ARTICLE
Auteur(s) : Mahmood Rasool1, Sadia
Nawaz1, Aysha Azhar1, Muhammad
Wajid1, Per Westermark2, Shahid M
Baig1, Joakim Klar1, Niklas Dahl2
1Human Molecular Genetics Laboratory, Health
Biotechnology Division, National Institute for Biotechnology
and Genetic Engineering (NIBGE), Faisalabad, Pakistan
2Department of Genetics and Pathology, The
Rudbeck Laboratory, Uppsala University and University
Hospital, 751 85 Uppsala, Sweden
accepté le 1 F�vrier 2010
Ectodermal dysplasia (ED) represents a heterogeneous group of
inherited and non-progressive disorders involving defects in at
least two of the ectodermal appendages, i.e. hair, nails, skin and
sweat glands [1]. More than 200 clinical entities of ED have
been delineated so far, of which only a minority have been
explained at the molecular level [2]. Ectodermal dysplasia
restricted to hair and nails (OMIM 602032) is rare and four
different types have been described with a variable degree of
severity [3-6]. These forms are inherited as either autosomal
recessive or autosomal dominant traits [3, 5]. The clinical
expression may vary from being restricted to hypotrichosis areata
to almost complete atrichosis of the entire body. The degree of
onychodysplasia is also variable between families but is usually
similar within multiplex families. The clinical expressions of both
hair and nails appear more severe in the autosomal recessive
form(s) but very few families are described [3-8]. One autosomal
recessive form of hair-nail dysplasia with total alopecia and
micronychia was recently found associated with a missense mutation
(R78H) in the KRTHB5 gene on chromosome 12 [7, 8]. To further
clarify the basis of pure hair nail ectodermal dysplasia we
investigated a consanguineous Pakistani kindred segregating a
variant form of autosomal recessive hair-nail ED with hypotrichosis
and mild onychodystrophy.
Material and methods
Case reports
The family comprises seven children, including one affected male
and three affected females (figure 1). The
consanguineous parents are unaffected which suggests an autosomal
recessive mode of inheritance. Informed consent was obtained from
all family members. Normal as well as affected family members
underwent medical examination with particular attention to skin,
dentition, nails, sweating, scalp, and body hair. The four affected
individuals have a generalised hypotrichosis present since birth.
Eyebrows, eyelashes and body hair are present but thin in all
affected individuals. The hairs appear brittle, thin and less
flexible with fragile shafts that break relatively easily upon
mechanical stress. The hair is sparse and evenly distributed over
the head in three affected individuals whereas one patient has
hypotrichosis which is more pronounced on the scalp (figure 2). Nail dystrophy
is present in the four affected individuals and with a similar,
relatively mild expression (figure 3). All fingers and
toes have a relative micronychia and mild distal onycholysis. The
growth of both hair and nails is reduced in the four cases compared
to the healthy siblings. Affected family members have a normal
dentition, no skin abnormalities and they reported normal sweating.
Linkage analysis and DNA sequencing
Blood samples were obtained from all seven siblings and their
parents. Genomic DNA was extracted by standard methods. The samples
were analysed using highly polymorphic microsatellite markers for
linkage to the disease. Six candidate gene regions were selected
from their association to ED phenotypes: The type I keratin gene
region on chromosome 17q12-q21; the type II keratin gene region on
chromosome 12q13; the ED2 locus on chromosome 13q12.11; the
ED3 locus at chromosome 2q11-q13; the ED4 locus at
chromosome 11q23.3; and the human hairless (HR) gene region on
chromosome 8p12. Four microsatellite markers were selected in each
gene region and amplified by polymerase chain reaction (PCR) with
incorporation of fluorescent labels. The physical location of each
marker was determined according to the National Center for
Biotechnology Information (NCBI) Build 36.3 sequence-based
physical map1. Pair-wise linkage analysis was performed
using MLINK program of FASTLINK computer package with inbreeding
loops broken [9]. For the analysis we assumed an autosomal
recessive mode of inheritance with complete penetrance and a
disease allele frequency of 0.001.
