The Molecular Basis of Chondrodysplasia Punctata in an Affected Female and her Unaffected Parents

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Information about The Molecular Basis of Chondrodysplasia Punctata in an Affected Female...

Published on June 8, 2016

Author: DanielleRobinson34


1. 1 CALIFORNIA STATE UNIVERSITY, NORTHRIDGE Molecular Basis of Chondrodysplasia Punctata in an Affected Female and her Unaffected Parents By Danielle Robinson May 2011 Table of Contents

2. 2 Abstract………………………………………………………………………………3 Chapter #1 Introduction Chondrodysplaysia Punctata (CDP)……………………………………..5 History of the X-linked Chondrodysplaysia Punctat………………….6 Clinical Presentation of X-linked Chondrodysplaysia Punctata……8 The EBP Gene………………………………………………………………..8 Treatment for X-Linked Dominant Chondrodysplaysia Punctata…..10 Objective………………………………………………………………….......10 Chapter # 2 Materials and Methods Patients Sample……………………………………………………………..11 Resuspending and Diluting Primers……………………………………..12 Amplification of Exons for Analysis………………………………………12 PCR Amplicon Purification………………………………………………...17 Chapter #3 Results Exon 3………………………………………………………………………….18 Exon 4 & 5……………………………………………………………………..19 Exon 2………………………………………………………………………….20 Chapter #4 Discussion Amplication of 3 Exons of the EBP Gene……………………………….20 Next Step……………………………………………………………………...21 X-Inactivation………………………………………………………………...22 Limitations….………………………………………………………………...23 Future Research……………………………………………………………..23

3. 3 References…………………………………………………………………………….24

4. 4 ABSTRACT The Molecular Basis of Chondrodysplasia Punctata in an Affected Female and her Unaffected Parents Danielle Robinson Chondrodysplasia Punctata (CDP) is a heritable disorder that results in skeletal abnormalities. Chondrodysplasia Punctata is characterized by the formation of small, hardened spots of calcium on the "growing portion" of the long bones (stippled epiphyses) or inside other areas of cartilage in the body. X-linked CDP, also known as Conradi-Hunermann syndrome, is the most well-characterized form of CDP, and is the most severe form (male-lethal). Chrondrodysplaysia Punctata is caused by mutation in the EBP gene. EBP gene stands for emopamil binding protein (sterol isomerase). The DNA of an Italian woman with skeletal abnormalities and skin discoloration was tested. Her mother and father were not affected by similar symptoms. Our hypothesis was that the families X-linked Chondrodysplaysia Punctata is caused by a mutation in the EBP gene. To test this hypothesis we will clone the exons of the EBP gene in the affected woman, including the splice junctions, and determine the DNA sequence (I did this work in collaboration with another student, Joaquin Zuluaga, and the work was directed by Dr. Aida Metzenberg). We are studying more than just the coding regions We are studying exons and part of the introns. Then we are going to determine the nucleotide sequence and compare to the wild-type.

5. 5 Thus far, we have isolated 2 of 3 amplicons that would amplify the coding region and splice junctions. The next step will be to amplify the third amplicon, the one we are having trouble with, and then clone each amplicon into pGem 3 vector. This will be sequenced by the CSUN DNA sequencing facility using universal primers . Afterwards, we will be doing bioinformatics comparing our sequence to the sequence in the Gene Bank database. Since we are looking at a female we will be looking for an unknown mutation in a female. We expect this affected female to be heterozygous for an EBP mutation. To test our prediction we are going to have to sequence multiple clones. Each clone has a 50% chance of being wild-type and a 50% chance of having the mutation. Therefore if we sequence multiple clones we expect at least one to have the mutation, which is what we are looking for. We are not sequencing amplicons directly, because the quality of sequence will be much better if we clone the amplicons first (A. Metzenberg personal communication).

6. 6 Figure 1: Punctate epiphyses contain small epiphyseal calcifications, are present at birth, and are classically associated with chondrodysplasia punctata. Source: Poznanski AK. Punctate epiphyses: a radiological sign not a disease. Pediatr Radiol. 1994;24(6):418-24, 436. Chapter #1: Introduction Chondrodysplaysia Punctata (CDP) Chondrodysplaysia Punctata, also known as CDP, is a collection of hereditary disorders that result in skeletal abnormalities. The main characteristic of CDP is punctuate calcification, which is the accumulation of hard calcium spots in the cartilage of, the growing ends of long bones. These alternate ends are known as stippled epiphyses. Calcifications are also found in other areas of the body including the larynx or trachea (Mueller et. al, 1985). Symptoms of CDP include formation of cataracts, large skin pores, growth retardation, limb shortening, patches of coarse dry hair, and dry, scaly skin. There are four forms of CDP : 1) autosomal recessive form, 2) autosomal dominant form, 3) X- linked recessive form, and 4) X- linked dominant form. The autosomal recessive form, also known as Rhizomelic Chondrodysplasia Punctata (RCDP1), is a peroxisome biogenesis disorder (PBD) that is characterized by severe

