Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics.

PURPOSE: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. METHODS: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. RESULTS: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. CONCLUSION: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

First report of OPA1 screening in Greek patients with autosomal dominant optic atrophy and identification of a previously undescribed OPA1 mutation.

PURPOSE: To describe the genotype-phenotype correlation in four Greek pedigrees with autosomal dominant optic atrophy (ADOA) and OPA1 mutations. METHODS: Seven patients from four unrelated families (F1, F2, F3, F4) were clinically assessed for visual acuity, color vision, ptosis, afferent pupillary defects, and visual fields and underwent orthoptic assessment, slit-lamp biomicroscopy, and fundus examination to establish their clinical status. Genomic DNA was extracted from peripheral blood samples from all participants. The coding region (exons 1-28), including the intron-exon boundaries of the OPA1 gene, was screened in the probands of the four families, as well as in seven additional family members (four affected and three unaffected) with PCR and direct DNA sequencing. RESULTS: All patients presented bilateral decrease in best-corrected visual acuity and temporal pallor of the optic disc. The visual fields of the adult patients showed characteristic scotomata. Other signs were present in some patients such as decreased color discrimination and a gray crescent within the neuroretinal rim. After the OPA1 gene was sequenced, a previously undescribed heterozygous splice-site mutation c.784-1G>T in intron 7 was detected in family F2. In families F1, F3, and F4, a previously reported in-frame deletion c.876_878delTGT/p.(Val294del), the frameshift c.2366delA/p.(Asn789Metfs*11), and splice-site c.1140+5G>C mutations were detected, respectively. CONCLUSIONS: This is the first report of molecular characterization of Greek patients with ADOA. Our findings provide additional information regarding the genotype-phenotype correlation and establish the role of the OPA1 gene in Greek patients with ADOA.

Wolfram Syndrome: A case report of two sisters Wolfram Syndrome: Case report of two sisters.

PURPOSE: To present a case of two siblings with optic atrophy associated with Wolfram Syndrome. OBSERVATIONS: Two young adult siblings presented with serious bilateral loss of vision and dyschromatopsia established in early adolescence. They were referred with a presumed diagnosis of Leber's Hereditary Optic Neuropathy. At baseline, visual acuity was 20/400 in the right eye and 20/200 in the left eye in patient A and 20/200 in both eyes in patient B, color perception tested with pseudo-isochromatic plates was 0/17 in each eye, optic discs were pale, visual field testing revealed diffuse scotomas bilaterally while electrophysiology showed delayed prominent positive deflection (P100) values in both patients. Personal history revealed Type 1 diabetes mellitus since early childhood. Patients were lost to follow-up and presented 4 years later with significant VA decrease (<20/400) and suspected hearing loss. At that point, genetic testing revealed a pathogenic variation in the WFS1 gene thus confirming the diagnosis of Wolfram syndrome. Treatment with idebenone was proposed, to which only one of the siblings agreed. The other patient remained under observation, as no known treatment for optic atrophy in Wolfram syndrome exists to date. CONCLUSIONS AND IMPORTANCE: Wolfram syndrome is a rare neurodegenerative genetic disease associated with diabetes mellitus, optic atrophy and deafness. Careful and detailed medical and family history led to appropriate testing that confirmed the diagnosis of Wolfram syndrome. To this day, there is no definite treatment for this disease, but the experimental use of idebenone has been suggested to improve visual function. Genetic testing of family members and offspring of patients is strongly recommended.

Bilateral adrenocortical carcinoma in a patient with multiple endocrine neoplasia type 1 (MEN1) and a novel mutation in the MEN1 gene.

The incidence of adrenal involvement in MEN1 syndrome has been reported between 9 and 45%, while the incidence of adrenocortical carcinoma (ACC) in MEN1 patients has been reported between 2.6 and 6%. In the literature data only unilateral development of ACCs in MEN1 patients has been reported. We report a 31 years-old female MEN1-patient, in whom hyperplasia of the parathyroid glands, prolactinoma, non functioning pancreatic endocrine carcinoma and functioning bilateral adrenal carcinomas were diagnosed. Interestingly, a not previously described in the literature data, novel germline mutation (p.E45V) in exon 2 of MEN1 gene, was detected. The association of exon 2 mutation of the MEN1 gene with bilateral adrenal carcinomas in MEN1 syndrome, should be further investigated.

