Loss-of-Function GRHL3 Variants Detected in African Patients with Isolated Cleft Palate.

In contrast to the progress that has been made toward understanding the genetic etiology of cleft lip with or without cleft palate, relatively little is known about the genetic etiology for cleft palate only (CPO). A common coding variant of grainyhead like transcription factor 3 ( GRHL3) was recently shown to be associated with risk for CPO in Europeans. Mutations in this gene were also reported in families with Van der Woude syndrome. To identify rare mutations in GRHL3 that might explain the missing heritability for CPO, we sequenced GRHL3 in cases of CPO from Africa. We recruited participants from Ghana, Ethiopia, and Nigeria. This cohort included case-parent trios, cases and other family members, as well as controls. We sequenced exons of this gene in DNA from a total of 134 nonsyndromic cases. When possible, we sequenced them in parents to identify de novo mutations. Five novel mutations were identified: 2 missense (c.497C>A; p.Pro166His and c.1229A>G; p.Asp410Gly), 1 splice site (c.1282A>C p.Ser428Arg), 1 frameshift (c.470delC; p.Gly158Alafster55), and 1 nonsense (c.1677C>A; p.Tyr559Ter). These mutations were absent from 270 sequenced controls and from all public exome and whole genome databases, including the 1000 Genomes database (which includes data from Africa). However, 4 of the 5 mutations were present in unaffected mothers, indicating that their penetrance is incomplete. Interestingly, 1 mutation damaged a predicted sumoylation site, and another disrupted a predicted CK1 phosphorylation site. Overexpression assays in zebrafish and reporter assays in vitro indicated that 4 variants were functionally null or hypomorphic, while 1 was dominant negative. This study provides evidence that, as in Caucasian populations, mutations in GRHL3 contribute to the risk of nonsyndromic CPO in the African population.

Association Studies and Direct DNA Sequencing Implicate Genetic Susceptibility Loci in the Etiology of Nonsyndromic Orofacial Clefts in Sub-Saharan African Populations.

Orofacial clefts (OFCs) are congenital dysmorphologies of the human face and oral cavity, with a global incidence of 1 per 700 live births. These anomalies exhibit a multifactorial pattern of inheritance, with genetic and environmental factors both playing crucial roles. Many loci have been implicated in the etiology of nonsyndromic cleft lip with or without cleft palate (NSCL/P) in populations of Asian and European ancestries, through genome-wide association studies and candidate gene studies. However, few populations of African descent have been studied to date. Here, the authors show evidence of an association of some loci with NSCL/P and nonsyndromic cleft palate only (NSCPO) in cohorts from Africa (Ghana, Ethiopia, and Nigeria). The authors genotyped 48 single-nucleotide polymorphisms that were selected from previous genome-wide association studies and candidate gene studies. These markers were successfully genotyped on 701 NSCL/P and 163 NSCPO cases, 1,070 unaffected relatives, and 1,078 unrelated controls. The authors also directly sequenced 7 genes in 184 nonsyndromic OFC (NSOFC) cases and 96 controls from Ghana. Population-specific associations were observed in the case-control analyses of the subpopulations, with West African subpopulations (Ghana and Nigeria) showing a similar pattern of associations. In meta-analyses of the case-control cohort, PAX7 (rs742071, P = 5.10 × 10(-3)), 8q24 (rs987525, P = 1.22 × 10(-3)), and VAX1 (rs7078160, P = 0.04) were nominally associated with NSCL/P, and MSX1 (rs115200552, P = 0.01), TULP4 (rs651333, P = 0.04), CRISPLD2 (rs4783099, P = 0.02), and NOG1 (rs17760296, P = 0.04) were nominally associated with NSCPO. Moreover, 7 loci exhibited evidence of threshold overtransmission in NSOFC cases through the transmission disequilibrium test and through analyses of the family-based association for disease traits. Through DNA sequencing, the authors also identified 2 novel, rare, potentially pathogenic variants (p.Asn323Asp and p.Lys426IlefsTer6) in ARHGAP29 In conclusion, the authors have shown evidence for the association of many loci with NSCL/P and NSCPO. To the best of this knowledge, this study is the first to demonstrate any of these association signals in any African population.

