Phenotype and genotype in females with POU3F4 mutations

X‐linked deafness is a rare cause of hereditary isolated hearing impairment estimated as at least 1% or 2% of the non‐syndromic hearing loss. To date, four loci for DFN have been identified and only one gene, POU3F4 responsible for DFN3, has been cloned. In males, DFN3 is characterized by a progressive deafness associated with perilymphatic gusher at stapes surgery and with a characteristic inner ear malformation. The phenotype of eight independent females carrying POU3F4 anomalies is defined, and a late‐onset hearing loss is found in three patients. Only one has an inner ear malformation. No genotype/phenotype correlation is identified.     

Molecular and clinical studies of X-linked deafness among Pakistani families

There are 68 sex-linked syndromes that include hearing loss as one feature and five sex-linked nonsyndromic deafness loci listed in the OMIM database. The possibility of additional such sex-linked loci was explored by ascertaining three unrelated Pakistani families (PKDF536, PKDF1132 and PKDF740) segregating X-linked recessive deafness. Sequence analysis of POU3F4 (DFN3) in affected members of families PKDF536 and PKDF1132 revealed two novel nonsense mutations, p.Q136X and p.W114X, respectively. Family PKDF740 is segregating congenital blindness, mild-to-profound progressive hearing loss that is characteristic of Norrie disease (MIM#310600). Sequence analysis of NDP among affected members of this family revealed a novel single nucleotide deletion c.49delG causing a frameshift and premature truncation (p.V17fsX1) of the encoded protein. These mutations were not found in 150 normal DNA samples. Identification of pathogenic alleles causing X-linked recessive deafness will improve molecular diagnosis, genetic counseling and molecular epidemiology of hearing loss among Pakistanis.     

New role of LRP5, associated with nonsyndromic autosomal‐recessive hereditary hearing loss

Human hearing loss is a common neurosensory disorder about which many basic research and clinically relevant questions are unresolved. At least 50% of hearing loss are due to a genetic etiology. Although hundreds of genes have been reported, there are still hundreds of related deafness genes to be found. Clinical, genetic, and functional investigations were performed to identify the causative mutation in a distinctive Chinese family with postlingual nonsyndromic sensorineural hearing loss. Whole‐exome sequencing (WES) identified lipoprotein receptor‐related protein 5 (LRP5), a member of the low‐density lipoprotein receptor family, as the causative gene in this family. In the zebrafish model, lrp5 downregulation using morpholinos led to significant abnormalities in zebrafish inner ear and lateral line neuromasts and contributed, to some extent, to disabilities in hearing and balance. Rescue experiments showed that LRP5 mutation is associated with hearing loss. Knocking down lrp5 in zebrafish results in reduced expression of several genes linked to Wnt signaling pathway and decreased cell proliferation when compared with those in wild‐type zebrafish. In conclusion, the LRP5 mutation influences cell proliferation through the Wnt signaling pathway, thereby reducing the number of supporting cells and hair cells and leading to nonsyndromic hearing loss in this Chinese family.     

Molecular characterization of a novel X-linked syndrome involving developmental delay and deafness

X-linked syndromes associated with developmental delay and sensorineural hearing loss (SNHL) have been characterized at the molecular level, including Mohr-Tranebjaerg syndrome and Norrie disease. In this study we report on a novel X-linked recessive, congenital syndrome in a family with developmental delay and SNHL that maps to a locus associated with mental retardation (MR) for which no causative gene has been identified. The X-linked recessive inheritance and congenital nature of the syndrome was confirmed by detailed clinical investigation and the family history. Linkage mapping of the X-chromosome was conducted to ascertain the disease locus and candidate genes were screened by direct sequencing and STRP analysis. The recessive syndrome was mapped to Xp11.3-q21.32 and a deletion was identified in a regulatory region upstream of the POU3F4 gene in affected family members. Since mutations in POU3F4 cause deafness at the DFN3 locus, the deletion is the likely cause of the SNHL in this family. The choroideremia (CHM) gene was also screened and a novel missense change was identified. The alteration changes the serine residue at position 89 in the Rab escort 1 protein (REP-1) to a cysteine (S89C). Prenylation of Rab proteins was investigated in patients and the location of REP-1 expression in the brain determined. However, subsequent analysis revealed that this change in CHM was polymorphic having no effect on REP-1 function. Although the causative gene at the MR locus in this family has not been identified, there are a number of genes involved in syndromic and nonsyndromic forms of MR that are potential candidates.     

