Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research.
Preimplantation genetic diagnosis (PGD) allows genetic diagnosis of embryos in very early stages, with the purpose of avoiding the transmission of genetic diseases to offspring. PGD represents an alternative to prenatal diagnosis and termination of pregnancy, in couples at risk of transmitting these disorders. Since the first PGD application [
Although, in theory, PGD could be accomplished using mutation analysis alone, this would be accompanied by a high error rate due to allele drop out (ADO) [
Most protocols use blastomere biopsy (single cells from a 6–8 cell, day 3 embryo) for embryo diagnosis. PGD using polar bodies (extruded by the oocytes) analysis has been shown to be an effective method for maternal autosomal dominant, X-linked, and, in some cases, recessive disorders [
Basic research for human disease is commonly studied in animal and cellular model systems. For many genetic disorders these models are not available. In these cases, diseased embryos obtained through PGD can provide a novel resource for the derivation of mutant human embryonic stem cell (HESC) lines. Such cell lines may be induced to differentiate different tissues and can be exceptionally useful for basic research studies as well as for drug screening and development.
Many clinicians are not aware of the new genetic methods for diagnosis and prevention of inherited lysosomal disorders. We present the development of PGD protocols for four lysosomal storage disorders: Tay Sachs, Gaucher type I, Fabry, and Hunter syndrome; two of them, Tay Sachs and Gaucher, have a very high carrier rate in the Ashkenazi Jewish population [
Tay Sachs (TSD, GM2 gangliosidosis type B, OMIM #272800) is a recessive neurodegenerative lysosomal storage disease caused by Hexosaminidase A isoenzyme deficiency. The most severe form of the disease is characterized by rapid progressive neurodegeneration, developmental regression, and death during age of 3-4 years [
Gaucher disease (GD, OMIM #230800), the most common lysosomal storage disorder (Goldblatt [
Mucopolysaccharidosis type II (MPS II) or Hunter syndrome is a lysosomal storage disease caused by a deficiency of the enzyme iduronate-2-sulfatase (
Fabry disease is caused by an X-linked (dominant) error of metabolism wherein deficiency of the lysosomal enzyme
We present 20 families who underwent preimplantation diagnosis for prevention of birth of children with inherited lysosomal disorders, without the need for invasive prenatal diagnosis procedure and termination of pregnancy. These methods are generally applicable for any disorder in which the genetic basis of the disease is known.
Of the eleven TS families that presented to our clinic, one of them was a double-carrier for Tay Sachs and Gaucher type 1, and in another, the male in addition to being a carrier of Tay Sachs was also a carrier of a balanced robertsonian translocation 45XYder(21;14). Details of the patients are presented in Table
(a) Characteristics of Tay Sachs carriers, (b) characteristics of Gaucher disease type 1 carriers, and (c) characteristics of couples in which females were carriers of Hunter syndrome.
Family number | Mutation female/male | Healthy/affected children prior to PGD | Female age (years) |
---|---|---|---|
1 | IVS12G>C/Gly269Ser | 0 | 25 |
2 | 1278insTATC/1278insTATC | 0 | 32 |
3 | 1278insTATC/1278insTATC | 1/0 | 29 |
4 | 1278insTATC/Gly269Ser | 1/0 | 31 |
5 | 1278insTATC/1278insTATC | 3/0 | 33 |
6 | 1278insTATC/Gly269Ser | 1/0 | 30 |
7 | 1278insTATC/Gly269Ser | 0 | 31 |
8 | 1278insTATC/1278insTATC | 3/0 | 34 |
9 | IVS12G>C/Gly269Ser | 0 | 28 |
10 | 1278insTATC/1278insTATC | 2/TOP* | 33 |
11 | 1278insTATC/1278insTATC | 0/1** | 29 |
TOP*: termination of pregnancy due to affected embryo.
**The male was also a carrier of a balanced Robertsonian translocation 45XYder(21;14).
Family number | Mutation female/male | Healthy/affected children prior to PGD | Female age (years) |
---|---|---|---|
1 | IVS2+1G>A/N370S | 0 | 25 |
2 | N370S/R359Q | 0/1* | 34 |
3 | N370 homozygous/84GG | 1/0 | 29 |
4 | Arg496His/84GG | 0 | 30 |
*The daughter died at the age of five due to severe pulmonary involvement.