7. 7 growth and mental deficiency (White, 2003). The autosomal dominant form, is the most common form of CDP that results in vitamin K deficiency or warfarin teratogenicity (Hall et al., 1980) . However this one is not well understood. The X-linked recessive form is known as, Brachytelephalangic Chondrodysplasia Punctata , and is characterized by underdevelopment of the hands and feet , and behavioral problems(Lachman,2007) . The X-linked dominant form is known as Conradi- Hunermann- Happle Syndrome or CDPX2. It is the most understood form of this rare disorder, but is also the most severe, because it is male lethal (Mueller et al., 1985). The X-linked dominant form is what this paper will be focusing on. Since the effects of X-inactivation on the phenotype of an X- linked disorder are better understood now that in the past, it is more common to refer to these as “X-linked” rather than “X-linked recessive” or “X-linked dominant” disorders. History of X-linked Chondrodysplaysia Punctata The first case of chondrodysplaysia punctata was described in 1914 by E. Conradi. Chondrodysplaysia punctata became known as “Condradi Syndrome”. In 1931 new findings by C. Hunermann in the disease led the changing of the name to “Condradi-Hunnermann Syndrome” (Gorlin , 2001). For many years chondrodysplaysia punctata was thought to be a single disorder. In 1971 , Spranger et al, suggested that there may be two different forms of chondrodysplaysia punctata. They proposed separating CDP into two groups, the mild form which we now call Conradi- Hunermann type and the lethal rhizomelic type (Gorlin, 2001). Rhizomelic form was considered lethal due to the the fact that this form resulted in death within the first year of the sufferers life (Spranger, 1971).

8. 8 1977 R. Happle made an additional contribution, when he saw the variation in phenotypes among the patients, with chondrodysplasia punctata, and proposed that it was limited to females. He termed it Chondrodysplaysia Punctata Type B and proposed in 1979 , that this type was inherited in an X-linked manner. Happle reached this conclusion after he found a 35:0 female versus male ratio in those that had the characteristic skin lesions of this form of chondrodysplaysia punctata. In1980 Happle observed that chondrodysplaysia punctata type B is lethal in hemizygous males, which is why it occurs exclusively in females (Happle, 1985). In1995, the first surviving male with X-linked dominant Chrondrodysplaysia Punctata is described. However he is found to have Klinefelter syndrome which results in the presence of an extra X chromosome, resulting in a 47, XXY genotype. His ability to survive was due to X-inactivation (Happle ,1995). X-inactivation is not restricted to females but, can also occur in males with Klinefelter syndrome who have more than one X chromosome. He was heterozygous and not hemizygous for the X-linked dominant form of chrondrodysplaysia punctata. There have also been cases reported of 46,XY males being affected with this disease, which is why many researchers do not consider it an X-linked dominant form of inheritance anymore. Conradi- Hunermann Syndrome was soon renamed to Conradi- Hunermann- Happle Syndrome, to acknowledge Happle for his significant contribution to the description of the disorder.

9. 9 Figure 2: (A) showing the right leg with erythroderma and linear ichthyosiform lesions. (B, C) showing asymmetric shortening of legs and numerous punctate calcifications at the thoracic and lumbar spines, respectively. Lower panel, photograph of patient 2 at 13 years old. (D) showing bilateral cataracts, a flat nasal bridge and coarse hair with patchy alopecia. (E, F) showing postaxial polydactyly on the left hand and marked scoliosis, respectively. Clinical Presentation of X-linked Dominant Chondrodysplaysia Punctata X-linked dominant chondrodysplaysia punctata (CDP) is characterized by growth retardation, shortening of limbs, cataracts , dry and scaly skin, large skin pores, alopecia, and patches of coarse, dry hair (Gorlin, 2001). What separates X-linked dominant chondrodysplaysia punctata from the other forms of chondrodysplaysia punctata is the presence of hyperkeratotic lesions (Blaschko, 1901). Examples of these skin lesions are shown in Figure2. The EBP Gene Cholesterol Is the basis of many important hormones, in the body. Some cholesterol is obtained from one’s diet, but when one cannot synthesize the rest of the

