Novel RETREG1 (FAM134B) founder allele is linked to HSAN2B and renal disease in a Turkish family

Abstract

Hereditary sensory and autonomic neuropathy type 2B (HSAN2B) is a rare autosomal recessive peripheral neuropathy caused by biallelic variants in FAM134B. HSAN2B is characterized by sensory impairment resulting in skin ulcerations, amputations, and osteomyelitis as well as variable weakness, spasticity, and autonomic dysfunction. Here, we report four affected individuals with recurrent osteomyelitis, ulceration and amputation of hands and feet, sensory neuropathy, hyperhidrosis, urinary incontinence, and renal failure from a family without any known shared parental ancestry. Due to the history of chronic recurrent multifocal osteomyelitis and microcytic anemia, a diagnosis of Majeed syndrome was considered, however sequencing of LPIN2 was negative. Family-based exome sequencing (ES) revealed a novel homozygous ultrarare FAM134B variant NM_001034850.2:c.321G>A;p.Trp107Ter. Electrophysiological studies of the proband demonstrated axonal sensorimotor neuropathy predominantly in the lower extremities. Consistent with the lack of shared ancestry, the coefficient of inbreeding calculated from ES data was low (F=0.002), but absence of heterozygosity (AOH) analysis demonstrated a 7.2 Mb AOH block surrounding the variant consistent with a founder allele. Two of the four affected individuals had unexplained renal failure which has not been reported in HSAN2B cases to date. Therefore, this report describes a novel FAM134B founder allele and suggests renal failure may be an unrecognized part of the FAM134B-disease spectrum.

INTRODUCTION

Hereditary sensory and autonomic neuropathy type 2 (HSAN2) is an autosomal recessive (AR) disease trait characterized by progressive sensory loss [Ota et al. 1973]. The prevalence of the disease worldwide is unknown. Age of onset is usually at birth or before puberty and predominant symptoms include sensory neuropathy in the distal lower extremities more than the upper extremities consistent with a length dependent distal symmetric polyneuropathy (DSP). The disease is progressive and in the advanced stage, unnoticed injuries, acral mutilations, infections and spontaneous amputations may develop, leading to significant morbidity. Recurrent osteomyelitis is a common finding. Autonomic dysfunction such as hyperhidrosis, tonic pupils, urinary incontinence, and trophic skin changes also occur [Axelrod and Gold-von Simson 2007]. Motor weakness and spasticity have also been reported and are less severe than sensory impairment [Ilgaz Aydinlar et al. 2014; Murphy et al. 2012; Park et al. 2019].

HSAN2 has four subtypes defined by molecular etiology: HSAN2A (WNK1, MIM# 201300), HSAN2B (FAM134B, MIM# 613115), HSAN2C (KIF1A, MIM# 614213), and HSAN2D (SCN9A, MIM# 243000) [Kurth 1993]. Although there is no clear clinical distinction among subtypes, autonomic disorders may be especially prominent in HSAN2B caused by biallelic loss-of-function (LoF) variants in FAM134B [Falcao de Campos et al. 2019; Kurth et al. 2009; Wakil et al. 2018]. FAM134B encodes a cis-Golgi transmembrane endoplasmic reticulum (ER) receptor regulating the ER turnover through reticulophagy [Islam et al. 2018]. FAM134B dysfunction disrupts ER turnover, leading to the accumulation of misfolded proteins. As a result, normal protein homestasis (i.e.proteostasis) is impaired in the ER and progressive neuronal dysfunction occurs [Islam et al. 2018; Kurth et al. 2009]

In the current report, we present the clinical and molecular findings of a Turkish family with a homozygous nonsense variant in FAM134B, c.321G>A;p.Trp107Ter, presenting with recurrent osteomyelitis, ulceration and amputation of hands and feet, sensory neuropathy, hyperhidrosis, urinary incontinence, and renal failure. Absence of heterozygosity (AOH) analysis of exome sequencing (ES) data suggests the variant is a founder allele. The renal failure observed within this family has not been previously reported in HSAN2B-related disease.

