- Email: [email protected]
PGD for familial hemiplegic migraine on polar bodies and blastomeres
Abstracts – PGDIS: 9th International Symposium on Preimplantation Genetics
bloody diarrhoea, intermittent or chronic petechiae and purpura, eczema, recurrent bacterial and viral infections and immune dysfunctions. A couple, where the woman had three affected WAS brothers and a genetic diagnosis of carrying WAS, attended genetic counselling asking about the feasibility of having healthy children. PGD was the chosen option. They were informed that a preclinical test with a familial study was necessary. Methods: An informativity study was carried out using direct diagnosis to detect the familial c.976delC mutation in WAS gene and haplotyping of seven microsatellite markers. The PGD test was carried out using hemi-nested fluorescent PCR to detect mutant and normal alleles by fragment size plus one informative microsatellite marker (DXS7125), also amplifying the amelogenine gene in order to know the number of expected X alleles. Results: Precycle workup was performed in some members of the family: the proband was a WAS carrier, her mother and her maternal aunt both were WAS carriers. Affected men were not available in the family since all were deceased because of this disease. The informativity study revealed a recombination event in the proband’s mother or aunt without being able to specify in which one of them it occurred. The PGD for WAS had to be performed without previous microsatellite allele association and only one of the eight microsatellite markers available was informative in the couple. Seven biopsied blastomeres (one from each embryo) were analysed. The allele dropout rate in the cycle was 9% and the amplification rate 100%. All embryos could be diagnosed and the 100% amplification rate allowed to assign the microsatellite allele linked to the disease, being all data concordant: Three embryos were diagnosed as carriers, three as healthy non-carriers and one as affected. Conclusion: Direct diagnosis in PGD with haplotyping of two linked microsatellite markers is the preferred option. Indeed, previous to the PGD cycle, microsatellite allele assignation is highly recommended. Nevertheless, these premises cannot always be fulfilled. In such cases, a robust protocol and great embryo numbers are necessary in the PGD cycle to obtain a correct diagnosis. PGD for familial hemiplegic migraine on polar bodies and blastomeres Pomerantseva E, Rechitsky S, Verlinsky Y Reproductive Genetics Institute, Chicago Familial hemiplegic migraine FHM1 (OMIM#141500) is a subtype of migraine with aura. Main symptoms include hemicranial pain and associated hemiparesis. Other symptoms, such as persistent cerebellar dysfunction, retinal degeneration, deafness, nystagmus, coma, fever and meningismus, recurrent episodes of acute paranoid psychosis with anxiety and visual hallucinations, vary in families. The pattern of inheritance of the condition is autosomal dominant. The FHM locus has been shown to be located on chromosome 19, though locus heterogeneity has been reported. R192Q mutation in conserved functional domains of the CACNL1A4 gene has been identified. We performed PGD for a patient affected with familial hemiplegic migraine, using nested and hemi-nested multiplex PCR protocol for the mutation and tightly linked short tandem repeats. Normal and mutant haplotypes were established by studying inheritance of the disease in the patient’s family. The final protocol included D19S906, D19S221, D19S914, D19S1150, D19S840, D19S226, D19S385 and D19S556
S-24 Reproductive BioMedicine Online, Vol. 18, Suppl. 3, May 2009
markers and mutation. Mutation was studied by restriction endonuclease digestion and electrophoresis, while polymorphic markers were analysed by fluorescent fragment analysis. First and second polar bodies from four oocytes were tested, of which two were found to be unaffected and two inherited mutation and were excluded from further analysis. Blastomere biopsy was also performed for five other embryos (polar bodies were not available). Two of them were found to be unaffected and three were chromosomally abnormal, having either one or three chromosome 19. Two out of four normal embryos were transferred, resulting in normal pregnancy with twins. PGD with blastomere analysis for Crigler–Najjar syndrome; UGT1A1 analysis with aneuploidy testing Ozen RS1,2, Karadayi H2, Kervancioglu E3, Hekim N2, Iannacone P1, Galat V1 1Children’s Memorial Research Center, Northwestern