Urs-Schnyder-Lecture ESPD Dubrovnik Prof Dr. Dr. Judith Fischer

Urs-Schnyder-Lecture

ESPD Dubrovnik 02.05.201

Prof Dr. Dr. Judith Fischer

University Medical Center Freiburg Institute of Human Genetics Discovery of a gene encoding SLURP-1 in Mal de Meleda Positional cloning

1998

2000 2001

Folie 2 • Milestones • Methods Positional Cloning Homozygosity Mapping Next Generation Sequencing • My Gene identifications - highlights

Folie 3 30 years ago… 1990

Department of Dermatology Prof. R. Happle

Folie 4 1994 Lutetia = Paris EU-grant: Training and mobility

550km

Genetics of psoriasis and genodermatoses

Pr. L. Dubertret Dr. C. Blanchet-Bardon Dr. E. Bourrat

Folie 5 1996 -1998 • Making tools available • Accelerate elucidation of diseases Genopole Evry • (Gene) Therapy / treatment

Dr. J-F. Prud‘homme Dr. J. Weissenbach Dr. S. Saker (DNA bank)

Folie 6 Positional Cloning – Reverse Genetics

TOOLS

MAPS COHORTS TECHNOLOGIES

Physical maps DNA Patients Genotyping facilities YACs, BACs Families microsatellite-panels SNP-microarrays Genetic maps Linkage studies Bioinformatics, Statistics Microsatellites /SNPs Homozygosity Linkage (LOD scores) Human genome project mapping Association studies (p- HapMap (consanguineous) values) GWAS 1000 genome project Sequencing facilities Positional Cloning Using maps, cohorts and technologies

2001 First draft of Human genome sequence

Gene1 Gene2

From: The general approach to positional cloning

Folie 8 1998-2010 Genopole - Evry

Gene identification • Positional cloning (< 2006) • Next Generation Sequencing NGS (> 2006) Whole Exome Sequencing

Pr. M. Lathrop Pr. J. Weissenbach

Folie 9 COHORTS DNA from > 1.000 families with genodermatoses

CNG/Généthon:

France Algeria Tunisia Morocco Turkey Italy Portugal UK Colombie Sub-Sahara Afrique Syria Spain Lebanon

Geneskin EU Sweden Danmark Switzerland Italy Active Participation day and night in the Algerian desert

11 HOMOZYGOSITY MAPPING * I. A chromosomal region which 2 1 3 4 contains the segregating allele from a same ancestor is transmitted via both sides of II. * * the family

Search for common

7 8 1 3 4 1 * homozygosity regions in * 5 6 affected children in consanguineous families III.

71 15 Strategy 3 times more

powerful for localizing a gene

* * * * Opportunity to localize genes IV. for very rare diseases

7 1 7 5 1 1 1 5 Discovery of a gene encoding SLURP-1 in Mal de Meleda Positional cloning

1998

2000 2001

Folie 13 Positional cloning of Mal de Meleda

Family cohorts: Consanguineous families with Mal de Melda from Algeria

Pr. B. Bouadjar

Bouadjar B, Benmazouzia S, Prud'homme J, Cure S, Fischer J. Clinical and Genetic Studies of 3 Large, Consanguineous, Algerian Families With Mal de Meleda. Arch Dermatol. 2000;136(10):1247–1252. doi:10.1001/archderm.136.10.1247

Folie 14 Positional cloning of Mal de Meleda

Family cohorts: Consanguineous families with Mal de Melda from Algeria

Folie 15 Genetic linkage of Meleda disease to chromosome 8qter Fischer et al. (1998) Homozygosity mapping

Fischer et al. (1998) Genetic linkage of Meleda disease to chromosome 8qter. Eur J Hum Genet Bouadjar et al. (2000) Clinical and Genetic Studies of 3 Large, Consanguineous, Algerian Families With Mal de Meleda Arch Dermatol.

Folie 16 Genetic linkage of Meleda disease to chromosome 8qter Fischer et al. (1998)

Folie 17 Mutations in the gene encoding SLURP-1 in Mal de Meleda Fischer et al. (2001) Hum Mol Genet

