Hana Lango Allen

Activating germline mutations in STAT3 cause early-onset multi-organ autoimmune disease.

Monogenic causes of autoimmunity provide key insights into the complex regulation of the immune system. We report a new monogenic cause of autoimmunity resulting from de novo germline activating STAT3 mutations in five individuals with a spectrum of early-onset autoimmune disease, including type 1 diabetes. These findings emphasize the critical role of STAT3 in autoimmune disease and contrast with the germline inactivating STAT3 mutations that result in hyper IgE syndrome.

GATA6 haploinsufficiency causes pancreatic agenesis in humans

Understanding the regulation of pancreatic development is key for efforts to develop new regenerative therapeutic approaches for diabetes. Rare mutations in PDX1 and PTF1A can cause pancreatic agenesis, however, most instances of this disorder are of unknown origin. We report de novo heterozygous inactivating mutations in GATA6 in 15/27 (56%) individuals with pancreatic agenesis. These findings define the most common cause of human pancreatic agenesis and establish a key role for the transcription factor GATA6 in human pancreatic development.

Recessive mutations in a distal PTF1A enhancer cause isolated pancreatic agenesis

The contribution of cis-regulatory mutations to human disease remains poorly understood. Whole-genome sequencing can identify all noncoding variants, yet the discrimination of causal regulatory mutations represents a formidable challenge. We used epigenomic annotation in human embryonic stem cell (hESC)-derived pancreatic progenitor cells to guide the interpretation of whole-genome sequences from individuals with isolated pancreatic agenesis. This analysis uncovered six different recessive mutations in a previously uncharacterized ∼400-bp sequence located 25 kb downstream of PTF1A (encoding pancreas-specific transcription factor 1a) in ten families with pancreatic agenesis. We show that this region acts as a developmental enhancer of PTF1A and that the mutations abolish enhancer activity. These mutations are the most common cause of isolated pancreatic agenesis. Integrating genome sequencing and epigenomic annotation in a disease-relevant cell type can thus uncover new noncoding elements underlying human development and disease.

The effect of early, comprehensive genomic testing on clinical care in neonatal diabetes: an international cohort study

Summary Background Traditional genetic testing focusses on analysis of one or a few genes according to clinical features; this approach is changing as improved sequencing methods enable simultaneous analysis of several genes. Neonatal diabetes is the presenting feature of many discrete clinical phenotypes defined by different genetic causes. Genetic subtype defines treatment, with improved glycaemic control on sulfonylurea treatment for most patients with potassium channel mutations. We investigated the effect of early, comprehensive testing of all known genetic causes of neonatal diabetes. Methods In this large, international, cohort study, we studied patients with neonatal diabetes diagnosed with diabetes before 6 months of age who were referred from 79 countries. We identified mutations by comprehensive genetic testing including Sanger sequencing, 6q24 methylation analysis, and targeted next-generation sequencing of all known neonatal diabetes genes. Findings Between January, 2000, and August, 2013, genetic testing was done in 1020 patients (571 boys, 449 girls). Mutations in the potassium channel genes were the most common cause (n=390) of neonatal diabetes, but were identified less frequently in consanguineous families (12% in consanguineous families vs 46% in non-consanguineous families; p<0·0001). Median duration of diabetes at the time of genetic testing decreased from more than 4 years before 2005 to less than 3 months after 2012. Earlier referral for genetic testing affected the clinical phenotype. In patients with genetically diagnosed Wolcott-Rallison syndrome, 23 (88%) of 26 patients tested within 3 months from diagnosis had isolated diabetes, compared with three (17%) of 18 patients referred later (>4 years; p<0·0001), in whom skeletal and liver involvement was common. Similarly, for patients with genetically diagnosed transient neonatal diabetes, the diabetes had remitted in only ten (10%) of 101 patients tested early (<3 months) compared with 60 (100%) of the 60 later referrals (p<0·0001). Interpretation Patients are now referred for genetic testing closer to their presentation with neonatal diabetes. Comprehensive testing of all causes identified causal mutations in more than 80% of cases. The genetic result predicts the best diabetes treatment and development of related features. This model represents a new framework for clinical care with genetic diagnosis preceding development of clinical features and guiding clinical management. Funding Wellcome Trust and Diabetes UK.

