A novel likely pathogenic CLCN5 variant in Dent's disease.

BACKGROUND: The majority of cases of Dent's disease are caused by pathogenic variants in the CLCN5 gene, which encodes a voltage-gated chloride ion channel (ClC-5), resulting in proximal tubular dysfunction. We present three members of the same family and one unrelated paediatric patient with the same insertion-deletion CLCN5 variant. The identification of these patients and positive familial segregation led to the re-classification of this variant from one of unknown significance to one of likely pathogenicity. CASE PRESENTATION: A 41 year old male presented with end stage kidney failure, proteinuria and haematuria. Whole genome sequencing identified an insertion-deletion variant in CLCN5, resulting in a missense change (c.1744_1745delinsAA p.(Ala582Lys)). His brother and nephew, who both exhibited renal impairment, haematuria, proteinuria, glycosuria and nephrocalcinosis, were found to have the same variant. In addition, genetic testing of an unrelated paediatric patient who presented with proteinuria and hypercalciuria, demonstrated the same variant. CONCLUSIONS: The identification of this novel variant in four individuals with features of Dent's disease, has led to the re-classification of the variant to one of likely pathogenicity. As a result, our patients and any future patients with the same variant can be offered a likely diagnosis, without the need for kidney biopsy, and their family members can be offered genetic screening.

Development, behaviour and sensory processing in Marshall-Smith syndrome and Malan syndrome: phenotype comparison in two related syndromes.

BACKGROUND: Ultrarare Marshall-Smith and Malan syndromes, caused by changes of the gene nuclear factor I X (NFIX), are characterised by intellectual disability (ID) and behavioural problems, although questions remain. Here, development and behaviour are studied and compared in a cross-sectional study, and results are presented with genetic findings. METHODS: Behavioural phenotypes are compared of eight individuals with Marshall-Smith syndrome (three male individuals) and seven with Malan syndrome (four male individuals). Long-term follow-up assessment of cognition and adaptive behaviour was possible in three individuals with Marshall-Smith syndrome. RESULTS: Marshall-Smith syndrome individuals have more severe ID, less adaptive behaviour, more impaired speech and less reciprocal interaction compared with individuals with Malan syndrome. Sensory processing difficulties occur in both syndromes. Follow-up measurement of cognition and adaptive behaviour in Marshall-Smith syndrome shows different individual learning curves over time. CONCLUSIONS: Results show significant between and within syndrome variability. Different NFIX variants underlie distinct clinical phenotypes leading to separate entities. Cognitive, adaptive and sensory impairments are common in both syndromes and increase the risk of challenging behaviour. This study highlights the value of considering behaviour within developmental and environmental context. To improve quality of life, adaptations to environment and treatment are suggested to create a better person-environment fit.

Phenotypic analysis of pneumococcal polysaccharide-specific B cells.

The phenotype of B cells responsible for the production of anti-pneumococcal polysaccharide Ab has been unclear. Although individuals that respond poorly to the 23-valent pneumococcal polysaccharide (PPS) vaccine, Pneumovax, such as children <2 y, the asplenic, and a subset of common variable immunodeficiency patients, are profoundly deficient or lack IgM memory cells (CD27(+)IgM(+)), they are also deficient in the switched memory (CD27(+)IgM(-)) compartment. Direct characterization of PPS-specific B cells has not been performed. In this study, we labeled PPS14 and PPS23F with fluorescent markers. Fluorescently labeled PPS were used in FACSAria flow cytometry to characterize the phenotype of PPS-specific B cells obtained from 18 young adults pre- and postimmunization with Pneumovax. The labeled PPS were capable of inhibiting binding of Ab to the native PPS. Similarly, the native PPS were able to inhibit binding of PPS-specific B cells in a flow cytometric assay demonstrating specificity and functionality. Phenotypic analysis of unselected B cells, pre- and postimmunization, demonstrated a predominance of naive CD27(-)IgM(+) cells accounting for 61.5% of B cells. Likewise, the PPS-specific B cells obtained preimmunization consisted primarily of naive, CD27(-) B cells, 55.4-63.8%. In contrast, the PPS-specific B cells obtained postimmunization were predominantly IgM memory cells displaying the CD27(+)IgM(+), 54.2% for PPS14 and 66% for PPS23F, significantly higher than both unselected B cells and PPS-specific B cells. There was no significant difference in switched memory B cell populations (CD27(+)IgM(-)) between groups. These results suggest a dominant role of IgM memory cells in the immune response to pneumococcal polysaccharides.

