2010 2

Editor in Chief
Maria Puiu Department of Medical Genetics University of Medicine and Pharmacy Victor Babes Timisoara, Romania Tel/fax: +40 256 220479 E-mail: maria_puiu@umft.ro
Scientific board
Adrian C. Nicolescu, Kingston, Canada, Adrian Lupescu, Tuebingen, Germany Alex Almasan, Cleveland, Ohio, USA Alice C. Ceacareanu, New York, USA Aurelia Szekely, Zalau, Romania Bogdan Iorga, Kln, Germany Calin Popoiu, Timisoara, Romania Cassian Sitaru, Freiburg, Germany Claudia Cosmineanu-Halaby, New York, USA Constantin Ilie, Timisoara, Romania Corin Badiu, Bucuresti, Romania Cristina Isar, Bucuresti, Romania Cristina Rusu, Iasi, Romania Cristina Skrypnyk, Oradea, Romania Curocichin Ghenadie, Chisinau, Moldavia Danalache Bogdan Alexandru, Montreal, Canada Dorin Bogdan Borza, Nashville, USA Dumitru Andrei Iacobas, New York, USA Dumitru Moldovan, Tg. Mures, Romania Emilia Severin, Bucuresti, Romania Eugen Boia, Timisoara, Romania Gabriela Anton, Bucuresti, Romania Gabriela Doros, Timisoara, Romania George-Lucian Moldovan, Boston, USA Gertrude-Emilia Costin, Maryland, USA Igor Cemortan, Chisinau, Moldavia Ionel Sandovici, Cambridge, UK Ionela Pascanu, Tg. Mures, Romania Katalin Csep, Tg. Mures, Romania Leonard Girnita, Stockholm, Suedia Marcel Ionita, Birmingham, USA Margit Serban, Timisoara, Romania Maria Puiu, Timisoara, Romania Marius Bembea, Oradea, Romania Mihai D. Niculescu, Chapel Hill, USA Minodora Dobreanu, Tg. Mures, Romania Mircea Covic, Iasi, Romania Mircea Ivan, Bloomington, USA Natalia Cucu, Bucuresti, Romania Peter Manu, New York, USA Sanda Marchian, Sibiu, Romania Sergiu P. Pasca, California, USA Sorin Ursoniu, Timisoara, Romania Tams Jnossy, Szeged, Hungary Tudor Oprea, New Mexico, USA Valerica Belengeanu, Timisoara, Romania Victor I. Pop, Cluj-Napoca, Romania Vlad Gorduza, Iasi, Romania
Assistant Editors
Cristina Rusu Cristina Skrypnyk Vlad Gorduza
Secretary
Vlad L. David
Editorial Board
Dorica Dan Mihai Gafencu Diter Atasie Dorina Stoicanescu Monica Stoian Vasilica Plaiasu Cristina Popa
Honorary Members
Sgolne Aym, France Gheorghe Benga, Romnia
Romanian Journal of Rare Diseases ISSN 2068 5882 rjrd@umft.ro www.rjrd.ro Publisher: Victor Babes Printing House Address: P-ta Eftimie Murgu 2, 300041 Timisoara, Romania Tel./fax: 0256 / 495 210 evb@umft.ro www.evb.umft.ro
ABOUT The Romanian Journal of Rare Diseases is an international journal addressing rare diseases and orphan drugs from the perspectives of basic and clinical genetics, molecular genetics, cytogenetics, epigenetics, population genetics, biotechnology, neurogenetics, cardiogenetics, oncogenetics, pharmacogenetics and related fields, by publishing original works, review articles, clinical reports and other contributions from all areas covered by The Romanian Journal of Rare Diseases. This publication is the international official journal of the National Committee for Rare Diseases, founded and started as part of the project of the Romanian Prader Willi Association, "The Norwegian-Romanian Partnership (NoRo) for progress in Rare Diseases" funded by the Norwegian Government granted by the Norwegian Cooperation Program for growth and sustainable development in Romania. The Romanian Prader Willi Association and the Victor Babes Publishing House, E. Murgu Square 2, 300041, Timisoara, tel./fax.0256220479, publish the journal quarterly
PURPOSE & AREA OF INTEREST For the journal are of interest articles from basic and clinical research. The journal publishes original articles, short reports, special communications, provided that they are based on adequate experimental evidence, clinical studies, case reports, images in rare diseases, letters to the editor, book reviews, reports of congresses and other articles that will be brought to Editorial Boards attention based on the publics for the journal. Editorials are published by invitation but we look forward to be offered such material from researchers with experience and results in the study of rare diseases. Requirements for publication in the Romanian Journal of Rare Diseases are in accordance with the requirements of "Uniform Requirements for Manuscripts Submitted to Biomedical Journals" 5th Edition. JAMA 1997, 277:927-934.
LEGAL DISCLAIMER The entire contents of the Romanian Journal of Rare Diseases are protected under international copyrights. The authors bare the entire responsibilities for the content of the article.
Romanian Journal of Rare Diseases (2010), 2
TABLE OF CONTENTS
TABLE OF CONTENTS Reflections on the ever changing face of ondines curse: a review of congenital central hypoventilation syndrome and of rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation Claudia Cosmineanu-Halaby and Mary Cataletto Multiple malformations in infant - case report T.Marcovici, I.Simedrea, L. Tunea, A.Militaru, O.Belei, D.Chiru, G.Brad, M.Puiu Laboratory strategies for diagnosis of Prader-Willi and Angelman syndromes Arsene Cosmin, Maria Puiu, Otilia Zarnescu, Alungulese Anca Loredana, Natalia Cucu Pathogenic gene responsible for the predisposition to Behets disease Cojocaru M, Inimioara Mihaela Cojocaru,Isabela Silosi Infertility due to genetic anomalies in both partners of the couple. Case report Atasie D, Chicea R, Ispasoiu F, Corina Ispasoiu
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16
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ANNOUNCEMENTS European Human Genetics Conference, May 28-31, 2011 - Amsterdam, Netherlands Genetics in Gastrointestinal and Liver Diseases, April 7-9, 2011 Cluj-Napoca, Romania MANUSCRIPT REQUIREMENTS 38
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REFLECTIONS ON THE EVER CHANGING FACE OF ONDINES CURSE: A REVIEW OF CONGENITAL CENTRAL HYPOVENTILATION SYNDROME AND OF RAPID-ONSET OBESITY WITH HYPOTHALAMIC DYSFUNCTION, HYPOVENTILATION, AND AUTONOMIC DYSREGULATION
Claudia Cosmineanu-Halaby and Mary Cataletto Winthrop University Hospital, NY, USA
..and so the water nymph cursed her unfaithful lover to a life of constant wakefulness. If he should fall asleep he would cease to breathe..German folk lore Abstract Currently referred to as Respiratory and Autonomic Disorders of Infancy, Childhood, and Adulthood (RADICA), these disorders form a group of respiratory control disorders with autonomic nervous system dysregulation (ANSD). This review is intended to update clinicians on the new guidelines and recommendations for the identification, differentiation and treatment of these two disorders. Key words: Congenital central hypoventilation syndrome, Respiratory and Autonomic Disorders of Infancy Introduction Once compared to the fateful story of Ondines curse, which condemned the unfaithful lover of a water nymph to a life where he would never sleep and breathe at the same time, we now realize that these central autonomic respiratory control syndromes are far more complex and involve additional organ systems than was previously known. Comparison to the mythologic tale has no relevance to todays understanding of these disorders. Currently referred to as Respiratory and Autonomic Disorders of Infancy, Childhood, and Adulthood (RADICA), these disorders form a group of respiratory control disorders with autonomic nervous system dysregulation (ANSD). Two distinct disorders emerge as having the most significant respiratory control deficits: Congenital Central Hypoventilation Syndrome (CCHS) and Rapid-Onset Obesity with Hypothalamic Dysfunction, Hypoventilation, and Autonomic Dysregulation (ROHHAD). 2 ROHHAD syndrome, first reported in 1965 described Late-onset Central Hypoventilation Syndrome with Hypothalamic Dysfunction (LO-CHS/HD) (1). Later in 1970 Mellins et al (2) reported the first case of congenital central alveolar hypoventilation (CCHS ). Although the clinical presentation of CCHS and ROHHAD overlap, ROHHAD distinguishes itself by exhibiting a wider spectrum of systemic involvement, a late onset of respiratory control deficit and lacks the characteristic CCHS mutation of PHOX2B gene. This review is intended to update clinicians on the new guidelines and recommendations for the identification, differentiation and treatment of these two disorders. Genetics In 2003, PHOX2B was found to be the diseasedefining gene for CCHS (5, 6). PHOX2B encodes a highly conservative homeodomain transcription factor that plays a key role in the development of autonomic nervous system (7,
8) and it is expressed only in several classes of differentiating neurons in both peripheral and central nervous system (9). In murine models, it was demonstrated that PHOX2B has a key role in autonomic nervous system reflex circuit development, by determining the cell fate of sympathetic, parasympathetis, or enteric neurons. There are two alanine-rich repeats in the Cterminus of the protein encoded by PHOX2B. The longer one, located on exon 3, is composed of 20 alanine residues. The majority of CCHSrelated PHOX2B mutations consist of an increased number of alanine in the poly-alanine repeat mutation (PARM) sequence from 20 to 24-335. There is a correlation between the size of the PHOX2B expanded allele and the severity of both the respiratory phenotype and associated symptoms. The longer alanine repeat sequence is associated with more severe symptoms (5,10). PHOX2B mutation was identified in a subgroup of patients that presented with central hypoventilation outside the newborn period, usually triggered by an environmental cofactorLate-Onset Central Hypoventilation syndrome (LO-CCHS)10. They usually have the fewest extra alanines (24-25) (10) and demonstrate a variable penetrance of the mutation. Mosaicism for the polyalanine expansion was found in parents of children with CCHS suggesting that not all CCHS-causing mutation occur de novo, 5-10% of PHOX2B mutations can be inherited from an unaffected parent5. Mosaic parents pass the same expanded allele to the affected child suggesting an authosomal dominant inheritance of CCHS and stable transmission of the PHOX2B mutation5. Non-polyalanine repeat mutations (NPARM), specifically: frameshift, nonsense and unisense mutations were described in 67 individuals with CCHS worldwide and were found to be associated with more severe phenotypes(11). Because of the heritability of the PHOX2B mutation, genetic counseling and parental 3
testing is essential for individuals diagnosed with CCHS(11). PHOX2B Screening Test, a clinically available method of detecting polyalanine repeat sequence is now widely used for prenatal diagnosis, family testing to detect mosaicism or disease, and diagnosis of individuals with relevant symptoms (12). Before 2003, when PHOX2B disease-defining gene for CCHS was discovered, many patients with ROHHAD were misdiagnosed due to clinical similarities between the two diseases. Patients with ROHHAD have no identified mutations in PHOX2B gene nor in NTRK2 or BDNF genes, two other genes suspected in relation with this syndrome based on their role in development and maintenance of neuronal populations involved in respiratory, food intake, and body weight control (5,12). Extensive genetic testing with high-resolution chromosome analysis, CGH, and subtelomeric FISH were negative in a subset of patients with ROHHAD (12). These findings suggest that alternative genes and pathways need to be considered. Respiratory Manifestations Described initially in a an infant boy who was cyanotic at nursery admission, during sleep, and with feeding, who had an attenuated ventilatory response and no change in mental status despite pCO2 values of 120-130mmHg1, CCHS was recognized to be a disorder of respiratory dysregulation. Patients with CCHS have monotonous respiratory rates awake and asleep, low tidal volumes, and profound alveolar hypoventilation mostly during the sleep (2). Due to the alveolar hypoventilation they become hypoxemic and hypercarbic but lack the normal ventilatory and arousal response to these endogenous challenges during sleep, and the perception of the asphyxia during wakefulness with and without exertion (2). However they maintain the ability to consciously alter the rate and depth of breathing (2).
The severity of central hypoventilation of patients with 20/27 to 20/33 genotype and nonpolyalanine repeat mutations requires aggressive management with mechanical ventilation and diaphragmatic pacing in the newborn period. Elucidating the mechanism of CCHS ventilatory dysregulation is the subject of ongoing research. The lack of ventilatory response to hypercarbia raises questions about the specifics of carbon dioxide chemoreception in patients with CCHS. Studies published to date are limited by missing such critical information as lack of phenotype analysis by PHOX2B genotype (14). A murine model with knock-in 20/27 PHOX2B genotype failed to fully answer the questions about the specifics of carbon dioxide chemoreception in CCHS was found to have site-specific loss of neurons in the retrotrapezoid nucleus and parafacial respiratory group in the medulla, regions important for integration/relay of chemosensory drive to respiratory rhythm and pattern generating circuits15. In contrast to patients with CCHS where many will present with alveolar hypoventilation in the newborn period. The onset of hypoventilation in patients with ROHHAD syndrome is usually after 2 years of age. Late onset central hypoventilation in a seemingly normal child explains the high incidence of cardiorespiratory arrest in patients with ROHHAD12. Evidences of abnormal respiratory control before the arrest, manifested as hemoglobin desaturation during sleep, cyanotic episodes during awakefulness, and obstructive sleep apnea were present from 2 months to a few days prior to the event (12). As in patients with CCHS, patients with ROHHAD have a blunted ventilatory response to hypercarbia and hypoxia during wakefulness and sleep12 and abnormal response to exogenous carbon dioxide challenge. As with CCHS, ROHHAD offers a unique opportunity to disentangle the mechanism of carbon dioxide responsiveness. 4
Obstructive sleep apnea was also described in patients with ROHHAD and can precede the onset of central hypoventilation and cardiorespiratory arrest (12). Many children with ROHHAD can be supported with nocturnal continuous positive airway pressure; a subset of patients with early onset of hypoventilation (median age of onset 3.8 years) (12) although may require 24h/day supported mechanical ventilation via tracheostomy due to the severity respiratory control deficit (12). The onset of obstructive sleep apnea is a potential marker for cardiorespiratory arrest (12). Hypothalamic Dysfunction As the acronym ROHHAD suggests, evidence of hypothalamic dysfunction is a hallmark of this syndrome. The most frequent presenting symptom reported is rapid-onset obesity early in life (median age of 3 years) (12). Hypothalamic dysfunction may also manifest as altered water balance (eg hypernatremia, polydipsia), diabetes insipidus, hyperprolactinemia, hypothyroidism, adrenal insufficiency, and alterations in pubertal development with delayed or precocious puberty (12, 19). While a defining characteristic for patients with ROHHAD syndrome, hypothalamic dysfunction was not recognized with increased frequency in patients with CCHS. Autonomic Dysregulation Autonomic dysfunction is frequently associated with and often complicates the central respiratory control anomalies. Conditions associated with CCHS include Hirschsprung disease, tumors of neural crest origin, and a large spectrum of symptoms compatible with autonomic nervous system dysregulation. Patients with CCHS can have diminished heart rate variability and transient abrupt asystoles, abnormal blood pressure response during sleep and with position change, decreased pupillary light response, esophageal dysmotility, breathholding spells, reduced basal body temperature,
sporadic profuse sweating, lack of perception to dyspnea, altered perception to anxiety, and lack of physiologic responsiveness to the challenges of exercise and environmental stressors (2, 11, 13, 17). An increased number of polyalanine repeats was associated with an increased number of symptoms of autonomic nervous system dysregulation (5). Autonomic dysregulation symptoms are present in all patients with ROHHAD. The most common reported manifestations were ophthalmologic: altered papillary response to light, strabismus, poor upward gaze, opsoclonus, oculomotor apraxia, and ptosis. Other frequent autonomic dysregulation symptoms described in patients with ROHHAD were: gastrointestinal dysmotility (constipation, chronic diarrhea, gastroesophageal reflux) and thermal dysregulation (hyperthermia or hypothermia). Neural Creast Origin Tumors Neural crest tumors develop in 5-10 % of pts with CCHS, most commonly neuroblastoma, ganglioneuroma or ganglioneuromas often multiple, located in either adrenals glands, chest, spinal cord or mediatinum. Tumors of neural crest origin occur more frequently among individuals with NPARMs (50%) than among those with PARMs (1%) and the majority is represented by neuroblastomas (18). Among patients with PARM, only subjects with the 20/29 and 20/33 genotypes have been identified to have tumors of neural crest origin (ganglioneuromas and ganglioneuroblastomas) (18).Patients with ROHHAD syndrome also have a high frequency of neural crest tumors. In a review of 23 patients with ROHHAD, five of