An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy

DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for lagging-strand DNA synthesis during DNA replication. It carries out this synthesis with high fidelity owing to its intrinsic 3′- to 5′-exonuclease activity, which confers proofreading ability. Missense mutations affecting the exonuclease domain of POLD1 have recently been shown to predispose to colorectal and endometrial cancers. Here we report a recurring heterozygous single-codon deletion in POLD1 affecting the polymerase active site that abolishes DNA polymerase activity but only mildly impairs 3′- to 5′-exonuclease activity. This mutation causes a distinct multisystem disorder that includes subcutaneous lipodystrophy, deafness, mandibular hypoplasia and hypogonadism in males. This discovery suggests that perturbing the function of the ubiquitously expressed POLD1 polymerase has unexpectedly tissue-specific effects in humans and argues for an important role for POLD1 function in adipose tissue homeostasis.

Analysis of Transcription Factors Key for Mouse Pancreatic Development Establishes NKX2-2 and MNX1 Mutations as Causes of Neonatal Diabetes in Man

Summary Understanding transcriptional regulation of pancreatic development is required to advance current efforts in developing beta cell replacement therapies for patients with diabetes. Current knowledge of key transcriptional regulators has predominantly come from mouse studies, with rare, naturally occurring mutations establishing their relevance in man. This study used a combination of homozygosity analysis and Sanger sequencing in 37 consanguineous patients with permanent neonatal diabetes to search for homozygous mutations in 29 transcription factor genes important for murine pancreatic development. We identified homozygous mutations in 7 different genes in 11 unrelated patients and show that NKX2-2 and MNX1 are etiological genes for neonatal diabetes, thus confirming their key role in development of the human pancreas. The similar phenotype of the patients with recessive mutations and mice with inactivation of a transcription factor gene support there being common steps critical for pancreatic development and validate the use of rodent models for beta cell development.

GATA4 mutations are a cause of neonatal and childhood-onset diabetes

The GATA family zinc finger transcription factors GATA4 and GATA6 are known to play important roles in the development of the pancreas. In mice, both Gata4 and Gata6 are required for pancreatic development. In humans, GATA6 haploinsufficiency can cause pancreatic agenesis and heart defects. Congenital heart defects also are common in patients with GATA4 mutations and deletions, but the role of GATA4 in the developing human pancreas is unproven. We report five patients with deletions (n = 4) or mutations of the GATA4 gene who have diabetes and a variable exocrine phenotype. In four cases, diabetes presented in the neonatal period (age at diagnosis 1–7 days). A de novo GATA4 missense mutation (p.N273K) was identified in a patient with complete absence of the pancreas confirmed at postmortem. This mutation affects a highly conserved residue located in the second zinc finger domain of the GATA4 protein. In vitro studies showed reduced DNA binding and transactivational activity of the mutant protein. We show that GATA4 mutations/deletions are a cause of neonatal or childhood-onset diabetes with or without exocrine insufficiency. These results confirm a role for GATA4 in normal development of the human pancreas.

Next generation sequencing of chromosomal rearrangements in patients with split-hand/split-foot malformation provides evidence for DYNC1I1 exonic enhancers of DLX5/6 expression in humans

Objective Split-hand/foot malformation type 1 is an autosomal dominant condition with reduced penetrance and variable expression. We report three individuals from two families with split-hand/split-foot malformation (SHFM) in whom next generation sequencing was performed to investigate the cause of their phenotype. Methods and results The first proband has a de novo balanced translocation t(2;7)(p25.1;q22) identified by karyotyping. Whole genome sequencing showed that the chromosome 7 breakpoint is situated within the SHFM1 locus on chromosome 7q21.3. This separates the DYNC1I1 exons recently identified as limb enhancers in mouse studies from their target genes, DLX5 and DLX6. In the second family, X-linked recessive inheritance was suspected and exome sequencing was performed to search for a mutation in the affected proband and his uncle. No coding mutation was found within the SHFM2 locus at Xq26 or elsewhere in the exome, but a 106 kb deletion within the SHFM1 locus was detected through copy number analysis. Genome sequencing of the deletion breakpoints showed that the DLX5 and DLX6 genes are disomic but the putative DYNC1I1 exon 15 and 17 enhancers are deleted. Conclusions Exome sequencing identified a 106 kb deletion that narrows the SHFM1 critical region from 0.9 to 0.1 Mb and confirms a key role of DYNC1I1 exonic enhancers in normal limb formation in humans.

