Defining the genotypic and phenotypic spectrum of X-linked MSL3-related disorder

Purpose We sought to delineate the genotypic and phenotypic spectrum of female and male individuals with X-linked, MSL3-related disorder (Basilicata–Akhtar syndrome). Methods Twenty-five individuals (15 males, 10 females) with causative variants in MSL3 were ascertained through exome or genome sequencing at ten different sequencing centers. Results We identified multiple variant types in MSL3 (ten nonsense, six frameshift, four splice site, three missense, one in-frame-deletion, one multi-exon deletion), most proven to be de novo, and clustering in the terminal eight exons suggesting that truncating variants in the first five exons might be compensated by an alternative MSL3 transcript. Three-dimensional modeling of missense and splice variants indicated that these have a deleterious effect. The main clinical findings comprised developmental delay and intellectual disability ranging from mild to severe. Autism spectrum disorder, muscle tone abnormalities, and macrocephaly were common as well as hearing impairment and gastrointestinal problems. Hypoplasia of the cerebellar vermis emerged as a consistent magnetic resonance image (MRI) finding. Females and males were equally affected. Using facial analysis technology, a recognizable facial gestalt was determined. Conclusion Our aggregated data illustrate the genotypic and phenotypic spectrum of X-linked, MSL3-related disorder (Basilicata–Akhtar syndrome). Our cohort improves the understanding of disease related morbidity and allows us to propose detailed surveillance guidelines for affected individuals.

Individual 11 -Total genomic DNA was extracted from the biological sample using a spin column method. DNA quality and quantity were assessed through gel electrophoresis and fluorometric analysis, respectively. DNA sample was then shipped to BGI* where whole exome capture and sequencing were performed. Exome enrichment was done, using Agilent SureSelect Human All Exon V6 kits. Libraries were sequenced on Illumina HiSeq platforms to a coverage >100x. Clean sequence reads of each sample was mapped to the human reference genome (GRCh37/hg19). Burrows-Wheeler Aligner (BWA) software was used to do the alignment. Local realignment around indels and base quality score recalibration were performed using GATK, with duplicate reads removed by Picard tools. Data analysis was performed at Blueprint Genetics. The exome variant data of the patient was filtered based on all possible modes of inheritance of the disorder.
Individual 14 -Double stranded capture baits against approximately 36.5MB of the human exome (targeting >98% of the coding RefSeq and Gencode v28 regions, which was obtained from the human genome build CRCH37/hg19 on May 2018) were used to enrich target regions from fragmented genomic DNA with the Twist Human Core Exome Plus kit. The generated library was sequenced on an Illumina platform to obtain at least 20x coverage depth for >98% of the targeted bases. An in-house bioinformatics pipeline, including read alignment to GRCH39/hg19 genome assembly, variant calling and annotation, and comprehensive variant filtering was applied. All disease causing variants reported in HGMD®, in ClinVar and in CentoMD® as well as all variants with a minor allele frequency (MAF) below 1% in the gnomAD database are considered. The investigation for relevant variants is focused on coding exons and flanking +/-20 intronic bases. All potential modes of inheritance are considered. In addition, provided family history and clinical information are used to evaluate identified variants with respect to their pathogenicity and causality, and are categorized into classes Individual 25 -Whole genome sequencing (WGS) was performed using the Illumina TruSeq DNA PCR-Free sample preparation kit (Illumina, Inc.) and an Illumina HiSeq 2500 sequencer, generating a mean depth of 30×. WGS reads were aligned using Isaac Genome Alignment Software and single variant nucleotides and indels called for chromosomes 1 to 22, X, and the mitochondrial genome using the Platypus variant caller, details as outlined in previous publication. 6 Genomics England bioinformatics tiers single nucleotide variants (SNVs) in the applied panels into Tier 1 high impact variants such as truncating variants and Tier 2 moderate impact variants such as missense changes. The remaining rare variants are in Tier 3 or untiered. The Exomiser tool is also applied to the data irrespective of panel. This tool uses HPO terms to prioritize variants according to frequency, pathogenicity, inheritance pattern of the variants and similarity of the patient phenotypes to reference genotype to phenotype associations in known human diseases, model organisms and protein-protein interactions to known human disease genes. Each variant is scored and ranked with the highest rank being 1.