Donor-derived breast cancer in a bone marrow transplantation recipient.

We present the case of a young lady who had been treated for acute myelocytic leukemia at the age of 14 by means of allogeneic bone marrow transplantation, the donor being her sister. At the age of 28 she underwent modified radical mastectomy for invasive breast adenocarcinoma. Genetic analysis revealed chimeric cellular populations on both the tumour and normal tissues of the patient with preponderance of donor-derived cells. We conclude that the patient's epithelia had been repopulated by donor-derived hemopoietic stem cells which gave rise to a malignant mammary neoplasm several years later. The donor remains healthy to date. This case adds weight to the theory of pluripotent normal and neoplastic stem cell histogenesis and emphasizes the pivotal role of supporting host stroma in carcinogenesis.

Novel germline mutations of the MEN1 gene in Greek families with multiple endocrine neoplasia type 1.

INTRODUCTION: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant hereditary disorder associated with mutations of the MEN1 gene and characterized by the combined occurrence of tumours of the parathyroid glands, the pancreatic islet cells and the anterior pituitary. AIM: To identify MEN1 gene mutations and characterize clinical manifestations in Greek patients with MEN1. PATIENTS AND METHODS: We studied four unrelated index patients with MEN1, 17 relatives and 100 control subjects. Among the relatives, seven were clinically and/or biochemically affected, while 10 were unaffected. DNA extraction, polymerase chain reaction (PCR) and direct sequencing of the MEN1 exons 2-10 and exon/intron boundaries were performed according to standard procedures. RESULTS: We identified novel MEN1 gene mutations in three out of four index patients (75%) and in all affected (100%) relatives. Novel mutations included: a frameshift mutation in exon 4 (c.684_685insG) at codon 229 (index patient A); a frameshift mutation in exon 8 (c.1160_1170dupAGGAGCGGCCG) involving codons 387-390 (index patient B); and a missense mutation in exon 4 (c.776T > C), which substitutes leucine with proline at codon 259 (L259P) (index patient C). In the fourth index patient, a common polymorphism (D418D) was detected. CONCLUSIONS: This is the first report to reveal a high prevalence of novel MEN1 gene mutations among Greek MEN1 patients with apparent absence of genotype-phenotype correlation. Because of the small number of patients examined, the high prevalence detected might be a chance phenomenon.

G1738R is a BRCA1 founder mutation in Greek breast/ovarian cancer patients: evaluation of its pathogenicity and inferences on its genealogical history.

We have performed screening in 287 breast/ovarian cancer families in Greece which has revealed that approximately 12% (8/65) of all index patients-carriers of a deleterious mutation in BRCA1 and BRCA2 genes, contain the base substitution G to A at position 5331 of BRCA1 gene. This generates the amino acid change G1738R for which based on a combination of genetic, in silico and histopathological analysis there are strong suggestions that it is a causative mutation. In this paper, we present further evidence suggesting the pathogenicity of this variant. Forty breast/ovarian cancer patients were reported in 11 Greek families: the above eight living in Greece, two living in Australia and one in USA, all containing G1738R. Twenty of these patients were screened and were all found to be carriers of the same base substitution. In addition, we have detected the same base change in five breast/ovarian cancer patients after screening 475 unselected patient samples with no apparent family history. The mean age of onset for all the above patients was 39.4 and 53.6 years for breast and ovarian cancer cases, respectively. A multi-factorial likelihood model for classification of unclassified variants in BRCA1 and BRCA2 developed previously was applied on G1738R and the odds of it being a deleterious mutation was estimated to be 11470:1. In order to explain the prevalence of this mutation mainly in the Greek population, its genealogical history was examined. DNA samples were collected from 11 carrier families living in Greece, Australia and USA. Screening of eight intragenic SNPs, three intragenic and seven extragenic microsatellite markers and comparison with control individuals, suggested a common origin for the mutation while the time to its most recent common ancestor was estimated to be 11 generations (about 275 years assuming a generational interval of 25 years) with a 1-lod support interval of 4-24 generations (100-600 years). Considering the large degree of genetic heterogeneity in the Greek population, the identification of a frequent founder mutation greatly facilitates genetic screening.

