A simultaneous next-generation sequencing approach to the diagnosis of couple infertility.

BACKGROUND: Infertility is a disorder of the male and/or female reproductive system, characterized by failure to establish a clinical pregnancy after 12 months of regular unprotected sexual intercourse. On a world basis, about one in six couples are affected by infertility during their reproductive lifespan. Despite a comprehensive diagnostic work-up, infertility in about 50% of couples remains idiopathic. In this context, a next-generation sequencing (NGS) approach has been suggested to increase diagnostic yield. Accordingly, this study aimed to evaluate the effectiveness of a custom-made NGS gene panel for the simultaneous genetic diagnosis of both partners of a large population of infertile couples. METHODS: We developed a custom-made NGS panel for 229 genes associated with male and female infertility. The panel targeted exons and their flanking regions and was used to screen 99 couples with idiopathic infertility. RESULTS: NGS sequencing revealed five pathogenic variants in six couples and 17 likely pathogenic variants or variants with uncertain significance (VUS). The pathogenic variants were identified in the following genes: GNRHR, CCDC39, DNAH5, and CCDC103; likely pathogenic variants were identified in TAC3, PROKR2, and CFTR; VUS were identified in CATSPER2, FGFR1, LRRC6, DNAH5, DNAH11, TGFBR3, and DNAI1. CONCLUSIONS: The panel of genes designed for this study allowed the identification of pathogenic gene mutations and the presence of VUS in 6.1% and 17.2%, respectively, of couples with idiopathic infertility. This is the first study to successfully apply an NGS-based genetic screening including 229 genes known to play a role in both male and female infertility.

Improving clinical interpretation of five KRIT1 and PDCD10 intronic variants.

Cerebral cavernous malformation (CCM) is a vascular malformation of the central nervous system which may occur sporadically or segregate within families due to heterozygous variants in KRIT1/CCM1, MGC4607/CCM2 or PDCD10/CCM3. Intronic variants are not uncommon in familial CCM, but their clinical interpretation is often hampered by insufficient data supporting in silico predictions. Here, the mRNA analysis for two intronic unpublished variants (KRIT1 c.1147-7 T > G and PDCD10 c.395 + 2 T > G) and three previously published variants in KRIT1 but without data supporting their effects was carried out. This study demonstrated that all variants can induce a frameshift with the lack of residues located in the C-terminal regions and involved in protein-protein complex formation, which is essential for vascular homeostasis. These results support the introduction of mRNA analysis in the diagnostic pathway of familial CCM and expand the knowledge of abnormal splicing patterning in this disorder.

Genetic analysis of genes associated with epilepsy.

BACKGROUND AND AIM: Epilepsy is a neurological disorder in which the altered activity of neurons causes convulsions, periods of unusual behavior and, sometimes, loss of consciousness. The aim of this mini-review is to summarize all the syndromes characterized by epilepsy and for which the associated gene is known. METHODS: We searched those syndromes in PubMed and OMIM database. RESULTS: Genetic causes underlie epilepsy in about 40% of individuals. Epilepsies are phenotypically and genetically heterogeneous. Inheritance can be autosomal dominant or recessive or X-linked recessive/dominant. CONCLUSION: Since epilepsy has high genetic heterogeneity, in diagnostics, the parallel sequencing of a panel of genes may speed up the determination of the molecular etiology and/or establish a risk of recurrence in family members for the purpose of planning appropriate preventive and/or therapeutic measures.

Complications related to in vitro reproductive techniques support the implementation of natural procreative technologies.

BACKGROUND AND AIM: Infertility affects ~20% of the couples in the world. Assisted reproductive technologies (ARTs) are currently the most common treatment option for infertility. Nevertheless, ARTs may be associated with complications for mothers and/or offspring. Natural procreative technology (NaProTechnology) is a natural treatment which minimizes these risks by seeking to identify the causes of infertility to enable better treatments. This narrative review summarizes the complications related to ARTs and clarifies how the NaProTechnology approach can help ARTs to achieve better results or be used in alternative to ARTs. METHODS: Data in the literature indicate that NaProTechnology is a natural approach for treating infertility. RESULTS: The percentage of live births obtained by NaProTechnology is similar to that of ARTs. CONCLUSIONS: An extensive search for the genetic defects causing infertility or subfertility through genetic testing can help both ARTs and NaProTechnology to achieve successful pregnancies. By discovering the underlying causes of infertility, genetic tests enable better family counseling, like the implications of transmitting risk- and disease-alleles to future generations.

