Detection of a novel gross deletion in the UNC13D gene ends the diagnostic odyssey for a family with familial hemophagocytic lymphohistiocytosis 3

doi:10.21203/rs.3.rs-2596196/v1

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Case Report

Detection of a novel gross deletion in the UNC13D gene ends the diagnostic odyssey for a family with familial hemophagocytic lymphohistiocytosis 3

https://doi.org/10.21203/rs.3.rs-2596196/v1

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published 11 Jan, 2024

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Background: Familial hemophagocytic lymphohistiocytosis (FHL) is an immunological disorder characterized by overactivation of macrophages and T lymphocytes. This autosomal recessive condition has been characterized into multiple types depending on the genetic etiology. FHL type 3 is associated with bi-allelic pathogenic variants in the UNC13D gene.

Case presentation: We present a 12-year diagnostic odyssey for a family with FHL that signifies the advances of FHL genetic testing in a diagnostic genetics laboratory with expertise in heritable immunodeficiencies. We describe the first case of a large UNC13D gross deletion in transto a sequence variant in a family with FHL3, which may have been mediated by Alu elements within introns 12 and 25 of the UNC13D gene.

Conclusions: This case highlights the importance of reevaluating past genetic testing for a patient and family as test technology evolves in order to end a diagnostic odyssey.

Familial hemophagocytic lymphohistiocytosis type 3

UNC13D

Deletion

Diagnostic odyssey

Familial hemophagocytic lymphohistiocytosis (FHL) is an immunological disorder characterized by overactivation of macrophages and T lymphocytes. Organs, such as the liver and spleen, are affected by this uncontrolled immune response and patients typically require a haematopoietic stem cell transplant. This autosomal recessive condition has been characterized into multiple types, with FHL type 3 being associated with pathogenic variants in the UNC13D gene [1, 2]. Currently, the two most common genes associated with FHL, PRF1 (FHL2 OMIM # 603553) and UNC13D (FHL3 OMIM # 608897), account for around 70% of FHL cases in some populations [3, 4].

To date, there have been over 180 variants reported in UNC13D in association with FHL3, with the vast majority being missense or nonsense variants that result in loss of function. Meeths et al. [5] reported a 253 kb inversion in UNC13D in association with FHL3. Gross deletions and duplications are far less common in the reported literature, with the first gross duplication being reported recently by Hiejima et al. [6]. The deletions in UNC13D that have been reported to date range from 24–90 base pairs [7–9].

Here, we present a 12-year diagnostic odyssey for a family with FHL that signifies the advances of FHL genetic testing technology in a diagnostic genetics laboratory with expertise in heritable immunodeficiencies. In this case we identified a gross deletion involving UNC13D in trans to a sequence variant. To the best of our knowledge, this is the first gross deletion reported in the UNC13D gene.

The proband (II-1) is the first born of a non-consanguineous couple with Indian (father, I-1) and Irish/ Scottish (mother, I-2) ancestry (Fig. 1). He was born in 2006 and presented early in infancy with fevers, pancytopenia, and upregulated cytotoxic proteins in cytotoxic cells. He was clinically diagnosed with hemophagocytic lymphohistiocytosis (HLH) and passed away at the age of 4 months. Genetic testing, comprising of PRF1, UNC13D, and SH2D1A sequencing, was performed posthumously using tissue sample at Laboratory of Genetics and Genomics (LGG) at Cincinnati Children’s Hospital Medical Center and identified variants in two of the genes. A heterozygous nonsense variant was identified in UNC13D, [Chr17(GRCh37): g.73839332G > T, NM_199242.2] c.169G > T (p.Glu57*), in 2006. A missense variant identified in PRF1, [Chr10(GRCh37): g.72358167G > A, NM_001083116.1] c.1310C > T (p.Ala437Val), was classified as a VUS in 2006 and still remains a VUS at this time. A second variant was not identified in either UNC13D or PRF1 at that time. Parental carrier testing ordered at LGG by Hudson Valley Perinatal Consulting (Lescale Maternal Fetal Medicine) revealed that both the UNC13D and PRF1 variants were maternally inherited.

The couple had a second son in 2008 (II-2) who has been healthy to date and had genetic testing in 2013 at LGG, which identified him to be a carrier of the familial UNC13D nonsense variant. He has been clinically monitored according to HLH protocol.

The couple had two pregnancies in 2010 (II-3 and II-4) and opted for prenatal exclusion testing for the familial UNC13D nonsense variant at LGG. Both pregnancies were positive for the UNC13D nonsense variant, putting them each at a 50% risk of FHL. The family chose to terminate the pregnancies.

The couple had another pregnancy in 2011 (II-5). Prenatal exclusion testing for the familial UNC13D variant performed at an external laboratory was positive for this pregnancy. The couple decided to proceed with this pregnancy and had a son, who was clinically diagnosed with HLH and passed away at the age of 4 years.

