A clinical and molecular investigation of two South African families with Simpson-Golabi-Behmel syndrome

Background. Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked recessive overgrowth syndrome manifesting primarily in boys and characterised by macrosomia, distinctive facial features and multiple congenital abnormalities. Although this rare condition is thought to be underdiagnosed, making a diagnosis is important as affected boys have a 7.5% risk of developing visceral tumours and surveillance is warranted. Mutations in GPC3 are found in up to 70% of boys affected with SGBS. Objectives. A clinical and molecular investigation of two boys with SGBS, probands B and S, and their mothers. Documentation of the clinical phenotype could assist with diagnosis in affected boys and will lead to early initiation of tumour surveillance.Methods. Hospital folders were reviewed and clinical consultations arranged for both probands and their mothers. Molecular investigations initially searched for whole-exon deletions in GPC3 followed by gene sequencing. Results. The clinical phenotype of both probands was consistent with that previously reported in the literature. The main features pointing towards the diagnosis were macrosomia, coarse facial features and macroglossia with a midline groove in the tongue. Proband B developed a Wilms tumour. He was found to have a novel mutation causing a premature stop codon.Conclusions. This research represents the first published report of SGBS in South Africa. Early recognition and confirmation of this condition is important in order to institute tumour surveillance and assist families with accurate recurrence risks.

Mutation profiling in South African patients with Cornelia de Lange syndrome phenotype

DATA AVAILABILITY STATEMENT : The variants described here were submitted to ClinVar and can be viewed under Organization ID 508172 or the ClinVar IDs recorded in Table 1. Data available on reasonable request from the corresponding author.BACKGROUND : Cornelia de Lange Syndrome (CdLS) presents with a variable multi-systemic phenotype and pathogenic variants have been identified in five main genes. This condition has been understudied in African populations with little phenotypic and molecular information available. METHODS AND RESULTS : We present a cohort of 14 patients with clinical features suggestive of CdLS. Clinical phenotyping was carried out and cases were classified according to the international consensus criteria. According to this criteria, nine patients had classical CdLS, one had non-classical CdLS and four presented with a phenotype that suggested molecular testing for CdLS. Each patient underwent mutation profiling using a targeted next generation sequencing panel of 18 genes comprising known and suspected CdLS causal genes. Of the 14 patients tested, pathogenic and likely pathogenic variants were identified in nine: eight variants in the NIPBL gene and one in the STAG1 gene. CONCLUSIONS : We present the first molecular data for a cohort of South African patients with CdLS. Eight of the nine variants identified were in the NIPBL gene, the most commonly involved gene in cases of CdLS. This is also the first report of a patient of African ancestry presenting with STAG1-related CdLS.The National Research Foundation and the South African Medical Research Council.http://www.wileyonlinelibrary.com/journal/mgg3hj2024BiochemistryGeneticsMicrobiology and Plant PathologySDG-03:Good heatlh and well-bein

Implications of direct-to-consumer whole-exome sequencing in South Africa

This editorial examines a number of vitally important ethical, legal and scientific concerns that have to be addressed to ensure proper and ethical implementation of direct-to-consumer whole-exome sequencing in South Africa. Individuals taking part in this endeavour must be fully informed of the positive and negative sequelae

Overlapping SETBP1 gain-of-function mutations in Schinzel-Giedion syndrome and hematologic malignancies

Schinzel-Giedion syndrome (SGS) is a rare developmental disorder characterized by multiple malformations, severe neurological alterations and increased risk of malignancy. SGS is caused by de novo germline mutations clustering to a 12bp hotspot in exon 4 of SETBP1. Mutations in this hotspot disrupt a degron, a signal for the regulation of protein degradation, and lead to the accumulation of SETBP1 protein. Overlapping SETBP1 hotspot mutations have been observed recurrently as somatic events in leukemia. We collected clinical information of 47 SGS patients (including 26 novel cases) with germline SETBP1 mutations and of four individuals with a milder phenotype caused by de novo germline mutations adjacent to the SETBP1 hotspot. Different mutations within and around the SETBP1 hotspot have varying effects on SETBP1 stability and protein levels in vitro and in in silico modeling. Substitutions in SETBP1 residue I871 result in a weak increase in protein levels and mutations affecting this residue are significantly more frequent in SGS than in leukemia. On the other hand, substitutions in residue D868 lead to the largest increase in protein levels. Individuals with germline mutations affecting D868 have enhanced cell proliferation in vitro and higher incidence of cancer compared to patients with other germline SETBP1 mutations. Our findings substantiate that, despite their overlap, somatic SETBP1 mutations driving malignancy are more disruptive to the degron than germline SETBP1 mutations causing SGS. Additionally, this suggests that the functional threshold for the development of cancer driven by the disruption of the SETBP1 degron is higher than for the alteration in prenatal development in SGS. Drawing on previous studies of somatic SETBP1 mutations in leukemia, our results reveal a genotype-phenotype correlation in germline SETBP1 mutations spanning a molecular, cellular and clinical phenotype