Case series: Pyramidal cataracts, intact irides and nystagmus from three novel PAX6 mutations.

PURPOSE: To investigate the association between novel PAX6 mutations to bilateral anterior pyramidal congenital cataracts (APyC), complete and intact irides, and nystagmus. OBSERVATIONS: This is a retrospective observational case series in a multi-center setting with genetic testing. Three female patients were diagnosed with bilateral APyC, intact irides and nystagmus. Genetic testing identified the three patients had novel missense mutations in PAX6 - c.128C > T; p.Ser43Phe (S43F), c. 197T > A; p.Ile66Asn (I66N) and c.781C > G; p.Arg261Gly (R261G). CONCLUSIONS AND IMPORTANCE: This study demonstrates a novel phenotype of bilateral APyC, intact irides, and nystagmus in whom genetic testing for PAX6 identified novel missense mutations (S43F, I66N, R261G) in highly conserved DNA-binding domains. Implications of understanding why the iris is present in these cases is discussed.

Corrigendum to "Mutation Spectrum of the ABCA4 Gene in a Greek Cohort with Stargardt Disease: Identification of Novel Mutations and Evidence of Three Prevalent Mutated Alleles".

[This corrects the article DOI: 10.1155/2018/5706142.].

MT-ND5 Mutation Exhibits Highly Variable Neurological Manifestations at Low Mutant Load.

Mutations in the m.13094T>C MT-ND5 gene have been previously described in three cases of Leigh Syndrome (LS). In this retrospective, international cohort study we identified 20 clinically affected individuals (13 families) and four asymptomatic carriers. Ten patients were deceased at the time of analysis (median age of death was 10years (range: 5·4months-37years, IQR=17·9years). Nine patients manifested with LS, one with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), and one with Leber hereditary optic neuropathy. The remaining nine patients presented with either overlapping syndromes or isolated neurological symptoms. Mitochondrial respiratory chain activity analysis was normal in five out of ten muscle biopsies. We confirmed maternal inheritance in six families, and demonstrated marked variability in tissue segregation, and phenotypic expression at relatively low blood mutant loads. Neuropathological studies of two patients manifesting with LS/MELAS showed prominent capillary proliferation, microvacuolation and severe neuronal cell loss in the brainstem and cerebellum, with conspicuous absence of basal ganglia involvement. These findings suggest that whole mtDNA genome sequencing should be considered in patients with suspected mitochondrial disease presenting with complex neurological manifestations, which would identify over 300 known pathogenic variants including the m.13094T>C.

Structural analysis and expression profile of a novel gene on chromosome 5q23 encoding a Golgi-associated protein with six splice variants, and involved within the 5q deletion of a Ph(-) CML patient.

We have identified a novel gene, upstream of the cytokine gene cluster region in 5q23-31, residing within one of the most common deleted segments associated with MDS. The novel gene exhibits significant alternative splicing generating at least six splice variants encoding four putative proline-rich protein isoforms, one of which is Golgi-associated. The gene is ubiquitously expressed and conserved among species with the C. elegans homologue being the most interesting, since it resides within an operon with two other genes, phospholipase D and dishevelled, a member of the Wnt pathway, suggesting a functional association. In addition, the novel gene and other key regulatory genes of the region, such IL3, Ril, AF5q31 and TCF-1, were found to be deleted in an atypical CML case, thus underscoring the significance of this subregion in the leukemogenesis process.

X-linked adrenoleukodystrophy: are signs of hypogonadism always due to testicular failure?

