Irene Y. Y. Szeto

DNA length analysis is essential for genomic workflows including next-generation sequencing and fragmentomics-based diagnostics. Conventional approaches typically require large, expensive instrumentation and sample-destructive protocols with long processing times. Here we present a rapid, label-free approach integrating vibrational spectroscopy with deep learning to quantify DNA fragment length distributions. We demonstrate that ATR-FTIR and Raman spectroscopy capture length-dependent spectral features arising from phosphate backbone, nucleobase, and structural vibrations. Machine learning models trained on spectra acquired from purified monodisperse DNA (50–300 bp) predicted DNA length with high accuracy (R2 = 0.92–0.94), with multimodal fusion improving performance to R2 = 0.96. A convolutional neural network trained on DNA mixtures comprising molecules of different lengths also successfully recovered their fragment length profile. Transfer learning enabled adaptation to biological samples, achieving low prediction error (RMSE = 0.18–7.2%, Δμ = 12 bp). Importantly, the method requires only 4 μL of sample and 15 min of passive drying, with no consumables beyond cleaning materials, and allows full sample recovery. These results demonstrate the initial feasibility of vibrational spectroscopy as a rapid, label-free, and nondestructive approach for estimating DNA fragment length distributions.

Haploinsufficiency for SOX9, the master chondrogenesis transcription factor, can underlie campomelic dysplasia (CD), an autosomal dominant skeletal malformation syndrome, because heterozygousSox9null mice recapitulate the bent limb (campomelia) and some other phenotypes associated with CD. However, in vitro cell assays suggest haploinsufficiency may not apply for certain mutations, notably those that truncate the protein, but in these cases in vivo evidence is lacking and underlying mechanisms are unknown. Here, using conditional mouse mutants, we compared the impact of a heterozygousSox9null mutation (Sox9^+/−) with theSox9^+/Y440XCD mutation that truncates the C-terminal transactivation domain but spares the DNA-binding domain. While someSox9^+/Y440Xmice survived, allSox9^+/−mice died perinatally. However, the skeletal defects were more severe and IHH signaling in developing limb cartilage was significantly enhanced inSox9^+/Y440Xcompared withSox9^+/−. ActivatingSox9^Y440Xspecifically in the chondrocyte–osteoblast lineage caused milder campomelia, and revealed cell- and noncell autonomous mechanisms acting on chondrocyte differentiation and osteogenesis in the perichondrium. Transcriptome analyses of developingSox9^+/Y440Xlimbs revealed dysregulated expression of genes for the extracellular matrix, as well as changes consistent with aberrant WNT and HH signaling. SOX9^Y440Xfailed to interact with β-catenin and was unable to suppress transactivation ofIhhin cell-based assays.We propose enhanced HH signaling in the adjacent perichondrium induces asymmetrically localized excessive perichondrial osteogenesis resulting in campomelia. Our study implicates combined haploinsufficiency/hypomorphic and dominant-negative actions of SOX9^Y440X, cell-autonomous and noncell autonomous mechanisms, and dysregulated WNT and HH signaling, as the cause of human campomelia.

The in vivo mechanisms underlying dominant syndromes caused by mutations in SRY-Box Transcription Factor 9 ( SOX9 ) and SOX10 ( SOXE ) transcription factors, when they either are expressed alone or are coexpressed, are ill-defined. We created a mouse model for the campomelic dysplasia SOX9 ^Y440X mutation, which truncates the transactivation domain but leaves DNA binding and dimerization intact. Here, we find that SOX9 ^Y440X causes deafness via distinct mechanisms in the endolymphatic sac (ES)/duct and cochlea. By contrast, conditional heterozygous Sox9 -null mice are normal. During the ES development of Sox9 ^Y440X/+ heterozygotes, Sox10 and genes important for ionic homeostasis are down-regulated, and there is developmental persistence of progenitors, resulting in fewer mature cells. Sox10 heterozygous null mutants also display persistence of ES/duct progenitors. By contrast, SOX10 retains its expression in the early Sox9 ^Y440X/+ mutant cochlea. Later, in the postnatal stria vascularis, dominant interference by SOX9 ^Y440X is implicated in impairing the normal cooperation of SOX9 and SOX10 in repressing the expression of the water channel Aquaporin 3, thereby contributing to endolymphatic hydrops. Our study shows that for a functioning endolymphatic system in the inner ear, SOX9 regulates Sox10 , and depending on the cell type and target gene, it works either independently of or cooperatively with SOX10. SOX9 ^Y440X can interfere with the activity of both SOXE factors, exerting effects that can be classified as haploinsufficient/hypomorphic or dominant negative depending on the cell/gene context. This model of disruption of transcription factor partnerships may be applicable to congenital deafness, which affects ∼0.3% of newborns, and other syndromic disorders.