An approach to integrating exome sequencing for fetal structural anomalies into clinical practice

PurposeWe investigated the diagnostic and clinical performance of trio exome sequencing (ES) in parent–fetus trios where the fetus had sonographic abnormalities but normal karyotype, microarray and, in some cases, normal gene-specific sequencing.MethodsES was performed from DNA of 102 anomalous fetuses and from peripheral blood from their parents. Parents provided consent for the return of diagnostic results in the fetus, medically actionable findings in the parents, and identification as carrier couple for significant autosomal recessive conditions.ResultsIn 21/102 (20.6%) fetuses, ES provided a positive-definitive or positive-probable diagnosis. In 10/102 (9.8%), ES provided an inconclusive-possible result. At least 2/102 (2.0%) had a repeat pregnancy during the study period and used the information from the study for prenatal diagnosis in the next pregnancy. Six of 204 (2.9%) parents received medically actionable results that affected their own health and 3/102 (2.9%) of couples received results that they were carriers for the same autosomal recessive condition.ConclusionES has diagnostic utility in a select population of fetuses where a genetic diagnosis was highly suspected. Challenges related to genetics literacy, variant interpretation, and various types of diagnostic results affecting both fetal and parental health must be addressed by highly tailored pre- and post-test genetic counseling.     

Genomic sequencing identifies secondary findings in a cohort of parent study participants

PurposeClinically relevant secondary variants were identified in parents enrolled with a child with developmental delay and intellectual disability.MethodsExome/genome sequencing and analysis of 789 “unaffected” parents was performed.ResultsPathogenic/likely pathogenic variants were identified in 21 genes within 25 individuals (3.2%), with 11 (1.4%) participants harboring variation in a gene defined as clinically actionable by the American College of Medical Genetics and Genomics. These 25 individuals self-reported either relevant clinical diagnoses (5); relevant family history or symptoms (13); or no relevant family history, symptoms, or clinical diagnoses (7). A limited carrier screen was performed yielding 15 variants in 48 (6.1%) parents. Parents were also analyzed as mate pairs (n = 365) to identify cases in which both parents were carriers for the same recessive disease, yielding three such cases (0.8%), two of which had children with the relevant recessive disease. Four participants had two findings (one carrier and one noncarrier variant). In total, 71 of the 789 enrolled parents (9.0%) received secondary findings.ConclusionWe provide an overview of the rates and types of clinically relevant secondary findings, which may be useful in the design and implementation of research and clinical sequencing efforts to identify such findings.     

Prenatal exome sequencing in 65 fetuses with abnormality of the corpus callosum: contribution to further diagnostic delineation

PurposeAbnormality of the corpus callosum (AbnCC) is etiologically a heterogeneous condition and the prognosis in prenatally diagnosed cases is difficult to predict. The purpose of our research was to establish the diagnostic yield using chromosomal microarray (CMA) and exome sequencing (ES) in cases with prenatally diagnosed isolated (iAbnCC) and nonisolated AbnCC (niAbnCC).MethodsCMA and prenatal trio ES (pES) were done on 65 fetuses with iAbnCC and niAbnCC. Only pathogenic gene variants known to be associated with AbnCC and/or intellectual disability were considered.ResultspES results were available within a median of 21.5 days (9–53 days). A pathogenic single-nucleotide variant (SNV) was identified in 12 cases (18%) and a pathogenic CNV was identified in 3 cases (4.5%). Thus, the genetic etiology was determined in 23% of cases. In all diagnosed cases, the results provided sufficient information regarding the neurodevelopmental prognosis and helped the parents to make an informed decision regarding the outcome of the pregnancy.ConclusionOur results show the significant diagnostic and prognostic contribution of CMA and pES in cases with prenatally diagnosed AbnCC. Further prospective cohort studies with long-term follow-up of the born children will be needed to provide accurate prenatal counseling after a negative pES result.     

