Clinical presentations of 23 half-siblings from a mosaic neurofibromatosis type 1 sperm donor

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1 Clin Genet 2016: 89: Printed in Singapore. All rights reserved Short Report 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: /cge Clinical presentations of 23 half-siblings from a mosaic neurofibromatosis type 1 sperm donor Ejerskov C., Farholt S., Skovby F., Vestergaard E.M., Haagerup A. Clinical presentations of 23 half-siblings from a mosaic neurofibromatosis type 1 sperm donor. Clin Genet 2016: 89: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2015 The Danish sperm donor number 7042 has fathered several offspring with neurofibromatosis type 1 (NF1) worldwide. NF1 is caused by loss-of-function mutations in the NF1 gene and more than 1000 NF1 mutations are identified. Analysis of the donor sperm demonstrated gonosomal mosaicism with an intragenic deletion involving exons in the NF1 gene. At the two Danish reference centres for NF1 patients, we evaluated 23 half-siblings from the donor. Nine were diagnosed with NF1. The severity grade of NF1 progressed from minimal to mild/moderate within 3 years of follow-up. The NF1 phenotype shows great variability in intraand inter-family expressivity and to date only two NF1 genotype phenotype correlations have been established. This rare possibility of a long-term follow-up of a cohort of half-siblings with NF1 makes further studies including phenotypic variability and search for modifier genes possible. To achieve this goal, we have initiated The International Donor 7042 NF1 Offspring Registry. Research facilitated via this registry may reveal important new knowledge of clinical characteristics and prognostics for the specific NF1 genotype and thereby contribute to future individualised targeted clinical follow-up and treatment. Conflict of interest The authors declare no conflict of interest. C. Ejerskov a,s.farholt a, F. Skovby b, E.M. Vestergaard c and A. Haagerup a a Centre for Rare Diseases, Department of Paediatrics, Aarhus University Hospital, Aarhus, Denmark, b Centre for Rare Diseases, Department of Clinical Genetics, Copenhagen University Hospital, Copenhagen, Denmark, and c Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark Key words: 17q11.2 genotype phenotype correlation gonosomal mosaicism neurofibromatosis type 1 Corresponding authors: Cecilie Ejerskov, MD, Centre for Rare Diseases, Department of Paediatrics, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, DK-8200 Aarhus N, Denmark Tel.: ; fax: ; cecilie@ejerskov.dk or Dr Annette Haagerup, Centre for Rare Diseases, Department of Paediatrics, Aarhus University Hospital, Palle Juul-Jensens Boulevard 99, DK-8200 Aarhus N, Denmark Tel.: ; fax: ; annehaag@rm.dk Received 10 January 2015, revised and accepted for publication 13 April 2015 An anonymous sperm donor, Nordic Cryobank ApS no 7042, has fathered several offspring with neurofibromatosis type 1 (NF1) worldwide (1). The first affected offspring was reported from Belgium in Following the detection of two additional reports in the United States, genetic analysis of donor sperm demonstrated an intragenic deletion involving exons (previous exon numbering ) (c.1642-?_3974+?del) in the NF1 gene with 20% gonosomal mosaicism (2). NF1 is an autosomal dominant multiorgan disease with complete penetrance and a variable expressivity (3). Worldwide, an incidence of 1 in 2500 to 1 in 3500 makes NF1 one of the most common monogenic disorders (4, 5). The NF1 gene encodes the GTPase-activating protein neurofibromin. Neurofibromin is a multidomain molecule with the capacity to regulate several intracellular processes, including the RAS-cAMP pathway and the mitogen-activated protein kinase cascade. 346