MATERIAL and METHODS

Clinical report

The proband (IV.7) is a 46-year-old Turkish female born to parents from the same small village but with no known shared ancestry (Figure 1a). Her developmental milestones were normal. She was referred to Medical Genetics outpatient clinic for progressive multifocal osteomyelitis that started at 7 years of age. On examination, there was swelling in her knees and severe amputations in her distal extremities, especially in her left foot. Her fingernails were dystrophic, and her toenails were autoamputated (Figures 1b and 1c). Some toes were removed surgically. On neurological examination, although atrophy was observed in lower extremity intrinsic foot muscles, strength was grade 5 in all leg and foot muscle groups according to Medical Research Council (MRC) Scale for Muscle Strength. Proprioception and vibration were normal, but there was loss of pain and light touch sensation in a glove and stocking distribution. There was no pain sensation in the legs and feet. Triceps and biceps reflexes were normoactive, brachioradial reflexes were hypoactive and others were absent. There was no spasticity or clonus. Cerebellar examination and cognition were intact.

Figure 1.

a. Pedigree of the family. The proband is highlighted by the black arrow. The phenotypes are shown in different colors, i.e. orange → HSAN-2B and blue → Renal failure.

b. Proband’s (patient IV.7) hands with multiple distal amputations.

c. Amputations in the proband’s feet more prominent in left foot/toes.

d. X-ray of the proband’s hands. Amputations and acrolysis are especially seen in the distal parts of the fingers

e. X-ray of the proband’s feet. Prominent total amputation in the left toes.

f. Prominent amputation on the fourth finger of the left hand of patient IV.5.

g. Patient IV.5’s multiple foot deformities, distal amputations and edematous appearance in the legs.

Laboratory examination demonstrated microcytic anemia. In limb radiographs, amputations especially in the distal fingers, and total amputations of the left toes were observed (Figures 1d and 1e). Electrophysiological study revealed normal nerve conduction velocities (NCVs) and low amplitudes of peroneal and posterior tibial nerve CMAP (compound muscle action potential). All sensory nerve conduction studies showed absent SNAPs (sensory nerve action potential). F-wave latencies were abnormal in the lower extremities and sympathetic skin responses were absent. Needle EMG of anterior tibial, lateral vastus, pronator teres muscles were normal, but there were some neurogenic changes in the right abductor pollicis brevis muscle. These studies were consistent with an axonal sensorimotor neuropathy. Magnetic resonance imaging (MRI) and scintigraphy showed osteomyelitis in the right knee and left foot. Her renal ultrasonography and kidney function tests were normal.

The proband had three additional family members with similar findings. Patient IV.5 is a 53-year-old woman. Her disease onset occurred at 6 years of age. On examination, there was an edematous appearance in both lower legs and skin ulcerations in both hands and feet (Figures 1f and 1g). There was partial spontaneous amputation in the fourth finger of the left hand and the third toe of the right foot. She had been using a wheelchair for 5-6 years. Also, she had urinary incontinence and hyperhidrosis. She died at the age of 53 due to renal failure. She also had microcytic anemia without hemolysis. Creatinine and blood urea nitrogen values were elevated. Single-photon emission computed tomography (SPECT)-Computed tomography (CT) demonstrated osteomyelitis in the left tibia, left distal fibula, and bones of the left ankle joint region. Renal Doppler ultrasonography showed bilateral renal parenchymal disease. Renal biopsy demonstrated inflammatory cell infiltration, diffuse eosinophilic accumulation, patchy fibrosis, tubular atrophy with hyaline casts, myointimal thickening of the arteries, and diffuse, global amyloid deposition. Immunohistochemistry revealed positive staining for amyloid A.

Patient IV.6 had a similar history of ulcerations and amputations. The age of onset was 8 years. She had undergone dialysis for two years due to chronic kidney failure before she died at the age of 47. Finally, only limited history for patient III.1 could be obtained, including that she had mild ulcerations in hands and feet and no renal complaints. She died at the age of 70.

Molecular analysis

Genomic DNA was isolated from peripheral blood samples of the affected patients (IV.5 and IV.7) and their unaffected relatives (III.3, IV.3, IV.4, IV.8 and IV.9). Due to concerns for Majeed syndrome, a recessive disorder associated with chronic recurrent multifocal osteomyelitis, LPIN2 coding exons were sequenced [Ferguson et al. 2005]. However, no LPIN2 variants were identified.