Determination of mosaicism of variants
Droplet digital PCR (ddPCR) specific to NM_078629.3 (MSL3) was used to assess allele specific copy number at MSL3: c.1382-1G>A. ddPCR was performed in the presence of a fluorescent interchelating dye, EvaGreen (BioRad), using the manufacturer's recommended conditions. Quantitation was carried out by normalizing the mutant allele to the wildtype allele. In a known hemizygous control (our ref 19FH3585K) this ratio was 100%. Samples were repeated in quadruplicate.

Supplementary Case Reports
Individual 1 -This girl is the 3rd child of healthy non-consanguineous parents with German-Turkish ancestry. Two older siblings are healthy. Pregnancy was complicated by polyhydramnios. Moreover, during prenatal ultrasound a shortening of fetal extremities was suspected. The child was born at 39+4 gestational weeks (weight 3232 g, lengths 51 cm, Head circumference 36 cm). Postnatally she had respiratory problems and neonatal pneumonia was suspected. The child was transferred to the ICU and received high-flow breathing support. During the following days the condition rapidly improved and the child was discharged home. At the age of 3 months the parents noted that the child was very unsettled with frequent crying episodes. Moreover, fixation was inconstant and she interacted less with the parents compared to the healthy siblings. During the following months developmental delay became evident. Diagnostic work-up with brain MRI revealed mild widening of inner cerebrospinal fluid spaces. Muscle ultrasound showed atrophy and increased fat content of her thighs. Because of lacking speech development, she was seen by the otorhinolaryngologists who diagnosed a hearing impairment (conductive hearing loss). Eye examination indicated cortical visual impairment. Laboratory work-up, including screening for different metabolic diseases, showed no abnormalities. At the current age of 14 months the girl shows clear motoric developmental delay with truncal muscular, hypotonia and reduced spontaneous movement pattern. She is unable to crawl or to sit without support and she shows no interest in grasping objects. So far there is no speech development and she produces hardly any babbling sounds. Her current growth parameters are as follows: weight 9.9 kg (51 st P), lengths 75.2 cm (18 th P), head circumference 46.7 cm (63 rd P).
Trio exome sequencing revealed a de novo heterozygous splice variant c.1466+1G>A, p.?) in MSL3 (NM_078629.3). The variant was classified as pathogenic according to the ACMG criteria. Individual 2 -Individual 2, a girl and the second child of unrelated, healthy Norwegian parents, was born GW after a normal pregnancy at 38+6 weeks of gestation with birth measurements within the normal range. The postnatal period was unremarkable. At the age of 10 months, she presented with gastroenteritis-like symptoms (vomiting, but no diarrhoea). She was hypotonic and was admitted to the hospital for 6 days for intravenous rehydration. Some weeks later she was again admitted with vomiting and subsequent hypotonia requiring parenteral feeding. She was seemingly healthy until the age of 15 months when she was again admitted with similar symptoms. Thereafter, she experienced cyclic episodes of vomiting, hypotonia and constipation about 1-3 times per month (duration 5-12 days). The last 3 months, she had increasing pain during those episodes with ventricular retention, urine retention, need of parenteral feeding, morphine iv, antiemetics and sleep inducers. A treatment with propranolol (prophylactic against cyclic vomiting) was started, however, as no effect was noticed it was discontinued. From the age of 19 months, dystonic movements (fingers, neck, arms) during those episodes with extended extremities (spastic like, but intermittent) were observed. Global developmental delay... Repeated abdominal ultrasound displayed normal findings except for ventricular/urine retention. Contrast X-ray of the oesophagus/stomach showed hiatal hernia with reflux. MRI brain (incl. brain spectroscopy), as well as several EEGs (incl 24hrs monitoring) were normal. Metabolic evaluation (urine/plasma/CSF), echocardiography and chest Xray showed normal findings. Trio exome sequencing revealed a de novo heterozygous frameshift variant c.1146del, p.(Lys383Serfs*22) in MSL3 (NM_078629.3).

