Rare Sequence Variants Research Articles

9242 SOX11 mutations cause hypogonadotropic hypogonadism through both hypothalamic and pituitary level deficits

Abstract Disclosure: B. Sun: None. S. Stockman: None. M. Stamou: None. L. Plummer: None. K. Salnikov: None. D. Kotan: None. K. Topaloglu: None. S.B. Seminara: None. R. Balasubramanian: None. AbstractPurpose: Human SOX11 mutations cause Coffin-Siris Syndrome (CSS) which is clinically characterized by aplasia or hypoplasia of the distal phalanx/nail of the fifth digit, developmental delay, cognitive impairment, and distinctive facial features. Recent studies show hypogonadotropic hypogonadism (HH) occurs in ∼20% of patients with SOX11 mutations. The genotypic-phenotypic spectrum of SOX11 mutations in patients presenting with Idiopathic Hypogonadotropic Hypogonadism (IHH) is unknown. Methods: Exome sequencing data from 1810 unrelated IHH probands (anosmic and normosmic forms) were reviewed for SOX11 rare sequence variants (RSVs) [minor allele frequency <0.1%]. Rare variant association analysis (RVAS) and domain-based enrichment for SOX11 RSVs was performed between the IHH and gnomAD population cohort. RSVs predicted as pathogenic/likely pathogenic by ACMG criteria were deemed as pathogenic RSVs and detailed phenotypic analysis was undertaken for these variants. Results: A total of 4 pathogenic SOX11 RSVs were identified in 5 IHH cases. RVAS analysis showed that SOX11 inactivating RSVs (frameshift/non-sense) across the entire protein was enriched in the IHH cohort (2 RSVs in 3 IHH cases (p.S303X [de-novo]; p p.S345Afs*13); p<0.0001) while SOX11 missense RSVs was enriched only within the SOX11-HMG domain (2 RSVs in 2 IHH cases (p.G84D [de-novo]; p.P114S; p=0.003). Phenotypic review of individuals with pathogenic SOX11-RSVs revealed a spectrum of endocrine defects including: anosmic and normosmic forms of IHH, growth-hormone (GH) deficiency, neuroimaging defects (pituitary microadenoma and hypothalamic mass), and hypothyroidism. Additionally, we also identified a pathogenic SOX11 HMG domain RSV in a patient with functional HH (p.R100Q). CSS-associated features were present in almost all individuals with pathogenic SOX11 RSVs. Conclusions: This study validates SOX11 mutations as causal for IHH and reiterates the critical importance of the SOX11-HMG domain in disease pathogenesis. The spectrum of endocrine abnormalities in SOX11-related human disorders includes IHH, functional HH, GH deficiency, pituitary lesions, and hypothyroidism. The association of congenital anosmic and normosmic forms of IHH, functional HH, and anatomic abnormalities of the pituitary gland suggest that the pathogenesis of HH in SOX11-disorder may stem from both hypothalamic and pituitary level deficits. Presentation: 6/1/2024

