The pathogenic p.Gln319Ter variant is not causing congenital adrenal hyperplasia when inherited in one of the duplicated CYP21A2 genes.

Objective: The study aimed to identify the pathogenic status of p.Gln319Ter (NM_000500.7: c.955C>T) variant when inherited in a single CYP21A2 gene (bimodular RCCX haplotype) and to discriminate between a non-causing congenital adrenal hyperplasia (CAH) allele when inherited in a duplicated and functional CYP21A2 gene context (trimodular RCCX haplotype). Methods: 38 females and 8 males with hyperandrogenemia, previously screened by sequencing and identified as carriers for the pathogenic p.Gln319Ter, were herein tested by multiplex ligation-dependent probe amplification (MLPA) and a real-time PCR Copy number Variation (CNV) assay. Results: Both MLPA and real-time PCR CNV analyses confirmed a bimodular and pathogenic RCCX haplotype with a single CYP21A2 in 19/46 (41.30%) p.Gln319Ter carriers and who in parallel all shared elevated 17-OHP levels. The remaining 27 individuals that also carried the p.Gln319Ter exhibited low 17-OHP levels as a result of their carriership of a duplicated CYP21A2 with a trimodular RCCX haplotype. Interestingly, all of these individuals also carried in linkage disequilibrium with p.Gln319Ter two single nucleotide polymorphisms, the c.293-79G>A (rs114414746) in intron 2 and the c.*12C>T (rs150697472) in the 3'-UTR. Therefore, these variants can be used to distinguish between pathogenic and non-pathogenic genomic contexts of the c.955T (p.Gln319) in the genetic diagnosis of congenital adrenal hyperplasia (CAH). Conclusion: The employed methodologies identified a considerable number of individuals with non-pathogenic p.Gln319Ter from the individuals that typically carry the pathogenic p.Gln319Ter in a single CYP21A2. Therefore, it is extremely important the detection of such haplotypes for the prenatal diagnosis, treatment and genetic counseling in patients with CAH.

Hereditary multiple exostoses caused by a chromosomal inversion removing part of EXT1 gene.

BACKGROUND: Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder characterized by the development of multiple, circumscript and usually symmetric bony protuberances called osteochondromas. Most HME are caused by EXT1 and EXT2 loss of function mutations. Most pathogenic mutations are nonsense followed by missense mutations and deletions. CASE PRESENTATION: Here we report on a patient with a rare and complex genotype resulting in a typical HME phenotype. Initial point mutation screening in EXT1 and EXT2 genes by Sanger sequencing did not reveal any pathogenic variants. The patient along with the healthy parents was subsequently referred for karyotype and array-Comparative Genomic Hybridization (CGH) analyses. Chromosomal analysis revealed two independent de novo apparently balanced rearrangements: a balanced translocation between the long arms of chromosomes 2 and 3 at breakpoints 2q22 and 3q13.2 and a pericentric inversion with breakpoints at 8p23.1q24.1. Both breakpoints were confirmed by Fluorescence In Situ Hybridization (FISH). Subsequently, array-CGH revealed a novel heterozygous deletion within the EXT1 gene at one of the inversion breakpoints, rendering the inversion unbalanced. The mode of inheritance, as well as the size of the deletion were further investigated by Quantitative Real-time PCR (qPCR), defining the deletion as de novo and of 3.1 kb in size, removing exon 10 of EXT1. The inversion in combination with the 8p23.1 deletion most likely abolishes the transcription of EXT1 downstream of exon 10 hence resulting in a truncated protein. CONCLUSIONS: The identification of a rare and novel genetic cause of HME, highlights the importance of additional comprehensive investigation of patients with typical clinical manifestations, even when EXT1 and EXT2 mutation analysis is negative.

Natural history of KBG syndrome in a large European cohort.

