Personal journeys to and in human genetics and dysmorphology.

Genetics has become a critical component of medicine over the past five to six decades. Alongside genetics, a relatively new discipline, dysmorphology, has also begun to play an important role in providing critically important diagnoses to individuals and families. Both have become indispensable to unraveling rare diseases. Almost every medical specialty relies on individuals experienced in these specialties to provide diagnoses for patients who present themselves to other doctors. Additionally, both specialties have become reliant on molecular geneticists to identify genes associated with human disorders. Many of the medical geneticists, dysmorphologists, and molecular geneticists traveled a circuitous route before arriving at the position they occupied. The purpose of collecting the memoirs contained in this article was to convey to the reader that many of the individuals who contributed to the advancement of genetics and dysmorphology since the late 1960s/early 1970s traveled along a journey based on many chances taken, replying to the necessities they faced along the way before finding full enjoyment in the practice of medical and human genetics or dysmorphology. Additionally, and of equal importance, all exhibited an ability to evolve with their field of expertise as human genetics became human genomics with the development of novel technologies.

An approach to rapid processing of camera trap images with minimal human input.

Camera traps have become an extensively utilized tool in ecological research, but the manual processing of images created by a network of camera traps rapidly becomes an overwhelming task, even for small camera trap studies.We used transfer learning to create convolutional neural network (CNN) models for identification and classification. By utilizing a small dataset with an average of 275 labeled images per species class, the model was able to distinguish between species and remove false triggers.We trained the model to detect 17 object classes with individual species identification, reaching an accuracy up to 92% and an average F1 score of 85%. Previous studies have suggested the need for thousands of images of each object class to reach results comparable to those achieved by human observers; however, we show that such accuracy can be achieved with fewer images.With transfer learning and an ongoing camera trap study, a deep learning model can be successfully created by a small camera trap study. A generalizable model produced from an unbalanced class set can be utilized to extract trap events that can later be confirmed by human processors.

A dyadic approach to the delineation of diagnostic entities in clinical genomics.

The delineation of disease entities is complex, yet recent advances in the molecular characterization of diseases provide opportunities to designate diseases in a biologically valid manner. Here, we have formalized an approach to the delineation of Mendelian genetic disorders that encompasses two distinct but inter-related concepts: (1) the gene that is mutated and (2) the phenotypic descriptor, preferably a recognizably distinct phenotype. We assert that only by a combinatorial or dyadic approach taking both of these attributes into account can a unitary, distinct genetic disorder be designated. We propose that all Mendelian disorders should be designated as "GENE-related phenotype descriptor" (e.g., "CFTR-related cystic fibrosis"). This approach to delineating and naming disorders reconciles the complexity of gene-to-phenotype relationships in a simple and clear manner yet communicates the complexity and nuance of these relationships.

Membrane-Induced Technique for the Management of Combined Soft Tissue and Osseous Defects.

The induced membrane technique is a simple, effective, and reproducible treatment method for segmental bone defects. It is a 2-stage approach that requires eventual autologous bone graft to manage the deficit. The first stage requires debridement of all nonviable tissue while preserving a healthy soft tissue envelope. A polymethylmethacrylate is implanted between the osseous segments to maintain length. The osseous defect can be stabilized internally or externally. During the second stage, a vascularized induced membrane is formed and produces multiple growth factors. The induced membrane technique is a valuable option for limb salvage in cases of segmental bone defects.

Primary Invasive Squamous Cell Carcinoma of the Foot.

Malignant transformation of wounds is a rare complication that if missed can lead to loss of life or limb. This case report presents a rare invasive variant of squamous cell carcinoma presenting in the setting of a chronic wound complicated by osteomyelitis. This aggressive form of squamous cell carcinoma has a high growth rate and a high propensity for metastasis and recurrence. Early intervention greatly decreases the risk of metastasis and recurrence. We present the systematic evaluation and surgical management of an aggressive primary tumor occurring in the forefoot.

