Survival of Male Patients with Incontinentia Pigmenti Carrying a Lethal Mutation Can Be Explained by Somatic Mosaicism or Klinefelter Syndrome

Incontinentia pigmenti (IP), or "Bloch-Sulzberger syndrome," is an X-linked dominant disorder characterized by abnormalities of skin, teeth, hair, and eyes; skewed X-inactivation; and recurrent miscarriages of male fetuses. IP results from mutations in the gene for NF-kappaB essential modulator (NEMO), with deletion of exons 4-10 of NEMO accounting for >80% of new mutations. Male fetuses inheriting this mutation and other "null" mutations of NEMO usually die in utero. Less deleterious mutations can result in survival of males subjects, but with ectodermal dysplasia and immunodeficiency. Male patients with skin, dental, and ocular abnormalities typical of those seen in female patients with IP (without immunodeficiency) are rare. We investigated four male patients with clinical hallmarks of IP. All four were found to carry the deletion normally associated with male lethality in utero. Survival in one patient is explained by a 47,XXY karyotype and skewed X inactivation. Three other patients possess a normal 46,XY karyotype. We demonstrate that these patients have both wild-type and deleted copies of the NEMO gene and are therefore mosaic for the common mutation. Therefore, the repeat-mediated rearrangement leading to the common deletion does not require meiotic division. Hypomorphic alleles, a 47,XXY karyotype, and somatic mosaicism therefore represent three mechanisms for survival of males carrying a NEMO mutation.     

A new mutation in exon 7 of NEMO gene: late skewed X-chromosome inactivation in an incontinentia pigmenti female patient with immunodeficiency

Incontinentia pigmenti is an X-linked genodermatosis, lethal in males. Affected females survive because of X-chromosome dizygosity and negative selection of cells carrying the mutant X-chromosome, and for this reason the skewed X inactivation pattern is often used to confirm the diagnosis. The most frequent mutation is a deletion of part of the NEMO gene (NEMOΔ4–10), although other mutations have been reported. Mutations of NEMO which do not abolish NF-κB activity totally permit male survival, causing an allelic variant of IP called hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). We present a non-classical IP female patient who also suffered transient immunodeficiency because of a late and progressive selection against peripheral blood cells carrying an active mutated X-chromosome. This finding suggests that in the absence of known mutation the X-inactivation studies used in genetic counselling can induce mistakes with some female patients. At the age of 3 years and 6 months, all immunodeficiency signs disappeared, and the X-chromosome inactivation pattern was completely skewed. The low T cell proliferation and CD40L expression corroborate the important role of NEMO/ NF-κB pathway in T cell homeostasis. The decreased NEMO protein amount and the impaired IkBα degradation suggest that this new mutation, NM_003639: c.1049dupA, causes RNA or protein instability. To our knowledge, this is the first time that selection against the mutated X-chromosome in X-linked disease has been documented in vivo.     

Skewed X-chromosome inactivation is a common feature of X-linked mental retardation disorders

Some deleterious X-linked mutations may result in a growth disadvantage for those cells in which the mutation, when on the active X chromosome, affects cell proliferation or viability. To explore the relationship between skewed X-chromosome inactivation and X-linked mental retardation (XLMR) disorders, we used the androgen receptor X-inactivation assay to determine X-inactivation patterns in 155 female subjects from 24 families segregating 20 distinct XLMR disorders. Among XLMR carriers, ~50% demonstrate markedly skewed X inactivation (i.e., patterns 80:20), compared with only ~10% of female control subjects (P<.001). Thus, skewed X inactivation is a relatively common feature of XLMR disorders. Of the 20 distinct XLMR disorders, 4 demonstrate a strong association with skewed X inactivation, since all carriers of these mutations demonstrate X-inactivation patterns 80:20. The XLMR mutations are present on the preferentially inactive X chromosome in all 20 informative female subjects from these families, indicating that skewing is due to selection against those cells in which the XLMR mutation is on the active X chromosome.     

Evidence against an X-linked visual loss susceptibility locus in Leber hereditary optic neuropathy.