KBG syndrome (KBGS) is characterized by distinctive facial gestalt, short stature and variable clinical findings. With ageing, some features become more recognizable, allowing a differential diagnosis. We aimed to better characterize natural history of KBGS. In the context of a European collaborative study, we collected the largest cohort of KBGS patients (49). A combined array- based Comparative Genomic Hybridization and next generation sequencing (NGS) approach investigated both genomic Copy Number Variants and SNVs. Intellectual disability (ID) (82%) ranged from mild to moderate with severe ID identified in two patients. Epilepsy was present in 26.5%. Short stature was consistent over time, while occipitofrontal circumference (median value: -0.88 SD at birth) normalized over years. Cerebral anomalies, were identified in 56% of patients and thus represented the second most relevant clinical feature reinforcing clinical suspicion in the paediatric age when short stature and vertebral/dental anomalies are vague. Macrodontia, oligodontia and dental agenesis (53%) were almost as frequent as skeletal anomalies, such as brachydactyly, short fifth finger, fifth finger clinodactyly, pectus excavatum/carinatum, delayed bone age. In 28.5% of individuals, prenatal ultrasound anomalies were reported. Except for three splicing variants, leading to a premature termination, variants were almost all frameshift. Our results, broadening the spectrum of KBGS phenotype progression, provide useful tools to facilitate differential diagnosis and improve clinical management. We suggest to consider a wider range of dental anomalies before excluding diagnosis and to perform a careful odontoiatric/ear-nose-throat (ENT) evaluation in order to look for even submucosal palate cleft given the high percentage of palate abnormalities. NGS approaches, following evidence of antenatal ultrasound anomalies, should include ANKRD11.

A de novo SFMBT1 pathogenic variant identified in a boy with Poland syndrome.

Poland syndrome is a rare developmental disorder characterized by unilateral, complete or partial, absence of the pectoralis major (and often minor) muscle, accompanied with ipsilateral hand malformations. To date, no clear genetic cause has been associated with Poland syndrome, although familial cases have been reported. We report the employment of trio exome investigation and the identification of a heterozygous de novo pathogenic variant in the SFMBT1 gene, a transcription factor associated with transcriptional repression during development, in a 14-yr-old boy with Poland syndrome. We further demonstrate by means of cDNA sequencing and western blot analysis that this variant results in SFMBT1 exon 10 skipping and a lower concentration of the SFMBT1 wild-type protein. To our knowledge, the heterozygous pathogenic SFMBT1 variant identified in association with this condition is novel as it has not been elsewhere described in the literature and it can be incorporated to the limited reported cases published.

Spectrum of Phenotypic, Genetic, and Functional Characteristics in Patients With Epilepsy With KCNC2 Pathogenic Variants.

BACKGROUND AND OBJECTIVES: KCNC2 encodes Kv3.2, a member of the Shaw-related (Kv3) voltage-gated potassium channel subfamily, which is important for sustained high-frequency firing and optimized energy efficiency of action potentials in the brain. The objective of this study was to analyze the clinical phenotype, genetic background, and biophysical function of disease-associated Kv3.2 variants. METHODS: Individuals with KCNC2 variants detected by exome sequencing were selected for clinical, further genetic, and functional analysis. Cases were referred through clinical and research collaborations. Selected de novo variants were examined electrophysiologically in Xenopus laevis oocytes. RESULTS: We identified novel KCNC2 variants in 18 patients with various forms of epilepsy, including genetic generalized epilepsy (GGE), developmental and epileptic encephalopathy (DEE) including early-onset absence epilepsy, focal epilepsy, and myoclonic-atonic epilepsy. Of the 18 variants, 10 were de novo and 8 were classified as modifying variants. Eight drug-responsive patients became seizure-free using valproic acid as monotherapy or in combination, including severe DEE cases. Functional analysis of 4 variants demonstrated gain of function in 3 severely affected DEE cases and loss of function in 1 case with a milder phenotype (GGE) as the underlying pathomechanisms. DISCUSSION: These findings implicate KCNC2 as a novel causative gene for epilepsy and emphasize the critical role of KV3.2 in the regulation of brain excitability.

Transcriptomic characterization of tissues from patients and subsequent pathway analyses reveal biological pathways that are implicated in spastic ataxia.