Unplanned Cesarean delivery is associated with risk for postpartum depressive symptoms in the immediate postpartum period.

PURPOSE: Postpartum depression (PPD) is a common pregnancy complication. The association between cesarean delivery (CD) and PPD has shown conflicting results in prior studies, although emergent CD appears to be a clear risk factor. Establishing PPD risk is critical and may, however, be related to the unplanned nature of the CD, rather than the surgery itself. Our objective was to determine whether women who underwent unplanned CD were more likely than those with vaginal delivery to have higher depressive symptoms and thus screen positive for PPD risk in the immediate postpartum period. MATERIALS AND METHODS: This cohort study was conducted at a community medical center using data for deliveries between 8/2015-1/2016. Women were screened in the hospital for depressive symptoms (PPD risk) using the Edinburgh Postnatal Depression Scale (EPDS) within 4 days post-delivery. Logistic regression, adjusting for maternal race/ethnicity and parity, was performed to evaluate the association between delivery route (vaginal vs planned vs unplanned CD) and PPD risk (EPDS ≥ 10). RESULTS: A total of 2094 women had complete data for analysis. Overall, 44 women (2.1%) screened positive for PPD risk. Logistic regression results showed that unplanned CD was significantly associated with PPD risk (OR = 2.28, 95% CI 1.13-4.57, p = .022), after adjusting for parity and race/ethnicity. Planned CD was not associated with PPD risk. CONCLUSION: Unplanned CD may be an independent risk factor for PPD risk in the immediate postpartum period. This finding might explain why some previous studies have demonstrated different results with regards to risk of CD where the unplanned nature of the delivery was not accounted for.

Barth syndrome: an X-linked cause of fetal cardiomyopathy and stillbirth.

OBJECTIVE: Barth Syndrome (BTHS) is an X-linked multisystem disorder (OMIM 302060) usually diagnosed in infancy and characterized by cardiac problems [dilated cardiomyopathy (DCM) ± endocardial fibroelastosis (EFE) ± left ventricular non-compaction (LVNC)], proximal myopathy, feeding problems, growth retardation, neutropenia, organic aciduria and variable respiratory chain abnormalities. We wished to determine whether BTHS had a significant impact on fetal and perinatal health in a large cohort of family groups originating from a defined region. METHOD: Case note review on 19 families originating from the UK and known to the Barth Syndrome Service of the Bristol Royal Hospital for Children. RESULTS: Details are presented on six kindreds (32%) with genetically and biochemically proven BTHS that demonstrate a wider phenotype including male fetal loss, stillbirth and severe neonatal illness or death. In these families, 9 males were stillborn and 14 died as neonates or infants but there were no losses of females. BTHS was definitively proven in five males with fetal onset of DCM ± hydrops/EFE/LVNC. CONCLUSION: These findings stress the importance of considering BTHS in the differential diagnosis of unexplained male hydrops, DCM, EFE, LVNC or pregnancy loss, as well as in neonates with hypoglycemia, lactic acidosis and idiopathic mitochondrial disease.

De novo variants in CNOT3 cause a variable neurodevelopmental disorder.