Prenatal diagnosis of cerebro‐oculo‐facio‐skeletal syndrome: Report of three fetuses and review of the literature

Cerebro‐oculo‐facio‐skeletal syndrome (COFS) is a rare autosomal recessive neurodegenerative disease belonging to the family of DNA repair disorders, characterized by microcephaly, congenital cataracts, facial dysmorphism and arthrogryposis. Here, we describe the detailed morphological and microscopic phenotype of three fetuses from two families harboring ERCC5/XPG likely pathogenic variants, and review the five previously reported fetal cases. In addition to the classical features of COFS, the fetuses display thymus hyperplasia, splenomegaly and increased hematopoiesis. Microencephaly is present in the three fetuses with delayed development of the gyri, but normal microscopic anatomy at the supratentorial level. Microscopic anomalies reminiscent of pontocerebellar hypoplasia are present at the infratentorial level. In conclusion, COFS syndrome should be considered in fetuses when intrauterine growth retardation is associated with microcephaly, arthrogryposis and ocular anomalies. Further studies are needed to better understand XPG functions during human development.     

Compound heterozygous Pkd1l1 variants in a family with two fetuses affected by heterotaxy and complex Chd

Heterotaxy and congenital heart defects associated with pathogenic variants in the PKD1L1 gene (autosomal visceral heterotaxy type 8, MIM 617205) has been reported in only four individuals from three unrelated families. We describe a further family with two affected fetuses and novel compound heterozygous pathogenic variants in PKD1L1.PKD1L1 has been shown to function in the ciliary sensation of nodal flow at the embryo primitive node and in the restriction of NODAL signalling to the left lateral.plate mesoderm, mechanisms involved in the development of laterality in vertebrates.Individuals affected with this autosomal recessive condition have variable thoracic and abdominal situs. Features of CHD and other anomalies vary between and within families.     

ABCA4-associated disease as a model for missing heritability in autosomal recessive disorders: novel noncoding splice,cis-regulatory,structural, and recurrent hypomorphic variants

PurposeABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability.MethodsBy locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4.ResultsWe identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells.ConclusionNoncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders.     

Diagnostic exome-based preconception carrier testing in consanguineous couples: results from the first 100 couples in clinical practice

PurposeConsanguineous couples are at increased risk of being heterozygous for the same autosomal recessive (AR) disorder(s), with a 25% risk of affected offspring as a consequence. Until recently, comprehensive preconception carrier testing (PCT) for AR disorders was unavailable in routine diagnostics. Here we developed and implemented such a test in routine clinical care.MethodsWe performed exome sequencing (ES) for 100 consanguineous couples. For each couple, rare variants that could give rise to biallelic variants in offspring were selected. These variants were subsequently filtered against a gene panel consisting of ~2,000 genes associated with known AR disorders (OMIM-based). Remaining variants were classified according to American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines, after which only likely pathogenic and pathogenic (class IV/V) variants, present in both partners, were reported.ResultsIn 28 of 100 tested consanguineous couples (28%), likely pathogenic and pathogenic variants not previously known in the couple or their family were reported conferring 25% risk of affected offspring.ConclusionES-based PCT provides a powerful diagnostic tool to identify AR disease carrier status in consanguineous couples. Outcomes provided significant reproductive choices for a higher proportion of these couples than previous tests.     

Mucolipidosis type II and type III: a systematic review of 843 published cases

PurposeMucolipidosis (ML) II, MLIII alpha/beta, and MLIII gamma are rare autosomal recessive lysosomal storage disorders. Data on the natural course of the diseases are scarce. These data are important for counseling, therapies development, and improvement of outcome. The aim of this study is to gain knowledge on the natural history of ML by obtaining data on survival, symptom onset, presenting symptoms, diagnosis, and pathogenic variants associated with the MLII or MLIII phenotype.MethodsA systematic review on all published MLII and MLIII cases between 1968 and August 2019 was performed.ResultsThree hundred one articles provided data on 843 patients. Median age at diagnosis: 0.7 for MLII and 9.0 years for MLIII. Median survival: 5.0 for MLII and 62.0 years for MLIIIII. Median age of death: 1.8 for MLII and 33.0 years for MLIII. Most frequent causes of death in all ML were pulmonary and/or cardiac complications. Pathogenic variants were described in 388 patients (GNPTAB: 571, GNPTG 179).ConclusionThis review provides unique insights into the natural history of MLII and MLIII, with a clear genotype–phenotype correlation with the most frequent pathogenic variant c.3503_3504del in MLII and in MLIII alpha/beta c.22A>G for GNPTAB. All pathogenic GNPTG variants resulted in MLIII gamma.     