Patient 1.9 1.11 1.6 2.12 3.19 6.12 7.10 9.5 10.10 11.4 12.9 14.3 16.3 17.3 18.15 4.17 5.9 8.10 13.3 15.6 19.3 Origin A A A A A A A A A A A C C C C A A A C C C Marker D8S1717 3 6 6 11 9 9 3 9 9 9 9 9 3 3 6 6 3 3 9 9 6 6 1 8 3 9 3 9 9 9 9 4 12 12 11 11 3 3 3 3 3 3 D8S1704 5 2 2 3 5 3 3 3 3 3 3 3 5 5 2 2 3 5 3 3 2 2 1 3 1 3 5 3 3 3 3 2 1 1 2 2 1 1 1 1 1 4 CNG001 7 3 3 4 4 5 11 5 7 7 5 5 2 2 3 3 5 2 5 5 3 3 4 6 4 6 1 5 6 6 2 5 6 6 4 4 4 4 4 4 4 8 D8S1727 3 4 - - 3 3 3 3 3 3 3 3 6 6 4 4 6 6 3 3 4 4 ------8 8 3 3 - - 8 8 8 8 8 8 CNG002 4 2 2 1 2 2 2 2 4 4 2 2 2 2 2 2 2 2 2 2 2 2 2 3 1 1 3 2 1 1 2 2 2 2 3 3 2 2 2 2 2 3 CNG003 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 7 7 7 7 7 7 8 8 8 8 8 8 D8S1751 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1 1 1 1 1 1 1 1 1 1 D8S1836 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 5 5 5 5 5 5 4 4 4 4 4 4 CNG004 7 7 7 7 8 11 5 5 9 9 11 11 6 6 7 7 6 6 11 11 4 4 6 6 6 6 6 6 6 6 9 9 7 9 9 9 6 6 6 6 6 6 CNG005 4 4 2 4 4 4 2 2 4 4 4 4 4 4 4 4 4 4 1 4 2 2 2 2 2 2 2 2 2 2 4 4 2 4 4 4 2 2 2 4 2 2 D8S2334 9 9 7 9 7 3 7 7 7 3 3 3 1 1 9 9 2 2 4 3 5 5 9 9 9 9 9 9 9 9 1 1 9 1 2 2 9 9 9 3 9 8 D8S1926 2 2 4 2 5 4 2 2 3 4 4 4 5 5 2 2 5 5 4 4 2 2 2 2 2 2 2 2 2 2 2 2 4 2 2 2 1 1 1 5 1 2 D8S1925 4 4 4 4 1 4 5 5 6 7 4 4 6 6 4 4 6 6 3 4 6 6 6 6 6 6 6 6 6 6 6 6 7 4 6 6 6 6 6 4 6 6 Mutation 82delT Altered Splicing C286T C28 32X 178+1g a R96X

• 19 families, all patients with a homozygous haplotype on 8qter • Smallest segregating interval 1 cM (recombination events, loss of homozygosity) • Contained only 3 genes • Maximum LOD score 24.19 for marker CNG003 at θ = 0.0. • 3 different haplotpes – 3 different mutations in gene ARS (component B) • ARS (component B) : 3 exons, 103 amino acids • coding for SLURP-1

Folie 18 International cooperations 2002 - Dubrovnik, Zagreb, Evry, Lausanne, Edinburgh and Netherlands

Folie 19 Marakchi et al., 2003 JID Large consanguineous pedigrees - Sfax Tunisia

Folie 20 New mutations – 5 Haplotypes – Sfax Tunisia Marakchi et al., 2003 JID

Folie 21 Slurping across the mare nostrum Commentary to Marrakchi et al. JID 2003, 120:351-355

Folie 22 Functional studies : group of Daniel Hohl

Folie 23 Mapping/Localization Papillon-Lefevre-Syndrom 1997

1997

2001

1999, two different groups (Hart et al., Toomes et al.) reported first PLS to be due to mutations in the gene encoding cathepsin C, a lysosomal cysteine protease of the papain type also known as dipeptidyl aminopeptidase (CTSC and DPP1)

Folie 24 Ichthyoses

Autosomal Recessive Congenital Ichthyoses ARCIs

Folie 25 Two new loci for autosomal recessive ichthyosis on Chromosomes 3p21 and 19p12-q12 and evidence for Further Genetic Heterogeneity (Fischer et al.) Positional cloning in 51 consanguineous families without TGM1 mutations 12 families linked to 2 new loci

Chrom. Chrom. 3q21 19p12-19q12

2000

Folie 26 Locus on chrom. 3p21 : Syndromic ichthyosis

Chanarin-Dorfman-Syndrom Neutral lipd storage syndrome with Ichthyosis NLSDI

2001

Folie 27 Jordan’s Chanarin- anomalies in Dorfman polynuclear granulocytes Syndrome NLSDI liver and muscle enzymes Two new mutations of the ABHD5 gene in a new adult case of Chanarin Dorfman syndrome: an uncommon lipid storage disease. Schleinitz et al 2005, Arch Dermatol.

HES Muscle biopsy atrophic (Sudan black) muscle

EM skin biopsy

EM muscle HES skin biopsy with vacuoles in keratinocytes Some families with typical Jordans anomalies had NO MUTATIONS in CGI58/ABDH5 (?? *)

Peripheral blood smear showing lipid vacuoles (arrows) in neutrophils (Jordans anomaly). Similar lipid deposition was also observed in hepatocyte nuclei. (Ciesek et al., 2006)

Folie 29 *2007

PNPLA2 = ATGL = Adipos triglyceride lipase

Folie 30 Mutations of ATGL/PNPLA2 in Neutral lipid storage disease with myopathy NLSDM Fischer et al., 2007 Mutations of ATGL/PNPLA2 in Neutral lipid storage disease with myopathy NLSDM Fischer et al., 2007

Comparaison of phenotypes, metabolic and biochemical defect between patients with NLSDM and CDS/NLSDI RT-PCR

Accumulation of lipids in muscle and skin NLSDM

Locus on 19p12-19q12

2006

Folie 34 Positional cloning of lamellar ichthyosis type 3 LI3 Fischer et al., 2000

6 consanguineous families

Genome-wide scan with microsatellites →Search for common homozygosity regions 5.9 cM in affected children (Homozygosity mapping)

Gene Localisation of LI3 on chromosome 19p21-q21 (Fischer et al 2000)

