Human complete NFAT1 deficiency causes a triad of joint contractures, osteochondromas, and B-cell malignancy.

The discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2, mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs∗18) and presented with joint contractures, osteochondromas, and recurrent B-cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in prosurvival and inflammatory genes. Systematic single-cell-omic analyses in PBMCs revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (enriched for oncogenic signatures MYC and JAK1), exhausted CD4+ T cells, impaired T follicular helper cells, and aberrant CD8+ T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further defines the detrimental effects associated with long-term use of calcineurin inhibitors.

Aberrant colon metabolome and the sudden infant death syndrome.

BACKGROUND: The Sudden Infant Death Syndrome (SIDS) has been associated with increased peripheral serotonin and an abnormal colonic microbiome, suggesting the colonic metabolome may also be abnormal. This study addresses this potential correlation by comparing colonic autopsy tissue from SIDS to age-matched non-SIDS controls. METHODS: Untargeted metabolomic analysis by mass spectrometry is used to assess human colonic metabolomic differences including serotonin. Expression of genes associated with colonic serotonin synthesis and transport (TPH1, TPH2, DDC, SCL6A4) is measured by qRT-PCR. Microbiome analysis is performed to compare the SIDS and non-SIDS colonic microbiome. RESULTS: Unsupervised hierarchical cluster and principal component analyses of metabolomic data shows increased variability in the SIDS cohort and separation of SIDS cases from the non-SIDS controls. There is a trend toward increased serotonin in the SIDS cohort but there is no significant difference in expression of the serotonin synthesis and transport genes between SIDS and non-SIDS control cohorts. Microbiome analysis shows no significant difference between the SIDS and non-SIDS control cohorts. CONCLUSIONS: This study demonstrates increased variability in the colonic metabolome and a trend towards increased colonic serotonin in SIDS. The underlying cause of colon metabolomic variability, and its potential role in SIDS pathogenesis, warrants further investigation. IMPACT STATEMENT: The key message of this article is that SIDS is associated with an aberrant colonic metabolome. This is a novel observation suggesting another component in the pathophysiology underlying SIDS. Investigation of why the colonic metabolome is aberrant may offer new insights to SIDS pathogenesis and new strategies to reduce risk.

Spatial Localization of Eubacterial 16S rRNA in Early Pregnancy Placenta and Decidua.

Bacteria derived from the maternal circulation have been suggested to seed the human placenta during pregnancy leading to development of an intrinsic placental microbiome; however, other data indicates these bacteria are artifactual contaminants. Limited research on the localization of bacteria in human placental tissue is available, which may help differentiate resident placental bacteria from contaminants. This study spatially localizes bacteria in situ in normal late first to early second trimester human placenta by 16S rRNA chromogenic in situ hybridization and demonstrates patterns consistent with both contaminants and intraparenchymal signals. These results suggest that placental microbiome studies may benefit from spatial strategies that can exclude surface contamination.

The Onset of Puberty Presents Unique Management Issues in Penile Chronic Graft-versus-Host Disease Requiring Circumcision in Male Pediatric Patients.

Chronic GvHD of the penile tract in male pediatric patients has not been described well in the literature and is often under-diagnosed. We report three cases of penile chronic GvHD in adolescent male patients who received HSCT before the onset of puberty. Their penile cGvHD became symptomatic upon the onset of penile growth associated with puberty in combination with the fibrotic changes in the foreskin. Symptoms did not respond to systemic chronic GvHD medication but require circumcision for alleviation of symptoms. This case series highlights the need for frequent monitoring of the prepubertal pediatric HSCT patient who has the presence of sclerotic cGvHD and enters puberty. This population is particularly reluctant to allow a thorough examination of the genitalia. In addition, optimization of systemic and topical immunosuppression treatment for patients with chronic GvHD of the penile tract potentially with the introduction of novel agents that target the tissue repair and fibrosis pathway is needed to prevent circumcision as the only option in the future.

NOTCH1 loss of the TAD and PEST domain: An antimorph?

