Estimating sex ratio biases in X-linked disorders: is there an excess of males in families with X-linked ichthyosis?

We derive the conditional probabilities for estimating the sex ratio in families ascertained through affected males for the study of X-linked recessive diseases. These conditional probabilities correct for the fact that the probability that a family will be ascertained increases with the number of males in the family. Data from four published studies for X-linked ichthyosis vulgaris are analyzed, three having an excess of males and one having a highly statistically significant excess of males. It is not known if this difference in the two samples represents a biological difference between the two populations or an unrecognized ascertainment bias.     

Harlequin ichthyosis — difficulties in prenatal diagnosis

Ichthyoses belong to the group of genodermatoses, characterized by hyperkeratosis and desquamation of the epidermis. Clinical manifestation is heterogeneous and depends on the type of the disease. Harlequin foetus is the most severe form of congenital ichtyosis, inherited as an autosomal recessive trait. The disfunction of the epidermis begins prenatally. Neonates are often born prematurely, in severe condition. At present better care and treatment prolong the length and quality of children's life. We report a case of harlequin ichthyosis. Parents were healthy and there was no history of ichthyosis or other congenital anomalies in the family. Sonography at the 26th week of gestation revealed anomalies of the fetal face; however, the diagnosis of harlequin ichthyosis was not established prenatally. The male child was born alive at the 37th week of the third pregnancy, with birth weight of 2900 g. Typical features of harlequin ichthyosis were present at birth. Intensive neonatological care was necessary. The child survived and at the time of the report was 6 months old and in good condition.     

Polymorphisms in the human X-linked pyruvate dehydrogenase E1α gene

Summary Pyruvate dehydrogenase E1 deficiency is an X-chromosome-linked disorder, often with fatal consequences. We have searched for genetically useful polymorphisms in or near this gene. No restriction fragment length polymorphisms were detected using a battery of 36 different restriction enzymes and probing with a fulllength cDNA fragment, or two single-copy genomic fragments located within intron 8, and 15 kb 3 of the coding region, respectively. The chemical cleavage method was then applied to the detection of base changes in or near the gene. One polymorphism was found in exon 8 of the coding region. However, no base changes were detected in intron 3 or in the part of intron 8 covered by fragment gB2. Three blocks of microsatellite DNA containing variable numbers of CA-repeats were isolated from the 5 end of the gene and characterized. Length polymorphisms in these microsatellite DNAs were analysed using the polymerase chain reaction. Although the three loci are tightly linked, the polymorphisms appear not to be in disequilibrium, making them useful markers in linkage studies of the pyruvate dehydrogenase E1 gene. Of 31 females analysed 12 (39%) were heterozygous for at least one length polymorphism of the three (CA)_n alleles.     

Mutations in the gene for the common gamma chain (γc) in X-linked severe combined immunodeficiency

X-linked severe combined immunodeficiency (XSCID) consitutes a disorder of the immune system caused by mutations in the gene encoding the common gamma chain (γ_c), a subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors, which are necessary for lymphocyte development and function. In this study the IL2RG gene of 31 patients with severe combined immunodeficiency (SCID) was examined by nonradioactive single-strand conformation polymorphism and sequence analysis. Among the 11 patients with XSCID, ten different mutations were identified in the IL2RG gene, including eight novel mutations. Ninety percent of the mothers of the XSCID patients are carriers of the mutated allele. One patient showed low numbers of B-cells, a striking deviation from the classical B-cell-positive and T-cell-negative phenotype. Received: 23 June 1998 / Accepted: 6 September 1998     

Nonspecific X-linked mental retardation—A review

Summary Mental retardation, in particular the X-linked type, has interested geneticists for many years. An increasing number of affected families have been to genetic counselling centres, and an effort is being made to find clinical and cytogenetic methods so a reliable diagnosis can be made. This would enable the detection of carriers and the opportunity to offer prenatal diagnosis. Many questions remain regarding X-linked mental retardation, its causes, diagnosis, and prevention. In this article we try to give an overview about the status of our present knowledge and the questions to be answered in the future.     