them were found to have ganglioneuroblastoma or ganglioneuroma. These were diagnosed before the hypothalamic dysfunction and hypoventilation (12). The term of ROHHADNET was proposed for patients with ROHHAD that have neural creast origin tumors. Psychiatric and Developmental Disorders Developmental delays, mild mental retardation, developmental regression, pervasive developmental disorders, ADHD, and Aspergers syndrome have been reported prior to the onset of hypoventilation in patients with ROHHAD (12). They also have an increased frequency of psychiatric disorders: depression, emotional lability and oppositional-defiant disorder. The need for medical and parenteral vigilance and frequent interventions impacts on both behavior and independence of these medically fragile children. Other Phenotype Characteristics Since the discovery of PHOX2B mutation, nearly 1000 of cases have been diagnosed with CCHS wordwide (14) enabling facial phenotype and dermatographic characterization of the syndrome. Todd at al. (16) described characteristic facies in CCHS, shorter and flatter (box shaped face), with an inferior inflection of the lateral segment of vermilion border on the upper lip. Dermatographic patterns with an increase number of arches in females and ulnar loops in males were observed in children with both CCHS and Hirschsprung s disease (16). Characteristic faces and dermatographic patterns are not yet described for patients with ROHHAD.
1. alveolar hypoventilation 2. absence of primary lung, cardiac, or neuromuscular disease or an identifiable brainstem lesion 3. identified PHOX2B gene mutation (chr. 4p12) - PARM (92%) - NPARM (8%) Table 1. Diagnosis Criteria for CCHS 5
Discussion Most of the cases with CCHS are diagnosed in the newborn period based on the presence of alveolar hypoventilation in the absence of primary lung, cardiac, or neuromuscular disease or an identifiable brainstem lesion and are confirmed by genetic testing (Table 1).
Confusion between the later onset- CCHS and ROHHAD was inevitable due to clinical similarities prior to 2003 when PHOX2B disease-defining mutation for CCHS was discovered. Table 2 reflects principal characteristics of the two disorders.
Genetics
Onset of central hypoventilation Hypothalamic dysfunction Autonomic dysregulation Tumors of neural crest Hirschsprung disease
CCHS PHOX2B gene mutation (chr. 4p12) - Polyalanine repeat expansion of PHOX2B (92%) - PHOX2B non-polyalanine repeat sequence variants (8%) newborn not present/not known present can be present can be present (increased frequency in patients with non-polyalanine repeat mutations)
ROHHAD Unknown (negative for PHOX2B mutation)
childhood, early adulthood present present can be present -/?
Table 2. principal characteristics of CCHS and ROHHAD Since the introduction of PHOX2B testing and of diagnosis criteria (table 3) there has been a dramatic increase to 75 reported cases of ROHHAD in the literature. Since there is no genetic testing available, the diagnosis of ROHHAD syndrome is based on clinical presentation. Clinician should have a high index of suspicion for the diagnosis in children older than 2 years of age who present with rapid-onset obesity followed by various hypothalamic disorders, autonomic dysregulation symptoms and tumors of neural origin. Since these children have a high prevalence of cardiorespiratory arrest with intercurrent viral infections, obstructive sleep apnea and alveolar 6 hypoventilation prompt evaluation in a specialized center is mandatory (18). Prompt identification and management of alveolar hypoventilation in patients with CCHS and ROHHAD is critical. Alveolar hypoventilation neither resolves nor improves with age. It does not respond to pharmacologic stimulants2. For these reasons patients will require chronic ventilatory management during the sleep. A limited number of children will require continuous ventilatory support. In order to secure the airway and ensure optimal ventilation and oxygenation positive pressure ventilators via tracheostomy (2, 20), bilevel positive airway pressure (2, 21-29), negative pressure ventilators (30, 31), or diaphragm
pacing (32-44) have been used. Negative pressure ventilation has been replaced with more efficient and adaptable forms of mechanical ventilatory support. Oxygen supplementation without ventilatory support
will improve the PaO2 levels, however the persistence of the hypoventilation will be followed by the development of pulmonary hypertension.
1. onset of rapid onset obesity and alveolar hypoventilation after the age of 1.5years 2. evidence of hypothalamic dysfunction, as defined by 1 of the following findings: rapid onset obesity, hyperprolactinemia, central hypothyroidism, disordered water balance, failed growth hormone stimulation test, corticotrophin deficiency, or delayed/precocious puberty 3. absence of PHOX2B mutation in cases reported after 2003 Table 3. Diagnosis Criteria for ROHHAD Syndrome Thoracic Society statement on CCHS recommends the following management for PHOX2B mutation-confirmed individuals with CCHS18 1. biannual then annual in-hospital evaluation with (i) physiologic studies during awake and asleep states to assess ventilatory needs during varying levels of activity and concentration, in all stages of sleep, with spontaneous breathing, and with artificial ventilation, and to assess ventilatory responsiveness to physiologic challenges while awake and asleep, (ii) 72-hour Holter monitoring, (iii) echocardiogram, (iv) assessment of autonomic nervous system dysregulation across all organ systems affected by the ANS, and (v) formal neurocognitive assessment; 2. barium enema or manometry and/or full thickness rectal biopsy for patients with a history of constipation; and 3. imaging for neural crest tumors in individuals at greatest risk based on PHOX2B mutation. These recommendations can be extended to the management of patients with ROHHAD who will also need extensive endocrinologic testing. Because of the autosomal dominant inheritance demonstrated in reported cases, a 5-10% chance of inheriting the disease from a mosaic typically unaffected parent, and a variable penetrance of some PHOX2B mutations, genetic testing and counseling is important for families of children 7 with diagnosed CCHS. Prenatal testing is available for babies of affected parents. The care of children with alveolar hypoventilation and autonomic dysregulation in specialized centers dramatically improved their quality of life and prolonged their lifeexpectancy. Many are able to participate in normal childhood activities including attending school, graduating from high school and college, getting married, having children, and finding steady employment (18). References 1. Mellins RB, Balfour HH, Turino GM, winters RW. Failure of autonomic control of ventilation (Ondines curse). Report of an infant born with this syndrome and review of the literature. Medicine (Baltimore) 1970; 49:487-504. 2. Weese-Mayer DE, Shannon DC, Keens TG, Silvestri JM. American Thoracic Society statement: idiopatic congenital central hypoventilation syndrome: diagnosis and management. Am J Respir Crit Care Med. 1999;160:368-373 3. Fishman LS, Samson JH, Sperling DR. Primary alveolar hypoventilation syndrome (Ondines curse). Am J Dis Child. 1965; 110:155-161 4. Katz ES, McGrath S, Marcus CL. Lateonset central hypoventilation with
hypothalamic dysfunction: a distinct clinical syndrome. Pediatr Pulmmonol. 2000; 29:62-68 5. Weese-Myer DE, Berry-Kravis EM, Zhou L, Maher BS, Silvestri JM, Curran ME, Marazita ML. Idiopatic congenital central hypoventilation syndrome: analysis of genes pertinent to early autonomic nervous system embryogenic development and identification of mutations in PHOX2B. Am J Med Genet A 2003; 123:267-278 6. Amiel J, Laudier B, Attie-Bitach T, Trang H, de Pontual L, Gener B, Trochet D, Etchevers H, Ray P, Simonneau M, Vekemans M, Munnich A, Gaultier C, Lyonnet S. Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome. Nat Genet 2003;33:459-461 7. Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF. Expression and interaction of the two closely related homeobox genes PHOX2A and PHOX2B during neurogenesis. Development 1997;124:4065-4075. 8. Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF. The homeobox gene PHOX2B is essential for the development of autonomic neural crest derivatives. Nature 1999;399:366-370. 9. Brunet JF, Pattyn A. PHOX2B genes- From patterning to connectivity. Curr Opin Genet Dev 2002;12:435-440 10. Matera I, Bachetti T, Puppo F, Di Duca M, Morandi F, Casiraghi GM, Cilio MR, Hennekam R, Hofstra R, Schober JG, Ravazzolo R, Ottonello G, Ceccherini I. PHOX2B mutations and polyalanine expansions correlate with the severity of the respiratory phenotype and associated symptoms in both congenital and late onset Ventral Hypoventilation syndrome. J Med Genet 2004; 41:373-380 8
11. Weese-Mayer DE, Rand CM, Berry-Kravis EM, Jennings LJ, Loghmanee DA, Patwari PP, Ceccherini I. Congenital Central Hypoventilation syndrome from Past to future: model for transitional autonomic medicine. Pediatric Pulmonology 2009; 44:521-535 12. Ize-Ludlow D, Gray JA, Sperling MA, Berry-Kravis EM, Milunsky JM, Sadaf Farooqi I, Rand CM, Weese-Mayer DE. Rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation presenting in childhood. Pediatrics 2007;120:179-188 13. Weese-Mayer DE, Silvestri JM, Huffman AD, Smok-Pearsall SM, Kowal MH, Maher BS, Cooper ME, Marazita ML. Case/control family study of autonomic nervous system dysfunction in idiopathic congenital central hypoventilation syndrome. Am J Med Genet 2001;100:237245 14. Carroll MS, Patwari PP, Weese-Mayer DE. Carbon dioxide chemoreception and hypoventilation syndrome with autonomic dysregulation. J Appl Physiol 2010; 15. Stornetta RL, Moreira TS, Takakura AC, Kang BJ, Chang DA, West GH, Brunet JF, Mulkey DK, Bayliss DA, Guyenet PG. Expression of PHOX2B by brainstem neurons involved in chemosensory integration in the adult rat. J Neurosci 2006;26:10305-10314 16. Todd ES, Weinberg S M, Berry-Kravis EM, Silvestri JM, Kenny AS, Rand CM, Zhou L, Maher BS, Marazita ML, WeeseMayer DE. Facial phenotype in children and young adults with PHOX2Bdetermined congenital central hypoventilation syndrome: quantitative pattern of dysmorphology. Pediatr Res 2006;59:39-45 17. Trang H, Boureghda S, Denjoy I, Alia M, Kabaker M. 24-hour BP in children with
congenital central hypoventilation syndrome. Chest. 2003; 124: 13939 18. Weese-Mayer,DE, BerryKravis,E,Ceccherini, I, et al, An Official ATS Clinical Policy Statement: Congenital Central Alveolar Hypoventilation Syndrome. Genetic Basis, Diagnosis and Management, Am J Respir Crit Care Med 2010; 181: 626-644. 19. Bougneres, P, Pantalone, L, Linglart, A, et al, Endocrine Manfestations of the Rapid onset Obsity with Hypoventilation, Hypothalamic, Autonomic Dysregulation and Neural Tumor syndrome in Childhood, J CLin Endocrinol Metab 2008; 93: 3971 3980. 20. Beckerman RC. Home positive pressure ventilation in congenital central hypoventilation syndrome: more than twenty years of experience. Pediatr Pulmonol 1997;23:154155. 21. Marcus CL. Ventilator management of abnormal breathing during sleep: continuous positive airway pressure and nocturnal noninvasive intermittent positive pressure ventilation. New York: Marcel Dekker, Inc.; 2000. 22. Kerbl R, Litscher H, Grubbauer HM, Reiterer F, Zobel G, Trop M, Urlesberger B, Eber E, Kurz R. Congenital central hypoventilation syndrome (Ondine's curse syndrome) in two siblings: delayed diagnosis and successful noninvasive treatment. Eur J Pediatr 1996;155:977980. 23. Costa Orvay JA, Pons Odena M, Jordan Garcia I, Caritg Bosch J, Cambra Lasaosa FJ, Palomeque Rico A. (Non-invasive ventilation in neonates with Ondine syndrome: a real indication?) An Pediatr (Barc) 2005;63:441443. 24. Fauroux B, Boffa C, Desguerre I, Estournet B, Trang H. Long-term noninvasive mechanical ventilation for children at home: a national survey. Pediatr Pulmonol 2003;35:119125. 9
25. Paditz E. (Nocturnal nasal mask ventilation in childhood.) Pneumologie 1994;48:744 749. 26. Simonds AK, Ward S, Heather S, Bush A, Muntoni F. Outcome of paediatric domiciliary mask ventilation in neuromuscular and skeletal disease. Eur Respir J 2000;16:476481. 27. Teague WG. Non-invasive positive pressure ventilation: current status in paediatric patients. Paediatr Respir Rev 2005;6:5260. 28. Tibballs J, Henning RD. Noninvasive ventilatory strategies in the management of a newborn infant and three children with congenital central hypoventilation syndrome. Pediatr Pulmonol 2003;36:544 548. 29. Villa MP, Dotta A, Castello D, Piro S, Pagani J, Palamides S, Ronchetti R. Bilevel positive airway pressure (BIPAP) ventilation in an infant with central hypoventilation syndrome. Pediatr Pulmonol 1997;24:6669. 30. Kajiura Y, Maeda H, Nishimura Y, Yahata T, Takatsuki K, Nakamura H, Yokoyama M. (A case of primary alveolar hypoventilation syndrome with a good response to nocturnal low-flow oxygen inhalation and negative pressure ventilation.) Nihon Kyobu Shikkan Gakkai Zasshi 1992;30:21512157. 31. Hartmann H, Jawad MH, Noyes J, Samuels MP, Southall DP. Negative extrathoracic pressure ventilation in central hypoventilation syndrome. Arch Dis Child 1994;70:418423. 32. Weese-Mayer DE, Hunt CE, Brouillette RT, Silvestri JM. Diaphragm pacing in infants and children. J Pediatr 1992;120:1 8. 33. Weese-Mayer DE, Silvestri JM, Kenny AS, Ilbawi MN, Hauptman SA, Lipton JW, Talonen PP, Garcia HG, Watt JW, Exner G, et al. Diaphragm pacing with a quadripolar
phrenic nerve electrode: an international study. Pacing Clin Electrophysiol 1996;19:13111319. 34. Weese-Mayer DE, Morrow AS, Brouillette RT, Ilbawi MN, Hunt CE. Diaphragm pacing in infants and children. A life-table analysis of implanted components. Am Rev Respir Dis 1989;139:974979. 35. Chen ML, Tablizo MA, Kun S, Keens TG. Diaphragm pacers as a treatment for congenital central hypoventilation syndrome. Expert Rev Med Devices 2005;2:577585. 36. Glenn WW, Phelps ML. Diaphragm pacing by electrical stimulation of the phrenic nerve. Neurosurgery 1985;17:974984. 37. Glenn WW, Brouillette RT, Dentz B, Fodstad H, Hunt CE, Keens TG, Marsh HM, Pande S, Piepgras DG, Vanderlinden RG. Fundamental considerations in pacing of the diaphragm for chronic ventilatory insufficiency: a multi-center study. Pacing Clin Electrophysiol 1988;11:21212127. 38. Hunt CE, Brouillette RT, Weese-Mayer DE, Morrow A, Ilbawi MN. Diaphragm pacing in infants and children. Pacing Clin Electrophysiol 1988;11:21352141. 39. Alonso Calderon JL, Garrido Garcia H, Perez Dominguez T, Mazaira J. (Simultaneous, bilateral and permanent ventilation with a diaphragm pacing in childhood: The implantation technique and indications.) Cir Pediatr 1994;7:37. 40. Shaul DB, Danielson PD, McComb JG, Keens TG. Thoracoscopic placement of phrenic nerve electrodes for diaphragmatic Coresponding author: Claudia Cosmineanu-Halaby Winthrop University Hospital 120 Mineola Blvd, Suite 210 Mineola, NY 11501 chalaby@winthrop.org
pacing in children. J Pediatr Surg 2002;37:974978, discussion 974978. 41. Hyland RH, Hutcheon MA, Perl A, Bowes G, Anthonisen NR, Zamel N, Phillipson EA. Upper airway occlusion induced by diaphragm pacing for primary alveolar hypoventilation: implications for the pathogenesis of obstructive sleep apnea. Am Rev Respir Dis 1981;124:180185. 42. Movahed MR, Jalili M, Kiciman N. Absence of device-device interaction (DDI) in a patient with cardiac and diaphragmatic pacemakers for congenital central hypoventilation syndrome. Pacing Clin Electrophysiol 2005;28:12381239. 43. Kolb C, Eicken A, Zrenner B, Schmitt C. Cardiac pacing in a patient with diaphragm pacing for congenital central hypoventilation syndrome (Ondine's curse). J Cardiovasc Electrophysiol 2006;17:789 791. 44. Ali, A, Flageole, H, Diagphragmatic pacing for the treatment of congenital central alveolar hypoventilation syndrome, J Ped Surg 2008; 43: 792-796 45. http://www.orpha.net/actor/EuropaNews/20 06/doc/French_National_Plan.pdf 46. http://eurlex.europa.eu/LexUriServ/LexUriServ.do?u ri=OJ:C:2009:151:0007:0010:RO:PDF 47. http://ec.europa.eu/health/rare_diseases/nati onal_plans/detailed/index_en.htm 48. http://www.raredis.org/pub/events/NPRD.p df 49. http://rarediseases.info.nih.gov/ 50. http://www.apwromania.ro/
10
MULTIPLE MALFORMATIONS IN INFANT - CASE REPORT

