Pathogenic mosaic variants in congenital hypogonadotropic hypogonadism

PurposeCongenital hypogonadotropic hypogonadism (CHH) is a rare disorder resulting in absent puberty and infertility. The genetic architecture is complex with multiple loci involved, variable expressivity, and incomplete penetrance. The majority of cases are sporadic, consistent with a disease affecting fertility. The current study aims to investigate mosaicism as a genetic mechanism for CHH, focusing on de novo rare variants in CHH genes.MethodsWe evaluated 60 trios for de novo rare sequencing variants (RSV) in known CHH genes using exome sequencing. Potential mosaicism was suspected among RSVs with altered allelic ratios and confirmed using customized ultradeep sequencing (UDS) in multiple tissues.ResultsAmong the 60 trios, 10 probands harbored de novo pathogenic variants in CHH genes. Custom UDS demonstrated that three of these de novo variants were in fact postzygotic mosaicism—two in FGFR1 (p.Leu630Pro and p.Gly348Arg), and one in CHD7 (p.Arg2428*). Statistically significant variation across multiple tissues (DNA from blood, buccal, hair follicle, urine) confirmed their mosaic nature.ConclusionsWe identified a significant number of de novo pathogenic variants in CHH of which a notable number (3/10) exhibited mosaicism. This report of postzygotic mosaicism in CHH patients provides valuable information for accurate genetic counseling.     

Genetic analysis of CHARGE syndrome identifies overlapping molecular biology

PurposeCHARGE syndrome is an autosomal-dominant, multiple congenital anomaly condition characterized by vision and hearing loss, congenital heart disease, and malformations of craniofacial and other structures. Pathogenic variants in CHD7, encoding adenosine triphosphate–dependent chromodomain helicase DNA binding protein 7, are present in the majority of affected individuals. However, no causal variant can be found in 5–30% (depending on the cohort) of individuals with a clinical diagnosis of CHARGE syndrome.MethodsWe performed whole-exome sequencing (WES) on 28 families from which at least one individual presented with features highly suggestive of CHARGE syndrome.ResultsPathogenic variants in CHD7 were present in 15 of 28 individuals (53.6%), whereas 4 (14.3%) individuals had pathogenic variants in other genes (RERE, KMT2D, EP300, or PUF60). A variant of uncertain clinical significance in KDM6A was identified in one (3.5%) individual. The remaining eight (28.6%) individuals were not found to have pathogenic variants by WES.ConclusionThese results demonstrate that the phenotypic features of CHARGE syndrome overlap with multiple other rare single-gene syndromes. Additionally, they implicate a shared molecular pathology that disrupts epigenetic regulation of multiple-organ development.     

Pathogenic TERT promoter variants in telomere diseases

PurposeThe acquisition of pathogenic variants in the TERT promoter (TERTp) region is a mechanism of tumorigenesis. In nonmalignant diseases, TERTp variants have been reported only in patients with idiopathic pulmonary fibrosis (IPF) due to germline variants in telomere biology genes.MethodsWe screened patients with a broad spectrum of telomeropathies (n = 136), their relatives (n = 52), and controls (n = 195) for TERTp variants using a customized massively parallel amplicon-based sequencing assay.ResultsPathogenic −124 and −146 TERTp variants were identified in nine (7%) unrelated patients diagnosed with IPF (28%) or moderate aplastic anemia (4.6%); five of them also presented cirrhosis. Five (10%) relatives were also found with these variants, all harboring a pathogenic germline variant in telomere biology genes. TERTp clone selection did not associate with peripheral blood counts, telomere length, and response to danazol treatment. However, it was specific for patients with telomeropathies, more frequently co-occurring with TERT germline variants and associated with aging.ConclusionWe extend the spectrum of nonmalignant diseases associated with pathogenic TERTp variants to marrow failure and liver disease due to inherited telomerase deficiency. Specificity of pathogenic TERTp variants for telomerase dysfunction may help to assess the pathogenicity of unclear constitutional variants in the telomere diseases.     

