Convergence of specified RUNX2, SALL1, and SAMD9 variants permits normal global immune and endocrine function despite bone marrow hypocellularity and reduced ovarian reserve

Abstract

Background

RUNX2, SALL1 and SAMD9 are independently assorted genes responsible for multisystem actions where disruption often results in severe developmental or functional impairment. Alteration in any of these ‘master regulators’ is usually diagnosed in early childhood when missed milestones, anatomical dysmorphia, or chronic infection/immune impairment initiate cross-disciplinary evaluation.

Methods

New variants were recently discovered in all three genes during SARS-CoV-19 hospitalization in an otherwise healthy 46,XX adolescent. Our research expands the clinical characterization for this unusual convergence based on medical records covering the 5 year interval before admission.

Results

Diffuse bone marrow hypocellularity without cytogenetic derangement was present, with no anemia or reduction in total immunoglobulin production. However, bone age was below mean by 1.5yrs and multiple dental anomalies were documented. Macrocytosis and elevated serum creatinine were consistent findings. Ovaries and uterus appeared undersized with collapse of endogenous estradiol output leading to secondary amenorrhea by age 13yrs. Besides oral contraceptives, no other daily hormone treatment was required. A TSH receptor gene mutation of uncertain significance was also identified.

Conclusions

This is the first work to correlate immunoglobulin patterns, bone marrow and dental morphology, hematology/renal screening, pelvic anatomy, ovarian reserve data and thyroid features with coincident variants in RUNX2, SALL1 and SAMD9. While the expected impact from each mutation alone would normally be adverse, this study suggests a milder phenotype can prevail when these three variants occur together. Nevertheless, focused monitoring is appropriate given the uncertain status of this unique combination of mutations.

Introduction

A ‘chief conductor’ driving cartilage formation and skeletal development, RUNX2 is a gene with at least 200 known mutations. Several such variants can manifest as cleidocranial dysplasia, a condition with delayed fontanel closure, enlarged head circumference, clavicular dysmorphia, abnormal dentition, short stature, or hand defects [1]. The frequency of RUNX2 variants in the general population may be as low as 0.4% [2]. Spalt-like transcription factor 1 gene (SALL1) broadly controls DNA packaging and chromatin remodeling. This gene also coordinates female urogenital structure, and Mullerian defects can occur with its mutation [3]. SALL1 also modulates human tumorigenesis with > 70 variants now associated with Townes-Brocks syndrome (i.e., otic, limb, renal, and anal anomalies) [4]. Such mutations occur with uncertain frequency, but from > 3,000 targets with putative association to single gene disorders, SALL1 was among the least common [5]. The gene for SAMD9 (sterile α motif domain-containing protein 9) localizes to a region frequently involved in myeloid malignancies, blood dyscrasias, and childhood immune impairments. That SAMD9 might be causative in certain hematologic disorders was first advanced by Asou et al [6] who linked microdeletions at this locus with myeloid malignancies. Germline missense SAMD9 changes were subsequently implicated in a constellation of features termed MIRAGE syndrome [7] consisting of myelodysplasia, infection, restricted growth, adrenal hypoplasia, genital anomaly, and enteropathy. Fewer than 50 cases of this syndrome have been described [8]. The severe pathobiological consequences of RUNX2, SALL1, or SAMD9 variants have limited opportunities for longitudinal follow-up, and alteration of all three in combination has only been encountered once [9]. The current project was undertaken to expand the clinical characterization of this atypical genetic confluence during the adolescent-to-adulthood transition.