The couple was pregnant again in 2018 (II-6). At this time, the family and their providers considered additional testing options that were available since the initial testing in an attempt to further understand the genetic etiology of the FHL diagnosis in the family. Additional testing on the father, which included sequencing of PRF1, UNC13D, STX11, STXBP2, RAB27A and deletion/duplication analysis by array comparative hybridization (aCGH) of UNC13D at LGG, identified a novel partial UNC13D deletion involving exons 13–25. Deletion/duplication analysis of UNC13D was negative for the mother. Prenatal testing for the paternal UNC13D deletion was positive for the current pregnancy. Prenatal testing for the familial UNC13D nonsense variant performed at an external laboratory was negative. The karyotype and microarray results of this pregnancy were 46, XX and negative for any clinically significant structural or copy number variations. Their asymptomatic son (II-2) has not been tested for the paternal UNC13D deletion to date.

Sanger sequencing of UNC13D on the proband’s genomic DNA revealed a heterozygous pathogenic nonsense variant, c.169G > T (p.Glu57*), which was later found to be maternally inherited through parental targeted variant analyses. However, the second disease causing variant was not detected in the proband by sequencing. Extended sequencing analyses of the entire coding regions, exon/intron boundaries, and the deep intronic c.118–308 mutation region, as well as the allele specific analysis for the pathogenic 253 kb inversion in UNC13D were performed on the father’s DNA, but no disease-causing variants were identified. Sequencing of PRF1 and SH2D1A on the proband’s DNA and sequencing of PRF1, STX11, STXBP2, and RAB27A on his father’s DNA did not reveal any pathogenic or likely pathogenic variants.

Targeted array comparative genomic hybridization (aCGH) was performed for the UNC13D gene on the father’s DNA according to previously reported protocols [10]. aCGH identified a partial heterozygous deletion of exons 13 to 25 of the UNC13D gene with breakpoints in introns 12 and 25 (Fig. 2). The deletion is between 7.63 kb and 8.19 kb in size, and has the following minimum breakpoints: chr17:73828213–73835841 and maximum breakpoints chr17: 73827722–73835910. To the best of our knowledge, this is the first report of a gross exonic deletion of the UNC13D gene.

This case highlights the importance of considering deletion/duplication analysis for patients with inconclusive or negative genetic testing results. This family was previously reported as a possible example of digenic inheritance since a second pathogenic variant in UNC13D had not been identified [11]. Clinicians should be aware that deletion/duplication analysis was not historically available in many laboratories and may be crucial for diagnosis in some cases [12, 13]. For instance, deletion/duplication analysis through aCGH was established in 2014 at LGG, 8 years after the proband in this case had inconclusive testing. In addition, next-generation sequencing copy number variant analysis, which has become more common, may miss some clinically significant copy number variants, particularly deletions of less than three exons [14]. Recent studies have identified gross deletions and duplications in other genes related to HLH, including PRF1, STX11, and STXBP2 [15–18]. In addition, a 253 kb UNC13D inversion has been identified that requires additional manual analysis steps [5]. Testing for this UNC13D inversion has been included in the UNC13D testing at LGG since December 2011.

It is hypothesized that the deletion on the father’s allele was mediated by Alu elements within introns 12 and 25 of the UNC13D gene. Hiejima et al. [6] described Alu elements within introns 6, 12, 25 and 30, and reported the first known in-frame intragenic duplication of UNC13D caused by aberrant recombination between the Alu elements within introns 6 and 12. Therefore, we suspect that the reported deletion was mediated by unequal crossing over of Alu elements, particularly the FRAM Alu within intron 12 and the AluSc within intron 25. This would be the second report of a copy number variant within the UNC13D gene mediated by Alu elements [6]. These events, yet rare, reiterate the importance of deletion/duplication testing in FHL3 cases when only one pathogenic allele has been identified.

Clinicians should periodically revisit genetic testing for patients with unsolved cases [19]. One limitation to continued evaluation is that the family may not present for care until another pregnancy is ongoing, which introduces time constraints. In addition, continuity of care may be disrupted when initial genetic testing was ordered for a proband who is now deceased. The family may next present for genetic testing when they see a maternal fetal medicine (MFM) specialist, OB/GYN, and/or prenatal genetic counselor who did not order the initial testing. We suggest that clinicians should periodically review all past genetic test results for patients with unsolved cases and should encourage these families to periodically contact them for updates. We also recommend that providers contact genetic testing labs to determine if any updates have been made to the variant classification for variants of uncertain significance and inquire if test updates or new technology is available. When possible, providers should send targeted testing to the same laboratory for all family members to ensure consistency and accuracy.

