Mother and daughter with 45,X/46,X,r(X)(p22.3q28) and mental retardation: Analysis of the x‐inactivation patterns

We report on a mother and daughter both with a 45,X/46,X,r(X)(p22.3q28) karyotype and mental retardation. Fluorescence in situ hybridization (FISH) and microsatellite analyses for 14 loci/region at Xp22.3 and seven loci/region at Xq28 indicated that the ring X chromosome was missing a roughly 12‐Mb region from Xp22.3 with the breakpoint between DXS85 and DXS9972, and another region of less than 100 kb from Xq28 with the breakpoint distal to the region defined by the FISH probe c8.2/1. X‐inactivation analysis, using the methylation status of the AR gene (exon 1) as an indicator, showed that the normal and ring X chromosomes in the X,r(X)(p22.3q28) cell lineage were randomly inactivated. The Xp22.3 deleted region partially overlaps with the regional intervals of MRX19, MRX21, MRX24, MRX37, MRX43, and MRX49 associated with heterozygote manifestation. Therefore, it is likely that one or more of these MRX genes, subject to X‐inactivation, are lost from the ring X chromosome, and that reduced expression of the MRX gene(s) caused by random X‐inactivation has resulted in mental retardation in the mother and daughter. Am. J. Med. Genet. 91:267–272, 2000. © 2000 Wiley‐Liss, Inc.     

Genes that escape from X‐chromosome inactivation: Potential contributors to Klinefelter syndrome

One of the two X chromosomes in females is epigenetically inactivated, thereby compensating for the dosage difference in X‐linked genes between XX females and XY males. Not all X‐linked genes are completely inactivated, however, with 12% of genes escaping X chromosome inactivation and another 15% of genes varying in their X chromosome inactivation status across individuals, tissues or cells. Expression of these genes from the second and otherwise inactive X chromosome may underlie sex differences between males and females, and feature in many of the symptoms of XXY Klinefelter males, who have both an inactive X and a Y chromosome. We review the approaches used to identify genes that escape from X‐chromosome inactivation and discuss the nature of their sex‐biased expression. These genes are enriched on the short arm of the X chromosome, and, in addition to genes in the pseudoautosomal regions, include genes with and without Y‐chromosomal counterparts. We highlight candidate escape genes for some of the features of Klinefelter syndrome and discuss our current understanding of the mechanisms underlying silencing and escape on the X chromosome as well as additional differences between the X in males and females that may contribute to Klinefelter syndrome.     

Mosaicism for a small supernumerary ring X chromosome in a dysmorphic, growth-retarded male: mos47,XXY/48,XXY, + r(X)

Supernumerary ring X [r(X)] chromosomes are often found in patients with Turner syndrome. The phenotypic effects of the r(X) chromosome are variable, and largely depend on the presence or absence of the X inactivation (XIST) locus. Ring(X) chromosomes in males are rare and have been previously reported in only four cases, with 47,XY, + r(X) or mos47,XY, + r(X)/46,XY karyotypes. These patients all had developmental delay and dysmorphic features. We describe a 2.5-year-old male patient with facial dysmorphia, growth retardation, microcephaly, global developmental delay, and microphallus. Cytogenetic analysis from peripheral blood lymphocytes and fibroblasts identified mosaicism for two cell lines: mos48,XXY, + r(?X)/47,XXY. Fluorescence in situ hybridization (FISH) with an X chromosome paint showed the ring chromosome to be X chromosome derived. This is the first case of an r(X) chromosome described in a 47,XXY patient. FISH analysis of the r(X) chromosome with an XIST probe showed that the XIST locus was absent. Functional disomy of genes in the r(X) chromosome most likely accounts for the abnormal phenotype in the proband.     