The Notch proteins play key roles in cell fate determination during development. Germline pathogenic variants in NOTCH1 predispose to a spectrum of cardiovascular malformations including Adams-Oliver syndrome and a wide variety of isolated complex and simple congenital heart defects. The intracellular C-terminus of the single-pass transmembrane receptor encoded by NOTCH1 contains a transcriptional activating domain (TAD) required for target gene activation and a PEST domain (a sequence rich in proline, glutamic acid, serine, and threonine), regulating protein stability and turnover. We present a patient with a novel variant encoding a truncated NOTCH1 protein without the TAD and PEST domain (NM_017617.4: c.[6626_6629del];[=], p.(Tyr2209CysfsTer38)) and extensive cardiovascular abnormalities consistent with a NOTCH1-mediated mechanism. This variant fails to promote transcription of target genes as assessed by luciferase reporter assay. Given the roles of the TAD and PEST domains in NOTCH1 function and regulation, we hypothesize that loss of both the TAD and the PEST domain results in a stable, loss-of-function protein that acts as an antimorph through competition with wild-type NOTCH1.

Patterns of Interferon γ Expression and C4d Deposition in Chronic Intervillositis of Unknown Etiology.

The pathogenesis of chronic intervillositis of unknown etiology (CIUE) may involve IFNγ overexpression. This study assesses the extent of IFNγ expression in CIUE by immunohistochemistry and compares it to spontaneous pregnancy losses. C4d deposition is also assessed to see whether IFNγ and C4d might represent separate diagnostic categories. Placenta from first to early second trimester with high grade CIUE (CHG; 17 cases) and low grade CIUE (CLG; 12 cases) is compared to euploid (SPLN; 18 cases), aneuploid spontaneous pregnancy losses (SPLA, 17 cases), normal placenta (NP, 13 cases). Protein level expression of IFNγ and C4d is assessed on whole tissue sections by immunohistochemistry. 35% of CHG and 42% of CLG show some level of IFNγ expression localized to the luminal surface of syncytiotrophoblast. 12% of SPLA and no SPLN or NP cases are IFNγ positive. C4d deposition is seen in 100% of CIUE, 88% of SPLA, 83% of SPLN, and 46% of NP samples. IFNγ overexpression occurs in approximately 40% of CIUE-related pregnancy losses. IFNγ expression restricted to a subgroup of CIUE implies that IFNγ may define a distinct disease process. The non-discriminatory pattern of C4d deposition suggests it is a non-specific phenomenon possibly related to placental damage.

Workload Measurement in Subspecialty Placental Pathology in Canada.

BACKGROUND: Workload measurement is important to help determine optimal staffing and workload distribution for pathology laboratories. The Level 4 Equivalent (L4E) System is the most widely used Anatomical Pathology (AP) workload measurement tool in Canada. However, it was initially not developed with subspecialties in mind. METHODS: In 2016, a Pan-Canadian Pediatric-Perinatal Pathology Workload Committee (PCPPPWC) was organized to adapt the L4E System to assess Pediatric-Perinatal Pathology workload. Four working groups were formed. The Placental Pathology Working Group was tasked to develop a scheme for fair valuation of placental specimens signed out by subspecialists in the context of the L4E System. Previous experience, informal time and motion studies, a survey of Canadian Pediatric-Perinatal Pathologists, and interviews of Pathologists' Assistants (PA) informed the development of such scheme. RESULTS: A workload measurement scheme with average L4E workload values for examination and reporting of singleton and multiple gestation placentas was proposed. The proposal was approved by the Canadian Association of Pathologist - Association canadienne des pathologistes Workload and Human Resources Committee for adoption into the L4E System. CONCLUSION: The development of a workload measurement model for placental specimens provides an average and fair valuation of these specimen types, enabling its use for resource planning and workload distribution.

Calretinin Staining in Anorectal Line Biopsies Accurately Distinguished Hirschsprung Disease in a Retrospective Study.