Herein we report the first large deletion of the UNC13D gene in trans to a nonsense sequence variant causing FHL type 3. This case highlights the importance of reevaluating past genetic testing for a patient and family as test technology evolves in order to end a diagnostic odyssey.

Ethics approval and consent to participate

According to Cincinnati Children’s Institutional Review Board, an ethics committee approval is not required for review and publication of a case report. Consent from the family for participation and publication was obtained, which is considered sufficient.

Consent for publication

The authors have written consent from the family to review and publish the case report.

Availability of data and materials

The datasets generated and/or analysed during the current study are available in the ClinVar repository, ID SCV003806696, https://www.ncbi.nlm.nih.gov/clinvar/variation/2442244/?oq=SCV003806696&m=Single+allele

Competing interests

The authors declare that they have no competing interests.

Funding

No funding to be declared.

Authors' contributions

Chinmayee B. Nagaraj, Diana S. Brightman, Hannah Rea, Emily Wakefield made substantial contributions to the conception and drafting of the manuscript, all authors interpreted the data and substantively revised the manuscript.

Acknowledgements

Not applicable

Gholam, C., et al., Familial haemophagocytic lymphohistiocytosis: advances in the genetic basis, diagnosis and management. Clin Exp Immunol, 2011. 163(3): p. 271-83.
Feldmann, J., et al., Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3). Cell, 2003. 115(4): p. 461-73.
Cetica, V., et al., Genetic predisposition to hemophagocytic lymphohistiocytosis: Report on 500 patients from the Italian registry. J Allergy Clin Immunol, 2016. 137(1): p. 188-196.e4.
Nagai, K., et al., Subtypes of familial hemophagocytic lymphohistiocytosis in Japan based on genetic and functional analyses of cytotoxic T lymphocytes. PLoS One, 2010. 5(11): p. e14173.
Meeths, M., et al., Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) caused by deep intronic mutation and inversion in UNC13D. Blood, 2011. 118(22): p. 5783-93.
Hiejima, E., et al., Characterization of a large UNC13D gene duplication in a patient with familial hemophagocytic lymphohistiocytosis type 3. Clin Immunol, 2018. 191: p. 63-66.
Poltavets, N.V., et al., Seven new mutations have been found in STX11, PRF1 and UNC13D genes in a group of Russian FHL patients. Genomic Med, 2008. 2: p. 241-252.
Sieni, E., et al., Genotype-phenotype study of familial haemophagocytic lymphohistiocytosis type 3. J Med Genet, 2011. 48(5): p. 343-52.
Zhang, K., et al., Hypomorphic mutations in PRF1, MUNC13-4, and STXBP2 are associated with adult-onset familial HLH. Blood, 2011. 118(22): p. 5794-8.
Dyer, L., et al., Gene dosage defects in primary immunodeficiencies and related disorders: a pilot study. Journal of Translational Genetics and Genomics, 2017. 1: p. 23-27.
Zhang, K., et al., Synergistic defects of different molecules in the cytotoxic pathway lead to clinical familial hemophagocytic lymphohistiocytosis. Blood, 2014. 124(8): p. 1331-4.
Giugliano, T., et al., Copy Number Variants Account for a Tiny Fraction of Undiagnosed Myopathic Patients. Genes (Basel), 2018. 9(11).
Truty, R., et al., Prevalence and properties of intragenic copy-number variation in Mendelian disease genes. Genet Med, 2019. 21(1): p. 114-123.
Välipakka, S., et al., Improving Copy Number Variant Detection from Sequencing Data with a Combination of Programs and a Predictive Model. J Mol Diagn, 2020. 22(1): p. 40-49.
Omoyinmi, E., et al., Clinical impact of a targeted next-generation sequencing gene panel for autoinflammation and vasculitis. PLoS One, 2017. 12(7): p. e0181874.
Tesi, B., et al., Targeted high-throughput sequencing for genetic diagnostics of hemophagocytic lymphohistiocytosis. Genome Med, 2015. 7: p. 130.
Tong, C.R., et al., [The study of gene mutations in unknown refractory viral infection and primary hemophagocytic lymphohistiocytosis]. Zhonghua Nei Ke Za Zhi, 2011. 50(4): p. 280-3.
zur Stadt, U., et al., Linkage of familial hemophagocytic lymphohistiocytosis (FHL) type-4 to chromosome 6q24 and identification of mutations in syntaxin 11. Hum Mol Genet, 2005. 14(6): p. 827-34.
Deignan, J.L., et al., Points to consider in the reevaluation and reanalysis of genomic test results: a statement of the American College of Medical Genetics and Genomics (ACMG). Genet Med, 2019. 21(6): p. 1267-1270.

No competing interests reported.

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published 11 Jan, 2024

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You are reading this latest preprint version

Detection of a novel gross deletion in the UNC13D gene ends the diagnostic odyssey for a family with familial hemophagocytic lymphohistiocytosis 3

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