Molecular and clinical profile of patients referred as Noonan or Noonan-like syndrome in Greece: a cohort of 86 patients.

Noonan syndrome (NS) is an autosomal dominant disorder characterized by clinical and genetic heterogeneity. It belongs to a wider group of pathologies, known as Rasopathies, due to the implication of genes encoding components of the Ras/MAPK signalling pathway. Recording the genetic alterations across populations helps assessing specific features to specific genes which is essential for better disease's recognition, prognosis and monitoring. Herein, we report the clinical and molecular data of a Greek cohort comprising of 86 NS or NS-like patients admitted at a single tertiary Centre in Athens, Greece. The analysis was performed using Sanger and next-generation sequencing, comprising 14 different genes. The mutational rates of the confirmed NS-associated genes in the Greek NS population are as follows: PTPN11 32.5%; RIT1 5.8%; SOS1 4.7%; BRAF 1.2%; CBL 1.2%; KRAS 1.2%; MAP2K1 1.2%; RAF1 1.2%; SHOC2 1.2%, corresponding to 50% of positivity in total NS population. The genotype-phenotype analysis showed statistically significant differences in craniofacial dysmorphisms (p = 0.005) and pulmonary valve stenosis (PS) (p < 0.001) frequencies between patients harbouring a pathogenic variant and patients without pathogenic variant in any of the tested genes. Patients with at least a pathogenic variant had 6.71 times greater odds to develop PS compared to pathogenic variant-negative patients (OR = 6.71, 95%; CI = (2.61, 17.27)). PTPN11 positive patients showed higher frequency of epicanthal folds (p = 0.004), ptosis (p = 0.001) and coarseness (p = 0.001) and lower frequency of neurological findings (p = 0.006), compared to patients carrying pathogenic variants in other genes. CONCLUSION: Craniofacial dysmorphism and PS prevail among pathogenic variant positive compared to pathogenic variant negative NS and NS-like patients while neurological defects are less common in PTPN11-affected NS patients compared to patients harbouring pathogenic variants in other genes. The significant prevalence of the Ras/MAPK pathogenic variants (17.4%), other than PTPN11, in Greek NS patients, highlights the necessity of a wider spectrum of molecular diagnosis. WHAT IS KNOWN: • Noonan syndrome (NS) has been associated with pathogenic variants in molecules-components of the Ras/MAPK pathway. • Clinical and genetic description of NS patients worldwide helps establishing personalized monitoring. WHAT IS NEW: • NS and NS-like mutational rate in Greece reaches 50% with pathogenic variants identified mostly in PTPN11 (32.5%), RIT1 (6%) and SOS1 (4.7%) genes. • The risk for pulmonary stenosis increases 6.71-fold in NS patients with a pathogenic variant compared to patients without genetic alterations.

High resolution Chromosomal Microarray Analysis (CMA) enhances the genetic profile of pediatric B-cell Acute Lymphoblastic Leukemia patients.

Acute Lymphoblastic Leukemia (ALL) is a malignancy of the immature lymphoid cells mainly associated with numerical and structural chromosomal aberrations. The current standard for profiling the diverse genetic background comprises a combination of conventional karyotype and FISH analysis for the most common translocations, albeit with many limitations. Chromosomal Microarray Analysis (CMA) is a high throughput whole genome method that is gradually implemented in routine clinical practice, but not many studies have compared the two methods. Here we aim to investigate the added benefits of utilizing the high resolution 2 x 400 K G3 CGH + SNP CMA platform in routine diagnostics of pediatric ALL. From the 29 bone marrow samples that were analyzed, CMA identified clinically relevant findings in 83%, while detecting chromosomal aberrations in 75% of the patients with normal conventional karyotype. The most common finding was hyperdiploidy (20%), and the most common submicroscopic aberration involved CDKN2A/B genes. The smallest aberration detected was a 9 kb partial NF1 gene duplication. The prognosis of the patients when combining conventional cytogenetics and CMA was either changed or enhanced in 66% of the cases. A rare duplication possibly indicative of a cryptic ABL1-NUP214 fusion gene was found in one patient. We conclude that CMA, when combined with conventional cytogenetic analysis, can significantly enhance the genetic profiling of patients with pediatric ALL in a routine clinical setting.

A dynamic trinucleotide repeat (TNR) expansion in the DMD gene.

Dystrophinopathies are allelic X-linked myopathies caused by large deletions/duplications or small lesions along the DMD gene. An unexpected dynamic trinucleotide (GAA) expansion, ranging from ∼59 to 82 pure GAA repeats, within the DMD intron 62, was revealed to segregate through three family generations. From the pedigree, two female patients were referred for DMD investigation due to chronic myopathy and a muscle biopsy compatible with dystrophinopathy. As the size of the GAA repeat is limited to 11-33 within the general population our findings may provide a novel insight towards a Trinucleotide Repeat Expansion. Whether this TNR has an impact on the reported phenotype remains to be resolved.

