Genotype-phenotype correlations of 39 patients with Cornelia De Lange syndrome: the Dutch experience

Background

Cornelia de Lange syndrome (CdLS) is a multiple congenital anomaly syndrome characterised by a distinctive facial appearance, prenatal and postnatal growth deficiency, psychomotor delay, behavioural problems, and malformations of the upper extremities. Recently mutations in NIPBL, the human homologue of the Drosophila Nipped‐B gene, were found to cause CdLS. Mutations have been found in 39% of reported cases.

Methods

Patients were enrolled in the study and classified into one of four groups based on clinical examination: classic, mild, possible, or definitively not CdLS. Three dimensional photography was taken of 20 subjects, and compared between groups. Behaviour was assessed with specific attention to autism. We searched for mutations in NIPBL and correlated genotype with phenotype.

Results

: We found mutations in 56% of cases.

Conclusions

Truncating mutations were generally found to cause a more severe phenotype but this correlation was not absolute. Three dimensional facial imaging demonstrated the potential for classifying facial features. Behavioural problems were highly correlated with the level of adaptive functioning, and also included autism. No correlation of behaviour with the type of mutation was found     

Characterization of Anti-Salmonella enterica Serotype Typhi Antibody Responses in Bacteremic Bangladeshi Patients by an Immunoaffinity Proteomics-Based Technology

Salmonella enterica serotype Typhi is the cause of typhoid fever and a human-restricted pathogen. Currently available typhoid vaccines provide 50 to 90% protection for 2 to 5 years, and available practical diagnostic assays to identify individuals with typhoid fever lack sensitivity and/or specificity. Identifying immunogenic S. Typhi antigens expressed during human infection could lead to improved diagnostic assays and vaccines. Here we describe a platform immunoaffinity proteomics-based technology (IPT) that involves the use of columns charged with IgG, IgM, or IgA antibody fractions recovered from humans bacteremic with S. Typhi to capture S. Typhi proteins that were subsequently identified by mass spectrometry. This screening tool identifies immunogenic proteins recognized by antibodies from infected hosts. Using this technology and the plasma of patients with S. Typhi bacteremia in Bangladesh, we identified 57 proteins of S. Typhi, including proteins known to be immunogenic (PagC, HlyE, OmpA, and GroEL) and a number of proteins present in the human-restricted serotypes S. Typhi and S. Paratyphi A but rarely found in broader-host-range Salmonella spp. (HlyE, CdtB, PltA, and STY1364). We categorized identified proteins into a number of major groupings, including those involved in energy metabolism, protein synthesis, iron homeostasis, and biosynthetic and metabolic functions and those predicted to localize to the outer membrane. We assessed systemic and mucosal anti-HlyE responses in S. Typhi-infected patients and detected anti-HlyE responses at the time of clinical presentation in patients but not in controls. These findings could assist in the development of improved diagnostic assays.Salmonella enterica serotype Typhi is a human-restricted pathogen that is the primary cause of enteric fever. It is estimated that S. Typhi infects over 20 million individuals and kills approximately 200,000 people globally each year (4). Currently, commercially available typhoid vaccines provide approximately 50 to 75% protection for 2 to 5 years (21), although an anti-typhoid Vi conjugate vaccine demonstrated 90% protection in 2- to 5-year-old children in a large field trial (23). Available and practical diagnostic tests for typhoid fever lack sensitivity and/or specificity (28). Identifying immunogenic S. Typhi antigens expressed during human infection could lead to improved diagnostic assays and vaccines.Infection with S. Typhi begins with the ingestion of contaminated water or food. The bacteria invade the gastrointestinal mucosa, translocate to the lymphoid follicles, where they survive and replicate within macrophages, and then disseminate via the bloodstream to the liver, spleen, and intestinal lymph nodes (14). The incubation period is typically 8 to 14 days (22), and symptoms include fever, abdominal pain, anorexia, weakness, potential complications of intestinal perforation, encephalopathy, and gastrointestinal bleeding (14, 34). Clinical studies demonstrate that S. Typhi infection stimulates both an intestinal mucosal and systemic humoral and cellular immune response (14, 34). S. Typhi is a facultative intracellular pathogen of macrophages, and both cellular and antibody-mediated immune responses are known to play roles in controlling and clearing S. Typhi infection (37). Despite this, there are limited data on antigen-specific cellular responses during wild-type S. Typhi infection in humans. Analyses of cellular immune responses during S. Typhi infection have largely used whole-cell preparations or flagellar antigens and have focused predominately on measuring immune responses in recipients of oral live attenuated typhoid vaccines, not in individuals with wild-type disease (24, 25, 40-42, 49).Antibody responses during wild-type infection have been better studied but have focused largely on a relatively small number of antigens, including O antigen (lipopolysaccharide [LPS]), H antigen (flagellar component), polysaccharide capsular antigen (Vi antigen), heat shock proteins such as GroEL, and outer membrane proteins such as OmpC and -F (13, 34). In addition, gut-derived IgA antibody-secreting cells that recognize LPS, a membrane preparation, or whole-killed S. Typhi organisms can be detected in the peripheral blood following natural S. Typhi infection or oral typhoid vaccination (16, 43, 50, 54). These cells eventually return home to the gastrointestinal mucosa, where they secrete secretory IgA antibody (36, 43).A number of immunoaffinity-based techniques that screen protein libraries of pathogens to identify immunogenic antigens have been developed (12, 17, 38), and we have previously reported using one such approach, in vivo-induced-antigen technology (IVIAT), to identify immunogenic S. Typhi antigens expressed during human infection (12). Another previously described technique, proteomics-based expression library screening (PELS), involves using antibody-charged columns to capture antigens produced by an Escherichia coli-based expression system containing an inducible library of a pathogen of interest, with subsequent elution and identification of bound proteins using mass spectrometric analysis (17). Here we describe using a modification of this approach that we have termed immunoaffinity proteomics-based technology (IPT). IPT involves directly screening the pathogen of interest using columns charged with IgG, IgM, or IgA antibody fractions recovered from the blood of infected humans. We applied IPT to S. Typhi to gain further insights into immunogenic antigens expressed in patients bacteremic with S. Typhi in Bangladesh.     