SCN3A ‐related neurodevelopmental disorder: A spectrum of epilepsy and brain malformation

Objective Pathogenic variants in SCN3A , encoding the voltage‐gated sodium channel subunit Nav1.3, cause severe childhood‐onset epilepsy and malformation of cortical development. Here, we define the spectrum of clinical, genetic, and neuroimaging features of SCN3A ‐related neurodevelopmental disorder. Methods Patients were ascertained via an international collaborative network. We compared sodium channels containing wild‐type vs. variant Nav1.3 subunits co‐expressed with β1 and β2 subunits using whole‐cell voltage clamp electrophysiological recordings in a heterologous mammalian system (HEK‐293 T cells). Results Of 22 patients with pathogenic SCN3A variants, most had treatment‐resistant epilepsy beginning in the first year of life (16/21, 76%; median onset, 2 weeks), with severe or profound developmental delay (15/20; 75%). Many, but not all (15/19; 79%), exhibited malformations of cortical development. Pathogenic variants clustered in transmembrane segments 4–6 of domains II‐IV. Most pathogenic missense variants tested (10/11; 91%) displayed gain of channel function, with increased persistent current and/or a leftward shift in the voltage dependence of activation, and all variants associated with malformation of cortical development exhibited gain of channel function. One variant (p.Ile1468Arg) exhibited mixed effects, with gain and partial loss of function. Two variants demonstrated loss of channel function. Interpretation Our study defines SCN3A‐ related neurodevelopmental disorder along a spectrum of severity, but typically including epilepsy and severe or profound developmental delay/intellectual disability. Malformations of cortical development are a characteristic feature of this unusual channelopathy syndrome, present in over 75% of affected individuals. Gain of function at the channel level in developing neurons is likely an important mechanism of disease pathogenesis

Implications of direct-to-consumer whole-exome sequencing in South Africa

Next-generation sequencing (NGS) has truly transformed human genetics and is now an integral discovery tool in the field. Whole-exome sequencing (WES) – an NGS application focused on the proteincoding regions of the human genome – has already bridged the bench-to-bedside divide internationally and is offered as a clinical test by several accredited laboratories. Clinical WES is not currently offered in South Africa (SA) for a number of reasons, including technological constraints, insufficient storage for the resulting large datasets, ethical considerations and limitations of our understanding of the impact of human genetic variants on health and in terms of clinical utility. The historical under-representation of individuals of black African descent in genomics research further complicates the interpretation of results obtained from WES data in black Africans. Concurrently, the application of WES for preventive healthcare in seemingly healthy individuals is progressing rapidly. WES offered as a direct-to-consumer (DTC) genetic test to healthy individuals in aid of wellness and future disease risk prediction raises many critical considerations, some of which were highlighted previously in the SAMJ by the Southern African Society for Human Genetics. This topic is currently back in the headlines as local health insurance company Discovery Health launched their suite of personalised medicine products, which includes WES.[5-7] This offering is presented in partnership with US-based company Human Longevity, Inc. (HLI) under the leadership of J Craig Venter.http://www.samj.org.zaam2017Immunolog

Overlapping SETBP1 gain-of-function mutations in Schinzel-Giedion syndrome and hematologic malignancies

Schinzel-Giedion syndrome (SGS) is a rare developmental disorder characterized by multiple malformations, severe neurological alterations and increased risk of malignancy. SGS is caused by de novo germline mutations clustering to a 12bp hotspot in exon 4 of SETBP1. Mutations in this hotspot disrupt a degron, a signal for the regulation of protein degradation, and lead to the accumulation of SETBP1 protein. Overlapping SETBP1 hotspot mutations have been observed recurrently as somatic events in leukemia. We collected clinical information of 47 SGS patients ( including 26 novel cases) with germline SETBP1 mutations and of four individuals with a milder phenotype caused by de novo germline mutations adjacent to the SETBP1 hotspot. Different mutations within and around the SETBP1 hotspot have varying effects on SETBP1 stability and protein levels in vitro and in in silico modeling. Substitutions in SETBP1 residue I871 result in a weak increase in protein levels and mutations affecting this residue are significantly more frequent in SGS than in leukemia. On the other hand, substitutions in residue D868 lead to the largest increase in protein levels. Individuals with germline mutations affecting D868 have enhanced cell proliferation in vitro and higher incidence of cancer compared to patients with other germline SETBP1 mutations. Our findings substantiate that, despite their overlap, somatic SETBP1 mutations driving malignancy are more disruptive to the degron than germline SETBP1 mutations causing SGS. Additionally, this suggests that the functional threshold for the development of cancer driven by the disruption of the SETBP1 degron is higher than for the alteration in prenatal development in SGS. Drawing on previous studies of somatic SETBP1 mutations in leukemia, our results reveal a genotype-phenotype correlation in germline SETBP1 mutations spanning a molecular, cellular and clinical phenotype

