Genotype-phenotype associations in a large PTEN Hamartoma Tumor Syndrome (PHTS) patient cohort

Background: Pathogenic PTEN germline variants cause PTEN Hamartoma Tumor Syndrome (PHTS), a rare disease with a variable genotype and phenotype. Knowledge about these spectra and genotype-phenotype associations could help diagnostics and potentially lead to personalized care. Therefore, we assessed the PHTS genotype and phenotype spectrum in a large cohort study. Methods: Information was collected of 510 index patients with pathogenic or likely pathogenic (LP/P) PTEN variants (n = 467) or variants of uncertain significance. Genotype-phenotype associations were assessed using logistic regression analyses adjusted for sex and age.Results: At time of genetic testing, the majority of children (n = 229) had macrocephaly (81%) or developmental delay (DD, 61%), and about half of the adults (n = 238) had cancer (51%), macrocephaly (61%), or cutaneous pathology (49%). Across PTEN, 268 LP/P variants were identified, with exon 5 as hotspot. Missense variants (n = 161) were mainly located in the phosphatase domain (PD, 90%) and truncating variants (n = 306) across all domains. A trend towards 2 times more often truncating variants was observed in adults (OR = 2.3, 95%CI = 1.5-3.4) and patients with cutaneous pathology (OR = 1.6, 95%CI = 1.1-2.5) or benign thyroid pathology (OR = 2.0, 95%CI = 1.1-3.5), with trends up to 2-4 times more variants in PD. Whereas patients with DD (OR = 0.5, 95%CI = 0.3-0.9) or macrocephaly (OR = 0.6, 95%CI = 0.4-0.9) had about 2 times less often truncating variants compared to missense variants. In DD patients these missense variants were often located in domain C2.Conclusion: The PHTS phenotypic diversity may partly be explained by the PTEN variant coding effect and the combination of coding effect and domain. PHTS patients with early-onset disease often had missense variants, and those with later-onset disease often truncating variants

