Hypomorphic Variants Research Articles

A novel pathogenic mutation of MeCP2 impairs chromatin association independent of protein levels.

Loss-of-function mutations in MECP2 cause Rett syndrome (RTT), a severe neurological disorder that mainly affects girls. Mutations in MECP2 do occur in males occasionally and typically cause severe encephalopathy and premature lethality. Recently, we identified a missense mutation (c.353G>A, p.Gly118Glu [G118E]), which has never been seen before in MECP2, in a young boy who suffered from progressive motor dysfunction and developmental delay. To determine whether this variant caused the clinical symptoms and study its functional consequences, we established two disease models, including human neurons from patient-derived iPSCs and a knock-in mouse line. G118E mutation partially reduces MeCP2 abundance and its DNA binding, and G118E mice manifest RTT-like symptoms seen in the patient, affirming the pathogenicity of this mutation. Using live-cell and single-molecule imaging, we found that G118E mutation alters MeCP2's chromatin interaction properties in live neurons independently of its effect on protein levels. Here we report the generation and characterization of RTT models of a male hypomorphic variant and reveal new insight into the mechanism by which this pathological mutation affects MeCP2's chromatin dynamics. Our ability to quantify protein dynamics in disease models lays the foundation for harnessing high-resolution single-molecule imaging as the next frontier for developing innovative therapies for RTT and other diseases.

AXIN1 bi-allelic variants disrupting the C-terminal DIX domain cause craniometadiaphyseal osteosclerosis with hip dysplasia

Sclerosing skeletal dysplasias result from an imbalance between bone formation and resorption. We identified three homozygous, C-terminally truncating AXIN1 variants in seven individuals from four families affected by macrocephaly, cranial hyperostosis, and vertebral endplate sclerosis. Other frequent findings included hip dysplasia, heart malformations, variable developmental delay, and hematological anomalies. In line with AXIN1 being a central component of the β-catenin destruction complex, analyses of primary and genome-edited cells harboring the truncating variants revealed enhanced basal canonical Wnt pathway activity. All three AXIN1-truncating variants resulted in reduced protein levels and impaired AXIN1 polymerization mediated by its C-terminal DIX domain but partially retained Wnt-inhibitory function upon overexpression. Addition of a tankyrase inhibitor attenuated Wnt overactivity in the AXIN1-mutant model systems. Our data suggest that AXIN1 coordinates the action of osteoblasts and osteoclasts and that tankyrase inhibitors can attenuate the effects of AXIN1 hypomorphic variants.

Novel homozygous variants in PRORP expand the genotypic spectrum of combined oxidative phosphorylation deficiency 54

Biallelic hypomorphic variants in PRORP have been recently described as causing the autosomal recessive disorder combined oxidative phosphorylation deficiency type 54 (COXPD54). COXPD54 encompasses a phenotypic spectrum of sensorineural hearing loss and ovarian insufficiency (Perrault syndrome) to leukodystrophy. Here, we report three additional families with homozygous missense PRORP variants with pleiotropic phenotypes. Each missense variant altered a highly conserved residue within the metallonuclease domain. In vitro mitochondrial tRNA processing assays with recombinant TRMT10C, SDR5C1 and PRORP indicated two COXPD54-associated PRORP variants, c.1159A>G (p.Thr387Ala) and c.1241C>T (p.Ala414Val), decreased pre-tRNAIle cleavage, consistent with both variants impacting tRNA processing. No significant decrease in tRNA processing was observed with PRORP c.1093T>C (p.Tyr365His), which was identified in an individual with leukodystrophy. These data provide independent evidence that PRORP variants are associated with COXPD54 and that the assessment of 5′ leader mitochondrial tRNA processing is a valuable assay for the functional analysis and clinical interpretation of novel PRORP variants.

Interface-guided phenotyping of coding variants in the transcription factor RUNX1 with SEUSS.