Individual 3 -
This female individual is the only daughter born to a non-consanguineous Caucasian couple. The family history was unremarkable except for her mother, who reported seizures in her first 2 years of life and a single episode when she was 15-year-old. The seizures had been attributed to difficult labour with forceps. Individual 3 was born at 40 weeks of gestation by spontaneous delivery after an uneventful pregnancy. At birth, weight was 2930 g (25 th centile), length was 48 cm (25 th centile) and occipitofrontal circumference (OFC) was 32.5 cm (10 th centile), the Apgar scores were 10 at the 1 st , and 10 at the 5 th minute, and perinatal period was physiological. At 7 months of life several episodes of brief psychomotor arrest characterized by gaze palsy, hypertonus, clonic movements of the limbs, perioral cyanosis, and drooling were noted, so electroencephalography (EEG) was performed showing diffuse epileptiform anomalies while falling asleep. Antiepileptic treatment was then started with valproic acid with benefit. A brain MRI was also performed showing normal morphology. She underwent several additional diagnostic procedures (abdominal and cardiac ultrasound, eye evaluation) that provided normal results. Psychomotor development of the patient was characterized by hypotonia, motor clumsiness, and speech delay. At 26 months of age a Griffiths Scales test was performed showing a General Quotient (GQ) of 60. Clinical examination at 4 years at our facility only showed some aspecific dysmorphic features, a long and smooth philtrum and thin upper lip, and pes planus. Growth was regular, since weight, length, and OFC were 16.5 kg (25 th centile), 98 cm (10 th centile), and 49.2 cm (10-25 th centile) respectively. Trio-based whole exome sequencing (WES) analysis was started, which detected the de novo heterozygous MSL3 Individual 4 -Individual 4 is a 5-year-old female with developmental delays, white matter abnormality on brain MRI, cerebral palsy and low muscle tone. She was born to unrelated healthy Caucasian parents after an uneventful pregnancy at 39 weeks of gestation. Acquisition of gross motor and fine motor skills were delayed. She sat at 14 months of age and walked independently at 19 months of age. First words were spoken by age on time. She receives occupational therapy and physiotherapy 30 minutes per week. She has normal vision and hearing. Trio exome sequencing revealed the de novo heterozygous splice variant c.589-4_591del, p.? in MSL3 (NM_078629.3) which was initially classified as variant of unknown significance and reclassified as pathogenic. In addition, a variant of uncertain significance (microduplication of 22q13.33) inherited from the healthy father was noticed. Mitochondrial DNA analysis was normal.
Individual 5 -Individual 5 is a female, born at 40 weeks of gestation to unrelated Caucasian parents.
Family history is unremarkable. No prenatal complications were reported. She was born by C-section due to absent fetal movement. APGARs were reported normal. She weighed 3033 g, length was 50.8 cm, and head circumference was reported within normal limits. She passed newborn hearing screening and newborn screening resulted normal. Neonatal concern was failure to thrive for 3-4 weeks addressed with feeding therapy. She did not require a NIUC stay. Primary care doctor noted that her fontanelles were not closing and around 4 months old she was referred to Genetics due to short limbs, poor growth, difficulty feeding, and macrocephaly. She sat independently around 1 year, walked at 2 years, and said first words around 2 years. Rightsided sensorineural hearing loss was noted, and she now has hearing aids. She can now speak in complete sentences and follow one and two-step directions. She has nonsensical utterances. She has diagnoses of expressive language disorder and sensory integration disorder. She can walk independently but tends to lead with left leg and fatigues easily. She has ankle-foot orthoses (AFOs) and uses wheelchair for long distances. She falls frequently. She tends to favor left hand for fine motor skills. She requires assistance with dressing and using toilet. She has frequent staring spells which are suspected to be behavioral rather than seizures. She has had a normal EEG. Brain MRI at 3 years was unremarkable. She was diagnosed with autism around 4 years. She has trouble with swallowing and video swallow study noted delayed swallowing initiation and shallow vestibular penetration when swallowing liquids, but no aspiration was seen. She has gastritis, severe constipation, and reflux. She has dynamic muscle tone with periods of "floppiness". She has spastic quadriplegic cerebral palsy. She has easy bruising, hyperhidrosis, and history of hemangioma on right shoulder which has resolved. Initial exome analysis was done around 4 years of age and showed the heterozygous de novo frameshift variant c.1319dup, p.(Gly441Argfs*2) in MSL3 (NM_078629. 3) which then was classified as variant of uncertain significance. Reanalysis of exome was done at 5 years 7 months with reclassification of the variant in MSL3 to pathogenic.