Initial smallest common interval 5.9 cM 1cM ~1Mb 12 consanguineous families linked to chrom. 19p12-q12 Homozygosity mapping Lefevre et al., 2006 (F1,2,6,8,9,10,11) haplotype a common Patients 7F shared in (F1,F8,F12) 3 families in 6 patients from Loss of homozygosity 3,06 interval: Mb common Smallest Q =0.00 15.83 LOD score D19S930 45 microsatellites Lefevre Patients’ haplotypes et al., 2006 12 11 10 9 7 3 4 5 8 6 2 1 Family N° E11 E11 E10 E10 E9 E9 E12 E12 E4 E4 E3 E3 E12 E12 E12 E12 E16 E16 E13 E13 E13 E13 E12 E12 E17 E17 E14 E14 E13 E13 E8 E8 E3 E3 E11 E11 E10 E10 E11 E11 E9 E9 Patient 0 0 0 0 0 0 6 6 6 7 2 7 4 6 6 6 3 3 5 6 5 4 5 4 0 0 0 0 0 0 6 6 7 7 7 7 7 5 6 3 0 0 3.17 a132zb9

0 0 0 0 0 0 6 6 0 0 4 6 3 4 0 0 0 0 0 0 0 0 0 0 2 6 6 6 7 6 6 6 6 6 1 5 1 6 4 8 3 8 8.04 b344zd1

0 0 0 0 0 0 5 5 9 8 9 8 9 9 9 9 0 0 0 0 0 0 0 0 6 8 6 9 9 9 1 1 1 4 8 9 8 3 7 3 1 3 8..05 a299zc5 7 8 9 3 0 0 8 8 6 6 7 7 7 7 4 4 6 6 6 6 6 6 4 4 4 4 8 8 8 8 8 8 8 8 8 8 7 7 7 7 7 8 12.57 224ye9

2 3 3 3 3 3 5 5 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 3 4 3 4 4 4 3 3 3 3 3 3 3 3 3 5 3 5 12.8 b312yb9

3 7 7 7 7 7 3 3 7 7 7 7 7 7 6 6 2 2 2 2 8 8 8 8 3 7 3 7 3 7 5 5 5 5 7 7 7 7 1 6 1 6 13.7 b022xb1 3 3 0 0 0 0 0 0 3 3 3 3 3 3 1 1 2 2 0 0 0 0 2 2 2 2 2 2 6 6 6 6 2 2 2 2 3 3 14.47 a357zd9 1 2 2 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 14.88 302yb9 0 0 0 0 0 4 4 4 4 4 4 4 4 3 3 3 3 3 3 4 4 4 4 0 0 0 0 0 0 0 0 1 1 1 1 4 4 3 3 0 0 0 15.02 a041zd1 * * * * * *

1 1 15.48 FLJ39501

6 6 6 6 4 4 1 1 5 5 5 5 1 1 0 0 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 15.58 GATA27C12 2 2 2 2 3 3 3 3 1 1 1 1 3 3 3 3 2 2 2 2 1 1 1 1 2 2 2 2 2 2 2 2 1 1 1 1 2 2 15.95 ATA37G08

5 5 5 5 2 2 3 3 2 2 2 2 4 4 2 2 2 2 5 5 5 5 5 5 3 3 3 3 5 5 5 5 1 1 1 1 16.07 a302ya5 10 10

8 8 8 8 1 1 8 8 8 8 6 6 3 3 3 3 3 3 3 3 3 3 1 1 1 1 1 1 1 1 3 3 3 3 16.22 b326zh5 4 4 4 4 1 1 1 1 2 2 2 2 3 3 3 3 2 2 2 2 4 4 2 2 4 4 4 4 0 0 1 1 4 4 4 4 4 2 16.76 ATA57F09

9 9 9 9 6 6 8 8 8 8 8 8 5 5 2 2 1 1 1 1 6 6 6 6 2 2 2 2 2 2 2 2 2 2 1 1 1 1 6 6 6 6 16.82 a50yc5 0 0 0 0 0 0 0 0 6 6 6 6 3 3 3 3 0 0 0 0 0 0 0 0 3 3 3 3 2 2 0 0 0 0 0 0 0 0 17.09 b021yb9

2 2 2 2 2 2 2 2 2 2 0 0 3 3 5 5 5 5 3 3 3 3 3 3 2 2 2 2 4 4 4 4 2 2 2 2 GATA64E12

4 4 4 4 2 2 7 2 4 4 4 4 4 4 1 1 4 4 4 4 6 6 6 6 6 4 4 4 4 4 6 6 6 6 4 4 4 4 2 2 2 2 17.25 278xc5 10 10 10 10 10 10 6 6 6 6 3 3 3 3 3 4 4 4 4 4 4 4 4 5 5 2 2 2 2 9 9 6 6 5 6 6 3 3 3 3 5 6 17.77 234xf4 2 2 2 2 3 4 3 3 4 4 4 4 2 2 2 2 2 2 2 2 2 2 1 2 1 1 1 1 3 3 2 2 3 6 3 6 1 1 1 1 2 2 18.2 143xe9 10 11 10 10 2 2 2 2 4 4 4 4 2 2 2 2 2 2 2 2 3 2 2 6 4 4 4 4 3 3 5 5 2 2 2 2 4 2 18.33 b019xe1