Methods

As none of the 3 newly discovered variants here were accessioned by any public genome library, a priori pathogenicity estimates could not be calculated. Prior to the current case, the only known nexus involving the three genes of interest was from experimental animal research [10]. Accordingly, precautionary bone marrow biopsy was advised to provide tissue for genome wide single nucleotide polymorphism (SNP) microarray analysis (Illumina Infinium CytoSNP-850Kv1.2 BeadChip; San Diego CA). With disease-associated gene centric content of > 800K SNP loci from chromosomes 1–22, > 29K and > 2K loci from X and Y chromosomes, respectively, this platform offered resolution of ~ 1 probe every 4kb with an extra 110,000 probes interrogating at least four pathology-associated genes, as per ABMG consortium opinion. Nucleotide positions were mapped using the February 2009 assembly of the human reference sequence [11].

Medical information for this intake was gathered after proband’s high school graduation to prepare for relocation to university. Laboratory tests, pathology slides, x-rays, and chart notes for the period corresponding to ages 13-18yrs were then matched with key health events based on patient narrative, contemporary notes, discussion with care team members, and parental interviews. Exome sequence analysis as part of a multispecialty workup related to Covid-19 hospitalization and ICU transfer was completed in 2021, followed by renal biopsy. Molecular characterization for the patient (and both non-consanguineous parents) provided an informative pedigree identifying novel variants in RUNX2, SALL1 and SAMD9, as previously described [9].

Results

Complete blood counts (CBC) were analyzed from routine check-ups between 2016 and 2021, when the patient’s most recent BMI was 17.8kg/m². While CBC data included marginally abnormal indices and occurred with no discernable pattern, the exception was mean corpuscular volume (MCV) which was consistently elevated. The patient had long taken multivitamin supplements for this macrocytosis, and serum folate was normal at 777ng/mL. Platelet (PLT) concentration remained within the standard range during the review period although mean PLT volume (MPV) was not measured at every visit. When available, MPV was slightly low (data not shown). Flow cytometry immune testing identified nominally elevated CD3⁺(ratio) and CD8⁺ (absolute) cells, an upward shift in the CD4⁺/CD8⁺ ratio, and markedly suppressed natural killer cells. Serum IgA and IgG levels were also marginally reduced, despite normal total immunoglobulin and IgM levels (see Table 1). Serum blood urea nitrogen (BUN) and creatinine levels were unrelated to any concurrent CBC fluctuations (see Fig. 1a & 1b).

Whole blood analysis on morning of bone marrow biopsy showed erythrocyte (RBC), hemoglobin, and hematocrit at 3.3 x10⁶/uL. 11.8g/dL, and 36.1%, respectively. Leukocyte count (WBC) was normal at 5.7K/uL (reference range = 4.4–9.7). Both MCV and mean corpuscular hemoglobin were elevated at 106.8fL and 34.9pg. PLT count was normal at 182K/uL. Generalized bone marrow hypocellularity was observed with trace storage iron, decreased erythroid precursors with mildly megaloblastoid maturation, decreased granulocyte precursors with full maturation, and normal megakaryocytes (see Fig. 2a & 2b). Myeloid:erythroid precursor ratio was normal on core analysis, and no increased fibrosis was evident on reticulin stain. Marrow sample showed high lymphocytes and monocytes (43.5 and 6%, respectively), low bands (5.5%; reference range = 17–33) and nominally elevated orthochromatic normoblasts (7.5%; reference range = 1–5). Fluorescent in situ hybridization demonstrated a normal signal with no copy number variance in nuclei (n = 200). Bone marrow cytogenetics verified a non-mosaic 46,XX karyotype, aligning with results obtained earlier from peripheral (somatic) samples. Fragile X carrier panel was negative.

By age 7yrs, all deciduous teeth were succeeded by permanent teeth, although anomalous non-descent of left cuspid was incidentally identified during pre-orthodontic screening. In addition, panoramic x-ray at age 17yrs revealed multiple underdeveloped roots and secondary loss of right lateral incisor. Surgery to remove all four impacted wisdom teeth (see Fig. 3) is scheduled.

