Human Gene Mutation Database Research Articles

Down-regulation of SORL1 is associated with Alzheimer's disease through activating ABC transporter pathway.

Alzheimer's disease (AD) is a common neurodegenerative disease with high morbidity among elderly people. A genetic attribution has been extensively proved. Here, we propose to further prioritize genes that harbor single nucleotide variation (SNV) or structural variation (SV) for AD and explore the underlying potential mechanisms through exploiting their expression and methylation spectra. A high-confidence AD-associated candidate gene list was obtained from the ClinVar and Human Gene Mutation Database (HGMD). Genome-wide methylation and expression profiles of AD and normal subjects were downloaded from the Gene Expression Omnibus (GEO). Through comprehensive comparison of expression and methylation levels between AD and normal samples, as well as different stages of AD samples, SORL1 was identified as the most plausible gene for AD incidence and progression. Gene Set Enrichment Analysis (GSEA) revealed significant activation of the ABC (ATP binding cassette) transporter with the aberrant up-regulation of SORL1 within AD samples. This study unfolds the expression and methylation spectra of previously probed genes with SNV or SV in AD for the first time, and reports an aberrant activation of the ABC transporter pathway that might contribute to AD progression. This should shed some light on AD diagnosis and precision treatment.

Interpretation of Maturity-Onset Diabetes of the Young Genetic Variants Based on American College of Medical Genetics and Genomics Criteria: Machine-Learning Model Development

Background Maturity-onset diabetes of the young (MODY) is a group of dominantly inherited monogenic diabetes, with HNF4A-MODY, GCK-MODY, and HNF1A-MODY as the three most common forms based on the causal genes. Molecular diagnosis of MODY is important for precise treatment. Although a DNA variant causing MODY can be assessed based on the criteria of the American College of Medical Genetics and Genomics (ACMG) guidelines, gene-specific assessment of disease-causing mutations is important to differentiate among MODY subtypes. As the ACMG criteria were not originally designed for machine-learning algorithms, they are not true independent variables. Objective The aim of this study was to develop machine-learning models for interpretation of DNA variants and MODY diagnosis using the ACMG criteria. Methods We applied machine-learning models for interpretation of DNA variants in MODY genes defined by the ACMG criteria based on the Human Gene Mutation Database (HGMD) and ClinVar database. Results With a machine-learning procedure, we found that the weight matrix of the ACMG criteria was significantly different between the three MODY genes HNF1A, HNF4A, and GCK. The models showed high predictive abilities with accuracy over 95%. Conclusions Our results highlight the need for applying different weights of the ACMG criteria in relation to different MODY genes for accurate functional classification. As proof of principle, we applied the ACMG criteria as feature vectors in a machine-learning model and obtained a precision-based result.