KAT6A Syndrome: genotype–phenotype correlation in 76 patients with pathogenic KAT6A variants

PurposePathogenic variants in KAT6Ahave recently been identified as a cause of syndromic developmental delay. Within 2 years, the number of patients identified with pathogenic KAT6A variants has rapidly expanded and the full extent and variability of the clinical phenotype has not been reported.MethodsWe obtained data for patients with KAT6A pathogenic variants through three sources: treating clinicians, an online family survey distributed through social media, and a literature review.ResultsWe identified 52 unreported cases, bringing the total number of published cases to 76. Our results expand the genotypic spectrum of pathogenic variants to include missense and splicing mutations. We functionally validated a pathogenic splice-site variant and identified a likely hotspot location forde novo missense variants. The majority of clinical features in KAT6A syndrome have highly variable penetrance. For core features such as intellectual disability, speech delay, microcephaly, cardiac anomalies, and gastrointestinal complications, genotype– phenotype correlations show that late-truncating pathogenic variants (exons 16–17) are significantly more prevalent. We highlight novel associations, including an increased risk of gastrointestinal obstruction.ConclusionOur data expand the genotypic and phenotypic spectrum for individuals with genetic pathogenic variants in KAT6A and we outline appropriate clinical management.     

A novel non-sense mutation in TDP2 causes spinocerebellar ataxia autosomal recessive 23 accompanied by bilateral upward gaze; report of a case and review of the literature

Pathogenic mutations in TDP2, encoding tyrosyl DNA phosphodiesterase 2, cause Spinocerebellar Ataxia autosomal recessive 23 (SCAR23). It is a rare autosomal recessive disorder and mainly has been reported in the European population. Thus far, merely eight patients harboring four TDP2 variants have been reported in the literature. In this study, a novel pathogenic variant (NM_016614: c.4G > T, p.Glu2*) was identified by Whole-Exome and confirmed by Sanger sequencing. The proband has both intellectual and developmental delay, dysphasia, elbow contracture, and upward gaze. The elbow contracture has not been previously described in previous SCAR23 cases. Lastly, we briefly review the phenotypic features of the patients with SCAR23 in the literature.     

Consensus interpretation of the p.Met34Thr and p.Val37Ile variants in GJB2 by the ClinGen Hearing Loss Expert Panel

PurposePathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants.MethodsThe ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case–control statistical analyses were performed.ResultsThe panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared with population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants.ConclusionResolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance.     

Implementation of fetal clinical exome sequencing: Comparing prospective and retrospective cohorts

PurposeWe compared the diagnostic yield of fetal clinical exome sequencing (fCES) in prospective and retrospective cohorts of pregnancies presenting with anomalies detected using ultrasound. We evaluated factors that led to a higher diagnostic efficiency, such as phenotypic category, clinical characterization, and variant analysis strategy.MethodsfCES was performed for 303 fetuses (183 ongoing and 120 ended pregnancies, in which chromosomal abnormalities had been excluded) using a trio/duo-based approach and a multistep variant analysis strategy.ResultsfCES identified the underlying genetic cause in 13% (24/183) of prospective and 29% (35/120) of retrospective cases. In both cohorts, recessive heterozygous compound genotypes were not rare, and trio and simplex variant analysis strategies were complementary to achieve the highest possible diagnostic rate. Limited prenatal phenotypic information led to interpretation challenges. In 2 prospective cases, in-depth analysis allowed expansion of the spectrum of prenatal presentations for genetic syndromes associated with the SLC17A5 and CHAMP1 genes.ConclusionfCES is diagnostically efficient in fetuses presenting with cerebral, skeletal, urinary, or multiple anomalies. The comparison between the 2 cohorts highlights the importance of providing detailed phenotypic information for better interpretation and prenatal reporting of genetic variants.     

