A homozygous frameshift variant expands the clinical spectrum of SAMD9 gene defects.

SAMD9, a ubiquitously expressed protein, is involved in several mechanisms, including endosome fusion, growth suppression and modulation of innate immune responses to stress and viral infections. While biallelic mutations in SAMD9 are linked to normophosphatemic familial tumoral calcinosis, heterozygous gain-of-function mutations in the same gene are responsible for MIRAGE, a multisystemic syndrome characterized by myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy. A two-and-a-half-year-old girl, from a consanguineous Lebanese family, was included in this study. She presents with pre- and post-natal growth retardation, recurrent fevers, persistent diarrhea, elevated CRP and intermittent hypoglycemia. Whole genome sequencing revealed a homozygous frameshift variant in SAMD9 (NM_017654.4: c.480_481del; p.Val162Ilefs*5) in the proband. Sanger sequencing confirms its segregation with the disease in the family, and immunoblotting showed that the detected variant abolishes SAMD9 expression in the patient. Our findings expand the clinical spectrum linked to SAMD9 and highlight the importance of investigating further cases with mutations in this gene, as this will pave the way towards the understanding of the pathways driving these diseases.

Genetic Polymorphisms Involved in Bladder Cancer: A Global Review.

Bladder cancer (BC) has been associated with genetic susceptibility. Single peptide polymorphisms (SNPs) can modulate BC susceptibility. A literature search was performed covering the period between January 2000 and October 2020. Overall, 334 articles were selected, reporting 455 SNPs located in 244 genes. The selected 455 SNPs were further investigated. All SNPs that were associated with smoking and environmental exposure were excluded from this study. A total of 197 genes and 343 SNPs were found to be associated with BC, among which 177 genes and 291 SNPs had congruent results across all available studies. These genes and SNPs were classified into eight different categories according to their function.

Analysis of G-quadruplex forming sequences in podocytes-marker genes and their potential roles in inherited glomerular diseases.

Nephrotic Syndrome is the most widespread pediatric kidney disorder. Genetic alterations in podocyte genes are thought to be responsible for the disease. G-quadruplexes are non-conventional guanine-rich DNA and RNA structures, which are commonly found in regulatory regions. This study examined the potential G-quadruplexes forming sequences in the promoters and gene bodies of podocyte-marker genes. High G-quadruplexes density was found in the vascular endothelial growth facto, cluster of differentiation-151, integrin subunit beta-4, metalloendopeptidase, Wilms tumor-1, integrin subunit beta-3, synaptopodin, and nephrin promoters. Vascular endothelial growth facto, cluster of differentiation-151 and integrin subunit beta-4 had the highest G-quadruplexes density in their gene bodies and promoters. Additionally, highly stable G-quadruplexes forming sequences were identified within all podocyte-marker genes. Furthermore, it is hypothesized that Wilms tumor-1 is capable of controlling the transcription of podocalyxin by binding to two possible G-quadruplexes forming motifs. We next analyzed the most frequently reported genetic mutations in the selected genes for their effect on DNA G-quadruplexes formation, and the thermodynamic stability of predicted RNA G-quadruplexes, using RNAfold. Importantly, the missense mutation c.121_122del in the nephrin gene reported in patients with NS type 1 affected DNA G-quadruplexes formation in this region as well as the thermodynamic stability of the corresponding RNA G-quadruplexes. Overall, we report the potential regulatory roles of G-quadruplexes in the etiology of nephrotic syndrome and their possible use as drug targets to treat kidney diseases.

G-quadruplex forming sequences in the genes coding for cytochrome P450 enzymes and their potential roles in drug metabolism.