Loss of HIF1A From Pancreatic Cancer Cells Increases Expression of PPP1R1B and Degradation of p53 to Promote Invasion and Metastasis.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) are hypovascular, resulting in the up-regulation of hypoxia inducible factor 1 alpha (HIF1A), which promotes the survival of cells under low-oxygen conditions. We studied the roles of HIF1A in the development of pancreatic tumors in mice. METHODS: We performed studies with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre (KPC) mice, KPC mice with labeled pancreatic epithelial cells (EKPC), and EKPC mice with pancreas-specific depletion of HIF1A. Pancreatic and other tissues were collected and analyzed by histology and immunohistochemistry. Cancer cells were cultured from PDACs from mice and analyzed in cell migration and invasion assays and by immunoblots, real-time polymerase chain reaction, and liquid chromatography-mass spectrometry. We performed studies with the human pancreatic cancer cell lines PATU-8988T, BxPC-3, PANC-1, and MiaPACA-2, which have no or low metastatic activity, and PATU-8988S, AsPC-1, SUIT-2 and Capan-1, which have high metastatic activity. Expression of genes was knocked down in primary cancer cells and pancreatic cancer cell lines by using small hairpin RNAs; cells were injected intravenously into immune-competent and NOD/SCID mice, and lung metastases were quantified. We compared levels of messenger RNAs in pancreatic tumors and normal pancreas in The Cancer Genome Atlas. RESULTS: EKPC mice with pancreas-specific deletion of HIF1A developed more advanced pancreatic neoplasias and PDACs with more invasion and metastasis, and had significantly shorter survival times, than EKPC mice. Pancreatic cancer cells from these tumors had higher invasive and metastatic activity in culture than cells from tumors of EKPC mice. HIF1A-knockout pancreatic cancer cells had increased expression of protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B). There was an inverse correlation between levels of HIF1A and PPP1R1B in human PDAC tumors; higher expression of PPP1R1B correlated with shorter survival times of patients. Metastatic human pancreatic cancer cell lines had increased levels of PPP1R1B and lower levels of HIF1A compared with nonmetastatic cancer cell lines; knockdown of PPP1R1B significantly reduced the ability of pancreatic cancer cells to form lung metastases in mice. PPP1R1B promoted degradation of p53 by stabilizing phosphorylation of MDM2 at Ser166. CONCLUSIONS: HIF1A can act a tumor suppressor by preventing the expression of PPP1R1B and subsequent degradation of the p53 protein in pancreatic cancer cells. Loss of HIF1A from pancreatic cancer cells increases their invasive and metastatic activity.

The Evolving Role of Metastasectomy for Patients with Metastatic Renal Cell Carcinoma.

Surgical metastasectomy continues to be utilized for patients with solitary or low-volume metastatic renal cell carcinoma (mRCC). Although few high-quality data are available to evaluate outcomes, local treatment is recommended when feasible because it may allow a subset of patients to delay or avoid systemic treatments. With the development of improved mRCC therapies, utilization of metastasectomy has increased because most patients have incomplete responses to systemic treatment of their metastases. This review discusses the rationale and history of metastasectomy, trends in utilization, prognostic factors for patient selection, site-specific considerations, alternatives for nonsurgical local treatment, and risk of morbidity associated with metastasectomy.

An apparent new syndrome of extreme short stature, microcephaly, dysmorphic faces, intellectual disability, and a bone dysplasia of unknown etiology.

We report on a 26-year-old male with extreme short stature, microcephaly, macroglossia, other dysmorphic features, severe intellectual disability, and a bone dysplasia. The patient had an extensive genetic and biochemical evaluation that was all normal or noninformative. Recently, the proband died following a period of not eating. He likely had a previously undescribed syndrome of unknown etiology.

Fatal hyperkeratosis syndrome in four siblings due to dolichol kinase deficiency.