Recording on genetic disorders and syndromes in the Greek-Cypriot population

The clinical genetics services were established in Cyprus in 1994. Patients were referred to this clinic from all over Cyprus for investigation of possible genetic disorders or for genetic counseling for a known or unknown diagnosis. Patients were referred from physicians of various specialties and sometimes from other health professionals. The population referred was mainly of Greek-Cypriot origin, but gradually as patients of other origin increased we included also patients of mixed descent, Turkish-Cypriots and others. A registry was created which reports on several demographic data and on medical data such as reason for referral and diagnosis. Reasons for referrals included all common indications such as: mental retardation, developmental delay, dysmorphic features, congenital anomalies, history of cancer, abnormalities of the skeleton, hearing of vision problems, history of infertility, recurrent miscarriages, consanguinity and some rare ones. This study reports on the diversity of diagnosis established for 3400 individuals in the years 1995-2009. 1007 patients were aetiologically diagnosed while 1099 were not. It has been shown that more than 50% of individuals remain without an aetiological diagnosis, a result that is in accordance with similar studies. Among the list of diagnosis it has been proved that monogenic disorders represented the largest category of diagnosis while chromosomal aberrations were less frequent. Among the monogenic disorders the largest group was neurofibromatosis type I while the commonest chromosomal abnormality was trisomy 21. 96 patients were reported with multifactorial congenital anomalies and exposures accounted for 24 patients. Congenital anomalies are probably under-represented. We assume that the main reason for this is the fact that there is no consistency in reporting congenital anomalies to a registry in Cyprus. Also in Cyprus there is a well established prenatal surveillance program (ultrasound and NT measurement of the first trimester, scan anomaly of the second trimester and biochemical markers) which has a very high uptake. Therefore we consider that many congenital anomalies are detected prenatally and several pregnancies are terminated. There is no communication of these outcomes to the genetics clinic or to a national registry. Apart from recording on “common” genetic disorders and syndromes, in the population of this study, several rare and very rare syndromes were recognized. It was also noted that the “Founders’ effect” is found in several areas or communities accounting for the high frequency of disorders such as Sandhof disease among the Maronites, Freidreich ataxia in Paphos etc. Genetic counseling is a new service established in Cyprus by the genetics clinic. This service is organized and offered based on the well established principles of practice for genetic counseling, in the western countries: confidentiality, non-directiveness and autonomy. Genetic counseling is now increasingly demanded and well accepted in Cyprus. 986 individuals were referred for genetic counseling for various common and rare indications with the exception of Thalassemia screening which is managed by the National Thalassemia center. Presymptomatic genetic counseling and testing is offered to individuals for hereditary cancer syndromes as well as for specific neurological disorders of late onset and the referrals for this service accounted for 290 individuals. In conclusion in this study we are recording on genetic disorders and syndromes in the greek-cypriot population, as these were diagnosed or represented through the clinical evaluation performed within the Clinical Genetics service, in the years 1995-2009.Η διδακτορική αυτή διατριβή είχε σαν στόχο να καταγράψει τα γενετικά νοσήματα στον Ελληνοκυπριακό πληθυσμό όπως αυτά παρουσιάστηκαν μέσα από την πορεία του ιατρείου κλινικής γενετικής από το 1995-2009 (πρώτο εξάμηνο). Μέσα από την καταγραφή αυτή επιχειρήθηκε μια αναδρομή στη διαδρομή του ιατρείου κλινικής γενετικής καθώς και συνολικά στην πορεία εξέλιξης της ιατρικής γενετικής στην Κύπρο. Το ιατρείο κλινικής γενετικής ιδρύθηκε τον Ιούλιο του 2004 και έχει έδρα στο Νοσοκομείο Αρχιεπίσκοπος Μακάριος ΙΙΙ και στο Ινστιτούτο Νευρολογίας και Γενετικής στη Λευκωσία της Κύπρου. Λειτουργεί σαν ιατρείο αναφοράς για όλα τα γενετικά νοσήματα και για ολόκληρη την Κύπρο καθώς είναι το μοναδικό στο είδος του. Παραπέμπονται ασθενείς όλων των ηλικιών, νεογνά, παιδιά, ενήλικες, ζεύγη και οικογένειες για οποιαδήποτε ένδειξη γενετικής αξιολόγησης καθώς και για οποιαδήποτε ένδειξη γενετικής συμβουλευτικής. Εξαίρεση αποτελεί η θαλασσαιμία για την οποία οι υπηρεσίες προσφέρονται από το αρμόδιο εθνικό κέντρο θαλασσαιμίας. Στις αιτίες παραπομπής καταγράφτηκαν οι πιο κάτω: νοητική υστέρηση, ψυχοκινητική καθυστέρηση, παλινδρόμηση, δυσμορφικά χαρακτηριστικά, συγγενείς ανωμαλίες στη διάπλαση, διαταραχές στη σωματική ανάπτυξη κ.α. Οι ειδικοί που παρέπεμψαν ασθενείς στο ιατρείο κλινικής γενετικής προέρχονται από όλες σχεδόν τις ιατρικές ειδικότητες, καθώς επίσης και από άλλους επαγγελματίες της υγείας (λογο-,εργο-, φυσιο-θεραπευτές) και από εκπαιδευτικούς της ειδικής εκπαίδευσης. Για την εργασία αυτή συστάθηκε αρχείο στο οποίο καταχωρήθηκαν όλοι οι ασθενείς που εξετάσθηκαν στο ιατρείο κλινικής γενετικής καθώς και τα άτομα που προσήλθαν για γενετική συμβουλευτική. Από πλευράς δημόσιας υγείας η σύσταση και συντήρηση ενός αρχείου για γενετικά νοσήματα και συγγενείς ανωμαλίες έχει συγκεκριμένες προϋποθέσεις και είναι απαραίτητη για την εφαρμογή προγραμμάτων αντιμετώπισης και πρόληψης. Ο πληθυσμός που μελετήθηκε συμπεριλάμβανε 3400 ασθενείς που παραπέμφθηκαν στο ιατρείο κλινικής γενετικής από το 1995-2009. Η συντριπτική πλειοψηφία αφορούσε Ελληνοκύπριους, πλην όμως καταγράφονται τα νοσήματα που διαγνώσθηκαν και σε παιδιά από μικτούς γάμους καθώς και σε μικρό αριθμό Τουρκοκυπρίων και άλλων με σπάνια γενετικά νοσήματα. Από την καταγραφή των αποτελεσμάτων της διαγνωστικής προσέγγισης προέκυψαν τα πιο κάτω: 986 άτομα (ποσοστό 29%) προσήλθαν στο ιατρείο γενετικής για γενετική συμβουλευτική για ποικίλους λόγους. 80 άτομα (ποσοστό 2%) δεν παρουσίαζαν λόγο διερεύνησης (φυσιολογικές παραλλαγές). 1007 ασθενείς (ποσοστό 29%) διαγνώσθηκαν με χρωμοσωμικό, μονογονιδιακό νόσημα η άλλο γνωστό γενετικό νόσημα. 8 ασθενείς είχαν μιτοχονδριακό νόσημα. 24 ασθενείς είχαν εκτεθεί σε τερατογόνα και 96 ασθενείς είχαν συγγενείς ανωμαλιες πολυπαραγοντικής αιτιολογίας. 1199 ασθενείς (ποσοστό 31%) παρέμειναν χωρίς αιτιολογική διάγνωση. Συνεπώς συγκριτικά η διαγνωστική προσπέλαση πέτυχε να δώσει αιτιολογική διάγνωση σε λιγότερους από τους μισούς ασθενείς που έτυχαν αιτιολογικής διερεύνησης. Το ποσοστό αυτό δεν αποκλείνει ιδιαίτερα από παρόμοιες μελέτες σε άλλους πληθυσμούς και ιατρεία κλινικής γενετικής και καταδεικνύει τις δυσκολίες στην αιτιολογική διερεύνηση των ασθενών με γενετικά νοσήματα και στην αποτύπωση σπάνιων φαινοτύπων παρά την μεγάλη πρόοδο της γενετικής στις μέρες μας. Οι διαγνώσεις που τέθηκαν κάλυψαν ένα εκτεταμένο φάσμα χρωμοσωμικών και μονογονιδιακών νοσημάτων. ...........................................................................................................

