Carrier detection in typical and atypical X-linked agammaglobulinemia

We have recently demonstrated that B cells from obligate carriers of typical X-linked agammaglobulinemia (XLA) exhibit nonrandom X chromosome inactivation. The active X is always the X that does not carry the gene defect. To determine if this were also true in carriers of atypical XLA and to provide carrier detection for all women at risk of being carriers of XLA, we developed a technique that permits analysis of X chromosome inactivation in cells from any woman. This technique combines the production of somatic cell hybrids that selectively retain the active X chromosome with the use of X-linked restriction fragment length polymorphisms that permit the distinction of the two X chromosomes. Three obligate carriers of typical XLA and four women whose sons might be considered to have atypical or sporadic XLA were studied. B cell hybrids from all seven women demonstrated exclusive use a single X as the active X. In addition, B cell hybrids from four of eight women at 25% or 50% risk of being carriers exhibited nonrandom X chromosome inactivation, indicating that these women were also carriers of X-linked forms of hypogammaglobulinemia. These results illustrate a technique that can be used both to help define XLA and to provide carrier detection for all women at risk of being carriers of this disorder.     

X chromosome inactivation analysis to distinguish sporadic cases of X-linked agammaglobulinaemia from common variable immunodeficiency

The X chromosome inactivation analysis of eight female relatives was performed to elucidate the X chromosome gene defect of six male hypogammaglobulinaemic individuals. The patients had diminished numbers of circulating B-cells and no relevant family history. The methylation status of three X-linked genes, phosphoglycerate kinase, hypoxanthine phosphoribosyl transferase and DXS255, was determined on DNA from Epstein-Barr virus-transformed B-cell lines established from the female relatives. The methylation pattern of at least one gene was informative in all eight females examined. While both alleles were equally methylated in four of eight females, the remaining four female relatives of three hypogammaglobulinaemia patients exhibited a non-random methylation pattern in their B-cells, suggesting that these three patients represented sporadic cases of X-linked agammaglobulinaemia (XLA). The clinical or immunological status of these three patients did not differ from the remaining two who had early onset hypogammaglobulinaemia and who were tentatively diagnosed as having common variable immunodeficiency. The sixth patient had recurrent infections after undergoing surgical removal of a brain tumour at 22 years of age, although his immunological features did not distinguish him from the other patients. X chromosome inactivation analysis can be useful in differentiating XLA from hypogammaglobulinaemia in male patients.     

Carrier Detection in Agammaglobulinemia by X Chromosome Inactivation Analysis

Using a recently developed strategy to analyze patterns of X chromosome inactivation in cell populations, we found that two mothers and a sister were carriers in three atypical or sporadic cases of patients with agammaglobulinemia, two of whom were brothers. In this study, a phosphogiycerate kinase 1 (PGK1) gene probe was used to detect patterns of methylation of X-chromosome genes. A random pattern of X inactivation was observed in isolated peripheral blood granulocytes. In contrast, one of the two X chromosomes was preferentially active in the Epstein-Barr virus (EBV)-transformed peripheral B cells of the family members of these patients. The volume of the blood specimen could be significantly reduced using EBV-transfomed B cell lines which contained multiple clones. The analysis described here can be used to distinguish between X-linked agammaglobulinemia (XLA) and other forms of a- or hypogammaglobulinemia as well as to detect the carrier state.     

Skewed X inactivation in healthy individuals and in different diseases

In female mammalian cells, one of the two X chromosomes is inactivated in early embryonic life. Females are mosaics for two cell populations, one with the maternal and one with the paternal X as the active chromosome. Skewed X inactivation is arbitrarily defined, often as a pattern where 80% or more of the cells show a preferential inactivation of one X chromosome. Inactivation is presumed to be permanent for all descendants of a cell; however, after about 55 years of age, the frequency of skewed X inactivation in peripheral blood cells increases, probably through selection. Unfavourable skewing of X inactivation, where the X chromosome carrying a mutant allele is the predominantly active X, has been found in affected female carriers of several X-linked disorders; however, for many X-linked disorders, a consistent relationship between the pattern of X inactivation and clinical phenotype has been difficult to demonstrate. One reason for this may be that peripheral blood cells are not a representative or relevant tissue in many disorders. In some severe X-linked disorders, post-inactivation selection takes place against the X chromosome carrying the mutant allele, leading to a completely skewed X-inactivation pattern. Skewed X inactivation has also been reported in young females with breast cancer, and may indicate an effect of X-linked genes on the development of this condition.Conclusion: The process of X inactivation and the resultant degree of skewing is clearly important for the expression of genetic diseases. It is also important to consider, however, that under normal conditions the frequency of skewed X inactivation increases with age in peripheral blood cells. Analysis of the expression of a large proportion of the genes on the X chromosome has revealed that X-chromosome inactivation is more heterogeneous than previously thought.     