Individual 6
This 10-year-old female is the second of two children born to a non-consanguineous Caucasian couple with a history of ADHD in the father, the brother, the paternal aunt and the paternal uncle, as well as learning difficulties in the father and two paternal cousins. Pregnancy was marked by polyhydramnios noted at 7 months of pregnancy. She was vaginally delivered at term weighing 3402g. She nursed poorly. She failed newborn hearing screen and was diagnosed with bilateral moderate to severe sensorineural hearing impairment. She had no eye contact until about 3 months of age, smiled at 6 to 7 months, sat at 8 to 9 months and walked at 23 months. She was able to feed with spoon and fork at 4 years and started communicating with sign language. She has a short attention span. By the age of 8 years, she presented with increasingly aggressive and self-abusive behavior; she was hitting her head when upset and developed tics. At 10 years, functioning was at 3 to 4-year level and she uses bilateral hearing aids. She can recognize alphabet letters and numbers as well as sight words including names of family members, but she is not able to read. She is able to sign words and phrases, but has difficulty writing due to abnormal grasp of writing implements with stiff hands. She is unable to do maths. She walks with her knees bent and seems to have difficulties walking with occasional falls. She continues to have major behavioral outbursts, requiring treatment with chlorpromazine. She is toilet trained. Current medications include methylphenidat, clonazepam, clonidine, melatonin, and chlorpromazine. She had myringotomy tubes twice. Her vision was not formally tested but appears normal. In the primary dentition, the right lateral incisor was fused with the first bicuspid and both were small. She has frequent staring spells that are felt to be behavioral in nature with a normal EEG. She has no respiratory problems. Constipation and straining with urination are present. At 10 years, her height was 132.2cm (10 th centile), weight 24.8 kg (3 rd to 10 th centile) and head circumference 51.8 cm (25th centile). Decreased facial expression and a deep hoarse voice were noticed. She has telecanthus with inner canthal distance at the 90 th centile and outer canthal distance at 25 th to 50 th centile, bilateral epicanthal folds and squared nasal root. Full extraocular movements with normal pupillary responses and creases under the eyes. Ears low set, posteriorly rotated, and small (10 th centile) with overfolded upper helices and anteverted earlobes. The philtrum seems to be poorly defined, and she has a small mouth with normal lips and a high narrow palate with intact uvula. Narrow chest with pectus carinatum and markedly underdeveloped pectoralis major muscles were observed. On examinations, a stiffness of movement of small joints of hands with limitation of extension were noticed. She has bilateral pes planus with hallux valgus, especially on the right foot. Unusual posture with a tendency to keep arms extended with hands clasped and knees bent with hyperlordosis. Deep tendon reflexes were 3+ at the knees and 2+ elsewhere. Individual 7 -Individual 7 is a 16-year-old female who is the younger of two children and was born to healthy non-consanguineous parents of British descent. Her older brother as well as her three paternal half-siblings are healthy. The family history is negative for known genetic problems.
