Evaluation of in silico predictors on short nucleotide variants in HBA1, HBA2, and HBB associated with haemoglobinopathies.

Haemoglobinopathies are the commonest monogenic diseases worldwide and are caused by variants in the globin gene clusters. With over 2400 variants detected to date, their interpretation using the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines is challenging and computational evidence can provide valuable input about their functional annotation. While many in silico predictors have already been developed, their performance varies for different genes and diseases. In this study, we evaluate 31 in silico predictors using a dataset of 1627 variants in HBA1, HBA2, and HBB. By varying the decision threshold for each tool, we analyse their performance (a) as binary classifiers of pathogenicity and (b) by using different non-overlapping pathogenic and benign thresholds for their optimal use in the ACMG/AMP framework. Our results show that CADD, Eigen-PC, and REVEL are the overall top performers, with the former reaching moderate strength level for pathogenic prediction. Eigen-PC and REVEL achieve the highest accuracies for missense variants, while CADD is also a reliable predictor of non-missense variants. Moreover, SpliceAI is the top performing splicing predictor, reaching strong level of evidence, while GERP++ and phyloP are the most accurate conservation tools. This study provides evidence about the optimal use of computational tools in globin gene clusters under the ACMG/AMP framework.

Whole Exome Sequencing Points towards a Multi-Gene Synergistic Action in the Pathogenesis of Congenital Combined Pituitary Hormone Deficiency.

Combined pituitary hormone deficiency (CPHD) is characterized by deficiency of growth hormone and at least one other pituitary hormone. Pathogenic variants in more than 30 genes expressed during the development of the head, hypothalamus, and/or pituitary have been identified so far to cause genetic forms of CPHD. However, the etiology of around 85% of the cases remains unknown. The aim of this study was to unveil the genetic etiology of CPHD due to congenital hypopituitarism employing whole exome sequencing (WES) in two newborn patients, initially tested and found to be negative for PROP1, LHX3, LHX4 and HESX1 pathogenic variants by Sanger sequencing and for copy number variations by MLPA. In this study, the application of WES in these CPHD newborns revealed the presence of three different heterozygous gene variants in each patient. Specifically in patient 1, the variants BMP4; p.Ala42Pro, GNRH1; p.Arg73Ter and SRA1; p.Gln32Glu, and in patient 2, the SOX9; p.Val95Ile, HS6ST1; p.Arg306Gln, and IL17RD; p.Pro566Ser were identified as candidate gene variants. These findings further support the hypothesis that CPHD constitutes an oligogenic rather than a monogenic disease and that there is a genetic overlap between CPHD and congenital hypogonadotropic hypogonadism.

Molecular Analysis of the CYP11B2 Gene in 62 Patients with Hypoaldosteronism Due to Aldosterone Synthase Deficiency.

CONTEXT: Isolated congenital hypoaldosteronism presents in early infancy with symptoms including vomiting, severe dehydration, salt wasting, and failure to thrive. The main causes of this rare autosomal recessive disorder is pathogenic variants of the CYP11B2 gene leading to aldosterone synthase deficiency. OBJECTIVE: To investigate the presence of CYP11B2 pathogenic variants in a cohort of patients with a clinical, biochemical, and hormonal profile suggestive of aldosterone synthase deficiency. DESIGN: Clinical and molecular study. SETTING: Tertiary academic Children's Hospital, Center for Rare Pediatric Endocrine Diseases. PATIENTS AND METHODS: Sixty-two patients (56 unrelated patients and 6 siblings), with hypoaldosteronism and their parents, underwent CYP11B2 gene sequencing after its selective amplification against the highly homologous CYP11B1 gene. In silico analysis of the identified novel variants was carried out to evaluate protein stability and potential pathogenicity. RESULTS: CYP11B2 gene sequencing revealed that 62 patients carried a total of 12 different pathogenic CYP11B2 gene variants, 6 of which are novel. Importantly, 96% of the 56 patients carried the previously reported p.T185I variant either in homozygosity or in compound heterozygosity with another variant. The 6 novel variants detected were: p.M1I, p.V129M, p.R141Q, p.A165T, p.R448C, and the donor splice site variant of intron 8, c.1398 + 1G > A. CONCLUSION: Molecular diagnosis was achieved in 62 patients with aldosterone synthase deficiency, the largest cohort thus far reported. Six novel genetic variants were identified as possibly pathogenic, extending the spectrum of reported molecular defects of the CYP11B2 gene.

