PhD, Faculty of Health Sciences, University of the Witwatersrand / HIV/AIDS continues to be one of the greatest health challenges which South Africa faces. The epidemic in children is closely linked to that in women, the prevalence of which continues to grow according to antenatal statistics from the South African Department of Health (DOH). HIV is known to invade the central nervous system at the time of infection, and causes widespead damage. In children, this leads to a well-researched condition known as HIV encephalopathy, which affects all areas of neurodevelopment. The effects of timely initiation of antiretroviral therapy on alleviating the impact of encephalopathy have been well described. Neurodevelopmental delay is a stage four disease indicator according to the World Health Organisation (WHO), and therefore is a criterion for initiation of Highly Active Antiretroviral Therapy (HAART). HAART is often only administered according to the virologic and immunologic status of a child, as standardised neurodevelopmental assessment tools are not widely available in South African clinics. When HAART initation is dependent on immunologic status, it is often too late to prevent encephalopahy. To date, the only means of prevention of this condition is early initation of HAART, which has not been widely available in South Africa. Stringent guidelines for the commencement of this therapy according to the WHO, and the South African Department of Health (DOH) have had to be followed, leading to late initiation of HAART, and widespread central nervous system encephalopathy. Studies which have been carried out in South African clinics have demonstrated the high prevalence of this condition. Once there is evidence of encephalopathy, children should be referred for assessments in all facets of development, and where necessary, for rehabilitation. A standardised developmental screening tool which is suitable for use in a developing country is therefore necessary in order to screen for neurodevelopmental delays to allow for further assessment and referral to rehabilitation services, as well as providing an additional assessment criterion for initiation of HAART. Paediatric HIV clinics in developing countries are understaffed, and children may be seen by junior staff or screened by nurses due to the high numbers of clinic attendees. This often results in neurodevelopment being inadequately assessed and children are therefore not referred for intervention services. A standardised screening tool, which The Development of a Screening Tool to Evaluate Infants who are HIV Positive could be administered by clinic staff in order to ensure correct and timely referral of children for further assessment and intervention is therefore necessary. This is of importance both locally and internationally where a screening tool, which has been developed specifically for this purpose, does not exist. The aim of this study was therefore to evaluate the agreement between the Bayley-III Screening Test and the Bayley Scales of Infant Development (3rd version) in a population of HIV positive infants in order to evaluate its appropriateness for use in South Africa. The Bayley Scales of Infant Development have long been considered the ‘gold standard’ in infant developmental assessment, which is why this tool was chosen to evaluate the Bayley-III Screening Test against. The developmental scores in each facet (cognitive, motor or language) were evaluated to determine which should be included in an assessment tool for this population. Further objectives for the study were to adapt the screening tool to the needs of the population, or to develop a new screening tool should the Bayley-III Screening Test not prove suitable for use in this population. In order to meet the aims and objectives, a cross-sectional study was conducted where 112 HIV positive infants between the ages of six and eighteen months were assessed using the Bayley-III Screening Test and the Bayley Scales of Infant Development (3rd version) (BSID III). The infants were stratified into four age groups namely 6-8 months, 9- 12 months, 13-16 months, and 17-18 months. Children were recruited from Harriet Shezi Children’s Clinic at Chris Hani Baragwanath Hospital in Soweto. The agreement between the Bayley-III Screening Test and the Bayley Scales of Infant Development (3rd version) was analysed using Kappa, for the overall group, and for each age group. Overall agreement between the tools was as follows: K=0.58 for the Cognitive facet, K=0.82 for the Expressive Communication facet, K=0.76 for the Receptive Communication facet, K=0.44 for the Fine Motor facet and K=0.57 for the Gross Motor facet. These values indicate that the Bayley-III Screening Test is therefore not acceptable for clinical use, as excellent agreement (k≥0.75) in all facets would be