8474 SOX11 mutations cause hypogonadotropic hypogonadism through both hypothalamic and pituitary level deficits

Abstract Disclosure: B. Sun: None. S. Stockman: None. M. Stamou: None. L. Plummer: None. K. Salnikov: None. D. Kotan: None. K. Topaloglu: None. S.B. Seminara: None. R. Balasubramanian: None. AbstractPurpose: Human SOX11 mutations cause Coffin-Siris Syndrome (CSS) which is clinically characterized by aplasia or hypoplasia of the distal phalanx/nail of the fifth digit, developmental delay, cognitive impairment, and distinctive facial features. Recent studies show hypogonadotropic hypogonadism (HH) occurs in ∼20% of patients with SOX11 mutations. The genotypic-phenotypic spectrum of SOX11 mutations in patients presenting with Idiopathic Hypogonadotropic Hypogonadism (IHH) is unknown. Methods: Exome sequencing data from 1810 unrelated IHH probands (anosmic and normosmic forms) were reviewed for SOX11 rare sequence variants (RSVs) [minor allele frequency <0.1%]. Rare variant association analysis (RVAS) and domain-based enrichment for SOX11 RSVs was performed between the IHH and gnomAD population cohort. RSVs predicted as pathogenic/likely pathogenic by ACMG criteria were deemed as pathogenic RSVs and detailed phenotypic analysis was undertaken for these variants. Results: A total of 4 pathogenic SOX11 RSVs were identified in 5 IHH cases. RVAS analysis showed that SOX11 inactivating RSVs (frameshift/non-sense) across the entire protein was enriched in the IHH cohort (2 RSVs in 3 IHH cases (p.S303X [de-novo]; p p.S345Afs*13); p<0.0001) while SOX11 missense RSVs was enriched only within the SOX11-HMG domain (2 RSVs in 2 IHH cases (p.G84D [de-novo]; p.P114S; p=0.003). Phenotypic review of individuals with pathogenic SOX11-RSVs revealed a spectrum of endocrine defects including: anosmic and normosmic forms of IHH, growth-hormone (GH) deficiency, neuroimaging defects (pituitary microadenoma and hypothalamic mass), and hypothyroidism. Additionally, we also identified a pathogenic SOX11 HMG domain RSV in a patient with functional HH (p.R100Q). CSS-associated features were present in almost all individuals with pathogenic SOX11 RSVs. Conclusions: This study validates SOX11 mutations as causal for IHH and reiterates the critical importance of the SOX11-HMG domain in disease pathogenesis. The spectrum of endocrine abnormalities in SOX11-related human disorders includes IHH, functional HH, GH deficiency, pituitary lesions, and hypothyroidism. The association of congenital anosmic and normosmic forms of IHH, functional HH, and anatomic abnormalities of the pituitary gland suggest that the pathogenesis of HH in SOX11-disorder may stem from both hypothalamic and pituitary level deficits. Presentation: 6/1/2024

Lack of a genetic risk continuum between pubertal timing in the general population and idiopathic hypogonadotropic hypogonadism.

Pubertal timing is a highly heritable trait in the general population. Recently, a large-scale exome-wide association study has implicated rare variants in six genes (KDM4C, MC3R, MKRN3, PDE10A, TACR3, and ZNF483) as genetic determinants of pubertal timing within the general population. Two of the genes (TACR3, MKRN3) are already implicated in extreme disorders of pubertal timing. This observation suggests that there may be a pervasive "genetic risk continuum" wherein genes that govern pubertal timing in the general population, by extension, may also be causal for rare Mendelian disorders of pubertal timing. Hence, we hypothesized that the four novel genes linked to pubertal timing in the population will also contribute to idiopathic hypogonadotropic hypogonadism (IHH), a genetic disorder characterized by absent puberty. Exome sequencing data from 1322 unrelated IHH probands were reviewed for rare sequence variants (RSVs) (minor allele frequency bins: <1%; <0.1%; <0.01%) in the six genes linked to puberty in the general population. A gene-based rare variant association testing (RVAT) was performed between the IHH cohort and a reference public genomic sequences repository-the Genome Aggregation Database (gnomAD). As expected, RVAT analysis showed that RSVs in TACR3, a known IHH gene, were significantly enriched in the IHH cohort compared to gnomAD cohort across all three MAF bins. However, RVAT analysis of the remaining five genes failed to show any RSV enrichment in the IHH cohort across all MAF bins. Our findings argue strongly against a pervasive genetic risk continuum between pubertal timing in the general population and extreme pubertal phenotypes. The biologic basis of such distinct genetic architectures' merits further evaluation.

Genetic architecture of congenital hypogonadotropic hypogonadism: insights from analysis of a Portuguese cohort.

