We present the first description of a male with X-chromosome mosaicism who has clinical features of the X-linked CGD carrier state and passed a CYBB mutation to a daughter and subsequently affected grandson: clinicians should be aware of this possibility

Braggins, H

Buckland, MS

Gilmour, K

Harris, S

Lowe, DM

Roos, D

van Leeuwen, K

UCL Discovery

1  Male X-chromosome Mosaicism leading to Carrier Phenotype and inheritance of Chronic Granulomatous Disease Stephanie Harris MD1, Helen Braggins2, Karin van Leeuwen MSc3, Kimberly Gilmour PhD2, Matthew S Buckland MD PhD2,4,5, Dirk Roos PhD3, David M Lowe MD PhD4,5  1 Department of Infectious Diseases, Royal Free London NHS Foundation Trust, London, UK 2 Department of Immunology, Great Ormond Street Hospital, London, UK 3 Sanquin Research, Amsterdam, the Netherlands  4 Institute of Immunity and Transplantation, University College London, London, UK 5 Department of Immunology, Royal Free London NHS Foundation Trust, London, UK   Corresponding author: Dr David Lowe     Institute of Immunity and Transplantation     University College London     Royal Free Campus     Pond Street     London     NW3 2QG     United Kingdom         d.lowe@ucl.ac.uk     +44 (0)207 794 0500  Funding: Nil  Word count: 946 words Clinical implications: We present the first description of a male with X-chromosome mosaicism who has clinical features of the X-linked CGD carrier state and passed a CYBB mutation to a daughter and subsequently affected grandson: clinicians should be aware of this possibility.  Key-words: Chronic Granulomatous Disease; CGD; mosaicism Conflicts of Interest: The authors declare no conflicts of interest. 2  To the Editor, 1  2 Chronic Granulomatous Disease (CGD) is a genetic primary immunodeficiency caused by 3 mutations in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme 4 complex. This leads to impaired production of reactive oxygen species and impaired killing 5 of ingested bacteria and fungi. About two thirds of cases are in males with X-linked CGD 6 caused by mutations in the CYBB gene, coding for gp91phox, found at Xp21.1-p11.4 [1]; in 7 approximately 10-12% of families these CYBB mutations are believed to arise de novo.  8 Female carriers of X-linked CGD exhibit a range of phenotypes, although in one series 37% 9 were completely asymptomatic and 52% had no features of CGD disease [2]. Common 10 manifestations in those who are symptomatic include recurrent oral ulceration, photosensitive 11 rashes and discoid lupus erythematosus (DLE) [2-4], the latter seen in between 9% [2] and 12 40% [3] depending on population. They can also be affected by gastrointestinal conditions 13 similar to classic inflammatory bowel disease (approximately 4% [2, 3]) and have an 14 increased predisposition to other lupus-like autoimmune manifestations. Female carriers can 15 also present with recurrent or suppurative infections (15% had severe infections in a recent 16 report [2]) and indeed with extremely skewed lyonisation they can essentially develop the full 17 CGD syndrome [1]. Thus the carriage state can also confer a significant burden of disease. 18 We present a patient who was identified after his grandson was diagnosed with X-linked 19 CGD. The grandson, who is the index case, was noted to have abnormal neutrophil functional 20 tests and a c.1253C>T (p.Ser418Phe) CYBB mutation, and has subsequently undergone 21 haematopoietic stem cell transplantation. During family screening his mother was found to be 22 a carrier of this variant, but not his maternal grandmother. It was noted that instead his 23 maternal grandfather had some features consistent with a carrier state. This person (the 24 3  maternal grandfather of the index case) is 51 years old with a history of asthma, epilepsy, 25 hypertension and a significant infection history with two previous episodes of pneumonia or 26 “pleurisy” and two episodes of cellulitis. He reported sun sensitivity, arthralgia and frequent 27 ‘blistering’ of the lips. He had previously been referred to dermatology with a rash on his 28 upper chest that was diagnosed on biopsy as Jessner’s lymphocytic infiltrate of the skin 29 (JLIS) (Figure 1A & 1B) treated successfully with hydroxychloroquine. JLIS demonstrates 30 very similar characteristics clinically and on histological examination to discoid lupus 31 erythematosus [5]. He reported recurrent lower respiratory tract infection requiring antibiotics 32 as a child and a requirement for antibiotics twice a year even in adulthood. A CT chest 33 showed mild left lower lobe bronchiectasis (Figure 1C).  