School of child & adolescent health

Title: Investigating the prevalence of selected MYO7A Mutations amongst a group of sub-Saharan African Patients with non- syndromic hearing loss

Authors: Noluthando Manyisa¹, Kamogelo Lebeko¹, Jean Jacques Noubiap Nzeale² Collet Dandara¹ and Ambroise Wonkam¹
Affiliation: ¹Division of Human Genetics, Department of Clinical Laboratory Sciences,² Department of Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa

Background:

Congenital hearing loss occurs in approximately 5.5/1000 birth in South Africa. Mutations in GJB2 and GJB6, that explain most of autosomal recessive non-syndromic hearing loss (ARNHL) in people of European and Asian descent, have been shown to be insignificant in African populations. In order to resolve ARNSHL amongst African patients, next generation sequencing (NGS) was employed thorough the use of OtoSCOPE®, a diagnostic platform for hearing loss, that investigated 66 genes of hearing loss. Compound heterozygous causative mutations (c.5806_5808delCTC and c.5880_5882delCTT) were identified in the MYO7A gene in one family. These mutations may have relevance in some cases of non-syndromic hearing loss among Africans.
Aim:

The aim of this project was to investigate two specific MYO7A deleterious mutations, in an African cohort with ARNSHL.

Methods:

Patients and controls: A previously well described group of 100 patients affected with ARNSHL together with 200 ethnically-matched normal hearing control samples.

Molecular Methods: Genetic screening will be performed using direct Sanger Sequencing.

Bioinformatics analysis: Pathogenicity of the mutation and influence of secondary variants were analysed using SHEsis program.

Preliminary Results:

The presence of c.5806_5808delCTC was detected, in heterozygosity, in 1 of the 100 patients and in none of the 100 controls population.

Similarly c.5880_5882delCTT was not detected in control populations; its detection among isolated case of hearing loss is ongoing.

Conclusions:

This study is a proof of concept of the use of NGS in resolving cases of ARNSHL amongst patients of African descent. The absence of the mutation in other cases of non-familial hearing loss could indicate that these are private mutations specific to the families’ studied and could not deserve routine investigation in isolated patient with hearing loss. The single patient carrier could carry a second MYO7A undiscovered mutation which affects the second allele that will need further investigations.

Address Correspondence to: A/Professor Ambroise Wonkam, Division of Human Genetics, Faculty of Health Sciences, University of Cape Town, Observatory, 7925, Cape Town, South Africa

Email: [e-mail]


Title: DISTRIBUTION OF SICKLE CELL DISEASE-RELATED GENETIC MARKERS IN MALARIA-FREE SUB-SAHARAN AFRICAN COUNTRIES
Authors: Khuthala Mnika¹, Gift Pule¹, Ambroise Wonkam¹
Affiliation: ¹Division of Human Genetics and ²Division of Haematology, Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa

Background:

Sickle Cell Disease (SCD) is a disease caused by a single nucleotide substitution (T > A) in the β-globin gene on chromosome 11. SCD occurs mostly in central Africa (sub-Saharan African countries) and co-occurs in populations where malaria is endemic. The β-gene has five haplotypes; Bantu (Central African Republic, CAR), Benin, Senegal, Cameroon and Indian - Arab haplotypes. There is an unusual combination of restriction polymorphisms which is called atypical haplotypes, different to the defined haplotypes by the change of one or more restriction sites.

Objective:

To calculate the frequency of SCD-related genetic markers (SCD mutation, haplotype and alpha-thalassemia gene deletion) in the cohorts from malaria-free countries (South Africa, Zimbabwe and Malawi).

Methods:

Molecular genotyping of the SCD mutation involved amplification of a 380 bp segment of the β-globin gene followed by restriction enzyme analysis using restriction endonuclease Del 1. Restriction fragment length polymorphism (RFLP)-PCR was also used to genotype and describe the haplotype background in the β-globin gene cluster. An expand long-template PCR system (Roche, UK) was used to assay the alpha-thalassemia deletion genotype in all cohorts. A total of 21 (SA), 11(ZIM), and 13 (MAL) SCD-unaffected were analysed from Xhosa (South Africa), Shona (Zimbabwe) and Chewa (Malawi), respectively.

