Female Hunter syndrome caused by a single mutation and familial XCI skewing: implications for other X-linked disorders

Familial X-chromosome inactivation (XCI) skewing was investigated in a family in which a female mucopolysaccharidosis type II (MPS II) (Hunter syndrome, an X-linked genetic disease) occurred. Among eight related females aged under 60 years from three generations who were tested, four revealed a non-random pattern of XCI. Detailed genetic analysis failed to find mutations in genes that were previously reported as important for the XCI process. Haplotype analysis excluded linkage of non-random XCI with genes localized on the X-chromosome. We propose that analysis of the XCI pattern should be taken into consideration when assessing risk factors for X-linked recessive genetic disorders.     

A non-ancestral RPGR missense mutation in families with either recessive or semi-dominant X-linked retinitis pigmentosa

Most X-linked diseases show a recessive pattern of inheritance in which female carriers are unaffected. In X-linked retinitis pigmentosa (XLRP), however, both recessive and semi-dominant inheritance patterns have been reported. We identified an Israeli family with semi-dominant XLRP due to a missense mutation (p.G275S) in the RPGR gene. The mutation was previously reported in two Danish families with recessive XLRP. Obligate carriers from the two Danish families had no visual complaints and normal to slightly reduced retinal function, while those from the Israeli family suffered from high myopia, low visual acuity, constricted visual fields, and severely reduced electroretinogram (ERG) amplitudes. The disease-related RPGR haplotype of the Israeli family was found to be different from the one found in the two Danish families, indicating that the mutation arose twice independently on different X-chromosome backgrounds. A series of genetic analyses excluded skewed X-inactivation pattern, chromosomal abnormalities, distorted RPGR expression level, and mutations in candidate genes as the cause for the differences in disease severity of female carriers. To the best of our knowledge, this is the first detailed analysis of an identical mutation causing either a recessive or a semi-dominant X-linked pattern of disease in different families. Our results indicate that an additional gene (or genes), linked to RPGR, modulate disease expression in severely affected carriers. These may be related to the high myopia concomitantly found in affected carriers from the Israeli family.     

Combined De-Novo Mutation and Non-Random X-Chromosome Inactivation Causing Wiskott-Aldrich Syndrome in a Female with Thrombocytopenia

Objective

Disorders linked to mutations in the X chromosomes typically affect males. The aim of the study is to decipher the mechanism of disease expression in a female patient with a heterozygous mutation on the X-chromosome.

Patients and Methods

Clinical data was extracted from the Canadian Inherited Marrow Failure Registry. Genomic ribonucleic acid (DNA) and complementary DNA (cDNA) underwent Sanger sequencing. Protein analysis was performed by flow cytometry. X-inactivation patterns were analyzed by evaluating the DNA methylation status and cDNA clonal expression of several genes on the X-chromosome. SNP array was used for molecular karyotyping of the X-chromosome.

Results

A female with thrombocytopenia, eczema and mild T-lymphocyte abnormalities with extensive negative diagnostic testing, was suspected to have Wiskott-Aldrich syndrome (WAS)/X-linked thrombocytopenia. Although the girl had a mutation (c.397G?>?A, p.E133K) in only one allele, she was found to have an extremely skewed X-inactivation pattern and no expression of the WAS protein. Family studies using DNA methylation analysis and cDNA clonal expression of several genes on the X-chromosome demonstrated that the patient developed de-novo non-random inactivation of the X-chromosome that does not carry the mutation. Genome-wide high-density molecular karyotyping excluded deletions and amplifications as a cause for the non-random inactivation of one X-chromosome.

Conclusions

Our study emphasizes the need to test selected female patients with complete or incomplete disease expression for X-linked disorders even in the absence of a family history.     

Two novel mutations identified in the Wiskott-Aldrich syndrome protein gene cause Wiskott-Aldrich syndrome and thrombocytopenia

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are rare X-linked genetic disorders caused by mutations of the Wiskott-Aldrich syndrome protein (WASP) gene. Both disorders are clinically characterized by chronic thrombocytopenia of small platelets. WAS is a more severe form of the disorder and also courses with eczema, and immune dysfunction. In the present study, we investigated two novel mutations of the WASP gene in two Spanish families with patients clinically diagnosed as having XLT and WAS, respectively. In one of the families a missense mutation in exon 12 (1488A>G), resulting in the highly conserved glutamic residue changing to glycine at position 485 (D485G), was identified in several members. Notably, a female of this family, with clinical signs of XLT, was determined as the carrier of the mutation and showed a skewed pattern of X-inactivation, preferentially inactivating the X-chromosome carrying the wild-type allele. In the case of the second family, we describe a WAS patient with a single base deletion in exon 2 (266-267delA), resulting in a frameshift (at codon 78) that creates a stop codon at amino acid 127. As a consequence, there was no WASP expression.     

