A bird's eye view on the use of whole exome sequencing in rare congenital ophthalmic diseases.

Phenotypic and genotypic heterogeneity in congenital ocular diseases, especially in anterior segment dysgenesis (ASD), have created challenges for proper diagnosis and classification of diseases. Over the last decade, genomic research has indeed boosted our understanding in the molecular basis of ASD and genes associated with both autosomal dominant and recessive patterns of inheritance have been described with a wide range of expressivity. Here we describe the molecular characterization of a cohort of 162 patients displaying isolated or syndromic congenital ocular dysgenesis. Samples were analyzed with diverse techniques, such as direct sequencing, multiplex ligation-dependent probe amplification, and whole exome sequencing (WES), over 20 years. Our data reiterate the notion that PAX6 alterations are primarily associated with ASD, mostly aniridia, since the majority of the cohort (66.7%) has a pathogenic or likely pathogenic variant in the PAX6 locus. Unexpectedly, a high fraction of positive samples (20.3%) displayed deletions involving the 11p13 locus, either partially/totally involving PAX6 coding region or abolishing its critical regulatory region, underlying its significance. Most importantly, the use of WES has allowed us to both assess variants in known ASD genes (i.e., CYP1B1, ITPR1, MAB21L1, PXDN, and PITX2) and to identify rarer phenotypes (i.e., MIDAS, oculogastrointestinal-neurodevelopmental syndrome and Jacobsen syndrome). Our data clearly suggest that WES allows expanding the analytical portfolio of ocular dysgenesis, both isolated and syndromic, and that is pivotal for the differential diagnosis of those conditions in which there may be phenotypic overlaps and in general in ASD.

Novel IGFALS mutations with predicted pathogenetic effects by the analysis of AlphaFold structure.

PURPOSE: According to the American College of Medical Genetics (ACMG) classification, variants of uncertain significance (VUS) are gene variations whose impact on the disease risk is not yet known. VUS, therefore, represent an unmet need for genetic counselling. Aim of the study is the use the AlphaFold artificial intelligence algorithm to predict the impact of novel mutations of the IGFALS gene, detected in a subject with short stature and initially classified as VUS according to the ACMG classification. METHODS: A short-stature girl and her parents have been investigated. IGFALS mutations have been detected through clinical exome and confirmed by Sanger sequencing. The potential presence of co-occurring gene alterations was investigated in the proband by whole exome and CGH array. Structure of the ALS protein (encoded by the IGFALS gene) was evaluated through the AlphaFold artificial intelligence algorithm. RESULTS: Two IGFALS variants were found in the proband: c.1349T > C (p.Leu450Pro) and c.1363_1365delCTC (p.Leu455del), both classified as VUS, according to ACMG. Parents' analysis highlighted the in trans position of the two variants. AlphaFold showed that the mutated positions were found the concave side a horseshoe structure of the ALS protein, likely interfering with protein-protein interactions. According to a loss of function (LoF) effect of the two variants, reduced levels of the IGF1 and IGFBP-3 proteins, as well as a growth hormone (GH) excess were detected in the proband's serum. CONCLUSIONS: By using the AlphaFold structure we were able to predict two IGFALS gene mutations initially classified as VUS, as potentially pathogenetic. Our proof-of-concept showed a potential application of AlphaFold as tool to a better inform VUS interpretation of genetic tests.

Dystonic tremor and blepharospasm in a patient with deletion of 18q.

18q- Syndrome is a rare chromosomic syndrome where neurological involvement is scarcely described. Movement disorders are rare and only one case with dystonia was described. In our paper, we describe the second report of a patient with 18q- Syndrome, blepharospasm, and dystonic tremor of his right hand and hyperthyroidism instead of hypothyroidism.

A novel de novo HDAC8 missense mutation causing Cornelia de Lange syndrome.

BACKGROUND: Cornelia de Lange syndrome (CdLS) is a rare and clinically variable syndrome characterized by growth impairment, multi-organ anomalies, and a typical set of facial dysmorphisms. Here we describe a 2-year-old female child harboring a novel de novo missense variant in HDAC8, whose phenotypical score, according to the recent consensus on CdLS clinical diagnostic criteria, allowed the diagnosis of a non-classic CdLS. METHODS: Clinical exome sequencing was performed on the trio, identifying a de novo heterozygous variant in HDAC8 (NM_018486; c. 356C>G p.Thr119Arg). Molecular modeling was performed to evaluate putative functional consequence of the HDAC8 protein. RESULTS: The variant HDAC8 c.356C>G is classified as pathogenic following the ACMG (American College of Medical Genetics and Genomics)/AMP (Association for Molecular Pathology) guidelines. By molecular modeling, we confirmed the deleterious effect of this variant, since the amino acid change compromises the conformational flexibility of the HDAC8 loop required for optimal catalytic function. CONCLUSION: We described a novel Thr119Arg mutation in HDAC8 in a patient displaying the major phenotypic traits of the CdLS. Our results suggest that a modest change outside an active site is capable of triggering global structural changes that propagate through the protein scaffold to the catalytic site, creating de facto haploinsufficiency.

