Epigenomic analysis of KLF1 haploinsufficiency in primary human erythroblasts

Haploinsufficiency for the erythroid-specific transcription factor KLF1 is associated with hereditary persistence of fetal hemoglobin (HPFH). Increased HbF ameliorates the symptoms of β-hemoglobinopathies and downregulation of KLF1 activity has been proposed as a potential therapeutic strategy. However, the feasibility of this approach has been challenged by the observation that KLF1 haploinsufficient individuals with the same KLF1 variant, within the same family, display a wide range of HbF levels. This phenotypic variability is not readily explained by co-inheritance of known HbF-modulating variants in the HBB, HBS1L-MYB and/or BCL11A loci. We studied cultured erythroid progenitors obtained from Maltese individuals in which KLF1 p.K288X carriers display HbF levels ranging between 1.3 and 12.3% of total Hb. Using a combination of gene expression analysis, chromatin accessibility assays and promoter activity tests we find that variation in expression of the wildtype KLF1 allele may explain a significant part of the variability in HbF levels observed in KLF1 haploinsufficiency. Our results have general bearing on the variable penetrance of haploinsufficiency phenotypes and on conflicting interpretations of pathogenicity of variants in other transcriptional regulators such as EP300, GATA2 and RUNX1.

The Study of Alternative Splicing Events in Human Induced Pluripotent Stem Cells From a Down's Syndrome Patient

Down's syndrome (DS) is one of the most commonly known disorders with multiple congenital disabilities. Besides severe cognitive impairment and intellectual disability, individuals with DS also exhibit additional phenotypes of variable penetrance and severity, with one or more comorbid conditions, including Alzheimer's disease, congenital heart disease, or leukemia. Various vital genes and regulatory networks had been studied to reveal the pathogenesis of the disease. Nevertheless, very few studies have examined alternative splicing. Alternative splicing (AS) is a regulatory mechanism of gene expression when making one multi-exon protein-coding gene produce more than one unique mature mRNA. We employed the GeneChip Human Transcriptome Array 2.0 (HTA 2.0) for the global gene analysis with hiPSCs from DS and healthy individuals. Examining differentially expressed genes (DEGs) in these groups and focusing on specific transcripts with AS, 466 up-regulated and 722 down-regulated genes with AS events were identified. These genes were significantly enriched in biological processes, such as cell adhesion, cardiac muscle contraction, and immune response, through gene ontology (GO) analysis of DEGs. Candidate genes, such as FN1 were further explored for potentially playing a key role in DS. This study provides important insights into the potential role that AS plays in DS.

Cellular models and therapeutic perspectives in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a clinically heterogeneous cardiac disease that is mainly characterized by left ventricular hypertrophy in the absence of any additional cardiac or systemic disease. HCM is genetically heterogeneous, inherited mainly in an autosomal dominant pattern, and so far pathogenic variants have been identified in more than 20 genes, mostly encoding proteins of the cardiac sarcomere. Based on its variable penetrance and expressivity, pathogenicity of newly identified variants often remains unsolved, underlining the importance of cellular and tissue-based models that help to uncover causative genetic alterations and, additionally, provide appropriate systems for the analysis of disease hallmarks as well as for the design and application of new therapeutic strategies like drug screenings and genome/base editing approaches. Here, we review the current state of cellular and tissue-engineered models and provide future perspectives for personalized therapeutic strategies of HCM.

A new murine Rpl5(uL18) mutation provides a unique model of variably penetrant Diamond Blackfan Anemia

Ribosome dysfunction is implicated in multiple abnormal developmental and disease states in humans. Heterozygous germline mutations in genes encoding ribosomal proteins (RPs) are found in the majority of individuals with Diamond Blackfan anemia (DBA) while somatic mutations have been implicated in a variety of cancers and other disorders. Ribosomal protein-deficient animal models show variable phenotypes and penetrance, similar to human DBA patients. Here we characterized a novel ENU mouse mutant (Skax23m1Jus) with growth and skeletal defects, cardiac malformations and increased mortality. Following genetic mapping and whole exome sequencing, we identified an intronic Rpl5 mutation, which segregated with all affected mice. This mutation was associated with decreased ribosome generation, consistent with Rpl5 haploinsufficiency. Rpl5Skax23-Jus/+ mutant animals had a profound delay in erythroid maturation and increased mortality at embryonic day E12.5, which improved by E14.5. Surviving mutant animals had a macrocytic anemia at birth as well as evidence of ventricular septal defect (VSD). Surviving adult and aged mice exhibited no hematopoietic defect or VSD. We propose that this novel Rpl5Skax23-Jus mutant mouse will be useful to study the factors influencing the variable penetrance that is observed in DBA.

