Prenatal Treatment of Genetic Diseases in the Unborn

Introduction: Benefits of Prenatal Genetic Diagnosis Beyond the Option of Termination

For several decades, prenatal genetic testing was available to pregnant women in three different situations. Traditional karyotyping was offered to a large group of women at increased risk for trisomy 21, based on population screening using maternal age, serum marker testing, ultrasound or a combination of these. Chorionic villus sampling (CVS) with DNA analysis was offered to couples at high risk for a fetus with dominant or recessive genetic condition. The third situation was karyotyping after the diagnosis of fetal structural anomalies on ultrasound examination. In the majority of cases in all three groups, an abnormal result led to the request by the parents to terminate the pregnancy. In case of potentially treatable fetal structural anomalies, an abnormal karyotype was considered a contraindication to fetal intervention.

Many people believed that with the completion of the Human Genome Project, in the year 2003, we would enter an era of diagnosing, and even curing, many or most genetic diseases. Although since then, enormous progress has been made in technology, leading to both much faster and much cheaper sequencing, clinical application is still mostly limited to testing predisposition for genetic diseases, including certain types of cancer. Major advances have been made in genetic testing methods, and most new modalities are now available for prenatal diagnosis and screening as well. Although a small role for traditional karyotyping remains, testing for fetal diseases is now mostly done using QF-PCR for rapid aneuploidy evaluation, and chromosomal microarray for detailed analysis. Whole exome sequencing is currently introduced in perinatal clinical practice as well, further refining our diagnostic capabilities. Hudecova published a method for noninvasive prenatal diagnosis of inherited single-gene disorders using droplet digital PCR from circulating cell-free DNA in maternal plasma . Noninvasive genetic diagnostic testing, safe and therefore more acceptable to offer to a broad group of pregnant women, is entering the clinic.

The development of treatment options often lag behind the advances in diagnostic testing. This is especially true for fetal diseases, where both high definition ultrasound and high-resolution chromosome analysis enable early and accurate detection of many pathologic conditions. If at all possible, fetal diseases are preferably treated after birth, when direct access to the patient is possible, and risks for the pregnant woman are absent. In a number of fetal conditions however, progressive disease may lead to demise before birth, or to irreversible damage and handicaps at the time of birth. In such diseases, prenatal intervention can be considered to save the child's life or to improve the prognosis for its future health. Any prenatal intervention has the potential to harm the mother, or to inadvertently harm the fetus in case of complications or failure. In particular, the possible harm to the mother makes fetal therapy a challenging subspecialty. In addition, unlike in most other areas of medicine, termination of the pregnancy may be an alternative to prenatal or postnatal treatment. Thus when considering fetal intervention, potential benefits to the fetus also need to be balanced against potential harm to the fetus and to risks to the pregnant woman. If an attempt to save the life of a fetus, or to prevent irreversible damage, leads to the birth of a severely handicapped child, either because of complications of failure of the prenatal treatment, the child and its family may suffer more than with expectant management. The risk of “trading mortality for morbidity” is a valid argument against some forms of fetal therapy. We will explore this aspect in more depth in the part on ethics.

The “classic” forms of fetal therapy are directed at acquired, and not genetic, fetal diseases, such as Rh-alloimmunization or twin-twin transfusion syndrome. In these conditions, the fetus is essentially healthy, only threatened to die from a “hostile environment.” Successful prenatal intervention in these conditions often leads to the birth of healthy children with a normal long-term outcome. In genetic diseases, the underlying cause, a DNA mutation, is in most cases present in every cell of the body. Until recently, treatment options were limited to reducing the consequences of the abnormal organ functions or alleviating symptoms. Early versions of gene- and stem-cell therapy were aimed mostly at addition of genes or cells with normal function to fetal organs, and/or inducing tolerance for safer postnatal transplantation. Repairing the DNA itself, gene correction , is now on the horizon, using DNA cutting techniques such as Cas9, zinc-finger, or TALE nucleases. In this chapter, we will focus on currently or likely soon available prenatal treatments for fetal genetic diseases.