7 7 7 7 4 4 1 5 2 2 2 2 4 4 2 2 3 3 4 3 2 5 5 5 5 5 1 1 1 1 1 1 1 1 3 3 3 3 18.61 b007xb9 11 11 11 11

1 1 8 2 3 3 3 3 3 3 4 4 6 6 6 6 3 4 4 4 4 4 2 5 5 5 4 4 4 4 7 7 7 7 19.02 a123xf1

0 0 0 0 0 0 4 4 0 0 0 0 4 4 0 0 0 0 0 0 1 5 5 5 5 5 2 7 2 2 5 5 5 5 2 2 2 2 19.92 214yf6

2 2 2 2 3 3 4 3 5 5 5 5 3 3 2 2 3 3 3 3 2 3 3 3 3 3 2 2 2 2 2 2 2 2 1 1 1 1 20.7 b293yg9

4 4 4 4 1 1 4 4 3 3 3 3 4 4 4 4 3 3 3 3 4 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 21.15 b355yd1

1 1 1 1 5 5 5 5 1 1 1 1 5 5 1 1 5 5 5 5 4 3 6 6 6 6 6 6 9 9 9 9 1 1 1 1 22.28 344yg5

4 4 4 4 2 2 3 3 5 5 5 5 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 3 3 3 3 3 3 3 3 22.55 GGAT4B07

0 0 0 0 0 0 1 1 3 3 3 3 1 1 1 1 3 3 3 3 3 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 23.04 GATA50H11

1 1 1 1 3 3 3 6 3 3 3 3 3 3 3 3 3 3 3 3 4 2 2 2 2 2 3 3 3 3 3 3 4 4 4 4 23.41 b292wf1

4 4 4 4 3 3 4 3 2 2 2 2 4 4 3 3 3 3 3 3 4 3 3 3 3 3 5 5 4 4 4 4 4 4 4 4 23.44 GATA86H02 24.4-34.5

1 1 1 1 2 2 2 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 3 3 3 3 32.98 326xb1

2 2 2 2 2 2 3 3 3 3 3 3 2 2 1 1 2 2 2 2 3 2 2 2 2 2 2 2 3 3 3 3 2 1 2 1 33.22 309zb9

0 0 0 0 0 0 8 2 2 2 2 2 2 2 8 8 0 0 0 0 8 6 6 6 6 6 7 7 6 6 6 6 1 1 1 1 33.31 a058xc5

5 5 5 5 6 1 3 2 3 3 3 3 2 2 4 4 6 6 6 6 2 5 5 5 6 6 3 3 3 3 3 3 2 2 2 2 33.33 a190yg9

2 2 2 2 3 3 1 1 1 1 1 1 0 0 3 3 2 2 2 2 2 1 1 1 1 1 2 2 3 3 3 3 2 2 2 2 34.18 b340va9

0 0 0 0 0 0 2 4 1 1 1 1 4 4 1 1 0 0 0 0 1 4 4 4 4 4 3 3 1 1 1 1 1 1 1 1 34.42 269xg5

3 3 3 3 3 3 6 2 1 1 1 1 3 3 1 1 1 1 1 1 2 1 1 1 1 1 3 3 3 3 3 3 3 3 3 3 34.46 a084xf1

8 8 8 8 3 3 1 1 6 6 6 6 7 7 6 6 1 1 1 1 6 6 6 6 6 6 6 6 8 8 8 8 5 5 5 5 34.48 a224wc9

4 4 4 4 2 2 2 2 3 3 3 3 4 4 2 2 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 3 3 3 3 34.6 b332xe5

2 2 2 2 4 4 3 3 3 3 3 3 3 3 3 3 6 6 6 6 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 35.41 205yf10

2 2 2 2 2 2 2 1 2 2 2 2 2 2 2 2 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 35.84 c021wd1

5 5 5 5 7 4 1 6 6 4 6 6 5 5 1 1 1 1 6 1 6 8 8 8 8 8 7 5 4 4 4 4 4 4 4 4 36.6 295xg9

0 0 0 0 0 0 2 1 4 2 2 2 2 2 2 2 0 0 0 0 2 2 2 2 2 2 2 3 3 3 3 3 2 2 2 2 37.11 a190zh5

0 0 0 0 0 0 1 2 4 3 3 3 2 2 4 4 0 0 0 0 5 4 4 4 4 4 3 5 5 5 5 5 3 3 3 3 37.51 248zc1 Lefevre et al., 2006

> 90 known genes in 3.06 Mb interval candidate gene selection: epidermal expression, domains/ function in skin lipid metabolism, 12-lipoxygenase pathway coding regions of 28 genes sequenced

Part of the 3.06 Mb interval including a gene cluster of 5 cytochrome P450 (CYP) subfamily F genes and a new homologous gene FLJ39501 = CYP4F22 Lefevre et al., 2006

