Functional and genetic characterization of two extremely rare cases of Williams-Beuren syndrome associated with chronic granulomatous disease

Abstract

Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder with multi-systemic manifestations, caused by a heterozygous segmental deletion of 1.55-1.83 Mb at chromosomal band 7q11.23. The deletion can include the NCF1 gene that encodes the p47(phox) protein, a component of the leukocyte NADPH oxidase enzyme, which is essential for the defense against microbial pathogens. It has been postulated that WBS patients with two functional NCF1 genes are more susceptible to occurrence of hypertension than WBS patients with only one functional NCF1 gene. We now describe two extremely rare WBS patients without any functional NCF1 gene, because of a mutation in NCF1 on the allele not carrying the NCF1-removing WBS deletion. These two patients suffer from chronic granulomatous disease with increased microbial infections in addition to WBS. Interestingly, one of these patients did suffer from hypertension, indicating that other factors than NADPH oxidase in vascular tissue may be involved in causing hypertension.

Figure 1

Characterization of the 7q11.23 microdeletions from patients 1 and 2 by FISH. FISH studies were performed on EBV immortalized B lymphocytes from patient 1 and patient 2 as described in Materials and methods section. LSI William's syndrome (elastin gene) region probe (orange, Abbott Molecular) shows only one signal on the metaphase chromosomes. 7q31 Telomeric probe (green, Abbott Molecular) was used as control. Slides were counterstained with DAPI/Vectashield and FISH signals were captured with an equipped fluorescence microscope.

Figure 2

CGH-array analysis of the microdeletion in chromosome 7 from patients 1 and 2. CGH-array profile of chromosome 7 shows the same heterozygous deletion in 7q11.23 detected in both patients, varying from 1.4 to 1.9 Mb, depending on the retained breakpoints. Genomic position of the normal centromeric (7:72 401 086) and the normal telomeric oligonucleotides (7:74 339 044) bordering the deletion are indicated with black arrows. Genomic position of the first centromeric deleted (7:72 726 578) and the last telomeric deleted oligonucleotides (7:74 139 390) are indicated with blue arrows. The NCF1 gene (red circle) is located between the last deleted and the first normal oligonucleotides.

Figure 3

Phenotypic profiles of neutrophils from patients 1 and 2. (a) The absence of NADPH oxidase activity was evidenced in the neutrophils of patients 1 and 2 by resorufin (Amplex Red) fluorescence kinetics as described in Materials and methods section. Parents of patient 1 exhibited normal activity as it is usually the case in AR CGD. (b) The absence of oxidase activity was confirmed by the absence of immunodetection of p47^phox expression in both patients, whereas the other oxidase components were present. The experiment was performed on 1% Triton X100 soluble extract of neutrophils subjected to SDS–PAGE and blotted onto nitrocellulose sheet. Proteins were detected by the ECL Advance Western Blotting Detection Kit (GE Healthcare, formerly Amersham Biosciences) for patient 1 and by infrared-based fluorometric detection by the Odyssey system (LI-COR Biosciences, Ltd,) for patient 2.

Figure 4

Gene-scan analysis of NCF1 and NCF1 pseudogenes (ψNCF1) regions around the beginning of exon2 for patients 1 and 2. Fragments of genomic DNA from AR47⁰CGD patient 1 (a) and patient 2 (b) and their parents were amplified with primers that anneal with regions in NCF1 as well as in ψNCF1 regions around the GTGT sequence at the start of exon 2 as described in Materials and methods section. The mixture of NCF1 and ψNCF1 PCR products was analyzed in a sequencer (Applied Biosystems, Foster City, CA, USA) to determine the ratio between the number of NCF1 and ψNCF1 genes. Both patients show only ψNCF1 PCR product; both fathers show a ψNCF1:NCF1 product ratio of 5; both mothers and both controls show a ψNCF1:NCF1 product ratio of 2.