Molecular and genetic diagnoses of the index case
First, the analyses of enzyme activities and oxygen consumption rates were performed for patients who were suspected to have mitochondrial diseases. Thereafter, whole mtDNA and whole exome sequencing and validation tests were performed. Genetic counseling before and after prenatal diagnosis, CVS, and AC were performed at the Department of Pediatrics and Department of Obstetrics and Gynecology, Saitama Medical University Hospital. The obtained samples were analyzed at Juntendo University.
Prenatal diagnosis
In total, 13 probands from 13 families were diagnosed with severe mitochondrial diseases caused by pathogenic variants in nDNA. Most of the probands had homozygous or compound heterozygous variants in the autosomal recessive genes (ACAD9 [MIM 611103] (n = 1), BOLA3 [MIM 613183] (n = 5), ECHS1 [MIM 602292] (n = 1), MPV17 [MIM 256810] (n = 2), NDUFAF6 [MIM 612392] (n = 1), RARS2 [MIM 611524] (n = 1), TUFM [MIM 602389] (n = 1)). Only one proband had a hemizygous variant in the X chromosome-linked gene (TAZ [MIM 300394] (n = 1)). The details of each family are described in the following section and Table 1. The 13 families (18 cases) decided to undergo prenatal diagnosis.
Among the 18 pregnancies, 2 resulted in early miscarriage. In the remaining 16 cases that reached the second trimester of pregnancy, 9 opted to have CVS, and 6 opted for AC. One case showed possible maternal cell contamination in the CVS sample and subsequently underwent AC. Each causative nuclear gene variant was confirmed by direct Sanger sequencing of PCR-amplified products.
Index case profiles (Table 1, Fig 2)
Family #1 (Fig 2-a): Proband (II-1) was a female born at full term (birth weight of 3.10 kg, within the normal range) as the first child to non-consanguineous healthy parents. She exhibited cardiomyopathy with lactic acidosis at 2 months of age, developed heart and liver failure, and died at 5 months of age. Genetic analysis revealed pathogenic variants in BOLA3 (c.287A>G/c.287A>G); additionally, both parents were heterozygous carriers of this variant.
Family #2 (Fig 2-b): Proband (II-1) was a female born at full term (birth weight of 3.29 kg, within the normal range). She exhibited feeding difficulty at 24 days of age. Laboratory studies revealed severe lactic acidosis, hyperglycemia, and metabolic acidosis; as a result, she was admitted to an intensive care unit. Moreover, she manifested cardiomyopathy, apnea, epilepsy, and liver failure and died of heart failure at 7 months of age. Genetic analysis revealed that she harbored pathogenic variants in BOLA3 (c.287A>G/c.287A>G), and both parents were heterozygous carriers of this variant.
Family #3 (Fig 2-c): Proband (II-3) was a male born at full term. His elder brother died at 4 months of age, but details were not available. The proband developed lactic acidosis, hyperglycemia, and an elevated creatine kinase level at 17 days of age. He subsequently showed muscle weakness, hypotonia, and cardiomyopathy and died at 17 months of age. Genetic analysis identified pathogenic variants in BOLA3 (c.287A>G/c.287A>G), and both parents were heterozygous carriers of this variant. Furthermore, he had homozygous variants in FKTN. The patient has been reported in a previous study.12
Family #4 (Fig 2-d): Proband (II-2) was a female born at full term (birth weight of 2.55 kg, within the normal range). Her elder sister, who had hypotonia with cardiomyopathy, died of acute bronchitis at 6 months of age. The proband was reported to have hypotonia at 5 months of age and exhibited lactic acidosis and hypertrophic cardiomyopathy. Thereafter, she died of acute myocarditis at 13 months of age. She had two different pathogenic variants in ACAD9 (c.1766-2A>G/c.811T>G), and both parents were carriers of either of these variants.
Family #5 (Fig 2-e): Proband (II-1) was a female born at full term (birth weight of 2.93 kg, within the normal range). Regression and lactic acidosis appeared at 7 months of age. She was diagnosed with Leigh syndrome and died of respiratory failure at 5 years of age. Genetic analysis identified two different pathogenic variants in NDUFAF6 (c.805C>G/c.226T>C), and both parents were carriers of either of these variants.
