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.     

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.     

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.     

X-linked mixed deafness (DFN3): cloning and characterization of the critical region allows the identification of novel microdeletions

We have found that the microsatellite marker AFM207zg5 (DXS995)maps to all previously described deletions which are associatedwith X-linked mixed deafness (DFN3) with or without choroideremiaand mental retardation. Employing this marker and pHU16 (DXS26)we have identified two partially overlapping yeast artificialchromosome clones which were used to construct a complete 850kb cosmid contig. Cosmids from this contig have been testedby Southern blot analysis on DNA from 16 unrelated males withX-linked deafness. Two novel microdeletions were detected inpatients which exhibit the characteristic DFN3 phenotype. Bothdeletions are completely contained within one of the known DFN3-deletions,but one of them does not overlap with two previously describeddeletions in patients with contiguous gene syndromes consistingof DFN3, chorolderemia, and mental retardation. Assuming thatonly a single gene is involved, this suggests that the DFN3gene spans a chromosomal region of at least 400 kb.     

PLP1 duplication at the breakpoint regions of an apparently balanced t(X;22) translocation causes Pelizaeus–Merzbacher disease in a girl

Fonseca ACS, Bonaldi A, Costa SS, Freitas MR, Kok F, Vianna‐Morgante AM. PLP1 duplication at the breakpoint regions of an apparently balanced t(X;22) translocation causes Pelizaeus–Merzbacher disease in a girl. PLP1 (proteolipid protein1 gene) mutations cause Pelizaeus–Merzbacher disease (PMD), characterized by hypomyelination of the central nervous system, and affecting almost exclusively males. We report on a girl with classical PMD who carries an apparently balanced translocation t(X;22)(q22;q13). By applying array‐based comparative genomic hybridization (a‐CGH), we detected duplications at 22q13 and Xq22, encompassing 487–546 kb and 543–611 kb, respectively. The additional copies were mapped by fluorescent in situ hybridization to the breakpoint regions, on the derivative X chromosome (22q13 duplicated segment) and on the derivative 22 chromosome (Xq22 duplicated segment). One of the 14 duplicated X‐chromosome genes was PLP1.The normal X chromosome was the inactive one in the majority of peripheral blood leukocytes, a pattern of inactivation that makes cells functionally balanced for the translocated segments. However, a copy of the PLP1 gene on the derivative chromosome 22, in addition to those on the X and der(X) chromosomes, resulted in two active copies of the gene, irrespective of the X‐inactivation pattern, thus causing PMD. This t(X;22) is the first constitutional human apparently balanced translocation with duplications from both involved chromosomes detected at the breakpoint regions.     

Investigation of the factor VIII intron 22 repeated region (int22h) and the associated inversion junctions

A region of intron 22 of the factor VIII gene, which containsfactor VIII-associated gene A (F8A), is repeated twice morenearer the Xq telomere. It has been proposed that intrachromosomalhomologous recombination occurs between the intron 22 repeatand either of the two extragenic copies, resulting in the recurrentinversions that cause almost half of all cases of severe haemophlliaA. We have precisely defined the repeated region as 9.5 kb ofDNA which we have termed int22h(intron 22 homologous region).The junctions of the inversions examined were shown to representprecise exchanges between the int22h repeats, thus providingconclusive evidence for homologous recombination. The threecopies of int22h were compared along 8 kb of their length, usingchemical mismatch analysis, and found to be 99.9% similar. Thepresence of such long, almost identical inverted repeats nearthe Xq telomere could account for the high frequency at whichthe inversions occur.     

Cloning and characterization of a murine brain specific gene Bpx and its human homologue lying within the Xic candidate region

The X inactivation centre (Xic) is a cis-acting locus thought to play a key role in the initiation of X-inactivation. We have cloned and characterized a new gene, Bpx, lying distal to the murine Xist. Bpx, which is specifically expressed in the brain, shows strong homology to genes encoding nucleosome assembly proteins and is normally X- inactivated in mice. Isolation and localization of BPX, its human homologue, has shown the gene to be located centromeric to XIST in man. The Xq13 region, whose orientation is apparently globally conserved between man and mouse, must therefore contain an inversion of at least 600 kb spanning the XIST sequence and including the CDX4 and BPX genes.      

Physical mapping of the Tec and Gabrb1 loci reveals that the Wsh mutation on mouse chromosome 5 is associated with an inversion

In the mouse, mutations in the c-Kit proto-oncogene, a memberof the receptor tyrosine kinase (RTK) gene family, have pleiotropiceffects on hematopoiesis, pigmentation and fertility (dominantspotting, W). However, in the W^sh allele the defect is confinedto abnormal pigmentation caused by the disruption of 5' regulatorysequences of Kit leaving an intact structural gene. In thisreport, the previously published physical map around the Pdgfra-Kit-Flk1RTK loci is extended by mapping the loci encoding the GABA_A(-aminobutyric acid) receptor subunit beta 1, Gabrb1 and a cytoplasmickinase (Tec) 3 Mb proximal to Kit. PFGE analysis of the wild-type(C57BL/6J) chromosome demonstrates the following gene order:cen-Gabrb1-Tec-Pdgfra-Kit, whereas the analysis of W^sh/W^sh DNAis consistent with the order: cen-Gabrb1-Pdgfra-Tec-Kit. Thisaltered physical map can be explained by an inversion on theW^sh chromosome located proximally to the Kit locus and spanningthe 2.8 Mb Pdgfra-Tec chromosomal segment. This high resolutionphysical mapping study identifies large DNA fragments that spanthe two inversion breakpoints and potentially carry Kit upstreamregulatory elements involved in the control of Kit expressionduring embryonic development.     