University, Chicago; 2International Reproductive Genetics Center (Interepgen), Istanbul; 3Dr Pakize I Tarzi IVF Center, Istanbul Crigler–Najjar syndrome types I and II are autosomal recessive disorders associated with near (type II) or complete (type I) absence of hepatic bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (UGT1A1; OMIM#191740) enzyme activity resulting in an impairment of the ability to conjugate and excrete bilirubin (estimated at 0.6–1.0 per million live births). The disorder results in an inherited form of nonhaemolytic jaundice, often leading to brain damage in infants. The UGT1A1 gene contains at least 12 different promoters/ first exons that are spliced to common exons 2 through 5 on chromosome 2q37. Here we present a family in which both parents (first cousins consanguinity) have the c.877T>A + c.878_890del mutation of UGT1A1 gene. We developed a multiplex nestedPCR protocol for single-cell analysis detecting the c.877T>A mutation with minisequencing and the c.878_890del mutation with fluorescent fragment analysis. The protocol included short tandem repeats (STR) closely linked to the UGT1A1 gene on both 5′ and 3′ sides; D2S331, D2S1279, D2S2348, D2S336, D2S2202 and D2S338. Single-sperm analysis was done to find the paternal haplotype because there was no affected child or relative. The maternal haplotype was inferred from paternal information since they were first cousins inheriting the same chromosome harbouring the same mutation with the same closely linked STR alleles. Aneuploidy testing set up for chromosomes 13, 18, 21, X and Y was done due to advanced maternal age (42 years old). Single blastomeres from eight embryos of one IVF–PGD cycle were tested. Three embryos were diagnosed as c.877T>A + c.878_890del mutation carriers (one of them was triploid). Two embryos were diagnosed as unaffected. Two embryos were found to have monosomy 2. One showed inconclusive results due to insufficient interpretable test data. Among the best developing embryos, one unaffected embryo and one carrier embryo were transferred resulting in a clinical singleton pregnancy. Prenatal diagnosis with chorionic villus sampling at week 11 of pregnancy showed the presence of an unaffected embryo and normal male karyotype. Spontaneous abortion occurred at week 13 (abortion material was not obtained).
bloody diarrhoea, intermittent or chronic petechiae and purpura, eczema, recurrent bacterial and viral infections and immune dysfunctions. A couple, where the woman had three affected WAS brothers and a genetic diagnosis of carrying WAS, attended genetic counselling asking about the feasibility of having healthy children. PGD was the chosen option. They were informed that a preclinical test with a familial study was necessary. Methods: An informativity study was carried out using direct diagnosis to detect the familial c.976delC mutation in WAS gene and haplotyping of seven microsatellite markers. The PGD test was carried out using hemi-nested fluorescent PCR to detect mutant and normal alleles by fragment size plus one informative microsatellite marker (DXS7125), also amplifying the amelogenine gene in order to know the number of expected X alleles. Results: Precycle workup was performed in some members of the family: the proband was a WAS carrier, her mother and her maternal aunt both were WAS carriers. Affected men were not available in the family since all were deceased because of this disease. The informativity study revealed a recombination event in the proband’s mother or aunt without being able to specify in which one of them it occurred. The PGD for WAS had to be performed without previous microsatellite allele association and only one of the eight microsatellite markers available was informative in the couple. Seven biopsied blastomeres (one from each embryo) were analysed. The allele dropout rate in the cycle was 9% and the amplification rate 100%. All embryos could be diagnosed and the 100% amplification rate allowed to assign the microsatellite allele linked to the disease, being all data concordant: Three embryos were diagnosed as carriers, three as healthy non-carriers and one as affected. Conclusion: Direct diagnosis in PGD with haplotyping of two linked microsatellite markers is the preferred option. Indeed, previous to the PGD cycle, microsatellite allele assignation is highly recommended. Nevertheless, these premises cannot always be fulfilled. In such cases, a robust protocol and great embryo numbers are necessary in the