A genetic link has now been found for some instances of Sudden Infant Death Syndrome, or SIDS.
The new UW Medicine research study is the first to make an explainable link tracking the mechanism between a genetic anomaly and some forms of the devastating syndrome, which claims the lives of more than 3,000 infants a year.
Hannele Ruohola-Baker, professor of biochemistry at the University of Washington School of Medicine, headed the multi-institutional study.
The findings are published in the Oct. 11 edition of Nature Communications. The research focused on mitochondrial tri-functional protein deficiency, a potentially fatal cardiac metabolic disorder caused by a genetic mutation in the gene HADHA.
Newborns with this genetic anomaly can’t metabolize the lipids found in milk, and die suddenly of cardiac arrest when they are a couple months old. Lipids are a category of molecules that include fats, cholesterol and fatty acids.
“There are multiple causes for sudden infant death syndrome,” said Ruohola-Baker, who is also associate director of the UW Medicine Institute for Stem Cell and Regenerative Medicine, where the lab she heads is located.
“There are some causes which are environmental.
But what we’re studying here is really a genetic cause of SIDS. In this particular case, it involves defect in the enzyme that breaks down fat.”
Jason Miklas, who earned his Ph.D. at the UW and is now a postdoctoral fellow at Stanford University, was the lead author on the paper. He said he first came up with the idea while researching heart disease.
He noticed a small study that had examined children who couldn’t process fats and who had cardiac disease that could not be readily explained.
So Miklas, along with Ruohola-Baker, started looking into why heart cells, grown to mimic infant cells, were dying in the petri dish where they were being grown.
“If a child has a mutation, depending on the mutation the first few months of life can be very scary as the child may die suddenly,” he noted.
“An autopsy wouldn’t necessarily pick up why the child passed but we think it might be due to the infant’s heart stopping to beat.”
“We’re no longer just trying to treat the symptoms of the disease,” Miklas added. “We’re trying find ways to treat the root problem. It’s very gratifying to see that we can make real progress in the lab toward interventions that could one day make their way to the clinic.”
In MTP deficiency, the heart cells of affected infants do not convert fats into nutrients properly, This results in a build-up of unprocessed fatty material that can disrupt heart functions.
More technically, the breakdown occurs when enzymes fail to complete a process known as fatty acid oxidation. It is possible to screen for the genetic markers of MTP deficiency; but effective treatments are still a ways off.
Newborns with this genetic anomaly can’t metabolize the lipids found in milk, and die suddenly of cardiac arrest when they are a couple months old.
Lipids are a category of molecules that include fats, cholesterol and fatty acids.
Ruohola-Baker sees the latest laboratory discovery as a big step towards finding ways to overcome SIDS.
“There is no cure for this,” she said. “But there is now hope, because we’ve found a new aspect of this disease that will innovate generations of novel small molecules and designed proteins, which might help these patients in the future.”
New genetic link found for some forms of SIDS. Credit: UW Medicine.
One drug the group is focusing on now is Elamipretide. It is used to stimulate hearts and organs that have oxygen deficiency, but was barely considered for helping infant hearts, until now. In addition, prospective parents can be screened to see if there is a chance that they could have a child who might carry this genetic mutation.
Ruohola-Baker has a personal interest in this research, as one of her friends in her home country of Finland had a baby who died of SIDS.
“It was absolutely devastating for that couple,” she said. “Since then, I’ve been very interested in the causes for Sudden Infant Death Syndrome.
It’s very exciting to think that our work may contribute to future treatments, and help for the heartbreak for the parents who find their children have these mutations.”
The incidence of stillbirth in Sweden, defined as fetal death occurring at completed gestational week 22 or later, has essentially remained constant at approximately 3–4 per 1 000 live births since the 1980’s [1]. Stillbirth can be caused by several factors, such as infections, placental insufficiency or abruption, maternal conditions (e.g. preeclampsia), chromosomal aberrations, malformations and umbilical cord complications [2]. In Stockholm County, all cases of stillbirth pass a thorough investigation, with the aim of identifying the underlying factor of fetal demise. The investigation includes physical examination and autopsy, infectious disease testing and chromosome analysis by conventional chromosome analysis by karyotyping, or, when it fails, by quantitative fluorescence polymerase chain reaction (QF-PCR). Using these methods, chromosomal abnormalities are identified in 6–17% of stillbirth cases [3,4]. We have previously shown that analysis with chromosomal microarray (CMA) increases both the analysis success rate, as well as the chromosomal aberration detection frequency, compared with conventional karyotyping [5]. However, many stillbirth cases remain unexplained, and determination of the underlying cause is important as a history of stillbirth is associated with an increased recurrence risk in following pregnancies [6].