10. 10 cholesterol then one is in trouble . CDP is usually caused by mutations in the emopamil binding protein ( EBP) gene, which is a sterol isomerase. EBP alleles are also known as CDPX2, CHO2, CPX, or CPXD. The protein encoded by this gene is a multipass protein found on the surface of the endoplasmic reticulum. The EBP gene is located on the short arm of the X chromosome and located between positions 11.23 and 11.22, as shown in Figure 3 The function of EBP protein is to catalyze the conversion of Delta(8)-sterols to their corresponding Delta(7)-isomers (Braverman, 1999). Delta8-delta7 sterol isomerase is an enzyme that catalyzes one step of eight in the biochemical synthetic process of making cholesterol. Defects of this enzyme lead to the accumulation of 8- dehydrocholesterol and cholest-8(9)-en 3-beta-ol in plasma and tissues. Therefore, chondrodysplaysia punctata is biochemically characterized by a defect in the cholesterol biosynthesis pathway. How mutation in EBP causes chondrodysplaysia punctata isn’t completely worked out , figure 4 shows the mutations in the EBP gene that have been characterized to date. Figure 3: The EBP gene is located on the short (p) arm of the X chromosome between positions 11.23 and 11.22.

11. 11 Investigations into patients that had point mutations in there EBP gene were shown to have defect in Delta(8), Delta(7) sterol isomerase (Braverman, 1999). However, their symptoms varied leading researchers to believe that X-inactivation occurs at random in affected individuals. Treatment for X-Linked Dominant Chondrodysplaysia Punctata There is currently no cure for X-linked dominant chondrodysplaysia punctata. Treatment is based on controlling the symptoms shown. Common treatments include dermatological care, to help with rough skin areas, operations to lengthen leg bones, via surgery, or genetic counseling, and prenatal diagnosis. Objective To learn about the molecular basis of chondrodysplasia punctata in an Italian woman with skeletal abnormalities and skin discoloration. Her mother and father were Figure 4: Mutations in CDPX2. Schematic diagram of the emopamil-binding protein with the mutations characterized to date. Source: Novel Mutations in X-Linked Dominant Chondrodysplasia Punctata (CDPX2) Neil V Whittock et al.

12. 12 not affected by similar symptoms. Gene mutation analysis is in the process of being performed on exons 2, 3, 4, and 5 of the EBP gene . The overall goal of the project was to clone exons of the EBP gene. We intend to clone splice junctions as well as coding sequence. We propose to sequence the cloned exons to look for a mutation in the EBP gene of the proposita. The nucleotide sequences will be compared tothat of allele. Our hypothesis was that X-linked Chondrodysplaysia Punctata in this family is caused by a mutation in the EBP gene. Chapter # 2 : Materials and Methods Patients Sample DNA of Italian woman suffering from skeletal abnormalities and skin discoloration was isolated and shipped. Her parents’ DNA was also collected even though neither one of them was shown to have any of their daughter’s symptoms. This study was approved by California State University Northridge Standing Committee for the Protection of Human Subjects (IRB). Figure 5: Image of patient depicting skeletal abnormalities and skin discoloration

13. 13 Resuspending and Diluting Primers IDT primers arrived desiccated, and were resuspended in 100 microliters H2O. The concentration of each primer is listed as follows:  ISO2F = 27,800 pmol  ISO2R = 26,000 pmol  ISO3F2 = 19,300 pmol  ISO3R2 = 25,800 pmol  ISO4F = 20,700 pmol  ISO5R = 29,800 pmol We then made the stock solution by adding :  5 μl ISO2F + 273 nH2O = 5 pmol / μl  5 μl ISO2R + 255 nH2O = 5 pmol / μl  5 μl ISO3F2 + 188 nH2O = 5 pmol / μl  5 μl ISO3R2 + 202 nH2O = 5 pmol / μl  5 μl ISO4F + 293 nH2O = 5 pmol / μl  5 μl ISO5R + 253 nH2O = 5 pmol / μl Working dilution for PCR = 5 pmol / μl . A 1 to 50 dilution of stock , therefore for a 25uL reaction, we used 1uL. The sequencing dilution 1 pmol/microliter = 1:5 dilution of working dilution for PCR (exactly). We then stored all at -20 degrees. Amplification of Exons for Analysis The coding exons 2,3,4,and 5 of the EBP gene were amplified using the Polymerase Chain Reaction (PCR). Oligonucleotide primers that were complementary

