- Neonatal-Onset Chronic Diarrhea Caused by Homozygous Nonsense WNT2B Mutations. [Journal Article]
- AJAm J Hum Genet 2018 Jun 04
- Homozygous nonsense mutations in WNT2B were identified in three individuals from two unrelated families with severe, neonatal-onset osmotic diarrhea after whole-exome sequencing was performed on trio...
Homozygous nonsense mutations in WNT2B were identified in three individuals from two unrelated families with severe, neonatal-onset osmotic diarrhea after whole-exome sequencing was performed on trios from the two families. Intestinal biopsy samples from affected individuals were used for histology and immunofluorescence and to generate enteroids ex vivo. Histopathologic evaluation demonstrated chronic inflammatory changes in the stomach, duodenum, and colon. Immunofluorescence demonstrated diminished staining for OLFM4, a marker for intestinal stem cells (ISCs). The enteroids generated from WNT2B-deficient intestinal epithelium could not be expanded and did not survive passage. Addition of CHIR-99021 (a GSK3A and GSK3B inhibitor and activator of canonical WNT/β-CATENIN signaling) could not rescue WNT2B-deficient enteroids. Addition of supplemental recombinant murine WNT2B was able to perpetuate small enteroids for multiple passages but failed to expand their number. Enteroids showed a 10-fold increase in the expression of LEF1 mRNA and a 100-fold reduction in TLR4 expression, compared with controls by quantitative RT-PCR, indicating alterations in canonical WNT and microbial pattern-recognition signaling. In summary, individuals with homozygous nonsense mutations in WNT2B demonstrate severe intestinal dysregulation associated with decreased ISC number and function, likely explaining their diarrheal phenotype. WNT2B deficiency should be considered for individuals with neonatal-onset diarrhea.
- Comprehensive Cancer-Predisposition Gene Testing in an Adult Multiple Primary Tumor Series Shows a Broad Range of Deleterious Variants and Atypical Tumor Phenotypes. [Journal Article]
- AJAm J Hum Genet 2018 Jun 12
- Multiple primary tumors (MPTs) affect a substantial proportion of cancer survivors and can result from various causes, including inherited predisposition. Currently, germline genetic testing of MPT-a...
Multiple primary tumors (MPTs) affect a substantial proportion of cancer survivors and can result from various causes, including inherited predisposition. Currently, germline genetic testing of MPT-affected individuals for variants in cancer-predisposition genes (CPGs) is mostly targeted by tumor type. We ascertained pre-assessed MPT individuals (with at least two primary tumors by age 60 years or at least three by 70 years) from genetics centers and performed whole-genome sequencing (WGS) on 460 individuals from 440 families. Despite previous negative genetic assessment and molecular investigations, pathogenic variants in moderate- and high-risk CPGs were detected in 67/440 (15.2%) probands. WGS detected variants that would not be (or were not) detected by targeted resequencing strategies, including low-frequency structural variants (6/440 [1.4%] probands). In most individuals with a germline variant assessed as pathogenic or likely pathogenic (P/LP), at least one of their tumor types was characteristic of variants in the relevant CPG. However, in 29 probands (42.2% of those with a P/LP variant), the tumor phenotype appeared discordant. The frequency of individuals with truncating or splice-site CPG variants and at least one discordant tumor type was significantly higher than in a control population (χ2 = 43.642; p ≤ 0.0001). 2/67 (3%) probands with P/LP variants had evidence of multiple inherited neoplasia allele syndrome (MINAS) with deleterious variants in two CPGs. Together with variant detection rates from a previous series of similarly ascertained MPT-affected individuals, the present results suggest that first-line comprehensive CPG analysis in an MPT cohort referred to clinical genetics services would detect a deleterious variant in about a third of individuals.
- Squalene Synthase Deficiency: Clinical, Biochemical, and Molecular Characterization of a Defect in Cholesterol Biosynthesis. [Journal Article]
- AJAm J Hum Genet 2018 Jun 01
- Mendelian disorders of cholesterol biosynthesis typically result in multi-system clinical phenotypes, underlining the importance of cholesterol in embryogenesis and development. FDFT1 encodes for an ...
Mendelian disorders of cholesterol biosynthesis typically result in multi-system clinical phenotypes, underlining the importance of cholesterol in embryogenesis and development. FDFT1 encodes for an evolutionarily conserved enzyme, squalene synthase (SS, farnesyl-pyrophosphate farnesyl-transferase 1), which catalyzes the first committed step in cholesterol biosynthesis. We report three individuals with profound developmental delay, brain abnormalities, 2-3 syndactyly of the toes, and facial dysmorphisms, resembling Smith-Lemli-Opitz syndrome, the most common cholesterol biogenesis defect. The metabolite profile in plasma and urine suggested that their defect was at the level of squalene synthase. Whole-exome sequencing was used to identify recessive disease-causing variants in FDFT1. Functional characterization of one variant demonstrated a partial splicing defect and altered promoter and/or enhancer activity, reflecting essential mechanisms for regulating cholesterol biosynthesis/uptake in steady state.