Atopic disorders in CHARGE syndrome: A retrospective study and literature review

Background

Atopic disorders have been reported in CHARGE syndrome, but the prevalence and underlying mechanisms are not known.

Tumor detection rates in screening of individuals with SDHx-related hereditary paraganglioma–pheochromocytoma syndrome

PurposeMinimal data exist regarding the efficacy of screening protocols for individuals with SDHx germline pathogenic variants with hereditary paraganglioma–pheochromocytoma syndrome. This study aimed to evaluate the SDHx-related tumor detection rate in individuals undergoing clinical screening protocols.MethodsA multicenter retrospective longitudinal observational study was conducted. Individuals with germline SDHx pathogenic variants underwent clinical whole-body imaging and biochemical testing.ResultsTwo hundred sixty-three individuals with SDHx germline pathogenic variants completed 491 imaging screens. Individuals with SDHB germline pathogenic variants were most common (n = 188/263, 72%), followed by SDHD (n = 35/263, 13%) and SDHC (n = 28/263, 11%). SDHx-related tumors were found in 17% (n = 45/263) of the cohort. Most SDHx-related tumors were identified on baseline imaging screen (n = 39/46, 85%). Individuals with SDHD pathogenic variants had the highest tumor detection rate (n = 14/35, 40%). Of imaging screens identifying SDHx-related paraganglioma/pheochromocytoma, 29% (n = 12/41) had negative biochemical testing. Secondary actionable findings were identified in 15% (n = 75/491) of imaging screens.ConclusionCurrent SDHx screening protocols are effective at identifying SDHx-related tumors. Tumor detection rates vary by SDHx gene and screening has the potential to uncover actionable secondary findings. Imaging is an essential part of the screening process as biochemical testing alone does not detect all disease.     

Genomic diagnostics within a medically underserved population: efficacy and implications

PurposeWe integrated whole-exome sequencing (WES) and chromosomal microarray analysis (CMA) into a clinical workflow to serve an endogamous, uninsured, agrarian community.MethodsSeventy-nine probands (newborn to 49.8 years) who presented between 1998 and 2015 remained undiagnosed after biochemical and molecular investigations. We generated WES data for probands and family members and vetted variants through rephenotyping, segregation analyses, and population studies.ResultsThe most common presentation was neurological disease (64%). Seven (9%) probands were diagnosed by CMA. Family WES data were informative for 37 (51%) of the 72 remaining individuals, yielding a specific genetic diagnosis (n = 32) or revealing a novel molecular etiology (n = 5). For five (7%) additional subjects, negative WES decreased the likelihood of genetic disease. Compared to trio analysis, “family” WES (average seven exomes per proband) reduced filtered candidate variants from 22 ± 6 to 5 ± 3 per proband. Nineteen (51%) alleles were de novo and 17 (46%) inherited; the latter added to a population-based diagnostic panel. We found actionable secondary variants in 21 (4.2%) of 502 subjects, all of whom opted to be informed.ConclusionCMA and family-based WES streamline and economize diagnosis of rare genetic disorders, accelerate novel gene discovery, and create new opportunities for community-based screening and prevention in underserved populations.     

Molecular analysis of the CHD7 gene in CHARGE syndrome: identification of 22 novel mutations and evidence for a low contribution of large CHD7 deletions

PurposeAutosomal dominant CHARGE syndrome (OMIM no. 214800) is characterized by choanal atresia or cleft lip or palate, ocular colobomas, cardiovascular malformations, retardation of growth, ear anomalies, and deafness, and is caused by mutations in the CHD7 gene. Here, we describe the outcome of a molecular genetic analysis in 18 Finnish and 56 German patients referred for molecular confirmation of the clinical diagnosis of suspected CHARGE syndrome.MethodsQuantitative real-time polymerase chain reaction or multiplex ligation-dependent probe amplification assays did not reveal deletions in mutation negative cases, suggesting that larger CHD7 deletions are not a major cause of CHARGE syndrome.ResultsIn this group of 74 patients, we found mutations in 30 cases. 22 mutations were novel, including 11 frameshift, 5 nonsense, 3 splice-site, and 3 missense mutations. One de novo frameshift mutation was found in the last exon and is expected to result in a minimally shortened CHD7 polypeptide. Because the mutation is associated with a typical CHARGE syndrome phenotype, it may indicate the presence of an as yet unknown functional domain in the very carboxyterminal end of CHD7.ConclusionsOur mutation detection rate of 40.5% is reflective of screening an unselected sample population referred for CHD7 testing based on suspected clinical diagnosis of CHARGE syndrome and not for having met strict clinical criteria for this disorder.     

