T Cell Repertoire Abnormality in Immunodeficiency Patients with DNA Repair and Methylation Defects

Both DNA damage response and methylation play a crucial role in antigen receptor recombination by creating a diverse repertoire in developing lymphocytes, but how their defects relate to T cell repertoire and phenotypic heterogeneity of immunodeficiency remains obscure. We studied the TCR repertoire in patients with the mutation in different genes (ATM, DNMT3B, ZBTB24, RAG1, DCLRE1C, and JAK3) and uncovered distinct characteristics of repertoire diversity. We propose that early aberrancies in thymus T cell development predispose to the heterogeneous phenotypes of the immunodeficiency spectrum. Shorter CDR3 lengths in ATM-deficient patients, resulting from a decreased number of nucleotide insertions during VDJ recombination in the pre-selected TCR repertoire, as well as the increment of CDR3 tyrosine residues, lead to the enrichment of pathology-associated TCRs, which may contribute to the phenotypes of ATM deficiency. Furthermore, patients with DNMT3B and ZBTB24 mutations who exhibit discrepant phenotypes present longer CDR3 lengths and reduced number of known pathology-associated TCRs.

Lack of GNAS re-methylation during oogenesis may be a cause of sporadic pseudohypoparathyroidism type Ib (PHP1B)

Context Pseudohypoparathyroidism type Ib (PHP1B) is characterized by hypocalcemia and hyperphosphatemia due to PTH-resistance in the proximal renal tubules. Maternal pathogenic STX16/GNAS variants leading to maternal epigenetic GNAS changes impair expression of the stimulatory G protein alpha-subunit (Gsα) thereby causing autosomal dominant PHP1B (AD-PHP1B). In contrast, genetic defects responsible for sporadic PHP1B (sporPHP1B) remain mostly unknown. Objective Determine whether PHP1B encountered after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) causes GNAS re-methylation defects similar to those in sporPHP1B. Design Retrospective analysis. Results Nine among thirty-six sporPHP1B patients investigated since 2000, all with LOM at the three maternal GNAS DMRs and gain-of-methylation (GOM) at the paternal NESP DMR, had been conceived through IVF or ICSI. Besides abnormal GNAS methylation, IVF/ICSI-PHP1B cases revealed no additional imprinting defects. Three of these PHP1B patients have dizygotic twins and four have IVF/ICSI-conceived siblings, all with normal GNAS methylation; two unaffected younger siblings were conceived naturally. Conclusion Sporadic and IVF/ICSI-conceived PHP1B patients revealed indistinguishable epigenetic changes at all four GNAS DMRs thus suggesting a similar underlying disease mechanism. Given that re-methylation at the three maternal DMRs occurs during oogenesis, male factors are unlikely to cause LOM post-fertilization. Instead, at least some of the sporPHP1B variants could be caused by a defect(s) in an oocyte-expressed gene that is required for fertility and for re-establishing maternal GNAS methylation imprints. It remains uncertain, however, whether lack of GNAS re-methylation alone impairs oocyte maturation because of insufficient Gsα expression, thus necessitating Assisted Reproductive Technology (ART) for conception.

Sporadic Pseudohypoparathyroidism Type 1B in Monozygotic Twins: Insights into the Pathogenesis of Methylation Defects