T.Marcovici1,2, I.Simedrea1,2, L. Tunea1,2, A.Militaru1,2, O.Belei1,2, D.Chiru1,2, G.Brad2, M.Puiu1,2 1 Victor Babes University of Medicine and Pharmacy, Timisoara, Romania 2 Louis Turcanu Childrens Emergency Hospital Timisoara, Romania
Abstract Background: Posterior urethral valves (PUV) are the most common cause of urethral obstruction in males. They are produced by mucosal folds in the posterior urethra. The pathological consequences of PUV are determined by the location and severity of obstruction. Increased resistance to urine outflow causes bladder muscle hypertrophy. Clinical presentation can be much diversified: from mild signs to end stage renal failure. Nearly 50% of the cases are diagnosed before the second month of life. The treatment is surgical. Methods: We report a 3 months old male infant admitted in our clinic for fever and rhinopharyngitis. He is the second child of a young, healthy and unrelated couple. No malformations are noticed in the family. Patient presented abdominal mass and difficulty voiding. Complete evaluation (anamnesis, clinical examination, imagistic and laboratory studies) was made. Results: The patient manifested straining during micturition and dribbling of urine, mild cardiac murmur and club feet. Imagistic exams (ultrasonography, intravenous urography, voiding cystourethrography) demonstrated crossed non-fused renal ectopia, both kidneys situated on the right side of the body; bilateral hydrouretheronephrosis; enlarged bladder, with irregular, grossly trabeculated walls and dilation of the posterior urethra. No vesico-ureteral reflux was found. Normal renal function and sterile urine were present. Cardiac ultrasonography revealed small atrial septal defect. Surgical ablation of the valves was performed in the pediatric urology department with good outcome. Conclusions: Posterior urethral valves are a component of a complex malformative syndrome in this patient. The ablation of the valves may preserve kidney function. Long term monitoring is required. Key Words: posterior urethral valves, congenital urethral obstruction, urinary malformation Introduction Posterior urethral valves (PUV) are the most common cause of urethral obstruction in males, with an incidence of 1:5000-1:8000 boys. (1, 2) The mucosal folds from the posterior urethra cause this problem. (2) The pathological consequences of PUV are determined by the location and the severity of obstruction. Increased resistance to urine leakage leads to urinary bladder muscle hypertrophy. Routine prenatal ultrasound identifies the consequences of subvesical obstacle such as ureterohydronephrosis, renal dysplasia, urinary bladder distension, etc. (2) Clinical manifestations may vary from mild signs to 11 terminal renal failure. (2, 3) Nearly 50% of cases are diagnosed before the second month of life. In cases prenatal diagnosed, the treatment is surgical, in mothers uterus and consists in ablation of PUV. (3, 4) The immediate prognosis, in general, is good and it depends on renal function at the time of intervention, while, at distance, it dependents on the age at which surgical correction is made. Evolution and prognosis depend by the recovery of kidney parenchyma. Without surgery immediate complications invariably appear such as repeated urinary tract infections, sepsis or electrolyte disorders and at distance like
hypertension and renal failure, while the prognosis is infaust. (1, 2, 3).
Fig. 1. Clinical aspect Materials and methods The authors want to present the case of a threemonth male infant, born from young and healthy genitors, from a rural area. The infant was admitted to the First Pediatric Clinic of Louis Turcanu Children Emergency Hospital, Timisoara for fever (38.5 C), frequent productive cough, nasal obstruction, rhinorrhea in April 2009. Complete evaluation (anamnesis, clinical examination, imaging and laboratory studies) was made. This case presentation has been approved by the Institution's Ethics Committee and has been performed in accordance to the ethical standards of the Declaration of Helsinki. Results The infant was from the second gestation with oligoamnios, born at term, in cranial presentation. His weight was 3100 g and had 51 cm in length. He was breastfeeding, vaccinated properly according to vaccination schedule, while the rickets prophylaxis was done with 2 drops per day of Vigantol. At hospital admission, the baby was feverish (37.6 C), but with preserved appetite. His skin was slightly pale with elastic turgor and discrete oral cyanosis. Nasal obstruction, 12
rhinorrhea, frequent productive cough and congested pharynx were associated too. A discrete systolic heart murmur was presented in his left second intercostals space. Bilateral clubfoot was found too. Inspection of the abdomen revealed the distension of the lower abdomen. (Figure 1) On midline, it can be palpated a tumor, with increased consistency, with the upper limit near the umbilicus, considered as being a distended urinary bladder. If a mild abdominal compression was done, he presented an interrupted urination stream with low pressure. The infant made repeatedly trunk flexion, with the contractions of abdominal muscles and thighs on the pelvis. Mother reported similar episodes in his first month of life, fact interpreted as abdominal colic. Abdominal ultrasound revealed the right kidney in a proper position but with marked dilatation of calyx and basins and the presence of a megaureter. The left kidney was undetectable at ultrasound. Urinary bladder wall was thickened and more relaxed. Urography identified both kidneys located on the right flank, ectopic left kidney in the right flank and bilateral grade III / IV ureterohydronephrosis. (Figure 2, Figure 3) Cistography revealed the presence of an oval urinary bladder with irregular outline, without active and passive vesicoureteral reflux. Posterior urethra had suggestive changes for subvesical urethral obstacle (large size of urinary bladder and increased postmictional residual urine). (Figure 4, Figure 5) The cardiopulmonary radiography revealed a normal aspect, while the echocardiography showed a patent foramen ovale. Biological investigation has identified a hypochromic anemia without BUN retention or positive bacteriological cultures, while the urine exam was normal. During hospitalization, his urinary bladder was distended, localized between umbilicus and symphysis, with spontaneous urination. He developed an episode of urinary tract infection with Enterobacter treated with Tazocin. After that, he was transferred to a pediatric urology department in order to complete the plan of investigations and surgical intervention.
Fig. 2. Urography
Fig. 3. Urography
Fig. 4. Voiding cystourethrography 13
Fig. 3. Voiding cystourethrography
Discussion Posterior urethral valves (PUV) are a congenital valve obstruction caused by folds of mucous membrane inside the posterior urethra, which cause various degrees of obstruction of bladder outlet tract and urinary tract dilatation proximally. It can be found predominantly in males, representing 80% of intrinsic urethral obstructions in childhood and it is responsible for 3-9% of all cases of prenatal hydronephrosis diagnosed. (1, 5) The severe cases associated oligoamnios, which was secondary to fetal posterior urethral obstruction. (2, 3) Mothers obstetrical history is also important in determining the postnatal risk factors. Male, oligoamnios and difficult urination, with significant contribution of abdominal press are important elements for the diagnosis of subvesical obstruction. (2) PUV cause urinary flow strength with bladder hypertrophy. (2) Vesicoureteral reflux is secondary to PUV in 2580% of affected patients. (2) Our patient presented no active or passive vesicoureteral reflux. (2) Medical literature showed that 15-20% of cases diagnosed with PUV associated renal dysplasia. (2, 5) In the case presented, the obstruction was associated with bilateral subvesical ureterohydronephrosis and ectopic left kidney, without kidneys fusion. Most PUV cases are prenatal diagnosed using ultrasound, but this fact was not done in our patient. (1, 3) The diagnosis of subvesical stenosis was done relatively early, at the age of 12 weeks of life , compared with other cases presented in medical literatures where children had more than three years old. (2) In undiagnosed patients, urinary tract infections can appear, similar to our case. (2) Cistography is the gold standard for the diagnosis of PUV. Some specific therapeutic measures are such as valve ablation, transurethral resection, percutaneous anterograde ablation or retrograde balloon catheter ablation. (2, 3) Cistography and the urological surgery was successfully completed.
Conclusions Subvesical barrier as part of a complex syndrome is a rare malformation. In our case, PUV is part of a complex reno-urinary malformation (left ectopic kidney on the right flank and megaureter) associated with a congenital heart malformation and an orthopedic abnormality. Identification of urethral obstruction based on clinical and imaging studies has been necessary for an optimal management of this case in order to preserve the functional kidney. Interdisciplinary long-term monitoring and genetic counseling should be part of it. Acknowledgments The authors want to thank the doctors from the Laboratory and Radiology Department of "Louis Turcanu Childrens Emergency Hospital Timisoara for their collaboration and help in solving this case. References 1. 1 SJ, Patel B, McLorie G, Atala A. Posterior urethral valves. TSWJ 2009; 9: 1119-26. 2. Kalhoro AS, Jamro S, Abbasi A, Mughal SA. A study of posterior urethral valves. JSP 2008; 13(3):112-6. 3. Quintero RA, Shukla AR, Hornsy YL, Bukkapatnma R. Successful in utero endoscopic ablation of posterior urethral valvas; a new dimension in fetal urology. Urology 2000; 55(5):774. 4. Holmes N, Harrison MR, Baskin LS. Fetal surgery of PUV: long term postnatal outcomes. Pediatrics 2001; 108(1): E7. 5. Cromie LK. Implication of prenatal ultrasound screening in the incidence of major genitourinary malformation. J Urol 2001; 165:1677-80.
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Corresponding author: Dr.Tamara Marcovici, C4 Mihail Kogalniceanu 300133, Timisoara, Romania Phone: 0723694950 E-mail: t_marcovici@yahoo.com
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LABORATORY STRATEGIES FOR DIAGNOSIS OF PRADER-WILLI AND ANGELMAN SYNDROMES