We present the clinical and hormonal findings of a young male with X-linked adrenoleukodystrophy (X-ALD), with special emphasis on the biochemical and clinical pattern of hypogonadism. A patient, with primary adrenal insufficiency since the age of 5 years, developed progressive neurological symptoms at the age of 29. Diagnosis of X-ALD was established by elevated serum very long chain fatty acids (VLCFAs) and genetic testing. His sexual body hair was sparse. Hormonal investigations revealed normal testosterone and inappropriately elevated LH levels. Androgen receptor gene analysis was negative for mutations or polymorphic variants associated with decreased receptor activity. Signs of hypogonadism in patients with confirmed X-ALD are not exclusively due to primary testicular failure. Tissue specific androgen resistance represents an alternative possibility. Since no loss-of-function mutations were detected in the androgen receptor, it is speculated that the patient's androgen resistance could be part of a functional defect mediated through VLCFA accumulation at the testosterone receptor and/or post-receptor levels.

Novel mutations of the HRAS gene and absence of hotspot mutations of the BRAF genes in oral squamous cell carcinoma in a Greek population.

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer in the world. The phosphatidylinositol 3 kinase (PI3K) signalling pathway has been reported to play an important role in OSCC. Since we have previously detected absence of hotspot PIK3CA gene mutations in the Greek population, we hypothesized that BRAF or HRAS may be activated as upstream effectors of the pathway. Furthermore, the status of the HRAS and BRAF mutations in OSCC has never been assessed before in the Greek population. Eighty-six primary paraffin-embedded tumors were screened for BRAF and HRAS hotspot mutations. In HRAS, two hotspot mutations in codon 12 (2.3%) and eight new genetic alterations were detected (8.6% overall). One new missense mutation, Alanine53Valine (Ala53Val), one silent mutation, two mutations in the 5'UTR region and four mutations in intron 1 were detected. No hotspot mutations in Braf were found. A new silent mutation/polymorphism T1803C was detected at a percentage of 30%. This study is the first to report HRAS mutations in the Greek population. The results suggest that RAS is an important member of the PI3K signalling pathway and may play a role in the tumorigenesis of OSCC.

Identification of two novel mutations in the RET proto-oncogene in the same family.

BACKGROUND: Activating germline mutations of the RET gene cause multiple endocrine neoplasia type 2 and familial medullary thyroid carcinoma (FMTC), conditions that are inherited in an autosomal dominant manner. In addition, somatic RET mutations have been identified in a variable proportion (about 30-70%) of sporadic (nonfamilial) MTC cases. METHODS: We describe a Greek family with two novel likely pathogenic sequence variants of the RET gene. The first is a C to T transition at position 2458 (c.2458C>T) that causes an arginine to cysteine substitution (p.R820C) in exon 14 in the intracellular region of the kinase. This sequence variant was identified in an apparently healthy woman who had a recently deceased sister with confirmed aggressive MTC (age of onset 37 years). To assess the pathogenicity of this novel missense sequence variant, screening was performed on all available relatives: her two sons, the mother, and a second sister, including an MTC tumor sample from the deceased sister of the proband. At the time of the investigation, no clinical symptoms suggestive of multiple endocrine neoplasia type 2 or MTC were present in any of the individuals screened. RESULTS: The c.2458C>T transition was found in one son, the living sister, and the mother. Interestingly, it was not present in the tumor sample from the deceased sister. Instead, an in-frame deletion of 54 nt in exon 10 resulting in a protein missing 18 amino acids from I590 to G608 (c.1766_1819del 54) was found. Both genetic alterations were present in heterozygous state. CONCLUSIONS: These data suggest that the novel in-frame deletion was the disease-causing mutation in the deceased sister. The effect of the 2458C>T mutation on the activity of the kinase is under investigation.

Absence of hotspot mutations in exons 9 and 20 of the PIK3CA gene in human oral squamous cell carcinoma in the Greek population.