Mucosal and systemic immune responses in patients with diarrhea due to CS6-expressing enterotoxigenic Escherichia coli

Colonization factor CS6 expressed by enterotoxigenic Escherichia coli (ETEC) is a nonfimbrial polymeric protein. A substantial proportion of ETEC strains isolated from patients in endemic settings and in people who travel to regions where ETEC is endemic are ETEC strains expressing CS6, either alone or in combination with fimbrial colonization factor CS5 or CS4. However, relatively little is known about the natural immune responses elicited against CS6 expressed by ETEC strains causing disease. We studied patients who were hospitalized with diarrhea (n = 46) caused by CS6-expressing ETEC (ETEC expressing CS6 or CS5 plus CS6) and had a disease spectrum ranging from severe dehydration (27%) to moderate or mild dehydration (73%). Using recombinant CS6 antigen, we found that more than 90% of the patients had mucosal immune responses to CS6 expressed as immunoglobulin (IgA) antibody-secreting cells (ASC) or antibody in lymphocyte supernatant (ALS) and that about 57% responded with CS6-specific IgA antibodies in feces. More than 80% of the patients showed IgA seroconversion to CS6. Significant increases in the levels of anti-CS6 antibodies of the IgG isotype were also observed in assays for ASC (75%), ALS (100%), and serum (70%). These studies demonstrated that patients hospitalized with the noninvasive enteric pathogen CS6-expressing ETEC responded with both mucosal and systemic antibodies against CS6. Studies are needed to determine if the anti-CS6 responses protect against reinfection and if protective levels of CS6 immunity are induced by vaccination.     