Acute myeloid leukemia associated with CHARGE syndrome

CHARGE syndrome is a malformation disorder with diverse phenotypes that shows autosomal dominance with heterozygous variants in the chromodomain helicase DNA-binding 7 (CHD7) gene. Only a few cases of CHARGE syndrome accompanied by neoplasm during childhood have been reported. We report the case of a girl with CHARGE syndrome who developed acute myelogenous leukemia at 12 years old. She had mild intellectual disability, and hearing loss with inner ear malformation, myopia, astigmatism, laryngotracheal malacia, hypogonadism, and clival hypoplasia, with a history of patent ductus arteriosus. The patient was genetically diagnosed with CHARGE syndrome based on the detection of a novel heterozygous frameshift pathogenic variant in the CHD7 gene. We review the reported pediatric cases of CHARGE syndrome with malignancy and suggest a possible molecular mechanism of carcinogenesis involving pathogenic variants of the CHD7 gene.     

Somatic TP53 variants frequently confound germ-line testing results

PurposeBlood/saliva DNA is thought to represent the germ line in genetic cancer-risk assessment. Cases with pathogenic TP53 variants detected by multigene panel testing are often discordant with Li-Fraumeni syndrome, raising concern about misinterpretation of acquired aberrant clonal expansions (ACEs) with TP53 variants as germ-line results.MethodsPathogenic TP53 variants with abnormal next-generation sequencing metrics (e.g., decreased ratio (<25%) of mutant to wild-type allele, more than two detected alleles) were selected from a CLIA laboratory testing cohort. Alternate tissues and/or close relatives were tested to distinguish between ACE and germ-line status. Clinical data and Li-Fraumeni syndrome testing criteria were examined.ResultsAmong 114,630 multigene panel tests and 1,454 TP53 gene-specific analyses, abnormal next-generation sequencing metrics were observed in 20% of 353 TP53-positive results, and ACE was confirmed for 91% of cases with ancillary materials, most of these due to clonal hematopoiesis. Only four met Chompret criteria. Individuals with ACE were older (50 years vs. 33.7; P = 0.02) and were identified more frequently in multigene panel tests (66/285; 23.2%) than in TP53 gene-specific tests (6/68; 8.8%, P = 0.005).ConclusionACE confounds germ-line diagnosis, may portend hematologic malignancy, and may provoke unwarranted clinical interventions. Ancillary testing to confirm germ-line status should precede Li-Fraumeni syndrome management.     

Racial/ethnic differences in multiple-gene sequencing results for hereditary cancer risk

PurposeWe examined racial/ethnic differences in the usage and results of germ-line multiple-gene sequencing (MGS) panels to evaluate hereditary cancer risk.MethodsWe collected genetic testing results and clinical information from 1,483 patients who underwent MGS at Stanford University between 1 January 2013 and 31 December 2015.ResultsAsians and Hispanics presented for MGS at younger ages than whites (48 and 47 vs. 55; P = 5E-16 and 5E-14). Across all panels, the rate of pathogenic variants (15%) did not differ significantly between racial groups. Rates by gene did differ: in particular, a higher percentage of whites than nonwhites carried pathogenic CHEK2 variants (3.8% vs. 1.0%; P = 0.002). The rate of a variant of uncertain significance (VUS) result was higher in nonwhites than whites (36% vs. 27%; P = 2E-4). The probability of a VUS increased with increasing number of genes tested; this effect was more pronounced for nonwhites than for whites (1.1% absolute difference in VUS rates testing BRCA1/2 vs. 8% testing 13 genes vs. 14% testing 28 genes), worsening the disparity.ConclusionIn this diverse cohort undergoing MGS testing, pathogenic variant rates were similar between racial/ethnic groups. By contrast, VUS results were more frequent among nonwhites, with potential significance for the impact of MGS testing by race/ethnicity.     