Arsene Cosmin1, Maria Puiu2, Otilia Zarnescu1, Alungulese Anca Loredana3, Natalia Cucu1 1. University of Bucharest, Faculty of Biology,Department of genetics, Epigenetics Center, Romania 2. Medical and Pharmaceutical University-Timisoara, Romania 3. University of Medicine and Pharmacy "Carol Davila" Bucharest, Romania
Abstract PraderWilli syndrome (PWS) and Angelman syndrome (AS) are two distinct neurodevelopmental disorders, each caused by several genetic and epigenetic mechanisms involving the proximal long arm of chromosome 15. Lack of a functional paternal copy within 15q11q13 causes PWS; lack of a functional maternal copy of ubiquitin protein ligase 3A gene (UBE3A), within 15q11q13, causes AS. The most sensitive single approach to diagnosing both PWS and AS is to study methylation patterns within this region; however many techniques exist for this purpose. Given the diversity of techniques available, there is a need for consensus testing and reporting guidelines. In this paper we describe the known mechanisms involved in the development of this syndromes, and the main tests used for a proper diagnostic, along with the advice on the assay sensitivity. A set of practice guidelines are offerd here for the diagnostic of PWS and AS. Key words: Prader-Willi Syndrome, Angelman Syndrome, laboratory diagnostic Introduction The PraderWilli syndrome and Angelman syndrome are the first known examples of human diseases involving imprinted genes. Each occurs with a frequency of ~1:15,0001:25,000 live births. De novo interstitial deletions of the same chromosome region 15q11-q13 in patients with PWS and AS, were first identified by highresolution chromosome banding and later confirmed by molecular studies (1-7). PWS is characterized by neonatal hypotonia, childhood onset obesity, cognitive impairment, distinctive behavioural characteristics, hypogonadism and a characteristic facial appearance (8). Phenotypic hallmarks of AS include motor dysfunction leading to an ataxic gait, frequent seizures that can be severe, profound learning disability coupled with a short attention span, absent speech, and characteristic happy demeanor (9). AS pacients also have an abnormal electroencephalogram 16 (EEG), with a characteristic pattern - large amplitude slow-spike waves - usually 2-3 Hz facilitated by eyes closure (10). There is not a single gene responsible for PWS, but most aspects of the PWS phenotype result from absence of paternal expression of a cluster of non-coding RNAs known as HBII-85 (11). In the great majority of PWS patients, the underlying molecular mechanism is either a 46 Mb chromosome deletion at 15q11.215q13 (70%) or maternal uniparental disomy (mat UPD) of chromosome 15 (25%). Epimutations causing PWS are very rare, accounting for 1% of PWS patients (12). Loss of UBE3A expression in AS patients occurs due to one of five classes of genetic abnormalities (fig. 2). 70% of patients suffer from a large deletion of the maternal allele of chromosome 15q11q13 and 2-5% of patients harbor a loss-of-function mutation in the UBE3A
Romanian Journal of Rare Diseases (2010), 2 gene (13). These two classes comprise the most common genetic etiologies of AS. The remainder of patients attribute AS to either paternal uniparental disomy (patUPD), an imprinting defect (ID), or an unknown molecular mechanism (9).
Figure 1. A map of the 15q11q13 subregion. Imprinted, paternally-expressed genes are denoted by yellow boxes, while imprinted, maternally-expressed genes are denoted by red boxes. Silent alleles are shown as black boxes, and genes expressed bi-allelically are shown as green boxes. The imprinting status of ATP10A varies among individuals, therefore is labeled with mosaic boxes. Dashed and dotted lines across the snoRNA clusters demarcate the broadly expressed and brain-specific snoRNA clusters, respectively. We have used a blue box to denote IPW, however, this transcript is part of the large, non-coding SNURFSNRPN transcript and not an independent gene. The PWS- and AS-ICs (imprinting centre - IC) are shaded in blue and red, respectively. Black circles on repressed alleles of SNURF-SNRPN, MKRN3, and NDN indicate methylated CpG islands and the white circles on corresponding expressed alleles indicate unmethylated CpG islands. BP1, BP2, and BP3 are the common deletion breakpoints and are represented by zigzag lines. The presumptive PWS critical region is contured in a box (after Chamberlain and Lalande 2010). The mechanisms involved in the development of PWS/AS PWS and AS can result from various chromosome 15q11q13 alterations, (14) including a typical 5-7 Mb de novo deletion, uniparental disomy of chromosome 15, an imprinting defect, or in cases of AS from, a mutation of the UBE3A gene (fig. 2).In contrast to PWS,~1015% of patients with AS phenotypic features have a genetic defect of unknown nature. An 57 Mb de novo interstitial deletion of the proximal region of chromosome 15, which includes the entire imprinted domain plus several non-imprinted genes, is found in the majority (70%) of patients with PWS and AS. The deletions are fairly common, occurring at a frequency of about 1/10,000 newborns. In PWS, the deletion is always on the paternal chromosome whereas in AS the deletion is always on the maternal chromosome. In some patients, the region is deleted as the result of an unbalanced translocation. The deletions result from nonhomologous recombination events mediated by 250400 kb repetitive sequence blocks which define the common breakpoint regions BP1-3 (fig. 1) (15-17).
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Figure 2. Molecular defects in PWS and AS. The frequency of the underlying defect in both disorders and the recurrence risk are given below. Methylation of paternally expressed genes is indicated by a red dot. Mat, maternal; pat, paternal (after Buiting 2010). Uniparental disomy (UPD) describes the inheritance of both homologues of a pair of chromosomes from only one parent (18). This chromosome composition stems from nondisjunction events during meiosis. Although some phenotypic differences between patients with PWS due to deletion 15q and UPD 15 were found in previous studies, no major difference that is likely to have a significant impact on prognosis of the disorder was previously detected (19-21). UPD in PWS mostly arises due to rescue of trisomic or monosomic zygotes, due to 3:1 segregation of balanced Robertsonian translocations involving chromosome 15 or due to 18 unequal crossing-over between the two homologous chromosomes 15 (22). IC deletions are found only in a small fraction of patients with PWS or AS. In the vast majority of patients (85% in PWS and 92% in AS) the imprinting defect represents a primary epimutation.(12,23). Such epimutations can occur during imprint erasure in primordial germ cells, imprint establishment during later stages of gametogenesis, or imprint maintenance after fertilization. If it occurs in the germ line, all cells of the patient are affected. If it occurs after fertilization, it results in somatic mosaicism (fig. 3) (24).
Figure 3. Model for imprinting errors in the PWS/AS region. In somatic cells (green rectangles) of males and females, the maternal PWS-SRO (smallest region of deletion overlap) carries a methylation mark (black circle). In normal imprinting cycle (A), methylation mark is erased in primordial germ cells (orange rectangles). A protein complex containing at least one protein specific to the oocyte lineage (star) associates with the PWS-SRO during oogenesis. Soon after fertilization (blue rectangle), this complex leads to CpG methylation of the maternal PWS-SRO. Imprinting errors can arise from failure to erase the methylation mark in the paternal germ line (B), from failure to establish methylation after oogenesis and fertilization (C), or from failure to maintain methylation after fertilization (D). Failure to maintain methylation leads to somatic mosaicism ( after Horsthemke and Wagstaff, 2008). mask the appearance of the deleted chromosome Mosaicism in Prader-Willi syndrome is rare, and 15. only a few cases have been described in the literature..Fluorescence in situ hybridization The laboratory diagnosis approach of AS and analysis proved useful in identifying PWS-like PWS patients who were mosaic for a deletion involving The most sensitive single approach to diagnosing 15q11-13. The use of traditional cytogenetics PWS and AS is to study methylation patterns and/or molecular genetics did not detect the within 15q11-q13 using molecular genetic mosaic deletion in these patients. Ascertainment techniques. These will detect deletions, UPD and of a mosaic deletion by either minisatellite repeats imprinting defects by establishing either a solely or methylation would be difficult due to the maternal methylated imprint (PWS) or paternal presence of the normal cell line, which would methylated imprint (AS) (25).
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Figure 4. Testing strategies for the molecular analysis of PWS and AS based upon an initial methylation analysis at the SNRPN locus and an initial MS-MLPA analysis (after Ramsden et al., 2010). The detection of methylation status solely at the SNRPN locus by use of methylation specific PCR (MS-PCR) or Southern blot analysis. This approach will confirm a diagnosis but will provide no further information regarding the disease mechanism necessitating follow up studies (FISH and/or microsatellite analysis). The simultaneous assessment of methylation status and genomic dosage at numerous sites across the 15q11-q13 region, by the use of methylation sensitive multiplex ligation20 dependent probe amplification (MS-MLPA). This approach will confirm the diagnosis and further identify the presence of a causative deletion. However, in the absence of a deletion follow up studies (microsatellite analysis) are required to distinguish between UPD and an imprinting defect (fig.4) (25). MS-PCR: This (PCR)-based assay allows rapid diagnosis of PWS and AS. Methylated cytosines in the CpG dinucleotide are resistant to chemical modification by sodium bisulfite. In contrast,
Romanian Journal of Rare Diseases (2010), 2 bisulfite treatment converts all unmethylated cytosines to uracil. Based on this differential effect, the bisulfite modified DNA sequence of a methylated allele was successfully distinguished from that of an unmethylated allele using 2 sets of allele-specific primer pairs: a methylated allelespecific primer pair (MET) and an unmethylated allele-specific primer pair (UNMET). Bisulfitemodified DNA from patients with PWS amplified only with the MET pair, while modified DNA from patients with AS amplified only with the UNMET pair (fig. 5) (26).
Figure 5. Outline of bisulfite conversion of genomic DNA. Nucleotides in blue are unmethylated cytosines converted to uracils by bisulfite, while methylated cytosines are resistant to conversion. Using 2 sets of allele-specific primer pairs for the methylated and the unmethylated sequence, the presence or the absence of functional maternal (methylated) and paternal (unmethylated) allele will be revealed. Normal (N)-both amplicons, PWS-only the maternal amplicon, AS- only the paternal amplicon. This method has the following significant advantages over conventional analysis using methylation-sensitive enzymes and Southern blotting: (1) MSPCR can be completed in 2 days. Rapid turnaround of the test result will be especially useful when evaluating hypotonic newborn infants among whom the incidence of PWS is high (27); (2) Testing can be performed with as little as 50 ng of genomic DNA. Thus, in addition to whole blood, other potential sources of genomic DNA for analysis include dried blood 21 spots and buccal cell smears; (3) MSPCR does not require use of radioactivity. MS-MLPA: The principle of MS-MLPA is almost similar to the previously described MLPA (28), except that the target sequences detected by MS-MLPA probes contain a restriction site recognized by endonucleases such as HhaI or HpaII that are sensitive to cytosine methylation of one CpG site in their recognition sequence. Upon digestion with one of these enzymes, a probe amplification product will only be obtained if the