Table.1. Origin of families and mutations

Family Number Origin Mutation Effect Exon of patients

F1 2 France 1303C→T H435Y 10 F2 2 Algeria 1303C→T H435Y 10 F6 2 Algeria 1303C→T H435Y 10 F8 3 Algeria 1303C→T H435Y 10 F10 2 Algeria 1303C→T H435Y 10 F11 2 Algeria 1303C→T H435Y 10 F3 2 Italy Large deletion 3-12 F4 1 Algeria 728G→A R243H 6 F5 1 Algeria 177C→G F59L 1 F7 1 Algeria 1114C→T R372W 8 F9 1 Lebanon 980delC frame shift 7 F12 2 Algeria 1306C→G H436D 10

FLJ39501 = CYP4F22 7 homozygous mutations: 5 missense mutations, 2 deletions CYP4F22 is a homolog of the leukotriene B4-w-hydroxylase 2002 LI5 - chr 17p13 Patients with deficiency in lipoxygenases ALOX12B or ALOXE3 Jobard et al 2002

Metabolic pathway of arachidonic acid C20H32O2 4-fold unsaturated Fatty acid (peanuts) synthetized from w-6-Fatty acid Linol acid C18H32O2

Brash et al, 2007 Linkage disequilibrium Jobard et al., 2002 A=ALOXE3

B=ALOX12 B B1 B2 B3 2003

Folie 43 Mutations in the transporter ABCA12 are associated with lamellar ichthyosis type 2 Lefèvre et al., 2003 Photos:PrBouadjar

LI2 on Chr.2q33q35: missense mutations in ABCA12

Diverse Functions: transport of lipids into the lamellar granule LG transport of LG to the cell membrane Harlequin Ichthyosis: Mutations leading to a truncated ABCA12 protein (Kelsell et al, Akiyama et al 2005)

Figure: Akiyama et al

Folie 45 2003

Folie 46 Kindler Syndrome Jobard et al., 2003

Folie 47 Kindler Syndrome Jobard et al., 2003

Folie 48 2004

Folie 49 Haplotypes of patients on chrom. 5q33 – Ichthyin /NIPAL4 (Lefèvre et al 2004)