As a result of exome-based sequencing work performed by the DDD study, de novo variants in CNOT3 have emerged as a newly recognised cause of a developmental disorder. This paper describes molecular and clinical details of 16 probands with developmental disorders and de novo CNOT3 variants. It is the first such description of the developmental phenotype associated with CNOT3 variants. Eight of these cases were discovered as part of the DDD study, while the other eight were found as a result of large-scale sequencing work performed by other groups. A highly specific phenotype was not recognised in these 16 cases. The most consistent phenotypic features seen in subjects with de novo variants in CNOT3 were hypotonia, relatively small stature, developmental delay, behavioural problems and intellectual disability. There is no easily recognisable facial phenotype, but some common dysmorphic features such as anteverted nares, thin upper lip and low set eyebrows were shared among some of the probands. Haploinsufficiency appears to be the most likely mechanism of action, with eight cases found to have protein-truncating variants. Of the other eight cases (all missense variants), three share an amino acid substitution at the same position which may therefore represent an important functional domain.

Phenotype of CNTNAP1: a study of patients demonstrating a specific severe congenital hypomyelinating neuropathy with survival beyond infancy.

CHN is genetically heterogeneous and its genetic basis is difficult to determine on features alone. CNTNAP1 encodes CASPR, integral in the paranodal junction high molecular mass complex. Nineteen individuals with biallelic variants have been described in association with severe congenital hypomyelinating neuropathy, respiratory compromise, profound intellectual disability and death within the first year. We report 7 additional patients ascertained through exome sequencing. We identified 9 novel CNTNAP1 variants in 6 families: three missense variants, four nonsense variants, one frameshift variant and one splice site variant. Significant polyhydramnios occurred in 6/7 pregnancies. Severe respiratory compromise was seen in 6/7 (tracheostomy in 5). A complex neurological phenotype was seen in all patients who had marked brain hypomyelination/demyelination and profound developmental delay. Additional neurological findings included cranial nerve compromise: orobulbar dysfunction in 5/7, facial nerve weakness in 4/7 and vocal cord paresis in 5/7. Dystonia occurred in 2/7 patients and limb contractures in 5/7. All had severe gastroesophageal reflux, and a gastrostomy was required in 5/7. In contrast to most previous reports, only one patient died in the first year of life. Protein modelling was performed for all detected CNTNAP1 variants. We propose a genotype-phenotype correlation, whereby hypomorphic missense variants partially ameliorate the phenotype, prolonging survival. This study suggests that biallelic variants in CNTNAP1 cause a distinct recognisable syndrome, which is not caused by other genes associated with CHN. Neonates presenting with this phenotype will benefit from early genetic definition to inform clinical management and enable essential genetic counselling for their families.

The effect of T cell independent and cross-reactive antigen on the immune response to pneumococcal conjugate vaccination.

The effect of priming with various antigens on subsequent vaccination with the pneumococcal conjugate vaccine (CPV) was determined using BALB/c mice. Priming with pneumococcal polysaccharide or cross-reactive polysaccharide did not inhibit the IgG response to CPV immunization. Additionally, live intranasal colonization by Streptococcus pneumoniae or cross-reactive organism resulted in higher IgG responses to CPV. These results suggest that colonization elicits immunological memory capable of boosting the immune response to CPV.

Orthopaedic manifestations and management of spondyloepimetaphyseal dysplasia Strudwick type.

Spondyloepimetaphyseal dysplasia (SEMD) Strudwick type is a rare autosomal dominant condition arising from defects in COL2A1 the genes responsible for the biosynthesis of procollagen type II. The orthopaedic manifestations of patients can be hypoplastic odontoid peg with atlantoaxial instability, severe kyphosis or lordosis of dorsal and lumbar spines, hip subluxation, coxa vara and early severe hip osteoarthritis, and malalignment of lower limbs like genu valgum or club foot. We report a mother and daughter with SEMD Strudwick Type and describe their orthopaedic problems, surgical management and clinical outcome after 30 years and 7 years of follow-up respectively.

Immune response to pneumococcal polysaccharides 4 and 14 in elderly and young adults. I. Antibody concentrations, avidity and functional activity.