2 Clinical presentations of 23 half-siblings NF1 is characterised by café-au-lait macules, skin fold freckling, Lisch nodules and neurofibromas. Some NF1 patients suffer from severe complications such as neurological and cognitive deficits, skeletal complications, and malignancies, while others have only mild cutaneous manifestations (6). The NF1 phenotype also shows great variability in intra- and inter-family expressivity (7 9). To date only two NF1 genotype phenotype correlations have been established: the NF1 microdeletion syndrome giving a relatively severe phenotype, and the del AAT exon 17 [3 base pair (bp) inframe deletion of exon 17 (c delaat)] giving a mild phenotype (10 13). More studies of geno phenotype correlations are needed to better understand the disease pathophysiology and the phenotypic diversity, which eventually may make future individualised follow-up and treatment possible. The total number worldwide of donor 7042 offspring is undisclosed. In September 2012, based on information from the Danish National Board of Health, the Danish Broadcasting Corporation (DR) reported on 43 children conceived in Danish fertility clinics, of whom 25 are Danish citizens and 18 are citizens in Norway and Sweden. Specimens of sperm from donor 7042 have been used also in United States, Canada, Belgium, Iceland, Georgia, Greece, Spain, Thailand and maybe in other countries. According to Cryos International Denmark ApS which exports sperm to more than 70 countries, one single donor can possibly father more than 200 children globally (14, 15). From personal communications with clinicians and patient associations, we know of children of donor number 7042 in Norway, Sweden, Iceland, United States and Belgium. Since 2010 the Centres for Rare Diseases, Aarhus University Hospital (CRD-AUH) and Copenhagen University Hospital (CRD-CUH), the two Danish reference centres for NF1, have examined 22 Danish and 1 Icelandic offspring of donor number 7042 of whom 9 have NF1. This is a short report of 23 half-siblings who are all offspring of a single sperm donor with gonosomal mosaicism for a NF1 deletion. To allow future follow-up, we have initiated The International Donor 7042 NF1 Offspring Registry. Materials and Methods Cohort By the end of December 2009, the initial referrals came to the Centres for Rare Diseases at AUH and CUH (CRDs). A total of 22 of the 25 reported Danish children and one Icelandic child conceived by use of Donor 7042 semen were referred to the CRDs from general practitioners or fertility clinics. Prior to the referrals, the families had been informed about the risk of NF1 via letter from the fertility clinics. During the period January 2010 to October 2012, 19 children had a physical examination done by one of the three senior CRDs physicians specialised in paediatrics or genetics and with extensive experience in NF1. A clinical diagnosis was made in nine cases based on the diagnostic criteria Table 1. Demographic data at referral and for clinical data at first Children with NF1 Children without NF1 Demographic data upon referral to CRDs Number 9 14 Females/males 7/2 8/6 Median age, months, 15 (4 38) 17 (5 58) a Clinical data from first Number 9 7 b Median age, months, 15 (4 38) 15.5 (5 29) Average height, SD c, 1.4 d ( 4 to 0.5) 0.1 ( 2 to1) Average weight, SD, 1.2 d ( 3 to1) 0.4 ( 2.5to0.5) [range] Average head circumference, SD, 1.7 ( ) 0.3 ( 1 to1) SD, standard deviation. a Median age of 11 of 14 children, because two children were tested prenatally and of one child tested post-natally we have no information about age. b Four children only had mutational analyses done and clinical data are missing for three children. c Standard deviation from median plotted on a female/male diagram of height, weight and head circumference. d Data missing for one child. of NF1 as stated by the National Institutes of Health Consensus Conference (16). Four children with normal pre- or post-natal genetic analysis of NF1 gene were not clinically examined. No clinical information is available on the remaining three Danish cases (3/25). Protocol The study is a prospective cohort study. According to the national follow-up guidelines for children with NF1 the patients are seen at the CRDs every 6 months or as needed and MRI cerebrum is performed when clinically indicated, i.e. neurological symptoms or pathological ophthalmological examination. The NF1 unaffected siblings were discharged after the