Novel likely pathogenic variant in the EYA1 gene causing Branchio oto renal syndrome and the exploration of pathogenic mechanisms.

OBJECTIVE: Branchio-oto-renal syndrome (BOR, OMIM#113,650) is a rare autosomal dominant disorder that presents with a variety of symptoms, including hearing loss (sensorineural, conductive, or mixed), structural abnormalities affecting the outer, middle, and inner ear, branchial fistulas or cysts, as well as renal abnormalities.This study aims to identify the pathogenic variants by performing genetic testing on a family with Branchio-oto-renal /Branchio-otic (BO, OMIM#602,588) syndrome using whole-exome sequencing, and to explore possible pathogenic mechanisms. METHODS: The family spans 4 generations and consists of 9 individuals, including 4 affected by the BOR/BO syndrome. Phenotypic information, including ear malformation and branchial cleft, was collected from family members. Audiological, temporal bone imaging, and renal ultrasound examinations were also performed. Whole-exome sequencing was conducted to identify candidate pathogenic variants and explore the underlying molecular etiology of BOR/BO syndrome by minigene experiments. RESULTS: Intra-familial variability was observed in the clinical phenotypes of BOR/BO syndrome in this family. The severity and nature of hearing loss varied in family members, with mixed or sensorineural hearing loss. The proband, in particular, had profound sensorineural hearing loss on the left and moderate conductive hearing loss on the right. Additionally, the proband exhibited developmental delay, and her mother experienced renal failure during pregnancy and terminated the pregnancy prematurely. Genetic testing revealed a novel heterozygous variant NM_000503.6: c.639 + 3 A > C in the EYA1 gene in affected family members. In vitro minigene experiments demonstrated its effect on splicing. According to the American College of Medical Genetics (ACMG) guidelines, this variant was classified as likely pathogenic. CONCLUSION: This study highlights the phenotypic heterogeneity within the same family, reports the occurrence of renal failure and adverse pregnancy outcomes in a female patient at reproductive age with BOR syndrome, and enriches the mutational spectrum of pathogenic variants in the EYA1 gene.

From clinical to molecular diagnosis: relevance of diagnostic strategy in two cases of branchio-oto-renal syndrome - case report.

BACKGROUND: Branchio-oto-renal syndrome (BOR) is an autosomal dominant disorder characterized by deafness, branchiogenic malformations and renal abnormalities. Pathogenic variants in EYA1, SIX1 and SIX5 genes cause almost half of cases; copy number variants (CNV) and complex genomic rearrangements have been revealed in about 20% of patients, but they are not routinely and commonly included in the diagnostic work-up. CASE PRESENTATION: We report two unrelated patients with BOR syndrome clinical features, negative sequencing for BOR genes and the identification of a 2.65 Mb 8q13.2-13.3 microdeletion. CONCLUSIONS: We highlight the value of CNV analyses in high level of suspicion for BOR syndrome but negative sequencing for BOR genes and we propose an innovative diagnostic flow-chart to increase current detection rate. Our report confirms a mechanism of non-allelic homologous recombination as causing this recurrent 8q13.2-13.3 microdeletion. Moreover, considering the role of PRDM14 and NCOA2 genes, both involved in regulation of fertility and deleted in our patients, we suggest the necessity of a longer follow-up to monitor fertility issues or additional clinical findings.

Genetic research progress in branchio-oto syndrome/ branchio-oto-renal syndrome. / 鳃耳综合征/é³ƒè€³è‚¾ç»¼åˆå¾çš„é—ä¼ å­¦ç ”ç©¶è¿›å±•.