Marker

IG52

323td1

311vf9

336xe9 343xe9 211yc7

210vg3 205yh9

c008zf9

a053yf1

D5S379 D5S378

b351xf9

UT2159 UT8057 UT6511 UT5972 UT5637

b027xc1 a083xb9 c033xh1

ichthyin

a233wc1

a212wg1

207wc11 198we11 a184wd5 b330wa9

GATA43C02

GATA68A01 GATA51G10

Position in Mb

155,4 156,9 158,6 159,6

151,36 152,91 153,58 154,38 155,23 155,25 155,29 155,61 155,64 155,72 155,97 156,66 156,88 157,15 157,34 157,43 157,69 157,84 158,18 158,52 158,64 162,13 162,27 162,81 163,33 165,04 166,49 Family Origin Child 1 TURK C1 2 3 4 3 2 1 2 2 3 2 1 4 3 G235R 3 4 5 5 1 0 5 3 2 2 3 8 2 3 1 1 4 1 TURK C1 2 3 4 3 2 1 2 2 3 2 1 4 3 G235R 3 4 5 5 1 0 5 3 2 2 3 8 2 3 1 1 7 1 TURK C2 2 2 3 3 2 1 2 2 3 2 1 4 3 G235R 3 4 5 5 1 0 5 3 2 2 3 8 2 3 1 1 4 1 TURK C2 2 3 4 3 2 1 2 2 3 2 1 4 3 G235R 3 4 5 5 1 0 5 3 2 2 3 8 2 3 1 1 7 1 TURK C3 2 3 4 3 2 1 2 2 3 2 1 4 3 G235R 3 4 5 5 1 0 5 3 2 2 3 8 2 3 1 1 4 1 TURK C3 2 3 4 3 2 1 2 2 3 2 1 4 3 G235R 3 4 5 5 1 0 5 3 2 2 3 8 2 3 1 1 7 2 ALG C1 1 2 3 2 2 2 2 5 4 1 2 2 9 R83X 8 4 2 4 2 0 8 5 3 2 7 2 2 3 1 2 4 2 ALG C1 2 7 6 2 2 2 2 5 4 1 2 2 9 R83X 8 4 2 4 2 0 8 5 3 2 7 4 2 2 2 1 2 3 ALG C1 2 2 3 2 0 1 1 1 4 1 3 2 6 R83X 4 4 4 5 2 0 4 6 2 1 11 6 3 3 4 4 4 3 ALG C1 2 2 3 2 0 1 1 1 4 1 3 2 6 R83X 4 4 4 5 2 0 4 6 2 1 11 6 3 3 4 4 4 4 ALG C1 2 2 3 2 0 1 1 1 4 1 3 2 6 R83X 4 4 4 5 2 0 4 6 2 1 7 2 1 4 1 1 7 4 ALG C1 2 2 3 2 0 1 1 1 4 1 3 2 6 R83X 4 4 4 5 2 0 4 6 2 1 8 4 2 2 4 3 6 5 COL C1 2 8 2 6 0 2 4 5 3 2 2 2 2 A114N 7 4 4 3 2 0 5 3 3 1 5 2 3 3 1 1 4 5 COL C1 1 2 2 5 0 8 4 3 4 2 1 1 9 A114N 7 4 4 3 2 0 5 3 3 1 5 2 3 3 1 1 4 5 COL C2 1 2 2 5 0 8 4 3 4 2 1 1 9 A114N 7 4 4 3 2 0 5 3 3 1 5 2 3 3 1 1 4 5 COL C2 1 2 2 5 0 8 4 3 4 2 1 1 9 A114N 7 4 4 3 2 0 5 3 3 1 5 2 3 3 1 1 4 6 TURK C1 3 6 9 6 7 1 4 3 4 1 2 1 2 A114N 7 4 4 3 1 0 1 1 4 2 8 8 2 3 1 4 3 6 TURK C1 3 6 9 6 7 1 4 3 4 1 2 1 2 A114N 7 4 4 3 1 0 1 1 4 2 8 8 2 3 1 4 3 7 TURK C1 1 1 2 4 0 6 1 6 3 2 3 0 4 A114N 7 4 4 2 1 0 3 1 1 2 9 6 2 3 1 6 7 7 TURK C1 1 1 2 4 0 6 1 6 3 2 3 0 4 A114N 7 4 4 2 1 0 3 1 1 2 9 4 2 2 4 1 3 7 TURK C2 1 1 2 4 0 6 1 6 3 2 3 1 4 A114N 7 4 4 2 1 0 3 1 1 2 9 4 2 2 4 1 3 7 TURK C2 1 1 2 4 0 6 1 6 3 2 3 1 4 A114N 7 4 4 2 1 0 3 1 1 2 9 4 2 2 4 1 3 7 TURK C3 1 1 2 4 0 6 1 6 3 2 3 1 4 A114N 7 4 4 2 1 0 3 1 1 2 9 4 2 2 4 1 3 7 TURK C3 1 1 2 4 0 6 1 6 3 2 3 1 4 A114N 7 4 4 2 1 0 3 1 1 2 9 4 2 2 4 1 3 8 TURK C1 3 10 7 2 0 8 4 4 4 1 1 5 2 A114N 7 4 4 4 1 0 8 5 2 2 7 4 2 3 1 4 3 8 TURK C1 3 10 7 2 0 8 4 4 4 1 1 5 2 A114N 7 4 4 4 1 0 8 5 2 2 7 4 2 3 1 4 4 9 ALG C1 2 1 2 6 0 2 2 4 3 2 2 3 2 A114N 7 4 3 5 2 0 4 5 2 2 4 4 2 6 5 1 4 9 ALG C1 1 1 2 6 0 2 2 4 3 2 2 3 2 A114N 7 4 3 5 2 0 4 5 2 2 7 8 2 4 3 6 3 10 ALG C1 4 2 5 3 0 2 1 5 4 1 2 2 2 A114N 7 3 5 2 3 0 7 4 2 2 8 4 2 2 1 7 7 10 ALG C1 2 4 2 3 0 2 1 5 4 1 2 2 2 A114N 7 3 5 2 3 0 7 4 2 2 8 4 2 2 1 7 7 11 ALG C1 2 2 5 3 0 2 1 5 4 1 2 2 2 A114N 7 3 5 2 3 0 7 4 2 2 8 4 2 2 1 7 4 Recombinations 11 ALG C1 2 2 5 3 0 2 1 5 4 1 2 2 2 A114N 7 3 5 2 3 0 7 4 2 2 8 4 2 2 1 7 4 12 ALG C1 1 1 5 2 6 1 4 5 2 3 4 2 7 S146F 7 2 4 3 2 0 8 5 3 0 5 5 2 2 2 6 0 Loss of homozygosity 12 ALG C1 1 1 5 2 6 1 4 5 2 3 4 2 7 S146F 7 2 4 3 2 0 8 5 3 0 5 5 2 2 2 1 0 13 SYR C1 3 1 4 3 0 8 2 6 4 1 2 3 6 H175N 3 3 2 0 2 0 3 5 3 1 7 2 1 2 0 4 5 Common haplotypes 13 SYR C1 3 1 3 3 0 8 2 6 4 1 2 3 6 H175N 3 3 2 0 2 0 3 5 3 1 7 2 2 1 0 2 8 13 SYR C2 3 1 4 3 0 8 2 6 4 1 2 3 6 H175N 3 3 2 0 2 0 3 5 0 1 7 2 1 2 1 4 5 Mutations 13 SYR C2 3 1 3 3 0 8 2 6 4 1 2 3 6 H175N 3 3 2 0 2 0 3 5 0 1 7 2 2 1 1 2 8 14 ALG C1 1 2 2 2 1 5 2 4 4 3 1 3 4 G80V 2 3 0 3 1 1 7 3 2 2 8 7 2 2 1 4 5 14 ALG C1 1 2 2 2 1 5 2 4 4 3 1 3 4 G80V 2 3 0 3 1 1 7 4 2 2 8 7 2 2 1 4 5 14 ALG C2 1 2 0 2 0 5 2 4 4 3 1 3 4 G80V 2 3 3 3 2 2 6 3 2 4 11 4 2 2 1 4 4 14 ALG C2 1 2 2 2 0 5 2 4 4 3 1 3 4 G80V 2 3 3 3 1 1 7 4 2 2 8 7 2 2 2 7 6 14 ALG C3 1 2 2 2 0 5 2 4 4 3 1 3 4 G80V 2 3 3 3 1 1 7 1 2 2 8 7 2 2 1 4 5 14 ALG C3 2 2 2 1 0 2 3 4 4 3 1 3 4 G80V 2 3 3 3 1 1 7 4 2 2 8 7 2 2 1 4 5 14 ALG C4 1 2 2 2 0 5 2 4 4 3 1 3 4 G80V 2 3 3 3 1 1 7 1 2 2 8 7 2 2 1 4 5 14 ALG C4 1 2 2 2 0 5 2 4 4 3 1 3 4 G80V 2 3 3 3 1 1 7 4 2 2 8 7 2 2 1 4 5 Haplotype Analysis or Genetic Sudoku ? First described as Self-Healing Congenital Verruciform Hyperkeratosis (Leaute-Labreze et al, 2004)