BACKGROUND: Spastic ataxias (SAs) encompass a group of rare and severe neurodegenerative diseases, characterized by an overlap between ataxia and spastic paraplegia clinical features. They have been associated with pathogenic variants in a number of genes, including GBA2. This gene codes for the non-lysososomal ß-glucosylceramidase, which is involved in sphingolipid metabolism through its catalytic role in the degradation of glucosylceramide. However, the mechanism by which GBA2 variants lead to the development of SA is still unclear. METHODS: In this work, we perform next-generation RNA-sequencing (RNA-seq), in an attempt to discover differentially expressed genes (DEGs) in lymphoblastoid, fibroblast cell lines and induced pluripotent stem cell-derived neurons derived from patients with SA, homozygous for the GBA2 c.1780G > C missense variant. We further exploit DEGs in pathway analyses in order to elucidate candidate molecular mechanisms that are implicated in the development of the GBA2 gene-associated SA. RESULTS: Our data reveal a total of 5217 genes with significantly altered expression between patient and control tested tissues. Furthermore, the most significant extracted pathways are presented and discussed for their possible role in the pathogenesis of the disease. Among them are the oxidative stress, neuroinflammation, sphingolipid signaling and metabolism, PI3K-Akt and MAPK signaling pathways. CONCLUSIONS: Overall, our work examines for the first time the transcriptome profiles of GBA2-associated SA patients and suggests pathways and pathway synergies that could possibly have a role in SA pathogenesis. Lastly, it provides a list of DEGs and pathways that could be further validated towards the discovery of disease biomarkers.

Exploring the Genetic Causality of Discordant Phenotypes in Familial Apparently Balanced Translocation Cases Using Whole Exome Sequencing.

Familial apparently balanced translocations (ABTs) are usually not associated with a phenotype; however, rarely, ABTs segregate with discordant phenotypes in family members carrying identical rearrangements. The current study was a follow-up investigation of four familial ABTs, where whole exome sequencing (WES) was implemented as a diagnostic tool to identify the underlying genetic aetiology of the patients' phenotypes. Data were analysed using an in-house bioinformatics pipeline alongside VarSome Clinical. WES findings were validated with Sanger sequencing, while the impact of splicing and missense variants was assessed by reverse-transcription PCR and in silico tools, respectively. Novel candidate variants were identified in three families. In family 1, it was shown that the de novo pathogenic STXBP1 variant (NM_003165.6:c.1110+2T>G) affected splicing and segregated with the patient's phenotype. In family 2, a likely pathogenic TUBA1A variant (NM_006009.4:c.875C>T, NP_006000.2:p.(Thr292Ile)) could explain the patient's symptoms. In family 3, an SCN1A variant of uncertain significance (NM_006920.6:c.5060A>G, NP_008851.3:p.(Glu1687Gly)) required additional evidence to sufficiently support causality. This first report of WES application in familial ABT carriers with discordant phenotypes supported our previous findings describing such rearrangements as coincidental. Thus, WES can be recommended as a complementary test to find the monogenic cause of aberrant phenotypes in familial ABT carriers.

A Novel CLN6 Variant Associated With Juvenile Neuronal Ceroid Lipofuscinosis in Patients With Absence of Visual Loss as a Presenting Feature.

The neuronal ceroid lipofuscinoses (NCLs), also known as Batten disease, are a group of autosomal recessive lysosomal storage disorders that are characterized by neurodegeneration, progressive cognitive decline, motor impairment, ataxia, loss of vision, seizures, and premature death. To date, pathogenic variants in more than 13 genes have been associated with NCLs. CLN6 encodes an endoplasmic reticulum non-glycosylated transmembrane protein, which is involved in lysosomal acidification. Mutations in CLN6 cause late-infantile juvenile NCL (JNCL) adult-onset NCL, and Kufs disease. Members from two available families with JNCL were clinically evaluated, and samples were collected from consenting individuals. The molecular investigation was performed by whole-exome sequencing, Sanger sequencing, and family segregation analysis. Furthermore, in silico prediction analysis and structural modeling of the identified CLN6 variants were performed. We report clinical and genetic findings of three patients from two Greek-Cypriot families (families 915 and 926) with JNCL. All patients were males, and the first symptoms appeared at the age of 6 years. The proband of family 926 presented with loss of motor abilities, ataxia, spasticity, seizure, and epilepsy. The proband of family 915 had ataxia, spasticity, dysarthria, dystonia, and intellectual disability. Both probands did not show initial signs of vision and/or hearing loss. Molecular analysis of family 926 revealed two CLN6 biallelic variants: the novel, de novo p.Tyr295Cys and the known p.Arg136His variants. In family 915, both patients were homozygous for the p.Arg136His CLN6 variant. Prediction analysis of the two CLN6 variants characterized them as probably damaging and disease-causing. Structural modeling of the variants predicted that they probably cause protein structural differentiation. In conclusion, we describe two unrelated Cypriot families with JNCL. Both families had variants in the CLN6 gene; however, they presented with slightly different symptoms, and notably none of the patients has loss of vision. In silico prediction and structural analyses indicate that both variants are most likely pathogenic.