Family number | Mutation female | Healthy/affected children prior to PGD | Female age (years) |
---|---|---|---|
1 | L410P* | 2/0 | 34 |
2 | L410P* | 1/0 | 25 |
3 | Del exons 4–7 | 0/TOP* | 24 |
TOP*: termination of pregnancy due to affected embryo.
Four GD families (including a couple also carrying TS mutations; Family number 1 (Tables
Two couples in which males were affected with Fabry disease presented to our clinic. In one of the couples, he also was affected with nonobstructive azoospermia (NOA) requiring micro-TESE for in vitro fertilization. Since in both couples the males were affected and since Fabry is an X-linked disorder, all the male embryos will be healthy while all the female embryos will be carriers. Since seventy percent of female carriers of Fabry disease manifest clinical symptoms during life (ref), both our couples chose not to transfer carriers.
Three HS couples presented to our clinic (Table
Controlled ovarian stimulation and IVF treatment were performed using the standard long downregulation protocol. For frozen-thawed embryo cycles, endometrium preparation was performed using oral estradiol valerate (Estrofem 4–8 mg daily (Novo Nordisk, Denmark)) and vaginal Utrogestan (micronized progesterone 900 mg/daily (CTS, France)). Polar body and blastomere (one cell of a six—eight cell embryo) biopsy, ICSI, and embryo cultures were performed as previously described [
DNA was extracted from peripheral blood cells from couples, affected children, and first-degree family members. For each disease, polymorphic microsatellite markers surrounding the diseased gene were identified and informative markers were used construct haplotypes in each family. These markers and the family mutations were then used for the development of single-cell multiplex assays. The panels of markers used for PGD analysis of each disease are presented in Table
Polymorphic microsatellite markers used in PGD/LSD analyses.
Disease | Markers |
---|---|
Tay Sachs | D15S204, D15S110, D15S197, TS-AT3 (chr15: 70157048-70157297), TS-TATT (chr15: 70231314-70231376), TS-TTTC (chr15: 70404391-70404690), TS-TC (chr15: 70408504-70408653), TS-ATCT (chr15: 70417649-70417798), TS-CA (chr15: 70420187-70420336), TS-TA (chr15: 70465534-70465733), TS-TG (chr15: 70172306-70172338), D15S215, D15S188, D15S169, D15S818 |
| |
Gaucher disease type 1 | D1S2715, D1S2858, D1S305, Gau-GT2 (chr1: 153399641-153399790), Gau-AC (chr1: 153726984-153727183), Gau-GT (chr1: 153425519-153425668), Gau-TTAT (chr1: 153461596-153461795), Gau-AAAG (chr1: 153462605-153462854), Gau-AAT (chr1: 153480717-153480916), Gau-TAT (chr1: 153519755-153519904), GAu-AAT2, (chr1: 153527305-153527554), D1S1153, Gau-AC3 (chr1: 153593444-153593643), D1S27777, Gau-AC4 (chr1: 153897882-153898031), D1S303, D1S2140, D1S2721, D1S2624 |
| |
Mucopolysaccharidosis II | DXS731, DXS1215, DXS1691, DXS8091, DXS6687, DXS2496, DXS1185, DXS1193, DXS457, DXS1123, Hunter-GAGG (chrX: 148394342-148394541), Hunter-AC (chrX: 148405052-148405251), DXS8086, DXS8377, DXS8069, DXS7423, DXS8011 |
| |
Fabry disease |
AmelogB96, SRY, DXS1254, DXS998, DXS1215, DXYS154, DXS566, DXS8377 |
Polymorphic microsatellite markers used in PGD/LSD analyses: location of markers is based on human USCS genome browser assembly March 2006, NCBI36/hg18 presented in the order of location on the chromosome (
The markers that do not have standard DS nomenclature were named arbitrarily and their physical chromosome and basepair location are indicated.
Derivation and maintenance of undifferentiated Shaare Zedek (SZ) Hunter and Gaucher cells were carried out according to routinely applied protocols on blastocysts diagnosed as mutant [
A total of 20 families carrying mutations in four genes causing lysosomal diseases presented to our PGD unit. We performed 56 PGD cycles and analyzed 329 oocytes/embryos with an overall pregnancy rate of 38%. All but three couples have so far delivered healthy children. The results and intricacies of these analyses are described below, a summary of the PGD cycles is presented in Table
IVF-PGD treatment outcome.