A novel locus for autosomal dominant nonsyndromic hearing loss identified at 5q31.1-32 in a Chinese pedigree

 Hearing impairment is an extremely heterogeneous disorder. A total of 35 loci and 17 related genes for autosomal dominant nonsyndromic hearing loss have been identified. In a Chinese pedigree characterized by autosomal dominant inheritance with bilateral, postlingual, progressive, and sensorineural nonsyndromic hearing impairment, the putative disease gene locus was localized to chromosome 5q31.1-32 by a genome-wide scan. Fine mapping indicated that the disease gene was located within an 8.8-cM region between markers D5S2056 and D5S638, with a maximum two-point logarithm of differences (LOD) score of 6.89 (θ = 0) at D5S2017. By the candidate gene approach, mutation screening of the DIAPH1 and POU4F3 genes at 5q31 was performed. No mutation was found, suggesting that this is a novel deafness locus, which has been named DFNA42. Received: May 8, 2002 / Accepted: October 1, 2002     

The molecular basis of X-linked deafness type 3 (DFN3) in two sporadic cases: Identification of a somatic mosaicism for a POU3F4 missense mutation

We have investigated two unrelated males with X-linked deafness type 3 (DFN3) for mutations in the POU3F4 gene. In one patient, we observed a mutation that is predicted to result in an Arg330Ser amino acid substitution. In another DFN3 patient, a somatic mosaicism for an Arg323Gly amino acid substitution was found. This mosaicism was detected in two independently established EBV immortalized B cells and peripheral blood lymphocytes (PBLs). Semiquantitative analysis showed that approximately 50% of the PBLs of this patient carry the mutation. We hypothesize that the Arg323Gly mutation occurred very early in embryogenesis, before the differentiation of cells involved in hematopoiesis and inner ear development. In both patients, the missense mutations are situated in the POU homeodomain and are predicted to disrupt the DNA binding of the POU3F4 protein. All nine point mutations thus far described were found in the POU domains of POU3F4. Since these domains constitute only 35% of the open reading frame of POU3F4, there is a statistically significant preference for mutations in the POU-specific and POU homeodomain. Hum Mutat 10:207–211, 1997. © 1997 Wiley-Liss, Inc.     

Novel form of X-linked nonsyndromic hearing loss with cochlear malformation caused by a mutation in the type IV collagen gene COL4A6

Hereditary hearing loss is the most common human sensorineural disorder. Genetic causes are highly heterogeneous, with mutations detected in >40 genes associated with nonsyndromic hearing loss, to date. Whereas autosomal recessive and autosomal dominant inheritance is prevalent, X-linked forms of nonsyndromic hearing impairment are extremely rare. Here, we present a Hungarian three-generation family with X-linked nonsyndromic congenital hearing loss and the underlying genetic defect. Next-generation sequencing and subsequent segregation analysis detected a missense mutation (c.1771G>A, p.Gly591Ser) in the type IV collagen gene COL4A6 in all affected family members. Bioinformatic analysis and expression studies support this substitution as being causative. COL4A6 encodes the alpha-6 chain of type IV collagen of basal membranes, which forms a heterotrimer with two alpha-5 chains encoded by COL4A5. Whereas mutations in COL4A5 and contiguous X-chromosomal deletions involving COL4A5 and COL4A6 are associated with X-linked Alport syndrome, a nephropathy associated with deafness and cataract, mutations in COL4A6 alone have not been related to any hereditary disease so far. Moreover, our index patient and other affected family members show normal renal and ocular function, which is not consistent with Alport syndrome, but with a nonsyndromic type of hearing loss. In situ hybridization and immunostaining demonstrated expression of the COL4A6 homologs in the otic vesicle of the zebrafish and in the murine inner ear, supporting its role in normal ear development and function. In conclusion, our results suggest COL4A6 as being the fourth gene associated with X-linked nonsyndromic hearing loss.     

