Toward robust clinical genome interpretation: Developing a consistent terminology to characterize Mendelian disease-gene relationships-allelic requirement, inheritance modes, and disease mechanisms.

PURPOSE: The terminology used for gene-disease curation and variant annotation to describe inheritance, allelic requirement, and both sequence and functional consequences of a variant is currently not standardized. There is considerable discrepancy in the literature and across clinical variant reporting in the derivation and application of terms. Here, we standardize the terminology for the characterization of disease-gene relationships to facilitate harmonized global curation and to support variant classification within the ACMG/AMP framework. METHODS: Terminology for inheritance, allelic requirement, and both structural and functional consequences of a variant used by Gene Curation Coalition members and partner organizations was collated and reviewed. Harmonized terminology with definitions and use examples was created, reviewed, and validated. RESULTS: We present a standardized terminology to describe gene-disease relationships, and to support variant annotation. We demonstrate application of the terminology for classification of variation in the ACMG SF 2.0 genes recommended for reporting of secondary findings. Consensus terms were agreed and formalized in both Sequence Ontology (SO) and Human Phenotype Ontology (HPO) ontologies. Gene Curation Coalition member groups intend to use or map to these terms in their respective resources. CONCLUSION: The terminology standardization presented here will improve harmonization, facilitate the pooling of curation datasets across international curation efforts and, in turn, improve consistency in variant classification and genetic test interpretation.

Beyond gene-disease validity: capturing structured data on inheritance, allelic requirement, disease-relevant variant classes, and disease mechanism for inherited cardiac conditions.

BACKGROUND: As the availability of genomic testing grows, variant interpretation will increasingly be performed by genomic generalists, rather than domain-specific experts. Demand is rising for laboratories to accurately classify variants in inherited cardiac condition (ICC) genes, including secondary findings. METHODS: We analyse evidence for inheritance patterns, allelic requirement, disease mechanism and disease-relevant variant classes for 65 ClinGen-curated ICC gene-disease pairs. We present this information for the first time in a structured dataset, CardiacG2P, and assess application in genomic variant filtering. RESULTS: For 36/65 gene-disease pairs, loss of function is not an established disease mechanism, and protein truncating variants are not known to be pathogenic. Using the CardiacG2P dataset as an initial variant filter allows for efficient variant prioritisation whilst maintaining a high sensitivity for retaining pathogenic variants compared with two other variant filtering approaches. CONCLUSIONS: Access to evidence-based structured data representing disease mechanism and allelic requirement aids variant filtering and analysis and is a pre-requisite for scalable genomic testing.

Towards robust clinical genome interpretation: developing a consistent terminology to characterize disease-gene relationships - allelic requirement, inheritance modes and disease mechanisms.

PURPOSE: The terminology used for gene-disease curation and variant annotation to describe inheritance, allelic requirement, and both sequence and functional consequences of a variant is currently not standardized. There is considerable discrepancy in the literature and across clinical variant reporting in the derivation and application of terms. Here we standardize the terminology for the characterization of disease-gene relationships to facilitate harmonized global curation, and to support variant classification within the ACMG/AMP framework. METHODS: Terminology for inheritance, allelic requirement, and both structural and functional consequences of a variant used by Gene Curation Coalition (GenCC) members and partner organizations was collated and reviewed. Harmonized terminology with definitions and use examples was created, reviewed, and validated. RESULTS: We present a standardized terminology to describe gene-disease relationships, and to support variant annotation. We demonstrate application of the terminology for classification of variation in the ACMG SF 2.0 genes recommended for reporting of secondary findings. Consensus terms were agreed and formalized in both sequence ontology (SO) and human phenotype ontology (HPO) ontologies. GenCC member groups intend to use or map to these terms in their respective resources. CONCLUSION: The terminology standardization presented here will improve harmonization, facilitate the pooling of curation datasets across international curation efforts and, in turn, improve consistency in variant classification and genetic test interpretation.