What is the contribution of genetic defects in Portuguese patients with congenital hypogonadotropic hypogonadism (CHH)? Approximately one-third of patients with CHH were found to have a genetic cause for their disorder, with causal pathogenic and likely pathogenic germline variants distributed among 10 different genes; cases of oligogenic inheritance were also included. CHH is a rare and genetically heterogeneous disorder characterized by deficient production, secretion, or action of GnRH, LH, and FSH, resulting in delayed or absent puberty, and infertility. Genetic screening was performed on a cohort of 81 Portuguese patients with CHH (36 with Kallmann syndrome and 45 with normosmic hypogonadotropic hypogonadism) and 263 unaffected controls. The genetic analysis was performed by whole-exome sequencing followed by the analysis of a virtual panel of 169 CHH-associated genes. The main outcome measures were non-synonymous rare sequence variants (population allele frequency <0.01) classified as pathogenic, likely pathogenic, and variants of uncertain significance (VUS). A genetic cause was identified in 29.6% of patients. Causal pathogenic and likely pathogenic variants were distributed among 10 of the analysed genes. The most frequently implicated genes were GNRHR, FGFR1, ANOS1, and CHD7. Oligogenicity for pathogenic and likely pathogenic variants was observed in 6.2% of patients. VUS and oligogenicity for VUS variants were observed in 85.2% and 54.3% of patients, respectively, but were not significantly different from that observed in controls. N/A. The identification of a large number of VUS presents challenges in interpretation and these may require reclassification as more evidence becomes available. Non-coding and copy number variants were not studied. Functional studies of the variants were not undertaken. This study highlights the genetic heterogeneity of CHH and identified several novel variants that expand the mutational spectrum of the disorder. A significant proportion of patients remained without a genetic diagnosis, suggesting the involvement of additional genetic, epigenetic, or environmental factors. The high frequency of VUS underscores the importance of cautious variant interpretation. These findings contribute to the understanding of the genetic architecture of CHH and emphasize the need for further studies to elucidate the underlying mechanisms and identify additional causes of CHH. This research was funded by the Portuguese Foundation for Science and Technology (grant numbers PTDC/SAU-GMG/098419/2008, UIDB/00709/2020, CEECINST/00016/2021/CP2828/CT0002, and 2020.04924.BD) and by Sidra Medicine-a member of the Qatar Foundation (grant number SDR400038). The authors declare no competing interests.

High microbiome and metabolome diversification in coexisting sponges with different bio-ecological traits

Marine Porifera host diverse microbial communities, which influence host metabolism and fitness. However, functional relationships between sponge microbiomes and metabolic signatures are poorly understood. We integrate microbiome characterization, metabolomics and microbial predicted functions of four coexisting Mediterranean sponges –Petrosia ficiformis, Chondrosia reniformis, Crambe crambe and Chondrilla nucula. Microscopy observations reveal anatomical differences in microbial densities. Microbiomes exhibit strong species-specific trends. C. crambe shares many rare amplicon sequence variants (ASV) with the surrounding seawater. This suggests important inputs of microbial diversity acquired by selective horizontal acquisition. Phylum Cyanobacteria is mainly represented in C. nucula and C. crambe. According to putative functions, the microbiome of P. ficiformis and C. reniformis are functionally heterotrophic, while C. crambe and C. nucula are autotrophic. The four species display distinct metabolic profiles at single compound level. However, at molecular class level they share a “core metabolome”. Concurrently, we find global microbiome-metabolome association when considering all four sponge species. Within each species still, sets of microbe/metabolites are identified driving multi-omics congruence. Our findings suggest that diverse microbial players and metabolic profiles may promote niche diversification, but also, analogous phenotypic patterns of “symbiont evolutionary convergence” in sponge assemblages where holobionts co-exist in the same area.

Genomic analysis of 116 autism families strengthens known risk genes and highlights promising candidates

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition with a strong genetic component in which rare variants contribute significantly to risk. We performed whole genome and/or exome sequencing (WGS and WES) and SNP-array analysis to identify both rare sequence and copy number variants (SNVs and CNVs) in 435 individuals from 116 ASD families. We identified 37 rare potentially damaging de novo SNVs (pdSNVs) in the cases (n = 144). Interestingly, two of them (one stop-gain and one missense variant) occurred in the same gene, BRSK2. Moreover, the identification of 8 severe de novo pdSNVs in genes not previously implicated in ASD (AGPAT3, IRX5, MGAT5B, RAB8B, RAP1A, RASAL2, SLC9A1, YME1L1) highlighted promising candidates. Potentially damaging CNVs (pdCNVs) provided support to the involvement of inherited variants in PHF3, NEGR1, TIAM1 and HOMER1 in neurodevelopmental disorders (NDD), although mostly acting as susceptibility factors with incomplete penetrance. Interpretation of identified pdSNVs/pdCNVs according to the ACMG guidelines led to a molecular diagnosis in 19/144 cases, although this figure represents a lower limit and is expected to increase thanks to further clarification of the role of likely pathogenic variants in ASD/NDD candidate genes not yet established. In conclusion, our study highlights promising ASD candidate genes and contributes to characterize the allelic diversity, mode of inheritance and phenotypic impact of de novo and inherited risk variants in ASD/NDD genes.

