D-chiro-inositol in clinical practice: A perspective from The Experts Group on Inositol in Basic and Clinical Research (EGOI).

BACKGROUND: D-chiro-inositol is a natural molecule that, in association with its well-studied isomer myo-inositol, may play a role in treating various metabolic and gynecological disorders. OBJECTIVES: This perspective seeks to explore the mechanisms and functions of D-chiro-inositol, laying the foundations to discuss its use in clinical practice, across dysmetabolism, obesity, and hormonal dysregulation. METHODS: A narrative review of all the relevant papers known to the authors was conducted. OUTCOME: D-chiro-inositol acts through a variety of mechanisms, acting as an insulin sensitizer, inhibiting the transcription of aromatase, in addition to modulating white adipose tissue/brown adipose tissue trans differentiation. These different modes of action have potential applications in a variety of therapeutic fields including: PCOS, dysmetabolism, obesity, hypoestrogenic/hyperandrogenic disorders, and bone health. CONCLUSIONS: D-chiro-inositol mode of action has been studied in detail in recent years, resulting in a clear differentiation between D-chiro-inositol and its isomer myo-inositol. The insulin sensitizing activities of D-chiro-inositol are well understood; however, its potential applications in other fields, in particular obesity and hyperestrogenic/hypoandrogenic disorders in men and women, represent promising avenues of research that require further clinical study.

Considerations on the use of carrier screening testing in human reproduction: comparison between recommendations from the Italian Society of Human Genetics and other international societies.

PURPOSE: Carrier screening (CS) is a term used to describe a genetic test performed on individuals without family history of genetic disorders, to investigate the carrier status for pathogenic variants associated with multiple recessive conditions. The advent of next-generation sequencing enabled simultaneous CS for an increasing number of conditions; however, a consensus on which diseases to include in gene panels and how to best develop the provision of CS is far to be reached. Therefore, the provision of CS is jeopardized and inconsistent and requires solving several important issues. METHODS: In 2020, the Italian Society of Human Genetics (SIGU) established a working group composed of clinical and laboratory geneticists from public and private fields to elaborate a document to define indications and best practice of CS provision for couples planning a pregnancy. RESULTS: Hereby, we present the outcome of the Italian working group's activity and compare it with previously published international recommendations (American College of Medical Genetics and Genomics (ACMG), American College of Obstetricians and Gynecologists (ACOG), and Royal Australian and New Zealand College of Obstetricians and Gynaecologists (RANZCOG)). We determine a core message on genetic counseling and nine main subject categories to explore, spanning from goals and execution to technical scientific, ethical, and socio-economic topics. Moreover, a level of agreement on the most critical points is discussed using a 5-point agreement scale, demonstrating a high level of consensus among the four societies. CONCLUSIONS: This document is intended to provide genetic and healthcare professionals involved in human reproduction with guidance regarding the clinical implementation of CS.

Expanding the Molecular Spectrum of ANKRD11 Gene Defects in 33 Patients with a Clinical Presentation of KBG Syndrome.

KBG syndrome (KBGS) is a neurodevelopmental disorder caused by the Ankyrin Repeat Domain 11 (ANKRD11) haploinsufficiency. Here, we report the molecular investigations performed on a cohort of 33 individuals with KBGS clinical suspicion. By using a multi-testing genomic approach, including gene sequencing, Chromosome Microarray Analysis (CMA), and RT-qPCR gene expression assay, we searched for pathogenic alterations in ANKRD11. A molecular diagnosis was obtained in 22 out of 33 patients (67%). ANKRD11 sequencing disclosed pathogenic or likely pathogenic variants in 18 out of 33 patients. CMA identified one full and one terminal ANKRD11 pathogenic deletions, and one partial duplication and one intronic microdeletion, with both possibly being pathogenic. The pathogenic effect was established by RT-qPCR, which confirmed ANKRD11 haploinsufficiency only for the three deletions. Moreover, RT-qPCR applied to six molecularly unsolved KBGS patients identified gene downregulation in a clinically typical patient with previous negative tests, and further molecular investigations revealed a cryptic deletion involving the gene promoter. In conclusion, ANKRD11 pathogenic variants could also involve the regulatory regions of the gene. Moreover, the application of a multi-test approach along with the innovative use of RT-qPCR improved the diagnostic yield in KBGS suspected patients.