An exome sequencing strategy to diagnose lethal autosomal recessive disorders.

Rare disorders resulting in prenatal or neonatal death are genetically heterogeneous. For some conditions, affected fetuses can be diagnosed by ultrasound scan, but this is not usually possible until mid-gestation. There is often limited fetal DNA available for investigation. We investigated a strategy for diagnosing autosomal recessive lethal disorders in non-consanguineous pedigrees with multiple affected fetuses. Exome sequencing was performed to identify genes where each parent is heterozygous for a rare non-synonymous-coding or splicing variant. Putative pathogenic variants were tested for cosegregation in affected fetuses and unaffected siblings. In eight couples of European ancestry, we found on average 1.75 genes (range 0–4) where both parents were heterozygous for rare potentially deleterious variants. A proof-of-principle study detected heterozygous DYNC2H1 variants in a couple whose five fetuses had short-rib polydactyly. Prospective analysis of two couples with multiple pregnancy terminations for fetal akinesia syndrome was performed and a diagnosis was obtained in both the families. The first couple were each heterozygous for a previously reported GLE1 variant, p.Arg569His or p.Val617Met; both were inherited by their two affected fetuses. The second couple were each heterozygous for a novel RYR1 variant, c.14130-2A>G or p.Ser3074Phe; both were inherited by their three affected fetuses but not by their unaffected child. Biallelic GLE1 and RYR1 disease-causing variants have been described in other cases with fetal akinesia syndrome. We conclude that exome sequencing of parental samples can be an effective tool for diagnosing lethal recessive disorders in outbred couples. This permits early prenatal diagnosis in future pregnancies.

Polygenic Risk Variants for Type 2 Diabetes Susceptibility Modify Age at Diagnosis in Monogenic HNF1A Diabetes

OBJECTIVE Mutations in the HNF1A gene are the most common cause of maturity-onset diabetes of the young (MODY). There is a substantial variation in the age at diabetes diagnosis, even within families where diabetes is caused by the same mutation. We investigated the hypothesis that common polygenic variants that predispose to type 2 diabetes might account for the difference in age at diagnosis. RESEARCH DESIGN AND METHODS Fifteen robustly associated type 2 diabetes variants were successfully genotyped in 410 individuals from 203 HNF1A-MODY families, from two study centers in the U.K. and Norway. We assessed their effect on the age at diagnosis both individually and in a combined genetic score by summing the number of type 2 diabetes risk alleles carried by each patient. RESULTS We confirmed the effects of environmental and genetic factors known to modify the age at HNF1A-MODY diagnosis, namely intrauterine hyperglycemia (−5.1 years if present, P = 1.6 × 10−10) and HNF1A mutation position (−5.2 years if at least two isoforms affected, P = 1.8 × 10−2). Additionally, our data showed strong effects of sex (females diagnosed 3.0 years earlier, P = 6.0 × 10−4) and age at study (0.3 years later diagnosis per year increase in age, P = 4.7 × 10−38). There were no strong individual single nucleotide polymorphism effects; however, in the combined genetic score model, each additional risk allele was associated with 0.35 years earlier diabetes diagnosis (P = 5.1 × 10−3). CONCLUSIONS We show that type 2 diabetes risk variants of modest effect sizes reduce the age at diagnosis in HNF1A-MODY. This is one of the first studies to demonstrate that clinical characteristics of a monogenic disease can be modified by common polygenic variants.