Deletions of a differentially methylated CpG island at the SNRPN gene define a putative imprinting control region.

To determine the molecular basis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS), we have isolated new transcripts from chromosome 15q11-q13. Two novel transcripts located within 300 kilobases telomeric to the small nuclear ribonucleoprotein-associated polypeptide N gene (SNRPN) were paternally expressed in cultured cells, along with SNRPN, defining a large imprinted transcriptional domain. In three PWS patients (two sibs), small deletions remove a differentially methylated CpG island containing a newly described 5' exon alpha of SNRPN, and cause loss of expression for the three imprinted transcripts and altered methylation over hundreds of kilobases. The smallest PWS deletion is familial and asymptomatic with maternal transmission. Our data imply the presence of a paternal imprinting control region near exon alpha.

A homeobox gene, HLXB9, is the major locus for dominantly inherited sacral agenesis.

Partial absence of the sacrum is a rare congenital defect which also occurs as an autosomal dominant trait; association with anterior meningocoele, presacral teratoma and anorectal abnormalities constitutes the Currarino triad (MIM 176450). Malformation at the caudal end of the developing notochord at approximately Carnegie stage 7 (16 post-ovulatory days), which results in aberrant secondary neurulation, can explain the observed pattern of anomalies. We previously reported linkage to 7q36 markers in two dominantly inherited sacral agenesis families. We now present data refining the initial subchromosomal localization in several additional hereditary sacral agenesis (HSA) families. We excluded several candidate genes before identifying patient-specific mutations in a homeobox gene, HLXB9, which was previously reported to map to 1q41-q42.1 and to be expressed in lymphoid and pancreatic tissues.

Increased skewing of X chromosome inactivation with age in both blood and buccal cells.

The X chromosome inactivation pattern in peripheral blood cells becomes more skewed after age 55, and a genetic effect on this age-related skewing has been reported. We investigated the effect of age on X inactivation phenotype in blood, buccal cells and tissue from duodenal biopsies in 80 females aged 19-90 years. The X inactivation pattern correlated positively with age in blood (r = 0.238, P = 0.034) and buccal cells (r = 0.260, P = 0.02). The mean degree of skewing was higher in the elderly (>/=55 years) than in the young (<55 years) in blood (70.1 and 63.5%, respectively, P = 0.013) and in buccal cells (64.7 and 59.0%, respectively, P = 0.004). Correlation of X inactivation between the different tissues was high in all tissues with a tendency to increase with age for blood and buccal cells (P = 0.082). None of the duodenal biopsies had a skewed X inactivation, and the mean degree of skewing was similar in the two age groups. The tendency for the same X chromosome to be the preferentially active X in both blood and buccal cells with advancing age is in agreement with a genetic effect on age-related skewing and indicates that genes other than those involved in hematopoiesis should be investigated in the search for genes contributing to age related skewing.

X chromosome inactivation in females with multiple sclerosis.

The aetiology of multiple sclerosis (MS) is unknown. Autoimmune mechanisms are most probably involved. Loss of immunological tolerance to self-antigens is a common feature of autoimmune disorders. Response to X-linked self-antigens could be influenced by X-chromosome inactivation, and contribute to the gender bias observed in autoimmune disorders. Previous studies have indicated an association between skewed X inactivation and autoimmune thyroid disease and scleroderma. To investigate a potential role of X inactivation in MS, we compared the X-inactivation pattern in 568 female MS patients with controls. We found no difference in degree of skewing between patients (median 64%) and controls (median 65%) (P = 0.474). The X-inactivation pattern did thus not explain the female predominance of MS patients in general. As the aetiology of different subgroups of MS may differ, patients were grouped according to disease course: relapsing-remitting (RR-MS), secondary progressive (SP-MS) and primary progressive (PP-MS). A comparison of the X-inactivation pattern between subgroups indicated a possible difference in degree of skewing between patients with a progressive versus a relapsing course (P = 0.05).

High incidence of skewed X chromosome inactivation in young patients with familial non-BRCA1/BRCA2 breast cancer.

