Arrest of Fetal Brain Development in ALG11-Congenital Disorder of Glycosylation

Abstract

Background:

Arrest of fetal brain development and the fetal brain disruption sequence describe a severe phenotype involving microcephaly, occipital bone prominence, and scalp rugae. Congenital disorders of glycosylation are a heterogeneous group of inherited disorders involved in glycoprotein and glycolipid biosynthesis which can cause microcephaly and severe neurodevelopmental disability.

Methods:

We report a case of fetal microcephaly diagnosed at 36 weeks gestation with a history of normal fetal biometry at 20 weeks gestation. Postnatal genetic testing was performed.

Results:

Fetal magnetic resonance imaging at 36 weeks gestational age showed severe cortical thinning with a simplified gyral pattern for gestational age, ventriculomegaly, and agenesis of the corpus callosum. The fetal skull had a posterior shelf at the level of the lambdoid suture, characteristic of fetal brain disruption sequence. Postnatal brain magnetic resonance imaging found no brain growth during the interval from the fetal to postnatal study. The infant was found to have biallelic pathological mutations in ALG11.

Conclusion:

Arrest of fetal brain development, with image findings consistent with fetal brain disruption sequence, is a previously unreported phenotype of congenital microcephaly in ALG11- congenital disorder of glycosylation. ALG-11 congenital disorder of glycosylation should be considered in the differential diagnosis of this rare form of congenital microcephaly.

INTRODUCTION

Fetal brain disruption sequence is a severe neurologic phenotype consisting of microcephaly, occipital bone prominence, and scalp rugae.[1] In congenital microcephaly resulting from fetal brain disruption sequence, the mechanism of the skull collapse is thought to result from decreased intracranial hydrostatic pressure associated with a disruption or “arrest” of brain growth usually in the second or third trimester when the fetal brain volume normally has significant increase.[1,2] This phenotype occurs from congenital infections including Zika virus,[3] fetal cerebral injury from vascular insults or maternal trauma, and in genetic conditions.[4] A similar term, “fetal brain arrest” has been proposed to refer to causes not associated with brain injury or infection, but which are felt to result from a genetic mutation.[4]

Congenital disorders of glycosylation (CDG) are a heterogeneous group of monogenic disorders involved in glycoprotein and glycolipid biosynthesis.[5,6] Affected infants have multisystemic abnormalities and often have neurologic phenotypes which include microcephaly, seizures, hypotonia, cerebellar hypoplasia, and severe intellectual disability.[7–8] ALG11-CDG results from a deficiency of GDP-mannose:Man₃GlcNAc₂-PP-dolichol/Man₄GlcNAc₂-PPdolichol α−1,2-mannosyltransferase, the enzyme that catalyzes the addition of the fourth and fifth mannoses to the oligosaccharide precursor.[9] It was first described in a patient in 2010 with neurocognitive delays and epilepsy (Table).[10]The phenotype of ALG11-CDG was further characterized as severe psychomotor disability, progressive microcephaly, and sensorineural hearing loss (Table).[11,12]

Table:

Clinical features, mutations, and brain MRI findings in 11 reported cases of ALG11-CDG.

Clinical Feature	Rind 2010 [10] (pt 1)	Rind 2010 [10](pt 2)	Thiel 2012 [11] (pt 1)	Thiel 2012 [11] (pt 2)	Thiel 2012 [11] (pt 3)	Regal 2015 [12] (pt 1)	Regal 2015 [12] (pt 2)	Al Teneiji 2017 [18] (pt 1)	Al Teneiji 2017 [18] (pt 2)	Pereira 2017 [19]	Present report
Age at presentation	1 week	6 weeks	Infancy	Infancy	Infancy	Birth at term	3 months of age	4 months of age	4 months of age	Early life	Fetal, 36 weeks gestation
Gender	Female#	Male#	Female	Female	Male	Male	Male	Female	Female	Female	Male
Microcephaly	+	NA	+	NA	−	+	+	+	+	+	+
Epilepsy	+	+	+	+	+	+	+	+	+	+	−
Hypotonia	Axial	+	Axial	Axial	Axial	Axial	Axial	+	+	+	+
Hypertonia	−	−	NA	NA	NA	Peripheral	Peripheral	NA	NA	NA	−
Global developmental delay	+	+	+	+	+	+	+	+	+	+	+
Eye/visual problems	+	NA	+	+	+	+	+	+	+	NA	+
Feeding problems	+	+	−	−	+	+	−	+	−	−	+
Outcome	Died at 2 years	NA	Alive at 7 years	Alive at 4.5 years	Alive at 8.5 years	Died at 3 years	Died at 4months	Alive at 7 years	Alive at 4 years	Alive at 6 years	Alive at 20 months
Mutations: cDNA (NM_001004127.2)/ protein	Hmz c.257T>C/ p.L86S	Hmz c.257T>C/ p.L86S	c.623_642de l/p.S208Yfs*4; c.836A>C/ p.Y279S	c.1142T>C/ p.L381S; c.1192G>A/ p.E398K	Hmz c.953A>C/ p.Q318P	c.479G>T/ p.G160V; c.45–2A>T	c.479G>T/ p.G160V; c.36dupG/ p.L13Vfs*44	Hmz c.1241T>A/ p.I414N	Hmz c.1123_1126de lAACA/ p.N375Ffs*6	NA	c.44G>C/ p.R15T; c.161C>T/ p.S54L
Brain MRI	NA	NA	Cerebral atrophy and abnormal white matter	NA	NA	Cerebral atrophy, subcortical heteromiddleia, delayed myelination, simplified gyral pattern	Cerebral atrophy	Hypomyelinati on, brain atrophy, thin corpus callosum	Cerebral atrophy	Enlarged subarach noid spaces; normal myelinati on and posterior fossa	Arrest of brain growth, agenesis of the corpus callosum, simplified gyral pattern
Other features	Temperature instability, deafness, inverted nipples, abnormal fat pads	Deafness		Oscillations of body temperature, inverted nipples		Sloping forehead, micrognathia	Burst suppression EEG, cochlear hearing loss	Inverted nipples, abnormal fat distribution		Epileptic spasms at 5 months of age	Non-epileptic breath-holding spells, inverted nipples