Genomic study of severe fetal anomalies and discovery of GREB1L mutations in renal agenesis

PurposeFetal anomalies represent a poorly studied group of developmental disorders. Our objective was to assess the impact of whole-exome sequencing (WES) on the investigation of these anomalies.MethodsWe performed WES in 101 fetuses or stillborns who presented prenatally with severe anomalies, including renal a/dysgenesis, VACTERL association (vertebral defects, anal atresia, cardiac defects, tracheoesophageal fistula, renal anomalies, and limb abnormalities), brain anomalies, suspected ciliopathies, multiple major malformations, and akinesia.ResultsA molecular diagnosis was obtained in 19 cases (19%). In 13 of these cases, the diagnosis was not initially suspected by the clinicians because the phenotype was nonspecific or atypical, corresponding in some cases to the severe end of the spectrum of a known disease (e.g., MNX1-, RYR1-, or TUBB-related disorders). In addition, we identified likely pathogenic variants in genes (DSTYK, ACTB, and HIVEP2) previously associated with phenotypes that were substantially different from those found in our cases. Finally, we identified variants in novel candidate genes that were associated with perinatal lethality, including de novo mutations in GREB1L in two cases with bilateral renal agenesis, which represents a significant enrichment of such mutations in our cohort.ConclusionOur study opens a window on the distinctive genetic landscape associated with fetal anomalies and highlights the power—but also the challenges—of WES in prenatal diagnosis.     

Eighth case of Li-Campeau syndrome in a Turkish patient caused by a novel pathogenic variant in UBR7 and expanding the phenotype

Li-Campeau syndrome (LICAS) is an autosomal recessive disorder characterized by developmental delay, intellectual disability, genital anomalies, congenital heart defects, and dysmorphic features. LICAS is caused by biallelic pathogenic variants in the UBR7 gene, acting as an E3 ubiquitin-protein ligase. Using exome sequencing (ES), we identified a homozygous novel pathogenic splice site variation c.1185+1G>C in UBR7 in a 32-month-old male from a nonconsanguineous Turkish family with clinical features of LICAS. Sanger sequencing revealed the heterozygous state of parents for this variant and confirmed the co-segregation study. The variant may lead to the loss of function of UBR7 and is in a highly conserved residue. Bioinformatic prediction analysis using in silico algorithms supports the pathogenic effect of the splice site variant in the UBR7.     

RNA sequencing solved the most common but unrecognized NEB pathogenic variant in Japanese nemaline myopathy

PurposeThe diagnostic rate for Mendelian diseases by exome sequencing (ES) is typically 20–40%. The low rate is partly because ES misses deep-intronic or synonymous variants leading to aberrant splicing. In this study, we aimed to apply RNA sequencing (RNA-seq) to efficiently detect the aberrant splicings and their related variants.MethodsAberrant splicing in biopsied muscles from six nemaline myopathy (NM) cases unresolved by ES were analyzed with RNA-seq. Variants related to detected aberrant splicing events were analyzed with Sanger sequencing. Detected variants were screened in NM patients unresolved by ES.ResultsWe identified a novel deep-intronic NEB pathogenic variant, c.1569+339A>G in one case, and another novel synonymous NEB pathogenic variant, c.24684G>C (p.Ser8228Ser) in three cases. The c.24684G>C variant was observed to be the most frequent among all NEB pathogenic variants in normal Japanese populations with a frequency of 1 in 178 (20 alleles in 3552 individuals), but was previously unrecognized. Expanded screening of the variant identified it in a further four previously unsolved nemaline myopathy cases.ConclusionThese results indicated that RNA-seq may be able to solve a large proportion of previously undiagnosed muscle diseases.     

