[Genetic analysis of a fetus with Coffin-Siris syndrome 2 due to a novel variant of ARID1A gene].

OBJECTIVE: To explore the genetic etiology of a fetus with Coffin-Siris syndrome (CSS). METHODS: A fetus with abnormal ultrasound findings detected at Luoyang Maternal and Child Health Care Hospital in July 2023 was selected as the study subject. Clinical data were analyzed retrospectively. Whole exome sequencing was carried out on fetal tissue and parental peripheral blood samples, and candidate variant was verified by Sanger sequencing and pathogenicity analysis. This study was approved by the Luoyang Maternal and Child Health Care Hospital (Ethics No. LYFY-YCCZ-2023011). RESULTS: Color Doppler ultrasound at 16+ gestational weeks revealed bilateral ventriculomegaly and cerebellar hypoplasia in the fetus. Trio-WES found that the fetus has harbored a heterozygous c.553C>T (p.Gln185Ter) variant of the ARID1A gene, which was verified by Sanger sequencing to have a de novo origin. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.553C>T (p.Gln185Ter) variant of the ARID1A gene was classified as pathogenic (PVS1+PS2_Supporting+PM2_Supporting). CONCLUSION: The fetus was diagnosed with CSS type 2, and the heterozygous c.553C>T (p.Gln185Ter) variant of the ARID1A gene probably underlay its brain malformations.

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome.

Camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP) is a rare autosomal recessive disease caused by mutation in proteoglycan 4 (PRG4) gene on chromosome 1q25-q31. We faced a dilemma and delay in diagnosis in two sisters. The elder sister had pericardial effusion with constrictive pericarditis, underwent pericardiectomy and received empirical treatment for suspected tuberculosis. After 2 years, she developed bilateral knee swelling with restriction of movement. At the same time, her younger sister also presented with bilateral knee swelling which aroused the suspicion of genetic disease. The whole-genome sequencing revealed homozygous PRG4 mutation suggestive of CACP syndrome.

TBX5 variants and cardiac phenotype: A systematic review of the literature and a novel variant.

T-Box Transcription Factor 5 (TBX5) variants are associated with Holt-Oram syndrome. Holt-Oram syndrome display phenotypic variability, regarding upper limb defects, congenital heart defects, and arrhythmias. To investigate the genotype-phenotype relationship between TBX5 variants and cardiac disease, we performed a systematic review of the literature. Through the systematic review we identified 108 variants in TBX5 associated with a cardiac phenotype in 277 patients. Arrhythmias were more frequent in patients with a missense variant (48% vs 30%, p = 0.009) and upper limb abnormalities were more frequent in patients with protein-truncating variants (85% vs 64%, p = 0.0008). We found clustering of missense variants in the T-box domain. Furthermore, we present a family with atrial septal defects. By whole exome sequencing, we identified a novel missense variant p.Phe232Leu in TBX5. The cardiac phenotype included atrial septal defect, arrhythmias, heart failure, and dilated cardiomyopathy. Clinical examination revealed subtle upper limb abnormalities. Thus, the family corresponds to the diagnostic criteria of Holt-Oram syndrome. We provide an overview of cardiac phenotypes associated with TBX5 variants and show an increased risk of arrhythmias associated to missense variants compared to protein-truncating variants. We report a novel missense variant in TBX5 in a family with an atypical Holt-Oram syndrome phenotype.

Syndromic Involvement of Patients Presenting With Congenital Upper Limb Anomalies: An Analysis of 4,317 Cases.