Identification and functional analysis of rare HECTD1 missense variants in human neural tube defects

Neural tube defects (NTDs) are severe malformations of the central nervous system that arise from failure of neural tube closure. HECTD1 is an E3 ubiquitin ligase required for cranial neural tube closure in mouse models. NTDs in the Hectd1 mutant mouse model are due to the failure of cranial mesenchyme morphogenesis during neural fold elevation. Our earlier research has linked increased extracellular heat shock protein 90 (eHSP90) secretion to aberrant cranial mesenchyme morphogenesis in the Hectd1 model. Furthermore, overexpression of HECTD1 suppresses stress-induced eHSP90 secretion in cell lines. In this study, we report the identification of five rare HECTD1 missense sequence variants in NTD cases. The variants were found through targeted next-generation sequencing in a Chinese cohort of 352 NTD cases and 224 ethnically matched controls. We present data showing that HECTD1 is a highly conserved gene, extremely intolerant to loss-of-function mutations and missense changes. To evaluate the functional consequences of NTD-associated missense variants, functional assays in HEK293T cells were performed to examine protein expression and the ability of HECTD1 sequence variants to suppress eHSP90 secretion. One NTD-associated variant (A1084T) had significantly reduced expression in HEK293T cells. All five NTD-associated variants (p.M392V, p.T801I, p.I906V, p.A1084T, and p.P1835L) reduced regulation of eHSP90 secretion by HECTD1, while a putative benign variant (p.P2474L) did not. These findings are the first association of HECTD1 sequence variation with NTDs in humans.

What have genetic studies of rare sequence variants taught us about the aetiology of schizophrenia?

With a population prevalence of 1%, schizophrenia is widespread, yet the aetiology of this psychiatric disorder remains elusive. There is an evident genetic component of schizophrenia, with heritability estimates lying at 60%-80%. While genome-wide association studies have identified 120 gene loci associated with schizophrenia risk, these involved common variants that confer only small effects on individual risk (median odds ratio &lt; 1.2). The recent emergence of whole exome sequencing (WES) technologies has facilitated the identification of rare sequence variants, including some protein-truncating variants that have significant effects on risk. Three key large-scale WES studies have demonstrated that rare sequence variants in the genes SETD1A , CACNA1G , CUL1 , GRIA3 , GRIN2A , HERC1 , RB1CC1 , SP4 , TRIO , XPO7 , and AKAP11 confer substantial risk for schizophrenia. These genes are highly expressed in central nervous system neurons and their products participate in diverse molecular functions including synaptic transmission, transcriptional regulation, and ubiquitin ligation. The understanding of these functional roles illuminates putative molecular mechanisms which may lead to schizophrenia-like phenotypes. It will also be possible to develop model systems in which the effects of impaired function of these genes can be further explored. Genetic studies of rare variants to date suggest that glutamatergic system dysregulation, chromatin modification, and the ubiquitin-proteasome system play key roles in schizophrenia aetiology.

Identifying subgroups of childhood obesity by using multiplatform metabotyping.

Introduction: Obesity results from an interplay between genetic predisposition and environmental factors such as diet, physical activity, culture, and socioeconomic status. Personalized treatments for obesity would be optimal, thus necessitating the identification of individual characteristics to improve the effectiveness of therapies. For example, genetic impairment of the leptin-melanocortin pathway can result in rare cases of severe early-onset obesity. Metabolomics has the potential to distinguish between a healthy and obese status; however, differentiating subsets of individuals within the obesity spectrum remains challenging. Factor analysis can integrate patient features from diverse sources, allowing an accurate subclassification of individuals. Methods: This study presents a workflow to identify metabotypes, particularly when routine clinical studies fail in patient categorization. 110 children with obesity (BMI > +2 SDS) genotyped for nine genes involved in the leptin-melanocortin pathway (CPE, MC3R, MC4R, MRAP2, NCOA1, PCSK1, POMC, SH2B1, and SIM1) and two glutamate receptor genes (GRM7 and GRIK1) were studied; 55 harboring heterozygous rare sequence variants and 55 with no variants. Anthropometric and routine clinical laboratory data were collected, and serum samples processed for untargeted metabolomic analysis using GC-q-MS and CE-TOF-MS and reversed-phase U(H)PLC-QTOF-MS/MS in positive and negative ionization modes. Following signal processing and multialignment, multivariate and univariate statistical analyses were applied to evaluate the genetic trait association with metabolomics data and clinical and routine laboratory features. Results and Discussion: Neither the presence of a heterozygous rare sequence variant nor clinical/routine laboratory features determined subgroups in the metabolomics data. To identify metabolomic subtypes, we applied Factor Analysis, by constructing a composite matrix from the five analytical platforms. Six factors were discovered and three different metabotypes. Subtle but neat differences in the circulating lipids, as well as in insulin sensitivity could be established, which opens the possibility to personalize the treatment according to the patients categorization into such obesity subtypes. Metabotyping in clinical contexts poses challenges due to the influence of various uncontrolled variables on metabolic phenotypes. However, this strategy reveals the potential to identify subsets of patients with similar clinical diagnoses but different metabolic conditions. This approach underscores the broader applicability of Factor Analysis in metabotyping across diverse clinical scenarios.

