Full clinical description of family 1
Family 1: see Figure 1a. This family lived in France and had both Vietnamese and
Martinique origins. The mothers of III.1 and III.12 were of European origin. The
proband (III.1) is describedin the main text of the paper.
His father (II.1) had complete greying (hair, eyebrows and eyelashes) at the age of
16 years, generalized hypopigmentation of the skin associated with patchy depigmented
macules, freckles in sun-exposed regions, lentigines, several cafe-au-lait macules, blue
irides with some brown partial heterochromia. Irides were not transilluminable. The
fundoscopic examination revealed a slightly diffuse hypopigmentation with small
papilla. There was no ocular abnormality ,W Index = 0.85. The audiometric examination
was normal.
A paternal uncle (II.8) had complete greying (hair, eyebrows and eyelashes) at the
age of 10 years, generalized hypopigmentation of the skin associated with freckles and
lentigines. He had progressive acquired bilateral perceptive hearing loss of both
occupational exposure to intense noises and cochlear sensitivity origins. He complained
with photophobia and had blue irides with partial brown heterochromia. The
fundoscopic examination revealed sectoral hypopigmentation with retinal pigment
clumps.
A cousin (III.12) was a 6-year-old boy with onset premature greying, freckles on
the face and several cafe-au-lait macules. He complained with photophobia. He had blue
irides with partial heterochromia and diffuse retinal hypopigmentation on fundoscopic
examination. The audiometric examination showed a small notch on 6 KHz on both ears
and tinnitus which could suggest acoustic trauma or some cochlear sensitivity in the
context.
Another paternal uncle (II.7) had complete greying at the age of 20 years affecting
hair, eyebrows and eyelashes, blue irides, diffuse hypopigmentation and freckles. He did
not have deafness. He had metastasic thoracic chondrosarcoma.
The grandmother (I.2) had Vietnamese origins. She was not clinically examinated
but her sons related history of premature greying, blue irides and late deafness.
Molecular analysis
Sequencing of the MITF-M (melanocytic) isoform coding exons and splice
consensus sequences was modified from Tassabehji et al.11 with the use of universal
primers UniSeq (N13). PCR was realised in standard conditions (annealing temperature
58° for exons 4, 9A and 9B, 60° for other exons) using primers listed in the Table
below. Sequencing was then performed using the UniSeq primers: forward
GTAGCGCGACGGCCAGT, reverse CAGGGCGCAGCGATGAC.
The absence of total or partial gene deletion was assessed by QMF-PCR
(Quantitative Multiplex Fluorescent PCR).12 The coding exons of the MITF-M isoform
(including a regulatory region RG that is located in the promoter13-14) and two control
fragment of other genes (F9 on chromosome X, DSCR1 on chromosome 21) were
amplified in a multiplex reaction using primers listed in the Table below. One primer of
each amplicon was labelled with the fluorescent phosphoramidite 6-FAM dye.
Amplifications were performed in duplicates in 25 l reactions using the QIAGEN
Multiplex PCR kit (Qiagen, France), with 75 ng of genomic DNA, a mix of primers
(concentration range 0.15 to 0.6 M), and 5% DMSO. The reaction started with an
initial denaturation of 10 min at 95°C followed by 22 cycles at 95°C for 30 sec, 55°C
for 30 sec, and 72°C for 45 sec with an increment of 3 sec per cycle, and a final
extension of 10 min at 72°C. Then, 4 l of the purified PCR products were processed as
previously described. Two normal DNAs (male and female) and one full deletion of
MITF were included in each experiment as controls.