Menarche was at age 11yrs but within two years menses had ceased. At age 15.5yrs, low-dose oral contraceptives were initiated to restore cyclicity and no other hormonal therapy was ever prescribed. Serum FSH was consistently > 100mIU/ml with anti-Mullerian hormone below measurement threshold (< 0.015ng/mL). Small ovaries were seen on abdominal ultrasound including sparse but active bilateral follicular response on clomiphene challenge test, and pre-replacement serum estradiol levels were regularly < 5pg/mL, all indicative of diminished ovarian reserve [12]. Anti-ovarian antibody test was negative. At age 15yrs, bone age via standard radiograph [13] was 2cm below mean, consistent with age 13.5yrs. At age 16, a solitary 2cm right anterior neck mass was evaluated; laboratory tests were negative for autoimmune involvement and found no evidence of abnormal thyroid or parathyroid function. The structure was non-tender and was palpable only after singing. Fine needle aspiration of the lesion showed Bethesda category III cytology; molecular testing [14] found a missense c.1358T > C mutation in the TSH receptor gene [15] with variant allele frequency of 46%, negative for gene fusions or copy number variants.

Discussion

Transcription factors set the pace and pattern of nuclear DNA conversion to mRNA. From amidst more than 1500 such factors, RUNX2, SALL1, and SAMD9 are especially prominent. The genes coding for these products thus control cell division, migration, body plan architecture, and apoptosis. While any disruption of these factors typically is poorly tolerated, often catastrophic, and usually apparent in childhood, this patient experienced an active, healthy childhood while carrying previously unreported variants in all three. Remarkably, the most dramatic highlight against a rather ordinary pediatric background was hospitalization for Covid-19. Attribution of findings discussed here to genetic etiologies vs. Covid-19 is challenging, given the unclassified status of these specified variants and the expansive, evolving nature of ‘Long Covid’. Curiously, the high MPV seen with Covid-19 infection documented by others [16] was absent here. Perhaps the three variants dampened MPV by altering individual platelet responsiveness, even though overall platelet output was unaffected. Likewise, the observed low serum IgA and IgG levels alongside normal total immunoglobulin may reflect a synthesis of coordinated action by these variants.

To date, some 150 mutations in SAMD9 have been associated with outcomes ranging from spontaneous remission to malignant progression [17]. Most SAMD9 variants presage early death from myeloid dysplasia, immune system imbalance, adrenal insufficiency, or chronic undernourishment from feeding difficulty [18]. SAMD9 mutations via germline transmission predispose to low platelets, acquired monosomy 7, constitutional abnormalities (e.g., ambiguous genitalia) and immune dysfunction [19]. Less is known about SAMD9 variants which appear as de novo events. Computer modeling has envisioned a protein-protein interaction network of differentially expressed genes, and human SAMD9 was among the ‘hub genes’ having special relevance after SARS-CoV-2 infection [20]. Thomas et al [21] assessed the functional impact of wild-type and mutant SAMD9 in primary mouse or human hematopoietic stem and progenitor cells. Using protein interactome analyses, transcriptional profiling, and functional validation, it was concluded that SAMD9 mutations tend to favor interference with DNA damage repair, and ultimately apoptosis, in hematopoietic cells [21]. The hypocellular terrain noted here on bone marrow biopsy may be a partial expression of this SAMD9 influence.

RUNX2 is considered to be a centrally regulating transcription factor for osteoblast and chondrocyte differentiation and overall skeletal architecture [22, 23]. Some 80 variants in RUNX2 have been identified [1, 24] and while heterozygous loss of function mutations can lead to cleidocranial dysplasia, this is inconsistent [25]. Triplication [26] or quadruplication [27] of RUNX2 accompanies more serious syndromic phenotypes, including coronal/sagittal synostosis or pan-craniosynostosis [26, 27] suggesting a dosage effect [28]. For our patient, sequence analysis did identify a 3’ duplication involving at least exons 6 through 9 of RUNX2 [9], so connecting osseous and dental changes observed here as related to this gene variant seems plausible.