14. 14 to coding exons and splice junctions of the EBP gene were designed to carry out the PCR protocol. PRIMER SEQUENCE (5’3’) Tm ISO2F ATA AGC TTA TTC GGT CCA TTT ACA TTT CTC 55.5̊C ISO2R ATG GAT CCC AAA TCC CAT CCC ACA GC 62.8 ̊C ISO3F2 ATA AGC TTG ACC CCC AGC TCT CAC AAG 63 ̊C ISO3R2 ATG GAT CCG ATG GTT TCC ATG CAC ACT G 62.6 ̊C ISO4F ATA AGC TTG GTG GTG AGT TGG GGA GCA 64.0 ̊C ISO5R ATG GAT CCA CCC CTG GAA GGG CAC CGT T 68.6 ̊C Table #1 Sequence of primers and melting points used in the PCR protocol for the coding exons of the EBP gene. The genomic sequence of the emopamil-binding protein gene is shown below. 1 cggagccagc gtgggaggcc gctgccgtcg cgcgccttgg tgagtgccct ccacccggcc 61 cctgctccct cccccagctc tccccggcta cgcggccagc cctcggcgtg ccagcgcgag 121 accctttgcc acccgccccc cccaccgccc ttttgcgcct gcgcgagacc cccagctacc 181 gcacggttgt ccagaggaca gaagatccgt cttctcattg ggcagcggga ctggagggtt 241 ctggttcgga ttgaccggct ttgtgttccg ttctagcgct gcacgccaga caccggcctt 301 tcaatatccg tctcttccct gcaggttgac ggcgttgcac gctctcgcgg ggaggctctg 361 gctttccaaa cgctggcacc gagggttgta gttctgattc gttttctttc ctctgtcccc 421 cagttgcagt tttcaggact acgtggggga gggaatagct ttttgttgaa cggtttaaga 481 cgctgacctg tgcaactgga ccacgcctgg ttcctgtgtc tcttaccaaa ccgtgacccc 541 ggagcacagg actgggtttg gtggtcgtga gcagtttgtg acccttgaag gaagacacca 601 ggcctggtcc tgtatctccc tcgccagact gtgaccaggg agggctggga tcaggtgttg 661 ttcccatgtg cctcccacca gactttgacc cccgagggct gagaccggga ctgtttctac 721 cacatcataa tatggcttct aaagggctgg aacaagcttt cagcctttct gtggccttgt 781 gtttcagtca tgttcttttt cttttctttt ttattttatt ttgagacaga gtctcgctct 841 gttgcccagg ctggagtgca gtggcatgac cccggctcac tgcagcctct gcctcccagg 901 ttcaagcgat tctcctgcct cagcctcctg agtagctgta actacaggcg cacgccacca 961 cacctggcta atttttgtat ttttagtaga gacggggttt tgccatgttg gccgggctgg 1021 tcttgaactc ctgagctcaa gtggtccact cgtcttggcc tcccaaagtg ctgggattac 1081 aggcgtgagc caccgcgccc ggccttcttt tttagaggca gagtctcaga ctggagggca

