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Novel germline CDK4 mutations in patients with head and neck cancer
© Sabir et al.; licensee BioMed Central Ltd. 2012
Received: 13 March 2012
Accepted: 13 August 2012
Published: 29 August 2012
Cyclin-dependent kinase 4 (CDK4) together with its regulatory subunit cyclin D1, governs cell cycle progression through G1 phase. Cyclin-dependent kinase inhibitors, including p16INK4A in turn regulate CDK4. In particular, deregulation of the p16/CDK4/cyclin D1 complex has been established in a variety of human tumors including gliomas, sarcomas, melanoma, breast and colorectal cancer. However, changes in CDK4 have rarely been observed.
In this study we used a combination of PCR-SSCP and direct sequencing for mutational screening of CDK4. DNA was isolated from peripheral blood leukocyte of patients with squamous cell carcinoma of head and neck, for screening germline mutations in coding regions of CDK4.
Variations observed in exon 2 and 5 were three missense mutations, g5051G > C (Ser52Thr), g5095G > C (Glu67Gln), g5906C > A, g5907C > G (Pro194Ser) and novel frame shift mutations g7321_23delTGA, g7121_7122insG, g7143delG in exon 7 and 3′UTR respectively.
In conclusion, two novel mutations were found in N terminal domain which indicates that CDK4 mutation may play a major role in the development and progression of squamous cell carcinoma of head and neck.
Cyclin-dependent kinases (Cdks) are serine/threonine kinases that regulate progression through cell cycle. CDK4 and CDK6 act early in the cell cycle and are involved in the transition from G1 to S phase . Loss of G1 control in cell cycle appears to be an important step contributing to tumorigenesis . D-type cyclin and their kinase partners, CDK4 and CDK6, coordinately phosphorylate the Rb protein, thereby releasing the transcription factor at G1 and then progressions into the S phase occurs . In addition to role in cell cycle, there is increasing evidence that Cdks, as well as cyclin and cyclin-dependent kinase inhibitors are important for other cellular functions, including cytoskeleton rearrangement and cell migration . CDK4 is a potential oncogene, located on chromosome 12q13; mechanisms of activation could include gene amplification, over-expression, decreased degradation, and activating point mutations. The CDK4 is amplified and over expressed in a number of human tumors including the gliomas, sarcomas, breast tumors and colorectal carcinomas . In humans, rare point mutations in the CDK4 have been described worldwide . All CDK4 reported mutations are located in exon 2, which codes for the p16INK4A binding site . Derailments of Rb pathway caused either by lack of Rb gene (pRb1-CycinD1-Cdk4/6-p16INK4) expression and over expression of Cdks are implicated in the deregulation of cell cycle machinery, resulting in uncontrolled growth, tumor heterogeneity, invasion and metastasis .
Aim of study was to investigate a possible disruption of Rb suppression pathway by germline mutational analysis in the p16-binding and cyclin D1 binding domain of CDK4 in squamous cell carcinoma of head and neck in Pakistani population. Furthermore, to determine association of novel mutations in CDK4 and risk of squamous cell carcinoma of head and neck, we performed polymerase chain reaction, single strand conformation polymorphism (PCR-SSCP) and sequence analysis.
Materials and methods
Sample collection and clinical data
The present study was conducted with a prior approval from ethical committees of both department and hospitals. Blood samples from total of 380 patients with histological confirmed squamous cell carcinoma of head and neck, including oral cavity, pharynx and larynx were collected from National Oncology and Radiotherapy Institute (NORI), Pakistan Institute of Medical Sciences (PIMS) and Military Hospital (MH). A total of 350 age, gender, and ethnicity matched cancer free healthy individuals were selected as controls. Patients and controls suffering from any other familial disease (diabetes, blood pressure and cardiovascular impairment) were excluded from this study. After obtaining informed consent, all individuals were personally interviewed using a specifically designed questionnaire.
Blood samples from patients and normal individuals (control), belonging to the same age and gender were collected in tubes containing EDTA and stored at 4°C. DNA was extracted from white blood cells, using standard phenol-chloroform extraction method [9, 10] and stored at -20°C for further processing. Electrophoresis was performed on isolated DNA in 1% ethidium-bromide stained agarose gel and photographed (BioDoc Analyze Biometra).