A diagnostic journey began in 1966 when a male was born with a lethal hyperkeratosis of undetermined etiology, only to be followed by three additional siblings with the same unknown disorder. All four siblings had unique circumferential skin constrictions on all of their digits. They died within 5 days after birth with no diagnosis or etiology established. The first author (BDH) maintained notes, partial medical records, photographs, and comments about one autopsy report. This information was regularly revisited in the hope of finding a literature match, but no etiological diagnosis was forthcoming. However, in 2017, Rush et al. reported two siblings with similar phenotype in whom they found dolichol kinase deficiency (DOLK). Ultimately, our family was relocated and DNA isolated from the pathology slides of the third affected infant showed compound heterozygous pathogenic variants in the DOLK gene. The variants were in trans, with different missense variants from the mother and father. This 52-year diagnostic pursuit, culminated in an answer that gave the family an explanation for their losses.

Glycopolycation-DNA Polyplex Formulation N/P Ratio Affects Stability, Hemocompatibility, and in Vivo Biodistribution.

Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose- block-2-methylaminoethyl methacrylate) [P(MAG- b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG- b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG- b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG- b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG- b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG- b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG- b-MAEMt) N/P = 5 and P(MAG- b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG- b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG- b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.

Prolonged Expression of Secreted Enzymes in Dogs After Liver-Directed Delivery of Sleeping Beauty Transposons: Implications for Non-Viral Gene Therapy of Systemic Disease.

The non-viral, integrating Sleeping Beauty (SB) transposon system is efficient in treating systemic monogenic disease in mice, including hemophilia A and B caused by deficiency of blood clotting factors and mucopolysaccharidosis types I and VII caused by α-L-iduronidase (IDUA) and ß-glucuronidase (GUSB) deficiency, respectively. Modified approaches of the hydrodynamics-based procedure to deliver transposons to the liver in dogs were recently reported. Using the transgenic canine reporter secreted alkaline phosphatase (cSEAP), transgenic protein in the plasma was demonstrated for up to 6 weeks post infusion. This study reports that immunosuppression of dogs with gadolinium chloride (GdCl3) prolonged the presence of cSEAP in the circulation up to 5.5 months after a single vector infusion. Transgene expression declined gradually but appeared to stabilize after about 2 months at approximately fourfold baseline level. Durability of transgenic protein expression in the plasma was inversely associated with transient increase of liver enzymes alanine transaminase and aspartate transaminase in response to the plasmid delivery procedure, which suggests a deleterious effect of hepatocellular toxicity on transgene expression. GdCl3 treatment was ineffective for repeat vector infusions. In parallel studies, dogs were infused with potentially therapeutic transposons. Activities of transgenic IDUA and GUSB in plasma peaked at 50-350% of wildtype, but in the absence of immunosuppression lasted only a few days. Transposition was detectable by excision assay only when the most efficient transposase, SB100X, was used. Dogs infused with transposons encoding canine clotting factor IX (cFIX) were treated with GdCl3 and showed expression profiles similar to those in cSEAP-infused dogs, with expression peaking at 40% wt (2 µg/mL). It is concluded that GdCl3 can support extended transgene expression after hydrodynamic introduction of SB transposons in dogs, but that alternative regimens will be required to achieve therapeutic levels of transgene products.

N-Acetylgalactosamine Block-co-Polycations Form Stable Polyplexes with Plasmids and Promote Liver-Targeted Delivery.

The liver is an ideal target for nucleic acid therapeutic applications (i.e., siRNA, gene therapy, and genome editing) due to its ability to secrete proteins into the blood. In this work, we present the first synthesis of a novel monomer derived from N-acetyl-d-galactosamine (GalNAc) and its polymerization as a facile route to create multivalent delivery vehicles with exceptional targeting efficiency to asialoglycoprotein receptors (ASGPRs) on liver hepatocytes. A series of cationic diblock GalNAc glycopolymers composed of a GalNAc-derived block of fixed length (n = 62) and cationic 2-aminoethylmethacrylamide (AEMA) blocks of varying lengths (n = 19, 33, and 80) have been synthesized and characterized. In addition, nontargeted control polymers consisting of either glucose or polyethylene glycol-derived neutral blocks with an AEMA cationic block were also created and examined. All polymeric vehicles were able to bind and encapsulate plasmids (pDNA) into polymer-pDNA complexes (polyplexes). The GalNAc-derived polyplexes were colloidally stable and maintained their size over a period of 4 h in reduced-serum cell culture media. The GalNAc-derived homopolymer effectively inhibited the uptake of Cy5-labeled asialofetuin (a natural ligand of ASGPRs) by cultured hepatocyte (HepG2) cells at lower concentrations (IC50 = 20 nM) than monomeric GalNAc (IC50 = 1 mM) and asialofetuin (IC50 = 1 µM), suggesting highly enhanced ASGPR binding due to multivalency. These polymers also showed cell type-specific gene expression in cultured cells, with higher protein expression in ASGPR-presenting HepG2 than HeLa cells, which lack the receptor. Biodistribution studies in mice show higher accumulation of pDNA and GalNAc-derived polymers in the liver compared with the glucose-derived nontargeted control. This study demonstrates the first facile synthesis of a multivalent GalNAc-derived block copolymer architecture that promotes enhanced delivery to liver and offers insights to improve targeted nanomedicines for a variety of applications.