Skewed X inactivation in healthy individuals and in different diseases

In female mammalian cells, one of the two X chromosomes is inactivated in early embryonic life. Females are mosaics for two cell populations, one with the maternal and one with the paternal X as the active chromosome. Skewed X inactivation is arbitrarily defined, often as a pattern where 80% or more of the cells show a preferential inactivation of one X chromosome. Inactivation is presumed to be permanent for all descendants of a cell; however, after about 55 years of age, the frequency of skewed X inactivation in peripheral blood cells increases, probably through selection. Unfavourable skewing of X inactivation, where the X chromosome carrying a mutant allele is the predominantly active X, has been found in affected female carriers of several X-linked disorders; however, for many X-linked disorders, a consistent relationship between the pattern of X inactivation and clinical phenotype has been difficult to demonstrate. One reason for this may be that peripheral blood cells are not a representative or relevant tissue in many disorders. In some severe X-linked disorders, post-inactivation selection takes place against the X chromosome carrying the mutant allele, leading to a completely skewed X-inactivation pattern. Skewed X inactivation has also been reported in young females with breast cancer, and may indicate an effect of X-linked genes on the development of this condition.
Conclusion: The process of X inactivation and the resultant degree of skewing is clearly important for the expression of genetic diseases. It is also important to consider, however, that under normal conditions the frequency of skewed X inactivation increases with age in peripheral blood cells. Analysis of the expression of a large proportion of the genes on the X chromosome has revealed that X-chromosome inactivation is more heterogeneous than previously thought.     

X-chromosome inactivation: role in skin disease expression

The occurrence of X inactivation in mammals has the consequence that all women are functional mosaics. In X-linked skin disorders, Lyonization usually gives rise to a mosaic pattern, as manifest by the appearance of the lines of Blaschko. This arrangement of lesions is observed in male-lethal X-linked traits, such as incontinentia pigmenti, focal dermal hypoplasia, Conradi-Hünermann-Happle syndrome, oral-facial-digital syndrome type 1 and MIDAS (microphthalmia, dermal aplasia and sclerocornea) syndrome, as well as in various X-linked non-lethal phenotypes, such as hypohidrotic ectodermal dysplasia of Christ-Siemens-Touraine, IFAP (ichthyosis follicularis-alopecia-photophobia) syndrome and X-linked dyskeratosis congenita. Analogous X-inactivation patterns have been documented in human bones, teeth, eyes and, possibly, the brain. Patterns that are distinct from the lines of Blaschko are also seen, such as the lateralization observed in CHILD (congenital hemidysplasia with ichthyosiform nevus and limb defects) syndrome, and the chequerboard pattern seen in women heterozygous for X-linked congenital hypertrichosis. Exceptional cases of either severe or absent involvement in a woman heterozygous for an X-linked trait can be explained by skewing of X inactivation. Some X-linked skin disorders are caused by genes that escape inactivation, which is why heterozygous female 'carriers' of these disorders do not show mosaicism. A well-known example is X-linked recessive ichthyosis due to steroid sulphatase deficiency, the locus for which is situated at the tip of the short arm of the X chromosome and does not undergo Lyonization. On the other hand, in the case of Fabry disease, the gene encoding alpha-galactosidase A is subject to inactivation. Remarkably, however, the skin lesions of women do not show a mosaic pattern.Conclusion: In the various X-linked skin disorders, affected women show quite dissimilar degrees of involvement and forms of manifestation because X inactivation may give rise to different patterns of functional mosaicism. Paradoxically, no such pattern is observed in women with Fabry disease. Like many X-linked diseases, Fabry disease should neither be called recessive nor dominant, because these dichotomous terms are obscured by the mechanism of X inactivation.     

