2.3.1. Skin examination
All probands and their sibling controls were invited to attend skin examinations at SCFA in Westmead or Darlinghurst, at Westmead hospital, or at outer metropolitan or rural hospitals for a few subjects who could not attend a city venue. The same measurement equipment was used at each site, and the same lights were used to enable standardized illumination. The candidate, a medical graduate, was trained to assess premalignant and malignant lesions and the markers of sun damage on subjects, according to a standard protocol as, described below.
Height and weight were recorded. Subjects were asked to identify the hair colour that was closest to their natural colour at age 18, using synthetic hair swatches (Clairol, USA) designed
for wig-making. Colours used in this study included: High-Lift golden blonde HL-G, Sable Brown 48D, Dusk Blonde 93D-N, Nightfall Brown 95D-N, Sunberry 72R, Reddest Fire Red 206RR, Golden Apricot 41G, Black Azure 52D. Skin colour was recorded using a reflectance spectrophotometer, as described in 2.3.3.
All subjects were tested for features of Gorlin Syndrome, including mandibular keratocysts (through the questionnaire) and examination of the palms for pitting. Each of these features would be expected to be seen in over 80% of patients with Gorlin syndrome [200].
Markers of sun exposure and damage were then assessed on a semi-quantitative scale and included: ephelides (freckles), solar lentigines, actinic keratoses, actinic damage/elastosis, seborrhoeic keratoses, and presence of any new skin cancers noted. If a lesion was suspicious of malignancy, the subject was referred to their local GP or dermatologist. Results were recorded on a patient data sheet and entered into a password-protected database in secure facilities. Data for analysis were deidentified.
All subjects undergoing skin examination also filled out a questionnaire (see section 7.1 for clinical protocol) regarding their skin type, hair colour, eye colour, childhood and adult freckling, and history of dental cysts.
The candidate undertook an informal audit of examining technique with an experienced dermatologist after the examinations were complete. Her technique was considered adequate for all variables except seborrhoeic keratoses, where fainter lesions were missed. Absolute values for this variable should be regarded as underestimates and were not interpreted, whereas categorisation of individuals with respect to number of these lesions was regarded as suitable for analysis.
2.3.2. Skin examination recoding variables
All skin phenotype variables, from examination and questionnaire, were subjected to an initial analysis to determine their distribution and informativeness. The number of categories was reduced by recoding in many cases in order to maximise the number of individuals in each recoded category. All recoded variables are described here.
Only one subject (1.1%) reported having black hair when asked to name their hair colour at age 21, and this subject chose a brown hair swatch when asked to select a representative hair
swatch colour visually: the category of ‘black’ was therefore combined with the category
‘dark brown’ for the analysis.
Few people (3.4% or 3/88) reported that they ‘go brown without any sunburn’ when asked
“which statement best describes what would happen if your skin were exposed to bright sunlight for the first time in summer for one hour in the middle of the day without any protection?” (see section 7.1). Therefore the answers for the ‘no sunburn/tanning’ were combined with the ‘mild sunburn’ category for analysis.
No subject out of 88 reported that they ‘never burn’ as a response to the question “in general, how does your skin react to the sun?” and therefore this phototype category was removed from the analysis.
Examination for presence of actinic keratoses (AKs) revealed that most subjects did not have many of these lesions on the regions of the body included (see section 4.2.4). The categories of ‘sparse’ and ‘moderate’ were therefore collapsed to the single category of ‘present’ or
‘absent’ for presence of any AK on the regions head/neck, chest, dorsum arms and dorsum hands examined. A large proportion of subjects had a high amount of solar elastosis, skewing the results. Therefore the score for each body region (Head/neck/anterior chest, periorbital, lips, dorsa forearms, and dorsa hands) were added up with a score of 0 for ‘absent’, 1 for
‘mild’ and 2 for ‘severe’. This was added up across all categories and each subject was given a score of ‘low’for a total body score between 0 and 9, and a score of ‘high’ for a total body score of 10.