PURPOSE: This study investigated the patterns of syndromic involvement for patients with congenital upper limb anomalies (CULAs). We hypothesize that patients with CULAs will present with predictable syndromic patterns. METHODS: This retrospective study queried the multicenter Congenital Upper Limb Differences (CoULD) Registry. Of the 4,317 patients enrolled, 578 (13%) reported one or more syndromes. Syndromes were confirmed to be recognized by the Online Mendelian Inheritance in Man. Demographics were reviewed and compared with the full CoULD registry group. Syndromes reported by five or more patients were examined to determine the type of CULA according to Oberg/Manske/Tonkin classifications. RESULTS: Of the 578 children with one or more reported syndromes, 517 had Online Mendelian Inheritance in Man recognized syndromes (cohort A), In cohort A, 58 syndromes were each represented by a single patient within the registry. Forty-eight syndromes in cohort A were reported by two or more patients, which accounted for 461 of the total patients with reported syndromes. However, VACTERL and Poland syndromes were the most commonly reported syndromes. Patients with CULAs and syndromes frequently exhibited bilateral involvement (61%), compared with the entire CoULD group (47%) and other orthopedic (50%) and medical conditions (61%) compared with the entire CoULD group (24% and 27%, respectively). Additionally, they exhibited a lower frequency of family history of a congenital orthopedic condition (21%) or a family member with the same CULA (9%) compared with the entire CoULD group (26% and 14%, respectively). CONCLUSIONS: Associated syndromes were recorded in 578 patients (13%) in the CoULD registry as follows: 58 syndromes represented by a single patient, 48 by 2 or more patients, and 23 syndromes by 5 or more patients. Rare syndromes that are only represented by a single patient are more likely to be unknown by a pediatric hand surgeon, and consultation with a geneticist is advised. TYPE OF STUDY/LEVEL OF EVIDENCE: Differential Diagnosis/Symptom Prevalence Study IV.

Thrombocytopenia-Absent Radius Syndrome: Descriptions of Three New Cases and a Novel Splicing Variant in RBM8A That Expands the Spectrum of Null Alleles.

Thrombocytopenia-absent radius (TAR) syndrome is a rare congenital disorder characterized by the bilateral absence of the radius and thrombocytopenia, and sometimes by other skeletal, gastrointestinal, cardiac, and renal abnormalities. The underlying genetic defect is usually the compound inheritance of a microdeletion in 1q21.1 (null allele) and a low-frequency, non-coding single nucleotide variant (SNV) in the RBM8A gene (hypomorphic allele). We report three new cases from two unrelated families. The two siblings presented the common genotype, namely the compound heterozygosity for a 1q21.1 microdeletion and the hypomorphic SNV c.-21G>A in RBM8A, whereas the third, unrelated patient presented a rare genotype comprised by two RBM8A variants: c.-21G>A (hypomorphic allele) and a novel pathogenic variant, c.343-2A>G (null allele). Of the eight documented RBM8A variants identified in TAR syndrome patients, four have hypomorphic expression and four behave as null alleles. The present report expands the RBM8A null allele spectrum and corroborates the particularities of RBM8A involvement in TAR syndrome pathogenesis.

Patient-Specific TBX5-G125R Variant Induces Profound Transcriptional Deregulation and Atrial Dysfunction.

BACKGROUND: The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt-Oram syndrome (also known as hand-heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. METHODS: We analyzed ECGs of an extended family pedigree of Holt-Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome (Tbx5G125R) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). RESULTS: We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt-Oram syndrome patients. Tbx5G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. CONCLUSIONS: Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.

Generation of a CRISPR/Cas edited human induced pluripotent stem cell line DHMi005-A-1 carrying a patient-specific disease-causing point mutation in the TBX5 gene.

A number of mutations in the human TBX5 gene have been described which cause Holt-Oram syndrome, a severe congenital disease associated with abnormalities in heart and upper limb development. We have used a prime-editing approach to introduce a patient-specific disease-causing TBX5 mutation (c.920_C > A) into an induced pluripotent stem cell (iPSC) line from a healthy donor. The resulting iPSC line provides a powerful tool to identify and analyze the biological and molecular impact of this specific TBX5 mutation in comparison to the isogenic control iPSC line during cardiac development.

A genotype-first analysis in a cohort of Mullerian anomaly.