Three novel mutations in the interleukin-2 receptor γ chain gene in four Japanese patients with X-linked severe combined immunodeficiency

Three novel mutations in the IL-2R chain gene were identified in four Japanese patients with X-linked severe combined immunodeficiency by direct sequence analysis of polymerase chain reaction (PCR) amplified DNA fragments.     

X-inactivation pattern in carriers of X-linked retinitis pigmentosa: A valuable means of prognostic evaluation?

In a large family with X-linked retinitis pigmentosa 2 (XLRP2), we reexamined 7 obligate carrier females and 6 daughters of obligate carriers, whose linkage relationships suggested that they carried the XLRP2 gene. The phenotype varied from totally normal eyes through mild retinal changes to complete loss of vision. The X-inactivation analysis was carried out with the highly informative probe M27 on DNA from blood lymphocytes. This probe detects a locus DXS 255 that is differentially methylated on the active and inactive X chromosomes. In 5 blind heterozygotes (aged 43 to 68 years), we found that the X chromosome carrying the RP2 gene was methylated and active in nearly all their cells. The opposite X inactivation pattern was found in a carrier female (aged 45 years) who gave normal findings on eye examination. Carriers with less skewed X inactivation had a less severe clinical outcome. However, we found little or no correlation between their phenotypes and the methylation status of their X chromosomes. Our results suggest that it may be possible to develop a predictive test that could identify cases with severe outcome and perhaps cases with normal outcome.     

CUL4B ubiquitin ligase in mouse development: A model for human X-linked mental retardation syndrome?