Family #6 (Fig 2-f): This family had two probands. Proband (II-1) was a male born at 34 weeks and 2 days (birth weight of 0.81 kg, –4.4 SD). On the first day, lactic acidosis and apnea were observed, and he died at 8 days of age. Proband (II-2) was the second male child born at 36 weeks and 6 days (birth weight of 1.06 kg; –5.1 SD). He had congenital heart disease and aortic coarctation and ventricular septal defect. He died of multiple organ failure at 20 days of age. Genetic analysis identified two different pathogenic variants in TUFM (c.440T>A/c.162delC), and both parents were carriers of either of these variants.
Family #7 (Fig 2-g): Proband (III-2) was a male born at full term (birth weight of 2.59 kg; within the normal range). Five maternal cousins of his mother (II-5, II-6, II-7, II-8, and II-9) died in the fetal period or during early infancy. Detailed information about them was not available. Hypertrophic cardiomyopathy with cyanosis and respiratory failure occurred in the proband at 8 months of age. Genetic analysis identified a pathogenic variant in TAZ (X chromosome-linked gene) (c.367C>T), and his mother was a carrier of this variant.
Family #8 (Fig 2-h): Proband (II-2) was a female born at 39 weeks of gestation (birth weight of 2.94 kg, within the normal range). Her elder sister died of respiratory failure with severe lactic acidosis at 1 day of age. The proband exhibited moderate lactic acidosis and hypotonia shortly after birth. Lactic acidosis was improved by medical treatment; however, she developed severe respiratory and cardiac failure resulting in death at 124 days of age. Metabolic profiling showed no abnormal findings. The brain magnetic resonance imaging at 8 days of age showed T2 high intensities in the cerebral white matter and diffuse brain atrophy at 58 days of age. An autopsy was performed after death. Genetic analysis identified two different pathogenic variants in ECHS1 (c176A>G/c.476A>G), and both parents were carriers of either of these variants.
Family #9 (Fig 2-i): Proband (II-1) was a male born at full term (birth weight of 3.2 kg, within the normal range). Poor weight gain and cholestasis were noticed at 1 month of age. The liver showed microvesicular steatosis with swollen hepatocytes and partial necrosis. His jaundice progressed and liver transplantation was performed at 11 months of age. He died of pulmonary hypertension at 2 years and 9 months of age. Genetic analysis identified pathogenic variant in MPV17 (c.451dupC/c.451dupC), and both parents were heterozygous carriers of this variant. The proband has been reported in a previous study13 (family 18, subject 20).
Family #10 (Fig 2-j): Proband (II-2) was a female born at full term (birth weight of 2.42 kg, within the normal range). She exhibited cardiomyopathy with lactic acidosis at 2 days post birth. Thereafter, her cardiomyopathy progressed to heart failure, and liver failure occurred. Genetic analysis identified pathogenic variant in RARS2 (c.944G>C/c.944G>C), and both parents were heterozygous carriers of this variant.
Family #11 (Fig 2-k): Proband (II-1) was a male born at full term (birth weight of 2.69 kg, within the normal range). He showed tachypnea due to respiratory distress syndrome shortly after birth and was treated with mechanical ventilation for 3 days. Hypotonia and poor weight gain was observed at 3 months of age. Laboratory examinations showed elevated levels of liver transaminases and direct bilirubin. He died of liver failure at 10 months of age. Genetic analysis identified two different pathogenic variants in MPV17 (c.451dupC/c.308_310del), and both parents were carriers of either of these variants.
Family #12 (Fig 1-l): Proband (II-1) was a male born at full term. He developed impaired consciousness and cardiomyopathy with lactic acidosis at 4 months of age. He died 15 days later. Genetic analysis identified pathogenic variant in BOLA3 (c.287A>G/ c.287A>G), and his mother was a carrier of this variant.
Family #13 (Fig 1-m): Proband (II-1) was a male born at full term (birth weight of 2.41 kg, within the normal range). Regression appeared at 4 months of age. Additionally, he showed hypotonia and horizontal nystagmus. Symmetrical white matter lesions and lactate peaks were observed in the brain magnetic resonance imaging and magnetic resonance spectroscopy, respectively. Cardiomyopathy had developed and progressed rapidly, and he died at 6 months of age. Genetic analysis identified two different pathogenic variants in BOLA3 (c.287A>G/ c.136C>T), and both parents were carriers of either of these variants.