Donor-type bone marrow aplasia following hematopoietic stem cell transplantation in a child with a novel SAMD9L variant

ABSTRACT

Pathogenic variants in the genes SAMD9 (sterile a-motif domain containing protein – 9) and SAMD9L (SAMD9-like) cause bone marrow failure with characteristic syndromic features. We report a case of a previously healthy, 3-year-old boy with no dysmorphology, who presented with severe aplastic anemia and a novel variant in the SAMD9L gene. His father, elder brother and sister who harbored the same variant were completely healthy. In the absence of a matched unrelated donor, he underwent a stem cell transplant from his sister, a 10/10 match. Almost 2 years later he developed donor type aplasia and succumbed to an invasive fungal infection after a failed haplograft from his mother. This case highlights the pathogenicity of this previously undescribed germline variation of uncertain significance in the SAMD9L gene and the value of comprehensive genetic testing for inherited bone marrow failures even in the absence of a positive family history or characteristic congenital abnormalities.

KEYWORDS:

• SAMD9
• SAMD9L
• aplastic anemia
• donor-type aplasia
• bone marrow failure syndromes

1. Introduction

Inherited bone marrow failure syndromes (BMFS)/myelodysplastic syndromes (MDS) represent an expanding list of disorders with considerable genetic and phenotypic heterogenicity. The genes SAMD9 (sterile a-motif domain containing protein – 9) and its paralog SAMD9L (SAMD9-like) are located on chromosome 7q21. Heterozygous mutations in these genes were first reported in myeloid malignancies by Asou et al, in an effort to discover molecular mechanisms responsible for monosomy 7 (-7) and interstitial deletions in 7q (del7q) [1]. Later, two distinct clinical presentations were identified that were attributed to germline mutations occurring in these two genes. Mutations in SAMD9 result in – ‘Myelodysplasia, Infection, Restriction of growth, Adrenal hypoplasia, Genital phenotypes, and Enteropathy’ (OMIM: *610456) coined MIRAGE syndrome [2] while SAMD9L mutations results in ‘Ataxia Pancytopenia syndrome’ (OMIM: *611170) an autosomal dominant condition associated with cerebellar degeneration and pancytopenia [3]. Bone marrow failure, the common feature of these two conditions has been successfully treated with allogeneic hemopoietic stem cell transplantation (HSCT) [4].

Here we report a case of a previously undescribed SAMD9L variant in a boy with bone marrow failure developing donor-type aplasia (DTA) after receiving a HSCT from a Human Leukocyte Antigen (HLA) matched sister, harboring the same variant.

2. Case report

A previously well, Sri Lankan boy, presented at the age of 3 years and 8 months with widespread ecchymosis and low-grade fever. He was born to non-consanguineous parents and had an uncomplicated antenatal and birth history. At presentation, he was an average built child with no dysmorphism. There was no organomegaly or lymphadenopathy and the review of systems was normal except for fever and bruising. Preliminary investigations revealed hemoglobin 88 g/L, total leukocyte count 7.3 × 10⁹/L, neutrophils 0.9 × 10⁹/L, lymphocytes 5.7 × 10⁹/L, platelets 12 × 10⁹/L, fetal hemoglobin (HbF) – 1.2% and normal serum biochemistry. Examination of the bone marrow revealed, cellularity of less than 10%, with no evidence of dysplasia, increase in blasts or fibrosis. (Figure 1) He had a normal karyotype, a negative paroxysmal nocturnal hemoglobinuria screen and a negative chromosomal fragility test. Possible infectious and autoimmune etiologies for secondary bone marrow aplasia were also excluded. Thereafter, targeted gene sequencing was performed on a platform of 93 genes that were specifically designed for the diagnosis of inherited BMFS. No pathogenic variants or likely pathogenic variants of genes known to cause bone marrow failure were found. However, a heterozygous missense variation in exon 5 of the SAMD9L gene (chr7:g.92762233C>T; Depth: 67x) that resulted in the amino acid substitution of Lysine for Glutamic acid at codon 1018 (p.Glu1018Lys; ENST00000318238.4) was detected (Figure 2). The p.Glu1018Lys variant was not reported in the 1000 genomes database and had a minor allele frequency of 0.002% and 0.005% in the Exome Aggregation Consortium (ExAC) and internal databases respectively. Based on the American College of Medical Genetics and Genomics, standards and guidelines, this SAMD9L variation was classified as a variant of uncertain significance due to lack of data to support a quantitative assignment to pathogenicity or benignity [5]. Sanger sequencing confirmed the mutation to be present in the father and two siblings. All three of them had normal haematological indices. As expected, the mother did not harbor the mutation (Figure 3).

Figure 1. Trephine biopsy showing bone marrow aplasia. Hematoxylin and Eosin stain. Magnification ×100.

Figure 2. Sanger sequencing from a peripheral leukocyte blood sample using PCR showing the variation in exon 5 of the SAMD9L gene (chr7:g.92762233C>T; c.3052G>A; p.Glu1018Lys).

Figure 3. Pedigree chart showing affected proband, heterozygous father and two siblings and unaffected mother.

Family screening for HLA compatibility showed the 12-year-old sister to be a 10/10 match. An unrelated donor search did not return any suitable donors. Considering the healthy status of the father and sister who harbored the same genetic variation, absence of congenital abnormalities in the proband or other family members and the uncertain significance of this previously unreported variation, he underwent an upfront matched sibling stem cell transplant from his sister. Conditioning was with fludarabine-cyclophosphamide-anti Thymocyte Globulin, Graft Versus Host Disease (GVHD) prophylaxis with methotrexate and tacrolimus. He engrafted on day +13 and went on to show 96.92% donor chimerism at day + 15. He showed no signs of acute or chronic GVHD. He was taken off of all immunosuppressants at one year post transplant at which time he showed full donor chimerism. This case was included in a previously reported case series of the first allogenic transplants for BMFS in Sri Lanka [6].

