Letters to the Editor / Journal of Dermatological Science 61 (2011) 129–150134

Letter to the Editor

Congenital cutis laxa syndrome maps to a novel locus onchromosome 9q13-q21.32

Congenital cutis laxa syndrome is a diverse group of inheritedabnormalities. It is characterized by loose, aged, wrinkled orinelastic skin associated with cardiovascular abnormalities, jointdislocation, low bone mass, developmental delay and mentalretardation. Both X-linked and autosomal forms of cutis laxa havebeen reported.

For the study, presented here, we ascertained a largePakistani family (Fig. 1a) segregating an autosomal recessiveform of cutis laxa syndrome. Affected subjects were clinicallyexamined at National Institute of Rehabilitation Medicine(NIRM), Islamabad, Pakistan after approval of the study fromInstitutional Review Board (IRB) of Quaid-i-Azam University,Islamabad, Pakistan.

At the time of the study the patients were one to eighteen yearsof age and showed phenotypes of equal intensity. Generalized skinlaxity and progeroid appearance were observed in all the patients.[()TD$FIG]

Fig. 1. (a) Pedigree of a family with cutis laxa syndrome. Filled symbols represent affected

individuals, genotypes are shown beneath each individual. The microsatellite markers an

interval of 16.94 cM is flanked by markers D9S1862 and D9S167. (b) Characteristic proger

cheeks, broaden nasal bridge and large pinnae in patient IV-2. Hyper-extensible and lo

The facial appearance was characterized by flat midface, saggingcheeks, broaden nasal bridge, hypertelorism, eyes with periorbitalswelling and slightly flattened elongated philtrum. The externalear morphology showed large pinnae with inconspicuous orflattened form of the strongest anthelical curvature (Fig. 1b).Wrinkled skin was more conspicuous that could easily be pulled byfew centimeters over the shoulders, neck and abdomen (Fig. 1c andd). Redundant skin folds were not observed. Pulmonary functiontests (PFTs) and electrocardiography (ECG) revealed normalfunctioning of the lungs and heart, respectively. Other laboratorytests showed normal levels of liver enzymes, blood proteins,leukocytes, cholesterol, follicle stimulating hormone (FSH) andluteinizing hormone (LH). Ophthalmological examinationsshowed normal functioning of eyes. Osteoporosis and jointdislocations were not observed in any patient of the family. Hair,teeth, nails, hearing, sweating were normal in all the patients. Thepatients had reduced body weight as compared to normalmembers of the family. They had normal prenatal developmenton scheduled ultrasound, delivery with normal cry but the

individuals, whereas clear symbols represent unaffected individuals. For genotyped

d their relative map positions in cM are shown along with the pedigree. The disease

oid facial appearance of the patient with hypertelorism, periorbital swelling, sagging

ose skin of the abdominal regions in patients IV-3 (c) and IV-6 (d).

Table 1Two-point and multipoint LOD score between disease and microsatellite markers of chromosome 9.