Individual 7 was diagnosed with autism and developmental delay in early childhood. She has received special education and additional supports. She is not fully toilet trained at the age of 16 years. She can follow simple commands and has a communication device, but she only used it to continually ask for food. She has not had a seizure in years but continues on her medication. She has episodes of behavioral outbursts, with crying and hitting. Those episodes occur a few times a week and often only resolve with the administration of Lorazepam. She is prone to wander and can get lost. She likes to fiddle with something in her hands and likes to rock. Moreover, she enjoys music and being in the water. She is mildly dysmorphic with a high forehead, arched eyebrows, a down turned mouth and a round face. Height was 156.8 cm (17 th centile), weight 69.4 kg (84 th centile) and head circumference 56 cm (94 th centile) at the last follow-up. Her fingers are tapered and her thumbs are proximally placed. Lungs were clear, heart sounds normal and abdomen was soft with no organomegaly. Trio exome sequencing revealed a heterozygous de novo deletion of exon 6-8 of MSL3 (NM_078629. 3) which was classified as pathogenic.

Individual 8 -This individual is a 17-year-old female, born to non-consanguineous Hispanic parents.
Her family history is significant for an older brother with ADHD, a younger sister who has mild neurocognitive delay, and two female paternal cousins who were described as "slow." Her mother was in a vehicular accident a week before delivery, but there was no apparent fetal distress.
Individual 8 was born at 39 weeks gestational age via an induced vaginal delivery with a birth length of 48 cm (29 th centile) and weight of 3.77 kg (85 th centile). After birth, she underwent a day of phototherapy for jaundice. She was discharged from the hospital on her second day of life. In early infancy she was noted to have feeding problems and hypotonia. Her gross motor milestones were reached within the upper limit of normal. Specifically, she was able to pull into a stand at 12 months of age and walk unassisted at 15 months of age. In contrast, her speech development was delayed. In her first year of life she only cooed. She was able to say "ma ma" and "da da" after she was a year old. She was putting two words together at 3 years of age and was speaking in short sentences at 4.5 years of age. Over time, she was diagnosed with autism spectrum disorder, intellectual disability, bradykinesia, parkinsonism, hypophonia and increased tone. She underwent bilateral lateral rectus recessions and currently has intermittent exotropia and wears glasses for myopia. An EEG and brain MRI performed at 14 years of age were normal. At 17 years 8 months of age her height was 148.5 cm Both of his parents are alive and well. He was born at 38+4 weeks of gestation with a birth weight of 4.47 kg and a birth length of 52.1 cm. He has a history of developmental delay, hypotonia, and macrocephaly. His parents first noted developmental concerns at 4 months of age because he was not holding his head up. On his most recent assessment, his height was 88 cm (10 th centile), weight was 13.2 kg (33 rd centile), and head circumference was 54.5 cm (>98 th centile). He has macrocephaly and plagiocephaly. His ears are small, and his nostrils flare out a bit. He has a broad forehead and short upturned nose. He holds his tongue outside of his mouth often. He is mildly coarse. He has stiff 5th fingers bilaterally, but no contractures. He has flat feet and low muscle tone. Furthermore, he previously had rectal prolapse. Trio exome sequencing revealed the de novo hemizygous nonsense variant c.913C>T, p.(Gln305*) in MSL3, classified as pathogenic.

Individual 10
Individual 10 is a 4-year-old male born at 39 weeks gestation to 41 year old, Caucasian, Non-Hispanic, non-consanguineous parents. A full older brother had no significant medical or cognitive concerns.
Delivery was by C-section at the time of spontaneous labor due to low transverse placental placement and a history of prior C-section. Non-invasive prenatal testing was normal. Birth parameters were within normal limits, (weight 3960 g, length 53.3 cm, head circumference 35.6 cm) and APGARS were 8 at 1 minute, 9 at 5 minutes. On newborn exam a heart murmur, umbilical hernia, bilateral hydroceles, and left lacrimal duct stenosis were noted. He was discharged to home on day three of life. The parents had concerns about his eye contact and social engagement at 2 months. As an infant, he was diagnosed with anisocoria and monitored for positional plagiocephaly (did not require helmet therapy). He was referred for developmental assessment by his pediatrician after concerning responses on his Ages & Stages Questionnaire at his 9 month well child checkup.