Novel Splice-Site Mutation in RASGRP2 Leading to Caldag-GEFI Deficiency

Introduction: CalDAG-GEFI (Calcium and diacyglycerol-regulated guanine exchange factor I) deficiency or platelet-type bleeding disorder-18 (BDPLT18) is a relatively new, rare hemorrhagic disorder with autosomal recessive inheritance (OMIM #615888). CalDAG-GEFI activates Rap1 and is essential for αIIbβ3 (GP IIb/IIIa) integrin activation in platelets, a key step in platelet aggregation. The deficiency is due to mutations of the encoding gene, RASGRP2 (HGNC ID 9879), which is located on chromosome 11q13.1. To date, 23 pathogenic variants in the RASGRP2 gene are listed in the Human Gene Mutation Database (HGMD®). Two other RASGRP2 splice-site mutations (c.74-1G>C and 372-3C>G) are described in the literature in patients suffering from CalDAG-GEFI loss of function. Patients and Methods: We investigated a 3 yr-old index patient who was referred to our outpatient clinic because of Glanzmann thrombasthenia (GT)-like symptoms and GT-typical pattern in platelet aggregometry analyses. The boy presented with hematomas and recurrent infections, postnatally, he had shown petechiae. After a tongue bite at the age of 1 yr, he needed tranexamic acid to stop bleeding. So far, no surgery has been performed. His family members including mother, father, elder sisters and brother had no history of bleeding. Informed consent for our study of biochemical and molecular genetic characterization for inherited platelet disorders using NGS was given by all family members. Platelet functions were assessed by light transmission aggregometry using citrated PRP. Membrane glycoprotein quantification (CD41, CD42a and CD42b), fibrinogen binding, von Willebrand factor (VWF) binding and the investigation of alpha- and delta- granule secretion were performed by flow cytometry. For the index patient and his parents we performed NGS panel sequencing (Nextera Rapid Custom Enrichment, Illumina). 80 genes known to be associated with platelet disorders were screened. The variants detected were classified based on the 2015 American College of Medical Genetics (ACMG) guidelines. ClinVar, Human Gene Mutation Database, and in silico pathogenicity prediction were used to assess the detected variants. The segregation of the putative mutation was confirmed by Sanger sequencing of three unaffected siblings. Results: The index patient presented with normal values for platelet count, aPTT, INR, FXIII and VWF parameters. ADP (4, 10 and 20µM/L), epinephrine (8, 16 and 40µM/L) and low concentration collagen (2µM/mL) and TRAP6 (5µM/mL) induced aggregation was markedly reduced. However, normal values after stimulation with 10µM/mL collagen and TRAP6 respectively were achieved. Agglutination after stimulation with ristocetin (1,2mg/mL) was normal. Flow cytometry revealed severely decreased fibrinogen binding after stimulation with ADP (0.25-5.0 μmol/l) compared to control platelets. For CD41 (GP IIb/IIIa-complex), CD42a/b (GP Ib/IX-complex), ristocetin-induced VWF-binding, CD62- and CD63-expression, normal values were measured. Molecular genetic analysis revealed a novel homozygous splice-site mutation of the RASGRP2 gene in the index patient. The mutation was heterozygous in the non- consanguineous parents (Tab.1). Conclusion: We have identified a novel recessive mutation of the RASGRP2 gene in a boy affected by a Glanzmann thrombasthenia-like disorder. Platelet aggregometry analysis showed typical failure of aggregation with low dose collagen and TRAP6, whereas aggregation after stimulation with higher concentration reached normal values. Flow cytometric measured severely reduced fibrinogen binding indicated a functional defect of the αIIbβ3 fibrinogen receptor with normal expression. Comprehensive platelet function analysis and panel sequencing allowed early and precise diagnosis and therapy of this ultra-rare platelet signaling defect. Disclosures Zieger: Biotest: Research Funding; DFG: Research Funding; CSL Behring: Research Funding; Takeda: Research Funding.

Reduced penetrance of pathogenic ACMG variants in a deeply phenotyped cohort study and evaluation of ClinVar classification over time

PurposeWe studied the penetrance of pathogenically classified variants in an elderly Dutch population from the Rotterdam Study, for which deep phenotyping is available. We screened the 59 actionable genes for which reporting of known pathogenic variants was recommended by the American College of Medical Genetics and Genomics (ACMG), and demonstrate that determining what constitutes a known pathogenic variant can be quite challenging. MethodsWe defined “known pathogenic” as classified pathogenic by both ClinVar and the Human Gene Mutation Database (HGMD). In 2628 individuals, we performed exome sequencing and identified known pathogenic variants. We investigated the clinical records of carriers and evaluated clinical events during 25 years of follow-up for evidence of variant pathogenicity. ResultsOf 3815 variants detected in the 59 ACMG genes, 17 variants were considered known pathogenic. For 14/17 variants the ClinVar classification had changed over time. Of 24 confirmed carriers of these variants, we observed at least one clinical event possibly caused by the variant in only three participants (13%). ConclusionWe show that the definition of “known pathogenic” is often unclear and should be approached carefully. Additionally variants marked as known pathogenic do not always have clinical impact on their carriers. Definition and classification of true (individual) expected pathogenic impact should be defined carefully.

Comprehensive analysis of genetic and clinical characteristics of 30 patients with X-linked juvenile retinoschisis in China.