Pedigree analysis of British families with Leber hereditary optic neuropathy (LHON) closely fits a model in which a pathogenic mtDNA mutation interacts with an X-linked visual loss susceptibility locus (VLSL). This model predicts that 60% of affected females will show marked skewing of X inactivation. Linkage analysis in British and Italian families with genetically proven LHON has excluded the presence of such a VLSL over 169 cM of the X chromosome both when all families were analyzed together and when only families with the bp 11778 mutation were studied. Further, there was no excess skewing of X inactivation in affected females. There was no evidence for close linkage to three markers in the pseudoautosomal region of the sex chromosomes. The mechanism of incomplete penetrance and male predominance in LHON remains unclear.     

X chromosome choice occurs independently of asynchronous replication timing

In mammals, dosage compensation is achieved by X chromosome inactivation in female cells. Xist is required and sufficient for X inactivation, and Xist gene deletions result in completely skewed X inactivation. In this work, we analyzed skewing of X inactivation in mice with an Xist deletion encompassing sequence 5 KB upstream of the promoter through exon 3. We found that this mutation results in primary nonrandom X inactivation in which the wild-type X chromosome is always chosen for inactivation. To understand the molecular mechanisms that affect choice, we analyzed the role of replication timing in X inactivation choice. We found that the two Xist alleles and all regions tested on the X chromosome replicate asynchronously before the start of X inactivation. However, analysis of replication timing in cell lines with skewed X inactivation showed no preference for one of the two Xist alleles to replicate early in S-phase before the onset of X inactivation, indicating that asynchronous replication timing does not play a role in skewing of X inactivation.     

NEMO/IKK gamma-deficient mice model incontinentia pigmenti

Disruption of the X-linked gene encoding NF-kappa B essential modulator (NEMO) produces male embryonic lethality, completely blocks NF-kappa B activation by proinflammatory cytokines, and interferes with the generation and/or persistence of lymphocytes. Heterozygous female mice develop patchy skin lesions with massive granulocyte infiltration and hyperproliferation and increased apoptosis of keratinocytes. Diseased animals present severe growth retardation and early mortality. Surviving mice recover almost completely, presumably through clearing the skin of NEMO-deficient keratinocytes. Male lethality and strikingly similar skin lesions in heterozygous females are hallmarks of the human genetic disorder incontinentia pigmenti (IP). Together with the recent discovery that mutations in the human NEMO gene cause IP, our results indicate that we have created a mouse model for that disease.     

Inhibition of the NF-kappaB survival pathway via caspase-dependent cleavage of the IKK complex scaffold protein and NF-kappaB essential modulator NEMO

Apoptosis is mediated by cysteine-dependent, aspartate-directed proteases of the caspase family that proteolyse strategic intracellular substrates to induce cell suicide. We describe here that engagement of apoptotic processes by Fas triggering or by staurosporine stimulation leads to the caspase-dependent inactivation of the nuclear factor kappa B (NF-kappaB) pathway after cleavage of IKK1 (IkappaB kinase 1) and NEMO (NF-kappaB essential modulator), which are needed to transduce NF-kappaB activation signals. In this study, we have analyzed in more detail, the role of NEMO cleavage, as NEMO, but not IKK1, is important for the pro-survival actions of NF-kappaB. We demonstrate that NEMO is cleaved after Asp355 to remove the last 64 C-terminal amino acids. This short form was unable to rescue NF-kappaB activation by tumor necrosis factor-alpha (TNF-alpha) when transfected in NEMO-deficient cells. Consequently, inactivation of NEMO resulted in an inhibition of the expression of antiapoptotic NF-kappaB-target genes coding for caspase inhibitors (cIAP-1, cIAP-2) or adaptors of the TNF receptor family. NEMO-deficient Jurkat cells transiently expressing a non-cleavable mutant of NEMO were less sensitive to TNF-alpha-induced apoptosis. Therefore, downmodulation of NF-kappaB activation via the proteolytic cleavage of NEMO could represent an amplification loop for apoptosis.     

