GDF6 Knockdown in a Family with Multiple Synostosis Syndrome and Speech Impairment

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 3′ 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 development of the joints of the carpals and tarsals, the pisiform, ears, larynx, mouth and face and an overlapping postnatal role in suppression of aberrant ossification and synostosis of the joints of the inner ear (otosclerosis), larynx and vertebrae. RNAseq gene expression analysis indicated >10 fold knockdown of NOMO3, RBMXL1 and NEIL2 in both primary fibroblast cultures and fresh white blood cells. Together these results provide greater insight into the role of GDF6 in skeletal joint development.

Transcriptional Interference Regulates the Evolutionary Development of Speech

“…GDF6 is known to have a distinct dose dependent inhibitory effect on ossification and mineralization in later-stage, differentiated chondrocytes and osteoblasts in vitro [28,29]. This dose-dependent ossification effect of GDF6 was also evident in vivo in the affected family [13] and in GDF6 knockout mice both of which displayed ossification and bony fusion of carpal and tarsal joints [10,13]. Notwithstanding, a broader ossification phenotype was evident within the affected family compared to the GDF6 knockout mice [13].…”

“…Together these findings indicated retarded and retrogressive postnatal positioning of pharyngeal and laryngeal elements in the affected family together with infantile tongue, mouth and mandible [12,13]. [13]: Filled symbols denote all those members of the family with evidence of SYNS4 and all of those affected that were tested had disruption of TOSPEAK/C8ORF37AS1 [12]. All but one of the affected family members were speech impaired and all of those affected that were tested had malformation of the larynx (Figure 4B,D).…”

“…We report the affected family harboured a breakpoint in a primate-specific gene TOSPEAK that blocked its transcription across long-range enhancers for the neighbouring GDF6 bone morphogenetic protein gene which regulate the transcription of GDF6 in the developing lar- Comparative rtPCR analysis of GDF6 expression in the speech impaired family. (A) GDF6 expression levels in fresh white blood cells expressed as the mean percentage change for two affected male members of the family compared with the mean for five age and gender matched unaffected controls independently normalised against the expression of two housekeeping reference genes GAPDH and 18sRNA [13]. (B) Transient Transcriptional Interference of the SMALLTALK-TOSPEAK-GDF6 overlapping speech gene complex: siRNA mediated knockdown of SMALLTALK concordant with siRNA mediated transient transcriptional interference of SMALLTALK.…”

“…In some affected family members, there was also restricted apposition of the thumbs that restricted grasping and writing. A discrete fusion was identified between the lunate and triquetrum in the carpals of some affected family members (Figure 5) and between select tarsals [13]. Unaffected male from the family of matching age with a (B) Severely speech impaired male member of the family (III-6) with malformation of the supra-laryngeal vocal apparatus including superior (infantile/retrograde) positioning of the tongue (red line), hyoid and laryngeal cartilages-relative to the spinal axis (white dotted lines) [3][4][5][6] and increased length of the mandible (blue line).…”

“…No other GDF6 enhancers were identified in or near the region of the genome spanned by TOSPEAK in primates (Table 2) and no GDF6 enhancers were either disrupted or relocated by the chromosomal breakpoint in the affected family. Notwithstanding, TOSPEAK transcription across the GDF6 enhancers had been blocked by the breakpoint in the affected family (Figure 6) and GDF6 expression was reduced (Figure 9A) [12,13]. fusion of the distal limb joints of the carpals and tarsals (DJE1 and DJE2-Figure 8ii,iii and Figure 5)) [14].…”