Long-read whole genome sequencing (lrWGS) has the potential to address the technical limitations of exome sequencing in ways not possible by short-read WGS. However, its utility in autosomal recessive Mendelian diseases is largely unknown.In a cohort of 34 families in which the suspected autosomal recessive diseases remained undiagnosed by exome sequencing, lrWGS was performed on the Pacific Bioscience Sequel IIe platform.TYMS as a novel candidate gene for lethal neonatal lactic acidosis, (2) a homozygous non-coding SV that we propose impacts STK25 expression and causes a novel neurodevelopmental disorder, (3) a compound heterozygous SV in RP1L1 with complex inheritance pattern in a family with inherited retinal disease, (4) homozygous deep intronic variants in LEMD2 and SNAP91 as novel candidate genes for neurodevelopmental disorders in two families, and (5) a promoter SNV in SLC4A4 causing non-syndromic band keratopathy. Surprisingly, we also encountered causal variants that could have been identified by short-read exome sequencing in 7 families. The latter highlight scenarios that are especially challenging at the interpretation level.Likely causal variants were identified in 13 (38%) of the cohort. These include (1) a homozygous splicing SV in Our data highlight the continued need to address the interpretation challenges in parallel with efforts to improve the sequencing technology itself. We propose a path forward for the implementation of lrWGS sequencing in the setting of autosomal recessive diseases in a way that maximizes its utility.The online version contains supplementary material available at 10.1186/s13073-023-01270-8. Mendelian diseases are defined by high-impact variants in single genes that are typically sufficient to cause the phenotype. The identification of these variants, therefore, represents an important diagnostic step that enables such important clinical actions as accurate counseling, reproductive planning, prognostication, and treatment. Variant identification, however, was a daunting task until the recent advent of next-generation sequencing, which allowed for exome-wide or genome-wide interrogation of candidate variants even when the correct clinical diagnosis  is lacking . The aveThe focus of exome sequencing on the coding part of the genome prompted many to investigate the diagnostic contribution of the non-coding genome using whole genome sequencing –5. SurprPKD1 locus ). TYMS (MIM 188350) encodes thymidylate synthase, a mitochondrial protein involved in the de novo and salvage dTTP pathways, deficiency of which leads to uracil misincorporation and mitochondrial dysfunction [TYMS expression in patient cells compared to controls  in family F6404:2- Deletion of ANO7. The deletion spans a strong peak for H3K27Ac enhancer mark, enriched for TF binding, and is DNAse hypersensitive. This deletion is within the region of 2q37 microdeletion syndrome characterized by microcephaly and intellectual disability, where STK25 was proposed to be a major contributor [STK25 (MIM 602255) is a germinal center kinase III (GCK III) that plays a role in serine-threonine liver kinase B1 (LKB1) signaling pathway. Its established role in neuronal and brain cortical development stems from its function in regulating neuronal polarization and morphology of the Golgi apparatus [STK25 expression given its close proximity . RT-qPCR data indeed confirmed a dramatic reduction of STK25 expression in both siblings compared to controls  shared an apparently novel syndromic association of intellectual disability, microcephaly, and hearing loss . One SV was inherited from the mother who is homozygote for this variant , while the other  was inherited from the healthy heterozygous father (Table F3981 consists of a mother 14DG0261) with late-onset retinitis pigmentosa and two daughters (17DG1097 and 14DG0524) with Leber congenital amaurosis (LCA). lrWGS in 17DG1097 (her sister 14DG0524 was confirmed to have the same finding) identified compound heterozygosity for two in-frame SVs in 61 with l4- A novel regulatory element variant in familial band keratopathy in family F7974:SLC4A4 (NM_001134742.2:c.-145C > T). SLC4A4 (MIM 603345) recessive variants are known to cause band keratopathy as part of a multisystem syndrome, which these two siblings lack. We hypothesized that this may represent an instance of variable expressivity caused by a specific transcriptional dysregulation [SLC4A4 expression in the two siblings compared to controls  that was associated with a significant reduction in transcript level of the gene compared to control by RT-qPCR  encodes synaptosomal-associated protein 91 which mediates endocytosis of synaptic vesicles (SVs). Snap91−/− mice have a compatible phenotype in the form of growth retardation, spasticity, altered behavior, impaired neurotransmission, epileptic seizures, and premature death [SNAP91 reduction on the number of vesicles, we performed transmission electron microscopy on patient-derived lymphoblastoid and fibroblast cell lines. Data show that the patient cells had fewer number of vesicles compared to control cells . RT-qPCR showed reduced expression of LEMD2 transcript in all four affected siblings compared to control . This canonical splicing variant was also identified on exome sequencing but was dismissed as an incidental finding because it had been reported in a family with adult-onset ataxia and retinitis pigmentosa, the only listed phenotype in OMIM [Flvcr1 is known to phenocopy Diamond-Blackfan anemia [FLVCR1 in humans. Indeed, this case will be part of a cohort of similar cases caused by severe biallelic variants in FLVCR1 . RT-PCR experiment showed that the variant causes a splicing defect (skipping of exon 9) and an early truncation of the protein ).PKHD1 variant causes lethal polycystic kidney disease in family F5543:2- A high-frequency 13DG1395 and 16DG0856 are two of three stillborn babies with microcephaly, intrauterine growth retardation, severe craniofacial dysmorphism with clefting and severe skeletal malformations in a pattern highly consistent with Diamond-Blackfan syndrome (Table  in OMIM . Howevern anemia . FurtherPKHD1 (MIM 606702). This missense variant was also identified on exome sequencing but was dismissed because of its unusually high local frequency (MAF 0.006196). However, an updated local exome database of 13,473 exomes revealed that this variant was observed in the homozygous state in 2 children who died with an identical phenotype and in the shared heterozygous state in a consanguineous couple who lost children with lethal polycystic kidney disease. Haplotype analysis confirmed that this is indeed a previously unrecognized common founder variant in the local population.3- Genetic heterogeneity of severe limb anomalies in a consanguineous family F5349:16DG0518 is a stillborn baby with severe bilateral polycystic kidney disease (Table BRIP1 (MIM 605882). This variant was also identified on exome sequencing but was ignored because it did not reside within a shared autozygous interval with the affected cousin. Indeed, segregation analysis of this pathogenic variant, which fully explains the phenotype, showed that the cousin is not homozygous, which confirms the genetic heterogeneity of the phenotype in this family. Rather, the cousin with split hand and foot malformation (SHFM) was subsequently found to have on chromosomal microarray a homozygous deletion in 12p11.23 . We propose this novel homozygous deletion as a potential cause through a position effect as has been demonstrated in other SHFM loci.STX3-related retinal dystrophy in family F5993:4- Isoform confusion in 20DG1379 is a 2.5-year child with radial ray deficiency as part of a syndromic presentation and has a similarly affected deceased brother and a cousin with severe limb malformations (Table STX3 (MIM 600876). Despite the deep intronic nature of this variant, it was also captured by exome sequencing but was ignored because in silico prediction and subsequent RT-PCR failed to show an abnormal impact on splicing. Further investigation of this variant revealed that it is exonic and truncating in one isoform that is only listed in Ensembl where the nomenclature is ). The same variant was subsequently identified in 2 unrelated patients with an identical phenotype, and haplotype analysis confirmed the founder nature of this variant  with compelling in silico predictions . NID1 is a member of the nidogen family of basement membrane glycoproteins, which play a role in cellular interactions with the extracellular matrix through interaction with several other components of basement membranes. Subsequent Sanger sequencing confirmed the homozygous status of the variant in the index child and absence in the unaffected siblings. To date, there is no phenotypic OMIM listing for NID1-related phenotype although we have previously published congenital stroke in two siblings who shared a novel homozygous splicing variant confirmed by RT-PCR [Nid1-/- mice were also noted to have abnormal motor control and this was tracked to the highly abnormal basement membrane of brain capillaries [NID1. This novel phenotypic aspect precluded the identification of this variant on the exome despite being fully captured and called.ABHD12 variant in family F7887:6- Atypical presentation in a case with 21DG0165 is a neonate who died of high throughput heart failure as a complication of vein of Galen malformation  with compelling in silico predictions . Although this variant was also called by exome sequencing, it was dismissed because of the atypical presentation compared to OMIM phenotype .7- C1orf109 as a candidate gene for a novel NDD in family F8544:20DG0198 is a 6-year-old child with developmental regression (Table C1orf109 (MIM 614799) with compelling in silico predictions . Although this variant was also called by exome sequencing, it was ignored because the analysis pipeline was erroneously set to exclude variants that are present in the homozygous state in the database. Subsequently, we identified the source of the error as a homozygous individual who shared the same phenotype. GeneMatcher submission revealed an ongoing study with a large cohort on C1orf109-related NDD (manuscript in preparation).20DG1533 and 20DG1534 are two siblings who shared a severe NDD in the form of progressive microcephaly, global developmental delay, and brain atrophy (Table CHMP5 (MIM 610900)  were available for testing  and the phenotypic challenge associated with NID1 as a novel candidate cause of autosomal recessive vein of Galen malformation.As expected, lrWGS demonstrated a clear advantage in detecting SVs, an important class of variants that remain challenging for short-read sequencing even with improved bioinformatic handling of the data. Nearly half of the identified candidate variants were SVs and SNVs that were missed by exome. It should be noted that the preselection of autosomal recessive phenotypes in our highly consanguineous cohort made it less likely for other classes of challenging variants to be identified, e.g., repeat expansion and chromosomal rearrangements, since these tend to be dominant in nature. What is surprising, however, is that the other half of the candidate variants we identified by lrWGS were similarly detected by exome sequencing and yet were not highlighted as likely candidates. This is despite the fact that all exome files were carefully reanalyzed for a long list of challenges that we identified based on a previously published study involving the systemic analysis of such challenges . For exaThere are limitations in this study. Despite being the largest to date on autosomal recessive phenotypes, we note the need for much larger cohorts. In order to limit the cost of the study, we resorted to a limited average depth of 10 × . Thus, it is possible that families that remained negative may benefit from a higher depth of sequencing. Indeed, as shown in Family F6440 that mapped to a single locus, low-depth lrWGS failed to identify the likely causal variant within this locus, which was a novel large insertion that may have been identified by higher-depth sequencing. Similarly, the SHFM duplication on chr10q24.3 was missed in F8280. Although our study specifically targeted phenotypes that are likely to be autosomal recessive in etiology, which may limit the generalizability of the findings, this can also be viewed as an advantage because it allowed us to showcase the added value of positional mapping when interpreting lrWGS just as we have shown for srWGS . FinallyOur data clearly demonstrate the important role of non-coding DNA as well as the continued need to address the interpretation challenge in parallel with efforts to address the detection challenge by improving the sequencing technology itself. We show a number of novel gene-disease relations (novel candidate genes and novel phenotypes of established genes) that await confirmation by future cohorts. We propose a path forward for the implementation of lrWGS in the setting of autosomal recessive diseases in a way that maximizes its utility by exploiting the power of positional mapping.Additional file 1: Fig. S1. Functional validation of TYMS, SNAP91, and SLC4A4 variants. Fig. S2. Comparing variant detection using different depths on the Pacific Bioscience Sequel IIe platform. Fig. S3. PacBio data describing variants highlighted in this study. Table S1. Sequences of primers used for the cloning-free reporter assay. Table S2. Coverage statistics for PacBio-sequenced samples. Table S3. List of variants identified within ROHs which were ultimately excluded. Table S4. Detailed listing of the study cohort. Table S5. List of transcription factors predicted to bind to the deleted region in F6404 with JASPER confidence scores.


ENT1−/−), a preclinical model of DISH, and behavioral indicators of pain.Diffuse idiopathic skeletal hyperostosis (DISH) is characterized by progressive calcification of spinal tissues; however, the impact of calcification on pain and function is poorly understood. This study examined the association between progressive ectopic spine calcification in mice lacking equilibrative nucleoside transporter 1 , microglia (IBA1), and nociceptive innervation (CGRP).A longitudinal study design was used to assess radiating pain, axial discomfort, and physical function in wild-type and ENT1−/− mice was associated with reductions in flexmaze exploration, vertical activity in an open field, and self-supporting behavior in tail suspension, suggesting flexion-induced discomfort or stiffness. Grip force during the axial stretch was also reduced in ENT1−/− mice at 6 months of age. Increased CGRP immunoreactivity was detected in the spinal cords of female and male ENT1−/− mice compared to wild-type. GFAP- and IBA1-immunoreactivity were increased in female ENT1−/− mice compared to wild-type, suggesting an increase in nociceptive innervation.Increased spine calcification in ENT1−/− mice experience axial discomfort and/or stiffness and importantly that these features are detected during the early stages of spine calcification.These data suggest that  Diffuse idiopathic skeletal hyperostosis (DISH) is a non-inflammatory spondyloarthropathy characterized by the formation of ectopic mineral along the spine and within the annulus fibrosus of the intervertebral disc (IVD) , 2. DISHOverall, the clinical features of DISH are poorly understood. Multiple co-morbidities have been associated with DISH including obesity, dyslipidemia, hypertension, and type 2 diabetes , 7 and rENT1−/−) develop ectopic calcification of the paraspinal tissues with remarkable resemblance to DISH . W. W32]. WENT1−/− mice at all time points  since in ENT1−/− mice the cervical region is the most severely affected by ectopic calcifications, which have been present for several months. Pain can be modulated by the neurotransmitter CGRP and increased expression is associated with nerve hyperexcitability and sensitization [ENT1−/− mice relative to wild-type  by ENT1−/− mice in open field suggests physical dysfunction and/or avoidance of discomfort-inducing activities. These behavioral measures are supported by molecular evidence of neuroplastic changes in ENT1−/− mice. Increased levels of CGRP in the cervical spinal cord of ENT1−/− mice confirm neuroplasticity in primary nociceptive neurons. Furthermore, elevated levels of GFAP and IBA1 detected in female ENT1−/− mice suggest alterations in the sensory nervous system. These changes are consistent with mouse models of IVD degeneration that show axial pain [ENT1−/− mice.The similarity between the performance of wild-type and ial pain  and suggENT1−/− mice despite similar levels of ectopic spine calcification. These findings are consistent with previous studies in rodent models and humans establishing sex-specific differences in pain perception [ENT1−/− mice showed significant differences in the grip force, tail suspension, and flexmaze assays at earlier stages of spine calcification than did male ENT1−/− mice. Since ectopic calcification was not different between female and male mice at any time point, these findings suggest that female ENT1−/− mice are more susceptible to discomfort or pain. While it is unclear what mechanism underlies the sex-specific differences in behavioral signs of pain, estrogen-modulated cyclooxygenase (COX)-2 activation has been proposed as a potential mediator [ENT1−/− mice in subsequent studies.In the current study, behavioral and neural changes were more robust in female than male rception . Female mediator  and coulENT1−/− mice, indicative of stiffness and/or discomfort, increased as spine calcification progressed. This suggests that the presence of small or focal ectopic calcifications may be insufficient to produce definitive symptoms. Instead, ectopic calcification may have to reach a certain extent and/or affect specific anatomy before symptoms can be detected in ENT1−/− mice. A similar threshold effect may also underlie the variability in pain reported in humans with DISH and underscores the importance of treatment strategies aimed at delaying the progression of ectopic calcification and maintaining physical function. In fact, a small study of exercise therapy to improve spinal range of motion for people living with DISH reported improvements in physical measures and reductions in self-reported pain [A strength of the current study is the longitudinal evaluation of behavioral indicators of physical function and discomfort with the initiation of spine calcification. Overall, significant differences between wild-type and ted pain .ENT1−/− mouse model differs from DISH in that the appendicular skeleton is not affected by ectopic calcification in the mouse. It is likely that calcification within appendicular joints would affect physical function and cause pain/discomfort similar to the spinal changes in ENT1−/− mice, leading to a different pain profile in people with DISH. Finally, further studies are required to differentiate symptoms of pain and physical dysfunction in ENT1−/− mice, for example by pharmacological targeting of inflammatory and nociceptive pain pathways at specific stages of spine calcification or by specifically investigating neuroplastic changes in mice at earlier time points associated with the onset of spine calcification and behavioral changes.There are several limitations that should be acknowledged in the interpretation of our findings. First, the assessment of pain in animal models using behavioral assays requires careful experimental design as assays are sensitive to different types of pain and no single assay conclusively demonstrates that an animal is experiencing pain. To address this limitation, we included multiple complementary assays that together inform on behavioral indicators of pain, as well as objective measurement of neuroplastic changes within the central nervous system. Second, the ENT1−/− mice. Importantly, behavioral changes were noted early in disease pathogenesis and increased as the severity of ectopic spine calcification progressed. These findings underscore that pain should be evaluated in people living with DISH throughout the progression of spine calcification, with emphasis on early-stage disease. To date, the most comprehensive case–control study concluded that chronic back pain, specifically in the cervical and thoracic regions of the spine, and upper extremity pain are significant clinical features of DISH [Taken together, our results provide the first evidence that impaired physical function, axial stiffness and/or discomfort are features associated with spine calcification in  of DISH . Althoug