Inositols: From Established Knowledge to Novel Approaches.

Myo-inositol (myo-Ins) and D-chiro-inositol (D-chiro-Ins) are natural compounds involved in many biological pathways. Since the discovery of their involvement in endocrine signal transduction, myo-Ins and D-chiro-Ins supplementation has contributed to clinical approaches in ameliorating many gynecological and endocrinological diseases. Currently both myo-Ins and D-chiro-Ins are well-tolerated, effective alternative candidates to the classical insulin sensitizers, and are useful treatments in preventing and treating metabolic and reproductive disorders such as polycystic ovary syndrome (PCOS), gestational diabetes mellitus (GDM), and male fertility disturbances, like sperm abnormalities. Moreover, besides metabolic activity, myo-Ins and D-chiro-Ins deeply influence steroidogenesis, regulating the pools of androgens and estrogens, likely in opposite ways. Given the complexity of inositol-related mechanisms of action, many of their beneficial effects are still under scrutiny. Therefore, continuing research aims to discover new emerging roles and mechanisms that can allow clinicians to tailor inositol therapy and to use it in other medical areas, hitherto unexplored. The present paper outlines the established evidence on inositols and updates on recent research, namely concerning D-chiro-Ins involvement into steroidogenesis. In particular, D-chiro-Ins mediates insulin-induced testosterone biosynthesis from ovarian thecal cells and directly affects synthesis of estrogens by modulating the expression of the aromatase enzyme. Ovaries, as well as other organs and tissues, are characterized by a specific ratio of myo-Ins to D-chiro-Ins, which ensures their healthy state and proper functionality. Altered inositol ratios may account for pathological conditions, causing an imbalance in sex hormones. Such situations usually occur in association with medical conditions, such as PCOS, or as a consequence of some pharmacological treatments. Based on the physiological role of inositols and the pathological implications of altered myo-Ins to D-chiro-Ins ratios, inositol therapy may be designed with two different aims: (1) restoring the inositol physiological ratio; (2) altering the ratio in a controlled way to achieve specific effects.

DNA methylation variations in familial female and male breast cancer.

In total, ~25% of familial breast cancer (BC) is attributed to germline mutations of the BRCA1 and BRCA2 genes, while the rest of the cases are included in the BRCAX group. BC is also known to affect men, with a worldwide incidence of 1%. Epigenetic alterations, including DNA methylation, have been rarely studied in male breast cancer (MBC) on a genome-wide level. The aim of the present study was to examine the global DNA methylation profiles of patients with BC to identify differences between familial female breast cancer (FBC) and MBC, and according to BRCA1, BRCA2 or BRCAX mutation status. The genomic DNA of formalin-fixed paraffin-embedded tissues from 17 women and 7 men with BC was subjected to methylated DNA immunoprecipitation and hybridized on human promoter microarrays. The comparison between FBC and MBC revealed 2,846 significant differentially methylated regions corresponding to 2,486 annotated genes. Gene Ontology enrichment analysis revealed molecular function terms, such as the GTPase superfamily genes (particularly the GTPase Rho GAP/GEF and GTPase RAB), and cellular component terms associated with cytoskeletal architecture, such as 'cytoskeletal part', 'keratin filament' and 'intermediate filament'. When only FBC was considered, several cancer-associated pathways were among the most enriched KEGG pathways of differentially methylated genes when the BRCA2 group was compared with the BRCAX or BRCA1+BRCAX groups. The comparison between the BRCA1 and BRCA2+BRCAX groups comprised the molecular function term 'cytoskeletal protein binding'. Finally, the functional annotation of differentially methylated genes between the BRCAX and BRCA1+BRCA2 groups indicated that the most enriched molecular function terms were associated with GTPase activity. In conclusion, to the best of our knowledge, the present study was the first to compare the global DNA methylation profile of familial FBC and MBC. The results may provide useful insights into the epigenomic subtyping of BC and shed light on a possible novel molecular mechanism underlying BC carcinogenesis.