How human genetic context can inform pathogenicity classification: FGFR1 variation in idiopathic hypogonadotropic hypogonadism.

Precision medicine requires precise genetic variant interpretation, yet many disease-associated genes have unresolved variants of unknown significance (VUS). We analyzed variants in a well-studied gene, FGFR1, a common cause of Idiopathic Hypogonadotropic Hypogonadism (IHH) and examined whether regional genetic enrichment of missense variants could improve variant classification. FGFR1 rare sequence variants (RSVs) were examined in a large cohort to (i) define regional genetic enrichment, (ii) determine pathogenicity based on the American College of Medical Genetics/Association for Molecular Pathology (ACMG/AMP) variant classification framework, and (iii) characterize the phenotype of FGFR1 variant carriers by variant classification. A total of 143 FGFR1 RSVs were identified in 175 IHH probands (n = 95 missense, n = 48 protein-truncating variants). FGFR1 missense RSVs showed regional enrichment across biologically well-defined domains: D1, D2, D3, and TK domains and linker regions (D2-D3, TM-TK). Using these defined regions of enrichment to augment the ACMG/AMP classification reclassifies 37% (20/54) of FGFR1 missense VUS as pathogenic or likely pathogenic (PLP). Non-proband carriers of FGFR1 missense VUS variants that were reclassified as PLP were more likely to express IHH or IHH-associated phenotypes [anosmia or delayed puberty] than non-proband carriers of FGFR1 missense variants that remained as VUS (76.9% vs 34.7%, p = 0.035). Using the largest cohort of FGFR1 variant carriers, we show that integration of regional genetic enrichment as moderate evidence for pathogenicity improves the classification of VUS and that reclassified variants correlated with phenotypic expressivity. The addition of regional genetic enrichment to the ACMG/AMP guidelines may improve clinical variant interpretation.

THU185 Molecular Etiologic Spectrum Of Patients With 46,XY And 46,XX Disorders Of Sex Development

Abstract Disclosure: J. Kim: None. S. Park: None. S. Hwang: None. J. Yoon: None. G. Kim: None. H. Yoo: None. J. Choi: None. Background: Disorders of sexual development (DSDs) is a group of congenital conditions associated with discordant development of chromosomal, gonadal, and anatomical sex. Sex development is a complex process with a precise synergistic temporal-spatial transcriptional regulation of multiple genes. Advances in next-generation sequencing (NGS) techniques have achieved genetic diagnosis for up to 40 % of 46,XY DSDs. This study investigated the etiological distribution of DSDs identified by NGS. Methods: This study included 70 patients with DSDs (56 patients with 46,XY DSD and 14 patients with 46,XX DSD). Molecular genetic analysis was performed using targeted gene panel sequencing (n = 43) and whole-exome sequencing (WES, n = 26), including 25 patients for the first-line test and one without sequence variants by targeted gene panel sequencing. Whole-genome sequencing (WGS) was performed on 7 patients, including two patients for the first-line test and five patients without sequence variants by targeted gene panel sequencing or WES. Results: Patients with DSDs presented in infancy with ambiguous genitalia and at older children with aberrant pubertal development or discordance between karyotype and external genitalia. Twenty-four of 56 patients (42.9%) with 46,XY DSD were reared female, and 5 of 14 patients (35.7%) with 46,XX DSD were raised as a male. Pathogenic or likely pathogenic variants were identified in 21 of 56 patients with 46,XY DSD (37.5%); however, no rare sequence variants were found in patients with 46,XX DSD. The AR variants were the most common (8/70, 11.4%), followed by CYP17A1 (4/70, 5.7%), SRD5A2 (4/70, 5.7%), NR5A1 (2/70, 2.8%), GATA4 (1/70, 1.4%), MYRF (1/70, 1.4%), and deletion of DMRT1/2 (1/70, 1.4%). Mutation-positive patients with 46, XY DSD were assigned as female more frequently than mutation-negative patients (19/21 vs. 6/35, P-value &amp;lt;0.001). The lower diagnostic yield was observed in patients with a less severe phenotype, such as hypospadias or micropenis. Conclusions: This study demonstrated that genetic etiologies could be identified in 37.5% of patients with 46,XY DSD, whereas there were no genetic variants in patients with 46,XX DSD. Patients with a more severe phenotype were more likely to have a conclusive genetic diagnosis. NGS is a helpful technique to improve diagnostic efficiency in patients with DSD because of phenotypic and genetic heterogeneity. Presentation: Thursday, June 15, 2023

A likelihood ratio approach for utilizing case-control data in the clinical classification of rare sequence variants: application to BRCA1 and BRCA2.