Throughout mitosis, a plethora of processes must be efficiently concerted to ensure cell proliferation and tissue functionality. The mitotic spindle does not only mediate chromosome segregation, but also defines the axis of cellular division, thus determining tissue morphology. Functional spindle orientation relies on precise actin dynamics, shaped in mitosis by the LIMK1-Cofilin axis. The kinase Haspin acts as a guardian of faithful chromosome segregation that ensures amphitelic chromosome attachment and prevents unscheduled cohesin cleavage. Here, we report an unprecedented role for Haspin in the determination of spindle orientation in mitosis. We show that, during mitosis, Haspin regulates Rho-ROCK activity through ARHGAP11A, a poorly characterized GAP, and that ROCK is in turn responsible for the mitotic activation of LIMK1 and stabilization of the actin cytoskeleton, thus supporting a functional spindle orientation. By exploiting 3D cell cultures, we show that this pathway is pivotal for the establishment of a morphologically functional tissue.  •Rho regulates mitotic spindle orientation through the LIMK1/Cofilin axis•ARHGAP11A limits Rho activity in M-phase•Haspin is required to prevent inactivation of Rho by ARHGAP11A•Loss of Haspin causes dysfunctional spindle orientation and tissue morphogenesis Molecular biology; Cell biology This is ensured thanks to an intricate integration between the mitotic spindle, responsible for mechanically pulling sister chromatids apart, and several checkpoints that delay anaphase onset until mandatory requirements are achieved. For example, centromeric cohesin must be protected from premature cleavage to sustain chromosome biorientation, and anaphase onset must be delayed until all chromosomes are correctly positioned on the metaphase plate. Both these processes rely on the activity of the evolutionarily conserved atypical protein kinase Haspin, which is emerging as a central factor in several types of cancers.,,,,,,,,,,,,,,,,,,,,,,,,,Sustaining tissue homeostasis and functionality requires proficient chromosome segregation and the proper orientation of cell division. Remarkably, both functions are based on a functional mitotic spindle, as the orientation of the mitotic apparatus defines the future site of cleavage and cell division pattern.,,,In previous works, we showed that budding yeast Haspin paralogues, Alk1 and Alk2,,A functional orientation of the mitotic spindle is a strict requirement for tissue organization, as it is instrumental to properly establish asymmetric versus symmetric cell divisions and to position the future site of cleavage.α/β-Tubulin) or centrioles (γ-Tubulin). The percentage of cells exhibiting non-planar spindles was measured . As shown in To investigate whether a similar function is present also in mammalian cells, we seeded HeLa cells on poly-Lysine (a substrate that does not direct a specific spindle orientation), fibronectin or collagen (substrates known to cause a planar orientation of the mitotic spindles),As mentioned, the orientation of the mitotic spindle is a function of actin cytoskeleton organization.,,,,,,,We then committed to the identification of the molecular pathway through which Haspin affects the reorganization of the actin cytoskeleton underlying mitotic round-up, which is under the control of the LIMK1-cofilin axis.We then analyzed the impact of Haspin loss on Cofilin in cells arrested in mitosis. As shown in ,,,,Considering the reported role for LIMK1 in Cofilin-Ser3 phosphorylation,Two main mechanisms oversee and cooperate in the regulation of LIMK1 activity, one orchestrated by Cdc42 and its effector PAK, and one relying on Rho and its effector ROCK. In yeast, we have shown that Haspin is required for the rerouting of Ras-GTP loaded vesicles from a polarized to an isotropic delivery,So far, our results position Haspin in the Rho-ROCK-dependent regulation of the LIMK1-Cofilin axis to modulate actin cytoskeleton and spindle orientation but provide no insights as to how Haspin might impact on Rho itself. The activity of GTPases is under control of different classes of proteins that have either activating (GEFs) or inhibiting (GAPs and GDIs) roles; a convenient mechanism by which such regulators are triggered or inactivated is through phosphorylation. We thus speculated that Haspin might impinge on Rho-GTP/Rho-GDP cycle modulation by phosphorylating one of its GEFs, GAPs or GDIs. Interestingly, Haspin regulates the phosphorylation of ARHGAP11A,We then assessed whether ARHGAP11A acts as a negative regulator of the Rho-ROCK pathway in mitotic cells. We depleted ARHGAP11A by RNA interference, synchronized cells in prometaphase by nocodazole treatment and monitored Cofilin-Ser3p levels. As shown in Having proved the involvement of Haspin kinase in the control of spindle orientation, we tested the physiological relevance of this pathway. CaCo2 cells can easily be cultured in 3D to form cysts embedded in Matrigel, and, if spindle orientation is proficient, they will generate a hollow sphere with a single lumen.,,,,,,,,,The atypical kinase Haspin has been reported to be central in chromosome segregation by acting at multiple levels. First, it phosphorylates H3-Thr3,,in silico analysis, ARHGAP11A was linked to mitotic nuclear division, cellular polarity, microtubule-based movements and chromosome organization, supporting our findings.,Saccharomyces cerevisiae, the decision regarding where the cell division plane will be built is taken in G1 and it is the mitotic spindle that needs to find the proper orientation. In human cells, the axis of cell division is determined in M following the assembly and orientation of the mitotic spindle. In this context, the misorientation of the spindle upon loss of Haspin activity causes an altered cell division pattern that comes with apparently limited impact on the single cell fitness (though it might severely affect cells that must undergo asymmetric division), but has a dramatic morphological effect when it comes to tissue.In most eukaryotes, the mitotic spindle defines the axis of cell division and thus its orientation is tightly regulated to properly shape tissue morphology. A major determinant of spindle orientation is the actin cytoskeleton, which undergoes extensive reshaping throughout mitosis to allow mitotic roundup and sustain the forces necessary for cell division. Work in budding yeast has shown that Haspin promotes a redistribution of polarity factors and a reshaping of the actin cytoskeleton, ensuring a functional orientation of the mitotic spindle.These findings are particularly interesting, as they extend the range of mitotic events orchestrated by Haspin from those strictly related to sister chromatids dynamics (cohesin stabilization and amphitelic attachment) to a much wider guardian of the whole segregation process. Failures in Haspin activity thus come with a complex impact on the M-phase, leading to cells susceptible to fail in several of the tight requirements to mitosis and thus prone to genome instability (due to unscheduled cohesin cleavage and errors in the buildup of a metaphase plate) or tissue outgrowth and cell migration (due to misoriented mitotic spindles and cell divisions), being overall primed for malignant transformation . SpindleOur work described a novel axis regulating spindle orientation in epithelial cells, highlighting an unprecedented involvement of the newly described Haspin-ARHGAP11A-Rho axis in such process. Beside the conceptual advance of this discovery in the elucidation of how epithelial tissues are shaped, some aspects remain to be addressed. How is this role of the Haspin kinase regulated and how does it integrate with previously described Haspin functions? What are the spatiotemporal dynamics of ARHGAP11A-mediated regulation of Rho? Does the Haspin-ARHGAP11A axis regulate other functions of Rho? All these questions remain open and will need further experimental investigation to be addressed.marco.muzifalconi@unimi.it).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Marco Muzi-Falconi  were mixed with 400μl of Matrigel and 200μl of Collagen I. 200μl of the mix were then plated on the bottom of an imaging dish and solidified by incubation at 37°C before medium and drug addition.Mitotic cells were obtained either by synchronization at the G2/M transition by treatment with CDK1 inhibitor RO-3306 (10μM) followed by 1 hour release, or by synchronization at prometaphase by a 16 hours incubation in the presence of 100nM Nocodazole. In both cases, mitotic cells were obtained by mitotic shake off.γ-tubulin and α-tubulin; while actin staining was performed by Alexafluor 488-conjugated phalloidin. Slides were mounted with a DAPI-containing mounting, before being analyzed by widefield (Leica DMRA2) or confocal microscopy (Leica SP2 or Nikon CSU-W1). Images were analyzed with FIJI.Fluorescence microscopy was exploited to assess spindle orientation and actin organization in mitotic cells; in both cases, cells were fixed with formaldehyde and then processed as follows. For spindle orientation analyses, cells were stained for Cells were transfected 48 hours before sample collection; plasmids for overexpression were transfected using Lipofectamine 3000 following provider’s manual; siRNAs were transfected using RNAiMAX following provider’s instruction.Cells were lysed in Laemmli buffer, samples were then boiled for 10 minutes and sonicated. After SDS-PAGE and transfer to nitrocellulose membrane, filters were blocked with 5% milk in PBS TWEEN-20 0.1% (PBST) and then incubated with given primary antibody. Filters were washed 3 times in PBST, incubated 1 hour at RT with secondary HRP-conjugated antibody, washed for further 3 times. ChemidocTouch (BIORAD) was used to acquire images.Samples were prepared for flow cytometry as previously described.To evaluate cellular proliferation in 2D cultures, cells were fixed with 3.7% formaldehyde at given time points and then stained with crystal violet. After washout of the dye, cells were lysed with 1% SDS and absorbance at 595 nm measured with a Perkin Elmer Ensight. CaCo2 proliferation in cysts was performed incubating cells embedded in Matrigel with MTS assay for 2 hours and then reading absorbance at 490 nm.Statistical significancy was evaluated by T-test, significancy: n.s.: not significant; ∗ p.value <0.05; ∗∗ p.value <0.01; ∗∗∗ p.value <0.005; ∗∗∗∗ p.value <0.001. Further details regarding number of experimental repeats or number of analyzed cells and graph description, are reported in figure legends.


