GNAS Heterozygous Inactivation Differentially Affects Osteoclast-Specific Calcitonin Receptor Bioactivity in a Mouse Model of Albright Hereditary Osteodystrophy Based Upon Parental Inheritance

Albright hereditary osteodystrophy (AHO) is caused by the heterozygous inactivation of GNAS, encoding the α-stimulatory subunit (Gαs) of G protein-coupled receptors. Skeletal manifestations of AHO include adult short stature, brachydactyly and subcutaneous ossifications. AHO patients with maternally-derived GNAS mutations develop pseudohypoparathyroidism type 1A (PHP1A) and are obese with resistance to hormones requiring Gαs (eg., PTH, TSH and GHRH) due to tissue-specific GNAS imprinting. Paternally-derived GNAS mutations cause pseudopseudohypoparathyroidism (PPHP) in which patients have AHO skeletal features but do not develop severe obesity or hormonal resistance. Mouse models have shown loss of Gα s signaling in osteoblasts or osteoclasts leads to osteopenia, and suggest AHO patients would display a reduced bone mineral density (BMD). Interestingly, PHP1A patients have been shown to have normal to increased BMD without any correlation to body mass index or serum PTH measurement. Based on the differences observed clinically and hormonally between PHP1A and PPHP, we hypothesize that there may also be distinctions in overall bone remodeling between these two disorders due to GNAS imprinting. This study addressed whether the heterozygous inactivation of Gnas differentially affects Gα s-receptor bioactivity within osteoclasts (OCs) based upon parental inheritance. Bone Marrow Macrophages (BMMs) were harvested from our laboratory’s AHO mouse model with either maternally-inherited (Gnas+/-m) mutations correlating to PHP1A or paternally-inherited (Gnas+/-p) mutations correlating to PPHP. BMMs were exposed to 10-7M salmon calcitonin (sCT), 10-5M forskolin or PBS for 6 hrs. OC receptor activity was measured by fluorescent microscopy to visualize actin ring morphology and RT-PCR analysis of Gα s-PKA signaling transcripts Crem and Ramp3. Forskolin treatment displayed no significant variations in OC ring morphology or Crem and Ramp3 mRNA expression between Gnas+/-m, Gnas+/-p and WT cultures. Both WT and Gnas+/-p OCs displayed appropriate responses to sCT, as indicated by a significant disruption in actin ring morphology and increased Crem and Ramp3 mRNA expression when compared to vehicle-treated controls. SCT-treated Gnas+/-m OCs, however, displayed only mild disruptions in actin ring morphology, and we observed significant reductions in Ramp3 expression compared to WT as well as reductions in Crem compared to WT and Gnas+/-p. These data suggest evidence of partial calcitonin resistance within Gnas+/-m OCs due to impaired Gα s- signaling. These data correlate with previous clinical observations of calcitonin resistance in PHP1A patients. Because these findings were observed only within Gnas+/-m cultures, future work is warranted to determine whether this impaired receptor activity may be attributed to partial Gnas imprinting within OCs or the myeloid lineage.

DeepTrio: Variant Calling in Families Using Deep Learning

Every human inherits one copy of the genome from their mother and another from their father. Parental inheritance helps us understand the transmission of traits and genetic diseases, which often involve de novo variants and rare recessive alleles. Here we present DeepTrio, which learns to analyze child-mother-father trios from the joint sequence information, without explicit encoding of inheritance priors. DeepTrio learns how to weigh sequencing error, mapping error, and de novo rates and genome context directly from the sequence data. DeepTrio has higher accuracy on both Illumina and PacBio HiFi data when compared to DeepVariant. Improvements are especially pronounced at lower coverages (with 20x DeepTrio roughly equivalent to 30x DeepVariant). As DeepTrio learns directly from data, we also demonstrate extensions to exome calling solely by changing the training data. DeepTrio includes pre-trained models for Illumina WGS, Illumina exome, and PacBio HiFi.

Improved molecular diagnosis of patients with neonatal diabetes using a combined next-generation sequencing and MS-MLPA approach

We evaluated a methylation-specific multiplex-ligation-dependent probe amplification (MS-MLPA) assay for the molecular diagnosis of transient neonatal diabetes mellitus (TNDM) caused by 6q24 abnormalities and assessed the clinical utility of using this assay in combination with next generation sequencing (NGS) analysis for diagnosing patients with neonatal diabetes (NDM).: We performed MS-MLPA in 18 control samples and 42 retrospective NDM cases with normal bi-parental inheritance of chromosome 6. Next, we evaluated 22 prospective patients by combining NGS analysis of 11 NDM genes and the MS-MLPA assay.: 6q24 aberrations were identified in all controls and in 19% of patients with normal bi-parental inheritance of chromosome 6. The MS-MLPA/NGS combined approach identified a genetic cause in ~64% of patients with NDM of unknown etiology.MS-MLPA is a reliable method to identify all known 6q24 abnormalities and comprehensive testing of all causes reveals a causal mutation in ~64% of patients.