The family was subsequently enrolled under the Institutional Review Board (IRB)-approved research protocol (H-29697) at the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) as part of a cohort of Turkish families with neurogenetic disorders and Ankara University School of Medicine Human Research Ethics Committee. Family-based duo exome sequencing (ES) (index-mother) was performed according to previously reported methods [Pehlivan et al. 2019]. A homozygous FAM134B variant (NM_001034850.2):c.321G>A; p.Trp107Ter was identified in the proband. This variant is absent in gnomAD (v2.1) and has a CADD score (v1.6) of 39. It has not been reported in the literature but is indexed in ClinVar as pathogenic in a clinical testing (RCV001045931.1). The variant is predicted to be pathogenic based on American College of Medical Genetics and Genomics (ACMG) criteria (PVS1, PM2, PP3) [Brnich et al. 2019]. Within the BHCMG database of >12,000 exomes, the variant is only found within this family. Segregation analysis and variant validation was performed by Sanger sequencing which showed that all available affected individuals (IV.5, IV.7) were homozygous whereas unaffected family members (III.3, IV.III, IV.4, IV.8, IV.9) were heterozygous consistent with Mendelian expectations for an autosomal recessive disease trait (Figure 2a).

Figure 2.

a. Sequence chromatograms. Proband and IV.5 are homozygous for the mutated allele. The mother (III.3) and the siblings (IV.3, IV.4, IV.8, IV.9) are heterozygous for the p.Trp107Ter mutation.

b. B-allele frequency for the proband calculated from exome variant data demonstrates a 7.2 Mb block of AOH on chromosome 17 (grey) around FAM134B variant (red line).

c. Schematic structure of FAM134B with two hydrophobic segments harboring reticulon-homology domain (RHD) (purple) and a C-terminal coiled-coil domain (yellow).

To determine identity-by-descent (IBD) intervals, unphased ES data was analyzed by BafCalculator (https://github.com/BCM-Lupskilab/BafCalculator) as previously described [Gonzaga-Jauregui et al. 2020]. The variant is located within a 7.2 Mb absence of heterozygosity (AOH) block with total autosomal AOH of 69.5 Mb (Figure 2b). The estimated inbreeding coefficient calculated from the total sum of AOH >1.5 Mb to the total length of the autosomal genome using the PLINK program (http://pngu.mgh.harvard.edu/purcell/plink/) was 0.002 [Purcell et al. 2007].

DISCUSSION

Hereditary sensory and autonomic neuropathy type 2B, caused by pathogenic variation in the FAM134B gene, is a rare syndrome characterized by autonomic dysfunction and impaired sensation of touch, heat and pain [Kurth et al. 2009]. Here, we report a family with HSAN2B due to a homozygous nonsense variant in FAM134B. Ten families with HSAN2B have been reported to date (Table 1). Despite phenotypic variability between families, almost all patients have osteomyelitis, ulcerations and spontaneous or surgical amputations resulting from profound sensory neuropathy. In the proband described here, the most prominent phenotypes on initial evaluation were progressive multifocal osteomyelitis and microcytic anemia, leading to concern for Majeed syndrome. Majeed syndrome is a recessive autoinflammatory disorder characterized by chronic recurrent multifocal osteomyelitis and congenital dyserthropoietic anemia caused by LPIN2 variants [Ferguson et al. 2005]. When targeted LPIN2 testing was unrevealing, ES identified a homozygous FAM134B nonsense variant, leading to consideration of the diagnosis of HSAN2B. Subsequent sensory examination and electrophysiologic studies confirmed the presence of an axonal sensorimotor neuropathy. Although limited clinical information is available regarding patient III-1, her seemingly milder phenotype compared with patients IV-5, IV-6, and IV-7 may demonstrate intrafamilial variability.

Table-1:

Comparison of features of HSAN-2B cases published so far with our index family.

