Regulation of epinephrine biosynthesis in HRAS-mutant paragangliomas.

The biochemical phenotype of paragangliomas (PGLs) is highly dependent on the underlying genetic background and tumor location. PGLs at extra-adrenal locations usually do not express phenylethanolamine N-methyltransferase (PNMT), the enzyme required for epinephrine production, which was explained by the absence of glucocorticoids. PGLs with pathogenic variants (PVs) in Harvey rat sarcoma viral oncogene homolog (HRAS) can occur in or outside of the adrenal, but always synthesize epinephrine independently of the localization. Here, we characterize the signaling pathways through which PVs in HRAS influence PNMT expression. Catecholamines, cortisol, and transcriptional features of PGL tissues with known genetic background were analyzed. Genetically modified rat pheochromocytoma cells carrying PVs in Hras were generated and analyzed for regulation of Pnmt expression. Elevated epinephrine contents in PGLs with PVs in HRAS were accompanied by enrichment in mitogen-activated protein kinase (MAPK) signaling compared to PGLs with PVs in genes that activate hypoxia pathways. In vitro, Hras PVs increased Pnmt expression and epinephrine biosynthesis through increased phosphorylation of stimulatory protein 1 via MAPK signaling. Here, we provide a molecular mechanism that explains the PV-dependent epinephrine production of PGLs.

Deep Molecular Characterization of Milder Spinal Muscular Atrophy Patients Carrying the c.859G>C Variant in SMN2

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic loss or pathogenic variants in the SMN1 gene. Copy number and modifier intragenic variants in SMN2, an almost identical paralog gene of SMN1, are known to influence the amount of complete SMN proteins. Therefore, SMN2 is considered the main phenotypic modifier of SMA, although genotype−phenotype correlation is not absolute. We present eleven unrelated SMA patients with milder phenotypes carrying the c.859G>C-positive modifier variant in SMN2. All were studied by a specific NGS method to allow a deep characterization of the entire SMN region. Analysis of two homozygous cases for the variant allowed us to identify a specific haplotype, Smn2-859C.1, in association with c.859G>C. Two other cases with the c.859G>C variant in their two SMN2 copies showed a second haplotype, Smn2-859C.2, in cis with Smn2-859C.1, assembling a more complex allele. We also identified a previously unreported variant in intron 2a exclusively linked to the Smn2-859C.1 haplotype (c.154-1141G>A), further suggesting that this region has been ancestrally conserved. The deep molecular characterization of SMN2 in our cohort highlights the importance of testing c.859G>C, as well as accurately assessing the SMN2 region in SMA patients to gain insight into the complex genotype−phenotype correlations and improve prognostic outcomes.

[Clinical and genetic aspects of ABCA4-associated inherited retinal diseases]. / Klinicheskie i geneticheskie aspekty ABCA4-assotsiirovannykh nasledstvennykh zabolevanii setchatki.

The clinical and genetic characteristics of ABCA4-associated inherited retinal diseases have been studied for more than 2 decades, since the identification of the ABCA4 protein in 1978 and the ABCA4 gene in 1997. ABCA4 mutations were initially associated with autosomal recessive Stargardt disease (STGD1). It has now been established that mutations in this gene can cause other inherited retinal diseases, such as cone-rod dystrophy and retinitis pigmentosa. In addition, the phenotypes of ABCA4-associated diseases can vary greatly from the classic presentation of Stargardt disease, from loss of central vision in adolescence to disease with early onset and rapid progression or late onset and milder course. ABCA4-associated diseases are inherited in autosomal recessive manner, i.e. the disease develops only if both alleles of the gene are damaged, one inherited from the father and the other inherited from the mother. As with many other recessive hereditary diseases, which are characterized by a variety of clinical manifestations, the diversity of the phenotypes of ABCA4-associated retinal diseases is explained by combinations of sequence variants in the ABCA4 gene inherited by patients from their parents. Despite the fact that in this respect inherited retinal diseases associated with mutations in the ABCA4 gene do not fundamentally differ from other autosomal recessive traits, due to the structure of the gene and the protein encoded by it, there are a number of features thatshould be taken into account when performing molecular diagnostics, predicting the possibility of manifestation and the course of the disease, and planning the approaches to treatment.

Beyond copy number: A new, rapid, and versatile method for sequencing the entire SMN2 gene in SMA patients.

