Massively Parallel Sequencing: Successes, Limitations and the Future for Inborn Errors of Metabolism

Abstract

Inborn errors of metabolism (IEM) are a diverse group of inherited diseases from a presentation, natural history and diagnostic perspective [1]. They remain one of the few groups of primary genetic diseases where treatment options are available and established. The management of IEM varies based on the associated dysfunctional metabolic pathway, and central to the management of IEM is multi-disciplinary team care. Amongst others, this includes medical, nursing, dietetics, social work and genetic counselling [2]. Common presentations of IEM include acute small molecule intoxication, multi-system malformation disorders [3], global developmental delay and developmental regression [2]. Given the broad nature of these disorders, the expansion of genomics with massively parallel sequencing (MPS) has revolutionised some aspects of IEM investigation and management [4] with the highest yield applying to multi-system malformation or complex molecule disorders. The most notable of these is the investigation of mitochondrial respiratory chain (MRC) defects. For these disorders, MPS has become the first-line clinical and health economic approach and has almost made the use of invasive tissue biopsies redundant [5, 6]. The clinical presentations of MRC defects are heterogeneous but commonly include global developmental delay, developmental regression, cardiomyopathy, lactic acidosis, liver failure or facial dysmorphism [7], many of which may be first referred for general paediatric review. Genomics with MPS includes exome sequencing (ES) and whole genome sequencing (WGS). ES is limited to exonic (protein-coding) information but is cheaper and faster to perform. WGS is more sensitive with full genome coverage, including coding and non-coding information, but is more costly, slower and can result in more challenging data interpretation with respect to relevant findings [7]. In Australia, ES is currently more accessible than WGS, but WGS is increasingly utilised overseas [6]. Through a series of cases, we aim to describe how MPS is influencing the diagnosis and management of IEM, including successes, limitations and looking to the future. MPS has revolutionised the diagnostic yield in some areas of paediatric care and reduced the diagnostic odyssey for many families, particularly those presenting with multi-system disorders [7]. Should families wish to pursue this, a genetic diagnosis can provide clarity, optimise management through established evidence, allow families to connect with support/family groups and provide timely reproductive planning information [8]. Dignity in a diagnosis is an important factor for families, as is reproductive confidence. The turnaround time for MPS has considerably improved, and options such as rapid and ultra-rapid ES have become more accessible in Australia. This has reduced the turnaround time to, in some cases, as fast as 2–5 days, opening early diagnostic possibilities in the appropriate setting [9]. As a result of reduced cost and improved technology, there has been rapid expansion in accessing MPS in general paediatric clinical practice. In Australia, in consultation with a clinical geneticist, a Medicare Benefits Schedule (MBS) item number for trio (parents and child) WGS or ES with mitochondrial DNA sequencing is now available to specialist paediatricians for paediatric patients younger than 11 years with multi-system disease and/or moderate to severe global developmental delay or intellectual disability [8]. An MBS item number is also available for suspected mitochondrial disease through WGS or ES and mitochondrial DNA sequencing. The expansion of MPS into general paediatric care highlights the critical need for the integration of genetic counsellors into the paediatric multi-disciplinary care team [8, 10], as the expertise and understanding of the broader implications for families with a new genetic diagnosis is critical [11]. Genetic counsellors bring a deep understanding of the broader implications for families receiving a new genetic diagnosis, and their role is pivotal in facilitating comprehensive discussions regarding reproductive planning options [10]. This is arguably the area with the greatest impact after a new genetic diagnosis in a family. Reproductive planning options might include preimplantation genetic testing or targeted invasive testing of subsequent pregnancies. This may influence pregnancy management or provide an antenatal genetic diagnosis, which allows for preparation for an appropriate delivery environment or potential postnatal complications for an affected newborn [12, 13]. This is described in Section 2.1. A 5-month-old boy presented with global developmental delay, central hypotonia and faltering growth. He had challenges with feeding in the newborn period and regular vomiting since infancy. His length and weight had been down-crossing centile lines despite regular feeds. He was globally developmentally delayed. He was fixing and following but not yet smiling. He was not yet cooing. He could not roll or sit independently. He also had significant