Disease | Family number | Total cycles | Number of oocytes retrieved | Number of oocytes/embryos biopsied | Wild type/ |
Number of transferable embryos | Number of embryos transferred/cycle | Treatment outcome |
---|---|---|---|---|---|---|---|---|
1* | 3 | 32 | 26 | 3/4/17 | 7 | 2 | 3 children from 3 cycles | |
2 | 3 | 16 | 16 | 8/4/4 | 12 | 2 | Twins | |
3 | 1 | 5 | 4 | 0/1/3 | 1 | 1 | No pregnancy | |
4 | 2 | 15 | 13 | 4/5/4 | 7 | 2 | Twins | |
5 | 1 | 12 | 10 | 3/7/0 | 10 | 2 | One child | |
Tay Sachs | 6 | 7 | 44 | 38 | 10/20/8 | 30 | 1-2 | One child |
7 | 1 | 5 | 5 | 2/0/3 | 2 | 2 | No pregnancy | |
8 | 4 | 26 | 18 | 4/7/3 | 11 | 1-2 | One child | |
9 | 1 | 11 | 10 | 2/5/3 | 7 | 1 | One child | |
10 | 1 | 8 | 6 | 0/4/2 | 4 | 1 | One child | |
11** | 3 | 30 | 24 | 3/8/13 | 3 | 3 | No pregnancy | |
| ||||||||
Gaucher | 2 | 11 | 28 | 23 | 6/10/7 | 16 | 2-3 | One spontaneous abortion week 10, one child, and one ongoing twin pregnancy week 22 |
3 | 2 | 15 | 13 | 0/6/7 | 6 | 1-2 | One child | |
4 | 1 | 6 | 6 | 2/2/2 | 4 | 1 | One child | |
| ||||||||
1 | 4 | 45 | 35 | 6/10/19 | 6 | 1-2 | One child | |
Hunter | 2 | 1 | 16 | 11 | 4/0/5 | 4 | 2 | Twins |
3*** | 7 | 35 | 33 | 9/0/19 | 9 | 1-2 | Twins | |
| ||||||||
Fabry | 1 | 3 | 38 | 23 | 6/0/17 | 7 | 1-2 | One child |
2 | 1 | 4 | 2 | 1/0/1 | 1 | 1 | One child |
*Double carriers for Tay Sachs and Gaucher disease.
**The male was also a carrier of a balanced robertsonian translocation 45XYder(21;14), therefore some embryos could not be transferred due to unbalanced karyotype.
***Female carrier of mutation in IDS gene; both partners are carriers of mutations in the Tyrosinase (
All eleven families were of Ashkenazi Jewish origin and carried one of the three common mutations in the HEXA gene c.1278insTATC, IVS12G>C, or p.Gly269Ser. Nine families achieved pregnancies and delivered between one and two (twin) healthy babies per cycle. Couple number 1, carried both TS and GD mutations necessitating analysis of both loci, they had three children from three separate PGD cycles. In couple number 3, of 10 screened microsatellite markers upstream from the HEXA gene, none were informative for the maternal wild-type alleles, and therefore only the wild-type paternal allele could be used to identify unaffected embryos. Only one embryo was transferred in one PGD cycle although no pregnancy was achieved. In couple number 7, of 16 microsatellite markers screened, only two were found to be fully informative for blastomere analysis, and since the partners presented different mutations, the mutations could not be used in blastomere analysis because of possible misdiagnosis due to ADO. However, by using polar body analysis (only maternal alleles are analyzed), six informative maternal markers were identified and used in one PGD cycle that did not yet result in pregnancy. Couple number 11, in which the male was also carrier of a balanced translocation, underwent three PGD cycles. In a total of 24 embryos analyzed, only three were found to be balanced/normal for the paternal translocation and nonaffected with TS, no pregnancy was achieved in two separate transfers. None of the couples agreed to perform prenatal confirmation by CVS or amniocentesis but all babies were tested for TS familial mutations after birth and were found to be unaffected and in accordance with the embryos transferred (wild type or carriers). A total number of 27 PGD cycles were performed with a pregnancy rate (per embryo transfer) of 40% and 11 healthy babies were born.