Reanalysis of eMERGE phase III sequence variants in 10,500 participants and infrastructure to support the automated return of knowledge updates

PurposeThe clinical genomics knowledgebase is dynamic with variant classifications changing as newly identified cases, additional population data, and other evidence become available. This is a challenge for the clinical laboratory because of limited resource availability for variant reassessment.MethodsThroughout the Electronic Medical Records and Genomics phase III program, clinical sites associated with the Mass General Brigham/Broad sequencing center received automated, real-time notifications when reported variants were reclassified. In this study, we summarized the nature of these reclassifications and described the proactive reassessment framework we used for the Electronic Medical Records and Genomics program data set to identify variants most likely to undergo reclassification.ResultsReanalysis of 1855 variants led to the reclassification of 2% (n = 45) of variants, affecting 0.6% (n = 67) of participants. Of these reclassifications, 78% (n = 35) were high-impact changes affecting reportability, with 8 variants downgraded from likely pathogenic/pathogenic to variants of uncertain significance (VUS) and 27 variants upgraded from VUS to likely pathogenic/pathogenic. Most upgraded variants (67%) were initially classified as VUS–Favor Pathogenic, highlighting the benefit of VUS subcategorization. The most common reason for reclassification was new published case data and/or functional evidence.ConclusionOur results highlight the importance of periodic sequence variant reevaluation and the need for automated approaches to advance routine implementation of variant reevaluations in clinical practice.     

Delineation of a KDM2B-related neurodevelopmental disorder and its associated DNA methylation signature

PurposePathogenic variants in genes involved in the epigenetic machinery are an emerging cause of neurodevelopment disorders (NDDs). Lysine-demethylase 2B (KDM2B) encodes an epigenetic regulator and mouse models suggest an important role during development. We set out to determine whether KDM2B variants are associated with NDD.MethodsThrough international collaborations, we collected data on individuals with heterozygous KDM2B variants. We applied methylation arrays on peripheral blood DNA samples to determine a KDM2B associated epigenetic signature.ResultsWe recruited a total of 27 individuals with heterozygous variants in KDM2B. We present evidence, including a shared epigenetic signature, to support a pathogenic classification of 15 KDM2B variants and identify the CxxC domain as a mutational hotspot. Both loss-of-function and CxxC-domain missense variants present with a specific subepisignature. Moreover, the KDM2B episignature was identified in the context of a dual molecular diagnosis in multiple individuals. Our efforts resulted in a cohort of 21 individuals with heterozygous (likely) pathogenic variants. Individuals in this cohort present with developmental delay and/or intellectual disability; autism; attention deficit disorder/attention deficit hyperactivity disorder; congenital organ anomalies mainly of the heart, eyes, and urogenital system; and subtle facial dysmorphism.ConclusionPathogenic heterozygous variants in KDM2B are associated with NDD and a specific epigenetic signature detectable in peripheral blood.     

Novel pathogenic variants in an Indian cohort with epidermolysis bullosa: Expanding the genotypic spectrum

BackgroundEpidermolysis bullosa (EB) is a genodermatosis characterized by skin fragility and blisters with variable severity. Patients with Dystrophic EB (DEB) or Junctional EB (JEB) mainly present to clinic due to greater functional impairment. Pathogenic sequence variations in COL7A1 are implicated in DEB.ObjectiveWe have tried to decipher the molecular spectrum and genotype phenotype correlation of 21 Indian patients with EB.MethodsNext generation sequencing (NGS) was performed to determine the pathogenic variants. Sanger sequencing was also done for validation of the variants in eleven individuals.ResultsPathogenic variants were detected in 20 individuals (diagnostic yield of 95%). Majority of them (90%) had sequence variation in COL7A1 while two had pathogenic variants in ITGB4 and KRT14 respectively. Out of the 18 patients confirmed to have DEB, 3 had Dominant DEB (DDEB) whereas 15 patients had Recessive DEB (RDEB). Amongst 23 sequence variations identified, 12 were found to be novel (3 were missense, 5 were premature termination codon variants while 4 were splice-site changes).ConclusionGenotype phenotype correlation was noted with milder manifestations in those with dominant inheritance types. Exact molecular diagnosis can be ascertained by NGS in majority of cases.