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Case Reports
. 2013 Jun;34(6):864-8.
doi: 10.1002/humu.22314. Epub 2013 Apr 12.

Deficiency of the cyclin-dependent kinase inhibitor, CDKN1B, results in overgrowth and neurodevelopmental delay

William Grey  1 , Louise Izatt, Wafa Sahraoui, Yiu-Ming Ng, Caroline Ogilvie, Anthony Hulse, Eric Tse, Roman Holic, Veronica Yu
Affiliations
Free PMC article
Case Reports

Deficiency of the cyclin-dependent kinase inhibitor, CDKN1B, results in overgrowth and neurodevelopmental delay

William Grey et al. Hum Mutat. 2013 Jun.
Free PMC article

Abstract

Germline mutations in the cyclin-dependent kinase inhibitor, CDKN1B, have been described in patients with multiple endocrine neoplasia (MEN), a cancer predisposition syndrome with adult onset neoplasia and no additional phenotypes. Here, we describe the first human case of CDKN1B deficiency, which recapitulates features of the murine CDKN1B knockout mouse model, including gigantism and neurodevelopmental defects. Decreased mRNA and protein expression of CDKN1B were confirmed in the proband's peripheral blood, which is not seen in MEN syndrome patients. We ascribed the decreased protein level to a maternally derived deletion on chromosome 12p13 encompassing the CDKN1B locus (which reduced mRNA expression) and a de novo allelic variant (c.-73G>A) in the CDKN1B promoter (which reduced protein translation). We propose a recessive model where decreased dosage of CDKN1B during development in humans results in a neuronal phenotype akin to that described in mice, placing CDKN1B as a candidate gene involved in developmental delay.

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Figures

Figure 1
Figure 1
A: Pedigree of the nonconsanguineous Bengali family. The proband (II:1) presented with overgrowth, autism, and severe developmental delay with absent speech at the age of 3 years. Array CGH demonstrated a deletion on 12p13.1 in the proband and his mother (I:2). Sanger sequencing of the CDKN1B gene demonstrated a SNP, rs34330: −79C>T, in the proband's father (I:1) and his sister (II:2). The proband harbors a de novo variant that is not seen in other members of the family at c.−73G>A. B: Array CGH analysis. Diagrammatic depiction of the maternally derived 12p13.1 deletion identified by array CGH (Agilent AMAID 017457). Coordinates displayed in build NCBI36/hg18. The position of probes showing normal copy number is shown in green, probes showing abnormal copy number are shown in red. Probe names, base-pair coordinates, and fluorescent ratios for the abnormal probes are shown (bottom right). For a patient: reference ratio of 1:2 (one allele deleted in the patient), the theoretical log2 value is −1. C: rs34330:C>T and c.−73G>A. Bidirectional Sanger sequencing of the promoter region of CDKN1B revealed the rs34330:C>T (−79C>T) in the proband's father and sister (I:1, II:2) (representative sequencing trace shown). In the reference sequence, the ancestral allele of T is shown but variant at this site is common in human populations (Supp. Fig. S3). The T allele has been linked to cancer [Chang, ; Landa et al., 2010]. A previously unreported SNP c.−73G>A was seen in the proband only (II:1). Sequencing primers are detailed in Supp. Figure S4A.
Figure 2
Figure 2
A: Quantification of CDKN1B mRNA expression by qPCR. Total RNA was extracted from peripheral blood. White blood cells from peripheral blood were spun down, lysed, and total RNA was purified using the Qiagen RNA easy extraction kit (cat. 52304). RNA was reversed transcribed, and quantitative PCR was carried using the Qiagen one-step SYBR green RT-PCR kit. Q-PCR primers (primer sequences are detailed in Supp. Fig. S4B) spanned the 3′ end of exon 1 (forward) of CDKN1B to the 5′ end of exon 2 (reverse). The amount of CDKN1B detected was normalized against the ubiquitously expressed ABL1 gene and expressed as a relative value Ratio (ABL1/CDKN1B) = 2CT(ABL1)-CT(CDKN1B). B: Quantification of CDKN1B minimal promoter activity. Region incorporating the 5′UTR of CDKN1B (encompassing the −79 and −73 residues) were amplified from patient DNA and subcloned into the pGL3 basic luciferase reporter vector (Promega cat. E1751). One microgram of pGL3−79C-73G, pGL3−79T-79G, pGL3−79C-73A, or an empty pGL3 vector was cotransfected with 20 ng of pRL-TK (Renilla reporter construct, Promega cat. E2241) into HEK293T cells with the ProFection mammalian transfection system (Promega cat. E1200) according to the manufacturer's protocol. Cells were transfected for 48 hr, followed by a 12 hr period of serum starvation. The dual-luciferase reporter assay system (Promega cat. E1910) was used to assay relative luciferase activity. The data represent the mean of three independent experiments with standard error bars. C: Western blot analysis of CDKN1B from white blood cells. Protein samples were extracted from peripheral blood samples by first isolating white blood cells through centrifugation. These cells were subsequently lysed using RIPA lysis buffer (50 mM Tris HCl pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) and run on a 12% SDS-PAGE. Western blotting was carried out using an anti-p27 antibody (BD Biosciences cat. 610242) and an anti-β-tubulin antibody (Cell Signaling cat. 2128) (left). Relative intensity of the signal obtained from the western blot was quantified using ImageJ (NIH). Bar chart shows the average of signals (and standard error) quantified from three blots normalized to β-tubulin level in each independent blood sample (right).
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