drooling requiring a bib and frequent mouth wiping by his parents. This was on a background of an unremarkable antenatal period and delivery, where he was born at full term with an adequate birth weight. Relevant metabolic biochemical investigations included a blood lactate level of 4.8 mmol/L (0.5–2.2) and a mild elevation of malate and fumarate, indicators of mitochondrial dysfunction, present on urine organic acid analysis. Trio ES including mitochondrial DNA sequencing was arranged after his review. Within 4 weeks, he was formally diagnosed with a MRC disorder with homozygous pathogenic variants in the SURF1 (OMIM 185620) gene identified (see Table 1) confirming mitochondrial complex IV deficiency (OMIM 220110). Most MRC defects arise from variants in the nuclear genome, thus the majority of these disorders will be detectable on ES alone. This provided diagnostic clarity and basic prognostic information for the family as well as allowing them to join a targeted family support group. Most importantly for the family, this information could be used for reproductive planning, opting for preimplantation genetic testing for a subsequent pregnancy. Despite the potential for improvement in diagnostic capabilities, the role of rapid MPS is limited in some IEM. This is most apparent in acute small molecule intoxication disorders, such as urea cycle disorders and organic acidurias, where metabolic biochemical investigations provide the most rapid diagnosis [4]. Management needs to be instigated well prior to even the speediest of genetic testing results. In these disorders, point-of-care metabolic investigations remain critical in providing fast and accurate diagnoses, which facilitate urgent management. These investigations might include venous blood gas, plasma amino acids, plasma acylcarnitine profile, plasma ammonia level and a urine metabolic screen including organic acids and amino acids. MPS comes with its own inherent limitations, including current genetic understanding or knowledge and the identification of inconclusive findings such as variants of uncertain significance (VUS) [7]. Caution should be advised as medical teams and families may place additional weight on MPS results in the hope of finding a clear cause of a presentation, which may ultimately yield a negative or inconclusive result [8]. This may delay important and timely clinical management decisions. Additionally, a clear genetic diagnosis should not solely influence the offering of an acute intervention, making deferral of management decisions unnecessary whilst awaiting genetic testing results [14]. It is critical that families are aware of these limitations through the consent process before genetic testing is requested [8]. Genetic counsellors hold key skills in these areas [11, 15]. This is described in Section 3.1. A term infant presented to the emergency department at 5 days of age with tachypnoea and increased work of breathing. Her grandmother was unwell with a viral upper respiratory tract infection leading to a presumed diagnosis of viral bronchiolitis. Multiple venous blood gases identified a high anion gap metabolic acidosis and raised suspicion for an inborn error of metabolism (see Table 2). An ammonia level confirmed severe hyperammonaemia of 1128 μmol/L (< 50 μmol/L). This led to a primary differential diagnosis of an acute small molecule intoxication disorder, and an organic aciduria was suspected. Severe hyperammonaemia is a metabolic emergency and fatal if untreated. The patient was managed with haemofiltration and ammonia scavenging medications. Intramuscular vitamin B12 was administered and she was commenced on levocarnitine. She clinically improved gradually over the following week. Metabolic biochemical investigations confirmed a diagnosis of methylmalonic aciduria (MMA), an organic aciduria, with raised 3-hydroxypropionate and methylcitrate on urine organic acid testing and raised propionylcarnitine (C3) on plasma acylcarnitine profile. The plasma methylmalonic acid level was elevated at 340 μmol/L (< 1.5 μmol/L). The patient's conventional newborn bloodspot screening (NBS) returned soon after presentation with a raised C3 suggesting possible MMA, with differential diagnoses extending to propionic aciduria and vitamin B12 deficiency. She was managed successfully with this rapid diagnosis and discharged from hospital on day 19 of life. Using MPS, an organic aciduria gene panel was performed and confirmed compound heterozygous variants in the MMAA (OMIM 607481) gene (see Table 3). This genetic testing returned 5 weeks after the initial presentation; however, the metabolic biochemistry had been diagnostic of MMA during her inpatient admission. The diagnosis was thus made of MMA on metabolic biochemical testing, with genetic confirmation of cobalamin A disease (OMIM 251100), a subtype of MMA involving the MMAA gene. Despite this result including a VUS in the MMAA gene, the metabolic biochemical evidence complements the genetic findings, confirming cobalamin A disease. This has since led to the upgrade of the VUS to a classification of pathogenic. Some of the limitations of genomic diagnosis in IEM can be addressed