Spinal muscular atrophy (SMA) is caused by bi-allelic loss or pathogenic variants in the SMN1 gene. SMN2, the highly homologous copy of SMN1, is considered the major phenotypic modifier of the disease. Determination of SMN2 copy number is essential to establish robust genotype-phenotype correlations and predict disease evolution, to stratify patients for clinical trials, as well as to define those eligible for treatment. Discordant genotype-phenotype correlations are not uncommon in SMA, some of which are due to intragenic SMN2 variants that may influence the amount of complete SMN transcripts and, therefore, of full-length SMN protein. Detection of these variants is crucial to predict SMA phenotypes in the present scenario of therapeutic advances and with the perspective of SMA neonatal screening and early diagnosis to start treatments. Here, we present a novel, affordable, and versatile method for complete sequencing of the SMN2 gene based on long-range polymerase chain reaction and next-generation sequencing. The method was validated by analyzing samples from 53 SMA patients who lack SMN1, allowing to characterize paralogous, rare variants, and single-nucleotide polymorphisms of SMN2 as well as SMN2-SMN1 hybrid genes. The method identifies partial deletions and can be adapted to determine rare pathogenic variants in patients with at least one SMN1 copy.

Biallelic KRT5 mutations in autosomal recessive epidermolysis bullosa simplex, including a complete human keratin 5 "knock-out".

Epidermolysis bullosa simplex (EBS) is usually inherited as an autosomal dominant disease due to monoallelic gain-of-function mutations in KRT5 or KRT14. Although autosomal recessive forms of EBS have been associated with mutations in at least 10 genes, recessive EBS due to homozygous biallelic KRT5 mutations has not been reported previously; it has been hypothesized that it would result in prenatal lethality. We sought the genetic causes of EB in a cohort of 512 distinct EB families by performing whole exome sequencing (WES) and using an EB-targeting next-generation sequencing (NGS) panel of 21 genes. The pathogenicity and consequences of the mutations were determined by expression profiling and at tissue and ultrastructural levels. Two pathogenic, homozygous missense variants of KRT5 in two patients with generalized EBS and a homozygous null mutation in a patient who died as a neonate from complications of EB were found. The two missense mutations disrupted keratin 5 expression on immunofluorescence microscopy, and the human "knock-out" of KRT5 showed no RNA and protein expression. Collectively, these findings identify biallelic KRT5 mutations with a phenotypic spectrum varying from mild, localized and generalized to perinatal lethal, expanding the genotypic profile of autosomal recessive EBS.

Characterization of large deletions of the MECP2 gene in Rett syndrome patients by gene dosage analysis.

BACKGROUND: Rett syndrome (RTT) is a developmental disorder with an early onset and X-linked dominant inheritance pattern. It is first recognized in infancy and is seen almost always in girls, but it may be seen in boys on rare occasions. Typical RTT is caused by de novo mutations of the gene MECP2 (OMIM*300005), and atypical forms of RTT can be caused by mutations of the CDKL5 (OMIM*300203) and FOXG1 (OMIM*164874) genes. METHODS: Approximately 5% of the mutations detected in MECP2 are large rearrangements that range from exons to the entire gene. Here, we have characterized the deletions detected by multiplex ligation-dependent probe amplification (MLPA) in the gene MECP2 of 21 RTT patients. Breakpoints were delineated by DNA-qPCR until the amplification of the deleted allele by long-PCR was possible. RESULTS: This methodology enabled us to characterize deletions ranging from 1,235 bp to 85 kb, confirming the partial or total deletion of the MECP2 gene in all these patients. Additionally, our cases support the evidence claiming that most of these breakpoints occur in some restricted regions of the MECP2 gene. CONCLUSION: These molecular data together with the clinical information enable us to propose a genotype-phenotype correlation, which is essential for providing genetic counseling.

Hereditary spastic paraplegias: time for an objective case definition and a new nosology for neurogenetic disorders to facilitate biomarker/therapeutic studies.

INTRODUCTION: Hereditary spastic paraplegias (HSPs) are heterogeneous neurodegenerative disorders characterized by progressive lower limb weakness and spasticity as core symptoms of the degeneration of the corticospinal motor neurons. Even after exclusion of infectious and toxic mimickers of these disorders, the definitive diagnosis remains tricky, mainly in sporadic forms, as there is significant overlap with other disorders. Since their first description, various attempts failed to reach an appropriate classification. This was due to the constant expansion of the clinical spectrum of these diseases and the discovery of new genes, a significant number of them was involved in overlapping diseases. Areas covered: In this perspective review, an extensive literature study was conducted on the historical progress of HSP research. We also revised the previous and the current classifications of HSP and the closely related neurogenetic disorders and analyzed the areas of overlap. Expert opinion: There is undeniable need for objective case definition and reclassification of all neurogenetic disorders including HSPs, a prerequisite to improve patient follow-up, biomarker identification and develop therapeutics. The challenge is to understand why mutations can give rise to multiple phenotypic presentations along this spectrum of diseases in which the corticospinal tract is affected.