A duplication/paracentric inversion associated with familial X-linked deafness (DFN3) suggests the presence of a regulatory element more than 400 kb upstream of the POU3F4 gene

X-linked deafness with stapes fixation (DFN3) is caused by mutationsin the POU3F4 gene at Xq21.1. By employing pulsed field gelelectrophoresis (PFGE) we identified a chromosomal aberrationin the DNA of a DFN3 patient who did not show alterations inthe open reading frame (ORF) of POU3F4. Southern blot analysisindicated that a DNA segment of 150 kb, located 170 kb proximalto the POU3F4 gene, was duplicated. Fluorescence in situ hybridization(FISH) analysis, PFGE, and detailed Southern analysis revealedthat this duplication is part of a more complex rearrangementincluding a paracentric inversion involving the Xq21.1 region,and presumably the Xq21.3 region. Since at least two DFN3-associatedminideletions are situated proximal to the duplicated segment,the inversion most likely disconnects the POU3F4 gene from aregulatory element which is located at a distance of at least400 kb upstream of the POU3F4 gene.     

Prenatal growth restriction,retinal dystrophy,diabetes insipidus and white matter disease: expanding the spectrum of PRPS1-related disorders

PRPS1 codes for the enzyme phosphoribosyl pyrophosphate synthetase-1 (PRS-1). The spectrum of PRPS1-related disorders associated with reduced activity includes Arts syndrome, Charcot–Marie–Tooth disease-5 (CMTX5) and X-linked non-syndromic sensorineural deafness (DFN2). We describe a novel phenotype associated with decreased PRS-1 function in two affected male siblings. Using whole exome and Sanger sequencing techniques, we identified a novel missense mutation in PRPS1. The clinical phenotype in our patients is characterized by high prenatal maternal α-fetoprotein, intrauterine growth restriction, dysmorphic facial features, severe intellectual disability and spastic quadraparesis. Additional phenotypic features include macular coloboma-like lesions with retinal dystrophy, severe short stature and diabetes insipidus. Exome sequencing of the two affected male siblings identified a shared putative pathogenic mutation c.586C>T p.(Arg196Trp) in the PRPS1 gene that was maternally inherited. Follow-up testing showed normal levels of hypoxanthine in urine samples and uric acid levels in blood serum. The PRS activity was significantly reduced in erythrocytes of the two patients. Nucleotide analysis in erythrocytes revealed abnormally low guanosine triphosphate and guanosine diphosphate. This presentation is the most severe form of PRPS1-deficiency syndrome described to date and expands the spectrum of PRPS1-related disorders.     

The expanding spectrum of PRPS1-associated phenotypes: three novel mutations segregating with X-linked hearing loss and mild peripheral neuropathy

Next-generation sequencing is currently the technology of choice for gene/mutation discovery in genetically-heterogeneous disorders, such as inherited sensorineural hearing loss (HL). Whole-exome sequencing of a single Italian proband affected by non-syndromic HL identified a novel missense variant within the PRPS1 gene ({"type":"entrez-nucleotide","attrs":{"text":"NM_002764.3","term_id":"164663865","term_text":"NM_002764.3"}}NM_002764.3:c.337G>T (p.A113S)) segregating with post-lingual, bilateral, progressive deafness in the proband''s family. Defects in this gene, encoding the phosphoribosyl pyrophosphate synthetase 1 (PRS-I) enzyme, determine either X-linked syndromic conditions associated with hearing impairment (eg, Arts syndrome and Charcot-Marie-Tooth neuropathy type X-5) or non-syndromic HL (DFNX1). A subsequent screening of the entire PRPS1 gene in 16 unrelated probands from X-linked deaf families led to the discovery of two additional missense variants (c.343A>G (p.M115V) and c.925G>T (p.V309F)) segregating with hearing impairment, and associated with mildly-symptomatic peripheral neuropathy. All three variants result in a marked reduction (>60%) of the PRS-I activity in the patients'' erythrocytes, with c.343A>G (p.M115V) and c.925G>T (p.V309F) affecting more severely the enzyme function. Our data significantly expand the current spectrum of pathogenic variants in PRPS1, confirming that they are associated with a continuum disease spectrum, thus stressing the importance of functional studies and detailed clinical investigations for genotype–phenotype correlation.     