Long-read genome sequencing identifies causal structural variation in a Mendelian disease

PurposeCurrent clinical genomics assays primarily utilize short-read sequencing (SRS), but SRS has limited ability to evaluate repetitive regions and structural variants. Long-read sequencing (LRS) has complementary strengths, and we aimed to determine whether LRS could offer a means to identify overlooked genetic variation in patients undiagnosed by SRS.MethodsWe performed low-coverage genome LRS to identify structural variants in a patient who presented with multiple neoplasia and cardiac myxomata, in whom the results of targeted clinical testing and genome SRS were negative.ResultsThis LRS approach yielded 6,971 deletions and 6,821 insertions > 50 bp. Filtering for variants that are absent in an unrelated control and overlap a disease gene coding exon identified three deletions and three insertions. One of these, a heterozygous 2,184 bp deletion, overlaps the first coding exon of PRKAR1A, which is implicated in autosomal dominant Carney complex. RNA sequencing demonstrated decreased PRKAR1A expression. The deletion was classified as pathogenic based on guidelines for interpretation of sequence variants.ConclusionThis first successful application of genome LRS to identify a pathogenic variant in a patient suggests that LRS has significant potential for the identification of disease-causing structural variation. Larger studies will ultimately be required to evaluate the potential clinical utility of LRS.     

Single-reviewer electronic phenotyping validation in operational settings: Comparison of strategies and recommendations

ObjectiveDevelop evidence-based recommendations for single-reviewer validation of electronic phenotyping results in operational settings.Material and methodsWe conducted a randomized controlled study to evaluate whether electronic phenotyping results should be used to support manual chart review during single-reviewer electronic phenotyping validation (N = 3104). We evaluated the accuracy, duration and cost of manual chart review with and without the availability of electronic phenotyping results, including relevant patient-specific details. The cost of identification of an erroneous electronic phenotyping result was calculated based on the personnel time required for the initial chart review and subsequent adjudication of discrepancies between manual chart review results and electronic phenotype determinations.ResultsProviding electronic phenotyping results (vs not providing those results) was associated with improved overall accuracy of manual chart review (98.90% vs 92.46%, p < 0.001), decreased review duration per test case (62.43 vs 76.78 s, p < 0.001), and insignificantly reduced estimated marginal costs of identification of an erroneous electronic phenotyping result ($48.54 vs $63.56, p = 0.16). The agreement between chart review and electronic phenotyping results was higher when the phenotyping results were provided (Cohen’s kappa 0.98 vs 0.88, p < 0.001). As a result, while accuracy improved when initial electronic phenotyping results were correct (99.74% vs 92.67%, N = 3049, p < 0.001), there was a trend towards decreased accuracy when initial electronic phenotyping results were erroneous (56.67% vs 80.00%, N = 55, p = 0.07). Electronic phenotyping results provided the greatest benefit for the accurate identification of rare exclusion criteria.DiscussionSingle-reviewer chart review of electronic phenotyping can be conducted more accurately, quickly, and at lower cost when supported by electronic phenotyping results. However, human reviewers tend to agree with electronic phenotyping results even when those results are wrong. Thus, the value of providing electronic phenotyping results depends on the accuracy of the underlying electronic phenotyping algorithm.ConclusionWe recommend using a mix of phenotyping validation strategies, with the balance of strategies based on the anticipated electronic phenotyping error rate, the tolerance for missed electronic phenotyping errors, as well as the expertise, cost, and availability of personnel involved in chart review and discrepancy adjudication.     