A paternally inherited 1.4 kb deletion of the 11p15.5 imprinting center 2 is associated with a mild familial Silver-Russell syndrome phenotype.

The Silver-Russell syndrome (SRS) is a rare disorder characterized by heterogeneous clinical features, including growth retardation, typical facial dysmorphisms, and body asymmetry. Genetic alterations causative of SRS mostly affect imprinted genes located on chromosomes 7 or 11. Hypomethylation of the Imprinting Center 1 (IC1) of the chromosome 11p15.5 is the most common cause of SRS, while the Imprinting Center 2 (IC2) has been more rarely involved. Specifically, maternally inherited 11p15.5 deletions including the IC2 have been associated with the Beckwith-Wiedemann Syndrome (BWS), while paternal deletions with a variable spectrum of phenotypes. Here, we describe the case of a girl with a mild SRS phenotype associated with a paternally inherited 1.4 kb deletion of IC2. The father of the proband inherited the deletion from his mother and showed normal growth, while the paternal grandmother had the deletion on her paternal chromosome and exhibited short stature. Together with previous findings obtained in mouse and humans, our data support the notion that deletion of the paternal copy of IC2 can cause SRS.

CACNA1C haploinsufficiency accounts for the common features of interstitial 12p13.33 deletion carriers.

We identified a de novo 44.7 Kb interstitial 12p13.33 micro-deletion that involves solely the first exon of the CACNA1C (MIM 114205), using microarray-based comparative genomic hybridization (aCGH). The associated main phenotype is characterized by expressive language impairment, tremors, fine motor-skills delay, muscular hypotonia, and joint laxity. A careful comparison between the clinical and genomic characteristics between our proband and 20 previously reported patients, led us to propose CACNA1C haploinsufficiency as the main cause of both expressive language delay and motor-skills impairment. Pathogenic variants of CACNA1C have been associated to a plethora of clinical phenotypes, such as Timothy syndrome (TS, OMIM 601005), Brugada syndrome (BRGDA3, OMIM 611875) and a variety of neuropsychiatric disorders (bipolar disorder, major depression, schizophrenia, autism spectrum disorder, psychotic manifestations). In this report we describe a 12p13.33 micro-deletion involving one coding gene only, in contrast with previous studies that mostly concluded that a multi-genes deletion in the 12p13.33 sub-telomeric region is responsible of the minimum clinical phenotype of patients with 12p13.33 monosomy. Certainly, larger deletions spanning multiple Mb in 12p13.33 are responsible for more severe phenotypes, associated to a variable degree of dysmorphic features.

Quantitative PCR evaluation of deletions/duplications identified by array CGH.

Genomic deletions/duplications detected by array comparative genomic hybridization (aCGH) should be confirmed by an independent technology. This approach allows also to test, at low cost, inheritance of the imbalance. In the present study we explored the use of quantitative PCR (qPCR) to confirm aCGH-detected potentially clinically relevant imbalances. Only samples with DLRS <0.2 were tested for confirmation. aCGH results were confirmed in 102/118 cases (86.5%). A major element for non-confirmation was the dimension (and the probe coverage) of the putative aberration. Imbalances detected by 10 or less probes in aCGH assay were not confirmed in 11 out of 41 cases (26.8%), while those ones detected by 20 or more probes were always confirmed (46 cases). Among not confirmed imbalances, no statistical difference was found between deletions and duplication. Our data indicate that validation should be required for imbalances detected by less than 10 probes in aCGH assays.

Genomic Deletion Involving the IMMP2L Gene in Two Cases of Autism Spectrum Disorder.

