G C A T T A C G G C A T genes Article GDF6 Knockdown in a Family with Multiple Synostosis Syndrome and Speech Impairment Raymond A. Clarke 1,*, Zhiming Fang 1, Dedee Murrell 2, Tabrez Sheriff 2 and Valsamma Eapen 1 1 Ingham Institute, School of Psychiatry, University of NSW, Sydney, NSW 2170, Australia; [email protected] (Z.F.); [email protected] (V.E.) 2 School of Medicine, St George Hospital, University of NSW, Sydney, NSW 2217, Australia; [email protected] (D.M.); [email protected] (T.S.) * Correspondence: [email protected] Abstract: Multiple synostoses syndrome type 4 (SYNS4; MIM 617898) is an autosomal dominant disorder characterized by carpal-tarsal coalition and otosclerosis-associated hearing loss. SYSN4 has been associated with GDF6 gain-of-function mutations. Here we report a five-generation SYNS4 family with a reduction in GDF6 expression resulting from a chromosomal breakpoint 30 of GDF6.A 30-year medical history of the family indicated bilateral carpal-tarsal coalition in ~50% of affected family members and acquired otosclerosis-associated hearing loss in females only, whereas vertebral fusion was present in all affected family members, most of whom were speech impaired. All vertebral fusions were acquired postnatally in progressive fashion from a very early age. Thinning across the 2nd cervical vertebral interspace (C2-3) in the proband during infancy progressed to block fusion across C2-7 and T3-7 later in life. Carpal-tarsal coalition and pisiform expansion were bilaterally symmetrical within, but varied greatly between, affected family members. This is the first report of SYNS4 in a family with reduced GDF6 expression indicating a prenatal role for GDF6 in regulating Citation: Clarke, R.A.; Fang, Z.; Murrell, D.; Sheriff, T.; Eapen, V. development of the joints of the carpals and tarsals, the pisiform, ears, larynx, mouth and face and GDF6 Knockdown in a Family with an overlapping postnatal role in suppression of aberrant ossification and synostosis of the joints Multiple Synostosis Syndrome and of the inner ear (otosclerosis), larynx and vertebrae. RNAseq gene expression analysis indicated Speech Impairment. Genes 2021, 12, >10 fold knockdown of NOMO3, RBMXL1 and NEIL2 in both primary fibroblast cultures and fresh 1354. https://doi.org/10.3390/ white blood cells. Together these results provide greater insight into the role of GDF6 in skeletal joint genes12091354 development. Academic Editors: Michele Cioffi and Keywords: multiple synostosis syndrome; vertebral fusion; GDF6; SYNS4; Klippel-Feil; progressive Maria Teresa Vietri ossification; pisiform; skeletal morphology Received: 22 July 2021 Accepted: 26 August 2021 Published: 29 August 2021 1. Introduction Publisher’s Note: MDPI stays neutral Bone morphogenetic proteins (BMPs) regulate skeletal development, bone morphol- with regard to jurisdictional claims in ogy and joint formation. Joints form when skeletal elements segment and develop articu- published maps and institutional affil- lations that provide flexibility, strength and versatility through specialised functions and iations. appendages [1]. Much of what we now know regarding the molecular basis of skeletal joint development derives from our understanding of genetic changes affecting BMP13 and BMP14, more commonly referred to as growth and differentiation factor 6 (GDF6) and 5 (GDF5), respectively [1–5]. GDF6 joint phenotypes overlap GDF5 joint phenotypes, including carpal, tarsal and Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. vertebral fusion [6–8]. GDF5 gain-of-function mutations increase the downstream sig- This article is an open access article nalling of GDF5 causing proximal symphalangism (SYM) and multiple synostoses syn- distributed under the terms and drome type 2 (SYNS2; MIM 610017) which is characterised by the fusion of the carpals, conditions of the Creative Commons tarsals and vertebrae [6–8]. By comparison, GDF5 loss-of-function mutations cause brachy- Attribution (CC BY) license (https:// dactyly (BDA and BDC) [8,9]. GDF6 gain-of-function mutations cause multiple synostoses creativecommons.org/licenses/by/ syndrome type 4 (SYNS4) [3–5] which is characterised by synostoses of the carpals and 4.0/). tarsals and otosclerosis-associated conductive hearing loss but not vertebral fusion [3–5]. Genes 2021, 12, 1354. https://doi.org/10.3390/genes12091354 https://www.mdpi.com/journal/genes Genes 2021, 12, x FOR PEER REVIEW 2 of 12 Genes 2021, 12, x FOR PEER REVIEW 2 of 12 Genes 2021, 12, x FOR PEER REVIEW 2 of 12 tarsals and otosclerosis-associated conductive hearing loss but not vertebral fusion [3–5]. 46 Genes 2021, 12, 1354 2 of 12 tarsals and otosclerosis-associated conductive hearing loss but not vertebral fusion [3–5]. 