Understanding the consequences of single amino acid substitutions in cancer driver genes remains an unmet need. Perturb-seq provides a tool to investigate the effects of individual mutations on cellular programs. Here we deploy SEUSS, a Perturb-seq like approach, to generate and assay mutations at physical interfaces of the RUNX1 Runt domain. We measured the impact of 115 mutations on RNA profiles in single myelogenous leukemia cells and used the profiles to categorize mutations into three functionally distinct groups: wild-type (WT)-like, loss-of-function (LOF)-like and hypomorphic. Notably, the largest concentration of functional mutations (non-WT-like) clustered at the DNA binding site and contained many of the more frequently observed mutations in human cancers. Hypomorphic variants shared characteristics with loss of function variants but had gene expression profiles indicative of response to neural growth factor and cytokine recruitment of neutrophils. Additionally, DNA accessibility changes upon perturbations were enriched for RUNX1 binding motifs, particularly near differentially expressed genes. Overall, our work demonstrates the potential of targeting protein interaction interfaces to better define the landscape of prospective phenotypes reachable by amino acid substitutions.

Phenotypic, molecular, and functional characterization of COQ7-related primary CoQ10 deficiency: Hypomorphic variants and two distinct disease entities.

Primary coenzyme Q10 (CoQ10) deficiency is a group of inborn errors of metabolism caused by defects in CoQ10 biosynthesis. Biallelic pathogenic variants in COQ7, encoding mitochondrial 5-demethoxyubiquinone hydroxylase, have been reported in nine patients from seven families. We identified five new patients with COQ7-related primary CoQ10 deficiency, performed clinical assessment of the patients, and studied the functional effects of current and previously reported COQ7 variants and potential treatment options. The main clinical features included a neonatal-onset presentation with severe neuromuscular, cardiorespiratory and renal involvement and a late-onset disease presenting with progressive neuropathy, lower extremity weakness, abnormal gait, and variable developmental delay. Baker's yeast orthologue of COQ7, CAT5, is required for growth on oxidative carbon sources and cat5Δ strain demonstrates oxidative growth defect. Expression of wild-type CAT5 could completely rescue the defect; however, yeast CAT5 harboring equivalent human pathogenic variants could not. Interestingly, cat5Δ yeast harboring p.Arg57Gln (equivalent to human p.Arg54Gln), p.Arg112Trp (equivalent to p.Arg107Trp), p.Ile69Asn (equivalent to p.Ile66Asn) and combination of p.Lys108Met and p.Leu116Pro (equivalent to the complex allele p.[Thr103Met;Leu111Pro]) partially rescued the growth defects, indicating these variants are hypomorphic alleles. Supplementation with 2,4 dihydroxybenzoic acid (2,4-diHB) rescued the growth defect of both the leaky and severe mutants. Overexpression of COQ8 and 2,4-diHB supplementation synergistically restored oxidative growth and respiratory defect. Overall, we define two distinct disease presentations of COQ7-related disorder with emerging genotype-phenotype correlation and validate the use of the yeast model for functional studies of COQ7 variants.

The Common PKD1 p.(Ile3167Phe) Variant Is Hypomorphic and Associated with Very Early Onset, Biallelic Polycystic Kidney Disease

Biallelic PKD1 variants, including hypomorphic variants, can cause very early onset polycystic kidney disease (VEO-PKD). A family with unexplained recurrent VEO-PKD and neonatal demise in one dizygotic twin was referred for clinical testing. Further individuals with the putative hypomorphic PKD1 variant, p.(Ile3167Phe), were identified from the UK 100,000 genomes project (100 K), UK Biobank (UKBB), and a review of the literature. We identified a likely pathogenic PKD1 missense paternal variant and the putative hypomorphic PKD1 variant from the unaffected mother in the deceased twin but only the paternal PKD1 variant in the surviving dizygotic twin. Analysis of 100 K cases identified a second family with two siblings with similar biallelic inheritance who presented at birth with VEO-PKD and reached kidney failure in their teens unlike other affected relatives. Finally, a survey of 618 UKBB cases confirmed that adult patients monoallelic for PKD1 p.(Ile3167Phe) had normal kidney function. Our data reveals that p.(Ile3167Phe) is the second most common PKD1 hypomorphic variant identified and is neutral in heterozygosity but is associated with VEO-PKD when inherited in trans with a pathogenic PKD1 variant. Care should be taken to ensure that it is not automatically filtered from sequence data for VEO cases.