The majority of drugs are metabolized by cytochrome P450 (CYP) enzymes, primarily belonging to the CYP1, CYP2 and CYP3 families. Genetic variations are the main cause of inter-individual differences in drug response, which constitutes a major concern in pharmacotherapy. G-quadruplexes (G4s), are non-canonical DNA and RNA secondary structures formed by guanine-rich sequences. G4s have been implicated in cancer and gene regulation. In this study, we investigated putative G4-forming sequences (PQSs) in the CYP genes. Our findings reveal a high density of PQSs in the full genes of CYP family 2. Moreover, we observe an increased density of PQSs in the promoters of CYP family 1 genes compared to non-CYP450 genes. Importantly, stable PQSs were also identified in all studied CYP genes. Subsequently, we assessed the impact of the most frequently reported genetic mutations in the selected genes and the possible effect of these mutations on G4 formation as well as on the thermodynamic stability of predicted G4s. We found that 4 SNPs overlap G4 sequences and lead to mutated DNA and RNA G4 forming sequences in their context. Notably, the mutation in the CYP2C9 gene, which is associated with impaired (S)-warfarin metabolism in patients, alters a G4 sequence. We then demonstrated that at least 10 of the 13 chosen cytochrome P450 G4 candidates form G-quadruplex structures in vitro, using a combination of spectroscopic methods. In conclusion, our findings indicate the potential role of G-quadruplexes in the regulation of cytochrome genes, and emphasize the importance of G-quadruplexes in drug metabolism.

Report on a Case with Moreno-Nishimura-Schmidt Overgrowth Syndrome: A Clinically Delineated Disease Yet of an Unknown Origin!

Introduction: Overgrowth syndromes are a heterogeneous group of genetic disorders characterized by excessive growth, often accompanied by additional clinical features, such as facial dysmorphism, hormonal imbalances, cognitive impairment, and increased risk for neoplasia. Moreno-Nishimura-Schmidt (M-N-S) overgrowth syndrome is a very rare overgrowth syndrome characterized by severe pre- and postnatal overgrowth, dysmorphic facial features, kyphoscoliosis, large hands and feet, inguinal hernia, and distinctive skeletal features. The clinical and radiological features of the disorder have been well delineated, yet its molecular pathogenesis remains unclear. Case Presentation: We report on a Lebanese boy with M-N-S syndrome, whose clinical manifestations were compared with those of previously reported 5 affected individuals. Whole-exome sequencing combined with comparative genome hybridization analysis failed to delineate the molecular basis of the phenotype. However, epigenetic studies revealed a different methylation status of several CpG sites between him and healthy controls, with methyltransferase activity showing the most significant enrichment. Conclusion: An additional case of M-N-S syndrome recapitulated the clinical and radiological manifestations described in the previous reports. The data in the epigenetic studies implicated that abnormal methylations might play an essential role in development of the disease phenotype. However, additional studies in a clinically homogeneous cohort of patients are crucial to confirm this hypothesis.

Genetic profile of borderline ovarian tumors in the Lebanese population by whole-exome sequencing.

OBJECTIVE: To assess the molecular profile of borderline ovarian tumors (BOT) in the Lebanese population by whole-exome sequencing and to correlate the results with the patients' clinical profiles. METHODS: We included in this retrospective study 33 tumors belonging to 32 Lebanese women presenting with BOT, diagnosed at Hôtel Dieu de France. A total of 234 genes involved in different germinal and somatic types of cancer were analyzed using next-generation sequencing. RESULTS: Molecular analysis of these tumors allowed us to detect mutations in genes involved in the mitogen-activated protein kinase cascade in 57.58% of BOT and to identify variants affecting the DNA repair mechanism in 63.89% of samples. Furthermore, our initial analysis revealed an association between defects in DNA double-strand break repair and the occurrence of mucinous BOT, in 75% of the cases. CONCLUSION: This study reports the molecular profiles of BOT in the Lebanese population and compares them to the literature. This is the first study associating the DNA repair pathway to BOT.

Spondyloocular Syndrome: A Report of an Additional Family and Phenotypic Spectrum Delineation.