Abbreviations: + = present; − = absent; NA = not available;

^# = siblings; hmz = homozygous

We present the first reported fetal imaging of arrest of fetal brain development in a patient with postnatal diagnosis of ALG11-CDG. This report shows the development of microcephaly in ALG11-CDG during the late second and third trimester and with a phenotype consistent with arrest of fetal brain development/fetal brain disruption sequence.

CASE PRESENTATION

A 30-year-old primigravida woman had a normal fetal anatomy obstetrical ultrasound (US) at 20 weeks gestation with fetal head circumference (HC) and biparietal diameter (BPD) both at the 75^th percentile for gestational age. Specifically, the cavum septum pellucidum was visualized and the lateral cerebral ventricles had a normal configuration with a measurement of 6mm at the level of the atria. At 36 0/7 weeks gestation she had a follow-up obstetrical US due to lower than expected fundal height which found microcephaly. She was referred to a maternal fetal medicine specialist, who by US described a male fetus in breech presentation with HC measuring 303 mm (consistent with 33 1/7 weeks) and BPD measuring 76 mm (consistent with 30 4/7 weeks), both measurements less than 3^rd percentile for gestational age. She was then referred to a Fetal Medicine Institute for fetal magnetic resonance imaging (MRI) and fetal neurology consultation.

Fetal US at the Fetal Medicine Institute (36 0/7 weeks) showed severe microcephaly, moderate lateral ventriculomegaly, redundant soft tissue around the occipital cranium, amniotic fluid index of 8, with an estimated fetal weight at the 39th percentile for gestational age. HC (282 mm, consistent with 30 6/7 weeks) and BPD (75 mm, consistent with 30 0/7 weeks) were <1^st percentile for gestational age. Fetal MRI revealed severe cortical hypoplasia with a simplified gyral pattern, under rotated left hippocampus, moderate lateral ventriculomegaly measuring 14 mm in diameter at the level of the atria, complete agenesis of the corpus callosum, a collapse of the posterior occipital skull (Figure 1). The posterior fossa structures and the brainstem were of normal configuration; however, measurements of the transverse cerebellar diameter and height of the vermis were smaller than expected for age. No other body abnormalities were described by MRI or US.

Figure 1. Clinical imaging of patient with ALG11-CDG.

(A) Sagittal single shot fast spinecho (SSFSE) T2-weighted image through the midline, fetus (36 weeks gestational age). The skull is severely microcephalic, with a posterior shelf at the level of the lambdoid suture, characteristic of fetal brain disruption sequence. (B) Coronal SSFSE T2, fetus. The corpus callosum is absent. The cerebral hemispheres appear severely hypoplastic; the cerebral biparietal diameter is approximately 53 mm (expected range for normal fetus of 25–26 weeks gestational age).[23] (C) Coronal fast spin-echo (FSE) T2 image at post-natal age of 16 days (3.5 weeks post fetal MRI). The cerebrum has a similar configuration to the fetal image (B), except for mild interval enlargement of temporal horns. The cerebral biparietal diameter is unchanged at approximately 53 mm, reflecting a complete lack of growth of the cerebrum since the fetal MRI. The sagittal diameters of the cerebrum were also unchanged (not shown). (D) Sagittal FSE T2 through the right cerebral hemisphere neonate. A band tissue with low T2 signal tissue (arrows) is evident between the subcortical white matter and in the lining of the lateral ventricle, consistent with neuronal elements of the germinal matrix that have failed to migrate. (E) Axial spin-echo T1 through the upper cerebral hemispheres, neonate. A band of mildly increased T1 signal is evident deep to the subcortical white matter bilaterally (arrows), which corresponds to the band of low T2 signal noted on the sagittal image (D), consistent with neuronal elements that have failed to migrate. A slightly hyperintense subdural collection is evident behind the left cerebral hemisphere. (F) Profile photo of infant at two weeks of age showing microcephaly, posterior skull protuberance, and scalp rugae.