Functional Characterization of Novel Lunatic Fringe Variants in Spondylocostal Dysostosis Type-III with Scoliosis

Scoliosis affects over four million Americans, with most cases having an idiopathic cause. Pathogenic variants in the LUNATIC FRINGE (LFNG) gene can cause spondylocostal dysostosis type-III (SCD3), which is a rare skeletal dysplasia characterized by the absence, fusion, or partial development of vertebrae and ribs. Acute restrictive lung disease and scoliosis may also be present in some cases. The variability in symptoms suggests that there may be other underlying pathological mechanisms that are yet to be discovered. We conducted an analysis of two novel LFNG variants, c.766G>A (p.G256S) and c.521G>A (p.R174H), that were observed in a patient with SCD3 phenotype and scoliosis. Characterizing these variants can help us better understand the relationship between genotype and phenotype. We assessed both variants for impaired glycosyltransferase activity, subcellular mislocalization, and aberrant pre-proprotein processing. Our results indicate that the p.G256S variant is enzymatically nonfunctional, while the p.R174H variant is functionally less effective. Both variants were correctly localized and processed. Our findings suggest that the hypomorphic variant (p.R174H) may have partially improved the patient’s stature, as evidenced by a lower arm span-to-height ratio, increased height, and more vertebrae. However, this variant did not appear to have any effect on the severity of vertebral malformations, including scoliosis. Further research is necessary to determine the extent to which variations in LFNG activity affect the presentation of SCD3.

Functional identification of two novel variants and a hypomorphic variant in ASS1 from patients with Citrullinemia type I.

Background: Citrullinemia type I (CTLN1) is a rare autosomal recessive inborn error of the urea cycle caused by mutations in the gene encoding the arginosuccinate synthetase (ASS1) enzyme. Classic CTLN1 often manifests with acute hyperammonemia and neurological symptoms. Molecular genetic testing is critical for patient diagnosis. Methods: Three unrelated families with clinically suspected CTLN1 were included in this study. Potential pathogenic variants were identified using whole exome sequencing (WES) and validated using Sanger sequencing. Western blotting, quantitative PCR, immunofluorescent staining, and ELISA were used to assess functional changes in candidate ASS1 variants. Results: Five variants were identified, two of which were novel, and one has been reported, but its pathogenicity was not validated. The novel variant c.649-651del (p.P217del) and the 5'UTR variant (c.-4C>T) resulted in a decrease in ASS1 expression at both the protein and transcription levels. The other novel variant, c.1048C>T (p.Q350*), showed a marked decrease in expression at the protein level, with the formation of truncated proteins but an increased transcription. Both c.649_651del (p.P217del) and c.1048C>T (p.Q350*) showed a highly significant reduction in enzyme activity, while c.-4C>T had no effect. Conclusion: We identified two novel variants and a hypomorphic non-coding variant in ASS1 and validated the pathogenicity using functional studies. Our findings contribute to expanding the spectrum of ASS1 variants and understanding the genotype-phenotype relationships of CTLN1.

Identifying p53 Target Genes Using Hypomorphic Variants.

The reduced tumor suppression activity of hypomorphic variants of the TP53 gene was used by Indeglia and colleagues to corroborate PADI4 as a p53 target. The study makes a noteworthy advancement in comprehending the downstream implications of TP53-PDI4, including potential predictions of survival and the efficacy of immunotherapy. See related article by Indeglia et al., p. 1696 (4).

Natural History of Stargardt Disease: The Longest Follow-Up Cohort Study.