Mild Phenotype in a Patient with a De Novo 6.3 Mb Distal Deletion at 10q26.2q26.3

We report on a 29-year-old Greek-Cypriot female with a de novo 6.3 Mb distal 10q26.2q26.3 deletion. She had a very mild neurocognitive phenotype with near normal development and intellect. In addition, she had certain distinctive features and postural orthostatic tachycardia. We review the relevant literature and postulate that certain of her features can be diagnostically relevant. This report illustrates the powerful diagnostic ability of array-CGH in the elucidation of relatively mild phenotypes

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

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

International audienceMultiple 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

Novel GLA Deletion in a Cypriot Female Presenting with Cornea Verticillata

Fabry disease is an X-linked lysosomal storage disorder resulting from a deficiency of the hydrolytic enzyme α-galactosidase A (α-Gal-A). It is characterized by progressive lysosomal accumulation of globotriaosylceramide (Gb3) and multisystem pathology, affecting the skin, nervous and cerebrovascular systems, kidneys, and heart. Heterozygous females typically exhibit milder symptoms and a later age of onset than males. Rarely, they may be relatively asymptomatic throughout a normal life span or may have symptoms as severe as those observed in males with the classic phenotype. We report on a 17-year-old female in whom cornea verticillata was found during a routine ophthalmological examination but with no other clinical symptoms. Leucocyte α-galactosidase activity was within the overlap range between Fabry heterozygotes and normal controls. Sanger sequencing of the GLA gene failed to reveal any pathogenic variants. Multiplex Ligation-dependent Probe Amplification (MLPA) analysis revealed a deletion of exon 7. Using a long-range PCR walking approach, we managed to identify the deletion breakpoints. The deletion spans 1182 bp, with its 5′ end located within exon 6 of the GLA gene and its 3′ end located 612 bp downstream of exon 7. This finding represents a novel deletion identified in the first reported Cypriot female carrier of Fabry disease

Position effect, cryptic complexity, and direct gene disruption as disease mechanisms in de novo apparently balanced translocation cases.

The majority of apparently balanced translocation (ABT) carriers are phenotypically normal. However, several mechanisms were proposed to underlie phenotypes in affected ABT cases. In the current study, whole-genome mate-pair sequencing (WG-MPS) followed by Sanger sequencing was applied to further characterize de novo ABTs in three affected individuals. WG-MPS precisely mapped all ABT breakpoints and revealed three possible underlying molecular mechanisms. Firstly, in a t(X;1) carrier with hearing loss, a highly skewed X-inactivation pattern was observed and the der(X) breakpoint mapped ~87kb upstream an X-linked deafness gene namely POU3F4, thus suggesting an underlying long-range position effect mechanism. Secondly, cryptic complexity and a chromothripsis rearrangement was identified in a t(6;7;8;12) carrier with intellectual disability. Two translocations and a heterozygous deletion disrupted SOX5; a dominant nervous system development gene previously reported in similar patients. Finally, a direct gene disruption mechanism was proposed in a t(4;9) carrier with dysmorphic facial features and speech delay. In this case, the der(9) breakpoint directly disrupted NFIB, a gene involved in lung maturation and development of the pons with important functions in main speech processes. To conclude, in contrast to familial ABT cases with identical rearrangements and discordant phenotypes, where translocations are considered coincidental, translocations seem to be associated with phenotype presentation in affected de novo ABT cases. In addition, this study highlights the importance of investigating both coding and non-coding regions to decipher the underlying pathogenic mechanisms in these patients, and supports the potential introduction of low coverage WG-MPS in the clinical investigation of de novo ABTs