A large number of variants identified through clinical genetic testing in disease susceptibility genes, are of uncertain significance (VUS). Following the recommendations of the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP), the frequency in case-control datasets (PS4 criterion), can inform their interpretation. We present a novel case-control likelihood ratio-based method that incorporates gene-specific age-related penetrance. We demonstrate the utility of this method in the analysis of simulated and real datasets. In the analyses of simulated data, the likelihood ratio method was more powerful compared to other methods. Likelihood ratios were calculated for a case-control dataset of BRCA1 and BRCA2 variants from the Breast Cancer Association Consortium (BCAC), and compared with logistic regression results. A larger number of variants reached evidence in favor of pathogenicity, and a substantial number of variants had evidence against pathogenicity - findings that would not have been reached using other case-control analysis methods. Our novel method provides greater power to classify rare variants compared to classical case-control methods. As an initiative from the ENIGMA Analytical Working Group, we provide user-friendly scripts and pre-formatted excel calculators for implementation of the method for rare variants in BRCA1, BRCA2 and other high-risk genes with known penetrance.

A population-based survey of FBN1 variants in Iceland reveals underdiagnosis of Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant condition characterized by aortic aneurysm, skeletal abnormalities, and lens dislocation, and is caused by variants in the FBN1 gene. To explore causes of MFS and the prevalence of the disease in Iceland we collected information from all living individuals with a clinical diagnosis of MFS in Iceland (n = 32) and performed whole-genome sequencing of those who did not have a confirmed genetic diagnosis (27/32). Moreover, to assess a potential underdiagnosis of MFS in Iceland we attempted a genotype-based approach to identify individuals with MFS. We interrogated deCODE genetics' database of 35,712 whole-genome sequenced individuals to search for rare sequence variants in FBN1. Overall, we identified 15 pathogenic or likely pathogenic variants in FBN1 in 44 individuals, only 22 of whom were previously diagnosed with MFS. The most common of these variants, NM_000138.4:c.8038 C > T p.(Arg2680Cys), is present in a multi-generational pedigree, and was found to stem from a single forefather born around 1840. The p.(Arg2680Cys) variant associates with a form of MFS that seems to have an enrichment of abdominal aortic aneurysm, suggesting that this may be a particularly common feature of p.(Arg2680Cys)-associated MFS. Based on these combined genetic and clinical data, we show that MFS prevalence in Iceland could be as high as 1/6,600 in Iceland, compared to 1/10,000 based on clinical diagnosis alone, which indicates underdiagnosis of this actionable genetic disorder.

Impacts to canine dermal microbiota associated with repeated bathing.

Working dogs routinely operate in environmental conditions which may necessitate daily bathing to remove contaminants or soilage. The impacts of frequent or repeated bathing on the canine dermal microbiota are unknown. The objective of this study was to characterize changes in canine dermal microbial populations following repeated daily bathing. Labrador retrievers (n = 16) were bathed daily using a dilute dish detergent solution (1.6% detergent solution) over the course of 14 days. Dermal microbial DNA was collected via sterile swabs (n = 142) taken at days 0, 7, 14, 16, 21, 28, 35, 42, and 49 and analyzed for alpha diversity, beta diversity and relative abundance to assess changes in the dermal microbiota via 16 s sequencing. Results indicate that daily bathing significantly increased Shannon diversity, Chao1, and several rare amplicon sequence variants. Although typically reported in highest abundance, relative abundance was decreased in the phyla Actinobacteria, Firmicutes, and Proteobacteria (p < 0.05). Repeated daily bathing with dilute dish detergent significantly reduced normal healthy dermal microbial taxa and created significant changes in the dermal microbiota of canines. Disruption to the canine dermal microbiota may cause negative impacts to canine dermal health and require further investigation.

Rare sequence variants associated with the risk of non-syndromic biliary atresia.

