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Riboflavin Transporter Deficiency Presentation, Genetics and Treatment

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Riboflavin Transporter Deficiency

Presentation, Genetics and Treatment

Keith Massey

Science Director, Cure RTD Foundation

Email: Keith.Massey@curertd.org

Riboflavin Transporter Deficiency (RTD)

• Early onset, life shortening, neurodegenerative

disorder.

• Autosomal recessive inheritance.
• Previously known as Brown-Vialetto-Van Laere

(BVVL) and Fazio-Londe syndromes.

• Sensory and/or motor neuropathy and cranial

neuropathy.

Brown, 1894

Gene Discovery

• 2008

2008-2010 2010 The SLC52A1, SLC52A2 and SLC52A3 genes first identified to code for riboflavin (Vitamin B2) transporters (RFVT1, RFVT2 and RFVT3) in humans.

• 2010

2010 SLC52A3 gene was found to cause BVVL. Daily high dose riboflavin found to help stabilize or improve clinical symptoms.

• 2012

2012 SLC52A2 gene was also found to cause BVVL and patients responded to riboflavin therapy.

RTD Disease Types

RTD Type 2

Riboflavin Transporter Deficiency, Type 2 SLC52A2-associated disease RFVT2 riboflavin transporter

RTD Type 3

Riboflavin Transporter Deficiency, Type 3 SLC52A3-associated disease RFVT3 riboflavin transporter

Flying under the radar?

Daughter SLC52A2 biallelic mutations c.155 C>T and c.935 T>C First Cousins Once Removed SLC52A2 biallelic mutations c.155 C>T and c.916 G>A

Prevalence

SLC5

C52A2 (R (RTD Type 2) 125 cases (biallelic mutations) 1 1 case e (heterozygous mutation)

• 21 deceased, 19 untreated
• Mean age of onset: 3.0

3.0 year ears SD=3.0, Range: 0-19 19 yea ears

• 55% Female

SLC5 C52A3 (R (RTD TD Type e 3) 3) 83 cases es (biallelic mutations) 12 12 cas ases es (heterozygous mutation - RF Responsive)

• 17 deceased, all untreated
• Mean age of onset: 7.6

7.6 year ears SD=8.2, Range: 0-34 34 yea ears

• 59% Female

Untreated: Mean age of death: 12.6 years (SD=14, Range: 0.9 - 55 years, n=31) Cause of death: Respiratory insufficiency

Cu Cure RTD D - Patie tients Reported sin ince ce 2010

• Carrier rate: ~1 in 500 people (single SLC52A2 or SLC52A3 known pathogenic mutation)
• Estimated Prevalence: ~1 in 1 million births (based on recessive inheritance)
• Estimated 50-100 new cases per year

Riboflavin (Vitamin B2)

• Precursor to flavin mononucleotide (FMN)

and flavin adenine dinucleotide (FAD) (active coenzyme)

• Riboflavin acquired via intestinal absorption with little storage in the

body.

• Human genome contains 90 genes encoding for flavin-dependent

proteins.

• FAD and FMN crucial for cell regulation and energy metabolism.

RVFT Human Tissue Expression

Yonezawa 2010

RVFT Function: Intestine and Blood Brain Barrier

RVFT: Transmembrane Protein (Apical or Basolateral Facing) Transports Riboflavin (RF) and small amounts of FMN/FAD Active Saturable Transporter

Transporter Kinetics

• RF

RFVT2 (S (SLC52A2) Km 0.33 +- 0.07 uM (330nM) Vmax 5.2 5.2 +- 0.7 0.7 pmol×mg protein-1×min-1

• RF

RFVT3 (S (SLC52A3) Km 0.98+- 0.11 uM Vmax 63 63.8 .8 +- 9.3 9.3 pmol×mg protein-1×min-1

Jin et. al 2017

Human Brain Gene Expression

GTEX Portal Cer Cerebellum

Clinical Features of RTD Untreated

SLC52A2

(RTD Type 2)

SLC52A3

(RTD Type 3)

N=38 N=18

Auditory Neuropathy

98% 95%

Pontobulbar Involvement

80% 82%

Respiratory Compromise

72% 82%

Muscle Weakness

87% (UL UL) 95%

Optic Atrophy w/o Nystagmus

100% 29%

Sensory Involvement / Gait Ataxia

100% 29% Untreated patients with over 10 years from onset of first symptom.