Epigenetic Determinants of Erythropoiesis: Role of the Histone Methyltransferase SetD8 in Promoting Erythroid Cell Maturation and Survival.

Erythropoiesis, in which committed progenitor cells generate millions of erythrocytes daily, involves dramatic changes in the chromatin structure and transcriptome of erythroblasts, prior to their enucleation. While the involvement of the master-regulatory transcription factors GATA binding protein 1 (GATA-1) and GATA-2 in this process is established, the mechanistic contributions of many chromatin-modifying/remodeling enzymes in red cell biology remain enigmatic. We demonstrated that SetD8, a histone methyltransferase that catalyzes monomethylation of histone H4 at lysine 20 (H4K20me1), is a context-dependent GATA-1 corepressor in erythroid cells. To determine whether SetD8 controls erythroid maturation and/or function, we used a small hairpin RNA (shRNA)-based loss-of-function strategy in a primary murine erythroblast culture system. In this system, SetD8 promoted erythroblast maturation and survival, and this did not involve upregulation of the established regulator of erythroblast survival Bcl-x(L). SetD8 catalyzed H4K20me1 at a critical Gata2 cis element and restricted occupancy by an enhancer of Gata2 transcription, Scl/TAL1, thereby repressing Gata2 transcription. Elevating GATA-2 levels in erythroid precursors yielded a maturation block comparable to that induced by SetD8 downregulation. As lowering GATA-2 expression in the context of SetD8 knockdown did not rescue erythroid maturation, we propose that SetD8 regulation of erythroid maturation involves multiple target genes. These results establish SetD8 as a determinant of erythroid cell maturation and provide a framework for understanding how a broadly expressed histone-modifying enzyme mediates cell-type-specific GATA factor function.

Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance.

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

A new case of a LUMBAR syndrome.

LUMBAR syndrome (lower body congenital infantile hemangiomas and other skin defects; urogenital anomalies and ulceration; myelopathy; bony deformities; anorectal malformations and arterial anomalies; and rectal anomalies) is a rare association between infantile hemangiomas of the lower half of the body and regional congenital anomalies. Since 1986, 53 cases have been reported and no etiology has been identified. We report on the 54th case in a male infant and review the literature concerning the manifestations of the LUMBAR syndrome.

Quantitative analysis of α-L-iduronidase expression in immunocompetent mice treated with the Sleeping Beauty transposon system.

The Sleeping Beauty transposon system, a non-viral, integrating vector that can deliver the alpha-L-iduronidase-encoding gene, is efficient in correcting mucopolysaccharidosis type I in NOD/SCID mice. However, in previous studies we failed to attain reliable long-term alpha-L-iduronidase expression in immunocompetent mice. Here, we focused on achieving sustained high-level expression in immunocompetent C57BL/6 mice. In our standard liver-directed treatment we hydrodynamically infuse mice with plasmids containing a SB transposon-encoding human alpha-L-iduronidase, along with a source of SB transposase. We sought to 1) minimize expression of the therapeutic enzyme in antigen-presenting cells, while avoiding promoter shutdown and gender bias, 2) increase transposition efficiency and 3) improve immunosuppression. By using a liver-specific promoter to drive IDUA expression, the SB100X hyperactive transposase and transient cyclophosphamide immunosuppression we achieved therapeutic-level (>100 wild-type) stabilized expression for 1 year in 50% of C57BL/6 mice. To gain insights into the causes of variability in transgene expression, we quantified the rates of alpha-L-iduronidase activity decay vis-a-vis transposition and transgene maintenance using the data obtained in this and previous studies. Our analyses showed that immune responses are the most important variable to control in order to prevent loss of transgene expression. Cumulatively, our results allow transition to pre-clinical studies of SB-mediated alpha-L-iduronidase expression and correction of mucopolysaccharidosis type I in animal models.