Branchio-oto syndrome (BOS)/branchio-oto-renal syndrome (BORS) is a kind of autosomal dominant heterogeneous disorder. These diseases are mainly characterized by hearing impairment and abnormal phenotype of ears, accompanied by renal malformation and branchial cleft anomalies including cyst or fistula, with an incidence of 1/40 000 in human population. Otic anormalies are one of the most obvious clinical manifestations of BOS/BORS, including deformities of external, middle, inner ears and hearing loss with conductive, sensorineural or mix, ranging from mild to profound loss. Temporal bone imaging could assist in the diagnosis of middle ear and inner ear malformations for clinicians. Multiple methods including direct sequencing combined with next generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), or array-based comparative genomic hybridization (aCGH) can effectively screen and identify pathogenic genes and/or variation types of BOS/BORS. About 40% of patients with BOS/BORS carry aberrations of EYA1 gene which is the most important cause of BOS/BORS. A total of 240 kinds of pathogenic variations of EYA1 have been reported in different populations so far, including frameshift, nonsense, missense, aberrant splicing, deletion and complex rearrangements. Human Endogenous Retroviral sequences (HERVs) may play an important role in mediating EYA1 chromosomal fragment deletion mutations caused by non-allelic homologous recombination. EYA1 encodes a phosphatase-transactivator cooperated with transcription factors of SIX1, participates in cranial sensory neurogenesis and development of branchial arch-derived organs, then regulates the morphological and functional differentiation of the outer ear, middle ear and inner ear toward normal tissues. In addition, pathogenic mutations of SIX1 and SIX5 genes can also cause BOS/BORS. Variations of these genes mentioned above may cause disease by destroying the bindings between SIX1-EYA1, SIX5-EYA1 or SIX1-DNA. However, the role of SIX5 gene in the pathogenesis of BORS needs further verification.

Growth hormone deficiency in a child with branchio-oto-renal spectrum disorder: Clinical evidence of EYA1 in pituitary development and a recommendation for pituitary function surveillance.

Branchio-oto-renal spectrum disorder (BORSD) is a rare autosomal dominant condition characterized by ear abnormalities with hard of hearing/deafness, second branchial arch malformations and renal anomalies. Pathogenic variations in EYA1 gene are found in the majority of clinically diagnosed individuals with BORSD. We describe an infant with BORSD related to a paternally inherited heterozygous pathogenic variation in EYA1 gene presenting with poor growth and hypoglycemia due to growth hormone deficiency. Magnetic resonance imaging revealed a diminutive pituitary gland and morphologically abnormal sella. Upon initiation of growth hormone therapy, the hypoglycemia resolved and catch up growth ensued. Pituitary abnormalities have not been reported previously in patients with BORSD. The zebrafish ortholog of eya1 is important for the development of adenohypophysis, suggesting that this patient's growth hormone deficiency and pituitary abnormality are part of BORSD. Inclusion of screening for pituitary hormone deficiency and pituitary imaging should be considered as a part of surveillance in patients with BORSD.

PAX1 is essential for development and function of the human thymus.

We investigated the molecular and cellular basis of severe combined immunodeficiency (SCID) in six patients with otofaciocervical syndrome type 2 who failed to attain T cell reconstitution after allogeneic hematopoietic stem cell transplantation, despite successful engraftment in three of them. We identified rare biallelic PAX1 rare variants in all patients. We demonstrated that these mutant PAX1 proteins have an altered conformation and flexibility of the paired box domain and reduced transcriptional activity. We generated patient-derived induced pluripotent stem cells and differentiated them into thymic epithelial progenitor cells and found that they have an altered transcriptional profile, including for genes involved in the development of the thymus and other tissues derived from pharyngeal pouches. These results identify biallelic, loss-of-function PAX1 mutations as the cause of a syndromic form of SCID due to altered thymus development.

Inherited glomerular diseases in the gilded age of genomic advancements.