CUL4B, a member of the cullin-RING ubiquitin ligase family, is frequently mutated in X-linked mental retardation (XLMR) patients. The study by Liu et al. showed that Cul4b plays an essential developmental role in the extra-embryonic tissues, while it is dispensable in the embryo proper during mouse embryogenesis. Viable Cul4b-null mice provide the first animal model to study neuronal and behavioral deficiencies seen in human CUL4B XLMR patients.CUL4 is a member of the cullin-RING ubiquitin ligase family, the largest E3 ligase family, which appears to account for ∼20% of total protein degradation by the ubiquitin-proteasome system^1,2,3. CUL4 is conserved during evolution from yeast to human. In yeast, CUL4 encodes a single gene, but mammalian cells express two closely related paralogs, CUL4A and CUL4B with about 82% sequence identity. CUL4A and CUL4B assemble structurally similar E3 complexes through binding to an adaptor protein (DDB1) and a substrate receptor protein (DCAF) at the N-terminus, and a RING protein RBX1 at the C-terminus (Figure 1), and share functional redundancy in targeting substrates such as p21 and Cdt1 for ubiquitination and degradation^1,2. The Cul4a-null mice are viable and display no abnormal development and growth phenotypes, likely due to functional compensation from Cul4b^4,5. The only phenotype associated with Cul4a abrogation is the reproductive defects seen with male but not female mice, resulting from differential non-overlapping expression patterns of the two Cul4 genes during male meiosis⁶. On the other hand, germline deletion of Cul4b resulted in embryonic lethality around E9.5⁷, indicating a unique function of Cul4b that cannot be compensated by Cul4a during embryogenesis.Open in a separate windowFigure 1Differential expression of Cul4a and Cul4b in the embryo proper and extra embryonic tissues determines their fate. Before implantation, both Cul4a and Cul4b are expressed in the blastocyst. Following implantation, Cul4a is expressed in the embryo proper, but not in extra-embryonic tissues. Upon Cul4b deletion, p21 accumulates in extra-embryonic tissues to induce G2/M arrest and eventually embryonic death due to degeneration of extra-embryonic tissues. Expression of Cul4a in embryo prevents p21 accumulation and subsequent embryonic death.Mental retardation (MR) affects approximately 1%-3% of the population and is about 30% more common in males than in females⁸, suggesting a causal relationship with gene mutations on the X chromosome. To date, mutations in about 100 genes have been identified in X-linked MR (XLMR), much more than those found on autosomes⁹. In 2007, two independent groups reported that mutations of CUL4B (Xq24) ubiquitin ligase gene are associated with XLMR^10,11. CUL4B-deficient patients display a syndrome of delayed puberty, moderate short stature, hypogonadism, relative macrocephaly, central obesity, fine intention tremor, brachydactyly, and large tongue^10,11. Similarly, the neuronal and developmental deficiencies found in XLMR patients with CUL4B mutations are not compensated by CUL4A. The studies of the molecular pathogenesis of human XLMR are lagging partly due to the lack of an animal model for the disease.In the most recent study published in Cell Research, Zhou and coworkers¹² attempted to generate conditional Cul4b knockout mice with targeted deletion of Cul4b at exons 4 and 5, giving rise to a non-functional Cul4b fragment lacking both the DDB1-binding domain and the cullin homology domain for RBX1 recruitment. The chicken-actin (CAG)-Cre was used, which drives Cre-mediated recombination at the early zygote stage, leading to Cul4b deletion in both the embryo proper and extra-embryonic tissues. Like human CUL4B, the mouse Cul4b is also located on the X-chromosome. Intercrossing of male CAG-Cre with female Cul4b^fl/+ revealed that hemizygous deletion of Cul4b causes embryonic lethality. No embryos with the genotype of Cul4b^−/y survived beyond E9.5. Interestingly, the heterozygous Cul4b^+/− embryos also die in the uterus before E13.5, suggesting that the paternal X chromosome undergoes imprinted inactivation with only trace amount, if any, of Cul4b expression remaining in extra-embryonic tissues. Detailed analysis of dissected embryos revealed that dying Cul4b^+/− embryos (E12.5) lack blood supply from the yolk sacs, whereas the Cul4b^−/y embryos (E8.5) showed remarkable reduction in proliferation with growth arrest at G2/M and enhanced apoptosis. The authors went on and investigated why Cul4a failed to compensate the loss of Cul4b, and found a dynamic expression pattern, differing between two forms, during early embryonic development. Prior to implantation, both Cul4 proteins are detectable in the blastocysts. Shortly after implantation, while both forms are expressed in the embryo proper, only Cul4b is expressed in the extra-embryonic tissues. Thus, upon Cul4b deletion, extra-embryonic tissues without Cul4a compensation degenerate, eventually leading to embryonic death. Consistently, when the authors deleted Cul4b in the epiblast using the Sox2-Cre (targeted Cul4b deletion in embryos proper only), viable Cul4b-null mice are produced likely due to Cul4a compensation. Thus, Cul4b is essential for the development of extra-embryonic tissues, but is dispensable for embryogenesis itself.To study the potential underlying mechanism(s) of embryonic lethality upon Cul4b deletion in extra-embryonic tissues, the authors used an extra-embryonic cell line (XEN). Cul4b knockdown induced a remarkable cell cycle arrest at the G2/M phase, consistent with observation made in Cul4b-null embryos, and robust accumulation of p21, a universal inhibitor of cyclin dependent kinase and a known substrate of Cul4¹. To determine whether accumulated p21 is responsible for the G2/M arrest, the authors simultaneously knocked down both Cul4b and p21 in XEN cells and observed a partial abrogation of growth arrest, suggesting that p21 plays a causal role, at least in part. Unfortunately, due to unavailability of anti-mouse p21 antibody specific for immunohistochemical staining, the authors were not able to show if p21 is indeed accumulated in extra-embryonic tissues upon Cul4b deletion. However, whether p21 indeed plays a causal role in embryonic death upon Cul4b deletion can be unequivocally determined by a rescuing experiment in which simultaneous deletion of p21 should abrogate or at least delay embryonic lethality, if it is causal. Nevertheless, the study by Zhou''s group can be summarized as follows. Before implantation, both Cul4a and Cul4b ubiquitin ligases are expressed in the blastocyst (inner cell mass and trophoblast cells). Following embryo implantation, while Cul4b is expressed in both the embryo proper and extra embryonic tissues, Cul4a is only expressed in the embryo proper. The CAG-Cre-driven Cul4b deletion (in both the embryo proper and extra-embryonic tissues) causes significant p21 accumulation in Cul4a non-expressing extra-embryonic tissues, resulting in G2/M arrest, followed by embryonic death due to degeneration of extra-embryonic tissues. On the embryo side, Cul4b deletion has no detrimental consequence, benefiting from the compensatory effect of Cul4a for p21 targeting. The same holds true when Cul4b is deleted driven by embryonic specific Sox2-Cre (Figure 1).It is noteworthy that the studies by Zhou''s group revealed two distinct differences between Cul4b KO mice and CUL4B-associated XLMR patients. First, Cul4b deletion at the zygote stage causes embryonic lethality, whereas XLMR patients with CUL4B mutations live to adulthood. Second, the Cul4b-null allele cannot be transmitted from the mother to the offspring, whereas human XLMR patients inherit X-linked CUL4B mutations from their mothers. Nevertheless, viable Cul4b-null mice (upon epiblast ablation by Sox2-Cre) provide the first mouse model for mechanistic study of human XLMR diseases associated with CUL4B mutations in the following three aspects:First, as noted earlier, human CUL4B XLMR patients have multiple neuronal and developmental defects. An obvious follow-up study will be to use this mouse model for neurological and behavioral analyses to determine whether Cul4b-null mice indeed present some of human XLMR symptoms.Second, this model can also be used to validate whether accumulation of Cul4b substrates during various stages of brain development indeed plays a pathogenic role and contributes to the clinical symptoms of XLMR patients. For instance, WDR5, a recently identified gene affecting general cognitive ability¹³, was found to be a novel nuclear substrate of CUL4B, but not CUL4A¹⁴. Investigation into whether WDR5 is abnormally accumulated upon Cul4b deletion in vivo would rule in or rule out its potential association with human XLMR, although it was not the case in this study using an extra-embryonic cell line in vitro.Third, the viability of Cul4b-null mice upon epiblast-specific deletion provides opportunities to study neuronal specific ablation of Cul4b in association with the pathogenesis of CUL4B-associated XLMR. For example, Cul4b is expressed at high levels in the hippocampus and cerebrum of mouse brains; both regions are affected in MR patients¹⁵. Thus, the use of Cre mouse lines that target the deletion of Cul4b in the entire brain, selected brain areas, or specific neuronal cells in both spatial and temporal manners¹⁶ would reveal potential contributions of particular regions and cell types to the development and symptoms of CUL4B-associated XLMR.A number of questions that warrant future investigation remain unanswered. First, in addition to p21, what are the other Cul4B substrates, which also contribute to degeneration of extra-embryonic tissues upon Cul4b deletion, since simultaneous deletion of p21 only partially rescues the growth defects? Second, besides the difference in tissue/cell specific expression seen in this study, are Cul4a and Cul4b targeting a unique set of substrates non-redundantly, thus differentiating their physiological functions? A related question will be why CUL4A cannot compensate for the loss of CUL4B in CUL4B-associated XLMR patients? Third, what is the pathogenic mechanism for CUL4B-associated XLMR? Is it mainly due to pathological accumulation of many CUL4B substrates? Answers to these questions may offer insights into potential therapeutic strategies for the treatment of CUL4B-associated XLMR patients.In summary, the findings reported by Zhou''s group provide the first convincing evidence that demonstrates an essential role of Cul4b in the development of extra-embryonic tissues during mouse embryogenesis. The viable Cul4b conditional knockout mice, generated in this study, may serve as the first mouse model for future mechanistic studies of neuronal and behavioral deficiencies of human XLMR associated with CUL4B mutations. We look forward to more exciting discoveries of how Cul4b deficiency leads to the development of XLMR in years to come.     