OBJECTIVE: Phosphatidylinositol-3 kinases (PI3K) are a group of heterodimeric lipid kinases that regulate many cellular processes. Recent studies have reported high frequencies of somatic hotspot mutations in the phosphatidylinositol-3 kinase catalytic alpha (PIK3CA) gene, which encodes for one of these kinases, in several human solid tumors, including oral squamous cell carcinoma (OSCC). The aim of this study was to determine the frequency of hotspot mutations in exons 9 and 20 of the PIK3CA gene in OSCC in the Greek population. STUDY DESIGN: Eighty-six formalin-fixed and paraffin-embedded primary tumor specimens were analyzed by direct genomic DNA sequencing. Chi-square was used for statistical analysis. RESULTS: No hotspot mutations were detected in any of the samples. Two intronic polymorphisms IVS8 and IVS9 were detected, mainly in patients with cancer of the buccal mucosa and lower gingival and alveolus respectively. CONCLUSIONS: PIK3CA hotspot mutations are unlikely to play a major role in the pathogenesis of OSCC in the Greek population.

A novel germline mutation of the VHL gene in a Greek family with Von Hippel-Lindau disease.

Von Hippel-Lindau disease (VHL) is an autosomal dominant disorder, caused by mutations of the VHL gene showing a strong genotype-phenotype correlation. The present report concerns a 16-year-old girl with VHL (retinal, spinal cord and cerebellar haemangioblastomas and pancreatic cysts), her father (retinal and spinal cord haemangioblastomas) and the phenotypically healthy mother and younger brother and sister. DNA extraction, PCR and direct sequencing of the VHL entire coding and intronic flanking sequences, were performed according to standard procedures. In the index patient and her father a novel heterozygous germline was identified; nonsense mutation (p.145X) in exon 2 of VHL, leading to a truncated VHL protein lacking the last 66 amino acids. This is the first report of a novel VHL mutation in patients with VHL associated with haemangioblastomas and pancreatic cysts but not renal cell carcinoma.

Multiple endocrine neoplasia type 2A in two families with the familial medullary thyroid carcinoma associated G533C mutation of the RET proto-oncogene.

INTRODUCTION: Multiple endocrine neoplasia type 2A (MEN2A) is an autosomal dominant hereditary disorder, associated with a cluster of germline gain-of-function mutations of the RET proto-oncogene (RET), mainly in exons 10-15. The G533C mutation in exon 8 of the RET is rare and has been mainly related to the familial medullary thyroid carcinoma. PATIENTS-METHODS: We describe the RET G533C mutation in exon 8 of the RET in two unrelated female index patients, with MEN2A phenotype, consisting of pheochromocytoma which was the presenting feature and medullary thyroid carcinoma. In addition, 12 family members were also studied. DNA extraction, PCR, and sequencing of RET was performed in exons 7-19 and 21, following standard procedures. RESULTS: The mutation was found in both index patients and in 6 out of 12 family members (50%). Three of them were biochemically affected with histologically proven medullary thyroid carcinoma in two of them while there are no certain clues regarding the other three members as they declined further evaluation. CONCLUSION: Patients with MEN2A should be also searched in exon 8 while positive carriers of this mutation should be screened annually for pheochromocytoma or other components of the syndrome.

Greek BRCA1 and BRCA2 mutation spectrum: two BRCA1 mutations account for half the carriers found among high-risk breast/ovarian cancer patients.

127 Greek breast/ovarian cancer families were screened for germline BRCA1/2 mutations by dHPLC followed by direct sequencing. Our results indicated 16 and 5 breast/ovarian cancer families bearing deleterious mutations in the BRCA1 and BRCA2 genes, respectively. Two novel BRCA2 germline mutations (G4X and 3783del10) are reported here for the first time. Subsequent compilation of our present findings with previously reported mutation data reveals that in a total of 287 Greek breast/ovarian cancer families, 46 and 13 carry a deleterious mutation in BRCA1 and BRCA2, respectively. It should be noted that two BRCA1 mutations, 5382insC and G1738R, both located in exon 20, account for 46% of the families found to carry a mutation. Based on our mutation analysis results, we propose here a hierarchical, cost-effective BRCA1/2 mutation screening protocol for individuals of Greek ethnic origin. The suggested protocol can impact on the clinical management of breast-ovarian cancer families on a national healthcare system level.