Semi-quantitative counts of solar lentigines were skewed across the 88 subjects examined with a disproportionate number falling into a low or high count category (see table 7.8 for raw scores). Therefore the median count category for each body region was determined and subjects re-categorised into ‘low’ or ‘high’ solar lentigines groups. For the face this meant that raw scores of 0 – 2 were recoded as ‘low’ with the remainder of scores 3 – 10 recoded as
‘high’. Similarly, the raw scores for solar lentigines on the forehead were recoded 0 – 1 ‘low’
and 2 – 10 ‘high’. For the upper limb raw scores, 0 – 4 became ‘low’ and 5 – 9 became ‘high’.
For the upper back and shoulders, 0 – 6 became ‘low’ and 7 – 9 became ‘high’.
Reporting of freckling in childhood and adulthood was skewed to include a greater proportion in the category ‘very few’ than the other positive freckling categories (see table 7.9 for raw scores). ‘Very few’ was therefore renamed ‘some’, and the ‘few’, ‘some’, and ‘many’
categories were collapsed into the single category of ‘many’. As with the analysis of semi-quantitative examination results for solar lentigines, subjects’ ephiledes (freckling) scores were skewed or biphasic (see table 7.9) and so the median (if skewed) or the score between the two peaks (if biphasic) for each region was taken, and subjects reclassified into high and low numbers of freckles based on this median division. For ephilides on the face, raw scores of 0 – 3 were recoded as ‘low’ and 4 – 10 as ‘high’. For raw scores of ephilides on the forehead, 0 – 3 were re-classified as ‘low’ and 4 – 10 as ‘high’. For the upper limbs, raw scores of 0 – 7 were scored as ‘low’ and 8 – 10 as ‘high’. For the upper back and shoulders, 0 – 6 raw scores were recoded as ‘low’ and 7 – 10 as ‘high’.
Examination results for numbers of seborrhoeic keratoses were skewed with many subjects scoring ‘nil’ on this variable for several of the body regions (see table 7.10). The results were reclassified into an absent/present dichotomy for each region examined to allow any real associations to be seen. The score for all the body regions examined (head/neck, upper limbs, chest, abdomen, back, lower limbs) were then summed to give a total body score, and this was also divided into ‘low’ (score of 0 – 1) and ‘high’ (score of 2 – 8) categories based on the median score.
2.3.3. Skin colour assessment
The apparent colour of a person’s skin is due to the aggregate of reflected light, the wavelengths of which depend on four biochromes: melanin, carotenoids, oxyhaemoglobin and reduced haemoglobin. Melanin and the carotenoids are brown and yellow respectively, and are found in the epidermis, the uppermost layer of the skin. Oxyhaemoglobin is bright red and found in the arterioles and capillaries of the upper dermis of the skin, just deep to the epidermis. Reduced haemoglobin is bluish-red and is found in the venous plexus of the dermis, just deep to the arterioles and capillaries [21].
Skin colour of each subject examined was assessed with a BYK Gardner spectrophotometer; a portable instrument that measures reflected colour and the colour difference using the Commission International de l’Eclairage (CIE; 1976) L*a*b* standard colour system. The L*
value (luminance) represents the relative lightness ranging from total black to total white; the a* value represents the balance between red and green; and the b* value the balance between yellow and blue. This instrument uses the same wavelengths of light (400 to 700nm at 20nm intervals) to measure skin reflectance as the Minolta 508, a spectrophotometer with
measurement of skin colour that correlates highly (r = 0.68) with melanin density seen histologically on biopsied skin from the site measured [391]. The Minolta does not separate the reflected light into the L, a and b axes, giving a single reading of reflected light for each 20nm interval of reflected light. Based on best correlation with melanin density over the spectrum of light used, researchers using the Minolta generally use the difference between the reflectance measurements at 400nm and 420nm of incident light as a single parameter.
The Minolta and BYK Gardner instruments are regarded as equivalent in commercial colour measurement applications. However, there are no published data on correlates between 420-400nm reflectance and L*a*b* measurements, or between L*a*b* measurements and melanisation. In this thesis, the raw L*a*b* measurements were used without further manipulation in order that all available information on skin colour should be used.
The spectrophotometer was recalibrated prior to each skin examination session to ensure comparable readings between subjects, and the mean of 6 consecutive readings was recorded.
The measurements were taken on the medial axillary wall (constitutional skin colour) and upper outer arm (sun exposed skin colour) of each subject examined. The mean L*a*b*
values was recorded on the subject’s data sheet.