The etiology of non-syndromic biliary atresia (BA) remains largely unknown. In this study, we performed genome-wide screening of genes associated with the risk of non-syndromic BA. We analyzed exome data of 15 Japanese patients with non-syndromic BA and 509 control individuals using an optimal sequence kernel association test (SKAT-O), a gene-based association study optimized for small-number subjects. Moreover, we examined the frequencies of known BA-related single nucleotide polymorphisms (SNPs) in the BA and control groups. SKAT-O showed that rare damaging variants of MFHAS1, a ubiquitously expressed gene encoding a toll-like receptor-associated protein, were more common in the BA group than in the control group (Bonferroni corrected p-value, 0.0097). Specifically, p.Val106Gly and p.Arg556Cys significantly accumulated in the patient group. These variants resided within functionally important domains. SKAT-O excluded the presence of other genes significantly associated with the disease risk. Of 60 known BA-associated SNPs, only eight were identified in the BA group. In particular, p.Ile3421Met of MYO15A and p.Ala421Thr of THOC2 were more common in the BA group than in the control group. However, the significance of these two variants is questionable, because MYO15A has been linked to deafness but not to BA and the p.Ala421Thr of THOC2 represents a relatively common SNP in Asia. The results of this study indicate that rare damaging variants in MFHAS1 may constitute a risk factor for non-syndromic BA, while the contribution of other monogenic variants to the disease predisposition is limited. This article is protected by copyright. All rights reserved.

Low frequency of AIP mutations in patients with young-onset sporadic pituitary macroadenomas

PurposeMutations in the aryl hydrocarbon receptor interacting protein (AIP) gene cause familial isolated pituitary adenomas (FIPA). AIP mutations have also been found in patients with apparently sporadic pituitary adenomas, particularly in young patients with large adenomas. The aim of this study was to determine the frequency of AIP germline mutations in patients with young-onset sporadic pituitary macroadenomas.MethodsThe AIP gene was sequenced in 218 Portuguese patients with sporadic pituitary macroadenomas diagnosed before the age of 40 years.ResultsHeterozygous rare sequence variants in AIP were identified in 18 (8.3%) patients. However, only four (1.8%) patients had pathogenic or likely pathogenic variants. These consisted of two already known mutations (p.Arg81* and p.Leu115Trpfs*41) and two novel mutations (p.Glu246*, p.Ser53Thrfs*36). All four patients had GH-secreting adenomas diagnosed between the ages of 14 and 25 years. The frequency of AIP pathogenic or likely pathogenic variants in patients under the age of 30 and 18 years was 3.4% and 5.0%, respectively.ConclusionThe frequency of AIP mutations in this cohort was lower than in other studies. Previous reports may have overestimated the contribution of AIP mutations due to the inclusion of genetic variants of uncertain significance. The identification of novel AIP mutations expands the known spectrum of genetic causes of pituitary adenomas and may help understand the role of AIP mutations in the molecular mechanisms underlying pituitary tumorigenesis.

Mutation spectrum and frequency of copy number variations of the ANOS1 gene in patients with Kallmann syndrome or normosmic isolated hypogonadotropic hypogonadism.

This study was performed to investigate the molecular characteristics and frequency of copy number variations (CNVs) of ANOS1 in patients with Kallmann syndrome (KS) or normosmic isolated hypogonadotropic hypogonadism (nIHH) using multiplex ligation-dependent probe amplification (MLPA) analysis and sequencing. Among 45 patients from 43 independent families, Sanger sequencing, next-generation sequencing (NGS), or microarray was performed in 24 patients from 23 families, and MLPA was performed in 19 patients who did not show rare sequence variants (n = 18) or ANOS1 amplification by PCR (n = 1). Seven patients (4 patients with KS, 1 patient with nIHH, one prepubertal boy with anosmia, and one newborn patient) from 6 families (6/43, 14%) harbored molecular defects in ANOS1 including a nonsense mutation [c.1140G>A (p.W380*)], a frameshift mutation [c.1260del (p.Q421Kfs*61)], a splice site mutation (c.1449+1G>A), an exon 7 deletion, a complete deletion, and 7.9 Mb-sized inversion encompassing ANOS1. The complete deletion of ANOS1 was identified in a neonate with a micropenis and cryptorchidism. Unilateral renal agenesis was found in three patients, whereas only one patient displayed both synkinesia and sensorineural hearing loss. There was no reversal of hypogonadotropic hypogonadism in any patient during 9.1 ± 2.9 years of treatment with testosterone enanthate. Molecular defects in the ANOS1 gene could be identified in 14% of probands including various types of CNVs (3/43, 7.0%). Comprehensive analysis using sequencing and analysis for CNVs is required to detect molecular defects in ANOS1.