•prematurity >6 weeks before term (A, B) •respiratory distress •Generalized hyperkeratosis, focal →accentuation on skalp, eyebrows •at 3 months of age: toad-like hyperkeratosis especially in the skin folds •Spontaneous improvement •mutations in FATP4 HI

PNPLA1 2012 CERS3 SULT2B1b 2013 2017 SDR9C7 2016 LI

NIPAL4/

BSI,SI, LI Figure from Fischer, JID, 2009

ARCI

PNPLA1 mutations cause autosomal recessive congenital ichthyosis in golden retriever dogs and humans (Grall et al., 2012)

Using a spontaneous dog model in the Golden retriever breed →identification of a new mutated human ARCI gene: PNPLA1

•Golden retriever with ichthyosis described in US (Mauldin et al., 2008) UK (Cadiergues et al., 2008) and France (Guaguere et al., 2009) •first signs of ichthyosis within a few weeks after birth, no collodion baby Folie 56 Patients with mutations in PNPLA1

CLINICAL •collodion baby •lamellar ichthyosis •in several cases: cyclic course of the disease with complete disappearance and recurrence of scaling every 6 weeks (Mauserung)

Zimmer et al. 2016: 17 novel mutations in PNPLA1 in patients with autosomal recessive congenital ichthyosis. submitted Photos: Dr. Bourrat, Paris; Dr. Oji Münster Dr. Stieler, Pr. Blume-Peytavi, Berlin HISTOLOGICAL •acanthosis •thickening of cornified and granular layer •cholsterol clefts in the cornified layer (as in TGM1 mutants) •accumulations of abnormal membranous and vesicular material around the nuclei of cells from the granular layer → degenerative process? normal mutant normal mutant mutant

HE Transmission electron micrographs from Grall et al, 2012 Folie 58 A contiguous gene deletion syndrome causes autosomal recessive congenital ichthyosis due to a partial deletion of CERS3 Radner et al., PLOS Genet 2013 Patients SNPs

• Genom-wide SNP-Genotyping analysis (homozygosity mapping) of 34 consanguineous families with ARCI (2008, CNG)

• 5 patients from 4 Tunisian families show a homozygous region on chromosome 15q26 with an identical haplotype in the smallest common interval of 1.67 Mb

1.67 Mb Genomic deletion on chromosome 15q26

Radner et al., PLOS Genet 2013 in 4 patients (3 from 4 families) 106,960 bp sized homozygous deletion encompasses ADAMTS17, lncRNA FLJ42289, and CERS3. Fluorescence in situ Hybridization

Radner et al., PLOS Genet 2013

FISH analysis confirmed homozygous deletion Red: 15q21.2 (control signal) Green: 15q26.3 CERS3 deficiency

CLINICAL Radner et al., PLOS Genet 2013 •collodion baby •lamellar ichthyosis: large brownish scales on the limbs and trunk •ichthyosiform erythrodermia with large, white scales •palmoplantar hyperlinearity and hyperkeratosis CERS3 mutations cause abnormal skin morphology

Radner et al., PLOS Genet 2013

HISTOLOGICAL •Acanthosis with •thickening of the stratum granulosum •psoriasiform epidermal hyperplasia •normal size of the stratum corneum

CERS3 localizes at the interface between stratum granulosum and stratum corneum in the epidermis Mutated CERS3 disturbs the epidermal sphingolipid profile

Radner et al., PLOS Genet 2013

Abbreviations: Cer, ceramide; DAG, diacylglycerol; NEFA, non- esterified fatty acid; TAG, triacylglycerol. * p<0.05, ** p<0.01, *** p<0.001 ➢reduced concentrations of very-long chain ceramides, acylceramides and glucosylceramides in patient’s keratinocytes Cers3-deficiency in mice Jennemann et al., Hum Mol Genet. 2012.

• absence of epidermal ultra-long chain ceramides (C>26) • defective skin barrier results in neonatal death of Cers3-deficient mice Biosynthesis of Ceramide

CERS3 shows a relatively broad substrate spectrum (acyl-CoAs of C18-28) Levy et al., IUBMB Life 2010 Positoinal Cloning

Disease- mutation at causing AMINO ACID Human genome mutation position 3x109 bp GENE

protein defect/ deficiency

Chromosome Candidate region DNA sequence linkage map 5x106 bp 10.000-100.000 bp 108 bp 1-100 genes

Folie 67 Since 2010 Freiburg

Folie 68 Next Generation Sequencing NGS

1. Multi-Gen-Panel Sequencing heterogene Group monogenetic disease 2. Whole-Exome-Sequencing WES (~95% coverage) 3. Whole-Genome-Sequencing WGS ➢ massive-parallel-sequencing