necessary for this purpose. A new screening tool therefore had to be developed. The infant’s developmental scores from the BSID III were analysed to determine which facets of development were most severely affected, and therefore which facets should be included in a new screening tool. Gross motor function was demonstrated to be the area which was most severely affected, followed by cognitive function. A gross motor screening tool would therefore be suitable for use in this population, as no equipment would be necessary. Gross motor development is the most universally similar aspect of development, which is not completely dependent on cultural or socioeconomic factors which often have an influence on language and cognitive development. Item selection from the BSID III was undertaken to determine which items should be included in a brief screening tool. In each of the four age groups, item selection occurred as follows: Two items which discriminated the At-Risk, from Emerging and Competent groups (less than 20% in the At-Risk group, and 100% in the other groups) were selected. Two items, which discriminated between children in the ‘Emerging’ and ‘Competent’ categories on the BSID III were selected (0-5% of children who were At-Risk obtained credit, 30-50% of the Emerging group obtained credit, and 100% of the Competent group obtained credit). Lastly, two items were selected which discriminated the Competent group from the other two groups (100% or as high as possible in the Competent group, and 0% in the other groups). The new gross motor screening tool was assembled using the selected items, scoring was allocated, and it was tested against the scores obtained on the Gross Motor facet of the BSID III for the initial 112 infants. Agreement between the tools was analysed using Kappa, and refinements were made according to the discrepancies. This was done three times, until the Kappa value revealed excellent agreement between the tools (k = 0.87). A panel of experts was then invited to examine the new gross motor screening tool, and to comment on it, and further adjustments were made accordingly. Preliminary concurrent validity testing of the new gross motor screening tool was then carried out against the Gross Motor facet of the BSID III on 60 children, who were recruited from the Harriet Shezi Children’s Clinic at Chris Hani Baragwanath Hospital in Soweto. Statistical analysis revealed that the agreement between the BSID III and the new screening tool was excellent (k=0.85). The diagnostic properties of the new gross motor screening tool were as follows: sensitivity 97.4%, specificity 85.7%, positive predictive value 92.7%, and negative predictive value 94.7%. These values indicate that the statistical properties of the tool are excellent, and the tool will not be predisposed to underreferrals or over-referrals. Preliminary reliability testing was carried out on 15 children for test-retest/intrarater reliability and 15 children for interrater reliability. Interrater, test-retest and intrarater reliability were excellent (r=1, r=0.98, r=0.98 respectively). Further testing of reliablity and validity should be undertaken in order to establish these properties, and standardisation should also be carried out on healthy children. Given the need for an assessment tool of this nature in South Africa and other developing countries, and the statistical properties thus far, the tool may be used clinically for the purposes for which it was developed.

Neurodevelopmental disorders (NDD) refer to a group of conditions resulting from disturbances of the developing brain with a typical onset in childhood before puberty. Genetic causes make up a large part of developmental delays, which is why genetic examinations play a decisive role in the clarification of the causes of NDD. Due to the development of Next Generation Sequencing (NGS) and the increased use of genome-wide analyses in recent years, it has become clear that a large proportion of cases are due to rare, monogenic alterations in each case. Meanwhile, 1534 genes have been currently associated with NDD. Nevertheless, half of the evaluated cases remain without a valid diagnosis. However, this is a prerequisite for personalized support and the estimation of the development prognosis, as well as the differentiated assessment of the risk of recurrence for family members. To decipher the genetics of NDD, I systematically analyzed the exome sequences of 104 individuals with NDD and their relatives (see Figure 2). In 10 of the 104 cases, I was able to find variants in already known genes that partially explain the phenotype. I intensively evaluated all the cases for new candidate genes and identified 89 candidate genes in 58 individuals (see Supplementary, Table S1). In the remaining 46 individuals, no candidate gene could be identified. I then