Studies have suggested that long QT syndrome (LQTS) might contribute to stillbirth in some cases [7,8]. LQTS is a channelopathy affecting cardiac ion channels, and is characterized by a prolonged Q-T interval on electrocardiogram. The condition is a common cause of sudden death postnatally, and is diagnosed in up to 9.5% of infant death syndrome cases [9]. Also other channelopathies, such as Brugada syndrome (BrS) and catecholaminergic polymorphic ventricular tachycardia (CPVT), as well as cardiomyopathies, such as hypertrophic cardiomyopathy (HCM), have been suggested to cause infant death [10–13]. Common for cardiac channelopathies is that the structure and function of ion channels are affected, which in turn leads to disrupted action potential propagation and thereby causes development of arrhythmias [14]. Cardiomyopathies, i.e. disorders of the heart muscle, are impairments of the ability of the myocardium to contract, which can result in heart failure [15]. A study including 47 cases of sudden unexpected death in infancy (SUDI) identified one or more genetic variants with likely functional effects in 34% of the cases, by investigation of 100 genes associated with cardiac channelopathies and cardiomyopathies [16]. It is reasonable to suspect that genetic variants associated with death in infancy might as well cause fetal death. However, this has not been extensively studied.
In this study, DNA from 290 stillbirth cases without chromosomal abnormalities was analyzed using a gene panel, including 70 genes associated with cardiac channelopathies and cardiomyopathies. We suggest that the results might provide clues to the underlying cause of stillbirth for a proportion of cases.
Results
The mean sequencing coverage for the complete HaloPlex gene panel was 99.5%. The mean coverage for each gene is displayed in S1 Table. Of the 290 investigated stillbirth cases, 35 (12.1%) had one (n = 31) or two (n = 4) variants with evidence supporting pathogenicity, i.e. LoF variants (nonsense, frameshift, splice site substitutions), evidence from functional studies, or previous identification of the variants in affected individuals (Table 2). The proportion of individuals harboring the same variants in ExAC NFE and SweGen was significantly lower, 4.8% (p<0.001) and 5.1% (p<0.001), respectively (Table 3). Twenty cases had a variant in a channelopathy gene (i.e. CACNB2, GPD1L, KCNH2, KCNJ8, KCNQ1, RYR2, SCN5A and TRPM4), whereas 15 cases had one (n = 11) or two (n = 1) variants in cardiomyopathy genes (i.e. ABCC9, BAG3, DES, DSG2, DSP, MYBPC3, NEBL, NEXN, TNNI3 and TTN). Three cases (34, 286 and 290) had one variant in a cardiomyopathy gene and one in a channelopathy gene (CSRP3 and TRPM4, PKP2 and ANK2, MYH7 and KCNH2, respectively). The proportion of individuals harboring putative pathogenic variants in the different categories are displayed in Table 3. As KCNQ1 was the gene in which most putative pathogenic SNVs were identified in the stillbirth cohort (n = 5), all missense and LoF variants recorded in ExAC NFE were systematically searched for in ClinVar, to get an approximation of how common pathogenic SNVs are in the European population for this gene. The results showed a significantly higher total number of observations of putative pathogenic alleles in KCNQ1 in relation to wild type alleles in the study cohort compared with ExAC NFE (5/580 (0.86%) vs. 219/66 740 (0.33%), p = 0.046). A Monte Carlo permutation test with 10 000 iterations was performed to compare the proportion of pathogenic SNVs in the stillbirth cohort to ExAC NFE across all 70 genes included in the gene panel. Groups of 50 variants from each cohort were drawn in each iteration. The average proportions of putative pathogenic SNVs did not differ between the stillbirth cohort and ExAC NFE, which were calculated as 3.1% and 3.2%, respectively.
Discussion
We have analyzed DNA from 290 stillbirth cases for prevalence of pathogenic SNVs in 70 genes associated with heart disease. To our knowledge, this is the first MPS-based study including a large stillbirth cohort, and according to our results, SNVs with evidence supporting pathogenicity was identified in as many as 12.1% of the cases. The proportion was significantly higher than the corresponding proportion for the same variants in ExAC NFE, (5.3%, p<0.001) as well as in SweGen (5.1%, p<0.001). When divided into the different disease categories, i.e. channelopathies and cardiomyopathies, the significant difference was seen only for channelopathies (Table 3).
Previous studies have mainly focused on stillbirth in association with LQTS. Crotti et al studied 91 stillbirth cases for SNVs in the most common LQTS susceptibility genes, i.e. KCNQ1, KCNH2 and SCN5A, and identified three putative pathogenic variants (3.3%) [8]. The proportion of putative pathogenic SNVs for the same genes in our study was 3.1% (n = 9), i.e. very similar to what was reported by Crotti. One variant, KCNQ1, p.(Arg397Trp), was identified in both studies. Crotti et al showed that this variant caused a significant reduction in current densities across the potassium channel encoded by KCNQ1, compared with the wild type channel [8]. Furthermore, the total number of pathogenic alleles in KCNQ1 observed in our cohort was significantly higher compared with ExAC NFE (p = 0.046), which supports that SNVs in this gene might play a role in stillbirth. In addition to the most common LQTS genes, one case in our cohort harbored a putative pathogenic SNVs in ANK2. Taken together, LQTS associated SNVs were identified in 10 cases (3.4%) of the stillbirth cases included in the present study.