PurposeTo provides the clinical and genetic characteristics of a series of Chinese patients with X‐linked juvenile retinoschisis (XLRS) through multimodal imaging and next‐generation sequencing.MethodsThirty patients (60 eyes) from 29 unrelated families of Chinese origin with XLRS were screened using multigene panel testing, and underwent a complete clinical evaluation. All variants identified in this study and reported in the Human Gene Mutation Database were analysed.ResultsTwenty‐five distinct variants in the retinoschisin gene were identified, of which eight were novel, and one was de novo. Missense mutations were the most prevalent type, and mutation hot spot was localized in the discoidin domain. The mean Snellen best‐corrected visual acuity was 0.28 ± 0.17. Of all eyes presenting with schisis, 92.86% had lamellar schisis and 62.5% had peripheral schisis. Schisis changes mostly involved inner and outer nuclear layers. X‐linked juvenile retinoschisis (XLRS) patients had a high incidence of complications, and peripheral schisis was a risk factor for it. No obvious genotype–phenotype association was observed.ConclusionThis study provides comprehensive analyses of the genetic and clinical characteristics of XLRS in a cohort of Chinese patients. The fourth de novo mutation in RS1 was identified. And we show that XLRS has a wide spectrum of clinical characteristics; hence, molecular diagnosis is crucial for its diagnosis, differential diagnosis and genetic counselling. Peripheral schisis is a risk factor for the high incidence of complications, and no clear genotype–phenotype correlations were found.

ADAMTS13 gene; a novel splicing site mutation in a case with thrombotic thrombocytopenic purpura

A plasma protease, ADAMTS13, cleaves the von Willebrand factor (VWF) and its deficiency is associated with the pathogenesis of thrombotic thrombocytopenic purpura (TTP). According to the Human Gene Mutation Database (HGMD), about 150 mutations have been identified in the ADAMTS13 gene. A 23-year-old man, with hematuria and gingival bleeding was admitted to our University Hospital. Four years ago he was diagnosed with a TTP history. During these years, he was under intermittent plasma exchange. A blood sample was taken for genetic study. He effectively responded to one session of fresh frozen plasma replacement and plasma exchange. Genetic study indicated that this case carries two heterozygous mutations in ADAMTS13 gene; a novel splicing variant (c.2610+5G>A) and a nonsense p.Arg910X mutation that previously is reported to relate to TTP. The novel variant predicted to result in an aberrant ADAMTS13 transcript processing.

Analysis of MYO15A variation in children with DFNB3

Objective: To analyze the genetic and clinical characteristics of MYO15A variants associated non-syndromic autosomal recessive deafness3 (DFNB3). Methods: The hearing test and high-throughput sequencing data of 108 families with non-syndromic hearing loss, who visited the Center of Genetics and Prenatal Diagnosis in the First Affiliated Hospital of Zhengzhou University from November 2016 to February 2019, were retrospectively analyzed to investigate the characteristics of MYO15A variation. Results: Compound heterozygous MYO15A variations were detected in nine patients from eight families, accounting for 7.4% of all 108 families. The variants were c.5910+1G>A/c.9417_9418insTA, c.4234T>G/c.8324G>T, c.3926A>T/c.5002delC, c.9690+1G>A/c.10257_10259delCTT, c.8324G>T/c.10419_10423delCAGCT, c.4519C>T/c.6454G>C, c.6177+1G>T/c.10257_10259delCTT and c.5692C>T/c.7396-1G>A. All patients had severe to profound hearing loss. Among the 14 variations, 12 variations were located in the main structural domains, including 5 in motor domain, 3 in FERM domain, 3 in MyTH4 domain and 1 in IQ motif. The c.3926A>T, c.4234T>G, c.4519C>T, c.5002delC, c.6454G>C, c.8324G>T, c.9417_9418insTA and c.10419_10423delCAGCT had not been reported in the Human Gene Mutation Database up to February 2020. According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), 6 reported variants and the first reported c.4519C>T, c.5002delC, c.9417_9418insTA and c.10419_10423delCAGCT were identified as pathogenic variants, while c.8324G>T was likely pathogenic variant, and c.3926A>T, c.4234T>G and c.6454G>C were variants of uncertain significance. Conclusions: The variations of MYO15A in patients with DFNB3 are mainly complex heterozygous. The clinical phenotypes are mostly severe to profound hearing loss, and the mutation loci are mainly in the motor, FERM and MyTH4 domains.

The Frequency of Discordant Variant Classification in the Human Gene Mutation Database: A Comparison of the American College of Medical Genetics and Genomics Guidelines and ClinVar.

