Background Heterozygous variants in Transient receptor potential melastatin type 7 (TRPM7), encoding an essential and ubiquitously expressed cation channel, may cause hypomagnesemia, but current evidence is insufficient to draw definite conclusions and it is unclear whether any other phenotypes can occur.Methods Individuals with unexplained hypomagnesemia underwent whole-exome sequencing which identified TRPM7 variants. Pathogenicity of the identified variants was assessed by combining phenotypic, functional and in silico analyses.Results We report three new heterozygous missense variants in TRPM7 (p.Met1000Thr, p.Gly1046Arg, p.Leu1081Arg) in individuals with hypomagnesemia. Strikingly, autism spectrum disorder and developmental delay, mainly affecting speech and motor skills, was observed in all three individuals, while two out of three also presented with seizures. The three variants are predicted to be severely damaging by in silico prediction tools and structural modeling. Furthermore, these variants result in a clear loss-of-function of TRPM7-mediated magnesium uptake in vitro, while not affecting TRPM7 expression or insertion into the plasma membrane.Conclusions This study provides additional evidence for the association between heterozygous TRPM7 variants and hypomagnesemia and adds developmental delay to the phenotypic spectrum of TRPM7-related disorders. Considering that the TRPM7 gene is relatively tolerant to loss-of-function variants, future research should aim to unravel by what mechanisms specific heterozygous TRPM7 variants can cause disease. Graphical Abstract

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains without disease trait associations. Using exome sequencing and family-based rare variant analysis, we identified 20 individuals with de novo , ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative HPO analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from a patient with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.(Gly411Glu) and p.(Arg761Gln), altered DHX9 ATPase activity. The severe NDD-associated variant p.(Arg141Gln) did not impact DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9 -/-mice exhibit hypoactivity in novel environments, tremor, and sensorineural hearing loss. Taken together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

TRPM3 encodes a transient receptor potential cation channel of the melastatin family, expressed in the central nervous system and in peripheral sensory neurons of the dorsal root ganglia. The recurrent substitution in TRPM3: c.2509G>A, p.(Val837Met) has been associated with syndromic intellectual disability and seizures. In this report, we present the clinical and molecular features of seven previously unreported individuals, identified by exome sequencing, with the recurrent p.(Val837Met) variant and global developmental delay. Other shared clinical features included congenital hypotonia, dysmorphic facial features (broad forehead, deep-set eyes, and down turned mouth), exotropia, and musculoskeletal issues (hip dysplasia, hip dislocation, scoliosis). Seizures were observed in two of seven individuals (febrile seizure in one and generalized tonic-clonic seizures with atonic drops in another), and epileptiform activity was observed in an additional two individuals. This report extends the number of affected individuals to 16 who are heterozygous for the de novo recurrent substitution p.(Val837Met). In contrast with the initial report, epilepsy was not a mandatory feature observed in this series. TRPM3 pathogenic variation should be considered in individuals with global developmental delays, moderate-severe intellectual disability with, or without, childhood-onset epilepsy.

Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is caused by de novo loss-of-function variants in the SON gene (MIM #617140). This multisystemic disorder is characterized by intellectual disability, seizures, abnormal brain imaging, variable dysmorphic features, and various congenital anomalies. The wide application and increasing accessibility of whole exome sequencing (WES) has helped to identify new cases of ZTTK syndrome over the last few years. To date, there have been approximately 45 cases reported in the literature. Here, we describe 15 additional individuals with variants in the SON gene, including those with missense variants bringing the total number of known cases to 60. We have reviewed the clinical and molecular data of these new cases and all previously reported cases to further delineate the most common as well as emerging clinical findings related to this syndrome. Furthermore, we aim to delineate any genotype-phenotype correlations specifically for a recurring pathogenic four base pair deletion (c.5753_5756del) along with discussing the impact of missense variants seen in the SON gene.

OBJECTIVE:Growing use of clinical exome sequencing (CES) has led to an increased burden of genomic education. Self-guided educational tools can minimize the educational burden for genetic counselors (GCs). The effectiveness of these tools must be evaluated. METHODS:Parents of patients offered CES were randomized to watch educational videos before their visit or to receive routine care. Parents and GCs were surveyed about their experiences following the sessions. The responses of the video (n = 102) and no-video (n = 105) groups were compared. RESULTS:GCs reported no significant differences between parents in the video and no-video groups on genetics knowledge or CES knowledge. In contrast, parents' scores on genetics knowledge questions were lower in the video than no-video group (p = 0.007). Most parents reported the videos were informative, and the groups did not differ in satisfaction with GCs or decisions to have CES. CONCLUSION:GCs and parents perceived the videos to be beneficial. However, lower scores on genetics knowledge questions highlight the need for careful development of educational tools. PRACTICE IMPLICATIONS:Educational tools should be developed and assessed for effectiveness with the input of all stakeholders before widespread implementation. Better measures of the effectiveness of these educational tools are needed.