Expression of the zebrafish CD133/prominin1 genes in cellular proliferation zones in the embryonic central nervous system and sensory organs

The CD133/prominin1 gene encodes a pentamembrane glycoprotein cell surface marker that is expressed in stem cells from neuroepithelial, hematopoietic, and various organ tissues. Here we report the analysis of two zebrafish CD133/prominin1 orthologues, prominin1a and prominin1b. The expression patterns of the zebrafish prominin1a and b genes were analyzed during embryogenesis using whole mount in situ hybridization. prominin1a and b show novel complementary and overlapping patterns of expression in proliferating zones in the developing sensory organs and central nervous system. The expression patterns suggest functional conservation of the zebrafish prominin1 genes. Initial analyses of prominin1a and b in neoplastic tissue show increased expression of both genes in a subpopulation of cells in malignant peripheral nerve sheath tumors in tp53 mutants. Based on these analyses, the zebrafish prominin1 genes will be useful markers for examining proliferating cell populations in adult organs, tissues, and tumors. Developmental Dynamics 239:1849–1857, 2010. © 2010 Wiley‐Liss, Inc.     

Clinical evaluation of DFN3 patients with deletions in the POU3F4 locus and detection of carrier female using MLPA

Song MH, Lee HK, Choi JY, Kim S, Bok J, Kim U‐K. Clinical evaluation of DFN3 patients with deletions in the POU3F4 locus and detection of carrier female using MLPA. X‐linked deafness type 3 (DFN3), the most prevalent X‐linked form of hereditary deafness, is caused by mutations of the POU3F4 locus in the Xq21 region. We evaluated two Korean families showing typical characteristics of DFN3, such as congenital hearing loss and pathognomonic inner ear anomalies. Genetic analysis of these families did not reveal any mutations in the POU3F4 coding sequence. Instead, one family carried a genomic deletion upstream of POU3F4 gene, where the regulatory element is predicted to reside, and the other family possessed a deletion of almost the entire Xq21 region. The lack of mutation in the POU3F4 coding sequence makes the detection of carrier females using conventional sequencing methods difficult. By applying the multiplex ligation‐dependent probe amplification (MLPA) method, we successfully determined the carrier status of female members in these families, demonstrating that MLPA is a rapid and accurate way to detect POU3F4 deletions in sporadic undiagnosed carriers of DNF3.     

Targeted and Genomewide NGS Data Disqualify Mutations in MYO1A,the “DFNA48 Gene”, as a Cause of Deafness

MYO1A is considered the gene underlying autosomal dominant nonsyndromic hearing loss DFNA48, based on six missense variants, one small in‐frame insertion, and one nonsense mutation. Results from NGS targeting 66 deafness genes in 109 patients identified three families challenging this assumption: two novel nonsense (p.Tyr740* and p.Arg262*) and a known missense variant were identified heterozygously not only in index patients, but also in unaffected relatives. Deafness in these families clearly resulted from mutations in other genes (MYO7A, EYA1, and CIB2). Most of the altogether 10 MYO1A mutations are annotated in dbSNP, and population frequencies (dbSNP, 1000 Genomes, Exome Sequencing Project) above 0.1% contradict pathogenicity under a dominant model. One healthy individual was even homozygous for p.Arg262*, compatible with homozygous Myo1a knockout mice lacking any overt pathology. MYO1A seems dispensable for hearing and overall nonessential. MYO1A adds to the list of “erroneous disease genes”, which will expand with increasing availability of large‐scale sequencing data.     