Polymerase chain reaction (PCR)
List of primers with annealing temperature and product size (bp)
Primer sequence (Forward) F
Primer sequence (Reverse) R
Product size (bp)
Annealing temperature °C
Mutational screening and sequence analysis
Amplification products were resolved on a 2% ethidium bromide containing agarose gel along with 100 bp DNA ladder. PCR products were analyzed by single stranded conformational polymorphism (SSCP)  and results were analyzed with the gel documentation system (BioDocAnalyze, Biometra). Samples with an altered mobility patterns were reamplified and than analyzed by direct sequencing to confirm and characterize the nature of mutations. Sequence analysis was carried out by MCLab (USA). Control (normal) samples were also sequenced along with cases to avoid false negative results and check the quality of sequencing.
Statistics and bioinformatic analysis
χ 2-test with Fisher exact test was used to evaluate the differences in selected demographic variables by using the SPSS 17.0 software, odd ratios (OR) and 95% confidence interval (CI) were calculated. Bioinformatic analysis for homology modeling was performed using ClustalW 2.1 multiple sequence alignment and UCSF Chimera 1.5.3 program.
Characteristics of head and neck cancer patients
Cases N = 380 (%)
Age at diagnosis
Family history of cancer
PCR-SSCP and direct sequence analysis
Frequency of CDK4 mutations in squamous cell carcinoma of oral cavity, pharynx & larynx
Region in gene
Change in amino acid
Frequency of mutations
g5051G > C*
g5095G > C
g5906C > A, g5907C > G
g7320 A > C, g7321_23delTGA
Distribution of CDK4 mutations with area of cancer and smoking status
Region in gene
Mutations/change in amino acid
Area of cancer
Oral cavity (N = 224)
Pharynx (N = 97)
Larynx (N = 59)
Non smoker (N = 238)
Smokers (N = 142)
g5051G > C
g5095G > C
g5906C > A, g5907C > G
g7320 A > C, g7321_23delTGA
p = 0.13
p = 0.03
OR 84.0, 95% CI (65.69 to 233.69)
OR 1.84, 95% CI (1.33 -2.54)
In our study three novel frame shift mutations were found on exon 7 and 3′UTR. Frame shift mutation observed in coding region g7321_23delTGA is changing the whole downstream sequence of gene. As a result translation of wrong reading frame continues and the resultant premature RNA stability is compromised so either mRNA will not stable or protein degradation happen . Although genetic alterations in 3′ UTR sequences can modify the binding properties of trans-acting factors and lead to deregulation in protein production . But most studies have sought to identify mutations in the coding region and very few naturally occurring mutations in non coding areas have been described to date. Pakistan harbors vast genetic, ethnic, cultural, social and life style diversity. In present study a group is characterized by onset of cancer frequently before the age of 40 year. Clinically, one key feature of genetic bases of cancer is early age at onset. Similar trends have been reported for familial melanoma, breast and colon cancers. Early disease onset has been associated with an increased prevalence of germline mutations .
Amplification and over expression of CDK4 has been detected in sarcoma and glioma, but in carcinoma the picture seems to be unclear. Only sporadic data are available in carcinoma where very low percentage of amplification was reported . CDK4 is altered in melanoma patients by a miscoding mutation (Arg24Cys) that blocks binding of INK4 inhibitors. However, the causal role of these alterations in tumor development is difficult to assess . Present study revealed that there was no germline mutation in codon 24 and 22 of CDK4 and these results support the findings of previous studies [20, 26, 27]. The potentially dominant Arg24Cys mutation of CDK4 was not detected in Indian patients with squamous cell carcinoma of head and neck . CDK4 mutations reported in the literature are rare and little is known about the functional implications of these changes . Study using in vitro site-directed mutagenesis analyzed that disruption of either codon 22 or codon 24 effectively abrogates interaction with cyclin Dl and p16INK4a. In vivo analysis of patients with the germ-line Arg24Cys mutation, revealed no documented changes in p16/CDKN2A binding site of CDK4.
In conclusion Arg24Cys mutation plays a key role in different cancers but in the current study we were unable to detect this mutation. This suggests that CDK4 novel germline mutations observed in study may play a different and important role in squamous cell carcinoma of head and neck cancer in Pakistani population. Potential of these novel mutations in head and neck carcinogenesis need to be explored in order to ascertain there potential importance.
This study was supported by grants from the higher education commission of Pakistan (HEC). Authors would like to acknowledge the patients and normal individual who contributed to this research work, we also acknowledge hospital staff (National Oncology and Radiotherapy Institute (NORI) and Pakistan Institute of Medical Sciences (PIMS) Islamabad, Pakistan for their cooperation.
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