Two siblings, one male and one female, ages 6 and 13 yr old, have similar clinical features of global developmental delay, multiple congenital anomalies affecting the cardiac, genitourinary, and skeletal systems, and abnormal eye movements. Whole-genome sequencing revealed a homozygous splice variant (NM_014462.3:c.231+4A>C) in LSM1 that segregated with the phenotype in the family. LSM1 has a role in pre-mRNA splicing and degradation. Expression studies revealed absence of expression of the canonical isoform in the affected individuals. The Lsm1 knockout mice have a partially overlapping phenotype that affects the brain, heart, and eye. To our knowledge, LSM1 has not been associated with any human disorder; however, the tissue expression pattern, gene constraint, and the similarity of the phenotype in our patients and the knockout mice models suggest it has a role in the development of multiple organ systems in humans.

Clinical exome sequencing (CES) is increasingly being used as an effective diagnostic tool in the field of pediatric genetics. We sought to evaluate the parental experience, understanding and psychological impact of CES by conducting a survey study of English‐speaking parents of children who had diagnostic CES. Parents of 192 unique patients participated. The parent's interpretation of the child's result agreed with the clinician's interpretation in 79% of cases, with more frequent discordance when the clinician's interpretation was uncertain. The majority (79%) reported no regret with the decision to have CES. Most (65%) reported complete satisfaction with the genetic counseling experience, and satisfaction was positively associated with years of genetic counselor (GC) experience. The psychological impact of CES was greatest for parents of children with positive results and for parents with anxiety or depression. The results of this study are important for helping clinicians to prepare families for the possible results and variable psychological impact of CES. The frequency of parental misinterpretation of test results indicates the need for additional clarity in the communication of results. Finally, while the majority of patients were satisfied with their genetic counseling, satisfaction was lower for new GCs, suggesting a need for targeted GC training for genomic testing.

Whole-exome sequencing of 13 individuals with developmental delay commonly accompanied by abnormal muscle tone and seizures identified de novo missense mutations enriched within a sub-region of GNB1, a gene encoding the guanine nucleotide-binding protein subunit beta-1, G beta. These 13 individuals were identified among a base of 5,855 individuals recruited for various undiagnosed genetic disorders. The probability of observing 13 or more de novo mutations by chance among 5,855 individuals is very low (p = 7.1 x 10(-21)), implicating GNB1 as a genome-wide-significant disease-associated gene. The majority of these 13 mutations affect known G beta binding sites, which suggests that a likely disease mechanism is through the disruption of the protein interface required for G alpha-G beta gamma interaction (resulting in a constitutively active G beta gamma) or through the disruption of residues relevant for interaction between G beta gamma and certain downstream effectors (resulting in reduced interaction with the effectors). Strikingly, 8 of the 13 individuals recruited here for a neurodevelopmental disorder have a germline de novo GNB1 mutation that overlaps a set of five recurrent somatic tumor mutations for which recent functional studies demonstrated a gain-of-function effect due to constitutive activation of G protein downstream signaling cascades for some of the affected residues.

Common FTO variants are associated with obesity. However, it has recently been shown that homozygous FTO c.947G>A variant, which predicts p.R316Q, and c.956C>T, which predicts p.S319F, are associated with a malformation syndrome inherited in an autosomal recessive pattern. We present a similar homozygous FTO c.965G>A variant that predicts p.R322Q, associated with a lethal malformation syndrome in a consanguineous Yemeni family. Functional studies showed that the p.R316Q, p.S219F, and p.R322Q variants render the FTO protein inactive. We further expand on the phenotype of homozygous FTO loss‐of‐function mutations to include eye abnormalities, gingival overgrowth, craniosynostosis, and cutaneous photosensitivity. © 2015 Wiley Periodicals, Inc.