Phenotypic expression of a spectrum of Neurofibromatosis Type 1 (NF1) mutations identified through NGS and MLPA.

Neurofibromatosis Type 1 (NF1) is caused by mutations of the NF1 gene. The aim of this study was to identify the genetic causes underlying the disease, attempt possible phenotype/genotype correlations and add to the NF1 mutation spectrum. A screening protocol based on genomic DNA was established in 168 patients, encompassing sequencing of all coding exons and adjoining introns using a custom targeted next generation sequencing protocol and subsequent confirmation of findings with Sanger sequencing. MLPA was used to detect deletions/duplications and positive findings were confirmed by RNA analysis. All novel findings were evaluated according to ACMG Standards and guidelines for the interpretation of sequence variants with the aid of in-silico bioinformatic tools and family segregation analysis. A germline variant was identified in 145 patients (86%). In total 49 known and 70 novel variants in coding and non-coding regions were identified. Seven patients carried whole or partial gene deletions. NF1 patients, present with high phenotypic variability even in cases where the same germline disease causing variant has been identified. Our findings will contribute to a better knowledge of the genetic causes and the phenotypic expression related to the disease.

Hb Souli, a 6 bp in-frame deletion on the HBA2 gene (HBA2: c.[41-46delCCTGGG]) leads to α-thalassemia intermedia, when in trans to a single α-globin gene deletion.

We report the case of a 5-year-old child with clinical and hematological findings consistent with the diagnosis of α-thalassemia intermedia (α-TI). Molecular analysis disclosed the common 3.7 kb deletion in the α-globin gene cluster in trans to a novel in-frame 6 bp deletion in the HBA2 gene. It removes the sequence CCTGGG (or GCCTGG) that normally encodes for alanine (codon 13) and tryptophan (codon 14). Even though several hemoglobin (Hb) variants with mutations affecting codons 13 or 14 have been described, Hb Souli (HBA2: c.[41-46delCCTGGG]) is, to the best of our knowledge, the first variant to be reported where both amino acid residues, α13Ala and α14Trp, are deleted, leading to unstable and rapidly degraded α-globin chains.

Are ALOX5AP gene SNPs a risk or protective factor for stroke?

ALOX5AP (5-lipoxygenase) has been recognized as a susceptibility gene for stroke. Using a case-control design, the whole coding and adjoining intronic regions of ALOX5AP were sequenced to study the role of SNPs and their interplay with other risk factors in Greek patients with stroke. Patients (n=213) were classified by the Trial of Org 10172 in Acute Stroke Treatment (TOAST). Their mean age of was 58.9 ± 14.64, comprising 145 males. The control group consisted of 210 subjects, ethnicity, sex and age matched, with no stroke history. Risk factors (hyperlipidemia, hypertension, atrial fibrillation, migraine, CAD, diabetes, smoking and alcohol consumption) were assessed as confounding factors and comparisons were done using logistic regression analysis. SNPs rs4769055, rs202068154 and rs3803277 located in intronic regions of the gene and according to in silico programs EX_SKIP and HSF possibly affecting splicing of exons 1 and 2 of ALOX5AP, showed significantly different frequencies between patients and controls. The genotype frequencies of rs4769055: AA, of rs202068154: AC and of rs3803277: CA were significantly higher (p<0.001, 0.058) in controls than in patients. The results were indicative of a protective role of the three SNPs either in homozygosity or heterozygosity for MAF and more specifically rs3803277: CA/AA genotypes were protective against SVO stroke subtype.

Sertoli cell tumor and gonadoblastoma in an untreated 29-year-old 46,XY phenotypic male with Frasier syndrome carrying a WT1 IVS9+4C>T mutation.