Symptomatic Treatment

Inherited or de novo mutations affecting erythropoiesis, red cell morphology, or the structure/function of hemoglobin may all lead to fetal anemia, which as a final common pathway can cause fetal heart failure, hydrops, and death. Irrespective of the underlying cause, fetal anemia can effectively and quite safely be treated by fetal blood transfusion, a method developed already in the early 1960s. This purely symptomatic treatment, often given multiple times during pregnancy, can be lifesaving. The diagnosis of genetic hematologic diseases in a fetus is made either by testing because of a family history or by workup after the incidental finding of fetal hydrops. To illustrate the complex choices and consequences doctors and parents can be confronted with, the example of fetal treatment of homozygous alpha-thalassemia will be explored further.

Alpha-thalassemia major or Hb Bart's disease is the most common inherited disorder of hemoglobin synthesis in Asia. It is caused by a deletion of all four “alpha genes” on chromosome 16. The gene mutation (a deletion) frequency in Southeast Asia is around 4.5%, resulting in a high prevalence of the homozygous mutation . Traditionally, this is considered a lethal disease due to deletion of all alpha-globin genes. In early fetal life, embryonic hemoglobin types can deliver sufficient oxygen to the tissues, however later on, hemoglobin consisting of four γ chains (Bart’s) becomes the dominant fetal hemoglobin. Hemoglobin Bart's has is a very high oxygen affinity, compromising oxygen delivery to the tissues. Screening programs identify carriers, and, in at risk couples, noninvasive fetal testing can be performed by ultrasonography aiming at finding early markers such as cardiomegaly, or by invasive testing and genetic analysis of chorionic villi. Cell-free DNA testing from maternal plasma for thalassemia is being developed . In the vast majority of cases, detection of fetal homozygous alpha-thalassemia within such screening programs will lead to termination of pregnancy.

Some early examples of fetal (intrauterine) intravascular blood transfusion for homozygous alpha-thalassemia were cases where the cause of fetal anemic hydrops was unknown, and diagnostic fetal blood sampling was combined with blood transfusion, to gain time for the diagnostic workup . Then when the diagnosis was made, parents requested continuation of the transfusion therapy, both prenatally and postnatally, to keep their children alive. Technically, this has been a relatively safe and, in fetal therapy centers, common procedure for fetal anemia treatment already for decades. Most often however, it is performed for red cell alloimmunization and parvovirus infection, two acquired fetal diseases carrying an excellent prognosis when treated timely. The main dilemma in alpha-thalassemia is the quality of life of the surviving children. As will be discussed in more detail later in this chapter, doctors feel a responsibility for the burden of the future child. The general view of fetal medicine specialists around the world is that homozygous alpha-thalassemia should still be regarded as a lethal disease, for which termination of pregnancy is justified.

A literature review identified 15 children with homozygous alpha-thalassemia surviving after fetal transfusions . In 5/15, hypospadia was present. Long-term neurodevelopmental outcome, with follow-up between 3 months and 7 years, revealed four children had mild and one had marked psychomotor impairment. Since insufficient tissue oxygenation is already present early in fetal life, fetal transfusion therapy, which at the earliest can be performed from 16 to 18 weeks’ gestation, may not be able to prevent neurologic damage . After birth all children required blood transfusions at 3–4-week intervals, and 9/15 were treated with bone marrow/stem cell transplantation. The morbidity related to iron overload and side effects of medication accompanying transplantation is considerable and significantly worse than in β-thalassemia.

This example of a technically feasible symptomatic treatment of fetuses with a serious gene mutation illustrates the main dilemma; the fetal symptomatic treatment may save the child's life, but it is no cure, and the survivors remain patients needing lifelong treatment, with far from normal lives.