The synchronized advent of high-throughput next-generation sequencing technology and knowledge of the human genome has rendered exponential contributions to our understanding of the pathophysiology of glomerular kidney diseases. A genetic diagnosis can now be made or confirmed in about two-thirds of the suspected inherited glomerular diseases. Next-generation sequencing is adept at identifying single nucleotide variations and small insertions or deletions that constitute majority of the disease-causing mutations. Description of the complete mutation spectrum in syndromic glomerulopathies may require the use of both sequencing and cytogenetic methods to detect large structural DNA variation in addition to single nucleotide changes. The enthusiastic application of genetic and genomic knowledge to inherited glomerular diseases has uncovered anticipated and unforeseen challenges mainly related to the biological interpretation of variants of uncertain significance and the limited benefit on clinical management for the individual patient when a diagnosis is obtained. To attain the ultimate goal of transforming clinical decision-making based on accurate genetic diagnosis using genomic information, these challenges need to be addressed. Till then, the glory of genomic medicine stands the test of time in this gilded age of genomic advancements.

Identification of ANLN as a new likely pathogenic gene of branchio-otic syndrome in a three-generation Chinese family.

BACKGROUND: Branchio-oto-renal (BOR) syndrome is one of the most common autosomal dominant hearing loss syndromes and features clinical and genetic heterogeneity. When there is no renal deformity, this disease can also be called branchio-otic (BO) syndrome. Though many genes have been reported, there are still many BO syndrome-related genes to be identified. To identify a hitherto unknown candidate gene causing BO syndrome in a three-generation Chinese family, clinical, genetic, and functional analyses were employed. METHODS: Whole-exome sequencing (WES) was conducted in three affected family members and two unaffected family members. PCR-Sanger sequencing was performed in all of the family members for segregation analysis and verification of the candidate variants. PCR-Sanger sequencing was also employed in 150 healthy people to examine the variants. In silico analysis was used to predict possible changes in the protein structure that may affect the phenotype. RESULTS: We identified a heterozygous missense variant in ANLN: NM_018685.4: c.G1105A; NP_061155.2: p.G369R that segregated in the pedigree with an autosomal dominant pattern. No variant was found in the 150 controls and normal family members at this site. The variant c.G1105A was located in a highly conserved F-actin binding site. The amino acid residue at position 369 in the ANLN protein was highly conserved across different species. CONCLUSION: In this study, we identified, for the first time, a heterozygous missense variant in ANLN (NM_018685.4: c.G1105A; NP_061155.2: p.G369R) that is likely to be a candidate causative gene of BO syndrome in a specific Chinese family.

The Unwound Cochlea: A Specific Imaging Marker of Branchio-Oto-Renal Syndrome.

BACKGROUND AND PURPOSE: Branchio-oto-renal syndrome is an important syndromic cause of hearing loss. Our aim was to determine the test characteristics of the unwound cochlea on temporal bone CT for the diagnosis of branchio-oto-renal syndrome in a cohort of children with hearing loss. MATERIALS AND METHODS: Patients were identified retrospectively with a clinical diagnosis of branchio-oto-renal syndrome and CT imaging of the temporal bones. Age-matched controls were also identified with sensorineural hearing loss not related to a diagnosis of branchio-oto-renal syndrome and CT imaging of the temporal bones. All examinations were reviewed by 2 neuroradiologists blinded to the diagnosis of branchio-oto-renal syndrome versus controls for the absence/presence of an unwound cochlea defined as anteromedial rotation and displacement of the middle and apical turns away from the basal turn. RESULTS: The final study group comprised 9 patients with branchio-oto-renal syndrome (age range, 1-14 years; mean age, 8.0 ± 4.3 years) and 50 control patients (age range, 1-16 years; mean age, 7.9 ± 4.1 years). The cochlea was subjectively abnormal in all 9 patients. In 8 patients (89%), imaging demonstrated a typical unwound cochlear morphology. By contrast, none of the control subjects demonstrated an unwound cochlea on either side. Statistically, the unwound cochlea was significantly more frequent in the branchio-oto-renal group compared with controls (P < .001). The unwound cochlea was 89% sensitive and 100% specific for the diagnosis of branchio-oto-renal syndrome. CONCLUSIONS: The unwound cochlea is a specific imaging marker of branchio-oto-renal syndrome. These findings further support the diagnostic accuracy and therefore the utility of temporal bone imaging in the diagnosis of this disorder.