Results:

For SCD diagnosis, all South African samples were homozygous unaffected (HbAA); Zimbabwean cohort 35 (89.74%) were HbAA and 4 (10.26%) heterozygous (HbAS); and Malawian cohort 39 (84.78%) were HbAA and 7 (15.23%) heterozygous HbAS. For the SCD haplotype background, a high frequency of atypical haplotype (SA = 53 (66.25%), ZIM = 42 (65.6%), MAL = 36 (51.5%) was observed in all cohorts. Benin haplotype had the second highest frequency (SA = 15 (18.75%), ZIM = 8 (12.5%), MAL = 19 (27.25%)). A total of 42 (SA), 22 (ZIM), 26 (MAL) chromosomes were analysed for the alpha-thalassemia deletion, of which all had the homozygous wild-type genotype (αα/αα).

Conclusions:

This study has confirmed that the frequency of sickle mutation is highest in malaria endemic regions due to positive selection and the partial resistance to the Plasmodium falciparum. Furthermore, the conservation of the haplotype background is also associated with regions of high SCD burden and thus malaria as well. Understanding SCD genetic markers in the absence of malaria could inform the global SCD community on the importance of gene/environment interaction, positive selection of traits and hopefully the clinical care for SCD in malaria-endemic and free regions.

Corresponding authors: A/Professor Ambroise Wonkam, Division of Human Genetics, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, 7925, Cape Town, Republic of South Africa. Tel: [phone]; Fax: [phone] e-mail: [e-mail]

1^st presenter (Khuthala Mnika): NRF Intern (Division of Human Genetics)

Title: HYDROXYUREA INDUCES γ-GLOBIN EXPRESSION THROUGH MICRO- RNAs-MEDIATED ACTIONS IN PRIMARY ERYTHROID AND K562 CELLS
Authors: Gift Pule¹, Shaheen Mowla², Nicholas Novitzky², Ambroise Wonkam¹
Affiliation: ¹Division of Human Genetics and ²Division of Haematology, Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.

Background:

Hydroxyurea is the only available drug treatment for Sickle Cell Disease (SCD); but the mechanism of fetal hemoglobin (HbF) induction is poorly understood. In particular, the role of microRNAs in the post-transcriptional regulation of HbF, through potent regulators such as MYB, BCL11A and KLF-1, is understudied.

Objectives:

We have investigated the role of microRNAs in the post-transcriptional regulation of HbF in erythroid cells treated with hydroxyurea.

Methods:

As models, we used 1) hematopoietic stem cells derived from umbilical cord blood (HSCs) and 2) the K562 human erythroleukemia cell line. Both primary erythroid and K562 cells were treated with hydroxyurea and changes in BCL11A, KLF-1, GATA-1, MYB, β- and γ-globin gene expression and 7 targeted miRNAs, previously shown to be modified by hydroxyurea or associated with basal γ-globin expression were also investigated.

Results:

We differentiated CD34+ stem cells into erythroid precursors using a single-phase expansion and differentiation protocol. BCL11A was down-regulated and there was a marked 7-fold increase (p < 0.003) in γ-globin expression in both primary human erythroid and K562 cells. Down-regulation of GATA-1 was also consistent with the above findings. Similarly, KLF-1 was down-regulated in both cell models, corresponding to the repressed expression of BCL11A and β-globin gene (p < 0.04). In both cell models, most miRNAs was significantly up-regulated by hydroxyurea with some level of variation. Down-regulation of, another potent negative regulator of γ-globin and direct target of the up-regulated miR-15a and miR-16-1, provides a post-transcriptional tier of HbF regulation. We also demonstrated that the inhibition of miR-26b; miR-151-3p; miR-451 and miR-494 resulted in an apparent decrease in HbF and combinatorial inhibition of miR-26b; miR-151-3p and miR-494 resulted in complete knock-down of HbF.

Conclusions:

The data provide evidence of a microRNAs-mediated post-transcriptional mechanism for γ-globin regulation under hydroxyurea treatment. These findings will add a unique piece to our understanding of HbF regulation and incite further investigation of the post-transcriptional regulatory network for HbF expression.