Marker Distance mpta Two-point LOD score at ub=

Mbc cMd ub = 0 0.0 0.01 0.05 0.1 0.2 0.3 0.4

D9S773 38.90 64.28 -inf -inf �2.81 �1.51 �0.94 �0.43 �0.19 �0.06

D9S238 64.48 65.84 -inf -inf �0.27 0.29 0.43 0.41 0.28 0.13

D9S1862 70.33 66.55 -inf -inf �1.68 �0.45 �0.06 0.14 0.11 0.04

D9S745 71.81 67.74 3.25 2.35 2.30 2.09 1.82 1.29 0.74 0.24

D9S301 72.99 68.13 3.25 2.82 2.76 2.53 2.23 1.62 1.01 0.42

D9S1876 74.42 69.40 3.25 2.72 2.66 2.43 2.14 1.55 0.95 0.38

D9S927 76.01 70.2 3.25 2.55 2.50 2.28 2.00 1.43 0.86 0.33

GATA165F10 77.21 71.93 3.25 2.55 2.50 2.28 2.00 1.43 0.86 0.33

D9S1807 78.07 73.84 3.25 2.35 2.30 2.09 1.82 1.29 0.74 0.24

D9S1122 78.87 75.87 3.25 2.88 2.82 2.59 2.28 1.67 1.05 0.45

D9S967 80.28 77.3 3.25 2.55 2.50 2.28 2.00 1.43 0.86 0.33

D9S153 80.77 78.04 3.25 2.78 2.72 2.48 2.19 1.59 0.98 0.41

D9S1867 81.63 79.66 3.25 2.72 2.66 2.43 2.14 1.55 0.95 0.38

D9S933 82.82 80.39 3.25 2.55 2.50 2.28 2.00 1.43 0.86 0.33

D9S245 83.50 82.08 3.25 2.35 2.30 2.09 1.82 1.29 0.74 0.24

D9S764 83.60 82.08 3.25 2.77 2.71 2.48 2.18 1.58 0.98 0.4

D9S2144 84.44 83.02 3.25 2.55 2.50 2.28 2.00 1.43 0.86 0.33

D9S167 84.97 83.49 -inf -inf �3.48 �1.55 �0.84 �0.3 �0.08 0.00

D9S1877 85.63 84.61 -inf -inf �3.18 �1.27 �0.59 �0.12 0.01 0.03

D9S1120 87.28 86.47 -inf -inf �0.86 �0.24 �0.04 0.06 0.06 0.03

a Multi-point LOD score.b Recombination fraction.c Physical distance (mega base pairs) according to the second-generation combined linkage physical map of the human genome [3].d Genetic distance (centiMorgans) according to the second-generation combined linkage physical map of the human genome [3].

Letters to the Editor / Journal of Dermatological Science 61 (2011) 129–150 135

pregnancy lasted for 10 months, however, body appeareddehydrated with scaphoid abdomen probably due to loss ofsubcutaneous fats. Walking and speech delay has been observed inall patients as compared to their normal brothers. Urogenitalsystem was normal with normal genitalia and both testes inscrotum. There was no evidence of pyloric stenosis and ultraso-nography revealed normal pylorus with normal pyloric canaldiameter. All the patients developed symptoms of gastro-esophageal reflux due to hiatal hernia in early infantile age.Fundoplication of all patients including IV-6 (Fig. 1d) at the age oftwo months was performed for diaphragmatic hernia withherniation of abdominal contents into right hemithorax. Laparo-tomic findings revealed type IV sliding hiatal hernia (gastro-esophageal junction, stomach, small intestine and transverse colonwere present in right hemithorax). A small mesentery with gutmalrotation was also present. Angiography to exclude the presenceof tortuous arteries and skin biopsy for electron microscopicexamination were not provided by the patients.

Before embarking on human genome scan, genetic mappingusing polymorphic microsatellite markers was performed totest linkage to 13 candidate genes including ATP7A (Xq13),ELN (7q11.23), ATP6V0A2 (12q24.23), FBLN4 (11q13.1), FBLN5

(14q32.12), P5CS (10q23.33), PYCR1 (17q25.3), GORAB (1q24.2),ADAMTS2 (5q35.3), LAMB1 (7q31.1), LOX (5q23.1), RIN2 (20p11.23),SLC2A10 (20q13.12), reported earlier in causing cutis laxaassociated syndromes. Genotyping results failed to show linkagein the family to known genes tested here.

Human genome scan using a total of 534 short tandem repeat(STR) markers from Linkage Mapping Set (Invitrogen, San Diego,CA, USA) revealed a common homozygous region of 14.6 Mb,flanked by markers D9S1862 and D9S167, on chromosome 9q13-21.32 in the affected individuals of the family (Fig. 1a). The highesttwo-point LOD score of 2.8 was obtained with two markers D9S301and D9S1122, using MLINK program of FASTLINK computerpackage [1] at (u = 0.00), while maximum multi-point LOD scoreof 3.2 was achieved, using ALLEGRO-2 [2] at several markers alongthe disease-interval (Table 1).