Prenatal genetic diagnosis: Fetal therapy as a possible solution to a positive test.

BACKGROUND: Fetal abnormalities cause 20% of perinatal deaths. Advances in prenatal genetic and other types of screening offer great opportunities for identifying high risk pregnancies. METHODS: Through a literature search, here we summarise what are the prenatal diagnostic technique that are being used and how those techniques may allow for prenatal interventions. RESULTS: Next generation sequencing and non-invasive prenatal testing are fundamental for clinical diagnostics because of their sensitivity and accuracy in identifying point mutations, aneuploidies, and microdeletions, respectively. Timely identification of genetic disorders and other fetal abnormalities enables early intervention, such as in-utero gene therapy, fetal drug therapy and prenatal surgery. CONCLUSION: Prenatal intervention is mainly focused on conditions that may cause death or lifelong disabilities, like spina bifida, congenital diaphragm hernia and sacrococcygeal teratoma; and may be an alternative therapeutic option to termination of pregnancy. However, it is not yet widely available, due to lack of specialized centers.

Propranolol for familial cerebral cavernous malformation (Treat_CCM): study protocol for a randomized controlled pilot trial.

BACKGROUND: Cerebral cavernous malformations (CCMs) are vascular malformations characterized by clusters of enlarged leaky capillaries in the central nervous system. They may result in intracranial haemorrhage, epileptic seizure(s), or focal neurological deficits, and potentially lead to severe disability. Globally, CCMs represent the second most common intracranial vascular malformation in humans, and their familial form (FCCMs) accounts for one-fifth of cases. Neurosurgical excision, and perhaps stereotactic radiosurgery, is the only available therapeutic option. Case reports suggest that propranolol might modify disease progression. METHODS: Treat_CCM is a prospective, randomized, open-label, blinded endpoint (PROBE), parallel-group trial involving six Italian clinical centres with central reading of brain magnetic resonance imaging (MRI) and adverse events. Patients with symptomatic FCCMs are randomized (2:1 ratio) either to propranolol (40-80 mg twice daily) in addition to standard care or to standard care alone (i.e. anti-epileptic drugs or headache treatments). The primary outcome is intracranial haemorrhage or focal neurological deficit attributable to CCMs. The secondary outcomes are MRI changes over time (i.e. de novo CCM lesions, CCM size and signal characteristics, iron deposition, and vascular leakage as assessed by quantitative susceptibility mapping and dynamic contrast enhanced permeability), disability, health-related quality of life, depression severity, and anxiety (SF-36, BDI-II, State-Trait Anxiety Inventory). DISCUSSION: Treat_CCM will evaluate the safety and efficacy of propranolol for CCMs following promising case reports in a randomized controlled trial. The direction of effect on the primary outcome and the consistency of effects on the secondary outcomes (even if none of them yield statistically significant differences) of this external pilot study may lead to a larger sample size in a definitive phase 2 trial. TRIAL REGISTRATION: ClinicalTrails.gov, NCT03589014. Retrospectively registered on 17 July 2018.

Generation of induced pluripotent stem cell line CSSi008-A (4698) from a patient affected by advanced stage of Dentato-Rubral-Pallidoluysian atrophy (DRPLA).

Dentato-Rubral-pallidoluysian atrophy (DRPLA) is a rare autosomal, dominant, progressive neurodegenerative disease that causes involuntary movements, mental and emotional problems. DRPLA is caused by a mutation in the ATN1 gene that encodes for an abnormal polyglutamine stretch in the atrophin-1 protein. DRPLA is most common in the Japanese population, where it has an estimated incidence of 2 to 7 per million people. This condition has also been seen in families from North America and Europe. We obtained a reprogrammed iPSC line from a Caucasian patient with a juvenile onset of the disease, carrying 64 CAG repeat expansion in the ATN1 gene.

Molecular diagnostic workflow, clinical interpretation of sequence variants, and data repository procedures in 140 individuals with familial cerebral cavernous malformations.