Autism and X-linked hypophosphatemia: A possible association?

We herein report the joint occurrence of an autistic disorder (AD) and X-linked hypophosphatemia. X-linked hypophosphatemia (XLH), an X-linked dominant disorder, is the most common of the inherited renal phosphate wasting disorders. Autism is a pervasive developmental disorder that occurs mainly due to genetic causes. In approximately 6-15% of cases, the autistic phenotype is a part of a broader genetic condition called syndromic autism.Therefore, reports of cases with the joint occurrence of a known genetic syndrome and a diagnosis of ASD by a child psychiatrist are relevant. A joint occurrence does not, however, mean that there is always a causal link between the genetic syndrome and the autistic behavioural phenotype. In this case, there are a number of arguments countering a causal link.     

A mouse model for intellectual disability caused by mutations in the X-linked 2′‑O‑methyltransferase Ftsj1 gene

Mutations in the X chromosomal tRNA 2′?O?methyltransferase FTSJ1 cause intellectual disability (ID). Although the gene is ubiquitously expressed affected individuals present no consistent clinical features beyond ID. In order to study the pathological mechanism involved in the aetiology of FTSJ1 deficiency-related cognitive impairment, we generated and characterized an Ftsj1 deficient mouse line based on the gene trapped stem cell line RRD143. Apart from an impaired learning capacity these mice presented with several statistically significantly altered features related to behaviour, pain sensing, bone and energy metabolism, the immune and the hormone system as well as gene expression. These findings show that Ftsj1 deficiency in mammals is not phenotypically restricted to the brain but affects various organ systems. Re-examination of ID patients with FTSJ1 mutations from two previously reported families showed that several features observed in the mouse model were recapitulated in some of the patients. Though the clinical spectrum related to Ftsj1 deficiency in mouse and man is variable, we suggest that an increased pain threshold may be more common in patients with FTSJ1 deficiency. Our findings demonstrate novel roles for Ftsj1 in maintaining proper cellular and tissue functions in a mammalian organism.     

Impaired Very Long-chain Acyl-CoA β-Oxidation in Human X-linked Adrenoleukodystrophy Fibroblasts Is a Direct Consequence of ABCD1 Transporter Dysfunction

X-linked adrenoleukodystrophy (X-ALD), an inherited peroxisomal disorder, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (adrenoleukodystrophy protein, ALDP). Biochemically, X-ALD is characterized by an accumulation of very long-chain fatty acids and partially impaired peroxisomal β-oxidation. In this study, we used primary human fibroblasts from X-ALD and Zellweger syndrome patients to investigate the peroxisomal β-oxidation defect. Our results show that the degradation of C26:0-CoA esters is as severely impaired as degradation of unesterified very long-chain fatty acids in X-ALD and is abolished in Zellweger syndrome. Interestingly, the β-oxidation rates for both C26:0-CoA and C22:0-CoA were similarly affected, although C22:0 does not accumulate in patient fibroblasts. Furthermore, we show that the β-oxidation defect in X-ALD is directly caused by ABCD1 dysfunction as blocking ABCD1 function with a specific antibody reduced β-oxidation to levels observed in X-ALD fibroblasts. By quantification of mRNA and protein levels of the peroxisomal ABC transporters and by blocking with specific antibodies, we found that residual β-oxidation activity toward C26:0-CoA in X-ALD fibroblasts is mediated by ABCD3, although the efficacy of ABCD3 appeared to be much lower than that of ABCD1. Finally, using isolated peroxisomes, we show that β-oxidation of C26:0-CoA is independent of additional CoA but requires a cytosolic factor of >10-kDa molecular mass that is resistant to N-ethylmaleimide and heat inactivation. In conclusion, our findings in human cells suggest that, in contrast to yeast cells, very long-chain acyl-CoA esters are transported into peroxisomes by ABCD1 independently of additional synthetase activity.     

Nonsyndromic X-linked mental retardation: where are the missing mutations?

Analysis of linkage intervals from 125 unrelated families with nonsyndromic X-linked mental retardation (NS-XLMR) has revealed that the respective gene defects are conspicuously clustered in defined regions of the human X-chromosome, with approximately 30% of all mutations being located on the proximal Xp. In 83% of these families, underlying gene defects are not yet known. Our observations should speed up the search for mutations that are still missing and pave the way for the molecular diagnosis of this common disorder.     