Novel ring chromosome composed of X‐ and Y‐derived material in a girl with manifestations of Ullrich‐Turner syndrome

We present a female infant who has a novel genetic variant of Ullrich‐Turner syndrome. Chromosome analysis on amniotic fluid cells obtained because of ultrasound observation of nuchal thickening showed 45,X in all cells. The infant was born with a low posterior hairline and moderate edema over hands and feet. Postnatal chromosome analysis demonstrated two cell lines—47% of the metaphases were 45,X, but 53% had a ring chromosome in addition to the normal X. FISH studies using alpha satellite probes, an X‐whole‐chromosome‐paint (WCP) probe, and a Y‐cocktail probe determined that the ring was composed of both X and Y sequences. FISH studies also determined that the KAL locus was present on the ring, but that XIST was absent. PCR‐based analysis of lymphocyte DNA documented that the ring contained sequences from both the short and the long arm of the Y chromosome. X‐chromosome analysis using a panel of highly polymorphic markers indicated that the ring contained material derived only from Xp22.1 to Xp21.3. No Xq material was identified on the ring, and androgen receptor‐based X‐inactivation studies suggested that the intact X chromosome was not subject to random X inactivation. Am. J. Med. Genet. 93:343–348, 2000. © 2000 Wiley‐Liss, Inc.     

Phenotypic variation in Melnick‐Needles syndrome is not reflected in X inactivation patterns from blood or buccal smear

Melnick‐Needles syndrome is a rare putative X‐linked dominant bone dysplasia. The patients have short stature, characteristic facial features, and a normal intelligence. The skeletal dysplasia includes S‐shaped curvature of tubular bones and sclerosis of the base of the skull. The phenotype of affected individuals varies, even within families. This could be related to X chromosome inactivation. We report here on a very mildly affected mother and her two severely affected daughters with characteristic features of Melnick‐Needles syndrome. In addition, the two daughters had very similar pigmented nevi on their back. X chromosome inactivation analysis of blood DNA revealed a skewed X inactivation pattern in all three affected females, with the normal X chromosome as the predominating active X chromosome. The X inactivation pattern was similar in buccal smear and blood DNA in the mother and one of the daughters, whereas the other daughter had a skewed pattern in blood only. X chromosome inactivation in blood and buccal smear DNA therefore does not explain the phenotypic variation in this family. The skewed X chromosome inactivation is in agreement with X‐linked inheritance of Melnick‐Needles syndrome and suggests a critical role of the Melnick‐Needles gene in hematopoietic cell proliferation. Clinical evidence indicates that Melnick‐Needles syndrome is allelic to the otopalatodigital syndromes, which have been assigned to Xq26‐28. Haplotype analysis of the X chromosomes in this family was in agreement with the localization of the gene for Melnick‐Needles syndrome to Xq25‐qtel. © 2002 Wiley‐Liss, Inc.     

Identification of Intellectual Disability Genes in Female Patients with a Skewed X‐Inactivation Pattern

Intellectual disability (ID) is a heterogeneous disorder with an unknown molecular etiology in many cases. Previously, X‐linked ID (XLID) studies focused on males because of the hemizygous state of their X chromosome. Carrier females are generally unaffected because of the presence of a second normal allele, or inactivation of the mutant X chromosome in most of their cells (skewing). However, in female ID patients, we hypothesized that the presence of skewing of X‐inactivation would be an indicator for an X chromosomal ID cause. We analyzed the X‐inactivation patterns of 288 females with ID, and found that 22 (7.6%) had extreme skewing (>90%), which is significantly higher than observed in the general population (3.6%; P = 0.029). Whole‐exome sequencing of 19 females with extreme skewing revealed causal variants in six females in the XLID genes DDX3X, NHS, WDR45, MECP2, and SMC1A. Interestingly, variants in genes escaping X‐inactivation presumably cause both XLID and skewing of X‐inactivation in three of these patients. Moreover, variants likely accounting for skewing only were detected in MED12, HDAC8, and TAF9B. All tested candidate causative variants were de novo events. Hence, extreme skewing is a good indicator for the presence of X‐linked variants in female patients.     

Favorably skewed X‐inactivation accounts for neurological sparing in female carriers of Menkes disease