Familial cerebral cavernous malformation (FCCM) is an autosomal dominant vascular disorder caused by heterozygous deleterious variants in KRIT1, CCM2 or PDCD10. In a previous study, we presented the clinical and molecular findings in 140 FCCM individuals. In the present work, we report supporting information on (a) applied diagnostic workflow; (b) clinical significance of molecular findings according to the American College of Medical Genetics and Genomics/Association for Molecular Pathology recommendations; (c) standardization of molecular and clinical data according to the Human Phenotype Ontology; (d) preliminary genotype-phenotype correlations on a subgroup of patients by considering sex, age at diagnosis, neurological symptoms, and number and anatomical site(s) of vascular anomalies; (e) datasets submitted to the Leiden Open Variation Database. An overview of the changes of our diagnostic approach before and after the transition to next-generation sequencing is also reported. This work presents the full procedure that we apply for molecular testing, data interpretation and storing in public databases in FCCM.

A Nonsense Mitochondrial DNA Mutation Associates with Dysfunction of HIF1α in a Von Hippel-Lindau Renal Oncocytoma.

The Von Hippel-Lindau (VHL) syndrome has been rarely associated with renal oncocytomas, and tumors usually show HIF1α chronic stabilization. By contrast, oncocytomas mainly associated with respiratory chain (RC) defects due to severe mitochondrial DNA (mtDNA) mutations are incapable of stabilizing HIF1α, since oxygen consumption by the RC is dramatically diminished and prolylhydroxylase activity is increased by α-ketoglutarate accumulation following Krebs cycle slowdown. Here, we investigate the cooccurrence of a pseudohypoxic condition with oncocytic transformation in a case of VHL-associated renal oncocytoma. While HIF1α was abundant in nuclei concordantly with defects in VHL, negative staining of its targets carbonic anhydrase IX (CAIX) and glucose transporter GLUT1, usually overexpressed in VHL-associated neoplasms, suggested HIF1α to be present in its inactive (hydroxylated) form. MtDNA sequencing and immunohistochemistry analyses revealed a MT-CO1 stop-gain mutation and cytochrome c oxidase loss. We suggest that a mitochondrial respiration impairment may lead to hyperhydroxylation of the transcription factor, which we confirmed by specific staining of hydroxylated HIF1α. Such inactive form hence accumulated in the VHL-deficient tumor, where it may contribute to the benign nature of the neoplasm. We propose that the protumorigenic role of HIF1α in VHL cancers may be blunted through drugs inhibiting mitochondrial respiratory complexes, such as metformin.

Mutational spectrum and clinical signatures in 114 families with hereditary multiple osteochondromas: insights into molecular properties of selected exostosin variants.

Hereditary multiple osteochondromas (HMO) is a rare autosomal dominant skeletal disorder, caused by heterozygous variants in either EXT1 or EXT2, which encode proteins involved in the biogenesis of heparan sulphate. Pathogenesis and genotype-phenotype correlations remain poorly understood. We studied 114 HMO families (158 affected individuals) with causative EXT1 or EXT2 variants identified by Sanger sequencing, or multiplex ligation-dependent probe amplification and qPCR. Eighty-seven disease-causative variants (55 novel and 32 known) were identified including frameshift (42%), nonsense (32%), missense (11%), splicing (10%) variants and genomic rearrangements (5%). Informative clinical features were available for 42 EXT1 and 27 EXT2 subjects. Osteochondromas were more frequent in EXT1 as compared to EXT2 patients. Anatomical distribution of lesions showed significant differences based on causative gene. Microscopy analysis for selected EXT1 and EXT2 variants verified that EXT1 and EXT2 mutants failed to co-localize each other and loss Golgi localization by surrounding the nucleus and/or assuming a diffuse intracellular distribution. In a cell viability study, cells expressing EXT1 and EXT2 mutants proliferated more slowly than cells expressing wild-type proteins. This confirms the physiological relevance of EXT1 and EXT2 Golgi co-localization and the key role of these proteins in the cell cycle. Taken together, our data expand genotype-phenotype correlations, offer further insights in the pathogenesis of HMO and open the path to future therapies.

A single-center study on 140 patients with cerebral cavernous malformations: 28 new pathogenic variants and functional characterization of a PDCD10 large deletion.