scored the candidate genes to prioritize them regarding the probability of being true NDD genes. From the detailed analysis of a relatively small cohort of NDD individuals (n=104) and the resulting 89 candidate genes, a total of 9 research collaborations have emerged (see Table 1). Of the candidate genes with further research in AutoCasC, six are in my top 20 candidate genes, which is a good indication of the efficiency of this systematic approach on deciphering the genetics of NDD. Studies on the candidate genes SKOR2, HCN2, SP9, CCDC66, and TANC1 are currently being worked on by collaborators worldwide and we could add our clinical and genetic data (see Table 1). Further, I was more substantially involved in the identification of the candidate gene ATP2B1, subsequently studied functionally, and published in the American Journal of Human Genetics (IF 11,0) with me as coauthor as a novel NDD gene, and the ongoing research on a genotype-phenotype correlation with functional lines of evidence of NDD-individuals with variants in DOCK4. Furthermore, I have led the efforts for the genes RIPPLY2 and PABPC1. I was able to describe three individuals from two families with compound-heterozygous variants in RIPPLY2 in two sisters and a homozygous nonsense variant in an 8-year-old boy. All individuals had multiple vertebral body malformations in the cervical and thoracic region, small or absent rib involvement, myelopathies, and common clinical features of spondylocostal dysostosis 6 (SCDO6) including scoliosis, mild facial asymmetry, spinal spasticity, and hemivertebrae. At this time, RIPPLY2 was only associated as a candidate gene with SCDO6 and had only been described in a small cohort of seven individuals from five families in two publications. I could confirm that bi-allelic variants in RIPPLY2 cause congenital cervical spine malformation in spondylocostal dysostosis type 6 and broaden the phenotype by adding myelopathy with or without spinal canal stenosis and spinal spasticity to the symptom spectrum as a first author in a publication published in Clinical Genetics (IF 4,4). In the study on PABPC1, I describe four individuals with an overlapping phenotype of developmental delay, expressive speech delay, autistic features, and heterozygous de novo variants that cluster in the PABP domain of PABPC1. Further symptoms are seizures and behavioral disorders. Molecular modeling predicted that the variants are pathogenic and would lead to decreased binding affinity to mRNA metabolism-related proteins such as PAIP2. Co-immunoprecipitation confirmed this as it demonstrated a significant weakening of the interaction between mutant PABPC1 and PAIP2. Electroporation of mouse embryo brains showed that Pabpc1 knockdown decreases the proliferation of neural progenitor cells. The wild type Pabpc1 could rescue this disturbance, while three of the four variants did not. Together with partners from the Central South University in Changsha, China, I was able to propose that pathogenic missense variants in the PABP-domain of PABPC1 lead to a novel form of developmental disorder and published my work in Genetics in Medicine (IF 8,9). Through this, I demonstrated that systematic trio exome analysis and identification and characterization of candidate genes followed by prioritizing the genes based on systematic scoring and by building international cooperation to gather further individuals, describe the phenotypes, and prove that the pathogenicity of the variants is an excellent way to decipher the genetics of NDD. With this approach, I was able to describe PABPC1 as a novel NDD-gene and confirm the association between RIPPLY2 and SCDO6. Moreover, I contributed as a co-author to the publication of ATP2B1 as a novel NDD-gene and to the ongoing research on SKOR2, SP9, HCN2, CCDC66, TANC1, and DOCK4. More might follow in the future. The continuation of this research in genetic diagnostics is important for creating personalized support and prevention programs for individuals with neurodevelopmental delays, to be able to estimate the developmental prognosis, and to be able to assess the recurrence risk of other family members in a more differentiated way.:1 Introduction 1.1 Genetics of neurodevelopmental disorders 1.2 Identification of neurodevelopmental delay genes 1.3 Assessment of candidate genes 1.4 Rationale 1.5 Results 1.5.1 Identification of neurodevelopmental delay genes 1.5.2 Scoring of the identified candidate genes 1.5.3 Candidate genes under further research 2 Publications 2.1 Congenital cervical spine malformation due to bi-allelic RIPPLY2 variants in spondylocostal dysostosis type 6 2.2 De novo variants in the PABP-domain of PABPC1 lead to developmental delay 3 Summary 4 References 5 Internet resources 6 Supplementary 7 Presentation of personal scientific contribution 8 Declaration of Authorship