Streptococcus pneumoniae is a serious worldwide pathogen and the focus of numerous vaccine development projects. Currently the most widely accepted surrogate marker for evaluating the efficacy of a given vaccine is to utilize ELISA. Measurement of antibody concentration by ELISA without reduction in cross-reactive antibodies causes an overestimation of antibody concentration and therefore protection, this is most notable in the aged, an at risk group for this infection. We compared the immune response to the pneumococcal polysaccharides (PPS) 4 and 14 of 20 young to 20 elderly adults. Pre-and post-vaccination IgG antibody concentrations and antibody avidity against PPS4 and PPS14 were measured using two different enzyme-linked immunosorbant assay (ELISA) absorption protocols. All sera were pre-absorbed with either cell-wall polysaccharide (CPS), or CPS and serotype 22F polysaccharide. Pre- and post-vaccination IgG antibody concentrations for serotype 4, but not 14, were significantly lowered with the additional absorption with serotype 22F polysaccharide in both age groups. Young and elderly demonstrated a significant increase from pre- to post-immunization antibody concentration, using either absorption method; and opsonophagocytic antibody titers in response to both PPS4 and PPS14. The correlation coefficients between ELISA and opsonophagocytic assays were improved by additional absorption with serotype 22F in response to serotype 4, but not serotype 14 in all age groups. Opsonophagocytic antibody titers in a sub-group of elderly (>77 years of age) were significantly lower than the opsonophagocytic antibody concentrations in young adults. These results suggest the importance of eliminating cross-reactive antibodies from ELISA measurements by absorption of serum and an age-related impairment in the antibody response to pneumococcal polysaccharides.

Molecular analysis of the heavy chain of antibodies that recognize the capsular polysaccharide of Neisseria meningitidis in hu-PBMC reconstituted SCID mice and in the immunized human donor.

The severe combined immunodeficient (SCID) mouse model, engrafted with human peripheral blood mononuclear cells (hu-PBMC) has proven to be useful in studying the human immune response. A major limitation of the hu-PBMC-SCID model has been the failure to consistently demonstrate a primary human immune response. Previously we developed a hu-PBMC-SCID mouse model in which we addressed both issues of adequate human lymphocyte engraftment and impaired differentiation. We demonstrated that a primary human immune response to the T-independent (TI-2) meningococcal group C capsular polysaccharide (MCPS) can be obtained in hu-PBMC-SCID mice by the administration of human cytokines. In this study we compared the V(H) sequence of the MCPS response generated by B cells derived from a volunteer in the SCID mouse model to those generated by the donors' B cells in vivo. Human peripheral blood mononuclear cells were recovered from MCPS immunized hu-PBMC-SCID mice and immunized donor. B cells with specificity for MCPS were isolated from these cell preparations using an anti-idiotypic monoclonal antibody which mimics MCPS. Immunoglobulin mRNA was isolated from single cells, amplified by the polymerase chain reaction, cloned and sequenced. We analysed a total of 15 V(H) regions from B cells obtained from SCID mice and a total of 13 V(H) regions from B cells obtained from the immunized donor. The response differed between SCID and in vivo cells, when studied at the genetric level. V, D and J gene usage was markedly different, however canonical structures of the hypervariable loops were conserved. The complementary determining region 3 (CDR3) varied, such that SCID-derived sequences encoded longer CDR3 s than those of the donor. However all CDR3 s were rich in hydrophobic amino acids, most notably tyrosine and tryptophan, a characteristic common to many carbohydrate binding antibodies.

Isolation and characterization of the urease gene (URE) from the pathogenic fungus Coccidioides immitis.

The urease (URE)-encoding gene from Coccidioides immitis (Ci), a respiratory fungal pathogen of humans, was cloned, sequenced, chromosome-mapped and expressed. Both the genomic and cDNA sequences are reported. The transcription start point and poly(A)-addition site were confirmed. The URE gene contains eight introns and a 2517-bp ORF that translates a 839-amino-acid (aa) protein of 91.5 kDa and pI of 5.5, as deduced by computer analysis of the nucleotide sequence. The translated protein revealed eight putative N-glycosylation sites. The deduced URE showed comparable levels of homology to reported URE of the jack bean plant (Canavalia ensiformis; 71.8%) and URE of several genera of bacteria (Bp, 71.7%; Hp, 68.3%; Ka, 71.6%; Pm, 71.9%). The URE gene was mapped to chromosome III of Ci and was shown to be a single copy gene by Southern hybridization. Expression of a 1687-bp fragment of the URE gene in E. coli resulted in the production of a 63-kDa recombinant protein that was recognized in an immunoblot by antiserum raised against the Ka URE homolog. This is the first report of a fungal URE gene.