Update of spectrum c.35delG and c.‐23+1G>A mutations on the GJB2 gene in individuals with autosomal recessive nonsyndromic hearing loss

Hearing loss (HL) is the most common birth defect and the most prevalent sensorineural condition worldwide. It is associated with more than 1,000 mutations in at least 90 genes. Mutations of the gap junction beta‐2 protein (GJB2) gene located in the nonsyndromic hearing loss and deafness (DFNB1) locus (chromosome 13q11‐12) are the main causes of autosomal recessive nonsyndromic hearing loss worldwide, but important differences exist between various populations. In the present article, two common mutations of the GJB2 gene are compared for ethnic‐specific allele frequency, their function, and their contribution to genetic HL in different populations. The results indicated that mutations of the GJB2 gene could have arisen during human migration. Updates on the spectrum of mutations clearly show that frequent mutations in the GJB2 gene are consistent with the founder mutation hypothesis.     

Genomic analysis of childhood hearing loss in the Yoruba population of Nigeria

Although variant alleles of hundreds of genes are associated with sensorineural deafness in children, the genes and alleles involved remain largely unknown in the Sub-Saharan regions of Africa. We ascertained 56 small families mainly of Yoruba ethno-lingual ancestry in or near Ibadan, Nigeria, that had at least one individual with nonsyndromic, severe-to-profound, prelingual-onset, bilateral hearing loss not attributed to nongenetic factors. We performed a combination of exome and Sanger sequencing analyses to evaluate both nuclear and mitochondrial genomes. No biallelic pathogenic variants were identified in GJB2, a common cause of deafness in many populations. Potential causative variants were identified in genes associated with nonsyndromic hearing loss (CIB2, COL11A1, ILDR1, MYO15A, TMPRSS3, and WFS1), nonsyndromic hearing loss or Usher syndrome (CDH23, MYO7A, PCDH15, and USH2A), and other syndromic forms of hearing loss (CHD7, OPA1, and SPTLC1). Several rare mitochondrial variants, including m.1555A>G, were detected in the gene MT-RNR1 but not in control Yoruba samples. Overall, 20 (33%) of 60 independent cases of hearing loss in this cohort of families were associated with likely causal variants in genes reported to underlie deafness in other populations. None of these likely causal variants were present in more than one family, most were detected as compound heterozygotes, and 77% had not been previously associated with hearing loss. These results indicate an unusually high level of genetic heterogeneity of hearing loss in Ibadan, Nigeria and point to challenges for molecular genetic screening, counseling, and early intervention in this population.Subject terms: Genetics research, Medical genomics     

Identification and expression patterns of members of the protease‐activated receptor (par) gene family during zebrafish development

Protease‐activated receptors (PARs) play critical roles in hemostasis in vertebrates including zebrafish. However, the zebrafish gene classification appears to be complex, and the expression patterns of par genes are not established. Based on analyses of genomic organization, phylogenetics, protein primary structure, and protein internalization, we report the identification of four zebrafish PARs: par1, par2a, par2b, and par3. This classification differs from one reported previously. We also show that these genes have distinct spatiotemporal expression profiles in embryos and larvae, with par1, par2a, and par2b expressed maternally and ubiquitously during gastrula stages and their expression patterns refined at later stages, and par3 expressed only in 3‐day‐old larvae. Notably, the expression patterns of zebrafish par1 and par2b resemble those of their mammalian counterparts, suggesting that receptor function is conserved among vertebrates. This conservation is supported by our findings that Par1 and Par2b are internalized following exposure to thrombin and trypsin, respectively. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

Mapping of DFN2 to Xq22

Non-syndromic X-linked deafness is a rare form of genetic deafness accounting for a small proportion of all hereditary hearing loss. It is both clinically and genetically heterogeneous and five loci have been described to date but only two of these have been mapped. DFN2 represents a locus for congenital profound sensorineural hearing loss that has yet to be mapped. We describe a four generation family with this phenotype in which female carriers have a mild/moderate hearing loss affecting the high frequencies. The mutant gene has been mapped to Xq22 using polymorphic microsatellite markers. A maximum two point lod score of 2.91 at theta = 0 was observed with a fully informative dinucleotide repeat at COL4A5, and flanking recombinations were observed at DXS990 and DXS1001.