Endocrine abnormalities in Townes-Brocks syndrome.

Townes-Brocks syndrome is a recognizable variable pattern of malformation caused by mutations to the SALL1 gene located on chromosome 16q12.1. Only three known cases of Townes-Brocks syndrome with proven SALL1 gene mutation and concurrent endocrine abnormalities have been previously documented to our knowledge [Kohlhase et al., 1999; Botzenhart et al., 2005; Choi et al., 2010]. We report on two unrelated patients with Townes-Brocks syndrome who share an identical SALL1 mutation (c.3414_3415delAT), who also have endocrine abnormalities. Patient 1 appears to be the first known case of growth hormone deficiency, and Patient 2 extends the number of documented mutation cases with hypothyroidism to four. We suspect endocrine abnormalities, particularly treatable deficiencies, may be an underappreciated component to Townes-Brocks syndrome.

Heterozygous missense mutations in SMARCA2 cause Nicolaides-Baraitser syndrome.

Nicolaides-Baraitser syndrome (NBS) is characterized by sparse hair, distinctive facial morphology, distal-limb anomalies and intellectual disability. We sequenced the exomes of ten individuals with NBS and identified heterozygous variants in SMARCA2 in eight of them. Extended molecular screening identified nonsynonymous SMARCA2 mutations in 36 of 44 individuals with NBS; these mutations were confirmed to be de novo when parental samples were available. SMARCA2 encodes the core catalytic unit of the SWI/SNF ATP-dependent chromatin remodeling complex that is involved in the regulation of gene transcription. The mutations cluster within sequences that encode ultra-conserved motifs in the catalytic ATPase region of the protein. These alterations likely do not impair SWI/SNF complex assembly but may be associated with disrupted ATPase activity. The identification of SMARCA2 mutations in humans provides insight into the function of the Snf2 helicase family.

Microdeletion 9q22.3 syndrome includes metopic craniosynostosis, hydrocephalus, macrosomia, and developmental delay.

Basal cell nevus syndrome (BCNS), also known as Gorlin syndrome (OMIM #109400) is a well-described rare autosomal dominant condition due to haploinsufficiency of PTCH1. With the availability of comparative genomic hybridization arrays, increasing numbers of individuals with microdeletions involving this locus are being identified. We present 10 previously unreported individuals with 9q22.3 deletions that include PTCH1. While 7 of the 10 patients (7 females, 3 males) did not meet strict clinical criteria for BCNS at the time of molecular diagnosis, almost all of the patients were too young to exhibit many of the diagnostic features. A number of the patients exhibited metopic craniosynostosis, severe obstructive hydrocephalus, and macrosomia, which are not typically observed in BCNS. All individuals older than a few months of age also had developmental delays and/or intellectual disability. Only facial features typical of BCNS, except in those with prominent midforeheads secondary to metopic craniosynostosis, were shared among the 10 patients. The deletions in these individuals ranged from 352 kb to 20.5 Mb in size, the largest spanning 9q21.33 through 9q31.2. There was significant overlap of the deleted segments among most of the patients. The smallest common regions shared among the deletions were identified in order to localize putative candidate genes that are potentially responsible for each of the non-BCNS features. These were a 929 kb region for metopic craniosynostosis, a 1.08 Mb region for obstructive hydrocephalus, and a 1.84 Mb region for macrosomia. Additional studies are needed to further characterize the candidate genes within these regions.