The treatment of metastatic pancreatic ductal adenocarcinoma (PDAC) is frequently characterized by significant toxicity and rapid development of resistance to current approved therapies. More reliable biomarkers of response are needed to guide clinical decision making.We evaluated cell-free DNA (cfDNA) using a tumor-agnostic platform and traditional biomarkers (CEA and CA19-9) levels in 12 patients treated at Johns Hopkins University on NCT02324543 “Study of Gemcitabine/Nab-Paclitaxel/Xeloda (GAX) in Combination With Cisplatin and Irinotecan in Subjects With Metastatic Pancreatic Cancer.” The pretreatment values, levels after 2 months of treatment, and change in biomarker levels with treatment were compared with clinical outcomes to determine their predictive value.KRAS and TP53 mutations in cfDNA after 2 months of treatment was predictive of progression-free survival (PFS) and overall survival (OS). In particular, patients with a lower-than-average KRAS VAF after 2 months of treatment had a substantially longer PFS than patients with higher posttreatment KRAS VAF (20.96 vs. 4.39 months). Changes in CEA and CA19-9 after 2 months of treatment were also good predictors of PFS.The variant allele frequency (VAF) of KRAS or TP53 VAF after 2 months of treatment to be better predictors of PFS and OS than CA19-9 or CEA. This pilot study requires validation but suggests cfDNA measurement is a useful adjunct to traditional protein biomarkers and imaging evaluation and could distinguish between patients who are likely to achieve prolonged responses versus those that will have early progression and may benefit from a change in treatment approach.Comparison via concordance index demonstrated We report on the association of cfDNA with response durability for patients undergoing treatment with a novel metronomic chemotherapy regimen  for metastatic PDAC. This investigation offers encouraging evidence that cfDNA may prove to be a valuable diagnostic tool to guide clinical management. Pancreatic cancer represents the fourth leading cause of cancer death in the United States with an estimated 48,220 pancreatic cancer–related deaths in 2021 which is driven in part by the highest case-fatality rate of any solid tumor malignancy . While tWhen one of these two combination regimens is no longer effective in controlling a patient's cancer, the current treatment paradigm involves transitioning the patient to whichever of these regimens was not yet utilized . HoweverWe hypothesized that combining the drug classes from the two most active first-line approaches into a five drug combination  would maximize therapeutic responses and delay the onset of therapeutic resistance and performance status deterioration. To investigate this combination, we launched a phase Ib/II trial in which 47 patients were treated with this approach including 30 patients at the stage II expansion cohort dosing. We used this trial as a platform to also explore the use of biomarker strategies to distinguish which patients are likely to achieve favorable clinical outcomes.Traditionally, protein-based biomarkers (CEA and CA19-9) have served as adjuncts to CT imaging for assessing the response of patients to treatment, an approach that has yielded limited incremental benefit in predicting patient outcomes. More recently, cell-free DNA (cfDNA) has emerged as a valuable tool to characterize and quantify tumor-specific genetic alterations shed into the bloodstream by cancer cells . This toThis study explored the utility of cfDNA to predict clinical treatment outcomes in patients with metastatic PDAC. The patients in this investigation were treated at Johns Hopkins University  on NCT02324543 “Study of Gemcitabine/Nab-Paclitaxel/Xeloda (GAX) in Combination With Cisplatin and Irinotecan in Subjects With Metastatic Pancreatic Cancer” with five different drugs active against PDAC in an effort to maximize depth and duration of response. Patients with untreated metastatic PDAC with Eastern Cooperative Oncology Group performance 0 or 1 were eligible for this trial and were excluded if they were considered candidates for surgery, or had received prior chemotherapy or radiation previously. The study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the JHU Institutional Review Board. All patients provided written informed consent prior to enrollment. From this cohort, we chose 12 patients  to perform an investigation into the ability of biomarkers to predict treatment outcomes. Demographic information is available in We obtained blood draws pretreatment and after 2 months of treatment (two cycles of GAX-CI) at the time of treatment evaluation with CT imaging. CEA and CA19-9 levels were assessed by the Johns Hopkins Pathology Department using standard laboratory practices and a Clinical Laboratory Improvement Amendments (CLIA)-approved Tosoh Bioscience Immunoassay method. cfDNA was obtained in cfDNA BCT blood collection tubes (Streck). cfDNA was stored at room temperature and isolated within 48 hours of collection using the Qiagen QIAam MinElute cfDNA Midi Kit (Qiagen). Following isolation, cfDNA was stored at −80°C prior to transfer to Tempus for use on the Tempus xF 105 gene liquid biopsy platform (Tempus). This assay is a hybrid-capture, next-generation sequencing assay that evaluates single-nucleotide variants and insertions and/or deletions at a minimum of 1,000× coverage . Reads wKRAS and TP53 alleles as well as the overall median VAF of all detected mutations in the cfDNA samples were used for analysis  and CT response.KRAS, TP53, and median tumor VAFs as continuous variables and survival outcomes. In addition, comparisons were made between patients with biomarker levels in the upper and lower half of those measured for each timepoint using log-rank tests. Kaplan–Meier curves were created for each biomarker's pretreatment, posttreatment, and change in biomarker levels for PFS and OS, respectively. Comparisons of predictive ability of cfDNA strategies and CEA or CA19-9 were made using the concordance index (c-index) for the same timepoints, or the percent changes pretreatment to posttreatment. Statistical tests were two sided, and P values equal to or less than 0.05 were considered statistically significant. Statistical analyses were performed using R version 4.1.0.Each of the respective biomarkers was correlated with clinical outcomes including PFS and OS. We performed Cox regression analysis to assess relationships between The data generated in this study are not publicly available due to patient privacy considerations but are available upon reasonable request from the corresponding author.KRAS mutations were detectable in the cfDNA of 11 of the 12 patients pretreatment and 7 of the 12 patients posttreatment, including the 1 patient without pretreatment positivity. TP53 mutations were detectable in the cfDNA of 8 of the 12 patients pretreatment, and 7 of the 12 posttreatment, including one that was negative pretreatment . There was a moderate correlation between CA19-9 and KRAS VAF at pretreatment and posttreatment and for the percent change . A similar pattern was observed for the correlation between CEA and TP53 VAF and between CA19-9 and TP53 at either timepoint and for the percent change between timepoints  and change in KRAS VAF  was compared to baseline KRAS and TP53 VAFs. This analysis revealed no significant correlation between either KRAS or TP53 VAF and baseline tumor burden . The perF KRASΔ; . There wesponses .To determine the association between biomarker measurements and duration of benefit to GAX-CI as measured by PFS and OS, patients were stratified in half between those with a biomarker level or VAF above or at median (high) versus those with below median for each timepoint. For percent change between two timepoints, patients were stratified by whether they had a percent change above or at median (high) versus below median (low). As the majority of patients experienced decreases in mutation VAFs after two cycles, the low group values were more negative denoting larger drops in mutation fractions. Survival outcomes were compared between two groups using log-rank test.While pretreatment and posttreatment levels of CEA and CA19-9 did not predict PFS and OS, the change in CEA with treatment significantly correlated with PFS but