Corresponding authors: A/Professor Ambroise Wonkam, Division of Human Genetics, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory, 7925, Cape Town, Republic of South Africa. Tel: [phone]; Fax: [phone] e-mail: [e-mail]
Ethics: HREC/REF: 507/2013
1^st presenter (GP): PhD student (Division of Human Genetics)


Title: A NOVEL APPROACH TO DUCTAL SPASM DURING PERCUTANEOUS DEVICE OCCLUSION OF PATENT DUCTUS ARTERIOSUS
Authors: Rik De Decker¹, George Comitis¹, Jenny Thomas¹, Elmarie van der Merwe¹, John Lawrenson²
Affiliation: ¹Red Cross War Memorial Children’s Hospital and University of Cape Town, ²Tygerberg Children’s Hospital and University of Stellenbosch


Objective:

Ductal spasm is a rare, yet important complication of percutaneous device occlusions of patent ductus arteriosus (PDA). Spasm may result in failure of the procedure, under-sizing of the device, or embolisation of the implanted device as the spasm resolves after the procedure. We have developed and describe for the first time a protocol that rapidly and completely reverses the ductal spasm that patients may experience during cardiac catheterisations for PDA occlusion.

Case Series & Methods:

Eight patients (constituting 3.3% of our PDA cases over the past 12 years) presented between 13 and 67 months of age to Red Cross Children’s Hospital for transcatheter PDA occlusion. All were preterm-born (“ex-prem babies”) with gestational ages of between 25 to 34 weeks. Seven of the eight patients experienced ductal spasm immediately before, during or soon after induction of anaesthesia. One experienced spasm only after entering the PDA with a catheter. After detection of the spasm, the anaesthetist, in consultation with the interventionist, in each case: 1) changed the mode of anaesthesia from inhaled sevoflurane to total intravenous anaesthesia (TIVA) and propofol infusion, 2) reduced the inhaled oxygen fraction to 21%, and, 3) initiated a continuous intravenous infusion of prostaglandin E₁ (PGE₁).

Results:

In all eight patients, the first 2 steps (TIVA and FiO₂ 0.21) resulted in only partial relaxation of the spasm, as confirmed on transthoracic echocardiography. Full relaxation, to the pre-catheterisation echocardiographically determined dimensions of the PDA, was attained after intravenous PGE₁ infusions of only 10-15 minutes’ duration. This was confirmed by anteroposterior and lateral angiography. While maintaining this anaesthetic protocol, six PDAs were successfully occluded. Two were considered to be unsuitable for device occlusion, and referred for surgery. A ninth patient suffered ductal spasm at another institution where intravenous PGE₁ was not available. The spasm resolved only partially after maintaining the first two steps of our protocol (TIVA and FiO₂ 0.21) for a period of 30 minutes. Consequently, an oversized occlusion device had to be implanted to avert the risk of device embolisation.

Conclusions:

1. We have shown for the first time that the PDAs of preterm born babies remain sensitive to the dilatory effect of intravenous prostaglandin for many years after pre-term delivery.

2. By utilising this observation, ductal spasm during transcatheter occlusion may be reliably resolved and the procedure safely completed by a simple anaesthetic protocol that includes the continuous infusion of intravenous prostaglandin E_1.

3. This is the first description of a robust protocol to avoid the complications of ductal spasm during percutaneous PDA occlusion.

UCT HREC No: R017/2014

Title: INCREASING THE EFFICIENCY OF A PAEDIATRIC CARDIOLOGY CLINIC USING SIMULATION OPTIMISATION
Authors: Claudi van Niekerk¹, Rachel Chater², Anthony Leiman¹, Rik De Decker³
Affiliation: ¹Honours Student, School of Economics, Faculty of Commerce, UCT; ²Bertha Centre for Social Innovation, Graduate School of Business, UCT; ³Division of Cardiology, Red Cross Children’s Hospital and UCT


Objective:

Often hospitals run inefficiently, not because of the lack of resources but due to the poor use of their allocated resources. Using an economics approach, we modeled the flow of patients through the cardiology outpatient (COPD) clinics at the Red Cross Children’s Hospital. Our aims were to determine its latent inefficiencies and the institutional (structural) dimensions constraining the system and then identify strategies that could improve patient flow. Our study explores how altering certain dimensions may increase the efficiency of the clinic. Economics modeling is a novel approach in the assessment of the dynamics of clinical health care delivery at the Red Cross Children’s Hospital.