Genetic and clinical characteristics of individuals with germline <i>SETBP1</i> mutations and Schinzel-Giedion syndrome.

<p><b>A</b>. Schematic representation of the SETBP1 protein, indicating changes found in SGS and in hematologic malignancies. The residues of the canonical degron are highlighted with arrows. Protein domains of SETBP1 are shown in different colors with green corresponding to three AT hooks, purple to the SKI homologous region, blue to the SET binding domain and orange to a repeat domain (modified from Piazza <i>et al</i>.). <b>B</b>. Sequence alignment of the region containing the degron of SETBP1 (in bold) in human (Uniprot accession number Q9Y6X0), chimpanzee (H2QEG8), mouse (Q9Z180), chicken (A0A1D5PT15), african clawed frog (F6TBV9) and zebrafish (B0R147). The consensus motif for βTrCP1 substrates is shown on top, with φ representing a hydrophobic residue and X any amino acid. Residues in which pathogenic germline mutations have been identified in classic SGS are highlighted in blue, while residues in which novel mutations leading to an atypical form of SGS are shown in green. <b>C</b>. Distinctive facial features encountered in classic SGS (current case 9 at 1,5 years of age). <b>D</b>. Typical question mark-shaped ear observed in current case 18. <b>E</b>. Characteristic hand posture with clenched fingers from current case 16. <b>F</b>. Facial features of current case 27 with a mutation in SETBP1 residue S867 at 4 years of age. Note the clenched fingers. <b>G</b>. Facial features of current case 28 with a mutation in SETBP1 residue E862 at 5 years of age. <b>H</b>. Facial features of current case 29 with a mutation in SETBP1 residue T873 at the age of 23 months.</p

On average, <i>SETBP1</i> mutations seen in cancer are more severe than those observed in SGS.

<p><b>A</b>. Distribution of mutations within the SETBP1 degron in SGS and in hematological malignancies. (** p<0.01, Fisher’s test and Bonferroni correction for multiple testing). <b>B</b>. ΔΔG values for protein stability (x-axis) and degron-βTrCP1 interaction (y-axis) for all mutations reported in SETBP1. The size of each circle is proportional to the frequency of the mutation in each condition. <b>C</b>. Difference in free energy of binding in the interaction between βTrCP1 and the degron of variants arising from germline or somatic SETBP1 mutations compared to that of the interaction between βTrCP1 and the wild-type degron (* p <0.05, Mann-Whitney’s U test). The median is highlighted by an arrow head.</p

Functional analysis of SETBP1 mutations identified in SGS.

<p><b>A</b>. Fluorescence measurements in live HEK293 cells expressing YFP-tagged SETBP1 variants. (*** p<0.001 versus wild-type and all mutants, ANOVA). All SETBP1 mutations studied displayed a statistically significant difference compared to wild-type and to all other mutations. This graph is representative of 3 independent experiments performed, with 6 technical replicates per experiment. Bars represent the standard error. <b>B</b>. Relative expression of SETBP1 protein variants in live HEK293 cells treated with MG132 proteasome inhibitor or vehicle only. Bars represent the standard error. (*** p<0.001, * p<0.05, NS: not significant, Student’s T test and Mann-Whitney U test). <b>C</b>. ΔΔG values for degron-βTrCP1 interaction for all germline mutations reported in SETBP1 per residue (** p<0.01 D868 versus other residues; ANOVA). <b>D</b>. Immunoblot of whole cell lysates of HEK293 cells expressing FLAG-tagged SETBP1 variants probed with anti-FLAG antibody. <b>E</b>. Immunoblot of whole-cell lysates of fibroblasts probed with anti-SETBP1 antibody. Fibroblasts were derived from two cases of SGS, one carrying the I871T variant and the other carrying the D868N variant, as well as from two unrelated controls. In D and E, blots were stripped and re-probed with anti-β-actin antibody.</p