Beyond gene-disease validity: capturing structured data on inheritance, allelic-requirement, disease-relevant variant classes, and disease mechanism for inherited cardiac conditions.

Background: As availability of genomic testing grows, variant interpretation will increasingly be performed by genomic generalists, rather than domain-specific experts. Demand is rising for laboratories to accurately classify variants in inherited cardiac condition (ICC) genes, including as secondary findings. Methods: We analyse evidence for inheritance patterns, allelic requirement, disease mechanism and disease-relevant variant classes for 65 ClinGen-curated ICC gene-disease pairs. We present this information for the first time in a structured dataset, CardiacG2P, and assess application in genomic variant filtering. Results: For 36/65 gene-disease pairs, loss-of-function is not an established disease mechanism, and protein truncating variants are not known to be pathogenic. Using CardiacG2P as an initial variant filter allows for efficient variant prioritisation whilst maintaining a high sensitivity for retaining pathogenic variants compared with two other variant filtering approaches. Conclusions: Access to evidence-based structured data representing disease mechanism and allelic requirement aids variant filtering and analysis and is pre-requisite for scalable genomic testing.

Redefining WILD syndrome: a primary lymphatic dysplasia with congenital multisegmental lymphoedema, cutaneous lymphovascular malformation, CD4 lymphopaenia and warts.

BACKGROUND: Primary lymphoedema (PL) syndromes are increasingly recognised as presentations of complex genetic disease, with at least 20 identified causative genes. Recognition of clinical patterns is key to diagnosis, research and therapeutics. The defining criteria for one such clinical syndrome, 'WILD syndrome' (Warts, Immunodeficiency, Lymphoedema and anogenital Dysplasia), have previously depended on a single case report. METHODS AND RESULTS: We present 21 patients (including the first described case) with similar clinical and immunological phenotypes. All had PL affecting multiple segments, with systemic involvement (intestinal lymphangiectasia/pleural or pericardial effusions) in 70% (n=14/20). Most (n=20, 95%) had a distinctive cutaneous lymphovascular malformation on the upper anterior chest wall. Some (n=10, 48%) also had hyperpigmented lesions resembling epidermal naevi (but probably lymphatic in origin). Warts were common (n=17, 81%) and often refractory. In contrast to the previous case report, anogenital dysplasia was uncommon-only found in two further cases (total n=3, 14%). Low CD4 counts and CD4:CD8 ratios typified the syndrome (17 of 19, 89%), but monocyte counts were universally normal, unlike GATA2 deficiency. CONCLUSION: WILD syndrome is a previously unrecognised, underdiagnosed generalised PL syndrome. Based on this case series, we redefine WILD as 'Warts, Immunodeficiency, andLymphatic Dysplasia' and suggest specific diagnostic criteria. The essential criterion is congenital multisegmental PL in a 'mosaic' distribution. The major diagnostic features are recurrent warts, cutaneous lymphovascular malformations, systemic involvement (lymphatic dysplasia), genital swelling and CD4 lymphopaenia with normal monocyte counts. The absence of family history suggests a sporadic condition, and the random distribution of swelling implicates mosaic postzygotic mutation as the cause.

Results from London Regional Clinical Genetics services over a 5-year period on germline TP53 testing in women diagnosed with breast cancer at <30 years.

BACKGROUND: The most common cancer diagnosed in germline TP53 pathogenic variant (PV) carriers is premenopausal breast cancer. An increased rate of breast tumour HER2 positivity has been reported in this group. Screening for breast/other cancers is recommended in PV carriers. OBJECTIVES: 1. To assess the frequency of germline TP53 PVs reported diagnostically in women with breast cancer at <30 years of age.2. To evaluate the impact of personal/family history and HER2 status on the likelihood of germline TP53 pathogenic/likely pathogenic variant (PV/LPV) identification. METHODS: Genetic test results from patients undergoing diagnostic germline TP53 tests between 2012 and 2017 in the four London Regional Clinical Genetics Services were reviewed. Clinical/pathology data and family history were extracted from genetics files for women diagnosed with breast cancer at <30 years. RESULTS: The overall germline TP53 PV/LPV variant detection rate was 9/270=3.3% in all women diagnosed with breast cancer at <30 years and 2/171=1.2% in those with no second/subsequent cancer diagnosis or family history of TP53-spectrum cancers. Breast cancers were significantly more likely to be HER2-positive in TP53 PV/LPV carriers than in non-carriers (p=0.00006). CONCLUSIONS: Germline TP53 PVs/LPVs are uncommon among women diagnosed with breast cancer aged <30 years without other relevant personal or family cancer history but have an important clinical impact when identified.