Detection of human herpesvirus-6 variants in pediatric brain tumors: Association of viral antigen in low grade gliomas

BackgroundHuman herpesvirus-6 (HHV-6) has been associated with a diverse spectrum of central nervous system (CNS) diseases and reported glial tropism.ObjectiveTo determine if HHV-6 is present in a series of pediatric brain tumors.Study designPediatric gliomas from 88 untreated patients represented in a tissue microarray (TMA) were screened for HHV-6 by nested polymerase chain reaction (PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) and compared to non-glial tumors (N = 22) and control brain (N = 32). Results were correlated with tumor grade and overall survival.ResultsHHV-6 U57 was detected by nested PCR in 68/120 (57%) tumors and 7/32 (22%) age-matched non-tumor brain (P = 0.001). HHV-6 U31 was positive in 73/120 (61%) tumors and 11/32 (34%) controls (P = 0.019). Seventy-two percent (43/60) of tumors were HHV-6 Variant A. HHV-6 U57 was confirmed by ISH in 83/150 (54%) tumors and 10/32 (31%) controls (P = 0.021), revealing a non-lymphocytic origin of HHV-6. HHV-6A/B gp116/64/54 late antigen was detected by IHC in 50/124 (40%) tumors and 6/32 (18%) controls (P = 0.013). Interestingly, 58% of low grade gliomas (N = 67) were IHC positive compared to 19% of high grade gliomas (N = 21, P = 0.002) and 25% of non-gliomas (N = 36, P = 0.001). HHV-6A/B gp116/64/54 antigen co-localized with glial fibrillary acidic protein, confirming the astrocytic origin of antigen. Overall, there was no primary association between HHV-6A/B gp116/64/54 antigen detection and survival (P = 0.861).ConclusionsWe provide the first reported series of HHV-6 detection in pediatric brain tumors. The predominance of HHV-6 in glial tumors warrants further investigation into potential neurooncologic disease mechanisms.     

Detection of CHD7 deletions by MLPA in CHARGE syndrome patients with a less typical phenotype

Bergman et al. performed a search for exon copy number alterations in the CHD7 gene using MLPA in CHARGE syndrome patients who did not have a CHD7 mutation. Based on their results they recommended to extend testing using MLPA solely in individuals with a typical CHARGE syndrome phenotype. However, since we have found deletions comprising the CHD7 gene in three patients with a less typical phenotype we recommend performing MLPA testing in all CHARGE syndrome patients without causal CHD7 mutations.     

Comparative assessment of gene-specific variant distribution in prenatal and postnatal cohorts tested for Noonan syndrome and related conditions

PurposeTo compare the pattern of gene-specific involvement and the spectrum of variants observed in prenatal and postnatal (mean ± SD, 8.9 ± 9.4 years) cohorts tested for Noonan syndrome and related conditions.MethodsOutcomes of sequencing panel testing were compared between prenatal (n = 845) and postnatal (n = 409) cohorts.ResultsPTPN11 and SOS1 harbored the majority of observed variants in both prenatal and postnatal cohorts, and BRAF, HRAS, KRAS, MAP2K1, MAP2K2, RAF1, and SHOC2 had similarities in their pattern of involvement in both cohorts. PTPN11 was the largest contributor of pathogenic variants and had the lowest frequency of variants of uncertain significance (VUS). SOS1 had the highest VUS frequency in both cohorts. The overall VUS frequency was twice as high in prenatal specimens (58.1 vs. 29.3%). PTPN11 and SOS1 had a 1.5-fold higher VUS frequency in the prenatal cohort (10.7 vs. 7.4% and 95 vs. 61.1%, respectively). The diagnostic yield was 3.7% for prenatal samples, with a higher yield of 12.3% in fetuses with cystic hygroma as a sole finding, and 21.3% for postnatal.ConclusionComparison of prenatal versus postnatal specimens demonstrates that the pattern of specific gene involvement is similar, whereas the classification spectrum of observed variants differs considerably.     