BACKGROUND: A higher frequency of skewed X chromosome inactivation has been reported in a consecutive series of young patients with breast cancer compared with controls of a similar age. OBJECTIVE: To investigate the X inactivation pattern in patients with familial non-BRCA1/BRCA2 breast cancer (n = 272), BRCA1/BRCA2 germline mutations (n = 35), and sporadic breast cancer (n = 292). METHODS: X inactivation pattern was determined by polymerase chain reaction analysis of the highly polymorphic CAG repeat in the androgen receptor (AR) gene. The X inactivation pattern was classified as skewed when 90% or more of the cells preferentially expressed one X chromosome. RESULTS: Young patients with familial breast cancer had a significantly higher frequency of skewed X inactivation (11.2%) than young controls (2.7%) (p = 0.001). There was also a strong tendency for middle aged patients with sporadic breast cancer to be more skewed than middle aged controls (13.6% v 4.4%) (p = 0.02). No association between skewed X inactivation and breast cancer was found for the BRCA1/BRCA2 patients . CONCLUSIONS: Skewed X inactivation may be a risk factor for the development of breast cancer in both sporadic and familial breast cancer and may indicate an effect of X linked genes.

High frequency of skewed X inactivation in young breast cancer patients.

INTRODUCTION: Patients with invasive ovarian cancer were recently shown to have a higher frequency of skewed X chromosome inactivation in peripheral blood cells compared to patients with borderline cancer and controls. In this study, we analysed the X inactivation pattern in peripheral blood from 216 breast cancer patients. METHODS: X inactivation analysis was performed using HpaII predigestion of DNA followed by PCR of the highly polymorphic CAG repeat of the androgen receptor gene (AR), which amplifies the undigested inactive X chromosome only. The X inactivation pattern was classified as skewed when 90% or more of the cells preferentially used one X chromosome. RESULTS: Young breast cancer patients (27-45 years) had a higher frequency of skewed X inactivation than young controls (13 and 1%, respectively) (p=0.009), whereas no difference was found for middle aged and older patients compared to controls of a similar age. CONCLUSIONS: A germline mutation in an X linked tumour suppressor gene may give a proliferative advantage to cells with this mutation on the active X chromosome, thus causing skewed X inactivation and an increased risk for developing cancer. Another possible explanation could be that females with a constitutionally skewed X inactivation pattern are more susceptible to develop breast cancer because of an X linked low penetrance susceptibility allele that is affected by the inactivation pattern.

Early and severe presentation of X-linked myotubular myopathy in a girl with skewed X-inactivation.

X-linked myotubular myopathy is a severe congenital myopathy in males, caused by mutations in the myotubularin (MTM1) gene on chromosome Xq28. In heterozygous carriers of MTM1 mutations, clinical symptoms are usually absent or only mild. We report a 6-year-old girl presenting at birth with marked hypotonia and associated feeding and respiratory difficulties. A muscle biopsy performed at 5 months suggested a diagnosis of myotubular myopathy. On examination at 6 years she had marked facial weakness with bilateral ptosis and external ophthalmoplegia, severe axial and proximal weakness and a mild scoliosis. Muscle magnetic resonance imaging showed a distinctive pattern of muscle involvement. Molecular genetic investigation of the MTM1 gene identified a heterozygous mutation in exon 12. X-inactivation studies in lymphocytes showed an extremely skewed pattern (97:3). This case emphasizes that investigation of the MTM1 gene and X-inactivation studies are indicated in isolated females with histopathological and clinical findings suggestive of myotubular myopathy.

X-inactivation patterns in carriers of X-linked myotubular myopathy.

X-linked myotubular myopathy is a rare severe muscle disorder in affected male neonates. Most female carriers are free from symptoms. Skewed X inactivation has been proposed to be responsible for the affected phenotype seen in some carriers. We have compared the X inactivation patterns in blood DNA with the clinical phenotype in carriers of X-linked myotubular myopathy. The X-inactivation analysis was performed using HpaII predigestion of DNA followed by polymerase chain reaction of the highly polymorphic CAG repeat of the androgen receptor (AR) gene. The frequency of skewed X inactivation was similar in the X-linked myotubular myopathy carriers (22%) and in 235 controls (18%). Three overtly affected carriers had skewed X inactivation with the mutated X as the predominantly active X in at least two of them. Four females with mild symptoms had random X inactivation. The unaffected X-linked myotubular myopathy carriers had either skewed X inactivation in favour of expression from the normal X or random X-inactivation. Thus, there was a tendency for females with a more severe phenotype to have a skewed pattern of X inactivation, while females with an intermediate phenotype had a random pattern of X-inactivation.