15. 15 1141 gtgatgtggt cactgctcac ttcagccttg aagtcctggg ctcaaagcga tcctcctgcc 1201 tcagcctacc tactagctgg gactacaggc ctttgtcacc acacctgact gattttctta 1261 tttttttaga gacagtttcg ctgtgttgcc caggccggac ttgaattcct ggcctcaggt 1321 gctgctcctg catcagcctc ccaaagcact ggaattacct gccacggtgc ctggccttta 1381 gtcatggtct taaaacagaa tgtaggccgg gcgcagtggc tcacgcctgt aatcccagca 1441 ctttgggagg ctgaggcggg cggatcgtga ggtcaggaga tcaagaccat cctggctaac 1501 acggtgaaac cccgtctcta ctaaaaatac aaaaaattag ctgggcgtgg tggcaggcac 1561 ctgtagtccc agctactcgg gaggctgagg caggagaatc gtttgaaccc ggaaggcaga 1621 ggttgcagtg agccgaggtc gcaccactcc actccagcct gggtgacaga gcaagactcc 1681 gtctgaaaaa aaaaaaaaaa aacggaatgt aattagaagt gttacaatcc tgtctgttaa 1741 ctggtaaatt cggtccattt acatttctca tgataataaa ctatttgaat ttgattttat 1801 tatctcattc tgtactttct atttgtccag gtttttctgt tccttttttt tttttttttt 1861 taacttcctg cctatacaca cgcagccatc agcccacaaa gacatgacta ccaacgcggg 1921 ccccttgcac ccatactggc ctcagcacct aagactggac aactttgtac ctaatgaccg 1981 ccccacctgg catatactgg ctggcctctt ctctgtcaca ggggtcttag tcgtgaccac 2041 atggctgttg tcaggtcgtg ctgcggttgt cccattgggg acttggcggc gactgtccct 2101 gtgctggttt gcagtgtgtg ggttcattca cctggtgatc gagggctggt tcgttctcta 2161 ctacgaagac ctgcttggag accaagcctt cttatctcaa ctctgtgagt cctgatttct 2221 ttcatatgct gtgggatggg atttgctggg cagggatcgg cttgcatgtt tacctatcca 2281 cctattcttc ttccattgat ttattttaaa tttttattta atcttttaaa aaatttatta 2341 taggccgggc acagtggctc acgcttgtaa tcccagcact ttgggaggcc gaggcaggtg 2401 gatcacttga ggtcaggaat ttgagaccag cctggtcaac atggcaaaac cccatctcta 2461 ctaaaaatac aaaaattagc caggcgcggt ggctcaagcc tgtaatccca gcactttggg 2521 aggccgaggc aggcagatca cgaggtcggg agttgaagac cagccaggcc aagatgatga 2581 aaccccatct ctactaaaaa tacaaaaatt agccgggtgt ggtggcacac gcctgtaatc 2641 ccagctacta gggaggctga ggcaggagaa ttgcttgaac ctgggaggcg gaggttgcag 2701 tgagccaaga ccatgccatt gcactccagc ctgggcaaca gagtgagact ttgtctcaaa 2761 aaaaaaatta ttatgtattg ttattattat tctttttgtt ttttgccctt ctctaaaact 2821 ggtagataac ttatttttga gacaaggtct tgctctgttg cccaggctgg agtgcagtgg 2881 tatgatgatt gctcactgca acctccaact cctgggttca agcgatcgtc ttgcctcagc 2941 ctccccagta gctgggacta tactataggt gcacaccttt atgcctggct aatattttca 3001 cttttttgta gagacaaggt cttgctatgt tgctcaggct gttctttaac tcctgggctc 3061 aagcaatcct tccacctcag cctcccaaaa ttctgggatg acaggtgcgc accaccgtgc 3121 ccagccttat ttaacttttt tgttgcatgt atattcacgt gggtcaaaaa accatttaaa 3181 gagatttgca gtgaaaatct gctttctatc cactaagttt ccccaaacgc tgttaggcac 3241 tgttgatagt tgtttatgtt tcttttcagt ttatttacac acgtaagtct caatttgctc 3301 aattgtaaaa ggaggataat agtaataacc aactatatgt aagatactta gaacagtgtt 3361 tggcaaacag tggttgccat gtaactgttc actattatta tcattatttg cacttttccc 3421 tttttacaca aatggtaata tgctatatat acttctaatc ttttctcttt ttctttcttt 3481 ccttacaaga aaaccaaaga agccattttc ttttttgaga cggggtctcg ctctgtcacc 3541 caggctagag tgcagtggca tgattatagc tcactgcaac cttgaactcc tgggcaatca 3601 agcaatcctc ctgcctcagc ctccctagta gctgggatta taggcatgtg ccaccccacc 3661 aagttatttt ttttttaaca actttttttt ttaagagatg gcgtcttgct ttgttgccca