Spondyloocular syndrome (SOS, OMIM # 605822) is a rare genetic disorder characterized by osseous and ocular manifestations, including generalized osteoporosis, multiple long bones fractures, platyspondyly, dense cataracts and retinal detachment, and dysmorphic facial features, with or without short stature, cardiopathy, hearing impairment, and intellectual disability. Biallelic mutations in the XYLT2 gene (OMIM * 608125), encoding the xylosyltransferase II, were shown to be responsible for this disease. To date, 22 cases with SOS have been described, with varying clinical presentations and a yet-to-be-established genotypic-phenotypic correlation. Two patients from a consanguineous Lebanese family that presented with SOS were included in this study. Whole exome sequencing revealed a novel homozygous nonsense mutation in XYLT2 (p.Tyr414*) in these patients. We review all previously reported cases with SOS, describe the second nonsense mutation in XYLT2, and contribute to a better delineation of the phenotypic spectrum of the disease.

NEK8-Associated Nephropathies: Do Autosomal Dominant Forms Exist?

INTRODUCTION: Nephronophthisis (NPHP) is a group of autosomal recessive renal diseases characterized by a reduced ability of the kidneys to concentrate solutes, chronic tubulointerstitial nephritis, and cystic kidney disease. It represents the most common genetic cause of childhood renal failure. To date, around 20 different genes, encoding primary cilia proteins, have been linked to NPHP. These contribute to one-third of cases with NPHP while the majority of patients remain molecularly undiagnosed. MATERIALS AND METHODS: Whole exome sequencing (WES) was carried out on a 2-year-old Lebanese boy with infantile NPHP characterized by multicystic kidney dysplasia, kidney insufficiency, and enlarged kidneys in addition to chronic anemia. The candidate variant, detected by WES, was then tested in the patient and his parents by Sanger sequencing. Copy number variation (CNV) analysis was subsequently performed in the proband. RESULTS: Our studies enabled the detection of a heterozygous de novo variant in NEK8 (NM_178170: p.Arg45Trp) in the proband. CNV analysis excluded the presence of big deletions or insertions in this gene. CONCLUSION: Here we report a de novo heterozygous variant in the NEK8 gene in infantile NPHP. This variant was previously detected at a de novo state in a patient presenting with the same clinical features as the proband. This suggests that autosomal dominant forms of NEK8-linked nephropathies may exist. Reporting further patients with NEK8 mutations is essential to confirm these findings and assess whether dominant forms of the disease are restricted to a specific mutational spot or are linked to variants scattered throughout the NEK8 gene.

Genetic susceptibility of bladder cancer in the Lebanese population.

BACKGROUND: Bladder cancer (BC) is the 10th most frequent tumor worldwide. Evidence shows an association between elevated risk of BC and various single nucleotide polymorphisms (SNP). BC incidence was the highest in Lebanon according to Globocan 2018 report, but little is known about the genetic susceptibility of Lebanese people to this disease. We aim to evaluate whether this prominent incidence of BC in Lebanon is attributable to known coding genetic variants. METHODS: A case-control study was conducted at Hotel-Dieu de France Hospital, Beirut. A cohort of 51 Lebanese patients with BC were recruited between 2017 and 2020. Whole Exome Sequencing (WES) was performed on peripheral blood samples to detect coding genetic variants in the patients. An in-house database including WES data from 472 Lebanese individuals served as control. Literature review of the genetic predisposition to BC was conducted to establish a database of variants known to influence the risk of BC. In-common SNPs were identified between cases and the aforecited database, and their allelic frequencies was quantified in the former and in controls. Comparative analysis of the allelic frequencies of each in-common SNP was carried out between cases, controls, and the genome aggregation database (gnomAD). Analysis was performed by applying the binomial law and setting the p-value to 10- 10. RESULTS: 484 polymorphisms associated with BC were extracted from the literature review ;151 of which were in-common with the 206 939 variations detected by WES in our cases. Statistically significant differences (p-value < 10- 10) in allelic frequencies was seen in 11 of the 151 in-common SNPs, but none of which corresponds with a higher BC risk. Moreover, rs4986782 variant in the NAT1 gene is not associated with BC in the Lebanese population. `. CONCLUSION: This is the first next-generation sequencing (NGS)- based study investigating BC risk in a Lebanese cohort of 51 patients. The majority of known exonic variants in the literature were not associated with BC in our patients. Further studies with larger sample sizes are warranted to explore the association of BC in our population with known non-coding genetic variants, and the remainder of WES-generated private Lebanese variants.