Whole exome sequencing identifies a mutation in EYA1 and GLI3 in a patient with branchio­otic syndrome and esophageal atresia: Coincidence or a digenic mode of inheritance?

Branchio­otic (BO) syndrome is a clinically and genetically heterogeneous disorder that presents with variable branchial arch and otic anomalies. Dominant mutations in the human homologues of the Drosophila eyes absent (EYA1) gene, and the Drosophila sine oculis homeobox 1 and 5 (SIX1 and SIX5, respectively) genes have been causally associated with BO syndrome. Esophageal atresia (EA), with or without tracheo­esophageal fistula (TEF), is the most common type of malformation of the upper digestive tract. To date, its causes are poorly understood. The present study investigated a family with three affected members who all presented with classic BO associated symptoms. Notably, the index patient also presented with the most common EA/TEF subtype type 3b. Whole exome sequencing (WES) was performed in the index patient, and prioritized genetic variants and their segregation in the family were analyzed by Sanger sequencing. WES demonstrated a known disease­causing heterozygous EYA1 splice variant in the patient, as well as his sister and mother; all of whom were affected with BO syndrome. A further GLI family zinc finger 3 (GLI3) splice variant of unknown significance, inherited from the unaffected father, was also detected in the index patient. EYA1 and GLI3 are involved in the Sonic Hedgehog transcriptional network and GLI3 seems to be involved in human foregut malformations. Therefore, one may hypothesize a digenic inheritance model involving EYA1 and GLI3, where the effect of the GLI3 variant observed here only emerges in the background of the EYA1 defect.

A novel PAX1 null homozygous mutation in autosomal recessive otofaciocervical syndrome associated with severe combined immunodeficiency.

Otofaciocervical syndrome (OFCS) is a rare disorder characterized by facial anomalies, cup-shaped low-set ears, preauricular fistulas, hearing loss, branchial defects, skeletal anomalies, and mild intellectual disability. Autosomal dominant cases are caused by deletions or point mutations of EYA1. A single family with an autosomal recessive form of OFCS and a homozygous missense mutation in PAX1 gene has been described. We report whole exome sequencing of 4 members of a consanguineous family in which 2 children, showing features of OFCS, expired from severe combined immunodeficiency (SCID). To date, the co-occurrence of OFCS and SCID has never been reported. We found a nonsense homozygous mutation in PAX1 gene in the 2 affected children. In mice, Pax1 is required for the formation of specific skeletal structures as well as for the development of a fully functional thymus. The mouse model strongly supports the hypothesis that PAX1 depletion in our patients caused thymus aplasia responsible for SCID. This report provides evidence that bi-allelic null PAX1 mutations may lead to a multi-system autosomal recessive disorders, where SCID might represent the main feature.

Anatomic and Quantitative Temporal Bone CT for Preoperative Assessment of Branchio-Oto-Renal Syndrome.

We describe the temporal bone computed tomography (CT) findings of an unusual case of branchio-oto-renal syndrome with ectopic ossicles that are partially located in the middle cranial fossa. We also describe quantitative temporal bone CT assessment pertaining to cochlear implantation in the setting of anomalous cochlear anatomy associated with this syndrome.

A clinical and molecular analysis of branchio-oculo-facial syndrome patients in Russia revealed new mutations in TFAP2A.

Branchio-oculo-facial syndrome (BOFS, OMIM# 113620) is a rare autosomal dominant disorder characterised by branchial cleft sinus defects, ocular anomalies and facial dysmorphisms, including lip or palate cleft or pseudocleft, and is associated with mutations in the TFAP2A gene. Here, we performed clinical analysis and mutation diagnostics in seven BOFS patients in Russia. The phenotypic presentation of BOFS observed in three patients showed high heterogeneity, including variation in its main clinical manifestations (linear loci of cervical cutaneous aplasia, ocular anomalies and orofacial cleft). In certain other cases, isolated ocular anomalies, or an orofacial cleft with accessory BOFS symptoms, were observed. In five BOFS patients, conductive hearing loss was diagnosed. Direct sequencing of the coding region of the TFAP2A gene revealed missense mutations in four BOFS patients. One patient was observed to have a previously described mutation (p.Arg251Gly), while three patients from two families were found to have novel mutations: p.Arg213Ser and p.Val210Asp. These novel mutations were not present in healthy members of the same family and therefore should be classified as de novo.