The linkage interval of the autosomal recessive cutis laxasyndrome locus on chromosome 9q13-21.32, mapped in the

present study, contains total of 83 known and predicted genes.Based on the expression data of the genes available at theUniversity of California-Santa Cruz (UCSC) human genomedatabase, similarity with the genes identified and their involve-ment in the pathways reported earlier and mode of action, weselected eight genes (PRKACG, TJP2, ALDH1A1, RFK, GCNT1, GNAQ,PSAT1, CHCHD9) for sequence analysis. Proteins encoded by thesegenes are involved in regulation of dynamics of many organelles,regulations of tight junction assembly, regulations of cortical actincytoskeletal components, and promote cell–cell adhesion. DNAsequence analysis of coding regions and exon–intron boundariesthese eight genes on ABI Prism 310 DNA sequencer (AppliedBiosystems, Foster City, CA, USA) failed to detect any pathogenicvariant in affected individuals of the family. However, five singlenucleotide polymorphisms (SNPs) in the coding regions of thegenes were determined including two synonymous (c.225C > T inALDH1A1 and c.228G > A in CHCHD9) and three non-synonymous(c.3T > G, p.I1 M in RFK, c.1446C > A, p.D482E and c.3029C > T,p.S1010F in TJP2).

Sequencing of the eight selected putative candidate genes inthe region 9q13-21.32 did not reveal any functional sequencevariant and therefore their involvement in causing cutislaxa syndrome phenotype at the present novel locus is notsupported. Further fine-mapping and sequencing work arerequired to identify the gene causing cutis laxa syndrome atthis locus.

Acknowledgements

We acknowledge the financial support provided by HigherEducation Commission (HEC), Islamabad, Pakistan, and co-opera-tion of the research volunteers of the family for the study.Musharraf Jelani is supported by indigenous PhD program ofHigher Education Commission (HEC), Islamabad, Pakistan.

References

[1] Cottingham Jr RW, Idury RM, Schaffer AA. Faster sequential genetic linkagecomputations. Am J Hum Genet 1993;53:252–63.

Letters to the Editor / Journal of Dermatological Science 61 (2011) 129–150136

[2] Gudbjartsson DF, Thorvaldsson T, Kong A, Gunnarsson G, Ingolfsdottir A. Allegroversion 2. Nat Genet 2005;37:1015–6.

[3] Matise TC, Chen F, Chen W, De La Vega FM, Hansen M, He C, et al. A second-generation combined linkage physical map of the human genome. Genome Res2007;17:1783–6.

Musharraf JelaniMuhammad Tariq

Department of Biochemistry,

Faculty of Biological Sciences,

Quaid-i-Azam University,

Islamabad, Pakistan

Iftikhar Ahmad JanHazrat Ullah

Department of Pediatric Surgery,

National Institute of Rehabilitation Medicine (NIRM),

Islamabad, Pakistan

Muhammad NaeemDepartment of Biotechnology,

Faculty of Biological Sciences,

Quaid-i-Azam University,

Islamabad, Pakistan

Wasim Ahmad*Department of Biochemistry,

Faculty of Biological Sciences,

Quaid-i-Azam University,

Islamabad, Pakistan

*Corresponding author. Tel.: +92 51 90643003;fax: +92 51 9205753

E-mail address: ahmad115@hotmail.comwahmad@qau.edu.pk (W. Ahmad)

30 June 2010

doi:10.1016/j.jdermsci.2010.11.014

Letter to the Editor

New insight into genotype/phenotype correlations in ABCA12

mutations in harlequin ichthyosis

Harlequin ichthyosis (HI) is a severe and often fatal congenitalichthyosis with an autosomal recessive inheritance pattern [1]. Theclinical features include thick, plate-like scales with ectropion,eclabium and flattened ears. ABCA12 mutations underlie HI [2,3]and it was clarified that HI is caused by severe functional defects inthe keratinocyte lipid transporter ABCA12 [2]. To date, variousABCA12 mutations have been reported in HI patients [4]. However,genotype/phenotype correlations in ABCA12 mutations have beenpoorly elucidated. In order to obtain clues to understand genotype/phenotype correlations in ABCA12 mutations, we report two HIpatients from two independent Japanese families, who werecompound heterozygotes for ABCA12 mutations.