Screening for mutations causing X-linked severe combined immunodeficiency in the IL-2Rγ chain gene by single-strand conformation polymorphism analysis

Mutations in the common gamma chain (_c or IL2RG) of the interleukin-2, –4, –7, –9 and –15 receptors have been found to cause X-linked severe combined immunodeficiency (SCIDX1). We report here on the mutations identified in a further ten families. Two of the mutations identified have occurred twice in unrelated families, indicating two possible mutational hotspots. Seven of the mutations, which were identified by single-strand conformational polymorphism (SSCP) analysis, are point mutations, and the eighth is a small deletion. We also report on the first use of assays based on these mutations within IL2RG for unambiguous carrier determination. The consequences for the _c proteins produced as a result of these mutations are discussed.     

A 25-kb deletion in the 5′ region of the cytochrome b558 heavy chain gene (CYBB) in a patient with X-linked chronic granulomatous disease

We performed molecular genetic analyses of the family of a boy suffering from chronic granulomatous disease (CGD) after immunocytochemically confirming him and his mother to be an X-linked CGD patient and a mosaic carrier, respectively. Southern blot hybridization using cDNA for the cytochrome b₅₅₈ heavy chain gene (CYBB) as a probe showed that the patient had a deletion in the 5′ region of the CYBB and his phenotypically normal mother was heterozygous for this deletion. Polymerase chain reaction analyses of all 13 exons of the patient’s CYBB gene demonstrated that the deletion extends from exon 7 or neighboring introns to 5′ upstream. The length of the deletion was determined by pulsed-field gel electrophoresis and Southern blotting of genomic DNA using CYBB cDNA and the genetic marker pERT55-5, centromeric to CYBB, as probes. Both probes recognized common SfiI-NotI fragments of 120 kb and 95 kb in normal individuals and the patient, respectively. These results revealed that the patient has a 25-kb deletion spanning from the middle of CYBB to 5′ upstream. This is the only report of a large 5′ deletion in CYBB and also the first observation that CYBB and pERT55-5 are within 120 kb in Xp21. Received: 24 September 1996 / Revised: 10 November 1996     

X-linked mental retardation with Marfanoid habitus: a changing phenotype with age?

In this report, we present two further examples of X-linked mental retardation with Marfanoid habitus. Follow-up data on these two patients reveal that the clinical diagnosis of this syndrome is extremely difficult, if not impossible before puberty, as the Marfanoid habitus only becomes strikingly evident during adolescence.     

MicroRNA-513a-5p mediates TNF-α and LPS induced apoptosis via downregulation of X-linked inhibitor of apoptotic protein in endothelial cells

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that have emerged as one of the central players of gene expression regulation. Endothelial cell apoptosis plays a fundamental role in the development of atherosclerosis. This study was designed to determine the effect of miR-513a-5p on apoptosis of human umbilical vein endothelial cells (HUVECs). HUVECs were treated with tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) and miR-513a-5p expression levels were determined. MiR-513a-5p target gene indentification, validation, and signalling pathways were investigated. Treatment of HUVECs with TNF-α and LPS up-regulated miR-513a-5p expressions more than 2-fold compared to control (P < 0.05). Inhibition of miR-513a-5p by antisense (AS) miR-513a-5p reversed TNF-α and LPS induced apoptosis (P < 0.01). Transfection of HUVECs with miR-513a-5p mimics also induced apoptosis (P < 0.01). Treatment of HUVECs with TNF-α and LPS attenuated X-linked inhibitor of apoptosis (XIAP) while increased caspase-3 expression, poly ADP-ribose polymerase (PARP) cleavage, and p53 expression. These effects were reversed by inhibition of miR-513a-5p. Of those miR-513a-5p candidate target genes, we identified and validated XIAP as a miR-513a-5p target gene. Targeting of the XIAP 3′-untranslated region by miR-513a-5p using luciferase reporter assay resulted in attenuated luciferase activity. Transfection of HUVECs with AS miR-513a-5p increased XIAP protein expression while miR-513a-5p mimics attenuated XIAP expression. These results together suggest that miR-513a-5p mediates TNF-α and LPS induced apoptosis via downregulation of XIAP in HUVECs.