34 This maternal grandfather of the index case was found to have an abnormal Nitro-blue 35 tetrazolium (NBT) test and dihydrorhodamine (DHR) oxidation, both of which demonstrated 36 two populations of neutrophils upon stimulation, one capable of respiratory burst and one 37 with an impaired burst (Figure 2A & 2B). This was confirmed on repeated testing. Gp91phox 38 expression in these cells showed one positive and one negative peak (Figure 2C). Genetic 39 analysis from blood and a salivary sample via Ion Torrent™ next generation sequencing 40 confirmed that he in fact has three X-chromosome variants, two of which carry a novel CYBB 41 mutation at position c.1253 (c.1253C>T  [p.Ser418Phe] in 44% of reads, c.1253C>G 42 [p.Ser418Cys] in 7-8% of reads and the remainder wild-type; Supplementary Figure 1). He 43 had undergone vasectomy and thus could not provide a semen sample for further analysis. 44 The brother and the three other daughters of the maternal grandfather of the index case 45 underwent genetic screening but all were negative for these mutations (Figure 2D).  46  47  48 Discussion 49 4  It has previously been reported that male patients with X-linked CGD can have small 50 populations of normal cells [6], suggesting mutation in early embryogenesis, or early 51 reversion in some cell lines after the mutation arose in the oocytes. There has also been a case 52 report of an X-linked CGD patient’s brother with Klinefelter’s syndrome having carrier status 53 [7], due to inheritance of both the mutated allele of CYBB and the normal allele. Case reports 54 of triple mosaic female carriers who had populations of neutrophils with two different 55 deletion mutations in CYBB and the normal variant have been described [8], as has a late-56 onset somatic mutation in CYBB with subsequent mosaicism in haematopoietic cells in a 57 female [9]. However, this is the first report to our knowledge of a fertile male acting as a true 58 ‘carrier’ for X-linked CGD with a compatible clinical syndrome (the ‘JLIS’ is likely to 59 represent discoid lupus as seen in the CGD carrier state) and inheritance of the disease-60 causing variant in offspring. Both wild-type and mutant forms were present in blood and 61 saliva: this raises the possibility of either early reversion of a germline mutation during 62 embryogenesis or a mutation arising during an early multicellular stage with both populations 63 of cells increasing proportionally. Given that there is no evidence that his brother has the 64 mutation or clinically that there are other carriers in the family before him we conclude it is 65 likely that this is a de novo mutation.  66 Male carriage of X-linked conditions in this manner would not be picked up via routine 67 genetic screening of affected families and can lead to unintentional inheritance of the 68 mutation without appropriate genetic counselling. Indeed in this case the maternal 69 grandfather passed the gene on to one of his four daughters who subsequently had an affected 70 child. There is a potential for grandfathers who are mosaics for X-linked conditions to pass 71 on the genetic mutation to future children and, in diseases such as CGD, carriers themselves 72 can develop symptoms and significant morbidity.  We therefore recommend that clinicians 73 5  are aware of this possibility and consider at least functional testing of maternal grandfathers 74 in the presence of compatible symptoms. 75   6   References   1. Roos D, Kuhns DB, Maddalena A, Roesler J, Lopez JA, Ariga T, et al. Hematologically important mutations: X-linked chronic granulomatous disease (third update). Blood Cells Mol Dis 2010,45:246-265. 2. Marciano BE, Zerbe CS, Falcone EL, Ding L, DeRavin SS, Daub J, et al. X-linked carriers of chronic granulomatous disease: Illness, lyonization, and stability. J Allergy Clin Immunol 2018,141:365-371. 3. Battersby AC, Braggins H, Pearce MS, Cale CM, Burns SO, Hackett S, et al. Inflammatory and autoimmune manifestations in X-linked carriers of chronic granulomatous disease in the United Kingdom. J Allergy Clin Immunol 2017,140:628-630 4. Battersby AC, Cale AM, Goldblatt D, Gennery AR. Clinical manifestations of disease in X-linked carriers of chronic granulomatous disease. J Clin Immunol 2013,33:1276-1284. 5. Lipsker D, Mitschler A, Grosshans E, Cribier B. Could Jessner's lymphocytic infiltrate of the skin be a dermal variant of lupus erythematosus? An analysis of 210 cases. Dermatology 2006,213:15-22. 