Results
Light- and polarization microscopy were used to investigate hair
derived from one affected family member (ind. V: 2, age
20 y.). Magnification of hair shafts at 4× and 10× showed a
shaggy structure when compared to the more homogeneous structure of
a gender- and age-matched control hair of Pakistani origin (figure 4). The
diameter of hair shafts was similar in the patient and the control.
Linkage to the nail-hair ED phenotype was excluded for
microsatellite markers at all candidate loci investigated except
for those located in the type II keratin gene cluster on chromosome
12. We then used additional microsatellite markers to refine the
linked interval and a maximum two point LOD score (Zmax)
of 2.92 was obtained with the marker D12S368 at zero
recombination fraction (table 1). The
linked region is flanked by markers D12S2080 and
D12S1686 spanning the type II hair keratin gene cluster
located between markers D12S368 and D12S398. We then performed
bidirectional sequencing on genomic DNA derived from two affected
individuals for all coding exons and splice junctions of the
KRTHB1, KRTHB3, KRTHB5 and KRTHB6 genes (protocols and primer
sequences are available upon request) [10]. No sequence variants
were identified when comparing the results to reference sequences
in public databases2.
Table 1 Two point lod score results between hair-nail
dysplasia and chromosome 12 marker loci
|
Marker
|
Genetic distance (cM)a
|
Physical position (kb)b
|
LOD score
|
|
D12S2080
|
55.58
|
33306
|
-∞
|
|
D12S297
|
-
|
37405
|
2.10
|
|
D12S291
|
58.15
|
42204
|
1.72
|
|
D12S368
|
66.55
|
50918
|
2.92
|
|
D12S398
|
64.72
|
51483
|
2.85
|
|
D12S90
|
72.76
|
56710
|
2.79
|
|
D12S1686
|
78.75
|
63951
|
-∞
|
|
D12S92
|
87.20
|
72420
|
-∞
|
Discussion
This is the first finding confirming autosomal recessive and “pure”
nail-hair ED caused by a gene on chromosome 12. The chromosome
12 region coincides with the keratin gene cluster and we
excluded mutations in the coding regions of the KRTHB1, KRTHB3,
KRTHB5 and KRTHB6 genes. The phenotype in our family appears
slightly milder than the clinical features reported previously in
association with a KRTHB5 gene mutation.
From our results, we cannot rule out the possibility of a
mutation in regulatory or intronic regions of the KRTHB5 gene. Yet
another explanation is genetic heterogeneity for autosomal
recessive HNED with a second gene for the disease located in the
linked region on chromosome 12. The candidate gene region on
chromosome 12p11.1-q14.3 is gene rich and contains about
478 genes. Among these are several additional keratin genes
expressed specifically in the hair cuticles. Examples are genes
encoding KRTHB2, KRTHB4, K6HF, K6irs1, K6irs2, k6irs3 and
K6irs4 remaining candidates for this phenotype.
A novel gene for HNED within the linked region may also be
supported by the fact that the phenotype in our family is
clinically different from the family associated with a KRTHB5
mutation. The KRTHB5 mutation is associated with total alopecia and
a more severe hyponychia whereas affected members in our family
have hypotrichosis, distal onycholysis and mild hyponychia. More
families with pure hair-nail ED are now required to further clarify
the genetic basis for this disease entity as well as to confirm the
structural abnormalities of the hair shafts observed in this study.
Identification of the underlying gene mutations will provide better
diagnostic tools for ED and novel insights into the formation of
hair and nails.
Acknowledgements
We wish to thank the family members for their cooperation.
Financial support: Mahmood Rasool is supported by a Ph.D.
fellowship from Higher Education Commission (HEC), Islamabad,
Pakistan. This study was supported by the Swedish Research Council
(ND) and Swedish Links (ND, SMB), The Indevelops fund (ND), Thuréus
fund, Torsten and Ragnar Söderbergs Foundation (ND), Uppsala
University and the Uppsala University Hospital. Conflict of
interest: none.
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1 http://www.ncbi.nlm.nih.gov
2 www.ensemble.org
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