Janus-faced EPHB4-associated disorders: novel pathogenic variants and unreported intrafamilial overlapping phenotypes.

PURPOSE: Several clinical phenotypes including fetal hydrops, central conducting lymphatic anomaly or capillary malformations with arteriovenous malformations 2 (CM-AVM2) have been associated with EPHB4 (Ephrin type B receptor 4) variants, demanding new approaches for deciphering pathogenesis of novel variants of uncertain significance (VUS) identified in EPHB4, and for the identification of differentiated disease mechanisms at the molecular level. METHODS: Ten index cases with various phenotypes, either fetal hydrops, CM-AVM2, or peripheral lower limb lymphedema, whose distinct clinical phenotypes are described in detail in this study, presented with a variant in EPHB4. In vitro functional studies were performed to confirm pathogenicity. RESULTS: Pathogenicity was demonstrated for six of the seven novel EPHB4 VUS investigated. A heterogeneity of molecular disease mechanisms was identified, from loss of protein production or aberrant subcellular localization to total reduction of the phosphorylation capability of the receptor. There was some phenotype-genotype correlation; however, previously unreported intrafamilial overlapping phenotypes such as lymphatic-related fetal hydrops (LRFH) and CM-AVM2 in the same family were observed. CONCLUSION: This study highlights the usefulness of protein expression and subcellular localization studies to predict EPHB4 variant pathogenesis. Our accurate clinical phenotyping expands our interpretation of the Janus-faced spectrum of EPHB4-related disorders, introducing the discovery of cases with overlapping phenotypes.

Refinement of the clinical and mutational spectrum of UBE2A deficiency syndrome.

UBE2A deficiency, that is, intellectual disability (ID) Nascimento type (MIM 300860), is an X-linked syndrome characterized by developmental delay, moderate to severe ID, seizures, dysmorphisms, skin anomalies, and urogenital malformations. Forty affected subjects have been reported thus far, with 31 cases having intragenic UBE2A variants. Here, we report on additional eight affected subjects from seven unrelated families who were found to be hemizygous for previously unreported UBE2A missense variants (p.Glu62Lys, p.Arg95Cys, p.Thr99Ala, and p.Arg135Trp) or small in-frame deletions (p.Val81_Ala83del, and p.Asp101del). A wide phenotypic spectrum was documented in these subjects, ranging from moderate ID associated with mild dysmorphisms to severe features including congenital heart defects (CHD), severe cognitive impairment, and pineal gland tumors. Four variants affected residues (Glu62, Arg95, Thr99 and Asp101) that contribute to stabilizing the structure of the E3 binding domain. The three-residue in-frame deletion, p.Val81_Ala83del, resulted from aberrant processing of the transcript. This variant and p.Arg135Trp mapped to regions of the protein located far from the E3 binding region, and caused variably accelerated protein degradation. By reviewing available clinical information, we revise the clinical and molecular profile of the disorder and document genotype-phenotype correlations. Pineal gland cysts/tumors, CHD and hypogammaglobulinemia emerge as recurrent features.

Non-catalytic Roles of Tet2 Are Essential to Regulate Hematopoietic Stem and Progenitor Cell Homeostasis.