Discordant variant classifications among public databases is one of the well-documented limitations when interpreting the pathogenicity of variants. The aim of this study is to investigate the level of germline variant misannotation from the Human Gene Mutation Database (HGMD) and the annotation concordance between databases. We used a total of 188,106 classified variants (disease-causing mutations [n = 179,454] and polymorphisms [n = 8652]) in 6466 genes from the HGMD. All variants were reanalyzed based on the American College of Medical Genetics and Genomics (ACMG) guidelines and compared to ClinVar database variants. When variants were classified based on the ACMG guidelines, misclassification was observed in 3.47% (2289/65,896) of variants. The overall concordance between HGMD and ClinVar was 97.62% (52,499/53,780) of variants studied. Variants in databases must be used with caution when variant pathogenicity is interpreted. This study reveals the frequency of misannotation of the HGMD variants and annotation concordance between databases in depth.

Abstract 207: A Catalogue of the Pathogenic Mutations of LDL Receptor Gene in Japanese Familial Hypercholesterolemia

Background: Familial hypercholesterolemia (FH), mainly caused by LDL receptor gene mutation is attracting a lot of attention associated with the development of novel therapies. However, rare mutations tend to be ethnicity-specific, hence, it is always difficult to determine pathogenicity of LDL receptor gene mutation in a single individual in a certain ethnicity. Currently, little data exist on the pathogenic mutations of LDL receptor in Japanese familial hypercholesterolemia (FH). Objective: We aimed to catalogue the pathogenic mutations of LDL receptor gene in the 2 major Japanese FH-care centers (Kanazawa University and National Cerebral and Cardiovascular Center Research Institute), where genetic testing of FH has been performed centrally upon requests from institutes all over Japan during more than past two decades. Methods: 796 FH subjects from 472 families who had nonsynonymous mutations in LDL receptor gene were included in this study. Genetic mutations were analyzed for mutations by Sanger sequencing as well as by multiplex ligation probe dependent amplification (MLPA) technique for large rearrangements. Pathogenic mutations were defined either as 1) protein truncated variants, 2) registered as pathogenic in Clinvar, or Human Gene Mutation Database (HGMD), or meet the criteria of American College of Medical Genetics and Genomics (ACMG) guideline, or 3) CADD-score > 10. Results: We found 138 different mutations. Among them, 132 mutations were considered as pathogenic, including 19 large rearrangement mutations. Whereas, 6 missense mutations were classified as variants of unknown significance (VUS). A single mutation accounted for as much as 41 % of the FH subjects recruited from Kanazawa University mainly due to founder gene effect, whereas, many singleton mutations were found from National Cerebral and Cardiovascular Center Research Institute located in Osaka. Conclusions: We provided the largest catalogue of pathogenic mutations of LDL receptor gene in Japanese FH. This could aid to determine the pathogenicity of the LDL receptor genetic mutations not only in Japanese but also in other ethnicities.

The role of polygenic susceptibility to obesity among carriers of pathogenic mutations in MC4R in the UK Biobank population.