Mutations/deletions of the IMMP2L gene have been associated with different cognitive/behavioral disturbances, including autism spectrum disorders (ASD). The penetrance of these defects is not complete since they often are inherited from a healthy parent. Using array-CGH in a cohort of 37 ASD patients, we found 2 subjects harboring a deletion inside the IMMP2L gene. In both cases, the IMMP2L gene deletion was inherited: from a healthy mother in one case and from a dyslectic father in the other. In the latter family, the IMMP2L deletion was also detected in the patient's brother, who showed delayed language development. In a cohort of 100 normal controls, no deletions including the IMMP2L gene were observed. However, a recent meta-analysis found no association between IMMP2L deletions and ASD. Our data would indicate that deletions involving the IMMP2L gene may contribute to the development of a subgroup of cognitive/behavioral disorders.

Evaluation of somatic genomic imbalances in thyroid carcinomas of follicular origin by CGH-based approaches.

Application of distinct technologies of cancer genome analysis has provided important information for the molecular characterization of several human neoplasia, including follicular cell-derived thyroid carcinoma. Among them, comparative genomic hybridization (CGH)-based procedures have been extensively applied to evaluate genomic imbalances present in these tumours, obtaining data leading to an increase in the understanding of their complexity and diversity. In this review, after a brief overview of the most commonly used CGH-based technichs, we will describe the major results deriving from the most influential studies in the literature which used this approach to investigate the genomic aberrations of thyroid cancer cells. In most studies a small number of patients have been analyzed. Deletions and duplications at different chromosomal regions were detected in all investigated cohorts. A higher number of genomic imbalances has been detected in anaplastic or poorly differentiated thyroid carcinomas compared to well differentiated ones. Limitations in the interpretation of the results, as well the potential impact in the clinical practice are discussed. Though a quite heterogeneous picture arises from results so far available, CGH array, combined with other methodologies as well as an accurate clinical management, may offer novel opportunities for a better stratification of thyroid cancer patients.

Familial 5q12.3 Microdeletion: Evidence for a Locus Associated with Epilepsy.

The clinical use of array comparative genomic hybridization (array CGH) has allowed the identification of very rare deletion and duplication disorders, such as 5q12 deletion syndrome (OMIM 615668) described as a contiguous gene deletion syndrome of chromosome 5q12. Chromosome microdeletions including band 5q12 have rarely been reported and have been associated with different phenotypes showing postnatal growth restriction, intellectual disability, epileptic seizures, hyperactivity, and ocular abnormalities. In this study, we describe a family in which array-CGH analysis revealed the presence of an interstitial microdeletion spanning approximately 2.9 Mb in the 5q12 region. The microdeletion is associated with epilepsy in the father and 2 siblings (a boy and a girl). So far, this is the first report in which a familial microdeletion 5q12 manifests in epilepsy. We suggest that this familial microdeletion could delineate a locus for susceptibility to epilepsy.

Microdeletion 15q26.2qter and Microduplication 18q23 in a Patient with Prader-Willi-Like Syndrome: Clinical Findings.

The small interstitial deletion in the long arm of chromosome 15 causing Prader-Willi/Angelman syndrome is well known, whereas cases that report terminal deletions in 15q in association with the Prader-Willi-like phenotype are very rare. By using GTG-banding analysis, metaphase FISH, MLPA analysis, and genome-wide array CGH, we detected an unbalanced translocation involving a microdeletion of the distal part of 15q and a microduplication of the distal part of 18q. The unbalanced translocation was found in a boy that was referred with clinical suspicion of Prader-Willi syndrome. In the 15q-deleted region, 23 genes have been identified, and 13 of them are included in the OMIM database. Among these, the deleted IGFR1, MEF2A, CHSY1, and TM2D3 genes could contribute to the patient's phenotype. Seven genes are included in the duplicated chromosome segment 18q, but only one (CTDP1) is present in the OMIM database. We suggest that the deleted chromosome segment 15q26.2qter may be responsible for the phenotype of our case and may also be a candidate locus of Prader-Willi-like syndrome.

Somatic amplifications and deletions in genome of papillary thyroid carcinomas.