- Recessive MYF5 Mutations Cause External Ophthalmoplegia, Rib, and Vertebral Anomalies. [Journal Article]
- AJAm J Hum Genet 2018 May 30
- MYF5 is member of the Myc-like basic helix-loop-helix transcription factor family and, in cooperation with other myogenic regulatory factors MYOD and MYF5, is a key regulator of early stages of myoge...
MYF5 is member of the Myc-like basic helix-loop-helix transcription factor family and, in cooperation with other myogenic regulatory factors MYOD and MYF5, is a key regulator of early stages of myogenesis. Here, we report three consanguineous families with biallelic homozygous loss-of-function mutations in MYF5 who define a clinical disorder characterized by congenital ophthalmoplegia with scoliosis and vertebral and rib anomalies. The clinical phenotype overlaps strikingly with that reported in several Myf5 knockout mouse models. Affected members of two families share a haploidentical region that contains a homozygous 10 bp frameshift mutation in exon 1 of MYF5 (c.23_32delAGTTCTCACC [p.Gln8Leufs∗86]) predicted to undergo nonsense-mediated decay. Affected members of the third family harbor a homozygous missense change in exon 1 of MYF5 (c.283C>T [p.Arg95Cys]). Using in vitro assays, we show that this missense mutation acts as a loss-of-function allele by impairing MYF5 DNA binding and nuclear localization. We performed whole-genome sequencing in one affected individual with the frameshift mutation and did not identify additional rare variants in the haploidentical region that might account for differences in severity among the families. These data support the direct role of MYF5 in rib, spine, and extraocular muscle formation in humans.
- Using Somatic Mutations from Tumors to Classify Variants in Mismatch Repair Genes. [Journal Article]
- AJAm J Hum Genet 2018 May 31
- Present guidelines for classification of constitutional variants do not incorporate inferences from mutations seen in tumors, even when these are associated with a specific molecular phenotype. When ...
Present guidelines for classification of constitutional variants do not incorporate inferences from mutations seen in tumors, even when these are associated with a specific molecular phenotype. When somatic mutations and constitutional mutations lead to the same molecular phenotype, as for the mismatch repair genes, information from somatic mutations may enable interpretation of previously unclassified variants. To test this idea, we first estimated likelihoods that somatic variants in MLH1, MSH2, MSH6, and PMS2 drive microsatellite instability and characteristic IHC staining patterns by calculating likelihoods of high versus low normalized variant read fractions of 153 mutations known to be pathogenic versus those of 760 intronic passenger mutations from 174 paired tumor-normal samples. Mutations that explained the tumor mismatch repair phenotype had likelihood ratio for high variant read fraction of 1.56 (95% CI 1.42-1.71) at sites with no loss of heterozygosity and of 26.5 (95% CI 13.2-53.0) at sites with loss of heterozygosity. Next, we applied these ratios to 165 missense, synonymous, and splice variants observed in tumors, combining in a Bayesian analysis the likelihood ratio corresponding with the adjusted variant read fraction with pretest probabilities derived from published analyses and public databases. We suggest classifications for 86 of 165 variants: 7 benign, 31 likely benign, 22 likely pathogenic, and 26 pathogenic. These results illustrate that for mismatch repair genes, characterization of tumor mutations permits tumor mutation data to inform constitutional variant classification. We suggest modifications to incorporate molecular phenotype in future variant classification guidelines.
- De Novo and Inherited Loss-of-Function Variants in TLK2: Clinical and Genotype-Phenotype Evaluation of a Distinct Neurodevelopmental Disorder. [Journal Article]
- AJAm J Hum Genet 2018 Jun 07; 102(6):1195-1203
- Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or in...
Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2.
- TRPV6 Variants Interfere with Maternal-Fetal Calcium Transport through the Placenta and Cause Transient Neonatal Hyperparathyroidism. [Journal Article]
- AJAm J Hum Genet 2018 Jun 07; 102(6):1104-1114
- Transient neonatal hyperparathyroidism (TNHP) is etiologically a heterogeneous condition. One of the etiologies is an insufficient maternal-fetal calcium transport through the placenta. We report six...
Transient neonatal hyperparathyroidism (TNHP) is etiologically a heterogeneous condition. One of the etiologies is an insufficient maternal-fetal calcium transport through the placenta. We report six subjects with homozygous and/or compound-heterozygous mutations in the gene encoding the transient receptor potential cation channel, subfamily V, member 6 (TRPV6), an epithelial Ca2+-selective channel associated with this condition. Exome sequencing on two neonates with skeletal findings consistent with neonatal hyperparathyroidism identified homozygous frameshift mutations before the first transmembrane domain in a subject born to first-cousins parents of Pakistani descent as well as compound-heterozygous mutations (a combination of a frameshift mutation and an intronic mutation that alters mRNA splicing) in an individual born to a non-consanguineous couple of African descent. Subsequently, targeted mutation analysis of TRPV6 performed on four other individuals (born to non-consanguineous Japanese parents) with similar X-rays findings identified compound-heterozygous mutations. The skeletal findings improved or resolved in most subjects during the first few months of life. We identified three missense variants (at the outer edges of the second and third transmembrane domains) that alter the localization of the TRPV6: one recurrent variant at the S2-S3 loop and two recurrent variants (in the fourth ankyrin repeat domain) that impair TRPV6 stability. Compound heterozygous loss-of-function mutations for the pathogenic frameshift allele and the allele with an intronic c.607+5G>A mutation resulted in the most severe phenotype. These results suggest that TNHP is an autosomal-recessive disease caused by TRPV6 mutations that affect maternal-fetal calcium transport.