Prevalence of human growth hormone-1 gene deletions among patients with isolated growth hormone deficiency from different populations.

Familial isolated growth hormone deficiency type IA results from homozygosity for either a 6.7-kb or a 7.6-kb hGH-1 gene deletion. Genomic DNA was extracted from circulating lymphocytes of 78 subjects with severe isolated growth hormone deficiency (height less than -4.5 SD score) and studied by polymerase chain amplification and by restriction endonuclease analysis looking for gene deletions within the hGH-gene cluster. The individuals analyzed were broadly grouped into three different populations (North-European, n = 32; Mediterranean, n = 22; and Turkish, n = 24). Ten out of 78 patients studied presented with an hGH-1 gene deletion; eight out of these 10 showed a 6.7-kb gene deletion, the remaining two a 7.6-kb hGH-1 gene deletion. Five of the 10 subjects developed anti-hGH antibodies to hGH replacement followed by a stunted growth response. Family studies of the affected patients were performed, revealing consanguinity in all the families, and the corresponding heterozygosity for the deletion was present in each of the parents. The results of our study revealed a prevalence for an hGH-1 gene deletion in three out of 32 North-European, three out of 22 Mediterranean, and four out of 24 Turkish patients with growth hormone deficiency (height less than 4.5 SD score). These data are important for prenatal diagnosis of at-risk pregnancies and for families at risk for recurrence and underline clearly the fact that the hGH-I gene deletion represents a common cause for growth hormone deficiency associated with severe growth retardation (height less than -4.5 SD score).     

X-linked agammaglobulinemia, growth hormone deficiency and delay of growth and puberty

Buzi F, Notarangelo LD, Plebani A, Duse M, Parolini O, Monteleone M, Ugazio AG. X-linked agammaglobulinemia, growth hormone deficiency and delay of growth and puberty. Acta Paediatr 1994;83:99–102. Stockholm. ISSN 0803–5253
Coinheritance of X-linked agammaglobulinemia and growth hormone deficiency (XLA/GHD) has been classified as an independent primary immune deficiency. We evaluated the pattern of growth and endocrine function in seven XLA subjects (ages 10.9–20.1 years); four belonged to two different XLA pedigrees and three represented sporadic XLA cases. Three had reached adulthood (final stature 176.0, 173.5 and 165.0 cm, respectively) and their retrospective growth showed delay in growth and puberty during adolescence. In the other four subjects, growth hormone production was measured by growth hormone pharmacological stimulation tests (clonidine, arginine): three of four patients had insufficient growth hormone responses (peak growth hormone < 10 μg/l); all three had delayed puberty; their growth hormone responses increased after "priming" with testosterone, reaching values > 10 μg/l in two of them and allowing diagnosis of "true" growth hormone deficiency in the third. The fourth was a normally growing subject who showed a normal growth hormone response both before and after testosterone priming. Six out of the seven subjects showed a growth pattern consistent with delay in growth and puberty. Our results suggest that true XLA/GHD is rarer than previously supposed and that subnormal responses to growth hormone stimulation tests may be found without sex steroid priming of the test in adolescence. The most probable growth pattern in XLA appears to be delay in growth and puberty, as has already been described for other chronic diseases.     