16. 16 3721 ggctgttctc caactcctgg cttcaagtga tctgcccacc tcagcctccc aaagtgctag 3781 gattacaagc atgagccacc gcacccagct gcttttttta atgaggtcac tcctcatgat 3841 ttcataaaga gctttcaaag catggacatt gtacagttgc ttgacttgtg tagtattgat 3901 gggcattttc cctcaatctt ctgccattat aaacaaggcc gggcagagtg gctcatgcct 3961 gtaatcccag cactttgtga ggcgaaggcg ggcagatcac ctgaggtcag gagtttgaga 4021 ccagtctggc caacatagtg aaaccccgtc tctactaaaa gtctaaaaat tagctgggcg 4081 tggtggcagg tgcctgtaat cccagctact caggaggctg aggcaggaga atctctggga 4141 cctgggaggc ggaggttgtg gtgagctgag attgcgccac tgcacactcc agcctgagcg 4201 ataagagcaa ggctccgtct caaaaaaaaa aaaaaaaaaa aaaaacatta caggcatgag 4261 ccattgtgcc cggctggtca gatgtttctt aaattttagt cattccagta gatgtgagtg 4321 atatctcatt atggttttaa tttgcttttc cttagtgact aatggaagtt tgaacatctt 4381 tcatatactt attggccatt tagaaatcct ctttgtgaag aacctattca agtcttctgc 4441 ctattttata ttggatgatc tttttctttc tttctttttt tttttttttt ttgagacagg 4501 gtcttaccct gttgtccagg ctgagcactg tggtgctatc atagctcact gcagcctcaa 4561 cctcctgggc tcaagacatc ctcccactgc agcctgggac tgtaggcacg tgccaccacg 4621 tccagctaat ttttgtatgt tttatagagt tgggggtctc actatattgt tcagactggt 4681 cttgaactcc tttatgcaag ccatcatccc acctcagcct cccaaagtgt tgggattaca 4741 ggcatgagcc atcgtgcccg gcggtctttt tctttctgat tctctgctca ctcttgtatt 4801 cactcatctg ttcacctaca ttctctctct ctcattggct acttcactca ctccatcatc 4861 tacacctgct tccttttata ccacacacac ttcttgagta ccaatcacag ccacactgtt 4921 ttgtgtcagg ctttacacat attaccttgt tttagagaaa taaaccacta gaatccaaac 4981 agttacccca tttcacggat gaggaagcag acgcatggga aggttatgaa gtctgtcttc 5041 ttgcagggac ccccagctct cacaagtgtg tgttcctttc actgccttta ttcttcatat 5101 ctctctcttc ttttcttcag ggaaagagta tgccaaggga gacagccgat acatcctgta 5161 agtgtttgcc tctgtcaatg gagactggca ttggttttcg gggggtggtg agttggggag 5221 cactaatggg ctaacctgta ggaagagcac accgatacca gtgtcccctc atgctttctc 5281 ctgcaggggt gacaacttca cagtgtgcat ggaaaccatc acagcttgcc tgtggggacc 5341 actcagcctg tgggtggtga tcgcctttct ccgccagcat cccctccgct tcattctaca 5401 gcttgtggtc tctgtgggta aggaaagggc actagagggg cactgggcac tagaggggtt 5461 gatgggggat ccacagacac agatgtatcc ctgtgggtgg gatctctcaa cggtgccctt 5521 ccaggggtaa gtcagactga atgacaaacc ccctgaggct ctggaaaggt catgcccttc 5581 tctgagcctg cactctcagg tggggagggt tcatttttct tcctcctcct ccttctccat 5641 cacaaagtct cctgtgaagg ttacatgagg tggcccaggg tggcaagaag cccctggaac 5701 ctccaggatc agggctctga gatcccttca tgtaagattc tgtcatattt tttttaaaac 5761 agggtctggc tctgtcaccc aggccagagt gctatggtgt gatctcggct cactgcaacc 5821 tccacctccc agactcaagc gatcttcctg cctcagtctc ctgagtagct gggactacag 5881 gcatgtgcca ccgtgcctgg ctaacttttg tatttttagt agagatgggg tttcgccatg 5941 ttgcccaggc tggtctcaag ctcctgagct caaacagtcc tcctggcttg gcctcccgaa 6001 gtactgggat tacaggtgtg agccactgtg cccggcctca gtcatcttgt gttattaaat 6061 tgttagcttt ctttagttct agcgatatga gttatcttta gtcccagaat cctaagctgt 6121 cttggatttg aggattttga acttccttaa atctgaaggt gtgagctctc ctgagttctg 6181 aaattctcag ctctctggtg tctggaatct cacttttctg atttctcaga ttttcagctc 6241 tgtgatttca gaatacttag ctctgagacc ttgaatgatg acttggaaag tgctttggaa

17. 17 6301 tagagaattg gcgaaagtgt ccccttcctc actggggctt ctccttcccc tcctgccacc 6361 cacaggccag atctatgggg atgtgctcta cttcctgaca gagcaccgcg acggattcca 6421 gcacggagag ctgggccacc ctctctactt ctggttttac tttgtcttca tgaatgccct 6481 gtggctggtg ctgcctggag tccttgtgct tgatgctgtg aagcacctca ctcatgccca 6541 gagcacgctg gatgccaagg ccacaaaagc caagagcaag aagaactgag gagtggtgga 6601 ccaggctcga acactggccg aggaggagct ctctgcctgc cagaagagtc tagtcctgct 6661 cccacagttt ggagggacaa agctaattga tctgtcacac tcaggctcat gggcaggcac 6721 aagaagggga ataaaggggc tgtgtgaagg cactgctggg agccattaga acacagatac 6781 aagagaagcc aggaggtcta tgatggtgac gatttttaaa atcaggaaat aaaagatctt 6841 gactctaa Each PCR tube contained .5 μl of dNTPs (10mM), .5 μl of Taq Polymerase (5units/ μL), 2.5 μl of 10x Taq Buffer (Mg + 2 Free), 2.5 μl of (5pmol/ μl) of each forward and reverse primer. The experimental tubes received 1 μl of genomic DNA, while the controls had 1 μl of nH2O. The concentration of magnesium chloride + nano pure water varied between 1.5M – 4M, in order to determine the optimal conditions for amplification. The amount of magnesium chloride + nano pure water added to each tube was 14.5 μl. All of these were added together bringing the total reaction volume to 25 μl. The PCR reactions were then placed in the PCR machine, the PCR conditions varied between exons. Each PCR reaction was set to 40 cycles. Figure#6. Genomic sequence of the emopamil-binding protein gene. Sequences in green = exons, in red = intron/exon boundaries. The intron/exon splicing sites (ag/gt) were configured by Bravermann et al. 1999. Exons 4 and 5 were amplified together. &to=48272048