Context Sporadic pseudohypoparathyroidism type 1B (sporPHP1B) is an imprinting disease without a defined genetic cause, characterized by broad methylation changes in differentially methylated regions (DMRs) of the GNAS gene. Objective This work aims to provide insights into the causative event leading to the GNAS methylation defects through comprehensive molecular genetic analyses of a pair of female monozygotic twins concordant for sporPHP1B who were conceived naturally i.e., without assisted reproductive techniques. Methods Using the leukocyte genome of the twins and family members, we performed targeted bisulfite sequencing, methylation-sensitive restriction enzyme (MSRE)-qPCR, whole-genome sequencing (WGS), high-density SNP array, and Sanger sequencing. Results Methylation analyses by targeted bisulfite sequencing and MSRE-qPCR revealed almost complete loss of methylation at the GNAS AS, XL, and A/B DMRs and gain of methylation at the NESP55 DMR in the twins, but not in other family members. Except for the GNAS locus, we did not find apparent methylation defects at other imprinted genome loci of the twins. WGS, SNP array, and Sanger sequencing did not detect the previously described genetic defects associated with familial PHP1B. Sanger sequencing also ruled out any novel genetic alterations in the entire NESP55/AS region. However, the analysis of 28 consecutive SNPs could not exclude the possibility of paternal heterodisomy in a span of 22 kb comprising exon NESP55 and AS exon 5. Conclusion Our comprehensive analysis of a pair of monozygotic twins with sporPHP1B ruled out all previously described genetic causes. Twin concordance indicates that the causative event was an imprinting error earlier than the timing of monozygotic twinning.

Pseudohypoparathyroidism-Ib due to novel heterozygous stop gain mutation in exon 8 of STX16 gene: A case report

We report a case of pseudohypoparathyroidism type 1b (PHP1b) manifesting in childhood with hypocalcemic seizures. Symptomatic hypocalcemia is a common emergency in the pediatric age group with vitamin D deficiency being a frequent underlying etiology and PHP is rare. Patients with PHP1b do not depict the Albright’s hereditary osteodystrophy (AHO) phenotype typical of patients with PHP1a and pseudopseudohypoparathyroidism (PPHP). The resistance to parathyroid hormone (PTH) is documented mostly at renal tubular site of action in patients with PHP1b. Hypothyroidism is reported occasionally, signifying resistance to thyroid-stimulating hormone (TSH). Individuals with autosomal dominant and maternally inherited form of PHP harbor methylation defects at GNAS exon A/B, while sporadic and non-familial cases harbor methylation defects at other locus sites, including differentially methylated regions (GNAS-DMR). A novel heterozygous stop gain mutation c.C910T/p.Arg304X in exon 8 of the STX16 gene (Syntaxin 16) was observed in our case. Resistance seems limited to the renal action of PTH alone as currently, TSH level is normal. Maternal STX16 gene analysis results confirmed the modality of inheritance.

Elevated S-adenosylhomocysteine induces adipocyte dysfunction to promote alcohol-associated liver steatosis

It has been previously shown that chronic ethanol administration-induced increase in adipose tissue lipolysis and reduction in the secretion of protective adipokines collectively contribute to alcohol-associated liver disease (ALD) pathogenesis. Further studies have revealed that increased adipose S-adenosylhomocysteine (SAH) levels generate methylation defects that promote lipolysis. Here, we hypothesized that increased intracellular SAH alone causes additional related pathological changes in adipose tissue as seen with alcohol administration. To test this, we used 3-deazaadenosine (DZA), which selectively elevates intracellular SAH levels by blocking its hydrolysis. Fully differentiated 3T3-L1 adipocytes were treated in vitro for 48 h with DZA and analysed for lipolysis, adipokine release and differentiation status. DZA treatment enhanced adipocyte lipolysis, as judged by lower levels of intracellular triglycerides, reduced lipid droplet sizes and higher levels of glycerol and free fatty acids released into the culture medium. These findings coincided with activation of both adipose triglyceride lipase and hormone sensitive lipase. DZA treatment also significantly reduced adipocyte differentiation factors, impaired adiponectin and leptin secretion but increased release of pro-inflammatory cytokines, IL-6, TNF and MCP-1. Together, our results demonstrate that elevation of intracellular SAH alone by DZA treatment of 3T3-L1 adipocytes induces lipolysis and dysregulates adipokine secretion. Selective elevation of intracellular SAH by DZA treatment mimics ethanol’s effects and induces adipose dysfunction. We conclude that alcohol-induced elevations in adipose SAH levels contribute to the pathogenesis and progression of ALD.