UL UL = Upper limb and axial predominant muscle weakness Other common feature: GI Distress, aggressive scoliosis (type 2), Upper motor neuron and cerebellar signs (type 3), Severe facial weakness (type 3)

Presenting Features

SL SLC5 C52A2 0-5 year ears SL SLC5 C52A2 6-19 19 years SL SLC5 C52A3 0-5 5 year ears SL SLC5 C52A3 6-33 years

N=77 N=16 N=31 N=28 Auditory Neuropathy

29% 63% 26% 75%

Pontobulbar Involvement

9%

• 61%

32%

Sensory Involvement / Ataxia

62% 32% 6% 4%

Optic Atrophy w/o Nystagmus

27% 38% 6%

• Muscle Weakness

29% (UL)

• 39%

11%

Respiratory Compromise

12% 6% 55% 7%

Megaloblastic Anemia

5%

• Symptoms, age at onset, time interval between symptoms, and severity and rate of

decline are all highly variable – even between siblings.

Laboratory Findings

Blo Blood

• Acylcarnitine profile abnormalities in 50-60% of patients. Selective

accumulation of selective short- and medium-chain acylcarnitines.

• Riboflavin (+FAD, FMN) levels may be low or normal.
• Signs of mild mitochondrial dysfunction in some patients.
• >10% - Low hemoglobin and serum iron and/or ferritin levels
• Anemia (macrocytic or normocytic)

Urin rine Organic ic Acid cid

• Elevated ethylmalonic acid in <50% of patients indicative of a defect in

riboflavin metabolism

Neurodiagnostic Tests

Ele lectromyogram (E (EMG) an and nerve con

• nduction stu

tudies (NC (NCS)

• chronic partial denervation
• conduction velocities are normal or mildly reduced
• indicative of axonal motor / sensory neuropathy

Brai ain mag agnetic resonance im imagin ing

• Most often unremarkable
• ~25% with RTD type 3 have cerebellar and brainstem

abnormalities Auditory ry brai ainstem resp sponse (AB (ABR / / BAEP)

• absent or abnormal

Audiologic Test Findings in RTD

Hearing loss consistent with auditory neuropathy pattern

• Absent or abnormal auditory brainstem response (ABR) with

cochlear microphonic present

• Otoacoustic emissions (OAE) are often present but found to

disappear over time in many patients.

• Acoustic reflexes often absent
• Magnetic resonance imaging (MRI) is generally normal without any

evidence of VIII cranial nerve hypoplasia

• fMRI with an auditory stimulation protocol showed integrity of the

central auditory pathways in patient (Salmina et al 2014)

Audiology Clinical Characteristics Reported in RTD

• Progressive hearing loss can occur from early infancy to 25+ years in age
• Pure tone thresholds ranging from mild to profound loss
• Hearing loss may progress to profound in months or slowly progressive over

decades

• Loss is bilateral but often asymmetric
• Disproportionately poor speech recognition abilities for the degree of hearing loss
• Difficulty hearing in noise and hearing fluctuation
• Patient with poor speech recognition have reported limited or no benefit from

hearing aids

• Vestibular deficits are rare

Cochlear Implantation

Over 40 RTD patients have received cochlear implants. (Age of implant: 2 to 45 years old) Results are variable but the large majority have received significant benefit and very pleased with CI decision. The few cases with less than optimal outcomes had:

• Long-term profound deafness
• Extended period of prelingual deafness

Cannot discount how much sound awareness can add to quality of life when dealing with multiple disabilities