18. 18  Exon 2 Temperature Time Cycles Holds 95̊ C 2 min 1 hold 96̊ C 1 min 52̊ C 1 min 72̊ C 1 min 72̊ C 10 min 1 hold  Exon 3, Exon 4& 5 Temperature Time Cycles Holds 95̊ C 2 min 1 hold 94̊ C 30 sec 52̊ C 30 sec 72̊ C 1.5 min 72̊ C 10 min 1 hold After completing all 40 cycles the reaction was held at 4̊C for convenience. PCR Amplicon Purification Electrophoresis was performed to see if we amplified the amplicons and to isolate the desired PCR fragments. A 7.5% polyacrylamide gel was electrophesed (PAGE). Each well contained 10 μl of PCR product and 2 μl of 6 X Dye. The first well

19. 19 was loaded with 1 μl of Phi X 174 DNA digested with HaeIII to produce size markers. These fragments range from 72 base pairs to 1353 base pairs in length. The gel was submerged in 1x TAE buffer and was electrophoresed for an hour and forty-five minutes to two hours at 94V to 96V. After electrophoresis, it was placed in ethidium bromide for staining, for 5 minutes, and then removed and placed in deionized water for another 5 minutes, for de-staining. The gels were then photographed using a UV transilluminator . The bands of interest were then cut out and placed into a 1.5ml tube containing, 350 μl of 100mM NaCL/1 mM EDTA. The samples set at room temperature overnight. Chapter #3: Results So far we have isolated 2 of 3 amplicons that would amplify the coding region and splice junctions. These amplified amplicons, are exons 3 and exon 4 & 5. Exon 3 Figure 7: 7.5% acrylamide gel showing successful amplification of exon 3. Gel was stained with ethidium bromide. Band in lane 2 shows amplification of exon 2

20. 20 Exon 3 was successfully amplified and is denoted here by the yellow box. Its nanodrop results were  260/.280 ratio = 1.84  Nucleic acid concentration = 17.7 ng/ μl We determined that concentration of the PCR products was not adequate enough to allow for sequencing. Exon 4 & 5 Exon 4 & 5 was successfully amplified and is denoted here by the yellow box. Exons 4 and 5 were amplified together since they are located very close to each other on the gene. Its nanodrop results were  260/.280 ratio= 1.19  Nucleic acid concentration = 29.7 ng/ μl Figure 8: 7.5% acrylamide gel showed using the gradient of magnesium concentrations in amplification of exons 4 and 5. Gel was stained with ethidium bromide.

21. 21 We determined that concentration of the PCR products was not adequate to allow DNA sequencing. Exon 2 Exon 2 was successfully amplified and is denoted here by the yellow box. Its nanodrop results were  260/.280 ratio = 1.84  Nucleic acid concentration = 17.7 ng/ μl However, we determined that the concentration of the PCR products were too low to allow for sequencing. In future, it will be important to determine conditions for amplifying this fragment more effectively. Chapter #4: Discussion Amplification of 3 exons of the EBP gene The PCR results differed. Cloning exon 2 was particularly difficult due to the fact that we have yet to figure out the optimal PCR conditions. Although we were Figure 9: 7.5% acrylamide gel showing the gradient of magnesium concentrations used to amplify exon2. Gel was stained with ethidium bromide.

22. 22 successfully able to amplify exon 2, we determined, by measuring the concentration of the PCR products, that the concentration of the exon 2 PCR product was too low to allow for sequencing. We then attempted to optimize PCR conditions by altering the magnesium concentrations, in our PCR master mix, and altering the annealing temperatures in the experiments. We are currently working very hard at improving the yield of the amplicon which includes exon 2 of the EBP gene in this individual with CDP. It was also speculated that the reason this amplicon was especially difficult could be due to the run of thymine’s near the beginning of the exon 2. This could be due to the fact that DNA polymerase , stuttered, and was unable to pinpoint where it is suppose to be when the same nucleotides are in a row. Next step Once we have worked out amplification of exon2 we will be doing restriction digest and ligating all of the amplicons into the multiple cloning sites on the pGEM3 vector. We will then perform bacterial transformation, grow up the clones and then isolate the DNA. Sequencing will be carried out by the CSUN DNA sequencing facility, using universal primers. We expect the affected individual to be heterozygous for an EBP mutation. We will be sequencing multiple clones so that the chances for missing a mutation is smaller, since each clone has a 50% chance of being wild type and a 50% chance of having the mutation. Bioinformatics will be used to compare our sequence with the wild type sequences in the GenBank database. We expect that one of the clones will have a mutation. If we do find a mutation, we will try to confirm that it is a deleterious mutation.