6. Yamada M, Okura Y, Suzuki Y, Fukumura S, Miyazaki T, Ikeda H, et al. Somatic mosaicism in two unrelated patients with X-linked chronic granulomatous disease characterized by the presence of a small population of normal cells. Gene 2012,497:110-115. 7. Gill HK, Kumar HC, Cheng CK, Ming CC, Nallusamy R, Yusoff NM, et al. X-linked chronic granulomatous disease in a male child with an X-CGD carrier, Klinefelter brother. Asian Pac J Allergy Immunol 2013,31:167-172. 8. de Boer M, Bakker E, Van Lierde S, Roos D. Somatic triple mosaicism in a carrier of X-linked chronic granulomatous disease. Blood 1998,91:252-257. 9.  Wolach B, Scharf Y, Gavrieli R, de Boer M, Roos D. Unusual late presentation of X-linked chronic granulomatous disease in an adult female with a somatic mosaic for a novel mutation in CYBB. Blood 2005;105:61-6.     7  Figure Legends Figure 1. A. Photograph of the rash diagnosed as Jessner’s lymphocytic infiltrate (diameter approximately 2cm). B. Histology from a skin lesion demonstrating superficial and deep dermal perivascular inflammation, normal epidermis on low power (left) and detail of the predominantly lymphocytic infiltrate on higher power (right). C. Axial section from CT thorax showing dilated bronchi larger than accompanying blood vessels, consistent with mild bronchiectasis (arrows).  Figure 2. A. NBT test (see Supplementary methods). The maternal grandfather of the index case (‘patient’, right) demonstrates two populations of cells, one able and one unable to reduce NBT and form blue formazan. B. Dihydrorhodamine test (see Supplementary methods). Rhodamine-1,2,3-FITC results are shown for a control (left) and the maternal grandfather of the index case (‘patient’, right) without stimulation (red histogram) and after stimulation with PMA (blue histogram), again indicating two cell populations in the maternal grandfather. C. gp91phox expression (see Supplementary methods) in neutrophils; isotype control (red histogram) and gp91phox stained (blue histogram) results are shown for a control (left) and the maternal grandfather of the index case (‘patient’, right) who has a population of gp91phox-negative cells. D. Family pedigree based on Ion Torrent™ next generation sequencing, indicating transmission of presumed deleterious CYBB mutation from the maternal grandfather to one daughter and then to a grand-son (index case) who presented with X-linked CGD.     8  Figure 1    9  Figure 2 10  Supplementary Information Supplementary Figure 1  Blood Saliva 11  Supplementary Methods NBT test. This was performed in accordance with published reports [1]. Briefly, 100 μl of EDTA whole blood was incubated with 100 μl of 0.2% NBT (Sigma) and 100 μl of 10 μg/ml phorbol 12-myristate 13-acetate (PMA) (Sigma) for 25 minutes at 37oC, then 25 minutes at room temperature before mixing with 500 μl of 2% acetic acid and trace crystal violet; 10-μl aliquots were added to counting slides and examined at x40 magnification. DHR test. This was performed in accordance with published reports [2]. The PhagoBurst™ kit (Glycotope Biotechnology) was used on heparinised whole blood according to manufacturers’ instructions and samples processed on a FACSCalibur flow cytometer. For analysis a DNA stain was used to identify live cells and then neutrophils were gated by forward and side scatter (Figure 2). Gp91phox expression. This was determined in neutrophils (gated by forward and side scatter; Figure 2) via flow cytometry using 7D5 mAb against gp91phox (IgG1, LifeSpan BioSciences) and a FITC-conjugated rabbit-anti-mouse IgG.    References 1. Meerhof LJ, Roos D. Heterogeneity in chronic granulomatous disease detected with an improved nitroblue tetrazolium slide test. J Leukoc Biol 1986,39:699-711. 2. Vowells SJ, Sekhsaria S, Malech HL, Shalit M, Fleisher TA. Flow cytometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes. J Immunol Methods 1995,178:89-97.  Supplementary Figure Legend Supplementary Figure 1. Results of Ion Torrent™ next generation sequencing of the CYBB gene in the patient’s blood and saliva.    

Male X-chromosome mosaicism leading to carrier phenotype and inheritance of chronic granulomatous disease

http://discovery.ucl.ac.uk/10050857/1/Lowe_Harris%20CGD%20carrier%20case%20report_combined.pdf

Male X-chromosome mosaicism leading to carrier phenotype and inheritance of chronic granulomatous disease

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