A new in silico approach to investigate molecular aspects of factor IX missense causative mutations and their impact on the hemophilia B severity.

Factor IX (encoded by F9) is a protein in the coagulation process, where its lack or deficiency leads to hemophilia B. This condition has been much less studied than hemophilia A, especially in Latin America. We analyzed the structural and functional impact of 54 missense mutations (18 reported by us previously, and 36 other mutations from the Factor IX database) through molecular modeling approaches. To accomplish this task, we examine the electrostatic patterns, hydrophobicity/hydrophilicity, disulfide, and H-bond differences of the Factor IX structures harboring the missense mutations found, correlating them with their clinical effects. The 54 mutated sequences were modeled and their physicochemical features were determined and used as input in clusterization tools. The electrostatic pattern seems to influence in disease severity, especially for mutations investigated in epidermal growth factors 1 and 2 (EGF1/2) domains. The combined use of all physicochemical information improved the clustering of structures associated to similar phenotypes, especially for mutations from GLA and EGF1-2 domains. The effect of mutations in the disease phenotype severity seems to be a complex interplay of molecular features, each one contributing to different impacts. This highlights that previous studies and tools analyzing individually single features for single mutations are missing elements that fulfill the whole picture.

[Inherited retinal diseases in patients with ABCA4 gene mutations]. / Nasledstvennye zabolevaniia setchatki pri mutatsiiakh gena ABCA4.

ABCA4 is one of the main genes which mutations are associated with various inherited retinal diseases (IRD) such as Stargardt disease, cone dystrophy, cone-rod dystrophy, and retinitis pigmentosa. Wide prevalence of IRD, high heterogeneity of ABCA4 gene mutations that lead to impaired function of the protein with varying expressiveness make studying of the clinical and genetic characteristics of retinal diseases relevant for further investigations into pathogenesis, prognosis and outcome of the disease. This article reviews the literature on incidence of IRD caused by mutations in the ABCA4 gene and characteristics of the clinical progression of retinal diseases associated with various types of mutations, and presents analysis of clinical and genetic correlations in terms of the effect the mutation has on the structure or function of the protein.

Correlation between SMA type and SMN2 copy number revisited: An analysis of 625 unrelated Spanish patients and a compilation of 2834 reported cases.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss or mutations in SMN1. According to age of onset, achieved motor abilities, and life span, SMA patients are classified into type I (never sit), II (never walk unaided) or III (achieve independent walking abilities). SMN2, the highly homologous copy of SMN1, is considered the most important phenotypic modifier of the disease. Determination of SMN2 copy number is essential to establish careful genotype-phenotype correlations, predict disease evolution, and to stratify patients for clinical trials. We have determined SMN2 copy numbers in 625 unrelated Spanish SMA patients with loss or mutation of both copies of SMN1 and a clear assignation of the SMA type by clinical criteria. Furthermore, we compiled data from relevant worldwide reports that link SMN2 copy number with SMA severity published from 1999 to date (2834 patients with different ethnic and geographic backgrounds). Altogether, we have assembled a database with a total of 3459 patients to delineate more universal prognostic rules regarding the influence of SMN2 copy number on SMA phenotype. This issue is crucial in the present scenario of therapeutic advances with the perspective of SMA neonatal screening and early diagnosis to initiate treatments.

[Clinical polymorphism of splice site mutations in the ABCA4 gene]. / Polimorfizm klinicheskikh proiavlenii pri mutatsiiakh saita splaisinga v gene AVSA4.

ABCA4 is one of the main genes whose mutations are associated with various inherited retinal diseases (IRD) such as Stargardt disease, cone dystrophy, cone-rod dystrophy, and retinitis pigmentosa. The severity of retinal dystrophy phenotype may be related to the degree of mutation pathogenicity, which depends on the localization in various regulatory regions of the gene and the effect on the amino acid composition of the protein molecule. The article describes two clinical cases of patients with splice site mutations in the compound heterozygous state with missense mutations in the ABCA4 gene with various phenotypic manifestations, which demonstrate the importance of molecular genetic analysis in patients with IRD. Such analysis allows determination and accumulation of data on phenotype-genotype correlations that can help predict the disease course.

RettBASE: Rett syndrome database update.