Pathogenic and Low-Frequency Variants in Children With Central Precocious Puberty.

Background: Central precocious puberty (CPP) due to premature activation of GnRH secretion results in early epiphyseal fusion and to a significant compromise in the achieved final adult height. Currently, few genetic determinants of children with CPP have been described. In this translational study, rare sequence variants in MKRN3, DLK1, KISS1, and KISS1R genes were investigated in patients with CPP. Methods: Fifty-four index girls and two index boys with CPP were first tested by Sanger sequencing for the MKRN3 gene. All children found negative (n = 44) for the MKRN3 gene were further investigated by whole exome sequencing (WES). In the latter analysis, the status of variants in genes known to be related with pubertal timing was compared with an in-house Cypriot control cohort (n = 43). The identified rare variants were initially examined by in silico computational algorithms and confirmed by Sanger sequencing. Additionally, a genetic network for the MKRN3 gene, mimicking a holistic regulatory depiction of the crosstalk between MKRN3 and other genes was designed. Results: Three previously described pathogenic MKRN3 variants located in the coding region of the gene were identified in 12 index girls with CPP. The most prevalent pathogenic MKRN3 variant p.Gly312Asp was exclusively found among the Cypriot CPP cohort, indicating a founder effect phenomenon. Seven other CPP girls harbored rare likely pathogenic upstream variants in the MKRN3. Among the 44 CPP patients submitted to WES, nine rare DLK1 variants were identified in 11 girls, two rare KISS1 variants in six girls, and two rare MAGEL2 variants in five girls. Interestingly, the frequent variant rs10407968 (p.Gly8Ter) of the KISS1R gene appeared to be less frequent in the cohort of patients with CPP. Conclusion: The results of the present study confirm the importance of the MKRN3-imprinted gene in genetics of CPP and its key role in pubertal timing. Overall, the results of the present study have emphasized the importance of an approach that aligns genetics and clinical aspects, which is necessary for the management and treatment of CPP.

Demographic characteristics, clinical and laboratory features, and the distribution of pathogenic variants in the CFTR gene in the Cypriot cystic fibrosis (CF) population demonstrate the utility of a national CF patient registry.

BACKGROUND: Specialized clinical care for cystic fibrosis (CF) in Cyprus, a small island country, has been implemented since the 1990s. However, only recently, a national CF patient registry has been established for the systematic recording of patients' data. In this study, we aim to present data on the epidemiological, genotypic and phenotypic features of CF patients in the country from the most recent data collection in 2019, with particular emphasis on notable rare or unique cases. RESULTS: Overall, data from 52 patients are presented, 5 of whom have deceased and 13 have been lost to follow-up in previous years. The mean age at diagnosis was 7.2 ± 12.3 years, and the mean age of 34 alive patients by the end of 2019 was 22.6 ± 13.2 years. Patients most commonly presented at diagnosis with acute or persistent respiratory symptoms (46.2%), failure to thrive or malnutrition (40.4%), and dehydration or electrolyte imbalance (32.7%). Sweat chloride levels were diagnostic (above 60 mmol/L) in 81.8% of examined patients. The most common identified mutation was p.Phe508del (F508del) (45.2%), followed by p.Leu346Pro (L346P) (6.7%), a mutation detected solely in individuals of Cypriot descent. The mean BMI and FEV1 z-scores were 0.2 ± 1.3 and - 2.1 ± 1.7 across all age groups, respectively, whereas chronic Pseudomonas aeruginosa colonization was noted in 26.9% of patients. The majority of patients (74.5%) were eligible to receive at least one of the available CFTR modulator therapies. In 25% of patients we recovered rare or unique genotypic profiles, including the endemic p.Leu346Pro (L346P), the rare CFTR-dup2, the co-segregated c.4200_4201delTG/c.489 + 3A > G, and the polymorphism p.Ser877Ala. CONCLUSIONS: CF patient registries are particularly important in small or isolated populations, such as in Cyprus, with rare or unique disease cases. Their operation is necessary for the optimization of clinical care provided to CF patients, enabling their majority to benefit from evolving advances in precision medicine.