Four Couples who were carriers of GS mutations underwent PGD and all families achieved healthy children. Family number 1, carriers of both TS and GD IVS2+1G>A/N370S mutations, was described above and had three healthy children through PGD. Couple number 2 was heterozygotes for two different mutations (p.N370S and p.R359Q) and underwent 11 PGD cycles. One cycle resulted in a spontaneous abortion in week 10, another in the birth of a healthy boy, and the most recent cycle resulted in an ongoing twin pregnancy in week 25. In Couple number 3, the female was homozygous for Gaucher disease type 1 (N370S/N370S) and the husband was a carrier of the 84GG mutation, therefore all embryos will at least be obligatory carriers of the maternal mutation. In one cycle of 7 embryos, four were found to be carriers and two of these were transferred resulting in birth of a healthy child. The wild-type paternal allele was confirmed on genomic DNA of the baby after birth. In family number 4, of six embryos two were found to be wild type and the remainder were mutant. One healthy child was born from this PGD cycle and the wild-type status was confirmed after birth.
Three couples presented to our center with HS and all had healthy babies. Couples number 1 and number 2 (sisters carrying the L410P mutation in the IDS gene) underwent four and one (resp.) PGD cycles. Since Hunter syndrome is an X-linked disorder and the females in both couples were carriers, indirect oocyte analysis by polar bodies 1 and 2 is sufficient for accurate diagnosis and was performed in these cases. Couple number 1 delivered a singleton and couple number 2 had twins. In couple number 3, the female was a carrier of a deletion of exons 4–7 in the IDS gene, and both members of the couple were also carriers of different mutations in the TYR gene, requiring simultaneous PGD for both Hunter syndrome and oculocutaneus albinism (a recessive disorder). Since in cases of autosomal recessive disorders, the information of both maternal and paternal contribution is required for complete diagnosis, a single blastomere was biopsied and used for PGD analysis for both diseases. This couple underwent seven PGD cycles, resulting in birth of healthy twins from a combined fresh and frozen blastomere analysis.
In both couples analyzed for FD, the males were affected, and since Fabry is an X-linked disorder, all male embryos are expected to be healthy; furthermore, 70% female carriers show clinical manifestations, we therefore used sex selection as a means to select for disease free embryos. Couple #1 underwent three PGD cycles. The male in this couple had nonobstructive azoospermia (NOA) requiring micro-testicular sperm extraction (TESE) for in vitro fertilization (IVF). Three cycles lead to a singleton pregnancy and subsequent birth of a healthy boy. Couple number 2 underwent one PGD cycle; analysis of two embryos led to the diagnosis of one male. Transfer of this embryo resulted in the birth of a healthy boy.
Mutant embryos from these 4 lysosomal diseases (after IRB approval and family consent) were donated for stem cell line derivation. Out of 28 embryos, two HESC lines were obtained (Table
Derivation of stem cell lines.
Disease | Number of mutant embryos received | Embryos plated | Cell line |
---|---|---|---|
Gaucher | 2 | No | No |
Gaucher | 2 | No | No |
Gaucher | 4 | 4 | 1 |
Hunter | 15 | 8 | 1 |
Tay Sachs | 5 | 3 | No |
| |||
Total | 28 | 15 (53.6%) | 2 (13.4%)* |
Characterization of SZ-Hunter HESCs for the expression of undifferentiated cell-specific markers, karyotype, and pluripotent potential. (a) RT-PCR products for the undifferentiated gene-specific markers OCT4, SOX2, NANOG, and REX-1, using cDNA-specific primers, in undifferentiated SZ-Hunter HESCs at passage P17. (b) Karyotype analysis for SZ-Hunter cells by Giemsa staining. (c) A typical morphology of an undifferentiated SZ-Hunter HESC colony. (d) A cystic embryoid body (EB) established from SZ-Hunter HESCs, grown for 20 days in culture of suspension.