Family 1 results with t(1;7) translocation.

<p>A) Family 1 pedigree showing the proband with intellectual disability, psychomotor delay and epilepsy, as well as the non-affected mother and father. The t(1;7)(p36.1;q22) translocation is maternally inherited. B) Ideograms showing the normal and derivative (der) chromosomes (chr) 1 and 7 (not to scale). Genetic material from chr1 and chr7 is shown with a solid, orange and dotted, blue frame line, respectively. The approximate breakpoint positions on 1p36.1 and 7q22 are indicated by arrows. C) UPD7 analysis results from one of the informative microsatellite markers (D71824) in the affected proband and non-affected parents; by comparing the peak sizes between all family members, normal biparental inheritance was concluded. D) Quantitative Real-Time PCR results demonstrating the paternal inheritance of the chr3 duplication predicted from the structural variant analysis in the affected proband.</p

Accurate Breakpoint Mapping in Apparently Balanced Translocation Families with Discordant Phenotypes Using Whole Genome Mate-Pair Sequencing

<div><p>Familial apparently balanced translocations (ABTs) segregating with discordant phenotypes are extremely challenging for interpretation and counseling due to the scarcity of publications and lack of routine techniques for quick investigation. Recently, next generation sequencing has emerged as an efficacious methodology for precise detection of translocation breakpoints. However, studies so far have mainly focused on <i>de novo</i> translocations. The present study focuses specifically on familial cases in order to shed some light to this diagnostic dilemma. Whole-genome mate-pair sequencing (WG-MPS) was applied to map the breakpoints in nine two-way ABT carriers from four families. Translocation breakpoints and patient-specific structural variants were validated by Sanger sequencing and quantitative Real Time PCR, respectively. Identical sequencing patterns and breakpoints were identified in affected and non-affected members carrying the same translocations. <i>PTCD1</i>, <i>ATP5J2-PTCD1</i>, <i>CADPS2</i>, and <i>STPG1</i> were disrupted by the translocations in three families, rendering them initially as possible disease candidate genes. However, subsequent mutation screening and structural variant analysis did not reveal any pathogenic mutations or unique variants in the affected individuals that could explain the phenotypic differences between carriers of the same translocations. In conclusion, we suggest that NGS-based methods, such as WG-MPS, can be successfully used for detailed mapping of translocation breakpoints, which can also be used in routine clinical investigation of ABT cases. Unlike <i>de novo</i> translocations, no associations were determined here between familial two-way ABTs and the phenotype of the affected members, in which the presence of cryptic imbalances and complex chromosomal rearrangements has been excluded. Future whole-exome or whole-genome sequencing will potentially reveal unidentified mutations in the patients underlying the discordant phenotypes within each family. In addition, larger studies are needed to determine the exact percentage for phenotypic risk in families with ABTs.</p></div

Translocation junction sequences identified in each family.

<p>Translocation junction sequences to the base-pair level as identified by mate-pair sequencing and verified by Sanger sequencing in A) family 1 with t(1;7)(p36.1;q22), B) family 2 with t(7;8)(q32;q24.13), C) family 3 with t(4;10)(q35;q11.2), and D) family 4 with t(1;20)(p35.3;q13.3). Translocation junction sequences (middle line) and matching reference sequences (top and bottom lines) are shown with a different colour depending on the chromosome involved (chr1-orange; chr4-purple; chr7-blue; chr8-red; chr10-yellow; chr20-green). Microhomology observed at the translocation breakpoint sites is highlighted in yellow, deleted sequences around the breakpoints are underlined, duplicated sequences are in bold, capital letters, and inserted sequences not aligning to either chromosome are in bold, lower-case letters.</p