Unravelling the genetic causes of multiple malformation syndromes: A whole exome sequencing study of the Cypriot population.

Multiple malformation syndromes (MMS) belong to a group of genetic disorders characterised by neurodevelopmental anomalies and congenital malformations. Here we explore for the first time the genetic aetiology of MMS using whole-exome sequencing (WES) in undiagnosed patients from the Greek-Cypriot population after prior extensive diagnostics workup including karyotype and array-CGH. A total of 100 individuals (37 affected), from 32 families were recruited and family-based WES was applied to detect causative single-nucleotide variants (SNVs) and indels. A genetic diagnosis was reported for 16 MMS patients (43.2%), with 10/17 (58.8%) of the findings being novel. All autosomal dominant findings occurred de novo. Functional studies were also performed to elucidate the molecular mechanism relevant to the abnormal phenotypes, in cases where the clinical significance of the findings was unclear. The 17 variants identified in our cohort were located in 14 genes (PCNT, UBE3A, KAT6A, SPR, POMGNT1, PIEZO2, PXDN, KDM6A, PHIP, HECW2, TFAP2A, CNOT3, AGTPBP1 and GAMT). This study has highlighted the efficacy of WES through the high detection rate (43.2%) achieved for a challenging category of undiagnosed patients with MMS compared to other conventional diagnostic testing methods (10-20% for array-CGH and ~3% for G-banding karyotype analysis). As a result, family-based WES could potentially be considered as a first-tier cost effective diagnostic test for patients with MMS that facilitates better patient management, prognosis and offer accurate recurrence risks to the families.

Assessing the utility of long-read nanopore sequencing for rapid and efficient characterization of mobile element insertions.

Short-read next generation sequencing (NGS) has become the predominant first-line technique used to diagnose patients with rare genetic conditions. Inherent limitations of short-read technology, notably for the detection and characterization of complex insertion-containing variants, are offset by the ability to concurrently screen many disease genes. "Third-generation" long-read sequencers are increasingly being deployed as an orthogonal adjunct technology, but their full potential for molecular genetic diagnosis has yet to be exploited. Here, we describe three diagnostic cases in which pathogenic mobile element insertions were refractory to characterization by short-read sequencing. To validate the accuracy of the long-read technology, we first used Sanger sequencing to confirm the integration sites and derive curated benchmark sequences of the variant-containing alleles. Long-read nanopore sequencing was then performed on locus-specific amplicons. Pairwise comparison between these data and the previously determined benchmark alleles revealed 100% identity of the variant-containing sequences. We demonstrate a number of technical advantages over existing wet-laboratory approaches, including in silico size selection of a mixed pool of amplification products, and the relative ease with which an automated informatics workflow can be established. Our findings add to a growing body of literature describing the diagnostic utility of long-read sequencing.

An unusual combination of an atypical maternally inherited novel 0.3 Mb deletion in Williams-Beuren region and a de novo 22q11.21 microduplication in an infant with supravalvular aortic stenosis.