High-Throughput Molecular Analysis of Pseudohypoparathyroidism 1b Patients Reveals Novel Genetic and Epigenetic Defects

Context Patients with pseudohypoparathyroidism type 1b (PHP1b) show disordered imprinting of the maternal GNAS allele or paternal uniparental disomy (UPD). Genetic deletions in STX16 or in upstream exons of GNAS are present in many familial but not sporadic cases. Objective Characterization of epigenetic and genetic defects in patients with PHP1b. Design and Patients DNA from 84 subjects, including 26 subjects with sporadic PHP1b, 27 affected subjects and 17 unaffected and/or obligate gene carriers from 12 PHP1b families, 11 healthy individuals, and 3 subjects with PHP1a was subjected to quantitative pyrosequencing of GNAS differentially methylated regions (DMRs), microarray analysis, and microsatellite haplotype analysis. Setting Academic medical center. Main Outcome Measurements Molecular pathology of PHP1b. Results Healthy subjects, unaffected family members and obligate carriers of paternal PHP1b alleles, and subjects with PHP1a showed normal methylation of all DMRs. All PHP1b subjects showed loss of methylation (LOM) at the exon A/B DMR. Affected members of nine PHP1b kindreds showed LOM only at the exon A/B DMR, which was associated with a 3-kb deletion of STX16 exons 4-6 in seven families and a novel deletion of STX16 and adjacent NEPEPL1 in one family. A novel NESP deletion was found in one of two other families with more extensive methylation defects. One sporadic PHP1b had UPD of 20q, two had 3-kb STX16 deletions, and five had apparent epigenetic mosaicism. Conclusions We found diverse patterns of defective methylation and identified novel or previously known mutations in 9 of 12 PHP1b families.

Defective folate metabolism causes germline epigenetic instability and distinguishes Hira as a phenotype inheritance biomarker

The mechanism behind transgenerational epigenetic inheritance is unclear, particularly through the maternal grandparental line. We previously showed that disruption of folate metabolism in mice by the Mtrr hypomorphic mutation results in transgenerational epigenetic inheritance of congenital malformations. Either maternal grandparent can initiate this phenomenon, which persists for at least four wildtype generations. Here, we use genome-wide approaches to reveal genetic stability in the Mtrr model and genome-wide differential DNA methylation in the germline of Mtrr mutant maternal grandfathers. We observe that, while epigenetic reprogramming occurs, wildtype grandprogeny and great grandprogeny exhibit transcriptional changes that correlate with germline methylation defects. One region encompasses the Hira gene, which is misexpressed in embryos for at least three wildtype generations in a manner that distinguishes Hira transcript expression as a biomarker of maternal phenotypic inheritance.

DNMT3A haploinsufficiency causes dichotomous DNA methylation defects at enhancers in mature human immune cells

DNMT3A encodes an enzyme that carries out de novo DNA methylation, which is essential for the acquisition of cellular identity and specialized functions during cellular differentiation. DNMT3A is the most frequently mutated gene in age-related clonal hematopoiesis. As such, mature immune cells harboring DNMT3A mutations can be readily detected in elderly persons. Most DNMT3A mutations associated with clonal hematopoiesis are heterozygous and predicted to cause loss of function, indicating that haploinsufficiency is the predominant pathogenic mechanism. Yet, the impact of DNMT3A haploinsufficiency on the function of mature immune cells is poorly understood. Here, we demonstrate that DNMT3A haploinsufficiency impairs the gain of DNA methylation at decommissioned enhancers, while simultaneously and unexpectedly impairing DNA demethylation of newly activated enhancers in mature human myeloid cells. The DNA methylation defects alter the activity of affected enhancers, leading to abnormal gene expression and impaired immune response. These findings provide insights into the mechanism of immune dysfunction associated with clonal hematopoiesis and acquired DNMT3A mutations.