INTRODUCTION: The absence of submucosal ganglion cells does not reliably distinguish Hirschsprung disease from non Hirschsprung disease in anorectal line biopsies. Calretinin staining might be helpful in these biopsies. To determine its value, we analyzed calretinin positive mucosal neurites in anorectal line biopsies. METHODS: Two pediatric pathologists, without access to patient data, evaluated calretinin positive mucosal neurites in anorectal line junctional mucosa in archival rectal biopsies contributed by 17 institutions. A separate investigator compiled patient information and sent data for statistical analysis. RESULTS: Biopsies with anorectal junctional mucosa from 115 patients were evaluated for calretinin positive mucosal neurites. 20/20 Hirschsprung disease biopsies were negative. 87/88 non Hirschsprung disease biopsies and 7/7 post pullthrough Hirschsprung disease neorectal biopsies were positive. Statistical analysis of the 108 non pullthrough biopsies yielded an accuracy of 99.1% (sensitivity 100%, specificity 98.9%). Age range was preterm to 16 years. Biopsy size was less than 1 mm to over 1 cm. CONCLUSIONS: Absence of calretinin positive mucosal neurites at the anorectal line was highly accurate in distinguishing Hirschsprung disease from non Hirschsprung disease cases in this blinded retrospective study. Calretinin staining is useful for interpreting biopsies from the physiologic hypoganglionic zone up to the anorectal line.

Orbital Teratoma: Case Report and Management Review.

Orbital teratoma is a rare, vision-threatening, congenital neoplasm derived from all germ cell layers. The management of orbital teratoma is uncertain because of its rarity and variable severity. We present a case with complete tumor excision with preservation of vision and cosmesis. A 4-day-old female presented with progressive proptosis of the OD. She was born term following an uneventful pregnancy and delivery. MRI of the orbits showed a large right-sided mass centered within the intraconal space with heterogeneous signal intensity characteristics consistent with a teratoma. She underwent a right posterior orbitotomy. The tumor was wrapped around the optic nerve but was gradually separated from it and all other normal structures and delivered intact. Histology was compatible with an immature teratoma without evidence of malignancy. The patient is now 6 months old, visually attentive with no evidence of optic neuropathy, and has essentially normal appearance.

Quantifying Proximal Collecting Tubule Deficiency in Angiotensin-Converting Enzyme Inhibitor and Angiotensin II Receptor Blocker Fetopathy.

INTRODUCTION: Angiotensin-Converting Enzyme Inhibitors and Angiotensin II Receptor Blockers (AAs) are used for several indications, with cessation recommended in pregnancy due to toxic effects. AA fetopathy phenotype is similar to renal tubular dysgenesis including reduced proximal convoluted tubules (PCTs). Our study aimed to quantify the reduction of PCTs in fetuses and infants with prenatal exposure to AAs. MATERIALS AND METHODS: We identified 5 fetal AA exposure cases that underwent autopsy at our institution between 2011 and 2018 and compared with 5 gestational age-matched controls. Immunohistochemistry with CD10 and epithelial membrane antigen (EMA) was utilized. RESULTS: CD10 and EMA identified a median PCT density of 19.0% ± 12.3% in AA fetopathy patients, significantly less than controls (52.8% ± 4.4%; p < 0.0001). One case with in utero cessation had a PCT density of 34.2% ± 0.2%. Among other AA fetopathy findings, 1 case demonstrated unilateral renal vein thrombosis and 4 had hypocalvaria. CONCLUSIONS: We have quantified the reduction in AA fetopathy PCT density, and demonstrated in utero cessation may recover PCT differentiation. Future studies may benefit from calculating PCT percentage as a potential biomarker to correlate with post-natal renal function and maternal factors including medication type, dosage, duration, and time from medication cessation.

Ulcerated amelanotic melanoma of the ear in an 11 year old with Fitzpatrick VI skin type: A case report.

Melanoma is rare in pediatric patients and even more so in those with darker Fitzpatrick skin types. Although risk factors for conventional melanoma are similar in both adult and pediatric cases, the presentation of melanoma in pediatric patients is often distinct from adults. Here, we describe a case of amelanotic ulcerated nodular melanoma with regional lymph node metastases treated with nivolumab in a patient with Fitzpatrick skin type VI.

Placental Expression of the Forelimb Patterning Transcription Factor MEIS2 in Trisomy 15.