An update on the use of inositols in preventing gestational diabetes mellitus (GDM) and neural tube defects (NTDs).

INTRODUCTION: Obstetric history and maternal body composition and lifestyle may be associated with serious complications both for the mother, such as gestational diabetes mellitus (GDM), and for the fetus, including congenital malformations such as neural tube defects (NTDs). AREAS COVERED: In view of the recent knowledge, changes in nutritional and physical activity habits ameliorate glycemic control during pregnancy and in turn improve maternal and neonatal health outcomes. Recently, a series of small clinical and experimental studies indicated that supplemenation with inositols, a family of insulin sensitizers, was associated with beneficial impact for both GDM and NTDs. EXPERT OPINION: Herein, we discuss the most significant scientific evidence supporting myo-inositol administration as a prophylaxis for the above-mentioned conditions.

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification.

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification.

Corrigendum to "Myoinositol: The Bridge (PONTI) to Reach a Healthy Pregnancy".

[This corrects the article DOI: 10.1155/2017/5846286.].

Genetic Testing and Clinical Management Practices for Variants in Non-BRCA1/2 Breast (and Breast/Ovarian) Cancer Susceptibility Genes: An International Survey by the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) Clinical Working Group.

PURPOSE: To describe a snapshot of international genetic testing practices, specifically regarding the use of multigene panels, for hereditary breast/ovarian cancers. We conducted a survey through the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium, covering questions about 16 non-BRCA1/2 genes. METHODS: Data were collected via in-person and paper/electronic surveys. ENIGMA members from around the world were invited to participate. Additional information was collected via country networks in the United Kingdom and in Italy. RESULTS: Responses from 61 cancer genetics practices across 20 countries showed that 16 genes were tested by > 50% of the centers, but only six (PALB2, TP53, PTEN, CHEK2, ATM, and BRIP1) were tested regularly. US centers tested the genes most often, whereas United Kingdom and Italian centers with no direct ENIGMA affiliation at the time of the survey were the least likely to regularly test them. Most centers tested the 16 genes through multigene panels; some centers tested TP53, PTEN, and other cancer syndrome-associated genes individually. Most centers reported (likely) pathogenic variants to patients and would test family members for such variants. Gene-specific guidelines for breast and ovarian cancer risk management were limited and differed among countries, especially with regard to starting age and type of imaging and risk-reducing surgery recommendations. CONCLUSION: Currently, a small number of genes beyond BRCA1/2 are routinely analyzed worldwide, and management guidelines are limited and largely based on expert opinion. To attain clinical implementation of multigene panel testing through evidence-based management practices, it is paramount that clinicians (and patients) participate in international initiatives that share panel testing data, interpret sequence variants, and collect prospective data to underpin risk estimates and evaluate the outcome of risk intervention strategies.

Myoinositol: The Bridge (PONTI) to Reach a Healthy Pregnancy.