Börjeson-Forssman-Lehmann syndrome in a woman with skewed X-chromosome inactivation

B?rjeson-Forssman-Lehmann (BFL) syndrome is an X-linked recessive disorder characterized by minor facial anomalies, obesity, epilepsy, and severe mental retardation. The phenotype of male patients is usually severe, whereas that of carriers is less severe, suggesting X-linked incompletely recessive inheritance. A recent linkage study mapped the BFL syndrome gene to Xq26-q27. The etiology of the condition in female patients with full manifestations is not known, although nonrandom X-chromosome inactivation has been considered. We recently developed an assay for X-inactivation studies based on the methylation-specific polymerase chain reaction (PCR) technique. Using the methylation-specific PCR assay, a woman with typical findings of this syndrome was shown to have an extremely skewed X-inactivation pattern. This finding suggests that the full manifestations of the BFL syndrome in carriers may be caused by skewed X inactivation with a high proportion of cells in which the X chromosome with a normal gene be inactivated, leaving the X chromosome with a mutant gene active.     

Familial Simpson-Golabi-Behmel syndrome: studies of X-chromosome inactivation and clinical phenotypes in two female individuals with GPC3 mutations

Simpson-Golabi-Behmel syndrome (SGBS) is an overgrowth/multiple congenital anomalies syndrome with an X-linked inheritance. Most cases of SGBS are attributed to mutations in the glypican 3-gene (GPC3), which is highly expressed in the mesodermal embryonic tissues and involves in a local growth regulation. Typical clinical features include pre/postnatal overgrowth, developmental delay, macrocephaly, characteristic facies with prominent eyes and macroglossia, diaphragmatic hernia, congenital heart defects, kidney anomalies, and skeletal anomalies. Obligate carrier females with GPC3 mutations are usually asymptomatic or with mild symptoms. It is thought that skewed X-inactivation is the underlining mechanism for the female patients to present with findings of SGBS. We identified three siblings with typical SGBS (two male and one female cases) and their mother with very mild symptoms in a family carrying c.256C>T (p.Arg86X) mutation in GPC3. X-inactivation studies on the androgen-receptor gene (AR) and the Fragile XE (FRAXE) gene were performed with blood, buccal swabs, and fibroblasts in the carrier females. The studies with blood showed moderately skewed X-inactivation with paternal X-chromosome being preferentially inactivated (71-80% inactivated) in the female patient with SGBS and no skewing was shown in the mother with very mild symptoms. The X-inactivation studies in the mother showed inactivation of the X-chromosome with the mutation by 57%. This suggests that loss of the functional GPC3 protein by 43% is closed to the threshold to develop the SGBS phenotype. Studies with buccal swabs and fibroblasts failed to show different X-inactivation patterns between the two female individuals.     

Reduced proportion of Purkinje cells expressing paternally derived mutant Mecp2308 allele in female mouse cerebellum is not due to a skewed primary pattern of X-chromosome inactivation

Rett syndrome (RTT) is an X-linked disorder caused by mutations in the methyl CpG binding protein 2 (MECP2) gene. The pattern of X-chromosome inactivation (XCI) is thought to play a role in phenotypic severity. In the present study, patterns of XCI were assessed by lacZ staining of embryos and adult brains of mice heterozygous for a X-linked Hmgcr-nls-lacZ transgene on a mutant mouse model of RTT. We found that there was no difference between the lacZ staining patterns in the brain of wild-type and heterozygous mutant embryos at embryonic day 9.5 (E9.5) suggesting that Mecp2 has no effect on the primary pattern of XCI. At 20 weeks of age, there was no significant difference between XCI patterns in the Purkinje cells in the cerebellum of heterozygous mutant and wild-type mice when the mutant allele was inherited from the mother. However, when the mutant allele was paternally inherited, a significant difference was detected. Thus, parental origin of the mutation may have a bearing on phenotype through XCI patterns. An estimation of the Purkinje cell precursor number based on XCI mosaicism revealed that, when the mutation was paternally inherited, the precursor number was less than that in the wild-type mice. Therefore, it is likely that the number of precursor cells allocated to the Purkinje cell lineage is affected by a paternally inherited mutation in Mecp2. We also observed that the pattern of XCI in cultured fibroblasts was significantly correlated with patterns in the Purkinje cells in mutant animals but not in wild-type mice.     