through a spectrum of approaches, termed ‘multi-omics’. This includes genomics with MPS, but also other data-driven components such as transcriptomics, metabolomics and proteomics. The ‘multi-omics’ approach adds diagnostic yield to genomics alone and aims to clarify inconclusive genomic findings such as VUS and new gene-disease associations [7], such as in this case with targeted metabolomic information. These methods examine transcript expression, metabolite production [16] and protein function to investigate gene function and the impact of variants at the cellular or protein level [7]. In IEM, metabolomics allows for a unique diagnostic contributor in addition to genomic information. Conventional NBS has a strong uptake in Australia, of over 99% [17], where IEMs are the main beneficiaries of current NBS programmes. NBS programmes are state-based and vary based on location but broadly screen for approximately 30 conditions [18]. These conditions are selected using the foundational principles of public health screening programmes devised by Wilson and Jungner. These include a specific and reliable test, well-understood health outcomes, and effectiveness and availability of treatment options [19]. The timely return of conventional NBS is critical in the management of neonates with certain IEMs as they require urgent intervention to avoid early metabolic decompensation. With pilot programmes of genomic newborn screening (gNBS) on the horizon, it is important that clinicians are aware of the possible outcomes of these. The stated goals of gNBS include early genetic diagnosis of treatable disorders to optimise early management [20]. The panel of disorders is much broader and, in some cases, encompasses hundreds of conditions. Critically, however, these target disorders must benefit from intervention and have available evidence-based management options [19]. Defining a treatable disorder is challenging [21] with differing views amongst clinicians and a differing strength of evidence across possible interventions. Factors such as the cost, availability and efficacy of treatments need to be considered carefully when defining these disorders, as does the scope of intervention burden such as administration route, frequency and risk of side effects of medications [22]. Consultation with appropriate sub-specialty expert clinician stakeholders is essential to ensure these are genuinely treatable conditions and that due weight is provided to the potential treatment burdens in balancing the benefits and harms of proposed interventions [23]. An example of this, for IEM, is the lysosomal storage disorder Krabbe disease (OMIM 245200). Krabbe disease is an autosomal recessive disease caused by biallelic pathogenic variants of the GALC (OMIM 245200) gene. This leads to a deficiency of the enzyme galactocerebrosidase, causing the accumulation of toxic metabolites in the neuronal myelin sheath with progressive central neurodegeneration and peripheral neuropathy [24]. The only intervention beyond symptomatic care currently offered is haematopoietic stem cell transplant (HSCT). HSCT aims to replace the deficient enzyme with that of the donor cells. It is a costly and intensive intervention. The evidence remains controversial on the balance of benefits and harms in treating Krabbe disease with HSCT, as HSCT comes with a 10%–20% risk of mortality and significant associated morbidity [25]. Treatment with HSCT for late-onset Krabbe disease appears to improve overall survival [25] but developmental outcomes remain significantly impacted, particularly in gross and fine motor skills [26]. Whilst intervention in the pre-symptomatic early-onset cohort does show some improved outcomes [27] compared to the late-onset group, the added complexity is that the identification with gNBS will not delineate early-onset compared to late-onset disease [24]. It is also recognised that the long-term outcomes for patients in the early-onset cohort remain uncertain [28]. Additionally, newborns with an IEM requiring urgent clinical management through dietary changes or the introduction of specialised formulas or medications are likely to present well before gNBS test results return, highlighting the importance that conventional NBS continues. Consent for participation in gNBS programmes must be thorough to ensure that trust in conventional newborn screening is not undermined in this process [29]. For IEM, the unique benefits of gNBS include the clarification of carrier status for conditions where conventional NBS results remain unclear. This is exemplified in certain fatty acid oxidation disorders such as very long-chain acyl-CoA dehydrogenase deficiency (VLCAD) (OMIM 609575) where conventional NBS can demonstrate a characteristic acylcarnitine profile pattern of raised C14:1 and C14, irrespective of whether the newborn is a genetic carrier or is affected by the disorder [30]. After a positive screen for VLCAD on conventional NBS, with equivalent results on second-tier biochemical investigations, MPS would usually be used for clarification. With gNBS, this outcome would be expedited. The potential benefits of gNBS also extend to reproductive planning