first visit at CRD. Demographic data at referral and for clinical data at first are displayed in Table 1. A registration form of clinical characteristics has been developed at CRD-AUH. At each visit the form is filled out by CRDs specialists in paediatrics or genetics. The following parameters are noted: (i) Demographics; age, height, weight, head circumference, (ii) Manifestations of the skin, the eyes, the central and peripheral nervous system, the cardiovascular system and the bones, (iii) Development; learning disabilities, a diagnosis of ADHD made by a specialist in child psychiatry, and current assessment of delayed development by a physiotherapist and/or a psychologist, and (iv) other NF1-related or non NF1-related disease. Furthermore, disease severity and visibility are assessed according to the Riccardi and 347

3 Ejerskov et al. Ablon scales, respectively, and the scales are presented in Table A1 (17, 18). Ethics The protocol follows Danish legislation on clinical research. According to The Danish Research Ethics Committee System no approval is needed. The International Donor 7042 NF1 Offspring Registry has been approved by the Danish Data Protection Agency (ID ). Genetic analysis Multiplex Ligation-dependent Probe Amplification (MLPA) analysis was performed on DNA extracted from blood according to the manufacturer s instructions. The kit used was SALSA MLPA probemix P081-B2/P082-B2 for NF1 (MRC-Holland BV, Amsterdam, The Netherlands). Products were run on an automated sequencer (ABI 3500XL, Applied Biosystems, Foster City, CA) and data were analysed using Genemarker version (Softgenetics, State College, PA). A positive control was included in all runs. We have used the exon numbering according to the NCBI mrna reference sequences NM_ (19). The variant was submitted to the LOVD, Leiden Open Variant Database, for NF1. Statistics The exact binomial test was used to calculate the NF1 transmission rate including the confidence interval (CI). p < 0.05 was considered statistically significant. Results In Table 2 we present the clinical characteristics of the nine half-siblings with the clinical diagnosis of NF1 at the first and at latest in Although the observational period is relative short, follow-up shows that the severity grade progressed from minimal in all children to the more severe grades. There was no progression of visibility grade. Three children each had one additional finding in the central nervous system, an arachnoid cyst, a failure of anterior pituitary gland with confirmed growth hormone deficiency, and a low-grade brainstem glioma. Furthermore, a fourth child underwent surgery for both scaphocephaly and a vascular ring; the child also had unilateral double kidney system. A fifth child was treated for orbital infantile haemangioma. These malformations are likely unrelated to NF1. Of the 23 half-siblings, 9 half-siblings [0.39, (95% CI: )] inherited NF1. In all nine half-siblings the variant with intragenic deletion of exons of NF1 (previous numbering ) was confirmed. The observed transmission rate of 39% (95% CI: 20 61%) does not differ significantly from the expected risk of Table 2. Clinical characteristics of nine half-siblings with NF1 At first At latest Median age, months, 15 (4; 38) 54 (28;75) Demographic data Average height, SD a, 1.4 b ( 4 to 0.5) 0.3 ( 3 to1.3) Average weight, SD, 1.2 b ( 3 to1) 0.2( 1.5 to 2.4) Average head circumference, SD, 1.7 ( ) 2.0 (0.5 4) NF1 diagnostic criteria, no. of children Café-au-lait macules 9 9 Skin fold freckling 3 9 Neurofibromas 2 3 Plexiform 2 2 neurofibromas Lisch nodules 1 3 Osseous dysplasia 0 1 Optic glioma, unilateral, 0 2 diagnosed on MRI Optic glioma, bilateral, 0 1 diagnosed on MRI Magnetic resonance 0 8 imaging (MRI), brain UBO 8 Intellect Ongoing assessment of 1 4 delayed development NF1 severity, Riccardi score Grade 1, minimal 9 2 Grade 2, mild 0 4 Grade 3, moderate 0 3 Grade 4, severe 0 0 NF1 visibility, Ablon score Grade 1, mild 9 9 Grade 2, moderate 0 0 Grade 3, severe 0 0 SD, standard deviation. a Standard deviation from median plotted on a female/male diagram of height, weight and head circumference. b Data missing for one child. 