Genotype-phenotype correlations and expansion of the molecular spectrum of AP4M1-related hereditary spastic paraplegia

We have studied a Greek family (Fig. 1) composed of three affected siblings born to healthy parents, who presented with complicated HSP. History of previous neurological disease was unremarkable in the family and the pedigree suggested an autosomal recessive inheritance. Thorough neurological examination and follow-up were carried out at the Departments of Neurology of the AHEPA Hospital and Papageorgiou Hospital by some of the authors (GX, TK, GD, MS, TB and HH). This study was approved by the UCLH institutional-research-board. After informed consent, we collected blood samples from the patients and their parents, and extracted DNA using standard procedures. Additional informed consent was obtained from all individual participants for whom identifying information is included in this manuscript.

To investigate the genetic cause of the disease in this family, whole-exome sequencing (WES) was performed in the proband (Fig. 1b: II-3) and his father (Fig.1b: I-1). Nextera Rapid Capture Enrichment kit (Illumina) was used according to the manufacturer instructions. Libraries were sequenced in an Illumina HiSeq3000 using a 100-bp paired-end reads protocol. Sequence alignment to the human reference genome (UCSC hg19), and variants call and annotation were performed using an in-house pipeline as described elsewhere [6]. The raw list of single nucleotide variants (SNVs) and indels was then filtered. Only exonic and donor/acceptor splicing variants were considered. In accordance with the pedigree and phenotype, priority was given to rare variants [<1% in public databases, including 1000 Genomes project, NHLBI Exome Variant Server, Complete Genomics 69, and Exome Aggregation Consortium (ExAC v0.2)] fitting a recessive model (i.e. homozygous in the proband but heterozygous in the father or compound heterozygous in the proband but not in the father), and located in genes previously associated with HSP [3, 7].

The three affected siblings (Fig. 1b: II-1, II-2 and II-3) have a phenotype consisting of complicated HSP. All of them showed early-onset and severe spastic lower limb weakness, brisk deep tendon reflexes, presence of Babinski sign and severe gait difficulties. The older male and the female (Fig. 1b: II-1 and II-2, respectively) need assistance to stand up. Only the younger male (Fig. 1b: II-3) is able to stand up independently and walk a few steps unassisted. The gait is characterized by feet dragging, shaking and leg scissoring. All three siblings have upper limbs weakness, more obvious in the proximal muscle groups, especially for the two older siblings. The facial muscles are hypotonic with reduced facial expressions. The facial appearance of the three siblings is characterized by coarse features, a prominent and bulbous nose and a wide mouth (Fig. 1a; left panel patient II-1, middle panel patient II-3, right panel patient II-2).

Interestingly, all affected siblings present hypometric and slow vertical saccades, especially at the upward gaze. Some limb ataxia is also present. There are no prominent extrapyramidal signs.

All three siblings have severe intellectual disability. Additional cognitive/behavioral abnormalities include apathy, reduced motor planning and/or initiation, attention-deficit disorder. An impairment of speech is also present, especially for the older male who has very poor language skills.

All siblings were born without any adverse perinatal events and had normal development during the first months of their lives. All three presented febrile tonic-clonic seizures during the first year of life, which were soon followed by epileptic non-febrile seizures. Developmental delay was noticed since early infancy. Only the younger brother (Fig. 1b; patient II-3) managed to walk independently during childhood but later his motor skills also started to gradually decline. The language and social skills of all three siblings were also lagging behind during their childhood and eventually showing some serious deficits by their puberty.

Spastic Paraplegia Rating Scale (SPRS) [8] was assessed for all 3 siblings: the older male and the female (Fig. 1b: II-1 and II-2, respectively) both scored 40 out of a maximum of 52, while the younger male (Fig. 1b: II-3) scored 35/52.

Other disorders presenting with spastic paraparesis (e.g. abetalipoproteinemia, funicular myelosis, multiple sclerosis, AIDS, lues, adrenoleucodystrophy, etc.) were excluded after an extensive diagnostic work-up that included brain magnetic resonance imaging (MRI), cerebrospinal fluid (CSF) analysis, nerve conduction and evoked potentials studies, electromyography (EMG), electroencephalography (EEG), serum vitamin B12 and E levels, serum arylsulphatase, galactocerebrosidase and very long chain fatty acids (VLCFA) levels, HTLV-1 antibodies, HIV and syphilis serology.

The brain MRI showed cerebellar hypoplasia/atrophy and downsloping splenium of the corpus callosum (Fig. 2).

Nerve conduction studies showed no evidence of peripheral neuropathy. Somatosensory evoked potentials (SSEPs) of median and tibial nerve were remarkable for prolongation of cortical latencies (N13-N20 and P40, respectively).

EEG was reported normal for the three siblings, without findings of focal slowing or seizure-like activity. More specifically, EEG recordings showed background of 9 to 10 hertz alpha activity, maximal over the posterior head region. These activities were symmetric on both sides and attenuated with eye opening. No focal slowing was seen and no seizure-like activity was observed during the recording. Patient entered into periods of drowsiness and light sleep. No abnormality was seen.

All clinical and radiological features are summarized in Table 1, and compared to other cases of HSP due to AP4M1 mutations previously reported in literature.

WES generated a total of 136,968,572 (proband, Fig. 1b: II-3) and 145,150,172 (father, Fig. 1b: I-1) unique reads, with an average on target depth over 160 reads, and >98% of the target bases covered at least 10X. A total of 24,872 (proband) and 25,228 (father) exonic/splicing variants were detected. After applying our filtering strategy described above, we identified two heterozygous AP4M1 (SPG50) variants in the proband, a novel frameshift insertion (c.521dupT; p.V174fs in exon 6), and a very rare (<2.5 × 10⁻⁵ in public databases) missense variant (c.955 T > C; p.C319R in exon 12). Only one of these variants was present in the father (c.955 T > C; p.C319R). No other rare homozygous or compound heterozygous variants were found in genes of relevance for the phenotype, including all know HSP genes. The AP4M1frameshift variant causes a very premature stop codon, truncating the protein (if expressed at all) to 179 amino acids (wild-type: 453 amino acids), just before the mu homology domain (MHD) which is an essential protein-protein interaction module (Fig. 1c). The missense variant lays on a highly conserved position (GERP++ score [9] of 4.75) of the MHD domain, and is predicted to be damaging by several prediction tools (including SIFT [10], PolyPhen2 [11], and MutationTaster [12]). Segregation analysis by Sanger sequencing showed that all three affected siblings have both variants, whereas the father carries only the missense and the mother carries only the frameshift (Fig. 1b), confirming that the compound heterozygous state of these variants is segregating with the phenotype.