Desai V, Donsante A, Swoboda KJ, Martensen M, Thompson J, Kaler SG. Favorably skewed X‐inactivation accounts for neurological sparing in female carriers of Menkes disease. Classical Menkes disease is an X‐linked recessive neurodegenerative disorder caused by mutations in ATP7A, which is located at Xq13.1‐q21. ATP7A encodes a copper‐transporting P‐type ATPase and plays a critical role in development of the central nervous system. With rare exceptions involving sex chromosome aneuploidy or X‐autosome translocations, female carriers of ATP7A mutations are asymptomatic except for subtle hair and skin abnormalities, although the mechanism for this neurological sparing has not been reported. We studied a three‐generation family in which a severe ATP7A mutation, a 5.5‐kb genomic deletion spanning exons 13 and 14, segregated. The deletion junction fragment was amplified from the proband by long‐range polymerase chain reaction and sequenced to characterize the breakpoints. We screened at‐risk females in the family for this junction fragment and analyzed their X‐inactivation patterns using the human androgen‐receptor (HUMARA) gene methylation assay. We detected the junction fragment in the proband, two obligate heterozygotes, and four of six at‐risk females. Skewed inactivation of the X chromosome harboring the deletion was noted in all female carriers of the deletion (n = 6), whereas random X‐inactivation was observed in all non‐carriers (n = 2). Our results formally document one mechanism for neurological sparing in female carriers of ATP7A mutations. Based on review of X‐inactivation patterns in female carriers of other X‐linked recessive diseases, our findings imply that substantial expression of a mutant ATP7A at the expense of the normal allele could be associated with neurologic symptoms in female carriers of Menkes disease and its allelic variants, occipital horn syndrome, and ATP7A‐related distal motor neuropathy.     

An assay for X inactivation based on differential methylation at the fragile X locus,FMR1

We describe an assay analyzing methylation at the fragile X mental retardation gene, FMR1, to examine patterns of random or non-random X chromosome inactivation. Digestion of genomic DNA with the methylation-sensitive enzyme HpaII cleaves two restriction sites near the CGG repeat of the FMR1 gene if they are unmethylated on the active X chromosome, but fails to digest these sites on the inactive chromosome. Subsequent PCR using primers that flank the sites and the variable CGG repeat within the FMR1 gene amplifies alleles only on undigested, methylated inactive X chromosomes. Amplification of the hypervariable CGG repeat distinguishes alleles in heterozygous samples, while the relative ratio of alleles within a HpaII-digested sample reflects the randomness or non-randomness of inactivation. To demonstrate that methylation of the HpaII sites within the amplified FMR1 fragment correlates strictly with the activity state of the X chromosome, we have tested the validity of this assay by comparing DNA from normal males and females, as well as DNA from mouse/human somatic cell hybrids carrying either active or inactive human X chromosomes. The data demonstrate that this assay provides a reliable means of assessing the inactivation status of X chromosomes in individuals with X-linked disorders or X chromosome abnormalities. © 1996 Wiley-Liss, Inc.     

Microduplication of Xp11.23p11.3 with effects on cognition,behavior, and craniofacial development

El‐Hattab AW, Bournat J, Eng PA, Wu JBS, Walker BA, Stankiewicz P, Cheung SW, Brown CW. Microduplication of Xp11.23p11.3 with effects on cognition, behavior, and craniofacial development. We report an ～1.3 Mb tandem duplication at Xp11.23p11.3 in an 11‐year‐old boy with pleasant personality, hyperactivity, learning and visual‐spatial difficulties, relative microcephaly, long face, stellate iris pattern, and periorbital fullness. This clinical presentation is milder and distinct from that of patients with partially overlapping Xp11.22p11.23 duplications which have been described in males and females with intellectual disability, language delay, autistic behaviors, and seizures. The duplicated region harbors three known X‐linked mental retardation genes: FTSJ1, ZNF81, and SYN1. Quantitative polymerase chain reaction from whole blood total RNA showed increased expression of three genes located in the duplicated region: EBP, WDR13, and ZNF81. Thus, over‐expression of genes in the interval may contribute to the observed phenotype. Many of the features seen in this patient are present in individuals with Williams‐Beuren syndrome (WBS). Interestingly, the SYN1 gene within the duplicated interval, as well as the STX1A gene, within the WBS critical region, co‐localize to presynaptic active zones, and play important roles in neurotransmitter release.     

Female factor IX deficiency due to maternally inherited X‐inactivation

Esquilin JM, Takemoto CM, Green, NS. Female factor IX deficiency due to maternally inherited X‐inactivation. X‐chromosome inactivation is normally a random event that is regulated by the X chromosome itself. Rarely, females are affected by X‐linked disorders from extremely skewed X‐chromosome inactivation. Here, we report a family with hemophilia B with female expression through inherited X skewing that appears to be independent of either X chromosome. This finding suggests the possibility of a dominant autosomal contribution to inherited skewed X inactivation.     