At 1 year and 9 months post transplant, he presented again with features of pancytopenia. Bone marrow biopsy showed a markedly hypoplastic marrow with some dysplastic changes in the erythroid and granulocytic lineages. Conventional karyotyping revealed a normal female karyotype and full donor chimerism of 98%. After the diagnosis of ‘donor-type aplasia’ he remained transfusion dependent. After approximately 9 months since the presentation of donor-type aplasia, and a failed second Matched Unrelated Donor search, a haploidentical transplant was attempted from his mother. As donor specific antibodies were detected at 800 MFI he was treated with rituximab, IV immunoglobulin and plasma exchange prior to initiation of conditioning. The second transplant went ahead with a Flu-Cy-ATG conditioning regimen with Post Transplant cyclophosphamide as T cell depletion. Unfortunately, he had no features of engraftment at 28 days and succumbed to a disseminated fungal infection soon after. His sister remains healthy to date at age 15, and is on regular hematology follow up.

3. Discussion

Proteins encoded by the genes SAMD9 and SAMD9L have a critical role in endosomal trafficking and cell proliferation amongst a host of other functions [7,8]. Mutations involving these two genes have provided remarkable insights into the plasticity of pediatric hemopoiesis [7]. Both these genes have been identified as possible candidate genes responsible for -7/del7q seen in familial cases of MDS [9]. It is believed, the cells that harbor the mutation develop a -7/del7q and loose the mutation in the process and with it, gain a survival advantage relative to the cells that contain the mutation, a process known as, ‘adaptation-by-aneuploidy’ or ‘somatic gene rescue’ [2,7]. However, these clones by virtue of the presence of -7/del7q will be at an increased risk of developing MDS/AML, especially upon accumulation of additional myeloid mutations. At times, this clonal escape mechanism can take the form of uniparental disomy of 7q (UPD7q) that result in reduplication and self-correction to the wild type and subsequent normal haemopoesis [7].

Differentiating inherited from acquired bone marrow failure is important in many aspects. These include selecting between immunosuppressive therapy and HSCT as treatment options, choosing the appropriate donor and conditioning regimen, planning for future cancer surveillance, appropriate counseling and familial genetic screening. There is a current trend to apply next generation sequencing methods in the diagnostic workup for inherited BMFS/MDS. These techniques allow large amounts of DNA to be analyzed in a single reaction saving on time and costs. Many authors have demonstrated improved diagnostic rates in IBMFS cohorts by the use of these platforms [10–12]. However, performing a comprehensive genetic screen in the absence of a positive family history of marrow failure or syndromic congenital abnormalities can lead to complicated clinical validation problems, as was demonstrated by this case. Therefore, whenever possible combining genetic screening with functional testing is recommended. Unfortunately, functional testing can only be performed in Fanconi anemia and telomeropathies. This highlights an unmet need in the diagnostic workup of inherited BMFS/MDS.

Transplant strategies differ between inherited and aquired aplastic anemia including choosing suitable donors. Avoiding matched relatives that harbor the same mutation as the donor remains the dogma in inherited BMFS/MDS [4,13]. In the index case the decision to choose the sister as the stem cell donor was taken after much deliberation. The fact that his sister, brother and father who shared the same variation were healthy suggested the observed nucleotide replacement was a variation rather than a pathogenic mutation. Sahoo et al have described the various challenges associated with predicting the pathogenicity of newly found SAMD9/9L variants [7]. Variants that are ultra-rare i.e. <0.005% have exhibited SAMD9/L- specific findings while variants that are less rare (0.005-0.1%) and occurring in the absence of other clinical features have shown unlikely to be disease drivers and more likely to be non-pathogenic [7]. Development of donor-type aplasia in the above case confirms the pathogenic nature of this variant in the SAMD9L gene. Interestingly, 15 months after the diagnosis of our index case, an unrelated 4-year-old girl also presented to a different hematology unit with severe aplastic anemia, a normal clinical phenotype and harboring the exact SAMD9L variation. She remains transfusion-dependent after 4 years to date.

This case describes a novel pathogenic variant in exon 5 of the SAMD9L gene that resulted in bone marrow failure in the absence of other organ involvement. It also highlights the importance of comprehensive genetic screening in the workup of inherited BMFS/MDS where, detection of variants of uncertain significance, especially in SAMD9/SAMD9L, should not be ignored as they could pose a significant impact on management.

Table

SAMD9	Sterile a-motif domain containing protein - 9
SAMD9L	SAMD9-like
BMFS	Inherited bone marrow syndromes
MDS	Myelodysplastic syndromes
(-7)	Monosomy 7
(del7q)	interstitial deletions in 7q
MIRAGE syndrome	Myelodysplasia, Infection, Restriction of growth, Adrenal hypoplasia, Genital phenotypes, and Enteropathy
HSCT	Hemopoietic stem cell transplantation
DTA	Donor-type aplasia
aAA	Acquired aplastic anemia
HLA	Human leukocyte antigen
ExAC	Exome Aggregation Consortium
HbF	Fetal hemoglobin

Acknowledgements

The authors would like to thank Prof. Vajira Dissanayake for his valuable feedback on the case report.

Disclosure statement

No potential conflict of interest was reported by the author(s).