The use of folic acid in the periconceptional period can prevent about 70% of neural tube defects (NTDs). In the remaining cases, no medical prevention is available, and those conditions should be defined as folate-resistant NTDs. Rodent models suggest that some folate-resistant NTDs can be prevented by inositol (myoinositol and chiroinositol) supplementation prior to pregnancy. Should folic acid be combined with myoinositol periconceptional supplementation to reduce the overall risk of NTDs even in humans? Hereafter, we discuss the results from the PONTI study that strongly support both the effectiveness and safety of myoinositol periconceptional supplementation in preventing human NTDs. We further report on the largest case series of pregnancies treated with myoinositol and folic acid. At our institution, a sequential study during 12 years involved mothers at risk of fetal NTDs, and 29 babies from 27 pregnancies were born after periconceptional combined myoinositol and folic acid supplementation. No case of NTDs was observed, despite the high recurrence risk in the mothers. Taken together, those data suggest that periconceptional folic acid plus myoinositol can reduce both the occurrence and recurrence risks of NTDs in a greater number of cases than folic acid alone.

Results from the International Consensus Conference on Myo-inositol and d-chiro-inositol in Obstetrics and Gynecology: the link between metabolic syndrome and PCOS.

In recent years, interest has been focused to the study of the two major inositol stereoisomers: myo-inositol (MI) and d-chiro-inositol (DCI), because of their involvement, as second messengers of insulin, in several insulin-dependent processes, such as metabolic syndrome and polycystic ovary syndrome. Although these molecules have different functions, very often their roles have been confused, while the meaning of several observations still needs to be interpreted under a more rigorous physiological framework. With the aim of clarifying this issue, the 2013 International Consensus Conference on MI and DCI in Obstetrics and Gynecology identified opinion leaders in all fields related to this area of research. They examined seminal experimental papers and randomized clinical trials reporting the role and the use of inositol(s) in clinical practice. The main topics were the relation between inositol(s) and metabolic syndrome, polycystic ovary syndrome (with a focus on both metabolic and reproductive aspects), congenital anomalies, gestational diabetes. Clinical trials demonstrated that inositol(s) supplementation could fruitfully affect different pathophysiological aspects of disorders pertaining Obstetrics and Gynecology. The treatment of PCOS women as well as the prevention of GDM seem those clinical conditions which take more advantages from MI supplementation, when used at a dose of 2g twice/day. The clinical experience with MI is largely superior to the one with DCI. However, the existence of tissue-specific ratios, namely in the ovary, has prompted researchers to recently develop a treatment based on both molecules in the proportion of 40 (MI) to 1 (DCI).

Results from the International Consensus Conference on myo-inositol and D-chiro-inositol in Obstetrics and Gynecology--assisted reproduction technology.

A substantial body of research on mammalian gametogenesis and human reproduction has recently investigated the effect of myo-inositol (MyoIns) on oocyte and sperm cell quality, due to its possible application to medically assisted reproduction. With a growing number of both clinical and basic research papers, the meaning of several observations now needs to be interpreted under a solid and rigorous physiological framework. The 2013 Florence International Consensus Conference on Myo- and D-chiro-inositol in obstetrics and gynecology has answered a number of research questions concerning the use of the two stereoisomers in assisted reproductive technologies. Available clinical trials and studies on the physiological and pharmacological effects of these molecules have been surveyed. Specifically, the physiological involvement of MyoIns in oocyte maturation and sperm cell functions has been discussed, providing an answer to the following questions: (1) Are inositols physiologically involved in oocyte maturation? (2) Are inositols involved in the physiology of spermatozoa function? (3) Is treatment with inositols helpful within assisted reproduction technology cycles? (4) Are there any differences in clinical efficacy between MyoIns and D-chiro-inositol? The conclusions of this Conference, drawn depending on expert panel opinions and shared with all the participants, are summarized in this review paper.

A case of neonatal Jeune syndrome expanding the phenotype.

KEY CLINICAL MESSAGE: We report the case of a premature, very low birth weight, newborn with stigmata of Jeune syndrome, a rare skeletal dysplasia, and marked renal involvement (i.e. remarkable prenatal oligohydramnios, histologic nephronophthisis-like pattern, macroscopic renal cysts, and renal failure), expanding the phenotype consistent with the continuum of syndromic ciliopathies.

Further genotype-phenotype correlation emerging from two families with PLP1 exon 4 skipping.