The Ten-eleven translocation (TET) enzymes regulate gene expression by promoting DNA demethylation and partnering with chromatin modifiers. TET2, a member of this family, is frequently mutated in hematological disorders. The contributions of TET2 in hematopoiesis have been attributed to its DNA demethylase activity, and the significance of its nonenzymatic functions has remained undefined. To dissect the catalytic and non-catalytic requirements of Tet2, we engineered catalytically inactive Tet2 mutant mice and conducted comparative analyses of Tet2 mutant and Tet2 knockout animals. Tet2 knockout mice exhibited expansion of hematopoietic stem and progenitor cells (HSPCs) and developed myeloid and lymphoid disorders, while Tet2 mutant mice predominantly developed myeloid malignancies reminiscent of human myelodysplastic syndromes. HSPCs from Tet2 knockout mice exhibited distinct gene expression profiles, including downregulation of Gata2. Overexpression of Gata2 in Tet2 knockout bone marrow cells ameliorated disease phenotypes. Our results reveal the non-catalytic roles of TET2 in HSPC homeostasis.

Genomics: the power, potential and pitfalls of the new technologies and how they are transforming healthcare.

Powerful new genomic technologies are transforming healthcare. The faster, cheaper generation of genomic data is driving the integration of genomics into all healthcare specialties. Within the next decade, healthcare professionals will be using genomic data to diagnose and manage their patients.However, despite these exciting advances, few clinicians are aware of or prepared for this genomics-based future. Through five patient-focused scenarios with accompanying interviews, this article showcases new genomic technologies while highlighting the inherent challenges associated with complex genomic data.

p.Val804Met, the Most Frequent Pathogenic Mutation in RET, Confers a Very Low Lifetime Risk of Medullary Thyroid Cancer.

Context: To date, penetrance figures for medullary thyroid cancer (MTC) for variants in rearranged during transfection (RET) have been estimated from families ascertained because of the presence of MTC. Objective: To gain estimates of penetrance, unbiased by ascertainment, we analyzed 61 RET mutations assigned as disease causing by the American Thyroid Association (ATA) in population whole-exome sequencing data. Design: For the 61 RET mutations, we used analyses of the observed allele frequencies in ∼51,000 individuals from the Exome Aggregation Consortium (ExAC) database that were not contributed via The Cancer Genome Atlas (TCGA; non-TCGA ExAC), assuming lifetime penetrance for MTC of 90%, 50%, and unbounded. Setting: Population-based. Results: Ten of 61 ATA disease-causing RET mutations were present in the non-TCGA ExAC population with observed frequency consistent with penetrance for MTC of >90%. For p.Val804Met, the lifetime penetrance for MTC, estimated from the allele frequency observed, was 4% [95% confidence interval (CI), 0.9% to 8%]. Conclusions: Based on penetrance analysis in carrier relatives of p.Val804Met-positive cases of MTC, p.Val804Met is currently understood to have high-lifetime penetrance for MTC (87% by age 70), albeit of later onset of MTC than other RET mutations. Given our unbiased estimate of penetrance for RET p.Val804Met of 4% (95% CI, 0.9% to 8%), the current recommendation by the ATA of prophylactic thyroidectomy as standard for all RET mutation carriers is likely inappropriate.

Compound heterozygous CASQ2 mutations and long-term course of catecholaminergic polymorphic ventricular tachycardia.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal inherited cardiac disorder characterized by episodic ventricular tachycardia during adrenergic stimulation. It is associated with significant morbidity and mortality. Knowledge of the underlying genetic cause, pathogenesis, and the natural history of the disease remains incomplete. Approximately 50% of CPVT cases are caused by dominant mutations in the cardiac ryanodine receptor (RYR2) gene, <5% of cases are accounted for by recessive mutations in cardiac calsequestrin (CASQ2) or Triadin (TRDN). METHODS: We report a family with two CASQ2 gene mutations. A research-based next-generation sequencing (NGS) initiative was used in a patient with a severe CPVT phenotype and her clinically unaffected son. Reverse transcription polymerase chain reaction (RT-PCR) from platelet RNA was used to assess the consequences of predicted splice variants. RESULTS: NGS revealed that the proband carried a novel c.199C>T (p.Gln67*) mutation and a previously reported splice site mutation c.532+1G>A in CASQ2. Her son is a heterozygous carrier of the c.199C>T (p.Gln67*) mutation alone and the proband was compound heterozygous at CASQ2. RNA analysis demonstrated that the splice site mutation results in the retention of intron 3 with no full-length CASQ2 mRNA. CONCLUSION: This study describes a novel CPVT genotype and further characterizes the effect of a previously reported CASQ2 splice site mutation. The long-term follow-up of 23 years since first symptom provides additional insight into the natural history of CASQ2-associated CPVT.