OBJECTIVE: Frasier syndrome (FS) phenotype in 46,XY patients usually consists of female external genitalia, gonadal dysgenesis, high risk of gonadoblastoma and the development of end stage renal failure usually in the second decade of life. FS is caused by heterozygous de novo intronic splice site mutations of the Wilms' tumor suppressor gene 1 (WT1), although a few cases with typical exonic WT1 Denys-Drash mutations that resemble an FS phenotype have been described. The aim of this study was to present further data on the spectrum of FS phenotypes through the evaluation of a 29-year-old patient with a predominantly male phenotype and coexistence of Sertoli cell tumor and gonadoblastoma. RESULTS: Genetic analysis using standard methods for DNA sequencing confirmed FS due to a WT1 gene mutation, IVS9+4C>T. CONCLUSIONS: This very rare case illustrates the natural course of FS over many years due to the neglect by the patient to address his need for follow-up, while adding further data on the spectrum of FS phenotypes associated with IVS9+4 C>T mutations. The coexistence of the rare Sertoli cell tumor and gonadoblastoma emphasizes that early clinical recognition and molecular identification facilitates appropriate patient management, especially with respect to the high risk of gonadal malignancy.

Broad and unexpected phenotypic expression in Greek children with steroid-resistant nephrotic syndrome due to mutations in the Wilms' tumor 1 (WT1) gene.

Mutations in the Wilms' tumor suppressor gene 1 (WT1), most commonly within exons 8 or 9 or intron 9, are found in cases with the overlapping conditions of Denys-Drash and Frasier syndromes, as well as in patients with steroid-resistant nephrotic syndrome (SRNS). This study investigated the presence of WT1 gene mutations in cases with childhood SRNS, along with an evaluation of their clinical outcome. Twenty-seven Greek children with sporadic (19 cases) and familial (8 cases) SRNS were tested. Four phenotypically female patients with sporadic SRNS were found to carry de novo WT1 mutations, including two cases with p.R394W, and one case each with p.R366H, or n.1228+5G>A. Karyotype analysis found 46XX in three cases, but 46XY in one. No phenotype-genotype correlations were apparent in the WT1 gene positive cases since their clinical presentation varied broadly. Interestingly, one patient with a pathological WT1 nucleotide variation responded fully to combined therapy with cyclosporine A and corticosteroids. This study further illustrates that investigation of WT1 gene mutations is clinically useful to support definitive diagnosis in children presenting with SRNS in order to direct the most appropriate clinical management.

Nucleotide variations in the NPHS2 gene in Greek children with steroid-resistant nephrotic syndrome.

Mutations in the NPHS2 gene, encoding podocin, are a major cause of autosomal-recessive steroid-resistant nephrotic syndrome (SRNS) in childhood, accounting for up to 30% of sporadic and 20-40% of familial cases. Among 22 Greek children with a clinical diagnosis of SRNS, mutation analysis was performed in all eight NPHS2 gene exons, using denaturing gradient gel electrophoresis and DNA sequencing. The frequency of all nucleotide variations found in patients was also evaluated in 100 unrelated samples (18-30 years) with no known history of nephrotic disease. Three pathogenic genotypes (R138Q/R138Q, R229Q/A295T, and R168H/R168H) accounted for 3/14 (21%) of sporadic patients; the A295T mutation in exon 8 (c.883G>A) is novel and predicted in silico to be pathogenic. Among the familial cases, a single patient was heterozygous for R229Q. Several known polymorphisms were found, including the in cis variants IVS3-46C>T plus IVS3-21C>T, IVS7+7A>G A and exonic variants S96S (c.288C>T), A318A (c.954T>C), and L346L (c.1038A>G), with allele frequencies comparable to those in other populations. A novel substitution (IVS3-17C>T) was found in two related patients, but in no controls. In conclusion, podocin mutations do not appear to be a major cause of SRNS in Greek children, although the study cohort was small. However, NPHS2 gene analysis could still be considered in Greek SRNS patients to support appropriate management. The present study also contributes potentially useful observations for the clinical management of SRNS patients.