The couple is Caucasian and are non-consanguineous. There was no family history of neurologic abnormalities or significant childhood illnesses. The couple had no recent travel to areas of endemic transmission of Zika virus. Maternal CMV IgM and IgG and Zika virus IgM and PCR were negative at 36 weeks gestation.

A boy infant was born via cesarean section due to oligohydramnios and breech presentation at 37 1/7 weeks gestation. Birth weight was 2879 grams and head circumference was 32 cm (<1^st percentile). Apgar scores were 9 and 9 at 1 and 5 minutes, respectively. On exam, he was microcephalic, the anterior fontanelle was open and flat, and there was a palpable bony protuberance at the back of his head. He had normal newborn reflexes and no abnormal activity. Hearing screen was abnormal in both ears. He breastfed and was discharged home at three days of age. Outpatient neurology examination at three days of age described a microcephalic infant with a flat and short forehead, a bony prominence at the occiput, and a small anterior fontanelle (Figure 1). There was redundant skin over the scalp and at the back of the neck. He was awake, had normal eye movements without nystagmus, pupils were equal and reactive to light, he had a normal suck and gag reflex, full facial movements, full strength with symmetric movements, some slip through and draping over on vertical and horizontal suspension, normal muscle stretch reflexes in upper and lower extremities, upgoing plantar response, no ankle clonus, and normal palmar and plantar grasp reflex. He also had downslanting palpebral fissures, anteverted nares, low set ears with prominent lobes, and inverted nipples.

Postnatal brain MRI at 16 days of age (3.5 weeks following the fetal MRI) showed microcephaly with cerebral hypoplasia, a simplified gyral pattern, agenesis of the corpus callosum, and no apparent growth of the cerebrum since the fetal MRI study (Figure 1). The ventral pons and cerebellar vermis, although normally formed, were smaller than expected for gestational age. The MRI also showed thin bands of decreased T1/T2 relaxation in the deep parietal and frontal white matter, extending into the subcortical regions, which may represent failed migration of neuronal elements.

At three weeks of age he had periods of fussiness accompanied by breath-holding spells (Table). The events were brief and self-resolved. An electroencephalogram was not performed at that time. At six weeks of age he had increasing irritability with feeding. A fluoroscopy swallow study showed dysfunctional and delayed swallowing with esophageal reflux and silent aspiration so he was transitioned to full nasogastric tube feedings. He has also had intermittent episodes of periodic breathing and poor control of body temperature.

Genetic Testing

Chromosomal microarray was negative. Trio whole exome sequencing found compound heterozygous variants in trans in ALG11; one variant was maternally inherited (NM_001004127.2:c.44G>C/p.Arg15Thr) and one variant paternally inherited (c.161C>T/p.Ser54Leu). Both variants are exceedingly rare, with an allele frequency for the former of 14/276,856 alleles, while the latter allele is not present in gnomAD. No other variants potentially associated with the phenotype had been reported. The R15T variant occurs at a position that is conserved in mammals, and in silico analysis predicted this variant is probably damaging to the protein structure; however, the S54L substitution occurs at a position that is not conserved, and in silico algorithms were inconsistent as to whether the variant is damaging to the protein. Thus, additional information was needed to validate the impact of these variants on protein glycosylation. Analysis of serum transferrin glycosylation status and total serum N-glycan analysis are often employed, but these broad biomarkers were both normal. Western blot analysis of ALG11 protein in patient fibroblasts was also normal.

Validation required direct functional analyses on patient fibroblasts in a research laboratory. The first step was to examine hypoglycosylation of the cellular biomarker, GP130.[13] The second step was a determination of the size of the lipid-linked oligosaccharide (LLO) precursor of N-glycans.[11] Functional mutations in ALG11 are predicted to generate a truncated LLO. In patient cells, GP130 was clearly underglycosylated and truncated LLO consistent with impaired ALG11 activity was also detected confirming the impact of the variant on protein glycosylation (Figure 2, A-C).[14,15]

Figure 2. LLO and western blot.

