Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-associated skin cancer. Robust cellular immune responses are associated with excellent outcomes in MCC patients, but these responses are typically absent. We determined the prevalence and reversibility of class I MHC (MHC-I) downregulation in MCC, a potentially reversible immune evasion mechanism. Cell surface MHC-I expression was assessed on 5 MCC cell lines using flow cytometry as well as immunohistochemistry on tissue microarrays representing 114 patients. Three additional patients were included that had received intralesional interferon treatment and had evaluable specimens before and after treatment. mRNA expression analysis of antigen presentation pathway genes from 35 MCC tumors was used to examine mechanisms of downregulation. 84% of MCCs (total n=114) demonstrated reduced MHC-I expression as compared to surrounding tissues, and 51% had poor or undetectable MHC-1 expression. Expression of MHC-I was lower in polyomavirus-positive MCCs as compared to virus-negative MCCs (p<0.01). The MHC-I downregulation mechanism was multifactorial and did not depend solely on HLA gene expression. Treatment of MCC cell lines with ionizing radiation, etoposide, or interferon (IFN) resulted in MHC-I upregulation, with IFNs strongly upregulating MHC-I expression in vitro and in 3 of 3 patients treated with intralesional IFNs. MCC tumors may be amenable to immunotherapy, but downregulation of MHC-I is frequently present in these tumors, particularly those that are polyomavirus-positive. This downregulation is reversible with any of several clinically available treatments that may thus promote the effectiveness of immune stimulating therapies for MCC.

Key Points Germ line biallelic loss-of-function THPO mutations cause BMF. Marrow failure due to THPO mutations is characterized by poor graft function after transplantation but responds to THPO receptor agonists.

Objectives Few genetic causes of exocrine pancreatic dysfunction have been described to date. We identified a family with multiple affected members manifesting exocrine pancreatic dysfunction. Additional associated features included facial rash, sparse hair, hypohidrosis, and swelling of the extremities. The transmission pattern of these clinical features was consistent with an autosomal dominant mode of inheritance. The two proband siblings also had transient elevated liver transaminases with hepatic steatosis early in life. This study identifies the genetic cause of exocrine pancreatic dysfunction in this family. Methods Whole exome sequencing was performed to identify the genetic cause of exocrine pancreatic dysfunction. Results A heterozygous germline in-frame deletion in the gene FAM111B (c.1261_1263delAAG, p.Lys421del) co-segregated with the phenotype: the variant was present in all affected relatives genotyped and absent in all unaffected relatives genotyped. The variant is also absent from public control sequence databases. Conclusions Our findings implicate FAM111B in autosomal dominantly inheritable exocrine pancreatic dysfunction.

Severe thrombocytopenia, characterized by dysplastic megakaryocytes and intracranial bleeding, was diagnosed in six individuals from a consanguineous kindred. Three of the individuals were successfully treated by bone marrow transplant. Whole-exome sequencing and homozygosity mapping of multiple family members, coupled with whole-genome sequencing to reveal shared non-coding variants, revealed one potentially functional variant segregating with thrombocytopenia under a recessive model: GALE p.R51W (c.C151T, NM_001127621). The mutation is extremely rare (allele frequency = 2.5 × 10−05), and the likelihood of the observed co-segregation occurring by chance is 1.2 × 10−06. GALE encodes UDP-galactose-4-epimerase, an enzyme of galactose metabolism and glycosylation responsible for two reversible reactions: interconversion of UDP-galactose with UDP-glucose and interconversion of UDP-N-acetylgalactosamine with UDP-N-acetylglucosamine. The mutation alters an amino acid residue that is conserved from yeast to humans. The variant protein has both significantly lower enzymatic activity for both interconversion reactions and highly significant thermal instability. Proper glycosylation is critical to normal hematopoiesis, in particular to megakaryocyte and platelet development, as reflected in the presence of thrombocytopenia in the context of congenital disorders of glycosylation. Mutations in GALE have not previously been associated with thrombocytopenia. Our results suggest that GALE p.R51W is inadequate for normal glycosylation and thereby may impair megakaryocyte and platelet development. If other mutations in GALE are shown to have similar consequences, this gene may be proven to play a critical role in hematopoiesis.

is essential for repair of DNA double-strand breaks by homologous recombination, and hence for survival. Complete loss of its function is lethal during early embryonic development. Patients who are compound heterozygous for truncating mutations and missense alleles that retain some DNA repair capacity may survive, albeit with very high risk of early onset breast or ovarian cancer and features of Fanconi anemia. However, a mechanism enabling survival of patients homozygous for truncating mutations has not been described. We studied two unrelated families in which four children presented with multiple congenital anomalies and severe chromosomal fragility. One child developed T cell acute lymphocytic leukemia (ALL), and a second child developed neuroblastoma. Each of the four children was homozygous for a nonsense mutation in exon 11. Homozygosity for the nonsense mutations was viable thanks to the presence of a naturally occurring alternative splice donor in exon 11 that lies 5' of the mutations. The mutations did not affect the alternative splice site, but transcription from it produced an in-frame message with deletion of 3,309 bp. The translated BRCA1 protein was only 40% of normal length, but with intact N- and C-terminal sequences. These patients extend the range of-related phenotypes and illustrate how naturally occurring alternative splicing can enable survival, albeit with severe consequences, of otherwise lethal genotypes of an essential gene.

This unit describes the selection of aptamers from a pool of single-stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate an ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription.

No abstract

This unit describes the selection of aptamers from a pool of single-stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate an ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription.