Novel COL2A1 variants in Japanese patients with spondyloepiphyseal dysplasia congenita.

Spondyloepiphyseal dysplasia congenita (SEDC) is a multisystemic skeletal disorder caused by pathogenic variants in COL2A1. Here, we report the genotype-phenotype correlations in five Japanese patients with SEDC based on their clinical and radiological findings. All five patients had novel missense variants resulting in glycine substitutions (G474V, G543E, G567S, G594R, and G1170R). Genetic testing is important for early intervention for the extraskeletal complications of SEDC. Spondyloepiphyseal dysplasia congenita (SEDC) (OMIM#183900) is an autosomal dominant chondrodysplasia characterized by disproportionate short stature, abnormal epiphyses, flattened vertebral bodies (skeletal abnormalities), and extraskeletal features, including myopia, retinal degeneration with retinal detachment, and cleft palate. SEDC is caused by a heterozygous variant in the collagen II alpha 1 (COL2A1) gene.

Deletion of ERF and CIC causes abnormal skull morphology and global developmental delay.

The ETS2 repressor factor (ERF) is a transcription factor in the RAS-MEK-ERK signal transduction cascade that regulates cell proliferation and differentiation, and pathogenic sequence variants in the ERF gene cause variable craniosynostosis inherited in an autosomal dominant pattern. The reported ERF variants are largely loss-of-function, implying haploinsufficiency as a primary disease mechanism; however, ERF gene deletions have not been reported previously. Here we describe three probands with macrocephaly, craniofacial dysmorphology, and global developmental delay. Clinical genetic testing for fragile X and other relevant sequencing panels were negative; however, chromosomal microarray identified heterozygous deletions (63.7-583.2 kb) on Chromosome 19q13.2 in each proband that together included five genes associated with Mendelian diseases (ATP1A3, ERF, CIC, MEGF8, and LIPE). Parental testing indicated that the aberrations were apparently de novo in two of the probands and were inherited in the one proband with the smallest deletion. Deletion of ERF is consistent with the reported loss-of-function ERF variants, prompting clinical copy-number-variant classifications of likely pathogenic. Moreover, the recent characterization of heterozygous loss-of-function CIC sequence variants as a cause of intellectual disability and neurodevelopmental disorders inherited in an autosomal dominant pattern is also consistent with the developmental delays and intellectual disabilities identified among the two probands with CIC deletions. Taken together, this case series adds to the previously reported patients with ERF and/or CIC sequence variants and supports haploinsufficiency of both genes as a mechanism for a variable syndromic cranial phenotype with developmental delays and intellectual disability inherited in an autosomal dominant pattern.

Discordant phenotype caused by CASK mutation in siblings with NF1.

With the advent of next-generation sequencing (NGS), a blended phenotype has been shown to be caused by multilocus molecular diagnosis. Here, we present siblings of neurofibromatosis type 1 (NF1) with discordant phenotypes. Further genetic investigation revealed that the younger sister had trisomy 8 mosaicism with a low ratio and a known pathogenic mutation in the CASK gene. This is the first report of a blended phenotype caused by NF1, CASK disorder, and trisomy 8 mosaicism.

Novel COL4A1 mutation in a fetus with early prenatal onset of schizencephaly.

Porencephaly and schizencephaly are congenital brain disorders that can be caused by COL4A1 mutations, though the underlying mechanism and developmental processes are poorly understood. Here, we report a patient with schizencephaly, detected by fetal ultrasonography and fetal magnetic resonance imaging, with a de novo novel mutation in COL4A1 (c.2645_2646delinsAA, p.Gly882Glu). Our results suggest that the onset of damage that potentially results in schizencephaly occurs mid-pregnancy.

Refining the clinical phenotype of Okur-Chung neurodevelopmental syndrome.

We describe an 8-year-old Japanese boy with a de novo recurrent missense mutation in CSNK2A1, c.593A>G, that is causative of Okur-Chung neurodevelopmental syndrome. He exhibited distinctive facial features, severe growth retardation with relative macrocephaly, and friendly, hyperactive behavior. His dysmorphic features might suggest a congenital histone modification defect syndrome, such as Kleefstra, Coffin-Siris, or Rubinstein-Taybi syndromes, which are indicative of functional interactions between the casein kinase II, alpha 1 gene and histone modification factors.

Maternal socio-demographic and psychological predictors for risk of developmental delays among young children in Mongolia.