Smith-Magenis syndrome and growth hormone deficiency

Smith-Magenis syndrome (SMS) is a multiple congenital anomaly/mental retardation syndrome including physical and neurobehavioural features. The disease is commonly associated with a ca. 3.7 Mb interstitial deletion of chromosome 17p11.2, while a 1.1 Mb critical region has been identified, containing about 20 genes expressed in multiple tissues. Haploinsufficiency of one of them, RAI1, seems to be responsible for the neurobehavioural, craniofacial and otolaryngological features of the syndrome, but not for short stature, commonly seen in SMS patients with chromosome deletion, implying the role of other genes in the 17p11.2 region. Growth failure is a final result of several different mechanisms involving decreased growth hormone (GH) production, reduced tissue response to GH, or impaired activity of epistatic factors. To our knowledge, the association of GH deficiency with SMS has never been reported and rarely investigated, despite the very short stature of SMS patients. We describe a girl with a full SMS phenotype and a typical 3.7 Mb deletion of 17p11.2 who also has GH deficiency. After starting replacement therapy, growth has significantly improved, her stature being now above both the 10th percentile and her genetic target. Conclusion:we suggest that an investigation of both growth hormone secretion and function is carried out in patients with Smith-Magenis syndrome and 17p11.2 deletion.Abbreviations FISH fluorescent in situ hybridisation - GH growth hormone - SMS Smith-Magenis syndrome     

Growth hormone releasing hormone receptor (GHRH-r) gene mutation in Indian children with familial isolated growth hormone deficiency: a study from western India

BACKGROUND: Various mutations of the growth hormone releasing hormone receptor (GHRH-R) gene have been recently described to cause familial isolated growth hormone (GH) deficiency (FIGHD), with the GHRH-R nonsense mutation E72X reported in patients with FIGHD from South Asia. The molecular genetic basis of FIGHD in Indian children is not known. Objective: To look for the GHRH-R E72X non-sense mutation in our patients with FIGHD and describe its clinical phenotype. PATIENTS AND METHOD: A total of 31 patients from 22 families diagnosed 4-20 years previously, 20 patients with familial IGHD-IB from 11 families and 11 patients with non-familial isolated GH deficiency (NFIGHD) (phenotypes IGHD-IB in eight patients and -IA in three) were included. Twenty-eight of 31 patients with IGHD-IB came from two states of Western India, 27 of them Hindus from 18 families (three consanguineous) and one from an inbred Moslem kindred. RESULTS: Twenty-two of the patients (71%) (18 FIGHD and four NFIGHD) had a homozygous G-->T transversion in exon 3, with this GHRH-R gene mutation E72X in 90% (18/20) of patients with FIGHD, 36% (4/11) of NFIGHD, altogether 78% (22/28) with phenotype IB. One parent pair with IGHD had homozygous E72X mutation, the rest were heterozygous carriers. Two siblings with IGHD due to homozygous E72X mutation were also heterozygous carriers for GH-1 gene 6.7 kb deletion, inherited from their mother, heterozygous for both GH-1 and GHRH-R mutations. Initial chronological age was 10.89 +/- 3.69 years, bone age 6.4 +/- 3.4 years, and mean height SDS was -5.83 +/- 1.41. The clinical phenotype, with sharp features, lean habitus, lack of frontal bossing or hypoglycemia, was characteristic. The mean peak GH was 1.25 +/- 0.75 ng/ml, IGF-I and IGFBP-3 below -2 SDS with no response to GHRH in those tested. MRI (n = 10) showed pituitary hypoplasia, mean vertical height 2.61 +/- 0.76 mm. Among the other 7/11 NFIGHD patients, four with phenotype IB were negative for genotypes tested in this study; of three patients with phenotype IA, two had the GH-1 gene 6.7 kb deletion, and one was a compound heterozygote with 6.7 and 7.6 kb deletions. CONCLUSIONS: The majority of patients with FIGHD from different communities belonged to non-consanguineous Hindu families from Western India. The GHRH-R gene E72X mutation was found in 71% of this series, in 90% of FIGHD, 36% of NFIGHD, and in 78% with phenotype IB. The characteristic phenotype helped in suspecting this mutation. GHRH-R gene mutations may be the most reasonable candidate for IGHD-IB with the E72X mutation predominating in the Indian subcontinent. More extensive studies need to be undertaken.     