Rosette formation in Plasmodium falciparum isolates and anti-rosette activity of sera from Gambians with cerebral or uncomplicated malaria.

The ability of Plasmodium falciparum-infected red blood cells (RBC) to form spontaneous erythrocyte rosettes was studied in 130 fresh isolates from Gambian children with cerebral or uncomplicated malaria from August to November 1990. All isolates (24 of 24) from patients with cerebral malaria formed rosettes, but only 61 of 106 isolates from children with uncomplicated malaria formed rosettes. The mean rate of rosette formation in isolates from children with cerebral malaria (28.3%) was significantly greater than that in isolates from children with uncomplicated malaria (8.5%). Giant rosettes were more frequently formed in isolates from patients with cerebral malaria than in those from patients with uncomplicated malaria. Sera of children with cerebral disease generally lacked anti-rosette activity, while many sera from children with uncomplicated malaria showed strong anti-rosette activity when tested against the patients' ow parasites. Some sera that were devoid of autologous rosette-disrupting activity were able to disrupt rosettes formed in other isolates, indicating the presence of different rosette formation mechanisms. Forty percent (6 of 15) of the sera from patients with cerebral malaria caused microagglutination of the patients' own uninfected and infected RBC, while only 10% (3 of 31) of sera from children with uncomplicated disease caused microagglutination. The ability of infected RBC to bind to melanoma cells grown in vitro did not differ between patients with cerebral or uncomplicated malaria. The results of this study, taken in conjunction with our previous findings, establish a strong association between rosette formation in P. falciparum-infected RBC and cerebral malaria.

Sensitivity of Plasmodium falciparum in The Gambia to co-trimoxazole.

In The Gambia co-trimoxazole is used widely to treat children with an acute respiratory infection (ARI). Because malaria may sometimes be mistaken for ARI, some children with malaria are treated with co-trimoxazole. Therefore, we investigated the sensitivity of Gambian isolates of Plasmodium falciparum to this drug. Six days after the start of treatment with co-trimoxazole 3.3% of blood films of 65 asymptomatic subjects were positive, and 7.7% were positive after 21 d. One of 10 patients with ARI and malaria treated with co-trimoxazole had a positive blood film 3 d after the start of treatment but was negative thereafter. All 10 patients recovered satisfactorily. Thirty 'wild' isolates of P. falciparum were tested in vitro against co-trimoxazole at a ration of 5 parts sulphamethoxazole (SMZ) to 1 part trimethoprim (TMP). The mean EC50s, using a 36 h assay, were 1.2 x 10(-7) and 2.5 x 10(-8) M for SMZ and TMP respectively. When a [3H]hypoxanthine incorporation assay was employed, values of 5.7 x 10(-7) M for SMZ and 1.2 x 10(-7) M for TMP were obtained. These values are well below the peak plasma concentration. Our findings suggest that co-trimoxazole is effective against falciparum malaria in The Gambia. However, if it were to be used widely, the parasite would be likely to develop resistance to this and other dihydrofolate reductase inhibitor antimalarials.

Local politicization of Primary Health Care as an instrument for development: a case study of community health workers in Zambia.