Craniofrontonasal syndrome in a male due to chromosomal mosaicism involving EFNB1: further insights into a genetic paradox

Craniofrontonasal syndrome (CFNS) is an X‐linked disorder caused by inactivating mutations in the gene for ephrin‐B1 (EFNB1). Paradoxically it shows a more severe phenotype in females than in males. As a result of X inactivation cell populations with and without EFNB1 expression are found in EFNB1+/? females. This is thought to initiate a process termed cellular interference which may be responsible for the phenotype in females. We present a boy with severe clinical features of CFNS. In ～42% of his blood cells we found a supernumerary ring X chromosome containing EFNB1 but lacking XIST. Mosaicism for cell populations with different levels of EFNB1 expression can explain the severe phenotype of this patient. In vitro experiments in Xenopus tissue showed that cells overexpress ephrinB1 cluster and sort out from wild‐type cells. Our report provides further evidence that cellular interference contributes to the paradoxical inheritance pattern of CFNS.     

A heterozygous mutation in RPGR associated with X‐linked retinitis pigmentosa in a patient with Turner syndrome mosaicism (45,X/46,XX)

Turner syndrome with retinitis pigmentosa (RP) is rare, with only three cases reported based on clinical examination alone. We summarized the 4‐year follow‐up and molecular findings in a 28‐year‐old patient with Turner syndrome and the typical features of short stature and neck webbing, who also had X‐linked RP. Her main complaints were night blindness and progressive loss of vision since the age of 9 years. Ophthalmologic examination, optical coherent tomographic imaging, and visual electrophysiology tests showed classic manifestations of RP. The karyotype of peripheral blood showed mosaicism (45,X [72%]/46,XX[28%]). A novel heterozygous frameshift mutation (c.2403_2406delAGAG, p.T801fsX812) in the RP GTPase regulator (RPGR) gene was detected using next generation sequencing and validated by Sanger sequencing. We believe that this is the first report of X‐linked RP in a patient with Turner syndrome associated with mosaicism, and an RPGR heterozygous mutation. We hypothesize that X‐linked RP in this woman is not related to Turner syndrome, but may be a manifestation of the lack of a normal paternal X chromosome with intact but mutated RPGR.     

Short stature in a mother and daughter caused by familial der(X)t(X;X)(p22.1‐3;q26)

Deletions of the terminal Xp regions, including the short‐stature homeobox (SHOX) gene, were described in families with hereditary Turner syndrome and Léri‐Weill syndrome. We report on a 10‐2/12‐year‐old girl and her 37‐year‐old mother with short stature and no other phenotypic symptoms. In the daugther, additional chromosome material was detected in the pseudoautosomal region of one X chromosome (46,X,add(Xp.22.3)) by chromosome banding analysis. The elongation of the X chromosome consisted of Giemsa dark and bright bands with a length one‐fifth of the size of Xp. The karyotype of the mother demonstrated chromosome mosaicism with three cell lines (46,X,add(X)(p22.3) [89]; 45,X [8]; and 47,X,add(X)(p22.3), add(X)(p22.3) [2]). In both daughter and mother, fluorescence in situ hybridization (FISH), together with data from G banding, identified the breakpoints in Xp22.1‐3 and Xq26, resulting in a partial trisomy of the terminal region of Xq (Xq26‐qter) and a monosomy of the pseudoautosomal region (Xp22.3) with the SHOX gene and the proximal region Xp22.1‐3, including the steroidsulfatase gene (STS) and the Kallmann syndrome region. The derivative X chromosome was defined as ish.der(X)t(X;X)(p22.1‐3;q26)(yWXD2540‐, F20cos‐, STS‐, 60C10‐, 959D10‐, 2771+, cos9++). In daughter and mother, the monosomy of region Xp22.1‐3 is compatible with fertility and does not cause any other somatic stigmata of the Turner syndrome or Léri‐Weill syndrome, except for short stature due to monosomy of the SHOX gene. © 2001 Wiley‐Liss, Inc.     

X‐chromosome duplications in males with mental retardation: pathogenic or benign variants?