PGD cycle to obtain a correct diagnosis. PGD for familial hemiplegic migraine on polar bodies and blastomeres Pomerantseva E, Rechitsky S, Verlinsky Y Reproductive Genetics Institute, Chicago Familial hemiplegic migraine FHM1 (OMIM#141500) is a subtype of migraine with aura. Main symptoms include hemicranial pain and associated hemiparesis. Other symptoms, such as persistent cerebellar dysfunction, retinal degeneration, deafness, nystagmus, coma, fever and meningismus, recurrent episodes of acute paranoid psychosis with anxiety and visual hallucinations, vary in families. The pattern of inheritance of the condition is autosomal dominant. The FHM locus has been shown to be located on chromosome 19, though locus heterogeneity has been reported. R192Q mutation in conserved functional domains of the CACNL1A4 gene has been identified. We performed PGD for a patient affected with familial hemiplegic migraine, using nested and hemi-nested multiplex PCR protocol for the mutation and tightly linked short tandem repeats. Normal and mutant haplotypes were established by studying inheritance of the disease in the patient’s family. The final protocol included D19S906, D19S221, D19S914, D19S1150, D19S840, D19S226, D19S385 and D19S556
S-24 Reproductive BioMedicine Online, Vol. 18, Suppl. 3, May 2009
markers and mutation. Mutation was studied by restriction endonuclease digestion and electrophoresis, while polymorphic markers were analysed by fluorescent fragment analysis. First and second polar bodies from four oocytes were tested, of which two were found to be unaffected and two inherited mutation and were excluded from further analysis. Blastomere biopsy was also performed for five other embryos (polar bodies were not available). Two of them were found to be unaffected and three were chromosomally abnormal, having either one or three chromosome 19. Two out of four normal embryos were transferred, resulting in normal pregnancy with twins. PGD with blastomere analysis for Crigler–Najjar syndrome; UGT1A1 analysis with aneuploidy testing Ozen RS1,2, Karadayi H2, Kervancioglu E3, Hekim N2, Iannacone P1, Galat V1 1Children’s Memorial Research Center, Northwestern University, Chicago; 2International Reproductive Genetics Center (Interepgen), Istanbul; 3Dr Pakize I Tarzi IVF Center, Istanbul Crigler–Najjar syndrome types I and II are autosomal recessive disorders associated with near (type II) or complete (type I) absence of hepatic bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (UGT1A1; OMIM#191740) enzyme activity resulting in an impairment of the ability to conjugate and excrete bilirubin (estimated at 0.6–1.0 per million live births). The disorder results in an inherited form of nonhaemolytic jaundice, often leading to brain damage in infants. The UGT1A1 gene contains at least 12 different promoters/ first exons that are spliced to common exons 2 through 5 on chromosome 2q37. Here we present a family in which both parents (first cousins consanguinity) have the c.877T>A + c.878_890del mutation of UGT1A1 gene. We developed a multiplex nestedPCR protocol for single-cell analysis detecting the c.877T>A mutation with minisequencing and the c.878_890del mutation with fluorescent fragment analysis. The protocol included short tandem repeats (STR) closely linked to the UGT1A1 gene on both 5′ and 3′ sides; D2S331, D2S1279, D2S2348, D2S336, D2S2202 and D2S338. Single-sperm analysis was done to find the paternal haplotype because there was no affected child or relative. The maternal haplotype was inferred from paternal information since they were first cousins inheriting the same chromosome harbouring the same mutation with the same closely linked STR alleles. Aneuploidy testing set up for chromosomes 13, 18, 21, X and Y was done due to advanced maternal age (42 years old). Single blastomeres from eight embryos of one IVF–PGD cycle were tested. Three embryos were diagnosed as c.877T>A + c.878_890del mutation carriers (one of them was triploid). Two embryos were diagnosed as unaffected. Two embryos were found to have monosomy 2. One showed inconclusive results due to insufficient interpretable test data. Among the best developing embryos, one unaffected embryo and one carrier embryo were transferred resulting in a clinical singleton pregnancy. Prenatal diagnosis with chorionic villus sampling at week 11 of pregnancy showed the presence of an unaffected embryo and normal male karyotype. Spontaneous abortion occurred at week 13 (abortion material was not obtained).