Proteolipid protein 1 (PLP1) gene-related disorders due to mutations in the PLP1 include a wide spectrum of X-linked disorders ranging from severe connatal Pelizaeus-Merzbacher disease (PMD) to spastic paraplegia 2 (SPG2). Duplications, deletions or point mutations in coding and noncoding regions of the PLP1 gene may occur. We report the clinical, neuroradiologic and molecular findings in six patients from two unrelated families. The affected males showed severe mental retardation, spastic tetraparesis, inability of walking and pes cavus at onset in early infancy. Brain magnetic resonance imaging (MRI) showed hypomyelination and brain atrophy. Nystagmus was never observed. The affected females showed adult-onset progressive spastic paraparesis leading to wheel-chair dependency and subtle white matter changes on brain MRI. Molecular studies in the two families identified two different intronic mutations, the novel c.622+2T>C and the known c.622+1G>A, leading to the skipping of PLP1-exon 4. The clinical presentation of the affected males did not consistently fit in any of the PLP1-related disorder subtypes (i.e., connatal or classic PMD, SPG2 and 'PLP1 null syndrome'), and in addition, the carrier females were symptomatic despite the severe clinical picture of their respective probands. This study provides new insight into the genotype-phenotype correlations of patients with PLP1 splice-site mutations.

Design and validation of a pericentromeric BAC clone set aimed at improving diagnosis and phenotype prediction of supernumerary marker chromosomes.

BACKGROUND: Small supernumerary marker chromosomes (sSMCs) are additional, structurally abnormal chromosomes, generally smaller than chromosome 20 of the same metaphase spread. Due to their small size, they are difficult to characterize by conventional cytogenetics alone. In regard to their clinical effects, sSMCs are a heterogeneous group: in particular, sSMCs containing pericentromeric euchromatin are likely to be associated with abnormal outcomes, although exceptions have been reported. To improve characterization of the genetic content of sSMCs, several approaches might be applied based on different molecular and molecular-cytogenetic assays, e.g., fluorescent in situ hybridization (FISH), array-based comparative genomic hybridization (array CGH), and multiplex ligation-dependent probe amplification (MLPA).To provide a complementary tool for the characterization of sSMCs, we constructed and validated a new, FISH-based, pericentromeric Bacterial Artificial Chromosome (BAC) clone set that with a high resolution spans the most proximal euchromatic sequences of all human chromosome arms, excluding the acrocentric short arms. RESULTS: By FISH analysis, we assayed 561 pericentromeric BAC probes and excluded 75 that showed a wrong chromosomal localization. The remaining 486 probes were used to establish 43 BAC-based pericentromeric panels. Each panel consists of a core, which with a high resolution covers the most proximal euchromatic ~0.7 Mb (on average) of each chromosome arm and generally bridges the heterochromatin/euchromatin junction, as well as clones located proximally and distally to the core. The pericentromeric clone set was subsequently validated by the characterization of 19 sSMCs. Using the core probes, we could rapidly distinguish between heterochromatic (1/19) and euchromatic (11/19) sSMCs, and estimate the euchromatic DNA content, which ranged from approximately 0.13 to more than 10 Mb. The characterization was not completed for seven sSMCs due to a lack of information about the covered region in the reference sequence (1/19) or sample insufficiency (6/19). CONCLUSIONS: Our results demonstrate that this pericentromeric clone set is useful as an alternative tool for sSMC characterization, primarily in cases of very small SMCs that contain either heterochromatin exclusively or a tiny amount of euchromatic sequence, and also in cases of low-level or cryptic mosaicism. The resulting data will foster knowledge of human proximal euchromatic regions involved in chromosomal imbalances, thereby improving genotype-phenotype correlations.

New T530C mutation in the aspartoacylase gene caused Canavan disease with no correlation between severity and N-acetylaspartate excretion.

