Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency.

We previously reported two siblings with inherited PD-1 deficiency who died from autoimmune pneumonitis at 3 and 11 years of age after developing other autoimmune manifestations, including type 1 diabetes (T1D). We report here two siblings, aged 10 and 11 years, with neonatal-onset T1D (diagnosed at the ages of 1 day and 7 wk), who are homozygous for a splice-site variant of CD274 (encoding PD-L1). This variant results in the exclusive expression of an alternative, loss-of-function PD-L1 protein isoform in overexpression experiments and in the patients' primary leukocytes. Surprisingly, cytometric immunophenotyping and single-cell RNA sequencing analysis on blood leukocytes showed largely normal development and transcriptional profiles across lymphoid and myeloid subsets in the PD-L1-deficient siblings, contrasting with the extensive dysregulation of both lymphoid and myeloid leukocyte compartments in PD-1 deficiency. Our findings suggest that PD-1 and PD-L1 are essential for preventing early-onset T1D but that, unlike PD-1 deficiency, PD-L1 deficiency does not lead to fatal autoimmunity with extensive leukocytic dysregulation.

Chromosome 20p11.2 deletions cause congenital hyperinsulinism via the loss of FOXA2 or its regulatory elements.

Persistent congenital hyperinsulinism (HI) is a rare genetically heterogeneous condition characterised by dysregulated insulin secretion leading to life-threatening hypoglycaemia. For up to 50% of affected individuals screening of the known HI genes does not identify a disease-causing variant. Large deletions have previously been used to identify novel regulatory regions causing HI. Here, we used genome sequencing to search for novel large (>1 Mb) deletions in 180 probands with HI of unknown cause and replicated our findings in a large cohort of 883 genetically unsolved individuals with HI using off-target copy number variant calling from targeted gene panels. We identified overlapping heterozygous deletions in five individuals (range 3-8 Mb) spanning chromosome 20p11.2. The pancreatic beta-cell transcription factor gene, FOXA2, a known cause of HI was deleted in two of the five individuals. In the remaining three, we found a minimal deleted region of 2.4 Mb adjacent to FOXA2 that encompasses multiple non-coding regulatory elements that are in conformational contact with FOXA2. Our data suggests that the deletions in these three children may cause disease through the dysregulation of FOXA2 expression. These findings provide new insights into the regulation of FOXA2 in the beta-cell and confirm an aetiological role for chromosome 20p11.2 deletions in syndromic HI.

Functional characterization of HNF4A gene variants identify promoter and cell line specific transactivation effects.

Hepatocyte nuclear factor-4 alpha (HNF-4A) regulates genes with roles in glucose metabolism and ß-cell development. Although pathogenic HNF4A variants are commonly associated with maturity-onset diabetes of the young (MODY1; HNF4A-MODY), rare phenotypes also include hyperinsulinemic hypoglycemia, renal Fanconi syndrome and liver disease. While the association of rare functionally damaging HNF1A variants with HNF1A-MODY and type 2 diabetes is well established owing to robust functional assays, the impact of HNF4A variants on HNF-4A transactivation in tissues including the liver and kidney is less known, due to lack of similar assays. Our aim was to investigate the functional effects of seven HNF4A variants, located in the HNF-4A DNA binding domain and associated with different clinical phenotypes, by various functional assays and cell lines (transactivation, DNA binding, protein expression, nuclear localization) and in silico protein structure analyses. Variants R85W, S87N and R89W demonstrated reduced DNA binding to the consensus HNF-4A binding elements in the HNF1A promoter (35, 13 and 9%, respectively) and the G6PC promoter (R85W ~10%). While reduced transactivation on the G6PC promoter in HepG2 cells was shown for S87N (33%), R89W (65%) and R136W (35%), increased transactivation by R85W and R85Q was confirmed using several combinations of target promoters and cell lines. R89W showed reduced nuclear levels. In silico analyses supported variant induced structural impact. Our study indicates that cell line specific functional investigations are important to better understand HNF4A-MODY genotype-phenotype correlations, as our data supports ACMG/AMP interpretations of loss-of-function variants and propose assay-specific HNF4A control variants for future functional investigations.