A genomics approach to male infertility

PurposeMale infertility remains poorly understood at the molecular level. We aimed in this study to investigate the yield of a “genomics first” approach to male infertility.MethodsPatients with severe oligospermia and nonobstructive azoospermia were investigated using exome sequencing (ES) in parallel with the standard practice of chromosomal analysis.ResultsIn 285 patients, 10.5% (n = 30) had evidence of chromosomal aberrations while nearly a quarter (n = 69; 24.2%) had a potential monogenic form of male infertility. The latter ranged from variants in genes previously reported to cause male infertility with or without other phenotypes in humans (24 patients; 8.4%) to those in novel candidate genes reported in this study (37 patients; 12.9%). The 33 candidate genes have biological links to male germ cell development including compatible mouse knockouts, and a few (TERB1 [CCDC79], PIWIL2, MAGEE2, and ZSWIM7) were found to be independently mutated in unrelated patients in our cohort. We also found that male infertility can be the sole or major phenotypic expression of a number of genes that are known to cause multisystemic manifestations in humans (n = 9 patients; 3.1%).ConclusionThe standard approach to male infertility overlooks the significant contribution of monogenic causes to this important clinical entity.     

Association of HHV-6 and HHV-7 reactivation with the development of chronic allograft nephropathy

BackgroundThe long-term effect of HHV-6 and HHV-7 infections on chronic allograft nephropathy (CAN) development after renal transplantation is uncertain.ObjectivesTo determine HHV-6 and HHV-7 reactivation during the post-transplantation period and to evaluate its effect on CAN development in renal transplant patients.Study designEighty-one renal allograft recipients (28 with CAN, 53 with normal transplant function) were studied to determine the frequency of HHV-6 and HHV-7 reactivation during 36.4 ± 7.8 months after renal transplantation using nested PCR. HHV-6 variants were identified using restriction endonuclease analysis. Patients were monitored for the development of CAN.ResultsThe frequency of HHV-6 and/or HHV-7 plasma DNA was significantly higher in CAN patients (25/28, 89.3%) compared to control patients (15/50, 30.0%, p = 0.0001). CAN patients also had an increased incidence of dual active infections (20/25, 80% and 2/15, 13.3%, p = 0.007, respectively). In all 34 HHV-6 positive cases, the HHV-6B variant was identified. The presence of HHV-7 DNA in plasma preceded the presence of HHV-6 DNA. Early development of CAN and graft loss was detected only in patients with simultaneous HHV-6 and HHV-7 plasma DNA.ConclusionsReactivation of HHV-6 and HHV-7 in renal graft recipients is a risk factor for CAN development. The presence of concurrent HHV-6 and HHV-7 DNA in the plasma is an unfavorable prognostic factor.     

Does HIV-1 co-receptor tropism correlate with fibrosis progression in HIV/HCV co-infected patients?

BackgroundIn HIV/HCV co-infected patients, HIV-1 gp120 activates human hepatic stellate cells (HSCs) which play a key role in fibrosis pathogenesis. It is still unclear whether pro-fibrogenic effects are more attributable to X4 or R5 strains in vivo.ObjectiveTo assess if HIV-1 X4 or R5 variants are associated with a different progression of fibrosis.Study designA total of 105 HIV/HCV co-infected patients were submitted to gp120 sequencing on proviral DNA and classified as X4 or R5 based on g2p (20% false positive rate). The fibrosis evolution was retrospectively determined by means of APRI and FIB-4 scores at 3-month intervals from the first anti-HCV-positive test. The association of co-receptor tropism with increased fibrosis scores was evaluated by linear mixed models.ResultsX4 variants were found in 41 patients (39%). The median observation period was similar in X4 and R5 patients (17 years). No difference was observed between the two groups of patients, except for nadir CD4 which was lower in X4 compared to R5 (percentage, p = 0.005, and absolute number, p = 0.005). X4 and R5 patients did not significantly differ for FIB-4 and APRI score over time (p = 0.5, and p = 0.1, respectively). No association between HCV-RNA levels over time and co-receptor tropism was noted (p = 0.9). Conversely, a significant correlation of fibrosis scores with gamma-glutamyl transferase levels, lower current CD4 count, HIV viremia and use of antiretrovirals was observed.ConclusionsThis retrospective analysis of fibrosis evolution did not support the evidence of a differing pro-fibrogenic activity for X4 and R5 HIV-1 variants in HIV/HCV co-infected patients.