not OS while change in CA19-9 was predictive of both .KRAS VAF was evaluated at different measurements including pretreatment, posttreatment with two cycles of GAX-CI, and change from pretreatment to posttreatment (KRAS VAF as a continuous variable and compared this with PFS and OS using Cox regression modeling (KRAS VAF was associated with OS  while KRAS2 was associated with PFS   with clinical outcomes was evaluated. Pretreatment values and change in VAF between these high and low groups did not show an association with OS or PFS whereas lower TP53-2 showed significantly improved OS and PFS (TP53 VAF as a continuous variable and compared this with PFS and OS using Cox regression modeling (TP53-2 was associated with PFS .The association of  and PFS . We lookmodeling . In thisKRAS and TP53 mutations were both found in the majority of our evaluated patients, there were other mutations identified in individual patients including RB1, SMAD4, and STK11 pathogenic mutations , there was also a trend toward these patients having a longer OS.As DNA damage repair pathway mutations have been previously been associated with responses to platinum-based chemotherapy in PDAC, we assessed for the presence of these mutations in our patient population with identification of 3 patients with previously characterized pathogenic mutations in these pathways, BRCA2 V2969fs, BRCA2 Y1655*, and PMS2 E705K . These pTP53, KRAS, and median tumor VAF after two cycles of treatment were correlated with PFS while the percent change in VAF during treatment was not predictive using this analysis. The predictive ability of biomarkers was compared against each other using c-index  chance of achieving an R0 resection at the time of PDAC removal  targetable by currently clinically FDA-approved agents . However in PDAC . The usede novo but does potentially allow for improved sensitivity for detection of minimal residual disease by incorporating more of tumor-specific mutations specifically found in the patient's cancer while also helping to avoid “false positives” from clonal hematopoesis. Another potential advantage to a tumor-informed assay is it more readily facilitates the development of a multiple allele VAF approach that incorporates a panel of mutations to assess disease status. While this can also be considered with tumor-agnostic approach this was not investigated in our study given the small sample size evaluated.Of note, the Tempus xF assay used for this investigation is a tumor-agnostic assay where a prespecified panel of genes was queried in the cfDNA specimen. This allowed for the identification of mutations even when these alterations are not detectable within the tissue specimen. A tumor-agnostic approach such as the one utilized in this investigation has several distinct advantages including not requiring a prior biopsy or resection specimen for completion which can be a limitation in sometimes challenging to biopsy pancreatic lesions. In contrast to a tumor-agnostic approach, tumor-informed approaches use tissue-based sequencing to identify molecular alterations in the tumor prior to development of a personalized cfDNA assay. This has a slower initial turnaround time as this assay has to be developed TP53-2 that were not present preoperatively in either the cfDNA or tissue evaluation. This raises the possibility that these represented clonal hematopoiesis of indeterminate potential but given the uncertainty these were counted as true mutations with VAF recorded as such. These 2 patients had among the longest PFS so the approach we used may lead to an underestimation of the predictive value of TP53 VAF. In addition, as this investigation was pursued in the context of a therapeutic trial, further studies are necessary to understand the associated predictive value in the standard-of-care treatment of PDAC or other treatment settings.This investigation has several limitations that must be considered when interpreting these results. First, this pilot study has only a limited number of patients with the intentional selection of 12 patients for evaluation which had disparate clinical outcomes (6 with short PFS and 6 with long PFS in the cohort). This increased our ability to demonstrate the utility of cfDNA in this disease context but means that these results particularly the magnitude of changes might not be representative to patients across the full range of clinical outcomes. Another limitation is 2 of our patients developed detectable KRAS and TP53) after 2 months of treatment predicted PFS. This observation adds to the growing body of literature suggesting cfDNA might provide an early marker of treatment outcomes in patients with metastatic cancer.In this investigation, we evaluated the ability of cfDNA to predict treatment outcomes in metastatic PDAC. This analysis identified that absolute cfDNA levels of the two most commonly identified mutations in our cohort  and % change (panel B). These data demonstrated good agreement between different cfDNA variables while the protein based showed variable correlations with cfDNA variables as outlined in Supplemental Table 3.Click here for additional data file.Figure S2Supplemental Figure 2. Scatterplots comparing baseline RECIST tumor measurements with baseline cell-free DNA (cfDNA) biomarkers. To determine the relationship between baseline tumor mutational variant allele frequency (VAFs) and total tumor burden we compared baseline tumor burden as measured by RECIST measurements of evaluable lesions (sum of diameters in mm) with KRAS (panel A) and TP53 (panel B) VAFs for these tumors at the same time point. These data demonstrated no statistically significant relationship between baseline cfDNA VAF levels and RECIST measurements on CT scan.Click here for additional data file.Figure S3Supplemental Figure 3. Post-treatment KRAS and change in KRAS VAF following treatment. To determine whether KRAS VAF following 2 cycles of treatment or the change in KRAS VAF from pre-treatment to after 2 cycles of treatment is correlated with CT response at 2 months, each patient’s CT response to treatment was plotted against KRAS VAF and calculated Spearman’s rank correlation coefficient. There was a significant correlation between post-treatment KRAS VAF and CT response (panel A) (R=0.58) and change in KRAS and CT response (panel B) (R=0.59)Click here for additional data file.Figure S4Supplemental Figure 4. Spaghetti plot of tumor mutation VAF changes with 2 cycles of GAX-CI. To determine the impact of GAX-CI on the allelic frequency of mutations detected in ctDNA, we measured the VAF of these mutations pre-treatment and after 2 cycles of GAX-CI. The mutations detected for a given patient were plotted using a spaghetti plot to provide a visual representation of the change in VAF with treatment.Click here for additional data file.Figure S5Supplemental Figure 5. Post-treatment Median VAF Predicts Progression-Free Survival. To assess whether Median mutation variant allele fraction (VAF) in cell-free DNA (cfDNA) predicted progression-free survival (PFS) and overall survival (OS), we sequenced cfDNA samples taken prior to treatment initiation and after 2 cycles of GAX-CI (on-treatment). The change in Median VAF was calculated was the percentage change from post- to pre-treatment. Patients were stratified by whether they had a higher (post-tx high) or lower (post-tx low) than the median post-treatment KRAS VAF, and a larger (change high) or smaller (change low) than the median of change in KRAS VAF with treatment. This demonstrated a statistically improved PFS with a lower post-treatment (after 2 cycles of treatment) Median mutation VAF (panel A). Post-treatment median mutation VAF did not predict OS (panel B). Change in Median mutation VAF with 2 cycles of treatment was not associated with a statistically improved PFS (panel C) or OS (panel D).Click here for additional data file.Tables S1-S4Table S1: Patient demographics. Table S2: Summary of TP53 VAF and KRAS VAF data for patient cohort (N=12). Table S3: Correlation between TP53 VAF, KRAS VAF, or median VAF and CEA and CA19-9 biomarkers using Spearman rank correlation coefficient (R). Table S4: Comparing predictive value of TP53 VAF, KRAS VAF, or median VAF versus CEA and CA19-9 for OS using C-indexClick here for additional data file.