Developmentally he had global delay, with less significant motor delay as compared to his language and social domains. He rolled and sat independently for brief periods at 11 months. Developmental assessment determined that he met DSM-V criteria for Autism Spectrum Disorder.
Performed due to periods of behavioral arrest, a sleep deprived EEG during this period was normal in both awake and asleep states. At age 4 years, he is functionally non-verbal, but does have a few signs, follows single step instructions and responds to his name. He continues to be well grown with normal growth parameters at 3 years and 5 months.  During pregnancy reduced fetal movements, oligohydramnios and cleft lip and suspected cleft were detected. No other structural abnormalities were present. He was born at term by emergency caesarean section due to fetal distress. Birth weight, length and OFC were 3380g (29 th centile), 53cm (59 th centile) and 35cm (32 nd centile). Incomplete left cleft lip was noted, but palate was intact. He had two pre-auricular tags on the right and his right external ear had a prominent serpiginous antihelix stem and a mild cleft. Global delay was first noticed at 14 months of age (unaided sitting at 7-8 months, unsupported standing for a few seconds around 24 months). By 18 months of age, he was diagnosed with autism spectrum disorder. At last follow-up at 4 2 12 years he walked unsupported.
So far, he has no expressive language. Intradural lipoma C6/7, but no other abnormalities were evident on cMRI. Cardiac and abdominal ultrasounds revealed normal results. Unilateral (right side) sensorineural hearing loss was present and he suffered from recurrent otitis media (grommets in situ). He had longstanding vomiting, possibly secondary due to esophageal sphincter dysfunction.
The patient developed postnatal short stature and his anthropometric data at 3

Individual 13 -Individual 13 is a 6 year 3 month old boy who was born at 40 weeks gestation via
Cesarean section for failure to progress to his then 28-year-old mother and 29-year-old father. The pregnancy was complicated by polyhydramnios, with prenatal ultrasound findings of short appearing limbs and macrocephaly. His birth weight was 3.95 kg (90 th centile), and there were no perinatal complications. He had poor weight gain in the first few weeks after birth and was treated for gastroesophageal reflux disease (GERD) with some improvement in weight. Due to his macrocephaly he had a head ultrasound at 7-months and a brain MRI at 13-months that were both normal. He had delayed milestones, rolling at 9 months, sitting at 12 months, and walking at 2 years of age. His first words were at 3 years of age and he started speaking in simple sentences around 4 years of age. He has had normal audiology evaluations. Initially he made poor eye contact and did not seek out interaction with parents or others, and he was diagnosed with autism spectrum disorder at 5 years of age. His social skills have improved over time, but he prefers to play by himself. He has a short attention span and is prone to anxiety when in crowds. He is not aggressive and does not exhibit selfharming behaviors. He has trouble sleeping at night, which has been helped somewhat by melatonin and physical activity during the day. He has hypotonia with frequent drooling, though sometimes will feel stiff, especially when getting picked up. He used to get sick a lot as a younger child but now seems to have a normal immune system, though he does have chronically loose stools. He receives physical, occupational and speech therapies and is in a special education classroom setting. At 6 years 3 month his weight was at the 87 th centile, height at the 66 th centile, and head circumference greater than the 98 th centile, with a Z-score of +3.9. He has dolichocephaly with frontal bossing, and thick, coarse, curly hair with a frontal upsweep. His ears are posteriorly rotated with slightly dysplastic helices, and he has down-slanting palpebral fissures. He has a prominent nasal bridge and mild retrognathia. He has a large, faint nevus flammeus on his forehead, and a smaller one at the nape of his neck. There is also a large, irregularly shaped hyperpigmented macule in his right groin. A chromosome microarray identified two variants of uncertain clinical significance: a 284 kb copy number gain at 6q27, including the MLLT4 and KIF25 genes, and a 107 kb copy number loss at Individual 18 -This male individual is a product of his mother's second pregnancy of five. The maternal age was 22 and the paternal age was 24 at the time of the