BACKGROUND: Factors influencing child development are not well studied in developing settings, and especially in Mongolia. This cohort study examined the relationship between maternal socio-demographic and psychological conditions on risk of young child developmental delay. METHODS: A total of 150 children aged between 13 ~ 24 months old participated in this study. The participants were randomly selected from a pre-existing cohort of 1297 children who were involved in a study on infant bilirubin nomogram development conducted at a tertiary health facility in Mongolia between 2012 and 2013. Child development was evaluated using the Mongolian Rapid Baby Scale (MORBAS), a validated scale for child development. The potential factors for child developmental delay were assessed using a pre-tested questionnaire comprising of 52 questions. Fisher's exact test and multivariable logistic regression analysis were conducted. RESULTS: Seventeen (11%) out of the 150 children that participated in the study were at risk of developmental delay. There was a negative association between the risk of child developmental delay and higher maternal education (AOR 0.15, 95% CI: 0.03-0.66). Increasing maternal age (AOR 1.12, 95%CI: 0.98-1.27), maternal depression symptoms (AOR 4.93, 95%CI: 0.93-26.10), child gender being female (AOR 0.25, 95%CI: 0.06-1.00) and being from single mother household (AOR 0.14, 95%CI: 0.01-1.11) were also predictors for risk of developmental delay - although the association was marginal. CONCLUSIONS: Our findings suggest that being of underprivileged social status, and poor psychological condition of mothers in Mongolia possibly increases the risk of child developmental delays. Interventions targeting these modifiable predictors are needed to develop prevention strategies for child developmental delay.

Novel SYNGAP1 variant in a patient with intellectual disability and distinctive dysmorphisms.

We describe a novel de novo heterozygous variant in SYNGAP1 (c.1741C>T, p.R581W), identified through targeted resequencing in an 8-year-old boy with intellectual disability, autism spectrum disorder, distinctive dysmorphic features, and no seizures. Our data strongly suggest that the SYNGAP1 variant is causative of intellectual disability in this patient.

Republication: Two Premature Neonates of Congenital Syphilis with Severe Clinical Manifestations.

Congenital syphilis (CS) is a public health burden in both developing and developed countries. We report two cases of CS in premature neonates with severe clinical manifestations; Patient 1 (gestational age 31 weeks, birth weight 1423 g) had disseminated idiopathic coagulation (DIC) while Patient 2 (gestational age 34 weeks and 6 days, birth weight 2299 g) had refractory syphilitic meningitis. Their mothers were single and had neither received antenatal care nor undergone syphilis screening. Both neonates were delivered via an emergency cesarean section and had birth asphyxia and transient tachypnea of newborn. Physical examination revealed massive hepatosplenomegaly. Laboratory testing of maternal and neonatal blood showed increased rapid plasma reagin (RPR) titer and positive Treponema pallidum hemagglutination assay. Diagnosis of CS was further supported by a positive IgM fluorescent treponemal antibody absorption test and large amounts of T. pallidum spirochetes detected in the placenta. Each neonate was initially treated with ampicillin and cefotaxime for early bacterial sepsis/meningitis that coexisted with CS. Patient 1 received fresh frozen plasma and antithrombin III to treat DIC. Patient 2 experienced a relapse of CS during initial antibiotic treatment, necessitating parenteral penicillin G. Treatment was effective in both neonates, as shown by reductions in RPR. Monitoring of growth and neurological development through to age 4 showed no evidence of apparent delay or complications. Without adequate antenatal care and maternal screening tests for infection, CS is difficult for non-specialists to diagnose at birth, because the clinical manifestations are similar to those of neonatal sepsis and meningitis. Ampicillin was insufficient for treating CS and penicillin G was necessary.

An hour-specific transcutaneous bilirubin nomogram for Mongolian neonates.

UNLABELLED: Transcutaneous bilirubin (TcB) nomograms have been developed for different populations. However, the TcB level, rate of rise and peak varies among countries and ethnicities. The aim of this study was to establish an hour-specific TcB nomogram for healthy term and late preterm Mongolian neonates during the first 144 h after birth. A total of 5084 TcB measurements from 1297 healthy neonates (gestational age ≥35 weeks, birth weight ≥2000 g) were obtained from October 2012 to October 2013. All measurements were performed using the Jaundice Meter, the JM-103 at 6 to 144 postnatal hours. Mongolian infants had the following characteristics: 27.1 % were delivered by cesarean section, 17.8 % had a birth weight >4000 g, and >90 % were being breastfed. TcB percentiles for each designated time point were calculated for the development of an hour-specific nomogram. TcB levels increased most rapidly in the first 24 h and less rapidly from 24 to 78 h, reaching a plateau after 78 h for the 50th percentile. TcB levels of Mongolian neonates for each time point were higher than those of previous studies. CONCLUSION: The higher values of the TcB nomogram for Mongolian neonates may be due to their Asian ethnicity and exclusive breastfeeding. WHAT IS KNOWN: • TcB nomograms for neonatal jaundice screening have been established for many countries and ethnicities. The pattern of the TcB nomogram varies by country and ethnicity. What is New: • A TcB nomogram for neonates of Mongolian ethnicity at 6-144 postnatal hours was created and it had higher values than those in previous studies.