Case Series & Methods:

Data were collected at all COPD clinics over a 3-month period (March – May 2015), including the number of patients seen by all part-time and full-time doctors. Hospital administration provided COPD patient numbers for the past three years. Interviews were also conducted with medical staff working at the cardiology clinic. With these data, a simulation optimization model was used to determine which structural changes could alter the clinic’s efficiency. The model allowed us to create a virtual clinic that mirrored reality as closely as possible. An increase in efficiency was defined as an increase in the number of patients seen by the clinic and/or a decrease of patient waiting times. First, we designed a base model, which reflected the current situation at the clinic. We then re-ran the model with various alterations to clinic structure to test how such changes impacted on efficiency.

Results:

We determined that the greatest obstacles to increased efficiency were the institutional dimensions constraining the system. The efficiency of the cardiology clinics may be improved by altering the following constraints:

1. 
   Updating the computer system and computerizing the folder system.
   
2. 
   Implementing a rotating roster system for full-time doctors.
   
3. 
   Locating cardiology facilities closer to one another.
   
4. 
   Implementing a pre-clinic triage system to sort patient cases according to complexity.
   
5. 
   Improving transitional services for patients that need to return to their referring clinics or need to be transferred to equivalent adult services.
   
6. 
   Combining the cardiology and rheumatic fever clinics.
   

Compared to the base model, we determined that these changes could increase the number of patients seen by 34.2% and decrease patient waiting times by 40.8%
Conclusions:

An economics approach and the use of simple simulation modeling of historical COPD data was successful in identifying the important constraints to efficiency of the cardiology clinic at Red Cross Children’s Hospital. This study has not looked at the feasibility and cost of the suggested proposed changes.

UCT HREC No: R017/2014

Title: Continuous flow peritoneal DIALYSIS (CFPD) use in children with AKI and inadequate ultrafiltration on convention Peritoneal DIALYSIS (PD)
Authors: Nourse P, Sinclair G, du Plessis M, Argent A
Criticism against the use of acute peritoneal dialysis has been its low clearance and low ultrafiltration (UF) volumes compared to extracorporeal techniques.
Aim:

To determine whether CFPD would improve ultrafiltration in children with acute kidney injury where UF on conventional PD was inadequate on maximum glucose concentration.

Methods:

CFPD was performed on five children with AKI who were fluid overloaded and ultrafiltration was deemed insufficient on conventional PD using 4.25% glucose concentration. CFPD was performed with two bedside-placed catheters using 2.50 % glucose concentration. After initial filling, dialysate flow rate (100 ml/1.73 m2 per minute) was maintained with an adapted continuous venovenous hemofiltration machine. Ultrafiltration flow rate was set at 2.5 ml/1.73 m2 per minute and adapted as necessary. Ultrafiltration and clearance rates were measured before and after implementation of CFPD.

Results:

Mean age of patients 4.7months (range 0.43-9); Mean weight: 5.6 kg (2.7-5.6). Mean ultrafiltration was 2.2ml/kg/hour with conventional PD versus 8ml/kg/hr with CFPD. Mean clearances of urea and creatinine were 6.48 and 7.43 ml/1.73 m2 per minute with conventional PD versus 22 and 24 ml/1.73 m2 per minute with CFPD, respectively. Ultrafiltration and clearance were significantly higher on CFPD than compared to conventional PD. Complications: The in and outflow lines needed to be swapped on one patient otherwise no complications occurred.

Conclusion:

CFPD may be an option to improve ultrafiltration of peritoneal dialysis in children with AKI in who ultrafiltration is deemed inadequate on maximally optimised conventional PD.

Title: Pediatric Lupus in South Africa: Treatment and Outcomes

Authors: Laura Lewandowski¹, Laura Schanberg¹, Peter Nourse², Graeme Spittal², Priya Gajjar², Nathan Thielmann¹, Chris Scott²
Affiliation: ¹Duke University, ²Red Cross War Memorial Hospital, University of Cape Town


Background:

Although African children with SLE may be at high risk for poor outcomes based on race, socioeconomic status, and age, little research has investigated this population. We have initiated the first registry of this high risk pediatric SLE (pSLE) population in South Africa (SA). Here, we present the first comparison of treatment and outcomes between a South African pSLE cohort (PULSE) and a North American pSLE cohort (Childhood Arthritis and Rheumatology Research Alliance (CARRA) Registry).