Structural and numerical changes of chromosome X in patients with esophageal atresia

Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) is a relatively common birth defect often associated with additional congenital anomalies such as vertebral, anal, cardiovascular, renal and limb defects, the so-called VACTERL association. Yet, little is known about the causal genetic factors. Rare case reports of gastrointestinal anomalies in children with triple X syndrome prompted us to survey the incidence of structural and numerical changes of chromosome X in patients with EA/TEF. All available (n=269) karyotypes of our large (321) EA/TEF patient cohort were evaluated for X-chromosome anomalies. If sufficient DNA material was available, we determined genome-wide copy number profiles with SNP array and identified subtelomeric aberrations on the difficult to profile PAR1 region using telomere-multiplex ligation-dependent probe amplification. In addition, we investigated X-chromosome inactivation (XCI) patterns and mode of inheritance of detected aberrations in selected patients. Three EA/TEF patients had an additional maternally inherited X chromosome. These three female patients had normal random XCI patterns. Two male EA/TEF patients had small inherited duplications of the XY-linked SHOX (Short stature HOmeoboX-containing) locus. Patients were small for gestational age at birth (<P5) and had additional, mostly VACTERL associated, anomalies. Triple X syndrome is rarely described in patients with EA/TEF and no duplications of the SHOX gene were reported so far in these patients. As normal patterns of XCI were seen, overexpression of X-linked genes that escape XCI, such as the SHOX gene, could be pathogenic by disturbing developmental pathways.     

Carrier detection of the X-linked primary immunodeficiency diseases using X-chromosome inactivation analysis

Carrier detection of three of the X-linked primary immunodeficiency diseases (X-linked agammaglobulinemia, X-linked severe combined immunodeficiency disease, and the Wiskott-Aldrich syndrome) is possible by analyzing patterns of X-chromosome inactivation in those cells affected by the disorder. Normal women have balanced patterns of X-chromosome inactivation; that is, in a given population of cells, approximately half of their active X chromosomes are of paternal origin and half of their active X chromosomes are of maternal origin. In contrast, female carriers of these X-linked immunodeficiency disorders have an unbalanced pattern of X-chromosome inactivation in those cell lineages that are affected by the disorder; that is, all the active X chromosomes in affected cell lineages are the X chromosomes that carry the normal allele. Two techniques are available for X-chromosome inactivation analysis. One technique depends on methylation differences between the active and inactive X chromosome, and the other technique uses somatic cell hybrids that selectively retain the active X chromosome. In either case, carrier detection can be performed in individuals from families in which only one member of the family has been affected, since neither of these methods depends on linkage analysis.     

Non-skewed X-inactivation may cause mental retardation in a female carrier of X-linked alpha-thalassemia/mental retardation syndrome (ATR-X): X-inactivation study of nine female carriers of ATR-X

X-linked alpha-thalassemia/mental retardation syndrome (ATR-X) is a syndromic form of X-linked mental retardation. We investigated the X-inactivation status of nine female ATR-X carriers by methylation-specific PCR of the HUMARA gene. Six carriers demonstrated a skewed X-inactivation pattern (>90:10) and one showed a non-skewed pattern (72:28), while two were uninformative because of homozygosity for the CAG repeat polymorphic alleles in the HUMARA. Only the carrier mother who showed non-skewed X-inactivation had moderate mental retardation. These findings suggest that mutations in ATRX may cause mental retardation in females, if the X chromosome carrying mutated ATRX is not properly inactivated.     

Expanding the molecular basis and phenotypic spectrum of X-linked Joubert syndrome associated with OFD1 mutations

Using a combination of linkage mapping and massively parallel sequencing of the X-chromosome exome, we identified an 18-bp deletion in exon 8 of the oral-facial-digital syndrome type 1 (OFD1) gene in a family with X-linked Joubert syndrome (JBTS10). The deletion results in an in-frame deletion of six amino acids. New features not noted in the two previously reported cases of X-linked Joubert syndrome include the presence of polycystic kidney disease, polymicrogyria and hydrocephalus. Our study further underlines the power of genetic mapping combined with massively parallel sequencing as a powerful tool for novel disease gene and mutation discovery.     

Father-to-daughter transmission of focal dermal hypoplasia associated with nonrandom X-inactivation: support for X-linked inheritance and paternal X chromosome mosaicism