Validation of a transcutaneous bilirubin meter in Mongolian neonates: comparison with total serum bilirubin.

BACKGROUND: Neonatal hyperbilirubinemia, especially kernicterus, can be prevented by screening for neonatal jaundice. The transcutaneous bilirubin (TcB) meter is a non-invasive medical device for screening neonates. The study aimed to investigate the validity of a TcB meter in a resource-limited setting such as Mongolia. METHODS: Term and late preterm neonates from the National Center for Maternal and Child Health of Ulaanbaatar in Mongolia who met the inclusion criteria (gestational age ≥35 weeks, birth weight ≥2000 g, postnatal age ≤ 1 month) were enrolled in the study. We used a TcB meter, JM-103 to screen for neonatal jaundice. TcB measurements at the infant's forehead and midsternum were performed within 3 h of obtaining samples for total serum bilirubin (TSB) measurement. We analyzed the correlation between TcB measurements and TSB measurements to validate the meter. RESULTS: A total of 47 term and six late preterm neonates were included in the study. TcB measured by the meter at both the forehead and the midsternum showed a strong correlation with TSB measured in the laboratory. The correlation equations were TSB = 1.409+0.8655 × TcB (R2=0.78871) at the forehead, and TSB = 0.7555+0.8974 × TcB (R2=0.78488) at the midsternum. Bland-Altman plots and the Bradley-Blackwood test showed no significant differences between the two methods at all measured ranges of bilirubin. The mean areas under the curves of TcB at the forehead and midsternum at three TSB levels (>10 mg/dL, >13 mg/dL, >15 mg/dL) of TcB were greater than 0.9, and all had high sensitivity and specificity. CONCLUSIONS: This study established the validity of the JM-103 meter as a screening tool for neonatal jaundice in term and late preterm infants in Mongolia. Future studies are needed, including the establishment of a TcB hour-specific nomogram, for more effective clinical practice to prevent severe hyperbilirubinemia.

A Japanese neonatal case of glucose-6-phosphate dehydrogenase deficiency presenting as severe jaundice and hemolytic anemia without apparent trigger.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is rare among Japanese ethnicity although it is known as one of the most common hereditary disorders of erythrocytes, causing intravascular hemolysis. It is well-known that G6PD deficiency may cause hemolysis even in the neonatal period. However, most cases are asymptomatic, and the frequency of severe anemia is low. FINDINGS: We describe a Japanese male neonatal case of G6PD deficiency presenting as severe, persistent indirect hyperbilirubinemia on day 2 and hemolytic anemia. He was born to non-consanguineous Japanese parents without any family history. We could not find any triggers that could have induced hemolysis during pregnancy. CONCLUSIONS: This case encouraged us to investigate G6PD deficiency as a differential diagnosis of severe neonatal jaundice and hemolytic anemia despite the low prevalence in Japan.

Early delayed-type hypersensitivity eosinophil infiltrates depend on T helper 2 cytokines and interferon-gamma via CXCR3 chemokines.

We investigated the role of T helper (Th)1- and Th2-type cytokines in delayed-type hypersensitivity to soluble protein antigens elicited early postimmunization. Mice were sensitized by intradermal injection without adjuvants, or subcutaneously with complete Freund's adjuvant, and subsequently ear challenged intradermally. As soon as day 3, antigen-specific eosinophil-rich responses were elicited in wild-type mice, but not in T-cell receptor-alpha-/- mice without adjuvant. Draining lymph node T cells stimulated with antigen secreted interleukin (IL)-4, IL-5 and interferon-gamma (IFN-gamma). IFN-gamma-dependent specific immunoglobulin G (IgG)2a and IL-4-dependent IgG1 were also generated. Delayed-type hypersensitivity ear swelling and local eosinophil recruitment were decreased in IL-5-/-, IL-4-/- and signal transducer and activator of transcription-6 (STAT-6)-/- mice, and with anti-IL-4 treatment of wild-type mice, suggesting Th2 mechanisms. Interestingly, responses were also decreased in IFN-gamma-/- mice, and IFN-gamma protein and the IFN-gamma-inducible CXC chemokine, IP-10, were present in 24-hr ear tissue extracts, suggesting Th1 effects. Finally, ear swelling, total histology and eosinophils were decreased in mice deficient in CXCR3, the chemokine receptor for IP-10. These results suggest that both a Th2-like (IL-5, IL-4 and STAT-6) and a Th1-like (IFN-gamma, IP-10, CXCR3) pathway contribute to eosinophil recruitment in early delayed-type hypersensitivity.