for families for subsequent pregnancies; however, the broader utility in this domain is preconception reproductive carrier screening where couples are screened for their genetic carrier status for multiple conditions, with many IEMs included, before pregnancy [12]. This has been shown to be feasible in a broad population group and an acceptable option for couples and practitioners [31]. The available MBS item number for reproductive carrier screening for couples who are pregnant or considering pregnancy currently includes spinal muscular atrophy, cystic fibrosis and Fragile X syndrome. This is limited in both the number of conditions and in the breadth of genetic variants tested. For example, for cystic fibrosis carrier screening, approximately 50 of the most common CFTR (OMIM 602421) gene variants are included out of many hundreds of known pathogenic variants [13]. This also leads to data biases with these variants being most common in the Caucasian population [32, 33]. There is scope for preconception reproductive carrier screening to be expanded, and equitable access will be an important focus [12]. This is the key target for reducing the future diagnosis of IEMs and should be offered to all families planning a pregnancy with appropriate counselling and understanding of the implications of the testing. Most importantly, parents should be able to decline participation in gNBS and remain interested in conventional NBS, with adequate understanding of the fundamental differences in these types of NBS programmes. This is described in Section 4.2. A newborn had gNBS performed in a research setting, returning a positive screen for Pompe disease (OMIM 232300) with variants in the GAA gene (OMIM 606800) flagged. Pompe disease is a glycogen storage disease that leads to progressive muscle weakness and cardiomyopathy. It is an autosomal recessive disorder caused by deficiency of the enzyme alpha-glucosidase [36]. However, it has a variable age of onset. It has two phenotypes, presenting with infantile-onset disease or adult-onset disease, with no differentiation possible based on genotype [37]. The newborn's parents are informed of this result and asked to present for review. The infant examines well with a normal cardiovascular examination. Biochemical investigations are requested, and he has a mildly elevated creatinine kinase (CK), indicating evidence of muscle involvement. Parental segregation testing was arranged, and this confirmed maternal inheritance of both variants (with no paternally inherited variant), confirming these variants are found to be in cis (on the same allele) for both the infant and the mother; thus, the infant is a carrier of Pompe disease only. He is not affected by the disease. The elevated CK normalised on repeat testing and was not further investigated. The parents have challenges bonding with their newborn baby and the newborn period has been considerably compromised. From a resourcing perspective, this required three specialist appointments, biochemical and genetic testing samples, and associated tangible and emotional costs. The family are now aware of his carrier status and the baby's right to an open future has been impacted. Additionally, if found to be affected by the disease, the variability in the age of onset would have led to uncertainty for the family and the baby's future. This also raises the consideration of pre-symptomatic enzyme replacement therapy, a regular intravenous infusion of considerable cost [36], which may not prevent the onset of disease. The evidence for efficacy is stronger in the prevention of progression of disease after the identification of symptoms, as compared to preventing the initial onset of symptoms in Pompe disease [36, 37]. The evidence remains unclear on the role of pre-symptomatic enzyme replacement therapy and comes at a cost and burden to the health system, patient and family. In summary, the expansion of MPS has revolutionised many parts of paediatric practice and will continue to play a key role in diagnosis in IEM, but is limited in some settings, such as in acute small molecule intoxication. Diagnosis of IEM includes a combination of clinical, phenotypic, biochemical and genetic information with a ‘multi-omics’ diagnostic approach complementing genomic findings and providing unique diagnostic information [7]. In this expanding use of MPS, remaining of limitations is including possible inconclusive and negative results [8]. This highlights the of genetic counsellors into the relevant multi-disciplinary as a benefit of MPS in IEM is the potential utility for reproductive planning [10]. IEM are the main beneficiaries of newborn screening, and the of gNBS has the potential to the clinical utility of current screening [29]. and consultation with key health and patient groups are to delineate is a genuinely treatable disorder for gNBS and to ensure due consideration of the relevant implications of gNBS Most importantly, the key focus for the future of IEM will be in access and equitable expansion of preconception reproductive carrier The no of is not to this as no new data or in this

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