PURPOSE:Reports of the use of whole-exome sequencing in clinical practice are limited. We report our experience with whole-exome sequencing in 115 patients in a single center and evaluate its feasibility and clinical usefulness in clinical care.METHODS:Whole-exome sequencing was utilized based on the judgment of three clinical geneticists. We describe age, gender, ethnicity, consanguinity, indication for testing, family history, insurance, laboratory results, clinician interpretation of results, and impact on patient care.RESULTS:Most patients were children (78.9%). The most common indications for testing were birth defects (24.3%) and developmental delay (25.2%). We identified four new candidate human disease genes and possibly expanded the disease phenotypes associated with five different genes. Establishing a diagnosis led to discontinuation of additional planned testing in all patients, screening for additional manifestations in eight, altered management in fourteen, novel therapy in two, identification of other familial mutation carriers in five, and reproductive planning in six.CONCLUSION:Our results show that whole-exome sequencing is feasible, has clinical usefulness, and allows timely medical interventions, informed reproductive choices, and avoidance of additional testing. Our results also suggest phenotype expansion and identification of new candidate disease genes that would have been impossible to diagnose by other targeted testing methods.

Leri-Weill syndrome (LWS) is a genetic disorder caused by deletions or mutations in the SHOX gene or by deletions downstream of the gene and is classically characterized by short stature, mesomelic shortening of forearms and legs, and Madelung deformity. Correct identification of short stature homeobox-containing gene (SHOX) deficiency in children with growth problems is vital for appropriate initiation of growth hormone therapy.

Methods. Levels of the uPA/uPAR system were assessed by ELISA in cord blood and immunohistological evaluation of autopsies.Results. uPA, uPAR and plasminogen levels were each significantly higher in cord blood from cardiac-NL (n = 35) compared with non-cardiac-NL (n = 26) anti-Ro-exposed neonates: 3.3 +/- 0.1 vs 1.9 +/- 0.05 ng/ml (P < 0.0001), 6.6 +/- 0.3 vs 2.1 +/- 0.2 ng/ml (P < 0.0001) and 435 +/- 34 vs 220 +/- 19 ng/ml (P < 0.0001), respectively. In three twin pairs discordant for cardiac-NL, the twin with cardiac-NL had higher levels of uPA, uPAR and plasminogen than the unaffected twin (3.1 +/- 0.1 vs 1.9 +/- 0.05 ng/ml; P = 0.0086, 6.2 +/- 1.4 vs 2.2 +/- 0.7 ng/ml; P = 0.147 and 412 +/- 61 vs 260 +/- 27 ng/ml; P = 0.152, respectively). Immunohistological evaluation of three hearts from fetuses dying with cardiac-NL revealed macrophages and giant cells expressing uPA and plasminogen in the septal region.Conclusion. Increased soluble uPA, uPAR and plasminogen in cord blood and expression in affected tissue of fetuses with cardiac-NL supports the hypothesis that fetal cardiac injury is in part mediated by plasmin generation initiated by anti-Ro binding to the apoptotic cardiocyte.

Objectives/Hypothesis: Orofacial clefts are the most common craniofacial birth defects in humans, with the majority of orofacial clefts occurring as nonsyndromic cleft lip with or without cleft palate (NSCLP). We previously demonstrated associations between single-nucleotide polymorphisms (SNPs) in the IRF6 gene and NSCLP in the Honduran population. Here we investigated other candidate genes and chromosomal regions associated with NSCLP identified from genome-wide association studies (GWAS), including MAFB, ABCA4, 8q24, 9q22, 10q25, and 17q22 in two independent Hispanic populations. Study Design: Case-control and family-based association testing. Methods: Honduran families with two or more members with NSCLP (multiplex) were identified. DNA was collected from affected and unaffected family members (488) and 99 gender-matched controls. NSCLP Colombian families were identified; DNA was collected from 26 proband-parent trios. All participants were genotyped for 17 SNPs in six chromosomal regions. Case-control association and family-based association testing (FBAT) analyses were conducted. Results: Seven SNPs demonstrated association in at least one model in the Honduran population. In the Colombian families, five SNPs demonstrated significance in FBAT when patients with isolated cleft palate (CP) were included; four overlapped with SNPs demonstrating significance in the Honduran population, two with the same allele. One SNP retained significance with CP excluded. Conclusions: This study supports the previous GWAS findings and is the first to suggest a role for FOXE1, ABCA4, and MAFB in orofacial clefting in two separate Hispanic populations.

