccharomyces cerevisiae.

The breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, are key players in the homologous recombination (HR) repair pathway and act as tumor suppressors by maintaining genome stability. The yeast Saccharomyces cerevisiae has no BRCA1/2 homolog; however, a number of HR genes are evolutionary conserved between human and yeast. Among them, RAD52 is involved in DNA double strand break (DSB) repair by HR, and promotes genome stability. We previously reported that the heterologous expression of cancer-associated BRCA1/2 missense variants in growing yeast cultures affects both spontaneous HR and gene reversion (GR) suggesting that yeast could be a reliable system to assess the functional impact of variants. Because inhibition of Rad52p is lethal in BRCA1/2 mutated tumors, and Rad52p is conserved between humans and yeast, we asked if the effect of BRCA1/2 variants on HR and GR could be affected by loss of RAD52. We found that the rad52∆ mutation predominantly suppressed the stimulation of HR in yeast by pathogenic BRCA1 variants but also facilitated increased GR by pathogenic variants. Conversely, the rad52∆ mutation stimulated HR by a pathogenic BRCA2 variant in yeast but had no effect on GR. These results demonstrate a functional interplay between the pathogenic BRCA1/2 variants and Rad52p in budding yeast, supporting the use of budding yeast as a suitable system for evaluating potential chemotherapeutic strategies.

Hypercalcemia due to CYP24A1 mutations: a systematic descriptive review.

BACKGROUND AND OBJECTIVES: CYP24A1 encodes a 24-hydroxylase involved in vitamin D catabolism, whose loss-of-function results in vitamin D-dependent hypercalcemia. Since the identification of CYP24A1 variants as a cause of idiopathic infantile hypercalcemia, a large body of literature has emerged indicating heterogeneity in penetrance, symptoms, biochemistry, and treatments. The objectives of the present research work were to investigate the clinical heterogeneity of the disease, the possibility of a relevant phenotype for monoallelic carriers, and to compare the hypocalcemic effect of the available therapies. METHODS: Two reviewers searched different databases for studies published between the identification of CYP24A1 variants and December 31, 2020. Eligible studies included clinical trials and reports describing carriers of CYP24A1 variants. RESULTS: Fifty eligible studies were identified, accounting for 221 patients. Genetic data were retrieved and allele frequencies were calculated. Acute hypercalcemia was the typical presentation during the first year of life (76%, P = 0.0005), and nephrocalcinosis was more frequent in infancy (P < 0.0001). Pregnancy was associated with symptomatic hypercalcemia in 81.8% and high rates of obstetric complications. Monoallelic carriers displayed significant rates of nephrolithiasis (19.4%), nephrocalcinosis (4.9%), and symptomatic hypercalcemia (5.6%). CONCLUSIONS: CYP24A1 loss-of-function results in an age-dependent phenotype, which can be exacerbated by triggering factors, such as pregnancy. Although biallelic carriers present more significant clinical and biochemical features, monoallelic carriers have an increased risk of calcium-related conditions. The highly variable tested therapeutic approaches did not allow to draw conclusions on preferable therapeutic regime.

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions.

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5'UTR and 3'UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband's group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3'UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.

Disorders/Differences of Sex Development Presenting in the Newborn With 46,XY Karyotype.

Differences/disorders of sex development (DSD) are a heterogeneous group of congenital conditions, resulting in discordance between an individual's sex chromosomes, gonads, and/or anatomic sex. The management of a newborn with suspected 46,XY DSD remains challenging. Newborns with 46,XY DSD may present with several phenotypes ranging from babies with atypical genitalia or girls with inguinal herniae to boys with micropenis and cryptorchidism. A mismatch between prenatal karyotype and female phenotype is an increasing reason for presentation. Gender assignment should be avoided prior to expert evaluation and possibly until molecular diagnosis. The classic diagnostic approach is time and cost-consuming. Today, a different approach may be considered. The first line of investigations must exclude rare life-threatening diseases related to salt wasting crises. Then, the new genetic tests should be performed, yielding increased diagnostic performance. Focused imaging or endocrine studies should be performed on the basis of genetic results in order to reduce repeated and invasive investigations for a small baby. The challenge for health professionals will lie in integrating specific genetic information with better defined clinical and endocrine phenotypes and in terms of long-term evolution. Such advances will permit optimization of counseling of parents and sex assignment. In this regard, society has significantly changed its attitude to the acceptance and expansion beyond strict binary male and female sexes, at least in some countries or cultures. These management advances should result in better personalized care and better long-term quality of life of babies born with 46,XY DSD.

Longitudinal Lung Volume Changes by Ultrastructure and Genotype in Primary Ciliary Dyskinesia.