Williams-Beuren syndrome (WBS) is a rare neurodevelopmental disorder characterized by supravalvular aortic stenosis (SVAS), intellectual disability, overfriendliness and dysmorphic features. It is typically caused by 1.5-1.8 Mb deletions on 7q11.23. The 22q11.21 microduplication syndrome has a variable phenotype and is frequently associated with congenital heart disease. Here we present a unique patient, carrying aberrations within both of the above syndrome regions, referred for possible diagnosis of WBS because of SVAS. The patient was a boy who died suddenly 47 days after birth, possibly due to cardiac complications. Genetic testing was carried out, including array Comparative Genomic Hybridization (aCGH), Fluorescence In situ Hybridization (FISH) and Multiplex Ligation-Dependent Probe Amplification (MLPA) showing that the proband was heterozygous for a novel and atypical 0.3 Mb deletion in WBS region (7q11.23) encompassing the ELN gene. In addition, he was found heterozygous for a 22q11.21 microduplication. Parental studies revealed that the 7q11.23 deletion was inherited from the mother who also exhibited a cardiovascular phenotype, however very mild. The same maternally inherited deletion was detected in one of the proband's siblings, born two years later with a less severe SVAS. The 22q11.2 microduplication was de novo in origin. Detection and investigation of atypical deletions within known syndrome regions are crucial for better genotype-phenotype correlations and more accurate characterization of critical regions. The combined effect of two different genetic defects - one in a known syndrome region and one with variable clinical significance, is valuable for revealing gene interactions and enabling more accurate predictions, especially in prenatal diagnosis.

NGS Panel Testing of Triple-Negative Breast Cancer Patients in Cyprus: A Study of BRCA-Negative Cases.

In Cyprus, approximately 9% of triple-negative (estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative) breast cancer (TNBC) patients are positive for germline pathogenic variants (PVs) in BRCA1/2. However, the contribution of other genes has not yet been determined. To this end, we aimed to investigate the prevalence of germline PVs in BRCA1/2-negative TNBC patients in Cyprus, unselected for family history of cancer or age of diagnosis. A comprehensive 94-cancer-gene panel was implemented for 163 germline DNA samples, extracted from the peripheral blood of TNBC patients. Identified variants of uncertain clinical significance were evaluated, using extensive in silico investigation. Eight PVs (4.9%) were identified in two high-penetrance TNBC susceptibility genes. Of these, seven occurred in PALB2 (87.5%) and one occurred in TP53 (12.5%). Interestingly, 50% of the patients carrying PVs were diagnosed over the age of 60 years. The frequency of non-BRCA PVs (4.9%) and especially PALB2 PVs (4.3%) in TNBC patients in Cyprus appears to be higher compared to other populations. Based on these results, we believe that PALB2 and TP53 along with BRCA1/2 genetic testing could be beneficial for a large proportion of TNBC patients in Cyprus, irrespective of their age of diagnosis.

Goldberg-Shprintzen syndrome is determined by the absence, or reduced expression levels, of KIFBP.

Goldberg-Shprintzen syndrome (GOSHS) is caused by loss of function variants in the kinesin binding protein gene (KIFBP). However, the phenotypic range of this syndrome is wide, indicating that other factors may play a role. To date, 37 patients with GOSHS have been reported. Here, we document nine new patients with variants in KIFBP: seven with nonsense variants and two with missense variants. To our knowledge, this is the first time that missense variants have been reported in GOSHS. We functionally investigated the effect of the variants identified, in an attempt to find a genotype-phenotype correlation. We also determined whether common Hirschsprung disease (HSCR)-associated single nucleotide polymorphisms (SNPs), could explain the presence of HSCR in GOSHS. Our results showed that the missense variants led to reduced expression of KIFBP, while the truncating variants resulted in lack of protein. However, no correlation was found between the severity of GOSHS and the location of the variants. We were also unable to find a correlation between common HSCR-associated SNPs, and HSCR development in GOSHS. In conclusion, we show that reduced, as well as lack of KIFBP expression can lead to GOSHS, and our results suggest that a threshold expression of KIFBP may modulate phenotypic variability of the disease.

GnRH Deficient Patients With Congenital Hypogonadotropic Hypogonadism: Novel Genetic Findings in ANOS1, RNF216, WDR11, FGFR1, CHD7, and POLR3A Genes in a Case Series and Review of the Literature.