	Kurth et al., 2009 P-1	Kurth et al., 2009 P-2	Kurth et al., 2009 P-3	Kurth et al., 2009 P-4	Murphy et al., 2012	Aydınlar et al., 2014	Wakil et al., 2018 F1/F2	Campos et al., 2019 F1/F2	Current family
Origin	Saudi Arabia	Turkey	Italy	Dubai	Saudi Arabia	Turkey	F1: Saudi Arabia F2: Saudi Arabia	F1: Turkey F2: Portuguese	Turkey
Consanguinity	+	Same village	ND	+	+	+	F1: + F2: +	F1: + F2: −	Same village
Age of onset	First-second decade	First decade	First years of life	Second decade	5 years	8 years	F1: 1-3 years F2: 4 years	F1: 9-12 years F2: 13 years	6-7 years
Age at the time of publication	ND	ND	ND	ND	10 years	41 years	F1: Three patients (28- nd-10 years) F2: 10 years	F1: Three patients (24-31-35 years) F2: 35 years	IV.5: 53 years IV.6: 47 years IV.7: 46 years
Ulcerations	Hands and mainly feet	+	Feet	Feet	Foot and tibia	Foot	F1: Big toes and soles F2: Feet and toes	F1: Feet F2: Feet	Several fingers and toes
Osteomyelitis	+	+	+	+	+	ND	F1: ND F2: +	F1: ND F2: +	+
Amputations	Hands and feet	Both lower legs	Several toes and metatarsals	+	Right forefoot	First toe of the right foot	F1: − F2: −	F1: Surgical forefoot amputation F2: −	Several fingers and toes
Sensory function	Severely disturbed	Impaired pain and temperature sensation	Severely disturbed (ankle fracture without pain sensation)	Severely disturbed	Sensory neuropathy	“Hypesthesia and hypoalgesia”	F1: Sensory loss F2: Sensory loss	F1: Thermo analgesia and proprioceptive hypoesthesia F2: Thermo-algic and proprioceptive deficit	Loss of pain and light touch sensation and no pain sensation in patients’ feet (analgesic)
Autonomic disturbances	Hyperhidrosis, urinary incontinence	+	Urinary incontinence, hyperhidrosis, slow pupillary reaction to light	Not known	None	Urge incontinence, orthostatic dizziness, excessive sweating	F1: + F2: None	F1: Recurrent vasovagal presyncope F2: None	Urinary incontinence and hyperhidrosis
Sural nerve biopsy	Not done	Axonal sensory neuropathy	Axonal neuropathy with a preference for small myelinated fibres	Not done	Severely reduced density of large myelinated fibers, loss of both myelinated and unmyelinated fibers	Loss of myelinated fibers	Not done	Not done	Not done
Nerve conduction velocities	Signs of axonal sensory neuropathy	Sensory axonal neuropathy, mild motor neuropathy	No sensory responses in lower limbs; severely reduced CMAP amplitudes in the lower limbs with reduced MNCVs	Axonal neuropathy in the legs	Axonal, sensory, motor, large fiber neuropathy	Axonal neuropathy involving motor and sensory fibers	F1: Sensory axonal polyneuropathy in the lower limbs F2: Normal	F1: Axonal sensorimotor neuropathy F2: Severe axonal sensorimotor neuropathy	Axonal sensorimotor neuropathy with lower extremity predominancy and right CTS
Motor involvement	None	None	Tetrapyramidal syndrome, Babinski signs, spastic gait and distal weakness in lower limbs	None	Motor weakness	Proximal distal weakness in lower extremities, spastic gait	F1: Mild weakness in feet and legs, clonus in lower limbs F2: Mild spasticity in the lower limbs, pyramidal weakness	F1: Brisk tendon reflexes, Babinski sign present F2: Mild lower limb weakness and absent Achilles DTR	Mute plantar response, hypoactive styloradial reflexes, absent patellar reflexes
Additional features	None	None	None	None	None	None	None	F1: Scoliosis F2: None	Renal failure Microcytic anemia
Variant (NM_001034850.2)	c.926C>A;p.Ser309Ter	c.433C>T;p.Gln145Ter	c.17_18delCT;p.Pro7GlyfsTer133	c.873+2T>C	c.433C>T;p.Gln145Ter	c.826delA;p.Ser276Valfs*8	F1 and F2: c.926C>A;p.Ser309Ter	F1: c.896_897delAA;p.Lys299Argfs*6 F2: c.1426del;p.Gln476Argfs*57)	c.321G>A;p.Trp107Ter