Factor IX alloantibodies shorten the bovine thromboplastin coagulation time of normal human plasma.

We have previously demonstrated that neutralization of factor IX in normal plasma by heterologous antisera shortens the one stage prothrombin time determined with bovine thromboplastin. In this study, a similar effect of homologous antibodies was demonstrated. Addition of plasma from two patients with hemophilia B- and an acquired inhibitor to factor IX gave a shortening of the prothrombin time of plasma from normal persons, compared to the prothrombin time determined after addition of control plasma from a patient with hemophilia B- and no inhibitor. Addition of inhibitor plasma had a similar effect on the prothrombin time of plasma from four patients with hemophilia B+ and one patient with hemophilia BM, but had no effect on the prothrombin time of plasma from ten patients with hemophilia B-. Complexes between factor IX and the human inhibitor could be demonstrated both before and after the coagulation with bovine thromboplastin. These complexes were demonstrated as a factor IX antigen with a reduced electrophoretic mobility in crossed immunoelectrophoresis against a rabbit antiserum to factor IX. The results demonstrate that normal factor IX loses the ability to act as an inhibitor in the coagulation with bovine thromboplastin after having formed a soluble complex with a homologous antibody, although the factor IX molecules still have antigenic determinants which are free to react with the rabbit antibodies.

Absence of correlation between X chromosome inactivation pattern and plasma concentration of factor VIII and factor IX in carriers of haemophilia A and B.

Haemophilia A and B are X-linked disorders which are due to a reduced activity of coagulation factor VIII or IX, respectively. Female carriers have a wide range of plasma concentration of factor VIII or factor IX, and may in rare cases have an affected phenotype. In order to investigate if this variation is related to X chromosome inactivation, we determined the X inactivation pattern in 31 haemophilia A and 15 haemophilia B carriers, using a PCR in the androgen receptor locus in blood DNA. Seven of the haemophilia A carriers and none of the haemophilia B carriers had a skewed pattern (> or =80:20). One of the skewed haemophilia A carriers had a low plasma concentration of factor VIII (0.15 U/ml), but the remaining 6 carriers did not differ in factor VIII concentration from that of carriers with a random X inactivation pattern. One carrier with a high factor VIII concentration (2.0 U/ml) did not have a skewed pattern. Similarly, for the haemophilia B carriers, there was no tendency to a more skewed X inactivation pattern in the carriers with low or high factor IX concentrations. In addition, we analysed a female with haemophilia B who was heterozygous for the mutation R180W in the factor IX gene. She had a random X chromosome inactivation pattern. We conclude that the wide range in plasma concentration of factor VIII and factor IX in haemophilia A and B carriers cannot in general be explained by the X chromosome inactivation pattern in peripheral blood cells.

Mutation spectrum in patients with Wiskott-Aldrich syndrome and X-linked thrombocytopenia: identification of twelve different mutations in the WASP gene.

Twelve different mutations in the WASP gene were found in twelve unrelated families with Wiskott-Aldrich syndrome (WAS) or X-linked thrombocytopenia (XLT). Four frameshift, one splice, one nonsense mutation, and one 18-base-pair deletion were detected in seven patients with WAS. Only missense mutations were found in five patients diagnosed as having XLT. One of the nucleotide substitutions in exon 2 (codon 86) results in an Arg to Cys replacement. Two other nucleotide substitutions in this codon, R86L and R86H, have been reported previously, both giving rise to typical WAS symptoms, indicating a mutational hot spot in this codon. The finding of mutations in the WASP gene in both WAS and XLT gives further evidence of these syndromes being allelic. The relatively small size of the WASP gene facilitates the detection of mutations and a reliable diagnosis of both carriers and affected fetuses in families with WAS or XLT.