Background: Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disease caused by Gonadotropin-Releasing Hormone (GnRH) deficiency. So far a limited number of variants in several genes have been associated with the pathogenesis of the disease. In this original research and review manuscript the retrospective analysis of known variants in ANOS1 (KAL1), RNF216, WDR11, FGFR1, CHD7, and POLR3A genes is described, along with novel variants identified in patients with CHH by the present study. Methods: Seven GnRH deficient unrelated Cypriot patients underwent whole exome sequencing (WES) by Next Generation Sequencing (NGS). The identified novel variants were initially examined by in silico computational algorithms and structural analysis of their predicted pathogenicity at the protein level was confirmed. Results: In four non-related GnRH males, a novel X-linked pathogenic variant in ANOS1 gene, two novel autosomal dominant (AD) probably pathogenic variants in WDR11 and FGFR1 genes and one rare AD probably pathogenic variant in CHD7 gene were identified. A rare autosomal recessive (AR) variant in the SRA1 gene was identified in homozygosity in a female patient, whilst two other male patients were also, respectively, found to carry novel or previously reported rare pathogenic variants in more than one genes; FGFR1/POLR3A and SRA1/RNF216. Conclusion: This report embraces the description of novel and previously reported rare pathogenic variants in a series of genes known to be implicated in the biological development of CHH. Notably, patients with CHH can harbor pathogenic rare variants in more than one gene which raises the hypothesis of locus-locus interactions providing evidence for digenic inheritance. The identification of such aberrations by NGS can be very informative for the management and future planning of these patients.

Identification of novel splice mutation in SMAD3 in two Cypriot families with nonsyndromic thoracic aortic aneurysm. Two case reports.

BACKGROUND: Thoracic aortic aneurysm and dissection (TAA/D) represents a potentially lethal disease group characterized by an increased risk of dissection or rupture. Only a small percentage (approximately 30%) of individuals with nonsyndromic familial TAA/D have a pathogenic variant in one of the genes that have been found to be associated with the disease. METHODS: A targeted sequencing panel and direct sequencing approach were used to identify causative mutations in the index patients and other family members. RESULTS: In this study we report two apparently unrelated Cypriot families with nonsyndromic familial TAA/D. The proband A is a female patient diagnosed with TAA/D and intracranial aneurysm and opted for an elective intervention. The proband B is a male patient who was diagnosed with TAA/D and underwent cardiac surgery. Sequencing analysis identified a novel splice site variant (c.871+1G>A) in SMAD3 which is shown to be associated with the disease. Analysis of mRNA from the patient's tissue confirmed aberrant splicing and exon 6 skipping. CONCLUSION: Our findings expand the mutation spectrum of variants that have been shown to be associated with nonsyndromic familial TAA/D. This study demonstrates the importance of a comprehensive clinical and genetic evaluation aiming at early diagnosis and intervention.

First reported case of Steel syndrome in the European population: A novel homozygous mutation in COL27A1 and review of the literature.

Steel syndrome is an autosomal recessive disorder that primarily affects the skeletal system causing a variety of manifestations. Sixteen individuals with Steel syndrome, mainly Puerto Ricans (11/16), were previously reported to carry bi-allelic mutations in the COL27A1 gene. Here, we present the first patient with Steel syndrome in Europe and the sixth non-Puerto Rican carrying a novel homozygous mutation in COL27A1. The patient is a 4-year-old boy born to non-consanguineous healthy parents, with dysmorphic facial features, absent hip ossification centres, external rotation of both feet, relatively short stature, mild skin syndactyly, short mid phalanges and bilateral sensorineural hearing loss. Whole exome sequencing (WES) revealed a novel homozygous missense variant p.(Gly802Glu) in COL27A1. The homozygous mutation was confirmed by Sanger sequencing in the proband and carrier status was confirmed in both parents and his unaffected sibling. According to online and in-house minor allele frequency (MAF) databases, this is the first COL27A1 mutation reported in the European population. Additional screening of healthy Greek-Cypriot individuals was thus performed, which did not reveal any additional carriers in the population for the variant in question.