pregnancy. At five months gestation, the mother had food poisoning and required IV fluids and promethazine, and also terbutaline for contractions. No other complications occurred during pregnancy. The spontaneous vaginal birth occurred at 37.5 weeks' gestation without complications. His birth weight was 2.6 kg and he did not have any dysmorphism noticed as an infant. He had significant vomiting from birth that was evaluated when it became bloody and he was found to have ulcers. He was also found to have a hiatal hernia. For these reasons he had a Nissen fundoplication. His GI issues resolved after that. This individual was last evaluated at 19 years of age and has a history of global developmental delay and episodes of encephalopathy with regression. He has macrocephaly, low hairline, and has some thickening of his facial features and also possibly some of his joints. His early development was not concerning until 2-3 years of age when he was noted to be falling behind his peers. He crawled at 6 months and walked at 10 months. Around the age of 2 years, he fine motor delays and speech/language delays became apparent, and he was still having vomiting and recurrent sinus infections and tonsillitis. He had vision problems and was diagnosed as being extremely near-sighted, and he was given glasses. He was found to have both conductive and sensorineural hearing loss in both ears. These were initially treated with PE tubes, which did not help. He received hearing aids at age 5, and his speech and language skills improved to some degree after this, although he still had delays in language. His fine motor skills continued to be delayed. He progressed through grade school and high school with an individualized educational plan in place throughout his entire schooling. He graduated from high school last year. His IQ post his 13-year-old episode was 55, he reads at about a grade 5 or 6 level and performs math at a similar grade level. He has had 2 episodes of developmental regression, occurring in association with acute encephalopathy, occurring at ages 13 and 18. He apparently recovered, partially, from the episode at age 13 over a period of 6 months. It took him 6 months to recover from the episode at age 18, but again only a partial recovery. This individual has a history of involuntary movements. They consist primarily of a left shoulder twitch. He also has some stuttering and some garbling of his speech. He has a tremor with reaching, fine motor skills, or action, but not at rest. There is no dystonia or chorea. He has never had seizures. The family has noticed coarsening of his facial features since age 13. He seems to have developed hairiness of the body more than they might have expected based on other family members. He also has required multiple surgeries for hammer toes. On neurological review of systems, this individual has moderate conductive and sensorineural hearing loss bilaterally and requires hearing aids. He has had migraines since age 13. Since age 13, they have noticed spasticity in the legs, and he is on baclofen for this. He has also had increased deep tendon reflexes and clonus. He has a high pain tolerance but normal sensation. Clinical testing has included MR Spectroscopy showing a relatively low NAA with a possible lactate peak. MRI brain showed nonspecific T2 hyperintensities in the white matter but no evidence of a leukodystrophy. EEGs have shown a slow background with no epileptiform activity. Biochemical evaluations for lysosomal, peroxisomal, inborn errors of metabolism, creatine disorders, CDG were unrevealing. He had an infectious workup in the CSF which was negative at the time of his encephalopathic episodes. His autoimmune workup has also been negative. Testing for lysosomal storage disorders including urine mucopolysaccharides and oligosaccharides and oligosaccharides in the white blood cells was unrevealing. He has had a negative carbohydrate-deficient transferrin test.
Oxysterols were normal ruling out Niemann-Pick C. Neurotransmitters raised the possibility of tetrahydrobiopterin metabolism defect based on low concentrations of tetrahydrobiopterin, homovanillic acid and 5-OH-indolacetic acid, but GCH1 gene encoding GTP cyclohydrolase was normal. Unrevealing genetic testing included normal chromosomal microarray, fragile X, an Individual 21 -Individual 21 is an 11-year-old male who was the 3345 g product of a 40 week gestation, born via cesarean section to a then 30 year old (gravida 2, para 1-2) mother with early prenatal care. Pregnancy complications included identification of cysts on the umbilical cord.
