Resolution of SLC6A1 variable expressivity in a multi-generational family using deep clinical phenotyping and Drosophila models

Abstract

Purpose: Variants in SLC6A1 result in a rare neurodevelopmental disorder characterized by a variable clinical presentation of symptoms including developmental delay, epilepsy, motor dysfunction, and autism spectrum disorder. SLC6A1 haploinsufficiency has been confirmed as the predominant pathway of SLC6A1-related neurodevelopmental disorders (NDDs), however, the molecular mechanism underlying the variable clinical presentation remains unclear.

Methods: Here, through work of the Undiagnosed Diseases Network, we identify an undiagnosed individual with an inherited p.(A334S) variant of uncertain significance. To resolve this case and better understand the variable expressivity with SLC6A1, we assess the phenotypes of the proband with a cohort of cases diagnosed with SLC6A1-related NDDs. We then create an allelic series in the Drosophila melanogaster to functionally characterize case variants.

Results: We identify significant clinical overlap between the unsolved case and confirmed cases of SLC6A1-related NDDs and find a mild to severe clinical presentation associated with missense variants. We confirm phenotypes in flies expressing SLC6A1 variants consistent with a partial loss-of-function mechanism.

Conclusion: We conclude that the p.(A334S) variant is a hypomorphic allele and begin to elucidate the underlying variability in SLC6A1-related NDDs. These insights will inform clinical diagnosis, prognosis, treatment and inform therapeutic design for those living with SLC6A1-related NDDs.

Figure 1

Comprehensive pedigrees and their genotypes for families with SLC6A1 variants. Standard pedigree structures are utilized—filled circles and squares denote clinically affected individuals, and probands are indicated by black arrows. (A) The UF1 pedigree is denoted with the phenotypes as noted in family members respectively. (B) Thirteen families diagnosed with SLC6A1-neurodevelopmental disorders recruited through the Brain Gene Registry. (C) Schematic diagram of SLC6A1 protein structure from conceptual translation of transcript NM_003042.3 with mapping location (circles) of the amino acid variants observed in this study. In total, we identified eleven different SNVs and indels that are distributed throughout the protein. Blue circles represent missense variants, red circle represent frameshift variants and yellow circle represents the splice variant observed in this study. Image created in Biorender.

Figure 2

Clinical phenotypic analyses. (A) SLC6A1 phenotypic grid of all individuals in the study. HPO terms from all individuals were categorized into 11 key phenotypic domains described in the draft SLC6A1 disease model. The frequency of terms per phenotypic domain is plotted. Neurological and cognitive domains show the highest frequency of terms. (B, C) Rapid neurodevelopmental assessment protocol (RNAP) profiles of individuals. The 804-item questionnaire in the RNAP was categorized into 7 domains namely: cognitive ability, adaptive behavioral function, social and emotional function, sensory-motor function, sleep symptoms, communication ability, physical features and neurologic symptoms. The RNAP domain score was generated for each individual, which was then normalized based on the number of questions answered by the individual in each domain. These scores are plotted to visualize the RNAP profile for each individual. (D) Phenotypic similarity heatmap for individuals with SLC6A1 variants. A heatmap was generated using proband phenotypic similarity scores and ordered based on Hierarchical Agglomerative Clustering of proband phenotypic similarity. Dendrograms showing clusters are present at the left and top sides of the heatmap. Green box highlights the two frameshift variants that cluster together with similar severe phenotypes. The UDN p.(A334S) variant clusters with other confirmed cases of SLC6A1.

Figure 3

Gat specific GAL-4 transcriptional activator enables spatial and temporal control of SLC6A1 variant expression. (A) The Trojan GAL-4 cassette is a strong loss-of-function allele that simultaneously truncates the Drosophila Gat protein and expresses a GAL-4 protein in under the regulatory control of the fly gene promoter. (B) Upstream activation sequence containing constructs were generated with the UDN and BGR variants p.(A288V), p.(S295L), p.(G297R), and p.(A334S) for functional analysis in the fly model. (C) Current lineage tracing identifies Gat^TG4 expression in the fly larval brain. (D) anti-Repo immunostaining labels glial cells. (E) Gat and Repo merge identifies that Gat^TG4 is expressed in Repo-positive glial cells.

Figure 4

Expression of SLC6A1^A334S results in decreased sleep latency. (A) Expression of a heterozygous loss-of-function allele Gat^TG4 / + results in a severe decrease in sleep latency compared to SLC6A1^Ref ;; Gat^TG4 / + (P<0.0001). Sleep latency is suppressed by expression of SLC6A1^Ref driven by Gat^TG4. Expression of variant lines also induces a significant decrease in sleep latency SLC6A1^A288V (P=0.067), SLC6A1^S295L (P=0.0006), SLC6A1^G297R (P=0.0001), SLC6A1^A334S (P=0.0008) compared to SLC6A1^Ref. (B) Total sleep is reduced for SLC6A1^G297R (P=0.0141) and SLC6A1^A334S (P=0.0064). (C) Sleep bout length is reduced in all variants SLC6A1^A288V (P<0.0001), SLC6A1^S295L (P<0.0001), SLC6A1^G297R (P<0.0001), SLC6A1^A334S (P<0.0001). (D) Sleep bout number is increased for all variants SLC6A1^A288V (P<0.0001), SLC6A1^S295L (P<0.0001), SLC6A1^G297R (P<0.0001), SLC6A1^A334S (P<0.0001). (E) Total activity is increased in SLC6A1^S295L (P=0.0004), SLC6A1^G297R (P=0.00075), SLC6A1^A334S (P=0.0002). Welch’s ANOVA with Dunnett’s T3 multiple comparisons test.

Figure 5

The UDN variant induces hypomorphic sleep phenotypes compared to expression of dSLC6A1. (A) Approach to replace the Gat^TG4 swappable insertion cassette with the Drosophila Gat sequence. This results in heterozygous expression of the transgene. (B) Expression of the loss-of-function control Gat^MIMIC (P=0.0121) and dSLC6A1^A334S (P=0.0110) results in seizure sensitivity compared to y¹ w^*. (C) There is a severe reduction in sleep latency when dSLC6A1 is expressed and there is a significant increase in sleep latency in dSLC6A1^A334S (P<0.0001) compared to dSLC6A1. (D) There is no significant difference between dSLC6A1 and dSLC6A1^A334S in total sleep. (E) There is a decrease in dSLC6A1^A334S sleep bout length (P=0.0085). (F) There is an increase in dSLC6A1^A334S sleep bout number (P=0.0003). (A) There is an increase in dSLC6A1^A334S total activity (P=0.0043). Welch’s ANOVA with Dunnett’s T3 multiple comparisons test.