In late adolescence or early adulthood third molar impaction is not unusual, and others have investigated differential expression of RUNX2 regarding tooth location before surgical removal [29]. While RUNX2 was not implicated, another transcription factor (MSX1) was differentially expressed depending on depth of molar impaction [29] and RUNX2 is itself partially controlled by MSX1 [30]. As alveolar bone remodeling is central to orthodontic tooth movement, a novel regulatory mechanism whereby FOXO3 induces osteocalcin transcription by promoter activation in concert with RUNX2 [31] could help explain dental features in this case.

The T > C mutation in codon 453 of the TSH receptor gene found here was previously reported in the setting of nonautoimmune hyperthyroidism [32] but without any RUNX2, SALL1, or SAMD9 involvement. Wide tissue expression of TSHRs is well established, and includes brain, bone marrow, lymphocytes, pituitary, thymus, testes, kidney, adipose tissue, and fibroblasts [33]. While oocyte growth is influenced via TSHR/cAMP signaling [34], there has been no reason to obtain ovarian tissue sampling in this patient. Pelvic ultrasound will be useful to identify any changes in gross ovarian anatomy. Similarly, tracking thyroid size, nodularity, and tenderness, as well as the thyroid laboratory panel will be important.

Concerning SALL1, some 50 different mutations are currently known [35]. SALL1 and RUNX2 may have special relevance in human reproduction. Mammalian estrogen receptor β (ERβ) is required for ovarian follicles to advance past the antral stage, and work in rat granulosa cells has placed RUNX2 within the ERβ-regulated genes directing folliculogenesis, oocyte maturation, and ovulation [36]. While SALL1 is essential for stem cell maintenance in kidney, heart, and spermatogonial progenitors [4, 37], its role in human ovarian tissue awaits better characterization. Should undetectable serum AMH persist and ovarian histology be normal, this patient may consider fertility treatment including platelet-rich plasma for ‘ovarian rejuvenation’ [38] as an alternative to donor egg IVF.

The Venn diagram for worldwide clinical experience with RUNX2, SALL1, and SAMD9 returned a null union set, prior to this patient. The detrimental consequences of mutation of these genes in isolation may have been mitigated by the chance occurrence of all three variants in concert—an offset possibly enabled by cross-gene effects or epigenetic silencing [9]. Monitoring for patients with a SAMD9 variant includes CBC with differential every six months, and repeat bone marrow aspirate/biopsy (and karyotype) should anemia, thrombocytopenia, or neutropenia develop [8]. Red blood cell dysplasia, agglutination, or fragmentation are unlikely given the low/normal red-cell distribution width measured here. Serum BUN and creatinine levels evidenced no discernable pattern whenever these were abnormal, but as cystatin-C may be superior to BUN/Cr to detect early-stage disease [39, 40] a screening panel including all three will guide the need for repeat renal biopsy. Since this case incorporates a de novo variant, the risk to siblings is considered somewhat greater than the general population [8] and cytogenetic testing for an older half-brother is pending. Clinical guidelines for RUNX2 or SALL1 have not been developed, probably due to the exceptionally low frequency of these variants. Because not all instances of marrow hypocellularity will render immediate effects [41] periodic reassessment of this patient is planned.

Declarations

Authors’ Contributions

ESS developed the research plan and organized initial drafts; ESS, CH, and SHW reviewed the literature; SHW supervised the project and editorial aspects. All authors read and approved the final manuscript.

Funding

No support was received from any funding agency in public, commercial, or nonprofit sectors.

Conflicts of Interest

The authors have no conflicts to disclose.

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Table 1

Table 1. Summary of complete blood count, differential and immunoglobulin data measured in healthy 46,XX with new variants in RUNX2, SALL1, and SAMD9.

Notes: ABS = absolute, NK =  natural killer cell, Ig = immunoglobulin, red = above normal, 

blue = below normal; (-) indicates reference range not specified/undefined.