Inheritance of skewed X chromosome inactivation in a large family with an X-linked recessive deafness syndrome.

A new X-linked recessive deafness syndrome was recently reported and mapped to Xq22 (Mohr-Tranebjaerg syndrome). In addition to deafness, the patients had visual impairment, dystonia, fractures, and mental deterioration. The female carriers did not have any significant manifestations of the syndrome. We examined X chromosome inactivation in 8 obligate and 12 possible carriers by using a polymerase chain reaction analysis of the methylation-dependent amplification of the polymorphic triplet repeat at the androgen receptor locus. Seven of 8 obligate carriers and 1 of 5 carriers by linkage analysis had an extremely skewed pattern in blood DNA not found in 30 normal females. The X inactivation pattern in fibroblast DNA from 2 of the carriers with the extremely skewed pattern was also skewed but to a lesser degree than in blood DNA. One obligate carrier had a random X inactivation pattern in both blood and fibroblast DNA. A selection mechanism for the skewed pattern is therefore not likely. The extremely skewed X inactivation in 8 females of 3 generations in this family may be caused by a single gene that influences skewing of X chromosome inactivation.

Non-random X chromosome inactivation in an affected twin in a monozygotic twin pair discordant for Wiedemann-Beckwith syndrome.

Wiedemann-Beckwith syndrome (WBS) is a syndrome including exomphalos, macroglossia, and generalized overgrowth. The locus has been assigned to 11p15.5, and genomic imprinting may play a part in the expression of one or more genes involved. Most cases are sporadic. An excess of female monozygotic twins discordant for WBS have been reported, and it has been proposed that this excess could be related to the process of X chromosome inactivation. We have therefore studied X chromosome inactivation in 13-year-old monozygotic twin girls who were discordant for WBS. In addition, both twins had Tourette syndrome. The twins were monochorionic and therefore the result of a late twinning process. This has also been the case in previously reported discordant twin pairs with information on placentation. X chromosome inactivation was determined in DNA from peripheral blood cells by PCR analysis at the androgen receptor locus. The affected twin had a completely skewed X inactivation, where the paternal allele was on the active X chromosome in all cells. The unaffected twin had a moderately skewed X inactivation in the same direction, whereas the mother had a random pattern. Further studies are necessary to establish a possible association between the expression of WBS and X chromosome inactivation.

Sibs with Ritscher-Schinzel (3C) syndrome and anal malformations.

Ritscher-Schinzel syndrome (cranio-cerebello-cardiac syndrome, 3C syndrome) is a recently delineated disorder with Dandy-Walker malformation, congenital heart defects, and characteristic face. Various other defects, including eye and kidney malformations, have been described in the few patients reported. Here we describe 3 sibs born to consanguineous Pakistani parents with 3C syndrome. All 3 children had atrial septal defects II and ventricular septal defects and died within 3 months. Two of them had a Dandy-Walker malformation, whereas 1 had only slightly dilated ventricles. One sib had anal atresia, and another a ventrally displaced anus. The findings in the 3 sibs demonstrate the intrafamilial variation in the Ritscher-Schinzel syndrome, because the second sib did not have a Dandy-Walker malformation. Anal anomalies have not been previously reported as a component manifestation of the disorder. The occurrence of 3 affected sibs in a consanguineous family confirms autosomal recessive inheritance.

VACTERL or MURCS association in a girl with neurenteric cyst and identical thoracic malformations in the father: a case of gonosomal mosaicism?

We report on a female infant with lethal congenital malformations including extreme hydrocephalus due to aqueductal stenosis, vertebral segmentation anomalies, fused costae, anal atresia, renal dysplasia, and bicornuate uterus with a double blind vagina. The VACTERL and the MURCS associations are possible diagnoses. Her father had a neurenteric cyst in infancy. He has identical vertebral and costal malformations as his daughter but is otherwise healthy. The possibility of dominant inheritance with gonosomal mosaicism in the father is discussed.

Aplasia cutis congenita associated with limb, eye, and brain anomalies in sibs: a variant of the Adams-Oliver syndrome?