Rett syndrome (RTT) is an X-linked progressive neurodevelopmental disorder that primarily affects females. Mutations in the MECP2 gene have been attributed as the major genetic cause of RTT. Recently, mutations in CDKL5 and FOXG1 genes have also been suggested to give rise to RTT, although subsequent more extensive studies suggest that diseases resulting from mutations in these two genes should be considered as distinct clinical entities. While the genetic basis for the RTT has been recognized, so far there is no effective cure for the disease and the treatments available are mainly aimed at ameliorating clinical problems associated with the disorder. The swift identification of the mutations in children is crucial for pursuing the best therapeutic care. RettBASE was created in 2002 as a MECP2 variant database and has grown to become a comprehensive variant database for RTT and related clinical phenotypes, containing a curated collection of variants for MECP2, CDKL5, and FOXG1 genes. Here, we describe the development and growth of RettBASE after its inception in 2001. Currently, RettBASE holds a total of 4,668 variants in MECP2, 498 variants in CDKL5, and 64 variants in FOXG1.

High Incidence of Noonan Syndrome Features Including Short Stature and Pulmonic Stenosis in Patients carrying NF1 Missense Mutations Affecting p.Arg1809: Genotype-Phenotype Correlation.

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients.

Phenotype-genotype correlations of facial width and height proportions in patients with Class II malocclusion.

OBJECTIVES: To characterize soft-tissue facial height and width variation in Class II malocclusion and test for correlations with genes HMGA2, AJUBA, and ADK. SETTING AND SAMPLE POPULATION: Nine facial proportions were estimated from 2D frontal repose photographs of 330 Caucasian adults with Class II malocclusion. MATERIAL AND METHODS: After adjustments for age and gender, the facial proportions were submitted to a principal component analyses (PCA). The most meaningful phenotypic variations were correlated with SNPs rs7924176 (ADK), rs17101923 (HMGA2), and rs997154 (AJUBA) genotyped in 106 individuals. RESULTS: Principal component analyses resulted in four principal components (PCs), which explained 75% of total variation. PC1 captured variation in the intercanthus distance and explained 28% of total variation. PC2 explained 21% of the variations in facial taper and facial index. PC3 explained 14% and reflected variations in the vertical dimension of the lower face. PC4 explained 12% and captured variations in distance between the eyes, width of the commissures, and the length of the superior aspect of the lower face height corresponding to the vertical dimension of the philtrum of the upper lip. A suggestive association (p<0.05) was observed between PC4 and rs997154 corroborating the role of AJUBA in variation of facial dimensions. CONCLUSION: 2D frontal photographs can be used to derive quantitative measures of soft-tissue phenotypes that are of clinical relevance. The methods described are suitable for discovery and replication of associations between genotypes and malocclusion phenotypes.

A Case of Familial Phaechromocytoma- Was It?

With advancement in genetic studies, familial phaeochromocytoma (PCC) and paraganglioma (PGL) are increasingly being recognized. Characteristically, correlations exist between genotypes and clinical and biochemical phenotypes. We report a phaeochromocytoma in a young patient with intriguing family histories, raising the possibility of his being a familial case.

[Phenotypic heterogeneity and phenotype-genotype correlations in dystrophinopathies: Contribution of genetic and clinical databases]. / Variabilité phénotypique et corrélations génotype-phénotype des dystrophinopathies : contribution des banques de données.

The objective of this work was to study the natural history of dystrophinopathies and the genotype-phenotype correlations made possible by the development of the clinical part of the French DMD database. The collection of 70,000 clinical data for 600 patients with an average longitudinal follow-up of 12years enabled clarification of the natural history of Duchenne and Becker muscular dystrophies and clinical presentations in symptomatic females. We were able to specify the phenotypic heterogeneity of motor, orthopedic and respiratory involvements (severe, standard and intermediary form), of the cardiac disorder (severe, standard or absent cardiomyopathy, absence of correlation between motor and cardiac involvements), and of brain function (mental deficiency in the patients with Becker muscular dystrophy, psychopathological disorders in dystrophinopathies). Phenotypic variability did not correlate with a specific mutational spectrum. We propose a model of phenotypic analysis based on the presence or not of muscular and cardiac involvements (described by age at onset and rate of progression) and brain involvement (described by the type and the severity of the cognitive impairment and of the psychological disorders). The methodology developed for the DMD gene can be generalized and used for other databases dedicated to genetic diseases. Application of this model of phenotypic analysis for each patient and further development of the database should contribute substantially to clinical research providing useful tools for future clinical trials.

Thalassemia and its relevance to personalized medicine.

Thalassemias are the most common monogenic gene disorders in the world. Patients present with a wide variability of clinical phenotypes ranging from severe phenotype (ß-thalassemia major) to a very mild, almost symptomless, condition. This variability is owing to the presence of a large number of genetic modifiers affecting the disease. Patients are treated with blood transfusions and iron chelation therapy. Pharmacological therapies have varying degrees of success depending on the genetic modifiers of the disease present in the patients. Studies undertaken to identify all the modifiers that affect ß-thalassemia will lead to more appropriate genetic counseling during prenatal diagnosis and enable targeted and personalized treatment regimens for patients in the future.