Melanocortin 4 receptor (MC4R) deficiency, caused by mutations in MC4R, is the most common cause of monogenic forms of obesity. However, these mutations have often been identified in small-scale, case-focused studies. Here, we assess the penetrance of previously reported MC4R mutations at a population level. Furthermore, we examine why some carriers of pathogenic mutations remain of normal weight, to gain insight into the mechanisms that control body weight. We identified 59 known obesity-increasing mutations in MC4R from the Human Gene Mutation Database (HGMD) and Clinvar. We assessed their penetrance and effect on obesity (body mass index [BMI] ≥ 30 kg/m2) in >450,000 individuals (age 40-69 years) of the UK Biobank, a population-based cohort study. Of these 59 mutations, only 11 had moderate-to-high penetrance and increased the odds of obesity by more than 2-fold. We subsequently focused on these 11 mutations and examined differences between carriers of normal weight and carriers with obesity. Twenty-eight of the 182 carriers of these 11 mutations were of normal weight. Body composition of carriers of normal weight was similar to noncarriers of normal weight, whereas among individuals with obesity, carriers had a somewhat higher BMI than noncarriers (1.44 ± 0.07 standard deviation scores [SDSs] ± standard error [SE] versus 1.29 ± 0.001, P = 0.03), because of greater lean mass (1.44 ± 0.09 versus 1.15 ± 0.002, P = 0.002). Carriers of normal weight more often reported that, already at age 10 years, their body size was below average or average (72%) compared with carriers with obesity (48%) (P = 0.01). To assess the polygenic contribution to body weight in carriers of normal weight and carriers with obesity, we calculated a genome-wide polygenic risk score for BMI (PRSBMI). The PRSBMI of carriers of normal weight (PRSBMI = -0.64 ± 0.18) was significantly lower than of carriers with obesity (0.40 ± 0.11; P = 1.7 × 10-6), and tended to be lower than that of noncarriers of normal weight (-0.29 ± 0.003; P = 0.05). Among carriers, those with a low PRSBMI (bottom quartile) have an approximately 5-kg/m2 lower BMI (approximately 14 kg of body weight for a 1.7-m-tall person) than those with a high PRS (top quartile). Because the UK Biobank population is healthier than the general population in the United Kingdom, penetrance may have been somewhat underestimated. We showed that large-scale data are needed to validate the impact of mutations observed in small-scale and case-focused studies. Furthermore, we observed that despite the key role of MC4R in obesity, the effects of pathogenic MC4R mutations may be countered, at least in part, by a low polygenic risk potentially representing other innate mechanisms implicated in body weight regulation.

Updating and identifying three novel variants of the CARD14 gene in Chinese Han patients with psoriasis

Psoriasis-2 (PSORS2) is caused by the heterozygous mutation of the caspase recruitment domain 14 (CARD14) gene on chromosome 17q25. To evaluate the contribution of CARD14 variants in psoriasis of the Chinese Han population, we performed deep sequencing of the CARD14 gene in 372 Chinese Han patients with psoriasis. The exonic nucleotide variants were confirmed by Sanger sequencing in the affected individuals and 1114 controls. In 27 patients with psoriasis, we identified 15 variations, including three novel variants: c.381C[G (p.Cys127Trp), c.712A[G (p.Met238Val) and c.2260_2261delinsGG (p.Gln754Gly). These findings could enrich and update the Human Gene Mutation Database of CARD14 variants for psoriasis.

Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1

BackgroundIn recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants.MethodsWe conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106−17T>G, c.393+4A>G, c.517−8A>G, c.517−3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results.ResultsWe successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106−17T>G).ConclusionWe found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.

New and Inherited STAT1 Mutations in a Chinese Family with Chronic Mucocutaneous Candidiasis: A Case Report

A mild clinical Chronic Mucocutaneous Candidiasis (CMC) phenotype with STAT1 transcription factor mutation has been identified in a Chinese family. It is a rare variant in STAT1 (NM_0073315.3c.1175T>C Met392Thr). Specifically, it is a heterozygous mutation. To date, the pathogenicity of this variant in STAT1 (NM_0073315.3c.1175T>C Met392Thr) for CMC has not been reported in the Human Gene Mutation Database. Thus, this is the first report about STAT1 mutation found in CMC patients from Chinese ethnic group. This study also shows the mutation on the DNA-binding domain of STAT1 for the first time. The findings will broaden the spectrum of STAT1 mutations and facilitate genetic diagnosis by the oral medicine specialists.