Romanian Journal of Rare Diseases (2010), 2 CpG site is methylated (fig.6). The level of methylation was determined by calculating the ratio of the relative peak area of each target probe from the digested sample and from the undigested sample. The DNA is first denatured, followed by the addition of MS-MLPA probes and a 16 h hybridization step. Subsequently, this probeDNA complex is simultaneously ligated and digested by methylation-specific enzymes. If the CpG site is methylated, a normal MLPA product will be detected. If the CpG site is not methylated, the DNAprobe complex will be digested by the methylation-sensitive enzyme and no amplification product is formed. The MS-MLPA method described here extends the MLPA method for multiplex copy number quantification to a method for simultaneous analysis of the copy number, as well as the methylation status of up to 40 sequences in a simple reaction. MS-MLPA has become the method of choice in many diagnostic laboratories as it investigates methylation status at several loci, thereby reducing the risk of a false positive or false negative result due to SNPs; in addition, if one probe fails there are four remaining probes with which to assess methylation status (25). Several aspects contribute to the benefit of MSMLPA: (1) a large number of genes can be studied using a minimum amount of only 20 ng sample DNA; (2) owing to its simple procedure, large number of samples can be analyzed simultaneously; (3) MLPA is quantitative and can discriminate between methylation of one, both or none of the alleles; and (4) the simultaneous ligation and digestion reaction enables MS-MLPA to be used on paraffin-embedded tissue samples, because DNA degradation and partial DNA denaturation during embedding of the tissues or longtime storage do not influence the results (29). Arguably the most important limitation of MLPA is that the relative signal intensities obtained in the assay vary according to the characteristics of the input DNA. Such characteristics may include the age of the DNA, the method of DNA isolation, the 22 solution in which the DNA has been stored, the degree of DNA degradation (if present), and the presence of various contaminants (isolation reagents, protein, RNA, salts, etc.). Another obvious limitation of MLPA is that in most cases it does not detect genomic rearrangements such as translocations or inversions, but rather provides copy number information only. However, if the breakpoint of such rearrangements is precisely defined and specific, an MLPA probe set may be designed for detection (30). FISH: Fluorescence in situ hybridization offers the possibility to use fluorochromes for specifically mark individual chromosomes over their entire length or defined chromosome regions in meta- and interphase preparations (31) then visualize their presence using fluorescence microscopy. The first step in FISH procedures is the procurement of cells. Unlike many other chromosomal visualization techniques, FISH can be conducted on currently dividing or terminally differentiated cells. Cells are grown to a specific culture density and fixed with formaldehyde and placed on a functionalized glass slide. This slide is then allowed to dry, dehydrated with ethanol, and then treated with the hybridization buffer. DNA probes are then added and the slide is allowed to incubate. This gives the DNA probes enough time to hybridize with their complementary sequences. Following hybridization, the slides are allowed to air dry and then are examined under microscope (32). Many PWS/AS deletions, are not detectable by G-banding. Even at higher band levels, variable G-banding quality, differences in homologue condensation/splitting of band 15q12, and possible presence of extra clinically benign Gbands in this region made interpretation difficult (33,34). Unreliability of G-banding for deletion detection in the region 15q11-q13 in comparison with FISH has been well documented (35-38). The development of molecular probes for the Prader-Willi syndrome and Angelman's syndrome region, enabled alternate or complementary means for the detection of deletions in patients (39-41).
Figure 6. Outline of the MS-MLPA procedure. An ordinary MLPA probe harbors two oligonucleotides, one short synthetic and one long M13-derived oligonucleotide and up to 50 probes can be added to each MLPA reaction. Both oligonucleotides contain universal primers sites. For each MLPA probe, the M13 oligonucleotide is cloned in a M13 vector that contains stuffer sequence that varies in length between the different probes. Subsequently, these long M13-oligonucleotides are obtained by restriction-digestion from the M13 clones. For MS-MLPA, the probe design is similar to an ordinary MLPA probe except that the sequence detected by the MS-MLPA probe contains a recognition sequence for HpaII or HhaI. Upon digestion of the DNA/MS-MLPA probe complex with one of the methylation-sensitive enzymes, probes of which the recognition sequence is methylated will generate a signal. If the CpG site is unmethylated the genomic DNAMS-MLPA probe complex will be digested and prevent exponential amplification and no signal will be detected after fragment analysis (after Errami et al. 2005) 23
For FISH analysis of 15q11-q13, probes for loci D15Sl1, SNRPN, D15S10, and GABRB3 within 15q11-13, and for identification of the chromosome 15 homologues an internal control probe for locus PML at 15q22, were cohybridized to chromosomes following protocols provided by the manufacturer (Oncor, Gaithersburg, MD). Although its expensive and time-consuming, this method confirms the diagnosis of ~ 70% PWS and AS, and reveals the mosaic and translocation cases. Microsatellite Analysis: When a diagnosis of AS or PWS is confirmed with any of the above techniques, microsatellite analysis will often be required to distinguish between the various disease mechanisms. There are many microsatellites suitable for this purpose (42,43). Once the diagnosis of PWS or AS has been confirmed using methylation analysis, the interpretation of the microsatellite results is as follows (25) : a) Uniparental inheritance inside the critical region, biparental outside. In this case the disease is due to a deletion of the critical region. In rare cases, microsatellites may be used to confirm a smaller deletion within the critical region, however, laboratories must not interpret results from a single informative microsatellite without supporting evidence. b) Uniparental inheritance both inside and outside the critical region. In this case, the disease is due to uniparental disomy. It is important to note that AS and PWS can be caused by either chromosomal isodisomy or heterodisomy. Further, heterodisomy or isodisomy at a single locus does not necessarily reflect the disomy status along the entire chromosome depending on the rate and level of crossing over and the meiotic stage at which non-segregation occurred. c) Biparental inheritance both inside and outside the critical region. In this case the disease is presumed to be due to an imprinting defect. 24
Interpretation of the analysis For the laboratory diagnostic of the PWS one should start with the methylation analysis because it is the most sensitive method, confirming over 99% of the cases. If we start the diagnostic test with the MS-PCR (it will confirm a diagnosis but will provide no further information regarding the disease mechanism deletion, UPD, ID) and the result is positive, for genetic counseling, the next step is to perform a FISH or MLPA analysis (asking for deletion) and microsatellites analysis (asking for UPD) to determine the genetic mechanism and recurrence risk. When the methylation test is negative, paternal deletion, uniparental disomy and an imprinting defect are excluded, and the presence of PWS highly unlikely. The other approach for the diagnosis of PWS is to start with the MS-MLPA assay. This test shows the methylation status and the dosage at 15q11q13, it is more precise then MS-PCR as it investigates methylation status at several loci, thereby reducing the risk of a false positive or false negative result due to SNPs, and can also identify microdeletions in the IC as it uses many probes in the same reaction (up to 40). If the test is positive, certain conclusions may be withdrawn: a) deletion indicates that the molecular cause of PWS is due to 15q11-q13 deletion; b) aberrant methylation suggests the molecular cause of PWS may be due to maternal UPD or an imprinting defect. Laboratories should recommend microsatellite studies to confirm or exclude UPD. If the results are negative (in either MS-PCR and MS-MLPA), paternal deletion, uniparental disomy or an imprinting defect are excluded, but to be more accurate, it is recommended to perform the FISH analysis to rule out the possibility of a translocation, or a mosaic form of the syndrome. For the laboratory diagnostic of the AS the starting test is the same used for the PWS the methylation analysis, which will confirm ~80% of the cases. Having a positive result of the MS-PCR
Romanian Journal of Rare Diseases (2010), 2 test, further investigations as FISH or microsatellites analysis are necessary for determining the mechanism involved in the development of the syndrome. In the case of a negative MS-PCR test, it may be appropriate to offer UBE3A analysis after a clinical reassessment of the patient (mutation in UBE3A, are responsible for ~5% of the AS cases). MS-MLPA assay confers both information about the methylation status and the dosage at 15q11q13, and as described earlier it has some characteristics which makes it more reliable than MS-PCR. A positive result suggests the following mechanisms: a) deletion - confirms a genetic cause of AS (15q11-q13 deletion); b) aberrant methylation - suggests either maternal UPD or an imprinting defect. As around 10% of patients with a clinical diagnosis of Angelman syndrome have no demonstrable abnormality at 15q11-q13 using the techniques described here, a careful review of the patient's history, clinical features and EEG findings is recommended (AS pacients have a characteristic, abnormal EEG). Differential diagnosis For PWS. There were described several disorders with a phenotype that can strongly resemble PWS, consisting in neonatal hypotonia and later onset obesity. Their associated mechanisms implied: (i) upd(14)mat, which can be caused by uniparental disomy 14 and imprinting defects or deletions affecting the DLK1/GTL2 locus in the chromosomal region 14q32 (44,45); (ii) a number of other conditions associated with obesity and developmental disability including Cohen syndrome, BardetBiedl syndrome, Alstrom syndrome, and (iii) the 1p36 microdeletion which characterize a specific syndrome (46). For AS. There are many molecular abnormalities with a wide spectrum of clinical features that overlap with AS (47): (i) the MowatWilson syndrome is caused by mutations affecting the ZFHX1B gene on chromosome 2, and is associated with severe mental retardation, microcephaly, seizures, short stature, and 25 characteristic facial features that resemble those of AS; (ii) Hirschsprung disease, characterized by congenital cardiac defects and agenesis of the corpus callosum, may be associated with mutations in ZFHX1B; (iii) some girls with features of AS present the Rett syndrome and a differential diagnosis should be considered, as in infants it can be difficult to distinguish between AS and Rett syndrome due to overlapping features such as microcephaly and ataxia; (iv) Pitt-Hopkins Syndrome (PHS) is a sporadic condition caused by mutations or deletions of the TCF4 gene on chromosome 18q, and patients present with absent speech, seizures and facial features resembling AS, together with a sociable personality; (v) mutations of the SLC9A6 have been reported in patients with an AS-like phenotype (48); (vi) single gene conditions mimicking AS are the ATR-X syndrome, Gurrierie syndrome and methylenetetrahydrofolate reductase (MTHFR) deficiency (49); (vii) the 22q13 microdeletion syndrome is another differential diagnosis with an AS-like condition. Conclusions The establishment of a practical set of molecular genetic testing guidelines for PWS and AS has been succeeded through numerous experiences linked with the technical performance, the complexity of the imprinting diseases and the basic concepts linked with the hereditary transmittance. The actual diagnosis scheme for the 2 sister syndromes suggests the methylation analysis to be performed first, as it confirms ~99% of PWS cases, and ~80% of AS cases. The diagnosis should be approached by MS-MLPA analysis as the first test, because a large number of genes can be analyzed simultaneously reducing the risk of a false positive or false negative result due to SNPs, and it also offers information of the copy number, as well as the methylation status of many sequences in a single reaction. In the case of AS, UBE3A analysis should be carried out if the result of the methylation test is negative. For genetic counseling, if the methylation analysis is