Müllerian anomaly (M.A.) is a group of congenital anatomic abnormalities caused by aberrations of the development process of the Müllerian duct. M.A. can either be isolated or be involved in Mendelian syndromes, such as Dandy-Walker syndrome, Holt-Oram syndrome and Bardet-Biedl syndrome, which are often associated with both uterus and kidney malformations. In this study, we applied a genotype-first approach to analyze the whole-exome sequencing data of 492 patients with M.A. Six potential pathogenic variants were found in five genes previously related to female urogenital deformities (PKD1, SON, SALL1, BMPR1B, ITGA8), which are partially overlapping with our patients' phenotypes. We further identified eight incidental findings in seven genes related to Mendelian syndromes without known association with reproductive anomalies (TEK, COL11A1, ANKRD11, LEMD3, DLG5, SPTB, BMP2), which represent potential phenotype expansions of these genes.

TBX3 and TBX5 duplication: A family with an atypical overlapping Holt-Oram/ulnar-mammary syndrome phenotype.

Holt-Oram syndrome (HOS) is a rare, autosomal dominant heart-hand syndrome caused by mutations in the TBX5 gene. A wide spectrum of TBX5 mutations have been reported previously, most resulting in a null allele leading to haploinsufficiency. TBX5 gene duplications have been previously reported in association with typical and atypical HOS phenotypes. Ulnar-Mammary syndrome (UMS) is a distinct rare, autosomal dominant condition caused by mutations in the TBX3 gene. TBX5 and TBX3 are physically linked in cis on human chromosome 12 and contiguous chromosome 12q24 deletions comprising both TBX5 and TBX3 genes have been previously reported but to our knowledge, duplications have never been described. We report on a large German family with at least 17 affected individuals over 6 generations bearing a duplication at 12q24.21 identified on array-CGH comprising both TBX5 and TBX3 genes. Affected patients are presenting with HOS and UMS symptoms, consisting of variable limb anomalies involving the radial and the ulnar rays and cardiac findings such as congenital heart defects, persistent arterial duct or aortic stenosis, and non-classical symptoms, such as supernumerary nipples and cardiomyopathy. Fluorescence in situ hybridisation confirmed a tandem duplication at the 12q24.21 locus. This is the first report of a contiguous TBX3/TBX5 duplication associated with HOS/UMS phenotype.

Functional analysis of two novel TBX5 variants present in individuals with Holt-Oram syndrome with different clinical manifestations.

Holt-Oram syndrome (HOS) is a rare disorder characterized by cardiac and upper-limb defects. Pathogenic variants in TBX5-a gene encoding a transcription factor important for heart and skeletal development-are the only known cause of HOS. Here, we present the identification and functional analysis of two novel TBX5 pathogenic variants found in two individuals with HOS presenting distinct phenotypes. The individual with the c.905delA variant has a severe cardiac phenotype but mild skeletal defects, unlike the individual with the c.246_249delGATG variant who has no cardiac problems but severe upper limbs malformations, including phocomelia. Both frameshift variants, c.246_249delGATG and c.905delA, generate mRNAs harbouring premature stop codons which, if not degraded by nonsense mediated decay, will lead to the production of shorter TBX5 proteins, p.Gln302Argfs*92 and p.Met83Phefs*6, respectively. Immunocytochemistry results suggest that both mutated proteins are produced and furthermore, like the wild-type protein, p.Gln302Argfs*92 mutant appears to be mainly localized in the nucleus, in contrast with p.Met83Phefs*6 mutant that displays a higher level of cytoplasmic localization. In addition, luciferase activity analysis revealed that none of the TBX5 mutants are capable of transactivating the NPPA promoter. In conclusion, our results provide evidence that both pathogenic variants cause a severe TBX5 loss-of-function, dramatically reducing its biological activity. The absence of cardiac problems in the individual with the p.Met83Phefs*6 variant supports the existence of other mechanisms/genes underlying the pathogenesis of HOS and/or the existence of an age-related delay in the development of a more serious cardiac phenotype. Further studies are required to understand the differential effects observed in the phenotypes of both individuals.