Molecular pathogenesis of hereditary lung cancer: a literature review.

Among all cancer types, pulmonary cancer has the highest mortality rate. Tobacco consumption remains the major risk factor for the development of lung cancer. However, many studies revealed a correlation between inherited genetic variants and predisposition to lung cancer, especially in nonsmokers. To date, genetic testing for the detection of germline mutations is not yet recommended in patients with lung cancer and testing is focused on somatic alterations given their implication in the treatment choice. Understanding the impact of genetic predisposition on the occurrence of lung cancer is essential to enable the introduction of accurate guidelines and recommendations that might reduce mortality. In this review paper, we describe familial lung cancer, and expose germline mutations that are linked to this type of cancer. We also report pathogenic genetic variants linked to syndromes associated with lung cancer.

Molecular profiling of basal cell carcinomas in young patients.

BACKGROUND: Basal cell carcinoma (BCC) represents by far the most common non-melanoma skin cancer (NMSC) in the world with an increasing incidence of 3% to 10% per year, especially in patients under the age of 40. While variants in the sonic Hedgehog and cell cycle regulation pathways account for the majority of BCC cases in adults, the molecular etiology of BCC in young patients is unelucidated yet. This study aims to investigate the molecular profile of BCC in the young population. METHODS: 28 tumors belonging to 25 Lebanese patients under the age of 40, presenting different stages of BCC and diagnosed at Hôtel Dieu de France-Saint Joseph University Medical Center were included in this study. A selected panel of 150 genes involved in cancer was analyzed by Next Generation Sequencing (NGS) in the 28 included tumors. RESULTS: Genetic variants detected in more than 5% of the reads, with a sequencing depth ≥ 50x, were selected. Two hundred and two genetic variants in 48 different genes were detected, with an overall average sequencing depth of 1069x. Among the 28 studied tumors, 18 (64.3%) show variations in the PTCH1 gene, 6 (21.4%) in TP53 and 3 (10.7%) in SMO. CONCLUSIONS: This is the first study reporting NGS-based analysis of BCC in a cohort of young patients. Our results highlight the involvement of the hedgehog and cell cycle regulation pathways in the genesis of BCC in the general population. The inclusion of a larger cohort of young patients is needed to confirm our findings.

Actionable Exomic Secondary Findings in 280 Lebanese Participants.

The expanded use of NGS tests in genetic diagnosis enables the massive generation of data related to each individual, among which some findings are of medical value. Over the last three and a half years, 280 unrelated Lebanese patients, presenting a wide spectrum of genetic disorders were referred to our center for genetic evaluation by WES. Molecular diagnosis was established in 56% of the cases, as was previously reported. The current study evaluates secondary findings in these patients in 59 genes, linked to conditions mostly responsive to medical interventions, as per the ACMG guidelines. Our analysis allowed us to detect 19 pathogenic/likely pathogenic variants in 24 individuals from our cohort. Dominant actionable variants were found in 17 individuals representing 6% of the studied population. Genes associated with dominant cardiac diseases were the most frequently mutated: variants were found in 2.1% of our cohort. Genetic predisposition to cancer syndromes was observed in 1.07% of the cases. In parallel to dominant disease alleles, our analysis identified a recessive pathogenic disease allele in 2.5% of the individuals included in this study. Of interest, some variants were detected in different patients from our cohort thus urging the study of their prevalence in our population and the implementation, when needed, of specific genetic testing in the neonatal screening panel. In conclusion, here we report the first study estimating the actionable pathogenic variant load in the Lebanese population. Communicating current findings to the patients will enable them to benefit from a multi-disciplinary approach. Furthermore, tailoring the ACMG guidelines to the population is suggested, especially in highly consanguineous populations where the information related to recessive alleles might be highly beneficial to patients and their families.