Romanian Journal of Rare Diseases (2010), 2 positive (by MS-MLPA), the next step is the microsatellites analysis, in order to confirm a UPD or an ID. If the results indicate an ID further tests are required to discriminate between a mutation or an epimutation. Acknowledgements: AC is a PhD fellow in POSDRU/88/1.5/S/61150. The financial support of the article was provided by the research project CNMP, 42-113/2008, National program II, head coordinator MP from UMF "Victor Babes", Timisoara, where NC is the coordinator of the University of Bucharest partner activities, and by project POSDRU/88/1.5/S/61150 Doctoral Studies in the field of life and earth sciences, project cofinanced through Sectorial Operational Program for the Development of Human Resources 20072013 from European Social Fund. References 1. Ledbetter DH, Riccardi VM, Airhart SD, Strobel RJ, Keenan BS, Crawford JD. Deletions of chromosome 15 as a cause of the Prader-Willi syndrome. N Engl J Med 1981;304:325329. 2. Kaplan LC, Wharton R, Elias E, Mandell F, Donlon T, Latt SA. Clinical heterogeneity associated with deletions in the long arm of chromosome 15: Report of 3 new cases and their possible genetic significance. Am J Med Genet 1987;28:4553. 3. Magenis RE, Brown MG, Lacy DA, Budden S, LaFranchi S. Is Angelman syndrome an alternate result of del(15)(q11q13)? Am J Med Genet 1987;28:829838. 4. Donlon TA. Similar molecular deletions on chromosome 15q11.2 are encountered in both the Prader-Willi and Angelman syndromes. Hum Genet 1988;80:322328. 5. Knoll JH, Nicholls RD, Magenis RE, Graham JM Jr, Lalande M, Latt SA. Angelman and Prader-Willi syndromes share a common chromosome 15 deletion but differ in parental 26 origin of the deletion. Am J Med Genet 1989;32:285290. 6. Tantravahi U, Nicholls RD, Stroh H, Ringer S, Neve RL, Kaplan L, Wharton R, WursterHill D, Graham JM Jr, Cantu ES, Frias JL, Kousseff BG, Latt SA. Quantitative calibration and use of DNA probes for investigating chromosome abnormalities in the Prader-Willi syndrome. Am J Med Genet 1989;33:7887. 7. Nicholls RD, Knoll JH, Glatt K, Hersh JH, Brewster TD, Graham JM Jr, Wurster-Hill D, Wharton R, Latt SA. Restriction fragment length polymorphisms within proximal 15q and their use in molecular cytogenetics and the Prader-Willi syndrome. Am J Med Genet 1989;33:6677. 8. Cassidy SB, Schwartz S. PraderWilli syndrome. GeneReviews at GeneTests: Medical Genetics Information Resource (database online). Seattle: University of Washington, 2006. 9. Lossie, A.C., Whitney, M.M., Amidon, D., Dong, H.J., Chen, P., Theriaque, D., Hutson, A., Nicholls, R.D., Zori, R.T., Williams, C.A., Driscoll, D.J. Distinct phenotypes distinguish the molecular classes of Angelman syndrome. J. Med. Genet. 2001;38:834845. 10. Guerrini R, Carozzo R, Rinaldi R, Bonanni P. Angelman syndrome: etiology, clinical features, diagnosis, and management of symptoms. Pediatr Drugs. 2003;5(10):647-61. 11. Sahoo T, del Gaudio D, German JR, Shinawi M, Peters SU, Person RE, Garnica A, Cheung SW, Beaudet AL. PraderWilli phenotype caused by paternal deficiency for the HBII-85 C/D box small nucleolar RNA cluster. Nat Genet 2008;40:719721. 12. Buiting K, Gross S, Lich C, GillessenKaesbach G, El-Maarri O, Horsthemke B. Epimutations in Prader-Willi and Angelman syndromes: A molecular study of 136 patients with an imprinting defect. Am J Hum Genet 2003;72:571577.
Romanian Journal of Rare Diseases (2010), 2 13. Buiting K. PraderWilli syndrome and Angelman syndrome. Am J Med Genet Part C Semin Med Genet 2010;154:365376. 14. Chamberlain, S.J., Lalande, M. Neurodevelopmental disorders involving genomic imprinting at human chromosome 15q11q13, Neurobiol. Dis. 2010;10:1016. 15. Christian SL, Bhatt NK, Martin SA, Sutcliffe JS, Kubota T, Huang B, Mutirangura A, Chinault AC, Beaudet AL, Ledbetter DH. Integrated YAC contig map of the PraderWilli/Angelman region on chromosome 15q11-q13 with average STS spacing of 35 kb. Genome Res 1998;8:146157. 16. Christian SL, Fantes JA, Mewborn SK, Huang B, Ledbetter DH. Large genomic duplicons map to sites of instability in the PraderWilli/Angelman syndrome chromosome region (15q11-q13). Hum Mol Genet 1999;8:10251037. 17. Amos-Landgraf JM, Ji Y, Gottlieb W, Depinet T, Wandstrat AE, Cassidy SB, Driscoll DJ, Rogan PK, Schwartz S, Nicholls RD. Chromosome breakage in the PraderWilli and Angelman syndromes involves recombination between large, transcribed repeats at proximal and distal breakpoints. Am J Hum Genet 1999;65:370386. 18. Engel E. A new genetic concept: uniparental disomy and its potential effect: isodisomy. Am J Med Genet 1980;6:137-142. 19. Gillessen-Kaesbach G, Robinson W, Lohmann D, Kaya-Westerloh S, Passarge E, Horsthemke B. Genotype-phenotype correlation in a series of 167 deletion and non-deletion patients with Prader-Willi syndrome. Hum Genet 1995;96:638643. 20. Mitchell J, Schinzel A, Langlois S, GillessenKaesbach G, Michaelis RC, Abeliovich D, Lerer I, Schuffenhauer S, Christian S, Guitart M, Mc- Fadden DE, Robinson WP. Comparison of phenotype in uniparental disomy and deletion Prader-Willi syndrome: Sex specific differences. Am J Med Genet 1996;65:133136. 27 21. Cassidy SB, Forsythe M, Heeger S, Nicholls RD, Schork N, Benn P, Schwartz S. Comparison of phenotype between patients with Prader-Willi syndrome due to deletion 15q and uniparental disomy 15. Am J Med Genet 1997;68:433440. 22. Horsthemke B, Buiting K. Imprinting defects on human chromosome 15. Cytogenet Genome Res 2006;113:292299. 23. Horsthemke B, Buiting K. Genomic imprinting and imprinting defects in humans. Adv Genet 2008;61:225246. 24. Horsthemke B, Wagstaff J. Mechanisms of imprinting of the PraderWilli/Angelman region. Am J Med Genet Part A 2008;146A:20412052. 25. Ramsden SC, Clayton-Smith J, Birch R and Buiting K.CPractice guidelines for the molecular analysis of Prader-Willi and Angelman syndromes. BMC Medical Genetics 2010;11:70 26. Jones KL, Kosaki K, McGinniss MJ, Veraksa AN and McGinnis WJ. Prader-Willi and Angelman Syndromes: Diagnosis With a Bisulfite-Treated Methylation-Specific PCR Method. American Journal of Medical Genetics 1997;73:308313. 27. Gillessen-Kaesbach G, Gross S, KayaWesterloh S, Passarge E, Horsthemke B. DNA methylation based testing of 450 patients suspected of having Prader-Willi syndrome. J Med Genet 1995;32:8892. 28. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F and Pals G. (2002) Relative quantification of 40 nucleic acid sequences by multiplex ligationdependent probe amplification. Nucleic Acids Res. 2002;30:57 29. Errami A, Nygren AOH, Ameziane N, Duarte HMB, Vijzelaar RNCP, Waisfisz Q, Hess CJ and Schouten JP. Methylation-Specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Research 2005;33:14-128.
Romanian Journal of Rare Diseases (2010), 2 30. Piotr K, Jasinska AJ, and Kwiatkowski DJ. New applications and developments in the use of multiplex ligation-dependent probe amplification. Electrophoresis 2008;29:4627 4636. 31. Chevret E, Volpi EV, Sheer D. Mini review: formand function in the human interphase chromosome. Cytogenet Cell Genet, 2000;90:13-21. 32. Langedijk PS, Schut F, Jansen GJ, Raangs GC, Kamphuis GR, Wilkinson MHF, Welling GW. Applied and Environmental Microbiology 1995;61:3069-3075. 33. Hoo J-J, Chao MC, Samuel IP, Morgan AM. Proximal 15q variant as possible pitfall in the cytogenetic diagnosis of Prader-Willi syndrome. Clin Genet 1990;37:161-166. 34. Ludowese CJ, Thompson KJ, Sekhon GS, Pauli RM. Absence of predictable phenotypic expression in proximal 15q duplications. Clin Genet 1991;40:194-201. 35. Delach JA, Rosengren SS, Kaplan L, Greenstein RM, Cassidy SB, Benn PA. Comparison of high resolution chromosome banding and fluorescence in situ hybridization (FISH) for the laboratory evaluation of Prader-Willi syndrome and Angelman syndrome. Am J Med Genet 1994;52:85-91. 36. Butler MG. High resolution chromosome analysis and fluorescence in situ hybridization in patients referred for Prader-Willi or Angelman syndrome. Am J Med Genet 1995;56:420-422. 37. Bettio D, Rizzi N, Giardino D, Grugni G, Briscioli V, Selicorni A, Carnevale F, Larizza L. FISH analysis in Prader-Willi and Angelman syndrome patients. Am J Med Genet 1995;56:224-228. 38. Smith A, Prasad M, Deng ZM, Robson L, Woodage T, Trent RJ. Comparison of high resolution cytogenetics, fluorescence in situ hybridisation, and DNA studies to validate the diagnosis of Prader-Willi and Angelmans syndromes. Arch Dis Child 1995;72:397-402. 28 39. Chan C-TJ, Clayton-Smith J, Cheng X-J, et al. Molecular mechanisms in Angelman syndrome: a survey of 93 patients. J Med Genet 1993;30:895-902. 40. Butler MG. Prader-Willi syndrome: current understanding of cause and diagnosis. Am J Med Genet 1990;35:319-32. 41. Knoll JH, Nicholls RD, Magenis RE, Graham JM, Lalande M, Latt SA. Angelman and Prader-Willi syndromes share a common chromosome 15 deletion but differ in parental origin of the deletion. AmJMed Genet 1989;32:285-290. 42. Mutirangura A, Greenberg F, Butler MG, Malcolm S, Nicholls RD, Chakravarti A, Ledbetter DH: Multiplex PCR of three dinucleotide repeats in the PraderWilli/Angelman critical region (15q11-q13): molecular diagnosis and mechanism of uniparental disomy. Hum Mol Genet 1993;2:143-151. 43. Glatt KA, Sinnett D, Lalande M. Dinucleotide repeat polymorphism at the GABAA receptor alpha 5 (GABRA5) locus at chromosome 15q11- q13. Hum Mol Genet 1992;1:348. 44. Temple IK, Shrubb V, Lever M, Bullman H, Mackay DJ. Isolated imprinting mutation of the DLK1/GTL2 locus associated with a clinical presentation of maternal uniparental disomy of chromosome 14. J Med Genet 2007;44:637-640. 45. Buiting K, Kanber D, Martn-Subero JI, Lieb W, Terhal P, Albrecht B, Purmann S, Gross S, Lich C, Siebert R, Horsthemke B, Gillessen-Kaesbach G. Clinical features of maternal uniparental disomy 14 in patients with an epimutation and a deletion of the imprinted DLK1/GTL2 gene cluster. Hum Mutat 2008;29:1141-1146. 46. Goldstone AP, Beales PL. Genetic Obesity Syndromes. Front Horm Res 2008;36:3760. 47. Williams CA, Lossie A, Driscoll D, R.C. Phillips Unit. Angelman syndrome:
Romanian Journal of Rare Diseases (2010), 2 mimicking conditions and phenotypes. Am J Med Genet 2001;101:5964. 48. Gilfillan GD, Selmer KK, Roxrud I, Smith R, Kyllerman M, Eiklid K, Kroken M, Mattingsdal M, Egeland T, Stenmark H, Sjholm H, Server A, Samuelsson L, Christianson A, Tarpey P, Whibley A, Stratton MR, Futreal PA, Teague J, Edkins S, Gecz J, Turner G, Raymond FL, Schwartz C, Stevenson RE, Undlien DE, Stromme P. SLC9A6 mutations cause X-linked mental retardation, microcephaly, epilepsy, and ataxia, a phenotype mimicking Angelman syndrome. Am J Hum Genet 2008;82:10031010. 49. Arn PH, Williams CA, Zori RT, Driscoll DJ, Rosenblatt DS. Methylenetetrahydrofolate reductase deficiency in a patient with phenotypic findings of Angelman syndrome. Am J Med Gen 1998;77:198-200.
Corresponding author: Arsene Cosmin University of Bucharest, Faculty of Biology, Department of Genetics, 1-3 Aleea Portocalelor, sector 6, 060101 Bucharest, Romania arsene_cosmin_sinaia@yahoo.com
29
PATHOGENIC GENE RESPONSIBLE FOR THE PREDISPOSITION TO BEHETS DISEASE