Branchio-oculo-facial syndrome: a three generational family with markedly variable phenotype including neonatal lethality.

Branchio-oculo-facial syndrome (BOFS) is a rare autosomal dominant condition with variable expressivity, caused by mutations in the TFAP2A gene. We report a three generational family with four affected individuals. The consultand has typical features of BOFS including infra-auricular skin nodules, coloboma, lacrimal duct atresia, cleft lip, conductive hearing loss and typical facial appearance. She also exhibited a rare feature of preaxial polydactyly. Her brother had a lethal phenotype with multiorgan failure. We also report a novel variant in TFAP2A gene. This family highlights the variable severity of BOFS and, therefore, the importance of informed genetic counselling in families with BOFS.

Recurrent 8q13.2-13.3 microdeletions associated with branchio-oto-renal syndrome are mediated by human endogenous retroviral (HERV) sequence blocks.

BACKGROUND: Human endogenous retroviral (HERV) sequences are the remnants of ancient retroviral infection and comprise approximately 8% of the human genome. The high abundance and interspersed nature of homologous HERV sequences make them ideal substrates for genomic rearrangements. A role for HERV sequences in mediating human disease-associated rearrangement has been reported but is likely currently underappreciated. METHODS AND RESULTS: In the present study, two independent de novo 8q13.2-13.3 microdeletion events were identified in patients with clinical features of Branchio-Oto-Renal (BOR) syndrome. Nucleotide-level mapping demonstrated the identical breakpoints, suggesting a recurrent microdeletion including multiple genes such as EYA1, SULF1, and SLCO5A1, which is mediated by HERV1 homologous sequences. CONCLUSIONS: These findings raise the potential that HERV sequences may more commonly underlie recombination of dosage sensitive regions associated with recurrent syndromes.

Branchio-oculo-facial syndrome in a newborn caused by a novel TFAP2A mutation.

We present an 18-day old boy with bilateral cervical cutaneous defect in the retroauricular region, low-set and posteriorly rotated ears, bilateral microphtalmia and bilateral pseudocleft of the upper lip. Histopathological evaluation of cervical cutaneous defect showed ulceration on the surface and ectopic thymus tissue in the deep dermis with cortex, medulla and Hassal's corpuscles. Clinical findings led to the diagnosis of Branchio-oculo-facial syndrome, characterized by branchial defects (erythematous cutaneous defects in cervical region), ocular anomalies (microphthalmia, anophthalmia, lacrimal duct obstruction, coloboma, cataract, ptosis) and facial defects (cleft lip and/or palate, pseudocleft or abnormal philtrum). DNA sequencing showed a novel heterozygous mutation, c.731T>C (p.L244P), in TFAP2A gene confirming the diagnosis of this rare autosomal dominant developmental disorder with variable clinical findings.

Identification of a novel nonsynonymous mutation of EYA1 disrupting splice site in a Korean patient with BOR syndrome.

The EYA1 gene is known as the causative gene of BOR (Branchio-oto-renal) syndrome which is a genetic disorder associated with branchial cleft cysts of fistulae, hearing loss, ear malformation, and renal anomalies. Although approximately 40% of patients with BOR syndrome have mutations in the EYA1 gene and over 130 disease-causing mutations in EYA1 have been reported in various populations, only a few mutations have been reported in Korean families. In this study, genetic analysis of the EYA1 gene was performed in a Korean patient diagnosed with BOR syndrome and his parents. A de novo novel missense mutation, c.418G>A, located at the end of exon 6, changed glycine to serine at amino acid position 140 (p.G140S) and was suspected to affect normal splicing. Our in vitro splicing assay demonstrated that this mutation causes exon 6 skipping leading to frameshift and truncation of the protein to result in the loss of eyaHR. To the best of our knowledge, this is the first report revealing that a missense mutation in the exon disturbs normal splicing as a result of a substitution of the last nucleotide of an exon in EYA1.