Long-term natural history studies are important in rare disease research. This study aimed to assess electrophysiological and fundus autofluorescence (FAF) progression rate in 18 genetically confirmed Stargardt disease (STGD1) patients with a minimum follow-up of 10 years. Age at the first and last exams, age at onset, Snellen decimal visual acuity (VA), electroretinography (ERG), and FAF images were evaluated. Patients were classified into four Fishman stages and three electroretinography groups, and areas of definitely decreased autofluorescence (DDAF) were measured. Patients were further substratified based on genotype, and phenotype-genotype correlations were performed. The median follow-up was 18 (range 10-26) years. The median yearly VA loss was 0.009 (range 0.002-0.071), while the median progression rate of the DDAF area was 0.354 (range 0.002-4.359) mm2 per year. Patients harbouring p.(Gly1961Glu) or p.(Asn1868Ile) allele had significantly slower DDAF area progression when compared to patients with other genotypes (0.07 mm2 vs. 1.03 mm2, respectively), as well as significantly later age at onset (20 years vs. 13 years, respectively). Results showed that structural and functional parameters, together with genotype, should be considered when counselling patients regarding prognosis and monitoring disease progression. Patients harbouring hypomorphic variants p.(Gly1961Glu) or p.(Asn1868Ile) presented with overall milder disease than patients with other genotypes.

Deoxyhypusine synthase mutations alter the post-translational modification of eukaryotic initiation factor 5A resulting in impaired human and mouse neural homeostasis.

DHPS deficiency is a rare genetic disease caused by biallelic hypomorphic variants in the Deoxyhypusine synthase (DHPS) gene. The DHPS enzyme functions in mRNA translation by catalyzing the post-translational modification, and therefore activation, of eukaryotic initiation factor 5A (eIF5A). The observed clinical outcomes associated with human mutations in DHPS include developmental delay, intellectual disability, and seizures. Therefore, to increase our understanding of this rare disease, it is critical to determine the mechanisms by which mutations in DHPS alter neurodevelopment. In this study, we have generated patient-derived lymphoblast cell lines and demonstrated that human DHPS variants alter DHPS protein abundance and impair enzyme function. Moreover, we observe a shift in the abundance of the post-translationally modified forms of eIF5A; specifically, an increase in the nuclear localized acetylated form (eIF5AAcK47) and concomitant decrease in the cytoplasmic localized hypusinated form (eIF5AHYP). Generation and characterization of a mouse model with a genetic deletion of Dhps in the brain at birth shows that loss of hypusine biosynthesis impacts neuronal function due to impaired eIF5AHYP-dependent mRNA translation; this translation defect results in altered expression of proteins required for proper neuronal development and function. This study reveals new insight into the biological consequences and molecular impact of human DHPS deficiency and provides valuable information toward the goal of developing treatment strategies for this rare disease.

Hypomorphic pathogenic variant in SFTPB leads to adult pulmonary fibrosis.

Biallelic pathogenic variants in the surfactant protein (SP)-B gene (SFTPB) have been associated with fatal forms of interstitial lung diseases (ILD) in newborns and exceptional survival in young children. We herein report the cases of two related adults with pulmonary fibrosis due to a new homozygous SFTPB pathogenic variant, c.582G>A p.(Gln194=). In vitro transcript studies showed that this SFTPB synonymous pathogenic variant induces aberrant splicing leading to three abnormal transcripts with the preservation of the expression of a small proportion of normal SFTPB transcripts. Immunostainings on lung biopsies of the proband showed an almost complete loss of SP-B expression. This hypomorphic splice variant has thus probably allowed the patients' survival to adulthood while inducing an epithelial cell dysfunction leading to ILD. Altogether, this report shows that SFTPB pathogenic variants should be considered in atypical presentations and/or early-onset forms of ILD particularly when a family history is identified.

Identification of USP9X as a leukemia susceptibility gene

AbstractWe recently reported that children with multiple birth defects have a significantly higher risk of childhood cancer. We performed whole-genome sequencing on a cohort of probands from this study with birth defects and cancer and their parents. Structural variant analysis identified a novel 5 kb de novo heterozygous inframe deletion overlapping the catalytic domain of USP9X in a female proband with multiple birth defects, developmental delay, and B-cell acute lymphoblastic leukemia (B-ALL). Her phenotype was consistent with female-restricted X-linked syndromic intellectual developmental disorder-99 (MRXS99F). Genotype-phenotype analysis including previously reported female probands (n = 42) demonstrated that MRXS99F probands with B-ALL (n = 3) clustered with subjects with loss-of-function (LoF) USP9X variants and multiple anomalies. The cumulative incidence of B-ALL among these female probands (7.1%) was significantly higher than an age- and sex-matched cohort (0.003%) from the Surveillance, Epidemiology, and End Results database (P < .0001, log-rank test). There are no reports of LoF variants in males. Males with hypomorphic missense variants have neurodevelopmental disorders without birth defects or leukemia risk. In contrast, in sporadic B-ALL, somatic LoF USP9X mutations occur in both males and females, and expression levels are comparable in leukemia samples from both sexes (P = .54), with the highest expressors being female patients with extra copies of the X-chromosome. Overall, we describe USP9X as a novel female-specific leukemia predisposition gene associated with multiple congenital, neurodevelopmental anomalies, and B-ALL risk. In contrast, USP9X serves as a tumor suppressor in sporadic pediatric B-ALL in both sexes, with low expression associated with poorer survival in patients with high-risk B-ALL.