Salmonella enterica Serovar Typhi-Specific Immunoglobulin A Antibody Responses in Plasma and Antibody in Lymphocyte Supernatant Specimens in Bangladeshi Patients with Suspected Typhoid Fever

Many currently available diagnostic tests for typhoid fever lack sensitivity and/or specificity, especially in areas of the world where the disease is endemic. In order to identify a diagnostic test that better correlates with typhoid fever, we evaluated immune responses to Salmonella enterica serovar Typhi (serovar Typhi) in individuals with suspected typhoid fever in Dhaka, Bangladesh. We enrolled 112 individuals with suspected typhoid fever, cultured day 0 blood for serovar Typhi organisms, and performed Widal assays on days 0, 5, and 20. We harvested peripheral blood lymphocytes and analyzed antibody levels in supernatants collected on days 0, 5, and 20 (using an antibody-in-lymphocyte-supernatant [ALS] assay), as well as in plasma on these days. We measured ALS reactivity to a serovar Typhi membrane preparation (MP), a formalin-inactivated whole-cell preparation, and serovar Typhi lipopolysaccharide. We measured responses in healthy Bangladeshi, as well as in Bangladeshi febrile patients with confirmed dengue fever or leptospirosis. We categorized suspected typhoid fever individuals into different groups (groups I to V) based on blood culture results, Widal titer, and clinical features. Responses to MP antigen in the immunoglobulin A isotype were detectable at the time of presentation in the plasma of 81% of patients. The ALS assay, however, tested positive in all patients with documented or highly suspicious typhoid, suggesting that such a response could be the basis of improved diagnostic point-of-care-assay for serovar Typhi infection. It can be important for use in epidemiological studies, as well as in difficult cases involving fevers of unknown origin.Salmonella enterica serovar Typhi (serovar Typhi) is the cause of typhoid fever, an illness that affects over 20,000,000 individuals worldwide each year, killing over 200,000 (5, 8, 16). The largest burden of typhoid fever is borne by impoverished individuals in resource-poor areas of the world. Serovar Typhi is a human-restricted invasive enteric pathogen which, after ingestion, crosses the intestinal mucosa, is taken up by gut-associated lymphoreticular tissues, and enters the systemic circulation. Both mucosal and systemic host immune responses are stimulated after infection. Serovar Typhi is an intracellular pathogen, and antibody and cell-mediated immune responses occur after infection or immunization with live oral attenuated typhoid vaccines (10, 25, 34).Diagnostic tests for typhoid fever often lack sensitivity and/or specificity, especially in areas of the world that are endemic for typhoid fever, where clinically distinguishing typhoid fever from other febrile illnesses is difficult (5, 17, 39). Microbiologic culturing of blood is approximately 30 to 70% sensitive, with the highest sensitivity being associated with an absence of prior use of antibiotics and the culturing of larger volumes of blood, features that complicate this mode of diagnosis in young children (5, 6, 8, 36). Microbiologic culturing of bone marrow aspirates is more sensitive than blood but often clinically impractical (1, 11, 12). Serum Widal assay titers are often nonspecific in endemic settings and are of limited value unless titers are markedly elevated or are analyzed for changes from acute to convalescent phases of illness (18, 33, 38). Molecular diagnostic assays including PCR are promising, but issues of practicality, contamination, and quality control have limited their use in many resource-poor areas of the world (14).Since serovar Typhi interacts with both the mucosal and the systemic immune systems, we were interested to determine whether analyses of mucosal immune responses would give improved insight into this human-restricted infection. Activated mucosal lymphocytes migrate from intestinal tissue and circulate within peripheral blood before rehoming to mucosal tissues (20, 31). This migration peaks 1 to 2 weeks after intestinal infection and may be measured by using peripheral blood mononuclear cells (PBMC) in an antibody-secreting cell (ASC) assay (19, 26) or in supernatants recovered from harvested PBMC (the “antibody in lymphocyte supernatant” [ALS] assay) (7, 31). Although ALS and ASC responses have previously been measured after immunization with oral live attenuated typhoid vaccines, detailed analyses of ALS or ASC responses in individuals with wild-type typhoid fever are lacking (21, 24). In order to gain further insight into mucosal immune responses during wild-type serovar Typhi infection, we undertook a study to characterize the serum and ALS responses to serovar Typhi among individuals with suspected typhoid fever in Bangladesh.