The mother was followed closely throughout the pregnancy. Prenatal testing included an amniocentesis for which the results were reportedly normal. Prenatal exposure and acute maternal illness during pregnancy was denied. Complications in the perinatal period include a NICU stay for five days due to apneic episodes and congenital contractures. He was discharged home on oxygen therapy and struggled with feeding from birth. He first sat at 2 years of age and walked at 3 years of age. He said his first word with meaning at 3 years of age and put 2-3 words together at 4 years.
Medical history is significant for limb contractures, atrial septal defect s/p closure, bilateral mild conductive hearing loss, dysphagia, kyphosis, and global developmental delays. Currently, he uses complex speech, and will have back and forth conversations. He will build on things other people say, and picks up things that other people are interested in. His articulation is challenging; his mother Individual 22 -Individual 22 is a 15-month-old male who was born at 37-weeks via vaginal delivery to 30-year-old mother. On ultrasound, mother was told there were "signs of dwarfism". Patient had nuchal cord at delivery, otherwise there were no complications. Growth parameters at birth were social pragmatic communication disorder, and generalized anxiety disorder. She had an EEG for staring episodes and repetitive blinking, and this demonstrated right-sided focal slowing during hyperventilation as well as eyelid closure blepharoclonus. A brain MRI at age 8 revealed non-specific T2 hyper intense foci in the bifrontal white mater. At her most recent outpatient visit, spasticity was noted at her knees bilaterally. She is mildly dysmorphic with diffuse hypotonia and an abnormal gait. Individual 24 -Individual 24 was born at 38 4/7 weeks after a pregnancy complicated by fetal ultrasound findings suggestive of short limbs, choroid plexus cyst, and hydronephrosis, of which the latter resolved. Amniocentesis was performed and karyotype and microarray were normal. At birth he was appropriately grown with a weigh of 3.6 kg, and length of 52.1 cm but had relative macrocephaly with a head circumference of 35.6 cm and was noted to have macroglossia. He did well neonatally with a brief period of transient tachypnea. At 2 months of age he was seen by the genetics service nearest to his hospital of birth. He was making normal developmental progress and growing well. Noted were a large anterior fontanelle (9x9 cm), coarse facial features, epicanthic folds, underrotated ears, broad nasal bridge, mild retrognathia, enlarged protuberant tongue, small umbilical hernia, mild rhizomelic limb shortening, no skeletal asymmetry, and capillary malformations over the glabella and occiput. Methylation testing for Beckwith-Wiedemann syndrome, very long chain fatty acids, urine mucopolysaccharide screen, radiographic skeletal survey, and abdominal ultrasound were all normal. In follow up, further testing included molecular genetic testing for metabolic disorders, which also yielded normal results. After moving to another state, he was seen by a second genetics service at just under 2 years of age. By that time, he was demonstrating global developmental delays, episodes concerning for seizures but normal EEG, and had been found to have first and second degree A-V block. Relative macrocephaly and macroglossia persisted. Whole exome sequencing was performed and revealed a de novo hemizygous pathogenic variant in MSL3 (NM_078629.3:c.973_974del, p.(Gln326Alafs*5)).
Individual 25 -Individual 25 is the second child of two children born to unrelated parents. She was born at term by spontaneous vaginal delivery weighing 3500g. A large head circumference was noted at birth. She sat at 8 months and walked at almost 2 years of age but her parents had concerns about her development from the age of a year. She was hospitalized at 6 months and 1 year for treatment of lower respiratory tract infections. Physiotherapy assessment noted low muscle tone. She has a speech and language delay with only a few single words at 2 years. Her voice sounded a bit hoarse with a nasal tone. At 3 years and 10 months she could follow single staged commands and there were concerns about her understanding. Her head circumference was 53.7cm (3.185 SD). Weight and height at 3 years and 6 months were 15.36kg (0.180 SD) and 95.8cm (-0.803 SD) respectively. On examination features included overfolded helices, beaked nose with broad nasal bridge, tapering of the fingers, narrow chest, short neck, flat feet and slim calf muscles. On review at 13 years and 5 months she was attending a special educational needs school and had a diagnosis of dyspraxia and poor motor planning, autistic spectrum disorder and a motor tic causing tilting of the head. Weight