Cojocaru M1, Inimioara Mihaela Cojocaru2,Isabela Silosi3 1. Dr Ion Stoia Center for Rheumatic Diseases, Department of Physiology, Faculty of Medicine,Titu Maiorescu University, Bucharest, Romania 2. Department of Neurology, Colentina Clinical Hospital, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania 3. Department of Immunology, University of Medicine and Pharmacy, Craiova, Romania
Abstract Behets disease (BD) is a rare, chronic, lifelong disorder that involves inflammation of blood vessels throughout the body. BD is a chronic inflammatory disorder characterized by recurrent oral and genital aphthous ulceration, uveitis and skin lesions. BD is a systemic inflammatory disorder of unknown etiology. Immunological abnormalities of BD are characterized by autoimmune responses against 60 kDa heat shock protein, neutrophil hyperfunction, and relative Th1 polarization with excessive production of inflammatory cytokines including tumor necrosis factor (TNF)-. Both genetic and environmental factors may be responsible for BD. Inheritance of BD refers to whether the condition is inherited from your patients or runs in families. The prevalence of familial cases in juvenile-onset BD indicates that there may be a genetic component to early expression of the disease. An association between BD and HLA-B51 has been regarded as the strongest evidence for involvement of genetic factors in the pathogenesis. The involvement of other genetic and/or environmental factors seems to be required and to be more important than B51 for the progression of BD. However, the pathogenic gene responsible for BD is as yet unknown. Key words: Behets disease, HLA-B51, disease activity Behets disease (BD) occurs throughout the world with varying prevalence. BD cases are distributed worldwide, however BD is especially prevalent in Japan, the Middle East and in some Mediterranean countries. Despite advances in genetics and genomics research, the diagnosis of BD is still determined by the clinical picture. BD is a systemic inflammatory disorder of unknown cause, which has a broad spectrum of clinical features. Four major symptoms, oral recurrent aphthous ulcer, genital ulcers, ocular lesions, and skin lesions, are commonly seen in patients with BD. Onset of disease can occur at any age, but is typically in the third decade of life. The disease is therefore a chronic multi-system one with spontaneous remissions and relapses similar to those of various autoimmune diseases (1-3). This condition was first recognized in ancient Greece by Hippocrates in the 5th century BC. The condition was later described by the Greek ophthalmologist Benedict Adamantiades (1931), and six years later by the Turkish dermatologist Hulusi Behet (4). The disease is serious and painful but it is not fatal. Although the etiology and pathogenesis of BD remains unknown, there are considerable data implicating abnormalities of the immune system, and in particular the T cells. It has been claimed that immunologic abnormalities triggered by some microbial agents or other environmental factors in genetically susceptible individuals play an 30
important role in the development of the disease. BD is not infectious, contagious, or sexually transmitted (5-8). Defects in immune responses have been reported in patients with BD. HLA class I molecules are primarily involved in the presentation of self or viral antigens to CD8+ T cells. Another function of HLA class I molecules has recently been described by the identification of a new family of receptors expressed mainly by natural killer (NK) cells, and also CD8+ cells, and T cell receptor (TCR)+ cells. These receptors, killer immunoglobulin-like receptors (KIR), bind to conserved epitopes shared by different allelic groups of HLA class I molecules, and engagement of these receptors has been shown to be associated with selective inhibition of NK cell or T cellmediated cytotoxicity. Behets disease is known to be associated with HLA-B51 in many ethnic groups. The stratification analysis indicates that this association results secondarily from a strong linkage disequilibrium with HLA-B51, and the real disease susceptibility gene which plays a part in the development of BD is most probably the HLA-B51 alleles itself. The level of inheritance of BD depends on how important genetics are to the disease. Researchers do not know how HLA-B51 increases the risk of this disorder (9-11). The strongest evidence of genetic susceptibility of BD is its association with a class I HLA antigen, HLA-B51, although the role of HLA-B51 in the pathogenesis of BD has not yet been classified. Although many people with BD have the HLAB51 variation, most people with this version of the HLA-B gene never develop the condition. The hypothesis may be presented that B51 molecules are primarily involved in BD development through specific antigen presentation (12,13). The association of HLA-B51 with specific manifestations of BD or a severe disease course has not been studied extensively and there are conflicting reports on the association of HLA-B51 with uveitis and the disease severity. HLA-B51 is more common in males than in females and is also associated with ophthalmic and more severe 31
disease. HLA-B51 heterozygosity or homozygosity is associated with familial aggregation in BD (14,15). Polymorphic analysis of the Tau- microsatellite between the HLA-B and TNF genes indicates that the pathogenic gene of BD is not the HLA-B51 gene itself but other gene located around the HLA-B gene. However, since HLA-B51 has been strongly associated with BD, relative contributions of other HLA-B alleles are possibly, being masked and cannot easily be determined using classical methods. Several novel genes have been identified by investigation of the region between the tumor necrosis factor (TNF) and HLA-B loci (16). The consistency of the strong association of HLAB51 and similar weaker positive and negative associations of some other HLA-B alleles with BD supports the hypothesis that HLA-B alleles may play a direct role in the pathogenesis of BD. Other HLA antigens have shown interesting relations with this condition. Thus, HLA-B27 is closely related to arthritic lesions and HLA-B12 to mucocutaneous lesions. Current research in this area includes investigation into new genetic factors and markers (HLA-DRw8 or HLA-B15, variants of ICAM-1 gene or major histocompatibility complex class I related gene A and many others) and to understand the role they play (17). HLA-A*2601 is possibly associated with ocular BD, independent of HLA-B*5101, indicating that HLA-A*2601 is an additional susceptibility allele candidate of ocular BD in Japan. HLA-A*2601 would also be a possible marker for poor visual prognosis (18). Further studies must be addressed to clarify the functional relevance of the different genes found to be associated with disease susceptibility and the potential interactions between genes located within and outside the MHC region. Conclusion Behets disease is now recognized as a systemic vasculitis also affecting the joints, all types and
sizes of blood vessels, lungs and the central nervous and gastrointestinal systems. It is well established that HLA-B51 is closely linked with BD. Molecular genetic studies suggest that HLAB51 might not be pathogenic itself, but indicate linkage disequilibrium with a putative susceptibility gene very close to the HLA-B locus. The strength of the association between BD and HLA-B51/B5, and its consistency across population of various ethnicities, lends further support to this allele being a primary and causal risk determinant for BD. HLA-B51 does not exhibit a strong association with a more severe disease course in BD. Weak association of HLAB antigens other than -B51 with BD have been reported. The pathogenic gene responsible for BD has not yet been clearly localized.
References 1. Al-Otaibi LM, Porter SR, Poate TWJ. Behets disease: a review. J Dent Res. 2005; 84: 209-22. 2. Yazici H, Yurdakul S, Hamuryudan V. Behet disease. Curr Opin Rheumatol. 2001; 13: 18-22. 3. Yurdakul S, Yazici H Behets syndrome. Best Pract Res Clin Rheumatol. 2008; 22(5): 793-809. 4. Yazici H, Seyahi E, Yurdakul S Behets syndrome is not so rare: why do we need to know? Arthritis Rheum. 2008; 58(12): 36403. 5. Mendes D, Correia M, Barbedo M, Vaio T, Mota M, Goncalves O, Valente J. Behets disease a contemporary review. Journal of Autoimmunity. 2009; 32: 178-88. 6. Gl A. - Behets disease: an update on the pathogenesis. Clin Exp Rheum. 2001; 19: (Suppl 24): S6-S12. 7. Bang D. - Clinical spectrum of Behets disease. J Dermatol. 2001; 28: 610-3.
1. Lawton G, Bhakta BB, Chamberlain MA, Tennant A. - The Behets disease activity index. Rheumatology 2004; 43: 73-8. 8. Mizuki N, Ota M, Yabuki K. Localization of the pathogenic gene of Behets disease by microsatellite analysis of three different populations. Invest Ophthalmol Vis Sci. 2000; 41: 3702-08. 9. Meguro A, Inoko H, Ota M, Katsuyama Y, Oka A, Okada E, Yamakawa R, Yuasa T, Fujioka T, Ohno S, Bahram S, Mizuki N. Genetics of Behet disease inside and outside the MHC. Ann Rheum Dis 2010; 69: 747-54. 10. Piga M, Mathieu A - Genetic susceptibility to Behets disease: role of genes belonging to the MHC region. Rheumatology (Oxford) 2011; 50: 299-310. 11. Bennani N, Atouf O, Benseffaj N, et al HLA polymorphism and Behets disease in Maroccan population. Pathol Biol (Paris) 2009; 57: 403-9. 12. Ahmad T, Wallace GR, James T, Neville M, Bunce M, Mulcahy-Hawes K, Armuzzi A, Crawshaw J, Fortune F, Walton R, Stanford MR, Welsh KI, Marshall SE, Jewell DP. Mapping the HLA association in Behets disease: a role for tumor necrosis factor polymorphisms? Arthritis Rheum. 2003; 48(3): 807-13. 13. Kurhan-Yavuz S, Direskeneli H, Bozkurt N. Anti-MHC autoimmunity in Behets disease. T cell responses to an HLA-B derived peptide cross-reactive with retinal-S antigen in patient with uveitis. Clin Exp Immunol. 2000; 120: 162-6. 14. Cohen R, Metzger S, Hahir M, Chajek-Shaul T. - Association of the MIC-A gene and HLA-B51 with Behets disease in arabs and non-Ashkenazi Jews in Israel. Ann Rheum Dis. 2002; 61(2): 157-60. 15. Ahn Jk, Park YG - Human leukocyte antigen B27 and B51 double-positive Behet uveitis. Arch Ophtalmol. 2007; 125: 1375-80. 16. Cohen R, Metzger S, Nahir M, Chajek-Shaul T. - Association of the MHC-A gene and 32
HLA-B51 with Behets disease in Arabs and non-Ashkenazi Jews in Israel. Ann Rheum Dis. 2002; 61: 157-60. 17. de Menthon M, Lavalley MP, Maldini C, Guillevin L, Mahr A. HLA-B51/B5 and the risk of Behets disease: a systematic review and meta-analysis of case-control genetic
association studies. Arthritis Rheum. 2009; 61: 1287-96 18. Mizuki N, Meguro A, Tohnai I, Gul A, Ohno S, Mizuki N. Association of major histocompatibility complex class I chainrelated gene A and HLA-B alleles with Behets disease in Turkey. Jpn J Ophthalmol. 2007; 51: 431-6.
Corresponding author: Manole Cojocaru Str. Thomas Masaryk No. 5 Sector 2 020983, Bucharest, Romania e-mail address: manole_cojocaru@yahoo.com
33
INFERTILITY DUE TO GENETIC ANOMALIES IN BOTH PARTNERS OF THE COUPLE. CASE REPORT
Atasie D1, Chicea R1, Ispasoiu F1, Corina Ispasoiu2 1.Polisano IVF Center Sibiu 2.Obstetrics&Gynecology Clinic Cluj-Napoca
Abstract Introduction: Genetic abnormalities are one of the causes of infertility but they are found rarely in both partners. Case report: This article presents an interesting case of an infertile couple with repeated IVF failure. The genetic testing revealed abnormalities in both partners. The female cariotype was a mozaicism 46,XX/47,XXX(83%/17%). The male cariotype showed a balanced translocation between chromosomes 9 and 15 and an inversion of the chromosome 9: 46,XY,t(9;15)(q21.2:p11.2),inv(9). The couple was counseled regarding the risks of these genetic abnormalities and decided to appeal to a sperm donor. Conclusions: although genetic abnormalities are implicated in infertility, the presence in both of the partners of the couple is a rare situation. Key words: infertility, IVF procedures, balanced translocation, mozaicism Introduction: Disorders of reproduction represent a significant social, medical, and economic burden for individuals and society. Approximately 1 in 10 couples in the United States are infertile, and each partner is equally likely to be affected. Although many causes of infertility can now be determined in both men and women, most couples still receive a diagnosis of idiopathic infertility. A subset of these patients is likely to have an underlying genetic disorder that is either inherited (germline) or acquired (somatic). Although the most severe genetic reproductive disorders cause dysgenetic gonads or abnormal hormonal profiles, milder phenotypes are being recognized with increasing frequency. Case report: This article presents an interesting case of an infertile couple with repeated IVF failure. The genetic testing revealed abnormalities in both partners. The female cariotype was a mozaicism 46,XX/47,XXX(83%/17%). The male cariotype showed a balanced translocation between 34 chromosomes 9 and 15 and an inversion of the chromosome 9: 46,XY,t(9;15)(q21.2:p11.2),inv(9). The couple was counseled regarding the risks of these genetic abnormalities and decided to appeal to a sperm donor. Discussions: The female is a carrier of a X triploidy mozaicism. Clinical female with 3 X chromosomes may be asymptomatic or have mental retardation, menstrual dysfunction, microcephaly, dental defects, strabismus, and hypertelorism; with 4 X, mental retardation is a rule, which may be accompanied by midfacial hypoplasia, micrognathia, radial synostosis, 5th finger clinodactyly, narrow shoulders, web neck; with 5 X, defects include growth retardation, FTT, mongoloid slant, saddle nose, PDA, colobomas, limb defects.(1,2,3)