Cutaneous immunization rapidly activates liver invariant Valpha14 NKT cells stimulating B-1 B cells to initiate T cell recruitment for elicitation of contact sensitivity.

T cell recruitment to elicit contact sensitivity (CS) requires a CS-initiating process mediated by B-1 cells that produce IgM, which activates complement to promote T cell passage into the tissues. We now show that Valpha14i NKT cells induce B-1 cell activation likely by releasing IL-4 early postimmunization. The CS initiation process is absent in Jalpha18-/- and CD1d-/- NKT cell-deficient mice and is reconstituted by populations enriched for Valpha14i NKT cells. Transfers are not effective if cells are derived from IL-4-/- mice. Staining with specific tetramers directly showed that hepatic Valpha14i NKT cells increase by 30 min and nearly double by 2 h postimmunization. Transfer of immune B-1 cells also reconstitutes CS responses in NKT cell-deficient mice. The B-1 cells act downstream of the Valpha14i NKT cells to restore CS initiation. In addition, IL-4 given systemically to Jalpha18-/- or CD1d-/- NKT cell-deficient mice reconstitutes elicitation of CS. Further, splenocytes from immune Jalpha18-/- mice produce less antigen (Ag)-specific IgM antibodies compared with sensitized WT mice. Together these findings indicate that very early after skin immunization Valpha14i NKT cells are stimulated to produce IL-4, which activates B-1 cells to produce Ag-specific IgM, subsequently needed to recruit effector T cells for elicitation of CS responses.

B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayed-type hypersensitivity.

We define the initiation of elicited delayed-type hypersensitivity (DTH) as a series of processes leading to local extravascular recruitment of effector T cells. Responses thus have two sequential phases: 1) 2-h peaking initiation required for subsequent recruitment of T cells, and 2) the late classical 24-h component mediated by the recruited T cells. We analyzed DTH initiation to protein Ags induced by intradermal immunization without adjuvants. Ag-spceific initiating cells are present by 1 day in spleen and lymph nodes. Their phenotypes, determined by depletion of cell transfers by mAb and complement, are CD5(+), CD19(+), CD22(+), B220(+), Thy1(+), and Mac1(+), suggesting that they are B-1 B cells. DTH initiation is absent in micro MT B cell and xid B-1 cell deficient mice, is impaired in mice unable to secrete IgM, and is reconstituted with 1 day immune serum, suggesting that early B-1 cell-derived IgM is responsible. Study of complement C5a receptor-deficient mice, anti-C5 mAb neutralization, or mast cell deficiency suggests that DTH initiation depends on complement and mast cells. ELISPOT assay confirmed production of Ag-specific IgM Abs at days 1 and 4 in wild-type mice, but not in B-1 cell-deficient xid mice. We conclude that rapidly activated B-1 cells produce specific IgM Abs which, after local secondary skin challenge, form Ag-Ab complexes that activate complement to generate C5a. This stimulates C5a receptors on mast cells to release vasoactive substances, leading to endothelial activation for the 2-h DTH-initiating response, allowing local recruitment of DTH-effector T cells.

B cell-dependent T cell responses: IgM antibodies are required to elicit contact sensitivity.

Contact sensitivity (CS) is a classic example of in vivo T cell immunity in which skin sensitization with reactive hapten leads to immunized T cells, which are then recruited locally to mediate antigen-specific inflammation after subsequent skin challenge. We have previously shown that T cell recruitment in CS is triggered by local activation of complement, which generates C5a that triggers C5a receptors most likely on mast cells. Here, we show that B-1 cell-derived antihapten IgM antibodies generated within 1 day (d) of immunization combine with local challenge antigen to activate complement to recruit the T cells. These findings overturn three widely accepted immune response paradigms by showing that (a) specific IgM antibodies are required to initiate CS, which is a classical model of T cell immunity thought exclusively due to T cells, (b) CS priming induces production of specific IgM antibodies within 1 d, although primary antibody responses typically begin by day 4, and (c) B-1 cells produce the 1-d IgM response to CS priming, although these cells generally are thought to be nonresponsive to antigenic stimulation. Coupled with previous evidence, our findings indicate that the elicitation of CS is initiated by rapidly formed IgM antibodies. The IgM and challenge antigen likely form local complexes that activate complement, generating C5a, leading to local vascular activation to recruit the antigen-primed effector T cells that mediate the CS response.