Maternal and neonatal outcomes of successful Kielland's rotational forceps delivery.

OBJECTIVE: To estimate the rates of early neonatal and maternal complications in a consecutive series of successful Kielland's rotational forceps deliveries. METHODS: This was a retrospective cohort study of consecutive cases of successful rotational forceps deliveries performed in singleton pregnancies at 36 weeks of gestation or more in a tertiary referral center in Scotland, UK, from 2001 to 2008 (n=873). We also compared outcomes associated with successful rotational forceps deliveries in 2008 (n=150) with those of nonrotational forceps delivery (n=873), ventouse delivery (n=159), spontaneous vertex delivery (n=3,494), and emergency cesarean delivery (n=947). RESULTS: There was one stillbirth associated with a rotational forceps delivery. This was diagnosed before application of forceps. After rotational forceps deliveries, 58 of 872 (6.7%) of live-born neonates were admitted to the neonatal unit. Twenty-seven of 872 (3.1%) neonates had one or more complications that could be attributable to traumatic delivery and seven neonates (0.8%) had a diagnosis of neonatal encephalopathy. When compared with alternative methods of delivery over a single year, neonatal admission rates after delivery by rotational forceps deliveries (5 of 150 [3.3%]) were not significantly different from spontaneous vertex delivery (128 of 3,494 [3.7%; P=1.00]) or ventouse delivery (6 of 159 [3.8%; P=1.00]) and lower than emergency cesarean delivery (106 of 947 [11.2%; P=.002). Postpartum hemorrhage rates after rotational forceps deliveries (8 of 150 [5.3%; P=.008]) were lower than those associated with emergency cesarean delivery (142 of 947 [15.0%; P=.008]). CONCLUSION: Rates of short-term neonatal and maternal complications after successful rotational forceps deliveries are low. LEVEL OF EVIDENCE: II.

Incidence and predictors of acute mountain sickness among trekkers on Mount Kilimanjaro.

We investigated the incidence of AMS amongst a general population of trekkers on Mount Kilimanjaro, using the Lake Louise consensus scoring system (LLS). Additionally we examined the effect of prophylactic acetazolamide and different ascent profiles. Climbers on 3 different ascent itineraries were recruited. At 2743 m we recruited 177 participants (mean age 31, range [18-71]) who completed LLS together with an epidemiological questionnaire. At 4730 m participants (n=189, male=108, female=68, mean age 33, range [1871]) completed LLS, 136 of whom had been followed up from 2730 m. At 2743 m, 3% (5/177) of climbers were AMS positive, and 47% (89/189) of climbers from all itineraries were AMS positive at 4730 m. Of climbers attempting the Marangu itineraries, 33% (45/136) were taking acetazolamide. This group had a similar rate of AMS and no statistical difference in severity of LLS when compared with those not taking prophylactic drugs. We also did not demonstrate a difference between the incidence of AMS in climbers who did or did not take a rest day at 3700 m. However, there was a significant reduction in the incidence of AMS amongst pre-acclimatized subjects. Consistent with previous work, we found that the rate of AMS on Mount Kilimanjaro is high. Furthermore, at these fast ascent rates, there was no evidence of a protective effect of acetazolamide or a single rest day. There is a need to increase public awareness of the risks of altitude sickness and we advocate a pragmatic "golden rules" approach (http://www.altitude.org/altitude_sickness.php).