Here we explore the use of a library of nine inducible genetic factors to augment the expansion of HSCs in vitro while maintaining their capacity to mature into functional neutrophils.Cellular therapies are of growing clinical interest, particularly for patients with deficiencies in white blood cell number related to chemotherapy or radiation exposure. Neutrophils are the predominant peripheral white blood cell, and their versatility and efficacy in fighting infections make them highly attractive for cellular therapy. As neutrophils have an inherent short lifespan and must be transfused in high numbers to be effective in protecting against infection, the development of neutrophil cellular therapy has been hindered by limitations in our ability to expand cells Stable cell lines were generated via electroporation with a transposase system. Factors were screened simultaneously in random combination and subsequent combinations were selected based on successful ‘hits’ from initial screens. Cells were characterized via flow cytometry to confirm maintenance of a progenitor state through expression of the cell surface marker CD34 and differentiation into neutrophil-like cells, identified by surface expression of CD11b/CD15/CD66b. Clonal populations were analyzed via qPCR to determine which of the nine factors are linked to conditional immortality.Methods & Experimental OutlineTimeline and methods for transfecting cells with our library and analyzing resultant cell lines.Two out of three transfections using the nine-factor library yielded a stably transfected population which grew to comprise at least 75% of viable cells in culture after 3 weeks. CD34 positivity was maintained through 8 weeks of culture in some clones, and neutrophil-like cells were generated from one cell line after 5 weeks. Several clones divided continuously for 11 weeks. Specific factors associated with these phenotypes have been identified.Flow Cytometric Analysis of Transfected CellsCells were transfected via single (1 Zap) or double (2 Zap) electroporation then cultured for 3 weeks. Transfected cells express the immature surface marker CD34 more highly than untransfected primary cells and are lower for CD11b, a mature myeloid marker.Flow Cytometric and Microscopic Analysis of Transfected Cells Ten Days After Induction of DifferentiationA ten-day differentiation assay was performed following 4 weeks in culture. A) Gating strategy used to identify a population of cells which is CD34(-), CD135(-), CD11b(+), CD15(+), and CD66b(+), and thus bear a neutrophil phenotype. B) Wright-Giemsa stain showing two cells with a neutrophil-like morphology.Enforced expression of a critical number of factors offers a developmental path for unlimited expansion of neutrophil precursors, making the generation of therapeutic neutrophil transfusions a realistic and feasible countermeasure for chemotherapy or radiation-induced neutropenia.Nathan E. Jeffries, BA, Safi Biotherapeutics: Salary Michael Mansour, MD, PhD, Thermofisher: Grant/Research Support


SGCB). Together, β-, α-, γ-, and δ-sarcoglycan form a 4-protein transmembrane complex (SGC) that localizes to the sarcolemma. Biallelic loss-of-function mutations in any subunit can lead to LGMD. To provide functional evidence for the pathogenicity of missense variants, we performed deep mutational scanning of SGCB and assessed SGC cell surface localization for all 6,340 possible amino acid changes. Variant functional scores were bimodally distributed and perfectly predicted pathogenicity of known variants. Variants with less severe functional scores more often appeared in patients with slower disease progression, implying a relationship between variant function and disease severity. Amino acid positions intolerant to variation mapped to points of predicted SGC interactions, validated in silico structural models, and enabled accurate prediction of pathogenic variants in other SGC genes. These results will be useful for clinical interpretation of SGCB variants and improving diagnosis of LGMD; we hope they enable wider use of potentially life-saving gene therapy.Genetic testing is essential for patients with a suspected hereditary myopathy. More than 50% of patients clinically diagnosed with a myopathy carry a variant of unknown significance in a myopathy gene, often leaving them without a genetic diagnosis. Limb-girdle muscular dystrophy (LGMD) type R4/2E is caused by mutations in β-sarcoglycan (  SGCB) cause limb-girdle muscular dystrophy type R4/2E (LGMDR4/2E), resulting in muscle wasting, progressive weakness, degeneration of skeletal muscle, and often premature death  , 16, 17.https://doi.org/10.1172/JCI168156DS1). Coexpression with the other 3 untagged SGC proteins , however, led to robust cell surface expression of WT YFP-SGCB-HA. To create a stable HEK293 cell line capable of reliably transporting SGCB to the cell surface, we transduced HEK293 cells with a mixture of lentiviruses designed to express SGCA, SGCD, and SGCG. Single clones from these transduced cells were isolated and tested at multiple passages for expression of the 3 stably expressing SGC proteins (We established a human cell system to model SGCB variant function using engineered HEK293 cells. Testing for protein expression for the 4 sarcoglycan (SGC) proteins verified that HEK293 cells lacked detectable protein expression of any of SGC gene. Furthermore, expression of a SGCB fusion protein (YFP-SGCB-HA) alone led to minimal cell surface expression, as measured by immunofluorescence with an anti-HA antibody  was divided into 6 overlapping sublibraries (204–225 bp) to allow full-length sequencing of each. The mutant cDNA library for each sublibrary was cloned into the WT lentiviral YFP-SGCB-HA vector. ADG-HEK cells were transduced at low multiplicity (<0.1) to yield a population in which each cell expressed either 0 or 1 SGCB variant, recapitulating a hemizygous-like state in which to test the effect of each variant. Sequencing of both plasmid libraries and integrated libraries from genomic DNA demonstrated that nearly all single-codon mutations (98%) and nearly all amino acid changes (99%) were present and transduced into cells and the average depth of each mutation was generally uniform . Each cell population transduced with a SGCB mutant lentivirus pool was first bulk-sorted for expression of YFP to select cells expressing SGCB. YFP-positive cells were then grown for an additional 5–7 days and split into 2 equal populations and stained either for HA to detect YFP-SGCB-HA surface expression or with an antibody against the extracellular domain of SGCA to detect cell surface of SGCA. Cells were sorted into 4 bins corresponding to the top and bottom 10% of cells for either YFP-SGCB-HA or SGCA cell surface expression. To determine the relative abundance of each mutation in each bin, we performed amplicon sequencing of each mutated sublibrary. Functional scores for each variant were calculated for YFP-SGCB-HA cell surface expression (bin 1 vs. bin 4) and SGCA cell surface expression (bin 1 vs. bin 4) as the log10 ratio of the variant’s frequency in bin 4 (high expression) divided by its frequency in bin 1 (low expression), such that deleterious variants should score negatively and neutral variants positively . We also investigated the functional scores of variants present in healthy populations. We used these databases to test the false positive rate of our functional assay. Presuming that no one in these cohorts had LGMD, we expected that there should be no homozygotes with nonfunctional alleles by our assay and no compound heterozygotes with 2 nonfunctional alleles by our assay in any of these populations. First, we utilized the UK Biobank  database. Among the UKBB population, there were 1,654 individuals with more than 0 nonsynonymous variants in SGCB. Of these, only 6 individuals harbored 2 nonsynonymous variants. In each instance, at least 1 of the 2 variants scored as functionally neutral/benign in our functional assay (https://gnomad.broadinstitute.org/) lists 198 SGCB missense variants, all rare . Of these variants, 20 variants  were deleterious in our assay  and present in non-Finnish Europeans, with 0 of these observed as homozygotes. The most frequent variant that scored as pathogenic (S114F) is a known pathogenic variant in ClinVar and was observed in 68 non-Finnish European individuals in gnomAD, with none observed as homozygotes. Three SGCB missense variants were observed in the homozygous state in at least one population in gnomAD , but each scored as functionally neutral in our assay. These results reflect the mild negative selection on pathogenic variants in recessive disease genes that allows genetic drift to increase the frequency of even disease-causing variants below the level that would frequently produce homozygotes. Furthermore, the collective frequency of missense variants that we predicted were pathogenic in these 2 databases suggests a carrier frequency around 1 in 1,250–2,400 and, therefore, a population prevalence of SGCB-deficient LGMD of approximately 0.2–0.6 per million, ignoring the de novo mutation rate for this gene, which is consistent with various disease prevalence estimates in the literature  with those with 2 fewer severe missense SGCB alleles . While there was no significant difference in the age at onset between these two groups, the age at loss of ambulation was significantly lower among patients with 2 severely nonfunctional SGCB alleles  , suggesting that the functional score derived here can not only predict pathogenicity but also disease severity.There is a wide range of phenotypes for patients with LGMDR4/2E; age at symptom onset ranges from less than 1 to more than 20 years of age, some patients require a wheelchair at as early as 7 years of age, and some patients never require a wheelchair . This ph< 0.001) , and theP < 4 × 10–6). A strong relationship between protein domain and functional score also was revealed when we compared the number of nonfunctional amino acid changes  in the cytoplasmic  or transmembrane domain  of SGCB with those in the extracellular topological domains of the protein . Because the SGCB protein has not yet been crystalized, no structural models of SGCB or the sarcoglycan protein complex have been published to our knowledge. In order to further determine the relationship between functional scores and protein structure, we produced a model of SGCB protein structure using the multimer function of AlphaFold2 in the presence of SGCA, SGCD, and SGCG. The model produced was substantially different than the AlphaFold2 model produced using SGCB alone and revealed a structure in which SGCB, SGCD, and