Rationale: Genotype-phenotype relationships are emerging in primary ciliary dyskinesia (PCD), but little is known about lung volume changes over time. Objectives: To investigate the evolution of static lung volumes with ultrastructural defects, gene mutations, body mass index, and specific infections in PCD. Methods: Prospective, longitudinal, single-center study in children and adults evaluated twice yearly for up to 10 years. Linear mixed-effects models were used to assess associations between ciliary morphology, genetic mutations, and clinical features. Results: A total of 122 patients had 1,096 visits. At enrollment, almost all spirometric and, especially in adults, plethysmographic parameters were significantly worse in absent inner dynein arms (IDAs), central apparatus (CA) defects, and microtubular disorganization (MTD) (IDA/CA/MTD) compared with patients with normal electron microscopy (EM) results. The mean trend increase with time for residual volume (RV) was significantly higher in IDA/CA/MTD group compared with groups with outer dynein arm defect and normal EM results. The mean trend of RV/total lung capacity in the IDA/CA/MTD group was significantly worse than in all other groups. The steepest rise in lung volumes was in CCDC39 and CCDC40, whereas hyperinflation increased less in DNAH5 and DNAH11 groups. RV/total lung capacity showed a significantly steeper rise in patients with Pseudomonas aeruginosa compared with patients with other infections or patients without infection. Conclusions: Patients with IDA/CA/MTD defects or CCDC39 and CCDC40 mutations had the greatest increase in hyperinflation, whereas those with outer dynein arm defect and normal EM results or DNAH11 and DNAH5 mutations had less severe changes. We have robustly confirmed the worse prognosis for some genetic and ultrastructural defects, which association hitherto rested solely on spirometry.

Nutrition, epigenetic markers and growth in preterm infants.

BACKGROUND AND AIM: Maternal diet and early nutrition of newborns may affect the phenotype later in adulthood. Susceptibility of epigenetic mechanisms to the nutritional environment is a critical element in neonatal development. Epigenetic mechanisms could be considered as a bridge between environmental stimuli and long lasting phenotype. IC2, a key region on 11p15, is involved in the control of growth and regulates CDKN1C, PHLDA2 and KCNQ1, growth inhibitor genes. Our aim was to investigate the relationship between epigenetic markers, nutrition and postnatal growth. METHODS: We enrolled 37 newborns (gestational age at birth was <34 weeks) admitted to Neonatal Intensive Care Unit at University Hospital of Pisa. RESULTS: We observed a relationship between reduced protein and lipid intake and IC2 hypermethylation (p = .003 and p = .001 respectively) and we also investigated the correlation between growth pattern and IC2 methylation. CONCLUSION: The reduced growth, in part related to a reduced intake of nutrients (lipids and proteins), might be due to IC2 hypermethylation, causing an increased expression of growth inhibitor genes. IC2 hypermethylation could be a marker of reduced infants' growth and may guides us to nutritional interventional strategies for a precocious prevention of extrauterine growth restriction (EUGR).

Do the Heterozygous Carriers of a CYP24A1 Mutation Display a Different Biochemical Phenotype Than Wild Types?

CONTEXT: Human cytochrome P450 24 subfamily A member 1 (CYP24A1) loss-of-function mutations result in impaired activity of the 24-hydroxylase involved in vitamin D catabolism, thus inducing a vitamin D-dependent hypercalcemia. Homozygotes often present an overt clinical phenotype named idiopathic infantile hypercalcemia (IIH), whereas it is debated whether heterozygotes display an abnormal phenotype. OBJECTIVE: To compare the clinical and biochemical features of heterozygous carriers of CYP24A1 variant and healthy wild-type controls sharing the same genetic and environmental exposure. METHODS: A large family harboring the nonsense c.667A>T, p.Arg223* pathogenic variant in the CYP24A1 gene was evaluated. All subjects underwent clinical and biochemical evaluation and complete analysis of vitamin D metabolites using mass spectroscopy including 1,24,25(OH)3D3. Subjects were divided into 2 groups according to their genotype: heterozygotes and wild-type for the CYP24A1 variant. RESULTS: The proband, a 40-year-old man, homozygous for p.Arg223* pathogenic variant, had a history of mild hypercalcemia with a seasonal trend, recurrent nephrolithiasis, and no episodes of acute hypercalcemia. He showed the highest serum levels of fibroblast growth factor 23, the highest 25(OH)D3/24,25(OH)2D3 ratio and undetectable levels of 1,24,25(OH)3D3, which represent indicators of a loss-of-function CYP24A1. Compared with the wild-types, heterozygotes had higher serum calcium and 25(OH)D3 concentrations (P = .017 and P = .025, respectively), without any difference in the other biochemical parameters and in the rate of nephrolithiasis. CONCLUSION: Heterozygotes exhibit a biochemical phenotype different from that of wild-type subjects. In clinical practice, these individuals might require surveillance because of the potential risk of developing hypercalcemia and related clinical manifestations if exposed to triggering factors.