SO094HUMAN URINE-DERIVED RENAL EPITHELIAL CELLS PROVIDE INSIGHTS INTO THE PATHOGENICITY OF PKHD1 VARIANTS

Abstract Background and Aims A 26 year old woman was referred for the investigation of polycystic kidney disease. There was no family history of renal disease and no obvious extra-renal manifestations. A GEMINI ciliopathy gene panel was performed which identified two heterozygous sequence changes (segregating from each parent) in PKHD1: a missense variant c.7964A>C, p.(His2655Pro), listed as a pathogenic variant in association with ARPKD on the Human Gene Mutation Database and a synonymous variant c.6900C>T, p.(Asn2300Asn). The pathogenicity of the synonymous PKHD1 variant is not clear. It is reported as a variant of unknown significance on ClinVar and has a low population frequency in the gnomAD cohort. The altered nucleotide is weakly conserved, but the same heterozygous change has been previously reported in an individual prenatally presenting with multicystic kidneys together with a missense variant on the other allele. We aim to confirm the pathogenicity of this variant in our patient, analysing its effects on the splicing of PKHD1 mRNA. Method PKHD1 is expressed at low levels in the leukocytes, therefore it can be difficult to analyse the splicing of PKHD1, using routine methods that involve RNA extraction from the blood. Urine-derived renal epithelial cells were isolated and cultured from two wild type individuals and from the patient and total RNA was extracted from these cells. RT-PCR and RT-qPCR were carried out to analyse the effects of the synonymous variant on the splicing of the PKHD1 gene in renal epithelial cells. Results RT-PCR revealed that PKHD1 is alternatively spliced both in the controls and in the patient and Sanger sequencing following T-cloning of PCR products revealed that both controls and the patient express an in-frame transcript and a shorter transcript with a 47 nucleotide loss in the exon 43 of PKHD1, that leads to a frame-shift and to the formation of a premature stop codon. We hypothesized that, although both mRNA isoforms are expressed in controls as well as in the patient URECs, the variant p.(Asn2300Asn) may shift the expression ratio between the two transcript isoforms in favour of the shorter, out-of-frame transcript. We designed and tested two sets of transcript-specific primers and performed a SYBR-green based RT-qPCR on controls and patient URECs cDNA. RT-qPCR, revealed that PKHD1 is expressed at lower levels in patient URECs, compared to controls. Specifically, the in-frame PKHD1 isoform is expressed at lower levels in patient URECs, compared to controls, whereas the levels of the shorter transcript leading to a frame-shift are higher in patient cells. Conclusion Using urine-derived renal epithelial cells as a source of kidney-specific RNA, we confirmed the pathogenicity of the PKHD1 synonymous variant p.(Asn2300Asn), which leads to an increased expression of an out-of-frame PKHD1 transcript, predicted to result in a truncated protein and expressed at lower levels also in control cells. The significance of this isoform in control renal epithelial cells is unclear.

InMeRF: prediction of pathogenicity of missense variants by individual modeling for each amino acid substitution.

In predicting the pathogenicity of a nonsynonymous single-nucleotide variant (nsSNV), a radical change in amino acid properties is prone to be classified as being pathogenic. However, not all such nsSNVs are associated with human diseases. We generated random forest (RF) models individually for each amino acid substitution to differentiate pathogenic nsSNVs in the Human Gene Mutation Database and common nsSNVs in dbSNP. We named a set of our models ‘Individual Meta RF’ (InMeRF). Ten-fold cross-validation of InMeRF showed that the areas under the curves (AUCs) of receiver operating characteristic (ROC) and precision–recall curves were on average 0.941 and 0.957, respectively. To compare InMeRF with seven other tools, the eight tools were generated using the same training dataset, and were compared using the same three testing datasets. ROC-AUCs of InMeRF were ranked first in the eight tools. We applied InMeRF to 155 pathogenic and 125 common nsSNVs in seven major genes causing congenital myasthenic syndromes, as well as in VANGL1 causing spina bifida, and found that the sensitivity and specificity of InMeRF were 0.942 and 0.848, respectively. We made the InMeRF web service, and also made genome-wide InMeRF scores available online (https://www.med.nagoya-u.ac.jp/neurogenetics/InMeRF/).

SUN-028 Concomitant Mutations in the POR and AR Genes in a Boy Presenting with Micropenis and Premature Adrenarche

