De novo mutations in FBRSL1 cause a novel recognizable malformation and intellectual disability syndrome

Abstract

We report truncating de novo variants in specific exons of FBRSL1 in three unrelated children with an overlapping syndromic phenotype with respiratory insufficiency, postnatal growth restriction, microcephaly, global developmental delay and other malformations. The function of FBRSL1 is largely unknown. Interestingly, mutations in the FBRSL1 paralogue AUTS2 lead to an intellectual disability syndrome (AUTS2 syndrome). We determined human FBRSL1 transcripts and describe protein-coding forms by Western blot analysis as well as the cellular localization by immunocytochemistry stainings. All detected mutations affect the two short N-terminal isoforms, which show a ubiquitous expression in fetal tissues. Next, we performed a Fbrsl1 knockdown in Xenopus laevis embryos to explore the role of Fbrsl1 during development and detected craniofacial abnormalities and a disturbance in neurite outgrowth. The aberrant phenotype in Xenopus laevis embryos could be rescued with a human N-terminal isoform, while the long isoform and the N-terminal isoform containing the mutation p.Gln163* isolated from a patient could not rescue the craniofacial defects caused by Fbrsl1 depletion. Based on these data, we propose that the disruption of the validated N-terminal isoforms of FBRSL1 at critical timepoints during embryogenesis leads to a hitherto undescribed complex neurodevelopmental syndrome.

Fig. 1

Clinical representation of the three patients. A Patient 1 at the age of six months: a part of the face b side view showing a pronounced neck fold, a flat back of the head and a dysmorphic auricle c side view of the right leg with wrinkles and d side view with a view of the back area with pronounced skin fold formation. B Patient 2 at the age of five months: a front view showing deep-set eyes, a round face and temporal indentations; b side view, which also shows a flat back of the head and a slightly dysplastic auricle. Due to the fixation of the tracheostoma, the pronounced neck fold is not visible; c view of the back with wrinkles; d view of the back of patient 2 at the age of two and a half years. A remarkable regression of the skin folds was observed. C Patient 1 at the age of 6 years and 7 months: a front-view and b side-view showing dysmorphic features. c Contractures on both hands and fingers are shown (d, e) view of the teeth. D front-view (a) and side-view (b) of patient 3 at the age of 12 years and 6 months. c, d facial appearance of patient 3 at the age of 14 years and 8 months. Contractures of both hands and fingers are observed, as well as wide-spaced teeth

Fig. 2

Scheme of validated human FBRSL1 isoforms and localization of the detected mutations. The scheme was created using the Exon–Intron-Graphic Maker available at https://wormweb.org/exonintron by Nikhil Bhatla (2012) (version 4)

Fig. 3

Western blot analysis of endogenous and transfected FBRSL1 isoforms. a Scheme representation of FBRSL1 isoforms detectable with an N-terminal antibody. The scheme was created using a domain architecture software (https://prosite.expasy.org/mydomains). Isoform 1, consisting of the AUTS2 domain, has a predicted molecular weight of 110 kDa, while isoform 3.1 has a predicted molecular weight of 66 kDa and isoform 3.2 of 55 kDa. Isoforms 3.1. and 3.2 lack the AUTS2 domain and contain a Ftsk DNA translocase domain with an unknown function. b Plasmids containing isoforms 1, 3.1 and 3.2 in fusion to an HA-taq were detected with either an HA-antibody (HA) or with the N-terminal FBRSL1 antibody (N-terminal) in comparison to the endogenous FBRSL1 expression of HEK293 cells. The approximately estimated sizes of 110 kDa, 66 kDa and 55 kDa of the three different isoforms were detected

Fig. 4

Immunofluorescence analysis performed on HEK293 cells (a) and human fibroblasts (b). The N-terminal antibody detected isoforms 1, 3.1 and 3.2 in the cytoplasm and nucleus. Interestingly, an association with centrosomes (white arrow) and kinetochores was detected. Staining with a C-terminal antibody, detecting the full-length isoform 1 and additional hypothetic short C-terminal isoforms, showed a mainly nuclear pattern without a co-localization with the mitotic spindle, centrosomes or kinetochores. α-Tubulin was used for cytoskeletal staining and nuclei were stained using DAPI. Images were obtained using a confocal laser microscope with × 600 magnification, and an additional software magnification as indicated in the respectively images