Complex preimplantation genetic diagnosis for beta-thalassaemia, sideroblastic anaemia, and human leukocyte antigen (HLA)-typing.

Preimplantation genetic diagnosis (PGD) to select histocompatible siblings to facilitate curative haematopoeitic stem-cell transplantation (HSCT) is now an acceptable option in the absence of an available human leukocyte antigen (HLA) compatible donor. We describe a case where the couple who requested HLA-PGD, were both carriers of two serious haematological diseases, beta-thalassaemia and sideroblastic anaemia. Their daughter, affected with sideroblastic anaemia, was programmed to have HSCT. A multiplex-fluorescent-touchdown-PCR protocol was optimized for the simultaneous amplification of: the two HBB-gene mutated regions (c.118C> T, c.25-26delAA), four short tandem repeats (STRs) in chr11p15.5 linked to the HBB gene, the SLC25A38 gene mutation (c.726C > T), two STRs in chr3p22.1 linked to the SLC25A38 gene, plus eleven informative STRs for HLA-haplotyping (chr6p22.1-21.3). This was followed by real-time nested PCR and high-resolution melting analysis (HRMA) for the detection of HBB and SLC25A38 gene mutations, as well as the analysis of all STRs on an automatic genetic analyzer (sequencer). The couple completed four clinical in vitro fertilization (IVF)/PGD cycles. At least one matched unaffected embryo was identified and transferred in each cycle. A twin pregnancy was established in the fourth PGD cycle and genotyping results at all loci were confirmed by prenatal diagnosis. Two healthy baby girls were delivered at week 38 of pregnancy. The need to exclude two familial disorders for HLA-PGD is rarely encountered. The methodological approach described here is fast, accurate, clinically-validated, and of relatively low cost.

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.

An unusual case of Cat-Eye syndrome phenotype and extragonadal mature teratoma: review of the literature.

BACKGROUND Cat-Eye syndrome (CES) with teratoma has not been previously reported. We present the clinical and molecular findings of a 9-month-old girl with features of CES and also a palpable midline neck mass proved to be an extragonadal mature teratoma, additionally characterized by array comparative genomic hybridization (aCGH). RESULTS High resolution oligonucleotide-based aCGH confirmed that the supernumerary marker chromosome (SMC) derived from chromosome 22, as was indicated by molecular cytogenetic analysis with fluorescence in situ hybridization (FISH). Additionally, aCGH clarified the size, breakpoints, and gene content of the duplication (dup 22q11.1q11.21; size:1.6 Mb; breakpoints: 15,438,946-17,041,773; hg18). The teratoma tissue was also tested with aCGH, in which the CES duplication was not found, but the analysis revealed three aberrations: del Xp22.3 (108,864-2788,689; 2.7 Mb hg18), dup Yp11.2 (6688,491-7340,982; 0.65 Mb, hg18), and dup Yq11.2q11.23 (12,570,853-27,177,133; 14.61 Mb, hg18). These results indicated 46 XY (male) karyotype of the teratoma tissue, making this the second report of mature extragonadal teratoma in a female neonate, probably deriving from an included dizygotic twin of opposite sex (fetus in fetu). CONCLUSIONS Our findings extend the phenotypic spectrum of CES syndrome, a disorder with clinical variability, pointing out specific dosage-sensitive genes that might contribute to specific phenotypic features.

FoSTeS, MMBIR and NAHR at the human proximal Xp region and the mechanisms of human Xq isochromosome formation.

The recently described DNA replication-based mechanisms of fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) were previously shown to catalyze complex exonic, genic and genomic rearrangements. By analyzing a large number of isochromosomes of the long arm of chromosome X (i(Xq)), using whole-genome tiling path array comparative genomic hybridization (aCGH), ultra-high resolution targeted aCGH and sequencing, we provide evidence that the FoSTeS and MMBIR mechanisms can generate large-scale gross chromosomal rearrangements leading to the deletion and duplication of entire chromosome arms, thus suggesting an important role for DNA replication-based mechanisms in both the development of genomic disorders and cancer. Furthermore, we elucidate the mechanisms of dicentric i(Xq) (idic(Xq)) formation and show that most idic(Xq) chromosomes result from non-allelic homologous recombination between palindromic low copy repeats and highly homologous palindromic LINE elements. We also show that non-recurrent-breakpoint idic(Xq) chromosomes have microhomology-associated breakpoint junctions and are likely catalyzed by microhomology-mediated replication-dependent recombination mechanisms such as FoSTeS and MMBIR. Finally, we stress the role of the proximal Xp region as a chromosomal rearrangement hotspot.

De novo interstitial duplication of the 15q11.2-q14 PWS/AS region of maternal origin: Clinical description, array CGH analysis, and review of the literature.