Current methods for detection of mutations by polymerase chain reaction (PCR) and sequence analysis frequently are not able to detect heterozygous large deletions. We report the successful use of a novel approach to identify such deletions, based on detection of apparent homozygosity of contiguous single-nucleotide polymorphisms (SNPs). The sequence analysis of genomic DNA PCR products containing all coding exons and flanking introns identified only a single heterozygous mutation (IVS18+2t-->a) in a patient with classic infantile-onset autosomal recessive glycogen storage disease type II (GSDII). Apparent homozygosity for multiple contiguous SNPs detected by this sequencing suggested presence of a large deletion as the second mutation; primers flanking the region of homozygous SNPs permitted identification and characterization by PCR of a large genomic deletion (8.26 kb) extending from IVS7 to IVS15. The data clearly demonstrate the utility of SNPs as markers for large deletions in autosomal recessive diseases when only a single mutation is found, thus complementing currently standard DNA PCR sequence methods for identifying the molecular basis of disease.

To the Editor: The dystrophic form of epidermolysis bullosa (DEB) can be inherited in both an autosomal dominant or a recessive fashion (Christiano et al., 1996Christiano A.M. Bart B.J. Epstein Jr, E.H. Uitto J. Genetic basis of Bart's syndrome: a glycine substitution mutation in the type VII collagen gene.J Invest Dermatol. 1996; 106: 1340-1342Crossref PubMed Scopus (27) Google Scholar). The recessive form can range from a very severe condition (Hallopeau-Siemens DEB) to a relatively mild disease, clinically indistinguishable from the dominantly inherited mild form of DEB, (DDEB) (Fine et al., 2000Fine J.D. Eady R.A. Bauer E.A. et al.Revised classification system for inherited epidermolysis bullosa: Report of the second international consensus meeting on diagnosis and classification of epidermolysis bullosa.J Am Acad Dermatol. 2000; 42: 1051-1066Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). A new case of mild DEB, therefore, always presents the problem of differentiating recessively inherited vs. dominantly inherited DEB due either to de novo mutations or to germline mosaicism. The latter was previously suggested in the junctional form of epidermolysis bullosa, which originates from mutations in the genes encoding laminin 5 (Kivirikko et al., 1996Kivirikko S. McGrath J.A. Pulkkinen L. Uitto J. Christiano A.M. Mutational hotspots in the LAMB3 gene in the lethal (Herlitz) type of junctional epidermolysis bullosa.Hum Mol Genet. 1996; 5: 231-237https://doi.org/10.1093/hmg/5.2.231Crossref PubMed Scopus (88) Google Scholar), but has not been demonstrated in the case of DEB. Here, we report a family with one child affected by mild DEB. At birth, blistering and denuded areas were present together with the syndactyly of the second and third toes. By the age of 11 mo, blistering was significantly reduced but the blisters healed with hyperpigmented and hypopigmented scars and milia. Both parents and an older sibling were clinically unaffected, and there was no family history of consanguinity or of a blistering disorder or skin fragility. Screening for mutations in the COL7A1 gene in the patient using previously described techniques (Christiano et al., 1997Christiano A.M. Hoffman G.G. Zhang X. Xu Y. Tamai Y. Greenspan D.S. Uitto J. Strategy for identification of sequence variants in COL7A1 and a novel 2-bp deletion mutation in recessive dystrophic epidermolysis bullosa.Hum Mutat. 1997; 10: 408-414Crossref PubMed Scopus (102) Google Scholar) revealed a single glycine substitution in the triple helical domain of the type VII collagen gene (G2003R), which has previously been associated with Bart's syndrome, a form of DDEB (Christiano et al., 1996Christiano A.M. Bart B.J. Epstein Jr, E.H. Uitto J. Genetic basis of Bart's syndrome: a glycine substitution mutation in the type VII collagen gene.J Invest Dermatol. 1996; 106: 1340-1342Crossref PubMed Scopus (27) Google Scholar). This mutation, however, was not found in the peripheral blood DNA of the clinically unaffected parents and the sibling Figure 1. As nonpaternity based on parental interviews was unlikely, we concluded that the mutation was most likely a de novo event in the patient, and would be associated with little risk of recurrence. Nevertheless, the parents were appropriately counselled that the possibility of having another affected child existed, if the mutation existed as a germline mosaic transmitted from either parent – a possibility that was considered remote. Shortly after completing the mutation analysis, the mother became pregnant and the parents requested prenatal diagnosis. DNA isolated from the amniotic fluid sample was used as template for polymerase chain reaction amplification and automated sequencing. Unexpectedly, the mutation G2003R was also present in the fetus, suggesting that the initial occurrence was in fact not a de novo event. We could only explain the second occurrence of the identical mutation in this family by invoking the possibility of germline mosaicism. To further investigate the parental transmission of the mutation, we performed haplotype analysis using chromosome 3 microsatellite markers Figure 1, synthesized based on publicly available oligonucleotide sequences (Cooperative Human Linkage Center and Genome Database). Polymerase chain reaction products containing microsatellite markers for chromosome 3 were analyzed on a 6% nondenaturing polyacrylamide gel. We determined that all three offspring carried the same maternal chromosome 3 haplotype. The clinically unaffected sister of the patient and the affected fetus shared the same paternal chromosome; however, the patient carried the other paternal chromosome. Thus, we concluded that the mutation is most likely present in a percentage of cells in the germline of the mother. To our knowledge, this study represents the first documented case of germline mosaicism in epidermolysis bullosa and serves as a reminder that germline mosaicism should be considered in every case when a mutation is found in the offspring but not in the parents. In the event of a de novo mutation, the family can be counselled that the likelihood of having another affected child is the same as in the general population. In the case of germline mosaicism, however, the likelihood of having another affected child is much higher, depending on the ratio of mutant to wild-type germ cells. This possibility has a significant impact on genetic counselling, and unfortunately, exact determination of the transmitting parent is not always possible. In the case of paternal transmission, a semen sample can be used for further studies to determine the percentage of mutant germ cells, and thus calculate an accurate risk of recurrence for the family. These findings heighten our awareness of the existence of unusual modes of inheritance in epidermolysis bullosa, which should be taken into account when counselling families about the recurrence risk for future offspring.