Cerebral cavernous malformation (CCM) is a capillary malformation arising in the central nervous system. CCM may occur sporadically or cluster in families with autosomal dominant transmission, incomplete penetrance, and variable expressivity. Three genes are associated with CCM KRIT1, CCM2, and PDCD10. This work is a retrospective single-center molecular study on samples from multiple Italian clinical providers. From a pool of 317 CCM index patients, we found germline variants in either of the three genes in 80 (25.2%) probands, for a total of 55 different variants. In available families, extended molecular analysis found segregation in 60 additional subjects, for a total of 140 mutated individuals. From the 55 variants, 39 occurred in KRIT1 (20 novel), 8 in CCM2 (4 novel), and 8 in PDCD10 (4 novel). Effects of the three novel KRIT1 missense variants were characterized in silico. We also investigated a novel PDCD10 deletion spanning exon 4-10, on patient's fibroblasts, which showed significant reduction of interactions between KRIT1 and CCM2 encoded proteins and impaired autophagy process. This is the largest study in Italian CCM patients and expands the known mutational spectrum of KRIT1, CCM2, and PDCD10. Our approach highlights the relevance of seeking supporting information to pathogenicity of new variants for the improvement of management of CCM.

Novel association of MEN1 gene mutations with parathyroid carcinoma.

Inactivating mutations of the multiple endocrine neoplasia 1 (MEN1) gene cause MEN1 syndrome, characterized by primary hyperparathyroidism (pHPT), and parathyroid and gastro-entero-pancreatic pituitary tumors. At present, only 14 cases of malignant parathyroid tumor have been associated with the syndrome, with 6 cases carrying an inactivating mutation of the MEN1 gene. The present study presents the case of a 48-year-old female who presented with multigland pHPT and multiple pancreatic lesions. The patient underwent surgery several times for the excision of parathyroid hyperplasia, carcinoma and adenoma. The MEN1 gene was screened, revealing three variants (in cis) at the intron/exon 3 boundary (IVS2-3G>C, c.497A>T and c.499G>T) detected on the DNA of the proband, not shared by her relatives. RNA sequencing revealed that the IVS2-3C>G variant caused the skipping of the exon 3. Therefore, the present study reports on a novel rare association of MEN1 syndrome and parathyroid carcinoma. The reported splicing mutation was previously identified in subjects who always developed malignant lesions; thus, a possible genotype-phenotype association may be considered.

High Mitochondrial DNA Copy Number Is a Protective Factor From Vision Loss in Heteroplasmic Leber's Hereditary Optic Neuropathy (LHON).

PURPOSE: Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease that typically causes bilateral blindness in young men. It is characterized by as yet undisclosed genetic and environmental factors affecting the incomplete penetrance. METHODS: We identified 27 LHON subjects who possess heteroplasmic primary LHON mutations. Mitochondrial DNA (mtDNA) copy number was evaluated. RESULTS: The presence of centrocecal scotoma, an edematous, hyperemic optic nerve head, and vascular tortuosity, as well as telangiectasia was recognized in affected subjects. We found higher cellular mtDNA content in peripheral blood cells of unaffected heteroplasmic mutation carriers with respect to the affected. CONCLUSIONS: The increase of cellular mtDNA content prevents complete loss of vision despite the presence of a heteroplasmic state of LHON primary mutation, suggesting that it is a key factor responsible for penetrance of LHON.

Osteoporosis-pseudoglioma syndrome: Report of two cases and a manifesting carrier.

BACKGROUND: Osteoporosis-pseudoglioma syndrome is a very rare disease mainly characterized by severe eye abnormalities and osteoporosis but also causing a broader range of clinical features. The syndrome is associated with homozygous or compound heterozygous variations in the LRP5 gene. In this report, we describe two children with a severe early-onset form of familial exudative vitreoretinopathy associated with skeletal abnormalities. MATERIALS AND METHODS: Two probands (4 and 7 years of age respectively) and their parents were assessed by genetic analysis and comprehensive ophthalmic examination. RESULTS: In both probands, the diagnosis of osteoporosis-pseudoglioma syndrome was confirmed by detection of three new pathogenic LRP5 variants: p.(Asp379Asn), found in the homozygous state in one proband, and p.(Asp203Ala) in the compound heterozygous state with p.(Cys612Valfs*25) in the other. The clinical and genetic study was extended to their parents, confirming that heterozygous carriers may also have incomplete clinical manifestation of this syndrome. CONCLUSIONS: To our knowledge, these are the first two cases of the syndrome described in Italy. Genetic testing proved to be fundamental for definition of the syndrome and confirms the importance of early detection of LRP5 variants for management of systemic features of the disease in patients and carrier relatives.