Supplementation Therapy

Genetic diseases generally described as inborn errors of metabolism often originate from (single) gene mutations leading to absent or abnormal enzyme, hormone, or protein function. The mode of inheritance is often autosomal recessive. In particular in metabolic diseases where one specific essential substance is lacking, replacement therapy may solve the clinical problem. In others, preventing the individual to take food containing substances the body cannot process normally, leading to dangerous levels of a toxic product, “nutrition management” may be the solution. The best-known example is phenylalanine hydroxylase deficiency, leading to phenylketonuria (PKU). During pregnancy, the unaffected (heterozygous) carrier mother may provide essential substances via the placenta, and in this way reduce the risk of harm for the fetus with this metabolic disorder. After birth however, completely asymptomatic but affected (homozygous) neonates may soon suffer from their metabolic disorder. Likewise pregnant women with PKU need strict dietary control at least 1 month prior to conception and during the whole of pregnancy, as well as monitoring of phenylalanine levels, to avoid the severe and irreversible teratogenic effects of hyperphenylalaninemia . Most of the rare diseases screened for with the neonatal dried blood spot test by the “heel prick” fall in this category. In some genetic mutations leading to errors of metabolism, the abnormal function of an enzyme or hormone may lead to irreversible damage already before birth. In such cases, prenatal intervention may improve the outcome and prognosis. Some have argued that several of the diseases screened for by the heel prick should actually be tested for already during pregnancy, to enable earlier intervention . Examples of prenatal fetal treatment of metabolic disorders such as methylmalonic acidemia and multiple carboxylase deficiency have already been given in the 1970s and 1980s . With our increasing ability to screen for fetal genetic alterations during pregnancy, it is expected that a much larger number of “inborn errors of metabolism” will be detected, followed by more research into suitable treatments. We speculate that very few parents (and Institutional Review Boards) are willing to go the uncertain route of experimental fetal treatment. The options and dilemmas will be illustrated by some examples.

Methylmalonic acidemia (MMA) is due to deficient synthesis of 5′-deoxyadenosylcobalamin, which can lead to fetal growth restriction and dilated cardiomyopathy. Children suffer from vomiting, failure to thrive, acidosis, and mental retardation. In 1975, Ampola et al. reported prenatal treatment by vitamin B12 in an attempt to diminish the accumulation of methylmalonic acid to a pregnant woman who lost a previous child to MMA . Dose and response were monitored using maternal urinary excretion of methylmalonic acid. Postnatally, the child was treated with dietary measures. At 19 months, the child was developing normally. In the following decades, a few more apparently successful cases have been published. In 2016, the long-term follow-up was published of a girl with Cobalamin C deficiency (leading to MMA as well as hyperhomocysteinemia) treated in utero by giving the pregnant mother a weekly dose of hydroxycobalamin in 2005, and her untreated older sister . At the age of 11 years the girl had a normal IQ of 103, attended secondary school, and only mild ophthalmic findings. The affected older sister, with the same genotype and similar residual enzymatic activity, received postnatal treatment only and was severely intellectually disabled, cannot walk or talk, shows aggressive behavior, and has severe ophthalmic symptoms. Due to the rarity of the disease and the many genetic variants, it remains difficult to establish optimal dosage and to predict clinical outcome, especially on the long term.

Multiple carboxylase synthetase deficiency presents in the newborn with severe metabolic acidosis. It is treated with lifelong biotin therapy. However, the fetus may already suffer from growth restriction as well as ventriculomegaly . The first case report of prenatal treatment, by maternal oral biotin supplementation, was published in 1982 . Dosage is still a problem, as illustrated by the latest published case where the investigators used 10 mg biotin daily based on a few previous reports. Fetal growth accelerated after biotin treatment, but the child was born with significant lactic acidemia and metabolic acidosis .

3-Phosphoglycerate-dehydrogenase deficiency is an amino acid (serine) synthesis disorder, associated with microcephaly, severe psychomotor retardation, and intractable seizures. In 2004, de Koning et al. from Utrecht, The Netherlands published a case of prenatal maternal administration of supplements of l -serine, three doses of 5 g l -serine (190 mg/kg) per day, starting at 26 weeks when the growth of the head circumference was decelerating . The child had low levels of serine at birth, but was born with a normal head size, and at the age of 2 developed normally, in sharp contrast with its two affected older siblings.