We present a reliable method for preventing birth of affected children for parents carrying mutations in genes causing lysosomal disorders and subsequent development of stem cell lines for research in the field of lysosomal diseases. When couples who are carriers of a genetic disorder wish to conceive they have the option to either not perform prenatal genetic testing and to accept the possibility of an affected child or to perform an invasive test (chorionic villus sampling or amniocentesis) in order to determine the genetic status of the embryo. Both of these invasive methods are accompanied by a small risk of abortion due to the procedure [
PGD is a technique that offers an alternative to pregnancy termination of an affected embryo by analyzing single blastomeres of biopsied embryos or polar bodies 1 and 2 from oocytes, and returning to the uterus only unaffected embryos. Since PGD was first performed in 1991 [
Screening tests for Tay Sachs disease prior to, or at the beginning of, pregnancy is performed free of charge in Israel for all Ashkenazi Jewish couples. All our couples found that both partners were carriers during the screening tests and while some approached our unit immediately for PGD, others performed CVS. One couple performed a pregnancy termination of an affected embryo prior to their arrival at our PGD clinic.
Since Gaucher disease can be asymptomatic in Ashkenazi Jews (who most commonly carry the N370S mutation), we most commonly perform PGD for this lysosomal disorder only in cases in which the phenotype is predicted to be severe based on the mutations carried by the couple. In the first family the couple were carriers of both Tay Sachs and Gaucher disease mutations and the main reason for PGD was Tay Sachs, a lethal disease, even though the Gaucher mutation combination of IVS2+1G>A/N370S predicted a possible more severe phenotype. In both couples number 2 and number 4 one of the partners was a carrier for the 84GG mutation predicting a possible severe phenotype. In couple number 3 the male was carrier of a private mutation R359Q that in combination with the N370S caused a severe pulmonary disease in their daughter leading to death at the age of 5 years despite high-dose enzyme replacement therapy. In the case of Hunter syndrome, the two sisters carrying mutations in the IDS gene had a brother who died of the disease. Both women had prior pregnancies and performed CVS testing prior to the PGD cycles. The third couple came to our PGD clinic after performing a termination of pregnancy due to an affected male embryo detected in prenatal testing.
In both couples with Fabry disease the male partners were affected, and since inheritance of this disease is X-linked, it is predicted that all their female offspring will be carriers and all males unaffected. One of these males also had severe oligospermia requiring IVF irrespective of PGD. In the second couple there was no known infertility but since more than half of female carriers of Fabry disease develop symptoms during life [
The mutant embryos derived from PGD are a valuable resource for disease research due to the possibility of developing mutant stem cell lines. In our unit, mutant embryos that are donated by the family for stem cell research undergo culturing for stem cell line development. Out of these four lysosomal disorders we have successfully established 2 lines. One Gaucher stem cell line was derived from mutant embryos caring 84GG and N370S mutations is of particular of interest due to recent evidence of correlations between Parkinson disease and Gaucher [
In summary, we have presented our PGD experience for 20 families with Tay Sachs, Gaucher, Fabry diseases, and Hunter syndrome. In 56 PGD cycles, 329 oocytes/embryos were analyzed, which led to the birth of 20 unaffected children, with an overall pregnancy rate of 38% per embryo transfer (the overall pregnancy rate reported in the ESHRE XI data collection was 27%). Surprisingly, we observed a significantly higher mutant embryo rate than we expected. In autosomal recessive disorders only 25% of embryos are expected to be mutant, however, of the 166 embryos we analyzed for Tay Sachs, 65 (39%) were mutant and not transferable, and analysis of embryos from Gaucher carrier couples yielded 33/66 (50%) mutant embryos. Inheritance of X-linked disorders expected to yield 50% wild-type offsprings, 25% carriers (females), and 25% affected males. In the case of Hunter and Fabry diseases, since carrier females are known to show some disease manifestation, these embryos are not transferred either. Embryo analysis of the X-linked disorders in our study yielded 53/72 (74%) mutant or carrier embryos in Hunter disease, and 18/25 (72%) nontransferrable embryos in Fabry couples. While we are not able to offer an explanation for the skewing towards mutant embryos observed here, these statistics confirm the importance of the role PGD has played in family planning for these couples.
Of the 20 unaffected children born, fourteen births were singletons and three were twins, the wild type or carrier disease status was confirmed in all cases after birth. Four unaffected children were born in families where PGD needed to be performed for 2 different disorders simultaneously, a circumstance which significantly lowers the number of unaffected embryos available for transfer. At present, of these 20 families, 17 have given birth to unaffected children (2 of the remaining 3 families have only undergone 1 PGD cycle), thus far the take-home baby rate per family in this study was 85%.