SGCG form a triple-helical quaternary protein structure with cobinding β sheets forming an interprotein β-barrel–like structure (P < 2.4 × 10–8) and SGCB-SGCG  interacting surfaces, the 3 proteins forming the triple-helical protein structure, and fewer for SGCB-SGCA interacting surfaces . The proportion of sites with at least one interprotein contact between SGCB and either SGCD or SGCG for amino acid positions with reported pathogenic variants in SGCB was also significantly greater than that of sites without known pathogenic variants , further supporting the claim that intermolecular interactions are critical for SGCB function. Similar to previous deep mutational scans, we observed that amino acid changes resulting in proline were also significantly enriched for deleterious functional scores compared with other amino acid changes . One region with strong mutational intolerance but unclear function is amino acids 90–99. Of particular note is position R91, which harbors 3 different known pathogenic variants . It may be that these positions on the cusp of the transmembrane domain (AA 60–90) are important for proper orientation of the protein complex in the membrane or are critical for initiating the helical structure of the 3 core SGC genes within the larger complex. It is unclear, however, why position R91 harbors more observed pathogenic variants than other sites in the SGCB protein.High-throughput functional screens, particularly those that test all possible amino acid changes, have the ability to inform our knowledge of the structure-function relationships present within a protein or protein complex. For example, as expected, we observed that amino acid changes that resulted in conservative changes  produced deleterious functional scores less often  than those that resulted in nonconservative changes , we observed a strong enrichment of deleterious functional scores among amino acid changes only possible from multinucleotide changes compared with amino acid changes possible from single nucleotide changes . To test more specifically that specific codons were selected by evolution to be maximally distant from deleterious amino acid changes, we calculated the average distance of possible codons at each position that encode the WT amino acid and compared it with the observed distance of the codon chosen by evolution. We observed that the difference between the utilized codon and the average of possible codons for functionally deleterious amino acid changes was significantly greater than for those that were functionally neutral , whereas there was no significant difference for neutral amino acid changes . Finally, when we restricted our analysis to only those amino acid changes possible by 1 nucleotide change, we saw that deleterious amino acid changes were more often encoded by a codon that requires a greater number of nucleotide changes to obtain the amino acid change compared with neutral amino acid changes , effectively maximizing the mutational distance from the current amino acid to the array of possible deleterious amino acids by codon choice.Unlike single nucleotide mutagenesis, which enables the measurement of all possible variants generally accessible during evolution, single amino acid saturation mutagenesis enables measurement of amino acid changes not easily accessible by evolution, i.e., codon changes requiring 2–3 nucleotide changes. We aimed to determine if amino acid changes that resulted from single nucleotide changes were more or less likely to lead to a nonfunctional protein compared with amino acid changes that are only possible following 2–3 adjacent nucleotide changes, consistent with the well-established finding that more similar codons tend to encode more biochemically similar amino acids , 25. WhiSGCB in particular are among the most frequently mutated genes underlying LGMD , particularly those whose cell surface expression is key to their function.Measured functional scores for variants are highly accurate in predicting the pathogenicity of known disease-causing variants, outperforming the newest prediction algorithms. This is the first time to our knowledge that a full-length muscular dystrophy gene has been subjected to DMS, suggesting that pooled functional screens are a viable method of large-scale functional assessment of protein-coding genetic variation in other muscular dystrophy genes , whereas only 1 known benign variant lies within this range.Our findings that disease severity, as measured by age at loss of ambulation, are related to functional score in our assay suggests that cell surface expression of SGCB and the SGC protein complex is potentially a quantitative trait that determines, in part, the stability of the sarcolemma and the integrity of muscle cells over the course of a lifetime. In the future, it will be interesting to determine if functional scores measured here correlate with more subtle muscle traits, such as sarcopenia or exercise intolerance in the general population, at least among individuals with protein-altering genetic variation in these genes.The pattern of deleterious amino acid changes across the SGCB gene closely mirrored the predicted protein structure produced using AlphaFold2 . The co-Overall, SGCB is moderately tolerant of protein-altering genetic variation, with 16% of single-nucleotide missense variants demonstrating nonfunctional scores . This number jumps to 30% when considering all possible amino acid changes, however, implying that evolutionary forces lead to the selection of specific amino acids and even specific codons with fewer nonfunctional alleles reachable by single nucleotide changes. We believe this is the first time this phenomenon has been reported in DMS data and that this helps to explain the relatively low rate of pathogenic variants observed in human populations despite the high degree of evolutionary conservation across species. These data suggest that proteins may have evolved a buffer against deleterious protein alterations by selecting amino acids and codons with a lower probability of producing nonfunctional proteins.SGCB missense variants demonstrating either a clear nonfunctional score or a neutral score. It is yet unknown, however, whether functional scores between the means of these functional classes represent true quantitative measures of protein function or experimental noise. For example, there are 13 VUSs with functional scores between those of the highest scoring known pathogenic variant  and the lowest scoring known benign variant . No known pathogenic or benign variants exist within these borderline functional scores; however, this may be due to sampling bias with only those patients with severe phenotypes and clear genetic findings entered into clinical databases. When more clinical data from patients with a wider range of disease severity are investigated in the context of these functional scores, we will likely identify individuals with more intermediate phenotypes and correspondingly intermediate functional scores. For variants that appear to act as hypomorphs in functional assays, it is unclear whether these are likely to be considered pathogenic in the strictest sense or rather should be considered factors contributing to a spectrum of limb-girdle muscle weakness present in the general population. The relationship we observe between functional score and age at loss of ambulation, even among patients with 2 alleles in the pathogenic range of functional scores, suggests that SGCB function is not purely normal or loss of function but a quantitative trait captured at least in part by measuring its ability to complex with other SGC genes and be transported to the cell surface as our assay measures. As large data sets of nonsyndromic individuals are sequenced and integrated with clinical information and physiological measurements, it may be possible to correlate more directly the role of SGCB genetic variation in the range of normal muscle function in addition to disease risk as explored here.The bimodal distribution of functional scores is similar to previous deep-mutational scanning reports, with most A limitation of our experimental design is the inability to measure the effects of variants on endogenous mRNA or protein levels, the effect of both coding and noncoding genetic variation on splicing, and the effects of structural variation . In ClinVar, there are 11 known pathogenic splicing mutations. In the future, splicing effects could be measured by mutagenizing splice regions in the endogenous locus and measuring cell surface expression of the SGC complex in primary muscle cells. It is clear that both small and large DNA insertions or deletions can disrupt SGCB function, with frame shift variants having a high likelihood of being pathogenic. Whole-exon deletions and in-frame indels are harder to predict, however, and would likely require functional assessment as we performed here. However, these are highly unique variants in terms of length, position, and composition, and, thus, they are better suited to individual testing if they are observed in an individual. Another potential limitation is that our assay does not measure muscle integrity or atrophy over time directly and would not detect defects in interactions between SGCB and other proteins in the membrane or extracellular space that are important for muscle function. Our results may be further improved through the use of orthogonal functional assays or specialized assays for protein function.SGCB. The map of functional effects we present has the ability to improve classification of variants observed in patients with LGMD and aid our understanding of the structure of an important member of the dystrophin-associated protein complex. When used together with available lines of evidence, these results will add confidence to variant interpretation and potentially allow patients with pathogenic variants to be treated with gene-specific therapeutics for which they would have otherwise been ineligible. Future work will explore these methods for additional SGC and muscular dystrophy genes with similar pathological mechanisms.Here, we demonstrate that high-throughput functional assays can accurately measure the effect of protein-coding genetic variation in the LGMD gene SGCA, SGCD, and SGCG was synthesized and individually cloned into a lentiviral vector by Genewiz. A YFP-SGCB construct was also synthesized and cloned into the same lentiviral vector expressing WT human β-sarcoglycan followed by YFP. A HA tag was further added to the C-terminal end of the SGCB by site-directed insertion mutagenesis (Takara). The new construct was termed YFP-SGCB-HA. Single or