Generation of a TBX5 homozygous knockout embryonic stem cell line (WAe009-A-45) by CRISPR/Cas9 genome editing.

Holt-Oram syndrome (HOS), which is caused by genetic changes in the TBX5 gene, affects the hands and heart. HOS patients have heart defects, including atrial septal defects (ASD), ventricular septal defects (VSD) and heart conduction disease. Here, we generated a homozygous TBX5 knockout human embryonic stem cell (hESC) line (TBX5-KO) using a CRISPR/Cas9 system. The TBX5-KO maintained stem cell like morphology, pluripotency markers, normal karyotype, and could differentiate into all three germ layers in vivo. This cell line can provide an in vitro platform for studying the pathogenic mechanisms and biological function of TBX5 in the heart development.

Growing up with a rare genetic disease: an interpretative phenomenological analysis of living with Holt-Oram syndrome.

BACKGROUND: Holt-Oram syndrome (HOS) is a rare genetic disease characterized by variable radial upper limb and cardiac defects. The aim of this research was to shed light on people's subjective perceptions of their diseases, how these perceptions provide meaning, and the consequences the syndrome can have in daily life and across all life stages. METHODS: Semistructured interviews with ten participants diagnosed with HOS were conducted in France and analyzed using interpretative phenomenological analysis. RESULTS: Participants' experiences fall under two main themes, namely, "stages of self-construction as different" and "when I am no longer the only one involved", each of which has three subthemes. From childhood onwards, symptoms monopolize the physical and psychological spheres. The feeling of being different is unavoidable until the patient can appropriate his or her condition, and by the end of adolescence, the patient generally feels that he or she has adapted to the syndrome. In adulthood, other concerns arise, such as the fear of rejection, the need to better understand the genetic issues of the condition and the desire for offspring to not experience the same life difficulties. CONCLUSION: The findings underscore the specific psychological issues associated with the syndrome at different life stages and the need for holistic genetic treatment with dedicated reference centers to improve care and further address these issues.IMPLICATIONS FOR REHABILITATIONHolt-Oram syndrome is a genetic disease characterized by abnormalities of the upper limbs and shoulder girdle and associated with a congenital heart defect.Specific issues arise at different stages of life: the physical consequences of the syndrome arise during childhood, the self-construction of pervasive difference during adolescence, the fear of being rejected as a young adult, and concerns about future parenthood and the transmission of the syndrome and the desire that one's child not be confronted with the same difficulties in adulthood.The complexity and entanglement of medical and existential issues related to HOS requires the development of multidisciplinary consultations that promote holistic care.The rarity of the syndrome and the lack of knowledge about HOS among health professionals and the general public make it necessary both to establish reference centers and to create patient associations to support patients.

T-Cell Acute Lymphoblastic Leukemia in a Young Adult With Thrombocytopenia-absent Radius Syndrome: A Case Report and Review of the Literature.

Thrombocytopenia-absent radius (TAR) syndrome is a rare inherited bone marrow failure syndrome not generally associated with acute leukemia. The authors report a case of T-cell acute lymphoblastic leukemia in an adult female individual newly diagnosed with TAR syndrome. A 347-kb microdeletion of chromosome 1q21.1 involving the RBM8A gene was detected within a gain of whole chromosome 1. Next-generation sequencing on fibroblasts confirmed germline heterozygous deletion of RBM8A but on the other allele, noncoding low-frequency regulatory single-nucleotide polymorphism of RBM8A (rs139428292; rs201779890) were not found. The tolerance of the treatment was unusual and mostly marked by a slow hematopoietic recovery leading to a 6-month delay at the beginning of the maintenance phase. Only 5 cases of acute leukemia were reported in patients with TAR syndrome in the literature: 4 acute myeloid leukemia and one B-cell acute lymphoblastic leukemia. This is the first report of T-cell acute lymphoid leukemia occurring in the context of TAR syndrome.