Methods:

We conducted a cross sectional analysis (retrospective chart review) of pSLE patients in Cape Town, South Africa from 1988-2014 meeting ACR criteria for pSLE. Patient age, gender, race, presenting features, clinical and serologic disease markers, and treatment were recorded and compared to an established North American pSLE cohort.

Results:

The SA cohort includes 72 patients; mean age 11.5 years, 83% female. The racial distribution was 68% Coloured*, 24% Black, 5% White, and 3% Asian/Indian. The SA cohort had increased rates and severity of lupus nephritis and high disease activity at enrollment (Table 1) SA patients had increased use of cyclophosphamide (942% vs 29%), methotrexate (30% vs 19%), and azathioprine (61% vs 16%). PSLE patients in the CARRA cohort had increased use of antimalarials and mycophenolate mofetil(Table 2). The PULSE cohort had high rates of end organ damage with 63% having a SLICC-DI score >0 (mean SLICC-DI 1.9. Within the SA cohort, 13% went on to develop ESRD, of which 9% required transplant, strikingly higher than North American peers. (Table 3)

Conclusions: The PULSE cohort is the largest registry of pSLE patients in Africa to date. SA children receive different therapy and progress to end organ damage at higher rates than pSLE cohorts in North America. The SA cohort demonstrates a striking increase in poor renal outcomes, end stage renal disease, and irreversible organ damage compared to North American peers. These differences may be due to treatment, health care access, racial predisposition to severe disease, environmental exposures, or a combination of factors. Further prospective research is required to determine the burden of pSLE in South Africa, and identify risk factors for poorer outcome in this high risk population.

*All race in South Africa is based on 5 standard categories used in SA population surveys: Coloured, White, Black, Asian/Indian, and Other.

Title: A STUDY OF THE AETIOLOGY AND OUTCOME OF CRESCENTIC GLOMERULONEPHRITIS IN CHILDREN PRESENTING TO THE RED CROSS CHILDRENS HOSPITAL
Authors: Dr Chisambo Mwaba, Komala Pillay, Dr Peter Nourse, Dr Priya Gajjar


Background:

Crescentic glomerulonephritis represents the extreme end of the spectrum of glomerular injury resulting from a wide array of disease conditions. It is marked by a rapid deterioration in renal function over days, weeks or months. Although rare it is important to recognize as prompt treatment can ameliorate outcome significantly.

Objective:

The objective of this study was to determine the prevalence, presentation, aetiology and outcome of histologically proven crescentic glomerulonephritis in children presenting to the Red Cross Children’s Hospital.

Methods:

This was a retrospective cross-sectional study that involved the review of renal biopsy results for children less than 18 years at the Red Cross children’s hospital that had been carried out between 2004 and July 2015. The clinical notes of patients found to have been diagnosed with crescentic glomerulonephritis were traced so as to extract demographic and clinical information which was then recorded onto the study data sheet. No attempt to contact patients or their families was made. Data analysis with regard to prevalence and associated clinical features was done using Epi Info version 3.3.2.

Results:

A total of 400 native kidney biopsies were performed in the period under review. Of these, 25 had crescentic glomerulonephritis, accounting for a prevalence of 6.2%. Most of the children (22) had class II crescentic glomerulonephritis, while 3 had type I and no child had type III crescentic glomerulonephritis. Of the children with type II crescentic glomerulonephritis 18 had post infectious glomerulonephritis. Thirteen (52%) of the children with crescentic glomerulonephritis were found to have either died, had residual renal dysfunction or been transplanted at the last clinical contact.

Conclusion:

Crescentic glomerulonephritis was diagnosed in 6.2 % of paediatric native renal biopsies which is consistent with what has been reported elsewhere. Unlike reports from other geographical areas the vast majority (88%) of the cases were type II with 80% being secondary to post-infectious glomerulonephritis a disease which can be prevented.