Abbreviations are as follows: P: patient; F: family; CMAP: compound muscle action potential; MNCV: motor nerve conduction velocities; CTS: carpal tunnel syndrome; DTR: deep tendon reflexes; ND: not documented

FAM134B is located on chromosome 5p15.1, contains nine exons, and encodes a 497 amino acid protein. Pathogenic variants identified in FAM134B include nonsense, frameshift, and splice site mutations (Table 1). No hotspot regions have been identified. The FAM134B protein has two hydrophobic segments harboring reticulon-homology domains (RHD) and a C-terminal coiled coil domain. Figure 2c demonstrates the FAM134B variants reported to date in HSAN2B patients [Bhaskara et al. 2019]. The p.Trp107Ter variant is predicted to either undergo nonsense-mediated decay (NMD) or result in a severely truncated protein with partial loss of the first RHD and complete loss of the second RHD and coiled-coil domain. While the family was unaware of shared ancestry, the identification of a homozygous pathogenic variant and their origin in a small village raised the possibility of unrecognized shared ancestry. However, the proband’s total AOH (69.5 Mb) and estimated inbreeding coefficient based on ES data was low (F=0.002), suggesting that clan genomics identity-by-descent (IBD) was not responsible [Lupski et al. 2011]. As the variant lies within a 7.2 Mb AOH block, the variant likely represents a founder allele homozygosed by population or geographic isolation [Gonzaga-Jauregui et al. 2020].

Two of the four affected individuals exhibited unexplained chronic renal failure. Neither individual had risk factors for chronic kidney disease, and there was no family history of renal disease. To date, renal failure has not been described in HSAN2B. Therefore, HSAN2B-related renal failure could represent a phenotypic expansion or a previously unappreciated feature of the condition. Indeed, if renal failure exhibits age-dependent penetrance in individuals with HSAN2B, it may be underreported as most reported individuals with HSAN2B were of younger ages at the time of publication (ranged from 24 to 41 in reported cases) and therefore may not have yet developed renal disease (Table 1). Age-dependent penetrance was indeed observed in this family as it was not present in the younger proband but was a terminal complication in the two older affected siblings, suggesting it may be an end-stage late complication of the disease. Similarly, motor involvement/weakness is not a defining feature of sensory and autonomic neuropathies; nonetheless it was observed in our subject, some previously reported subjects with HSAN2B, and in some autosomal dominant HSANs. Weakness is not a primary presenting symptom of HSAN and typically occurs in later disease stages as potentially a chronic complication.

Renal disease is associated with several hereditary neuropathies, including HSAN3 (MIM #223900) and Charcot-Marie-Tooth disease (CMT) dominant intermediate E (MIM #610982). Patients with HSAN3 secondary to ELP1 variants develop global ischemic-type glomerulosclerosis due to chronic volume depletion, postural hypotension, and supine hypertension [Rekhtman et al. 2010]. In contrast, patients with heterozygous pathogenic variants in INF2, a regulator of actin filaments, have focal segmental glomerulosclerosis and CMT [Boyer et al. 2011]. Exome analysis did not identify any potential candidate that can explain the renal phenotype in our subjects. The mechanism by which loss of FAM134B might cause renal dysfunction is unclear. Kidney biopsy was performed in patient IV-5 and demonstrated AA amyloidosis. AA amyloidosis results from chronic inflammation [Westermark et al. 2015]. Since FAM134B is involved in the clearance of misfolded proteins via reticulophagy and the unfolded protein response induces inflammation, FAM134B deficiency might cause a pro-inflammatory state [Watkin et al. 2015]. Alternatively, chronic osteomyelitis itself or a combination of both the proinflammatory state plus recurrent infections could be responsible.

In summary, we describe a novel FAM134B founder allele in a Turkish family with HSAN2B. The observation that renal failure co-segregated with disease within the family suggests renal dysfunction may be a late and end-stage feature of HSAN2B, potentially due to autoinflammation related to perturbation of the ER to Golgi axis, as has been found for COPA/STING related inflammatory disease [Konno et al. 2018; Lepelley et al. 2020; Watkin et al. 2015]. Additional case identification and long-term patient follow up is needed to substantiate this observation.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in dbGaP at https://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000711.v7.p2.