Neuroblastoma Amplified Sequence (NBAS) mutation in recurrent acute liver failure: Confirmatory report in a sibship with very early onset, osteoporosis and developmental delay.

BACKGROUND: Recently, biallelic mutations in the Neuroblastoma Amplified Sequence NBAS gene have been identified in ten patients that present recurrent acute liver failure (RALF) in early infancy. In addition to severe liver dysfunction, some of these individuals also suffered from other comorbidities including cardiomyopathy, neurologic phenotypes and gastrointestinal immune defects. Here we report on a consanguineous Lebanese family with three siblings affected by RALF. Of note, neonatal spontaneous fractures, developmental delay, prominent eyes, generalized hirsutism, gum hypertrophy, and hepato-splenomegaly ​were also present. METHODS: Whole-genome SNP genotyping in all the patients, followed by exome sequencing was performed in one of the affected siblings. RESULTS: A homozygous c.409C > T (p.Arg137Trp) missense mutation in NBAS was identified in all patients. CONCLUSION: Overall, our findings confirm the involvement of NBAS in the pathogenesis of this condition characterized by severe liver dysfunction and help expand its phenotypical spectrum.

Exome sequencing reveals a mutation in DMP1 in a family with familial sclerosing bone dysplasia.

INTRODUCTION: Hypophosphatemic rickets (HR) comprises a rare group of inherited diseases. Very recently, mutations in the dentin matrix protein 1 (DMP1) gene were identified in patients with an extremely rare autosomal recessive form of HR (ARHR). To date, very few cases of these mutations were reported. MATERIALS AND METHODS: A Lebanese consanguineous family with 2 affected sisters was studied. Patients aged 45 and 47years old presented with short stature, severe genu varum, cranial hyperostosis and a very high bone density that led to a diagnosis of a familial sclerosing bone dysplasia. Molecular analysis of known genes involved in osteopetrosis showed normal results. A combination of genotyping and exome sequencing was performed in order to elucidate the genetic basis of this pathology. RESULTS: Biochemical analysis was consistent with normal serum calcium and 1-25(OH)2D levels, low to normal serum phosphorus and elevated PTH values. Serum c-terminal FGF-23 was elevated in one of the two patients. A homozygous mutation disrupting the initiation codon of the DMP1 gene (OMIM 600980), NM_001079911.2: c.1A>G, p.Met1Val, was identified by exome sequencing and confirmed by Sanger sequencing. CONCLUSION: We report here a family of ARHR secondary to a DMP1 mutation located in the first coding exon of the gene. Our cases show that some ARHR cases may develop with age an unaccountable increase in bone density and bone overgrowth.

The impairment of MAGMAS function in human is responsible for a severe skeletal dysplasia.

Impairment of the tightly regulated ossification process leads to a wide range of skeletal dysplasias and deciphering their molecular bases has contributed to the understanding of this complex process. Here, we report a homozygous mutation in the mitochondria-associated granulocyte macrophage colony stimulating factor-signaling gene (MAGMAS) in a novel and severe spondylodysplastic dysplasia. MAGMAS, also referred to as PAM16 (presequence translocase-associated motor 16), is a mitochondria-associated protein involved in preprotein translocation into the matrix. We show that MAGMAS is specifically expressed in trabecular bone and cartilage at early developmental stages and that the mutation leads to an instability of the protein. We further demonstrate that the mutation described here confers to yeast strains a temperature-sensitive phenotype, impairs the import of mitochondrial matrix pre-proteins and induces cell death. The finding of deleterious MAGMAS mutations in an early lethal skeletal dysplasia supports a key role for this mitochondrial protein in the ossification process.

A second family with autosomal recessive spondylometaphyseal dysplasia and early death.