Focal dermal hypoplasia (FDH) is a rare syndrome of severe developmental anomalies of the tissues and organs derived from ectoderm and mesoderm. Though data have suggested that FDH is an X-linked dominant trait associated with male hemizygote lethality, a hypothesis supported by the observation of three unrelated infants with FDH manifestations and de novo chromosome rearrangements involving Xp22, observations of father-to-daughter transmission have suggested possible genetic heterogeneity and autosomal dominant inheritance with sex limitation. We hypothesize that, if FDH is an X-linked disorder, cells expressing an active disease locus might experience a selective disadvantage resulting in a nonrandom pattern of X-inactivation in patient tissue. To test this hypothesis, we studied one of the two previously described families demonstrating father-to-daughter inheritance of FDH. To determine if the affected daughter had a skewed pattern of X-inactivation consistent with X-linked inheritance of FDH, somatic cell hybrids were constructed by fusing hypoxanthine phosphoribosyl transferase (HPRT)-deficient rodent fibroblasts with either patient dermal fibroblasts or peripheral white blood cells (WBCs); hybrid clones retaining an active X chromosome were analyzed to determine the parental origin of the active X chromosome. Analyses of resulting hybrid clones showed that while hybrids constructed from skin fibroblasts contained an active X chromosome inherited from either of the patient's parents, hybrids constructed from WBCs showed a skewed pattern of X-inactivation; 11 of 11 hybrids contained an active maternal X chromosome (chi 2 = 12.2, P = .001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Birth of healthy female twins after preimplantation genetic diagnosis of cystic fibrosis combined with gender determination

Two healthy sisters with a familial history of mental retardation were referred to our centre for preimplantation genetic diagnosis (PGD). Their two brothers showed severe mental retardation. The molecular basis for their disorder could not be identified, but one of the sisters and the mother presented a highly skewed pattern of X-inactivation reinforcing the likelihood of an X-linked mode of inheritance. Both sisters requested PGD to avoid the abortion of potentially affected male fetuses. PGD for sex by fluorescent in-situ hybridization was carried out for the first sister and resulted in the birth of a female child. The second sister and her partner, whose niece had cystic fibrosis (CF), were tested for CF mutations, and were both found to be deltaF508 heterozygous. We developed an efficient single cell PCR protocol for the simultaneous amplification of the CF (deltadeltaF508) locus as well as the X-linked amelogenin gene and its highly homologous pseudogene on the Y chromosome. Two PGD cycles were carried out to screen against male and deltaF508 homozygous deleted embryos. In each case several embryos could be selected for transfer and the second cycle resulted in a twin pregnancy followed by the birth of two healthy female infants.     

DNA methylation profiles of human active and inactive X chromosomes

X-chromosome inactivation (XCI) is a dosage compensation mechanism that silences the majority of genes on one X chromosome in each female cell. To characterize epigenetic changes that accompany this process, we measured DNA methylation levels in 45,X patients carrying a single active X chromosome (X(a)), and in normal females, who carry one X(a) and one inactive X (X(i)). Methylated DNA was immunoprecipitated and hybridized to high-density oligonucleotide arrays covering the X chromosome, generating epigenetic profiles of active and inactive X chromosomes. We observed that XCI is accompanied by changes in DNA methylation specifically at CpG islands (CGIs). While the majority of CGIs show increased methylation levels on the X(i), XCI actually results in significant reductions in methylation at 7% of CGIs. Both intra- and inter-genic CGIs undergo epigenetic modification, with the biggest increase in methylation occurring at the promoters of genes silenced by XCI. In contrast, genes escaping XCI generally have low levels of promoter methylation, while genes that show inter-individual variation in silencing show intermediate increases in methylation. Thus, promoter methylation and susceptibility to XCI are correlated. We also observed a global correlation between CGI methylation and the evolutionary age of X-chromosome strata, and that genes escaping XCI show increased methylation within gene bodies. We used our epigenetic map to predict 26 novel genes escaping XCI, and searched for parent-of-origin-specific methylation differences, but found no evidence to support imprinting on the human X chromosome. Our study provides a detailed analysis of the epigenetic profile of active and inactive X chromosomes.     

X-chromosome inactivation patterns in monozygotic twins and sib pairs discordant for nonsyndromic cleft lip and/or palate

Nonsyndromic clefts of the lip and/or palate are common birth defects with a strong genetic component. Based on unequal gender ratios for clefting phenotypes, evidence for linkage to the X chromosome and the occurrence of several X-linked clefting syndromes, we investigated the role of skewed X chromosome inactivation (XCI) in orofacial clefts. Our samples consisted of female monozygotic (MZ) twins (n = 8) and sister pairs (n = 152) discordant for nonsyndromic clefting. We measured the XCI pattern in peripheral blood lymphocyte DNA using a methylation based androgen receptor gene assay. Skewing of XCI was defined as the deviation in inactivation pattern from a 50:50 ratio. Our analysis revealed no significant difference in the degree of skewing between twin pairs (P = 0.3). However, borderline significant differences were observed in the sister pairs (P = 0.02), with the cleft lip with cleft palate group showing the most significant result (P = 0.01). We did not find evidence for involvement of skewed XCI in the discordance for clefting in our sample of female MZ twins. However, results from the paired sister study suggest the potential contribution of skewed XCI to orofacial clefting, particularly cleft lip and palate.