50% for autosomal dominant disorders (exact binomial test p = 0.4). The 23 half-siblings were from 17 families. When assessing the relationship between the nine children with NF1, none were full siblings. The cohort consists of 13 one-child families, two families with two children, and two families with three children. Discussion We present a relatively large group of NF1-affected half-siblings. The severity scores during diagnosis were of minimal grade. As the patients grew older, there was a disease progression to more severe degrees in seven of nine children, which underlines the increase in 348

4 Clinical presentations of 23 half-siblings severity grade over time and the phenotypic variability. A noticeable number of children have neurofibromas and gliomas, their young ages taken into consideration (20). We do not yet see an increase in visibility grade, but as the children get older, this would be expected. Our cohort is a non-selected group since all 23 siblings were referred before the diagnosis of NF1 was confirmed or excluded clinically or genetically. Thereby we are able to illuminate the clinical spectrum in the affected offspring from infancy. At present we are not able to state whether the spectrum of phenotypes in this group of NF1-affected children differs from other NF1 phenotypes. Also, we must take into account a potential genetic contribution from yet unknown genes modifying the NF1 phenotype. The Nordic Cryobank ApS interpreted 20% of the gonosomal mosaicism as a 10 20% risk of NF1 among the offspring (21). In our data the observed transmission rate of 9 of 23 (39%) does not differ significantly from the expected risk of 50% in any autosomal disorder. The rate of transmission is in accordance with two previous reports where three of six and two of three siblings had NF1 in spite of a germ line mosaicisms of 10% and 10 17%, respectively (22, 23). We have to take into account the limited size of our dataset. However, we believe that we have included close to all Danish offspring of donor 7042 as an information letter from the fertility clinics to the families were sent at a stage of unknown disease status, and the number is consistent with the report from the Danish National Board of Health. The conception of a cohort of half-siblings with NF1 was unintended and unexpected for the sperm donor and the involved families. A cohort of half-siblings with NF1 gives new possibilities to investigate NF1 pathophysiology and genetics. The NF community in Denmark is supportive of pursuing this work. Therefore we have initiated The International Donor 7042 NF1 Offspring Registry to secure future scientific follow-up. Moreover, the registry will facilitate research in phenotypic variability and the search for NF1 modifier genes. This may reveal important new knowledge of clinical characteristics and prognostics for the specific NF1 genotype, and thereby contribute to future individualised targeted clinical follow-up and treatment. We call for international collaboration from physicians worldwide regarding offspring with NF1 from sperm donor The corresponding authors will provide information about the registry, participation, informed consent, and sample shipment upon request from physicians of donor offspring. Acknowledgements We thank our many clinical colleagues who contributed to the NF1 clinical set-up at Aarhus University Hospital and Copenhagen University Hospital and the board of NF Denmark for ongoing collaboration. No support or funds were given for this study. References 1. Haagerup A, Pedersen C, Farholt S. A mosaic sperm donor fathers a large group of half sibs with NF1 2012, from Accessed on October 7, Callum P, Messiaen LM, Bower PV et al. Gonosomal mosaicism for an NF1 deletion in a sperm donor: evidence of the need for coordinated, long-term communication of health information among relevant parties. Hum Reprod 2012: 27 (4): OMIM. Online Mendelian Inheritance in Man, OMIM. Johns Hopkins University, Baltimore, MD. MIM Number: {162200} 2013, from Accessed on December 