Uniparental disomy of the entire X chromosome in a female with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked muscle-wasting disorder with an incidence of approximately 1/3,500 male births. Females are also affected, in rare instances. The manifestation of mild to severe symptoms in female carriers of dystrophin mutations is often the result of the preferential inactivation of the X chromosome carrying the normal dystrophin gene. The severity of the symptoms is dependent on the proportion of cells that have inactivated the normal X chromosome. A skewed pattern of X inactivation is also responsible for the clinical manifestation of DMD in females carrying X;autosome translocations, which disrupt the dystrophin gene. DMD may also be observed in females with Turner syndrome (45,X), if the remaining X chromosome carries a DMD mutation. We report here the case of a karyotypically normal female affected with DMD as a result of homozygosity for a deletion of exon 50 of the dystrophin gene. PCR analysis of microsatellite markers spanning the length of the X chromosome demonstrated that homozygosity for the dystrophin gene mutation was caused by maternal isodisomy for the entire X chromosome. This finding demonstrates that uniparental isodisomy of the X chromosome is an additional mechanism for the expression of X-linked recessive disorders. The proband's clinical presentation is consistent with the absence of imprinted genes (i.e., genes that are selectively expressed based on the parent of origin) on the X chromosome.     

Evidence that a dodecamer duplication in the gene HOPA in Xq13 is not associated with mental retardation

A recent study suggested that a dodecamer duplication in exon 42 of the HOPA gene in Xq13 may be a significant factor in the etiology of X-linked mental retardation. In an effort to investigate this possibility, we determined the incidence of the dodecamer duplication in cohorts of non-fragile X males with mental retardation from three countries, cohorts of fragile X males from two countries, 43 probands from families with X-linked mental retardation and control cohorts from three countries. The duplication was found in 3.6-4.0% of male patients from two non-fragile X groups (Italy and South Carolina), in 1.2% from another non-fragile X group (South Africa), but in no male patients from families with X-linked mental retardation (South Carolina). The dodecamer duplication was also found in several white males with fragile X syndrome from France (5%) and South Africa (22.2%). Additionally, the duplication was found in 1.5% of South Carolinian newborn males, 2.5% South Carolinian male college students, 5% Italian male controls and 4.5% of the white South African controls. None of the black South African non-fragile X individuals with mental retardation, the fragile X or the control samples tested carried the duplication, suggesting that the duplication is rare in the black South African population. The incidence of the duplication was not significantly different between any of the groups in the study. Therefore, results of our studies in four different populations do not corroborate the findings of the previous study, and indicate that the HOPA dodecamer duplication does not convey an increased susceptibility to mental retardation.     

Application of carrier testing to genetic counseling for X-linked agammaglobulinemia.

Bruton X-linked agammaglobulinemia (XLA) is a phenotypically recessive genetic disorder of B lymphocyte development. Female carriers of XLA, although asymptomatic, have a characteristic B cell lineage-specific skewing of the pattern of X inactivation. Skewing apparently results from defective growth and maturation of B cell precursors bearing a mutant active X chromosome. In this study, carrier status was tested in 58 women from 22 families referred with a history of agammaglobulinemia. Primary carrier analysis to examine patterns of X inactivation in CD19+ peripheral blood cells (B lymphocytes) was conducted using quantitative PCR at the androgen-receptor locus. Obligate carriers of XLA demonstrated > 95% skewing of X inactivation in peripheral blood CD19+ cells but not in CD19- cells. Carrier status for mothers of isolated affected males could be assessed in 10 of 11 families: 7 women showed skewing, and 3 did not. Five carriers were found in six families in which there were no living affected males. Among all those tested, one individual's carrier status was considered to be indeterminate and five women were noninformative for the carrier test. Results obtained by the carrier test were congruent with linkage analysis (where applicable) using the RFLPs DXS178 and DXS94 and two newly developed polymorphic microsatellite markers, DXS178CA and DXS101AAT. Refinements in techniques for primary carrier testing and genetic mapping of XLA now make possible an ordered approach to diagnosis, prenatal diagnosis, and genetic counseling.     

Deletion of a DNA sequence in eight of nine families with X-linked ichthyosis (steroid sulphatase deficiency).

Deficiency of steroid sulphatase (STS) is associated with ichthyosis, with failure of the placental production of oestriol in late pregnancy and with difficulties in childbirth. The STS gene has been localised by deletion mapping to the distal tip of the snort arm of the X chromosome, and is of interest in that it appears to escape X-inactivation. We have constructed an X-specific DNA library and screened it for single copy DNA sequences which lie at the distal end of Xp. The sequence GMGX9 was found to map in the interval Xp22.3-pter and to detect a frequent HindIII polymorphism. We have used GMGX9 in linkage studies in families with classical X-linked ichthyosis and this has not only shown tight linkage with STS deficiency but has also revealed that the sequence is deleted in affected males in eight of nine families. GMGX9 is present in all of 26 normal male individuals so far examined. Our findings suggest that a high proportion of the mutations at the STS locus leading to enzyme deficiency are deletions, presumably generated by unequal cross-over events in female meiosis or by illegitimate X-Y interchange in male meiosis.     