Three cases (1%) harbored putative pathogenic SNVs in RYR2, associated with CPVT. CPVT is one of the most severe cardiac channelopathies, and is characterized by ventricular arrhythmias causing syncope, cardiac arrest and sudden cardiac death, predominantly in young patients including infants [11]. Thereby, these SNVs are good candidates for being associated with stillbirth. BrS associated SNVs were identified in 5.2% (n = 15) of the cases in the cohort. As BrS has mainly been described in association with sudden death in adults, its role in stillbirth is difficult to interpret. However, one of the BrS associated SNVs are worth highlighting, namely GPD1L, p.(Ile124Val). This variant was identified in four cases, and has been associated with sudden infant death syndrome (SIDS) in a previous study [24]. According to ExAC NFE, it has a minor allele frequency (MAF) of 0.24% in the European population, whereas the MAF in our cohort is 0.69%, i.e. almost three times as high (p = 0.054). Although not statistically significant, this might indicate that this variant is a risk factor for stillbirth as well as SIDS.
In our cohort, SNVs with evidence supporting pathogenicity in genes associated with cardiomyopathies (HCM, DCM and ARVC) were identified in 12 cases. As cardiomyopathies are progressive disorders which are generally not detected during the early years of life, they have not been studied in association with stillbirth previously. However, increasing evidence suggests that they might play a role in SIDS [13,25]. Brion et al studied 286 SIDS cases for variants in genes associated with HCM and found variants with possibly damaging effects in 4% of the cases [13]. One of their identified SNVs, MYBPC3, p.(Ala833Thr), was detected in two of our cases. Brion et al hypothesized that their identified variants might cause sudden cardiac death even in the absence of a cardiac phenotype, but they do also emphasize the possibility that the variants could be non-disease causing rare variants [13]. Furthermore, the proportion of putative pathogenic variants associated with cardiomyopathies identified in this study was not significantly higher than the corresponding proportion in ExAC NFE and SweGen. The Monte Carlo permutation test revealed no significant difference in proportions of putative pathogenic variants between the study cohort and ExAC NFE, which probably reflects that the majority of the 70 genes included in the panel are indeed not associated with stillbirth.
Although the putative pathogenic SNVs identified in this study had a significantly higher prevalence in in the stillbirth cohort compared with ExAC NFE data, the results should be interpreted with caution. As the cohort included in this study is substantially smaller than the one included in ExAC NFE, there is a high probability that MAFs of rare alleles are overestimated, and do not reflect the true MAFs in all cases. Nonetheless, to our knowledge, this is the largest cohort of stillbirth cases investigated for pathogenic SNVs in a large set of genes associated with heart disease that has been analyzed to date. Thereby it provides some insight to the frequency of putative pathogenic SNVs in stillbirth cases. However, there are additional limitations to the study which need to be addressed. No parental DNA samples were available, and hence it is unknown whether the identified variants are inherited or of de novo origin. This information would otherwise have provided additional support for or against a clinical significance of the variants. Additionally, no clinical information regarding the parents was available, and therefore it is unknown whether there is a history of heart disease or recurrent stillbirth in any of the families. Furthermore, we did not perform any functional studies on the identified variants. Because of the inherent limitations of the study, most of the putative pathogenic variants identified in this study only qualify as variants of unknown significance (VUS) when classified in accordance to the current state-of-the-art guidelines used in a clinical setting, formulated by ACMG [21]. Hence, the SNVs displayed in Table 2 should not be interpreted as verified pathogenic variants that, without further investigation, could be used for carrier testing and/or prenatal testing in a clinical laboratory. Although we have based our classifications on damage prediction of the variants and previously published data, studies suggest that pathogenicity of several reported LQTS and cardiomyopathy associated variants is overestimated [26–28]. Indeed, recent data reveals that the importance of several cardiomyopathy- and BrS related genes—some of which are included in our gene panel—is probably not as high as previously thought [29,30]. Conversely, some of the 144 missense variants of unknown significance displayed in S2 Table might be associated with heart disease, but have not yet been reported as such. Additional research is required to further clarify the clinical impact of the SNVs identified in this study.
Better knowledge of the etiology of stillbirth is needed in order to achieve a reduction in the stillbirth rate. Our results give further support to the hypothesis that cardiac channelopathies might contribute to stillbirth. Screening for pathogenic SNVs in genes associated with heart disease might be valuable in cases of stillbirth where today’s conventional investigation does not reveal the underlying cause of fetal demise.
Source:
University of Washington
Media Contacts:
Leila Gray – University of Washington
Image Source:
The image is in the public domain.
Original Research: Open access
“TFPa/HADHA is required for fatty acid beta-oxidation and cardiolipin re-modeling in human cardiomyocytes”. Jason W. Miklas, Elisa Clark, Shiri Levy, Damien Detraux, Andrea Leonard, Kevin Beussman, Megan R. Showalter, Alec T. Smith, Peter Hofsteen, Xiulan Yang, Jesse Macadangdang, Tuula Manninen, Daniel Raftery, Anup Madan, Anu Suomalainen, Deok-Ho Kim, Charles E. Murry, Oliver Fiehn, Nathan J. Sniadecki, Yuliang Wang & Hannele Ruohola-Baker.
Science doi:10.1038/s41467-019-12482-1.