A novel genetic variant in DNAI2 detected by custom gene panel in a newborn with Primary Ciliary Dyskinesia: case report.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a highly heterogeneous genetic disorder caused by defects in motile cilia. The hallmark features of PCD are the chronic infections of the respiratory tract, moreover, clinical manifestations include also laterality defects and risk of male infertility. Clinical phenotypes of PCD are the result of mutations in genes encoding components of axonema or factors involved in axonemal assembly. Recent studies have identified over 45 PCD-associated genes, therefore, molecular analysis represents a powerful diagnostic tool to confirm and uncover new genetic causes of this rare disease. CASE PRESENTATION: Here, we describe a female infant of Moroccan origin with normal pressure hydrocephalus (NPH) in addition to most common PCD symptoms. Transmission Electron Microscopy (TEM) and molecular tests, such as a Next generation Sequencing panel and a custom array CGH, were performed for diagnosis of PCD. TEM revealed outer dynein arm (ODA) defects, whilst molecular analyses detected a novel 6,9 kb microdeletion in DNAI2 gene. CONCLUSIONS: Since DNAI2 mutations are very rare, this case report contributes to better delineate the important role of DNAI2 as causative of PCD phenotype, suggesting, furthermore, that the variations in DNAI2 may be as a new genetic risk factor for NPH. Indeed, although the association of hydrocephalus with PCD has been well documented, however, only a small number of human patients show this defect. Furthermore, this study highlights the importance of high-throughput technologies in advancing our understanding of heterogeneous genetic disorders.

Effect of BRCA1 missense variants on gene reversion in DNA double-strand break repair mutants and cell cycle-arrested cells of Saccharomyces cerevisiae.

Evaluation of the functional impact of germline BRCA1 variants that are likely to be associated to breast and ovarian cancer could help to investigate the mechanism of BRCA1 tumorigenesis. Expression of pathogenic BRCA1 missense variants increased homologous recombination (HR) and gene reversion (GR) in yeast. We thought to exploit yeast genetics to shed light on BRCA1-induced genome instability and tumorigenesis. We determined the effect on GR of several neutral and pathogenic BRCA1 variants in the yeast strain RSY6wt and its isogenic DSB repair mutants, such as mre11∆, rad50∆ and rad51∆. In the RSY6wt, four out of five pathogenic and two out of six neutral variants significantly increased GR; rad51∆ strain, the pathogenic variants C61G and A1708E induced a weak but significant increase in GR. On the other hand, in rad50∆ mutant expressing the pathogenic variants localised at the BRCT domain, a further GR increase was seen. The neutral variant N132K and the VUS A1789T induced a weak GR increase in mre11∆ mutant. Thus, BRCA1 missense variants require specific genetic functions and presumably induced GR by different mechanisms. As DNA repair is regulated by cell cycle, we determined the effect on GR of BRCA1 variants in cell cycle-arrested RSYwt cells. GR is highly BRCA1-inducible in S-phase-arrested cells as compared to G1 or G2. Sequence analysis of genomic DNA from ILV1 revertant clones showed that BRCA1-induced ilv1-92 reversion by base substitution when GR is at least 6-fold over the control. Our study demonstrated that BRCA1 may interfere with yeast DNA repair functions that are active in S-phase causing high level of GR. In addition, we confirmed here that yeast could be a reliable model to investigate the mechanism and genetic requirements of BRCA1-induced genome instability. Finally, developing yeast-based assays to characterise BRCA1 missense variants could be useful to design more precise therapies.

Mouse mammary tumor virus (MMTV) - like exogenous sequences are associated with sporadic but not hereditary human breast carcinoma.

The inheritance of mutated suppressor genes, such as BRCA1 and BRCA2, is acknowledged as an etiological factor in hereditary breast carcinoma (HBC). Two different molecular mechanisms are possible; the Knudson's "two hits" or the gene haploinsufficiency. Etiology of sporadic breast carcinoma (SBC) is not known, although data support the possible role of the betaretrovirus Mouse Mammary Tumor Virus (MMTV). This study analyzes the presence of MMTV exogenous sequences in two representative groups of HBC and SBC, excluding any contamination by murine and retroviral material and endogenous betaretroviruses. The 30.3% of 56 SBC contained MMTV sequences, against the 4.2% of 47 HBC (p < 0.001). Cases positive for viral sequences showed the presence of p14, signal peptide of the MMTV envelope precursor. This result was expected based on the fact that HBCs, having a specific genetic etiology, do not need the action of a carcinogenetic viral agent. Moreover, the striking results obtained by comparing two groups of vastly different tumors represent an additional element of quality control: the distinction between HBC and SBC is so well-defined that results cannot be ascribed to mere coincidence. This paper strengthens the hypothesis for a viral etiology for human sporadic breast carcinoma.