The 15q11-q13 PWS/AS critical region involves genes that are characterized by genomic imprinting. Multiple repeat elements within the region mediate rearrangements, including interstitial duplications, interstitial triplications, and supernumerary isodicentric marker chromosomes, as well as the deletions that cause Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Recently, duplications of maternal origin concerning the same critical region have been implicated in autism spectrum disorders (ASD). We present a 6-month-old girl carrying a de novo duplication of maternal origin of the 15q11.2-q14 PWS/AS region (17.73 Mb in size) [46,XX,dup(15)(q11.2-q14)] detected with a high-resolution microarray-based comparative genomic hybridization (array-CGH). The patient is characterized by severe hypotonia, obesity, microstomia, long eyelashes, hirsutism, microretrognathia, short nose, severe psychomotor retardation, and multiple episodes of drug-resistant epileptic seizures, while her brain magnetic resonance imaging (MRI) documented partial corpus callosum dysplasia. In our patient the duplicated region is quite large extending beyond the Prader-Willi-Angelman critical region (PWACR), containing a number of genes that have been shown to be involved in ASD, exhibiting a severe phenotype, beyond the typical PWS/AS clinical manifestations. Reporting of similar well-characterized clinical cases with clearly delineated breakpoints of the duplicated region will clarify the contribution of specific genes to the phenotype.

A clinical study of Sotos syndrome patients with review of the literature.

Sotos syndrome is characterized by tall stature, advanced bone age, typical facial abnormalities, and developmental delay. The associated gene is NSD1. The study involved 22 patients who fulfilled the clinical criteria. Phenotypic characteristics, central nervous system findings, and cardiovascular and urinary tract abnormalities were evaluated. Meta-analysis on the incidence of cardinal clinical manifestations from the literature was also performed. Macrocephaly was present in all patients. Advanced bone age was noted in 14 of 22 patients (63%), and its incidence presented significant statistical difference in the meta-analysis of previous studies. Some patients had serious clinical manifestations, such as congenital heart defects, dysplastic kidneys, psychosis, and leukemia. Clinical and laboratory examinations should be performed to prevent and manage any unusual medical aspect of the syndrome. Facial gestalt and macrocephaly, rather than advanced bone age, are the strongest indications for clinical diagnosis.

Three novel mutations in greek sotos patients with rare clinical manifestations.

BACKGROUND: Sotos syndrome is an autosomal dominant disease characterized by tall stature, advanced bone age, typical morphological abnormalities of the face and developmental delay. It is caused by mutations in the NSD1 gene located on chromosome 5. NSD1 mutations are detected in the majority of the Sotos patients, and include intragenic NSD1 mutations and microdeletions in the 5q35 region. Cardiovascular and urogenital symptoms are more frequent in the microdeletion group. METHODS: Mutation analysis was performed in 4 patients with Sotos syndrome with typical phenotypic characteristics. RESULTS: In each of the 4 patients a NSD1 mutation was found (2 frame shifts, 1 nonsense and 1 missense mutation). Two of our patients presented dysplastic kidneys with cysts and psychosis, respectively. CONCLUSIONS: We describe 4 Greek patients with Sotos syndrome. Apart from the typical phenotypic characteristics, 2 of our patients presented rare clinical manifestations such as dysplastic kidneys and psychosis. The 3 detected mutations are novel.

Tumor development in three patients with Noonan syndrome.

The diagnosis of Noonan syndrome is essentially clinical, based upon the distinct phenotype and the involvement of the cardiovascular system. Tumor development is a rare manifestation of Noonan syndrome but can be explained by the molecular pathophysiology involved in the disorder. We present three Noonan patients who developed solid tumors. The first patient, a 4-year-old girl, developed granular cell tumors as did her mother in childhood. The second patient, a 1-year-old boy, had a low grade pilocytic astrocytoma, the clinical expression of which was persistent headache. MRI showed a pituitary mass in the posterior lobe. It was surgically removed. The third patient, a 7-year-old boy was found to have Sertoli tumors in his right cryptorchid testis. All three patients fulfilled the clinical criteria for Noonan syndrome. However, genetic testing was negative in patients 1 and 3. The diagnosis of Noonan syndrome was made based on distinct phenotypic findings in three patients who had different types of tumors.

Rhabdomyosarcoma in a patient with Noonan syndrome phenotype and review of the literature.

An increased risk of different types of malignancy has been reported in patients with Noonan syndrome (NS). We describe a patient with short stature, dysmorphic features, developmental delay, and congenital cardiomyopathy. At 5 years old, he presented with abdominal pain, constipation, and evaluation with ultrasound and computed tomography scan demonstrated the presence of an abdominal mass. Total resection of the mass and consequent histology revealed an embryonal rhabdomyosarcoma. Rhabdomyosarcoma is a rare tumor in NS patients and to the best of our knowledge only 2 cases have been reported so far. The presentation underlines the importance of frequent follow-up of patients with NS, since the incidence of malignancy is low but existing.