Primers used in this study
PCR-sequence
sequence
Exon 1M
Forward primer
Reverse primer
GTAGCGCGACGGCCAGTGGATACCTTGTTTATAGTACCTTC
CAGGGCGCAGCGATGACAAAAGAGCAGATTTATACTTATTG
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
Exon 9(1)
Exon 9(2)
GTAGCGCGACGGCCAGTTCTGAAACTCACAAATAACAGCGC
CAGGGCGCAGCGATGACTATTCAACAGACAAGTTATTTAGC
GTAGCGCGACGGCCAGTCCATCAGCTTTGTGTGAACAGGTC
CAGGGCGCAGCGATGACTTTCAGGAAGGTGTGATCCACCAC
GTAGCGCGACGGCCAGTAACTAAAGACCATTATTGCTTTGG
CAGGGCGCAGCGATGACAGAAAAGAACCCTGGAAACACCTC
GTAGCGCGACGGCCAGTATAAATCCTAGAGTAGGATATAGG
CAGGGCGCAGCGATGACACTTTGTCTTATCAGGAAATGGAC
GTAGCGCGACGGCCAGTTCAAGTCAAATAAGCTTCTGTATG
CAGGGCGCAGCGATGACGTAGGAATCAACTCTCCTCTACAG
GTAGCGCGACGGCCAGTGTGCTAAATGCATACATGGCACTG
CAGGGCGCAGCGATGACTTAGGAATAGAACCAAAGGGAGAG
GTAGCGCGACGGCCAGTTTCATTGAGCCTCAAATCCTAAAG
CAGGGCGCAGCGATGACCTGTTTCTACTGTCTTGAAGTCGG
GTAGCGCGACGGCCAGTAGTCCTCTGTGCTCTGCCTATTTC
CAGGGCGCAGCGATGACGCTGCTTGTTTTGGAAGCTC
GTAGCGCGACGGCCAGTGGGATCCAAACTGGAAGACA
CAGGGCGCAGCGATGACAAGCTAAAGTCTGTGGTGAATTC
QMF-PCR
RG
Exon 1M
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Exon 7
Exon 8
Exon 9(1)
Exon 9(2)
F9
DSCR1
Forward primer
Reverse primer
TTAGATGATGTCTCCTCCAA
AAATGTTGATATCAATTTTTCC
GGATACCTTGTTTATAGTACCTTC
AAAAGAGCAGATTTATACTTATTG
TCTGAAACTCACAAATAACAGCGC
TATTCAACAGACAAGTTATTTAGC
CCATCAGCTTTGTGTGAACAGGTC
TTTCAGGAAGGTGTGATCCACCAC
AAAGACCATTATTGCTTTGGGTAA
AGAAAAGAACCCTGGAAACACCTC
ATAAATCCTAGAGTAGGATATAGG
TGCACATCACTATATCAATAAC
TCAAGTCAAATAAGCTTCTGTATG
GTAGGAATCAACTCTCCTCTACAG
GGAGAAGTTAATATGCACATGC
TTAGGAATAGAACCAAAGGGAGAG
TTCATTGAGCCTCAAATCCTAAA
TAAGGATTGACTGTGTTAGGATC
AGTCCTCTGTGCTCTGCCTATTTC
TCCGGGGGACACTGAGGAAAG
TCGGTGTCACTGATCCACTC
AAGCTAAAGTCTGTGGTGAATTC
AAATGATGCTGTTACTGTCTA
GAAGTTTCAGATACAGATTTTC
GCGACGAGGACGCATTCCAA
GTCCTTGTGCGATCACCACA
Size
220 bp
270 bp
343 bp
245 bp
259 bp
290 bp
281 bp
300 bp
320 bp
390 bp
369 bp
213 bp
237 bp
The underlined sequence corresponds to the universal sequence primers N13. Exon 9(1) stands for the proximal part of exon 9, exon 9(2) the
distal part. RG is for regulatory region.
Supplementary Figure 3D conformational representation of the basic domain. (a) General
representation of Srebp1-A conformation (PDB accession number 1AM9) using the Swiss-Pdb
Viewer software. In its bound conformation, the basic domain is an α-helix that is closely
inserted within the DNA major groove, residues pointing outside of the α-helix The DNA
target is in light blue. One of the two Srebp1-A molecules is in dark blue for clarity, while the
other one is showed in yellow and its basic domain in red. In the second picture, a different
view of the basic domain α-helix, showing its close insertion within the DNA major groove,
residues pointing outside of the α-helix. (b) Sequence alignment between MITF and SREBP1A basic domains. Conserved residues are in red; residues mutated in patients are underligned.
(c) These residues are represented in pink in the 3D view. All of them have their side-chain
pointing towards DNA. None is located on the externally half the α-helix of the basic domain.