Familial chromosome X structural aberrations - case report

The X chromosome contains about 1000 genes with different functions, both general and specialized, among which are genes responsible for normal function of the ovaries in women and normal growth in both sexes. Patients with chromosome X aberrations present a large spectrum of phenotypes depending on a variable size of chromosome X regions which are involved in aberrations, differences in genes located in/around given break-points, number of X chromosomes in cells and preferential X inactivation. We report on two families diagnosed with chromosome X aberration. The familial dupXq/delXp and the familial Xp deletion were diagnosed using GTG-banding techniques and confirmed by FISH analysis. In both families chromosome X aberrations were observed in healthy, fertile women, without a history of recurrent miscarriages or mentally retarded children. In one of the cases (family A) a mother with a mosaic X chromosome aberration [mos 45,X/46,X,der(X)(qter→q2? 7::p22.2→qter)], with previously undiagnosed Turner's syndrome, gave birth to her third daughter with a karyotype that contained only the structural X chromosome aberration, of maternal origin [46,X,der(X)(qter→q2? 7::p22.2→→ qter).ish der(X) (wcpX+, pter-, KAL-, qter++]. In the second case (family B) a mother [46,X,del(X)(p22.1p22.2).ish del(X)(wcpX+, pter+, KAL-, qter+)] gave birth to a girl with Down syndrome (21 trisomy), who has also had an additional chromosome X aberration [47,X,del(X)(p22.1p22.2),+21.ish del(X)(wcpX+, pter+, KAL-, qter+)]. Conclusions: Chromosome X aberrations found in women may sometimes not influence the incidence of phenotypic abnormalities and their fertility. In such cases, the diagnosis usually occurs incidentally.     

Autoimmune polyendocrinopathy associated with ring chromosome 18

Phenotypic and clinical features of individuals with ring chromosome 18 [r(18)] vary with the extent of deletion of the short (18p-) or long arm (18q-). Most patients with r(18), therefore, demonstrate a clinical spectrum of both 18p- and 18q- deletions. Short stature, microcephaly, mental and motor retardation, craniofacial dysmorphism and extremity abnormalities are the most commonly reported features in patients with r(18). Abnormalities of chromosome 18, especially 18p- syndrome, are often reported with autoimmune thyroid disease and growth hormone deficiency, but reports of endocrine abnormalities associated with r(18) are rare. Here, we report a case of an African-American female with hyperthyroidism, type 1 diabetes mellitus, vitiligo and IgA deficiency associated with a r(18) chromosome complement. This patient additionally had mild intellectual disability and dysmorphic features. Karyotype analysis showed a de novo ring chromosome 18 (deletion 18q23-18qter and deletion 18p11.3-18pter). Although this unique association of autoimmune polyglandular endocrinopathy with ring chromosome 18 could be coincidental, we speculate that a gene or genes on chromosome 18 might play a role in the autoimmune process.     

Duchenne muscular dystrophy, glycerol kinase deficiency, and adrenal insufficiency associated with Xp21 interstitial deletion

We report an interstitial deletion in the short arm of the X chromosome in a 6-year-old boy with Duchenne muscular dystrophy, glycerol kinase deficiency, adrenal insufficiency, intermittent hypoglycemia, spasticity, psychomotor retardation, and growth delay. His mother also has this deletion in an X chromosome. From our findings, we propose that the human glycerol kinase locus and the human X-linked adrenal hypoplasia locus are in the Xp21 band.     

Growth hormone deficiency associated with 22q11.2 deletion: a case report

The 22q11.2 microdeletion produces many syndromes, characterized by similar phenotypical features. The most known syndromes are: the DiGeorge syndrome, the velocardiofacial syndrome, the conotruncal anomaly face syndrome. The hallmark features are represented by cardiac anomalies, palate defects, immune and cognitive deficiencies, facial dysmorphisms. Less common disorders include: genito-urinary abnormalities, visual defects, autoimmune disorders and pituitary anomalies, being the last represented by growth hormone and/or insulin growth factor-I deficiency. We present the case of a 8 years old male admitted to our Division for failure to thrive. We found growth hormone deficiency and pituitary hypoplasia associated with some of the anomalies shown above, thus we suspected and confirmed the 22q11.2 deletion syndrome. In literature few cases of associated 22q11.2 deletion syndrome with growth hormone deficiency are described, while short stature between children with and children without cleft palate is reported to be more frequent in the first ones, suggesting that the 22q11.2 deletion syndrome remains undetected in many affected children and that the growth hormone deficiency prevalence in affected children has to be investigated. The wide phenotypical presentation of 22q11.2 deletion syndrome requires a multidisciplinary approach to the affected subject and, from the auxologic point of view, is good to monitoring the growing trend and, if short stature is present, check for the growth hormone deficiency.     

Disease manifestations and X inactivation in heterozygous females with Fabry disease

Aim: Fabry disease is an X-linked lysosomal storage disorder characterized by an accumulation of neutral glycosphingolipids in multiple organ systems caused by α-galactosidase A deficiency due to mutations in the GLA gene. The majority of heterozygous females show the characteristic signs and symptoms of the disease, and some of them are severely affected. The current hypothesis for the occurrence of disease manifestations in females is skewed X inactivation favouring the mutant GLA allele.
Method: We analyzed the patterns of X inactivation in the leukocytes of 28 biochemically and genetically characterized symptomatic Fabry disease heterozygotes and their correlation with clinical and biochemical disease expression.
Results: X inactivation patterns in symptomatic females who are heterozygous for Fabry disease did not differ from those of female controls of the same age ( p = 0.669). Thirteen (46%) of the 28 females with Fabry disease showed random X inactivation, ten (36%) moderate skewing, and five (18%) highly skewed X inactivation. Segregation analysis was performed in the families of six females who had highly or moderately skewed X inactivation. In four of these females, skewing favoured the wild-type GLA allele and in the other two skewing favoured the mutant allele. Patterns of X inactivation or the extent of skewing were not related to the severity of clinical manifestations or to residual enzyme activity.
Conclusion: In this study we provide evidence that heterozygous females with Fabry disease show random X inactivation. Our data do not support the hypothesis that the occurrence and severity of disease manifestations in the majority of Fabry heterozygotes are related to skewed X inactivation.     

X-linked recessive chondrodysplasia punctata. Cytogenetic study and role of molecular biology]

Among the many acquired or constitutional causes of chondrodysplasia punctata, the X-linked recessive form is well individualized. CASE REPORT: A male newborn presented a dysmorphic syndrome with a marked nasal hypoplasia, a macroglossia and a short neck. The diagnosis of chondrodysplasia punctata was made by radiography whereas the chromosomal chart revealed the existence of an additional Y fragment in Xpter, effectuating a partial disomy Yp and a monosomy Xpter. Molecular biology showed a deletion of very small size, isolated and located between the gene missing aryl sulfatase E and the microsatellite DXS 1233, sping gene MRX2 (non-specific gene of mental retardation), and making it possible to give the reassuring elements as regards the psychomotor prognosis, sometimes compromised in this disorder. CONCLUSION: In case of chondrodysplasia punctata with dysmorphy, it is important to execute a chromosomal chart in the search for a chromosomal reorganization on the X and a study in molecular biology.     

Disease manifestations and X inactivation in heterozygous females with Fabry disease

AIM: Fabry disease is an X-linked lysosomal storage disorder characterized by an accumulation of neutral glycosphingolipids in multiple organ systems caused by alpha-galactosidase A deficiency due to mutations in the GLA gene. The majority of heterozygous females show the characteristic signs and symptoms of the disease, and some of them are severely affected. The current hypothesis for the occurrence of disease manifestations in females is skewed X inactivation favouring the mutant GLA allele. METHOD: We analyzed the patterns of X inactivation in the leukocytes of 28 biochemically and genetically characterized symptomatic Fabry disease heterozygotes and their correlation with clinical and biochemical disease expression. RESULTS: X inactivation patterns in symptomatic females who are heterozygous for Fabry disease did not differ from those of female controls of the same age (p = 0.669). Thirteen (46%) of the 28 females with Fabry disease showed random X inactivation, ten (36%) moderate skewing, and five (18%) highly skewed X inactivation. Segregation analysis was performed in the families of six females who had highly or moderately skewed X inactivation. In four of these females, skewing favoured the wild-type GLA allele and in the other two skewing favoured the mutant allele. Patterns of X inactivation or the extent of skewing were not related to the severity of clinical manifestations or to residual enzyme activity. CONCLUSION: In this study we provide evidence that heterozygous females with Fabry disease show random X inactivation. Our data do not support the hypothesis that the occurrence and severity of disease manifestations in the majority of Fabry heterozygotes are related to skewed X inactivation.