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from 2q11.1-q12.1 associated with fetal bilateral radial dysplasia.

OBJECTIVE: We present prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome (sSMC) derived from 2q11.1-q12.1 associated with fetal bilateral radial dysplasia. CASE REPORT: A 27-year-old woman underwent amniocentesis at 18 weeks of gestation because of club hands on fetal ultrasound. The internal organs of the fetus were normal. Amniocentesis revealed a karyotype of 47,XY,+mar [13]/46,XY [11]. The parental karyotypes were normal. Simultaneous array comparative genomic hybridization (aCGH) analysis of the DNA extracted from uncultured amniocytes revealed the result of arr 2q11.1q12.1 (95,529,039-102,825,556) × 3.0 [GRCh37 (hg19)]. The pregnancy was terminated at 20 weeks of gestation, and a malformed fetus was delivered with isolated bilateral radial dysplasia. The cord blood had a karyotype of 47,XY,+mar[24]/46,XY[16]. Polymorphic DNA marker analysis of the DNAs extracted from umbilical cord and parental bloods excluded uniparental disomy for chromosome 2. Metaphase fluorescence in situ hybridization analysis confirmed an sSMC derived from chromosome 2q11.1-q12.1 in cultured amniocytes. CONCLUSION: High-level mosaicism for an sSMC derived from chromosome 2q11.1-q12.1 can be associated with fetal abnormalities.

Familial dilated cardiomyopathy associated with pathogenic TBX5 variants: Expanding the cardiac phenotype associated with Holt-Oram syndrome.

Holt-Oram syndrome (HOS) is a rare, autosomal dominant disorder caused by heterozygous pathogenic variants in cardiac T-box transcription factor, TBX5. Classically, it is associated with upper limb malformations and variable cardiac abnormalities. Limb manifestations are considered to be invariably present, ranging in severity from limitation in movement, to triphalangeal thumbs, absent thumbs, shortened forearms, or phocomelia. Cardiac involvement is characterized by congenital heart defects, most commonly septal structural malformations, and conduction system disease. Recently, novel TBX5 variants have also been reported in association with dilated cardiomyopathy (DCM). In this context, we report eight individuals from four unrelated families, in whom pathogenic variants in TBX5 segregated with an atypical HOS phenotype. Affected individuals exhibit relatively mild skeletal features of HOS, with a predominant cardiac phenotype, which includes several individuals affected by non-ischaemic DCM. To our knowledge, these represent the first reported cases of DCM in families with skeletal features of HOS, some of whom also harbored variants previously linked to a classical HOS phenotype (p. Arg279* and p.Arg237Gln). This finding supports diverse roles of TBX5 in cardiovascular development and function, and confirms the importance of long-term cardiac surveillance for individuals affected by HOS. Furthermore, these families highlight the wide phenotypic variability of HOS, which may include comparatively mild upper limb findings in respect to cardiac manifestations.

TAR syndrome: Clinical and molecular characterization of a cohort of 26 patients and description of novel noncoding variants of RBM8A.

Thrombocytopenia-absent radius (TAR) syndrome is characterized by radial defect and neonatal thrombocytopenia. It is caused by biallelic variants of RBM8A gene (1q21.1) with the association of a null allele and a hypomorphic noncoding variant. RBM8A encodes Y14, a core protein of the exon junction complex involved in messenger RNA maturation. To date, only two hypomorphic variants have been identified. We report on a cohort of 26 patients affected with TAR syndrome and carrying biallelic variants in RBM8A. Half patients carried a 1q21.1 deletion and one of the two known hypomorphic variants. Four novel noncoding variants of RBM8A were identified in the remaining patients. We developed experimental models enabling their functional characterization in vitro. Two variants, located respectively in the 5'-untranslated region (5'-UTR) and 3'-UTR regions, are responsible for a diminished expression whereas two intronic variants alter splicing. Our results bring new insights into the molecular knowledge of TAR syndrome and enabled us to propose genetic counseling for patients' families.