Identification of disease-relevant modulators of the SHH pathway in the developing brain

ABSTRACT Pathogenic gene variants in humans that affect the sonic hedgehog (SHH) pathway lead to severe brain malformations with variable penetrance due to unknown modifier genes. To identify such modifiers, we established novel congenic mouse models. LRP2-deficient C57BL/6N mice suffer from heart outflow tract defects and holoprosencephaly caused by impaired SHH activity. These defects are fully rescued on a FVB/N background, indicating a strong influence of modifier genes. Applying comparative transcriptomics, we identified Pttg1 and Ulk4 as candidate modifiers upregulated in the rescue strain. Functional analyses showed that ULK4 and PTTG1, both microtubule-associated proteins, are positive regulators of SHH signaling, rendering the pathway more resilient to disturbances. In addition, we characterized ULK4 and PTTG1 as previously unidentified components of primary cilia in the neuroepithelium. The identification of genes that powerfully modulate the penetrance of genetic disturbances affecting the brain and heart is likely relevant to understanding the variability in human congenital disorders.

Acral Lentiginous melanoma arising in Brauer-Buschke-Fischer plantar keratoderma

Type I Punctuate palmoplantar keratoderma or Brauer-Buschke-Fischer keratoderma (BBFK) is a rare autosomal dominant keratinisation disorder with variable penetrance. \r\n The emergence of acral lentiginous melanoma on palmoplantar keratoderma is rarely reported. Herein, we present a case of a 53-year-old woman with a history of BBFK in both palms and soles, who developed an acral lentiginous melanoma. The clinical examination showed multiple tiny hyperkeratotic and translucent papules on both palms and soles. On the right sole, she developed an acral lentiginous melanoma over a hyperkeratoic area. This case presentation illustrates a rare association of malignant melanoma and BBFK and seeks to enhance further investigations to determine its pathophysiological mechanism.\r\n

Description of a SDHD c.129G>A (p.W43X) Mutation With Variable Presentation in Multiple Family Members

Approximately 40% of paragangliomas and pheochromocytomas are attributed to hereditary mutations. SDHD mutations account for 7% of inherited mutations (PGL1 syndrome), is maternally imprinted and has variable penetrance. SDHD pathogenic variants (PV) have been previously described extensively in Dutch pedigrees, with a varying lifetime risk for tumor development. Here we report a large family (Fig 1) displaying a SDHD c.129G>A (p.W43X) variation in 12 family members, 10 of whom had screening or tumor history available. The presentation and age of diagnosis in family members showed variable penetrance. Age at first diagnosis of a pheochromocytoma/paraganglioma ranged from 10 - 45 years. Family members displayed bilateral pheochromocytomas, bilateral carotid body tumors, and paragangliomas of the head, neck and trunk with variable recurrence rates (none to multiple). No malignant lesions were detected to date. Pheochromocytomas were norepinephrine producing. Paragangliomas ranged from non-functional to dopamine and norepinephrine producing. Compared to previous reports of other SDHD mutations, the SDHD c.129G>A (p.W43X) variation displayed an earlier age at first diagnosis with a highly variable phenotype ranging from one benign, non-secreting paraganglioma to bilateral pheochromocytomas and recurrent parganagliomas along the parasympathetic chain from head to abdomen. This report contributes to the evolving understanding of the phenotypic presentations of various genetic mutations. We propose that expert guidelines that suggest screening family members with the SDHD c.129G>A (p.W43X) variation at the age of 8 for early detection of pheochromocytomas and paragangliomas is beneficial.

Two Cases of Tuberous Sclerosis With Multiple Rhabdomyoma Succumbing to Sudden Cardiac Death

Tuberous sclerosis complex is an autosomal dominant condition with variable penetrance. It is characterized by tuberose deposits in various organ systems. Although clinical features predominate neurocutaneous manifestations, cardiac, kidney, and lung involvement are common. Cardiac involvement is marked by the presence of multiple rhabdomyomas and in some cases arrhythmias. In the absence of symptoms, rhabdomyomas require no specific treatment. However, cardiac arrhythmias are unpredictable and may be the cause of sudden cardiac death in some cases. Although treatment is mainly symptomatic, drugs like rapamycin have shown promise in the regression of astrocytomas and angiofibromas. Here, we are reporting two cases of tuberous sclerosis of which one succumbed to arrhythmias and the other to possible sudden cardiac death.