Inherited thrombocytopenia associated with mutation of UDP-galactose-4-epimerase (GALE).

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.

Mechanism for survival of homozygous nonsense mutations in the tumor suppressor gene BRCA1.

BRCA1 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 BRCA1 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 BRCA1 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 BRCA1 exon 11. Homozygosity for the nonsense mutations was viable thanks to the presence of a naturally occurring alternative splice donor in BRCA1 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 BRCA1 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 BRCA1-related phenotypes and illustrate how naturally occurring alternative splicing can enable survival, albeit with severe consequences, of otherwise lethal genotypes of an essential gene.

Bone marrow failure unresponsive to bone marrow transplant is caused by mutations in thrombopoietin.

We report 5 individuals in 3 unrelated families with severe thrombocytopenia progressing to trilineage bone marrow failure (BMF). Four of the children received hematopoietic stem cell transplants and all showed poor graft function with persistent severe cytopenias even after repeated transplants with different donors. Exome and targeted sequencing identified mutations in the gene encoding thrombopoietin (THPO): THPO R99W, homozygous in affected children in 2 families, and THPO R157X, homozygous in the affected child in the third family. Both mutations result in a lack of THPO in the patients' serum. For the 2 surviving patients, improvement in trilineage hematopoiesis was achieved following treatment with a THPO receptor agonist. These studies demonstrate that biallelic loss-of-function mutations in THPO cause BMF, which is unresponsive to transplant due to a hematopoietic cell-extrinsic mechanism. These studies provide further support for the critical role of the MPL-THPO pathway in hematopoiesis and highlight the importance of accurate genetic diagnosis to inform treatment decisions for BMF.

FAM111B Mutation Is Associated With Inherited Exocrine Pancreatic Dysfunction.

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 2 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) cosegregated 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.

Downregulation of MHC-I expression is prevalent but reversible in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-associated skin cancer. Robust cellular immune responses are associated with excellent outcomes in patients with MCC, but these responses are typically absent. We determined the prevalence and reversibility of major histocompatibility complex class I (MHC-I) downregulation in MCC, a potentially reversible immune-evasion mechanism. Cell-surface MHC-I expression was assessed on five MCC cell lines using flow cytometry as well as immunohistochemistry on tissue microarrays representing 114 patients. Three additional patients were included who had received intralesional IFN 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 the mechanisms of downregulation. Of note, 84% of MCCs (total n = 114) showed reduced MHC-I expression as compared with surrounding tissues, and 51% had poor or undetectable MHC-I expression. Expression of MHC-I was lower in polyomavirus-positive MCCs than in polyomavirus-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 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 positive for polyomavirus. This downregulation is reversible with any of several clinically available treatments that may thus promote the effectiveness of immune-stimulating therapies for MCC.