Localization of human phosphoribosylpyrophosphate synthetase subunit I and II genes (PRPS1 and PRPS2) to different regions of the X chromosome and assignment of two PRPS1-related genes to autosomes

Complementary DNA clones for phosphoribosylpyrophosphate synthetase subunits I and II (PRS I and PRS II) were used to determine the chromosomal localization of the corresponding human genes. Southern blot analysis of genomic DNAs isolated from human placenta and a panel of humanmouse somatic cell hybrids revealed that the rat PRS I cDNA probe detected at least five human specific DNA segments (23, 20, 14.5, 6.7, and 4.3 kb) in BamHI digests. The 23-, 14.5-, and 6.7-kb DNA segments were detected only if the hybrids contained human chromosome X or translocation chromosome 7p ⁺ (7qter>7p22::Xq21>Xqter), indicating the location of these segments to Xq21-qter (PRPS1). The 20- and 4.3-kb DNA segments did not cosegregate with the other three segments, and spot blot hybridization analysis using flow-sorted human chromosomes indicated that these are the PRPS1-related genes (PRPS1L1 and PRPS1L2) and could be assigned to chromosomes 7 and 9, respectively. The human-specific PRS II cDNA probe revealed a BamHI DNA segment (17 kb), which segregated condordantly with the X chromosome but not with the PRPS1 gene. We surmise that the gene for PRS II (PRPS2) is located at a different region of the X chromosome, namely Xpter-q21.Preliminary report of this research was presented at Ninth International Workshop on Human Gene Mapping, Abstract supplement p. 5 (1987).     

An Xq22.3 duplication detected by comparative genomic hybridization microarray (Array-CGH) defines a new locus (FGS5) for FG syndrome

FG syndrome is an X-linked multiple congenital anomalies (MCA) syndrome. It has been mapped to four distinct loci FGS1-4, through linkage analysis (Xq13, Xp22.3, and Xp11.4-p11.3) and based on the breakpoints of an X chromosome inversion (Xq11:Xq28), but so far no gene has been identified. We describe a boy with FG syndrome who has an inherited duplication at band Xq22.3 detected by comparative genomic hybridization microarray (Array-CGH). These duplication maps outside all four loci described so far for FG syndrome, representing therefore a new locus, which we propose to be called FGS5. MID2, a gene closely related to MID1, which is known to be mutated in Opitz G/BBB syndrome, maps within the duplicated segment of our patient. Since FG and Opitz G/BBB syndromes share many manifestations we considered MID2 a candidate gene for FG syndrome. We also discuss the involvement of other potential genes within the duplicated segment and its relationship with clinical symptoms of our patient, as well as the laboratory abnormalities found in his mother, a carrier of the duplication.     

Opposite deletions/duplications of the X chromosome: two novel reciprocal rearrangements

Paralogous sequences on the same chromosome allow refolding of the chromosome into itself and homologous recombination. Recombinant chromosomes have microscopic or submicroscopic rearrangements according to the distance between repeats. Examples are the submicroscopic inversions of factor VIII, of the IDS gene and of the FLN1/emerin region, all resulting from misalignment of inverted repeats, and double recombination. Most of these inversions are of paternal origin possibly because the X chromosome at male meiosis is free to refold into itself for most of its length. We report on two de novo rearrangements of the X chromosome found in four hypogonadic females. Two of them had an X chromosome deleted for most of Xp and duplicated for a portion of Xq and two had the opposite rearrangement (class I and class II rearrangements, respectively). The breakpoints were defined at the level of contiguous YACs. The same Xp 11.23 breakpoint was found in the four cases. That of the long arm coincided in three cases (Xq21.3) and was more proximal in case 4 (Xq21.1). Thus class I rearrangements (cases 1 and 2) are reciprocal to that of case 3, whilst that of case 4 shares only the Xp breakpoint. The abnormal X was paternal in the three cases investigated. Repeated inverted sequences located at the breakpoints of rearrangements are likely to favour the refolding of the paternal X chromosome and the recombination of the repeats. The repeat at the Xp11 may synapse with either that at Xq21.3 or that at Xq21.1. These rearrangements seem to originate as the Xq28 submicroscopic inversions but they are identifiable at the microscopic level and result from a single recombination event.     

Sandwiching of a gene within 12 kb of a functional telomere and alpha satellite does not result in silencing

Pericentrlc heterochromatin and telomeres have been shown tobe capable of repressing the expression of genes located Inclose proximity. The effect of adjacent structural sequenceson gene expression will be important in the design of mammalianartificial chromosomes. In the process of using telomere-containlngconstructs to generate a deletion panel of the long arm of thehuman X chromosome, several cell lines were produced which appearedby in situ hybridization to be broken In Xq at or near the centromere.After analysis of end clones rescued from these cell lines,only two produced data consistent with breaks In the alpha satellitearray without accompanying rearrangements. The mitotic stabilityof an X chromosome, with at least 750 kb of the alpha satellitearray deleted, was compared to controls where the alpha satellitearray remained Intact. No significant change In the stabilityof the chromosome was observed, suggesting that the truncatedchromosome has a fully functional mitotic centromere. Therewas no detectable change in the expression of the hygromycinresistance gene, which is located between a functional centromereand telomere, In this cell line. This study indicates that structuralelements flanking a mammalian selectable marker do not resultin silencing.     

Inversion of the IDS gene resulting from recombination with IDS-related sequences in a common cause of the Hunter syndrome

We have recently described the identification of a second IDSlocus (IDS-s) located within 90 kb telomeric of the IDS gene(Bondeson et al. submitted). here, we show that this regionis involved in a recombination with the IDS gene in about 13%of patients with the Hunter syndrome. Analysis of the resultingaearrangement at the molecular level should that these patientshave suffered a recombination event that results in a disruptionof the IDS gene is intron 7 with an inversion of the interveningDNA. Inerestingly, all of the six cases with a similar typeof rearrangement showed recombination between intron 7 of theIDS gene and sequences close to exon 3 at the IDS-2 locus implyingthat these regions are hot spots for recombination. Analysisby uncleotide sequencing should that the inversion is causedby recombination between homologous sequences present in theIDS gene and the IDS-2 locus. No detectable deletions or insertionswere observed as a result of the recombination event. The resultsin this study have practical implications for diagnosis of theHunter syndrome.     

Molecular analysis of the t(8;14)(q24;q11) chromosomal breakpoint junctions in the T-cell leukemia line MOLT-16

The MOLT-16 cell line was established from the leukemic cells of a patient with T-cell acute lymphoblastic leukemia and contains a t(8;14)(q24;q11) resulting in juxtaposition of sequences downstream of the MYC gene on chromosome 8 and the J region of the T-cell receptor alpha chain gene (TCRA) on chromosome 14. The reciprocal translocation involved a complex rearrangement with two chromosome breakpoints within the TCRA J region on chromosome 14, resulting in inversion of a 1.4 kb DNA fragment between the two breakpoints. The 5′ border of the inversion joins with another segment of chromosome 14, whereas the 3′ border joins with a region of chromosome 8 located at least 257 kb downstream of MYC. Extensive deletions have occurred on both chromosomes 8 and 14 in conjunction with the translocation. To investigate the possible involvement of the V(D)J recombinase in this translocation, we analyzed the nucleotide sequences surrounding the translocation breakpoints. The breakpoint on chromosome 14 occurs between a segment coding for a TCRA J sequence and its heptamer-nonamer signal. Heptamer-nonamer consensus sequences are also identified on chromosome 8 adjacent to the breakpoint. Inserted N and P nucleotides are observed at the breakpoint junctions. Genes Chromosomes Cancer 20:363–371, 1997. © 1997 Wiley-Liss, Inc.     

Brachymesomelic dysplasia with Peters anomaly of the eye results from disruptions of the X chromosome near the SHOX and SOX3 genes

We report on a mother and son affected with an unusual skeletal dysplasia and anterior segment eye abnormalities. Their skeletal phenotype overlaps with the SHOX-related skeletal dysplasias and is intermediate between Leri-Weill dyschondrosteosis (LWD) and Langer Mesomelic dysplasia (LMD). The mother has bilateral Peters anomaly of the eye and was reported as having a new syndrome; the son had severe bilateral sclerocornea. Chromosome analysis showed that the mother has a pericentric inversion of the X chromosome [46,X,inv(X)(p22.3q27)] and the son, a resultant recombinant X chromosome [46,Y,rec(X)dup(Xq)inv(X)(p22.3q27)]. The observed skeletal and ophthalmologic abnormalities in both patients were similar in severity. The additional features of developmental delay, growth retardation, agenesis of the corpus callosum, cryptorchidism and hypoplastic scrotum in the son are consistent with Xq28 duplication. Analysis of the son's recombinant X chromosome showed that the Xp22.33 breakpoint lies 30-68 kb 5' of the SHOX gene. This finding suggests that the skeletal dysplasia in both mother and son is allelic with LWD and LMD and results from a novel misexpression of SHOX. Analysis of the Xq27.1 breakpoint localized it to a 90 kb interval 3' of the SOX3 gene, supporting a novel role of SOX3 misexpression in the development of Peters anomaly of the eye.