Preimplantation Genetic Diagnosis For Single Gene Disorders

The father was also born with an anal stricture (see Figure 3.34, III 3) requiring anal dilatation, and also had sacral defect detected by x-ray, involving a central anomaly from S2 downward. One of his two sisters (see Figure 3.34, III 1) was born with imperforate anus and anterior meningocoele and also had rectovaginal fistula, and a vesicoureteric reflux resulted in the need for renal transplantation; a sacral x-ray showed the same central defect with absence of the distal one third of the sacrum. The other sister (see Figure 3.34, III2) was clinically asymptomatic, but sacral x-ray revealed no coccyx and a MRI scan disclosed an anterior meningocoele. His mother had undeveloped coccyx and urinary tract bilateral ureteropelvic junction obstruction (see Figure 3.34, II 4). Of her three siblings, only one of two brothers had anal stricture (see Figure 3.34, II 1), while the asymptomatic sister (see Figure 3.34, II2) was identified as a carrier of the mutation because of the finding of imperforate anus in her grandson. As seen from the pedigree, the father inherited the mutation from his grandmother (see Figure 3.34, I 2), who was probably the first affected member of the family, known to have constipation but normal sacral x-ray.

DNA analysis in this family demonstrated the presence of homeobox HLXB9 mutation due to frameshift insertion of a cytosine into a stretch of six cytosines at positions 125-130 in exon 1 of the gene, leading to the introduction ofpremature termination codon [74]. The primer sequences for mutation testing and their positions are presented in Figure 3.35 and listed in Table 3.20.

PGD cycle was performed using single blas-tomeres, removed from the eight-cell embryos and tested by multiplex nested PCR analysis, involving specific mutation testing simultaneously with linked marker analysis. PCR product was identified by fragment length analysis using capillary elecrophoresis (see Figure 3.35).

Prior to initiating PGD cycle, a single sperm testing was performed to establish paternal haplo-types. To be able to identify a possible ADO in the mutation analysis, four closely linked microsatellite dinucleotide DNA markers, D7S559, D7S550, D7S637, and D7S594, were tested in the same reaction with the HLXB9 gene [75]. As seen from Figure 3.34 and Table 3.21, the mutant allele was linked to 119, 155, 124, and 162 bp, and the normal allele to 123, 159, 116, and 168 bp repeats of D7S559, D7S550, D7S637, and D7S594 markers, respectively. Testing for these markers in the af fected child and the mother showed that the parents shared the same site of two of four markers linked to normal allele making theses markers of limited value for preselection of mutation-free embryos for transfer.

A total of 17 embryos were available for testing in a single PGD cycle. Single blastomeres were removed from these embryos, of which three failed to amplify either HLXB9 alleles or polymorphic markers (embryos #1, 9, and 14), suggesting the lack of a nucleus in these blastomeres, and two showed amplification of polymorphic markers only with no signal detected for the HLXB9 gene (embryos #8 and 12) (see Table 3.21). Of a total of 12 embryos with results for the mutation and linked marker analysis, 11 were with conclusive results, of which 5 were predicted to contain the mutant allele in agreement with the presence of repeat markers (embryos #3, 5, 6, 11, and 13). One of these embryos contained only paternal alleles (embryo #13), which maybe explained by the absence of the maternal chromosome 7, which may be due to mosaicism, known to be very frequent at the cleavage stage.

The remaining six embryos were predicted to be free of mutation, but only in three of them (embryos #2, 4, and 16) could ADO of the mutant allele be excluded based on the linked marker analysis (see Table 3.21 and Figure 3.35). Two of these embryos were transferred, yielding a singleton pregnancy and birth of a mutation-free child, following confirmation of diagnosis by amniocentesis (Figure 3.36). In the other three embryos predicted to contain the normal allele (embryos #7, 10, and 15), ADO of the mutant gene could not be excluded, because of the parents' sharing the same site of polymorphic markers linked to the normal allele. For example, one of these embryos (embryo #15) was informative only for one linked marker, D7S637 (116/122 bp), which may have suggested the presence of both paternal and maternal normal alleles, assuming that both of these alleles could not be of maternal origin. However, this may have been also due to the uniparental disomy 7 of maternal origin, which was not supported by the other linked markers.

The probability of ADO of the mutant gene in the other two embryos (embryos #7 and 10) could have not been excluded either, despite the fact that these embryos were heterozygous for the two linked markers, D7S637 (116/122) and D7S550 (159/155), because the detected alleles (116 and 159 bp), linked to paternal normal gene, may have

Figure 3.35. Preimplantation diagnosis for homeobox gene HLXB 9 mutation in exon 1 causing Currarino syndrome. {Top) location of the mutation in HLXB9 gene and linked markers on chromosome 7. The other three panels show capillary elecrophoregarms of fluorescently labelled PCR products of HLXB9 alleles and each of the four linked markers. Paternally derived mutant allele is shown by arrow in embryo #5 {bottom), in agreement with paternally derived markers (CA repeats) linked to the mutant gene. The mutant allele is absent in embryos #4 and 16 {middle), also in agreement with all four markers. These embryos have been transferred back to the patient resulting in the birth of mutation-free baby. According to marker analysis, the baby originates from the transfer of embryo #16.

Figure 3.35. Preimplantation diagnosis for homeobox gene HLXB 9 mutation in exon 1 causing Currarino syndrome. {Top) location of the mutation in HLXB9 gene and linked markers on chromosome 7. The other three panels show capillary elecrophoregarms of fluorescently labelled PCR products of HLXB9 alleles and each of the four linked markers. Paternally derived mutant allele is shown by arrow in embryo #5 {bottom), in agreement with paternally derived markers (CA repeats) linked to the mutant gene. The mutant allele is absent in embryos #4 and 16 {middle), also in agreement with all four markers. These embryos have been transferred back to the patient resulting in the birth of mutation-free baby. According to marker analysis, the baby originates from the transfer of embryo #16.

Constipation Prescription

Constipation Prescription

Did you ever think feeling angry and irritable could be a symptom of constipation? A horrible fullness and pressing sharp pains against the bladders can’t help but affect your mood. Sometimes you just want everyone to leave you alone and sleep to escape the pain. It is virtually impossible to be constipated and keep a sunny disposition. Follow the steps in this guide to alleviate constipation and lead a happier healthy life.

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