Lipid-linked Oligosaccharide (Fig 2 A-B): Decreased Glc₃Man₉GlcNAc₂-PP-dolichol and increased Man₃GlcNAc₂-PP-dolichol and Man₄GlcNAc₂-PP-dolichol in patient (B-CDG-1077) as compared to control (A-GM-3348), indicating a block in the addition of the fourth and fifth mannoses to the lipid-linked oligosaccharide— reactions catalyzed by ALG11. [GlcNAc (N), Mannose (M), Glucose (G)](Fig 2 C) Western Blot + GP130: ALG11 protein expression was unaltered. Hypoglycosylation of GP130 was detected in fibroblasts from the patient as judged by SDS-PAGE migration detected by Western blot. Control = GM3348; patient = CDG1077.

DISCUSSION

We report a patient with arrest of fetal brain development in a pregnancy with prior normal fetal biometry, for an infant found to have ALG11-CDG. Given the phenotype of skull collapse with occipital bone prominence and significant simplification of the gyral pattern seen at 36 weeks, the suspected onset of the arrested fetal brain development was during the latter part of the second trimester, around 28 weeks gestation. The end of the second trimester coincides with a time of expansion of the volume of the cerebral cortex and increasing complexity of the cortical folding pattern. The failure of this occurrence in this patient with ALG11-CDG is also supported by the MRI findings of thin bands of decreased T1/T2 relaxation in the deep parietal and frontal white matter, which may represent disrupted neuronal cells. The additional finding of agenesis of the corpus callosum indicates a severe disruption of brain development. During the 3.5 week interval from the fetal MRI to the postnatal brain MRI, the brain did not increase in size indicating an arrested growth pattern. This should be differentiated from a decrease in size, or atrophy, which did not occur during that same time period. It is not known how a deficiency of ALG11 activity caused an arrest of fetal brain development at this particular stage, although it can be postulated that the mutation disrupted cellular maturation of immature oligodendrocytes resulting in an arrest of brain growth at this particular stage. No additional postnatal neuroimaging was performed in this case. Other reported cases of ALG11-CDG report findings of cerebral atrophy (i.e. low cerebral volume compared to normative values) and microcephaly (Table), [11,12] and based on our case we anticipate that the other cases similarly have an arrested growth pattern.

Although the cerebellar volume was low for gestational age in our case, it was normally formed, and the low measurement was felt to be secondary to reduced cerebral volume rather than primary hypoplasia. Cerebellar atrophy with significant loss of Purkinje and granular cells appears to be a prominent neurologic feature and has been found by neuropathology in PMM2-CDG (formerly CDG 1a).[16] While cerebellar atrophy is usually found by postnatal imaging in PMM2-CDG, a prenatal diagnosis has been reported from cordocentesis at 27 weeks gestation in a fetus with non-immune hydrops.[17] Cerebellar atrophy to the extent seen in PMM2-CDG, does not seem to be a consistent neuroimaging feature of ALG11-CDG (Table).

The skull finding associated with arrest of fetal brain development has been seen in other suspected autosomal recessive genetic conditions. Abdel-Salam et al, reported four patients with severe microcephaly and developmental impairment in consanguineous families with this skull finding; however a precise genetic mutation was not identified.[4] Similar cases likely with potential genetic origin were reported by Moore CA et al, with varying degrees of cerebral destruction.[2] The phenotype of skull collapse/occipital bone prominence is infrequently seen in cases of congenital microcephaly likely due to most genetic conditions causing a more slowly progressive reduction in fetal brain growth trajectory. This case may represent a more severe phenotype of microcephaly in ALG11-CDG since the onset of microcephaly was prior to birth, whereas other reported cases of ALG11-CDG have a postnatal presentation of microcephaly (Table).[10–12, 18, 19]

Our patient presented at the height of the Zika epidemic and despite absence of an epidemiological link for Zika infection in the mother, Zika testing was performed and was negative. In cases of microcephaly from congenital Zika syndrome an irregular skull contour, similar to that seen in our case, could be detected even prior to 26 weeks gestation.[20] Brain abnormalities in congenital ZIKV infection are often more destructive and associated with cortical dysplasia and polymicrogyria, which is not a feature of ALG11-CDG. Thus, given the similarity in the brain findings with congenital Zika syndrome, genetic etiologies should be considered even if there is an epidemiologic link to Zika infection in the absence of positive maternal/newborn testing.

Interestingly, serum N-glycan analysis and carbohydrate deficient transferrin were normal, but the novel GP130 biomarker revealed cellular hypoglycosylation, while LLO analysis was consistent with ALG11-CDG. It is important to note that a normal screening test via carbohydrate deficient transferrin does not rule out an N-linked CDG, and in fact even the most common form of the disease, PMM2-CDG, can be associated with a normal serum result.[21,22]

Conclusion

The finding of severe microcephaly, scalp rugae, occipital bone prominence with skull collapse is in-line with the description by Russell et al. of fetal brain disruption sequence which has not previously been shown to occur in a CDG.[1] ALG11-CDG should therefore be considered in the differential diagnosis of this infrequent skull finding in a patient with severe microcephaly.