BackgroundObservations of first trimester human trisomy 15 (T15) embryos have identified meromelic changes in the upper limbs. These changes are similar to those observed in animal studies investigating the effects of overexpression of Meis2, a signaling transcription factor expressed during forelimb development. Although it would be exceedingly difficult to assess MEIS2 expression in the human embryonic arm, MEIS2 has been reported as consistently expressed in first trimester placental villus stroma. Methods: This study addresses whether gene dosage effect might underlie meromelia in T15 by comparing MEIS2 expression in placentas from T15 and euploid spontaneous abortions employing manual and automated assessment of MEIS2 immunohistochemical scoring. Results: Average MEIS2 expression is increased in T15 first trimester placental tissue compared to euploid controls but that the difference is marginal. Manual and automated scoring showed moderately strong correlation. Conclusion: Extrapolation of these results suggests MEIS2 overexpression may not be required for meromelia in T15.

Reproducibility of Grading in Chronic Intervillositis of Unknown Etiology.

Chronic intervillositis of unknown etiology (CIUE) is a rare placental inflammatory process associated with pregnancy loss and recurrence. We conducted a quality assurance study to assess the diagnostic accuracy and reproducibility of CIUE grading at our institution. Hematoxylin and eosin-stained slides from 20 CIUE cases (31 slides) were reviewed by 7 perinatal pathologists in 2 sequential rounds. Reviewers were instructed to use the diagnostic criteria they were presently following for CIUE and to grade each slide according to the Rota scheme. In the first round, 20 slides were assessed. The diagnostic accuracy was 94%, the average percent agreement of Rota grade was 79%, and the Fleiss' kappa value for interobserver variability was 0.54. The results were reviewed by all pathologists with diagnostic and grading criteria agreed upon prior to the second round. In round 2, the remaining 11 slides were assessed. Diagnostic accuracy was 83%, the average percent agreement on Rota grade was 70%, and the Fleiss' kappa value for interobserver variability was 0.36. Overall, diagnostic accuracy was high and agreement on Rota grade was moderate. Group review did not appear to improve accuracy. Simplifying CIUE grading to a low-grade/high-grade scheme (<50% or ≥50%) might improve grading reproducibility.

Prenatal Autoimmune Disease, Multisystem, Infantile Onset-like Phenotype and Proximal Renal Tubular Dysplasia Associated With STAT3 Mutation.

Activating heterozygous germline mutations in the signal transducer and activator of transcription 3 (STAT3) gene are associated with the rare autoimmune disorder autoimmune disease, multisystem, infantile onset (ADMIO). The phenotype of ADMIO is typified by hypogammaglobulinemia and onset of autoimmune phenomena during early childhood that include diabetes and autoimmune enteritis. This case report describes in utero onset of precocious lymphocyte maturation, autoimmune enteropathy-like inflammation, and proximal renal tubular dysplasia associated with a novel de novo heterozygous STAT3 mutation. The findings expand the phenotype associated with activating STAT3 mutations and suggest that the impact of the immunological abnormalities associated with ADMIO can begin prior to birth.

Association of a placental Interleukin-6 genetic variant (rs1800796) with DNA methylation, gene expression and risk of acute chorioamnionitis.