Mild generalised pustular psoriasis patient with a heterozygous hypomorphic MPO variant successfully treated with granulocyte and monocyte adsorption apheresis.

Pathogenic variants in MPO, which encodes the myeloperoxidase, were reported as causative genetic defects in several cases of generalised pustular psoriasis (GPP) in addition to patients with myeloperoxidase deficiency in 2020. However, which clinical subtypes of GPP patients have pathogenic variants in MPO remains largely undetermined, and elucidating this is clinically important. The present report outlines a mild case of GPP with a rare missense heterozygous variant, c.1810C>T p.(Arg604Cys), in MPO. Our structural analysis and functional assays to measure myeloperoxidase activity suggest that the present MPO substitution is a hypomorphic variant in MPO. Thus, the mild phenotype of the present GPP patient might be associated with an incomplete hypomorphic loss-of-function variant in MPO. Additionally, the severe intractable edematous pustules and erythema improved dramatically after five rounds of granulocyte and monocyte adsorption apheresis (GMA) therapy. This is the first report of GMA treatment for GPP associated with a pathogenic variant in MPO, as far as we know. Our findings suggest that GMA might be a useful and powerful tool for controlling GPP in patients with myeloperoxidase deficiency.

The genetic landscape and clinical spectrum of nephronophthisis and related ciliopathies

Nephronophthisis (NPH) is an autosomal-recessive ciliopathy representing one of the most frequent causes of kidney failure in childhood characterized by a broad clinical and genetic heterogeneity. Applied to one of the worldwide largest cohorts of patients with NPH, genetic analysis encompassing targeted and whole exome sequencing identified disease-causing variants in 600 patients from 496 families with a detection rate of 71%. Of 788 pathogenic variants, 40 known ciliopathy genes were identified. However, the majority of patients (53%) bore biallelic pathogenic variants in NPHP1. NPH-causing gene alterations affected all ciliary modules defined by structural and/or functional subdomains. Seventy six percent of these patients had progressed to kidney failure, of which 18% had an infantile form (under five years) and harbored variants affecting the Inversin compartment or intraflagellar transport complex A. Forty eight percent of patients showed a juvenile (5-15 years) and 34% a late-onset disease (over 15 years), the latter mostly carrying variants belonging to the Transition Zone module. Furthermore, while more than 85% of patients with an infantile form presented with extra-kidney manifestations, it only concerned half of juvenile and late onset cases. Eye involvement represented a predominant feature, followed by cerebellar hypoplasia and other brain abnormalities, liver and skeletal defects. The phenotypic variability was in a large part associated with mutation types, genes and corresponding ciliary modules with hypomorphic variants in ciliary genes playing a role in early steps of ciliogenesis associated with juvenile-to-late onset NPH forms. Thus, our data confirm a considerable proportion of late-onset NPH suggesting an underdiagnosis in adult chronic kidney disease.