Branchio-otic syndrome caused by a genomic rearrangement: clinical findings and molecular cytogenetic studies in a patient with a pericentric inversion of chromosome 8.

Branchio-oto-renal (BOR) syndrome is an autosomal dominantly inherited developmental disorder, which is characterized by anomalies of the ears, the branchial arches and the kidneys. It is caused by mutations in the genes EYA1,SIX1 and SIX5. Genomic rearrangements of chromosome 8 affecting the EYA1 gene have also been described. Owing to this fact, methods for the identification of abnormal copy numbers such as multiplex ligation-dependent probe amplification (MLPA) have been introduced as routine laboratory techniques for molecular diagnostics of BOR syndrome. The advantages of these techniques are clear compared to standard cytogenetic and array approaches as well as Southern blot. MLPA detects deletions or duplications of a part or the entire gene of interest, but not balanced structural aberrations such as inversions and translocations. Consequently, disruption of a gene by a genomic rearrangement may escape detection by a molecular genetic analysis, although this gene interruption results in haploinsufficiency and, therefore, causes the disease. In a patient with clinical features of BOR syndrome, such as hearing loss, preauricular fistulas and facial dysmorphisms, but no renal anomalies, neither sequencing of the 3 genes linked to BOR syndrome nor array comparative genomic hybridization and MLPA were able to uncover a causative mutation. By routine cytogenetic analysis, we finally identified a pericentric inversion of chromosome 8 in the affected female. High-resolution multicolor banding confirmed the chromosome 8 inversion and narrowed down the karyotype to 46,XX,inv(8)(p22q13). By applying fluorescence in situ hybridization, we narrowed down both breakpoints on chromosome 8 and found the EYA1 gene in q13.3 to be directly disrupted. We conclude that standard karyotyping should not be neglected in the genetic diagnostics of BOR syndrome or other Mendelian disorders, particularly when molecular testing failed to detect any causative alteration in patients with a convincing phenotype.

Analysis of TFAP2A mutations in Branchio-Oculo-Facial Syndrome indicates functional complexity within the AP-2α DNA-binding domain.

Multiple lines of evidence indicate that the AP-2 transcription factor family has an important regulatory function in human craniofacial development. Notably, mutations in TFAP2A, the gene encoding AP-2α, have been identified in patients with Branchio-Oculo-Facial Syndrome (BOFS). BOFS is an autosomal-dominant trait that commonly presents with facial clefting, eye defects and branchial skin anomalies. Examination of multiple cases has suggested either simple haploinsufficiency or more complex genetic causes for BOFS, especially as the clinical manifestations are variable, with no clear genotype-phenotype correlation. Mutations occur throughout TFAP2A, but mostly within conserved sequences within the DNA contact domain of AP-2α. However, the consequences of the various mutations for AP-2α protein function have not been evaluated. Therefore, it remains unclear if all BOFS mutations result in similar changes to the AP-2α protein or if they each produce specific alterations that underlie the spectrum of phenotypes. Here, we have investigated the molecular consequences of the mutations that localize to the DNA-binding region. We show that although individual mutations have different effects on DNA binding, they all demonstrate significantly reduced transcriptional activities. Moreover, all mutant derivatives have an altered nuclear:cytoplasmic distribution compared with the predominantly nuclear localization of wild-type AP-2α and several can exert a dominant-negative activity on the wild-type AP-2α protein. Overall, our data suggest that the individual TFAP2A BOFS mutations can generate null, hypomorphic or antimorphic alleles and that these differences in activity, combined with a role for AP-2α in epigenetic events, may influence the resultant pathology and the phenotypic variability.