BACKGROUND: Acute chorioamnionitis (aCA), inflammation of the placenta and fetal membranes, is a frequently reported lesion in preterm deliveries. Genetic variants in innate immune system genes such as Interleukin-6 (IL6) may contribute to the placenta's inflammatory response, thus predisposing some pregnancies to aCA. These genetic variants may modulate molecular processes such as DNA methylation and gene expression, and in turn might affect susceptibility to aCA. Currently, there is remarkably little research on the role of fetal (placental) genetic variation in aCA. We aimed to explore the associations between genetic variants in candidate immune-system genes and susceptibility towards inflammatory responses in the placenta, which is linked to a strong inflammatory response in the newborn. METHODS: DNA samples from 269 placentas (72 aCA cases, 197 non-aCA cases) were collected for this study. Samples were genotyped at 55 ancestry informative markers (AIMs) and 16 additional single nucleotide polymorphisms (SNPs) in 12 candidate innate immune system genes using the Sequenom iPLEX Gold Assay. Publicly available datasets were used to obtain DNA methylation (GSE100197, GSE74738, GSE115508, GSE44667, GSE98224) and gene expression data (GSE44711, GSE98224). RESULTS: Differences in IL6 placental allele frequencies were associated with aCA (rs1800796, p = 0.04) with the CC genotype specifically implicated (OR = 3.1; p = 0.02). In a subset of the placental samples (n = 67; chorionic villi), we showed that the IL6 SNP (rs1800796) was associated with differential DNA methylation in five IL6-related CpG sites (cg01770232, cg02335517, cg07998387, cg13104385, and cg0526589), where individuals with a CC genotype showed higher DNA methylation levels than individuals carrying the GG genotype. Using two publicly available datasets, we observed that the DNA methylation levels at cg01770232 negatively correlated with IL6 gene expression in the placenta (r = - 0.67, p < 0.004; r = - 0.56, p < 2.937e-05). CONCLUSIONS: We demonstrated that the minor C allele at the IL6 SNP (rs1800796), which is largely limited to East Asian populations, is associated with the presence of aCA. This SNP was associated with increased DNA methylation at a nearby MEPC2 binding site, which was also associated with decreased expression of IL6 in the placenta. Decreased expression of IL6 may increase vulnerability to microbial infection. Additional studies are required to confirm this association in Asian populations with larger sample sizes.

Morphology of early intrauterine deaths with full trisomy 15.

OBJECTIVE: The morphologic features of embryos with full trisomy 15 are described. METHOD: A total of 1195 pregnancy losses were examined embryoscopically and cytogenetically. RESULTS: Of 1173 successfully karyotyped specimens, full trisomy 15 was diagnosed cytogenetically in 59 cases (5%). All 59 trisomy 15 embryos were diagnosed cytogenetically in the group of 962 embryonic miscarriages (6%). Trisomy 15 was not registered in 171 anembryonic or yolk sac miscarriages, and no case of full trisomy 15 was observed in 62 fetal miscarriages. Fifty-eight embryos with full trisomy 15 showed structural defects on embryoscopic examination. The most common defects were craniofacial anomalies (n = 73), retarded development of the limbs (n = 39), and abnormally short umbilical cords closely attaching the embryo to the chorionic plate (n = 27). Seven embryos were classified as growth disorganized. Limb reduction defects with a prevalence of 5.6/10 000 births, all affecting upper limb development (10 terminal transverse limb reduction defects and 3 embryos with split hand), were registered in 13 (22%) trisomy 15 embryos. CONCLUSION: Limb reduction defects and craniofacial abnormalities are a typical feature of trisomy 15. Gene dosage imbalances related to trisomy 15 might be the main molecular mechanism underlying the developmental defects observed in the present study and require further investigation.

The importance of considering monogenic causes of autoimmunity: A somatic mutation in KRAS causing pediatric Rosai-Dorfman syndrome and systemic lupus erythematosus.

OBJECTIVES: Clinicians need to be aware of the growing list of defined monogenic etiologies of autoimmune diseases. This is particularly relevant when evaluating children, as these rare monogenic forms of autoimmunity tend to present very early in life. METHODS AND RESULTS: By harnessing the transformative power of next generation sequencing, we made the unifying diagnosis of RAS-associated autoimmune leukoproliferative disease (RALD), caused by the somatic gain-of-function p.G13C KRAS mutation, in a boy with the seemingly unrelated immune dysregulatory conditions of Rosai-Dorfman and systemic lupus erythematosus (SLE). CONCLUSIONS: This case expands our understanding of the clinical phenotypes associated with the extremely rare condition of RALD, and emphasizes the importance of always considering the possibility of a monogenic cause for autoimmunity, particularly when the disease manifestations begin early in life and do not follow a typical clinical course.

Disruption of IKAROS activity in primitive chronic-phase CML cells mimics myeloid disease progression.

Without effective therapy, chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized, but biologically poorly characterized, accelerated phase (AP). Here, we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts, which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML, we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML, including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients.