Bruton tyrosine kinase gene mutations in Turkish patients with presumed X‐linked agammaglobulinemia

X‐linked agammglobulinemia (XLA) is a ptototypical humoral immunodeficiency caused by mutations in the gene coding for Bruton tyrosine kinase (BTK). The genetic defect in XLA impairs early B cell development resulting in marked reduction of mature B cells in the blood. Studies from different countries have demonstrated that approximately 90% of males with presumed XLA bear mutations in BTK. In this study, we report for the first time the occurrence of BTK mutations in Turkey. We performed mutational analysis of the BTK gene in 16 Turkish male patients from 13 separate families with presumed XLA based on abnormally low peripheral blood B‐cell numbers (lt; 1%), hypogammaglobulinemia, and recurrent bacterial infections. We found that in nine of the 13 families (69%) a Btk mutation caused XLA. Two of the mutations were previously described, but seven novel mutations were identified: two missense (Y39C, G584R), one nonsense (Q343X), and 4 deletions (1800‐1821del, 1843‐1847del, 1288‐1292del, 291del) resulting in frameshift and premature stop codon. By contrast, no mutations in the BTK gene were identified in the other 4 families. A consanguinity in three of these families raises the possibility that mutations in other autosomal genes which affect early B cell development may contribute to their phenotype resembling XLA. Hum Mutat 18:356, 2001. © 2001 Wiley‐Liss, Inc.     

Identification of the bruton tyrosine kinase (BTK) gene mutations in 20 Australian families with X-linked agammaglobulinemia (XLA)

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by mutations in the Bruton tyrosine kinase (BTK) gene. Twenty Australian patients with an XLA phenotype, from 15 unrelated families, were found to have 14 mutations. Five of the mutations were previously described c.83G>A (p.R28H), c.862C>T (p.R288W), c.904G>A (p.R302G), c.1535T>C (p.L512P), c.700C>T (p.Q234X), while nine novel mutations were identified: four missense c.82C>A (p.R28S), c.494G>A (p.C165Y), c.464G>A (p.C155Y), c.1750G>A (p.G584E), one deletion c.142_144delAGAAGA (p.R48_G50del), and four splice site mutations c.241-2A>G, c.839+4A>G, c.1350-2A>G, c.1566+1G>A. Carrier analysis was performed in 10 mothers and 11 female relatives. The results of this study further support the notion that molecular genetic testing represents an important tool for definitive and early diagnosis of XLA and may allow accurate carrier status and prenatal diagnosis.     

Carrier identification by FISH analysis in isolated cases of X‐linked ichthyosis

X‐linked ichthyosis (XLI) is an inborn error of metabolism due to steroid sulfatase (STS) deficiency. STS assay and FISH are useful in diagnosing carrier status of XLI. Biochemical analysis appears to indicate that most sporadic cases are inherited. Since this method does not seem to be completely reliable in recognizing XLI‐carriers, the aim of the present study was to corroborate by FISH whether or not most sporadic cases of XLI had de novo mutations. XLI patients were classified through STS assay and PCR amplification of 5′‐3′ ends of the STS gene. XLI patients had undetectable levels of STS activity and complete deletion of the STS gene. Patients' mothers were studied through STS assay and FISH. Nine out of 12 mothers presented an STS activity compatible with XLI‐carrier state. These mothers also had only one copy of the STS gene, indicating that they carry the primary gene defect. One mother had normal STS activity but only one copy of the STS gene. This data corroborated that most sporadic cases do not represent de novo mutations, and that FISH must be included in the analysis of mothers of sporadic cases when they present with normal STS activity, in order to correctly diagnose the XLI carrier state. © 2001 Wiley‐Liss, Inc.     

X‐chromosomal inactivation directly influences the phenotypic manifestation of X‐linked protoporphyria

X‐linked protoporphyria (XLP), a rare erythropoietic porphyria, results from terminal exon gain‐of‐function mutations in the ALAS2 gene causing increased ALAS2 activity and markedly increased erythrocyte protoporphyrin levels. Patients present with severe cutaneous photosensitivity and may develop liver dysfunction. XLP was originally reported as X‐linked dominant with 100% penetrance in males and females. We characterized 11 heterozygous females from six unrelated XLP families and show markedly varying phenotypic and biochemical heterogeneity, reflecting the degree of X‐chromsomal inactivation of the mutant gene. ALAS2 sequencing identified the specific mutation and confirmed heterozygosity among the females. Clinical history, plasma and erythrocyte protoporphyrin levels were determined. Methylation assays of the androgen receptor and zinc‐finger MYM type 3 short tandem repeat polymorphisms estimated each heterozygotes X‐chromosomal inactivation pattern. Heterozygotes with equal or increased skewing, favoring expression of the wild‐type allele had no clinical symptoms and only slightly increased erythrocyte protoporphyrin concentrations and/or frequency of protoporphyrin‐containing peripheral blood fluorocytes. When the wild‐type allele was preferentially inactivated, heterozygous females manifested the disease phenotype and had both higher erythrocyte protoporphyrin levels and circulating fluorocytes. These findings confirm that the previous dominant classification of XLP is inappropriate and genetically misleading, as the disorder is more appropriately designated XLP.     

Maternal germinal mosaicism of X-linked agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an immunodeficiency caused by abnormalities in tyrosine kinase (BTK), and is characterized by a deficiency of peripheral blood B cells. We studied cytoplasmic expression of BTK protein and analyzed the BTK gene (BTK) in peripheral blood mononuclear cells from two siblings with XLA and additional family members. Cytoplasmic expression of BTK protein in monocytes was not detected in either patient with XLA. A single base deletion (C563) in BTK-exon 6, which encodes the TH domain, was identified in both XLA patients. However, normal cytoplasmic expression of BTK protein in monocytes was detected in their mother without any BTK mutation. These results strongly suggest germinal mosaicism in the mother.     

Identification of mutations of Bruton's tyrosine kinase gene (BTK) in Brazilian patients with X-linked agammaglobulinemia

Mutations in the Bruton tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA), which is characterized by recurrent bacterial infections, profound hypogammaglobulinemia, and decreased numbers of mature B cells in the peripheral blood. We evaluated 17 male Brazilian patients from 13 unrelated families who showed markedly reduced numbers of blood B cells and hypogammaglobulinemia. BTK gene analysis detected mutations in 10 of the 13 presumed XLA families. Seven mutations (Q196X, G613D, R28L, 251-273del, Q234X, H364P, and R13X) had been reported previously, whereas the remaining three mutations (M501T, IVS15+1G>C, and IVS14+1G>A) were novel. Mutation IVS15+1G>C occurred in a splice donor site and caused exons 15 and 16 to be skipped, and IVS14+1G>A might cause exon 14 to be skipped. Flow cytometry revealed deficient expression of BTK protein in 10 of the 13 families. This is the first report of the diagnosis of XLA by analysis of mutations of the BTK gene in Brazilian patients.     

Non‐syndromic anophthalmia/microphthalmia can be caused by a PORCN variant inherited in X‐linked recessive manner

Anophthalmia and microphthalmia (A/M) represent severe developmental ocular malformations, corresponding, respectively, to absent eyeball or reduced size of the eye. Both anophthalmia and microphthalmia may occur in isolation or as part of a syndrome. Genetic heterogeneity has been demonstrated, and many genes have been reported to be associated with A/M. The advances in high‐throughput sequencing have proven highly effective in defining the molecular basis of A/M. Nevertheless, there are still many patients with unsolved genetic background of the disease, who pose a significant challenge in the molecular diagnostics of A/M. Here we describe a family, with three males affected with the non‐syndromic A/M. Whole exome‐sequencing performed in Patient 1, revealed the presence of a novel probably pathogenic variant c.734A>G, (p.[Tyr245Cys]) in the PORCN gene. Pedigree analysis and segregation of the identified variant in the family confirmed the X‐linked recessive pattern of inheritance. This is the first report of X‐linked recessive non‐syndromic A/M. Until now, pathogenic variants in the PORCN gene have been identified in the patients with Goltz syndrome, but they were inherited in X‐linked dominant mode. The ocular phenotype is the only finding observed in the patients, which allows to exclude the diagnosis of Goltz syndrome.     

Characterization of All Possible Single‐Nucleotide Change Caused Amino Acid Substitutions in the Kinase Domain of Bruton Tyrosine Kinase

Knowledge about features distinguishing deleterious and neutral variations is crucial for interpretation of novel variants. Bruton tyrosine kinase (BTK) contains the highest number of unique disease‐causing variations among the human protein kinases, still it is just 10% of all the possible single‐nucleotide substitution‐caused amino acid variations (SNAVs). In the BTK kinase domain (BTK‐KD) can appear altogether 1,495 SNAVs. We investigated them all with bioinformatic and protein structure analysis methods. Most disease‐causing variations affect conserved and buried residues disturbing protein stability. Minority of exposed residues is conserved, but strongly tied to pathogenicity. Sixty‐seven percent of variations are predicted to be harmful. In 39% of the residues, all the variants are likely harmful, whereas in 10% of sites, all the substitutions are tolerated. Results indicate the importance of the entire kinase domain, involvement in numerous interactions, and intricate functional regulation by conformational change. These results can be extended to other protein kinases and organisms.     

The RBMX gene as a candidate for the Shashi X‐linked intellectual disability syndrome

A novel X‐linked intellectual disability (XLID) syndrome with moderate intellectual disability and distinguishing craniofacial dysmorphisms had been previously mapped to the Xq26‐q27 interval. On whole exome sequencing in the large family originally reported with this disorder, we identified a 23 bp frameshift deletion in the RNA binding motif protein X‐linked (RBMX) gene at Xq26 in the affected males (n = 7), one carrier female, absent in unaffected males (n = 2) and in control databases (7800 exomes). The RBMX gene has not been previously causal of human disease. We examined the genic intolerance scores for the coding regions and the non‐coding regions of RBMX; the findings were indicative of RBMX being relatively intolerant to loss of function variants, a distinctive pattern seen in a subset of XLID genes. Prior expression and animal modeling studies indicate that loss of function of RBMX results in abnormal brain development. Our finding putatively adds a novel gene to the loci associated with XLID and may enable the identification of other individuals affected with this distinctive syndrome.     

Twin carriers of X‐linked agammaglobulinemia (XLA) due to germline mutation in the Btk gene

We report on an X‐linked agammaglobulinemia (XLA) family in which mothers of two affected cousins were monozygotic twins. We analyzed the Btk gene of several members in three generations of the family by SSCP analysis, DNA sequencing, and RFLP analysis following polymerase chain reaction‐amplification of the individual exons. We identified a missense point mutation, G1817C (R562P), in exon 17 of the Btk gene in the affected cousins. The same mutation was also present in both mothers (twin sisters) of the cousins identifying them as carriers. However, the mutation was absent in all other relatives including the grandmother of the cousins (mother of the twin sisters). This strongly suggests that the mutation in the Btk gene had originated in one of the germ lines or in the zygote. This may be the first demonstration of a germ line (or zygotic) mutation in XLA. Am. J. Med. Genet. 90:229–232, 2000. © 2000 Wiley‐Liss, Inc.     

Osteopathia striata cranial sclerosis: Non‐random X‐inactivation suggestive of X‐linked dominant inheritance

Osteopathia striata with cranial sclerosis (OS‐CS) is a rare syndrome comprising macrocephaly, minor anomalies, conductive hearing loss, and mild mental retardation. The diagnosis is based on radiological findings, including cranial sclerosis and longitudinal striations of metaphyses of long bones. Here we report on 10 new cases of OS‐CS, including two sporadic cases and three families, with an excess of affected females (9F/1M). Phenotypic variability was observed in our patients as well as several unusual findings. Hirschsprung disease, Pierre Robin sequence, coronal craniostenosis, and laryngotracheomalacia were associated with a poor prognosis. The X‐inactivation pattern of peripheral blood lymphocytes in a mildly affected mother and her severely affected boy demonstrated a non‐random X‐inactivation in the mother. This finding, in combination with a sex ratio in favor of females and an increased morbidity and mortality in males, is highly suggestive of X‐linked dominant inheritance. © 2001 Wiley‐Liss, Inc.     

Novel insertions of Bruton tyrosine kinase in patients with X-linked agammaglobulinemia

Mutations in the gene encoding Bruton tyrosine kinase (BTK) result in X-linked agammaglobulinemia (XLA), an immunodeficiency of antibody defect. By using base excision sequence scanning method (BESS) followed by direct sequencing we found in seven unrelated families with a classical XLA phenotype various mutations including six novel mutations (g.64512_64513insC, c.108_109insG, c.1700_1701insACTACAG, g.51375_51376GC>TG, g.63991_63992insGGTAGAAAAAA, c.1956_1957insCA) and a previously known silent polymorphism (c.2031C>T). Except for two mutations, the alterations affect the kinase domain. There was exceptionally high proportion of insertions in the cohort. Frameshift insertion was found altogether in five patients, three of which are on introns, one in upstream region, and one in exon 18 leading to frameshift mutation and truncation of the protein. In the intron 4 there is a substitution of two bases. Carrier detection was performed in four families. In one case the mutation was found to be de novo.     

Phenotypic heterogeneity and mutational spectrum in a cohort of 45 Italian males subjects with X‐linked ectodermal dysplasia

Ectodermal dysplasias (EDs) are a group of genetic disorders characterized by the abnormal development of the ectodermal‐derived structures. X‐linked hypohidrotic ectodermal dysplasia, resulting from mutations in ED1 gene, is the most common form. The main purpose of this study was to characterize the phenotype spectrum in 45 males harboring ED1 mutations. The study showed that in addition to the involvement of the major ectodermal tissues, the majority of patients also have alterations of several minor ectodermal‐derived structures. Characterizing the clinical spectrum resulting from ED1 gene mutations improves diagnosis and can direct clinical care.     

Genetic heterogeneity of syndromic X‐linked recessive microphthalmia‐anophthalmia: Is Lenz microphthalmia a single disorder?

Nonsyndromic congenital microphthalmia or anophthalmia is a heterogeneous malformation with autosomal dominant, autosomal recessive, and X‐linked modes of inheritance. Lenz microphthalmia syndrome comprises microphthalmia with mental retardation, malformed ears, skeletal anomalies, and is inherited in an X‐linked recessive pattern. Prior studies have shown linkage of both isolated (or nonsyndromic) anophthalmos (ANOP1, [MIM 301590]) and Lenz syndrome [MIM 309800] to Xq27–q28. Nonsyndromic colobomatous microphthalmia [MIM 300345] has been linked to Xp11.4–Xq11.1. We describe a five‐generation African‐American family with microphthalmia or anophthalmia, mental retardation, and urogenital anomalies, in an X‐linked recessive inheritance pattern, consistent with Lenz syndrome. Initial linkage analysis with microsatellite markers excluded the region in Xq27–q28 previously reported as a candidate region for ANOP1 [MIM 301590]. An X‐chromosome scan revealed linkage to a 10‐cM region between markers DXS228 and DXS992 in Xp11.4–p21.2. Multipoint analysis gave a maximum LOD score of 2.46 at marker DXS993. These data show that X‐linked recessive syndromic microphthalmia exhibits genetic heterogeneity. In addition, it suggests that Lenz microphthalmia syndrome, previously thought to be a single disorder, may represent an amalgam of two distinct disorders. © 2002 Wiley‐Liss, Inc.     

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.