We report on a consanguineous Lebanese family in which two sibs had pre- and post-natal growth retardation, developmental delay, large anterior fontanel, prominent forehead, low-set ears, depressed nasal bridge, short nose, anteverted nares, increased nasal width, prominent abdomen, and short limbs. Radiographs disclosed the presence of wormian bones, platyspondyly, decreased interpedicular distance at the lumbar vertebrae, square iliac bones, horizontal acetabula, trident acetabula, hypoplastic ischia, partial agenesis of the sacrum, ribs with cupped ends, short long bones with abnormal modeling, slight widening of the distal femoral metaphyses, and delayed epiphyseal ossification. Both sibs had a severe cardiomegaly and died at around 24 months from a heart failure. Differential diagnosis suggests that this is a second family presenting a newly described early lethal chondrodysplasia first reported by [Mégarbané et al. (2008); Am J Med Genet Part A 146A:2916-2919].

The identification of MAFB mutations in eight patients with multicentric carpo-tarsal osteolysis supports genetic homogeneity but clinical variability.

Multicentric carpo-tarsal osteolysis (MCTO) with or without nephropathy is a rare osteolysis disorder beginning in early childhood and involving mainly carpal and tarsal bones. Renal disease appears later in life in the majority of cases and evolves quickly to end stage renal failure. Autosomal dominant (AD) inheritance has been demonstrated, with a high frequency of sporadic cases. Recently, mutations in a highly conserved region of the MAFB gene (v-maf musculoaponeurotic fibrosarcoma oncogene ortholog B) have been identified in MCTO patients by exome sequencing. MafB, known as a regulator of various developmental processes, is essential for osteoclastogenesis and renal development. We report here the molecular screening of MAFB in eight MCTO patients from six families. We identified MAFB mutations in all, including three novel missense mutations clustering within the hot spot mutation region. Among the eight patients, six only presented renal disease. Our report confirms the genetic homogeneity of MCTO and provides data underlying the clinical variability of this disorder.

Marfanoid habitus, inguinal hernia, advanced bone age, and distinctive facial features: a new collagenopathy?

We report on two sibs, a girl, and a boy, with tall stature, long, and triangular faces, prominent foreheads with high frontal hairlines, telecanthus, downward slanting of the palpebral fissures, ptosis of the eyelids, everted lower eyelids, large ears, long noses, full, and everted vermilions, highly arched and narrow palates, tooth crowding, thin and long uvulae, coloboma of the alae, hyperextensible joints, long digits, positive thumb signs, flat feet, slightly diminished muscle strength, myopia, astigmatia, inguinal hernia, and vesical diverticula. Total body X-rays showed the presence of advanced bone age in both sibs and bilateral hallux valgus in the girl. Array-CGH did not reveal any pathological CNV. Molecular analysis of FBN1, FBN2, TGFBR1, TGFBR2, and CHST14 gene was normal, and SNP linkage analysis excluded more candidate genes. Differential diagnoses and the possibility that we might be reporting on a hitherto unreported syndrome are discussed.

Identification of a novel causative mutation in the ROR2 gene in a Lebanese family with a mild form of recessive Robinow syndrome.

Autosomal recessive Robinow syndrome (OMIM 268310) is a condition caused by mutations in the ROR2 gene, the receptor tyrosine kinase-like orphan receptor 2. The main characteristic features are: a face resembling that of a fetus, cleft lip and palate, mesomelic limb shortening, a micropenis in males, hydronephrosis or urinary tract infections, and skeletal and vertebral anomalies. This study reports two sisters from a consanguineous Lebanese family with an autosomal recessive Robinow syndrome. Both presented with short stature, dysmorphic facial features, and mild bone abnormalities. One of the affected girls had a malformation of her right hand: a mesoaxial polydactyly combined with a syndactyly of the 3rd and 4th fingers, and a short right 3rd metacarpal bone. Molecular analysis of the ROR2 gene revealed the presence of a previously undescribed missense mutation: p.R272C (c.814C>T), in the cysteine-rich domain of the protein. These patients are compared with other cases, and a phenotype-genotype correlation is discussed.