Background: Micropenis is caused either by a defect in androgen synthesis, conversion to Dihydrotestosterone, or action due to mutant androgen receptor. Premature adrenarche is caused by excess of adrenal androgens. Clinical case: A 6,5-year-old boy presented to our pediatric endocrinology clinic due to increased body weight and hyperthyreotropinemia. Physical examination revealed premature adrenarche with testicular volume 1,5 ml, micropenis (4cm, <-2 SDS) and acanthosis nigricans. His linear growth presented an acceleration the last 2 years from +0.85 to +1.81 SD and his bone age was advanced by 2 years with a statural height at -0.5 SD, not in accordance with the mean parental height (+0.90 SD). At the same time, his BMI was increased from +1,36 to +3,64 SDS.Laboratory investigations for premature adrenarche with synacthen test revealed mild elevation of compound-S 15,4 ng/dl at 60’, which could be theoretically attributed to partial 11-β-hydroxylase deficiency (CYP11B1). DHEA was also elevated 2,1 ng/ml with an increased DHEA/Δ4 ratio at 20 (normal < 10) which could be explained in the case of partial 3-β-Hydroxysteroid dehydrogenase deficiency. Method: Whole exome sequencing was preformed targeted to a gene panel related to premature adrenarche. Results: A heterozygous mutation c.[1174C>T]+[1174C>T]; p.[Pro329Ser]+[Pro329Ser] in exon 1 in Androgen Receptor (AR) gene (X-linked) was found to the patient and his mother. The mutation, according to Human Gene Mutation Database, can cause disorder of sex development (DSD), partial androgen insensitivity syndrome (PAIS), infertility and hypospadias. Additionally, sequencing of the coding region of the P450 oxyreductase (POR) gene revealed a heterozygous mutation c.[642-5C>G] in exon 7, which in homozygosity can cause steroidogenesis disorder due to oxyreductase deficiency. This heterozygous POR mutation was found to the patient and his father. Discussion: Our patient caries mutations in the AR and in POR genes. The AR mutation is obviously responsible for micropenis in our patient. CYP11B1 and 3β-Hydroxysteroid dehydrogenase genes were normal. POR deficiency is a disorder of steroidogenesis with phenotypic spectrum ranging from PCOS to ambiguous genitalia and glycocorticosteroid deficiency or even to classic Antley-Bixler syndrome (1). Conclusion: The genetic and hormonal results in our case imply a role for POR in the CYP11B1 gene expression, which is reported for the first time to our knowledge.1.Concomitant Mutations in the P450 Oxidoreductase and Androgen Receptor Genes Presenting with 46,XY Disordered Sex Development and Androgenization at Adrenarche. J Clin Endocrinol Metab. 2010 Jul; 95(7): 3418-3427

A novel nonsense variant in SLC24A4 causing a rare form of amelogenesis imperfecta in a Pakistani family

BackgroundAmelogenesis imperfecta (AI) is a highly heterogeneous group of hereditary developmental abnormalities which mainly affects the dental enamel during tooth development in terms of its thickness, structure, and composition. It appears both in syndromic as well as non-syndromic forms. In the affected individuals, the enamel is usually thin, soft, rough, brittle, pitted, chipped, and abraded, having reduced functional ability and aesthetics. It leads to severe complications in the patient, like early tooth loss, severe discomfort, pain, dental caries, chewing difficulties, and discoloration of teeth from yellow to yellowish-brown or creamy type. The study aimed to identify the disease-causing variant in a consanguineous family.MethodsWe recruited a consanguineous Pashtun family of Pakistani origin. Exome sequencing analysis was followed by Sanger sequencing to identify the pathogenic variant in this family.ResultsClinical analysis revealed hypomaturation AI having generalized yellow-brown or creamy type of discoloration in affected members. We identified a novel nonsense sequence variant c.1192C > T (p.Gln398*) in exon-12 of SLC24A4 by using exome sequencing. Later, its co-segregation within the family was confirmed by Sanger sequencing. The human gene mutation database (HGMD, 2019) has a record of five pathogenic variants in SLC24A4, causing AI phenotype.ConclusionThis nonsense sequence variant c.1192C > T (p.Gln398*) is the sixth disease-causing variant in SLC24A4, which extends its mutation spectrum and confirms the role of this gene in the morphogenesis of human tooth enamel. The identified variant highlights the critical role of SLC24A4 in causing a rare AI type in humans.

Functional Characterization of PHEX Gene Variants in Children With X-Linked Hypophosphatemic Rickets Shows No Evidence of Genotype-Phenotype Correlation.