double variants in β-sarcoglycan were introduced by site-directed mutagenesis according to the manufacturer’s protocol (Takara). All constructs were verified by Sanger sequencing (Genewiz).The full-length cDNA of human WT 2 at 37°C. During routine passaging, cells were washed with PBS and dissociated with trypsin-EDTA 0.05%. HEK293 cells were tested for endogenous α-, β-, γ-, and δ-sarcoglycan by Western blot assay; all were undetectable. To generate monoclonal stable cell lines coexpressing α-, γ-, and δ-sarcoglycan, HEK293 cells were tri-transduced with lentiviruses containing WT SGCA, SGCD, and SGCG as described above. Colonies formed by expansion of single cells were screened by PCR and Western blot for the presence of human sarcoglycans  were cultured in DMEM , supplemented with 10% fetal bovine serum . Cells were maintained in a humidified atmosphere of 5% COoglycans . Clone 1oglycans .Cells were lysed by the addition of RIPA buffer  and protease inhibitor cocktail (MilliporeSigma). Protein concentrations of lysates were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were run on a 10% gel and transferred to a nitrocellulose membrane. The blots were blocked with 5% nonfat dry milk in PBS for 1 hour and probed with anti–α-sarcoglycan , β-sarcoglycan , γ-sarcoglycan , and δ-sarcoglycan  at 4°C overnight. Secondary antibodies included horseradish peroxidase–labeled anti-mouse IgG . Blots were developed with ECL . Images were taken using the G: Box Chemi XT4, Genesys . For immunocytochemistry, cells were fixed using 4% paraformaldehyde but not permeabilized. These cells were then stained for cell surface expression using an anti-HA antibody  followed by staining with an Alexa Fluor 647 goat anti-mouse secondary antibody , an Alexa Fluor 647–conjugated anti-HA antibody , a Pacific Blue anti-HA antibody , or an Alexa Fluor 647–conjugated anti-α-sarcoglycan antibody . Images were taken using a fluorescence microscope (Nikon 80i).To achieve all possible amino acid substitutions of the entire SGCB , the complete library was divided into 6 sublibraries , and eacFor cloning of each sublibrary into lentiviral vector YFP-SGCB-HA (described above), the synthesized oligonucleotide pools were dissolved and diluted at 1:100 and used as template for PCR amplification with CloneAmp HiFi Polymerase . The product was purified using the Nucleospin Gel and PCR Clean-up kit (Takara). Backbone plasmid YFP-SGCB-HA was PCR amplified with primers that are complementary to the WT SGCB homologues sequences at the ends of each sublibrary. The resulting linearized backbone that carried the remaining template sequence was digested with DpnI to deplete starting plasmid, separated from nonspecific fragments by electrophoresis on a 1% agarose gel, and purified using the gel clean-up kit (Nucleospin). Each sublibrary was inserted into YFP-SGCB-HA plasmids via Gibson assembly (NEBuilder HiFi DNA assembly Master Mix) according to the manufacture’s protocol, so that it replaces the equivalent part of the WT SGCB cDNA sequence. The assembly reactions were purified and transformed into E. cloni DUO Electrocompetent cells (Lucigen) with 5% of cells plated to estimate cloning efficiency and 95% of cells grown in liquid media for 12–16 hours at 37°C in LB supplemented with 100 mg/mL ampicillin. We obtained an estimated >40,000 colonies per section to ensure >10× colonies per potential sequence present within each library. Each library was isolated using an Endo-free Maxiprep kit (Qiagen). Library diversity was assessed by sequencing on an Illumina MiSeq 2 × 250 bp lane such that the entire mutated section was sequenced in both directions and a portion of the nonmutagenized section was sequenced as an internal control for background mutations.6 cells were plated onto each of six 100 mm dishes and transfected with 10 μg total plasmid (2.0:1.0:1.3 transfer/envelope/packaging ratio), using Lipofectamine 3000 (Thermo Fisher Scientific). Media were replaced after 6 hours, and viral supernatants were collected at 24 hours, filtered using a 0.45 mm filter (Millipore), and stored at –80°C. Viral titer was determined by qPCR and empirically estimated with a dilution series of transduced cells followed by FACS to measure the proportion of YFP-positive cells. For each biological replicate for each section, ADG-HEK cells were transduced with mutant library at low multiplicity of infection (<0.1), by applying 1.5 mL viral supernatant (with 8 mg/mL polybrene) to a 100 mm dish (TPP) containing approximately 5 × 106 cells, with three 100 mm dishes per replicate to maintain library diversity. After 72 hours, transduced cells were bulk sorted for the presence of YFP using a FACS Aria II (BD) into DMEM containing 100 U/mL penicillin and 100 g/mL streptomycin. Cells were grown for 5–7 days and then stained for cell surface expression of YFP-SGCB-HA or SGCA.SGCB cDNA subsection libraries (A–F) were packaged into lentivirus at the Hope Center Viral Core facility at Washington University by cotransfecting HEK293T cells (ATCC) with the transfer plasmid pool plus envelope and packaging vectors . For each pool, 4 × 10Three measurements of function were obtained for each mutagenized section of SGCB. First, transduced cells were harvested during passaging to obtain a measurement of the frequency of each mutation present in the unselected pool of transduced cells. A sample of transduced cells after selection for YFP expression was obtained to measure the abundance of each mutation in YFP-positive cells compared with all transduced cells to measure the effect, if any, on total protein level. Third, YFP-positive sorted cells were further labeled with Alexa Fluor 647–conjugated antibody for FACS. Briefly, live cells were collected using trypsin-EDTA 0.05%. Thereafter, cells were blocked in blocking buffer (0.5% BSA-PBS) for 20 minutes on ice; half the cells were incubated with Alexa Fluor 647–conjugated anti-HA, and the other half were stained with Alexa Fluor 647–conjugated anti–α-sarcoglycan antibody on ice for 30 minutes. After incubation, cells were washed twice with PBS. The cells were fixed with 2% paraformaldehyde on ice for 20 minutes. Next, cells were resuspended in ClearFlow Sheath Fluid (Leinco Technologies) and sorted using FACS Aria II into bins according to the intensity of Alexa Fluor 647 signal. After the single cells were selected using forward and side scatter, a histogram of the FITC was created to select for YFP-positive cells. For those YFP-positive cells, a histogram of the Alexa Fluor 647 was further created and gates were drawn across the library based on red intensity: low , medium low , medium high , and high . Cells harboring single or double variants in β-sarcoglycan were labeled with Alexa Fluor 647–conjugated antibody in the same ways as described above. The single cells were gated and analyzed for YFP expression. Furthermore, the YFP-positive population was analyzed for the expression of Alexa Fluor 647 by MACS Flow Cytometer (Miltenyi Biotec). The data analysis was performed using FlowJo software (TreeStar Inc). Each assay was performed in duplicate starting from transduction.After selections, genomic DNA was isolated from more than 500,000 cells using a DNeasy Blood and Tissue DNA extraction kit (Qiagen) and sequenced for the mutated region. For each sample, 16 replicate PCR reactions were performed and pooled, each with 200 ng genomic DNA as template. Mutated sections from integrated inserts were PCR amplified with NEBnext High-Fidelity polymerase (NEB). Each set of 16 replicate amplicons was pooled and purified with SPRI beads. Mutated sections were then amplified for 15 cycles using primers with 5′ overhangs of Illumina adapter handles and then secondarily amplified for 25 cycles using dual-indexed illumina adapters. The resulting Illumina libraries contained inserts ranging from 200 to 240 bp, enabling full read coverage in both directions for 2 × 250 bp sequencing and more than 50 bp overlap for 2 × 150 bp sequencing. Illumina libraries were either sequenced using 2 × 250 bp flow cells on a MiSeq or 2 × 150 bp flow cells on a NovaSeq.10 ratio of the frequency in bin 4 (high expression) over that in bin 1 (low expression) and then normalized across chunks such that the functional scores for synonymous mutations in each chunk have an average of 0 and a standard deviation of 0.5. For amino acid substitutions represented by more than one equivalent codon substitution, the median was taken across those codons’ functional scores to yield an amino acid–level score. The median score was taken for each amino acid functional score across biological replicates.Within each sample, raw read counts for each mutant codon were counted and per-mutation frequencies were calculated; any variant read counts under 50 per 1 million reads across the 4 sorted bins were removed from further analysis. For both HA-SGCB and SGCA, functional scores were taken as the logn = 500,000).SGCB missense variant classifications were obtained from ClinVar on September 6, 2022; the Leiden database; or extracted from the literature . If a vaSGCB missense variants were scored by REVEL (All by REVEL , CADD (3by REVEL , and Polby REVEL . Scores The SGCB predicted structure was generated using AlphaFold2  using tht test. A 2-sample Student’s t test was used to compare the differences between 2 groups. Kaplan-Meier method was used to estimate the probability of survival, and the log-rank test was used to compare the overall survival difference between groups. Correlation between variables and significance was determined using linear regression with the lm function in R. All statistical tests were 2 sided, and the differences were considered significant when P was less than 0.05.Functional scores were defined as the log ratio of mutant read counts in cells with high cell surface SGC protein level and cells with low cell surface SGC protein level. For all quantitative measurements, mean ± SEM are reported. Normal distribution of the sample sets was determined before application of 2-tailed Student’s Collection of clinical information from the Institutional Review Boards of Washington University School of Medicine, Fundación Canaria Instituto de Investigación Sanitaria de Canarias, and John Walton Muscular Dystrophy Research Center. Written informed consent was obtained from all participants for participation in these studies.GH and CCW designed research studies. CL, JW, SP, and JD conducted experiments. GH provided statistics analysis. JAP and JDM provided clinical data. CL, GH, and CCW wrote the manuscript. CL, GH and CCW analyzed data. CL, JW, SP, JD, GH, CCW, JAP, and JDM revised the manuscript