Aplasia cutis congenita (ACC) may occur in isolation or with other congenital malformations. Peripheral limb anomalies and ACC are major elements of the Adams-Oliver syndrome, which is usually inherited as an autosomal dominant disorder. We report on a sister and brother with ACC and brain, eyes, and transverse limb anomalies. The phalanges of the hands and feet were either short or absent. The girl also had absence of right patella, was severely mentally retarded and blind with retinal nonattachment. The boy had a falciform fold in the left eye. He died at age one week and autopsy showed partial agenesis of corpus callosum. The findings in the sibs may represent a severe variant of the Adams-Oliver syndrome, or a previously unrecognized syndrome involving vascular disruption.

Sibs with anencephaly, anophthalmia, clefts, omphalocele, and polydactyly: hydrolethalus or acrocallosal syndrome?

Major characteristics of the acrocallosal syndrome include severe mental retardation, agenesis or hypoplasia of the corpus callosum, and polydactyly of fingers and toes. In the past few years, anencephaly has also been noted, together with other midline defects. We report on a nonconsanguineous, Norwegian couple with a history of two pregnancies with a male and a female fetus, respectively, with anencephaly, median cleft lip and palate, omphalocele, and preaxial polydactyly, suggesting the diagnosis of the acrocallosal syndrome. Both fetuses also lacked eyes and nose, a finding not previously reported in the acrocallosal syndrome. Microphthalmia has been reported in the hydrolethalus syndrome, which may be caused by mutations in the same gene as the acrocallosal syndrome. The present report adds support to the hypothesis that the acrocallosal and hydrolethalus syndromes may be allelic conditions. The family history is consistent with autosomal recessive inheritance.

Severe craniofacial malformations and deglutition dysfunction in a brother and sister: new syndrome?

We report seemingly unique craniofacial malformations and deglutition dysfunction in a sib pair. The boy had right maxillomandibular alveolar synechae, ankylosis of right temporomandibular joint, hypoplasia of the zygomatico-maxillary region, nasal deviation to the left, choanal stenosis, and exophthalmos due to shallow orbita. His ears were apparently low-set with prominent lobules. He had severe gastroesophageal reflux and increasing respiratory problems and died at age 11 months. Psychomotor development was normal. His 10-year-old sister had similar craniofacial malformations and a cleft soft palate. She also had a severe deglutition dysfunction and developed a thoracolumbar kyphoscoliosis. Psychomotor development was normal. The parents were healthy and non-consanguineous. The malformations in the sibs do not fit any reported craniofacial malformation syndrome and may represent a previously unrecognized monogenic disorder. This may be an autosomal recessive or dominant trait with gonadal mosaicism in one of the parents.

Prader-Willi syndrome in a brother and sister without cytogenetic or detectable molecular genetic abnormality at chromosome 15q11q13.

We report on a 12-year-old boy and his 7-year-old sister with the Prader-Willi syndrome. They both had severe initial hypotonia with feeding problems and later developed an increasing appetite. Both sibs have almond-shaped eyes, triangular mouth, hypogonadism, retarded growth, and mental retardation. An older brother suffered from severe hypotonia and died at 7 days of age. The children have normal chromosomes by high-resolution technique and have inherited the same chromosomes 15 short arm polymorphisms from their parents. The family was informative for one of four DNA markers specific for the 15q11q13 region. No deletion was found using this marker. The parents were healthy and unrelated. Autosomal recessive inheritance or a paternally inherited submicroscopic deletion are possible explanations for the sib occurrence in this family.

The X chromosome and the female survival advantage: an example of the intersection between genetics, epidemiology and demography.

Despite differences in research traditions, the disciplines of genetics, epidemiology, and demography are becoming increasingly integrated in health-related research. The enormous development within genetic technology, with the possibility of genotyping thousands of variants from small samples of biological material obtained by non-invasive methods, now makes it feasible to include genetic information in epidemiologic and demographic studies. Simultaneously, new insight can be obtained from hybrids of methods and data from the three disciplines. This paper illustrates how a genetic observation combined with demographic insight and a modified genetic-epidemiologic design (a twin study) provides evidence that part of the sex difference in survival can be attributed to the fact that females have two X chromosomes and males have only one, a result that is of potential interest for genetics, epidemiology, and demography.