Bi-allelic variants in HMGCR cause an autosomal-recessive progressive limb-girdle muscular dystrophy

Statins are a mainstay intervention for cardiovascular disease prevention, yet their use can cause rare severe myopathy. HMG-CoA reductase, an essential enzyme in the mevalonate pathway, is the target of statins. We identified nine individuals from five unrelated families with unexplained limb-girdle like muscular dystrophy and bi-allelic variants in HMGCR via clinical and research exome sequencing. The clinical features resembled other genetic causes of muscular dystrophy with incidental high CPK levels (>1,000U/L), proximal muscle weakness, variable age of onset, and progression leading to impaired ambulation. Muscle biopsies in most affected individuals showed non-specific dystrophic changes with non-diagnostic immunohistochemistry. Molecular modeling analyses revealed variants to be destabilizing and affecting protein oligomerization. Protein activity studies using three variants (p.Asp623Asn, p.Tyr792Cys, and p.Arg443Gln) identified in affected individuals confirmed decreased enzymatic activity and reduced protein stability. In summary, we showed that individuals with bi-allelic amorphic (i.e., null and/or hypomorphic) variants in HMGCR display phenotypes that resemble non-genetic causes of myopathy involving this reductase. This study expands our knowledge regarding the mechanisms leading to muscular dystrophy through dysregulation of the mevalonate pathway, autoimmune myopathy, and statin-induced myopathy.

An African-Specific Variant of TP53 Reveals PADI4 as a Regulator of p53-Mediated Tumor Suppression.

We analyze the African-centric Y107H hypomorphic variant and show that it confers increased cancer risk; we use Y107H in order to identify PADI4 as a key tumor-suppressive p53 target gene that contributes to an immune modulation signature and that is predictive of cancer survival and the success of immunotherapy. See related commentary by Bhatta and Cooks, p. 1518. This article is highlighted in the In This Issue feature, p. 1501.

Somatic genetic reversion of large duplication event in IL2RG in siblings

BackgroundDeleterious mutations in the common gamma chain (γC, CD132) are associated with X-linked severe combined immunodeficiency (X-SCID) characterized by a T-B+NK- phenotype. Hypomorphic variants in the IL2RG gene have also been associated with a milder or atypical SCID phenotype. Milder phenotypes can also occur when somatic genetic rescue occurs in the setting of severe IL2RG mutations. Spontaneous somatic variants particularly in hematologic disorders can occur particularly in genes with known selection pressure including IL2RG. ResultsHere, we report the clinical and molecular findings from two brothers with atypical X-SCID phenotype including hypogammaglobulinemia, elevated IgM, T cell lymphopenia and extensive verruca plana. One brother required splenectomy secondary to a benign vascular tumor and had recurrent pancreatitis consistent with primary sclerosing cholangitis. Both brothers developed recurrent suppurative pneumonia resulting in early bronchiectasis in their first decade. Next generation sequencing initially was negative, yet reanalysis found a large duplication event in exon 3 of IL2RG gene with somatic genetic rescue events that occurred twice in two brothers. Functional studies revealed a unique mosaic pattern in each affected sibling when stimulating peripheral blood mononuclear cells with IL-2 or IL-21 resulting in bimodal phospho-STAT5 or phospho-STAT3 respectively. The genetic analysis revealed the maternal allele was heterozygous as expected, although the ratio of these events and clonal analysis revealed cell type skewing. ConclusionOur data demonstrate an atypical SCID phenotype in two brothers secondary to the combination of a large duplication event in the IL-2RG gene partially rescued by somatic genetic rescue. Although previous somatic reversion cases have been reported involving the IL2RG genetic locus, our cases demonstrate a large duplication reversion event with recurrence. Our case highlights the unique selection pressure to amplify rare genetic events at the IL2RG locus.

Hypomorphic splice site IL2RG variants associated with Cryptosporidium liver disease