Sequence Characterization of ITS Regions of Immortelle Helichrysum italicum (Roth) G. Don from the East Adriatic Coast

The immortelle (Helichrysum italicum (Roth) G. Don) is a typical perennial plant of natural vegetation in the Mediterranean region, and due to secondary metabolites with several biological properties (anti-inflammatory, antioxidant, antimicrobial, and anti-proliferative), it has become an important species for essential oil production, especially in the cosmetic industry. To increase the production of highly priced essential oils, it has been moved to cultivated fields. However, due to the lack of highly characterized planting material, there is a great need for genotype identification, and to provide a link with chemical profiles and geographic origin as a basis for the identification of local superior genotypes. The aims of the study were to characterize the ITS (ribosomal internal transcribed spacer) regions, ITS1 and ITS2, in samples from the East Adriatic region to determine the possibility of using these regions for plant genetic resources identification. Genetic variation was observed when comparing the ITS sequence variants of samples from the North-East Adriatic and the South-East Adriatic. Some rare and unique ITS sequence variants can be helpful for identifying specific populations from different geographical regions.

The moderate substitution of Astragalus sinicus returning for chemical fertilizer improves the N cycle function of key ecological bacterial clusters in soil.

Astragalus sinicus (Chinese milk vetch) is a well-established resource of organic fertilizer widely used in paddy soil to partially replace chemical fertilizers. However, the influence of returning A. sinicus to fields on the soil bacterial community remains poorly understood. Here, we used different amounts of A. sinicus partially replacing chemical fertilizers and investigated the changes in soil physicochemical factors and the soil bacterial community structure responses. Returning A. sinicus to the field significantly increased the soil total nitrogen and available phosphorus content (p < 0.05). Weighted gene correlation network analysis (WGCNA) was applied to detect significant associations between the soil microbiome data and physicochemical factors. Two key ecological bacterial clusters (MEturquoise and MEgreen), mainly containing Acidobacteria, Proteobacteria, and Chloroflexi, were significantly correlated with soil nitrogen (N) levels. A. sinicus partially replacing chemical fertilizers reduced the normalized stochasticity ratio (NST) of rare amplicon sequence variants (ASVs), abundant ASVs, MEturquoise, and MEgreen (p < 0.05). Our results further indicated that a moderate amount of A. sinicus returned to the soil effectively mitigated the trend of reduced relative abundance of N fixation function of key ecological clusters caused by chemical fertilizer. However, a large amount of A. sinicus led to a significant increase in relative abundance of denitrification function and a significant decrease in relative abundance of N fixation function of key ecological clusters. This implies that the moderate substitution of A. sinicus returning for chemical fertilizer improves the N cycling function of key ecological bacterial clusters in soil. From the perspective of the bacterial community in paddy soil, this study provides new insight and a reference on how to find a good balance between the amount of A. sinicus returned to the soil and ecological safety.

Genetic variants of G-protein coupled receptors associated with pubertal disorders.

The human hypothalamic-pituitary-gonadal (HPG) axis is the regulatory center for pubertal development. This axis involves six G-protein coupled receptors (GPCRs) encoded by KISS1R, TACR3, PROKR2, GNRHR, LHCGR, and FSHR. Previous studies have identified several rare variants of the six GPCR genes in patients with pubertal disorders. In vitro assays and animal studies have provided information on the function of wild-type and variant GPCRs. Of the six GPCRs, those encoded by KISS1R and TACR3 are likely to reside at the top of the HPG axis. Several loss-of-function variants in the six genes were shown to cause late/absent puberty. In particular, variants in KISS1R, TACR3, PROKR2, and GNRHR lead to hypogonadotropic hypogonadism in autosomal dominant, recessive, and oligogenic manners. Furthermore, a few gain-of-function variants of KISS1R, PROKR2, and LHCGR have been implicated in precocious puberty. The human HPG axis may contain additional GPCRs. The six GPCRs in the HPG axis govern pubertal development through fine-tuning of hormone secretion. Rare sequence variants in these genes jointly account for a certain percentage of genetic causes of pubertal disorders. Still, much remains to be clarified about the molecular network involving the six GPCRs.