Fig. 1: Cariotyp 46,XX Gonadal function is normal in majority patients, however, premature ovarian failure might occur too. They usually produce normal children, though there may be a slightly increased risk of non-disjunction occurred among them, which lead to birth of children with other abnormalities such as Down syndrome and Patau syndrome. In this case because of the predominant normal cell line with 46,XX cariotype, no phenotipic changes were observed.(2,3,5) In our case the male partner carries a balanced reciprocal translocation 46,XY,t(9;15)(q21.2:p11.2),inv(9) which can explain repeated conception failure. Reciprocal translocations are usually an exchange of material between nonhomologous 35 chromosomes. They are found in about 1 in 625 human newborns. Such translocations are usually harmless and may be found through prenatal diagnosis. However, carriers of balanced reciprocal translocations have increased risks of creating gametes with unbalanced chromosome translocations leading to miscarriages or children with abnormalities. Most of balanced translocation carriers don't have any symptoms and are healthy. But about 6% of them have many symptoms including autism, intellectual disability, congenital anomalies. A gene disrupted or disregulated at the breakpoint of the translocation carrier is likely the cause of these symptoms.
Figura 2: Cariotyp 47, XXX There are studies indicating that in spermatogenesis, there is a strict checkpoint in gametogenesis which stops meiosis when unbalanced chromosome complements are detected, leading to a reduced number of altered spermatozoa although the arrest may be overcomed and result in the production of diploid or aneuploid sperm. An inversion is a chromosome rearrangement in which a segment of a chromosome is reversed end 36 to end. An inversion occurs when a single chromosome undergoes breakage and rearrangement within itself. Inversions usually do not cause any abnormalities in carriers as long as the rearrangement is balanced with no extra or missing genetic information. However, in individuals which are heterozygous for an inversion, there is an increased production of abnormal chromatids (this occurs when crossingover occurs within the span of the inversion). This
leads to lowered fertility due to production of unbalanced gametes. The most common inversion seen in humans is on chromosome 9, at inv(9)(p11q12). This inversion is generally considered to have no deleterious or harmful effects, but there is some evidence it leads to an increased risk for miscarriage for about 30% of affected couples.(4,5,6) Conclusions: although genetic abnormalities are implicated in infertility, the presence in both of the partners of the couple is a rare situation. Also, the genetic anomaly in the male is a rare situation; we found a few papers related to this condition in literature. References: 1. Covic M.,Stefanescu D, Sandovici I. "Genetica medicala",Ed. Polirom 2004. 2. Emery Alan E.M., Alan Eglin Heathcote
2.
3.
4.
5.
Elements of medical genetics, Edinburg: Churchill Livingstone, 1992 Garver Henneth L., Marchese Sandra G. Genetic counseling for clinicians Chicago: Year Book Medical Publishers inc. S.U.A., 1986. Israil Anca-Mihaela Biologie molecular. Prezent i perspective. Ed. Humanitas, Bucureti, 2000 Mc Kussik V. A.- Mendelian inheritance in man, 11th edition, John Hopkins, University Press, Baltimore, 1994. Rooney D.E., Czelpulkowski B.H.- Human Citogenetics, A Practical Approach, vol.I-II, Oxford University, 1992.
Corresponding author: Diter Atasie diter_ro@yahoo.com
37
ESHG 2011
European
HUMAN GENETICS
Conference 2011
Amsterdam, The Netherlands, May 2831, 2011
www.eshg.org
The European Society of Human Genetics promotes research in basic and applied human and medical genetics and facilitates contact between all persons who share these aims.
A bs N tra ew c t E su du bm ca is tio sio na n l T is ra op ck en !
General Information
ESHG Scientic Programme Committee 2010 - 2011 as per date of printing Brunhilde Wirth, Chair Corinne Antignac Frank Baas Antonio Baldini Jeffrey Barrett Alexis Brice Han Brunner The-Hung Bui Bert de Vries Manolis Dermitzakis Robert Hofstra, Local Host Gunnar Houge, Secretary General-elect Helena Kriinen, Secretary General Stefan Mundlos Carla Oliveira Peter Scambler Michael Speicher Eduardo Tizzano Draga Toncheva Miikka Vikkula Cisca Wijmenga Cologne, Germany Paris, France Amsterdam, The Netherlands Naples, Italy Cambridge, United Kingdom Paris, France Nijmegen, The Netherlands Stockholm, Sweden Nijmegen, The Netherlands Geneva, Switzerland Groningen, The Netherlands Bergen, Norway Helsinki, Finland Berlin, Germany Porto, Portugal London, United Kingdom Graz, Austria Barcelona, Spain Soa, Bulgaria Brussels, Belgium Utrecht, The Netherlands
ESHG Annual Meetings Committee 2010 - 2011 President: Milan Macek Jr., CZ President-elect: Jrg Schmidtke, DE Vice-President: Jean-Jacques Cassiman, BE Secretary General: Helena Kriinen, FI Secretary General-elect: Gunnar Houge, NO Chair of the AMC: Andrew Read, UK Local Host: Robert Hofstra, NL
General Information
This international conference (now in its 42nd year) is a forum for all workers in human and medical genetics to review advances and develop research collaborations. The conference has become one of the premier events in the eld of human genetics with 2.000 delegates, more than 100 oral presentations, 12 workshops, and 8 educational sessions. The ESHG conference is where the latest developments in human genetics are discussed, and where professionals from all parts of human genetics meet.
Scientic Programme
Invited Plenary lectures and Symposia ESHG Award and Mendel lectures Educational Track throughout the meeting Workshops Concurrent Sessions of submitted abstracts Poster presentations of submitted abstracts Young Scientist and Poster Awards Conference Fellowships for young researchers from central and eastern Europe, as well as Fellowships for National Societies are available
Further details Abstract submission Scientic & Administrative Conference Secretariat Exhibition, Sponsoring, Corporate Satellites
Please consult the website www.eshg.org Online abstract submission via www.eshg.org Closing date: February 18, 2011 ESHG 2011 c/o Vienna Medical Academy Alser Strasse 4, 1090, Vienna, Austria Tel: +43 1 405 13 83 16 Email: conference@eshg.org Rose International P.O.Box 93260, 2509 AG The Hague The Netherlands Tel: +31 70 383 8901 Email: eshg@rose-international.com
Further information on programme, registration and abstract submission on
www.eshg.org
Invitation to the 42nd European Human Genetics Conference
Dear Colleagues, The next ESHG meeting will take place in Amsterdam, for the 4th time after 1972, 2000 and 2006. On behalf of the Dutch and the European Societies of Human Genetics I would like to invite you to join us in 2011. This meeting will continue in the successful tradition of excellent conferences that cover the latest developments in the eld of human genetics that are of interest for both clinicians and research scientists. Amsterdam is the largest city and the nominal capital of The Netherlands and well known for its cultural diversity, its world famous museums and universities and of course its architecture. The meeting will be lled with exciting and up-to-date scientic sessions, educational lectures and distinguished speakers, hopefully making the conference a success both from the scientic as well as from the social point of view. I hope that you will seize the opportunity to join us in Amsterdam and look forward to seeing you in May 2011.
With best regards,
Robert M.W. Hofstra Local host ESHG 2011 President of the Dutch Society of Human Genetics
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Manuscripts should be submitted to the Editor-in-Chief. The Romanian Journal of Rare Disease (RJRD) is publishing high quality peer-reviewed articles from the entire spectrum of human rare disease. Any manuscript submitted to the journal must not already have been published in another journal or be under consideration by any other journal. Manuscripts submitted for publication must contain a statement mentioning that the institutions ethics committee has approved all human and animal studies and all human studies have therefore been performed in accordance with the ethical standards laid down in an appropriate version of the 1964 Declaration of Helsinki. This statement must appear in the Methods section of the manuscript. For all articles that include information or photographs relating to individual patients, written and signed consent must be obtained from every patient prior to publish. The manuscripts will be submitted electronically using the online submission system. Before submission, please make sure that the article is prepared according to manuscript requirements stated below. Two independent experts will review submitted articles. Article types Original articles: Should not exceed 4,000 words, including references and abstract. Case reports: Up to 2,000 words, including references and abstract. Review articles: Up to 6,000 words, including references and abstract. Letter to the Editor: Up to 1,000 words. Announcements of conferences, meetings, courses, awards, and other items likely to be of interest should be submitted with contact data. Up to 100 words. Manuscript requirements The manuscript must be in English, typed single space, one column on A4 paper, with margins: top - 3 cm, bottom - 2,26 cm, left -1,5 cm, right - 1,7cm. A 12-point font Times New Roman is required. The manuscripts should be prepared according to the "Uniform requirements for manuscripts submitted to biomedical journals" (www.icmje.org). Each of the following components should begin on a separate page: Title Page, Abstract, Text, Acknowledgments, References, Tables and Figures. 1. Title page should include the following information: Article title Authors names and institutional affiliations connected trough Arabic numbers. The name of the department(s) and institution(s) to which the work should be attributed Contact information for corresponding authors Source(s) of support in the form of grants, equipment, drugs, or all of these 2. Abstract should state the purpose, basic procedures, main findings and principal conclusions of the study. The abstract must not exceed 2,000 characters (including spaces). 3. Text should be structured as followed: Introduction Material and methods Results Discussion and Conclusions 4. Acknowledgements of people, grants, funds, etc. 5. References. Citations in the text should be identified by numbers in square brackets. At the end of the paper they should be listed in numerical order corresponding to the order of citation in the text. The list of references should only include works that are cited in the text and that have been published or accepted for publication. The reference list should be structured in Vancouver style: names and initials of the first three authors, the title, source (journal abbreviations should conform to those in Index Medicus), year, volume and page numbers. Example: Citing a Journal article: Russell FD, Coppell AL, Davenport AP. In vitro enzymatic processing of radiolabelled big ET-1 in human kidney as a food ingredient. Biochem Pharmacol 1998;55:697-701. Citinq a boock: Lodish H, Baltimore D, Berk A, Zipursky SL, Matsudaira P, Darnell J. Molecular cell biology. 3rd ed. New York: Scientific American; 1995. 6. Tables and Figures should be followed by a legend numbered with Arabic numerals in order of appearance in the text and must include a brief, specific, descriptive title after the figure number.
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ROHHAD Unknown (negative for PHOX2B mutation)

childhood, early adulthood present present can be present -/?

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MULTIPLE MALFORMATIONS IN INFANT - CASE REPORT

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Fig. 4. Voiding cystourethrography 13

Fig. 3. Voiding cystourethrography

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LABORATORY STRATEGIES FOR DIAGNOSIS OF PRADER-WILLI AND ANGELMAN SYNDROMES

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PATHOGENIC GENE RESPONSIBLE FOR THE PREDISPOSITION TO BEHETS DISEASE

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Corresponding author: Diter Atasie diter_ro@yahoo.com

A bs N tra ew c t E su du bm ca is tio sio na n l T is ra op ck en !

Further information on programme, registration and abstract submission on

Invitation to the 42nd European Human Genetics Conference

With best regards,

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