The integrated approach of the Primary Health Care Concept has obvious implications for development. In view of Zambia's commitment to Primary Health Care it is important to evaluate the effectiveness of present institutional frameworks and the problems that may arise in shifting towards community responsibility for the provision of health. It is often assumed that the Primary Health Care approach of working through the community should be free of serious implementation problems. However, experience from community participation projects in a wide variety fields carried out in many countries, including Zambia has shown that failure to account for local institutional arrangements and political interests has hindered success. This article presents the theoretical issues involved in community participation research, reviews relevant literature and presents a case study of a community health worker in Western Province, Zambia. The case study derives from an on-going UNICEF/Government of Zambia sponsored project which is monitoring and evaluating the impact of child health and nutrition services in rural areas. The study illustrates some of the problems encountered by a CHW because of clashes with local political interests. An alternative model is proposed which if implemented can help alleviate and/or avoid these types of conflicts.

TNF concentration in fatal cerebral, non-fatal cerebral, and uncomplicated Plasmodium falciparum malaria.

Plasma levels of tumour necrosis factor (TNF) were significantly higher in 178 Gambian children with uncomplicated malaria due to Plasmodium falciparum than in 178 children with other illnesses. 110 children with cerebral malaria were studied shortly after admission to hospital; 28 subsequently died. Compared with the children with uncomplicated malaria, mean plasma TNF levels were twice as high in cerebral malaria survivors and ten times as high in the fatal cases. Although high TNF levels were associated with high parasitaemia and with hypoglycaemia, they predicted fatal outcome in cerebral malaria independently of parasitaemia and glucose concentrations. Concentrations of interleukin-1 alpha, but not interferon gamma, were also related to the severity of malaria. We conclude that increased TNF production is a normal host response to P falciparum infection, but that excessive levels of production may predispose to cerebral malaria and a fatal outcome.

Induction of immunologic memory in Gambian children by vaccination in infancy with a group A plus group C meningococcal polysaccharide-protein conjugate vaccine.

Two hundred twenty-one Gambian children vaccinated previously with one, two, or three doses of a meningococcal conjugate vaccine or two doses of polysaccharide vaccine before the age of 6 months were revaccinated at the age of 18-24 months with either meningococcal polysaccharide, conjugate, or inactivated polio vaccines. Children who had previously received one, two, or three doses of conjugate vaccine had significantly (P < .001) higher anti-group C meningococcal antibody levels following revaccination than did children vaccinated with a polysaccharide vaccine for the first time. Children vaccinated previously with two doses of polysaccharide vaccine had a lower group C antibody response than did control children. Group A antibody responses following revaccination of children who had previously received polysaccharide or conjugate vaccine were not significantly higher than those in control children. Thus, immunologic memory was probably induced by the group C but not by the group A component of the conjugate vaccine.

Common west African HLA antigens are associated with protection from severe malaria.

A large case-control study of malaria in West African children shows that a human leucocyte class I antigen (HLA-Bw53) and an HLA class II haplotype (DRB1*1302-DQB1*0501), common in West Africans but rare in other racial groups, are independently associated with protection from severe malaria. In this population they account for as great a reduction in disease incidence as the sickle-cell haemoglobin variant. These data support the hypothesis that the extraordinary polymorphism of major histocompatibility complex genes has evolved primarily through natural selection by infectious pathogens.

A trial of a group A plus group C meningococcal polysaccharide-protein conjugate vaccine in African infants.

The safety and immunogenicity of a group A plus group C meningococcal polysaccharide-CRM197 conjugate vaccine was evaluated in 304 8- to 10-week-old Gambian infants. Infants were immunized with one, two, or three doses of conjugate vaccine or with two doses of a meningococcal A plus C polysaccharide vaccine. The conjugate vaccine produced few systemic side effects, and local reactions were similar to those produced by the polysaccharide vaccine. Postvaccination group A meningococcal polysaccharide antibody levels, measured by ELISA, increased progressively after one, two, or three doses of conjugate vaccine. However, one dose of conjugate vaccine given at the age of 6 months induced a higher group C meningococcal antibody response than did two doses of conjugate vaccine given at 2 and 6 months. Two doses of conjugate vaccine induced higher levels of antibody than did two doses of polysaccharide vaccine. Thus, this new meningococcal conjugate vaccine proved to be safe and immunogenic.