Extreme skewing of X chromosome inactivation in mothers of homosexual men

Human sexual preference is a sexually dimorphic trait with a substantial genetic component. Linkage of male sexual orientation to markers on the X chromosome has been reported in some families. Here, we measured X chromosome inactivation ratios in 97 mothers of homosexual men and 103 age-matched control women without gay sons. The number of women with extreme skewing of X-inactivation was significantly higher in mothers of gay men (13/97=13%) compared to controls (4/103=4%) and increased in mothers with two or more gay sons (10/44=23%). Our findings support a role for the X chromosome in regulating sexual orientation in a subgroup of gay men.     

Histone macroH2A1.2 is concentrated in the XY-body by the early pachytene stage of spermatogenesis

The pairing of sex chromosomes during meiosis in male mammals is associated with ongoing heterochromatinization and X inactivation. This process occurs in a specific area of the nucleus that can be discerned morphologically: the sex vesicle or XY-body. In contrast to X inactivation in the somatic cells of female mammals the reasons for X inactivation in the male germline remain obscure. We have recently demonstrated that the inactive X chromosome in somatic cells of female mammals is marked by a high concentration of histone macroH2A. Here we investigate X inactivation in the meiotic cells of the male germline. We demonstrate here that macroH2A1.2 is present in the nuclei of germ cells starting first with localization that is largely, if not exclusively, to the developing XY-body in early pachytene spermatocytes. Our results suggest that inactivation of sex chromosomes in the male germ cell includes a major alteration of the nucleosomal structure.     

Mutation analysis of PMP22 in Slovak patients with Charcot-Marie-Tooth disease and hereditary neuropathy with liability to pressure palsies

Charcot-Marie-Tooth disease (CMT) and related peripheral neuropathies are the most commonly inherited neurological disorders in humans, characterized by clinical and genetic heterogeneity. The most prevalent clinical entities belonging to this group of disorders are CMT type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP). CMT1A and HNPP are predominantly caused by a 1.5 Mb duplication and deletion in the chromosomal region 17p11.2, respectively, and less frequently by other mutations in the peripheral myelin protein 22 (PMP22) gene. Despite being relatively common diseases, they haven't been previously studied in the Slovak population. Therefore, the aim of this study was to identify the spectrum and frequency of PMP22 mutations in the Slovak population by screening 119 families with CMT and 2 families with HNPP for causative mutations in this gene. The copy number determination of PMP22 resulted in the detection of CMT1A duplication in 40 families and the detection of HNPP deletion in 7 families, 6 of which were originally diagnosed as CMT. Consequent mutation screening of families without duplication or deletion using dHPLC and sequencing identified 6 single base changes (3 unpublished to date), from which only c.327C>A (Cys109X) present in one family was provably causative. These results confirm the leading role of PMP22 mutation analysis in the differential diagnosis of CMT and show that the spectrum and frequency of PMP22 mutations in the Slovak population is comparable to that seen in the global population.     

Characterization of the Ikappa B-kinase NEMO binding domain

Proinflammatory activation of NF-kappaB requires an upstream kinase complex (IkappaB-kinase; IKK) composed of two catalytic subunits (IKKalpha and IKKbeta) and a noncatalytic regulatory component named NEMO (NF-kappaB essential modulator). NEMO interacts with a COOH-terminal sequence within both IKKs termed the NEMO-binding domain (NBD), and a cell-permeable NBD peptide blocks NEMO/IKKbeta interactions and inhibits tumor necrosis factor-alpha-induced NF-kappaB. We report here that a peptide encompassing the NBD not only blocked association of both IKKs with NEMO but also disrupted preformed NEMO/IKK complexes in vitro. Furthermore, peptide blocking and alanine-scanning mutation studies revealed differences between the NBDs of IKKalpha and IKKbeta, and mutational analysis of the IKKbeta NBD identified the physical properties required at each position to maintain association with NEMO. Finally, we demonstrate that loss of NEMO-binding by IKKbeta through deletion of the NBD renders it catalytically active and that potential phosphorylation within the IKKbeta NBD may serve as a signal to down-regulate IKK activity. Our findings therefore provide critical insight into the physical properties of the NBD that will be valuable for the design of drugs aimed at disrupting the IKK complex and also reveal potential regulatory mechanisms controlling the function of the IKK complex.     