OBJECTIVE: Canavan disease (OMIM 271900) is a severe autosomal recessive neurodegenerative disorder characterized by spongy degeneration of the brain and caused by mutations in the gene encoding for aspartoacylase (ASPA). The enzyme is responsible for the catalyses of the brain-specific compound N-acetylaspartate (NAA). DESIGN AND METHODS: We report the case of two Egyptian sibling patients suspected of Canavan disease (CD) showing clinical deterioration, white matter degeneration, megalencephaly and severe intellectual impairment. The patients underwent magnetic resonance imaging (MRI) and biochemical analysis of NAA in biological fluid samples (serum and urine). Subsequently, in order to determine the mutation responsible for CD in these two sibs, a molecular biological examination was performed. RESULTS: MRI findings and quantification of high NAA excretion (1378.5 and 680.1µmolNAA/mmolcreatinine in urine of 4months and 4years old patients, respectively) confirmed the diagnosis of CD and prompted a search for the responsible mutation. The molecular biological analysis revealed homozygosity for the substitution T530C (Ile177Thr) in the exon 4 of the ASPA gene in both sibs. A total loss of enzymatic activity was also recorded. CONCLUSIONS: The substitution T530C (Ile177Thr) results in a novel missense mutation causing a CD phenotype with severe clinical characteristics. This mutation was not previously described in the literature. In these two sibs, urinary concentration of NAA appears to correlate inversely to symptom severity and CD progression.

The future of prenatal diagnosis: karyotype, microarray or both? Technical and ethical considerations.

Prenatal diagnosis is now offered to high-risk pregnancies, including advanced maternal age, ultrasound anomalies and positive Down's syndrome screening, and karyotype on cultured fetal material is the test of choice to screen these pregnancies. However, microscope analysis can only detect gross chromosome abnormalities, highlighting the need for more sensitive techniques. It has recently been established that the higher resolution of microarray-based platforms can increase the diagnostic yield, offering more information to couples, and it is being discussed as a replacement to the standard karyotype. Conversely, the very high sensitivity of microarray-based analysis allows us to detect small microdeletions/microduplications (copy number variations) with unknown functional role and difficult genotype/phenotype correlation. In addition, the new copy number variation syndromes are often associated with variable outcomes, ranging from normal to severely affected individuals. This means that the microarray-based analysis introduced routinely in prenatal diagnosis needs to answer the question: are laboratory staff, clinical geneticists and counselors really experienced enough to manage these new scenarios?

Myo-inositol soft gel capsules may prevent the risk of coffee-induced neural tube defects.

OBJECTIVE: Neural tube defects (NTDs) are classified as folate sensitive (about 70%) and folate resistant (about 30%); although folic acid is able to prevent the former, several data have shown that inositol may prevent the latter. It has recently been proposed that coffee intake might represent a risk factor for NTD, likely by interfering with the inositol signaling. In the present study, we tested the hypothesis that, beside affecting the inositol signaling pathway, coffee also interferes with inositol absorption. RESEARCH DESIGN AND METHODS: In order to evaluate coffee possible negative effects on inositol gastrointestinal absorption, a single-dose bioavailability trial was conducted. Pharmacokinetics (PK) parameters of myo-inositol (MI) powder and MI soft gelatin capsules swallowed with water and with a single 'espresso' were compared. PK profiles were obtained by analysis of MI plasma concentration, and the respective MI bioavailability was compared. RESULTS: Myo-inositol powder administration was negatively affected by coffee intake, thus suggesting an additional explanation to the interference between inositol deficiency and coffee consumption. On the contrary, the concomitant single 'espresso' consumption did not affect MI absorption following MI soft gelatin capsules administration. Furthermore, it was observed that MI soft gelatin capsule administration resulted in improved bioavailability compared to the MI powder form. CONCLUSIONS: Myo-inositol soft gelatin capsules should be considered for the preventive treatment of NTDs in folate-resistant subjects due to their higher bioavailability and to the capability to reduce espresso interference.