Cloning and characterisation of a gene encoding a cuticle-degrading protease from the insect pathogenic fungus Metarhizium anisopliae.

Metarhizium anisophilae (Ma) secretes a range of proteases when grown in vitro on insect cuticle. A trypsin-like serine protease, PR2, was purified from culture filtrates by anion exchange chromatography and the N-terminal sequence determined. Using oligodeoxyribonucleotide probes based on this sequence and that of the highly conserved trypsin active site, a gene was isolated from a lambda EMBL3 genomic library of Ma isolate ME1. Sequencing of the gene and RT-PCR revealed that the gene contains two introns which are 94 and 40 bp long. The deduced protein consists of 254 amino acids, has a putative signal sequence to allow transport into the endoplasmic reticulum and probably undergoes a second proteolytic processing step at its N terminus to yield the mature enzyme. The putative mature enzyme has extensive homology with other serine proteases of the trypsin subclass and, in particular, with the trypsin characterised from Fusarium oxysporum.

Two different methods result in the selection of peptides that induce a protective antibody response to Neisseria meningitidis serogroup C.

Neisseria meningitidis is a leading cause of morbidity and mortality worldwide. The presently available capsular polysaccharide vaccine is poorly immunogenic in children under the age of 2 due to its T-independent (TI) nature. Efforts to overcome the TI response elicited by the polysaccharide vaccine have led to the development of polysaccharide-protein conjugate vaccines. Although a T-dependent (TD) response can be achieved in young children, the response to the polysaccharide still retains characteristics of a TI antibody response. An alternative method of potentially inducing a TD response to a carbohydrate antigen is through peptides that mimic the capsular polysaccharide. Our laboratory, through the production of an anti-idiotypic (anti-id) monoclonal antibody, designated 6F9, has previously identified a peptide mimic of the meningococcal serogroup C polysaccharide (MCPS). Using the same selecting monoclonal antibody (mAb), 1E4, we have screened a phage display library and identified 13 unique peptides that bound specifically to mAb1E4. Two peptides, Pep1C and Pep2C, that demonstrated the highest binding to mAb1E4, were selected, complexed to proteosomes, and used to immunize Balb/c mice. Of the 13 peptide motifs, only one peptide motif, that of Pep2C, was found to resemble the immunogenic peptide sequence of the anti-id selected with the same mAb, although many contained several similar amino acid residues. Immunization with Pep2C, but not Pep1C, induced a significant and functional anti-MCPS antibody response that conferred protection from a lethal challenge with meningococci. Our results indicate that immunization with a peptide of N. meningitidis serogroup C, screened with the same mAb that selected an anti-id of MCPS, induces a functional and protective anti-MCPS immune response similar to that of the anti-id. This study demonstrates that two different selection methods, production of an anti-id and biopanning using a phage display library, can be used to select functional and protective peptides of MCPS with similar moieties.

Development and characterization of anti-idiotype based peptide and DNA vaccines which mimic the capsular polysaccharide of Neisseria meningitidis serogroup C.

Anti-idiotypic antibodies and peptides which mimic antigens may offer an alternate strategy for converting a thymus-independent (TI) antigen to a thymus dependent (TD) antigen. We have developed an anti-idiotype based peptide mimic of the capsular polysaccharide of N. meningitidis serogroup C (MCPS) which induces a T-dependent protective immune response in mice. Subsequent studies have demonstrated that immunization of severe combined immunodeficient mice reconstituted with human B cells respond to immunization with the MCPS peptide mimic with bactericidal anti-polysaccharide directed antibody response. We hypothesized that administration of a DNA vaccine resulting in endogenous expression of this carbohydrate peptide mimic would induce anti-MCPS antibodies.