Sporadic Pseudohypoparathyroidism 1B in Monozygotic Twins: Insights Into the Pathogenesis of Methylation Defects

Background: Pseudohypoparathyroidism (PHP) 1B is an imprinting disorder characterized by renal resistance to parathyroid hormone (PTH) without Albright Hereditary Osteodystrophy (AHO). PHP1B is associated with methylation defects at the GNAS differentially methylated regions (DMRs). In sporadic cases with PHP1B, the mechanistic basis of methylation defects remains to be solved, except in rare cases with uniparental disomy of chromosome 20. In addition, to date, monozygotic twin cases with sporadic PHP1B have not been reported. Clinical Case: The patients were 26-year-old Japanese female monozygotic twins. They had been born to nonconsanguineous parents after an uneventful pregnancy. Both twins had common biochemical features, including hypocalcemia, hyperphosphatemia, elevated PTH levels, and impaired urinary excretion of phosphorus and cAMP in response to teriparatide. They showed no signs of AHO. The serum calcium levels of their parents and brother were within the normal range, and family history was unremarkable. Based on these findings, the twins were diagnosed with PHP1B. Targeted bisulfite sequencing of the GNAS DMRs in all family members revealed almost complete gain-of-methylation at the NESP55 DMR, and loss-of-methylation at the AS, XL, and A/B DMRs in the twins, but not in other family members. Except for the GNAS locus, we did not find clear methylation defects in other imprinted genome loci in the twins. Methylation defects at the GNAS locus were further confirmed by methylation-sensitive restriction enzyme-qPCR. Whole-genome sequencing of the twins showed no pathogenic variants in the GNAS exons encoding the Gs alpha subunit. No large deletions or insertions were found at the STX16 locus or in the region from AS exon 5 to XL. Based on the SNP genotyping results, large paternal isodisomies in the GNAS DMRs were unlikely. Collectively, these results suggested that the twins had concordant methylation defects that are seen in the sporadic form of PHP1b. We speculate that an early developmental event before the twin splitting is responsible for the abnormal methylation of the GNAS DMRs. Conclusion: We report, for the first time, monozygotic twins with sporadic PHP1B who were phenotypically and epigenetically concordant. Our comprehensive molecular genetic analyses have thus far ruled out the previously described genetic defects underlying PHP1B. The current findings provide new insights into the mechanistic basis of the GNAS methylation defects in sporadic PHP1B.

Clinical and Molecular Diagnosis of Beckwith-Wiedemann Syndrome with Single- or Multi-Locus Imprinting Disturbance

Beckwith-Wiedemann syndrome (BWS) is a clinically and genetically heterogeneous overgrowth disease. BWS is caused by (epi)genetic defects at the 11p15 chromosomal region, which harbors two clusters of imprinted genes, IGF2/H19 and CDKN1C/KCNQ1OT1, regulated by differential methylation of imprinting control regions, H19/IGF2:IG DMR and KCNQ1OT1:TSS DMR, respectively. A subset of BWS patients show multi-locus imprinting disturbances (MLID), with methylation defects extended to other imprinted genes in addition to the disease-specific locus. Specific (epi)genotype-phenotype correlations have been defined in order to help clinicians in the classification of patients and referring them to a timely diagnosis and a tailored follow-up. However, specific phenotypic correlations have not been identified among MLID patients, thus causing a debate on the usefulness of multi-locus testing in clinical diagnosis. Finally, the high incidence of BWS monozygotic twins with discordant phenotypes, the high frequency of BWS among babies conceived by assisted reproductive technologies, and the female prevalence among BWS-MLID cases provide new insights into the timing of imprint establishment during embryo development. In this review, we provide an overview on the clinical and molecular diagnosis of single- and multi-locus BWS in pre- and post-natal settings, and a comprehensive analysis of the literature in order to define possible (epi)genotype-phenotype correlations in MLID patients.