HiSeq Illumina® Why only Exome-sequencing? Principal of Next Generation Sequencing NGS

• massively parallel sequencing • perform simultaneous millions of independent sequencing reactions • depending on the technology*, on millions of beads, droplets of an emulsion, or individual micropores on a chip

Work flow ✓ DNA-fragmentation ✓ library preparation ✓ library amplification/immobilization

✓ sequencing Myllykangas et al. 2012 ✓ bioinformatic analysis

Folie 71 Bioinformatic Analysis of WES

27,774 variants • Quality filtering • Remove synonymous • - 26,218 Only coding regions • dbSNP MAF <=0.05 • ESP6500 MAF <=0.05 1,556 variants

MAF : minor allel frequency -430 (~28%) • ExAC database MAF <=0.05 1,126 variants

Trio data 158 variants 968 variants (hom) (het)

22 variants (hom) 42 variants (het) Am J Hum Genet 2017;100(6):926-939 74 Mutations in SULT2B1 Cause Autosomal-Recessive Congenital Ichthyosis in Humans Clinical features of family I: Tunisian family

• consanguineous parents • 3 affected individuals born as collodion babies • here: patient 1 at the age of 15 years • presents a lamellar form of ARCI with large, dark scales • hyperkeratotic plaques over the trunk varied from mild to severe grade (C)

• to note: Axilla (A), face and ears (B), and part of the soles (D) were not affected! → Overlap with X-linked ichthyosis?

(Heinz et al. 2017) 75 Mutations in SULT2B1 Cause Autosomal-Recessive Congenital Ichthyosis in Humans (Heinz et al. 2017) Clinical features of family III: Kurdish family

• consanguineous parents

• 2 affected individuals not born as collodion babies

• here: patient 5 (aged 10 years) and patient 6 (aged 6 years)

• present dry, scaly skin with severe itching and erythema at birth

• itching and scaling improved with age

• thicker dry, greyish skin on the knees (E), elbow, and dorsal feet (G) and hands (H)

• lichenification of the popliteal fossa (F) 76 Mutations in SULT2B1 Cause Autosomal Recessive Congenital Ichthyosis in Humans Identification of SULT2B1b mutations in three families

(Heinz et al. 2017)

• 2 missense mutations clinical pictures from patients 1, 5, 6 (arrows)

• 1 nonsense mutation * skin biopsies obtained from patients 5, 6 (asterisks)

• 1 splice-site mutation 77

Histological and ultrastructural analysis of skin biopsies

• pronounced hyperkeratosis with a massive orthokeratotic cornified layer (B) • perivascular lymphocytic infiltrations with eosinophilic granulocytes (C) • horny lamellae of patient 5 with numerous small vesicular inclusions (E) 78 SULT2B1b mRNA and protein expression in patients is disturbed

• RT-PCR with total RNA

→ absence of SULT2B1b expression at the mRNA level

• Western blot analysis of SULT2B1b in control and patient keratinocytes:

→ SULT2B1b was not detectable in keratinocytes of patient 5

• immunofluorescence staining of paraffin sections:

→ absence of SULT2B1b expression in the epidermis of patient 5 (red) 79

A 3D in vitro skin model reproduces the characteristics of patients with SULT2B1b mutations

• organotypic models formed a multilayered stratified epithelium composed of layers which are representative for in vivo skin of patient

→ stratum corneum was significantly thickened in the patient’s organotypic model (A)

→ Western blot analysis of these models confirmed the absence of SULT2B1b in the patient’s model (B)

→ Immunofluorescence analysis confirmed the increased expression of the differentiation markers loricrin and involucrin in the patient’s model (C) 80

SULT2B1b is involved in cholesterol sulfate cycle

• SULT2B1b catalyzes the transfer of the 5’ sulfate from the donor 3’- phosphoadenosine 5’-phosphosulfate (PAPS) to the hydroxyl group (OH) of cholesterol

• STS catalyzes the counter reaction converting cholesterol sulfate into cholesterol 81

Mutations in SULT2B1b cause absence of cholesterol sulfate and accumulation of cholesterol

→ Thin layer chromatography of polar lipids revealed the absence of cholesterol sulfate in organotypic cultures from SULT2B1b-patient, but accumulation of cholesterol sulfate in those of STS-patient

→ Thin layer chromatography of neutral lipids revealed a statistically significant increase in the cholesterol amount in organotypic cultures from patient 6 82

Distribution of gene mutations in ARCI

19% of cases Novel forms of Ichthyoses only a few patients/families

Neues Gen: SDR9C7 ~ 2%

SULT2B1b ~ 0,52% 2019 in Freiburg: Ongoing - News

Folie 83 Hunting or fishing?

GeneHunter or GeneFischer?

Folie 84 A novel monoallelic two-hit mechanism in PLCD1 explains the genetic pathogenesis of hereditary trichilemmal cyst formation

Hörer et al. accepted for publication in JID

Folie 85 Institut für Humangenetik

Research Lab Thanks !

Diagostics Labs for Genetics and Zytogenetics NGS-Analysis and Validation Bioinformatics All collaboration partners Genetic councelling Germany, France, Italy, Spain…. Europe North Africa US ….

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