Gijsbers ACJ, den Hollander NS, Helderman‐van de Enden ATJM, Schuurs‐Hoeijmakers JHM, Vijfhuizen L, Bijlsma EK, van Haeringen A, Hansson KBM, Bakker E, Breuning MH, Ruivenkamp CAL. X‐chromosome duplications in males with mental retardation: pathogenic or benign variants? Studies to identify copy number variants (CNVs) on the X‐chromosome have revealed novel genes important in the causation of X‐linked mental retardation (XLMR). Still, for many CNVs it is unclear whether they are associated with disease or are benign variants. We describe six different CNVs on the X‐chromosome in five male patients with mental retardation that were identified by conventional karyotyping and single nucleotide polymorphism array analysis. One deletion and five duplications ranging in size from 325 kb to 12.5 Mb were observed. Five CNVs were maternally inherited and one occurred de novo. We discuss the involvement of potential candidate genes and focus on the complexity of X‐chromosomal duplications in males inherited from healthy mothers with different X‐inactivation patterns. Based on size and/or the presence of XLMR genes we were able to classify CNVs as pathogenic in two patients. However, it remains difficult to decide if the CNVs in the other three patients are pathogenic or benign.     

Evidence of skewed X‐chromosome inactivation in 47,XXY and 48,XXYY Klinefelter patients

Klinefelter (47,XXY) syndrome occurs in approximately 1:800 male births and accounts for about 10‐20% of males attending infertility clinics. Recent studies have shown no obvious phenotypic differences between subjects in which the extra X‐chromosome is of paternal or maternal origin; however, a minority of Klinefelter patients are adversely affected clinically and intellectually to an exceptional level, and the underlying basis of this phenotypic variation is not known. We hypothesize that skewed X‐inactivation and possibly parental origin of the X‐chromosomes is involved. In this study, we determined parental origin and inactivation status of the X‐chromosomes in 17 cytogenetically confirmed 47,XXY cases, two 48,XXYY cases and one mosaic 46,XY/47,XXY case. Eight highly polymorphic markers specific to the X‐chromosome and the polymorphic human androgen‐receptor (HUMARA) methylation assay were used to determine the parental origin and X‐inactivation status of the X‐chromosomes, respectively. Overall, 17 cases were fully informative, enabling parental origin to be assigned. In 59% of cases, both X‐chromosomes were of maternal origin (X^m); in the remaining 41%, one X was of maternal (X^m) and one was of paternal origin (X^p). In 5 of 16 (31%) cases informative at the HUMARA locus, skewed X‐inactivation was observed as defined by greater than 80% preferential inactivation involving one of the two X‐chromosomes. The two 48,X^mX^pYY cases both showed preferential paternal X‐chromosome (X^p) inactivation. Three 47,X^mX^mY cases also showed preferential inactivation in one of the two maternal X‐chromosomes. These results suggest that skewed X‐inactivation in Klinefelter (47,XXY and 48,XXYY) patients may be common and could explain the wide range of mental deficiency and phenotypic abnormalities observed in this disorder. Further studies are warranted to examine the role of X‐inactivation and genetic imprinting in Klinefelter patients. © 2001 Wiley‐Liss, Inc.     

Expanding the clinical spectrum of the ‘HDAC8‐phenotype’ – implications for molecular diagnostics,counseling and risk prediction

Cornelia de Lange syndrome (CdLS) is a clinically heterogeneous disorder characterized by typical facial dysmorphism, cognitive impairment and multiple congenital anomalies. Approximately 75% of patients carry a variant in one of the five cohesin‐related genes NIPBL, SMC1A, SMC3, RAD21 and HDAC8. Herein we report on the clinical and molecular characterization of 11 patients carrying 10 distinct variants in HDAC8. Given the high number of variants identified so far, we advise sequencing of HDAC8 as an indispensable part of the routine molecular diagnostic for patients with CdLS or CdLS‐overlapping features. The phenotype of our patients is very broad, whereas males tend to be more severely affected than females, who instead often present with less canonical CdLS features. The extensive clinical variability observed in the heterozygous females might be at least partially associated with a completely skewed X‐inactivation, observed in seven out of eight female patients. Our cohort also includes two affected siblings whose unaffected mother was found to be mosaic for the causative mutation inherited to both affected children. This further supports the urgent need for an integration of highly sensitive sequencing technology to allow an appropriate molecular diagnostic, genetic counseling and risk prediction.