Somatic gene copy number variation contributes to tumor progression. Using comparative genomic hybridization (CGH) array, the presence of genomic imbalances was evaluated in a series of 27 papillary thyroid carcinomas (PTCs). To detect only somatic imbalances, for each sample, the reference DNA was from normal thyroid tissue of the same patient. The presence of the BRAF V600E mutation was also evaluated. Both amplifications and deletions showed an uneven distribution along the entire PTC cohort; amplifications were more frequent than deletions (mean values of 17.5 and 7.2, respectively). Number of aberration events was not even among samples, the majority of them occurring only in a small fraction of PTCs. Most frequent amplifications were detected at regions 2q35, 4q26, and 4q34.1, containing FN1, PDE5A, and GALNTL6 genes, respectively. Most frequent deletions occurred at regions 6q25.2, containing OPMR1 and IPCEF1 genes and 7q14.2, containing AOAH and ELMO1 genes. Amplification of FN1 and PDE5A genomic regions was confirmed by quantitative PCR. Frequency of amplifications and deletions was in relationship with clinical features and BRAF mutation status of tumor. In fact, according to the American Joint Committee on Cancer stage and American Thyroid Association (ATA) risk classification, amplifications are more frequent in higher risk samples, while deletions tend to prevail in the lower risk tumors. Analysis of single aberrations according to the ATA risk grouping shows that amplifications containing PDE5A, GALNTL6, DHRS3, and DOCK9 genes are significantly more frequent in the intermediate/high risk group than in the low risk group. Thus, our data would indicate that analysis of somatic genome aberrations by CGH array can be useful to identify additional prognostic variables.

The PARP inhibitor PJ34 modifies proliferation, NIS expression and epigenetic marks in thyroid cancer cell lines.

Since PARP-1 is supposed to be part of a multimeric repressor of sodium iodide symporter (NIS) expression, in this study the effect of the PARP inhibitor PJ34 on several properties of thyroid cancer cell lines was investigated. In TPC1, BCPAP, FRO, WRO cell lines PJ34 induced a strong increase in NIS mRNA levels. In BCPAP and TPC1 cells also significant increase of radio-iodine uptake was induced. Accordingly, in transfection experiments performed in TPC1 cells, treatment with PJ34 increased NIS promoter activity without affecting PARP-1 binding to the promoter sequence. We also investigated the epigenetic status of NIS promoter after PJ34 treatment in TPC1 cell line: in addition to an increase of histone modification activation marks (H3K9K14ac, H3K4me3), surprisingly we observed also an increase of H3K27me3, a classical repressive mark. Our data demonstrate that in various thyroid cancer cell lines PARP inhibition increases NIS gene expression through a particular modulation of transcriptional regulatory mechanisms. Therefore, we suggest that PARP inhibitors may deserve future investigations as tools for medical treatment of thyroid cancer.

A new germline VHL gene mutation in three patients with apparently sporadic pheochromocytoma.

CONTEXT: Germline mutations in four genes (RET, VHL, SDHB and SDHD) are detected in about 17% of patients with apparently sporadic pheochromocytoma. Thus, genetic screening of all patients with this disease is suggested for a rational diagnostic approach and management. OBJECTIVE: To report the clinical, biochemical and genetic analysis of three unrelated patients affected by pheochromocytoma. DESIGN AND PATIENTS: All the coding regions and exon-intron boundaries of RET, VHL, SDHB and SDHD genes were sequenced in three unrelated patients with intra-adrenal pheochromocytoma: a 17-year-old girl, a 15-year-old boy and a 73-year-old man. The family history of all three cases was negative for von Hippel-Lindau lesions or other types of endocrine tumours. Structural modelling of the VHL protein was then performed. RESULTS: We identified a novel germline VHL gene point mutation, a G to A nucleotide substitution in exon 3, leading to an aspartate to asparagine amino acid change in codon 197 (D197N). No mutations were found in RET, SDHB and SDHD genes. Structural modelling of the VHL protein suggests that the D197N mutation could have a functional role. CONCLUSIONS: Our study expands the number of VHL gene known mutations and indicates the usefulness of performing the genetic analysis in all patients with apparently sporadic pheochromocytoma.

Sequence and copy number analyses of HEXB gene in patients affected by Sandhoff disease: functional characterization of 9 novel sequence variants.

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.

Cyclic AMP-response element modulator inhibits the promoter activity of the sodium iodide symporter gene in thyroid cancer cells.

BACKGROUND: Comprehension of the regulatory mechanism involved in the sodium iodide symporter (NIS) expression is of great relevance for thyroid cancer. In fact, restoration of NIS expression would be a strategy to treat undifferentiated thyroid cancer. Previous in vitro findings suggest that the cyclic AMP-response element (CRE) modulator (CREM) is involved in control of NIS expression. In this work, we examined the expression of CREM in a series of thyroid cancer tissues and its action on NIS promoter in human thyroid cancer cells. METHODS: Expression of mRNA levels for CREM, PAX8 and NIS was measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in 6 normal thyroid tissues, 22 papillary, 12 follicular and 4 anaplastic thyroid cancers. The effect of CREM on transcriptional activity of the NIS promoter was investigated by transient transfection of human thyroid cell lines. RESULTS: Compared to normal tissues, NIS and PAX8 mRNA levels were significantly reduced in all types of thyroid cancer. As expected, the maximal decrease was detected in anaplastic thyroid cancer. Conversely, CREM mRNA levels were increased in all types of thyroid cancer, reaching statistical significance for follicular and anaplastic thyroid carcinoma (p=0.0157 and 0.0045, respectively). Transfection experiments showed an inhibitory effect of CREM on NIS promoter activity in various thyroid cancer cell lines. CONCLUSIONS: These data demonstrate that CREM expression is increased in thyroid cancer tissue and may play a role in the downregulation of NIS expression in thyroid cancer acting at the transcriptional level.