23. 23 Two ways to go about doing this are to, 1) See if someone else with X-linked dominant chondrodysplaysia punctata has the same one, 2) Seeing whether the mutation is present in 100 random X chromosomes of normal individuals, using a database, or doing the work ourselves. Polymorphism is defined as more than 3% of the population having nucleotide changes that do not cause CDP. If a mutation is not present in the samples studied, it is likely to be a polymorphism rather a deleterious mutation. A mutation is defined as a deleterious nucleotide change. If we do find a mutation this does not prove we have a deleterious mutation, but it does support it. X-inactivation Mary Lyons’ “X-inactivation Theory” is a phenomenon when one of a females paternally or maternally derived X-chromosome is randomly inactivated in a cell. Since females affected with X-linked dominant chondrodysplaysia punctata are heterozygous, they are only affected if cells with the mutation outnumber the cells without the mutation. This might be responsible for observations that, if a woman is an obligate heterozygote for a mutation, then the phenotype can range from absent to severe. It all depends upon the degree of X-inactivation in the individual. Even if the mom was unaffected, she can still have a mutation that’s not active in other cells, which is why she has a normal phenotype. Skewed inactivation results when most cells with the active x, is the one without the mutation, and the inactive x is the one with the mutation. Skewed X-inactivation is defined as the state where 95% of cells only express wild-type, while the other 5% express mutant. Thus, phenotype can be normal even though the person is heterozygous, if it is skewed toward wild- type.

24. 24 Skewing is possible in either direction (wild-type of mutant, so the phenotype of a heterozygote ranges from normal to severely affected). In theory, the affected individual could have gotten a mutation from either parent. Her father would have had to have a new mutation occur well after the first cell division of the fertilized egg that became him. However, a new mutation might not show up in his blood DNA. The mother could have random or skewed x-inactivation. Another possibility is that the daughter could have a new mutation not inherited from parents. Resulting in a redundancy seeing that there might be another gene involved that can do similar work as the EBP gene. Limitations We are only sequencing the exons in the splice junctions and coding regions. The mutation in this family could be due to a mutation that is not in either region. For example, the mutation could be in the promoter region upstream of the gene itself. Future Research What we have been doing is molecular diagnosis. We would like there to be a molecular treatments which might involve 1) enzyme replacement therapy 2) gene therapy or 3) stem cell therapy. Gene therapy is an ultimate goal, however it is not efficient right now, and is only used for a few disorders. Since the affected individual will already have problems by the time he or she is born, the current treatment is focused on making everything better going forward.

25. 25 References Blaschko, A. 1901. Die Neventerteilung in der Haut in ihrer Beziehung zu den Erkrankungen der Haut. Wien and Leipzig, Braumuller. Braverman N, Lin P & Moebius FF et al. Mutations in the gene encoding 3 beta- hydroxysteroid-delta 8, delta 7-isomerase cause X-linked dominant Conradi– Hunermann syndrome. Nat Genet (1999) 22: 291–294. Gorlin, Robert J., Cohen, Meyer, Hennekam. Syndromes of the head and neck. Oxford, Oxford Univ. Press, 2001 Hall, J. G., Pauli, R. M., Wilson, K. M. Maternal and fetal sequelae of anticoagulation during pregnancy. Am. J. Med. 68: 122-138, 1980. [PubMed: 6985765] Happle, R. 1985. Lyonization and the lines of Blaschko. Human Genetics, 70 (3), 200- 206. Happle, R. 1995. X-linked dominant chondrodysplaysia punctata/ichytosis/cataract syndrome in males. (Letter). Am J Med Genet, (57), 493. Lachman R. Chondrodysplasia punctata. In Taybi and Lachman’s Radiology of Syndromes, Metabolic Disorders and Skeletal Dysplasias, R Lachman (ed.). Mosby Elsevier: Philadelphia, PA, 2007; 900–910. Mueller, R.F., P.M Crowle, R.A.K. Jones, B.C.C. Davison. X-linked dominant chondrodysplasia punctata: A case report and family studies. Am J Med Genet. Volume 20, Issue 1, pages 137–144, January 1985 Spranger, J. W., Opitz, J. M., Bidder, U. Heterogeneity of Chondrodysplasia punctata. Human Genetics. 190-212 September 1971 White AL, Modaff P, Holland-Morris F, Pauli RM. Natural history of rhizomelic chondrodysplasia punctata. Am J Med Genet.2003;118A:332–42.

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