46 tarsals and otosclerosis-associated conductive hearing loss but not vertebral fusion [3–5]Leber. 46 congenital amaurosis-17 (LCA17, MIM 615360) and isolated cases of microphthal- 47 Leber congenital amaurosis-17 (LCA17, MIM 615360) and isolated cases of microphthal- 47 Leber congenital amaurosis-17 (LCA17, MIM 615360) and isolated cases of microphthal-mia (MCOP4,47 MIM 613094) have also been reported in association with GDF6 loss-of- 48 mia (MCOP4, MIM 613094) have also been reported in association with GDF6 loss-of- 48 mia (MCOP4, MIM 613094) have also been reported in association with GDF6 loss-of-function 48 mutations [10,11]. In contrast, increased levels of GDF6 expression, which derive 49 Leberfunction congenital mutations amaurosis-17 [10,11]. In (LCA17, contrast, MIM increased 615360) levels and isolatedof GDF6cases expression, of microphthalmia which derive 49 function mutations [10,11]. In contrast, increased levels of GDF6 expression, which derivefrom 49 the recurrent duplication of the GDF6 gene in Leri’s pleonosteosis are characterised 50 (MCOP4,from the MIM recurrent 613094) duplication have also of been the reportedGDF6 gene in associationin Leri’s pleonosteosis with GDF6 loss-of-functionare characterised 50 from the recurrent duplication of the GDF6 gene in Leri’s pleonosteosis are characterisedby vertebral 50 fusion, reduced joint flexibility and mobility and flexion contractures of the 51 mutationsby vertebral [10 ,fusion,11]. In reduced contrast, joint increased flexibility levels and of mobilityGDF6 expression, and flexion which contractures derive from of the 51 by vertebral fusion, reduced joint flexibility and mobility and flexion contractures of theinterphalangeal 51 joints [12]. 52 theinterphalangeal recurrent duplication joints [12] of. the GDF6 gene in Leri’s pleonosteosis are characterised by 52 interphalangeal joints [12]. 52In this study we report a SYNS4 family with classical carpal-tarsal coalition and ac- 53 vertebralIn this fusion, study reduced we report joint a SYNS4 flexibility family and with mobility classical and carpal flexion-tarsal contractures coalition ofand the ac- 53 In this study we report a SYNS4 family with classical carpal-tarsal coalition and ac-quired 53 otosclerosis-related conductive hearing loss [3–5]. All affected family members 54 interphalangealquired otosclerosis joints-related [12]. conductive hearing loss [3–5]. All affected family members 54 quired otosclerosis-related conductive hearing loss [3–5]. All affected family memberspresented 54 with variable degrees of vertebral fusion and most were speech impaired. 55 presentedIn this with study variable we report degrees a SYNS4 of vertebral family fusion with classicaland most carpal-tarsal were speech coalition impaired. and 55 presented with variable degrees of vertebral fusion and most were speech impaired. 55 acquired otosclerosis-related conductive hearing loss [3–5]. All affected family members2. Materials and Methods 56 presented2. Materials with and variable Methods degrees of vertebral fusion and most were speech impaired. 56 2. Materials and Methods 56Clinical Investigation: Radiology of the wrists, feet and spine were performed using 57 Clinical Investigation: Radiology of the wrists, feet and spine were performed using 57 2. MaterialsClinical Investigation: and Methods Radiology of the wrists, feet and spine were performed usingroutine 57 practices [13]. We conducted a long-term review of radiographs and reports com- 58 routine practices [13]. We conducted a long-term review of radiographs and reports com-piled over 58 30 years. We performed a detailed review of radiographs of the hands, feet and 59 routineClinical practices Investigation: [13]. We conducted Radiology a long of the-term wrists, review feet of and radiographs spine were and performed reports com- using 58 piled over 30 years. We performed a detailed review of radiographs of the hands, feet andspine from59 four affected family members IV-5, IV-9, IV-10 and IV-12 (Figure 1 and Table 60 piledroutine over practices 30 years. [We13]. performed We conducted a detailed a long-term review of review radiographs of radiographs of the hands, and feet reports and 59 spine from four affected family members IV-5, IV-9, IV-10 and IV-12 (Figure 1 and Table1). Other 60 members of this family have been described previously with what was misdiag- 61 spinecompiled from overfour affected 30 years. family We performed members IV a detailed-5, IV-9, IV review-10 and of IV radiographs-12 (Figure of1 and the hands,Table 60 1). Other members of this family have been described previously with what was misdiag-nosed as61 congenital vertebral fusion (Klippel-Feil anomaly; MIM 118100) [13]. Hearing 62 1).feet Other and members spine from of this four family affected