29, Shen MH, Harper PS, Upadhyaya M. Molecular genetics of neurofibromatosis type 1 (NF1). J Med Genet 1996: 33 (1): Williams VC, Lucas J, Babcock MA et al. Neurofibromatosis type 1 revisited. Pediatrics 2009: 123 (1): Ferner RE. Neurofibromatosis 1. Eur J Hum Genet 2007: 15 (2): Szudek J, Joe H, Friedman JM. Analysis of intrafamilial phenotypic variation in neurofibromatosis 1 (NF1). Genet Epidemiol 2002: 23 (2): Rieley MB, Stevenson DA, Viskochil DH et al. Variable expression of neurofibromatosis 1 in monozygotic twins. Am J Med Genet A 2011: 155A (3): Sabbagh A, Pasmant E, Laurendeau I et al. Unravelling the genetic basis of variable clinical expression in neurofibromatosis 1. Hum Mol Genet 2009: 18 (15): Upadhyaya M, Huson SM, Davies M et al. An absence of cutaneous neurofibromas associated with a 3-bp inframe deletion in exon 17 of the NF1 gene (c delaat): evidence of a clinically significant NF1 genotype-phenotype correlation. Am J Hum Genet 2007: 80 (1): Mautner VF, Kluwe L, Friedrich RE et al. Clinical characterisation of 29 neurofibromatosis type-1 patients with molecularly ascertained 1.4 Mb type-1 NF1 deletions. J Med Genet 2010: 47 (9): De Raedt T, Brems H, Wolkenstein P et al. Elevated risk for MPNST in NF1 microdeletion patients. Am J Hum Genet 2003: 72 (5): Pasmant E, Sabbagh A, Spurlock G et al. NF1 microdeletions in neurofibromatosis type 1: from genotype to phenotype. Hum Mutat 2010: 31 (6): E1506 E DR. Syge børn i udlandet fra donor 7042 og Donorsæd er ude af kontrol 2012, from DR. Dansk sædbank dækkede over arvelig sygdom 2012, from Stumpf D. Neurofibromatosis Conference statement. National Institutes of Health Consensus Development Conference. Arch Neurol 1988: 45 (5): Ablon J. Gender response to neurofibromatosis 1. Soc Sci Med 1996: 42 (1): Riccardi VM, Kleiner B. Neurofibromatosis: a neoplastic birth defect with two age peaks of severe problems. Birth Defects Orig Artic Ser 1977: 13 (3C): van Minkelen R, van Bever Y, Kromosoeto J et al. A clinical and genetic overview of 18 years neurofibromatosis type 1 molecular diagnostics in the Netherlands. Clin Genet 2013: 8: Kaufmann D, (ed). Neurofibromatoses, Vol. 16. Basel: Karger, Paper from Nordic Cryobank to the Danish Parliament 2012, from Bottillo I, Torrente I, Lanari V et al. Germline mosaicism in neurofibromatosis type 1 due to a paternally derived multi-exon deletion. 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5 Ejerskov et al. Appendix Table A1. Disease severity and visibility scores assessed using the Riccardi and Ablon scales, respectively NF1 severity score: the Riccardi scale (19) Minimal Mild Moderate Severe NF1 visibility score: the Ablon scale (18) Mild Moderate Severe The presence of few features of NF with no compromise of health or well-being. The presence of enough stigmata to make the disease obvious and a source of concern, but without significant compromise of health. For example, the patient may exhibit facial café-au-lait spots or a modest number of cutaneous or deep neurofibromas. An unequivocal compromise of health and well-being, but the compromise can be reasonably well managed, is not intractable, and will not invariably lead to a shortened life span. Because many features of NF can lead to this level of clinical problems, the category of severity grade 3 is necessarily broad, spanning a relatively large age range. The presence of serious compromise that is intractable, is managed or treated only with difficulty, or is associated, at least statistically, with a shortened life span. Mental retardation, drug-resistant seizures, brain tumours, and malignant tumours contribute to this category of severity. Essentially no tumours are visible outside of normal Western clothing areas; gait and posture appear unremarkable when casually observed. Some tumours appear on the neck, face, and hands, and mild scoliosis or other skeletal features are present but without a noticeable limp. Numerous tumours appear on the face; optic glioma (tumour) affects sight and the eye socket; severe scoliosis or skeletal features are present causing a noticeable limp. 350