Unexpected X chromosome skewing during culture and reprogramming of human somatic cells can be alleviated by exogenous telomerase

Somatic tissues in female eutherian mammals are mosaic due to random X inactivation. In contrast to mice, X chromosome reactivation does not occur during the reprogramming of human female somatic cells to induced pluripotent stem cells (iPSCs), although this view is contested. Using balanced populations of female Rett patient and control fibroblasts, we confirm that all cells in iPSC colonies contain an inactive X, and additionally find that all colonies made from the same donor fibroblasts contain the same inactive X chromosome. Notably, this extreme "skewing" toward a particular dominant, active X is also a general feature of primary female fibroblasts during proliferation, and the skewing seen in reprogramming and fibroblast culture can be alleviated by overexpression of telomerase. These results have important implications for in?vitro modeling of X-linked diseases and the interpretation of long-term culture studies in cancer and senescence using primary female fibroblast cell lines.     

Polymorphic X-chromosome inactivation of the human TIMP1 gene.

X inactivation silences most but not all of the genes on one of the two X chromosomes in mammalian females. The human X chromosome preserves its activation status when isolated in rodent/human somatic-cell hybrids, and hybrids retaining either the active or inactive X chromosome have been used to assess the inactivation status of many X-linked genes. Surprisingly, the X-linked gene for human tissue inhibitor of metalloproteinases (TIMP1) is expressed in some but not all inactive X-containing somatic-cell hybrids, suggesting that this gene is either prone to reactivation or variable in its inactivation. Since many genes that escape X inactivation are clustered, we examined the expression of four genes (ARAF1, ELK1, ZNF41, and ZNF157) within approximately 100 kb of TIMP1. All four genes were expressed only from the active X chromosome, demonstrating that the factors allowing TIMP1 expression from the inactive X chromosome are specific to the TIMP1 gene. To determine if this variable inactivation of TIMP1 is a function of the hybrid-cell environment or also is observed in human cells, we developed an allele-specific assay to assess TIMP1 expression in human females. Expression of two alleles was detected in some female cells with previously demonstrated extreme skewing of X inactivation, indicating TIMP1 expression from the inactive chromosome. However, in other cells, no expression of TIMP1 was observed from the inactive X chromosome, suggesting that TIMP1 inactivation is polymorphic in human females.     

Mutations in NDUFB11, Encoding a Complex I Component of the Mitochondrial Respiratory Chain,Cause Microphthalmia with Linear Skin Defects Syndrome

Microphthalmia with linear skin defects (MLS) syndrome is an X-linked male-lethal disorder also known as MIDAS (microphthalmia, dermal aplasia, and sclerocornea). Additional clinical features include neurological and cardiac abnormalities. MLS syndrome is genetically heterogeneous given that heterozygous mutations in HCCS or COX7B have been identified in MLS-affected females. Both genes encode proteins involved in the structure and function of complexes III and IV, which form the terminal segment of the mitochondrial respiratory chain (MRC). However, not all individuals with MLS syndrome carry a mutation in either HCCS or COX7B. The majority of MLS-affected females have severe skewing of X chromosome inactivation, suggesting that mutations in HCCS, COX7B, and other as-yet-unidentified X-linked gene(s) cause selective loss of cells in which the mutated X chromosome is active. By applying whole-exome sequencing and filtering for X-chromosomal variants, we identified a de novo nonsense mutation in NDUFB11 (Xp11.23) in one female individual and a heterozygous 1-bp deletion in a second individual, her asymptomatic mother, and an affected aborted fetus of the subject’s mother. NDUFB11 encodes one of 30 poorly characterized supernumerary subunits of NADH:ubiquinone oxidoreductase, known as complex I (cI), the first and largest enzyme of the MRC. By shRNA-mediated NDUFB11 knockdown in HeLa cells, we demonstrate that NDUFB11 is essential for cI assembly and activity as well as cell growth and survival. These results demonstrate that X-linked genetic defects leading to the complete inactivation of complex I, III, or IV underlie MLS syndrome. Our data reveal an unexpected role of cI dysfunction in a developmental phenotype, further underscoring the existence of a group of mitochondrial diseases associated with neurocutaneous manifestations.