Expression of Dicer and Drosha in triple-negative breast cancer.

AIMS: Dicer and Drosha are components of the miRNA-producing machinery and their altered expression may play a role in cancer progression. The main purpose of this study was a detailed investigation of Dicer and Drosha expression and localisation in triple-negative breast cancers. METHODS: Thirty-one triple-negative breast cancers and several breast cancer cell lines were investigated. Expression of Dicer and Drosha was evaluated at the mRNA level by quantitative reverse transcription PCR and at the protein level by immunohistochemistry or western blot. RESULTS: Compared with normal breast tissues, a wide variation of Dicer and Drosha mRNA levels was detected in triple-negative breast cancers. As a group, Drosha mRNA levels in triple-negative breast cancers were significantly higher than those in normal breast tissues. Immunohistochemical data confirmed higher expression of Drosha protein in triple-negative breast cancers. In normal breast tissues Dicer was detectable predominantly in the cytoplasm of basal/myoepithelial cells only. In contrast, in the majority of triple-negative breast cancers, intense Dicer staining was detectable also in the nuclear compartment. Detection of Dicer and Drosha mRNA and protein levels in breast cancer cell lines confirmed the nuclear localisation of Dicer, suggesting, in addition, that the steady-state protein levels could be controlled by post-mRNA regulatory events. CONCLUSIONS: These findings indicate that Dicer and Drosha expression is deregulated in triple-negative breast cancers.

Histone post-translational modifications associated to BAALC expression in leukemic cells.

BAALC expression is an indicator of aggressiveness in acute myelogenous leukemia (AML). Overexpression of this gene is associated to poor of clinical outcome. It is known that post-translational histone modifications control gene transcription. Thus, here we have investigated BAALC expression and post-translational histone modifications in leukemia cell lines. We show that Kasumi-6 and Kyo cells have high and low BAALC mRNA levels, respectively. Moreover, we demonstrate that these cell lines present distinct profiles in terms of histone post-translational modifications (H3K9K14 acetylation, H3K4 trimethylation and H3K23 trimethylation) at the level of BAALC promoter. These findings, in light of recent data on how histone post-translational modifications control gene expression, indicate that BAALC gene is "paused" and that in leukemia cells its transcription can be activated or repressed by mechanisms acting on epigenetic marks.

Levels of histone acetylation in thyroid tumors.

Histone acetylation is a major mechanism to regulate gene transcription. This post-translational modification is modified in cancer cells. In various tumor types the levels of acetylation at several histone residues are associated to clinical aggressiveness. By using immunohistochemistry we show that acetylated levels of lysines at positions 9-14 of H3 histone (H3K9-K14ac) are significantly higher in follicular adenomas (FA), papillary thyroid carcinomas (PTC), follicular thyroid carcinomas (FTC) and undifferentiated carcinomas (UC) than in normal tissues (NT). Similar data have been obtained when acetylated levels of lysine 18 of H3 histone (H3K18ac) were evaluated. In this case, however, no difference was observed between NT and UC. When acetylated levels of lysine 12 of H4 histone (H4K12ac) were evaluated, only FA showed significantly higher levels in comparison with NT. These data indicate that modification histone acetylation is an early event along thyroid tumor progression and that H3K18 acetylation is switched off in the transition between differentiated and undifferentiated thyroid tumors. By using rat thyroid cell lines that are stably transfected with doxycyclin-inducible oncogenes, we show that the oncoproteins RET-PTC, RAS and BRAF increase levels of H3K9-K14ac and H3K18ac. In the non-tumorigenic rat thyroid cell line FRTL-5, TSH increases levels of H3K18ac. However, this hormone decreases levels of H3K9-K14ac and H4K12ac. In conclusion, our data indicate that neoplastic transformation and hormonal stimulation can modify levels of histone acetylation in thyroid cells.