A novel approach to study variable heavy chain gene usage in response to the capsular polysaccharide of Neisseria meningitidis serogroup C.

The molecular mechanisms involved in the relatively poor immune response in the elderly are not clearly understood. Qualitative aspects of the immune response could be a possible explanation for the differential response to T-independent antigens in young adults and elderly. This study is directed towards elucidating the differential usage of variable heavy chain by young adult and elderly derived sequences in response to the capsular polysaccharide of Neisseria meningitidis serogroup C. We currently report findings of a preliminary study designed to test the feasibility of a novel approach to isolate antigen-specific B cells. Paramagnetic beads coated with an anti-idiotypic antibody, which mimics the capsular polysaccharide of N. meningitidis serogroup C, were used to select B cells. Analysis of the gene usage data indicates some unexpected differences in the use of variable chain heavy chain in the case of young adult versus elderly sequences. The elderly derived sequences use a more diverse array of V(H) gene families in contrast to the young adult sequences, where the V(H) gene family usage is restricted. Nearly half the young adult sequences utilize V(H)3-15 germline sequence while only 25% of the elderly sequences use this germline sequence. There were interesting differences in the types of JH chain and the composition and length of CDR3 utilized by the two groups. Together, these significant differences may contribute towards the poor immune response to T-independent antigens in the elderly. These data validate the techniques used for these studies and suggest that it is pertinent to use this approach towards future investigations to elucidate gene usage in response to an antigen.

Daycase cataract surgery.

The increasing demand for cataract surgery, combined with limited resources, has created renewed interest in daycase admission. We have audited the results of all daycase and inpatient cataract surgery in a large unit over a six-month period to determine the factors influencing daycase admission and surgical outcome; 34% of cataract patients were admitted as daycases in the study period. Neither distance travelled by the patient nor age appear to influence daycase admission. However, the differing policies of the individual consultant surgeons resulted in a wide variation in the number of operations performed on a daycase basis by each firm. The rate of preoperative complications was unaffected by daycase admission, by the grade of surgeon operating, or by the type of anaesthetic employed. Only 2.1% of daycase admissions resulted in unplanned inpatient admission on the day of surgery, with another 2.1% being readmitted within six months of surgery. Late cancellation of surgery was much lower for daycases (0.4%) than for inpatients (5.1%).

Protective effect of inactivated bovine herpesvirus-1 in calves experimentally infected with bovine herpesvirus-1 and Pasteurella haemolytica.

The protective effect of an inactivated whole-virion bovine herpesvirus-1 (BHV-1) immunising inoculum, without adjuvant, against viral-bacterial respiratory disease was studied in three experimental treatment groups of five calves each. One group was boosted 14 days after the first vaccination and at this time the second group received their initial inoculation. Seven days later, calves were challenged with BHV-1 in aerosol and four days after this challenge all calves were exposed to Pasteurella haemolytica A1 in aerosol. Among the three groups, differences in rectal temperature responses four days after viral challenge (P less than 0.01) did not relate to protection. However the main response variable, viral-bacterial pneumonia, was reduced in boosted calves (P less than 0.05).

A short incubation serum neutralization test for bovine viral diarrhea virus.

A three day serum neutralization (SN) test for the detection of antibodies to bovine viral diarrhea virus (BVDV), which is an improvement on the existing five day test, is described. The improved test results in a more rapid viral cytopathic effect and utilizes Madin Darby kidney (MDBK) cells, and horse serum as a medium supplement. A comparison of tests utilizing the NADL and the Singer strains of BVDV and the use of either secondary bovine kidney cells with calf serum (BKCS) or continuous MDBK cells with horse serum (MDHS) was performed. Analysis of the SN results of 685 serum samples from 445 Quebec and Ontario cattle showed that there was no difference, as expected, in the means of the SN antibody titers when the NADL strain was used in either the BKCS or MDHS system but SN antibody titers were elevated (p less than 0.01) when the Singer strain was used in the MDHS system. The SN test with the Singer strain also yielded significantly higher titers for sera from 200 Alberta cattle.