BackgroundX-linked severe combined immunodeficiency (X-SCID) is caused by mutations in IL2RG encoding for the common gamma chain (γc). This disease is typically fatal within the first year without immune reconstitution. We present two patients with IL2RG variants leading to a phenotype of Cryptosporidium-related sclerosing cholangitis. MethodsMedical history and laboratories were reviewed for two patients with IL2RG variants at c.270G. For one patient, we examined cDNA, γc expression, TCR repertoire, cytokine signaling, in vitro B-cell plasmablast differentiation, and in vitro T-cell differentiation of peripheral blood CD34+ cells. ResultsTwo unrelated male patients (17 and 21 years old) were identified with liver disease, recurrent childhood pneumonia and resultant bronchiectasis. P1 has persistent warts. Elevated transaminases in P1 led to liver imaging and biopsy consistent with sclerosing cholangitis. Stool PCR was positive for Cryptosporidium. P2 was diagnosed at age 6 with cryptogenic cirrhosis. He underwent orthotopic liver transplant at age 19; diarrhea and cholestatic liver disease developed 4 months post-transplant, and cryptosporidium was found in stool and liver. Cryptococcus meningitis also developed on immunosuppression. Both patients were hemizygous for an IL2RG variant resulting in p.W90C (P1:c.270G>T; P2:c.270G>C). P1 had mildly reduced CD4+ lymphocytes, CD4+ and CD8+ central/ effector memory T-lymphocytes, memory B-lymphocytes, and normal NK counts. P1 cDNA revealed a leaky splicing defect with a mix of exons 2–4 skipping along with full-length transcript encoding p.W90C. Cells from heterozygous mother expressed wild-type sequence suggesting skewed X-inactivation. Surface protein expression and distribution of TCR V-Beta repertoire was largely normal. IL-2-mediated STAT5 phosphorylation was impaired in T- and NK-cells. P1’s B-lymphocytes proliferated in response to in vitro IL-21/CD40L co-stimulation but failed to differentiate into CD27+CD38+ plasmablasts (< 1%). In vitro T-cell differentiation showed impaired survival of developing thymocytes, despite capability of maturing intoTCRaB+ CD3+ T cells. ConclusionsWe present two patients hemizygous for IL2RG variants leading to milder clinical phenotype compared to classic X-SCID. Consistent with other diseases of impaired IL-21 signaling, there was significant Cryptosporidium-related liver disease in addition to clinical manifestations of hypomorphic IL-2 signaling defect. Further work is necessary to address restoring IL-21 signaling to improve liver disease.

Treatment of Omenn syndrome using multiple immunosuppressive strategies: a case series

Introduction: Omenn syndrome represents a therapeutic challenge as a state of florid immune dysregulation born out of autoreactive oligoclonal T cells due to hypomorphic variants in genes associated with severe combined immunodeficiency (SCID).Aims: To describe the therapeutic strategies used in the care of three patients with Omenn syndrome from 2013 to 2022. All had negative maternal engraftment studies and received immunosuppression to control autoreactive T cells. All patients ultimately received myeloablative conditioning with anti-thymocyte globulin (ATG), busulfan, and fludarabine for transplant. All patients are currently alive and well.Case 1: 2-month-old female with adenosine deaminase (ADA)-deficient SCID on pegylated ADA replacement developed respiratory distress, rash, and diarrhea in the setting of rising CD3+/CD4+/CD45RO+ cell count. Treated with high-dose systemic steroids. Due to continued CD3+ increase, systemic tacrolimus was added. She clinically improved and underwent matched unrelated donor cord blood transplant at 5 months of age.Case 2: A male with RAG1-SCID developed generalized erythroderma and rapidly rising CD3+/CD4+/CD45RO+ cell and absolute eosinophil counts soon after birth. Systemic steroids were given with clinical improvement. With taper of steroid, ATG 1 mg/kg was given. Initially, the absolute lymphocyte count (ALC) improved; however, within 5 days, his rash worsened and ALC rose. This necessitated re-initiation of high-dose steroids and addition of tacrolimus until matched related donor bone marrow transplant (MRD BMT) at 2 months old.Case 3: 9-day-old male with RAG1-SCID developed diffuse maculopapular rash, lymphadenopathy, and elevated memory T cells and eosinophils. Initiated high-dose systemic steroids and cyclosporine A (CSA) with quick clinical improvement. CSA discontinued due to toxicity and rising ALC and eosinophils. Given ATG 3 mg/kg/dose for 4 doses over 2 weeks with pattern of quick drop then rapid rise in ALC. Treated with steroids and tacrolimus until MRD BMT at 2 months old. ConclusionsWe describe consistent disease control with immunosuppression by systemic steroids and tacrolimus prior to transplant and suggest ATG as a potential adjunctive consideration in poorly-controlled disease. A growing body of knowledge and experience in optimal Omenn syndrome treatment strategies is imperative to improving outcomes.