- Quantifying the Impact of Rare and Ultra-rare Coding Variation across the Phenotypic Spectrum. [Journal Article]
- AJAm J Hum Genet 2018 Jun 07; 102(6):1204-1211
- There is a limited understanding about the impact of rare protein-truncating variants across multiple phenotypes. We explore the impact of this class of variants on 13 quantitative traits and 10 dise...
There is a limited understanding about the impact of rare protein-truncating variants across multiple phenotypes. We explore the impact of this class of variants on 13 quantitative traits and 10 diseases using whole-exome sequencing data from 100,296 individuals. Protein-truncating variants in genes intolerant to this class of mutations increased risk of autism, schizophrenia, bipolar disorder, intellectual disability, and ADHD. In individuals without these disorders, there was an association with shorter height, lower education, increased hospitalization, and reduced age at enrollment. Gene sets implicated from GWASs did not show a significant protein-truncating variants burden beyond what was captured by established Mendelian genes. In conclusion, we provide a thorough investigation of the impact of rare deleterious coding variants on complex traits, suggesting widespread pleiotropic risk.
- βIV Spectrinopathies Cause Profound Intellectual Disability, Congenital Hypotonia, and Motor Axonal Neuropathy. [Journal Article]
- AJAm J Hum Genet 2018 Jun 07; 102(6):1158-1168
- βIV spectrin links ankyrinG (AnkG) and clustered ion channels at axon initial segments (AISs) and nodes of Ranvier to the axonal cytoskeleton. Here, we report bi-allelic pathogenic SPTBN4 variants (t...
βIV spectrin links ankyrinG (AnkG) and clustered ion channels at axon initial segments (AISs) and nodes of Ranvier to the axonal cytoskeleton. Here, we report bi-allelic pathogenic SPTBN4 variants (three homozygous and two compound heterozygous) that cause a severe neurological syndrome that includes congenital hypotonia, intellectual disability, and motor axonal and auditory neuropathy. We introduced these variants into βIV spectrin, expressed these in neurons, and found that 5/7 were loss-of-function variants disrupting AIS localization or abolishing phosphoinositide binding. Nerve biopsies from an individual with a loss-of-function variant had reduced nodal Na+ channels and no nodal KCNQ2 K+ channels. Modeling the disease in mice revealed that although ankyrinR (AnkR) and βI spectrin can cluster Na+ channels and partially compensate for the loss of AnkG and βIV spectrin at nodes of Ranvier, AnkR and βI spectrin cannot cluster KCNQ2- and KCNQ3-subunit-containing K+ channels. Our findings define a class of spectrinopathies and reveal the molecular pathologies causing nervous-system dysfunction.
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- Functional Assays Are Essential for Interpretation of Missense Variants Associated with Variable Expressivity. [Journal Article]
- AJAm J Hum Genet 2018 Jun 07; 102(6):1062-1077
- Missense DNA variants have variable effects upon protein function. Consequently, interpreting their pathogenicity is challenging, especially when they are associated with disease variability. To dete...
Missense DNA variants have variable effects upon protein function. Consequently, interpreting their pathogenicity is challenging, especially when they are associated with disease variability. To determine the degree to which functional assays inform interpretation, we analyzed 48 CFTR missense variants associated with variable expressivity of cystic fibrosis (CF). We assessed function in a native isogenic context by evaluating CFTR mutants that were stably expressed in the genome of a human airway cell line devoid of endogenous CFTR expression. 21 of 29 variants associated with full expressivity of the CF phenotype generated <10% wild-type CFTR (WT-CFTR) function, a conservative threshold for the development of life-limiting CF lung disease, and five variants had moderately decreased function (10% to ∼25% WT-CFTR). The remaining three variants in this group unexpectedly had >25% WT-CFTR function; two were higher than 75% WT-CFTR. As expected, 14 of 19 variants associated with partial expressivity of CF had >25% WT-CFTR function; however, four had minimal to no effect on CFTR function (>75% WT-CFTR). Thus, 6 of 48 (13%) missense variants believed to be disease causing did not alter CFTR function. Functional studies substantially refined pathogenicity assignment with expert annotation and criteria from the American College of Medical Genetics and Genomics and Association for Molecular Pathology. However, four algorithms (CADD, REVEL, SIFT, and PolyPhen-2) could not differentiate between variants that caused severe, moderate, or minimal reduction in function. In the setting of variable expressivity, these results indicate that functional assays are essential for accurate interpretation of missense variants and that current prediction tools should be used with caution.