Design and Validation of a New MLPA-Based Assay for the Detection of RS1 Gene Deletions and Application in a Large Family with X-Linked Juvenile Retinoschisis.

AIMS: X-linked juvenile retinoschisis (XLRS) is a severe ocular disorder that can evolve to blindness. More than 200 different disease-causing mutations have been reported in the RS1 gene and approximately 10% of these are deletions. Since transmission is X-linked, males are always affected and females are usually carriers. The identification of female carriers is always important and poses a technical challenge. Therefore, we sought to develop a multiplex ligation dependent probe amplification (MLPA)-based method to identify deletions or duplications in this gene. We then used our assay to study a large XLRS family. METHODS: We designed six probes specific for each RS1 exon and then optimized and validated our method using control samples with known gene deletions. In the XLRS family, RS1 gene copy number variation was assessed by "home-made" MLPA analysis and by single nucleotide polymorphism (SNP) array analysis using the CytoScan HD Array. Direct sequencing was used for deletion breakpoint mapping. RESULTS: Our assay detected all deletions in control samples. All affected males of the family were positive for a deletion of exon 2 of the RS1 gene (RS1:NM_000330:c.53-?_78+?del). Carrier females were also identified. CONCLUSION: Our method is easily replicated, reliable, and inexpensive and allows female carriers to be detected. This is the first report of deep characterization of a whole exon deletion in the RS1 gene.

Leber's hereditary optic neuropathy (LHON) in an Apulian cohort of subjects.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder that causes severe loss of sight in young adults, and is typically associated to mitochondrial DNA (mtDNA) mutations. Heteroplasmy of primary LHON mutations, presence of 'ancillary' mtDNA mutations, and mtDNA copy number are probably correlated with the penetrance and the severity of the disease. In this study, we performed a mutational screening in an Apulian cohort of LHON patients and we found that 41 out of 54 subjects harbored the m.11778G>A mutation, and 13 harbored the m.3460G>A mutation. Whole mtDNA sequencing was performed in three affected subjects belonging to three unrelated m.11778G>A pedigrees to evaluate the putative synergistic role of additional mtDNA mutations in determining the phenotype. Our study suggests to include haplogroup T as a possible genetic background influencing LHON penetrance and to consider the increase of mtDNA copy number as a protective factor from vision loss regardless the hetero/homoplasmic status of LHON primary mutations.

Increased prevalence of the GCM2 polymorphism, Y282D, in primary hyperparathyroidism: analysis of three Italian cohorts.

CONTEXT: Glial cells missing-2 (GCM2) is key for parathyroid gland organogenesis. Its persistent expression in the adult parathyroid raises the possibility that overactive forms play a role in the evolution of parathyroid hyperactivity or tumorigenesis. A GCM2 c.844T → G; p.Y282D missense variant has been described within a transactivation inhibitory domain (amino acids 263-352). OBJECTIVE: The aims of the study were to 1) assess the frequency of Y282D in Italian primary hyperparathyroidism (PHPT) and control (C) populations, 2) test for association of 282D with PHPT and its phenotypic features, and 3) compare the transactivation potency of GCM2 282D relative to wild-type Y282. SUBJECTS AND METHODS: Subjects included a large southern Italian cohort (310 PHPT and 433 C) and 2 replication cohorts from northern Italy. Association of 282D with PHPT was tested in all cohorts and with phenotypic features in the larger PHPT cohort. An in vitro GCM promoter-luciferase reporter assay was conducted in HEK293 cells. RESULTS: 282D was significantly increased in the PHPT group, with a minor allele frequency of 0.066 compared with 0.029 in the C group (P = .0008), in the discovery cohort and was more prevalent in the replication cohorts. Combined analysis (510 PHPT and 665 C) yielded a likelihood ratio of 2.27 (95% confidence interval = 1.50-3.42; P < .0001). The 282D variant was not associated with serum calcium, phosphate, creatinine, or PTH levels or with bone mineral density, fractures, or renal stones in the PHPT group. The 282D variant had significantly greater transcriptional activity than the wild-type Y282 (17× basal vs 12× basal; P < 0.05). CONCLUSION: The higher frequency of GCM2 282D in PHPT and enhanced transcriptional activity of this variant supports the notion that it could contribute causally to parathyroid tumorigenesis.