RTD Type 2: Cochlear Implant Case Example

AGE

3mths – 2 years Vision loss, anemia, muscle weakness, sensory gait ataxia, feeding difficulties 2 years Language development normal and first sign of hearing loss became apparent 2.5 years RTD diagnosis and started riboflavin treatment (75mg/kg QID + mitococktail) 3.8 years Received unilateral cochlear implant. ~10-20% accuracy on close word speech set 5.7 years ~95% accuracy on closed word set in CI ear / still ~10-20% in non-implanted ear 7.4 years Received bilateral cochlear implants. 8.0 years ~95% accuracy on open word set using both CIs 2.8 years old

• ld

4 months on Riboflavin 3.7 years old

• ld

1.5 years on Riboflavin 5.7 years old

• ld

3.5 years on Riboflavin

Pathogenic Mutations

SLC52A2 (RTD Type 2)

• 53 Mutations

SLC52A3 (RTD Type 3)

• 52 Mutations
• Mutations distributed throughout all coding exons (2 to 5)
• Mostly missense mutations where RFVT retains some function
• Combined >15% of mutations across both SLC52A2 and SLC52A3

are:

• Nonsense mutations
• Single/double nucleotide insertions/deletions causing

frameshift

• Biallelic “Loss of Function” mutations generally not compatible with

life.

Impact of Pathogenic Mutations on RVFT Function

• Reduced RFVT cell surface expression
• RFVT ability to transport riboflavin is

impaired

• Reduction in mRNA stability (1 mutation)

Overall consequence is reduced riboflavin transport across the cell membrane.

WT SLC52A2 Mutations

Foley et al. 2013 Riboflavin Cellular Uptake

SLC52A2 Gene Structure

~50% of patients identified have 1 or 2 mutations in: c.9 c.916 G> G>A (Population frequency 14 / ~250,000) c.1 c.1016 T> T>C (Population frequency 18 / ~250,000)

Transmembrane Topology Of RFVT2

Predicted Using TMHMM

Pathomechanisms?

Mitochondria Dysfunction

• ETC complex 1 and 2 deficiency (Fibroblasts, Muscle, Animal models)
• Reduced mitophagy and fusion (human iPSc-derived motor neurons)
• Abnormal mitochondrial morphology (human iPSc MN, Drosophila knockdown)
• Impaired mitochondrial β-oxidation of fatty acids (A3 Mouse knockout)

Other:

• Cytoskeletal abnormities (human iPSc MN)
• Neurite outgrowth / axon guidance gene expression changes (human iPSc MN)

Protein-Protein Interaction?

STRING Consortium

SLC52A2 SLC52A1 SLC52A3

Riboflavin Treatment

• Early treatment can slow disease progression and

improve symptoms.

• Oral Riboflavin – 20 to 80mg/kg/day (2-6 per day)
• Optimal dosing and frequency unknown.

Plasma Riboflavin profile after large oral dose of riboflavin

Riboflavin levels peak 1-2 hours after oral dose. Rapidly expelled in Urine. Back near baseline in 6-9 hour after oral dose.

Complementary Treatments

Given evidence of mitochondrial dysfunction:

• Coenzyme Q10 / Idebenone
• L-Carnitine
• Alpha Lipoic Acid
• Vitamin C/E
• B Complex

Flavin mononucleotide (FMN) Esterified derivative of riboflavin?

Formal studies of effectiveness of complimentary

treatments have not yet been performed

Treatment Outcome

• Treatment with high dose oral riboflavin is both effective and

lifesaving in many patients.

• Short-term: 75%+ have clinical improvement or stabilization of

symptoms.

• The most positive responses are when treatment is received

shortly after disease onset.

• Long-term: Many patients still report new or worsening

symptoms on riboflavin treatment alone.

• More research and complementary novel therapeutic strategies

are needed.

Keith Massey

Science Director, Cure RTD Foundation

Email: Keith.Massey@curertd.org

2 year old patient with RTD type 2 with severe phenotype on 100mg/kg RF/FMN QID. Riboflavin is reactive under UV light. Discolored patches suggests that the pigment is within the stratum corneum, which is mostly keratin