1 INTRODUCTION

Recently, Fliedner et al. (2020) published a study describing heterozygous loss-of-function (LoF) variants in the SCAF4 gene associated with a novel autosomal dominant neurodevelopmental disorder characterized by intellectual disability (ID), seizures, behavioral abnormalities, facial dysmorphisms, and skeletal anomalies. Nine LoF SCAF4 variants classified as likely pathogenic were reported, eight of which were de novo and one inherited. An additional LoF variant present in two siblings with neurodevelopmental phenotypes was classified as a variant of unknown significance (VUS) because it mapped to the C-terminal part of the protein. A missense variant was also described by Fliedner et al. as a VUS.

Here, we report a de novo frameshift SCAF4 variant mapped to exon 5/20 in a Brazilian patient with clinical features overlapping those described by Fliedner et al. (2020). This is the second publication supporting the involvement of SCAF4 in a new Mendelian neurodevelopmental disease.

2 CASE REPORT

The patient was first referred to the Institute of Biosciences of the University of Sao Paulo for genetic evaluation at the age of 4.4 years due to presentation of microcephaly and speech delay. He was born at term gestation from consanguineous parents (double first cousins) and exhibited feeding difficulties in early infancy, apparently without a significant degree of neonatal hypotonia. His motor development progressed without significant delay (seated and walking without support at the ages of 9 months and 1.5 years, respectively). His physical examination confirmed the presence of microcephaly (occipitofrontal circumference 45.5 cm, −3.43 SD) and slightly long protruding ears with a moderate degree of cartilage hypoplasia, epicanthus, slight convergent strabismus, bulbous nasal tip, high palate, joint hyperextensibility, pes planus, and obesity (20 kg, 1.03 m—BMI z-score 2.38); the patient did not exhibit brachydactyly (Figure 1a). Magnetic resonance imaging and cranial tomography were performed, both with normal results. The patient has no history of kidney problems, although renal ultrasound was not performed. Brazil has a high degree of ethnic admixture, but the patient is phenotypically Caucasian. His family has no previous history of individuals with ID or syndromic characteristics.

The family reported that at 6 years of age, he underwent cardiac surgery to correct a congenital atrial communication. Currently, he has mild mitral and tricuspid regurgitation. At the age of 8 years, he presented with epileptic seizures that are currently well-controlled with anticonvulsants.

Further clinical evaluation was performed at 16.6 years, in which facial dysmorphisms (convergent strabismus, epicanthus, prominent nasal bridge, bulbous nasal tip, long philtrum, and long ears—Figure 1b) and obesity (87 kg, 1.7 m—BMI z-score 2.27—Figure 1c) were observed. Notably, his father and brother also presented obesity. Microcephaly was no longer present (occipitofrontal circumference 54 cm, −0.74 SD).

The patient exhibited ID associated with speech impairment and behavioral problems, including anxiety and aggressiveness, particularly related to feeding. The following genetic tests were performed previously, all with normal results: G-banding karyotype, chromosome microarray analyses (CMA—Agilent 180K), and PCR for Fragile-X syndrome investigation (Haddad et al., 1996).

Genomic libraries for exome sequencing were produced from the proband's blood DNA using SureSelect QXT Human All Exon V6 technology (Agilent). The raw paired-end reads (Fastq) were aligned with the reference genome GRCh38 (hg38) using the Burrows–Wheeler Aligner (BWA; Li & Durbin, 2009) and processed with GATK tools (Depristo et al., 2011). The annotation was performed using Varseq software (Golden Helix), and the filtering involved the following criteria: read depth, population frequency—1KG Phase3 (The 1000 Genomes Project Consortium, 2015), gnomAD (Cummings et al., 2020), and ABraOM (Naslavsky et al., 2022)-, clinical data—ClinVar 2018 (Landrum et al., 2018)-, OMIM (Amberger et al., 2015), functional prediction—Ensembl (Hunt et al., 2018)-, and pathogenicity prediction algorithms. Filtered variants were classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines (Richards et al., 2015) using Varsome with manual review (Kopanos et al., 2019).

Segregation analyses were performed by Sanger sequencing with genomic DNA samples extracted from saliva from the patient's parents and unaffected brother.

3 RESULTS

We identified a pathogenic variant in the SCAF4 gene [SCAF4(NM_020706.2):c.374_375dup(p.Glu126LeufsTer20)] of a patient with syndromic ID. Figure 1d shows the presence of the variant in the proband and the absence in his parents and unaffected brother. The variant, which leads to a frameshift and consequent premature stop codon, was classified as pathogenic (ACMG criteria: PVS1, PM2, and PM6). Other clinically relevant variants were not identified.

4 DISCUSSION

There is a significant overrepresentation of epigenetic machinery-encoding genes in ID etiology in general (Scandaglia & Barco, 2019), with SCAF4 falling into this category. SCAF4 is an exclusively nuclear anti-terminator mRNA protein associated with RNA polymerase II; it inhibits the use of early alternative polyA sites to diminish the production of nonfunctional mRNAs and proteins (Gregersen et al., 2019). An RNAseq assay performed by Fliedner et al. (2020) using blood lymphocytes of SCAF4-deficient individuals revealed a broad deregulation of more than 9000 genes and a significant differential splicing of more than 2900 genes.

Our patient presents a partial clinical overlap with those described by Fliedner et al. (2020) carrying likely pathogenic SCAF4 variants (Table 1). Variable levels of cognitive and speech impairment were present in all cases. Other features described in some patients reported by Fliedner et al. (2020), including epilepsy and heart malformations, were also present in this patient. Behavioral problems were frequent, and our patient exhibited anxiety and aggressiveness. Notably, we report the first observations of microcephaly (in early development) associated with SCAF4 LoF variant.

Patients 7 and 11 from the study of Fliedner et al. (2020) also had obesity, but Patient 11 carried a SCAF4 VUS. Obesity is a common feature and has a high prevalence among people with cognitive impairment (Carvalho et al., 2022; Emerson et al., 2016; Hsieh et al., 2014; Melville et al., 2008). There are many factors that may contribute to this higher prevalence in people with ID, such as low levels of physical activity, poor nutritional habits (Melville et al., 2017), and the use of psychiatric drugs (de Winter et al., 2012). In particular, our patient does not exercise, is very anxious regarding food and uses some medications that can lead to weight gain (currently: chlorpromazine, periciazine, and clobazam; in previous years: risperidone). In addition, he has a family history of obesity, which is a condition with high heritability (Sandholt et al., 2012).

This publication substantiates the role of SCAF4 LoF variants as a cause of syndromic ID and contributes to broadening the associated clinical spectrum.

AUTHOR CONTRIBUTIONS

Laura Machado Lara Carvalho performed NGS data analysis, Sanger sequencing, and the interpretation of genetic variants, was responsible for the initial writing of the manuscript and produced the figures under the guidance of Carla Rosenberg and Ana Cristina Victorino Krepischi, who conceived and designed the study. Carla Franchi Pinto and Paulo A. Otto clinically evaluated the patient and contributed to the interpretation of the results. Marília de Oliveira Scliar contributed to bioinformatics processing (alignment to the reference genome and calling of variants). All authors have read, edited, and approved the final manuscript.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.