Patient 1 is the second child of healthy, unrelated Japaneseparents. The skin of the baby girl was covered with white, diamondshaped plaques at birth (Fig. 1a). After therapy with oral retinoidsand local application of white petrolatum, in a humid incubator,the scales gradually detached and passive and spontaneousmobility of the joints increased. Now at the age of 1 year and 7months, her general condition is good, although she still has whiteto grey scales on a background of erythematous skin over herentire body. Patient 2 is the fourth child of healthy, unrelatedJapanese parents. Her older brother had a history of congenitalichthyosis and died in early infancy. The skin of the newbornshowed serious symptoms with thick, white, diamond shapedplaques, partly bordered by bleeding fissures (Fig. 1c). Althoughshe had therapy with oral retinoids and local application of whitepetrolatum, in a humid incubator, her clinical symptoms failed toshow any apparent improvement and she died when she was 5months old.

Skin biopsies showed thick stratum corneum in both patients(Fig. 1d–g). In Patient 2, parakeratosis was observed in theepidermis and a sparse inflammatory cell infiltration was seen inthe superficial dermis (Fig. 1e inset). Electron microscopy (Hitachi,Tokyo, Japan) revealed a large number of abnormal, variously sizedlipid droplets that accumulated in the cornified cells of bothpatients’ epidermis.

Mutational analysis of ABCA12 was performed in both patientsand their families. Each genomic DNA sample was subjected to PCR

amplification, followed by direct automated sequencing. Oligonu-cleotide primers and PCR conditions used for amplification of allexons 1–53 of ABCA12 were originally derived from the report byLefevre et al. [5] and were partially modified for the present study.The entire coding region including the exon/intron boundaries forboth forward and reverse strands from the patients, their parentsand 50 healthy Japanese controls were also sequenced. Bothpatients had the same paternal novel nonsense mutationp.Arg1515X (Fig. 1h) which leads to truncation of the first ATP-binding cassette within ABCA12 likely resulting in ABCA12 loss offunction (Fig. 2a). On the other allele, Patient 1 had a maternalrecurrent splice acceptor site mutation c.3295-2A>G (Fig. 1h). Thissplice site mutation was reported in an unrelated Japanese familywith HI and was shown to lead to comparable amounts of 2 splicepattern variants [2]. The first mutant transcript would result in a 3amino acids deletion (1099_1101delYMK). These 3 amino acids arelocated in the first transmembrane domain and are highlyconserved (Fig. 2b). The second mutant transcript lost a 170-bpsequence from exon 24, which led to a frameshift. Expression of asmall amount of ABCA12 protein, although mutated, was detectedin the granular layer keratinocytes of the patient’s epidermis andcultured keratinocytes by immunofluorescent staining [2]. Thus, itis possible that Patient 1 expresses some mutated ABCA12 proteinwith a partial function. This might be the reason why Patient 1survived beyond the perinatal and neonatal period and is still alivealthough this might also be in part due to the prompt oral retinoidtreatment.

Patient 2 carried a maternal missense mutation p.Gly1179Arg onthe other locus (Fig. 1h). To confirm the presence of the mutationp.Gly1179Arg in Patient 2, we performed restriction enzymedigestion analysis using BclI (NEW ENGLAND BioLabs). Restrictionenzyme digestion of PCR products was carried out according to themanufacture’s protocols. The 255-bp PCR products from wild typealleles were not digested by BclI, although the PCR products from theallele with the mutation p.Gly1179Arg were digested into 173- and82-bp fragments. The father’s PCR product after BclI digestionshowed a single 255-bp band, which indicated he had only normalalleles. In contrast, the PCR product after BclI digestion from themother of Patient 2 showed 255-, 173- and 82-bp bands, whichindicated that she was heterozygous for the p.Gly1179Arg missensemutation (supplementary Fig. S1). This mutation was reported in a