X-linked hypophosphatemia (XLHR) is caused by loss-of-function mutations in the phosphate regulating endopeptidase homolog X-linked (PHEX) gene. Considerable controversy exists regarding genotype-phenotype correlations in XLHR. The present study describes the clinical features and molecular genetic bases of 53 pediatric patients with XLHR. Overall, 47 different mutations were identified, of which 27 were not previously described in the literature or entered in the Human Gene Mutation Database (HGMD). A high prevalence (72.34%) of truncating variants was observed in XLHR patients. The clinical presentation and severity of XLHR did not show an evident correlation between the truncating and non-truncating mutation types in our cohort. To further delineate the characteristics of PHEX variants underlying this nonsignificant trend, we assessed the effects of 10 PHEX variants on protein expression, cellular trafficking, and endopeptidase activity. Our results showed that the nonsense mutations p.Arg567*, p.Gln714*, and p.Arg747* caused a reduction of protein molecular weight and a trafficking defect. Among seven non-truncating mutations, the p.Cys77Tyr, p.Cys85Ser, p.Ile281Lys, p.Ile333del, p.Ala514Pro, and p.Gly572Ser mutants were not secreted into the medium and remained trapped inside cells in an immature form, whereas the p.Gly553Glu mutant was terminally glycosylated and secreted into the medium. We further assessed the endopeptidase activity of the p.Gly553Glu mutant using a quenched fluorogenic peptide substrate and revealed that the activity of p.Gly553Glu significantly reduced to 13% compared with the wild type, which indicated disruption of catalytic function. These data not only support the clinical results showing no correlation between disease severity and the type of PHEX mutation but also provide helpful molecular insights into the pathogenesis of XLHR. © 2020 American Society for Bone and Mineral Research.

Mutational analysis and prenatal diagnosis of TMPRSS3 gene in two Chinese families affected with deafness

Objective: To detect potential mutations in two Chinese families affected with deafness, so as provide prenatal diagnosis for them. Methods: Two Chinese families affected with deafness were identified at the genetic and prenatal diagnosis center of the First Affiliated Hospital of Zhengzhou University from March 2018 to December 2018.Mutation analyses were carried out by next generation sequencing (NGS),suspected mutations were verified by Sanger sequencing in the probands, unaffected relatives. Prenatal diagnosis for high-risk fetus were carried out through Sanger sequencing. Results: The proband of family 1 carried a c.432delA and a c.617-2_617-1insTC mutation of the TMPRSS3 gene, the proband of family 2 carried a c.271C>T(p.R91X) and a c.147dupTmutation ofthe TMPRSS3 gene, both parents of the two probands were carriers of heterozygous variants. Conclusions: Mutations in the TMPRSS3 gene are the suspected cause of deafness in two families. Application of next generation sequencing technologies make gene diagnosis of deafness efficiently and accurately and the molecular findings increase our understanding of the function of TMPRSS3 gene and enrich the human gene mutation database. It is helpful for recurrent genetic counseling and prenatal diagnosis for these families.

A catalog of the pathogenic mutations of LDL receptor gene in Japanese familial hypercholesterolemia

Little data exist on the pathogenic mutations of LDL receptor in Japanese familial hypercholesterolemia (FH). We aimed to catalog the pathogenic mutations of LDL receptor gene in the 2 major Japanese FH-care centers (Kanazawa University and National Cerebral and Cardiovascular Center Research Institute), where genetic testing of FH has been performed centrally on requests from institutes all over Japan during more than past 2 decades. 796 FH subjects from 472 families who had nonsynonymous mutations in LDL receptor gene were included in this study. Genetic mutations were analyzed for mutations by Sanger sequencing as well as by multiplex ligation probe dependent amplification technique for large rearrangements. Pathogenic mutations were defined either as 1) protein truncated variants, 2) registered as pathogenic in ClinVar, or Human Gene Mutation Database (HGMD), or meet the criteria of American College of Medical Genetics and Genomics guideline, or 3) CADD score >10. We found 138 different mutations. Among them, 132 mutations were considered as pathogenic, including 19 large rearrangement mutations. However, 6 missense mutations were classified as variants of unknown significance. A single mutation accounted for as much as 41% of the FH subjects recruited from Kanazawa University mainly due to founder gene effect, whereas many singleton mutations were found from National Cerebral and Cardiovascular Center Research Institute located in Osaka. We provided the largest catalog of pathogenic mutations of LDL receptor gene in Japanese FH. This could aid to determine the pathogenicity of the LDL receptor genetic mutations not only in Japanese but also in other ethnicities.