EchocardiographyVolume 6, Issue 3 p. 271-275 Doppler Velocimetry in the Fetus with Abnormal Karyotype BURTON ROCHELSON M.D., Corresponding Author BURTON ROCHELSON M.D. Departments of OB/GYN, University Hospital, Stony Brook, New YorkAddress for correspondence: Dr. Burton Rochelson, M.D., 1320 Stony Brook Rd., Stony Brook, NY 11790.Search for more papers by this authorCYNTHIA KAPLAN M.D., CYNTHIA KAPLAN M.D. Departments of OB/GYN, University Hospital, Stony Brook, New YorkSearch for more papers by this authorCAROLYN TRUNCA PH.D., CAROLYN TRUNCA PH.D. Departments of OB/GYN, University Hospital, Stony Brook, New YorkSearch for more papers by this authorHAROLD SCHULMAN M.D., HAROLD SCHULMAN M.D. Winthrop University Hospital, Mineola, New YorkSearch for more papers by this authorEDWIN GUZMAN M.D., EDWIN GUZMAN M.D. Winthrop University Hospital, Mineola, New YorkSearch for more papers by this authorADIEL FLEISCHER M.D., ADIEL FLEISCHER M.D. University of Arizona, Health Sciences Center, Tuscon, ArizonaSearch for more papers by this authorKATHRYN REED M.D., KATHRYN REED M.D. Long Island Jewish Medical Center, New Hyde Park, New YorkSearch for more papers by this author BURTON ROCHELSON M.D., Corresponding Author BURTON ROCHELSON M.D. Departments of OB/GYN, University Hospital, Stony Brook, New YorkAddress for correspondence: Dr. Burton Rochelson, M.D., 1320 Stony Brook Rd., Stony Brook, NY 11790.Search for more papers by this authorCYNTHIA KAPLAN M.D., CYNTHIA KAPLAN M.D. Departments of OB/GYN, University Hospital, Stony Brook, New YorkSearch for more papers by this authorCAROLYN TRUNCA PH.D., CAROLYN TRUNCA PH.D. Departments of OB/GYN, University Hospital, Stony Brook, New YorkSearch for more papers by this authorHAROLD SCHULMAN M.D., HAROLD SCHULMAN M.D. Winthrop University Hospital, Mineola, New YorkSearch for more papers by this authorEDWIN GUZMAN M.D., EDWIN GUZMAN M.D. Winthrop University Hospital, Mineola, New YorkSearch for more papers by this authorADIEL FLEISCHER M.D., ADIEL FLEISCHER M.D. University of Arizona, Health Sciences Center, Tuscon, ArizonaSearch for more papers by this authorKATHRYN REED M.D., KATHRYN REED M.D. Long Island Jewish Medical Center, New Hyde Park, New YorkSearch for more papers by this author First published: May 1989 https://doi.org/10.1111/j.1540-8175.1989.tb00306.xCitations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume6, Issue3May 1989Pages 271-275 RelatedInformation