Fig. 4

Immunofluorescence analysis performed on HEK293 cells (a) and human fibroblasts (b). The N-terminal antibody detected isoforms 1, 3.1 and 3.2 in the cytoplasm and nucleus. Interestingly, an association with centrosomes (white arrow) and kinetochores was detected. Staining with a C-terminal antibody, detecting the full-length isoform 1 and additional hypothetic short C-terminal isoforms, showed a mainly nuclear pattern without a co-localization with the mitotic spindle, centrosomes or kinetochores. α-Tubulin was used for cytoskeletal staining and nuclei were stained using DAPI. Images were obtained using a confocal laser microscope with × 600 magnification, and an additional software magnification as indicated in the respectively images

Fig. 5

Fbrsl1 loss-of-function causes craniofacial defects in Xenopus laevis development. a Scheme of the 5′ region of Xenopus laevis fbrsl1 with indicated exons, introns and the fbrsl1 E1/I1 splice-blocking Morpholino target side and possible outcome (mRNA with intron 1 inclusion) after splicing. Xenopus laevis fbrsl1 consists of 19 exons. The Morpholino sequence is given in the red dashed square. Arrows under exon 1 and exon 7 indicate the locations of the forward and reverse primer used for RT-PCR. b RT-PCR analysis of temporal fbrsl1 expression in the oocyte and different developmental stages of Xenopus embryos. RT-PCR analysis of histone H4 serves as loading control. Ma: Marker, O: Oocyte. c Stage 40 wild-type and control Morpholino (10 ng) injected embryos developed normal craniofacial structures and eyes. Injection of 10 ng fbrsl1 MO results in severe craniofacial defects and a reduction of the eye on the fbrsl1 MO injected side (marked by *). d Anti-Collagen Type II immunofluorescence staining of stage 44 Xenopus embryos showing cartilage defects in embryos injected with fbrsl1 MO but not in wild-type or Co MO injected embryos. M: Meckel’s, Q: quadrate, C: ceratohyal, BH: basihyal, BA: branchial arches. Scale bar represents 500 µm. e Anti-Ncam immunofluorescence staining of stage 40 Xenopus embryos shows normal brain development in control embryos, but reduced Ncam expression in embryos injected with fbrsl1 MO. f, g, h The graphs summarize craniofacial, cartilage and brain defects of at least three independent experiments; number of embryos (n, above each bar) and standard errors of the mean are given. ***p < 0.001 in a Student’s t-test and a one-way ANOVA test

Fig. 6

Neuronal migration is disturbed in fbrsl1 depleted embryos. a Anti-Ncam immunofluorescence staining of stage 40 Xenopus laevis embryos indicate normal neuronal migration of cranial nerves and motor neurons in wild-type and 10 ng Co MO injected embryos, but disturbed neuronal migration in embryos injected with 10 ng fbrsl1 MO (arrow). b The graph summarizes three independent experiments, number of embryos (n, above each bar)) and standard errors of the mean are given. ***p < 0.001 in a Student’s t-test and a one-way ANOVA test

Fig. 7

The short human N-terminal FBRSL1 isoform 3.1 can rescue craniofacial malformations induced by Fbrsl1 depletion in Xenopus laevis. a Injection of 10 ng fbrsl1 MO caused a reduction of craniofacial structures and the eye, while wild-type and 10 ng Co MO injected embryos developed normally. b–e Co-injection of the human FBRSL1 isoform 3.1 significantly rescues craniofacial malformations. In contrast, co-injection of the human FBRSL1 isoform 1 or the mutated human isoform 3.1-p.Gln163* with 10 ng fbrsl1 MO does not rescue craniofacial malformations caused by Fbrsl1 depletion. Embryos injected with 300 pg of the human FBRSL1 isoforms are shown in (b). c–e Graphs summarizing the percentage of craniofacial defects of at least three independent experiments after co-injection of increasing concentrations (100, 200 and 300 pg) of the indicated human FBRSL1 isoforms with fbrsl1 MO. ± s.e.m. and numbers of embryos are indicated (n, above each bar). Scale bar: 500 µm. ***p < 0.001 in a Student’s t-test and one-way ANOVA with Dunnett’s multiple comparisons test