Widening of the genetic and clinical spectrum of Lamb-Shaffer syndrome, a neurodevelopmental disorder due to SOX5 haploinsufficiency

Abstract

Purpose: Lamb-Shaffer syndrome (LAMSHF) is a neurodevelopmental disorder described in just over two dozen patients with heterozygous genetic alterations involving SOX5, a gene encoding a transcription factor regulating cell fate and differentiation in neurogenesis and other discrete developmental processes. The genetic alterations described so far are mainly microdeletions. The present study was aimed at increasing our understanding of LAMSHF, its clinical and genetic spectrum, and the pathophysiological mechanisms involved.

Methods: Clinical and genetic data were collected through GeneMatcher and clinical or genetic networks for 41 novel patients harboring various types ofSOX5 alterations. Functional consequences of selected substitutions were investigated.

Results: Microdeletions and truncating variants occurred throughout SOX5. In contrast, most missense variants clustered in the pivotal SOX-specific high-mobility-group domain. The latter variants prevented SOX5 from binding DNA and promoting transactivation in vitro, whereas missense variants located outside the high-mobility-group domain did not. Clinical manifestations and severity varied among patients. No clear genotype-phenotype correlations were found, except that missense variants outside the high-mobility-group domain were generally better tolerated.

Conclusions: This study extends the clinical and genetic spectrum associated with LAMSHF and consolidates evidence that SOX5 haploinsufficiency leads to variable degrees of intellectual disability, language delay, and other clinical features.

Keywords: autism; developmental delay; epilepsy; intellectual disability; missense variants..

Fig. 1. SOX5 variant spectrum associated with Lamb–Shaffer syndrome (LAMSHF).

(a) Location of genetic alterations identified in patients in this study. SOX5 transcript isoforms are labeled with National Center for Biotechnology Information (NCBI) accession numbers. Boxes 1to 15, coding exons of isoform NM_006940. 5’ and 3’UTR: 5’ and 3’ untranslated sequences. p1 to p11 represent SOX5 promoters listed in the Fantom5 database; p1 and p2 (in bold) are the main promoters driving SOX5 expression in brain. CC, coiled-coil domain. Double-arrowed lines, deletions in patients D1–D8. Point variants, labeled as indicated. (b) Location of point variants reported here (above) and previously (below) on the longest SOX5 isoform. Protein and domain residue boundaries are indicated underneath the schematic. Red, nonsense and frameshift variants. Blue and green, missense variants within and outside the HMG domain, respectively. Superscripts, references.

Fig. 2. Patients exhibit similar clinical features regardless of the SOX5 alteration type.

Box plots showing comparative distribution of ages at (a) sitting unsupported, (b) walking unsupported, and (c) first words for patients with deletion, truncating, and missense variants. (d) Number of patients with normal to borderline cognitive abilities and various degrees of intellectual disability (ID). (e) Number of patients with autism spectrum disorder (ASD) or other behavioral disturbances. (f) Number of patients with seizures. (g) Facial profiles of individuals with de novo SOX5 variants. Above: D2 at age 10 years; D6 at age 26 years; D8 at age 24 years. Center: P1 at age 2 years, and his mother (41 years old); P6 at age 19 years; P10 at age 8 years. Below: P13 at ages 2 years, 6 months, and 11 years, 4 months, respectively; P14 at ages 2 years, 4 months and 8 years, respectively; P25 at age 4 years; P28 at age 5 years. Common facial features include broad or full nasal tip, thin upper lip and/or full lower lips, small jaw or prominent chin, prominent upper incisors and epicanthus.

Fig. 3. Human SOX5 is under tight conservation constraint.

(a) Distribution of synonymous and missense variants in SOX5 in gnomAD individuals. CC, coiled coil. (b) Percentages of residues carrying at least one synonymous or missense variant in the functional and other domains of SOX5 in gnomAD individuals. T-tests were performed to calculate the statistical significance of differences between protein domains. P values are indicated. (c) Alignment of all human SOX protein HMG domain sequences, with indication of residues altered in Lamb–Shaffer syndrome (LAMSHF) patients (red) and altered only in gnomAD individuals (purple). Asterisks, fully conserved residues. Dots, semiconserved residues. Colored triangles, residues important for DNA binding and bending. Brackets, H1, H2, and H3 α-helices. Continued lines linked with dotted lines, key amino acids in nuclear localization signal sequences (NLS) and nuclear export signal sequence (NES). (d) Alignment of human SOXD protein sequences outside the HMG domain that encompass residues altered in LAMSHF patients.

Fig. 4. Subcellular localization and activities of SOX5 variants.

(a) Western blots of cytoplasmic (C) and nuclear (N) extracts from HEK-293 cells transfected with plasmids encoding no protein (−), wild-type SOX5 (WT), or SOX5 variants. Blots were incubated with SOX5 antibody. Red boxes, SOX5specific protein signals. Numbers, Mr of protein standards. (b) Representative images of SOX5 immunostaining (green signal) in HEK-293 cells transfected with plasmids encoding wild-type SOX5 (WT) or the indicated variants. Nuclei are seen in blue and plasma membranes in red. Scale bars: 20 μm. (c) Test of the abilities of SOX5 variants to synergize with SOX9 in transactivation. HEK-293 cells were transfected with Acan and pSV2βGal reporter plasmids and plasmids encoding no protein, SOX9, and/or SOX5. The WT SOX5 plasmid was used in the indicated amounts, and the variant plasmids at 150 ng. Reporter activities are presented as the mean ± standard deviation obtained for triplicates in one representative experiment. They were normalized for transfection efficiency and are reported as increase over the activity of SOX9 alone. (d) Test of the abilities of SOX5 variants to interfere with WT SOX5 in transactivation. HEK-293 cells were transfected essentially as described above. SOX5 variant plasmids were tested at 150 ng with 150 ng SOX5 WT plasmid. Reporter activities were calculated and are presented as described above. (e) Test of the abilities of SOX5 variants to bind DNA in electrophoretic mobility shift assay (EMSA). Extracts from HEK-293 cells transfected with empty, WT SOX5, or SOX5 variant plasmid were incubated with a 2HMG DNA probe. Top, X-ray film images. SOX5/DNA complexes migrated more slowly than nonspecific protein (non-sp.)/DNA complexes. Bottom, western blot showing similar amounts of all SOX5 proteins. (f) Dimerization assay with the same extracts as in (c) for no protein, WT SOX5, and the R235C variant. Western blots were performed using SOX5 antibody. SOX5 dimers ran in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent Mr twice as large as that of monomers.